Protective Effects Of M2 Macrophages On Acute Lung Injury In Mice And Its Mechanisms

Acute lung injury / acute respiratory distress syndrome(ALI / ARDS)is the primary performance of multiple organ dysfunction(MODS)in the lungs in intensive care unit(ICU).Acute Lung Injury is usually the result of multiple causes including trauma,burns,infection,shock and some other factors.Acute Lung Injury is often a severe disease which is rapidly progressed.In recent years,a wide range of research referring to the pathogenesis and therapeutic strategies of ALI / ARDS have been carried out all over the world by many scholars,but rapid and efficient treatment protocols are still in urgent need,and the morbidity and mortality are far from a low incidence.Macrophages are one of the most important immunocells in the body's immune defense system,playing an important role in immune surveillance and initiation and the clearance of pathogen.Lots of studies have found that macrophage activation and apoptosis are all closely linked with pathological changes in the development of acute lung injury.At the early stage of ALI,affected by various pathogenic stimuli in vitro and in vivo,M1 macrophages activated massively and manifested as M1-polarized,abundant pro-inflammatory cytokines and mediators are secreted,followed by the migration of neutrophils into the inflammatory site,which all together promote the lung inflammation and tissue damage,as well as the damage of pulmonary vascular permeability,resulting in pulmonary edema.Some scholars believe that the phenotype and functional characteristics of macrophages change when the phase and microenvironment of inflammation change.At the later stage,macrophages usually manifested as M2-polarized,aim at inhibiting the inflammatory response through phagocytizing neutrophils and secreting anti-inflammatory cytokines,promoting collagen synthesis and wound repair.However,if the over-activated inflammatory response gets out of control,the function of macrophages lose the balance between the pro-inflammatory and anti-inflammatory responses.The function of macrophage decline after phagocytosis of neutrophils,and the number of macrophages decreased with the occurrence of apoptosis or pyroptosis,the antiinflammatory function decreased thus the lung inflammation and tissue damage further aggravated.Therefore,it is of great importance to confirm the function and mechanism of M2 macrophages during the development of acute lung injury,so as to provide new revelation in the treatment strategies of acute lung injury.In the present study,we chose C57BL/6 mice to induce and differentiate bone marrow derived M2 macrophage,then the harvested M2 macrophages were administrated intrapulmonarily into ALI mice.The change in cytokine secretion,inflammatory cell infiltration,total protein leakage and pathological lung injury were evaluated to assess the effect of M2 macrophages on ALI.Finally,we further explored the potential mechanisms of M2 macrophages in the protection against LPS-induced ALI.Part 1 The differentiation of Bone Marrow Derived M2 MacrophagesObjective To induce the differentiation of Bone Marrow-Derived M2 Macrophages from primary Bone Marrow cells and identify the purity.Methods 1.Bone Marrow Cells were harvested from the lower limb and proximal tibial of 4-5w C57BL/6 mice.The original BM cells were cultured with M-CSF for 7 days before replaced with IL-4 for 1 day to obtain M2 macrophages.2.M2 macrophages was collected and labeled with F4/80 and CD206 antibody.The purity of M2 macrophages was detected by flow cytometry.Results With the induction of differentiation of M-CSF and IL-4,M2 macrophages grew in good condition and displayed satisfactory microscopic morphology.M2 macrophages met the experimental requirements with 90% purity after flow cytometry detection.Conclusion The culture of Bone Marrow-Derived M2 macrophages was successful,subsequent experiments can be carried out smoothly.Part 2 The effect of Bone Marrow Derived M2 Macrophages on LPS-induced acute lung injuryObjective To investigate the effect of Bone Marrow-Derived M2 Macrophages on pulmonary inflammatory response and pathological damage in LPS-induced acute lung injury in mice.Methods 6-8w C57 BL / 6 mice were randomly divided into 3 groups: sham(Sham)group,model(LPS)group and M2 macrophage treatment(M2)group.Mice were intrapulmonary administrated with sterile M2 macrophages 3 h after lipopolysaccharide(LPS)injection.Mice were sacrificed at 6 h and 24 h after ALI.Lung tissues and bronchoalveolar lavage fluid(BALF)were obtained for follow-up detection 1.The number of neutrophils and T cells in BALF were counted by flow cytometry.2.The total protein concentration in BALF was detected with BCA method.3.The levels of TNF-?,IL-6,IL-1? and IL-10 in BALF were detected by ELISA.4.The Wet/Dry weight ratio of lung tissue was calculated.5.The lung tissues were stained with HE and then pathologically scored..Results 1.At 6h and 24 h after ALI,the number of neutrophils in BALF of M2 group were significantly lower than that in LPS group.At 24 hours after ALI,the number of T cells in BALF of M2 group increased compared with LPS group(P <0.05).2.The protein concentration in BALF of M2 group was significantly lower than that in LPS group at 6 h and 24 h after lung injury(P <0.05).3.The levels of proinflammatory cytokines TNF-?,IL-6,IL-1? in BALF of M2 group were significantly lower than those in LPS group at 6h and 24 h,while the level of antiinflammatory cytokines IL-10 was higher instead(P <0.05).4.There was no difference between M2 group and LPS group at 6 hours after lung injury.However,at 24 hours after operation,the W/D Ratio of M2 group was markedly lower than LPS group,which proved that pulmonary edema was alleviated(P <0.05).5.The pathological damage of the lungs in M2 group was significantly lower than that in ALI group at 6 h and 24 h after lung injury,and the pathological score was significantly decreased(P <0.05).Conclusion Bone Marrow-Derived M2 Macrophages have protective effects against ALI.M2 Macrophages could alleviate lung pathological damage and the leakage of protein-rich solution,as well as inhibiting neutrophil infiltration and pro-inflammatory cytokine secretion while increasing the anti-inflammatory cytokine secretion.Part 3 The potential mechanisms of Bone Marrow Derived M2 Macrophages in the protection against ALIObjective To explore the possible mechanism of Bone Marrow-Derived M2 Macrophages in the protection against acute lung injury in vitro and in vivo.Methods 1.The M0 and M2 macrophages were digested and counted before co-cultured at different proportions of [1:10],[1:1] and [10:1],24 hours after LPS stimulation,the supernatant was collected and the expression of TNF-?,IL-6,IL-1? and IL-10 were detected by ELISA.2.The expression of PD-L1 on M2 macrophages was detected by Flow cytometry.3.The expression of PD-1 on T cells in BALF after M2 macrophage administration was detected by Flow cytometry.4.Mice were randomly divided into four groups: sham group,model(LPS)group,Isotype antibody(Isotype)group and Anti-PD-L1 antibody(Anti-PD-L1)group.3 hours after LPS modeling,Anti-PD-L1 antibody was injected before infusion with M2 macrophages.24 hours after ALI,BALF was harvested and the concentration of cytokines TNF-?,IL-6,IL-1? and IL-10 were measured.Results 1.M0/M2 co-culture results showed that the secretion of proinflammatory cytokines TNF-?,IL-6 and IL-1? decreased gradually with the increase of M2 macrophage proportion.Meanwhile,anti-inflammatory cytokine IL-10 also showed a decreasing trend when the number of M2 cells increased(P <0.05).2.Flow cytometry detection showed that the expression of PD-L1 on M2 macrophages was about 99%±1.8%.3.Flow cytometry detection showed that the expression of PD-1 on T cells in BALF after M2 macrophage administration was nearly 95%±4.1%.4.Antibody-blocking results showed that the inflammatory cytokines in BALF were not reduced by M2 macrophages but given a rising trend after blocking the PD-L1 pathway.The secretion of TNF-?,IL-6,IL-1? were increased by PD-L1 blockade(P <0.05).Conclusion M2 macrophages can regulate the secretion of cytokines.Anti-inflammatory cytokine IL-10 may not be the main mechanism by which M2 macrophages exert anti-inflammatory effects.The protective effect of M2 macrophages against ALI may be related to PD-1 / PDL1 signaling pathway.