Roles And Mechanisms Of Rheb1 In Macrophages In OVA-induced Allergic Asthma In Mice

Background and aimsAsthma is a chronic inflammatory lung disease that is characterized by airway hyper-responsiveness to allergens,airway remodeling,and increased mucus secretion.T-helper type 2(Th2)cells are dominant in the airway reactions and Th2 cytokines such as IL-4,IL-5 and IL-13 play a pivotal role in the pathophysiology of asthma,and are involved in the differentiation of alternatively activated(M2)macrophages.These macrophages can generate several proinflammatory factors,such as chemokines,chitinase-like molecules and ’found in inflammatory zone 1’(FIZZ1,also known as Relm-α),which all contribute to the inflammation and remodeling of airways in asthma.Markers of M2 macrophages correlate with the severity of allergic airway disease in humans and mice,suggesting that M2 macrophages contribute to the disease.M1 macrophages are differentiated by interferon(IFN)-y and lipopolysaccharide(LPS)derived from y-negative bacteria in both mice and humans.These macrophages release inflammatory cytokines and chemokines(IL-12,IL-6,TNF-α,and CXCL10,CCL3),produce high levels of nitric oxide,and play an important protective role against intracellular pathogens.In short,the polarization status of macrophages plays a vital role in asthma,but the relevant mechanism by which M2 macrophages reduce the Th2 cell response has not been fully investigated.Rheb(ras homolog enriched in brain)belongs to the ras superfamily of GTPases,and it is essential for development of both flies and mice,and a potent activator of mTORC1 as well.Two Rheb family members,Rheb1 and Rheb2,are found in mammals,among which Rheb1 is found to be the essential isoform in mice and appears to be the dominant regulator of mTORC1.Rheb1 can bind directly to the active kinase domain of mTOR,but Rheb1 mutants with nucleotide-deletion trap mTOR in a catalytically inactive state.Nevertheless,Rheb-GTP targets other than mTOR,such as FKBP38(FK506-binding protein 38)and/or PLD1(phospholipase D1),may also contribute to mTOR activation.Rheb1 acts epistatically to exert an inhibitory effect of the TSC1-TSC2 heterodimer on mTORCl signaling,and the relationship is explained by the finding that TSC is an activator of Rhebl GTPase activity.However,the role of Rhebl in regulation of allergic asthma and macrophage polarization is still not fully understood.In this study,we found increased activity of Rhebl and mTORCl in myeloid cells of C57BL/6 mice with ovalbumin(OVA)-induced allergic inflammation.We utilized a mouse model with myeloid-specific deletion of Rhebl to study the role of Rhebl in OVA-induced allergic asthma.Methods1、Lys-MCre mice were first mated with Rheb1flox/flox mice to generate Lys-MCre,Rheblflox/+mice.Their offspring were then backcrossed to Rheb1flox/flox mice to generate Lys-MCre,Rheblflox/flox mice,which was reffered to as knock-out mice and Rhe1flox/flox mice generated in the same house as wild-type mice.2、Building the model of OVA-induced allergic asthma mice.Mice of about 20g weight were separated into two group:the saline group(control group)and OVA-induced group(experimental group).Firstly,all experimental mice were injected intraperitoneally(i.p.)with 20μg OVA mixed with 2 mg aluminum hydroxide in 0.2 mL of saline on day 0 and day 7.The mice in the control group received 0.2 mL of saline.From day 24 to 30,the OVA-sensitized mice were challenged with 2%aerosolized OVA,and the control group mice were challenged with saline for 30 min per day.3、Assessment of airway hyper-responsiveness.Calm down the mice in the box after 10 minutes before the experiment.Mice in each group were exposed to a series of incremental doses of aerosolized methacholine(6.25,12.5,25,50,100,200 mg/mL)for three minutes at each concentration,and airway hyper-responsiveness was recorded as enhanced pause(Penh).4、We use immunohistochemical and immunofluorescence to find out the expression of pS6 in mice of lung tissue,in order to determine the activation level of mTORCl.Finding out the the expression of F4/80、iNOS and CD206 in mice of lung tissue,in order to determine the inflammation infilting of macrophages.What’s more,using Western blotting to determine the expression level of the protein that correlated with mTORCl activity.Using Trizol reagent extract the total mRNA of lung tissue to determin the changes in transcript levels of inflammatory factors IL-1β and IL-6 in mice.Results1、Increased activity of Rheb1 and mTORCl is found in BALF cells of C57BL/6 mice with OVA-induced allergic inflammation.we found that Rheb1 1.7 times expressed in the asthma group higher than control group after OVA treatment.In addition,the pS6(s235/236)expression was observed,we analysis that pS6(s235/236)3.2 times expressed in the asthma group higher than control group.This suggested that Rhebl and pS6(s235/236)were both much more highly expressed in the asthma group than in the control group.Thus,we can preliminarily conclude that Rhebl expression and mTORCl activity are both markedly increased in the OVA-induced allergic asthma model group compared with the control group,suggesting that mTORC1 and Rheb1 may play a vital role in regulating allergic asthma.2、Rheb1 deletion in myeloid cells aggravates OVA-induced allergic asthma in mice.(1)Airway hyperresponsiveness analysis that Penh level showed a significant difference between the two groups(P<0.05).The OVA-treated Rhebl-KO mice showed a significant increase compared with WT mice,which was not observed in KO or WT mice with saline treatment(P>0.05).(2)Wright-Giemsa staining analysis that mice treated with OVA displayed an obvious increase in the total cell number,compared with the saline-treated group.Furthermore,in the OVA-treated group,BALF from Rhebl-KO mice contained more eosinophils and neutrophils than that from WT mice.(3)HE staining analysis that the OVA-challenged group displayed typical pathologic features of allergic airway inflammation in the lung tissue,compared with the saline-challenged controls.OVA-challenged Rheb1-KO mice showed a marked pathologic features of allergic airway inflammation compared with WT mice.(4)PAS staining analysis that mice the OVA-challenged group displayed numerous inflammatory cells infiltrating around the bronchioles,compared with the saline-challenged controls.OVA-challenged Rheb1-KO mice showed a marked increase in inflammation score compared with WT mice.(5)In immunohistochemical(IHC)staining,compared with saline-treated WT mice,OVA-treated mice showed increased levels of α-SMA and Muc5ac,and the expression levels of α-SMA and Muc5ac were highest in the OVA-treated KO mice.Based on all these data,we clarified that knockout of Rhebl in mouse myeloid cells would aggravate OVA-induced allergic asthma.3、Rhebl deletion induces Th2 cell response and inhibits Th1 cell response to OVA sensitization.(1)ELISA analysis that some sorts of Th1 cytokines and Th2 cytokines in serum were significantly elevated in the OVA-challenged group compared with the control group.In addition,in the OVA-challenged group,levels of Th1 cytokines in KO mice were lower than those in WT mice,while levels of Th2 cytokines of KO mice were higher than in WT mice.(2)ELISA analysis that some sorts of Th1 cytokines and Th2 cytokines in BALF were significantly elevated in the OVA-challenged group compared with the control group.In addition,in the OVA-challenged group,levels of Th1 cytokines in KO mice were lower than those in WT mice,while levels of Th2 cytokines of KO mice were higher than in WT mice.Overall,our data show that OVA-challenged,Rhebl-KO mice displayed diminished production of Th1 cytokines and enhanced production of Th2 cytokines compared with WT mice.4.Rheb1 deletion in myeloid cells promotes M2 but inhibits M1 macrophage polarization.(1)mRNA analysis that macrophages from Rhebl-KO mice showed obviously lower mRNA levels of TNF-α,iNOS and IL-6 in comparison with those of WT mice.Meanwhile macrophages of Rhebl-KO mice displayed increased mRNA levels of Argl,CD206,Fizzl,and Ym1.This suggested that macrophages of Rhebl-KO mice toward to polarization of M2 macrophages,decreaced M1 macrophages.(2)According to the results of flow cytometric analysis of BMMs in WT and KO mice,the percentage of cells with double positive of F4/80 and CD206 in total BMMs was 37.72%,12.03%in WT mice.The expresstion was higher in Rhebl KO mice than in WT mice.This suggested that macrophages of Rheb1-KO mice was more toward to polarization of M2 macrophages.5、Rheb1-KO mice displayed increased M2 polarization and decreased M1 polarization in BALF cells of OVA-induced asthma mice.(1)mRNA analysis that,in the control group,the mRNA expressions of M2 markers such as Argl,Fizzl,IL-10 and Ym1 in Rhebl-KO mice were greatly enhanced compared with those in WT mice,while in contrast,differences in mRNA expressions of M2 markers were more significant between Rhebl-KO mice and WT mice in the asthma group.M2 macrophages were enhanced in Rhebl-KO mice compared with WT mice in two groups.The asthma group was more significant than the control.(2)IF staining of F4/80 and CD206 revealed that Rhebl-KO mice had higher expression of CD206 in F4/80 positive cells than WT mice,in both the control and asthma group.As expected,the difference was more marked in the asthma group than in the saline-treated group.(3)mRNA analysis that,in the control group,the mRNA expressions of M1 markers such as IL-6,iNOS and TNF-α in Rhebl-KO mice were greatly reduced compared with those in WT mice,while in contrast,differences in mRNA expressions of M1 markers were more significant between Rhebl-KO mice and WT mice in the asthma group.M1 macrophages were reduced in Rhebl-KO mice compared with WT mice in two groups.The asthma group was more significant than the control.(4)IF staining of F4/80 and iNOS revealed that Rhebl-KO mice had lower expression of iNOS in F4/80 positive cells than WT mice,in both the control and asthma group.As expected,the difference was more marked in the asthma group than in the saline-treated group.We therefore concluded that Rhebl knockout in myeloid cells increases M2 polarization and decreases M1 polarization in macrophages,and these differences are increased in OVA-induced asthma.Conclusion1、Increased activity of Rheb1 and mTORC1 is found in BALF cells of C57BL/6 mice with OVA-induced allergic inflammation2、Rheb1 deletion in myeloid cells aggravates OVA-induced allergic asthma in mice3、Rheb1 deletion induces Th2 cell response and inhibits Th1 cell response to OVA sensitization4、Rheb1 deletion in myeloid cells promotes M2 but inhibits M1 macrophage polarization5、Rheb1-KO mice displayed increased M2 polarization and decreased M1 polarization in BALF cells of OVA-induced asthma mice...