The Effect Of The TLR9 Ligand CpG-oligodeoxynucleotide On The Protective Immune Response To Radiation-induced Lung Fibrosis In Mice

Radiation-induced lung injury (RILI) is a common complication of thoracic radiotherapy, seriously affecting the efficacy and quality of life of patients. Its clinical manifestations are early radiation-induced pneumonia and late irreversible radioactive pulmonary fibrosis. However, related theories can't fully elucidate its pathogenesis. Th1/Th2 disorders of the immune system are correlated with tumor microenvironment and radiation injury.Toll-like receptor 9 (TLR9) agonist, CpG-ODN, has stronger immunomodulato-ry effects, which can effectively inhibit the immune response to Th2. It is commonly used in clinical and basic researches on airway hyperreactivity disease. In addition, CpG-ODN also has anti-cancer effects, and several ?-? clinical trials have been successively launched. Recent years, it was reported that the CpG-ODN combined with radiotherapy effectively inhibited the growth of lung cancer, but how CpG-ODN influences the common complications of lung cancer, RILI, and its radiotherapy outcomes is unclear.Through the construction of tumor bearing and RILI tumor-free mice model, this paper observed the expression of Th1/Th2 immune-related factors in lung tissue of each group at different time points, in order to understand the immune status of RILI Th1/Th2 in tumor bearing mice, and explore the laws of RILI development in tumor constitution and normal constitution, which made it clear the influence of Th1/Th2 immune to RILI in tumor model. At the same time, this paper explored the influence of CpG-ODN to formation of pulmonary fibrosis in RILI mice under the different immune status, and discussed the possible immune regulating mechanisms. It can provide theoretical basis for maintaining the immune balance as a new method for the prevention and treatment of RILI, and put forward new ideas for which for the future to carry out the radiotherapy combined immunotherapy more safely and effectively.Part I Effect of tumor constitution on lung tissue Th1/Th2 immune balance in mice after X-ray radiationObjective:Building lung cancer a tumor-burdened mice model, get to know the immune state of a tumor-burdened Thl/Th2, discussing the different laws of the occurrence and development of radioactive lung injury under tumor physique mice and normal physical mice.Method:Selected 18 to 25 g C57BL/6 female mice, Lewis lung cancer cell lines is grown in the right armpit, when the tumor grows to diameter with 0.5-1.0 cm, and with the non tumor mice randomly get into the experimental group. With 6 MV X-ray, single 12 Gy on the whole lung and tumor, we recorded tumor size and weight. Then we sacrificed them and preserved the lung tissues and serums at corresponding time points. HE staining and Masson staining and the content detection of hydroxyproline were used to evaluate under lung tumor immunity, the changes of the early lung tissue inflammation and fibrosis degree of a tumor bearing mice and tumor-free mice after radiotherapy. By the technologies of RT-PCR, Western blot, BDTMBA multiple cytokines detection techniques, etc, we analyzed the expression of the Thl/Th2 transcription factor T-bet in the lung tissue, the gene of the GATA-3, protein expression level and serum IFN-y, IL-13 after radiotherapy in the tumor bearing mice and tumor-free mice respectively.Results:Lung tissue HE staining of lung anatomy showed a few cancer cell infiltrations, whose termination of observation time was till 3 weeks. Tumor-free mice had not yet appeared breathing within 3-week radiotherapy. At the observation time point, HE staining (200*) of lung tissue showed no obvious inflammatory congestion, which was mainly characterized as interstitial thickening, alveolar normal structures disappeared in the tumor bearing mice; But the interstitial congestion and inflammatory cell aggregated in the tumor-free mice, obvious in the third days and continued for a long time. Masson staining results (200*) showed that there was collagen deposition early after radiotherapy in two groups, and increased with the time. Notably, collagen deposition was more obvious in the tumor bearing mice than the tumor-free mice, same as the results of hydroxyproline content (P< 0.05). Real-time quantitative PCR results showed that Th2 transcription factor GATA-3 gene expression levels were higher in tumor bearing mice at different time points after radiotherapy compared with rumor-free mice, and rapidly rose in 1 week. Moreever, the Thl transcription factor T-bet gene expression in tumor bearing mice was decreased rapidly after peak at early radiotherapy (the first day), while it showed early high response and still increased after 2 weeks in the tumor-free mice.Western blot results are similar to the results of real-time quantitative PCR, Th2 transcription factor GATA-3 protein expression in two groups gradually increased with the passage of time, and Th2 transcription factor GATA-3 protein expression was higher in the tumor bearing mice than the tumor-free mice at each time point of radiotherapy; However, Thl transcription factor T-bet expression tumor in the bearing mice decreased with the time of radiotherapy, lowest at 3 weeks, but T-bet was still on the rise with the time within 3 weeks of radiotherapy in the tumor-free group. BDTMCBA multiple cytokines technology results showed that the content of serum Th2 cytokine and IL-13 protein in tumor bearing mice gradually increased with time and peak in the 3 week after radiotherapy. In addition to the third days of high expression, the expression level of Thl cytokine INF-y protein was lower in tumor bearing mice than that in tumor-free mice.Conclusion:Under tumor constitution, pulmonary fibrosis occurred earlier and severer in tumor-free mice than in tumor bearing mice. There was also Thl/Th2 immune imbalance existing in tumor bearing mice with RILI, but tumor-free mice with RILI were more inclined to Th2 reaction and pulmonary fibrosis. This may be partially interpreted as the irreversibility RILI pulmonary fibrosis in patients with clinical tumors. Serum Thl/Th2 cytokines INF-y, IL-13 can predict the extent of RILI.Part II:the effect of CpG-ODN on the prognosis of RILI in mice with lung cancerObjective:To study the effects of CpG-ODN on the formation of pulmonary fibrosis in mice by using its characteristics of immune regulation.Methods:Introduction of CpG-ODN into intervention group (CRT group),1 weeks before radiotherapy and after radiotherapy 3 weeks, continuous intermittently administration, and evaluated the safety and effectiveness of CpG-ODN intervention (anti-tumor effects, the immune status of Thl/Th2 intervention). BDTMBA multiple cytokines technology detected the changes in serum Thl/Th2 related cytokines ((IFN-?, IL-12, IL-5, IL-13 and etc) expression. Take the method in the first part to assess the effects of CpG-ODN on early lung tissue inflammation and fibrosis degree intracellular in tumor bearing mice after radiotherapy. Western blot and ELISA detected the expression of primary intracellular signaling molecules in the activation pathway of T helper cells, p-STAT1, p-STAT4, pSTAT6 and transcription factor T-bet/GATA-3 protein.Results:From the tumor growth curve, we observed that after undergoing chest X-ray irradiation, the growth of tumor in tumor bearing mice with CpG-ODN treatment group (CRT group) was slower than tumor bearing mice of non-intervention (LRT group). BDTMBA multiple cytokines test showed in CRT group that the expression of Thl-type cytokines, IL-12 and IFN-y, was rapidly increased after administration, but the expression of Th2-type cytokines, IL-5 and IL-13, was decreased. The fibrosis score showed that CpG-ODN intervention treatment significantly reduced the pulmonary fibrosis in tumor bearing mice after radiotherapy for 14 days and 21 days compared with LRT group, P<0.05. Hydroxyproline detection results showed that collagen deposition n CRT group was lower than that of LRT group at each observational time point, P< 0.05. RT-PCR and WB results showed that the expression of T-bet protein in lung tissue of LRT group was significantly lower than that of CRT group, the gray value analysis showed that the difference of 7d,14d,21d between two groups was statistically significant (P<0.05). Double antibody sandwich ELISA was used for analysis of the expression of pSTAT-1 protein, and its expression slightly decreased with time in 7d and 21d. But from each time point of comparison between the two groups of mice, CRT group receiving CpG-ODN therapy had higher pSTAT-1 protein expression in 7d and 21d compared with mice in RT group, and the difference was statistically significant (P< 0.05). In LRT group, group the expression of pSTAT-4 slightly decreased in 7d and 21d, but it showed a rising trend in CRT group. From the comparison between the two groups at each time point, the expression of pSTAT-4 protein in 7d and 21 d in group was higher than that in RT group, and the difference was statistically significant (P< 0.05). The expression of pSTAT-6 in 7d and 21d was increased with time. From the comparison between the two groups at each time point, pSTAT-6 protein expression was lower in 21 d CRT group than that in RT group.Conclusion:CpG-ODN can reduce the lung inflammation and pulmonary fibrosis in mice post-irradiation, as well as the expression of type Th2 inflammatory cytokines in the serum of mice with lung injury, and ultimately reduced the occurrence of radiation-induced pulmonary fibrosis.Part III:the molecular mechanism of TLR9 agonist CpG-ODN in the early stage of radiation-induced lung injury in miceObjective:To study the effect of CpG-ODN on the formation of pulmonary fibrosis in radiation-induced lung injury, and to explore the possible mechanisms of immune regulation.Methods:C57BL/6 mice were randomly divided into 4 groups, respectively (1) RT group:single irradiation of 12Gy X-ray to the whole chest; (2) CpG group: intraperitoneal injection of 0.5ml CpG-ODN for 4 weeks, once a week; (3) RT+CpG group:Immediately after single radiotherapy on chest, combination with intraperitoneal injection of CpG-ODN, whose schemes are same as above (4) CTL group:blank control. After 3 months, the mice were sacrificed for specimens from lung tissue and blood. Pathological staining was used to assess lung fibrosis after radiotherapy; hydroxyproline acid was used to test the deposition of collagen; and the immunohistochemical technology was used to detect macrophages, change of macrophage M1/M2 subsets, as well as to construct macrophage model after radiation detection, to test the macrophage proliferation and differentiation, immunohistochemical detection of lung fibrosis molecular TGF-? expression. Multiple cytokines detection technology was used to test the expression of I/II type cytokines. ELISA and Western blot were used to detect TLR9, the main intracellular signaling molecules in the upstream and downstream of T cell activated JAK-STAT pathway, p-STATl, p-STAT4, pSTAT6, JAK, SOCS3 protein expression.Results:The pulmonary fibrosis was significantly lighter in RT+CpG than RT group. The fibrosis index score in RT group was 5.2+0.7, while it was 3.4+0.7 RT+CpG, group, which was statistically significant. The pulmonary fibrosis in the other two groups were not obvious, and the fibrosis index score between 0-1. Collagen deposition and the expression of fibrosis molecular TGF-? and a-SMA in RT CpG group also showed a lower level (P< 0.05). Compared with the CTL group, macrophage accumulated in the other three groups of mice, the average optical density analysis showed that the macrophages RT group were mainly M2 subtype, while in RT+CpG were mainly M1 subtype (P< 0.05). Ml macrophages in the CPG group were slightly higher than that of CTL control group. The expression levels of serum II cytokines IL-4, IL-13 was higher than Thl type cytokines INF-?? IL-12 in RT. After treatment with CpG-ODN, the expression of p-STAT6 was significantly decreased. The expression of p-STAT6 in RT group was significantly higher than that of CTL group (P<= 0.05). However, p-STAT1, p-STAT4 expression was lower; on the contrary, in CpG+RT group and RT group respectively, the expression level of TLR9, JAK protein was significantly increased, while that of p-STAT6, SOCS3 was significantly decreased (P< 0.05).Conclusion:One of the mechanisms for CpG-ODN reversed the immune imbalance of Thl/Th2 and relieved radiation-induced lung injury is to induce TLR9 through activating jak-statl signaling pathway, enhance immune response to Thl, promote the proliferation and differentiation of macrophages into M1 macrophages, and release I cytokine.