Cigarette smoke suppresses TLR-7 stimulation in response to virus infection in plasmacytoid dendritic cells

Toxicology in Vitro 25 (2011) 1106–1113

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Cigarette smoke suppresses TLR-7 stimulation in response to virus infection in plasmacytoid dendritic cells Shawn M. Castro, Krishnendu Chakraborty, Antonieta Guerrero-Plata ⇑ Department of Pathobiological Sciences, Louisiana State University, Baton Rouge, LA 70803, United States

a r t i c l e

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Article history: Received 29 October 2010 Accepted 17 March 2011 Available online 22 March 2011 Keywords: Cigarette smoke Plasmacytoid dendritic cells Respiratory syncytial virus Toll-like receptors

a b s t r a c t Exposure to environmental tobacco smoke (ETS) is associated with an increase in the frequency and severity of respiratory infections, including bronchiolitis, a clinical syndrome of infancy caused by viruses such as respiratory syncytial virus (RSV). The mechanisms by which ETS increases the risk of viral respiratory infections are largely unknown. A major effector integrating early antiviral and immunostimulatory activities is interferon-a (IFN-a), which is highly produced by plasmacytoid dendritic cells (pDC). In this work, we determined the effect of cigarette smoke extract (CSE) on human pDC immunity in response to a respiratory viral infection. We found that CSE inhibited RSV-induced IFN-a in pDC as well as the release of IL-1b, IL-10 and CXCL10. However, the production of additional cytokines and chemokines such as IL-6, TNF-a, CCL2, CCL3, CCL5 and CXCL8 was not altered. Quantitative RT-PCR analysis indicated that CSE decreased the expression of toll-like receptor (TLR)-7 and interferon regulatory factor (IRF)-7 in RSV-infected pDC. Furthermore, determination of IRF-7 phosphorylation by flow cytometry showed that CSE prevented IRF-7 activation. These data provide evidence that cigarette smoke suppresses key pDC functions upon viral infection by a mechanism that involves downregulation of TLR7 expression and decreased activation of IRF-7. Ó 2011 Elsevier Ltd. All rights reserved.

1. Introduction Exposure to environmental tobacco smoke (ETS) is associated with an increase in the frequency and severity of lower respiratory tract symptoms induced by viral infections (Arcavi and Benowitz, 2004; Bradley et al., 2005; Gilliland et al., 2003). The mechanisms of increased susceptibility to infections after smoke exposure are multifactorial and include suppression or modulation of the immune system (Arcavi and Benowitz, 2004; Sopori, 2002). However, the effect of smoking on the host immunity is not well understood and the specific mechanism by which tobacco smoke increases the risk of viral respiratory infections have not been fully characterized. Respiratory syncytial virus (RSV), a single stranded RNA paramyxovirus pathogen, is the major cause of lower respiratory tract infection (LRTI) in infants, young children, elderly and immunocompromised patients (Duncan et al., 2009; Glezen, 2009). Around 75% of children acquire RSV infection during their first year of life and by the age of two nearly 100% have been infected with this virus (Glezen, 2009). Several studies have suggested that exposure ⇑ Corresponding author. Address: Department of Pathobiological Sciences and Center for Experimental Infectious Disease Research, Louisiana State University, Skip Bertman Drive, Baton Rouge, LA 70803, United States. Tel.: +1 225 578 9678; fax: +1 225 578 9701. E-mail address: [email protected] (A. Guerrero-Plata). 0887-2333/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.tiv.2011.03.011

to ETS may occur in up to 60% of the infants with RSV bronchiolitis and LRTI in the United States (Bauman et al., 2002; Bradley et al., 2005), suggesting that ETS contributes to the severity of RSV infection. A major effector integrating both early antiviral and immunostimulatory activities is the type I interferon (IFN) system. Plasmacytoid dendritic cells (pDC) have the primary function and unique property of secreting type I IFN in response to virus and/or nucleic acids (Cella et al., 1999; Fitzgerald-Bocarsly et al., 2008; Siegal et al., 1999). When stimulated by viruses or Toll-like receptors (TLRs) agonists, pDC are capable of producing IFN-a 10–100-fold more efficient than other cell types. The TLRs expressed by pDC are restricted to those that enable recognition of RNA and DNA viruses by TLR7 and TLR9, respectively. Transcription of type I IFN genes is primarily controlled by members of the interferon regulatory factor (IRF) family. pDC are capable of rapidly secreting IFN-a due to constitutive expression of IRF-7 and the ability of MyD88 to recruit IRF-7, but not IRF-3 (McCartney and Colonna, 2009). Thus, engagement of TLRs in pDC triggers a signaling cascade that rapidly activates IRF-7 and transcription of IFN-a genes independent of IRF-3 activation. In addition to IFN-a, pDC are also efficient producers of other cytokines and chemokines in response to virus infection, as we have previously reported, RSV induces also a robust response of IL-6, IL-10, TNF-a and IL-1b in human pDC (Guerrero-Plata et al., 2006). Despite RSV induces the influx of sev-

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eral DC subsets including pDC, cDC and IKDC in mouse (GuerreroPlata et al., 2009) and pDC and myeloid DC to mucosal sites in infected children (Gill et al., 2005), based on our data and those from other groups, RSV fails to induce type I IFN production in several cells including human monocyte-derived DC and mouse cDC (Guerrero-Plata et al., 2006, 2009; Hornung et al., 2004). Therefore, pDC represent a suitable model in vitro to determine the effect of environmental factors on the innate immune response to respiratory virus infections such as RSV. In this work, we analyzed the effect of cigarette smoke extract (CSE) on IFN-a production and other cytokines in response to RSV infection in human pDC. The mechanisms by which CSE alters the function of human pDC were also explored. Our data indicate that cigarette smoke inhibits the production of IFN-a, IL-10, IL1b, CXCL10 and downregulates TLR7 expression and stimulation in RSV–infected pDC. 2. Material and methods 2.1. Culture medium and reagents Human pDC were cultured in RPMI 1640 supplemented with 2 mM L-glutamine, 10% FBS, 1000 U/I penicillin–streptomycin and 10 ng/ml IL-3 (R&D Systems) (herein referred as complete RPMI). TLR agonists (loxoribine; TLR-7 and CpG oligodeoxynucleotide 2006; TLR-9) were purchased from Invivogen. 2.2. Viral stocks preparation RSV A2 [American Type Culture Collection (ATCC), Manassas, VA] was grown in HEp-2 cells (ATCC) and purified by polyethylene glycol precipitation, followed by centrifugation on 35–65% discontinuous sucrose gradients as described elsewhere (Ueba, 1978). 2.3. Isolation and infection of pDC Human pDC were isolated from buffy coats obtained from healthy adult blood donors (Our Lady of the Lake Blood Donor Center, Baton Rouge, LA) and were used under Institutional Biosafety Committee-approved protocols. Cells were purified from peripheral blood mononuclear cells (PBMC) as previously described (Guerrero-Plata et al., 2006). Briefly, human pDC were purified from PBMC using magnetic microbeads coated with anti-BDCA-4 Mab (Miltenyi Biotec) according to the manufacturer’s instructions. For intracellular staining, untouched pDC were isolated from PBMC by negative depletion using the pDC Isolation Kit (Miltenyi Biotec). Flow cytometry analysis revealed a pDC population with a purity P97% based on the positive expression of cell surface markers BDCA-2 and CD123. Cell viability after isolation using the AutoMacsPro™ was P98% as determined by trypan blue. However, only live cell counts were considered for the experiments. To infect pDC, 105 cells were resuspended in 100 ll of complete RPMI medium and seeded in a 96-well plate. Cells were then exposed to different concentrations of CSE for 30 min at 37 °C followed by subsequent infection with sucrose-purified RSV at a multiplicity of infection (MOI) of 1 for 24 h as previously described (Guerrero-Plata et al., 2006). In a separate set of experiments, pDC were stimulated with TLR agonists in the presence or absence of CSE. Cells were stimulated with 500 lM of loxoribine or 1 lg/ml of CpG-ODN 2006. 2.4. Preparation of CSE Aqueous CSE was prepared from Kentucky research cigarettes 3R4F as previously described (Castro et al., 2008; Smelter et al., 2010). Briefly, CSE was prepared using a modification of method

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Blue and Janoff (1978) by drawing 35 ml of cigarette smoke into a syringe and then slowly bubbling the smoke into RPMI medium. One cigarette was smoked per 10 ml of medium. Following preparation of CSE, the solution was filtered to remove bacteria and large particles and used immediately. Initially, human pDC were exposed to increasing concentrations of CSE. However, based on the reduced cell viability at concentrations >2%, in most of the experiments performed in this work we used CSE at a concentration of 1%. Cell viability in CSE-treated cells was determined by quantitative measures of lactate dehydrogenase (LDH), a stable cytosolic enzyme that is released upon cell lysis (Promega Corporation, Madison WI). 2.5. Measurement of cytokines and IFN-a To determine the production of cytokines and IFN-a, cell-free supernatant from virus- and mock-infected pDC was collected after 24 h of culture. Samples were tested for multiple cytokines using the Milliplex™ Map Human Cytokine/Chemokine Panel (Millipore Corp., Billerica, MA) according to manufacturer’s instructions. The panel included the following cytokines: IL-1b, IL-6, IL-10, TNF-a, CCL2 (MCP-1), CCL3 (MIP-1a), CCL5 (RANTES), CXCL10 (IP-10) and CXCL8 (IL-8). The range of the sensitivity of the assay is 3.2– 10,000 pg/ml. IFN-a (isoforms IFN-aA, a2, aA/D, aD, aK and a4b) was measured by ELISA (PBL Interferon Source, Piscataway, NJ), according to the manufacturer’s instructions. 2.6. Intracellular staining of IRF-7 After 5 h of culture, cells were washed and immediately fixed with 2% paraformaldehyde for 15 min at 37 °C. Cells were stained with cell surface markers BDCA-2 and CD123 followed by permeabilization with PermBuffer III (BD Biosciences, Franklin Lakes, NJ) for 30 min on ice, and stained with either PE anti-human IRF-7 (pS477/pS479) or PE-isotype control (all from BD Biosciences) for 30 min at 4 °C and analyzed with a FACScan flow cytometer equipped with BD CellQuest software (both from BD Biosciences Immunocytometry Systems). Analysis was performed using FlowJo software v7.5 (Tree Star Inc., Ashland, OR, USA). 2.7. Quantitative real time polymerase chain reaction (qRT-PCR) Total RNA was extracted from pDC using the RNeasy Mini Kit (Qiagen, Valencia CA). RNA was then quantified and quality tested by UV spectrophotometry. In a two-step process cDNA was synthesized from RNA using TaqManÒ Reverse Transcription Reagents and duplicate target gene amplification reactions were carried out in single-plex format using the Taqman Gold RT-PCR kit and Gene Expression Assay for each target gene (Applied Biosystems, Foster City, CA). All assays for IRF-7, TLR7, TLR9 and 18S were run on the 7500 Fast Real-Time PCR System following suggested manufacturer’s cycling parameters (Applied Biosystems, Foster City, CA). The comparative CT (DDCT) method was used to quantitate the expression of target genes which were normalized to endogenous reference (18S) expression in reference to transcripts from uninfected and untreated control cells. 2.8. Statistical analysis Statistical analysis was performed using the InStat 3.05 biostatistics package (GraphPad, San Diego, CA). Unless otherwise indicated, mean ± SEM is shown. Intergroup comparisons were performed using a one way ANOVA to ascertain differences between groups, followed by a Tukey–Kramer test to correct for multiple comparisons. P values were considered significant if less than 0.05.

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3.2. Production of RSV-induced IL-1b, IL-10 and CXCL10 in pDC is reduced by cigarette smoke

3. Results 3.1. Cigarette smoke inhibits virus-induced IFN-a production The effect of cigarette smoke on IFN-a production by pDC in response to virus was determined. Human pDC were treated for 30 min with increasing concentrations of CSE prior to infection with RSV. Concentrations of IFN-a were determined by ELISA after 24 h of culture. As shown in Fig. 1a, a robust amount of IFN-a was released by infected pDC. However, the addition of CSE in the culture resulted in a dose dependent inhibition of IFN-a release. Uninfected pDC had nominal IFN-a levels measured below the assay limit of detection (data not shown). Treatment of RSV-infected cells with 0.1% CSE resulted in a marginal decrease in the production of IFN-a (25% reduction). Concentration of 1% CSE significantly inhibited IFN-a production in viral infected pDC (>87%), while concentrations of 2% and 4% CSE completely abolished IFN-a production to levels comparable to uninfected control cells. Although a broad range of CSE concentrations was used to determine CSE effects on RSV-induced IFN-a, based on cellular viability assays, we found that concentrations of CSE >2% produced significant cell death (33%) when compared to those treated with 0% of CSE (Fig. 1b). Based on these results, in further experiments we used a concentration of CSE of 1%, a concentration where we observed a lower cell death and a significant effect of IFN-a inhibition.

α (pg IIFN-α g/mll)

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In order to investigate whether cigarette smoke had an effect on the activation of TLR7 and TLR9 in pDC, we further determined the production of IFN-a in response to TLR agonists in cells exposed to CSE. pDC were stimulated with loxoribine, a TLR7 agonist, and CpG-ODN 2006, a TLR9 agonist, in the presence or absence of 1% of CSE for 24 h. Production of IFN-a was assessed by ELISA. As Fig. 3a shows, CSE reduced the capacity of pDC to produce IFN-a in response to loxiribine by 96%. Similarly, CSE reduced IFN-a production by 92% in pDC stimulated with ODN-CpG. Production of TNF-a and IL-6 in response to TLR agonists were also downregulated by the effect of CSE (data not shown). These data suggest that CSE inhibits the activation of the TLR7 and TLR9 pathway in pDC. Since we observed a reduced IFN-a production induced by TLR agonists in pDC, the expression of TLRs in pDC treated with CSE was also determined. Quantitative RT-PCR data show that CSE reduced the expression of TLR7 (Fig. 3b) and TLR9 (Fig. 3c) in pDC in response to TLR agonists by 66% and 51%, respectively.

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a pathway, qRT-PCR experiments were performed to assess the 4.0

expression of TLRs in RSV-infected pDC in the presence and absence of CSE. Human pDC were infected with RSV with or without CSE 1% for 24 h. As shown in Fig. 4a, RSV induced a 4-fold increase in TLR7 gene expression in pDC. However, the presence of CSE decreased RSV-induced TLR7 expression to levels comparable to those in uninfected cells. On the other hand, expression of TLR9 was not altered by RSV infection alone or the combination of RSV + CSE in human pDC (Fig. 4b).

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3.5. Transcriptional activation of viral-induced IRF-7 is downregulated by cigarette smoke

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3.3. Cigarette smoke inhibits TLR-7 and -9 expression and stimulation by specific TLR agonists in pDC

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The next set of experiments were designed to determine whether the inhibitory effect of CSE in pDC was extended to the production of cytokines other than IFN-a in response to viral infection. The production of cytokines and chemokines in pDC infected with RSV in the presence of CSE was determined using a luminex multiplex assay. As shown in Fig. 2, we observed that except for the production of IL-10, no differences were observed in the levels of cytokine production between untreated cells and pDC exposed to CSE alone. However, in response to viral infection, CSE inhibited the production of IL-1b, IL-10 and CXCL10. On the other hand, the robust induction of IL-6 and TNF-a by RSV infection was not affected by the presence of cigarette smoke. Likewise, the production of chemokines such as CCL2, CCL3, CCL5 and CXCL8 in uninfected and RSV-infected pDC remained unchanged.

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CSE % Fig. 1. CSE decreases IFN-a production in RSV-infected pDC. Human pDC were infected with RSV in the presence of increasing concentrations of CSE. (a) Cell-free supernatants were collected after 24 h of culture and IFN-a production was measured by ELISA. (b) Cell viability was determined by lactate dehydrogenase release. Bar graphs represent IFN-a values and viable cells percentage. Each value represents mean ± SEM (n = 6 donors). ⁄P < 0.05, ⁄⁄P < 0.01.

Since engagement of TLRs in pDC triggers a signaling cascade that rapidly activates IRF-7, we further investigated the effect of cigarette smoke on the expression and activation of IRF-7 in RSVinfected pDC. Cells were exposed to CSE and infected with RSV as mentioned above. Total RNA was isolated at 24 h and IRF-7 gene expression was measured by qRT-PCR. As Fig. 5a shows, there were no differences observed in IRF-7 gene expression between CSE exposed and untreated cells, suggesting that CSE does not interfere with the constitutive levels of IRF-7 in pDC. On the other hand, RSV induced a greater than 15-fold increase in IRF-7 gene expression in comparison to untreated control cells and that increase is

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Fig. 2. Modulation of cytokine profile by CSE in virus-infected pDC. Cell-free supernatant was collected from pDC exposed to CSE 1% after 24 h of culture and tested for different cytokines by a multiplex bead assay. Bar graphs represent cytokine concentrations of untreated (open bars) and CSE-exposed (black bars) cells. Data show mean ± SEM (n = 5 donors). ⁄P < 0.05. ns = no significant.

inhibited by the addition of CSE to the culture. To further assess the effect of CSE on the transcriptional activation of IRF-7, we determined the phosphorylation of IRF-7 (p-IRF-7) using specific antibody and analyzed by flow cytometry. Due to the sensitivity of the assay, pDC were isolated by negative selection to prevent cell activation previous to the addition of the stimulus. Cells were labeled for CD123 and BDCA2 surface markers in combination with intracellular staining with anti-human IRF-7 fluorescent antibody (IRF-7 pS477/pS479). As shown in the histograms and in the mean fluorescence intensity (MFI) bar graph in Fig. 5b, RSV induced the phosphorylation of IRF-7 and the presence of CSE abolished that induction to values similar to untreated control cells. Based on the above results, the interactions between CSE, RSV and IFN-a production in human pDC are summarized in Fig. 6. 4. Discussion Accumulating evidence supports that cigarette smoke alters several functions of human dendritic cells. It is currently unknown whether cigarette smoke is capable of inhibiting IFN-a production in human pDC in response to respiratory viral infections. The experiments presented in this work were therefore designed to answer this question. In the present study, we found that exposure to CSE impaired the capacity of human pDC to produce IFN-a and other cytokines after RSV infection. IFN-a is induced in human pDC upon viral infection and act on as yet uninfected cells to activate a global antiviral state. In addition, we have previously demonstrated that IFN-a plays a fundamental role in RSV infection both in an experimental mouse model (Guerrero-Plata et al., 2005) and in vitro in human dendritic cells (Guerrero-Plata et al.,

2006). The inhibition of IFN-a by CSE was dose-dependent as the decrease of the production of this cytokine in pDC was directly proportional to the concentration of CSE added to the culture. However, nonlethal concentration of 1% CSE significantly inhibited RSV-induced IFN-a release. These findings are in accordance with those recently reported by Mortaz and colleagues, where human pDC treated with 1.5% CSE, had a reduced capacity to produce IFN-a in response to synthetic ODN-CpG (Mortaz et al., 2009). However, in primary epithelial cells infected with RSV, it has been observed that CSE had no effect on type I IFN and interferon-inducible genes (Groskreutz et al., 2009) suggesting that cigarette smoke alters different activation pathways perhaps attributed to cell type specificity and/or the characteristics of the stimulus. In order to exclude that the decreased production of RSV-induced IFN-a in pDC was due to cell death by the effect of CSE, the toxicity of CSE on human pDC viability was evaluated. We observed that CSE decreased pDC viability in a dose-dependent manner. When compared to 0%, concentrations of 0.1% and 1% of CSE induced less than 16% mortality in pDC cultures, while concentrations of 2% and 4% resulted in considerable cell death >30%. Therefore, all parameters measured here were evaluated in the presence of the concentration of 1% CSE. Our findings are comparable to those reported in an earlier study where pDC exposed to 1.5% CSE decreased cell viability by 10% after 8 h of exposure (Mortaz et al., 2009). However, the small difference in the level of CSE toxicity in pDC is due perhaps to the length of time of CSE exposure since in our system virus-infected pDC were cultured for longer period of time (24 h). Cytokines and chemokines are important mediators that orchestrate inflammatory and immunologic effector mechanisms.

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Upon viral infection, pDC rapidly produce the pro-inflammatory cytokines TNF-a and IL-6 (Fitzgerald-Bocarsly et al., 2008) as well as IL-10 (Guerrero-Plata et al., 2006; Parcina et al., 2009). pDC also produce chemokines in response to virus stimulation, including CCL2, CCL3, CCL5, CXCL10 and CXCL8 (Banchereau et al., 2000; Pulendran et al., 2001). Data shown in Fig. 2, indicate that RSV induced the production of IL-1b, IL-10, CXCL10, IL-6 and TNF-a in pDC, but not that of CCL2, CCL3, CCL5 and CXCL8. However, we demonstrate that CSE decreased the release of IL-1b, a proinflammatory cytokine that plays an important role in the host defense against viral infections (Dinarello, 2002). Our data are in accordance with those reported in vitro using PBMC (Ouyang et al., 2000) and ex vivo using alveolar macrophages (Brown et al., 1989; Yamaguchi et al., 1989) where IL-1b release was suppressed

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Fig. 5. RSV-induced IRF-7 phosphorylation is inhibited by CSE. (a) Total RNA was isolated from pDC infected with RSV in the presence of 1% CSE. IRF-7 gene expression was determined by qRT-PCR and reported as fold changes over control pDC. Each value represents mean ± SEM of n = 4 donors. (b) IRF-7 phosphorylation was determined by intracellular staining of IRF-7(pS477/pS479) in pDC cultured under different conditions. Expression of IRF-7 was determined in gated pDC population identified by the expression of BDCA2 and CD123. pDC from untreated (gray shaded histogram), RSV-(open histogram) and RSV + CSE (dotted line histogram) are shown. IRF-7 phosphorylation was also quantified in mean fluorescent intensity (MFI) values as displayed in the bar graph. Data shown represent mean ± SEM of n = 3 donors. ⁄P < 0.05, ⁄⁄P < 0.01.

by the effect of cigarette smoke. The production of IL-10 was also decreased by the effect of cigarette smoke exposure in RSV-infected pDC. IL-10 is an immunoregulatory cytokine that inhibits proinflammatory responses from innate and adaptive immunity and plays a central role during the resolution phase of inflammation (Ouyang et al., 2010). In support to our findings, other studies

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Fig. 3. CSE inhibits TLR response in human pDC. (a) pDC were stimulated with either TLR7 agonist loxoribine (500 lM) or TLR9 agonist ODN-CpG (1 lg/ml) in the presence of 1% CSE for 24 h. IFN-a was measured in the cell-free supernatants by ELISA. Values (n = 3 donors) represent mean ± SEM. Total RNA was isolated from TLR-activated pDC in a two-step process, RNA was first converted to cDNA and amplified gene transcripts were measured by qRT-PCR. (b) TLR7 and (c) TLR9 expression were determined. Target gene expression was normalized to 18S gene expression and reported as fold changes over control pDC (untreated). Values (n = 3) represent mean ± SEM. ⁄P < 0.05, ⁄⁄P < 0.01.

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Fig. 4. Cigarette smoke inhibits RSV-induced TLR7 expression in pDC. pDC infected with RSV in the presence of CSE 1% were cultured for 24 h. Total RNA was isolated, converted to cDNA and amplified gene transcripts were measured by qRT-PCR. (a) TLR7 and (b) TLR9 gene expression was normalized to 18S gene expression and reported as fold changes over control pDC (untreated). Bar graph represent mean ± SEM. n = 4 donors ⁄⁄P < 0.01.

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RSV

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Fig. 6. Schematic representation of the effect of cigarette smoke on RSV-infected pDC. RSV products such as viral RNA (vRNA) or virus intermediate (iRNA) may access TLR containing endosomes via the process of autophagy. In the endosomes cigarette smoke can prevent that these nucleic acids trigger the activation of TLR7 and lead to the recruitment of MyD88 and eventually to the phosphorylation of IRF7. If IRF7 is not phosphorylated, it cannot translocate into the nucleus where it initiates the transcription of the type I IFN genes. Cigarette smoke may also alter the activation of TLR7 upon trafficking into late endosomes/lysosomes that leads to the activation of nuclear factor (NF)jB which, thus preventing the production of inflammatory cytokines such as IL-1b. Since the effect of cigarette smoke on cytokine profile produced by RSV-infected pDC was not generalized, we cannot discard the possibility that RSV activates additional pathways in the cytosol that could compensate the production of those cytokines that were induced by RSV infection but not altered by the effect cigarette smoke exposure.

have reported an association between tobacco smoke and a diminished IL-10 production in infants (Gentile et al., 2004) and monkeys (Wang et al., 2008). We also observed that RSV-induced CXCL10 in pDC was decreased by the effect of CSE. CXCL10 (also called interferon-c-inducible protein 10, IP-10) regulates immune responses and inflammation by activation and recruitment of T cells, monocytes, eosinophils and NK cells (Lee et al., 2009). Reduced production of CXCL10 by CSE has also been reported in bronchial epithelial cells (16-HBE) stimulated with LPS (Pace et al., 2008). Interestingly, it has been shown that virus-induced IFN-a acts to induce CXCL10 production by pDC (Megjugorac et al., 2004). Although not addressed in this work, there is the possibility that the reduced release of CXCL10 by CSE in RSV-infected pDC could be associated to the production of IFN-a, also downregulated by the effect of CSE. Other studies in pDC stimulated with synthetic ODN-CpG differ from the current report since CSE did not have any effect on IL-1b, IL-10 and CXCL10 release (Mortaz et al., 2009). This discrepancy may be related to the different experimental conditions. In particular, the different type of stimulus and the culture conditions of the in vitro systems. TLRs are evolutionarily conserved innate receptors expressed in pDC and many other mammalian cells. TLRs play a crucial role in defending against pathogenic microbial infection through the induction of inflammatory cytokines and type I IFN. The TLR repertoire of human pDC is composed of TLR7 and TLR9, both located in the endosomal membrane. TLR7 recognizes viral single-stranded RNA (Diebold et al., 2004; Heil et al., 2004) as well as imidazoquinolines such as imiquimod and resiquimod (R848) and guanosine analogs (Akira and Takeda, 2004). In contrast, TLR9 recognizes bac-

terial or viral DNA (Akira and Takeda, 2004), including synthetic CpG-ODN (Hartmann et al., 1999). In this work we observed that CSE decreased the production of IFN-a and the expression of TLR7 and TLR9 in pDC stimulated with TLR agonists. Our findings are in agreement with previous findings reported by Mortaz et al. where CSE decreased the production of IFN-a in pDC stimulated with ODN-CpG (Mortaz et al., 2009). Viruses induce IFN-a production in human pDC through activation of TLR7/9 signaling pathway (Cao and Liu, 2007). RSV is a single stranded RNA virus that induces a robust amount of IFN-a in human pDC (GuerreroPlata et al., 2006; Hornung et al., 2004) and therefore likely to activate the production of IFN-a through the activation of TLR7 pathway. However, no current information is available that substantiate the role of TLR7 in RSV-infected pDC. Based on the RSV-induced upregulation of TLR7 expression, the current data suggest that RSV induces IFN-a by the activation of the TLR7 signaling pathway. However, the molecular mechanism(s) by which RSV induces IFN-a in human pDC needs further characterization beyond the scope of this work. Given the complex regulation of IFN-a/b gene, RSV infection could activate or interfere (GuerreroPlata et al., 2005, 2006, 2009; Schlender et al., 2005) with one, multiple, and in a virus- or cell-specific manner of the signal transduction pathways leading to IFN-a/b gene transcription. A novelty of the current study lies in the finding that cigarette smoke inhibits TLR7 expression in pDC. Similarly, the suppressive effect of cigarette smoke exposure on the expression or activation of other TLRs such as TLR2, TLR4 and TLR9 has been previously reported in different cell types including pDC (Mortaz et al., 2009) and macrophages (Droemann et al., 2005; Sarir et al., 2009). Opposite

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results, however, have been reported in epithelial cells exposed to cigarette smoke where TLR4 expression was rather upregulated (Pace et al., 2008). It is not clear whether this discrepancy is due to inherent differences of the cell type or the conditions of cigarette smoke exposure. The stimulation of TLRs activates multiple downstream intracellular signaling molecules that regulate the induction of IFN-a. IFN-a stimulates infected cells in autocrine or paracrine manner to upregulate the expression of IRF-7, which in turn is phosphorylated and translocated to the nucleus, leading to the full expression of the IFN-a genes (Kumar et al., 2009). Our data indicate that RSV infection in pDC induced IRF-7 phosphorylation and that CSE inhibits that effect. IRF-7 activation has been reported to be specific for the IFN-a pathway in pDC but not of that of other cytokines such as IL-6 or IL-12 (Honda et al., 2005). This may explain in part why the production of proinflammatory cytokines showed in the present study such as IL-6 were unaltered by the effect of CSE while a striking reduction of IFN-a release was observed. However, we cannot exclude that additional pathway(s) are activated by the virus since the production of cytokines/chemokines in pDC was differentially affected by CSE. Current work is in progress to determine the effect of CSE in NF-jB pathway in human DC subsets including pDC. In summary, the current study demonstrates that CSE decreased the expression of specific receptors in the innate immune system such as TLR7, reduced the activation of IRF-7 and the production of IFN-a, IL-1b, IL-10 and CXCL10 during RSV infection. Decreased levels of IFN-a may lead to a compromised anti-viral response and likely to increase the susceptibility and pathogenesis of respiratory viral infections while the reduced levels of the other cytokines may compromise the specific response to the infection as well as the regulation of the inflammatory response in the lung. While there remain considerable gaps in our knowledge of the complex interaction between viruses, immune cells and the host environment, the current observations may suggest a potential mechanism by which ETS exposure contributes to the severity of RSV-induced bronchiolitis and LTRI. More studies are needed to better understand the cellular and molecular mechanisms of cigarette smoke-induced lung disease and its role in exacerbated respiratory viral infections. Acknowledgments This work was supported by the Flight Attendant Medical Research Institute (FAMRI) Young Clinical Scientist Award to A. G-P. References Akira, S., Takeda, K., 2004. Toll-like receptor signalling. Nat. Rev. Immunol. 4, 499– 511. Arcavi, L., Benowitz, N.L., 2004. Cigarette smoking and infection. Arch. Intern. Med. 164, 2206–2216. Banchereau, J., Briere, F., Caux, C., Davoust, J., Lebecque, S., Liu, Y.T., Pulendran, B., Palucka, K., 2000. Immunobiology of dendritic cells. Annu. Rev. Immunol. 18, 767–811. Bauman, L.J., Wright, E., Leickly, F.E., Crain, E., Kruszon-Moran, D., Wade, S.L., Visness, C.M., 2002. Relationship of adherence to pediatric asthma morbidity among inner-city children. Pediatrics 110, e6. Blue, M.L., Janoff, A., 1978. Possible mechanisms of emphysema in cigarette smokers. Release of elastase from human polymorphonuclear leukocytes by cigarette smoke condensate in vitro. Am. Rev. Respir. Dis. 117, 317–325. Bradley, J.P., Bacharier, L.B., Bonfiglio, J., Schechtman, K.B., Strunk, R., Storch, G., Castro, M., 2005. Severity of respiratory syncytial virus bronchiolitis is affected by cigarette smoke exposure and atopy. Pediatrics 115, e7–e14. Brown, G.P., Iwamoto, G.K., Monick, M.M., Hunninghake, G.W., 1989. Cigarette smoking decreases interleukin 1 release by human alveolar macrophages. Am. J. Physiol. 256, C260–C264. Cao, W., Liu, Y.J., 2007. Innate immune functions of plasmacytoid dendritic cells. Curr. Opin. Immunol. 19, 24–30. Castro, S.M., Kolli, D., Guerrero-Plata, A., Garofalo, R.P., Casola, A., 2008. Cigarette smoke condensate enhances respiratory syncytial virus-induced chemokine

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