Investigating immunomodulating activities of recombinant horse IL-2, IL-18 and IFN-ã in peripheral blood mononuclear cells (PBMCs)

Cytokines and chemokines are instrumental for mounting host defense and controlling intracellular replication of equine pathogens such as Burkholderia mallei, Rhodococcus equi and equine infectious anemia virus (Berghaus et al., 2018, Lin et al., 2011, Saikh and Mott 2017). Th1 cytokines like IL-2, IL-18 and IFN-γ directs cell-mediated immunity and phagocyte-dependent killing of infected cells (Kim et al., 2002, Silva and Dow 2013). Increased Th1 cytokines profile was also observed during early pregnancy in cows (Yang et al., 2016). 
       
In comparison to bovine and ovine, only limited studies were undertaken to assess proliferative response and antiviral activity of recombinant horse cytokines. Recombinant horse IL-2 produced in COS cells augmented the proliferative response of equine PBMCs (Vandergrifft and Horohov 1993). Horse IL-2 and IL-4, transiently expressed in CHO cells, were able to induce proliferation of horse PBMCs (Dohmann et al., 2000). Mammalian cells derived horse IFN-γ showed profound antiviral activity against various equine viruses (Steinbach et al., 2002). Similarly, insect cells derived recombinant equine IFN-γ showed strong antiviral effect against RNA viruses (Sentsui et al., 2010). However, low level of protein expression, high operational cost and less protein yield are major drawbacks of eukaryotic expression system.
       
In the present study, recombinant horse IL-2, IL-18 and IFN-γ were produced in prokaryotic expression system and immune modulating activities of these purified cytokines were evaluated in PBMCs.

Cloning and expression of horse IL-2, IL-18 and IFN-γ
 
Coding sequences of horse mature IL-2 and IL-18 were  previously cloned and sequenced (Singha et al., 2015). Similarly, coding sequence of mature IFN-γ was cloned in pGEMT-easy vector. For protein expression, IL-2, IL-18 and IFN-γ encoding gene was cloned into Sac I and Hind III restricted pQE30 expression vector. The recombinant pQE30 vectors with respective cytokine gene were transformed in E. coli M15 competent cells. Positive colonies were selected on Luria Bertani (LB) agar plates supplemented with kanamycin (30 μg/ml) and ampicillin (50 μg/ml) and screened by SDS-PAGE. Briefly, 10 colonies were randomly picked and grown in 5 ml of LB broth. The log phase cultures were induced by 1mM isopropyl-β-D-thiogalactoside (IPTG). Different incubation temperatures (30°C, 37°C, 42°C and 45°C) and induction period (3-5 h) were evaluated for optimum expression of recombinant cytokines. The uninduced cells were used as negative control. Samples from bacterial cells were prepared and run on 12 % SDS-PAGE.
 
Purification of recombinant IL-2, IL-18 and IFN-γ
 
Recombinant cytokines were purified from 300 ml culture by Ni-NTA agarose column under denaturing condition. Different purification fractions (eluted fractions, the washing fractions, unbound fractions) were collected and checked by 12% SDS-PAGE. The concentrations of recombinant cytokines were measured by the Bradford method (1976). Similarly, M15 E.coli cells with empty pQE30 vector (vector control) were also induced with IPTG and same purification protocol was followed. Eluates from the vector control cells (sham purification) were used as sham control in PBMCs culture to rule out residual effect of bacterial products if any obtained during the purification process.
 
Verification of recombinant horse cytokines by western blot and sequencing of recombinant clones
 
The purified recombinant cytokines were electroblotted to PVDF membrane and probed with mouse anti-horse IL-2, IL-18 and IFN-γ monoclonal antibody. The membrane was incubated with alkaline phosphatase conjugated antibody at 1:2000 dilutions for 1 h at room temperature. The membrane was developed by chromogenic substrate NBT/BCIP. For further verification, two positive recombinant clones of each cytokine were sequenced commercially and sequences were confirmed by NCBI-BLAST (https://blast.ncbi.nlm.nih.gov/Blast.cgi).
 
Isolation of PBMCs and stimulation with recombinant cytokines
 
Blood sample (5 ml) was collected aseptically from healthy horses (n=6) and PBMCs were isolated by Histopaque-1077 density-gradient centrifugation. The PBMCs were cultured in triplicate in 96-well microtitre plates at 2 × 10⁵ cells/well. Different concentrations of recombinant cytokines ranging from 250-1500 ng/ml were used for stimulation of PBMCs. Unstimulated equine PBMCs and PBMCs stimulated with sham eluates were used as negative control and sham control, respectively. PBMCs treated with Con A (5 µg/ml) and PHA (10 µg/ml) were used as positive controls. The cells were harvested at 72 h for lymphocyte proliferation, 48 h for cytokine ELISA assay and 3 h, 12 h, 24 h, 72 h for real time PCR.
 
Lymphocyte proliferation assay
 
Lymphocyte proliferation was assessed by XTT reagent. Briefly, XTT solution was prepared by adding 10 mg of XTT reagent and 100 μl of phenazine metho sulphate (PMS) in 10 ml of RPMI media without phenol red. After 72 h of stimulation, 50 μl of XTT solution was added in each experimental well and incubated at 37°C for 4 h. Absorbance was measured at 500 nm and at 600 nm for reference. Stimulation index (S.I) was calculated by following formula.

 
 

Measurement of IL-4, IL-10, IFN- γ and TNF-α in PBMCs culture supernatant
 
After 48 h, the cell culture supernatants from each well were harvested and IL-4, IL-10, IFN-γ and TNF-α were quantified by the ELISA. For estimation of IL-10 and IFN- γ sandwich ELISA kit (Ray Bio®, USA) and for IL-4 and TNF-α competitive ELISA kit (BlueGene, Shanghai) was used according to manufacturer’s instructions. Optical density was measured at 450 nm and standard curve was plotted. Concentration of cytokines in culture supernatants was calculated with respect to standard curve.
 
Quantification of cytokine mRNA in PBMCs by real time qPCR
 
PBMCs were harvested at 3 h, 12 h, 24 h and 72 h interval and RNA was isolated using Tri reagent. The purity and quantity of RNA were assessed using a NanoDrop spectrophotometer. Subsequently, cDNA was synthesized using SmartScribe^TM reverse transcriptase cDNA kit. Real time qPCR was performed in a CFX96^TM fluorescence detection system. Primers used for amplification of equine IL-2, IL-4, IL-10, IL-18, IFN-γ, IL-6, TNF-α, IL-12p35 and ß-actin are shown in Table 1. Optimization of PCR protocol and standard curve preparation was previously described (Saini et al., 2019). PCR reaction consisted of 5 µl of a 2x SYBR Green master mix, 1µl of primer, 1 µl of plasmid DNA in a final volume of 10µl. Thermal programme was set at 95°C for 3 min for initial denaturation and 40 cycles of 95°C for 5 s and 60°C for 30 s. A non template control and no-RT control were included for every PCR run. Copy number of cytokine mRNAs was calculated by extrapolating the Ct value to the standard curves.
 

 
Statistical analysis
 
Statistical significance for XTT assay and cytokine ELISA was evaluated by one-way analysis of variance followed by Turkey’s multiple comparison using GraphPad Prism Software 5.0. Significant differences between copy numbers of different cytokines were evaluated by two way analysis of variance (ANOVA).

Expression and purification of IL-2, IL-18 and IFN-γ and western blotting
 
Molecular weight of recombinant IL-2 and IFN-γ were 17 KDa and IL-18 was 18 KDa (Fig 1 A, B and C). Optimal incubation temperature and induction time for IL-18 and IFN-γ was 37°C and 4 h, respectively. For IL-2, stable and optimum expression was found at 42°C after 3 h of IPTG induction. Requirement of relatively higher incubation temperature (42°C) was also reported for human IL-2 expression (Sengupta et al., 2008). However, high temperature dependant expression of IL-2 warrants further research. Recombinant cytokines showed specific reactivity with monoclonal horse IL-2, IL-18 and IFN-γ antibody (Fig 1.D).
 

 
Lymphocyte proliferation assay
 
Stimulation indices (SI) of proliferation assay for each cytokine are shown in Fig 2. Recombinant IL-2 induced peak cell proliferation at 250 ng/ml concentration (p<0.05, 250 vs 750/1000/1500 ng/ml) with stimulation index of 1.79. Recombinant IL-2 was found to be dominant growth stimulant than IL-18 and IFN-γ (p>0.05) at 250 ng/ml. Previous report indicated that IL-2 supports growth and proliferation of T cells, NK cells and B cells (Olejniczak and Kasprzak 2008). Both recombinant IL-18 and IFN-γ required double concentration (500 ng/ml) for induction of cellular proliferation which corresponds to SI 1.57 and 1.34, respectively. Optimum stimulation dose (500ng/ml) of recombinant IL-18 observed in the present study was in agreement with previous findings where equine IL-18 and human IL-12 were used in combination for cell proliferation (Tong et al., 2010).
 
 
 
Quantification of IL-4, IL-10, IFN-γ and TNF-α in culture supernatants by ELISA and cytokine mRNA by real time RT-PCR
 
Characteristic statistical value of real time PCR are shown in Table 1. Cytokines quantification by ELISA is shown in Table 2. Peak amplification of cytokines mRNA was observed at 3h and transcripts were barely detected after 12 h and 24 h. Several previous findings also indicated that expression of human TNF-α and IFN-γ by PHA (Stordeur et al., 2002); ovine IL-1β, IL-4 and IFN-γ by LPS (Budhia et al., 2006) and rabbit TNF-α, IFN-γ, IL-2, IL-4 and IL-10 by Con A was seen after 3-6 h (Godornes et al., 2007).
 

       
Recombinant IL-2 enhanced secretion of IL-10 (228.9 pg/ml, p<0.001), TNF-α (198.10 pg/ml) and IFN-γ (178.7 pg/ml) in culture supernatant and up regulated copy number (Cn) for IFN-γ (Cn=17620), IL-4 (Cn=2197), IL-12p35 and TNF-α. However, less copy number (Cn<1000) was observed for IL-10, IL-18 and IL-6 (Fig 3.A). Together, ELISA and real-time PCR revealed that recombinant horse IL-2 enhanced IFN-γ, IL-4, TNF-α, IL-10 and IL-12p35 production. Earlier reports showed that IL-2 induces TNF-α and IFN-γ secretion in murine macrophages (Puddu et al., 2005) and TNF-α in human macrophages and monocytes (Strieter et al., 1989). It was also demonstrated that IL-2 modulate expression of IL-4, IL-5, IL-6 and IL-10 (Cote-Sierra et al., 2004).
 

       
Recombinant IL-18 induced secretion of TNF-α (188.9 pg/ml), IL-10 (108.7 pg/ml) and IFN-γ (97.25 pg/ml) in culture supernatants. But it had very strong upregulatory effect on IFN-γ (Cn=27064.91) and moderate effect on IL-4 (Cn=1731.70). However, no significant up regulation of IL-6, IL-2, IL-12p35, TNF-α and IL-10 mRNA (Cn<500) was detected (Fig 3.B). One of the most important immunological functions of IL-18 is induction of IFN-γ (Okamura et al., 1995). Induction of Th2 and pro-inflammatory cytokines by IL-18 was also observed in other animal species (Xu et al., 2000, Nakanishi et al., 2001).
       
Recombinant horse IFN-γ had significant effect on TNF-α secretion (208 pg/ml) in culture supernatants. However, it had little effect on other cytokine mRNA (Fig 3.C). Earlier studies suggested that bacterially derived recombinant equine and human IFN-γ was less active and had less antiviral activity in comparison to mammalian and baculovirus originated IFN-γ (Steinbach et al., 2002, Razaghi et al., 2016). Recently, Bai et al., (2010) demonstrated that the horse IFN-γ expressed in E. coli had significant antiviral activity against vesicular stomatitis virus.
       
Recombinant cytokines and Con A and PHA were equally capable of inducing TNF-α secretion (188-208 pg/ml). However, Con A and PHA were more potent inducer of IL-4(p <0.01) in culture supernatant. Con A and PHA treated cells showed up regulation of IL-12p35, IFN-γ, TNF-α and IL-6 mRNA (Fig 3 D and E).
       
Structural and functional studies indicated that integrity of at least three regions of the human IL-2 molecule like N-terminus (residues 1-20), C-terminus (residues 121-133) and 2 of the 3 cysteine residues are required for full biological activity (Ju et al., 1987). Non-glycosylated and glycosylated version of recombinant human IL-2 showed similar functional activity (Smith 1984). Equine IL-2 has one N-linked glycosylation site and four conserved cysteine residues and mature equine IL-18 do not have glycosylation site (Singha et al., 2015). Present study demonstrated that bacterially derived horse IL-2 and IL-18 had significant immuno-modulating activity.

Here we have reported low cost production of biologically active recombinant horse IL-2 and IL-18 in E. coli. The future experiments may be designed to determine the therapeutic effects of these cytokines against equine infectious diseases. 

We sincerely acknowledge Science and Engineering Research Board (SERB), Department of Science and Technology, New Delhi for funding this study (SERB/F/3326/2012–13) and providing fellowship to first author for Ph.D work.