RESUMO
Casein, the major allergen in cow's milk, presents a significant challenge in providing nutritional support for children with allergies. To address this issue, we investigated a composite enzyme, comprising papain and chymotrypsin, to reduce the allergenicity of casein. Enzymatic hydrolysis induced substantial structural changes in casein, diminishing its affinity for specific IgE and IgG antibodies. Additionally, in a BALB/c mouse model, casein hydrolysate alleviated allergic symptoms, evidenced by lower serum IgE and IgG levels, reduced plasma histamine, and decreased Th2 cytokine release during cell co-culture. Peptidomic analysis revealed a 52.38% and 60% reduction in peptides containing IgE epitopes in casein hydrolyzed by the composite enzyme compared to papain and chymotrypsin, respectively, along with a notable absence of previously reported T cell epitopes. These results demonstrate the potential of enzyme combinations to enhance the efficiency of epitope destruction in allergenic proteins, providing valuable insights into the development of hypoallergenic dairy products.
Assuntos
Alérgenos , Caseínas , Quimotripsina , Hipersensibilidade a Leite , Papaína , Animais , Bovinos , Feminino , Humanos , Camundongos , Alérgenos/imunologia , Alérgenos/química , Caseínas/imunologia , Caseínas/química , Quimotripsina/química , Quimotripsina/imunologia , Epitopos de Linfócito B/imunologia , Epitopos de Linfócito B/química , Epitopos de Linfócito T/imunologia , Epitopos de Linfócito T/química , Imunoglobulina E/imunologia , Camundongos Endogâmicos BALB C , Leite/química , Leite/imunologia , Hipersensibilidade a Leite/imunologia , Hipersensibilidade a Leite/prevenção & controle , Papaína/imunologia , Papaína/químicaRESUMO
Osteoarthritis (OA) is a chronic age-related progressive joint disorder. Degradation of the cartilage extracellular matrix (ECM) is considered a hallmark of OA and may be a target for new therapeutic methods. Schisandrae Fructus (SF) has been shown to be effective in treating OA. The major active components of SF are lignans. However, the targets of SF and the pharmacological mechanisms underlying the effects of SF lignans in the treatment of OA have not been elucidated. Therefore, based on network pharmacology, this research predicted the treatment targets of six lignans in SF, constructed a protein-protein interaction network and identified 15 hub genes in the OA-target protein-protein interaction network. Through Gene Ontology function and pathway analyses, the gene functions of lignans in the treatment of OA were determined. Finally, the anti-OA effects of lignans and underlying mechanisms identified in the network pharmacology analysis were verified by molecular docking, real-time PCR and western blotting in vitro. The biological processes of the genes and proteins targeted by lignans in the treatment of OA included the immune response, inflammatory response, cell signal transduction and phospholipid metabolism. Moreover, 20 metabolic pathways were enriched. Network pharmacology, molecular docking and in vitro and in vivo experimental results revealed that SF, schisanhenol and gamma-schisandrin inhibited EGFR and MAPK14 gene expression by inhibiting SRC gene expression and activity and then decreased MMP 13 and collagen II protein and gene expression. This research provides a basis for further study of the anti-OA effects and mechanisms of SF, schisanhenol and gamma-schisandrin.
Assuntos
Artrite Experimental/tratamento farmacológico , Lignanas/farmacologia , Osteoartrite/tratamento farmacológico , Extratos Vegetais/farmacologia , Schisandra/química , Animais , Artrite Experimental/imunologia , Frutas/química , Humanos , Lignanas/isolamento & purificação , Lignanas/uso terapêutico , Masculino , Camundongos , Simulação de Acoplamento Molecular , Farmacologia em Rede , Osteoartrite/imunologia , Papaína/administração & dosagem , Papaína/imunologia , Extratos Vegetais/isolamento & purificação , Extratos Vegetais/uso terapêutico , Mapas de Interação de Proteínas/efeitos dos fármacosRESUMO
INTRODUCTION: Epicutaneous (e.c.) allergen exposure is an important route of sensitization toward allergic diseases in the atopic march. Allergen sources such as house dust mites contain proteases that involve in the pathogenesis of allergy. Prostanoids produced via pathways downstream of cyclooxygenases (COXs) regulate immune responses. Here, we demonstrate effects of COX inhibition with nonsteroidal anti-inflammatory drugs (NSAIDs) on e.c. sensitization to protease allergen and subsequent airway inflammation in mice. METHODS: Mice were treated with NSAIDs during e.c. sensitization to a model protease allergen, papain, and/or subsequent intranasal challenge with low-dose papain. Serum antibodies, cytokine production in antigen-restimulated skin or bronchial draining lymph node (DLN) cells, and airway inflammation were analyzed. RESULTS: In e.c. sensitization, treatment with a nonspecific COX inhibitor, indomethacin, promoted serum total and papain-specific IgE response and Th2 and Th17 cytokine production in skin DLN cells. After intranasal challenge, treatment with indomethacin promoted allergic airway inflammation and Th2 and Th17 cytokine production in bronchial DLN cells, which depended modestly or largely on COX inhibition during e.c. sensitization or intranasal challenge, respectively. Co-treatment with COX-1-selective and COX-2-selective inhibitors promoted the skin and bronchial DLN cell Th cytokine responses and airway inflammation more efficiently than treatment with either selective inhibitor. CONCLUSION: The results suggest that the overall effects of COX downstream prostanoids are suppressive for development and expansion of not only Th2 but also, unexpectedly, Th17 upon exposure to protease allergens via skin or airways and allergic airway inflammation.
Assuntos
Alérgenos/imunologia , Ciclo-Oxigenase 1/metabolismo , Ciclo-Oxigenase 2/metabolismo , Inibidores de Ciclo-Oxigenase/farmacologia , Peptídeo Hidrolases/imunologia , Células Th17/imunologia , Células Th2/imunologia , Animais , Anti-Inflamatórios não Esteroides/farmacologia , Diferenciação Celular , Feminino , Imunização , Camundongos , Papaína/imunologia , Hipersensibilidade Respiratória/imunologia , Hipersensibilidade Respiratória/metabolismo , Hipersensibilidade Respiratória/patologia , Pele/efeitos dos fármacos , Pele/imunologia , Pele/metabolismo , Subpopulações de Linfócitos T/efeitos dos fármacos , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismo , Células Th17/efeitos dos fármacos , Células Th17/metabolismo , Células Th2/efeitos dos fármacos , Células Th2/metabolismoRESUMO
Environmental allergen sources such as house dust mites contain proteases, which are frequently allergens themselves. Inhalation with the exogenous proteases, such as a model of protease allergen, papain, to airways evokes release and activation of IL-33, which promotes innate and adaptive allergic airway inflammation and Th2 sensitization in mice. Here, we examine whether epicutaneous (e.c.) vaccination with antigens with and without protease activity shows prophylactic effect on the Th airway sensitization and Th2-medated airway inflammation, which are driven by exogenous or endogenous IL-33. E.c. vaccination with ovalbumin restrained ovalbumin-specific Th2 airway sensitization and/or airway inflammation on subsequent inhalation with ovalbumin plus papain or ovalbumin plus recombinant IL-33. E.c. vaccination with papain or protease inhibitor-treated papain restrained papain-specific Th2 and Th9 airway sensitization, eosinophilia, and infiltration of IL-33-responsive Th2 and group 2 innate lymphoid cells on subsequent inhalation with papain. However, e.c. vaccination with papain but not protease inhibitor-treated papain induced Th17 response in bronchial draining lymph node cells. In conclusions, we demonstrated that e.c. allergen vaccination via intact skin in mice restrained even protease allergen-activated IL-33-driven airway Th2 sensitization to attenuate allergic airway inflammation and that e.c. vaccination with protease allergen attenuated the airway inflammation similar to its derivative lacking the protease activity, although the former but not the latter promoted Th17 development. In addition, the present study suggests that modified allergens, of which Th17-inducing e.c. adjuvant activity such as the protease activity was eliminated, might be preferable for safer clinical applications of the e.c. allergen administration.
Assuntos
Inflamação/imunologia , Ovalbumina/imunologia , Papaína/antagonistas & inibidores , Papaína/imunologia , Células Th17 , Células Th2/imunologia , Vacinação/métodos , Administração por Inalação , Animais , Feminino , Imunoglobulina E/imunologia , Inflamação/prevenção & controle , Mediadores da Inflamação/imunologia , Interleucina-33/administração & dosagem , Interleucina-33/imunologia , Camundongos , Ovalbumina/administração & dosagem , Ovalbumina/sangue , Papaína/administração & dosagem , Células Th17/imunologiaRESUMO
Background: Eosinophils develop from CD34+ progenitor cells in the bone marrow under the influence of interleukin (IL)-5. Several cell types produce IL-5, including type 2 innate lymphoid cells (ILC2s). The alarmin cytokine IL-33 is known to activate ILC2s in mucosal tissues, but little is known about IL-33-responsive ILC2s in the bone marrow in allergen-induced airway inflammation. Methods: Wild type (WT) and Rag1 deficient (Rag1-/-) mice, which lack mature T and B cells, received intranasal doses of papain to induce acute allergic inflammation. In some experiments, mice were pre-treated with anti-IL-5 prior to the papain challenge. Furthermore, recombinant IL-33 was administered to WT mice, Rag1-/- mice, lymphocyte deficient mice (Rag2-/-Il2rg-/-) and to ex vivo whole bone marrow cultures. Bone marrow eosinophils and ILC2s were analyzed by flow cytometry. Eosinophil count was assessed by differential cell count and secreted IL-5 from bone marrow cells by ELISA. Results: Intranasal administration of papain or IL-33 increased the number of mature eosinophils in the bone marrow despite the absence of adaptive immune cells in Rag1-/- mice. In parallel, an increased number of eosinophils was observed in the airways together with elevated levels of Eotaxin-2/CCL24. Bone marrow ILC2s were increased after papain or IL-33 administration, whereas ILC2s was found to be increased at baseline in Rag1-/- mice compared to WT mice. An upregulation of the IL-33 receptor (ST2) expression on bone marrow ILC2s was observed after papain challenge in both Rag1-/- and WT mice which correlated to increased number of bone marrow eosinophilia. Furthermore, an increased number of ST2+ mature eosinophils in the bone marrow was observed after papain challenge, which was further dependent on IL-5. In addition, bone marrow-derived ILC2s from both mouse strains produced large amounts of IL-5 ex vivo after IL-33 stimulation of whole bone marrow cultures. In contrast, IL-33-induced bone marrow and airway eosinophilia were abolished in the absence of ILC2s in Rag2-/-Il2rg-/- mice and no production of IL-5 was detected in IL-33-stimulated bone marrow cultures. Conclusion: These findings establish bone marrow ILC2s and the IL-33/ST2 axis as promising targets for modulation of uncontrolled IL-5-dependent eosinophilic diseases including asthma.
Assuntos
Eosinofilia/imunologia , Proteína 1 Semelhante a Receptor de Interleucina-1/imunologia , Interleucina-33/imunologia , Imunidade Adaptativa , Alérgenos/administração & dosagem , Alérgenos/imunologia , Animais , Asma/etiologia , Asma/imunologia , Células da Medula Óssea/imunologia , Modelos Animais de Doenças , Eosinofilia/etiologia , Feminino , Imunidade Inata , Interleucina-5/biossíntese , Linfócitos/imunologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Papaína/administração & dosagem , Papaína/imunologia , Eosinofilia Pulmonar/etiologia , Eosinofilia Pulmonar/imunologiaRESUMO
BACKGROUND: Epithelial cells (ECs) play a crucial role in allergic sensitization to inhaled protease allergens by instructing type 2 innate lymphoid cells (ILC2) and dendritic cells (DCs) via release of pro-type 2 cytokines, particularly interleukin-33 (IL-33). Leukotriene B4 (LTB4) is a well-known leukocyte chemoattractant via engagement of its receptor 1 (BLT1). However, the role of LTB4-BLT1 axis in allergic sensitization via activation of ECs is still unknown. METHODS: We evaluated the effect of LTB4-BLT1 axis on IL-33 expression and ILC2 activation in vivo and in vitro. Chimeric mice were established to evaluate the contribution of BLT1 expression in nonimmune cell to allergic sensitization. RESULTS: Genetical or pharmacological interruption of LTB4-BLT1 axis during sensitization phase markedly reduced papain-induced IL-33 expression, decreased ILC2 activation and DC migration, thereby impairing the priming of allergic Th2 responses. Furthermore, papain inhalation induced a rapid release of LTB4 preceding IL-33, and intranasal administration of LTB4 to naïve WT mice significantly increased IL-33 expression and ILC2 activation in lung, which was absent in Il33-/- or Ltb4r1-/- mice. Furthermore, BLT1 was expressed in primary mouse ECs or normal human bronchial ECs (NHBE), and papain induced LTB4 release by NHBE, which in turn amplified IL-33 production dependent on Akt activation via BLT1. Consequently, bone marrow chimeric mice lacking BLT1 in radio-resistant structural cells failed to develop allergic lung inflammation to papain. CONCLUSION: Our study reveals a functional role of LTB4-BLT1 axis in nonimmune cells, most likely lung ECs, in controlling allergic sensitization as an upstream regulator of IL-33.
Assuntos
Células Epiteliais/metabolismo , Hipersensibilidade/imunologia , Hipersensibilidade/metabolismo , Interleucina-33/biossíntese , Receptores do Leucotrieno B4/metabolismo , Transdução de Sinais , Alérgenos/imunologia , Animais , Citocinas/metabolismo , Hipersensibilidade/genética , Imunização , Interleucina-33/genética , Leucotrieno B4/metabolismo , Ativação Linfocitária/imunologia , Linfócitos/imunologia , Linfócitos/metabolismo , Camundongos , Papaína/imunologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismoRESUMO
Eosinophil infiltration, a hallmark of allergic asthma, is essential for type 2 immune responses. How the initial eosinophil recruitment is regulated by lung dendritic cell (DC) subsets during the memory stage after allergen challenge is unclear. Here, we show that the initial eosinophil infiltration is dependent on lung cDC1s, which require nitric oxide (NO) produced by inducible NO synthase from lung CD24-CD11b+ DC2s for inducing CCL17 and CCL22 to attract eosinophils. During late phase responses after allergen challenge, lung CD24+ cDC2s inhibit eosinophil recruitment through secretion of TGF-ß1, which impairs the expression of CCL17 and CCL22. Our data suggest that different lung antigen-presenting cells modulate lung cDC1-mediated eosinophil recruitment dynamically, through secreting distinct soluble factors during the memory stage of chronic asthma after allergen challenge in the mouse.
Assuntos
Asma/imunologia , Células Dendríticas/imunologia , Eosinófilos/imunologia , Alérgenos/imunologia , Animais , Quimiocina CCL17/imunologia , Quimiocina CCL17/metabolismo , Quimiocina CCL22/imunologia , Quimiocina CCL22/metabolismo , Doença Crônica , Células Dendríticas/metabolismo , Modelos Animais de Doenças , Feminino , Humanos , Pulmão/citologia , Pulmão/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Óxido Nítrico/metabolismo , Óxido Nítrico Sintase Tipo II/metabolismo , Papaína/imunologia , Fator de Crescimento Transformador beta1/genética , Fator de Crescimento Transformador beta1/metabolismoAssuntos
Alérgenos/administração & dosagem , Hiper-Reatividade Brônquica/imunologia , Inflamação/imunologia , Papaína/administração & dosagem , Papaína/imunologia , Administração Intranasal , Alérgenos/química , Animais , Inflamação/induzido quimicamente , Camundongos , Camundongos Endogâmicos BALB C , Pele/efeitos dos fármacos , Pele/imunologiaRESUMO
There was a significant amount of non-specific, but not of allergen (e.g., papain, mite feces and four kinds of pollen)-specific, IgE antibodies (Abs) in the sera of normal mice. An i.n. injection of each allergen without adjuvant into mice caused an increase in total IgE Ab titers with a similar time course in the serum. However, the stage of initiation of allergy varied from allergen to allergen. Submandibular lymph node cells from normal mice contained papain-, but not mite feces- or pollen-specific IgE+ cells and an i.n. injection of papain induced papain-specific IgE Abs in the serum. In contrast, one (i.n.) or two (i.n. and s.c) injections of mite feces induced neither mite feces-specific IgE+ cells in the lymph nodes nor mite feces-specific IgE Abs in the serum. I.n. sensitization with cedar pollen induced cedar pollen-specific IgE+ small B cells in the lymph nodes on Day 10, when non-specific IgE Ab titers reached a peak in the serum, implying induction of related allergen-specific IgE+ small cells as well. In fact, a second (s.c.) injection of ragweed (or cedar) pollen into mice sensitized i.n. once with cedar (or ragweed) pollen, but not with mite feces, induced a large amount of ragweed (or cedar) pollen-specific IgE Abs in the serum. These results indicate that when firstly-sensitized non-specific IgE+ small B cells in mouse lymph nodes include some secondly-sensitized allergen-specific ones, mice produce IgE Abs specific for the secondly-injected allergen.
Assuntos
Alérgenos/imunologia , Formação de Anticorpos/imunologia , Linfócitos B/imunologia , Imunoglobulina E/imunologia , Adjuvantes Imunológicos , Animais , Proteínas de Artrópodes/imunologia , Sobrevivência Celular , Fezes , Imunoglobulina E/sangue , Linfonodos/imunologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Ácaros , Papaína/imunologia , Pólen/imunologiaRESUMO
Coronaviruses (CoVs), such as human coronavirus NL63 (HCoV-NL63), severe acute respiratory syndrome CoV (SARS-CoV), murine hepatitis virus (MHV), porcine epidemic diarrhea virus (PEDV), and Middle East Respiratory Syndrome Coronavirus (MERS-CoV), encode papain-like (PL) proteases that inhibit Sendai virus- (SeV-) induced interferon (IFN-ß) production. Recently, the crystal structure of transmissible gastroenteritis virus (TGEV) PL1 has been solved, which was similar to that of SARS-CoV PL2pro, which may antagonize host innate immunity. However, very little is known about whether TGEV PL1 can antagonize host innate immune response. Here, we presented evidence that TGEV PL1 encoded by the replicase gene could suppress the IFN-ß expression and inhibit the nuclear translocation of interferon regulatory factor 3 (IRF3). The ability to antagonize IFN-ß production was dependent on the intact catalytic activity of PL1. Furthermore, TGEV PL1 exerted deubiquitinase (DUB) activity which strongly inhibited the retinoic acid-induced gene I- (RIG-1-) and stimulator of interferon gene- (STING-) dependent IFN expression. Our data collectively suggest that TGEV PL1 can inhibit the IFN-ß expression and interfere with RIG-1- and STING-mediated signaling through a viral DUB activity. Our study has yielded strong evidence for the TGEV PL1 mechanisms that counteract the host innate immunity.
Assuntos
Interações Hospedeiro-Patógeno/genética , Imunidade Inata/genética , Interferon beta/genética , Papaína/genética , Vírus da Gastroenterite Transmissível/genética , Animais , Proteases Semelhantes à Papaína de Coronavírus , Proteína DEAD-box 58/genética , Enzimas Desubiquitinantes/genética , Células HEK293 , Humanos , Fator Regulador 3 de Interferon/genética , Interferon beta/biossíntese , Proteínas de Membrana/genética , Papaína/química , Papaína/imunologia , RNA Polimerase Dependente de RNA/genética , Receptores Imunológicos , Suínos , Vírus da Gastroenterite Transmissível/química , Vírus da Gastroenterite Transmissível/patogenicidade , Ubiquitina/genéticaRESUMO
The genital mucosa is a barrier that is constantly exposed to a variety of pathogens, allergens, and external stimuli. Although both allergen exposure and parasite infections frequently occur in the genital area, the mechanism by which immune responses-particularly type 2 immunity-are induced has rarely been studied in the genital mucosa. Here, we demonstrate the induction of T helper type 2 (Th2) immunity in the genital mucosa in response to a model allergen, the protease papain. Intravaginal papain immunization induced type 2 immunity in a manner that was dependent on protease activity and the estrous phase of the mice. In addition, IL-33 was released from the vaginal epithelia after intravaginal papain immunization, leading to the activation of type 2 innate lymphoid cells (ILC2s). Moreover, the IL-33-MyD88 (myeloid differentiation primary response gene 88) signaling pathway was critical for the induction of type 2 immunity. We also found that Th2 differentiation in response to intravaginal papain treatment requires a specific dendritic cell (DC) subset that is controlled by interferon regulatory factor 4 (IRF4). These findings suggest that type 2 immunity is induced by a unique mechanism in the genital tract, which is an important, but often overlooked, barrier surface.
Assuntos
Genitália Feminina/imunologia , Imunização/métodos , Papaína/imunologia , Células Th2/imunologia , Animais , Células Cultivadas , Citocinas/imunologia , Citocinas/metabolismo , Feminino , Genitália Feminina/metabolismo , Interleucina-33/imunologia , Interleucina-33/metabolismo , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Fator 88 de Diferenciação Mieloide/imunologia , Fator 88 de Diferenciação Mieloide/metabolismo , Papaína/metabolismo , Células Th2/metabolismo , Vagina/imunologia , Vagina/metabolismoRESUMO
Allergic asthma is characterized by a strong Th2 response with inflammatory cell recruitment and structural changes in the lung. Papain is a protease allergen disrupting the airway epithelium triggering a rapid inflammation with eosinophilia mediated by innate lymphoid cell activation (ILC2) and leading to a Th2 immune response. In this study, we focused on inflammatory responses to a single exposure to papain and showed that intranasal administration of papain results in the recruitment of inflammatory cells, including neutrophils and eosinophils with a rapid production of IL-1α, IL-1ß, and IL-33. The inflammatory response is abrogated in the absence of IL-1R1 and MyD88. To decipher the cell type(s) involved in MyD88-dependent IL-1R1/MyD88 signaling, we used new cell-specific MyD88-deficient mice and found that the deletion of MyD88 signaling in single cell types such as T cells, epithelial cells, CD11c-positive or myeloid cells leads to only a partial inhibition compared to complete absence of MyD88, suggesting that several cell types contribute to the response. Importantly, the inflammatory response is largely ST2 and IL-36R independent. In conclusion, IL-1R1 signaling via MyD88 is critical for the first step of inflammatory response to papain.
Assuntos
Alérgenos/imunologia , Imunidade Inata , Pulmão/imunologia , Fator 88 de Diferenciação Mieloide/metabolismo , Papaína/imunologia , Pneumonia/imunologia , Receptores Tipo I de Interleucina-1/metabolismo , Alérgenos/administração & dosagem , Animais , Eosinófilos/imunologia , Interleucina-1alfa/metabolismo , Interleucina-1beta/metabolismo , Interleucina-33/metabolismo , Pulmão/fisiopatologia , Camundongos , Fator 88 de Diferenciação Mieloide/deficiência , Fator 88 de Diferenciação Mieloide/genética , Fator 88 de Diferenciação Mieloide/imunologia , Neutrófilos/imunologia , Papaína/administração & dosagem , Receptores de Interleucina-1/imunologia , Receptores de Interleucina-1/metabolismo , Receptores Tipo I de Interleucina-1/imunologia , Transdução de Sinais , Células Th2/imunologiaRESUMO
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.
Assuntos
Alérgenos/administração & dosagem , Alérgenos/imunologia , Hipersensibilidade/imunologia , Interleucina-33/imunologia , Mastócitos/imunologia , Absorção Nasal , Peptídeo Hidrolases/imunologia , Absorção Subcutânea , Animais , Asma , Hiper-Reatividade Brônquica/imunologia , Hiper-Reatividade Brônquica/patologia , Eosinófilos/imunologia , Hipersensibilidade/patologia , Imunoglobulina E/sangue , Imunoglobulina G/sangue , Inflamação , Interleucina-33/deficiência , Pulmão/imunologia , Camundongos , Papaína/administração & dosagem , Papaína/imunologia , Peptídeo Hidrolases/administração & dosagem , Eosinofilia Pulmonar/imunologia , Eosinofilia Pulmonar/patologia , Pele/imunologia , Pele/patologia , Células Th2/imunologiaRESUMO
Allergen sources such as mites, insects, fungi, and pollen contain proteases. Airway exposure to proteases induces allergic airway inflammation and IgE/IgG1 responses via IL-33-dependent mechanisms in mice. We examined the epicutaneous sensitization of mice to a model protease allergen, papain; the effects of tape stripping, which induces epidermal barrier dysfunction; and the atopic march upon a subsequent airway challenge. Papain painting on ear skin and tape stripping cooperatively promoted dermatitis, the skin gene expression of proinflammatory cytokines and growth factors, up-regulation of serum total IgE, and papain-specific IgE/IgG1 induction. Epicutaneous sensitization induced T helper (Th) 2 cells and Th17 differentiation in draining lymph nodes. Ovalbumin and protease inhibitor-treated papain induced no or weak responses, whereas the co-administration of ovalbumin and papain promoted ovalbumin-specific IgE/IgG1 induction. Wild-type and IL-33-deficient mice showed similar responses in the epicutaneous sensitization phase. The subsequent airway papain challenge induced airway eosinophilia and maintained high papain-specific IgE levels in an IL-33-dependent manner. These results suggest that allergen source-derived protease activity and mechanical barrier damage such as that caused by scratching cooperatively promote epicutaneous sensitization and skin inflammation and that IL-33 is dispensable for epicutaneous sensitization but is crucial in the atopic march upon a subsequent airway low-dose encounter with protease allergens.
Assuntos
Alérgenos/imunologia , Dermatite/imunologia , Hipersensibilidade/imunologia , Pele/imunologia , Pele/lesões , Animais , Diferenciação Celular , Citocinas/metabolismo , Ensaio de Imunoadsorção Enzimática , Feminino , Imunoglobulina E/imunologia , Imunoglobulina G/imunologia , Inflamação , Interleucina-33/genética , Interleucina-33/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Ovalbumina , Papaína/imunologia , Inibidores de Proteases/química , Reação em Cadeia da Polimerase em Tempo Real , Pele/efeitos dos fármacos , Estresse Mecânico , Células Th17/citologia , Células Th2/citologia , Ferimentos e Lesões/metabolismoRESUMO
BACKGROUND: This paper reports the case of an egg-allergic pediatric patient who, once desensitized to egg following a successful rush oral immunotherapy protocol, could also tolerate Lizipaina®, a drug containing lysozyme (LYS) and papain, which had previously caused him a severe allergic reaction. Because the LYS amount that elicited the anaphylactic reaction (5 mg) was much lower than that tolerated during a double-blind placebo-controlled food challenge (corresponding to approximately 60 mg of LYS), the possibility that the presence of papain could increase the allergenic potential of LYS was investigated. METHODS: Lizipaina, LYS and LYS hydrolyzed with papain were analyzed by SDS-PAGE under reducing and nonreducing conditions, and Western blotting of sera from egg-allergic patients was performed in order to detect IgE-binding fragments. Finally, sequence identification of the IgE-reactive bands was carried out by MALDI-TOF/TOF. RESULTS: The SDS-PAGE pattern of LYS treated with papain under nonreducing conditions showed the presence of intact LYS that partially disappeared following reduction with ß-mercaptoethanol, releasing IgE-reactive fragments as determined by Western blotting. MALDI-TOF/TOF revealed that papain degraded LYS, giving rise to three IgE-binding fragments: LYS (22-129), LYS (34-96) and LYS (62-128) that likely remained linked through the disulfide bonds present in the LYS molecule. CONCLUSION: The combined administration of LYS with proteolytic enzymes such as papain may have developed a severe allergic reaction in the patient studied, underlining the importance of considering all the components and their interactions when drugs are to be consumed by allergic persons.
Assuntos
Anafilaxia/imunologia , Hipersensibilidade a Drogas/imunologia , Efeitos Colaterais e Reações Adversas Relacionados a Medicamentos/imunologia , Muramidase/imunologia , Papaína/imunologia , Adolescente , Sequência de Aminoácidos , Hipersensibilidade a Ovo/imunologia , Hipersensibilidade a Ovo/terapia , Humanos , Imunoglobulina E/sangue , Imunoglobulina E/imunologia , Masculino , Dados de Sequência Molecular , Muramidase/química , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/imunologiaRESUMO
Allergic diseases represent a significant burden in industrialized countries, but why and how the immune system responds to allergens remain largely unknown. Because many clinically significant allergens have proteolytic activity, and many helminths express proteases that are necessary for their life cycles, host mechanisms likely have evolved to detect the proteolytic activity of helminth proteases, which may be incidentally activated by protease allergens. A cysteine protease, papain, is a prototypic protease allergen that can directly activate basophils and mast cells, leading to the production of cytokines, including IL-4, characteristic of the type 2 immune response. The mechanism of papain's immunogenic activity remains unknown. Here we have characterized the cellular response activated by papain in basophils. We find that papain-induced IL-4 production requires calcium flux and activation of PI3K and nuclear factor of activated T cells. Interestingly, papain-induced IL-4 production was dependent on the immunoreceptor tyrosine-based activation motif (ITAM) adaptor protein Fc receptor γ-chain, even though the canonical ITAM signaling was not activated by papain. Collectively, these data characterize the downstream signaling pathway activated by a protease allergen in basophils.
Assuntos
Alérgenos/farmacologia , Basófilos/metabolismo , Interleucina-4/biossíntese , Papaína/farmacologia , Transdução de Sinais/efeitos dos fármacos , Subunidades do Complexo de Proteínas Adaptadoras/fisiologia , Animais , Basófilos/efeitos dos fármacos , Basófilos/imunologia , Bloqueadores dos Canais de Cálcio/farmacologia , Canais de Cálcio/fisiologia , Sinalização do Cálcio/efeitos dos fármacos , Sinalização do Cálcio/imunologia , Células Cultivadas , Inibidores de Cisteína Proteinase/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Imunização , Interleucina-13/biossíntese , Interleucina-13/genética , Interleucina-33 , Interleucina-4/genética , Interleucinas/farmacologia , Leucina/análogos & derivados , Leucina/farmacologia , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Sistema de Sinalização das MAP Quinases/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fatores de Transcrição NFATC/metabolismo , Papaína/imunologia , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/fisiologia , Receptores de IgE/genética , Receptores de IgE/fisiologia , Receptores de IgG/genética , Receptores de IgG/fisiologia , Transdução de Sinais/imunologia , Organismos Livres de Patógenos EspecíficosRESUMO
Autophagy plays important roles in modulating viral replication and antiviral immune response. Coronavirus infection is associated with the autophagic process, however, little is known about the mechanisms of autophagy induction and its contribution to coronavirus regulation of host innate responses. Here, we show that the membrane-associated papain-like protease PLP2 (PLP2-TM) of coronaviruses acts as a novel autophagy-inducing protein. Intriguingly, PLP2-TM induces incomplete autophagy process by increasing the accumulation of autophagosomes but blocking the fusion of autophagosomes with lysosomes. Furthermore, PLP2-TM interacts with the key autophagy regulators, LC3 and Beclin1, and promotes Beclin1 interaction with STING, the key regulator for antiviral IFN signaling. Finally, knockdown of Beclin1 partially reverses PLP2-TM's inhibitory effect on innate immunity which resulting in decreased coronavirus replication. These results suggested that coronavirus papain-like protease induces incomplete autophagy by interacting with Beclin1, which in turn modulates coronavirus replication and antiviral innate immunity.
Assuntos
Proteínas Reguladoras de Apoptose/imunologia , Coronavirus Humano NL63/imunologia , Regulação da Expressão Gênica , Interações Hospedeiro-Patógeno/imunologia , Proteínas de Membrana/imunologia , Proteínas Associadas aos Microtúbulos/imunologia , Papaína/imunologia , Proteínas Reguladoras de Apoptose/antagonistas & inibidores , Proteínas Reguladoras de Apoptose/genética , Autofagia , Proteína Beclina-1 , Coronavirus Humano NL63/genética , Proteases Semelhantes à Papaína de Coronavírus , Células HEK293 , Células HeLa , Humanos , Evasão da Resposta Imune , Imunidade Inata , Interferon gama/genética , Interferon gama/imunologia , Lisossomos/metabolismo , Lisossomos/virologia , Células MCF-7 , Fusão de Membrana , Proteínas de Membrana/antagonistas & inibidores , Proteínas de Membrana/genética , Proteínas Associadas aos Microtúbulos/genética , Papaína/genética , Fagossomos/metabolismo , Fagossomos/virologia , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/imunologia , Transdução de Sinais , Replicação ViralRESUMO
Papain, a cysteine protease allergen with inherent adjuvant activity, induces potent IL-4 expression by T cells in the popliteal lymph nodes of mice following footpad immunization. In this study, we identify a novel, non-BCR-mediated capacity for B cells to rapidly bind and internalize papain. B cells subsequently regulate the adaptive immune response by enhancing ICOS expression on CD4(+) T cells and amplifying Th2 and follicular helper T cell induction. Ab blockade of ICOS ligand, expressed by popliteal lymph node B cells, but not dendritic cells, at the peak of the response inhibits IL-4 responses in wild-type mice but not B cell-deficient mice. Thus, B cells play a critical role in amplifying adjuvant-dependent Th2 polarization following noncanonical acquisition and internalization of the cysteine protease papain.
Assuntos
Linfócitos B/imunologia , Linfócitos T CD4-Positivos/imunologia , Papaína/imunologia , Receptores de Antígenos de Linfócitos B/imunologia , Animais , Linfócitos B/metabolismo , Linfócitos T CD4-Positivos/metabolismo , Células Cultivadas , Citometria de Fluxo , Imunização/métodos , Proteína Coestimuladora de Linfócitos T Induzíveis/imunologia , Proteína Coestimuladora de Linfócitos T Induzíveis/metabolismo , Interleucina-4/imunologia , Interleucina-4/metabolismo , Linfonodos/citologia , Linfonodos/imunologia , Linfonodos/metabolismo , Camundongos , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Microscopia Confocal , Microscopia de Fluorescência por Excitação Multifotônica , Papaína/administração & dosagem , Papaína/metabolismo , Ligação Proteica/imunologia , Receptores de Antígenos de Linfócitos B/metabolismo , Células Th2/imunologia , Células Th2/metabolismoRESUMO
BACKGROUND: Epicutaneous sensitization to allergens is important in the pathogenesis of not only skin inflammation such as atopic dermatitis but also "atopic march" in allergic diseases such as asthma and food allergies. We here examined antibody production and skin barrier dysfunction in mice epicutaneously administered papain, a plant-derived occupational allergen belonging to the same family of cysteine proteases as mite major group 1 allergens. METHODS: Papain and Staphylococcus aureus V8 protease were patched on the backs of hairless mice. Transepidermal water loss was measured to evaluate the skin barrier dysfunction caused by the proteases. Papain or that treated with an irreversible inhibitor specific to cysteine proteases, E64, was painted onto the ear lobes of mice of an inbred strain C57BL/6. Serum total IgE levels and papain-specific IgE and IgG antibodies were measured by ELISA. RESULTS: Papain and V8 protease patched on the backs of hairless mice caused skin barrier dysfunction and increased serum total IgE levels, and papain induced the production of papain-specific IgG1, IgG2a, and IgG2b. Papain painted onto the ear lobes of C57BL/6 mice induced papain-specific IgE, IgG1, IgG2c, and IgG2b, whereas papain treated with E64 did not. IgG1 was the most significantly induced papain-specific IgG subclass among those measured. CONCLUSIONS: We demonstrated that the epicutaneous administration of protease not only disrupted skin barrier function, but also induced IgE and IgG responses in a manner dependent on its protease activity. These results suggest that protease activity contained in environmental sources contributes to sensitization through an epicutaneous route.
Assuntos
Cisteína Proteases/metabolismo , Hipersensibilidade Imediata , Imunoglobulina E/imunologia , Imunoglobulina G/imunologia , Papaína/imunologia , Alérgenos/imunologia , Alérgenos/metabolismo , Animais , Especificidade de Anticorpos/imunologia , Dermatite Atópica/imunologia , Dermatite Atópica/metabolismo , Dermatite Atópica/terapia , Modelos Animais de Doenças , Feminino , Imunoglobulina E/sangue , Imunoglobulina G/sangue , Camundongos , Papaína/administração & dosagem , Papaína/metabolismo , Pele/imunologia , Pele/metabolismo , Pele/patologiaRESUMO
Although type 2 immune responses to environmental Ags are thought to play pivotal roles in asthma and allergic airway diseases, the immunological mechanisms that initiate the responses are largely unknown. Many allergens have biologic activities, including enzymatic activities and abilities to engage innate pattern-recognition receptors such as TLR4. In this article, we report that IL-33 and thymic stromal lymphopoietin were produced quickly in the lungs of naive mice exposed to cysteine proteases, such as bromelain and papain, as a model for allergens. IL-33 and thymic stromal lymphopoietin sensitized naive animals to an innocuous airway Ag OVA, which resulted in production of type 2 cytokines and IgE Ab, and eosinophilic airway inflammation when mice were challenged with the same Ag. Importantly, upon exposure to proteases, uric acid (UA) was rapidly released into the airway lumen, and removal of this endogenous UA by uricase prevented type 2 immune responses. UA promoted secretion of IL-33 by airway epithelial cells in vitro, and administration of UA into the airways of naive animals induced extracellular release of IL-33, followed by both innate and adaptive type 2 immune responses in vivo. Finally, a potent UA synthesis inhibitor, febuxostat, mitigated asthma phenotypes that were caused by repeated exposure to natural airborne allergens. These findings provide mechanistic insights into the development of type 2 immunity to airborne allergens and recognize airway UA as a key player that regulates the process in respiratory mucosa.