In the present study, we found that both CS exposure systems induced a similar degree of lung function impairment and morphological alterations, but lung inflammation was more severe with the whole-body CS exposure system as shown by an enhanced lymphoid presence in BAL, lymph nodes and the upregulation of cytokine (IL-6) and chemokine (KC) mRNA expression in the lung. However, this lung inflammation did not translate into enhanced immunoglobulins in the BAL or serum, which was solely observed in the nose-only CS exposure system.
Up to now there have been no studies comparing whole-body with nose-only CS exposure within the same study while using a standardized exposure protocol especially at early stage of COPD. Indeed, most of the time, CS exposure models are compared with each other without having a clear knowledge regarding the effect of the CS exposure method used. Moreover, the fact that those studies have been performed separately makes it even more difficult to compare the results, notably because the CS exposure protocols differ between studies. In the present study, the CS exposure protocol was adapted so that particle matter of the CS and the carbon monoxide (CO) levels were similar in the whole-body and the nose-only systems, ensuring that mice in each exposure system were submitted to the same levels of CS. Total particulate matter was in the range of 188 ± 28.4 mg/m3 and CO levels were 817 ± 49 and 937 ± 50 ppm/cigarette measured in each housing compartment of respectively the nose-only and whole-body system. However, one should be aware that with the whole-body CS exposure system, it is difficult to fully control the amount of CS absorbed through the skin or gastrointestinal tract. This has been proven to induce inflammation and to alter the microflora and mucin production in the gut when using the whole-body exposure system [14, 15]. Conversely, the nose-only CS exposure system allows a better controllability on the amount of CS inhaled and circumvents the influence of its absorption through the skin.
Currently, only one study has performed a comparison between the nose-only and whole-body system, but with some major differences compared to our study . Shu et al. used a mouse model combining CS exposure with lipopolysaccharides (LPS), where mice were exposed two times to an LPS-instillation at day 0 and 14 followed with an intense CS exposure protocol using 18 cigarettes twice a day for 10 weeks (6 days/week). In our model, we found after 14 weeks of CS exposure alone (6 cigarettes twice/day) a 25% less increase in FRC and no enhanced chord compliance compared to the data reported by Shu et al. However, in agreement with Shu et al.’s data, no differences in lung function were observed between the two CS exposure systems. Although only discrete alterations were found in the lung histology in our study compared to the severe changes such as bronchial wall thickening, emphysema, goblet cell hyperplasia and even pulmonary bullae formation reported in Shu et al.’s study , both studies indicated no differences between the two CS exposure systems. It is worth to mention, that this type of severe alterations in lung histology as shown in Shu et al.’s study usually occur after 24 weeks of CS exposure [17–19]. The intense CS exposure protocol combined with LPS instillations might explain the presence of these alterations at an earlier time point. Finally, our study indicated that the mean airspace enlargement was slightly enhanced in the nose-only exposure system compared to whole-body, but this enlargement was not significantly different compared to control. Taken together, these data suggest that nose-only and whole-body CS exposure systems lead to the same degree of lung function impairment and histological alterations.
Interestingly, we also looked at desmosine, a crosslinker between elastin fibers that is usually used as a biomarker for elastin breakdown, and that has been found to be enhanced in the BAL fluid at this stage of emphysema [20, 21]. Desmosine in BAL has also been reported to correlate with mean airspace size and with BAL neutrophils [22, 23]. Up to now, no studies have compared (iso-)desmosine levels in nose-only and whole-body CS exposure systems. In our experiment, while desmosine was undetectable in the BAL fluid with both CS exposure systems, a significant increase in iso-desmosine and total desmosine in serum was observed with the whole-body CS exposure system compared to the nose-only CS exposure system and to control. However, since the MMP12/TIMP1 mRNA expression ratio in the lungs were similar between both CS exposure systems, this suggests that lung proteolysis was similar and that the enhanced serum desmosine, in the whole-body CS exposure system, is likely to come from another source i.e. elastin degradation of the skin .
Regarding inflammation, in agreement with Shu et al. , no differences in BAL macrophages or neutrophils were noticeable between the two CS exposure systems. By contrast, the increase in BAL lymphocytes was significantly more pronounced with the whole-body CS exposure system compared to nose-only exposure and controls. This enhanced lymphocyte presence with the whole-body CS exposure system might suggest a progressive implication of the adaptive immunity with further worsening of lung remodeling and inflammation. mRNA expression of KC and IL-6 in the lung was already more increased in the whole-body CS exposure system in comparison with nose-only, but BAL and serum protein levels of pro-inflammatory cytokines, particular KC and IL-6, were similarly enhanced in both CS exposure systems, as shown also by Shu et al. . Overall, these data indicate an enhanced lymphocyte inflammation in the lungs with the whole-body CS exposure system.
Furthermore, both CS exposure systems demonstrated an equal amount of lymphoid inflammation and aggregates. In an attempt to categorize the lung inflammation of each CS exposure system, cytokines specific for Th1 (IFN-γ, IL-12p40), Th2 (IL-4, IL-13), Th17 (IL-17, IL-23) and regulatory T lymphocytes (Treg; IL-10, TGF-β) response were measured via mRNA expression in lung homogenate. Actually, most animal models of emphysema with CS exposure reported a type 1 mediated inflammation with Th1/Th17 immunity [25, 26], while other studies that use CS extracts  for in vivo or in vitro experiments showed a polarization towards a type 2 mediated inflammation with Th2 immunity (reviewed in ). In fact, CS can suppress the expression of IL-12 and IL-23 in dendritic cells, needed for a Th1/Th17 differentiation . In addition, CS can also be used as an adjuvant to provoke an enhanced Th2 immune response when combined with allergens [27, 30]. In our study, we showed a similar enhanced expression of IL-4, IL-13, IL-10 and TGF-β in the lungs with no changes in IFN-γ and IL-17 with both CS exposure systems. This corresponds to a type 2 mediated inflammation with a Th2 and Treg immune response which is surprising in the presence of IL-12 and IL-23. It stands to fact that this balance can later shift towards a full Th1/Th17 response with production of IFN-γ and IL-17, when CS exposure will induce more damage to the lungs [25, 26, 31] and might even persist after smoking cessation [32–34]. In human COPD, lymphocyte inflammation is mainly driven by a Th1 immune response with an enhanced IFN-γ production in both helper (CD4+) and cytotoxic (CD8+) T lymphocytes, that increased with COPD severity [35, 36]. The reason why this was not observed with any of our CS exposure systems might be related to the duration and intensity (number of cigarettes, filtered versus unfiltered cigarettes) of the CS exposure protocol. Nevertheless, both IFN-γ and IL-13 inflammation can induce emphysema [37, 38]. In summary, in our study both CS exposure systems induce a Th2/Treg induced inflammation in the lungs, likely related to the duration and intensity of our CS exposure protocol.
Our data shows an enhanced presence of (CD3+) T- and (CD19+) B-lymphocytes in the cervical and mediastinal lymph nodes with the whole-body CS exposure system. This suggests a role for sensitization at the skin or gastrointestinal tract. Indeed, Pollaris et al. demonstrated for toluene diisocyanate that dermal sensitization, before repeated intranasal challenges, could induce an aggravated lymphocyte proliferation in lymph nodes and even airway hyperreactivity . Thus, in our study, sensitization to CS with the whole-body system might have provoked an aggravated inflammatory response in the lungs during CS exposure. However, for the time being, there are no data proving if this assumption is valid. On the other hand, opposite immunological effects of CS exposure have been reported between the CS exposure systems. Robbins et al. demonstrated, in a nose-only CS exposure system, a decrease in number of dendritic cells (DC) in the lungs and an inhibition of their maturation, which suppresses the proliferation of lymphocytes in the lymph nodes . However, Demoor et al. showed, in a whole-body CS exposure system, an increase in lung DCs already at 4 weeks followed by an enhanced lymphocyte proliferation in the lymph nodes which was almost 3 times higher than normal after 24 weeks of CS exposure . These data let us postulate that CS exposure with the whole-body system would induce an immunological response presumably involving more lymph nodes and systemic activation. However, it remains difficult to fully support this postulate with the current data as the smoking protocols were different and it seems not fair to compare data obtained with 4 against 20 unfiltered cigarettes/day [40, 41].
Humoral immunological markers (e.g. IgA and IgG) were enhanced in BAL and serum of mice exposed to the nose-only CS exposure system in comparison with the whole-body system and control. These data are, however, inconsistent with the increased B-lymphocytes in the lymph nodes of mice exposed to the whole-body CS exposure system and could be related to the maturation or migration of B-lymphocytes in the two systems as we only measured the general CD19 + B-lymphocyte marker and not the markers for plasma B lymphocytes. However, a significant increase in IgA, IgM and IgG in BAL, but not in serum was previously reported when using the whole-body exposure system with CS from cigarettes without filter . In our study, this increase in Ig’s might be related to the fact that CS in the nose-only system is likely denser inhaled than in the whole-body system.