TLR4 Up-regulation and Reduced Foxp3 Expression in Mechanically Ventilated Smokers with Obstructive Chronic Bronchitis

Figures & data

Table 1.  Demographic and clinical characteristics of the subjects

Characteristic	C	S	COPD	P-value
No. of subjects	10	8	18
Age (years)	73 (72–78)	79.5 (76–82)	76 (71–81)	n.s.
Male/Female	7/3	5/3	11/7	n.s.
Cigarette smoke Pack-years	–	45 ± 20	50 ± 21	n.s.
FEV1 (% of predicted normal)	85 ± 2.5	87 ± 3.9	67 ± 5*	*p < 0.05 COPD vs C and S
FEV1/FVC (% of predicted normal)	77 ± 3	76 ± 2	65 ± 2*	*p < 0.05 COPD vs C and S
CPIS	2.3 ± 1.1	3.3 ± 1.4	3.7 ± 0.9*	*p < 0.05 COPD vs C
SOFA	9.1 ± 1.2	8.7 ± 1.3	8.9 ± 1	n.s.
SAPS II	53.6 ± 7.8	59.5 ± 11	56 ± 7	n.s.


Data are expressed as percentiles (age) or as mean ± SD.

Table 2.  Total and differential cell counts of mini-BAL

	C = 10	S = 8	COPD = 18	P-value
Mini-BAL cells	536 ± 269	919 ± 167*	3.2 ± 2,400*	* p < 0.05
Total number (X1000)/ml				COPD and S vs C
% Neutrophils	46 ± 30	48 ± 17	83 ± 13*	*p < 0.05 COPD vs C and S
% Macrophages	51 ± 29	52 ± 16	17 ± 13*	*p < 0.05 COPD vs C and S
% Lymphocytes	2.7 ± 1.3	0*	0*	*p < 0.05 COPD and S vs C


Results are expressed as mean ± SD.

Table 3.  Microbiology of mini-BAL

	C with Mini-BAL cultures Positive (n = 4)	C with Mini-BAL cultures Negative (n = 6)	CFU
Patient#1		X
Patient#2		X
Patient#3	Candida		≥10^3/ml
Patient#4	Candida		≥10^3/ml
Patient#5		X
Patient#6		X
Patient#7	Candida		≥10^3/ml
Patient#8		X
Patient#9		X
Patient#10	Candida		≥10^3/ml
	S with Mini-BAL cultures Positive (n = 3)	S with Mini-BAL cultures Negative (n = 5)	CFU
Patient#1		X
Patient#2		X
Patient#3	Candida		≥10^3/ml
Patient#4	Candida		≥10^3/ml
Patient#5		X
Patient#6		X
Patient#7		X
Patient#8	Acinetobacter Baumannii		≥10^3/ml
	COPD with Mini-BAL cultures positive (n = 7)	COPD with Mini-BAL cultures negative (n = 11)	CFU
Patient#1	Candida		≥10^3/ml
Patient#2		X
Patient#3		X
Patient#4	Acinetobacter Baumannii		≥10^3/ml
Patient#5		X
Patient#6	Candida		≥10^3/ml.
Patient#7		X
Patient#8	Pseudomonas Aeruginosa		≥10^3/ml
Patient#9		X
Patient#10	Acinetobacter Baumannii		≥10^3/ml
Patient#11		X
Patient#12		X
Patient#13	Candida		≥10^3/ml
Patient#14		X
Patient#15		X
Patient#16	Pseudomonas Aeruginosa		≥10^3/ml
Patient#17		X
Patient#18		X

Figure 1.  Increased expression of TLR4 in mini-BAL but not in the peripheral blood of S and of COPD. Mini-BAL cells and paired blood samples were recovered from C (n = 10), from S (n = 8) and from COPD (n = 18) patients. The expression of TLR4 was assessed by immunocytochemistry using an anti-TLR4 polyclonal antibody; *p < 0.05 vs C; **p < 0.05 vs S; #p < 0.05 vs autologous peripheral blood. Data are expressed as median (25-75 percentiles).

Table 4.  Total and differential cell counts of mini-BAL

	C = 10	S = 8	COPD = 18	p-value
TLR4 expression in Mini-BAL cells (% of positive cells)	21.9 ± 6.4	39.8 ± 3.8	64.88 ± 4.8	*p < 0.05 COPD and S vs C
TLR4 expression in peripheral blood cells (% of positive cells)	25.4 ± 6.5	17.5 ± 1.76	26.8 ± 6.37	n.s.
Foxp3 expression in Mini-BAL cells (arbitrary units)	2.5 ± 0.24	1.83 ± 0.12*	1.39 ± 0.41*	*p < 0.05 COPD and S vs C
Foxp3 expression in peripheral blood cells (arbitrary units)	3.79 ± 0.3	2.36 ± 0.36	1.75 ± 0.53	*p < 0.05 COPD and S vs C


Results are expressed as mean ± SE.

*Mann–Whitney test.

Figure 2.  Absence of differences in the concentrations of IL-8 in mini-BAL and in the peripheral blood of S and of COPD. Mini-BAL supernatants and paired blood samples were recovered from C (n = 10), from S (n = 8) and from COPD (n = 18) patients. IL-8 concentrations were measured by ELISA as described in “materials and methods” and are expressed as pg/ml. Data are expressed as median (25-75 percentiles).

Figure 3.  Reduced concentrations of IP-10 in mini-BAL but not in the peripheral blood of COPD. Mini-BAL supernatants and paired blood samples were recovered from C (n = 10), from S (n = 8) and from COPD (n = 18) patients. IP-10 concentrations were measured by ELISA as described in “materials and methods” and are expressed as pg/ml. *p < 0.05 vs C; **p < 0.05 vs S; #p < 0.05 vs autologous peripheral blood. Data are expressed as median (25-75 percentiles).

Figure 4.  Reduced expression of Foxp3 in mini-BAL and in the peripheral blood of S and of COPD. Mini-BAL cells and paired blood samples were recovered from C (n = 10), from S (n = 8) and from COPD (n = 18) patients. Total proteins were extracted and analysed for Foxp3 expression by western blot analysis. Membranes were then stripped and incubated with goat polyclonal anti–ß-actin. A. Densitometric analysis of Foxp3 expression. Signals corresponding to Foxp3 on the various western blots were semiquantified by densitometric scanning, normalized and expressed after correction with the density of the band obtained for beta-actin (mean±SD). * p < 0.05. B. Representative Western blot analysis for Foxp3 expression from C, S and COPD subjects. Data are expressed as median (25-75 percentiles).

Figure 5.  Correlations between the expression of TLR4 in mini-BAL and CPIS score. The expression of TLR4 in mini-BAL of C (n = 10), S (n = 8) and COPD (n = 18) was correlated with CPIS score by Spearman Correlation test.