Frontiers in Immunology

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.

A viral exposure signature (VES) has been previously described predicting the development of Hepatocellular carcinoma (HCC) in at-risk patients. This has been achieved by a serological profiling of the viral infection history using a synthetic human virome including >100k epitopes (VirScan). In the present study we applied the same VirScan strategy to identify a differential serum binding pattern (DSBP) for classifying patients of different cancer types from healthy individuals. In particular, the healthy group included both age-matched (ADULTS) as well as elderly (ELDERS) individuals, the latter counting also nonagenarians and centenarians. The class comparison performed with serological data show DSBPs supporting class predictions, as confirmed by the receiver operating characteristic (ROC) curve analysis. Antibody responses supporting the class predictions are specific to peptides from persistent herpesviruses, acute-infecting viruses and, consistently in all comparisons, human respiratory syncytial virus (HRSV). Strikingly, the DSB of the ELDERS vs. CANCER comparison is characterized by higher titers in the healthy subjects; on the contrary, the DSB of the ADULTS vs. CANCER comparison is characterized by lower titers in the healthy subjects. Overall, the results show a differential serological binding pattern predicting healthy individuals (ADULTS or ELDERS) from patients with different types of cancer. Such results provide the first evidence suggesting a close link between anti-microbial immunity and cancer development. They may be of the highest relevance in terms of predictive, diagnostic and/or prognostic impact in oncology.

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.

Many intracellular pathogens have evolved to evade immune responses and establish a secure niche inside host cells. One such stealth pathogen is the obligate intracellular bacterium Coxiella burnetii, the causative agent of Q-fever. Coxiella translocates an array of bacterial proteins ( effectors) into the host cell through a type IVB secretion system (T4BSS) that mediates suppression of pathogen sensing and innate immunity. Yet, at a systemic level, immunocompetent hosts often restrict pathogens through Th1-mediated and cell-autonomous immunity through the expression of immune-inducible genes. However, the expression and regulation of chemokines, particularly, the CXC-ligands (CXCL9,-10,-11) that are considered biomarkers of Q-fever, is poorly understood. We observed minimal to no CXCL10 transcript levels during Coxiella infection. However, Coxiella-infected cells robustly augmented IFN{gamma}-activated expression of CXCL10 in both phagocytic and non-phagocytic cells, and this process was dependent on viability and T4BSS in epithelial cells. This phenomenon extends to other highly pro-inflammatory cytokines and other pathogens including Salmonella, Mycobacteria (H37Ra) and Toxoplasma. Synergistic increase in CXCL10 expression in Coxiella-infected, IFN{gamma}-activated cells requires ISRE and NF-{kappa}B transcriptional elements in the promoter, and the transcription factors STAT1, STAT3 and IRF9. Inhibition of STAT3 by small molecule inhibitors potently decreased the excess promoter activity of CXCL10. In addition, treatment of Coxiella-infected cells with IFN{gamma} is associated with decreased expression of SOCS1, a negative regulator of the IFN{gamma} signaling axis and relatively higher detection of extracellular bacteria. Altogether, these data demonstrate that intracellular pathogens including those conventionally considered to be "immunologically silent", robustly synergize with IFN{gamma} signaling, with STAT3 activation emerging to be a nodal point for promoting both persistent infection as well as synergism in the expression of immune genes. Author summaryAcute host immune response is often associated with production of soluble messenger molecules called cytokines/chemokines which direct the migration, recruitment and activation of leukocytes and serve as biomarkers in infectious and inflammatory diseases. The regulation of expression of these molecules and their influence on the infection process is not well-understood. In particular, interferon-gamma (IFN{gamma}), a potent pro-inflammatory cytokine produced by activated T and NK cells, activates signaling pathways involved in host defense and inflammation in macrophages and other cell types. We observed that infection with many intracellular bacterial/parasitic pathogens that employ sophisticated immune evasion strategies, synergize with IFN{gamma} signaling and significantly amplify the levels of pro-inflammatory mediators implicating the origin of adverse immune pathologies. We investigated the mechanistic basis of this seemingly counter-intuitive phenomenon, underlying host and bacterial factors involved in distinct cell types, and identified the small molecule-targetable-transcription factor STAT3 as a host determinant in promoting excess cytokine synthesis.

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.

BackgroundBevacizumab resistance severely limits long-term efficacy in metastatic colorectal cancer (CRC). This study aimed to develop and validate a bevacizumab resistance-associated gene signature for prognosis prediction and immune microenvironment characterization in CRC. MethodsTwo GEO datasets (GSE19862, GSE86582) with bevacizumab response data and TCGA-COAD/READ RNA-seq data were analyzed. Overlapping differentially expressed genes (DEGs) linked to both CRC progression and bevacizumab resistance were identified. An 8-gene signature (AXIN2, PSORS1C1, KRT74, SLC2A3, STIL, IL33, GALNT6, HSD11B2) was constructed via univariate Cox and LASSO-Cox regression. ResultsIn the TCGA cohort, high-risk patients had shorter overall survival (OS; log-rank P < 0.0001). Time-dependent ROC yielded 1-year AUC = 0.638, 3-year AUC = 0.657, and 5-year AUC = 0.757. Multivariate Cox regression confirmed the risk score as an independent prognostic factor. External validation in GSE39582 (optimal cutoff = -1.49) replicated these findings: high-risk patients had inferior OS (P = 0.0016) with acceptable 1/3/5-year AUCs and retained independent prognostic value (HR = 1.634, P = 0.00415). CIBERSORT and ESTIMATE analyses showed that the high-risk group was characterized by increased M2 macrophages and neutrophils, higher immune and stromal scores, and reduced activated memory CD4+ T cells, monocytes, and activated dendritic cells (all P < 0.05). GSEA highlighted enrichment of TNF-/NF-{kappa}B, IL-6/JAK/STAT3, and immune checkpoint pathways in the high-risk group. AXIN2 (HR = 0.829, P = 0.032) was an independent protective factor, while PSORS1C1 (HR = 1.356, P = 0.048) was an independent risk factor. ConclusionThe 8-gene bevacizumab resistance signature robustly predicts prognosis and reflects an immunosuppressive microenvironment closely linked to bevacizumab failure in CRC. These findings provide novel insights into immune-mediated resistance and support clinical risk stratification.

Background: Chronic rhinosinusitis (CRS) is a heterogeneous inflammatory airway disease associated with impaired mucociliary clearance and persistent inflammation. While prior work has focused on inflammatory and molecular pathways, the physicochemical properties of mucus itself remain poorly characterized. This study aimed to define compositional and biophysical features of CRS mucus that may contribute to dysfunction. Methods: A prospective cross-sectional study was conducted in 15 adults undergoing endoscopic sinus surgery (11 CRS, 4 controls). Mucus was collected from the middle meatus. Hydration was measured by lyophilization. Ionic composition was quantified using mass spectrometry. Viscoelasticity was assessed via oscillatory shear rheology. Total protein, total carbohydrate, sialic acid (Sia) and fucose (Fuc) content were quantified using enzymatic and chemical assays. Statistical comparisons were performed using nonparametric tests. Results: CRS mucus exhibited significantly higher Ca2+; and Mg2+; concentrations (approximately two-fold; p<0.05) and increased variability in hydration and ion content compared to controls. Rheology showed greater heterogeneity and a non-significant trend toward increased viscoelasticity in CRS. Total protein and carbohydrate content were not significantly different; however, the carbohydrate-to-protein ratio was significantly reduced in CRS (p=0.04). Sia content and Sia-to-carbohydrate ratio were significantly elevated in CRS (p=0.04 and p=0.002), particularly in CRS with nasal polyps. Fuc content did not differ between groups. Conclusions: CRS mucus demonstrates coordinated alterations in ionic composition and glycosylation, characterized by increased cation content, hypersialylation, and reduced carbohydrate-to-protein ratios. These changes may contribute to altered mucus properties and impaired mucociliary clearance, highlighting mucus composition as a potential therapeutic target in CRS.

Tuberculosis (TB) continues to pose a significant global public health challenge with substantial patient morbidity and mortality. Current TB patient biomarkers lack sufficient resolution to inform treatment response and patient stratification. This necessitates the development of sensitive and reliable host biomarkers. We previously demonstrated the efficacy of TruCulture whole blood stimulation for differentiating asymptomatic TB from active pulmonary TB disease patients in endemic regions. Our systems immunology study expands upon this previous work by evaluating the potential of TruCulture to monitor longitudinal responses to TB treatment in patients from the Predict-TB trial before, during, and after 6 months of antibiotic therapy. We stimulated whole blood from TB patients (n=40) using TruCulture under four conditions (Null, Mycobacterium tuberculosis-antigen, LPS, and IL-1{beta}) at baseline (week 0), during treatment (weeks 16 and 24), and one-year follow-up post- treatment (week 72). 20/25 measured cytokines exhibited significant changes throughout treatment, with several continuing to evolve during post-therapy follow-up. Machine learning based analysis identified Mtb-Ag-induced IL-1RA (AUC = 0.90, 0.92, 0.95 at weeks 16, 24, 72) and LPS-induced NLRP3 (AUC = 0.94 at week 16) as the best protein and transcriptional biomarkers for distinguishing treated from untreated patients, strongly implicating the inflammasome response. Combining these results with the extent of lung disease assessed by FDG PET/CT scans, we showed direct disease relevance for these blood-based biomarkers. The identified biomarker profiles hold promise for improving TB patient care through early prediction of treatment responses, real-time therapy monitoring, and informed development of host-directed therapeutic strategies for clinical decision-making. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=146 HEIGHT=200 SRC="FIGDIR/small/723467v1_ufig1.gif" ALT="Figure 1"> View larger version (45K): org.highwire.dtl.DTLVardef@14a32eforg.highwire.dtl.DTLVardef@55f3d4org.highwire.dtl.DTLVardef@fb0137org.highwire.dtl.DTLVardef@10cf39e_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOGraphical abstractC_FLOATNO Predict-TB clinical study overview and summary of TB-specific biomarkers identified from TruCulture whole blood stimulation system. C_FIG

ProblemThe anti-microbial protein granulysin is present in vaginal secretions during the follicular phase of the menstrual cycle but nearly disappears during the luteal phase. The reason for this change is unknown. Method of studyParticipants (n = 23) with regular menstrual cycles collected daily vaginal swabs for granulysin ELISAs. Endocervical cytobrushes, ectocervical biopsies, vaginal biopsies, and PBMC were collected across the cycle to enumerate granulysin-expressing cells by flow cytometry. Cycle phase was determined by daily urinary luteinizing hormone testing and confirmed by serum progesterone levels. ResultsGranulysin levels in secretions were up to 10,000 times higher during menstruation than during the luteal phase (menstruation, median 3,924 pg/mL [IQR 400-17,280]; luteal, median and IQR undetectable [<7.81 pg/mL]). In the endocervical canal, granulysin-expressing cells were much more abundant during menstruation than during the mid-follicular or mid-luteal phases. In contrast, the number of granulysin-expressing cells in the ectocervix and vagina remained stable during the cycle. The most abundant granulysin-expressing cell types in the mucosa were CD8 T cells and NK cells. In a minority of participants, granulysin was consistently detected in luteal-phase swabs; this phenomenon was associated with parity. ConclusionsGranulysin in vaginal secretions is associated with menstruation, which also drives a spike in granulysin-expressing cells in the endocervical canal. This result explains the much higher granulysin levels in secretions during the follicular than the luteal phase. In contrast, immune cells from ectocervical and vaginal biopsies express granulysin independently of the menstrual cycle, indicating their continuous ability to respond to microbial infection.

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.

Kawasaki disease (KD) is an acute pediatric vasculitis characterized by dysregulated host immune responses and risk of coronary artery injury. Although a two-transcript IFI27-MCEMP1 axis has been clinically validated to distinguish KD from other febrile illnesses, the long noncoding RNA (lncRNA) context of this interferon-myeloid imbalance remains incompletely understood. We evaluated whether peripheral blood mononuclear cell (PBMC)-derived lncRNAs are altered in KD and associated with the interferon and myeloid components of the IFI27-MCEMP1 transcriptomic axis. Children younger than 8 years with suspected KD were prospectively enrolled at the Children's Hospital of Fudan University from 2024 to 2025. The newly enrolled cohort included 55 children with KD and 48 febrile controls. For integrated immune-transcript association analyses, these data were combined with two previously characterized same-site cohorts, yielding 188 children with KD and 175 febrile controls. Expression of IFI27, MCEMP1, CHROMR, MALAT1, and NEAT1 was measured by reverse transcription quantitative PCR and normalized to GAPDH using {Delta}Ct values. In the newly enrolled cohort, the IFI27-MCEMP1 axis reproduced discrimination between KD and febrile controls, with an area under the receiver operating characteristic curve of 0.88; performance was similar in the integrated cohort, with an area under the curve of 0.89. In PBMC lncRNA analyses, CHROMR and MALAT1 {Delta}Ct values were significantly higher in KD than in febrile controls, indicating lower relative expression, whereas NEAT1 did not show a significant KD-specific differential-expression signal. CHROMR showed the strongest association with the IFI27 interferon-associated component, while MALAT1 showed weaker but directionally informative associations with both IFI27 and MCEMP1, including an inverse association with MCEMP1. These findings support an lncRNA-associated interferon-myeloid immune architecture in KD, marked by coordinated attenuation of IFI27, CHROMR, and MALAT1 together with increased MCEMP1. This PBMC RNA pattern provides a biologically interpretable framework for KD immune dysregulation and generates testable hypotheses regarding RNA-regulatory programs in KD vasculitis.

NK cells can form clonal populations demonstrating features of adaptive immunity, including long-term memory and at least partial antigenic specificity. Given the limited individual diversity of activating receptors, the nature of NK cell antigenic specificity remains elusive. To explore this riddle, we combined scRNA-Seq of ex vivo FACS-sorted NK cell subsets expressing specific KIR receptors, single-cell cloning and bulk RNA-Seq of in vitro cultured KIR2DS4 NK cell clones, transcriptomic profiling of antigen-stimulated NK cells, and in silico modeling of glycosylated KIR2DS4-peptide-HLA complexes. scRNA-Seq resolved 12-15 clusters per KIR subset with highly heterogeneous KIR, KLRC and NCR expression patterns, consistent with clonal lineages. Notably, those clusters demonstrated over 30 differentially expressed glycosyltransferase genes, potentially involved in post-translational modification of NK cell receptors. Single-cell-derived KIR2DS4 cultures exhibited clone-specific cytotoxic, chemokine and KIR receptor genes, and transcriptional differences in > 40 glycosyltransferases. In peptide culturing autologous assays, SARS-CoV-2 (KTFPPTEPK) and EBV (CRAKFKHLL) peptides elicited NK cell proliferation and distinct transcriptional programs linking cytotoxicity genes, KIR2DS4 and glycosyltransferases. Structural modeling revealed that N-linked glycosyl residues in specific regions of KIR2DS4 may alter its contacts and interaction with MHCI and the presented peptide. We conclude that KIR human NK cells comprise clonally imprinted populations with distinct glycosyltransferase expression profiles, and site-specific KIR2DS4 glycosylation may modulate interaction with peptide-MHCI complexes, suggesting a post-translational layer of clonal NK cell diversification as a clue to their antigenic specificity.

Abstract: Background: More than half of metastatic melanomas arise from patients initially diagnosed with early-stage melanoma. Objective biomarkers are needed to better identify high-risk patients. Objective: To evaluate the prognostic value of multiple histopathological characteristics in predicting distant metastasis risk, in early-stage melanoma. Methods: Using data from discovery set (n=442) and a population-based validation cohort (n=306, sampled from 5,815 patients) of the Dutch Early-Stage Melanoma (D-ESMEL) study, we investigated 14 histopathological characteristics of melanoma and their tumor micro-environment (TME) in an unprecedented integration, by expert pathologist scoring and automated quantitative measurements derived from a validated automated segmentation. Results: Increased immune infiltrates (40% in cases vs. 50% in controls) were associated with lower risk of metastasis. Automated immune cell density was predictive in both the discovery set and the validation cohort, outperforming the manual pathological tumor infiltrating lymphocytes. The remaining histopathological features, including mitotic activity, did not retain independent value after controlling for current staging variables. Limitations: TME evaluation in standard Hematoxylin-Eosin slides. Conclusion: TME reaction is an important determinant of melanoma progression. The automated quantification of immune cell density appears to be a biomarker for distant metastasis risk. Further investigation into specific immune cell subtypes is required to facilitate clinical integration.

Osteolytic bone diseases are driven by excessive osteoclast formation and bone resorption. While cGAS-STING signaling is known to regulate bone homeostasis via macrophage-intrinsic mechanisms, its role in osteoblast-mediated control of osteoclastogenesis remains poorly defined. Here, we show that cGAS-STING activation of macrophages suppresses their osteoclastogenic potential while promoting immune activation. In osteoblasts, cGAS-STING triggers IRF3-mediated IFN-{beta} production and, notably, shifts the OPG-RANKL axis toward increased osteoprotegerin. In transwell co-culture, pre-activated osteoblasts reduce osteoclast differentiation of strain-matched macrophages. Mechanistically, osteoblast-derived IFN-{beta} is sufficient to inhibit osteoclastogenesis in a paracrine manner. Furthermore, autocrine IFN-{beta} signaling appears to modulate the OPG-RANKL axis, although additional regulatory factors may contribute. Together, these findings identify cGAS-STING-IFN-{beta} signaling as a dual regulator of osteoclastogenesis, acting directly on macrophages and indirectly via osteoblast-derived anti-osteoclastogenic mediators. This highlights osteoblasts as cGAS-STING-responsive bystander cells within the bone microenvironment that can be targeted as an alternative strategy to limit pathological bone resorption. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=127 SRC="FIGDIR/small/724040v1_ufig1.gif" ALT="Figure 1"> View larger version (70K): org.highwire.dtl.DTLVardef@167dfcorg.highwire.dtl.DTLVardef@a95477org.highwire.dtl.DTLVardef@e88c77org.highwire.dtl.DTLVardef@15de567_HPS_FORMAT_FIGEXP M_FIG C_FIG

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: Synthetic oligopeptides provide a rapid and cost-efficient approach to developing antibodies and diagnostics for emerging viral variants. Methods: This study computationally and experimentally characterized a synthetic peptide analog of the SARS-CoV-2 spike subdomain 2 major disulfide loop (SD2MDL), designated S621 (CPVAIHADQLTPTWRVYSTC). Binding affinity was computationally estimated using the Heuristic Affinity Prediction Tool for Immune Complexes (HAPTIC), while experimental validation was performed using enzyme-linked immunosorbent assay (ELISA) with rabbit-derived antipeptide antibodies. Clinical diagnostic accuracy testing was done using plasma samples from RT-PCR-confirmed COVID-19 patients and pre-COVID-19 controls. Results: S621 demonstrated nanomolar binding affinity (Kdapp = 1.14 nM) and high avidity (3.67 nM), closely matching HAPTIC predictions (3.54 nM). Diagnostic evaluation yielded a sensitivity of 89.92% and specificity of 27.79%, corresponding to an overall accuracy of 71.79%. Discussion: These findings demonstrate that a single synthetic peptide derived from a conserved spike subdomain can function as a high-affinity surrogate for full-length antigens, supporting its potential application in rapid peptide-based immunodiagnostics.

WHIM (warts, hypogammaglobulinemia, infections, and myelokathexis) syndrome is a primary immunodeficiency caused by gain-of-function in CXCR4 chemokine receptor (CXCR4GOF) in response to its chemokine ligand CXCL12. The patients suffering from this syndrome display lymphopenia and neutropenia, and most of them show exacerbated susceptibility to human papillomavirus pathogenesis. In a mouse model harboring a WHIM-associated CXCR4 mutation and expressing HPV16 oncoproteins in keratinocytes, we previously reported reduced circulating plasmacytoid dendritic cells (pDCs), mirroring patients blood, and impaired dendritic cell (DC) trafficking from the skin to lymphoid organs, with the few migrating DCs displaying an overactivated phenotype. Given the promising results of CXCR4-targeted therapies in WHIM patients, we investigated whether and how the orally available CXCR4-specific antagonist, X4-136, affects DC localization, activation, and trafficking at the subset level, as well as skin immune landscape. CXCR4GOF inhibition corrected defects in circulating myeloid cells and pDCs, as well as in lymph node-resident DCs. Furthermore, it rescued skin DC migration to lymph nodes in WHIM mice, in a context- and subset-dependent manner, by promoting their activation and relocation within the dermis. Taken together, these findings indicate that inhibiting CXCR4GOF may restore skin immunity in WHIM syndrome by rescuing DC counts and functions. Key pointsO_LICXC R4 gain-of-function inhibition promotes subset-selective dermal dendritic cell migration to lymph nodes in a WHIM syndrome mouse model. C_LIO_LIInhibiting CXCR4 corrects migratory WHIM dendritic cell hyperactivation with subset-specific effects tied to the inflammatory context. C_LI

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

The study investigated the interaction between estrogen deprivation and periodontitis, systemically, in the bone marrow, and locally in periodontal tissues using a mouse model. MethodsWe used the ligature-induced periodontitis (LIP) model concurrently with ovariectomy-induced estrogen deprivation. Bone marrow was assessed for myeloid cell proportion by flow cytometry. The femur metaphysis was examined histologically and by micro-CT. Cytokine responses of CD11b+ myeloid cells to lipopolysaccharide stimulation were investigated ex vivo across ovary-intact (Sham), ovariectomized (OVX), and estrogen-replaced (OVX+E2) mice with or without periodontitis. Estrogen-related alterations in periodontitis, including microbiome composition and transcriptomic changes in the gingiva and dentoalveolar complex, were investigated by 16S rRNA sequencing and bulk RNA sequencing, respectively. ResultsOvariectomy increased osteoblast-like and adipocyte-like cell numbers in femoral marrow, whereas LIP reduced both populations (p = 0.020 and p = 0.029, respectively). LIP increased the bone marrow CD45+ hematopoietic fraction in Sham mice. LPS-stimulated bone marrow CD11b+ cells from OVX mice showed lower Tnf, Ccl2, and Il10 expression than Sham mice (p = 0.003, p = 0.005, and p = 0.001, respectively). OVX exacerbated LIP-associated alveolar bone loss, reducing BV/TV (p = 0.003) and increasing osteoclast numbers (p = 0.012). Neither OVX nor E2 replacement significantly altered ligature-associated microbial composition in 16S rRNA sequencing. Bulk RNA sequencing demonstrated estrogen-responsive transcriptomic changes in both the gingiva and dentoalveolar complex, including OVX-associated gene-expression changes that returned toward Sham levels in OVX+E2 mice. These included genes related to stromal regulation (Acan, Igfbp3, Erbb3) and immunity (Gp2, Spib, B2m). ConclusionPeriodontitis and estrogen deprivation exert combined effects on the bone marrow niche. Estrogen deprivation modulates immune- and healing-related gene expression in the gingiva and remaining dentoalveolar tissues during periodontitis.

MicroRNAs (miRNAs) are small noncoding RNAs that play critical roles in regulating immune responses and have emerged as potential biomarkers and therapeutic targets in complex diseases. Leishmaniasis is a neglected disease that compromises host immunity and is associated with challenging treatments regimens. Leishmania amazonensis (L. amazonensis), an intracellular protozoan parasite, causes cutaneous leishmaniasis by replicating inside mammalian macrophages to establish infection. In this context, miRNAs have emerged as vital post-transcriptional factors that regulate the inflammatory landscape during infection. In this study, we aimed to analyze the function of miR-721 in macrophages during L. amazonensis infection by integrating in silico miR-721 target prediction with RNAseq data from macrophages of two distinct mouse genotypes, resistant C57BL/6 and susceptible BALB/c. We found that miR-721 is induced in macrophages infected with L. amazonensis, but is not in LPS-stimulated macrophages, suggesting a TLR4-independent activation. Integrating miR-721 target prediction with comparative transcriptomic analyses in resistant C57BL/6 and susceptible BALB/c models revealed the TNF-IRF1 axis as a primary miR-721-associated regulatory network. Specifically, miR-721 is predicted to target the 3UTRs of Tnf and Irf1 to suppress the inflammatory response. Functional inhibition of miR-721 successfully restored Tnf and Irf1 expression and reduced the amastigote burden over 24 hours. Furthermore, we showed that the miR-721/TNF-IRF1 axis regulates downstream genes associated with macrophage response, such as Serpine1, Csf1, Cd69 and Maf. Our work demonstrated that Leishmania induces miR-721, which negatively modulates the TNF-IRF1 axis, thereby suppressing the immune response and favoring parasite persistence. While C57BL/6 macrophages exhibit a robust activation of the TNF-IRF1 network, promoting inflammatory response, BALB/c macrophage showed a breakdown of this network. This was associated with post-transcriptional suppression of inflammatory responses, thereby favoring parasite persistence. These findings link miR-721 to the establishment of macrophage polarization, providing relevant insights into the mechanisms of parasite subversion of the host immune response.