Browse Articles

HaloCAR - a Universal Chimeric Antigen Receptor (CAR) Programmable by Chloroalkane-Conjugated Adaptors 2241611

Abstract Introduction Chimeric Antigen Receptor (CAR) T cell therapy has demonstrated clinical success against hematologic cancers. However, to date, it has failed against solid tumors due to several issues, including antigen heterogeneity, which leads to incomplete tumor elimination. To address this limitation, we developed a universal CAR T cell system, HaloCAR, that can target multiple tumor antigens via antibody and small molecule “adaptors”. Instead of containing an scFv that binds to an antigen of interest, our CAR contains Halotag, a highly efficient self-labeling haloalkane dehalogenase enzyme. When co-administered with tumor-targeting adaptors containing a chloroalkane (CA) motif, the adaptors covalently attach to the HaloCAR, arming it to attack tumor cells. Methods We generated antibody-based adaptors targeting antigens (CD20, HER2, EGFR, and CAIX). We also synthesized small molecule-based adaptors targeting CAIX and folate receptor-alpha and -beta. In vitro, we assessed the antigen-targeting activity of HaloCAR T cells via dose titration of one or more adaptors on antigen-positive and antigen-negative target cells. We tested the ability of antibody adaptors to conjugate to HaloCAR T cells in vivo in a mouse model. Results Potent and specific T cell activation and tumor cell lysis were observed for both single- and multi-antigen targeting studies. This activity was titratable and dependent on the dose of the adaptor(s). In vivo, we detected robust conjugation of the antibody-adaptor to Halo CAR-T cells, and assembled CAR was detectable for at least 72 hours post-adaptor injection. Conclusion We successfully generated and tested a new covalent universal CAR system targeted by CA-conjugated adaptors. In vitro assays highlight the multi-targeting and tunable capabilities of the platform. In vivo labeling of HaloCAR T cells with antibody adaptors is promising for therapeutic feasibility. We are currently evaluating anti-tumor efficacy in both solid and hematological human tumor xenograft mouse models. Funding Source NIH 1R01CA290866 Topic Categories Tumor Immunology: Checkpoints, Prevention, and Treatment (TIPT)

IL-11-induced NLRP3 inflammasome activation mediates pyroptotic cell death in human neurons 2260831

Abstract Introduction IL-11, a member of the IL-6 cytokine family, is significantly increased in the serum and cerebrospinal fluid (CSF) of patients with relapsing-remitting multiple sclerosis (RRMS), and serum IL-11 levels correlate with clinical relapses. This study examined the neurotoxic effect of IL-11 and its role in the NLRP3 inflammasome activation in human neurons. S1PR1 signaling promotes NLRP3/IL-1b expression. We propose that inhibiting IL-11-induced NLRP3 inflammasome activation in neurons using anti-IL-11 mAb, NLRP3 inhibitor MCC950, and an S1PR inhibitor Ozanimod, may provide a neuroprotective effect. Methods Human inducible pluripotent stem cell (iPSC)-derived neurons were labeled with DRAQ7 dye, which incorporates in the nuclei of dead cells. We examined the neurotoxicity of IL-11 (100 and 200 ng/ml), CSF from 10 untreated RRMS patients (1:25 dilution) and 10 control donors in the absence or presence of anti-IL11 mAb, MCC950 and Ozanimod. Dead cells were scored by visualization of DRAQ7-positive neurons. Results We identified the expression of IL-11R, NLRP3 and caspase-1 in iPSC-derived human neurons by immunofluorescent (IF) staining. IL-11 induced neuronal death in a dose-dependent manner. The effect was inhibited by the selective NLRP3 inhibitor MCC950, which blocks the inflammasome activation, indicating that IL-11-NLRP3 inflammasome activation mediates the neuronal cell death. CSF samples from 10 RRMS patients induced a similar degree of neuronal cell death (57.8%), which was significantly higher in comparison to the CSF from matched control donors or PBS (15.3%). The neurotoxic effect of the MS CSF was detected at day 7, and it was inhibited by human anti-IL-11 mAb (12.8%), MCC950 (17.2%) and Ozanimod (11.3%). Conclusion IL-11 within the CSF induces neuronal cell death, which is mediated via NLRP3 inflammasome-induced pyroptosis. Anti-IL11 mAb, MCC950, and Ozanimod demonstrated a neuroprotective effect. Funding Source NIH, R01 Topic Categories Neuroimmunology (NEUR)

Regulatory B cells ameliorate experimental periodontitis through promoting macrophage-mediated pro-resolving functions 2300751

Abstract Introduction This study aimed to determine the role of B10 in the induction of pro-resolving macrophage function and the resolution of experimental periodontitis. Methods A combination of ligation and P. gingivalis infection was used to establish experimental periodontitis in C57BL/6 mice. Enriched B10 cells were adoptively transferred to recipient mice via tail vein injection. In some experiments, macrophages were depleted by intraperitoneal injection of clodronate liposomes. Alveolar bone resorption was evaluated using Micro-CT. TRAP staining was performed to identify osteoclasts. In vivo imaging was used to track the macrophages and adoptively transferred-B10 cells in mice. The expression of specialized pro-resolving mediators was detected by Mass Spectrometry (LC-MS/MS). The CD68+CD206+ pro-resolving macrophages, CD68+LC3B+ autophagic cells and CD68+Ly6G+ neutrophils were detected by immunofluorescence. Results B10 transfer inhibited alveolar bone resorption and osteoclast activation. A significant increase in IL-10, Arg-1, and YM-1 expression in periodontal tissues following B10 transfer, accompanied by a marked decrease in IL-17A, TNF-α, and IL-1β expression. B10 transfer promoted the expression of pro-resolving macrophages compared with the control groups. The expressions of ATG5+, LC3B+, CD68+LC3B+ and CD68+Ly6G+ cells were elevated in the B10 transfer group. However, the B10-mediated suppression of inflammation and alveolar bone resorption was attenuated upon macrophage depletion. B10 transfer enhanced the production of the SPMs in periodontitis, such as RvD4 and RvD2. Notably, B10 cells promoted RvD2 synthesis in a macrophage-dependent manner. Conclusion This study demonstrated that B10 promoted macrophage-mediated release of specialized pro-resolving mediators and autophagy function, which represent as novel mechanism in B10-induced inhibition of inflammation and bone loss in experimental periodontitis. Funding Source NIH NIDCR R01DE025255 Topic Categories Immune Response Regulation: Cellular Mechanisms (IRC)

ERK promotes miR34a/ISOC1 signaling to enhance all trans retinoic acid-induced myeloid differentiation in acute promyelocytic leukemia

Factors associated with venous thromboembolism in ICU patients with sepsis: A retrospective analysis using the MIMIC-IV database

Pathogenic and protective lung lipid predictors of tuberculosis in Rhesus macaques 2308411

Abstract Introduction Lipids are critical in pathogenesis and immunity. We hypothesize that Mycobacterium tuberculosis (M.tb) infection induces global lipid profile alterations in lung tissues to regulate host immunity. Methods To test the hypothesis, lipids from lung tissues of Bacillus Calmette-Guérin (BCG)-vaccinated (n = 4) and unvaccinated (n = 4) Rhesus macaques with M.tb infection and from uninfected controls (n = 4) were extracted with a modified Matyash method. Samples were profiled on a Vanquish HPLC-coupled Thermo Orbitrap IQ-X Tribrid mass spectrometer by heated electrospray ionization in positive and negative modes, with untargeted MS1/MS2 and data-dependent MS2/MS3 analyses. Mass profiles were annotated using the LipidSearch program. Results From over 1000 annotated lipid species of over 50 classes, the cholesterol esters and triacylglycerols, which are associated with lipid droplet and granuloma caseum formation, and the oxidized lipids that potentially contribute to ferroptosis, were detected more abundantly with active infection in unvaccinated animals. For ether-linked lipids with metabolic and immune regulatory functions as ligands for transcription factors and CD1 antigen-presentation molecules, plasmalogens and non-plasmalogens were enhanced, respectively, in vaccinated macaques exhibiting protection from subsequent M.tb challenge, and unvaccinated controls with active M.tb infection. Sphingosines and hexosylsphingosines were highly expressed in vaccine-protected animals but low in active infection, as sphingosines stimulate macrophage responses. Conclusion Our lipidomic platform displays superior sensitivity and accuracy in detecting essential lipid classes altered in lung tissues of Rhesus macaques with BCG vaccination and M.tb infections. The altered lipid classes, such as glycerolipids, phospholipids, sphingolipids, ether-lipids, and cholesterol, with various chain lengths and unsaturation, provide candidate lipids and metabolic pathways to induce protective anti-tuberculosis immunity. Funding Source N/A Topic Categories Innate Immune Responses and Host Defense: Molecular Mechanisms (INM)

Functional dichotomy of GC-Derived Memory B Cells in Lupus: A Role for FCRL5+ MBCs in ASC Differentiation 2260895

Abstract Introduction In systemic lupus erythematous (SLE), germinal center (GC)-derived autoreactive memory B cells (MBCs) are a critical reservoir for pathogenic autoantibodies. However, the functional specialization of distinct GC-derived MBC subsets remains poorly defined, creating a major barrier to developing targeted therapies that can selectively deplete pathogenic B cell lineages. Methods We employed a fate-reporter mouse model (S1PR2-tdTomato) within a spontaneous lupus background to precisely track GC-derived B cells. Using this system, we isolated two key MBC subsets, FCRL5+ and CD23+ for functional assessment in vitro and via adoptive transfer. To probe the role of the GC niche, we performed transient ablation of follicular dendritic cells (FDCs) in mixed bone marrow (BM) chimeric animals using the Cxcl13Cre-DTR model. Additionally, we evaluated the therapeutic impact of CD21 blockade on MBC subset dynamics and function. Results We identified a fundamental functional dichotomy. FCRL5+ MBCs demonstrated a superior capacity to differentiate into antibody-secreting cells (ASCs) and produce autoantibodies, while CD23+ MBCs were refractory to ASC differentiation. Depleting FDCs, thereby ablating the GC niche, caused a preferential accumulation of FCRL5+ MBCs and a concurrent loss of the CD23+ subset, demonstrating the GC niche’s essential role in maintaining the CD23+ lineage. Therapeutically, CD21 blockade also ablated GCs and led to the accumulation of a CD21low/- MBC subset with impaired ASC potential. Conclusion Our findings establish a model where FCRL5+ MBCs act as dedicated antibody effectors, while CD23+ MBCs serve as a niche-dependent reservoir for GC re-entry, a process that likely fuels epitope spreading and chronicity. CD21-targeted therapy limits autoimmunity by dismantling the active GC and, crucially, by restricting the supportive niche required for pathogenic, ASC-genic MBC development. Funding Source Department of Defense, Lupus research Alliance (LRA) Topic Categories Therapeutic Approaches to Autoimmunity (THER)

Allergen-specific regulatory T cells form lung resident memory capable of suppressing Th2 recall responses to allergen 2267598

Abstract Introduction Intranasal house dust mite (HDM) exposure elicits allergen-peptide (p):MHCII-specific Th2 and Tregs in the airways of mice. The balance between opposing pro- and anti-inflammatory functions of these cell populations may influence the outcome of airway re-exposure to HDM. However, the number of cells specific for known HDM derived epitopes in B6 mice is low, making longitudinal studies of these cell populations difficult. Methods To overcome this obstacle, we developed an approach using adoptive transfer of a physiologically relevant number of CD4+ T cells from a fixed TCR beta chain transgenic (1-DERβ) mouse and corresponding HDM p:MHCII tetramer to simultaneously interrogate the trafficking, memory formation, and functional properties of HDM-specific Th2 and Treg cells. Results Naïve 1-DERβ CD4+ T cells generated similar frequencies of Th2 and Treg fates as endogenous HDM-specific cells. Parabiosis experiments demonstrated HDM-specific Tregs form stable tissue-resident memory (Trm) cells in the lungs, similar to their previously described Th2 counterparts. Single cell RNA sequencing of these cell populations revealed shared expression of Trm genes, including the chemokine receptors CXCR6 and CCR8. Co-adoptive transfer of wild type and chemokine receptor deficient 1-DERβ cells revealed differential roles for CXCR6 and CCR8 in the generation and maintenance in the lung. Depletion of Tregs in the memory phase led to increased Th2 responses in the lung upon allergen rechallenge. Conclusion Thus, our findings are consistent with a model where lung-resident allergen-specific Tregs persist long term and are capable of suppressing resident Th2 cells following allergen rechallenge. Funding Source NIH K08 KAI171176 Topic Categories Immediate Hypersensitivity, Asthma, and Allergic Responses (HYP)

Microbiota-dependent Interferon Lambda Controls Uterine Immunity and Placental Anti-bacterial Responses 2304493

Abstract Introduction Tight regulation of uterine immunity is necessary to prevent infection and promote reproduction. How local tissue factors, including the cervicovaginal microbiota, influence uterine immunity is poorly understood. At other barrier sites, interferon lambda (IFN-L) is induced by the microbiota and controls barrier function, tissue immunity, and anti-viral immunity. IFN-L is constitutively expressed in the uterus and placenta and protects the placenta from virus infection. However, the source of immune stimulation that drives basal IFN-L expression and the additional functions of IFN-L outside of antiviral immunity in the uterus and placenta are largely unknown. Methods We leveraged spectral flow cytometry to investigate the effect of the cervicovaginal microbiota on uterine immunity using gnotobiotic mice and a model of new intravaginal commensal exposure. We performed spectral flow cytometry and single cell RNA sequencing (scRNAseq) on IFN-L signaling deficient mice to investigate the effect of IFN-L on uterine immunity. Next, we evaluated the anti-bacterial effect of IFN-L in nulliparous and pregnant mice using Streptococcus agalactiae. Results We found that IFN-L regulates uterine type 17 immune cell abundance in a microbiota-dependent manner. Our scRNAseq data revealed that loss of homeostatic IFN-L signaling resulted in decreased expression of genes involved in the defense responses to viruses and bacteria. We found that IFN-L did not influence bacterial burden in nulliparous mice. But, during pregnancy, IFN-L protects the placenta and fetus from ascending bacterial infection and from infection induced necrosis. Furthermore, loss of IFN-L signaling during bacterial infection in pregnancy had long term impacts on offspring health and fitness. Conclusion Overall, we find that IFN-L is a key immunoregulator in nulliparous animals and a key anti-bacterial factor in pregnant animals. Future work will explore how the microbiota influences uterine IFN-L signaling. Funding Source NIGMS F12 GM150424; intramural NIH funding Topic Categories Mucosal and Regional Immunology (MUC)

Enhance Anti-Tumor Immunity by CD004-induced Reprogramming of IL-17-Producing CD8+ T Cells Toward Cytotoxic Tc1-Like Phenotypes 2237759

Abstract Introduction While IL-17-producing CD8+ T cells (Tc17) are conventionally considered tumor-promoting, recent studies have highlighted their functional plasticity. Tc17 cells can be reprogrammed into IFN-γ-producing, cytotoxic Tc1-like phenotype, offering new opportunities for their application in immunotherapy. Through database screening, this study investigates the potential of a candidate drug, CD004, to induce Tc17 reprogramming and enhance anti-tumor immunity. Methods Polarized Tc17 cells were treated with CD004, the compound identified from database screening with anti-inflammatory properties. The expression of IL-17A, IFN-γ, T-bet, Eomes, and granzyme B was analyzed by flow cytometry. Cytotoxicity was evaluated against E.G7-OVA lymphoma cells, and metabolic profiling was performed to explore underlying mechanisms. An adoptive transfer model using E.G7-OVA-bearing mice was employed to assess the in vivo anti-tumor efficacy. Results CD004 treatment significantly upregulated IFN-γ, T-bet, and granzyme B expression in Tc17 cells, accompanied by a reduction in IL-17A expression. Cytotoxicity assays confirmed enhanced tumor cell killing after treatment in vitro. Metabolic analysis suggested that Tc17 reprogramming may involve mitochondrial metabolic rewiring. In the in vivo model, adoptive transfer of CD004-treated Tc17 significantly reduced tumor volume compared with the control group. Conclusion CD004 treatment reprogrammed Tc17 cells toward a Tc1-like phenotype, enhancing their cytotoxicity and anti-tumor function both in vitro and in vivo. These findings highlight a potential therapeutic strategy for cancer immunotherapy through pharmacological modulation of T cell plasticity. Funding Source This study is partially supported by the National Science and Technology Council, Taiwan, and the Chinese Medicine Research Center of China Medical University, under the Featured Areas Research Center Program within the framework of the Higher Education S Topic Categories Tumor Immunology: Cellular Responses and Tumor Microevironment (TIME)

CD22 Defines a Core Follicular Regulatory T Cell Subset that Controls the Antibody Response 2307652

Abstract Introduction Germinal centers (GCs) formation and the associated antibody responses within B-cell follicles are dependent on the interaction of GC B cells with T Follicular Helper (TFH) and T Follicular Regulatory (TFR) cells. Follicular regulatory T (TFR) cells control antibody responses, yet the mechanisms underlying their differentiation and function remain incompletely understood. Methods We used flow cytometry to analyze the phenotype of TFR cells in both mouse and human samples. To investigate the role of CD22, we generated Treg cell-specific Cd22 knockout mice and assessed their antigen-specific immune responses following NP-OVA immunization. Furthermore, we performed in vitro co-culture assays to evaluate the suppressive function of TFR cells. Results We identified that both mouse and human TFR cells express CD22, representing a functionally distinct subset with a highly differentiated T follicular cell phenotype. Transcriptional analysis revealed that CD22+ TFR cells are enriched in a core T follicular cell gene signature distinct from their CD22— counterparts. Using a Treg cell-specific Cd22 deletion model, we observed dysregulated homeostatic and antigen-specific antibody responses, as well as age-dependent autoantibody production. Furthermore, CD22 deficiency impaired mTORC1-pSTAT3 activation, a pathway implicated in TFR cell function. Conclusion Our findings thus establish CD22 as critical to the differentiation of a core subset of TFR cells that regulates the magnitude and quality of the humoral immune response. Funding Source National Institutes of Health R01AI128976 Topic Categories Immune Response Regulation: Cellular Mechanisms (IRC)

Molecular mechanisms controlling the growth and functional characteristics of the neonatal and juvenile thymus microenvironments 2267575

Abstract Introduction Unlike most organs, thymus growth is untethered to body size, following an independent trajectory across the lifespan, peaking during the perinatal period, then leveling off and then declining at a juvenile age. These changes in thymus size correlate to changes in thymus function and the generation of stage-specific subsets of T cells which persist throughout the lifespan, resulting in the full complexity of the adult T cell complement. Methods We used single-cell RNAseq and functional and phenotypic validation approaches to identify and test molecular mechanisms that control the dynamic changes in thymus growth and function during the perinatal to juvenile period , working first in mice, and validating results in human. Results Differentially expressed gene and intercellular network analyses of temporally resolved scRNA-seq data revealed fibroblast-derived insulin-like growth factor 2 (IGF2) as a candidate driving neonatal thymic expansion. We demonstrated IGF2 activity promotes a cortical TEC-specific proliferation and is tightly regulated at the thymic growth transition, acting at least partly via regulation of the CyclinD1-RB-E2F-Foxn1 molecular pathway in thymic epithelial cells (TECs). We further demonstrated using both gain and loss of function genetic models that this pathway impacts not only proliferation but also function, tying the dynamics of growth to TEC differentiation and production of stage-specific T cells. We demonstrated that multiple components of the IGF2 pathway are differentially regulated across this time period in multiple cell types, implicating an extrinsic mechanism responsible for the timing of these transitions. Conclusion We have identified IGF2 as a critical regulator of thymus growth and function across the perinatal to juvenile stages, likely controlled by a factor(s) extrinsic to the thymus. Current efforts are focused on identifying the upstream mechanisms controlling these dynamic changes that are required for propoer immune system development. Funding Source NIH/NIAID Topic Categories Hematopoiesis and Immune System Development (HEM)

Unlocking Lewis‐Acid Catalysis and Crystalline Polyselenide Evolution for Ultra‐Stable Sodium‐Ion Batteries

ABSTRACT Metal selenides (MSes) are promising anodes for sustainable sodium‐ion batteries (SIBs), but their practical application is fundamentally hindered by sluggish kinetics, severe sodium‐polyselenide (Na x Se y ) dissolution, and structural degradation. Herein, we uncover that the poor cycling reversibility of SnSe 2 originates from localized electron distribution and high energy barriers, which hinder complete conversion during cycling. We further demonstrate that the in‐situ generated Sn intermediates function as stage‐selective catalysts, preferentially promoting the conversion of Na 2 Se 6 into soluble Na 2 Se 4 , leading to the accumulation of shuttle‐active intermediates and rapid capacity decay. Guided by theoretical calculations, a bimetallic selenide composite (Cu 2 SnSe 4 @NC) was rationally designed, where copper incorporation delocalizes electrons and weakens Cu─Se bonding, thereby accelerating the initial conversion reaction. Crucially, the in situ generated Cu/Sn heterostructure enables Lewis‐acid‐regulated and stepwise crystalline evolution of Na x Se y from Na 2 Se 6 to the final Na 2 Se, thereby substantially suppressing the solvation and shuttling of soluble intermediates. Consequently, the Cu 2 SnSe 4 @NC electrode achieves excellent cycling stability, retaining 95% of its capacity after 7000 cycles at 5.0 A g −1 in half‐cells and sustaining over 5000 cycles at 1.0 A g −1 in full cells. This work establishes a new design paradigm for fabricating ultra‐long lifespan MSes anodes toward scalable SIBs.

ARMH3 acts as a central scaffold at the Golgi/TGN through interactions with Arl5, GBF1, and PI4KB

Performance analysis of U-shaped viscoelastic seismic brace for pipelines

Interleukin 15 stimulation augments myeloid-derived suppressor cell immunosuppression in cross-species models of sarcoma 2220048

Abstract Introduction Myeloid-derived suppressor cells (MDSCs) are a heterogenous immune cell population expanded in cancer, with immunosuppressive effects on natural killer (NK) and T cells, inhibiting antitumor immunity. Cytokine immunotherapy with interleukin 15 (IL-15) has shown promise in canine and human sarcomas. Although IL-15 is classically associated with heightened NK and T cell antitumor response, MDSC expression of IL-15 receptor alpha (IL-15Rα) may be associated with shorter disease-free intervals in dogs receiving IL-15 for osteosarcoma (OSA). We sought to characterize the effects of pre-conditioning MDSCs in vitro with IL-15 in cross-species models. Methods Murine MDSCs (CD11b+ Gr1+) were isolated from OSA-bearing BALB/c spleens, pre-treated +/- IL-15, and cultured with wild type splenocytes (anti-CD3/28 stimulated), then evaluated by flow cytometry. Human MDSCs were differentiated in vitro from CD33+ PBMCs with IL-6 and GM-CSF, then pre-treated +/- IL-15 and TTI-101 (STAT3 inhibitor). MDSCs were cultured with donor-matched CD33- PBMCs (anti-CD3/28 and IL-2 stimulated), then evaluated by flow cytometry. Results IL-15 pre-treatment increased the suppressive effects of murine MDSCs on wild type splenic CD8 T cell and NK cell proliferation after in vitro stimulation. IL-15 pre-treatment increased the suppressive effects of human MDSCs on T cell (CD4 and CD8) and NK cell proliferation and cytotoxicity. Pre-treatment with TTI-101 reduced the IL-15-induced increase in suppressive effects of MDSCs. Conclusion The IL-15/IL-15Rα axis appears to regulate MDSC function with potential pro-tumor effects. Better understanding of this pathway is likely to have translational impact in cancer and autoimmunity. Funding Source NIH Training Grant T32GM136559, UC Davis Center for Companion Animal Health Endowment Funds Topic Categories Tumor Immunology: Cellular Responses and Tumor Microevironment (TIME)

Myeloid STAT4 at the Intersection of Sleep Disruption and Hyperlipidemia: A Molecular Link Between Lifestyle Stress and Atherogenesis 2261152

Abstract Introduction Neutrophils, crucial regulators of antimicrobial defense, also play a role in atherosclerosis. STAT4 controls key neutrophil functions, including ROS generation, NETosis, and migration. Myeloid STAT4 deficiency reduces atherogenesis but impairs antimicrobial defense. Sleep fragmentation (SF), a widespread health concern, increases cardiovascular risk. SF accelerates atherosclerosis in a neutrophil-dependent manner by hyperactivating neutrophil functions. Methods To test the role of STAT4 in SF-accelerated atherosclerosis, myeloid-specific STAT4-deficient low-density lipoprotein receptor-deficient Stat4fl/fl LysMcre Ldlr-/- and Stat4fl/fl Ldlr-/- mice were subjected to chronic SF and HFD for 16 weeks. We used Kaplan—Meier analysis to monitor survival, Oil Red O staining to quantify aortic plaque burden and spectral flow cytometry to assess neutrophil effector functions. Intestinal oxidative stress and microbial translocation were evaluated via histology and ELISA. Results Myeloid-specific STAT4 deficiency reduced neutrophil production, activation, and atherosclerosis, but also caused peritonitis and increased mortality after SF and HFD feeding in Stat4fl/flLysMcreLdlr-/- vs Stat4fl/flLdlr-/- mice. Premature death was linked to peritonitis, possibly due to intestinal inflammation and barrier dysfunction. Conclusion Thus, STAT4 critically balances immune responses during sleep fragmentation by driving inflammatory neutrophil activation that promotes atherosclerosis while simultaneously preserving essential host defense mechanisms, underscoring its dual role in modulating inflammation and survival under physiological stress. Funding Source AHA Predoctoral Fellowship Grant, AHA Innovation Grant Topic Categories Immune Mechanisms of Human Disease (HUM)

LOX-1 promotes efferocytosis and oxLDL-mediated immune reprogramming of alveolar macrophages during pneumonia 2267460

Abstract Introduction Pneumonia is a worldwide public health concern, necessitating improved understanding of host response to lung pathogens. We recently reported that lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) limits inflammatory injury, partially mediated by alveolar macrophages (AMs). While oxidized low-density lipoprotein (oxLDL), the primary ligand for LOX-1, elicits inflammation in extrapulmonary macrophages, it is unknown whether oxLDL or other LOX-1 ligands alter AM function, thereby regulating the host response to lung infection. Methods Primary AMs or MH-S cells were cultured with oxLDL or apoptotic cells (± anti-LOX-1 or control IgG) to determine primary outcomes and, subsequently, LPS or GFP-labeled E. coli to determine secondary outcomes. WT mice were treated intranasally with oxLDL (± anti-LOX-1 or control IgG) for 24H or after one week, infected with E. coli. WT mice were treated intratracheally (± anti-LOX-1 or control IgG) with apoptotic cells or GFP-labeled E. coli to determine the impact of LOX-1 on efferocytosis or phagocytosis, respectively. Outcomes were measured by live-cell imaging, flow cytometry, RT-qPCR or ELISA. Results Earlier work showed that LOX-1 limits injury and inflammation during pneumonia, potentially via AMs. Here, we found that LOX-1 promotes efferocytosis, while limiting phagocytosis in AMs. Moreover, pre-stimulation of AMs with oxLDL reduced iNOS and IL-1B expression in response to LPS or E. coli, while promoting IL-6 and CXCL2 expression. Arginase-1, while suppressed in AMs, was more highly expressed in neutrophils following administration of oxLDL and infection with E. coli, independent of LOX-1. Conclusion LOX-1 has emerged as a unique immunoregulatory receptor in the lungs during infection. Our data show that this likely occurs through increased AM efferocytosis and oxLDL-dependent regulation of inflammatory factors, such as iNOS. Future studies will further interrogate LOX-1-dependent and independent mechanisms of protection against lung injury. Funding Source NHLBI Topic Categories Microbial, Parasitic, and Fungal Immunology (MPF)

Spatiotemporal Regulation of RIG-I-Like Receptor Signaling by Endosomes 2305200

Abstract Introduction RIG-I-like receptors are cytosolic viral RNA sensors that are critical for type I interferon (IFN)-driven antiviral defense against RNA virus infections. RIG-I has been shown to survey viral RNA in subcellular compartments, including stress granules, mitochondria-associated membranes, microsomes, and the nucleus, after virus invasion. Our recent work revealed a novel RIG-I action mode in which RIG-I is recruited onto endosomes, the “gateway” of virus entry into a host cell, to detect viral RNA upon viral breaching the endosomes. Endosomes serve as signaling platforms for the ubiquitination and activation of RIG-I. To better understand this novel RIG-I action mode, we further explored the spatiotemporal regulation of RIG-I signaling by endosomes, as well as the potential interplay between MDA5 and endosomes. Methods Biochemical and microscopic analyses are employed to investigate the interaction between RLRs and endosomes. Genetic approaches are employed to investigate the role of the endosomal adaptor TAPE in antiviral defense in vivo. Results Our data suggest that endosomes act as signaling platforms for MDA5 ubiquitination and activation. Conclusion These novel action modes of RIG-I-like receptors reveal the host’s early engagement with viruses to trigger antiviral defense. Funding Source Taiwan National Science and Technology Council Topic Categories Innate Immune Responses and Host Defense: Cellular Mechanisms (INC)

STEMprep™ Automated Tissue Dissociation System: Maximize Cell Viability and Yield 2252190

Abstract Introduction Tissue environments are specialized to support distinct functions, but isolating high-quality cells in sufficient quantities is a major challenge. Manual dissociation methods are labor-intensive and limit throughput, while balancing enzymatic and mechanical approaches is time-consuming and technically demanding. Methods The STEMprep™ Tissue Dissociation System is an automated platform developed to standardize the preparation of single-cell suspensions and homogenates. Results The system consistently produces single-cell suspensions with high viability and yield from various mouse tissues, including spleen (96.9±1.2% viability; 1.1E8 ±3.6E7 cells/tissue), lung (90.3±3.8%; 2.0E7±4.0E6 cells/tissue), liver (89.7±2.4%; 2.8E7±8.9E6 cells/tissue), brain (88.5±2.8%; 2.8E6±1.2E6 cells/tissue), 4T1 tumors (89.5±3.0%; 2.5E4±1.0E4 cells/mg), and B16 melanomas (90.7±3.1%; 1.8E4±6.9E3 cells/mg). Importantly, cell surface marker integrity is preserved, with 88.8% of over 200 epitopes remaining stable post-dissociation. Processed cells are compatible with downstream applications such as EasySep™ immunomagnetic isolation, and the isolated cells remain functional in assays including microglial phagocytosis and T cell proliferation. The system also supports a homogenization protocol for extracting high-quality RNA. Conclusion In summary, the STEMprep™ system provides a robust, automated solution that enhances consistency, throughput, and efficiency to accelerate tissue research and discovery. Funding Source n/a Topic Categories Technological Innovations in Immunology (TECH)