title: Macrophage genetic reprogramming during chronic peritonitis is augmented by LPS pretreatment.
abstract: BACKGROUND
  Persistent and tertiary chronic peritonitis is a clinically challenging problem especially in those who are critically ill. This could be attributed to a state of immune-paralysis, known as microbial tolerance. Microbial tolerance is the diminished pro-inflammatory protein response following repeated stimulation by numerous pathogen-associated molecular patterns (PAMPs) of varying origins, which we have shown in this novel model of chronic peritonitis. We aimed in this study to investigate the molecular mechanisms behind microbial tolerance and the early innate immune response resolution in this model.
  
  
  METHODS
  C57BL/6 mice were pretreated intra-peritoneally (IP) with saline or endotoxin LPS 10 mg/kg (LPS 10). Following pretreatment, peritonitis was induced 24 h later injecting 10(3)Klebsiella pneumonia CFU IP. Gentamicin was administered 4 h prior to infection and BID thereafter. Peritoneal exudate cells (PEC) were obtained through peritoneal lavage and RNA was isolated (n = 3) at 4, 24, and 48 h following infection. SA Biosciences© RT2-Profiler PCR array mouse Toll-like receptor signaling pathway (PAMM_018A) data were further analyzed by Ingenuity Pathway Inc. analysis (IPA).
  
  
  RESULTS
  Of the 89 genes studied, 26 were significantly up-regulated (fold change > 1.2 and P value < 0.05) in the saline pretreated group at 4, 24, and 48 h after infection. There were no down-regulated genes. In the LPS-pretreated group, 35 genes were significantly up-regulated; of these genes, 13 were not increased in the saline pretreated infected mice. This left 22 up-regulated genes in both infected groups. At 4 h, 6 of these 22 genes (CHUK, HMGB1, HSPD1, IRAK2, LY96, and TLR4) were further 2-fold increased in the LPS pretreatment group compared with the saline pretreatment group. Only IRAK2 was 2-fold increased at 24 h. By 48 h, no LPS effect was seen. When applying IPA analysis, six main canonical pathways were constantly dysregulated in the same significance order in both the saline and LPS group at 4, 24, and 48 h. These were: Toll-like receptor and NF-κB signaling, hepatic cholestasis, interleukin-6, and LPS-mediated MAPK signaling pathways, and pattern recognition receptors of bacterial pathway.
  
  
  CONCLUSIONS
  Peritonitis increased PEC gene expression associated with sepsis and a pro-inflammatory response, which was further augmented by LPS pretreatment over 24 h only. Prior exposure to LPS did not induce PEC gene tolerance to subsequent infection with Klebsiella at the mRNA level. Post-transcriptional modification as microRNA down-regulation of inflammatory cytokines could possibly explain such phenomena.
authors.affiliation: Department of Surgery, University of Louisville, School of Medicine, Louisville, Kentucky 40292, USA.
authors.forename: Ziad
authors.initials: Z
authors.lastname: Kanaan
authors.forename: Sarah
authors.initials: S
authors.lastname: Gardner
authors.forename: Christopher
authors.initials: C
authors.lastname: Carruba
authors.forename: Jameson
authors.initials: J
authors.lastname: Mattingly
authors.forename: Devin
authors.initials: D
authors.lastname: Druen
authors.forename: William G
authors.initials: WG
authors.lastname: Cheadle
mesh_terms: D000818:Animals; D018834:Chaperonin 60 / Q000235:genetics / Q000378:metabolism; D002908:Chronic Disease; D004195:Disease Models, Animal; D020869:Gene Expression Profiling; D005786:Gene Expression Regulation / Q000187:drug effects*; D024243:HMGB1 Protein / Q000235:genetics / Q000378:metabolism; D051550:I-kappa B Kinase / Q000235:genetics / Q000378:metabolism; D007108:Immune Tolerance / Q000187:drug effects*; D007113:Immunity, Innate / Q000187:drug effects / Q000502:physiology; D053592:Interleukin-1 Receptor-Associated Kinases / Q000235:genetics / Q000378:metabolism; D007711:Klebsiella pneumoniae / Q000302:isolation & purification; D008070:Lipopolysaccharides / Q000494:pharmacology*; D051218:Lymphocyte Antigen 96 / Q000235:genetics / Q000378:metabolism; D017737:Macrophages, Peritoneal / Q000187:drug effects / Q000276:immunology* / Q000378:metabolism*; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D024101:Mitochondrial Proteins / Q000235:genetics / Q000378:metabolism; D010537:Peritoneum / Q000382:microbiology / Q000473:pathology; D010538:Peritonitis / Q000276:immunology* / Q000378:metabolism* / Q000473:pathology; D051197:Toll-Like Receptor 4 / Q000235:genetics / Q000378:metabolism; D015854:Up-Regulation / Q000502:physiology
chemical_list: D018834:Chaperonin 60; D024243:HMGB1 Protein; C557066:Hspd1 protein, mouse; D008070:Lipopolysaccharides; C411728:Ly96 protein, mouse; D051218:Lymphocyte Antigen 96; D024101:Mitochondrial Proteins; C493487:Tlr4 protein, mouse; D051197:Toll-Like Receptor 4; D053592:Interleukin-1 Receptor-Associated Kinases; C496564:Chuk protein, mouse; D051550:I-kappa B Kinase