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

Background: Cigarette smoking is associated with increased frequency and duration of viral respiratory infections, but the underlying mechanisms are incompletely defined. We investigated whether smoking reduces expression by human lung macrophages (Mo) of receptors for viral nucleic acids and, if so, the effect on CXCL10 production. Methods: We collected alveolar macrophages (AMo) by bronchoalveolar lavage of radiographically-normal lungs of subjects undergoing bronchoscopies for solitary nodules (n= 16) and of volunteers who were current or former smokers (n= 7) or never-smokers (n= 13). We measured expression of mRNA transcripts for viral nucleic acid receptors by real-time PCR in those AMo and in the human Mo cell line THP-1 following phorbol myristate acetate/ vitamin D3 differentiation and exposure to cigarette smoke extract, and determined TLR3 protein expression using flow cytometry and immunohistochemistry. We also used flow cytometry to examine TLR3 expression in total lung Mo from subjects undergoing clinically-indicated lung resections (n= 25). Of these, seven had normal FEV1 and FEV1/FVC ratio (three former smokers, four current smokers); the remaining 18 subjects (14 former smokers; four current smokers) had COPD of GOLD stages I-IV. We measured AMo production of CXCL10 in response to stimulation with the dsRNA analogue poly(I:C) using Luminex assay. Results: Relative to AMo of never-smokers, AMo of smokers demonstrated reduced protein expression of TLR3 and decreased mRNA for TLR3 but not TLR7, TLR8, TLR9, RIG-I, MDA-5 or PKR. Identical changes in TLR3 gene expression were induced in differentiated THP-1 cells exposed to cigarette smoke-extract in vitro for 4 hours. Among total lung Mo, the percentage of TLR3-positive cells correlated inversely with active smoking but not with COPD diagnosis, FEV1% predicted, sex, age or pack-years. Compared to AMo of never-smokers, poly(I:C)-stimulated production of CXCL10 was significantly reduced in AMo of smokers. Conclusions: Active smoking, independent of COPD stage or smoking duration, reduces both the percent of human lung Mo expressing TLR3, and dsRNA-induced CXCL10 production, without altering other endosomal or cytoplasmic receptors for microbial nucleic acids. This effect provides one possible mechanism for increased frequency and duration of viral lower respiratory tract infections in smokers.