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-γ 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.


Introduction
Galectins, animal lectins, are known to be widely distributed from lower invertebrates, such as nematodes and sponges, to higher vertebrates [1]. It has been demonstrated that the interaction of

Ethical Statement
All experimental protocols were approved by the Science and Technology Agency of Jiangsu Province (Approval ID: SYXK (SU) 2010-0005).

Animals and Parasites
Local crossbred 5-6 months old goats were purchased from the local market of Jiangsu province and housed in an animal house of Nanjing Agricultural University under controlled conditions. Levamisole (8 mg/kg body weight) was orally administered to all goats to remove natural parasitic infections at two-week intervals. Microscopic analysis was performed for detection of helminth eggs in feces samples collected at week intervals. Furthermore, infected larvae of H. contortus (L3) were obtained from experimental infection by a conventional method [26]. Briefly, feces from H. contortus infected-goat were collected, crushed, mixed with water, and combined with vermiculite to make the mixture moist. The pan was covered with aluminum foil having several holes to allow air flow and put at room temperature for ten days. The larvae mixture was filtered through cheesecloth to recover larvae, which were identified microscopically and preserved at 4 • C in penicillin G treated water until use.
Female Sprague Dawley rats (body weight: 150 g) (n = 6) were purchased from the Experimental Animal Center of Jiangsu, PR China (Certified: SCXK 2008-0004). To collect polyclonal antibodies, rats were divided into experimental (n = 3) and control (n = 3) groups and housed in sterilized experimental animal house Nanjing Agricultural University.

Cloning and Sequence Analysis of Hc-GDC
Total RNA was extracted from adult H. contortus parasites using Trizol reagent (Invitrogen, Shanghai, China) as described previously [30]. Briefly, the cDNA was synthesized using cDNA Kit (Takara Biotechnology, Dalian, China) as per the manufacturer's instructions and stored at −20 • C until use. The complete ORF (open reading frame) of GDC was amplified by RT-PCR (reverse transcription-polymerase chain reaction) using specific primers designed from CDS (conserved domain sequences) of H. contortus (Genbank: CDJ98063.1). The sequence of specific primers carrying EcoR I and Hind III digestion site was as follows: Sense Primer: (5 -GAATTCATGTCTTGCGTTCAATTCAG -3 ) and antisense primer: (5 -AAGCTTTTAGCCGATATGAATAGAATGC -3 ). The total 25 µL PCR product was made up of 12.5 µL 2× Taq Master Mix (Takara Biotechnology), 2 µL of cDNA, 8.5 µL ddH 2 O, and 1 µL of each primer. PCR amplification was performed as follows: initial denaturing (1 cycle) 94 • C for 5 min, followed by 35 cycles of 94 • C for 30 s, 55 • C for 30 s and 72 • C for 1-2 min, and finally extension at 72 • C for 10 min. E.Z.N.A. Gel Extraction Kit (Omega Bio-tech, Norcross, GA, USA) was used according to instructions of manufacturer to purify the PCR products and followed by ligation into cloning vector, pMD19-T (Takara, Dalian, China). Transformation of recombinant plasmid (pMD19-T/GDC) into E. coli DH 5α strain (Invitrogen) was performed and cultured in ampicillin containing Luria Bertini (LB) medium. The recombinant plasmid, pMD19-T/GDC, was identified by restriction enzyme digestion with EcoR I and Hind III. GDC gene was sub-cloned into pET32a, followed by confirmation of successful insertion in the proper reading frame by sequencing (Invitrogen) and blast online.

Generation of Polyclonal Antibodies
Complete Freund's adjuvant mixed equally with rHc-GDC (300 µg) was injected subcutaneously in SD rats of experimental group to obtain polyclonal antibodies. After 2 weeks, three more doses of this protein mixed with Freund's incomplete adjuvant were injected at a 1-week interval. After one week of the last dose, SD rats were anesthetized with 25% isoflurane (inhaling anesthesia) by an open drop method [33] and blood samples were collected from the eye. Finally, SD rats were euthanized by head dislocation. The serum samples from the experimental group and control group were prepared and stored at −80 • C until use.

Western Blot Assay
Western blotting played a preliminary role in selection of target protein to evaluate the immunogenicity and immuno-reactivity of antigen [34]. Antigenic characteristics of rHc-GDC were also evaluated in sera of H. contortus infected goats as described previously [35,36]. Firstly, purified rHc-GDC was separated on 12% SDS-PAGE and semi dry system (Novablot Hoefer, Holliston, MA, USA) was used to transfer onto PVDF (polyvinyl difluoride membrane, Millipore, Billerica, MA, USA) with a transfer solution (Tris 48 mM, glycine 39 mM, SDS 0.0375%, methanol 20%). Blocking buffer (5% skimmed milk) diluted in TBS-T (Tris-Buffered Saline containing 0.05% Tween 20) was used to block the membrane at 37 • C for 2 h. The PVDF membrane was washed three times, cut into strips and incubated at 37 • C with 1:100 diluted primary antibody (anti-H. cortortus goat serum/anti-rHcGDC polyclonal antibody) for 2 h. After another three washes with TBS-T, the strips were incubated with 1:4000 diluted secondary antibodies (Horseradish Peroxidase conjugated rabbit anti-goat IgG; Sigma, Hilden, Germany). Finally, immunoreactions were observed with the help of chromogenic substrate, 3-diaminobenzidine tetrahydrocholoride (DAB; Tiangen Biotech, Beijing, China).

Localization of rHc-GDC in Adult H. contortus Worms
H. contortus adult worms were fixed on glass slides (poly-l-lysine hydrobromide) with 4% formaldehyde-0.2% glutaraldehyde in PBS for 45 min and suspended in TISSUE-TEK ® O.C.T. compound (Sakura, Torrance, CA, USA) and blast freeze in liquid nitrogen. Worms were cut into pieces (10-µm thickness) with a cryotome (CM1950, Wetzlar, Germany) and washed with PBS. The slides were treated with 5% Bovine Serum Albumin (BSA) to block non-specific bindings followed by incubation with 1:100 dilutions of rat-anti-rHc-GDC antiserum (experimental group) and normal rat serum (control group) as first antibody at 37 • C. After 2 h incubation, slides of both groups were incubated with 1:3000 dilutions of Cy3-labeled Goat Anti-Rat as second antibody (Beyotime, Shanghai, China) at 37 • C for 1 h. DAPI (diamidino-2-phenylindole) was used to stain the nucleus of worm cells and anti-Fade Fluoromount Medium (Beyotime, Shanghai, China) was used before observing under confocal laser scanning microscope to prevent fading.

PBMC Binding Assay
Immunofluorescence assay was performed to evaluate the binding ability of rHc-GDC to goat PBMCs as described previously [30]. Briefly, fresh goat PBMCs were inoculated with 10 µg/mL rHc-GDC and PBS separately in a 24-well plate (1 mL/well). Plate was incubated at 37 • C in a humidified atmosphere with 5% CO2 for 1 h. Washed cells were permitted to settle on poly-L-lysine coated glass slides for 20 min and fixed with 4% phosphate-buffered paraformaldehyde at room temperature for 30 min. The slides were blocked with PBS containing 5% BSA at 37 • C for 1 h. Subsequently, slides were incubated with 1:100 dilution primary antibodies, rat anti-rHc-GDC sera, and normal sera (control slide) for two hours. After three washes, slides were incubated in dark with secondary antibodies, and rat anti-goat IgG coupled with Cy3 (Beyotime Institute of Biotechnology, Shanghai, China; 1:1000 dilutions) for 30 min. DAPI (Sigma, St. Louis, MO, USA) was subsequently added and slides were incubated for 5 min. Finally, after washing, slides were covered with coverslip and immersed in Anti-Fade Fluoromount solution (Beyotime Institute of Biotechnology, Shanghai, China). PBMCs were observed by confocal microscope with laser scanner (PerkinElmer, Waltham, MA, USA) at 100× magnification and digital pictures were taken using Nikon microscope software packages (Nikon, Tokyo, Japan).

Cell Apoptosis Assay
Fresh goat PBMCs (1 × 10 6 cells/mL) were cultured into 24-well plates and incubated with consecutive concentrations of rHc-GDC (10, 20, 40 and 80 µg/mL), PBS, and purified pET32a protein (10 µg/mL) at 37 • C in a humidified atmosphere with 5% CO2 for 24 h. After washing, cells were re-suspended in binding buffer, and Annexin V-FITC (Miltenyi Biotec, Bergisch Gladbach, Nordrhein-Westfalen, Germany) was added to the cells and incubated for 15 min in the dark at room temperature [39]. Flow cytometer (BD Biosciences, San Jose, CA, USA) was used after addition of Propidium Iodide (PI, Sigma-Aldrich, Shanghai, China) to the cell suspension.

Nitric Oxide Production Assay
Goat PBMCs (1 × 10 6 cells/mL) were washed twice and 100 µL of cells were incubated with serial concentrations of rHc-GDC (10, 20, 40 and 80 µg/mL), PBS and purified pET32a protein (positive control), and in Dulbecco's Modified Eagle Medium in 96-well plate at 37 • C in a humidified atmosphere with 5% CO2. Nitric oxide (NO) production assay was performed in triplicate by using Griess assay as the instructions given by Total Nitric Oxide Assay Kit (Beyotime Biotechnology, Jiangsu, China). The absorbance values of colored solution were measured using a micro plate reader at 450 nm (OD 540 ) and converted these values to micro moles per liter (µmol/L) with a standard curve obtained by adding 0-80 (µmol/L) of sodium nitrate in fresh culture media. This experiment was performed in triplicate individually.

Determination of Cytokine Expression
Quantitative real-time polymerase chain reaction (QRT-PCR) was used to determine the expression of IFN-γ, IL-4, and IL-9. Initially, collected goat PBMCs were activated with 10 ng/ml phorbol myristate acetate (PMA) and 1 µg/mL ionomycin (Sigma) and treated with different concentrations of rHc-GDC (10, 20, 40, 80 µg/mL), PBS (Negative control) and purified pET32a protein (10 µg/mL). Subsequently, total RNA was extracted using Trizol reagent (Invitrogen, Shanghai, China) as described above and cDNA was synthesized using cDNA Kit (Takara Biotechnology, Dalian, China). Actin-β was used as reference gene ad sequence details of primers used for QRT-PCR are given below (Table A1 in Appendix A).

Statistical Analysis
Statistical analysis of the data was evaluated by using the Graph Pad Prism™ version 6.01software. All data obtained from the above experiments were displayed as mean ±SEM. One-way ANOVA followed by Tukey's post-hoc test was employed to compare the variances between groups and considered statistically significant at * p < 0.05, ** p < 0.01, *** p < 0.001. QRT-PCR data were analyzed based on raw cycle threshold (Ct), obtained from the ABI Prism 7500 software (Applied Biosystems, Foster City, CA, USA) by the comparative Ct (2 −∆∆ Ct ) method [43]. Flowcytometry data was analyzed using Cytometrists Expert software.

Molecular Cloning, Expression, and Purification of Hc-GDC
The fragment size of Hc-GDC gene (438) was detected between 500 and 250 ( Figure 1A) and confirmed by sequence analysis. Multiple sequence alignment showed that all sequences were closely related with conserved domain residues ( Figure 1B). Recombinant plasmid (pET32a + Hc-GDC) was induced with IPTG and analyzed by SDS-PAGE that showed the highest concentration after five hours of induction (Figure 2  Putative alternate dimerization interfaces based on amino acids sequences were marked as red boxes and sugar binding pockets based on amino acids sequences were marked as blue boxes. The residue that matches the majority is indicated by "-".

Western Blot Assay of rHc-GDC
The purified His tagged fusion rHc-GDC protein was resolved on 12% SDS-PAGE and showed that rHc-GDC protein could be recognized by serum from goat experimentally infected with H. contortus kDa (Figure 3; Lane 1). Western blotting analysis revealed that rHc-GDC could be recognized by antibodies generated against rHc-GDC protein in SD rats' sera. The molecular mass of the native GDC protein was 14.6 kDa, which was the same molecular weight as that of the rHc-GDC after reduction from pET32a protein (Figure 3; Lane 2). No protein was detected when membranes were incubated with normal goat sera (Figure 3; Lane 3) and normal rat sera (Figure 3; Lane 4). The results indicated that rHc-GDC had good antigenicity and it is one of the ESPs from H. contortus that could be recognized by the immune system of the host.

Binding of rHc-GDC to Goat PBMCs
The cultured goat PBMCs were analyzed by immunofluorescence assay (IFA) using a confocal microscope. DAPI was used to stain nuclei (blue fluorescence) and a secondary antibody was labeled with Cy3 to stain the cells (red fluorescence). Confocal microscopy results revealed that rHc-GDC was merged with the cell surface and showed red and blue combined fluorescence (Figure 4, upper Section), whereas no combined fluorescence was observed in cells treated with PBS ( Figure 4, lower Section). The dense concentration of red around goat PBMCs revealed that rHc-GDC could strongly bind to the surface of the cell.

Localization of rHc-GDC on surface of Adult H. contortus Worm
Immunofluorescence assay was performed to detect localization of rHc-GDC using partial body sections of H. contortus adult. Clusters of blue spots inside the body of worms are indicating the position of nuclei along the gut structure in both merge and DAPI sections. IFA results indicated that Hc-GDC (red color) might be localized outer and inner surfaces of the membrane and in the gut region of the parasite as well. No protein was detected in the control group ( Figure 5).

Effect of rHc-GDC on PBMC Viability and Apoptosis
Cell viability assessed by means of the Trypan blue exclusion test was consistently >95%. To explore the impact of different concentrations of rHc-GDC on early and late apoptosis of PMBCs, cell apoptosis assay was performed. The results showed that there was no significant change between annexin V positive-purified pET32a protein and PBS (negative control). Moreover, there was no significant impact (p > 0.05) of rHc-GDC observed on the early and late apoptosis of goat PBMCs at concentrations of 10 µg/mL, 20 µg/mL, 40 µg/mL, and 80 µg/mL.

PBMC Migration Assay
Cell migration assay was performed to evaluate the impacts of rHc-GDC on cell migration. The percentage of migrated cells through Millipore polycarbonate membrane (Millicell ® Cell Culture Inserts, Corning, NY, USA) into the lower chamber was calculated, and results indicated that 13.15 ± 1.02% with control and 13.08 ± 1.21% cells with pET32a group were migrated. Significant migration into a lower chamber was observed in a group treated with 10 µg/mL (20.61 ± 2.83%), 20 µg/mL (21.84 ± 3.21%), 40 µg/mL (32.44 ± 2.77%), and 80 µg/mL (34.5 ± 2.51%) protein concentration was observed as compared to the control and pET32a group (Figure 7).

Figure 7.
Effects of rHc-GDC on PBMC migration. Cells were treated with PBS (control), purified pET32a protein, and serial concentrations of rHc-GDC. Then, the random migration was determined. Statistical differences between standard error mean of three independent experiments (n = 5) were calculated using one-way ANOVA and marked ** when p < 0.01, *** when p < 0.001 and ns when no significant difference was found.

Nitric Oxide Production Assay
The total nitric oxide assay kit was used to evaluate the effect of rHc-GDC on nitric oxide production by PBMCs. Results revealed that the rHc-GDC with 10 µg/mL, 20 µg/mL, 40 µg/mL, and 80 µg/mL concentration showed significant (ANOVA, F (5, 12) = 25.03, p < 0.001) NO production in PBMCs as compared to control group, whereas no significant difference in NO production was observed between control and pET32a ( Figure 8). The nitrate concentration in the PBMCs was measured by using the Griess assay. Statistical differences between standard error mean of three independent experiments were calculated using one way ANOVA and marked * when p < 0.05, ** when p < 0.01, *** when p < 0.001, and ns when no significant difference was found.

Protein rHc-GDC Modulated Cytokine Secretion by PBMCs
QRT-PCR assay was performed to assess the cytokine production by PBMCs that had been incubated with different concentrations of rHc-GDC. As shown in Figure 9, the production of IL-4 and IL-9 increased significantly (p < 0.001) when BFA treated PBMCs were cultured with 20, 40, and 80 µg/mL rHc-GDC, while the expression of IFN-γ and IL-10 did not show obvious changes (p > 0.05) when compared with purified pET-32a protein and PBS groups.

Discussion
Galectins are an evolutionarily ancient family of proteins which are closely related to carbohydrate binding proteins, located either intracellularly or extracellularly and can be produced by both the parasite and the host [44]. Previous studies suggested that parasite galectin might play an important role in host-parasite interactions [7]. Previously, the ability of recombinant galectins to exert various in vitro activities by engaging glycans on the extracellular matrices as well as cell surface has been convincingly and extensively documented [45,46]. In this study, recombinant GDC protein from H. contortus was characterized and localization of this protein by immunofluorescence assay confirmed the binding with host immune cells, in surface ligand complex shape, which is a characteristic feature of ESPs to modulate the immune functions of host PBMCs [47]. Mammalian galectins have been identified in various cells and tissue; however, the digestive tract is particularly rich in galectins [48]. Similar to its mammalian homologues, immunohistochemical investigation of this study also demonstrated that the native GDC protein was predominantly covering the internal gut surface of adult H. contortus worm. In our previous proteomic analysis, galectin was found at luminal surface of the adult worm of H. contortus [49]. In another study, a 32 kDa galectin was found most abundantly in the adult cuticle of Caenorhabditis elegans [50].
Proteomic analysis of ESPs indicated that galectins were expressed numerously in H. contortus, Teladorsagia circumcincta, and Ostertagia ostertagi [51]. In this study, we found that rHc-GDC could be recognized by the antiserum from goats experimentally infected with H. contortus. All of these findings indicated that the galectin domain containing protein of H. contortus is one of the excretory secretory antigens and interacts with the host immune system during infection. Galectins from the parasite source have evidenced an increased expression during the infection and hence they are considered as key players in host-parasite interactions [51,52].
In previous studies, immuno-regulatory roles of galectins have been reported in different cellular processes such as cell adhesion, cell proliferation [53], apoptosis [54], and immune responses [55]. Cell proliferation and migration are very important for the development of immune responses and tissue regeneration. Cell proliferation is regulated by both T cells and antigen-presenting cells (APCs) [56], and increases the number of immune effector cells. These effector cells migrate to the surrounding tissue or infection sites through the cell migration process and sometimes resulted in tissue damage [52,57]. In this study, it was identified that goat PBMC proliferation was increased at a significant level, which ultimately showed constant apoptosis of PBMCs in response to rHc-GDC treatment. Moreover, increased migration rate of immune effector cells was observed in vitro. The proliferation results of this study are in line with the results of the previous study [58]. In contrast, cell proliferation was suppressed after PBMCs were treated with rHcARF1 [59]. Specific immuno-regulatory properties of antigen might be the possible reason for these differences. The possible reason may be the dependency of migration on cell proliferation as cells need more room to migrate. Moreover, further study is needed to investigate the actual mechanism of cell migration.
Nitric oxide (NO) has been documented as an important immunomodulator in various infections including H. contortus by mediating host protection, parasite killing, or suppressing the growth. Previously, it was documented that NO had been involved in nonspecific defense mechanism against H. contortus and various other parasites [60]. In the present study, immunomodulatory effects of the rHc-GDC on the NO production by goat PBMCs were evaluated. Cells incubated with different concentrations of rHc-GDC significantly increased the NO production in a dose dependent manner. A constant increase in NO production indicated that rHc-GDC involved in the immunomodulatory regulation of NO on goat PBMCs. A previous study supports these results in which one of the ESPs, recombinant Arginine kinase, was used. Insights of NO production mechanism are still lacking [39].
PBMCs consist of several immune cells, including lymphocytes and monocytes that play important roles in innate and adaptive immunity [26]. The current study focused on evaluating the effects of rHc-GDC protein on PBMCs-derived CD4+ T helper cells. CD4+ T helper cells play an important role in the host defense of pathogens [19]. T helper cells mainly consist of Th1, Th2, Th17, and newly described Th9 cells. Th1 cells induce delayed-type hypersensitivity reactions and Th2 cells are excellent helpers for B-cell antibody secretion [14]. A previous study reported that T cell subsets are closely related to dyslipidemia and kidney injury during preeclampsia disease [61]. Moreover, reduced T cell activity has been reported during Parkinson's disease [62]. Immune cells secrete cytokines that enable communication and regulation of the immune system [49]. Previously, increased levels of CD4+ T cells-derived IL-2 and IL-21 have been reported in inflammatory bowel disease [63]. Moreover, previous studies documented that IL-9 is a signature cytokine for Th9 cells with diverse functions [64,65]. In this study, PBMCs incubated with different concentrations (20,40,80; µg/mL) of rHc-GDC increased Th9 levels in a dose-dependent manner and, in turn, the surge of signature cytokine was recorded in vitro. The rHc-GDC didn't show a significant effect on the production of suppressive cytokine IL-10, Th1 cells, and associated immune response IFN-γ, but the secretion of IL-4 cytokine and production of Th2 cells were up-regulated. One of the possible reasons is that Th2 cells in combination with IL-4 promoted the differentiation of Th9 cells directly. Th2 lymphocytes and mast cells also play important roles during nasal congestion, over sneezing, rhinorrhea, and itching [66]. The physiological function of IL-9 seems to be in some way connected with a Th2 response, as IL-9 is important in the immune defense against helminth infection but also appears during allergic reactions in the lung, both scenarios that depend on Th2 responses [67]. More recent studies revealed the multifunctional activities of IL-9 cytokine. IL-9 production can modulate immune response in both manners as IL-9 promotes allergic and inflammatory reactions by recruiting mast cells, macrophages, and eosinophils [68,69]. Conversely, IL-9 enhances the immunosuppressive activity of natural regulatory T-cells (16). Previously, promotion of mast cells expansion was reported by IL-9 during some allergic responses in the lungs [70][71][72]. Similarly, in food allergy, effects of IL-9 production were also reported in mastocytosis and intestinal permeability [73]. Additionally, involvement of IL-9 has also been observed in the development of autoimmune diseases and its deficiency lessens the disease progression [68,74]. On the contrary, IL-9Rα-deficient mice had developed more severe experimental autoimmune encephalomyelitis (EAE) [75]. In addition, high levels of IL-9+TH17 cells were found in diabetic patients' blood [76]. Furthermore, parasitic removal from the gastro intestinal tract can be promoted by IL-9. In these conditions, it is thought that Th9 cells produce IL-9 that stimulate the mucosal mast cells production that contribute in mucus production and increase the muscle contraction as well as intestinal permeability [21,77], while IL-9-deficient mice during lung parasitic infection caused by Schistosoma mansoni only decreased the mucus production and no effect on granuloma formation for parasitic egg removal [78]. As far as the tissue grafting is concerned, IL-9 increases the capability of T Regulatory cells for reduction of immune responses at the graft site [64,79]. The functions of IL-9 cytokine in immunity are still controversial and further studies are needed to understand it completely.

Conclusions
This study revealed that rHc-GDC is an important active protein of H. contortus ESPs that played crucial roles in the immune regulations. Results indicated that the interaction of rHc-GDC with host cells increased the production of Th9 cells, signature cytokine IL-9, nitric oxide, and cell migration in dose-dependent manner. The findings concluded that rHc-GDC has the potential to enhance the protective immunity by inducing Th2 and Th9 immune response in vitro, which will help to elucidate the host-parasite interaction. However, further studies are needed to evaluate the effects of rHc-GDC on Th9 immune response during different stages of H. contortus infection in goats.

Acknowledgments:
We greatly thankful to Syed Sajjad Hussain Naqvi and all the people whose hard work in laboratories contributes to this work.