IL-17诱导炎性因子及iNOS表达参与宿主抗衣原体免疫保护
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摘要
目的：
针对IL-17对某些细胞因子/趋化性细胞因子及一氧化氮合成酶(inducible nitric oxide synthase, iNOS)表达的调节,探讨IL-17在小鼠沙眼衣原体肺炎菌株(Chlamydia muridarum, Cm)呼吸道感染中的作用及机制。
方法：
BALB/c小鼠鼻腔吸入1×103 IFU Cm,建立小鼠Cm呼吸道感染模型。于感染后不同天数处死小鼠。利用RT-PCR及EL ISA技术检测小鼠肺组织IL-17mRNA及蛋白的表达,并利用细胞内细胞因子染色技术检测小鼠脾脏IL-17+ CD4+T细胞即Th17细胞百分率,确定IL-17及Th17在小鼠衣原体肺感染中的产生及诱导。利用抗鼠IL-17单克隆抗体鼻腔吸入中和Cm感染小鼠内源性IL-17,以抗鼠IL-17单克隆抗体独特型(IgG2a)作为对照。通过检测小鼠体重变化、肺组织衣原体生长及病理学改变,确定IL-17在衣原体肺感染中的保护作用。对支气管肺泡灌洗液内炎症细胞进行染色及分类计数,确定IL-17对肺组织浸润的炎细胞类型的影响。最后,利用RT-PCR检测IL-17对肺组织及小鼠肺上皮细胞内细胞因子/趋化性细胞因子及iNOS表达的影响,探讨IL-17在小鼠衣原体肺感染中的保护作用机制。
结果：
Cm呼吸道感染可诱导小鼠肺组织IL-17产生及脾脏Th17细胞扩增。与给予独特型抗体IgG2a的对照组比较,IL-17中和小鼠显示严重的疾病状态,主要包括：明显的体重降低、肺组织衣原体持续大量生长及更为严重的肺组织病理学改变。IL-17中和小鼠支气管肺泡灌洗液中性粒细胞(PMN)百分率及绝对数量显著下降,而淋巴及单核细胞百分率及绝对数量升高。IL-17中和小鼠肺组织与PMN趋化作用相关的因子,MIP-2及IL-6 mRNA表达显著下降,而T细胞趋化因子RANTES表达上调；体外实验显示,IL-17可协同TNF-a显著上调小鼠肺上皮细胞(TC-1) MIP-2及IL-6表达,但下调TNF-a诱导的RANTES表达。此外,IL-17可显著上调小鼠肺组织及TC-1细胞iNOS表达,并增强细胞内NO产生,参与细胞内抗衣原体反应。
结论：
衣原体呼吸道感染可诱导小鼠体内IL-17产生及Th17细胞扩增。IL-17在衣原体呼吸道感染中发挥重要保护作用。其作用机制包括：IL-17调节细胞因子/趋化性细胞因子在小鼠肺组织,尤其是肺上皮细胞内的表达,诱导不同类型炎细胞在肺组织浸润,参与炎性应答的调节；其次,IL-17通过增强衣原体感染小鼠肺组织及肺上皮细胞iNOS表达及NO产生,直接参与细胞内衣原体抑制。
Objective:
To investigate the role and mechanism of IL-17 during Chlamydia muridarum (Cm) infection.
Methods:
BALB/c mice were inoculated intranasally with 1×103 inclusion-forming units (IFUs) of Cm to induce the chlamydia pneumonitis. The mice were killed at the different days following infection. In order to determine the production of IL-17 and Th17 expansion, RT-PCR was used to detect the mRNA expression of IL-17 in the lung, and intracellular cytokine staining was used to detect the expansion of IL-17+ CD4+ T (Th17+). Anti-mouse IL-17 mAb was used to neutralize endogenous IL-17 following Cm infection. To evaluate the role of IL-17 during infection, the body weight change, the growth of organisms and histopathology in the lung were monitored and detected. A differential cell count in bronchoalveolar lavage (BAL) fluids was used to initially evaluate the changes of types of infiltrated inflammatory cells influenced by IL-17. Finally, RT-PCR was used to detect the expression of cytokine/chemokines and iNOS in the lung and the mouse pulmonary epithelial cell line (TC-1), to investigate the role mechanism of IL-17 during chlamydial infection.
Results:
Intranasal infection with 1×103 IFU in mice significantly induced IL-17 production in the lung and Th17 expansion in spleen cells. IL-17-neutralized mice exhibited a more severe state of disease, including greater body weight loss, higher organism growth, and much more severe pathological changes in the lung compared with IgG2a-treated mice. The percentage of neutrophils in the IL-17-neutralized mice was significantly lower than IgG2a-treated mice in BAL cells (p<0.001), and the percentages of lymphocyte and monocyte were relatively higher in the IL-17-neutralized mice than IgG2a-treated mice (p<0.01). Compared with IgG2a-treated mice, the expression of MIP-2 and IL-6 in the lung of IL-17-neutralized mice significantly decreased, however, the expression of RANTES significantly increased. IL-17 significantly increased TNF-a-induced MIP-2 and IL-6 expression in pulmonary epithelial cells but repressed RANTES expression. The expression of iNOS in the lung of IL-17-neutralized mice was significantly lower than that of IgG2a-treated mice. IL-17 potently induced IFN-γmediated iNOS expression in un-infected and Cm-infected pulmonary epithelial cells, and significantly increased IFN-γmediated intracellular Cm inhibition.
Conclusion:
Chlamydial lung infection can induce IL-17 production and Th17 expansion. IL-17 contributes to immune protection against chlamydial lung infection. The mechanisms include that IL-17 affects types of infiltrated inflammatory cells through regulating corresponding cytokine/chemokine expression, and IL-17 directly participates in inhibition on intracellular Cm, partially through enhancing iNOS expression and NO production in the lung of mice following chlamydial infection.
针对IL-17对某些细胞因子/趋化性细胞因子及一氧化氮合成酶(inducible nitric oxide synthase, iNOS)表达的调节,探讨IL-17在小鼠沙眼衣原体肺炎菌株(Chlamydia muridarum, Cm)呼吸道感染中的作用及机制。
方法：
BALB/c小鼠鼻腔吸入1×103 IFU Cm,建立小鼠Cm呼吸道感染模型。于感染后不同天数处死小鼠。利用RT-PCR及EL ISA技术检测小鼠肺组织IL-17mRNA及蛋白的表达,并利用细胞内细胞因子染色技术检测小鼠脾脏IL-17+ CD4+T细胞即Th17细胞百分率,确定IL-17及Th17在小鼠衣原体肺感染中的产生及诱导。利用抗鼠IL-17单克隆抗体鼻腔吸入中和Cm感染小鼠内源性IL-17,以抗鼠IL-17单克隆抗体独特型(IgG2a)作为对照。通过检测小鼠体重变化、肺组织衣原体生长及病理学改变,确定IL-17在衣原体肺感染中的保护作用。对支气管肺泡灌洗液内炎症细胞进行染色及分类计数,确定IL-17对肺组织浸润的炎细胞类型的影响。最后,利用RT-PCR检测IL-17对肺组织及小鼠肺上皮细胞内细胞因子/趋化性细胞因子及iNOS表达的影响,探讨IL-17在小鼠衣原体肺感染中的保护作用机制。
结果：
Cm呼吸道感染可诱导小鼠肺组织IL-17产生及脾脏Th17细胞扩增。与给予独特型抗体IgG2a的对照组比较,IL-17中和小鼠显示严重的疾病状态,主要包括：明显的体重降低、肺组织衣原体持续大量生长及更为严重的肺组织病理学改变。IL-17中和小鼠支气管肺泡灌洗液中性粒细胞(PMN)百分率及绝对数量显著下降,而淋巴及单核细胞百分率及绝对数量升高。IL-17中和小鼠肺组织与PMN趋化作用相关的因子,MIP-2及IL-6 mRNA表达显著下降,而T细胞趋化因子RANTES表达上调；体外实验显示,IL-17可协同TNF-a显著上调小鼠肺上皮细胞(TC-1) MIP-2及IL-6表达,但下调TNF-a诱导的RANTES表达。此外,IL-17可显著上调小鼠肺组织及TC-1细胞iNOS表达,并增强细胞内NO产生,参与细胞内抗衣原体反应。
结论：
衣原体呼吸道感染可诱导小鼠体内IL-17产生及Th17细胞扩增。IL-17在衣原体呼吸道感染中发挥重要保护作用。其作用机制包括：IL-17调节细胞因子/趋化性细胞因子在小鼠肺组织,尤其是肺上皮细胞内的表达,诱导不同类型炎细胞在肺组织浸润,参与炎性应答的调节；其次,IL-17通过增强衣原体感染小鼠肺组织及肺上皮细胞iNOS表达及NO产生,直接参与细胞内衣原体抑制。
Objective:
To investigate the role and mechanism of IL-17 during Chlamydia muridarum (Cm) infection.
Methods:
BALB/c mice were inoculated intranasally with 1×103 inclusion-forming units (IFUs) of Cm to induce the chlamydia pneumonitis. The mice were killed at the different days following infection. In order to determine the production of IL-17 and Th17 expansion, RT-PCR was used to detect the mRNA expression of IL-17 in the lung, and intracellular cytokine staining was used to detect the expansion of IL-17+ CD4+ T (Th17+). Anti-mouse IL-17 mAb was used to neutralize endogenous IL-17 following Cm infection. To evaluate the role of IL-17 during infection, the body weight change, the growth of organisms and histopathology in the lung were monitored and detected. A differential cell count in bronchoalveolar lavage (BAL) fluids was used to initially evaluate the changes of types of infiltrated inflammatory cells influenced by IL-17. Finally, RT-PCR was used to detect the expression of cytokine/chemokines and iNOS in the lung and the mouse pulmonary epithelial cell line (TC-1), to investigate the role mechanism of IL-17 during chlamydial infection.
Results:
Intranasal infection with 1×103 IFU in mice significantly induced IL-17 production in the lung and Th17 expansion in spleen cells. IL-17-neutralized mice exhibited a more severe state of disease, including greater body weight loss, higher organism growth, and much more severe pathological changes in the lung compared with IgG2a-treated mice. The percentage of neutrophils in the IL-17-neutralized mice was significantly lower than IgG2a-treated mice in BAL cells (p<0.001), and the percentages of lymphocyte and monocyte were relatively higher in the IL-17-neutralized mice than IgG2a-treated mice (p<0.01). Compared with IgG2a-treated mice, the expression of MIP-2 and IL-6 in the lung of IL-17-neutralized mice significantly decreased, however, the expression of RANTES significantly increased. IL-17 significantly increased TNF-a-induced MIP-2 and IL-6 expression in pulmonary epithelial cells but repressed RANTES expression. The expression of iNOS in the lung of IL-17-neutralized mice was significantly lower than that of IgG2a-treated mice. IL-17 potently induced IFN-γmediated iNOS expression in un-infected and Cm-infected pulmonary epithelial cells, and significantly increased IFN-γmediated intracellular Cm inhibition.
Conclusion:
Chlamydial lung infection can induce IL-17 production and Th17 expansion. IL-17 contributes to immune protection against chlamydial lung infection. The mechanisms include that IL-17 affects types of infiltrated inflammatory cells through regulating corresponding cytokine/chemokine expression, and IL-17 directly participates in inhibition on intracellular Cm, partially through enhancing iNOS expression and NO production in the lung of mice following chlamydial infection.
引文
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