Y, expression of IL-6 was also significantly reduced in ST1-infected TLR22/2 mice compared to WT mice (Fig. 3F). In contrast, no significant differences were observed between ST7-infected groups of mice for these chemokines/cytokines (Fig. 3A ). TNF gene upregulation was not significantly different between groups of mice infected with either of the strains, confirming microarray results (Fig. 3G). Production of high levels of IFN-c has been associated to the STSLS induced by the ST7 strain [13,24]. Expression of this cytokine was not significantly dependent on the presence of TLR2 for either strain of S. suis (Fig. 3H).TLR2-Independent Activation by S. suisFigure 3. Proinflammatory cytokines and chemokines gene expression in TLR22/2 or 166518-60-1 site wild-type mice infected with ST1 or ST7 strain. Proinflammatory cytokine and chemokine gene expression in TLR22/2 mice infected with S. suis highly virulent strain ST1 is lower than wild-type counterparts, whereas no major differences are observed between epidemic strain ST7-infected groups. Quantitative PCR analysis of cytokine and chemokine gene expression in C57BL/6 and TLR22/2 mice infected with 16107 CFU of either S. suis strain P1/7 (ST1) or SC84 (ST7). Total RNA was isolated from spleen samples at 6 h post-infection. Tested cytokines and chemokines are indicated at the top of each panel. Data represent mean mRNA Title Loaded From File relative fold induction values 6 SEM. * P,0.05, indicates TLR22/2 group significantly different from corresponding C57BL/6 mice as determined by t-test. doi:10.1371/journal.pone.0065031.gTLR2-Independent Activation by S. suisST1 and ST7 differences in TLR2 dependency are also observed at chemokine and cytokine protein levelsProtein expression of proinflammatory chemokines and cytokines is tightly regulated at transcriptional and post-transcriptional levels. A Luminex assay was thus performed to confirm qPCR results. Similarly to qPCR and/or microarray results, protein levels of CCL2, CCL3, CCL4, CXCL1 and IL-6 were significantly lower only in TLR22/2 mice infected with the ST1 strain than in WT counterparts (Fig. 4A , E, F). CXCL2 expression was also significantly different between these two groups of mice. However, a slight reduction of this chemokine expression was also noticeable in ST7-infected TLR22/2 mice when compared to WT mice (Fig. 4D). Although not detected either by microarray or qPCR, TNF protein expression was clearly and significantly reduced in ST1-infected TLR22/2 mice, whereas no significant changes were observed in mice infected with the ST7 strain (Fig. 4G). Protein expression of IFN-c was also monitored and no significant differences were observed in infected TLR22/2 mice when compared to WT mice, confirming gene expression results (Fig. 4H).DiscussionInflammation plays an important role in the pathogenesis of S. suis-induced septicemia and meningitis [3]. Cases in humans and pigs with presence of shorter incubation time, more rapid disease progression, and a higher rate of mortality have been lately described and may be due, in part, to enhanced induction of inflammation. TLRs are critical sensors in detecting infections and initially activate the innate immune system. S. suis is a pathogen that, in acute bacterial infections, will induce an exacerbated inflammatory reaction that contributes to the disease [23]. Within the family of TLRs, TLR2 has been implicated as the major pattern recognition receptor for ligands derived from Grampositive bacteria [27?9]. In the cas.Y, expression of IL-6 was also significantly reduced in ST1-infected TLR22/2 mice compared to WT mice (Fig. 3F). In contrast, no significant differences were observed between ST7-infected groups of mice for these chemokines/cytokines (Fig. 3A ). TNF gene upregulation was not significantly different between groups of mice infected with either of the strains, confirming microarray results (Fig. 3G). Production of high levels of IFN-c has been associated to the STSLS induced by the ST7 strain [13,24]. Expression of this cytokine was not significantly dependent on the presence of TLR2 for either strain of S. suis (Fig. 3H).TLR2-Independent Activation by S. suisFigure 3. Proinflammatory cytokines and chemokines gene expression in TLR22/2 or wild-type mice infected with ST1 or ST7 strain. Proinflammatory cytokine and chemokine gene expression in TLR22/2 mice infected with S. suis highly virulent strain ST1 is lower than wild-type counterparts, whereas no major differences are observed between epidemic strain ST7-infected groups. Quantitative PCR analysis of cytokine and chemokine gene expression in C57BL/6 and TLR22/2 mice infected with 16107 CFU of either S. suis strain P1/7 (ST1) or SC84 (ST7). Total RNA was isolated from spleen samples at 6 h post-infection. Tested cytokines and chemokines are indicated at the top of each panel. Data represent mean mRNA relative fold induction values 6 SEM. * P,0.05, indicates TLR22/2 group significantly different from corresponding C57BL/6 mice as determined by t-test. doi:10.1371/journal.pone.0065031.gTLR2-Independent Activation by S. suisST1 and ST7 differences in TLR2 dependency are also observed at chemokine and cytokine protein levelsProtein expression of proinflammatory chemokines and cytokines is tightly regulated at transcriptional and post-transcriptional levels. A Luminex assay was thus performed to confirm qPCR results. Similarly to qPCR and/or microarray results, protein levels of CCL2, CCL3, CCL4, CXCL1 and IL-6 were significantly lower only in TLR22/2 mice infected with the ST1 strain than in WT counterparts (Fig. 4A , E, F). CXCL2 expression was also significantly different between these two groups of mice. However, a slight reduction of this chemokine expression was also noticeable in ST7-infected TLR22/2 mice when compared to WT mice (Fig. 4D). Although not detected either by microarray or qPCR, TNF protein expression was clearly and significantly reduced in ST1-infected TLR22/2 mice, whereas no significant changes were observed in mice infected with the ST7 strain (Fig. 4G). Protein expression of IFN-c was also monitored and no significant differences were observed in infected TLR22/2 mice when compared to WT mice, confirming gene expression results (Fig. 4H).DiscussionInflammation plays an important role in the pathogenesis of S. suis-induced septicemia and meningitis [3]. Cases in humans and pigs with presence of shorter incubation time, more rapid disease progression, and a higher rate of mortality have been lately described and may be due, in part, to enhanced induction of inflammation. TLRs are critical sensors in detecting infections and initially activate the innate immune system. S. suis is a pathogen that, in acute bacterial infections, will induce an exacerbated inflammatory reaction that contributes to the disease [23]. Within the family of TLRs, TLR2 has been implicated as the major pattern recognition receptor for ligands derived from Grampositive bacteria [27?9]. In the cas.
