NLRP3 inflammasome activity is upregulated in an in-vitro model of COPD exacerbation
NLRP3 inflammasome activity is upregulated in an in-vitro model of COPD exacerbation
Noy Nachmias 0 1
Sheila Langier 1
Rafael Y. Brzezinski 1
Matan Siterman 1
Moshe Stark 1
Sara Etkin 1
Avital Avriel 0 1
Yehuda Schwarz 1
Shani Shenhar-Tsarfaty 1
Amir Bar-ShaiID 1
? These authors contributed equally to this work. 1
0 The Division of Pulmonary Medicine, Barzilai Medical Center, Faculty of Health Sciences, Ben-Gurion University , Ashkelon , Israel , 2 Department of Internal Medicine "C, "D and "E, The Tel Aviv Sourasky Medical Center, Tel Aviv, affiliated to the Sackler Faculty of Medicine, Tel Aviv University , Tel Aviv , Israel , 3 The Pulmonary Institute, Tel-Aviv Sourasky Medical Center and Sackler Faculty of Medicine, Tel Aviv University , Tel Aviv , Israel , 4 Neufeld Cardiac Research Institute, Sackler Faculty of Medicine, Tel Aviv University, Israel; Tamman Cardiovascular Research Institute, Leviev Heart Center, Sheba Medical Center , Tel-Hashomer , Israel
1 Editor: David M. Ojcius, University of the Pacific , UNITED STATES
Cells exposed to CSE present an increase in inflammatory cytokines (IL-8 and MCP-1)
production in a dose-dependent manner. Incubation with LPS to these cells results in higher
levels of IL-8 and MCP-1 compared to stimulation of CSE alone. NLRP3 inflammasome
activity and IL-1? levels were significantly increased in cells exposed to both CSE and LPS
compared to CSE alone.
Data Availability Statement: All relevant data are
within the manuscript and its Supporting
Funding: The authors received no specific funding
for this work.
Competing interests: The authors have declared
that no competing interests exist.
in vitro model.
NLRP3 inflammasome is upregulated in an in-vitro model of COPD and COPD
exacerbation. Our findings provide novel biomarkers for COPD exacerbation and may present new
targets for future research.
Chronic obstructive pulmonary disease (COPD) is a common respiratory condition associated
with cigarette smoke exposure and characterized by airflow limitation that is usually
progressive. COPD is also associated with an enhanced chronic inflammatory response in the airways,
lungs, and serum [
]. The clinical pattern of COPD is characterized by episodes of symptom
worsening termed exacerbations. COPD exacerbations have a major role in deterioration in
health status and disease progression, as well as a decline in lung function after each
exacerbation . Furthermore, COPD exacerbations are the most prevalent cause of mortality and
cardiovascular events in COPD patients, and the risk of mortality is increasing with each
hospitalization due to an exacerbation episode [
The inflammatory process and its regulation during COPD exacerbation are remarkably
complex. The leading cause to exacerbation is the immune response to bacterial pathogens
such as Staphylococcus aureus, Streptococcus pneumoniae, Haemophilus influenzae,
Klebsiella pneumoniae, Chlamydia pneumoniae, Mycobacterium tuberculosis and viral pathogens,
such as influenza A and respiratory syncytial virus [
]. However, since there is a clinical
heterogeneity in COPD exacerbations, researchers try to find biomarkers that would help
clinicians to tailor the most appropriate treatment. Many biomarkers were investigated including
C- reactive protein (CRP), sputum or blood eosinophils, IL-1 ?, caspase-1, IL-18, CXCL10,
IL8\CXCL8 and MCP-1 [
Recently many studies investigated the role of NLR (Nod-like receptors) family in COPD
and the NLRP-3 (NLR containing a Pyrin domain 3) [
]. Briefly, the activation of
NLRP3 inflammasome complex leads to the activation of Caspase-1 which causes the cleavage and
maturation of the pro-inflammatory cytokines IL-1? and IL-18 [
]. Likewise, the signaling
pathway of NLRP-3 is found to be a major mediator of immune response to exposure of
cigarette smoke and airborne insults, through activation of toll-like receptors on epithelial cells
and activation of immunological cells such as neutrophils and dendritic cells [
While there is abundant information regarding the NLRP-3 role in stable COPD by using
animal models [
11, 12, 13
] and COPD patients?sputum [
], there is limited information
about NLRP-3 during COPD exacerbation [
Here, we aim to investigate the role of NLRP3 in an in-vitro model which was designed to
mimic COPD exacerbation.
A549 cells, pulmonary adenocarcinoma derived cell line [
], purchased directly from
SigmaAldrich (catalog number 86012804) and cultured in Dulbecco?s modified eagle?s medium
(DMEM), (Biological Industries, Beit Haemek, Israel) with 10% fetal calf serum (FCS), 100
units/ml penicillin, and 100 mg/ml streptomycin, under a humidified atmosphere (5% CO2
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plus 95% air) at 37?C. Treatments included 2%, 4% and 10% CSE. LPS was added to the
cultures to stimulate COPD exacerbation condition [
Preparation of aqueous cigarette smoke extract (10%)
One research cigarette (3R4F, Kentucky tobacco research center) was bubble into 10 ml of
culture media supplemented with 1% FBS through a smoking apparatus at a rate of one cigarette/
minute. CSE was sterile filtered through a 0.22 ?m filter. CSE preparation was standardized by
measuring the absorbance (OD 0.74 ? 0.05) at a wavelength of 320 nm [
Evaluation of inflammatory response of IL-8, MCP-1 and IL-1? levels
To evaluate CSE and LPS cytotoxicity during COPD exacerbation model, we measured levels
of IL-8 and MCP-1. These cytokines were found to be increased in BAL (Broncho-Alveolar
Lavage) of COPD patients [
5, 7, 8
]. Briefly, Enzyme-Linked immunosorbent assay (ELISA)
was performed in the supernatant of the cell cultures for human IL-8, MCP-1 and IL-1?,
according to the manufacturer (e-bioscience coating ELISA kit).
Evaluation of NLRP3 activity
NLRP3 protein expression was measured by flow cytometry, according to the instructions of
the manufacturer. Briefly, A-549 cell suspension adjusted to a concentration of 1?5 x 106 cells/
mL in ice-cold PBS, 10% FCS 1% sodium azide. Cells were stained in an Anti-Human NLRP3
conjugated monoclonal antibody. To facilitate intracellular staining, cells were fixed with Flow
Cytometry Fixation Buffer and permeabilized with Flow Cytometry wash Buffer. Then, cells
centrifuged sufficiently, and the supernatant fluid was removed with minimum loss of cells.
The results are presented as means ? SEM. Analysis of variance (ANOVA) was conducted
with the Bonferroni post hoc comparison tests and used to compare the levels of inflammatory
biomarkers and to evaluate the differences between each group. P value < 5% was considered
Exposure to cigarrete smoke cause a depletion in cell viability
According to Fig 1, exposure of A-549 cells to CSE showed reduced cell viability compared to
the non-exposed cells (control group). The magnitude of cell depletion was partially correlated
with the concentration of cigarette smoke extract (Fig 1).
In order to evaluate the inflammatory response during COPD exacerbation, we added LPS
to the cell cultures, as an in vitro model of the disease. While the addition of LPS caused a
significant cell depletion in all CSE concentration (p value<0.001) it was not in a dose-dependent
manner, with no significant difference between CSE of 2% and 4% (p value = 0.0649). The
most profound effect was in cells exposed to 10% CSE with the addition of LPS (Fig 1).
The inflammatory response in COPD and COPD exacerbation model
The levels of IL-8 and MCP-1 were measured to evaluate the inflammatory effect in COPD.
In cells exposed to CSE solely, the concentration of IL-8 and MCP-1 increased significantly,
(P <0.001) (Fig 2A and 2B), though the differences between 2% and 4% concentration in the
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Fig 1. The effect of cigarette smoke exposure with or without LPS exposure on cell?s viability: A-549 cells were
incubated for 24 hours with CSE of 2%, 4% and 10% or with the combination of LPS (0.1 ?g\ml). Control group
incubated with medium alone (FCS 1%). Cell viability evaluated by using XTT test. The data represent the
mean ? SEM of 3 experiments P value<0.0001.
production of IL-8 (Fig 2A) and between 4% and 10% in the MCP-1 group (Fig 2B) were not
The addition of LPS exposure (Fig 3) induced an increase in IL-8 and MCP-1 (Fig 3A and
3B). The increment in levels of both cytokines was significant (P value<0.001 for both). Post
hoc analysis reveals that the most profound effect was observed in CSE 10% and LPS, while
there was no significant difference between control group to 2% CSE with LPS treatment.
Inflammasome activity in COPD and COPD exacerbation in vitro model
Levels of intra-cellular NLRP3 protein, were measured in cells exposed to CSE. No change was
observed in NLRP3 expression between the control group and CSE 2% (Fig 4A). However, a
significant increase in the inflammasome response was found in cells exposed to CSE in
concentrations of 4% or higher (Fig 4A). The addition of LPS increase NLRP3 activation in a
dose-dependent manner (Fig 4B), the mean NLRP3 counts were 21.63 (+ 0.18), 25.69 (?0.21),
32.54 (?0.28) 47.07 (?0.69) for control, 2%,4%.10% CSE, respectively.
Fig 2. The effect of CSE in different concentrations on the inflammatory response in a COPD in-vitro model:
A549 cells were incubated for 24 hours with CSE of 2%, 4% and 10% or with medium alone (FCS 1%, control
group). The culture supernatants were collected and the concentrations of IL-8 (A) and MCP-1(B) were measured by
ELISA. The data represent the mean ? SEM of 3 experiments. P value<0.0001.
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Fig 3. The effect of CSE in different concentrations on the inflammatory response in a COPD Exacerbation in
vitro model: A-549 cells were incubated for 24 hours with LPS (0.1?g\ml) and CSE of 2%, 4% and 10% or with LPS
alone (control group). The culture supernatants were collected and the concentrations of IL-8 and MCP-1 were
measured by ELISA. The data represent the mean of 3 experiments ? SEM. P value < 0.0001.
Since IL-1? production depends on NLRP-3 expression [
], we measured its levels in
the supernatant of the cell cultures. High levels of IL-1? were measured in cells exposed to CSE
4% and 10% versus control (A-549 cells without exposure to CSE) (Fig 5A).
Concordant to NLRP3 activity, the concentration of IL-1? also increased following
exposure to CSE+ LPS (Fig 5A and 5B).
Our study offers new in vitro model for COPD and COPD exacerbation, our model
demonstrates two major findings: first, NLRP3 and its functional products are up-regulated in an in
vitro model of COPD. Second, this up-regulation is augmented in COPD exacerbation.
Numerous in-vitro and animal models for stable COPD are in use [
to the limited data for COPD exacerbation models [
]. In our in vitro model, we exposed
A-459 cells to cigarette smoke in different concentrations, as previously established as an in
vitro model for COPD [
6, 19, 20
]. A-549 cell line is derived from a human pulmonary
adenocarcinoma with biochemical and morphological features of an alveolar cell and is frequently
used in experimental models [
6, 20, 21
]. The addition of LPS, designed to mimic COPD
exacerbation as a part of our in vitro model. This addition is based on the fact that bacterial
pathogens are the leading triggers to COPD exacerbation. and on other published studies [6,
Fig 4. The effect of CSE with different concentrations on the activity of inflammasome as a part of COPD and
COPD exacerbation in vitro model: A-549 cells were incubated for 24 hours with the following treatments: (A)- CSE
of 2%, 4% and 10% or with medium alone (control group), (B)- CSE of 2%, 4% and 10% and LPS (0.1?g\ml) or with
LPS (0.1?g\ml) alone (control group), then culture supernatants were collected. NLRP3 level was evaluated by Flow
cytometry. The data represent the mean of 3 experiments ? SEM. P value < 0.0001.
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Fig 5. The effect of CSE with different concentrations on the activity of IL-1? as a part of COPD and COPD
exacerbation in vitro model: A-549 cells were incubated for 24 hours with the following treatments:(A)- CSE of 2%,
4% and 10% or with medium alone (FCS 1%, control group), (B)- LPS (0.1?g\ml) and CSE of 2%, 4% and 10% or with
LPS alone (control group). The culture supernatants were collected. The concentration of IL-1? was measured by
ELISA. The data represent the mean of 3 experiments ? SEM. P value < 0.0001.
Our study supports the principal pathophysiology of cigarette smoke-induced
inflammation in COPD which damage lung tissue by depletion of cell viability [
]. Although there
wasn?t a significant difference between exposure to CSE 4% and CSE 10%, the overall effect on
viability was found to be dose dependent, as CSE concentration increases. This observation
may imply that the effect of CSE on cell viability reaches its maximal effect in the
concentration of 4%.
Since MCP-1 and IL-8 are important mediators in the inflammatory response in COPD
], our results strengthen the in-vitro model in this experiment for COPD and are in line
with previous studies demonstrating an increase in these cytokines in stable COPD [
We found high levels of MCP-1 and IL-8 after exposure to CSE, and IL8 levels were also in
correlation to CSE concentration. Concordant to cell viability experiment, this correlation was
not found in MCP-1 concentration: the highest concentration of MCP-1 was in cells exposed
to 4% of CSE and did not show further increase when exposing the cells to CSE 10%. We
predict this is due to the maximal production level.
The inflammasome pathway in respiratory diseases and particularly in COPD was
increasingly investigated in the last years, with a focus on stable COPD. The activation of NLRP-3 was
found to be a key modulator of respiratory infections and airway inflammation and the
assembly of NLRP-3 components?triggers a pro-inflammatory cell death mode [
Pauwels et al. showed that IL-1? is an important component in cigarette smoke- induced
inflammation and in COPD [
]. Similar to these results we found that exposing cell lines to
CSE, representing stable COPD, results in activation of the inflammasome pathway. In our
model, in addition to the increase in cell death and inflammatory cytokines, a higher NLRP-3
activity was observed in cells exposed to CSE. Furthermore, levels of IL-1? increased in a
similar manner, suggesting that the production of IL-1? is a direct result of the NLRP-3 activation.
Given that exacerbation in COPD is an episode of an enhancement of the immune
response, which causes an amplification of cell death [
], we found a greater decline in
viability of cells after LPS exposure, an augmentation in the production of IL-8 and MCP-1, as
well as a significant increase in NLRP3 activity and its product IL-1?.
Faner et al. found a high activity of NLRP3 and IL-1? in sputum samples taken from
patients with infectious COPD exacerbation [
]. Two human studies looked at biomarkers
during COPD exacerbation. Bafadel et al. investigated the expression of inflammatory
biomarkers in patients?sputum during COPD exacerbation and found that IL-1? levels correlate
significantly with COPD exacerbation due to bacterial infection [
], and recently, in a small
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cohort of COPD patients, levels of serum IL-1? and IL-17 were found elevated in COPD
exacerbation compared to stable COPD and healthy controls [
]. Altogether, these studies
validate the important role of NLRP3 during COPD exacerbation and are concordant with the
results shown by our in -vitro model of COPD exacerbation.
Our study has some limitations which originate, primarily since it is based on an in vitro
model. As such it cannot represent the multi-aspects of COPD exacerbation in real life. Other
medical illnesses in COPD patients, response to therapies, different etiologies for exacerbation
which may influence the results in the clinical settings. In addition, our model included only
epithelial cells, without their factual environment, thus cannot evaluate downstream products,
such as activation of innate immune cells which can further enhanced the upregulation of
NLRP3.These effects should be addressed in future studies.
In conclusion, our study presents a possible role of epithelial cells? inflammasome in an in
vitro model of COPD and COPD exacerbation. Further investigation in in-vivo models and in
COPD patients is needed to support our findings in the clinical setting. Nevertheless, our
results may lead to the foundation of NLRP-3 system as a novel biomarker in the diagnosis of
COPD exacerbation and as a new target for therapies.
S1 Table. The effect of cigarette smoke exposure with or without LPS exposure on cell?s
viability: A-549 cells were incubated for 24 hours with CSE of 2%, 4% and 10% or with the
combination of LPS (0.1 ?g\ml). Control group incubated with medium alone (FCS 1%). Cell
viability evaluated by using XTT test. The data represent the mean ? SEM of 3 experiments
S2 Table. The level of IL-8 and MCP-1 in response to cigarrete smoke exposure as an in
vitro model of COPD: A-549 cells were incubated for 24 hours with CSE of 2%, 4% and 10%
or with medium alone (FCS 1%, control group). The culture supernatants were collected and
the concentrations of IL-8 (A) and MCP-1(B) were measured by ELISA. The data represent
the mean of 3 experiments ? SEM.
S3 Table. The level of IL-8 and MCP-1 in response to cigarrete smoke exposure and LPS as
an in vitro model of COPD exacerbation: A-549 cells were incubated for 24 hours with LPS
(0.1?g\ml) and CSE of 2%, 4% and 10% or with LPS alone (control group). The culture
supernatants were collected and the concentrations of IL-8 and MCP-1 were measured by ELISA.
The data represent the mean of 3 experiments ? SEM.
S4 Table. The effect of CSE with different concentrations on the activity of IL-1? as a part
of COPD and COPD exacerbation in vitro model: A-549 cells were incubated for 24 hours
with the following treatments:(A)- CSE of 2%, 4% and 10% or with medium alone (FCS 1%,
control group), (B)- LPS (0.1?g\ml) and CSE of 2%, 4% and 10% or with LPS alone (control
group). The culture supernatants were collected. The concentration of IL-1? was measured by
ELISA. The data represent the mean of 3 experiments ? SEM.
S5 Table. The level of NLRP3 inflammasome in different concentrations of CSE with or
without exposure to LPS, as a part of an in vitro model for COPD and COPD exacerbation:
A-549 cells were incubated for 24 hours with the following treatments: (A)- CSE of 2%, 4%
and 10% or with medium alone (control group), (B)- CSE of 2%, 4% and 10% and LPS (0.1?g
7 / 9
\ml) or with LPS (0.1?g\ml) alone (control group), then culture supernatants were collected.
NLRP3 level was evaluated by Flow cytometry. The data represent the mean of 3
experiments ? SEM.
Conceptualization: Amir Bar-Shai.
Data curation: Noy Nachmias, Shani Shenhar-Tsarfaty.
Formal analysis: Noy Nachmias, Rafael Y. Brzezinski, Moshe Stark.
Investigation: Noy Nachmias, Sheila Langier, Matan Siterman, Sara Etkin.
Methodology: Noy Nachmias, Sheila Langier, Moshe Stark.
Project administration: Noy Nachmias.
Resources: Matan Siterman, Yehuda Schwarz.
Supervision: Amir Bar-Shai.
Validation: Avital Avriel, Shani Shenhar-Tsarfaty, Amir Bar-Shai.
Visualization: Noy Nachmias, Rafael Y. Brzezinski.
Writing ? original draft: Noy Nachmias.
Writing ? review & editing: Noy Nachmias, Amir Bar-Shai.
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1. Lopez AD , Murray CCJL . "The global burden of disease, 1990 - 2020 . Nature Medicine . 1998 ; 4 : 1241 . https://doi.org/10.1038/3218 PMID: 9809543
2. Bade G , Khan MA , Srivastava AK , Khare P , Solaiappan KK , Gulerai R et al. Serum cytokine profiling and enrichment analysis reveal the involvement of immunological and inflammatory pathways in stable patients with chronic obstructive pulmonary disease . Int J Chron Obstruct Pulmon Dis . 2014 ; 9 : 759 - 773 . https://doi.org/10.2147/COPD.S61347 PMID: 25125975
3. Wedzicha JA , Mackay A , Richa S. COPD exacerbations: impact and prevention . Breathe . 2013 ; 9 ( 6 ): 434 - 440 .
4. Brusselle GG , Joos GF , Bracke KR . New insights into the immunology of chronic obstructive pulmonary disease . Lancet 2011 ; 378 : 1015 - 1026 . https://doi.org/10.1016/S0140- 6736 ( 11 ) 60988 - 4 PMID: 21907865
5. Brusselle GG , Provoost S , Bracke KR , Kuchmiy A , Lamkanfi M. Inflammasomes in Respiratory Disease- From Bench to Bedside . CHEST 2014 ; 145 : 1121 - 1133 . https://doi.org/10.1378/chest.13-1885 PMID: 24798836
6. Victoni T , Gleonnec F , Lanzetti M , Tenor H , Valenca S , Porto LC , et al. Roflumilast N-Oxide Prevents Cytokine Secretion Induced by Cigarette Smoke Combined with LPS through JAK/STAT and ERK1/2 Inhibition in Airway Epithelial Cells . PLoS ONE 2014 ; 9(1): e85243 . https://doi.org/10.1371/journal. pone. 0085243 PMID: 24416369
7. Sin DD , Hollander Z , DeMarco ML , McManus BM , Ng RT . Biomarker Development for Chronic Obstructive Pulmonary Disease . From Discovery to Clinical Implementation. American Journal of Respiratory and Critical Care Medicine . 2015 ; 192 ( 10 ): 1162 - 70 . https://doi.org/10.1164/rccm.201505-0871PP PMID: 26176936
8. Bafadhel M , McKenna S , Terry S , Mistry V , Reid C , Halder P , et al. Acute exacerbations of chronic obstructive pulmonary disease: identification of biologic clusters and their biomarkers . Am J Respir Crit Care Med 2011 ; 184 : 662 - 671 . https://doi.org/10.1164/rccm.201104-0597OC PMID: 21680942
9. Lopez-Campos JL , Agusti A . Heterogeneity of chronic obstructive pulmonary disease exacerbations: a two-axis classification proposal , Lancet Respir Med 2015 ; 3 : 729 - 734 . https://doi.org/10.1016/S2213- 2600 ( 15 ) 00242 - 8 PMID: 26165134
10. Pauwels NS , Bracke KR , Dupont LL , Van Pottelberge GR , Provoost S , Vanden Berghe T , et al. Role of IL-1a and the Nlrp3/caspase-1/IL-1b axis in cigarette smoke-induced pulmonary inflammation and COPD . European Respiratory Journal 2011 ; 38 : 1019 - 1028 . https://doi.org/10.1183/09031936. 00158110 PMID: 21622588
11. Lee S , Suh GY , Ryter SW , Choi AM . Regulation and Function of the Nucleotide Binding Domain Leucine-Rich Repeat-Containing Receptor, Pyrin Domain-Containing-3 Inflammasome in Lung Disease . Am J Respir Cell Mol Biol . 2016 ; 54 ( 2 ): 151 - 60 . https://doi.org/10.1165/rcmb.2015 -0231TR PMID: 26418144
12. Faner R , Sobradillo P , Noguera A , Gomez C , Cruz T , Lo? pez-Giraldo A , et al. The inflammasome pathway in stable COPD and acute exacerbations . ERJ Open Res . 2016 ; 2 ( 3 ): 00002 - 2016 . Published 2016 Jul 11 . https://doi.org/10.1183/23120541. 00002 -2016 PMID: 27730204
13. Di Stefano A. , Caramori G. , Barczyk A. , Vicari C. , Brun ., Zanini A , et al. Innate immunity but not NLRP3 inflammasome activation correlates with severity of stable COPD . Thorax 2014 ; 69 : 516 - 524 . https:// doi.org/10.1136/thoraxjnl-2012 -203062 PMID: 24430176
14. Gesner C , Scheibe R , Wotzel M , Hammerschmidt S , Kuhn H , Engelmann L , et al. Exhaled breathe condensate cytokine patterns in chronic obstructive pulmonary disease . Respiratory Medicine 2005 ; 99 : 1229 - 1240 . https://doi.org/10.1016/j.rmed. 2005 . 02 .041 PMID: 16140223
15. Lamkanfi M , Dixit VM . Inflammasomes and their roles in health and disease . Annu Rev Cell Dev Biol . 2012 ; 28 : 137 - 161 . https://doi.org/10.1146/annurev-cellbio- 101011 -155745 PMID: 22974247
16. Hirota JA , Hirota SA , Warner SM , Stefanowicz D , Shaheen F , Beck PL , et al. The airway epithelium nucleotide-binding domain and leucine-rich repeat protein 3 inflammasome is activated by urban particulate matter . Journal of Allergy and Clinical Immunology 2012 , 129 .4: 1116 - 1125 . e6 . https://doi.org/ 10.1016/j.jaci. 2011 . 11 .033 PMID: 22227418
17. Mortaz E , Henricks PAJ , Kraneveld AD , Givi ME , Garssen J , Folkerts G . Cigarette smoke induces the release of CXCL-8 from human bronchial epithelial cells via TLRs and induction of the inflammasome . Biochimica et Biophysica Acta (BBA)-Molecular Basis of Disease 2011 : 1812 (9): 1104 - 1110 .
18. Giard DJ , Aaronson SA , Todaro GJ , Arnstein P , Kersey JH , Dosik H , et al. In vitro cultivation of human tumors: establishment of cell lines derived from a series of solid tumors . Journal of the National Cancer Institute . 1973 ; 51 ( 5 ): 1417 - 23 . PMID: 4357758
19. Basu S , Wiklund L . Experimental Models to Study Cigarette Smoke-Induced Oxidative Stress . Studies on Experimental Models . New-York, NY; Humana Press, 2011 : 401 - 402 .
20. Adamson J , Haswell LE , Phillips G , Gac?a MD. In vitro models of chronic obstructive pulmonary disease (COPD) . In Bronchitis. IntechOpen; 2011 .
21. Smith BT . Cell Line A 549: A Model System for the Study of Alveolar Type II Cell Function . American Review of Respiratory Disease . 1977 ; 115 ( 2 ): 285 - 93 . https://doi.org/10.1164/arrd. 1977 . 115 .2.285 PMID: 842942
22. Rotta detto Loria J , Rohmann K , Droemann D , Kujath P , Rupp J , Goldmann T , et al. Nontypeable Haemophilus Influenzae Infection Upregulates the NLRP3 Inflammasome and Leads to Caspase-1-Dependent Secretion of Interleukin-1b- A Possible Pathway of Exacerbations in COPD . PLoS ONE 2013 ; 8 (6): e66818 . https://doi.org/10.1371/journal.pone. 0066818 PMID: 23840534
23. Mullerova H , Maselli DJ , Locantore N , Vestbo J , Hurst JR , Wedzicha JA , et al. for the ECLIPSE Investigators, Hospitalized Exacerbations of COPD Risk Factors and Outcomes in the ECLIPSE Cohort , CHEST, 2015 ; 147 : 999 - 1007 .
24. Zou Y , Chen X , Liu J , Zhou DB , Kuang X , Xiao J , et al. Serum IL-1? and IL-17 levels in patients with COPD: associations with clinical parameters . Int J Chron Obstruct Pulmon Dis 2017 ; 12 : 1247 - 54 . https://doi.org/10.2147/COPD.S131877 PMID: 28490868