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DR3 Stimulation Expands Functionally Suppressive Tregs During EAU 2257177
Abstract Introduction Autoimmune uveitis (AU) is a potentially blinding ocular inflammatory disorder that can be modeled in mice (Experimental Autoimmune Uveitis). While AU requires chronic treatment to preserve vision and prevent relapse, EAU mice do not spontaneously relapse. We previously identified Tregs that are induced during EAU in an adenosine 2A receptor (A2aR)-dependent manner and protect against EAU relapse. Of note, we also found A2aR stimulation failed to induce Tregs in uveitis patients. Following reports of death receptor 3 (DR3)-mediated Treg expansion in other inflammatory models, we asked if DR3 sustains A2aR-dependent Tregs in EAU. Methods Wildtype or A2aR-/- EAU mice were treated with a DR3 agonist (4C12) at peak disease and disease severity was monitored by Fundus exam until resolution. Separately, DR3+ Tregs in post-EAU eyes of CD4-CreERT2; A2aRfl/fl EAU mice were quantified by flow cytometry. The role of DR3 in Treg survival was assessed by treating ocular antigen-primed T cells with 4C12 in vitro and identifying surviving cells by flow cytometry. The function of DR3-expanded cells was assessed by isolating and adoptively transferring primed T cells from 4C12 or vehicle-treated mice that were immunized with ocular antigen. Results We demonstrate that DR3 agonism during peak EAU reduces disease severity in A2aR-/- mice, and show a CD4 T cell-intrinsic dependence on A2aR for the accumulation of DR3+ Tregs in the eye at peak EAU. Additionally, we find that in vitro and in vivo DR3 stimulation promotes the accumulation of functionally suppressive Tregs. Conclusion Taken together, our findings identify a role for DR3 in promoting Treg accumulation during EAU and provide mechanistic insight into how A2aR-dependent Tregs are maintained. Funding Source n/a Topic Categories Mucosal and Regional Immunology (MUC)
Spatial transcriptome mapping to decode Cannabinoid-mediated neuroprotection in HIV-1 gp120—induced degenerating mouse brain 2255864
Abstract Introduction HIV-associated neurocognitive disorders (HAND) remain a major complication in AIDS patients and are thought to arise from chronic neuroinflammation. Our previous work established that Δ9-tetrahydrocannabinol (THC) exerts neuroprotective effects in HIV gp120 transgenic (Tg) mice by downregulating gp120 expression and reducing astrocytic activation. Methods In this study, we employed spatial transcriptomics to map the neuroimmune landscape of the gp120 Tg brain and to define the regional effects of THC treatment. Results Soluble gp120 protein was distributed throughout the hippocampus, corpus callosum, cortex, and thalamus, coinciding with increased reactive astrocytes–an effect markedly diminished by THC. While reactive microglia were evenly distributed across brain regions, THC treatment enhanced oligodendrocyte populations and induced IL-33 expression in the corpus callosum and stria. Notably, THC increased excitatory neurons in the cortex and thalamus but decreased inhibitory neurons primarily in the hypothalamus, suggesting restoration of neural balance underlying cognition and memory. T cells and dendritic cells localized around the subfornical organ and lateral ventricles decreased after THC exposure. Conclusion These spatial data align with our prior single-cell RNA-seq findings, collectively revealing that THC modulates the neuroimmune and cellular architecture of the HIV-compromised brain. Our findings highlight spatial transcriptomics as a powerful approach to uncover region-specific neuroimmune remodeling and support the therapeutic potential of cannabinoids in HAND. Funding Source Supported by NIH grants P20GM103641, P30GM154631, R01ES030144, and R01AI160896 Topic Categories Neuroimmunology (NEUR)
Thermal‐Responsive Self‐Assembly of Organic Crystals With Asymmetric Architectures
ABSTRACT The miniaturization and integration of electronic/photonic devices demand precise control over light at the micro‐scale. However, achieving tailored optical anisotropy through intrinsic material design, rather than external components, remains a significant challenge. Herein, we report a general and programmable strategy for the growth of one‐dimensional organic crystals with precisely tunable asymmetric architectures via a spatially defined temperature gradient. By leveraging the competitive, facet‐dependent growth kinetics under a thermal bias, continuous and precise control over the structural asymmetry is achieved in single crystals, with a tunable morphological anisotropy ranging from 9% to 81%. The resulting asymmetric crystals exhibit a pronounced direction‐dependent optical response, yielding a photoluminescence intensity contrast ratio as high as 113.3, which scales directly with the degree of structural asymmetry. This work establishes a material‐based platform for applying direction‐dependent photonic properties directly into crystal morphology, paving the way for advanced organic photonic materials with built‐in anisotropy.
Gut microbiota modulation potentiates anti-tumor immunity and immunotherapy in hepatocellular carcinoma 2308568
Abstract Introduction The gut microbiota functions as a critical extrinsic regulator along the gut-liver axis, shaping hepatocarcinogenesis and anti-tumor immunity and therapy. However, the specific microbial determinants and mechanisms underlying these effects remain incompletely defined. Methods Motivated by the observed impact of gut microbiota on anti-PD-1 therapeutic efficacy in hepatocellular carcinoma (HCC) patients, we employed our unique and clinically relevant mouse HCC models to comprehensively characterize tumor-associated microbial signatures using 16S rRNA gene sequencing. Application of a non-hepatotoxic antibiotic cocktail 3 (ABX-3), targeted microbial supplementation, and recolonization with selected bacterial species following gut sterilization with ABX-5 enabled us to delineate the causal relationship between microbiota modulation, intrahepatic immune activation, and immunotherapeutic responses. Results Bacteroides-enriched gut microbiota derived from anti-PD-1 responder HCC patients significantly suppressed HCC growth in our mouse model. In parallel, ABX-3 administration, implemented as both a preventive and therapeutic intervention, attenuated tumor initiation and progression by selectively enriching specific gut bacterial taxa. Functionally, ABX-3 enhanced tumor antigen-specific T cell activation and boosted intrahepatic anti-tumor immunity via microbial remodeling. Among enriched taxa, Bacteroides thetaiotaomicron (B.th) emerged as a key microbial effector driving these immunomodulatory and anti-tumor effects. These findings prompted further investigation into additional enriched microbial members with the capacity to activate hepatic immunity and potentiate immunotherapy either individually or combination with B.th. Conclusion ABX-3-mediated modulation of gut microbiota promotes robust intrahepatic anti-HCC immune activation. Identifying microbial species with ability to enhance hepatic immune responses provides a foundation for developing microbiome-based immunotherapeutic strategy for HCC. Funding Source National Institutes of Health (NIH) R01CA274959, R01CA250536, R01 DK130340, R01CA208396; partly supported by VA merit Award I01 BX004065-1. Topic Categories Tumor Immunology: Cellular Responses and Tumor Microevironment (TIME)
Comparative Analysis of Nasopharyngeal Mast Cells Across Pediatric Viral Infections 2309423
Abstract Introduction Mast cells are long-lived, myeloid-lineage immune cells enriched at mucosal surfaces, where they reside to sense and respond to foreign antigens. While well studied in allergic disease, their roles in viral immunity remain incompletely defined. By defining the cellular states of mast cells in response to rhinovirus (RV), respiratory syncytial virus (RSV), and SARS-CoV-2, we aimed to evaluate the hypothesis that mast cells in the nasopharyngeal mucosa adopt virus-specific inflammatory states during pediatric upper respiratory tract infection (URTI). Methods Nasopharyngeal swabs were collected from pediatric patients infected with RV, RSV, or SARS-CoV-2, and uninfected controls (UC) at Boston Children’s Hospital. Single-cell RNA sequencing (10X Genomics) was performed on swab-dissociated cell suspensions, followed by integrated analysis to identify mast cell subsets and compare virus-associated transcriptional programs. Results 684 mast cells were isolated from a dataset of approximately 290,000 cells from 97 participants. Unsupervised clustering revealed four distinct mast cell states: 1) Resting, 2) Inflammatory, 3) Interferon-stimulated gene-positive (ISG+, TNF-), and 4) ISG+, TNF+. Across all viral infections, mast cell frequency was increased; however, discrete cell states displayed virus-specific associations. SARS-CoV-2 mast cells were predominantly defined by the Inflammatory state; RV by the ISG+, TNF- state; RSV by the ISG+, TNF+ state; and UCs by the Resting state. Gene-set enrichment analysis reflected these virus-specific differences. Conclusion These data suggest that mast cells mount unique, virus-specific inflammatory responses in the nasopharyngeal mucosa during pediatric viral URTI and thus may be a future target for modulation of virus-specific mucosal inflammation in children. Funding Source NIH R01 DE031928 Topic Categories Mucosal and Regional Immunology (MUC)
Neuregulin-1 Prevents RSV Induced Disruption of Human Airway Epithelium Polarity 2257854
Abstract Introduction Respiratory viruses cause significant morbidity and mortality in very young and old. Mechanism of mortality to respiratory viruses are unknown. We have studied Neuregulin-1 (NRG1), a ligand for ERBb receptor tyrosine kinases (ERBb2 -4) and found pre-treatment with NRG1 provides ∼75% survival for mice infected with normally lethal doses of influenza A or Sendai virus (a rodent para-influenza virus). Administering NRG1 to human bronchial epithelial cells (hBEC) increased epithelial proliferation and repair following infection with RSV. Epithelial barrier function closely links to epithelial polarity; polarized epithelial cells have an apico-basal axis maintained through 3 polarity complexes: PAR (Par3/Par6/aPKC) and CRB (Crumbs/Pals/Patj), apically, and SCRIB (Scrib/Dlg1/Lgl), basolaterally. Reactive oxygen species (ROS) play a role in airway epithelium permeability also affecting polarity. The current study determined the effect of NRG1 on polarity when given prophylactically before RSV infection of hBEC. Methods hBEC grown in transwell were pretreated on the basal side for 5 days with human NRG1 (100 ng) before RSV-gfp (4000 pfu) was added to the apical side. Anti-ERBb2 (10mcg) and anti-ERBb3 (0.1 mcg) antibodies were added for 5 days to the basal side. Immunohistochemistry was performed on paraffin sections for polarity markers (CRB3 and DLG1) d5 post infection. In addition, hBEC were treated for 30 mins at 37oC in CO2 incubator with MitoSOX superoxide indicators to detect mitochondrial superoxide production in live cells. Results NRG1 prevented disruption of CRB3 localization by RSV, an effect blocked by anti-ERBb3. Blocking ERBb2 reduced epithelial proliferation (something ERBb3 antagonism did not do), but did not affect localization of polarity proteins. NRG1 treatment reduced mitoROS in RSV infected cells. Conclusion NRG1 prevents disruption of airway epithelium apico-basal polarity and reduces excessive ROS thus stabilizing hBEC during infection. Funding Source This work was supported by National Institutes of Health (NIH) grants R01AI171027 (to Mitchell H Grayson (MHG)); R01 AI093848 and U19AI42733 (to Mark E Peeples)), the Robert F Wolfe & Edgar T Wolfe Foundation (to MHG). Topic Categories Viral Immunology (VIR)
Diverse Microbial Exposure Enhances CD8+ T Cell Effector Memory Output and Function 2310008
Abstract Introduction Mice with normalized microbial exposure (NME) harbor an immune system that more accurately reflects that of humans compared to mice maintained in specific pathogen-free (SPF) facilities. When comparing the immune compartment of NME mice to SPF controls, major shifts are observed in many different immune cell subsets, including expansion of activated T cells. A mechanism for the observed alterations in the composition of the T cell compartment in NME mice has not been reported. Thus, in collaboration with immgenT, we set out to profile the T cell compartment of NME mice on a single cell level. Methods Here, we compared the splenic T cell landscape in NME versus SPF mice at baseline and after acute LCMV infection via single cell RNA sequencing. Additionally, we compared immune cell populations from the blood of mice with increasing levels of NME conversion. We then confirmed the changes we saw in our sequencing data with flow cytometry and adoptive transfers. Results Using the immgenT dataset as a reference, we found that there are no new T cell populations in NME, but the landscape shifts towards more activated T cells with increased propensity for effector functions and improved pathogen clearance capacity. In particular, CD8+ KLRG1+ long-lived effector T cells are significantly expanded in NME mice and showed increased cytokine production. The shift to long lived effector T cells in NME mice is a result of increased formation and the conversion of other memory populations. Conclusion By leveraging the immgenT atlas we characterized T cells from the spleen and blood of NME mice, providing insight into the compositional and functional differences between SPF and NME mouse T cell compartments. We found improved LLEC function and pathogen clearance by NME-derived T cells. Finally, we showed that the numeric shifts in T cell populations in NME are likely to be promoted by continued inflammatory cues rather than only antigenic stimulation. Funding Source n/a Topic Categories Lymphocyte Differentiation and Peripheral Maintenance (LYM)
Development of a sensitive and specific assay to detect the presence of immune complex in human serum 2253742
Abstract Introduction In several autoimmune diseases, such as systemic lupus erythematosus (SLE), glomerulonephritis or vasculitis, autoantibodies recognize and bind self-protein, forming immune complexes (IC). IC deposition or improper clearance can lead to inflammation and tissue damage. Here, we describe the development of a plate-based assay to detect presence of IC in serum samples. Methods Employing a FcgRIIb reporter cell line, we observed that IC formed in vitro in assay media induced signal over antigen or antibody alone. However, in vitro formed IC spiked in serum completely abrogate luciferase signal. This effect was caused by an unknown serum component and was independent of IgG levels, since similar results were obtained by spiking IC into IgG depleted serum. To optimize this assay, we formed IC, spiked it into neat or multiple serum dilutions in assay media and conclude that a dilution factor of 1000 is required to overcome matrix interference. Due to the matrix interference, we developed a more sensitive assay that could detect IC formation in a more concentrated serum. Results Therefore, we developed a plate-based assay using streptavidin coated plates with biotinylated recombinant FcgRIIb to capture IC and a sulfo-tagged anti-light chain antibody to detect. We observed in vitro formed IC spiked into healthy serum, IgG depleted serum or assay buffer is detected with no matrix interference and the signal to background (antibody or antigen alone) of 13. Conclusion All together, we describe an assay that specifically detects immune complexed IgG that can be used to screen the presence of IC in serum from patients. Funding Source n/a Topic Categories Basic Autoimmunity (BA)
Effect of small molecule inhibitors of IRF3 and TBK1 signaling pathways on ovarian cancer cells 2257743
Abstract Introduction Ovarian cancer (OvCa) is a highly lethal gynecologic cancer, often due to late diagnosis and therapy resistance. High-grade serous ovarian cancer (HGSOC) is the most prevalent and deadly subtype. In OvCa, the tumor microenvironment is highly immunosuppressive, which aids in the immune system evasion of cancer cells. The TBK1-IRF3 signaling pathway is relevant in the innate immune response, especially against viruses. Upon activation, TBK1 phosphorylates IRF3, which allows IRF3 to dimerize and move into the nucleus. There, it acts as a transcription factor to produce type I interferons, cytokines, and chemokines In tumors, depending on its level of activation, TBK1-IRF3 signaling pathway may either promote or repress malignant tumors. We are interested in the expression of this signaling pathway in tumor cells, and the possibility of targeting it as a therapeutic approach. Methods In these studies, we treated mouse (ID8 and BPPNM) and human (A2780, SKOV3) OvCa cells with inhibitors of IRF3 (BXT-795, C10 [phenylmethimazole]), TBK1(GSK-8612, MRT-67307); and TBK1 and IKKe (Amlexanox) at different concentrations. DMSO served as the vehicle control. We investigated the effect of the drugs on tumor cell viability, migration, generation of spheroids and their synergism with oncolytic herpesvirus (DNA genome) and reovirus (RNA genome). Results We observed that of all the drugs studied, BXT-795 and MRT-67307, had the highest capability to inhibit, in a dose-dependent fashion, the proliferation, migration (as determined by a scratch assay) and the size of spheroids (3D cultures) of mouse and human OvCa cells. In addition, BXT-795 was able to act synergistically with oncolytic herpesvirus and reovirus to kill human ovarian cancer cells. Conclusion These findings suggest that IRF3/TBK1 inhibition may influence metastatic potential and proliferative capacity of ovarian cancer cells and make them more prone to the action of oncolytic viruses. Funding Source Ohio University-HCOM RSAC grant RP2404 (FB) Topic Categories Tumor Immunology: Checkpoints, Prevention, and Treatment (TIPT)
Allograft Inflammatory Factor 1 expression in cDC1 is required to direct Tbet—driven T cell immunity to defend against intracellular pathogens in vivo 2256292
Abstract Introduction Allograft Inflammatory Factor‑1 (AIF1) is a calcium‑binding protein enriched in conventional dendritic cells, with selective expression in cDC1. We hypothesized that AIF1 programs cDC1 immunity to intracellular pathogens by sustaining their activation and T cell‑priming capacity. Methods To directly address, AIF1 was selectively deleted in CD11c+ cells prior to infection with the intracellular or extracellular pathogens in vivo. DC and T cell subsets were then analyzed by multiparametric flow cytometry. Additionally Single-cell RNA sequencing (scRNA-seq) under steady-state conditions was analyzed by differential expression and gene set enrichment (GSEA). Results Conditional deletion of AIF1 in DCs resulted in a modest (∼20%) reduction of cDC1, without affecting cDC2 populations, indicating a partial lineage dependency. During infection with intracellular pathogens Listeria monocytogenes and LCMV, AIF1‑deficient cDC1 showed impaired activation and defective pathogen control, leading to markedly reduced CD4+ T cell activation, loss of Th1 polarization (CD4+T‑bet+), and diminished CD8+CD54+GranzymeB+ cytotoxic responses. In contrast, infection with extracellular pathogens Helicobacter pylori and Nippostrongylus brasiliensis, eliciting Th2-biased immunity, induced no observable impairment in T cell responses. This aligns with AIF1’s unique expression within cDC1, but not cDC2. Conclusion These findings identify AIF1 as a critical regulator of cDC1 immunogenic identity and protective T cell immunity to intracellular infection, defining a pathway where AIF1 loss reprograms DC—T cell communication toward a regulatory, non-protective outcome. Funding Source National Institutes of Health (NIH) Topic Categories Classical and Non-Classical Antigen Presenting Cells (APC)
Ambient Light‐Activatable Luminescent Particle‐Embedded Conformal Patch for Photochemical Tissue Bonding and Photobiomodulated Healing
ABSTRACT Biophotonic healthcare devices have been widely investigated for photochemical tissue bonding (PTB), photobiomodulation (PBM), photodynamic therapy (PDT), and photothermal therapy (PTT). However, their clinical applications remain limited due to the short light penetration depth and the incompliant light source, including externally powered light‐emitting diodes (LEDs). Here, we present a stretchable photonic patch embedded with biocompatible room light‐ and sunlight‐activatable luminescent particles (RSLPs) in an elastomeric matrix. This patch enables ambient light‐activatable phototherapy without an external light source by absorbing broadband ambient light and converting it into red luminescence. The emission promotes PTB, fibroblast proliferation, and M1/M2 macrophage polarization for tissue regeneration, activating photosensitizers to induce collagen crosslinking and wound sealing. The conformal patch maintains optical performance even under movement, enabling continuous phototherapy during daily life activity. Remarkably, after PTB‐driven sealing, wound healing is accelerated in both linear and circular wounds, mediated by PBM‐induced tissue regeneration. A further clinical study on normal human facial skins demonstrates that a single PBM treatment with the patch acutely improves viscoelastic recovery and attenuates superficial erythema. This ambient light‐activatable biophotonic patch platform would pave a big avenue toward PTB, PBM, and various biomedical applications.
MARCO is an IFN-restricted immunometabolic decoy LPS receptor 2308885
Abstract Introduction Cytosolic sensing of bacterial lipopolysaccharide (LPS) is mediated by caspase-11 (caspase-4/5 in humans), which triggers non-canonical inflammasome activation. This leads to gasdermin D cleavage, pyroptosis, and release of pro-inflammatory cytokines. Severe gram-negative bacterial infections cause endotoxic septic shock, with systemic inflammation, organ failure, and potential death. Bacterial derived LPS gains access to the cytosol through extracellular vesicles and CD14 receptor mediated endocytosis. However, how this process is controlled during infection to maintain homeostasis is unknown. Methods We performed an unbiased proteomic screen with biotinylated LPS and identified MARCO as an LPS-binding protein by mass spectrometry. Using macrophages from transgenic mice, we defined the mechanism by which MARCO facilitates LPS clearance to limit activation of the non-canonical inflammasome. Results MARCO is a scavenger receptor expressed on tissue resident macrophages (TRMs) and is known to promote clearance of pathogens and apoptotic debris. However, there is a lack of genetic studies and a paucity in our understanding of the functional role MARCO plays in innate immunity and how MARCO expression is regulated on TRMs. Here, we show that activation of the non-canonical inflammasome is preceded by a priming step by which type I interferon (IFN) suppresses MARCO expression on TRMs to allow for optimal recognition of LPS and activation of caspase-11. Remarkably, the metabolite itaconate and transcription factor NRF2 mediate MARCO expression. IFN signaling inhibits NRF2 stabilization, reducing surface MARCO levels. In vivo, MARCO-deficient mice show increased sensitivity to LPS-induced septic shock due to enhanced caspase-11 and non-canonical inflammasome activation. Conclusion IFN-mediated suppression of MARCO expression is a previously unknown innate checkpoint that facilitates the recognition of LPS by caspase-11, activation of the non-canonical inflammasome, and progression of septic shock. Funding Source n/a Topic Categories Innate Immune Responses and Host Defense: Molecular Mechanisms (INM)
Inflammatory hematopoiesis drives intestinal inflammation and is modulated by anti-TNF therapy 2309660
Abstract Introduction Chronic inflammatory diseases often originate in local tissues yet display systemic immune abnormalities, suggesting durable changes in upstream immune regulation. Crohn’s disease (CD), while centered in the intestine, is a systemic inflammatory disorder. Hematopoietic stem and progenitor cells (HSPCs) can retain memory of inflammatory cues, leading to sustained changes in hematopoiesis and immune responsiveness. Here, we investigated how chronic intestinal inflammation in CD reprograms HSPCs and contributes to disease. Methods We analyzed a longitudinal CD cohort using paired pre- and post—anti-TNF blood samples with healthy controls. Circulating HSPCs and mature immune cells were profiled by single-nucleus RNA-seq and ATAC-seq with progenitor enrichment, enabling integrated multiomic and gene regulatory analyses. Functional relevance was assessed using bulk RNA-seq and ATAC-seq of HSPCs from a murine DSS colitis model, together with bone marrow chimera and transplantation experiments. Results HSPCs from individuals with CD exhibited loss of quiescence, metabolic activation, epigenetic priming of inflammatory and stress-responsive programs, and shifts in progenitor composition. These molecular programs were conserved in murine colitis HSPCs, with altered progenitor subtype frequencies and pathogenic hematopoiesis, as inflammation-experienced HSPCs exacerbated disease upon transplantation. Circulating monocytes in CD also shifted toward inflammatory subpopulations sharing transcriptional and regulatory features with reprogrammed HSPCs. A central TNF—NF-κB regulatory axis underpinned these alterations, and anti-TNF therapy largely normalized HSPC composition, metabolic state, and inflammatory gene regulatory networks. Conclusion Inflammatory hematopoiesis represents a durable, therapy-responsive driver of CD. HSPCs act as a systemic reservoir of inflammatory memory, while TNF blockade partially restores hematopoietic homeostasis, highlighting progenitor reprogramming as a therapeutic target. Funding Source NA Topic Categories Hematopoiesis and Immune System Development (HEM)
QS-21 Modulates Macrophage Activation Through Synergistic Crosstalk with Pattern Recognition Receptors 2258356
Abstract Introduction QS-21, a saponin-based adjuvant, is a key component of several licensed vaccines and is commonly co-formulated with monophosphoryl lipid A (MPLA) to enhance adaptive immune responses. Although its adjuvant efficacy is well established, the broader potential of QS-21 in combination with other pattern recognition receptor (PRR) agonists remains insufficiently characterized. In this work, we systematically investigated the immunomodulatory properties of QS-21 paired with five different PRR agonists: MPLA (TLR4 agonist), L18-MDP (NOD2 agonist), R848 (TLR7/8 agonist), CpG ODN (TLR9 agonist), and Poly I:C (TLR3 agonist). Methods Cytotoxicity assays were first conducted to define the lowest concentrations of QS-21 and each agonist, that were non-toxic yet capable of stimulating cells. Differentiated THP-1 macrophages were subsequently treated with individual agonists, QS-21 combinations, or MPLA combinations, and activation was assessed through surface activation marker expression and cytokine/chemokine secretion profiles. To further delineate the molecular mechanisms underlying these responses, we performed RNA sequencing and metabolomics profiling to identify distinct immunological signatures associated with each treatment. Results Our findings reveal that, although the PRR agonists exhibited variable activation capacities, the combinations of QS-21+MPLA with either Poly I:C or L18-MDP produced the strongest macrophage activation, surpassing the effects of single agents. Conclusion Collectively, these results expand the understanding of QS-21’s synergistic potential with diverse PRR agonists and suggest new avenues for adjuvant design. Notably, Poly I:C emerges as a particularly promising co-adjuvant with QS-21, capable of engaging complementary innate immune pathways to enhance vaccine-induced immunity. Funding Source NIH Topic Categories Vaccines and Immunotherapy (VAC)
Colonic Complement Factor B Production Is Required To Respond To Tissue Injury 2310076
Abstract Introduction The complement system is an evolutionarily conserved arm of immunity. Key components of this system such as C3 and Factor B are activated in inflammatory bowel disease (IBD), however coding variant in Factor B (rs4151651) is associated with severe IBD complications, thus, suggesting two sides to Factor B in IBD. First, its chronic upregulation is associated with excessive inflammation, but second its absence correlates with increased risk for developing colitis. Although these proteins are primarily derived from the liver and operative in the circulation, we and others have shown that they are active in the gut. Yet, major knowledge gaps remain regarding the production, activation and regulation of Factor B in IBD in order to develop tailored therapies for modulating complement activity in the gut. Methods We conducted transcriptomic and proteomic analyses of human and mouse colon, and generated conditional knockout mice lacking systemic (liver-derived) Factor B, and cell type-specific inducible Factor B deletion to alter colonic Factor B production. To determine contribution of tissue derived Factor B, we induced chemical-induced colitis in these models and assessed the severity of mucosal injury. Results Transcriptomic and proteomic analysis of colonic tissue from patients with ulcerative colitis reveal an increase in activators of the complement cascade, notably Factor B. Additionally, local Factor B production is upregulated in colonic structural cells such as epithelial cells and fibroblasts upon stimulation with inflammatory cytokines such as rIL-1β and rTNF-α. In vivo studies reveal that local Factor B production protects against chemical-induced colitis, independent of circulating Factor B. Conclusion These observations indicate that local Factor B production by intestinal cells is required for mucosal response to injury, and creates the precedent for its regulation locally, rather than inhibition, in IBD. Funding Source Institutional funding, foundation award Topic Categories Mucosal and Regional Immunology (MUC)
Ketogenic diet-mediated immunometabolic regulation improves airway clearance of Klebsiella pneumoniae 2253582
Abstract Introduction Multidrug-resistant Klebsiella pneumoniae (MDR Kp) is a global health threat, causing persistent, subacute airway infections that are often fatal in healthcare settings. The failure of antimicrobials underscores an urgent need for alternative host-targeted therapies. Kp evades airway immune responses by perturbing host metabolism, inducing mitochondrial oxidative phosphorylation (OXPHOS) and oxidative stress, thus establishing a disease-tolerant microenvironment. We hypothesized that a ketogenic diet (KD), which improves OXPHOS efficiency via ketone production, would restore immune control of Kp by enhancing the bioenergetics and function of innate immune cells. Methods C57BL/6 mice were fed a KD or control diet prior to intranasal Kp infection. Airway bacterial burden was enumerated and immunometabolic changes were assessed by scRNA-seq, flow cytometry, and spatial metabolomics. Results KD significantly enhanced Kp airway clearance and host survival. ScRNA-seq of lung tissue revealed markedly higher numbers of neutrophils and alveolar macrophages at day 2 post-infection in KD-fed mice, as well as an upregulation in phagolysosome signaling. By day 7 in the KD group, the neutrophil population contracted while B and T cells concomitantly expanded. Notably, IFN-γ-responsive genes were upregulated across all immune cell subsets. Exogenous ketones or drug-induced ketone accumulation bypassed the need for dietary intervention, likewise, conferring protection against Kp airway infection. Conclusion Ketones promote effective immunity to Kp by sustaining cellular energy production, thereby supporting immune cell survival and effector function. The ketone-enhanced innate immune response is followed by adaptive immune cell expansion and a broad IFN-γ transcriptional signature. Together, these findings suggest a novel therapeutic strategy for energetically demanding bacterial infections via diet-induced metabolic reprogramming. Funding Source NIH K99/R00 HL157550 (NHLBI) NIH R35 GM160393 (NIGMS) Both awarded to Tania Wong (Principal Investigator) Topic Categories Innate Immune Responses and Host Defense: Cellular Mechanisms (INC)
Mast Cell and TGF-β interactions in a model of Metabolic Dysfunction-Associated Steatotic Liver Disease and responses to cannabinoid intervention 2250623
Abstract Introduction Mast cells (MCs) are best known for their role in allergies, inflammation, and fibrosis, yet their contribution to metabolic associated steatotic liver disease (MASLD) remains unknown. MASLD is a progressive condition characterized by lipid accumulation, hepatocellular injury, and fibrosis. Only one approved therapy for MASLD exists with moderate effect. Methods To address this, we investigated MC and T cell populations in the livers of BALB/c mice with methionine and choline deficient diet (MCD) induced MASLD compared to healthy controls. Using ex vivo flow cytometry and cytokine multiplex assays, we quantified T cell response to the model. Using immunofluorescence microscopy, we identified and quantified MCs to assess whether their population size changes in MASLD. Results Our MASLD model successfully induced liver steatosis and liver injury with increases in pro-fibrotic and pro-inflammatory cytokines, validating its use for immunological studies. Ex vivo stimulation from control and MASLD livers demonstrated that cannabigerol (CBG) or CBG + cannabidiol (CBD), but not CBG alone, mitigated both helper and cytotoxic T cell activation (assessed by CD69, CD25, and IL-2 expression). TGF-β1 production was enhanced nearly 3-fold in the MASLD model, as expected, and this was restored to nearly control levels with CBG intervention. A similar pattern was observed with MC infiltration into the liver of MASLD mice with CBG intervention. Conclusion MC are a highly leverageable therapeutic target due to their unique expression of FceRI in solid tissue. These findings highlight MCs as potential targets of liver pathology and as an avenue for more affordable intervention via cannabinoids in a disease space that has relatively few effective alternatives. Funding Source NIDDK Grant 1R15DK131507-01A1 Topic Categories Immune Mechanisms of Human Disease (HUM)
Oral farnesol treatment of gastrointestinal inflammation models highlights the need for developing novel delivery methods 2257415
Abstract Introduction Farnesol (FOL) is a sesquiterpene alcohol produced as an intermediate of the mevalonate pathway. FOL has immunoregulatory effects in experimental models of cancer, neuroinflammation, and gastrointestinal (GI) inflammation. In addition, FOL contributes to microbiome homeostasis, suggesting FOL’s therapeutic effects in GI inflammation have a multifaceted approach. FOL is highly hydrophobic, which hinders its delivery. Corn oil (CO) is a commonly used vehicle in disease studies; however, CO, and other oil types, is shown to have confounding effects on disease models and microbiome modulation. We propose to test the impact of CO on FOL’s effectiveness as a therapeutic when administered as a delivery vehicle in both in vitro and in vivo GI inflammation models. We also provide preliminary data for a novel liposome preparation to improve efficacy of oral FOL delivery. Methods Experimental colitis was induced with one dose of TNBS, and FOL or CO control were orally delivered daily. Weights were collected daily and fecal samples were collected for microbiome analysis. Naive fecal samples were cultured in varying conditions to study effects of FOL in CO and CO alone. Model intestinal cell co-cultures were utilized to study intestinal barrier and immune responses to FOL treatment. FOL and control liposomes were prepared via a modified thin-film method. Results FOL and CO alone both have significant impact on TNBS-associated pathology/mortality. The microbiome composition from in vivo and in vitro studies show FOL and CO have unique modulatory activity on the microbiome, highlighting a need for less influential delivery methods. We designed a preliminary FOL liposome that provides a more targeted approach for FOL delivery without the use of CO. Conclusion Our study highlights the need for optimized delivery methods due to the impact CO has on immunological and microbial studies. We propose a liposome-based therapeutic approach to better characterize FOL as a natural therapeutic approach in future studies. Funding Source National Institutes of Health #1R15AI191202-01 Idaho INBRE Technology Access Grant #P20GM103408 Topic Categories Mucosal and Regional Immunology (MUC)
Persistent NK Cell Dysfunction May Be Linked to Gut Dysbiosis in HIV Despite Long-Term Antiretroviral Therapy (ART) 2308898
Abstract Introduction Natural killer (NK) cells are critical for controlling HIV and other infectious and non-infectious diseases. Although antiretroviral therapy (ART) suppresses HIV viral loads and restores CD4+T cells, NK cell dysfunction persists, potentially compromising long-term health in people living with HIV(PLWH). Methods We compared immune signatures in peripheral blood mononuclear cells and gut integrity biomarkers between age-matched individuals without HIV (PWoH) (n = 15) and adult PLWH (n = 13) during viremia and ART-mediated viral suppression (< 50c/mL; ∼5 years post-ART) using high dimensional flow cytometry and plasma biomarker profiling. Pearson correlation and one-way ANOVA were used for analyses. Results Compared to PWoH, PLWH exhibited a reduction in CD56briCD16- and CD56dimCD16- NK cells during viremia (pre-ART), with these levels remaining low post-ART. In contrast, CD56dimCD16+ NK cells expanded during pre-ART and remained elevated post-ART. There were sustained increases in activation markers (CD69, NKp44, HLA-DR) and memory markers (NKG2C, CD57) in these NK subsets both before and after ART. Despite ART, dysregulation of chemokine receptors (CCR2, CCR5, CCR7, CXCR5) in these NK subsets did not return to the levels seen in PWoH. Many NK subsets in PLWH showed reduced pro-inflammatory (IFNγ, IL-1β) and anti-inflammatory (TGFβ, IL-10) cytokine responses, as well as reduced polyfunctionality to both HIV-specific and non-specific stimuli, with no recovery despite ART. Plasma levels of intestinal fatty acid binding protein (IFAB), a marker of microbial translocation, were elevated in PLWH during viremia, and were positively correlated with memory-like (CD57+, NKG2C+) and activated (CD69+) in NK cell subsets in PLWH. (p < 0.05 for all). Conclusion These findings suggest that persistent NK cell dysregulation may be linked to gut dysbiosis in PLWH, underscoring the need for improved therapeutic strategies that focus on immune restoration through gut health. Funding Source n/a Topic Categories Viral Immunology (VIR)
Development of in vitro models to study CD137 agonist antibody mediated hepatotoxicity 2331460
Abstract Introduction Immune-mediated hepatotoxicity from biotherapeutics is a growing concern in drug development due to the poor translatability of preclinical safety findings and the lack of suitable in vitro models to predict in vivo risks. Urelumab, a CD137 agonist antibody, presented with severe liver toxicity in clinical trials. In contrast utomilumab, demonstrated a clean safety profile. This difference offers a unique compound set to investigate mechanisms contributing to immune-mediated hepatotoxicity and to develop in vitro models to forecast human risks. Methods In house versions of urelumab and utomilumab were produced with their original and altered Fc backbones. Effector CD8+ T cells were cultured with FcγRIIb expressing CHO cells and CD137 agonist antibodies with or without anti-CD3. Effector functions were quantified via IFN-γ, TNF-α, Granzyme B, and Perforin measurements by MSD or ELISA. Incucyte Sx5 was utilized to quantify hepatoxicity (caspase 3/7) induced from urelumab and utomilumab using a co-culture of labeled HepG2 cells (WT or β2M KO) and human effector CD8+ T cells. Results Urelumab induced cytokine release from effector CD8+ T cells was enhanced by FcγRIIb crosslinking and T cell receptor (TCR) engagement but was not dependent on these factors, like utomilumab. Additionally, urelumab mediated cytotoxic granule release required parallel TCR engagement and was enhanced by FcγRIIb crosslinking. Using an in vitro co-culture of human effector CD8+ T cells and HepG2 cells, we modeled urelumab mediated hepatotoxicity, demonstrating a lack of dependence on TCR engagement and a correlation between cytokine production and hepatocyte death, which was absent with non-toxic utomilumab. Conclusion This fully human in vitro model is a sensitive tool for evaluating immune mediated hepatotoxicity risk for immunostimulatory biotherapeutics. Funding Source Boehringer Ingelheim Topic Categories Translational and Interventional Immunology (TI)