cobl

Subhashini Arimilli a,, Brad E. Damratoski a, G.L. Prasad b

bR&D Department, R.J. Reynolds Tobacco Company, Winston-Salem, NC 27102, United States

a r t i c l e i n f o

Article history:Received 12 April 2013Accepted 29 June 2013Available online 11 July 2013

tive immunity are active areas of investigation and subjects ofseveral recent reviews (Goncalves et al., 2011; Lee et al., 2012;Yao and Rahman, 2011). Several clinical studies show that chronicsmoking induces inammation as seen by increases in white blood

n and function oft al., 2003; Roos-arly, in vitro stud-componened by decactor-alpha

a) secretion (Ouyang et al., 2000). Another characteristic reto exposure to cigarette smoke (or its constituent phasescreased secretion of interleukin-8 (IL-8) in several cell(example, human aortic and bronchial epithelial cells) (Nordskoget al., 2005; Parsanejad et al., 2008).

Cigarette smoking adversely affects the functions of compo-nents of innate immunity, including natural killer cells (NKs) andToll-like receptors (TLRs). NK cells are a vital part of the innateimmune system and play an important role against microbialinfections and tumor surveillance through secretion of an array

Corresponding author. Address: Department of Microbiology & Immunology,Wake Forest Baptist Health, Wake Forest Biotech Place, Room 2N-052, 575Patterson Avenue, Winston Salem, NC 27101, United States. Tel.: +1 336 7131390; fax: +1 336 716 9928.

Toxicology in Vitro 27 (2013) 19922004

Contents lists available at

Toxicology

elsE-mail address: sarimill@wakehealth.edu (S. Arimilli).1. Introduction

Chronic cigarette smoking is known to affect immune responsesand compromise host defense against microbial infections and tu-mor surveillance (Holt and Keast, 1977). The mechanisms ofimmunomodulatory effects of cigarette smoke on innate and adap-

cells (Frost-Pineda et al., 2011) and the distributioleukocyte subsets are altered in smokers (Hoser eEngstrand et al., 2010; Rumora et al., 2008). Similies show that exposure to cigarette smoke (or itssults in altered immune responses as measurinterferon-gamma (IFN-c) and tumor necrosis f0887-2333/$ - see front matter 2013 Elsevier Ltd. All rights reserved.http://dx.doi.org/10.1016/j.tiv.2013.06.015ts) re-reased(TNF-

sponse) is in-typesmay contribute to increased microbial infections and cancer incidence among smokers. 2013 Elsevier Ltd. All rights reserved.Keywords:Tobacco product preparations (TPPs)EC50T cellsNK cellsPBMCsPoly I:CLPSCytotoxicity assaya b s t r a c t

Natural killer (NK) cells and T cells play essential roles in innate and adaptive immune responses inprotecting against microbial infections and in tumor surveillance. Although evidence suggests that smok-ing causes immunosuppression, there is limited information whether the use of smokeless tobacco (ST)products affects immune responses. In this study, we assessed the effects of two preparations of cigarettesmoke, ST extract and nicotine on T cell and NK cell responses using Toll-like receptor-ligand stimulatedhuman peripheral blood mononuclear cells (PBMCs). The tobacco product preparations (TPPs) testedincluded whole smoke conditioned media (WS-CM), total particulate matter (TPM) and a ST productpreparation in complete articial saliva (ST/CAS). The PBMCs were stimulated with polyinosinic:polycytidylic acid (poly I:C) and lipopolysaccharide (LPS). A marked reduction of the expression ofintracellular IFN-c and TNF-a was evident in NK cells and T cells treated with WS-CM and TPM.Consistently, attenuation of ligand-induced secretion of cytokines (IL-1b, IL-10, IL-12 and TNF-a) fromPBMCs treated with WS-CM and TPM were observed. While the treatment with TPPs did not alter theexpression of the maturation marker CD69, WS-CM and TPM inhibited the cytolytic activity of humanPBMCs. Suppression of perforin by WS-CM was also detected. Although interference from the vehicleconfounded the interpretation of effects of ST/CAS, some effects were evident only at high concentrations.Nicotine treatment minimally impacted expression of cytokines and cytolytic activity. Data presentedherein suggests that the function of NK cells and T cells is inuenced by exposure to TPPs (based onequi-nicotine units) in the following order: WS-CM > TPM > ST/CAS. These ndings are consistent withthe hypothesis put forward by others that chronic smoking leads to immunosuppression, an effect thataDepartment of Microbiology & Immunology, Wake Forest University School of Medicine, Winston-Salem, NC 27101, United StatesCombustible and non-combustible tobacdifferentially regulate human peripheralfunctions

journal homepage: www.product preparationsood mononuclear cell

SciVerse ScienceDirect

in Vitro

evier .com/locate / toxinvi t

The purpose of this study was to determine how the combusti-

and acid phosphatase).We presented the above information at the 64th Tobacco Science

in Vitro 27 (2013) 19922004 1993of cytokines including IFN-c and TNF-a and by cytolysis of infectedor neoplastic cells (Bancroft, 1993; Biron, 1997; Biron et al., 1999;Cerwenka and Lanier, 2001; Mian et al., 2008; Nadigel et al., 2011).NK cell functions have also been shown to be signicantly reducedby cigarette smoke (Mehta et al., 2008; Mian et al., 2008). TLRs areinvolved in innate immunity by recognizing molecules that arebroadly shared by pathogens, and they activate a cascade of signal-ing pathways, triggering NF-jB and type-1 interferon production(Reimer et al., 2008).

T cells are involved in the initiation and regulation of innate andadaptive immune responses. T cell subsets play specic roles ineliciting immune response, and smoking has been reported to de-crease CD4/CD8 cell ratios (Runo et al., 2007). Furthermore, expo-sure to cigarette smoke alters T cell function including cytokinesecretion, anergy and proliferation (Chang et al., 1990; Lambertet al., 2005; Petro and Zhang, 1997; Sopori and Kozak, 1998).

Cigarette smoke extracts inhibit the secretion of cytokines suchas IL-1b, IL-2, IFN-c and TNF-a by PBMCs (Ouyang et al., 2000).These proinammatory cytokines are capable of driving NK celland cytotoxic T cell responses that are critical to tumor suppres-sion (Ouyang et al., 2000; Yoneda et al., 1993). Given the impor-tance of these cell types in the immune response, it has beensuggested that T cell and NK cell anergy resulting from smokingcould signicantly impair the ability to ght viral and bacterialinfections (Mehta et al., 2008; Phipps et al., 2010) as well as tumorsurveillance (Hogan et al., 2011b).

While cigarette smoking represents the most common form oftobacco consumption, non-combustible tobacco such as, moistsnuff, snus, and other smoke-free products, exist in the market-place. Relative to cigarettes, health risks associated with the useof smokeless tobacco (ST) products have shown to be lower(Hatsukami et al., 2002; Zeller et al., 2009). While the effects of cig-arette smoking on immune function has been subject of active re-search, relatively less is known of how the use of ST alters immuneresponse. For example, some in vitro studies suggest ST elicitsimmunostimulatory responses as observed by altered cytokinesecretion (Goud et al., 1993; Johnson et al., 1994, 1996; Petroet al., 1999; Petro and Zhang, 1997). Therefore, we have initiatedinvestigation into the relative effects of combustible and non-com-bustible tobacco products using HL60 cells, PBMCs, and oral cavitycells as representative in vitro models for systemic and local(mucosal) exposure, respectively (Arimilli et al., 2012a; Gaoet al., 2013).

Cigarette smoke contains a particulate phase and a gasvaporphase. The particulate phase, dissolved in DMSO (or alternate sol-vent) is known as total particulate matter (TPM) and is commonlyused in cell culture studies. A different method of exposing cells tocigarette smoke involves the use of smoke-conditioned mediumgenerated by passing cigarette smoke through it; this preparationis referred to as cigarette smoke extract (Sopori, 2002), whole-smoke conditioned medium (WS-CM) (Arimilli et al., 2012a) oraqueous extract. Each of these TPPs is chemically distinct andtherefore could elicit different responses. Smokeless tobacco ex-tracts may be generated in several different ways: example, directextraction in cell culture medium (Mitchell et al., 2010; Petro,2003) or extraction in complete articial saliva (CAS) (Arimilliet al., 2012a; Gao et al., 2013).

We have prepared WS-CM and TPM from 3R4F cigarettes andextracts from 2S3 reference moist snuff ST in CAS, respectively.Since these TPPs are chemically distinct, we used the nicotine con-centration of the TPPs as a common measure to determine theexposure of different TPPs. The biological effects due to TPP expo-sure were compared based on the nicotine content of the TPPs used

S. Arimilli et al. / Toxicologyin various treatments in in vitro and ex vivo studies, and we termedit equi-nicotine unit paradigm (Arimilli et al., 2012a; Gao et al.,2013).Research Conference held in Hilton Head, SC in 20101. WS-CM wasprepared by passing smoke from four 3R4F cigarettes through 20 mLof RPMI 1640 medium (Invitrogen, Grand Island, NY) without phenolred. Nicotine free base (SigmaAldrich, Milwaukee, WI) was used as areference. Aliquots of frozen TPPs were analyzed for nicotine, tobaccospecic nitrosamines (TSNAs) and polycyclic aromatic hydrocarbons(PAHs) at Labstat International (Kitchener, Ontario, Canada) usingpublished methods (Rickert et al., 2009; Wu et al., 2008).

We tested TPPs and nicotine at different doses based on theequi-nicotine unit paradigm. The EC50 values of different TPPs weredetermined by 7-aminoactinomycin D (7AAD) positive staining ofPBMCs. The EC50 is dened as the concentration at which 50% ofthe cells were no longer viable in a 24 h assay and the values areexpressed as lg of equi-nicotine units/mL (Arimilli et al., 2012a).The EC50 values of combustible preparations TPM andWS-CMweredetermined to be 2.58 lg/mL and 1.56 lg/mL of equi-nicotine units,respectively (Arimilli et al., 2012a). As discussed in a previouspublication Arimilli et al. (2012a), EC50 values for non-combustibleST/CAS could not be determined due to its low cytotoxicity, whichrequired addition of substantial (>30%) volumes of ST/CAS into cellble and non-combustible TPPs modulate select immune responses.We used PBMCs collected from non-smoking donors, exposedthem to TPPs and determined their responses upon stimulationwith polyinosinic:polycytidylic acid (poly I:C) and lipopolysaccha-ride (LPS). Poly I:C, a synthetic double-stranded RNA analog, andlipopolysaccharide (LPS), a major component of gram-negativebacteria cell wall, have been used extensively to study the variouspathways of innate immune activation associated with specicTLRs (Reimer et al., 2008). Poly I:C and LPS bind to TLRs and acti-vate NF-jB, TNF-a and other signaling molecules. The ex vivo cul-ture model has been used to investigate the mechanisms ofimmunosuppression in smokers which are linked to the increasedsusceptibility of smokers to infections and to tumors (Chen et al.,2007; Lambert et al., 2005; Lee et al., 2012; Ouyang et al., 2000).

In this manuscript, we investigated the functional responses ofPBMCs, NK cells and T cells pre-treated acutely with cigarettesmoke-derived (combustible) and smokeless (non-combustible)TPPs followed by stimulation with poly I:C and LPS. We also com-paratively assessed the effects of nicotine pre-treatment on theresponsiveness of PBMCs to TLR ligand stimulation.

2. Materials and methods

2.1. Tobacco product preparations and EC50 values

The TPPs were prepared as described previously (Arimilli et al.,2012a). Briey, TPM was prepared by smoking 3R4F reference cig-arettes using the standard ISO method (Johnson et al., 2009b)(35 mL, 60 s, 2 s; puff volume, frequency and duration, respec-tively) and dissolving the particulate phase, trapped on the Cam-bridge lter pad, in DMSO. Smokeless tobacco extract wasprepared by extracting 2S3 reference smokeless tobacco (ST)(North Carolina State University Tobacco Services Analytical Labo-ratory) for 2 h in complete articial saliva (CAS) using a publishedmethod (Chou and Hee, 1994). The CAS consists of mucin, salts(potassium chloride, sodium chloride, calcium chloride dehydrate,di-potasium hydrogen phosphate and magnesium chloride hexa-hydrate), urea, glucose and enzymes (alpha-amylase, lysozyme1 Standardization of the Preparation of Smokeless Tobacco Extracts for Assessmentof Biological Effects. Kathy Fowler, Jo Ann Hill, Betsy Bombick and G.L. Prasad.Research and Development, R.J. Reynolds Tobacco Company, Winston-Salem, NC USA.

for combustible TPPs. The nicotine EC50 value was determined to be1650 lg/mL (Arimilli et al., 2012a).

were washed after 3 h treatment and re-plated in a 48-well plateat 1 million cells/well/mL with and without the presence of

on ice. After washing the cells, they were re-suspended in a totalvolume of 200 lL of 2% paraformaldehyde for FACS analysis. For-

TPPs and stimulated with either poly I:C or LPS. Stimulation with

themselves induced basal number of TNF-a+ T cells. A dose-depen-

in Vward scatter, side scatter, uorescence intensity and percent posi-tivity were measured after acquiring 100,000 cells per sample byow cytometry (BD Biosciences, San Jose, CA), and the data wereanalyzed using Cell Quest (BD Biosciences, San Jose, CA) and FlowJo (Tree Star, Ashland, OR) software.

2.4. Cytometric bead array assay

PBMC culture supernatants were harvested after 67 h of poly I:Cor LPS stimulation from the above experiments. A Cytometric BeadArray (CBA) for human cytokine secretion was used as described10 lg/mL poly I:C or LPS. After a 67 h incubation, supernatantswere taken for secreted cytokine analysis. Cells were plated ontoa 96-well plate at 106/mL and further incubated for 4 h with200 lL of media with Golgiplug (1 lg/mL) (BD Biosciences, SanJose, CA) to measure intracellular cytokines (Arimilli et al., 2006;Mian et al., 2008). Cells were then harvested, washed and surfacestained for CD56-PE and CD2-FITC, CD69-APC monoclonal antibod-ies (mAB) for 30 min at 4 C.

After washing, cells were xed and permeabilized with BD Bio-sciences Cytox/Cytoperm buffer for 20 min on ice in the dark.Cells were then stained with anti-IFN-c-PE, anti-TNF-a-APC andanti-perforin-FITC mAB and incubated for an additional 30 minA range of lower doses of TPPs and nicotine were also used insome of the experiments. The numbers in the parentheses in eachgure indicate the dose of lg/mL nicotine units used in thoseexperiments. Equivalent volumes of media, DMSO and CAS wereused as controls for WS-CM, TPM and ST/CAS, respectively.

2.2. Isolation of PBMCs

Fresh blood was collected from healthy non-smoking donorsafter written informed consent (who were non-consumers of to-bacco products) at a local Clinical Contract Research Organization(Piedmont Medical Group, Winston-Salem, NC) under IRB ap-proval. PBMCs were isolated from fresh blood as described earlier(Arimilli et al., 2012b) under Wake Forest Baptist Health IRB ap-proval. Briey, PBMCs were isolated by standard density gradientcentrifugation by using Isolymph (CTL Scientic Supply Corp., DeerPark, NY). Isolated PBMCs were cryopreserved for further use.

2.3. Cell staining and ow cytometry

PBMCs were treated with indicated concentrations (nicotineunits) of WS-CM, TPM, ST/CAS and neat nicotine for 3 h in a 24-well plate at 3 million cells/well in 2 mL RPMI complete media.Dosing was based on an equi-nicotine exposure paradigm. Cellsculture medium. Separately, CAS at higher volumes altered basaland TLR ligand-induced immune response which interfered withthe assessment of the effects of treatment at higher doses of ST/CAS (data not shown). Hence, we present results with ST/CAS at2 lg/mLof equi-nicotineunits,which is comparable to thedoseused

1994 S. Arimilli et al. / Toxicologypreviously (Arimilli et al., 2007). Briey, we measured IL-1b, IL-6,IL-8, IL-10, IL-12, and TNF-a (BD Biosciences, San Jose, CA) by owcytometry according to the manufacturers instructions.dent suppression of TNF-a+ T cells in stimulated PBMCs by WS-CMis evident (Fig. 1B, top panel). Although a 26% reduction from con-trol with WS-CM at 0.5 lg/mL was detected, a statistically signi-cant reduction in number of TNF-a+ T cells (72%) was observed at1.56 lg/mL of equi-nicotine units of WS-CM. Treatment with TPMdid not result in statistically signicant differences at the dosestested. Treatment with ST/CAS at 2 lg/mL of equi-nicotine unitsdid not alter the number of TNF-a producing T cells.

T cells treated with nicotine (101650 lg/mL) did not yield sta-tistically signicant differences in their TNF-a+ T cell numbers topoly I:C and LPS stimulation. Exposure to 1650 lg/mL of nicotineresulted in higher basal levels of TNF-a+ T cells, which were unal-tered by stimulation with the TLR ligands.

We next evaluated changes in the number of IFN-c+ T cells(Fig. 2) with TPP treatment followed by poly I:C and LPS stimula-tion. A set of representative ow cytometric images is includedTLR ligands resulted in robust and statistically signicant increasesin TNF-a+ and IFN-c+ cells compared to unstimulated controls(basal).

First, we assessed whether the pretreatment of PBMCs withTPPs compromised their ability to produce TNF-a+ cells when stim-ulated with poly I:C and LPS (Fig. 1). Fig. 1A presents representativeow cytometric data of T cells positive for TNF-a after treatmentwith the TPPs followed by no stimulation (top panel), poly I:C stim-ulation (middle panel) and LPS stimulation (bottom panel). TPPs by2.5. Cytotoxicity assay

K562 cells were purchased from ATCC (Manassas VA, USA) andgrown in RPMI complete media. One vial of cryopreserved PBMCswas thawed and washed, and a live cell count was taken. Differentconcentrations of TPPs were prepared in 100 lL media. 1.5 millionPBMCs in a 50 lL volume were added to each well and incubatedfor 1.5 h at 37 C in 96-well round bottom plates. K562 target cellswere labeled with carboxy uorescein succinimidyl ester (CFSE)and added at a density of 100,000 cells/well (target:effector ratiois 1:15). Cell co-cultures were incubated at 37 C for an additional4 h. Immediately after the incubation period, cells were stainedwith 7AAD (which labels the dead cells) and the killing of CFSE-la-beled K562 target cells was evaluated after 7AAD staining by owcytometric analysis (BD Biosciences, San Jose, CA). Flow data wereanalyzed using Flow Jo software (Tree Star, Ashland, OR).

2.6. Statistical comparisons

The results were presented as the mean the standard error ofthe mean (four donor samples). The t-test between treatment anduntreated control samples was performed using Sigma Plot (ver-sion 9) for all treatments with their corresponding controls. Thestatistical signicance was indicated by: , P < 0.05; , P < 0.005;, P < 0.0005.

3. Results

3.1. Suppression of TNF-a+ and IFN-c+ T cell populations in PBMCstreated with TPPs

We sought to evaluate intracellular cytokine levels in T cellsafter treating with combustible and non-combustible TPPs. In therst set of experiments, we measured the induction of intracellularTNF-a and IFN-c by ow cytometry after PBMCs were treated with

itro 27 (2013) 19922004in Fig. 2A and summary data from four donors is presented inFig. 2B. The TPPs induced a minimal number of IFN-c+ T cells underbasal (non-stimulated) conditions. Control and DMSO treated cells

Fig. 1. Reduction of intracellular TNF-a+ T cells by different TPPs. PBMCs were exposed to varying concentrations of WS-CM, TPM, ST/CAS, nicotine and their correspondingvehicle controls for 3 h with no stimulation (top panel) or stimulated with poly I:C (middle panel) and LPS (bottom panel) for 67 h. The vehicle controls for WS-CM is medium,for TPM it is DMSO and for ST/CAS it is CAS; the volumes of vehicle control correspond to the volumes used for the TPPs. Intracellular TNF-a+ T cells were quantied by owcytometry. (A) Representative ow cytometric data after acquiring 100,000 cells per sample. (B) Combined data from four independent experiments using four differentdonors PBMCs and indicated concentrations of TPPs and nicotine. The statistical signicance was indicated by: , P < 0.005; , P < 0.0005.

S. Arimilli et al. / Toxicology in Vitro 27 (2013) 19922004 1995

in V1996 S. Arimilli et al. / Toxicologyshowed an increase in the number of IFN-c+ T cells after poly I:Cand LPS induction. Treatment with WS-CM resulted in near com-plete suppression of IFN-c+ T cells induced by poly I:C and LPS,while incubation with TPM resulted in statistically signicant dosedependent reduction of IFN-c+ T cells.

Fig. 2. Suppression of intracellular IFN-c+ T cells by different TPPs. PBMCs were exposedto Fig. 1. Intracellular IFN-c+ T cells were quantied by ow cytometry. (A) Representativfour different donors PBMCs and indicated concentrations of TPPs and nicotine. The staitro 27 (2013) 19922004In terms of equi-nicotine units, the combustible TPPs, WS-CMand TPM, were far more potent suppressors of IFN-c+ T cells(Fig. 2B, top panel) than ST/CAS or nicotine (Fig. 2B, bottom panel).Treatment with combustible TPPs resulted in statisticallysignicant suppression of IFN-c+ T cells at or below 2.58 lg/mL

to TPPs as indicated and stimulated with poly I:C and LPS as described in the captione ow cytometric data. (B) Combined data from four independent experiments usingtistical signicance was indicated by: , P < 0.05; , P < 0.005.

and shows a statistically signicant decrease of K562 cell killing

in Vof equi-nicotine units. Measurable suppression of the cytokine-po-sitive cells was observed only at the highest concentrations of nic-otine, although these changes were not statistically signicant.

Thus, a dose-dependent suppression of IFN-c+ T cells in re-sponse to TPM treatment was observed, whereasWS-CMwas morepotent in suppressing TNF-a+ and IFN-c+ T cells at both dosestested. The combustible TPPs exerted more pronounced effects rel-ative to ST/CAS and nicotine. Furthermore, IFN-c+ T cells appear tobe compromised more severely by combustible TPPs and nicotine(1650 lg/mL) than TNF-a+ T cells in the stimulated PBMCs.

3.2. Suppression of TNF-a+ NK cell populations in PBMCs treated withTPPs

Because of the importance of NK cells in innate immunity, wenext tested the effect of TPPs on the induction of TNF-a+ NK cellsin poly I:C-and LPS-stimulated PBMCs. Fig. 3A shows the owcytometry data of differential suppression of TNF-a in NK cellsby various TPPs. Consistent with the data obtained with T cells,NK cells treated with WS-CM showed the lowest number of TNF-a+ NK cells after stimulation with poly I:C (Fig. 3A, middle panel)and LPS (Fig. 3A, lower panel) when compared to the control cells.Fig. 3B summarizes four experiments and demonstrates that theinhibition of TNF-a+ NK cells are more pronounced with combusti-ble TPPs than ST/CAS at equi-nicotine units (WS-CM > TPM > ST/CAS). PBMCs treated with WS-CM showed 90% reduction andTPM showed 64% reduction of intracellular TNF-a+ NK cells, whichwere statistically signicant, upon stimulation with TLR ligands.However, pre-treatment with ST/CAS (2) did not result in statisti-cally signicant changes relative to vehicle control. While pre-treatment with lower doses (10 and 500 lg/mL) of nicotine didnot induce statistically signicant changes in TNF-a+ NK cells, ata higher dose (1650 lg/mL) produced a statistically signicantreduction after LPS stimulation (Fig. 3B).

3.3. Effect of TPPs on CD69 expression in NK cells

Since the induction of TNF-a+ NK cells is reduced by TPPs in thestimulated NK cells, we further investigated whether TPPs inter-fered with NK cell differentiation and function. CD69 is an earlyinducible cell surface glycoprotein expressed during lymphoidactivation and is involved in NK cell function (Borrego et al.,1999). PBMCs were treated with TPPs, and CD69 expression onNK cell population was analyzed by ow cytometry after poly I:Cand LPS stimulation (Fig. 4). Pre-treatment of PBMCs with the TPPsresulted in increased CD69 expression upon stimulation with polyI:C and LPS. It is interesting to note that the WS-CM (1.56) treat-ment increased CD69 expressing NK cells under basal conditionsby 5-fold, whereas poly I:C or LPS did not further enhance theCD69 expression. Exposure to nicotine (1650 lg/mL) did not in-crease the basal levels of the induction of CD69 relative to the un-treated cells.

3.4. Inhibition of secreted cytokines in TPP-treated PBMCs

Next we assessed the effect of TPPs on the secreted cytokines.PBMCs were treated with different TPPs and stimulated with eitherpoly I:C (Fig. 5) or LPS (Fig. 6). Cell culture supernatants were usedto measure the levels of a panel of cytokines. Among the TPPstested, WS-CM exerted statistically signicant and profound inhib-itory effect (which was statistically signicant, P < 0.0005) on thesecretion of the cytokines. Levels of IL-1b, IL-6, IL-10 and TNF-awere profoundly reduced in WS-CM-treated PBMCs stimulated

S. Arimilli et al. / Toxicologywith poly I:C or LPS. While IL-8 and IL-12 secretion was reducedby 94% and 68% with poly I:C, respectively, they were reduced by91% and 67% with LPS, respectively.ability with WS-CM (65%) treatment. Treatment with ST/CAS (2)or nicotine (1650) did not reduce the target cell killing ability(Fig. 7B).

3.6. Effect of TPP exposure on perforin levels in total PBMCs and NKcells

We next investigated whether the altered cytolytic activity ofPBMCs and NK cells is due to differences in the endogenous perfo-rin levels. Perforin, a cytoplasmic granular protein, is known tomediate target cell killing. We therefore determined perforin levelsin TPP-treated PBMCs (Fig. 8, upper panel) and NK cells (Fig. 8, low-er panel).

WS-CM (1.56)-treated PBMCs and NK cells showed a statisti-cally signicant reduction (70% and 78%, respectively) in perforinlevels. Treatment with TPM, ST/CAS and nicotine did not result instatistically signicant changes in perforin levels in PBMC popula-tions or NK cells.

4. Discussion

The purpose of this work is to assess how the exposure of com-bustible and non-combustible TPPs inuences immune responses;particularly induction, secretion of cytokines and cytolytic activityby PBMCs. Cigarette smoking has been known to elicit inamma-tion and alter a wide range of innate and adaptive immune re-sponses (Goncalves et al., 2011). Such altered immune responseshave been linked to compromised host defense against microbialinfections and tumor surveillance (Cerwenka and Lanier, 2001).The key nding of our study is that the exposure to combustibleTPPs (WS-CM and TPM), on an equi-nicotine unit basis, caused po-tent immunosuppression relative to ST and nicotine, as measuredby NK cell and T cell functions.

Due to their critical role in modulating immune functions in re-sponse to microbial pathogens and in tumor surveillance, NK cellsand T cells have been studied for possible adverse effects of smok-ing. Our ndings are in general agreement with the previouslypublished work which showed that the responsiveness of T cellsand NK cells to poly I:C, a TLR3 ligand, is signicantly attenuatedby WS-CM (Mian et al., 2008, 2009a,b).Treatment with TPM also reduced the secretion of several cyto-kines upon stimulation with the TLR ligands. Statistically signi-cant reductions were observed in IL-10 (>60%), IL-12 (>68%) andTNF-a (>80%) secretion with both TLR ligands (Figs. 5 and 6).TPM also suppressed IL-1b and IL-6 levels in LPS-stimulated cells(Fig. 6). Pre-treatment with ST/CAS (2) resulted in statistically sig-nicant reductions in IL-6 levels with poly I:C, but not with LPSstimulation (Figs. 5 and 6).

3.5. Suppression of PBMC cell target killing ability

As the intracellular and secreted cytokines are suppressed bypre-treatment with TPPs to varying degrees, we tested whetherthe functional properties, particularly target cell killing by NK cellsand CD8+ T cells in the PBMC pool was compromised from theexposure to TPPs. These two cell types are known as cytolytic effec-tor cells that confer innate protection against viral and bacterialinfections and are involved in anti-tumor surveillance. We utilizedthe K562 cell line as a target in the cytolytic assay and a set of rep-resentative ow cytometric images of cytolysis is shown in Fig. 7A.The cytolytic data on multiple donor PMBCs is presented in Fig. 7B

itro 27 (2013) 19922004 1997That chronic cigarette smoking causes increased inammationand yet results in compromised immune responses has beenwidely known (Lee et al., 2012; US Department of Health and Hu-

in V1998 S. Arimilli et al. / Toxicologyman Services, 2010). A number of different experimental models,similar to that described herein, have been utilized to dissect themechanisms. However, the effects of ST on immune responsesare incompletely understood, and the results appear to be

Fig. 3. Suppression of intracellular TNF-a+ NK cells with different TPPs. PBMCs were exvehicle controls as described in the caption to Fig. 1. Intracellular TNF-a+ NK cells were qdata from four independent experiments using four different donors PBMCs and indicateP < 0.005; , P < 0.0005.itro 27 (2013) 19922004dependent on the cell types and other experimental conditions.For example, ST exposure was shown to cause immunostimulatoryand proinammatory effects (Goud et al., 1993; Petro, 2003), aswell as immunosuppression (Fine et al., 2002; Johnson et al.,

posed to different concentrations of WS-CM, TPM, ST/CAS and their correspondinguantied by ow cytometry. (A) Representative ow cytometric data. (B) Combinedd concentrations of TPPs. The statistical signicance was indicated by: , P < 0.05; ,

in VS. Arimilli et al. / Toxicology2009a). Some of the widely characterized consequences of expo-sure to cigarette smoke (or its constituent phases) are cytotoxicityand inammation, as measured by increased secretion of cytokinessuch as IL-8. Recently we reported that exposure to combustible

Fig. 4. Stimulation of CD69 expression in NK cells with different TPPs. PBMCs werecorresponding vehicle controls for 3 h and stimulated with poly I:C, LPS or no stimulatiocombined from four independent experiments using PBMCs from ve different donors).

Fig. 5. Attenuation of cytokine secretion by TPPs following poly I:C stimulation. PBMcorresponding vehicle controls for 3 h and stimulated with poly I:C for 67 h. Levels of cyand ow cytometer. The statistical signicance was indicated by: , P < 0.05; , P < 0.00itro 27 (2013) 19922004 1999TPPs results in cytotoxicity relative to ST/CAS and increased secre-tion of IL-8 in PBMCs and other cultured cells (Arimilli et al.,2012a). Further, we showed that T-helper (CD4+) cells appear tobe more sensitive to the exposure to TPPs compared to other

exposed to different concentrations of WS-CM, TPM, ST/CAS, nicotine and theirn for 67 h. CD69 expressing NK cells were quantied by ow cytometry (data wereThe statistical signicance was indicated by: , P < 0.0005.

Cs were exposed to different concentrations of WS-CM, TPM, ST/CAS and theirtokines in the culture supernatants were determined using a cytometric bead array5; , P < 0.0005.

weated

in VFig. 6. Attenuation of cytokine secretion by TPPs following LPS stimulation. PBMCsmeasured as described in the caption of Fig. 5. The statistical signicance was indic

2000 S. Arimilli et al. / Toxicologyleukocyte subsets tested in that study. Although we exposedPBMCs in this study at the EC50 equi-nicotine units of WS-CMand TPM, the pre-treatment was for only 3 h and no cytotoxicitywas evident either at the end of pre-treatment or stimulation withTLR ligands for 67 h (data not shown).

One of the strengths of this study is that, for the rst time, theeffects of exposure to combustible and non-combustible TPPs arecomparatively evaluated in a single system. We have utilized awell characterized TLR stimulated PBMC system to assess the cel-lular and cytokine secretion in response to TPP exposure. Further,we have reported the biological responses in equi-nicotine unitsfor comparison of the effects of TPPs.

The cytokines secreted by NK cells and T cells play a crucial rolein modulating immune responses and subsequent cell killing(Mian et al., 2008). Our nding that WS-CM exposure markedlysuppresses T cell and NK cell responsiveness is in agreement withprevious work (Mian et al., 2008, 2009a,b). Further, we demon-strate that the WS-CM also suppresses the secretion of severalcytokines, which is consistent with published data (Ouyang et al.,2000). For example, IFN-c is key in both innate and adaptive im-mune responses and is secreted by NK cells, cytotoxic T cells andT helper cells to induce multiple cellular effects such as inhibitionof cell proliferation, immunoregulation and apoptotic activity(Schoenborn and Wilson, 2007; Schroder et al., 2004).

Pretreatment of PBMCs with commercial cigarette smoke ex-tracts have previously been shown to suppress the production ofIL-1b, IL-2, IFN-c and TNF-a by greater than 90% without signi-cant loss of cell viability (Lambert et al., 2005; Ouyang et al.,2000). Further, smoke extracts from 1R3 reference cigarettes havebeen shown to impair NK cell functions (Mian et al., 2008). Ourdata with WS-CM (which other authors referred to as smoke ex-tracts) are in agreement with these ndings and also show the rel-ative effects of different TPPs tested herein. Acrolein, catechol andhydroquinone have been shown to contribute to the observedre exposed to TPPs, stimulated with LPS for 67 h, and the secreted cytokines wereby: , P < 0.05; , P < 0.005; , P < 0.0005.

itro 27 (2013) 19922004impairment of the immune responses (Lambert et al., 2005; Ouy-ang et al., 2000).

Similarly, TNF-a is important in suppression of tumorigenesisand infection as well as inducing immune cell activation and pro-liferation. TNF-a is critical for activation of the NF-jB pathway(Van Antwerp et al., 1996) and thus near complete abolition ofsecretion by WS-CM and markedly diminished secretion of TNF-a by TPM, could signicantly contribute to immunosuppression(Figs. 1, 2, 5 and 6). Consistent with our results, cigarette smoke ex-tract was shown to signicantly reduce the production of TNF-aand IFN-c levels of invariant NK cells stimulated with a-galactosyl-ceramide (Hogan et al., 2011a).

Besides TNF-a, secretion of IL-1b, IL-6, IL-8, IL-10 in poly I:C(Fig. 5) and LPS-stimulated PBMCs (Fig. 6) was essentiallyblocked by treatment with WS-CM, whereas secretion of IL-12was markedly reduced. Treatment with TPM, however, resultedin a signicant reduction in IL-10, IL-12 and TNF-a, with noreductions in the secretion of the other cytokines. There isoverall directional concordance between the levels of theintracellular cytokine and the secreted cytokine (Figs. 5 and 6)in response to WS-CM. Thus, a qualitatively important differencebetween the effects of WS-CM and TPM (and ST) on IL-1b,zIL-6 and IL-10 cytokine secretion in stimulated PBMCs wasevident.

To investigate the effect of TPPs on early anti-inammatory re-sponses we assessed the expression of CD69 after treatment withTPPs. CD69 is a pleiotropic immune regulator, involved in the acti-vation and differentiation of a wide variety of hematopoietic cells,including NK cells (Ziegler et al., 1994), and the expression of mac-rophage CD69 is known to increase upon cigarette smoke exposure(Tsuyusaki et al., 2011). Data presented in Fig. 4 show that CD69expression increases markedly in WS-CM-treated NK cells withoutpoly I:C or LPS stimulation, and stimulation with poly I:C or LPS didnot further enhance CD69 in cells treated with WS-CM. TPM and

in VS. Arimilli et al. / ToxicologyST/CAS (2)-treated cells, although exhibiting higher basal levels ofCD69, responded to the ligand stimulation.

Cigarette smoke contains many different classes of chemicals.The identity of the chemicals in cigarette smoke which are respon-sible for immune suppression is not completely established. For

Fig. 7. Reduction of PBMCs cytolytic ability with different TPPs. PBMCs were treated withthen added as target cells and incubated for additional 4 h. Cells are stained with 7AADcytometry results with percent killing shown in the gated boxes. (B) Combined datastatistical signicance was indicated by: , P < 0.05.itro 27 (2013) 19922004 2001example, a, b unsaturated aldehydes, such as acrolein and croton-aldehyde have been shown to cause cytokine suppression, but notthe saturated aldehydes (acetaldehyde, propionaldehyde andbutyraldehyde) (Lambert et al., 2005, 2007). Our ongoing workshows that the WS-CM used in the current study contains both

varying concentrations of TPPs and nicotine for 1.5 h. CFSE-labeled K562 cells wereand ow cytometry was used to gauge killing ability. (A) The representative ow

from four independent experiments using PBMCs from four different donors. The

425 lg/mL equi-nicotine dose (data not shown). Consistent with

In contrast to the effects of the combustible TPPs and ST/CAS,

the innate and adaptive immune responses and how in vitro treat-

in V2002 S. Arimilli et al. / Toxicologythe a, b unsaturated aldehydes and the saturated aldehydes. It ispossible that the cytokine suppression observed in this study alsomay be mediated by the a, b unsaturated aldehydes. However,additional work remains to be done in determining the compo-nents of the WS-CM that are responsible for suppression of the tar-get cell killing.

NK cells and CD8+ T cells play critical roles in tumor surveil-lance and defending against microbial infections through cytoly-sis of target cells. Several investigators have assessed NK cellfunction in response to cigarette smoke exposure and reporteda decrease in target cell killing by the exposed cells (Fersonet al., 1979; Hogan et al., 2011b; Mehta et al., 2008; Mianet al., 2008). Consistent with those ndings, we report here thatexposure to WS-CM signicantly inhibits target cell killing, withWS-CM being the most potent of the TPPs tested in this study(Fig. 7). One possible mechanism through which WS-CM inter-feres with the NK cell and CD8+ cell function is likely throughits ability to block the expression of perforin (Fig. 8). Perforinis largely responsible for mediating the cytolysis of targeted cells(Smyth et al., 1999). Although TPM exposure did not interferewith the induction of perforin (Fig. 8), a near 50% decrease incell killing was observed, suggesting the involvement of addi-tional proteases in NK cell-mediated cytolysis. Previous studieshave suggested that anti-tumor cell activity of NK cells is re-duced in smokers compared to non-smokers (Ferson et al.,1979). Thus TPPs, particularly WS-CM, diminishes perforin basal

Fig. 8. Inhibition of perforin levels in PBMCs and NK cells with different TPPs. Cellswere exposed to varying concentrations of WS-CM, TPM, ST/CAS, nicotine and theircorresponding vehicle controls. Upper panel shows percent perforin-positivePBMCs from four different donors PBMCs. Lower panel shows percent perforinpositive NK cells in the PBMC population. The statistical signicance was indicatedby: , P < 0.05; , P < 0.0005.ment with TPPs can impact the immune system. In this manuscriptwe have used both combustible and non-combustible tobaccoproduct preparations to treat PBMCs and then stimulated withpoly I:C and LPS. We showed a reduction of multiple cytokinesand the cytolytic ability of PBMCs which are important for fullyfunctional immune responses. We have demonstrated that humanNK and T cell function was reduced with TPPs in an order of WS-CM > TPM > ST/CAS. Our ndings indicate that combustible TPPs,most notably WS-CM, attenuate cellular functions by suppressingcytokine production and cytolytic ability to a greater extent at low-er nicotine equivalent units than smokeless TPPs. These ndingsmay provide a mechanistic explanation why smokers are moreprone to viral and bacterial infections, and cancers. Investigatingthe molecular mechanisms underlining these effects and effectson other cytokines regulating PBMC functions will provide furtherinsight into the potential impact of cigarette smoke on the immunesystem.

Conict of Interest

The authors declare that there are no conicts of interest.

Acknowledgments

We sincerely thank Dr. Peter Chen for his statistical analysis ofthe work. We also thank Dr. Evan Gregg for his help in manuscriptpreparation. This work is funded by R.J. Reynolds Tobacco Com-pany (RJRT) under a collaborative research agreement with WakeForest University School of Medicine. G.L. Prasad is a full time em-ployee of RJRT.

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2004 S. Arimilli et al. / Toxicology in Vitro 27 (2013) 19922004

Combustible and non-combustible tobacco product preparations differentially regulate human peripheral blood mononuclear cell functions1 Introduction2 Materials and methods2.1 Tobacco product preparations and EC50 values2.2 Isolation of PBMCs2.3 Cell staining and flow cytometry2.4 Cytometric bead array assay2.5 Cytotoxicity assay2.6 Statistical comparisons

3 Results3.1 Suppression of TNF-+ and IFN-+ T cell popu3.2 Suppression of TNF-+ NK cell populations in3.3 Effect of TPPs on CD69 expression in NK cells3.4 Inhibition of secreted cytokines in TPP-treated PBMCs3.5 Suppression of PBMC cell target killing ability3.6 Effect of TPP exposure on perforin levels in total PBMCs and NK cells

4 DiscussionConflict of InterestAcknowledgmentsReferences