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. 2015 Dec 22:11:31-41.
doi: 10.2147/COPD.S96412. eCollection 2016.

Cigarette smoke-induced lung inflammation in COPD mediated via LTB4/BLT1/SOCS1 pathway

Ran Dong  1 , Liang Xie  1 , Kaishun Zhao  2 , Qiurui Zhang  1 , Min Zhou  1 , Ping He  3
Affiliations

Cigarette smoke-induced lung inflammation in COPD mediated via LTB4/BLT1/SOCS1 pathway

Ran Dong et al. Int J Chron Obstruct Pulmon Dis. .

Abstract

Background: Evidence suggests that suppressor of cytokine signaling 1 (SOCS1) is crucial for the negative regulation of inflammation. We investigated the relationship between smoking, SOCS1, and leukotriene B4 (LTB4) in vitro and in clinical samples of COPD; besides which we detected the impact of LTB4 receptor 1 (BLT1) antagonist on inflammation.

Methods: SOCS1 expression in bronchial mucosa was determined by immunohistochemistry and real-time polymerase chain reaction. We also detect SOCS1 and BLT1 expression in alveolar macrophages from bronchoalveolar lavage fluid (BALF) by real time-PCR, in addition to measuring the level of cytokines in BALF using enzyme-linked immunosorbent assay. In vitro, we investigated the expression of SOCS1 in cigarette smoke extract-induced mouse macrophage cell line RAW264.7 by real-time polymerase chain reaction and Western blot, and detected the level of cytokines in the supernatant by enzyme-linked immunosorbent assay. Then, we investigated the effects of BLT1 antagonist U-75302 on SOCS1 expression in these cells.

Results: We obtained endobronchial biopsies (15 COPD patients and 12 non-COPD control subjects) and BALF (20 COPD patients and 20 non-COPD control subjects), and our results showed that SOCS1 expression significantly decreased in lung tissues from COPD patients. Inflammatory cytokines in BALF were higher in COPD and these inflammatory cytokines negatively correlate with SOCS1 levels. Further, the BLT1 antagonist restored SOCS1 expression and in turn inhibited inflammatory cytokine secretion in vitro.

Conclusion: Long-term cigarette smoke exposure induced SOCS1 degradation and LTB4 accumulation, which was associated with emphysema and inflammation. A BLT1 antagonist might be a potential therapeutic candidate for the treatment of COPD.

Keywords: BALF; LTB1 antagonist; endobronchial biopsies; inflammatory cytokines; treatment.

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Figures

Figure 1
Figure 1
The distribution and expression levels of SOCS1 in lung tissue. Notes: Immunohistochemistry of lung tissues with antibodies against SOCS1. Representative SOCS1 expression (brown staining) in sections from control (A and C) and COPD (B and D). (E) Quantification of the immunohistochemistry results, and expressed as integral optical density of brown staining in the different view of COPD and controls. The results are presented as mean ± SEM (n=12). Original magnification ×200 or ×400. ****P<0.0001. Abbreviations: SOCS1, suppressor of cytokine signaling 1; SEM, standard error of the mean.
Figure 2
Figure 2
SOCS1 mRNA expression levels in lung tissue. Notes: RT-qPCR detection of SOCS1 mRNA expression of lung tissue. COPD patients showed a significant low level of SOCS1 mRNA compared with the control sample. The results are presented as mean ± SEM (n=12–15; ****P<0.0001 vs the control group). Abbreviations: RT-qPCR, real-time reverse transcription quantitative polymerase chain reaction; SOCS1, suppressor of cytokine signaling 1; SEM, standard error of the mean.
Figure 3
Figure 3
mRNA expression of SOCS1 and BLT1 in alveolar macrophages. Notes: RT-qPCR detection of mRNA expression of alveolar macrophages. COPD patients showed a significant low level of SOCS1 mRNA compared with control sample (A). In contrast, BLT1 had an opposite result (B). The results are presented as mean ± SEM (*P<0.05 vs the control group). Abbreviations: BLT1, leukotriene B4 receptor 1; RT-qPCR, real-time reverse transcription quantitative polymerase chain reaction; SOCS1, suppressor of cytokine signaling 1; SEM, standard error of the mean.
Figure 4
Figure 4
Cytokine concentration in bronchoalveolar lavage fluid (BALF). Notes: BALF was assayed by ELISA, and COPD patients showed a significant high level of LTB4 (A), IL-8 (B), and TNF-α (C) compared with the control group. Data are expressed as mean ± SEM (n=15–25; **P<0.01 vs the control group, ***P<0.001 vs the control group, ****P<0.0001 vs the control group as indicated in the figure). Abbreviations: ELISA, enzyme-linked immunosorbent assay; IL-8, interleukin-8; LTB4, leukotriene B4; TNF-α, tumor necrosis factor-α; SEM, standard error of the mean.
Figure 5
Figure 5
Correlations between SOCS1 mRNA levels, LTB4 expression level, and FEV1 %predicted. Notes: There were significant negative correlations between SOCS1 mRNA level in lung tissues and LTB4 concentration in COPD patients (A). In addition, there were significant positive correlations between SOCS1 mRNA level of lung tissues and FEV1 %predicted (B). Data are expressed as mean ± SEM. Abbreviations: LTB4, leukotriene B4; SOCS1, suppressor of cytokine signaling 1; FEV1, forced expiratory volume in 1 second; SEM, standard error of the mean.
Figure 6
Figure 6
Expression of SOCS1, LTB4, TNF-α, and IL-6 in CSE-treated RAW264.7 cells. Notes: Total RNA isolated from RAW264.7 cells after 0, 6, 12, and 24 hours treated with CSE was examined. The expression of mRNA was assessed by real-time PCR (A). The supernatant was collected at 0, 6, 12, and 24 hours, and the amount of TNF-α, LTB4, and IL-6 released was measured by ELISA (B–D). Whole-cell extracts from 0, 12, and 24 hours cultures were isolated and subjected to Western blot to detect the expression of SOCS1 (E). The SOCS1 expression had increased when treated with U-75302 at 24 h (F). Data were expressed as mean ± SEM, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001 vs control group. Abbreviations: CSE, cigarette smoke extract; ELISA, enzyme-linked immunosorbent assay; IL-6, interleukin-6; LTB4, leukotriene B4; SOCS1, suppressor of cytokine signaling 1; TNF-α, tumor necrosis factor-α; SEM, standard error of the mean.
Figure 7
Figure 7
Effects of U-75302 on SOCS1 expression and cytokines’ secretion. Notes: The figure shows that SOCS1 expression increased with U-75302 treatment (A); at the same time, IL-6 and TNF-α secretion decreased (C and D); however, the level of LTB4 had notable increase (B). Data are expressed as mean ± SEM, *P<0.05, **P<0.01 vs control. Abbreviations: LTB4, leukotriene B4; IL-8, interleukin-8; SOCS1, suppressor of cytokine signaling 1; TNF-α, tumor necrosis factor-α; SEM, standard error of the mean.
Figure 7
Figure 7
Effects of U-75302 on SOCS1 expression and cytokines’ secretion. Notes: The figure shows that SOCS1 expression increased with U-75302 treatment (A); at the same time, IL-6 and TNF-α secretion decreased (C and D); however, the level of LTB4 had notable increase (B). Data are expressed as mean ± SEM, *P<0.05, **P<0.01 vs control. Abbreviations: LTB4, leukotriene B4; IL-8, interleukin-8; SOCS1, suppressor of cytokine signaling 1; TNF-α, tumor necrosis factor-α; SEM, standard error of the mean.
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