ASD events cause deterioration of pulmonary function in asthmatic patients and aggravation of their symptoms [16, 17]. Therefore it is important to investigate the series of manifestations in allergic airway disease caused by co-exposure to allergens and ASD for 14 weeks when deciding on a clinical strategy in the treatment of ASD-stimulated allergic airway disease.
The four-time treatment of ASD alone (6-week study) caused bronchitis and alveolitis, and clearly increased neutrophils along with its relevant chemokines MIP-1α, KC (IL-8 in human) and other cytokines IL-12, TNF-α in BALF. These chemokines and cytokines may play an important role in a neutrophilic inflammation processes. The eight-time exposure of ASD alone (14-week study) deteriorated bronchitis and alveolitis, which accompanied with further increase of cytokines IL-1β, IL-6, IL-12, IL-17A and TNF-α; and chemokines KC, MIP-1α, and RANTES in BALF. However, thickening of the subepithelial layer in the airway was slight in both the four-time and the eight-time exposure to ASD alone.
LPS and β-glucan presented in ASD may contribute to cause the neutrophilic inflammation and the production of these cytokines and chemokines, because a previous study reported that LPS and β-glucan induced the expression of their pro-inflammatory molecules . The induction of IL-17A, which was secreted from T-helper 17 (Th17) cells, may be an infection defense reaction to the pathogens such as fungi that adhere to ASD .
OVA alone causes a slight proliferation of goblet cells and an infiltration of eosinophils and lymphocytes, and slight thickening of the airway wall, which are the pathology correlatives seen in human asthma. The four-time co-exposure to OVA and ASD enhanced thickening of the subepithelial layer, eosinophil and neutrophils infiltration and the proliferation of goblet cells in the airway, which was evidenced by pathological examination. As an overall trend, these changes and the cellular profile of BALF were paralleled by the expression of Th2-associated effector molecules and eosinophil and neutrophil relevant cytokines/chemokines in BALF as well as the production of OVA-specific IgE and IgG1. It was reported that eosinophils, Th2 lymphocytes and their released inflammatory mediators, such as IL-5 and IL-13, played a crucial role in human allergic asthma . IL-5 has been shown to attract and activate eosinophils, which were implicated in tissue destruction in allergic asthma . IL-13, also released from Th-2 lymphocytes, has been shown to stimulate B cells and to lead to the production of antigen specific antibodies  and promote mucous secretion and production of mucous cells, such as goblet cells, in the bronchial epithelium . IL-17A contributes to neutrophil infiltration in the airway inflammation of allergic asthma . Therefore, the airway injury under the co-exposure may be due to enhanced airway inflammation by eosinophilis and neutrophils.
TGF-β1 is well known as a repair and profibrotic cytokine . Hyperplasia of bronchial structural cells, like fibroblasts and smooth muscle cells in the airway, is a typical feature of airway remodeling; hence TGF-β1 plays an important role in the development of airway remodeling . Fibroblast growth factor (FGF)-2 and platelet-derived growth factor (PDGF)-BB also have a significant role in airway remodeling in asthma . However these fibrogenic parameters were found only at low levels or not detected in the four-time co-exposure in spite of the fibrous thickening in the airway. Further increases of these fibrogenic parameters in BALF may be required for serious remodeling of the asthmatic airway to occur.
On the other hand, the eight-time co-exposure to OVA and ASD attenuated fibrous thickening of the subepithelial layer, eosinophil infiltration in the airway as well as eosinophil number and the relevant cytokines IL-5, L-13, and chemokine MCP-3 in BALF compared with the four-time co-exposure group. Oppositely TGF-β1 was significantly increased only in the eight-time co-exposure group.
TGF-β1 is also known to play an important role as an immunosuppressive cytokine . The differentiation of Th1 and Th2 cells is blocked by TGF-β-induced Foxp3+Treg cells, which play an important role in immunological tolerance [33, 34]. In fact, TGF-β1 can suppress OVA-induced eosinophilic airway inflammation . Over-expression of TGF-β in T cells resulted in the suppression of allergic asthma in a murine asthma model . In contrast, impairment of TGF-β signaling led to increased allergic airway responses in transgenic mouse models compared to wild-type mice . From these reports, we speculate that the attenuation of eosinophil recruitment in the airway under eight-time co-exposure to ASD and OVA may be due to the suppression of Th2 cytokine (IL-13, IL-5) production, which operates by blocking of the differentiation of Th2 cell by TGF-β-induced Foxp3+Treg cells. TGF-β induced by the eight-time co-exposure may have an important role in the self-defense reaction for repairing the severe airway injury and for weakening the eosinophilic inflammation enhanced by ASD in an early stage.
Foxp3+Treg cells or Type 1 regulatory T (Tr1) cells can suppress the Th2 cell-driven response to allergen through producing IL-10 [37, 38], whereas IL-10 was not detected in BALF in this study.
IFN-γ released from Th-1 lymphocytes can suppress Th-2-driven allergic airway responses . However, no increase of IFN-γ was observed in the eight-time co-exposure to OVA and ASD, suggesting that the eight-time sensitization did not cause skewing of the immune response from a Th2 to Th1.
On the other hand, the eight-time combined treatment did not cause the suppression of neutrophil number in BALF compared with the four-time combined treatment. The induction levels of neutrophil relevant cytokines IL-12, IL-17A, TNF-α and chemokines MIP-1α, KC in BALF of the 8-OVA+ASD group were almost similar or higher compared with the 4-OVA+ASD group. The allergic inflammation in the 8-OVA+ASD group may shift to neutrophil-dominant inflammation induced by ASD.
Regarding OVA-specific immunoglobulin production, adjuvant effect of ASD on IgG1 and IgE production was detected in both four-time and eight-time exposure. However, the eight-time exposure could not attenuate the IgE and IgG1 production than those of the four-time exposure. Although a relatively high concentration of Al2O3 is contained in the ASD, Al2O3 may not contribute to the adjuvant effect because the adjuvant effect of Al2O3 particle on their immunoglobulin productions was not detected in our previous study, whereas the possibility of an adjuvant may be in SiO2. The augmentation of TGF-β-induced Treg cell differentiation reportedly causes the suppressive effect of IgE production and bronchial hyper responsiveness . From these findings, the suppression of antigen-specific immunoglobulin in serum may require further induction of TGF-β1.