Cholinergic Anti-inflammatory Pathway Alleviates Lipopolysaccharide-induced Acute Lung Injury By Regulating Alveolar Macrophage Polarization

Background:Acute lung injury(ALI)is a common complication of sepsis with high morbidity and mortality.Alveolar macrophages(AM)play key roles in defense,resolution in ALI.The polarization of AMs is dependent on micro environmental stimuli and might influence the progression of ALI.GTS-21,a selective a7 nicotinic acetylcholine receptor agonist of the cholinergic anti-inflammatory pathway(CAP),has recently been established to be promising in the treatment of ALI.However,the molecular mechanism underlying the GTS-21-mediated suppression of inflammatory responses has been explored only partially.High mobility group box1(HMGB1)was reported to activate the Absent in melanoma 2(AIM2)inflammasome complex,which are both essential in acute lung injury(ALI).However,their interaction mechanism remains unknown.Macrophages are known to express the AIM2 inflammasome complex and the main receptors:receptor for advanced glycation end product(RAGE),toll-like receptor 2/4(TLR2/4)of HMGB1 to transmit intracellular signals.Objective:1.To explore the anti-inflammatory mechanism of GTS-21 in lipopolysaccharides(LPS)-induced ALI through regulating the function of AM.2.To explore the role of HMGB1 in the inflammatory response of LPS induced ALI by regulating the function of AM.MethodsPart 1:The AM was extracted from the mouse alveolar lavage fluid(BALF)and was polarized to M1 type AM and M2 AM.The M1 and M2 type AM were injected into the mice airway in vitro.Then the LPS induced ALI model was established,and the lung injury scores,the degree of pulmonary edema and the effect of pulmonary inflammatory factors were observed.Part 2:The ALI model induced by LPS was established and divided into control group,LPS group,GTS-21 group and GTS-21 + LPS group.AM was cultured in vitro experiment and divided into the same four groups under the stimulation of with or without LPS and GTS-21.The effects of GTS-21 on the polarization of AM and the secretion of HMGB1 in ALI induced by LPS were observed in vivo and in vitro.Part 3:In vivo experiment,anti-HMGB1 or rHMGB1 were used to intervene ALI mice induced by LPS,and the protein and mRNA expression of AIM2 inflammasome complex in lung tissue were measured.Flow cytometry was used to detect the expression of Ml type AM surface related factors(MHCII,CD80,CD86 and CD40)and M2 type AM surface related factors(CD206,IL-10).Bone marrow-derived macrophages were cultured in vitro to observe the protein and mRNA expression of AIM2 inflammasome complex and the expression of M1 and M2 AM surface related factors after anti-HMGB1 or rHMGB1 stimulation.Part 4:HMGB1 antibody or rHMGB1 was applied to LPS induced ALI mice to observe the changes of TLR2/4,Rage receptor and NF-?B expression.TLR2/4 and RAGE receptor antagonists were applied to LPS induced ALI mice to observe the changes of pulmonary inflammatory and AIM2 inflammasome complexe.To observe the changes of AIM2 inflammasome complex,the above intervention measures were added to the cultured macrophages in vitro.Results(1)M 1 type AM aggravated the pro-inflammatory response of ALI induced by LPS.The main manifestations were as follows:the activity of lung MPO and W/D was increased,and the level of TNF-?,IL-6,IL-1?,MCP-1 in lung were increased significantly.M2 type AM reduced the inflammatory response of ALI mice induced by LPS.The main manifestations were the decrease of lung injury score,the decrease of lung MPO activity,lung W/D,and the decrease of MCP-1 level.(2)In ALI mice induced by LPS,GTS-21 reduced the inflammation of lung tissue,decreased the number of AM and the expression of inflammatory factors.The secretion and expression of HMGB1 in plasma and BALF was decreased after GTS-21.GTS-21 decreased the levels of M1 type AM bioactive molecules iNOS,TNF-?,IL-6,IL-12 and increased the levels of Arg1,ym1,CCL17,CCL22,CCL24 both in ALI mice induced by LPS and in vitro.(3)HMGB 1 antibody alleviated the lung inflammation induced by LPS and inhibit ed the activity of AIM2 inflammasome complex in ALI mice and Inhibited AM to M1 polarizing.rHMGB1 aggravated the lung tissue inflammation induced by LPS in ALI mice,enhanced the activity of AIM2 inflammasome complex and promoted M1 polarization.(4)HMGB 1 antibody down-regulated the expression of TLR 2,TLR4 and RAGE/NF-?B in ALI induced by LPS.rHMGB1 up-regulated the expression of them.TLR2,TLR4 receptor antagonist(LPS-RS),and RAGE antagonist(FPS-ZM1)inhibited ALI induced by LPS and decreased the expression of AIM 2 inflammasome complex.Conclusions(1)M1 AM aggravated the pulmonary inflammation of ALI induced by LPS and M2 AM reduced the pulmonary inflammation of ALI induced by LPS.(2)CAP reduced the LPS induced ALI by decreasing the number of AM,inhibiting the polarization of M1 type AM and the secretion of HMGB1 in AM.(3)HMGB1 participated in the inflammatory response of ALI induced by LPS by activating the inflammasome complex of AIM2 and promoting Ml polarization of AM.(4)In ALI induced by LPS,HMGB1 activated the inflammasome complex of AIM2 and promoted the M1 polarization of macrophages,which could be mediated by the TLR 2,TLR4,RAGE and NF-?B pathway.