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

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 snRNA-treated 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.

Galectin Domain Containing Protein from Haemonchus contortus Modulates the Immune Functions of Goat PBMCs and Regulates CD4+ T-Helper Cells In Vitro.

Galectins are glycan-binding proteins that are widely expressed and distributed in mammalian tissues as well as cells of innate and adaptive immune responses. CD4+ T-helper cells differentiate into effector subsets in response to cytokines. T helper 9 cells are one of the recently described subsets of effector T cells that are relatively new and less studied. In this study, galectin domain containing protein from Haemonchus contortus (Hc-GDC) was cloned, expressed in pET32a, and immunoblotting was performed. Localization of recombinant (r)Hc-GDC on outer and inner surface of H. contortus worm and binding with goat Peripheral Blood Mononuclear cells (PBMCs) were performed using immunofluorescence assay. Moreover, effects of rHc-GDC on proliferation, apoptosis, cell migration, and the nitric oxide production in goat PBMCs were evaluated. Furthermore, modulatory effects of rHc-GDC on production of Th1, Th2, and Th9 cells were evaluated by flowcytometry and on interferon gamma, interleukin (IL)-4 and IL-9 were evaluated by quantitative real-time polymerase chain reaction. The results demonstrated that rHc-GDC was successfully cloned, expressed in expression vector as well as in the gut surface of adult H. contortus worm and successful binding with PBMCs surface were observed. Immunoblotting results revealed that rHc-GDC is an important active protein of H. contortus excretory and secretory products. Moreover, the interaction of rHc-GDC with host cells increased the production of Th2, Th9 cells, IL4, IL-9, PBMC proliferation, nitric oxide, and cell migration. No effects of rHc-GDC were observed on PMBC apoptosis, production of Th1 cells, and secretions of IFN- and IL-10 cytokines. These findings indicate that recombinant GDC protein from H. contortus modulates the immune functions of goat PBMCs and has the potential to enhance protective immunity by inducing T helper-9-derived IL-9 in vitro.

Immunodiagnostic potential of recombinant tropomyosin during prepatent Haemonchus contortus infection in goat.

Excretory and secretory products (ESPs) are released by the parasites during Haemonchus contortus (H. contortus) infection. In this study, Tropomyosin (TpMy), one of these ESPs was used to develop western blotting and optimized Enzyme Linked immunosorbent assay (ELISA) for detection of H. contortus during early infection in goat. Microscopic examination was performed parallel for comparison. Recombinant tropomyosin protein was purified successfully. Western blotting results revealed that anti-recombinant H. contortus Tropomyosin (rHc-TpMy) antibodies could recognize the natural proteinand rHc-TpMy antigen did not show any cross-reaction with goat anti-sera of Fasciola hepatica, Trichinella spiralis, and Toxoplasma gondii. Moreover, initial antibodies were detected by both western blotting and indirect ELISA at 14 days post infection (DPI) and persisted till 30 DPI but fecal eggs count couldn't detect the eggs in feces at early stage (7 and 14 DPI). The optimized antigen coating concentration was calculated as 10 µg/ml (P/N Optimum Density450 = 4.165) with optimized dilution of serum (1:50) and secondary antibody (1:2500). Positive and negative cutoff value of the indirect-ELISA assay was calculated as 0.392 and 0.344, respectively. Receiver operating characteristic curve analysis validated the cutoff value (0.392) based on a high specificity and sensitivity. Indirect ELISA showed 90% diagnostic sensitivity and 100% diagnostic specificity. In comparison of serological and conventional method, rHc-TpMy based indirect ELISA showed more positive results (30%; 9/30) than microscopic examination (20%; 6/30). These results demonstrated that rHc-TpMy is a potential immunodiagnostic antigen to detect specific antibodies at early stage of infection in goat and serological methods are more reliable as compared to microscopic examination.

αv Integrin expression by DCs is required for Th17 cell differentiation and development of experimental autoimmune encephalomyelitis in mice.

Th17 cells are a distinct lineage of T helper cells that protect the body from bacterial and fungal infection. However, Th17 cells also contribute to inflammatory and autoimmune disorders such as multiple sclerosis. Th17 cell generation requires exposure of naive T cells to the cytokine TGF-ß in combination with proinflammatory cytokines. Here we show that differentiation of Th17 cells is also critically dependent on αv integrins. In mice, lack of integrin αv in the immune system resulted in loss of Th17 cells in the intestine and lymphoid tissues. It also led to protection from experimental autoimmune encephalomyelitis (EAE). Further analysis indicated that αv integrins on DCs activated latent TGF-ß during T cell stimulation and thereby promoted differentiation of Th17 cells. Furthermore, pharmacologic inhibition of αv integrins using cyclic RGD peptides blocked TGF-ß activation and Th17 cell generation in vitro and protected mice from EAE. These data demonstrate that activation of TGF-ß by αv-expressing myeloid cells may be a critical step in the generation of Th17 cells and suggest that αv integrins could be therapeutic targets in autoimmune disease.