Over the last decade, proteomic based mapping of the protein expression profiles of complex biological samples has been well established for getting a comprehensive molecular insight in biological processes underlying disease pathology. To our knowledge, this is the first study on quantitative proteomic profiling of lung-derived specimen in experimental eosinophilic and neutrophilic asthma. We hypothesised that protein expression patterns of BAL fluid would reflect the mechanistic differences between asthma phenotypes. In this study, we therefore investigated BAL proteome dynamics from experimental eosinophilic and neutrophilic asthma using an integrated proteomics approach based on high resolution mass spectrometry and multiplexed ELISA.
We demonstrated that the protein expression levels of several acute phase proteins such as S100-A9, complements (CO3, CFAB) and immunoglobulins (IGJ, IGH, PIGR) were increased in the BAL from mice with OVA + LPS-induced airway inflammation compared to mice with OVA-induced airway inflammation, and that these up regulations could be nearly completely averted by pre-treatment with glucocorticoid therapy (Additional file 2: Figure S1 and S2). Our major findings show that the eosinophilic (OVA-induced) and the neutrophilic (OVA + LPS induced) asthma models encompass significant and relevant differences in their protein patterns, which could not be delineated by common techniques used for characterization of airway inflammation, such as inflammatory cell counts and lung mechanics (Figures 2 and 3). Using multivariate data analysis allowed for discriminating the two asthma models from each other, as well as from healthy control and steroid treated animals (Figures 5).
The most characteristic protein regulations associated with neutrophilic experimental asthma included increased levels of acute phase reactants. The adaptive immune response is traditionally expected to be steroid sensitive, while the innate is expected to be steroid resistant . The Th17 driven response has been suggested to play a critical role for the innate host defence against bacteria in mammalian lungs through its ability to indirectly mobilise neutrophils . In line with this, our findings show an increased production of IL-17 as a result of the accumulated neutrophils after bacterial endotoxin stimulation in vivo, as well as a considerable decrease of IL-17 after steroid treatment. T cells have been reported to release IL-17 after endotoxin exposure, but only in the presence of macrophages . IL-17 is suggested to be the strongest recruiter of neutrophils in lung tissue. In agreement with this, neutrophils and macrophages were increased in BAL from the NA group compared to the EA group (p < 0.05), in our model (Figure 3). Moreover, neutrophils and macrophages displayed strong positive correlations with other proteins elevated in the NA model (Table 3). The NA model resembles severe human asthma more than the more conventional EA model in that it shows Th17 response related characteristics such as IL17 expression and neutrophil recruitment. However, as previously demonstrated for LPS induced IL17 expression, effects of the NA model used in this study were attenuated upon steroid treatment , which in turn highlights the difficulties in creating experimental models of severe steroid-resistent human asthma.
The EA group could be delineated from the NA group based on the protein species; including TPPP3, IL-3, IFN-γ and eotaxin, which were found significantly elevated in the EA group compared to the NA group. In asthma, it is known that reducing histone deacetylases (HDAC) increases asthmatic inflammation and that glucocorticoids down regulate the inflammatory response in turn by modulating HDAC activity . TPPP3 has been described to inhibit HDAC , possibly regulating the immune reaction towards the steroid sensitive Th2 response. Similarly, IL-3 has been associated with atopic asthma and Th2 response [13–15].
IFN-γ is traditionally distinguished as an essential Th1 response cytokine, but has been described to have a dual role and even protective effects in other disease models . In the present study, IFN-γ was significantly increased in the EA group compared to the NA group. In addition, IFN-γ as well as eotaxin correlated strongly with eosinophil count.
As expected, the EA group had a marked increase of eotaxin expression. Eotaxin selectively attracts allergic effector cells, eosinophils and basophils, due to the requirement of a high affinity receptor (CCR3) [17, 18]. Its synthesis is stimulated in different cell types by IL-4, IL-5 and IL-13, which is released mainly by Th2 lymphocytes . Interestingly, eosinophilic cationic protein 2 (ECP2) was more pronounced in BAL from the NA group. This protein is localised in the cytoplasmic granula of eosinophils, with the main function to selectively attract dendritic cells to the source of infection. In spite the low cell count of eosinophils in BAL from the NA group, our data provide evidence that eosinophils indeed are present, but in the case of NA instead of recruiting more eosinophils, rather regulating the inflammation away from a Th2 response. Group specific protein regulations are therefore suitable markers for delineating different immune response mechanisms in between these models.
Complementally lung mechanics parameters, including elastance (H), tissue damping (G) and newtonian resistance (RN), showed a significant increase in the asthma models compared to the control group. While this verifies the animal model, both lung mechanics as well as BAL counts that are commonly used for characterizing asthma phenotypes, did not allow delineating the asthma models. However, correlation of lung mechanic data with the protein regulations revealed differences in peripheral and central parameters of airway responsiveness (Table 4). Here, strong correlation of peripheral parameters, elastance and tissue damping, correlated strongly with proteins elevated in NA. These correlations were found to be very similar to protein correlations observed for neutrophil and macrophage cell counts. Indeed, direct correlation analysis revealed a strong positive correlation for G (R = 0.99) and H (R = 0.97) with recruited neutrophils but not for other BAL cells. Conversely, Newtonian resistance as a central parameter for airway responsiveness displayed no correlation with any inflammatory cell count. This supports the theory that lung mechanics in the peripheral airways plays an important role in asthma pathophysiology due to exaggerated airway closure . Thus, protein species associated with the NA phenotype also reflected peripheral airway closure. If confirmed, these proteins could serve as biomarkers indicating inflammation of distal airways.
Moreover, RN was found to correlate with chitinase 3, a common biomarker in asthma. Chitinase 3 did not differentiate the two models of inflammation, although it has been suggested to play a key role in Th2 driven inflammatory response . Similarly, further Th2 associated proteins, IL-5 and IL-13, correlated positively with RN. This suggests that commonly used markers for asthma, including IL-13 and chitinase, do in fact only reflect central airway inflammation.