Browse Articles

PD-L1 facilitates transcription of anti-apoptotic genes IL-8, Bcl3 and STAT1, resulting in increased proliferation of ovarian cancer cells 2247762

Abstract Introduction Blocking cell surface expression of PD-L1 has revolutionized immunotherapies, but only a subset of patients achieves durable responses. Indeed, recent studies have also demonstrated PD-L1 nuclear localization that has been associated with cancer progression, but the specific nuclear functions of PD-L1 are just emerging. Here, we investigated the nuclear function of PD-L1 in ovarian cancer (OC) cells. Methods Using gene suppression/overexpression studies, chromatin immunoprecipitation, and cell proliferation and apoptosis studies, we examined whether nuclear PD-L1 regulates apoptosis and expression of anti-apoptotic genes IL-8, Bcl3 and STAT1 in OC cells. We also analyzed the gene co-expression profiles of PD-L1, IL-8 and Bcl3 in OC and pan-cancer tissues using TCGA database. Results Our results demonstrate that PD-L1 overexpression induces PD-L1 nuclear accumulation and increases proliferation of OC cells. Nuclear PD-L1 is recruited to the promoters of IL-8, Bcl3, and STAT1, resulting in their increased expression. Intriguingly, PD-L1 promotes histone acetylation and recruitment of RNA pol II to IL-8, Bcl3 and STAT1 promoters. Suppression of nuclear PD-L1 decreases IL-8, Bcl3 and STAT1 expression and increases apoptosis in OC cells. In addition, analysis of TCGA database demonstrates a positive correlation between PD-L1, IL-8 and Bcl3 gene expression in OC as well as pan-cancer tissues. Conclusion These findings demonstrate that the immune checkpoint PD-L1 facilitates transcription of anti-apoptotic genes IL-8, Bcl3 and STAT1 by inducing promoter acetylation and RNA pol II recruitment, thus providing a novel function of PD-L1 in cancer cells. The increased expression of IL-8, Bcl3 and STAT1 mediated by nuclear PD-L1 might contribute to the limited effectiveness of cancer immunotherapies that target only the surface expression of PD-L1. Funding Source NIH Grant R16GM149263 Topic Categories Tumor Immunology: Checkpoints, Prevention, and Treatment (TIPT)

BCG vaccine induces distinct metabolic shifts in monocytes in vitro and peripheral blood plasma in vivo: Implications for neonatal trained immunity 2308689

Abstract Introduction BCG protects infants against tuberculosis as well as unrelated infections via incompletely understood mechanisms, including metabolic reprogramming of innate immune cells. We sought to characterize age-specific shifts in extracellular metabolism of human monocytes (Mo) induced by BCG stimulation in vitro, determine how they contribute to metabolic reprogramming, and integrate observations in vitro with results from human newborns in vivo. Methods In a human in vitro Mo training platform, newborn (NB = 10) and adult (AD = 12) CD33+ Mo from US-based BCG-naive participants were stimulated with RPMI (control) or BCG for 24h, washed, cultured for 6 days and stimulated with LPS for 24h to assess trained immunity. Day 1 (D1) and D7 supernatants were subjected to mass-spectrometry-based global metabolomics and data integrated with those from an in vivo prospectively randomized newborn cohort from The Gambia (NCT03246230), where BCG was given early (birth) vs delayed (D7) and peripheral blood collected before and 1 or 7 days post-BCG (N = 44-50/group). Results Arginine biosynthesis was highly and significantly enriched in vitro after both primary BCG stimulation and training in both NB and AD. NB BCG trained immunity was characterized by significant upregulation of female sex steroids and downregulation of most other lipid pathways compared to AD. In vivo, early vs delayed BCG resulted in significant downregulation of amino acid (AA) and upregulation of acylcholine pathways on D1, followed by increases in lysophospholipids and acylcholines and decreases in pyruvate, lactate and α-ketoglutarate on D7. In vivo-in vitro integration revealed overlapping signatures featuring decreases in key AA, phospholipids and acylglycines on D1, and sphingolipids, N-acetylglutamate, α-ketoglutarate and leucine/valine metabolites on D7. Conclusion Human in vitro modeling recapitulates distinct signatures of BCG primary and trained immunity in newborns that may contribute to BCG immunogenicity and off-target effects in vivo. Funding Source NIH/NIAID K08AI168487, U19AI118608, U19Al168643 Topic Categories Translational and Interventional Immunology (TI)

T Cells at the Maternal—Fetal Interface: Placental Dynamics in Healthy and Complicated Pregnancy 2260996

Abstract Introduction Deciphering the cellular dynamics that sustain tolerance and placental function in health and disease is essential for better therapies. We built a maternal—fetal placental atlas to characterize common immune-mediated pregnancy complications. Methods We collected 69 placenta samples from patients with pregnancy complications including spontaneous preterm birth (n = 10), fetal growth restriction (n = 9), pre-term preeclampsia (n = 10), term preeclampsia (n = 8), type 1 diabetes (n = 10) and healthy controls (n = 21). We profiled samples via multi-modal10x Genomics single cell sequencing: gene expression, CITE-seq and paired T cell receptor, with spatial transcriptomics (Visium HD) in a subset for validation. Results We profiled over 1.2 million placental cells, the largest atlas to date. We identified 114 cell populations, among them 15 T cell subsets. Using genetic demultiplexing we determined fetal or maternal origin of each cell. We found that the majority of the placental T cells are maternal and that the fetal T cells are only naïve or unconventional T cells (γδT cell subsets and MAIT cells). Using paired TCR-seq data we identified T cell clones and associated clonal expansions to specific T cell subsets. Conclusion This comprehensive atlas redefines the placental cellular landscape. Uncovering the different roles that the maternal and fetal T cells have in the cellular micro-environment is key for understanding mechanisms of placental dysregulation. Funding Source NIH/NIAID 1U19AI167899 Topic Categories Immune Mechanisms of Human Disease (HUM)

Investigating the CXCR4/CXCL12 Axis to Predict Multiple Sclerosis Symptom Onset 2259236

Abstract Introduction Radiologically Isolated Syndrome (RIS) is a pre-symptomatic form of Multiple Sclerosis (MS), where lesions are present in the central nervous system (CNS). Although RIS can now be diagnosed as MS, not all people will progress. Accessible approaches to prognose pwRIS remain to be developed. We propose to study peripheral blood cell (PBMC) and cerebrospinal fluid (CSF) samples of pwRIS, pwMS and healthy (HC) individuals. Selected for this study is the lymphocyte migration molecule CXCR4 and its ligand CXCL12. Methods A cohort of 48 pwRIS with 4 years follow up, as well as age- and sex-matched pwMS and HC were used for the study. We further divided pwRIS based on disease activity within a year of sample collection. An Ella assay was performed on CSF to titer levels of CXCL12. PBMCs were phenotyped using CyTOF, and flow cytometry following stimulation, focusing on T cell migration and activation. Single-cell RNA and TCR sequencing was performed to correlate transcriptomic and TCR repertoire changes with disease activity. Results CXCL12 levels in pwRIS and pwMS CSF were increased significantly in comparison to HC. These levels positively correlated to GFAP but not NfL. Upregulation of CXCR4 was observed on multiple lymphocyte subsets, particularly on CD8 effector T cells in pwRIS with disease activity, as found by CyTOF and flow cytometry. CXCR4 expression also correlated to cytokine production in stimulated PBMCs. Conclusion We have shown that CXCL12, a proposed ligand for early CNS chemotaxis and entry, is upregulated in the CSF of pwRIS and pwMS. CXCL12 correlates with other markers of MS progression, suggesting a potential use for early prognosis. CXCR4 is upregulated on CD8 effectors, suggesting an aberrant immune response leading to early CNS trafficking, which is correlated with increased T cell reactivity and disease activity. These findings will be central in the improvement in clinical care by providing a prognostic tool, while understanding the pathophysiology of early disease. Funding Source Brain Canada Canadian Institutes for Health Research - Doctoral Research Award (536121) Topic Categories Immune Mechanisms of Human Disease (HUM)

Peri-Hippocampal Mast Cells Regulate Hippocampal Neurogenesis and Blood-Brain Barrier Permeability During Early Postnatal Development 2307916

Abstract Introduction Inflammation in early life is an established risk factor for the diagnosis of neurodevelopmental disorders, highlighting the consequences of immune activation during neurodevelopment. While microglial involvement has been studied extensively, the contributions of other immune cells are poorly defined. Using Sprague Dawley rats, we identified a population of mast cells proximal to the hippocampal neurogenic niche (dentate gyrus), termed peri-hippocampal mast cells (phMCs). phMCs are highly proliferative, peak in number at postnatal day (PN) 7, steadily decline until PN14, and are no longer detected by the third week of life, a pattern reminiscent of a critical developmental window. This temporal profile coincides with the peak of hippocampal neurogenesis. Thus, we hypothesize that phMCs contribute to hippocampal neurogenesis through two complementary mechanisms: 1) the release of histamine that directly binds to neural precursor cells and 2) histamine-dependent induction of blood-brain barrier (BBB) permeability, permitting peripheral growth factors access to the neurogenic niche. Methods To test this, hippocampal neurogenesis and BBB permeability were evaluated following pharmacological phMC degranulation. BBB permeability was assessed by measuring leakage of peripherally injected tracers in brain homogenates. Results Degranulation at PN2 caused region-specific, histamine-dependent leakage of 70-kDa molecules into the hippocampus but not the cerebellum, whereas at PN7 leakage was restricted to molecules ≤10-kDa. phMC degranulation at PN5—7 significantly increased BrdU+ neurons in the dentate gyrus at PN14. Conclusion These findings indicate that phMCs have the functional capacity to promote hippocampal neurogenesis and regulate BBB permeability in a developmentally and regionally constrained manner. Early degranulation at PN2 permits large-molecule entry, whereas later degranulation induces limited, size-restricted permeability, consistent with BBB maturation. Funding Source 5R01MH052716-27 to MMM (PI); 3R01MH052716-28S1 to AWA (trainee) Topic Categories Neuroimmunology (NEUR)

Overexpression of FGF21 in thymic stromal cells enhances T cell responses to viral infection and immune checkpoint inhibition efficacy during aging 2259870

Abstract Introduction Age-associated thymus atrophy diminishes T cell repertoire diversity, diminishing responsiveness to vaccination, viral infection, and tumor surveillance. Age-associated T cell dysregulation includes the persistence of virus-specific lung tissue resident memory T (Trm) cells in aged mice after influenza infection, driving chronic non-resolving inflammation and lung pathology 60 days post-infection (d.p.i.) Our recent work demonstrated that sustained thymic overexpression of fibroblast growth factor 21 (FGF21) in LPOFGF21 knock-in (KI) mice promotes increased thymic size and naïve T cell export in aged mice, improving age-related declines in CD8+ T cell responses to influenza in the acute phase, at 10 d.p.i. Methods To further test the impact of thymic overexpression of FGF21 on lung Trm and pathology in older mice, we examined immune responses to primary and heterosubtypic secondary challenge in 12—15-month-old LPOWT and LPOFGF21 mice at 7 and 60 d.p.i. Mice were infected intranasally with IAV PR8 only, or with IAV PR8 20 days after primary IAV X31 infection, and were analyzed at 7 or 60 d.p.i. Results Sustained FGF21 overexpression in LPOFGF21 mice mitigated the age-associated accumulation of CD8+ Trm, and the associated chronic lung pathology at 60 d.p.i. It also increased the frequency of T follicular helper and class-switched B cells in mediastinal lymph node at 7 and 60 d.p.i. We also tested the efficacy of immune checkpoint inhibition (ICI, anti-PD-L1 antibody) treatment in young and aged LPOFGF21 KI mice and age-matched controls using a B16 melanoma tumor model. We found that aging impaired tumor control and ICI efficacy in aged control mice, and that these age-associated impairments were ameliorated in age-matched LPOFGF21 KI mice. Conclusion These results suggest that intrathymic FGF21 overexpression mitigates age-associated declines in anti-viral and anti-tumor CD8+ T cell responses, revealing a potential therapeutic target for improving T cell function in aging. Funding Source NIH R01 AG086271 Topic Categories Lymphocyte Differentiation and Peripheral Maintenance (LYM)

Synergistic Crystallization Regulation by Substituent Electronic Effects of Polyfluoroarenes Toward Large Area Perovskite Solar Module Fabrication

ABSTRACT Achieving uniform and high‐quality perovskite crystallization across large substrates is a prerequisite for transitioning perovskite solar cells (PSCs) from laboratory scale to industrial production. Herein, we systematically regulate perovskite crystallization kinetics by using polyfluoroarene molecules featured with distinct electronic effects. We demonstrate that 3,4,5‐trifluorobenzonitrile (TFBN) containing a strong electron‐withdrawing cyano group creates an electron‐deficient conjugation system. This configuration enhances anion‐π interactions with I − and forms strong coordination with Pb 2+ framework. Synergizing with intermolecular hydrogen bonding, TFBN increases the effective nucleation barrier, converting rapid nucleation into a controlled, uniform growth process. Consequently, this multidimensional regulation yields pinhole‐free perovskite films with significantly suppressed non‐radiative recombination. The TFBN‐optimized small area device (aperture area: 0.09 cm 2 ) achieves a champion power conversion efficiency (PCE) of 27.01% along with a low non‐radiative voltage loss of only 55 mV. Furthermore, we demonstrate excellent scalability, achieving PCEs of 25.56% for 1 cm 2 (aperture area) cells, 24.51% for 14.63 cm 2 (active area) modules, and 21.05% for 58.51 cm 2 (active area) modules, respectively. Moreover, the resulting devices exhibit improved long‐term operational stability under maximum power point tracking and thermal stress. Overall, this synergistic regulation approach provides molecular‐level design principles for scalable fabrication of efficient and durable perovskite photovoltaics.

Zein‐Ceria Hybrid Microparticles Enable Long‐Term ROS‐Scavenging Oxygenation for Osteogenic Microtissues Engineering

ABSTRACT Oxygen‐ and biological cue‐deprived microenvironments formed during tissue regeneration severely limit cell survival and differentiation, resulting in long‐term structural and functional deficits. However, conventional oxygen‐releasing biomaterials often exhibit burst releases, with the vast majority of oxygen released during the first few days, which is associated with high levels of concomitant reactive oxygen species (ROS)‐derived oxidative stress and a lack of bioactive factors. Here, we report a hierarchically engineered zein‐ceria hybrid microparticle that enables sustained ROS‐neutral oxygenation for over 40 days and supplies an osteoinductive factor. A hydrophobic zein core stabilizes the oxygen source and suppresses burst release, while a ceria nanozyme‐integrated shell continuously scavenges excess ROS via redox cycling. Biocompatible surface engineering enables the seamless integration of these microparticles within stem cell spheroids, which markedly enhances cell survival under anoxia. Their biofunctional surface supports enzymatic protein immobilization under physiological conditions, enabling spontaneous osteogenesis of engineered bone microtissues. In a severely oxygen‐deprived mouse calvarial defect model, the engineered microtissues accelerated bone regeneration. Our biomaterial design enables control of burst oxygen release, ROS modulation, and growth factor release, built on a zein‐ceria double‐layer architecture, offering a modular platform that broadens the utility of oxygenating and bioactive micromaterials in regenerative medicine.

Sequential plastic and terminal tolerogenic states limit helper T cell responses towards ingested antigen 2309592

Abstract Introduction T cell tolerance is the process by which the immune system is trained to ignore harmless antigens. When it breaks down, uncontrolled responses to self, commensal and/or environmental antigens result in pathological disbalance between pro- and anti-inflammatory T cell states. Understanding cell intrinsic mechanisms that set or shift this balance is key to development of therapeutics that invoke or subvert tolerance in clinical settings. Methods To study T cell tolerance, we leveraged a mouse model of oral tolerization whereby naive, Ovalbumin-responsive T cells are transferred to host mice which are fed Ovalbumin in water. Donor cells are then recovered from draining lymph nodes and subjected to high-content, single-cell resolution analyses. The capacity to genetically manipulate donor T cells is a key feature; congenic markers enable us to unambiguously pinpoint donors, FOXP3 reporter alleles enable us to track Treg and gene ‘knockouts’ enable us to assess putative tolerogenic factors. Results Based on the finding that anergy, a tolerogenic state that restricts inflammatory functions, and ‘peripheral’ regulatory T cells (pTreg) are sequentially engaged, we have devised a two-stage model. First, T cells pass through a multipotent ‘proto-anergic’ state bearing elements of T follicular helper responses (Stage 1), then proceed to terminal tolerogenic states, becoming either terminally anergic or pTreg (Stage 2). Each stage bears a distinct transcriptional program, with BCL6 and PD1 emerging as drivers of anergy, IL-2Ra and STAT5 as drivers of pTreg. Both BCL6 and STAT5 promote stemness and are required for maintenance of tolerogenic responses. Conclusion T cell tolerance to ingested antigen involves sequential stages of regulation. Stage 1 is a temporary, plastic state that we term ‘proto-anergy’ where PD1 keeps cells at bay until Stage 2, where they proceed to terminal anergy or pTreg differentiation. BCL6 and STAT5 are critical for both induction and maintenance of two-stage tolerance. Funding Source University of Miami Intramural Funding Topic Categories Immune Response Regulation: Molecular Mechanisms (IRM)

Complement expression in mesenchymal cells across bacterial and viral infections in the lung 2248926

Abstract Introduction We have recently identified the lung mesenchyme as a potent source of complement proteins during homeostasis. However, the changes in the complement transcriptomic profile in the mesenchyme in the context of infections has not yet been elucidated. Here, we show that mesenchymal cells express a defined pattern of complement genes during respiratory bacterial and viral infections. Methods Single cell RNA-sequencing data from selected published studies in murine experimental lung infection with Streptococcus pneumoniae, Pseudomonas aeruginosa, Klebsiella pneumoniae, influenza, and SARS-CoV-2 that included mesenchymal cells were analyzed. The raw counts data were retrieved from NCBI GEO and imported into RStudio/R (v4.3.1) and processed using Seurat (v5.0.1). Results Uninfected mesothelial cells exhibited high expression of classical pathway genes such as C1ra, C1s, C3, Serping1 (C1-INH), and Cfh, while fibroblasts highly expressed Serping1 and Cfh, consistent with our previous findings. During pneumococcal pneumonia, mesothelial cells showed downregulation of C2, C4b and Cfh expression, and upregulation of Cfb, whereas fibroblasts showed elevated expression of C3, Cfb and C4b, and repression of Cfh versus uninfected cells. Similar expression patterns in these cell types, though not always statistically significant, were observed in the other bacterial and viral infection models analyzed. As mesenchymal cells modulate inflammation and tissue repair through extracellular matrix (ECM) remodeling, we also explored ECM, collagen and cytokine expression. Across all infection models except Klebsiella, fibroblasts expressed higher levels of Timp1 (up to 100-fold), Col4a1, and Col4a2, while Stat1 was elevated in S. pneumoniae and viral infections. Conclusion Our study elucidates a cell-specific pattern of complement, ECM and cytokine/chemokine gene expression within lung mesenchymal cells during bacterial and viral infections and highlights a potential role for complement-driven ECM remodeling. Funding Source This work was funded by the National Institutes of Health (R01HL166588 to M.B.). Topic Categories Immune Response Regulation: Molecular Mechanisms (IRM)

SLC15A4 Inhibition by NTX-348 Suppresses Endolysosomal Toll-like Receptor Signaling, IRF5 Activation, and Type I Interferon Induction 2249463

Abstract Introduction Recent studies have revealed that nucleic acid sensing by the endolysosomal TLRs (TLR7, TLR8, and TLR9) requires coordinated activity of the lysosomal peptide transporter SLC15A4. This peptide transporter interacts with the adaptor protein TASL and is necessary for the recruitment and activation of the transcription factor IRF5. The importance of this signaling pathway in the pathogenesis of autoimmune disease has been highlighted by human genetic variants in SLC15A4, TASL, and IRF5 that are associated with elevated risk of developing systemic lupus erythematosus (SLE), while pre-clinical studies in mouse have shown that genetic knock-out of these genes is protective in multiple autoimmune or inflammatory disease models. Methods Using structure-based drug design, we have developed a series of novel small molecule SLC15A4 inhibitors. Further optimization of the potency and drug-like properties of these molecules has led to discovery of potential first-in-class lead compounds that exhibit nanomolar cellular potency, favorable in vitro ADME and off-target profiles, and suitable PK properties in mouse that allow for once-daily oral dosing. Results In vitro studies show that SLC15A4 inhibition results in the intracellular degradation of the TASL adapter protein and blockade of inflammatory cytokine production in response to TLR7/8/9 agonists. Using in vivo mouse models of TLR-driven acute cytokine production, treatment with an advanced tool compound NTX-348 results in complete suppression of IRF5 phosphorylation and type I interferon production in response to TLR7 or TLR9 agonist treatment. Conclusion These results highlight our early progress developing novel, drug-like inhibitors of SLC15A4. SLC15A4 inhibition represent a novel therapeutic approach that may provide clinical benefit as an oral therapy for SLE and other immune-mediated diseases. Funding Source All authors are employees or contractors of Nimbus Therapeutics Topic Categories Therapeutic Approaches to Autoimmunity (THER)

Oral Pathogenic Bacterial Products Differently Modulate Responses in HIV+ and HIV— Peripheral Blood Immune Cells 2260611

Abstract Introduction Porphyromonas gingivalis plays key roles in the human oral microbiome and disease. Oral dysbiosis and bacterial products such as lipopolysaccharide from P. gingivalis (Pg-LPS) and lipoteichoic acid from Staphylococcus aureus (Sa-LTA) can activate peripheral immune cells, which may cross the blood brain barrier and trigger inflammation and neuronal dysfunction, leading to cognitive decline. These processes occur in people with HIV (PWH), nearly half of whom develop HIV-associated neurocognitive disorders despite antiretroviral treatment. Since oral dysbiosis affects PWH, this study characterizes the responses of HIV positive (HIV+) and HIV negative (HIV—) peripheral blood mononuclear cells (PBMCs) to Pg-LPS and Sa-LTA stimulation in vitro. Methods PBMCs from HIV+ and HIV— donors were cultured and exposed to Pg-LPS or Sa-LTA (0.1 or 1 µg/mL) for 24 or 48 hours (h). iNOS expression was assessed by western blot. IL-6 was quantified by ELISA, while TNF-α and IL-10 were measured via Ella multiplex system. Results Pg-LPS increased TNF-α in HIV— PBMCs at 48h (p = 0.033); in HIV+ PBMCs, TNF-α increased at 24h (p = 0.026) but declined by 48h (p = 0.011). HIV+ PBMCs, 24h stimulation with Pg-LPS induced a slight increase in IL-6 secretion, which decreased by 48h (p = 0.025). IL-10 levels in HIV— PBMCs remained unchanged, while in HIV+ PBMCs IL-10 was higher at 24h compared to 48h (p = 0.006). No significant changes in iNOS were detected. Sa-LTA did not induce notable effects. Conclusion HIV+ PBMCs exhibit a pro-inflammatory response to Pg-LPS at 24h, which is not sustained for 48h. This activation appears to be counterbalanced by an anti-inflammatory response at 24h exposure. These findings suggest that oral pathogen toxins trigger distinct immune activation dynamics in PWH, potentially contributing to neuroinflammatory mechanisms. Ongoing studies include shorter stimulation times and PBMC—brain organoid co-cultures to further explore their contribution to neuronal dysfunction and cognitive impairment. Funding Source NIHMHD U54 MD007600, NINDS K22NS118975, 5R21NS131061-02, NIGMS PR-INBRE 5P20GM103475, The Alliance U54GM133807, HiREC S21MD001830, and Título V RCM PO31S200104. Topic Categories Mucosal and Regional Immunology (MUC)

A Six-month Ketogenic Diet Alters The Immune And Metabolic Landscape In Multiple Sclerosis 2261201

Abstract Introduction A large body of preclinical research supports the immunomodulatory effects of diet, and dietary strategies for multiple sclerosis (MS) remain of major interest to clinicians and people with MS. Ketogenic diets produce anti-inflammatory effects in animal models of MS, but the effect in humans remains unknown. Methods Cryopreserved PBMCs and plasma were analyzed at baseline and after six months of MAD in 39 patients with relapsing MS who completed a previously-published phase 2 study of MAD. PBMCs were analyzed using single cell RNA sequencing, flow cytometry, and ex vivo stimulation assays. Plasma samples were subjected to metabolomics and multiplex ELISA. Results Six months of MAD produced substantial changes in the composition and transcriptional profiles of peripheral immune subsets associated with both innate and adaptive immunity. These changes included reduced pro-inflammatory phenotypes in myeloid cells, a shift from memory to naïve CD8 cells, increased abundance and suppressive activity of regulatory T (Treg) cells, and decreased B cell activation. Multiplex ELISA revealed that MAD significantly reduced plasma levels of pro-inflammatory cytokines and chemokines, such as IL-6 and CCL2. As a low carbohydrate/high fat diet, we hypothesized that MAD might shift the balance between glycolysis and fatty acid oxidation. As predicted, gene and protein expression patterns revealed metabolic reprogramming from glycolysis to fatty acid oxidation. These changes were corroborated by plasma metabolomics, which demonstrated a decrease in glycolytic products and an increase in fatty acid oxidation intermediates. Conclusion Our findings support the immunomodulatory potential of ketogenic diets in MS, demonstrating the capacity of MAD to reprogram immune cell metabolism and promote anti-inflammatory phenotypes. These results provide a rationale for larger, randomized studies comparing dietary interventions and evaluating clinical outcomes in MS. Funding Source n/a Topic Categories Therapeutic Approaches to Autoimmunity (THER)

“Listen to your nerves”: Skin Sensory Neurons promote Immunity against Parasitic Worms 2266688

Abstract Introduction Skin employs interdependent cellular networks for barrier integrity and host immunity, but most underlying mechanisms remain obscure. While bacteria and fungi often cause painful and itchy cutaneous reactions, host skin invasion by the human parasitic helminth Schistosoma mansoni frequently goes unnoticed. Methods Our studies interrogated whether sensory neurons that transmit itch and pain contributed to the detection and initiation of skin immunity against S. mansoni. Results Herein, we demonstrate that S. mansoni exposure inhibited itch evoked by afferents bearing the MrgprA3 receptor in mice. In vitro approaches revealed that S. mansoni was directly recognized by MrgprA3+ neurons, but this interaction prevented calcium influx and neuropeptide release induced by the MrgprA3 ligand chloroquine. S. mansoni infection also significantly reduced thermal pain sensitivity evoked by TRPV1+ neurons and neuropeptide release induced by the TRPV1 agonist capsaicin. Data show that MrgprA3+ or TRPV1+ neurons promoted IL-17+ gd T cell expansion and host resistance against S. mansoni through shaping cytokine expression in cutaneous macrophages. MrgprA3+ neuron secretions increased chromatin accessibility and expression of inflammatory cytokines in macrophages, partially through the neuropeptide CGRP. Conclusion Taken together, this work suggests that S. mansoni may have evolved to manipulate MrgprA3+ or TRPV1+ neuron activation as a countermeasure to limit IL-17-mediated inflammation, facilitating systemic dissemination and chronic parasitism. We will define the mechanisms underlying these interactions in my independent research program that will define the contributions of itch-inducing neurons to skin anti-helminth immunity. Funding Source NIH-NIAID Topic Categories Microbial, Parasitic, and Fungal Immunology (MPF)

Macrophage ALOX15B restricts CIITA-dependent antigen presentation via MHC II through lipid peroxide-driven NRF2 activation 2308504

Abstract Introduction Macrophage major histocompatibility complex (MHC) II-dependent antigen presentation is essential for activating CD4+ T cells. Transcriptionally, macrophage (Mφ) MHC II genes hinge on the constitutively expressed transactivator CIITA. While IFNγ strongly boosts CIITA and thereby MHC II gene expression, intracellular signals controlling constitutive CIITA expression remain largely unknown. Methods Human CD4+ T cells and monocytes were isolated from blood of anonymous donors. Genes of interest were silenced in monocyte-derived Mφ using siRNA. Changes in gene expression and protein abundance were quantitatively analyzed using RNA-seq, RT-qPCR, Western analysis and flow cytometry. Intracellular protein localization was assessed by confocal microscopy. Pull-down of thiol-oxidized proteins was achieved through BIAM (biotinylated iodoacetamide) switch assays. Following Mφ-CD4+ T cell co-culture, T cell proliferation and cytokine release were investigated via flow cytometry. Results The lipid peroxidizing enzyme ALOX15B (Arachidonate 15-Lipoxygenase Type B) is constitutively expressed in primary human Mφ and drives lipid peroxide-mediated nuclear translocation and thus activation of NRF2 (NFE2 Like BZIP Transcription Factor 2), the master regulator of cellular antioxidant response. We noticed that under resting conditions, ALOX15B dampens the constitutive expression of CIITA through NRF2 activation. Following ALOX15B KD, an increase in CIITA expression enhanced total and cell surface MHC II proteins as well as MHC II-dependent CD4+ T cell activation. While MHC II upregulation in ALOX15B KD Mφ was attenuated through NRF2 stabilization, MHC II-dependent CD4+ T cell activation increased significantly upon NRF2 KD. Investigations into its relevance in lung pathologies are ongoing. Conclusion In Mφ, MHC II-dependent antigen presentation along with CD4+ T cell activation is controlled through ALOX15B-driven repression of CIITA via lipid peroxide-mediated NRF2 activation. Funding Source Deutsche Forschungsgemeinschaft SFB1039 TP B06 Topic Categories Classical and Non-Classical Antigen Presenting Cells (APC)

Racial Disparity in Immune Response: Prospective Study in Obesity-Associated Lupus Patients 2260048

Abstract Introduction Systemic lupus erythematosus (SLE) is a chronic autoimmune disease characterized by systemic inflammation and the production of autoantibodies (Ab), leading to multiple organ damage. Our recent retrospective data showed that obese patients have exacerbated lupus symptoms and overactive autoimmunity than non-obese lupus patients (pts). Here we further investigated the racial differences in immune cell profile and clinical features in obese-associated SLE pts. Methods Peripheral blood was collected from 60 consented SLE pts grouped by race and BMI levels. Immune cell profile was identified by flow cytometry. Clinical data including demographic information, BMI, SLE disease activity (SLEDAI), anti-dsDNA Ab, creatinine, urine protein, CRP, ESR, and complement levels were extracted from EPIC. Unpaired Student’s t-test was used for statistical analysis among the groups. Results SLE pts in this study were 75% Africa American (AA), 20% Caucasian American (CA), 2% Asian, 3% Hispanic/Latino, with 76.7% overweight/obese. Although there is no difference in CRP and complement levels, AA pts had significantly higher positive percentages of dsDNA Ab, skin rash, and nephritis history in all BMI groups comparing to CA pts. In addition, significantly higher levels of ESR (p < 0.05) and urine creatinine/protein (p < 0.05) were observed in AA pts than CA pts. Interestingly, significant increases of circulating T follicular helper (Tfh) cells (p < 0.005) and Th17 cells (p < 0.05) were found in overweight/obese pts comparing to non-obese pts, but no difference of these cells between AA and CA lupus pts. Conclusion Our data showed that AA (especially obese) lupus patients demonstrated similar T cell subsets to CA pts, but carried a significantly disproportionate burden of lupus, including higher incidence, more severe manifestations, and worse outcomes. Further study may provide biological insights into the pathogenesis of lupus racial diversity and potentially tailored therapy for high-risk lupus pts. Funding Source Collaborative Intramural Reseach Program (CIRP) in LSUHS and Ochsner Topic Categories Immune Mechanisms of Human Disease (HUM)

Plasmonic Enhancement of Fluorescence and Protein Dynamics in Living Mammalian Cells (Adv. Mater. 45/2026)

Gut bacteria-specific IgG regulates CNS neuroinflammation 2309610

Abstract Introduction The gut microbiota is an emerging regulator of neuroinflammatory diseases like Multiple Sclerosis (MS), but the mechanisms underlying microbiota-immune crosstalk are not fully understood. We previously showed that commensal bacteria-specific IgG antibodies shape the gut microbiota and restrain intestinal inflammation by facilitating the clearance of pathogenic gut bacteria. However, the role of bacteria-specific IgG in regulating neuroinflammation via the gut-brain-immune axis remains undefined. Methods Flow cytometry was used to profile immune cells in the intestinal lamina propria and brain parenchyma of IgG-deficient (IgG KO) and WT mice. Mice were immunized with MOG35-55 to induce Experimental Autoimmune Encephalomyelitis (EAE). To study the role of B cells, we treated mice with B cell-depleting αCD20 or adoptively transferred naïve splenic B cells from WT and IgG KO mice into B cell-deficient μMT recipients. To assess how IgG-targeted bacteria affect neuroinflammation, we transplanted IgG-coated bacteria isolated from human stool into germ-free (GF) mice. Results At steady state, IgG KO mice have increased inflammatory T cells in the CNS and elevated systemic levels of CNS-reactive autoantibodies. Consistently, IgG KO mice exhibit increased EAE susceptibility and disease severity. B cell depletion rescues EAE severity in IgG KO mice and adoptive transfer of IgG KO, but not WT, B cells promotes more severe EAE, suggesting a pathogenic role for B cells in driving neuroinflammation in the absence of IgG. Finally, GF mice given human IgG-coated gut bacteria recapitulate immune activation in the CNS and EAE severity, suggesting that IgG controls neuroinflammation through the microbiota. Conclusion Together, our findings demonstrate a critical role for IgG-mediated control of the gut microbiota and adaptive immune responses in the regulation of CNS neuroinflammation. This work will inform the development of novel gut microbiome-targeting therapies to treat neuroinflammatory diseases like MS. Funding Source n/a Topic Categories Neuroimmunology (NEUR)

An epigenetic polarity governing T cell tolerance for commensal microbiota 2250947

Abstract Introduction T cells differentiate into subtypes to maintain immune tolerance or mount inflammatory response upon antigen stimulation. This raises questions about whether and how T cell subtypes rely on fundamentally distinct epigenetic programs. Methods Using Wdr82, a component of the Set1/COMPASS histone H3K4 methyltransferase complex, as a model, we discover that this pathway is broadly required for the activation and function of both Te and Treg cells. Results Counterintuitively, T cell-specific deletion of Wdr82 leads to Te activation and lethal spontaneous colitis. This dysregulation is nearly completely prevented by microbiome depletion or wild-type Treg transfer. Mechanistically, Set1/COMPASS complex interacts with Foxp3 in a TCR-signaling dependent manner. H3K4me3 pathway is preferentially required for Treg induction and for the expression of Treg functional genes such as Il10 and Rorc. Conclusion Thus, the generic H3K4me3 pathway plays a biased role in Treg-dependent immune homeostasis particularly in the presence of commensal microbiota. Our study depicts an epigenetic polarity governing Treg-mediated immune tolerance, highlighting a fundamental asymmetry at the epigenetic level that differentially regulates Treg and conventional T cells. Perturbation of this asymmetry by genetic and environmental factors would lead to autoimmune dysregulation. Funding Source National Institute of Allergy and Infectious Diseases Topic Categories Immune Response Regulation: Molecular Mechanisms (IRM)

ATF3 Regulates Cholesterol Trafficking in Macrophages 2259886

Abstract Introduction Metabolic dysfunction-associated steatohepatitis (MASH) is an inflammatory liver disease linked to type 2 diabetes and cardiovascular disease. During MASH, Kupffer cells and other liver macrophages exacerbate liver inflammation in response to elevated lipids and immune stimuli. In a mouse model, Kupffer cells had increased expression and genomic occupancy of activating transcription factor 3 (ATF3)–a known transcriptional repressor of macrophage inflammation. Methods Kupffer cells and other liver macrophages from Clec4f-Cre Atf3-fl/fl or Atf3-fl/wt controls were analyzed by flow cytometry and RNA-seq. Bone marrow derived macrophages from Lyz2-Cre Atf3-fl/fl mice or Cre negative controls were treated with oxidized low-density lipoprotein, lipopolysaccharide, and/or methyl-beta-cyclodextrin. Genotype-dependent effects were assessed by ELISA, RNA-seq and cholesterol assays. Results ATF3 binds cholesterol-associated genes, and its loss in Clec4f+ Kupffer cells increased neutral lipid levels with limited transcriptional differences compared to wild type controls. In vitro, ATF3 restricted baseline cellular cholesterol, while its deficiency elevated basal interferon-stimulated genes (ISG) expression and exaggerated type 1 interferon and cytokine responses to TLR stimulation. Lipid loading reduced ISG expression, potentially explaining limited in vivo differences. However, inflammatory stimuli in lipid loaded cells amplified gene expression changes, suggesting ATF3 function is both lipid- and inflammation-dependent. ATF3-deficient macrophages upregulated Gramdb1 (Aster B), a membrane to ER cholesterol transporter, and downregulated Cav1 (Caveolin-1), a potential efflux mediator. These changes may increase membrane cholesterol. Depletion of plasma membrane cholesterol with methyl-beta-cyclodextrin negated the genotype-dependent cytokine response. Conclusion Together, our data suggest ATF3 supports cholesterol trafficking from the plasma membrane to preserve membrane homeostasis and limit inflammation. Funding Source This research was supported by PHS Grant P30 DK078392, AASLD PNC23-216751 and the Cincinnati Children’s Research Foundation. Topic Categories Innate Immune Responses and Host Defense: Molecular Mechanisms (INM)