RESUMEN
Multiple sclerosis (MS) is an autoimmune disease driven by lymphocyte activation against myelin autoantigens in the central nervous system leading to demyelination and neurodegeneration. The deoxyribonucleoside salvage pathway with the rate-limiting enzyme deoxycytidine kinase (dCK) captures extracellular deoxyribonucleosides for use in intracellular deoxyribonucleotide metabolism. Previous studies have shown that deoxyribonucleoside salvage activity is enriched in lymphocytes and required for early lymphocyte development. However, specific roles for the deoxyribonucleoside salvage pathway and dCK in autoimmune diseases such as MS are unknown. Here we demonstrate that dCK activity is necessary for the development of clinical symptoms in the MOG35-55 and MOG1-125 experimental autoimmune encephalomyelitis (EAE) mouse models of MS. During EAE disease, deoxyribonucleoside salvage activity is elevated in the spleen and lymph nodes. Targeting dCK with the small molecule dCK inhibitor TRE-515 limits disease severity when treatments are started at disease induction or when symptoms first appear. EAE mice treated with TRE-515 have significantly fewer infiltrating leukocytes in the spinal cord, and TRE-515 blocks activation-induced B and T cell proliferation and MOG35-55 -specific T cell expansion without affecting innate immune cells or naïve T and B cell populations. Our results demonstrate that targeting dCK limits symptoms in EAE mice and suggest that dCK activity is required for MOG35-55 -specific lymphocyte activation-induced proliferation.
Asunto(s)
Encefalomielitis Autoinmune Experimental , Esclerosis Múltiple , Animales , Ratones , Desoxicitidina Quinasa/genética , Linfocitos/metabolismo , Modelos Animales de Enfermedad , Ratones Endogámicos C57BLRESUMEN
BACKGROUND: Chimeric antigen receptor (CAR)-T cell therapies targeting glioblastoma (GBM)-associated antigens such as interleukin-13 receptor subunit alpha-2 (IL-13Rα2) have achieved limited clinical efficacy to date, in part due to an immunosuppressive tumor microenvironment (TME) characterized by inhibitory molecules such as transforming growth factor-beta (TGF-ß). The aim of this study was to engineer more potent GBM-targeting CAR-T cells by countering TGF-ß-mediated immune suppression in the TME. METHODS: We engineered a single-chain, bispecific CAR targeting IL-13Rα2 and TGF-ß, which programs tumor-specific T cells to convert TGF-ß from an immunosuppressant to an immunostimulant. Bispecific IL-13Rα2/TGF-ß CAR-T cells were evaluated for efficacy and safety against both patient-derived GBM xenografts and syngeneic models of murine glioma. RESULTS: Treatment with IL-13Rα2/TGF-ß CAR-T cells leads to greater T-cell infiltration and reduced suppressive myeloid cell presence in the tumor-bearing brain compared to treatment with conventional IL-13Rα2 CAR-T cells, resulting in improved survival in both patient-derived GBM xenografts and syngeneic models of murine glioma. CONCLUSION: Our findings demonstrate that by reprogramming tumor-specific T-cell responses to TGF-ß, bispecific IL-13Rα2/TGF-ß CAR-T cells resist and remodel the immunosuppressive TME to drive potent anti-tumor responses in GBM.
RESUMEN
25-Hydroxyvitamin D (25D) circulates bound primarily to serum vitamin D binding protein (DBP), with DBP showing higher binding affinity for 25D3 than 25D2. We therefore hypothesized that vitamin D2 (D2) promotes higher serum levels of unbound 25D (free 25D), with different functional responses, relative to vitamin D3 (D3). Week 3 C56BL/6 mice were placed on diets containing either D2 or D3 alone (both 1000 IU/kg). At week 8 and week 16, D2 mice had only 25D2 in circulation (26.6 ± 1.9 and 33.3 ± 4.4 ng/mL), and D3 mice had only 25D3 (28.3 ± 2.0 and 31.7 ± 2.1 ng/mL). At week 8 (44.5 ± 6.4 vs 62.4 ± 11.6 pg/mL, P < .05) and week 16 (78.4 ± 12.6 vs 95.5 ± 11.6), D2 mice had lower serum 1,25-dihydroxyvitamin D relative to D3 mice. By contrast, measured free 25D was significantly higher in D2 mice at week 8 (16.8 ± 0.65 vs 8.4 ± 0.63 pg/mL, P < .001) and week 16 (17.4 ± 0.43 vs 8.4 ± 0.44, P < .001). A two-way ANOVA of bone histomorphometry showed that week 8 D2 mice had significantly higher osteoclast surface/bone surface, eroded surface/bone surface, and mineral apposition rate compared with D3 mice. Osteoblast surface/bone surface was higher in week 8 D2 females but not week 8 D2 males. At week 16, D2 mice had significantly higher bone volume/total volume and trabecular number compared with D3 mice. Differences in bone phenotype were observed despite D2 mice reaching similar serum 25D levels and lower 1,25D levels compared with D3 mice. These data indicate that 25D2 binds less well to DBP than 25D3, with resulting higher levels of free 25D promoting differential effects on bone in mice exposed to D2 alone.
Asunto(s)
Colecalciferol/farmacocinética , Ergocalciferoles/farmacocinética , Vitamina D/análogos & derivados , Animales , Huesos/anatomía & histología , Línea Celular , Femenino , Humanos , Riñón/metabolismo , Masculino , Ratones Endogámicos C57BL , Bazo/metabolismo , Vitamina D/sangreRESUMEN
Osteoporosis is a disease characterized by low bone mass, leading to an increased risk of fragility fractures. GATA4 is a zinc-finger transcription factor that is important in several tissues, such as the heart and intestines, and has recently been shown to be a pioneer factor for estrogen receptor alpha (ERα) in osteoblast-like cells. Herein, we demonstrate that GATA4 is necessary for estrogen-mediated transcription and estrogen-independent mineralization in vitro. In vivo deletion of GATA4, driven by Cre-recombinase in osteoblasts, results in perinatal lethality, decreased trabecular bone properties, and abnormal bone development. Microarray analysis revealed GATA4 suppression of TGFß signaling, necessary for osteoblast progenitor maintenance, and concomitant activation of BMP signaling, necessary for mineralization. Indeed, pSMAD1/5/8 signaling, downstream of BMP signaling, is decreased in the trabecular region of conditional knockout femurs, and pSMAD2/3, downstream of TGFß signaling, is increased in the same region. Together, these experiments demonstrate the necessity of GATA4 in osteoblasts. Understanding the role of GATA4 to regulate the tissue specificity of estrogen-mediated osteoblast gene regulation and estrogen-independent bone differentiation may help to develop therapies for postmenopausal osteoporosis.
Asunto(s)
Proteínas Morfogenéticas Óseas/metabolismo , Receptor alfa de Estrógeno/metabolismo , Factor de Transcripción GATA4/metabolismo , Osteoblastos/metabolismo , Osteogénesis/fisiología , Transducción de Señal/fisiología , Factor de Crecimiento Transformador beta/metabolismo , Animales , Proteínas Morfogenéticas Óseas/genética , Diferenciación Celular/fisiología , Células Cultivadas , Receptor alfa de Estrógeno/genética , Factor de Transcripción GATA4/genética , Regulación de la Expresión Génica/fisiología , Ratones , Ratones Transgénicos , Osteoblastos/citología , Proteínas Smad/genética , Proteínas Smad/metabolismo , Factor de Crecimiento Transformador beta/genéticaRESUMEN
The benefits of estrogens on bone health are well established; how estrogens signal to regulate bone formation and resorption is less well understood. We show here that 17ß-estradiol (E2)-induced apoptosis of bone-resorbing osteoclasts is mediated by cleavage and solubilization of osteoblast-expressed Fas ligand (FasL). U2OS-ERα osteoblast-like cells expressing an EGFP-tagged FasL at the C-terminus showed decreased fluorescence after E2 treatment, indicative of a cleavage event. Treatment of U2OS-ERα cultures with a specific MMP3 inhibitor in the presence of E2 blocked FasL cleavage and showed an increase in the number of EGFP-FasL+ cells. siRNA experiments successfully knocked down MMP3 expression and restored full-length FasL to basal levels. E2 treatment of both human and murine primary osteoblasts showed upregulation of MMP3 mRNA expression, and calvarial organ cultures showed increased expression of MMP3 protein and colocalization with the osteoblast-specific RUNX2 after E2 treatment. In addition, osteoblast cell cultures derived from ERαKO mice showed decreased expression of MMP3 but not MMP7 and ADAM10, two known FasL proteases, demonstrating that ERα signaling regulates MMP3. Also, conditioned media of E2-treated calvarial osteoblasts showed an approximate sixfold increase in the concentration of soluble FasL, indicating extensive cleavage, and soluble FasL concentrations were reduced in the presence of a specific MMP3 inhibitor. Finally, to show the role of soluble FasL in osteoclast apoptosis, human osteoclasts were cocultured with MC3T3 osteoblasts. Both a specific MMP3 inhibitor and an MMP inhibitor cocktail preserved osteoclast differentiation and survival in the presence of E2 and demonstrate the necessity of MMP3 for E2-induced osteoclast apoptosis. These experiments further define the molecular mechanism of estrogen's bone-protective effects by inducing osteoclast apoptosis through upregulation of MMP3 and FasL cleavage.
Asunto(s)
Receptor alfa de Estrógeno/metabolismo , Proteína Ligando Fas/metabolismo , Metaloproteinasa 3 de la Matriz/metabolismo , Osteoclastos/citología , Osteoclastos/enzimología , Transducción de Señal , Animales , Apoptosis/efectos de los fármacos , Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , Células Cultivadas , Estradiol/farmacología , Proteína Ligando Fas/genética , Proteínas Fluorescentes Verdes/metabolismo , Humanos , Inhibidores de la Metaloproteinasa de la Matriz/farmacología , Ratones , Ratones Noqueados , Modelos Biológicos , Osteoblastos/citología , Osteoblastos/efectos de los fármacos , Osteoblastos/enzimología , Osteoclastos/efectos de los fármacos , Unión Proteica/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Cráneo/citología , Solubilidad , Transcripción Genética/efectos de los fármacosRESUMEN
Estrogens regulate osteoblast differentiation and mineralization. We identified GATA4 as a transcription factor expressed in osteoblasts and directly regulated by 17ß-estradiol in this cell type but not in breast cancer cells, another estrogen-responsive tissue. Chromatin immunoprecipitation sequencing (chromatin immunoprecipitation sequencing) reveals that estrogen receptor α (ERα) binds to chromatin near GATA4 at five different enhancers. GATA4 and ERα are both recruited to ERα binding sites near genes that are specifically expressed in osteoblasts and control osteoblast differentiation. Maximal binding of GATA4 precedes ERα binding, and GATA4 is necessary for histone 3 lysine 4 dimethylation at ERα binding sites, suggesting that GATA4 is a pioneer factor for ERα. As such, knockdown of GATA4 reduced recruitment of ERα to DNA. Our study illustrates that GATA4 is a pioneer factor for ERα recruitment to osteoblast-specific enhancers.