Clinical Immunology

Systemic lupus erythematosus (SLE) is a chronic autoimmune disease characterised by sustained type I interferon signalling and widespread immune dysregulation. Low-density neutrophils (LDNs) are expanded in SLE and display pro-inflammatory and tissue-damaging properties. However, their metabolic phenotype remains poorly defined. Here, we performed a comprehensive metabolic characterisation of circulating LDNs and normal-density neutrophils (NDNs) from patients with SLE and matched healthy individuals (HC). Neutrophil subsets were isolated from peripheral blood of SLE patients and HC donors using a two-step protocol of negative selection and Percoll density centrifugation. Immunophenotyping phenotype was carried out by flow cytometry to assess phenotypic expression of common neutrophil markers CD15, CD16, CD10, CD66b, CD62L, MPO, and IL-1{beta}. Bioenergetic profiling of LDNs and NDNs was performed in situ using the Seahorse MitoStress test to measure oxygen consumption rate (OCR) and extracellular acidification rate (ECAR). Metabolic flexibility and phenotypic alterations were assessed in LDNs and NDNs following inhibiting mitochondrial metabolism with oligomycin and glycolysis with 2DG. We found that SLE LDNs exhibit an immature phenotype compared with autologous and healthy NDNs, as determined transcriptionally by C/EBP{varepsilon} and by surface protein expression levels of CD10. Both LDNs and NDNs from SLEDAI[≥]4 patients demonstrated significantly elevated ECAR relative to HC neutrophils. Further, SLE LDNs displayed enhanced metabolic flexibility, with the capacity to switch towards a glycolytic phenotype under metabolic stress conditions. Inhibition of glycolysis altered the inflammatory and maturation-associated phenotype of both SLE neutrophil subsets, indicating a direct link between cellular metabolism and pathogenic neutrophil function. Collectively, these findings identify fundamental metabolic alterations in SLE neutrophil subsets and support neutrophil immunometabolism as a potential therapeutic target in SLE.

Systemic lupus erythematosus (SLE) is associated with infection susceptibility and altered innate immune function. Monocyte metabolism is linked to appropriate cytokine release and bacterial containment. We investigated cytokine production and metabolic programming in the monocyte population from SLE patients and healthy controls following lipopolysaccharide (LPS) stimulation. SLE monocytes displayed increased IL-10, TNF, and IL-8 production, with impaired IL-1{beta} induction. Metabolic profiling revealed altered substrate use, with increased glucose dependence and reduced fatty acid and amino acid oxidation after LPS stimulation. SLE patients exhibited reduced numbers of classical monocytes, expansion of intermediate monocytes, and dysregulated subset-specific metabolic reprogramming in response to LPS. This descriptive study provides a cornerstone for (i) understanding infection susceptibility in SLE, (ii) subset-resolved immunometabolic profiling as a tool in autoimmunity, and (iii) developing future metabolic-targeted therapeutic strategies HighlightsO_LIDescriptive mapping shows SLE monocytes are proinflammatory with glucose dependence after LPS C_LIO_LIClassical and intermediate SLE subsets show divergent baseline metabolic preferences versus healthy C_LIO_LISLE subsets display aberrant LPS responses, i.e.. increased glucose and reduced fatty acid oxidation C_LIO_LIThis study provides a cornerstone for subset-resolved immunometabolism in infection susceptibility. C_LI

ObjectiveSystemic sclerosis (SSc) predominantly affects females but exhibits greater disease severity in males, suggesting sex differences underlying SSc pathogenesis. We sought to define sex-associated alterations in the peripheral immune landscape of patients with SSc. MethodsWe performed high-dimensional immune profiling of PBMCs from 37 healthy donors (68% female) and 37 patients with SSc (11 limited, 26 diffuse; 68% female) using 30-color spectral flow cytometry, quantifying 56 immune cell subsets per donor. We conducted sex-stratified comparisons and correlation analysis, and used principal component analysis followed by linear discriminant analysis to derive a sex-discriminant immune cellular module. ResultsDiffuse cutaneous SSc (dcSSc) was associated with a distinct immune landscape characterized by increased monocyte and decreased natural killer-like and B cell frequencies, suggesting a myeloid-skewed peripheral immunophenotype. Males exhibited greater enrichment of innate immune subsets, including monocyte and dendritic cell subsets, while females exhibited greater enrichment of adaptive immune subsets. Among T cells, dcSSc was associated with coordinated remodeling across CD4+ and CD8+ subsets, including expansion of stem cell memory T cells (Tscm), and increased regulatory T cells, Th17 skewing, and decreased effector-memory CD8+ subsets. Females exhibited greater proportions of naive- and Tscm, and males exhibited higher proportions of effector-memory subsets. Integrating these data, we identified a sex-discriminant immune module comprised of 20 cell types that distinguishes males and females with dcSSc. ConclusionsSSc is associated with sex-specific differences in the peripheral immune landscape. A sex-associated immune program, further amplified in disease, may contribute to the paradox of female-biased susceptibility and male-biased severity in SSc.

Long-lived plasma cells (LLPC) sustain humoral immunity but also contribute to the persistence of pathogenic autoantibodies in autoimmune diseases. New therapies targeting LLPC are therefore desirable. Recent studies have shown increased expression of Slc12a2, encoding the Na+ -K+ -Cl- cotransporter (NKCC1), in LLPC. This study investigated whether NKCC1 activity was required for plasma cell survival, persistence or secretion of antibodies. Across in vitro and in vivo settings, mouse plasma cell survival was undiminished by treatment with the NKCC1 inhibitor bumetanide. Acute in vivo bumetanide treatment did not diminish plasma cell numbers, nor show any demonstrable impact on the survival of phenotypically mature I-A/I-EloSLAMF6lo plasma cells. With genetic plasma cell timestamping, even the survival of persistent LLPC was unaffected by bumetanide. Plasma cell secretory capacity, assessed by measuring IgM and IgG2b secretion in culture over three days, was also unaltered by bumetanide. Overall, these results show that pharmacological inhibition of NKCC1 is not sufficient to impair plasma cell survival, persistence or antibody secretion. Despite elevated Slc12a2 mRNA expression in LLPC, NKCC1 alone does not represent a critical plasma cell survival pathway, highlighting the resilience of plasma cells and the challenges associated with therapeutically targeting LLPC.

Pediatric Autoimmune Neuropsychiatric Disorders Associated with Streptococcal Infections (PANDAS) is characterized by prepubertal abrupt onset of obsessive-compulsive disorder (OCD). The sine qua non is group A streptococcus (GAS) infection, which is hypothesized to elicit an IgG-class anti-GAS antibody response that cross-reacts with antigens in the basal ganglia. However, the association between GAS antibody (GAS-IgG) levels and PANDAS has been inconsistent, and qualitative differences in GAS-IgG profiles have not been carefully evaluated in well-phenotyped cohorts. Moreover, independent studies have yet to converge on anti-neural autoantibodies that are specific to PANDAS. Here, we used phage display immunoprecipitation sequencing (PhIP-Seq) to perform ultra-deep anti-pathogen antibody repertoire profiling of serum from definitive pediatric PANDAS patients (N = 34) collected as part of a prior double-blind, placebo-controlled clinical trial of intravenous immunoglobulin (IVIg). PANDAS cases were compared to pediatric controls without a history of neuropsychiatric illness (N = 31). To assess for objective evidence of neuroglial injury, serum neurofilament light (NfL) and glial fibrillary acidic protein (GFAP) levels were compared to healthy pediatric controls. Within PANDAS, NfL and GFAP levels were compared between pre- and post-treatment sera. To evaluate for central autoantibodies, a subset of baseline cerebrospinal fluid (CSF) samples (N = 25) was profiled by full-length human protein microarray. Though GAS reactivity by PhIP-Seq was well correlated with clinical anti-DNaseB and anti-streptolysin O titers, there were no quantitative or qualitative differences in GAS-IgG profiles between PANDAS and controls. Furthermore, NfL and GFAP levels did not differ between cases and controls. Within PANDAS, changes in NfL or GFAP levels at six weeks did not differ between placebo and IVIg groups. However, CSF autoantibody profiling by protein microarray revealed infrequent but notable candidate autoantibodies. In one patient, we identified autoantibodies against Argonaute family proteins (AGO-IgG), a marker of autoimmune sensory neuropathy. Longitudinal measurement of AGO-IgG in sera revealed that titers were unchanged after placebo, but decreased after IVIg, coinciding with symptomatic improvement, including a decrease in that patients CY-BOCS score. Overall, these results do not support an etiologic role for GAS-IgG in PANDAS. However, some individuals diagnosed with PANDAS may harbor anti-neural autoantibodies.

Germinal matrix-intraventricular hemorrhage (GM-IVH) is one of the most frequent and severe neurological complications in preterm infants (PT). It triggers an inflammatory response accompanied by neuronal and glial injury and may progress to post-hemorrhagic ventricular dilatation (PHVD), thereby increasing long term disability and cognitive deficits. Nevertheless, the characteristics and evolution of the associated pathology is poorly understood. To assess neuroimmune response and neuropathology induced by GM-IVH, we quantified cytokines, glial activation and neurodegeneration makers in cerebrospinal fluid collected from 12 patients with grades III/IV GM-IVH and PHVD and 5 controls neonates from the onset of pathology up to 2 months of age. Additionally, to evaluate long-term deficits and behavioral outcomes, we used standard behavioral test including Bayley Scales of Infant and Toddler Development, Third Edition (Bayley-III) at 2 years of age. Interestingly, we found that while pathology markers such as ubiquitin carboxy-terminal hydrolase L1 (UCHL1), alpha II spectrin breakdown product 145 (SBDP145) and myelin basic protein (MBP) are elevated in PT, their level decline over time. Furthermore, cytokine profiling identified two divergent temporal trajectories (i.e., diminishing or sustained) that correspond with either neuronal or astrocytic markers. Specifically, diminishing cytokines including IL-6, IL-8, and IP-10 decreased with age and were correlated with neuronal markers such as SBDP145, UCH-L1, and MBP. In contrast, sustained cytokines such as IFN-{gamma}, IL-7, IL-13, and MCP-1 remained elevated or unchanged throughout the study period and were positively correlated with astrocyte reactivity marker GFAP. Notably, sustained cytokines were consistent with worse motor function and behavioral outcome. Together, longitudinal CSF analysis in PT with severe GM-IVH and PHVD identifies a cytokine profile that declines and correlates with neuronal and glial injury markers, and another that remains sustained and correlates with gliosis and adverse neurodevelopmental outcomes. These findings highlight potential CSF biomarkers associated with disease progression and long-term neurological impairment, providing a foundation for future evaluation of candidate therapeutic interventions.

Immune phenotyping represents a pillar in diagnostics, characterization of new genetic defects, and understanding mechanisms of diseases. Cell population distribution often does not cover the intrinsic function changes that may contribute to disease. Outcome of signaling activation can be used as proxy for cell function. To overcome the limitation of sample availability and standardization of signaling assays, we developed a multiplex full spectrum cytometry phosphoflow assay allowing the study of 6 phospho-proteins representing BCR/TCR, MAPK, PI3K/Akt/mTOR and canonical NF-{kappa}B signaling pathways in 18 immune cell subpopulations. Maximal stimulation and temporal dynamics were studied in response to pan-stimuli, activating cells regardless of receptor, and targeted stimuli for T, B, and innate immune cells. We studied healthy individuals between 1-69 years and discovered subpopulations-specific responses. Furthermore, pediatric donors showed broad differences in B cell and T cell function compared to adults. Hence, we established a tool to assess multiple signaling pathways at once and provide age- and subpopulation-specific references for signaling outcome. SummaryMultiplex full spectrum flow cytometry-based phosphoflow assay across 18 immune cell subpopulations, 6 phospho-proteins in response to 6 stimuli at 4 time points in individuals aged 1-69 years, reveals distinct age- and subpopulation-associated signaling patterns in magnitude and dynamics of pathways activation.

Abnormalities in the gut microbiome, intestinal permeability, and the gut-immune-brain axis are increasingly linked to neuropsychiatric disorders, neurodegenerative disorders, inflammatory bowel disease (IBD), and other immunologic/autoimmune conditions. We investigated these phenomena in 128 youth with Pediatric Acute-Onset Neuropsychiatric Syndrome (PANS) and individuals with autism spectrum disorder (ASD) and other neurodevelopmental disorders (NDD) characterized by profound, unexplained deteriorations/regressions in developmental, neuropsychiatric, and behavioral functioning. Previous studies we have carried out showed that immune dysregulation and DNA damage response (DDR) gene mutations are implicated in a subset of these patients. The current study examines the role of genetic variants affecting intestinal homeostasis. We report a series of patients exhibiting both neuropsychiatric deterioration and gastrointestinal symptoms. Genetic analysis identified ultrarare (minor allele frequency < 0.001) pathogenic or likely pathogenic variants in eight genes primarily expressed in the intestines and associated with IBD, dysbiosis, or intestinal permeability. Across thirteen patients, mutations were identified in DUOX2 (n=4), SLC10A2 (n=2), UNC45A, TTC7A, LGALS4, SI, CCR9, MEP1B, and BACH2. While these findings suggest a potential role for genetic variants governing intestinal homeostasis in these cases of neuropsychiatric decline, their presence in only a small subgroup necessitates larger, prospective cohorts to determine whether these variants are statistically significant and play a definitive role in the pathogenesis of these disorders.

BackgroundCTLA-4 haploinsufficiency (CHAI) and LRBA deficiency cause severe immune dysregulation including enteropathy. Abatacept, a CTLA-4-immunoglobulin fusion protein, targets the underlying pathway defect, but its impact on the gut microbiome remains undefined. MethodsWe performed longitudinal shotgun metagenomics (MetaPhlAn4/HUMAnN3) on stool samples from patients enrolled in the ABACHAI clinical trial, collected at pre-treatment baseline and months 3, 6, and 12. Healthy individuals from the same household served as controls. Compositional and functional microbiome changes were analyzed using linear mixed-effects models and MaAsLin3, and correlated with organ-specific CHAI Morbidity Scores. ResultsAt baseline, patients showed significantly reduced alpha diversity (Shannon index, p=0.0029) and distinct community composition (PERMANOVA p=0.0001) compared to healthy controls, characterised by enrichment of oral-associated taxa (Veillonella, Streptococcus, Lacrimispora) and depletion of butyrate-producing commensals (Ruminococcus, Oscillibacter, Dysosmobacter). Functionally, the baseline metagenome exhibited broad reductions in amino acid and SCFA biosynthesis alongside enrichment of purine salvage and folate pathways. During treatment, beta diversity shifted significantly with treatment duration (Aitchison PERMANOVA R2=0.103, p=0.015), with within-patient community turnover peaking at month 6 ({Delta}=0.216, p=0.006). Longitudinal analyses demonstrated progressive decreases in disease-enriched taxa (Veillonella, Lacrimispora) and recovery of commensals (Collinsella, Adlercreutzia). FDR-significant reductions in microbial folate and purine biosynthesis pathways were observed over the treatment course. Gut CHAI domain severity correlated inversely with butyrate-producer abundance and positively with oral taxon enrichment. ConclusionIn CTLA-4 pathway insufficiency patients, abatacept therapy is associated with an improvement of enteropathy and a progressive, measurable gut microbiome restructuring, positioning microbiome dynamics as a candidate biomarker of treatment response in this monogenic immune dysregulation disorder.

BackgroundGastrointestinal (GI) involvement in systemic sclerosis (SSc) affects up to 90% of patients and is a major driver of morbidity and mortality. Despite its clinical importance, GI disease in SSc is highly heterogeneous, with upper and lower GI manifestations representing distinct phenotypic extremes whose underlying immunologic basis remains poorly defined. MethodsWe performed unbiased, proteome-wide autoantibody profiling using a human protein microarray comprising >21,000 full-length proteins (>80% of the human proteome). Sera from patients with SSc and isolated upper GI dysmotility (n=23), isolated lower GI dysmotility (n=17), and non-SSc controls (n=20) were analyzed. Enriched autoantibodies were identified using Fishers exact test, and unsupervised clustering was applied to define serology-based patient subsets and relate immune signatures to clinical phenotypes. ResultsDistinct autoantibody profiles differentiated patients with upper versus lower GI disease. Upper GI-predominant SSc was characterized by enrichment of previously unreported autoantibodies, including those targeting TiSSc1/2 (newly identified proteins encoded within the MIRLET7BHG locus), FAM9C, SPATA20, FAM110D, EMILIN1, CARD14, SMN1, KCTD7, and PHYHD1, whereas lower GI disease was associated with antibodies against HAO2, KLHL7, SUFU, APPL1, BNIP2, UCHL3, ZNF385A, LIMD1, MAGEA9, and PPP2R3C. Serology-driven clustering identified four reproducible subgroups with distinct patterns of GI, pulmonary, vascular, and autonomic involvement, defining clinically meaningful disease phenotypes that extend beyond traditional anatomic classification. ConclusionsProteome-scale serological profiling reveals previously unrecognized autoimmune signatures underlying GI heterogeneity in SSc. These findings support a shift from anatomy-based to serology-defined classification of SSc GI disease and provide a foundation for biomarker development, patient stratification, and precision medicine approaches in this population.

Anti-melanoma differentiation-associated protein 5 (anti-MDA5) autoantibodies identify a distinct dermatomyositis subset frequently associated with rapidly progressive interstitial lung disease (RP-ILD). While these antibodies are established disease markers, their direct contribution to pulmonary fibrosis is poorly defined. This study investigated the pathogenic effects of patient-derived polyclonal anti-MDA5 antibodies on IMR-90 human lung fibroblasts. Recombinant human MDA5 protein was produced in HEK293F cells and utilized to selectively isolate autoantibodies from a patients plasma via affinity chromatography. Fibroblasts were stimulated with MDA5, anti-MDA5 antibodies, or both. Real-Time Cell Analysis (RTCA) showed a statistically significant increase in cell impedance following treatment with an MDA5-anti-MDA5 mixture compared with controls, accompanied by a reduction in cell doubling time. MTT assays showed that neither MDA5 nor anti-MDA5, nor their immunocomplex, exerted acute cytotoxic effects in cell culture. Direct cell counting revealed a significant increase in fibroblast proliferation in response to the MDA5-anti-MDA5 combination. Molecular characterization by RT-qPCR revealed a significant alteration of TLR2, TLR7, and endothelin-1 (ET-1) mRNA levels. ELISA assays detected an increased secretion of pro-collagen and type I interferons in culture supernatants. All these results were mainly, but not only, observed in the MDA5/anti-MDA5-exposed cells. Our results suggest that anti-MDA5 autoantibodies and MDA5 antigen complex are not merely disease biomarkers, but active pathogenic drivers that stimulate proliferation and pro-fibrotic responses in lung fibroblasts. This mechanism may contribute to the rapid tissue remodeling characteristic of RP-ILD, supporting the development of targeted therapeutic strategies to mitigate fibrosis in this high-mortality patient subset.

Objective: To characterize pregnancy outcomes and menstrual irregularities in Mexican women with systemic lupus erythematosus (SLE) and identify clinical factors associated with adverse pregnancy outcomes and early-onset menopause. Methods: We conducted a cross-sectional study of women with SLE enrolled in the Mexican Lupus Registry (LupusRGMX) between May 2021 and September 2024. Clinical and reproductive data were collected using standardized questionnaires. Menopause was defined as the absence of menstruation for [&ge;]12 consecutive months, and early menopause as onset before age 40. Univariable and multivariable logistic regression analyses were used to identify factors associated with pregnancy complications and early menopause. Results: A total of 210 women were included. Median age was 38 years (IQR 29-46) and median disease duration was 4 years (IQR 1-10). Among women with a history of pregnancy (47%), full-term delivery predominated (61%), while pregnancy loss occurred in 26% and preterm delivery in 13%. Pregnancy complications were reported in 9.6%, most commonly preeclampsia (6.7%). Younger maternal age was independently associated with pregnancy complications (OR 0.89, 95% CI 0.83-0.95) and adverse outcomes (OR 0.95, 95% CI 0.92-0.98). Higher disease activity was associated with complications in univariable analysis. Most pregnancies (68.3%) occurred before diagnosis. Early menopause was observed in 6.2% and independently associated with longer disease duration and older age. Conclusion: Younger maternal age was independently associated with adverse pregnancy outcomes, whereas disease activity showed an association in univariable analysis. Most pregnancies occurred prior to SLE diagnosis. Early menopause was associated with longer disease duration, suggesting impact of cumulative disease burden on ovarian function.

Background & AimsCeliac disease (CeD) is an autoimmune disorder triggered by dietary gluten in genetically predisposed individuals, but environmental factors contributing to disease onset remain incompletely defined. Epidemiological studies implicate enterovirus infections as potential triggers. Here, we investigated the epithelial-intrinsic mechanisms by which coxsackievirus B1 (CVB1) infection may prime the intestine for CeD. MethodsHuman intestinal organoids were infected with CVB1 and analyzed using single-cell RNA sequencing to resolve lineage-specific responses. Interferon signaling and transglutaminase 2 (TG2) regulation were interrogated using type I interferon stimulation and pharmacologic JAK inhibition. ResultsCVB1 infection induced a robust epithelial antiviral program dominated by type I interferon signaling. This response was accompanied by marked upregulation of TG2 expression and enzymatic activity. Single-cell analysis localized TG2 induction to immature goblet-lineage cells, which exhibited strong interferon-stimulated gene activation and epithelial stress signatures. Mechanistically, IFN-/{beta} stimulation was sufficient to induce TG2 via JAK-STAT signaling, while JAK inhibition effectively suppressed both TG2 expression and activity. In parallel, CVB1 infection triggered coordinated mucin remodeling, including induction of MUC5AC, indicating interferon-linked epithelial reprogramming. Notably, these effects occurred independently of immune cell involvement, highlighting a cell-intrinsic pathway. ConclusionOur findings identify a direct epithelial mechanism linking enterovirus infection to TG2 activation via interferon-driven JAK-STAT signaling. This pathway provides a mechanistic bridge between viral infection and gluten peptide modification, a critical step in the onset of CeD. The reversibility of TG2 induction by JAK inhibition suggests a potential strategy to prevent virus-mediated priming of celiac disease.

Introduction: Incomplete recovery from relapses contributes to long-term disability accumulation in relapsing remitting multiple sclerosis (RRMS), yet the relationship between immune regulation and relapse recovery remains poorly defined. Objective: To longitudinally characterize regulatory/effector immune cell dynamics in untreated RRMS patients and assess their association with immune balance and relapse recovery. Methods: Monocytic myeloid-derived suppressor cells (M MDSCs), regulatory T cells (Treg), and effector CD4 T cell subsets were measured in blood from 69 untreated RRMS patients sampled during relapse or remission and reevaluated after 12 months. Associations with clinical recovery after relapse were examined. Results: During relapse, patients exhibited higher M MDSC and Treg frequencies than in remission, while effector T cell subsets remained unchanged. Over one year, M-MDSCs increased consistently regardless of baseline clinical status, whereas Treg frequencies remained stable. Effector to M MDSC ratios were markedly elevated during relapse and declined over time, while effector-to-Treg ratios showed minimal variation. M MDSC levels during relapse were associated with sustained regulatory features at 12 month follow up. Importantly, higher baseline M MDSC levels, but not Treg frequencies, were associated with complete relapse recovery at one year. Conclusion: These findings suggest that circulating M-MDSCs, but not Treg, reflect interindividual differences in immune regulation and clinical recovery after relapse in early RRMS.

BackgroundImmunoglobulin E (IgE) plays a fundamental role in the pathogenesis of allergic disease, including asthma. The IgE-producing plasma cells (PCs) are thought to persist indefinitely, providing a sustained source of allergen-specific IgE. Although these cells can accumulate in the bone marrow (BM), after prolonged allergen exposure, their frequency remains remarkably low, and the mechanisms that regulate their migration are poorly understood. ObjectiveTo investigate the chemokine receptor profile and the migration potential of the human IgE-producing cells. MethodsTonsil B cells were stimulated with IL-4 and anti-CD40 to induce class switching to IgE and IgG1. The chemokine receptor profile of IgE+ and IgG1+ switched cells was determined using flow cytometry and migration towards relevant chemokines was quantified using transwell chemotaxis assays. Chemokine expression was also validated by re-analysis of a published single cell RNA sequencing (scRNAseq) dataset of PCs isolated from nasal polyps (NP) of patients with allergic fungal rhinosinusitis. ResultsIgE PCs exhibit significantly reduced expression of the BM-homing chemokine receptor CXCR4 and impaired migration towards its ligand, CXCL12. While IgE+ PCs can upregulate CCR10 and respond to its ligand, CCL28, this behaviour is similar to IgG1+ PCs. Strikingly, however, IgE PCs selectively upregulate CCR2 and migrate robustly towards its ligand CCL2. Re-analysis of NP scRNAseq data confirmed that IgE PCs express significantly higher levels of CCR2 compared with PCs of all other isotypes. ConclusionsThese findings identify CCR2 as a key regulator of IgE PC migration and provide insights into their homing preferences that may shape the nature of the IgE responses.

High levels of IL-18 have been causally implicated in IBD risk and may represent a unique mechanism driving IBD yet to be therapeutically targeted. To identify individuals predisposed to increased levels of IL-18, we implemented a polygenic approach to predict IL-18 plasma protein levels. Using a dataset with over 50,000 individuals with both genetic and plasma protein levels from Olink, we developed a 27 SNP polygenic score that predicts IL-18 levels and IBD risk. Further, we identified a threshold to classify patients as 'IL-18 High' using a data-driven approach that optimized prediction of both IL-18 and IBD risk. We show that ~30% of the overall IBD patient population is 'IL-18 High', meaning a genetic predisposition towards higher protein levels. The IL-18 PGS and corresponding threshold have the potential to identify IBD patients with IL-18-driven IBD that may respond more effectively to a therapy targeting this mechanism.

Abstract: Objective This study aimed to systematically screen for potential candidate biomarkers and identify therapeutic targets associated with gouty arthritis (GA) through integrated analyses of single-cell and bulk RNA sequencing (RNA-seq) data. Methods The single-cell dataset GSE211783 and the bulk RNA-seq dataset GSE160170 were analyzed using a series of bioinformatic approaches, including cell clustering, differential expression analysis, immune cell infiltration assessment, protein-protein interaction network construction, gene set enrichment analysis, as well as drug sensitivity evaluation. To establish an animal model of GA, monosodium urate crystals were injected intra-articularly into experimental mice. Joint swelling was evaluated, and morphological changes in joint tissues were analyzed through hematoxylin-eosin staining. The presence of TREM1-positive cells was detected by immunohistochemistry and the level of TREM1 protein expression in joint tissues were assessed by Western blotting. Results We identified 102 differentially expressed genes (DEGs) and 14 signaling pathways associated with GA. The PPI network revealed 25 hub genes, of which 17 (including TREM1, TNF, PTGS2, and NLRP3) were highly expressed and 8 (including FCGR3B and CXCR6) showed low expression in the GA samples. These genes correlated significantly with the infiltration levels of macrophages. Among the hub genes, TREM1 was selected for further validation because it correlated significantly with all 14 differential pathways. In animal experiments, GA mice developed marked joint swelling and inflammatory tissue injury, along with a significant increase in TREM1-positive cells and TREM1 protein expression. Conclusion Integrative analysis of single-cell and bulk RNA-seq data identified 102 GA-related DEGs and 14 key pathways, from which 25 hub genes were screened. TREM1 is significantly upregulated in GA and may be linked to macrophage function, providing new insights into biomarker and therapeutic target discovery for GA.

Celiac disease (CD) is strongly associated with specific HLA DQ heterodimers, formed by HLA DQA1 and HLA DQB1 proteins. In particular DQ2.5 (DQB1*02 associated to DQA1*05) and DQ8 (DQB1*03:02 with DQA1*03) are present in virtually all celiac patients. HLA DQB1*02 is considered the main single genetic susceptibility marker and has been reported in 90 to 95% of CD patients. However, the distribution of these alleles may vary across populations, potentially impacting the performance of genetic screening strategies. In this study, we evaluated the prevalence of HLA DQ2.5 and DQ8 genotypes in celiac patients (n = 41) and an unbiased general population cohort (n = 60) from San Luis, Argentina, using a PCR-based genotyping approach. In addition, we assessed the feasibility of a simplified saliva direct PCR protocol for large scale testing. Overall, 95.1% of CD patients carried DQ2.5 and/or DQ8. Notably, 41.5% of patients were DQ8(+)/DQ2.5(-), and 36.6% lacked the DQB1*02 allele, indicating that DQB1*02 based screening alone would have reduced sensitivity in this population. In the general population, 53.3% of individuals carried CD associated genotypes, with a markedly higher prevalence of DQ8 compared to European cohorts. Genotype distributions deviated from Hardy Weinberg equilibrium in CD patients but not in the general population. We show that DQB1*03:02 is a reliable proxy for DQ8, allowing simplification of genotyping strategies, whereas DQA1*05 typing remains essential to discriminate DQ2.5 from other lower risk DQB1*02 carrying heterodimers. We also describe a saliva direct PCR approach showing a performance comparable to purified DNA based assays. These findings highlight the importance of population specific genetic data for optimizing CD screening strategies and foster the development of simplified, cost effective genotyping approaches for large scale applications.

Introduction: Myalgic Encephalomyelitis (ME)/Chronic Fatigue Syndrome (CFS) is a debilitating condition characterised by severe fatigue and post-exertional malaise (PEM). Reported neuropsychophysiological abnormalities suggest ME/CFS is multifactorial, but current knowledge remains fragmented. This study protocol outlines a multimodal investigation designed to (1) compare neuropsychophysiological mechanisms between ME/CFS patients and healthy participants, (2) test an integrative model of ME/CFS, (3) identify neuropsychophysiological subgroups within the patient population, and (4) identify predictors of symptom response during rehabilitation. Methods and analysis: This study will enroll 115 ME/CFS patients and 55 healthy participants. Groups will be comparable in age, sex, and education level, with a larger patient sample enabling subgroup and longitudinal analyses. A cross-sectional assessment at baseline will be carried out in both groups. Patients will then be evaluated longitudinally throughout a standardized cognitive-behavioral therapy rehabilitation program delivered as routine care. Baseline measures include systemic inflammation and general health biomarkers, measures of autonomic and central nervous system function, neuroinflammation (magnetic resonance spectroscopy, [18F]DPA714 PET in a subsample), serum short-chain fatty acid levels, gut microbiota composition and function, and neuroendocrine and self-reported responses to psychosocial stress. Fatigue severity (physical and cognitive) and PEM will be assessed through validated questionnaires, ecological momentary assessment, and laboratory tasks. These will be re-evaluated during therapy, and all non-neuroimaging measures will be repeated after the rehabilitation program. Statistical analyses will comprise multivariate analysis of variance, general linear models, classification algorithms, structural equation models, least absolute shrinkage selection operator principal component regression (LASSO-PCR), cluster analysis and latent class growth analysis (LCGA).

BackgroundT helper 2 (Th2) lymphocytes orchestrate type-2 immunity and drive allergic diseases that disproportionately affect females. Sexual dimorphism in Th2 responses is well-documented, yet current models attribute sex differences exclusively to circulating gonadal hormones and sex chromosomes. Whether cell-intrinsic steroidogenesis, mediated by the enzyme Cyp11a1, contributes to female-biased Th2 differentiation and function remains unknown. MethodsTranscriptomes of in vitro generated Th2 cells from male and female T cell-specific Cyp11a1-knockout (Cyp11a1fl/fl;Cd4Cre) and control (Cyp11a1fl/fl) mice were compared. Differential expression, hallmark pathway analysis, transcription factor activity scoring, and functional assays were performed across sexes and genotypes. Cyp11a1-dependent differentially expressed genes were integrated with sex-stratified human Th2 transcriptomes obtained from the type-2 inflammatory skin disease atopic dermatitis. ResultsCyp11a1 deletion markedly reduced the transcriptional signature distinguishing female from male Th2 cells. Female Cyp11a1-knockout Th2 cells underwent extensive transcriptomic reprogramming converging toward the male profile, while male cells were largely unaffected. Female-specific pathway changes included reduced inflammatory signatures and enhanced cell-cycle programmes. Functionally, female Cyp11a1-deficient Th2 cells exhibited significantly increased proliferation and elevated IL-13 production; male knockout cells showed no comparable changes. These effects were developmentally stage-specific, emerging during Th2 differentiation but not in naive precursors. Cross-species analysis identified a conserved gene module shared between Cyp11a1-deficient female mouse Th2 cells and female-biased human Th2 cells in atopic dermatitis. ConclusionsCyp11a1-mediated steroidogenesis is a cell-intrinsic regulator of the female-biased Th2 transcriptional and functional state, identifying de novo steroidogenesis as a mechanism of immunological sexual dimorphism with direct relevance for female-predominant allergic disease.