Role And Mechanisms Of Bronchial Epithelial Shp2 In OVA-and LPS-Induced Expression Of IL-25 And Asthmatic Inflammation

Backgroud and ObjectivesAsthma is a T lymphocyte-controlled disease of the airway wall caused by inflammation,overproduction of mucus and airway wall remodeling,leading to bronchial hyperreactivity and airflow obstruction.Recently,the research of the airway epithelial cells in the pathogenesis was hot.Airway epithelium is considered as more than just a structural barrier.It is also considered as an essential controller in asthmatic inflammation.Epithelial cells express pattern recognition receptors(PRRs)that detect environmental stimuli and secrete epithelium-devived cytokines(EDCs),bridging innate and adaptive immunity.EDCs,including IL-25,IL-33,TSLP,promote the initiation and the survival of type 2 responses,contributing to the pathogenesis of asthma.How IL-25 is produced and the related regulatory mechanisms are still poorly understood.Allergens,airborne pathogens and helminth infection have been reported to increase IL-25 expression in lung epithelial cells.However,the downstream factors responsible for regulating IL-25 expression remain poorly characterized.Analysis of the genomic sequence upstream of the IL-25 encoding region revealed a putative signal transducer and activator of transcription 6(STAT6),GATA-3,and NF-?B binding sites.However,the functional requirement of these transcription factors in the induction of IL-25 has not been investigated.LPS(lipopolysaccharide),a kind of pathogen-associated molecular patterns(PAMPs),is a cell wall component of Gram-negative bacteria and ubiquitous in the environment and is often present in polluted air as well as in household dust.TLR4(Toll-like receptor 4),expressed by epithelium,is a receptor for LPS.Stimulation of TLR4 is essential for downstream cellular events that are mediated by mitogen-activated protein kinases(MAPK)p38 and c-Jun N-terminal kinases(JNK)leading to nuclear trans-location of NF-?B,resulting in cytokine mRNA transcription and release of proinflammatory cytokines.We hypothesized that LPS induced the secretion of IL-25 in bronchial epithelial cells dependent of MAPK.Src homology 2 domain-containing phosphatase 2(Shp2)is a member of intracellular classical protein tyrosine phosphatases(PTPs).Recently,Shp2 has been shown to play an important role in a wide variety of diseases.Of interest,Shp2 is known to be universally expressed in the lungs.It was reported that Shp2 played an important role in acute cigarette smoke-mediated IL-8 overproduction,and that loss of Shp2 in alveoli epithelia induced deregulated surfactant homeostasis,resulting in spontaneous pulmonary fibrosis.Our previous work revealed that Shp2 regulated TGF-?1 production in airway epithelia and asthmatic airway remodeling in mice-However,whether Shp2 regulates other essential pro-inflammatory cytokines,such as IL-25,in asthmatic bronchial epithelial cells still needs further exploration.Our research aimed to explore the role and the mechanism of Shp2 in ovalbumin(OVA)-and LPS-mediated IL-25 production.Firstly,we determined whether IL-25 expression was increased in OVA-induced asthmatic mice,and whether OVA and LPS induced the production of IL-25 in bronchial epithelial cells.Secondly,we characterized the role of Shp2 in regulating IL-25 production in bronchial epithelial cells.Finally,we discovered the role of bronchial epithelial Shp2 in OVA-induced asthmatic inflammation.Materials and MethodsIn vitro,IL-25 mRNA,IL-25 protein concentration,the level of P-p38 and P-JNK were detected in OVA-or LPS-treated Beas-2Bs and mouse tracheal epithelial cells(MTECs).To confirm that the secretion of IL-25 depended on p38,JNK and Shp2,we used p38 inhibitor SB202190,JNK inhibitor SP600125,Shp2 inhibitor PHPS-1 and Shp2 siRNA.In vivo the mRNA levels of IL-25 and IL-33 were measured in the lungs of C57BL/6 mice which were administrated with OVA to induce allergic asthma.Finally,we established triple transgenic mice CC10-rtTA/(tetO)7-Cre/Shp2flox/flox,in which Shp2 was deleted specifically in bronchial epithelial cells when exposed to DOX,trying to deeply confirm the role of bronchial epithelial Shp2 in OVA-induced asthmatic mice.Compared to mice without DOX,we observed the expression of IL-25 in the lungs of transgenic mice and IL-25 priming events,including the expression of type 2 cytokines,the ratio of TH subsets,and infiltration of eosinophils in BALF.ResultsIn vitro,different dose of OVA(2 mg/ml and 20 mg/ml),and LPS(100 ng/ml)induced the production of IL-25 in Beas-2Bs(P<0.05).Different dose of OVA(2 mg/ml and 20 mg/ml),and LPS(100 ng/ml)activated p38 and JNK,but not extracellular regulated protein kinases(ERK)in Beas-2Bs.When we pretreated Beas-2Bs with SB202190 or/and SP600125,LPS-induced IL-25 production was significantly decreased.To explore the regulatory role of Shp2 in the secretion of IL-25,Shp2 inhibitor PHPS-1 and Shp2 siRNA were employed,both of which inhibited the secretion of IL-25 and selectively inhibited the activation of JNK stimulated by LPS in bronchial epithelial cells.In vivo,we demonstrated that IL-25 was significantly increased in OVA-adsministrated C57BL/6 mice,and this phenomenon lasted at least from 8h(P<0.001)to 24h(P<0.05)after the last challenege.However,in OVA-treated CC10-rtTA/(tetO)7-Cre/Shp2flox/flox mice with(Shp2?/?)or without DOX(Shp2F/F),we discovered that the expression of IL-25 mRNA in Shp2?/? mice was not significantly changed to that in Shp2F/F(P>0.05)and the IL-25 priming events,including the expression of type 2 cytokines(P>0.05),the ratio of TH subsets(P>0.05),infiltration of eosinophils in BLAF(P>0.05)were also slightly influenced,compared to Shp2F/F mice.ConclusionsThe presence of LPS in OVA induced the production of IL-25.IL-25 induced by OVA and LPS depended on activation of p38 and JNK.Shp2 positively regulated LPS-induced IL-25 expression,selectively via activation of JNK.However,in vivo,deficiency of Shp2 in bronchial epithelium had slightly effects on OVA-induced asthmatic inflammation.