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Can immune cells be recruited to an area of inflammation, and later go on to a second?

Can immune cells be recruited to an area of inflammation, and later go on to a second?


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With regards to innate or adaptive immune cells… can naive/immature cells such as neutrophils, monocytes, macrophage/dendritic cells or adaptive T-cells be recruited to an area of inflammation, for instance an infection in the gastric mucosa, then leave that area and be recruited to another different area of inflammation? Or once recruited to a site are they unresponsive to other events?


Newly Recruited Faculty Bring Expertise in Key Research Areas

Thanks to a successful first year of Translating Duke Health (TDH), twelve new faces have popped up around the School of Medicine’s labs, clinics and meeting rooms. These new faculty members—representing a spectrum of career stages—were recruited to Duke because of their expertise in numerous fields including transplantation, neurosurgery, gene therapy, HIV, and more. Known as Translating Duke Health Scholars, they will work with faculty across the School to advance research aimed at addressing major health challenges in five key areas: cardiovascular disease, children’s health, brain metastasis, brain resilience, and immunology. These hires represent the first wave of a large recruitment effort that will continue as the TDH initiative moves into its second year.

Meet the Translating Duke Health Scholars:

Priyamvada Acharya, PhD
Department of Surgery
Director, Division of Structural Biology, Duke Human Vaccine Institute

This is an exciting time for HIV-1 vaccine research with multiple lines of approach showing promise for developing an effective HIV-1 vaccine. This is an exciting time for structural biology as well, with rapid and continuing progress in cryo-electron microscopy (cryo-EM) allowing unprecedented throughput and ability to determine structures of complex samples. Because my lab has expertise in both cryo-EM and x-ray crystallography, we are well-poised to take advantage of cutting edge structural techniques to further our research on HIV-1 entry and vaccine design.

The primary focus of my research is to understand how HIV enters cells in the body, and to use this basic knowledge for vaccines and therapeutics development. We use cryo-EM and x-ray crystallography to determine the structures involved in HIV-1 entry at atomic level resolutions. We are also interested in understanding the interactions of the HIV-1 Envelope (Env) protein with the human immune system. We do this by structural determination of complexes of HIV-1 Env with receptors and antibodies. Atomic level understanding of these interactions helps drive vaccine development.

The Duke Human Vaccine Institute is a world leader in HIV-1 vaccine research. In addition to this, Duke has a strong structural biology community, emerging strengths in cryo-EM that include a state-of-the-art microscopy suite equipped with a Titan Krios microscope, and a supportive environment for new faculty. These were the major deciding factors for me in coming to Duke University.

Aravind Asokan, PhD
Departments of Surgery, Molecular Genetics and Microbiology
Director of Gene Therapy, Division of Surgical Sciences, Surgery

This is an incredibly exciting time for gene therapy. Our lab focuses on understanding non-pathogenic viruses and tinkering with them so they can deliver therapeutics to different tissues in the body. We are particularly interested in applying these engineered viruses for gene therapy. The rationale behind gene therapy is that the root cause of many diseases is at the genetic level. No matter what modality you are looking at – whether it is gene replacement, gene editing, or gene silencing – essentially gene therapy is delivering the tools or the information that is going to correct that situation, in the form of DNA.

There are already a couple of approved products with more on the horizon next year. We are on the verge of a whole new class of medicines. The exciting long-term research questions/breakthroughs are going to be: where else can we use this [gene therapy] approach beyond monogenic diseases? Can we use these viral tools successfully for genome editing? In regenerative medicine, could we genetically manipulate tissues of interest and then use them for transplantation into patients? Are we going to be able to reprogram tissues to regenerate?

We are tremendously excited to be exploring these new paradigms, and Duke is the place to do it. The spectrum of research opportunities, outstanding scientific community, and visionary administration at Duke can’t be beat.

Mihai Azoitei, PhD
Department of Medicine

After many years of research, the field of HIV vaccine design is at a point where promising pathways have been identified to develop an effective vaccine. Therefore, my work is focused on engineering immunogens that could become part of an HIV vaccine.

I work on building proteins that will be effective as vaccines against HIV. Despite many efforts over the last 30 years, no vaccine exists that prevents HIV infection. I hope that the molecules developed in my lab and in collaboration with the other groups at the Duke Human Vaccine Institute, will “train” the immune system to fight and neutralize the virus in case of infection.

I came to Duke because of the excellent scientific environment that encourages collaborations and provides access to the resources necessary to do high impact research. My lab is part of the Duke Human Vaccine Institute, a world leader in vaccine research and development, where basic scientific discoveries can be rapidly advanced from the bench into the clinic.

Francis Chan, PhD
Department of Immunology

Cell death and inflammation are found in many human diseases. My passion is to understand how these two processes are connected with each other. Prior to joining Duke, my lab discovered a form of cell death called “necroptosis” that can powerfully stimulate inflammation. I believe this knowledge is not only interesting at a basic level, but also has the potential to lead to better understanding of how we treat patients suffering from different forms of inflammatory diseases.

In the last few years, there have been numerous reports implicating necroptosis in inflammatory diseases from all tissue and organ origin. These findings point to great promise for understanding how to treat these diseases better in the clinic. However, effective therapeutic approach will require comprehensive understanding of the molecular mechanisms that regulate necroptosis. This is the area where we hope to make the most impact and contribution.

There are strong interests from pharmaceutical companies to pursue necroptosis inhibitors as therapeutic agents for inflammatory diseases including inflammatory bowel diseases and rheumatoid arthritis. The strength of clinical and translational research at Duke makes it an attractive place to merge our basic research program with more clinically-relevant investigation.

Sherika Hill, PhD
Department of Psychiatry and Behavioral Sciences

This is an exciting time to be studying biopsychosocial factors related to adolescent mental health and wellbeing because it is no longer cost prohibitive to conduct large-scale, whole-genome genetic and epigenetic studies. Also, there is a great amount of human genomic data available through free public databases.

Within this spectrum, I am cultivating a research agenda that seeks to understand the development of later psychopathology and chronic illnesses in adolescence based on exposures to early pediatric stress or trauma. In particular, I am curious how these adverse experiences correlate with epigenetic markers, such as DNA methylation and accelerated biological aging, which can disrupt normal biological processes and result in measurable clinical vitals including overweight/obesity and elevated blood pressure. In turn, these clinical indicators of poor or progressively worse outcomes over time substantially increase risk for chronic physical and mental health conditions. Examining these factors longitudinally and prospectively from birth is essential for both preventative strategies and timely clinical interventions.

I chose to come to Duke because of its commitment to innovative, interdisciplinary, collaborative research to promote healthy childhoods by bridging clinical care, basic sciences, social sciences, informatics and policy.

Annette Jackson, PhD
Department of Surgery

The immune system has the ability to discriminate proteins as self versus non-self in order to protect the body from infection. Approximately one third of patients on transplantation waiting lists have generated antibody immune responses to non-self proteins as a result of exposure to blood transfusions, pregnancies, or previous transplants. These antibodies can prevent a patient from finding an acceptable donor or, if the patient is transplanted, these antibodies can significantly reduce the long-term survival of the transplanted organ. The Duke Transplant Center has identified a new treatment strategy that silences these antibody responses and holds the potential of creating safe and successful pathways to transplantation for many waitlisted patients. Leveraging my previous experience at Johns Hopkins, we will develop algorithms for monitoring the efficacy of this new treatment, aid in donor selection to avoid immune memory, and utilize new post-transplant immune monitoring tools to identify the earliest signs of rejection.

The Translating Duke Health initiative has recruited a prestigious group of transplant clinicians and researchers to the Duke Transplant Center with the goal of developing innovative strategies to improve transplant rates and longevity of transplanted organs. I wanted to be a part of this elite team to bring new molecular, serologic and bioinformatics tools into current medical practice and personalize immune monitoring strategies to reduce rejection incidence and improve long-term transplant survival.

Leadership within Duke University School of Medicine has created opportunities to ignite cross-discipline research and facilitate the translation of basic science discovery into improvements in human health. In doing so, we are improving the quality of life for transplant recipients and creating a sense of hope and security for their families and loved ones.

Xunrong Luo, MD, PhD
Department of Medicine

It is an exciting time to be working in transplantation research. My primary research focus is to understand and find ways to control the body's immune responses against transplant organs, so that life-long immunosuppression in organ transplant recipients will no longer be necessary. Ultimately, our research will identify the individual needs in controlling such immune responses, so that strategies to minimize and/or eliminate immunosuppression for each individual transplant recipient can be precisely predicted and optimally personalized.

The past 50 years of clinical transplantation and related research have already built a strong foundation and a mature platform from which innovations can now sprout. Rapid advances in technologies have now made it possible for highly granulated histological, cellular and molecular details of transplant immunobiology to be obtained and archived at individual levels. Our capacity for computational analysis of large data banks is also rapidly advancing and is setting the perfect stage for processing high dimensional data sets and deriving complex algorithms for individualizing treatment options for transplant recipients. Therefore, with necessary resources and seamless collaborations, we are entering an exciting new era of personalized transplantation.

My decision to return to Duke is two-fold. First, I obtained both of my graduate and medical degrees from Duke in 1990s. Therefore, Duke is truly the institution that trained me to be the physician scientist I am today. With my past 20 years of experiences in transplant clinical and basic research, I feel that the timing is now mature for me to return to Duke to contribute my knowledge and expertise to advancing the academic mission of Duke. Secondly, Duke has the nation’s leading experts in transplant surgery, nephrology, pathology and immunology, all of which form the basis of the collaborative network for my research. Integrating into such a collaborative network with its collectively expertise, we are perfectly positioned to accelerate scientific discoveries in transplantation and to translate scientific innovations into transplant clinical practice.

Ashley Moseman, PhD
Department of Immunology

We are hoping to learn how olfactory barrier defense juggles its requirement to protect the brain, yet also smells efficiently. In addition, we are interested in further understanding how the brain and hematopoietic system are able to sometimes work cooperatively to drive infections from the brain, and yet other times, they fail. My goal is to understand the mechanisms that pathogens use to breach the olfactory barrier how immune cells work in concert with the parenchymal cells of the olfactory epithelium to form a barrier and how this barrier can be compromised. Once the olfactory barrier has been compromised, pathogens can access the central nervous system (CNS).

The School of Medicine has invested in a state-of-the art intravital multiphoton microscope that will allow us to visualize cellular interactions within the CNS of living animals. In particular, this microscope will allow our group to better understand the host-pathogen interactions that drive central nervous system diseases, like PAM. Our hope is that insights from understanding basic anatomical mechanisms of olfactory to CNS communication and barrier function will have implications for a number of human diseases including infectious diseases, neurodegeneration, as well as cancer therapies.

Single cell RNA sequencing technology is providing us the ability to gather enormous amounts of information on cell populations we would not have been able to decipher without years of guesswork and labor. These data sets generate nearly limitless genomic foundations for hypothesis driven research. In addition, systems biology and the ability to render massive data sets into workable data sets is transforming basic research. At Duke in particular, there is a great deal of support for cross-disciplinary collaboration which fosters opportunities for different specialists to come up with ideas they’d not typically even considered.

Duke obviously has a reputation for excellence. One of the big attractions was the relatively centralized nature of the medical research facilities. At some institutions the hospital and clinical departments are quite physically separated from graduate and undergraduate campuses. But the tight proximity of things at Duke was appealing because it’s obviously easier to find and interact with collaborators when you are nearby. The youthful vigor that you find at Duke and Durham have been exactly what we were looking for.

John Pearson, PhD
Department of Biostatistics and Bioinformatics

I'm interested in data. More specifically, I'm interested in what data can tell us about how the brain functions, and how new ways of looking at that data can give rise to new theories and suggest new experiments. My lab works on new analysis methods for neuroscience data, from the dynamics of social interaction to vision in the retina.

The amount of data collected by neuroscience researchers is growing exponentially. Machine learning is constantly in the news, and much of the hype is justified. The BRAIN Initiative began under President Obama in 2013 with the ambitious goal of recording the activity of every cell in the brain, but the real challenge is what to do with that information when we have it. My goal is to provide mathematical and computational tools that help experimenters better test new ideas, whether that be analyzing data in real time or understanding rich and complex behaviors.

I came to Duke for my postdoctoral training. I decided to stay at Duke both because my family loves Durham and because Duke provides a real home for the kind of interdisciplinary research my lab does. I'm in a quantitative department in the medical school, but my lab is located directly between the cognitive neuroscientists and the neurobiologists. That wouldn't be possible many other places.

Derek Southwell, MD, PhD
Department of Neurosurgery

I am interested in understanding how brain cells (neurons) develop during early life, and how their dysfunction results in disease conditions. I am also studying the transplantation of neurons as a prospective treatment for nervous system disease and injury.

Advances in imaging, device manufacturing and informatics have allowed neuroscientists to measure and characterize brain function in numerous dimensions. We can now observe the brain's microscopic cellular components in action, both individually and in large aggregates of thousands or millions of cells. We are beginning to grasp how patterns of cellular activity represent what we see and hear, and how they give rise to our movements, speech and moods. This knowledge has potential to drastically improve medical care for patients with neurologic disorders and injuries. As a physician with a strong interest in neuroscience, my intention is to see that the promise of this research is realized for patients.

It takes significant resources and inspiration to pursue translational research. Through TDH, Duke is directing its strengths in science and engineering towards the development of transformative clinical therapies. One of my professional goals is to advance patient care through translational neuroscience, and Duke is one of the few universities that can really support this type of work.

Neil Surana, MD, PhD
Departments of Pediatrics, Molecular Genetics and Microbiology

Work over the past 15 years has demonstrated a clear association between the human microbiota—the trillions of bacteria, viruses, fungi, and Archaea that live in and on each of us and outnumber human cells by at least 10-fold— and a variety of different disease states, ranging from inflammatory bowel disease to Parkinson’s disease to cancer. The field of microbiome research has made great strides in cataloguing the “normal” microbiota, and it now stands at the precipice of an ability to treat the fundamental basis of many diseases. Given its putative central role in driving disease pathogenesis, the microbiota is considered to represent an untapped treasure-trove of therapeutics.

My research seeks to understand how the microbiota influences susceptibility to inflammatory disease. More specifically, I seek to identify and characterize “healthy” bacteria that can modulate the immune system, with the ultimate goal of translating these bacteria and/or their products into clinical practice to treat various inflammatory and autoimmune conditions.

Duke is widely known as a clinical powerhouse, and it also has burgeoning strength in microbiome-related research that is supported by the recent advent of the Duke Microbiome Center. Given my interest to work on translational aspects of the microbiome, the decision to come to Duke was an easy one. My hope is that I will be able to help cross-pollinate ideas between the clinical and basic science worlds to ultimately accelerate the development of microbiota-based therapeutics.

Andrew West, PhD
Department of Pharmacology and Cancer Biology

The last 20 years of research has pinpointed the critical action of just a few proteins in the most common neurodegenerative diseases, and tools are just becoming available to test whether these proteins are as important as we think they are in the progression of disease. Many of us are optimistic that soon Parkinson’s disease and Alzheimer’s disease will become problems of the past.

Our lab focuses on mechanisms underlying neurodegenerative diseases and experimental therapeutics that might slow or halt disease progression. I chose to come to Duke because it offers substantive research infrastructure and a multidisciplinary collaborative environment.


Vaccines are an important health policy tool and have changed the history of infectious diseases. In recent years, the number of vaccines injected to infants have increased, and many doses are administered during the first year of life, when the immune system and the central nervous system have yet to complete their development. Moreover, the immune system and the brain are bonded for life, dependent upon each other in sickness and in health [1]. In addition, at the same time, each immunological challenge is a challenge for the brain, and each vaccination is a challenge for both. Each injection of vaccine, regardless of the type, is followed by the production of variable amounts of pro-inflammatory cytokines, which exert both local effects and at a distance from the production site.

Since the peripheral cytokines, produced after the injection of the vaccines, are able to reach the central nervous system, we have hypothesized, in our recent paper [2], that these cytokines can have effects on the microglia (macrophages of the central nervous system). Microglia are the primary responders to an immune challenge and the primary producers of cytokines and chemokines within the brain. Microglial activation is the initial cellular event that occurs during acute neuroinflammation [3]. Furthmore, timing of a developmental immune challenge can be critical in determining the long-term outcomes on brain and behavior [4], since cognitive function and immune function are inextricably linked [1].

Since the post-vaccination adverse events (AEs) are related to the different period of life (childhood or adolescence), and to the different nervous area involved (brain or spinal cord) in this article we will deal with the regressive form of ASDs and present our hypothesis of a new post-vaccination inflammatory syndrome triggered by HPV vaccines. In our discussion we will only manage with molecular biology and our work is not suitable for improbable comparisons with epidemiological studies on vaccines. In this case, our task will be to describe the biological plausibility that links vaccine injections to these two clinical entities. Therefore, we will also proceed with a review of the specific scientific literature published on the two topics to find the evidence that supports our scientific hypothesis.


Introduction

Stroke is one of the major diseases threatening physical and mental health, with the characteristics of high morbidity, mortality, disability rate and recurrence rate, and brings heavy burden to families and society (1). Stroke is divided into two types, namely ischemic stroke (cerebral infarction) and hemorrhagic stroke (cerebral hemorrhage), of which 85% is ischemic stroke. Cerebral ischemia may result in secondary brain injury and neuronal death, producing inflammatory mediators and leading to inflammation in brain tissue (2).

Following cerebral ischemia-reperfusion, reactive oxygen species (ROS) cause initial breakdown of the blood brain barrier (BBB) by upregulating inflammatory mediators and activating matrix metalloproteinases (MMPs). The initial BBB breakdown occurs within 3 h after stroke onset and is accompanied by vasogenic edema. Destruction of the BBB induced by stroke promotes the migration of immune cells to the brain (3). Acute brain injury causes the release of damage associated molecular patterns (DAMPs) and the contents from dying and necrotic neurons into the extracellular environment, and subsequently trigger intense innate immune response involving microglia and infiltrating leukocytes (4). Ischemic stroke results in up-regulated expression of integrin on the leukocyte surface and of the corresponding adhesion molecules on the endothelium. Leukocytes wrap around the vascular endothelium before being activated by chemokines. Subsequently, the leukocytes firmly adhere to the endothelium and undergo either transcellular or paracellular diapedesis through the endothelial layer (5). In the early stages of cerebral ischemia, leukocytes are recruited by cell-adhesion molecules expressed on endothelial cells and enter parenchyma at a later stage. Recruitment of leukocytes, including neutrophils, monocytes and lymphocytes, is a continuous process and has a significant impact on the pathogenesis of ischemic brain injury (6).

Microglia in central nervous system (CNS) and peripheral immune cells, including blood-derived monocytes/macrophages, neutrophils, and lymphocytes, are recruited into the ischemic cerebral hemisphere which induce inflammatory response (4, 7). The inflammatory cascade in brain tissue could accelerate, expand or delay, alleviate ischemic brain injury (8). There are two main sources of macrophages infiltrating into ischemic brain tissue after stroke: microglia derived macrophages (MiDM) and monocytes derived macrophages (MoDM). Microglia originate from the migration and differentiation of macrophages during the primitive hematopoiesis of the fetal yolk sac, and they are resident into the brain in the early stage of fetal development, and maintain the proliferation ability during the postnatal development. By contrast, granulocyte-monocyte progenitors are the precursors of macrophages during development and adulthood (9). After ischemic stroke, peripheral monocytes are migrated through the BBB to the ischemic brain under the action of chemokines and cell adhesion molecules. Therefore, a series of changes of macrophages after stroke and their effects on disease progression are extremely complex.

In this review, we will discuss the role of different types of immune cells in the secondary inflammatory responses after stroke. We focus on the different phenotypes and functions of macrophages in ischemic stroke and briefly introduce the anti-cerebral ischemia therapy targeting macrophages.


Inflammation Explained

Our understanding of inflammation dates back to the 1st century, when Roman medical writer Aulus Cornelius Celsus described the four cardinal features of inflammation: heat, swelling, pain and redness. A fifth feature, loss of function, was added later in the century by the Greek medical researcher Galen. Whether you stub your toe, burn a finger or get hit with the coronavirus, your body sends a flood of immune cells to the scene, where they gobble up bacteria, viruses, dead cells and debris.

Eduardo Marbán, MD, PhD
Executive Director, Smidt Heart Institute Mark Siegel Family Foundation Distinguished Chair

White blood cells called neutrophils rush to the area to fight infection (they constitute pus). Blood-borne cells called monocytes take up residence inside the tissue. And cells called macrophages (which means “big eaters” in Greek) begin releasing compounds called cytokines, which then sound the alarm for reinforcements. Soon, troops of immune cells flood the site, destroying foreign invaders and damaged tissue in equal measure. Once all of the pathogens are annihilated and the last troops of cytokines attack, the inflammatory process recedes and makes way for healing.

"Inflammation is largely the body's defense mechanism against things that should not be in the body," says Eduardo Marbán, MD, PhD, executive director of the Smidt Heart Institute. "But as with any complicated defense system, any misstep can lead to friendly fire." The same physiological process that reddens the skin around an insect bite and causes swelling in a bum knee can also lead to a host of ailments, ranging from cancer and depression to diabetes and severe cases of COVID-19.

Usually, inflammation enables our bodies to fight off bacteria, viruses and other toxins. But if that immune response continues unchecked, even after the threat has passed, the immune system can turn on healthy tissue. "We have to find a way to target inflammation so that we block its harmful effects without interfering with the beneficial effects," says Prediman K. Shah, MD, director of the Oppenheimer Atherosclerosis Research Center at the Smidt Heart Institute.

It’s that relentless, harmful type of inflammation that’s capturing the attention of scientists and the public. Study protocols and news headlines are increasingly focusing on the deleterious effects of chronic inflammation. It shakes up cholesterol deposits in our arteries, increasing the risk of heart attacks. It gobbles up healthy nerve cells in the brain, leading to memory loss and Alzheimer’s disease. It may even encourage cancerous cells to grow and thrive. Inflammation, as it turns out, could be the engine that drives the most feared diseases, including COVID-19.

Getting to The Heart of Disease

In the 1800s, a German pathologist named Rudolf Virchow suggested that atherosclerosis was an inflammatory disease. “The idea got lost in translation,” Shah says. “That is, until we realized that cholesterol buildup and the subsequent activation of inflammatory cascades within the body are actually what does the most damage to the arteries and organs, whether that’s the heart, the brain or other tissues.”

The immune system views plaque buildup in the arteries as a foreign invader and sends immune cells and other molecules to the scene of the suspected crime. Instead of healing, though, the cells become trapped inside the plaques. It’s likely that chronic inflammation can make these plaques more vulnerable to rupturing.

“We used to think that atherosclerosis was really like buildup of rust in a pipe—a passive process that results from cholesterol deposits in the artery,” Shah says. “Now we know there’s an active component to plaque buildup that involves the immune system and that actually orchestrates the evolution of the plaque, its progression and its eventual destabilization.”

Inflammation can trigger LDL cholesterol (the “bad” type) to latch onto arterial walls, which in turn causes the plaques to become unstable and even burst, leading to clots that cut off the heart’s blood supply. Of course, once a heart attack occurs, inflammatory cells come in to clean up the debris. The body has to strike a perfect balance between clearing up the dead tissue and producing normal, not excessive, healing.

Moshe Arditi, MD
Director, Infectious and Immunologic Disorders Translational Research Center Academic Director, Division of Infectious Diseases Executive Vice Chair of Research, Department of Pediatrics GUESS?/Fashion Industries Guild Chair in Community Child Health

As it turns out, staving off infection could be at the heart of preventing a whole host of diseases.

“People who have gum disease, chronic lung infections and inflammatory diseases, even autoimmune inflammatory diseases, such as rheumatoid arthritis and lupus, are at higher risk of developing heart disease,” says Moshe Arditi, MD, director of the Infectious and Immunologic Disorders Translational Research Center. “Even a mild infection, such as influenza, can make things a lot worse for people who have coronary plaques.”

The reason, he suspects, is that their bodies are already in an inflammatory state. And it may work both ways. An initial infection can set off chronic inflammation that is tied to all sorts of diseases, including inflammatory bowel disease, Alzheimer’s disease and cancer.

Scientists are hard at work trying to devise a way to block the molecules that induce inflammation. The hope, of course, is that one day they’ll be able to prescribe a medication that prevents inflammatory processes from taking a dangerous turn. To some degree, a class of medications called statins already plays that role. “In addition to reducing cholesterol, these drugs also work on reducing inflammation, so it’s a dual pathway,” Arditi says.

Studies show that aspirin, too, not only can protect against heart attacks but also may play a protective role in colon cancer and Alzheimer’s disease by reducing inflammation in the digestive tract and brain. Lifestyle factors, such as diet, exercise and sufficient sleep also keep inflammatory processes in check. So it’s no surprise that these same lifestyle factors can help curb the rate of the nation’s second most common killer: cancer.

Scientists now think that mutation and inflammation are mutually reinforcing processes that can transform normal cells into deadly tumors. “Low-level inflammation feeds cancer cells, encouraging them to grow, thrive and proliferate,” explains Neil Bhowmick, PhD, director of the Cancer Biology Program in the Samuel Oschin Comprehensive Cancer Institute.

Great Communicator

The body’s immune system acts as a gatekeeper, detecting sources of harm and obliterating them before they can do damage. Inflammation is the immune system’s messenger, sharing information between organ systems, including one of the most sensitive and responsive areas: the gut.

Suzanne Devkota, PhD
Director, Microbiome Research, F. Widjaja Foundation Inflammatory Bowel and Immunobiology Research Institute

"The largest immune network in the body resides in the gut," says Suzanne Devkota, PhD, director of microbiome research in the F. Widjaja Foundation Inflammatory Bowel and Immunobiology Research Institute. As such, the brain and gut send signals back and forth, sounding an alarm when they sense a threat. "It turns out that the gut microbiome—that is, the trillions of microorganisms that live within us and on our skin—profoundly affects both immune responses. We believe it affects brain function, too."

Like security guards at the gates of the digestive system, the immune system’s role is to allow the good guys (like vitamins, minerals, proteins and fatty acids) to enter the body and the bad guys (like toxins and other pathogens) to be turned away.

"Our gastrointestinal tract is essentially a hollow tube from our mouth to our anus, so our bodies are really shaped like a donut," Devkota says. "That means the gut is equally exposed to the outside of our bodies as the inside—and it sees all foreign exposure first. So it makes sense that systemic inflammation likely originates in the gut."

When the gut is operating well, the toxins can’t get past the intestinal cells and their tight junctions. In a healthy digestive tract, the white blood cells never even see the invaders. But if too much bad bacteria infiltrates the gut and the balance of good bacteria to bad is out of whack, the lining of the digestive tract can be damaged—loosening those once-tight junctions.

"But if we take care of our gut by eating a diversity of whole foods, especially fiber-rich fruits and vegetables, and avoiding unnecessary antibiotics, we can potentially delay or stave off systemic inflammation."

With the intestinal barrier that separates microorganisms from the rest of the body is compromised, particles, toxins and bacteria can enter the bloodstream and cause further damage. That breakdown of the gut’s intestinal lining causes the immune system to go into overdrive while trying to take out foreign invaders, which can lead to generalized inflammation that wreaks havoc on every organ system in the body.

"But if we take care of our gut by eating a diversity of whole foods, especially fiber-rich fruits and vegetables, and avoiding unnecessary antibiotics, we can potentially delay or stave off systemic inflammation," Devkota says. In theory, that means we can also make a dent in the incidence of disease states that affect the heart, lungs, joints and brain.

Like the gut, the brain has a gatekeeper to prevent toxins from gaining access to healthy tissue. Called the blood-brain barrier, it acts as a physical barricade while cells called microglia travel through the brain to keep a lookout for possible danger. If the brain is confronted with Parkinson’s disease, Alzheimer’s disease or even an infection like COVID-19 that dumps inflammatory proteins into the blood, the microglia react.

With Alzheimer’s disease, for example, the immune system mistakenly assumes the disease’s plaques and tangles need to be cleared out, so it overreacts, sending in inflammatory cytokines that damage the brain. Unfortunately, healthy brain cells can get caught in friendly fire. Just like the plaques that build up in vessel walls and begin churning out inflammatory proteins, the influx of toxic proteins in the brain transforms protective microglia into disease-like cells.

"Instead of protecting healthy brain cells, these disease-associated microglia begin pumping out high levels of inflammatory proteins that exacerbate neurodegenerative processes," says Maya Koronyo, PhD, an Alzheimer’s disease and neuroimmunology research scientist. "That’s why tackling neuroinflammation early in the disease process is key to fighting the disease."

The idea is to return diseased-associated microglia to their protective state. Then those healthy microglia can communicate with other cells in the body that the threat has passed, ultimately restoring blood flow to once-diseased vessels, in a sort of positive snowball effect.

"What we need is a multi-targeted approach to strengthen the types of cells that are protective and dump some cells that could be overly reactive," Koronyo says.

Illustration: Jason Holley


Trafficking of immune cells in the central nervous system

1 Division of Biomedical Sciences, University of California, Riverside, California, USA. 2 The Centenary Institute for Cancer Medicine and Cell Biology, Newtown, New South Wales, Australia. 3 Department of Pathobiology, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, USA.

Address correspondence to: Emma H. Wilson, Division of Biomedical Sciences, University of California, Riverside, Riverside, California 92521, USA. Phone: 951.827.4328 Fax: 951.827.5504 E-mail: [email protected]

1 Division of Biomedical Sciences, University of California, Riverside, California, USA. 2 The Centenary Institute for Cancer Medicine and Cell Biology, Newtown, New South Wales, Australia. 3 Department of Pathobiology, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, USA.

Address correspondence to: Emma H. Wilson, Division of Biomedical Sciences, University of California, Riverside, Riverside, California 92521, USA. Phone: 951.827.4328 Fax: 951.827.5504 E-mail: [email protected]

Find articles by Weninger, W. in: JCI | PubMed | Google Scholar

1 Division of Biomedical Sciences, University of California, Riverside, California, USA. 2 The Centenary Institute for Cancer Medicine and Cell Biology, Newtown, New South Wales, Australia. 3 Department of Pathobiology, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, USA.

Address correspondence to: Emma H. Wilson, Division of Biomedical Sciences, University of California, Riverside, Riverside, California 92521, USA. Phone: 951.827.4328 Fax: 951.827.5504 E-mail: [email protected]

The CNS is an immune-privileged environment, yet the local control of multiple pathogens is dependent on the ability of immune cells to access and operate within this site. However, inflammation of the distinct anatomical sites (i.e., meninges, cerebrospinal fluid, and parenchyma) associated with the CNS can also be deleterious. Therefore, control of lymphocyte entry and migration within the brain is vital to regulate protective and pathological responses. In this review, several recent advances are highlighted that provide new insights into the processes that regulate leukocyte access to, and movement within, the brain.

The observations of Paul Ehrlich in the late 19th century that water-soluble vital dyes injected into the peripheral circulation would stain all organs except the brain provided the first indication that the CNS was anatomically separated from the rest of the body ( 1 ). Subsequent studies by Edwin Goldmann, showing that dye injected into the spinal fluid did not stain peripheral tissues, confirmed the idea that the brain was a unique anatomical compartment ( 2 ). We now know that this feature is a consequence of the existence of the blood-brain barrier (BBB), which limits access of soluble factors to the CNS and restricts access of immune cells to this site ( 3 – 5 ). Combined with the lack of an obvious lymphatic system, low constitutive levels of MHC class I and II molecules, local production of suppressive factors, and, in the normal state, limited numbers of professional antigen-presenting cells, these features all reinforced the concept of the CNS as an immune-privileged site ( 6 , 7 ).

The work of Peter Medawar in 1948 on graft rejection provided some of the first experimental evidence indicating that the brain might not be an immunologically pristine site ( 8 ). Those experiments demonstrated that skin transplants in the brain of naive animals did not provoke an immune response, but if animals were first exposed to graft antigens, such that immune cells in the periphery were “educated” beforehand, grafts would be rejected. It is now appreciated that these events involve the ability of a graft-specific adaptive immune response that is primed in the periphery to access the CNS and mediate rejection of the foreign tissue ( 9 ). It is also apparent that cells of the immune system have access to the three distinct anatomical compartments (i.e., cerebrospinal fluid [CSF], meninges, and parenchyma of the brain) that are relevant to the CNS under physiological circumstances and disease states. Several neurodegenerative, physical, and infectious diseases can be modeled in the mouse, allowing direct analysis of inflammatory processes in the brain (Table 1) to corroborate observations from human postmortem tissue analysis, CSF samples, and biopsies. Thus, the presence in the brain of neutrophils in the context of bacterial meningitis ( 10 ), eosinophils associated with migrating helminths ( 11 ), T cells in post-vaccinal or post-infectious CNS autoimmunity ( 12 ), and plasma cells (Mott cells) during African sleeping sickness, which is caused by Trypanosoma spp. ( 13 ), illustrate that innate and adaptive immunity are operational at this site. Indeed, immune cells are required to control certain viral, bacterial, fungal, and parasitic pathogens that affect the brain. For example, infection with the parasite Toxoplasma gondii leads to a latent infection in the CNS, and T cells are essential for its long-term control. This is illustrated by the development of toxoplasmic encephalitis in previously infected individuals that acquire defects in T cell functions ( 14 ). This can be recapitulated in experimental models in which chronically infected mice depleted of T cells develop uncontrolled parasite replication in the brain ( 15 , 16 ). A similar requirement for immune cells in the CNS has been shown for the control of many pathogens, including the human polyomavirus JC virus, which causes progressive multifocal leukoencephalopathy ( 17 ) Cryptococcus spp., which can cause meningitis and cytomegalovirus ( 18 ), which can cause encephalitis. These examples illustrate the importance of immune surveillance in the CNS.

Modeling inflammation and cell trafficking in the CNS

Although the ability to recognize infections in the CNS is required to limit pathogen replication, this response is not always beneficial. The presence of the rigid bone casing of the brain makes the classic features of an inflammatory response, such as swelling and expansion, a dangerous prospect. Similarly, the development of pathology associated with meningitis and/or encephalitis can lead to reduced neuronal function and survival ( 19 , 20 ). In addition, although the immune system can contribute to the successful resolution of tissue damage associated with many CNS disorders ( 21 , 22 ), there is also an appreciation that inflammation in the brain may contribute to the pathogenesis of multiple neurodegenerative conditions, including Parkinson disease, Alzheimer disease, and lysosomal storage diseases ( 23 ).

This association of inflammation and adverse events may explain why the brain seems to be governed by a unique set of immunological guidelines. A clear understanding of these “rules” may inform the design of strategies to augment protective immune responses to infection while minimizing collateral damage. Similar principles would apply to tumors in the CNS and may allow for the design of rational treatments that provide better access for T cells to this relatively immune-privileged site. Conversely, in the case of autoimmune conditions of the CNS, such as MS, the ideal therapeutic strategy would ameliorate the pathological response while still allowing normal immune surveillance.

The past two decades have seen remarkable advances in understanding how cells of the immune system can access the CNS, and several recent studies have highlighted the basis for immune surveillance of this organ ( 24 – 29 ). More recently, the ability to directly image immune cells in the context of live tissue has been possible using multi-photon microscopy. This technique, originally used to visualize neuronal morphology in the brain ( 30 – 32 ), has allowed the observation of fluorescently labeled immune cell populations and their migratory and interactive behavior in primary and secondary lymphoid organs, as well as peripheral tissues, during thymic selection, priming, and activation ( 33 , 34 ). Imaging of the brain presents unique challenges because the skull prevents direct access to the tissue, but brain slices and explanted tissues can be kept viable in warmed and aerated media, and partial removal or thinning of the skull in anesthetized mice can be conducted. These techniques have allowed imaging of CNS-resident cells — neurons ( 30 , 32 , 35 , 36 ), microglia ( 37 , 38 ), and astrocytes ( 28 , 39 , 40 ) — over the course of hours and days. In addition, imaging of inflamed brains and spinal cords has provided unprecedented insights into the behavior of immune cell populations in the CNS. In this review, we discuss these advances in the context of the trafficking and behavior of immune cells during protective and pathological immune responses in the CNS.

The presence of the BBB, which limits the entry of cells and pathogens to the brain, in addition to the lack of obvious lymphatics in the brain, indicate that there are a limited number of portals to and from the CNS. To appreciate the barriers that exist for immune cells accessing the brain, it is important to recognize that the brain has three membranes — the dura mater (outer), the arachnoid membrane (middle), and the pia mater (inner) — that enclose the parenchyma of the CNS (Figure 1). The BBB encompasses the capillaries and postcapillary venules in the brain and spinal cord and is composed of specialized endothelial cells, linked by complex tight junctions comprised of transmembrane adhesion molecules including cadherins, junctional adhesion molecules, occludin, and claudins ( 41 , 42 ). This structure limits the transport of specific factors and solutes, including >98% of antibodies and small molecules, into the parenchyma, while ensuring the efflux of others ( 7 , 43 ). The structure of this endothelial barrier acts to limit leukocyte trafficking directly across the BBB ( 44 ). Importantly, following injury to the CNS, the activation of endothelial cells and associated cells such as astrocytes can lead to reduced tight junction integrity and formation of transendothelial cell channels ( 42 , 45 , 46 ), thereby facilitating the migration of leukocytes across and through the BBB into the brain ( 43 , 47 , 48 ). The migration of leukocytes into the CSF is thought to occur through the choroid plexus and into the subarachnoid space (which contains the CSF), and their migration from the blood directly into the brain parenchyma occurs across the BBB via the perivascular space (Figure 1). In addition to the differences in barrier properties, the vasculature itself differs between CNS compartments, and this may influence immune cell access. Thus, the capillaries of the meninges have a simple one-layer structure, whereas the postcapillary venules of the parenchyma require cells to transition across inner and outer basement membranes (for detailed reviews of these processes see refs. 4 , 7 , 41 , 49 ). In the following sections we discuss the circumstances and mechanisms facilitating access to these specific compartments.

The structure of the brain and routes of leukocyte entry. Beneath the skull lie three membranes that enclose the parenchyma of the brain: the dura mater, the arachnoid membrane, and the pia mater. The latter two enclose the subarachnoid space. (i) Leukocytes can enter across the choroid plexus, where CSF is produced by the choroid plexus epithelium in the ventricles. CSF containing leukocytes then enters the subarachnoid space, circulates around the brain, and (ii) exits via the venous sinus to be resorbed by the blood via the arachnoid villi. (iii) Blood supply to the brain enters in the subarachnoid space over the pia mater, generating the perivascular space (or Virchow-Robin space). Main arterial branches divide into capillaries, which terminate deep within the brain, supplying the parenchyma with blood. Leukocytes can potentially enter from the blood (iii), which requires them to cross the tightly regulated vascular endothelium (i.e., the BBB: the glia limitans, the subarachnoid space, and the pia mater). Cells can adhere to the endothelium and arrest at any point during this process.

As highlighted earlier, there are numerous pathogens that invade the CNS and/or establish latent infection with the potential to cause disease. Consequently, there is a need for immune surveillance — a continuous process whereby the peripheral immune system is able to monitor the brain for signs of infection or tissue damage. This is likely distinct from the events involved in the recruitment of various immune populations to sites of ongoing infection or inflammation. In the context of immune surveillance, the compartment that has been best studied is the CSF. The choroid plexus, which is located in the ventricles of the brain (Figure 1), has secretory epithelium that produces the CSF. Unlike the BBB, the fenestrated endothelial cells of the choroid plexus lack tight junctions that would normally limit diapedesis of leukocytes. Therefore, although immune cells still have to negotiate the tight junctions of the choroid plexus epithelium, it appears that this site is specialized to allow lymphocytes more ready access to the CSF (Figure 2). Instructively, the composition of immune cells in the blood and CSF differs. Under normal circumstances, the CSF contains few innate immune cells but a much higher percentage of memory or antigen-experienced CD4 + T cells than the blood ( 50 , 51 ). This observation suggests that it is these cells that are specifically involved in immune surveillance.

Immune surveillance via the choroid plexus. The choroid plexus is composed of highly invaginated loops of capillaries and pia mater that reach into the ventricles of the brain. Cells from the blood and under the influence of chemokines undergo adhesion, rolling, and diapedesis across the fenestrated capillary endothelium and pia mater of the choroid plexus. The basement membrane and tight junctions of the choroid plexus epithelium provide a further barrier, the brain-CSF barrier. These modified epithelial cells (Kolmer cells) have bulbous microvilli that secrete the CSF. Infiltrating leukocytes migrating through these cells enter the ventricles that contain CSF and circulate around the CNS. The chemokine CCL20 is expressed on the basolateral side of the choroid plexus epithelial cells, attracting CCR6-expressing CD4 + T cells. Chemokines and their receptors demonstrated to be involved in the trafficking of immune cells into the CSF are provided in Table 2.

Activated T cells upregulate many integrins and adhesion molecules, enabling their rolling and adhesion to vessel walls. However, deciphering which of these molecules are necessary for immune surveillance is complex. Multiple integrins, chemokine receptors, and adhesion molecules expressed on circulating and CNS-resident cells have been implicated in this process ( 50 – 52 ). The most likely candidates are those expressed constitutively in the CNS in the absence of ongoing inflammation. These include the adhesion molecule P-selectin ( 51 , 53 ) the adhesion molecules vascular cell adhesion molecule 1 (VCAM1) and intercellular adhesion molecule (ICAM1), which bind to very late antigen-4 (VLA-4, also known as α4β1 integrin) and lymphocyte function–associated-1 (LFA-1), respectively ( 54 ) and the chemokines CCL19 and CCL20 ( 29 , 55 , 56 ), all of which are constitutively expressed by epithelial cells of the choroid plexus (Figure 2). One recent study suggested a model in which, as part of normal immune surveillance, CD4 + T cells specific for the autoantigen myelin oligodendrocyte glycoprotein (MOG) had to express CCR6, a receptor for CCL20, for optimal access to the CSF before they could initiate experimental autoimmune encephalomyelitis (EAE) ( 29 ). However, other studies have indicated that the main contribution of CCR6 is to the priming of myelin-specific CD4 + T cells in the periphery and that CCR6 is not actually required for the ability of effector cells to access the CNS ( 57 ). Similarly, Th1 cells, which are characterized by the production of IFN-γ and are required for resistance to multiple viral, bacterial, and parasitic pathogens that affect the CNS, do not express CCR6 and can be recruited to a site of ongoing inflammation in the CNS independently of CCR6 ( 29 ). This is consistent with reports that the majority of T cells in the CSF express CXCR3, a receptor normally associated with Th1 cells ( 58 , 59 ). Clearly, further studies are required to determine whether pathogen-specific (or autoantigen-specific) Th1, Th2, or Th17 and/or central memory T cells are involved in this route of immune surveillance as well as whether there are specific trafficking requirements for Tregs as a mechanism to limit inflammation ( 60 – 62 ).

Although the ability of lymphocytes to enter the CSF has been studied in the context of autoimmune inflammation and during homeostasis, many studies on immune cell access to the meninges have been carried out in the context of infection. Inflammation of the meninges is associated with viral (herpes simplex virus [HSV], varicella zoster virus, and HIV), bacterial (Neisseria meningitidis, Streptococcus spp., Haemophilus spp., and Mycobacterium tuberculosis), fungal (Cryptococcus spp.), and parasitic (apicomplexa, trypanosomes, and amoebae) infections as well as with various noninfectious causes such as cancer or as a consequence of certain drugs or immunoglobulin therapy.

In mouse models of Theilier’s murine encephalomyelitis virus (TMEV) and lymphocytic choriomeningitis virus (LCMV) infection, intracranial injection leads to leukocyte accumulation in the meninges and a fatal pathology ( 63 , 64 ). Although it has long been known that CD8 + T cells are required for this to occur, it has recently been highlighted that this is independent of their cytolytic function, and intravital imaging of this process provided an unprecedented view of these events ( 64 ). In these studies, intracerebral LCMV challenge led to the infection of stromal cells in the meninges and adjacent astrocytes present in the parenchyma. This in turn resulted in the accumulation in the meninges of virus-specific CD8 + T cells that had a low migratory velocity. Blockade of MHC class I molecules in the subarachnoid space substantially increased the average T cell velocity (from approximately 3 μm/min to approximately 5 μm/min) and decreased both the time and the proportion of T cells that remained stationary, suggesting that T cell behavior was influenced by MHC class I–dependent recognition of infected cells. Thus, in addition to integrins and adhesion molecules, antigen recognition may be a further level of control required for T cell entry into the CNS and/or retention of these cells at that site ( 28 , 65 – 67 ). However, meningeal LCMV-specific CD8 + T cells did not appear to arrest or form long-term interactions associated with efficient CTL killing of target cells ( 26 , 68 ). Rather, these cells mediated the recruitment of neutrophils and monocytes to the meninges. Imaging of these events revealed that these latter populations crossed the meningeal endothelium in such numbers that they elicited BBB breakdown and vascular leakage, which was the main cause of death ( 64 , 69 ). In contrast to the random migration of T cells that have not yet encountered antigen ( 70 ), the swarming behavior of neutrophils and monocytes at the meningeal surface involved highly directed migration, with many cells clumping, localizing to the subarachnoid space, and migrating continuously over the same area ( 64 ). Similar behavior by neutrophils in the periphery has been described during parasite-induced inflammation, where neutrophil clusters are associated with infected cells ( 71 , 72 ). Regardless, in the LCMV model, the leakiness of the vasculature could be observed following i.v. injection of fluorescent quantum dots and coincided with neutrophil extravasation. These studies suggest a model in which infected cells, including stromal cells of the meninges and closely associated astrocytes below the pia mater, promote the recruitment and activation of CD8 + T cells, likely via the release of chemokines and cytokines. These activated lymphocytes can then cross the meningeal vessel walls and encounter infected cells, which promotes T cell production of chemokines and leads to the recruitment of neutrophils and monocytes, which cause vascular leakage. Similar events occur during infection with mouse hepatitis virus (MHV) ( 63 ) and are consistent with data that neutrophils promote a breakdown in vascular integrity in the CNS ( 73 ). The distinct behavior of T cells and neutrophils in this microenvironment suggests that it might be possible to selectively alter the recruitment of individual cell populations to the CNS to prevent immune pathology, without compromising protective antipathogen responses.

Adhesion molecules. Several studies that directly visualized the molecular steps that mediate the access of T cells to various compartments associated with the CNS have been performed by imaging the superficial vessels in the meninges that are associated with the spinal cord as well as pial vessels and parenchymal branches in a variety of model systems ( 74 – 78 ). In the context of EAE, several reports have described the initial tethering and rolling of leukocytes along the endothelium prior to firm adhesion ( 25 , 79 ) and migration against the blood flow ( 74 ). Similarly, intravital microscopy studies of HSV-infected mice have demonstrated increased leukocyte rolling and adhesion in the microvasculature of the pia mater of infected mice ( 80 ), and similar neutrophil behaviors in the meninges have been identified during LCMV infection ( 64 ). These observations are consistent with the idea that inflammation in the CNS, either due to autoimmune responses or infection, leads to increased expression of adhesion molecules on endothelial cells of the BBB and choroid plexus, including members of the selectin family cell adhesion molecules of the immunoglobulin superfamily, for example, ICAM1, VCAM1, and PECAM1 and members of the integrin family (Figure 3) ( 51 , 53 , 75 , 81 – 83 ). This topic has been reviewed extensively elsewhere ( 41 , 49 , 76 ) and therefore is not discussed in detail here. Nevertheless, the biology of the integrin dimer VLA-4 (α4β1 integrin) and its ligand VCAM1, as well as their association with the development of MS and EAE, are particularly instructive in thinking about the need to balance immune access to the brain.

Leukocyte trafficking across the glia limitans into the parenchyma of the brain. Activated leukocytes expressing adhesion molecules and integrins roll and attach to the vascular endothelium. Successful diapedesis requires appropriate ligation of adhesion molecules, selectins, and integrins, signaling to both the infiltrating leukocyte and the brain endothelium. Expression of CXCL12 on the basolateral surface of endothelial cells recruits CXCR4 + T cells. However, retention of cells in the perivascular space occurs in the presence of high concentrations of CXCL10. Continued migration puts cells in contact with the glia limitans, which is composed of a highly structured wall of astrocytes. Further positive migratory signals, including chemokines, from these and surrounding cells may allow leukocyte migration into the parenchyma.

Although it is controversial as to whether VCAM1 is expressed in human vasculature, the finding that blockade of VLA-4/VCAM1 interactions delayed the onset and/or decreased the severity of EAE implicated this molecule as a target for the treatment of MS ( 84 ). This led to the clinical development of a monoclonal antibody (known as natalizumab) that targets α4 integrin (a component of VLA-4) natalizumab was successfully used in clinical trials to manage this condition ( 85 ). However, a small number of patients treated with this reagent developed progressive multifocal leukoencephalopathy associated with the reactivation of latent JC polyomavirus infection ( 20 ). This observation has been paralleled by the recent withdrawal of an antibody (known as efalizumab) that blocks LFA-1, which was used for the treatment of psoriasis and also led to the reactivation of JC polyomavirus in the brain ( 86 ). Whether JC polyomavirus persists in a latent form in the CNS or these events are a consequence of reactivation of the virus in peripheral tissues and subsequent spread to the CNS is unclear ( 87 ). However, recent reports that detected the presence of JCV in normal brain tissue support the former notion ( 88 – 90 ). Regardless, blockade of α4 integrin has also been shown to antagonize protective immune responses to multiple pathogens in the brain, including T. gondii ( 28 ), simian immunodeficiency virus–induced AIDS encephalitis ( 91 ), and Borna virus–induced progressive encephalitis ( 92 ). Other promising strategies to block cell trafficking to the CNS ( 77 ) may encounter similar problems. Despite all of these potential complications, the number of adverse events associated with natalizumab therapy has been lower than might have been predicted from studies in experimental systems, and this therapy continues to be used for the treatment of MS. Importantly, recent studies involving the generation of bone marrow–chimeric mice in which the hematopoietic compartment lacked β1-integrin but retained α4β7 integrin signaling indicated that T cell accumulation in the CNS during EAE required α4β1-integrin but that migration of granulocytes and macrophages into the CNS was independent of β1 integrin ( 93 ). These findings highlight the potential to control the trafficking of specific immune populations and perhaps even subsets of lymphocytes into the brain.

Astrocytes and immune cell trafficking in the parenchyma. Astrocytes provide an important structural component of the BBB ( 94 ) and are thought to restrict access of immune cells to the CNS. So, after rolling along, adhering to, and finally crossing the endothelial cells of the BBB and their associated basement membrane, the migrating leukocytes reach their next barrier, the glia limitans (Figure 3). This structure surrounds the blood vessel, is composed of astrocytic foot processes, is linked to the basal membrane by the transmembrane receptor dystroglycan, and forms its own molecularly distinct basement membrane composed of laminin, fibronectin, and type IV collagen ( 95 , 96 ). There are very few studies that have considered how leukocytes cross this second barrier, although several reports have provided evidence that production of MMPs by immune cells is required for cleavage of dystroglycan and the breakdown of BBB during EAE and neurocysticercosis associated with tapeworm infection ( 96 , 97 ).

Reactive astrocytes are a hallmark of most inflammatory responses in the brain and this activation corresponds with increased astrocyte numbers, changes in their morphology, and upregulated expression of glial fibrillary acidic protein, an astrocyte-specific structural protein ( 98 ). The seminal studies by Fontana et al. ( 99 ), which suggested that astrocytes could present antigen to CD4 + T cells, highlighted the possible role of these glial cells in the immune response. Although the ability of astrocytes to present antigen through MHC class II remains controversial ( 100 ), the formation of synapses between astrocytes and antigen-specific CD8 + T cells in vivo is consistent with their ability to present antigen through MHC class I ( 101 , 102 ). Nevertheless, reactive astrocytes are frequently associated with migrating T cells and act as a source of multiple cytokines and chemokines during inflammation ( 103 ), which may actively promote cell trafficking into and within the CNS. However, in the majority of experimental systems it has not yet been defined how these interactions affect the coordination of antimicrobial immune responses.

The possible contribution of astrocytes to immune responses within the brain has been described in several settings, including those involving the targeted overexpression of cytokines — such as TNF, IFN-α, TGF-β, IL-6, and IL-12 — by astrocytes, which leads to chronic inflammation and progressive neurodegeneration ( 104 – 108 ). More recent studies analyzing mice in which the ability of astrocytes to participate in immune function is compromised through the specific loss of a cytokine receptor such as gp130 or reduced NF-κB signaling, have shown that this alters the course of immune responses in the CNS ( 109 – 112 ). Thus, in a mouse model of spinal cord injury, astrocyte-specific inhibition of NF-κB (which is necessary for the activation of many cytokine genes) resulted in a reduction in the number of reactive astrocytes in the CNS, in lower levels of chemokines, and in reduced infiltration of T cells and macrophages ( 111 ). Consequently, this led to improved spinal cord healing. Future challenges include determining how individual cytokines, adhesion molecules, and chemokines produced by astrocytes influence the development of inflammation and the behavior of infiltrating immune cell populations.

Chemokines and migration in the parenchyma. Once cells have crossed all the membrane barriers and gained access to the parenchyma of the brain, what molecular cues guide their migration? There is an extensive list of chemokines that are either expressed constitutively or upregulated in the brain during inflammation, and infiltrating immune cells express a wide array of chemokine receptors associated with chemotaxis and/or effector function. The use of mice lacking specific chemokines or chemokine receptors and treatment with antagonists of these interactions has provided useful insights into which interactions are likely important in the brain. However, one of the frequently raised caveats is that this may not distinguish between their role in the development of immunity versus trafficking of cells to the CNS (compare conclusions of refs. 29 and 57 ). For example, the increased susceptibility of mice that lack CCL3 to viral infection in the brain may be due to poor activation and priming of dendritic cells rather than to a failure of T cells to traffic to and migrate within the CNS ( 113 ).

Regardless, the relevance of chemokines to immune cells in the CNS remains an area of active research and has been covered extensively in other articles ( 76 , 114 ), and therefore we only provide a summary of their role in various model systems (Table 2). However, it is helpful to highlight the range of pathologies in which these molecules appear to have critical roles. The chemokine receptors CCR2 and CCR5, which are expressed on many monocytes and T cells and, despite difficulties in detecting these receptors, in MS lesions ( 115 – 117 ), have been implicated in CNS inflammation because blockade of their interactions leads to a reduction in inflammation in mouse models of immune-mediated demyelination ( 118 – 123 ). These findings are broadly consistent with the ability of chemokines to mediate their activities through chemotaxis and activation of integrins ( 124 ), but they can also have more complex effects on cell behavior. The majority of T cells found in the uninflamed CNS express CXCR3 ( 59 ), and this receptor has been implicated in cerebral malaria pathology ( 125 , 126 ) and recruitment of protective CTLs during viral infection ( 113 , 127 ). Furthermore, the recruitment of CXCR3 + T cells to neurons infected with West Nile virus has been attributed to the localized production of CXCL10 by the infected cells ( 128 ). However, the role of CXCR3 and one of its ligands, CXCL10, during EAE appears more complex ( 60 ). During this autoimmune condition, expression of CXCR3, rather than inducing a chemotactic response, is implicated in the retention of autoimmune cells and Tregs in the perivascular space ( 60 ). It is also implicated in the retention of antiviral CD8 + T cells during LCMV infection ( 129 ). Similarly, CXCL12, the ligand for CXCR4, is constitutively expressed in the CNS on the basolateral surface of endothelial cells and is upregulated during neuronal inflammation, and the absence of CXCR4 signaling during EAE leads to perivascular accumulation of mononuclear cells in the spinal cord ( 130 ). These studies suggest that multiple chemokines regulate access from the perivascular space to the parenchyma.

Chemokines and their receptors implicated in the trafficking of immune cells into the CNS

Kinetics and behavioral analysis of lymphocytes within the brain parenchyma. Related to the themes of this review, CCR7 has an important role for T cell and dendritic cell recruitment to the lymph node, where its ligands CCL19 and CCL21 provide motogenic signals required for efficient T cell and dendritic cell migration ( 131 , 132 ). It has been suggested that expression of CCL19 in the uninflamed parenchyma has a role in immune surveillance by CCR7 + CD4 + memory T cells ( 56 , 133 ), but this expression is elevated in MS lesions ( 56 ). CCL21 is also upregulated in other models of CNS inflammation ( 28 ), and whether these chemokines also provide motogenic signals in the parenchyma of the brain is unknown. Multiphoton microscopy of the spinal cord has been used to image the behavior of encephalitogenic cells within the white and grey matter during the induction of EAE ( 25 , 74 ). A recent detailed study has pinpointed three distinct phases for encephalitogenic CD4 + T cell entry into the brain: (a) arresting to leptomeningeal vessels and scanning of the luminal surface against the blood flow (b) diapedesis and scanning of the pial membrane for antigen being presented by perivascular macrophages and (c) successful antigen-dependent activation of T cells, triggering effector capacity and resulting in tissue invasion ( 74 ). This study challenges the notion that the choroid plexus is the major route of cell entry during EAE and solidifies data suggesting an antigen-dependent mechanism. Following activation, during the initial disease process, myelin-specific cells enter the CNS in a rapid wave and can migrate deep into the parenchyma ( 134 ). These cells could be divided into two main populations based on migratory velocities (ranging from 6–25 μm/min). After entry into the perivascular space, many of the cells displayed a restrained or stationary phenotype, suggesting that they were forming long-term contacts with resident cells. This type of arrested behavior is associated with MHC/TCR interactions, although chemokines have also been implicated in mediating cell-cell interactions ( 135 , 136 ). Nevertheless, transfer of myelin-specific (encephalitogenic) CD4 + T cells led to substantially more stationary cells in the brain than did the transfer of T cells not specific for CNS proteins ( 25 ). Somewhat unexpectedly, these studies revealed that T cell migration in this microenvironment was, at the population level, random, indicating that local migration was not regulated by chemokine gradients. Thus, although the arrest of encephalitogenic CD4 + T cells was antigen specific, their migration did not seem to be directional and was more like the random motility of naive T cells found in the lymph node ( 25 ).

In contrast to EAE induced by the transfer of autoimmune T cells and intracerebral injection of LCMV, a condition with well-defined localized acute inflammatory events, mice infected with T. gondii have provided a model of chronic CNS inflammation to study the behavior of pathogen-specific CD8 + T cells ( 28 , 137 ). Unlike the rapid burst of infiltration during EAE, in this experimental system, there was a continuous recruitment of antigen-specific T cells that could be observed over a prolonged period of time (1–8 weeks) ( 28 , 137 ). Various migratory behaviors including clustering and homotypic T cell interactions were observed. Slowing of CD8 + T cells and clustering of antigen-specific cells were seen around actively replicating parasites but not latent cysts ( 137 ). With no correlation between the amount of antigen present and the confinement ratio (or meandering index) of cells, the pattern of motility seemed to represent a “search and destroy mission” to find infected cells, rather than directional migration in response to a chemokine gradient.

Despite extensive investigations, we still have a limited understanding of exactly how chemokines and other chemotactic factors contribute to the migratory behavior of T cells, whether in meninges, CSF, or parenchyma. Since the initial studies describing the random behavior of T cell migration in the lymph node, it has been established that cells follow chemokine-coated conduits and thus remain “directed” ( 138 ). Previously, intravital imaging of peripheral lymph nodes indicated that naive T cells migrate at speeds greater than 10 μm/min and are guided by conduits formed by follicular dendritic cells, fibroblastic reticular cells, and stromal cells expressing the fibroblast marker ERTR7 ( 138 ). ERTR7 + cells have also been detected at distinct but confined areas of the brain (such as the meninges, vasculature, and sulci) during inflammation caused by LCMV ( 64 ) and T. gondii infection ( 28 ). There is little understanding of the role that ERTR7 + stromal cells have in the CNS, but it is tempting to speculate that these cells promote trafficking or retain migratory leukocytes in these distinct compartments of the brain.

While the presence of a haptotaxic mechanism of migration (i.e., migration in response to chemokine bound to matrix molecules) has given rise to the idea of random exploration, it has not excluded the role of soluble chemokine gradients, nor has it been shown that gradients exist in an immobilized fashion on stromal networks ( 139 ). However, this has led to investigations into the existence of similar networks in non-lymphoid organs. Indeed, the ECM in the CNS may have a similar role to that of the constitutive structures in the lymph node ( 28 , 138 ). Inflammation in the brain and in the periphery induces the production of ECM molecules that are known to support cell migration in the context of neural development ( 140 , 141 ). A proteomics study demonstrated the production of many ECM molecules by astrocytes ( 142 ), and increased expression of collagen and laminins associated with myelin-containing macrophages is present in perivascular lesions of patients with MS ( 143 ). The use of second harmonic generation signals during multi-photon microscopy led to the visualization of a reticular network of fibers in the inflamed brain that closely associated with migrating T cells ( 28 ). These fibers were not present in the brains or spinal cord of naive mice but were upregulated during T. gondii infection and following EAE induction. This network may be the functional equivalent of the fibroblastic reticular cell network in the lymph node ( 138 ) and might not only provide structural support for migration but also display bound chemotactic signals for directional migration of lymphocytes. This model needs to be rigorously tested, but it may explain how lymphocytes can reach migratory velocities in this dense tissue that are comparable to those of naive T cells within lymph nodes.

It is clear that the use of intravital microscopy has advanced our understanding of the immune response in the CNS from visualizing how immune cells interact with endothelium to the more recent studies showing how immune cells behave during meningitis and parenchymal inflammation. Perhaps at one point there was the expectation that some of the adhesion molecules and chemokines that have been identified in the CNS might be specific for neuroinflammation, but to date all the molecular interactions that allow lymphocytes to access the brain are also relevant to other tissues. Thus, although natalizumab was developed specifically as a potential therapy for MS ( 84 ), it has also been approved by the FDA for the treatment of individuals with moderate to severe Crohn disease ( 144 ). However, it remains possible that there are mechanisms that provide a tissue-specific signal to lymphocytes to traffic to the brain, and there may be molecular equivalents of vitamin D or retinoic acid, which program skin- and gut-homing populations of T cells, respectively ( 145 ).

Presently, the constrained migratory behavior of T cells reported in EAE, LCMV infection, and toxoplasmic encephalitis has not yet been linked to effector function. Although it is reminiscent of the productive interactions between T cells and antigen-presenting cells in the lymph node, there is no information about whether the cytolytic activity, antigen presentation, cytokine production, or regulatory mechanisms of T cells (all of which are known to occur in the CNS) are linked to these events. Similarly, whether these stalled T cells interact with different accessory cell populations present in the inflamed brain has not been explored. Of particular interest is the finding that increased numbers of dendritic cells are present in the brain during neuroinflammation and have been associated with the regulation of local disease processes ( 146 – 148 ). Moreover, vessel-associated dendritic cells have been implicated in the regulation of T cell entry to the CNS ( 149 ), and introducing different dendritic cell subsets directly into the brain can have distinct effects on local inflammation ( 150 ). Nevertheless, there remain fundamental questions about how these professional antigen-presenting cells access and behave within the CNS. The development of strategies to deplete these populations or modify their function while in the brain will add to our understanding of how dendritic cells contribute to immune surveillance and promote or resolve ongoing local inflammation. The application of available reporters for dendritic cells ( 151 – 153 ), cytokine production ( 154 ), and ways to visualize cytolytic activity ( 155 ), combined with the development of new approaches such as the ability to deliver small interfering RNAs into the brain ( 156 ), should allow a dissection of how the behavior of immune cells relates to their function in the CNS. Ultimately this will help in the design of therapies that will allow for better management of the immune response in the brain.

Thanks to M.J. Carson, T.H. Harris, B. John, P. Kennedy, T. van Winkle, A. Durham, U. Von Andrian, and R. Germain for discussions at multiple times. The authors wish to acknowledge funding from the State of California (to E.H. Wilson), funding from the State of Pennsylvania and grants from the NIH (to C.A. Hunter), and grants from the National Health and Medical Research Council and New South Wales government, Australia (to W. Weninger).

Conflict of interest: C.A. Hunter has received support for his research from Centocor Ortho Biotech Inc.

Reference information: J Clin Invest. 2010120(5):1368–1379. doi:10.1172/JCI41911


Wound healing: cellular mechanisms and pathological outcomes

Wound healing is a complex, dynamic process supported by a myriad of cellular events that must be tightly coordinated to efficiently repair damaged tissue. Derangement in wound-linked cellular behaviours, as occurs with diabetes and ageing, can lead to healing impairment and the formation of chronic, non-healing wounds. These wounds are a significant socioeconomic burden due to their high prevalence and recurrence. Thus, there is an urgent requirement for the improved biological and clinical understanding of the mechanisms that underpin wound repair. Here, we review the cellular basis of tissue repair and discuss how current and emerging understanding of wound pathology could inform future development of efficacious wound therapies.

1. Introduction

Millennia of evolution have created our skin, a highly adaptive, multifunctional organ that protects us from a daily onslaught of chemical, physical and ultraviolet radiation challenge. This harsh external environment often results in injury to the skin, and it will therefore come as no surprise that our skin possesses sophisticated reparative processes that allow it to heal quickly and efficiently. Despite considerable innate reparative ability, multiple cellular aspects of an individual's injury response can become attenuated, compromising wound closure. This attenuation is most often a result of pathological systemic changes, such as those associated with advanced age or uncontrolled diabetes. Indeed, age and diabetes are primary risk factors for developing a chronic wound (i.e. a wound that takes longer than 12 weeks to heal). Unfortunately, these chronic wounds (primarily venous ulcers, pressure sores and diabetic foot ulcers) are a major area of unmet clinical need, increasing significantly on a global scale [1]. Here, we discuss the current understanding of skin repair and illustrate impaired cellular behaviours that underpin chronic wound healing pathology. Application of emerging research technologies will be essential in further elucidating the underlying cellular and molecular basis of acute and pathological repair.

2. Cellular aspects of acute wound repair

Our skin is specialized to interface with the external environment and provides a variety of important homeostatic functions, from regulating thermostability to sensing extrinsic stimuli. Crucially, the skin acts as a primary defence barrier, preventing desiccation and mechanical, chemical, thermal and photic damage to internal structures [2]. This defence extends to a sophisticated immune barrier response that protects against pathogenic infection, while supporting commensal microorganisms via an elegantly adapted host–microbiota axis [3]. The skin has also evolved efficient and rapid mechanisms to close breaches to its barrier in a process collectively known as the wound healing response. Wound repair is classically simplified into four main phases: haemostasis, inflammation, proliferation and dermal remodelling [4], which result in architectural and physiological restoration following damage (figure 1). The following sections describe these stages in detail.

Figure 1. The stages of wound repair and their major cellular components. Wound repair begins with haemostasis, where a platelet plug prevents blood loss and a preliminary fibrin matrix is formed. Inflammation then ensues to remove debris and prevent infection, commencing with neutrophil influx, which is promoted by histamine release from mast cells. Monocytes arrive later and differentiate into tissue macrophages to clear remaining cell debris and neutrophils. During the proliferative phase, keratinocytes migrate to close the wound gap, blood vessels reform through angiogenesis, and fibroblasts replace the initial fibrin clot with granulation tissue. Macrophages and regulatory T cells (Tregs) are also vital for this stage of healing. Finally, the deposited matrix is remodelled further by fibroblasts, blood vessels regress and myofibroblasts cause overall wound contraction.

2.1. Haemostasis

Immediately after injury, damaged blood vessels rapid contract and a blood clot forms preventing exsanguination from vascular damage [5]. Platelets, principle contributors to haemostasis and coagulation, are activated when they encounter the vascular subendothelial matrix. Platelet receptors (e.g. glycoprotein VI) interact with extracellular matrix (ECM) proteins (e.g. fibronectin, collagen and von Willebrand factor), promoting adherence to the blood vessel wall. Thrombin subsequently triggers platelet activation, inducing a conformational change, and release of alpha and dense granules containing bioactive molecules which reinforce coagulation (reviewed in [6]). An insoluble clot (eschar) of fibrin, fibronectin, vitronectin and thrombospondin forms [7], primarily serving to plug the wound and prevent bleeding. The eschar also fulfils a number of secondary functions, including shielding against bacterial invasion, providing a scaffold for incoming immune cells and harbouring a reservoir of cytokines and growth factors to guide the behaviour of wound cells in early repair [8].

Platelets are crucial in the recruitment of immune cells to the injury site, by either directly capturing immune cells in the eschar, or by releasing a secretome of chemokine attractants upon degranulation [6]. In fact, the platelet secretome also contains growth factors that stimulate resident skin cells, including fibroblasts and keratinocytes [9]. As the most abundant cell type during early repair, platelets play an active role in the early inhibition of bacterial infection. They express a number of toll-like receptors (TLRs) [10,11], which regulate the production of antimicrobial peptides [12]. Once a sufficient clot has formed, the coagulation process is switched off, preventing excessive thrombosis. Here, platelet aggregation is inhibited by prostacylin, thrombin inhibited by antithrombin III, and coagulation factors V and VII degraded by activated protein C [13]. At the same time, the injured vessel wall is repaired by smooth muscle cells and endothelial cells that proliferate in response to released platelet-derived growth factor (PDGF) [14]. Endothelial progenitors are also recruited to aid this process as mature endothelial cells show limited proliferative capacity [15].

2.2. Inflammation

Innate inflammation evolved as the primary defence against pathogenic wound invasion. This immune response is initiated by injury-induced signals damage-associated molecular patterns (DAMPs) released by necrotic cells and damaged tissue, and pathogen-associated molecular patterns (PAMPs) from bacterial components. These PAMPs and DAMPs activate resident immune cells, such as mast cells, Langerhans cells, T cells and macrophages, by binding pattern recognition receptors to elicit downstream inflammatory pathways [16]. A subsequent release of pro-inflammatory cytokines and chemokines attracts circulating leucocytes to the site of injury (reviewed in [17]). Pro-inflammatory molecules also stimulate vasodilatation, which, along with the expression of endothelial cell adhesion molecules, such as selectins, facilitates neutrophil and monocyte adhesion and diapedesis [18]. In fact, the importance of selectins in immune cell recruitment has been clearly demonstrated, with genetic [19] and pharmacological [20] blockade of E- and P-selectin significantly impairing both immune cell infiltration and wound healing.

Neutrophils, which arrive early after injury, are recruited into the wound from damaged vessels, attracted by chemoattractants, including interleukin 1 (IL-1), tumour necrosis factor-alpha (TNF-α) and bacterial endotoxins, such as lipopolysaccharide (LPS) [21]. In response to pro-inflammatory signals, and activation of inflammatory signalling pathways (e.g. NF-κB [21]), neutrophils (and other wound cells) release their own cytokines. Neutrophils remove necrotic tissue and pathogens via phagocytosis and the release of reactive oxygen species (ROS), antimicrobial peptides, eicosanoids and proteolytic enzymes [22]. They also trap and kill pathogens in an extruded web of DNA coated with antimicrobial peptides and cytotoxic histones, termed extracellular traps [23].

The inflammatory response is complex, modulated by a multitude host of intrinsic and extrinsic factors. Uncontrolled and excessive inflammation promotes tissue injury and delays healing (as in diabetic mice [24]). However, insufficient immune cell recruitment, for example in TLR3 knockout mice, also hinders repair [25]. Thus, immune cell responses must be situational, increasing to respond appropriately to infection, yet clearing effectively to allow wound resolution. In the absence of infection, wound neutrophils decline within a few days of injury onset [26]. Most neutrophils are extruded from the wound site as they adhere to the fibrin scab, while others are removed by innate clearance mechanisms such as macrophage efferocytosis [17]. Remaining neutrophils are cleared by apoptosis, necrosis or phagocytosis, or may leave inflamed tissue and return to the circulation through reverse transendothelial migration, as observed in zebrafish [27], mice [28] and human neutrophils in vitro [29].

Circulating monocytes enter the wound tissue where, in response to the local milieu, they differentiate into macrophages. Although it is generally suggested that macrophages are recruited following neutrophils, an initial wave of monocytes has been observed entering the wound simultaneously with neutrophils [30]. Macrophages are master effector cells in tissue repair, displaying both versatility and high plasticity (reviewed in [31]). They reach peak wound infiltration 72 h after injury in mice and 7 days post-injury in humans [32]. Like neutrophils, macrophages engulf necrotic cellular debris and pathogenic material through evolutionarily conserved receptors, but also exhibit differential behaviours and morphological changes in response to cytokines [33].

Wound macrophages are traditionally separated into two main subsets: M1-stimulated and M2-stimulated. However, this dichotomous classification has become outdated, with both human [34] and murine [35] macrophages now known to show diverse transcriptional and phenotypic responses to different stimuli (reviewed in [36]). Hence, the macrophage repertoire should be viewed as a spectrum of phenotypes governed by tissue status and environmental signals [37,38]. For simplicity, we will herein refer to classically activated (pro-inflammatory) and alternatively activated (anti-inflammatory) groups.

Classically activated macrophages are induced by pro-inflammatory stimuli, such as LPS and interferon-gamma (IFN-γ), and promote inflammation by releasing ROS, inflammatory cytokines (e.g. IL-1, IL-6 and TNF-α) and growth factors (e.g. vascular endothelial growth factor, VEGF and PDGF). These macrophages phagocytose apoptotic neutrophils, replacing them as the main inflammatory mediator [8]. Later stages of inflammation are characterized by a transition to alternative activation, which occurs through neo-differentiation of newly recruited monocytes, or via switching of existing macrophages in situ to an anti-inflammatory phenotype. Although not widely characterized, this phenotypic switch can be stimulated by environmental changes in cytokines [39] and efferocytosis [40]. It may additionally be driven by miRNAs [31], transcription factors [41], and modulation of pro-inflammatory and anti-inflammatory receptors [41,42].

Alternatively activated macrophages express pro-resolutory cytokines (IL-4, IL-10, IL-13 [43,44]) and arginase, a key factor for effective wound repair [45]. Anti-inflammatory macrophages also release a myriad of growth factors to promote re-epithelialization, fibroplasia [8] and angiogenesis [46]. More recently, macrophages have been shown to be crucial in the stabilization and remodelling of blood vessels in mice and fish [47].

The importance of macrophages is further demonstrated in selective ablation studies, where Cd11b-specific deletion of macrophages leads to delayed wound repair and increased inflammation [48]. Similarly, inducible knockdown of macrophages during early healing caused delayed re-epithelialization, angiogenesis and granulation tissue formation, while knockdown of macrophages mid-way through healing led to endothelial cell damage, severe haemorrhage and immature granulation [49]. Thus, the collective behaviours of macrophages promote scavenging of debris, bacteria and pro-inflammatory cells, while also stimulating reparative processes to allow effective wound resolution.

The overwhelming presence of neutrophils and macrophages in wounds has potentially masked the importance of other myeloid cells in wound repair. However, recent studies have revealed that resident T cells are critical for the early injury response, while circulating T cells are recruited to resolve inflammation [50]. Indeed, aged and diabetic mice show reduced resident dendritic epidermal T cells and a delayed healing phenotype, whereas subcutaneous administration of dendritic epidermal T cells can restore healing [51,52]. Moreover, the removal of anti-inflammatory regulatory T cells delays tissue repair in mice [50]. Mast cells also play a role in wounds, releasing histamine to aid neutrophil recruitment during early inflammation [53].

2.3. Proliferation

The proliferative phase of healing is characterized by extensive activation of keratinocytes, fibroblasts, macrophages and endothelial cells to orchestrate wound closure, matrix deposition and angiogenesis. As early as 12 h post-injury, keratinocytes are activated by changes in mechanical tension and electrical gradients, and exposure to hydrogen peroxide, pathogens, growth factors and cytokines [54]. This activation causes keratinocytes at the wound edge to undergo partial epithelial–mesenchymal transition, where they develop a more invasive and migratory phenotype [55]. Front-to-rear polarity replaces top-to-bottom polarity, allowing the leading-edge keratinocytes to migrate laterally across the wound to reform the epidermal layer, a process termed re-epithelialization [56]. Keratinocytes behind the leading edge modulate their cell adhesion via PCKα-mediated changes in desmosome adhesiveness [57] and Eph-mediated changes in adherens junctions [58], allowing them to rearrange their order with the migrating epithelial sheet [54]. Keratinocytes in the neo-epidermis release matrix metalloproteinases (MMPs) to aid their path of migration, while laying down new ECM proteins to reconstitute the basement membrane [59].

Hair follicle stem cells are induced to proliferate, with progeny epidermal cells streaming out of the follicle to meet the cellular demand required to resurface the wound [60]. These cells sprout from damaged appendages in shallow wounds, or arrive from the epidermal edge in full-thickness wounds. Only specific stem cell compartments are activated or recruited to the re-epithelialization process [61]. For example, Krt15+ve [62] and Krt19+ve [63] bulge region stem cells appear dispensable for re-epithelialization, while Lgr5- and Lgr6-expressing cells from the follicle and interfollicular epidermis respond to wound cues, contributing to re-epithelialization [64]. A key characteristic of full-thickness wounds in mice is that appendages, including follicles, are absent from re-formed scar tissue [2]. However, under specific circumstances wound-induced follicle neo-genesis can occur, seemingly via re-activation of developmental Wnt and Shh signalling [60].

Keratinocytes negotiate through debris and necrotic tissue of the wound bed through their interactions with structural proteins of the preliminary matrix via integrin receptors [65]. MMPs, particularly MMP-1 and MMP-9, are vital for keratinocyte migration as they aid integrin receptor dissociation [56]. The production of other proteases, such as plasmin, further facilitates keratinocyte migration by degrading the provisional fibrin-rich wound bed [59]. When keratinocytes from opposing edges meet, migration terminates (via an undetermined mechanism), a thin epithelial layer is established and keratinocytes form new adhesions to the underlying matrix. Keratinocytes then fully reform the basement membrane and undergo terminal differentiation, to stratify and regenerate the epidermis [32].

Fibroblasts are the main cell type responsible for replacing the provisional fibrin-rich matrix with a more substantial granulation tissue. Resident and mesenchymally derived fibroblasts respond to a milieu of signalling molecules from platelets, endothelial cells and macrophages, including transforming growth factor (TGF-β) and PDGF. These signals direct fibroblasts to either become pro-fibrotic, laying down ECM proteins, or differentiate into myofibroblasts which drive wound contraction [55]. It is important to note that this is again a simplification, as in reality fibroblasts exhibit functional diversity, assisting dermal repair in different ways. In a seminal study Driskell et al. [66] demonstrated that skin fibroblasts originate from two distinct lineages, where the upper lineage aids re-epithelialization while the lower lineage contributes to ECM deposition. Recent findings have further challenged conventional understanding of wound fibroblast origin, showing that two-thirds of granulation tissue fibroblasts are actually myeloid derived [67], and are thus likely to stem from wound macrophages. Fibroblasts degrade the provisional matrix by producing MMPs and replace it with a granulation tissue rich in fibronectin, immature collagens and proteoglycans [68]. This granulation tissue acts as a scaffold for the migration and differentiation of wound cells, supporting both the formation of new blood vessels and the deposition of mature ECM.

New blood vessels are created during the process of angiogenesis to meet the metabolic demands of the highly proliferative healing tissue. Angiogenesis is triggered by hypoxia, which in turn drives the expression of hypoxia-inducible factors (HIFs) and cyclooxygenase 2, and subsequent release of VEGF and other factors [69]. In response to these changes, microvascular endothelial cells proliferate and migrate into the wound bed, sprouting new vessels that fuse with others to develop stable, tubular networks [70]. VEGF prevents endothelial cell apoptosis by upregulating anti-apoptotic proteins such as BCL-2 [71], while the fibrin matrix promotes angiogenesis by triggering phenotypic changes in endothelial cells to stimulate their migration [72].

Macrophages play a significant role in angiogenesis by aiding microvascular endothelial cell behaviours. They produce proteases such as MMPs to degrade the dense fibrin network and chemotactic factors (e.g. TNF-α, VEGF and TGF-β) to drive endothelial migration (reviewed in [73]). Willenborg et al. [74] demonstrated the importance of macrophage-derived factors in angiogenesis, where myeloid-specific deletion of VEGF-A reduced capillary formation in murine wounds. Macrophages also participate in the remodelling of new vasculature, by guiding vessel tips together [75], phagocytosing superfluous vessels [47,76] and dampening the angiogenic response to prevent excessive vascularization [77].

The skin houses a dense network of sensory and autonomous nerve fibres which allow sensation and movement. Nerve fibre regeneration is therefore essential following injury. Despite the principle role of diabetic skin denervation in wound pathogenesis (reviewed in [78]), wound innervation per se remains an understudied area. Neuropeptides, such as substance P, are known to be released from sprouting neurons and immune cells during repair, influencing diverse cellular processes (e.g. proliferation and angiogenesis [79,80]). Notably, substance P is reduced in delayed healing in diabetic wounds, where topical restoration restores healing [81,82] and contributes to nerve regeneration [83]. Wound-activated glial cells are also an important component of the repair response, shown to express factors important for chemotaxis, while the loss of glial cells delays healing in wild-type mice [84]. These and other studies suggest that innervation plays a substantial role in effective repair.

2.4. Matrix remodelling

Remodelling of the ECM spans the entire injury response, beginning with the initial deposition of a fibrin clot, and ending several years later with the formation of a mature, type I collagen-rich scar [55]. Fibroblasts are the major cell type responsible for wound ECM remodelling, replacing the initial fibrin clot with hyaluronan, fibronectin and proteoglycans, and forming mature collagen fibrils later in repair [85]. Proteoglycans aid construction of mature, cross-linked collagen fibrils and act as a conduit for cell migration [86]. The collagen composition of uninjured adult skin is approximately 80% collagen type I: 10% collagen type III. By contrast, granulation tissue predominantly comprises of the embryo-associated collagen type III (approx. 30%), with only 10% collagen type I [87]. As healing progresses, collagen type III is replaced by collagen type I, directly increasing the tensile strength of the forming scar [88]. The integrity and architecture of scar ECM never fully returns to that of unwounded skin. Collagen fibrils in scar dermis adopt large parallel bundles, while in uninjured skin fibrils adopt a basket weave orientation. Thus, wound scar tissue confers only up to 80% of pre-wounding strength post-injury [87,89].

These sequential changes in the ECM require a fine balance between collagen degradation and synthesis, achieved through temporal regulation of key MMPs. These collagenases, expressed by anti-inflammatory macrophages, fibroblasts and keratinocytes, cleave native helical collagens throughout repair [85]. Elastin, another key dermal ECM component, must reform elastic fibres to retain skin elasticity. Interestingly, the degradation of normal dermal matrix causes the release of elastin fragments, or elastokines, which act as signalling molecules [90]. Elastin is formed from its precursor, tropoelastin, and early in healing shows the aberrant arrangement. In fact, mature elastin fibres are often only apparent in scar tissue many months after injury [91,92].

Heightened expression of TGF-β and mechanical tension stimulate myofibroblast differentiation in vivo and in vitro [93]. Myofibroblasts are characterized by an abundance of alpha-smooth muscle actin (α-SMA), associated with an ability to generate strong contractile forces and focal adhesions [85]. Curiously, mice lacking the gene encoding α-SMA, Acta2, heal normally with no obvious change in fibroblast contraction [94]. This apparent redundancy, with compensation by other microfilaments, highlights the importance of wound contraction. Myofibroblast contraction is facilitated by pseudopodial extensions that allow cytoplasmic actin to bind to fibronectin in the matrix scaffold [55]. Myofibroblasts adhere to one another via desmosomes, binding to matrix fibrils and drawing the matrix together by a process termed contracture [95]. The wound healing response abates when macrophages, endothelial cells and fibroblasts undergo apoptosis or exit the injury site, leaving a scar [96].

3. When healing fails—factors influencing chronic wound healing

Acute wound repair is a highly dynamic cascade of cellular signalling and behavioural events that ensures rapid closure of the skin barrier. High levels of redundancy and compensatory mechanisms ensure that small alterations to this response seldom cause problems in healing wounds [97]. For example, the ablation of specific subsets of hair follicle stem cells [63], MMPs [98], fibroblast growth factors [99], TGF-α [100] and VEGFR2 [101] each individually fail to significantly impair wound closure. However, like any biological process, sufficient perturbation to the system leads to aberrations, which in the case of wounds manifest as excessive scarring at one extreme or failure to heal entirely at the other. Wounds that fail to heal (defined as generally remaining unhealed after 12 weeks) are termed chronic wounds. They primarily affect the elderly and diabetic, are highly prevalent and a major socioeconomic burden [102,103]. More effective clinical management would prevent a proportion of these wounds [104], yet many remain refractory to current treatment, highlighting the need to better understand the cellular basis of wound pathology in order to develop therapeutically viable treatments.

Susceptibility to injury remains understudied. We know that the skin of aged and diabetic mammals is more predisposed to injury, as it undergoes atrophy, with altered skin barrier and reduced hydration [105,106]. Both ageing and diabetes lead to the gradual loss of dermal matrix, with corresponding changes in tissue mechanics, loss of resilience and increased susceptibility to friction damage [107,108]. Once an injury occurs, a range of molecular and cellular perturbations contribute to overall healing impairment. One factor widely implicated in aged and diabetic wound pathology is cellular senescence (reviewed in [109]). Mitotic cells become senescent and non-proliferative in response to a host of intrinsic and extrinsic factors. Senescent cells acquire a hypersecretory phenotype, producing a secretome rich in pro-inflammatory cytokines and tissue-degrading proteases (reviewed in [110]). The chronic wound environment is the perfect platform for senescent cell induction due to the high levels of inflammation and oxidative stress [111]. Indeed, we recently demonstrated that high senescent cell burden contributes to wound pathology, where blockade of the proposed senescence receptor, CXCR2, dampens macrophage senescence and improves healing in diabetic mice [112].

A key contributor to wound pathology is excessive inflammation, which perpetuates chronicity through the continued destruction of wound tissue. Chronic wounds are characterized by high numbers of Langerhans cells [113,114], neutrophils [115], pro-inflammatory macrophages [116,117] and proteases [118–120], linked to clinical ulcer severity [121]. Along with elevated infiltration of specific immune cell subsets [122], pathological immune cell function is perturbed and collectively contributes to poor healing. Here, neutrophils are excessively primed to produce neutrophil extracellular traps, which are cytotoxic [123] and delay wound healing [124]. In diabetic mice, neutrophils are more resistant to apoptosis, and less effectively cleared by macrophages [125], furthering their excessive presence in pathological wounds. Diabetic macrophages also exhibit defective efferocytosis of apoptotic cells [126], impaired phagocytosis of bacteria [127,128] and reduced ability to polarize to an anti-inflammatory state [129]. Interestingly, even prior to ulceration, the skin of diabetic humans and mice exhibits higher numbers of mast cells and macrophages primed to the pro-inflammatory state [130]. By contrast, T cell receptor diversity [131] and the number of CD4+ T cells [116,131] are reduced in diabetic foot ulcers. Together, these aberrant features of chronic wound immune cells not only prevent the shift from inflammation to resolution, but greatly increase vulnerability to infection. Heightened inflammation may also persist due to chronic wound infection, thus maintaining the wound in a continuous cycle of infection, inflammation and inadequate repair.

Cellular impairment is not only restricted to inflammation, but also extends to re-epithelialization and dermal remodelling. Non-healing diabetic foot ulcers are typically characterized by an epidermal wound edge that is hyperkeratotic and parakeratotic [132]. Keratinocytes at the chronic wound edge show abnormal nuclear presence of β-catenin and elevated c-myc, which directly delays migration in vitro [132] and prevents healing in mice [133]. Ulcer wound edge epidermis additionally displays the misexpression of a number of cell cycle, differentiation and desmosomal markers [134], impaired growth factor receptor signalling [135], and lacks hair follicles [136]. This aberrant activation phenotype, with seemingly uncontrolled wound edge proliferation, is thought to directly inhibit keratinocyte-mediated chronic wound closure.

At the same time, dermal reconstitution is significantly inhibited by the high wound protease levels, which not only break down dermal ECM components, but also degrade growth factors (e.g. VEGF and TGF-β [137,138]) and cytokines (e.g. TNF-α [139]). Chronic wound fibroblasts are highly senescent, further compromising ECM deposition [140–142], and are unresponsive to ECM-stimulating factors such as TGF-β [143,144]. Interestingly, we recently demonstrated that deficiency in wound iron may underpin reduced ECM deposition in diabetic mice, as iron loading of fibroblasts directly stimulates ECM deposition and remodelling [145]. Macrophages are key to this reparative response, where iron sequestration causes alternatively activated macrophages to produce ECM-stimulating factors [146]. Note that disparities exist in the reported role(s) of iron in wound repair. Sindrilaru et al. [117] suggest that iron deposition caused delayed healing in diabetic foot ulcers, promoting an unrestrained M1-like macrophage phenotype, increased oxidative stress and senescence. Similarly, others have shown that the iron chelator, deferoxamine, improves wound healing in pressure ulcers of diabetic [147] and aged [148] mice. Thus, the cellular effects of iron are probably context-dependent and wound-type-specific, exacerbating tissue damage in an already pro-inflammatory environment, while promoting alternatively activated macrophage- and fibroblast-mediated wound resolution in late-stage repair.

Sustained hyperglycaemia in diabetes directly contributes to defective healing, compromising leucocyte function [149], inducing cellular senescence [150] and causing non-enzymatic glycation of ECM and the formation of advanced glycation end products (AGEs) [151]. AGEs not only alter the dermal structural architecture, but also trigger inflammation and ROS via their receptor, RAGE [152]. These effects impair neovascularization, in part by preventing HIF-1α transactivation and subsequent upregulation of VEGF and stromal-derived factor 1 (SDF-1) [153,154]. At the macroscopic level, uncontrolled diabetes causes long-term damage to the microvasculature, which results in local tissue hypoxia, arterial vasculopathy and/or lower limb neuropathy—all extreme risk factors for chronic wound development [155].

In diabetes, stem cell populations that would usually participate in vascularization are depleted (e.g. bone marrow [156]) or show impaired neovascular potential (in adipose tissue [157]). A reduction in SDF-1, which aids recruitment of endothelial progenitor cells to wounds, is also observed, while topical administration of SDF-1 accelerates diabetic wound repair [158]. Slowing AGE formation in diabetic mice improves the neovascular potential of bone marrow progenitors [159], confirming functional relevance and further demonstrating the important contribution of uncontrolled diabetes in wound pathology.

It is crucial to note that the causes of delayed healing, while simplified above, are often multifactorial and complex. Wound chronicity is influenced by local and systemic defects [160], along with imbalances in hormones, cytokines and growth factors (e.g. reduced PDGF [161]). However, in recent years, the presence and persistence of wound infection has been widely discussed as a major contributor to chronicity [162]. Indeed, high abundance of common wound pathogens, such as Staphyloccoccus aureus and Pseudomonas aeruginosa, is reported in chronic wounds [163,164], with a wound's microbial profile strongly linked to healing outcome [165]. These pathogens often develop into polymicrobial aggregates (biofilms) encapsulated in a protective matrix of extracellular polymeric substances that confers resistant to traditional antibiotics and host defences (reviewed in [166]).

The microbiome profiles of aged and diabetic skin differ considerably from their young and non-diabetic counterparts, in each case displaying reduced α-diversity [167,168]. Although critical wound colonization occurs as a result of inadequate immune cell function, poor perfusion and the presence of a persistent open wound, it is likely (though yet to be proven) that aged and diabetic skin is intrinsically predisposed to infection by an altered microbiome. Diabetic wounds also show altered expression of pattern recognition receptors responsible for eliciting a host response, which may link to poor healing [169]. Interestingly, knockout of the pattern recognition receptor, Nod2, impaired wound closure [170] and altered the skin microbiome [171] of mice. Curiously, wild-type mice cross-fostered into Nod2−/− litters adopted an altered microbiome and acquired a delayed healing phenotype [171], therefore directly demonstrating the impact of skin microbiota dysbiosis on repair. Key factors in chronic wound pathology are summarized in figure 2.

Figure 2. Factors contributing to chronic wound healing. Chronic wounds become infiltrated with bacteria that exacerbates inflammation. Chronic wound keratinocytes show aberrant activation causing hyperproliferation and impaired migration. A large proportion of chronic wound cells (e.g. macrophages and fibroblasts) become senescent, producing a senescence-associated secretory phenotype (SASP) that perpetuates senescence, triggers reactive oxygen species (ROS) release and heightens inflammation. High amounts of advanced glycation end products (AGEs) also contribute to inflammation and cellular senescence in the wound environment. Together these features cause excessive tissue breakdown and impair cellular functions to prevent normal healing. re-ep = re-epithelialization.

4. Translational techniques to enhance clinical understanding of wounds

Our knowledge of the mechanisms underlying chronic wound healing is constantly improving, largely due to the development and refinement of wound models and diagnostic tools. For example, until the advent of sequencing technologies, wound bacterial profiling was restricted to simple culture methods, limiting speciation to only organisms capable of expansion in culture. Further analysis was then required to gather complete diagnostic information about a clinical isolate (reviewed in [172]). The emergence of short-read 16S sequencing provided new insight into clinical bacterial communities, but bacterial identification was limited to genus level based on inference from sequence homology [173], with little information about their virulence or clinical significance. Novel genomic technologies are now emerging to allow rapid molecular identification of microorganisms to the sub-species level. Simultaneous characterization of antibiotic resistance and virulence profiles [173,174] provides unprecedented insight into the role of bacterial, fungal and viral ecosystems in wound pathology. Combining these techniques with host genomic, metabolomic and proteomic approaches promises to deliver in depth understanding of the myriad of factors influencing wound repair, while ultimately facilitating a true ‘personalised medicine' approach to clinical wound management.

Historically, wound studies have relied on the use of in vivo models to address the complexity of the multifactorial wound response. However, it is widely accepted that between-species differences have hindered translational wound research efforts. We are now moving towards the development of more dynamic in vitro approaches, such as three-dimensional skin equivalents [175], allowing closer modelling of native human cell behaviours, and moving away from artificial single-cell monolayer culture. While cultured three-dimensional skin equivalents still lack many skin features, such as glands, immune cells and blood vessels, current research is beginning to address this deficit [176,177]. The development of three-dimensional-printed skin equivalents is particularly exciting, offering profound implications in translational research. Indeed, a recently developed vascularized three-dimensional-printed skin model reflected many aspects of native skin, including tissue maturation, and epidermal stratification and stemness [178].

Porcine and human ex vivo models are also gaining traction, with the advantage that they provide native skin tissue architecture and the full gamut of resident skin cells to recapitulate important aspects of the human chronic wound healing response [179,180]. Ex vivo models are not without their caveats, lacking immune cell infiltration and maintaining viability for a limited time-frame [181]. It is likely that novel culture methods, such as microfluidics [182], will extend tissue viability and allow skin perfusion with biologically relevant factors (and immune cells) to increase the relevance of ex vivo wound models.

In vivo models are still widely used, with mice favoured for mechanistic studies [183]. The multitude of available transgenic mouse lines (including reporter lines) allows temporal and spatial investigation of the molecular basis of in vivo wound healing. Nevertheless, strain- and species-specific differences must be considered, especially when extrapolating conclusions for translational research purposes. Pigs, though used far less frequently, provide a useful translational model with skin that closely resembles that of humans. Wounding in mice involves full-thickness incisions or excisions, yet variability can be introduced between laboratories by the methods used to apply wounds, the analgesics and anaesthetics used, and how the wounds are treated (e.g. splinted, occluded or left to heal by secondary intention [184,185]). Continued efforts to standardize in vivo methodology will be essential to increase experimental validity and progress current and future wound research.

An array of pre-clinical delayed healing models are used to better recapitulate human chronic wounds, from pressure ulcers in mice using magnets [186], to infected wounds in pigs [187]. As those primarily at risk of developing chronic wounds are elderly or diabetic, it follows that the most widely used chronic healing models involve aged and diabetic rodents [188]. Type I and type II diabetes mellitus (T1DM and T2DM) can be modelled in mice. T1DM-mediated delayed healing is commonly stimulated through streptozocin injection [189,190], where timing post-injection is critical to the delayed healing phenotype [192]. Genetically altered mice are used to mimic T2DM through leptin or leptin receptor deficiency. These mice are morbidly obese by 6–8 weeks of age, go on to show hallmarks of T2DM (reviewed in [193]), and display substantially delayed healing versus their non-diabetic, heterozygous littermates [194]. There remains some controversy as to whether delayed healing in diabetic mice is a result of hyperglycaemia, leptin deficiency or obesity [184].

To mimic age-associated healing pathology, mice are wounded at 18 plus months of age (reviewed in [195]). Young ovariectomized mice provide an alternative accelerated ageing model, where surgical removal of the ovaries mimics the human menopause [196]. Here, the loss of circulating sex hormones, particularly 17β-estradiol, produces a delayed healing phenotype that is largely comparable to that of aged mice (reviewed in [197]). Unlike diabetic models, limited to comparison against diabetic wounds, aged models have the advantage that they emulate a more generalized underlying risk factor for all chronic wounds, advanced age [198].

5. Current therapies and future opportunities

Wound management begins with an assessment of wound aetiology and a patient-centric approach to managing systemic and lifestyle factors. In the case of diabetic foot ulcers, local management often starts with debridement, the removal of necrotic, infected or hyperkeratotic tissue via surgical or less invasive modalities [5,199]. Extracting the chronic tissue back to less affected epidermis, while triggering an acute injury response, is thought to kick-start normal reparative healing pathways [200]. Wounds are then irrigated with saline or antibacterial solution and a tailored dressing is applied [201]. Contemporary dressings contain a myriad of material properties to aid tissue repair and incorporate substances with known pro-healing or antimicrobial effects [202,203]. More advanced solutions are available, including the continually evolving negative pressure wound therapy modality [204]. Despite numerous available treatments, current best practice wound management is almost exclusively aimed at addressing secondary causes of chronicity, while also relying heavily on patient compliance. These two factors result in up to 40% of chronic wounds persisting for many months or years despite extensive treatment [102]. There remains a clinical unmet need to address this shortfall with novel therapies that are financially, physiologically and practically viable for the wound care setting.

A major contributor to chronic wound recalcitrance is persistent, antibiotic-resistant biofilm infection. It is therefore unsurprising that a large proportion of recent wound research has focused on the development of novel antimicrobial and anti-biofilm therapies. Traditional non-antibiotic antimicrobials, such as silver salts, alleviate bacterial burden but are cytotoxic to the host, while modern formulations (e.g. nanoparticles) have lower cytotoxicity and may also promote wound healing (reviewed in [205]). Emerging antimicrobial treatments that may also show beneficial roles in tissue repair include cold atmospheric plasma [206,207] and bioactive glass [179,208].

Most antimicrobials display broad effects and are not targeted to specific pathogenic species and strains. This is important, as commensal bacteria have a positive role in skin maintenance and wound repair (reviewed in [209]), and unlike their pathogenic counterparts, commensal biofilms do not cause persistent delayed healing in diabetic wounds [166]. As a result, more directed treatments for pathogenic bacteria, such as phage therapy [210] or pharmacological inhibition of bacterial virulence mechanisms such as quorum sensing [211], may confer higher specificity and efficacy. Moreover, most treatments focus on the bacterial component of infection, but the fungal diversity of wounds is also linked to healing outcome [212]. Thus, to elucidate the role of host–microorganism interactions in pathological repair, prospective research should acknowledge the wound ecosystem in its entirety.

Experimental studies are providing new insight into the underlying molecular and cellular correlates to chronic wound pathology. This in turn offers exciting new avenues for future therapeutic prevention and intervention. For example, chronic wounds are burdened by high levels of cellular senescence [141,142]. Senolytic drugs such as quercetin target senescent cells, and have already shown promise in reducing senescent cell burden in pathology [213,214] and ameliorating symptoms of diabetes, including inflammation and hyperglycaemia (reviewed in [215]). Further, blockade of the senescence-linked receptor, CXCR2, directly accelerates diabetic wound repair in vivo [112]. Repurposing these existing treatments (a number of senolytics drugs and CXCR2 antagonists have been tested in clinical trials [216,217]) offers an attractive approach for wound management. Other cell-targeted strategies include the administration of stem cells (reviewed in [218]), growth factors (reviewed in [219]) and gene therapies (reviewed in [220]). The major reparative effects of emerging and potential chronic wound therapies are outlined in figure 3.

Figure 3. Traditional and novel chronic wound treatments and their major tissue effects. Debridement of infected and necrotic tissue, followed by tailored dressing use, is common in wound treatment, with the aim of reducing microbial burden, dampening inflammation and providing a more suitable environment for healing. Antimicrobial therapies are emerging to disrupt biofilms and selectively remove pathogenic, rather than commensal, organisms. EPS = extracellular polymeric substance. QS = quorum sensing. ABs = antibiotics. Cell therapies such as mesenchymal stem cells (MSCs) can benefit multiple aspects of wound repair. re-ep = re-epithelialization. Finally, targeting chronic wound senescence with senolytics (e.g. metformin or CXCR2 antagonists) may be a viable option to reduce inflammation and promote healing.

6. Conclusion

The high cellular diversity, complexity and plasticity of wound healing provide a considerable challenge to comprehensively elucidate. While this remains a perplexing goal, it is essential that we continue to strive to more fully understand the mechanisms that underpin both normal and pathological healing. While not without their limitations, emerging wound models provide an unprecedented opportunity to further explore the molecular and cellular features of wound repair. Combining these approaches with novel tissue, cell and molecular ‘omics' technologies will considerably advance our understanding of wound pathology. Indeed, the future holds great promise for the development of innovative new therapeutic strategies for advanced wound care.


University of Minnesota, Mayo report COVID-fighting success with anti-aging therapy

Tests of an anti-aging therapy in mice are boosting hopes at Mayo Clinic and the University of Minnesota about a potential COVID-19 treatment that could reduce deaths and hospitalizations and improve vaccine effectiveness.

Survival increased in mice with COVID-like illnesses when they received drugs that removed senescent cells — sometimes called "retirement" or "zombie" cells that no longer divide or grow, but persist in the body, according to research published Tuesday by Mayo and U researchers in the journal Science.

While success in mice doesn't guarantee success in people, the results give the researchers confidence as they proceed with two human clinical trials in which they remove senescent cells from COVID-19 patients using high doses of the supplement fisetin. Senescent cells increase with age and chronic disease and could explain why older and unhealthier people make up more than 90% of Minnesota's 7,477 COVID-19 deaths.

"If you've got a lot of senescent cells, what's going to happen is you're going to have an exaggerated response . and you're going to get all of these things that happen in older people that kill them with COVID," said Dr. James Kirkland, director of Mayo's Kogod Center on Aging and a lead author of the Science study.

Kirkland and colleagues were among the first to hypothesize how infectious agents prompt senescent cells to increase harmful inflammation in the body. They also discovered how substances such as fisetin — a coloring agent in fruits and vegetables — clear out senescent cells.

The latest finding comes amid substantial declines in COVID-19 activity in Minnesota, which on Wednesday reported that its rate of new infections fell below the state's pandemic caution threshold for the first time since April 2020. Hospitalizations for COVID-19 in Minnesota fell to 192 on Tuesday, the lowest number since spring last year. The state has reported 603,144 infections in the pandemic, adding 150 more infections on Wednesday along with eight more COVID-19 deaths.

While vaccination progress has slowed, nearly 3 million people 12 and older in Minnesota have received a shot and 2.7 million have completed the one- or two-dose series. Mayo and U researchers said a new therapy would be critical even if the pandemic dissipates in Minnesota. Other parts of the world haven't received broad access to vaccine and are in need of treatments to combat the pandemic, which also could rise again in the U.S. if variants of the coronavirus become more severe or vaccine-resistant.

Senescent research at Mayo and the U started years before COVID-19 but was adapted to see if it could make a difference in the pandemic. Discoveries from this work could lead to senescent cell therapies for people with various aging conditions such as arthritis or dementia or to treatments for the next pandemic.

"This approach is improving the resilience to pathogen exposure — one being coronaviruses like SARS-CoV-2 — in the elderly," said Paul Robbins, a co-director of the Institute on the Biology of Aging and Metabolism at the U Medical School. "This will just increase the chances of survival whether it's pneumonia or COVID-19 or COVID-24 or whatever is going to be next."

A key U contribution was its "dirty mouse facility," mixing pristine lab mice with ordinary mice carrying a coronavirus similar to the one that causes COVID-19 in people. All the older lab mice died after exposure, while the younger ones survived. However, when the older mice received treatment to reduce senescent cells, more than half survived.

It made intuitive sense that removing senescent cells could address related conditions of aging, said Dr. Laura Niedernhofer, the other co-director of the U aging center and a lead author of the Science report, but it was surprising to see its impact on a new and unrelated infection.

"That is just pretty amazing that this approach to treating biology of aging protects you from [bad outcomes from] infection," she said.

One of Mayo's trials has recruited 51 out of a goal of 70 patients hospitalized with COVID-19 to compare outcomes and antibody levels between those who receive fisetin and those who don't.

Mayo and the U are starting a second trial using fisetin to try to prevent severe COVID-19 illness and complications in nursing home residents, whose ages and illnesses can make vaccines less effective.

"Senescent cells reduce the ability of normal cells to fight off viruses," Kirkland said, and they "upregulate" the processes in which viruses bind to and enter healthy cells.

Drugs such as metformin are already being tested at the U and other institutions to interfere with those processes in people with COVID-19. While fisetin is available over the counter as an anti-aging supplement, Kirkland discouraged people from taking it on their own to protect against COVID-19.

The dosage level is probably too low in supplements to have an impact, he said, compared with the clinical-grade doses being used in the trials. However, there are unknown risks to taking high doses of fisetin — which Mayo received permission to administer after filing the kind of investigational new drug application with the U.S. Food and Drug Administration that is usually reserved for experimental new drugs.

"We're playing with a fundamental aging mechanism," Kirkland said. "We don't know what all the potential downsides could be."

Jeremy Olson is a Pulitzer Prize-winning reporter covering health care for the Star Tribune. Trained in investigative and computer-assisted reporting, Olson has covered politics, social services, and family issues.


The treatment of precancerous cells will again depend upon the location of the cells. Sometimes close monitoring is all that is recommended to see if the level of dysplasia progresses or resolves without treatment.

Often the precancerous cells will be removed by a procedure such as cryotherapy (freezing the cells) or surgery to remove the region in which the abnormal cells are located.  

Even if the abnormal cells are removed, it’s important to keep in mind that whatever caused the cells to become abnormal in the first place may affect other cells in the future, and careful monitoring over the long term is important.

If abnormal cervical cells are treated with cryotherapy, it will still be important to monitor for recurrent problems with Pap smears in the future.   And if Barrett’s esophagus is treated with cryotherapy, you will still need to have your esophagus monitored at intervals in the future.  

For some abnormalities, your doctor may recommend chemoprevention. This is the use of a medication that reduces the risk of cells' becoming abnormal in the future.  

An example of this is to treat an infection with the H. pylori bacteria in the stomach. Ridding the body of the bacteria appears to reduce precancerous cells and the development of stomach cancer.  

Researchers are looking at the use of several medications and vitamins to see if their use in former and current smokers will lower their risk of developing lung cancer in the future.  

A last and important point to make is a reminder that, in some cases, the progression of precancerous changes may be altered by our environment: the foods we eat, the exercise we get, and the lifestyle choices we make. A diet rich in foods containing certain vitamins, for example, may help the body clear the HPV virus more rapidly.  

Similarly, avoiding substances that may be responsible for precancerous changes (such as tobacco) may reduce the risk of precancerous cells progressing or the formation of further precancerous cells in the future.

An example is the situation with smoking and cervical cancer. While smoking does not appear to cause cervical cancer, combining smoking with an HPV infection increases the chance that a cancer will develop.  


Introduction

Peripheral versus Central Serotonin

Serotonin [5-hydroxytryptamine (5-HT)] has two lifes: as a neurotransmitter, it regulates sleep, appetite, mood, and other important brain functions and—separated by the blood𠄻rain barrier and synthesized in a different way—it plays a central role in many other organ systems as a peripheral hormone (Figure 1) (1, 2). In fact, most of the body’s serotonin is circulating in the bloodstream, transported by blood platelets (3). Most of the peripheral serotonin is synthesized by TPH1 in the enterochromaffin cells of the intestine, secreted into the bloodstream, and then taken up by circulating platelets (4). Platelets store serotonin at very high concentrations in their dense granules (at 65 mM) and secrete it upon activation (5). Resting plasma serotonin concentrations (around 10 nM) can rapidly increase to 10 µM or more when platelets become activated at the site of thrombus formation or inflammation (6, 7).

Figure 1. The effects of peripheral and central serotonin.

Discovered by Rapport et al. in 1948 as a vasoconstrictor (8), new functions of serotonin have since been described continuously. These functions are mediated by members of the 7 known mammalian serotonin receptor subtype classes (15 known subtypes), the serotonin transporter (SERT), and by covalent binding of serotonin to different effector proteins—named “serotonylation” by Walther, Bader, and colleagues (3, 9). Peripheral serotonin is involved in the regulation of hemostasis, heart rate, vascular tone, intestinal motility, cell growth in liver, bone, and pulmonary arteries, and the development of heart, brain, and mammary gland (3). In addition, a number of immunoregulatory functions have been ascribed to serotonin (as described below).

Theoretically, either peripheral—i.e., predominantly platelet-derived—or central—i.e., neuronal—serotonin (or both) could modulate immune responses. In their review article, from 1998, Mössner and Lesch discussed the possibility of a neural-immune interaction via the autonomic nervous system, but found only two of four criteria to be fulfilled in the case of serotonin (6): serotonin receptors are present on immune cells and serotonin has immunoregulatory effects. Two other criteria do not apply in the case of serotonin, though. One criterion is the local association of neurotransmitter-specific nerve fibers with immune cells (although serotonin can be taken up by noradrenergic terminals on smooth muscle cells, similar to the adrenal medulla) (10, 11). The other criterion is the exclusive neurotransmitter supply of the immune target cells/organ by neurons, i.e., that the target organ could be depleted of serotonin by denervation. It is hence more likely that serotonin derived from non-neuronal sources exerts most of the immunoregulatory effects. In accordance, Roszman et al. concluded from several studies that the immunomodulatory effects of serotonin are mediated primarily through peripheral mechanisms directed toward circulating immune cells (2). Possible sources for peripheral serotonin are plasma (at rather stable, nanomolar levels), monocytes/macrophages, lymphocytes, vascular smooth muscle cells, adipocytes, mast cells (although human mast cells were long thought not to contain serotonin), and platelets (6, 12�). Local mast cells (probably rodent as well as human) produce, store, and release serotonin into the extravascular space—in part, even under neural control (6, 16, 17). Still, the vast majority of total peripheral serotonin is stored in platelets and released upon platelet activation (reaching micromolar levels) (3, 5). At least intravascular effects are, therefore, certainly mediated by platelet serotonin.

Platelet Serotonin in Immune Responses

In 1960, Davis et al. observed that serotonin, platelets, and inflammation were closely linked: within the first minute after injection of a lethal dose of E. coli endotoxin, they observed a sharp decrease in platelet count and serum serotonin, accompanied by a transient increase in plasma serotonin in dogs (18). It is now known that platelets (as transport vehicles) ensure the targeted release of serotonin in platelet-activating environments like a thrombus or an inflammatory reaction. At inflammatory sites, not only soluble factors like platelet-activating factor, complement anaphylatoxin C5a, and IgE-containing immune complexes but also bacteria or parasites as well as platelet𠄾ndothelial interactions activate platelets, resulting in serotonin secretion (6, 19�). Serotonin was shown to exert functions in innate as well as adaptive immunity. Serotonin stimulates monocytes (23) and lymphocytes (24) and hence influences the secretion of cytokines. Vascular smooth muscle cells respond to serotonin by synthesizing interleukin (IL)-6, a possibly atherogenic mechanism (25). In contrast to these descriptions of a pro-inflammatory function of serotonin, specific activation of the 5-HT2A receptor subtype in primary aortic smooth muscle cells presents a superpotent inhibition of tumor necrosis factor (TNF)-α-mediated inflammation (26). This effect was also shown in vivo in an animal model. The systemic selective activation of the 5-HT2A receptor with (R)-DOI blocks the systemic inflammatory response by downregulating the expression of pro-inflammatory genes and preventing the TNF-α-induced increase of circulating IL-6 (27).

Several other seemingly contradictory findings underline the complexity of peripheral serotonin effects. Two conflicting reports describe the interaction between leukocytes and inflamed endothelium upon serotonergic intervention. Kubes and Gaboury showed in 1996 that perivascular mast cells, which are believed to rapidly internalize serotonin and also to synthesize serotonin via TPH1, secrete serotonin to induce an early, leukocyte-independent phase of edema formation (16, 28, 29). The recruitment of leukocytes did not seem to depend on (mast cell-derived) serotonin. In 2007, Walther et al. found that leukocyte adhesion to inflamed endothelium after injection of endotoxin depended on the activation of serotonin receptors as shown by pharmacological blockade (30). Müller et al. found in 2009 that dendritic cell migration and cytokine release was modulated by serotonin (31). In our recent studies, leukocyte recruitment to sites of inflammation is impaired in the absence of (platelet-derived) serotonin and enhanced if plasma serotonin levels rise (32, 33).

In 1999, Gershon commented the complexity of peripheral serotonin effects in an ironic way (34): 𠇅-HT has delighted every pharmacologist who ever applied it to a gastrointestinal preparation something always happens, no matter what the experimental circumstances. For example, depending on the conditions, 5-HT can make the bowel contract or relax, secrete, or not secrete. The problem that has bedeviled attempts to determine what 5-HT actually does for the gut has been that it is able to do too much.” In 2009, Berger even counted a “Myriad effects of serotonin outside the central nervous system” (3). The same complexity seems to apply also to the role of serotonin in immunity [Figure 3 (40)]. In conclusion, to date, the knowledge in this field remains incomplete but assigns a variety of important immunomodulatory functions to peripheral serotonin.