| Backgroud: Lung cancer is one of the most common malignancies in the world, they have always already metastasized by the time the tumors are diagnosed. Recent years, increasing researches were focused on the role of tumor microenvironment in lung cancer. American biologist Shreigber proposed "tumor immunoediting theory" in 2002, which believed that 3 phases- immune elimination, immune equilibrium and immune escape were included in tumor process. With the proliferation of tumor cells, local organization structure destroyed, which affected locally lymphocyte recruitment, phenotype and function.As one of the earliest subsets to sense the process of immunoediting, stromal cells were seldom studied. This study focused on the function and mechanisms of how stromal cells modulate immune system by using cytokines and chemokines. IL-17 family is one of the most sensitive danger sensors for tissue damage especially mucosa tissue. IL-17 D is a member of IL-17 family and recently it was described to highly expressed in lung, skeletal muscle and brain. Our preliminary study suggested that IL-17 D was mainly expressed in CD45- cells in lung. This study aimed to investigate the effect and explore the mechanisms of IL-17 D in lung cancer microenvironment. Besides, we aimed to clarify the mechanisms of IL-17 D in modulating migration, phenotype and function of NK, CD8+ T cells in lung.Purpose: 1. IL-17 D was mainly expressed in CD45- cells in lung tissue as shown in our previous study. To identify the main cell origin and characterize of IL-17 D during mice lung cancer.2. To understand the migration, surface marker and function of NK and CD8+ T cell in lung, and identify the impact of IL-17 D deletion on migration, phenotype and function of NK cells in lung.Methods and Materials: 1. B16 lung metastatic lung cancer, carcinoma of skin and small intestine were established. Mice were offered exogenous IL-17 D by inhalation.2. The m RNA of IL-17 D in lung, skin, small intestine were detected by quantitative real-time PCR. The m RNA of CXCL9?CXCL10?CXCL11?S1Pr5 and S1Pr1?IL-12?IL-15?GM-CSF?IL-22?IL-23?IL-1??TNF-a were also detected by real-time PCR.3. IL-17 D secreted by fibroblasts and the proportions of CD4+ T, CD8+ T and NK cells in lung of mice were detected by flow cytometry.4. Primary mice lung fibroblasts and human embryo lung fibroblasts were separated and cultured by using collagenase or organization adhesion. The primary cells were identified by flow cytometry.5. Tumor cells supernatants and lung fibroblasts were cocultured in different number, the m RNA of IL-17 D, CXCL9, CXCL10 in lung fibroblasts were detected by RT-PCR.6. Lung fibroblasts were seeded in lower chamber of Transwell plates, and were stimulated by lung cancer cell LLC cell supernatant and r m IL-17 D. And lymphocytes from mice peripheral blood, lung or spleen were seeded in upper chamber of Transwell plates. 5.5 hours later, flow cytometry was applied to detect the proportions of NK and CD8+ T cells in lower chamber. RT-PCR was used to detect the m RNA expression of IL-17 D, CXCL10 and IL-15 of lung fibroblasts in lower chamber.Results: 1. IL-17 D expression was decreased in tumor.As shown by real-time PCR and flow cytometry, IL-17 D was mainly secreted in CD45-cells in lung; IL-17 D expression in B16 lung metastasis lung cancer, skin cancer, small intestine cancer were significantly down-regulated. The m RNA expression of IL-17 D in lung cancer was decreased in time-dependent way.2. The cell origin of IL-17 D in lung tissue.Flow cytometry showed that, lung fibroblasts(CD45-CD140a+) take a percent of 40~80% in CD45-IL-17D+ cells in lungs. Besides, flow cytometry intracellular staining suggested that, IL-17 D secreted by lung fibroblasts were obviously decreased in B16 lung metastasis lung cancer mice(P<0.05).3. Tumor infiltrating lymphocytes profile in lung cancer.As shown by flow cytometry, the proportions of CD3+ T, CD8+ T and NK cells in B16 lung metastasis lung cancer mice were significantly decreased(P<0.05). However, CD4+ T cells were not affected. Besides, IFN-? produced by NK cells were accordingly down-regulated(P<0.05).4. Chemokines and cytokines profile in lung cancer.Reports suggested that lung fibroblasts not only recruited amount of lymphocytes to local tissue, but also regulated the phenotype and function of local lymphocytes by secreting cytokines like CXCL9, CXCL10, GM-CSF, IL-1?, TNFa and IL-23.As shown by real-time PCR, the m RNA expression of CXCL9, CXCL10 and S1Pr1, IL-12, IL-15, GM-CSF in the lungs of B16 lung metastasis lung cancer mice were declined.5. IL-17 D suppress tumor lung metastasis in mice.A significantly reduction of lung tumor colonies was observed in mice with exogenous IL-17 D inhalation compared with control PBS(P<0.05). Besides, the proportions of tumor infiltrating CD8+ T and NK cells were increased as shown by q-PCR. Furthermore, the m RNA expression of CXCL9, CXCL10, GM-CSF were up-regulated in IL-17 D inhalation mice(P<0.05). These results suggested that IL-17 D may recruited NK, CD8+ T cells to local microenvironment kill tumor cells through CXCL9/ CXCL10 pathway.6. Lung tumor cells suppress IL-17 D expression of primary lung fibroblasts.B16 tumor cells and mice primary lung fibroblasts were cocultured, and then were collected. The m RNA expression were detected by RT-PCR, as the results showed, B16 cells can effectively suppress the expression of IL-17 D and CXCL9, CXCL10. Furthermore, a similar result was observed when the cell supernatants of human lung cancer cell line A549 were cocultured with primary human embryo lung fibroblasts.7. The effect of IL-17 D on the recruitment of NK and CD8+ T cells by lung fibroblasts recruited in vitro.Compared with control group(no cells plated in lower chamber), lung fibroblasts effectively recruited NK and CD8+ T cells to lower chamber. However, lung fibroblasts recruited a significantly decreased proportions of NK and CD8+ T cells when lung fibroblasts was stimulated by LLC cell supernatant in lower chamber, but this result can be reversed if r m IL-17 D were simultaneously added in lower chamber, and this was dose- dependent.Besides, lung fibroblasts in lower chamber were collected its IL-17 D, CXCL10 and IL-15 m RNA expression were detected by RT-PCR. As it shown, the m RNA expression of IL-17 D, CXCL10 and IL-15 by lung fibroblasts following LLC cell supernatant stimulation were significantly down-regulated while the m RNA expression were obviously up-regulated with simultaneous r m IL-17 D stimulation.Conclusions: 1. IL-17 D from lung fibroblasts play important role in antitumor immunity. That is, The reduction of IL-17 D produced by fibroblasts in lung cancer induced a reduction of CXCL9, CXCL10 and IL-12, IL-15, GM-CSF, which then caused a decreased proportions and numbers of NK, CD8+ T cells, and this finally promoted tumor growth and metastasis.2. IL-17 D produced by lung fibroblasts recruited NK and CD8+ T cells by CXCL9 and CXCL10 to local tumor microenvironment to strengthen antitumor immunity.Background: According to a recent report by the United Nations scientific committee on atomic radiation, medical irradiation take about 20 percent of total irradiation the world's population suffers. Irradiation induced damage of patients and radiologist during radiotherapy by production of reactive oxygen species and free radicals, which causes severe morphological and functional lesions in living cells, especially in lymphocytes. Besides, Radiotherapy can induce tumor-promoting cell subsets accumulation such as Regulatory cells(Treg), Th17 cells and myeloid-derived suppressor cells(MDSC).T cell is a highly heterogeneous subset, and CD4+ T-cell subsets including Th1, Th2, Th17 and Treg cells are defined by their pattern of cytokine production and function. Accordingly, the Th1/Th2/Th17/Treg paradigm plays a central role in maintaining immune homeostasis based on their cytokine production, and the cytokine environment they produce determines the fate of the host antitumor response. Cinnamon has been used as spices and condiments world widely for centuries, and it was thought to nourish liver and kidney in clinical practice of traditional Chinese medicine. Besides, it was a common herb in the recipe to treat diabetes, arthritis and tumors. However, little is known about the effects of cinnamon on reconstitution of T-cell subsets after irradiation.Puepose: 1. To understand the profile of T-cell reconstitution after a single low-dose total body irradiation(SLTBI), and thus provide theoretical and experimental bases for radiotherapy.2. Inefficient T-cell reconstitution from x-ray-induced immune damage reduces antitumor response. This study aimed to identify the recovery profile of T-cell subsets following x-ray irradiation and to highlight the role of cinnamon on efficient T-cell restoration post exposure in the antitumor response, which may provide the basic data for research of clinical drug treatment.Methods: 1. Preparation water extraction of cinnamon: air-dried cinnamon decocted with water, dissolved in ethanol as traditional extraction methods.2. Irradiated mice were given a single dose of 2.5 Gy for total body x-ray irradiation; Irradiated mice for cinnamon treatment and for tumor induction in the cinnamon therapy model were given 0.1% water extraction of cinnamon.3. Some mice that were either treated or not treated with irradiation and cinnamon for induction of B16 lung melanoma were injected intravenously(i.v.) with 2×105 B16 melanoma cells.4. A complete blood count(CBC) of mice was performed using a Hematology Analyzer in different time point following SLTBI.5. CD3+, CD8+, and CD4+ T cells in peripheral blood and spleen and CD80, CD86, MHC-I, MHC-II in spleen in different time point after SLTBI were analyzed by flow cytometry analysis.6. Tc1, Th1, Th2, Th17, and regulatory T(Treg) cells were evaluated at different time points after SLTBI by intracellular staining.7. T-bet, GATA3, ROR?t, and Foxp3 signaling specific for Th1, Th2, Th17, and Treg cells were also analyzed by reverse transcription PCR assay. The m RNA expression of IL-12, IL-15, IL-23 were detected by RT-PCR assay.8. All lung lobes were evaluated using a stereomicroscope.Results: 1. Lymphocytes were highly sensitive to x-ray irradiation.In all major cell populations of peripheral blood, lymphocytes were seriously reduced both in number and in percentage in 3-5 day post irradiation(P<0.05), while monocytes and granulocytes were slightly decreased only in their numbers(P>0.05). Lymphocytes have been restored from x-ray-induced impairment starting from day 5 post-SLTBI.2. T-cell reconstitution after SLTBI.Along with irradiation-induced lymphocyte reduction, T lymphocytes, as the main population of lymphocytes, were also damaged after SLTBI. Both the cell counts and the frequencies of total T cells(CD3+ T) cells from the spleen were significantly decreased within 3 to 5 days post-SLTBI, and have been restored to normal level within 10 days. The decreased proportion of CD4+ Tcells in total CD3+ T cells on day 5 was concomitant with the reduction of CD3+ T cells. In contrast, the CD8+ T cells were found at slightly increased number at the same time point. CD4+ T cells have a delayed reconstitution than CD8+ T cells.MHC-II and CD80, CD86 expressed on DCs and were decreased at day 5, and were restored to normal level at day 10 post irradiation. Accordingly, the m RNA expression of IL-12, IL-15 and IL-23 were down-regulated at day 5, and were recovered 10 days post irradiation.3. Functional reconstitution of CD4+ T-cell subsets after SLTBI.IFN-? production released by Th1 or by Tc1 was still at a lower level in irradiated mice than in control mice(P<0.05), whereas the proportions of Th2, Th17, and Treg cells were significantly increased in the IR group(P<0.05), even 10 days post exposure. And antineoplastic IFN-?+IL-17A+ Th17 cell subsets were significantly decreased in IR group(P<0.05).4. Cinnamon rescues the efficient reconstitution of T-cell subsets.Th1 and Tc1 cells were significantly up-regulated(P<0.05) and Th17 and Treg cells were down-regulated(P<0.05), based on their proportions and numbers in cinnamon-treated mice. However, cinnamon had no effect on Th2 cells. And the proportions of IFN-?+IL-17A+ Th17 cells in cinnamon-treated mice were obviously increased(P<0.05).5. Cinnamon modulates specific signaling of distinct T-cell subsets.The m RNA expression of T-bet was down-regulated, whereas Foxp3 was up-regulated in the IR group(P<0.05). After administration of cinnamon, increased transcription of T-bet and decreased expression of Foxp3 were detected. The transcription levels of ROR?t and GATA-3 were not changed significantly.6. Cinnamon improves the host protection from tumor metastasis of irradiated mice.Lung tumor colonies in irradiated mice were significantly increased compared with control mice(P<0.05), while the tumor foci was obviously decreased in irradiated mice with cinnamon treatment(P<0.05).7. The profile of T cell infiltrated in tumor cite following SLTBI.Th1 and Tc1 cells were decreased in tumor-loaded lungs in irradiated mice compared with control mice, while cinnamon effectively increased the proportions of Th1 and Tc1 cells in lungs of irradiated mice.Conclusions: 1. T cells decreased sugnificantly in 3-5 days, and restored to standard level within 10 days post SLTBI. CD4+ T cells showed a delayed reconstitution than CD8+ T cells.2. T-cell reconstitution from SLTBI was characterized by impaired Th1 and elevated Th17 and Treg cells, which promoted tumor metastasis. Cinnamon effectively improved the imbalance of T-cell subsets by promoting the proliferation of Th1 and by suppressing expansions of Th17 and Tregs. The role of cinnamon in efficient T-cell reconstitution from SLTBI is effective in antitumor immunity. |
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