The Impact of Cytokines and HSV-1 on Rab5 Protein Expression, F-actin Cytoskeleton Rearrangement, and Cell Viability of Uninfected and Virus-Infected M0, M1, and M2 RAW264.7 Murine Macrophages

The endocytic pathway in all eukaryotic cells is necessary to maintain cellular functions, such as initiating transport of intracellular cargos and ingesting pathogens. The main regulator of this process is a member of small GTPase family, Rab5 protein. Rab5 protein plays a key role in the endocytic dynamic delivery of molecules, receptors, and pathogens from the cell membrane to cytoplasmic vesicles.as well as in the exocytic delivery of cellular products to the cell\u27s exterior (Bonifacino & Glick, 2004). Many pathogens have exploited this protein to enter cells. Herpes Simplex Virus Type 1 (HSV-1) enters most cells by fusion or utilizes the endocytic pathway using Rab5 protein (Spearman, 2017). Also, HSV-1 depends on Rab5 in the enveloping process to produce mature viral particles (Hollinshead, 2012). F-actin is a major microfilament of the cell\u27s cytoskeleton, aiding migration to the site of infection, muscles contraction, and cell division (Khadijeh, Amir, & Maryam, 2015). As a barrier, F-actin also protects the cellular organelles within the cytoplasm (Vitale, Seward, & Trifaro, 1995). The goals of this study were to determine the effect of the HSV-1 infection on Rab5 expression in RAW 264.7 murine macrophages and on F-actin distribution at 2, 4, 6, and 24 hours or late time at 24 hours after polarization of the macrophages to M1, M2a, and M2c phenotypes. M1 macrophages were polarized by interferon gamma (IFN-¿) and lipopolysaccharides (LPS). Unpolarized cells (M0) were converted to M2a phenotype by treating them with interleukin 4 (IL-4). M2c phenotype was polarized with interleukin 10 (IL-10). HSV-1 infection upregulated Rab5 protein expression at 2-6 hours in both M2a and M2c phenotypes but not in M1 polarized macrophages. The Impact of Cytokine Treatments and HSV-1 on Rab5 Protein Expression, F-actin Cytoskeleton Rearrangement, and Cell Viability of Uninfected and Virus- Infected M0, M1, and M2 RAW 264.7 Murine Macrophages. increase in Rab5 expression was seen at 24 hours after virus infection in any of the polarized macrophages but was seen in HSV-1 infected unpolarized M0 cells. Both M1 and M2 polarizing agents caused an upregulation in Rab5 expression from 2 to 6 hours after polarization. HSV-1 infection caused a decrease F-actin distribution (staining intensity) levels among test groups at most time points. Polarization caused a decrease in cell viability of M1 macrophage; HSV-1 infection did not enhance this decrease in cell viability after 24 hours. M2 phenotypes, uninfected or HSV-1-infected, did not exhibit any decrease in cell viability at 24 hours. Treatment of HSV-1 phenotypes with M2 polarizing anti-inflammatory cytokines, Il-4 and Il-10, as well as with SOCS3, an inducer of IL-10 expression, enhanced expression of Rab5 and F-actin distribution