Immunology & Cell Biology

Sarcoidosis is a multisystem disorder that primarily affects the lungs and is characterizedby granulomatous inflammation. However, much of the underlying disease mechanisms remain poorly understood. Extracellular vesicles (EVs) are small membrane-bound particles released by all cells and carry various cargos including metabolites. They are involved in intercellular communication that can be dysregulated in diseases.This study characterizes the metabolic cargo of EVs isolated from bronchoalveolar lavage fluid (BALF), using liquid chromatography-mass spectrometry (LC-MS)-based metabolomic analysis, in patients with sarcoidosis (n=37), compared to healthy controls (n=10). Additionally, the sarcoidosis signature was compared to another pulmonary disorder, anti-synthetase syndrome (ASyS, n=10). Arachidonic acid (AA) results were verified by ELISA. A total of 1202 metabolites were detected, with 111 annotated ones further analyzed. EVs from sarcoidosis patients showed distinct metabolomic profiles compared to both ASyS patients and healthy controls, with 38 annotated metabolites differentially expressed in any of the groups. In both annotated and non-annotated data, sarcoidosis patients clustered separately from ASyS patients and healthy individuals. Furthermore, sarcoidosis patients clustered in 3 subgroups, whereof one was similar to ASyS patients and one stood out as showing higher cell counts in BALF. Higher AA levels were found in sarcoidosis patient EVs by LC-MS, and AA results were verified by ELISA. Our data show that BALF EV metabolites are disease-dependent and support the notion thatsarcoidosis patients should be further subgrouped for better diagnosis and treatment.

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.

Resident dermal macrophages (DMs) play essential roles in maintaining skin homeostasis and initiating inflammatory responses during tissue injury and against infectious agents. However, studies of their cellular mechanisms have been limited by their low abundance in steady-state skin and by technical challenges in isolating resident DMs. Here, we describe the generation and characterization of a novel DM cell line, termed SB89. F4/80+ skin-resident DMs were sorted and immortalized using J2 retroviral transduction. SB89 cells display a stable, homogeneous macrophage phenotype and distinct surface markers compared with Langerhans cells and alveolar macrophages. Functionally, SB89 cells efficiently phagocytose methicillin-resistant Staphylococcus aureus (MRSA), Escherichia coli, zymosan particles, and apoptotic cells, and effectively kill MRSA. Importantly, SB89 cells respond to LPS, as evidenced by production of IL-6, TNF, and IL-10, and by MRSA-induced production of inflammatory cytokines, chemokines, and eicosanoids. RNA-seq and gene ontology analyses revealed that SB89 cells elicit stronger responses in innate immunity, cell signaling, and epigenetic regulation than immortalized bone marrow-derived macrophages. SB89 cells are genetically tractable, amenable to gene silencing via RNAi and gene introduction via plasmid transfection. Overall, SB89 cells provide a renewable, dermis-imprinted macrophage model that preserves key functional and transcriptional features of resident DMs while reducing reliance on primary cells and animal models. This cell line represents a powerful platform for mechanistic, genetic, and translational studies in skin immunobiology.

Neutrophils recruited to the airways are important for innate lung defense and can release neutrophil extracellular traps (NETs) to capture and eliminate microbes. While NETs are not abundant in healthy airways, uncontrolled NETosis is a known pathological feature and contributor to both chronic and acute respiratory diseases. Prior studies have shown that mucin glycoproteins secreted in the oral cavity and cervicovaginal tract can modulate NETosis, but it remains unknown whether mucins secreted in the respiratory tract influence NET formation. In these studies, we discovered that human airway mucus strongly inhibits NETosis in primary human neutrophils in a sialic acid dependent manner. In comparison, mucus produced by human airway epithelial cells genetically engineered to lack either MUC5B or MUC5AC secreted airway mucins showed a reduced ability to suppress NETosis. To assess how the lung microenvironment in obstructive lung diseases may influence mucus-dependent NET formation, we engineered a synthetic, mucin-laden hydrogel model with physical properties resembling that of mucus in a healthy lung and a disease-affected lung. When neutrophils were cultured on these gel substrates, we found that increasing gel stiffness led to a significantly greater extent of NETosis. Together these data demonstrate a new functional role of airway mucus in modulating neutrophil homeostasis in the respiratory tract and provide evidence that mucus dysfunction in disease can impair its ability to regulate NETosis.

Alveolar macrophages (AMs) are tissue-resident and the primary immune cells in the airspace. Following perturbations in the lungs, these AMs that are derived from the fetal liver, become depleted and are transiently replaced by myeloid cells that use lung-specific cues to differentiate into myeloid-derived AMs. While these myeloid-derived AMs are critically important in a range of pulmonary diseases, including post-influenza bacterial pneumonia, it remains challenging to fully understand their function due to a lack of ex vivo models that recapitulate key differences observed in vivo between AMs and myeloid-derived AMs. Here, we overcome this limitation by expanding our recently developed model of fetal liver-derived alveolar macrophages (FLAMs) to differentiate myeloid progenitors in the presence of GM-CSF and TGF{beta}, key cytokines that drive tissue resident AM functions. These myeloid-derived alveolar-like macrophages (MAMs) express AM surface markers and look similar morphologically to FLAMs, however, they remain more inflammatory than FLAMs. Mechanistic studies found that differential CpG methylation at inflammatory loci, basal transcriptional expression, and metabolic flux all contribute to the hyperinflammatory state of MAMs. Importantly, we find that while FLAMs are highly dependent of lipid metabolism, MAMs are more glycolytic and this hardwired metabolism is not easily overcome to mute their inflammatory state. Finally, we found that MAMs and FLAMs both function within the lung environment following transfer into mice lacking AMs. While both MAMs and FLAMs stably seed the lungs and reverse pulmonary proteinosis, MAMs remain highly inflammatory in the lungs following an LPS model of acute lung injury. Taken together our results find that MAMs are a reproducible model of myeloid-derived AMs and lays the groundwork to better understand how these important immune cells contribute to pulmonary homeostasis and responses to lung perturbations. These future studies will help to identify new targets that can be modulated to prevent severe pulmonary disease outcomes.

BackgroundTrained immunity is a durable functional reprogramming of innate immune cells characterized by enhanced responsiveness upon secondary challenge. While metabolic rewiring and epigenetic remodeling are well-established features of this process, the contribution of ubiquitin-mediated post-translational regulation remains poorly defined. MethodsWe performed an integrative analysis of publicly available human transcriptomic datasets derived from monocytes, macrophages, and PBMCs exposed to established training stimuli ({beta}-glucan, Bacillus Calmette-Guerin [BCG], and hemin-{beta}-glucan) followed by secondary stimulation. A curated panel of deubiquitinating enzymes (DUBs) and E3 ubiquitin ligases with established immune functions was analyzed for differential expression. Gene Ontology (GO) and KEGG pathway enrichment analyses were conducted to evaluate higher-order convergence across independent datasets. ResultsAcross multiple trained immunity models, we identified reproducible transcriptional remodeling of ubiquitin-modifying enzymes. USP25, OTUB1, and TRIM25 were consistently upregulated following restimulation, whereas several chromatin- and cytokine-regulatory DUBs--including USP3, USP4, USP7, USP16, MYSM1, and USP38--were downregulated. Normalization to RPMI-restimulated controls reduced many activation-associated signals; however, USP25 remained persistently elevated, suggesting a stable training-associated signature. Pathway enrichment analysis independently demonstrated significant engagement of ubiquitin-related functional categories across datasets, supporting coordinated reorganization of ubiquitin regulatory networks. ConclusionThese findings identify selective transcriptional remodeling of the ubiquitin- proteasome system as a recurring feature of trained immunity. Integrating ubiquitin signaling into the established metabolic-epigenetic framework expands the conceptual model of innate immune memory and suggests that ubiquitin-modifying enzymes function as modulatory rheostats shaping immune amplitude and stability. Future functional and proteomic studies are required to determine whether these transcriptional signatures directly mediate trained immunity phenotypes.

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.

Mesenchymal stromal cells exhibit potent immunomodulatory properties and are under active investigation for the treatment of immune-mediated disorders. However, their clinical translation is hindered by the lack of standardized potency assays. Here, we established a reproducible mixed lymphocyte reaction platform by systematically optimizing peripheral blood mononuclear cell donor composition, culture conditions, and co-culture ratios to define a robust activation window. Using this system, we compared bone marrow and adipose derived Mesenchymal stromal cells across independent donor batches. Both sources effectively suppressed T cell proliferation, with the adipocyte derived source consistently showing greater inhibitory activity, while a conserved lower threshold of suppression was observed across both sources. Mesenchymal stromal cells reduced early (CD25+) and late (CD25+HLA-DR+) T cell activation, with downregulation of these markers emerging as a sensitive correlate of functional potency. Notably, bone marrow derived mesenchymal stromal cells exerted stronger suppression on late-stage activation and preferentially suppressed CD8+ T cell expansion. Mechanistically, this immunosuppression was associated with modulation of the PD-1 pathway, characterized by decreased soluble PD-1, increased PD-L1, and induction of mesenchymal stromal cells derived PD-L2. PD-L2 levels inversely correlated with T cell proliferation, identifying a PD-1/PD-L2 regulatory axis linked to the cells potency. These findings define a standardized and mechanistically informed potency assay framework for assessing mesenchymal stromal cell immunomodulatory function.

Aspergillus fumigatus is a ubiquitous environmental mould and a leading cause of chronic fungal-associated respiratory disease, yet the mechanisms by which persistent airway colonisation drives immune adaptation and lung pathology remain poorly understood. Progress in this area has been limited by the lack of in vivo models that recapitulate stable, non-invasive fungal persistence without immunosuppression. Here, we developed and optimised a murine model of chronic airway colonisation using agar bead-embedded A. fumigatus conidia delivered intratracheally. Embedding did not impair fungal germination or hyphal growth, and the agar matrix was immunologically inert, supporting its use as a neutral scaffold. This approach established stable fungal persistence in the airways for at least three weeks in immunocompetent mice without inducing invasive disease or systemic morbidity. Colonisation elicited a transient, airway-restricted innate immune response characterised by early neutrophil and monocyte recruitment and increased CXCL1, MIP-1, MIP-1{beta}, and TNF production, which resolved over time. Histopathological analysis revealed a progressive sequence of disease-relevant features, including initial immune containment, followed by mucus hypersecretion, and airway remodelling. At the adaptive level, persistent colonisation induced a dynamic T cell response that transitioned from an early polyfunctional profile to a sustained Th17-dominant phenotype. Importantly, application of this model in CFTR-deficient mice uncovered enhanced collagen deposition and fibrotic remodelling without altered fungal burden, demonstrating its utility in modelling disease-relevant outcomes in susceptible hosts. Together, this study establishes a robust and physiologically relevant platform for investigating host-fungal interactions during chronic airway colonisation. This model provides new opportunities to dissect mechanisms of immune adaptation, fungal persistence, and tissue remodelling, and to identify therapeutic strategies targeting chronic Aspergillus-associated lung disease.

BackgroundObesity disrupts type 2 immune cell populations in white adipose tissue, replacing the homeostatic network of group 2 innate lymphoid cells (ILC2s), eosinophils, T helper 2 (Th2) cells, and alternatively activated macrophages (AAMs) with pro-inflammatory type 1 populations. Whether this remodelling reflects permanent immune impairment or a reversible shift in cellular equilibrium, and to what extent bariatric surgery restores type 2 immunity, remain incompletely understood. MethodsWe performed comprehensive immunophenotyping of visceral white adipose tissue (WAT) and peripheral blood from persons with severe obesity (people with obesity, PWO) scheduled for or having undergone bariatric surgery (sleeve gastrectomy, gastric bypass), combined with lean controls. Using flow cytometry, quantitative PCR, and in vitro polarization assays, we assessed immune cell frequencies, transcription factor expression, cytokine profiles, and functional polarization capacity across lean, pre-operative, and post-operative states. ResultsObesity was associated with decreased eosinophil and CD8+ T cells frequencies in WAT, accompanied by an increase in CD4+ frequency and a shift from Th2 toward Th1 predominance, as well as elevated PD-1 expression on T cell subsets. Bariatric surgery partially normalised peripheral immune cell composition, reducing CD8+ T cell frequencies while increasing CD4+ T cells. Macrophage polarization capacity, dampened in pre-operative PWO, recovered after surgery. Conversely, Th2 polarization capacity and IL-13 production were reduced in post-operative T cells despite preserved function pre-operatively, indicating divergent trajectories of innate and adaptive immune reconstitution. ConclusionType 2 immune cells retain functional plasticity in human obesity despite reduced frequency. Bariatric surgery differentially reconstitutes immune function, restoring macrophage plasticity while paradoxically reducing Th2 polarization capacity, arguing against uniform immune normalisation after weight loss. FundingGerman Federal Ministry of Research, Technology and Space (BMFTR, FKZ 01KI2109), Interdisciplinary Center for Clinical Research (IZKF, Faculty of Medicine, Friedrich-Alexander Universitat (FAU) Erlangen-Nurnberg).

Mounting evidence indicates that T cells can operate in an innate-like mode challenging the classical description of T cells as strictly adaptive immune effectors. T cells can engage innate pattern recognition receptors to mount rapid but antigen-nonspecific responses to infection or cellular stress. This study observed that CD8+ T cells, and to a lesser extent also CD4+ T cells, responded to viral proteins in the mouse lung quickly in an innate-like fashion. We employed intravital lung microscopy to visualize infiltration of CD8+ T cells into the lung following intratracheal instillation of the SARS-CoV-2 envelope (E)-protein. Here, we demonstrate acute recruitment of CD8+ from the pulmonary microcirculation into the lung as early as 4 and 24 hours after (E)-protein instillation. The acute infiltration of CD8+ T cells was not observed in Tlr2-/- mice. Immunohistochemistry analysis of mouse lungs revealed T cell accumulation in nodular inflammatory foci (NIF) of the lung at perivascular regions and around large airways. Stimulating spleen-derived CD8+ T cells from wild-type mice with (E)-protein ex vivo in combination with cytokines or TCR agonists significantly upregulated CD69 and activated secretion of interferon (IFN){gamma} which was not observed with CD8+ T cells isolated from Tlr2-/- mice. These findings indicate rapid bystander activation of CD8+ T cells by the SARS-CoV-2 envelope (E)-protein that depends on (E)-protein sensing by TLR2. This innate-like CD8+ T cell response to SARS-CoV-2 (E)-protein may offer novel opportunities for diagnostic and therapeutic development, warranting further investigation.

ObjectiveTo determine whether pyroptosis-related transcription in systemic sclerosis skin forms a translayer spatial architecture rather than a single coextensive inflammatory program. MethodsWe reanalyzed public Visium formalin-fixed paraffin-embedded skin sections (4 healthy controls, 9 systemic sclerosis) from a discovery cohort and tested prespecified endpoints in 10 independent systemic sclerosis sections. The tissue section was the inferential unit. Epidermal versus dermal contrasts within each section were evaluated for inflammasome-related and gasdermin genes, followed by cell2location spatial deconvolution and partial correlation adjusted for endothelial context in the dermis. ResultsNLRP1, PYCARD, and CASP4 displayed epidermal bias in all 13 discovery sections, whereas GSDMD displayed dermal bias in all 13. This spatial separation was detectable in healthy skin and appeared stronger in systemic sclerosis. A tier 1 triad captured the epidermal signal better than broader composites (dilution 35.5%; P = 0.0002). In an independent systemic sclerosis cohort, the dermal gasdermin endpoint retained its direction in 8 of 10 sections and the epidermal inflammasome-related endpoint in 10 of 10. Spatial deconvolution indicated that dermal GSDMD associated most strongly with estimated endothelial abundance in both healthy and systemic sclerosis skin. The IFN{gamma}-GSDMD association remained positive after endothelial adjustment across sections, compatible with an additional IFN{gamma} component. ConclusionSystemic sclerosis skin harbors a reproducible translayer pyroptosis-related transcriptional architecture in which upstream epidermal inflammasome-related transcription and dermal GSDMD expression are spatially dissociated. This organization, detectable in healthy skin and often stronger in SSc, may warrant future mechanistic and therapeutic interrogation by compartment.

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.

BackgroundTryptophan (Trp) metabolism is a central immunometabolic axis in inflammatory bowel disease (IBD) and has been linked to inflammatory activity and immune regulation. While individual Trp metabolites have been associated with disease severity and treatment response, systems-level frameworks to define metabolic subtypes in IBD are lacking. ObjectiveTo identify reproducible Trp-related metabolic subtypes ("metabotypes") in IBD and assess their association with disease activity, clinical outcomes, and early disease development. DesignWe applied unsupervised clustering to serum concentrations of 16 Trp-related metabolites in a discovery cohort of patients with IBD undergoing biologic induction therapy (n=134). Metabotypes were validated in three independent IBD cohorts (total n>2,800), a healthy reference population, and a prospective cohort of first-degree relatives at risk for Crohns disease. Associations with disease activity, longitudinal outcomes, and metabolic pathways were assessed using multivariable regression and survival analysis. ResultsFour reproducible metabotypes with distinct metabolite profiles were identified across cohorts: Low Kyna, High Kyna, High Quin, and Balanced. Low Kyna and High Quin metabotypes were consistently associated with increased inflammatory activity and adverse clinical outcomes, including increased risk of treatment escalation and disease progression. Pathway-level analyses revealed alterations in NAD-related, lipid, and amino acid pathways between inflammatory metabotypes. A metabotype resembling inflammatory disease states was enriched in individuals who later developed Crohns disease in a prospective pre-disease cohort. ConclusionTrp-linked metabotypes define reproducible immunometabolic states in IBD that associate with disease activity and clinical outcomes and may precede disease onset. These findings provide a framework for metabolic stratification and biomarker-guided clinical trials targeting immunometabolic pathways. What is already known on this topicTryptophan metabolism through the kynurenine pathway is a central immunometabolic axis in inflammatory bowel disease (IBD) and has been linked to inflammatory activity and immune regulation. Individual tryptophan metabolites have been associated with disease severity and treatment response, but their clinical utility for patient stratification remains limited. Systems-level approaches to define clinically meaningful metabolic subtypes in IBD are lacking. What this study addsWe identify four reproducible tryptophan-related metabolic subtypes ("metabotypes") that are consistently associated with disease activity across multiple independent IBD cohorts. Inflammation-associated metabotypes show distinct pathway-level alterations, including differences in NAD-related metabolism and broader metabolic programs. A metabotype resembling inflammatory disease states is detectable before clinical diagnosis in individuals who later develop Crohns disease. How this study might affect research, practice or policyMetabotype-based classification provides a framework for molecular stratification of patients in mechanistic studies and clinical trials targeting immunometabolic pathways. This approach may support biomarker-guided monitoring of disease activity and disease progression in IBD. Identification of preclinical metabolic states highlights the potential of metabolomics for early disease detection and prevention-oriented research strategies.

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

Background & Aims: Metabolic Dysfunction Associated Steatotic Liver Disease (MASLD) is the leading cause of chronic liver disease in children. However, accurate, noninvasive diagnostic tools remain limited. Current screening methods are invasive or lack sensitivity. Breath-based volatile organic compound (VOC) analysis offers a simple approach with potential for point of care screening. This study aimed to identify and validate breath VOC signatures of pediatric MASLD. Approach & Results: We conducted a prospective IRB approved cohort study at the Childrens Hospital of Philadelphia (CHOP). Children aged between 7 and 20 years with MASLD (n=22), as defined by hepatic steatosis either by liver biopsy or imaging and 1 cardiometabolic risk factor, and a control group without MASLD (n=20) were enrolled. Breath samples were collected using a standardized protocol and analyzed by untargeted comprehensive two-dimensional gas chromatography-mass spectrometry (GCGCMS). Machine learning and unsupervised clustering were applied to identify discriminatory VOCs and assess heterogeneity. Untargeted GCGCMS analysis identified a distinct breath VOC signature in children with MASLD compared with non MASLD controls. A Random Forest model achieved a sensitivity of 73% and specificity of 65%, with AUC of 0.84. The VOC 2,4-dimethyl-1-heptene demonstrated strong diagnostic performance in the discovery cohort with a sensitivity of 85%, specificity of 77% and an AUC of 0.81. Unsupervised clustering revealed four MASLD subgroups with distinct volatile phenotypes associated with differences in liver enzymes and metabolic parameters. External validation in a second pediatric cohort confirmed reproducible reductions in o/p-xylene in subjects with MASLD. Conclusions: Pediatric MASLD is associated with a reproducible breath VOC signature identified by untargeted GCGCMS. These findings support breath analysis as a scalable, noninvasive screening and stratification tool for pediatric MASLD and warrant validation in larger, longitudinal studies.

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

Specific anaerobic species within the penile microbiome - Bacteria Associated with Seroconversion, Inflammation and Immune Cells (BASIC) - have been linked to increased HIV-1 susceptibility. These bacteria can directly disrupt epithelial integrity and are believed to increase local inflammation, resulting in an increased density of HIV-susceptible T cells in the inner foreskin. It is currently unknown whether other immune cells bearing the HIV entry receptors, CD4 and CCR5, are also elevated in individuals with a high abundance of BASIC species. Using inner foreskin tissues and penile swabs from males undergoing voluntary medical male circumcision, we performed a retrospective cross-sectional study to assess the relationship between BASIC species and the tissue density of such immune cells, including CD68+ macrophages, CD11c+ dendritic cells, and CD207+ Langerhans cells. The most abundant cells in the inner foreskin expressing the HIV co-receptors were CD11c+ dendritic cells (48.6% of CD4+/CCR5+ cells), followed by CD68+ macrophages (28.6%), CD3+ T cells (18.8%), and CD207+ Langerhans-like (8.8%) cells. The absolute abundance of BASIC species was associated with elevated tissue densities of both CD4+/CCR5+ T cells (as previously reported) and a heterogeneous population of CD3-/CD4+/CCR5+ cells of myeloid origin. In the dermis, BASIC species abundance was linked to elevated densities of cells expressing CD11c, CD68, and CD207, as well as those co-expressing CD11c and CD207; furthermore, CD11c+ and CD207+ cells were farther from the basement membrane in participants with a high abundance of BASIC species. Myeloid cells were not elevated in participants with a high abundance of control taxa. In an integrated analysis including previously published data from this same cohort, myeloid-cell densities clustered tightly together, positively correlated with BASIC species and pro-inflammatory cytokines, and had trends to negative correlations with control taxa (significant for CD207+ cell density). Overall, our findings suggest that BASIC species are associated with a broader foreskin immune phenotype marked by increased densities of HIV-susceptible myeloid and T cells, alongside epithelial disruption.

Since the 1950s, micro- and nanoplastics (MNPs) have become omnipresent, representing a novel environmental hazard which continually deposits in our airways. Pulmonary macrophages (pMacs) orchestrate the balance between inflammation and tolerance required for homeostasis of the lung and are among the first immune cells to encounter inhaled MNPs. Yet, how pMacs react to plastic deposition in the lung and implications for disease remain unknown. Here, we exposed mice in vivo, human precision-cut lung slices (hPCLS) ex vivo, and monocyte-derived macrophages and cell lines to polystyrene MNPs in vitro. MNP deposition in the lung and extrapulmonary tissues was determined over a 1-week period and pMacs from MNP-laden lungs isolated for RNA-sequencing. We compared the effects of MNPs or diesel exhaust particulate exposures on hPCLS viability and metabolism, monocyte-derived macrophage transcription, and macrophage mitochondrial function, inflammation, and antigen presentation. MNPs readily translocated the lung and were observed in all organs examined within 1-day. pMacs from MNP-exposed mice expressed transcriptional pathways associated with endocrine system disorders, tissue remodeling, and malignant disease. Macrophage phagocytosis was impaired through decreased mitochondrial function which could be rescued pharmacologically. MNPs inhibited the ability of macrophages to effectively present OVA-antigen preventing TCR-specific activation, an effect that could be restored by blocking PD-1/PD-L1. These findings indicate that MNPs impair macrophages via unique mechanisms linking phagocytic and bioenergetic dysfunction. Loss of antigen-presenting capabilities in MNP-laden macrophages may compromise immunosurveillance. As such, MNPs have the potential to increase susceptibility to lung disease independent of the conventional mechanisms of inflammation and oxidative stress. Clinical relevanceO_LIBioaccumulation of micro- and nanoplastics in macrophages impairs their ability to function as antigen-presenting cells increasing susceptibility to pathogenic and malignant disease. C_LIO_LIPulmonary macrophages residing in micro- and nanoplastic laden lungs possess transcriptional profiles associated with endocrine system disorders, gastrointestinal disease, and cancers. C_LI

IgA nephropathy (IgAN) is a common primary glomerulonephritis characterized by glomerular immune-complex deposits with (co)dominant IgA. These deposits are enriched for IgA1 glycoforms with some O-glycans deficient in galactose (Gd-IgA1). Circulating Gd-IgA1 is bound by IgG autoantibodies to form immune complexes, some of which deposit in glomeruli. Genomic and immunologic studies indicate involvement of pro-inflammatory signaling pathways in the production of Gd-IgA1 in IgAN. Genomic studies identified multiple genetic loci associated with IgAN and suggested a convergence on the NF-{kappa}B pathway, including RELA, the gene encoding the NF-{kappa}B subunit p65. However, the mechanisms by which NF-{kappa}B pathways may affect O-glycosylation in IgA1-producing cells are unknown. Using EBV-immortalized B cells derived from peripheral-blood mononuclear cells of IgAN patients and healthy controls that have constitutively activated NF-{kappa}B, we report that inhibition of NF-{kappa}B/p65 by a selective IKK{beta} inhibitor TPCA-1 reduced phosphorylation of NF-{kappa}B/p65 at S536 and decreased production of IgA1 and, conversely, increased Gd-IgA1 production. This was likely related to reduced expression of C1GALT1 gene that encodes the enzyme responsible for galactosylation of IgA1 O-glycans. Flow-cytometry imaging revealed changes in nuclear translocation and co-localization of the NF-{kappa}B/p65 with co-transcriptional factor SP1, a transcriptional activator of C1GALT1, suggesting that NF-{kappa}B pathway affects IgA1 O-glycosylation via SP1 transcriptional control of C1GALT1 expression. Furthermore, prolonged IKK{beta} inhibition altered B cell subpopulations, enhancing generation of cells with a plasmablast-like phenotype, characterized by high SSC MFI and CD138 expression. Together, these findings provide functional evidence for involvement of NF-{kappa}B/p65 and its transcriptional partners in IgA1 O-glycosylation. HighlightsO_LIIKK{beta} inhibition reduced C1GALT1 expression and thereby increased galactose-deficient IgA1 (Gd-IgA1) production in immortalized human B cells. C_LIO_LISP1+ subpopulations, a transcriptional activator of C1GALT1, declined after sustained NF-{kappa}B inhibition. C_LIO_LINF-{kappa}B inhibition shifted a subpopulation of B cells into a plasmablast-like phenotype. C_LIO_LIThis study links NF-{kappa}B signaling with the GWAS-identified RELA susceptibility locus and IgA1 O-glycosylation. C_LI