Abstract
CD274, one of two co-stimulatory ligands for programmed death 1 and widely expressed in the mononuclear phagocyte system (MPS), may co-stimulate T cells and regulates inflammatory responses. However, changes in CD274 gene expression and the underlying molecular mechanism are poorly understood during inflammatory responses. Therefore, delineation of the complex mechanisms regulating CD274 expression is critical to understand this immunoregulatory system during inflammatory responses. The purpose of this study was to assess the molecular mechanisms regulating CD274 expression in an in vitro monocyte model of inflammatory response. Firstly, CD274 expression levels in human primary monocytes after lipopolysaccharide (LPS) treatment were observed and correlated with NF-κB activation. Secondly, based on the distribution of putative NF-κB binding sites, 5′ truncated human CD274 promoter reporters were constructed, transfected into U937 cells and critical promoter regions for basal (nt −570 to +94) and LPS-induced (nt −1735 to −570) transcription were identified by dual luciferase assays. Finally, a key NF-κB binding site (nt −610 to −601) for LPS-inducible CD274 transcriptional activity was characterized by point mutation analysis and chromatin immunoprecipitation analysis assays (ChIP). Thus, the present study establishes a molecular basis to understand the mechanisms governing CD274 expression in certain infections and inflammatory disorders.
Highlights
The primary inflammatory response induced by gram-negative bacteria involves activation of the innate immune system
The CD274 mRNA level increased between 0 and 2 h and continued to increase to a maximum after 12 h (Fig. 1A). These data suggested that the up-regulation of CD274 transcription in monocytes was in a time-dependent manner after LPS treatment
Data showed that only NF-kB inhibitor BAY pre-treatment markedly inhibited CD274 mRNA accumulation in primary human monocytes after LPS treatment for 12 h (Fig. 1B), which indicated that NF-kB may be involved in LPS-induced CD274 expression, the protein expression assayed by western blotting has consist results with mRNA level data, further corroborating that NF-kB plays a significant role in LPS induced CD274 gene expression (Fig. 1C)
Summary
The primary inflammatory response induced by gram-negative bacteria involves activation of the innate immune system. This activation can trigger systemic inflammatory response syndrome (SIRS) by releasing a cascade of proinflammatory cytokines, thereby causing high morbidity and mortality [1,2]. As an important class of antigen presenting cells for T cells and the major component of the innate immune system, the mononuclear phagocyte system (MPS) regulates the inflammatory response and immune functions via membrane proteins and secreted cytokines such as B7, TNF-a (tumor necrosis factor-alpha), IL-6 (interleukin-6) and IL-10 (interleukin-10) [4]. After binding to its cognate receptor, CD274 exerts inflammation regulatory functions via a negative co-stimulatory effect on T cell functions to inhibit cytokine secretion, facilitate apoptosis of activated T cells and induce T cell anergy [7,8]
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