Effects And Mechanisms Of HMGXB44,A Novel Member Of The High Mobility Protein Family,on Systemic Inflammation In Mice

Background and aims:Sepsis is a life-threatening inflammatory disorder.Sepsis usually caused by bacterial infection or sterile injury induced systemic inflammatory response,leading to multi-organ damage and ultimately died of organ failure.Despite decades of research,sepsis is still the most common cause of death in hospital intensive care units,and there still no effective anti-sepsis treatments available,and remains sepsis as a tremendous public health burden in the world.High mobility group box(HMGB)protein family is a class of nuclear proteins,possessing a unique DNAbinding domain,the HMG-Box,which can bind non-B type DNA structure with high affinity,and distort DNA by bending and unwinding,and plays role in maintaining chromatin structure,DNA repairing,regulating transcription or facilitating ribosome biogenesis.However,an unexpected discovery in 1999 revealed that HMGB1 can be secreted from the nucleus to the extracellular during systemic inflammatory response,as a pro-inflammatory cytokine-mediated inflammatory response.Blocking HMGB1 secretion or using specific antibody anti-HMGB1 in the blood can significantly improve the survival rate during sepsis.Multiple members of HMGB family proteins have been implicated in mediating inflammatory responses.However,a possible involvement of novel HMGB protein,HMG-Box containing 4(HMGXB4)that we previously cloned,remains unknown.Thus,this study aims to explore the functions and mechanisms of Hmgxb4 gene in systemic inflammatory responseand provide new targets for further understanding and treating systemic inflammation-induced sepsis.Methods:1.Exploring the response of Hmgxb4 to LPS treated murine peritoneal macrophage:1)Cultured murine peritoneal macrophages harvested from WT mice were treated with or without LPS for the indicated time or dose,and total RNA were harvested for q RT-PCR and Western blot analysis.2)Using immunofluorescence stainingto test the location of HMGXB4 in the macrophages with or without LPS treatment.3)Separating the cytoplasmic and nuclear proteins to further test the location of HMGXB4 in the macrophages with or without LPS treatment.2.Establishment of a systemic inflammatory response model and analyzing the role of Hmgxb4 in mice: 1)Generation and characterization of global Hmgxb4 gene knockout mice.2)Analyzing the following phenotypes in WT and Hmgxb4 gene KO mice after administrated with lethal dose LPS or subjected to CLP to induce sepsis.a.Analysis the survival curve;b.HE staining to analyze the lung and kidney tissues damage;c.Using ELISA kitto measure the pro-inflammatory cytokines in the serum.d.TUNEL staining to test the apoptosis cells in the lung tissues.e.Immunofluorescence staining to analyze the macrophages infiltration in the lung tissues.f.Injection of EBD from the tail vein to analyze the pulmonary vascular permeability3.The mechanism study of Hmgxb4 gene in systemic inflammatory response : 1)Identification the differentially expressed genes in LPS-stimulated Hmgxb4 gene KO macrophages using RNA-seq.2)Identification of the potential target genes directly regulated by HMGXB4 by combining the RNA-seq data and the HMGXB4 Ch IP-seq data in the ENCODE database.3)Using q RT-PCR and western blot to validate the transcriptional regulation of HMGXB4 on the potential target Nos2 gene and analysis its biological function.4)Dual luciferase reporter assay to testthe transcriptional regulation role of HMGXB4 on Nos2 gene promoter.5)Using high dose LPS(20 mg/kg)to induce acute lung injury to validate the transcriptional regulation role of HMGXB4 on Nos2 gene using q RTPCR and western blot.6)In LPS induced systemic inflammatory response in WT and Hmgxb4 gene KO mice,using NOS2 inhibitor 1400 W to block NOS2 activity,and then injection of EBD from the tail vein to analyze the pulmonary vascular permeability.Results:1.The nuclear expression of HMGXB4 was induced by LPS in mice peritoneal macrophages:1)The results of q RT-PCR and Western blot revealed that HMGXB4 was significantly induced by LPS treated macrophage at both m RNA and protein levels in timeand dose-dependent manner.2)Immunofluorescence and Western blot analysis of HMGXB4 expression in cytoplasm or nucleus.The results indicated that HMGXB4 expression was induced by LPS treatment and exclusively restricted to the nucleus in macrophages.2.Hmgxb4 gene deficiency ameliorates LPS-induced lethality and tissue injury: 1)Successfully generated the Hmgxb4 gene knockout mice,and the Hmgxb4 global knockout mice appear to be viable and fertile with no obvious difference.2)The survival rate of Hmgxb4-deficient mice was significantly higher than that of WT mice in both endotoxin(LPS)induced sepsis orpoly-microbial(CLP)induced sepsis models.3)The HE staining results revealed that LPS-induced lung and kidney tissues damage were significantly ameliorated in Hmgxb4 gene KO mice.4)The ELISA results showed that there were no significant differences in pro-inflammatory cytokines in the serum between the Hmgxb4 gene KO and WT mice.5)TUNEL staining results showed that the apoptosis in the lungs induced by LPS administration were significantly attenuated in Hmgxb4 gene KO mice.6)The LGALS3(Mac2)immunofluorescence results showed that macrophages’ infiltration following LPS administration were significantly attenuated in Hmgxb4 gene KO mice.7)The albumin-bound EBD in lung tissues showed that Hmgxb4 deficiency drastically ameliorated LPS-mediated pulmonary vascular hyper-permeability.3.Hmgxb4 deletion attenuates LPS-induced systemic inflammatory response by inhibiting LPS-induced NOS2 expression and NO production.1)The analysis of RNAseq results showed that HMGXB4 modulates LPS-induced gene expression.2)Identification of Nos2 gene is a potential target of HMGXB4 by combining the RNA-seq data and the HMGXB4 Ch IP-seq data in the ENCODE database.3)q RT-PCR and Western blot results showed that Hmgxb4 deficiency inhibited LPS-induced transcription and expression of Nos2 gene in macrophage in vitro and attenuated NO production.4)The dual luciferase reporter assay showed that HMGXB4 trans-activates the enhancer activity on the Nos2 gene promoter to regulate the transcription and expression of Nos2 gene.5)q RT-PCR and Western blot results showed that Hmgxb4 deficiency inhibited LPS-induced transcription and expression of Nos2 gene in LPS-induced acute lung injury.6)The albumin-bound EBD in lung tissues showed that Hmgxb4 deficiency or using NOS2 inhibitor 1400 W drastically ameliorated LPS-mediated pulmonary vascular hyper-permeability.Conclusions:1.Hmgxb4 was induced in LPS-elicited macrophages inflammation and exclusively restricted to the nucleus;HMGXB4 modulated the LPS-induced gene expression;HMGXB4 regulated Nos2 gene transcription and expression by trans-activating the enhancer region on the promoter of Nos2 gene.2.Hmgxb4 deficiency attenuated systemic inflammation-induced tissues injury and lethality through inhibition NOS2 expression and NO production.