Values of 0.05 were regarded as significant. Open in a separate window Fig 10 Compound E567 inhibits both LCMV and other TLR ligands induced cytokine production and viral replication in cell culture and in the mouse. While TLRs are important in initiating the protective innate immune response, TLRs have also been demonstrated to cause disease by virtue of their ability to stimulate inflammatory responses (Baenziger et al., 2008; Kurt-Jones et al., 2004; Wang et al., 2004). mouse cell cultures. We also demonstrated that E567 inhibits cytokine responses in the mouse. Remarkably, E567 is also capable of inhibiting LCMV replication in mice. This is a new model for developing drugs for use in treating viral illnesses. and studies comparing the effect of E567 on cytokine responses, the differences between E567 and DMSO were evaluated using the two-tailed Student`s test. For the study examining the effects of E567 on virus replication in individual mice (Fig 10C), means, medians and percentiles were estimated to describe the distribution of titer values. An F-test was used to compare variances between groups (Pagano)(Gauvreau and Pagano, 1993). Two sample t-tests using unequal variances were used to test means between groups. Quantile regression with bootstrapped standard errors was used to compare 25 percentiles (Gould). Results were expressed as means+/-standard deviation. Values of 0.05 were regarded as significant. Open in a separate window Fig 10 Compound E567 inhibits both LCMV and other TLR ligands induced cytokine production and viral replication in cell culture and in the mouse. While TLRs are important in initiating the protective innate immune response, TLRs have also been demonstrated to cause disease by virtue of their ability to stimulate inflammatory responses (Baenziger et al., 2008; Kurt-Jones et al., 2004; Wang et al., 2004). TLR-mediated responses to the invading microbial pathogens can lead to the production of excessive chemokines and cytokines and cause immunopathology (Finberg and Wang, 2009; O’Neill, 2006). Thus TLR signaling inhibitors might have potential therapeutic beneficial effects in treating viral infection-associated diseases (O’Neill, 2006). TLR antagonists to TLR2, TLR4, nd TLR9 have been developed for clinical use (Czarniecki, 2008; Nakamura et al., 2007; Yamada et al., 2005). TLR4 antagonists have been reported to be able to inhibit TLR4 agonist LPS-induced inflammation in vitro as well as suppress LPS-induced disease in a mouse model (Nakamura et al., 2007). Lipolanthionine peptides have been developed based on the structural characteristics of TLR2 agonist Pam3CSK4. These peptides have been shown to suppress Pam3CSK4-induced inflammation in cell culture, however, the effect of these TLR2 antagonists in vivo has not been reported (Seyberth et al., 2006). The role of TLR inhibitors in inhibition of viral infection-associated inflammation had not been previously explored. Macrophages and monocytes play critical roles in the host response to infection with various microbial pathogens. Both cell types express various TLRs, including high levels of TLR2 (Hornung et al., 2005). Expression of TLR2 is involved in both LCMV and HSV-1-induced production of cytokines / chemokines in these two cell types (Kurt-Jones et al., 2004; Zhou et al., 2005). Our studies demonstrated that Cevimeline hydrochloride compound E567 is able to block both LCMV and HSV-1-induced inflammatory responses in these cell Cevimeline hydrochloride types. Our results revealed that treatment with compound E567 also blocked both Pam2CSK4 (TLR2 ligand) and LPS (TLR4 ligand) induced inflammatory responses in these cell types, but it did not affect the production of cytokines / chemokines induced by either recombinant human TNF- or poly IC in human monocytes (Fig 9CCD) and mouse primary macrophages (Fig 7C, ?,8C).8C). Moreover, we demonstrated that compound E567 blocks both the TLR2 agonist Pam2CSK4 and the TLR4 agonist LPS induced cytokine responses in mice (Fig 10DCF). This is consistent with other published results indicating that a TLR2 signaling inhibitor could affect both TLR2 Cevimeline hydrochloride and TLR4 signaling pathways (Czarniecki, 2008; Nakamura et al., 2007; Yamada et al., 2005). Although the exact mechanisms involved in the effect of E567 on cytokine/chemokine responses in both mouse and human primary cells are not yet fully defined, certain findings are likely to be relevant. Since the adapter proteins MyD88 and MyD88 adapter-like (MAL) (Fitzgerald et al., 2001) are involved in both CASP12P1 the TLR2 and TLR4 signaling pathways, it is possible that compound E567 might target these adapter proteins. Alternatively, E567 could act on other molecules downstream of TLR2 but shared by TLR4 signaling pathways. Compound E567 blocks LCMV-induced cytokine response in a mouse model. The compound E567 inhibited LCMV replication (Fig 10C) and modulated TLR2 signaling. Either or both of the effects could be responsible for the ability of the compound to decrease the cytokine response in vivo in response to LCMV infection. We have previously demonstrated that LCMV induced a potent dose dependent cytokine response that requires live virus (Zhou et al., 2005). Although TLR2 is required for LCMV-induced inflammation, expression of TLR2 is not required for LCMV infectivity (Zhou et al., 2005). So far, although a few publications have described TLR inhibitor small molecule compounds, none of these compounds have been reported to be able to inhibit viral replication both in vitro and in vivo (Czarniecki, 2008; Nakamura et al., 2007; Yamada et.