RESUMO
IgE induced by type 2 immune responses in atopic dermatitis is implicated in the progression of atopic dermatitis to other allergic diseases, including food allergies, allergic rhinitis, and asthma. However, the keratinocyte-derived signals that promote IgE and ensuing allergic diseases remain unclear. Herein, in a mouse model of atopic dermatitis-like skin inflammation induced by epicutaneous Staphylococcus aureus exposure, keratinocyte release of IL36α along with IL-4 triggered B cell IgE class-switching, plasma cell differentiation, and increased serum IgE levels-all of which were abrogated in IL-36R-deficient mice or anti-IL36R-blocking antibody-treated mice. Moreover, skin allergen sensitization during S. aureus epicutaneous exposure-induced IL-36 responses was required for the development of allergen-specific lung inflammation. In translating these findings, elevated IL36 cytokines in human atopic dermatitis skin and in IL36 receptor antagonist-deficiency patients coincided with increased serum IgE levels. Collectively, keratinocyte-initiated IL36 responses represent a key mechanism and potential therapeutic target against allergic diseases.
Assuntos
Dermatite Atópica/imunologia , Imunoglobulina E/imunologia , Interleucina-1/imunologia , Queratinócitos/imunologia , Plasmócitos/imunologia , Staphylococcus aureus/imunologia , Animais , Diferenciação Celular/genética , Diferenciação Celular/imunologia , Dermatite Atópica/genética , Dermatite Atópica/microbiologia , Humanos , Switching de Imunoglobulina , Imunoglobulina E/genética , Interleucina-1/genética , Interleucina-4/genética , Interleucina-4/imunologia , Queratinócitos/microbiologia , Camundongos , Camundongos Knockout , Plasmócitos/patologiaRESUMO
BACKGROUND & AIMS: Intestinal fibrosis is a long-term complication in inflammatory bowel diseases (IBD) that frequently results in functional damage, bowel obstruction, and surgery. Interleukin (IL) 36 is a group of cytokines in the IL1 family with inflammatory effects. We studied the expression of IL36 and its receptor, interleukin 1 receptor like 2 (IL1RL2 or IL36R) in the development of intestinal fibrosis in human tissues and mice. METHODS: We obtained intestinal tissues from 92 patients with Crohn's disease (CD), 48 patients with ulcerative colitis, and 26 patients without inflammatory bowel diseases (control individuals). Tissues were analyzed by histology to detect fibrosis and by immunohistochemistry to determine the distribution of fibroblasts and levels of IL36R ligands. Human and mouse fibroblasts were incubated with IL36 or control medium, and transcriptome-wide RNA sequences were analyzed. Mice were given neutralizing antibodies against IL36R, and we studied intestinal tissues from Il1rl2-/- mice; colitis and fibrosis were induced in mice by repetitive administration of DSS or TNBS. Bone marrow cells were transplanted from Il1rl2-/- to irradiated wild-type mice and intestinal tissues were analyzed. Antibodies against IL36R were applied to mice with established chronic colitis and fibrosis and intestinal tissues were studied. RESULTS: Mucosal and submucosal tissue from patients with CD or ulcerative colitis had higher levels of collagens, including type VI collagen, compared with tissue from control individuals. In tissues from patients with fibrostenotic CD, significantly higher levels of IL36A were noted, which correlated with high numbers of activated fibroblasts that expressed α-smooth muscle actin. IL36R activation of mouse and human fibroblasts resulted in expression of genes that regulate fibrosis and tissue remodeling, as well as expression of collagen type VI. Il1rl2-/- mice and mice given injections of an antibody against IL36R developed less severe colitis and fibrosis after administration of DSS or TNBS, but bone marrow cells from Il1rl2-/- mice did not prevent induction of colitis and fibrosis. Injection of antibodies against IL36R significantly reduced established fibrosis in mice with chronic intestinal inflammation. CONCLUSION: We found higher levels of IL36A in fibrotic intestinal tissues from patients with IBD compared with control individuals. IL36 induced expression of genes that regulate fibrogenesis in fibroblasts. Inhibition or knockout of the IL36R gene in mice reduces chronic colitis and intestinal fibrosis. Agents designed to block IL36R signaling could be developed for prevention and treatment of intestinal fibrosis in patients with IBD.
Assuntos
Colite Ulcerativa/metabolismo , Colágeno Tipo VI/metabolismo , Colo/patologia , Doença de Crohn/metabolismo , Interleucina-1/metabolismo , Mucosa Intestinal/patologia , Intestino Delgado/patologia , Receptores de Interleucina-1/metabolismo , Actinas/metabolismo , Animais , Anticorpos Neutralizantes/farmacologia , Estudos de Casos e Controles , Células Cultivadas , Colite/induzido quimicamente , Colite/patologia , Colite Ulcerativa/patologia , Doença de Crohn/patologia , Sulfato de Dextrana , Fibroblastos/efeitos dos fármacos , Fibrose , Expressão Gênica/efeitos dos fármacos , Perfilação da Expressão Gênica , Humanos , Interleucina-1/farmacologia , Ligantes , Camundongos , Camundongos Knockout , Receptores de Interleucina-1/antagonistas & inibidores , Receptores de Interleucina-1/genética , Transdução de Sinais , Transcriptoma , Ácido TrinitrobenzenossulfônicoRESUMO
Signaling by the interleukin-36 receptor (IL-36R) is linked to inflammatory diseases such as psoriasis. However, the regulation of IL-36R signaling is poorly understood. Activation of IL-36R signaling in cultured cells results in an increased polyubiquitination of the receptor subunit, IL-1Rrp2. Treatment with deubiquitinases shows that the receptor subunit of IL-36R, IL-1Rrp2, is primarily polyubiquitinated at the K63 position, which is associated with endocytic trafficking and signal transduction. A minor amount of ubiquitination is at the K48 position that is associated with protein degradation. A focused siRNA screen identified RNF125, an E3 ubiquitin ligase, to ubiquitinate IL-1Rrp2 upon activation of IL-36R signaling while not affecting the activated IL-1 receptor. Knockdown of RNF125 decreases signal transduction by the IL-36R. Overexpression of RNF125 in HEK293T cells activates IL-36R signaling and increases the ubiquitination of IL-1Rrp2 and its subsequent turnover. RNF125 can coimmunoprecipitate with the IL-36R, and it traffics with IL-1Rrp2 from the cell surface to lysosomes. Mutations of Lys568 and Lys569 in the C-terminal tail of IL-1Rrp2 decrease ubiquitination by RNF125 and increase the steady-state levels of IL-1Rrp2. These results demonstrate that RNF125 has multiple regulatory roles in the signaling, trafficking, and turnover of the IL-36R.
Assuntos
Inflamação/imunologia , Subunidade alfa de Receptor de Interleucina-18/metabolismo , Lisossomos/metabolismo , Psoríase/imunologia , Receptores de Interleucina/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Endocitose , Células HEK293 , Humanos , Subunidade alfa de Receptor de Interleucina-18/genética , Mutação/genética , Transporte Proteico , RNA Interferente Pequeno/genética , Transdução de Sinais , Ubiquitina-Proteína Ligases/genética , UbiquitinaçãoRESUMO
Deficiency of interleukin (IL)-36 receptor antagonist (DITRA) syndrome is a rare autosomal recessive disease caused by mutations in IL36RN. IL-36R is a cell surface receptor and a member of the IL1R family that is involved in inflammatory responses triggered in skin and other epithelial tissues. Accumulating evidence suggests that IL-36R signaling may play a role in the pathogenesis of psoriasis. Therapeutic intervention of IL-36R signaling offers an innovative treatment paradigm for targeting epithelial cell-mediated inflammatory diseases such as the life-threatening psoriasis variant called generalized pustular psoriasis (GPP). We report the discovery and characterization of MAB92, a potent, high affinity anti-human IL-36 receptor antagonistic antibody that blocks human IL-36 ligand (α, ß and γ)-mediated signaling. In vitro treatment with MAB92 directly inhibits human IL-36R-mediated signaling and inflammatory cytokine production in primary human keratinocytes and dermal fibroblasts. MAB92 shows exquisite species specificity toward human IL-36R and does not cross react to murine IL-36R. To enable in vivo pharmacology studies, we developed a mouse cross-reactive antibody, MAB04, which exhibits overlapping binding and pharmacological activity as MAB92. Epitope mapping indicates that MAB92 and MAB04 bind primarily to domain-2 of the human and mouse IL-36R proteins, respectively. Treatment with MAB04 abrogates imiquimod and IL-36-mediated skin inflammation in the mouse, further supporting an important role for IL-36R signaling in epithelial cell-mediated inflammation.
Assuntos
Anticorpos Monoclonais/imunologia , Receptores de Interleucina/antagonistas & inibidores , Animais , Especificidade de Anticorpos , Humanos , Camundongos , Psoríase/imunologiaRESUMO
Improper signaling of the IL-36 receptor (IL-36R), a member of the IL-1 receptor family, has been associated with various inflammation-associated diseases. However, the requirements for IL-36R signal transduction remain poorly characterized. This work seeks to define the requirements for IL-36R signaling and intracellular trafficking. In the absence of cognate agonists, IL-36R was endocytosed and recycled to the plasma membrane. In the presence of IL-36, IL-36R increased accumulation in LAMP1+ lysosomes. Endocytosis predominantly used a clathrin-mediated pathway, and the accumulation of the IL-36R in lysosomes did not result in increased receptor turnover. The ubiquitin-binding Tollip protein contributed to IL-36R signaling and increased the accumulation of both subunits of the IL-36R.
Assuntos
Endocitose/fisiologia , Interleucina-1/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Lisossomos/metabolismo , Receptores de Interleucina/metabolismo , Transdução de Sinais/fisiologia , Linhagem Celular , Humanos , Interleucina-1/genética , Peptídeos e Proteínas de Sinalização Intracelular/genética , Lisossomos/genética , Transporte Proteico/fisiologia , Receptores de Interleucina/genéticaRESUMO
Herein, we describe the generation and characterization of BI 655066, a novel, highly potent neutralizing anti-interleukin-23 (IL23) monoclonal antibody in clinical development for autoimmune conditions, including psoriasis and Crohn's disease. IL23 is a key driver of the differentiation, maintenance, and activity of a number of immune cell subsets, including T helper 17 (Th17) cells, which are believed to mediate the pathogenesis of several immune-mediated disorders. Thus, IL23 neutralization is an attractive therapeutic approach. Designing an antibody for clinical activity and convenience for the patient requires certain properties, such as high affinity, specificity, and solubility. These properties were achieved by directed design of the immunization, lead identification, and humanization procedures. Favorable substance and pharmacokinetic properties were established by biophysical assessments and studies in cynomolgus monkeys.
Assuntos
Anticorpos Monoclonais/farmacologia , Anticorpos Monoclonais/farmacocinética , Anticorpos Neutralizantes/farmacologia , Sistemas de Liberação de Medicamentos , Subunidade p19 da Interleucina-23/antagonistas & inibidores , Animais , Anticorpos Monoclonais/imunologia , Anticorpos Neutralizantes/imunologia , Doença de Crohn/tratamento farmacológico , Doença de Crohn/imunologia , Humanos , Subunidade p19 da Interleucina-23/imunologia , Macaca fascicularis , Psoríase/tratamento farmacológico , Psoríase/imunologia , Células Th17/imunologiaRESUMO
Benzamide 1 demonstrated good potency as a selective ITK inhibitor, however the amide moiety was found to be hydrolytically labile in vivo, resulting in low oral exposure and the generation of mutagenic aromatic amine metabolites. Replacing the benzamide with a benzylamine linker not only addressed the toxicity issue, but also improved the cellular and functional potency as well as the drug-like properties. SAR studies around the benzylamines and the identification of 10n and 10o as excellent tools for proof-of-concept studies are described.
Assuntos
Benzimidazóis/síntese química , Química Farmacêutica/métodos , Inibidores Enzimáticos/síntese química , Proteínas Tirosina Quinases/antagonistas & inibidores , Animais , Benzimidazóis/farmacologia , Complexo CD3/biossíntese , Desenho de Fármacos , Inibidores Enzimáticos/farmacologia , Feminino , Hepatócitos/metabolismo , Humanos , Concentração Inibidora 50 , Camundongos , Camundongos Endogâmicos BALB C , Ratos , Ratos Sprague-Dawley , Relação Estrutura-AtividadeRESUMO
Identification and optimization of two classes of CB2 selective agonists are described. A representative from each class is profiled in a murine model of inflammation and each shows similar efficacy to prednisolone upon oral dosing.
Assuntos
Morfolinas/síntese química , Receptor CB2 de Canabinoide/agonistas , Analgésicos/síntese química , Analgésicos/farmacologia , Animais , Anti-Inflamatórios/farmacologia , Linhagem Celular , Química Farmacêutica/métodos , Desenho de Fármacos , Humanos , Inflamação , Camundongos , Modelos Químicos , Estrutura Molecular , Morfolinas/farmacologia , Receptor CB2 de Canabinoide/química , EstereoisomerismoRESUMO
A series of novel 5-aminomethyl-1H-benzimidazole based inhibitors of Itk were prepared. Structure-activity relationships, selectivity and cell activity are reported for this series. Compound 2, a potent and selective antagonist of Itk, inhibited anti-CD3 antibody induced IL-2 production in vivo in mice.
Assuntos
Benzimidazóis/administração & dosagem , Benzimidazóis/química , Benzimidazóis/síntese química , Química Farmacêutica/métodos , Proteínas Tirosina Quinases/antagonistas & inibidores , Proteínas Tirosina Quinases/química , Administração Oral , Animais , Benzimidazóis/farmacologia , Complexo CD3/biossíntese , Desenho de Fármacos , Humanos , Concentração Inibidora 50 , Ativação Linfocitária , Camundongos , Camundongos Endogâmicos BALB C , Modelos Químicos , Relação Estrutura-Atividade , Linfócitos T/citologiaRESUMO
A series of novel potent benzimidazole based inhibitors of interleukin-2 T-cell kinase (Itk) were prepared. In this report, we discuss the structure-activity relationship (SAR), selectivity, and cell-based activity for the series. We also discuss the SAR associated with an X-ray structure of one of the small-molecule inhibitors bound to ITK.
Assuntos
Amidas/química , Benzimidazóis/química , Ácidos Carboxílicos/química , Química Farmacêutica/métodos , Inibidores Enzimáticos/síntese química , Microssomos Hepáticos/metabolismo , Proteínas Tirosina Quinases/química , Animais , Benzimidazóis/síntese química , Ácidos Carboxílicos/síntese química , Cristalografia por Raios X , Desenho de Fármacos , Inibidores Enzimáticos/farmacologia , Concentração Inibidora 50 , Camundongos , Modelos Químicos , Conformação Molecular , Relação Estrutura-AtividadeRESUMO
The design and synthesis of dipeptidyl disulfides and dipeptidyl benzoylhydrazones as selective inhibitors of the cysteine protease Cathepsin S are described. These inhibitors were expected to form a slowly reversible covalent adduct of the active site cysteine of Cathepsin S. Formation of the initial adduct was confirmed by mass spectral analysis. The nature and mechanism of these adducts was explored. Kinetic analysis of the benzoyl hydrazones indicate that these inhibitors are acting as irreversible inhibitors of Cathepsin S. Additionally, the benzoylhydrazones were shown to be potent inhibitors of Cathepsin S processing of Class II associated invariant peptide both in vitro and in vivo.