title: MRP8/14 enhances corneal susceptibility to Pseudomonas aeruginosa Infection by amplifying inflammatory responses.
abstract: OBJECTIVE
  We explored the role of myeloid-related protein 8 and 14 (MRP8/14) in Pseudomonas aeruginosa (PA) keratitis.
  
  
  METHODS
  MRP8/14 mRNA levels in human corneal scrapes and mouse corneas infected by PA were tested using real-time PCR. MRP8/14 protein expression in C57BL/6 (B6) corneas was confirmed using Western blot assay and immunohistochemistry. B6 mice were injected subconjunctivally with siRNA for MRP8/14, and then infected with PA. Bacterial plate counts and myeloperoxidase assays were used to determine the bacterial load and polymorphonuclear neutrophil (PMN) infiltration in infected B6 corneas. Pro-inflammatory cytokine levels in vivo and in vitro were examined with PCR and ELISA. In murine macrophage-like RAW264.7 cells, phagocytosis and bacterial killing were assessed using plate count assays, and reactive oxygen species (ROS) and nitric oxide (NO) levels were tested with flow cytometry and Griess assay, respectively.
  
  
  RESULTS
  MRP8/14 expression levels were increased significantly in human corneal scrapes and B6 corneas after PA infection. Silencing of MRP8/14 in B6 corneas significantly reduced the severity of corneal disease, bacterial clearance, PMN infiltration, and pro-inflammatory cytokine expression after PA infection. In vitro studies demonstrated further that silencing of MRP8/14 suppressed pro-inflammatory cytokine production, bacterial killing, and ROS production, but not phagocytosis or NO production.
  
  
  CONCLUSIONS
  Our study demonstrated a dual role for MRP8/14 in bacterial keratitis. Although MRP8/14 promotes bacterial clearance by enhancing ROS production, it functions more importantly as an inflammatory amplifier at the ocular surface by enhancing pro-inflammatory cytokine expression, thus contributing to the corneal susceptibility.
authors.affiliation: Department of Immunology, Zhongshan School of Medicine, Institute of Human Virology, Sun Yat-sen University, Guangzhou, China.
authors.forename: Qiuchan
authors.initials: Q
authors.lastname: Deng
authors.forename: Mingxia
authors.initials: M
authors.lastname: Sun
authors.forename: Kun
authors.initials: K
authors.lastname: Yang
authors.forename: Min
authors.initials: M
authors.lastname: Zhu
authors.forename: Kang
authors.initials: K
authors.lastname: Chen
authors.forename: Jin
authors.initials: J
authors.lastname: Yuan
authors.forename: Minhao
authors.initials: M
authors.lastname: Wu
authors.forename: Xi
authors.initials: X
authors.lastname: Huang
mesh_terms: D000328:Adult; D000818:Animals; D040501:Calgranulin A / Q000235:genetics / Q000378:metabolism*; D040502:Calgranulin B / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D003315:Cornea / Q000276:immunology / Q000378:metabolism / Q000382:microbiology; D016207:Cytokines / Q000276:immunology / Q000378:metabolism; D005260:Female; D015870:Gene Expression / Q000276:immunology; D020868:Gene Silencing / Q000276:immunology; D006801:Humans; D007634:Keratitis / Q000276:immunology / Q000378:metabolism*; D008264:Macrophages / Q000166:cytology / Q000276:immunology / Q000378:metabolism; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D008875:Middle Aged; D009000:Monocytes / Q000166:cytology / Q000276:immunology / Q000378:metabolism; D009569:Nitric Oxide / Q000276:immunology / Q000378:metabolism; D010587:Phagocytosis / Q000276:immunology; D011552:Pseudomonas Infections / Q000276:immunology / Q000378:metabolism*; D011550:Pseudomonas aeruginosa / Q000254:growth & development* / Q000276:immunology; D012333:RNA, Messenger / Q000378:metabolism; D017382:Reactive Oxygen Species / Q000276:immunology / Q000378:metabolism
chemical_list: D040501:Calgranulin A; D040502:Calgranulin B; D016207:Cytokines; D012333:RNA, Messenger; D017382:Reactive Oxygen Species; D009569:Nitric Oxide