| Objective:Uncontrolled inflammation is a central issue of acute respiratory distress syndrome(ARDS),which is characterized by leukocyte infiltration and lung injury.However,signals that initiate these events remain barely understood.We evaluate the role of the nuclear alarmin Interleukin(IL)-33 in lung damage and immune response triggered by lipopolysaccharide(LPS)induced ARDS.Methods:In this study,an intra-airway injection of lipopolysaccharide(LPS)was used to induce the ARDS model in mice.The protein concentration of IL-33 in lung tissue and bronchoalveolar lavage fluid(BALF)of wild-type(WT)C57BL/6 mice were first measured by immunoblotting and ELISA to assess the changes of IL-33 in the ARDS mouse model.Then immunohistochemistry was applied to localize IL-33 expression sites in lung tissue.Next,IL-33 and ST2 knockout mice were used to explore the effect of the IL-33/ST2 axis on uncontrolled inflammatory response in ARDS.The severity of lung injury was assessed by HE staining and injury scoring.Flow cytometry was used to detect changes in the proportion and activation of invariant natural killer T(i NKT)cells.Further,CD1d knockout mice and Vα14 transgenic(Vα14Τg)mice were used to clarify the role of NKT cells in the uncontrolled inflammatory response in ARDS.Finally,a reversal assay was applied to explore the relationship between IL-33 and NKT cells.Results:In WT mice,this study found that IL-33 was released immediately after the onset of ARDS,peaking at 1 h,followed by a significant decrease at 3 h and then at 24 h.Immunohistochemical results showed that IL-33 was localized in the nucleus of type II alveolar epithelial cells.Compared to wild-type mice,IL-33 and ST2 knockout mice showed a protective effect in ARDS in terms of reduced neutrophil infiltration,alveolar capillary leakage and lung injury.Simultaneous lung tissue flow cytology results showed a reduced percentage of i NKT in the lungs with reduced CD69 geometric mean fluorescence intensity.Then,applying CD1d~-/~-and Vα14Τg mice,it was further verified that increased i NKT cells could promote inflammatory response and exacerbate pathological injury in ARDS.The results showed that Vα14Τg mice exhibited increased neutrophil infiltration,increased alveolar-capillary leakage,and increased lung injury scores in ARDS compared to WT mice.In contrast,CD1d~-/~-mice were the opposite of Vα14Τg mice.Finally,this study applied ST2pretreatment to WT and Vα14Τg mice 1 hour before LPS administration to neutralize IL-33.We further validate that IL-33 promotes inflammation via NKT cells in ARDS.Conclusions:In conclusion,our data provide clear evidence that the IL-33/ST2 axis promotes ARDS early uncontrolled inflammatory response by activation and recruitment of i NKT cells.Thus,IL-33 and NKT cells may be targetable molecules and immune cells to administrate early cytokine storm in ARDS.Part I: IL-33/ST2 axis is involved in an early uncontrolled inflammatory response in ARDSObjective: The aim of this study was to investigate the expression changes and tissue localization of the nuclear alarm protein interleukin-33(IL-33)in lipopolysaccharide(LPS)-induced ARDS model in mice.Methods: In this study,a mouse ARDS model was induced by intra-airway injection of LPS 20 mg/Kg.The experiment was divided into 4 groups: control group,ARDS 1 h group,ARDS 3 h group and ARDS 24 h group.After successful modeling,lung tissues and alveolar lavage fluid were collected at different time points,and the relative expression of IL-33 m RNA in lung tissues and bronchoalveolar lavage fluid(BALF)was measured by q RT-PCR,and the protein concentration of IL-33 in lung tissues and BALF was measured by Western Blot and ELISA.Immunohistochemistry was applied to detect IL-33 lung tissue localization.Finally,Western Blot assay was used to detect ST2 protein expression level in lung tissue.Results: In this study,we found that IL-33 m RNA levels in ARDS model group lung tissues were significantly increased at 1 h after modeling and then decreased at 3 h and 24 h compared with the control group.In line with the changes in IL-33 m RNA levels,Western Blot showed a significant increase in IL-33 protein concentration at 1 h,followed by a decrease at 3 h and 24 h(p<0.05).ELISA quantification again verified this change in IL-33 protein concentration in lung tissue.Meanwhile,to observe the change of IL-33 protein concentration in BALF,we quantified the IL-33 protein level in BALF by ELISA,which showed a significant increase in the ARDS 1 h group,followed by a significant decrease in the ARDS 3 h and ARDS 24 h groups.Immunohistochemical staining revealed that IL-33 was localized in the nucleus of alveolar II epithelial cells,and Western Blot assay showed a slight but not statistically significant decrease in ST2 protein concentration at 1 h and a significant increase at 3 h and 24 h(p<0.05).Conclusion: IL-33 was increased at 1 h in ARDS and then decreased significantly at 3 h and 24 h.It was mainly expressed in the nuclei of type II alveolar epithelial cells.Part II: Inhibition of the IL-33/ST2 axis inhibits uncontrolled inflammatory response and NKT cell recruitment activation in early ARDSObjective: This study was conducted to verify the effect of the interleukin-33(IL-33)/ST2 axis on the inflammatory response in acute respiratory distress syndrome(ARDS)and its possible mechanisms.Methods: In this study,a mouse ARDS model was induced by intra-airway injection of lipopolysaccharide(LPS)20 mg/Kg.Wild-type C57BL/6 mice,IL-33 knockout mice and ST2 knockout mice(all C57BL/6 background)were used to divide the experiment into 6 groups: WT Ctr group,WT ARDS group,IL-33 Ctr group,IL-33 ARDS group,ST2 Ctr group,ST2 ARDS group.24 h later,serum,lung tissue,spleen and bronchial alveolar lavage fluid(BALF)were collected.The severity of lung tissue injury was assessed by HE staining and injury scoring.IL-6,MCP-1,and TNF-α in serum and BALF were measured by cytometric bead array(CBA).Results: In this study,we found a significant increase in D/W(p<0.05)and a decrease in the number of neutrophils in BALF(p<0.05)in the IL-33 and ST2 knockout mice group compared to the WT ARDS group.Meanwhile,ARDS mice lacking IL-33 and ST2 had less lung tissue damage,as evidenced by reduced leukocyte infiltration,reduced alveolar congestion/hemorrhage,reduced area of alveolar collapse and alveolar wall thickening,and lower lung injury scores.The levels of IL-6,MCP-1,and TNF-α factors were significantly reduced in the serum and BALF of ST2 knockout mice.In IL-33 knockout mice,only serum concentrations of IL-6,MCP-1,and TNF-α were reduced.Results at the cellular mechanistic level showed that the proportion of i NKT cells and the geometric mean fluorescence intensity of CD 69 were significantly reduced in the lungs of IL-33 and ST2-deficient mice,while activation of CD4+T lymphocytes and CD8+T lymphocytes in the lung was attenuated.Conclusion: Inhibition of the IL-33/ST2 axis attenuates the early inflammatory response in ARDS while inhibiting i NKT cell recruitment and activation and conventional T lymphocyte activation in the lung.Part III: NKT cell increase promotes early uncontrolled inflammatory response in ARDSObjective: To investigate the effect of natural killer T(NKT)cells on the early inflammatory response in acute respiratory distress syndrome(ARDS).Methods: In this study,lipopolysaccharide(LPS)20 mg/Kg was applied to induce ARDS model in mice by intra-airway injection.Wild-type C57BL/6 mice,CD1 d knockout mice(NKT cell knockout),and Vα14 transgenic(Vα14Τg,NKT cell increase)mice were used to divide the experiment into 6 groups: WT Ctr group,WT ARDS group,CD-1d Ctr group,CD-1d ARDS group,Vα14Τg Ctr group,and Vα14Τg ARDS group.Serum,lung tissue,spleen and bronchoalveolar lavage fluid(BALF)were collected after 24 hours.The severity of lung tissue injury was assessed by HE staining and lung injury score,protein concentration in BALF was measured by BCA,and neutrophil ratio in BALF was measured by flow cytometry.IL-6,MCP-1,and TNF-α in serum and BALF were measured by cytometric bead array(CBA).Results: The D/W ratio was significantly increased and Vα14Tg was decreased in the CD1 d KO group compared to the WT ARDS group(p<0.05).However,total protein concentration and neutrophils were reduced in BALF in the CD 1d KO group,while Vα14Tg was significantly increased in the Vα14Tg group(p<0.05).Meanwhile,inflammatory injury was reduced in the CD 1d ARDS group compared with the WT ARDS group,and lung injury scores were significantly lower.In contrast,the Vα14Tg ARDS group had alveolar congestion and hemorrhage,increased inflammatory infiltration in the lungs,and significantly higher lung injury scores.Levels in serum and BALF were significantly higher in the Vα14Tg mice compared to the WT ARDS group.However,there were no significant differences in IL-6,MCP-1 and TNF-α levels in serum and BALF in CD 1d KO mice.Conclusion: The increase of NKT cells in the lung promotes the inflammatory response and pathological injury in the early stage of ARDS.Part IV: IL-33/ST2 axis-dependent NKT cells promote an uncontrolled inflammatory response in ARDSObjective: To verify the effect of IL-33/ST2 axis natural killer T(NKT)cells on the inflammatory response in acute respiratory distress syndrome(ARDS).Methods: In this study,IL-33 neutralizing antibody ST2 was administered intraperitoneally to WT and Vα14Tg mice 1 hour before administration of lipopolysaccharide(LPS)via intraairway injection to induce ARDS model in mice.Wild-type C57BL/6 mice and Vα14 transgenic(Vα14Tg with increased NKT cells)mice were used to divide the experiment into four groups: WT Ctr group,WT ARDS group,CD1 d Ctr group,CD1 d ARDS group,Vα14Tg Ctr group,Vα14Τg ARDS group.24 hours later,serum,lung tissue,spleen and bronchoalveolar lavage fluid(BALF)were collected.The severity of lung tissue injury was assessed by HE staining and lung injury score,protein concentration in BALF was measured by BCA,and neutrophil ratio in BALF was measured by flow cytometry.IL-6,MCP-1,and TNF-α in serum and BALF were measured by cytometric bead array(CBA).Results: In this study,mice were pretreated with IL-33 neutralizing antibody ST2 by intraperitoneal injection 1 hour prior to LPS administration.The results showed that compared with the WT ARDS group,mice in the Vα14Tg ARDS group had increased alveolar-capillary leakage and increased pulmonary edema,as evidenced by increased total protein concentration and neutrophil infiltration in BALF and decreased D/W.In addition,lung injury was more severe in the Vα14Tg ARDS group,as evidenced by increased leukocyte infiltration,alveolar congestion/hemorrhage,alveolar collapse,and alveolar wall thickening with increased lung injury scores.Also,protein concentrations of proinflammatory cytokines(IL-6,TNF-α)and chemokines(MCP-1)were higher in plasma and BALF in Vα14Tg mice.Conclusion: The IL-33/ST2 axis promotes early uncontrolled inflammatory response in ARDS via NKT cells. |
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