Blocking IL-10 signaling with soluble IL-10 receptor restores in vitro specific lymphoproliferative response in dogs with leishmaniasis caused by Leishmania infantum

rIL-10 plays a major role in restricting exaggerated inflammatory and immune responses, thus preventing tissue damage. However, the restriction of inflammatory and immune responses by IL-10 can also favor the development and/or persistence of chronic infections or neoplasms. Dogs that succumb to canine leishmaniasis (CanL) caused by L. infantum develop exhaustion of T lymphocytes and are unable to mount appropriate cellular immune responses to control the infection. These animals fail to mount specific lymphoproliferative responses and produce interferon gamma and TNF-alpha that would activate macrophages and promote destruction of intracellular parasites. Blocking IL-10 signaling may contribute to the treatment of CanL. In order to obtain a tool for this blockage, the present work endeavored to identify the canine casIL-10R1 amino acid sequence, generate a recombinant baculovirus chromosome encoding this molecule, which was expressed in insect cells and subsequently purified to obtain rcasIL-10R1. In addition, rcasIL-10R1 was able to bind to homologous IL-10 and block IL-10 signaling pathway, as well as to promote lymphoproliferation in dogs with leishmaniasis caused by L. infantum.


Introduction
Cytokines are polypeptides that participate in communication between cells and the orchestration of immune system responses. In response to tissue damage and/or stimulation, these molecules may be secreted and bind to the extracellular domains of cognate receptors on target cells. This results in the phosphorylation of intracytoplasmic domains of cytokine receptors and activation of transcription factors, which migrate to the nucleus and implement cellular transcription and functions [1]. Some cytokines mainly promote pro-inflammatory activities, while others are anti-inflammatory or immunosuppressive in nature [2]. Proinflammatory cytokines are predominantly produced by activated macrophages and lymphocytes, including interleukein-1β (IL-1β), IL-6, tumor necrosis factor alpha (TNF-α), granulocyte-macrophage colony stimulating factor (GM-CSF), interferon gamma (IFN-γ) and IL-12 [1,2]. Anti-inflammatory cytokines are produced mainly by lymphocytes or non-classically activated macrophages, e.g. IL-4, IL-10, IL-11, IL-13 and TGF-β [2][3][4]. IL-10, the most important anti-inflammatory and immunosuppressive cytokine, can be produced by a variety of immune cells, including CD4+ and CD8+ T cells, B lymphocytes, natural killer (NK) cells, monocytic and dendritic cells, as well as eosinophils and neutrophils [5,6]. IL-10 signaling occurs via a receptor consisting of two distinct polypeptide chains, subunits IL-10Rα (IL-10R1) and IL-10Rβ (IL-10R2). Accordingly, IL-10 initially binds to the extracytoplasmic domain (ECD) of IL-10R1 with high affinity, followed by low affinity interactions with the IL-10R2 subunit by both IL-10 and IL-10R1 [7]. Next, JAK1 and TYK2 respectively interact with IL-10R1 and IL-10R2, become phosphorylated and mainly activate STAT3 [8], leading to the implementation of gene transcription programs and consequent cellular responses [5,6,8].
Canine leishmaniasis (CanL) caused by Leishmania infantum (synonymous with L. chagasi in the Americas) is a serious disease caused by the obligate intracellular protozoan [30,31]. Following natural inoculation with L. infantum, dogs may or may not develop disease [32]. Those that are susceptible may present mild signs or even develop severe and fatal disease [32,33]. Dogs that develop the symptomatic form of leishmaniasis may exhibit higher IL-10 and lower IFN-γ concentrations in the blood, while the inverse it true in asymptomatic dogs [34,35]. A positive correlation is evidenced between the expression of IL-10 and L. infantum parasitic load in the lymph nodes and spleens of infected canines. Under stimulation with leishmania antigens, susceptible animals exhibit an inability to mount a cellular immune response, as evidenced by the lack of lymphoproliferative response and cytokine production (IFN-γ and TNFα), which stimulates microbicidal mechanisms in macrophages. The addition of IL-10 to cultures of peripheral blood mononuclear cells (PBMC) has been shown to inhibit the lymphoproliferative response to leishmania antigens [36].
The immunization of animals with antigens concomitantly with the blocking of IL-10 signaling may favor the induction of a cellular immune response (Th1), even in the course of infection. This may represent a valid strategy in the development of preventive or therapeutic vaccines, as well as immunotherapeutic protocols, against canine diseases, including leishmaniasis caused by L. infantum [37][38][39][40].
The present work aimed to manipulate immune responses in dogs by producing recombinant casIL-10R1 (rcasIL-10R1) in a baculovirus-insect cell system, and evaluated this generated molecule's ability to bind to IL-10 inhibit signaling, and restore a lymphoproliferative response in dogs with leishmaniasis.

Recombinant protein production
Rcas-IL10R1 was produced following a previously described method [43]. Briefly, High Five cells were cultured in Express-Five SFM medium supplemented with L-glutamine, grown to exponential phase, and then infected with the recombinant baculovirus (AcBacΔcc-GP64-ca-sIL-10R1-6H) with a multiplicity of infection (MOI) of 5 for 72 hours (TOI 72 h). The cell suspension was centrifuged at 3,000 x g for 15 minutes at 4˚C to remove cell debris, and the supernatant was spun down at 30,000 x g for 1 hour at 4˚C. The resulting supernatant was stored at -70˚C until use. For purification, the thawed supernatant was dialyzed against PBS with 30 mM imidazole, pH 7.2 (binding buffer) and applied to a Sepharose-Nickel column (HisTrap HP, General Eletrics Healthcare) equilibrated with binding buffer. A HisTrap column was eluted with PBS-500 mM imidazole, pH 7.2. After analysis by SDS-PAGE, the chromatographic fractions containing rcasIL-10R1 were pooled together, submitted to dialysis against PBS, aliquoted and stored at -70˚C until use. Protein concentrations were determined by Micro BCA (Thermo Fisher Scientific, Rockford, USA). Endotoxin concentration was determined using Limulus Amebocyte Lysate (Gel-clot Method, Pyrotell, USA) [44]. Purified recombinant protein was confirmed by Western blot assay using anti-his antibodies, as previously described [43].

Animals
This study was approved by the Brazilian Society of Science on Laboratory Animals/Brazilian College of Animal Experimentation (SBCAL/COBEA), and the Committee for Animal Care and Use-São Paulo State University (UNESP), protocol no. 00765-2017. The approved license covered the use of 5 healthy and 10 diseased dogs. A previous report characterized these animals, including clinical data [45]. Out of the 15 dogs, five were healthy (negative controls, two males and three females, two mongrels, one blue heeler, one cocker spaniel and one golden retriever) and 10 (CanL) were diagnosed with leishmaniasis (Leishmania infantum) (six males and four females, seven mongrels, two poodles and one blue heeler). All control dogs tested negative for Leishmania DNA and Leishmania-specific antibodies by real-time PCR and ELISA, respectively, and presented complete blood counts and mean serum biochemistry parameters within reference ranges [45]. The 10 CanL dogs selected from the Araçatuba Zoonosis Control Center presented at least three of the following characteristic clinical signs of leishmaniasis: onychogryphosis, cachexia, ear-tip injuries, periocular lesions, alopecia, skin lesions or lymphadenopathy. Leishmania DNA was detected in the peripheral blood of each diseased dog by real-time PCR [45].

Statistical analysis
Statistical analysis was performed using GraphPad Prism v6 software (GraphPad Software, Inc., La Jolla, CA, USA). Statistical variables were tested for normality using the Shapiro-Wilk test. Friedman's test with Dunn's post-test was used to compare lymphoproliferation rates. The Mann-Whitney test was used to compare results among groups. Values were considered significant when p <0.05.

Cloning and production of rcasIL-10R1
Initially, the amino acid sequence of the extra-cytoplasmic domain of canine IL-10 receptor alpha chain (R1) (casIL-10R1) was identified. This was carried out by comparing the entire canine IL-10R1 predicted protein (Genbank accession number XM_005620306.1), full-length human IL-10R1 (GeneBank, accession number NM_001558) and, human IL-10R1 extra-cytoplasmic domain [41] using the Basic Local Align Search Tool (BLAST, https://blast.ncbi.nlm. nih.gov/Blast.cgi). In addition, canine IL-10R1 extra-cytoplasmic domain was confirmed by analyzing full-length human and canine IL-10R1 using an online tool https://tmdas.bioinfo.se/ DAS/. This analysis revealed all domains (protein signal peptide, extra-cytoplasmic, transmembrane, intracytoplasmic) of the human protein and allowed us to infer the corresponding domains in the canine sequence (Fig 1), therefore, allowing identification of canine extra-cytoplasmic domain. CasIL-10R1 was defined as an array of 215 amino acids which exhibited 74% similarity to homologous region of human protein. Molecular weight and isoelectric point predicted for mature rcasIL-10R1 were 25.7 kDa and PI 8.3, respectively. A DNA construct was synthesized to encode in tandem the AcMNPV GP64 signal peptide, casIL-10R1, and a 6-histidine tag. RcasIL-10R1 was purified from supernatant of High-five cells infected with the recombinant baculovirus by affinity chromatography. Purified rcasIL-10R1 showed one strong and two weak bands of 40, 28 and 23 kDa in SDS-PAGE, respectively (Fig 2A), however only the highest band was detected using anti-histidine antibodies by Western blotting ( Fig 2B). The yield of the purified rcasIL-10R1 was 2.8 mg/L of High-five cell culture and the endotoxin concentration was less than 0.03 EU/mg of protein. Together, these data indicate that the recombinant protein was successfully produced.

Evaluation of binding between rcasIL-10R1 and canine IL-10
To assess binding between canine rcasIL-10R1 and IL-10, rcasIL-10R1 was covalently immobilized to carboxymethylated dextran matrix (CM5 chip) activated by EDC/NHS to achieve 1000 resonance response units (RU) in a Biacore T100 device. After blocking the remaining reactive chemical groups on the matrix, two samples of either PBS containing 1% BSA and 0.05% Tween 20 (to determine the baseline signal), various concentrations of canine IL-4 (cytokine irrelevant to the system, negative control) or various canine concentrations of IL-10, were applied on the matrix and RU readings were taken, and X ± SD of RU were calculated from them. When samples of PBS containing 1% BSA and 0.05% Tween 20 or IL-4 in concentrations of 125, 250 or 500 ng/ml were applied to the matrix, 1.0 ± 1.2, 17 ± 6, 15.1 ± 4.4, and 510 ± 132 RU were observed, respectively (Fig 3). On the other hand, when samples of canine

PLOS ONE
IL-10 were applied, there was a progressive increase in signal starting at 211 ± 52 RU for 31.2 ng/mL and reaching a plateau at 1720 ± 352 RU for 125 ng/mL, indicating a strong binding between rcasIL-10R1 and IL-10 (Fig 3). Binding equilibrium constant (EC 80 ) between rcasIL-10R1 and IL-10 was determined as 51.4 nM.

RcasIL-10R1 induces peripheral blood lymphocyte proliferation in dogs with leishmaniasis caused by Leishmania infantum
Dogs with leishmaniasis exhibit limited specific-cellular immune response and increase in IL-10 production [34,47], to determine if blocking IL-10 signaling would revert Leishmania-specific lymphoproliferative unresponsiveness, CFSE labeled-PBMCs from healthy or infected dogs were cultured together with, or without, rcasIL-10R1, and with or without the addition of SLA, or in the presence of PHA alone for five days. The Mean Fluorescence Intensities (MFI) of CFSE-labeled lymphocytes was determined under each condition. Reductions in CFSE-fluorescence were considered an indicator of cell proliferation [48]. The data described herein was previously reported in the context of testing combinations of several recombinant canine proteins [45]. In healthy dogs, lymphoproliferation was observed when PBMCs were cultured with PHA (median, interquartile 25, and 75, 256, 176, and 337) (Fig 5A), as compared with medium alone (101, 82, and 225). In diseased dogs, although CFSE-labeled lymphocytes cultured with PHA showed reductions in MFI, these were not statistically significant (Fig 5B). Lymphocytes from diseased dogs showed proliferative response when cultured with rcasIL-10R1, regardless of the addition of SLA to cultures (without SLA addition, rcasIL-10R1: 2.9, 2.0, and 10.2 vs medium: 128, 117, and 205; with SLA addition, rcasIL-10R1: 3.8; 1.3, and 12.1 vs medium: 121, 87, and 176) (Fig 5B). L. infantum parasites are transported throughout blood circulation mainly inside mononuclear leucocytes [49] and these parasites were detected in the blood samples of every single diseased dog by real-time PCR in the current studies. One probable explanation for lymphoproliferation stimulation by rcasIL-10R1, regardless of the addition of SLA to the cell cultures of diseased dogs, was the presence of Leishmania within the PBMCs used in experimentation. These results suggest that blocking IL-10 signaling using rcasIL-10R1 restores specific lymphoproliferative response in dogs with leishmaniasis.

Discussion
IL-10 can restrict exaggerated inflammatory and immune responses, thus preventing tissue damage and promoting homoeostasis [5,6,9]. However, by downregulating these responses, IL-10 may favor the development and/or persistence of chronic infections [50][51][52]. Therefore, blocking IL-10 signaling may contribute to the establishment of adequate immune responses for the treatment of chronic infections [37,50,53]. Blocking IL-10 signaling can also be useful in immunization protocols that aim to induce immune responses against intracellular pathogens [50,54].
IL-10 signaling can be blocked in vitro by the use of IL-10 or IL-10R reactive molecules, including antibodies, oligonucleotide or peptide aptamers, as well as soluble IL-10 receptor [39,41,53,[55][56][57]. However, to date, only blocking antibodies to canine IL-10 or IL-10R have been developed (https://www.rndsystems.com R&D Systems), [38]. Such antibodies were produced in mice or goats, therefore, their administration in dogs could result in humoral responses to heterologous proteins [58], that would limit their use as blocking agents to IL-10 signaling in these animals.
The present work endeavored to identify the canine casIL-10R1 amino acid sequence, generate a recombinant baculovirus chromosome encoding this molecule, which was expressed in insect cells and subsequently purified to obtain rcasIL-10R1. In addition, rcasIL-10R1 was evaluated in vitro with respect to its binding ability and blocking of the homologous IL-10 signaling pathway, as well as promoting lymphoproliferation in dogs with leishmaniasis caused by L. infantum.
Initially, casIL-10R1 was identified by comparing the amino acid sequences of caIL-10R1, huIL-10R1, husIL-10R1, and then detecting the extracellular domains in the first two proteins using an online tool for transmembrane domain prediction. Next, a DNA construct encoding casIL-10R1 was synthesized and transferred to a baculovirus artificial chromosome used to produce the protein in High five cells. Chromatographic affinity protein purification from cell culture supernatants indicated an adequate yield, reaching 2.8 mg/L. The generated recombinant protein presented a high degree of purity, as evidenced by a main band of 42 kDa when evaluated on SDS-PAGE and Western blotting assays. Since the predicted molecular weight of rcasIL-10R1 was 28 kDa, and considering its six canonical N-linked glycosylation motives (NXS/T), it follows that the protein must have been produced in a heavily glycosylated form. Similarly, a discrepancy was noted between the predicted molecular weight (24 kDa) and relative mobility (35-45 kDa) of rhusIL-10R1 produced in myeloma cells on SDS-PAGE analysis [41]. Moreover, these authors reported that treatment with N-glycanase promoted a reduction in molecular weight back to 24 kDa, indicating that the produced rhusIL-10R1 was highly glycosylated [41]. In addition, our analysis of the purified protein in solution presented a low concentration of endotoxin [59].
To assess its binding ability, rcasIL-10R1 was immobilized on a dextran matrix and resonance was recorded following the application of different concentrations of canine IL-10. In comparison to diluent alone or canine IL-4 (negative control), much higher resonance values were observed for IL-10, indicating specific binding between rcasIL-10R1 and IL-10. The equilibrium constant (EC80) concentration was determined to be 51.4 nM. In a previous report, the established equilibrium constant (EC50) for human IL-10 and hus-IL-10R1 binding was 0.47 nM [60], which is much lower than that found herein. These observed discrepancies can be at least partially attributed to divergencies in the experimental conditions used, including the use of EC80 and native dimeric IL-10 in our protocols in comparison to EC50 and a mutated monomeric protein, in addition to differences in the methods of immobilization employed.
In combination with IL-4 and/or IL-3, IL-10 has been shown to induce mast cell proliferation [46], as demonstrated by growth in the mouse mast cell line (MC/9) through the concomitant stimulation of homologous IL-4 and IL-10. Moreover, MC/9 cells and a subcloned line, so-called MC/9.2, which expresses a lower amount of growth factor mRNA [61], have been used by several authors and biotechnology companies to demonstrate the functional activity of IL-10 in many animal species [46,[62][63][64], including Canis familiaris (https://www.rndsystems. com/ R&D systems catalog number 735-CL-010 data sheet). To test the ability of rcasIL-10R1 in the blockade of the cognate signaling pathway, MC/9 cells were stimulated with canine IL-10 and IL-4 in the presence or absence of rcasIL-10R1 (4 μg/mL). In the presence of the recombinant protein, an incomplete reduction was observed in the proliferation of MC/9 cells, indicating the partial blocking of this signaling pathway. Tan et al. (1995) [41] showed that 15-20 nM of rhusIL-10R1 induced a 50% inhibition in the maximal proliferation of Ba8.1 cells (murine pro-B lymphocytes transfected with the gene encoding huIL-10R1) under stimulation with human IL-10 at 100 pM. In the present study, rcasIL-10R1 (95 nM) was found to promote a 45% reduction in the proliferation of MC/9 cells stimulated with 6 nM of canine IL-10.
Dogs naturally infected with L. infantum that remain asymptomatic have been shown to mount a specific lymphoproliferative response. However, dogs that succumb to the disease evolve with T cell exhaustion, involving both CD4+ and CD8+ T lymphocytes [34,47], which implies the loss of these cells' ability to perform effector functions. One of the first functions lost due to this exhaustion is the capacity of lymphocytes to proliferate intensely under antigenic stimulation [65]. In the current study, the blocking of IL-10 signaling with rcasIL-10R1 for 5 days in infected canine PBMCs, under stimulation or not by SLA, resulted in the restoration of a lymphoproliferative response. Since Leishmania DNA was detected in the peripheral blood of these dogs, the observed lymphoproliferation was quite likely specific. By contrast, Esch et al., (2013) [47] carried out assays in PBMCs from dogs with leishmaniasis caused by L. infantum to evaluate the impact of blocking IL-10 signaling with anti-IL-10 antibodies. In these assays, the authors assessed the percentage of T CD4 or T CD8 lymphocytes that incorporated EdU (5-ethynyl-2'-deoxyuridine) at 7 days of culture after stimulation with leishmania antigens in the presence of anti-IL-10 antibodies or an isotype control. They observed no increases in the percentages of either CD4 or CD8 T lymphocytes after the blockade of IL-10 signaling, suggesting the absence of lymphocyte proliferation. The discrepancies between these authors' results and those herein likely occurred due to differences in the methodology used to evaluate lymphocyte proliferation. In consonance with our results, the blocking of this signaling cascade with anti-IL-10 antibodies in PBMCs from human patients with visceral leishmaniasis using a method similar to that employed herein was also shown to result in lymphoproliferation [66].
Future investigations designed to determine the conditions in which rcasIL-10R1 would block IL-10 in vivo in a wider context will be of great scientific interest, and could be applied to induce a Th1 immune response in the development of vaccines and immunotherapeutic protocols against chronic infection and cancer in dogs.

Conclusion
The rcasIL-10R1 produced in this baculovirus-insect cell system demonstrated the blockade of the IL-10 signaling pathway and the restoration of in vitro lymphoproliferative response in dogs with leishmaniasis caused by L. infantum.