RESUMEN
BACKGROUND: New medications for Rheumatoid Arthritis (RA) have emerged in the last decades, including Disease Modifying Antirheumatic Drugs (DMARDs) and biologics. However, there is no known cure, since a significant proportion of patients remain or become non-responders to current therapies. The development of new mode-of-action treatment schemes involving combination therapies could prove successful for the treatment of a greater number of RA patients. METHODS: We investigated the effect of the Tyrosine Kinase inhibitors (TKIs) dasatinib and bosutinib, on the human TNF-dependent Tg197 arthritis mouse model. The inhibitors were administered either as a monotherapy or in combination with a subtherapeutic dose of anti-hTNF biologics and their therapeutic effect was assessed clinically, histopathologically as well as via gene expression analysis and was compared to that of an efficient TNF monotherapy. RESULTS: Dasatinib and, to a lesser extent, bosutinib inhibited the production of TNF and proinflammatory chemokines from arthritogenic synovial fibroblasts. Dasatinib, but not bosutinib, also ameliorated significantly and in a dose-dependent manner both the clinical and histopathological signs of Tg197 arthritis. Combination of dasatinib with a subtherapeutic dose of anti-hTNF biologic agents, resulted in a synergistic inhibitory effect abolishing all arthritis symptoms. Gene expression analysis of whole joint tissue of Tg197 mice revealed that the combination of dasatinib with a low subtherapeutic dose of Infliximab most efficiently restores the pathogenic gene expression profile to that of the healthy state compared to either treatment administered as a monotherapy. CONCLUSION: Our findings show that dasatinib exhibits a therapeutic effect in TNF-driven arthritis and can act in synergy with a subtherapeutic anti-hTNF dose to effectively treat the clinical and histopathological signs of the pathology. The combination of dasatinib and anti-hTNF exhibits a distinct mode of action in restoring the arthritogenic gene signature to that of a healthy profile. Potential clinical applications of combination therapies with kinase inhibitors and anti-TNF agents may provide an interesting alternative to high-dose anti-hTNF monotherapy and increase the number of patients responding to treatment.
Asunto(s)
Antirreumáticos , Artritis Reumatoide , Dasatinib , Inhibidores del Factor de Necrosis Tumoral/uso terapéutico , Animales , Antirreumáticos/uso terapéutico , Artritis Reumatoide/tratamiento farmacológico , Dasatinib/uso terapéutico , Humanos , Infliximab/uso terapéutico , RatonesRESUMEN
BACKGROUND: The transmembrane-TNF transgenic mouse, TgA86, has been shown to develop spontaneously peripheral arthritis with signs of axial involvement. To assess similarity to human spondyloarthritis, we performed detailed characterization of the axial, peripheral, and comorbid pathologies of this model. METHODS: TgA86 bone pathologies were assessed at different ages using CT imaging of the spine, tail vertebrae, and hind limbs and characterized in detail by histopathological and immunohistochemical analysis. Cardiac function was examined by echocardiography and electrocardiography and bone structural parameters by µCT analysis. The response of TgA86 mice to either early or late anti-TNF treatment was evaluated clinically, histopathologically, and by µCT analysis. RESULTS: TgA86 mice developed with 100% penetrance spontaneous axial and peripheral pathology which progressed with time and manifested as reduced body weight and body length, kyphosis, tail bendings, as well as swollen and distorted hind joints. Whole-body CT analysis at advanced ages revealed bone erosions of sacral and caudal vertebrae as well as of sacroiliac joints and hind limbs and, also, new ectopic bone formation and eventually vertebral fusion. The pathology of these mice highly resembled that of SpA patients, as it evolved through an early inflammatory phase, evident as enthesitis and synovitis in the affected joints, characterized by mesenchymal cell accumulation, and neutrophilic infiltration. Subsequently, regression of inflammation was accompanied by ectopic bone formation, leading to ankylosis. In addition, both systemic bone loss and comorbid heart valve pathology were evident. Importantly, early anti-TNF treatment, similar to clinical treatment protocols, significantly reduced the inflammatory phase of both the axial and peripheral pathology of TgA86 mice. CONCLUSIONS: The TgA86 mice develop a spontaneous peripheral and axial biphasic pathology accompanied by comorbid heart valvular dysfunction and osteoporosis, overall reproducing the progression of pathognomonic features of human spondyloarthritis. Therefore, the TgA86 mouse represents a valuable model for deciphering the role of transmembrane TNF in the pathogenic mechanisms of spondyloarthritis and for assessing the efficacy of human therapeutics targeting different phases of the disease.
Asunto(s)
Osteogénesis , Espondiloartritis , Animales , Humanos , Inflamación , Imagen por Resonancia Magnética , Ratones , Articulación Sacroiliaca , Espondiloartritis/diagnóstico por imagen , Inhibidores del Factor de Necrosis TumoralRESUMEN
Anti-TNF agents have been in the first line of treatment of various inflammatory diseases such as Rheumatoid Arthritis and Crohn's Disease, with a number of different biologics being currently in use. A detailed analysis of their effect at transcriptome level has nevertheless been lacking. We herein present a concise analysis of an extended transcriptomics profiling of four different anti-TNF biologics upon treatment of the established hTNFTg (Tg197) mouse model of spontaneous inflammatory polyarthritis. We implement a series of computational analyses that include clustering of differentially expressed genes, functional analysis and random forest classification. Taking advantage of our detailed sample structure, we devise metrics of treatment efficiency that take into account changes in gene expression compared to both the healthy and the diseased state. Our results suggest considerable variability in the capacity of different biologics to modulate gene expression that can be attributed to treatment-specific functional pathways and differential preferences to restore over- or under-expressed genes. Early intervention appears to manage inflammation in a more efficient way but is accompanied by increased effects on a number of genes that are seemingly unrelated to the disease. Administration at an early stage is also lacking in capacity to restore healthy expression levels of under-expressed genes. We record quantifiable differences among anti-TNF biologics in their efficiency to modulate over-expressed genes related to immune and inflammatory pathways. More importantly, we find a subset of the tested substances to have quantitative advantages in addressing deregulation of under-expressed genes involved in pathways related to known RA comorbidities. Our study shows the potential of transcriptomic analyses to identify comprehensive and distinct treatment-specific gene signatures combining disease-related and unrelated genes and proposes a generalized framework for the assessment of drug efficacy, the search of biosimilars and the evaluation of the efficacy of TNF small molecule inhibitors.
Asunto(s)
Artritis Reumatoide/tratamiento farmacológico , Artritis/genética , Perfilación de la Expresión Génica/métodos , Adalimumab/farmacología , Animales , Artritis/tratamiento farmacológico , Biosimilares Farmacéuticos , Certolizumab Pegol/farmacología , Modelos Animales de Enfermedad , Femenino , Humanos , Inflamación/inmunología , Infliximab/farmacología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Transcriptoma/genética , Resultado del Tratamiento , Factor de Necrosis Tumoral alfa/inmunología , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
We have developed a composite hydrogel for improved topical delivery of the poorly soluble drug Tacrolimus (TAC) to psoriasis lesions. TAC is efficiently solubilized in methoxy poly- (ethylene glycol) hexyl substituted poly-(lactic acid) (mPEGhexPLA) based nanocarriers. For convenient and patient-friendly topical administration, TAC loaded polymeric nanocarriers were incorporated in a Carbopol® based hydrogel, to yield a composite hydrogel formulation (TAC composite hydrogel). TAC composite hydrogel was designed to have superior pharmaceutical formulation properties, delivery efficiency and local bioavailability, compared to currently available paraffin-based TAC ointments. Composite hydrogel formulations had good local tolerance and showed no signs of immediate toxicity after repeated topical administration in healthy mice. Skin delivery of TAC composite hydrogel in an imiquimod-induced psoriasis mouse model was found to be twice as high as for the commercial formulation Protopic™, used as benchmark. TAC composite hydrogel showed significant improvement in the in vivo and histopathological features of the imiquimod-induced psoriasis model.
Asunto(s)
Sistemas de Liberación de Medicamentos , Inmunosupresores/administración & dosificación , Psoriasis/tratamiento farmacológico , Tacrolimus/administración & dosificación , Administración Cutánea , Aminoquinolinas , Animales , Disponibilidad Biológica , Química Farmacéutica , Modelos Animales de Enfermedad , Portadores de Fármacos/química , Hidrogeles , Imiquimod , Inmunosupresores/farmacocinética , Inmunosupresores/farmacología , Ratones , Ratones Endogámicos C57BL , Polímeros/química , Piel/metabolismo , Absorción Cutánea , Solubilidad , Tacrolimus/farmacocinética , Tacrolimus/farmacologíaRESUMEN
BACKGROUND: Novel molecules that specifically target human TNFα in rheumatoid arthritis pose problems for preclinical assessment of efficacy. In this study collagen antibody-induced arthritis (CAIA) has been induced in human TNFα transgenic mice to provide a novel model that has been optimised for the evaluation of molecules targeting human TNFα. METHODS: Tg1278TNFko mice lack murine TNFα and are heterozygous for multiple copies of the human TNFα transgene that is expressed under normal physiological control. To establish CAIA, a collagen II monoclonal antibody cocktail (CAb) at 2, 4 or 8 mg was injected i.p. on Day 0 followed by a lipopolysaccharide (LPS) boost (10 or 100 µg) i.p. on Day 1 or Day 4. Animals were assessed for arthritis symptoms using a clinical score, cytokine levels (human TNFα, IL-1ß and IL-6) in sera and joints, and histopathology. The dependence of the model on human TNFα was determined by dosing animals with etanercept. RESULTS: Tg1278TNFko animals treated with 2, 4 or 8 mg CAb on Day 0, with 100 µg LPS on Day 4, had more severe arthritis and earlier symptoms than wild type animals at all doses of CAb tested. Subsequently it was found that the transgenic model did not require LPS at all for arthritis development but a lower dose of LPS (10 µg) was found necessary for reproducible and robust disease (close to 100% incidence, well-synchronised, with high arthritis scores). Furthermore the LPS challenge could be brought forward to Day 1 so that its' actions to facilitate disease could be separated temporally from the arthritis phase (beginning about Day 4). Etanercept, administered immediately after the serum spike of cytokines associated with LPS had subsided, was able to dose-dependently inhibit arthritis development and this was associated with a marked protection of the joints histologically on Day 14. Etanercept was also able to reverse the signs of arthritis when given therapeutically allowing animals to be matched for disease burden before dosing begins. CONCLUSIONS: The features of CAIA in Tg1278TNFko animals make the model well-suited to testing the next generation of therapeutics that will target human TNFα in rheumatoid arthritis.
Asunto(s)
Anticuerpos/administración & dosificación , Artritis Experimental/tratamiento farmacológico , Artritis Reumatoide/tratamiento farmacológico , Colágeno Tipo II/antagonistas & inhibidores , Factor de Necrosis Tumoral alfa/antagonistas & inhibidores , Animales , Artritis Reumatoide/metabolismo , Etanercept/uso terapéutico , Humanos , Ratones , Ratones Noqueados , Ratones TransgénicosRESUMEN
TNF has remarkable antitumor activities; however, therapeutic applications have not been possible because of the systemic and lethal proinflammatory effects induced by TNF. Both the antitumor and inflammatory effects of TNF are mediated by the TNF receptor p55 (p55TNFR) (encoded by the Tnfrsf1a gene). The antitumor effect stems from an induction of cell death in tumor endothelium, but the cell type that initiates the lethal inflammatory cascade has been unclear. Using conditional Tnfrsf1a knockout or reactivation mice, we found that the expression level of p55TNFR in intestinal epithelial cells (IECs) is a crucial determinant in TNF-induced lethal inflammation. Remarkably, tumor endothelium and IECs exhibited differential sensitivities to TNF when p55TNFR levels were reduced. Tumor-bearing Tnfrsf1aâºâº/â» or IEC-specific p55TNFR-deficient mice showed resistance to TNF-induced lethality, while the tumor endothelium remained fully responsive to TNF-induced apoptosis and tumors regressed. We demonstrate proof of principle for clinical application of this approach using neutralizing anti-human p55TNFR antibodies in human TNFRSF1A knockin mice. Our results uncover an important cellular basis of TNF toxicity and reveal that IEC-specific or systemic reduction of p55TNFR mitigates TNF toxicity without loss of antitumor efficacy.
Asunto(s)
Antineoplásicos/farmacología , Mucosa Intestinal/efectos de los fármacos , Melanoma Experimental/tratamiento farmacológico , Receptores Tipo I de Factores de Necrosis Tumoral/genética , Factor de Necrosis Tumoral alfa/farmacología , Animales , Anticuerpos Monoclonales/farmacología , Antineoplásicos/uso terapéutico , Antineoplásicos/toxicidad , Apoptosis , Citocinas/genética , Citocinas/metabolismo , Ensayos de Selección de Medicamentos Antitumorales , Endotelio/patología , Expresión Génica , Técnicas de Sustitución del Gen , Humanos , Inflamación/inducido químicamente , Interferón gamma/fisiología , Mucosa Intestinal/metabolismo , Mucosa Intestinal/patología , Melanoma Experimental/metabolismo , Melanoma Experimental/patología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Trasplante de Neoplasias , Receptores Tipo I de Factores de Necrosis Tumoral/antagonistas & inhibidores , Receptores Tipo I de Factores de Necrosis Tumoral/metabolismo , Transducción de Señal , Factor de Necrosis Tumoral alfa/uso terapéutico , Factor de Necrosis Tumoral alfa/toxicidadRESUMEN
Heme oxygenase-1 (HO-1) is a key cytoprotective, antioxidant, and antiinflammatory molecule. The pathophysiological functions of HO-1 have been associated with its enzymatic activities in heme catabolism. We have examined the immune functions of HO-1 by its conditional ablation in myeloid cells (HO-1(M-KO) mice). We demonstrate that myeloid HO-1 is required for the activation of interferon (IFN) regulatory factor (IRF) 3 after Toll-like receptor 3 or 4 stimulation, or viral infection. HO-1-deficient macrophages show reduced expression of IFN-beta and of primary IRF3 target genes encoding RANTES, IP-10 and MCP-1. In the presence of polyI:C, myeloid HO-1 knockout mice infected with Listeria monocytogenes, a model dependent on IFN-beta production, showed enhanced bacterial clearance and survival, whereas control mice succumbed to infection. Moreover, after induction of experimental autoimmune encephalomyelitis, mice with myeloid-specific HO-1 deficiency developed a higher incidence and an exacerbated, nonremitting clinical disease correlating with persistent activation of antigen-presenting cells, enhanced infiltration of Th17 cells, and a nonregressing myelin-specific T cell reactivity. Notably, these defects were rectified by exogenous administration of IFN-beta, confirming that HO-1 functions directly upstream of this critical immune pathway. These results uncover a novel direct function for myeloid HO-1 in the regulation of IFN-beta production, establishing HO-1 as a critical early mediator of the innate immune response.
Asunto(s)
Hemo-Oxigenasa 1/inmunología , Interferón beta/inmunología , Células Mieloides/enzimología , Animales , Autoinmunidad/genética , Cruzamientos Genéticos , Hemo-Oxigenasa 1/deficiencia , Hemo-Oxigenasa 1/genética , Homeostasis/inmunología , Inmunidad Innata/genética , Integrasas/genética , Interferón beta/biosíntesis , Interferón beta/genética , Listeria monocytogenes , Listeriosis/inmunología , Listeriosis/mortalidad , Ratones , Ratones Noqueados , Ratones Transgénicos , Sobrevivientes , Receptor Toll-Like 3/inmunología , Receptor Toll-Like 4/inmunologíaRESUMEN
Tumor necrosis factor receptor 1 (TNFR1) and Toll-like receptors (TLRs) regulate immune and inflammatory responses. Here we show that the TNFR1-associated death domain protein (TRADD) is critical in TNFR1, TLR3 and TLR4 signaling. TRADD deficiency abrogated TNF-induced apoptosis, prevented recruitment of the ubiquitin ligase TRAF2 and ubiquitination of the adaptor RIP1 in the TNFR1 signaling complex, and considerably inhibited but did not completely abolish activation of the transcription factor NF-kappaB and mitogen-activated protein kinases 'downstream' of TNFR1. TRIF-dependent cytokine production induced by the synthetic double-stranded RNA poly(I:C) and lipopolysaccharide was lower in TRADD-deficient mice than in wild-type mice. Moreover, TRADD deficiency inhibited poly(I:C)-mediated RIP1 ubiquitination and activation of NF-kappaB and mitogen-activated protein kinase signaling in fibroblasts but not in bone marrow macrophages. Thus, TRADD is an essential component of TNFR1 signaling and has a critical but apparently cell type-specific function in TRIF-dependent TLR responses.
Asunto(s)
Transducción de Señal , Proteína de Dominio de Muerte Asociada a Receptor de TNF/deficiencia , Factor 1 Asociado a Receptor de TNF/metabolismo , Péptidos y Proteínas Asociados a Receptores de Factores de Necrosis Tumoral/fisiología , Animales , Ratones , Proteínas Quinasas Activadas por Mitógenos/metabolismo , FN-kappa B/metabolismo , Transducción de Señal/efectos de los fármacos , Proteína de Dominio de Muerte Asociada a Receptor de TNF/metabolismo , Péptidos y Proteínas Asociados a Receptores de Factores de Necrosis Tumoral/genética , Ubiquitina/metabolismoRESUMEN
Tumour necrosis factor-alpha (TNF-alpha) plays a critical role in the recruitment and activation of mononuclear cells in mycobacterial infection. The role of membrane TNF, in host resistance against Mycobacterium bovis bacille Calmette-Guérin (BCG), was tested in knock-in mice in which the endogenous TNF was replaced by a non-cleavable and regulated allele (Delta1-12, TNF(tm/tm)). While 100% of mice with complete TNF deficiency (TNF(-/-)) succumbed to infection, 50% of TNF(tm/tm) mice were able to control M. bovis BCG infection and survived the experimental period. Membrane expressed TNF allowed a substantial recruitment of activated T cells and macrophages with granuloma formation and expression of bactericidal inducible nitric oxide synthase (iNOS). Using virulent Mycobacterium tuberculosis infection we confirm that membrane TNF conferred partial protection. Infection in TNF(tm/tm) double transgenic mice with TNF-R1 or TNF-R2 suggest protection is mediated through TNF-R2 signalling. Therefore, the data suggest that membrane-expressed TNF plays a critical role in host defence to mycobacterial infection and may partially substitute for soluble TNF.
Asunto(s)
Infecciones por Mycobacterium/inmunología , Mycobacterium bovis , Factor de Necrosis Tumoral alfa/inmunología , Animales , Vacuna BCG/farmacología , Líquido del Lavado Bronquioalveolar/inmunología , Recuento de Células , Citocinas/inmunología , Femenino , Citometría de Flujo , Inmunohistoquímica , Lipopolisacáridos , Activación de Linfocitos , Linfocitos/inmunología , Macrófagos/inmunología , Ratones , Ratones Endogámicos C57BL , Ratones Mutantes , Ratones Transgénicos , Neutrófilos/inmunología , Choque Séptico/inmunología , Factor de Necrosis Tumoral alfa/genéticaRESUMEN
Using targeted mutagenesis in mice, we have blocked shedding of endogenous murine TNF by deleting its cleavage site. Mutant mice produce physiologically regulated levels of transmembrane TNF (tmTNF), which suffice to support thymocyte proliferation but cannot substitute for the hepatotoxic activities of wild-type TNF following LPS/D-galactosamine challenge in vivo and are not sufficient to support secondary lymphoid organ structure and function. Notably, however, tmTNF is capable of exerting anti-Listerial host defenses while remaining inadequate to mediate arthritogenic functions, as tested in the tristetraprolin-deficient model of TNF-dependent arthritis. Most interestingly, in the EAE model of autoimmune demyelination, tmTNF suppresses disease onset and progression and retains the autoimmune suppressive properties of wild-type TNF. Together, these results indicate that tmTNF preserves a subset of the beneficial activities of TNF while lacking detrimental effects. These data support the hypothesis that selective targeting of soluble TNF may offer several advantages over complete blockade of TNF in the treatment of chronic inflammation and autoimmunity.
Asunto(s)
Autoinmunidad , Infecciones Bacterianas/inmunología , Inflamación/inmunología , Factor de Necrosis Tumoral alfa/inmunología , Animales , Artritis Experimental/inmunología , Northern Blotting , Membrana Celular/metabolismo , Encefalomielitis Autoinmune Experimental/inmunología , Ensayo de Inmunoadsorción Enzimática , Citometría de Flujo , Inmunohistoquímica , Ganglios Linfáticos/citología , Ratones , Ratones Transgénicos , Factor de Necrosis Tumoral alfa/genética , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
In experimental autoimmune encephalomyelitis (EAE), an animal model for multiple sclerosis, immunization with myelin Ags leads to demyelination and paralysis. To investigate which molecules are crucial for the pathogenesis of EAE, we specifically assessed the roles of the death receptors Fas and TNF-R1. Mice lacking Fas expression in oligodendrocytes (ODCs) were generated and crossed to TNF-R1-deficient mice. To achieve specific deletion of a loxP-flanked fas allele in ODCs, we generated a new insertion transgene, expressing the Cre recombinase specifically in ODCs. Fas inactivation alone as well as the complete absence of TNF-R1 protected mice partially from EAE induced by the immunization with myelin ODC glycoprotein. The double-deficient mice, however, showed almost no clinical signs of EAE after immunization. Histological analysis revealed that demyelination was suppressed in CNS tissue and that lymphocyte infiltration was notably reduced. We conclude that the death receptors Fas and TNF-R1 are major initiators of ODC apoptosis in EAE. Although only moderate reduction of lymphocyte infiltration into CNS tissue was observed, the absence of these receptors appears to confer protection from demyelination and development of clinical disease.