Caveolin-1 Promotes the Imbalance of Th17/Treg in Chronic Obstructive Pulmonary Disease by Regulating Hsp70 Expression

Figures & data

Table 1 Demographic and Clinical Characteristics of All Subjects

Feature	HC (n=26)	SCOPD (n=28)	AECOPD (n=30)
Demographics
Age (years)	60.55 ± 8.01	61.08 ± 9.35	62.24 ± 7.98
Male/Female	14/12	16/12	16/14
BMI (kg/m^2)	23.98 ± 2.71	24.01 ± 2.12	24.45 ± 2.11
Lung Function
FEV1 (L)	3.31 ± 0.42	1.34 ± 0.78^a	1.13 ± 1.17^a,c
FEV1 (pred%)	95.33 ± 6.23	50.36 ± 11.91^a	36.19 ± 14.78^a,b
FEV1/FVC%	83.83 ± 4.17	57.09 ± 9.74^a	39.47 ± 15.08^a,b
RV/TLC	31.13 ± 4.97	52.45 ± 7.88^a	41.52 ± 14.82^a,b

Notes: ^ap < 0.01 versus the HC group. ^bp < 0.01 versus the SCOPD group. ^cp < 0.05 versus the SCOPD group.

Abbreviations: BMI, body mass index; FEV₁, forced expiratory volume at first second; FVC, forced vital capacity; RV, residual volume; TLC, total lung capacity.

Figure 1 The serum levels of caveolin-1 (Cav-1) and Heat shock protein 70 (Hsp70) and frequencies of circulating Th17 and Treg cells in patients with chronic obstructive pulmonary disease (COPD) and healthy controls (HC) subjects. Cytokine-specific sandwich ELISA of serum from patients with COPD and HC were performed to measure the levels of circulating (a) Cav-1 and (b) Hsp70. Representative dot plots of CD4⁺ IL-17⁺ Th17 cells (c) and CD4⁺ CD25⁺ Foxp3⁺ Treg cells (e). CD25⁺ Foxp3⁺ T regulatory (Treg) cells were gated from CD4⁺ subset of CD3⁺ T cells. (d and f) Frequencies of CD4⁺ IL-17⁺ Th17 (Th17) cells and CD25⁺ Foxp3⁺ Tregs. (g) The ratio of Th17/Treg cells. Each symbol represents an individual subject. Data are shown mean ± SD; *p < 0.05, **p < 0.01 (HC, n=26; SCOPD, n=28; AECOPD, n=30).

Figure 2 Correlation of serum Hsp70 levels with serum Cav-1 expression and immune parameters of patients with COPD. Correlation between serum levels of Hsp70 and Cav-1 expression, Th17 or Treg cells frequency and the ratio of Th17/Treg cells in (a) HC group (n=26), (b) SCOPD group (n=28), and (c) AECOPD group (n=30). Each symbol represents an individual subject.

Figure 3 Effect of Cav-1 levels on Hsp70 expression. PBMCs were transfected with Cav-1-plasmid or mock, Cav-1 siRNA or control siRNA as described in the “Materials and Methods” section. (a) After 36 h, Cav-1 expression in the cells was detected by Western blot. Representative blots of three independent experiments are shown. GAPDH was used as a loading control. o/e: overexpression. (b) After 48 h, Hsp70 levels in supernatants were measured by ELISA. Each data point represents an individual subject; data are shown as mean ± SD; *p<0.05, **p<0.01 (SCOPD, n=30; AECOPD, n=28). (c) Relative Hsp70 mRNA in PBMCs were transfected with Cav-1 siRNA for indicated times. Data are shown mean ± SD (n=3); *p < 0.05, **p < 0.01. Similar results were obtained in at least three independent experiments.

Figure 4 Cav-1 regulates the frequencies of Th17 and Treg cells by modulating Hsp70 levels in PBMCs from COPD patients and healthy control subjects. PBMCs were transfected with Cav-1-plasmid or mock, Hsp70-plasmid or mock as described in (4a). After 36 h, the cells were transfected with Hsp70-specific siRNA or control siRNA and with Cav-1-specific siRNA or control siRNA as indicated. After 36 h, (a and d) Cav-1 and Hsp70 expression in part cells were determined by Western Blot. Representative blots of three independent experiments are shown. GAPDH was used as a loading control. o/e: overexpression. After 48 h, another part was stimulated with PMA (50 ng/mL) and Ionomycin (1 μg/mL) for 4 h. Subsequently, cells were examined (b and e) frequencies of Th17 and (c and f) Treg cells by FCM. Data are shown mean ± SD (n=3); *p < 0.05, **p < 0.01. Similar results were obtained in at least three independent experiments.