Involvement of Dectin-2 in the Innate Inflammatory Response Triggered by Influenza Virus Hemagglutinin

C-type lectin receptors (CLRs) are representative pattern recognition receptors that recognize microbial polysaccharides expressed on antigen-presenting cells. In the present study, we carried out further detailed analysis on the involvement of Dectin-2, a CLR that senses high mannose polysaccharide, in innate immune responses induced by influenza virus hemagglutinin (HA). Treatment of HA with periodate or PNGase F induced lower interleukin (IL)-12p40 secretion by conventional dendritic cells (DCs) compared with the untreated group. In contrast, treatment with O-glycosidase did not affect cytokine production. Green fluorescent protein expression in canonical Dectin-2-transducing cells was approximately 3% - 12% following HA stimulation, except with the A/H1N1pdm09 subtype HA. This expression was markedly reduced in cells possessing mutated amino acids in the carbohydrate recognition domain of Dectin-2, especially following stimulation with HA derived from the A/H3N2 subtype. Interferon (IFN)-α production from CD11c+Siglec-H+PDCA-1+ plasmacytoid DCs was significantly increased in Dectin-2 knockout mice compared with wild-type mice upon stimulation with HA except for the B/Yamagata lineage HA. These results suggested that Dectin-2 is involved in initiating inflammatory responses via mannose polysaccharide on HA. However, other mechanisms may function in the antiviral response, including the type I IFN axis.

Disruption of the TWEAK/Fn14 pathway prevents 5-fluorouracil-induced diarrhea in mice

AIM To clarify the roles of TWEAK and its receptor Fn14 in 5-fluorouracil(5-FU)-induced diarrhea.METHODS Diarrhea was induced in wild-type(WT), Fn14 knockout(KO), and IL-13 receptor(IL-13R)α1 KO BALB/c mice using a single injection of 5-FU. Histological analysis, cytokine analysis, and flow cytometry was performed on ileal tissues and cells. Murine colon carcinomabearing mice were co-treated with an anti-TWEAK antibody and 5-FU. Embryonic fibroblast response to cytokines was also analyzed.RESULTS5-FU induced high Fn14 expression in epithelial cells. The severity of 5-FU-induced diarrhea was lower in Fn14 KO mice compared with WT mice. Administration of anti-TWEAK antibody reduced 5-FU-induced diarrhea without affecting the antitumor effects of 5-FU in vivo. 5-FU-induced expression of IL-13, IL-17 A, TNF-α, and IFN-γ in the ileum was Fn14 dependent. The severity of 5-FU-induced diarrhea was lower in IL-13Rα1 KO mice, indicating major role for IL-13 signaling via IL-13Rα1 in pathogenesis. We found that IL-13Rα2, an IL-13 neutralizing/cell protective receptor, was strongly induced by IL-33 in vitro and in vivo. IL-13Rα2 was upregulated in the ileum of 5-FU-treated Fn14 KO mice. Thus, the deletion of Fn14 upregulated IL-13Rα2 expression, which reduced IL-13 expression and activity. CONCLUSION Disruption of the TWEAK/Fn14 pathway affects several interconnected pathways, including those associated with IL-13, IL-33, and IL-13Rα2, to attenuate 5-FUinduced intestinal side effects.

Efficient isolation of specific genomic regions by insertional chromatin immunoprecipitation (iChIP) with a second-generation tagged LexA DNA-binding domain

Comprehensive understanding of mechanisms of epigenetic regulation requires identification of molecules bound to genomic regions of interest in vivo. We have developed a novel method, insertional chromatin immunoprecipitatin (iChIP), to isolate specific genomic regions retaining molecular interaction in order to perform non-biased identification of interacting molecules in vivo. Here, we developed a second-generation tagged LexA DNA-binding domain, 3xFNLDD, for the iChIP analysis. 3xFNLDD consists of 3 x FLAG tags, a nuclear localization signal (NLS), the DNA-binding domain (DB) and the dimerization domain of the LexA protein. Expression of 3xFNLDD can be detected by immunoblot analysis as well as flowcytometry. We showed that iChIP using 3xFNLDD is able to consistently isolate more than 10% of input genomic DNA, several-fold more efficient compared to the first-generation tagged LexA DB. 3xFNLDD would be a useful tool to perform the iChIP analysis for locus-specific biochemical epigenetics.

A simple and efficient method for the preparation of live leukocytes from peripheral blood using the LeukoCatch^TMsystem

Leukocytes from peripheral blood (PB) are of great value for diagnosis as well as basic and clinical research. However, no easy, centrifugation-free method is available for the isolation of live leukocytes from blood. We here develop a simple and quick method for the purification of viable leukocytes from whole blood using novel tools, named tLeukoCatch (tip-type) or sLeukoCatch (syringe-type), which is equipped with three Pall filter layers and captures leukocytes but not red blood cells (RBCs) in whole blood. Indeed, we showed that several million leukocytes per mL (~35% of the recovery rate) were captured and eluted from whole blood. The number of contaminant RBCs decreased from several million to several thousand. When mouse blood was hemolysed, almost all of the lysed RBC fragments were removed by passage through sLeukoCatch. Optical microscopic observation confirmed that the recovered leukocytes were sufficiently healthy to respond to growth stimuli. Efficient leukocyte recovery was also confirmed for hemolysed human blood. These results suggest that the LeukoCatchTM system is useful for bedside diagnosis and basic research with blood samples.

Single-cell analysis for identification of T-cell clonotypes associated with IgG4 production of autoimmune pancreatitis

Introduction Autoimmune pancreatitis(AIP)is one of the recently established immunoglobulin G4-related diseases(IgG4-RD)[1].The detailed pathogenic mechanisms have been an intensive research area for prophylactic and therapeutic purposes because aberrant immune activation and tissue fibrosis in AIP are the major factors that worsen the disease outcomes in these patients.

Regulation of leptin on insulin secretion and sulfonulurea receptor 1 transcription level in isolated rats pancreatic islets

Objective To investigate the regulation of leptin on insulin secretion and expression of ATP-sensitive potassium channel subunit sulfonulurea receptor 1 (SUR1) mRNA, and to determine whether the effects of leptin are mediated through known intracellular signaling transduction.Methods Pancreatic islets were isolated by the collagenase method from male SD rats. The purified islets were incubated with different concentrations of leptin for 2 h in the presence of different concentrations of glucose. Insulin release was measured using radioimmunoassay. Expression of SUR1 mRNA was detected by RT-PCR.Results In the presence of leptin 2 nmol/L, insulin release was significantly inhibited at either 11.1 or 16.7 mmol/L glucose concentration (both P<0.05), but insulin release was not altered at glucose of 5. 6 mmol/L physiological concentration. The dose-response experiment showed that the maximal effect of leptin on insulin secretion achieved at 2 nmol/L. Exposure of islets to 2 nmol/L leptin induced a significant increase of SUR1 transcription levels by 71% (P<0. 01) at 11.1 mmol/L glucose and by 56% (P<0. 05) at 16. 7 mmol/L glucose concentration. Selective phosphatidylinositol 3-kinase (PI 3-kinase) inhibitor wortmannin significantly prevented the leptin effect on insulin secretion and SUR1 mRNA expression.Conclusions Regulatory effects of leptin on insulin secretion could be biphasic at different concentrations of glucose and leptin. The stimulatory regulation of SUR1 transcription levels may be mediated through activation of PI 3-kinase pathway, which may be a possible mechanism of leptin in regulating insulin secretion.