title: Toll-like receptor 2 and 4 ligation results in complex altered cytokine profiles early and late after burn injury.
abstract: BACKGROUND
  Toll-like receptors (TLR) 2 and TLR4 expressed on innate immune cells are important mediators of the immune response to pathogens. In this study, we hypothesized that burn injury results in altered cytokine secretion profiles after TLR2 or TLR4 ligation that is associated with altered TLR expression on innate immune cells.
  
  
  METHODS
  Female C56BL/6 mice were subjected to 20% full thickness burn or sham injury. Three or 14 days after injury whole splenocytes or purified splenic macrophages were cultured with TLR2 ligand peptidoglycan or TLR4 ligand lipopolysaccharide. Supernatants were assayed for TNF-alpha, MCP-1, IL-6 and IL-10. Cell death was assessed using flow cytometry. Innate CD11b F4/80 macrophages were sorted 14 days after burn injury and TLR2 and 4 expression was determined by quantitative reverse-transcriptase polymerase chain reaction and flow cytometry.
  
  
  RESULTS
  Burn injury results in a steady accumulation in the periphery of CD11bF4/80 macrophages. Macrophages purified early after burn injury upregulated TLR2 and 4, followed by a decrease of TLR2 and TLR4 expression late after burn injury. TLR2 and TLR4 ligation of an equivalent number of purified macrophages 3 days after burn injury revealed no significant differences in cytokine secretion compared with sham. Stimulation 14 days after burn injury revealed a significant reduction in tumor necrosis factor-alpha secretion by macrophages compared with sham mice. In contrast, interleukin-10 was significantly increased (mean, approximately 1.8-fold) late after burn injury after either TLR2 or TLR4 stimulation. Interleukin-6 and monocyte chemotactic protein-1 secretion was unchanged from sham levels. In contrast, whole splenocyte stimulation resulted in increased cytokine 3 days and 14 days after burn injury. This effect is likely caused by the accumulation of TLR macrophages, which are resistant to TLR-induced cell death.
  
  
  CONCLUSIONS
  Cytokine secretion profiles after TLR2 and TLR4 ligation after burn injury are altered in a manner not clearly reflective of an anti-inflammatory or proinflammatory state and are associated with unique changes in the macrophage population. TLR2 and TLR4 ligation have complex and varied roles in mediating the immune response to burn injury.
authors.affiliation: Department of Surgery and North Carolina Jaycee Burn Center, Chapel Hill, North Carolina, USA. bruce_cairns@med.unc.edu
authors.forename: Bruce A
authors.initials: BA
authors.lastname: Cairns
authors.forename: Carie M
authors.initials: CM
authors.lastname: Barnes
authors.forename: Stefan
authors.initials: S
authors.lastname: Mlot
authors.forename: Anthony A
authors.initials: AA
authors.lastname: Meyer
authors.forename: Robert
authors.initials: R
authors.lastname: Maile
mesh_terms: D000818:Animals; D002056:Burns / Q000276:immunology* / Q000503:physiopathology; D016923:Cell Death; D002478:Cells, Cultured; D016207:Cytokines / Q000032:analysis / Q000378:metabolism*; D004195:Disease Models, Animal; D005260:Female; D005434:Flow Cytometry; D007113:Immunity, Innate / Q000502:physiology*; D018836:Inflammation Mediators / Q000378:metabolism; D008070:Lipopolysaccharides / Q000494:pharmacology*; D008264:Macrophages / Q000166:cytology; D051379:Mice; D008810:Mice, Inbred C57BL; D010457:Peptidoglycan / Q000494:pharmacology*; D011897:Random Allocation; D012016:Reference Values; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012680:Sensitivity and Specificity; D013154:Spleen / Q000166:cytology; D013997:Time Factors; D051195:Toll-Like Receptor 2 / Q000276:immunology / Q000378:metabolism; D051197:Toll-Like Receptor 4 / Q000276:immunology / Q000378:metabolism
chemical_list: D016207:Cytokines; D018836:Inflammation Mediators; D008070:Lipopolysaccharides; D010457:Peptidoglycan; D051195:Toll-Like Receptor 2; D051197:Toll-Like Receptor 4