Oxidative stress-mediated NKT cell activation is involved in COPD pathogenesis.
Muriel Pichavant, Gaëlle Rémy , Sandrine Bekaert, Olivier Le Rouzic, Gwénola Kervoaze,
Eva Vilain, Nicolas Just, Isabelle Tillie-Leblond, François Trottein, Didier Cataldo,
Philippe Gosset.
Online Data Supplement Figures
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Supplemental Figure 1
Supplemental Figure 1- Gating strategy for pulmonary APC sorting in murine lung.
Large cells were selected based on the FSC/SSC dot plot. Alveolar macrophages (AM) and
dendritic cell (DC) were identified as CD11c+ F4/80+ cells and CD11c+ F4/80- cells
respectively. Pulmonary DC were next separated into 2 subsets: CD11b+ CD103- and CD11bCD103+ DC. DC phenotype was checked after cell sorting (lower panel).
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Supplemental Figure 2
Supplemental Figure 2. Characterization of cytokines in human fluids of control donors
and COPD patients.
A, Lung inflammation in COPD patients compared to controls. CXCL-8 and TNF-α levels
were evaluated by ELISA in sputum of control donors (white bars) and COPD patients (black
bars). Data represent the mean ± SEM of 7 controls and 28 COPD patients. * p < 0.05, ** p <
0.01. B, Production of IL-22 and IFN-γ was evaluated by ELISA in blood mononuclear cells
from controls and COPD patients, after 24 hour culture. Data represent the mean ± SEM. * p
< 0.05, ** p < 0.01.
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Supplemental Figure 3
Supplemental Figure 3- CS exposure enhances the number of activated NKT cells in the
lungs, but not in the spleen in mice. A and B, The frequency and the expression level of
CD69 on CD45+ CD5+ NK1.1+ NKT-like cells in lung tissues were determined 12 wk post
CS-exposure. Data are representative of one experiment out of three performed. C and D, The
frequency and the number of splenic NKT cells was determined in a kinetic manner. Data are
expressed as mean ± SEM. E, The expression level of CD69 on splenic NKT cells is shown
(12 wk post CS-exposure).
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Supplemental Figure 4
Supplemental Figure 4- iNKT cells are essential to promote COPD features in mice.
A, Analysis of lung function decline induced by CS in (i)NKT-cell deficient mice. Agematched WT, Ja18-/- and CD1d-/-mice were exposed to CS, 5 days a week, over a period of 12
weeks. Changes in dynamic compliance and airway elastance were measured on mechanically
ventilated mice, in response to increasing doses of methacholine (week 12 post-CS exposure).
Data are expressed as mean ± SEM. Results are representative of three independent
experiments. **, p ≤ 0.01; *, p ≤ 0.05 (WT mice vs iNKT-cell deficient mice). B, Percentage
of weight loss after 12 weeks of CS exposure compared to the air groups in WT, Ja18-/- and
CD1d-/-mice. Data are expressed as mean ± SEM (n= 7-10 per group). C, Lungs were
collected 12 weeks post-exposure to CS, IL-1b mRNA expression were determined by
quantitative RT-PCR. Data are normalized to expression of GAPDH and are expressed as fold
increased over average gene expression in air-exposed WT mice or J18-/- mice, respectively
(n=5). * p < 0.05, ** p < 0.01.
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Supplemental Figure 5
Supplemental Figure 5- Priming of respiratory APC in CS-exposed mice.
A, Total lung cell numbers were evaluated in WT mice after CS exposure (black bars) and
control mice (white bars). Data are representative of one experiment out of three performed
(mean ± SEM, n = 6). B, Expression of I-A on CD45+ CD11c+ F4/80+ alveolar macrophages
(AM) and CD45+ CD11c+ F4/80- Dendritic cell (DC) in the lung of WT mice was determined
in WT mice after CS exposure (black bars) and control mice (white bars). Data are
representative of one experiment out of three performed (mean ± SEM, n = 6). C,
Representative histograms of CD40 and I-A expression on pulmonary AM and DC from Ja18/-
mice after 12 wk CS-exposure are shown. MFI are represented on the dot plots. * p < 0.05,
** p < 0.01.
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Supplemental Figure 6
Supplemental Figure 6- Il-23p19 and St3gal5 mRNA levels in total lungs. Mice were
exposed or not to CS for 12 weeks. Lung tissues were collected. Il-23p19 and St3gal5 mRNA
expression were determined by quantitative RT-PCR. Data are normalized to expression of
GAPDH and are expressed as fold increased over average gene expression in air-exposed WT
mice (n=10). * p < 0.05.
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Supplemental Figure 7
Supplemental Figure 7- Mouse airway epithelial cells (AEC) in vitro activate iNKT cells.
Mouse AEC were exposed to -GalCer (100 ng/ml) overnight and, after extensive washing,
were co-cultutred with DN32 iNKT hybridoma (panel A) or sorted iNKT cells (panel B) for
48 hours. IL-2, and IL-4 and IFN-γ were respectively measured by ELISA in coculture
supernatants. Data represent the mean ± SEM of three independent experiments performed in
triplicate. * p < 0.05, ** p < 0.01, *** p < 0.001.
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Supplemental Figure 8
Supplemental Figure 8- CSE triggers oxidative stress in human APC. Human DC and
human AEC were exposed to CSE. mRNA were prepared at different time-points. NAD(P)H
dehydrogenase quinone -1 (NQO-1) and heme-oxygenase -1 (HO-1) mRNA copy numbers
were determined by quantitative RT-PCR. Data are normalized to expression of β-actin and
are expressed as fold increased over average gene expression in unstimulated cells. Data
represent one out of three independent experiments. * p < 0.05, ** p < 0.01.
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Supplemental Figure 9
Supplemental Figure 9- Oxidative stress is involved in changes of lung function.
Mice were exposed to CS, 5 days a week, over a period of 12 weeks. Some mice received the
treatment with the anti-oxidant NAC in their drinking water all along the CS exposure.
Changes in dynamic compliance and airway elastance were measured on mechanically
ventilated mice, in response to increasing doses of methacholine (week 12 post-CS exposure).
Data are expressed as mean ± SEM. Results are representative of three independent
experiments (n=5 mice per group). * p < 0.05, ** p < 0.01.
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