Study On The Role Of TAM/Gas6 Signaling Pathway In Silica Dust Induced Pulmonary Inflammation And Fibrosis

Long exposure to silica dust can cause excessive inflammatory responses and chronic fibrosis in lung and finally lead to the happening of silicosis, the pathological process of which is closely related to the imbalance of immune system. The TAM receptors, including Tyro3 receptor, Axl receptor and Mer receptor, and the growth arrest specific protein 6 (Gas6) ligand constitute the TAM/Gas6 signaling pathway, which has been found can suppress the activation levels of immune responses. In this present study, we intended to investigate possible role of TAM/Gas6 pathway in silica induced pulmonary inflammation and fibrosis by both in vivo and epidemic logic studies.Part I The role of TAM/Gas6 pathway in silica dust induced pulmonary inflammation in miceObjectives:To investigate the possible role of TAM/Gas6 pathway in silica dust induced pulmonary inflammation.Methods:Based on the method of intratracheal instillation, we established eight lung exposure models in mice:? normal saline (NS) control group, in which mice were intratracheally instillated with stroke-physiological normal saline solution; ?silica exposure group, in which mice were intratracheally instillated with silica dust suspensions; ? nonspecific antibody group, in which mice were intratracheally instillated with silica and nonspecific antibody; ?Tyro3, Axl or Mer receptor blocking groups, in which mice were intratracheally instilled with silica and blocking antibodies of Tyro3, Axl or Mer respectively; ? supplying or blocking groups of Gas6, in which mice were intratracheally instilled with silica and the recombinant mice Gas6 (rmGas6) or silica and blocking antibody of Gas6. After the intratracheal instillations, mice were intraperitoneally injected with corresponding blocking ant bodies or proteins, and then killed on the day 7,28 and 84. The left lungs were isolated for immunohistochemistry (IHC) and hematoxylin-eosin staining to observe the stimulation levels of TAM/Gas6 pathway and the pulmonary inflammatory levels (inflammatory scores were caculated). The bronchoalveolar lavage fluid (BALF) was collected and detected for the levels of cell injury indexes (lactate dehydrogenase and total protein), inflammatory cells (macrophages and neutrophils) and inflammatory cytokines (IL-1?, TNF-?).Results:The IHC results showed that the TAM receptor stimulation levels in lung tissue were higher in silica exposure group than those in the NS control group. Compared with silica exposure group or nonspecific antibody group, the lactic dehydrogenase and total protein levels, macrophage and neutrophil counting, IL-1? and TNF-a expression levels, and pathology inflammatory scores were significantly increased in TAM/Gas6 blocking groups and decreased in Gas6 supplying group (P<0.05 or P<0.01). Compare pulmonary inflammatory levels among Tyro3, Axl and Mer blocking groups, we found that mice in Mer blocking group had the highest inflammatory indexes.Conclusions:Silica exposure enhanced the stimulation levels of TAM/Gas6 pathway in lung tissue. It might be possible that the TAM/Gas6 pathway, especially Mer receptor, could negatively regulate the pulmonary inflammatory responses induced by silica dust in mice.Part II The role of TAM/Gas6 pathway in silica dust induced pulmonary fibrosis in miceObjectives:To investigate possible role of TAM receptors and Gas6 ligand played in sifica induced pulmonary fibrosis in mice.Methods:In this part, we established a total of 8 groups, including TAM/Gas6 pathway interference groups and reference groups. Grouping details were same to Part ?. On days 7,28 and 84 after intratracheal instillation, mice in different groups were killed. The right lungs were collected to measure the mRNA levels (RT-PCR method) and protein levels (Western blot method) of fibronectin and Collagen I. The left lungs were isolated to make pathology slice and then treated with Masson staining to observe fibrotic changes and calculate fibrotic scores.Results:On day 84, compared with mice in silica exposure group or nonspecific antibody group, mice in Tyro3, Axl, Mer or Gas6 blocking groups had higher levels of fibronectin and collagen I expression levels (mRNA and protein) and fibrosis scores in lung, while mice in the Gas6 supplying group had lower levels of the above indexes in lung (P<0.05 or P<0.01).Conclusions:The TAM/Gas6 pathway might be a negative regulative way during the process of silica induced pulmonary fibrosis.Part ? The peripheral blood expression levels and potential roles of TAM/Gas6 among a silicosis case-control populationObjectives:To evaluate the expression levels of TAM/Gas6 in peripheral blood, and its possible relationships with the risk of silicosis happening and progression.Methods:All subjects recruited in this case-control study came from a mine from Huangshi area. The cases were 50 male workers with silicosis, which included 19 class I patients,21 class II patients and 10 class III patients. The controls were divided into two groups, the healthy workers with silica exposure history (48 male individuals) and the healthy workers without silica exposure history (49 male individuals). All individuals in the three groups were matched in age, body mass index, smoking and drinking status. Fasting peripheral blood were collected for each individual in early morning. The Gas6 protein levels in plasma were evaluated by enzyme-linked immuno sorbent assay, and the mRNA levels of Tyro3, Ax1, Mer and Gas6 in peripheral blood leukocytes were isolated and measured by RT-PCR. The multiple Logistic regression analysis was adopted to investigate relationships between peripheral blood TAM/Gas6 levels and the risk of silicosis happening or progression.Results:Compared with healthy workers without silica exposure history, individuals in the silicosis group and the silica exposure healthy group had significantly higher Tyro3 and Ax1 leukocyte mRNA levels (P<0.01) and lower Gas6 leukocyte mRNA levels (P<0.05). Through the multiple Logistic regression analysis, we found that the higher Tyro3 and Axl leukocyte mRNA levels and the lower Gas6 mRNA level in peripheral leukocytes were associated with higher risk of silicosis [Tyro3 mRNA OR-5.13 (2.65-9.95); Axl mRNA OR=1.90 (1.01-3.58); Gas6 mRNA OR=0.19 (0.10-0.37)]. Comparing the peripheral blood TAM/Gas6 levels among individuals of different silicosis stages, we found that the Gas6 mRNA level in peripheral leukocytes were significantly lower in silicosis patients of class ? than that in silicosis patients of class I and II. However, there were no obvious relationships found between TAM/Gas6 peripheral blood levels and the risk of silicosis developmentConclusions:The higher Tyro3 and Axl mRNA levels and the lower Gas6 mRNA level in peripheral leukocytes were associated with higher risk of silicosis.The main advantages of this study were as follows. (1) This was the first in vitro study to investigate the negatively regulative role of TAM/Gas6 pathway in silica induced pulmonary inflammation and fibros is, which should be helpful for further understanding the mechanisms of silica induced pulmonary injuries. (2) The regulative roles of Tyro3, Axl and Mer in silica induced pulmonary inflammatuin and fibrosis were evaluated simultaneously, and Mer receptor was found to be more functional than Tyro3 and Axl receptors. (3) This study combined both in vivo and epidemiobgic studies simultaneously to verify possible relationships between TAM/Gas6 pathway and silicosis.The limitations for this present study were as follows. (1) For the case-control study, we only detected the expression levels of TAM/Gas6 in peripheral blood but not in lung. If it is possible, TAM/Gas6 expression levels in BALF or lung tissue obtained by bronchial biopsies should be detected. (2) Limited by the small size of study population, the conclusions we drew from this study should be further verified among larger populations or prospective cohort studies.