Browse Articles

Nrf2-mediated epigenetic priming of osmoprotective genes enhances cellular adaptation to hyperosmotic stress

Assessment of AAV3B transduction efficiency in a liver normothermic machine perfusion model

Male-Biased Macrophage Signaling and CD8 T Cell Dysfunction Drive Immunosuppression in Pulmonary Fibrosis 2258616

Abstract Introduction Idiopathic pulmonary fibrosis (IPF) is a progressive and fatal lung disease with marked sex-based disparities, as males show higher prevalence and worse outcomes than females. However, the cellular and immune mechanisms underlying these differences remain poorly understood. Methods To investigate sex-specific immune mechanisms in pulmonary fibrosis, we performed single-cell RNA sequencing (scRNA-seq) on lung tissues from male and female mice following bleomycin injury. Major immune and stromal cell populations were identified, and pathway enrichment analysis was used to define sex-biased signaling pathways. Results Autologous co-culture studies with precision-cut lung slices (PCLS) demonstrated greater collagen deposition in male PCLS, consistent with enhanced fibrotic activity. Similarly, in the bleomycin model, male mice developed more severe fibrosis and had poorer survival than females. scRNA-seq analysis revealed broad enrichment of pro-fibrotic and immunoregulatory pathways in male immune compartments. Macrophages in males showed activation of IL-8 signaling and PD-L1/PD-1 checkpoint pathways, indicative of immunosuppression and neutrophil recruitment. CD8 T cells displayed enrichment of apoptosis, TGF-β signaling, unfolded protein response, and negative regulation of type I interferon production. Moreover, male fibroblasts and epithelial cells expressed IL-4/IL-13, JAK/STAT3, and TGF-β pathways, programs likely to promote macrophage polarization and suppress effective T cell responses. Conclusion Male lungs exhibit upregulated immune checkpoint signaling, neutrophil-recruiting chemokines, and T cell exhaustion, reinforced by stromal-epithelial signaling. This immune dysregulation drives a pro-fibrotic, immunosuppressive environment and emphasizes sex-based disparities in IPF, pointing to therapeutic opportunities targeting male-biased immune pathways. Funding Source This work was supported by the NIH grant R01HL146802, the UCOP TRDRP grant T32IR5347, and the DoD PRMRP grant W81XWH2110086 (#PR202411). Topic Categories Immune Mechanisms of Human Disease (HUM)

Interrogating mechanisms of a novel anti-tumor CD4 response 2308216

Abstract Introduction Agonists of the STING pathway, such as cyclic di-nucleotides (CDNs), exhibit powerful antitumor activity in preclinical models. CDN-driven tumor control has been primarily attributed to the activity of CD8 T cells, which recognize tumor-antigens presented on MHC I. We have found that CDN treatment also elicits strong responses against CD8-resistant MHC I-deficient tumors, including RMA-B2m-/- lymphoma cells, where both NK cells and CD4 T cells play crucial and at least partly independent roles in tumor control. NK cell-mediated clearance of B2m-/- tumors is expected, but little is known about how CD4 T cells mediate tumor clearance, particularly of MHC I-deficient tumors where CD4 T cell help of CD8 T cells is demonstrably irrelevant. Methods To identify the mechanisms by which CD4 T cells mediate tumor control, we conducted in vivo tumor growth experiments probing various mechanisms and phenotyped tumor-infiltrating immune cells under different conditions. Results The results rule out a role of direct MHC II recognition or death receptor signaling on tumor cells. Furthermore, they argue against a role for innate-like receptors on CD4 T cells. IFN-g produced by T cells after CDN treatment is crucial for tumor control but contrary to published paradigms, IFN-g receptor signaling in macrophages is dispensable for the anti-tumor response as are CD64+ macrophages. Notably, IFN-g receptor signaling in T cells contributes to tumor control, and is associated with a reduced frequency of tumoral regulatory T cells. However, the major impact of IFN-g in tumor control is on radioresistant cells. Alterations in the TME associated with IFN-g signaling on radioresistant cells will be discussed. Conclusion IFN-g produced by CD4 T cells plays a critical role in controlling MHC I-deficient tumors, independent of CD8 T cells, NK cells, CD64+ macrophages or expression of MHC II or death receptors on tumor cells. Its main impact on tumor control is on non-hematopoietic cells where it alters tumor progression. Funding Source R01CA270790 Topic Categories Tumor Immunology: Cellular Responses and Tumor Microevironment (TIME)

FCRL3 is an immunoregulatory receptor that restrains the activation of human memory T lymphocytes 2328576

Abstract Introduction Genetic variants in the FCRL3 gene are associated with multiple autoimmune diseases, yet the function and regulation of FCRL3 in human T lymphocytes remain poorly understood. We aimed to define the cellular identity, regulation, and functional role of FCRL3-expressing human T cells and to elucidate the mechanisms by which FCRL3 modulates T cell responses. Methods We combined multiparametric flow cytometry, transcriptomic analyses, mass spectrometry and functional assays to characterize FCRL3+ human T lymphocytes. FCRL3 expression was analyzed across naïve, memory, and effector T cell subsets. T cell activation, cytokine production, and cytotoxic features were assessed following TCR stimulation. Gain-of-function approaches were used to evaluate the direct impact of FCRL3 expression on T cell responses, and biochemical analyses were performed to investigate associated signaling pathways. Results FCRL3 expression was enriched in cytotoxic CD4+ T cells and CD8+ effector memory TEMRA cells. FCRL3+ T cells displayed a transcriptional program associated with cytotoxicity but showed reduced activation capacity upon TCR engagement. FCRL3 expression was induced by repetitive TCR stimulation and was sufficient to attenuate T cell activation. Mechanistically, the cytoplasmic domain of FCRL3 engaged inhibitory signaling molecules, resulting in dampened downstream TCR signaling. Conclusion Our findings identify FCRL3 as a functional immunoregulatory receptor that restrains activation of highly differentiated human T cells with cytotoxic potential. This work provides mechanistic insight into how genetic associations at the FCRL3 locus may contribute to T cell functions and highlights the importance of inhibitory pathways in maintaining immune balance. Funding Source Swiss National Science Foundation Topic Categories Immune Response Regulation: Molecular Mechanisms (IRM)

Optimizing Cancer Antibody Effectiveness: Pharmacology and Mathematical Modeling to Achieve Better Clinical Outcomes 2257606

Abstract Introduction A longstanding practice for therapeutic dosing of monoclonal antibodies is to identify the maximally tolerated dose (MTD). However, this approach has had limited success with some antibodies, especially in cancer trials. Here we report on an alternative approach using novel antibodies against tumor necrosis factor receptor 2 (TNFR2), an oncology target. TNFR2 expression is largely restricted to cancer cells and to regulatory T cells (Treg) in the tumor microenvironment; TNFR2 inhibition with antibodies reduces immunosuppressive Tregs with subsequent activation of effector T cells and can directly kill cancer cells. Methods Using data from cytotoxicity assays in a human lymphoma cell line (JeKo-1) and a pharmacokinetic (PK) study in cynomolgus monkeys, we developed a semi-mechanistic PK/receptor occupancy model for the tumor microenvironment and simulated effective doses of novel anti-TNFR2 antagonist antibodies for humans. Results TNFR2 antagonistic signaling was best when the antibody stabilized an anti-parallel dimer of two adjacent TNFR2 proteins on the cell surface. Studies in culture showed a bell-shaped, rather than sigmoidal, dose-response curve. Thus, maximal activity occurred at doses far below the MTD. Dose modeling in monkeys confirmed that the antibody dose-response followed a bell-shaped curve. Due to lower peak-to-trough ratio, a subcutaneous (SC) route was found more effective than an intravenous (IV) route at reaching the desired cell surface bicomplexes. Conclusion These findings define the complex biology of bivalent antibodies and help explain a vast literature on failed cancer antibody clinical trials. For such antibodies, modeling of cell surface clustering antibodies predicts potency with low doses and SC administration. A low dose of antibodies, delivered subcutaneously, allows for consistent, optimal concentration of drug with a low peak-to-trough ratio that maximizes bicomplex formation, and is therefore recommended over higher-dose IV administration. Funding Source n/a Topic Categories Translational and Interventional Immunology (TI)

Therapeutic antigen-specific PLGA nanoparticles tolerance induces detectable changes in the spleen, blood, and disease-associate tissue 2260904

Abstract Introduction Autoreactive T cell infiltration, tissue destruction, and spread epitope-specific T cell activation underly CD4+ T cell-mediated autoimmune disease pathogenesis. Antigen (Ag)-containing biodegradable poly(lactide-co-glycolide) nanoparticle, i.e. tolerogenic immune-modifying particles/Cour Nanoparticles (TIMP/CNPs), treatment is both safe and efficacious for Ag-specific tolerance induction in mice and Phase I/IIa clinical trials in celiac disease and primary biliary cholangitis. Methods The present studies utilized the transfer of congenically marked TCR transgenic T cells (5B6 T cells) into wildtype SJL mice. These recipient mice were primed with PLP139-151/CFA and followed for disease. At the peak of acute disease, the mice were treated with either unloaded CNP or CNP-PLP139-151. At various time points post treatment, spleen, liver, CNS, and blood were collected to quantify and phenotype the 5B6 T cells. Results In continuing to identify the mechanism by which Ag-specific CNP treatment induces tolerance, the present data show that cDC2s are the APCs that present the CNP-associated Ag in a tolerogenic manner to Ag-specific CD4+ T cells. Additionally, we identify that therapeutic treatment with Ag-specific CNP significantly decreases the number of effector CD4+ T cells within the disease-associate tissue, while increasing the ratio of Ag-specific CD4+ regulatory cells (Tregs) to IFN-γ+ cells. Consequently, treatment induced an increase in the number of Ag-specific Tregs, anergic CD4+ T cells, and the ratio of Ag-specific Treg to IFN-γ+ cells present within the spleen and blood. Additionally, we have identified that the percentage of both Ag-specific and host-derived Tregs increased within the blood post Ag-specific CNP dosing. Conclusion Therefore, these data are the first to show treatment-induced increases in Ag-specific CD4+ T cells post dosing and cellular alterations across treatment relevant tissues. Funding Source This work was supported by grants to S.D.M. from Cour Pharmaceutical Development Company, NIH grant R01AI148076; and by gifts from the Johnnie Walkers MS Foundation, the Amy and David Fulton Foundation, the Crammer Family Foundation, the Thomas and Deige McLaughlin Foundation, and the Rottering Family Foundation. Topic Categories Therapeutic Approaches to Autoimmunity (THER)

Sex difference in viral reservoir and intestinal immune responses during simian HIV infection and antiretroviral therapy in rhesus macaques 2308802

Abstract Introduction Despite effective viral suppression by antiretroviral therapy (ART), HIV persists in tissues, particularly in gut-associated lymphoid tissue (GALT). Previously, we showed sex difference in rectal immune responses and disease outcomes in rhesus macaques (RMs) following intrarectal SHIV-C109 challenge. Here, we examined viral burden and intestinal immune responses during infection and ART in RMs with intravenous (iv) SHIV infection. Methods Sixteen adult RMs (8 males, 8 females) were infected intravenously with SHIV-C109P4 (1 × 106 TCID50). Blood, rectal, and jejunal biopsies were collected before and during acute, chronic infection, and ART. Gene expression was assessed by RNA-seq. Viral RNA was detected by smFISH. Rectal T cells and neutrophils were quantified by immunofluorescence using anti-CD3 Ab and myeloperoxidase staining and analyzed with QuPath. Results Rectal viral RNA was higher in males during chronic infection on ART. Immune responses were tissue- and stage-specific: acute infection featured type I interferon signaling in the jejunum and combined type I/II interferon and remodeling pathways in the rectum, while chronic infection was marked by neutrophil-associated pathways in both tissues. ART revealed divergent responses, with wound healing and oxidative stress in rectal tissue and enhanced protein translation in the jejunum. Sex-specific mucosal responses were minimal during acute infection after iv challenge but emerged during chronic infection and ART. Rectal T cell loss was similar in both sexes, while neutrophil dynamics differed, higher in females during acute infection and in males during chronic infection on ART. Overall, sex differences were most pronounced during chronic infection with ART. Conclusion Our findings demonstrate sex-dependent regulation of intestinal immunity and viral persistence during chronic SHIV infection and ART, with potential implications for long-term disease outcomes. Funding Source NIH Topic Categories Viral Immunology (VIR)

Antibody-Coating of Vaginal Bacteria in Women with Cervical Squamous Intraepithelial Lesions 2257486

Abstract Introduction A healthy human vaginal microbiome is characterized by low microbial diversity and is dominated by Lactobacillus spp. Notably, a Lactobacillus-dominant vaginal microbiome has been associated with reduced risk of human papillomavirus (HPV) infection and persistence. Although HPV infections are prevalent, it is unclear why some high-risk HPV-infected individuals develop squamous intraepithelial lesions (SIL) and cervical cancer. A clinical study revealed that the proportion of Lactobacillus inversely correlated with SIL grade, suggesting that the vaginal microbiome may influence epithelial oncogenesis. Antibody-coating of bacteria plays an important role in regulating microbial composition and activity. In the vagina, L. crispatus-dominant microbiomes are associated with higher levels of IgA-coated bacteria. In this study, we aim to define the associations between the proportion and composition of antibody-coated and uncoated bacteria in women with low-grade SIL (L-SIL) and high-grade SIL (H-SIL). Methods We utilized fluorescence-activated cell sorting to sort vaginal bacteria from women with L-SIL or H-SIL into IgA+IgG-, IgA-IgG+, IgA-IgG-, and IgA+IgG+ subsets. Additionally, we visualized antibody-coated vaginal bacteria with a spectral flow cytometer. Results The proportion of antibody-coated bacteria subsets described above did not differ between women with HSIL and LSIL. However, gating on IgG+ and total IgA+ populations revealed that women with LSIL have significantly higher levels of IgG-coated bacteria than women with HSIL. Additionally, we found that IgA and IgG exhibit distinct and non-overlapping patterns on vaginal bacteria. Conclusion The reduced IgG coating observed in HSIL may reflect the underlying epithelial disruption and dysbiosis associated with advanced lesion progression. The divergent IgG and IgA coating patterns potentially reflect differences in epitope recognition. Future work will use 16S rRNA sequencing to identify bacterial taxa associated with IgA and IgG binding. Funding Source The National Cancer Institute (Award: 1R21CA289927-01, S. Langel) and Case Comprehensive Cancer Center Research Scholar Award (YG Jiang) funded by VeloSano. Topic Categories Mucosal and Regional Immunology (MUC)

Treatment Effect of CD40 Inhibition in Combination with Duoconjugates in a Mouse Model of Myasthenia Gravis 2306252

Abstract Introduction Muscle weakness and fatigue in autoimmune myasthenia gravis (MG) result from neuromuscular junction damage caused by autoantibody- and complement-mediated destruction of the acetylcholine receptor (AChR). In our ongoing efforts to develop a new and effective treatment for MG, we evaluated the effect of CD40 inhibition combined with duoconjugate treatments (BAFF receptor and B-cell maturation antigen-specific) in a mouse model of MG. Methods Conjugates consisted of target-specific siRNA linked to monoclonal antibodies that target pathogenic plasma cells and mature B cells. We evaluated this treatment using a Torpedo AChR/Complete Freund’s Adjuvant-immunized mouse model of MG. Results Our research shows that conjugate treatment disrupts pathogenic B cells, reducing autoantibody levels and improving clinical outcomes. Additionally, combining CD40 with duoconjugates boosts treatment effectiveness, but only with specific combinations, not when used alone. Conclusion Our results suggest that CD40 inhibition, whether alone or in combination with other treatments, may not be effective for treating myasthenia gravis or other antibody-mediated autoimmune disorders. Funding Source AFM Telethon Topic Categories Therapeutic Approaches to Autoimmunity (THER)

Phase Engineering of Iridium Oxides Enables Direct Coupling of Proton Exchange Membrane Water Electrolysis With Intermittent Electrical Energy

ABSTRACT Proton exchange membrane water electrolysis (PEMWE) coupled with intermittent renewables is a leading technology for green hydrogen production, but its large‐scale deployment is impeded by the sluggish kinetics and high iridium cost of the anodic oxygen evolution reaction (OER). Crystal phase regulation offers a rational approach to enhance catalyst intrinsic activity, yet a clear phase‐activity correlation for IrO 2 under realistic PEMWE conditions remains lacking. Here, we synthesize four crystalline phases of iridium oxide (metastable 1T‐, 3R‐, Tri‐, and conventional Rutile‐IrO 2 ) and demonstrate a strict phase‐dependent OER activity trend: 1T‐IrO 2 > 3R‐IrO 2 > Tri‐IrO 2 > Rutile‐IrO 2 . The 1T‐IrO 2 catalyst achieves a PEMWE performance of 3 A cm −2 at only 1.75 V with an Ir loading of 0.4 mg Ir cm −2 , exceeding the U.S. DOE 2026 target. It also shows stable operation for 2000 h at 2 A cm −2 and maintains durability during 1000 h of dynamic current cycling. In situ XANES/EXAFS analyses link the enhanced activity to a higher Ir oxidation state, while in situ Raman spectroscopy identifies the reaction pathway through characteristic Ir‐*OH, Ir‐*O, and Ir‐*OOH intermediates. This work establishes a direct phase‐activity relationship for IrO 2 catalysts and highlights the promise of phase engineering for efficient energy conversion.

Upcycling of Commodity Thermoset Polyurethane Towards Smart Materials with End‐User‐On‐Demand Programmable Properties and Adaptable Applications

ABSTRACT Polyurethane is indispensable to our life, but its end‐of‐life wastes bring severe environmental problems due to the bulky volume and thermoset nature. The regenerated products based on current recycling strategies normally have fixed mechanical properties, limiting adaptability to diverse applications. Here, we present a chemical recycling strategy that can transform polyurethane waste into smart materials with end‐user‐on‐demand programmable properties and adaptable applications. It is achieved through inserting a thermal‐triggered network topology transformation switch in the polyurethane. The network topology transformation switch endows the regenerated materials with on‐demand mechanical programmability, which could achieve a 54‐fold modulus enhancement from 5 MPa (soft rubber) to 270 MPa (rigid/semi‐rigid plastic). More importantly, the thermal‐triggered feature empowers end‐users not only to tailor material stiffness post‐production, but also to benefit from enhanced portability, functional adaptability for multi‐scenario applications. Our strategy of recycling waste into products with programmable properties and adaptable applications is not only environmentally friendly but also highly appealing to the end‐users, providing a promising direction in the future reuse of plastic waste.

Saturated cardiolipins are potent disruptors of inner mitochondrial membrane structure and function

Whole-genome sequencing data confirms the uniqueness of Ireland’s native honey bee

Abstract Recent evidence suggests a rise in the importation of non-native honey bee subspecies into Ireland with an associated potential rise in hybridisation followed by introgression. The purity of the Apis mellifera mellifera (A. m. mellifera) population in Ireland, given its decline elsewhere in Europe, makes the Irish population a very important biodiversity, and food security, resource that will also play an important role in conserving the genetic diversity of A. m. mellifera . To investigate if the Irish A. m. mellifera population can be distinguished from other European populations and to assess the introgression levels within Irish honey bees, we sampled 30 worker bees from each of 153 honey bee colonies for a pool-sequencing analysis. The assembled genomic data, also including pool sequencing data from other European studies, allowed us to confirm that pure Irish A. m. mellifera colonies are distinct from other A. m. mellifera populations and that introgression has become a serious issue.

Filament formation drives activation of NLR-like proteins 2249724

Abstract Introduction The nucleotide-binding domain (NBD) and leucine-rich repeat (LRR)-containing protein (NLR) family plays a key role in immune defense across domains of life. Prior studies on inflammasomes and resistosomes revealed that NLRs tend to form ring-like structure for activation. Here, we focused on a unique family of NLR called NLR-SIR2 that comprises SIR2, NBD, and TPR domains, uncovering a filamentous assembly. This discovery has broad implication in the activation of many other NLRs, including NLRC4 and NLRP6. Methods We determined Cryo-EM structures of NLR-SIR2 in three states and combined them with biochemical and cellular assays to link structures with functions. Results NLR-SIR2 adopts a closed monomer in the resting state. At higher concentration it forms a symmetric dimer via SIR2—SIR2 contacts without major domain rearrangement. Incubation at 37 °C promotes concentration-dependent filament formation driven by SIR2-mediated interfaces, in contrast to the NBD-mediated oligomerization observed for other NLRs. ATP hydrolysis accompanies the transition from apo to filament, indicating a regulated assembly step. Filament formation is required for robust NAD+ hydrolysis, which is essential for anti-phage defense in cells. Thus, NLR-SIR2 activation proceeds through SIR2-guided polymerization that licenses NAD+ cleavage. Conclusion NLR-SIR2 reveals an alternative NLR activation pathway in which oligomerization is mediated by SIR2 domain rather than NBD. Coupling of ATP turnover, filament formation, and NAD+ hydrolysis provides a mechanistic framework for NLR immunity and suggests conserved yet diversified assembly logics across NLRs. Funding Source N/A Topic Categories Innate Immune Responses and Host Defense: Molecular Mechanisms (INM)

Spatial Transcriptomics to Study Tissue-Specific Pathways Linked to Secondary Anti-TNF Resistance in Crohn’s Disease 2307998

Abstract Introduction Crohn’s disease is a chronic inflammatory disorder of the gastrointestinal tract affecting millions worldwide. TNF-α—blocking antibodies are widely used to control inflammation and induce remission; however, more than half of patients either fail to respond initially or develop secondary loss of response. While anti-drug antibodies (ADA) account for a subset of cases, some patients develop treatment resistance through mechanisms that remain poorly understood and cannot be overcome by dose escalation. Methods To investigate these mechanisms, we assembled a cohort of archival intestinal biopsies from patients with either ADA-negative secondary loss of response or sustained response to TNF-α inhibitors. Samples were incorporated into tissue microarrays and analyzed using Xenium spatial transcriptomics with a customized panel of intestinal and inflammation-associated genes. The same tissue sections were subsequently profiled by high-plex immunofluorescence using PhenoCycler staining with a panel of over 20 markers. Results Integrated spatial transcriptomic and proteomic analyses identified inflammation-associated cell populations and molecular pathways linked to secondary loss of response in the ileum and colon. Conclusion These signatures suggest pharmacodynamic mechanisms underlying ADA-negative treatment failure and highlight candidate prognostic biomarkers for future loss of response. Funding Source The Leona M. and Harry B. Helmsley Charitable Trust Topic Categories Mucosal and Regional Immunology (MUC)

Therapeutic Targeting of the IL-22/IL-22Rα Axis to Suppress Tumor Proliferation in Gynecologic Cancers 2330046

Abstract Introduction This study explores the potential of Interleukin-22 receptor alpha (IL-22Rα) as a therapeutic target for gynecologic cancer. Gynecologic cancers, including cervical cancer and ovarian cancer, are closely associated with chronic inflammation. Interleukin (IL)-22, a pro-inflammatory cytokine involved in epithelial regeneration, has emerged as a critical mediator linking inflammatory signaling to oncogenic processes. Methods We investigated the functional role of IL-22/IL-22Rα signaling in cervical and ovarian cancer models by CCK-8 assay, cell cycle analysis, and Western blotting. Furthermore, IL-22Rα–knockdown cell lines were established to determine whether the proliferative effects were specifically mediated through IL-22/IL-22Rα signaling. Results IL-22 stimulation enhanced proliferation of cervical cancer cell lines (CaSki and HeLa) and ovarian cancer cell lines (SK-OV-3 and NIH:OVCAR-3) by promoting S and G2/M phase progression. This effect was accompanied by upregulation of cell cycle–associated cyclins, including A, B1, D1, and E1, without distinct changes in cyclin–dependent kinase expressions. Genetic suppression of IL-22Rα abrogated IL-22–induced signaling activation, cell cycle progression, and proliferation in vitro. Consistently, IL-22Rα suppression markedly inhibited IL-22–driven tumor growth in cervical cancer and ovarian cancer xenograft models. Conclusion Our findings identify the IL-22/IL-22Rα axis as a key inflammation–driven regulator of tumor proliferation in gynecologic cancers and reveal the potential therapeutic target of IL-22Rα. Funding Source n/a Topic Categories Tumor Immunology: Cellular Responses and Tumor Microevironment (TIME)

Siglec-F+ neutrophils are pro-inflammatory and exacerbate C. difficile colitis 2257588

Abstract Introduction Neutrophils exhibit a dual role in C. difficile infection (CDI), contributing to tissue homeostasis by containing pathogens while potentially causing tissue damage through excessive inflammatory mediator release. We identified a unique population of neutrophils expressing the eosinophil marker Siglec-F that rapidly accumulates in the colon during CDI. While Siglec-F+ neutrophils have been observed in other disease contexts, their specific role in CDI pathogenesis remains unclear. Methods We used CDI mouse models to characterize colon-infiltrating neutrophils. Flow cytometry and staining confirmed Siglec-F expression on mature neutrophils, distinguishing them from SiglecFhigh eosinophils. Functional depletion employed anti-Siglec-F antibodies in eosinophil-deficient mice. RNA sequencing compared gene expression profiles to identify differences. Further metabolic studies were done by using a Flow cytometry-based assay, SCENITH. Results We verified that Siglec-F+ cells are bona fide mature neutrophils distinct from eosinophils and other myeloid populations. These neutrophils demonstrated significant metabolic alterations (Glycolytic capacity, 5.153776494 %, 66.72 %) compared to conventional neutrophils. Depletion of Siglec-F+ neutrophils using anti-Siglec-F antibodies reduced CDI (p = 0.049) in eosinophil-deficient mice. RNA sequencing revealed that Siglec-F+ neutrophils exhibit enhanced phagocytic activity, increased extracellular trap formation, and elevated production of proinflammatory TH1-associated genes. Conclusion Siglec-F+ neutrophils represent a metabolically distinct neutrophil subset that contributes to CDI pathogenesis through enhanced inflammatory functions. Their depletion ameliorates disease severity, suggesting these cells play a detrimental role in CDI. This population may serve as a therapeutic target for managing severe CDI, as their unique metabolic profile and enhanced inflammatory capacity distinguish them from protective conventional neutrophils in the infected colon. Funding Source n/a Topic Categories Innate Immune Responses and Host Defense: Cellular Mechanisms (INC)

Spatially resolved gene programs and cell neighborhoods of scleroderma fibroblasts 2261024

Abstract Introduction Systemic sclerosis (SSc, scleroderma) is a chronic autoimmune disease with high mortality, characterized by progressive fibrosis of the skin and internal organs. Skin involvement is among the earliest manifestations of disease, and changes in skin pathology can mirror systemic progression. While single-cell studies have revealed striking transcriptional diversity among dermal fibroblasts in SSc, the spatial organization and immune signals that govern their function remain unclear. Methods To address this, we performed high-resolution spatial transcriptomic profiling of clinically affected and unaffected skin from SSc donors alongside healthy controls. Results We identify known disease-associated fibroblast subsets, including COMP+ myofibroblasts, which localize within cell neighborhoods enriched in activated endothelial and immune cells. In contrast, LGR5+/PI16+ fibroblasts — abundant in healthy skin and previously shown to contain a T cell-inducible gene program — are significantly reduced in both clinically involved and uninvolved SSc skin and occupy distinct microenvironments from disease-associated cells. Conclusion Our findings provide a spatial framework for understanding fibroblast heterogeneity and identify potential therapeutic strategies aimed at reinforcing protective fibroblast programs to prevent or delay fibrosis in scleroderma. Funding Source R21AI185642 NIAID/NIH, National Scleroderma Foundation Topic Categories Immune Mechanisms of Human Disease (HUM)

Involvement of ICOS and ICOSL System in Platelet Function 2327673

Abstract Introduction Platelets are non-nucleated, megakaryocyte-derived cell fragments involved in hemostasis and thrombosis. Beyond this activity, they express T cell co-stimulatory/co-inhibitory molecules, including CD40L, CD86, and PD-L1. ICOS and ICOSL are T-cell co-stimulatory molecules that belong to the CD28 and B7 families, respectively. Recent studies show that the ICOS-ICOSL axis also influences non-immune processes including wound healing and endothelial cell function, but its role in platelet biology is poorly defined. This study aimed to evaluate the role of ICOS and ICOSL in platelets formation and function in mice. Methods ADP-induced platelet aggregation was assessed by light transmission aggregometry using platelet-rich plasma from wild-type (WT) mice and mice deficient for ICOS (ICOS⁻/⁻) or ICOSL (ICOSL⁻/⁻). Then, platelet activation induced by thrombin (0.05 and 0.1u/mL) was assessed by staining the activation markers CD62P and GPIIb/IIIa and flow cytometry analysis. Differentiation of primary bone marrow megakaryocytes was evaluated by DNA ploidy analysis by flow cytometry. Results Platelets from ICOS-/- or ICOSL-/- mice exhibited significantly impaired aggregation compared to that detected in WT mice. Thrombin fully activated wild-type platelets at both 0.1 and 0.05 U/ml, whereas platelets from ICOS-/- and ICOSL-/- mice were fully activated by 0.1 U/ml thrombin only. ICOSL-/- mice displayed a reduction in the 2N and 4N megakaryocyte populations compared to WT (p < 0.05), indicating impaired megakaryocyte maturation, whereas maturation was normal in ICOS-/- mice. Primary mouse megakaryocytes were found to express both ICOS and ICOSL in wild-type mice, as determined by confocal microscopy. Conclusion These findings show that ICOS and ICOSL are involved in platelet function and early megakaryocyte development. The mechanisms of this activity deserve further investigation. Funding Source this project is funded by AIRC -Italian Association for Cancer Research Topic Categories Immune Response Regulation: Molecular Mechanisms (IRM)