efeito do laser em baixa intensidade sobre a expressão gênica e proteica de mediadores de macrófagos J774 induzidos a diferenciação

Abstract

The interactions between tissue and macrophages that migrate to the injury area are crucial to the repair process. In this context, Low-level laser therapy (LLLT) has been extensively used in the treatment of tissue damage in different clinical and experimental conditions; however, its effects on macrophages are still unclear The objective of this study was to evaluate the effects of LLLT (660 nm and 780 nm) on gene expression and protein synthesis of 12 products of macrophages induced to M1, M2a and M2c phenotypes. J774 macrophages were activated/polarized for the three phenotypes for 24 h, irradiated with red laser (660 nm) and infrared (780 nm) using the same dosimetric parameters (70 mW, 17,5 J/ cm2, 1J). Following incubation periods of 4 and 24 hours, PCR array was performed to investigate gene expression profile of chemokines (CXCL2, CCL2, CCL3, CCL4); cytokines (IL-1β, IFNγ, IL-6, TNFα, IL-13) and growth factors (TGFβ1, IGF1 e VEGFA). Culture supernatant, for the different experimental groups (after 24 hours of irradiation), was used to assess the production of the different proteins according to the characteristic profile of each phenotype. In each experimental situation, non-irradiated and non- activated cells were used as controls. Three independent experiments were performed for protein acess and two independent experiments were performed to evaluate gene expression. All results were analyzed statistically. The macrophages induced for M1 phenotype (treatment with LPS+ IFN-γ), showed an increase in IL-6 gene expression and a decrease in expression of CCL2, CCL4, CXCL2 e TNFα genes in relation to non-activated macrophages in the 24h period. At the protein level, activated macrophages showed an increase in the production of CCL4 and IL-6 when compared to non-activated macrophages. In addition, macrophages induced for M2a phenotype (treatment with IL-4), showed an increase in TGFβ1 (24 h) and CCL3, CCL4, CXCL2 e TNFα (4h) gene expression when compared to the control group. Additionally, the polarization of the M2c phenotype (treatment with IL- 10 + Dexamethasone), induced an increase in CXCL2 (4 h), CCL3 and IL-1β (24 h) gene expression. After treatment with red laser (660 nm), a decrease in CCL3, CXCL2 and TNFα (4 h) as well as an increase in IL-1β (4 h), CCL2, CXCL2 and TNFα (24 h) gene expression was observed in J774 cells. In M2a macrophages, red

laser irradiation caused a decrease in CCL3, CXCL2 and TNFα (4 h) gene expression. In this same context, macrophages induced for M2c phenotype and irradiated with 660 nm showed a decrease in CXCL2, TNFα and IL-1β (4 h) and TNFα (24 h) gene expression. Moreover, the treatment with infrared (780 nm) was able to cause an increase in CCL2, IL-1β and TGFβ1 (4 h) followed by a decrease in CCL3, IL-6 and TGFβ1 (24 h) gene expression. Regarding protein synthesis, a decrease in IL-6 and TNFα production by activated and irradiated macrophages in comparison with the non-irradiated cells was observed. Irradiation with infrared laser resulted in increased expression levels of TGFβ1 (4 h) as well as decreased mRNA expression levels of CCL4 (4 h), CCL3 and TGFβ1 (24 h). In addition, M2c phenotype trated with infrared laser showed an increase in TNFα (4h) gene expression and a decrease in CCL2, CCL3, CCL4, TNFα, IL-1β and TGFβ1 (24h) gene expression. Although the J774 cell line has been extensively used to evaluate the effects of different therapies in macrophages phenotypes, in the experimental conditions performed in this study, J774 showed distinct pattern of chemokines, cytokines and growth factors expression after activation to M1, M2a or M2c phenotype. Regarding LLLT treatment, both parameters (660 nm and 780 nm) demonstrated ability to modulate the gene expression in all phenotypic profiles. However, only the infrared laser (780 nm) was able to decrease the gene and protein expression of pro-inflammatory mediators when applied to M1 phenotype macrophages. Additionally, this irradiation was able to modulate TGFβ1 gene expression in M2a macrophages, helping to better understand the mechanisms involved in the decrease of inflammatory response and fibrosis observed in the injured tissues irradiated with LLLT.