Distinct Sustained Structural And Functional Effects Of Interleukin-33 And Interleukin-25 On The Airways In A Murine Asthma Surrogate

Objective Interleukin-25(IL-25) and IL-33, which belong to distinct cytokine families, induce and promote T helper type 2 airways inflammation. Both cytokines probably play a role in the pathogenesis of asthma, but there is a lack of direct evidence to clarify distinctions between their functions and how they might contribute to distinct ‘endotypes’ of disease. To address this, we made a direct comparison of the effects of IL-25 and IL-33 on airways inflammation and pathophysiological changes in our established murine asthma surrogate.Materials and Methods Chronic murine asthma models(up to70 days) were established. The animals were divided into 4 subgroups:(1) Saline group(negative control);(2) OVA-sensnsitization and challenged group(positive control);(3) IL-25-inhalation challenged group(experimental group).(4) IL-33-inhalation challenged group(experimental group). Bronchoalveolar lavage fluid(BALF) was collected for cellular differential counts. Paraffin-embedded lung tissues were stained with hematoxylin-eosin(HE) and Periodic acid-Schiff(PAS) to assess the infiltration of inflammatory cells and mucus secretion, respectively. Enzyme-linked immunosorbent assay(ELISA) was used to measure the expressions of soluble collagen and T helper type2(Th2) cytokines in BALF and lung homogenate. Immunohistochemistry(IHC) of α–smooth muscle actin(α-SMA) and CD31 was used to determine the airways smooth muscle hyperplasia and neovascularization, respectively. Airways hyperresponsiveness(AHR) and lung functions were measured and assessed using the Flexi Vent system. And using flow cytometry, analysis of the number of type 2 innate lymphoid cells(ILC2) in bone marrow, mesenteric lymph nodes, spleen, thymus, mediastinal lymph nodes, lung tissues and BALF.Results 1. Total BALF cells and differential cellular counts in murine airways: IL-25 and IL-33 significantly increased the total numbers of BALF cells, the numbers of eosinophils and neutrophils in BALF. Thereafter, the infiltration of inflammatory cells was gradually reduced. IL-25-induced inflammatory cells peaked at day 36, then rapidly declined while IL-33-induced inflammatory cells also peaked at day 36 and persisted until day 54. 2. IL-25- and IL-33-induced pathological changes of murine lungs: On days 24~70, IL-33 induced significant infiltration of inflammatory cells(in particular of eosinophils) into murine alveolar walls and lumens as well as the peribronchial and perivascular areas. Increased/extended bronchial mucosal fold, hyperplasia of airway mucus gland and a large number of mucus bolts were also found in IL-33- challenged groups. Compared with IL-25, IL-33-induced pathological changes were more durable. 3. Production of cytokines and Ig E levels induced by IL-25 and IL-33: Both IL-25 and IL-33 significantly induced the expressions of eotaxin and Th2 cytokines, including IL-4, IL-5 and IL-13 in lung homogenate. However, compared with IL-25, the role of IL-33 was more significant and lasting. Meanwhile, IL-25 and IL-33 failed to augment the concentrations of serum Ig E. 4. IL-25- and IL-33-induced airways remodelling changes of murine lungs: On days 48~70, IL-25 and IL-33 induced significant collagen deposition around the airways and vasculature, smooth muscle hypertrophy/hyperplasia and significant submucosal neovascularization as indicated by immunohistochemical staining with CD31. All of these changes were significantly different from saline control group, however, on day 70, the effect of IL-25 was less remarkable than IL-33. 5. IL-25 and IL-33 induced AHR: Mice in IL-25 and IL-33-challenged group showed significant AHR to methacholine stimulation compared with saline treated mice. However, compared with IL-25, IL-33-induced increased tissue damping was more prolonged. 6. ILC2 s counts in murine bone marrow, mesenteric lymph nodes, spleen, thymus, mediastinal lymph nodes, lung tissues and BALF: IL-25 and IL-33 significantly increased the numbers of ILC2 s in bone marrow, mesenteric lymph nodes, spleen, mediastinal lymph nodes, lung tissues and BALF. But IL-33 also significantly increased the number of ILC2 s in thymus. And the role of IL-33 was more significant and lasting.Conclusion The data suggest that chronic airways exposure to IL-25 and IL-33 alone is sufficient to induce allergen-and Ig E-independent, asthma-like airways inflammation, remodeling, hyperresponsiveness and accumulation of ILC2 s in multi-organ tissue in murine model. But the role of IL-33 was more significant and lasting. Hence, both IL-25 and IL-33 may contribute significantly and independently to asthma ‘endotypes’ when considering molecular targets for the treatment of human disease.