Analysis of the role of Dirofilaria repens macrophage migration inhibitory factors in host–parasite interactions

Complementary DNA encoding two proteins (GenBank accession numbers MT071087.1 and MT071088.1) was amplified using gene-specific primers containing restriction enzyme sites and cloned in the E. coli transformation pET28a plasmid (Novagen, San Diego, CA, USA) following BamHI and XhoI digestion. The cloned insert was sequenced using the Sanger technique to confirm that no amino acids had changed by mutation and verify that the open reading frame was appropriate. The plasmids containing the verified insert sequences were transformed to two E. coli expression strains: SoluBL21 and BL21. To induce protein expression, isopropyl-1-thio-β-d-galactopyranoside (IPTG) at a final concentration of 1mM was added to the bacterial culture at the log phase of growth. After 2 h, cells were centrifuged at 5,000 × g for 10 min at room temperature. Bacterial pellets were either used immediately for protein purification or stored at −20°C.

The pellets were sonicated to disintegrate cell membranes and centrifuged at 10,000 × g for 20 min at 4°C. The supernatants containing recombinant fusion proteins with 6 × His tags were collected and the proteins were purified using a Ni²⁺-charged affinity chromatography column (Cytiva, Little Chalfont, UK) according to the manufacturer’s protocol. The purity of the eluted proteins was assessed using sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and Perfect Tricolor Protein Ladder molecular weight marker was used for protein sizing (EURx, Gdańsk, Poland). The two elution fractions with the highest protein concentrations were pooled, dialysed against Dulbecco’s phosphate-buffered saline (Biowest, Nuaillé, France) using 2 mL Zeba Spin 7 kDa molecular weight cut-off (7K MWCO) Desalting Columns (Thermo Scientific, Waltham, MA, USA) and assessed for recombinant protein content by Western blotting using Anti-polyHistidine-Peroxidase antibody (Sigma-Aldrich, St. Louis, MO, USA). The purity and concentration of the recombinant protein solutions were determined using SDS-PAGE and a bicinchoninic acid BCA Protein Assay Kit (Pierce Biotechnology, Rockford, IL, USA), respectively.

Alignment of human MIF (hMIF), dog MIF (dMIF), Dre-MIF-1 and Dre-MIF-2 protein sequences was performed using the Multalin tool (10). The GenBank accession numbers of the analysed sequences were CAG30406.1 (hMIF), XP_038293371.1 (dMIF), MT071087.1 (Dre-MIF-1) and MT071088.1 (Dre-MIF-2). The protein identity (%) between them was calculated using ClustalW (25). The tertiary structures of Dre-MIF-1 and Dre-MIF-2 were predicted using Phyre2 (18). The structures were visualised and superimposed using Protein Imager (26).

Total RNA was isolated from microfilariae and adult worms using a Total RNA purification kit (A&A Biotechnology, Gdansk, Poland) according to the manufacturer’s instructions. Genomic DNA contamination was removed using DNAseI (Thermo Scientific) and the efficiency of DNA cleavage was assessed using PCR. When free of DNA contamination, the RNA solution was used for cDNA synthesis using a RevertAid RT Reverse Transcription Kit (Thermo Scientific).

The qPCR was performed in a 12 μL volume in a 96-well PCR plate. The reaction components were as follows: 2 μL of cDNA template, 1 × Maxima SYBR Green qPCR Master Mix (Thermo Scientific), ROX passive reference dye (10 nM) and a mixture of the primers Dre-mif-1_F and Dre-mif-1_R or Dre-mif-2_F and Dre-mif-2_R at 0.3 μM (Table 1). Due to the lack of data regarding a suitable reference gene for quantitative real-time PCR analyses in D. repens, the direct copy number was estimated for reaction evaluation. To achieve standard curves for both genes, pET28a/Dre-mif-1 or pET28a/Dre-mif-2 recombinant plasmid was 10-fold serially diluted to contain from 10⁸ to 10² copies per reaction and used as a matrix for qPCR. The PCR was performed as follows: 50°C for 2 min, 95°C for 10 min, 45 cycles of 95°C for 15 s and 60°C for 1 min and a disassociation curve stage. The reaction was performed in triplicate.

The antibodies were generated by subcutaneous injection into the neck tissue of two 10-week-old male BALB/c mice of 100 μg of either rDre-MIF-1 or rDre-MIF-2 precipitated with Imject Alum (Thermo Scientific) in a final volume ratio of 1 : 2, followed by two boosts at 14-day intervals. The experiments were conducted following the guidelines and regulations of the 2^nd Local Ethics Committee for Animal Experimentation in Warsaw (Permit No. WAW2/ 142/2021). Polyclonal sera were collected from mice 14 days after the last immunisation.

Serum reactivity against the recombinant proteins was assessed using ELISA and Western blot. Ninety-six-well plates (Wuxi NEST Biotechnology, Wuxi, China) were incubated with rDre-MIF-1 or rDre-MIF-2 (2.5 μg/mL) diluted in bicarbonate buffer (100 μL/well of 0.015 M Na2CO3 and 0.035 M NaHCO3 at pH 9.5) overnight at 4°C. The plates were rinsed three times with 250 μL of phosphate-buffered saline (PBS) supplemented with 0.05% Tween-20 and then blocked with 200 μL of 5% filtered skimmed milk in PBS buffer for 90 min at 20°C. The plates were rinsed as described above, and subsequently 100 μL of anti-rDre-MIF-1, anti-rDre-MIF-2 or negative control mouse serum (n = 2) appropriately diluted in PBS (1 : 51,200 for total IgG, IgG1, IgG2 and IgM and 1 : 5,120 for IgE) was added to the wells. Dilutions were established based on preliminary data obtained using a serum dilution series. The plates were incubated at room temperature for 1.5 h and washed three times. The subsequent step was a 1-h incubation with horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG solution (1 : 30,000), goat anti-mouse IgG1 (1 : 4,000), goat anti-mouse IgG2 (1 : 4,000), goat anti-mouse IgM (1 : 4,000) or goat anti-mouse IgE (1 : 4,000) (all from AbD Serotec, now Bio-Rad, Kidlington, UK). The reaction was developed with the TMB Substrate Kit (Thermo Scientific) and stopped after 30 min using 2 M H2SO4. Absorbance readings were recorded at 450 nm using a Synergy H1 microplate reader (BioTek, Winooski, VT, USA).

To confirm the cross-reactivity between the sera, Western blot analyses were performed. A 5-μg mass of either rDre-MIF-1 or rDre-MIF-2 was resolved in polyacrylamide gel and transferred to a nitrocellulose membrane. The membrane was blocked with 5% skimmed milk in PBS (w/v) overnight with continuous shaking at 4°C. The membranes were probed with anti-rDre-MIF-1 or anti-rDre-MIF-2 sera (diluted 1 : 5,000), rinsed with PBS/0.05% Tween 20 buffer and incubated for 60 min with HRP-conjugated anti-mouse IgG solution (1 : 5,000, Sigma-Aldrich). The immunoreactive bands were developed using West Pico Chemiluminescent substrate (Thermo Scientific) and visualised on radiography films.

Samples from naturally infected and uninfected dogs were collected during the study by Wysmołek et al. (29). Infected dogs were classified for the study based on a positive Knott’s test result. The reactivity of rDre-MIF-1 and rDre-MIF-2 with sera from infected (n = 17) and uninfected (n = 6) dogs with total IgG, IgG1, IgG2, IgE and IgM antibodies was measured using an indirect ELISA.

The secondary antibodies for this ELISA were HRP-conjugated rabbit anti-dog IgG (total) (Jackson ImmunoResearch, Cambridge, UK), goat anti-dog IgG1, goat anti-dog IgG2, goat anti-dog IgM and goat anti-dog IgE (all from AbD Serotec). The ELISA procedure was the same as for the mouse sera except that the dog sera were diluted 1 : 400 for total IgG, IgG1 and IgG2; 1 : 20 for IgE; and 1 : 3,200 for IgM, and the secondary antibodies were diluted 1 : 30,000 for rabbit anti-dog IgG; 1 : 10,000 for goat anti-dog IgG1 and IgG2; and 1 : 1,000 for goat anti-dog IgM and IgE.

All statistical analyses were performed using GraphPad Prism 9 software (GraphPad, Boston, MA, USA).

Expression studies were conducted in two distinct E. coli strains: BL21 and SoluBL21. In both cases, cells produced soluble recombinant proteins with an approximate size of 15 kDa–17 kDa. However, there were differences between the purification efficiency from one strain and the efficiency from the other. Purification of rDre-MIF-1 was more efficient from the SoluBL21 strain, while better results for rDre-MIF-2 were achieved using the BL21 strain. Analyses by SDS-PAGE indicated that the purified rDre-MIF-1 and rDre-MIF-2 were electrophoretically homogeneous and without impurities (Fig. 1).

Fig. 1.

Sodium dodecyl sulphate–polyacrylamide gel electrophoresis analysis of purified recombinant Dirofilaria repens (rDre)-macrophage migration inhibitory factor (MIF)-1 (left) and rDre-MIF-2 (right). Lane M – molecular weight marker; Lane 1 – rDre-MIF-1 (10 μL); Lane 2 – rDre-MIF-2 (10 μL)

A significantly higher expression of both genes was observed in the adult stage. Interestingly, the expression level of Dre-mif-1 was double that of Dre-mif-2 in the adult stage (Fig. 2).

Fig. 2.

Dirofilaria repens macrophage migration inhibitory factor (Dre-mif)-1 and Dre-mif-2 gene expression in microfilariae and the adult stage of D. repens. The fold increase in expression is shown over the level of expression of Dre-mif-1 in microfilariae. Statistical analysis was performed using one-way analysis of variance; **** – P-value <0.001

The sequence comparison analysis (Fig. 3) revealed that Dre-MIF-1 had higher identity (40 %) to host MIFs (human and dog) than Dre-MIF-2 (27%). Despite Dre-MIF-1 and Dre-MIF-2 amino acid sequences having shown low identity of 27.8 % (Table 2), their potential tertiary structures showed a high level of similarity (Fig. 4).

Fig. 3.

Alignment of human macrophage migration inhibitory factor (hMIF), dog MIF (dMIF), Dirofilaria repens (Dre)-MIF-1 and Dre-MIF-2 amino acid sequences

Fig. 4.

The visualisation of superimposed potential structures of Dirofilaria repens (Dre)-macrophage migration inhibitory factor (MIF)-1 (red) and Dre-MIF-2 (blue). The complete structures are shown on fragments A) and C), whereas matching helices and strands are respectively shown on fragments B) and D)

The results suggest that rDre-MIF-1 was more immunogenic, as the levels of anti-rDre-MIF-1 total IgG, IgG2 and IgE antibodies were much higher than those of anti-rDre-MIF-2 immunoglobulins (Fig. 5). At the same time, antibodies raised against rDre-MIF-1 were less specific than these produced after rDre-MIF-2 immunisation. Western blot analysis revealed that antibodies specific to rDre-MIF-1 recognised both molecules with similar affinity, whereas anti-rDre-MIF-2 IgG antibodies showed only weak reactions with rDre-MIF-1 (Fig. 6). However, as shown in Fig. 5, only IgG1 antibodies were responsible for this cross-reactivity. Our results show that immunisation with MIFs favours the production of IgG1 subclass antibodies. In turn, IgG2 antibodies are produced only after rDre-MIF-1 immunisation and do not cross-react with rDre-MIF-2 molecules. A similar observation was made for the IgE class. Antibodies of the IgM class were found to be the least specific and the most cross-reactive of all the analysed classes.

Fig. 5.

Reactivity of mouse anti-recombinant Dirofilaria repens (rDre)-macrophage migration inhibitory factor (MIF)-1 and anti-rDre-MIF-2 sera with rDre-MIF-1 and rDre-MIF-2. Serum dilutions for detection: immunoglobulin (Ig)G, IgG1, IgG2 and IgM – 1 : 51,200; IgE – 1 : 5,120. OD – optical density

Fig. 6.

Western blot cross-reactivity analysis of mouse anti-recombinant Dirofilaria repens (rDre)-macrophage migration inhibitory factor (MIF)-1 (A) and anti-rDre-MIF-2 (B) antibodies with rDre-MIF-1 and rDre-MIF-2

The reactivity of rDre-MIF-1 and rDre-MIF-2 was tested with sera of naturally infected and uninfected dogs. An elevated level of IgG1-subclass antibodies recognising both rDre-MIF-1 and rDre-MIF-2 was noted in infected dogs’ sera (Fig. 7). No significant differences were observed in the levels of total IgG, IgG2, IgE or IgM antibodies between infected dogs’ and uninfected dogs’ sera.

Fig. 7.

Reactivity of different serum antibody classes in infected and non-infected dogs with recombinant Dirofilaria repens (rDre)-macrophage migration inhibitory factor (MIF)-1 and rDre-MIF-2. Serum dilutions for detection: immunoglobulin (Ig) G, IgG1, and IgG2 – 1 : 400; IgM – 1 : 3,200; IgE – 1 : 20. Statistical analysis was performed using the Mann–Whitney test; * – P-value <0.05; *** – P-value <0.001