The Impact of HSV-1 Infection on Cell Viability, Morphology, and CD Markers Expression by Unpolarized and Cytokine-Polarized J774A.1 Mouse Macrophages
Year: 2015
Type: article
Abstract: The Impact of HSV-1 Infection on Cell Viability, Morphology, and CD Markers Expression by Unpolarized and Cytokine-Polarized J774A.1 Mouse MacrophagesMacrophages play an important role in the immune system, particularly in neutralizing pathogens via phagocytosis and the production of multiple cytokines and chemokines that control infection after exposure to specific stimuli.Macrophages exhibit two different phenotypes, M1 and M2.This study evaluated the role of Suppressors of cytokine signaling (SOCS)1 and SOCS3 on Herpes Simplex Virus (HSV-1) infection of polarized macrophages, cell viability, cell morphology, and the expression of cell surface CD markers.I hypothesized that J774A.1 murine macrophages cells in the naïve state (M0), and M1 and M2 phenotypes would display differences in CD markers CD80,CD163, and CD200R, cell morphology, and viability following HSV-1infection and that the SOCS3 peptide mimetic would reduce the cytotoxicity associated with the M1 phenotype or HSV-1 infection.Unpolarized J774A.1 macrophages (M0) or polarized M1 and M2 phenotypes displayed differences in CD surface proteins at 18, 24, and 48 hours following HSV-1 infection and there was ~22% decrease in viability in M1 macrophages regardless of whether the macrophages were uninfected or infected with HSV-1.M2 macrophages demonstrated ~10% reductions in viability compared with infected M0 macrophage.Differences in cell morphology were noticed in uninfected polarized and unpolarized cells.M1 macrophages displayed irregular shapes and intracellular vacuoles were more pronounced at 24 and 48 hours compared to 18 hours.M0 appeared rounded or elongated, and M2 macrophages induced by IL-4, IL-10, or IL-13 showed elongation with the majority of cells appearing rounded.HSV-1-infected M0, M1 or M2 macrophages all became rounded and aggregated following HSV-1 infection.We found CD80 expression (by flow cytometry) was slightly deceased on uninfected M1 macrophages and M2 macrophages compared to uninfected M0 macrophages and infected M1 macrophages and IL-10-induced M2 macrophages also exhibited a minor increase in CD80.A reduction of CD163 expression occurred on M1 macrophages uninfected or infected with HSV-1.The level of CD163 on infected M2 macrophages was increased compared to uninfected M2 macrophage cells with the level being higher on infected IL-10-induced M2 macrophages.These observations were not statistically significant; however, the level of CD200R significantly increased on infected M2 macrophages polarized by IL-13 compared to CD200R expression in virus-infected M0 macrophages.Treatment with either SOCS3 peptide mimetic or pJAK2 increased the viability of polarized J774A.1 M1 macrophages.Production of pro-inflammatory cytokines TNF-α and IL-6 were reduced in polarized M1 macrophage treated with SOCS3 or pJAK2 peptide mimetic compared with polarized M1 untreated cells.Also, the SOCS3 peptide mimetic increased cell viability in M1 and HSV-1-infected M1 RAW264.7 and J774A.1 macrophages, but this increase was only significant in the RAW264.7 cells.This result suggests that SOCS3 mimetic exerts an anti-inflammatory effect by diminishing the lytic effect of M1 polarization.M1 cells treated with SOCS3 behave similar to the M2 phenotype and produce the anti-inflammatory cytokine IL-10.This study highlights the events that lead to M1 versus M2 polarization and the knowledge gained regarding SOCS3's ability to reduce inflammation may make it a potential therapeutic target for inflammatory diseases. v SPECIFIC AIMS and HYPOTHESISA previous study by Reichard (2012) showed that J774A.1 murine macrophages exhibited variability in cell morphology, viability, and surface proteins expression 24 hours after exposure to IL-4 or IFN-γ and LPS and /or HSV-1 infection.Based on these previous observations, in the present study we evaluate the impact of HSV-1 on macrophages of different polarization phenotypes and the impact of SOCS1 and SOCS3 during HSV-1 infection.Aim 1 is to examine the effects of HSV-1 infection and macrophage polarization on cell viability, morphology, and surface expression of CD80, CD163, and CD200R.We hypothesize that J774A.1 murine macrophages cells in the naïve state (M0), and M1 and M2 phenotypes will display differences in cell morphology, viability, and surface proteins at 18, 24, 48 hours following HSV-1infection.Aim 2 is to quantitate cell viability and measure cytokine secretion for HSV-1-infected M1 macrophages supplemented with SOCS1, SOCS3, or phosphorylated-JAK2.We hypothesize that a SOCS3 peptide mimetic will reduce the cytotoxicity associated with the M1 phenotype or HSV-1 infection.Macrophages treated with either SOCS3 or SOCS1 inhibitor will produce more antiinflammatory cytokine (IL-10) and less pro-inflammatory cytokines (IL-6 and TNF-α) than untreated M1 macrophages.We further anticipate that macrophages treated with SOCS1 will increase the production of TNF-α.vi
Author: Sarah Alsharif
Institution: Wright State University
Language: English
Subfield: Infectious Diseases
Field: Medicine
Domain: Health Sciences
Open Access status: green
OpenAlex ID: W2545575477