Expression Analysis Of MrgprX2(B2) In The Skin Lesion Of Atopic Dermatitis And Dock5 In The Regulation Of BCR Signaling

PART Ⅰ EXPRESSION OF NON-IGE ACTIVATION RECEPTOR MRGPRX2(B2)ON MAST CELLS IN THE SKIN LESION OF AD PATIENTS AND MOUSE MODELSObjective:To observe the expression difference of MrgprX2(B2)in skin lesions between intrinsic or extrinsic atopic dermatitis and healthy controls.Methods: The skin from patients with atopic dermatitis,AD mouse models and controls were collected.The pathological morphology and mast cells infiltration were observed by HE staining and TB staining.The expression of FcεRI and MrgprX2 on mast cells was detected by immunofluorescence technique.RNA was extracted from skin tissues and MrgprX2(B2)mRNA was examined at the transcriptional level.Skin suspension was obtained by using enzymatic digestion,and the expression of MrgprX2 on mast cells was detected by flow cytometry.ELISA was used to detect the serum total-IgE and OVA-sIgE in the mouse models.Real time PCR to detect the expression level of IL-4,IL-17 A,IFN-γ,TSLP and IL-33 in the skin lesions.Results: H&E staining of skin from patients with atopic dermatitis showed obvious epidermal hyperplasia than normal controls(P<0.001),focal keratosis or hyperkeratosis,partial sponge-like edema of the epidermis,and a large number of lymphocytes and histiocytes around the dermal papilla and superficial perivascular vessels.The number of infiltrated mast cells stained as blue-violet was also significantly increased(P<0.001).The number of mast cells and the expression of surface FcεRI in intrinsic atopic dermatitis did not have much difference compared with extrinsic atopic dermatitis,but both significantly higher than that in normal controls(P<0.001,P <0.001);MrgprX2 was expressed on the mast cell membrane,and its expression in patients with intrinsic atopic dermatitis was not significantly different from patients with extrinsic atopic dermatitis,but both the number and percentage of MrgprX2-positive mast cells were much higher than normal children(P<0.001,P <0.05);The expression levels of MrgprX2 at the RNA level and protein level were not significantly different between intrinsic and extrinsic atopic dermatitis,but both higher than that of normal children(P<0.01,P<0.05).In the mouse models induced with OVA,the total-IgE and OVA-sIgE in the OVA-sensitized mice group were higher than those in the control group(P<0.005 and P<0.01);the epidermal inflammation was found in the OVA-treated group,and the number of infiltrated mast cells was higher than that of the control group(P<0.01);there was no significant difference in IFN-γ levels between the two groups at the RNA level(P>0.05),but the RNA levels of IL-4,IL-17 A,TSLP,IL-33 and MrgprB2 were higher than those of the control group(P<0.05,P<0.05,P<0.05,P<0.01 and P<0.01).Conclusion: Expression of MrgprX2(B2)on mast cells in skin lesions of atopic dermatitis is increased compared to normal controls,but no obvious difference in expression of MrgprX2 was observed between the intrinsic and extrinsic atopic dermatitis.PART Ⅱ DOCK5 CONTROLS THE PERIPHERAL B CELL DIFFERENTIATION VIA REGULATING BCR SIGNALING AND ACTIN REORGANIZATIONObjective: To study the role of Dock5 on B cell development,differentiation and BCR signaling.Methods: We generated a Dock5 knockout mouse model.The bone marrow and PBMC from spleen of the mice were extracted.The development of B cells in the bone marrow and periphery was detected by flow detection;then spleen B cells and protein were extracted,Western Blot was used to detect the positive upstream BCR signaling molecules;TIRFm was used to detect the changes of positive upstream BCR signaling molecules and cellular fibronectin(F-actin)on the B cell membrane.Results: The Dock5 knockout mouse models were established effectively.We found that the absence of Dock5 leads to a moderate effect on bone marrow B cell development,but reduces follicular(FO)and marginal zone(MZ)B cells.Mechanistically,the key positive upstream B-cell receptor(BCR)signaling molecules,CD19 and Brutons’ s tyrosine kinase(Btk),whose activation determines the fate of FO and MZ B cells,is reduced in Dock5 KO B cells upon antigenic stimulation by using total internal reflection fluorescence microscopy(TIRFm)and immunoblot.Interestingly we found that the cellular filamentous actin(F-actin)is also decreased in Dock5 KO B cells upon stimulation,which,in turn,offers feedback to BCR signaling.Conclusion: Dock5 regulates the peripheral B cell differentiation via controlling the CD19-Btk signaling axis as well as actin reorganization.