No abstract

Protein kinase D (PKD), also called protein kinase C (PKC)μ, is a serine-threonine kinase that is involved in diverse areas of cellular function such as lymphocyte signaling, oxidative stress, and protein secretion. After identifying a putative PKD phosphorylation site in the Toll/IL-1R domain of TLR5, we explored the role of this kinase in the interaction between human TLR5 and enteroaggregative Escherichia coli flagellin in human epithelial cell lines. We report several lines of evidence that implicate PKD in TLR5 signaling. First, PKD phosphorylated the TLR5-derived target peptide in vitro, and phosphorylation of the putative target serine 805 in HEK 293T cell-derived TLR5 was identified by mass spectrometry. Furthermore, mutation of serine 805 to alanine abrogated responses of transfected HEK 293T cells to flagellin. Second, TLR5 interacted with PKD in coimmunoprecipitation experiments, and this association was rapidly enhanced by flagellin treatment. Third, pharmacologic inhibition of PKC or PKD with Gö6976 resulted in reduced expression and secretion of IL-8 and prevented the flagellin-induced activation of p38 MAPK, but treatment with the PKC inhibitor Gö6983 had no significant effects on these phenotypes. Finally, involvement of PKD in the p38-mediated IL-8 response to flagellin was confirmed by small hairpin RNA-mediated gene silencing. Together, these results suggest that phosphorylation of TLR5 by PKD may be one of the proximal elements in the cellular response to flagellin, and that this event contributes to p38 MAPK activation and production of inflammatory cytokines in epithelial cells.

Together, these data indicate that stress, in the form of extracellular ATP, reshapes both the inflammatory response of flagellin-stimulated IECs and downstream adaptive immunity, representing a possible strategy by which these cells differentiate between commensal and pathogenic bacteria.

Mycobacterium leprae-specific and polyclonal immunoglobulin G (IgG) subclass and IgE antibodies in leprosy patients across the histopathological spectrum were determined by using a quantitative enzyme-linked immunosorbent assay. Antibody responses to M. leprae sonicates were detected only in IgG1,-2, and-3 subclasses. Even at 100-times-lower dilutions, very little IgG4 and IgE antibody activity against M. leprae was detected in any group of leprosy patients. Quantitatively, antibody responses were highest at the lepromatous pole and decreased towards the tuberculoid pole. The greatest quantitative difference in antibodies between the lepromatous and tuberculoid poles was observed with IgG1 (140-fold), this was followed by the difference with IgG3 antibodies (32-fold). Polyclonal antibodies, on the other hand, were elevated for all four IgG subclasses as well as IgE in both lepromatous and tuberculoid leprosy patients compared with healthy controls from a leprosy-endemic area. Selective elevation of M. leprae-specific antibody responses in IgG1 and IgG3 subclasses, therefore, could not be attributed to selective polyclonal activation in these particular subclasses. Furthermore, polyclonal activation for IgE was observed in both lepromatous and tuberculoid leprosy patients, with higher levels in the tuberculoid group, which does not support selective TH2 activation in lepromatous leprosy patients. IgG1 and IgG3 antibodies also showed the highest Spearman rank correlation with the bacterial index in these patients (rho ‫؍‬ 0.748 and P < 0.001 for IgG1; rho ‫؍‬ 0.721 and P < 0.001 for IgG3). Thus, disease progression in leprosy showed a significant correlation with selective increases in IgG1 and IgG3 responses.