International Immunopharmacology

Cyclooxygenase-1 (COX-1) is classically regarded as a constitutive enzyme that produces eicosanoids with housekeeping functions, but recent evidence indicates that it may also be involved in the acute phase of severe systemic inflammation. There is evidence indicating that COX-1 is selectively activated in the spleen via post-translational mechanisms early the course of LPS-induced systemic inflammation. However, the mechanistic link between COX-1 and the spleen has not yet been demonstrated in direct experiments. The present study was conducted to fill this gap. The effects of the COX-1 inhibitor SC-560 on the LPS-induced severity triad (hypotension, hypothermia and acidosis) were evaluated in rats subjected to splenectomy or in sham-operated controls. In the sham-operated group, SC-560 significantly attenuated the severity triad independently of changes in plasma cytokines (TNF and IL-1{beta}). In the splenectomized rats, SC-560 completely lost its ability to attenuate the hypotension and the acidosis induced by LPS. The effect of SC-560 on LPS-induced hypothermia was also impaired by splenectomy, though not completely. We then conducted a lipidomic screening to identify which COX-1-derived eicosanoids might be responsible for mediating the severity triad. Based on spleen-blood correlations, the screening identified PGE2 and PGD2 as putative candidates. In conclusion, the present study provides direct evidence for a mechanistic link between the spleen and COX-1 in the mediation of severity in systemic inflammation, and identifies PGE2 and PGD2 as putative candidates involved.

Background: Chronic and non-healing wounds remain a major clinical challenge with limited therapeutic options. Angiogenesis and inflammation are central to tissue repair, and mesenchymal stem cells (MSC) contribute to these processes through their trophic and immunomodulatory secretome. Cannabidiol (CBD) exhibits antioxidant and immunomodulatory properties. However, whether CBD-rich Cannabis sativa extract stimulate MSC toward a pro-angiogenic secretome remains unclear. Purpose: This study aims to determine whether purified CBD or a phytochemically CBD-rich full spectrum extract stimulate umbilical cord-derived human MSC (UC-hMSC) to secrete pro-angiogenic factors and enhance endothelial responses relevant to wound healing. Methods: UC-hMSC were preconditioned with either purified CBD or a CBD-rich full-spectrum extract. Transcriptional changes were assessed by qPCR. The functional impact of the resulting secretome was evaluated in vitro using HUVEC-based proliferation and tube formation assays, and in vivo through the chick chorioallantoic membrane assay. To explore underlying mechanisms, we examined HIF-1 stabilization and VEGFA release in UC-hMSC, and VEGFR-2/ERK signaling in HUVEC. Results: Purified CBD and full-spectrum CBD extract preconditioned UC-hMSC secretomes, increased HUVEC proliferation, tube formation, and enhanced vascular branching in the CAM assay. Mechanistic analyses indicated activation of the HIF-1/VEGF axis in UC-hMSC, and ERK1/2 activation in HUVEC that was sensitive to VEGFR-2 blockade. Conclusion: Purified CBD and CBD-rich full-spectrum extract prime UC-hMSC toward a pro-angiogenic secretome that promotes endothelial activation and neovascularization. These findings suggest that cannabinoid-based preconditioning of UC-hMSC involves the HIF-1/VEGF axis and VEGFR-2/ERK signaling pathways in endothelial cells, supporting further investigation of this approach in wound healing and regenerative therapies.

Thymosin {beta}4 (T{beta}4) is a conserved acidic polypeptide with 43-amino acids participating in multiple pathophysiological processes. In this study in vivo effects of T{beta}4 on liver regeneration are investigated in carbon-tetrachloride (CCL4) induced rodent animal liver jury models. Results illustrate that exogenous T{beta}4 treatment significantly reduced CCL4-rendered liver necrosis around central vein. At 48 hours after CCL4 insults hepatocytes proliferation occur mainly around the periportal area, while hepatocytes proliferation around the necrosis area is prominently increased by exogenous T{beta}4 treatment. The holistic proliferation level of liver tissues are also enhanced by exogenous T{beta}4. Hepatocyte proliferation activities negatively correlate with the necrosis extent of the liver tissue. These results suggested firstly exogenous T{beta}4 treatment could enhance liver regeneration and exhibit prosperous potential for application in clinical conditions such as liver transplantation.

Objectives; The role of nuclear receptor subfamily 4, group A, member 3 (NR4A3) in hepatic steatosis, inflammation, and insulin resistance (IR) within the context of metabolic dysfunction-associated steatotic liver disease (MASLD) remains largely underexplored. Consequently, this study aimed to examine NR4A3's impact on MASLD and the potential underlying mechanisms. Methods; We aimed to elucidate the functional role of NR4A3 in MASLD through its knockdown in cell culture and animal models. To establish the cell culture model of MASLD, LO2 cells were treated with free fatty acids (FFAs), while male C57BL/6 mice were fed a high-fat diet (HFD) to create the animal model. NR4A3 knockdown was achieved using specific short hairpin RNA (NR4A3-shRNA) in the mice model and three small interfering RNAs (NR4A3-siRNAs) in the cell culture model. The lipids content, fatty acid synthesis, inflammatory factors, and IR were then assessed with and without NR4A3 knockdown. Furthermore, the underlying mechanism through which NR4A3 exerts its influence was explored by analyzing the interaction between NR4A3 and activating transcription factor 3 (ATF3). Results: In the cell culture experiments, the knockdown of NR4A3 significantly decreased the lipids content, fatty acid synthesis, and inflammatory factors in the LO2 cells treated with FFAs in the NR4A3-shRNA group compared with those in the NC-shRNA control group. In the animal model experiments, NR4A3 knockdown in the HFD male C57BL/6 mice significantly ameliorated HFD-induced hepatic steatosis, inflammation, and IR. Mechanistically, the knockdown of NR4A3 downregulated the expression and transcriptional activity of ATF3, resulting in an impaired ATF3 function. ATF3 overexpression significantly reversed lipid accumulation decline and reduced inflammation after NR4A3 knockdown. Conclusion: The downregulation of NR4A3 alleviates MASLD by modulating ATF3, suggesting this may be a promising therapeutic target.

BACKGROUND: Perfluorooctane sulfonate (PFOS) is a widely distributed persistent organic pollutant in the environment and has been associated with an increased risk of atherosclerosis. However, the underlying pathogenic mechanisms remain largely unclear. This study aimed to investigate the effects of PFOS on atherosclerosis and its associated gut-vascular axis. METHODS: Pseudo-germ-free mouse models and fecal microbiota transplantation (FMT) were used to determine the role of the gut microbiota in PFOS-induced atherosclerosis. Metagenomic sequencing was performed to characterize alterations in gut microbial composition following PFOS exposure, and targeted metabolomics was used to assess bile acid profiles in the ileum and plasma. Transcriptomic analysis of Bacteroides caecimuris (B.caecimuris) was conducted to explore the reasons for the increased abundance of B.caecimuris after PFOS exposure. In addition, intestinal transcriptomics and ChIP-qPCR were performed to validate transcriptional regulation within the FXR-TLR3 signaling axis. RESULTS: Among 127 participants with paired serum and fecal samples, including 82 patients undergoing coronary angiography with Gensini scores (GS score), fecal PFOS levels were significantly associated with lipid profiles and GS score, whereas serum PFOS showed no such association. Mechanistically, PFOS exposure promotes intestinal enrichment of B. caecimuris by upregulating its tolC gene, thereby enhancing efflux capacity. This microbial shift was accompanied by reduced levels of tauro-ursodeoxycholic acid (TUDCA) and aberrant activation of intestinal FXR signaling. Further analyses demonstrated that FXR activation upregulated TLR3 expression and promoted inflammatory responses and atherosclerosis progression via the TLR3-NF-{kappa}B signaling axis. Both intestinal epithelial-specific FXR deficiency (Fxr{Delta}IE) and TUDCA supplementation significantly suppressed pathway activation and alleviated disease phenotypes.Functional experiments identified TLR3 as a key downstream effector of FXR. Overexpression of TLR3 abolished the protective effects observed in Fxr{Delta}IE mice. Moreover, pharmacological inhibition of TLR3 using CU CPT-4a significantly improved established atherosclerotic lesions in vivo. CONCLUSIONS: This study identifies a gut microbiota-driven FXR-TLR3 signaling axis that mediates PFOS-induced atherosclerosis. These findings provide new mechanistic insights into environmentally induced cardiovascular disease and suggest potential targets for risk assessment and therapeutic intervention.

BackgroundAtherosclerosis is a chronic inflammatory vascular disorder with persistent residual inflammation even after standard lipid-lowering therapy. Mounting evidence from bench to bedside suggests that diabetes and obesity accelerate atherosclerosis development. Tirzepatide (TZP), a dual Glucagon-Like Peptide-1 Receptor/Glucose-Dependent Insulinotropic Polypeptide Receptor (GLP-1R/GIPR) agonist approved for treating diabetes and obesity, has demonstrated proven cardiometabolic efficacy in large cardiovascular outcome trials. However, it remains largely uncertain whether TZP attenuates atherosclerosis independent of its anti-diabetic and anti-obese effects through direct actions on the vasculature. MethodsWe established atherosclerotic mouse models under diabetic, obese, and non-diabetic/non-obese conditions. Analysis of covariance (ANCOVA) and pair-feeding experiments were applied to experimentally decouple weight-dependent metabolic improvement from intrinsic vasculoprotection. Molecular and cell biological assays in human umbilical vein endothelial cells (HUVECs) and human aortic endothelial cells (HAECs) were performed to dissect the underlying signaling mechanisms. ResultsTZP markedly reduced aortic plaque burden and inflammation, restrained necrotic core enlargement, and improved plaque stability across all experimental mouse models. Both ANCOVA and pair-feeding experiments confirmed that these atheroprotective effects were independent of food intake and body weight loss. Furthermore, TZP attenuated systemic and vascular inflammation in Tumor Necrosis Factor- (TNF)-treated C57BL/6J mice, and this protection occurred without changes in body weight or blood glucose levels. Mechanistically, TZP directly targeted endothelial cells and activated the cyclic adenosine monophosphate (cAMP)/protein kinase A (PKA)/endothelial nitric oxide synthase (eNOS) pathway, increased eNOS phosphorylation and nitric oxide bioavailability, consequently downregulating the expression of the pro-inflammatory adhesion molecules Vascular Cell Adhesion Molecule-1 (VCAM-1) and Intercellular Adhesion Molecule-1 (ICAM-1). ConclusionsTZP arrests atherosclerosis progression through weight loss-independent anti-inflammatory mechanisms. These findings implicate TZP as a promising therapeutic drug for mitigating residual vascular inflammation in patients with atherosclerotic cardiovascular disease (ASCVD), irrespective of glycemic status or obesity. Clinical PerspectiveO_ST_ABSWhat Is New?C_ST_ABSO_LITirzepatide exerts direct anti-atherosclerotic effects in preclinical mouse models of atherosclerosis under diabetic, obese, and non-obese conditions. C_LIO_LITirzepatide directly targets endothelial GLP-1R/GIPR and downstream cAMP/PKA/eNOS signaling pathway to suppress NF-{kappa}B-driven vascular inflammation, thereby uncovering a previously unrecognized vasculoprotective mechanism underlying its cardiovascular benefits C_LI What Are the Clinical Implications?O_LITirzepatide exerts direct vascular protective effects independent of body weight reduction, suggesting that its cardiovascular benefits may extend beyond glycemic control and obesity management. C_LIO_LITirzepatide may represent a promising therapeutic drug for addressing residual vascular inflammation in ASCVD patients, including those without overt diabetes or obesity C_LI

Type 1 diabetes (T1D) is caused by T cell-mediated autoimmune destruction of insulin-producing islet beta-cells. Treatment with T-cell depleting therapies delays the progression of stage 2 and 3 T1D, but these agents exert broad immunosuppressive effects on T cell populations, including T regulatory cells (Tregs), which are key in promoting immune tolerance. We evaluated non-obese diabetic (NOD) mice and recently diagnosed T1D patients and identified CD38 as a marker for pathogenic T cell populations. Using adoptive T-cell transfer in Recombination Activating Gene 1 knockout NOD mice and in a humanized mouse model of autoimmune diabetes, we demonstrated that CD38-expressing autoreactive T cells drive diabetes pathogenesis. Furthermore, we found that selective depletion of CD38+ cells, using an anti-CD38 monoclonal antibody (mAb), prevents insulitis and diabetes onset without depleting CD4+CD25+ Tregs. Administration of anti-CD38 mAb did not adversely affect islet function and may selectively eliminate immunogenic senescent islet beta-cells. These results support the strategy of selectively depleting diabetogenic T cells using an anti-CD38 mAb to treat T1D and restore immune tolerance. Therefore, transient depletion of autoreactive T cells using anti-CD38 mAb may provide a novel strategy to prevent or abrogate autoimmunity in T1D.

BackgroundSuccinate has emerged as an immunometabolic mediator of cardiovascular diseases. However, the enzymatic mechanisms linking macrophage succinate metabolism to aortic dissection remain incompletely understood. This study investigated whether Suclg2, which encodes the GDP-forming {beta}-subunit of succinyl-CoA ligase, regulates succinate-associated macrophage remodelling and aortic dissection progression. MethodsSuclg2 expression was examined in BAPN-induced AD and human acute type A aortic dissection tissues by Western Blot and immunofluorescence. Myeloid- and smooth muscle cell-specific Suclg2 conditional knockout mice were subjected to BAPN treatment to evaluate survival, aortic outcomes, histological injury and aortic morphology. Aortic RNA-seq was used to discover transcriptional changes. Bone marrow-derived macrophages were analysed under basal, M1-like and M2-like conditions to assess macrophage-intrinsic transcriptional responses. Plasma succinate levels and untargeted metabolomic profiles were further examined. ResultsSuclg2 was increased in murine and human dissected aortas and partially localized to CD68 cells. Myeloid Suclg2 deletion markedly reduced BAPN-induced aortic rupture and dissection, whereas smooth muscle cell Suclg2 deletion did not confer comparable protection. Aortic transcriptomic analysis showed that myeloid Suclg2 deficiency attenuated inflammatory adhesion and matrix-destructive programmes. In macrophages, Suclg2 deletion did not induce a simple M1/M2 polarization shift; instead, it remodelled lipid-handling, phagolysosomal, adhesive and matrix-remodelling pathways across stimulation states. Metabolic profiling showed reduced circulating succinate and broader changes in central carbon, lipid-associated, nucleotide and redox-related metabolites after myeloid Suclg2 deletion. ConclusionsMyeloid Suclg2 is a succinate-associated immunometabolic regulator of aortic dissection. Its deficiency protects against aortic dissection by reshaping macrophage inflammatory-remodelling programmes and the systemic metabolic environment.

Rupture of the fibrous cap is the primary cause of clinical complications from atherosclerosis. Smooth muscle cells (SMCs) are a major contributor to fibrous cap development and stability through de-differentiation to extracellular matrix-producing cells. We previously showed that the antioxidant enzyme cytoglobin (CYGB) is expressed in vascular SMCs and regulates SMC dependent vascular remodeling and gene expression. In the present study, we investigated the function of SMC-CYGB in atherosclerosis. To this end, we generated a mouse line with SMC-specific deletion of Cygb and simultaneous SMC-lineage tracing. We found that SMC specific deletion of CYGB increased fibrous cap thickness in a 17-week PCSK9-AAV8 gain of function combined with Western diet mouse model of atherosclerosis. SMC specific deletion of CYGB increased collagen deposition and SMC cellularity of the fibrous cap in the absence of changes in total plaque and necrotic core sizes. CYGB expression in SMCs was associated with transdifferentiation towards a fibroblast-like, matrix remodeling phenotype. Finally, CYGB was expressed in the fibrous cap of human coronary atherosclerotic lesions and was associated with ACTA2 positive cells. These results provide first-time evidence that SMC-CYGB reduces plaque stability by decreasing cap thickness, collagen deposition, and SMC cellularity.

Abdominal aortic aneurysms (AAAs) occur predominantly in the elderly population and currently there is no effective pharmacological therapy for mitigating AAA growth and preventing impending rupture. Proprotein subtilisin kexin type 9 (PCSK9) gene has been identified as a specific risk-locus for AAA development. However, the mechanistic and clinical role of PCSK9-mediated signaling in AAAs has not been delineated. We demonstrate that treatment with PCSK9 inhibitors, such as Evolocumab, mitigates vascular inflammation and remodeling, resulting in attenuated aneurysm growth in clinical datasets as well as experimental models of AAA and aortic rupture. Mechanistically, Evolocumab immunomodulates macrophage reprogramming to enhance clearance of apoptotic smooth muscle cells via MerTK-dependent efferocytosis that ameliorates aortic inflammation and vascular remodeling. Furthermore, Evolocumab increases the expression of oxidized phosphatidylserine species and decreases expression of lysophospholipids, succinate, and glycolytic intermediates within the aortic wall compared to untreated controls, further enhancing the pro-resolving functions of macrophages. Collectively, our data demonstrates the ability of PCSK9 inhibition to regulate macrophage-specific efferocytosis that limits AAA progression and prevents aortic rupture.

Reliable serological tools are needed to measure mpox virus (MPXV) exposure, evaluate vaccine-induced immunity, and support population-level surveillance. Using a previously established six-antigen serological reference framework, in which seropositivity was defined as reactivity to [≥]4 of 6 MPXV antigens, we evaluated the diagnostic performance of individual antigens and all 15 pairwise combinations. B6R demonstrated the highest overall individual discriminatory performance, whereas A35R showed maximal sensitivity and M1R the highest specificity. The A35R+B6R combination most closely approximated the full multiplex assay (AUC 0.93), supporting simplified, scalable MPXV serological assays for surveillance and vaccine evaluation.

Background and aimsAlcohol-associated hepatitis (AH) is characterized by excessive inflammation and blunted antiviral interferon (IFN) responses. We hypothesized that specific gut microbiome-derived metabolites could selectively enhance interferon signaling while limiting NF-{kappa}B mediated inflammation, thereby restoring immune balance in AH. Our goal is to identify microbiome-derived metabolites that differentially regulate the NF-{kappa}B and IFN signaling pathways. Methods and resultsWe used human monocytic THP1-Dual cells, which secrete reporters for NF-{kappa}B and IFN signaling, to model innate immune responses and screened a library of 152 gut microbiome-derived metabolites. From the metabolite screen, 4-hydroxyphenylacetic acid (4-HPAA) emerged as a unique immunomodulator: in LPS-challenged cells, 4-HPAA selectively increased IFN signaling with minimal NF-{kappa}B activation. 4-HPAA was evaluated in vivo using a NIAAA-model, with 4-HPAA supplementation (0.4mg/ml) added to the diet. In the NIAAA-model, dietary 4-HPAA did not induce liver injury and was associated with enhanced interferon-stimulated gene expression. Simultaneously, 4-HPAA reduced pro-inflammatory markers such as Il1{beta}, Ly6g and F4/80 compared to the group exposed to ethanol alone. Metabolomic profiling of mouse cecal contents revealed 4-HPAA supplementation counteracted ethanols metabolic effects, selectively reducing triglyceride-associated lipids that had accumulated with ethanol feeding. Conclusions4-HPAA enhances interferon signaling and antiviral gene induction while dampening NF-{kappa}B-driven inflammation in the presence of LPS, both in vitro and in vivo. In an acute-on-chronic alcohol injury model, 4-HPAA attenuated hepatic inflammation, reduced immune cell recruitment, and activated antioxidant defenses, reflecting a shift toward a more hepatoprotective effect. 4-HPAA treatment was associated with reduced pro-inflammatory markers and modest attenuation of ethanol-induced liver injury. Additionally, 4-HPAA reversed ethanol-induced lipid-dysregulation, particularly triglyceride accumulation, highlighting its metabolic benefit in alcohol-fed mice. In conclusion, 4-HPAA rebalances immune and metabolic pathways by enhancing IFN signaling, suppressing NF-{kappa}B inflammation, and reversing alcohol-induced hepatic injury and lipid accumulation.

Cross-reactive antibodies against dengue virus are known to cause antibody-dependent enhancement (ADE) of infection or disease severity under specific conditions. In our previous study, we showed that primary immunization with the COVID-19 vaccine induces induces cross-reactive IgG causing ADE against dengue. In the present study, we investigated the influence of IgG Fc-glycosylation (analyzed by LC-MS/MS) on ADE mediated by cross-reactive IgG against dengue from IgG against SARS-CoV-2. We found a clear correlation between anti-DENV2 E IgG2 galactosylation and the ADE capacity of cross-reactive IgG against dengue in individuals vaccinated against COVID-19. IgG2 sialylation increased over time; however, it was not correlated with ADE capacity. This phenomenon was restricted to IgG2, whereas anti-DENV2 E IgG1 Fc-glycosylation remained stable after COVID-19 vaccination.

The IFN-I (type I interferon) signaling pathway is the first line of defence against foreign pathogens. Stringent control of signalling pathways is necessary to maintain host immune responses and homeostasis. However, the underlying mechanism for its tight regulation is yet completely understood. In this study, we demonstrated that the TRIM family protein tripartite motif-containing 52 (TRIM52) is a novel negative regulator of IFN-{beta} production. Ectopically expressed TRIM52 markedly inhibited the activation of the IFN-{beta} promoter by ectopic expression of cGAS/STING, RIG-IN, or TRIF, MAVS, STING, and TBK1 but not by IRF3/5D, indicating that TRIM52 targets TBK1. TRIM52 also significantly inhibited the IFN-{beta}, ISG54, and ISG56 production, the dimerization of IRF3 and the nuclear localization of IRF3-YFP induced by ectopic expression of TBK1. Co-immunoprecipitation experiment revealed that TRIM52 specifically interacted with TBK1. Furthermore, the TBK1 protein, but not its mRNA, decreased considerably with increasing expression of TRIM52, and TRIM52 did not decrease the expression of the cGAS, STING, or IRF3 proteins. In addition, proteasome inhibitor MG-132 blocked the reduced TBK1 induced by TRIM52, indicating that TRIM52 caused TBK1 degradation via the proteasome pathway. Co-IP and ubiquitination assays demonstrated that TRIM52 promotion of K48-linked ubiquitination of TBK1, which depends on its E3 ubiquitin ligase. Collectively, our findings identify a previously unrecognized role of TRIM52 in regulating the IFN-I signalling pathway through targeting TBK1 for polyubiquitination and degradation.

Interleukin-38 (IL-38) is a cytokine of the IL-1 cytokine family that promotes the resolution of inflammation. Resolution mechanisms comprise the induction or recovery of immune tolerance that is lacking in various acute and chronic inflammatory pathologies, including Graft-versus-Host Disease (GvHD). The role of IL-38 in the context of immune tolerance, its primary immune cell targets and underlying molecular mechanisms are not defined. In this study, we investigated the impact of IL-38 on human alloreactivity and in a mouse model of acute GvHD. Our data suggests that monocytes differentiating into macrophages are the main cellular target of IL-38. Specifically, IL-38 reduces antigen presentation capacity in differentiating monocytes through an IL-1 family receptor-independent mechanism, which subsequently avoids T-cell activation. In parallel, IL-38 ameliorates inflammation in allogeneic settings in human and murine GvHD models by promoting the expansion of regulatory T-cells. Our findings indicate that IL-38 promotes immune tolerance during alloreactivity by affecting myeloid cells and T-cells.

RNA-binding protein LARP4 plays an important role in T cell activation and differentiation, but its role in B cell biology and the pathogenesis of systemic lupus erythematosus (SLE) remains unclear. This study found that LARP4 was specifically highly expressed in B cells of SLE patients and was positively correlated with disease activity. By constructing T cell-specific and B cell-specific conditional knockout mice, we found that deletion of LARP4 in B cells, but not in T cells, significantly alleviated pristane-induced and Bm12-induced lupus nephritis. Further analysis showed that LARP4 deletion selectively inhibited B cell differentiation into plasma cells, but did not affect germinal center B cell formation. Integrated transcriptomic and metabolomics analyses revealed that this effect is due to reduced phosphatidic acid synthesis and decreased mTORC1 activity caused by mitochondrial oxidative phosphorylation dysfunction. Furthermore, we used LIPEP, a LARP4 inhibitory peptide that effectively mimicked the therapeutic effects of LARP4 gene knockout in the MRL/lpr spontaneous lupus model and outperformed cyclophosphamide in reducing glomerular immune complex deposition and improving extrarenal dermatitis. These results indicates that LARP4 is a key metabolic checkpoint regulating B cell differentiation into Plasma cells and suggest that it may be a potential therapeutic target for SLE.

Acute ischemic stroke (AIS) induces profound systemic immune alterations that contribute to infection susceptibility. Here, we identify lipocalin-2 (LCN-2) as a rapidly induced and conserved regulator of stroke-associated immunosuppression. Using 3-MACE-Seq, cytokine profiling, and immunofluorescence in C57BL/6J mice subjected to transient middle cerebral artery occlusion (tMCAO), we show that LCN-2 is strongly upregulated in splenic red pulp macrophages (RPMs) within 24 hours and again 7 days post-tMCAO. LCN-2-expressing RPMs form immunological synapses with CD3+ T cells, thereby impacting T cell trafficking. Recombinant LCN-2 directly reprogrammed T cells and monocytes toward hyporesponsive, tolerogenic phenotypes by suppressing inflammatory cytokines, impairing chemotaxis, enhancing phagocytosis, and uncoupling oxidative burst. Human spleens likewise displayed LCN-2-expressing CD68+ RPMs, and LCN-2 preconditioning of monocytes reproduced reduced HLA-DR, CD80, CD206, and ROS with increased uptake of E. coli bioparticles. These findings identify LCN-2 signaling as a central orchestrator of stroke-induced peripheral immunoreprogramming and a potential therapeutic target to mitigate post-stroke immunodepression. SummaryAcute ischemic stroke induces LCN-2 in splenic red pulp macrophages, which reprogram T cells and monocytes toward tolerogenic, hyporesponsive states. Mouse and human data identify LCN-2 as a driver of peripheral immunodepression and a potential target to reduce infection risk. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=143 SRC="FIGDIR/small/733904v1_ufig1.gif" ALT="Figure 1"> View larger version (47K): org.highwire.dtl.DTLVardef@149bd46org.highwire.dtl.DTLVardef@2974aaorg.highwire.dtl.DTLVardef@1aa7a52org.highwire.dtl.DTLVardef@144dbe5_HPS_FORMAT_FIGEXP M_FIG Graphical abstract C_FIG

Background: Hypoxia develops within growing atherosclerotic lesions, inducing nuclear translocation of hypoxia-inducible factor-1 (HIF1) and metabolic reprogramming. Its role in plaque macrophages and endothelial cells has been studied, but the hypoxic plaque interior is dominated by smooth muscle cell (SMC)-derived cells, for which the role of hypoxia signaling remains unclear. Here, we investigated how loss of Hif1a in SMC lineage cells impacts plaque progression and cell phenotype in murine atherosclerosis. Methods: Atherosclerosis was induced in mice with inducible SMC-specific deletion of Hif1a (Hif1aSMC-KO) and lineage tracing of SMC-derived plaque cells. Plaque size, necrotic core size, calcification, and SMC-derived cell phenotypes were quantified in aortic root sections and gene expression changes mapped by single-cell RNA sequencing. In parallel, a cultured SMC line with or without siRNA-mediated Hif1a knockdown was exposed to hypoxia for assessments of mitochondrial function and reactive oxygen species production. Results: Hif1aSMC-KO mice developed larger plaques, with expanded necrotic cores and increased calcification, compared with littermate controls. SMC-derived plaque cells were more abundant with a higher fraction of Col2a1+ chondromyocytes, and showed elevated markers of proliferation and apoptosis, whereas macrophage and endothelial cell numbers were unaffected. Single-cell RNA sequencing analysis revealed strong dysregulation of mitochondrial genes, including electron transport chain transcripts, along with upregulation of protein folding, proteasome, and oxidative stress response pathways. In cultured SMCs subjected to hypoxia, Hif1a silencing increased cell counts, aggravated mitochondrial proton leak, and led to the accumulation of depolarized, reactive oxygen species-generating mitochondria. Further analysis of SMC-derived cells in plaques from Hif1aSMC-KO mice confirmed increased oxidative stress by 8OHdG staining. Conclusions: HIF1 maintains mitochondrial function and restrains oxidative stress in SMC-derived plaque cells in murine atherosclerosis. Its chronic loss destabilizes redox homeostasis and promotes maladaptive SMC responses, leading to SMC-driven plaque expansion, necrosis, and calcification.

Background: This study aimed to elucidate B cell subset pathology in COPD, a poorly characterized area, with a focus on its similarities to and differences from classical autoimmune disorders. Methods: Single-cell RNA-sequencing (scRNA-seq) data from COPD and autoimmune diseases were obtained from the Gene Expression Omnibus (GEO) for comparative analyses of B cell subsets and functions via differentially expressed genes (DEGs), KEGG, protein-protein interaction (PPI), and cell-cell communication analyses. Serum IgG4 was measured by ELISA and correlated with clinical parameters. Peripheral blood B cells were sorted by flow cytometry for single-cell B cell receptor (BCR) sequencing. A v-Abl-Bcl2 pro-B cell line was stimulated with cigarette smoke extract (CSE) to assess abnormal development in vitro. Results: In lung tissue, IgG4 plasma cells were enriched and expressed BCR activation and inflammatory genes and TNF-NF-kB-MAPK pathways. Serum IgG4 concentrations correlated negatively with pre- and post-bronchodilator FEV1-FVC. B cells interacted with monocytes, macrophages, fibroblasts, and endothelial cells via IL-1B-IL-6, integrin, and chemokine signaling, contributing to chronic inflammation and remodeling. In peripheral blood, transitional T1 B cells were increased, accompanied by lambda-chain enrichment and increased IGLV1-47 usage, as well as enrichment of autoimmune pathways. In the bone marrow, the numbers of pre-B I cells were increased while those of small pre-B III cells were reduced, with altered expression of BCR development genes. CSE stimulation of the pro-B cell line reduced lambda expression in a concentration-dependent manner. Conclusions: The autoimmune abnormalities in COPD appear more restricted, although IgG4 antibody generation may contribute to immune-mediated lung damage.

The cellular origin of smooth muscle cell (SMC)-derived populations in vascular lesions remains unresolved. Here we show, using single-cell transcriptomic analyses spanning carotid ligation injury, Myh11-CreERT{superscript 2}-traced aortic homeostasis, and LDLR- and ApoE-deficient atherosclerosis, that a rare progenitor-like "Primed" SMC compartment pre-exists at baseline in all models and in the healthy human aorta. Relative to contractile SMCs, Primed SMCs attenuate sarcomeric and contractile programmes while inducing matricellular, progenitor-niche and chondrogenic-poised developmental programmes, resolving into conserved niche/progenitor (Cd34, Fst, Tnfrsf11b) and matricellular (Vcam1, Thbs1, Timp1) cores overlaid by vessel-specific signatures, on a retained SMC identity. Multiple orthogonal computational lineage-inference approaches indicate that this compartment expands predominantly through autonomous self-renewal and is the dominant inferred source of cycling and lesion fibrochondrocyte populations, while contractile SMCs are consistently depleted as a feeder source. These findings reframe lesional SMC cellularity as expansion of a pre-existing Primed compartment rather than widespread phenotypic switching of contractile SMCs.