The chronic inflammatory disease, asthma is characterized by increased airway hyper responsiveness to irritants such as smoke, dust or other allergens causing wheezing, discomfort in the chest due to difficulty in breathing and mucus accumulation in airway (Lee et al. 2010). The pathophysiology behind these symptoms is due to increased infiltration of mast cells, and eosinophils which cause inflammation in the airway due to activation of these immune cells by allergens. The activated immune cells thus cause an increase in mucus secretion contributing to these symptoms. In our immuno-histological results, we have shown the severity of the onset of asthma in the OVA-induced asthmatic group and such infiltration of inflammatory cells is absent, or the effects were less in the SAC-co-administered group. The effect of such decrease in the infiltration was seen as decreased mucus secretion (Bochner and Busse 2004; Bochner and Busse 2005) and the symptoms associated in the aftermath of mucus secretion were also less. SAC relives the alveoli of the animals affected by asthma by decreasing the infiltration of eosinophils and monocytes into them and decreasing the number of goblet cells in the airway that is responsible for mucus secretion.
Infiltration of eosinophils, lymphocytes and mast cells into the airways has been the hallmark of asthma. In order to find the extent of onset of inflammation in the OVA-induced asthmatic rats, we have tested the BALF for the eosinophils and mast cells in relation to the disease severity and subsequent airway inflammation and mucus secretion due to hyperresponsiveness (Wu et al. 2017). These numbers were high in the OVA-induced asthmatic rats and got reduced with the treatment of SAC in the other group. Also, histological analysis of the pulmonary region indicates that SAC could inhibit the eosinophil numbers in the airway and hence the inflammation. Such results would improve the reliability of SAC in its use against asthma and airway inflammation.
Asthma has been well associated with the hemostatic imbalance of Th1/Th2, and TNF-α expression is observed (Nam et al. 2009). Mice studies have indicated that anti-TNF-α antibodies have reduced the intensity of antigen-induced airway hyperresponsiveness and inflammation due to eosinophilic infiltration of airway (Rudmann et al. 2000; Zuany-Amorim et al. 2004). TNF-α produced by eosinophils and T-cells effectively damages the airway by activating the expression of adhesion molecules for sustained presence of eosinophils in the airways (Proceedings of the ATS workshop 2000). TNF-alpha also increases the goblet cell metaplasia to increase the excretion of mucus in the lungs (Thomas 2001). These effects observed in our asthmatic rats were reduced when SAC was co-administered in the OVA-induced asthmatic rats. Infiltration of inflammatory cells would generate ROS (Sahiner et al. 2011) and increased eosinophils have produced superoxide anions to react with nitric oxide (Barnes and Kharitonov 1996) and cause oxidative stress in the affected animals. Hence the NO levels were higher in the asthmatic animals and got reduced in their levels in SAC-co-administered animals. A typical Th2-type immune response was observed in asthmatic animals which are the increased expression of IgE against OVA (Ano et al. 2013).
In the development and spreading of allergic inflammation, the hemostatic imbalance is important and can be detected from Fibrinogen level (FIB), prothrombin time (PT), thrombin time (TT), activated partial thromboplastin time (APTT). The OVA-induced asthma group has significantly higher FIB, and TT and the trend were reversed in the SAC-co-administered rats. Thus the hemostatic imbalance in the OVA-induced rats was reversed in the treated group. Also, the coagulation factors activity was tested in all the groups indicating the asthmatic group had higher FXII activity and decreased in the activity of FII and FV factors. Fibrinogen, generated through airway inflammation would impair the hemostatic imbalance, and such observations were not present in the SAC-group.
We observed an increase in the expression of IL-13 in the BALF, peripheral blood, mast cells in the OVA-induced asthmatic animals and is responsible for the inflammation in the airway, remodeling and acute hyperresponsiveness (Wills-Karp et al. 1998; Zhu et al. 1999). IL-13 plays a major role in the pathogenesis of acute hyper-responsiveness (Brightling et al. 2003; Berry et al. 2004) by increasing the mucous secretion through goblet cell metaplasia (Doran et al. 2017). Such observation has well correlated with the number of eosinophils observed in the asthmatic animals, without treatment, that are having airway inflammation (Truyen et al. 2006) as overexpression of IL-13 is very important for the survival of eosinophils, their activation (Doran et al. 2017) and for chemotaxis the eosinophils to the site of inflamed or damaged airway tissues (Wynn 2003). Our OVA-induced animal models showed the eosinophil infiltration, airway inflammation, mucus secretion, and hyperresponsiveness which were not observed with the SAC-co-administered OVA models. Hence, IL-13 is important in playing a major role in the pathogenesis of allergen-induced asthma, and SAC is inhibiting primarily the airway inflammation, which is a probable small molecule candidate to be used similar to the inhibition of eosinophil inflammation as observed here (Wills-Karp et al. 1998).
IL-17 is significantly higher in the airway of asthmatic animals which is similar to the mice experimental results of asthma which were induced with OVA (He et al. 2009). It is present at a level that is significant when compared to the normal animals in our experimental animal groups and is characteristic of the onset of severe asthma (Wong et al. 2001; Bullens et al. 2006). An increase in the airway inflammation of OVA-induced rats has been partially attributed to IL-17 since the implication of overexpression of IL-13 in this model has a deep impact on the airway inflammation and hold prime responsibility in the airway inflammation of lung in asthma-induced rats as the route of OVA was induced through inhalation and not through the skin. This is in-line with the skin-induced OVA induction observed in research done here (He et al. 2009) where the infiltration of IL-17 secreting cells would occur in the lung only in the absence of expression of IL-13.
With the administration of allergens, the airway epithelium is infected and has undergone inflammation (Allard et al. 2006; Allard et al. 2009), and elevated expression of IL-6 in the lungs is observed (Neveu et al. 2009). IL-6 is known to be a pro-inflammatory mediator and is involved in the synthesis of prostaglandin E2 (PGE2) and promotes infiltration of eosinophils in the airway. The expression of COX-2 high in asthmatic animals indicates that the inflammatory cytokine, IL-6 would mediate the immune cells to generate PGE2 (Sousa 1997). The levels of IL-6 is increased in our asthmatic groups of animals, and the animals are known to be clinically asthmatic (Rincon and Irvin 2012). IL-6 also promotes IL-13 production (Neveu et al. 2009) in asthmatic animals and such synergistic action has been observed in human patients who are allergic. The co-administration of SAC has decreased the IL-6 levels and also IL-13 indicating that such effects are not due to complete blockage of IL-6R but by increasing the expression of Treg cells and reducing expression of CD4 T effector cells (Doganci et al. 2005; Finotto et al. 2007).
The simultaneous increase in the expression of IL-1beta indicates that IL-1beta, along with IL-6 would aid in the differentiation of Th17 cells (Ghoreschi et al. 2010) that contribute to the airway inflammation in asthmatic allergies (Nakae et al. 2003; Schnyder-Candrian et al. 2006). Such events did not occur in our SAC-coadministered rats where the airway inflammation was nullified. Lung epithelial cells could not produce IL-6 due to decreased IL-17 in our SAC-group as in, and hence no positive feedback mechanism (Rincon and Irvin 2012) occurred to raise IL-6 and hence the sequence of inflammatory events triggered by IL-13, goblet cell activation, mucus production, hyperresponsiveness and airway inflammation. Treatment of SAC has increased the anti-inflammatory molecule IL-10 in effectively controlling the inflammation.
The consequence of inflammation in the airway would be the oxidant damage with the assistance of antioxidants and reactive oxygen species (ROS) having primary role thereafter in sustaining the inflammation (Wood et al. 2003) to cause tissue damage (Doelman and Bast 1990). Accumulation of ROS would lead to peroxidation of arachidonic acid to produce many isomers of isoprostanes, of which, 8-isoprostane is involved in the constriction of smooth muscles and airway obstruction (Okazawa, Kawikova et al. 1997) that cause discomfort in breathing during asthma. Leukotriene members such as cysteinyl Leukotrienes, Leukotriene B4, also derived from arachidonic acid by another pathway (Bisgaard 2001). In eosinophils, act as potent broncho constrictors and causing airway smooth muscle constriction and increased mucous secretion. The increase in such lipid peroxidation products has been observed in naïve children (Zanconato et al. 2004) alike in our neonatal rats induced with OVA rather than in other subjects. The reduction in the levels of 8-isoprostanes in our SAC-co-administered animal model indicates that SAC would inhibit the inflammation and the effect is long-lasting, unlike conventional corticosteroid therapy where residual inflammation (Kharitonov et al. 2002) in asthmatic individuals resides.
In order to understand the cellular events and alterations occurring at the molecular level, due to IL-13, in the lungs, we have elucidated the levels of chemokines that are stimulated by IL-13 (Zhu et al. 2002). In that order, we have checked the levels of MCP-1, MIP-1β, RANTES, and Eotaxin in the lungs. IL-13 plays as an effector in the Th2 inflammation (Ma et al. 2004) and regulates the inflammation into remodeling of the lung tissues (Chiaramonte et al. 1999; Zheng et al. 2000) and thus contributing to hyper-responsiveness (Gonzalo et al. 1998; Kunkel et al. 1999; Teran 2000). The coordinated response of the various chemokines such as Eotaxin, RANTES, MIP1-β and MCP-1 results in the Th2 inflammatory response in the affected lungs which would eventually increase trafficking of eosinophils from the blood circulation into the airway (Wen et al. 2013), upregulating the adhesion molecules, to attach them to the airway epithelial cells. We did not necessarily discuss the molecular mechanism into activation of Th2 immune response in asthma but we speculate that the expression of chemokines with IL-13 activation would be to enhance the pre-effector events of ‘sensitization, Th2 cell generation, and Th2 cytokine elaboration in the airway inflammation (Zhu et al. 2002).
In summary, IL-13 is implicated in the pathogenesis of airway inflammation in OVA-induced asthma and is a stimulator of various cytokines and chemokines to attract the eosinophils and neutrophils by chemotaxis into the airway to cause a respiratory burst and thereby increasing the inflammation and tissue injury. Our molecule, SAC is a potent drug candidate to inhibit the airway inflammation by decreasing the expression of various inflammatory cytokines, in cases where even corticosteroids have failed to give an improvement in the treatment. This study has a shortcoming in that we have failed to address the molecular pathway that SAC undertakes in inhibiting the airway inflammation which future research would address.