RESUMEN
DNA sensor pathways can initiate inflammasome, cell death, and type I interferon (IFN) signaling in immune-mediated inflammatory diseases (IMIDs), including type I interferonopathies. We investigated the involvement of these pathways in the pathogenesis of ulcerative colitis (UC) by analyzing the expression of DNA sensor, inflammasome, and type I IFN biomarker genes in colonic mucosal biopsy tissue from control (n = 31), inactive UC (n = 31), active UC (n = 33), and a UC single-cell RNA-Seq dataset. The effects of type I IFN (IFN-ß), IFN-γ, and TNF-α on gene expression, cytokine production, and cell death were investigated in human colonic organoids. In organoids treated with cytokines alone, or in combination with NLR family pyrin domain-containing 3 (NLRP3), caspase, or JAK inhibitors, cell death was measured, and supernatants were assayed for IL-1ß/IL-18/CXCL10. The expression of DNA sensor pathway genes-PYHIN family members [absent in melanoma 2 (AIM2), IFI16, myeloid cell nuclear differentiation antigen (MNDA), and pyrin and HIN domain family member 1 (PYHIN1)- as well as Z-DNA-binding protein 1 (ZBP1), cyclic GMP-AMP synthase (cGAS), and DDX41 was increased in active UC and expressed in a cell type-restricted pattern. Inflammasome genes (CASP1, IL1B, and IL18), type I IFN inducers [stimulator of interferon response cGAMP interactor 1 (STING), TBK1, and IRF3), IFNB1, and type I IFN biomarker genes (OAS2, IFIT2, and MX2) were also increased in active UC. Cotreatment of organoids with IFN-ß or IFN-γ in combination with TNFα increased expression of IFI16, ZBP1, CASP1, cGAS, and STING induced cell death and IL-1ß/IL-18 secretion. This inflammatory cell death was blocked by the JAK inhibitor tofacitinib but not by inflammasome or caspase inhibitors. Increased type I IFN activity may drive elevated expression of DNA sensor genes and JAK-dependent but inflammasome-independent inflammatory cell death of colonic epithelial cells in UC.NEW & NOTEWORTHY This study found that patients with active UC have significantly increased colonic gene expression of cytosolic DNA sensor, inflammasome, STING, and type I IFN signaling pathways. The type I IFN, IFN-ß, in combination with TNF-α induced JAK-dependent but NLRP3 and inflammasome-independent inflammatory cell death of colonic organoids. This novel inflammatory cell death phenotype is relevant to UC immunopathology and may partially explain the efficacy of the JAKinibs tofacitinib and upadacitinib in patients with UC.
Asunto(s)
Colitis Ulcerosa , Interferón Tipo I , Inhibidores de las Cinasas Janus , Humanos , Inflamasomas/metabolismo , Interleucina-18 , Proteína con Dominio Pirina 3 de la Familia NLR/metabolismo , Factor de Necrosis Tumoral alfa , Inhibidores de Caspasas , Organoides/metabolismo , Pirina , Caspasa 1/metabolismo , Nucleotidiltransferasas/metabolismo , ADN , Muerte Celular , Proteínas de Unión al ADN/metabolismo , Antígenos de DiferenciaciónRESUMEN
CD40 is a costimulatory receptor on APCs that is critical for the induction and maintenance of humoral and cell-mediated immunity. Accordingly, CD40 and its ligand, CD40L, have long been considered targets for the treatment of autoimmune diseases. We developed a rat/mouse chimeric anti-mouse CD40 antagonist mAb, 201A3, and evaluated its ability to alleviate murine lupus. Treatment of NZB/W-F1 mice with 201A3 after the onset of severe proteinuria rapidly reversed established severe proteinuria and nephritis and largely restored normal glomerular and tubular morphology. This coincided with a normalization of the expression of genes associated with proteinuria and injury by kidney parenchymal cells. Anti-CD40 treatment also prevented and reversed loss of saliva production and sialadenitis. These effects on kidney and salivary gland function were confirmed using mice of a second strain, MRL/Mp-lpr/lpr, and extended to alleviating joint inflammation. Immunologically, anti-CD40 treatment disrupted multiple processes that contribute to the pathogenesis of systemic lupus erythematosus (SLE), including autoreactive B cell activation, T effector cell function in target tissues, and type I IFN production. This ability to disrupt disease-critical immunological mechanisms, to reverse glomerular and tubular injury at the cellular and gene expression levels, and to confer exceptional therapeutic efficacy suggests that CD40 is a central disease pathway in murine SLE. Thus, a CD40 antagonist Ab could be an effective therapeutic in the treatment of SLE.
Asunto(s)
Anticuerpos Bloqueadores/uso terapéutico , Linfocitos B/inmunología , Antígenos CD40/inmunología , Inmunoterapia/métodos , Glomérulos Renales/patología , Lupus Eritematoso Sistémico/terapia , Proteínas Recombinantes de Fusión/uso terapéutico , Linfocitos T/inmunología , Animales , Autoantígenos/inmunología , Células Cultivadas , Modelos Animales de Enfermedad , Humanos , Interferón Tipo I/metabolismo , Lupus Eritematoso Sistémico/inmunología , Ratones , Ratones Endogámicos MRL lpr , Ratones Endogámicos NZB , Proteinuria , Ratas , Eliminación SalivalRESUMEN
BACKGROUND & AIMS: Although tumor necrosis factor (TNF) antagonists reduce many clinical features of inflammatory bowel disease, complete mucosal healing occurs in fewer than 50% of patients. The Fc-region of monoclonal antibodies against TNF has immunosuppressive properties via effects on macrophage polarization. We examined the interaction between the anti-TNF Fc-region and Fcγ receptors (FcγR), and whether the absence of the Fc core fucose (which increases binding to FcγRIIIa) increases the efficacy of anti-TNF in mice with colitis. METHODS: We generated Rag1-/- mice that lack all activating FcγRs (FcγRI, FcγRIII, and FcγRIV; called FcγR-/-Rag1-/- mice). We produced hypo-fucosylated antibodies against mouse and human TNF (adalimumab). Colitis was induced in mice by transfer of CD4+CD45RBhi to FcγR-/-Rag1-/- or Rag1-/- littermates; mice were given different antibodies against TNF or isotype (control) antibodies and disease activity index scores were determined. Colon tissues were collected and analyzed by histology. Human peripheral blood mononuclear cells (PBMCs) were isolated from blood of healthy donors. T-cell proliferation and proportions of CD206+ (immune regulatory) macrophages were measured in mixed lymphocyte reactions. Human PBMCs were genotyped for FCGR3A158 (the FcγRIIIa-158F allotype displays a lower Fc binding affinity) using the TaqMan single nucleotide polymorphism genotype assay. RESULTS: Rag1-/- mice with colitis given anti-TNF had near complete mucosal healing and Rag1-/- mice given an isotype control antibody developed severe colitis. In contrast, FcγR-/-Rag1-/- mice were refractory to the effects of anti-TNF: their histological colitis scores were as severe as those from FcγR-/-Rag1-/- mice given a control antibody. Colons from Rag1-/- mice that received anti-TNF had an increased number of CD206+ macrophages compared with Rag1-/- mice given control antibody; in FcγR-/-Rag1-/- mice given anti-TNF these numbers were as low as FcγR-/-Rag1-/- given the control antibody. In human PBMCs, anti-TNF increased the number of CD206+ macrophages: this required expression of FcγRIIIa; numbers of these cells were reduced in PBMCs with the low-affinity FcγRIIIa-158F genotype. A hypo-fucosylated form of adalimumab bound human FcγRIIIa with a higher affinity than control adalimumab. When hypo-fucosylated adalimumab was added to PBMCs, a larger number of CD206+ macrophages formed and T-cell proliferation was reduced, compared with addition of a control adalimumab. Hypo-fucosylated adalimumab increased the number of CD206+ macrophages in PMBCs that expressed the low-affinity FcγRIIIa. In mice with colitis, hypo-fucosylated anti-TNF significantly increased the number of CD206+ macrophages in the colon compared with control anti-TNF and was more effective in reducing colitis severity as measured by histology. CONCLUSIONS: In a study of the in vitro and in vivo mechanisms of anti-TNF, we found FcγR engagement by anti-TNF to be required for reduction of colitis in mice and development of CD206+ macrophages. A hypo-fucosylated form of anti-TNF binds FcγRIIIa with higher affinity and induces development of CD206+ macrophages in human PBMCs, especially PBMCs that express low-affinity FcγRIIIa. Hypo-fucosylated anti-TNF might be more effective in patients with inflammatory bowel disease.
Asunto(s)
Adalimumab/farmacología , Anticuerpos Monoclonales/farmacología , Colitis/tratamiento farmacológico , Colon/efectos de los fármacos , Inmunosupresores/farmacología , Mucosa Intestinal/efectos de los fármacos , Factor de Necrosis Tumoral alfa/antagonistas & inhibidores , Traslado Adoptivo , Animales , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Colitis/genética , Colitis/inmunología , Colitis/metabolismo , Colon/inmunología , Colon/metabolismo , Colon/patología , Modelos Animales de Enfermedad , Predisposición Genética a la Enfermedad , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Mucosa Intestinal/inmunología , Mucosa Intestinal/metabolismo , Mucosa Intestinal/patología , Lectinas Tipo C/inmunología , Lectinas Tipo C/metabolismo , Activación de Linfocitos/efectos de los fármacos , Macrófagos/efectos de los fármacos , Macrófagos/inmunología , Macrófagos/metabolismo , Receptor de Manosa , Lectinas de Unión a Manosa/inmunología , Lectinas de Unión a Manosa/metabolismo , Ratones Endogámicos C57BL , Ratones Noqueados , Fenotipo , Receptores de Superficie Celular/inmunología , Receptores de Superficie Celular/metabolismo , Receptores de IgG/deficiencia , Receptores de IgG/genética , Linfocitos T/efectos de los fármacos , Linfocitos T/inmunología , Linfocitos T/metabolismo , Linfocitos T/trasplante , Factores de Tiempo , Factor de Necrosis Tumoral alfa/inmunología , Cicatrización de Heridas/efectos de los fármacosRESUMEN
In multiple sclerosis, long-term disability is caused by axonal and neuronal damage. Established therapies target primarily the inflammatory component of the disease, but fail to prevent neurodegeneration. Fingolimod (codenamed FTY720) is an oral sphingosine 1-phosphate (S1P) receptor modulator with promising results in phase II trials in multiple sclerosis patients and is under further development as a novel treatment for multiple sclerosis. To evaluate whether FTY720 has neuroprotective properties, we tested this drug in a rat model of myelin oligodendrocyte glycoprotein-induced optic neuritis. FTY720 exerted significant anti-inflammatory effects during optic neuritis and reduced inflammation, demyelination, and axonal damage; however, FTY720 treatment did not prevent apoptosis of retinal ganglion cells (RGCs), the neurons that form the axons of the optic nerve. Consistent with this lack of effect on RGC survival, FTY720 treatment did not improve visual function, nor did it prevent apoptosis of RGCs in vitro. We observed a persistent activation of apoptotic signaling pathways in RGCs under FTY720 treatment, a possible underlying mechanism for the lack of neuroprotection in the presence of strong anti-inflammatory effects, Furthermore, FTY720 shifted the remaining inflammation in the optic nerve toward neurotoxicity by modest up-regulation of potential neurotoxic cytokines. We conclude that FTY720-induced anti-inflammation and axon protection did not of itself protect neurons from apoptotic cell death.
Asunto(s)
Antiinflamatorios/farmacología , Neuronas/citología , Neuritis Óptica/metabolismo , Glicoles de Propileno/farmacología , Esfingosina/análogos & derivados , Animales , Apoptosis , Citocinas/metabolismo , Modelos Animales de Enfermedad , Electrofisiología/métodos , Encefalomielitis Autoinmune Experimental/inmunología , Femenino , Clorhidrato de Fingolimod , Glicoproteínas/metabolismo , Inmunosupresores/farmacología , Vaina de Mielina/metabolismo , Oligodendroglía/metabolismo , Ratas , Esfingosina/farmacologíaRESUMEN
Inflammatory bowel disease (IBD) is characterized by a dysregulated intestinal epithelial barrier leading to breach of barrier immunity. Here we identified similar protein expression changes between IBD and Citrobacter rodentium-infected FVB mice with respect to dysregulation of solute transporters as well as components critical for intestinal barrier integrity. We attribute the disease associated changes in the model to the emergence of undifferentiated intermediate intestinal epithelial cells. Prophylactic treatment with IL-22.Fc in C. rodentium-infected FVB mice reduced disease severity and rescued the mice from lethality. Multi-omics and solute analyses revealed that IL-22.Fc treatment prevented disease-associated changes including disruption of the solute transporter machinery and restored proper physiological functions of the intestine, respectively. Taken together, we established the disease relevance of the C. rodentium-induced colitis model to IBD, demonstrated the protective role of IL-22 in amelioration of epithelial dysfunction and elucidated the molecular mechanisms with IL-22's effect on intestinal epithelial cells.
Asunto(s)
Colitis , Infecciones por Enterobacteriaceae , Enfermedades Inflamatorias del Intestino , Interleucinas , Animales , Ratones , Citrobacter rodentium/fisiología , Colitis/tratamiento farmacológico , Colitis/microbiología , Infecciones por Enterobacteriaceae/tratamiento farmacológico , Enfermedades Inflamatorias del Intestino/tratamiento farmacológico , Enfermedades Inflamatorias del Intestino/microbiología , Mucosa Intestinal/metabolismo , Intestinos , Ratones Endogámicos C57BL , Interleucinas/farmacología , Interleucina-22RESUMEN
The Bcl-2 family of proteins plays a critical role in controlling immune responses by regulating the expansion and contraction of activated lymphocyte clones by apoptosis. ABT-737, which was originally developed for oncology, is a potent inhibitor of Bcl-2, Bcl-x(L), and Bcl-w protein function. There is evidence that Bcl-2-associated dysregulation of lymphocyte apoptosis may contribute to the pathogenesis of autoimmunity and lead to the development of autoimmune diseases. In this study, we report that ABT-737 treatment resulted in potent inhibition of lymphocyte proliferation as measured by in vitro mitogenic or ex vivo Ag-specific stimulation. More importantly, ABT-737 significantly reduced disease severity in tissue-specific and systemic animal models of autoimmunity. Bcl-2 family antagonism by ABT-737 was efficacious in treating animal models of arthritis and lupus. Our results suggest that treatment with a Bcl-2 family antagonist represents a novel and potentially attractive therapeutic approach for the clinical treatment of autoimmunity.
Asunto(s)
Autoinmunidad/efectos de los fármacos , Compuestos de Bifenilo/farmacología , Nitrofenoles/farmacología , Proteínas Proto-Oncogénicas c-bcl-2/antagonistas & inhibidores , Sulfonamidas/farmacología , Animales , Presentación de Antígeno/efectos de los fármacos , Artritis Experimental/inducido químicamente , Artritis Experimental/inmunología , Artritis Experimental/patología , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Hemocianinas/inmunología , Humanos , Hipersensibilidad Tardía/inmunología , Interferón-alfa/farmacología , Nefritis Lúpica/inducido químicamente , Nefritis Lúpica/inmunología , Nefritis Lúpica/patología , Linfocitos/citología , Linfocitos/efectos de los fármacos , Linfocitos/inmunología , Ratones , Piperazinas/farmacología , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Especificidad por SustratoRESUMEN
AIMS: Ulcerative colitis and Crohn's disease, collectively known as inflammatory bowel disease (IBD), are chronic inflammatory disorders of the intestine for which key elements in disease initiation and perpetuation are defects in epithelial barrier integrity. Achieving mucosal healing is essential to ameliorate disease outcome and so new therapies leading to epithelial homeostasis and repair are under investigation. This study was designed to determine the mechanisms by which IL-22 regulates intestinal epithelial cell function. MAIN METHODS: Human intestinal organoids and resections, as well as mice were used to evaluate the effect of IL-22 on stem cell expansion, proliferation and expression of mucus components. IL-22 effect on barrier function was assessed in polarized T-84 cell monolayers. Butyrate co-treatments and organoid co-cultures with immune cells were performed to monitor the impact of microbial-derived metabolites and inflammatory environments on IL-22 responses. KEY FINDINGS: IL-22 led to epithelial stem cell expansion, proliferation, barrier dysfunction and anti-microbial peptide production in human and mouse models evaluated. IL-22 also altered the mucus layer by inducing an increase in membrane mucus but a decrease in secreted mucus and goblet cell content. IL-22 had the same effect on anti-microbial peptides and membrane mucus in both healthy and IBD human samples. In contrast, this IL-22-associated epithelial phenotype was different when treatments were performed in presence of butyrate and organoids co-cultured with immune cells. SIGNIFICANCE: Our data indicate that IL-22 promotes epithelial regeneration, innate defense and membrane mucus production, strongly supporting the potential clinical utility of IL-22 as a mucosal healing therapy in IBD.
Asunto(s)
Células Epiteliales/fisiología , Homeostasis/fisiología , Interleucinas/fisiología , Interleucinas/uso terapéutico , Mucosa Intestinal/fisiología , Animales , Línea Celular , Técnicas de Cocultivo , Colitis Ulcerosa/tratamiento farmacológico , Colitis Ulcerosa/patología , Células Epiteliales/efectos de los fármacos , Homeostasis/efectos de los fármacos , Humanos , Interleucinas/farmacología , Mucosa Intestinal/citología , Mucosa Intestinal/efectos de los fármacos , Ratones , Ratones Endogámicos C57BL , Organoides/efectos de los fármacos , Organoides/fisiología , Interleucina-22RESUMEN
Rewiring of host cytokine networks is a key feature of inflammatory bowel diseases (IBD) such as Crohn's disease (CD). Th1-type cytokines-IFN-γ and TNF-α-occupy critical nodes within these networks and both are associated with disruption of gut epithelial barrier function. This may be due to their ability to synergistically trigger the death of intestinal epithelial cells (IECs) via largely unknown mechanisms. In this study, through unbiased kinome RNAi and drug repurposing screens we identified JAK1/2 kinases as the principal and nonredundant drivers of the synergistic killing of human IECs by IFN-γ/TNF-α. Sensitivity to IFN-γ/TNF-α-mediated synergistic IEC death was retained in primary patient-derived intestinal organoids. Dependence on JAK1/2 was confirmed using genetic loss-of-function studies and JAK inhibitors (JAKinibs). Despite the presence of biochemical features consistent with canonical TNFR1-mediated apoptosis and necroptosis, IFN-γ/TNF-α-induced IEC death was independent of RIPK1/3, ZBP1, MLKL or caspase activity. Instead, it involved sustained activation of JAK1/2-STAT1 signalling, which required a nonenzymatic scaffold function of caspase-8 (CASP8). Further modelling in gut mucosal biopsies revealed an intercorrelated induction of the lethal CASP8-JAK1/2-STAT1 module during ex vivo stimulation of T cells. Functional studies in CD-derived organoids using inhibitors of apoptosis, necroptosis and JAKinibs confirmed the causative role of JAK1/2-STAT1 in cytokine-induced death of primary IECs. Collectively, we demonstrate that TNF-α synergises with IFN-γ to kill IECs via the CASP8-JAK1/2-STAT1 module independently of canonical TNFR1 and cell death signalling. This non-canonical cell death pathway may underpin immunopathology driven by IFN-γ/TNF-α in diverse autoinflammatory diseases such as IBD, and its inhibition may contribute to the therapeutic efficacy of anti-TNFs and JAKinibs.
Asunto(s)
Caspasa 8/metabolismo , Células Epiteliales/patología , Interferón gamma/metabolismo , Intestinos/patología , Janus Quinasa 1/metabolismo , Factor de Transcripción STAT1/metabolismo , Factor de Necrosis Tumoral alfa/metabolismo , Apoptosis , Biopsia , Muerte Celular , Línea Celular Tumoral , Colon/patología , Citoprotección , Células Epiteliales/metabolismo , Humanos , Janus Quinasa 2/metabolismo , Mitocondrias/metabolismo , Organoides/patología , Interferencia de ARN , Receptores Tipo I de Factores de Necrosis Tumoral/metabolismo , Transducción de SeñalRESUMEN
Intestinal permeability and neutrophil activity are closely linked to inflammatory bowel disease (IBD) pathophysiology. Here we discuss two techniques for assessing permeability and neutrophil activity in mouse IBD models using near infrared (NIR) detection. To address the limitation of visible light readouts-namely high background-IRDye 800CW was used to enable rapid, non-terminal measurements of intestinal permeability. The increased sensitivity of NIR readouts for colon permeability is shown using dextran sulfate sodium (DSS) and anti-CD40 murine colitis models in response to interleukin-22 immunoglobulin Fc (IL22Fc) fusion protein and anti-p40 monoclonal antibody treatments, respectively. In addition to enhanced permeability, elevated levels of neutrophil elastase (NE) have been reported in inflamed colonic mucosal tissue. Activatable NIR fluorescent probes have been extensively used for disease activity evaluation in oncologic animal models, and we demonstrate their translatability using a NE-activatable reagent to evaluate inflammation in DSS mice. Confocal laser endomicroscopy (CLE) and tissue imaging allow visualization of spatial NE activity throughout diseased colon as well as changes in disease severity from IL22Fc treatment. Our findings with the 800CW dye and the NE probe highlight the ease of their implementation in preclinical IBD research.
Asunto(s)
Enfermedades Inflamatorias del Intestino/diagnóstico , Enfermedades Inflamatorias del Intestino/metabolismo , Mucosa Intestinal/metabolismo , Mucosa Intestinal/patología , Imagen Óptica/métodos , Animales , Transporte Biológico , Biomarcadores , Modelos Animales de Enfermedad , Inmunohistoquímica , Enfermedades Inflamatorias del Intestino/etiología , Elastasa de Leucocito/metabolismo , Ratones , Microscopía Confocal , Permeabilidad , Espectroscopía Infrarroja CortaRESUMEN
BACKGROUND: CD40 is a 48 kDa type I transmembrane protein that is constitutively expressed on hematopoietic cells such as dendritic cells, macrophages, and B cells. Engagement of CD40 by CD40L expressed on T cells results in the production of proinflammatory cytokines, induces T helper cell function, and promotes macrophage activation. The involvement of CD40 in chronic immune activation has resulted in CD40 being proposed as a therapeutic target for a range of chronic inflammatory diseases. CD40 antagonists are currently being explored for the treatment of autoimmune diseases and several anti-CD40 agonist mAbs have entered clinical development for oncological indications. RESULTS: To better understand the mode of action of anti-CD40 mAbs, we have determined the x-ray crystal structures of the ABBV-323 (anti-CD40 antagonist, ravagalimab) Fab alone, ABBV-323 Fab complexed to human CD40 and FAB516 (anti-CD40 agonist) complexed to human CD40. These three crystals structures 1) identify the conformational CD40 epitope for ABBV-323 recognition 2) illustrate conformational changes which occur in the CDRs of ABBV-323 Fab upon CD40 binding and 3) develop a structural hypothesis for an agonist/antagonist switch in the LCDR1 of this proprietary class of CD40 antibodies. CONCLUSIONS: The structure of ABBV-323 Fab demonstrates a unique method for antagonism by stabilizing the proposed functional antiparallel dimer for CD40 receptor via novel contacts to LCDR1, namely residue position R32 which is further supported by a closely related agonist antibody FAB516 which shows only monomeric recognition and no contacts with LCDR1 due to a mutation to L32 on LCDR1. These data provide a structural basis for the full antagonist activity of ABBV-323.
Asunto(s)
Anticuerpos Monoclonales/química , Complejo Antígeno-Anticuerpo/química , Antígenos CD40/agonistas , Antígenos CD40/antagonistas & inhibidores , Antígenos CD40/química , Células HEK293 , Humanos , Fragmentos Fab de Inmunoglobulinas/química , Modelos Moleculares , Transducción de Señal , Electricidad EstáticaRESUMEN
BACKGROUND: Fibrosis in patients with Crohn's disease (CD) results from an imbalance toward excessive fibrous tissue formation driven by fibroblasts. Activation of fibroblasts is linked to the B-cell lymphoma 2 (BCL2) family, which is involved in the induction of apoptosis. We investigated the impact of BCL2 repression on fibrogenesis. METHODS: The model of dextran sodium sulfate (DSS)-induced chronic colitis and the heterotopic transplantation model of fibrosis were used. Following the administration of the BCL2 antagonist (ABT-737, 50 mg/kg/d), collagen layer thickness and hydroxyproline (HYP) content were determined. Fibroblasts were stimulated with the BCL2 antagonist (0.01-100 µM). BCL2, alpha smooth muscle actin (αSMA), and collagen I (COL1A1) were determined by quantitative polymerase chain reaction (qPCR), immunofluorescence microscopy (IF), and western blot (WB). mRNA expression pattern was determined by next-generation sequencing (NGS). RESULTS: Collagen layer thickness was significantly decreased in both DSS-induced chronic colitis and the transplantation model of fibrosis upon BCL2 antagonist administration compared with vehicle. Decreased HYP content confirmed the preventive effects of the BCL2 antagonist on fibrosis. In vitro, a significant increase in PI+/annexin V+ human colonic fibroblasts was determined by fluorescence-activated cell sorting upon treatment with high-dose BCL2 antagonist; at a lower dose, αSMA, COL1A1, and TGF were decreased. NGS, IF, and qPCR revealed decreased expression and nuclear translocation of GATA6 and SOX9, known for reprogramming fibroblasts. CONCLUSION: BCL2 antagonist administration partially prevented fibrogenesis in both fibrosis models. The BCL2 antagonist reduced the expression of TGFß-induced factors involved in differentiation of myofibroblasts, and therefore might represent a potential treatment option against CD-associated fibrosis.
Asunto(s)
Compuestos de Bifenilo/administración & dosificación , Diferenciación Celular/genética , Fibroblastos/efectos de los fármacos , Intestinos/patología , Nitrofenoles/administración & dosificación , Proteínas Proto-Oncogénicas c-bcl-2/antagonistas & inhibidores , Sulfonamidas/administración & dosificación , Anciano , Animales , Técnicas de Cultivo de Célula , Sulfato de Dextran , Femenino , Fibrosis/inducido químicamente , Fibrosis/tratamiento farmacológico , Fibrosis/genética , Humanos , Mucosa Intestinal , Intestinos/citología , Ratones , Ratones Endogámicos C57BL , Persona de Mediana Edad , Piperazinas/administración & dosificaciónRESUMEN
A HTS campaign identified compound 1, an excellent hit-like molecule to initiate medicinal chemistry efforts to optimize a dual ROCK1 and ROCK2 inhibitor. Substitution (2-Cl, 2-NH2, 2-F, 3-F) of the pyridine hinge binding motif or replacement with pyrimidine afforded compounds with a clean CYP inhibition profile. Cocrystal structures of an early lead compound were obtained in PKA, ROCK1, and ROCK2. This provided critical structural information for medicinal chemistry to drive compound design. The structural data indicated the preferred configuration at the central benzylic carbon would be ( R), and application of this information to compound design resulted in compound 16. This compound was shown to be a potent and selective dual ROCK inhibitor in both enzyme and cell assays and efficacious in the retinal nerve fiber layer model after oral dosing. This tool compound has been made available through the AbbVie Compound Toolbox. Finally, the cocrystal structures also identified that aspartic acid residues 176 and 218 in ROCK2, which are glutamic acids in PKA, could be targeted as residues to drive both potency and kinome selectivity. Introduction of a piperidin-3-ylmethanamine group to the compound series resulted in compound 58, a potent and selective dual ROCK inhibitor with excellent predicted drug-like properties.
Asunto(s)
Inhibidores de Proteínas Quinasas/química , Inhibidores de Proteínas Quinasas/farmacología , Quinasas Asociadas a rho/antagonistas & inhibidores , Administración Oral , Animales , Disponibilidad Biológica , Cristalografía por Rayos X , Inhibidores del Citocromo P-450 CYP2C9/química , Inhibidores del Citocromo P-450 CYP2C9/farmacología , Inhibidores del Citocromo P-450 CYP3A/química , Inhibidores del Citocromo P-450 CYP3A/farmacología , Diseño de Fármacos , Evaluación Preclínica de Medicamentos/métodos , Semivida , Humanos , Ratones Endogámicos C57BL , Traumatismos del Nervio Óptico/tratamiento farmacológico , Traumatismos del Nervio Óptico/patología , Ratas Sprague-Dawley , Relación Estructura-Actividad , Quinasas Asociadas a rho/químicaRESUMEN
BACKGROUND AND AIMS: Anti-tumour necrosis factor [TNF] monoclonal antibodies [infliximab, adalimumab] induce complete mucosal healing in a proportion of patients with Crohn's disease whereas a TNF receptor fusion protein [etanercept] is not effective and the anti-TNF F[ab']2 fragment [certolizumab] shows a very low rate of complete mucosal healing. In contrast, all four TNF-neutralising drugs have demonstrated efficacy in the treatment of rheumatoid arthritis. These observations suggest that factors other than neutralisation of TNF may contribute to clinical outcomes in Crohn's disease. Here we tested the hypothesis that Fc receptor [FcR]-mediated effects may contribute to the therapeutic response of anti-TNF antibodies in inflammatory bowel disease. METHODS: We modified an IgG2c mouse anti-TNF antibody that binds the high-affinity FcRs to generate an IgG1 isotype with strongly diminished binding. We examined the therapeutic effects of both antibodies in the T cell transfer model of inflammatory bowel disease and the collagen-induced arthritis model. RESULTS: The IgG2c anti-TNF antibody prevented colonic inflammation in the T cell transfer model of colitis, whereas the IgG1 anti-TNF did not. Conversely, both the IgG2c and IgG1 anti-TNFs were similarly effective in reducing the severity of articular inflammation in mouse collagen-induced arthritis. CONCLUSION: These data support the concept that the mechanism of action for TNF-neutralising drugs may differ across immune-mediated diseases and, potentially, between therapeutics within a particular disease. Our data suggest a specific role of Fc-mediated immune regulation in the resolution of intestinal inflammation by anti-TNF monoclonal antibodies.
Asunto(s)
Anticuerpos Monoclonales Humanizados/administración & dosificación , Enfermedades Inflamatorias del Intestino/tratamiento farmacológico , Enfermedades Inflamatorias del Intestino/inmunología , Receptores Fc/efectos de los fármacos , Factor de Necrosis Tumoral alfa/antagonistas & inhibidores , Animales , Artritis Experimental/tratamiento farmacológico , Artritis Experimental/inmunología , Artritis Experimental/patología , Biomarcadores/metabolismo , Colitis Ulcerosa/tratamiento farmacológico , Colitis Ulcerosa/inmunología , Colitis Ulcerosa/patología , Enfermedad de Crohn/tratamiento farmacológico , Enfermedad de Crohn/inmunología , Enfermedad de Crohn/patología , Modelos Animales de Enfermedad , Femenino , Inmunohistoquímica , Enfermedades Inflamatorias del Intestino/patología , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos DBA , Ratones SCID , Terapia Molecular Dirigida/métodos , Distribución Aleatoria , Receptores Fc/metabolismo , Sensibilidad y Especificidad , Factor de Necrosis Tumoral alfa/administración & dosificaciónRESUMEN
The Src family kinases Lck and Fyn play an important role in T cell development and function. We have synthesized a novel small molecule, A-420983, which inhibits Lck and Fyn, as well as other Src family kinases, but has selectivity with respect to non-Src family kinases. A-420983 completely inhibited antigen-stimulated production of IFN-gamma and IL-4 by mouse Th1 and Th2 cells, respectively. Antigen-induced T cell proliferation was also blocked by treatment with A-420983. In contrast, IL-15-induced proliferation was unaffected by A-420983, suggesting that TCR-independent pathways of T cell activation were not impaired. When mice were dosed orally, A-420983 inhibited TCR-mediated c-jun and ZAP-70 phosphorylation in CD4+ T cells and suppressed the disease course of established EAE. Treatment with A-420983 for 7 days resulted in a block in thymocyte development at the CD4- CD8- stage, consistent with inhibition of Lck and Fyn in vivo. These results demonstrate that a small molecule inhibitor of Lck and Fyn can block TCR-induced T cell activation in vitro and in vivo. Furthermore, CNS demyelination mediated by activated encephalitogenic CD4+ T cells is dependent upon the kinase activity of these Src family members. We conclude that inhibition of Src family kinases may represent a promising strategy for the treatment of T cell-mediated disorders.
Asunto(s)
Linfocitos T CD4-Positivos/efectos de los fármacos , Activación de Linfocitos/efectos de los fármacos , Pirazoles/farmacología , Pirimidinas/farmacología , Familia-src Quinasas/antagonistas & inhibidores , Animales , Línea Celular , Proliferación Celular/efectos de los fármacos , Femenino , Ratones , Ratones Endogámicos , Timo/citología , Timo/efectos de los fármacosRESUMEN
USPIO-enhanced MRI allows non-invasive visualization of mononuclear cell infiltration into CNS lesions in MS and EAE. Herein, we show a distinct spatiotemporal pattern of CNS lesions that reveals the involvement of spino-olivocerebellar pathways in MOG-induced EAE rats using USPIO-enhanced MRI. Specifically, lesions of the inferior olives were observed primarily in the acute phase whereas lesions of cerebellum or spinal cord/brainstem were observed during the relapse phase. Further, behavioral deficits observed from these animals are consistent with the functional role of spino-olivocerebellar pathways in coordination and movement. Collectively, our results provide new insights into the pathophysiology of this animal model of MS.
Asunto(s)
Encéfalo/patología , Encefalomielitis Autoinmune Experimental/patología , Imagen por Resonancia Magnética , Vías Nerviosas/patología , Médula Espinal/patología , Animales , Cerebelo/patología , Dextranos , Femenino , Óxido Ferrosoférrico , Inmunohistoquímica , Hierro , Imagen por Resonancia Magnética/métodos , Nanopartículas de Magnetita , Actividad Motora/fisiología , Proteínas de la Mielina , Glicoproteína Asociada a Mielina/inmunología , Glicoproteína Asociada a Mielina/toxicidad , Glicoproteína Mielina-Oligodendrócito , Núcleo Olivar/patología , Óxidos , Radiofármacos , RatasRESUMEN
OBJECTIVE: To assess the role of interleukin-18 (IL-18) in the evolution of septic arthritis induced by group B streptococci (GBS) in mice. METHODS: CD1 mice were inoculated intravenously with 8 x 10(6) colony-forming units (CFU) of type IV GBS (strain 1/82), and administered intraperitoneally 1 hour before infection with anti-IL-18 monoclonal antibodies (0.25 mg/mouse). In a subsequent set of experiments, mice infected with a suboptimal arthritogenic dose of GBS (4 x 10(6) CFU/mouse) were administered different doses of recombinant IL-18 for 4 days, starting 1 hour after infection. Mortality, evolution of arthritis, bacterial clearance, joint histopathology, and cytokine production were examined in infected mice that did or did not receive treatment with anti-IL-18 antibodies or IL-18. RESULTS: IL-18 was produced during GBS infection. Neutralization of IL-18 resulted in a decrease in mortality rates, and in the incidence and severity of arthritis. Amelioration of arthritis was accompanied by a dramatic reduction in local IL-1 beta, IL-6, macrophage inflammatory protein 1 alpha (MIP-1 alpha) and MIP-2 production, and reduced bacterial burden. Administration of exogenous IL-18 resulted in increased mortality rates and increased incidence and severity of GBS arthritis, concomitant with a higher number of GBS and increased levels of IL-6, IL-1 beta, MIP-1 beta, and MIP-2 production in the joints. CONCLUSION: The present study indicated some involvement of IL-18 in the pathogenesis of GBS-induced arthritis. The role of IL-18 in joint pathology is shown by a regulatory effect on inflammatory mediator levels and local cell influx. Thus, IL-18 should be regarded as a potential therapeutic target in GBS infection and arthritis.