Scientific Reports (Reino Unido)

Beneficial effects of Red Light-Emitting Diode treatment in experimental model of acute lung injury induced by sepsis

Publicado em 03 outubro 2017

Por Silvia Goes Costa

Sepsis is a severe disease with a high mortality index and it is responsible for the development of acute lung injury (ALI). We evaluated the effects of light-emitting diode (LED) on ALI induced by sepsis. Balb-c mice were injected with lipopolysaccharide or saline and then irradiated or not with red LED on their tracheas and lungs for 150?s, 2 and 6?h after LPS injections. The parameters were investigated 24?h after the LPS injections. Red LED treatment reduced neutrophil influx and the levels of interleukins 1ß, 17?A and, tumor necrosis factor-a; in addition to enhanced levels of interferon ? in the bronchoalveolar fluid. Moreover, red LED treatment enhanced the RNAm levels of IL-10 and IFN-?. It also partially reduced the elevated oxidative burst and enhanced apoptosis, but it did not alter the translocation of nuclear factor ?B, the expression of toll-like receptor 4 (TLR4), as well as, oedema or mucus production in their lung tissues. Together, our data has shown the beneficial effects of short treatment with LED on ALI that are caused by gram negative bacterial infections. It is suggested that LED applications are an inexpensive and non-invasive additional treatment for sepsis.

Acute lung injury (ALI) is a severe and serious disease. It is multifactorial and characterized by diffused alveolar damage, lung inflammatory cell infiltrations, as well as a loss of alveolar epithelium, together with oedema and an impaired gas exchange1. ALI is associated with a high mortality incidence around the world (200,000 patients per year in the US alone, resulting in a mortality rate of 40%)1.

ALI is divided in exudative and fibro proliferative phases. The first one is characterized by an activation of the coagulation system, with a production of pro-inflammatory cytokines by the epithelial tissues, together with the resident mast cells, the macrophages, and the recruitment of neutrophils, monocytes and macrophages, as well as lymphocytes, into the lung injury. Furthermore, the release of cytotoxic mediators, reactive oxygen species (ROS), reactive nitrogen species (RNS), proteolytic enzymes, as well as metalloproteinases (MMPs), cause an endothelial and epithelial lung injury, impacting in the loss of functions of lung2,3. The fibro proliferative phase occurs approximately 3 days after the injury, with a predominant proliferation of mesenchymal cells, such as myofibroblasts, fibroblasts, and pluripotent cells.

Sepsis is an important clinical condition that is considered to be the most important cause of ALI developments. Sepsis is a systemic response to infection that is manifested by changes in the body temperature and tachycardia, together with elevations in the respiratory frequency, leukocytosis in the lungs, and blood leukopenia4. ALI has been experimentally induced by systemic administration of lipopolysaccharide (LPS), which interacts with toll-like receptors (TLR), mainly of type 4 (TLR4)5. The activation of TLR4 downstream leads to complex signal transduction pathways that induce the translocation of nuclear factor ? B (NF-?B) into the nuclei. NF-?B is responsible for the transcription of the pivotal inflammatory genes6. Indeed, a pharmacological inhibition of the NF-?B nuclear translocation leads to an impairment of the inflammatory responses to LPS7.

Neutrophils are target cells in ALI that are evoked by sepsis, as circulating cells are recruited into the lung in the early phases of the syndrome8. As a consequence, the activated neutrophils secrete cytokines and chemokines, releasing granule contents as proteases, producing reactive oxygen (ROS) and nitrogen (RNS) species, which contribute to the amplification of the inflammation and tissue damage9. When considering the crucial role of neutrophils in an ALI, some studies have proposed to induce neutrophil apoptosis as a strategy of treatment10,11.

The treatment of ALI is a clinical problem, as anti-inflammatory drugs are inefficient3,12,13,14. Additional approaches have been integrated into the therapy, such as mechanical ventilations with a low volume, a prone position (face down), as well as an extracorporeal membrane oxygenation (ECMO). Nevertheless, these kinds of treatments all require high costs and they are not sufficiently effective3,12,13,14. As a result, treatments that are more efficient and therapies with lower costs are required in order to treat ALI.

Photobiomodulation is a treatment that is based on the effects of light on damage tissues, such as lasers, light-emitting diode (LED), among others. Photobiomodulation has been pointed out as an interesting tool for the treatment of lung diseases, as experimental studies have shown that a low level of laser therapy reduces the inflammation and oxidative stress in lung disorders15,16,17,18,19,20. However, the beneficial effect of LED in the lung diseases, such as experimental model of asthma21 and lung fibrosis22 has been shown by our group. The red LED treatment in asthmatic mice reduced the lung cell infiltration, mucus production, oedema, and tracheal’s contractile response by IL-10, IFN-? and mast cells mechanisms-involved21. We also showed that the red LED treatment reduced the number of inflammatory cells in the alveolar space, collagen production, interstitial thickening, and static and dynamic pulmonary elastance in experimental model of lung fibrosis. In addition, reduced levels of IL-6 and CXCL1/KC released by cultured pneumocytes as well as decreased secretion of CXCL1/KC by fibroblasts in culture22.

Moreover, recent data has shown that LED treatment have inhibited the release of inflammatory mediators in experimental model of arthritis. They have abrogated the mechanical and thermal hyperalgesia that was modulated by tumor necrosis factor-a (TNF-a), interleukins IL1-ß (IL1-ß) and 10 (IL-10), in murine experimental models with chronic inflammatory hyperalgesia. They have also inhibited cytokine secretions in human fibroblasts23,24,25.

Therefore, based on the beneficial effects of LED treatment in inflammatory diseases and in our previous studies, we have hypothesized that the LED treatment could act as anti-inflammatory agent in the lungs and be employed as an additional treatment for sepsis. Using an experimental model of sepsis that was induced by LPS in mice, we have investigated the effects of LED treatment on the initial course of ALI. The research has focused on leukocyte influxes into the lung, taking note of the local gene expression, the secretion of cytokines, oedema, mucus production, NF-?B nuclear translocation, oxidative burst, phagocytosis, as well as the gene expression of TLR4.

Data presented in Fig. 1 (Panel A) show that i.p. injection of LPS caused lung inflammation, characterized by elevated number of leukocytes, which include macrophages, lymphocytes and neutrophils in the BAL in comparison to number of cells found in non-manipulated mice (Basal). The treatment with LED in LPS injected mice reduced the number of leukocytes in the BAL, and numbers were rescued to those equivalent in Basal group (Fig. 1A). Moreover, LED treatment did not modify the number of cells in the BAL in absence of inflammation (Fig. 1A).

LED treatment decreases lung cell recruitment in the bronchoalveolar lage (BAL) and myeloperoxidase (MPO) activity in the lung tissue in ALI experimental model. Groups of mice were induced to ALI by ip injection of LPS and treated or not with LED 2 and 6?h after the induction. Non-manipulated and LED treated mice were used as control. After 24?hours of ALI induction, the cellular recruitment and the MPO activity were determined. Data mean?±?SEM of 6 animals per group. *P??P?Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP). Adriana Lino-dos-Santos-Franco is a research fellow from FAPESP (2015/00830-9).

A.L.-d.-S.-F., S.H.P.F. conceived the experiments; A.L.-d.-S.-F. supervised and coordinated the studies; S.G.C., E.D.B., A.I., J.A., A.S.D. and L.B.V. conducted the experiments; S.G.C., L.B.V., C.P., A.S.D. and A.L.-d.-S.-F. analyzed the data; S.G.C., A.L.S.F., A.L.-d.-S.-F., and N.O.S.C. wrote the manuscript. All authors reviewed the manuscript.

The authors declare that they have no competing interests.

Correspondence to Adriana Lino-dos-Santos-Franco.