International Immunopharmacology

BackgroundReactive oxygen species (ROS) overproduction, inflammatory cytokines, and plasma mediators disrupt alveolar tight junction integrity and perpetuate oxidative injury in acute respiratory distress syndrome (ARDS). Biological heterogeneity of ARDS, reflected by hyper- and hypoinflammatory subphenotypes with distinct plasma mediator profiles, may contribute to heterogeneity of treatment effect. Stem cell-derived EVs have been shown to exert regenerative and immunomodulatory effects by horizontal transfer of proteins, lipids and genetic material to injured target cells. This study investigated whether extracellular vesicles derived from bone marrow (MSC-EVs) and adipose tissue (ASC-EVs) mesenchymal stem cells restore barrier integrity and redox homeostasis, and whether efficacy differs across subphenotypes. MethodsBronchial epithelial cells (BEAS-2B), microvascular endothelial cells (HMEC-1), and small airway epithelial cells (SAEC) were challenged with plasma from patients with hyperinflammatory or hypoinflammatory ARDS subphenotypes, including those treated with venovenous ECMO. MSC-EVs or ASC-EVs were added concomitantly with plasma exposure or six hours later, at a dose of 5,000 particles/cell. After 24 h, the expression of actin and tight junction proteins, zonula occludens (ZO)-1, occludin, and vascular endothelial (VE)-cadherin, was evaluated by immunofluorescence. Oxidative stress parameters, antioxidant defenses, and antioxidant enzyme activities, were measured. ResultsPlasma from patients with the hyperinflammatory ARDS subphenotype induced marked disruption of ZO-1, occludin, and VE-cadherin expression, together with actin cytoskeletal rearrangement, across BEAS-2B, HMEC-1, and SAEC cells. Barrier disruption was accompanied by increased oxidative stress markers and altered antioxidant enzyme activity. Both MSC-EVs and ASC-EVs restored tight junction protein expression and cytoskeletal organization when administered either concurrently with ARDS plasma or 6 hours after exposure, and both effectively modulated redox parameters. Delayed administration 6 hours after injury produced greater effects for several oxidative stress endpoints, particularly in cells challenged with hypoinflammatory plasma. ConclusionARDS-associated plasma mediators induce subphenotype-dependent impairment of epithelial-endothelial barrier integrity through oxidative mechanisms. Both MSC-EVs and ASC-EVs mitigate this injury, with efficacy that varies according to ARDS subphenotype and timing of administration.

Type 1 diabetes (T1D) is a chronic autoimmune disease characterized by T-cell mediated destruction of pancreatic islet {beta}-cells with genetic, environmental, and molecular triggers involved in disease pathogenesis. Patients with T1D have elevated serum levels of microRNA146a (miR146a). Polymorphisms in the miR146a gene that result in reduced expression of miR146a are associated with protection from T1D. We studied physiological regulators of miR146a expression and found that both hyperglycemia and elevated O-GlcNAcylation increased miR146a expression in T cells. Peripheral blood mononuclear cells (PBMCs) from T1D patients showed increased miR146a and O-GlcNAc transferase (OGT) expression, suggesting increased O-GlcNAcylation may promote miR146a expression in T1D patients. To determine the genetic and developmental role of miR146a in T1D, we generated miR146a-knockout (KO) non-obese diabetic (NOD) mice and found that absence of miR146a significantly protected NOD mice from spontaneous autoimmune diabetes. While we found no impact of miR146a knockout on general hematopoietic parameters and immune cell populations, remarkably, immune cell infiltration into the pancreas was significantly attenuated. Protection from autoimmune diabetes in miR146a-KO NOD mice was associated with increased regulatory T (Treg) cells in the spleen and pancreatic lymph node. Mechanistically, absence of miR146a increased NF-{kappa}B c-Rel expression in Treg cells and enhanced c-Rel binding at the Forkhead box protein P3 (FOXP3) promoter, which positively regulated Treg cell development and suppressor function, offering protection from T1D in miR146a-KO NOD mice. Our findings reveal miR146a as a key regulator of Treg cell-mediated immune tolerance through controlling NF-{kappa}B c-Rel-dependent FOXP3 expression and suggest targeting miR146a as a potential strategy to restore peripheral tolerance in T1D.

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 Mortality in sepsis is often considered a direct consequence of infection and organ dysfunction, although many deaths occur after withdrawal of life sustaining treatment. The clinical factors leading to these decisions and their implications for interpreting sepsis mortality are not well characterized. Methods We performed a secondary analysis of a prospective observational cohort of adults with physician adjudicated sepsis at a tertiary academic medical center. Patients who transitioned to comfort measures only (CMO) during the index hospitalization underwent structured chart review to characterize baseline health and functional status, clinical severity, care preferences, anticipated outcomes following survival, and the primary reason for CMO transition. Results Among 239 patients with adjudicated sepsis, 42 (17.6%) transitioned to CMO. These patients had substantial underlying disease, including malignancy in 76% and immunocompromised status in 62%. However, 88% lived at home before hospitalization, 52% were independent in activities of daily living, an additional 26% were partially independent, and 90% were full code on admission. Progressive multi organ failure (19%), refractory septic shock (19%), and irreversible respiratory failure (12%) accounted for half of CMO transitions, while advanced frailty (5%) and poor baseline functional status (2%) were infrequent primary reasons. Median time from admission to CMO was 16 days (IQR 7 to 33), and median time from admission to death was 18 days (IQR 11 to 35). Anticipated ventilator dependence (62%), dialysis dependence (36%), and cognitive impairment frequently influenced goals of care discussions. Overall, 38 patients died during the index hospitalization, and 35 (92%) of these deaths followed transition to CMO. Conclusions Most CMO transitions in this cohort occurred after an evolving course of severe sepsis and were driven primarily by progressive organ dysfunction and failure to recover rather than advanced frailty or poor baseline function. These findings identify failure to recover after the acute septic insult as an important clinical trajectory and highlight the need to account for treatment limitation when interpreting sepsis mortality. Understanding why some patients recover from sepsis associated organ dysfunction while others progress to irreversible organ failure may help identify patients and mechanisms for future therapeutic intervention.

Arteries are among the first tissues to exhibit age-related dysfunction, yet the metabolic mechanisms driving vascular senescence remain poorly understood. Here, analysis of human aortic transcriptomic data identified HCAR1, encoding the lactate receptor GPR81, as one of the genes most significantly downregulated with age. We therefore investigated whether age-associated loss of GPR81 contributes to cellular senescence within the vessel wall. Senescent human endothelial cells and vascular smooth muscle cells accumulated neutral and oxidized lipids and exhibited increased labile iron and ferroptosis. Silencing GPR81 in early-passage cells recapitulated this metabolic phenotype together with multiple hallmarks of cellular senescence. Moreover, endothelial-specific deletion of GPR81 in young mice was sufficient to induce senescent cell accumulation, impaired lipid homeostasis, endothelial dysfunction, and elastin disorganization. Conversely, pharmacological activation of GPR81 with the agonist CHBA restored fatty acid metabolism, promoted glycolytic reprogramming, and attenuated ferroptotic stress and senescence-associated phenotypes. In lamin A knock-in (LAKI) progeroid mice, CHBA reduced arterial lipid accumulation and cellular senescence, shifted vascular cell composition toward a youthful state, improved endothelial integrity, and restored extracellular matrix homeostasis. Together, these findings identify age-associated loss of GPR81 as a driver of vascular metabolic dysfunction and cellular senescence and establish pharmacological GPR81 activation as a promising therapeutic strategy for preserving vascular homeostasis and mitigating age-associated cardiovascular disease.

Pathogenic variants of COL4A1 and COL4A2, which encode the 1 and 2 chains of type IV collagen in vascular basement membranes, cause cerebral small vessel disease and intracerebral hemorrhage, but the signaling mechanisms that precede vascular damage remain incompletely defined. Here, we investigated cerebral arterial function in mice heterozygous for the Col4a2+/G646D mutation. Cerebral pial arteries from aged (12-month-old), but not young (3-month-old), Col4a2+/G646D mice exhibited impaired pressure-induced myogenic tone, whereas downstream parenchymal arterioles retained normal myogenic responsiveness, revealing a segment-selective vascular defect. The frequency of subcellular Ca2+ signals (Ca2+ sparks) generated by the release of Ca2+ through ryanodine receptors (RyR2s) on the sarcoplasmic reticulum (SR) was increased, and large-conductance Ca2+-activated K+ (BK) channel activity was elevated, in cerebral pial artery smooth muscle cells (SMCs) from aged Col4a2+/G646D mice. SR Ca2+ store load and Ryr2 transcript abundance were unchanged in these cells, but Stimulated Emission Depletion super-resolution microscopy revealed that RyR2 protein clusters were enlarged compared with controls. Protein kinase A (PKA) activation reproduced RyR2 cluster enlargement, whereas PKA inhibition normalized RyR2 cluster organization and BK channel activity in SMCs from aged Col4a2+/G646D mice. These findings identify age-associated, PKA-dependent, nanoscale remodeling of RyR2 clusters as a previously unknown mechanism underlying cerebral arterial dysfunction in COL4A2-related disease.

Background: Calcific aortic valve disease (CAVD) lacks disease-modifying therapy. LDL-cholesterol lowering does not attenuate valvular calcification, suggesting cholesterol-independent drivers. Thus, we tested whether ceramide accumulation drives CAVD and whether inhibiting sphingolipid de novo synthesis is therapeutic. Methods: We integrated lipidomics and proteomics of calcified and non-calcified human aortic valves with matched plasma. Mechanistic effects of ceramides were tested in primary human valvular interstitial cells (VICs). Therapeutic efficacy of serine palmitoyltransferase (SPT) inhibition with Myriocin was evaluated in ApoE-/- mice with PCSK9DY-AAV-driven hyperlipidemia on Western diet using preventive treatment and therapeutic initiation after echocardiographically verified gradient elevation. Myriocin was additionally tested in a normolipidemic wild-type aortic valve wire-injury model. Hemodynamics, valve structure, lipid composition, and cell-state transitions were assessed by echocardiography, histology, lipidomics, and single-cell RNA sequencing. Results: Calcified human valves showed enrichment of long-chain ceramides linked to NF-{kappa}B, endothelial-to-mesenchymal transition (EndMT), and ER-stress/unfolded protein response (UPR) programs, whereas matched plasma sphingolipids were not elevated. In hVICs, long-chain C18 ceramide induced GRP78/BiP, CHOP, and ATF4, activated NF-{kappa}B, and promoted dose-dependent calcification. In vivo, SPT inhibition with Myriocin lowered valvular ceramides, reduced leaflet inflammation and mineralization, and improved cusp separation and transvalvular hemodynamics without lowering plasma LDL-cholesterol. In a therapeutic protocol initiated after gradient elevation, Myriocin halted further hemodynamic progression, providing the first evidence that de novo sphingolipid synthesis can be targeted after established CAVD onset in vivo. Myriocin also attenuated injury-induced stenosis in normolipidemic mice. Single-cell RNA sequencing showed reduced NF-{kappa}B/EndMT and ER-stress signatures and shifted VIC states away from osteogenic phenotypes. Conclusions: Human valve lipidomics and human VIC experiments identify ceramide-driven lipotoxicity as a driver of an ER-stress-NF-{kappa}B-EndMT axis in CAVD. In vivo, de novo sphingolipid synthesis is an actionable, LDL-independent target capable of modifying established hemodynamic disease progression, supporting SPT inhibition as a tractable strategy for medical CAVD therapy.

Background: Macrophage foam cell formation is a central event in atherosclerosis. Although protein ubiquitination is increasingly recognized as a regulator of macrophage lipid handling, whether deubiquitinating enzymes directly control lipid uptake and metabolic remodeling during atherogenesis remains unclear. Methods and Results: Here, transcriptomic profiling identified ubiquitin-specific protease 20 (USP20) as a previously unrecognized regulator of macrophage foam cell formation. Genetic and pharmacological studies demonstrated that USP20 promoted macrophage lipid accumulation and foam cell formation, whereas macrophage-specific Usp20 deficiency markedly attenuated atherosclerotic lesion formation, lipid deposition, and macrophage accumulation in mice. To elucidate the underlying mechanism, integrative analysis of USP20-interacting proteins and proteomic alterations in Usp20-deficient macrophages identified fatty acid-binding protein 5 (FABP5) as a substrate of USP20. Mechanistically, USP20 interacted with FABP5 and stabilized its protein abundance by preventing ubiquitin-dependent proteasomal degradation. Consequently, USP20-mediated stabilization of FABP5 enhanced PPAR{gamma} occupancy at the Cd36 promoter, thereby increasing Cd36 transcription, ox-LDL uptake, and foam cell formation. Accordingly, Fabp5 knockdown abolished USP20-induced CD36 expression, ox-LDL uptake, and foam cell formation, whereas pharmacological activation of PPAR{gamma} restored CD36 expression in Fabp5-deficient macrophages. Finally, macrophage-targeted delivery of the USP20 inhibitor GSK2643943A markedly reduced atherosclerotic burden without evident systemic toxicity, supporting the therapeutic potential of targeting USP20 in atherosclerosis. Conclusions: USP20 promotes macrophage foam cell formation by stabilizing FABP5 and enhancing PPAR{gamma}-dependent Cd36 transcription. Targeting the USP20-FABP5-PPAR{gamma}-CD36 axis may represent a therapeutic strategy to limit macrophage lipid accumulation, metabolic dysfunction, and atherosclerosis progression.

Drug targets and vaccines for bacterial diseases are challenging to develop. Computational subtractive genomics and reverse vaccinology offer a viable substitute for conventional drug and vaccine development approaches. In recent years, Pasteurella multocida, a Gram-negative pathogen responsible for severe infections in humans along with major diseases in livestock and poultry, has developed resistance to multiple drugs. As a result, there is an urgent demand for research into reliable, efficient, highly potent, and cost-effective strategies to combat the rise of Pasteurella multocida infections. To address this, the present study integrated an in silico subtractive genomics and reverse vaccinology workflow to pinpoint proteins with potential value as both drug and vaccine targets. From the complete Pasteurella multocida Pm70 proteome, we identified 446 proteins that had no similarities with human proteins, out of which 129 were found to be essential for the pathogens survival. These 129 proteins are therefore potential candidates for drug development. Further metabolic pathway analysis, virulence potential, subcellular localization, and structural analyses revealed two outer membrane proteins, namely, 47 kDa outer membrane protein and lipopolysaccharide-assembly protein LptD, as promising candidates for therapeutic development. Subsequent antigenicity analysis revealed both outer membrane proteins as potential vaccine targets, exhibiting highly favorable antigenicity scores. Candidate B- and T-cell epitopes were systematically mapped from these two targets based on antigenicity, immunogenicity, toxicity, and allergenicity potential and only high-confidence epitopes were assembled using adjuvants and class-specific linkers to construct the final multi-epitope vaccine (MEV). The designed MEV construct was demonstrated to be highly antigenic, non-toxic and non-allergenic to humans, while possessing favorable structural and physicochemical properties, and exhibited strong binding interactions with human toll-like receptors with minimal molecular deformability. Although further experimental validation is recommended to fully assess their efficacy, the present study establishes a solid foundation for the development of novel therapeutics and vaccine candidates against P. multocida, ultimately contributing to the reduction of its rising infections.

Abstract Background: Chronic subdural hematoma (cSDH) remains a significant and increasingly prevalent cause of morbidity in older adults. Disease progression is sustained by de novo formed highly vascularized pseudomembranes and a persistent cycle of fluid accumulation and hematoma expansion. Although inflammatory and angiogenic pathways have been implicated in cSDH pathogenesis, the endothelial mechanisms underlying persistent vascular leak remain poorly understood. We investigated the cSDH vascular microenvironment in human patients and examined the role of vascular endothelial cadherin (VECAD) remodeling in cSDH endothelial dysfunction. Methods: Human cSDH membranes and fluid were evaluated by multiplex profiling, histology, immunofluorescence, immunoprecipitation, western blotting and exploratory bulk RNA sequencing. A human brain endothelial cell (HBEC) line was exposed to patient-derived cSDH fluid to assess VECAD remodeling, endothelial barrier permeability, and proliferation. Results: Multiplex profiling of patient-derived cSDH fluid detected mediators associated with endothelial cell (EC) activation, extracellular matrix remodeling, inflammation, and angiogenesis. In patient pseudomembranes, discontinuous VECAD coincided with erythrocyte extravasation. Two hyperdense (high-risk) specimens showed more extensive disruption than the hypodense (low-risk) specimen while vessel counts appeared similar. Bulk RNA sequencing identified an angiogenic environment and enrichment in cadherin regulation. A soluble VECAD (sVECAD) isoform was detected in cSDH fluid. Exposure of HBECs to patient-derived cSDH fluid increased EC proliferation, cytoplasmic VECAD (cVECAD) accumulation, and EC barrier permeability. Concurrent dexamethasone or minocycline treatment attenuated VECAD cleavage. Conclusions: VECAD disruption is a prominent feature of cSDH and is associated with EC barrier dysfunction. These findings support further evaluation of soluble VECAD as a candidate biomarker and of junction-preserving pathways as potential therapeutic targets.

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.

Objective: Autoimmune hepatitis (AIH) and primary biliary cholangitis (PBC) frequently present as overlap syndromes, characterized by shared sexual dimorphism and elevated intestinal Veillonella dispar (V. dispar) abundance, yet exhibiting distinct hallmark pathological manifestations. This study aimed to elucidate the shared pathogenesis, phenotypic differentiation mechanisms and sex-specific predisposition of AIH and PBC induced by V. dispar. Methods: Clean-grade and germ-free (GF) mice were employed to establish AIH and PBC models. Model induction involved oral gavage of V. dispar and intraperitoneal injection of either liver-specific antigen S100 (for AIH) or a pyruvate dehydrogenase complex E2 subunit (PDC-E2) mimetic peptide (for PBC). Single-cell RNA sequencing, metabolomics, transmission electron microscopy, western blotting, and flow cytometry were utilized to elucidate the underlying mechanisms. Results: V. dispar successfully induced autoimmune liver disease-like features in mice, characterized by significant intrahepatic B cell infiltration. Mechanistically, V. dispar caused systemic arginine reduction, leading to intrinsic apoptosis of gonadal stromal cells and a subsequent reduction in testosterone. This decreased testosterone weakened its inhibitory effect on B cell activating factor (BAFF) secretion, thereby amplifying B cell-mediated immune responses. Furthermore, in the presence of S100 or PDC-E2 mimetic peptide, V. dispar also facilitated Th17 cell activation, which guided the phenotypic differentiation into AIH or PBC, respectively. Consistent with these findings, our clinical observations revealed that patients with AIH and PBC tended to harbor enriched V. dispar, along with lower serum arginine and testosterone. In addition, elevated serum levels of BAFF and IL-17 were also observed in these patients. Conclusion: V. dispar induces reduced testosterone levels and upregulation of BAFF, leading to intrahepatic B cell expansion, which serves as the common pathogenic basis for AIH and PBC. The presence of S100 and PDC-E2 subsequently drives further maturation of these expanded B cells and promotes Th17 cell activation, thereby determining the phenotypic divergence between AIH and PBC.

Background: Host response and biomarker research to advance sepsis therapeutics requires well-characterized cohorts with longitudinal biospecimens across a range of hosts and infections. Automated screening based on SIRS criteria is sensitive but nonspecific and identifies more candidates than research teams can approach, sample, and follow longitudinally. We evaluated a structured enrichment review by non-physician study staff, done after automated screening in a hospital-wide prospective sepsis cohort. Methods: The CISID cohort is a prospective observational study of hospitalized patients with suspected sepsis, enrolling adults from the ED, inpatient wards, and ICUs at a tertiary academic medical center. Patients were identified through: (1) automated electronic health record screening for two or more SIRS criteria with a 24-hour lookback, documentation of microbiologic culture or PCR, and receipt of a new antibiotic; (2) a sepsis enrichment step of structured chart review by trained non-physician study staff; and (3) discussion with the primary clinical team. Enrolled patients underwent physician adjudication into four diagnostic categories. To evaluate the enrichment step, we analyzed all patients screened during a 9-week period, assigned a composite "sepsis-likely" variable (CDC Adult Sepsis Event criteria or ICD-10 code R65.20/R65.21 within 48 hours before or after screening), and assessed mortality across each stage of screening. We also assessed concordance of the composite with physician adjudication among enrolled patients. Results: From November 2023 through February 2026, 4,318 patients screened in through EHR-based automated screening; 1,220 (28%) were flagged for enrollment, and 288 were enrolled. Physician adjudication classified 209 (73%) as sepsis, 46 (16%) as infection without sepsis, 14 (5%) were classified as undifferentiated critical illness, and 19 (6.6%) as non-infectious sepsis mimics. Patients with sepsis were severely ill (median SOFA score, 9; ICU admission, 80%; shock, 48%), and 51% were immunocompromised; mortality was 14% in hospital and 27% at 90 days. In a 9-week sample of 907 screened encounters, 21% of those deferred and 70% of those flagged for enrollment met the sepsis-likely composite, a 3.3-fold increase in prevalence after structured study team review. Conclusions: A brief structured enrichment review after automated screening tripled the prevalence of EHR-based sepsis indicators among patients selected for enrollment, while concentrating enrollment efforts on roughly one-quarter of screened-in patients in this hospital-wide, all-source suspected-sepsis cohort. This approach may help prospective cohorts direct limited biospecimen resources toward patients most informative for host-response and biomarker research.

Psoriasis is a chronic skin disease characterized by red, scaly plaques mainly driven by dysregulated T cells that induce keratinocytes (KCs) proliferation and inflammation. Despite significant advancements in disease management through biologics and topical therapies, these treatments offer only temporary relief and fail to prevent disease recurrence. Considering the current therapeutic needs, we described herein a novel small molecule TEM1657, derived from 3,5-dicaffeoylquinic acid with anti-inflammatory potency in imiquimod-induced psoriasis mouse model as well as in Th17-stimulated KCs. Mechanistically, its action is mediated through modulation of mitochondrial dynamics, specifically via OPA1, an inner mitochondrial membrane protein that alters mitochondrial calcium influx, and further impacts cellular bioenergetics. Moreover, TEM1657 rapidly suppresses transcription of NFKBIZ and various other psoriasis-associated genes. These observations highlighted a pathway that represents an innovative target in psoriasis and other NFKBIZ-related disorders.

Aims: Premature coronary artery disease (PCAD) lacks specific molecular drivers, and the role of immunosenescence is unclear. We investigated whether aging-related gene dysregulation in T cells contributes to PCAD. Methods: We combined bulk transcriptomics of PBMCs from 12 PCAD patients and 21 controls, single-cell RNA sequencing of PBMCs and human atherosclerotic plaques, weighted gene co-expression network analysis, gene perturbation network analysis, and molecular docking. Results: KRAS was identified as a hub gene intersecting PCAD-associated genes and aging-related genes. Single-cell analysis showed KRAS upregulation predominantly in effector CD8+ T cells, which exhibited the highest senescence scores that were further elevated in disease. Network perturbation of KRAS strongly impacted the cell killing pathway. KRAS-high effector CD8+ T cells were detected in coronary and carotid plaques, displaying enhanced cytotoxicity, exhaustion, and senescence features. Additionally, a candidate small molecule was computationally predicted to bind inactive KRAS. Conclusions: Elevated KRAS expression in senescent, cytotoxic CD8+ T cells is associated with PCAD, bridging immunosenescence and premature atherosclerosis. This finding provides a novel biomarker candidate and potential therapeutic entry point, awaiting further functional validation.

Background: Autophagy is a homeostatic pathway supporting stress adaptation and is dysregulated in atherosclerosis. Its potential as a biomarker or therapeutic target in atherosclerotic vascular disease (ASVD) remains incompletely defined. We measured autophagic flux in peripheral blood mononuclear cells (PBMCs) from patients with peripheral arterial disease (PAD) or carotid stenosis (CS), compared with healthy controls, and explored clinical outcome associations. Methods: Ninety-four patients with PAD or CS and 19 healthy controls were studied. Autophagic flux was quantified from fresh blood using a validated ex vivo chloroquine inhibition ELISA measuring LC3BII accumulation. Major adverse cardiovascular events (MACE) and major adverse limb events (MALE) were ascertained over a median follow up of 828 days. Results: The ASVD cohort comprised claudication (n = 16), chronic limb threatening ischemia (CLTI; n = 49), and CS (n = 29). Autophagic flux was higher in ASVD than controls (mean 281.4 vs. 182.3 ng LC3BII/mg protein/h; p < 0.0001) and remained independently associated after multivariable adjustment. Within CLTI, concurrent infection was associated with lower flux (p = 0.001), approaching control levels (p = 0.327). In CLTI, higher flux quartiles were associated with lower MACE risk, most strongly for quartile 3 (hazard ratio 0.07 vs. quartile 1, 95% CI 0.01 to 0.50; p = 0.009). Conclusion: Autophagic flux is elevated in PBMCs from ASVD patients, independent of age and sex. Attenuated flux in CLTI with concurrent infection may indicate autophagic exhaustion in advanced disease. The association between higher flux and lower MACE in CLTI suggests prognostic utility, warranting evaluation in larger prospective studies.

Japanese Encephalitis Virus (JEV) belongs to the Flavivirus family, and the RNA dependent RNA polymerase (RdRp) domain located at the C terminus of non structural protein 5 (NS5) regulates de novo viral genome synthesis. The conserved priming loop inserted in the thumb domain of RDRP initiates de novo genome synthesis. Interestingly, the reported replication process initiates with an interaction between NS5 (methyltransferase/RDRP) and NS3 (protease/helicase), and inhibiting this interaction directly correlates with the inhibition of viral replication. The function of the priming loop in the context of the NS5 NS3 interaction is not yet known. In this study, we studied the priming loop function in the NS5 NS3 interaction and viral replication. Using a structure based drug design approach, we screened the Maybridge compound library against the RdRp priming loop and identified 15 candidate compounds based on binding energy. Among them, DSHS00151 showed significant dose dependent inhibition of the NS5 NS3 interaction with an IC50 of 3.5 micromolar in the mammalian two hybrid assay, inhibited viral infectivity with an IC50 of 2.54 micromolar, and reduced viral RNA load with an IC50 of 2.9 micromolar, compared to CD10712. Molecular Dynamics (MD) simulations revealed that DSHS00151 exhibits stronger and more stable binding with the priming loop residues (790 to 812) compared to CD10712. The molecular mechanism suggested by the docking of the NS5 and NS3 proteins showed that the priming loop tends to rotate toward the NS3 protein to stabilize the complex, and compound binding restricts this loop rotation, thus compromising the stability of the NS5 and NS3 complex and affecting viral replication. Overall, our study validated the function of the NS5 priming loop as an allosteric site, and compounds binding to this allosteric site belong to the Non Nucleoside Reverse Transcriptase Inhibitors (NNRTI) class of inhibitors, which disrupt the NS5 and NS3 interaction and could be developed as novel anti JEV therapeutics.

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.

Mesaconate (MES, an immunosuppressive metabolite) but not itaconate is upregulated in stool of Crohns Disease (CD) patients, albeit below therapeutic levels.. However, colonic MES level is difficult to maintain because of its high absorption rate, limiting its therapeutic potential for CD. Herein, we developed two strategies that elevate MES level and suppress inflammation: EcN-CAD, an engineered probiotic Escherichia coli Nissle 1917 (EcN) that overexpresses cis-aconitate decarboxylase (CAD, produces itaconic acid that is converted to MES); and MES+Zn, a Zinc-MES ionically crosslinked formulation. Mechanistically, MES modulates T cell function by downregulating glycolytic and mitochondrial activity and decrease ROR{gamma}t expression. In preclinical SAMP1/YitFC dexamethasone-withdrawal model and TNFDARE model, MES+Zn decreased gut permeability significantly compared to controls and reduced myeloperoxidase (MPO, an inflammatory marker) 1000-fold compared to controls. EcN-CAD probiotics decreased ROR{gamma}t expression in CD4+ and CD8+ T cells, gut permeability and inflammation in preventive SAMP1/YitFC model, and SAMP1/YitFC dexamethasone-withdrawal model. Overall, MES rewires T cell responses and technologies that enhance MES levels can be effective in gut-related inflammatory diseases.

Introduction: Mast cells are central effectors of IgE-mediated allergic responses and reside in barrier tissues, where they encounter diverse environmental compounds. How naturally occurring bioactive compounds influence mast cell function under resting versus allergic conditions remains poorly defined. Methods: We compared the effects of cholera toxin, aconitine, and sennoside A on viability, degranulation, and cytokine production in murine bone marrow-derived mast cells. We then examined the acute physiological effects of oral sennoside A in an ovalbumin-induced mouse model of food allergy. Results: None of the compounds reduced mast cell viability at the concentrations tested. Sennoside A, but not cholera toxin or aconitine, induced modest degranulation in resting mast cells without increasing TNF- or IL-6 secretion. In contrast, sennoside A markedly suppressed IgE-dependent antigen-induced degranulation, TNF- and IL-6 secretion, and Il1b expression. Acute oral sennoside A induced dose-dependent diarrhea and hypothermia and increased colonic Tnfa, Il1b, Il33, and Hdc expression. In allergic mice, sennoside A attenuated allergen-induced hypothermia, reduced duodenal Tpsab1 expression, and decreased circulating total IgE, OVA-specific IgE, and OVA-specific IgG1, while increasing Il5 expression. Conclusion: Sennoside A differentially regulates mast cell responses according to activation state and attenuates features of experimental food allergy. These findings identify a previously unrecognized immunomodulatory effect of sennoside A on IgE-driven mast cell activation. However, the mechanisms and the contribution of mast cells to the in vivo phenotype remain to be defined.