Microbes and metabolites in immunity.

The immune system has a vital, albeit complex, relationship with the microbes residing within us, one that we are only beginning to understand. We asked investigators what they felt were the fundamental challenges we currently face in unraveling the impacts of microbes and their metabolites on host immunity and to discuss key opportunities toward achieving future insights and innovation.

Epigenetic control of commensal induced Th2 Responses and Intestinal immunopathology.

Understanding the initiation of T-helper (Th)-2 immunity is crucial for addressing allergic diseases that have been linked to the commensal microbiota. However, Th2 responses are notably absent from known host-microbiota intestinal immune circuits. Notably, the commensal protist Tritrichomonas induces a transient innate ILC2 circuit rather than a chronic Th2 circuit. Canonical Th2 responses rely on the induction of IL-4 production by innate cells. This study shows that the absence of Tet2 , a DNA demethylase, reprograms naïve T cells to autonomously produce IL-4 upon T cell receptor stimulation, bypassing the need for IL-4 from innate cells for Th2 differentiation. Loss of this checkpoint induces chronic Th2 responses to Tritrichomonas , associated with IL-25-dependent barrier dysfunction and increased susceptibility to allergic pathology in response to dietary antigens. Sentence Summary: Regulation of cell autonomous IL-4 in T cells is critical to prevent dysregulated Th2 immunity to commensals and predisposition to allergy.

Dietary Commensal Wrestles Iron from Tumor Microenvironment to Activate Antitumoral Macrophages.

The microbiome dictates the response to cancer immunotherapy efficacy. However, the mechanisms of how the microbiota impacts therapy efficacy remain poorly understood. In a recent issue of Nature Immunology, Sharma and colleagues elucidate a multifaceted, macrophage-driven mechanism exerted by a specific strain of fermented food commensal plantarum strain IMB19, LpIMB19. LpIMB19 activates tumor macrophages, resulting in the enhancement of cytotoxic cluster differentiation 8 (CD8) T cells. LpIMB19 administration led to an expansion of tumor-infiltrating CD8 T cells and improved the efficacy of anti-PD-L1 therapy. Rhamnose-rich heteropolysaccharide, a strain-specific cell wall component, was identified as the primary effector molecule of LplMB19. Toll-like receptor 2 signaling and the ability of macrophages to sequester iron were both critical for rhamnose-rich heteropolysaccharide-mediated macrophage activation upstream of the CD8 T-cell effector response and contributed to tumor cell apoptosis through iron deprivation. These findings reveal a well-defined mechanism connecting diet and health outcomes, suggesting that diet-derived commensals may warrant further investigation. Additionally, this work emphasizes the importance of strain-specific differences in studying microbiome-cancer interactions and the concept of "nutritional immunity" to enhance microbe-triggered antitumor immunity.

Melanoma and microbiota: Current understanding and future directions.

Over the last decade, the composition of the gut microbiota has been found to correlate with the outcomes of cancer patients treated with immunotherapy. Accumulating evidence points to the various mechanisms by which intestinal bacteria act on distal tumors and how to harness this complex ecosystem to circumvent primary resistance to immune checkpoint inhibitors. Here, we review the state of the microbiota field in the context of melanoma, the recent breakthroughs in defining microbial modes of action, and how to modulate the microbiota to enhance response to cancer immunotherapy. The host-microbe interaction may be deciphered by the use of "omics" technologies, and will guide patient stratification and the development of microbiota-centered interventions. Efforts needed to advance the field and current gaps of knowledge are also discussed.

Altered hepatic and intestinal homeostasis in a neonatal murine model of short-term total parenteral nutrition and antibiotics.

Parenteral nutrition (PN) prevents starvation and supports metabolic requirements intravenously when patients are unable to be fed enterally. Clinically, infants are frequently provided PN in intensive care settings along with exposure to antibiotics (ABX) to minimize infection during care. Unfortunately, neonates experience extremely high rates of hepatic complications. Adult rodent and piglet models of PN are well-established but neonatal models capable of leveraging the considerable transgenic potential of the mouse remain underdeveloped. Utilizing our newly established neonatal murine PN mouse model, we administered ABX or controlled drinking water to timed pregnant dams to disrupt the maternal microbiome. We randomized mouse pups to PN or sham surgery controls +/- ABX exposure. ABX or short-term PN decreased liver and brain organ weights, intestinal length, and mucosal architecture (vs. controls). PN significantly elevated evidence of hepatic proinflammatory markers, neutrophils and macrophage counts, bacterial colony-forming units, and evidence of cholestasis risk, which was blocked by ABX. However, ABX uniquely elevated metabolic regulatory genes resulting in accumulation of hepatocyte lipids, triglycerides, and elevated tauro-chenoxycholic acid (TCDCA) in serum. Within the gut, PN elevated the relative abundance of Akkermansia, Enterococcus, and Suterella with decreased Anaerostipes and Lactobacillus compared with controls, whereas ABX enriched Proteobacteria. We conclude that short-term PN elevates hepatic inflammatory stress and risk of cholestasis in early life. Although concurrent ABX exposure protects against hepatic immune activation during PN, the dual exposure modulates metabolism and may contribute toward early steatosis phenotype, sometimes observed in infants unable to wean from PN.NEW & NOTEWORTHY This study successfully established a translationally relevant, murine neonatal parenteral nutrition (PN) model. Short-term PN is sufficient to induce hepatitis-associated cholestasis in a neonatal murine model that can be used to understand disease in early life. The administration of antibiotics during PN protects animals from bacterial translocation and proinflammatory responses but induces unique metabolic shifts that may predispose the liver toward early steatosis.

Detection of viable commensal bacteria in murine melanoma tumors by culturomics.

Emerging evidence suggests the tumor microbiome at gut-distal sites can modulate tumor immunity and response to cancer immunotherapy. However, detection of commensal bacteria at gut-distal tumor sites is challenging given their low abundance. Here, we present a culturomics approach to facilitate recovery of phylogenetically diverse live commensal bacteria within gut-distal melanoma tumors. We describe steps for media preparation, tissue isolation, tissue homogenization, and host cell lysis. We then detail broth expansion culture followed by agar culture and single-colony 16S rRNA sequencing. For complete details on the use and execution of this protocol, please refer to Bender and McPherson et al. (2023).1.

The gut protist Tritrichomonas arnold restrains virus-mediated loss of oral tolerance by modulating dietary antigen-presenting dendritic cells.

Loss of oral tolerance (LOT) to gluten, driven by dendritic cell (DC) priming of gluten-specific T helper 1 (Th1) cell immune responses, is a hallmark of celiac disease (CeD) and can be triggered by enteric viral infections. Whether certain commensals can moderate virus-mediated LOT remains elusive. Here, using a mouse model of virus-mediated LOT, we discovered that the gut-colonizing protist Tritrichomonas (T.) arnold promotes oral tolerance and protects against reovirus- and murine norovirus-mediated LOT, independent of the microbiota. Protection was not attributable to antiviral host responses or T. arnold-mediated innate type 2 immunity. Mechanistically, T. arnold directly restrained the proinflammatory program in dietary antigen-presenting DCs, subsequently limiting Th1 and promoting regulatory T cell responses. Finally, analysis of fecal microbiomes showed that T. arnold-related Parabasalid strains are underrepresented in human CeD patients. Altogether, these findings will motivate further exploration of oral-tolerance-promoting protists in CeD and other immune-mediated food sensitivities.

Dietary tryptophan metabolite released by intratumoral Lactobacillus reuteri facilitates immune checkpoint inhibitor treatment.

The use of probiotics by cancer patients is increasing, including among those undergoing immune checkpoint inhibitor (ICI) treatment. Here, we elucidate a critical microbial-host crosstalk between probiotic-released aryl hydrocarbon receptor (AhR) agonist indole-3-aldehyde (I3A) and CD8 T cells within the tumor microenvironment that potently enhances antitumor immunity and facilitates ICI in preclinical melanoma. Our study reveals that probiotic Lactobacillus reuteri (Lr) translocates to, colonizes, and persists within melanoma, where via its released dietary tryptophan catabolite I3A, it locally promotes interferon-γ-producing CD8 T cells, thereby bolstering ICI. Moreover, Lr-secreted I3A was both necessary and sufficient to drive antitumor immunity, and loss of AhR signaling within CD8 T cells abrogated Lr's antitumor effects. Further, a tryptophan-enriched diet potentiated both Lr- and ICI-induced antitumor immunity, dependent on CD8 T cell AhR signaling. Finally, we provide evidence for a potential role of I3A in promoting ICI efficacy and survival in advanced melanoma patients.

IFNγ-induction of TH1-like regulatory T cells controls antiviral responses.

Regulatory T (Treg) cells are an immunosuppressive population that are required to maintain peripheral tolerance and prevent tissue damage from immunopathology, via anti-inflammatory cytokines, inhibitor receptors and metabolic disruption. Here we show that Treg cells acquire an effector-like state, yet remain stable and functional, when exposed to interferon gamma (IFNγ) during infection with lymphocytic choriomeningitis and influenza A virus. Treg cell-restricted deletion of the IFNγ receptor (encoded by Ifngr1), but not the interleukin 12 (IL12) receptor (encoded by Il12rb2), prevented TH1-like polarization (decreased expression of T-bet, CXC motif chemokine receptor 3 and IFNγ) and promoted TH2-like polarization (increased expression of GATA-3, CCR4 and IL4). TH1-like Treg cells limited CD8+ T cell effector function, proliferation and memory formation during acute and chronic infection. These findings provide fundamental insights into how Treg cells sense inflammatory cues from the environment (such as IFNγ) during viral infection to provide guidance to the effector immune response. This regulatory circuit prevents prolonged immunoinflammatory responses and shapes the quality and quantity of the memory T cell response.

The microbiota control the neonatal WNT-ernet.

The microbiome plays a fundamental role in maintaining intestinal stem cell (ISC)-niche equilibrium. In this issue of Immunity, Kim and colleagues uncover a mechanism by which the microbiota drives macrophage WNT ligand-release to maintain ISC-niche homeostasis during early postnatal development.

Team victory, yellow helmets for a computational tour de force.

Changes in the gut microbiota are associated with the etiopathogenesis of complex diseases, such as multiple sclerosis. In this issue of Cell, the international Multiple Sclerosis Microbiome Study consortium deployed a multi-omics approach to profile the composition and function of the gut microbiome in an extensive cohort of MS patients.

Adenovirus-vectored SARS-CoV-2 vaccine expressing S1-N fusion protein.

Additional COVID-19 vaccines that are safe and immunogenic are needed for global vaccine equity. Here, we developed a recombinant type 5 adenovirus vector encoding for the SARS-CoV-2 S1 subunit antigen and nucleocapsid as a fusion protein (Ad5.SARS-CoV-2-S1N). A single subcutaneous immunization with Ad5.SARS-CoV-2-S1N induced a similar humoral response, along with a significantly higher S1-specific cellular response, as a recombinant type 5 adenovirus vector encoding for S1 alone (Ad5.SARS-CoV-2-S1). Immunogenicity was improved by homologous prime-boost vaccination, and further improved through intramuscular heterologous prime-boost vaccination using subunit recombinant S1 protein. Priming with low dose (1 × 1010 v.p.) of Ad5.SARS-CoV-2-S1N and boosting with either wild-type recombinant rS1 or B.1.351 recombinant rS1 induced a robust neutralizing response, which was sustained against Beta and Gamma SARS-CoV-2 variants. This novel Ad5-vectored SARS-CoV-2 vaccine candidate showed promising immunogenicity in mice and supports the further development of COVID-19-based vaccines incorporating the nucleoprotein as a target antigen.

Tet2 deficiency drives liver microbiome dysbiosis triggering Tc1 cell autoimmune hepatitis.

The triggers that drive interferon-γ (IFNγ)-producing CD8 T cell (Tc1 cell)-mediated autoimmune hepatitis (AIH) remain obscure. Here, we show that lack of hematopoietic Tet methylcytosine dioxygenase 2 (Tet2), an epigenetic regulator associated with autoimmunity, results in the development of microbiota-dependent AIH-like pathology, accompanied by hepatic enrichment of aryl hydrocarbon receptor (AhR) ligand-producing pathobionts and rampant Tc1 cell immunity. We report that AIH-like disease development is dependent on both IFNγ and AhR signaling, as blocking either reverts ongoing AIH-like pathology. Illustrating the critical role of AhR-ligand-producing pathobionts in this condition, hepatic translocation of the AhR ligand indole-3-aldehyde (I3A)-releasing Lactobacillus reuteri is sufficient to trigger AIH-like pathology. Finally, we demonstrate that I3A is required for L. reuteri-induced Tc1 cell differentiation in vitro and AIH-like pathology in vivo, both of which are restrained by Tet2 within CD8 T cells. This AIH-disease model may contribute to the development of therapeutics to alleviate AIH.

Systemic Immunoregulatory Consequences of Gut Commensal Translocation.

One major determinant of systemic immunity during homeostasis and in certain complex multifactorial diseases (e.g. cancer and autoimmune conditions), is the gut microbiota. These commensals can shape systemic immune responses via translocation of metabolites, microbial cell wall components, and viable microbes. In the last few years, bacterial translocation has revealed itself as playing a key, and potentially causal role in mediating immunomodulatory processes in nongastrointestinal diseases. Moreover, recent observations regarding the presence of complex microbial communities and viable bacteria within gut-distal tissues during homeostasis challenge the current paradigm that healthy mammals are entirely sterile at nonmucosal sites. This review discusses our current understanding of how the gut microbiota orchestrates systemic immunity during noninfectious extraintestinal diseases and homeostasis, focusing on the translocation of viable bacteria to gut-distal sites.

High-sensitivity C-reactive protein is associated with clonal hematopoiesis of indeterminate potential.

Clonal hematopoiesis of indeterminate potential (CHIP) is predictive of hematological cancers and cardiovascular diseases, but the etiology of CHIP initiation and clonal expansion is unknown. Several lines of evidence suggest that proinflammatory cytokines may favor mutated hematopoietic stem cell expansion. To investigate the potential link between inflammation and CHIP, we performed targeted deep sequencing of 11 genes previously implicated in CHIP in 1887 subjects aged >70 years from the Montreal Heart Institute Biobank, of which 1359 had prior coronary artery disease (CAD), and 528 controls did not. We assessed association of CHIP with log transformed high-sensitivity C-reactive protein (hs-CRP), a validated biomarker of inflammation. CHIP was identified in 427 of the 1887 subjects (22.6%). CHIP mutations were more frequently identified in DNMT3A (11.6%) and TET2 (6.1%), with a higher proportion of TET2 mutations occurring in controls than in patients with CAD (9.0% vs 4.9%, P < .001). CHIP carriers had 21% higher hs-CRP levels compared with their noncarrier counterparts (eß = 1.21, 95% confidence interval [CI]: 1.08 to 1.36; P = .001). A similar effect was observed in the subgroup of patients with known CAD (eß = 1.22, 95% CI: 1.06 to 1.41; P = .005). These findings confirm the association between inflammation and CHIP. This association may open investigational avenues aimed at documenting mechanisms linking inflammation to clonal progression and ultimately supports prevention interventions to attenuate CHIP's impact on cardiovascular disease and cancer.

Duodenal bacterial proteolytic activity determines sensitivity to dietary antigen through protease-activated receptor-2.

Microbe-host interactions are generally homeostatic, but when dysfunctional, they can incite food sensitivities and chronic diseases. Celiac disease (CeD) is a food sensitivity characterized by a breakdown of oral tolerance to gluten proteins in genetically predisposed individuals, although the underlying mechanisms are incompletely understood. Here we show that duodenal biopsies from patients with active CeD have increased proteolytic activity against gluten substrates that correlates with increased Proteobacteria abundance, including Pseudomonas. Using Pseudomonas aeruginosa producing elastase as a model, we show gluten-independent, PAR-2 mediated upregulation of inflammatory pathways in C57BL/6 mice without villus blunting. In mice expressing CeD risk genes, P. aeruginosa elastase synergizes with gluten to induce more severe inflammation that is associated with moderate villus blunting. These results demonstrate that proteases expressed by opportunistic pathogens impact host immune responses that are relevant to the development of food sensitivities, independently of the trigger antigen.

Mechanisms by which gut microorganisms influence food sensitivities.

Finely tuned mechanisms enable the gastrointestinal tract to break down dietary components into nutrients without mounting, in the majority of cases, a dysregulated immune or functional host response. However, adverse reactions to food have been steadily increasing, and evidence suggests that this process is environmental. Adverse food reactions can be divided according to their underlying pathophysiology into food intolerances, when, for instance, there is deficiency of a host enzyme required to digest the food component, and food sensitivities, when immune mechanisms are involved. In this Review, we discuss the clinical and experimental evidence for enteric infections and/or alterations in the gut microbiota in inciting food sensitivity. We focus on mechanisms by which microorganisms might provide direct pro-inflammatory signals to the host promoting breakdown of oral tolerance to food antigens or indirect pathways that involve the metabolism of protein antigens and other dietary components by gut microorganisms. Better understanding of these mechanisms will help in the development of preventive and therapeutic strategies for food sensitivities.

Microbial signals drive pre-leukaemic myeloproliferation in a Tet2-deficient host.

Somatic mutations in tet methylcytosine dioxygenase 2 (TET2), which encodes an epigenetic modifier enzyme, drive the development of haematopoietic malignancies1-7. In both humans and mice, TET2 deficiency leads to increased self-renewal of haematopoietic stem cells with a net developmental bias towards the myeloid lineage1,4,8,9. However, pre-leukaemic myeloproliferation (PMP) occurs in only a fraction of Tet2-/- mice8,9 and humans with TET2 mutations1,3,5-7, suggesting that extrinsic non-cell-autonomous factors are required for disease onset. Here we show that bacterial translocation and increased interleukin-6 production, resulting from dysfunction of the small-intestinal barrier, are critical for the development of PMP in mice that lack Tet2 expression in haematopoietic cells. Furthermore, in symptom-free Tet2-/- mice, PMP can be induced by disrupting intestinal barrier integrity, or in response to systemic bacterial stimuli such as the toll-like receptor 2 agonist. PMP was reversed by antibiotic treatment and failed to develop in germ-free Tet2-/- mice, which illustrates the importance of microbial signals in the development of this condition. Our findings demonstrate the requirement for microbial-dependent inflammation in the development of PMP and provide a mechanistic basis for the variation in PMP penetrance observed in Tet2-/- mice. This study will prompt new lines of investigation that may profoundly affect the prevention and management of haematopoietic malignancies.

Natural polyreactive IgA antibodies coat the intestinal microbiota.

Large quantities of immunoglobulin A (IgA) are constitutively secreted by intestinal plasma cells to coat and contain the commensal microbiota, yet the specificity of these antibodies remains elusive. Here we profiled the reactivities of single murine IgA plasma cells by cloning and characterizing large numbers of monoclonal antibodies. IgAs were not specific to individual bacterial taxa but rather polyreactive, with broad reactivity to a diverse, but defined, subset of microbiota. These antibodies arose at low frequencies among naïve B cells and were selected into the IgA repertoire upon recirculation in Peyer's patches. This selection process occurred independent of microbiota or dietary antigens. Furthermore, although some IgAs acquired somatic mutations, these did not substantially influence their reactivity. These findings reveal an endogenous mechanism driving homeostatic production of polyreactive IgAs with innate specificity to microbiota.

Antibiotic-induced perturbations in microbial diversity during post-natal development alters amyloid pathology in an aged APPSWE/PS1ΔE9 murine model of Alzheimer's disease.

Recent evidence suggests the commensal microbiome regulates host immunity and influences brain function; findings that have ramifications for neurodegenerative diseases. In the context of Alzheimer's disease (AD), we previously reported that perturbations in microbial diversity induced by life-long combinatorial antibiotic (ABX) selection pressure in the APPSWE/PS1ΔE9 mouse model of amyloidosis is commensurate with reductions in amyloid-ß (Aß) plaque pathology and plaque-localised gliosis. Considering microbiota-host interactions, specifically during early post-natal development, are critical for immune- and neuro-development we now examine the impact of microbial community perturbations induced by acute ABX exposure exclusively during this period in APPSWE/PS1ΔE9 mice. We show that early post-natal (P) ABX treatment (P14-P21) results in long-term alterations of gut microbial genera (predominantly Lachnospiraceae and S24-7) and reduction in brain Aß deposition in aged APPSWE/PS1ΔE9 mice. These mice exhibit elevated levels of blood- and brain-resident Foxp3+ T-regulatory cells and display an alteration in the inflammatory milieu of the serum and cerebrospinal fluid. Finally, we confirm that plaque-localised microglia and astrocytes are reduced in ABX-exposed mice. These findings suggest that ABX-induced microbial diversity perturbations during post-natal stages of development coincide with altered host immunity mechanisms and amyloidosis in a murine model of AD.