Diesel exhaust particles modify natural killer cell function and cytokine release

Abstract

Background: Natural killer (NK) cells are an important lymphocyte population in the nasal mucosa and play important roles in linking the innate and the adaptive immune response. Their two main functions are direct cell-mediated cytotoxicity and the release of cytokines. They are important during viral infections and cancer. Due to their location in the nasal mucosa, NK cells are likely exposed to inhaled pollutants, such as diesel exhaust. Whether and how exposure to diesel exhaust particles (DEP) affects NK cell function in the context of viral infections has not been investigated.

Methods: NK cells were isolated from peripheral blood obtained from normal healthy volunteers and subsequently stimulated with the viral mimetic polyinosinic:polycytidylic acid (pI:C), DEP, or pI:C+DEP for 18 hours. NK cells were subsequently analyzed for changes in surface marker expression, cytokine production, gene expression changes, and cytotoxic function using flow cytometry, ELISA, qRT-PCR, and cell-mediated cytotoxicity assay, respectively.

Results: Stimulation of NK cells with pI:C and pI:C+DEP, but not DEP alone, increased the release of IL-1β, IL-2, IL-4, IL-8, IL-10, IL-12p70, IFN-γ and TNF-α. As compared to pI:C alone or pI:C+DEP, the release of IL-1β, IL-8 and TNF-α was significantly lower after DEP stimulation alone. Stimulation with pI:C alone increased the gene and protein expression of granzyme B and perforin, which was completely blunted by adding DEP. Addition of DEP further reduced CD16 expression in pI:C stimulated cells. Similarly, cell-mediated cytotoxicity was significantly reduced by the addition of DEP.

Conclusions: In the context of viral infection, DEP potentially reduces NK cells' ability to kill virus-infected host cells, in spite of normal cytokine levels, and this may increase susceptibility to viral infections . This reduction in the potential ability of NK cells to kill virus-infected host cells may increase the susceptibility to viral infections after DEP exposure.

Figure 1

RNA levels of cytotoxicity related factors in NK cells after stimulation with pI:C, DEP and pI:C+DEP. (A) Gene expression of granzyme B and (B) gene expression of perforin expressed as fold induction over untreated control. (C) Protein levels of granzyme B and (D) protein levels of perforin expressed as fold induction over untreated control. Data are presented as mean±SEM. *significantly different from the untreated control, ^# significantly different between each other (both tested with a Wilcoxon signed rank test, *^,#p<0.05, n=9).

Figure 2

NK Cell phenotypes assessed by flow cytometry after exposure to pI:C, DEP and pI:C+DEP. (A) Gating strategy for the phenotype analysis. (B) Representative histogram showing that CD16 expression decreased after p:IC+DEP exposure compared to pI:C alone and the subset of CD16- NK cells increased. (C&D) Surface markers presented as fold induction over untreated control of percent of positive total NK cells (C) or mean fluorescecne intensity (MFI) (D). Data are presented as mean±SEM. *significant different from the untreated control, ^# significant different between each other (both tested with a Wilcoxon signed rank test, *^,#p<0.05, n=9).

Figure 3

Controls for the cell-mediated cytotoxic potential of NK. (A) Representative dot plot of the NK-target cell mixture. Target cells were gated based on side scatter (SSC) properties and CFSE staining. (B-E) NK cell viability is not affected signifcantly by treatment DEP. Only cells gated for NK cells (based on SSC and negative CFSE staining) are shown. (F&G) The live-dead dye 7-AAD is not adsorbed by DEP. (F) 7-AAD pre-incubated with DEP for 15 min before staining a control sample of NK-target cell mixture after 4 hrs of incubation. (G) Control sample stained with 7-AAD normally after 4 hrs of incubation. (H&I) DEP do not kill target cells. (H) Target cells only stained with 7-AAD. (I) Target cells only incubated with DEP and stained with 7-AAD.

Figure 4

Cell-mediated cytotoxicity potential of NK cells exposed to pI:C, DEP or pI:C+DEP. Results were assessed as percentage of killed target cells (K562 cancer cell line) and are expressed as fold induction over untreated NK cells. (A-E) Representative dot plots of all four conditions plus IL-12 stimulated NK cells as positive control to enhance cytotoxicity. (F) Summary of 5 individual experiments. Data are presented as mean±SEM. *significantly different from control, ^#significantly different between each other, *^,#p<0.05.