Abstract
BackgroundUpon initial infection with mycobacteria, macrophages secrete multiple cytokines and chemokines, including interleukin-6 (IL-6), IL-8 and tumor necrosis factor-α (TNF-α), to mediate host immune responses against the pathogen. Mycobacteria also induce the production of IL-10 via PKR activation in primary human monocytes and macrophages. As an anti-inflammatory cytokine, over-expression of IL-10 may contribute to mycobacterial evasion of the host immunity. Radix Paeoniae Rubra (RPR, Chishao), a Chinese medicinal herb with potentials of anti-inflammatory, hepatoprotective and neuroprotective effects, is used to treat tuberculosis. This study investigates the immunoregulatory effects of RPR on primary human blood macrophages (PBMac) during mycobacterial infection.MethodsThe interaction of Bacillus Calmette-Guerin (BCG) with PBMac was used as an experimental model. A series of procedures involving solvent extraction and fractionation were used to isolate bioactive constituents in RPR. RPR-EA-S1, a fraction with potent immunoregulatory effects was obtained with a bioactivity guided fractionation scheme. PBMac were treated with crude RPR extracts or RPR-EA-S1 before BCG stimulation. The expression levels of IL-6, IL-8, IL-10 and TNF-α were measured by qPCR and ELISA. Western blotting was used to determine the effects of RPR-EA-S1 on signaling kinases and transcriptional factors in the BCG-activated PBMac.ResultsIn BCG-stimulated macrophages, crude RPR extracts and fraction RPR-EA-S1 specifically inhibited IL-10 production while enhanced IL-8 expression at both mRNA and protein levels without affecting the expressions of IL-6 and TNF-α. Inhibition of BCG-induced IL-10 expression by RPR-EA-S1 occurred in a dose- and time-dependent manner. RPR-EA-S1 did not affect the phosphorylation of cellular protein kinases including MAPK, Akt and GSK3β. Instead, it suppressed the degradation of IκBα in the cytoplasm and inhibited the translocation of transcription factor NF-κB1 p50 to the nucleus.ConclusionRPR crude extracts and its fraction RPR-EA-S1 inhibited anti-inflammatory cytokine IL-10 and enhanced pro-inflammatory chemokine IL-8 expression in BCG-activated PBMac. The inhibitory effects of RPR-EA-S1 on IL-10 expression in BCG-activated PBMac may be due to the reduced nuclear translocation of NF-κB1 p50.
Highlights
Upon initial infection with mycobacteria, macrophages secrete multiple cytokines and chemokines, including interleukin-6 (IL-6), IL-8 and tumor necrosis factor-a (TNF-a), to mediate host immune responses against the pathogen
The Enzyme-linked immunosorbent assay (ELISA) assays found that primary human blood macrophages (PBMac) pretreated with RPR-EA showed 68% reduction in IL-10 production induced by Bacillus Calmette-Guerin (BCG) while RPR-H, RPR-Bu and RPR-W did not show any inhibitory effect (Figure 2A)
Differential effects of RPR extracts on BCG-induced cytokine and chemokine expression Since cytokines and chemokines secreted by macrophages play a critical role in the host immune responses against mycobacteria, we examined the RPR’s regulatory effects on BCG-induced IL-6, IL-8, IL-10 and TNF-a expression in PBMac
Summary
Upon initial infection with mycobacteria, macrophages secrete multiple cytokines and chemokines, including interleukin-6 (IL-6), IL-8 and tumor necrosis factor-a (TNF-a), to mediate host immune responses against the pathogen. Upon Mtb infection, multiple immune cells including macrophages/monocytes [3], dendritic cells (DCs) [4], neutrophils [5], natural killer cells [6] and T cells [6] are activated to mediate host defense against the pathogen. Among these cell types, macrophages are the main immunocytes in initiating innate immunity against Mtb. Macrophages are professional antigen presenting cells that are important in bridging innate and adaptive immunity [7]. Macrophages exert their direct antimicrobial effects through phagocytosis [8], generation of reactive oxygen intermediates (ROIs) and reactive nitrogen intermediates (RNIs) [9], autophagy [10], activation of vitamin D pathway [11], apoptosis [12] and secretion of cytokines and chemokines [13]
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