title: Nasal lavage natural killer cell function is suppressed in smokers after live attenuated influenza virus.
abstract: BACKGROUND
  Modified function of immune cells in nasal secretions may play a role in the enhanced susceptibility to respiratory viruses that is seen in smokers. Innate immune cells in nasal secretions have largely been characterized by cellular differentials using morphologic criteria alone, which have successfully identified neutrophils as a significant cell population within nasal lavage fluid (NLF) cells. However, flow cytometry may be a superior method to fully characterize NLF immune cells. We therefore characterized immune cells in NLF by flow cytometry, determined the effects of live attenuated influenza virus (LAIV) on NLF and peripheral blood immune cells, and compared responses in samples obtained from smokers and nonsmokers.
  
  
  METHODS
  In a prospective observational study, we characterized immune cells in NLF of nonsmokers at baseline using flow cytometry and immunohistochemistry. Nonsmokers and smokers were inoculated with LAIV on day 0 and serial nasal lavages were collected on days 1-4 and day 9 post-LAIV. LAIV-induced changes of NLF cells were characterized using flow cytometry. Cell-free NLF was analyzed for immune mediators by bioassay. Peripheral blood natural killer (NK) cells from nonsmokers and smokers at baseline were stimulated in vitro with LAIV followed by flow cytometric and mediator analyses.
  
  
  RESULTS
  CD45(+)CD56(-)CD16(+) neutrophils and CD45(+)CD56(+) NK cells comprised median 4.62% (range 0.33-14.52) and 23.27% (18.29-33.97), respectively, of non-squamous NLF cells in nonsmokers at baseline. LAIV did not induce changes in total NK cell or neutrophil percentages in either nonsmokers or smokers. Following LAIV inoculation, CD16(+) NK cell percentages and granzyme B levels increased in nonsmokers, and these effects were suppressed in smokers. LAIV inoculation enhanced expression of activating receptor NKG2D and chemokine receptor CXCR3 on peripheral blood NK cells from both nonsmokers and smokers in vitro but did not induce changes in CD16(+) NK cells or granzyme B activity in either group.
  
  
  CONCLUSIONS
  These data are the first to identify NK cells as a major immune cell type in the NLF cell population and demonstrate that mucosal NK cell cytotoxic function is suppressed in smokers following LAIV. Altered NK cell function in smokers suggests a potential mechanism that may enhance susceptibility to respiratory viruses.
authors.affiliation: Curriculum in Toxicology, The University of North Carolina at Chapel Hill, Chapel Hill, NC 27599-7127, USA.
authors.forename: Katherine M
authors.initials: KM
authors.lastname: Horvath
authors.forename: Margaret
authors.initials: M
authors.lastname: Herbst
authors.forename: Haibo
authors.initials: H
authors.lastname: Zhou
authors.forename: Hongtao
authors.initials: H
authors.lastname: Zhang
authors.forename: Terry L
authors.initials: TL
authors.lastname: Noah
authors.forename: Ilona
authors.initials: I
authors.lastname: Jaspers
mesh_terms: D000281:Administration, Intranasal; D000328:Adult; D000704:Analysis of Variance; D019002:CD56 Antigen / Q000378:metabolism; D016022:Case-Control Studies; D002478:Cells, Cultured; D003602:Cytotoxicity, Immunologic; D005260:Female; D005434:Flow Cytometry; D058851:GPI-Linked Proteins / Q000378:metabolism; D053804:Granzymes / Q000378:metabolism; D006801:Humans; D007113:Immunity, Innate; D007150:Immunohistochemistry; D016130:Immunophenotyping / Q000379:methods; D007252:Influenza Vaccines / Q000008:administration & dosage*; D007694:Killer Cells, Natural / Q000276:immunology* / Q000821:virology; D017493:Leukocyte Common Antigens / Q000378:metabolism; D008137:Longitudinal Studies; D008297:Male; D055655:NK Cell Lectin-Like Receptor Subfamily K / Q000378:metabolism; D017817:Nasal Lavage Fluid / Q000276:immunology / Q000821:virology; D009657:North Carolina; D009975:Orthomyxoviridae / Q000276:immunology*; D011446:Prospective Studies; D054367:Receptors, CXCR3 / Q000378:metabolism; D017452:Receptors, IgG / Q000378:metabolism; D012907:Smoking / Q000276:immunology*; D013997:Time Factors; D014613:Vaccines, Attenuated / Q000008:administration & dosage; D055815:Young Adult
chemical_list: D019002:CD56 Antigen; C515187:CXCR3 protein, human; C428200:FCGR3B protein, human; D058851:GPI-Linked Proteins; D007252:Influenza Vaccines; C527085:KLRK1 protein, human; C522918:NCAM1 protein, human; D055655:NK Cell Lectin-Like Receptor Subfamily K; D054367:Receptors, CXCR3; D017452:Receptors, IgG; D014613:Vaccines, Attenuated; D017493:Leukocyte Common Antigens; C516769:PTPRC protein, human; C509213:GZMB protein, human; D053804:Granzymes