IL-17-mediated inflammation promotes cigarette smoke-induced genomic instability

doi:10.21203/rs.3.rs-182920/v1

PDF

Research

IL-17-mediated inflammation promotes cigarette smoke-induced genomic instability

https://doi.org/10.21203/rs.3.rs-182920/v1

This work is licensed under a CC BY 4.0 License

Journal Publication

published 31 Mar, 2016

Read the published version in Chest →

Version 1

posted 23 Feb, 2021

You are reading this latest preprint version

Background

Chronic inflammation has been regarded as a risk factor for the onset and progression of human cancer, but the critical molecular mechanisms underlying this pathological process has yet to be elucidated.

Methods

In this study, we investigated whether interleukin (IL)-17-mediated inflammation was involved in cigarette smoke-induced genomic instability.

Results

Higher levels of both IL-17 and the DNA damage response (DDR) were found in the lung tissue of smokers than that of non-smokers. Similarly, elevated levels of IL-17 and the DDR were observed in mice after cigarette smoke exposure, and a positive correlation was observed between IL-17 expression and the DDR. In line with these observations, the DDR in the mouse lung was diminished in IL-17 KO when exposed to cigarette smoke. Besides, the treatment of human bronchial epithelium cells with IL-17 led to increased levels of the DDR and chromosome breakage.

Conclusions

These results suggest that cigarette smoke induces genomic instability at least partially through IL-17 mediated inflammation, implying that IL-17 could play an important role in the development of lung cancer.

Cancer Biology

IL-17

Inflammation

Cigarette smoke

DNA damage response

It is well known that chronic obstructive pulmonary disease (COPD) is a significant risk factor for lung cancer ¹⁴. Both COPD and lung cancer are primarily caused by cigarette smoke (CS), and are frequently presented as co-morbidities ^{14, 4}. A better understanding of the relationship between these two diseases could lead to significant advances in the development of new treatments, so the identification of common mechanisms has become a priority in tobacco-related illness research. In a recent study, it was demonstrated that smoking increases the mutational burden, is associated with multiple mutational signatures that contribute to different types of cancer, and increases cancer risk mainly due to the mis-repair of DNA damage caused by tobacco carcinogens ¹. The maintenance of genomic stability is critical for the viability of all organisms. DNA repair plays a fundamental role in the maintenance of genomic stability against the threats posed by both exogenous and endogenous stress. This process prevents the accumulation of DNA damage and its detrimental consequences on chromosomal rearrangements, sensitivity to genotoxins, and cell viability ⁷. Nevertheless, thus far, there is only a limited body of data pertaining to the underlying DNA repair mechanisms protecting cells against cigarette smoke (CS)-induced cancers. Moreover, the interplay between DNA damage response (DDR) and inflammation, and the link to cancer, remain largely unexplored. In our previous study, we demonstrated that cigarette smoke-exposure promoted interleukin (IL)-17 secretion and upregulated IL-17 expression ¹⁷. Additionally, we showed that IL-17 increased cigarette smoke-induced lung injury. We therefore performed the present study to explore (i) whether inflammation promotes CS-induced genomic instability, and (ii) whether IL-17 is involved in CS-induced genomic instability.

Patient samples

Benign lung disease samples were collected for the analysis CS-induced DDR and airway inflammation. 10 patients with smoking and 6 non-smokers were included in our studt. Patients were excluded if they had a previous history of cancer. The study protocol was approved by the Research Ethics Committees of the local ethics committee.

Cell culture

Human bronchial epithelial cells (16HBE cells) were purchased from the American Type Culture Collection (ATCC, USA) and were cultured in RPMI 1640 (Sigma-Aldrich, St. Louis, USA) supplemented with 10% fetal calf serum. Cells were seeded at 1×10⁴ cells/well in 24-well plates and cultured at 37 °C in a humidified atmosphere containing 5% CO₂/95% air for 24 h before exposure.

Mice

C57BL/6 mice (male, aged 8-10 weeks) were purchased from Slac Laboratory Animal Center (Shanghai, China). IL-17 KO mice (C57BL/6 background) were purchased from the Center for Experimental Medicine and Systems Biology (Institute of Medical Science, University of Tokyo, Japan). Mice were randomly assigned to different treatments (air or cigarette smoke) at the time of purchase to minimize any potential bias. All protocols in this study were approved by the Ethics Committee for Animal Studies of Zhejiang University, China.

CS exposure and CS extract (CSE) preparation

Mice were exposed to cigarette smoke in a chamber using a smoking machine (Model TE-10, Teague Enterprises). The total particulate matter concentrations of the chamber atmosphere were 160–180 mg/m³. Mice were exposed 2 hours a day for 5 d/week in consecutive 12 or 24 weeks. The control group was exposed to filtered air under identical conditions. To prepare CSE, smoke of 1 cigarette was bubbled slowly through 10 ml of RPMI 1640, which was considered as 100% CSE solution, and then sterilized.

Immunohistochemistry

Immunohistochemical staining was performed on formalin-fixed, paraffin-embedded tissue sections. Briefly, 4 µm thick sections were deparaffinized in xylene, rehydrated in graded ethanol, and washed twice with PBS. Endogenous peroxidase activity was blocked by incubating sections with 3% hydrogen peroxide in the dark for 10 min. Slides were incubated overnight at 4°C with the primary antibody (gH2AX, 1:200, Abcam; IL-17, 1:400, Cell Signaling). The sections were washed and incubated at room temperature for 1 h with the secondary antibodies. Finally, the slides were exposed to a substrate chromogen mixture and counterstained with Hematoxylin & Eosin (H&E). Stained slides were analysed on an Olympus optical microscope and scored according to the number of positively stained cells.

Immunofluorescence

2×10⁴ HBE cells were treated with or without CSE or IL-17 as indicated concentrations. The cells were washed 3 times with phosphate-buffered saline (PBS) and fixed in 4% paraformaldehyde for 30 min at room temperature and permeabilized with 0.2% Triton X-100 for 5 min at 4°C. After blocking with 5% bovine serum albumin PBS for 30 min, the cells were incubated with primary antibody against gH2AX diluted in PBS containing 5% BSA (1:1,000) overnight at 4°C, washed three times in PBS, and then incubated with secondary antibody diluted in PBS containing 5% bovine serum albumin for 30 min at room temperature. DNA was counterstained with 1 mg/ml 4’,6-diamidino-2-phenylindole (DAPI) for 10 min at 37°C. Cells mounted on cover slips were observed with a fluorescence microscope or confocal laser scanning microscope.

Western blot analysis

Cells were lysed in RIPA buffer. Equal amounts of protein were loaded onto SDS-polyacrylamide gels (PAGE), fractionated by electrophoresis, and transferred to nitrocellulose membranes (Bio-Rad). The membranes were blocked for 1h with 5% fat-free milk prepared in PBS containing 0.05% Tween-20. Membranes were incubated overnight at 4°C with IL-17 antibody (Cell Signaling) or anti-β Actin antibody (Cell Signaling). Then membranes were then blotted with corresponding secondary antibodies (1:2000 dilution) for 2 hours and protein bands were visualized using enhanced chemiluminescence.

Statistical analysis

Prism version 5 (GraphPad Software, Inc. La Jolla, CA) and SPSS for Windows, version 13.0 (SPSS Inc., Chicago, IL, USA) was used for data collection and presentation. The data shown are presented as mean ± standard error. Statistical significance was considered at P<0.05 using Student’s t test. Different levels of significance are indicated as *P<0.05, **P<0.01, ***P<0.001.

Smoking induced a DNA Damage Response and airway inflammation in human tissue

We first quantified the degree to which the so-called DDR was associated with smoking, as measured using immunohistochemistry for gH2AX in human lung tissue (Figure 1A). The results showed a significant difference in gH2AX expression in the lungs of smokers and non-smokers (t = 5.190, P = 0.0001; Figure 1B). In our prior study, we demonstrated that Tc17 cells are associated with CS-induced lung inflammation and emphysema ¹⁷. The main function of IL-17 is to coordinate local tissue inflammation via the regulation of pro-inflammatory cytokines ¹⁶. Based on these premises, we investigated the expression of IL-17 in smokers and non-smokers. Interesting, IL-17 was observed mainly in the lung specimens of smokers (Figure 1C). The expression of IL-17 was significantly increased in the lung (t = 6.334, P < 0.0001; Figure 1D) of smokers compared to that of non-smokers. Moreover, IL-17 expression was strongly correlated with gH2AX (r = 0.9313, P < 0.0001; Figure 1E).

Cigarette smoke induced a DNA damage response in C57BL/6 mice

C57BL/6 mice were exposed to smoke for 12 weeks. A higher level of gH2AX expression in bronchial epithelial cells of CS-exposure mice than in control mice was observed (50.7 ± 2.5% vs. 18.5 ± 1.5%; P < 0.0001), indicating the presence of damaged DNA (Figure 2A, B). In addition, we observed that there was an increase in inflammatory cells infiltrating in airway of CS-exposure mice (Figure 2C). Hematoxylin and eosin (HE) staining revealed the cellularity of the lungs was disrupted, and the number of lymphocytes and neutrophils were increased. Next, we assessed the inflammatory cell infiltrate in the lung using previously published criteria (on a 0 to 4+ scale) ⁸. The inflammatory score was 1.79 ± 0.26 for the CS group and 0.50 ± 0.15 for controls (Figure 2D). Interestingly, there was a relationship between inflammation scores and DDR in the lung (Figure 2E). A higher IL-17 expression was also observed in CS-exposure mice (Figure 2F, G). Consistent with the findings from human specimens, IL-17 expression was strongly correlated with gH2AX in the lung (r = 0.6537, P = 0.0013; Figure 2H).

IL-17 induced genomic instability in bronchial epithelial cells

To assess whether IL-17 can trigger DNA damage and genomic instability, we investigated the chromosome breakages in 16HBE cells after IL-17 treatment (Figure 3A). Fragile site expression was 2.2 ± 0.5% breaks per chromosome, which was higher than that of controls (0.7 ± 0.3%; P <0.05) (Figure 3B). The DNA damage of bronchial epithelial cells by IL-17 was observed in a dose-dependent manner (e-Figure 1A, B). This finding was also confirmed by western blotting (Figure 3C). We further studied whether the increased DDR following IL-17 treatment was due to the induction of a cell cycle arrest (Figure 3D). In the 24 h period after IL-17 treatment, the majority of gH2AX foci were observed in cyclin A positive cells and were therefore in S/G2 phase (Figure 3E), which indicates that IL-17 treatment induces replication stress in 16HBE cells. In order to identify the genes involved in IL-17 induced genomic instability, we performed RNA-seq experiment. The RNA was isolated from 16HBE cells after treatment with or without IL-17 for 24 h. The RNA samples were reverse transcribed into cDNA and cDNA libraries were constructed using the mRNA-sequencing assay. Based on the expression levels of known genes, we identified 583 up- and 704 down-regulated genes. Several genes specifically related to the DDR were identified in IL-17 induced genomic instability in bronchial epithelial cells (e-Table 1).

CS induced airway inflammation and DDR in IL-17 KO mice

IL-17 knockout (KO) mice and C57BL/6 mice were exposed to smoke for 12 weeks. The inflammation scores of IL-17 KO and C57BL/6 mice in the CS treated group were 0.63 ± 0.15 and 1.53 ± 0.28, respectively (Figure 4A, B). In addition, the DDR was decreased in IL-17 KO mice when exposed to CS (Figure 4C), and fewer gH2AX positive cells were observed in IL-17 KO compared to C57BL/6 after CS-exposure (27.3 ± 5.3% vs. 1.2 ± 2.5%; P = 0.02) (Figure 4D). These findings further demonstrate that IL-17 increases CS-induced airway inflammation, and is involved in CS-induced genomic instability.

It is well established that CS is a major preventable risk factor for many diseases including cancer and heart disease. The mechanisms by which CS leads to disease, however, are still not fully understood, but it is known that smoke exposure triggers cellular DNA damage, causing the accumulation of somatic cell mutations, which contribute to disease development. The current study demonstrated that CS-exposure induced DDR in vitro (in HBE cells) and in vivo (in lung tissue of both mice and human), suggesting an increased genomic instability. In addition, we observed that IL-17-mediated inflammation promoted this CS-induced genomic instability. These data are the first to suggest a link between IL-17, CS induced DDR and genomic instability.

In the present study, we found that CS-exposure induced the DDR in the lung, which could be explained by the pro-inflammatory effect of CS. HE staining revealed a large number of infiltrated inflammatory cells in the lung of CS-exposed mice and an increase in IL-17 expression. The main function of IL-17 is to coordinate local tissue inflammation via the regulation of pro-inflammatory cytokines ¹⁶. Our study showed that IL-17 induced, and was positively correlated with, the DDR and genomic instability in HBE cells, and its knock-out led to both reduced airway inflammation and a reduced DDR in the lung of mice after CS-exposure. This suggests that IL-17-mediated inflammation promotes the CS-induced DDR.

We found it surprising that there was a positive correlation between IL-17 expression and the DDR. The increase in IL-17 may be a critical process in the bioactivation of the CS-induced DDR in the lung. We found that IL-17 triggered the DDR and chromosome breakages in vitro. After IL-17 addition, the majority of gH2AX foci were observed in cells expressing cyclin A, which indicated that increased DDR seen with IL-17 was due to cell cycle arrest.

COPD is a chronic airway inflammatory disease and tobacco smoke is by far the most important risk factor for COPD worldwide ⁹. It is reported that smoking causes inflammatory response in the lung and leads to the characteristic pathological lesions of COPD ³. Patients with COPD have a greater risk of lung cancer than smokers without COPD with any level of tobacco exposure ¹⁵. An increased DDR in circulating white blood cells has been observed in smokers and COPD patients ^{2, 5, 12}, providing evidence for widespread systemic inflammatory and confirming the pro-inflammatory effects of smoking. Interesting, inhaled corticosteroids reduce local and systemic inflammation among patients with COPD ⁶. Additionally, inhaled corticosteroids may have a potential role in lung cancer prevention in patients with COPD ¹³.

The results from our study take us a step closer to understanding how smoke exposure causes disease, including lung cancer and COPD. CS contains a large number of chemical compounds, many of which can directly cause DNA damage and genomic instability. In response to DNA damage, cells either repair the damage or activate pathways leading to programmed cell death to maintain genome integrity ¹¹. The potency of cigarettes may result from their ability to induce DNA damage while failing to trigger the apoptosis, which results in epigenetic changes or somatic cell mutation potentially leading to the development of disease ¹⁰.

In summary, we identified IL-17 as a link between inflammation and genomic instability. Our study results suggest that the CS induced DDR in the lung is due to its pro-inflammatory effect. CS-exposure was found to promote IL-17 expression and induce a substantial inflammatory cell infiltrate in the mouse lung. Secondly, we showed that IL-17 directly induced the DDR and genomic instability in HBE cells and that the CS-induced DDR and inflammatory cell infiltration into the lung were decreased in IL-17 KO mice. Together, these results suggest for the first time that IL-17 promotes CS-induced airway inflammation, DDR and genomic instability and imply that the targeting of IL-17 could be a promising approach for the treatment of CS-related diseases.

COPD Chronic obstructive pulmonary disease

CS Cigarette smoke

CSE Cigarette smoke extract

DAPI 4’,6-diamidino-2-phenylindole

DDR DNA damage response

HBE Human bronchial epithelial

HE Hematoxylin and eosin

IL-17 Interleukin-17

ACKNOWLEDGEMENTS: This work was supported by the Key Laboratory of Respiratory Disease of Zhejiang Province, the Key Science-Technology Innovation Team of Zhejiang Province (2011R50016), the National 1000 Talents Program for Young Scholars, the National Natural Science Foundation of China (81700023, 81700025, 81422031, and 31370901), Medical and Health Science and Technology Project of Zhejiang (2019KY156), and the Natural Science Foundation of Ningbo (2016A610193, 2018A610269).

DATA ACCESSIBILITY STATEMENT: All data generated or analyzed during this study are included either in this article or in the supplementary information files.

CONFLICT OF INTEREST: The authors declare that they have no conflict of interest.

AUTHOR CONTRIBUTIONS: CC, BT and XG carried out the data collection, alignment and analysis. SY and HS carried out the algorithm design and development. CC, BT, XG, HZ, ZX, TL, YW, ZB, ZC, WL, HS and SY carried out the result correction and integration. CC, BT and XG performed figure production and wrote the manuscript. All authors read and approved the final manuscript.

ETHICS APPROVAL AND CONSENT TO PARTICIPATE: Written informed consent was obtained from all patients and the study was approved by the local Institutional Review Board. The clinical information was retrieved from the medical records.

Supplementary information

The e-Figure and e-Table can be found in the Supplemental Materials section of the online article.

Alexandrov LB, Ju YS, Haase K, et al. Mutational signatures associated with tobacco smoking in human cancer. Science. 2016;354(6312):618–22.
Aoshiba K, Zhou F, Tsuji T, Nagai A. DNA damage as a molecular link in the pathogenesis of COPD in smokers. Eur Respir J. 2012;39(6):1368–76.
Celli BR, MacNee W. Standards for the diagnosis and treatment of patients with COPD: a summary of the ATS/ERS position paper. Eur Respir J. 2004;23(6):932–46.
Durham AL, Adcock IM. The relationship between COPD and lung cancer. Lung Cancer. 2015;90(2):121–7.
Ghorbanihaghjo A, Safa J, Alizadeh S, et al. Protective effect of fish oil supplementation on DNA damage induced by cigarette smoking. J Health Popul Nutr. 2013;31(3):343–9.
Hattotuwa KL, Gizycki MJ, Ansari TW, Jeffery PK, Barnes NC. The effects of inhaled fluticasone on airway inflammation in chronic obstructive pulmonary disease: a double-blind, placebo-controlled biopsy study. Am J Respir Crit Care Med. 2002;165(12):1592–6.
Hays LE, Zodrow DM, Yates JE, et al. Cigarette smoke induces genetic instability in airway epithelial cells by suppressing FANCD2 expression. Br J Cancer. 2008;98(10):1653–61.
Henderson WR Jr, Lewis DB, Albert RK, et al. The importance of leukotrienes in airway inflammation in a mouse model of asthma. J Exp Med. 1996;184(4):1483–94.
Laniado-Laborin R. Smoking and chronic obstructive pulmonary disease (COPD). Parallel epidemics of the 21 century. Int J Environ Res Public Health. 2009;6(1):209–24.
Liu X, Conner H, Kobayashi T, et al. Cigarette smoke extract induces DNA damage but not apoptosis in human bronchial epithelial cells. Am J Respir Cell Mol Biol. 2005;33(2):121–9.
Liu X, Togo S, Al-Mugotir M, et al. NF-kappaB mediates the survival of human bronchial epithelial cells exposed to cigarette smoke extract. Respir Res. 2008;9:66.
Oit-Wiscombe I, Virag L, Soomets U, Altraja A. Increased DNA damage in progression of COPD: a response by poly(ADP-ribose) polymerase-1. PLoS One. 2013;8(7):e70333.
Parimon T, Chien JW, Bryson CL, McDonell MB, Udris EM, Au DH. Inhaled corticosteroids and risk of lung cancer among patients with chronic obstructive pulmonary disease. Am J Respir Crit Care Med. 2007;175(7):712–9.
Sekine Y, Katsura H, Koh E, Hiroshima K, Fujisawa T. Early detection of COPD is important for lung cancer surveillance. Eur Respir J. 2012;39(5):1230–40.
Skillrud DM, Offord KP, Miller RD. Higher risk of lung cancer in chronic obstructive pulmonary disease. A prospective, matched, controlled study. Ann Intern Med. 1986;105(4):503–7.
Swaidani S, Bulek K, Kang Z, et al. The critical role of epithelial-derived Act1 in IL-17- and IL-25-mediated pulmonary inflammation. J Immunol. 2009;182(3):1631–40.
Zhou H, Hua W, Jin Y, et al. Tc17 cells are associated with cigarette smoke-induced lung inflammation and emphysema. Respirology. 2015;20(3):426–33.

Supplement.docx

PDF

Journal Publication

published 31 Mar, 2016

Read the published version in Chest →

Version 1

posted 23 Feb, 2021

You are reading this latest preprint version

IL-17-mediated inflammation promotes cigarette smoke-induced genomic instability

Status:

Journal Publication

Version 1

Abstract

Background

Methods

Results

Conclusions

Figures

Introduction

Materials And Methods

Results

Discussion

Abbreviations

Declarations

References

Supplementary Files

Status:

Journal Publication

Version 1