Journal of Leukocyte Biology

Background. The cholinergic anti-inflammatory pathway regulates inflammation through vagus signaling that activates the 7 nicotinic acetylcholine receptor (7nAChR) on leukocytes. Suppression of macrophage inflammatory cytokine production through 7nAChR-mediated inhibition of NF-{kappa}B activation is considered a central mechanism of this pathway. However, the effects of cholinergic activation of 7nAChR on neutrophils remain poorly understood. Here, we investigated whether and how activation of 7nAChR in neutrophils regulates neutrophil migration, defined the underlying molecular mechanism, and evaluated its contribution to anti-inflammatory protection during endotoxemia. Results. The ex vivo swarming assays revealed that activation of 7nAChR with the selective agonists PNU-282987 and AR-R17779 significantly inhibited human and mouse neutrophil migration. The inhibitory effect of PNU-282987 was abolished in 7nAChR-deficient neutrophils, confirming receptor specificity. Mechanistically, 7nAChR activation reduced integrin M{beta}2 activation, decreased F-actin polymerization, and impaired neutrophil polarization. These effects required M{beta}2, as PNU-282987 failed to inhibit actin remodeling in M-deficient neutrophils. Modulation of M{beta}2 activation with an activating antibody (MEM48) demonstrated that 7nAChR regulates neutrophil migration by limiting excessive M{beta}2 activation and maintaining an optimal adhesive state. In vivo, adoptively transferred fluorescently labeled PNU-282987-treated neutrophils exhibited reduced recruitment to the lungs compared with vehicle-treated neutrophils during LPS-induced endotoxemia. Furthermore, neutrophil depletion markedly diminished the protective effect of PNU-282987 on survival, demonstrating that neutrophils contribute to 7nAChR-mediated protection during systemic inflammation. Conclusions. Our findings identify neutrophils as an important cellular target of the cholinergic anti-inflammatory pathway and demonstrate that 7nAChR activation limits neutrophil migration by suppressing integrin M{beta}2 activation and actin cytoskeletal remodeling. Together, these findings establish M{beta}2 as a critical downstream effector of 7nAChR signaling in neutrophils and identify regulation of integrin activation as a previously unrecognized mechanism of the cholinergic anti-inflammatory pathway.

Innate immune cells, including macrophages and DCs, are critical for controlling infection by Cryptococcus neoformans. Although C. neoformans is phagocytosed equally by each pulmonary phagocyte subset, CD11b+ DCs support fungal growth, whereas Ly6c- monocyte-like macrophages are antifungal. The metabolic programs underlying these contrasting responses remain incompletely understood, but our previous work points to increased glycolysis in antifungal cells. Therefore, we hypothesized that metabolic reprogramming in phagocytes following cryptococcal exposure leads to different outcomes. We examined ten metabolic pathways using flow cytometry, metabolic inhibition, antifungal assays, and pulmonary infection. Cryptococcal exposure increased GLUT-1, HK, HIF-1, and lactate production in J774A.1 macrophages, consistent with enhanced glycolytic metabolism in antifungal cells. In contrast, ATP5a and IDH2 were reduced, suggesting diminished mitochondrial metabolism. Cryptococcus also decreased G6PD, ACAC, ASS1, and PRDX2, indicating alterations in PPP activity, fatty acid synthesis, arginine metabolism, and antioxidant defense. Pharmacologic inhibition of PPP further reduced G6PD expression and significantly decreased intracellular cryptococcal burden, supporting a role for glucose-derived metabolic pathways in regulating fungal survival. In vivo, infection increased GLUT-1 and HK expression in antifungal female Ly6c- monocyte-like macrophages but not in permissive CD11b+ DCs, whereas other metabolic markers showed limited or subset-specific changes. Collectively, these findings demonstrate that antifungal cells use a predominantly glycolytic program accompanied by reduced mitochondrial, anabolic, and antioxidant-associated protein expression following interaction with C. neoformans. This phagocyte subset-dependent metabolic response during pulmonary infection may influence antifungal activity and identify metabolic pathways that could be targeted to strengthen host defense.

The human host defence peptide LL-37 mediates pleiotropic immunomodulatory functions that can be both pro- and anti-inflammatory. Under inflammatory conditions in the lungs, LL-37 is susceptible to the post-translational modification (PTM) citrullination. The impact of this PTM on LL-37's immunomodulatory functions is not fully understood. Therefore, we characterized the transcriptome and cytokine profile in response to LL-37 and citrullinated-LL-37 (citLL-37), in human bronchial epithelial cells (HBEC). Cells were stimulated with either LL-37 or citLL-37 (0.5 M), or a scrambled peptide (sLL-37). RNA after 4 hours (h) was analyzed with NanoString nCounter Host Response Panel, and 96 cytokines were examined (after 24 h) in tissue culture supernatants (Luminex platform). Genes and cytokines with a Log2 fold-change [&ge;]0.5 with p<0.05 compared to unstimulated cells were considered differentially expressed (DE). These studies revealed overlapping and distinct biosignatures. There were 118 DE genes with LL-37 and 71 with citLL-37, of which magnitude of change was significantly different for 32 genes. 16 out of 17 DE genes altered by both peptides were directly related to inflammation, and these were significantly less (by ~45%) with citLL-37 compared to LL-37. Similarly, inflammatory cytokines enhanced in response to LL-37 were significantly less (by 37-94%) with citLL-37. Overall, our findings indicate that citrullination of LL-37 does not abrogate cellular response in HBEC, instead dampens the inflammatory biosignature induced by LL-37.

Background: Neutrophils migrate to the respiratory epithelium during rhinovirus (RV) infection, but whether RV directly activates neutrophils or whether activation arises from epithelial responses remains unclear. Objective: To determine whether RV directly activates human neutrophils or indirectly activates them through RV infected airway epithelial cells. Methods: Human neutrophils were exposed to purified RV, and activation was assessed by myeloperoxidase activity, CD11b/CD66b expression, and reactive oxygen species (ROS) production. Neutrophils were co-cultured with RV-infected air liquid interface (ALI) bronchial epithelium evaluated for effects on epithelial integrity. At 48 to 72 hours post-infection, we used live-cell imaging to measure ROS responses and neutrophil-tracking assays to measure migration velocity and ROS responses to basolateral supernatants, clarified apical washes, and apical debris from RV-infected epithelial cells. Plaque assays were used to assess for RV infectivity after incubation with apical neutrophils. Results: RV did not directly activate neutrophils. Adding neutrophils to the apical surface of airway epithelial cells increased neutrophil ROS production at 48 to 72 hrs post-infection but did not alter epithelial cytotoxicity. Conversely, basolateral supernatants from infected cells enhanced neutrophil migration but not ROS, consistent with cytokine-mediated priming. Clarified apical washes had no effect on migration or ROS. By contrast, apical debris from infected cultures triggered a robust ROS response. Basolateral fractions from medium control or RV infected cultures did not induce ROS. Infectivity is not impacted by apical neutrophil presence. Conclusion: RV does not directly activate neutrophils. Instead, RV infected epithelial cells release basolateral factors that enhance neutrophil migration, while apical debris from infected cells drives ROS activation. These findings indicate that epithelial injury, not RV itself, is the dominant trigger of neutrophil activation during infection.

Somatic mutations in the de novo DNA methyltransferase DNMT3A are frequent in clonal hematopoiesis, but how they alter the function of differentiated myeloid cells remains unclear. Here, we used isogenic human embryonic stem cell-derived macrophage models to define the consequences of DNMT3A dysfunction during myeloid differentiation. DNMT3A-mutant macrophages selectively upregulated bivalent Polycomb target genes, accompanied by DNA hypomethylation, reduced H3K27me3 and increased promoter accessibility. Despite broad epigenetic changes across bivalent promoters, transcriptional derepression occurred at a subset of loci characterized by lower baseline DNA methylation and higher H3K27me3, identifying pre-existing chromatin state as a determinant of transcriptional response. This Polycomb-associated program was conserved in murine macrophages and enriched for genes involved in cell migration and wound repair. DNMT3A-mutant macrophages exhibited enhanced migration and preferential early recruitment to injured tissue. These findings link clonal hematopoiesis-associated epigenetic alterations to selective transcriptional reprogramming and altered function of differentiated myeloid cells.

Interleukin-15 (IL-15) trans-presentation (TP) by B cells is an important mechanism of T-cell activation; however, the spatial organisation of interleukin-15 receptor (IL-15R) relative to major histocompatibility complex (MHC) molecules on B-cell microvilli remains poorly understood. As microvilli protrude from the B-cell surface and may serve as sites of initial B cell-T-cell contact, the distribution of IL-15R and MHC molecules within these structures may be important during the earliest stages of T-cell recognition and activation. Here, we investigated the spatial association and molecular proximity of IL-15R with MHC class I and class II molecules on B-cell microvilli before immunological synapse formation, using confocal microscopy, stimulated emission depletion (STED) microscopy, stochastic optical reconstruction microscopy (STORM), and fluorescence lifetime imaging microscopy-based Forster resonance energy transfer (FLIM-FRET). Both MHC class I and class II molecules showed significant spatial association with IL-15R; however, the extent of colocalisation decreased as spatial resolution increased. STED microscopy revealed significant colocalisation between IL-15R and MHC class I, whereas STORM did not detect this association. In contrast, IL-15R and MHC class II remained significantly colocalised at both resolutions. FLIM-FRET further demonstrated molecular proximity between IL-15R and both MHC class I and class II molecules, with higher FRET efficiency observed for MHC class II. Collectively, these findings indicate that IL-15R is spatially organised in proximity to both MHC class I and class II molecules on B-cell microvilli before immunological synapse formation. This arrangement at potential sites of initial B-cell-T-cell contact may facilitate the coordination of IL-15 trans-presentation and antigen presentation during the earliest stages of B-cell-T-cell interactions.

Sepsis mimics resemble sepsis clinically but may require different treatments. We evaluated whether inflammatory protein profiles could distinguish these conditions in a retrospective case-control study using 4 prospective Japanese cohorts. The study included 34 adults with sepsis and 46 with sepsis mimics. We measured 92 proteins using Olink, assessed between-group differences with false discovery rate (FDR) adjustment, and evaluated 3 prediction models using repeated nested cross-validation. No protein met the FDR threshold; the lowest FDR-adjusted P value was 0.345. The primary elastic-net model had an area under the receiver operating characteristic curve of 0.442 (95% confidence interval, 0.315-0.574), based on cross-validation predictions averaged for each participant. Random forest and gradient boosting also showed no clear evidence of discrimination above chance. Confidence intervals included chance performance in both the all-serum and quality-control-Pass sensitivity analyses. In this cohort, a single measurement of an inflammation-focused protein panel showed limited discrimination between sepsis and sepsis mimics. Integration with clinical information and longitudinal measurements warrants further investigation.

Background. Persistent SARS-CoV-2 antigen is a leading hypothesis for post-acute sequelae of SARS-CoV-2 infection (PASC), commonly known as Long COVID. Impaired Fc-mediated antiviral antibody function could contribute to inefficient clearance of infected cells and viral antigen despite preserved antibody levels. Therefore, we tested whether PASC is associated with altered Fc{gamma} receptor-mediated antibody function and differences in IgG1 Fc fucosylation, a major regulator of Fc effector function. Methods. Serum samples from RECOVER-Adult participants with prior SARS-CoV-2 infection (99 PASC, 139 non-PASC) were analyzed for anti-SARS-CoV-2 spike receptor-binding domain (RBD) IgG and subclasses, Fc{gamma}RIIIa- and Fc{gamma}RIIa-mediated reporter activity as surrogate measures of antibody-dependent cellular cytotoxicity (ADCC) and antibody-dependent cellular phagocytosis (ADCP), anti-RBD IgG1 Fc glycosylation, and circulating fucose-modifying enzymes. Results. Despite higher anti-RBD IgG and IgG1 abundance, PASC sera showed lower ADCC- and ADCP-associated Fc{gamma}R reporter activity, including after normalization for antibody abundance. Anti-RBD IgG1 showed greater fucosylation in PASC samples (30 PASC, 34 non-PASC). Serum FUCA2 was lower in PASC samples (24 PASC, 34 non-PASC), whereas FUCA1 and soluble CD16a levels did not differ between the two groups. Conclusion. PASC is associated with impaired antiviral IgG Fc function, increased IgG1 Fc fucosylation, and reduced circulating FUCA2, identifying a glycoimmune axis that may contribute to impaired Fc-dependent antigen clearance and represents a potentially targetable pathway in Long COVID.

Platelet-leukocyte aggregates (PLAs) are a proposed interface between coagulation and immune regulation, but their distribution across leukocyte subsets beyond monocytes and neutrophils, and their behavior in acute disorders of immune regulation, such as sepsis and severe trauma, remain largely undefined. The study of thromboimmune mechanisms in these acute conditions has relied heavily on murine sepsis models, whose species differences in platelet and leukocyte biology limit translation to human disease. Direct measurement in human whole blood is needed yet reported PLA frequencies vary widely because quantification is highly sensitive to sample handling. Here we present a robust, minimally manipulative 27-color spectral flow cytometry protocol that allows simultaneous deep immunophenotyping and classification of PLAs across >40 leukocyte subpopulations. In healthy donors, PLA frequency ranged from 1.7% on memory CD4 T cells to over 50% on monocyte subsets, with monocytes contributing 25% of all PLAs. We also found novel platelet-binding leukocyte populations, including Th9 and Th22 CD4+ T cells. Optimization identified anticoagulant choice, centrifugation conditions, dilution, and sample processing time as key determinants of reproducible measurement; PLA quantification was validated by imaging flow cytometry. In a small validation cohort of sepsis and trauma patients, the assay recapitulated established immunophenotypic features of critical illness, including neutrophilia and myeloid-derived suppressor cells (MDSC) expansion, and quantified PLAs within each subset. This protocol provides a reference framework for studying thromboinflammation and other dysregulated immune response diseases directly in human blood, and a foundation for further studies, defining the mechanisms and clinical relevance of platelet-leukocyte interactions in disease.

Antiviral monoclonal antibodies (mAbs) rely on Fc-mediated effector functions, which depend on interactions with Fc receptors (FcRs) expressed on immune cells such as monocytes and dendritic cells (DCs). While combinations of antiviral mAbs with type I interferons (IFN-I) or IFN inducers are being explored to enhance therapeutic efficacy, the impact of these immunomodulators on FcR expression remains poorly understood. In this study, we investigated the effects of IFN-2, IFN-{beta}, and the IFN-inducer TAK-981 (a SUMOylation inhibitor) on FcR modulation and functional activation in human monocyte subsets and monocyte-derived DCs (moDCs). Using flow cytometry and imaging techniques, we mapped the expression of activating Fc{gamma}Rs (CD64, CD32A, CD16A) and neonatal FcR (FcRn) under steady-state and stimulated conditions. We found that IFN-I selectively downregulates CD16A expression in classical monocytes while preserving other FcRs, whereas TAK-981 reduces FcRn expression in classical monocytes without affecting CD16A. Additionally, IFN-{beta} and TAK-981 but not IFN-2, selectively enhanced Fc{gamma}RI expression in moDC. IFN-{beta} also induced a stronger maturation and functional activation program in monocytes and moDCs compared with IFN-2, whereas TAK-981 largely reproduced the IFN-{beta}-driven phenotype and secretory profile. Furthermore, we demonstrated that IFN-I signaling alters the spatial distribution of FcRs, with IFN-{beta} and TAK-981 promoting distinct redistribution patterns of Fc{gamma}Rs in monocytes and moDCs. These findings reveal a novel layer of FcR regulation, highlighting that IFN-I and IFN inducers differentially modulate FcR expression, localization, and functional activation in a cell-type- and stimulus-specific manner. Overall, this work provides a framework for understanding how IFN-I-based immunotherapies may influence Fc-mediated immune functions while preserving FcRn-dependent antibody persistence.

Core fucosylation (alpha1,6-fucosylation) of N-glycans, catalyzed solely by fucosyltransferase 8 (FUT8), plays important roles in development, immunity, and antibody effector functions. Here we report the generation of Fut8 floxed mice using a CRISPR/Cas9-based Extreme Genome Editing (EGE) system, in which loxP sites flank exon 8 of the Fut8 gene and a LoxP-STOP-LoxP-RFP cassette was knocked in downstream of the promoter, rendering the targeted allele functionally null before Cre-mediated recombination. Crossing floxed mice with CMV-Cre transgenic mice yielded complete knockout mice carrying the Cre-recombined allele. A PCR-based genotyping method was established that unambiguously distinguishes the wild-type, heterozygous, and homozygous genotypes (+/+, delta/+, and delta/delta). Intercrossing of heterozygous mice revealed that homozygous knockout mice die during the early postnatal period, consistent with previous reports, whereas heterozygous mice are viable. Flow cytometric analysis of AAL binding to splenic B cells and AAL lectin blotting both demonstrated markedly reduced core fucosylation in heterozygous mice (60.0 +/- 6.8% and approximately 48% of wild-type levels, respectively). These results show that disruption of a single Fut8 allele substantially reduces core fucosylation, and the mouse model described here provides a practical tool for functional studies of core fucosylation.

The skull bone marrow axis has recently emerged as an important source of immune cells during neuroinflammation, including in the experimental autoimmune encephalomyelitis (EAE) , the mouse model of multiple sclerosis. The cribriform plate bone marrow (cpBM), is a portion of the ethmoid bone of the skull of mice, and is positioned next to nasal regions. At the cribriform plate olfactory nerves pass through the skull and recent evidence supports that it represents an important immune niche with links to CSF outflow and connections to cpBM. Previous data supports cellular composition of the ethmoid bone marrow in mice is altered in neuroinflammation suggesting it senses inflammatory cues, but the exact nature of these changes is still unclear. Here using scRNAseq and flow cytometry of the ethmoid bone marrow region we test how the region responds to EAE. In addition we also compare the cpBM region with systemic bone marrow in the femur to understand region specific differences. We report that neuroinflammation creates a regulatory response at these sites and implicates the cpBM in controlling these effects. A better understanding of the cribriform niche may offer new treatments for neuroinflammatory disease

Aspergillus fumigatus is the most common cause of invasive aspergillosis (IA), an opportunistic pulmonary infection of immunocompromised patients that can be fatal, despite treatment with modern antifungal drugs. During infection, myeloid cells, such as neutrophils, monocytes, and monocyte-derived dendritic cells (Mo-DCs) are recruited to the lung and are critical for phagocytosing and killing A. fumigatus spores. Recent evidence has shown that intracellular crosstalk between these professional immune cells and other resident pulmonary cell types is required for an effective immune response to A. fumigatus, and that signaling via the cytokine granulocyte macrophage-colony stimulating factor (GM-CSF) is part of this intracellular communication. In this study, we sought to determine a role for GM-CSF signaling on monocytes and their progeny. We harnessed a mouse model of deletion of GM-CSF receptor {beta} chain (encoded by Csf2rb) in CCR2-expressing cells. Using this model, we found that GM-CSF signaling is required on CCR2+ cells for killing of A. fumigatus and for host survival. This observation expands the importance of GM-CSF in host defense during A. fumigatus pulmonary infection and extends its role in pathways of intracellular crosstalk that mediate effective pulmonary antifungal immunity.

Dendritic cells (DCs) are central orchestrators of antiviral immunity but can be exploited by HIV-1 to facilitate systemic dissemination. HIV-1 recognition by CD169 triggers massive endocytosis (MEND)-related internalization of viral particles into sac-like compartments in monocyte-derived dendritic cells (MDDCs), thereby promoting trans-infection of CD4+ T cells. However, MDDCS represent only one DC subpopulation, and whether other physiologically relevant DC subsets exhibit similar behavior remains unclear. Moreover, endogenous CD169 expression and viral capture have not been fully characterized in cDC1, DC2, or pDCs, while remain unknown in DC3. We therefore investigated the contribution of these DC subsets to CD169-mediated HIV-1 capture and subsequent viral dissemination. We used flow cytometry to assess CD169 expression and viral capture across DC subsets differentiated from CD34 hematopoietic stem and progenitor cells (HSPC) obtained from bone marrow (BM) and cord blood (CB), two sources that differ in cellular maturity. Confocal microscopy was used to evaluate sac-like compartment formation following immune activation with IFN- or LPS and treatment with MEND inhibitors. Trans-infection capacity was quantified by measuring luciferase activity in TZM-bl reporter cells. We identified cDC1, DC2, DC3, moDC (similar to MDDCs), and pDC subsets following the differentiation of CD34 HSPC from both CB and BM. Among these populations, DC2, DC3, and moDC displayed high CD169 expression, which was significantly upregulated by IFN- or LPS stimulation. BM-derived DCs exhibited higher basal CD169 expression than CB-derived DCs, whereas stimulation induced greater CD169 upregulation in CB-derived DCs. DC2, DC3, and moDC efficiently captured HIV-1 particles in cultures derived from both sources, and viral uptake was further enhanced by immune activation. Viral internalization into sac-like compartments depended on CD169 binding and MEND-related mechanisms, ultimately facilitating HIV-1 transfer to target cells. DC2 and DC3 are key dendritic cell subsets that support CD169 dependent HIV-1 capture and are associated with MEND-driven internalization into sac-like compartments and subsequent viral transfer to target cells, as was previously described for moDCs. We further observed distinct basal and inducible CD169 expression profiles in BM- and CB-derived DCs, consistent with source-dependent maturation states. These results identify DC2 and DC3 as key cellular contributors to HIV-1 dissemination.

Prostaglandin E2 (PGE2) is recognized as a major mediator of inflammatory pain. However, the intracellular signaling mechanisms by which PGE2 mediates pain remain unclear. Here, we show that pain-like responses evoked by PGE2 in dorsal root ganglion (DRG) nociceptors are mediated by internalization and compartmentalized signaling of the EP4 receptor. The EP4-selective agonist L-902,688 induced immediate nocifensive behavior and prolonged mechanical allodynia in mice and sensitized isolated DRG nociceptors. Pharmacological inhibition of clathrin- and dynamin-mediated endocytosis (using pitstop2 and dyngo4a) and siRNA knockdown of Dnm1 prevented EP4-induced nociception and sensitization, implicating receptor trafficking in pain signaling. Using genetically-encoded biosensors, we monitored EP4 trafficking and downstream signaling within subcellular compartments of HEK293 cells. PGE2 stimulated dynamin- and {beta}-arrestin-dependent EP4 trafficking from the plasma membrane to early, late and recycling endosomes and Golgi apparatus, and mobilized intracellular EP4 pools from the endoplasmic reticulum. PGE2 induced the assembly of EP4, G proteins and {beta}-arrestin signaling complexes in endosomes and the Golgi apparatus. Inhibition of EP4 endocytosis suppressed intracellular cAMP production and ERK activation. Our findings reveal that EP4-mediated nociceptive signaling originates from intracellular compartments and suggest that modulating EP4 internalization and subcellular signaling dynamics could offer a novel strategy for inflammatory pain management.

Macrophage-driven inflammation underlies many inflammatory diseases, yet the mechanisms that restrain macrophage activation within inflamed tissues remain poorly understood. Using mouse models of foreign body granuloma formation and macrophage activation syndrome, we show that neutrophils accumulate at sites of inflammation and metabolically restrain macrophage-driven pathology. Neutrophil depletion or genetic impairment exacerbated tissue pathology, whereas induced neutrophilia attenuated inflammatory damage. Tissue-infiltrating neutrophils acquired an alternatively polarized state characterized by high arginase 1 activity that was regulated by an IL4Ra-STAT3-NOX2 signaling axis. Functionally, polarized neutrophils depleted extracellular arginine in an arginase 1-dependent manner, thereby limiting macrophage effector programs. Human neutrophils exhibited conserved regulation of arginase activity, and alternatively polarized neutrophils were detected in patients with sarcoidosis and macrophage activation syndrome. Together, these findings identify an arginine-dependent metabolic mechanism through which alternatively polarized neutrophils restrain macrophage-driven inflammation and reveal a previously unrecognized immune-regulatory role for these cells in chronic inflammatory disease.

Sepsis causes approximately 11 million deaths globally each year. Whole-genome sequencing (WGS) of founder populations offers a unique opportunity for discovery of genes related to susceptibility of sepsis. We performed GWAS and EXWAS on WGS data for n=210 Arab ICU patients (n=68 sepsis cases, n=142 controls, other ICU-related disorders) and prioritized n=40 associated deleterious founder variants in n=31 genes (REGENIE Firth regression p < 0.05; CADD score > 20; enriched AAF vs AllofUs/gnomAD), including a novel start-loss variant in UBE2L3 (c.2T>G; p.Met1?; CADD = 25.6; SIFT = 0.001) in sepsis cases (3/68 vs 0/142 controls; Fisher's exact P = 0.026). In All of Us (AoU), UBE2L3 missense burden was associated with bacterial infection (n=26,939 cases; SKAT-O P = 2.38x10-3; OR=23.10; 95% CI = [0.63-845.27]). LLM AI models fine-tuned for drug development scored UBE2L3 a 7.8 of 10 as a drug target for sepsis, consistent with mouse UBE2L3 KO showing increased IL-1Beta mediated inflammation in sepsis.

BACKGROUND: Severe influenza pneumonia with secondary bacterial infection is complicated by progressive pulmonary thrombus exacerbation, a key contributor to respiratory failure, yet anticoagulant therapies show limited efficacy and bleeding risks. Although platelet-monocyte crosstalk initiates thrombosis, whether and how it drives thrombus exacerbation via procoagulant platelets and monocyte efferocytosis remains unclear. METHODS: Clinical samples, mouse models, and isolated platelets challenged with influenza A virus followed by methicillin-resistant Staphylococcus aureus (MRSA) were analyzed. Procoagulant platelet, platelet programmed cell death and monocyte efferocytosis were assessed, and pharmacological inhibition, platelet depletion and systemic/platelet-specific Gsdmd knockout were used. Platelet proteomics and exogenous C1qa supplementation identified C1qa as a key mediator. RESULTS: We showed that elevated procoagulant platelet-driven thrombus exacerbation, rather than initial thrombus formation, was a critical driver of disease progression in influenza pneumonia with secondary bacterial infection, whereas influenza alone caused milder illness. Procoagulant platelet formation was induced via MLKL-mediated necroptosis and GSDMD-mediated pyroptosis. These platelets exacerbated pulmonary thrombosis and lung injury by inhibiting monocyte efferocytosis via complement C1qa. Platelet depletion reduced monocyte efferocytosis and worsened pneumonia, while pharmacological inhibition or platelet-specific Gsdmd knockout decreased procoagulant platelet levels, restored monocyte efferocytosis, and alleviated thrombotic and pulmonary injury. Mechanistically, C1qa impaired efferocytosis both by directly suppressing monocyte function and by reducing the proportion of reparative (M2-like) monocytes. Clinical relevance was confirmed by detection of MLKL/GSDMD-dependent procoagulant platelets and reduced efferocytosis receptor levels on monocytes in bronchoalveolar lavage fluid from severe pneumonia patients. CONCLUSIONS: Necroptosis/pyroptosis-driven procoagulant platelets exacerbate pulmonary thrombosis by suppressing monocyte efferocytosis in a C1qa-dependent manner. These findings extend platelet-monocyte crosstalk from thrombus initiation to thrombus exacerbation, identifying modulation of the interaction between procoagulant platelets and monocyte efferocytosis as a potential therapeutic strategy for thrombus-exacerbating diseases, especially in subpopulations of patients with severe pneumonia and progressive pulmonary thrombosis.

Atherosclerosis is a chronic inflammatory disease primarily initiated by the sub-endothelial retention and modification of low-density lipoproteins (LDL), such as oxidation and aggregation. While oxidized LDL (oxLDL) exists in atherosclerotic lesions, aggregated lipoproteins are also prominent. Aggregated LDL (agLDL), for instance, is viewed as a passive substrate for macrophages to uptake and form foam cells. Recent studies show that agLDL, through toll-like receptor 4 (TLR4), induces formation of lysosomal synapses, which drive agLDL degradation by macrophages, a process described as "digestive exophagy". As TLR4 is one of the pattern-recognition receptors for inflammatory responses in macrophages, we postulated that agLDL could initiate macrophage inflammation along with lipoprotein degradation/internalization, thereby contributing to a low degree of chronic inflammation in atherosclerosis. Here, we demonstrate that agLDL directly activates the canonical NF-{kappa}B signaling pathway and triggers a pro-inflammatory transcriptional program through TLR4, which closely resembles classical lipopolysaccharide (LPS)-mediated TLR4 activation. In addition, agLDL also activates TLR4 to generate inflammatory macrophages that recruit immune cells, a key process in the development of atherosclerotic lesions. Our findings therefore support a novel dual functionality of agLDL: a lipid donor and an inflammatory agonist. This establishes the agLDL-TLR4-NF-{kappa}B axis as a direct link between LDL aggregation in the intima and the initiation of sterile vascular inflammation. It also strengthens the understanding of the role of digestive exophagy with new mechanistic details.

ABSTRACT Endothelial cells (ECs) display marked vascular bed-specific heterogeneity, and their inflammation-induced activation is a central feature of vascular pathology. However, whether inflammatory responses are primarily shaped by intrinsic organotypic endothelial identity or by the inflammatory stimulus itself remains largely unknown. To address this question, we compared the temporal responses of brain- (hCMEC/D3), skin- (HMEC-1), and lung-derived (HuLEC-5a) human microvascular ECs to lipopolysaccharide (LPS) and interleukin-6 (IL-6/IL-6R). Across all conditions, endothelial origin was the dominant determinant of transcriptional variation, outweighing the effects of both the inflammatory stimulus and exposure time. Brain-, skin- and lung-derived ECs exhibited distinct basal inflammatory and innate immune gene expression programs and responded differently to both LPS and IL-6/IL-6R. LPS induced a rapid but largely transient response, whereas IL-6/IL-6R elicited more sustained inflammatory changes. Although transcriptional profiling identified a shared inflammatory signature across EC populations, most differentially expressed genes were vascular bed specific. These molecular differences translated into distinct functional outcomes: IL-6/IL-6R increased endothelial permeability and promoted more sustained metabolic adaptations, whereas LPS induced robust inflammatory activation without persistent barrier dysfunction. Collectively, these findings demonstrate that while inflammatory stimuli determine which signaling pathways are activated, intrinsic endothelial identity dictates the magnitude, kinetics, and functional consequences of the inflammatory response. Our results highlight the importance of incorporating vascular bed specificity into experimental models and the development of therapies targeting endothelial dysfunction and inflammation.