We showed that a down-regulated TLR4 expression in the lung can be a predictor or biomarker of emphysema in smokers or biomarker of emphysema, as patients with down-regulated TLR4 expression had a greater probability of developing emphysema and airflow limitation. Differences in TLR4 expression were attributable mainly to expression levels in bronchial and alveolar epithelial cells. Smoke-induced emphysema itself did not appear to alter TLR4 expression; therefore TLR4 expression seems to affect emphysema development or progression, rather than vice versa.
Several studies have shown that defects in innate TLR4-mediated immunity can be associated with emphysema. TLR4 knockout causes spontaneous emphysema in animals
. The presence of the TLR4-T339I polymorphism is associated with a 2.4-fold increased risk for developing COPD, highlighting the relationship between impaired innate immunity and COPD development
. The Gly299 allele is present at a decreased frequency among patients with COPD and may be absent from COPD patients who have never smoked
. Our findings can make an important contribution on the relationship between innate immunity and COPD, because they show that down-regulated TLR4 expression in the lung may be related to emphysema in humans, as in animals.
An association between TLR4 expression and COPD has also been noted previously. Most studies have suggested that TLR4 expression is decreased in the blood of patients with COPD. The peripheral blood TH1 cell response to lipopolysaccharide (LPS) is impaired in patients with COPD compared with the response in never-smokers, and TLR4 overexpression via transfection restores this impairment
. Compared with less severe disease, severe COPD is associated with reduced TLR4 expression in the nasal epithelium
. In contrast to some positive results in blood, cells in a sputum analysis failed to show this relationship. TLR4 expression on sputum neutrophils was not different in COPD patients
, and TLR4 mRNA in induced sputum did not differ significantly between COPD patients and healthy controls
. A previous study showed no significant difference in TLR4 expression in epithelial cells between COPD and normal subjects, but several factors such as smoking history, age and sex were not controlled and small number subjects limited the power of test
. This study is meaningful because we compare the TLR4 expression among smokers. Previous studies compare the expression between smoker COPD and never-smoker controls
Theoretically, TLR4 deficiency may partially protect against smoke-induced emphysema, because TLR4 deficiency partially prevents smoke-induced influx of dendritic cells, lymphocytes, and neutrophils
. Clear correlations have been found between the numbers of these inflammatory cells and the severity of COPD
[34, 35]. However, previous studies and our study have shown that TLR4 deficiency does not protect against the development of emphysema. To the contrary, it appears to be related with emphysema development, which can be explained by the role of TLR4 in the respiratory system. In TLR4 deficient mouse, NADPH oxidase was up-regulated in lungs, resulting in increased oxidant generation and elastolytic activity
. Expression of the autophagic protein LC3B and markers of cell death in response to cigarette smoke exposure were increased in epithelial cells from TLR4 deficient mice
. Overall, TLR4 exerts a protective role with respect to smoke-induced emphysema development. It is not still evident whether the same mechanism is applied to human and further study is necessary. TLR4 expression seemed associated with emphysema severity which was reported to be more correlated with airflow limitation (FEV1/FVC) than COPD stage defined by FEV1 (% predicted)
Several TLR4-dependent mechanisms are likely to be involved in cigarette smoke-induced pulmonary inflammation. Smoke may activate TLR4 signaling on pulmonary epithelial cells and transmigrated resident cells such as macrophages, which act as the first line of defense against external threats
[16, 37]. Innate immunity mediated by TLR4 also triggers the first-line host defense response to Gram-negative bacterial infections and is crucial for initiating subsequent T cell-mediated adaptive immune responses
. TLR4 expression on the respiratory epithelium allows for rapid activation of host defenses against outside stimuli such as smoke and bacteria, resulting in the induction of inflammatory mediators and antimicrobial peptides. The increased risk for bacterial infection in patients with COPD may be caused by an inability to effectively clear bacteria and by misguided inflammatory responses. Emphysema develops due to chronic inflammation and impaired matrix and cellular repair. Thus, an impaired defense owing to TLR4 deficiency combined with repeated inflammation may result in the development of emphysema. However, it is unclear whether the reduced TLR4 expression in patients with COPD is an adaptive response to increased exposure to external threats such as Gram-negative bacteria or smoke, as part of the phenomenon of endotoxin tolerance.
Several limitations of this study should be noted. First, the causal relationship between down-regulated TLR4 expression and emphysema is ambiguous. To make a firm conclusion, smokers should be followed for several decades after obtaining lung tissues or respiratory cells; however, such studies are not practical and present ethical issues. Similar experiment using other tissues or blood-born cells could have clarified this issue more, but we could not because they were not available. However, our data showed that smoke-induced emphysema itself did not affect TLR4 expression, providing more evidence that down-regulated TLR4 expression results in emphysema. Previous reports showing that TLR4 knockdown induces spontaneous emphysema also support this idea
. Second, TLR4 expression can be induced or decreased by LPS stimulation
[29, 38]. Thus, TLR4 expression levels can vary with different clinical conditions such as Gram-negative bacterial infections. In our study, lung tissues were taken during elective lung resection, when patients were clinically stable without evidence of pulmonary infection or exacerbation. Third, we could not find a causable gene that could explain the differential TLR4 expression. Although we examined several single-nucleotide polymorphisms in the TLR4 gene, including minor alleles with frequencies >5% in the HAP map of Asians, no significant results were identified (data not shown). Epigenetic factors may be associated with the differential expression of TLR4. Fourth, functional aspects by down-regulation of TLR4 expression, such as cytokine secretion, could not be evaluated and because of the retrospective nature of this study.