In this study we aimed to investigate the effects of cigarette smoke (CS) on upper airways and lower airways, in a mouse model of subacute and chronic CS exposure. We here demonstrate for the first time that the inflammatory response upon CS exposure clearly differs between nose and lungs in mice. The nature and kinetics of both the neutrophil and monocyte/macrophage inflammation differ in both airways compartments. This indicates the involvement of different regulatory mechanisms, which is reflected by the observed differences in FoxP3 increase after CS exposure. The suppressive mechanisms arise earlier and appear to be more efficient in nose than in lungs. Although increased levels of MCP-1, MIP-3α and GCP-2 are found both in nose and lungs after subacute CS exposure, the neutrophilic influx and increase in neutrophilic chemoattraction signals are transient in upper airways while they remain constant in lower airways. Consequently, chronic upper airway CS exposure results in a non-inflammatory status with a significant downregulation of inflammation, while lower airway inflammation is clearly present and ongoing.
Neutrophilic inflammation in the nasal turbinate tissue was not present after 2-wk CS exposure, likely due to the absence of a neutrophilic chemoattraction signal, as both GCP-2 and KC levels were not increased in the CS group. However, prolonged (4-wk) exposure caused a significant GCP-2 increase in the CS group, which correlates with the immunohistochemistry, showing a higher number of neutrophils per field in the CS group compared to the air group, but only after 4-wk. To our surprise, chronic (24-wk) CS exposure did not cause a further increase in neutrophil accumulation in the nasal turbinate tissue. Moreover, GCP-2 levels and KC levels in the CS group did not differ and were significantly down regulated from controls respectively. This was again confirmed by IHC, where we found a significant decrease in the number of neutrophils per field in the CS group compared to controls. This may be interpreted as a clear sign of down-regulation of the neutrophilic inflammatory long-term response in the nasal turbinates. Evaluation of neutrophilic inflammation in upper airways was done in nasal turbinate tissue, because nasal lavage did not yield sufficient cells allowing a reliable cell differentiation. As a consequence, compartmentalization of inflammation in both upper and lower airways may influence the interpretation of these findings. Indeed, cigarette smoke causes an increase of neutrophil numbers in BAL (mouse studies), or sputum (human studies), whereas its effect in lung tissue or biopsies is less pronounced.
Our findings on neutrophilic inflammation in upper airways are in sharp contrast with the data obtained from experiments in the lung, where CS exposure resulted in a significant increase in both GCP-2 and KC at all time points, accounting for to the observed influx of neutrophils in the BAL fluid of these mice .
We have shown a remarkable change over time in the nasal mRNA MCP-1 levels of CS exposed mice, showing an initial increase, followed by a significant decrease in MCP-1 levels in the nasal turbinate upon chronic exposure. In the lungs of these mice however, we detected a consistent increase in MCP-1 levels in CS exposed mice on each time point . This is another sign of the different inflammatory response to CS in the upper airway.
The role of pro-inflammatory T helper 17 phenotype in the pathogenesis of COPD is increasingly studied, and it is suggested that COPD might be better explained by the Th17 phenotype . These Th17 cells, which require the up-regulation of the orphan nuclear receptor RORgammat (encoded by RORc) for differentiation from naïve T cells , account for the production of several members of the IL-17 family of cytokines, which have proven abilities to recruit and activate neutrophils . Here, nasal mRNA levels of RORc and IL-17 in the nose were significantly down-regulated after 2-wk CS exposure, but not upon longer (4-wk and 24-wk) exposure. In lungs however, the response of Th17 cells appears to be opposite, as 2-wk and 4-wk CS exposure resulted in a significant up-regulation of IL-17, and chronic (24-wk) exposure showed a similar tendency. These differences in IL-17 levels between nose and lungs, can explain the observed differences in neutrophil accumulation, as described above.
T regulatory cells expressing FoxP3 are thought to play a role in controlling CS induced inflammation [15, 26], amongst others via the immunomodulatory cytokine TGF-β1 . In nose, FoxP3 mRNA expression was increased already after 2-wk, and was mainly found - at least at 4-wk - in the subepithelium, possibly due to invading Tregs expressing FoxP3. In lungs, FoxP3 was only increased after 4-wk, which is in line with increased Tregs in lungs after CS exposure . Interestingly, these infiltrating Tregs in lungs are thought to have a weak functionality, as they are unable to control inflammation in lungs . It is tempting to speculate that Tregs act early and adequately in nose to suppress CS-induced inflammation, but that they invade later and have weaker functionality in lungs, allowing inflammation to persist. Alternatively, the CS exposure of the nose might be higher in mice - obligatory nose-breathing animals - compared to lungs, allowing tolerazation or change in cell populations to occur earlier. Indeed, upon 24-wk CS exposure the number of neutrophils shows a decreasing tendency compared to 4-wk CS exposed mice.
Although in vivo cigarette smoke-exposed mice can offer valuable information on several aspects of the pathogenesis of COPD, such as the time course of upper and lower airway inflammation, there are also limitations that need to be taken into account. Firstly, a number of anatomical and physiological differences exist between the respiratory tract of mice and humans. For example, mice are obligate nose breathers that filter tobacco smoke inefficiently, and they have less branching of the bronchial tree. Furthermore, the profile of inflammatory mediators is also slightly different in the mouse. And lastly, there is no mouse model that mimics all the hallmarks of COPD pathology, including exacerbations and extrathoracic manifestations.
Another possible limitation to this study is the fact that not only T cells are able to produce either IL-17, TGF-β or FoxP3, but a number of other cells like neutrophils or epithelial cells can do so. Furthermore, the suppressive capacity of the FoxP3 producing Tregs in upper airways stills remains to be elucidated.
Although the inflammatory answer of nose and lungs is clearly different upon CS exposure, possible confounding factors might influence the data interpretation in this model. Above, we have described the issue of compartmentalization of inflammation, and the relative dosage exposure, with higher deposition of CS in the nose vs. lungs. Furthermore, physiologic temporal changes are seen in the inflammatory readouts: levels of inflammatory cells and mediators of unexposed control mice vary over time, as shown in Fig. 3, 4, 5. By using age-matched control mice in our experiments, we have corrected for these physiologic temporal changes. Altogether, the above mentioned limitations of this model remain to be elucidated.