Periostin promotes chronic allergic inflammation in response to Th2 cytokines

Abstract

Allergic inflammation triggered by exposure of an allergen frequently leads to the onset of chronic inflammatory diseases such as atopic dermatitis (AD) and bronchial asthma. The mechanisms underlying chronicity in allergic inflammation remain unresolved. Periostin, a recently characterized matricellular protein, interacts with several cell surface integrin molecules, providing signals for tissue development and remodeling. Here we show that periostin is a critical mediator for the amplification and persistence of allergic inflammation using a mouse model of skin inflammation. Th2 cytokines IL-4 and IL-13 stimulated fibroblasts to produce periostin, which interacted with αv integrin, a functional periostin receptor on keratinocytes, inducing production of proinflammatory cytokines, which consequently accelerated Th2-type immune responses. Accordingly, inhibition of periostin or αv integrin prevented the development or progression of allergen-induced skin inflammation. Thus, periostin sets up a vicious circle that links Th2-type immune responses to keratinocyte activation and plays a critical role in the amplification and chronicity of allergic skin inflammation.

Figure 1. Allergic skin inflammation is STAT6/periostin dependent in the HDM mouse model.

(A) Ear thickness of HDM-painted mice at the indicated times. n = 4 (vehicle-treated WT and HDM-painted Stat6^–/–); 11 (HDM-painted WT and Postn^–/–). (B–E) Mice were sacrificed at day 50, and ear skin tissues were prepared. Experiments were done at least 3 times. (B) Epidermal thickness, measured in 10 views selected randomly. Red lines indicate means. (C) Staining with H&E, Masson trichrome, and anti-periostin Ab. Scale bars: 50 μm. (D) mRNA expression of the indicated cyto­kines, normalized to Actb. ND, not detected. (E) Western blot analysis of periostin and GAPDH. *P < 0.05, **P < 0.01, ^#P < 0.001.

Figure 2. Periostin-induced proliferation, differentiation, and activation of keratinocytes in the 3-dimensional organotypic coculture system.

(A) 3-dimensional organotypic coculture system. Keratinocytes were cocultured with Postn^–/– or WT mouse–derived fibroblasts in the presence or absence of 10 ng/ml IL-13. (B) Histological staining with H&E, anti-CK10, and anti-CK14 of the keratinocytes cultured for 7 days. Scale bars: 25 μm. (C) Number of cells positive or negative for TUNEL, PCNA, and phospho-Akt in histological staining, counted in 10 ×200 views per section. (D) Amount of TSLP, TNF-α, GM-CSF, and IL-1α in the supernatant in keratinocytes from day 5 to day 7. (E) Mixed lymphoid reaction system. Allogeneic CD4⁺ T cells were cocultured with bone marrow–derived DCs treated with the supernatants from the culture as in A for 5 days. (F) Quantitative RT-PCR analysis for Il4, Il13, Ifng, and Il17a in mRNA samples prepared from allogeneic CD4⁺ T cells. Experiments were done at least 3 times. *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 3. Induction of TSLP production by periostin through the NF-κB pathway in keratinocytes.

Keratinocytes were cultured on precoated periostin for 72 (A) or 24 (B and C) hours. (A) TSLP production in the culture medium. (B) Confocal microscopic imaging of keratinocytes. Green and magenta represent p65 and nucleus, respectively. Scale bars: 10 μm. (C) Expression of Tslp mRNA with the indicated concentrations of the NF-κB inhibitor BAY 11-7082. Experiments were done at least 3 times. *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 4. Requirement of the integrin/periostin interaction on keratinocytes for generation of allergic skin inflammation.

(A) H&E staining of keratinocytes cocultured with WT mouse–derived fibroblasts (2 × 10⁵ cells) in the presence of 10 ng/ml IL-13 and control, anti–α_v integrin, and/or anti–β₃ integrin Abs in the 3-dimensional organotypic coculture system. Scale bars: 25 μm. (B) TSLP production in keratinocytes cultured on a dish coated with 10 μg/ml recombinant periostin in the presence of IL-13 and anti–α_v integrin Ab. Experiments were done at least 3 times. (C–F) Preventive protocol. BALB/c mice were treated with vehicle (n = 3 per group) or HDM (n = 5 per group) once per week for 4 weeks and injected subcutaneously with control or anti–α_v integrin Ab twice per week, then examined on day 29. (C) Ear swelling. (D) Epidermal thickness. (E) Skin histology with H&E. Scale bars: 50 μm. (F) mRNA expression of the indicated cytokines. *P < 0.05, **P < 0.01, ^#P < 0.001.

Figure 5. Blockage of the integrin/periostin interaction is effective in the mouse model of established skin inflammation.

(A–F) Therapeutic protocol. WT mice treated with vehicle (n = 4 per group) or HDM (n = 6 per group) were administered control or anti–α_v integrin Ab by local injection twice weekly for 3 weeks from day 21. Mice were harvested 24 hours after the final painting of HDM (day 43). (A) Ear swelling. (B) Skin histology with H&E. Scale bars: 100 μm. (C) Total number of infiltrated cells. (D) Number of infiltrated eosinophils. (E) Epidermal thickness. (F) Serum IgE. NT, not tested. *P < 0.05, **P < 0.01.

Figure 6. Periostin is highly expressed in skin tissues of AD patients, correlated with disease severity.

(A) Skin tissues stained with H&E and anti-periostin Ab of a control donor and an AD patient with marked periostin expression. Scale bars: 200 μm. (B) Inflammation score, lymphocyte number in the dermis, and epidermal thickness in healthy controls and AD patients with periostin expression classified as mild (n = 3), moderate (n = 11), or marked (n = 13). Lymphocyte numbers were counted and epidermal thickness measured in 10 ×400 fields for each sample. (C) Periostin levels in sera obtained from control donors (n = 66) and AD patients (n = 29) using ELISA. Red lines in B and C indicate means. **P < 0.01, ***P < 0.001.

Figure 7. Vicious circle in allergic skin inflammation, composed of IL-4/IL-13, periostin, and proinflammatory cytokines from keratinocytes.

The exposure of APCs, including DCs, to allergens induces Th2-type responses. IL-4 and IL-13 derived from Th2 cells stimulate fibroblasts to produce periostin. Periostin binds to α_v integrin on keratinocytes, inducing proinflammatory cytokines, including TSLP. The proinflammatory cytokines derived from keratinocytes enhance Th2 inflammation.