Background
Idiopathic interstitial pneumonia is an umbrella term used to classify a group of lung diseases of unknown etiology characterized by the accumulation of inflammatory cells and fibrosis in the pulmonary parenchyma and interstitium. Among them, idiopathic pulmonary fibrosis (IPF) is characterized by irreversible fibrosis, resulting in gradual deterioration and poor prognosis, for which few effective therapeutics exist [
1]. A variety of factors in combination with a permissive genetic background such as
MUC5B are thought to play a role in the development of pulmonary fibrosis [
2]. The initial injury process and secondary fibrosis development likely begins as the result of alveolar epithelial injury due to exposure to environmental triggers, including cigarette smoke [
3], particles [
4], occupational dust [
5], or chemical fumes [
5], radiotherapy [
6], chemotherapy [
6]. Endogenous triggers, include obstructive sleep apnea, chronic graft-versus-host disease and connective tissue disease followed by abnormal repair. These triggers are capable of inducing both innate and adaptive immune responses [
7].
The epithelium of the respiratory tract forms a large surface area that maintains intimate contact with the environment, serving as a primary participant in innate immunity [
8,
9]. IL-33, IL-25 and Thymic stromal lymphopoietin (TSLP) are the main cytokines involved in the innate immune response by the epithelium [
8]. Recent studies have demonstrated a clear role for these cytokines in both the induction and amplification of Th2-mediated immunity [
10‐
12] and lineage-negative type 2 innate lymphoid cells (ILC2s), which also produce IL-13: the dominant inducer of fibrosis in several chronic lung diseases [
13].
In animal models, the lungs of bleomycin-treated mice exhibit a substantial accumulation of IL-33-positive cells [
14] along with over-production of mature IL-33 by the ILC of the lungs [
15]. IL-33 protein and mRNA levels are significantly higher in the BAL fluids of patients with IPF relative to healthy controls [
14,
16]. In addition to IL-33, TSLP has emerged as a candidate cytokine in the pathogenesis of pulmonary fibrosis by the elevated TSLP levels seen in both systemic sclerosis [
17,
18]. TSLP and its receptor are highly upregulated in IPF lung [
19]. Like IL-33 and TSLP, expression of IL-25 is also increased in human IPF lung tissue and IL-25 levels were significantly increased in the BAL fluids of patients with IPF [
20].
Despite the relations of the pulmonary fibrosis with IL-25, IL-33, and TSLP, the clinical implications of these cytokine remain poorly defined due to the small number of subjects (less than 15) examined in the previous studies [
14,
16,
18‐
20]. Here, we measured the IL-25, IL-33, and TSLP levels in a relatively large cohort of patients with IPF and other interstitial lung diseases, including non-specific interstitial pneumonia (NSIP), hypersensitivity pneumonitis (HP), and sarcoidosis in order to evaluate the significance of these cytokines on clinical outcomes of IPF.
Discussion
In this study, we demonstrated that IL-33 and TSLP were significantly elevated in the lungs of patients with IPF. These observations strongly support the notion of accentuated innate immune activation in the development of IPF, an effect commonly seen in animal models of acute lung injury and fibrosis. A wide variety of studies have demonstrated the importance of Th2 cells: IL-4, IL-5, and IL-13 have been causally linked to the development of fibrosis [
13]. Recent studies have demonstrated a clear role of IL-33 and TSLP in both the adaptive and innate immune response. IL-13 is highly detected in the bronchoalveolar lavage (BAL) fluid of patients with IPF, and IPF fibroblasts are hyper-responsive to IL-13. Furthermore, the combined over-expression of both IL-13 and IL-13 receptor α1 directly correlates with disease severity [
26]. TGF-β and IL-13 are essential for the development of pulmonary fibrosis by promoting differentiation of myofibroblast and stimulating production of extracellular matrix, such as collagens. Although IL-33, TSLP, and IL-25 might exert no direct induction of epithelial-mesenchymal transition, these cytokines are obviously involved in the process of fibrosis via regulation of TGF-β and IL-13. IL-33 polarizes M2 macrophages to produce IL-13 and TGF-β and expansion of ILC2s to producing IL-13 [
15]. The interaction of these cytokines has been well documented in allergic inflammation. IL-33 stimulates the rapid expansion of type 2 innate lymphoid cells (ILC2) producing IL-13 [
29], independently from the canonical CD4
+ T helper 2 cells responses [
30]. In addition to ILC2, eosinophils are additional sources of IL-13. Bone marrow-derived eosinophils secrete IL-13 in response to IL-33 stimulation in cutaneous fibrosis [
31]. While IL-33 mainly affects the upstream of IL-13, TSLP acts both upstream and downstream of IL-13. TSLP induce conventional Th2 cell priming through the costimulatory action of OX40 ligand [
32,
33] and an activation of ILC2 [
34,
35] . In addition, TSLP is a downstream target of IL-13/Stat6 pathway [
36]. Thus, the elevation of IL-33 and TSLP in IPF of our study suggest that these cytokines stimulate the upstream and downstream signals of IL-13 in concert to potentiate the process of fibrosis in the lung.
In animal models, bleomycin-mediated injury exerted a synergistic effect with IL-33 on collagen accumulation in the lung [
14]. Bleomycin enhanced the production of the mature form of IL-33 in lung tissue, while deletion of the IL-33 receptor (IL-1 receptor-like 1; ST2) attenuated the bleomycin-induced lung inflammation and fibrosis [
15]. Similar effects were seen following intranasal administration of a lentiviral construct expressing soluble ST2, resulting in markedly lower levels of pro-inflammatory and pro-fibrotic mediators, such as IL-4, IL-13, IL-33, and TGF-β1 along with improved survival rates in bleomycin-treated mice [
37]. Although IL-33 is constitutively expressed in both epithelial and endothelial cells, IL-33 is clearly induced in the other lung tissues of patients with IPF. The infiltrating cells in and around the inflammatory lesions and the fibrotic foci, composed primarily of lymphocytes with occasional macrophages, neutrophils, and eosinophils, express IL-33 [
14,
16]. In addition to the findings from animal models, elevated IL-33 levels have been observed in the BAL fluid from 10 patients with IPF compared with those from 5 healthy controls [
16]. The authors also demonstrated that primary pulmonary fibroblasts derived from the lungs of the patients with IPF and systemic sclerosis, showed enhanced expression of IL-33 mRNA [
16], suggesting that the fibroblasts may be an important source of IL-33.
Ever since TSLP was first implicated as a driver of Th2 responses in the airways [
12], aberrant levels of TSLP have been observed in a range of airway diseases, such as asthma, COPD, and nasal polyps [
38,
39]. Recently, TSLP has also emerged as an important cytokine in the pathogenesis of non-allergic diseases, including both cutaneous and lung fibrotic conditions of systemic sclerosis and IPF [
17‐
19]. While a recent comparison of TSLP demonstrated no difference in BAL fluids between the IPF and HP groups [
40], the number of subjects was too small (10 in each group) to draw any meaningful conclusions. Beyond this work, few studies have attempted to address the clinical relevance of IL-33 and TSLP expression in the context of IPF or other interstitial lung diseases. Our study clearly demonstrate that both IL-33 and TSLP levels were robustly increased in the patients with IPF compared with patients afflicted with other interstitial lung diseases, including HP, NSIP, and sarcoidosis, suggesting that the enhanced innate immune responses may be more related with the development of IPF than the other interstitial lung diseases.
Significant differences have been observed among IPF and other chronic interstitial lung diseases at both the mRNA and protein expression levels. More than 1000 genes were shown to be differentially expressed among IPF and other interstitial lung diseases, including HP and NSIP [
41]; a meta-analysis of four representative datasets on IPF and sarcoidosis revealed 708 differentially expressed genes [
42]. Interestingly, the expression of TSLP and IL-33 was not significantly different among the disease groups in any of these studies. The most likely explanation for the discrepancy between these mRNA expression studies is the small number of lung tissues used in the gene expression studies compared to the large number of BAL fluids in ours.
Similarly, no correlation was found between the TSLP and IL-33 concentrations in patients with IPF. This may indicate different cell sources for each of these two cytokines. IL-33 is constitutively expressed in both epithelial and endothelial cells [
43], and can be expressed by several other cell types within active lesions [
14,
15]. TSLP is highly expressed by lung epithelial cells [
44], but is also expressed by non-epithelial cell types, including mesenchymal cells and fibrocytes. Additionally, we observed no correlations between the levels of the two cytokines and the clinical and physiological parameters, such as lung function deterioration or cell numbers in the BAL fluid. These data indicate that IL-33 and TSLP may be more likely to be related to the development of IPF rather than its severity or progression.
In the present study, IL-25 in BAL fluid was not statistically different between the patients with IPF and the normal controls. We therefore did not perform any subsequent assessments of IL-25 in other interstitial lung diseases. Pulmonary expression of IL-25 has been shown to be increased in patients with IPF, and is essential in the generation of experimental pulmonary fibrosis: IL-25 levels were shown to be higher in the BAL fluid of patients with IPF compared with a small number of controls [
20]. The reason for the discrepancy between this study and ours is not clear. Prostaglandin E2 (PGE2) exhibits potent anti-fibrotic activity in the IPF lung [
45]. PGE2 directly inhibits major pathobiologic functions of effector fibroblasts including chemotaxis, proliferation, collagen synthesis, and differentiation to myofibroblasts via E prostanoid receptor 2– mediated increases in intracellular cyclic AMP [
46]. Derangements of PG synthesis are present in fibrotic diseases in humans and animal models of pulmonary fibrosis. Fibroblasts from IPF patients are unable to upregulate the COX-2 enzyme and are thereby deficient in PGE2 production [
47]. Thus, diminished PGE2 production and/or signaling characterize lung fibrosis and are likely to be pathophysiologically significant. It is unknown whether IL-33, TSLP and IL-25 are related with PGE2 production although these cytokines stimulate mast cells to produce a large quantity of PGD2 [
48]
.
One limitation of our study was the small number of patients with other interstitial lung diseases, including those with NSIP, HP, and sarcoidosis. Additionally, long-term follow up will be necessary to fully address the clinical implications of these cytokines, particularly the survival rate of IPF. Furthermore, validation studies using other cohorts’ samples are mandatory to improve the diagnostic values of IL-33 and TSLP. Finally, the lack of transcriptome analysis in conjunction with these cytokines data limited our ability to assess some of the potential mechanisms underlying the changes in protein expression.
Acknowledgements
The BAL fluids were generously provided by Soonchunhyang University, Bucheon Hospital Biobank, a member of the National Biobank of Korea, supported by the Ministry of Health, Welfare, and Family Affairs, Republic of Korea.