A vitamin D3 analog augmented interleukin-8 production by human monocytic cells in response to various microbe-related synthetic ligands, especially NOD2 agonistic muramyldipeptide
Highlights
► A vitamin D3 analog, OCT, affects human monocytic cells to produce higher IL-8. ► NOD2 agonistic MDP was the highest inducer of IL-8 in OCT-treated cells. ► OCT up-regulated the expression of NOD2 in THP-1cells. ► MDP induced higher IL-8 via NOD2 even in OCT-treated cells. ► TAK1, NF-κB and MAPK are involved in the activity of OCT on MDP.
Introduction
In the innate immune system, pattern recognition receptors (PRRs) recognize the pathogen-associated molecular patterns (PAMPs), the distinctive molecular structure of microbes and the wide distribution of specified groups of microbes, to protect the host from microbial infections [1]. Toll-like receptors (TLRs) and nucleotide-binding oligomerization domain (NOD)-like receptor (NLRs) are representative PRRs in innate immunity [1]. In particular, NOD containing 2 (NOD2) is an important NLR family molecule that recognizes the ubiquitous molecular pattern of bacterial cell-wall peptidoglycan, muralyldipeptide (MDP: N-acetylmuramyl-l-alanyl-d-isoglutamine or d-glutamic acid) and is expressed in the cytoplasm of various types of host cells, especially in monocyte/macrophage lineage cells [2], [3]. When NOD2 detects MDP, it recruits receptor-interacting protein 2 (RIP2), followed by the activation of transforming growth factor β-activated kinase 1 (TAK1), which in turn phosphorylates IκBα/β, finally resulting in the activation of NF-κB [3], [4]. Further, NOD2 activates the mitogen-activated protein kinase (MAPK) pathway, containing ERK, p38, and JNK signaling molecules [5], [6]. There have been numerous reports concerning the immunobiological activities of MDP, especially immunomodulating activities [7].
Vitamin D3 exhibits pleiotropic functions [8] and is an essential factor in human physiology. Originally, vitamin D3 was discovered as a preventive agent against nutritional rickets and plays an important role in bone metabolism [9]. Vitamin D3 is also capable of regulating the differentiation and proliferation of various cells [10], [11], modulating immune responses and central nervous system functions, and exerting therapeutic functions against various malignant tumors [8], [12]. Recently, it has been proven that vitamin D3 up-regulates innate immune responses [13]. Rook et al. [14], [15] in their pioneer studies described that vitamin D3 caused human monocytes to differentiate into macrophages, which exhibited enhanced production of tumor necrosis factor-β in response to LPS and Mycobacterium tuberculosis and might inhibit the growth of M. tuberculosis. Many reports have since suggested the involvement of vitamin D3 in human susceptibility to M. tuberculosis [16]. Liu et al. [17], [18], [19] showed that human macrophages recognized M. tuberculosis via TLR2/1 heterodimer, and the recognition triggered TLR activation, which in turn sensitized the cells to vitamin D3, resulting in the increased production of an anti-microbial peptide, cathelicidin, in response to vitamin D3, and the produced cathelicidin inhibited intracellular M. tuberculosis. The possible involvement of TLR4 and TLR9 as well as TLR2 in anti-mycobacterial activity is also suggested [20]. Further, vitamin D3 plays an important role in adoptive immunity [21]. However, the molecular mechanisms of the regulation of the immune response by vitamin D3 have not been understood.
In this context, Wang et al. [22] reported that vitamin D3 up-regulated NOD2 expression at both gene and protein levels in monocytes and epithelial cells. They also found that primary human keratinocytes pretreated with vitamin D3 showed enhanced activation of NF-κB and expression of defensin β2 in response to MDP stimulation. The detailed mechanisms, however, were not clarified in the report. Here, we found that pretreatment of human monocytic THP-1 cells with a vitamin D3 analog, 1α,25-dihydroxy-22-oxavitamin D3 (22-oxacalcitriol; OCT), markedly enhanced the production of interleukin (IL)-8 in response to various ligands to TLRs and NLRs, although treatment of the cells with OCT alone resulted in little enhancement of IL-8 production. Among the microbe-related synthetic ligands used in this study, the clearest augmenting effect of OCT was observed in combination with MDP. Therefore, our study was carried out with special focus on the possible regulation of NOD2 signaling by OCT treatment to present novel functions of vitamin D3 in innate immunity.
Section snippets
Reagents
An analog of the active form of vitamin D3 (1,25-dihydroxyvitamin D3 [1α,25(OH)2D3]), 1α,25-dihydroxy-22-oxavitamin D3 (22-oxacalcitriol; OCT) [23], supplied by Chugai Pharmaceutical Co. (Tokyo, Japan) was diluted in ethanol. In this study, we used only chemically-synthesized ligands to avoid the influence of minor components that contaminate widely used specimens prepared from microbes. A synthetic NOD2 agonist, MDP, and an E. coli-type lipid A (LA-15-PP) were purchased from the Protein
Human monocytic cells pre-treated with OCT produced higher IL-8 production in response to chemically synthesized microbe-related compounds than non-treated cells
To examine the possible augmenting effects of the vitamin D3 analog OCT, we first analyzed IL-8 production in response to various microbe-related innate immune ligands: FSL-1 (TLR 2/6 ligand), poly:IC (TLR3 ligand), lipid A (TLR4 ligand), sspoly U (TLR7 ligand), CpG DNA (TLR9 ligand), FK565 (NOD1 ligand), and MDP (NOD2 ligand) by human monocytic THP-1 cells pretreated with OCT. With these ligands, except sspoly U and CpG DNA, OCT-treated THP-1 cells exhibited higher IL-8 production than
Discussion
Vitamin D3 is capable of differentiating monocytic cells to macrophage-like cells [25]. Vitamin D3 induced expression of CD14 in THP-1 cells [26]. In relation to the regulation of innate immune responses by vitamin D3, the induction of CD14 by vitamin D3 analog, OCT, in human monocytic cells was reported by Yang et al. [27]. Moeenrezakhanlou et al. [28] reported that the cAMP response element (CRE) in the CD14 promoter was bound by CRE binding protein (CREB) in a downstream of
Acknowledgments
The authors express their thanks to D. Mrozek (Medical English Service, Kyoto, Japan) for reviewing the paper.
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