Role Of Toll-like Receptor2 In Allergic Inflammation And PI3k/Akt Autophagious Pathway In Mice

Background Asthma is a chronic respiratory disease with a significant morbidity and mortality.Approximately 300 million people worldwide currently suffer from this disease.Its incidence and economic burden are on the rise throughout the world..Toll-like receptors(TLRs)are primary pattern recognition receptors that play critical roles in innate and acquired immunity.TLRs have been proved to play a pivot role in the allergic inflammatory response.However,their involvement in autophagy in the mechanism of asthma remains poorly defined.TLR2,one of the TLRs,has been reported involved in asthma.Autophagy is an evolutionarily conserved process by which cellular components are degraded and cellular homeostasis maintained.Emerging evidences demonstrate that autophagy exerts significant impact on immunity and antiviral response,and may be involved in asthma.Phosphoinositide3-kinases(PI3Ks)are a family of closely related enzymes.In cells,PI3 Ks play a important role in a diverse array of cellular mechanisms,including survival,activation,differentiation and proliferation.Recent studies found that PI3 Ks contribute to the pathogenesis of asthma by influencing the proliferation of airways smooth muscle,the recruitment of eosinophils and granulocyte recruitment,cell activation and apoptosis.Meanwhile,the exact role of PI3 K in the inflammation and autophagy in asthma is largely unknown.The regulation of PI3 K by TLRs has been either poorly investigated.In this study,we explored the roles of TLR2 in allergic inflammation using an ovalbumin induced mice model of airway inflammation,focusing on the role of TLR2on inflammation and PI3K/Akt autophagious signaling pathway.Aims:1.To investigate the role of TLR2 in the allergic inflammation in mice.2.To investigate the effects of TLR2 on PI3 K autophagious signaling pathway.Methods:1.Establishment of allergic inflammatory mice model and detection of TLR2 in lung tissue WT mice were ovalbumin(OVA)-sensitized and-challenged.On day 21,the mice were executed,lung tissue samples and bronchial-alveolar lavage fluid were collected.Hematoxylin and eosin(H&E)staining were performed to observe the inflammation changes and periodic acid-schiff(PAS)staining was applied to decide the mucous secretion and number of goblet cells in lung tissue slices.Expression of TLR2 in lung tissue was determined by Western blotting.2.Effect of TLR2-knockout on allergic inflammation and PI3 K autophagious signaling pathway in mice WT and TLR2-knockout mice were OVA-sensitized and-challenged.Mice were executed and bronchial-alveolar lavage fluid were collected for cell counting using a Weigh's staining,IL-13 and INF-? levels were detected by ELISA.The lung tissue was collected for pathologic examination using H&E and PAS staining.Western blotting was used to detect the expression of My D88,PI3 K,P-Akt,Beclin-1 and LC3-II.3.Targeted inhibitory effects of 3-methyladenine(3-MA)on PI3 K signaling pathway in WT and TLR2-knockout mice WT and TLR2-knockout mice were OVA-sensitized and-challenged with a concomitant treatment of 3-methyladenine(3-MA).Mice were intraperitoneal injected with low or high doses of 3-MA.ELISA was used to determine the change of cytokines including IFN-? and IL-13 levels in bronchiole alveolar lavage fluid.Western blot analysis was used to detect the expression of My D88,PI3 K,P-Akt,Becline-1 and LC3-II in the lung tissue lysates.Results:1.Inflammatory changes in the lung tissue accompanied by an increased expression of TLR2 after OVA-sensitization and-challenge In OVA-sensitized and-challenged WT mice,bronchial cavity narrowing,remarkable cell infiltrations,and mucous production in the airway were observed.A remarkable increase of cell numbers including the esosinophiles,neutrophiles,and monocytes in the bronchoalveolar lavage fluid was observed.TLR2 expression enhanced significantly in the lung tissues.2.Weaker inflammatory and cytokine changes in TLR2 knockout mice In OVA-sensitized and-challenged TLR2 knockout mice,a similar but weaker inflammatory changes were found.The increases of inflammatory cells and IL-13,and reduce of IFN-? levels in BALF were either not as remarkable as those in the WT ones.3.Decreased protein expression of PI3 K autophagious signaling pathway in TLR2-knockout mice The expression of PI3 K,P-Akt,Beclin-1 and LC3-II increased significantly in OVA-sensitized and-challenged WT mice.While in OVA-sensitized and –challenged TLR2 knockout mice,the expression of PI3 K,P-Akt,Beclin-1 and LC3-II was also increased,but weaker than that in the WT mice.A significant difference between the WT and TLR2 knockout mice was observed.4.3-MA remarkably inhibited the allergic inflammation in WT mice In 3-MA treated OVA-sensitized and-challenged WT mice,the bronchial cavity narrowing,remarkable cell infiltrations,and mucous production in the airway were decreased.The increase of IL-13 level and decrease of IFN-? level was reversed partially.Contrarily,in 3-MA treated inflammaory TLR2-knockout mice,similar tendency was found but not statistically significant.5.3-MA inhibited the PI3 K autophaious signaling pathway In 3-MA treated OVA-sensitized and –challenged WT mice,the enhanced expression of PI3 K,P-Akt,Beclin-1 and LC3 was inhibited by 3-MA treatement significantly.A high dose of 3-MA resulted in a stronger inhibition.While in OVA-sensitized and-challenged TLR2-knockout mice,neither low nor high doses of 3-AM treatment led to statistically significant changes as those in the WT ones.Conclusions1.TLR2 confers a important role in OVA-induced airway inflammation in mice.2.PI3K/Akt signaling pathway might play a critical role in the autophagy in OVA-induced inflammatory mice,and this survival pathway migh be TLR2 dependent..