pestisstrain lacking LcrV (lcrv Y. (reviewed in (Kalinski et al., 1999)). For instance, differentiation of pro-inflammatory IFN- Th1 cell or anti-inflammatory T regulatory type 1 (Tr1) cells producing selectively IL-10 (Groux, 2003) is linked to the ratio of IL-12 to IL-10 produced by DC (reviewed in (Kalinski et al., 1999)). The concept, that production of high levels of IL-10 by DC is not sufficient to induce Tr1 cells, is well illustrated by the observation that DC stimulated by LPS promote differentiation of Th1 cells even though they secrete high levels of IL-10 (Pulendran et al., 2001;Re and Strominger, 2004). In contrast TLR2 ligands, such as zymosan, induce high IL-10 producing tolerogenic DC which promote differentiation of Tr1 cells (Dillon et al., 2006). However, intriguingly, other groups have shown that TLR2 ligands provide a strong inflammatory signal and promote the development of Th1 cells (Cleveland et al., 1996). The signals leading to the differentiation of tolerogenic DC and the induction of Tr1 cells hence remain poorly understood. Interestingly, TLR2 is a promiscuous TLR that can form heterodimers with TLR6 and TLR1 (Takeda et al., GSK-2033 2003;Triantafilou et al., 2006) recognizing triacylated or diacylated lipoproteins, respectively (Takeda et al., 2002;Takeuchi et al., 2001). Whether the ability of TLR2 to induce tolerogenic DC and Tr1 cells is determined by the TLR with which it associates remains to be determined. An effective evasion strategy by a pathogen would be to target DC and educate them to become tolerogenic and prime regulatory IL-10 response that block inflammation and allow the pathogen to multiply without restraint.Yersinia pestis, the causative agent of bubonic plague, must replicate to a high density in the blood such that fleas can take up sufficient numbers of bacteria and enable transmission to a new host. Interestingly, induction of IL-10 by theYersiniavirulence factor LcrV (V-antigen) is reported to suppress macrophage activation (Overheim et al., 2005b;Sing et al., 2002) and production of inflammatory cytokines (Brubaker, 2003;Nakajima et al., 1995). However the immune-modulatory role of LcrV remains controversial for several reasons. LcrV is a multi-functional virulence factor that is encoded on a 70kB virulence plasmid (pYV), which is part of the type III secretion machinery (T3SS) that allows for the injection ofYersiniaOuter Proteins (YOPs) directly into the host cell cytosol. TNK2 These YOPS inhibit phagocytosis, block NFkB activation and prevent the release of chemoattractants by immune cells (Cornelis, 2002). Thus it is difficult in that context to determine whether LcrV has direct immune-modulatory functions. Furthermore, it is also argued that the synthesis of GSK-2033 a lipopolysaccharide (LPS)-lipid A with poor TLR4 stimulating activity is the dominant strategy used byY. pestisto prevent GSK-2033 the development of a protective inflammatory response (Montminy et al., 2006b). Finally,in vitroexperiments suggest that LcrV interacts with TLR2 and CD14 to induce IL-10 (Sing et al., 2002). However, TLR2/ mice show no resistance to plague (Pouliot et al., 2007). Using complementary approaches we address the controversial issue as to whether LcrV may play immune-modulatory functions that contribute to plague pathogenesis. Furthermore, using LcrV as a model, we establish the molecular basis underlying TLR-mediated education of tolerogenic DC and the apparently contradictory pro- and anti-inflammatory functions of TLR2. Ultimately, our results reveal an unexpected role of TLR6 in the education of tolerogenic DC andYersinia pestispathogenesis. == RESULTS == == LcrV blocks the development of an inflammatory response during plague infection == To evaluate the role of regulatory cytokines in a model of bubonic plague we subcutaneously (s.c.) infected mice deficient for IL-10, IL-4 and their appropriate littermate controls with 10LD50 plague strain Colorado-92 (CO92). Interestingly, mice lacking IL-10, but not IL-4, were fully protected from disease. This protection correlated with a significant reduction in bacterial burden and splenic IFN- levels (Fig. 1A). Altogether these results suggest that IL-10 plays a significant role in plague pathogenesis by blocking the development of a Th1 response. == Figure 1. IL-10 induction byY. pestisLcrV contributes to virulence. == (A) IL-10/, IL-4/ or littermate controls were infected s.c. with 10LD50 (10cfu)Y. pestisstrain CO92. Survival was monitored. *,p<0.002 by Wilcoxon rank test. At Day 3 post-infection the amount of plague and IFN- levels.