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.
Subject(s)
Interleukin-10/metabolism , Leishmaniasis/drug therapy , Receptors, Interleukin-10/metabolism , Animals , Cell Line , Cytokines/metabolism , Dog Diseases/genetics , Dogs , Female , Immunity, Cellular/immunology , Immunity, Cellular/physiology , Interferon-gamma/genetics , Interleukin-10/agonists , Interleukin-12/genetics , Leishmania infantum/immunology , Leishmania infantum/pathogenicity , Leishmaniasis/immunology , Macrophages/metabolism , Male , Mice , Receptors, Interleukin-10/drug effects , Signal Transduction , T-Lymphocytes/immunology , Tumor Necrosis Factor-alphaABSTRACT
BACKGROUND: Interleukin-12 is an important cytokine in mediating cellular immune responses. RESULTS: Recombinant single-chain canine IL-12 was produced in a baculovirus-insect cell system with the aim of conducting further studies on modulation of immune responses in dogs. To optimize the production of recombinant canine IL-12, a classical baculovirus and a modified vector (chitinase A and v-cathepsin knockout) were used containing a native or an optimized insert of canine IL-12. The optimized IL-12 construct contained the GP64 signal peptide and was synthesized with optimized codons for expression in Trichoplusia ni cells. Dot-blot and Western blot analysis showed the highest production levels of recombinant IL-12 protein by the use of the modified baculovirus vector containing the optimized insert, at a multiplicity of infection of five and at 48 h after infection. The recombinant cytokine was successfully purified and showed a good degree of purity, integrity, folding, and yield, with very little endotoxin contamination. Recombinant canine IL-12 induced IFN-γ in canine lymphocytes, indicating that it was biologically active. CONCLUSION: Therefore, this study describes an efficient method to produce adequate amounts of biologically active canine IL-12, useful for immunomodulation studies in dogs.
Subject(s)
Baculoviridae/genetics , Genetic Engineering/methods , Genetic Vectors/metabolism , Interleukin-12/biosynthesis , Animals , Baculoviridae/metabolism , Cathepsins/genetics , Cathepsins/metabolism , Cell Line , Chitinases/genetics , Chitinases/metabolism , Cloning, Molecular , Dogs , Gene Expression , Genetic Vectors/chemistry , Interleukin-12/genetics , Interleukin-12/pharmacology , Lymphocytes/cytology , Lymphocytes/drug effects , Lymphocytes/metabolism , Moths , Primary Cell Culture , Protein Folding , Protein Sorting Signals , Recombinant Proteins/biosynthesis , Recombinant Proteins/genetics , Recombinant Proteins/pharmacology , Sf9 Cells , SpodopteraABSTRACT
BACKGROUND: Very few studies have been carried out so far aiming at modulating cellular immune responses in dogs. In this study, we evaluated the ability of recombinant canine IL-2 (rcaIL-2) and IL-12, in the form of a single-chain fusion protein (rsccaIL-12), to stimulate peripheral blood mononuclear cells (PBMC) of healthy mongrel dogs. RESULTS: Recombinant canine IL-2 purified from Escherichia coli or present in the supernatant of COS-7 cells transfected with pcDNA3.1-caIL-2 (COS-7 caIL-2 supernatant) was able to induce proliferation of CTLL-2 cells, thus showing their functional activity. In addition, purified rcaIL-2 and COS-7 caIL-2 supernatant stimulated resting canine PBMC proliferation to a level higher than baseline level. Neither COS-7 sccaIL-12 supernatant nor COS-7 caIL-2 supernatant alone was able to induce significant production of interferon gamma by resting PBMC. However, COS-7 sccaIL-12 supernatant in combination with COS-7 caIL-2 supernatant induced production of IFN-γ by those cells. CONCLUSIONS: The data shown herein suggest that the combination of canine recombinant IL-12 and IL-2 can be useful to promote cellular immune responses in dogs.
