Cathelicidin-WA polarizes E. coli K88-induced M1 macrophage to M2-like macrophage in RAW264.7 cells

Highlights

• RAW264.7 cells showed a pro-inflammatory response induced by E. coli K88.

• The pro-inflammatory macrophage is analogous to M1 macrophage.

• CWA effectively suppresses the inflammation in E. coli K88-induced macrophages.

• CWA might exert its immunomodulatory activities by switching M1 macrophages to M2 macrophages.

Abstract

Immune cells - macrophages induced by E. coli K88 will lead to a pro-inflammatory response, which is important in host defense. Cathelicidin-WA (CWA) is an efficient antimicrobial peptide (AMP) and can exert immunomodulatory properties. Many studies have demonstrated that AMP can modulate cellular subsets but whether CWA can regulate macrophage polarization by transferring E. coli K88-induced M1 macrophage towards M2 one that of anti-inflammation remains unclear. In this study, E. coli K88 increased the expression of pro-inflammatory cytokines interleukin-6, interleukin-1β, tumor necrosis factor-α and chemokine CCL3 in RAW264.7 cells with a time-dependent manner, as well as the expression of reactive oxygen species (ROS) and inducible nitric oxide synthase (iNOS). On this basis, CWA significantly decreased the pro-inflammatory molecules but increased the anti-inflammatory mediators interleukin-4, interleukin-10 and other M2-related genes in E. coli K88-induced macrophages. Western blot analysis indicated that CWA suppressed the expression of TLR-4 and the phosphorylation of STAT1 and NF-κB which modulated M1 macrophage while induced the phosphorylation of STAT6 which activated M2 macrophage. Double staining of M1-specific CD86 and M2-specific CD206 also proved the hypothesis. These results suggested that CWA might dampen the inflammation by modulating M1 phenotype to M2 phenotype in E. coli K88-induced macrophages.

Introduction

Macrophages are critical immune cells in host defense and inflammation, participating in the handling of infection and keeping homeostasis [1]. In response to different environmental signals, macrophages can display different functional phenotypes including classically activated (M1 or pro-inflammatory) and alternatively activated (M2 or anti-inflammatory) phenotypes [2], [3]. M1 macrophage can be generated by toll-like receptor (TLR) ligands such as IFN-γ and/or lipopolysaccharides (LPS), which expresses pro-inflammatory cytokines, reactive oxygen and nitrogen species to exert antimicrobial properties [4], [5], [6]. In contrast, stimuli like IL-4 or IL-13 activates M2 macrophage which is considered to serve as the role of dampening inflammatory response, keeping metabolic homeostasis and promoting tissue repair [5], [6]. We all know that excessive inflammation can also cause damage, so editing macrophage activation to tuning inflammation by polarizing M1 macrophages to M2 macrophages is of high interest [7].

The molecular mechanisms that determine M1 or M2 polarization involve specific transcription factors as well as posttranscriptional changes. Upon pathogen/damage-associated molecular patterns (PAMPs/DAMPs), signal transducer and activator of transcription 1 (STAT1) and nuclear factor-κB (NF-κB) are two critical transcription factors which result in M1 polarization [8], [9], [10]. M1 macrophage expresses pro-inflammatory mediators including IL-1β, IL-6, TNF-α, ROS and chemokines [10], [11]. Besides, M1 macrophage specifically regulates the expression of iNOS, inducible enzyme cyclooxygenase-2 (COX-2) and can be marked by CD86 and CD80 [12]. On the other hand, signal transducer and activator of transcription 6/3 (STAT6/STAT3) are key proteins for M2 polarization and the transcription factors up-regulate M2-associated genes such as IL-4, IL-10, transforming growth factor-β (TGF-β), arginase-1 (Arg-1) and mannose receptor CD206 [13], [14], [15]. Although many findings have widely proved the stand-alone responses to macrophage, such as LPS or IL-4, more complex responses are observed in the presence of bacterias [16]. As a typical enterotoxigenic Escherichia coli, E. coli K88 will lead to severe infection diseases but whether it can induce macrophage polarization remains unclear.

Antimicrobial peptides (AMPs) are short cationic molecules that act as a host defense against microbial infection, also called host defense peptides (HDPs) [17]. These peptides not only directly kill microbes but also modulate the immune system of the host [18]. Biochemical and immunological studies indicate AMPs are important in immunomodulation, participating in p38, ERK1/2, NF-κB and other signaling pathways [19], [20]. Moreover, some evidences have proved that one consequence of AMPs modulation events is cellular differentiation, which is observed for macrophages and neutrophils [19], [21]. For example, when present during monocyte-macrophage differentiation, peptide IDR-1018 induced distinctive macrophage profiles which were intermediate between M1 and M2, with M2-like characteristic and potential M1 immune activation feature [22]. CWA is an effective antimicrobial peptide derived from the endemic genera Bungarus fascia [23]. Previous studies have showed that CWA could protect the epithelial barrier and inhibit inflammation in the intestine of both mice and weaned piglets models [24], [25], [26]. The aim of our study is to investigate whether CWA can modulate cell polarization in E. coli K88-induced pro-inflammatory macrophages.

In this study, we found that E. coli K88 induced M1 macrophages with the activation of STAT1/NF-κB signaling pathways and pro-inflammatory mediators. CWA effectively killed all microbes and meantime suppressed the inflammation in E. coli K88-induced macrophages. We hypothesised that CWA switched M1 macrophage towards an immunomodulatory phenotype similar to M2 macrophage via STAT6 and other M2-related mediators. In conclusion, our study demonstrated that CWA could dampen the inflammation by regulating macrophage polarization upon E. coli infection.