Immune dysregulation is increasingly recognized as a key contributor to male infertility, yet the role of the efferent ductules in reproductive immune homeostasis remains unexplored. Located between the immune-privileged testis and the epididymis, efferent ductules are essential for male fertility by reabsorbing over 90% of testicular fluid and maintaining sperm in suspension through the coordinated activity of reabsorptive primary ciliated cells (RPCCs) and multiciliated cells (MCCs). Using complementary in vivo and in vitro approaches, we investigated the contribution of efferent ductule epithelial cells to innate immunity. In a mouse model of lipopolysaccharide (LPS)-induced epididymitis, efferent ductules displayed significant interstitial neutrophil infiltration and abnormal sperm accumulation, indicating disrupted local homeostasis. Both mouse and human epithelial cells expressed Toll-like receptor 4, supporting a role in pathogen sensing. Transcriptomic analyses showed MCCs enriched in cilia-related genes, while RPCCs preferentially expressed genes involved in signaling and inflammation. To further explore epithelial immune function, we developed 3D organoid models recapitulating key features of efferent ductules. Upon LPS stimulation, these organoids mounted a robust innate immune response, secreting cytokines, chemokines, and growth factors, consistent with in vivo observations. Together, these findings identify efferent ductule epithelial cells as an upstream immunological checkpoint and establish organoids as a valuable model to study efferent ductule epithelial cells function in male reproductive health.
EoE is an inflammatory condition characterised by tissue lesions and significant activation of immune cells in the oesophageal mucosa. Phenotypic and functional analyses in this study revealed widespread accumulation and activation of multiple immune cell types, with eosinophils showing the most notable expansion and activation. MAIT cells were the only activated T cell population associated with eosinophil activation, and this activation correlated with eosinophil infiltration in oesophageal tissue. Functional tests indicated that eosinophils increase MR1 expression in response to bacterial ligands and can present microbial antigens to and activate MAIT cells through an MR1- and TCR-dependent process, which, in turn, promotes cytokine release and further eosinophil activation. These findings reveal a previously unrecognised eosinophil-MAIT cell interaction that might enhance local immune responses by presenting microbial antigens that penetrate the lesioned mucosa and sustain inflammation in active EoE.
Early life is characterized by heightened susceptibility to respiratory pathogens, yet the immune mechanisms that predispose infants to infection remain poorly defined. Using an infant mouse model of Streptococcus pneumoniae (Spn) colonization, we identify an age-dependent delay in IL-17 A production that was associated with prolonged bacterial colonization in infant mice. Consistent with a critical role for this pathway, IL-17 receptor A (IL-17RA)-deficient infant mice exhibited persistent Spn colonization of the upper respiratory tract (URT). Transcriptomic profiling of the URT from Spn-colonized IL-17RA-deficient infant mice revealed an enrichment of antiviral responses compared to WT controls and included increased expression of the interferon-stimulated gene Cxcl10, a chemokine whose expression is positively associated with Spn carriage in humans. These results suggested that IL-17RA signaling constrains the interferon response during URT Spn colonization. WT infant mice exhibited progressively increased expression of Cxcl10 during Spn colonization and was dependent on type 1 interferon (IFNAR1) signaling and the pore forming bacterial toxin, pneumolysin. Genetic deletion of Ifnar1 or Cxcl10, including myeloid cell-specific deletion of Ifnar1, accelerated bacterial clearance in infant mice. In contrast, adult mice exhibited earlier induction of IL-17 A, minimal induction of Cxcl10 expression, and no alteration in bacterial clearance rates following Ifnar1 or Cxcl10 deficiency. Taken together, our results reveal a developmental window in which delayed IL-17 A production postpones the IL-17RA-dependent restraint of the type I interferon/CXCL10 axis, thereby prolonging pneumococcal carriage through impaired myeloid cell-mediated bacterial clearance.
SARS-CoV-2 primarily targets epithelial cells in the respiratory and intestinal tracts where its cognate receptor ACE2 and obligate processing enzymes furin and TMPRSS2 are richly expressed. However, compared with severe inflammation and tissue damage in the lungs of a COVID-19 patient, clinical lesions in the intestine are rare, suggesting an effective intestinal mucosal immunity against SARS-CoV-2 infection. Here, we report that MMP7-/-/hACE2 hybrid mice lacking mature enteric α-defensins or cryptdins were more susceptible to SARS-CoV-2 infection in the intestine than K18-hACE2 transgenic mice. The mouse α-defensin cryptdin-5 (Crp5) displayed potent and broad antiviral activity in vitro and in vivo by two distinct mechanisms, (1) directly targeting the RBD of the spike (S) protein to antagonize its interactions with ACE2, thus blocking viral attachment, membrane fusion and cell-to-cell transmission, and (2) binding to the 630 loop of the S protein to induce its multimerization, thereby impairing proteolytic processing, membrane fusion and, ultimately, viral infectivity. Our findings imply that enteric α-defensins help alleviate, as host protective factors, Covid-19 symptoms in the intestine despite higher ACE2 expression in the gut than in the lungs, and that Crp5 may be developed as a broad-spectrum antiviral for the treatment of coronavirus infection irrespective of virus type and variant.
B cells play a crucial role in maintaining immune homeostasis at mucosal surfaces, including those of the gastrointestinal (GI) tract. Perturbed humoral immunity and antibody-mediated functions of B cells are well described in patients with chronic GI inflammatory diseases. However, our understanding of antibody-independent B cell functions such as antigen presentation, immunoregulatory cytokine production and tissue remodeling are more limited. Comprehensively defining the immunobiology of B cells beyond humoral immunity during GI inflammation will improve our ability to develop effective, targeted treatments that modulate B cell pro- versus anti-inflammatory activity in the clinical management of GI inflammatory diseases. In this review, we discuss the emerging roles of B cells using antibody-independent mechanisms to maintain intestinal homeostasis, and how this evolves to B cells becoming active players in immunopathology underlying GI inflammatory conditions and specific GI cancers.
Sex-based differences in respiratory disease outcomes are well recognized. However, the underlying immunological mechanisms driving this dimorphism remain incompletely understood. While sex hormones influence immune cell development and function, the role of commensal microbes in shaping sex-specific lung immunity has not been explored. Here, we used single-cell RNA sequencing (scRNAseq) and flow cytometry to profile lung immune cells in male and female mice housed under specific pathogen-free (SPF) or germ-free (GF) conditions. Under SPF conditions, males exhibited a striking myeloid bias, with increased monocytes and macrophages, along with broad upregulation of inflammatory mediators, including S100a8, S100a9, and Il1b, across multiple cell types, and enrichment of TNF and interferon (IFN) signaling pathways. In contrast, females displayed lymphocyte-skewed profiles, with higher frequencies of T cells, B cells, and natural killer (NK) cells. Interestingly, these sex-based differences in immune composition and inflammatory programs were largely absent in GF mice, suggesting that microbial exposure influences baseline immunological dimorphism between males and females. Notably, select sex-associated immune differences, including female-biased NK cell enrichment, persisted irrespective of microbial status, suggesting intrinsic, microbiota-independent programming. Together, these findings indicate that commensal microbes may modulate sex-specific lung immunity, potentially amplifying pre-existing intrinsic differences, highlighting the intersection of extrinsic (microbial) and intrinsic (sex-linked) factors in shaping baseline mucosal immunity.
Pulmonary fibrosis is a complex disease with poorly understood, multifactorial triggers. Gammaherpesviruses (γHVs), including Epstein-Barr virus in humans and Murid herpesvirus 4 (MuHV-4) in mice, have been linked to pulmonary fibrosis exacerbation, although the underlying mechanisms remain unclear. This study explores how γHV infection modulates bleomycin (bleo)-induced lung fibrosis in mice, using flow cytometry and multiplex spectral immunofluorescence (MSI) to analyze cell dynamics, localization, and intercellular crosstalk. Despite the substantial contribution of monocytes (MOs) to the alveolar macrophage (AM) pool following MuHV-4 infection, these cells do not exhibit overt profibrotic properties upon bleo treatment. In contrast, MuHV-4 infection led to the accumulation of activated, IFNγ-producing memory CD8+ T cells in the lungs of bleo-treated mice, with MSI revealing their enrichment and colocalization with fibroblasts. Notably, fibroblasts showed increased IFNγ-induced PD-L1 expression, while T cells upregulated PD-1, co-localizing in fibrosis-rich regions. In vivo neutralization of the PD-L1/PD-1 axis after bleo treatment significantly improved the resolution of fibrosis in MuHV-4-infected mice, highlighting the key pathogenic role of this interaction in sequelae persistence. Compared to fibroblasts cocultured with T cells from naïve mice, those cocultured with MuHV-4-induced T cells exhibited significantly increased collagen expression, while this effect was abrogated by PD-1 blockade. Similar results were observed in cocultures of human fibroblasts and CD8+ T cells isolated from EBV-infected patients, underscoring the translational potential of these findings. Collectively, our findings identify interactions between PD-1+ CD8+ T cells and PD-L1+ fibroblasts as central drivers of persistent lung fibrosis associated with γHV infection.
BACKGROUND:Mucus obstructs the airways in respiratory diseases where MUC5B is the major gel-forming mucin in COPD and MUC5AC-rich mucus dominates in asthma. Mucin production changes in response to inflammatory signals, but whether mucin dysregulation drives inflammation is less studied. OBJECTIVE:We sought to identify if MUC5B and MUC5AC affect immune cell composition during homeostasis and inflammation. METHODS:Immune cells in airways and distal compartments from wild type (WT), Muc5ac-/- and Muc5b-/- mice were assessed by flow cytometry and morphological examination. Epithelial permeability was assessed via intranasal dextran administration, and inflammation was induced by IL-33. Gene expression and inflammatory mediators were analyzed by qPCR and ELISA, respectively. RESULTS:Mucin expression increased with age in WT mice, where Muc5b remained 40 times more abundant than Muc5ac, however, single-mucin deficiency resulted in compensatory increase of the other. MUC5B protected mice from increased bacterial load with neutrophil infiltration in airways, but MUC5B and MUC5AC were similarly important to prevent eosinophilia in lungs and distal compartments. Airway inflammation correlated with epithelial shedding, aberrant expression of epithelial integrity genes, increased epithelial permeability, and altered alarmins along with activation of innate lymphoid cells upon mucin disruption. Finally, IL-33 airway challenge increased Muc5b and Muc5ac where both mucins were required for normal granulocyte recruitment to lungs. CONCLUSION:MUC5B and MUC5AC play nonredundant yet complementary roles in maintaining airway immune homeostasis and regulating inflammation. Loss or imbalance of either mucin disrupts normal epithelial responses and compromises barrier integrity, potentially driving downstream changes in immune cell composition locally and systemically. It highlights an underappreciated immunomodulatory function of airway mucus which sheds new light on its role in asthma and COPD.
Idiopathic pulmonary fibrosis is a progressive lung disease characterized by the rapid scarring of the lung parenchyma resulting in impaired gas exchange and early mortality. Current treatment options are limited; lung transplantation remains the only definitive treatment. The adaptive immune system has been increasingly evoked as a potential contributor to disease initiation or progression. Using spectral flow cytometry, immunofluorescence imaging, and in-vitro functional assays we studied T cells obtained from explanted lungs and lung draining lymph nodes (HLN) from patients with IPF and non-diseased controls. We found the accumulation of granzyme K producing, hypofunctional CD8+ T cells in the lungs and HLN from IPF compared to controls. We also showed the accumulation of regulatory T cells in both lung and HLN in patients with IPF. CD4+ and CD8+ T cells were found to accumulate around areas of active fibrosis in IPF lung sections. Finally, when exposed to extracellular granzyme K, epithelial cells from human lungs showed increased expression of genes related to fibrosis, proliferation, and inflammation. Together, these show that IPF lungs and lymph nodes are characterized by the accumulation of granzyme K producing T cells and that granzyme K can promote pro-fibrotic effects of lung epithelial cells, providing a potential means whereby T cells might contribute to lung fibrosis.
After respiratory infection, the lung does not simply return to the prior baseline but adopts a new distinct post-infection steady state involving durable imprints that can shape tissue responses to subsequent challenges. This review introduces the concept of lung tissue memory, whereby prior infection leaves long-term changes across immune and structural compartments, with consequences for tissue repair and disease severity during later, antigenically distinct challenges. Pre-existing and newly emerging cell populations establish networks of cell-cell crosstalk that can initiate and maintain altered cellular states. We discuss to what degree these persistent adaptations reflect shifts in cellular composition, cell-intrinsic reprogramming, changes in inflammatory tone and altered tissue niches. Finally, we propose that infection-experienced models provide a more translationally relevant framework to understand immune networks operating in the lung during infection in humans, and we present how a better understanding of tissue memory informs therapeutic strategies and improved design of antigen-specific and more broadly protective mucosal vaccines.
BACKGROUND:Early postnatal gut colonization, microbial metabolite production, and immune maturation proceed in parallel. Although disruption has been linked to later allergic disease, cellular immune patterns remain insufficiently defined. Very and extremely preterm infants frequently develop eosinophilia, providing a setting to relate eosinophil dynamics to exposures and gut microbial-metabolic features. METHODS:We analyzed 15,795 CBC measurements from 734 preterm infants, together with fecal 16S profiling, metabolomics, eosinophil follow-up, and mouse perturbation experiments. Antibiotic and feeding exposures, microbiota, and metabolites were evaluated by longitudinal and integrated analyses; mouse experiments examined antibiotic responses with or without sodium butyrate. RESULTS:Eosinophils showed a transient postnatal trajectory, with highest levels at 3-4 weeks. Higher eosinophil levels were associated with greater antibiotic exposure, delayed feeding tolerance, and higher eosinophil counts during the first year of follow-up. During the peak window, eosinophil levels were inversely associated with gut microbial diversity, Clostridium abundance, and fecal short-chain fatty acids, particularly butyrate. In neonatal mice, antibiotics coincided with higher splenic eosinophils and intestinal ILC2/type-2 signatures, whereas sodium butyrate yielded profiles closer to controls. CONCLUSION:Early eosinophil expansion in these infants represents a time-restricted immune trajectory associated with NICU exposures and microbial-metabolic features. Clostridium-butyrate signatures are candidate correlates warranting mechanistic and outcome validation.
Licensure of influenza vaccines relies on serum hemagglutination inhibition (HAI) titers, a correlate of protection (CoP) that was developed more than 50 years ago and which is only poorly predictive of protection. This is especially true of immunity induced by intranasal live attenuated influenza vaccines (LAIVs). Unlike intramuscular inactivated influenza vaccines (IIVs), LAIV and natural infection selectively stimulate mucosal immunity. Developments in mucosal immunology now enable measurement of diverse aspects of mucosal immunity, including the frequencies and functions of nasal antibodies, T cells, and B cells. This review assesses the potential of new sampling and assay approaches that may overcome inconsistent findings from previous methodologies. Standardization of the collection and assessment of mucosal samples is essential in developing new CoPs for vaccine development and licensure of novel vaccines that induce nasal protection and are more effective in prevention of viral transmission.
Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a cytokine that predominantly acts on myeloid cells. Although decades of research have revealed diverse functions of GM-CSF during inflammation across tissues, it exerts important homeostatic functions, most notably in the lung. Extensive studies have established the essential requirement of GM-CSF for the development and function of alveolar macrophages (AMs), while also uncovering more nuanced effects on additional lung immune cell populations. As these cell types actively participate in pulmonary inflammatory processes, the homeostatic and inflammatory roles of GM-CSF are closely intertwined, positioning GM-CSF as a central regulator of lung immune cell composition and function. In this review, we discuss the multifaceted biology of GM-CSF in pulmonary immunity, ranging from its foundational role in alveolar macrophage biology to its contributions to lung inflammation and pathology.
A central challenge faced by the immune system is not simply detecting microbes but determining how to respond to them. In the intestine, where commensals and pathogens coexist, this decision is neither binary nor dictated by a single receptor. Instead, it emerges from the integration of signals across multiple pattern recognition receptors (PRRs), which together encode microbial identity and context. Among these, C-type lectin receptors (CLRs) occupy a unique position. By recognizing diverse ligands present on bacteria, fungi, viruses, and even host derived molecules, CLRs extend microbial sensing beyond pathogen detection to include signals of tissue state and environmental context. In doing so, CLRs function not merely as detectors of specific microbes, but as regulators that calibrate immune responses across the spectrum of tolerance and defense. Many reviews exist that detail how CLRs regulate pathogen defense, so here we will focus on the recent literature that demonstrates how these molecules influence maintenance of homeostasis with our commensal microbiota.
Multiple sclerosis (MS) is an autoimmune disorder of the central nervous system associated with alterations in gut commensals, including Akkermansia muciniphila (A. muciniphila). However, its role in MS remains unclear. Here, we report elevated serum lipopolysaccharide (LPS) and anti-LPS IgG levels in patients with relapsing-remitting MS (RRMS), indicating compromised gut barrier integrity. Notably, RRMS patients also exhibited increased serum anti-A. muciniphila IgA and enhanced A. muciniphila-induced Th17 responses in peripheral blood mononuclear cells (PBMCs). Using experimental autoimmune encephalomyelitis (EAE), a mouse model of MS, we found that A. muciniphila colonization worsened EAE severity, with increased infiltration of GM-CSF+CD4+ and IL-17A+CD4+ T cells in spinal cord. Mechanistically, A. muciniphila colonization enhanced tryptophan metabolism and elevated levels of aryl hydrocarbon receptor (AhR) agonists, including indole derivatives, during EAE. Although A. muciniphila does not directly metabolize tryptophan, it promotes expansion of tryptophan-utilizing bacterium Alistipes onderdonkii (A. onderdonkii) through mucin degradation. We further demonstrate that A. onderdonkii utilizes mucin-derived metabolites, including galactose and N-acetylneuraminic acid (NANA). Importantly, dietary tryptophan restriction significantly attenuated EAE severity. Collectively, these findings reveal a cross-feeding mechanism in which A. muciniphila supports growth of A. onderdonkii, thereby enhancing microbial tryptophan metabolism and production of AhR agonists that drive Th17-mediated neuroinflammation.
Cataloging the gut microbiome and understanding its interactions and association with the host immune system remains relevant to identify potential treatments for complex chronic diseases, including cancer. Studying the gut microbiome is more accessible than ever, thanks to the dramatic cost reduction of sequencing and the continuous improvements in bioinformatics and machine learning approaches. A plethora of bioinformatics and statistical methods are currently available to analyze gut microbiome sequencing data and evaluate its interactions with the host immune system across health and disease. This review summarizes different approaches and selected bioinformatics tools for gut microbiome data analysis. Furthermore, we underline methods for integrating and correlating sequencing data with other biological datasets, such as those resulting from metabolomics and immunological assays. Overall, this review provides a roadmap to help researchers with limited programming experience understand the approaches available to study the gut microbial community in relation to their topic of interest.
The intestine is a multifunctional tissue relying on multipotent stem cells to establish a repertoire of epithelial cell lineages. We have previously shown that the enteric parasitic nematode Heligmosomoides polygyrus bakeri (Hpb) directly regulates the intestinal stem cell compartment, tuning the epithelium to a regenerative fetal-like state, marked by the expansion of Clusterin-expressing revival stem cells (revSCs) and inhibiting goblet and tuft cell differentiation. However, the host signaling pathway driving this response remained obscure. Here, we demonstrate that TGFβ receptor (TGFβR) engagement is critical for helminth-induced epithelial reprogramming. Specifically, we show that Hpb induces potent epithelial TGFβR signaling in both mouse and human intestinal epithelium and that Hpb-mediated revSC expansion is TGFβR-dependent. In addition, we show that the loss of TGFβR signaling in the intestinal epithelium enhances goblet cell expansion while compromising Hpb egg production. Finally, we identify the TGFβ mimic, TGM, as the Hpb-secreted protein responsible for the induction of the fetal-like transcriptional program and revSC expansion. Collectively, our study reveals how a parasitic helminth hijacks epithelial TGFβR signaling to expand a pro-regenerative stem cell population and support host-helminth mutualism.
Seasonal epidemics and the persistent threat of a pandemic provide a strong impetus to understand mechanisms of protection against influenza infection. T cell intrinsic signaling through the TNFR superfamily member 4-1BB is critical for the accumulation of antigen-specific CD8+ effector and memory T cells in the lung and protection from influenza A virus (IAV). However, the APCs that provide 4-1BB ligand (4-1BBL) have not been definitively characterized. Here, using single-cell RNA sequencing and multiparameter flow cytometry, we define murine monocyte lineage cell (MC) and classical dendritic cell (cDC) populations in the lung during acute IAV infection and show that 4-1BBL is more highly expressed on CD64+MAR-I+CD26- inflammatory MCs than on MHCIIhiCD11c+CD26+ cDCs following IAV infection. Mixed bone marrow chimeras, in which 4-1BBL is only absent on Ccr2-dependent cells, and Cre-driven deletion using Ccr2- or Zbtb46-cre demonstrate that 4-1BBL on MCs rather than cDCs is important for the accumulation of nucleoprotein (NP)-specific CD8+ effector and tissue-resident memory T cells. Importantly, 4-1BBL on Ccr2-dependent cells is critical for mouse survival following severe IAV infection. These findings reveal a division of labor between cDC and MCs, with infMCs uniquely providing 4-1BBL to increase CD8+ T cell accumulation in the lung and protect against severe IAV infection.
Inflammatory bowel disease (IBD), including ulcerative colitis (UC) and Crohn's disease affects approximately 7 million individuals worldwide. Its pathogenesis involves genetic susceptibility, alterations in the gut microbiota, and immune system imbalance, with both innate and adaptive components contributing through aberrant cytokine signaling. The Transforming Growth Factor-β (TGFβ) pathway plays a central role in this context, controlling the differentiation of regulatory T-cells (Tregs) and Th17 lymphocytes within the intestinal mucosa. Given the importance of these populations in maintaining intestinal homeostasis, we investigated the role of the TGFβ inhibitor BAMBI (BMP and Activin Membrane-Bound Inhibitor) in UC progression. In addition, due to the increased risk of colorectal cancer associated with chronic intestinal inflammation, we evaluated the effects of genetic ablation of Bambi (BAMBI-KO) in chronic inflammation-associated colon cancer progression. BAMBI expression is predominantly localized in the colonic epithelium, and either its absence or pharmacological inhibition confers protection against Dextran Sodium Sulfate-induced colitis. Mechanistically, this effect is TGFβ-dependent, independent of immune suppression and is associated with enhanced epithelial cell renewal and improved barrier function. Moreover, chronic UC induction in BAMBI-KO mice results in reduced colorectal tumor multiplicity, size and severity. Collectively, these findings identify BAMBI as a critical regulator of intestinal homeostasis and a potential therapeutic target in colorectal disease.
Bacille Calmette-Guérin (BCG) is the only licensed vaccine against tuberculosis (TB) but provides inconsistent protection against disease. Alveolar macrophages (AM) are widely considered the primary myeloid mediators of BCG-induced lung immunity, whereas the contribution of lung interstitial macrophages (IM) remains poorly defined. Here, we investigated pulmonary macrophage remodeling following BCG vaccination delivered by three distinct routes in mice, using flow cytometry, single cell RNA sequencing and spatial transcriptomics. We show that the route of vaccine administration dictates which macrophage subset is rewired. Intratracheal (IT) BCG preferentially reprograms IM, which form spatially organized immune hubs with CD4 T cells, while AM retain a transcriptional state closer to homeostasis. In contrast, intravenous (IV) BCG induces transcriptional remodeling of AM. Importantly, IT BCG conferred superior protection against both Mycobacterium tuberculosis and the heterologous pathogen Pseudomonas aeruginosa. Collectively, these findings identify IM as key mediators of mucosal vaccine-induced protection and highlight macrophage subset targeting as a framework for optimizing vaccines against respiratory pathogens.
