Fig 2: The presence of functional TLR4 facilitates CHIKV infection in host macrophages, in vitro. RAW264.7 and TLR4KO RAW cells were subjected to CHIKV infection at 5 MOI and harvested at 8 hpi (A, B) The flow cytometry dot plot analysis depicts comparative CHIKV-E2 expression. (C, D) Western blot analysis showing comparative E2 level. Normalization of E2 expression was done using β-actin as a housekeeping gene. (E) The bar diagram showing comparative CHIKV titre obtained from plaque assay (F–K) The flow cytometry dot plot-based bar diagram analysis showing percent positive cells expressing surface TLR4, total TLR4, CD14, CD86, MHC-II and p- NF-κB respectively in mock and CHIKV infected TLR4KO RAW cells. (L–N) Bar diagrams depicting ELISA-based TNF-α, IL-6 and MCP-1 quantification respectively in RAW 264.7 and TLR4KO RAW cells. Data represent the Mean ± SEM of three independent experiments. p< 0.05 was considered as a statistically significant difference between the groups (ns: non-significant, *p <0.05; **p ≤0.01; ***p ≤0.001; ****p ≤0.0001.

Fig 3: TLR4 promotes viral entry at the early stages of CHIKV infection in host macrophages, in vitro. (A) TLR4 inhibition before CHIKV infection is most effective to regulate viral copy number at 8 hpi (B, C) Viral entry assay in RAW 264.7 cells showing the internalization of around 24% and 11% less virus in TAK-242 treated condition using plaque assay-based viral titre determination and q-RT PCR based viral copy number determination respectively. (D) Time of addition assay in RAW 264.7 cells showed no significant decrease in viral infection during post-infection treatment. (E) TAK-242 pre-treatment decreases CHIKV copy number in different time points inside the RAW264.7 macrophage cells. (F) Post-infection TLR4 inhibition (TAK-242 was added at 0 hpi) does not have a role in CHIKV E1 gene transcription in the RAW264.7 cells. (G, H) Post-infection TLR4 inhibition (TAK-242 was added at 0 hpi) does not have a role in CHIKV-E2 translation in the RAW264.7 cells. The densitometry was performed with respect to the corresponding GAPDH expression. Data represent the Mean ± SEM of three independent experiments. p< 0.05 was considered as a statistically significant difference between the groups (ns: non-significant, *p <0.05; **p ≤0.01; ***p ≤0.001; ****p ≤0.0001).

Fig 4: TLR4 inhibition lowers p38 and SAPK-JNK phosphorylation in host macrophages, in vitro. RAW264.7 cells were either pre-treated with DMSO or TAK-242 for 3 h before CHIKV infection. The CHIKV infection was given at 5 MOI for 2 h followed by the cells were harvested at 8 hpi. (A–D) Western blot analysis showing differential expression of p-P38, p-SAPK-JNK, E2 and their quantification normalized against GAPDH, in respective order. (E, F) Western blot analysis showing TLR4 expression with the corresponding quantification normalized against GAPDH. Data represent the Mean ± SEM of three independent experiments. p< 0.05 was considered as a statistically significant difference between the groups (ns: non-significant, *p <0.05; **p ≤0.01; ***p ≤0.001).

Fig 5: Pre-incubation with anti-TLR4 antibody alleviates CHIKV infection in host RAW264.7 macrophages, in vitro. Before pre-incubation of the RAW264.7 cells with either DMSO or TAK-242, the anti-TLR4 antibody or anti-R-IgG antibody was added in the pre-incubation volume in respective conditions at 4 μg/ml concentration and the cells from all conditions were preincubated for 3 h. The CHIKV infection was given at 5 MOI for 2 h and the cells were harvested at 8 hpi. (A, B) The flow cytometry dot plot analysis shows comparative CHIKV-E2 levels at different conditions. (C, D) Western blot analysis shows E2 expression in different experimental conditions. (E, F) Western blot analysis shows differential E1 expression. All densitometric quantifications were performed with respect to GAPDH. (G) The bar diagram represents ELISA-based cytokine analysis of TNF.