RESUMEN
Preventing the biogenesis of disease-relevant proteins is an attractive therapeutic strategy, but attempts to target essential protein biogenesis factors have been hampered by excessive toxicity. Here we describe KZR-8445, a cyclic depsipeptide that targets the Sec61 translocon and selectively disrupts secretory and membrane protein biogenesis in a signal peptide-dependent manner. KZR-8445 potently inhibits the secretion of pro-inflammatory cytokines in primary immune cells and is highly efficacious in a mouse model of rheumatoid arthritis. A cryogenic electron microscopy structure reveals that KZR-8445 occupies the fully opened Se61 lateral gate and blocks access to the lumenal plug domain. KZR-8445 binding stabilizes the lateral gate helices in a manner that traps select signal peptides in the Sec61 channel and prevents their movement into the lipid bilayer. Our results establish a framework for the structure-guided discovery of novel therapeutics that selectively modulate Sec61-mediated protein biogenesis.
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Proteínas de la Membrana , Señales de Clasificación de Proteína , Animales , Ratones , Transporte de Proteínas , Proteínas de la Membrana/metabolismo , Canales de Translocación SEC/química , Canales de Translocación SEC/genética , Canales de Translocación SEC/metabolismo , Biosíntesis de ProteínasRESUMEN
Zetomipzomib (KZR-616) is a selective inhibitor of the immunoproteasome currently undergoing clinical investigation in autoimmune disorders. Here, we characterized KZR-616 in vitro and in vivo using multiplexed cytokine analysis, lymphocyte activation and differentiation, and differential gene expression analysis. KZR-616 blocked production of >30 pro-inflammatory cytokines in human peripheral blood mononuclear cells (PBMCs), polarization of T helper (Th) cells, and formation of plasmablasts. In the NZB/W F1 mouse model of lupus nephritis (LN), KZR-616 treatment resulted in complete resolution of proteinuria that was maintained at least 8 weeks after the cessation of dosing and was mediated in part by alterations in T and B cell activation, including reduced numbers of short and long-lived plasma cells. Gene expression analysis of human PBMCs and tissues from diseased mice revealed a consistent and broad response focused on inhibition of T, B, and plasma cell function and the Type I interferon pathway and promotion of hematopoietic cell lineages and tissue remodeling. In healthy volunteers, KZR-616 administration resulted in selective inhibition of the immunoproteasome and blockade of cytokine production following ex vivo stimulation. These data support the ongoing development of KZR-616 in autoimmune disorders such as systemic lupus erythematosus (SLE)/LN.
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Leucocitos Mononucleares , Nefritis Lúpica , Humanos , Animales , Ratones , Leucocitos Mononucleares/metabolismo , Citocinas/metabolismo , InmunidadRESUMEN
KZR-616 is an irreversible tripeptide epoxyketone-based selective inhibitor of the human immunoproteasome. Inhibition of the immunoproteasome results in anti-inflammatory activity in vitro and based on promising therapeutic activity in animal models of rheumatoid arthritis and systemic lupus erythematosus KZR-616 is being developed for potential treatment of multiple autoimmune and inflammatory diseases. The presence of a ketoepoxide pharmacophore presents unique challenges in the study of drug metabolism during lead optimization and clinical candidate profiling. This study presents a thorough and systematic in vitro and cell-based enzymatic metabolism and kinetic investigation to identify the major enzymes involved in the metabolism and elimination of KZR-616. Upon exposure to liver microsomes in the absence of NADPH, KZR-616 and its analogs were converted to their inactive diol derivatives with varying degrees of stability. Diol formation was also shown to be the major metabolite in pharmacokinetic studies in monkeys and correlated with in vitro stability results for individual compounds. Further study in intact hepatocytes revealed that KZR-616 metabolism was sensitive to an inhibitor of microsomal epoxide hydrolase (mEH) but not inhibitors of cytochrome P450 (P450) or soluble epoxide hydrolase (sEH). Primary human hepatocytes were determined to be the most robust source of mEH activity for study in vitro. These findings also suggest that the exposure of KZR-616 in vivo is unlikely to be affected by coadministration of inhibitors or inducers of P450 and sEH. SIGNIFICANCE STATEMENT: This work presents a thorough and systematic investigation of metabolism and kinetics of KZR-616 and related analogs in in vitro and cell-based enzymatic systems. Information gained could be useful in assessing novel covalent proteasome inhibitors during lead compound optimization. These studies also demonstrate a robust source in vitro test system that correlated with in vivo pharmacokinetics for KZR-616 and two additional tripeptide epoxyketones.
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Cisteína Endopeptidasas/inmunología , Sistema Enzimático del Citocromo P-450/metabolismo , Epóxido Hidrolasas/metabolismo , Morfolinas/farmacología , Complejo de la Endopetidasa Proteasomal/inmunología , Proteínas/inmunología , Animales , Enfermedades Autoinmunes/tratamiento farmacológico , Células Cultivadas , Cisteína Endopeptidasas/metabolismo , Epóxido Hidrolasas/inmunología , Hepatocitos/metabolismo , Humanos , Inactivación Metabólica , Inflamación/tratamiento farmacológico , Macaca fascicularis , Inhibidores de Proteasoma/farmacologíaRESUMEN
Inhibitors of the proteolytic activity of the 20S proteasome have transformed the treatment of multiple B-cell malignancies. These agents have also been employed with success in the treatment of patients with autoimmune diseases and immune-mediated disorders. However, new agents are needed to fully unlock the potential of proteasome inhibitors as immunomodulatory drugs. The discovery that selective inhibitors of the immunoproteasome possess broad anti-inflammatory activity in preclinical models has led to the progression of multiple compounds to clinical trials. This review focuses on the anti-inflammatory potential of immunoproteasome inhibition and the early development of KZR-616, the first selective inhibitor of the immunoproteasome to reach clinical testing.
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Desarrollo de Medicamentos , Descubrimiento de Drogas , Complejo de la Endopetidasa Proteasomal/inmunología , Inhibidores de Proteasoma/farmacología , Animales , Antiinflamatorios/farmacología , Humanos , Morfolinas/farmacología , Inhibidores de Proteasoma/químicaRESUMEN
Selective immunoproteasome inhibition is a promising approach for treating autoimmune disorders, but optimal proteolytic active site subunit inhibition profiles remain unknown. We reveal here our design of peptide epoxyketone-based selective low molecular mass polypeptide-7 (LMP7) and multicatalytic endopeptidase complex subunit-1 (MECL-1) subunit inhibitors. Utilizing these and our previously disclosed low molecular mass polypeptide-2 (LMP2) inhibitor, we demonstrate a requirement of dual LMP7/LMP2 or LMP7/MECL-1 subunit inhibition profiles for potent cytokine expression inhibition and in vivo efficacy in an inflammatory disease model. These and additional findings toward optimized solubility led the design and selection of KZR-616 disclosed here and presently in clinical trials for treatment of rheumatic disease.
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Antiinflamatorios no Esteroideos/farmacología , Artritis Reumatoide/tratamiento farmacológico , Morfolinas/farmacología , Inhibidores de Proteasoma/farmacología , Administración Intravenosa , Animales , Antiinflamatorios no Esteroideos/química , Antiinflamatorios no Esteroideos/farmacocinética , Artritis Experimental/tratamiento farmacológico , Línea Celular Tumoral , Cisteína Endopeptidasas/metabolismo , Citocinas/metabolismo , Diseño de Fármacos , Femenino , Humanos , Ratones Endogámicos BALB C , Morfolinas/química , Morfolinas/farmacocinética , Oligopéptidos/farmacología , Complejo de la Endopetidasa Proteasomal/inmunología , Complejo de la Endopetidasa Proteasomal/metabolismo , Inhibidores de Proteasoma/química , Inhibidores de Proteasoma/farmacocinética , Relación Estructura-ActividadRESUMEN
Building upon the success of bortezomib (VELCADE) and carfilzomib (KYPROLIS), the design of a next generation of inhibitors targeting specific subunits within the immunoproteasome is of interest for the treatment of autoimmune disease. There are three catalytic subunits within the immunoproteasome (low molecular mass polypeptide-7, -2, and multicatalytic endopeptidase complex subunit-1; LMP7, LMP2, and MECL-1), and a campaign was undertaken to design a potent and selective LMP2 inhibitor with sufficient properties to allow for sustained inhibition in vivo. Screening a focused library of epoxyketones revealed a series of potent dipeptides that were optimized to provide the highly selective inhibitor KZR-504 (12).
RESUMEN
Multiple sclerosis (MS) is a chronic demyelinating immune mediated disease of the central nervous system. The immunoproteasome is a distinct class of proteasomes found predominantly in monocytes and lymphocytes. Recently, we demonstrated a novel function of immunoproteasomes in cytokine production and T cell differentiation. In this study, we investigated the therapeutic efficacy of an inhibitor of the immunoproteasome (ONX 0914) in two different mouse models of MS. ONX 0914 attenuated disease progression after active and passive induction of experimental autoimmune encephalomyelitis (EAE), both in MOG35-55 and PLP139â151-induced EAE. Isolation of lymphocytes from the brain or spinal cord revealed a strong reduction of cytokine-producing CD4(+) cells in ONX 0914 treated mice. Additionally, ONX 0914 treatment prevented disease exacerbation in a relapsing-remitting model. An analysis of draining lymph nodes after induction of EAE revealed that the differentiation to Th17 or Th1 cells was strongly impaired in ONX 0914 treated mice. These results implicate the immunoproteasome in the development of EAE and suggest that immunoproteasome inhibitors are promising drugs for the treatment of MS.
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Encefalomielitis Autoinmune Experimental/tratamiento farmacológico , Encefalomielitis Autoinmune Experimental/inmunología , Complejo de la Endopetidasa Proteasomal/inmunología , Inhibidores de Proteasoma/uso terapéutico , Animales , Encéfalo/efectos de los fármacos , Encéfalo/patología , Diferenciación Celular/inmunología , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Encefalomielitis Autoinmune Experimental/prevención & control , Femenino , Factor Estimulante de Colonias de Granulocitos y Macrófagos/biosíntesis , Humanos , Inflamación/patología , Ratones , Ratones Endogámicos C57BL , Glicoproteína Mielina-Oligodendrócito , Oligopéptidos/farmacología , Oligopéptidos/uso terapéutico , Fragmentos de Péptidos , Complejo de la Endopetidasa Proteasomal/deficiencia , Complejo de la Endopetidasa Proteasomal/metabolismo , Inhibidores de Proteasoma/farmacología , Médula Espinal/efectos de los fármacos , Médula Espinal/patología , Linfocitos T/efectos de los fármacos , Linfocitos T/metabolismoRESUMEN
OBJECTIVE: To investigate the hypothesis that proteasome inhibition may have potential in the treatment of SLE, by targeting plasmacytoid dendritic cells (PDCs) and plasma cells, both of which are critical in disease pathogenesis. METHODS: Lupus-prone mice were treated with the nonselective proteasome inhibitors carfilzomib and bortezomib, the immunoproteasome inhibitor ONX 0914, or vehicle control. Tissue was harvested and analyzed by flow cytometry using standard markers. Nephritis was monitored by evaluation for proteinuria and by histologic analysis of kidneys. Serum anti-double-stranded DNA (anti-dsDNA) levels were measured by enzyme-linked immunosorbent assay (ELISA), and total IgG and dsDNA antibody-secreting cells (ASCs) by enzyme-linked immunospot assay. Human peripheral blood mononuclear cells or mouse bone marrow cells were incubated with Toll-like receptor (TLR) agonists and proteasome inhibitors, and interferon-α (IFNα) levels were measured by ELISA and flow cytometry. RESULTS: Early treatment of lupus-prone mice with the dual-targeting proteasome inhibitors carfilzomib or bortezomib or the immunoproteasome-specific inhibitor ONX 0914 prevented disease progression, and treatment of mice with established disease dramatically abrogated nephritis. Treatment had profound effects on plasma cells, with greater reductions in autoreactive than in total IgG ASCs, an effect that became more pronounced with prolonged treatment and was reflected in decreasing serum autoantibody levels. Notably, proteasome inhibition efficiently suppressed production of IFNα by TLR-activated PDCs in vitro and in vivo, an effect mediated by inhibition of both PDC survival and PDC function. CONCLUSION: Inhibition of the immunoproteasome is equally efficacious as dual targeting agents in preventing lupus disease progression by targeting 2 critical pathways in disease pathogenesis, type I IFN activation and autoantibody production by plasma cells.
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Células Productoras de Anticuerpos/efectos de los fármacos , Ácidos Borónicos/uso terapéutico , Interferón Tipo I/antagonistas & inhibidores , Lupus Eritematoso Sistémico/tratamiento farmacológico , Nefritis Lúpica/tratamiento farmacológico , Oligopéptidos/uso terapéutico , Inhibidores de Proteasas/uso terapéutico , Pirazinas/uso terapéutico , Animales , Células Productoras de Anticuerpos/inmunología , Autoanticuerpos/inmunología , Ácidos Borónicos/farmacología , Bortezomib , Progresión de la Enfermedad , Humanos , Leucocitos Mononucleares/efectos de los fármacos , Leucocitos Mononucleares/inmunología , Lupus Eritematoso Sistémico/inmunología , Nefritis Lúpica/inmunología , Ratones , Oligopéptidos/farmacología , Inhibidores de Proteasas/farmacología , Pirazinas/farmacologíaRESUMEN
PURPOSE: Bortezomib (Velcade), a dipeptide boronate 20S proteasome inhibitor and an approved treatment option for multiple myeloma, is associated with a treatment-emergent, painful peripheral neuropathy (PN) in more than 30% of patients. Carfilzomib, a tetrapeptide epoxyketone proteasome inhibitor, currently in clinical investigation in myeloma, is associated with low rates of PN. We sought to determine whether PN represents a target-mediated adverse drug reaction (ADR). EXPERIMENTAL DESIGN: Neurodegenerative effects of proteasome inhibitors were assessed in an in vitro model utilizing a differentiated neuronal cell line. Secondary targets of both inhibitors were identified by a multifaceted approach involving candidate screening, profiling with an activity-based probe, and database mining. Secondary target activity was measured in rats and patients receiving both inhibitors. RESULTS: Despite equivalent levels of proteasome inhibition, only bortezomib reduced neurite length, suggesting a nonproteasomal mechanism. In cell lysates, bortezomib, but not carfilzomib, significantly inhibited the serine proteases cathepsin G (CatG), cathepsin A, chymase, dipeptidyl peptidase II, and HtrA2/Omi at potencies near or equivalent to that for the proteasome. Inhibition of CatG was detected in splenocytes of rats receiving bortezomib and in peripheral blood mononuclear cells derived from bortezomib-treated patients. Levels of HtrA2/Omi, which is known to be involved in neuronal survival, were upregulated in neuronal cells exposed to both proteasome inhibitors but was inhibited only by bortezomib exposure. CONCLUSION: These data show that bortezomib-induced neurodegeneration in vitro occurs via a proteasome-independent mechanism and that bortezomib inhibits several nonproteasomal targets in vitro and in vivo, which may play a role in its clinical ADR profile.
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Ácidos Borónicos/efectos adversos , Efectos Colaterales y Reacciones Adversas Relacionados con Medicamentos/etiología , Oligopéptidos/efectos adversos , Inhibidores de Proteasoma , Pirazinas/efectos adversos , Animales , Antineoplásicos/administración & dosificación , Antineoplásicos/efectos adversos , Ácidos Borónicos/administración & dosificación , Bortezomib , Células Cultivadas , Cisteína Endopeptidasas/administración & dosificación , Cisteína Endopeptidasas/efectos adversos , Sistemas de Liberación de Medicamentos , Células Hep G2 , Humanos , Masculino , Modelos Biológicos , Oligopéptidos/administración & dosificación , Complejo de la Endopetidasa Proteasomal/metabolismo , Pirazinas/administración & dosificación , Ratas , Ratas Sprague-DawleyRESUMEN
The immunoproteasome, a distinct class of proteasome found predominantly in monocytes and lymphocytes, is known to shape the antigenic repertoire presented on class I major histocompatibility complexes (MHC-I). However, a specific role for the immunoproteasome in regulating other facets of immune responses has not been established. We describe here the characterization of PR-957, a selective inhibitor of low-molecular mass polypeptide-7 (LMP7, encoded by Psmb8), the chymotrypsin-like subunit of the immunoproteasome. PR-957 blocked presentation of LMP7-specific, MHC-I-restricted antigens in vitro and in vivo. Selective inhibition of LMP7 by PR-957 blocked production of interleukin-23 (IL-23) by activated monocytes and interferon-gamma and IL-2 by T cells. In mouse models of rheumatoid arthritis, PR-957 treatment reversed signs of disease and resulted in reductions in cellular infiltration, cytokine production and autoantibody levels. These studies reveal a unique role for LMP7 in controlling pathogenic immune responses and provide a therapeutic rationale for targeting LMP7 in autoimmune disorders.
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Artritis Experimental/tratamiento farmacológico , Citocinas/biosíntesis , Complejos Multienzimáticos/antagonistas & inhibidores , Oligopéptidos/farmacología , Inhibidores de Proteasoma , Animales , Presentación de Antígeno/efectos de los fármacos , Progresión de la Enfermedad , Femenino , Humanos , Virus de la Coriomeningitis Linfocítica/inmunología , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Endogámicos DBA , Complejos Multienzimáticos/fisiología , Oligopéptidos/uso terapéutico , Complejo de la Endopetidasa ProteasomalRESUMEN
Proteasome inhibition has been validated as a therapeutic modality in the treatment of multiple myeloma and non-Hodgkin's lymphoma. Carfilzomib, an epoxyketone currently undergoing clinical trials in malignant diseases, is a highly selective inhibitor of the chymotrypsin-like (CT-L) activity of the proteasome. A chemistry effort was initiated to discover orally bioavailable analogues of carfilzomib, which would have potential for improved dosing flexibility and patient convenience over intravenously administered agents. The lead compound, 2-Me-5-thiazole-Ser(OMe)-Ser(OMe)-Phe-ketoepoxide (58) (PR-047), selectively inhibited CT-L activity of both the constitutive proteasome (beta5) and immunoproteasome (LMP7) and demonstrated an absolute bioavailability of up to 39% in rodents and dogs. It was well tolerated with repeated oral administration at doses resulting in >80% proteasome inhibition in most tissues and elicited an antitumor response equivalent to intravenously administered carfilzomib in multiple human tumor xenograft and mouse syngeneic models. The favorable pharmacologic profile supports its further development for the treatment of malignant diseases.
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Dipéptidos/síntesis química , Dipéptidos/farmacología , Diseño de Fármacos , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/farmacología , Oligopéptidos/síntesis química , Oligopéptidos/farmacología , Inhibidores de Proteasoma , Tiazoles/síntesis química , Tiazoles/farmacología , Administración Oral , Animales , Antineoplásicos/química , Antineoplásicos/farmacología , Disponibilidad Biológica , Línea Celular , Dipéptidos/química , Dipéptidos/farmacocinética , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacocinética , Humanos , Cinética , Ratones , Oligopéptidos/química , Oligopéptidos/farmacocinética , Relación Estructura-Actividad , Especificidad por Sustrato , Tiazoles/química , Tiazoles/farmacocinéticaRESUMEN
BACKGROUND: Nuclear factor-kappaB (NF-kappaB) is a key transcriptional regulator of inflammatory bowel disease (IBD). AIM: To investigate the therapeutic potential of a locally administered "non-viral" nuclear factor-kappaB decoy (NFkappaBD) in multiple experimental models of IBD. METHODS: A fully phosphorothioated decoy oligonucleotide with improved stability that specifically binds NF-kappaB and blocks inflammatory mediators regulated by this transcription factor without the help of viral envelope-assisted delivery was developed. The therapeutic effects of NFkappaBD were studied in the trinitrobenzene sulphonic acid, oxazolone and dextran sodium sulphate induced colitis models. RESULTS: Intracolonic administration of NFkappaBD results in the delivery of NFkappaBD to inflammatory cells and a reduction of NF-kappaB heterodimers. In the T helper cell 1-driven trinitrobenzene sulphonic acid-induced colitis model, mice receiving NFkappaBD treatment exhibit a dose-dependent reduction in disease severity and a more rapid recovery to normal body weight, similar to a clinically relevant dose of budesonide. Clinical efficacy was corroborated by considerable reductions in colitis pathology and tissue levels of several pro-inflammatory markers, including tumour necrosis factor alpha, interleukin 6, interleukin 1beta and monocyte chemotactic protein 1. NFkappaBD also mitigates disease activity in the T helper cell 2-like oxazolone colitis and epithelial injury-related acute dextran sodium sulphate colitis models. Interestingly, restoration of tissue homeostasis is observed in NFkappaBD-treated animals with the rapid re-emergence of functional goblet cells and a return to normal patterns of cell proliferation in the mucosal epithelium and smooth muscle cell layers. CONCLUSIONS: These data support the potential use of "naked" NFkappaBD as a cross-functional therapeutic in IBD, and show for the first time that it can facilitate the restoration of colon homeostasis and function.
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Terapia Genética/métodos , Enfermedades Inflamatorias del Intestino/terapia , Oligodesoxirribonucleótidos/administración & dosificación , Animales , Colon/metabolismo , Sulfato de Dextran , Modelos Animales de Enfermedad , Femenino , Técnicas de Transferencia de Gen , Homeostasis/efectos de los fármacos , Mediadores de Inflamación/metabolismo , Enfermedades Inflamatorias del Intestino/inducido químicamente , Enfermedades Inflamatorias del Intestino/metabolismo , Enfermedades Inflamatorias del Intestino/patología , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Microscopía Confocal , FN-kappa B/metabolismo , Oligodesoxirribonucleótidos/uso terapéutico , Oxazolona , Ácido TrinitrobencenosulfónicoRESUMEN
Atopic dermatitis (AD) is a common chronic skin inflammatory disease. Long-term use of topical corticosteroids in skin inflammation poses risks of systemic and local side effects. The NF-kappaB transcription factor family plays a central role in the progression and maintenance of AD. This study explores the possibility of using topical NF-kappaB Decoy as a novel therapeutic alternative for targeting Th1/Th2-driven skin inflammation in experimental AD. A high-affinity, topical NF-kappaB Decoy developed for human efficacy demonstrates: (i) efficient NF-kappaB Decoy penetration in pig skin, (ii) NF-kappaB Decoy nuclear localization in keratinocytes and key immune cells, and (iii) potent "steroid-like" efficacy in a chronic dust-mite antigen skin inflammation treatment model. NF-kappaB Decoy exerts its anti-inflammatory action through the effective inhibition of essential regulators of inflammation and by induction of apoptosis of key immune cells. Unlike betamethasone valerate (BMV), long-term NF-kappaB Decoy treatment does not induce skin atrophy. Moreover, topical NF-kappaB Decoy, in contrast to BMV, restores compromised stratum corneum integrity and barrier function. Steroid withdrawal causes rapid rebound of inflammation, while the NF-kappaB Decoy therapeutic benefit was maintained for weeks. Thus, topical NF-kappaB Decoy provides a novel mechanism of reducing chronic skin inflammation with improved skin homeostasis and minimal side effects.
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Dermatitis Atópica/tratamiento farmacológico , Inmunosupresores/administración & dosificación , Oligodesoxirribonucleótidos/administración & dosificación , Piel/efectos de los fármacos , Administración Tópica , Animales , Apoptosis/efectos de los fármacos , Apoptosis/inmunología , Atrofia , División Celular/efectos de los fármacos , División Celular/inmunología , Dermatitis Atópica/inmunología , Dermatitis Atópica/patología , Oído Externo , Edema/tratamiento farmacológico , Edema/inmunología , Masculino , Ratones , Ratones Endogámicos , Pomadas , Permeabilidad/efectos de los fármacos , Piel/inmunología , Piel/patologíaRESUMEN
We have biologically characterized two new members of the IL-17 cytokine family: IL-17F and IL-25. In contrast to conventional in vitro screening approaches, we have characterized the activity of these new molecules by direct in vivo analysis and have compared their function to that of other IL-17 family members. Intranasal administration of adenovirus expressing IL-17, IL-17C, or IL-17F resulted in bronchoalveolar lavage neutrophilia and inflammatory gene expression in the lung. In contrast, intranasal administration of IL-25-expressing adenovirus or IL-25 protein resulted in the production of IL-4, IL-5, IL-13, and eotaxin mRNA in the lung and marked eosinophilia in the bronchoalveolar lavage and lung tissue. Mice given intranasal IL-25 also developed epithelial cell hyperplasia, increased mucus secretion, and airway hyperreactivity. IL-25 gene expression was detected following Aspergillus and Nippostrongylus infection in the lung and gut, respectively. IL-25-induced eosinophilia required IL-5 and IL-13, but not IL-4 or T cells. Following IL-25 administration, the IL-5(+) staining cells were CD45R/B220(+), Thy-1(+/-), but were NK1.1-, Ly-6G(GR-1)-, CD4-, CD3-, and c-kit-negative. gamma-common knockout mice did not develop eosinophilia in response to IL-25, nor were IL-5(+) cells detected. These findings suggest the existence of a previously unrecognized cell population that may initiate Th2-like responses by responding to IL-25 in vivo. Further, these data demonstrate the heterogeneity of function within the IL-17 cytokine family and suggest that IL-25 may be an important mediator of allergic disease via production of IL-4, IL-5, IL-13, and eotaxin.
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Sustancias de Crecimiento/administración & dosificación , Sustancias de Crecimiento/fisiología , Interleucina-17/fisiología , Interleucinas , Homología de Secuencia de Aminoácido , Células TH1/inmunología , Células TH1/metabolismo , Células Th2/inmunología , Células Th2/metabolismo , Adenoviridae/genética , Adenoviridae/inmunología , Administración Intranasal , Secuencias de Aminoácidos/genética , Secuencias de Aminoácidos/inmunología , Secuencia de Aminoácidos , Animales , Aspergilosis/genética , Aspergilosis/inmunología , Basófilos/inmunología , Hiperreactividad Bronquial/inmunología , Movimiento Celular/inmunología , Femenino , Vectores Genéticos , Granulocitos/inmunología , Sustancias de Crecimiento/química , Sustancias de Crecimiento/genética , Células Madre Hematopoyéticas/inmunología , Células Madre Hematopoyéticas/metabolismo , Humanos , Inflamación/genética , Inflamación/inmunología , Interleucina-13/fisiología , Interleucina-17/biosíntesis , Interleucina-17/química , Interleucina-17/genética , Interleucina-5/biosíntesis , Interleucina-5/fisiología , Parasitosis Intestinales/genética , Parasitosis Intestinales/inmunología , Células Asesinas Naturales/inmunología , Leucocitosis/genética , Leucocitosis/inmunología , Leucocitosis/virología , Pulmón/inmunología , Pulmón/metabolismo , Pulmón/patología , Subgrupos Linfocitarios/inmunología , Masculino , Mastocitos/inmunología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Noqueados , Datos de Secuencia Molecular , Moco/metabolismo , Neutrófilos/inmunología , Neutrófilos/virología , Eosinofilia Pulmonar/genética , Eosinofilia Pulmonar/inmunologíaRESUMEN
Ocular inflammation leads to vision loss through the destruction and scarring of delicate tissues along the visual axis. To identify inflammatory mediators involved in this process, we used real time RT-PCR to quantify the expression of mRNA transcripts of 34 cytokines, 26 chemokines, and 14 chemokine receptors at certain time points during T cell-mediated ocular inflammation. We induced disease by adoptive transfer of Ag-specific Th1 or Th2 cells into recipients expressing the target Ag in their eyes. We also compared the mediator expression patterns seen in adoptive transfer-induced inflammation with that seen in mouse eyes developing experimental autoimmune uveoretinitis. In addition, we used laser capture microdissection to examine chemokine mRNA production by both retinal pigment epithelium cells and infiltrating leukocytes in inflamed eyes. Major findings included the following: 1) Three patterns of expression of the inflammation-related molecules were seen in recipients of adoptively transferred Th cells: preferential expression in Th1 recipients, or in Th2 recipients, or similar expression in both recipient groups. 2) In experimental autoimmune uveoretinitis, the inflammatory mediator expression pattern largely paralleled that seen in Th1-induced disease. 3) Both retinal pigment epithelium and infiltrating leukocytes expressed chemokine transcripts in distinct, but overlapping patterns in inflamed eyes. 4) Interestingly, transcripts of multiple cytokines, chemokines, and chemokine receptors were constitutively expressed in high levels in mouse eyes. Seven of these molecules have not been previously associated with the eye. These data underscore the multiplicity of mediators that participate in the pathogenesis of eye inflammation and point to upstream cytokines as potential therapeutic targets.