ILC subsets in control subjects versus patients with COPD.

<p><b>A,</b> The frequency of ILC1 (CD45⁺, Lin^-, CD127⁺, CD56^-, IL12Rβ2⁺), ILC2 (CD45⁺, Lin^-, CD127⁺, CRTH2⁺), NCR⁺ ILC3 (CD45⁺, Lin^-, CD127⁺, CD117⁺, NKp44⁺) and NCR^- ILC3 (CD45⁺, Lin^-, CD127⁺, CD117⁺, NKp44^-) in digested human lung from control (n = 5) and COPD patients (n = 11) was determined by flow cytometry. ILC numbers were expressed as percentages (%) of the CD45⁺ population (mean ± SEM). <b>B,</b> Pie chart of the relative abundance of ILC1, ILC2, NCR⁺ ILC3 and NCR^- ILC3 subsets in control subjects and patients with COPD.</p

Overview of innate lymphoid cell subsets in human lung tissue.

<p>The presence of CD45⁺, Lin^- (i.e. CD3, CD19, CD11c, CD11b) and CD127⁺ ILC in pulmonary tissue was demonstrated. These ILC were further subdivided in a CD56^- IL12Rβ2⁺ ILC1 subset, CRTH2⁺ ILC2 subset, CD117⁺ NKp44⁺ (NCR⁺) ILC3 subset and CD117⁺ NKp44^- (NCR^-) ILC3 subset. Further, expression of signature transcription factors (i.e. T-bet, GATA-3 and RORγT) within the specific ILC subset and cytokine production (i.e. IFN-γ, IL-5, IL-17A, IL-22 and GM-CSF) within the pulmonary ILC population was demonstrated.</p

Intracellular staining of transcription factors in pulmonary ILC subsets.

<p>The developmental transcription factors were determined in the specific ILC subsets on single cell suspensions of digested human lung. <b>A,</b> Expression of T-bet (black line) versus isotype control (grey line) in the ILC1 (CD45⁺, Lin^- CD127⁺, CD56^-, CRTH2^-, CD117^-), ILC2 (CD45⁺, Lin^-, CD127⁺, CRTH2⁺), ILC3 (CD45⁺, Lin^-, CD127⁺, CD117⁺) population. <b>B,</b> GATA-3 expression (black line) versus isotype control (grey line) in the different ILC subsets. <b>C,</b> Expression of RORγT (black line) versus isotype control (grey line) in ILC1, ILC2 and ILC3 population.</p

Intracellular cytokine production in the pulmonary ILC population.

<p>Several signature cytokines in pulmonary ILC (gated as CD45⁺, Lin^- CD127⁺ cells) were determined on single cell suspensions of digested human lung (n = 8). Since NK cells could contaminate the non-toxic ILC1 subset, CD56⁺ cells were excluded to investigate the IFN-γ production. For the production of these cytokines, lung cells were first stimulated for 15 hours with PMA/ionomycin (+ transport inhibitors). <b>A,</b> IFN-γ production in ILC. <b>B,</b> Production of IL-5 in the pulmonary ILC population. <b>C,</b> IL-17 production in ILC. <b>D,</b> ILC production of IL-22. <b>E,</b> Production of GM-CSF in the ILC population. The bottom panels represent the isotype controls of the specific cytokine staining. <b>F,</b> Frequency of IFN-γ, IL-5, IL-17A, IL-22 and GM-CSF positive cells within the ILC (CD45⁺, Lin^- CD127⁺) population (n = 8, mean ± SEM).</p

BAL fluid cytokines in wild type versus Grx1 KO mice exposed to air and cigarette smoke.

<p>Measurement of cytokines in the BAL fluid by multiplex: IL 12 (A), GCSF (B), MCP1 (C), KC (D), RANTES (E) and MIP1α (F) expressed in pg/ml and represented as mean ± SD. * represents p<0.05 between air and smoke exposed mice, # represents p<0.05 between WT smoke and KO smoke and $ represents p<0.05 between WT air and Grx1 KO air.</p

Grx1 expression in lung tissue and primary macrophages of mice exposed to cigarette smoke.

<p>(A) Grx1 mRNA expression corrected for HPRT mRNA expression in lung tissue of mice exposed to air and cigarette smoke, represented as mean ± SD. (B) Fluorescent Grx1 staining in primary macrophages after 24 hours of control or 0.5% cigarette smoke extract exposure. The green staining represents Grx1 protein expression, whereas blue represents the nuclear DAPI staining. Quantification of fluorescent Grx1 staining is expressed as RFI in (C).</p

BAL fluid cell counts and differentials in wild type versus Grx1 KO mice exposed to air and smoke.

<p>Total BAL fluid cells (A), total numbers of macrophages (B), neutrophils (C), dendritic cells (D), CD3+ cells (F), CD4+ cells (G) and CD8+ cells (H) in BAL fluid represented as mean ± SD. * represents p<0.05 between air and smoke exposed mice, # represents p<0.05 between WT smoke and KO smoke.</p

S-glutathionylation in lungs and BAL fluid cells and BAL fluid of Grx1 KO compared to wild type mice exposed to cigarette smoke and air.

<p>S-glutathionylation of proteins quantified by the DTNB assay in lung tissue of Grx1 KO compared to wild type mice exposed to cigarette smoke and air, data shown in nmol GSH/mg protein and represented as mean ± SD (A). S-glutathionylation of proteins visualised by the biotin switch staining in BAL fluid cells of Grx1 KO compared to wild type mice exposed to cigarette smoke and air, the outer right panel shows the quantification of staining expressed as relative fluorescent intensity of red staining (S-glutathionylated proteins) compared to blue, nuclear DAPI staining and represented as mean ± SD (B). S-glutathionylation of proteins quantified by the DTNB assay in BAL fluid of Grx1 KO compared to wild type mice exposed to cigarette smoke and air, data shown in nmol GSH/µg protein and represented as mean ± SD (C). * represents p<0.05 between air and smoke exposed mice, # represents p<0.05 between WT smoke and KO smoke.</p

Lung tissue differential cell counts in wild type versus Grx1 KO mice exposed to air or smoke.

<p>Total lung cells (A), total numbers of lung macrophages (B), dendritic cells (C), CD3+ cells (D), CD4+ CD69+ cells (E) CD8+ CD69+ cells (F) and GR1 cells (G), represented as mean ± SD. * represents p<0.05 between air and smoke exposed mice.</p

KC in primary macrophages versus primary epithelial cells from wild type and Grx1 KO mice after cigarette smoke extract exposure.

<p>KC in medium of primary macrophages (A) and of mouse tracheal epithelial cells (B) from wild type and Grx1 KO mice exposed to air or 0.1% cigarette smoke extract for 24 hours expressed in pg/ml.</p