Experimental and genetic evidence for the impact of CD5 and CD6 expression and variation in inflammatory bowel disease.

Crohn's disease (CD) and ulcerative colitis (UC) are inflammatory bowel diseases (IBD) resulting from the interaction of multiple environmental, genetic and immunological factors. CD5 and CD6 are paralogs encoding lymphocyte co-receptors involved in fine-tuning intracellular signals delivered upon antigen-specific recognition, microbial pattern recognition and cell adhesion. While CD5 and CD6 expression and variation is known to influence some immune-mediated inflammatory disorders, their role in IBD remains unclear. To this end, Cd5- and Cd6-deficient mice were subjected to dextran sulfate sodium (DSS)-induced colitis, the most widely used experimental animal model of IBD. The two mouse lines showed opposite results regarding body weight loss and disease activity index (DAI) changes following DSS-induced colitis, thus supporting Cd5 and Cd6 expression involvement in the pathophysiology of this experimental IBD model. Furthermore, DNA samples from IBD patients of the ENEIDA registry were used to test association of CD5 (rs2241002 and rs2229177) and CD6 (rs17824933, rs11230563, and rs12360861) single nucleotide polymorphisms with susceptibility and clinical parameters of CD (n=1352) and UC (n=1013). Generalized linear regression analyses showed association of CD5 variation with CD ileal location (rs2241002CC) and requirement of biological therapies (rs2241002C-rs2229177T haplotype), and with poor UC prognosis (rs2241002T-rs2229177T haplotype). Regarding CD6, association was observed with CD ileal location (rs17824933G) and poor prognosis (rs12360861G), and with left-sided or extensive UC, and absence of ankylosing spondylitis in IBD (rs17824933G). The present experimental and genetic evidence support a role for CD5 and CD6 expression and variation in IBD's clinical manifestations and therapeutic requirements, providing insight into its pathophysiology and broadening the relevance of both immunomodulatory receptors in immune-mediated disorders.

Lysophosphatidic Acid-Mediated GPR35 Signaling in CX3CR1+ Macrophages Regulates Intestinal Homeostasis.

Single-nucleotide polymorphisms in the gene encoding G protein-coupled receptor 35 (GPR35) are associated with increased risk of inflammatory bowel disease. However, the mechanisms by which GPR35 modulates intestinal immune homeostasis remain undefined. Here, integrating zebrafish and mouse experimental models, we demonstrate that intestinal Gpr35 expression is microbiota dependent and enhanced upon inflammation. Moreover, murine GPR35+ colonic macrophages are characterized by enhanced production of pro-inflammatory cytokines. We identify lysophosphatidic acid (LPA) as a potential endogenous ligand produced during intestinal inflammation, acting through GPR35 to induce tumor necrosis factor (Tnf) expression in macrophages. Mice lacking Gpr35 in CX3CR1+ macrophages aggravate colitis when exposed to dextran sodium sulfate, which is associated with decreased transcript levels of the corticosterone-generating gene Cyp11b1 and macrophage-derived Tnf. Administration of TNF in these mice restores Cyp11b1 expression and intestinal corticosterone production and ameliorates DSS-induced colitis. Our findings indicate that LPA signals through GPR35 in CX3CR1+ macrophages to maintain TNF-mediated intestinal homeostasis.

The Copper/Zinc Ratio Correlates With Markers of Disease Activity in Patients With Inflammatory Bowel Disease.

BACKGROUND: Zinc (Zn) and copper (Cu) are trace elements that serve as cofactors in catalytic processes with impact on immune responses. In patients with inflammatory bowel disease (IBD), decreased levels of serum Zn and Cu have been observed. Here, we investigated the effect of inflammation on serum concentrations of these trace elements in patients with IBD. METHODS: In this cross-sectional study, 98 patients with Crohn disease (CD) and 56 with ulcerative colitis (UC) were prospectively enrolled. Disease activity parameters, such as C-reactive protein (CRP) and fecal calprotectin (FC) were compared to serum Zn, Cu, and Cu/Zn ratio. RESULTS: Zinc insufficiency was observed in 11.2% of patients with CD and 14.3% with UC, Cu insufficiency in 20.4% with CD and 7.1% with UC. Anemia, hypoalbuminemia, increased FC, and elevated CRP were more frequently present in Zn-insufficient patients with IBD. In contrast, lower serum CRP values and a trend to lower FC were observed in Cu-insufficient patients. In multiple linear regression models adjusted for age, gender, and serum albumin, CRP positively correlated with serum Cu (P < 0.001) and the Cu/Zn ratio in both CD and UC (P < 0.001) but not with serum Zn concentrations. FC levels correlated only with the Cu/Zn ratio in patients with UC (P < 0.038). CONCLUSION: Systemic inflammation inversely affected the serum Zn and Cu concentrations and, consequently, resulted in an increased Cu/Zn ratio.

Loss of the branched-chain amino acid transporter CD98hc alters the development of colonic macrophages in mice.

Comprehensive development is critical for gut macrophages being essential for the intestinal immune system. However, the underlying mechanisms of macrophage development in the colon remain elusive. To investigate the function of branched-chain amino acids in the development of gut macrophages, an inducible knock-out mouse model for the branched-chain amino acid transporter CD98hc in CX3CR1+ macrophages was generated. The relatively selective deletion of CD98hc in macrophage populations leads to attenuated severity of chemically-induced colitis that we assessed by clinical, endoscopic, and histological scoring. Single-cell RNA sequencing of colonic lamina propria macrophages revealed that conditional deletion of CD98hc alters the "monocyte waterfall"-development to MHC II+ macrophages. The change in the macrophage development after deletion of CD98hc is associated with increased apoptotic gene expression. Our results show that CD98hc deletion changes the development of colonic macrophages.

Extracellular ATP and Purinergic P2Y2 Receptor Signaling Promote Liver Tumorigenesis in Mice by Exacerbating DNA Damage.

Release of ATP to the extracellular compartment and subsequent activation of purinergic receptors is a conserved mechanism mediating inflammatory responses and cell fate decisions in various organs including the liver. Previous findings suggest that extracellular ATP may promote liver tumorigenesis, however, the underlying mechanisms are poorly understood. Therefore, our aim was to dissect the functions of extracellular ATP and P2Y2 receptors (P2Y2R) during hepatocarcinogenesis. Liver tumors were induced in wild-type and P2y2r -/- knockout mice by intraperitoneal diethylnitrosamine (DEN) injection. Tumorigenesis was analyzed after 8 to 10 months and molecular analyses were performed at different stages of tumorigenesis in vivo, as well as in primary mouse hepatocytes in vitro. Liver tumor incidence and tumor numbers were strongly reduced in P2y2r -/- mice, whereas tumor size and morphology were comparable to wild-type controls, suggesting that P2Y2R contributes to tumor initiation. Mechanistically, hepatocyte proliferation in DEN-treated P2y2r -/- mice was reduced, which correlated with reduced c-JUN and CCND1 but increased p21 expression. Moreover, DNA damage as determined by hepatocellular expression of γH2A.X and of genes related to genotoxic stress, as well as STAT3 phosphorylation, was reduced in the absence of P2y2r. Administration of genotoxic agents to primary hepatocytes in vitro confirmed that DNA damage was indeed exacerbated by extracellular ATP, subsequent P2Y2R activation, and downstream intracellular calcium-dependent signal transduction. In conclusion, our data reveal that extracellular ATP and subsequent P2Y2R function stimulate DNA damage responses and hepatocyte proliferation, thereby promoting hepatocarcinogenesis. Targeting this pathway may be an attractive approach for chemoprevention of hepatocellular carcinoma. SIGNIFICANCE: Extracellular ATP and subsequent P2Y2 receptor function stimulate DNA damage responses and hepatocyte proliferation, thereby promoting hepatocarcinogenesis in mice. Targeting this pathway may be an attractive approach for chemoprevention of hepatocellular carcinoma. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/80/4/699/F1.large.jpg.

NLRP6 Deficiency in CD4 T Cells Decreases T Cell Survival Associated with Increased Cell Death.

The nucleotide-binding oligomerization domain (NOD)-like receptors belong to the family of pattern recognition receptors (PRRs). NOD-like receptors play a role in regulation of innate immune response by recognition of both pathogen-associated molecular patterns that are engulfed during phagocytic process and danger-associated molecular patterns that are mainly byproducts of cell stress mediated response. NOD-like family pyrin domain containing 6 (NLRP6) is one of the 14 pyrin domain-containing receptors. NLRP6 is highly expressed by epithelial and goblet cells to regulate epithelial renewal and mucus production in mice and humans, but its function in T cells is rather unknown. Increased caspase-1 activation and cell death were observed in mouse Nlrp6-deficient T cells following adoptive transfer into Rag2-deficient mice, indicating that Nlrp6 deficiency in CD4+ T cells led to decreased survival.

Metabolite-Sensing G Protein-Coupled Receptors Connect the Diet-Microbiota-Metabolites Axis to Inflammatory Bowel Disease.

Increasing evidence has indicated that diet and metabolites, including bacteria- and host-derived metabolites, orchestrate host pathophysiology by regulating metabolism, immune system and inflammation. Indeed, autoimmune diseases such as inflammatory bowel disease (IBD) are associated with the modulation of host response to diets. One crucial mechanism by which the microbiota affects the host is signaling through G protein-coupled receptors (GPCRs) termed metabolite-sensing GPCRs. In the gut, both immune and nonimmune cells express GPCRs and their activation generally provide anti-inflammatory signals through regulation of both the immune system functions and the epithelial integrity. Members of GPCR family serve as a link between microbiota, immune system and intestinal epithelium by which all these components crucially participate to maintain the gut homeostasis. Conversely, impaired GPCR signaling is associated with IBD and other diseases, including hepatic steatosis, diabetes, cardiovascular disease, and asthma. In this review, we first outline the signaling, function, expression and the physiological role of several groups of metabolite-sensing GPCRs. We then discuss recent findings on their role in the regulation of the inflammation, their existing endogenous and synthetic ligands and innovative approaches to therapeutically target inflammatory bowel disease.

Purinergic receptor type 6 contributes to airway inflammation and remodeling in experimental allergic airway inflammation.

RATIONALE: Extracellular nucleotides have recently been identified as proinflammatory mediators involved in asthma pathogenesis by signaling via purinergic receptors, but the role of the purinergic receptor type 6 (P2Y6R) has not been previously investigated. OBJECTIVES: To investigate the role of P2Y6R in asthma pathogenesis. METHODS: Acute and chronic OVA model and also HDM model of allergic inflammation in C57Bl/6 mice treated with specific P2Y6R antagonist and P2Y6R(-/-) mice were evaluated for classical features of asthmatic inflammation. In addition, primary epithelial cell culture from human and epithelial cell lines from mouse and human were stimulated with P2Y6R agonist and treated with P2Y6R antagonist and assessed for IL-6, IL-8/CXCL8 and KC levels. Experiments with P2Y6R(-/-) and P2Y6R(+/+) chimera were performed to discriminate the role of P2Y6R activation in structural lung cells and in cells from hematopoietic system. MEASUREMENTS AND MAIN RESULTS: We observed that the intratracheal application of a P2Y6R antagonist (MRS2578) and P2Y6R deficiency inhibited cardinal features of asthma, such as bronchoalveolar lavage eosinophilia, airway remodeling, Th2 cytokine production, and bronchial hyperresponsiveness in the ovalbumin-alum model. MRS2578 was also effective in reducing airway inflammation in a model using house dust mite extracts to induce allergic lung inflammation. Experiments with bone marrow chimeras revealed the importance of the P2Y6R expression on lung structural cells in airway inflammation. In accordance with this finding, we found a strong up-regulation of P2Y6 expression on airway epithelial cells of animals with experimental asthma. Concerning the underlying mechanism, we observed that MRS2578 inhibited the release of IL-6 and IL-8/KC by lung epithelial cells in vivo, whereas intrapulmonary application of the P2Y6R agonist uridine-5'-diphosphate increased the bronchoalveolar levels of IL-6 and KC. In addition, selective activation of P2Y6 receptors induced the release of IL-6 and KC/IL-8 by murine and human lung epithelial cells in vitro. CONCLUSIONS: P2Y6R expression on airway epithelial cells is up-regulated during acute and chronic allergic airway inflammation, and selective blocking of P2Y6R or P2Y6R deficiency on the structural cells reduces cardinal features of experimental asthma. Thus, blocking pulmonary P2Y6R might be a target for the treatment of allergic airway inflammation.

BDNF and its receptors in human myasthenic thymus: implications for cell fate in thymic pathology.

Here we show that in myasthenic thymus several cell types, including thymic epithelial cells (TEC) and immune cells, were the source and the target of the neurotrophic factor brain-derived growth factor (BDNF). Interestingly, many actively proliferating medullary thymocytes expressed the receptor TrkB in vivo in involuted thymus, while this population was lost in hyperplastic or neoplastic thymuses. Furthermore, in hyperplastic thymuses the robust coordinated expression of BDNF in the germinal centers together with the receptor p75NTR on all proliferating B cells strongly suggests that this factor regulates germinal center reaction. Finally, all TEC dying of apoptosis expressed BDNF receptors, indicating that this neurotrophin is involved in TEC turnover. In thymomas both BDNF production and receptor expression in TEC were strongly hindered. This may represent an attempt of tumour escape from cell death.