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Galectin Hco-gal-m from Haemonchus contortus modulates goat monocytes and T cell function in different patterns.

ABSTRACT: BACKGROUND: Monocytes and T cells are two major subpopulations of peripheral blood mononuclear cells (PBMC) and play an essential role in the innate and adaptive immune systems. Different members of the galectin family show multiple and distinct regulatory effects on different cell types. Previous studies have demonstrated that the galectin from Haemonchus contortus (Hco-gal-m) performed immunomodulatory effects on goat PBMC, however, which subpopulation of PBMC is the primary target of Hco-gal-m and whether the immune modulations share the same mechanism remain unclear. METHODS: In this study, the developmental expression of Hco-gal-m was analyzed by RT-PCR and Western blot analysis. The distribution of Hco-gal-m in adult worm was detected by an immunohistochemical test. The binding activity of the recombinant Hco-gal-m (rHco-gal-m) on goat monocytes and T cells were assessed by flow cytometry. The immunomodulatory effects of Hco-gal-m on cytokine secretion, cell activation and apoptosis were observed by co-incubation of rHco-gal-m with goat monocytes and T cells. RESULTS: Hco-gal-m was expressed in L4 as well as adult worms and predominantly localized at the internal surface of the worm guts. rHco-gal-m could bind to both monocytes and T cells. The engagement of rHco-gal-m decreased the production of IL-6, IL-10 and TNF-? in T cells, however, it significantly increased the secretion of IL-10 in monocytes. After rHco-gal-m exposure, the expression of MHC-II on monocytes and that of CD25 on T cells were restricted. Consequently, T cell proliferations were potently inhibited by rHco-gal-m. In addition, rHco-gal-m induced apoptosis in T cells, but not significantly in monocytes. CONCLUSIONS: Our results indicated that rHco-gal-m modulated goat monocytes and T cell function in different patterns.

SUBMITTER: Wang W

PROVIDER: S-EPMC4117971 | biostudies-literature | 2014

REPOSITORIES: biostudies-literature

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Galectin Hco-gal-m from Haemonchus contortus modulates goat monocytes and T cell function in different patterns.

Wang Wang W Wang Shuai S Zhang Hui H Yuan Cheng C Yan RuoFeng R Song XiaoKai X Xu LiXin L Li XiangRui X

Parasites & vectors 20140723

<h4>Background</h4>Monocytes and T cells are two major subpopulations of peripheral blood mononuclear cells (PBMC) and play an essential role in the innate and adaptive immune systems. Different members of the galectin family show multiple and distinct regulatory effects on different cell types. Previous studies have demonstrated that the galectin from Haemonchus contortus (Hco-gal-m) performed immunomodulatory effects on goat PBMC, however, which subpopulation of PBMC is the primary target of H ...[more]

PMID: 25056558

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Project description:BackgroundRecombinant galectins of male and female Haemonchus contortus (rHco-gal-m/f) have been recognized as significant regulators of the functions of goat peripheral blood mononuclear cells (PBMC). In previous research, transmembrane protein 63A (TMEM63A) was identified as a partner protein in the regulation associated with H. contortus infection. However, in the identification of binding partners for galectins of male and female H. contortus (Hco-gal-m/f) by yeast two-hybrid (YTH) screening, it was found that the transmembrane protein 147 (TMEM147) could also bind to Hco-gal-m/f. In this study, the functions of TMEM147 in the regulations of H. contortus galectin on the goat PBMC were investigated.MethodsTo identify Hco-gal-m/f-interacting proteins, a yeast two-hybrid system to detect interactions was used. Co-immunoprecipitation and immunoblotting were used to validate the interaction between recombinant galectins of male H. contortus (rHco-gal-m) and candidate binding protein. The localization of TMEM147 in PBMC was explored by immunofluorescence in confocal imaging studies. Flow cytometry was used to determine the distribution of TMEM147 in T cells, B cells and monocytes in PBMC. The modulatory effects of rHco-gal-m and TMEM147 on cell proliferation, phagocytosis, nitric oxide production, migration, apoptosis and cytokine mRNA transcription were observed by co-incubation of rHco-gal-m and knockdown of the tmem147 gene.ResultsIn this research, it was demonstrated that TMEM147 could bind to rHco-gal-m/f. Immunofluorescence assays showed that TMEM147 was localized to the cell membrane and within the cell membrane in goat PBMC. Flow cytometric analysis revealed that TMEM147 was expressed in all B cells and monocytes in goat PBMC. However, 3.8 % of T cells did not express this protein. Knockdown of the tmem147 gene using RNA interference (RNAi) showed that the interaction of galectin with TMEM147 mainly mediated cell proliferation, cell apoptosis, transcription of interleukin-10 (IL-10) and transforming growth factor-β1 (TGF-β1) of goat PBMC. This membrane protein, together with TMEM63A, was also related to the regulation of galectin on phagocytosis and nitric oxide production of goat PBMC. However, it might not be involved in the regulation of galectin on the migration and interferon-γ (IFN-γ) transcription of goat PBMC.ConclusionsOur results showed that TMEM147 was a binding partner of Hco-gal-m/f and mediated the immunological regulation of Hco-gal-m/f on goat PBMC in a manner different to that of TMEM63A.

Project description:BackgroundHaemonchosis is a disease of the small ruminant caused by a nematode parasite Haemonchus contortus, and it is most important and alarming challenges to the small ruminant's production. The infection of the H. contortus could cause high economic losses worldwide. H. contortus is a blood feeding parasite which penetrates into the abomasal mucosa to feed the blood of the host and causing the anemia and decreased total plasma protein. Modulation and suppression of the immune response of the host by nematode parasites have been reported extensively, and the cysteine protease inhibitor (cystatin) is identified as one of the major immunomodulators.MethodsThe recombinant protein of HCcyst-3 was expressed in a histidine-tagged fusion soluble form in Escherichia coli, and its inhibitory activity against cathepsin L, B, as well as papain, were identified by fluorogenic substrate analysis. Native HCcyst-3 protein was localized by an Immunohistochemical test. The immunomodulatory effects of HCcyst-3 on cytokine secretion, MHC molecule expression, NO production and phagocytosis were observed by co-incubation of rHCcyst-3 with goat monocytes.ResultsWe cloned and produced recombinant cystatin protein from H. contortus (rHCcyst-3) and investigated its immunomodulatory effects on goat monocyte. The rHCcyst-3 protein is biologically functional as shown by its ability to inhibit the protease activity of cathepsin L, cathepsin B, and papain. The immunohistochemical test demonstrated that the native HCcyst-3 protein was predominantly localized at the body surface and internal surface of the worm's gut. We demonstrated that rHCcyst-3 could be distinguished by antisera from goat experimentally infected with H. contortus and could uptake by goat monocytes. The results showed that the engagement of rHCcyst-3 decreased the production of TNF-α, IL-1β and IL-12p40. However, it significantly increased the secretion of IL-10 and TGF-β1 in goat monocytes. After rHCcyst-3 exposure, the expression of MHC-II on goat monocytes was restricted. Moreover, rHCcyst-3 could upregulate LPS induced NO production of goat monocytes. Phagocytotic assay by FITC-dextran internalization showed that rHCcyst-3 inhibited the phagocytosis of goat monocytes.ConclusionsOur results suggested that the recombinant cystatin from H. contortus (rHCcyst-3) significantly modulated goat monocyte function in multiple aspects.

Project description:BackgroundHco-gal-m and -f were two isoforms of galectin cloned from male and female Haemonchus contortus, respectively, and it was demonstrated that recombinant Hco-gal-m and -f could act as immune suppressors. However, little is known about the receptors or binding partners of these galectins in the host. The research of the molecular mechanisms that govern the interactions between these galectins and host molecules will fill a gap in our understanding how parasite galectins interact with host cells.MethodsA yeast two-hybrid system was used to identify the binding partners of Hco-gal-m and -f in this research. The interaction between rHco-gal-m and candidate binding protein was validated by co-immunoprecipitation. The localization of transmembrane protein 63A (TMEM63A) in peripheral blood mononuclear cells (PBMCs) was detected by immunofluorescence. The distribution of TMEM63A in T cells, B cells and monocytes in PBMCs was detected by flow cytometry. The immunomodulatory effects of Hco-gal-m and TMEM63A on cell proliferation, migration, apoptosis, nitric oxide production and cytokine secretion were observed by co-incubation of rHco-gal-m and TMEM63A-siRNA with goat PBMCs and monocytes.ResultsWe found that TMEM63A, a functionally unknown protein, from goat PBMCs could bind to Hco-gal-m and -f. Immunofluorescence showed that TMEM63A was localized to the cell membrane. Flow cytometric analysis revealed that TMEM63A was expressed in the majority of goat PBMCs. After using RNA interference to knockdown expression of TMEM63A, the PBMC proliferation and migration were significantly increased, while the influence of rHco-gal-m on monocyte phagocytosis, PBMC nitric oxide production and migration were potently blocked. In addition, the production of IL-10, IFN-γ and TGF-β induced by rHco-gal-m were also altered.ConclusionsOur results show that TMEM63A is a binding partner of Hco-gal-m/f, and involved in the immune responses of host PBMCs induced by Hco-gal-m for the first time.

Project description:BACKGROUND:Smad proteins are essential cellular mediators within the transforming growth factor-β (TGF-β) superfamily. They directly transmit incoming signals from the cell surface receptors to the nucleus. In spite of their functional importance, almost nothing is known about Smad proteins in parasitic nematodes including Haemonchus contortus, an important blood-sucking nematode of small ruminants. METHODS:Based on genomic and transcriptome data for H. contortus and using bioinformatics methods, a Smad homologue (called Hco-daf-8) was inferred from H. contortus and the structural characteristics of this gene and its encoded protein Hco-DAF-8 established. Using real-time PCR and immunofluorescence assays, temporal transcriptional and spatial expression profiles of Hco-daf-8 were studied. Gene rescue in Caenorhabditis elegans was then applied to assess the function of Hco-daf-8 and a specific inhibitor of human Smad3 (called SIS3) was employed to evaluate the roles of Hco-DAF-8 in H. contortus development. RESULTS:The features of Hco-DAF-8 (502 amino acids), including conserved R-Smad domains and residues of the L3-loop that determine pathway specificity, are consistent with a TGF-β type I receptor-activated R-Smad. The Hco-daf-8 gene was transcribed in all developmental stages of H. contortus studied, with a higher level of transcription in the fourth-stage larval (L4) females and the highest level in adult males. Hco-DAF-8 was expressed in the platymyarian muscular cells, intestine and reproductive system of adult stages. Gene rescue experiments showed that Hco-daf-8 was able to partially rescue gene function in a daf-8 deficient mutant strain of C. elegans, leading to a resumption of normal development. In H. contortus, SIS3 was shown to affect H. contortus development from the exsheathed third-stage larvae (L3s) to L4s in vitro. CONCLUSIONS:These findings suggest that Hco-DAF-8, encoded by the gene Hco-daf-8, is an important cellular mediator of H. contortus development via the TGF-β signalling pathway. They provide a basis for future explorations of Hco-DAF-8 and associated pathways in H. contortus and other important parasitic nematodes.

Project description:Resistance to the anthelmintic drug monepantel (Zolvix®) has emerged in parasitic worms infecting sheep and goats. The mechanism of resistance in these cases is unknown. The drug targets nicotinic acetylcholine receptors belonging to the nematode-specific DEG-3 subfamily. We examined the receptor gene, Hco-mptl-1, in a highly Zolvix®-resistant and a -susceptible isolate of the parasitic nematode Haemonchus contortus. cDNA coding for the full length receptor protein (Hco-MPTL-1) was present in all clones prepared from a pool of susceptible larvae (21/21 clones) and approximately 50% of those from the resistant isolate (17/33). On the other hand, the remaining clones from the resistant isolate showed various mutations that resulted in truncated predicted proteins, missing at least one transmembrane domain. The most common mutation (11/33 clones) resulted in the retention of intron 15, a premature stop codon, and a truncated protein. Sequencing of intron 15 genomic DNA showed very few SNPs in susceptible larvae and in 12/18 clones from resistant larvae, alongside the presence of at least 17 SNPs in the remaining resistant clones. The present study shows that the highly resistant isolate has a number of mutations in the drug target gene that would most-likely result in a non-functional receptor, thus rendering the larvae insensitive to the drug. The presence of many wild-type sequences in this highly-resistant population suggests that there was a significant presence of heterozygotes in the survivors of the field drench treatment from which the isolate was derived, and hence that at least some of the mutations may be dominant. Alternatively, their presence may be due to the additional influence of mutations at another locus contributing to the resistance phenotype. The presence of multiple separate mutations in the Hco-mptl-1 gene in this viable field-derived worm isolate may at least partly explain why resistance to Zolvix® has arisen rapidly in the field.

Dataset Information

Galectin Hco-gal-m from Haemonchus contortus modulates goat monocytes and T cell function in different patterns.

Publications

Galectin Hco-gal-m from Haemonchus contortus modulates goat monocytes and T cell function in different patterns.

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