Transcription factor ISX mediates the cross talk between diet and immunity.

The intestinal epithelium is a major site for the conversion of dietary ß-carotene to retinaldehyde by the enzyme BCO1. The majority of retinaldehyde is further metabolized to retinol (vitamin A), esterified and packaged into triacylglycerol-rich chylomicrons for bodily distribution. Some serve on-site for the synthesis of retinoic acid, a hormone-like compound, which exerts pleiotropic and dominant effects on gastrointestinal immunity. We report here that the intestine-specific homeobox protein ISX is critical to control the metabolic flow of ß-carotene through this important branching point of vitamin A metabolism. This transcription factor represses Bco1 gene expression in response to retinoic acid signaling. In ISX-deficient mice, uncontrolled Bco1 gene expression led to increased retinoid production in the intestine. Systemically, this production resulted in highly elevated hepatic retinoid stores. In the intestine, it increased the expression of retinoic acid-inducible target genes such as Aldh1a2, Dhrs3, and Ccr9 The ß-carotene-inducible disruption of retinoid homeostasis affected gut-homing and differentiation of lymphocytes and displayed morphologically in large lymphoid follicles along the intestine. Furthermore, it was associated with an infiltration of the pancreas by gut-derived lymphocytes that manifested as a pancreatic insulitis with ß-islet cell destruction and systemic glucose intolerance. Thus, our study identifies an important molecular interlink between diet and immunity and indicates that vitamin A homeostasis must be tightly controlled by ISX to maintain immunity and tolerance at the intestinal barrier.

A mouse model of the protease-activated receptor 4 Pro310Leu variant has reduced platelet reactivity.

BACKGROUND: Protease-activated receptor 4 (PAR4) mediates thrombin signaling on platelets and other cells. Our recent structural studies demonstrated that a single nucleotide polymorphism in extracellular loop 3 and PAR4-P310L (rs2227376) leads to a hyporeactive receptor. OBJECTIVES: The goal of this study was to determine how the hyporeactive PAR4 variant in extracellular loop 3 impacts platelet function in vivo using a novel knock-in mouse model (PAR4-322L). METHODS: A point mutation was introduced into the PAR4 gene F2rl3 via CRISPR/Cas9 to create PAR4-P322L, the mouse homolog to human PAR4-P310L. Platelet response to PAR4 activation peptide (AYPGKF), thrombin, ADP, and convulxin was monitored by αIIbß3 integrin activation and P-selectin translocation using flow cytometry or platelet aggregation. In vivo responses were determined by the tail bleeding assay and the ferric chloride-induced carotid artery injury model. RESULTS: PAR4-P/L and PAR4-L/L platelets had a reduced response to AYPGKF and thrombin measured by P-selectin translocation or αIIbß3 activation. The response to ADP and convulxin was unchanged among genotypes. In addition, both PAR4-P/L and PAR4-L/L platelets showed a reduced response to thrombin in aggregation studies. There was an increase in the tail bleeding time for PAR4-L/L mice. The PAR4-P/L and PAR4-L/L mice both showed an extended time to arterial thrombosis. CONCLUSION: PAR4-322L significantly reduced platelet responsiveness to AYPGKF and thrombin, which is in agreement with our previous structural and cell signaling studies. In addition, PAR4-322L had prolonged arterial thrombosis time. Our mouse model provides a foundation to further evaluate the role of PAR4 in other pathophysiological contexts.

A Mouse Model of the Protease Activated Receptor 4 (PAR4) Pro310Leu Variant has Reduced Platelet Reactivity.

Background: Protease activated receptor 4 (PAR4) mediates thrombin signaling on platelets and other cells. Our recent structural studies demonstrated a single nucleotide polymorphism in extracellular loop 3 (ECL3), PAR4-P310L (rs2227376) leads to a hypo-reactive receptor. Objectives: The goal of this study was to determine how the hypo-reactive PAR4 variant in ECL3 impacts platelet function in vivo using a novel knock-in mouse model (PAR4-322L). Methods: A point mutation was introduced into the PAR4 gene, F2rl3, via CRISPR/Cas9 to create PAR4-P322L, the mouse homolog to human PAR4-P310L. Platelet response to PAR4 activation peptide (AYPGKF), thrombin, ADP, and convulxin was monitored by αIIbß3 integrin activation and P-selectin translocation using flow cytometry or platelet aggregation. In vivo responses were determined by the tail bleeding assay and the ferric chloride-induced carotid artery injury model. Results: PAR4-P/L and PAR4-L/L platelets had a reduced response to AYPGKF and thrombin measured by P-selectin translocation or αIIbß3 activation. The response to ADP and convulxin was unchanged among genotypes. In addition, both PAR4-P/L and PAR4-L/L platelets showed a reduced response to thrombin in aggregation studies. There was an increase in the tail bleeding time for PAR4-L/L mice. The PAR4-P/L and PAR4-L/L mice both showed an extended time to arterial thrombosis. Conclusions: PAR4-322L significantly reduced platelet responsiveness to AYPGKF and thrombin, which is in agreement with our previous structural and cell signaling studies. In addition, PAR4-322L had prolonged arterial thrombosis time. Our mouse model provides a foundation to further evaluate the role of PAR4 in other pathophysiological contexts.