Study On The Formation Function Of Neutrophil Extracellular Traps In Chronic Granulomatous Disease And Clinical And Molecular Features Of Partial Deficiency Of Interferon-? Receptor 1 Disease

PART ? THE EFFECT OF RECOMBINANT HUMAN INTERFERON-? ON THE FORMATION OF THE NEUTROPHIL EXTRACELLULAR TRAPS IN CHRONIC GRANULOMATOUS DISEASEObjective:We explored the formation function of neutrophil extracellular traps(NETs)in chronic granulomatous disease(CGD)and the effect of recombinant human interferon-?(rhIFN-?)on ROS and NETs in 16 children with CGD.Methods: Sixteen patients diagnosed with CGD were enrolled into this study.The clinical manifestations,laboratory results and gene analysis of these patients were summarized.The production of intracellular(ic)and extracellular(ec)ROS of PMA,Tamoxifen(Tam)or ionomycin stimulation of neutrophils treated by rhIFN-? was detected by luminol/isoluminol chemiluminescence assay.The NETs production of PMA or Tamoxifen stimulation of neutrophils after rhIFN-? treatment was detected by enzyme-labeled instrument.The morphology of NETs of PMA or Tamoxifen stimulation of neutrophils after rhIFN-? treatment was observed under confocal laser scanning microscope.Results:1.Clinical features: The mean onset age of 16 male patients was 3 months old,and the mean diagnostic age was 4 years and 1 months old.The main clinical manifestations of these patients including recurrent fever(100%),pneumonia(100%),lymph node enlargement(75%),BCG disease(62.5%),diarrhea(43.75%),abscess(43.75%)and enlargement of liver and spleen(25%).The NBT after stimulation with LPS range from 0% to 1%.The range of SI(0.99±0.34)of the function of neutrophil respiratory burst was 0.49~1.68,and all patients were X-linked-CGD with CYBB gene mutation.2.ROS production of neutrophils after rhIFN-? treatment: The lack of ROS production of neutrophils with or without rhIFN-? treatment stimulated by PMA,Tam or ionomycin in CGD patients were found compared to healthy controls(HC).The ic ROS production of neutrophils with rhIFN-? treatment(3567.75±1486.6)stimulated by ionomycin in HC was significantly increased compared to the untreated group(2627.75±996.43)(P<0.05).However,the ROS production of the groups with rhIFN-? treatment(ic:69.31±28.44,ec:103.5±61.4)and without rhIFN-? treatment(ic:71.81±31.21,ec:85.19±28.08)stimulated by Tam were significantly lower than the rhIFN-? treated group(ic:399.38±138.79,ec:460.54±267.41)and untreated group(ic:297.44±118.39,ec:399.65±238.97)(P<0.0001)without Tam stimulation in HC.3.NETs production of neutrophils after rhIFN-? treatment: The lack of NETs production of neutrophils with or without rhIFN-? treatment stimulated by PMA in CGD patients were found compared to HC,and the NETs production of neutrophils with rhIFN-? treatment(1198.35±342.5)stimulated by PMA in HC was significantly increased compared to the untreated group(954.85±301.86)(P<0.05).However,the NETs production of group with rhIFN-? treatment(2120.04±703.18)stimulated by Tam in CGD patients was significantly increased compared to the untreated group(1615.82±526.28)(P<0.05),and the NETs production of group with rhIFN-? treatment(2203±607.09)compared with the untreated group(1827.02±453.34)in HC was also significantly increased(P<0.05).In addition,there was no significant difference in the NETs production of group with/without rhIFN-? treatment between patients and HC(P values were 0.331 and 0.771,respectively).The lack of NETs was found in CGD in the group treated with rhIFN-? alone(139.11±78.21)compared with the rhIFN-? plus Tam group(2120.04±703.18)(P<0.0001).4.The morphology of NETs of PMN after rhIFN-? treatment under the laser confocal scanning microscope: After PMA stimulation for 3h,lack of NETs production in CGD were found,and the group of rhIFN-? treatment was significantly increased compared to untreated group in HC.After Tam stimulation for 3h,the NETs production of groups with rhIFN-? treatment in CGD and HC were significantly increased compared with that in the untreated groups,which was consistent with the results of NETs by Sytox Green staining detection.Conclusion: Children with CGD lack the function of NETs formation.rhIFN-? treatment of neutrophils in children with CGD in vitro does not promote ROS production,nor does it promote the production of ROS-dependent NETs,but it can enhance the production of ROS-independent formation of NETs stimulated by Tam,which may be a possible mechanism for rhIFN-? to reduce the incidence of recurrent infections in CGD.This also provides a new idea for the clinical use of rhIFN-? and Tam in the prevention and treatment of infections in CGD.PART ? THE CLINICAL AND MOLECULAR CHARACTERISTICS OF 2 CHILDREN WITH VARIANT X-LINKED CHRONIC GRANULOMATOUS DISEASEObjective: To investigate the clinical characteristics,function of respiratory burst of neutrophils,expression level of gp91 phox and gene mutations of 2 children with variant X-linked chronic granulomatous disease(XL-CGD).Methods: This study collected and summarized the clinical data and laboratory results of 2 children(P17 and P18)with variant XL-CGD.Flow cytometry was used to detect the respiratory burst function of neutrophils,and the neutrophil stimulation index was calculated.Luminol/isoluminol chemiluminescence assay was used to detect the ic and ec ROS production of the neutrophils stimulated by PMA,FMLF,WKYMVM or ionomycin.Flow cytometry was used to detect the gp91 phox expression on PMN.Sanger sequencing was used to analyze the DNA of CYBB gene.TA-cloning and sequencing was used to analyze the c DNA of CYBB gene.Results: 1.Clinical features: The onset age of P17 and P18 were eight years old and 4 years and 3 months old,respectively.The diagnostic age of P17 and P18 were 8 years and 4months old and 4 years and 7 months old,respectively.Both were significantly later than classical XL-CGD.P17 suffered lymph node enlargement,lobar pneumonia and anal abscess.The main performance of P18 included fever,interstitial pneumonia and lymph node enlargement.Burkholderia cepacian and Mycoplasma pneumoniae infection were found in P17.Only mycoplasma pneumoniae infection was found in P18.2.Neutrophil respiratory burst: SI of P17(2.52)and P18(3)were both significantly lower than that of HC(100.35±43.72),but were slightly higher than the classic XL-CGD(0.99±0.34).3.Detection of luminol/isoluminol chemiluminescence assay of neutrophil ROS production: The ec ROS production of PMN stimulated by PMA,FMLP or WKYMVM in P17 and P18 were significantly lower than that of HC,but higher than that of classic XL-CGD.However,the production of ic ROS of PMN in P17 nd P18 stimulated by PMA or ionomycin were significantly lower than that of HC,but were similar to classic XL-CGD.4.Expression level of gp91 phox protein of PMN in peripheral blood: Expression levels of gp91 phox protein of P17(11.9%)and P18(3.14%)were significantly lower than those of HC(87.82%±6.9%),but were both higher than that of classic XL-CGD(0%).5.CYBB gene Sanger sequencing: A splicing mutation c.483+5G>A in the exon 5 of P17 and a synonymous mutation c.252G>A(p.A84A)in the exon 3 of P18 were found.TA clone sequencing: Exon 5 and exon 3 were deleted in P17 and P18,respectively.Conclusion: Variant XL-CGD(X91)have a late onset and milder infections compared to classic XL-CGD,and residual ROS production can be detected in their neutrophils.It's difficult to diagnose the variant XL-CGD at its early course.The production of ic and ec ROS,expression of gp91 phox of neutrophils should be detected and gene mutation should be analyzed to confirm it.PART ? CLINICAL AND MOLECULAR FEATURES OF A CHILD WITH PARTIAL INTERFERON-? RECEPTOR 1 DEFICIENCYObjective: To explore the clinical and molecular features in a child with partial deficiency of IFNGR1.Methods: The clinical manifestations of patients with partial IFNGR1 gene defects were reviewed.We detected the function of the IL-12-IFN-? axis by Q-RT-PCR,measured the expression of IFNGR1 protein by flow cytometry and analyzed the IFNGR1 gene mutation by Sanger sequencing.Results: There were no abnormalities for routine immune functions in the patient.The production of IL-12 B of the patient in response to BCG plus rh IFN-? was slightly increased compared to stimulation with BCG alone(P>0.05),but was significantly decreased than healthy control(P<0.05).The expression of IFNGR1 protein on the surface of T cells was higher than that of healthy control.A heterozygous deletion mutation c.819-822 del4(p.N274Hfs*2)in the exon 6 of IFNGR1 gene was found and her parents were normal.Conclusion: Partial defects of autosomal recessive IFNGR1 gene were extremely rare and prone to suffer disseminated mycobacterium infections.The function of IL-12-IFN-?-axis,the expression of related proteins and gene analysis should be detected to confirm the diagnosis.