Characterization of TLR-induced inflammatory responses in COPD and control lung tissue explants

Figures & data

Table 1 Demographics of patients used in this study

	COPD (n=33)	Smokers (n=19)
Sex (M/F)	17/16	7/12
Age, years	68.7±7.9	63.7±8.9
FEV1	1.84±0.60	2.53±0.61
FEV1 % predicted	73.4±17.5	99.3±20.0
FEV1/FVC % ratio	59.6±11.1	75.1±0.1
Smoking status (current/ex)	21/12	7/12
Smoking history (pack-years)	42 (13–126)	37 (2–71)
ICS use? (yes/no)	8/25	0/19
GOLD stage (I/II/III/IV)	10/20/3/0	NA

Note: Data are presented as mean ± standard deviation or mean (range).

Abbreviations: FEV₁, forced expiratory volume in 1 second; FVC, forced vital capacity; ICS, inhaled corticosteroid; M, male; F, female; ex, ex-smoker; GOLD, Global Initiative for Chronic Obstructive Lung Disease; COPD, chronic obstructive pulmonary disease; NA, not applicable.

Figure 1 Time course of cytokine release from poly(I:C)- and R848-stimulated lung tissue.

Notes: The release of TNFα (A and D), CCL5 (B and E), and IL-6 (C and F) from poly(I:C)- (100 μg/mL) (A–C) and R848-stimulated (10 μg/mL) (D–F) whole tissue explants from smokers and COPD patients (pooled, n=13) were measured after 1, 6, 24, and 48 hours. Data presented as mean with SEM (TNFα and IL-6) or median with range (CCL5). ^{*, **, ***}Refer to significantly above time-matched unstimulated control (P<0.05, 0.01, 0.001, respectively). Results were obtained using paired t-tests (TNFα and IL-6) and Wilcoxon matched-pairs test (CCL5).

Abbreviations: TNFα, tumor necrosis factor α; IL-6, interleukin 6; COPD, chronic obstructive pulmonary disease; SEM, standard error of the mean; h, hour(s).

Figure 2 Time course of cytokine mRNA expression in poly(I:C)- and R848-stimulated lung tissue.

Notes: The mRNA expression of TNFα (A and D), CCL5 (B and E), and IL-6 (C and F) in poly(I:C)- (100 μg/mL) (A–C) and R848-stimulated (10 μg/mL) (D–F) whole tissue explants from smokers and COPD patients (pooled, n=13) was measured after 1, 6, 24, and 48 hours. Data presented as median with range. ^{*, **, ***}Refer to significantly above time-matched unstimulated control (P<0.05, 0.01, 0.001, respectively). Horizontal line at y=1 represents unstimulated control. Results were obtained using a Wilcoxon matched-pairs test.
Abbreviations: TNFα, tumor necrosis factor α; IL-6, interleukin 6; COPD, chronic obstructive pulmonary disease; h, hour(s).

Figure 3 Concentration-dependent induction of cytokine release from poly(I:C)- and R848-stimulated lung tissue.

Notes: The release of TNFα (A and D), CCL5 (B and E), and IL-6 (C and F) from poly(I:C)- (A–C) and R848-stimulated (D–F) whole tissue explants from smokers (n=13) and COPD patients (n=13, poly(I:C); n=12, R848) after 24 hours. Data are presented as mean with SEM (TNFα and IL-6) or as median with range (CCL5). ^{#, ##, ###}Refer to significantly above time-matched unstimulated control (P<0.05, 0.01, 0.001, respectively). ^{*, **}Refer to significant difference between subject groups (P<0.05, 0.01, respectively). Results were obtained using one way ANOVA with Dunnett’s post-test (TNFα and IL-6) and Friedman test with Dunn’s post-test (CCL5) ^#P<0.05 and an unpaired t-test (TNFα and IL-6) and Mann-Whitney U test (CCL5) *P<0.05.
Abbreviations: TNFα, tumor necrosis factor α; IgG, immunoglobulin G; IL-6, interleukin 6; COPD, chronic obstructive pulmonary disease; SEM, standard error of the mean.

Figure 4 Effect of simultaneous activation of TLR3 and TLR7/8 on pro-inflammatory cytokine release from lung tissue.

Notes: The effect of simultaneous TLR3 and TLR7/8 activation (poly(I:C) =100 μg/mL, R848=10 μg/mL, respectively) on the release of TNFα (A), CCL5 (B), and IL-6 (C) from whole tissue explants from smokers (n=10) and COPD patients (n=12) after 24 hours. Data are presented as mean with SEM (TNFα and IL-6) or as median with range (CCL5). Sum represents the predicted additive result of poly(I:C) + R848 cytokine release. ^{*, **, ***}Refer to significant difference between conditions (P<0.025, 0.005, 0.0005, respectively). ^{#, ##}Refer to significantly above predicted sum (P<0.05, 0.01, respectively). Results are in response to paired t-tests (TNFα and IL-6) and Wilcoxon tests (CCL5).
Abbreviations: TNFα, tumor necrosis factor α; IL-6, interleukin 6; COPD, chronic obstructive pulmonary disease; SEM, standard error of the mean; Comb, combination.

Figure 5 Effect of TNFα neutralization on pro-inflammatory cytokine release from lung tissue.

Notes: The effect of pre-exposure to neutralizing TNFα IgG antibody (nTNFα IgG) and control IgG antibody (Isotype IgG) (both 1 μg/mL) on CCL5 (A) and IL-6 (B) release from whole tissue explants from COPD patients (n=6) that were either unstimulated (control) or stimulated with poly(I:C) (100 μg/mL) or R848 (10 μg/mL) for 24 hours. Data are presented as median with range (CCL5) or mean with SEM (IL-6). ^{*, **}Refer to significantly below untreated levels (P<0.05, 0.01, respectively). Results are in response to one way ANOVA with Dunnett’s post-test (IL-6) and Friedman test with Dunn’s post-test (CCL5).
Abbreviations: TNFα, tumor necrosis factor α; IgG, immunoglobulin G; IL-6, interleukin 6; COPD, chronic obstructive pulmonary disease; SEM, standard error of the mean; ANOVA, analysis of variance.

Figure 6 Effect of dexamethasone on pro-inflammatory cytokine release from TLR-stimulated lung tissue.

Notes: The effect of dexamethasone (1 μM) on poly(I:C)- (100 μg/mL), R848- (10 μg/mL), and combination-induced release of TNFα (A), CCL5 (B), and IL-6 (C) after 24 hours from whole tissue explants from COPD patients (n=6). Data are presented as mean with SEM (TNFα and IL-6) or median with range (CCL5). ^{*, **, ***}Refer to significantly below DMSO control (P<0.05, 0.01, 0.001, respectively). Results are in response to paired t-tests (TNFα and IL-6) and Wilcoxon tests (CCL5).
Abbreviations: TNFα, tumor necrosis factor α; IL-6, interleukin 6; COPD, chronic obstructive pulmonary disease; SEM, standard error of the mean; Dex, dexamethasone; DMSO, dimethyl sulfoxide.