TRIF-GEFH1-RhoB pathway is involved in MHCII expression on dendritic cells that is critical for CD4 T-cell activation.

Dendritic cells (DC) play a central role in immune responses by presenting antigenic peptides to CD4+ T cells through MHCII molecules. Here, we demonstrate a TRIF-GEFH1-RhoB pathway is involved in MHCII surface expression on DC. We show the TRIF (TIR domain-containing adapter inducing IFNbeta)- but not the myeloid differentiation factor 88 (MyD88)-dependent pathway of lipopolysaccharide (LPS)-signaling in DC is crucial for the MHCII surface expression, followed by CD4+ T-cell activation. LPS increased the activity of RhoB, but not of RhoA, Cdc42, or Rac1/2 in a manor dependent on LPS-TRIF- but not LPS-Myd88-signaling. RhoB colocalized with MHCII+ lysosomes in DC. A dominant-negative (DN) form of RhoB (DN-RhoB) or RhoB's RNAi in DC inhibited the LPS-induced MHCII surface expression. Moreover, we found GEFH1 associated with RhoB, and DN-GEFH1 or GEFH1's RNAi suppressed the LPS-mediated RhoB activation and MHCII surface expression. DN-RhoB attenuated the DC's CD4+ T-cell stimulatory activity. Thus, our results provide a molecular mechanism relating how the MHCII surface expression is regulated during the maturation stage of DC. The activation of GEFH1-RhoB through the TRIF-dependent pathway of LPS in DC might be a critical target for controlling the activation of CD4+ T cells.

Fas ligand induces cell-autonomous IL-23 production in dendritic cells, a mechanism for Fas ligand-induced IL-17 production.

Fas ligand (FasL) has the potential to induce inflammation accompanied by massive neutrophil infiltration. We previously reported that FasL rapidly induces the production of various inflammatory cytokines including IL-1beta and IL-17. In this study, we investigated the mechanism of the FasL-induced IL-17 production. We found that the culture supernatant of mouse resident peritoneal exudate cells (PEC) cocultured with FasL-expressing tumor (FFL) cells induced IL-17 production in freshly isolated resident PEC. Anti-IL-1beta Ab strongly inhibited the IL-17-inducing activity. However, rIL-1beta by itself induced only weak IL-17 production. Intriguingly, anti-IL-12 Ab but not an IL-15-neutralizing agent, IL15R-Fc, strongly inhibited the FasL-induced IL-17-inducing activity. IL-23, which shares the p40 subunit with IL-12, but not IL-12 itself, induced IL-17 production synergistically with IL-1beta in resident PEC. FasL induced the production of IL-23 in PEC in vivo and in vitro, and IL-17 production following the i.p. injection of FFL cells was severely impaired in p40-/- mice, indicating that IL-23 plays an important role in the FasL-induced IL-17 production. FFL also induced the production of IL-23 in bone marrow- or PEC-derived dendritic cells (DCs). Finally, FasL induced only weak p40 production in a mixture of p40-/- and Fas-/- DC, indicating that FasL induces IL-23 production in DC mainly in a cell-autonomous manner.

Involvement of IL-17 in Fas ligand-induced inflammation.

Fas ligand (FasL) has been well characterized as a death factor. However, recent studies revealed that ectopic expression of FasL induces inflammation associated with massive neutrophil infiltration. We previously demonstrated that the neutrophil infiltration-inducing activity of FasL is partly dependent on, but partly independent of, IL-1beta. Here we investigated the cytokine profile of peritoneal lavage fluid obtained from mice that received i.p. injections of FFL, a FasL-expressing tumor cell line. We found that FFL injection caused a marked increase of not only IL-1beta but also IL-6, IL-17, IL-18, KC/chemokine CXC ligand 1 and macrophage inflammatory protein (MIP)-2, but not of IL-1alpha, IFN-gamma, TGF-beta or TNF-alpha. The FFL-induced cytokine production was not observed in Fas-deficient lpr mice. Among cells transfected to express individually IL-1beta, IL-6, IL-17, or IL-18, only those expressing IL-1beta and IL-17 induced neutrophil infiltration. In these analyses, as little as 20 pg of peritoneal IL-17 induced neutrophil infiltration. The peritoneal IL-17 levels after FFL-injection were greatly diminished in IL-1-deficient mice. However, the IL-17 level was still above the threshold for neutrophil infiltration. Consistent with this, co-administration of the anti-IL-17 antibody with FFL diminished the peritoneal KC levels and neutrophil infiltration in IL-1-deficient mice. In addition, the expression of IL-17 by the tumor cells inhibited tumor growth in wild-type and nude mice. These results indicate that FasL is an upstream inflammatory factor that induces a variety of other inflammatory cytokines in vivo, and suggest that IL-17 is involved in FasL-induced inflammation in the absence of IL-1beta.

Pathogen-associated molecular patterns sensitize macrophages to Fas ligand-induced apoptosis and IL-1 beta release.

Antigenic stimulation activates T cells and simultaneously destines them to die by Fas-mediated apoptosis. In this study, we demonstrated that various pathogen-associated molecular patterns up-regulated Fas expression in macrophages and sensitized them specifically to Fas ligand (FasL), but not to other apoptosis-inducing agents such as TNF-alpha, etoposide (VP-16), and staurosporine. Toll-like receptor, NF-kappaB, and p38 mitogen-activated protein kinase mediated these responses. LPS stimulation induced the expression of Fas, caspase 8, cellular FLIP Bfl-1/A1, and Bcl-x, but not FasL, TNFR p55, Bak, Bax, and Bad at the transcriptional level. Thus, LPS selectively induced the expression of apoptotic molecules of the Fas death pathway (except for cellular FLIP) and antiapoptotic molecules of the mitochondrial death pathway. However, the kinetics of macrophage disappearance following Escherichia coli-induced peritonitis was similar between wild-type and Fas-deficient mice, suggesting that Fas is not essential for the turnover of activated macrophages in T cell-independent inflammation. In contrast, LPS-activated macrophages produced a large amount of IL-1beta upon FasL stimulation. Thus, unlike the activation-induced cell death of T cells, the sensitization of macrophages to FasL by pathogen-associated molecular patterns seems to be a proinflammatory rather than an anti-inflammatory event.