Effect Of Recombinant Human B-type Natriuretic Peptide On THP-1 Derived Macrophages Polarization Shifting

Objective: Neutrophil infiltration in infarct area immediately after myocardial infarction ( MI ) followed by mononuclear-macrophages infiltration plays an important role in post-infarct inflammation and repair. Results from recent researches have have indicated that macrophages can be divided into pro-inflammatory phenotype M1 and anti-inflammatory phenotype M2. In the process of inflammation after MI, macrophages exhibited a time-dependent phenotype shift. In the setting of excessive expression of pro-inflammatory phenotype, adverse reactions emerge, including extension of inflammation, tissue over-injury, increased scar formation and poor repair. B-type natriuretic peptide (B-type natriuretic peptides, BNP) is a multiple functional polypeptide secreted mainly by ventricular myocytes. Its receptor is widely expressed in heart, kidney, brain, blood vessels and other tissues and cells, regulating many physiological functions through NPRA / GC / cGMP / PKG pathway. After acute MI, BNPmRNA content in ventricular tissue increases dramatically. So does plasma BNP levels. It has shown that use of exogenous recombinant human B-type natriuretic peptide in the short and long duration after MI can prevent and reduce ventricular remodeling, and improve cardiac function. But the mechanism of BNP on post-infarction ventricular remodeling is still uncoverd. Recent studies have confirmed the existence of BNP receptors in macrophage, indicating the regulatory role of BNP on macrophage function. Therefore, in the present work, we were going to study the effect of exogenous BNP on LPS induced macrophage phenotype shift, to provide novel evidence underlying BNP's beneficial effect on post-infarction healing process.Method:We stimulated human monocytic THP-1 cell line with phorbol myristate acetate to induce macrophage differentiation. Then differentiated macrophages were incubated with lipopolysaccharide (LPS) to activate macrophage through M1 polarization. The expression of BNP receptors was analyzed by real-time quantitative PCR (qPCR) before and after LPS stimulation. Exogenous BNP was then added with a concentration gradient from 10-13 to 10-9 mol/L to LPS-activated macrophages. The effect of BNP via its receptor was confirmed by measuring cytosolic cGMP production via commercially available ELISA kit. Total RNA of macrophages was isolated then reverse transcripted to cDNA for quantification of markers for M1 (TNF-α, RANTES, IL-6, IL-12 and MCP-1) macophge polarization and for M2 polarization (TGF-β1, IL-10 and CCL-18). In addition, TNF-α, IL-12 and TGF-β1 concentration in supernatant was determined by ELISA..Results:1 The morphological change of THP-1 cells before and after the induction of LPS.THP-1 cells before induction were round, suspended growth; After 48h treatment of 100nmol/L PMA (phorbol myristoyl acetate, PMA), followed by 3h incubation of 1ng/mL LPS, the cells were adherent, deformed or stretching out pseudopodia like the macrophages.2 Expressions of BNP and its related receptors in lipopolysaccharide-induced THP-1 macrophage cells:After THP-1 was induced into macrophages by LPS, the mRNA expression levels of the BNP and its type 1 receptor (Natriuretic peptides receptor 1, NPR1) increased significantly, and reached the peak after 3h's incubation of LPS3 cGMP.in LPS-induced THP-1 derived macrophages were significantly elevated by adding exogenous BNP.4 A dose-dependent phenotypic shift from M1 to M2 at the mRNA level was observed in LPS-induced THP-1 macrophages. The down-regulated mRNA in this paper including TNF-α,MCP-1,RANTES,IL-6,IL-12.5 A dose-dependent phenotypic shift from M1 to M2 at the protein level was observed in LPS-induced THP-1 macrophages. The down-regulated protein in this paper is IL-12Conclusion:1 After macrophages were activated, BNP expression upregulated as well as its major receptor, which manifested that BNP may play a role in autocrine and paracrine effects on macrophage.2 Upon macrophages activation, exogenous BNP may, sharing the same signaling pathway with the endogenous BNP, elevate intracellular cGMP levels to regulate gene transcription, and related protein expression.3 Exogenous BNP has a dose-dependent effect on the phenotypic shift from M1 to M2, resulting in down-regulation of pro-inflammatory cytokines and up-regulation of anti-inflammatory cytokines.