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The R-spondin family of proteins has recently been described as secreted enhancers of β-catenin activation through the canonical Wnt signaling pathway. We previously reported that Rspo2 is a major determinant of susceptibility to Citrobacter rodentium-mediated colitis in mice and recent genome-wide association studies have revealed RSPO3 as a candidate Crohn’s disease-specific inflammatory bowel disease susceptibility gene in humans. However, there is little information on the endogenous expression and cellular source of R-spondins in the colon at steady state and during intestinal inflammation. RNA sequencing and qRT-PCR were used to assess the expression of R-spondins at steady state and in two mouse models of colonic inflammation. The cellular source of R-spondins was assessed in specific colonic cell populations isolated by cell sorting. Data mining from publicly available datasets was used to assess the expression of R-spondins in the human colon. At steady state, colonic expression of R-spondins was found to be exclusive to non-epithelial CD45- lamina propria cells, and Rspo3/RSPO3 was the most highly expressed R-spondin in both mouse and human colon. R-spondin expression was found to be highly dynamic and differentially regulated during C. rodentium infection and dextran sodium sulfate (DSS) colitis, with notably high levels of Rspo3 expression during DSS colitis, and high levels of Rspo2 expression during C. rodentium infection, specifically in susceptible mice. Our data are consistent with the hypothesis that in the colon, R-spondins are expressed by subepithelial stromal cells, and that Rspo3/RSPO3 is the family member most implicated in colonic homeostasis. The differential regulation of the R-spondins in different models of intestinal inflammation indicate they respond to specific pathogenic and inflammatory signals that differ in the two models and provides further evidence that this family of proteins plays a key role in linking intestinal inflammation and homeostasis. [ABSTRACT FROM AUTHOR]

Cerebral malaria (CM) is a lethal neurological complication of malaria. We implemented a genome-wide screen in mutagenized mice to identify host proteins involved in CM pathogenesis and whose inhibition may be of therapeutic value. One pedigree (P48) segregated a resistance trait whose CM-protective effect was fully penetrant, mapped to chromosome 8, and identified by genome sequencing as homozygosity for a mis-sense mutation (W81R) in the FERM domain of Janusassociated kinase 3 (Jak3). The causative effect of Jak.W81R was verified by complementation testing in Jak3W81R/- double heterozygotes that were fully protected against CM. Jak3W81R homozygotes showed defects in thymic development with depletion of CD8+ T cell, B cell, and NK cell compartments, and defective T cell-dependent production of IFN-γ. Adoptive transfer of normal splenocytes abrogates CM resistance in Jak3W81Rhomozygotes, an effect attributed to the CD8+ T cells. Jak3W81R behaves as a dominant negative variant, with significant CM resistance of Jak3W81R/+ heterozygotes, compared to CM-susceptible Jak3+/+ and Jak3+/- controls. CM resistance in Jak3W81R/+ heterozygotes occurs in presence of normal T, B and NK cell numbers. These findings highlight the pathological role of CD8+ T cells and Jak3-dependent IFN-γ-mediated Th1 responses in CM pathogenesis. [ABSTRACT FROM AUTHOR]

Cranberry-derived compounds, including a fraction known as proanthocyanidins (PACs) exhibit anti-microbial, anti-infective, and anti-adhesive properties against a number of disease-causing organisms. In this study, the effect of cranberry proanthocyanidins (CPACs) on the infection of epithelial cells by two enteric bacterial pathogens, enteropathogenic Escherichia coli (EPEC) and Salmonella Typhimurium was investigated. Immunofluorescence data showed that actin pedestal formation, required for infection by enteropathogenic Escherichia coli (EPEC), was disrupted in the presence of CPACs. In addition, invasion of HeLa cells by Salmonella Typhimurium was significantly reduced, as verified by gentamicin protection assay and immunofluorescence. CPACs had no effect on bacterial growth, nor any detectable effect on the production of bacterial effector proteins of the type III secretion system. Furthermore, CPACs did not affect the viability of host cells. Interestingly, we found that CPACs had a potent and dose-dependent effect on the host cell cytoskeleton that was evident even in uninfected cells. CPACs inhibited the phagocytosis of inert particles by a macrophage cell line, providing further evidence that actin-mediated host cell functions are disrupted in the presence of cranberry CPACs. Thus, although CPAC treatment inhibited Salmonella invasion and EPEC pedestal formation, our results suggest that this is likely primarily because of the perturbation of the host cell cytoskeleton by CPACs rather than an effect on bacterial virulence itself. These findings have significant implications for the interpretation of experiments on the effects of CPACs on bacteria-host cell interactions. [ABSTRACT FROM AUTHOR]

Enteropathogenic and enterohemorrhagic Escherichia coli (EPEC and EHEC) are food-borne pathogens that colonize the small intestine and colon, respectively. To cause disease, these pathogens must overcome the action of different host antimicrobial peptides (AMPs) secreted into these distinct niches. We have shown previously that EHEC expresses high levels of the OmpT protease to inactivate the human cathelicidin LL-37, an AMP present in the colon. In this study, we investigate the mechanisms used by EPEC to resist human α-defensin 5 (HD-5), the most abundant AMP in the small intestine. Quantitative PCR was used to measure transcript levels of various EPEC surface structures. High transcript levels of gfcA, a gene required for group 4 capsule (G4C) production, were observed in EPEC, but not in EHEC. The unencapsulated EPEC ∆gfcA and EHEC wild-type strains were more susceptible to HD-5 than EPEC wild-type. Since the G4C is composed of the same sugar repeats as the lipopolysaccharide O-antigen, an -antigen ligase (waaL) deletion mutant was generated in EPEC to assess its role in HD-5 resistance. The ∆waaL EPEC strain was more susceptible to HD-5 than both the wild-type and ∆gfcA strains. The ∆gfcA∆waaL EPEC strain was not significantly more susceptible to HD-5 than the ∆waaL strain, suggesting that the absence of -antigen influences G4C formation. To determine whether the G4C and -antigen interact with HD-5, total polysaccharide was purified from wild-type EPEC and added to the ∆gfcA∆waaL strain in the presence of HD-5. The addition of exogenous polysaccharide protected the susceptible strain against HD-5 killing in a dose-dependent manner, suggesting that HD-5 binds to the polysaccharides present on the surface of EPEC. Altogether, these findings indicate that EPEC relies on both the G4C and the -antigen to resist the bactericidal activity of HD-5. [ABSTRACT FROM AUTHOR]

Enteropathogenic and enterohemorrhagic Escherichia coli (EPEC and EHEC) are food-borne pathogens that colonize the small intestine and colon, respectively. To cause disease, these pathogens must overcome the action of different host antimicrobial peptides (AMPs) secreted into these distinct niches. We have shown previously that EHEC expresses high levels of the OmpT protease to inactivate the human cathelicidin LL-37, an AMP present in the colon. In this study, we investigate the mechanisms used by EPEC to resist human α-defensin 5 (HD-5), the most abundant AMP in the small intestine. Quantitative PCR was used to measure transcript levels of various EPEC surface structures. High transcript levels of gfcA, a gene required for group 4 capsule (G4C) production, were observed in EPEC, but not in EHEC. The unencapsulated EPEC ∆gfcA and EHEC wild-type strains were more susceptible to HD-5 than EPEC wild-type. Since the G4C is composed of the same sugar repeats as the lipopolysaccharide O-antigen, an -antigen ligase (waaL) deletion mutant was generated in EPEC to assess its role in HD-5 resistance. The ∆waaL EPEC strain was more susceptible to HD-5 than both the wild-type and ∆gfcA strains. The ∆gfcA∆waaL EPEC strain was not significantly more susceptible to HD-5 than the ∆waaL strain, suggesting that the absence of -antigen influences G4C formation. To determine whether the G4C and -antigen interact with HD-5, total polysaccharide was purified from wild-type EPEC and added to the ∆gfcA∆waaL strain in the presence of HD-5. The addition of exogenous polysaccharide protected the susceptible strain against HD-5 killing in a dose-dependent manner, suggesting that HD-5 binds to the polysaccharides present on the surface of EPEC. Altogether, these findings indicate that EPEC relies on both the G4C and the -antigen to resist the bactericidal activity of HD-5.