title: Interferon regulatory factor-1 in flagellin-induced reprogramming: potential protective role of CXCL10 in cornea innate defense against Pseudomonas aeruginosa infection.
abstract: OBJECTIVE
  We previously showed that pre-exposure of the cornea to Toll-like receptor (TLR)5 ligand flagellin induces strong protective innate defense against microbial pathogens and hypothesized that flagellin modulates gene expression at the transcriptional levels. Thus, we sought to determine the role of one transcription factor, interferon regulatory factor (IRF1), and its target gene CXCL10 therein.
  
  
  METHODS
  Superarray was used to identify transcription factors differentially expressed in Pseudomonas aeruginosa-challenged human corneal epithelial cells (CECs) with or without flagellin pretreatment. The expression of CXCL10, IRF1, LI-8(CXCL2), and IFNγ was determined by PCR, immunohistochemistry, Western/dot blotting, and/or ELISA. IRF1 knockout mice, CXCL10 and IFNγ neutralization, and NK cell depletion were used to define in vivo regulation and function of CXCL10. The severity of P. aeruginosa was assessed using clinical scoring, slit-lamp microscopy, bacterial counting, polymorphonuclear leukocytes (PMN) infiltration, and macrophage inflammatory protein 2/Chemokine (C-X-C motif) ligand 2 (MIP-2/CXCL2) expression.
  
  
  RESULTS
  Flagellin pretreatment drastically affected P. aeruginosa-induced IRF1 expression in human CECs. However, flagellin pretreatment augmented the P. aeruginosa-induced expression of Irf1 and its target gene Cxcl10 in B6 mouse corneas. Irf1 deficiency reduced infection-triggered CXCL10 expression, increased keratitis severity, and attenuated flagellin-elicited protection compared to values in wild-type (WT) controls. CXCL10 neutralization in the cornea of WT mice displayed pathogenesis similar to that of IRF1⁻/⁻ mice. IFNγ receptor neutralization and NK cell depletion prevented flagellin-augmented IRF1 and CXCL10 expression and increased the susceptibility to P. aeruginosa infection in mouse corneas.
  
  
  CONCLUSIONS
  IRF1 plays a role in the corneal innate immune response by regulating CXCL10 expression. IFNγ-producing NK cells augment the epithelial expression of IRF1 and CXCL10 and thus contribute to the innate defense of the cornea against P. aeruginosa infection.
authors.affiliation: Department of Anatomy and Cell Biology, Kresge Eye Institute, Wayne State University, Detroit, Michigan.
authors.forename: Gi Sang
authors.initials: GS
authors.lastname: Yoon
authors.forename: Chen
authors.initials: C
authors.lastname: Dong
authors.forename: Nan
authors.initials: N
authors.lastname: Gao
authors.forename: Ashok
authors.initials: A
authors.lastname: Kumar
authors.forename: Theodore J
authors.initials: TJ
authors.lastname: Standiford
authors.forename: Fu-Shin X
authors.initials: FS
authors.lastname: Yu
mesh_terms: D000818:Animals; D015153:Blotting, Western; D002478:Cells, Cultured; D054357:Chemokine CXCL10 / Q000378:metabolism*; D054426:Chemokine CXCL2 / Q000378:metabolism; D015169:Colony Count, Microbial; D003320:Corneal Ulcer / Q000276:immunology / Q000378:metabolism / Q000517:prevention & control*; D004797:Enzyme-Linked Immunosorbent Assay; D019573:Epithelium, Corneal / Q000187:drug effects / Q000378:metabolism / Q000382:microbiology; D015818:Eye Infections, Bacterial / Q000276:immunology / Q000378:metabolism / Q000517:prevention & control*; D005408:Flagellin / Q000494:pharmacology*; D006801:Humans; D007113:Immunity, Innate / Q000502:physiology; D007150:Immunohistochemistry; D050856:Interferon Regulatory Factor-1 / Q000502:physiology*; D007371:Interferon-gamma / Q000378:metabolism; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D011552:Pseudomonas Infections / Q000276:immunology / Q000378:metabolism / Q000517:prevention & control*; D011550:Pseudomonas aeruginosa / Q000502:physiology*; D060888:Real-Time Polymerase Chain Reaction
chemical_list: C114753:CXCL10 protein, human; C499706:CXCL2 protein, human; D054357:Chemokine CXCL10; D054426:Chemokine CXCL2; D050856:Interferon Regulatory Factor-1; D005408:Flagellin; D007371:Interferon-gamma
keywords: CXCL10; IRF1; cell reprogramming; inflammation; toll-like receptors