Comparison of pro- and anti-inflammatory responses in paired human primary airway epithelial cells and alveolar macrophages

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1 Murk et l. Respirtory Reserch (2018) 19:126 RESEARCH Comprison of pro- nd nti-inflmmtory responses in pired humn primry irwy epithelil cells nd lveolr mcrophges Reem Al Murk 1, Nicole Roerts 1, Roert J. Mson 1, Scott Alper 2,3* nd Hong Wei Chu 1* Open Access Astrct Bckground: Airwy epithelil cells nd lveolr mcrophges (AMs) re the first line of defense in the lung during infection. Toll-like receptor (TLR) gonists hve een extensively used to define the regultion of inflmmtion in these cells. However, previous studies were performed in non-pired irwy epithelil cells nd AMs. The mjor gol of our study ws to compre the pro- nd nti-inflmmtory responses of pired humn primry irwy epithelil cells nd AMs to TLR3 nd TLR4 gonists. Methods: Trcheoronchil epithelil cells (TBEC) nd AMs from four smokers nd four non-smokers without lung disese were cultured with or without Poly(I:C) (PIC) ( TLR3 gonist) or LPS ( TLR4 gonist) for 4, 24 nd 48 h. The immune responses of pired cells were compred. Results: TBEC nd AMs showed stronger pro-inflmmtory cytokine (e.g., IL-8) responses to PIC nd LPS, respectively. TLR3 nd TLR4 mrna levels were similr in non-stimulted TBEC nd AMs. However, PIC stimultion in AMs led to sustined up-regultion of the immune negtive regultors Tollip nd A20, which my render AMs less sensitive to PIC stimultion thn TBEC. Unlike AMs, TBEC did not increse NF-κB ctivtion fter LPS stimultion. Interestingly, smoking sttus ws correlted with less TLR3 nd IRAK-M expression in non-stimulted TBEC, ut not in AMs. PIC-stimulted TBEC nd LPS-stimulted AMs from smokers vs. non-smokers produced more IL-8. Finlly, we show tht expression of A20 nd IRAK-M is strongly correlted in the two pired cell types. Conclusions: By using pired irwy epithelil cells nd AMs, this study revels how these two criticl types of lung cells respond to virl nd cteril pthogen ssocited moleculr ptterns, nd provides rtionle for modulting immune negtive regultors to prevent excessive lung inflmmtion during respirtory infection. Keywords: Trcheoronchil epithelil cells, Alveolr mcrophges, Immune negtive regultors, Inflmmtion, Toll-like receptor, Pthogen ssocited moleculr ptterns, Cigrette smoke Bckground Airwy epithelil cells long with lveolr mcrophges serve s the first line of host innte immune defense ginst irorne pthogens nd other irorne environmentl hzrds [1]. These lung cells re le to recognize pthogen ssocited moleculr ptterns (PAMPs) using receptors tht include the Toll-like * Correspondence: lpers@njhelth.org; chuhw@njhelth.org 2 Deprtment of Biomedicl Reserch nd Center for Genes, Environment, nd Helth, Ntionl Jewish Helth, University of Colordo, 1400 Jckson Street, Denver, CO 80206, USA 1 Deprtment of Medicine, Ntionl Jewish Helth, 1400 Jckson Street, Room A639, Denver, CO 80206, USA Full list of uthor informtion is ville t the end of the rticle receptor (TLR) fmily. TLR-medited recognition of PAMPs leds to the ctivtion of downstrem signling cscdes, the susequent ctivtion of pro-inflmmtory trnscription fctors including NF-κB, nd ultimtely the production of pro-inflmmtory cytokines including CXCL8 (IL-8), CXCL10 (IP-10) nd TNF-α. While this inflmmtory response is importnt for comting the pthogens, inflmmtion must e ppropritely regulted to prevent excessive inflmmtion nd tissue dmge. One regultory mechnism tht ensures tht inflmmtion is self-limiting is the PAMP-medited induction of negtive regultors. Numerous negtive regultors of TLR-medited signling hve een identified The Author(s) Open Access This rticle is distriuted under the terms of the Cretive Commons Attriution 4.0 Interntionl License ( which permits unrestricted use, distriution, nd reproduction in ny medium, provided you give pproprite credit to the originl uthor(s) nd the source, provide link to the Cretive Commons license, nd indicte if chnges were mde. The Cretive Commons Pulic Domin Dediction wiver ( pplies to the dt mde ville in this rticle, unless otherwise stted.

2 Murk et l. Respirtory Reserch (2018) 19:126 Pge 2 of 14 including Toll-intercting protein (Tollip), TNF lphinduced protein 3 or TNFAIP3 (A20) nd interleukin-1 receptor-ssocited kinse 3 (IRAK-M). These negtive regultors down-regulte the trnscription nd trnsltion of TLR-induced genes during infection nd inflmmtion [2]. Hosts re protected from hyper-inflmmtion nd utoimmunity y the inhiitory effect of these negtive regultors [2]. It hs een proposed tht irwy epithelil cells nd lveolr mcrophges my respond similrly or differently to vrious microes nd microil PAMPs [1, 3, 4]. However, few studies hve een conducted tht directly compre the response of these two cell types. Notly, negtive regultors of TLR signling pthwys hve not een previously investigted in irwy epithelil cells nd lveolr mcrophges from the sme humn suject (pired cells) to clrify their effect on inflmmtory responses. In the current study, we used pired irwy epithelil cells nd lveolr mcrophges from the sme helthy donors to test the hypothesis tht functionl differences exist etween irwy epithelil cells nd lveolr mcrophges with respect to pro-inflmmtory cytokine relese fter TLR stimultion. In prticulr, we tested if the response to TLR3 nd TLR4 gonists differs in these two cell types nd if this difference my e explined y ltered expression of negtive regultors of TLR signling. We lso explored the effect of smoking sttus in these pired cells on the immune response fter TLR stimultion. A full understnding of how inflmmtion is regulted y these negtive regultors in different host cell types will fcilitte the design of new therpeutics to lnce the eneficil nd detrimentl effects of inflmmtion in vrious lung diseses. Methods Mterils Bronchil epithelil cell growth medium (BEGM) with ntiiotics ws purchsed from Lonz (Wlkersville, MD). The BEGM ws prepred following mnufcturer s guideline, which contined ll the supplements (BPE, hegf, epinephrine, trnsferrin, insulin, retinoic cid, triiodothyronine, GA) except hydrocortisone to void ny inhiitory effect of corticosteroids on cell pro-inflmmtory responses. RNA lysis uffer RLT ws from Qigen (Hilden, Germny). RIPA western lysis uffer ws purchsed from Thermo-Fisher Scientific (Wlthm, MA). DMEM (high glucose) for mking D10 (DMEM +10% FBS+1% Pen/Strep +1% Amphotericin B + 1% L-Glutmine+ 0.5% Gentmicin) ws from GE Life Sciences (Logn, UT). The nucler extrction kit nd TrnsAM NF-κB p65 ssy kit were from Active Motif (Crlsd, CA). IL-8, IP-10 nd TNF-α ELISA kits were otined from R&D systems (Minnieplois, MN). Humn donor informtion To isolte humn primry irwy epithelil cells nd lveolr mcrophges, we otined humn lungs from de-identified orgn donors whose lungs were not suitle for trnsplnttion nd were donted for medicl reserch through the Ntionl Disese Reserch Interchnge (Phildelphi, PA), the Interntionl Institute for the Advncement of Medicine (Edison, NJ), or Donor Allince of Colordo. The Institutionl Review Bord (IRB) t Ntionl Jewish Helth deemed this reserch s non-humn sujects reserch. Donors were chosen sed on lung function with P O2 /Fi O2 rtio of > 225, no history of clinicl lung diseses, chest rdiogrph indicting no infection, nd time on the ventiltor of < 5 dys. The sex, ge, rce, nd smoking history of donors were vrile nd were not selection criteri. Isoltion nd culture of humn trcheoronchil irwy epithelil cells To isolte trcheoronchil epithelil cells (TBEC), trchel nd min ronchil tissue ws digested with 0.1% protese in DMEM overnight t 4 C, nd processed s previously descried [5 7]. TBEC t pssge 1 from the frozen stock were cultured nd expnded in collgen-coted 60-mm tissue culture dishes in BEGM medium t 37 C, 5% CO 2. When the cells were 80% confluent, they were trypsinized nd seeded onto 12-well pltes for sumerged culture. In our culture model (primry sumerged culture), TBEC grown in monolyer did not differentite into the mucociliry phenotype, ut showed the feture of sl cells expressing KRT5 (Fig. 1, ). Sumerged culture nd stimultion of TBEC TBEC t cells/well in BEGM medi were seeded into 12-well pltes. After 48 h, the medium ws chnged to refresh BEGM, nd LPS (10 ng/ml) or Poly(I:C) (PIC) (1 μg/ml) ws dded. LPS ws used to mimic cteril infection s use of live cteri could cuse cell deth, thus compromising dt interprettion. We chose to use Poly I:C (PIC) s dsrna mimic of RNA viruses to rodly test the pro-inflmmtory response in pired irwy epithelil cells nd lveolr mcrophges. Given the vrying susceptiility of the two types of cells to common respirtory viruses such s influenz A viruses nd rhinoviruses, it would hve een difficult to compre their responses to the live viruses. It hs een reported tht humn AMs re less susceptile to infection y live influenz A viruses or rhinoviruses compred to epithelil cells [8]. PAMP doses were chosen following LPS nd PIC dose response experiments; the lowest concentrtions yielding pro-inflmmtory response in oth cell types were chosen. The doses we chose were comprle to previous studies using LPS nd PIC

3 Murk et l. Respirtory Reserch (2018) 19:126 Pge 3 of 14 c d Fig. 1 Chrcteriztion of primry humn trcheoronchil epithelil cell (TBEC) nd lveolr mcrophges (AM) under sumerged cultures. TBEC showing fetures of sl cells with positive stining for cytokertin-5 (KRT5 in red, DAPI in lue, 400X.); Negtive control for KRT5 stined with DAPI in lue (400X); c Primry lveolr mcrophges (AM) stined positive for CD68 (red) nd DAPI (lue, 400X); d Negtive control for CD68, stined with DAPI in lue (400X) stimultion in cell culture experiments [9, 10]. The cells were hrvested fter 4, 24 nd 48 h in RNA lysis uffer (RLT) or in RIPA uffer with protese nd phosphtse inhiitors. The superntnts were collected nd stored t 80 C for ELISA. Isoltion, culture, nd stimultion of humn lveolr mcrophges Broncholveolr lvge (BAL) ws performed on the right middle loe or lingul of the donor lungs y completely filling the loe three times with lnced slt solution nd EDTA, nd then three times with the slt solution lone [11, 12]. After ech instilltion, lvge fluid ws drined from the lung, collected, pooled, nd centrifuged to otin BAL cells including lveolr mcrophges. The BAL cells were frozen in 90% FBS nd 10% DMSO. Bsed on the protocols estlished y us [12, 13] nd others [8, 14], mcrophges were enriched from the BALF fter lysis of the RBCs, nd dhered onto the plstic surfce in 12-well culture pltes, nd then wshed to remove non-dherent cells. By using the CD68 immunofluorescent stining s shown in Fig. 1c, d nd in our previous puliction [13], nerly 99% of cells were positive for CD68. Isolted lveolr mcrophges were thwed nd seeded into 12-well pltes t densities of either cells/well or cells/well in D10 medi t 37 C in 5% CO 2. After 48 h, the medium ws chnged to remove the non-dherent cells nd tretments with LPS (10 ng/ml) or PIC (1 μg/ml) in D10 medi were strted on dhered AM. After 4, 24 nd 48 h, the superntnts were collected nd stored t 80 C for ELISA. The cells were hrvested t these sme time points in RNA lysis uffer (RLT) or in RIPA uffer with protese nd phosphtse inhiitors. Chrcteriztion of the TBEC cells nd BAL mcrophges with immunofluorescence Primry pired TBEC nd lveolr mcrophges were cultured on coverslips. After 48 h, the TBEC were stined with n nti-cytokertin ntiody (KRT5) (Acm, 1:500) nd, AM were stined with n nti-cd68 ntiody (Bioscience, 1:200) following pulished protocol [6] Fig.1. NF-κB ctivity ssy Following the tretments of the pired cells s descried ove, nucler proteins t ech of the time points (4, 24, 48 h) were extrcted using the Active Motif kit s per the mnufcturer s instructions. The extrcted nucler proteins (10 μg/condition) were tested for NF-κB p65 trnscription fctor ctivity using the TrnsAM NF-κB

4 Murk et l. Respirtory Reserch (2018) 19:126 Pge 4 of 14 p65 kit following the mnufcturer s instructions. The dt were expressed s opticl density (OD) vlue. ELISA for humn IL-8 (CXCL8), IP-10 (CXCL10) nd TNF-α IL-8, IP-10 nd TNF-α protein levels were mesured in cell superntnts using specific DuoSet ELISA kits (R&D Systems, Minnepolis, MN) s per the mnufcturer s instructions. RNA extrction nd RT-PCR for humn TLRs, Tollip, A20, IRAK-M, IL-8 nd IP-10 RNA ws extrcted from cells stored in RLT using n RNesy Plus kit (Qigen). RNA ws reverse trnscried using the High Cpcity cdna Reverse Trnscription Kit (Applied Biosystems, Cliforni). Rel-time PCR ws performed on the CFX96 (Bio-Rd) using TqMn gene expression ssys from Applied Biosystems (Life Technologies, Foster City, CA, USA). An identicl threshold cycle (Ct) ws pplied for ech gene of interest (TLR3, TLR4, Tollip, A20, IRAK-M, IL-8 nd IP-10). Reltive mrna expression nd fold chnge levels were clculted using the delt Ct method for trget genes nd GAPDH. Trget gene expression ws normlized to GAPDH [15]. Western lot nlysis Equl mounts of protein from smples with different tretments were seprted y electrophoresis on 10% SDS-polycrylmide gels. The proteins were then trnsferred onto nitrocellulose memrnes nd proed with either got polyclonl nti-tollip ntiody (sc27315), mouse monoclonl nti-a20 ntiody (sc69980) or mouse nti-β-ctin ntiody (sc47778) (Snt Cruz Biotechnology, Inc.). Blots were then incuted with pproprite HRP-linked secondry ntiodies nd developed with the ECL Western lotting sustrte. The lots were scnned using Fotodyne imging system, nd densitometry ws performed using the NIH Imge-J softwre. Sttisticl nlysis Pirwise comprisons were performed using the Student s t-test. For multiple comprisons, nlysis of vrince (ANOVA) or the Kruskl-Wllis test ws used for normlly distriuted dt or nonprmetric dt, respectively. Flse discovery rte (FDR) ws used for correction (Benjmini nd Hocherg). Single vrile liner regression ws used to test for n ssocition etween gene expression in AMs nd TBECs. All sttisticl nlyses nd grphs were performed using GrphPd Prism softwre (GrphPd Softwre, L Joll, CA, USA). P-vlues less thn 0.05 were considered significnt. Results Trcheoronchil epithelil cells (TBEC) nd lveolr mcrophges disply different responses to poly(i:c) nd LPS The pro-inflmmtory cytokines IL-8 (CXCL8) nd IP-10 (CXCL10) were mesured in the superntnts of cultured epithelil cells nd mcrophges from eight helthy sujects (four smokers nd four non-smokers) s descried in Tle 1. TBEC responded to PIC stimultion y producing IL-8 strting t 4 h, peking t 24 h (P < 0.001), nd then mintining cytokine production t 48 h. (Fig. 2). Similrly, TBEC incresed IP-10 production fter PIC stimultion (Fig. 2c). In contrst to PIC, LPS stimultion in TBEC did not significntly increse IL-8 production t ny of the time points exmined (Fig. 2). Alveolr mcrophges responded to oth PIC nd LPS y producing IL-8, ut the induction of IL-8 ws stronger fter LPS t 24 nd 48 h (P = 0.01) (Fig. 2d nd e). Alveolr mcrophges lso responded to PIC y producing IP-10 t 24 nd 48 h, ut the increse did not rech sttisticl significnce (Fig. 2f). Although the two cell types were initilly seeded t the sme cell density ( /well), lveolr mcrophges, unlike the TBEC, do not proliferte. As complementry pproch to monitoring cytokine protein levels produced y the two cell types, IL-8 nd IP-10 mrna levels were monitored t the pek time (24 h) of cytokine induction. Consistent with the protein dt, in TBEC, IL-8 mrna incresed (> 20-fold) fter PIC stimultion (P = 0.01), ut did not increse fter LPS stimultion (Fig. 3). Mcrophges showed significnt increse in IL-8 mrna expression (> 15-fold) fter LPS stimultion t 24 h (P = 0.01). Mcrophges lso incresed IL-8 mrna fter PIC, ut the induction level ws out 50% of tht in TBEC (Fig. 3). Similr to IL-8, IP-10 mrna levels fter PIC stimultion t 24 h were higher in stimulted TBEC (out 3000-fold) thn mcrophges tht showed out 1000-fold induction (Fig. 3). Interestingly, IP-10 protein ws not detectle fter LPS stimultion in oth lveolr mcrophges nd Tle 1 Chrcteristics of Reserch Sujects Sujects Gender Age (yrs) Smoking sttus 1 Femle 64 Smoker: 1pck/dy 43 yers 2 Mle 58 Smoker: < 1/2 pck/dy 20 yers 3 Femle 57 Smoker: < 1pck/dy 30 yers 4 Mle 58 Smoker: 1/2 pck/dy 37 yers 5 Femle 45 Non-smoker 6 Mle 45 Non-smoker 7 Femle 75 Non-smoker 8 Mle 63 Non- smoker

5 Murk et l. Respirtory Reserch (2018) 19:126 Pge 5 of 14 d e c f Fig. 2 Humn trcheoronchil epithelil cells (TBEC) nd lveolr mcrophges disply different responses to different pthogen ssocited moleculr ptterns (PAMPs). IL-8 nd IP-10 protein production in superntnts of cultured humn TBEC (, nd c) nd cultured lveolr mcrophges (d, e nd f) in the sence ( ) nd presence of Poly(I:C) (PIC) or LPS tretment for 4, 24 nd 48 h. N = 8 donor sujects Fig. 3 PAMP stimultion increses inflmmtory cytokine mrna levels in primry humn lung cells. mrna expression of IL-8 () nd IP-10 () in humn trcheoronchil epithelil cells (TBEC) nd lveolr mcrophges in the sence ( ) nd presence of LPS nd Poly(I:C) (PIC) t 24 h. N = 8 donor sujects. The horizontl lines indicte the medins

6 Murk et l. Respirtory Reserch (2018) 19:126 Pge 6 of 14 irwy epithelil cells, nd LPS did not significntelly induce IP-10 mrna in oth cell types (Fig. 3). Together, our dt suggest tht TBEC re etter responder to PIC, nd lveolr mcrophges hve stronger response to LPS. Nucler fctor-kppb (NF-κB) ctivtion differs in pired TBEC nd lveolr mcrophges stimulted with Poly(I:C) or LPS To determine the possile mechnism of vrying pro-inflmmtory cytokine responses of the pired irwy epithelil cells nd lveolr mcrophges, NF-κB ctivity ws mesured with or without LPS or PIC stimultion. We chose to monitor NF-κB ctivtion ecuse NF-κB is ctivted y LPS nd PIC nd plys key role in induction of pro-inflmmtory cytokines. We monitored NF-κB ctivtion y mesuring NF-κB levels in nucler extrcts using n ELISA. Consistent with the IL-8 dt, levels of NF-κB in the nuclei were incresed in TBEC fter stimultion with PIC (P < 0.05) t 24 nd 48 h, ut not LPS (Fig. 4). On the other hnd, LPS, ut not PIC, significntly incresed NF-κB ctivtion in lveolr mcrophges t 24 h (P = 0.01) (Fig. 4). Levels of TLR4 nd TLR3 expression do not explin the differences in IL-8 production nd NF-κB ctivtion in TBEC nd lveolr mcrophges TLR4 nd TLR3 re the respective receptors for LPS nd PIC. Binding of TLRs to their lignds ctivtes TLR signling pthwys, leding to ctivtion of NF-κB nd other trnscriptionl fctors, nd thus pro-inflmmtory cytokine production [16]. In order to determine why TBEC nd lveolr mcrophges respond more strongly to PIC nd LPS, respectively, we compred the seline (untreted) expression of TLR3 nd TLR4 in the two cell types. No significnt differences of TLR3 or TLR4 mrna expression were found etween the two cell types (Additionl file 1: Figure S1). This suggests tht the differing responsiveness of TBEC nd lveolr mcrophges to PIC nd LPS my e regulted y something other thn TLR expression. One possiility tht we explored elow is tht ltered expression of negtive regultors of TLR signling my ccount for this difference. Differences in PAMP-medited induction of negtive regultors in TBEC To potentilly explin the different pro-inflmmtory responses to PIC nd LPS in TBEC nd mcrophges, we exmined the expression of Tollip, A20 nd IRAK-M, which re known to down-regulte TLR3 nd TLR4 signling pthwys. In TBEC, there ws no significnt chnge in Tollip mrna expression fter LPS or PIC stimultion t ll the time points mesured (Fig. 5). In contrst, the other two negtive regultors did exhiit ltered expression following PAMP chllenge. A20 mrna levels were not significntly chnged fter LPS stimultion in TBEC. In contrst, PIC up-regulted A20 mrna expression (p < 0.005) fter 4, 24 nd 48 h (Fig. 5). A20 mrna expression in TBEC ws highest t 4 h fter PIC stimultion nd remined significntly incresed, leit t lower level, 24 nd 48 h fter PIC chllenge (Fig. 5). LPS tretment of TBEC did led to slight ut not sttisticlly significnt increse in IRAK-M mrna expression t 4 nd 24 h fter chllenge ut not t 48 h (Fig. 5c). In contrst, PIC tretment did not lter IRAK-M mrna levels t the erliest time point ut did significntly increse IRAK-M mrna t the lter 24 nd 48 h time points (Fig. 5c). Thus, A20 expression ws induced erly fter PIC chllenge while IRAK-M expression ws induced lter fter PIC chllenge. Tollip expression ws not ltered t ny time point exmined. Fig. 4 Differentil ctivtion of NF-κB in humn trcheoronchil epithelil cells (TBEC) nd lveolr mcrophges in responses to PAMPs. NF-κB p65 levels were mesured in nucler extrcts from pired TBEC () nd lveolr mcrophges () in the sence ( ) nd presence of tretments with LPS nd Poly(I:C) (PIC) t 4, 24 nd 48 h. N = 3 donor sujects

7 Murk et l. Respirtory Reserch (2018) 19:126 Pge 7 of 14 c d e Fig. 5 PAMP stimultion induces expression of negtive regultors of TLR signling in humn trcheoronchil epithelil cells (TBEC). mrna expression of negtive regultors Tollip (), A20 () nd IRAK-M (c) in TBEC in the sence ( ) nd presence of LPS or Poly(I:C) (PIC) t 4, 24 nd 48 h. N = 8 donor sujects. Medin vlues re shown s horizontl lines. Densitometric nlysis of Tollip (d) nd A20 (e) western lots on lystes of TBEC in the sence ( ) or presence of tretments with LPS, PIC t 4, 24 nd 48 h. β-ctin ws included s protein loding control to normlize Tollip or A20 expression. N = 3 donor sujects (dditionl lots in Additionl file 1: FigureS2) We lso exmined Tollip nd A20 protein levels y western lot in the suset of sujects (n = 3) with reltively undnt cells tht llowed us to perform dditionl cell culture studies due to limited vilility of lveolr mcrophges in other sujects. In support of the mrna dt, Tollip protein levels were not chnged significntly fter LPS or PIC stimultion (Fig. 5d). Consistent with the mrna dt, A20 protein ws induced significntly y PIC stimultion in TBEC with significnt increse t 4 nd 24 h ut not 48 h fter chllenge (Fig. 5e). Western lots for two other sujects re shown in Additionl file 1:FigureS2A-C. PAMP-medited induction of negtive regultors in lveolr mcrophges overlps ut is distinct from tht in TBEC In the pired lveolr mcrophges, Tollip mrna ws incresed fter 24 nd 48 h of PIC tretment; in contrst, Tollip mrna levels were not ltered significntly y LPS tretment (Fig. 6). Thus, Tollip expression is induced y PIC in lveolr mcrophges ut not in TBEC. A20 regultion in lveolr mcrophges ws similr to tht in TBEC: PIC ut not LPS induced A20 mrna expression in mcrophges t ll the time points (4, 24 nd 48 h) (Fig. 6). IRAK-M mrna mrginlly incresed t 24 h post LPS nd PIC stimultion ut returned to seline levels y 48 h (Fig. 6c). To confirm the mrna expression dt, we lso monitored Tollip nd A20 protein levels in the mcrophges fter LPS nd PIC stimultion. We found tht oth Tollip nd A20 protein levels were incresed fter PIC chllenge, ut this only reched sttisticl significnce for A20 fter 4 h of chllenge (Fig. 6d, e). Western lots for two other sujects re shown in Additionl file 1: Figure S2 A-C. The more roust effects t the mrna level rther thn t the protein level my e due to the lrger vriility oserved in the protein dt. Together, the ove dt showed tht fter PIC stimultion, Tollip, A20 nd IRAK-M were ll up-regulted in lveolr mcrophge, while only A20 nd IRAK-M were incresed in TBEC. Moreover, there were temporl differences in this induction. LPS, on the other hnd, did not increse production of ny of these three negtive regultors in either cell type.

8 Murk et l. Respirtory Reserch (2018) 19:126 Pge 8 of 14 c d e Fig. 6 PAMP stimultion induces expression of negtive regultors of TLR signling in lveolr mcrophges. mrna expression of the negtive regultors Tollip (), A20 () nd IRAK-M (c) in mcrophges in the sence ( ) nd presence of LPS, Poly(I:C) (PIC) t 4, 24 nd 48 h. N = 8 donor sujects. Medin vlues re shown s horizontl lines. Densitometric nlysis of Tollip (d) nd A20 (e) western lots on lystes of mcrophges in the sence ( ) or presence of tretments with LPS, PIC t 4, 24 nd 48 h. β-ctin ws included s protein loding control to normlize Tollip or A20 expression. N = 3 donor sujects (dditionl lots in Additionl file 1: Figure S2) Correltion in expression of negtive regultors etween pired irwy epithelil cells nd lveolr mcrophges We nd others hve oserved sustntil vriility in the response of AMs nd TBECs from different donors to PAMP stimultion [3, 4]. However, to our knowledge, prior studies hve not ddressed if AMs nd TBECs from individul donors cted similrly regrding their responses to PAMPs. We therefore nlyzed our cytokine, TLR, nd negtive regultor expression dt to see if expression correlted in the two cell types from individul donors. We found tht IL-8, IP-10, TLR3 nd TLR4 expression in these pired nlyses did not correlte (Additionl file 1: Tle S1 with p vlues). In contrst, the negtive regultors A20 nd IRAK-M showed striking positive correltions fter 24 h of PIC stimultion (P < 0.01) (Fig. 7). The impct of smoking sttus on TLR expression nd pro-inflmmtory responses to PIC nd LPS To determine if smoking sttus lters the regultion of inflmmtion in TBEC or lveolr mcrophges, we compred cytokine production following PIC or LPS stimultion, the levels of TLR3 nd TLR4, nd the levels of negtive regultors in cells from donors with or without smoking history. PIC tretment induced more IL-8 in TBEC from smokers thn non-smokers t the protein (Fig. 8) nd mrna levels (Additionl file 1: Figure S3). Likewise, LPS tretment induced more IL-8 in lveolr mcrophges from smokers thn non-smokers t the protein (Fig. 8) nd mrna levels. (Additionl file 1: Figure S3) Thus, smoking enhnced IL-8 production in oth cell types. While smoking enhnced IL-8 production in oth cell types, this effect ws not oserved for other pro-inflmmtory cytokines. Smoking wekened LPS nd PIC-induced TNF-α production in lveolr mcrophges (Fig. 8), nd smoking did not significntly lter IP-10 production in either PIC-stimulted TBEC or lveolr mcrophges (Additionl file 1: Figure S3). Although TNF-α ws incresed in superntnts of lveolr mcrophges stimulted with TLR gonists, it ws not detectle in irwy epithelil cell superntnts

9 Murk et l. Respirtory Reserch (2018) 19:126 Pge 9 of 14 Fig. 7 Expression of the negtive regultors A20 nd IRAK-M re strongly correlted etween pired humn trcheoronchil epithelil cells (TBEC) nd lveolr mcrophge smples from the sme individul donors. A20 nd IRAK-M mrna expression showed significnt correltion etween the two cell types fter 24 h of PIC stimultion. N = 8 donor sujects under ny conditions. Thus, we cnnot compre the production of TNF-α etween lveolr mcrophges nd irwy epithelil cells stimulted with TLR gonists. To determine how smoking ltered the inflmmtory response, we monitored expression of TLR3 nd TLR4 nd negtive TLR regultors. Unstimulted TBEC from non-smokers hd greter mrna expression of TLR3 nd TLR4 thn TBEC from smokers (P < 0.05) (Fig. 9). Moreover, fter LPS stimultion, TLR3 nd TLR4 mrna levels significntly incresed t 4 h in TBEC from non-smokers compred with smokers (P < 0.05) (Fig. 9, ). In contrst, PIC did not lter TLR3 or TLR4 mrna levels significntly in either smokers or non-smokers (Fig. 9, ). In lveolr mcrophges, TLR3 nd TLR4 mrna levels were not ltered significntly y smoking sttus in the sence or presence of PAMP stimultion (Fig. 9c, d), lthough there ws wek trend towrds more TLR3 nd TLR4 t 4 h fter LPS stimultion (Fig. 9c, d). Thus, chnges in TLR expression could not ccount for ll the effect of smoking on inflmmtory cytokine production in these two cell types. We lso mesured the levels of the negtive regultors in non-stimulted TBEC nd mcrophges from smokers nd non-smokers. No differences in Tollip nd A20 expression were found etween cells from the smokers nd non-smokers (dt not shown). However, IRAK-M mrna expression in TBEC ws lower in the smokers thn the non-smokers t 24 h (P = 0.01) (Additionl file 1:FigureS4). Discussion The present study leverges the use of pired irwy epithelil cells nd lveolr mcrophges from the sme donors in order to clerly demonstrte how these two types of criticl innte immune cells respond to two mjor TLR gonists (PIC nd LPS) tht re relevnt to cteril nd virl lung infections. Pired TBEC nd mcrophges showed differentil immune responses to PIC nd LPS stimultion. While Fig. 8 Smoking history lters IL-8 expressiont the protein level in humn trcheoronchil epithelil cells (TBEC) nd mcrophges, nd decreses TNF-α production in mcrophges treted with LPS. IL-8 production ws mesured in TBEC nd lveolr mcrophges in the sence ( ) or presence of LPS or poly(i:c) (PIC) t 24 h, nd compred etween smokers (S, n = 4) nd non-smokers (NS, n = 4). These dt re re-nlysis of the dt displyed in Fig. 3. There is significnt induction of IL-8 in smokers TBEC fter PIC stimultion, nd in smokers mcrophges fter LPS stimultion. TNF-α production in superntnts of cultured lveolr mcrophges. The cells from smokers (S, n = 4) nd non-smokers (NS, n = 4) were treted in the sence ( )orpresence of LPS or PIC for 4, 24 nd 48 h. NS trend to hve higher levels of TNF-α t ll the time points (P =0.1)

10 Murk et l. Respirtory Reserch (2018) 19:126 Pge 10 of 14 c d Fig. 9 Smoking history lters TLR expression in humn trcheoronchil epithelil cells (TBEC) ut not mcrophges. TLR3 nd TLR4 mrna expression in TBEC ( nd ) nd lveolr mcrophges (c nd d) tht were not treted, or treted with LPS or Poly(I:C) (PIC) for 4 nd 24 h. N = 8 donor sujects including four smokers (S) nd four non-smokers (NS) TBEC hve greter pro-inflmmtory response (IL-8, IP-10) to the TLR3 gonist PIC, lveolr mcrophges re stronger responder to LPS, TLR4 gonist. These differentil responses t the inflmmtory cytokine level were likely driven y differences in NF-κB ctivtion in the two cell types. In the TBEC, NF-κB ctivity ws higher fter PIC stimultion; in contrst, mcrophges showed higher NF-κB ctivity fter LPS stimultion. In order to understnd the mechnisms ehind this differentil immune response in the pired cells fter LPS nd PIC stimultion, we monitored their TLR3 nd TLR4 expression. There were no significnt differences in the mrna expression of TLR3 or TLR4 etween the pired cells tht were not stimulted with either PIC or LPS. Thus seline expression levels of TLR3 nd TLR4 my not e responsile for the differentil cell type-specific responses to TLR gonists. Becuse of the limited vilility of primry humn lveolr mcrophges, we nlyzed TLR mrna ut not protein expression. Previous studies indicte tht mrna expression of TLRs such s TLR3 in epithelil cells is consistent with protein expression determined using flow cytometry [17], suggesting tht TLR mrna is resonle surrogte for TLR protein levels. Nevertheless, this is one limittion of our study. It hs een found tht TLR4 in AEC is normlly loclized in the endosoml comprtment, ut is trnslocted to the cell surfce to recognize pthogens or environmentl LPS exposure [18, 19]. In contrst, mcrophges express TLR4 primrily t the cell surfce memrne [19]. While TLR3 in mcrophges is usully oserved intrcellulrly, it is found on the cell surfce s well s in the cytoplsm of AEC [17, 20]. The different locliztion of TLR3 nd TLR4 in vrious types of lung cells my provide n dditionl explntion for their different responses to TLR gonists. Despite the fct tht the two cell types showed no differences in TLR4 nd TLR3 expression following LPS or PIC stimultion, their NF-κB ctivity nd susequent inflmmtory cytokine production differed. These dt suggest the involvement of other mechnisms or regultors in the differentil ctivtion of epithelil cells nd mcrophges exposed to TLR gonists. One possiility tht we explored ws expression of negtive regultors of TLR signling. The literture suggests tht TLR-induced expression of immune negtive regultors represents criticl negtive feedck mechnism to prevent excessive TLR signling. These negtive regultors function t multiple levels in the TLR signling pthwy rnging from inhiition of receptor complex protein formtion to NF-κB

11 Murk et l. Respirtory Reserch (2018) 19:126 Pge 11 of 14 ctivtion [21 24]. Previous studies hve elucidted the role of immune negtive regultors such s A20, Tollip nd IRAK-M in the regultion of LPS-medited pro-inflmmtory responses of unpired irwy epithelil cells nd lung mcrophges. For exmple, A20 ws found to ttenute irwy epithelil cell responses to TLR2 nd TLR4 gonists [25, 26] s well s endotoxin tolernce in mcrophges [27]. The role of A20 in ntivirl responses hs not een well studied. To the est of our knowledge, our study is the first one to investigte the time course of immune negtive regultor expression in the response of pired lung cells (AM, AEC) to the virl mimic PIC s well s LPS. This llowed us to determine the potentil mechnisms of differentil responses of irwy epithelil cells nd lveolr mcrophges to vrious TLR gonists. We found tht the expression of these negtive regultors differed with respect to time nd cell type. Alveolr mcrophges up-regulted multiple immune negtive regultors including Tollip, A20 nd IRAK-M fter PIC stimultion. While A20 mrna nd protein were induced rpidly fter PIC stimultion, Tollip nd IRAK-M were induced t lter times. Importntly, A20 induction ws mintined throughout the entire 48 h post PIC stimultion. In contrst, TBEC showed significnt induction of A20 only t the erliest time point fter PIC stimultion, ut t lter times (24 nd 48 h), A20 expression significntly declined. Moreover, unlike mcrophges, PIC-stimulted TBEC did not significntly chnge the expression of Tollip. We speculte tht the lck of PIC-induced Tollip induction coupled with the trnsient A20 induction in TBEC my contriute to their more roust pro-inflmmtory responses thn the lveolr mcrophges. The erly induction nd lte reduction of A20 in PIC-stimulted TBEC re consistent with previous puliction performed y Gu et l. in the humn cytomeglovirus (HCMV) infection model [28]. Together, our dt indicte the possiility tht these immune negtive regultors could ffect the differentil response of vrious types of lung immune cells to PAMP stimultion. Future functionl studies using the RNA interference my further our understnding of these immune negtive regultors in modulting TLR gonist-medited pro-inflmmtory responses in pired lveolr mcrophges nd irwy epithelil cells. The design of our current study ws lso imed to ddress the effect of smoking sttus on pro-inflmmtory response in pired TBEC nd lveolr mcrophges s smoking hs een linked to chnges in the immune response in irwy cells [29, 30]. Mcrophges from smoking sujects produced less TNF-α ut more IL-8 fter LPS stimultion. However, in TBEC, IL-8 induction ws significntly higher in smoker s cells thn non-smoker s cells fter PIC ut not LPS stimultion. One possile explntion for these differences in the cells previously exposed to cigrette smoke ws the difference in TLR3 nd TLR4 expression. As oserved previously [31 34], smoking down-regulted TLR3 nd TLR4 expression in TBEC with nd without stimultion. The down-regultion of TLRs y smoking ws consistent with the decresed TNF-α nd IP-10 production in the smokers, ut not the incresed IL-8 production following stimultion. Mny studies hve shown tht cigrette smoking cn inhiit inflmmtory cytokine production (TNF-α, IL-6) nd host defense responses (IFN-β, IP-10) in response to TLR stimultion [30, 35, 36]. However, there re discrepncies in the literture regrding the effect of smoking on IL-8 production. While some studies hve demonstrted reduction in IL-8 production or no effect from smoking [37], other studies hve reported incresed production of IL-8 nd other neutrophil chemokines from smoker s irwy cells fter TLR gonist stimultion [33, 34, 38]. The signl trnsduction mechnisms controlling TNF-α nd IL-8 production pper to e different in the lveolr mcrophge. In mcrophges, smoking hs een shown to ctivte p38 MAPK tht controls trnscription, stiliztion of mrna nd secretion of IL-8 ut not TNF-α [34]. The induction of the negtive regultors oserved in our study my lso contriute to the differences in cytokine production in cells from smokers compred to non-smokers. Our dt showed tht smoking decresed expression of IRAK-M without stimultion in TBEC, which is consistent with the incresed IL-8 production tht we oserved in smokers. A significnt strength of our study ws our nk of pired TBECs nd AMs, which llowed us to determine if the response to TLR gonists in different cell types correlted within individul donors. We did not oserve ny correltion in inflmmtory cytokine production etween the two pired cell types. Thus, the roustness of n individul s immune response in one key lung innte immune cell type did not correlte with the roustness of tht response in the other cell type. Despite this oservtion, we did identify very strong correltion in PIC-induced expression of negtive regultors in the two cell types. Consistent with the mrna dt, in preliminry studies on smll numer of smples, we lso oserved trend of positive correltions of A20 nd Tollip protein levels etween the two cell types (dt not shown). This indictes tht n individul s ility to restrin inflmmtion in the lung my extend to multiple cell types. One limittion of our study is tht cultured cells my not mintin the phenotype of the in vivo cells, including the loss of the full differentition sttus. Unfortuntely, this is n inherent issue for every cell culture study. Nevertheless, one dvntge of the cell culture model is to llow us to quntittively nlyze the impct

12 Murk et l. Respirtory Reserch (2018) 19:126 Pge 12 of 14 Fig. 10 Proposed immune regultory mechnisms of pired irwy epithelil cells nd lung mcrophges in responses to Poly(I:C) nd LPS stimultion of multiple TLR gonists. In the future study, we pln to vlidte our results y using other culture methods such s ir-liquid interfce to mintin the differentition sttus of irwy epithelil cells. Also, it will e interesting in the future to study how irwy epithelil cells nd lveolr mcrophges cooperte in the effective lung defense ginst ny environmentl hzrds using the co-culture model. Conclusions In summry, we find tht TBEC nd lveolr mcrophges exhiit different responses to different TLR gonists. While these cells did not exhiit different levels of TLR mrnas, they did exhiit different expression of negtive regultors of TLR signling, which could impct the extent of the immune response in these two cell types. Addtionlly, our dt suggest mild impct of smoking sttus on the pro- (e.g., IL-8 nd IP-10) nd nti-inflmmtory (e.g., A20 nd Tollip) responses t the seline nd/or fter TLR gonist stimultion. We propose the involvement of potentil pthwys in regulting the different responses of irwy epithelil cells nd lveolr mcrophges to TLR gonists in Fig. 10. Understnding the role of immune negtive regultors in primry humn lung cells my provide new nd promising therpeutic strtegies to control pulmonry inflmmtion nd infection. Additionl file Additionl file 1: Figure S1. Comprison of seline (untreted) expression of TLR3 nd TLR4 in humn trcheoronchil epithelil cells (TBEC) nd lveolr mcrophges (AM). No significnt differences of TLR3 or TLR4 mrna expression were found etween the two cell types. Figure S2. Detection of A20, Tollip in humn trcheoronchil epithelil cells (TBEC) nd lveolr mcrophges efore nd fter PAMP stimultion t different time points. Western lot showing A20, Tollip, nd β-ctin proteins from TBEC nd lveolr mcrophges efore ( ) ndfter tretments with LPS, Poly(I:C) (PIC) for 4, 24 nd 48 h (N =3).These lots were lso used for desitometry in Figs. 5 nd 6. Figure S3. Comprison of IL-8 nd IP-10 expression in smokers nd non-smokers humn trcheoronchil epithelil cells (TBEC) nd mcrophges. mrna expression of IL-8 nd IP-10 in TBEC nd lveolr mcrophges in the sence ( ) orpresenceoflpsorpoly(i:c)(pic)t24hws compred etween smokers (S, n = 4) nd non-smokers (NS, n =4). These dt re re-nlysis of the dt displyed in Fig. 2. Figure S4. The effect of smoking sttus on IRAK-M expression. IRAK-M mrna expression ws exmined fter 24 h of culture in non-stimulted humn trcheoronchil epithelil cells from smokers (S, n =4)nd non-smokers (NS, n = 4). NS hve higher IRAK-M expression thn S. Tle S1. Correltion nlysis etween pired irwy epithelil cells nd lveolr mcrophges from the sme donors with P-vlues. (PPTX 367 k) Arevitions AMs: Alveolr mcrophges; BAL: Broncholveolr lvge; IRAK-M: Interleukin-1 receptor-ssocited kinse 3; LPS: Lipopolyscchride; PAMPs: Pthogen ssocited moleculr ptterns; PIC: Poly(I:C); TBEC: Trcheoronchil epithelil cells; TLR: Toll-like receptor; TNF-α: Tumor necrosis fctor-lph; TNFAIP3 (A20): TNF lph-induced protein 3; Tollip: Toll-intercting protein

13 Murk et l. Respirtory Reserch (2018) 19:126 Pge 13 of 14 Acknowledgements Ntionl Jewish Helth Mucosl Inflmmtion nd Immunity Progrm. Dr. Jnssen W., nd Dr. Seiold M. for providing ntiodies for immunofluorescent stining of irwy epithelil cells nd lveolr mcrophges. Funding This study ws supported y NIH grnts R01 HL122321, R01 AI106287, R01 HL125128, U19AI125357, 1R01 ES025161, nd the Ntionl Jewish Helth Mucosl Inflmmtion nd Immunity Progrm. Avilility of dt nd mterils The dt tht support the findings of this study re ville from the corresponding uthor upon resonle request. Authors contriutions All uthors red nd pproved the finl mnuscript. RAM nd NR contriuted to the design nd performnce of the experiments nd mnuscript writing. RJM, SA nd HWC were involved in experimentl design, mnuscript writing nd editing. Ethics pprovl nd consent to prticipte The study protocols were pproved y the IRB t Ntionl Jewish Helth. Consent for puliction Not pplicle. Competing interests The uthors declre tht they hve no competing interests. Pulisher s Note Springer Nture remins neutrl with regrd to jurisdictionl clims in pulished mps nd institutionl ffilitions. Author detils 1 Deprtment of Medicine, Ntionl Jewish Helth, 1400 Jckson Street, Room A639, Denver, CO 80206, USA. 2 Deprtment of Biomedicl Reserch nd Center for Genes, Environment, nd Helth, Ntionl Jewish Helth, University of Colordo, 1400 Jckson Street, Denver, CO 80206, USA. 3 Deprtment of Immunology nd Microiology, University of Colordo, 1400 Jckson Street, Denver, CO 80206, USA. Received: 6 April 2018 Accepted: 8 June 2018 References 1. Bsset C, Holton J, O Mhony R, Roitt I. Innte immunity nd pthogen-host interction. Vccine. 2003;21(SUPPL. 2):S12 S Mogensen TH. Pthogen recognition nd inflmmtory signling in innte immune defenses. Clin Microiol Rev. 2009;22(2): Hiriw K, vn Eeden SF. Contriution of lung mcrophges to the inflmmtory responses induced y exposure to ir pollutnts. 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