Fasciola gigantica Recombinant Abelson Tyrosine Protein Kinase (r Fg Abl) Regulates Various Functions of Buffalo Peripheral Blood Mononuclear Cells

Abstract

Fasciola gigantica can modulate host immune mechanisms through excretory-secretory products (ESP). As one of the components of ESP, it is unknown whether Abelson tyrosine protein kinase (Abl) is involved in parasite-host immune interaction. To investigate the immunoregulatory function of Abl in Fasciola gigantica, we cloned and expressed the Fasciola gigantica Abl protein and assessed its effect on specific immune functions of buffalo peripheral blood mononuclear cells (PBMCs). Recombinant F. gigantica Abelson tyrosine protein kinase (rFgAbl) was expressed in Escherichia coli. Western blot analysis was performed to assess the reactivity of anti-rFgAbl antibodies with rFgAbl, serum from F. gigantica-infected buffalo, and excretion and secretion products of F. gigantica. Immunohistochemical analysis was conducted to determine the localization of FgAbl in tissues from larval stages and adult worms of F. gigantica. Furthermore, immunofluorescence analysis was utilized to evaluate the binding ability of the rFgAbl protein to buffalo peripheral blood mononuclear cells (PBMCs), as well as to investigate the effects of varying concentrations of rFgAbl protein (5, 10, 20, 40, and 80 μg/mL) on the functional responses of PBMCs. Anti-rFgAbl antibodies specifically recognize rFgAbl, serum from buffalo infected with F. gigantica, and FgESP. rFgAbl is localized in the cecum and capsule of juvenile worms, as well as in the testis and viellaria of adult worms. Additionally, rFgAbl enhances cell proliferation, migration, nitric oxide (NO) production, and phagocytosis, while also increasing the transcription levels of cytokines (IFN-γ, IL-12, TNF-α, IL-4, IL-10, and TGF-β). The results indicate that rFgAbl can influence the immune function of PBMCs. Further investigation into the immunomodulatory properties of the rFgAbl protein will enhance our understanding of the immune interaction mechanisms between trematodes and their hosts.

Keywords: Abelson tyrosine protein kinase; Fasciola gigantica; host–parasite interactions; immune regulation.

Figure 1

Cloning, expression, and Western blot analysis of Fasciola gigantica (Fg) Abelson tyrosine protein kinase (Abl). (A,B) M: DNA molecular weight standard. (A) 1: Amplification of the Abl gene from F. gigantica cDNA. (B) 1: Double enzyme restriction digest of the rpET28a-FgAbl plasmid. (C,D) M: Protein molecular weight standard. (C) 1: Supernatant of bacterial cell lysate prior to induction; 2: Pellet of bacterial cell lysate prior to induction; 3: Supernatant of cell lysate induced at 37 °C for 10 h; 4: Cell lysates were pelleted after induction at 37 °C for 10 h. (D) 1: rFgAbl was transferred to PVDF membrane and recognized by the His tag antibody.

Figure 2

Western blot analysis of rFgAbl. (A–C) M: protein molecular weight standard; (A) 1: FgAbl recombinant protein, which was detected by rabbit anti-rFgAbl serum, revealing a band at approximately 50 kDa; 2: FgAbl recombinant protein. No bands were observed in serum incubation of healthy rabbits (B) 1: FgAbl recombinant protein reacted with serum from fluke-infected buffalo, resulting in the detection of a band at ~50 kDa; 2: FgAbl recombinant protein. FgAbl recombinant protein. Healthy bovine serum incubation is band-free. (C) 1: F. gigantica native protein reacted with rabbit anti-rFgAbl serum, yielding a band at ~50 kDa; 2: FgESP reacted with rabbit anti-rFgAbl serum, also detecting a band at ~50 kDa. 3-4: Fg natural protein and FgESP. Incubation with healthy rabbit serum without bands. The final form of the image is stitched together.

Figure 3

Distribution of FgAbl in juvenile (42 dpi) and adult worms. Tg: tegument; Ms: muscle; Pc: parenchyma; Ca: cecum; Ti: Testis. Vi: viellaria. (A) The localization of FgAbl protein in juvenile worms (42 dpi) is observed in the capsule, muscles, and cecal epithelium. (B) The FgAbl protein in adult worms is primarily located in the testis and viellaria. Red indicates the target antigen stained by a Cy3-coupled secondary antibody, while blue represents the nucleus stained by DAPI. The term ‘Merge’ refers to the combined effect of DAPI and Cy3 staining. Scale bars represent 200 μm.

Figure 4

The recombinant F. giganticaAbelson tyrosine protein kinase (rFgAbl) binds to the surface of buffalo peripheral blood mononuclear cells (PBMCs). PBMCs treated and untreated with rFgAbl were incubated with rabbit anti-rFgAbl antibody and stained with Cy3-conjugated goat anti-rabbit IgG. PBMCs surface staining was observed in cells treated with rFgAbl, whereas no staining was detected in untreated cells. Scale bar: 50 μm.

Figure 5

rFgAbl promotes the proliferation and migration of buffalo PBMCs. Buffalo PBMCs were treated with phosphate-buffered saline (PBS), concanavalin A (10 μg/mL), and various concentrations of rFgAbl (μg/mL) before being incubated at 37 °C for 48 h. Cell proliferation was assessed using the CCK-8 method. The results indicated that rFgAbl significantly enhanced the proliferation of PBMCs. PBMCs were treated with phosphate-buffered saline (PBS) and various concentrations of rFgAbl (μg/mL) to assess the percentage of cell migration (%). Graphs represent means ± standard deviations of data from 3 independent biological replicates; Asterisks denote significant differences between control and treated cells (* p < 0.05; *** p < 0.001; **** p < 0.0001; ns, not significant).

Figure 6

Effect of rFgAbl on NO Production and Cellular phagocytosis in PBMCs. After treating PBMCs with PBS and various concentrations of rFgAbl (μg/mL) for 24 h, the total concentration of NO produced by the PBMCs was measured using the Griess method. PBMCs were treated with PBS and varying concentrations of rFgAbl (μg/mL), after which the cellular uptake of FITC-dextran was measured. Graphs represent means ± standard deviations of data from 3 independent biological replicates; Asterisks denote significant differences between control and treated cells (*** p < 0.001; **** p < 0.0001; ns, not significant).

Figure 7

Effect of rFgAbl on cytokines in PBMCs. After incubating PBMCs with rFgAbl for 48 h, the transcription levels of IFN-γ, IL-12, TNF-α, IL-4, IL-10, and TGF-β were measured using real-time fluorescence PCR. Graphs represent means ± standard deviations of data from 3 independent biological replicates. Asterisks indicate significant differences between control and treated cells (** p < 0.01; **** p < 0.0001).