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Updated: Dec 31, 2025
Depletion and Reconstitution of Macrophages in Mice
Published on: August 1, 2012
Clonorchis sinensis MF6p/HDM (CsMF6p/HDM) induces pro-inflammatory immune response in RAW 264.7 macrophage cells via
Jung-Mi Kang1,2, Won Gi Yoo3, Hương Giang Lê1,2
1Department of Parasitology and Tropical Medicine, and Institute of Health Sciences, Gyeongsang National University College of Medicine, Jinju, 52727, Republic of Korea.
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.
Insights
The Clonorchis sinensis MF6p/host defense molecule (HDM) binds to lipopolysaccharide (LPS) but does not neutralize its inflammatory effects. Instead, it triggers pro-inflammatory responses via NF-κB signaling, potentially contributing to hepatobiliary diseases.
Area of Science:
- Parasitology
- Immunology
- Molecular Biology
Background:
- Host defense molecules (HDMs) are secreted by helminth parasites.
- MF6p/HDMs' role in trematodes, including heme homeostasis and immune modulation, is under investigation.
- Clonorchis sinensis is a parasitic trematode implicated in hepatobiliary diseases.
Purpose of the Study:
- To clone and characterize the MF6p/HDM from Clonorchis sinensis (CsMF6p/HDM).
- To investigate the biochemical and immunological properties of recombinant CsMF6p/HDM (rCsMF6p/HDM).
- To elucidate the role of CsMF6p/HDM in modulating host inflammatory responses.
Main Methods:
- Gene cloning and recombinant protein expression of CsMF6p/HDM in E. coli.
- Analysis of rCsMF6p/HDM binding to lipopolysaccharide (LPS).
- Assessment of pro-inflammatory cytokine (TNF-α, IL-6) induction in RAW 264.7 cells via cytokine array, RT-PCR, and ELISA.
- Investigation of signaling pathways (JNK, p38 MAPK, NF-κB) involved in the inflammatory response.
- Structural analysis and molecular docking simulations of CsMF6p/HDM with LPS.
Main Results:
- CsMF6p/HDM shares sequence similarity with other trematode orthologs and is expressed across developmental stages.
- rCsMF6p/HDM binds to LPS but fails to neutralize LPS-induced inflammation in macrophages.
- rCsMF6p/HDM actively induces pro-inflammatory cytokines TNF-α and IL-6 in RAW 264.7 cells.
- The induced inflammation is mediated by JNK and p38 MAPKs and regulated by NF-κB.
- Structural analysis suggests insufficient LPS binding affinity for effective neutralization.
Conclusions:
- rCsMF6p/HDM binds LPS, but the affinity is insufficient for stable complex formation and neutralization.
- rCsMF6p/HDM induces a pro-inflammatory response characterized by IL-6 and TNF-α release.
- The inflammatory cascade is mediated through an NF-κB-dependent MAPK signaling pathway.
- CsMF6p/HDM likely contributes to C. sinensis-induced inflammation and subsequent hepatobiliary diseases.
