The Impact Of Coactosin-like Protein On Allergic Asthma And Mechanism Research

In this paper,the model of allergic asthma induced by house dust mite?HDM?was used to explore the regulatory effects of coactosin-like protein?CLP?on allergic asthma,and the possible mechanism was discussed,which provided a theoretical basis for the target screening and drug development.Male C57BL/6J and CLP knockout(Cotl1^-/-)mice aged about 6-8 weeks were randomly divided into control group?n=6?and asthma model group?n=6?.Mice were sensitized by intranasal injection of 50?L phosphate buffer solution?PBS?or 50?L HDM?1?g HDM/?L?on days 1,3 and 5,respectively,and the same dose of PBS or HDM was injected on days 10,12,14,16,18,20 and 22.On the 23rd day,mice were injected with lethal dose of pentobarbital,and bronchoalveolar lavage?BALF?and lung tissue were collected.The immune cells in BALF were stained by Diff-Quik staining,the level of Th2 cytokines were determined by reverse transcription polymerase chain reaction,the pathological sections of lung tissue were observed by hematoxylin-eosin and periodic acid-schiff staining,the level of leukotrienes?LTs?and prostaglandins?PGs?in BALF was determined by liquid chromatography–mass spectrometry,and the expression of eicosanoids-related protein in lung tissue was detected by western blotting.The subtypes of macrophages and the proportion of Th2 cells,regulatory T cells and eosinophils in lung tissue were detected by flow cytometry,and the co-localization of CLP and hematopoietic prostaglandin D synthase?HPGDS?was detected by immunofluorescence.In addition,bone marrow chimera and alveolar macrophage?AM?chimera mice were constructed to explore the effect of Cotl1 gene expression in different cells on the pathogenesis of asthma.The results showed that CLP knockout could significantly aggravated allergic asthma in mice.In the HDM-induced asthma model,the total cell count,eosinophils,neutrophils and lymphocytes in BALF of Cotl1^-/-mice were significantly increased,Th2 type inflammatory mediators,interleukin-4?IL-4?,IL-5 and IL-13 were significantly up-regulated,bronchoconstriction and mucin secretion were significantly surged.At the same time,the expression of PGD₂ in BALF of Cotl1^-/-mice was significantly increased,and the expression of HPGDS and chemoattractant receptor-homologous molecule expressed on TH2 cells?CRTH2?was also significantly up-regulated.Through further screening,it was determined that the main source of PGD₂ was AM.High expression of HPGDS was also found in the isolated AM.By collecting lung tissue at different time points,it was found that there was a significant difference in the polarization of AM after Cotl1 knockout.AM was more inclined to pro-inflammatory M1 subtype,while anti-inflammatory M2c subtype decreased significantly.Through the construction of AM chimeric mice,it was confirmed that Cotl1^-/-AM could secrete more PGD₂,promoted the infiltration of Th2 cells and eosinophils through CRTH2,and ultimately promoted the deterioration of asthma.CRTH2 inhibitor OC000459 could alleviate adverse effects caused by Cotl1^-/-AM,inhibit the occurrence of inflammation.This study confirmed for the first time the effect of knockout of CLP on allergic asthma in mice,which is related to the regulation of macrophage polarization and PGD₂ synthesis,and the promotion of downstream inflammatory response through CRTH2.Although CLP can be used as an important target to inhibit the synthesis of leukotriene,its regulatory effect on AM polarization makes it unsuitable as a target for the treatment of allergic asthma.