RESUMEN
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.
Asunto(s)
Interleucina-10/metabolismo , Leishmaniasis/tratamiento farmacológico , Receptores de Interleucina-10/metabolismo , Animales , Línea Celular , Citocinas/metabolismo , Enfermedades de los Perros/genética , Perros , Femenino , Inmunidad Celular/inmunología , Inmunidad Celular/fisiología , Interferón gamma/genética , Interleucina-10/agonistas , Interleucina-12/genética , Leishmania infantum/inmunología , Leishmania infantum/patogenicidad , Leishmaniasis/inmunología , Macrófagos/metabolismo , Masculino , Ratones , Receptores de Interleucina-10/efectos de los fármacos , Transducción de Señal , Linfocitos T/inmunología , Factor de Necrosis Tumoral alfaRESUMEN
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.
Asunto(s)
Baculoviridae/genética , Ingeniería Genética/métodos , Vectores Genéticos/metabolismo , Interleucina-12/biosíntesis , Animales , Baculoviridae/metabolismo , Catepsinas/genética , Catepsinas/metabolismo , Línea Celular , Quitinasas/genética , Quitinasas/metabolismo , Clonación Molecular , Perros , Expresión Génica , Vectores Genéticos/química , Interleucina-12/genética , Interleucina-12/farmacología , Linfocitos/citología , Linfocitos/efectos de los fármacos , Linfocitos/metabolismo , Mariposas Nocturnas , Cultivo Primario de Células , Pliegue de Proteína , Señales de Clasificación de Proteína , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/genética , Proteínas Recombinantes/farmacología , Células Sf9 , SpodopteraRESUMEN
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.
