cytokine

From B-cell decisions to antibody repertoires

PROJECT SUMMARY/ABSTRACT Vaccine responses are highly variable across the population and not without risk for debilitating side-effects. Antibody-mediated immunity is generated by a Darwinian process to generate B-cells that contain B-cell receptors (BCR) that have high affinity for the pathogen-derived antigen, while also eliminating B-cells that happen to react to self-antigens. This process depends on cell fate decisions such as (i) death vs survival, (ii) entry into a proliferative program, (iii) differentiation into antibody-secreting plasma cells. According to clonal selection theory, B-cell fate decisions are made based on the genetically encoded affinity of the the BCR to the antigen (Signal 1) and the cognate T-cells’ TCR to the antigen peptide (Signal 2). However, single-cell resolution studies have revealed that fate decisions of genetically identical B-cells are remarkably heterogeneous. Our studies of the previous funding period revealed that B-cell epigenetic heterogeneity is in fact dynamically controlled: it is generated during the selection process but remains largely stable during the proliferative burst. This leads to our newly proposed Aim 1 to examine how the dynamic control of epigenetic state variability affects antibody responses. An innovative multi-scale model of Darwinian evolution directs and interprets experimental studies by life cell video microscopy in vitro and in immunization studies in vivo. Our previous studies also found that B-cells are capable of sensing the time gap between signal 1 and 2, suggesting a temporal proofreading mechanism for negative selection. This leads to newly proposed Aim 2 which seeks to identify the regulatory circuits that control the stringency of negative selection, as well as contextual germinal center (GC) cytokines that could be manipulable in vivo. These in silico and in vitro studies are followed by in vivo immunization to extend their physiological relevance. Finally, in Aim 3, we will ask what determines the time-gap of signal1 and signal 2, which occur in the immune- induced structure of the GC. We will develop a new model that simulates B-cell fate decisions as a function of their interactions with antigen-presenting stromal cells and T-cells that may be cognate or non-cognate. Model simulations will be used to interpret spatial transcriptomic data to test different adjuvants and predictions will be tested in in vivo immunization studies. With mouse models of inflammation and aging we will examine how adjuvants alter vaccine efficacy and risk.

The role of endogenous chimeric mRNA encoded GasderminD fusion proteins in immunity

Project Summary: Programmed inflammatory cell death, or pyroptosis, is a crucial innate defense mechanism that protects hosts against infection and orchestrates subsequent immune responses. Central to this process is Gasdermin D (GSDMD), a protein that forms plasma membrane pores upon activation, enabling the release of pro- inflammatory cytokines such as IL-1β and driving cell lysis. Although GSDMD-mediated pyroptosis has been conventionally understood to be controlled mainly at the post-translational level, through proteolytic cleavage by inflammatory caspases, we have discovered compelling evidence that alternative RNA processing may introduce additional, previously unappreciated complexity in GSDMD regulation. Our laboratories have developed and optimized a highly innovative long-read direct RNA sequencing pipeline, which bypasses conventional cDNA synthesis to avoid artifacts and enables unbiased discovery of native chimeric mRNA (chRNA) in mammalian cells. Using this approach, we have uncovered a remarkably diverse repertoire of chRNA species, including over a thousand unique fusions in murine macrophages and more than two thousand in human inflamed tissues. Among the chRNA found in mice, we identified a chRNA joining the effector domain of GSDMD with a novel C-terminal region encoded by Tmem106a, giving rise to the GSDMD:TMEM106A fusion protein. Functional studies demonstrate that GSDMD:TMEM106A is not only produced in response to inflammatory signals in macrophages but is critical for GSDMD-dependent cytokine release and optimal pyroptosis. Genetic loss of GSDMD:TMEM106A in mice results in reduced cytokine secretion and increased susceptibility to bacterial infection, while in vivo delivery of Gsdmd:Tmem106a mRNA is sufficient for protective immunity. Intriguingly, we have also identified a putative human counterpart, GSDMD:S100A6, which is highly inducible in colon biopsies from patients with inflammatory bowel disease. In this application, we propose a comprehensive exploration of this newly defined class of naturally occurring GSDMD fusion proteins. The specific aims are: (1) to elucidate the subcellular localization, protein-protein interactions, and pore-forming function of GSDMD:TMEM106A during canonical and non-canonical inflammasome activation; (2) to determine the transcriptomic, proteomic, and physiological consequences of GSDMD chRNA expression in vivo during infection, sepsis, and inflammatory disease, and to validate and functionally characterize GSDMD:S100A6 in relevant immune and barrier cell populations. Collectively, this work will establish chimeric splicing as a fundamental source of immunoregulatory protein diversity, redefining the landscape of cell death control in the immune system. By revealing new layers of gasdermin regulation and function, our studies have the potential to identify novel therapeutic strategies for infectious, auto-inflammatory, and immune-mediated diseases.

Eosinophils promote persistence and transmission during Bordetella spp. infections

ABSTRACT Despite widespread vaccination, Bordetella spp., the causative agents of whooping cough, continue to circulate globally. Resurgent outbreaks contribute to significant healthcare burdens and costs estimated up to $79 million annually. This persistence and reemergence highlight a critical need for new therapies and prevention methods. Our laboratory investigates bacterial and host drivers that enable Bordetella success, defined as enhanced persistence, reinfection, and transmission. We have identified the Bordetella sigma factor BtrS as a regulator of immunosuppressive pathways that modulate eosinophil function. Leveraging genetically tractable Bordetella strains, advanced murine models, and immunological tools, we are uniquely positioned to dissect how eosinophils contribute to respiratory bacterial infections. Our preliminary data reveal that eosinophils promote Bordetella persistence. Our results also show that the anti-inflammatory cytokine IL1 receptor antagonist (IL1Ra) also contribute to persistence. However, the contribution of eosinophil-derived immunosuppressors remains unclear and will be investigated in Specific Aim 1. Moreover, we have evidence that eosinophils are required for nasal shedding, through mucus enhancement, and paroxysmal coughing, via exacerbation of bronchoconstriction, during Bordetella spp. infection, two key metrics of transmission. The eosinophil-effectors that promote shedding, coughing, and transmission, will be investigated in Specific Aim 2. Based on our data, we hypothesize that eosinophils contribute to Bordetella pathogenesis by (1) promoting persistent infection and (2) enhancing transmission through mucus-driven shedding and cough reflex induction. This proposal will test this hypothesis through two specific aims: Aim 1: Delineate the immunosuppressive role of eosinophils in modulating host responses and enabling Bordetella persistence. Aim 2: Define the mechanisms by which eosinophils facilitate Bordetella spp. transmission. By reframing eosinophils as active modulators of bacterial pathogenesis, this research challenges traditional views of eosinophils as terminal effector cells and positions them as novel targets for therapeutic intervention, that might be applicable to other mucosal pathogens. The outcomes will contribute to our understanding of eosinophil biology in infection and may lead to innovative strategies to halt bacterial persistence and transmission.

Borrelia burgdorferi genotypic diversity, pathogenesis, and host cellular responses

PROJECT SUMMARY Lyme disease is the most common tick-borne illness in the United States, with an estimated 476,000 cases annually, and Pennsylvania (PA) consistently reports one of the highest case numbers nationwide. Borrelia burgdorferi sensu stricto (Bb) is a causative agent of Lyme disease in the US and is transmitted by Ixodes spp. ticks. Bb produces various outer surface proteins (Osp) and other mechanisms to survive in vectors, evade host immune systems, and to propagate infection within a host. Over 35 OspC genotypes have been characterized, which fluctuate in abundance in natural vector and host populations, suggesting host adaptation. While many Lyme-infected patients recover following antibiotic treatment, some may experience neurological symptoms, Lyme neuroborreliosis (LNB), which may be associated with specific genotypes. While previous studies focused on clinical manifestations, pathogenicity, genetic variations, and host immune responses using mouse models or patient samples, the genotype-specific immune responses that contribute to disease progression in humans remain poorly understood. Our central hypothesis is that certain Bb OspC genotypes, maintained in natural populations, are associated with distinct host immune responses that influence disease severity, progression, and persistence. Aim 1 will define the dynamics of OspC genotypes in tick and small mammal populations over time in Western PA to establish a 16-year longitudinal tick study and an 8-year longitudinal small mammal study. Using deep amplicon sequencing, we will quantify genotype diversity, detect low-abundance genotypes, and identify potential host-adapted genotypes. These empirical data will inform a compartmental mathematical model to evaluate OspC genotype prevalence, distribution, and public health risks, including LNB, across space and time. Aim 2 will assess how distinct Bb OspC genotypes affect the host immune landscape and cellular responses using human samples. To determine how Bb genotype contributes to disease phenotype, we will perform immune profiling studies which will include microscopy-based assessment of infected cell cultures, flow cytometric analysis of immune cell phenotypes, and measurement of genotype-specific cytokine, chemokine, and antigen production (sub-Aim2a). We will also employ multi-omics approaches that integrate single cell RNA sequencing with antibody-based protein profiling (scRNA-seq/Ab-seq) to characterize transcriptional and functional changes in immune cell populations exposed to different Bb genotypes (sub-Aim2b). This work is innovative in its integration of long-term ecological data with advanced immune profiling and single cell multi- omics to uncover genotype-specific mechanisms of Bb pathogenicity and human immune response—an approach not previously applied in Lyme disease research. These studies will clarify how specific genotypes influence immune responses and disease severity. Together, the proposed aims will identify critical genetic and immunological mechanisms that drive Bb pathogenicity and human susceptibility, informing the development of improved diagnostics, targeted therapies, and public health interventions to reduce the burden of Lyme disease.

Investigating the nonlinear complex dynamics of the tuft cell-microbiome cross-talk: the impact of feedback loops on immune regulation, microbial modulation and response to tissue insults

Project Abstract Tuft cells (TCs) are specialized chemosensory epithelial cells that are emerging as critical regulators of intestinal homeostasis. Named over 70 years ago based on their distinct morphology, a defined function for TCs was only elucidated in the last decade. TCs in the small intestine sense succinate from helminths to initiate type 2 immune responses that mediate parasite expulsion. Recently, we discovered a novel physiologic function for TCs in the colon, where their role had been considered minimal. Succinate, a key microbial metabolite, is produced by colonic microbiota as both a precursor to other metabolites and a cross-feeding fuel source for pathogens. TCs respond to succinate by secreting interleukin-25 (IL-25), which activates type 2 cytokine- producing lymphocytes (T2Ls), amplifying TC expansion and reinforcing barrier function. We recently demonstrated that this SPB–TC–IL-25–T2L feedback loop is essential for protection against pathogen-induced colitis. Our preliminary data further suggest that TCs actively promote colonization by succinate-producing bacteria (SPBs), establishing positive feedback on TC-supporting microbes, while other epithelial cells such as goblet cells (GCs) and Paneth cells (PCs) may exert complementary or counterbalancing influences. Supported by new modeling insights, we hypothesize that these epithelial–immune–microbiome interactions form coordinated feedback loops that collectively optimize intestinal resilience. These loops may create a dynamic, multi-stable system that flexibly transitions between homeostatic and hyperplastic states, buffering against microbial fluctuations and pathogenic insults while preventing uncontrolled type 2 inflammation. Using a combination of mathematical modeling and experimental validation, we will develop a multi- layered systems framework to explore how epithelial–immune–microbial feedbacks shape resilience or breakdown in clinically relevant models of colonic infection and inflammation. Our three Aims will (1) develop, calibrate, and validate a mathematical model that integrates TCs, GCs, PCs, SPBs, and SCBs; (2) define the immunological circuits governing epithelial–microbiome equilibrium; and (3) determine how epithelial feedbacks regulate microbial community structure and resilience. In line with NIH’s new initiative to prioritize human-based research, our proposal combines computational modeling, human colonic organoids, and complementary mouse models. Organoid experiments will provide human-relevant data for model calibration, while in vivo studies validate systemic predictions, ensuring both rigor and translational relevance while minimizing reliance on animal models. This work will generate interoperable models that integrate epithelial, microbial, and immune networks, providing predictive insight into intestinal outcomes under homeostatic, infectious, and inflammatory conditions and informing therapeutic strategies for microbiome-targeted interventions.

Systems Biology of Early Atopy: Role of Human Milk (SunBEAm-Milk)

Surprisingly little is known about the effect of breastfeeding (BF) on infant immune system development besides an effect on the gut microbiome, but its impact on metabolites and Tregs could support protection against food allergy (FA). BF is currently recommended to prevent the development of allergic diseases, especially asthma/recurrent wheezing and AD in early childhood, but firm conclusions could not be drawn regarding FA due to high heterogeneity and low quality of studies. Reverse causation, recall bias and the poor accuracy of outcome assessment are significant limitations. Most are inadequately powered to specific FA; however, a recent study showed that exclusively BF infants had lower odds of egg, sesame, and peanut allergies. Importantly, immunomodulatory composition of HM varies between mothers, which has not been taken into consideration. For over two decades we have been developing methods to assess immunomodulatory factors in the complex matrix of HM and their association with infant FA. We have shown that high levels of HM total and specific IgA are associated with protection against cow’s milk allergy, but it is unclear whether HM IgA is responsible for or is a biomarker of the vertical transfer of protection. Infant fecal and systemic IgA levels during breastfeeding and after weaning are also elevated in infants at low risk for atopic disease raising the question of whether HM factors such as cytokines can promote IgA production in infants. Consistent with this, we showed that HM cytokines, such as APRIL, induce IgA production in naïve infant B cells, and infants receiving HM with higher levels of APRIL had lower incidence of allergic disease. Finally, lower levels of several HM fatty acids including short-chain fatty acids and DHA were associated with FA. While some these factors were are associated with maternal atopic disease, several of them are not and suggest a role for diet instead. The System Biology of Early Atopy (SunBEAm) population-based cohort of 2500 mother-infant pairs is >50% recruited and provides an unprecedented opportunity to assess association of HM feeding and immune factors in HM with development of infant immune system and FA/AD. The Common Sample comprises a subset of 100 dyads with FA, 100 with FA+AD, 100 with AD, 100 with no FA or AD and more extensively profiled biological data. Utilizing all 2-month HM samples available in the Common Sample, we will assess levels of immune factors in HM and their association with maternal/infant characteristics (Aim 1). Utilizing data from the whole cohort, we will assess the association between HM vs formula feeding on well-defined FA/AD further adjusted based on high vs low levels of HM immune components in the Common Sample (Aim 2b). Finally, we will examine the immune cell and epithelial effects of HM on infant immune markers and intestinal organoids (Aim 3). Key findings will be validated in an independent birth cohort. The ultimate goal is to uncover protective properties of BF and HM in FA and subsequent design of policies and prevention strategies to address the increasing rates of FA.

Mechanisms of age-related inflammatory dysregulation in the pathogenesis of periodontal disease

Periodontal disease is a chronic inflammatory condition that affects the supporting tissues of the dentition. Similar to other chronic inflammatory conditions, the prevalence of periodontal disease increases with age. Dysregulation of the host inflammatory response is central to the pathogenesis of periodontal disease and other age-related diseases. Therefore, an improved understanding of the pathologic mechanisms that contribute to age-related inflammatory dysregulation is needed to better manage periodontal disease in older adults. Towards understanding a mechanism of age-related inflammatory dysregulation in periodontal disease, we will investigate the role of triggering receptor expressed on myeloid cells 2 (TREM2). TREM2 is a potent immunoregulator expressed on macrophages. Signaling through TREM2 downregulates inflammation, in part, through inhibition of inflammatory cytokine expression. Dysregulation of TREM2 has been implicated in chronic inflammatory disease and age-related conditions, such as Alzheimer’s disease, liver disease, and osteoarthritis. However, the role of TREM2 in periodontal disease is understudied. Therefore, we propose to study TREM2 in the pathogenesis of periodontal disease and age-related inflammatory dysregulation. Our preliminary work has demonstrated that TREM2 is critical in macrophage immunoregulatory processes in the periodontium and TREM2 dysregulation contributes to periodontal disease in mice. We have shown that Trem2 is expressed in macrophages isolated form the periodontium in mice. We demonstrated that old mice expressed less Trem2 in the periodontium compared to young, which was associated with local inflammatory dysregulation and increased periodontal disease severity. Interestingly, Trem2 depletion in young mice resulted in increased inflammatory dysregulation and periodontal disease severity, similar to what is observed in old mice. From the preliminary data, we hypothesize that TREM2 modulates macrophage activity in the periodontium and age-related dysregulation of TREM2 drives a pathologic inflammatory response in periodontal disease. In Aim 1, we will demonstrate the extent to which TREM2 modulates inflammation and periodontal disease severity using old, young, and Trem2-/- mouse models of periodontal disease. In Aim 2, we will develop tissue-specific, single cell map of the immune cells in the periodontium and understand the effect of age and Trem2 on immune cell phenotypes and subpopulations. Findings from this proposal will elucidate a novel mechanism in age-related inflammatory dysregulation in the pathogenesis of periodontal disease and further advance our understanding of the role of TREM2 within oral tissues. This proposal was designed to generate a novel body of work that will be used to develop the independent research program of an early stage investigator and to support an R01 proposal to be submitted at the completion of this project period.

Circulating and Mucosal Predictors and Effects of Therapeutic Interleukin-23 Blockade in Crohn's Disease

PROJECT SUMMARY/ABSTRACT Since its discovery 20 years ago, the cytokine interleukin (IL)-23 has increasingly been implicated in the pathogenesis of immune mediated diseases, such as Crohn’s disease (CD). Consequently, four monoclonal antibodies that block IL-23 are currently approved CD therapies, including risankizumab. Although suppression of pathogenic Th17 cells has been widely cited as the mechanism by which IL-23 blockade controls disease, there is a paucity of data to indicate that this is how such therapy works, and a few other immune cell populations expressing the IL-23 receptor could instead be its target. We therefore propose to study how risankizumab affects not only Th17 cells, but also mucosa-associate invariant T (MAIT) cells γδ T cells and (in the colon) type 3 innate lymphoid cells (ILC3s). In addition to quantifying these cells, we will study their gene expression to detect phenotypic differences in treated patients, and in the case of T cells, track their clonal expansion and deletion through their unique T cell receptor sequences. In colon samples, we will use a combination of single cell sequencing of sort-enriched immune cell populations and spatial transcriptomics to characterize cells in situ, at the site of disease, and determine how IL-23 blockade affects their microenvironment in vivo. By contrasting results in patients who do or do not respond therapeutically to IL-23 blockade, we will reveal valuable insights into how this treatment succeeds or fails in CD, in the process identifying predictive biomarkers to guide treatment decisions, and potentially identifying future molecular targets with which to prevent treatment failure.

Autoreactive T cells in lupus

The autoimmune disease systemic lupus erythematosus (SLE) is characterized by loss of adaptive immune tolerance in conjunction with innate immune system hyperactivity. Autoantibodies, produced by plasma cells derived from activated B cells, form proinflammatory immune complexes. These immune complexes drive feed forward loops that sustain a systemic inflammatory environment and deposit in tissues leading to potentially fatal organ damage. B cells receive help from T cells to produce antibodies. They also contribute to disease by shaping T cell responses and secreting cytokines. Recent case reports in which SLE patients were treated with anti-CD19 CAR-T cell therapy to deplete B cells highlight the pathogenic role of B cells in lupus and their value as a therapeutic target. However, a better understanding of how autoreactive B cells interact with autoreactive T cells may reveal more targeted points of therapeutic intervention that specifically block autoreactive responses while sparing protective ones. Antigen specific interactions between CD4+ T cells and B cells are required for the development of autoimmune disease in lupus. However, whether these critical interactions occur in germinal centers, where competition for CD4+ T cell help selects high affinity B cells, or in extrafollicular responses, where B cells may avoid peripheral tolerance checkpoints, is unclear. Gene expression profiles and pathways specific to autoreactive CD4+ T cells, and how they are shaped by their interaction with autoreactive B cells, are also ill defined. CD8+ T cells, which recognize antigen presented on MHC Class I, have also been suggested to modulate the fate of autoreactive B cells. They can directly kill autoreactive B cells as a means of tolerance, and a subset of CD8+ T cells has recently been shown to have B cell helper function. Whether and how such interactions between B and CD8+ T cells enhance or suppress the development of lupus is unknown. Here, we will use genetic and in vivo proximity labeling approaches to address these knowledge gaps. In Aim 1, we will test the hypothesis that antigen specific interactions between B and CD8+ T cells promote B cell activation and autoantibody production in lupus. We will prevent B cells, but not other cells, from undergoing cognate interactions with CD8+ T cells via B cell-specific deletion of B2M, a component of the MHC Class I complex, in two lupus models. In Aim 2, will use the uLIPSTIC in vivo proximity system to label all T cells interacting with B cells in lupus models compared to wild type controls. Features specific to these autoreactive T cells will be defined by flow cytometry, scRNA Seq, and scTCR-Seq. These studies will provide valuable molecular and cellular insight into the mutual activation of B and T cells in lupus. They will set the stage for future mechanistic studies defining the role of autoreactive T cell specific genes and pathways and potentially highlight new therapeutic targets specific to autoreactive B/T interactions.

Optimizing gamma-delta T cell receptor-mediated signaling to improve cancer immunotherapy

PROJECT SUMMARY The recent development of T cell-based cancer immunotherapies, including checkpoint blockade (anti-PD-1, anti-CTLA-4 and others) or adoptive cell therapy (ACT) using modified patient T cells, has led to improved patient outcomes for a variety of cancers. However, durable responses are observed in only a fraction of patients. Further progress can be made by studying and targeting different T cell subpopulations, such as the gd T cells which are known to possess antitumor activities. Further, gd T cells are mostly independent of MHC-restriction, unconstrained by neoantigen burden, preferential homing to peripheral tissues and possess unique properties of T cells as well as natural killer cells making them an extremely attractive cancer immunotherapy target. One way of gd T cell activation involves the gd T cell receptor (gdTCR)-CD3 signaling pathway. gd T cell recognition of antigen by the gdTCR and the resulting proximal signaling through surrounding CD3 subunits are key steps of gd T cell activation. Even though the individual components of the gdTCR-CD3 and abTCR-CD3 complexes remain the same except for the TCRs, the complete gdTCR-CD3 complex extracellular structure is unknown. Identification of the specific extracellular interactions between the gdTCR and CD3 subunits could offer precise guidance for the development of immunotherapeutic strategies that modulate gdT cell immunity by targeting signaling through the gdTCR-CD3 complex. Our previous data showed that mutating residues in the constant domain of the abTCR resulted in altered ab T cell cytokine responses. Based on this data, our hypothesis is that gdTCR-CD3 signaling can also be modulated by targeting specific regions of the gdTCR by mutagenesis to improve gd T cell antitumor activities. To test our hypothesis, in Aim 1, we will use a novel photo-crosslinking and computational docking methodology to solve the complete extracellular structure of a gdTCR-CD3 complex. Further, we will use an in silico structure-based TCR design approach to identify gdTCR mutants that enhance signaling. In Aim 2, we will use an in vitro retroviral TCR display method using degenerate primers to create gdTCR mutant libraries at specific gdTCR sites such as Cg helix 3 and connecting peptide (CP) regions. In both instances, identified mutants will be tested for improved functionalities in an MHC-independent gd TCR (G115 Vg9Vd2 TCR) using in vitro cytokine and tumor-killing assays. Overall, the newly identified enhanced gd T cell clones could potentially lead to a new wave of effective cancer immunotherapy strategy by leaning into the largely untapped potential of gd T cells.

A Novel Mitochondrial-Targeted Inhibitor of NLRP3 Inflammasome Activation

PROJECT ABSTRACT Inflammasomes are multiprotein complexes of the innate immune system that assemble upon detecting specific molecular patterns associated with pathogens and cellular damage. Once assembled, activated inflammasomes trigger a cascade of downstream events that culminate in cell death and inflammation. Aberrant activation of the NLRP3 inflammasome contributes to the pathogenesis of numerous inflammatory and degenerative diseases, including gout, atherosclerosis, type 2 diabetes, and Alzheimer’s disease. Despite its central role in innate immunity and inflammation, there are no FDA-approved therapies that directly target the NLRP3 inflammasome. Current strategies rely on biologics that inhibit downstream pro-inflammatory cytokines produced from inflammasome activation, such as interleukin-1β (IL-1β), but do not block upstream inflammasome assembly or pyroptotic cell death, highlighting a critical unmet need for selective small-molecule inhibitors with novel mechanisms of action. To address this gap, we identified a covalent small molecule, Compound-2 (C-2), that robustly inhibits NLRP3 inflammasome activation in murine and human immune cells. C-2 suppresses multiple downstream events triggered by inflammasome activation, including IL-1β secretion and pyroptosis, with no apparent toxicity. Chemoproteomic profiling revealed that C-2 interacts with SLC25A3, a mitochondrial phosphate and copper transporter, suggesting a previously unrecognized regulatory node in inflammasome signaling. This R21 project aims to (1) elucidate the mechanism by which C-2 suppresses NLRP3 activation and (2) define the molecular interaction between C-2 and SLC25A3 and its functional consequences. Our studies will integrate biochemical, cellular, and in vivo approaches to uncover a novel mitochondrial mechanism of inflammasome regulation and validate C-2 as a first-in-class inflammasome inhibitor. Successful completion of this project will lay the foundation for future therapeutic development targeting mitochondrial- inflammasome crosstalk in inflammatory disease.

Targeted Prodrug Cytokines for Metastatic Breast Cancer Immunotherapy

Project Summary. Our approach directly addresses key limitations in targeting and treating metastatic breast cancer, where we propose the selective activation of modular immune-modulating cytokines within the hypoxic and ROS-active TME for delivery across the BBB, providing the necessary pre-clinical data for future clinical translation. The in vitro and in vivo investigations of this novel immunotherapeutic in immunocompetent models will allow our team to study the interplay between tumor-driven immune activation, cytokine signaling, and anti-tumor immunity in both primary and metastatic sites, and establish a robust groundwork for subsequent clinical validation within the OSUCCC. This proposal addresses two key challenges in developing a novel immunotherapy strategy for breast cancer by answering two hypotheses: (1) can a modular immunotherapy platform with tumor-selective activation of prodrug recombinant cytokines overcome these limitations in drug delivery, and (2) can the development of nanobody-cytokine fusions that can selectively target primary breast cancer tumors and cross the BBB to reach metastatic tumor sites? The first hypothesis focuses on achieving tumor environment-specific activation of prodrug-based recombinant cytokines. Protein cytokines are highly potent, and while others have tried to block their activity using a fused genetic linker to ‘mask’ functionality, no one has yet attempted to use a non-canonical-based chemical strategy to achieve this inhibition. Immune-modulating cytokines will be recombinantly expressed with integrated ncAAs that block cytokine activity until the function is regenerated in the breast cancer TME. Once the cytokine activity is controlled, our second hypothesis will be to achieve selective delivery of the cytokine via fusion to nanobodies. While success has been found in targeting primary tumors in drug and protein delivery, a key challenge remains in reaching secondary metastatic tumors in hard-to-reach sites (i.e., brain). Engineered nanobodies, with affinity for breast cancer tumors and the ability to bind to BBB transcytosis receptors, will enable selective delivery to metastatic breast-to-brain tumors, resulting in tumor- specific activation, immune responses, and improved therapeutic outcomes. This system can significantly improve therapeutic outcomes for patients with mBC by integrating selective activation and delivery mechanisms to reduce off-target effects and enhance tumor-specific immune responses in both primary and secondary metastatic tumor sites. Optimizing drug delivery systems to tune immune responses could offer more effective and less invasive treatment options when compared to traditional and engineered cell-based approaches. Our momentum towards precision medicine and targeted therapies holds significant promise for improving outcomes for mBC patients, and has the potential to serve as a pan-cancer treatment for aggressive metastatic cancers from the following aims: (1) generating a modular platform for tumor-specific activation of prodrug cytokines, (2) evaluating cytokine delivery and anti-cancer immune phenotypes in mBC.

The multiciliation cycle: a variant cell cycle coordinating centriole biogenesis and ciliogenesis

Project summary/Abstract Differentiating multiciliated cells line the mammalian airway and are critical for protecting the lungs from inhaled pathogens and particulates. Multiciliated cells have a distinct architecture from other cell types, having hundreds of centrioles, each of which matures into a basal body and nucleates a motile cilium. Defects in multiciliation cause a form of Primary Ciliary Dyskinesia (PCD), a lung disease. Most cells generate two centrioles and one cilium per cell cycle. We found that differentiating multiciliated cells redeploy cell cycle regulators into a novel cell cycle variant, which we refer to as the multiciliation cycle, to break these rules, generate hundreds of centrioles and cilia, and coordinate their differentiation. The multiciliation cycle redeploys many mitotic cell cycle regulators, including cyclin-dependent kinases (CDKs) and their cognate cyclins. For example, Cyclin D1-CDK4/6, regulators of mitotic G1 to S progression, is required for multiciliated cell fate initiation and entry into the multiciliation cycle. While we have focused on lung multiciliated cells, others have found that cell cycle regulators similarly participate in multiciliation of ependymal cells of the brain. Some cells, such as mammalian trophoblast giant cells, employ cell cycle variants like the endocycle to bypass mitosis. We propose that the multiciliation cycle is another cell cycle variant that augments some aspects of the canonical cell cycle, such as centriole synthesis, and blocks others, such as DNA replication. During the multiciliation cycle, E2F7, a transcriptional regulator of canonical S to G2 progression, is expressed at high levels. During multiciliated cell differentiation, E2F7 directly dampens expression of genes encoding DNA replication machinery and terminates the S phase-like gene expression program. Loss of E2F7 causes a reacquisition of DNA synthesis in multiciliated cells and dysregulation of multiciliation cycle progression, disrupting centriole maturation and ciliogenesis. We propose that multiciliated cell differentiation is coordinated by an alternative cell cycle that organizes, instead of cell proliferation, the steps of cell differentiation. In this project, we investigate how the multiciliation cycle redeploys the mitotic cell cycle regulatory framework to generate many centrioles without undergoing DNA synthesis or cytokinesis. More specifically, we seek to uncover how CDKs and cyclins are regulated to control the amount and timing of basal body synthesis, how Retinoblastoma (RB) protein controls the transcriptional program of multiciliation, and how E2Fs advance the multiciliation cycle. This work will test the hypothesis that multiciliation is organized by a variant cell cycle that uncouples centriole synthesis from DNA replication and mitosis. We propose that his variant cell cycle orchestrates progression through sequential phases required to construct the multiciliated cells that protect the lungs.

Tapeworm larvae in the brain: cellular mechanisms of epilepsy in neurocysticercosis

Cerebral infection by the larvae of the cestode, Taenia solium (neurocysticercosis), is thought to be the leading cause of adult-acquired epilepsy worldwide. Despite this, little is known about the cellular mechanisms that underlie seizure development in this condition. In this talk I will present our recent data exploring multiple interactions between cestode larvae, neuroinflammatory processes and network excitability. We find that viable cestode larvae are able to strongly suppress microglial activation and inflammatory cytokine release with consequences for the modulation host neuroinflammatory responses and seizure development in vivo. At the same time, larvae produce and release glutamate, with acute excitatory effects on neuronal circuits. We hope that an improved understanding of epileptogenic mechanisms in neurocysticercosis will one day improve the management of this condition as well as other inflammatory causes of epilepsy.

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.

Carnosine negatively modulates pro-oxidant activities of M1 peripheral macrophages and prevents neuroinflammation induced by amyloid-β in microglial cells

Carnosine is a natural dipeptide widely distributed in mammalian tissues and exists at particularly high concentrations in skeletal and cardiac muscles and brain. A growing body of evidence shows that carnosine is involved in many cellular defense mechanisms against oxidative stress, including inhibition of amyloid-β (Aβ) aggregation, modulation of nitric oxide (NO) metabolism, and scavenging both reactive nitrogen and oxygen species. Different types of cells are involved in the innate immune response, with macrophage cells representing those primarily activated, especially under different diseases characterized by oxidative stress and systemic inflammation such as depression and cardiovascular disorders. Microglia, the tissue-resident macrophages of the brain, are emerging as a central player in regulating key pathways in central nervous system inflammation; with specific regard to Alzheimer’s disease (AD) these cells exert a dual role: on one hand promoting the clearance of Aβ via phagocytosis, on the other hand increasing neuroinflammation through the secretion of inflammatory mediators and free radicals. The activity of carnosine was tested in an in vitro model of macrophage activation (M1) (RAW 264.7 cells stimulated with LPS + IFN-γ) and in a well-validated model of Aβ-induced neuroinflammation (BV-2 microglia treated with Aβ oligomers). An ample set of techniques/assays including MTT assay, trypan blue exclusion test, high performance liquid chromatography, high-throughput real-time PCR, western blot, atomic force microscopy, microchip electrophoresis coupled to laser-induced fluorescence, and ELISA aimed to evaluate the antioxidant and anti-inflammatory activities of carnosine was employed. In our experimental model of macrophage activation (M1), therapeutic concentrations of carnosine exerted the following effects: 1) an increased degradation rate of NO into its non-toxic end-products nitrite and nitrate; 2) the amelioration of the macrophage energy state, by restoring nucleoside triphosphates and counterbalancing the changes in ATP/ADP, NAD+/NADH and NADP+/NADPH ratio obtained by LPS + IFN-γ induction; 3) a reduced expression of pro-oxidant enzymes (NADPH oxidase, Cyclooxygenase-2) and of the lipid peroxidation product malondialdehyde; 4) the rescue of antioxidant enzymes expression (Glutathione peroxidase 1, Superoxide dismutase 2, Catalase); 5) an increased synthesis of transforming growth factor-β1 (TGF-β1) combined with the negative modulation of interleukines 1β and 6 (IL-1β and IL-6), and 6) the induction of nuclear factor erythroid-derived 2-like 2 (Nrf2) and heme oxygenase-1 (HO-1). In our experimental model of Aβ-induced neuroinflammation, carnosine: 1) prevented cell death in BV-2 cells challenged with Aβ oligomers; 2) lowered oxidative stress by decreasing the expression of inducible nitric oxide synthase and NADPH oxidase, and the concentrations of nitric oxide and superoxide anion; 3) decreased the secretion of pro-inflammatory cytokines such as IL-1β simultaneously rescuing IL-10 levels and increasing the expression and the release of TGF-β1; 4) prevented Aβ-induced neurodegeneration in primary mixed neuronal cultures challenged with Aβ oligomers and these neuroprotective effects was completely abolished by SB431542, a selective inhibitor of type-1 TGF-β receptor. Overall, our data suggest a novel multimodal mechanism of action of carnosine underlying its protective effects in macrophages and microglia and the therapeutic potential of this dipeptide in counteracting pro-oxidant and pro-inflammatory phenomena observed in different disorders characterized by elevated levels of oxidative stress and inflammation such as depression, cardiovascular disorders, and Alzheimer’s disease.

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