Portal de Periódicos da CAPES

Protease Activity Enhances Production of Thymic Stromal Lymphopoietin and Basophil Accumulation in Flaky Tail Mice

2013; Elsevier BV; Volume: 182; Issue: 3 Linguagem: Inglês

10.1016/j.ajpath.2012.11.039

1525-2191

Catharina Sagita Moniaga, Se Kyoo Jeong, Gyohei Egawa, Saeko Nakajima, Mariko Hara‐Chikuma, Jeong Eun Jeon, Seung Hun Lee, Toshihiko Hibino, Yoshiki Miyachi, Kenji Kabashima,

Food Allergy and Anaphylaxis Research

Epidermal barrier abnormality due to filaggrin deficiency is an important predisposing factor in the development of atopic dermatitis (AD). In addition, the expression of thymic stromal lymphopoietin (TSLP) in keratinocytes (KCs), induced by barrier disruption, can promote type 2 helper T-cell polarization. Protease activity, including protease-activated receptor-2 (PAR-2), is also known to be involved in epidermal barrier function in AD. However, to our knowledge, the relationship between protease activity and filaggrin deficiency from the perspective of AD has not been elucidated. Flaky tail (Flgft) mice, known to have a mutation in the filaggrin gene, were used to assess the role of protease in KCs in the steady state and the mite-induced AD-like skin inflammation model. In the steady state, the expression and activity levels of endogenous proteases, kallikreins 5, 7, and 14, in the skin and TSLP were higher in Flgft than in control mice. In addition, activation of PAR-2 by its agonist induced the production of TSLP in KCs of Flgft mice, which was abrogated by a newly developed PAR-2 antagonist. Application of the PAR-2 antagonist improved symptoms and basophil accumulation in Flgft mice treated with mite extracts. These results suggest that possibly through the PAR-2 activation in KCs, filaggrin deficiency induces TSLP production and basophil accumulation, which play important roles in the establishment of AD. Epidermal barrier abnormality due to filaggrin deficiency is an important predisposing factor in the development of atopic dermatitis (AD). In addition, the expression of thymic stromal lymphopoietin (TSLP) in keratinocytes (KCs), induced by barrier disruption, can promote type 2 helper T-cell polarization. Protease activity, including protease-activated receptor-2 (PAR-2), is also known to be involved in epidermal barrier function in AD. However, to our knowledge, the relationship between protease activity and filaggrin deficiency from the perspective of AD has not been elucidated. Flaky tail (Flgft) mice, known to have a mutation in the filaggrin gene, were used to assess the role of protease in KCs in the steady state and the mite-induced AD-like skin inflammation model. In the steady state, the expression and activity levels of endogenous proteases, kallikreins 5, 7, and 14, in the skin and TSLP were higher in Flgft than in control mice. In addition, activation of PAR-2 by its agonist induced the production of TSLP in KCs of Flgft mice, which was abrogated by a newly developed PAR-2 antagonist. Application of the PAR-2 antagonist improved symptoms and basophil accumulation in Flgft mice treated with mite extracts. These results suggest that possibly through the PAR-2 activation in KCs, filaggrin deficiency induces TSLP production and basophil accumulation, which play important roles in the establishment of AD. A recently discovered link between the incidence of atopic dermatitis (AD) and loss-of-function mutations in the gene encoding filaggrin (FLG) has demonstrated that skin barrier dysfunction is a critical driving force in the initiation and exacerbation of AD.1Palmer C.N. Irvine A.D. Terron-Kwiatkowski A. Zhao Y. Liao H. Lee S.P. Goudie D.R. Sandilands A. Campbell L.E. Smith F.J. O’Regan G.M. Watson R.M. Cecil J.E. Bale S.J. Compton J.G. DiGiovanna J.J. Fleckman P. Lewis-Jones S. Arseculeratne G. Sergeant A. Munro C.S. El Houate B. McElreavey K. Halkjaer L.B. Bisgaard H. Mukhopadhyay S. McLean W.H. Common loss-of-function variants of the epidermal barrier protein filaggrin are a major predisposing factor for atopic dermatitis.Nat Genet. 2006; 38: 441-446Crossref PubMed Scopus (2190) Google Scholar In addition to serving as a physical barrier between the environment and the body, keratinocytes (KCs) are thought to play a vital role in both innate and adaptive immune responses.2Hammad H. Lambrecht B.N. Dendritic cells and epithelial cells: linking innate and adaptive immunity in asthma.Nat Rev Immunol. 2008; 8: 193-204Crossref PubMed Scopus (494) Google Scholar Stimulated KCs can trigger and modify the activation and differentiation of dendritic cells, B cells, and T cells through the production of cytokines, such as thymic stromal lymphopoietin (TSLP) and IL-33, which induce type 2 helper T-cell (Th2)–polarized immune responses in patients with AD.3Schleimer R.P. Kato A. Kern R. Kuperman D. Avila P.C. Epithelium: at the interface of innate and adaptive immune responses.J Allergy Clin Immunol. 2007; 120: 1279-1284Abstract Full Text Full Text PDF PubMed Scopus (254) Google Scholar The critical association between the abnormal barrier and Th2 polarization in AD may, in part, be explained by the expression of TSLP.4Ebner S. Nguyen V.A. Forstner M. Wang Y.H. Wolfram D. Liu Y.J. Romani N. Thymic stromal lymphopoietin converts human epidermal Langerhans cells into antigen-presenting cells that induce proallergic T cells.J Allergy Clin Immunol. 2007; 119: 982-990Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar, 5Nakajima S. Igyártó B.Z. Honda T. Egawa G. Otsuka A. Hara-Chikuma M. Watanabe N. Ziegler S.F. Tomura M. Inaba K. Miyachi Y. Kaplan D.H. Kabashima K. Langerhans cells are critical in epicutaneous sensitization with protein antigen via thymic stromal lymphopoietin receptor signaling.J Allergy Clin Immunol. 2012; 129: 1048-1055.e6Abstract Full Text Full Text PDF PubMed Scopus (188) Google Scholar TSLP is mainly produced by epithelial cells, including KCs, and is highly expressed in the epidermis of patients with AD.6Ziegler S.F. The role of thymic stromal lymphopoietin (TSLP) in allergic disorders.Curr Opin Immunol. 2010; 22: 795-799Crossref PubMed Scopus (134) Google Scholar, 7Soumelis V, Reche PA, Kanzler H, Yuan W, Edward G, Homey B, Gilliet M, Ho S, Antonenko S, Lauerma A, Smith K, Gorman D, Zurawski S, Abrams J, Menon S, McClanahan T, de Waal-Malefyt Rd R, Bazan F, Kastelein RA, Liu YJ: Human epithelial cells trigger dendritic cell mediated allergic inflammation by producing TSLP. Nat Immunol 2002, 3:673–680Google Scholar In addition, keratinocyte-specific overexpression of TSLP leads to the development of a spontaneous Th2-type skin inflammation in mice8Yoo J. Omori M. Gyarmati D. Zhou B. Aye T. Brewer A. Comeau M.R. Campbell D.J. Ziegler S.F. Spontaneous atopic dermatitis in mice expressing an inducible thymic stromal lymphopoietin transgene specifically in the skin.J Exp Med. 2005; 202: 541-549Crossref PubMed Scopus (460) Google Scholar and accumulation of basophils in the skin.9Siracusa M.C. Saenz S.A. Hill D.A. Kim B.S. Headley M.B. Doering T.A. Wherry E.J. Jessup H.K. Siegel L.A. Kambayashi T. Dudek E.C. Kubo M. Cianferoni A. Spergel J.M. Ziegler S.F. Comeau M.R. Artis D. TSLP promotes interleukin-3-independent basophil haematopoiesis and type 2 inflammation.Nature. 2011; 477: 229-233Crossref PubMed Scopus (368) Google Scholar Recently, it has been shown that patients with Netherton syndrome, in which affected individuals experience a significant predisposition for AD, exhibit an elevated level of TSLP in their skin.10Briot A. Deraison C. Lacroix M. Bonnart C. Robin A. Besson C. Dubus P. Hovnanian A. Kallikrein 5 induces atopic dermatitis-like lesions through PAR2-mediated thymic stromal lymphopoietin expression in Netherton syndrome.J Exp Med. 2009; 206: 1135-1147Crossref PubMed Scopus (377) Google Scholar In patients with Netherton syndrome, the lack of the protease inhibitor, lymphoepithelial kazal–type–related inhibitor, results in the up-regulation of epidermal protease kallikreins (KLKs), which, in turn, activates protease-activated receptor 2 (PAR-2) on KCs, causing overexpression of TSLP in the skin. Activated PAR-2 has also induced the expression of TSLP by airway epithelial cells.11Kouzaki H. O’Grady S.M. Lawrence C.B. Kita H. Proteases induce production of thymic stromal lymphopoietin by airway epithelial cells through protease-activated receptor-2.J Immunol. 2009; 183: 1427-1434Crossref PubMed Scopus (267) Google Scholar The protease activity in particular allergens, such as papain, has been shown to be required for the induction of Th2 and IgE responses.12Kheradmand F. Kiss A. Xu J. Lee S.H. Kolattukudy P.E. Corry D.B. A protease-activated pathway underlying Th cell type 2 activation and allergic lung disease.J Immunol. 2002; 169: 5904-5911Crossref PubMed Scopus (250) Google Scholar, 13Gough L. Schulz O. Sewell H.F. Shakib F. The cysteine protease activity of the major dust mite allergen Der p 1 selectively enhances the immunoglobulin E antibody response.J Exp Med. 1999; 190: 1897-1902Crossref PubMed Scopus (186) Google Scholar It has been reported that papain directly activated basophils and recruited them to the lymph nodes during primary immune responses.14Sokol C.L. Chu N.Q. Yu S. Nish S.A. Laufer T.M. Medzhitov R. Basophils function as antigen-presenting cells for an allergen-induced T helper type 2 response.Nat Immunol. 2009; 10: 713-720Crossref PubMed Scopus (479) Google Scholar, 15Tang H. Cao W. Kasturi S.P. Ravindran R. Nakaya H.I. Kundu K. Murthy N. Kepler T.B. Malissen B. Pulendran B. The T helper type 2 response to cysteine proteases requires dendritic cell-basophil cooperation via ROS-mediated signaling.Nat Immunol. 2010; 11: 608-617Crossref PubMed Scopus (259) Google Scholar Despite the fact that basophils represent 2 minutes. For the histological analysis, the dorsal skin was stained with H&E. The histological findings were evaluated as previously reported.30Nakajima S. Honda T. Sakata D. Egawa G. Tanizaki H. Otsuka A. Moniaga C.S. Watanabe T. Miyachi Y. Narumiya S. Kabashima K. Prostaglandin I2-IP signaling promotes Th1 differentiation in a mouse model of contact hypersensitivity.J Immunol. 2010; 184: 5595-5603Crossref PubMed Scopus (52) Google Scholar In brief, samples were scored for the severity and character of the inflammatory response using a subjective grading scale. Responses were graded as follows: 0, no response; 1, minimal response; 2, mild response; 3, moderate response; and 4, marked response. The total histological score was calculated as the sum of the scores concerning inflammation, neutrophils, mononuclear cells, edema, and epithelial hyperplasia. The number of basophils was calculated as the average from three different fields of each sample (original magnification, ×40). mRNA was isolated using TRIzol (Invitrogen, Tokyo, Japan), per the manufacturer’s protocol, and subjected to quantitative real-time PCR analysis, using the housekeeping gene GAPDH as an internal control.20Moniaga C.S. Egawa G. Kawasaki H. Hara-Chikuma M. Honda T. Tanizaki H. Nakajima S. Otsuka A. Matsuoka H. Kubo A. Sakabe J. Tokura Y. Miyachi Y. Amagai M. Kabashima K. Flaky tail mouse denotes human atopic dermatitis in the steady state and by topical application with Dermatophagoides pteronyssinus extract.Am J Pathol. 2010; 176: 2385-2393Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar All data are presented as fold change against each control (mean of non-stimulated cells or control mice). For the enzyme-linked immunosorbent assay (ELISA), skin samples were chopped and homogenized using a Polytron RT-3000 instrument (Kinematica AG, Bohemia, NY) in cell lysis buffer (Epitomics, Burlingame, CA). The protein concentration was quantified using a Bio-Rad Protein assay (Bio-Rad, Marnes-la-Coquette, France). The concentrations of TSLP protein were measured using a TSLP DuoSet Kit (R&D Systems). To induce murine AD-like skin lesions, 40 mg of 0.5% Dp (Biostir, Kobe, Japan) in white petrolatum was topically applied to the ears and upper back three times a week for 7 weeks.20Moniaga C.S. Egawa G. Kawasaki H. Hara-Chikuma M. Honda T. Tanizaki H. Nakajima S. Otsuka A. Matsuoka H. Kubo A. Sakabe J. Tokura Y. Miyachi Y. Amagai M. Kabashima K. Flaky tail mouse denotes human atopic dermatitis in the steady state and by topical application with Dermatophagoides pteronyssinus extract.Am J Pathol. 2010; 176: 2385-2393Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar Petrolatum without Dp was used as a control. Alternatively, mice were treated with 0.5% Dp and 1% PAR-2 antagonist in petrolatum. Dp body product (1 g) contained 1.78 mg total protein with 2.47 μg of Dp protein (Der p1). Unless otherwise indicated, data were presented as the means ± SD. P values were calculated according to the two-tailed Student’s t-test. P < 0.05 were considered to indicate significant difference between the indicated groups. In the stratum corneum, filaggrin is proteolytically degraded into a pool of free amino acids, including histidine and glutamine, which are further converted to urocanic acid and pyrrolidine carboxylic acid, respectively. These metabolites are thought to play a key role in maintaining the acidic pH of the stratum corneum.31Krien P.M. Kermici M. Evidence for the existence of a self-regulated enzymatic process within the human stratum corneum: an unexpected role for urocanic acid.J Invest Dermatol. 2000; 115: 414-420Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar, 32O’Regan G.M. Sandilands A. McLean W.H. Irvine A.D. Filaggrin in atopic dermatitis.J Allergy Clin Immunol. 2008; 122: 689-693Abstract Full Text Full Text PDF PubMed Scopus (265) Google Scholar It is already known that the skin surface pH is higher in patients with AD than in healthy controls.33Eberlein-Konig B. Schafer T. Huss-Marp J. Darsow U. Mohrenschlager M. Herbert O. Abeck D. Kramer U. Behrendt H. Ring J. Skin surface pH, stratum corneum hydration, trans-epidermal water loss and skin roughness related to atopic eczema and skin dryness in a population of primary school children.Acta Derm Venereol. 2000; 80: 188-191Crossref PubMed Scopus (172) Google Scholar, 34Choi S.J. Song M.G. Sung W.T. Lee D.Y. Lee J.H. Lee E.S. Yang J.M. Comparison of transepidermal water loss, capacitance and pH values in the skin between intrinsic and extrinsic atopic dermatitis patients.J Korean Med Sci. 2003; 18: 93-96Crossref PubMed Scopus (48) Google Scholar In addition, filaggrin concentrations in the stratum corneum are significantly lower in FLG mutation carriers than in healthy donors.32O’Regan G.M. Sandilands A. McLean W.H. Irvine A.D. Filaggrin in atopic dermatitis.J Allergy Clin Immunol. 2008; 122: 689-693Abstract Full Text Full Text PDF PubMed Scopus (265) Google Scholar, 35Kezic S. Kammeyer A. Calkoen F. Fluhr J.W. Bos J.D. Natural moisturizing factor components in the stratum corneum as biomarkers of filaggrin genotype: evaluation of minimally invasive methods.Br J Dermatol. 2009; 161: 1098-1104Crossref PubMed Scopus (111) Google Scholar Given this information, we evaluated the skin surface pH and the amino acid content of Flgft mice. We found that skin surface pH in Flgft mice was similar to that of control mice during the neonatal period when mice did not exhibit any clinical dermatitis, but increased with age and with the development of clinical dermatitis (eg, scaling and erythema) (Figure 1A). These data suggest a parallel correlation between skin inflammatory status and the surface pH. Histidine content, meanwhile, decreased in Flgft mice, although glutamine content in Flgft mice was similar to that in control mice (Figure 1B), suggesting that a low histidine level might be involved, at least in part, in the elevation of skin surface pH under filaggrin-deficient conditions. The elevation of skin surface pH induces the