We could not confirm the inhibitory effect of Th3 cells on immune responses at inflammatory sites, as TGF-β1 mRNA expression did not correlate with the frequency of sensitization or dose in this antigen induced inflammation model. CD4+CD25+T cells
express cytotoxic T-lymphocyte antigen 4 (CTLA-4) with membrane-associated TGF-β on the cell surface, which suppresses multiplication of positive effector T cells by direct cytoadherence [33, 34]. Foxp3, a master regulatory gene is constitutively expressed in CD4+CD25+T cells , and both Tr1 and Th3 cells AZD2171 are negative for Foxp3 [36, 37]. It was assumed that intrapulmonary Foxp3 mRNA expression is not increased as drastically in comparison with IL-10, as frequent and large quantity sensitization with M. pneumoniae antigens induced CD4+CD25+T cell translocation from thymus to the
lung. LY3023414 mouse Additionally, we performed an in vitro analysis aimed to evaluate the specificity of immuno-inducibility and Th17-differentiation enhancability of M. pneumoniae antigens. It was reported that IL-6 and TGF-β1 are necessary for early differentiation of the Th17 cell from naïve T cells . Therefore, mouse lymphocytes were primed with M. pneumoniae antigens in the presence of IL-6 and TGF-β1. Furthermore, in order to simulate the presentation of M. pneumoniae antigens by dendritic cells in vitro, we added VS-4718 anti-CD3 antibodies and anti-CD28 antibodies. Compared to saline control, 50 μg protein/ml of M. pneumoniae antigen stimulation significantly induced IL-17A production by mouse lymphocytes from day 2 to 5, with greater than sixfold production observed on day 3 (Figure 4a). Additionally, IL-10 production showed a significant increase from day 1 to 5 (Figure 4b). This showed that IL-17A and IL-10 production in vitro induced by M. pneumoniae antigen sensitization mirrored the in vivo antigen induced inflammation model. When we compared viable cell count at the peak of IL-17A and IL-10 production on day 4, 50 μg protein/ml of M. pneumoniae antigens induced multiplication of mouse lymphocytes approximately twofold compared to saline control. Though mildly increased growth rates were observed
in the presence of IL-6 and TGF-β1, higher concentrations of M. pneumoniae antigens induced activation and Teicoplanin proliferation of lymphocytes (Table 1). IL-17A and IL-10 production were enhanced in a concentration-dependent manner by M. pneumoniae antigens, and the presence of IL-6 and TGF-β1 led to further production of IL-17A and IL-10 (Figures 5a, 6a), showing induction of the two genes under a Th17 dominant immune balance both in vivo and in vitro. With respect to the effects of antigens prepared from bacteria causing a classical pneumonia, 50 μg protein/ml of S. pneumoniae sonicated antigens imposed a lethal effect on lymphocytes, with decreased viability to 18% of saline control, possibly through the effect of pneumolysin (Table 1). S.