Immunology & Cell Biology

Fibroblasts are traditionally considered structural cells that maintain tissue homeostasis and facilitate repair. However, accumulating evidence suggests they also participate in innate immunity, although their pattern recognition capabilities remain incompletely characterized. Here, we systematically assessed the innate immune responses of commercially available primary human dermal fibroblasts from a male and a female donor. Fibroblasts were stimulated with a panel of microbe-associated molecular patterns (MAMPs) targeting various pattern recognition receptors (PRRs), including Toll-like receptors (TLRs), NOD-like receptors (NODs), Alpha kinase 1 (ALPK1) and STING. Innate immune activation was quantified by measuring the nuclear translocation of NF-{kappa}B via high content microscopy and cytokines and chemokines secretion by ELISA; baseline PRRs expression was determined by quantitative PCR. Only a restricted subset of agonists, specifically E. coli LPS (TLR4), Poly I:C (TLR3 / RIG-I) and unexpectedly ADP heptose (ALPK1) induced robust NF-{kappa}B activation and secretion of the chemokines IL-8 and MCP-1. Apart from IL-6 and RANTES, which were produced exclusively following Poly I:C stimulation, pro-inflammatory cytokines (IL-1{beta}, TNF, IFN-{beta}) and the anti-inflammatory cytokine IL-10 remained undetectable. Consistent with this limited reactivity, qPCR of PRRs revealed basal expression of TLR4 and ALPK1, whereas most other receptors were expressed at very low or undetectable levels. Notably, NOD1 was highly expressed although no cell activation was observed with several NOD1 agonists. Dose-response analysis revealed surprisingly high sensitivity to LPS. In conclusion, primary human dermal fibroblasts exhibit a highly selective but sensitive innate immune response, largely restricted to chemokine production upon PRR activation. This unexpected dissociation between chemokine and cytokine responses suggests that fibroblasts function as sentinel cells in early skin defense, capable of detecting key microbial patterns at low concentrations, to orchestrate local immune surveillance. Further investigation into interindividual variability and context-dependent activation is needed.

BackgroundMalnutrition compromises mucosal immunity, especially in the respiratory tract, increasing susceptibility to pathogens like Streptococcus pneumoniae. This study assessed whether nasal administration of Lacticaseibacillus rhamnosus CRL1505 or its peptidoglycan could promote the recovery of nasopharynx-associated lymphoid tissue (NALT) structure and functionality, thereby enhancing resistance to S. pneumoniae infection in protein-malnourished mice. MethodsMale Swiss albino mice were fed to a protein-free diet to induce malnutrition, followed by nutritional repletion with or without nasal supplementation of CRL1505 or its peptidoglycan. Resistance to S. pneumoniae infection, NALT architecture, immune cell composition in NALT and regional lymph nodes, and nasal cytokine production were evaluated. ResultsProtein deficiency caused marked NALT atrophy, immune cell depletion, and heightened susceptibility to S. pneumoniae. Nutritional repletion alone partially reversed these effects. In contrast, nasal supplementation with CRL1505 or its postbiotic fully restored NALT structure and cellularity, normalized lymphoid and myeloid populations, and reduced pathogen burden. Both treatments increased B and T lymphocytes, immature B cells, dendritic cells, and macrophages. The postbiotic also enhanced MHCII expression and balanced neutrophil-like Gr-1 cells. Notably, immune enhancement was evident even before infection, indicating a mucosal priming effect. Cytokine levels in nasal fluids remained largely unchanged. ConclusionsNasal delivery of L. rhamnosus CRL1505 or its postbiotic effectively reestablished NALT integrity and mucosal immunity in malnourished mice, providing significant protection against respiratory pathogens. These findings support the development of nasal immunobiotic formulations as non-invasive interventions to bolster respiratory defenses in immunocompromised hosts.

Acquired immunity in mammals depends upon capture and presentation of antigens by specialised macrophage populations in splenic marginal zone and lymph node sinuses and follicular dendritic cells (FDC) within germinal centres. Cells referred to as FDC in chickens express CSF1R, the receptor for macrophage colony-stimulating factor (CSF1) and IL34. We utilised single cell RNA-seq on CSF1R+ cells from chicken spleen to identify monocytes and two distinct populations of macrophages. TIMD4/C1Q/MAFB+ macrophages were enriched for expression of genes involved in iron metabolism. A MARCO/VSIG4+ population expressed SPIC, a transcription factor associated with red pulp macrophages in mammals but also expressed receptors (CR2) and trophic factors (TNFSF13, CXCL13) associated with mammalian FDC. SPIC+ cells were located within follicles in spleen, caecal tonsil and bursa. We generated a CSF1R knockout in the chicken germ line. Mutant birds lack macrophages in the embryo. They were indistinguishable from wild type at hatch and behaved and fed normally but from day 5-6 post hatch they failed to thrive. Loss of CSF1R function in hatchlings led to monocytopenia and granulocytosis and the loss of macrophage subpopulations in lymphoid organs. Consistent with their expression of B cell trophic factors, the loss of follicular macrophages in the bursa was associated with involution and severe B cell deficiency in the circulation and spleen. In summary, lymphoid tissues of chickens contain specialised macrophage populations with distinct expression profiles. The details of regulation by CSF1R, specialised functions and underlying transcriptional regulation are quite different between birds and mammals.

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.

BackgroundEmerging evidence suggests that the oral microbiome may contribute to aberrant gut immune responses in Inflammatory Bowel Disease (IBD). MethodsWe performed a comprehensive, harmonised analysis of aggregated oral microbiome 16S rRNA datasets across multiple cohorts. Data were processed using a unified bioinformatics pipeline including DADA2 for taxonomic assignment, PICRUSt2 for functional prediction, MaAsLin2 for multivariable modelling, and machine learning. ResultsAcross 25 studies (n = 1,136 IBD; n = 759 controls), meta-analysis showed significantly reduced oral microbial Shannon diversity in IBD (standardised mean difference -0.31, p = 0.007). Secondary bioinformatics analysis of six datasets plus in-house data confirmed this reduction (Shannon diversity; Hedges SMD = -0.372, p < 0.001), driven primarily by Crohns disease (CD). Beta diversity demonstrated global compositional shifts, with CD demonstrating greater divergence from controls than ulcerative colitis (UC). Multivariable modelling identified reproducible taxa enriched in IBD, including Corynebacterium, Serratia and Streptoccocus, while Porphyromonas and Ruminococcaceae.G1 were enriched in controls. Functional pathway prediction indicated reduced butyrate metabolism in IBD sub-types and increased aromatic amino acid and related metabolite degradation pathways. Machine learning classifiers achieved modest discrimination (mean AUC [~]0.67), supporting the potential of saliva-based microbiome profiling to study dysbiosis in IBD. ConclusionsThese findings demonstrate that the oral microbiome in IBD is characterised by reduced diversity and reproducible structural community reorganisation. Together, these data support a contributory role for the oral-gut axis in CD pathogenesis and provide a rationale for targeted mechanistic and longitudinal studies to define causal links between oral dysbiosis and intestinal inflammation. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=200 SRC="FIGDIR/small/26351936v1_ufig1.gif" ALT="Figure 1"> View larger version (38K): org.highwire.dtl.DTLVardef@57306corg.highwire.dtl.DTLVardef@2c0ef0org.highwire.dtl.DTLVardef@88b0b3org.highwire.dtl.DTLVardef@8ed62_HPS_FORMAT_FIGEXP M_FIG C_FIG

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.

ObjectivesCirculating monocytes from rheumatoid arthritis (RA) patients are pre-primed for inflammatory activation, but their disease-intrinsic features have not been systematically characterized. Given the important role of metabolism in shaping immune cell function, we aimed to determine how this pre-primed state is underpinned metabolically and whether these changes persist across different activation states, using an unbiased multi-omics approach. MethodsPeripheral blood CD14 monocytes from RA patients and matched healthy donors were analyzed in an undifferentiated state (M0) and after differentiation into classically activated M(IFN{gamma}+LPS) and alternatively activated M(IL-4) macrophages, followed by acute lipopolysaccharide (LPS) stimulation. Metabolomic (untargeted LC-MS/MS), transcriptomic (RNA-seq), and proteomic (label-free mass LC-MS/MS) profiling were performed. Data was comprehensively analyzed by weighted gene correlation network analysis, differential analysis, gene set enrichment analysis, multi-omics factor analysis and metabolic flux modeling. ResultsRA monocytes exhibited a stable disease-driven signature across activation states. Integration of metabolomic, transcriptomic and proteomic data revealed an unexpected convergence on metabolic-secretory coupling, with depletion of nucleotide and redox metabolites, downregulation of mitochondrial and translational pathways, and remodeling of the secretory apparatus, including loss of cis-Golgi components. Consistently, metabolic modeling predicted reduced glycosylation fluxes, connecting metabolic changes to altered secretory capacity. ConclusionsRA monocytes adopt a stable, disease-intrinsic state that persists across activation conditions. Multi-omics data identify a linked metabolic and secretory defect, with reduced glycosylation capacity as a potential functional consequence. This metabolic-secretory coupling represents a defining feature of RA monocyte dysfunction and a potential therapeutic target.

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.

Unbiased classification of equine dendritic cells (DC) is necessary to address various research questions such as the role of DC subsets in immune-mediated diseases of horses. We applied single-cell RNA sequencing (scRNA-seq) on DC enriched from the blood of two horses. All main DC subsets were detected by key gene expression, including conventional DC type 1 (cDC1; XCR1) and type 2 (cDC2; FCER1A, CD1E) as well as plasmacytoid DC (pDC; TCF4). In addition, we detected a small cluster of hematopoietic progenitors, as well as transitional DC (tDC; FCER1A, TCF4) and putative DC type 3 (DC3; FLT3, CD163). Our data confirms the previously reported phenotype of equine pDC (Flt3+MHC-IIlowCADM1lowCD172aint), cDC1 (Flt3+MHC-IIhighCADM1highCD172alow-int) and cDC2 (Flt3+MHC-IIhighCADM1intCD172ahigh), while also highlighting considerable CD14 expression for cDC2. Two subclusters of equine cDC2 were found to be enriched in FCER1A or CX3CR1 transcripts (cDC2.1 and cDC2.2, respectively), with suggested enhanced extravasation and T-cell stimulatory capacities of the latter. Conservation of DC subsets across species (horse, pig, human, mouse) was illustrated by enrichment analyses with subset-specific gene signatures and by cross-species data integration with publicly available scRNA-seq datasets. Our atlas of equine blood DC is a valuable resource for comparative analyses, and it forms the foundation for understanding the involvement of distinct DC subsets in infections and immune-mediated pathologies.

Autophagy has been implicated in several lung diseases, either protecting tissues or driving pathology. Hypersensitivity pneumonitis (HP) is a complex inflammatory lung disease, and autophagy is heavily involved in regulating inflammation. The role of autophagy in HP remains unclear. The aim of our study was to understand the role of autophagy in HP pathogenesis. GFP-LC3 transgenic mice were exposed intranasally to Saccharopolyspora rectivirgula (SR) to induce HP and follow autophagy activation in the lung. Then, we take advantage of our Atg4b-deficient mouse model to assess how autophagy disruption impacts lung inflammation in response to SR antigen challenge. Increased autophagy activation was observed in epithelial and inflammatory cells after SR antigen exposure in GFP-LC3 transgenic lungs. GFP-LC3 puncta colocalized with ATG4B and ATG5 in epithelial and inflammatory cells after antigen exposure. Autophagy impairment limits the inflammatory response after SR antigen exposure in the lungs from the Atg4b-deficient mice when compared to WT mice. To evaluate whether lipopolysaccharide (LPS) exacerbates the inflammatory response in the Atg4b-deficient, a SR+LPS combined treatment was developed and we discovered that LPS aggravates the SR-induced HP in WT but not in Atg4b-deficient mice. Reduced HP severity in Atg4b-deficient mice was associated with decreased expression of NFkB, CCL1, CCL25, CXCL1, TNFR1, IL-13, and IL-17A, diminished CD4+ T cell recruitment and expansion, reduced M2-like macrophages, and decreased granuloma and iBALT development. Our findings highlight autophagy as a critical driver in HP pathogenesis and as a potetial target for novel theraphy development.

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.

Inflammation is a key driver of hematopoietic dysfunction in myeloid malignancies, but its role in the context of hypomethylating therapy remains incompletely understood. Although 5-Azacytidine is used posttransplant in high-risk myelodysplastic syndrome (MDS), only 50% of patients show a clinical response. We provide evidence that inherent inflammatory properties of healthy donor CD34+ stem cells exist that are likely to contribute to the "response" seen in MDS patients. These are linked to epigenetic priming of the myeloid niche, resulting in S100A8/A9-driven inflammatory program that promotes functionality of immature NK cells. Using in vitro differentiation systems, multi-omic profiling, and a S100A9-/- mouse model, we find that 5-AzaC modulates inflammatory transcriptional programs through epigenetic rewiring of upstream regulatory elements. Loss of S100A9 disrupts myeloid differentiation, impairs NK cell maturation, and alters key developmental regulators including CEBPB, JUN, and NFIL3. In vivo, 5-AzaC restores these defects and primes NK cells in a time- and context-dependent manner. Re-analysis of the published Australian MDS/CMML cohort shows that "responders" display increased S100A8/A9 expression together with enhanced IFN-{gamma}, IL6-JAK-STAT3, and TNF signaling. These findings suggest that inflammatory myeloid programs may serve as predictive biomarkers and therapeutic targets to enhance NK cell-mediated graft-versus-leukemia activity posttransplant. SummaryO_LIWe provide compelling evidence that inherent properties of healthy donor CD34+ hematopoietic stem cells (SCs) exist that are likely to contribute to the "response" seen upon pre-emptive posttransplant 5-AzaC therapy of patients with high-risk myelodysplastic syndrome (MDS). C_LIO_LIThese properties are linked to a distinct form of epigenetic plasticity at upstream-located transcription factor (TF) binding sites. This may indirectly contribute to acute S100A8/A9-driven inflammation, which is demonstrable in distinct monocyte subsets and, importantly, also in NK cells thereby determining the characteristics of inflammatory monocyte-NK cell crosstalk. C_LIO_LIMice with a targeted deletion of S100A9 fail to upregulate CEBPB / JUN and NFIL3 which results in impaired myeloid priming and dysfunctional NK cell maturation, respectively. C_LIO_LIRe-analysis of the Australian MDS/CMML cohort confirms that MDS patients that "respond" to 5-AzaC exhibit activated IFN-{gamma}, IL6-JAK-STAT3, and TNF-signaling pathways in the context of upregulated S100A8/A9 after six months of treatment. C_LIO_LIOur study indicates that screening of healthy donors SCs for specific inflammatory markers in early developing monocytes could be used as a marker to predict which donor will have the potential of generating a S100A8/A9-driven inflammatory response. This may help identify patients with MDS as well as AML who are likely to benefit from low-dose, short-term 5-AzaC therapy as early as day 7 after transplantation, potentially resulting in increased graft-versus-leukemia (GvL) activity. C_LI

Background and AimsAlterations in immunoglobulin G (IgG) N-glycosylation are implicated in inflammatory bowel disease (IBD); however, the robustness of IgG glycan signatures across IBD cohorts with diverse demographics and geographic origins remains underexplored. We aimed to determine whether compositional data analysis (CoDA) and machine learning (ML) can identify IBD-related IgG N-glycan signatures and whether these signatures capture disease-associated acceleration of biological aging. MethodsWe analyzed the IgG glycome profiles of 1,367 plasma samples collected from healthy controls (HC), symptomatic controls (SC), and people with newly diagnosed Crohns (CD), and ulcerative colitis (UC) across four cohorts (UK, Italy, United States, and Netherlands). IgG glycosylation was analyzed by ultra-high-performance liquid chromatography, yielding 24 total-area-normalized glycan peaks (GPs). Analyses were performed using cross-sectional data obtained at baseline. CoDA-powered association analyses were used to identify disease-related effects on GPs while controlling for demographic covariates. ML models were trained and evaluated to assess generalizability to unseen cohorts and demographic subgroups, with a focus on discrimination and reliability. ResultsAcross all cohorts, people with IBD demonstrated accelerated biological aging as quantified by the GlycanAge index. This was accompanied by consistent reductions in IgG galactosylation, with effects partially modulated by age. Classification models trained on glycomics and demographics achieved robust discrimination (AUROC{approx}0.80) between non-IBD (HC+SC) and IBD across cohorts. ConclusionThese findings reveal accelerated biological aging in people with IBD and support the translational potential of IgG glycans as biomarkers and a novel route toward clinically interpretable personalized risk estimates.

{gamma}{delta} T cells provide mucosal defense against infection while also contributing to tissue repair. However, data regarding the effect of the human lung environment on {gamma}{delta} T cell functionality remains limited. To address whether lung inflammation impacts {gamma}{delta} T cell functionality, we analyzed lung and matched hilar lymph node (LN) tissue from deceased donors and patients with interstitial lung disease (ILD). We performed high-parameter spectral flow cytometry to examine the expression pattern of phenotypic biomarkers and assess ex vivo function. We identified lung-specific enrichment of {gamma}{delta} T cells with an effector memory phenotype relative to matched regional LN. We then used an ex vivo stimulation approach to interrogate the capacity to protect against infection (granzyme B [GzmB], interferon-{gamma} [IFN{gamma}] and tumor necrosis factor [TNF]) and promote epithelial cell proliferation (amphiregulin [AREG]). We found that {gamma}{delta} T cells in lung and LN from deceased donors had similar functional properties. While {gamma}{delta} T cell populations from ILD lungs largely maintained cytokine production capacity, expression was diminished relative to LN counterparts. Importantly, lung {gamma}{delta} T cells maintained polyfunctional GzmB, IFN{gamma} and TNF expression across cohorts. Overall, we report human lung {gamma}{delta} T cells are regionally distinct with conserved functionality in a fibrotic environment.

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.

Sarcoidosis is a heterogenous disease of unknown etiology characterized by non-caseating granulomas. Disease prevalence and presentation vary significantly by ancestry and ranges from acute, self-resolving disease to severe, chronic disease. Following previous reports suggesting B cells in the development and pathogenesis of sarcoidosis, we present here results of single-cell RNA sequencing, supporting B cell involvement in sarcoidosis through altered immediate early response, rewiring of MAPK signaling, and ancestry-specific preferential expansion of B cell receptors. Peripheral blood mononuclear cells were obtained from individuals of African or European Ancestry (AA and EA, respectively) including 48 healthy controls, 59 sarcoidosis patients, and 28 systemic lupus erythematosus (SLE) patients. SLE samples were used as a disease control. Differential expression analysis highlighted many differentially expressed genes (DEGs) with almost 5x more in the AA sarcoidosis versus AA control group compared to the EA sarcoidosis versus EA control group. B cells had the most DEGs of all cell types and expression patterns were similar between ancestries, however, sarcoidosis had an opposite transcription pattern than SLE, demonstrating an alternative immune response to acute activation than that seen in a prototypical autoinflammatory disease. This trend was maintained when examining specialized B cell subsets, with the most pronounced effect in the AA sarcoidosis versus AA control comparison. Our results strongly support further investigation of the role of humoral immune response in sarcoidosis and the potential to highlight patient groups likely to benefit from existing B cell therapies.

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.