Microbiota-dependent activation of the myeloid calcineurin-NFAT pathway inhibits B7H3- and B7H4-dependent anti-tumor immunity in colorectal cancer.

Bacterial sensing by intestinal tumor cells contributes to tumor growth through cell-intrinsic activation of the calcineurin-NFAT axis, but the role of this pathway in other intestinal cells remains unclear. Here, we found that myeloid-specific deletion of calcineurin in mice activated protective CD8+ T cell responses and inhibited colorectal cancer (CRC) growth. Microbial sensing by myeloid cells promoted calcineurin- and NFAT-dependent interleukin 6 (IL-6) release, expression of the co-inhibitory molecules B7H3 and B7H4 by tumor cells, and inhibition of CD8+ T cell-dependent anti-tumor immunity. Accordingly, targeting members of this pathway activated protective CD8+ T cell responses and inhibited primary and metastatic CRC growth. B7H3 and B7H4 were expressed by the majority of human primary CRCs and metastases, which was associated with low numbers of tumor-infiltrating CD8+ T cells and poor survival. Therefore, a microbiota-, calcineurin-, and B7H3/B7H4-dependent pathway controls anti-tumor immunity, revealing additional targets for immune checkpoint inhibition in microsatellite-stable CRC.

SETDB1 is required for intestinal epithelial differentiation and the prevention of intestinal inflammation.

OBJECTIVE: The intestinal epithelium is a rapidly renewing tissue which plays central roles in nutrient uptake, barrier function and the prevention of intestinal inflammation. Control of epithelial differentiation is essential to these processes and is dependent on cell type-specific activity of transcription factors which bind to accessible chromatin. Here, we studied the role of SET Domain Bifurcated Histone Lysine Methyltransferase 1, also known as ESET (SETDB1), a histone H3K9 methyltransferase, in intestinal epithelial homeostasis and IBD. DESIGN: We investigated mice with constitutive and inducible intestinal epithelial deletion of Setdb1, studied the expression of SETDB1 in patients with IBD and mouse models of IBD, and investigated the abundance of SETDB1 variants in healthy individuals and patients with IBD. RESULTS: Deletion of intestinal epithelial Setdb1 in mice was associated with defects in intestinal epithelial differentiation, barrier disruption, inflammation and mortality. Mechanistic studies showed that loss of SETDB1 leads to de-silencing of endogenous retroviruses, DNA damage and intestinal epithelial cell death. Predicted loss-of-function variants in human SETDB1 were considerably less frequently observed than expected, consistent with a critical role of SETDB1 in human biology. While the vast majority of patients with IBD showed unimpaired mucosal SETDB1 expression, comparison of IBD and non-IBD exomes revealed over-representation of individual rare missense variants in SETDB1 in IBD, some of which are predicted to be associated with loss of function and may contribute to the pathogenesis of intestinal inflammation. CONCLUSION: SETDB1 plays an essential role in intestinal epithelial homeostasis. Future work is required to investigate whether rare variants in SETDB1 contribute to the pathogenesis of IBD.

CD1d-Restricted pathways in hepatocytes control local natural killer T cell homeostasis and hepatic inflammation.

Invariant natural killer T (iNKT) cells recognize lipid antigens presented by CD1d and play a central role in regulating immunity and inflammation in peripheral tissues. However, the mechanisms which govern iNKT cell homeostasis after thymic emigration are incompletely understood. Here we demonstrate that microsomal triglyceride transfer protein (MTP), a protein involved in the transfer of lipids onto CD1d, regulates liver iNKT cell homeostasis in a manner dependent on hepatocyte CD1d. Mice with hepatocyte-specific loss of MTP exhibit defects in the function of CD1d and show increased hepatic iNKT cell numbers as a consequence of altered iNKT cell apoptosis. Similar findings were made in mice with hepatocyte-specific loss of CD1d, confirming a critical role of CD1d in this process. Moreover, increased hepatic iNKT cell abundance in the absence of MTP is associated with susceptibility to severe iNKT cell-mediated hepatitis, thus demonstrating the importance of CD1d-dependent control of liver iNKT cells in maintaining immunological homeostasis in the liver. Together, these data demonstrate an unanticipated role of parenchymal cells, as shown here for hepatocytes, in tissue-specific regulation of CD1d-restricted immunity and further suggest that alterations in lipid metabolism may affect iNKT cell homeostasis through effects on CD1d-associated lipid antigens.

XIAP variants in male Crohn's disease.

OBJECTIVE: The genetic basis of inflammatory bowel disease (IBD) is incompletely understood. The aim of this study was to identify rare genetic variants involved in the pathogenesis of IBD. DESIGN: Exome sequencing and immunological profiling were performed in a patient with early onset Crohn's disease (CD). The coding region of the gene encoding X-linked inhibitor of apoptosis protein (XIAP) was sequenced in samples of 275 paediatric IBD and 1047 adult-onset CD patients. XIAP genotyping was performed in samples of 2680 IBD patients and 2864 healthy controls. Functional effects of the variants identified were investigated in primary cells and cultured cell lines. RESULTS: Our results demonstrate the frequent occurrence of private variants in XIAP in about four percent of male patients with paediatric-onset CD. While XIAP mutations are known to be associated with the primary immunodeficiency (PID) X-linked lymphoproliferative disease type 2 (XLP2), CD patients described here exhibited intestinal inflammation in the absence of XLP2 and harboured a spectrum of mutations partially distinct from that observed in XLP2. The majority of XIAP variants identified was associated with a selective defect in NOD1/2 signalling, impaired NOD1/2-mediated activation of NF-κB, and altered NF-κB-dependent cytokine production. CONCLUSIONS: This study reveals the unanticipated, frequent occurrence of XIAP variants in male paediatric-onset CD. The link between XIAP and NOD1/2, and the association of XIAP variants with XLP2, support the concept of PID in a subset of IBD patients. Moreover, these studies provide a rationale for the implementation of XIAP sequencing in clinical diagnostics in male patients with severe CD.

Tumor-infiltrating plasmacytoid dendritic cells are associated with survival in human colon cancer.

BACKGROUND: Plasmacytoid dendritic cells (pDCs) play a key role in the induction and maintenance of antitumor immunity. Conversely, they can act as tolerogenic DCs by inhibiting tumor-directed immune responses. Therefore, pDCs may profoundly influence tumor progression. To gain novel insights into the role of pDCs in colon cancer, we investigated the frequency and clinical relevance of pDCs in primary tumor tissues from patients with colon cancer with different clinicopathological characteristics. METHODS: Immunohistochemical stainings were performed to explore the frequency of tumor-infiltrating BDCA-2+ pDCs in patients with colon cancer. Statistical analyses were conducted to determine an association between the pDC density and clinicopathological characteristics of the patients. Furthermore, we used multiplex immunofluorescence stainings to evaluate the localization and phenotype of pDCs in stroma and tertiary lymphoid structures (TLS) of colon cancer tissues. RESULTS: An increased density of infiltrating pDCs was associated with lower Union for International Cancer Control (UICC) stages. Furthermore, a higher pDC frequency was significantly correlated with increased progression-free and overall survival of patients with colon cancer. Moreover, a lower number of coloncancer-infiltrating pDCs was significantly and independently linked to worse prognosis. In addition, we found that a proportion of pDCs shows a nuclear expression of the transcription factor interferon regulatory factor 7 (IRF7), which is characteristic for an activated phenotype. In various tumor stroma regions, IRF7+ pDCs were located in the neighborhood of granzyme B-expressing CD8+ T cells. Moreover, pDCs were identified as a novel component of the T cell zone of colon cancer-associated TLS, which are major regulators of adaptive antitumor immunity. A proportion of TLS-associated pDCs displayed a nuclear IRF7 expression and was preferentially located close to CD4+ T cells. CONCLUSIONS: These results indicate that higher densities of tumor-infiltrating pDCs are associated with prolonged survival of patients with colon cancer. Moreover, colon cancer-infiltrating pDCs may represent a novel prognostic factor. The colocalization of activated pDCs and T cells in tumor stroma and within TLS may contribute to the correlation between higher pDC densities and better prognosis. In addition, our findings may have implications for the design of novel immunotherapeutic strategies that are based on targeting colon cancer-infiltrating pDCs.

Long-Term Overconsumption of Fat and Sugar Causes a Partially Reversible Pre-inflammatory Bowel Disease State.

Nutrition appears to be an important environmental factor involved in the onset of inflammatory bowel diseases (IBD) through yet poorly understood biological mechanisms. Most studies focused on fat content in high caloric diets, while refined sugars represent up to 40% of caloric intake within industrialized countries and contribute to the growing epidemics of inflammatory diseases. Herein we aim to better understand the impact of a high-fat-high-sucrose diet on intestinal homeostasis in healthy conditions and the subsequent colitis risk. We investigated the early events and the potential reversibility of high caloric diet-induced damage in mice before experimental colitis. C57BL/6 mice were fed with a high-fat or high-fat high-sucrose or control diet before experimental colitis. In healthy mice, a high-fat high-sucrose diet induces a pre-IBD state characterized by gut microbiota dysbiosis with a total depletion of bacteria belonging to Barnesiella that is associated with subclinical endoscopic lesions. An overall down-regulation of the colonic transcriptome converged with broadly decreased immune cell populations in the mesenteric lymph nodes leading to the inability to respond to tissue injury. Such in-vivo effects on microbiome and transcriptome were partially restored when returning to normal chow. Long-term consumption of diet enriched in sucrose and fat predisposes mice to colitis. This enhanced risk is preceded by gut microbiota dysbiosis and transcriptional reprogramming of colonic genes related to IBD. Importantly, diet-induced transcriptome and microbiome disturbances are partially reversible after switching back to normal chow with persistent sequelae that may contribute to IBD predisposition in the general population.

Deficiency in X-linked inhibitor of apoptosis protein promotes susceptibility to microbial triggers of intestinal inflammation.

Inflammatory bowel disease (IBD) is characterized by inappropriate immune responses to the microbiota in genetically susceptible hosts, but little is known about the pathways that link individual genetic alterations to microbiota-dependent inflammation. Here, we demonstrated that the loss of X-linked inhibitor of apoptosis protein (XIAP), a gene associated with Mendelian IBD, rendered Paneth cells sensitive to microbiota-, tumor necrosis factor (TNF)­, receptor-interacting protein kinase 1 (RIPK1)­, and RIPK3-dependent cell death. This was associated with deficiency in Paneth cell­derived antimicrobial peptides and alterations in the stratification and composition of the microbiota. Loss of XIAP was not sufficient to elicit intestinal inflammation but provided susceptibility to pathobionts able to promote granulomatous ileitis, which could be prevented by administration of a Paneth cell­derived antimicrobial peptide. These data reveal a pathway critical for host-microbial cross-talk, which is required for intestinal homeostasis and the prevention of inflammation and which is amenable to therapeutic targeting.

Thirty-eight-negative kinase 1 mediates trauma-induced intestinal injury and multi-organ failure.

Dysregulated intestinal epithelial apoptosis initiates gut injury, alters the intestinal barrier, and can facilitate bacterial translocation leading to a systemic inflammatory response syndrome (SIRS) and/or multi-organ dysfunction syndrome (MODS). A variety of gastrointestinal disorders, including inflammatory bowel disease, have been linked to intestinal apoptosis. Similarly, intestinal hyperpermeability and gut failure occur in critically ill patients, putting the gut at the center of SIRS pathology. Regulation of apoptosis and immune-modulatory functions have been ascribed to Thirty-eight-negative kinase 1 (TNK1), whose activity is regulated merely by expression. We investigated the effect of TNK1 on intestinal integrity and its role in MODS. TNK1 expression induced crypt-specific apoptosis, leading to bacterial translocation, subsequent septic shock, and early death. Mechanistically, TNK1 expression in vivo resulted in STAT3 phosphorylation, nuclear translocation of p65, and release of IL-6 and TNF-α. A TNF-α neutralizing antibody partially blocked development of intestinal damage. Conversely, gut-specific deletion of TNK1 protected the intestinal mucosa from experimental colitis and prevented cytokine release in the gut. Finally, TNK1 was found to be deregulated in the gut in murine and porcine trauma models and human inflammatory bowel disease. Thus, TNK1 might be a target during MODS to prevent damage in several organs, notably the gut.

Epithelial calcineurin controls microbiota-dependent intestinal tumor development.

Inflammation-associated pathways are active in intestinal epithelial cells (IECs) and contribute to the pathogenesis of colorectal cancer (CRC). Calcineurin, a phosphatase required for the activation of the nuclear factor of activated T cells (NFAT) family of transcription factors, shows increased expression in CRC. We therefore investigated the role of calcineurin in intestinal tumor development. We demonstrate that calcineurin and NFAT factors are constitutively expressed by primary IECs and selectively activated in intestinal tumors as a result of impaired stratification of the tumor-associated microbiota and toll-like receptor signaling. Epithelial calcineurin supports the survival and proliferation of cancer stem cells in an NFAT-dependent manner and promotes the development of intestinal tumors in mice. Moreover, somatic mutations that have been identified in human CRC are associated with constitutive activation of calcineurin, whereas nuclear translocation of NFAT is associated with increased death from CRC. These findings highlight an epithelial cell-intrinsic pathway that integrates signals derived from the commensal microbiota to promote intestinal tumor development.

Deciphering µ-opioid receptor phosphorylation and dephosphorylation in HEK293 cells.

BACKGROUND AND PURPOSE: The molecular basis of agonist-selective signalling at the µ-opioid receptor is poorly understood. We have recently shown that full agonists such as [D-Ala(2)-MePhe(4)-Gly-ol]enkephalin (DAMGO) stimulate the phosphorylation of a number of carboxyl-terminal phosphate acceptor sites including threonine 370 (Thr(370)) and serine 375 (Ser(375)), and that is followed by a robust receptor internalization. In contrast, morphine promotes a selective phosphorylation of Ser(375) without causing rapid receptor internalization. EXPERIMENTAL APPROACH: Here, we identify kinases and phosphatases that mediate agonist-dependent phosphorylation and dephosphorylation of the µ-opioid receptor using a combination of phosphosite-specific antibodies and siRNA knock-down screening in HEK293 cells. KEY RESULTS: We found that DAMGO-driven phosphorylation of Thr(370) and Ser(375) was preferentially catalysed by G-protein-coupled receptor kinases (GRKs) 2 and 3, whereas morphine-driven Ser(375) phosphorylation was preferentially catalysed by GRK5. On the functional level, inhibition of GRK expression resulted in enhanced µ-opioid receptor signalling and reduced receptor internalization. Analysis of GRK5-deficient mice revealed that GRK5 selectively contributes to morphine-induced Ser(375) phosphorylation in brain tissue. We also identified protein phosphatase 1γ as a µ-opioid receptor phosphatase that catalysed Thr(370) and Ser(375) dephosphorylation at or near the plasma membrane within minutes after agonist removal, which in turn facilitates receptor recycling. CONCLUSIONS AND IMPLICATIONS: Together, the morphine-activated µ-opioid receptor is a good substrate for phosphorylation by GRK5 but a poor substrate for GRK2/3. GRK5 phosphorylates µ-opioid receptors selectively on Ser(375), which is not sufficient to drive significant receptor internalization.