Relationship Between Th17Cells And Neutrophilic Airway Inflammtion In Childhood Neutrophilic Asthma

Bronchial asthma (Asthma) is one of the most common chronic airwaydiseases worldwide. Airway inflammation plays a key role in the pathogenesis ofasthma. Many cells (such as eosinophils, mast cells, T lymphocytes, neutrophils,and airway epithelial cells, etc.) and cellular elements are involved in thepathogenesis of airway inflammation in asthma.Airway inflammation witheosinophils is accepted as a fundamental characteristic of allergic asthma.However, current studies show there are different subtypes in the airwayinflammation of asthma. According to phenotype of airway inflammation,asthma can be classified into different subtypes: eosinophilic asthma (EA) andnon-eosinophilic asthma（NEA）, and more than half of non-eosinophilic asthmaare characterized by neutrophilic asthma. But the pathogenetic mechanisms ofneutrophilic asthma are not well understood now.T cells are critical to thepathogenesis of asthma. Typically, it is known that Th2cells can promoteeosinophilic airway inflammation in atopic asthma,but recent studies suggestthere are differences in the immunopathology between atopic asthma andnonatopic asthma.Recently, a new T helper cell subset is identified, namely, Th17cells, which characterized by the secretion of IL-17and the expression ofretinoic acid-related orphan receptor γt (RORγt), can promote neutrophilicinflammation.Some studies show that Th17cells and IL-17also seem to beinvolved in asthma pathogenesis.What is the role of Th17cells in neutrophilicasthma? Our previous animal studies have shown that both Th17cells and Th2cells are involved in the pathogenesis of neutrophilic asthma and eosinophilicasthma in mice. Th17cells and IL-17may mediate neutrophilic airwayinflammation in the mouse model of neutrophilic asthma by up-regulatingRORγt expression. And Th17cells may be maintain their survival throughactivation of the JAK/STAT5signaling pathway. However, whether Th17-drivenimmunity contributes to the pathogenesis of neutrophilic asthma in human isunclear,and remains to be investigated.The efficacy of inhaled glucocorticosteroids in reducing airwayinflammation and the symptoms of asthma has led to their widespread use asinitial therapy in the treatment of asthma. Although inhaled glucocorticosteroidsare effective for most of asthmatic patients, it is uncertain whether their use canreduce neutrophilic airway inflammation in some patients with neutrophilicasthma.Recent studies suggest that Th17cell-mediated airway inflammation issteroid resistant. On the contrary,some researches show that glucocorticosteroidscan decrease the level of IL-17in asthmatic patients.What is the effect ofglucocorticosteroid on Th17cell-mediated neutrophilic airway inflammation inneutrophilic asthma? Our previous animal studies have shown thatDexamethasone can reduce Th17cell-mediated neutrophilic airwayinflammation in the mouse model of neutrophilic asthma by decreasingpercentage of Th17cells, level of IL-17and down-regulating RORγt expression.And Dexamethasone has no significant effect on apoptosis of airway neutrophils and survival of Th17cells.But the effect of glucocorticosteroid on Th17cell-mediated neutrophilic airway inflammation in patients with neutrophilic asthmaremains to be investigated. To better understand the role of Th17cells inasthma may improve the understanding of the pathogenesis of asthma.In thisstudy,we will explore the relationship between Th17cells and neutrophilicairway inflammation in childhood neutrophilic asthma, and identify the possiblerole of Th17cells in pathogenesis of neutrophilic asthma in children. Our studywas divided into two parts.Part oneRelationship between Th17cells and neutrophilic airwayinflammation in childhood neutrophilic asthmaObjective To explore relationship between Th17cells and neutrophilicairway inflammation in childhood neutrophilic asthma.Methods Twenty-eight children with exacerbated asthmatics withoutusing any corticosteroids were divided into three groups: eosinophilic asthmagroup (EA group)(n=12), neutrophilic asthma group (NA group)(n=10) andnon-eosinophilic asthma and non-neutrophilic asthma (NEA+NNA group)(n=6) according to induced sputum cytology.Ten healthy children were recruited ashealthy control group (HC group)(n=10). Th17cells,Th2cells in peripheralblood and Ki-67,STAT5, BCL-2expressed in Th17cells were detected by flowcytometry. Expression of RORγt-mRNA in PBMC was detected by Real-timePCR.Concentrations of IL-17, IL-5and IL-8in induced sputum supernatant, aswell as concentrations of IL-17in plasma and in the culture supernatant fromPMA-stimulated PBMC were measured by ELISA Results1.The percentage of Th17cells in peripheral blood wassignificantly higher in NA group than in EA group, NEA+NNA group and HCgroup (both P<0.01), and was significantly higher in EA group and NEA+NNAgroup than in HC group (both P<0.01), but there was no significant differencebetween EA group and NEA+NNA group (P>0.05).The percentage of Th2cellsin peripheral blood was significantly higher in EA group than in NA group,NEA+NNA group and HC group (both P<0.01), and was significantly higher inNA group and NEA+NNA group than in HC group (both P<0.01), but there wasno significant difference between NA group and NEA+NNA group (P>0.05).2.The expression level of Ki-67in Th17cells was significantly higher in NAgroup than in EA group, NEA+NNA group and HC group (both P<0.01). andwas significantly higher in EA group and NEA+NNA group than in HC group(both P<0.01), but there was no significant difference between EA group andNEA+NNA group (P>0.05). The expression level of STAT5in Th17cells wassignificantly higher in NA group than in EA group, NEA+NNA group and HCgroup (both P<0.01). and was significantly higher in EA group and NEA+NNAgroup than in HC group (both P<0.01), but there was no significant differencebetween EA group and NEA+NNA group (P>0.05). The expression level ofBCL-2in Th17cells was significantly higher in NA group than in EA group,NEA+NNA group and HC group (both P<0.01),and there was no significantdifference among EA group,NEA+NNA group and HC group (P>0.05).3. Theexpression level of RORγt-mRNA in PBMC was significantly higher in NAgroup than in EA group, NEA+NNA group and HC group (both P<0.01), andwas significantly higher in EA group and NEA+NNA group than in HC group(both P<0.01), but there was no significant difference between EA group andNEA+NNA group (P>0.05).4. The level of IL-17in sputum supernatant was significantly higher in NA group than in EA group, NEA+NNA group and HCgroup (both P<0.01),and was significantly higher in EA group and NEA+NNAgroup than in HC group (both P<0.01), but there was no significant differencebetween EA group and NEA+NNA group (P>0.05). The level of IL-5in sputumsupernatant was significantly higher in EA group than in NA group, NEA+NNAgroup and HC group (both P<0.01), and was significantly higher in NA groupand NEA+NNA group than in HC group (both P<0.01), but there was nosignificant difference between NA group and NEA+NNA group (P>0.05). Thelevel of IL-8in sputum supernatant was significantly higher in NA group than inEA group, NEA+NNA group and HC group (bothP<0.01), and was significantlyhigher in EA group and NEA+NNA group than in HC group (both P<0.01), butthere was no significant difference between EA group and NEA+NNA group(P>0.05).5.The level of IL-17in plasma was no significant difference amongNA group,EA group,NEA+NNA group and HC group (P>0.05). The level ofIL-17in the culture supernatant from PMA-stimulated PBMC was significantlyhigher in NA group than in EA group, NEA+NNA group and HC group (bothP<0.01), and was significantly higher in EA group and NEA+NNA group than inHC group (both P<0.01), but there was no significant difference between EAgroup and NEA+NNA group (P>0.05). The levels of IL-17in the culturesupernatant from PMA-stimulated PBMC were significantly higher than thosein plasma in NA group,EA group and NEA+NNA group (both P<0.01),but therewas no significant difference between plasma level of IL-17and PBMC culturesupernatant level of IL-17in HC group(P>0.05).6. The percentage of Th17cellsin peripheral blood, the levels of IL-17in sputum and PBMC culturesupernatant,and the level of IL-8in sputum supernatant were all correlatedpositively with the percentage of neutrophils in sputum, Both FEV1%pred and PEF%pred were correlated negatively with the percentage of neutrophils insputum. The percentage of Th2cells in peripheral blood and the level of IL-5insputum supernatant were correlated positively with the percentage ofeosinophils in sputumConclusions Both Th17cells and Th2cells are involved in thepathogenesis of asthma in children. Th17cells and IL-17may mediateneutrophilic airway inflammation in asthma，indicating an important role forTh17cells in childhood neutrophilic asthma. Th17cells may be maintain theirsurvival through activation of the JAK/STAT5signaling pathway. Part twoEffect of inhaled glucocorticosteroid on Th17cell-mediatedneutronphilic airway inflammation inchildhood neutrophilic asthmaObjective To explore the effect of inhaled glucocorticosteroid on Th17cell-mediated neutrophilic airway inflammation in childhood neutrophilicasthma.Methods Children with neutrophilic asthma from NA group (n=10) inthe part one inhaled glucocorticosteroid over three months,and then underwentlung function tests and sputum induction. Sputum was assessed differential forcell count.Th17cells in peripheral blood and Ki-67expressed in Th17cells weredetected by flow cytometry. Expression of RORγt-mRNA in PBMC wasdetected by Real-time PCR. Concentrations of IL-17in induced sputum supernatant and in the culture supernatant from PMA-stimulated PBMC weremeasured by ELISA.Results1. FEV1%pred and PEF%pred were significantly higer aftertreatment than those before treatment in neutrophilic asthma(both p <0.01),butwere lower than those of HC group (p<0.05, p<0.01, respectively).2. Thepercentage of neutrophil in induced sputum was significantly lower aftertreatment than that before treatment in neutrophilic asthma (p<0.01), but washiger than that of HC group (p<0.05).3. The percentage of Th17cellsin peripheral blood was significantly lower after treatment than that beforetreatment in neutrophilic asthma (p<0.01), but was higer than that of HC group(p <0.01). The expression level of Ki-67in Th17cells was significantly lowerafter treatment than that before treatment in neutrophilic asthma (p<0.01), butwas higer than that of HC group (p<0.01).4. The expression level ofRORγt-mRNA in PBMC was significantly lower after treatment than thatbefore treatment in neutrophilic asthma (p<0.01), was no significant differentfrom that of HC group (P>0.05).5. The levels of IL-17in sputum and PBMCculture supernatant were significantly lower after treatment than those beforetreatment in neutrophilic asthma (both p<0.01), but were higer than those of HCgroup (p<0.05, p <0.01,respectively).Conclusions Inhaled glucocorticosteroid may reduce Th17cell-mediatedneutrophilic airway inflammation in childhood neutrophilic asthma bydecreasing percentage of Th17cells, sputum level of IL-17and down-regulatingRORγt expression.