Enzymatically Active Papain Preferentially Induces an Allergic Response in Mice

102

The major proteolytic allergen derived from the house dust mite Dermatophagoides pteronyssinus, Der p1, is one of the most clinically relevant allergens worldwide. In the present study, we evaluate the contribution of the proteolytic activity and structure of a highly purified rDer p 1 to immune responses. Mice were i.p. immunized with three forms of rDer p 1 adsorbed to Alum: one enzymatically active, one treated with an irreversible cysteine protease-specific inhibitor, E-64, and one heat denatured. Immunization with E-64-treated or heat-denatured rDer p 1 elicited much less production of serum total IgE and not only rDer p 1-specific IgE but also IgGs compared with immunization with active rDer p 1. Assays for Ab-binding and its inhibition and structural analyses indicated that E-64-treated rDer p 1 retained its global structure and conformational B cell epitopes. A proliferative response and production of IL-5 by spleen cells restimulated with rDer p 1 were observed on immunization with the active rDer p 1 but not E-64-treated rDer p 1. The cells from mice immunized with heat-denatured rDer p 1 exhibited the highest levels of proliferation and production of IL-5 and IFN-γ. The results indicate that the proteolytic activity of the highly purified rDer p 1 crucially commits to the sensitization process, including both IgE and IgG responses. Additionally, we demonstrated immunogenic differences by functional or structural manipulations of the rDer p 1. The findings have implications for sensitization to this relevant allergen in humans and for the design of modified allergen-vaccines for future allergen-specific immunotherapy.

Section: Discussionsupporting

confidence: 47%

Crucial Commitment of Proteolytic Activity of a Purified Recombinant Major House Dust Mite Allergen Der p1 to Sensitization toward IgE and IgG Responses

Kikuchi

Takai²,

Kuhara³

et al. 2006

102

“…immunization of mice with natural (7) or rDer p 1 plus Alum (8) or rDer f 1 with no adjuvants (9) are dependent on the protease activity of these HDM major protease allergens. Partial reduction (15,46) or elimination (15) of Ab production has been reported for E64-treated or heat-denatured papain, respectively, when s.c. administered with no adjuvants. The cysteine protease inhibitor we used, E64, irreversibly binds to the catalytic site of papain-like cysteine proteases without altering their tertiary structure (8,47).…”

Section: Discussionmentioning

confidence: 97%

Subcutaneous Allergic Sensitization to Protease Allergen Is Dependent on Mast Cells but Not IL-33: Distinct Mechanisms between Subcutaneous and Intranasal Routes

Kamijo

Suzuki

Hara

et al. 2016

Protease activity of papain, a plant-derived occupational allergen homologous to mite major allergens, is essential to IgE/IgG1 production and lung eosinophilia induced by intranasal papain administration in mice, and IL-33 contributes to these responses. In this work, we investigate skin and Ab responses induced by s.c. papain administration into ear lobes and responses induced by subsequent airway challenge with papain. Subcutaneous papain injection induced swelling associated with increased epidermal thickness, dermal inflammation, serum IgE/IgG1 responses, and Th2 cytokine production in draining lymph node cells restimulated in vitro. These responses were markedly less upon s.c. administration of protease inhibitor-treated papain. Results obtained by using mast cell–deficient mice and reconstitution of tissue mast cells suggested the contribution of mast cells to papain-specific IgE/IgG1 responses and eosinophil infiltration. The responses were equivalent between wild-type and IL-33−/− mice. After the subsequent airway challenge, the s.c. presensitized wild-type mice showed more severe lung eosinophilia than those without the presensitization. The presensitized IL-33−/− mice showed modest lung eosinophilia, which was absent without the presensitization, but its severity and IgE boost by the airway challenge were markedly less than the presensitized wild-type mice, in which protease activity of inhaled papain contributed to the responses. The results suggest that mechanisms for the protease-dependent sensitization differ between skin and airway and that cooperation of mast cell–dependent, IL-33–independent initial sensitization via skin and protease-induced, IL-33–mediated mechanism in re-exposure via airway to protease allergens maximizes the magnitude of the transition from skin inflammation to asthma in natural history of progression of allergic diseases.

“…administered with Alum to mice. Partial reduction (14,16) or elimination (14) of Ab production has been reported in E64-treated or heat-denatured papain, respectively, when s.c. administered with no adjuvants. In the current study, we established a mouse allergic airway inflammation model associated with IgE/IgG1 Ab production and lung eosinophilia, which were induced by repeated airway exposure of naive mice to protease allergens without using the Th2-skewing adjuvants such as Alum (Figs.…”

Section: Discussionmentioning

confidence: 97%

“…Protease activity of house dust mite (HDM) major group 1 allergens (Der f 1 and Der p 1), two of the most important allergens common worldwide, has been shown to cause barrier dysfunction, to induce the production of proinflammatory cytokines in epithelial cells and keratinocytes, to cleave various molecules, to modulate functions of various cell types, and to induce Th2 responses (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11). Papain, a papaya fruit-derived occupational protease allergen, stimulates epithelial cells (12,13) and mouse basophils in vitro (14,15) and induces Th2 responses (14)(15)(16)(17) and IL-33-dependent innate responses of lung eosinophilia in mice (18). HDM-derived Der f 1 and Der p 1 and plantderived papain belong to the same family of cysteine proteases, family C1 of clan CA (19,20).…”

mentioning

confidence: 99%

IL-33–Mediated Innate Response and Adaptive Immune Cells Contribute to Maximum Responses of Protease Allergen–Induced Allergic Airway Inflammation

Kamijo

Takeda

Tokura

et al. 2013

151

164

How the innate and adaptive immune systems cooperate in the natural history of allergic diseases has been largely unknown. Plant-derived allergen, papain, and mite allergens, Der f 1 and Der p 1, belong to the same family of cysteine proteases. We examined the role of protease allergens in the induction of Ab production and airway inflammation after repeated intranasal administration without adjuvants and that in basophil/mast cell stimulation in vitro. Papain induced papain-specific IgE/IgG1 and lung eosinophilia. Der f 1 induced Der f 1–specific IgG1 and eosinophilia. Although papain-, Der f 1–, and Der p 1–stimulated basophils expressed allergy-inducing cytokines, including IL-4 in vitro, basophil-depleting Ab and mast cell deficiency did not suppress the papain-induced in vivo responses. Protease inhibitor–treated allergens and a catalytic site mutant did not induce the responses. These results indicate that protease activity is essential to Ab production and eosinophilia in vivo and basophil activation in vitro. IL-33–deficient mice lacked eosinophilia and had reduced papain-specific IgE/IgG1. Coadministration of OVA with papain induced OVA-specific IgE/IgG1, which was reduced in IL-33–deficient mice. We demonstrated IL-33 release, subsequent IL-33–dependent IL-5/IL-13 release, and activation of T1/ST2-expressing lineage−CD25+CD44+ innate lymphoid cells in the lung after papain inhalation, suggesting the contribution of the IL-33–type 2 innate lymphoid cell–IL-5/IL-13 axis to the papain-induced airway eosinophilia. Rag2-deficient mice, which lack adaptive immune cells, showed significant, but less severe, eosinophilia. Collectively, these results suggest cooperation of adaptive immune cells and IL-33–responsive innate cells in protease-dependent allergic airway inflammation.