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. 2015 Nov;129(9):785-96.
doi: 10.1042/CS20140703. Epub 2015 Jul 1.

Innate cellular sources of interleukin-17A regulate macrophage accumulation in cigarette- smoke-induced lung inflammation in mice

Steven Bozinovski  1 , Huei Jiunn Seow  2 , Sheau Pyng Jamie Chan  2 , Desiree Anthony  2 , Jonathan McQualter  2 , Michelle Hansen  2 , Brendan J Jenkins  3 , Gary P Anderson  2 , Ross Vlahos  4
Affiliations

Innate cellular sources of interleukin-17A regulate macrophage accumulation in cigarette- smoke-induced lung inflammation in mice

Steven Bozinovski et al. Clin Sci (Lond). 2015 Nov.

Abstract

Cigarette smoke (CS) is the major cause of chronic obstructive pulmonary disease (COPD). Interleukin-17A (IL-17A) is a pivotal cytokine that regulates lung immunity and inflammation. The aim of the present study was to investigate how IL-17A regulates CS-induced lung inflammation in vivo. IL-17A knockout (KO) mice and neutralization of IL-17A in wild-type (WT) mice reduced macrophage and neutrophil recruitment and chemokine (C-C motif) ligand 2 (CCL2), CCL3 and matrix metalloproteinase (MMP)-12 mRNA expression in response to acute CS exposure. IL-17A expression was increased in non-obese diabetic (NOD) severe combined immunodeficiency SCID) mice with non-functional B- and T-cells over a 4-week CS exposure period, where macrophages accumulated to the same extent as in WT mice. Gene expression analysis by QPCR (quantitative real-time PCR) of isolated immune cell subsets detected increased levels of IL-17A transcript in macrophages, neutrophils and NK/NKT cells in the lungs of CS-exposed mice. In order to further explore the relative contribution of innate immune cellular sources, intracellular IL-17A staining was performed. In the present study, we demonstrate that CS exposure primes natural killer (NK), natural killer T (NKT) and γδ T-cells to produce more IL-17A protein and CS alone increased the frequency of IL17+ γδ T-cells in the lung, whereas IL-17A protein was not detected in macrophages and neutrophils. Our data suggest that activation of innate cellular sources of IL-17A is an essential mediator of macrophage accumulation in CS-exposed lungs. Targeting non-conventional T-cell sources of IL-17A may offer an alternative strategy to reduce pathogenic macrophages in COPD.

Keywords: chronic lung disease; chronic obstructive pulmonary disease (COPD); cigarette smoke; innate immunity; interleukin-17; macrophage.

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Figures

Figure 1
Figure 1. Effect of CS exposure on BALF cellularity in WT and IL-17A-/- mice
IL-17A−/− mice have reduced BALF total cell number (A), macrophages (B) and neutrophils (C) in response to 4 days of CS exposure. Data are shown as means±S.E.M. for eight to 12 mice per treatment group. White bars represent sham-exposed mice and black bars represent smoke-exposed mice. *P<0.001 compared with corresponding sham, †P<0.001 compared with WT smoke.
Figure 2
Figure 2. Effect of 4 days of CS exposure on whole lung gene expression in WT and IL-17A-/- mice
mRNA expression for all genes was measured simultaneously under identical conditions using QPCR. Responses are shown as fold expression relative to 18S rRNA. Data are shown as means±S.E.M. of three replicates, as previously described [34]. White bars represent sham-exposed mice and black bars represent CS-exposed mice.
Figure 3
Figure 3. Effect of anti-IL-17A mAb on BALF cellularity in CS-exposed mice
Anti-IL-17A mAb inhibits CS (4 days)-induced increases in BALF total cell number (A), macrophages (B) and neutrophils (C) in BALB/c mice. Data are shown as means±S.E.M. for eight mice per treatment group. White bars represent isotype control-treated mice and black bars represent anti-IL-17A mAb (50 μg/mouse)-treated mice. *P<0.001 compared with respective sham, †P<0.001 compared with isotype-treated CS-exposed mice.
Figure 4
Figure 4. Effect of anti-IL-17A mAb on whole lung gene expression in CS-exposed mice
Anti-IL-17A mAb inhibits CS (4 days)-induced increases in BALB/c whole lung IL-17A (A), CCL2 (B), CCL3 (C) and MMP-12 (D) mRNA. mRNA expression for all genes was measured simultaneously under identical conditions using QPCR. Responses are shown as fold expression relative to 18S rRNA. Data are shown as means±S.E.M. of three replicates, as previously described [34]. White bars represent isotype control-treated mice and black bars represent anti-IL-17A mAb (50 μg/mouse)-treated mice.
Figure 5
Figure 5. Effect of CS exposure on NOD SCID mouse BALF cellularity
NOD SCID mice were exposed to CS for 4 days, 2 weeks and 4 weeks. Sham mice were not exposed to CS. DiffQuik-stained cytospins were generated from BAL cells isolated from NOD SCID (SCID) mice (A). IL-17A expression was measured using QPCR in NOD SCID mice at the specified time points over the 4-week CS-exposure period (B). The total number of BAL macrophages (C) and neutrophils (D) presented as means±S.E.M. (n=8–10). ††P<0.01, ‡P<0.001 compared with sham; ***P<0.001 compared with BALB/c mice.
Figure 6
Figure 6. Alternative innate cellular sources of IL-17A in CS-exposed lungs
Individual immune cell populations were isolated from whole lung homogenates from sham- and CS (4 days)-exposed mice as detailed in the Materials and methods section. Gene expression was determined by QPCR analysis using an IL-17A Taqman assay in sorted macrophages/DCs (A), neutrophils (B), NK/NKT cells (C) and lymphocytes (D). For analysis of macrophage/DC and neutrophil expression, ΔCT (IL-17A–18S CT) values are presented as IL-17A expression was not detected (n.d.) in sham-exposed mice. For expression in CD4+ T-cells and NK cells, the ΔΔCT method was used as IL-17A transcript expression was detected in sham-exposed mice.
Figure 7
Figure 7. Effect of short-term CS exposure on NK, NKT and γδT-cell frequency in the lungs
Lungs were harvested from mice following exposure to CS or sham for 4 days. Representative dot plots gating on (A) lung NK (CD49b+, TCRβ−) and NKT (CD49b+, TCRβ+) cells and (B) γδ T-(γδ-TCR+, CD3+) cells. Numbers represent the frequency of each cell population relative to single CD45+ lymphocytic events. (C) Single- cell suspensions from CS-exposed mice were cultured for 4 h in medium containing brefeldin with or without PMA/ionomycin and stained for intracellular IL-17A. The frequency (percentage of total IL-17A+ cells) of IL-17+ innate T-cells in innate immune cellular sources (that include NK, NKT and γδ T-cells) is compared with IL-17A+ conventional CD4+ T-cells.
Figure 8
Figure 8. The frequency of intracellular IL-17A+ innate T-cell subsets following CS exposure
Lungs were harvested from mice exposed to CS or sham for 4 days; single-cell suspensions were generated and cultured for 4 h in medium containing brefeldin with or without PMA/ionomycin prior to staining for intracellular IL-17A. Representative dot plots gating on IL-17A+ cells in (A) lung NK, (C) NKT cells and (E) γδ T-cells. Numbers represent the percentage of IL-17+ cells relative to each cell subset. The adjacent histograms show the frequency of IL-17A+ NK (B), NKT (D) and γδ T- cells (F) relative to all single CD45+ cells. Pooled data from n=4 mice per group. *P<0.05, as determined by two-way ANOVA.
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