Exposure to cigarette smoke impacts myeloid-derived regulatory cell function and exacerbates airway hyper-responsiveness

Abstract

Cigarette smoking enhances oxidative stress and airway inflammation in asthma, the mechanisms of which are largely unknown. Myeloid-derived regulatory cells (MDRC) are free radical producing immature myeloid cells with immunoregulatory properties that have recently been demonstrated as critical regulators of allergic airway inflammation. NO (nitric oxide)-producing immunosuppressive MDRC suppress T-cell proliferation and airway-hyper responsiveness (AHR), while the O2(•-) (superoxide)-producing MDRC are proinflammatory. We hypothesized that cigarette smoke (CS) exposure may impact MDRC function and contribute to exacerbations in asthma. Exposure of bone marrow (BM)-derived NO-producing MDRC to CS reduced the production of NO and its metabolites and inhibited their potential to suppress T-cell proliferation. Production of immunoregulatory cytokine IL-10 was significantly inhibited, while proinflammatory cytokines IL-6, IL-1β, TNF-α and IL-33 were enhanced in CS-exposed BM-MDRC. Additionally, CS exposure increased NF-κB activation and induced BM-MDRC-mediated production of O2(•-), via NF-κB-dependent pathway. Intratracheal transfer of smoke-exposed MDRC-producing proinflammatory cytokines increased NF-κB activation, reactive oxygen species and mucin production in vivo and exacerbated AHR in C57BL/6 mice, mice deficient in Type I IFNR and MyD88, both with reduced numbers of endogenous MDRC. Thus CS exposure modulates MDRC function and contributes to asthma exacerbation and identifies MDRC as potential targets for asthma therapy.

Figure 1. In vitro differentiation and characterization of Ly-6C⁺MDRC from bone marrow precursors

(A) Flow cytometry plots of differentiated BM-MDRC showing forward and side scatter plots and percentage of Ly-6C⁺ and F4/80⁺ cells within the Gr-1⁺CD11b⁺ gated cells (B) Overlaid histogram flow cytometry plots showing levels of expression of cell surface and intracellular markers that define the phenotype of MDRC. Black lines represent isotype controls and gray lines represent cells positive for the indicated marker.

Figure 2. Cigarette smoke exposure inhibits NO production, enhances T cell proliferation and induces a proinflammatory cytokine signature in MDRC

(A) In vitro differentiated Ly-6C⁺ MDRC were immunosorted and were exposed to cigarette smoke as described in methods. Cell culture supernatants were collected at 0-24 hours after culture. Levels of nitrite and nitrate in the culture supernatants were measured in triplicates using Griess assay. Data are Mean ± S.D., **p<0.001 from statistical comparisons of each time point to unexposed controls using ANOVA (B) Flow cytometry plots of CFSE labeled 10⁵ OVA transgenic T cells co-cultured with 10⁵ CS exposed or unexposed MDRC with 10⁴ BMDC pulsed with OVA peptide. CFSE dilution was assessed at 72 hours after culture. Right panel shows flow cytometry analyses of CFSE dilution from triplicate experiments of co-cultures similar to that described in (B) was carried out for n=3 samples /group/experiment. Data are Mean ± S.D., **p<0.001 in comparison of co-cultures with unsmoked MDRC versus smoked MDRC. (C) Protein levels of IL-6, TNF-α, IL-1β and IL-10 in cell culture supernatants collected at 24 hours of culture from smoke exposed and unexposed MDRC were determined by ELISA. Measurements were made from triplicate wells for each condition. Data are Mean ± S.D., representative of three independent experiments. **p<0.001 from comparisons of unexposed versus exposed to CS.

Figure 3. Cigarette smoke exposure activates NF-κB pathway in BM-MDRC

(A-C) Bone marrow cells were cultured in the presence of GM-SCF (10 ng/ml) and LPS (1 μg/ml) for 7 days. Immunosorted MDRC cells were exposed to cigarette smoke as described in methods. Western blot analyses was carried out and relative expression of p-IKKα/β versus IKKα, IKKβ or β-actin was calculated by densitometry and quantified with ImageJ software; relative expression of p-IKBα versus β-actin and IKBα versus β-actin was evaluated and relative expression of p-NF-ĸB p65 was normalized with NF-ĸB p65 or β-actin. Data are presented as mean ± SEM of triplicates. * p < 0.05, ** p < 0.01.

Figure 4. Cigarette smoke exposure increases superoxide production in MDRC via NF-κB dependent mechanism

(A) Fold change in superoxide production was determined by spectrophotometric quantitation and monitoring of kinetics of reduction cytochrome c as described before. **p<0.001 from statistical comparisons of smoked MDRC compared to unsmoked MDRC activated by PMA in presence or absence of PDTC (NF-κB inhibitor), in presence or absence of JSH-23, an inhibitor of transcriptional activation of NF-κB or with SOD (B) Percentage of cells with ROS producing potential determined by flow cytometry analyses after staining with DHE, a fluorescent indicator for ROS (C) FACS plots showing percentages of in vitro differentiated BM-MDRC from WT, IL-1β^−/−, IFNAR^−/−, MyD88^−/− mice (D) Immunofluorescence analysis of lung sections showing NF-κB expression in WT recipients of adoptively transferred (a) unsmoked MDRC (b) smoked MDRC (c) IFNAR^−/− recipients of unsmoked MDRC (d) IFNAR^−/− recipients of smoked MDRC (e) MyD88^−/− recipients of unsmoked MDRC (f) MyD88^−/− recipients of smoked MDRC

Figure 5. Intratracheal adoptive transfer of smoke exposed MDRC enhanced Th2 responses, BAL IgE and mucin levels in vivo

(A) Percentages of lung cells producing ROS and NO were determined from collagenase extracted immune infiltrates in lung tissue following staining with fluorescent indicators, DHE for ROS and DAF-FM-DA for NO and flow cytometry analyses. Lung tissues were harvested from mice with asthma which were adoptive transfer recipients of either control MDRC or smoked MDRC. **p<0.001 from statistical comparisons of adoptive transfer recipients of control versus smoked MDRC. (B) Percentages of total CD4⁺ T lymphocytes and subsets of Th2 (IL-4⁺), Th1 (IFN-γ⁺) and Th17 (IL-17⁺) in the lung tissues of mice described in (A) determined by flow cytometry analyses. **p<0.001 from statistical comparisons of adoptive transfer recipients of control versus smoked MDRC. (C- Left Panel) Levels of Muc5Ac in BAL determined by ELISA. Data are Mean ± S.D. from n=3 mice/group, versus OVA/OVA, OVA/OVA + smoke exposed MDRC compared to both OVA/OVA + unsmoked MDRC and OVA/OVA groups using ANOVA. (C-Right Panel) Levels of OVA IgE in BAL determined by ELISA. Data are Mean ± S.D. from n=3 mice/group, **p<0.001 from statistical comparisons of OVA/PBS versus OVA/OVA, OVA/OVA + smoke exposed MDRC compared to both OVA/OVA + unsmoked MDRC and OVA/OVA groups using ANOVA

Figure 6. Smoke exposure promotes IL-33 production in MDRC in vitro and in vivo

(A) Western blot of control and smoke exposed BM-MDRC probed with anti-IL-33 and anti-β actin antibodies showing increased IL-33 expression after CS exposure. (B) Western blot analyses of perfused lung tissue samples of ovalbumin sensitized wild type C57BL/6 mice harvested at day 5 after intratracheal transfer of control and smoke exposed BM-MDRC and 3 days after intranasal ovalbumin challenge probed with anti-IL-33 and anti-β actin antibodies showing increased IL-33 expression after CS exposure. (C) Levels of IL-33 detected by ELISA in BAL fluid harvested at 5 days after adoptive transfer and 3 days after ovalbumin or PBS challenge from adoptive transfer recipients of unsmoked and smoked MDRC compared to controls. ** p < 0.01 compared to controls. (D) Immunofluorescence analysis of lung sections harvested at 5 days after adoptive transfer and 3 days after ovalbumin challenge showing IL-33 expression in (a) WT ovalbumin sensitized mice (b) WT ovalbumin sensitized and challenged mice, WT ovalbumin sensitized and challenged mice which are recipients of adoptively transferred (c) unsmoked MDRC (d) smoked MDRC.

Figure 7. Intratracheal adoptive transfer of CS exposed MDRC exacerbates airway hyper-responsiveness in antigen challenged mice with asthma

Change in airway resistance was measured using flexiVent in OVA sensitized mice at Day3 after intranasal challenge with OVA (5 days after intratracheal transfer of smoke exposed and unexposed MDRC) in n=6-8 animals/group for OVA/PBS (OVA sensitized and PBS challenged mice), OVA/OVA +PBS (OVA sensitized and OVA challenged with intratracheal transfer of PBS), OVA/OVA + MDRC (OVA sensitized and OVA challenged with intratracheal transfer of unsmoked MDRC) and OVA/OVA + smoke exposed MDRC (OVA sensitized and OVA challenged with intratracheal transfer of smoked MDRC). ** p < 0.01 in comparison with transfer of unsmoked MDRC and controls, * p < 0.05 in comparison with adoptive transfer of unsmoked MDRC and controls.