immunology

The Role of the Intestinal Microbiota in Sepsis Mortality

Project Summary/Abstract Sepsis is a life-threatening condition characterized by a dysregulated host response to infection that can cause multi-organ damage and death. As the leading cause of in-hospital mortality, sepsis mortality rates reach up to 50%, and account for approximately 270,000 deaths and $38 billion annually in health care costs in the United States. Notably, patients with similar medical backgrounds can have vastly different sepsis outcomes— some survive with medical treatment while others die. The reasons for this dichotomy are unknown but is seen across all forms of bacterial bloodstream infections, is not specific to any strain-level differences in the infecting pathogen and cannot be explained by human genetic differences. Human microbiota studies suggest that gut microbial dysbiosis is associated with sepsis mortality and that these alterations influence gut barrier breakdown, leading to gram-negative bacteremia—one of the most common causes of sepsis and mortality. However, there are a lack of studies that investigate the causal role of the intestinal microbiota in sepsis mortality. This K08 proposal will elucidate the role of the intestinal microbiota in sepsis mortality. Utilizing the well- established murine model of sepsis by intraperitoneal injection of lipopolysaccharide (LPS), we combine microbiota taxonomic sequencing and metagenomics, advanced bioinformatic techniques and prediction modeling, with knowledge of mucosal immunity and germ-free mouse systems to characterize the microbiota features and members that correlate with, predict, and cause sepsis mortality. This proposal is organized into two specific aims: (1) identify baseline stool microbial features associated with and predictive of sepsis outcomes and (2) determine how colonization with immunostimulatory microbes heightens sepsis mortality. In this work, I will holistically characterize the host immunologic and microbiota features that are associated with and predictive of mortality and experimentally identify microbes and microbial pathways that cause death in our model. These findings will reveal new microbial and host biomarkers of sepsis mortality and identify novel targets for sepsis prevention and treatment to reduce the overall mortality rate of this deadly disease. My long-term goal is to become an independent physician-scientist who integrates cutting-edge computational methods with experimental biology to identify predictive biomarkers of disease onset and outcomes, investigate how they influence disease processes, and develop novel therapeutic and preventive strategies to improve patient care. This proposal details specific research aims and a structured career development and training plan that will allow me to acquire focused, in-depth and multidisciplinary training under the guidance of an internationally recognized team of experts in clinical infectious diseases, host-microbiota interactions, immunology, immunometabolism, and computational biology. The knowledge generated will address the fundamental role of the microbiota in sepsis outcomes and inform future preventative and therapeutic strategies that will lower the sepsis mortality rate worldwide.

Mechanisms of antigen-specific T cell activation in MOGAD

PROJECT SUMMARY / ABSTRACT The overarching goal of this application is to train Dr. Carson E. Moseley, MD, PhD, who is a clinical neurologist and a research immunologist, to become an independent investigator studying and treating neuroimmunologic disorders. Myelin oligodendrocyte glycoprotein (MOG) antibody-associated disease (MOGAD) is a recently described, severe, neuroinflammatory syndrome of the central nervous system (CNS) with no approved therapies. Although MOG-specific antibodies helped define the disease, MOG antibodies alone are not clearly pathogenic and our understanding of MOGAD immunopathology is limited. CD4+ T cells are a dominant lymphocyte population in MOGAD lesions, yet the targets of T cell responses to MOG and how T and B cells interact to drive pathogenic immune response in MOGAD are unknown. This proposal uses a complementary approach of human and mouse immunology along with new technologies in T cell repertoire mapping and genome editing to dissect MOG-specific CD4+ T cell responses in MOGAD. Additionally, it will use new models to investigate how B cells promote pathogenic T cell differentiation and select pathogenic T cell receptors. The proposed training plan involves mentored training, seminars, formal learning, and advising to ensure completion of the proposed research and Dr. Moseley’s career development. He will train at UCSF, which is an outstanding institute for research and environment for physician-scientists. He will receive training in human immunology and CRISPR-based gene editing technologies. He will be mentored by Dr. Scott Zamvil, a leader in identifying antigen-specific T cell responses in neuroimmunologic disorders, and co-mentored by Dr. Alexander Marson, an expert in CRISPR gene editing to understand lymphocyte function. This application will provide Dr. Moseley with the long-term skills needed to become an independent investigator leading efforts to study and treat neuroimmunologic disorders.

Administrative Core

CORE A: PROJECT SUMMARY/ABSTRACT Administrative Core The administrative core will be led by Dr. Jordan Pober, the overall PI of this P01 application. Dr. Pober has had past experience as PI of an NHLBI P01 focused on allograft vasculopathy. He also has administrative experience at Yale as the founder and director of two Yale interdepartmental programs: Vascular Biology and Therapeutics and Human and Translational Immunology. The co-leader of the Core is Dr. Marie Robert, a surgical pathologist with extensive expertise in celiac disease (CeD) who has served in the recent past as the head of the scientific advisory board to the Beyond Celiac organization. The principal task of the Core will be to facilitate interactions among Project, Core and Collaborating Site personnel to foster synergies to address the overall aims of the proposal. Specific tasks include (1) organizing an executive committee of all Project, Core and Site Leaders with advisory and review responsibilities; (2) organizing monthly review meetings, each meeting focused on an individual project and site and (sometimes) core activities involving all program personnel and our internal advisors; (3) organizing an external advisory committee of experts to participate in an annual review of the whole program; and (4) managing budgetary and regulatory functions of the program. The innovative aspects of Core A is its prioritization of team science, bringing together the insights and knowledge of clinical-based and laboratory-based investigators.

Bridging Local and System-Wide Autoreactive, Extrafollicular B Cell Signatures in a TLR7-Driven Model

Project Summary A substantial body of literature has described the development of autoreactive humoral responses in the context of autoimmune disease and recently discerned an exciting new avenue for investigation. While early work focused on canonical mechanisms of activation through the germinal center (GC) response, recent studies have found GC infrastructure to be dispensable for the onset of chronic autoimmunity. It has become clear that an alternative pathway of B cell activation, the extrafollicular (EF) pathway, can drive the onset of new autoreactivity in multiple human disorders including rheumatoid arthritis and systemic lupus erythematosus (SLE). In comparison to the GC pathway, the EF pathway represents a less stringent method for B cell activation, leads to accelerated antibody-secreting cell (ASC) formation, and thus has a higher propensity for the production of autoreactive B cell effectors and ASCs. Recently, our group has identified a similar skew toward the EF response in the context of severe viral infection, tied to acute tolerance loss, increased disease severity, and complicated recovery from infection. These findings highlight how further study of the EF response is crucial to our understanding of autoimmune induction across multiple areas of disease. Toll-like receptor 7 (TLR7) stimulation has been identified as a key contributor to EF B cell development in SLE, and several studies have now linked TLR7 overstimulation to chronic autoimmune disease. While EF effector B cell populations have now been identified in both murine models and humans, substantial gaps in our knowledge remain to be answered concerning i) the origins of these cells and ii) the system-wide and microenvironmental signaling and organization that drive this differentiation pathway. We propose to address these gaps, here, by utilizing a TLR7 agonist (R848) in a murine model to characterize the autoreactive response within the blood and draining lymph node through innovative high-throughput analytical techniques. Systemic shifts in proteomic signatures and immune cell phenotype will be monitored in the blood throughout the induction of autoreactivity, using novel applications of machine-learning based classification. These signatures will then be connected to developing inflammatory microenvironments identified within the draining lymph node by applying a customized set of software tools to spatial transcriptomic data. This work will deepen our understanding of the immunologic mechanisms by which the EF pathway can lead to “run-away” autoreactive B cell development, with the added potential for identification of early blood-based biomarkers for this developing autoreactivity. The above proposed work will provide an ideal training opportunity for the candidate to develop experience with advanced immunologic laboratory techniques, rigorous bioinformatic analysis, a systems-level view of immunology, and scientific communication. The Woodruff and Sanz Labs are highly experienced within the autoimmune disease space with extensive experience with the required techniques and established routes for clinical collaboration to act on these findings.

Facilitating the Advancement of Research and Education for Undergraduate Students by Incorporating Laser Scanning Confocal Microscopy (FAREUS-LSCM)

PROJECT SUMMARY/ABSTRACT The University of Puerto Rico at Aguadilla (UPR-Aguadilla) requests funding to acquire a Nikon AX Galvo Confocal Laser Scanning Microscope (LSCM) with a TI2-E inverted platform and a four- laser configuration (405/488/561/640 nm) to establish transformative imaging capabilities at our resource-limited institution serving 96% Pell Grant recipients. This state-of-the-art instrument addresses a critical infrastructure gap, enabling high-resolution fluorescence imaging, live-cell microscopy, and quantitative analysis essential for competitive biomedical research and undergraduate education. The LSCM will directly support four active research projects spanning parasitology (monogenean host-specificity studies), plant pathology (coffee biocontrol development), environmental chemistry (metalloprotein biomarkers), and neuroscience (astrocyte dysfunction in diabetic epilepsy) while integrating into core laboratory courses including Immunology (BIOL 4009) and Undergraduate research courses (BIOL 3108 and QUIM 4999). Our multidisciplinary faculty, in partnership with the Neuroimaging and Electrophysiology Facility (NIEF) Excellence Imaging Center, offers expertise in confocal microscopy, encompassing advanced imaging and specialized sample preparation techniques. This collaboration ensures effective implementation of the technology, sustained technical support, and high-quality training programs that will enhance research productivity and broaden educational impact. The broad, long-term objective is to transform UPR-Aguadilla from a primarily teaching institution into a research-active campus capable of producing graduate-school-ready students equipped with cutting-edge technical skills. Access to advanced confocal microscopy will stimulate new research collaborations, enhance faculty productivity, and provide 30-40 students annually with hands-on experience in modern imaging technologies currently absent from our curriculum. The instrument will strengthen our partnership with the emerging Natural History Museum of Puerto Rico for specimen digitization and support comprehensive outreach programs targeting 25-50 high school students annually through "Seeing Science Up Close" workshops. Expected outcomes include 1- 2 peer-reviewed publications within three years, establishment of 1-2 new institutional collaborations, and measurable enhancement of biomedical research capacity. This investment will significantly advance STEM education and research opportunities at UPR-Aguadilla while expanding access to cutting-edge scientific instrumentation for students pursuing biomedical careers and contributing to the development of skilled researchers in the biomedical sciences.

How the brain barriers ensure CNSimmune privilege”

Britta Engelhard’s research is devoted to understanding thefunction of the different brain barriers in regulating CNS immunesurveillance and how their impaired function contributes toneuroinflammatory diseases such as Multiple Sclerosis (MS) orAlzheimer’s disease (AD). Her laboratory combines expertise invascular biology, neuroimmunology and live cell imaging and hasdeveloped sophisticated in vitro and in vivo approaches to studyimmune cell interactions with the brain barriers in health andneuroinflammation.

Cerebrospinal fluid and the meninges : Understanding brain immunology from its borders

The immunopathogenesis of autoimmune seizure disorders

Immune-mediated mechanisms are increasingly recognised as a cause of epilepsy even in the absence of an immune response against a specifical neuronal antigen. In some cases, these autoimmune processes are clearly pathogenic, for example acute seizures in autoimmune encephalitis, whereas in others this is less clear, for example autoimmune-associated epilepsy. Recent research has provided novel insights into the clinical, paraclinical and immunopathogenetic mechanisms in these conditions. I will provide an overview of clinical and paraclinical features of immune-associated seizures. Furthermore, I will describe specific immunopathogenic examples implicating lymphoid follicular autoimmunisation and intrathecal B cells in these conditions. These insights into immunopathogenesis may help to explain the role of current and immunotherapies in these conditions.

The role of CNS microglia in health and disease

Microglia are the resident CNS macrophages of the brain parenchyma. They have many and opposing roles in health and disease, ranging from inflammatory to anti-inflammatory and protective functions, depending on the developmental stage and the disease context. In Multiple Sclerosis, microglia are involved to important hallmarks of the disease, such as inflammation, demyelination, axonal damage and remyelination, however the exact mechanisms controlling their transformation towards a protective or devastating phenotype during the disease progression remains largely unknown until now. We wish to understand how brain microglia respond to demyelinating insults and how their behaviour changes in recovery. To do so we developed a novel histopathological analysis approach in 3D and a cell-based analysis tool that when applied in the cuprizone model of demyelination revealed region- and disease- dependent changes in microglial dynamics in the brain grey matter during demyelination and remyelination. We now use similar approaches with the aim to unravel sensitive changes in microglial dynamics during neuroinflammation in the EAE model. Furthermore, we employ constitutive knockout and tamoxifen-inducible gene-targeting approaches, immunological techniques, genetics and bioinformatics and currently seek to clarify the specific role of the brain resident microglial NF-κB molecular pathway versus other tissue macrophages in EAE.

Aging promotes reactivation from metastatic melanoma dormancy

How does the primary tumor imprint a dormancy signature in disseminated tumor cells?

T cells specific for alpha-myosin drive immunotherapy-related myocarditis

CD8+ T cell activation in cancer comprises an initial activation phase in lymph nodes followed by effector differentiation within the tumor

Immune regulation by fungal strain diversity in inflammatory bowel disease

Nociceptor neurons direct goblet cells via a CGRP-RAMP1 axis to drive mucus production and gut barrier protection

Lifestyle, cardiovascular health, and the brain

Lifestyle factors such as sleep, diet, stress, and exercise, profoundly influence cardiovascular health. Seeking to understand how lifestyle affects our biology is important for at least two reasons. First, it can expose a particular lifestyle’s biological impact, which can be leveraged for adopting specific public health policies. Second, such work may identify crucial molecular mechanisms central to how the body adapts to our environments. These insights can then be used to improve our lives. In this talk, I will focus on recent work in the lab exploring how lifestyle factors influence cardiovascular health. I will show how combining tools of neuroscience, hematology, immunology, and vascular biology helps us better understand how the brain shapes leukocytes in response to environmental perturbations. By “connecting the dots” from the brain to the vessel wall, we can begin to elucidate how lifestyle can both maintain and perturb salutogenesis.

Emerging Treatment Options in Psychiatry

The World Health Organization (WHO) estimates that untreated mental disorders accountfor 13% of the total global burden of disease, and by 2030, depression alone will be the leadingcause of disability around the world – outpacing heart disease, cancer, and HIV. This grim pictureis further compounded by the mental health burden delivered by the coronavirus pandemic.The lack of novel treatment options in psychiatry is restricted by a limited understanding in theneuroscience basis of mental disorders, availability of relevant biomarkers, poor predictability inanimal models, and high failure rates in psychiatric drug development. However, theannouncement in 2019 from the Federal Drug Administration (FDA) for approvals of newinterventions for treatment-resistant depression (intranasal esketamine) and postpartumdepression (i.v. brexanolone), demand critical attention. Novel public-private partnerships indrug discovery, new translational data on co-morbid biology, in particular the ascendance ofpsycho-immunology, have highlighted the arrival of a new frontier in biological psychiatryresearch for depressive disorders.

Reflex Regulation of Innate Immunity

Reflex circuits in the nervous system integrate changes in the environment with physiology. Compact clusters of brain neuron cell bodies, termed nuclei, are essential for receiving sensory input and for transmitting motor outputs to the body. These nucelii are critical relay stations which process incoming information and convert these signals to outgoing action potentials which regulate immune system functions. Thus, reflex neural circuits maintain parameters of immunological physiology within a narrow range optimal for health. Advances in neuroscience and immunology using optogenetics, pharmacogenetics, and functional mapping offer a new understanding of the importance of neural circuitry underlying immunity, and offer direct paths to new therapies.

Neuro-Immune Coupling: How the Immune System Sculpts Brain Circuitry

In this lecture, Dr Stevens will discuss recent work that implicates brain immune cells, called microglia, in sculpting of synaptic connections during development and their relevance to autism, schizophrenia and other brain disorders. Her recent work revealed a key role for microglia and a group of immune related molecules called complement in normal developmental synaptic pruning, a normal process required to establish precise brain wiring. Emerging evidence suggests aberrant regulation of this pruning pathway may contribute to synaptic and cognitive dysfunction in a host of brain disorders, including schizophrenia. Recent research has revealed that a person’s risk of schizophrenia is increased if they inherit specific variants in complement C4, gene plays a well-known role in the immune system but also helps sculpt developing synapses in the mouse visual system (Sekar et al., 2016). Together these findings may help explain known features of schizophrenia, including reduced numbers of synapses in key cortical regions and an adolescent age of onset that corresponds with developmentally timed waves of synaptic pruning in these regions. Stevens will discuss this and ongoing work to understand the mechanisms by which complement and microglia prune specific synapses in the brain. A deeper understanding of how these immune mechanisms mediate synaptic pruning may provide novel insight into how to protect synapses in autism and other brain disorders, including Alzheimer’s and Huntington’s Disease.

Regenerative Neuroimmunology - a stem cell perspective

There are currently no approved therapies to slow down the accumulation of neurological disability that occurs independently of relapses in multiple sclerosis (MS). International agencies are engaging to expedite the development of novel strategies capable of modifying disease progression, abrogating persistent CNS inflammation, and support degenerating axons in people with progressive MS. Understanding why regeneration fails in the progressive MS brain and developing new regenerative approaches is a key priority for the Pluchino Lab. In particular, we aim to elucidate how the immune system, in particular its cells called myeloid cells, affects brain structure and function under normal healthy conditions and in disease. Our objective is to find how myeloid cells communicate with the central nervous system and affect tissue healing and functional recovery by stimulating mechanisms of brain plasticity mechanisms such as the generation of new nerve cells and the reduction of scar formation. Applying combination of state-of-the-art omic technologies, and molecular approaches to study murine and human disease models of inflammation and neurodegeneration, we aim to develop experimental molecular medicines, including those with stem cells and gene therapy vectors, which slow down the accumulation of irreversible disabilities and improve functional recovery after progressive multiple sclerosis, stroke and traumatic injuries. By understanding the mechanisms of intercellular (neuro-immune) signalling, diseases of the brain and spinal cord may be treated more effectively, and significant neuroprotection may be achieved with new tailored molecular therapeutics.

Microglia, memories, and the extracellular space

Microglia are the immune cells of the brain, and play increasingly appreciated roles in synapse formation, brain plasticity, and cognition. A growing appreciation that the immune system involved in diseases like schizophrenia, epilepsy, and neurodegenerative diseases has led to renewed interest in how microglia regulate synaptic connectivity. Our group previously identified the IL-1 family cytokine Interleukin-33 (IL-33) as a novel regulator of microglial activation and function. I will discuss a mechanism by which microglia regulate synaptic plasticity and long-term memories by engulfing brain extracellular matrix (ECM) proteins. These studies raise the question of how these pathways may be altered or could be modified in the context of disease.

Associations between brain interoceptive network dysconnectivity and heightened peripheral inflammation in depression

Are the immune system, brain, mind and mood related? Could this explain why chronic low-grade peripheral inflammation is also noted in approximately 1/3 of those with major depressive disorder (MDD)? The field recognized today as immunopsychiatry was founded on scientific evidence that germinated over 30 years ago. Since, it has been understood that (i) there could be a causal link between inflammation and depression, (ii) select blood immune markers show robust potential as biomarkers for inflammation-linked depression, and more generally, (iii) Descartes' theories on mind-body dualism were biologically erroneous. Nonetheless, the mechanistic brain-immune axis in the trinity formulating inflammation-linked depression i.e. psycho-neuro-immunology, still remains unclear. This talk will discuss findings from our recent investigation endeavored to unpack this by linking functional connectivity abnormalities with peripheral immune markers.

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Sexual dimorphism of microglia

Sex differences in brain structure and function are of substantial scientific interest because of sex-related susceptibility to psychiatric and neurological disorders. Neuroinflammation is a common denominator of many of these diseases and thus microglia as the brain´s immunocompetent and instrumental cells has come into focus in sex specific studies. We and others show that male microglia are more frequent in specific brain areas and appear to have a higher potential to respond to stimuli, whereas female microglia seem to acquire a more “protective” phenotype.

The immunopathology of advanced multiple sclerosis

We recently analyzed a large cohort of multiple sclerosis (MS) autopsy cases of the Netherlands Brain Bank (NBB) and showed that 57% of the lesion in advanced MS is active (containing activated microglia/macrophages). These active lesions correlated with disease severity and differed between males and female MS patients.1 Already in normal appearing white matter microglia show early signs of demyelination.5 T cells are also frequently present in advanced stages of MS and have a tissue resident memory (Trm) phenotype, are more frequently CD8+ then CD4+, are located perivascular, enriched in active and mixed active/inactive MS lesions and correlated with lesion activity, lesion load and disease severity.2-4 Like Trm cells, B cells are located perivascular and were also enriched in active MS lesions but in lower numbers and a proportion of the MS patients had almost no detectable B cells in the regions analyzed. MS patients with limited presence of B cells had less severe MS, and less active and mixed active /inactive lesions. We conclude that advanced MS is characterize by a high innate and adaptive immune activity which is heterogeneous and relates to the clinical disease course.

Meningeal lymphatics and peripheral immunity in brain function and dysfunction

Immune cells and their derived molecules have major impact on brain function. Mice deficient in adaptive immunity have impaired cognitive and social function compared to that of wild-type mice. Importantly, replenishment of the T cell compartment in immune deficient mice restored proper brain function. Despite the robust influence on brain function, T cells are not found within the brain parenchyma, a fact that only adds more mystery into these enigmatic interactions between T cells and the brain. Our results suggest that meningeal space, surrounding the brain, is the site where CNS-associated immune activity takes place. We have recently discovered a presence of meningeal lymphatic vessels that drain CNS molecules and immune cells to the deep cervical lymph nodes. This communication between the CNS and the peripheral immunity is playing a key role in neurophysiology and in several CNS disorders. Interestingly, meningeal lymphatics are impaired in aging and their dysfunction may be related to age-related cognitive decline as well as to Alzheimer’s pathology. In addition to providing new insights into age-related disorders, meningeal lymphatics may also serve as a novel therapeutic target for these diseases and are worth of in-depth mechanistic exploration.

Novel immunotherapy to treat Alzheimer’s disease and Dementia: from curiosity-driven research to prospect of therapy