The principal results of this analysis of TLR molecules on human lung T cells, NK cells and NKT cells demonstrate for the first time several observations relevant to COPD pathogenesis. First, expression of multiple TLRs on lung CD8+ T cells (but not on simultaneously analyzed lung CD4+ T cells, NK cells or NKT cells) was increased in COPD patients relative to smokers with preserved lung function. Second, TLR2/1 molecules expressed by lung CD8+ T cells were functional, as shown by the markedly enhanced production of cytokines linked to lung inflammation and COPD progression following co-stimulation by the TLR2/1 agonist in vitro. Third, expression by lung CD8+ T cells of TLR 2/1 and TLR5 correlated significantly with quantitative emphysema scores in univariate analysis. Collectively, these findings strengthen the evidence linking lung CD8+ T cells to smoking-induced lung destruction in susceptible cigarette smokers.
The finding that an increased percentage of lung CD8+ T cells express TLR1, TLR2, TLR2/1, TLR4 and TLR6 in COPD subjects extends to human lung parenchyma and to a specific disease state the results of previous studies of peripheral blood from healthy humans
[16, 19, 20, 26, 27]. Although many TLRs have multiple ligands, it is noteworthy that increased expression was seen in the cell surface receptors most strongly associated with bacterial recognition (TLR1, TLR2, TLR2/1, TLR4 and TLR6), and not in the endosomal receptors TLR3 or TLR9, which recognize viruses and endogenous danger signals
. Furthermore, the very strong correlation between TLR5 or TLR2/1 and emphysema severity suggests that TLRs on lung CD8+ T cells may be specifically contributing to the pathogenesis of emphysema and highlights the importance of defining specific COPD phenotypes in selecting patients for personalized therapies
. Additional experiments will be needed to determine whether the co-stimulatory effect of TLR ligation is additive or synergistic when multiple TLRs are stimulated simultaneously, as would plausibly occur during exacerbations of COPD. Neither the presence of lung cancer nor recent infections appeared to contribute to the difference in TLR expression that we observed between healthy subjects and COPD subjects. Little is known about the effect of glucocorticoids on TLRs, but it has been shown that dexamethasone, in combination with IFN-γ or TNF-α, was able to synergistically enhance TLR2 expression on respiratory epithelial cells
; however in a separate study, dexamethasone was shown to downregulate TLR4 expression in an airway epithelial cell line
. The effect of steroids on TLRs has not been investigated in T cells. In our study, we saw no difference in TLR expression when ICS use was evaluated.
Although we also detected expression of multiple TLRs on lung CD4+ T cells, the near complete lack of their increased expression on this cell type in COPD, relative to age-matched smokers with preserved spirometry, implies either that the regulation or significance of TLR expression on CD4+ lung T cells, or the role of that subset itself, differs in COPD from that of lung CD8+ T cells. It is interesting that on the CD56+ NK cells, TLR5, TLR6, and TLR2/1 only showed a correlation with emphysema severity but showed no difference between COPD subjects and controls. These finding are compatible with the suggestion by Borchers and colleagues
 that NK cells are particularly crucial in emphysema pathogenesis. Collectively, our findings support previous studies
[33–37] on the importance of adaptive immune responses in emphysematous lung destruction. Whether TLR expression is restricted to specific CD8+ T cell clones, especially those with autoreactivity, will require further study, although the rather low fraction of total lung CD8+ expressing TLRs in the current study would be compatible with that possibility.
Lung CD8+ T cells in COPD appear especially sensitive to stimulation through the TLR2/1 heterodimer, as shown by the results of co-stimulation by the specific agonist Pam3CSK4. Our findings extend to the human system the finding that murine ovalbumin-specific OT-1 cytotoxic T lymphocytes responded to TLR2/1 stimulation with increased levels of IFN-γ at both the RNA and protein levels
. TLR2 is known to recognize the most diverse repertoire of microbe-associated patterns, in part through its unique ability to heterodimerize with either TLR1 or TLR6. TLR2/1 heterodimers are primarily responsible for recognizing triacylated lipoproteins, such as the outer membrane proteins of nontypeable Haemophilus influenzae (NTHI)
. This organism is one of the predominant bacterial pathogens associated with airway infection in COPD, both in stable disease and as an important infectious trigger of exacerbations
[40, 41]. Although the presence of TLR2/1 on lung CD8+ T cells might play a role in host defense in early COPD, repeated cycles of infection could swing the balance from host defense to inappropriate activation and subsequent tissue damage. Importantly, NTHI can be an intracellular pathogen of respiratory epithelial cells and macrophages
[42, 43], suggesting the possibility for presentation both of NTHI-derived antigens in the context of class I MHC molecules and of their lipoproteins to TLR2/1 on lung CD8+ T cells. A recent study by King and colleagues used live NTHI to stimulate T cells from the lungs of COPD subjects and control subjects
. They found that both CD4+ and CD8+ T cells from COPD subjects produced significantly higher levels of TNF-α, IL-17, and IL-13. Additional experiments will be needed to confirm whether TLRs have a role in this response.
The range of effector molecules significantly increased by co-stimulation via TLR2/1 (IFN-γ, TNF-α and the chemokine CCL3) is also intriguing. Transgenic overexpression of IFN-γ in the lungs induces production of matrix metalloproteinases by macrophages and development of emphysema in a murine model
, Similarly, a central role for TNF-α in smoking-induced emphysema development is well-supported by data from murine models
[46–48], likely reflecting in part the ability of TNF-α to activate endothelial cells to increase recruitment of inflammatory cells. CCL3 (previously known as MIP-1α) is a ligand for the chemokine receptors CCR1 and CCR5, and we previously showed that expression of CCR5 by lung CD8+ T cells increases with spirometrically-defined COPD severity
. Hence, stimulation via TLR2/1 and possibly other TLRs could generate a positive feed-back loop via CCR5 inducing the accumulation of lung CD8+ T cells seen in COPD.
Collectively, the current results agree with our previous finding that lung CD8+ T cells in COPD have a Tc1 phenotype and lack Tc2 cytokine secretion, even following TCR-dependent or TCR-independent stimulation
. The practical importance of the small, statistically insignificant increases in IL-13 protein production following stimulation with flagellin is questionable, because its concentration was so much lower than that of IFN-γ (100-fold less) and TNF-α (10-fold less). Because we had previously shown that perforin and granzyme B transcripts from lung CD8+ T cells were increased in severe COPD
, we were surprised to see that TLR-stimulation did not increase production of perforin or granzyme B. It is possible that perforin and granzyme were increased in the intracellular granules of the CD8+ T cells but release of these molecules into the supernatant requires CD8+ degranulation. If the CD8+ T cells did not degranulate, then any increases in perforin or granzyme B would only be detectable with intracellular flow cytometry, which we did not perform in these particular experiments.
Our results differ in several respects from a previous study by Nadigel and colleagues, which found that both TLR4 and TLR9 were increased on lung CD8+ T cells from COPD patients
, relative to healthy control subjects with normal spirometry who included two ex-smokers and three never-smokers. Although our study confirmed the finding of increased TLR4 expression, we did not see an increase in TLR9 expression in COPD relative to our reference population, who were entirely active smokers or ex-smokers. Furthermore, in our analysis, the percentage of lung CD8+ T cells expressing TLR4 only modestly increased in COPD, whereas they saw an increase from 20% in the control lung tissue to 90% in the COPD lung tissue
. These differences can likely be attributed to the tissue sample and techniques that were used. Nadigel et al. used immunofluorescence microscopy to analyze TLR expression of CD8+ T cells in endobronchial biopsies (i.e., relatively large airways)
. We used flow cytometry, which permits very objective quantification of specific staining relative to isotype control antibody, and we analyzed dispersed tissue from the distal lung compartment. CD8+ T cells are present in the central and peripheral airways and lung parenchyma in COPD, but the number of CD8+ T cells in the distal airways negatively correlates with airflow obstruction in patients with COPD
, suggesting that this is a key location in COPD pathogenesis
It is well documented that TLRs are expressed prominently on antigen-presenting cells, such as dendritic cells, and therefore play an important, if indirect, role in the initiation of adaptive T cell responses. TLR expression by macrophages and B cells can also contribute to production of antibodies and of chemokines. It is less clear whether the direct signaling of TLRs on T cells has the same physiological importance. On the one hand, the requirement for stimulation via the TCR, shown in this study and others, implies that TLRs are serving a primarily co-stimulatory role in CD8+ T cells. Indeed, numerous studies have shown that a number of TLR ligands can provide co-stimulatory signals to T cells, even in the absence of CD28 engagement
[16–20]. Additional studies have suggested that TLR signaling through myeloid differentiation protein 88 (MyD88) may increase the clonal expansion and survival of activated T cells
[50, 51]. Conversely, the other lung cell types that express TLRs (dendritic cells, macrophages, B cells and epithelial cells) cannot produce IFN-γ, a unique cytokine that has both protective and potentially damaging properties. Thus, the current findings collectively suggest that lung CD8+ T cells use TLRs to sense and respond to microenvironmental conditions and to receive additional signals at the site of injury.