Smoking decreases the response of human lung macrophages to double-stranded RNA by reducing TLR3 expression

Studies and consent procedures were performed in accordance with the Declaration of Helsinki at the VA Ann Arbor Healthcare System (all bronchoscopies and some clinically-indicated lung resections) and the University of Michigan Health System (some clinically-indicated lung resections, including all lung transplants and lung volume reduction surgeries) and were approved by their Institutional Review Boards (FWA 00000348 & FWA 00004969, respectively). All subjects understood the purpose of the study and gave written consent before any research procedures. All subjects underwent a complete history and physical examination by a Pulmonologist, spirometry, chest imaging, prospective collection of medication history, and complete blood count with differential, coagulation studies and chemistry panel.

We recruited 13 healthy never-smoker volunteers and 23 subjects with a smoking history who either were scheduled to undergo bronchoscopy in the evaluation of solitary pulmonary nodules (n = 16) or who volunteered (n = 7). Among the 23 subjects who had ever smoked, 18 were active smokers (<six months since quitting) and 5 were former smokers (>six months since quitting). Of those with any smoking history, subjects (n =10) with ≥10 pack years, a ratio of forced expiratory volume in 1 second to forced vital capacity (FEV₁/FVC) >0.7, normal spirometry, and no clinical diagnosis of COPD represent control smokers. Subjects (n =13) with a smoking history, FEV₁/FVC <0.7 and abnormal spirometry were considered to have COPD. All subjects were without evidence of lung infection, interstitial lung disease or collagen vascular disease.

Importantly, not all types of experiments were performed on cells from every subject in this cohort, and conversely, some subjects were used for more than one type of experiment. The characteristics of the BAL subjects used in each type of experiments are summarized in Tables in the Results sections; demographic and clinical data for this entire cohort are shown in Additional file 1: Table S1.

Lung tissue was collected from consented subjects undergoing clinically-indicated resections for pulmonary nodules, lung volume reduction surgery, or lung transplantation (n = 25). Using the same definitions as in the BAL cohort, this cohort comprised seven control smokers and 18 subjects with COPD. This cohort was used exclusively for flow cytometric analysis of total lung Mø. Characteristics of this cohort are summarized in the Results section, and demographic and clinical data for individual subjects are shown in Additional file 2: Table S2.

BAL was performed in the right middle lobe and lingula of the volunteers and in whichever of these sites was contralateral to the nodule in the clinically-indicated bronchoscopies, in which the research BAL was performed as early as feasible, and always before any biopsies or brushings. We instilled 100 ml normal saline per site, using a 30 ml syringe and gentle manual suction. BAL fluid was filtered through sterile gauze to remove mucous, and cells were washed thrice with PBS, with centrifugation between washes.

BAL cells (>95% AMø by Wright-Giemsa-stained cytospins) were either immediately processed for flow cytometry and immunohistochemistry or were cultured briefly to purify AMø by adherence. For this purpose, cells were resuspended in complete medium (RPMI 1640 containing 25 mM HEPES, 2 mM l-glutamine, 1 mM pyruvate, 100 U/ml penicillin/streptomycin, 10% heat-inactivated AB human serum (all from Invitrogen, Carlsbad, CA), and 55 μM 2-ME (Sigma Chemical, St. Louis, MO), and were plated at 2 × 10⁵ cells/well in sterile 24-well plates. Cells were incubated for 1.5 h in 5% CO₂ at 37°C, washed to remove nonadherent cells, then incubated in AIM-V serum-free medium (Invitrogen) under experimental conditions. For TLR3 stimulation, adherence-purified AMø were cultured in AIM-V alone or with poly(I:C) at 50 μg/ml for 24 h.

Lung tissue was dissected free of any areas containing nodules, cancers or evidence of infection by a Pathologist before being released to the study. Lung sections weighing approximately 3 g were dispersed using a Waring blender in a biosafety cabinet without enzyme treatments, which we have previously shown produces single cell suspensions of high viability and functional capacity [27, 28]. Cells were filtered through a 40 μm strainer to remove debris and were resuspended in staining buffer (2% FBS in PBS) for flow cytometery.

THP-1 cells obtained from ATCC (Manassas, VA) were grown according to the supplier’s guidelines. Cells were plated at 4 × 10⁵ cells/well in 24-well plates, and after adherence, were differentiated by exposure to 150 ng/ml phorbol myristate acetate (PMA) and 0.01 μM vitamin D3 for 48 hrs, and then were washed with fresh complete medium before exposure to various concentrations of cigarette smoke-extract (CSE) for 4 hr. Fresh CSE was prepared by bubbling the mainstream smoke of two University of Kentucky Tobacco Health Research Institute standardized cigarettes (lot 2R4F), using a Jaeger-Baumgartner Cigarette Smoking Machine (C.H. Technologies, New Jersey) driven by dry compressed air, into 20 ml RPMI 1640; the result was defined as 10% CSE. CSE was filter-sterilized using a 0.22 μm membrane and was used the same day.

RNA was isolated using RiboPure kits (Applied Biosystems, Foster City, CA), TURBO DNA-free (Applied Biosystems) to remove genomic DNA, and Retroscript kits (Applied Biosystems) for reverse-transcription. We performed quantitative real-time PCR using the Stratagene Mx3000P (LaJolla, CA), with human glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as the endogenous reference, and commercial primer-probe sets (Taqman chemistry, Applied Biosystems). Transcript levels were reported relative to a calibrator of unstimulated THP cells, or AMø from never smokers, as appropriate, as calculated on the thermocycler.

BAL cells were resuspended in 100 μL staining buffer {1% FA Buffer (BD Bioscience, San Jose, CA), 1% FCS, 0.01% sodium azide} per tube. We used antibodies against the following antigens (clones in parentheses): CD45 (2D1) (eBioscience, San Diego, CA), TLR3 (40C1285.6), TLR7 (polyclonal), TLR9 (26C593.2) (Imgenex Corp., San Diego, CA), and TLR8 (303F1.14) (Dendritics, Lyon, France). To detect intracellular receptor expression, cells were treated with Fixation and Permeabilization Buffers (eBioscience). In all experiments, we used isotype-matched controls, analyzed cells on an LSR II flow cytometer (BD Bioscience), and collected a minimum of 10,000 CD45+ events per sample, using FACS Diva software with automatic compensation, and FlowJo analysis software (Tree Star, Inc., Ashland, OR). Details of instrument setup have been described recently [29].

Cytospins were immersed in cold acetone followed by methanol plus hydrogen peroxide. The slides were stained with biotinylated anti-human TLR3 (Imgenex) or isotype control antibodies, followed by Universal ABC peroxidase complex and 3-amino-9-ethylcarbazole peroxidase substrate. Slides were counter-stained with hematoxylin and photographed using an Olympus BX51 digital camera.

CXCL10 levels were determined using Biosource Multiplex Assays (Invitrogen) and a Luminex 200™ (Luminex Corporation, Austin, TX).

To test whether cigarette smoking impacts expression by human AMø of TLRs implicated in defense against respiratory viruses, we first analyzed adherence-purified AMø obtained by BAL of 11 current or former smoking subjects and six never-smokers (Table 1) using quantitative real-time PCR. Results showed significantly decreased TLR3 mRNA transcripts in smokers compared with never-smokers (p = 0.0015; Mann–Whitney test) (Figure 1A), but no significant differences in transcripts for TLR7, TLR8 or TLR9, which are also found in the endosome (Figure 1A). We also found no significant differences between smokers and never-smokers in mRNA transcripts for the cytoplasmic dsRNA receptors RIG-I, MDA-5 and PKR (Additional file 3: Figure S1).

These findings were supported by two measures of AMø protein expression. Flow cytometry (Additional file 4: Figure S2) permitted identification of specific staining for TLR3 and TLR9 amongst AMø of never-smokers, although expression of TLR7 was very low (Additional file 4: Figure S2, middle panel) in both groups, and was not analyzed further. Comparing BAL samples from smokers (n = 13), and never-smokers (n = 10) (Table 2), we found a significantly decreased percentage (p < 0.0001, Mann–Whitney test) of AMø positive for intracellular TLR3 in AMø of smokers (Figure 1B). Note that these experiments contain some of the same subjects studied in Table 1, as well as other BAL subjects, as described in Methods; for additional details about individual subjects, see Additional file 1: Table S1). TLR3 was not detected on the surface of unpermeabilized AMø of either group (not shown). There were no differences between smokers (n = 6) and never-smokers (n = 5) in the percentage of AMø expressing intracellular TLR9 (Figure 1B) (or TLR7 and TLR8, data not shown). Similarly, immunohistochemical staining of BAL cytospins (Additional file 1: Table S1) demonstrated greatly reduced TLR3 expression in the AMø from smokers, relative to AMø from the never-smokers (Figure 1C). These independent data indicate that reduction in TLR3 protein expression measured by flow cytometry did not result simply from greater autofluorescence in AMø of smokers.

In univariate analyses including both smokers and never-smokers, TLR3 RNA transcripts correlated inversely with both FEV₁ % predicted and subject age (Figure 2A-B). Considering only those with a history of smoking, there was no correlation with pack-years (Figure 2C). However, in a linear regression, none of the variables (smoker versus never-smoker, age, sex, FEV₁ % predicted) reached statistical significance for mRNA transcripts. AMø positivity for TLR3 protein did not correlate in univariate analyses with FEV₁ % predicted (Figure 2D) or pack-years of smoking exposure (Figure 2F), but did correlate with subject age (Figure 2E). In a linear regression, smoking status (smoker vs. never-smoker) was strongly associated with TLR3+ AMø (p < 0.0001) and subject age was also significant (p = 0.035), but sex and FEV₁ % predicted remained insignificant. When we analyzed smokers with COPD versus smokers without COPD, no significant differences were seen in TLR3 mRNA transcripts (COPD, 0.23 ± 0.16 vs. no COPD, 0.49 ± 0.49, mean ± SEM dRn; p = 0.52, unpaired t test) or flow cytometric results (COPD, 12.2 ± 12.2 vs. non-COPD, 10.4 ± 5.2; mean ± SEM % TLR3-positive AMø, p = 0.87, unpaired t test). Despite the use of inhaled corticosteroids (ICS) by ~50% of the smoking subjects, the decrease in the fraction of TLR3+ AMø of smokers was not a result of steroid usage (p = 0.79, determined by t-test comparing TLR3 mRNA transcript expression between ICS users and non-users; p = 0.92 when comparing TLR3 protein measurements by flow cytometry).

Thus, these secondary analyses supported a significant effect of smoking on AMø expression of TLR3. However, due to the distribution of our subjects, we were unable to exclude the possibility that our findings resulted from the preponderance of women in the never-smoker group and of men among the smokers in this cohort, or from the significant difference in ages between the volunteer and clinical subjects.

In apparent contradiction to our findings, a recent paper primarily using immunohistochemistry in lung tissue sections reported that the percentage of TLR3-positive lung Mø were significantly increased in smokers compared with never-smokers [30]. To address this discrepency and the questions about the possible effects of age and sex in our BAL subjects, we studied a different cohort of subjects (n = 25) undergoing clinically-indicated lung resection procedures (Table 3). This cohort also had the advantage of having more nearly balanced ratios of male and female subjects, as well as a wide range of spirometry values. We used mechanical disaggregation of tumor-free lung parenchyma to produce a single cell suspension of high viability containing both AMø and interstitial lung Mø, which we identified as autofluorescent, CD45+, high side scatter cells.

Flow cytometric analysis permitted objective quantification of the percentage of individual lung Møs positive for intracellular TLR3, relative to simultaneously analyzed isotype-control monoclonal antibodies (Figure 3A, B). We found the percentage of TLR3-positive lung Møs from current smokers was significantly reduced (p = 0.006) compared to the percentage of TLR3-positive lung Møs from former smokers (Figure 3C). By contrast, considering these same individuals, there were no significant differences in percentages of lung Mø expressing TLR3 between those with normal pulmonary function and those with COPD (Figure 3D). Nor was there a correlation between the percentage of TLR3-positive lung Mø by flow cytometry and either FEV1 % predicted or duration of smoking in pack-years (Additional file 5: Figures S3A & B). In a linear regression, smoking status (current vs. former) was significantly associated with TLR3 positivity (p = 0.016) and was not affected by age, FEV_{1 %} predicted, sex, or pack-years. These independent data agree with and extend results of our BAL experiments and collectively show that active smoking reduces TLR3 expression by resident human lung Mø.

To explore the effect of acute smoke-exposure in vitro, we used the well-characterized system of differentiating the human Mø cell line THP-1 by treatment with PMA plus vitamin D3. In four independent experiments, we found that CSE at concentrations of 1.25% and 2.5% significantly decreased expression of TLR3 transcripts (Figure 4A), but had no effect on transcripts of TLR7, TLR8 or TLR9 (Figures 4B-D). CSE at these concentrations had no effect on viability as assessed by trypan blue exclusion (not shown). CSE also had no significant effect on mRNA transcripts for RIG-I, MDA-5 or PKR at concentrations up to 2.5% CSE (Figure 4E-G). Thus, the effect of smoke exposure on TLR3 mRNA levels can be induced within as little as four hours in THP-1 cells differentiated to a mature Mø phenotype. These findings indicate that smoking reduces Mø TLR3 expression directly, and not secondarily due to an effect on another lung cell type or on the lung microbiome.

Finally, we investigated whether decreased TLR3 expression on the AMø of smokers would affect CXCL10 production following poly(I:C) stimulation. These experiments were performed using AMø from subjects in the BAL cohort (Additional file 2: Table S2). CXCL10 concentrations were close to the level of detection in unstimulated cells from both smokers (n = 5) and never-smokers (n = 4) (Table 4) and did not differ significantly (Figure 5). Following poly(I:C) stimulation, AMø from never-smokers showed a 1000-fold increase in CXCL10 production, and differed significantly from the response of AMø from smokers, which showed only a 10-fold increase from unstimulated levels (Figure 5). These results illustrate a functional consequence of the difference in TLR3 expression between the two groups.

Supported by R01 HL082480 (JLC, FJM), R01 HL056309 (JLC) and U01 HL098961 (JMB, JLC) from the USPHS; a Career Development Award (CMF) and a Merit Review Award (CMF) and a Research Enhancement Award Program (SWC, JMB, JLC) from the Biomedical Laboratory Research & Development Service, Department of Veterans Affairs. These investigations were also supported in part by the Tissue Procurement Core of the University of Michigan Comprehensive Cancer Center, Grant P30 CA46952, and by the Lung Tissue Research Consortium (Clinical Centers), Grant N01 HR046162.

Current e-mail addresses: James M. Beck, M.D.: http://​james.​beck@denver.​edu.