SOCS1/3 Expression Levels in HSV-1-Infected, Cytokine-Polarized and -Unpolarized Macrophages

Abstract

Macrophage subtypes are characterized as proinflammatory (M1) or immunomodulatory and tissue remodeling (M2). Since macrophages play a pivotal role in controlling Herpes simplex virus type-1 (HSV-1) replication, effects of HSV-1 by 24 h of infection were determined in murine J774A.1 macrophages unpolarized (M0) or polarized to either an M1 or M2 phenotype. Morphology, cell viability, and expression of CD14 (co-receptor for lipopolysaccharide), CD86 (B7.2-immune co-stimulatory molecule), and suppressors of cytokine signaling (SOCS1 and SOCS3) were determined. M1 macrophages were flattened and vacuolated, while M2 cells appeared elongated with a few vacuoles. Compared with unpolarized M0 cells, M1 cells showed a 31% decrease in viability, a 2-fold increase in the number of CD14 + -CD86 + cells, no change in SOCS1 expression, and an 11-fold decrease in SOCS3 expression. M2 cells exhibited a 9% decrease in viability, a 26.0% decrease in the number of CD14 + -CD86 + cells, and no change in SOCS1/SOCS3 expression levels compared with M0 cells. After HSV-1 infection, all phenotypes appeared rounded, cell viabilities decreased as did numbers of M1 cells expressing CD14 and CD86. At 24 h after infection, M0 control and M2 cells showed greater virus yield than did the M1 cells, presumably reflecting the loss of viable M1 cells. SOCS1 expression was predominant in uninfected M1-polarized cells and in virus-infected control (M0) cells. SOCS1/SOCS3 expression ratio was 7:1 in uninfected M1 macrophages and approached 1:1 in M1 cells at 24 h after infection with HSV-1. In contrast, little differences were seen in SOCS1/SOCS3 expression ratios in uninfected M2-polarized cells or virusinfected M2 cells. These observations suggest that SOCS1/SOCS3 expression ratios can be used to characterize HSV-1-infected and uninfected macrophages.

Roles of M1 and M2 Macrophages in Herpes Simplex Virus 1 Infectivity

“…In this study, we have shown that M1 macrophages derived from both the RAW264.7 cell line and PM of C57BL/6 mice were refractory to HSV-1 replication compared to M2 macrophages or unpolarized macrophages. These results are consistent with the report that the yields of HSV-1 from M1 macrophages derived from the murine J774A.1 macrophage line were lower than the yields from the unpolarized macrophages and M2 macrophages (79). We also showed that after infection M1 macrophages derived from either RAW264.7 cells or PM produce significantly larger amounts of proinflammatory cytokines and chemokines than the M2 macrophages, which is consistent with previous reports (26, 28-30).…”

Oxidized LDL phagocytosis during foam cell formation in atherosclerotic plaques relies on a PLD2–CD36 functional interdependence

“…Macrophage foam cells were incubated with Agg‐Ox‐LDL in a time‐course experiment to measure phagocytic cup formation, similar to . Samples were fixed, permeabilized, blocked and then incubated with fluorescent antibodies specific to WASP, growth factor receptor‐bound protein 2 (Grb2), and PLD2 using AlexaFluor350‐, FITC‐, or tetramethylrhodamine isothiocyanate (TRITC) labeling of 2 out of the 3 total proteins at any one time, allowing us to visualize fluorescent Agg‐Ox‐LDL and 2 target proteins . Photomicrographs at 100x magnification were rendered using a Nikon Eclipse 50i inverted fluorescence microscope, Infinity2 Lumenera digital camera and Infinity Analyze software (v. 6.2).…”

“…40 Samples were fixed, permeabilized, blocked and then incubated with fluorescent antibodies specific to WASP, growth factor receptorbound protein 2 (Grb2), and PLD2 using AlexaFluor350-, FITC-, or tetramethylrhodamine isothiocyanate (TRITC) labeling of 2 out of the 3 total proteins at any one time, allowing us to visualize fluorescent Agg-Ox-LDL and 2 target proteins. 40,55 Photomicrographs at 100x magnification were rendered using a Nikon Eclipse 50i inverted fluorescence microscope, Infinity2 Lumenera digital camera and Infinity Analyze software (v. 6.2). The immunofluorescence colocalization of protein pairs was quantified using ImageJ software with ∼2-4 cells per field; 6 fields; n = 3 independent experiments, by determining the Pearson's coefficient, 56,57 which was then plotted as % of colocalization (yellow = TRITC-PLD2 and FITC-Grb2; or TRITC-PLD2 and FITC-Actin; or TRITC-PLD2 and FITC-WASP).…”

Assessment of Herpes Simplex Virus Type 1 and 2 by IHC in Association with CD14 Antigen in Placental Tissues from Women with Miscarriage

“…HSV-1 infection caused most macrophage cells to cluster as well as to be attached to each other. In addition , HSV-1 infection changed the expression levels of CD14 [24], the results of current study revealed that collectively, herpes simplex virus use an inhibitory immune receptor to enter and invasion into the cells and that is beneficial to the virus because binding to inhibitory receptors may provide entry. Regarding the expression of the CD14 in relation to the HSV2 infection the results suggested that there is a considerable role for CD14 in association with HSV-2 in the studied groups since it has been detected in both miscarriage and healthy control study groups., although there has been a lack of association between HSV-2 and CD14 since J. Arii et.al, denoted that HSV-2 was not able to infect primary human CD14-positive -mononuclear cells [25], which predominantly consist of monocytes or macrophages, the results presented herein are in accordance with Ellermann who claimed a possibility for different susceptibilities of these cells to the HSV-2.…”