Relationship Between The Expression Of GITR-GITRL And The Activity Of ILC2 Cells In Lung Tissue Of Asthmatic Mice

Objective:To detect the changes of numbers of ILC2 s and levels of GITR-GITRL in the lung tissue of asthma mice, analyze the effect of GITR-GITRL signaling on the frequencies and function of IL-5/IL-13 producing ILC2 s, and then analyze the influence of them on immune microenvironment in development process of asthma.Methods:⑴ Mice asthma model: Mice were sensitized intraperitoneally(i.p.) on day 1 and day11 with 50 μg OVA and 2 mg aluminum hydroxide gel in 0.1ml saline. Then sensitized mice were exposed to OVA(10 mg/ml in saline) inhalation challenges for 30 min every day from day 22 to day 26. Control animals received saline.⑵ Evaluation of allergic asthma: Lung tissue was obtained sterilely, then used to extract total RNA sterilely by Trizol. c DNA, reverse transcripted from RNA, was used to detect the expression of Th2 cytokines in asthmic mice lung by using q RT-PCR. Peripheral blood Ig E was detected by ELISA and histological inflammation by HE staining.⑶ The frequency of ILC2 s and GITR positive cells in lung tissue: For ILC2 s quantification, single-cell suspension was prepared and stained with Lineage antibody-FITC, ICOS(inducible costimulatory molecule)-Per CP/Cy5.5, T1/ST2-PE, then fixed in 4% paraformaldehyde for Flow cytometry. For GITR positive cells, single-cell suspension was stained with GITR-APC. Meanwhile, ILC2 s related molecules and GITR/GITRL expression was detected by q RT-PCR.⑷ The expression of murine GITRL(m GITR) fusion protein and its effect on ILC2s: m GITRL fusion protein was expressed in E.coli BL-21(Escherichia coli BL21), induced by Isopropyl β-D-1- thiogalacto- pyranoside(IPTG). Then the protein was identified by SDS-PAGE, purified by IMAC column, endotoxin removed by Endotoxin Removal Kit. The asthma mice were injected with m GITRL protein or control protein respectively, the frequency of ILC2 s was measured by Flow cytometry.⑸ Statistical analysis: The mice were assigned randomly without investigator blinding. All the experiments were replicated at least 3 times. The data was analyzed using Graph Pad software, independent-sample t test was used to campare data from 2 groups, and the Figures was displayed as means±s.e.m. P<0.05 was considered statistically significant. * means P<0.05, ** P<0.01, *** P<0.001, **** P<0.0001.Results:⑴ Murine asthma model showed obvious pathological changes in lung: excess inflammatory cells and eosinophil cells infiltration and mucus-production. They also shows increased secretion of Ig E.⑵ The expression of GITR/GITRL system in the lung tissue from asthma mice was significantly higher than that in control mice lung tissue. Also murine asthma model showed increased numbers of ILC2 s and levels of ILC2 s related molecules in lung than control mice.⑶ According to correlation analysis results, there were significant positive correlations between expression of GITR and levels of ILC2 characteristic molecules in asthma mice lung, and also between GITRL and ILC2-related transcription factor and receptors.⑷ The m GITRL protein was expressed in E.coli BL-21 induced by IPTG, and then was injected into asthma mice. The result showed m GITRL fusion protein can induce the expansion of ILC2.Conclusion:This study revealed that the expression of GITR and GITRL was obviously up-regulated in the lung of asthma mice, accompanied by expansion of ILC2 s. It suggest that the frequencies and activity of GITR-expressing ILC2 s may can be enhanced by GITR-GITRL interaction, which then contribute to pathogenesis of asthma.