T cells in harmony: Aligning the TCR repertoire pool to identify microbiota recognizing T cells.

Microbiota-specific T cell responses have been identified for select microbes, but individual T cell receptor repertoire differences make characterizing responses across populations difficult. In this issue of Immunity, Muschaweck et al. establish a system allowing for reproducible responses between individual mice, a powerful tool for characterizing microbiota directed immunity.

Identifying the Patterns of Pattern Recognition Receptors.

Intestinal homeostasis requires microbial recognition that results in appropriate responses to commensals and pathogens. In this issue of Immunity, Price et al. (2018) map the in vivo expression of five toll-like receptors (TLR) in intestinal epithelia, revealing distinct spatio-temporal expression patterns that shape responses to TLR ligands.

Critical Role for the Microbiota in CX3CR1+ Intestinal Mononuclear Phagocyte Regulation of Intestinal T Cell Responses.

The intestinal barrier is vulnerable to damage by microbiota-induced inflammation that is normally restrained through mechanisms promoting homeostasis. Such disruptions contribute to autoimmune and inflammatory diseases including inflammatory bowel disease. We identified a regulatory loop whereby, in the presence of the normal microbiota, intestinal antigen-presenting cells (APCs) expressing the chemokine receptor CX3CR1 reduced expansion of intestinal microbe-specific T helper 1 (Th1) cells and promoted generation of regulatory T cells responsive to food antigens and the microbiota itself. We identified that disruption of the microbiota resulted in CX3CR1+ APC-dependent inflammatory Th1 cell responses with increased pathology after pathogen infection. Colonization with microbes that can adhere to the epithelium was able to compensate for intestinal microbiota loss, indicating that although microbial interactions with the epithelium can be pathogenic, they can also activate homeostatic regulatory mechanisms. Our results identify a cellular mechanism by which the microbiota limits intestinal inflammation and promotes tissue homeostasis.

Microbiota-Induced TNF-like Ligand 1A Drives Group 3 Innate Lymphoid Cell-Mediated Barrier Protection and Intestinal T Cell Activation during Colitis.

Inflammatory bowel disease (IBD) results from a dysregulated interaction between the microbiota and a genetically susceptible host. Genetic studies have linked TNFSF15 polymorphisms and its protein TNF-like ligand 1A (TL1A) with IBD, but the functional role of TL1A is not known. Here, we found that adherent IBD-associated microbiota induced TL1A release from CX3CR1+ mononuclear phagocytes (MNPs). Using cell-specific genetic deletion models, we identified an essential role for CX3CR1+MNP-derived TL1A in driving group 3 innate lymphoid cell (ILC3) production of interleukin-22 and mucosal healing during acute colitis. In contrast to this protective role in acute colitis, TL1A-dependent expression of co-stimulatory molecule OX40L in MHCII+ ILC3s during colitis led to co-stimulation of antigen-specific T cells that was required for chronic T cell colitis. These results identify a role for ILC3s in activating intestinal T cells and reveal a central role for TL1A in promoting ILC3 barrier immunity during colitis.

Thymic development of gut-microbiota-specific T cells.

Humans and their microbiota have coevolved a mutually beneficial relationship in which the human host provides a hospitable environment for the microorganisms and the microbiota provides many advantages for the host, including nutritional benefits and protection from pathogen infection1. Maintaining this relationship requires a careful immune balance to contain commensal microorganisms within the lumen while limiting inflammatory anti-commensal responses1,2. Antigen-specific recognition of intestinal microorganisms by T cells has previously been described3,4. Although the local environment shapes the differentiation of effector cells3-5 it is unclear how microbiota-specific T cells are educated in the thymus. Here we show that intestinal colonization in early life leads to the trafficking of microbial antigens from the intestine to the thymus by intestinal dendritic cells, which then induce the expansion of microbiota-specific T cells. Once in the periphery, microbiota-specific T cells have pathogenic potential or can protect against related pathogens. In this way, the developing microbiota shapes and expands the thymic and peripheral T cell repertoire, allowing for enhanced recognition of intestinal microorganisms and pathogens.

Bona fide dendritic cells are pivotal precursors for osteoclasts.

OBJECTIVES: Osteoclasts (OCs) are myeloid-derived multinucleated cells uniquely able to degrade bone. However, the exact nature of their myeloid precursors is not yet defined. METHODS: CD11c-diphtheria toxin receptor (CD11cDTR) transgenic mice were treated with diphtheria toxin (DT) or phosphate buffered saline (PBS) during serum transfer arthritis (STA) and human tumour necrosis factor transgenic (hTNFtg) arthritis and scored clinically and histologically. We measured cytokines in synovitis by quantitative polymerase chain reaction (qPCR). We performed ovariectomy in CD11cDTR mice treated with PBS or DT. We analysed CD11cDTR, CD11c-Cre/CX3CR1-STOP-DTR and Zbtb46-DTR-treated mice with DT using histomorphometry and OC of CD11c and Zbtb46 fate reporter mice by fluorescent imaging. We sorted murine and human OC precursors and stimulated them with macrophage colony-stimulating factor (M-CSF) and receptor activator of nuclear factor kappa-B ligand (RANKL) to generate OCs. RESULTS: Targeting CD11c+ cells in vivo in models of inflammatory arthritis (STA and hTNFtg) ameliorates arthritis by reducing inflammatory bone destruction and OC generation. Targeting CD11c-expressing cells in unchallenged mice removes all OCs in their long bones. OCs do not seem to be derived from CD11c+ cells expressing CX3CR1+, but from Zbtb46+conventional dendritic cells (cDCs) as all OCs in Zbtb46-Tomato fate reporter mice are Tomato+. In line, administration of DT in Zbtb46-DTR mice depletes all OCs in long bones. Finally, human CD1c-expressing cDCs readily differentiated into bone resorbing OCs. CONCLUSION: Taken together, we identify DCs as important OC precursors in bone homeostasis and inflammation, which might open new avenues for therapeutic interventions in OC-mediated diseases.

Skin IL-17-Producing T Cells Support Repair 2!

In a recent study, Harrison et al. (Science 2019;363;eaat6280) report that RORγt-expressing skin commensal-specific T cells rapidly respond to tissue wounding by producing type 2 T helper cell (Th2) cytokines in mice. The cells constitutively coexpress GATA-3 and type 2 cytokine mRNAs that are translated after injury. These T cells act as sentinels, linking T cell receptor (TCR) recognition of commensals, tissue damage, and wound repair.

Interleukin 23 production by intestinal CD103(+)CD11b(+) dendritic cells in response to bacterial flagellin enhances mucosal innate immune defense.

Microbial penetration of the intestinal epithelial barrier triggers inflammatory responses that include induction of the bactericidal C-type lectin RegIIIγ. Systemic administration of flagellin, a bacterial protein that stimulates Toll-like receptor 5 (TLR5), induces epithelial expression of RegIIIγ and protects mice from intestinal colonization with antibiotic-resistant bacteria. Flagellin-induced RegIIIγ expression is IL-22 dependent, but how TLR signaling leads to IL-22 expression is incompletely defined. By using conditional depletion of lamina propria dendritic cell (LPDC) subsets, we demonstrated that CD103(+)CD11b(+) LPDCs, but not monocyte-derived CD103(-)CD11b(+) LPDCs, expressed high amounts of IL-23 after bacterial flagellin administration and drove IL-22-dependent RegIIIγ production. Maximal expression of IL-23 subunits IL-23p19 and IL-12p40 occurred within 60 min of exposure to flagellin. IL-23 subsequently induced a burst of IL-22 followed by sustained RegIIIγ expression. Thus, CD103(+)CD11b(+) LPDCs, in addition to promoting long-term tolerance to ingested antigens, also rapidly produce IL-23 in response to detection of flagellin in the lamina propria.

Microbiota restricts trafficking of bacteria to mesenteric lymph nodes by CX(3)CR1(hi) cells.

The intestinal microbiota has a critical role in immune system and metabolic homeostasis, but it must be tolerated by the host to avoid inflammatory responses that can damage the epithelial barrier separating the host from the luminal contents. Breakdown of this regulation and the resulting inappropriate immune response to commensals are thought to lead to the development of inflammatory bowel diseases such as Crohn's disease and ulcerative colitis. We proposed that the intestinal immune system is instructed by the microbiota to limit responses to luminal antigens. Here we demonstrate in mice that, at steady state, the microbiota inhibits the transport of both commensal and pathogenic bacteria from the lumen to a key immune inductive site, the mesenteric lymph nodes (MLNs). However, in the absence of Myd88 or under conditions of antibiotic-induced dysbiosis, non-invasive bacteria were trafficked to the MLNs in a CCR7-dependent manner, and induced both T-cell responses and IgA production. Trafficking was carried out by CX(3)CR1(hi) mononuclear phagocytes, an intestinal-cell population previously reported to be non-migratory. These findings define a central role for commensals in regulating the migration to the MLNs of CX(3)CR1(hi) mononuclear phagocytes endowed with the ability to capture luminal bacteria, thereby compartmentalizing the intestinal immune response to avoid inflammation.

Fas-associated death domain (FADD) is a negative regulator of T-cell receptor-mediated necroptosis.

Cell death is an important mechanism to limit uncontrolled T-cell expansion during immune responses. Given the role of death-receptor adapter protein Fas-associated death domain (FADD) in apoptosis, it is intriguing that T-cell receptor (TCR)-induced proliferation is blocked in FADD-defective T cells. Necroptosis is an alternate form of death that can be induced by death receptors and is linked to autophagy. It requires the death domain-containing kinase RIP1 and, in certain instances, RIP3. FADD and its apoptotic partner, Caspase-8, have also been implicated in necroptosis. To accurately assess the role of FADD in mature T-cell proliferation and death, we generated a conditional T-cell-specific FADD knockout mouse strain. The T cells of these mice develop normally, but lack FADD at the mature stage. FADD-deficient T cells respond poorly to TCR triggering, exhibit slow cell cycle entry, and fail to expand over time. We find that programmed necrosis occurs during the late stage of normal T-cell proliferation and that this process is greatly amplified in FADD-deficient T cells. Inhibition of necroptosis using an inhibitor of RIP1 kinase activity rescues the FADD knockout proliferative defect. However, TCR-induced necroptosis did not appear to require autophagy or involve RIP3. Consistent with their defective CD8 T-cell response, these mice succumb to Toxoplasma gondii infection more readily than wild-type mice. We conclude that FADD constitutes a mechanism to keep TCR-induced programmed necrotic signaling in check during early phases of T-cell clonal expansion.

Acute high-fat diet impairs macrophage-supported intestinal damage resolution.

Chronic exposure to high-fat diets (HFD) worsens intestinal disease pathology, but acute effects of HFD in tissue damage remain unclear. Here, we used short-term HFD feeding in a model of intestinal injury and found sustained damage with increased cecal dead neutrophil accumulation, along with dietary lipid accumulation. Neutrophil depletion rescued enhanced pathology. Macrophages from HFD-treated mice showed reduced capacity to engulf dead neutrophils. Macrophage clearance of dead neutrophils activates critical barrier repair and antiinflammatory pathways, including IL-10, which was lost after acute HFD feeding and intestinal injury. IL-10 overexpression restored intestinal repair after HFD feeding and intestinal injury. Macrophage exposure to lipids from the HFD prevented tethering and uptake of apoptotic cells and Il10 induction. Milk fat globule-EGF factor 8 (MFGE8) is a bridging molecule that facilitates macrophage uptake of dead cells. MFGE8 also facilitates lipid uptake, and we demonstrate that dietary lipids interfere with MFGE8-mediated macrophage apoptotic neutrophil uptake and subsequent Il10 production. Our findings demonstrate that HFD promotes intestinal pathology by interfering with macrophage clearance of dead neutrophils, leading to unresolved tissue damage.

Intestinal toxicity to CTLA-4 blockade driven by IL-6 and myeloid infiltration.

Immune checkpoint blockade (ICB) has revolutionized cancer treatment, yet quality of life and continuation of therapy can be constrained by immune-related adverse events (irAEs). Limited understanding of irAE mechanisms hampers development of approaches to mitigate their damage. To address this, we examined whether mice gained sensitivity to anti-CTLA-4 (αCTLA-4)-mediated toxicity upon disruption of gut homeostatic immunity. We found αCTLA-4 drove increased inflammation and colonic tissue damage in mice with genetic predisposition to intestinal inflammation, acute gastrointestinal infection, transplantation with a dysbiotic fecal microbiome, or dextran sodium sulfate administration. We identified an immune signature of αCTLA-4-mediated irAEs, including colonic neutrophil accumulation and systemic interleukin-6 (IL-6) release. IL-6 blockade combined with antibiotic treatment reduced intestinal damage and improved αCTLA-4 therapeutic efficacy in inflammation-prone mice. Intestinal immune signatures were validated in biopsies from patients with ICB colitis. Our work provides new preclinical models of αCTLA-4 intestinal irAEs, mechanistic insights into irAE development, and potential approaches to enhance ICB efficacy while mitigating irAEs.

Microbiota manipulation to increase macrophage IL-10 improves colitis and limits colitis-associated colorectal cancer.

Inflammatory bowel disease (IBD) is a chronic life-long inflammatory disease affecting almost 2 million Americans. Although new biologic therapies have been developed, the standard medical treatment fails to selectively control the dysregulated immune pathways involved in chronic colonic inflammation. Further, IBD patients with uncontrolled colonic inflammation are at a higher risk for developing colorectal cancer (CRC). Intestinal microbes can impact many immune functions, and here we asked if they could be used to improve intestinal inflammation. By utilizing an intestinal adherent E. coli that we find increases IL-10 producing macrophages, we were able to limit intestinal inflammation and restrict tumor formation. Macrophage IL-10 along with IL-10 signaling to the intestinal epithelium were required for protection in both inflammation and tumor development. Our work highlights that administration of immune modulating microbes can improve intestinal outcomes by altering tissue inflammation.

Interleukin-1ß secretion induced by mucosa-associated gut commensal bacteria promotes intestinal barrier repair.

The gut microbiota is essential for maintenance and repair of the intestinal epithelial barrier. As shifts in both intestinal epithelial barrier function and microbiota composition are found in inflammatory bowel disease patients, it is critical to understand the role of distinct bacteria in regulating barrier repair. We identified a mouse commensal E. coli isolate, GDAR2-2, that protects mice from Citrobacter rodentium infection and dextran sulfate sodium-induced colitis. Colonization with GDAR2-2 in mice resulted in expansion of CX3CR1+ mononuclear phagocytes, including CX3CR1+ macrophages/dendritic cells and monocytes, along with IL-22-secreting type 3 innate lymphoid cells and improved epithelial barrier function. In vitro co-culture of macrophages with GDAR2-2 resulted in IL-1ß production. In vivo, protection after GDAR2-2 colonization was lost after depletion of CX3CR1+ MNPs, or blockade of IL-1ß or IL-22. We further identified human commensal E. coli isolates that similarly protect mice from C. rodentium infection through CX3CR1+ MNP and IL-1ß production. Together, these findings demonstrate an unexpected role for commensal bacteria in promoting IL-1ß secretion to support intestinal barrier repair.

Healthy Intestinal Function Relies on Coordinated Enteric Nervous System, Immune System, and Epithelium Responses.

During both health and disease, a coordinated response between the epithelium, immune system, and enteric nervous system is required for proper intestinal function. While each system responds to a number of common stimuli, their coordinated responses support digestion as well as responses and recovery following injury or pathogenic infections. In this review, we discuss how individual responses to common signals work together to support these critical functions.

Adherent-invasive E. coli metabolism of propanediol in Crohn's disease regulates phagocytes to drive intestinal inflammation.

Adherent-invasive E. coli (AIEC) are enriched in the intestinal microbiota of patients with Crohn's disease (CD) and promote intestinal inflammation. Yet, how AIEC metabolism of nutrients impacts intestinal homeostasis is poorly defined. Here, we show that AIEC encoding the large subunit of propanediol dehydratase (PduC), which facilitates the utilization of fucose fermentation product 1,2-propanediol, are increased in the microbiome of CD patients and drive AIEC-induced intestinal T cell inflammation. In murine models, CX3CR1+ mononuclear phagocytes (MNP) are required for PduC-dependent induction of T helper 17 (Th17) cells and interleukin-1ß (IL-1ß) production that leads to AIEC-induced inflammatory colitis. Activation of this inflammatory cascade requires the catalytic activity of PduC to generate propionate, which synergizes with lipopolysaccharide (LPS) to induce IL-1ß by MNPs. Disrupting fucose availability limits AIEC-induced propionate production and intestinal inflammation. These findings identify MNPs as metabolic sensors linking AIEC metabolism with intestinal inflammation and identify microbial metabolism as a potential therapeutic target in Crohn's disease treatment.

Intestinal Microbes in Autoimmune and Inflammatory Disease.

Autoimmune diseases and chronic inflammatory disorders are characterized by dysregulated immune responses resulting in excessive and uncontrolled tissue inflammation. Multiple factors including genetic variation, environmental stimuli, and infection are all thought to contribute to continued inflammation and pathology. Current evidence supports the microbiota as one such factor with emerging data linking commensal organisms to the onset and progression of disease. In this review, we will discuss links between the microbiota and specific diseases as well as highlight common pathways that link intestinal microbes with multiple autoimmune and inflammatory diseases.

Nramp1 expression by dendritic cells modulates inflammatory responses during Salmonella Typhimurium infection.

Host resistance against Salmonella enterica serovar Typhimurium (S. Typhimurium) is mediated by natural resistance-associated macrophage protein 1 (Nramp1/Slc11a1). Nramp1 is critical to host defence, as mice lacking Nramp1 fail to control bacterial replication and succumb to low doses of S. Typhimurium. Despite this crucial role, the mechanisms underlying Nramp1's protective effects are unclear. Dendritic cells (DCs) that sample the intestinal lumen are among the first cells encountered by S. Typhimurium following oral infection and act as a conduit for S. Typhimurium to cross the intestinal epithelial barrier. We report that DCs, including intestinal, splenic and bone marrow-derived DCs (BMDCs), express Nramp1 protein. In the small intestine, Nramp1 expression is greater in a subset of DCs (CD11c(+)CD103(-)) characterized by the elevated expression of pro-inflammatory cytokines in response to bacterial products. While Nramp1 expression did not affect S. Typhimurium replication in BMDCs, infected Nramp1+/+ BMDCs and intestinal CD11c(+)CD103(-) DCs secreted more inflammatory cytokines (IL-6, IL-12 and TNF-alpha) than Nramp1-/-, suggesting that Nramp1 expression may promote a more rapid inflammatory response following infection. Collectively, these findings reveal a new role for DCs and Nramp1 in modulating the host inflammatory response to S. Typhimurium.

Intestinal microbes direct CX3CR1+ cells to balance intestinal immunity.

Intestinal damage driven by unrestricted immune responses against the intestinal microbiota can lead to the development of inflammatory diseases including inflammatory bowel disease. How such breakdown in tolerance occurs alongside the mechanisms to reinforce homeostasis with the microbiota are a focus of many studies. Our recent work demonstrates coordinated interactions between intact microbiota and CX3CR1 expressing intestinal antigen presenting cells (APCs) that limits T helper 1 cell responses and promotes differentiation of regulatory T cells (Treg) against intestinal antigens including pathogens, soluble proteins and the microbiota itself. We find a microbial attachment to intestinal epithelial cells is necessary to support these anti-inflammatory immune functions. In this addendum, we discuss how our findings enhance understanding of microbiota-directed homeostatic functions of the intestinal immune system and implications of modulating this interaction in ameliorating inflammatory disease.

Cigarette Smoke Induces Intestinal Inflammation via a Th17 Cell-Neutrophil Axis.

Epidemiological evidence finds cigarette smoking is a common risk factor for a number of diseases, not only in the lung but also in other tissues, such as the gastrointestinal tract. While it is well-documented that smoking directly drives lung inflammatory disease, how it promotes disease in peripheral tissues is incompletely understood. In this study, we utilized a mouse model of short-term smoke exposure and found increased Th17 cells and neutrophilia in the lung as well as in the circulation. Following intestinal inflammatory challenge, smoke exposed mice showed increased pathology which corresponds to enhanced intestinal Th17 cells, ILC3 and neutrophils within intestinal tissue. Using cellular depletion and genetic deficiencies, we define a cellular loop by which IL-17A and downstream neutrophils drive cigarette smoke-enhanced intestinal inflammation. Collectively, cigarette smoke induced local lung Th17 responses lead to increased systemic susceptibility to inflammatory insult through enhanced circulating neutrophils. These data demonstrate a cellular pathway by which inflammatory challenge in the lung can sensitize the intestine to enhanced pathological innate and adaptive immune responses.