Regulation of Viral Replication, Apoptosis and Pro-Inflammatory Responses by 17-AAG during Chikungunya Virus Infection in Macrophages

Abstract

Chikungunya virus (CHIKV) infection has re-emerged as a major public health concern due to its recent worldwide epidemics and lack of control measures. Although CHIKV is known to infect macrophages, regulation of CHIKV replication, apoptosis and immune responses towards macrophages are not well understood. Accordingly, the Raw264.7 cells, a mouse macrophage cell line, were infected with CHIKV and viral replication as well as new viral progeny release was assessed by flow cytometry and plaque assay, respectively. Moreover, host immune modulation and apoptosis were studied through flow cytometry, Western blot and ELISA. Our current findings suggest that expression of CHIKV proteins were maximum at 8 hpi and the release of new viral progenies were remarkably increased around 12 hpi. The induction of Annexin V binding, cleaved caspase-3, cleaved caspase-9 and cleaved caspase-8 in CHIKV infected macrophages suggests activation of apoptosis through both intrinsic and extrinsic pathways. The pro-inflammatory mediators (TNF and IL-6) MHC-I/II and B7.2 (CD86) were also up-regulated during infection over time. Further, 17-AAG, a potential HSP90 inhibitor, was found to regulate CHIKV infection, apoptosis and pro-inflammatory cytokine/chemokine productions of host macrophages significantly. Hence, the present findings might bring new insight into the therapeutic implication in CHIKV disease biology.

P38 and JNK Mitogen-Activated Protein Kinases Interact With Chikungunya Virus Non-structural Protein-2 and Regulate TNF Induction During Viral Infection in Macrophages

“…MTT assay was performed to assess the cytotoxicity of SB and SP according to the methods described before (37). Briefly, the Raw264.7 cells were seeded in 96 well plates at a density of 5 × 10 3 cells per well before 18-20 h of drug treatment.…”

“…Raw 264.7 cell line has been well-reported to study CHIKV infection, replication and associated altered host immune responses (31,37). The Raw264.7 cells were seeded in six-well cell culture plates before 18-20 h of infection with around 70% confluency.…”

“…The Raw264.7 cells were seeded in six-well cell culture plates before 18-20 h of infection with around 70% confluency. The cells were infected with the DRDE-06 strain of CHIKV with multiplicity of infection (MOI) 5 as reported previously (37). Briefly, after washing the cells in 1X PBS, the virus was added over confluent monolayer for 2 h in the incubator with manual shaking at an interval of 15 min.…”

“…Recent studies on CHIKV induced immune responses suggest that the host immune system is found to be both beneficiary in one hand by controlling viral infection, whereas deleterious on the other hand by promoting severe inflammatory responses (24)(25)(26)(27)(28). Studies have shown that CHIKV induces different inflammatory cytokines/chemokines (TNF, IL-1β, IL-6, IFNγ, IL-8, and MCP-1) (24,(29)(30)(31)(32)(33)(34)(35)(36)(37), which might be associated with arthritis like pathogenesis during CHIKV infection. In different in vivo systems (both mouse and non-human primates), predominant cellular infiltration of macrophages, monocytes, NK cells and T cells to the site of inoculation and other tissues have been observed (38,39).…”

“…We have shown earlier that CHIKV triggers robust TNF production in the host macrophages, which might be a key mediator of virus induced inflammation (37) and macrophages are identified as the major cellular reservoirs during the late stages of CHIKV infection in macaques (21). However, the precise role of MAPK activation pathways in terms of CHIKV infection and associated robust TNF induction in macrophages (immune cell) remains largely unknown.…”