Nicotine and Cotinine Induce Neutrophil Extracellular Trap Formation-Potential Risk for Impaired Wound Healing in Smokers

Abstract

Smoking undoubtedly affects human health. Investigating 2318 representative patients at a level 1 trauma center identified delayed wound healing, tissue infections, and/or sepsis as main complications in smokers following trauma and orthopedic surgery. Therefore, smoking cessation is strongly advised to improve the clinical outcome in these patients, although smoking cessation often fails despite nicotine replacement therapy raising the need for specific interventions that may reduce the complication rate. However, the underlying mechanisms are still unknown. In diabetics, delayed wound healing and infections/sepsis are associated with increased neutrophilic PADI4 expression and formation of neutrophil extracellular traps (NETs). The aim was to investigate if similar mechanisms hold for smokers. Indeed, our results show higher PADI4 expression in active and heavy smokers than non-smokers, which is associated with an increased complication rate. However, in vitro stimulation of neutrophils with cigarette smoke extract (CSE) only moderately induced NET formation despite accumulation of reactive oxygen species (ROS). Physiological levels of nicotine and its main metabolite cotinine more effectively induced NET formation, although they did not actively induce the formation of ROS, but interfered with the activity of enzymes involved in anti-oxidative defense and NET formation. In summary, we propose increased formation of NETs as possible triggers for delayed wound healing, tissue infections, and/or sepsis in smokers after a major trauma and orthopedic surgery. Smoking cessation might reduce this effect. However, our data show that smoking cessation supported by nicotine replacement therapy should be carefully considered as nicotine and its metabolite cotinine effectively induced NET formation in vitro, even without active formation of ROS.

Keywords: cotinine; neutrophil extracellular traps; nicotine; reactive oxygen species; smoking.

Figure 1

Overview of the patients prospectively collected to analyze the effect of smoking on neutrophilic PADI4 expression. (A) CONSORT diagram on the patients analyzed in this study. Smoking was recorded as packyears (PY). The complications rates in non-smokers and smokers are summarized as (B) pie chart and additionally as (C) VENN diagram to show multiple complications in one individual.

Figure 2

Inter-individual stability of the housekeeper genes. Five different housekeeping genes (B2M, HPRT, RPL13, 18S, and EF1α) were tested with cDNA from 10 different donors. The stability of the housekeeper genes was analyzed by ΔCt, BestKeeper, GENorm, and NormFinder methods. The results are presented as spider-web diagram with the stability values as axis. In summary, the housekeeping genes with the best inter-individual stability were RPL13 and EF1α.

Figure 3

Effect of smoking on the complication rate in patients following orthopedic and trauma surgery. Data on smoking behavior and post-surgical complications from 2318 representative patients at a level 1 trauma center have been obtained from the hospital information system. (A) Smoking was recorded as packyears (PY): 51.3% of the patients claimed to be non-smokers (0 PY), 29.5% of the patients were considered as moderate smokers (<20 PY), and 18.8% of the patients were considered as heavy smokers (≥20 PY). (B) Complications were summarized as groups and are presented in a cord diagram (made with SankeyMATIC, https://sankeymatic.com/build/, accessed on 20 October 2022), where the cord width equals the complication rate in % within the individual group (non-, moderate, or heavy smokers).

Figure 4

Influencing factors on neutrophilic PADI4 expression in orthopedic and trauma patients. Neutrophilic PADI4 expression was analyzed by qRT-PCR using the 2^−ΔΔCt method with EF1α and RPL13a as housekeeping genes and non-smokers as reference. To investigate the effect of smoking, samples were divided according to the patients smoking behavior, differentiating (A) non-smokers (0 PY), moderate smokers (<20 PY), and heavy smokers (≥20 PY); (B) non-smokers, former smokers and active smokers, and (C) non-smokers and smokers that did or did not develop complications. To identify other factors that possibly affect neutrophilic PADI4 expression, samples were additionally grouped by (D) age, (E) body mass index, number of (F) comorbidities and (G) daily drugs, and (H) alcohol consumption. Data are presented as violin blots with median and interquartile range. Groups were compared by non-parametric Kruskal–Wallis test followed by Bonferroni multiple comparison test. A p < 0.05 was considered as significant as is presented in the graphs.

Figure 5

Induction of NET formation by CSE, nicotine, and cotinine. Neutrophils were isolated from healthy volunteers and stimulated for NET formation with freshly prepared (A) CSE (10, 20, and 30%), (B) nicotine (100, 200, and 300 ng/mL), or (C) cotinine (100, 200, and 300 ng/mL). As a positive control, cells were stimulated with 100 nM PMA. NET formation was quantified with the SYTOX^TM Green assay (N ≥ 5, n = 6). Data are presented as box blots with individual measurement points, median, and interquartile range. Groups were compared by non-parametric Kruskal–Wallis test followed by Bonferroni multiple comparison test. * p < 0.05, ** p < 0.01, and *** p < 0.001 as compared to the respective control group (Ctrl). NET formation was additionally confirmed by immuno-fluorescent imaging for DNA (Hoechst 33342—blue) and MPO (green). Representative images are given in (D). Scale bar = 400 µm.

Figure 6

Influence of CSE, nicotine, and cotinine on neutrophilic ROS formation and anti-oxidative defense mechanisms. Neutrophils were isolated from healthy volunteers, loaded with DCFH-DA, and then stimulated with freshly prepared (A) CSE (10, 20, and 30%), (B) nicotine (100, 200, and 300 ng/mL), or (C) cotinine (100, 200, and 300 ng/mL) to quantify formed ROS. The effects of 20% CSE, 200 ng/mL nicotine (NIC), or 200 ng/mL cotinine (COT) on the activity of isolated (synthetic) enzymes of the anti-oxidative defense were determined. Enzyme activities analyzed were (D) superoxide-dismutase (SOD), (E) catalase (CAT), (F) glutathione peroxidase (GPx), and (G) glutathione reductase (GR). (N = 6, n = 6). Data are presented as box blots with individual measurement points, median, and interquartile range. Groups were compared by non-parametric Kruskal–Wallis test followed by Bonferroni multiple comparison test. * p < 0.05 and ** p < 0.01 as compared to the respective control group (Ctrl).

Figure 7

Influence of CSE, nicotine, and cotinine on activity of enzymes involved in NET formation. The effects of 20% CSE, 200 ng/mL nicotine (NIC), or 200 ng/mL cotinine (COT) on the activity of isolated enzymes involved in NET formation were determined. Enzyme activities analyzed were (A) peptidyl-arginine-deiminase 4 (PADI4), (B) myeloperoxidase (MPO), (C) neutrophilic elastase (ELANE). (N = 4, n = 2). Data are presented as box blots with individual measurement points, median, and interquartile range. Groups were compared by non-parametric Kruskal–Wallis test followed by Bonferroni multiple comparison test. * p < 0.05 and ** p < 0.01 as compared to the respective control group (Ctrl).