Precise regulation of uterine immunity is required to support fundamental processes including reproduction and pathogen protection. How the local milieu and constitutive stressors, including the cervicovaginal microbiota, shape the delicate balance underlying uterine immunity is poorly understood. Here, we found that the cervicovaginal microbiota promotes both local immunity and the immunoregulatory activity of interferon lambdas (IFN-λ) in the uterus. Using murine models, we found a keystone role for IFN-λ in constraining the immune tone of this compartment, more specifically of innate lymphoid cells and Th17 cells. Conversely, in the context of pregnancy, IFN-λ promotes antibacterial responses at maternal-fetal barriers, and as a result controlling fetal and neonatal transmission. Collectively, this work demonstrates how IFN-λ integrates microbial signals under both steady state and pregnancy conditions thereby orchestrating key functions of the uterine immune system, namely immunoregulation and antibacterial protection.

Conventional dendritic cells (cDCs) are key sentinels at epithelial barriers, regulating immunity to microbial pathogens and commensals while preserving tissue integrity. NOTCH2 deficiency in CD11c-expressing cells (Notch2cKO) disrupts type 2a DC (cDC2a) development, impairs intestinal TH17 immunity and increases susceptibility to enteropathogenic bacteria. This defect leads to persistent dysbiosis in Notch2cKO mice, characterized by low-grade inflammation and systemic autoimmune features, including elevated autoantibody titers and renal immune complex deposition. Dysbiosis precedes expansion of highly inflammatory AXL-expressing type 3 DCs (AXL+inf-DC3), promoting chronic inflammation and tertiary lymphoid structures driving adaptive immune responses. Notably, dysbiosis is defined by three dominant pathobionts and is transferable to wild-type mice, recapitulating the autoimmune features observed in Notch2cKO mice. Here these findings identify a microbiota-DC axis linking intestinal pathobionts to systemic autoimmunity, establishing inflammatory DC3 as the cellular bridge between dysbiosis, chronic inflammation and autoimmune pathogenesis.

Immune responsiveness at barrier surfaces is tailored to the exposures of each tissue. In the oral mucosa, mechanisms by which a permeable epithelium coexists with diverse microbiota and maintains integrity during inflammatory pathology remain poorly understood. We compile a multiomics spatial map of this exposed mucosal microenvironment and uncover remarkable immune zonation with organization that is preserved even during inflammatory disease. At the tooth interface, we identify a dynamic epithelium underlined by a layer of neutrophils and a zone of antigen-presenting cell-lymphocyte aggregates. During disease, inflammatory zones expand and organize into immature tertiary lymphoid structures, suggesting local antibody production. Location-specific transcriptomes support a role for the stromal compartment in the spatial organization of immunity. This preserved immune zonation meets the demands for continuous protection of this vulnerable interface and suggests unique tissue-specific wiring of immunity at the human oral mucosal barrier.

Abstract Introduction Nutrition influences all host physiological processes, yet its role in regulating tissue repair remains poorly understood. Although both the immune system and the microbiota have been implicated in tissue repair, how diet reshapes host physiology, microbial function, or host—microbe interactions to promote regeneration has not been investigated. Methods To test this, we manipulated the nutritional status of mice using either dietary restriction or nutrient-enriched diets and assessed wound closure following injury. Results Here, we show that the ketogenic diet (KD), a diet enriched in fats and low in carbohydrates, feeding reshapes the metabolic environment of the skin and enhances both the abundance and metabolic output of the commensal Staphylococcus epidermidis. In vivo metatranscriptomics revealed KD-induced increases in microbial glycolysis, nucleotide synthesis, and riboflavin-pathway activity. Lipidomic profiling further showed that KD elevated bacterial sphingomyelinase-dependent ceramides in the epidermis. These diet-responsive microbial metabolites amplified cutaneous γδ T cell and mucosal-associated invariant T (MAIT) cell responses and directly promoted keratinocyte activation, collectively accelerating wound repair. Genetic disruption of microbial ribD or sphingomyelinase impaired these effects, demonstrating that host nutritional status drives tissue regeneration by rewiring commensal metabolic programs. Conclusion Together, our findings reveal a diet-sensitive metabolic axis through which skin microbes coordinate tissue regeneration. Funding Source Supported by NIAID; 1ZIA-AI001115 and 1ZIA-AI001132; JSPS CPD Reseach Fellowship for Young Scientist Topic Categories Mucosal and Regional Immunology (MUC)

Hidradenitis suppurativa (HS) is a chronic inflammatory skin disease associated with a polybacterial dysbiosis devoid of a known pathogen. Here, we report that HS patients mount IgA and IgG responses against skin colonizers, notably Porphyromonas uenonis (Pu), a rare species selectively enriched in severe disease. In these patients, Pu foci are detected in the epidermis, surrounded by IgA deposits, and anti-Pu IgGs cross-react with self-antigens expressed by healthy keratinocytes. Using healthy human skin explants, we demonstrate that patient-derived Pu can cross an intact epidermal barrier, infect and persist within keratinocytes, triggering their expression of pro-inflammatory mediators. In contrast, topical application of Pu on immunocompetent mice elicit cutaneous and systemic humoral immune responses without tissue infection. These findings uncover an impaired innate immune control of Pu in HS patients, linking keratinocyte infection to skin inflammation and humoral autoimmunity. They underscore the potential of targeting cutaneous dysbiosis as a strategy to limit HS progression.

Metastasis remains the primary cause of cancer-related deaths and is characterized by complex reprogramming of systemic processes. Emerging evidence indicates that extraosseous tumors can rewire bone marrow physiology and disrupt hematopoiesis, thereby compromising effective systemic immune responses. However, how tumor-induced immune alterations in bone marrow contribute to skeletal metastasis remains poorly defined. Here, using immunocompetent mouse models of mammary tumor bone metastasis, we show that mammary cancer cells precondition the bone marrow niche prior to metastatic colonization, driving early remodeling of the microenvironment and depleting bone marrow lymphoid populations. Specifically, cancer cells induce a dramatic B cell reduction, the most abundant lymphoid subset in bone marrow, resulting from dysregulated cell cycle gene expression in pre-B cells, along with impaired B-cell proliferation and differentiation. These findings are further validated in breast cancer bone metastasis patients, who exhibit significant bone marrow B-cell loss alongside disrupted molecular and developmental programs. A causal role for B cells in restraining skeletal metastasis is supported by the finding that experimental B-cell depletion significantly increases both incidence and severity of bone metastasis. Mechanistically, we find that B-cell loss is driven by systemic elevation of G-CSF. Accordingly, pharmacological neutralization of G-CSF significantly reduces both B-cell depletion and bone metastasis susceptibility. Collectively, our data reveal that breast cancer cells can distantly hijack B-cell developmental trajectories, promoting skeletal metastasis. This work identifies B cells and G-CSF as potential therapeutic targets in bone metastasis and highlights the importance of targeting early bone marrow immune dysregulation to prevent or limit skeletal metastasis. HIGHLIGHTS:Mammary tumor cells reshape the bone marrow niche inducing B cell lossBone marrow B cell development is impaired in mammary tumor metastasisExperimental depletion of B cells promotes bone metastasisG-CSF mediates B cell loss in mammary tumor metastasis.

Abstract Early-life microbiota represent an indispensable factor for the proper development and function of host metabolism and the immune system. We have demonstrated that neonatal exposure to antibiotics for the first 3 weeks (NeoATB) leads to obesity in adulthood, characterized by gut microbiota dysbiosis and dysregulated immune responses. Here, we demonstrate that feeding D-mannose suppresses NeoATB-induced obesity, accompanied by improved glucose tolerance and decreased insulin resistance. Mechanistically, D-mannose feeding decreased hypoxia and increased oxygenation and recovery of metabolic activity of adipocytes. D-mannose restored CD4 + Foxp3 + ST2 + Tregs, leading to a reduction of Th1 pro-inflammatory cells in the adipose tissue of NeoATB mice. Significantly, we revealed that D-mannose treatment reversed the dysregulated ratios of phylum Firmicutes to phylum Bacteroidetes in obese NeoATB mice, which was surprisingly attributed to D-mannose-mediated suppression of the growth of Firmicutes rather than an increase in the growth of Bacteroidetes. These findings should have therapeutic implications for the treatment of obesity in human patients.

Allergic asthma is driven by type 2 immune responses, including type 2 innate lymphoid cells (ILC2s). Although ILC2s are activated by the tissue alarmins interleukin (IL)-33 and IL-25, these signals do not intrinsically enforce type 2 identity and the mechanisms that maintain type 2 cytokine expression remain unclear. Here we show that allergen-induced IL-33 and IL-25 rapidly induce IL-9, which in turn upregulates the transcriptional repressor Blimp-1 in ILC2s. Blimp-1 sustains type 2 immunity by directly repressing type 1 inflammatory programs, including expression of interferon-γ and tumor necrosis factor. Deletion of Blimp-1 in ILC2s increased type 1 cytokine production and reduced IL-5 and IL-13 expression, eosinophil recruitment and mucus production in the lung. In contrast, IL-9 expression was enhanced in the absence of Blimp-1, leading to increased mast cell recruitment. Together, these findings identify Blimp-1 as a key regulator of ILC2 transcriptional fidelity that stabilizes type 2 inflammation while constraining divergent inflammatory programs during allergic responses.

Nutrition influences host physiological processes, yet how diets reshape host physiology, microbial functions, or host-microbe interactions to promote regeneration remains poorly explored. Here, we show that a ketogenic diet (KD), enriched in fats and low in carbohydrates, reprograms both skin microbial and immune functions to promote tissue repair. KD enhances IL-17A activity in γδ T cells and mucosal-associated invariant T (MAIT) cells, accelerating tissue repair, while KD-induced skin lipidomic alterations enhance both the abundance and metabolic output of Staphylococcus epidermidis. Metatranscriptomic and lipidomic analyses revealed increased riboflavin biosynthesis and sphingomyelinase (Sph)-dependent ceramide production in S. epidermidis under KD conditions. Genetic depletion of microbial ribD, a key enzyme for riboflavin biosynthesis, or of sph compromised the ability of the bacteria to promote tissue repair. Thus, host nutritional status drives tissue regeneration by synergistically rewiring host and microbial functions, providing new insights into how diet can be harnessed to regulate host physiology.

Intestinal immunity defends against enteric pathogens, mediates symbiotic relationships with the resident microbiota, and provides tolerance to food antigens, safeguarding critical nutrient absorption and barrier functions of this mucosal tissue. Despite the abundance of tissue resident activated T cells, their contributions to these various roles remain poorly understood. Here, we identify a dominant population of IL-10 producing, T-bet-expressing Tr1 T cells, residing in the small intestinal lamina propria at homeostasis. Remarkably, these intestinal Tr1 cells emerge at the time of weaning and accumulate independently of the microbiota displaying similar abundance, function, and TCR repertoire under germ-free conditions. Instead, the small intestinal T-bet+ Tr1 program is driven and shaped by dietary antigens, and accumulates in a cDC1-IL-27-dependent manner. Upon activation, these cells robustly express IL-10 and multiple inhibitory receptors, establishing a distinct suppressive profile. Altogether, this work uncovers a previously unappreciated dominant player in homeostatic small intestinal immunity with the potential to play critical suppressive roles in this tissue, raising important implications for the understanding of immune regulation in the intestine.

Precise regulation of uterine immunity is required to support fundamental processes including reproduction and pathogen protection. How the local milieu and constitutive stressors, including the cervicovaginal microbiota, shape the delicate balance underlying uterine immunity is poorly understood. Here, we found that the cervicovaginal microbiota promotes both local immunity and the immunoregulatory activity of interferon lambda (IFN-L) in the uterus. Using murine models, we found a keystone role for IFN-L in constraining the immune tone of this site, in particular of innate lymphoid cells and Th17 cells. Further, in the context of pregnancy, IFN-L enhanced antibacterial responses at maternal-fetal barriers to Streptococcus agalactiae infection, thereby controlling fetal and neonatal transmission. Collectively, this work uncovered how IFN-L integrates microbial signals under both steady state and pregnancy conditions and mediates the essential functions of the uterine immune system - antimicrobial protection and immunoregulation.

Abstract Introduction Tight regulation of uterine immunity is necessary to prevent infection and promote reproduction. How local tissue factors, including the cervicovaginal microbiota, influence uterine immunity is poorly understood. At other barrier sites, interferon lambda (IFN-L) is induced by the microbiota and controls barrier function, tissue immunity, and anti-viral immunity. IFN-L is constitutively expressed in the uterus and placenta and protects the placenta from virus infection. However, the source of immune stimulation that drives basal IFN-L expression and the additional functions of IFN-L outside of antiviral immunity in the uterus and placenta are largely unknown. Methods We leveraged spectral flow cytometry to investigate the effect of the cervicovaginal microbiota on uterine immunity using gnotobiotic mice and a model of new intravaginal commensal exposure. We performed spectral flow cytometry and single cell RNA sequencing (scRNAseq) on IFN-L signaling deficient mice to investigate the effect of IFN-L on uterine immunity. Next, we evaluated the anti-bacterial effect of IFN-L in nulliparous and pregnant mice using Streptococcus agalactiae. Results We found that IFN-L regulates uterine type 17 immune cell abundance in a microbiota-dependent manner. Our scRNAseq data revealed that loss of homeostatic IFN-L signaling resulted in decreased expression of genes involved in the defense responses to viruses and bacteria. We found that IFN-L did not influence bacterial burden in nulliparous mice. But, during pregnancy, IFN-L protects the placenta and fetus from ascending bacterial infection and from infection induced necrosis. Furthermore, loss of IFN-L signaling during bacterial infection in pregnancy had long term impacts on offspring health and fitness. Conclusion Overall, we find that IFN-L is a key immunoregulator in nulliparous animals and a key anti-bacterial factor in pregnant animals. Future work will explore how the microbiota influences uterine IFN-L signaling. Funding Source NIGMS F12 GM150424; intramural NIH funding Topic Categories Mucosal and Regional Immunology (MUC)

Abstract Introduction CNS autoimmune diseases such as multiple sclerosis and uveitis are driven by IL-17—producing Th17 cells, whose pathogenic conversion is promoted by IL-23 through the induction of T-bet and its downstream target IFN-γ. However, the CNS-specific signals that contribute to the pathogenic reprogramming of Th17 cells remain poorly understood. Methods We generated a dual fluorescent reporter mouse that marks the Th17 lineage and IFN-γ production to perform single-cell transcriptomic profiling of ocular T cells in experimental autoimmune uveitis (EAU). We used flow cytometry to validate candidate regulators in CNS and peripheral tissues in both wild-type and CRISPR—Cas9-engineered mice targeting genes enriched in pathogenic Th17 cells. We then evaluated disease progression in mutant mice and performed scRNA-seq analysis of ocular CD4+ T cells. We applied computational analyses to predict Th17 differentiation trajectories and disease relevance. Results Transcriptomic profiling revealed that, compared with non-pathogenic Th17 cells, pathogenic Th17 cells upregulated not only canonical inflammatory genes but also a set of neuronal genes, including neurogranin (Ng), a postsynaptic calmodulin-binding protein implicated in calcium signaling and synaptic plasticity. Using flow cytometry, we found that Ng protein is preferentially expressed by ocular Th17 cells, with minimal expression in the periphery, indicating that Ng is selectively induced in CNS-infiltrating Th17 cells. Ng-KO mice developed more severe EAU, consistent with scRNA-seq analysis of ocular CD4+ T cells showing enrichment of immune activation pathways in Ng-deficient eyes. Conclusion We propose that Ng restrains Th17 pathogenicity within the CNS, potentially by modulating TCR—driven calcium signaling. Our findings uncover an unexpected convergence of neuronal and immune programs and suggest that tissue-adapted T cells can repurpose neuronal gene networks to fine-tune local autoimmune responses in the CNS. Funding Source n/a Topic Categories Basic Autoimmunity (BA)

Abstract Introduction The neonatal dysbiosis of gut microbiota represents a critical environmental factor promoting obesity, but the underlying immunological mechanisms remain unknown. Methods Neonatal mice (from day1) were treated with broad-spectrum antibiotics for the first 3 weeks of life (NeoATB) only . The mice were then hosted under a normal environment for rest of their life. Results We show here that neonatal treatment of mice with broad-spectrum antibiotics for the first 3 weeks of life (NeoATB) resulted in the development of obesity and metabolic abnormalities in adulthood. The NeoATB mice exhibited a permanent dysbiosis of gut microbiota, decreased CD4+Foxp3+ regulatory T cells (Tregs), and increased proinflammatory Th1 cells in visceral adipose tissue (VAT). Mechanistically, neonatal antibiotic treatment resulted in increased intestinal permeability that allowed bacterial translocation into the VAT and liver and an increase in systemic LPS levels. Consequently, VAT dendritic cells were activated via the TLR4 pathway to increase IL-12-triggered Th1-inflammation. Moreover, the decrease in VAT Tregs in NeoATB mice was attributed to reduced infiltration of Tregs from the periphery, decreased expansion of adipose IL-33-mediated ST2+ Tregs, and reduced conversion of local Tregs from VAT CD4+CD25- Foxp3- T cells. Conclusion Thus, we have revealed a previously unrecognized immunological link between neonatal microbiota dysbiosis and the development of obesity. Funding Source NIH, NIDCR IRP Topic Categories Mucosal and Regional Immunology (MUC)

Endogenous retrovirus (ERVs) result from accumulated germline infections by ancient exogenous retroviruses and constitute a sizable proportion of the mammalian genome. Recent findings support the idea that these elements control tissue immune threshold of activation and inflammation. Within this context, our lab has recently described that, in the skin, the beneficial immunity to the microbiota depends on endogenous retrovirus expression. How the crosstalk between ERVs and the microbiota controls tissue immunity at other barrier sites and how dysregulation of this dialogue impairs tolerance responses remain unknown. Our results reveal that antiretroviral (anti-RT) treatment (blocking the conversion of ERV into cDNA) impairs gut regulatory T cell homeostasis and differentiation and compromises the induction of oral tolerance to food antigens. Consistently, our data demonstrate that antiretroviral treatment impairs type I IFN signaling in mucosal dendritic cells involved in the induction of regulatory T cells. Further, our results show that epithelial ERV expression is highly conserved across tissues and seems to be only partially affected by the microbiota in the small intestine. Altogether, our results propose that ERV control immunoregulation within the gastrointestinal tract by acting as a tonic signal required for Treg induction and differentiation thereby promoting oral tolerance and preventing allergic responses. This work was supported in part by intramural funds of NIAID, NIH. C.A.R is supported by Damon Runyon Fellowship program (DRG-2496-23).

Background:Cesarean section (CS) delivery is associated with an increased risk of inflammatory diseases, hypothesized to be driven by differences in the microbiome acquired at birth compared to vaginally delivered (VD) infants. How delivery mode associated differences in initial colonizers directly modulate early life immune education and metabolic development is poorly understood. Objective:First, to investigate how differences in pioneering colonizers associated with delivery mode directly modulate early life immune education and metabolic programming. Second, to examine the effect of "vaginal seeding", an intervention aimed to restore the microbiome of CS infants to a VD state. Design:Germ-free mice were colonized with transitional stool from VD, CS or CS-delivered and vaginally seeded neonates. Immune cell populations, serum immunoglobulin levels, and fecal microbiome and metabolome profiles were analyzed. Results:Mice colonized with stool from VD neonates displayed increased numbers of myeloid cells at barrier tissues, whereas CS microbiome colonized mice exhibited decreased Th1/Th2 ratios and increased serum IgE levels. Key amino-acid pathways including tryptophan metabolism, riboflavin co-enzymes and carbohydrate metabolites were significantly enriched in the murine VD fecal metabolome and correlate with the increased abundance of Escherichia typically observed in the VD microbiome. Mice colonized with stool from CS neonates who received vaginal seeding, resulted in increased regulatory T cells and serum IgA in mice, suggesting potential benefits of vaginal seeding. Conclusion:Collectively, our studies demonstrate the ability of pioneering colonizers to set the immune and metabolic tone that could have long-lasting effects and provide avenues for microbiome-mediated interventions.

Hydrogen sulfide is a gaseous, reactive molecule specifically enriched in the gastrointestinal tract. Here, we uncover a non-redundant role for sulfide in the control of both microbial and immune homeostasis of the gut. Notably, depletion of sulfide via both pharmaceutical and dietary interventions led to a profound collapse of CD4 T cells in the ileum of the small intestine lamina propria and significant impact on microbial ecology. As a result, mice with reduced sulfide within the gut were deficient in their ability to mount T cell dependent antibody responses to oral vaccine. Mechanistically, our results support the idea that sulfide could act directly on CD4 T cells via enhanced AP-1 activation, leading to heightened proliferation and cytokine production. This study uncovers sulfides as keystone components in gut ecology and provides mechanistic insight between diet, gut sulfide production and mucosal immunity.

The interaction between the immune system and the somatosensory system plays a fundamental role in the regulation of diverse biological processes. Chronic itch is a common yet hard-to-treat symptom of many inflammatory skin conditions. One hallmark of chronic itch is the hyperinnervation of the skin by sensory fibers, yet what drives this aberrant nerve growth or how it contributes to disease progression remains unclear. Here, we identify IL-17A and immunity to skin microbiota as key triggers of sensory neuron plasticity and pruritus. In a murine model of psoriatic itch, we show that exposure to Staphylococcus aureus prior to experimental psoriasis results in heightened skin inflammation, increased itch, and marked hyperinnervation of CGRPα+ sensory neurons. Accordingly, single-nuclei RNA sequencing of dorsal root ganglia reveals that microbiota-driven inflammation induces a regenerative transcriptional program in sensory neurons, including upregulation of axonal growth, injury response, and IL-17RA signaling pathways. Mechanistically, we show that IL-17A/IL-17RA signaling within TRPV1+ sensory neurons drives hyperinnervation and pruritus, establishing a causal link between IL-17A and microbiota-driven immune responses in sensory circuit remodeling. Further, we identify sensory hyperinnervation as a key driver of chronic itch and inflammation. Collectively, we reveal that aberrant IL-17A signaling in sensory neurons, triggered by dysregulated microbiota immunity, promotes neuronal remodeling that amplifies itch and inflammation. These findings provide a framework for targeting microbiota-neuroimmune interactions as a therapeutic strategy for pruritus.

Bone is the most common site of breast cancer metastasis, occurring in 70% of patients, and an indication of short-term prognosis. Bone colonization by cancer cells is a stepwise process that relies on interactions between tumor cells and the bone microenvironment. While it is well established that host immunity plays a fundamental role in tumorigenesis, the role of immune cells in bone metastasis remains poorly understood. Using immunocompetent mouse models of breast cancer bone metastasis, we found that breast cancer profoundly transforms the bone marrow lymphoid microenvironment before the arrival of tumor cells and induces bone lymphoid cell loss, with a dramatic reduction of B cells, the most abundant bone marrow lymphoid subset. Additionally, breast cancer induces an expansion of lymphoid progenitors with a reprogrammed transcriptional landscape and impaired differentiation capacity. Of note, single cell analysis showed that the remaining B cells present in the bone marrow exhibit reduced activation, maturation, antigen processing and presentation. Importantly, depletion of mature B cells increases bone metastasis incidence, suggesting that B cell loss creates a vulnerable bone environment prone to tumor cell colonization. Overall, our data reveals that breast cancer cells distantly shape B cell trajectories and imprint specific molecular programs in B cell progenitors that enhance metastasis, and identifies B cells as potential therapeutic targets in bone metastasis. This work was supported by the Division of Intramural Research of NIAID (NIAID; 1ZIA-AI001115 and 1ZIA- AI001132). A.T. was supported by the EMBO Postdoctoral Fellowship ALTF 1014-2021. Hematopoiesis and Immune System Development (HEM)

Inbred mice used for biomedical research display an underdeveloped immune system compared with adult humans, which is attributed in part to the artificial laboratory environment. Despite representing a central component of adaptive immunity, the impact of the laboratory environment on the B cell compartment has not been investigated in detail. Here, we performed an in-depth examination of B cells following rewilding, the controlled release of inbred laboratory mice into an outdoor enclosure. In rewilded mice, we observed B cells in circulation with increased signs of maturation, alongside heightened germinal center responses within secondary lymphoid organs. Rewilding also expanded B cells in the gut, which was accompanied by elevated systemic levels of immunoglobulin G (IgG) and IgM antibodies reactive to the microbiota. Our findings indicate that exposing laboratory mice to a more natural environment enhances B cell development to better reflect the immune system of free-living mammals.