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- Materials and methods
- Supporting Information
Asthma is a common disease characterized by reversible airflow obstruction, airway hyperresponsiveness (AHR), airway inflammation, mucus hypersecretion and sub-epithelial fibrosis . The cellular and molecular mechanisms underlying asthma remain incompletely understood, but new insights are being uncovered from the application of genomic technologies to human airway biospecimens. For example, in high-density microarray studies of gene expression in the airway epithelium in asthmatic subjects and healthy controls, we recently found that periostin is among the most highly up-regulated genes in asthma and that its expression in airway epithelial cells is regulated by interleukin 13 (IL-13), a Th2 cytokine . We went on to show that periostin gene expression in airway epithelial cells is a marker of an asthma subphenotype driven by excessive Th2-type inflammation and characterized by high levels of serum IgE, systemic and lung eosinophilia, increased thickness of the reticular basement membrane and responsiveness to corticosteroids .
Periostin is a matricellular protein first identified in osteoblasts and later found more widely expressed in mesenchymal cells in other organs [4-7]. Periostin null mice show aberrant type 1 collagen fibrillogenesis in skin and poor integrity of the periodontal ligament in response to mechanical stress [8, 9]. Further studies demonstrate the essential role of periostin in tissue repair in both the skin and heart following injury [10-12]. Its interaction with type 1 collagen, fibronectin and tenascin C as well as integrins, αvβ3 and αvβ5, within the extracellular matrix (ECM) are thought to underlie its function in ECM organization and tissue repair [13-15]. We have demonstrated that in airway epithelial cell culture models periostin increases levels of active TGF-β and that it has roles in regulating collagen synthesis and collagen gel elasticity . To begin to explore the biological roles of periostin in the airway in asthma, we took advantage of a periostin deficient mouse previously described  to determine how deficiency of periostin modulates airway responses to allergic airway inflammation. We anticipated that allergen challenge in mice would up-regulate periostin in the airway and model our previous findings in human asthma. Because we have found that periostin up-regulates TGF-β, we further anticipated that upregulation of lung periostin in these mice would be associated with an increase in TGF-β. TGF-β has multiple effects in the lung, including anti-inflammatory activity attributable to its induction of T regulatory cells and pro-fibrotic activity attributable to its effects on fibroblasts [17-23], so that its influence on asthma outcomes, such as AHR, airway eosinophilia and airway remodelling (peribronchial fibrosis and epithelial mucins), can vary depending on the specifics of the mouse model and the mechanism of TGF-β manipulation. Thus, we included a range of outcomes in our study design, including markers of AHR, eosinophils, T regulatory cells, peribronchial fibrosis and epithelial mucins.
Our results provide new insights into the roles of periostin in allergic airway inflammation.
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- Materials and methods
- Supporting Information
Periostin expression is increased in the airways of asthmatic subjects [2, 34], but its role in asthma pathogenesis is unknown. Herein we explore the biological role of periostin in asthma using a mouse model and periostin deficient mice. We anticipated that periostin deficiency would protect from allergen-induced inflammation and hyperresponsiveness, but we found the opposite effect. Specifically, we found that compared to wild-type controls, periostin deficient mice have increased AHR and markedly increased serum IgE levels following repeated intranasal challenge with A. fumigatus antigen without differences in IL-4 producing-Th2 cells or in two outcomes of airway remodelling (epithelial mucin stores and peribronchial fibrosis). We also found evidence that periostin deficient mice have blunted TGF-β responses to allergen leading us to propose that the protective effects of epithelial cell-derived periostin are mediated by TGF-β-induced differentiation of T regulatory cells. In support of this hypothesis, we find that airway epithelial cell-derived periostin, but not recombinant periostin, induces conversion of CD4+ CD25− T cells into CD25+ Foxp3+ T cells in vitro in a TGF-β dependent manner. These data suggest an important role for airway epithelial cell-derived periostin and local TGF-β activation on the regulation of allergic immune responses in the airway.
Periostin deficient mice challenged with Aspergillus allergen were significantly more hyperresponsive to acetylcholine than wild-type controls and demonstrated marked increases in systemic IgE responses. It should be noted that C57B6 mice traditionally demonstrate less hyperresponsiveness in response to allergen than other mouse strains , and the large changes in AHR reported herein likely relate to the aspergillus allergen model used. Aspergillus antigen challenge [36, 37] typically yields higher AHR measurements in C57BL6 mice than the ovalbumin model [38-40]. The mechanism of the effects of periostin on AHR and serum IgE levels may relate to the role of locally produced TGF-β and its anti-inflammatory effects within the airway. For example, we recently reported that periostin is a product of airway epithelial cells that activates TGF-β in a mechanism involving matrix metalloproteinases . Consistent with those findings, we demonstrate that TGF-β1 gene expression and total protein are decreased in periostin deficient mice following allergen challenge.
A negative regulatory role for periostin and TGF-β in allergic airway responses is plausible. TGF-β is a complex and pleiotropic cytokine with numerous cellular functions, including pro and anti-inflammatory effects that depend on the context of its activation [16, 33, 41]. TGF-β induces the development of CD4+ CD25+, Foxp3+ T regulatory cells [33, 42], which suppress IgE [43-45] and CD4+ CD25− T cell proliferation [46-48] via IL-10 production. CD4+ CD25+ T regulatory cells have also been shown to significantly reduce AHR in mouse models of asthma [49-51]. A reduction in T regulatory cells in the periostin deficient mice might therefore explain the enhanced serum IgE responses and AHR following airway allergen challenge in these mice. We found evidence for this mechanism in our experiments. TGF-β1 was decreased in the lungs of the periostin deficient mice following allergen challenge, as were Foxp3 transcripts. The reduction in Foxp3 transcripts suggests reduced numbers of T regulatory cells, and we would expect concomitant reductions in IL-4 producing CD4+ T effector cells or decreased levels of Th2 cytokines. The absence of these findings leads us to speculate that reductions in other TGF-β responsive cell types, such as Th17 or Th9 cells, might explain these findings.
In complimentary in vitro experiments, we demonstrate that epithelial cell-derived periostin, but not recombinant periostin alone, can promote development of CD25+ Foxp+ T cells from CD4+ CD25− T cells. This suggests that while periostin plays a role in the development of a T regulatory cell phenotype in this model, other epithelial cell-derived factors such as latent TGF-β, matrix metalloproteinase, or epithelial integrins, are required for the induction of Foxp3 gene expression. Furthermore, we show that epithelial cell-derived periostin up-regulates Foxp3 gene expression in a mechanism dependent on TGF-β1 activity. While these data do not preclude the possibility that other cell types such as fibroblasts are also a source of periostin in the asthmatic lung, they suggest a role for epithelial cell-derived periostin in TGF-β-mediated regulation of airway immune responses via epithelial-T cell cross-talk.
We did not find differences in BAL eosinophilia between the periostin null mice and wild-type controls following Aspergillus antigen challenge, a finding that differs from recent reports that periostin deficiency exacerbates oesophageal eosinophilia in a mouse model of allergic oesophagitis . However, our experiments were performed in mice backcrossed into a C57BL/6 background, whereas the allergic oesophagitis studies were done in the 129SVEV background , and it is possible that eosinophil responses in different model systems are strain specific.
The effects of periostin deficiency on allergen-induced AHR and serum IgE occurred without any effect on allergen-induced changes in peribronchial fibrosis. Periostin has been implicated in mechanisms of wound repair and collagen fibrillogenesis [10, 12, 14]; thus, periostin deficiency with a concomitant decrease in TGF-β1 might have been expected to inhibit allergen-induced airway fibrosis. For example, periostin null mice exhibit a significant decrease in the thickness of the collagenous dermal layer of the skin and display abnormal collagen fibrillogenesis and a reduced level of collagen cross-linking [10, 14]. It is possible that non-TGF-β1 dependent pathways are recruited in the development of peribronchial fibrosis in this model of allergic inflammation. It is also possible that aberrations in peribronchial collagen matrix deposition or collagen cross-linking are indeed present in the periostin deficient mice following allergen challenge and may even contribute to the observed increase in bronchial hyperresponsiveness but that these differences are not readily measured using stereological techniques.
We found that airway challenge with Aspergillus allergen caused a large increase in epithelial mucin stores that was not affected by periostin deficiency. This result differs from a recent article showing that periostin deficiency results in enhanced ovalbumin-induced mucus production and increased mucin gene expression in the airway . Multiple differences in methods could explain these divergent outcomes for mucin outcomes in periostin deficient mice, including differences in allergen (Aspergillus antigen vs. ovalubumin), mouse strain (mixed vs. C56BL/6) and methods of quantification (semi-quantitative vs. stereology-based). In the systems we used, we found no evidence that periostin regulates airway epithelial mucin stores.
In summary, we find that periostin's role in the airway is to act as a brake on allergen-induced IgE production and AHR. The mechanism of this effect may be explained by periostin's regulation of TGF-β and the anti-inflammatory effects of TGF-β-induced T regulatory cell differentiation. Overall, our data provide support for a novel paradigm in which periostin mediates a role for the epithelium in regulating T cell responses to inhaled allergens.