Browse Articles

SOCS1-derived mimetic peptides attenuate cytokine-induced Microglial Activation via Jak2 inhibition 2308895

Abstract Introduction Recent evidence suggests that microglia can initiate autoimmune uveitis (AU). Notably, the JAK/STAT pathway is a central driver of both microglial function and AU progression. While our lab has shown that peptides mimicking the suppressor of cytokine signaling 1-kinase inhibitory region (SOCS1-KIR) can mitigate JAK/STAT-associated AU in induced rodent disease models and equine recurrent uveitis patients, the ability of the peptide to modulate microglial function is unknown. The goal of this study was to assess the tolerability and efficacy of SOCS1-KIR mimetics on microglia through immune regulation and Jak2 inhibition. Methods Human microglial (HMC3) cells were stimulated with TNFα in the presence or absence of SOCS1-KIR mimetics to evaluate the effects on viability (MTT assay) and inflammatory gene expression (qPCR). In vitro Jak2 kinase assays were conducted to assess SOCS1-KIR mediated modulation of kinase activity. Results SOCS1-KIR mimetics showed IC50 values of 11.5µM and 3.2µM for inhibiting Jak2 kinase activity. SOCS1-KIR mimetics were well tolerated by HMC3 cells, with the monomer showing no toxicity from 0-33µM and the dimer from 0-11µM. TNF-α induced a robust pro-inflammatory response, with IL-1β, IL-6, and MCP-1 transcripts increasing 10- to 50-fold. Preliminary data suggest that the peptides reduce TNFα-induced IL-6 mRNA expression, indicating potential attenuation of microglial activation at the transcriptional level. Conclusion SOCS1-KIR mimetics are non-toxic and exert immunomodulatory effects in TNFα-stimulated microglia. Jak2 inhibition supports the mechanism of JAK/STAT pathway suppression. Future studies will examine IFNγ-driven activation to further define the role of SOCS1-KIR in modulating JAK/STAT-associated inflammation. These findings will not only clarify the immunomodulatory mechanism of SOCS1-KIR but also advance development of targeted therapies to restore immune homeostasis in sight-threatening ocular diseases. Funding Source The University of Florida Topic Categories Therapeutic Approaches to Autoimmunity (THER)

IL-15 induces durable resident memory T cell formation and function following genetically attenuated Plasmodium vaccination in mice 2306464

Abstract Introduction Due to their immediate effector function and position in tissues, resident memory T (TDRM) cells are uniquely equipped to provide early defense against invading pathogens. Malaria is a major public health threat in need of a highly efficacious and durable vaccine. Following an infected mosquito bite, Plasmodium sporozoites (SPZ) travel to the liver and infect hepatocytes. Stopping infection at the liver stage prevents the disease-causing blood stage infection and transmission. Whole sporozoite vaccines, including genetically attenuated parasites (GAPs), induce liver CD8+ TDRM cells and are highly efficacious. However, manufacturing and deployment of these vaccines is incredibly resource intensive. Since liver TDRM formation depends on IL-15, we investigated the ability of IL-15 complex (IL-15C, IL-15 combined with IL-15Rα) to augment TDRM-mediated immunity in a mouse model of GAP vaccination. Methods We tested the impact of IL-15C on liver TDRM formation in the absence and presence of GAP vaccination by flow cytometric analysis of T cells in the spleen, liver and liver-draining lymph node. We tested the impact of IL-15C on vaccine efficacy by measuring liver parasite load following challenge with live SPZ. Results IL-15C transiently increases the number of liver CD8+ and CD4+ TDRM cells in the absence of antigen. In the context of GAP vaccination, IL-15C treatment results in a sustained increase in the number of liver CD8+ and CD4+ TDRM cells. Additionally, IL-15C enhances liver T cell functionality as measured by IFN-γ production following ex vivo simulation. Importantly, IL-15C treatment decreases parasite burden in the liver following SPZ challenge. Conclusion Overall, these findings improve our understanding of the mechanisms that increase TDRM cell numbers and function, which will inform vaccine strategy and facilitate greater control of malaria. Funding Source NIH R21 R21AI182639; Falk Medical Research Trust Catalyst Award Topic Categories Vaccines and Immunotherapy (VAC)

Ultrasound‐Mediated OFF‐to‐ON Switching of Bone Regeneration via a Bioorthogonal Cell‐Capture Hydrogel, Enabling Spatiotemporal Control and Monitoring

ABSTRACT Repairing critical‐sized bone defects remains a formidable challenge in orthopedics, primarily due to the limitations of current stem cell‐based therapies, including poor engraftment, limited real‐time monitoring of cell fate, and a lack of spatiotemporal control over therapeutic activity. To address these challenges, in this study, we present a bioorthogonal hydrogel platform engineered for precise stem cell capture and ultrasound‐triggered bone regeneration. Bone marrow mesenchymal stem cells (BMSCs) were dual‐functionalized with trans‐cyclooctene (TCO) and biogenic gas vesicles (GVs) via a metabolic glycoengineering approach. The resulting BMSCs‐GV‐TCO were efficiently and covalently captured within tetrazine‐functionalized hydrogels at the defect site through a rapid inverse electron‐demand Diels–Alder reaction. This strategy ensured targeted cell homing and retention. The GVs served as a potent acoustic contrast agent, enabling noninvasive and real‐time ultrasound monitoring of cell delivery and localization. Furthermore, the unique ultrasound‐responsive property of GVs enabled their on‐demand collapse, acting as a remote‐controlled switch to precisely upregulate osteogenic transcription factors and enhance bone regeneration, as validated in a rat critical‐sized calvarial defect model. This study presents a novel, efficient, and traceable strategy for stem cell‐based therapy, with broad potential to advance the treatment of bone defects and the clinical translation of regenerative medicine.

TRAIP promotes the development of papillary thyroid cancer by inhibiting TRAF2-mediated BRAF ubiquitination

Rocaglates and pateamines target the DEAD-box RNA helicase eIF4A from Schistosoma mansoni and demonstrate anti-schistosomal activity in vitro

Abstract For decades, infections with the parasite Schistosoma mansoni have been treated with praziquantel, the only available drug, which highlights the urgent need of treatment alternatives. Natural compounds including rocaglates from plants of the genus Aglaia and pateamines isolated from the marine sponge Mycale hentscheli have exhibited promising anti-viral, anti-cancer, and anti-pathogenic properties. These compounds target the eukaryotic translation initiation factor 4A (eIF4A), which is a crucial factor for unwinding RNA structures in the 5’-UTR of selected mRNAs, and prevent the unwinding activity of eIF4A, which interferes with protein synthesis. Preliminary data indicated anthelminthic activity of the rocaglate silvestrol against S. mansoni in vitro. In this study, we confirmed the interaction of rocaglates and pateamines with recombinant S. mansoni eIF4A and analyzed the activity of these compounds against adult S. mansoni in vitro. Our findings showed reduced parasite vitality and stem-cell proliferation as well as impaired embryogenesis after rocaglate or pateamine treatment in the nanomolar range. Furthermore, we observed that rocaglate-treated S. mansoni recovered during washout experiments, while pateamine treatment displayed irreversible effects finally killing most of the worms. Although some properties of the used compounds impede their suitability as anti-schistosomal candidates, eIF4A remains a promising drug target in this parasite.

Non-linear Generalizable Machine Learning Re-Analysis of Plasma Proteomics and Single-Cell Immune Network for Classification of COVID-19 Severity 2262757

Abstract Introduction Severe respiratory viral infections (i.e., flu, COVID-19) trigger complex cellular and protein immune dysregulation. Linear models may overlook non-linear immune patterns important for risk stratification. We re-analyzed plasma omics and single-cell immune data to benchmark a non-linear machine-learning pipeline, derived mechanistic markers, and position an approach extensible to environmental-viral immunology cohorts. Methods From ImmProt (study ID SDY2129, https://www.immport.org), we merged patient characteristics, plasma proteomics, and CyTOF data for 81 COVID-19 cases (Mild, Moderate, Severe). After median imputation, scaling, and one-hot encoding, features with high missingness, low variance, or strong collinearity were removed; 150 features were retained. We evaluated Random Forest, Gradient Boosting, and Elastic Net in a stratified train/test split and nested cross-validation. We validated and interpreted results with PCA, ROC curves, confusion matrices, and RF importance. Results RF captured non-linear interactions and outperformed GB and EN (accuracy = 83%, AUC = 0.94). Cross model drivers increased with severity, including LGALS1 (galentcin-1), CCL7 (MCP-3), TNFRS10A, FURIN, PLAUR (in Olink NPX), and stimulus-responsive STAT/ERK/NF-κB axes. (CyTOF), while pDA resting measures declined–findings concordant with original network biology (dysregulated JAK/STAT, MAPK/mTOR, NF-κB) but via a compact, prefiltered pipeline. Mechanistically, LGALS1 suggests T-cell dampening and stromal remodeling; CCL7 indicates monocyte recruitment and lung immunopathology. Conclusion This ImmPort re-analysis corroborates published biology and shows that a non-linear ML pipeline enables effective three-class severity classification with interpretable, cross-platform markers–positioned for respiratory viral immunology beyond COVID-19. Future steps involve batch correction, pathway analysis, a small marker panel, external validation, and integration into environmental—viral severity modeling efforts. Funding Source n/a Topic Categories Computational and Systems Immunology (COMP)

Epstein-Barr virus reprograms autoreactive B cells as antigen-presenting cells in systemic lupus erythematosus 2305557

Abstract Introduction Systemic lupus erythematosus (SLE) is a systemic autoimmune disease characterized by anti-nuclear antibodies (ANA). Epstein-Barr virus (EBV) infection has been epidemiologically associated with SLE, yet its role in pathogenesis remains incompletely defined. Methods We developed an EBV-specific single-cell RNA-sequencing platform and used it to sequence EBV-infected B cells directlry isolated form the patient. Using combined EBV-seq, CITE-seq, BCR-seq, and scRNA-seq, we sequenced the EBV genes, B cell receptor (BCR) repertoires, and transcriptomes of blood B cells from patients with SLE . Results We demonstrated that, in SLE, EBV+ B cells are predominantly CD27+CD21low memory B cells that are present at increased frequencies and express ZEB2, TBX21, and antigen presenting cell transcriptional pathways. Integrative analysis of chromatin immunoprecipitation sequencing (ChIP-seq), assay for transposase-accessible chromatin (ATAC-seq), and RNA Polymerase II occupancy data revealed EBNA2 binding at the transcriptional start sites and regulatory regions of CD27, ZEB2, and TBX21 (Tbet), as well as the antigen presenting cell genes demonstrated to be up-regulated in SLE EBV+ B cells. We expressed recombinant antibodies from SLE EBV+ B cells and demonstrated that they bind prototypical SLE nuclear autoantigens, whereas those from healthy individuals do not. We further found that SLE EBV+ B cells can serve as antigen presenting cells to activate T peripheral helper cells with concomitant activation of related EBV- anti-nuclear double negative 2 B cells and plasmablasts. Conclusion Our results provide a potential mechanistic basis for EBV being a driver of SLE through infecting and reprograming nuclear antigen reactive B cells to become activated antigen presenting cells with the potential to promote systemic disease-driving autoimmune responses. Funding Source Lupus Research Alliance Topic Categories Basic Autoimmunity (BA)

Decoding Antigen-specific Tolerance Mechanisms Imposed by an MHC-II targeted Nanobody-based Therapy for Multiple Sclerosis 2331736

Abstract Introduction We developed a therapy to induce antigen-specific tolerance in multiple sclerosis (MS). The MS therapy involves an antibody fragment, called a nanobody, that recognizes class II major histocompatibility complex antigens (VHHMHCII), conjugated to a myelin oligodendrocyte glycoprotein (MOG) peptide. MHC II is expressed by antigen presenting cells (APCs). When antigen presenting cells encounter antigen under non-inflammatory conditions, they are tolerogenic. Therefore, if MS-associated peptides are delivered to APCs, antigen-specific tolerance should be induced. The lab has demonstrated that a single dose of VHHMHCII-MOG provides enduring protection from induction of experimental autoimmune encephalitis (EAE). When VHHMHCII-MOG was conjugated to dexamethasone, a small molecule anti-inflammatory drug, this trimodal construct (VHHMHCII-MOG-DEX) effectively halted and reversed the severity of EAE. We are now investigating the mechanisms that explain the efficacy of this therapy. One major aim is to understand the therapeutic’s influence on the interaction between T-cells and APCs. Methods Using maleimide-thiol click chemistry and the theory of uLIPSTIC (universal labelling of immune partnerships by SorTagging intercellular contacts), we developed an ex-vivo strategy to look at immune cell interactions. Results Maleimide-thiol click chemistry can be used to label proteins at the cells surface, including T-cells which are functional post-labeling. We were able to monitor T-cell interactions with antigen-presenting cells ex vivo. We determined that LysM+ cells (e.g. macrophages) interact with MOG-specific T cells after incubation with VHHMHCII-MOG-DEX. Conclusion We will continue by investigating the interactions of antigen-specific T-cells with other APCs including dendritic cells and B-cells. Elucidating the treatment’s mechanism of action will enable further modification and improvement of MS treatments, and translation to other autoimmune diseases. Funding Source National Multiple Sclerosis Society Topic Categories Therapeutic Approaches to Autoimmunity (THER)

Fever Temperatures Differentially Alter Ebola-Antibody Functionality Depending on Affinity Maturation 2306819

Abstract Introduction In 2013-16, Western Africa experienced the largest known outbreak of the highly lethal virus, Ebolavirus (EBOV) and ongoing sporadic outbreaks have occurred since. EBOV is the causative agent of EBOV hemorrhagic fever, characterized by a high-grade fever. Antibody responses to EBOV have been correlated with protection against EBOV disease. However, it is unclear how antibody functions result in protection from repeat exposure. Understanding antibody-mediated mechanisms of protection against Ebola infection is critical to maximize long-term protection against infection. Methods We utilize a systems serology approach to analyze a cohort of 60 survivors of the 2013-16 Ebola epidemic in Guinea and 81 individuals of pygmy ancestry in the Republic of Congo who did not have documented EBOV infection, but do experience frequent zoonotic exposures to characterize the impact of fever conditions on affinity matured EBOV-directed antibody responses. Antibody binding and cellular activation were measured at both physiologic (37C) and high grade fever (40C) temperatures. Results We show that neutralization of EBOV is impaired under fever temperatures, both for affinity-matured and non-specific antibody responses. In contrast, effector-mediated functions, particularly against the EBOV soluble glycoprotein (sGP, a signature previously linked to enhanced protection in animal vaccine challenge models), are enhanced at febrile temperatures. This temperature-sensitive enhancement of effector function correlates with full length GP and sGP-directed IgG subclasses, IgA, FcγR-binding- and FcαR-binding antibodies, demonstrating a highly associated network of humoral features that are maintained during periods of febrile temperatures. Conclusion Collectively, our findings suggest that although neutralization plays a key role in surveillance in a non-fever state, fever modulates affinity-matured antibody responses to the hemorrhagic fever-inducing EBOV, moving antibody profiles to a pro-effector function phenotype. Funding Source NIH NIAID U19 AI135995 Topic Categories Viral Immunology (VIR)

Immune Regulatory Markers in Post-Acute Sequelae of COVID-19: A Clinical Case Report 2253140

Abstract Introduction The interplay between apoptotic versus non-apoptotic programmed cell death (PCD) mechanisms may have a significant role in post-acute sequelae of COVID-19 (PASC). The objective of the current study is to investigate PCD markers in serum and plasma samples derived from PASC patient cases in comparison with unaffected controls. Methods The study protocol was approved by the Hackensack Meridian Health (HMH) Biorepository Research Integrity Committee and is an IRB-exempt study for deidentified samples. Serum and plasma samples derived from PASC cases and unaffected controls (N = 15) were analyzed by Enzyme-Linked ImmunoSorbent Assays (ELISAs) for PCD markers and antiviral immune-responsive cytokines. One-way analysis of variance (ANOVA) combined with Tukey’s multiple comparisons statistical analyses of the data was performed. Statistical significance level set at p < 0.05 and data presented as mean-fold increase in PASC cases compared to controls. Results Preliminary ELISA results indicated no significant differences in serum or plasma levels of caspase-3, serum cleaved caspase-3 and serum caspase-1 between PASC cases and unaffected controls (p > 0.05). However, plasma levels of cleaved caspase-3 and caspase-1 were significantly higher in PASC cases versus unaffected controls (2.5-fold, *p=0.02 and 4-fold, ****p< 0.0001, respectively). SQSTM1/p62 showed significantly higher levels in serum and plasma from PASC cases compared to controls (90-fold, **p=0.006 and 60-fold, *p=0.03, respectively). Serum and plasma antiviral immune responsive cytokines IL-1-beta (over 10-fold, ****p< 0.0001), IFN-gamma (over 5-fold, ****p< 0.0001) and TNF-alpha (over 10-fold, **p< 0.01) were significantly elevated in PASC cases compared to controls. Conclusion Significant elevation of non-apoptotic PCD markers in serum and/or plasma samples derived from PASC cases compared to unaffected controls might indicate the diagnostic utility of non-apoptotic PCD markers in PASC cases. Funding Source New Jersey Health Foundation Topic Categories Translational and Interventional Immunology (TI)

STING Gain-of-Function in Myeloid and Non-Hematopoietic Cells Compromises Lung Function 2309151

Abstract Introduction Gain-of-function (GOF) mutations in the dsDNA sensing adaptor STING lead to a severe auto-inflammatory syndrome known as STING-Associated Vasculopathy with onset in Infancy (SAVI). Patients develop interstitial lung disease (ILD) resulting in respiratory failure. Mice heterozygous for the most common SAVI mutation, STING V154M/WT (VM), develop bronchus-associated lymphoid tissue (BALT) that is driven by VM-expressing endothelial cells (ECs). However, EC-specific VM expression is not sufficient for myeloid cell infiltration of lung tissue nor compromised lung function. Thus, we hypothesized that both non-hematopoietic cells (ECs) and VM-expressing myeloid cells are required to cause the lung disease phenotype exhibited by the original VM mice. Methods To better understand the role of hematopoietic cells in both BALT formation and lung dysfunction, we generated bone marrow radiation chimeras such that lethally irradiated WT or VM mice were reconstituted with bone marrow stem cells isolated from either WT mice or mice where VM expression was limited to myeloid cells. The VM mutation was restricted to myeloid cells by crossing VM conditional knock-in (CKI) mice to LysM-Cre mice (CKI x LysM-Cre). Results VM mice reconstituted with either WT or CKI x LysM-Cre stem cells developed extensive BALT formation. However, only the mice reconstituted with CKI x LysM-Cre stem cells exhibited significantly compromised lung function and this was associated with significantly increased myeloid cell infiltration of the lungs. WT mice reconstituted with CKI x LysM-Cre stem cells did not develop BALT, lung myeloid cell infiltration, or show compromised lung function. Conclusion STING GOF mutation in both myeloid and non-hematopoietic cells is required for compromised lung function that phenocopies the original VM mouse, highlighting a critical synergy between VM-expressing myeloid cells and VM-expressing non-hematopoietic cells in the development of fulminant lung disease. Funding Source R01HL165787, R21AI78978, T32A132152, 1F31HL180004-01 Topic Categories Immune Response Regulation: Cellular Mechanisms (IRC)

Aging disrupts the affinity threshold for naïve B cell germinal center participation 2306948

Abstract Introduction Vaccination is a crucial public health measure that provides protection from infectious pathogens, but protection is reduced in aged individuals due to waning immune responses. One correlate of protective immunity is the production of high affinity antibodies by germinal centers, which are known to be severely impaired in aged organisms due primarily to a reduction in T cell help. Less is known about how aging affects naïve B cells prior to an immune response. Here, we sought to address how naïve B cells in old (2 years of age) mice performed in an immune response compared to B cells in young mice (8 weeks of age). Methods We studied naïve B cells from old or young mice using adoptive transfer into young B cell knockout mice, followed by immunization with nitrophenyl-chicken gamma globulin (NP-CGG) in alum. B cell responses were characterized using flow cytometry, scRNA-seq, and scBCR-seq. Individual antibodies were cloned from responding B cells and their affinities were measured using biolayer interferometry. Results Upon immunization, mice that received B cells from old donors generated significantly more germinal center B cells than those that received young donor cells, but a smaller percentage of the old cells were specific for NP. scBCR-seq of responding old GC B cells revealed increased usage of less common heavy and light chain pairings. Recombinant antibodies produced by these cells had reduced affinity for NP than those produced by young GC B cells. Furthermore, scRNA-seq of GC B cells and naïve B cells suggested that old B cells have a signature of activation even prior to immunization, which was supported by increased phosphorylation of protein kinases in old naïve B cells. Conclusion We found that during aging, naïve B cells had increased basal stimulation that allowed the selection of lower affinity B cells into the germinal center reaction, resulting in the production of lower quality antibodies throughout the immune response. Funding Source National Institute on Aging Intramural Research Program Topic Categories Immune Response Regulation: Molecular Mechanisms (IRM)

Ligand‐Mediated Shape Engineering of SWIR InAs Colloidal Quantum Dots for Photodetector Applications

ABSTRACT Indium arsenide colloidal quantum dots (InAs CQDs) are promising candidates for next‐generation short‐wave infrared (SWIR) optoelectronics; however, synthesis using non‐pyrophoric amino‐arsine precursors has faced limitations in reaching long wavelengths (>1300 nm) due to intrinsic growth stagnation and challenging surface control. Herein, we report a robust strategy to synthesize high‐quality SWIR InAs CQDs by introducing bulky oleic acid (OA) ligands to overcome these kinetic barriers. By modulating the interplay between the steric hindrance of OA ligands and the stabilization of (111) facets, we induce a controlled shape evolution from initial kinetically stabilized spheres to thermodynamically stable tetrahedrons. This morphological transition enables the achievement of the same absorption wavelength followed by a nearly fourfold reduction in nanocrystal volume compared to spherical counterparts. This mechanism offers a distinct pathway for the bandgap tunability of InAs CQDs enabling access to the SWIR region (>1700 nm) under mild reaction conditions (260–280°C). Furthermore, the OA‐mediated surface chemistry unlocks the formulation of conductive halide‐based inks, enabling photodetector development based on amino‐arsine‐synthesized InAs CQDs extending their spectral coverage to 1.6 um. The resulting photodetectors exhibit an external quantum efficiency of 19.1% at 1300 nm and D * of 3.4 × 10 10 Jones.

Tamoxifen induces region-specific osteocytic recombination and transiently alters bone structure in Dmp1-Cre-ERT2 mice

Numerical simulation on water migration in compacted loess subgrade under extreme rainfall conditions

Macrophage regulation of inflammation in type 2 diabetes 2265746

Abstract Introduction Type 2 diabetes is a metabolic disease characterized by chronic, low-grade inflammation that leads to comorbidities that significantly impact quality of life, such as susceptibility to infections and non-healing wounds. Studies in mouse models suggest that macrophages from diabetic contexts exhibit hyper-inflammatory states, but the mechanisms behind this phenomenon are poorly explored and have not yet been investigated in human macrophages. We hypothesize that diabetic macrophages have impaired response mechanisms and are more sensitive to metabolic flux, limiting their ability to effectively resolve inflammation. Methods To study this, we isolated and cultured bone marrow-derived macrophages from WT and db/db mice and stimulated them with inflammatory and resolving cues. We assessed their functional inflammatory response through phenotypic marker changes and cytokine release, and their sensitivity to metabolic flux by culturing in the presence or absence of extracellular glutamine. Results Both healthy and diabetic mature macrophages displayed similar levels of inflammatory response cytokines, inflammatory gene expression changes, and phenotypic marker expression, which was blunted in the absence of glutamine. Interestingly, we observed differences in the differentiation timeline between the two genotypes, which prompted further investigation in their response to maturation cues. Notably, diabetic macrophages lag behind healthy macrophages in expression of CD45 and F4/80, but eventually acquire a similar level of mature cell surface markers by day 7-8 in culture. In addition, diabetic cells have a higher proportion of Ly6Chi cells earlier in culture, which are generally more poised to be pro-inflammatory. Conclusion Ongoing experiments are assessing the inflammatory response of monocytes from diabetic contexts, both mouse and human. This research begins to explore the cell-mediated inflammatory aberrations in type 2 diabetes and may reveal targetable mechanisms to improve patient outcomes. Funding Source TL1TR001864, R01AR084558 Topic Categories Innate Immune Responses and Host Defense: Cellular Mechanisms (INC)

Substance P regulates the immune response to promote coronavirus clearance in the airways 2306173

Abstract Introduction The sensory neuropeptide Substance P helps to protect against respiratory viral infection by promoting key defense reflexes like cough and bronchoconstriction. It can also promote pathogenic neurogenic inflammation by regulating cytokine production and immune cell activity. We thus asked whether Substance P plays a pro-viral or anti-viral role during coronavirus infection. Methods Male C57BL/6 mice 8-12 weeks were infected with the mouse β-coronavirus MHV-A59 (1,000 PFU in 40 µL PBS delivered intranasally) and treated with the Substance P-receptor TacR1 antagonist fosaprepitant (i.p. 20 mg/kg in PBS) or vehicle (meglumine 9.37 mg/kg) twice daily over the course of infection, specifically post-infection days zero through nine. We profiled and quantified pulmonary and splenic immune cells in infected vehicle-treated and fosaprepitant-treated mice using flow cytometry. Serum, BAL fluid, and lung samples were collected at various post-infection timepoints to assess cytokine concentration, antibody titers, and viral load. Results Substance P levels were significantly increased in the lungs of MHV-A59 infected mice and were dependent on sensory neuron signaling. Administration of fosaprepitant promoted weight loss, an increase in pulmonary viral titers and significantly decreased survival in infected mice. Immune dysregulation was observed in the lungs and spleen of infected fosaprepitant-treated versus vehicle-treated animals. Overall, blocking Substance P signaling during active infection significantly dysregulated the pulmonary and splenic immune response and increased disease severity. Conclusion Substance P receptor signaling plays a key early role in regulating the immune response to significantly improve viral clearance during coronavirus infection. Future experiments will examine the effects of blocking Substance P early vs. late in the infection cycle, and will determine how Substance P signaling affects immune cell function to promote viral clearance in infected animals. Funding Source NIH F31 Topic Categories Neuroimmunology (NEUR)

Batf3 Promotes a Distinct Chromatin Accessibility Program During T cell Activation 2334000

Abstract Introduction CD8+ T cell activation involves dynamic chromatin accessibility regulation at AP-1 sites. This study examines the regulation of AP-1 transcription factor Batf3 during TCR stimulation. Methods Naïve CD8+ T cells from P14, P14 Il2ra-/-, P14 Batf3-/- mice were activated in culture (CD3/CD28) and harvested at 0-48h for nascent RNA and ATAC sequencing. For adoptive transfers, 50,000 activated (or 5,000 naïve) cells per genotype/construct were transferred to naïve C57BL/6 recipients, followed by LCMVArm/C13 infection. Spleens were harvested at 5-35d post-infection for analysis. Results Analysis of RNA expression in IL-2Rα-/- and Runx3fl/fl CD8+ T cells revealed Batf3 upregulation upon TCR stimulation requires Runx3 and IL-2R stimulation. Chromatin sites with STAT motifs near the Batf3 locus gained accessibility during activation. Retroviral transduction of constitutively active STAT5a induced Batf3 protein expression. Chromatin accessibility analysis of Batf3- and Batf-deficient cells shows these paralogs operate at distinct timepoints within 48h post-TCR stimulation. Batf3-dependent sites are enriched for AP-1, STAT, and IRF motifs, and are distinct from Batf-dependent sites. Batf3-dependent sites exhibit greater accessibility in early effector and tissue-resident memory CD8+ T cells. P14+ Batf3-/- and P14+ Il2ra-/- cells show decreased survival during effector-to-memory transition in acute LCMVarm infection and reduced contribution to re-challenge. Retroviral Batf3 expression in Il2ra-deficient cells enhanced their proliferation during secondary LCMVarm response. Batf3-promoted sites also showed higher accessibility in progenitor versus exhausted CD8+ T cells. Mixed adoptive transfers revealed Batf3 ablation reduces cell proliferation and contribution to chronic LCMVC13 response. Conclusion These data demonstrate that IL-2R-induced Batf3 promotes accessibility at sites distinct from Batf during CD8+ T cell activation, associated with enhanced long-term survival of antigen-experienced effectors. Funding Source n/a Topic Categories Immune Response Regulation: Molecular Mechanisms (IRM)

Cell Intrinsic Programmed Death-Ligand 1 (PD-L1) Signaling Limits Low Affinity T cell Responses during Acute Infection with Listeria Monocytogenes 2306749

Abstract Introduction Programmed Death-1 (PD-1)/Programmed Death-Ligand 1 (PD-L1) signaling through the extracellular domain is responsible for T cell downregulation following acute infection and prevents excessive tissue damage. Although the role of extracellular PD-1/PD-L1 signaling is well defined during infection, signaling via the cytoplasmic domain, termed PD-L1 intrinsic signaling, is less understood. Utilizing a novel mouse model containing mutations in the cytoplasmic tail of PD-L1 (Pdl1CyMt), our lab defined that bone marrow-derived dendritic cells (BMDCs) obtained from Pdl1CyMt mice exhibit heightened activation compared to control BMDCs following treatment with TLR agonists. Methods To investigate if heightened DC activation leads to exacerbated T cell responses, we infected control and Pdl1CyMt mice with Listeria monocytogenes (Lm). Pdl1CyMt mice exhibit decreased liver pathology at day 7 post infection (p.i.) compared to control mice, and increased CD8+ T cell cytokine production. To address if increased CD8+ T cell responses resulted from lower affinity T cells responding in the Pdl1CyMt mice we infected mice with Lm expressing H2-Kb peptides of OVA with altered amino acids (altered peptide ligands) that bind MHCI equally but vary in their ability to stimulate OT-I cells. Results We found that in Pdl1CyMt mice, OT-I CD8+ T cells expanded more to low-affinity ligands compared to controls and displayed reduced tissue pathology. Conclusion These data suggest that intrinsic PD-L1 signaling decreases the threshold of activation for lower affinity T cell responses and limits liver pathology associated with Lm infection. Intriguingly, Pdl1CyMt mice exhibit enhanced bacteremia at day 7 when control mice have cleared the infection. These studies suggest that both the intracellular and extracellular domain of PD-L1 contribute to tissue specific responses that modulate T cell activation. Funding Source n/a Topic Categories Innate Immune Responses and Host Defense: Cellular Mechanisms (INC)

Discovery of a novel innate immune cell death pathway, mitoxyperilysis 2253951

Abstract Introduction Innate immune activation and metabolic dysregulation are common features of disease. Specifically, mitochondrial dysfunction and oxidative stress contribute to pathogenesis during infection, inflammation, and cancer. However, the molecular pathways that connect these processes to disease remain poorly understood. Methods Using complementary techniques under in vitro and in vivo experimental settings, we sought to investigate how the synergism of innate immune activation and metabolic dysregulation affects the cell fate mechanistically. Results We discovered a distinct form of lytic cell death induced by the synergism of innate immune activation and metabolic disruption, which was independent of caspase activity and known regulated cell death pathways. Instead, mitochondria that were damaged by oxidative stress persisted in contact with the plasma membrane, leading to localized membrane oxidation and rupture–a process we term mitoxyperilysis. Mechanistically, mTOR activity was required for cell death following mitochondrial damage, and mTOR inhibition restored cytoskeletal dynamics, enabling lamellipodia retraction and mitochondrial repositioning away from the plasma membrane to preserve membrane integrity. Upstream, we identified BAX/BAK and SLC7A11 as key mediators of this pathway. In tumor models, simultaneous activation of innate immune signaling and metabolic perturbation induced tumor cell death exhibiting hallmarks of mitoxyperilysis. Conclusion Collectively, our findings define a new cell death modality driven by mitochondrial oxidative damage and subcellular localization in response to immune—metabolic stress. Given the central role of mitochondrial stress, inflammation, and cell death in disease, characterization of this pathway offers new therapeutic strategies for cancer and inflammatory disorders. Funding Source n/a Topic Categories Innate Immune Responses and Host Defense: Molecular Mechanisms (INM)