Dataset Information

Trichinella spiralis cathepsin L induces macrophage M1 polarization via the NF-κB pathway and enhances the ADCC killing of newborn larvae.

ABSTRACT:

Background

During the early stages of Trichinella spiralis infection, macrophages predominantly undergo polarization to the M1-like phenotype, causing the host's inflammatory response and resistance against T. spiralis infection. As the disease progresses, the number of M2-type macrophages gradually increases, contributing to tissue repair processes within the host. While cysteine protease overexpression is typically associated with inflammation, the specific role of T. spiralis cathepsin L (TsCatL) in mediating macrophage polarization remains unknown. The aim of this study was to assess the killing effect of macrophage polarization mediated by recombinant T. spiralis cathepsin L domains (rTsCatL2) on newborn larvae (NBL).

Methods

rTsCatL2 was expressed in Escherichia coli strain BL21. Polarization of the rTsCatL2-induced RAW264.7 cells was analyzed by enzyme-linked immunosorbent assay (ELISA), quantitative PCR (qPCR), western blot, immunofluorescence and flow cytometry. The effect of JSH-23, an inhibitor of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), on rTsCatL2-induced M1 polarization investigated. Cytotoxic effects of polarized macrophages on NBL were observed using in vitro killing assays.

Results

Following the co-incubation of rTsCatL2 with RAW264.7 murine macrophage cells, qPCR and ELISA revealed increased transcription and secretion levels of inducible nitric oxide synthase (iNOS), interleukin (IL)-6, IL-1β and tumor necrosis factor alpha (TNF-α) in macrophages. Western blot analysis showed a significant increase in iNOS protein expression, while the expression level of arginase-1 protein remained unchanged. Flow cytometry revealed a substantial increase in the number of CD86-labeled macrophages. The western blot results also indicated that rTsCatL2 increased the expression levels of phospho-NF-κB and phospho-nuclear factor-κB inhibitor alpha (IκB-α) proteins in a dose-dependent manner, while immunofluorescence revealed that rTsCatL2 induced nuclear translocation of the p65 subunit of NF-κB (NF-κB p65) protein in macrophages. The inhibitory effect of JSH-23 suppressed and abrogated the effect of rTsCatL2 in promoting M1 macrophage polarization. rTsCatL2 mediated polarization of macrophages to the M1-like phenotype and enhanced macrophage adhesion and antibody-dependent cell-mediated cytotoxicity (ADCC) killing of NBL.

Conclusions

The results indicated that rTsCatL2 induces macrophage M1 polarization via the NF-κB pathway and enhances the ADCC killing of NBL. This study provides a further understanding of the interaction mechanism between T. spiralis and the host.

SUBMITTER: Liu RD

PROVIDER: S-EPMC10666456 | biostudies-literature | 2023 Nov

REPOSITORIES: biostudies-literature

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Publications

Trichinella spiralis cathepsin L induces macrophage M1 polarization via the NF-κB pathway and enhances the ADCC killing of newborn larvae.

Liu Ruo Dan RD Meng Xiang Yu XY Le Li Chen C Xu Qiu Yi QY Lin Xin Zhi XZ Dong Bo Rang BR Ye Chu Yan CY Miao Tian Tian TT Si Xin Yi XY Long Shao Rong SR Cui Jing J Wang Zhong Quan ZQ

Parasites & vectors 20231122 1

<h4>Background</h4>During the early stages of Trichinella spiralis infection, macrophages predominantly undergo polarization to the M1-like phenotype, causing the host's inflammatory response and resistance against T. spiralis infection. As the disease progresses, the number of M2-type macrophages gradually increases, contributing to tissue repair processes within the host. While cysteine protease overexpression is typically associated with inflammation, the specific role of T. spiralis cathepsi ...[more]

PMID: 37993938

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Project description:BackgroundTrichinellosis is a serious zoonosis with a worldwide distribution. Fecund adult worms in the intestine release newborn larvae (NBL) that enter the general circulation from 4 days post infection (dpi). Alternatively activated macrophages in the peritoneal cavities and the diaphragms in Trichinella spiralis infected mice have been reported. However, a role of newborn larvae is poorly understood.MethodsThe total numbers of peritoneal macrophages in mice infected with 500 muscle-stage larvae were counted during early infection and then total RNA was extracted. Peritoneal macrophages from uninfected C57BL/6 mice were incubated with IL-4 or LPS as a control, or co-cultured with live NBL, and peritoneal macrophages were obtained from mice injected with live or frozen dead NBL into peritoneal cavity. Total RNA was extracted from these macrophages. Two types of gene expression, classical and alternative activation, were examined in the macrophages and diaphragms of the infected mice using semi-quantitative reverse transcription-PCR.ResultsThe number of peritoneal macrophages in T. spiralis infected mice increased significantly. mRNA peak expression of alternative activation markers, Ym1 and arginase-1 (Arg1), was confirmed in the peritoneal macrophages and in diaphragm of mice around 15 dpi, while mRNA expression of classical activation markers, TNFα, IP-10, and iNOS was not detected. Injection of live NBL into the peritoneal cavities induced mRNA expression of Ym1 and Arg1 in the peritoneal macrophages of mice 9 dpi. However, dead NBL did not induce such gene expression. Alternative activation was not detected in the peritoneal macrophages co-cultured with NBL in vitro.ConclusionGene expression of alternative activation makers, Ym1 and Arg1, was confirmed in the peritoneal macrophages and diaphragms of mice infected with T. spiralis. However, gene expression of classical activation markers was not detected. Live NBL induced an alternative activation of peritoneal macrophages in vivo, but not in vitro.

Project description:BackgroundTrichinellosis is a re-emerging infectious disease, caused by Trichinella spp. Cathepsin F belongs to cysteine protease that is a major virulence factor for parasitic helminths, and it may be a potential anti-helminth drug target and vaccine candidate. The aim of this study was to clone, express and identify a cathepsin F-like protease in Trichinella spiralis and to investigate its biochemical characteristics.MethodsThe full-length cDNA encoding a putative cathepsin F-like protease in T. spiralis, TsCF1, was cloned and its biochemical characterization and expression profile were analyzed. Transcription of TsCF1 at different developmental stages of T. spiralis was observed by RT-PCR. The recombinant TsCF1 protein was expressed by prokaryotic expression system and recombinant TsCF1 (rTsCF1) was analyzed by western blotting. And expression of TsCF1 at muscle larvae stage was performed by immunofluorescent technique. Molecular modeling of TsCF1 and its binding mode with E-64 and K11777 were analyzed. Enzyme activity and inhibitory test with E-64 as inhibitor were investigated by using Z-Phe-Arg-AMC as specific substrate.ResultsSequence analysis revealed that TsCF1 ORF encodes a protein of 366 aa with a theoretical molecular weight of 41.9 kDa and an isoelectric point of 7.46. The cysteine protease conserved active site of Cys173, His309 and Asn333 were identified and cathepsin F specific motif ERFNAQ like KLFNAQ sequence was revealed in the propeptide of TsCF1. Sequence alignment analysis revealed a higher than 40 % identity with other cathepsin F from parasitic helminth and phylogenetic analysis indicated TsCF1 located at the junction of nematode and trematode. RT-PCR revealed the gene was expressed in muscle larvae, newborn larvae and adult stages. SDS-PAGE revealed the recombinant protein was expressed with the molecular weight of 45 kDa. The purified rTsCF1 was used to immunize rabbit and the immune serum could recognize a band of about 46 kDa in soluble protein of adult, muscle larvae and ES product of muscle larvae. Immunolocalization analysis showed that TsCF1 located on the cuticle and stichosome of the muscle larvae. After renaturation rTsCF1 demonstrated substantial enzyme activity to Z-Phe-Arg-AMC substrate with the optimal pH 5.5 and this activity could be inhibited by cysteine protease inhibitor E-64. Further analysis showed the kinetic parameters of rTsCF1 to be Km = 0.5091 μM and Vmax = 6.12 RFU/s μM at pH 5.5, and the IC50 value of E64 was 135.50 ± 16.90 nM.ConclusionTsCF1 was expressed in all stages of T. spiralis and localized in the cuticle and stichosome. TsCF1 might play a role in the life cycle of T. spiralis and could be used as a potential vaccine candidate and drug target against T. spiralis infection.

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Dataset Information

Trichinella spiralis cathepsin L induces macrophage M1 polarization via the NF-κB pathway and enhances the ADCC killing of newborn larvae.

Background

Methods

Results

Conclusions

Publications

Trichinella spiralis cathepsin L induces macrophage M1 polarization via the NF-κB pathway and enhances the ADCC killing of newborn larvae.

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