Clonorchis sinensis MF6p/HDM (CsMF6p/HDM) induces pro-inflammatory immune response in RAW 264.7 macrophage cells via NF-κB-dependent MAPK pathways - PubMed (original) (raw)
Clonorchis sinensis MF6p/HDM (CsMF6p/HDM) induces pro-inflammatory immune response in RAW 264.7 macrophage cells via NF-κB-dependent MAPK pathways
Jung-Mi Kang et al. Parasit Vectors. 2020.
Abstract
Background: MF6p/host defense molecules (HDMs) are a broad family of small proteins secreted by helminth parasites. Although the physiological role of MF6p/HDMs in trematode parasites is not fully understood, their potential biological function in maintaining heme homeostasis and modulating host immune response has been proposed.
Methods: A gene encoding the MF6p/HDM of Clonorchis sinensis (CsMF6p/HDM) was cloned. Recombinant CsMF6p/HDM (rCsMF6p/HDM) was expressed in Escherichia coli. The biochemical and immunological properties of rCsMF6/HDM were analyzed. CsMF6p/HDM induced pro-inflammatory response in RAW 264.7 cells was analyzed by cytokine array assay, reverse transcription polymerase chain reaction, and enzyme-linked immunosorbent assay. The structural feature of CsMF6p/HDM was analyzed by three-dimensional modeling and molecular docking simulations.
Results: The CsMF6p/HDM shares a high level of amino acid sequence similarity with orthologs from other trematodes and is expressed in diverse developmental stages of the parasite. The rCsMF6p/HDM bound to bacteria-derived lipopolysaccharide (LPS), without effectively neutralizing LPS-induced inflammatory response in RAW 264.7 macrophage cells. Rather, the rCsMF6p/HDM induced pro-inflammatory immune response, which is characterized by the expression of TNF-α and IL-6, in RAW 264.7 cells. The rCsMF6p/HDM-induced pro-inflammatory immune response was regulated by JNK and p38 MAPKs, and was effectively down-regulated via inhibition of NF-κB. The structural analysis of CsMF6p/HDM and the docking simulation with LPS suggested insufficient capture of LPS by CsMF6p/HDM, which suggested that rCsMF6p/HDM could not effectively neutralize LPS-induced inflammatory response in RAW 264.7 cells.
Conclusions: Although rCsMF6p/HDM binds to LPS, the binding affinity may not be sufficient to maintain a stable complex of rCsMF6p/HDM and LPS. Moreover, the rCsMF6p/HDM-induced pro-inflammatory response is characterized by the release of IL-6 and TNF-α in RAW 264.7 macrophage cells. The pro-inflammatory response induced by rCsMF6p/HDM is mediated via NF-κB-dependent MAPK signaling pathway. These results collectively suggest that CsMF6p/HDM mediates C. sinensis-induced inflammation cascades that eventually lead to hepatobiliary diseases.
Keywords: Clonorchis sinensis; Docking; Lipopolysaccharide; MF6p/host defense molecule; Pro-inflammatory immune response; Structure.
Conflict of interest statement
The authors declare that they have no competing interests.
Figures
Fig. 1
Sequence conservation of CsMF6p/HDM and other flukes’ homologues. The deduced amino acid sequences of CsMF6p/HDM (GenBank: AAM55183.1) was aligned with MF6p/HDMs of O. viverrini (GenBank: ES416124.1), F. hepatica (GenBank: CCA61804.1), P. westermani (GenBank: AT007125.1). The shading represented the degree of identity among the sequences, ranging blue (100%) to white (0%). Sequence identities were represented in the right. The signal peptide is indicated by the box with dotted red line. Closed circle in red indicated the C-terminal conserved motif, as reported previously [9]
Fig. 2
Production of recombinant CsMF6p/HDM and anti-CsMF6/HDM. a Expression and purification of recombinant CsMF6p/HDM. Lane 1: E. coli lysate control; Lane 2: IPTG-induced E. coli lysate; Lane 3; CsMF6p/HDM purified by Ni-NTA affinity column. b Validation of anti-CsMF6p/HDM. The anti-CsMF6p/HDM was collected from mice immunized with CsMF6p/HDM and confirmed by immunoblot. Lane 1: normal serum; Lane 2: anti-CsMF6p/HDM
Fig. 3
Expression pattern of CsMF6p/HDM. a Transcriptional profiles of CsMF6p/HDM genes at various developmental stages of C. sinensis. The C. sinensis actin gene was amplified as an internal control. b Immunoblot. Each developmental stage of C. sinensis worm extract was proved with anti-CsMF6p/HDM antibody. Abbreviations: M, metacercariae; 2W, 2-week-old juveniles; 4W, 4-week-old adults; 6W, 6-week-old adults; 9W, 9-week-old adult worm; Re; Recombinant CsMF6p/HDM
Fig. 4
LPS-binding assay. Serial concentrations of CsMF6p/HDM (1 to 20 μg) were incubated with LPS (10 μg) coated or non-coated blot and proved with anti-CsMF6p/HDM
Fig. 5
Cytokine profile analysis of RAW 264.7 cells stimulated by CsMF6p/HDM. a RAW 264.7 cells were stimulated with CsMF6p/HDM (10 μg/ml) for 12 h and the culture supernatants were collected and subjected to cytokine array analysis. The cells treated with PBS were used as a negative control. The results of one of two independent experiments which revealed similar expression patterns are shown. b Quantitative analysis of mean pixel density from the cytokine array assay. Data are representative of two independent experiments. Abbreviations: G-CSF, granulocyte-colony stimulating factor; IP-10, interferon gamma-induced protein 10; IL-1ra, interleukin-1 receptor antagonist; IL-6, interleukin 6; MIP-1, macrophage inflammatory protein; MIP-2, macrophage inflammatory protein 2; RANES, regulated upon activation normal T cell expressed and secreted; sICAM-1, soluble intercellular adhesion molecule-1; TNF-α, tumor necrosis factor_-_α
Fig. 6
Neutralizing effect of CsMF6p/HDM in LPS-induced inflammatory response. Transcriptional activities of IL-6 and TNF-α were measured in RAW 264.7 cells, which were treated with the mixture of CsMF6p/HDM (5 or 10 μg) and LPS (1 μg)
Fig. 7
Effect of p38, JNK and ERK inhibitors on the expression of TNF-α and IL-6 in RAW 264.7 cells stimulated by CsMF6p/HDM. a RAW 264.7 cells were pretreated with different concentration (5 or 10 μM) of JNK inhibitor (SP600125), p38 inhibitor (SB203580), or ERK inhibitor (U0126) for 3 h before the cells were incubated with CsMF6p/HDM (10 μg) and LPS (0.5 μg) for 12 h. The cells were harvested and the expression levels of TNF-α and IL-6 were analyzed by RT-PCR. Graphs show the densitometric ratios of TNF-α and IL-6 to GAPDH. b ELISA analysis. Cytokine production profiles were analyzed by quantitative ELISA
Fig. 8
Effect of NF-κB and AP-1 inhibitors on the expression of TNF-α and IL-6 in RAW 264.7 cells stimulated with CsMF6p/HDM. a RAW 264.7 cells were pretreated with different concentrations (0.1, 0.5, 1.0 μM) of NF-κB inhibitor (MG132) or AP-1 inhibitor (SR11302) for 3 h, and the cells were then incubated with CsMF6p/HDM (10 μg) and LPS (0.5 μg) for 12 h. The cells were harvested and the expression levels of TNF-α and IL-6 were analyzed by RT-PCR. Graphs show the densitometric ratios of TNF-α and IL-6 to GAPDH. b ELISA analysis. Cytokine production profiles were analyzed by quantitative ELISA
Fig. 9
Structural comparison between CsMF6p/HDM and FhMF6/HDM. Two 3D models, CsMF6p/HDM in yellow (a) and FhMF6p/HDM in blue (c), were superposed (b). The superposed structure is in front view (top) and perpendicular view (bottom). Hydrophobic regions of CsMF6p/HDM (d) and FhMF6p/HDM (e) models are visualized from grey to red according to the hydrophobicity value
References
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