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.

Figure 1

Nasal Lavage Cells. NLF cells from nonsmokers at baseline were stained with hematoxylin and eosin. Squamous epithelial cells (black arrows) and neutrophils (dashed arrows) can be positively identified. n = 12. A representative image is shown. Bar = 10 μm.

Figure 2

Nasal Lavage Immune Cell Flow Cytometry. NLF cells were collected from nonsmokers at baseline and analyzed by flow cytometry for leukocyte markers. The majority of NLF squamous epithelial cells are gated out by FSC and SSC settings. CD45+ NLF leukocytes are identified in NLF cells. CD45+ NLF cells (P1) are negative for surface markers CD3 (T lymphocytes) CD4 (Helper T lymphocytes), and CD14 (monocytes). CD45+ NLF cells contained populations positive for CD16 (neutrophils, NK cells, P2) and CD56 (NK cells, P3). A representative image at baseline is shown. Open histograms: isotype matched control, P1. Shaded histograms: surface markers, P1.

Figure 3

Characterization of NK Cells in the Nasal Lavage by Flow Cytometry. NK cells were identified in CD45+ NLF cell populations of nonsmokers at baseline. A) Representative flow cytometric plot depicting NK cells (CD56+) and neutrophils (CD56-CD16+). B) Percentages of total NK cells are greater than neutrophils in non-squamous NLF cell populations. C) Percentages of CD16- NK cells are greater than percentages of CD16+ NK cells in non-squamous NLF cell populations. **p < 0.01, * p < 0.05. Nonsmoker n = 11.

Figure 4

Characterization of NK cells in the Nasal Lavage by Immunohistochemistry. NLF cells were characterized using immunohistochemistry. A) NLF cells are stained with anti-CD56-HRP to identify NK cells and B) NLF cells are stained with anti-perforin-HRP to identify cytotoxic NK cells. Bar = 10 μm.

Figure 5

Schematic of LAIV Study Design. Nonsmokers and smokers were given a baseline nasal lavage followed by inoculation with LAIV on Day 0. Serial nasal lavages were obtained on Days 1-4 and again on Day 9. Urine was collected throughout to study to measure cotinine, a metabolite of nicotine, as a marker of cigarette smoke exposure.

Figure 6

LAIV Does Not Significantly Affect Total NK Cell or Neutrophil Percentages in Nonsmokers or Smokers. Using flow cytometry we identified NK cells and neutrophils in nonsmoker and smoker non-squamous NLF cells after LAIV inoculation. Neither A) NK cell nor B) neutrophil percentages in total NLF cells change following LAIV in either group. Nonsmokers n = 12 (■, solid line), smokers n = 9 (▲, dashed line).

Figure 7

Cytotoxic NK Cell Activity is Suppressed in Smokers Following LAIV. Cytotoxic NK cell percentages and activity were analyzed in NLF of nonsmokers and smokers after LAIV inoculation. A) CD16+ cytotoxic NK cell percentages of total NK cells were decreased in the NLF of smokers following LAIV. Kruskal-Wallis p = 0.09, *p < 0.05 nonsmoker vs smoker posttest. Nonsmokers n = 12 (■, solid line), smokers n = 9 (▲, dashed line). B) Granzyme B activity was decreased in NLF of smokers following LAIV inoculation. Kruskal-Wallis p < 0.01, *p < 0.05 nonsmoker vs smoker post-test. Nonsmokers n = 13 (■, solid line), smokers n = 13 (▲, dashed line).

Figure 8

Effect of LAIV On Peripheral NK Cell Activity. Peripheral blood NK cells were isolated from nonsmokers and smokers and stimulated in vitro with LAIV. LAIV did not increase A) percent CD16+ cytotoxic NK cells or B) granzyme b activity in either nonsmokers or smokers. LAIV increased expression of C) NK cell activation receptor NKG2D and D) chemokine receptor CXCR3 in both nonsmokers and smokers. Nonsmokers n = 6 (black bars), smokers n = 6 (white bars). *** p < 0.001 vs vehicle control.