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

Abstract

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.

Fig 1. Identification and design of DNA construct encoding soluble canine IL-10R1 production.

Identification of the extra-cytoplasmic domain of canine IL-10 receptor alpha chain was performed by comparing the amino acid sequences of human (huIL-10R1, GeneBank, accession number NM_001558) and canine (caIL-10R1, Genbank accession number XM_005620306.1) IL-10 receptor alpha chain, and husIL-10R1, previously described by Tan et al. 1991, using Basic Local Align Search Tool, as well as defining signal peptide and transmembrane domains (TM) (defined by vertical bars) using an online tool (https://tmdas.bioinfo.se/DAS/) (A). The DNA construct was designed to encode the following elements in tandem: a) Autographa californica multiple nuclear polyhedrosis virus (AcMNPV) GP64 leader sequence, b) casIL-10R1, and c) six histidines (B).

Fig 2. Evaluation of purified rcasIL-10R1 by SDS–PAGE and Western blot.

RcasIL-10R1 was produced in High-five cells infected with the AcBacΔcc-GP64-casIL-10R1-6H baculovirus construct at MOI 5 for 72 h. Then, rcasIL-10R1 was purified from cell-free and virus-free culture supernatant (SN) by Ni-Sepharose affinity chromatography column. Samples of purified protein were evaluated by SDS–PAGE (A): molecular weight markers (lane 1), cell culture SN applied to the chromatographic column (lane 2), flow through (lane 3), and purified protein (lane 4) or Western blot developed by anti-his antibodies (B): SN from cells infected with baculovirus devoid of insert (negative control) (lane 1), SN from cells infected with AcBacΔcc-GP64-casIL-10R1-6H baculovirus construct (lane 2) or sample of purified rcasIL-10R1. Arrows indicate a band around 42 kDa corresponding to rcasIL-10R1-6H.

Fig 3. Evaluation of binding between rcasIL-10R1 and canine IL-10.

Purified rcasIL-10R1 was immobilized on a CM5 chip in a Biacore T100 analyzer by applying 800 μL of the recombinant protein (0.5 μg/mL) at (50 μL/minute) to the chip matrix activated by 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC)/N-Hydroxysuccinimide (NHS) to achieve 1000 resonance response units (RU). Then remaining reactive chemical groups on the chip were blocked by applying Ethanolamine hydrochloride-NaOH for one minute. The following samples were applied to the matrix for two minutes and 30 seconds: a) PBS containing 1% BSA and 0.05% Tween 20 (open triangle); b) canine IL-4 at 125, 250 or 500 ng/mL (open square); c) canine IL-10 at 31.2, 62.5, 125, 250 or 500 ng/ml (open circle). IL-4 and IL-10 were diluted with PBS containing 0.05% Tween 20. After each analyte binding evaluation, matrix regeneration was performed by applying regeneration buffer for 30 seconds. Each sample was evaluated twice and results are presented as means and standard deviations (X±SD) of RU.

Fig 4. Blocking canine IL-10 signaling by rcasIL-10R1 reduces MC/9 cell proliferation.

The murine mast cell line MC/9 was cultured at 1 x10⁵/mL in triplicate wells (100 μL/well) on a 96-well flat-bottomed microtiter plate with: a) complete DMEM alone (negative control) or complete DMEM containing: b) 0.625% of supernatant from Con-A-stimulated rat splenocytes (Con-A-SRS, assay positive control), c) rcaIL-4, 180 ng/mL, d) rcaIL-10, 20 ng/mL, e) rcaIL-4 (180 ng/mL) and rcaIL-10 (20 ng/mL), or f) rcaIL-4 (180 ng/mL), rcaIL-10 (20 ng/mL) and rcasIL-10R1 (4 μg/mL). The plate was kept for 48 h under 5% CO₂ at 37°C. Then, 10 μL of Alamar Blue were added to each well. Cells were cultured for an additional 24 hours and optical density (OD) was read at 570 nm and 600 nm wavelengths. Differences in mean OD values were used to estimate MC/9 cell proliferation rates. Symbols and bars represent replicates and means. RasIL-10R1 inhibited proliferation of MC/9 cells stimulated with IL-10 and IL-4 by 45%.

Fig 5. Blocking canine IL-10 signaling by rcasIL-10R1 restores in vitro specific lymphoproliferative response in dogs with VL.

CFSE-labeled PBMCs from healthy negative control dogs (n = 5) (A) and dogs with leishmaniasis (n = 10) (B) were cultured in medium alone (Medium), medium with soluble leishmania antigens (SLA) or phytohemagglutinin (PHA). In addition, PBMCs cultured in medium alone or with SLA were stimulated with rcasIL-10R1. After 5 days, the mean fluorescence intensity (MFI) of CFSE-labeled lymphocytes was assessed by flow cytometry. Bars represent MFI median values and 25th and 75th percentile interquartile range. Symbols represent data from individual animals. Asterisks indicate significant differences (Friedman’s test with Dunn’s multiple comparison, p < 0.05).