The Role Of Eosinophil In A Murine Lung Iniury Model Induced By Cigarette Smoke

Chronic obstructive pulmonary disease (COPD) is a common inflammatory airway disease, which is characterized by inflammatory cells infiltration, not fully reversible airflow limitation, mucus overproduction and airway remodeling. As is known to all, cigarette smoke is the most important risk factor of COPD, but the underlying mechanisms have not been fully elucidated. However, it is widely accepted that local immune dysregulation plays an important role in the pathogenesis of this disease. Clinical studies suggested Tc1and Th17cells were tightly associated with COPD. In addition, animal experiments also confirmed that Thl and Th17cells were involved in cigarette smoke induced lung inflammation.Persistent inflammatory response is the key point of COPD, with large number of leukocytes aggravation in the airway, including macrophage, neutrophil and lymphocyte. So far, it has not been clear that whether eosinophils take part in the pathogenesis of COPD. Although eosinophils are always linked with allergic diseases (i.e. asthma) and parasites infection, clinical researches indicated an increase of eosinophil proportion in peripheral blood and induced sputum from COPD patients. Glucocortcoid, a kind of routine drug for asthma control, displayed rather disappointing effect for COPD therapy. However, a part of COPD patients with relative higher eosinophils number presented better response to glucocortcoid, which suggested eosinophils played a role in at least part of COPD patients. Traditionally, eosinophils is considered to be a kind of terminal differentiated cell, and participate in innate immune response by releasing some inflammatory media, accompanied with process of degranulation. However, it was found in recent studies that eosinophil was also a regulator of adaptive immune response. In a classical OVA model, Th2immune response was significantly inhibited in eosinophil-deficiency mice. Moreover, adoptive transfer of OVA activated dendritic cells failed to restore immune response. This response could not be restored except for transferring both dendritic cells and eosinophils to the mice. Given the fact that COPD is a kind of Th1/Th17dominant disease, we put forward the hypothesis that if eosinophils affect airway inflammation via regulating Th1/Th17response.In order to testify our hypothesis, we made use of a strain of eosinophils deficient mice-PHIL-to establish a lung injury model induced by cigarette smoke, observed airway inflammation and T cell subpopulations in lung and spleen. Compared with WT mice, PHIL mice developed less airway inflammation, accompanied with the reduction of Th17/Tc17proportion in both organs. However, eosinophil deficiency seemed not affect Th1/Tc1cells, which was evidenced by the similar elevation of IFN-γ concentrations in BALF and lung homogenate in both strains of mice after cigarette smoke exposure. In contrast, we only found a little increase of IL-17A in cigarette smoke exposed PHIL mice. Our data supported the idea that eosinophil deficiency alleviated airway inflammation induced by cigarette smoke via regulating Th17/Tc17immune response.Part Ⅰ the establishment of murine lung injury model induced by cigarette smokeObjective:To establish a murine lung injury model by cigarette smoke exposure, and observe the kinetics of airway inflammation and T cell mediated immune response.Methods:183C57BL/6mice (male,8-10weeks) were randomly divided into2groups, control group (CON) and cigarette smoke group (CS).93mice in CS group were exposed to cigarette smoke in whole body exposure manner (100cigarettes/day,5days/week), while90mice in CON group were exposed to filtered air. The mice in each group were further divided into six subgroups according to six time points (1week,2weeks,4weeks,8weeks,12weeks and24weeks). At each time point, some mice in each group were sacrificed to analyze brochoalveolar lavage fluid (BALF), including total cell number and different cell counting, detect mRNA expression of cytokines, chemokines and transcriptional factors in lung tissue at by Real-time PCR. For some cytokines (i.e. IFN-γ and IL-17A), the concentrations in BALF and lung homogenate were measured by ELISA. In addition, left lung was inflated by gravity with4%paraformaldehyde, held at a pressure of30cmH20for15minutes and made pathological sections. Tissue samples were stained with haematoxylin and eosin (H&E) to examine peribronchial inflammation and emphysematous change. Other mice were sacrificed to obtain pulmonary mononuclear cell suspension by PBS perfusion, collagenase type I digestion, grinding and separation and splenocytes suspension by direct grinding. All the cell suspension was applied to detect the frequencies of T cell subpopulations by flowcytometry after PMA and Ionomycin stimulation.Results:cigarette smoke exposure led to large number of inflammatory cells infiltration in the airway. Inflammatory cells aggravated near the large airways at the early stage, while they infiltrated around small airways following long term exposure. The most evident inflammation could be observed at week12, as the inflammation score in CS group was much higher than that in CON group (2.25±0.22vs.1.00±0.21, P<0.001). When the mice were exposed to cigarette smoke for24weeks, mean linear chord (MLI) value increased in CS group in spite of reduced inflammation (94.38±6.45vs.70.20±4.45μ m, P<0.05). Cigarette smoke exposure induced large amount of leukocytes in BALF, especially of neutrophils, with their number reaching peak level at week12((2.77±1.07)×104/ml vs.(9.75±1.60)×104/ml, P<0.01). In addition, mid-and long-term exposure elevated the proportion of Tc1, Th17and Tc17in lung, and the most significant elevation also occurred at week12(Tc1:(43.63±3.25)%vs.(56.58±2.28)%, P<0.05; Th17:(11.25±1.92)%vs. (19.58±2.31)%, P<0.05; Tc17:(5.96±0.17)%vs.(8.53±0.78)%, P<0.05). The similar condition could be seen in spleen. In accordance with flowcytometry results, IFN-γ and IL-17A expression in lung was upregulated, and the concentrations of both cytokines in BALF and lung homogenate also increased. Moreover, cigarette smoke was also contributed to the induction of various kinds of chemokines and proinflammatory cytokines. Conclusion:cigarette smoke exposure led to airway inflammation and emphysema at different time points. Mid-and long-term exposure induced the expansion of Tc1, Th17and Tc17subpopulations in lung and spleen, which not only changed local proportion of T cell subtypes, but also affected T cell subpopulations distally.Part Ⅱ the role of eosinophils in cigarette smoke induced murine lung injuryObjective:To observe the effect of eosinophils on murine lung injury model induced by cigarette smoke.Methods:PHIL mice, as well as WT littermates, were randomly divided into four groups, WT/CON group, WT/CS group, PHIL/CON group and PHIL/CS group. CS group and CON group were exposed to cigarette smoke and filtered air for12weeks, respectively. The mice were sacrificed for the analysis of total leukocytes number and’ cell types in both BALF and peripheral blood, examination of peribronchial inflammation and emphysematous change, detection of T cell subpopulations in lung and spleen, observation of cytokine and chemokines expression in lung tissue and measurement of their secretion in BALF and lung homogenate.Results:Compared with WT/CS group, the mice in PHIL/CS developed less severe inflammation, but similar alveolar destruction. In line with pathological change, the cell total number in BALF from WT/CS group was much higher than that from PHIL/CS group ((54.25±2.41)×104/ml vs.(30.22±2.99)×104/ml, P<0.001). So did macrophage ((42.47±1.71)×104/ml vs.(25.75±2.39)×104/ml, P<0.001), neutrophil ((6.59±1.32)×104/ml vs.(2.34±0.63)×104/ml, P<0.01) and lymphocyte numbers ((5.18±0.65)×104/ml vs.(2.33±0.42)×104/ml, P<0.001). Reduced proportion of Th17and Tc17cells were found in both lung (Th17:(4.31±0.36)%vs.(8.44±0.56)%, P<0.01; Tc17:(4.66±0.73)%vs.(10.43±0.51)%, P<0.01) and spleen (Th17:(1.15±0.20)%vs.(2.13±0.31)%, P<0.05; Tc17:(0.80±0.08)%vs.(1.66±0.34)%, P<0.05) from PHIL mice after cigarette smoke exposure. However, there was no difference in the percentage of Th1and Tc1cells between WT/CS and PHIL/CS mice, whether in lung or in spleen. In parallel, a dramatically increase of IL-17expression level in lung tissue and its concentrations in both BALF (31.06±4.36vs.16.96±2.18pg/ml, P<0.01) and lung homogenate (11.11±0.86vs.5.77±1.10pg/mg protein, P<0.001) was only found in WT/CS mice, but not in PHIL/CS mice. On the other hand, similar elevation of IFN-γ at mRNA and protein level could be observed in both WT/CS and PHIL/CS mice. In addition, high expression of some chemokines (CXCL1, CXCL2, and CCL2) and proinflammatory factors (IL-6) only existed in WT/CS mice, while other proinflammatory factors were induced to the similar degree in both mice strains.Conclusion:Eosinophils deficiency alleviated airway inflammation induced by cigarette smoke, but not altered the formation of emphysema. Eosinophils positively regulated Th17/Tc17mediated immune response, but had minor effect on Th1/Tc1cellsPart Ⅲ the potential mechanisms of reduced airway inflammation and T cell mediated immune response due to eosinophils deficiencyObjective:To study the potential mechanisms of reduced airway inflammation and T cell mediated immune response due to eosinophils deficiency via airway epithelial cells and dendritic cells. Methods:The primary mouse tracheal epithelial cells (MTEC) were isolated from both WT and PHIL mice. After14-day air-liquid culture, MTEC were treated by2%cigarette smoke extract (CSE) for0,3,6,12and24hours. Some chemokines and cytokines mRNA expressions were detected by Real-time PCR. For the study of dendritic cells (DC), pulmonary and splenic leukocytes were obtained with the use of red blood cell lysis buffer. DC and costimulator molecules on its surface were detected by flowcytometry..Results:2%CSE treatment led to a kinetic change of mRNA expressions and secretion of chemokines and cytokines. For some genes, the expression level in MTEC derived from WT mice was much higher than that from PHIL mice following3h CSE exposure (CXCL2:8.22vs.3.04fold, P<0.05; CCL2:13.06vs.0.60fold, P<0.001; IL-6:7.24vs.0.77fold, P<0.01). There was almost no change of DC proportion after cigarette smoke exposure. However, cigarette smoke could significantly reduce the expression of MHC molecules in both mouse strains (MHC Ⅰ:3.340±0.036vs.2.965±0.027, P<0.001(WT),3.424±0.049vs.3.007±0.012, P<0.001(PHIL); MHC Ⅱ:3.395±0.079vs.2.721±0.079, P<0.001(WT),3.432±0.100vs.3.010±0.072, P<0.01(PHIL)). With regard to costimulators on DC surface, CD80and CD86expression was more significantly inhibited by cigarette smoke in WT mice (CD80:2.881±0.027vs.2.727±0.048, P<0.01; CD86:3.050±0.023vs.2.957±0.015, P<0.05), compared with PHIL mice.Conclusion:The MTEC from PHIL mice developed a weaker response to CSE. Eosinophils did not participate in the regulation of DC maturation and activation in this cigarette smoke exposure model.