[Effect of Pp2cm Gene Silencing on Mouse Macrophage Resistance Against Staphylococcus aureus Infection via TLR Pathway]
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Published on: November 3, 2014
Kaiteng Luo1,2, Maodi Xie2, Wei Yang1,2
1Department of Anesthesiology, West China Hospital, Sichuan University, Chengdu 610041, China.
Objective:
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-1 β), 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 Pp2cmgene 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-1 β 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.
Insights
Silencing the protein phosphatase 2cm (Pp2cm) gene exacerbates Staphylococcus aureus infection-induced inflammation in macrophages. The Toll-like receptor (TLR) pathway is crucial for this heightened inflammatory response.
Area of Science:
- Immunology
- Molecular Biology
Context:
- Macrophages play a critical role in innate immunity and host defense against bacterial infections.
- Staphylococcus aureus (S. aureus) is a common pathogen that can cause severe infections.
- Understanding the molecular mechanisms regulating macrophage inflammatory responses is essential for developing new therapeutic strategies.
Purpose:
- To investigate the effect of silencing the protein phosphatase 2cm (Pp2cm) gene on the expression of inflammatory factors in macrophages infected with S. aureus.
- To elucidate the role of the Toll-like receptor (TLR) signaling pathway in this process.
Summary:
- Silencing the Pp2cm gene in RAW 264.7 macrophages led to increased expression of inflammatory cytokines, tumor necrosis factor-alpha (TNF-α) and interleukin-1β (IL-1β), following S. aureus infection.
- Knockdown of Pp2cm also upregulated the expression of TLR2, TLR4, Tirap, and Myd88, key components of the TLR signaling pathway.
- No significant differences in cell proliferation or apoptosis were observed between groups.
Impact:
- These findings suggest that Pp2cm acts as a negative regulator of the inflammatory response in macrophages during S. aureus infection.
- Targeting Pp2cm or the TLR pathway could be a potential therapeutic approach for managing S. aureus infections.
