Figures & data
Figure 1. Hsp70 treatment decreases basal levels of TNF-α, IFN-γ, MCP-1 and down-regulates C/EBPβ and C/EBPδ. BMDCs were treated with OVA (30 μg/mL), Hsp70 (30 μg/mL) or LPS (500 ng/mL) for 24 h. Supernatants were analysed for (A) TNF-α, (B) IFN-γ, (C) MCP-1 using a CBA mouse inflammation kit. (D) C/EBPβ and (E) C/EBPδ expression evaluation by qPCR in BMDCs treated as described in A. β-actin was used as a normaliser as described in Materials and methods. *p < 0.05; **p < 0.01; ***p < 0.001. Experiments were performed three times in triplicates.
Figure 2. Pro-inflammatory cytokine inhibition and C/EBPβ and C/EBPδ down-regulation induced by Hsp70 is dependent on TLR2. WT or TLR2 KO BMDCs were treated with OVA, TBHsp70 or PGN for 24 h. Cytokines in the supernatants were analysed using a CBA mouse inflammation kit: (A) TNF-α, (B) IFN-γ and (C) MCP-1; (D) C/EBPβ and (E) C/EBPδ expression evaluation by qPCR in WT or TLR2 KO BMDCs treated with Hsp70 or left unstimulated for 24 h. β-actin was used as a normaliser as described in Materials and methods. *p < 0.05; **p < 0.01; ***p < 0.001. Experiments were performed three times in triplicates.
Figure 3. ERK and STAT3 are required for Hsp70-induced pro-inflammatory cytokines inhibition and C/EBPβ and C/EBPδ down-regulation. BMDCs from WT mice were treated with inhibitors of ERK PD98059 or JAK2/STAT3 AG490 for 1 h prior to stimulation. After that, cells were treated with OVA, Hsp70, LPS or left unstimulated for 24 h. Culture supernatants were analysed for the presence of (A) TNF-α, (B) IFN-γ, and (C) MCP-1 using a CBA inflammation kit, (D) C/EBPβ and (E) C/EBPδ expression evaluation by qPCR. β-actin was used as a normaliser as described in Materials and methods. *p < 0.05; **p < 0.01; ***p < 0.001. #p < 0.05, ##p < 0.01 when compared with Hsp70. Experiments were performed three times in triplicates.
Figure 4. IL-10 is necessary for Hsp70 anti-inflammatory effects. BMDCs from WT or IL-10 KO mice were treated with OVA, Hsp70, PGN or left unstimulated for 24 h. Cytokines in the supernatants were analysed using a CBA mouse inflammation kit: (A) TNF-α, (B) IFN-γ and (C) MCP-1; (D) C/EBPβ and (E) C/EBPδ expression evaluation by qPCR. β-actin was used as a normaliser as described in Materials and methods. *p < 0.05; **p < 0.01; ***p < 0.001. #p < 0.05 and ##p < 0.01 when compared with Hsp70. Experiments were performed two or three times in triplicates.
