Biomarkers Of Omalizumab For Treatment Of Severe Asthma And Its Effect On Immune Cells

OBJECTIVETo discover the biomarkers of the efficacy of omalizumab in treating severe asthma patients and its impact on immune system cells and provide a basis for the individualized precision treatment of omalizumab in severe asthma patients.METHODSThe first part used bioinformatics methods such as xCell evaluation of immune cell infiltration,weighted gene co-expression network analysis,GO enrichment analysis,KEGG pathway analysis,PPI network analysis,and logistic regression methods to analyze the transcriptome microarray data and clinical information in the GSE134544 dataset.We were looking for biomarkers that can predict omalizumab treatment response.In order to find predicting biomarkers and clinical models of omalizumab responsiveness,the second part used clinical statistical methods such as chi-square test,logistic regression,and subgroup analysis to retrospectively analyze the baseline characteristics and biomarker levels of patients with severe asthma who received omalizumab treatment for at least 16 weeks in China-Japan Friendship Hospital from January 2018 to December 2020.The guidelines recommend choosing omalizumab for treatment based on the clinical phenotype.The third part used density gradient centrifugation separation of peripheral blood mononuclear cells,intervention with omalizumab in vitro,flow cytometry to detect immune cells' phenotype,and protein antibody chip technology to detect the concentration of cytokines and antibodies in the cell supernatant.RESULTSBioinformatics analysis of the GSE134544 dataset shows CD3E,CD19,CD34,CD79A,CD5,MME,CR2,CD40,CD28,CD40LG,MS4A1,CXCR5,CCR7,ADORA3,CCR3,GPR55,GPR18,PNOC,C5AR1,CCR4 are hub genes in the red module.We compared the expression of 20 hub genes in the response group and non-response group by Wilcoxon test.Only CD3E and CD79A are statistically significant,with a P-value of 0.014 and 0.037,respectively.CD3E and CD79A were analyzed by univariate and multivariate logistic regression with age,sex,ACT score,FEV1%predicted value,and serum IgE.CD3E is only significant in univariate and multivariate logistic regression with P values of 0.0188 and 0.0116,respectively.The area under the ROC curve of the reactivity of CD3E to omalizumab is 0.758,which is the area's maximum value under the other curves.Then,we retrospectively analyzed the baseline clinical characteristics and baseline biomarkers of omalizumab response in 32 severe asthma patients who received omalizumab treatment in China-Japan Friendship Hospital at least 16 weeks from January 2018 to December 2020.The results show that the response rate of omalizumab to severe asthma is 71.875%.In univariate logistic regression,factors not affecting the responsiveness of omalizumab in severe asthma included age(P=0.6720),gender(P=0.6436),body mass index(P=0.6029),FeNO(P=0.2700),percentage of induced sputum eosinophils(P=0.8347),total serum IgE(P=0.7438),the absolute number of blood eosinophils(P=0.2166),percentage of blood eosinophils(P=0.1588),the absolute number of blood basophils(P=0.3822),percentage of blood basophils(P=0.2111),FEV1(P=0.3813),FEV1%predicted(P=0.5950),whether bronchial thermoforming surgery(P=0.2061),whether oral corticosteroids(P=0.7610),Whether there is allergic rhinitis(P=0.1521),whether there are nasal polyps(P=0.8335),whether there is sinusitis(P=0.4989),whether there is gastroesophageal reflux(P=0.2061)and whether there is dermatitis(P=0.9952).In subgroup analysis,the difference in the above indicators is still not statistically significant with P values greater than 0.05.Finally,we isolated PBMC cells from the peripheral blood of 9 patients with severe asthma who met the inclusion criteria by density gradient centrifugation and treated them with 125?g/ml omalizumab for 18 hours and then added ?g/ml rabbit anti-human IgE for stimulation.After 96 hours of culture,the phenotype of immune cells was detected by flow cytometry.The results show that the frequency of naive CD4+T lymphocytes to CD4+T lymphocytes in the omalizumab group(27.49±10.59)%was higher than that in the control group(25.91±9.967)%with a P-value of 0.0292.The frequency of effector CD4+T lymphocytes to CD4+T lymphocytes was lower in the omalizumab group(23.65±7.89)%than the control group(25.04±8.38)%,and the P-value was 0.0104.The frequency of activated CD8+T lymphocytes to CD8+T lymphocytes in the omalizumab group was 6.11%[1.96%-8.02%]higher than the control group with 3.03%[1.69%-5.03%],P=0.0391.The frequency of classical monocytes to monocytes in the omalizumab group was 99.64%[99.01%-99.84%]lower than the control group 99.82%[99.62%-99.90%],while the non-classical monocytes were 0.36%[0.16%-0.99%]in the omalizumab group higher than the control group by 0.18%[0.105%-0.385%],and both P values were 0.0117.Although there is no statistical significance in the differences of the frequency of plasmacytoid dendritic cells(P=0.2351)and myeloid dendritic cells(P=0.0977)to dendritic cells between the two groups with both P values greater than 0.05,each indicator of the omalizumab group {(19.23±11.10)%,17.5%[7.27%-37.72%]} was higher than that of the control group {(16.0016.65)%,12.93%[7.80%-22.81%]}.As for other cells,such as helper T lymphocytes,regulatory T lymphocytes,B lymphocytes,and NK cells,no statistically significant differences were noted between the omalizumab group and control group,where all P-values are greater than 0.05.The protein antibody chip detected the secretion of cytokines and antibodies in the cell supernatant,with results showing that only the concentration of IL-2 in the omalizumab group(53.23±36.24)pg/ml is lower than that of the control group(70.28±49.93)pg/ml,the P-value is 0.0273.The remaining cytokines and antibodies are not statistically significant between the two groups.CONCLUSIONS1.CD3E can be used as an independent predictive biomarker of omalizumab treatment response in patients with moderate to severe asthma.2.Response to omalizumab treatment in patients with severe asthma was independent of baseline biomarkers,and baseline clinical characteristics including peripheral blood eosinophils,peripheral blood basophils,total serum IgE,percentage of induced sputum eosinophils,FeNO,FEV1%predicted,age,sex,body mass index,whether undergoing a bronchial thermoforming surgery,whether using oral corticosteroids and whether comorbidities are containing allergic rhinitis,sinusitis,nasal polyps,gastroesophageal reflux,and dermatitis.3.In Vitro,omalizumab can increase the frequency of naive CD4+T lymphocytes and that of activated CD8+T lymphocytes and non-classical mononuclear cells and decrease the frequency of effective CD4+T lymphocytes as well as that of classical mononuclear cells decreased.4.The secretion of IL-2 was decreased by omalizumab,but the secretion of residual cytokines was not affected by omalizumab.5.Omalizumab does not affect the frequency of CD3+T cells,CD4+T cells,CD8+T cells,B cells,and NK cells in vitro as well as that of helper T cells,including Th1,Th2,Th9,Th17,Th22,and TFH,and regulatory T cells in CD4+T cells.Omalizumab has no effect on dendritic cells and the ratio of plasma dendritic cells and myeloid dendritic cells.It does not affect the secretion of antibodies.