Cigarette smoking leads to vast array of illnesses including cardiac diseases. The focus of our laboratory is to investigate the influence of cigarette smoke exposure on Cardiac Stem Cell (CSC) functions and study underlying mechanisms. Cardiac stem cell therapy is one of the emerging treatment options to treat myocardial damage. Therefore, studying cigarette smoke-induced modulations of CSC functions and responsible mechanisms are of furthest importance. The current study investigated whether CSCs produce interleukin 1 beta (IL-1ß), which is a potent pro-inflammatory cytokine, in response to exposure to cigarette smoke, with the view of understanding the role of interleukins as one of the causative agents in cigarette smoke- induced modulation of cardiac stem cell functions. Cigarette Smoke Extracts (CSE) were created by combusting camel filter-free cigarettes and dissolving the smoke puff in cell culture media based on a published method. No smoke control (0% CSE) and three different concentrations of CSE, which are 2%, 5%, and 10%, were utilized. CSCs were cultured and treated with CSE for 15 minutes, 1 hour, and 24 hours. Then from each treatment group media on top of the CSCs (supernatant) were removed and protein samples were quantified using badford protein assay. Equal amount of protein from each treatment was subjected to IL-1ßELISA based on manufacturer’s instructions. Compared to no smoke control, in 5% and 10% CSE treated CSCs, IL-1ßsecretion were significantly upregulated within 15 minutes (p < 0.05). Based on the results it can be concluded that acute cigarette smoke exposure promotes secretion of IL-1ß at 5% and10% concentrations.
Cigarette smoking is a public health crisis and a leading cause of death around the world. Smoking leads to numerous illnesses including several Cardiovascular Diseases (CVD) . Cigarette smoking related CVD include coronary artery disease / atherosclerosis [2-5], hypertension  and stroke . Cigarette smoking-induced atherosclerosis occurs due to many mechanisms including modulation of prostaglandin levels , modification of the function of the endothelial cells, platelets, fibrinogen, and coagulation factors [8-10]. In addition, activation of endothelial inflammatory response, Il-8 , IL-6  and IL-1ß production  leading to downstream signaling such as activation of Cyclooxygenase 2 (COX 2) enzyme  play a role in cigarette smoke-induced atherosclerosis. Cigarette Smoke (CS) caused attenuation of mitochondrial function both in-vitro (H9C2 heart cells)  and in-vivo (wild type FVB mice) . In addition, CS induced endothelial cell death  and modulation of cardiac contractility  have been reported, all of these will lead to CS induced -CVD.
Although abundant literature is available linking CS exposure with CVD, there is limited literature on CS-induced changes in Cardiac Stem Cells (CSCs). Our focus is to investigate CS-induced modulations of Cardiac Stem Cell (CSC) function and responsible mechanisms. CS-induced adverse functional effects of c-kit+ Cardiac Stem Cells (CSCs) were reported for the first time by our group . Cardiac Stem Cell Therapy (CSCT) is currently being used in clinical trials . Adverse changes of CSCs caused by CS might lead to decreased potential of resident CSCs to respond to cardiac injury or to be used in CSCT. Therefore, studying CS-induced modulations of CSC functions and responsible mechanisms are of uttermost importance.
Our previous study reported that exposure to Cigarette Smoke Extract (CSE) caused adverse functional effects in CSCs. Those CSE- induced CSC modulations included increased apoptosis, decreased proliferation, increased cytotoxicity and gap junctional permeability . The current study investigated whether CSCs produce interleukin 1 beta (IL-1ß) in response to exposure to CSE, with the view of understanding the role of interleukins as one of the causative agents in CSE-induced modulation of CSC signaling and functional effects.
IL-1ß and IL-18 are inflammatory cytokines that mature in inflammasomes, which are usually not present in healthy cells but are assembled upon infection, cellular stress, cell and tissue injury . IL-1ß is one of the key mediators of inflammation, which is necessary to elicit host response to pathogens . Upon stimulation by an insult, IL-1ß is secreted continuously from inflammasomes and the secretion depends on the stimulus strength and the extracellular IL-1 beta requirement . IL-1ß plays a role in progression of cardiovascular disease. Increased secretion of Il-1ß was linked to the progression of many cardiovascular diseases including heart failure , ventricular arrhythmias due to QT interval prolongation  and coronary artery disease / atherosclerosis . Our laboratory focuses on studying cigarette smoke induced cardiovascular effects and the focus of this project is to investigate whether cigarette smoke induces IL-1ß production in cardiac stem cells.
Tobacco induced production of inflammatory cytokines was observed in both in-vitro and in-vivo studies. Cigarette smoke induced inflammation was seen in several different cell types leading to many different diseases. Upregulation of IL-6 and IL-1ß due to cigarette smoke exposure have been reported in human gingival cells . Increased interleukin 8 production due to cigarette smoke exposure was seen in dendritic cells  and endothelial cells . Mice exposed to cigarette smoke as well as waterpipe tobacco smoke demonstrated elevated levels of pro-inflammatory markers such as TNF-α and IL-6 in the lung alveoli .
In a cardiac transplant study, it has been reported that pre-exposure of donor and recipient rats to cigarette smoke caused allograft rejection due to inflammation/interleukin production . However to date the cigarette smoke-induced production of interleukin 1ß has not been studied in c-kit+ CSCs. The role of these interleukins in cigarette smoke-induced modulations of cardiac stem cell functions remains elusive. The current study tested the hypothesis that exposure to Cigarette Smoke Extract (CSE) will cause increased production of IL-1ß in CSCs.
Aqueous extracts of cigarette smoke were prepared using the smoking apparatus, a 60 ml syringe attached to an adapter to hold the cigarette (Figure 1). CSCs were treated with these freshly prepared CSE for different time periods. Acute (either 15 minutes or 1 hour) CSE exposure to CSCs caused an increase in IL-1ß secretion in a dose dependent manner. At 15 minute CSE exposure, IL-1ß production of CSCs over no smoke control was significant at 5% and 10% CSE concentrations (Figure 2). Following 1 hr CSE exposure, CSE caused significant increase in IL-1ß secretion at 10% CSE concentrations (Figure 2). In addition, at 10% CSE treatment, IL-1ß secretion after 1 hour exposure was significantly higher than that of 15 min exposure. Long term (24 hour) exposure of CSCs to CSE showed significant increase in IL-1ß production only at 2% concentration (Figure 3).
Figure 2: Detection of IL-1β production due to acute exposure to CSE. c-kit+ CSCs were treated with either the no smoke control or three different concentrations (2%, 5% and 10%) of CSE dissolved in 10 ml of serum free HAM’s F-12 media for 15 minutes and 1 hr. After CSE treatment, equal amount of protein from treatment groups (supernatants) were subjected to IL-1β ELISA. Secreted IL-1β was quantified, and the amount present in the no smoke control was taken as 1 and CSE-induced production of IL-1β relative to the no smoke control was plotted against the CSE concentration.
Tobacco smoke is an intricate mixture that contains thousands of chemicals , many of which are water soluble and present in aqueous extracts. A study that analyzed the water-soluble portion of cigarette smoke utilizing IR, mass and NMR spectra reveled that there were close to 500 water-soluble components in cigarette smoke . These include groups such as acids, lactones, esters, amides, imides, aldehydes, ketones, alcohols, imidazole and pyridine derivatives . Aqueous extracts of cigarette smoke contain chemicals including tar, which contains a mixture of semiquinones, hydroquinones and quinones that can produce free radicals, which were detected by Electron Paramagnetic Resonance (EPR) spectroscopy and Gas Chromatography/Mass Spectrometry (GC/MS) . Analysis of the aqueous extraction from the tobacco that were used to fill cigarettes by Electrospray Ionization-Ion Trap Mass Spectrometry (ESI-ITMS) revealed that the tobacco extract contained quinic acid, citric acid and malic acid .
It was difficult to determine the exact chemical components of the CSE in our aqueous preparation since there were so many of them. However, it is very likely that all water soluble contents in particulate matter and gas phase must be dissolved in the culture medium. Our aqueous extract of cigarette smoke should contain all the water soluble components of cigarette smoke, including cotinine, hydralazine, heavy metals, anthracenes, tobacco specific nitrosamines, alkaloid, ammonia, dioxins, nicotine and pyrenes including benzo (a) pyrene; many of which are known toxicants and carcinogens [38,42].
Although CSE induced-cytotoxicity of many cells including cardio myoblasts  and endothelial cells  have been reported, our group was the first ones to report the CSE-induced malfunction of c-kit+CSCs . We have reported that CSE exposure has led to attenuated CSC proliferation and migration, increased CSC apoptosis, cytotoxicity and caused damage in CSC membrane resulting in dysfunctional CSCs .
In 2015, based on an in-vitro differentiation assay, palpant and colleagues reported that CSE (both tobacco cigarettes and e-cigarettes) caused malfunction of human embryonic stem cells leading to malformed hearts . This study also investigated CSE induced in-vivo effects using zebra fish. They have reported that CSE caused severe heart malformation and reduced heart function of zebra fish, and the toxic effects were more severe in tobacco cigarettes compared to e cigarettes . CSE also caused reduced heart rate and decreased expression of cardiac transcription factors and channels including GATA4 and L-type calcium channels .
The focus of our laboratory is to investigate the CSE induced damage to c-kit+ CSCs and to unveil the possible mechanism. Properly functioning CSCs are required for cardiac stem cell therapy. Cardiac stem cell therapy is one of the promising therapeutic options available to treat a damaged heart following Myocardial Infarction (MI) . Stem cell therapy using c-kit+ cardiac stem cells has been conducted in post MI patients and proven to increase cardiac function of the stem cell therapy recipients .
Since we have previously reported CSE induced functional changes of CSCs, the current study was focused on investigating the possibility of CSE induced modulation of IL-1ß secretion as a possible mechanism for CSE induced changes in CSCs. IL-1ß is a potent pro-inflammatory cytokine and it is upregulated in the infarcted myocardium due to MI- induced activation of cardiomyocytes and interstitial cell inflammasomes . In addition to cytokines, ischemic damage due to MI leads to recruitment of huge amount of leukocytes. A recent study utilizing Apolipoprotein -E (ApoE) knock out atherosclerotic mice, suggests that IL-1ß was involved in hematopoietic stem cell proliferation leading to large amount of leukocyte production. Targeting IL-1ß provides a way to decrease inflammatory damage after MI . There were some reports discussing CSE induced IL-1ß production in other cell models [48,49]. However, the current study is the first report discussing CSE induced IL-1ß release in c-kit+ CSCs. Here we tested the hypothesis that CSE caused an increase in IL-1ß secretion in CSCs.
Based on the results of the current study, it can be concluded that cigarette smoke (5% and 10%) promotes production of IL-1ß in c-kit+ CSCs within a short period of time (Figure 2). It is a novel finding and we are the first ones to report CSE induced IL-1ß recruitment in c-kit+ CSCs. IL-1ß is a secretory cytokine and 2% CSE may not be sufficient to cause significant increase in IL-1ß in CSCs for exposures of 15min or 1 hr. However, when exposed to CSCs for 24 hours, it increases Il-1ß production significantly (Figure 3).
Following 1 hr exposure of CSE, there is a dose dependent increase in IL-1ß release from CSCs, which is significant at 10% CSE concentration. In agreement with our results, the CS-induced production of IL-1ß has been reported in macrophages  and blood mononuclear cells . CS induced IL-8 has also been reported in Human Umbilical Vein Endothelial Cells (HUVEC) [11,25].
Based on an inflammatory transcriptome profiling study using CSE and Human Monocytes (THP-1 cells), it has been reported that numerous genes, which are involved in innate immune system, were activated by 10% CSE exposure. Following 8 hr exposure to 10% CSE, pro-inflammatory cytokines such as TNF and IL-12 genes were upregulated . Following 24 hours of exposure to 10% CSE, the gene profile has somewhat changed and genes involved in adaptive immune system were activated, no longer showing an increase in pro inflammatory IL- 12. Their data was in agreement with the recent hypothesis of “monocytes and macrophages change their profile from a pro-inflammatory phenotype (caused by innate immunity) to a tissue remodeling phenotype (caused by adaptive immunity) when in diseased tissue” . Both innate and adaptive immunity play a crucial role in cardiac injury and repair . Cardiac injury induced gene profile switch from pro-inflammatory genes to tissue repair genes occurred with the long term exposure of 10% CS .
This is in agreement with our results. In our study, 24 hr exposure of both 10% and 5% CSE did not cause significant increase in IL-1ß production in CSCs. The reason for non-significant IL-1ß production at 24 hr exposure to higher CSE concentrations could be due to more dead cells causing the switch in pro-inflammatory genes to tissue repair genes, as seen by wright and colleagues in their experiments . Our group previously reported the increased apoptotic death of the same cell line at 5% and 10% CSE concentrations .
Any of the other pro-inflammatory genes in interleukin family (Il-1ß, IL-6, or IL-8) were not upregulated or downregulated by CSE treatment ininflammatory transcriptome profiling study . According to another report, 6 hr and 24 hr. CSE exposure to cultured carotid arteries resulted in significant increase in IL-1ß and IL-6 mRNA . Since we have observed significant increase in CSE induced Il-1ß production in CSCs just after 15 minutes, it is very unlikely that this Il-1ß production is a result of transcription, because transcription takes long time (hours). Therefore, ELISA was chosen over RT-PCR, so we can detect CSE-induced modulation of IL-1ß protein level.
IL-1ß plays a major role in inflammatory damage after myocardial infarction and it is upregulated in the infarcted myocardium . Since c-kit+ CSCs are currently being used in CSCT trials it is important to investigate the ways to improve function of c-kit+ CSCs. The current study will provide mechanistic insight for CSE induced malfunction of c-kit+ CSCs reported earlier . CSE induced IL-1ß production is just one of the possible mechanisms for CSE induced CSC malfunction. CSE induced modulation of other cytokines, cell signaling, and oxidative stress may serve as other possible mechanisms. Those mechanisms and the effects of IL-1ß inhibition on CSC functions will be investigated in the near future.
Funding was provided by the Sullivan University Faculty Development Grant (RG_1_PS_2012_03).
Special thanks go to Dr. Anversa and Dr. Rokosh for providing c-kit+ CSCs. Dr. Rokosh also provided the smoking apparatus and also served as a collaborator of the funded grant. We would like to thank Dr. Zhao, Sullivan University College of Pharmacy for serving as a collaborator for the funded grant. We acknowledge Dr. Houston Acha for his help with preparation of chemicals and Thimira Sumanasekera for editing the manuscript.
Citation: Sumanasekera W, Waingeh B (2016) Does Cigarette Smoke Cause Interleukin 1 - Beta (Il-1ß) Production in Cardiac Stem Cells? J Cell Biol Cell Metab 3: 012.
Copyright: © 2016 Wasana Sumanasekera, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.