Effect Of Free SiO₂ Exposure Dendritic Cells Derived From Rat Bone Marrow On The Differentiation Of T Cells

Background and objectiveSince the mechanisms underlying silicosis,caused by occupational inhalation of free silica dusts,are unclear,there are no effective treatments.Many studies,conducted to explore the pathogenesis of silicosis,showed that the pathological process of silicosis involved in the abnormalities of cellular immunity,humoral immunity and innate immune responses.However,those studies were used to focusing on the roles of immune cells including alveolar macrophages,neutrophils,T lymphocytes and B lymphocytes.etc.Dendritic cell（DC）is the most important antigen presenting cell during immune responses,but its role in the development of silicosis has not been explored.Our previous studies indicated that the maturation of DC derived from rat peripheral blood could be inhibited and the differentiation of Th1/Th2 would be affected when exposed to free SiO₂.The mononuclear cells derived from bone marrow are the main source of DC,but the changes of DC from rat bone marrow exposed to free SiO₂ have not been explored.For a comprehensive understanding of the correlation between DC and silica dusts and further interpretation of the immunological machanisms related to silica,firstly,we established the culture method of DC derived from rat rat bone marrow to attain large numbers of steady state DC.Subsequently,the effect of free SiO₂ exposure on phenotype and function of DC derived from rat bone marrow was explored.Moreover,the distribution of DC,Treg and Th1/Th2 in coalminer’s pneumoconiosis patients was also detected.Materials and Methods1.Effect of free SiO₂ exposure DCs derived from rat bone marrow on the differentiation of T cells in vitro.The observation of phenotype and function of DC induced by different concentrations of cytokines:rat bone marrow mononuclear cells were induced by different concentrations of cytokines.The morphology of DC was observed by inverted phase contrast microscope;the yield and phenotype of DC were detected by flow cytometry;the phagocytosis ability of DC was detected by fluorescent microsphere,and the antigen presenting ability of DC was detected by mixed lymphocyte reaction.The observation of the characteristics of different maturity states of DC:the immature DC（iDC）was stimulated with TNF-αand LPS for 48h,respectively.The morphology of DC was observed by inverted phase contrast microscope;the phagocytosis ability of DC was detected by fluorescent microsphere;the phenotype of DC was detected by flow cytometry;the expression of IL-12 were detected by Elisa;the antigen presenting ability of DC was detected by mixed lymphocyte reaction and the level of proteins was measured by western blot.The effect of free SiO₂ exposure on the phenotype of mDC:CCK8 experiment was used to measure the activity of alveolar macrophages（AM）,iDC and mDC exposed to different concentrations of free silica dusts to determine the optimum level of free SiO₂ exposure.And the co-culture models were established with AM and mDC.The experiment is divided into four groups:（1）control group（mDC）;（2）directly exposure group（SiO₂/mDC）;（3）co-culture group（AM/mDC）;（4）indirectly exposure group（SiO₂+AM/mDC）.Using flow cytometry to detect the expression of MHCⅡ,CD80 and CD86.The effect of free SiO₂ exposure on the process of i DC to mDC:iDC was exposed to free SiO₂ through directly exposure and indirectly exposure for 48h,then iDC was collected and stimulated with TNF-αfor anther 48h.Flow cytometry was used to detect the expression of MHCⅡ,CD80 and CD86.The effect of DC exposed to free SiO₂ on the differentiation of T cells:T cells collected from spleen of rat were co-cultured with DC exposed to free SiO₂ at a ratio of 10:1 for 48h.Using flow cytometry to detect the number of Th1（CD4⁺IFN-γ⁺）,Th2（CD4⁺IL-4⁺）and Treg（CD4⁺CD25⁺）,and the ratio of Th1/Th2 was also calculated.2.The detection of DC,Treg and Th1/Th2 in peripheral blood of coalminer’s pneumoconiosis patients.The investigation on occupational people was carried out.The exposed workers（43 cases）were used as control group and the coalminer’s pneumoconiosis patients as case group.Flow cytometry was used to detect the number of DC（CD11c⁺）,Th1（CD4⁺IFN-γ⁺）,Th2（CD4⁺IL-4⁺）and Treg（CD4⁺CD25⁺）,and the ratio of Th1/Th2was also calculated.3.Statistical analysisSPSS 21.0 was used to analysis the data.The quantitative data was presented with mean±SD.The comparison of means between two groups were used by Student’t test.The one-way ANOVA analysis was used to compare multiple groups,and the LSD test were used to further analysis of each two groups.The comparison of rates was used by chi-square test（?² test）.The Pearson correlation analysis was used to analysis the correlation between two variables.Inspection level of alpha was 0.05.Results1.Effect of free SiO₂ exposure DCs derived from rat bone marrow on the differentiation of T cells in vitro.The observation of different cytokine concentrations on the phenotype and function of DC:GM-CSF and IL-4 were combined to induce the generation of DC in vitro.Large numbers of DC were harvested on 6d to 8d.With the increase of cytokine concentrations,the harvest rate,MHC-II positive rate and the expression of costimulatory factors of DC were increased;and the antigen presenting ability of DC was also increased as the increased cytokine concentrations.The observation of the characteristics of different maturity of DC:both TNF-αand LPS could obviously promote the immature DCs to mature DC.Compared with the iDC,mDC presented a higher expression of MHCII,CD80 and CD86;the secretion of IL-12 was increased,and the ability to stimulate T cells was also significantly enhanced.However,the phagocytosis of mDC was decreased obviously.The results of the quantitative detection of protein found that the expression of HO-1was significantly inhibited in the process of DC maturation（P<0.05）.The effect of free SiO₂ exposure on the phenotype of DC:the optimum level of free SiO₂ exposure to AMs,iDC and mDC determined by CCK8 was 100ug/mL.The study showed that compared with the control group,mDC,exposed to free SiO₂through directly exposure or indirectly exposure,had a lower expression of MHCII,CD80 and CD86（P<0.05）,suggesting that the exposure of free SiO₂ dusts may inhibit the mature process of DC.Further study showed that iDC,pre-exposed to free SiO₂,presented a lower expression of MHCII,CD80 and CD86 when stimulated by TNF-αagain（P<0.05）.The effect of DC exposed to free SiO₂ on the differentiation T cells:T cells were co-cultured with DC exposed to free SiO₂.Compared to the control group,the results from the directly exposure group showed Th1 cells declined,Th2 and Treg cells increased,a significant reduction in the proportion of Th1/Th2 were presented.Compared to the co-culture group,the same trend was found in the indirectly exposure group（P<0.05）.2.The ditribution of DC,Treg and Th1/Th2 in peripheral blood of coalminer’s pneumoconiosis patientsThe results showed that compared with the control group,the number of DC,Th2 and Treg was significantly up-regulated in the case group（P<0.05）,however,no statistically significant difference was showed in the number of Th1 cells（P>0.05）.A significant reduction in the proportion of Th1/Th2 was presented in the case group（P<0.05）.Correlation analysis showed that there was a negative cprrelation between DC and Th1/Th2 ratio and a positive correlation is found between DCs and Treg ratio.（r=-0.656,P=0.003;r=0.597,P=0.009）.Conclusion1.In vitro,the maturation of DC derived from rat bone marrow could be inhibited,leading to the differentiation of T cells when exposed to free SiO₂.2.Coalminer’s pneumoconiosis patients showed a shift of Th1/Th2,DC and Treg,moreover,the shift of DC have a correlation with the shift of Th1/Th2 and Treg.