TLR4 is one of the receptors for Chikungunya virus envelope protein E2 and regulates virus induced pro-inflammatory responses in host macrophages

by Selleckchem | Jun 11 2023

Toll like receptor 4 (TLR4), a pathogen-associated molecular pattern (PAMP) receptor, is known to exert inflammation in various cases of microbial infection, cancer and autoimmune disorders. However, any such involvement of TLR4 in Chikungunya virus (CHIKV) infection is yet to be explored. Accordingly, the role of TLR4 was investigated towards CHIKV infection and modulation of host immune responses in the current study using mice macrophage cell line RAW264.7, primary macrophage cells of different origins and in vivo mice model. The findings suggest that TLR4 inhibition using TAK-242 (a specific pharmacological inhibitor) reduces viral copy number as well as reduces the CHIKV-E2 protein level significantly using p38 and JNK-MAPK pathways. Moreover, this led to reduced expression of macrophage activation markers like CD14, CD86, MHC-II and pro-inflammatory cytokines (TNF, IL-6, MCP-1) significantly in both the mouse primary macrophages and RAW264.7 cell line, in vitro. Additionally, TAK-242-directed TLR4 inhibition demonstrated a significant reduction of percent E2-positive cells, viral titre and TNF expression in hPBMC-derived macrophages, in vitro. These observations were further validated in TLR4-knockout (KO) RAW cells. Furthermore, the interaction between CHIKV-E2 and TLR4 was demonstrated by immuno-precipitation studies, in vitro and supported by molecular docking analysis, in silico. TLR4-dependent viral entry was further validated by an anti-TLR4 antibody-mediated blocking experiment. It was noticed that TLR4 is necessary for the early events of viral infection, especially during the attachment and entry stages. Interestingly, it was also observed that TLR4 is not involved in the post-entry stages of CHIKV infection in host macrophages. The administration of TAK-242 decreased CHIKV infection significantly by reducing disease manifestations, improving survivability (around 75%) and reducing inflammation in mice model. Collectively, for the first time, this study reports TLR4 as one of the novel receptors to facilitate the attachment and entry of CHIKV in host macrophages, the TLR4-CHIKV-E2 interactions are essential for efficient viral entry and modulation of infection-induced pro-inflammatory responses in host macrophages, which might have translational implication for designing future therapeutics to regulate the CHIKV infection.

Comments:

The study investigated the role of Toll-like receptor 4 (TLR4) in Chikungunya virus (CHIKV) infection and its impact on host immune responses. The researchers used a mouse macrophage cell line (RAW264.7), primary macrophages from different sources, and a mouse model to examine the involvement of TLR4 in CHIKV infection.

The findings of the study demonstrated that inhibiting TLR4 using a specific pharmacological inhibitor called TAK-242 resulted in a significant reduction in viral copy number and CHIKV-E2 protein levels. This inhibition was achieved by modulating the p38 and JNK-MAPK pathways. Furthermore, the expression of macrophage activation markers (CD14, CD86, MHC-II) and pro-inflammatory cytokines (TNF, IL-6, MCP-1) was significantly reduced in both mouse primary macrophages and the RAW264.7 cell line when TLR4 was inhibited.

The researchers also investigated the effects of TAK-242-mediated TLR4 inhibition on CHIKV infection in human peripheral blood mononuclear cell (hPBMC)-derived macrophages in vitro. They observed a significant reduction in the percentage of E2-positive cells, viral titers, and TNF expression upon TLR4 inhibition.

To further validate the involvement of TLR4 in CHIKV infection, the researchers utilized TLR4-knockout (KO) RAW cells. The results confirmed that TLR4 is necessary for the early stages of CHIKV infection, particularly during viral attachment and entry into host macrophages. However, TLR4 was found to be not involved in the post-entry stages of CHIKV infection.

The study also provided evidence for the interaction between CHIKV-E2 and TLR4 through immuno-precipitation studies in vitro and molecular docking analysis in silico. Additionally, an anti-TLR4 antibody-mediated blocking experiment supported the role of TLR4 in viral entry.

In the in vivo experiments using a mouse model, administration of TAK-242 significantly reduced CHIKV infection, improved survivability (approximately 75%), and decreased inflammation.

In conclusion, this study is the first to report TLR4 as a novel receptor involved in facilitating the attachment and entry of CHIKV into host macrophages. The TLR4-CHIKV-E2 interactions were found to be crucial for efficient viral entry and modulation of infection-induced pro-inflammatory responses in host macrophages. These findings may have translational implications for the development of future therapeutics targeting TLR4 to regulate CHIKV infection.