The Experimental Study On The Mechanism Of Pulmonary Allergic Responses Drived By Intestinal Microflora Disruption

Background and Objectives Bronchial asthma is the most common chronic allergic disease with the increasing trend of incidence rate over the past three decades, which affects the children's life quality a lot and becomes a global concerned public health issue. Intestinal microflora is important to the organism, but antibiotics usage lead to intestinal microflora disruption in clinic usually. In recent years, antibiotics abuse is generally existed in the medical institutions, so the intestinal microflora disruption becomes a common problem. Epidemiologic study showed that people used antibiotics in their infant period, they would have higher risk of asthma later. Animal experiment also suggested that intestinal microflora disruption caused by antibiotics correlated to allergic disease.Th17 cell is the third kind of responsive T cell discovered recently after Th1 and Th2 cells. Both clinical study and animal experiment suggested that Th17 was a key factor to lead asthma. In recent years, disbalance of immunocyte effection-regulation function has become a key subject of allegic diseases study. Deficient of regulatory T cell (Treg) subset regulating function may be the key mechanism of allegic diseases such as asthma. Furthermore, dendritic cell (DC) is significant in the immune system. Being a full-time antigen presenting cell, DC can recognize pathogen-associated molecular pattern (PAMP) produced by various microorganism through toll-like receptor (TLR) in their membrane surface. Through their presenting antigen and secreting related cytokine, they played an important role in guiding Th0 cells to differentiate to Th1, Th2, Th17 and Treg cells.Based on the previous study, we adopted ovalbumin (OVA) atomization challenge to further confirm the Th2 predominance effect; to identify whether there is Th1/Th2/Th17/Treg unbalance during the process that lung tissue allergy caused by intestinal microflora disruption; to observe the function change of DC in lung tissue when intestinal microflora disruption. The results will further clarify the effect that lung tissue allergy caused by T lymphocyte subsets (especially Th17/Treg balance) and DC when intestinal microflora disruption, which provide new ideas and theories for the prevention and control of asthma and other allergic diseases. Part one Pulmonary allergic responses were drived by atomization with ovalbumin in BALB/c mice with intestinal microflora disruptionObjective The mice in which the intestinal microflora disruption resulted from antibiotic therapy were challenged by atomization with ovalbumin (OVA), to investigate the relation of allergic airway response and intestinal microflora disruption, so that to provide a good animal model for the further experiment.Methods A total of one hundred and twelve female BALB/c mice were devided at random into 6 groups. They were microbiota disruptionⅠgroup, controlⅠgroup, microbiota disruptionⅡgroup, microbiota disruption and OVA challenge group, OVA challenge group and controlⅡgroup. Cecal contents were collected for quantitative analysis of the intestinal microflora in mice in the former two groups and in mice in the latter four groups on day 6 and day 14, respectively. On day 14 in the latter four groups, the bronchoalveolar lavage fluid (BALF) was collected for cells counting, parts of lung tissue were collected for histopathology and detection of Th1 and Th2 cell percent by flow cytometry, and OVA-specific IgE level in BALF and sera was detected by ELISA assay.Results Compared with controlⅠgroup, the mice drinking antibiotics freely were suffered from intestinal microbiota disruption. On day 6, there were no obvious intestinal bacterial colony growth. On day 14, it has not returned to normal intestinal microflora. In microbiota disruption and OVA challenge group, eosinophil and lymphocyte infiltration was significant and mucus secretion was increased in lung. The total cells number, eosinophils, lymphocytes, neutrophils and OVA-specific IgE level were increased in BALF in microbiota disruption and OVA challenge group. Th2 cell percent were increased and Th1 cell percent were not significantly different in microbiota disruption and OVA challenge group compared with those in the controlⅡgroup, but the Th1/Th2 ratio decreased.Conclusions The allergic (Th2) immune response can be induced by atomization with ovalbumin in the mice in which the intestinal microflora disruption is resulted from antibiotic therapy. The result suggests that the intestinal microflora disruption may be a risk factor for asthma and other allergic diseases.Part two Effects of ovalbumin airway challenge on the T lymphocyte subsets of BALB/c mice with intestinal microflora disruptionObjective To observe the effects of ovalbumin airway challenge on the T lymphocyte subsets (Th1/Th2/Th17/Treg) in lung and spleen of BALB/c mice with intestinal microflora disruption resulted from antibiotic therapy. To further explore the mechanism of intestinal microflora disruption induced pulmonary allergic response in T cell levels.Methods A total of ninety six female BALB/c mice were devided at random into 4 groups with twenty four mice in each group. They were microbiota disruption group, microbiota disruption and OVA challenge group, OVA challenge group and control group. On day 14, ELISA assay was adopted for cytokines IFN-γ, IL-4, IL-17, IL-10 levels detection in BALF, RT-PCR were adopted for the detection of lung tissue specific transcription factor mRNA expression levels of RORγt and Foxp3, and the levels of Th1, Th2, Th17 and CD4+ CD25+ Foxp3+ regulatory T cell (Treg) in lung and spleen were detected by flow cytometry.Results IL-4 and IL-17 levels in BALF increased in the microbiota disruption and OVA challenge group, while IFN-γand IL-10 levels showed no difference among the groups. Mice lung tissue RORγt mRNA expression increased but Foxp3 mRNA expression decreased in microbiota disruption group, and microbiota disruption and OVA challenge group. In microbiota disruption and OVA challenge group, Th2 and Th17 cell levels in lung were increased and no difference was found of Th1 cell levels. However, Th1, Th2 as well as Th17 cell levels in spleen showed no difference among the groups. Furthermore, the levels of Treg were both decreased in lung and spleen of the mice in microbiota disruption group as well as microbiota disruption and OVA challenge group.Conclusions The allergic airway response driven by Th2 and Th17 cells polarization can be induced by airway challenge with ovalbumin in mice with intestinal microflora disruption resulted from antibiotic therapy, and the lung immune state potentially changed in mRNA level. It's maybe associated with decreased production quantity and insufficient function of Treg cells.Part three Effects of functional change of dendritic cells on the lung allergic responses in BALB/c mice with intestinal microflora disruptionObjective To observe the effect of intestinal microflora disruption caused by antibiotics to the dendritic cell (DC) maturation and toll-like receptor 2, 4 in mouse lung tissue, in order to explore the mechanism of lung allergic reactions caused by OVA airway stimulation in antigen presenting cell levels.Methods A total of sixty four female BALB/c mice were devided at random into 4 groups. They were microbiota disruption group, microbiota disruption and OVA challenge group, OVA challenge group, and control group, with each group sixteen mice. On day 14, flow cytometry was adopted to measure the DC subtypes and maturation (MHC-Ⅱmolecules), and its surface expression of toll-like receptor 2, 4. Immunohistochemical method was adopted to detect the lung tissue TGF-β1, IL-6 protein expression in situ, and to detect the TGF-β1, IL-6 production level in BALF by ELISA assay.Results Compared with control group and OVA challenge group, DC surface MHC-Ⅱmolecules and TLR2, 4 expression of mice lung tissue were increased in both microbiota disruption group, and microbiota disruption and OVA challenge group, but there was no statistical significance in the latter two groups. Compared with the other 3 groups, mice lung tissue TGF-β1, IL-6 protein expression in situ were increased in microbiota disruption and OVA challenge group, and TGF-β1, IL-6 levels also increased simultaneously in BALF.Conclusions Disruption of intestinal microflora caused the increasing of lung DC maturity and the antigen presentation capacity. When the airway were stimulated by allergen OVA, the mature DC can regulate Th0 cells differentiate to Th17 cells, which may cause lung allergic reactions.