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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 anti-inflammatory pathways including IL10, which was lost after acute HFD feeding and intestinal injury. IL10 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.

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-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.

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 CX(3)CR1 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.

Immunotherapies such as anti-CTLA-4 immune checkpoint blockade (ICB) have revolutionized cancer treatment, yet quality of life and continuation of therapy can be constrained by off-target tissue damage or immune-related adverse events (irAEs). At present, there is limited understanding of irAE mechanisms, hampering development of approaches to mitigate their damage. We addressed this problem by generating animal models of intestinal irAE. Our results show that disruption of homeostatic immunity by genetic predisposition to intestinal inflammation or acute gastrointestinal infection sensitizes mice to anti-CTLA-4-mediated intestinal toxicity. Inflammation-prone mice treated with anti-CTLA-4 showed neutrophil accumulation, systemic interleukin-6 (IL-6) release, and dysbiosis. Significantly, IL-6 blockade combined with antibiotic treatment improved anti-CTLA-4 therapeutic efficacy and reduced intestinal irAEs. Immune signatures were validated in biopsies from patients who developed colitis during ICB, supporting the utility of our models. This study provides new pre-clinical models, mechanistic insight into irAEs, and potential approaches to enhance ICB efficacy while mitigating irAEs. Citation Format: Yifan Zhou, Yusra B. Medik, Bhakti Patel, Daniel B. Zamler, Sijie Chen, Thomas Chapman, Sarah Schneider, Rachel L. Babcock, Taylor T. Chrisikos, Laura M. Kahn, Allison M. Dyevoich, Elizabeth M. Park, Alexandria P. Cogdill, Daniel H. Johnson, Sarah B. Johnson, Khalida M. Wani, Debora A. Ledesma, Courtney W. Hudgens, Jingjing Wang, Md Abdul Wadud Khan, Aron Y. Joon, Weiyi Peng, Haiyan S. Li, Reetakshi Arora, Ximing Tang, Maria Gabriela Raso, Xuegong Zhang, Wai Chin Foo, Michael T. Tetzlaff, Gretchen E. Diehl, Karen Clise-Dwyer, Elizabeth M. Whitley, Matthew M. Gubin, James P. Allison, Patrick Hwu, Nadim J. Ajami, Adi Diab, Jennifer A. Wargo, Stephanie S. Watowich. Intestinal toxicity to CTLA-4 blockade driven by IL-6 and myeloid infiltration [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5545.

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.

While exposure to microbial antigens in peripheral sites causes expansion of antigen-specific T cells, a role for antigen-specific expansion had not been identified during T cell development in the thymus. We recently found that intestinal colonization induced intestinal dendritic cells migration to the thymus, driving thymic expansion of microbiota-specific T cells early in life. In contrast, colonization in adulthood leads to expansion of peripheral but not thymic microbiota-specific T cells. Thymic microbiota-specific T cells developed early in life were undifferentiated and caused intestinal inflammation in immunocompromised hosts upon antigen reencounter. We sought to understand regulation of this developmental window and define if thymic expansion of microbiota-specific T cells could be restarted in adult mice. We found that microbiota manipulation reestablished expansion of microbiota-specific T cells in the thymus of adult mice. We are in the process of characterizing these T cells in adulthood to assess their impact on intestinal inflammation. Our data suggests that intestinal colonization controls the generation of thymic microbiota-specific T cells which has the potential to modulate inflammatory processes.

Inflammatory bowel disease (IBD) patients with poorly controlled intestinal inflammation are at an elevated risk for colorectal cancer (CRC). IBD patients exhibit intestinal dysbiosis with expanded proteobacteria such as E. coli. Here, we find that colonization with an E. coli isolated from the intestine of an IBD patient (E. coli 541-15) prevents tumorigenesis in an inflammation-related model of CRC. Colonization increased tumor infiltration of T helper 1 (Th1) cells, cytotoxic T lymphocytes (CTLs), and type 1 innate lymphoid cells (ILC1s) and decreased myeloid derived suppressor cells (MDSCs) and regulatory T cells (Tregs). Prevention of tumorigenesis occurs if colonization takes place before induction of inflammation. Intestinal inflammation in colitis models was ameliorated by E. coli 541-15 and this protection depended on IL-10 production by macrophages and IL-10 signaling to the intestinal epithelium. Colonization with E. coli 541-15 also promotes these IL-10 pathways if colonization occurs after tumorigenesis is established. However, this leads to worse CRC outcome, with increased tumor burden alongside decreased tumor infiltration of Th1 cells, CTLs, and ILC1s and increased MDSCs and Tregs. These results identify activation of an IL-10 signaling loop between immune cells and the intestinal epithelium after E. coli colonization that modulates intestinal inflammation and CRC. Importantly, these pathways can be protective or pathogenic depending on timing of activation. Supported by 2021 Ludwig Center Basic and Translational Research Award

Microbiota interactions with host tissue support proper intestinal functions. In diseases such as inflammatory bowel disease, microbial shift along with epithelial dysfunctions are found. Understanding how intestinal epithelial functions are regulated by distinct commensal bacteria is key to improving outcomes in such diseases. Here, we identified a mouse commensal E. coli isolate, GDAR2-2, that promotes intestinal barrier repair in mouse colitis models including Citrobacter rodentium infection and dextran sulfate sodium-induced colitis. In mice, protection after GDAR2-2 colonization depends on CX3CR1+ mononuclear phagocytes (MNPs), which induce expansion of IL-22-secreting type 3 innate lymphoid cells. We detect upregulation of markers of transit amplifying cells, proliferation of which is downstream of IL-22 signaling. Protection is lost if IL-22 is blocked, indicating IL-22 driven epithelial regeneration is downstream of GDAR2-2 colonization. By co-culturing bone marrow derived macrophages with live GDAR2-2, we found GDAR2-2 promotes IL-1b production and the in vivo protection was lost after blockade of IL-1b. We further identified a subset of human commensal E. coli isolates that similarly induce CX3CR1+ MNP expansion and IL-1b mediated protection from C. rodentium infection in mice. This study reveals an unexpected role for IL-1b that promotes intestinal barrier repair and is induced by select commensal bacteria. Supported by NIH (AI125264, P30DK056338)

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. In young mice, antigens from the gut microbiota are trafficked by CX3CR1+ dendritic cells from the gut to the thymus, where they induce the expansion of T cells that are specific to commensal microorganisms.

Somatic mutations can promote malignant transformation of airway epithelial cells and induce inflammatory responses directed against resultant tumors. Tumor-infiltrating T lymphocytes (TIL) in early-stage non–small cell lung cancer (NSCLC) secrete distinct proinflammatory cytokines, but the contribution of these TILs to tumor development and metastasis remains unknown. We show here that TILs in early-stage NSCLC are biased toward IL17A expression (Th17) when compared with adjacent tumor-free tissue, whereas Th17 cells are decreased in tumor infiltrating locoregional lymph nodes in advanced NSCLC. Mice in which Pten and Smad4 (Pts4d/d) are deleted from airway epithelial cells develop spontaneous tumors, that share genetic signatures with squamous- (SQ.2b), and adeno- (AD.1) subtypes of human NSCLC. Pts4d/d mice globally lacking in IL17a (Pts4d/dIl17a–/–) showed decreased tumor latency and increased metastasis. Th17 cells were required for recruitment of CD103+ dendritic cells, and adoptive transfer of IL17a-sufficient CD4+ T cells reversed early tumor development and metastasis in Pts4d/dIl17a–/– mice. Together, these findings support a key role for Th17 cells in TILs associated with the Pts4d/d model of NSCLC and suggest therapeutic and biomarker strategies for human SQ2b and AD1 lung cancer. Cancer Immunol Res; 6(6); 645–57. ©2018 AACR.

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.

Airborne pathogens commonly trigger severe respiratory failure or death in smokers with lung disease. Cigarette smoking compromises the effectiveness of innate immunity against infections but the underlying mechanisms responsible for defective acquired immune responses in smokers remains less clear. We found that mice exposed to chronic cigarette smoke recovered poorly from primary Influenza A pneumonia with reduced type I and II interferons (IFNs) and viral-specific immunoglobulins, but recruited γδ T cells to the lungs that predominantly expressed interleukin 17A (IL-17A). Il-17a−/− mice exposed to smoke and infected with Influenza A also recruited γδ T cells to the lungs, but in contrast to wild-type mice, expressed increased IFNs, made protective influenza-specific antibodies, and recovered from infection. Depletion of IL-17A with blocking antibodies significantly increased T-bet expression in γδ T cells and improved recovery from acute Influenza A infection in air, but not smoke-exposed mice. In contrast, when exposed to smoke, γδ T cell deficient mice failed to mount an effective immune response to Influenza A and showed increased mortality. Our findings demonstrate a protective role for γδ T cells in smokers and suggest that smoke-induced increase in IL-17A inhibits the transcriptional programs required for their optimal anti-viral responses. Cigarette smoke induces IL-17A expression in the lungs and inhibits γδ T-cell-mediated protective anti-viral immune responses.

Signaling by Toll-like receptors (TLRs) on intestinal epithelial cells (IECs) is critical for intestinal homeostasis. To visualize epithelial expression of individual TLRs in vivo, we generated five strains of reporter mice. These mice revealed that TLR expression varied dramatically along the length of the intestine. Indeed, small intestine (SI) IECs expressed low levels of multiple TLRs that were highly expressed by colonic IECs. TLR5 expression was restricted to Paneth cells in the SI epithelium. Intestinal organoid experiments revealed that TLR signaling in Paneth cells or colonic IECs induced a core set of host defense genes, but this set did not include antimicrobial peptides, which instead were induced indirectly by inflammatory cytokines. This comprehensive blueprint of TLR expression and function in IECs reveals unexpected diversity in the responsiveness of IECs to microbial stimuli, and together with the associated reporter strains, provides a resource for further study of innate immunity.

Defective tissue repair is a hallmark of many inflammatory disorders including, inflammatory bowel disease (IBD). While it is clear that high fat diets (HFD) can exacerbate inflammatory disease by increasing inflammation, the direct effect of lipids on tissue homeostasis and repair remains undefined. We show here that short term exposure to HFD directly impairs barrier repair after intestinal epithelial damage by interfering with recognition and uptake of apoptotic neutrophils by intestinal macrophages. Apoptotic neutrophil uptake induces macrophage IL-10 production, which is lacking after intestinal damage in the context of HFD. Overexpression of IL-10 rescues repair defects after HFD treatment, but not if epithelial cells lack the IL-10 receptor, highlighting the key role of IL-10 in barrier repair. These findings demonstrate a previously unidentified mechanism by which dietary lipids directly interfere with homeostatic processes required to maintain tissue integrity.

Alteration of innate immune cells in the lungs can promote loss of peripheral tolerance that leads to autoimmune responses in cigarette smokers. Development of autoimmunity in smokers with emphysema is also strongly linked to the expansion of autoreactive T helper (Th) cells expressing interferon γ (Th1), and interleukin 17A (Th17). However, the mechanisms responsible for enhanced self-recognition and reduced immune tolerance in smokers with emphysema remain less clear. Here we show that C1q, a component of the complement protein 1 complex (C1), is downregulated in lung CD1a(+) antigen-presenting cells (APCs) isolated from emphysematous human and mouse lung APCs after chronic cigarette smoke exposure. C1q potentiated the function of APCs to differentiate CD4(+) T cells to regulatory T cells (Tregs), while it inhibited Th17 cell induction and proliferation. Mice deficient in C1q that were exposed to chronic smoke exhibited exaggerated lung inflammation marked by increased Th17 cells, whereas reconstitution of C1q in the lungs enhanced Treg abundance, dampened smoke-induced lung inflammation, and prevented the development of emphysema. Our findings demonstrate that cigarette smoke–mediated loss of C1q could play a key role in reduced peripheral tolerance, which could be explored to treat emphysema.

Summary 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-1β (IL-1β) 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-1β 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.

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 CX

Peripheral spondyloarthritis (SpA) is a common extraintestinal manifestation in patients with active inflammatory bowel disease (IBD) characterized by inflammatory enthesitis, dactylitis, or synovitis of nonaxial joints. However, a mechanistic understanding of the link between intestinal inflammation and SpA has yet to emerge. We evaluated and functionally characterized the fecal microbiome of IBD patients with or without peripheral SpA. Coupling the sorting of immunoglobulin A (IgA)-coated microbiota with 16S ribosomal RNA-based analysis (IgA-seq) revealed a selective enrichment in IgA-coated Escherichia coli in patients with Crohn's disease-associated SpA (CD-SpA) compared to CD alone. E. coli isolates from CD-SpA-derived IgA-coated bacteria were similar in genotype and phenotype to an adherent-invasive E. coli (AIEC) pathotype. In comparison to non-AIEC E. coli, colonization of germ-free mice with CD-SpA E. coli isolates induced T helper 17 cell (TH17) mucosal immunity, which required the virulence-associated metabolic enzyme propanediol dehydratase (pduC). Modeling the increase in mucosal and systemic TH17 immunity we observed in CD-SpA patients, colonization of interleukin-10-deficient or K/BxN mice with CD-SpA-derived E. coli lead to more severe colitis or inflammatory arthritis, respectively. Collectively, these data reveal the power of IgA-seq to identify immunoreactive resident pathosymbionts that link mucosal and systemic TH17-dependent inflammation and offer microbial and immunophenotype stratification of CD-SpA that may guide medical and biologic therapy.

Interleukin (IL)-22–producing group 3 innate lymphoid cells (ILC3) promote mucosal healing and maintain barrier integrity, but how microbial signals are integrated to regulate mucosal protection offered by these cells remains unclear. Here, we show that in vivo depletion of CX3CR1+ mononuclear phagocytes (MNPs) resulted in more severe colitis and death after infection with Citrobacter rodentium. This phenotype was rescued by exogenous IL-22, which was endogenously produced by ILC3 in close spatial proximity to CX3CR1+ MNPs that were dependent on MyD88 signaling. CX3CR1+ MNPs from both mouse and human tissue produced more IL-23 and IL-1β than conventional CD103+ dendritic cells (cDCs) and were more efficient than cDCs in supporting IL-22 production in ILC3 in vitro and in vivo. Further, colonic ILC3 from patients with mild to moderate ulcerative colitis or Crohn’s disease had increased IL-22 production. IBD-associated SNP gene set analysis revealed enrichment for genes selectively expressed in human intestinal MNPs. The product of one of these, TL1A, potently enhanced IL-23– and IL-1β-induced production of IL-22 and GM-CSF by ILC3. Collectively, these results reveal a critical role for CX3CR1+ mononuclear phagocytes in integrating microbial signals to regulate colonic ILC3 function in IBD.

Summary 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.

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.

The vertebrate intestinal tract is colonized by hundreds of species of bacteria that must be compartmentalized and tolerated to prevent invasive growth and harmful inflammatory responses. Signaling initiated by commensal bacteria shapes antigen-specific mucosal and systemic adaptive immunity. A distinct type of effector CD4(+) T cells, Th17 cells, have a key role in coordinating the inflammatory immune responses that afford protection to pathogens at the mucosal interface. Balancing this powerful inflammatory response, regulatory T cells limit collateral damage and provide antigen-specific tolerance to both food and microbial antigens. Here, we discuss the implications for how the microbiota as a whole contributes to compartmentalization from the host and how individual constituents of the microbiota influence the functions and repertoire of effector T cells and organ-specific autoimmune disease.

Peripheral spondyloarthritis (SpA) is a common extraintestinal manifestation in patients with active inflammatory bowel disease (IBD) characterized by inflammatory enthesitis, dactylitis, or synovitis of nonaxial joints. However, a mechanistic understanding of the link between intestinal inflammation and SpA has yet to emerge. We evaluated and functionally characterized the fecal microbiome of IBD patients with or without peripheral SpA. Coupling the sorting of immunoglobulin A (IgA)-coated microbiota with 16

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.

TRAIL receptor (TRAIL-R) signaling has been implicated in inducing apoptosis in tumor cells, but little is understood about its physiological function. Here, we report the generation and characterization of TRAIL-R(-/-) mice, which develop normal lymphocyte populations but possess enhanced innate immune responses. TRAIL-R(-/-) mice exhibited increased clearance of murine cytomegalovirus that correlated with increased levels of IL-12, IFN-alpha, and IFN-gamma. Stimulation of macrophages with Mycobacterium and Toll-like receptor (TLR)-2, -3, and -4, but not TLR9, ligands resulted in high levels of TRAIL upregulation and enhanced cytokine production in TRAIL-R(-/-) cells. The immediate-early TLR signaling events in TRAIL-R(-/-) macrophages and dendritic cells are normal, but I kappa B-alpha homeostatic regulation and NF-kappa B activity at later time points is perturbed. These data suggest that TRAIL-R negatively regulates innate immune responses.

Loss of TRAIL-R does not affect thymic or intestinal tumor development in p53 and adenomatous polyposis coli mutant mice

Retinol plays a vital role in the immune response to infection, yet proteins that mediate retinol transport during infection have not been identified. Serum amyloid A (SAA) proteins are strongly induced in the liver by systemic infection and in the intestine by bacterial colonization, but their exact functions remain unclear. Here we show that mouse and human SAAs are retinol binding proteins. Mouse and human SAAs bound retinol with nanomolar affinity, were associated with retinol in vivo, and limited the bacterial burden in tissues after acute infection. We determined the crystal structure of mouse SAA3 at a resolution of 2 Å, finding that it forms a tetramer with a hydrophobic binding pocket that can accommodate retinol. Our results thus identify SAAs as a family of microbe-inducible retinol binding proteins, reveal a unique protein architecture involved in retinol binding, and suggest how retinol is circulated during infection.

Interleukin (IL)-22-producing group 3 innate lymphoid cells (ILC3) promote mucosal healing and maintain barrier integrity, but how microbial signals are integrated to regulate mucosal protection offered by these cells remains unclear. Here, we show that in vivo depletion of CX₃CR1⁺ mononuclear phagocytes (MNPs) resulted in more severe colitis and death after infection with Citrobacter rodentium. This phenotype was rescued by exogenous IL-22, which was endogenously produced by ILC3 in close spatial proximity to CX₃CR1⁺ MNPs that were dependent on MyD88 signaling. CX₃CR1⁺MNPs from both mouse and human tissue produced more IL-23 and IL-1β than conventional CD103(+) dendritic cells (cDCs) and were more efficient than cDCs in supporting IL-22 production in ILC3 in vitro and in vivo. Further, colonic ILC3 from patients with mild to moderate ulcerative colitis or Crohn's disease had increased IL-22 production. IBD-associated SNP gene set analysis revealed enrichment for genes selectively expressed in human intestinal MNPs. The product of one of these, TL1A, potently enhanced IL-23- and IL-1β-induced production of IL-22 and GM-CSF by ILC3. Collectively, these results reveal a critical role for CX₃CR1⁺ mononuclear phagocytes in integrating microbial signals to regulate colonic ILC3 function in IBD.

TRAIL (tumor necrosis factor-related apoptosis-inducing ligand) is a member of the tumor necrosis factor family that can kill a wide variety of tumor cells but not normal cells. TRAIL-induced apoptosis in humans is mediated by its receptors DR4 (TRAIL-R1) and DR5 (TRAIL-R2). What constitutes the signaling molecules downstream of these receptors, however, remains highly controversial. Using the FADD dominant negative molecule, several groups have reached different conclusions with respect to the role of FADD in TRAIL-induced apoptosis. More recently, usingFADD-deficient (−/−) mouse embryonic fibroblasts, Yehet al. (Yeh, W.-C., Pompa, J. L., McCurrach, M. E., Shu, H.-B., Elia, A. J., Shahinian, A., Ng, M., Wakeham, A., Khoo, W., Mitchell, K., El-Deiry, W. S., Lowe, S. W., Goeddel, D. V., and Mak, T. W. (1998) Science279, 1954–1958) concluded that DR4 utilizes a FADD-independent apoptotic pathway. The latter experiment, however, involved transient overexpression, which often leads to nonspecific aggregation of death domain-containing receptors. To address this issue in a more physiological setting, we stably transfected mouse DR4/5, human DR4, or human DR5 intoFADD−/− mouse embryonic fibroblast cells. We showed that FADD−/− MEF cells stably transfected with TRAIL receptors are resistant to TRAIL-mediated cell death. In contrast, TRAIL receptors stably transfected into heterozygous FADD+/− cells orFADD−/− cells reconstituted with a FADD retroviral construct are sensitive to the TRAIL cytotoxic effect. We conclude that FADD is required for DR4- and DR5-mediated apoptosis.

This report describes the production of a monoclonal antibody raised against Bcl-xl, and includes an initial study of bcl-xl expression in neuropathology including Alzheimer's disease (AD). Bcl-xl is a potent apoptotic inhibitor and is known to be the predominant Bcl-x isoform in brain. To examine the expression of bcl-xl in aged brain and neurodegenerative disease, we raised a Bcl-xl-specific monoclonal antibody. In aged human brain, the highest bcl-xl expression was observed in cerebellum. By immunohistochemistry, significant bcl-xl expression was detected in reactive microglia of patients with AD and other neurological diseases such as progressive supranuclear palsy. Bcl-xl-positive microglia frequently colocalized with β-amyloid plaques in AD and with activated astrocytes in non-AD and AD brains, suggesting a general role for Bcl-xl in regions of pathology. High levels of Bcl-xl protein might render microglia more resistant to cytotoxic environments such as areas of neurodegeneration and astrogliosis. J. Neurosci. Res. 47:98–108, 1997. © 1997 Wiley-Liss, Inc.

The vertebrate intestinal tract is colonized by hundreds of species of bacteria that outnumber the total cells in the host, yet must be compartmentalized and tolerated to prevent invasive growth and harmful inflammatory responses. A key function of commensal microbes is to contribute to the adaptive immune repertoire and to diverse lymphocyte effector functions. T cell responses against non-invasive commensals, as exemplified by responses elicited by the segmented filamentous bacteria (SFB) and Helicobacter hepaticus, contribute to shaping the repertoire of effector/memory and regulatory T cells. SFB adhere to the epithelium in the terminal ileum of mice and induce differentiation of Th17 cells that normally protect the mucosal barrier, but contribute to autoimmune disease in susceptible mice. H. hepaticus colonizes the large intestine and induces Treg cells in wild type mice and inflammatory Th17 cells in IL-10-deficient mice. How T cells elicited by commensal bacteria can influence autoimmunity is a central question that remains unsolved. We are studying the antigenic specificity of microbiota-induced T cells and the mechanisms by which their functions are acquired upon interaction with distinct commensal species. We find that induction of SFB-specific Th17 cells and H. hepaticus-specific Treg cells requires priming and polarization of antigen-specific RORgt-expressing CD4+ T cells (also expressing Foxp3 for Tregs) in the draining lymph nodes. Induction of IL-17 in lamina propria Th17 cells occurs only in regions with adjacent SFB colonization. Acquisition of Th17 effector functions requires the local activation of IL-22-producing innate lymphoid cells (ILC3). These, in turn, act on intestinal epithelial cells to induce secretion of serum amyloid A proteins, SAA1 and SAA2, that activate primed RORgt+ T cells. Expression of the Th17 cell effector program requires formation of a complex of RORgt with a DEAD-box RNA helicase and a long noncoding RNA that, together, confer transcriptional activity at target loci. Requirements for induction of Treg versus Th17 cells will be discussed, as will be the potential role of SAAs in inflammation. These studies in mice are not only relevant for human autoimmune diseases, many of which have Th17 cell involvement, but may also provide insights into how commensal microbe-specific T cell responses could be harnessed for mucosal vaccination and cancer immunotherapy. Indeed, it has recently become evident that the microbiota plays an important role in patient responses to cancer chemotherapy and checkpoint immunotherapy. A better understanding of how individual or communities of bacteria (or commensal viruses and fungi) regulate tumor-specific T cell responses may pave the way for microbiota-based cancer therapies. Citation Format: Teruyuki Sano, Mo Xu, Lin Wu, June-Tong Lee, Wendy Huang, Yi Yang, Gretchen Diehl, Dan R. Littman. The commensal microbiota as instructors and arbiters of homeostatic and pathogenic immune responses [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr IA09.

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.

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.

During fetal development, lymphoid tissue inducer cells (LTis) seed the de- veloping lymph node and Peyer's patch anlagen and initiate the formation of both types of lymphoid organs. In the adult, a similar population of cells, termed lym- phoid tissue inducer-like cells (LTi-like cells), supports the formation of organized gut-associated lymphoid tissue (GALT) in the intestine, including both isolated lym- phoid follicles (ILFs) and cryptopatches (CPs). Both LTi and LTi-like cells require expression of the transcription factor RORγt for their differentiation and function, and mice lacking RORγt lack lymph nodes, Peyer's patches, and other organized GALT. In ILFs and cryptopatches, LTi-like cells are in close contact with different populations of intestinal dendritic cells (DCs), including a subpopulation recently shown to extend dendrites and sample luminal microflora. This interaction may allow for communica- tion between the intestinal lumen and the immune cells in the lamina propria, which is necessary for maintaining homeostasis between the commensal microflora and the intestinal immune system. The potential functional implications of the organization of LTi-like cells, DCs, and lymphocytes in the lamina propria are discussed in the context of maintenance of homeostasis and of infectious diseases, particularly HIV infection.

cells, imprinting gut homing expression on responding lymphocytes, and facilitating IgA production. How CD103+ LP-DCs become imprinted to produce ATRA is not well understood. In vitro studies have shown that epithelial cells can condition DCs to express retinaldehye dehydrogenase (ALDH1), the enzyme required for the final irreversible step in ATRA production, and in vivo studies have shown that luminal retinoids from the diet or bile, as opposed to systemic retinoids, are required to condition intestinal LP-DCs with the capacity to generate ATRA. Through the formation of goblet cell associated antigen passages (GAPs), the small intestine epithelium provides a portal for CD103+ LP-DCs to acquire soluble substances from the intestinal lumen. Moreover during the transfer of luminal substances, CD103+ LP-DCs physically interact with goblet cells (GCs) and acquire GC proteins. These observations suggest a role for GCs and GAPs in imprinting CD103+ LP-DCs. Using an in vivo two-photon (2P) imaging approach, we evaluated if and how LP-DCs acquire luminal retinoids. We observed that GAPs delivered luminal beta-carotene and the enzyme required to metabolize beta-carotene to LP-DCs. Moreover in the absence of GCs, LP-DCs are not imprinted with ALDH activity. We observed that LP-DCs express CCR6, which was required for LP-DCs to associate with the small intestine epithelium. As a consequence of impaired association with the epithelium, CCR6 deficient LP-DCs do not acquire GC proteins or luminal antigens, do not become conditioned with ALDH activity, and are impaired at inducing ATRA dependent events in responding lymphocytes. These findings demonstrate that GAPs deliver luminal retinoids and the enzymes required to metabolize these retinoids to CD103+ LP-DCs, and accordingly GCs play a critical role in imprinting CD103+ LPDCs. Thus GAPs deliver luminal antigens, immunomodulatory dietary substances, and epithelial proteins simultaneously to CD103+ LP-DCs to shape intestinal immune responses to small soluble luminal antigens.