Figure 3.

11^th domain of LigA (A₁₁) produces pro-inflammatory cytokines via signalling through MAP kinase involving p38 and JNK pathway. (A) A₁₁ signals through TLR4 involving the MyD88 adapter. WT, MyD88 KO, TRIF KO and TMDKO bone marrow-derived macrophages cell lines were treated with LAV or A₁₁ or A∆₁₁ (2μg/ml) for 24h at 37°C in the presence of 5%CO₂ and levels of IL-6 and TNF-α in the supernatants were measured by ELISA. (B) A₁₁ signals through TLR4 via the MAP kinase pathway involving p38 and NFkb. WT or TLR2KO or TLR4KO or DKO macrophages cell lines were stimulated with A₁₁ (2μg/ml) for 24h at 37°C in the presence of 5%CO₂. Levels of phosphorylated p38, JNK, and ERK1/2 induced by A₁₁ were analyzed by western blot as described in materials and methods. (C) Pharmacological inhibitors of p38 and NFkb significantly reduces A₁₁ mediated cytokine response. RAW 264.7 cells were pre-treated for 30min with NF-kB inhibitor (SN50; 20μM), JNK inhibitor (SP600125; 40μM) or p38MAPK inhibitor (SB203580; 30μM) or ERK (U0126; 50µM) or all four inhibitors together and then stimulated with A₁₁ (2μg/ml) for 24h at 37°C in the presence of 5%CO₂ and supernatant was collected to measure levels of IL-6 and TNF-α by ELISA. (D) Analysis of expression of immune response-related genes in mouse macrophages stimulated with A₁₁. WT, TLR2KO, TLR4KO and DKO mouse macrophage cell lines were treated with 2μg/ml A₁₁ for 4h or 24h or 48h. Cells were recovered, RNA was isolated, converted to cDNA and gene expression was analyzed by RT-PCR as described in material and methods. The data were presented as fold changes between stimulated cells vs control and normalized to GAPDH. E. coli LPS (500ng/ml) or PAM3CSK4 (20ng/ml) as TLR4 and TLR2 ligands respectively were used as positive controls in all experiments wherever indicated. All data are representative of three independent experiments. Significant differences were calculated using the one or two way ANOVA (****, *, ns indicates P < 0.0001, P < 0.05 and non-significant respectively).