F European and North American strains [22]. So far, the production of

F European and North American strains [22]. So far, the production of superantigens could not be proved for the epidemic strain [13]. However, a recent report showed some differences in receptor recognition in vitro between these two strain types [20]. Results observed in the Title Loaded From File present study indicate that TLR2 does not seem to be mainly implicated in the severe symptoms and lethality associated with the ST7 strain. Bacterial loads and induction of most proinflammatory cytokines and chemokines were independent of TLR2. We have recently reported that the higher mortality observed with the ST7 strain compared to ST1 strain was mainly due to a massive secretion of IFN-c by Natural killer cells [24]. In the present study, levels of mRNA and protein of IFN-c were significantly higher in ST7 compared to ST1-infected mice (P ,0.05), confirming our previous observations. However, levels of this mediator were induced at Title Loaded From File similar levels by WT and TLR22/2 ST7-infected mice, which may explain, at least in part, similar levels of mortality in both mouse groups. It is quite possible that this particular strain of S. suis has a better capacity to stimulate other host receptors besides TLR2 during a systemic infection. As mentioned above, other TLR receptors (such as TLR3, TLR6 and TLR9) as well as other non-TLR receptors, such as NLR families, may also be involved in cell activation of this particular strain. So far, virulence factors that would be present in this strain (but absent in ST1 strains) that may be responsible for such differences have not been clearly identified [8]. The implication of lipoteichoic acid (LTA) in the activation of TLRs is still controversial [29,34]. For GBS for example, it has been clearly demonstrated that secreted lipoproteins but not LTA are essential for TLR2 activation [35]. Cell-wall associated lipoproteins of S. suis have been suggested as being implicated on TLR2/6 but not TLR1/2 cell activation and this effect varies depending on the strain and serotype [30]. Further studies using isogenic mutants clearly demonstrated that lipoproteins are potent and dominant innate immunity activating molecules of S. suis [36]. These lipoproteins can be released after penicillin treatment or directly released in the supernatant [36]. However, receptors involved in the activation of these released bacterial components have not been elucidated. A recent study showed that differences on in vitroTLR2-Independent Activation by S. suisFigure 4. Production of proinflammatory cytokines and chemokines in TLR22/2 or wild-type mice infected with ST1 or ST7 strain. Production of proinflammatory cytokines and chemokines in TLR22/2 mice infected with S. suis highly virulent strain ST1 is lower than in wild type counterparts, whereas no differences are observed when animals are infected with epidemic strain ST7. Plasma levels of cytokines and chemokines in C57BL/6 and TLR22/2 mice infected with 16107 CFU of S. suis strain P1/7 (ST1) or SC84 (ST7) for 6 h, as quantified by Luminex. Plasma samples were tested for the cytokines and chemokines indicated at the top of each panel. Data represent mean values 6 SEM in pg/ml. * P,0.05 indicates statistically significant differences between infected TLR22/2 group and its wild-type infected counterpart as determined by t-test. doi:10.1371/journal.pone.0065031.gTLR activation between an European ST1 and the ST7 epidemic strain were observed also with washed heat killed suspensions, indicating a lower implicat.F European and North American strains [22]. So far, the production of superantigens could not be proved for the epidemic strain [13]. However, a recent report showed some differences in receptor recognition in vitro between these two strain types [20]. Results observed in the present study indicate that TLR2 does not seem to be mainly implicated in the severe symptoms and lethality associated with the ST7 strain. Bacterial loads and induction of most proinflammatory cytokines and chemokines were independent of TLR2. We have recently reported that the higher mortality observed with the ST7 strain compared to ST1 strain was mainly due to a massive secretion of IFN-c by Natural killer cells [24]. In the present study, levels of mRNA and protein of IFN-c were significantly higher in ST7 compared to ST1-infected mice (P ,0.05), confirming our previous observations. However, levels of this mediator were induced at similar levels by WT and TLR22/2 ST7-infected mice, which may explain, at least in part, similar levels of mortality in both mouse groups. It is quite possible that this particular strain of S. suis has a better capacity to stimulate other host receptors besides TLR2 during a systemic infection. As mentioned above, other TLR receptors (such as TLR3, TLR6 and TLR9) as well as other non-TLR receptors, such as NLR families, may also be involved in cell activation of this particular strain. So far, virulence factors that would be present in this strain (but absent in ST1 strains) that may be responsible for such differences have not been clearly identified [8]. The implication of lipoteichoic acid (LTA) in the activation of TLRs is still controversial [29,34]. For GBS for example, it has been clearly demonstrated that secreted lipoproteins but not LTA are essential for TLR2 activation [35]. Cell-wall associated lipoproteins of S. suis have been suggested as being implicated on TLR2/6 but not TLR1/2 cell activation and this effect varies depending on the strain and serotype [30]. Further studies using isogenic mutants clearly demonstrated that lipoproteins are potent and dominant innate immunity activating molecules of S. suis [36]. These lipoproteins can be released after penicillin treatment or directly released in the supernatant [36]. However, receptors involved in the activation of these released bacterial components have not been elucidated. A recent study showed that differences on in vitroTLR2-Independent Activation by S. suisFigure 4. Production of proinflammatory cytokines and chemokines in TLR22/2 or wild-type mice infected with ST1 or ST7 strain. Production of proinflammatory cytokines and chemokines in TLR22/2 mice infected with S. suis highly virulent strain ST1 is lower than in wild type counterparts, whereas no differences are observed when animals are infected with epidemic strain ST7. Plasma levels of cytokines and chemokines in C57BL/6 and TLR22/2 mice infected with 16107 CFU of S. suis strain P1/7 (ST1) or SC84 (ST7) for 6 h, as quantified by Luminex. Plasma samples were tested for the cytokines and chemokines indicated at the top of each panel. Data represent mean values 6 SEM in pg/ml. * P,0.05 indicates statistically significant differences between infected TLR22/2 group and its wild-type infected counterpart as determined by t-test. doi:10.1371/journal.pone.0065031.gTLR activation between an European ST1 and the ST7 epidemic strain were observed also with washed heat killed suspensions, indicating a lower implicat.