The Role And Mechanism Of IFIH1 For Macrophage M1 Polarization In Early Stage Of ARDS

Part One: Screening the crucial molecules for macrophage M1 polarization in early stage of ARDSObjective: There still lack robust evidence that the uncovered molecules involved in macrophage M1 polarization were associated with ARDS.We aim to screen the crucial regulatory molecules for macrophage M1 polarization in early ARDS via high throughput experiment.Methods:(1)The current study is a single center,prospective and observational study.We screened all patients with ARDS(the Berlin Definition of ARDS)who admitted in the ICU in Zhongda Hospital,Southeast University and whose blood sample were collected for further research from January 2017 to December 2017.We only included the adult patients whose blood samples were collected within 48 hours after onset of ARDS.Gender,age,major diagnosis,severity of ARDS,acute physiology and chronic health(APACHE II)score,sequential organ failure score were recorded after admission.Peripheral blood samples were collected within 24 hours.(2)The candidate molecules involved in macrophage M1 polarization would be screened based on alveolar macrophage transcriptome data from GEO public database.(3)Venn diagram would be plotted to catch the overlap of molecules significantly associated with the severity of ARDS and molecules specially expressed in macrophage M1.Then,the crucial molecules were determined by hub gene algorithm.(4)External m RNA datasets from GEO public database were determined as validation cohorts to confirm whether 5 hub genes may be involved in macrophage M1 polarization or not.(5)Furthermore,ARDS patiets and mechanical ventilated patients without ARDS from November 2018 to March 2019 who admitted in the ICU in Zhongda Hospital were enrolled in the validation cohort study.To validate the hub genes whether or not involved in ARDS,the m RNA levels of IFIH1,IRF1,IFIT3,GBP1 and STAT1 were measured by q RT-PCR in bronchoalveolar lavage fluid from patients with ARDS and ventilated patients without ARDS.Meanwhile,the m RNA levels of Ifih1,Irf1,Ifit3,Gbp1 and Stat1 were measured in lung tissue homogenate from ARDS mice model,compared with control mice.(6)In order to select the molecule with strongest ARDS-related correlation involved in macrophage M1 polarization,we utilized Robust Rank Aggreg algorithm to calculate the interrelated P value based on human and animal experiments.Results: 32 ARDS patients were finally enrolled in current study.To screen molecules associated with the severity of ARDS,m RNA microarray were utilized to measure the transcriptome varitation on the peripheral blood from 26 ARDS patients.In addition,6 ARDS patients and 6 mechanical ventilated patients without ARDS were enrolled in external cohort to validate the crucial molecules involved in ARDS.(1)Transcriptome microarray data from 26 ARDS patients’ peripheral blood indicated that 234 molecules were significantly associated with the severity of ARDS(P= 6×10^5,Spearman correlation coefficient = 0.71);(2)100 M1-distinct genes were determined via differently expressed genes analysis on human alveolar macrophages transcriptome data from GEO public database(Log FC>2,FDR<0.05);(3)The interrelated analysis was utilized to screen 36 candidate molecules significantly associated with the severity of ARDS and specially expressed in Macrophage M1.STAT1,IFIH1,GBP1,IFIT3 and IRF1 were identified as the crucial molecules based on hub gene algorithm;(4)External validation cohorts from GEO database indicated that 5 hub genes markedly up-regulated in M1-polarized macrophage(FDR<0.05);(5)The clinical experiment confirmed that IFIH1,IRF1,GBP1 and STAT1 were significantly up-regulated in ARDS patients’ BALF(P<0.05).Animal experiments validated that the m RNA levels of Ifih1,Irf1,Ifit3,Gbp1 and Stat1 were obviously up-regulated in lung tissue from ARDS mice model(P<0.05);(6)IFIH1 was selected because IFIH1 had the minimum interrelated P value and maximum Spearman correlation coefficient.Conclusions: IFIH1 may be the crucial molecule involved in macrophage M1 polarization in ARDS.Part Two: Effect of IFIH1 on macrophage M1 polarizationObjective: To evaluate the influence of IFIH1 on macrophage M1 polarization.Methods: LPS and Poly(I:C)were utilized as activators for macrophage M1 polarization.The lentivirus vectors(sh IFIH1)and IFIH1 overexpression vector were used to knock down and over express IFIH1 expression in RAW264.7 cells and bone marrow–derived macrophages(BMDMs).The efficiency of IFIH1 knocking down and over-expressing was evaluated by q PCR and western blot.CD86 was measured by flow cytometry;i NOS expression was detected by western blot;IL1 and CCL2 was assessed by ELISA.Results:(1)Construct the low and high IFIH1 expressed RAW264.7 cell line: Compared with Ctrl-RAW264.7 cells,IFIH1 m RNA and protein level were significantly down-regulated in sh IFIH1-RAW264.7 cells,P<0.05;the efficiency of IFIH1 protein knocking down was 93% and the efficiency of IFIH1 protein over-expressing was 533%.(2)Construct the low and high IFIH1 expressed BMDMs: Compared with Ctrl-BMDMs,IFIH1 m RNA and protein level were significantly up-regulated in sh IFIH1-BMDMs,P<0.05;the efficiency of IFIH1 protein knocking down was 93% and the efficiency of IFIH1 protein over-expressing was 533%.(3)Down-regulating IFIH1 markedly decreased macrophage M1 polarization: Compared with control group,western blot indicated that both LPS-induced and Poly(I:C)-induced i NOS expression in RAW264.7 cells and BMDMs were markedly decreased after silencing of IFIH1.To confirm our findings,we examined other the expression of M1-phenotype markers.Flow cytometry results illuminated that expression of CD86 in both LPS-induced and Poly(I:C)-induced M1 polarization cell models were markedly decreased after silencing of IFIH1.Additionally,ELISA results revealed that expression of IL-1β protein and CCL2 were markedly decreased in M1 polarization cell models after silencing of IFIH1,P<0.05.(4)Over-expressing IFIH1 obviously increased macrophage M1 polarization: Compared with control groups,both LPS-induced and Poly(I:C)-induced i NOS expression in RAW264.7 cells and BMDMs was significantly increased after over expressing of IFIH1.Meanwhile,CD86 in both LPS-induced and Poly(I:C)-induced M1 polarization cell models were markedly increased after over expressing of IFIH1.Similarly,expression of IL-1β and CCL2 were significantly increased M1 polarization cell models after over expressing of IFIH1.In addition,only IFIH1 expression without LPS or Poly(I:C)stimulation could not influence M1 macrophage polarization.Conclusions: IFIH1 contributes to both LPS induced and Poly(I:C)induced macrophage M1 polarization,but only IFIH1 expression could not affect macrophage M1 polarization.Part Three: The molecular mechanism of MyD88/IFIH1/IRF3 on macrophage M1 polarizationObjective: To explore the molecular mechanism of IFIH1 on macrophage M1 polarization.Methods:(1)Predict the molecular mechanisms underlying the regulatory role of IFIH1: GSEA was performed respectively on m RNA dataset of human alveolar macrophages and mononuclear macrophages.The interrelated analysis was utilized to screen the common mechanisms;(2)Validate the conclusion from GSEA prediction in Vitro: The cells were divided into groups as follow: sh IFIH1-macrophage,sh Ctrl-macrophage,IFIH1-macrophage,Ctrl-macrophage.The crucial molecule phosphorylation was detected by western blot.The translocation into the nucleus of transcription factor was measured by nuclear western blot;(3)Explore whether LPS and Poly(I:C)induced IFIH1 activation dependent on MyD88 pathway: ST 2825(a specific MyD88 dimerization inhibitor)was utilized to further explore whether LPS and Poly(I:C)are involved in MyD88-dependent or independent mechanism activation of IRF3.The crucial molecule phosphorylation was detected by western blot.The translocation into the nucleus of transcription factor was measured by nuclear western blot.Results:(1)GSEA predicted the underlying mechanism of IFIH1: in alveolar macrophage,GSEA indicated that IFIH1 regulated macrophage M1 polarization through activation of metabolic pathway of linolenic acid,metabolic pathway of linoleic acid and RIG-I pathway;and in monocyte macrophages,GSEA indicated that IFIH1 regulated macrophage M1 polarization through activation of RIG-I pathway,TOLL like receptor pathway and intestinal immune network pathway.The interrelated analysis uncovered that RIG-I pathway is the common mechanism.(2)The influence of knocking down IFIH1 on RIG-I pathway: Compared with control group,the phosphorylated western blot indicated that p-IRF3 was markedly down-regulated in sh IFIH1 groups,P<0.05.Compared with control group,the nucleoprotein western blot indicated that IRF3 in nuclear was significantly down-regulated in sh IFIH1 group,P<0.05.(3)The influence of over-expressing IFIH1 on RIG-I pathway: Compared with control group,the phosphorylated western blot indicated that p-IRF3 was markedly up-regulated in over-expressed IFIH1 groups,P<0.05.Compared with control group,the nucleoprotein western blot indicated that IRF3 in nuclear was significantly up-regulated in over-expressed IFIH1 group,P<0.05.In addition,only IFIH1 expression without LPS or Poly(I:C)stimulation could not affect IRF3 activation and M1 macrophage polarization.(4)Explore whether LPS and Poly(I:C)induced IFIH1 activation dependent on MyD88 pathway: Western blot indicated that blocking the MyD88 pathway did not affect the Poly(I:C)-induced phosphorylation of IRF3,but the MyD88 inhibitor significantly decreased the LPS-induced phosphorylation of IRF3 compared with the control treatment in RAW264.7 cells and BMDMs,P<0.05.Quantitative analysis of IRF3 in the cell nucleus indicated that blocking the MyD88 pathway did not affect the Poly(I:C)-induced expression of IRF3 in the RAW264.7 cell and BMDMs nucleus,but the MyD88 inhibitor significantly decreased the expression of IRF3 in the RAW264.7 cell and BMDMs nucleus compared with the control treatment,P<0.05.Conclusions: IFIH1 regulates macrophage M1 polarization via activating IRF3;LPS-induced activation of IFIH1-IRF3 depends on MyD88;Poly(I:C)-induced activation of IFIH1-IRF3 independ on MyD88.