Cigarette smoking induces human CCR6+Th17 lymphocytes senescence and VEGF-A secretion

Chronic exposure to environmental pollutants is often associated with systemic inflammation. As such, cigarette smoking contributes to inflammation and lung diseases by inducing senescence of pulmonary cells such as pneumocytes, fibroblasts, and endothelial cells. Yet, how smoking worsens evolution of chronic inflammatory disorders associated with Th17 lymphocytes, such as rheumatoid arthritis, psoriasis, Crohn’s disease, and multiple sclerosis, is largely unknown. Results from human studies show an increase in inflammatory CD4+ Th17 lymphocytes at blood- and pulmonary level in smokers. The aim of the study was to evaluate the sensitivity of CD4+ Th17 lymphocytes to cigarette smoke-induced senescence. Mucosa-homing CCR6+ Th17- were compared to CCR6neg -and regulatory T peripheral lymphocytes after exposure to cigarette smoke extract (CSE). Senescence sensitivity of CSE-exposed cells was assessed by determination of various senescence biomarkers (β-galactosidase activity, p16Ink4a- and p21 expression) and cytokines production. CCR6+ Th17 cells showed a higher sensitivity to CSE-induced senescence compared to controls, which is associated to oxidative stress and higher VEGFα secretion. Pharmacological targeting of ROS- and ERK1/2 signalling pathways prevented CSE-induced senescence of CCR6+Th17 lymphocytes as well as VEGFα secretion. Altogether, these results identify mechanisms by which pro-oxidant environmental pollutants contribute to pro-angiogenic and pathogenic CCR6+Th17 cells, therefore potential targets for therapeutic purposes.

Substantial epidemiologic evidence links environmental pollutants to adverse health effects through an inflammatory pathway. As such, cigarette smoking plays dual role in regulating immunity by either attenuation of defensive immunity or exacerbation of pathogenic immune responses 1 . Prevalence of smoking is higher in patients with inflammatory diseases such as chronic obstructive pulmonary disease (COPD) and various pathogenic Th17-associated autoimmune disorders such as rheumatoid arthritis, psoriasis, Crohn's disease, and multiple sclerosis; moreover, smoking worsens the evolution of these diseases 2 . While the mechanism of cigarette smoke affecting the lung is well known, its effect on CD4 + T helper (Th) lymphocytes is still incompletely understood. CD4+ Th lymphocytes differentiate upon combined activation through the T cell receptor for antigen (TCR), costimulatory signals through CD28 receptor and appropriate polarizing cytokines into different subsets (e.g. Tfh, Th1, Th2, Th17, regulatory T cell). Those are characterized mainly by their distinct master transcription factors as well as the cytokines they produce and ultimately their functions against invading pathogens or in mediating tolerance 3 . IL-17-producing Th17 cells protect against extracellular bacterial and fungi infections by secreting cytokines IL-17A, IL-17F and IL-22. However, so-called "pathogenic" Th17 have been described as contributing to the pathogenesis of several human chronic inflammatory diseases via excessive and/or aberrant cytokine production 3 . The chemokine receptor CCR6 (CD196) was identified as the main surface marker characterizing the Th17 lineage, and regulating the recruitment of Th17 cells into inflamed tissues under physiological as well as inflammatory conditions 3 . Data from human studies show an increase in CD4 + Th17 cells absolute number and 1 Université Paris-Est, Créteil, 94000, France. 2 Inserm, UMR 955, équipe 4, Créteil, F-94010, France. 3 Inserm, UMR 955, équipe 7, Créteil, F-94010, France. 4 Institut Curie, PSL Research University, Paris, France. 5 Inserm, UMR 932, F-75005, Paris, France. 6 Inserm, UMR 955, équipe 16, Créteil, 94000, France. 7 Sorbonne Université, UPMC Univ-Paris 06, CIMI-Paris-INSERM UMR U 1135, Paris, France. 8 Inserm, UMR 955, équipe 3, Créteil, F-94010, France. 9 AP-HP, Hôpital H. Mondor-A. Chenevier, Pôle de Biologie-Pathologie, Créteil, 94000, France. 10 These authors contributed equally: Indoumady Baskara and Stéphane Kerbrat. *email: sabine.le-gouvello@aphp.fr proportion, in the blood and lungs of smokers [4][5][6][7] . Accordingly, cigarette smoke-induced pulmonary inflammation is attenuated in CCR6-deficient mice by preventing mucosa-homing of CCR6 + Th17 cells 8 . However, the direct influence of cigarette smoke exposure towards CCR6 + Th17 cells and its pathogenic consequences, remain largely unknown.
Various studies show an increased number of senescent cells in lungs and peripheral leucocytes from smokers with normal lung function and even higher in patients with COPD [9][10][11] . Cellular senescence is recognized as a cell-cycle arrest and is induced by replicative proliferation or various stresses, including oxidative stress 12 . Replicative senescence as well as stress-induced senescence show similar phenotypic features such as increased activity of senescence-associated-β-galactosidase (SA-β-Gal), relative resistance to apoptosis, and activation of the ATM/ATR-p53-p21 Cdkn1a and/or the p16 INK4a -retinoblastoma protein (pRb) pathways. Senescent cells usually secrete a complex mix of mostly pro-inflammatory factors termed as senescence-associated secretory phenotype (SASP), which can have detrimental effects on organs function 12 . In the present study, we hypothesized that cigarette smoke could induce senescence of resting Th17 cells, which in turn will promote pathogenic secretory protein profiles in smokers, independently of any infectious challenge. In order to examine this hypothesis, we first analyzed the sensitivity of non-reactivated memory Th17 cells to senescence. To do so, we used an in vitro standardized method 13 by testing acute effects of cigarette smoke extract (CSE) on CD4 + CD45RO + CCR6 + Th17 memory cells from healthy donors. Secondly, we examined the molecular mechanisms involved during this process, by focusing on oxidants and ERK1/2 pathway.

Results
CCR6 + Th17 cells are highly susceptible to cigarette smoke-induced senescence. To analyze the senescence susceptibility of Th17 cells to CSE, we evaluated three hallmarks of senescence: SA β-gal activity, p16 INK4a and p21 Cdkn1a expression 14 . Resting CCR6 + CD45RO+ Th17 memory cells (henceforth referred to as "CCR6 + Th17") were treated with non-toxic doses of CSE (Fig. S1A), and compared to CSE-exposed resting CCR6 neg T effector/memory lymphocytes (henceforth referred to as "CCR6 neg Th") and regulatory T cells (Treg). CSE exposure induced SA β-gal activity both in CCR6 + Th17 and CCR6 neg Th cells, but not in Treg. The proportion of SA β-gal positive cells was significantly more important among CCR6 + Th17 compared to CCR6 neg Th cells (Fig. 1A). Similar effects were observed for the expression of p16 INK4a in CCR6 + Th17 and CCR6 neg Th cells, after 5% CSE treatment (Fig. 1B). Conversely, p21 Cdkn1a expression in CCR6 + Th17 and CCR6 neg Th cells was not significantly different compared to controls, after CSE exposure (Fig. 1C). The proportion of live CCR6 + Th17 cells, observed after 48 hours treatment with CSE 5%, attested their absence of proliferation (Figs. 1D & S1A). In addition, the level of transcripts such as CTLA4, ICOS and PD-1, associated with replicative senescence-induced exhaustion 15 was unchanged after CSE exposure (Fig. 1E). As resistance to apoptosis is one of the hallmarks of senescence 14 , we analyzed the expression of pro-and anti-apoptotic effectors (Bcl2, Bcl-xL, Bcl-xS, Bim, Fas, TNFR2) after exposing cells to CSE. Only CCR6 + Th17 cells showed a decrease in the pro-apoptotic gene Fas expression, and only CCR6 neg Th cells presented a decrease in the anti-apoptotic Bcl2 gene expression compared to controls, after treatment of cells with CSE (Fig. 2); no modulation of the other genes expression was observed after CSE exposure.
Reactive oxygen species (ROS) modulate ERK1/2 activity and its regulation is required for T cell survival, proliferation and cytokine production 19 . To investigate the direct involvement of ROS in CSE-induced senescence of CCR6 + Th17 cells, we examined the effect of pan free-radical scavenger N-acetyl-cysteine (NAC) on CSE-dependent ERK1/2 activation. Pretreatment with NAC prevented nuclear staining of phospho-ERK1/2 induced in CCR6 + Th17 cells by CSE exposure. Similarly, CCR6 + Th17, pretreated with NAC, presented similar level of cytosolic phospho-ERK1/2 staining compared to control cells, regardless CSE doses used (Fig. 4B). www.nature.com/scientificreports www.nature.com/scientificreports/ High sensitivity of CCR6+ Th17 cells to cigarette smoke-induced senescence is associated with higher ROS production. CSE contains more than 4500 chemicals, in which some of them are well-known sources of exogenous ROS 20 . Moreover, endogenous cellular production of ROS is increased in response to CSE exposure, in smokers 21 . To further investigate the involvement of ROS in our system of interest, we first treated CCR6 + Th17 and CCR6 neg Th cells with exogenous H 2 O 2 . p16 INK4a expression was increased in H 2 O 2 -treated CD4+ T cells compared to non-treated cells suggesting the contribution of ROS in CSE-induced senescence of CCR6 + Th17 and CCR6 neg Th cells. A greater sensitivity of CCR6 + Th17 cells to ROS-associated senescence is also observed in comparison with CCR6 neg Th cells (Fig. 5A). Then, we tested the effect of NAC pretreatment on CCR6 + Th17 cells and the induction of p16 INK4a expression and VEGFα production after CSE exposure. Since mitochondrial metabolism is a critical component of T cell activation 22 , we also tested the effects of pretreatment with rotenone and/or antimycin A (Ant), two inhibitors of mitochondrial complex I and complex III respectively. NAC pretreatment mitigated CSE-induced increase in p16 INK4a expression in both CCR6 + Th17 and CCR6 neg Th cells. However, in NAC-pretreated cells, CSE exposure induced a higher proportion of p16 INK4a -positive CCR6 + Th17 cells compared to controls (Fig. 5B). Pretreatment with Ant prevented this effect on p16 INK4a expression in CCR6 + Th17 and CCR6 neg Th cells (Fig. 5C). Simultaneous pretreatment with rotenone and Ant induced similar results (data not shown). Pretreatment with NAC inhibited VEGFα production induced by CSE treatment in both cell subtypes, whereas Ant pretreatment had no effect (Fig. 5D). We further explored the ROS involvement in CSE-induced senescence of Th cells, by testing the effect of CSE exposure on H 2 DCF-DA probe oxidation, which is mostly dependent on the presence of H 2 O 2 23 . Basal H 2 DCF-specific fluorescence was higher in resting CCR6 + Th17 compared to resting CCR6 neg Th cells (Fig. 6A). This difference of basal fluorescence intensity between the two resting cellular subpopulations was deleted by performing a pretreatment with either Ant or NAC or both (Fig. 6A). In response to CSE exposure, H 2 DCF -specific fluorescence intensity was increased www.nature.com/scientificreports www.nature.com/scientificreports/ in both sub-populations and the intensity enhancement was similar in CCR6 + Th17 and in CCR6 neg Th cells (1.5 fold). Independent pretreatment of cells with either Ant or NAC or both reduced H 2 DCF-specific fluorescence in CSE-exposed CCR6 + Th17 and CCR6 neg Th cells. These pretreatments were also associated with an abolition of the difference in H 2 DCF-specific fluorescence observed between 5% CSE-exposed CCR6 + Th17 and CCR6 neg Th cells; however, H 2 DCF-specific fluorescence remained higher in CCR6 + Th17 cells pretreated with Ant and after 10% CSE exposure, compared to controls (Fig. 6A). As mitochondrial-derived superoxide (O 2 − ) production could be converted in H 2 O 2 , we measured mitochondrial O 2 − using the mitochondria-specific probe MitoSOX Red 23 . After treatment with H 2 O 2 or rotenone/Ant 24 , MitoSOX-specific fluorescence was increased more markedly in CCR6 + Th17 cells compared to CCR6 neg Th cells, suggesting a higher mitochondrial basal activity in CCR6 + Th17 cells (Fig. 6B). Mitochondrial O 2 − production was not altered in CSE-exposed CCR6 + Th17 and CCR6 neg Th cells; CSE exposure decreased MitoSOX-specific fluorescence in rotenone/Ant-pretreated CCR6 + Th17 and CCR6 neg Th cells, and abolished the difference in MitoSOX-specific fluorescence intensity observed between these two subpopulations. Coherently, the expression of NAD(P)H:quinone oxidoreductase (NQO1) and heme oxygenase-1 (HO-1), two major NF-E2-related factor 2 (Nrf2) regulated anti-oxidant phase II genes 25 , was more abundant in CSE-exposed CCR6 + Th17 cells than in CCR6 neg Th cells (Fig. 6C).

Discussion
Although the literature is rich in defining the immunosuppressive role of cigarette smoke exposure on TCR-reactivated antigen-specific Th cells 3 , its effect on memory resting Th cells in a quiescent state is not known. Through the study of peripheral CCR6 + Th17 cells 26 exposed to cigarette smoke independently of TCR stimulation, we provide evidences that higher sensitivity to CSE-induced premature senescence of resting CCR6 + Th17 cells compared to other memory CD4 + T cells relies on higher basal ROS production and dependent signalling in these cells. Moreover, CSE-induced premature senescence of CCR6 + Th17 is associated with VEGFα production, which could confer pathogenicity to CCR6 + Th17 cells and contribute to worsen chronic inflammation in smokers with autoimmune diseases when homing to mucosal tissues.
Increased number and ratio of CCR6 + Th17 cells have been found in healthy smokers and patients with COPD, and associated with the capacity of dendritic cells of smokers to bias differentiation of naïve CD4 + T cells toward a Th17 phenotype 27 . Considering elevated levels of senescent pulmonary cells in smokers with COPD 9-11,28 , our current findings that acute CSE exposure induces CCR6 + Th17 cells premature senescence can at least partially also explain the resultant systemic accumulation of CCR6 + Th17 cells [4][5][6][7] , and contribute to higher CCR6 + /CCR6 − T cells ratio in healthy smokers. Our data are in accordance with other reports showing that accumulation of p16 INK4a -expressing T cells in human peripheral blood is a biomarker of physiologic age as opposed to chronologic age, and independently correlates with gerontogenic behaviors including smoking 29,30 . The composition of the secretome of senescent cells depends on the stimuli triggering senescence, and is also specific of cell type 12 . This could explain the restricted secretory phenotype of isolated CCR6 + Th17 cells exposed to CSE, consisting of  www.nature.com/scientificreports www.nature.com/scientificreports/ www.nature.com/scientificreports www.nature.com/scientificreports/ combined VEGFα and IL-8 expression, contrasting with FoxP3 + regulatory T cells induced-senescent CD4 + T cells which produce other classical SASP factors such as TNFα, Il-1β and IL-6 17 .
We further demonstrated that higher sensitivity of CCR6 + Th17 cells to premature senescence relied on a higher basal production of ROS resulting in ROS dependent -senescence after CSE exposure, as already shown for various cell types 28 . Higher basal production of ROS could contribute to the TCR-independent higher tonic ERK1/2 signalling in resting CCR6 + Th17 18 , leading to p16 INK4a expression after ERK1/2 activity reinforcement by CSE-induced ROS overproduction 31 .
By using different pharmacological inhibitors, our results suggest a dichotomic regulation of expression of p16 INK4a , a growth arrest-related characteristics of senescence, and VEGFα production by CSE-induced senescent CCR6 + Th17 cells, as already reported for the SASP of other cell types 32,33 . In support of this is the selective inhibition of CSE-induced production of VEGFα by NAC pretreatment, in comparison with the antimycin A -sensitivity of CSE-stimulated p16 INK4a expression. Of note, CSE-modulation of antimycin A-sensitivity in CCR6 + Th17 cells was not associated with enhancement of mitochondrial superoxide formation as already shown in epithelial cells 34 suggesting that basal respiratory chain activity of CCR6 + Th17 cells was not sensitive to CSE. In contrast, antimycin A+ rotenone treatment increased mitochondrial superoxide formation in CCR6 + Th17 cells, and this mitochondrial activity was suppressed by CSE exposure, suggesting that CSE induced dysfunction of respiratory chain activity in CCR6 + Th17 cells, only when respiratory chain was activated beforehand. Moreover, our results suggest that CSE-induced VEGFα production could result from ROS-activated ERK1/2 signalling cooperating with overactivation of the antioxidant Nrf2 axis 35,36 . Overactivation of the antioxidant Nrf2 axis in CSE-exposed CCR6 + Th17 cells could also account for the repression of TNFα-, IL-6 & IL-1β expression as recently described in macrophage 37 . In conclusion, the functional characteristics of CSE-exposed CCR6 + Th17 cells, both inflammatory and angiogenic, could contribute to the pathogenic potential of cigarette smoking in susceptible patients, as described for circulating angiogenic T cells in encephalomyelitis, systemic lupus erythematosus, systemic sclerosis and rheumatoid arthritis 38,39 . Confirming our previous data on radiation-induced senescent CCR6 + Th17 cells 40 , our results also suggest that secretion of VEGFα by mucosa-homing CCR6 + Th17 cells submitted to any oxidative microenvironments, independently of TCR-reactivation, could represent a potential damage response altering insulted and nearby tissue. This damage response should be addressed Figure 6. Higher mitochondrial activity is critical for cigarette smoke-driven p16 INK4a expression in CCR6 + Th17 cells. (A) Cells were exposed (or not) to 5% cigarette smoke extract (CSE) or 10% CSE or 250 µM TBHP (positive control) for 30 minutes after (or not) pretreatment with 10 mM NAC for 1h30 or 0,5 µM Ant for 30 minutes, or the combination of Ant and NAC. ROS production was measured by flow cytometry using H 2 DCF-DA probe oxidation (n = 5). (B) Flow cytometry analysis of MitoSOX red probe oxidation in CCR6 + Th17 and CCR6 neg Th cells, pretreated by a combination of 0,5 µM Ant and 2 µM Rotenone for 30 minutes, exposed (or not) to 5% CSE or 125 µM H 2 O 2 (positive control) for 3 h (n = 5). Data shown (mean ± SEM) are expressed in mean fluorescence intensity (MFI) of the probe in the different cell types and conditions, normalized to the MFI in total CD4 + T cells in medium condition. (C) HO-1 and NQO1 gene expression was analyzed by qRT-PCR in 5% CSE -exposed cells for 6 h (n = 5). Statistical analysis by Mann-Whitney test; # p < 0.05, ## p < 0.01: comparison between 2 sub-populations; *p < 0.05, **p < 0.01: comparison to medium condition; $ p < 0.05, $$ p < 0.01: comparison to "medium + CSE condition"; § p < 0.05, § § p < 0.01: comparison to corresponding condition w/o CSE. www.nature.com/scientificreports www.nature.com/scientificreports/ independently from cell-cycle regulation or senescence per se, for discovering new targets controlling senescence effector response in diverse chronic inflammatory disorders both environmental-and age-related.
Cells treatment with cigarette smoke extract and pharmacologic inhibitors. Cigarette smoke extract (CSE) was generated from research-grade cigarettes (3R4F; University of Kentucky, Lexington, KY USA) by repeating bubbling cycles (30 s duration) of 40 mL puff volumes mainstream smoke from one cigarette into 10 mL of serum free culture medium. The CSE was collected (100% concentration) supplemented with 10% human AB serum or 2.5% FBS, adjusted to pH 7.4 and then passed through a 0.22 μm pore size filter (Millipore Corporation, Billerica, MA, USA). CSE preparations were standardized by measuring the absorbance at 320 nm; only preparations with optical density >0.42 were used. CSE were prepared shortly (<1 h) before being added to cells cultures. Non-toxic doses of CSE were CSE doses with >80% live cells remaining, at the time of experiment: 5% and/or 10% CSE if CSE treatment ≤24 h, 5% CSE for 48 h treatment (Fig. S1A). According to figure legends, cells were pretreated (or not) with 10 mM N-Acetyl-L-cysteine (NAC, Sigma Aldrich) for 1h30, 5 μM U0126 (Merck Millipore), 0.5 µM antimycin A or 2 µM rotenone (Sigma Aldrich) for 45 min before CSE exposure.

RNA isolation and real time quantitative RT-PCR.
Total mRNA isolation and qRT-PCR analysis were performed, as previously published 41 . The expression of the indicated target transcripts was measured by the relative quantification of real-time PCR using a mix of each cDNA sample as a calibrator sample, according to the ΔΔCt method 42 .
Cytokine secretion assays. Culture supernatants were analyzed by Luminex assay (PROCARTAPLEX, Thermo Fisher Scientific, Waltham, MA USA) for the analytes listed in Fig. S1B according to the manufacturer's instructions.
Detection of intracellular reactive oxygen species. Intracellular ROS levels were assessed by analyzing oxidation of H 2 DCF-DA (2′7′-dichlorofluorescin diacetate diacetate, Thermo Fisher Scientific), probed in enriched CD4 + T cells from PBMCs. Cells were incubated with 0.25 μM H 2 DCF-DA for 45 min at 4 °C in the dark, then exposed or not to CSE in the presence of 0.25 μM H 2 DCF-DA for 30 min. 250 µM tert-butyl H 2 O 2 (TBHP, Sigma Aldrich) treatment was used as a positive control. Finally, cells were stained with the fluorochrome conjugated mAbs directed against specific cell surface markers and DCF. Mean fluorescence intensity (MFI) was measured in the different CD4 + T cells subpopulations by flow cytometric analysis using Cyan ADP LX7 and Summit software (Beckman Coulter). Mitochondrial ROS production was measured by analyzing MitoSOX Red (Thermo Fisher) fluorescence. CCR6 + Th17 and CCR6 negative Th sorted cells were exposed or not to CSE at 10 6 cells/ml, in sealed tubes containing PBS Ca ++ , Mg ++ and BSA (0.5%), for 3 h. MitoSOX Red, at the final concentration of 2.5 µM, was added to cells for the last 30 min of the culture. MitoSox MFI was acquired on LSR Fortessa X20 and Diva software (BD Biosciences). Statistical analysis of flow cytometry data were performed with FlowJo software (Tree Star, San Carlos, CA, USA). DCF/MitoSox MFI of the different CD4 + T cells subpopulations in the different experimental conditions are expressed as percentage of DCF/MitoSox MFI of total CD4 + T cells in unstimulated condition.