Effect of Pp2cm Gene Silencing on Mouse Macrophage Resistance Against Staphylococcus aureus Infection via TLR Pathway

LUO Kaiteng, XIE Maodi, YANG Wei, LI Tao, JIANG Chunling

Abstract

To investigate the effect of silencing protein phosphatase 2cm (Pp2cm) gene on the expression of inflammatory factors in macrophages infected with Staphylococcus aureus (S. aureus) and the mechanisms involved.   Methods   The effects of Pp2cm knockdown on inflammatory factors, proliferation, apoptosis, and Toll-like receptor (TLR) signaling were analyzed in RAW 264.7 cells, a murine macrophage cell line, transfected with adenovirus (Ad). The cells were divided into four groups, including Ad-Ctrl group, Ad-Pp2cm group, Ad-Ctrl+S. aureus group and Ad-Pp2cm+S. aureus group. Cell transfection was achieved by separately introducing control adenovirus (Ad-Ctrl) or adenovirus targeting the Pp2cm gene (Ad-Pp2cm) and inflammation or the absence of inflammation was induced by applying or not applying S. aureus. The expression of tumor necrosis factor-alpha (TNF-α), interleukin-1β (IL-1β), TLR2, TLR4, Toll-like receptor adaptor protein (Tirap) and myeloid differentiation factor 88 (Myd88) was determined by real-time fluorescent quantitative polymerase chain reaction (RT-qPCR). PP2Cm protein expression was determined by Western blot. Cell proliferation was determined by cell counting kit-8 (CCK-8) assay and cell apoptosis was measured by flow cytometry.   Results  The expression of Pp2cm gene and PP2Cm protein was downregulated in the Ad-Pp2cm group when compared to the Ad-Ctrl group, with the diference showing statistical significance (P<0.05). When compared to those of the Ad-Ctrl+S. aureus group, macrophages in the Ad-Pp2cm+S. aureus group showed significantly increase in the TNF-α and IL-1β gene levels (P<0.01). Furthermore, the Ad-Pp2cm group demonstrated elevated gene expression levels of TLR2, TLR4, Tirap and Myd88 in macrophages when compared to the Ad-Ctrl group, with the difference showing statistical significance (P<0.05). There were no statistically significant differences in cell apoptosis and proliferation between the Ad-Ctrl and Ad-Pp2cm groups.  Conclusions   Silencing Pp2cm gene promotes the inflammatory response of macrophages to S. aureus infection. Moreover, the TLR pathway plays an important role in the inflammatory activation of macrophages.


Keywords: Staphylococcus aureus, Protein phosphatase 2Cm, Toll-like receptor

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