Recombinant <i>Haemonchus contortus</i> 24 kDa excretory/secretory protein (rHcES-24) modulate the immune functions of goat PBMCs <i>in vitro</i>

Gadahi, Javaid Ali; Li, Baojie; Ehsan, Muhammad; Wang, Shuai; Zhang, Zhenchao; Wang, Yujian; Hasan, Muhammad Waqqas; Ren, Yan; Song, Xiaoling; Xu, Lixin

doi:10.18632/oncotarget.13487

Cited by 29 publications

(50 citation statements)

References 59 publications

(67 reference statements)

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“…The mixtures were stimulated with 10 µg/mL of concanavalin A (ConA) (Sigma-Aldrich) for 72 h in RPMI 1640. Supernatants were collected by centrifugation at 200× g for 10 min and the concentration of interleukin-4 (IL-4), interleukin-10 (IL-10), interleukin-17 (IL-17), Interferon gamma (IFN-γ) and transformation growth factor-β1 (TGF-β1) were measured by commercially available goat ELISA kits (Jiancheng Biotechnology, Nanjing, China) [33] according to the manufacturer's instructions. Each experiment was performed in triplicate.…”

Section: Elisa Dependent Cytokines Secretion By Pbmcs Treated With Rhmentioning

confidence: 99%

Tropomyosin: An Excretory/Secretory Protein from Haemonchus contortus Mediates the Immuno-Suppressive Potential of Goat Peripheral Blood Mononuclear Cells In Vitro

Ehsan

Haseeb

et al. 2020

Vaccines

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During host-parasite interactions, binding of excretory/secretory proteins (ESPs) on the host immune cells is considered the fundamental phase for regulation of immune responses. In this study, gene encoding Haemonchus contortus tropomyosin (Hc-TpMy), was successfully cloned and expressed, and the recombinant protein after host cell surface attachment was evaluated for immune functional analysis with goat peripheral blood mononuclear cells (PBMCs) in vitro. The isopropyl-β-D-thiogalactopyranoside (IPTG)-induced recombinant protein was successfully recognized by the sera of rat experimentally infected with rHc-TpMy. The immunofluorescence assay detected attachment of rHc-TpMy on the surface of host PBMCs. Furthermore, immunoregulatory roles of rHc-TpMy on cytokines expression, PBMC proliferation, migration, nitric oxide (NO) production, apoptosis and monocytes phagocytosis were observed. The results showed that expression of IL-4 and IFN-γ cytokines, cell proliferation, NO production and PBMC migration were significantly suppressed by goat PBMCs after co-incubation with rHc-TpMy protein. However, the productions of IL-10, IL-17 and TGF-β1 cytokines, PBMCs apoptosis and monocytes phagocytosis were elevated at dose dependent manner. Our findings indicated that rHc-TpMy is an important ES binding protein exhibit distinct immuno-suppressive roles on goat PBMCs which might be a potential molecular target to control haemonchosis in future.

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Section: Elisa Dependent Cytokines Secretion By Pbmcs Treated With Rhmentioning

confidence: 99%

Tropomyosin: An Excretory/Secretory Protein from Haemonchus contortus Mediates the Immuno-Suppressive Potential of Goat Peripheral Blood Mononuclear Cells In Vitro

Ehsan

Haseeb

et al. 2020

Vaccines

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“…Modulation of immune responses during the infection process is also linked with active stimulation and recruitment of eosinophils and other lymphocytes by helminths, resulting in stability of worms within the host [46,47]. Previously, we determined stimulatory roles of HcESPs collectively and individually in the case of rHcftt-2 and rHcES-24 to induce the migration of goat PBMCs [20,48,49]. Here, we also found that rHcMTF-12, as an interacting protein of HcESPs, showed significant enhancement ability to the migration of host PBMCs.…”

Section: Discussionmentioning

confidence: 99%

Identification of a novel methyltransferase-type 12 protein from Haemonchus contortus and its effects on functions of goat PBMCs

et al. 2020

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Background: Methyltransferases (MTFs) are broad range of enzymes, which are ubiquitously expressed in diverse organisms ranging from bacteria to animals. MTFs proteins have been associated with various biological/cellular processes including transcriptional regulation, subcellular protein and RNA localization, signal transduction and DNA-damage repair. However, the role of MTFs in immune mechanism during host-parasite interaction has not been addressed yet. Results: An open reading frame (764 bp) of methyltransferase-type 12 gene of H. contortus denoted as HcMTF-12, was successfully cloned using reverse transcriptase-polymerase chain reaction (RT-PCR) followed by prokaryotic expression in Escherichia coli BL21 (DE3 strain). The recombinant HcMTF-12 protein (rHcMTF-12) was about 47 kDa along with a fusion vector protein of 18 kDa. Immunoblot results identified the native protein MTF-12 with antibodies produced in rats against rHcMT-12, whereas rHcMTF-12 protein was recognized with sera of goat experimentally infected with H. contortus. Immunohistochemical analysis revealed that the native MTF-12 protein was mainly located in the periphery (cuticle) of parasite sections as well as within the pharynx and intestinal region. An immunofluorescence assay validated that rHcMTF-12 attached to the surface of goat PBMCs. Furthermore, the cytokines transcription of IL-2, IFN-γ and IL-4 transcripts of PBMCs incubated with rHcMTF-12 were enhanced in a dose-dependent manner. The secretion of TGF-β1 and IL-10 was significantly decreased. However, IL-6 production was not significantly different as compared to the control groups. Moreover, the migration activity and nitric oxide (NO) production by PBMCs were induced considerably, whereas the proliferation of PBMCs cells was negatively affected when incubated with the rHcMTF-12 protein. Conclusions: Our findings suggest that HcMTF-12 significantly mediated the functions of PBMCs, and it might be a potential candidate for therapeutic interventions against haemonchosis.

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“…Total RNA was extracted from adult H. contortus using TRIzol reagent (Invitrogen, Shanghai, China) to clone the MMP-12 gene [30]. The cDNA was transcribed as per manufacturer's instructions of the cDNA Kit used (Takara, Dalian, China) and stored at − 20 °C until use.…”

Section: Molecular Cloning Of Hc-mmp-12mentioning

confidence: 99%

“…An immunofluorescence assay was performed to evaluate the binding ability of rHc-MMP-12 to goat PBMCs as described previously [30]. Briefly, freshly collected goat PBMCs were inoculated with 10 μg/ml rHc-MMP-12 and 10 μg/ml purified pET32a tag protein separately in a 24-well plate (1 ml/well).…”

Section: Pbmcs Binding Assaymentioning

confidence: 99%

Haemonchus contortus: siRNA mediated knockdown of matrix metalloproteinase 12A (MMP-12) results in reduction of infectivity

Naqvi

Gao

et al. 2020

Parasites Vectors

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Background: RNA interference (RNAi) is an important tool to determine the role of genes. RNAi has been widely used to downregulate target molecules, resulting in the reduction of mRNA for protein expression. Matrix metalloprotease 12A (MMP-12) is known to have important roles during embryonic development, organ morphogenesis and pathological processes in animals. However, MMP-12 from Haemonchus contortus has not been characterized.Methods: Haemonchus contortus MMP-12 gene was cloned and recombinant protein of MMP-12 (rHc-MMP-12) was expressed. Binding activities of rHc-MMP-12 to goat peripheral blood mononuclear cells (PBMCs) were assessed by immunofluorescence assay (IFA) and the immuno-regulatory effects of rHc-MMP-12 on cell proliferation and nitric oxide production were observed by co-incubation of rHc-MMP-12 with goat PBMCs. Furthermore, a soaking method was used to knockdown the expression of Hc-MMP12 gene using three siRNA, targeting different regions of the gene and infectivity of effective siRNA on the development of H. contortus was evaluated in goat.Results: rHc-MMP-12 was successfully expressed in an expression vector as well as the tissues of the cuticle of adult H. contortus worms and a successful binding with PBMCs surface were observed. Increased cellular proliferation and nitric oxide production by goat PBMCs was observed in a dose-dependent manner. Quantitative real time PCR (qRT-PCR) results confirmed the successful silencing of Hc-MMP-12 gene in siRNA of 1, 2 and 3 treated third-stage larvae (L3) of H. contortus in vitro. The most efficient qRT-PCR-identified siRNA template was siRNA-2, with a 69% suppression rate compared to the control groups. Moreover, in an in vivo study, silencing of the Hc-MMP-12 gene by siRNA-2 reduced the number of eggs (54.02%), hatchability (16.84%) and worm burden (51.47%) as compared to snRNAtreated control group. In addition, a shorter length of worms in siRNA-2-treated group was observed as compared to control groups. Conclusions:Our results indicate that siRNA-mediated silencing of Hc-MMP-12 gene in H. contortus significantly reduce the egg counts, larval hatchability, and adult worm counts and sizes. The findings of the present study demonstrate important roles of Hc-MMP-12 in the development of H. contortus.

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Recombinant Haemonchus contortus 24 kDa excretory/secretory protein (rHcES-24) modulate the immune functions of goat PBMCs in vitro

Cited by 29 publications

References 59 publications

Tropomyosin: An Excretory/Secretory Protein from Haemonchus contortus Mediates the Immuno-Suppressive Potential of Goat Peripheral Blood Mononuclear Cells In Vitro

Tropomyosin: An Excretory/Secretory Protein from Haemonchus contortus Mediates the Immuno-Suppressive Potential of Goat Peripheral Blood Mononuclear Cells In Vitro

Identification of a novel methyltransferase-type 12 protein from Haemonchus contortus and its effects on functions of goat PBMCs

Haemonchus contortus: siRNA mediated knockdown of matrix metalloproteinase 12A (MMP-12) results in reduction of infectivity

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