Genes & Immunity

Background. Polygenic scores (PRS) for coronary artery disease (CAD) are derived almost entirely from European-ancestry data. Their portability to Southeast Asian populations, including the Vietnamese, is largely uncharacterised and clinically consequential when scores are used with risk thresholds. Methods. We evaluated four independent European-derived CAD scores from the PGS Catalog (PGS000058, PGS000349, PGS002809, PGS004198; 70 - 5,723 variants) in 2,504 individuals from the 1000 Genomes Project, focusing on the Vietnamese Kinh (KHV) and Dai (CDX) samples. Per-individual scores were computed with PLINK2 and standardised. We assessed (i) the cross-ancestry distribution (calibration) and (ii) a clinically-relevant consequence: the proportion of each population flagged high genetic risk when the European top-20% threshold is applied (20% if perfectly calibrated). Results. For the primary score (PGS000058) the standardised PRS differed across super-populations (ANOVA F(4, 2499) = 121.1, p < 0.001); the Vietnamese Kinh mean was +0.47 SD above the European mean (Welch t = 7.77, p = 2.0 x 10^ -14). Applying the European top-20% high-risk threshold, the fraction of Vietnamese Kinh flagged ranged from 22.2% to 57.6% across the four scores, and of Dai from 21.5% to 43.0%, versus the intended 20%. Three of the four scores over-flagged Vietnamese (25-58%); the largest score (PGS004198) was approximately calibrated for East/Southeast Asians ([~]22%) but markedly over-flagged Africans (69.3%). Conclusions. European-derived CAD polygenic scores are inconsistently calibrated in Vietnamese and other Southeast Asian samples, and most substantially over-flag high genetic risk when a European threshold is applied. The magnitude and even the direction of miscalibration depend on the specific score, so no such score can be assumed transferable without local validation and recalibration. Distribution shift bounds, but does not by itself quantify, loss of predictive accuracy, which requires phenotyped data.

Background and Aims: The molecular pathogenesis of Inflammatory Bowel Disease (IBD) remains unclear. We aimed to establish a high-resolution immune landscape of treatment-naive IBD to identify central drivers of disease onset and early pathogenic signalling. Methods: We generated a single-cell atlas using intestinal biopsies from a large adult inception cohort of 137 individuals, including treatment-naive Crohn's disease (CD), ulcerative colitis (UC), and symptomatic non-IBD controls. We integrated scRNA-seq (1 million cells) with co-varying neighbourhood analysis (CNA) and unbiased tensor decomposition of cell-cell communication (CCC) networks. Findings were validated in vitro macrophage stimulation model and using serum from patients. Results: The inception cohort exhibited significantly more homogenous compartmental diversity compared to benchmark reference studies (p < 0.001). Inflammation in both CD and UC was characterized by a marked expansion of inflammatory monocytes. Unbiased CCC analysis identified a dominant disease-specific signalling module centred on the Galectin family (LGALS1 and LGALS9). Galectin-9 expression was specifically enriched in inflammatory monocytes, which exhibited distinct. transcriptional programs linked to antigen presentation and microbial sensing. In vitro, Galectin-9 acted as a potent stimulus, driving macrophages toward a pro-inflammatory phenotype. Clinically, serum Galectin-9 levels were significantly elevated in IBD patients and correlated with systemic inflammatory markers and treatment response. Conclusions: Our data identify a galectin-monocyte signalling axis as a unifying inflammatory hallmark of early IBD. Galectin-9 serves as both a functional driver of mucosal inflammation and a dynamic biomarker, offering new opportunities for therapeutic targeting and disease monitoring from diagnosis. Keywords: Inflammatory Bowel Disease; Crohn's Disease; Ulcerative Colitis; Single-cell RNA sequencing; Galectin-9; Inflammatory monocytes.

Immunity to P. falciparum malaria develops slowly, requiring repeated infection in areas of high transmission, and wanes rapidly in the absence of parasite exposure. Key to this immunity, is the development of antibodies which is supported by CD4 T follicular helper (Tfh) cells that drive robust germinal centre responses. However, in malaria, the malaria-specific CD4 T cell compartment in peripheral blood is dominated by Type 1 regulatory T cells (Tr1), which produce high levels of IL-10 in response to parasites. Tr1-like Tfh cells (Tfh10) have been reported in several settings of repeated antigen stimulation but have not been investigated in malaria. Here we used single-cell RNA sequencing and multiparameter flow cytometry to characterise malaria-specific Tfh and Tr1 cells in a longitudinal cohort of highly exposed individuals and assessed their persistence after transmission interruption. Malaria-specific Tfh and Tr1 cells shared overlapping profiles, and Tr1 cell-like transcriptional signatures and phenotypes were detectable within the Tfh cell compartment. Tfh10 cell subsets were the dominant phenotype of malaria-specific Tfh cells. Following disruption of malaria transmission, the frequencies of malaria-specific Tr1 and Tfh10 cells declined. These findings highlight a close relationship between Tfh and Tr1 cells and show that the Tfh cell compartment in malaria is dominated by Tfh10 cells. The rapid waning of these cells in the absence of continuous exposure is consistent with requirements of persistent antigen in maintaining regulatory CD4 T cell phenotypes.

Background Complex traits are influenced by numerous variants, most of which have regulatory effects on gene expression that can be mediated by DNA methylation. Molecular QTL mapping is an approach that aims to dissect these effects. However, obtaining molecular phenotypes on a large scale is challenging, particularly in livestock species. In cattle, an epigenotyping array called EpiChip has recently been developed in the European RUMIGEN project. The EpiChip, which contains 43,317 CpG sites distributed all over the bovine genome, enables large-scale measurement of DNA methylation. This study aims to characterize the genetic determinism of blood DNA methylation in cows by estimating heritability and mapping cis- and trans-methylation QTLs (meQTLs). Results Whole blood samples from 4,457 genotyped Holstein cows were epigenotyped. Across all CpG sites, the heritability estimates averaged 24.6%. The local meQTL mapping at sequence-level for variable CpG sites (SD > 2.5%; n = 28,806) detected cis-meQTLs for 80.1% of the CpG sites, with sentinel SNPs located close to their associated CpGs. A two-step analysis was also conducted to identify long-range associations, with a particular focus on trans-meQTL hotspots. First, we identified CpG-SNP trans-associations using medium-density genotypes (50k SNPs) that revealed 31,846 SNPs with significant effects on 1 to 530 trans-CpG sites. Then, regions associated with at least 34 independent trans-CpGs were retained defining 31 hotpots. For each hotspot, a local sequence-level GWAS was conducted using the first principal component derived from the associated trans-CpGs. Out of the 31 detected hotspots, three were located close to transcription factor genes (RUNX1, NFIC and FOXA3) for which the associated trans-CpGs were enriched for the corresponding binding motif. Two other hotspots were located within KDM5A and KDM5B, and their corresponding trans-CpGs were strongly overrepresented in H3K4me3 narrow peaks in blood as well as in other tissues. Conclusions By identifying functional candidate genes associated with blood DNA methylation in cattle, these findings provide new insights into the regulatory architecture of DNA methylation in mammals, highlighting the value of large-scale molecular data from livestock populations.

Background Pneumococcal disease is a leading cause of paediatric pneumonia and meningitis. Pneumococcal colonisation is the fundamental step to pneumococcal disease causation. We aimed to identify genetic loci associated with pneumococcal colonisation amongst children. Methods We conducted a genome-wide association study on 2111 Nepalese children, comprising 1346 cases carrying pneumococcus and 765 controls. We tested 8.1 million imputed variants using logistic regression and ten principal components as covariates. Fine mapping and functional evidence were used to identify suspected causal variants and related genes of interest. Findings A cluster of 22 variants of genome-wide significance (p<5x10-8) were identified on chromosome 12q21.31, eight of which were within PPFIA2. Fine mapping of this region identified 5 variants within 0.1 Mb of the 5-prime region of PPFIA2 all of which are significant eQTLs for PPFIA2. We further describe three loci (10q23.31, 12q23.1, and 20p11.21) which had variants with highly suggestive associations (p<5x10-7)with pneumococcal carriage. Interpretation Our study demonstrate human susceptibility to pneumococcal carriage to be polygenic with genetic variations which regulate PPFIA2 expression playing a key role in the ability for pneumococcus to colonise children. Targeting these genetic factors and the associated pathways are a means for preventing pneumococcal disease. Funding This study was supported by funding from Gavi - the vaccine alliance, the European Unions Horizon 2020 research and innovation program under grant agreement number 668303 (PERFORM), and a Robert Austrian Research Award.

The Developmental Origins of Health and Disease (DOHaD) hypothesis proposes that the perinatal environment shapes susceptibility to complex traits across life [1]. The placenta, a transient organ mediating maternal-fetal exchange, plays a central role in this process and has emerged as a key molecular archive in utero [2-4]. Placental DNA methylation (DNAm) is a unique mediator between prenatal exposures, fetal genetics and later-life outcomes [5-9]. DNAm quantitative trait loci (mQTL) have helped disentangling causal mechanisms underlying GWAS loci for complex diseases [10-15]. Despite growing evidence that placental genomic regulation has broad and profound effects on the developmental programming of early- and later-life health outcomes [17], existing placental studies remain limited in scale and largely focused on growth- and neuro-related traits [12-16]. Here, we construct a high-resolution placental mQTL resource and systematically investigate how placental DNAm relates to early- and later-life traits, and to shared vulnerability and complex interactions among them.

The human leukocyte antigen (HLA) region is the strongest genetic contributor to many immune-mediated diseases, yet whether HLA architecture is shared across ancestries remains unclear. We analyzed high-resolution HLA variation in 390,823 participants from the All of Us Research Program spanning six genetic ancestry groups, including 262,915 with linked electronic health records. Using whole-genome sequencing and graph-based inference, we genotyped 20 HLA genes at G-group resolution and identified 4,780 distinct alleles. Analyses accounting for disparate sample sizes demonstrated that ancestry-private allelic variation reflected unequal discovery depth rather than ancestry-population specificity. A meta-analysis of ancestry-stratified phenome-wide association analyses with 363 HLA alleles with frequency > 0.001 and 3,430 clinical phenotypes identified 1,461 significant HLA-phenotype associations (FDR < 0.05). Although many associations reached significance in only one ancestry group, effect directions were largely concordant, highlighting differences in allele frequency, linkage disequilibrium, and statistical power among ancestry groups. Stepwise conditional modeling demonstrated that common complex trait variation could be concurrently explained by five to seven independent HLA allele signals. These findings demonstrate that a multi-ancestry, phenome-wide study can distinguish true biological heterogeneity from sampling-driven detectability differences in HLA.

Multiple sclerosis (MS) is a chronic T cell-mediated autoimmune disease characterized by blood-brain barrier (BBB) disruption, neuroinflammation, and demyelination of the central nervous system (CNS). Emerging evidence links gut microbiota to disease pathogenesis, but the microbial factors that regulate pathogenic microRNA (miRNA) programs are largely unknown. Here, using experimental autoimmune encephalomyelitis (EAE, a MS mouse model), we investigated whether gut microbiota exacerbate EAE pathogenesis by modulating host miRNA expression. Antibiotic-induced depletion of the gut microbiota markedly attenuated EAE scores and reduced circulating inflammatory miRNAs, with miR-21 emerging as the dominant pathogenic candidate. Functional inhibition of miR-21 significantly ameliorated disease severity and reduced CNS T-cell infiltration. Mechanistically, miR-21 enhanced IL-17 and GM-CSF production by CD4 T cells and promoted immune-cell entry into the CNS through endothelial activation and blood-brain barrier dysfunction. We identified a transient expansion of Akkermansia muciniphila during the prodromal phase of EAE that positively correlated with circulating miR-21 levels. Colonization of antibiotic-treated mice with A. muciniphila exacerbated EAE and increased serum miR-21, whereas monocolonization of germ-free mice was insufficient to induce systemic miR-21, indicating a requirement for an inflammatory host environment. Further analyses revealed that atypical lipopolysaccharides (LPS) derived from A. muciniphila induce epithelial miR-21 production through coordinated TLR2/TLR4 signaling. Circulating miR-21 subsequently promoted endothelial dysfunction through the TIMP3-ADAM17 pathway, facilitating pathogenic T-cell migration into the CNS. Importantly, circulating miR-21 was also elevated in patients with MS. Collectively, these findings identify a previously unrecognized A. muciniphila-LPS-miR-21 axis linking gut dysbiosis to neuroinflammation and suggest that host-derived miRNAs function as systemic mediators through which microbial signals influence CNS autoimmunity.

Abstract Background: The Chikungunya virus (CHIKV), a re-emerging mosquito-borne alphavirus, is responsible for acute febrile illness and severe polyarthralgia. Although both innate and adaptive immune responses influence the disease outcomes, the detailed cellular immunopathogenesis of CHIKV in peripheral blood is not yet fully elucidated. Methods: We conducted single-cell RNA sequencing (scRNA-seq) on peripheral blood mononuclear cells (PBMCs) obtained from patients acutely infected with CHIKV and from healthy control subjects. Cellular interactions were inferred, and the transcriptomic results were orthogonally validated through quantitative real-time PCR (qPCR) and Enzyme-Linked Immunosorbent Assay (ELISA) to assess systemic interferon-stimulated responses. Furthermore, a comparative analysis was performed using publicly available single-cell data from Dengue virus (DENV) infections. Results: CHIKV infection significantly altered the immune system, increasing monocytes and dendritic cells while reducing T and B lymphocytes. Monocytes and NK cells showed strong activation of interferon-stimulated genes (ISGs). Monocytes were identified as key in driving inflammatory and immune responses. In adaptive immunity, CHIKV led B cells to become plasmablasts with antiviral immunoglobulins and caused T cells and NK-like T cells to show signs of cytotoxicity and exhaustion. Validation showed increased levels of IFN-{gamma}, IFN-{beta}1, MX1, and ISG15. CHIKV triggered a more intense, monocyte-driven interferon response than DENV. Conclusions: Acute CHIKV infection induces a systemic interferon response predominantly centered on monocytes, accompanied by significant alterations in adaptive immunity. Circulating ISG products, including MX1 and ISG15, reflect the transcriptomic activation and may serve as potential biomarkers for assessing the early intensity of innate antiviral responses.

Background Endometriosis is a complex, estrogen-dependent disease with a strong genetic component. Although genome-wide association studies (GWAS) have identified multiple susceptibility loci, most associated variants reside in noncoding regions, limiting biological interpretation and causal gene identification. Moreover, GWAS gene prioritization is limited by incomplete tissue-specific annotation coverage (e.g., GTEx, ENCODE, fine-mapping, Mendelian randomization, and network-based methods). We therefore applied the AlphaGenome artificial intelligence framework to prioritize endometriosis-associated variants based on predicted uterus-specific regulatory effects. Methods We analysed the top 10,000 endometriosis-associated single-nucleotide polymorphisms (SNPs) identified by previously published GWAS by Rahmioglu et al, using AlphaGenome across multiple genomic output types. Uterus-specific predictions with high-confidence effects (quantile score| [&ge;] 0.90) were grouped into major regulatory modalities. AlphaGenome-prioritized SNPs within {+/-}500 kb of known GWAS loci were classified into tiers based on the number of supported regulatory modalities, with broader support indicating stronger multilayer regulatory evidence. Effect allele frequency, linkage disequilibrium (LD), and overlap with previously published endometriosis-associated variants were also assessed. Results AlphaGenome generated uterus-specific, 147,033 high-confidence signals across 10,000 endometriosis-associated variants, spanning six regulatory modalities including gene expression, promoter activity, chromatin accessibility, transcription factor binding, histone modification, and RNA splicing. Within the 42 established endometriosis GWAS loci, AlphaGenome identified 42 alternative sub-threshold SNPs with stronger predicted uterus-specific regulatory effects than the published GWAS lead variants. Nineteen AlphaGenome-prioritized SNPs were classified as tier 1, showing support across all six regulatory modalities, compared with five GWAS lead SNPs. Linkage disequilibrium analysis identified eight tier 1 SNPs with weak-to-low LD (r<2> < 0.5) relative to the corresponding GWAS lead variants, regulating majority of genes involved in estrogen-driven proliferation and inflammatory signalling, highlighting their potential relevance to endometriosis pathogenesis. Additionally, we identified 167 genome-wide significant SNPs outside 42 published GWAS lead SNP loci including six tier 1 SNPs (rs1482061, rs7772579, rs6557140, rs2982571, rs12631337 and rs79626929), encompassing genes nearby ESR1/6q25.1, substantiating biological relevance for endometriosis pathogenesis. Conclusions AlphaGenome-based regulatory prioritization refined endometriosis-associated genome-wide association study loci by identifying variants with stronger predicted uterus-specific functional relevance. These findings provide a regulatory framework for prioritizing candidate variants and genes for downstream functional validation in endometriosis.

Women are predisposed to systemic lupus erythematosus (SLE) with a prevalence ratio of up to 9:1 over men. Multiple mouse strains including NZM2328 exhibit strong female dominance in developing spontaneous lupus as in humans with SLE. While lupus-prone mice can develop disease under germ free (GF) condition, the role of gut microbiota in female bias for lupus nephritis is not investigated systematically. Here, using specific pathogen free (SPF) and GF NZM2328 mice, and employing microbiota-depletion and microbial-association strategies, we show that microbiota influences lupus-like disease outcomes differently in males and females. Female NZM2328 mice with intact microbiota presents higher inflammation factor expression, including X-chromosome linked TLRs, in the distal gut and systemic compartments, and higher activation of genes and biological pathways such as neutrophil extracellular trap (NET) formation and complement and coagulation cascade (CCC) pathways, associating with their higher disease susceptibility. Gut microbiota-depletion as well as GF derivation eliminated not only the modest differences in the serum and fecal antibody levels and nAg reactivity, but also the gender bias in the timing of clinical stage disease onset as well as systemic NET and CCC pathway activation. Reciprocally, conventionalization of GF NZM2328 mice at juvenile age restored the female bias in intestinal and systemic autoantibody levels, pro-inflammatory immune pathway activation, and the timing of clinical stage disease onset. Overall, our observations show that, while genetic susceptibility appears to be the cause of lupus-like disease in NZM2328 mice, differential activation of NET and CCC pathways in males and females upon exposure to gut microbes, in combination with host-factors, causes gender bias in disease outcomes. We conclude that microbiota exposure-dependent protection of males and overactivation of NET and CCC pathways in females could be contributing to the female bias in lupus-like disease in NZM2328 mice.

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

CD4+FOXP3+ regulatory T (Treg) cells maintain self-tolerance, restrain immune responses during inflammatory stimuli, and promote tissue function and repair. Treg cell lineage identity, stability, and function depend on specific DNA methylation patterns maintained by the epigenetic regulator, UHRF1. Aging disrupts DNA methylation patterns necessary for Treg cell-mediated lung repair in a cell-autonomous manner. Nevertheless, whether maintenance DNA methylation is necessary for age-related Treg cell transcriptional and methylation programs is unknown. Here, we performed transcriptional and DNA methylation profiling on young and old Treg cells isolated from mice with chimeric Treg cell-specific loss of UHRF1. We observed cell-autonomous, age-related alterations in transcriptional and DNA methylation signatures that were dependent on UHRF1. We conclude that maintenance DNA methylation is required for age-related alterations in Treg cell transcriptional and DNA methylation signatures.

Immune effector cell-associated neurotoxicity syndrome (ICANS) is a major complication after CAR T cell therapy, but its underlying mechanisms remain poorly understood. We performed longitudinal immune profiling of paired whole blood and serum samples from patients with relapsed or refractory diffuse large B cell lymphoma (DLBCL) treated with CD19 CAR T cells. At peak neurotoxicity, high-dimensional mass cytometry and serum proteomics identified the expansion of CD163 monocytes and immature CD10lowCD101low neutrophils correlated with elevated serum ST2 and IL-2RA concentrations. Integrative immune module analysis identified these features among the strongest predictors of ICANS severity. Independent single-cell transcriptomic profiling validated the emergence of immunoregulatory CD163 monocytes and identified CD177 as a biomarker of ICANS-associated immature neutrophils. Together, these findings reveal a coordinated myeloid inflammatory network associated with ICANS and nominate candidate biomarkers and therapeutic targets for improving the safety of CAR T cell therapy. Significance: We demonstrate that immunoregulatory CD163+ monocytes and immature, activated CD177hiCD10lowCD101low neutrophils emerge in patients with moderate to severe ICANS at peak toxicity following CD19 CAR T cell therapy. These findings identify an uncharacterized myeloid network potentially contributing towards ICANS pathogenesis.

Chikungunya virus (CHIKV) infection causes acute febrile illness and debilitating arthralgia, with up to 40-80% of patients developing chronic chikungunya disease characterized by persistent arthralgia and fatigue lasting months to years. The immunological mechanisms underlying this transition remain poorly understood. We performed longitudinal immune profiling of CHIKV-infected patients stratified by clinical outcome. Single-cell RNA sequencing of peripheral blood mononuclear cells collected during acute infection and six months post-infection was combined with multiplex plasma cytokine and flow cytometric analysis. Patients who later developed chronic symptoms were characterized by early innate immune hypoactivation during acute infection, characterized by reduced monocyte and dendritic cell frequencies, lower circulating IFN-, IL-6 and IL-8, and reduced antigen-presentation and interferon-associated transcriptional programs. This was accompanied by diminished adaptive immune activation, including reduced IL-17 and IL-21 and lower HLA-DR expression across multiple T-cell subsets. Cell-cell communication analysis further indicated impaired acute monocyte-centered immune coordination in chronic progressors, whereas non-chronic patients displayed stronger monocyte-driven innate-to-adaptive signaling. By six months, chronic patients showed persistent innate remodeling, including increased non-classical monocytes, altered plasmacytoid dendritic cell and natural killer cell transcriptional programs, elevated CXCL10, reduced IL-6 and MMP8, and emergence of NK-centered predicted communication networks. Longitudinal transcriptomic analysis further identified divergent immune recovery trajectories, most prominently in non-classical monocytes and plasmacytoid dendritic cells. Together, these findings suggest that chronic chikungunya disease is associated with early innate immune hypoactivation followed by persistent innate immune remodeling, providing insight into immune mechanisms that may contribute to post-viral chronic inflammatory syndromes.

Inflammatory bowel disease (IBD) arises from dysregulated crosstalk between innate immune, adaptive immune, and stromal compartments, yet the compartment-specific mechanisms driving tissue injury and tumorigenesis remain incompletely defined. To address this gap, we used conditional knockout and overexpression mouse models together with human IBD biopsy analysis to dissect the compartment-specific roles of lamin A/C in intestinal inflammation and colitis-associated tumorigenesis. Pan-hematopoietic lamin A/C deletion attenuated acute dextran sulfate sodium (DSS)-induced colitis. Myeloid-specific lamin A/C deletion ameliorated chronic colitis and was associated with altered dendritic cell (DC) programs, enhanced regulatory T cell (Treg) responses, and reduced effector T cell activation. Adoptive transfer of lamin A/C-deficient bone marrow-derived DCs recapitulated this reduced-damage phenotype in DSS colitis, while proteomic profiling revealed reduced antigen-processing and inflammatory programs together with enhanced metabolic and mucosal defense pathways. T cell-specific lamin A/C deletion reduced the Th1/Treg ratio and limited tumor development by suppressing chronic inflammation, whereas T cell-specific lamin A/C overexpression promoted severe Th1-skewed pathology, sustained intestinal inflammation, and increased colitis-associated tumor burden. Stromal fibroblast-specific lamin A/C deletion generated a tissue-protective niche characterized by enhanced epithelial barrier gene expression, regulatory cytokine production, and remodeling of the local immune milieu. Human IBD biopsies revealed compartment-specific lamin A/C alterations consistent with the murine findings. In lamina propria CD3+; T cells, lamin A/C levels were blunted in IBD and associated with local histological severity rather than IBD diagnosis, whereas epithelial lamin A/C showed a steeper crypt-axis spatial gradient in a Crohn's disease-specific pattern. Together, these findings identify lamin A/C as a cell-type- and context-dependent regulator of intestinal inflammation and tumorigenesis.

BACKGROUNDMetabolic dysfunction-associated steatotic liver disease (MASLD) is a systemic immunometabolic disorder rapidly increasing worldwide, affecting nearly 38% of adults. Gut dysbiosis and host genetic factors, such as PNPLA3 I148M variant, modulate disease development and progression. Through the gut-liver axis, increased intestinal permeability enables microbial translocation to the liver, promoting inflammation and metabolic disruption. However, the composition and functional potential of the hepatic microbiome remain poorly characterized. Understanding its relationship with histological injury and genetic susceptibility may provide novel mechanistic insights. We hypothesized that the hepatic microbiome composition and function are associated with histological severity and PNPLA3 genotype in this disease. AIMTo characterize the hepatic microbiome and assess its association with histological severity and PNPLA3 genotype. METHODSThis cross-sectional observational study included 30 patients with MASLD from a tertiary care hospital. Liver tissue underwent shotgun metagenomic sequencing. Histological severity was assessed using the NAFLD Activity Score (NAS). PNPLA3 genotype was determined by PCR. Differential abundance and functional enrichment analyses were performed using MaAsLin2. Somatic variants were identified using Mutect2. Correlation networks were constructed using Spearmans correlation coefficients. RESULTSPatients with advanced histological injury (NAS [&ge;]5) and PNPLA3 I148M carriers showed a trend toward higher somatic mutational load and a markedly reduced microbial abundance. Analyses revealed broad compositional shifts across bacterial, fungal, viral, and eukaryotic taxa, affecting both commensal and context-dependent pathobiont lineages. Pseudomonas species were enriched, whereas Siphoviridae phages were depleted in advanced disease and PNPLA3 I148M carriers. Functional analysis revealed enrichment of pathways related to nutrient transport and metabolic stress adaptation, while TonB-associated functions were enriched in advanced liver injury but depleted in PNPLA3 I148M carriers. Network analysis identified Sphingomonas leidyi as a keystone node associated with hexosamine metabolism. Salmonella enterica abundance positively correlated with somatic variant burden, suggesting a link between microbial signatures and genomic instability. Histological progression and the risk PNPLA3 genotype were accompanied by marked topological simplification, reflecting less resilient community structures. CONCLUSIONSThe hepatic microbiome in MASLD is a low-biomass, polymicrobial ecosystem shaped by the host genetic background. Its functional activity, taxonomic composition and system architecture bidirectionally relate to liver DNA instability and the severity of histological damage. Core tipThis study characterizes the multi-kingdom hepatic microbiome in MASLD using FFPE-derived metagenomics. We demonstrate that microbial abundance-including bacteria, fungi, protozoa, and viruses- significantly decreases with increased histological severity and the PNPLA3 risk genotype. Rather than global diversity shifts, results showed that disease progression could be linked to specific functional adaptations and simplified microbial network connectivity. In addition, we described associations between specific taxa and somatic mutational burden, suggesting a link between microbial signals and genomic instability. These findings indicate that changes in the liver microbiome as a whole, rather than specific taxonomic modifications, influence MASLD pathophysiology.

Vaccination is one of the most effective public health interventions. However, vaccine efficacy varies widely among individuals, as immunity arises from complex interplay between genetic, pathogen, and immunological factors. To date, most systems vaccinology studies have remained pathogen-specific, precluding the discovery of potential shared immune architectures underlying durable antibody responses. To address this gap, we leveraged transcriptomic data from 1,032 participants receiving influenza, hepatitis B, or yellow fever vaccines to develop an interpretable machine learning framework for comparative analysis across diverse vaccine platforms. Pathogen-specific models using Blood Transcriptional Module-based feature aggregation accurately predicted high antibody responders and consistently outperformed gene-level models. Distinct predictive immune architectures identified across vaccines were further resolved for dominant hierarchical immune programs using surrogate decision trees. This approach identified the dominant decision boundaries underlying each vaccine model, highlighting leukocyte migration and Th2 differentiation in Hepatitis B, CD4+ T cells, M2 macrophages, and c-MYC signaling in Influenza, and B-cell receptor signaling with B-cell developmental pathways in Yellow Fever. Cross-pathogen concordance analyses further identified four shared transcriptional modules, suggesting partially conserved immune architectures across diverse vaccines. Together, these findings provide new insights into the immune mechanistic underpinnings of durable vaccine responses across vaccines and provide an interpretable framework for comparative systems vaccinology that may guide the rational design of next-generation vaccines. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=77 SRC="FIGDIR/small/737303v1_ufig1.gif" ALT="Figure 1"> View larger version (28K): org.highwire.dtl.DTLVardef@1b5be56org.highwire.dtl.DTLVardef@e323dborg.highwire.dtl.DTLVardef@470eb4org.highwire.dtl.DTLVardef@116759f_HPS_FORMAT_FIGEXP M_FIG C_FIG

Black-footed ferrets (Mustela nigripes) are highly susceptible to sylvatic plague caused by Yersinia pestis, but the genetic basis of this vulnerability remains poorly understood. Here, comparative immunogenomic analyses across Carnivora species identified a conserved class of immunoglobulin lambda variable (IGLV) genes with unusually long antigen-binding sites (CDRL1) that are common among Caniformia species but absent in Feliformia species. First discovered in the domestic ferret (Mustela putorius furo), these genes encode tyrosine-rich and anionic motifs resembling the chemokine receptor CCR5 and contain experimentally validated sulfotyrosines previously associated with pathogen-interacting interfaces. Evolutionary analyses revealed distinct selective pressures across Caniformia lineages and showed strong purifying selection acting on long-CDRL1 IGLV genes in mustelids and bears. Antibody repertoire sequencing demonstrated that these genes are actively utilized in expressed repertoires and that their usage correlates with evolutionary conservation. Functional analyses of monoclonal antibodies derived from the long-CDRL1 IGLV gene identified an antibody that significantly reduced intracellular Y. pestis survival in macrophages and revealed a positive correlation between anti-plague activity and sulfotyrosine signal. Notably, all analyzed black-footed ferrets carried a frameshifting deletion in the long-CDRL1 IGLV gene resulting in loss of its expression in antibody repertoires. Together, these findings uncover a germline-encoded immunoglobulin feature conserved across dog-like carnivores and suggest a potential link between antibody germline variation and immune responses to plague.

Dysregulated gut homing of leukocytes drives chronic inflammation in Crohns disease (CD). We employed phage display selection campaigns with libraries of stabilized, constrained peptides against endogenous conformation states of the cannabinoid receptor CB2R on human T cells, to discover novel receptor antagonists with potential to inhibit gut homing. Cluster and frequency analysis of 50,000 enriched sequences resulted in expression and functional characterisation of 10 protein candidates using assays of glucose uptake, ERK phosphorylation (pERK) and beta-arrestin recruitment. Each candidate antagonised CB2R activity with recorded IC50 values of between 5-10 nM. Cannabinoid receptor nanodisc binding experiments and SPR confirmed CB2R selectivity. SLKC_09 with an IC50 of 5.4 nM, was studied in a mouse model of chronic ileitis where it significantly inhibited gut homing of CD4+ & CD8+ naive, effector and memory cell types. Our findings highlight an alternative route to therapeutic inhibition of leukocyte trafficking in CD with a biologic inhibitor of CB2R.