RESUMEN
Neuropsychiatric lupus (NPSLE), the nervous system presentation of systemic lupus erythematosus (SLE), remains challenging to treat due to its unclear pathogenesis and lack of available targeted therapies. A potential contributor to disease progression is brain tertiary lymphoid structures (TLS); these ectopic lymphoid follicles that can develop tissue-targeted antibodies have recently been described in the MRL/lpr lupus mouse strain, a classic model for studying NPSLE. The brains of MRL/lpr mice show a significant increase of CXCL13, an important chemokine in lymphoid follicle formation and retention that may also play a role in the disease progression of NPSLE. The aim of the present study was to inhibit CXCL13 and examine the effect of this intervention on lymphoid formation and the development of neurobehavioral manifestations in lupus mice. Female MRL/lpr mice were injected with an anti-CXCL13 antibody, an IgG1 isotype-matched antibody, or PBS either three times a week for 12 weeks intraperitoneally (IP) starting at 6-8 weeks of age, or continuously intracerebroventricularly (ICV) with an osmotic pump over a two-week period starting at 15 weeks of age. Cognitive dysfunction and depression-like behavior were assessed at the end of treatment. When treatment was delivered IP, anti-CXCL13 treated mice showed significant improvement in cognitive function when compared to control treated mice. Depression-like behavior was attenuated as well. Furthermore, mice that received anti-CXCL13 by the ICV route showed similar beneficial effects. However, the extent of lymphocyte infiltration into the brain and the general composition of the aggregates were not substantively changed by anti-CXCL13 irrespective of the mode of administration. Nevertheless, analysis of brain gene expression in anti-CXCL13 treated mice showed significant differences in key immunological and neuro-inflammatory pathways that most likely explained the improvement in the behavioral phenotype. Our results indicate that CXCL13 affects the behavioral manifestations in the MRL/lpr strain and is important to the pathogenesis of murine NPSLE, suggesting it as a potential therapeutic target.
Asunto(s)
Quimiocina CXCL13/antagonistas & inhibidores , Vasculitis por Lupus del Sistema Nervioso Central/tratamiento farmacológico , Animales , Encéfalo/patología , Quimiocina CXCL13/fisiología , Cognición/efectos de los fármacos , Femenino , Inyecciones Intraventriculares , Vasculitis por Lupus del Sistema Nervioso Central/patología , Vasculitis por Lupus del Sistema Nervioso Central/psicología , Ratones , Ratones Endogámicos MRL lpr , Factor de Transcripción STAT3/fisiologíaRESUMEN
Neuropathic pain is an unneglectable pain condition with limited treatment options owing to its enigmatic underlying mechanisms. Long noncoding RNA small nucleolar RNA host gene 5 (SNHG5) is involved in the progression of a spectrum of human cancers. However, its role in neuropathic pain remains undiscovered. In the present study, we established a mouse spinal nerve ligation (SNL) model, and a significant upregulation of SNHG5 was observed. Then we knocked down SNHG5 level in mouse L5 dorsal root ganglion (DRG) by delivering specific short hairpin RNA against SNHG5 with adenovirus vehicle. Mouse paw withdrawal threshold (PWT) and paw withdrawal latency (PWL) in response to mechanical stimuli was increased after SNHG5 knockdown, accompanied with decreased protein levels of glial fibrillary acidic protein (GFAP) and ionized calcium binding adapter molecule 1 (IBA-1). Besides, SNHG5 directly modulated the expression of miR-154-5p, which was downregulated in SNL mice. MiR-154-5p inhibition abolished the effect of SNHG5 knockdown on mouse behavioral tests and GFAP and IBA-1 levels. In addition, we validated that C-X-C motif chemokine 13 (CXCL13) was a novel downstream target of miR-154-5p, and CXCL13 level was positively related to that of SNHG5 in SNL mice. In conclusion, our study demonstrated that SNHG5 knockdown alleviated neuropathic pain and inhibited the activation of astrocytes and microglia by targeting the miR-154-5p/CXCL13 axis, which might be a novel therapeutic target for neuropathic treatment clinically.
Asunto(s)
Quimiocina CXCL13/metabolismo , Técnicas de Silenciamiento del Gen/métodos , MicroARNs/metabolismo , Neuralgia/metabolismo , Neuralgia/terapia , ARN Largo no Codificante/metabolismo , Adenoviridae/genética , Animales , Quimiocina CXCL13/antagonistas & inhibidores , Quimiocina CXCL13/genética , Células HEK293 , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , MicroARNs/antagonistas & inhibidores , MicroARNs/genética , Neuralgia/genética , ARN Largo no Codificante/antagonistas & inhibidores , ARN Largo no Codificante/genéticaRESUMEN
Myasthenia gravis (MG) is an autoimmune neuromuscular disorder, affecting the quality of life of millions of people worldwide. The present study aims to determine the relationship between micro-RNA-143 (miR-143) and C-X-C motif chemokine 13 (CXCL13) and whether it influences the pathogenesis of myasthenia gravis (MG). Thymus specimens were resected from patients with thymic hyperplasia combined with MG and then infused into normal mouse cavities to establish MG mouse models. Immunohistochemistry, reverse transcription-quantitative PCR, in situ hybridization detection, and Western blot analysis were employed to identify the expression of miR-143 and CXCL13 in MG and normal mice. The obtained thymocytes were cultured in vitro and transfected with a series of miR-143 mimic, miR-143 inhibitor, overexpression of CXCL13, or siRNA against CXCL13. MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] and flow cytometry assays were employed to assess cell viability, cycle entry, and apoptosis of the thymocytes. Dual-luciferase reporter assay provided verification, confirming that CXCL13 was the target gene of miR-143. Low miR-143 expression in the thymus tissues of the MG mice was detected, which presented with a reciprocal relationship with the expression rate of CLCX13. Observations in relation to the interactions between miR-143 mimic or siRNA-CXCL13 exposure showed reduced cell viability, with a greater number of cells arrested at the G0/G1 phase and a greater rate of induced apoptosis. Furthermore, overexpression of CXCL13 rescued miR-143 mimic-induced apoptosis. The findings have identified the potential role of miR-143 as a MG development mediator by targeting CXCL13. The key results obtained provide a promising experimental basis for targeted intervention treatment with miR-143.
Asunto(s)
Proliferación Celular/fisiología , Quimiocina CXCL13/biosíntesis , Modelos Animales de Enfermedad , MicroARNs/biosíntesis , Miastenia Gravis/metabolismo , Timocitos/metabolismo , Adolescente , Adulto , Animales , Apoptosis/fisiología , Células Cultivadas , Quimiocina CXCL13/antagonistas & inhibidores , Femenino , Humanos , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Persona de Mediana Edad , Miastenia Gravis/patología , Timocitos/patología , Adulto JovenRESUMEN
OBJECTIVE: Treatment of the high-risk triple negative breast cancer (TNBC) is a critical clinical challenge. Here we aimed to explore a novel strategy for TNBC treatment by blocking the tumor-associated chemokine CXCL13 in the MDA-MB-231 TNBC cells. MATERIALS AND METHODS: MDA-MB-231 cells were treated with anti-CXCL13 antibodies (inhibition group), or phosphate-buffered saline (PBS) (control group), followed by determining the levels of interleukin-1 (IL-1), tumor necrosis factor-alpha (TNF-α) and transforming growth factor beta-1 (TGF-ß1) with enzyme-linked immunosorbent assay (ELISA). The effects of CXCL13 inhibition on cell proliferation and apoptosis were assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay and flow cytometry, respectively. Quantitative Real Time-PCR (qRT-PCR) and Western blot were used to compare the levels of CXCL13, CXCR5, extracellular signal-regulated kinase (ERK). The levels of cyclin D1 and cleaved caspase-9 were detected by Western blot. RESULTS: The levels of IL-1, TNF-α and TGF-ß1 in MDA-MB-231 cells treated with anti-CXCL13 antibodies were significantly downregulated (p<0.05). Meanwhile, CXCL13 blockade decreased the cell proliferation and increased the apoptosis rate of MDA-MB-231 cells. The inhibition of CXCL13 led to marked reduction in CXCL13 and CXCR5 mRNA and an increase in ERK mRNA. The inhibition of CXCL13 resulted in the downregulation of CXCL13, CXCR5, p-ERK/ERK, cyclin D1 and upregulation of cleaved caspase-9 proteins. CONCLUSIONS: CXCL13 blockade effectively suppressed the proliferation of MDA-MB-231 cells by promoting cell apoptosis. This effect is presumably associated with the downregulation of CXCL13 and suppression of the CXCR5/ERK signaling pathway.
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Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Quimiocina CXCL13/antagonistas & inhibidores , Transducción de Señal/efectos de los fármacos , Neoplasias de la Mama Triple Negativas/tratamiento farmacológico , Antineoplásicos/uso terapéutico , Línea Celular Tumoral , Quimiocina CXCL13/metabolismo , Ensayos de Selección de Medicamentos Antitumorales , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Femenino , Humanos , Receptores CXCR5/metabolismo , Neoplasias de la Mama Triple Negativas/patologíaRESUMEN
BACKGROUND The aim of this study was to investigate the role of chemokine (C-X-C motif) ligand 13 (CXCL13) in morphine tolerance in rats with cancer-induced bone pain (CIBP). MATERIAL AND METHODS We established a rat CIBP model and a rat CIBP-morphine tolerance (BM) model. BM rats were intrathecally administered rmCXCL13, neutralizing anti-CXCL13, and normal saline, while the control group rats underwent a sham operation and were injected with normal saline. The morphine analgesia was assessed by measuring mechanical withdrawal threshold (MWT) and mechanical withdrawal duration (MWD) at various time points. The co-expressions of CXCL13 and NeuN were measured by immunofluorescence double-staining. CXCL13 protein and mRNA expressions were detected by Western blot and quantitative real-time polymerase chain reaction (RT-qPCR), respectively. RESULTS Compared to the sham-operation (S) group, the BM group showed obviously decreased MWT and increased MWD on Day 9 after CIBP, but obviously increased MWT and decreased MWD on Day 3 after morphine administration; subsequently, the MWT was decreased and MWD was increased (all P<0.05). In comparison with the S+saline group, increased MWT and decreased MWD were observed in BM rats on Day 3 after anti-CXCL13 administration, and obviously decreased MWT and increased MWD were found in BM rats on Day 3 after rmCXCL13 administration (all P<0.05). CONCLUSIONS Up-regulated CXCL13 has a negative role in morphine analgesia in relief of CIBP, which may provide a new target for the management of CIBP.
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Dolor en Cáncer/tratamiento farmacológico , Quimiocina CXCL13/biosíntesis , Morfina/farmacología , Manejo del Dolor/métodos , Analgesia/métodos , Animales , Anticuerpos Neutralizantes/inmunología , Anticuerpos Neutralizantes/farmacología , Neoplasias Óseas/tratamiento farmacológico , Neoplasias Óseas/patología , Neoplasias Óseas/fisiopatología , Dolor en Cáncer/fisiopatología , Quimiocina CXCL13/antagonistas & inhibidores , Quimiocina CXCL13/inmunología , Modelos Animales de Enfermedad , Tolerancia a Medicamentos , Femenino , Neoplasias Mamarias Experimentales/tratamiento farmacológico , Neoplasias Mamarias Experimentales/patología , Neoplasias Mamarias Experimentales/fisiopatología , ARN Mensajero/biosíntesis , ARN Mensajero/genética , Distribución Aleatoria , Ratas , Ratas Sprague-Dawley , Médula Espinal/metabolismo , Regulación hacia Arriba/efectos de los fármacosRESUMEN
Sjögren's syndrome (SS) is a debilitating autoimmune disease. Patients with SS may develop xerostomia. This process is progressive, and there are no therapeutics that target disease etiology. We hypothesized BAFF receptor (BAFFR) blockade would mitigate SS disease development, and neutralization of CXCL13 and BAFF signaling would be more efficacious than BAFFR blockade alone. We treated NOD/ShiLtJ SS mice with soluble BAFF receptor (BAFFR-Fc) or anti-CXCL13/BAFFR-Fc in combination, prior to the development of clinical disease. Our results show treatment with BAFFR-Fc reduced peripheral B cell numbers and decreased sialadenitis. In addition, this treatment reduced total serum immunoglobulin as well as IgG and IgM specific anti-nuclear autoantibodies. NOD/ShiLtJ mice treated with BAFFR-Fc and anti-CXCL13 antibody were protected from salivary deficits. Results from this study suggest blockade of CXCL13 and BAFFR together may be an effective therapeutic strategy in preventing salivary hypofunction and reducing autoantibody titers and sialadenitis in patients with SS.
Asunto(s)
Quimiocina CXCL13/antagonistas & inhibidores , Sialadenitis/prevención & control , Síndrome de Sjögren/prevención & control , Animales , Anticuerpos/inmunología , Receptor del Factor Activador de Células B/antagonistas & inhibidores , Receptor del Factor Activador de Células B/inmunología , Quimiocina CXCL13/inmunología , Modelos Animales de Enfermedad , Femenino , Ratones , Ratones Endogámicos NOD , Saliva/metabolismo , Glándulas Salivales/patología , Glándulas Salivales/fisiología , Sialadenitis/inmunología , Sialadenitis/patología , Síndrome de Sjögren/inmunología , Síndrome de Sjögren/patologíaRESUMEN
Fully-human single-chain Fv (scFv) proteins are key potential building blocks of bispecific therapeutic antibodies, but they often suffer from manufacturability and clinical development limitations such as instability and aggregation. The causes of these scFv instability problems, in proteins that should be theoretically stable, remains poorly understood. To inform the future development of such molecules, we carried out a comprehensive structural analysis of the highly stabilized anti-CXCL13 scFv E10. E10 was derived from the parental 3B4 using complementarity-determining region (CDR)-restricted mutagenesis and tailored selection and screening strategies, and carries four mutations in VL-CDR3. High-resolution crystal structures of parental 3B4 and optimized E10 scFvs were solved in the presence and absence of human CXCL13. In parallel, a series of scFv mutants was generated to interrogate the individual contribution of each of the four mutations to stability and affinity improvements. In combination, these analyses demonstrated that the optimization of E10 was primarily mediated by removing clashes between both the VL and the VH, and between the VL and CXCL13. Importantly, a single, germline-encoded VL-CDR3 residue mediated the key difference between the stable and unstable forms of the scFv. This work demonstrates that, aside from being the critical mediators of specificity and affinity, CDRs may also be the primary drivers of biotherapeutic developability.
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Productos Biológicos/química , Quimiocina CXCL13/antagonistas & inhibidores , Modelos Moleculares , Anticuerpos de Cadena Única/química , Sustitución de Aminoácidos , Afinidad de Anticuerpos , Especificidad de Anticuerpos , Complejo Antígeno-Anticuerpo/química , Complejo Antígeno-Anticuerpo/metabolismo , Sitios de Unión de Anticuerpos , Productos Biológicos/metabolismo , Quimiocina CXCL13/química , Quimiocina CXCL13/metabolismo , Regiones Determinantes de Complementariedad/química , Regiones Determinantes de Complementariedad/genética , Regiones Determinantes de Complementariedad/metabolismo , Humanos , Cinética , Mutación , Agregado de Proteínas , Conformación Proteica , Estabilidad Proteica , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Anticuerpos de Cadena Única/genética , Anticuerpos de Cadena Única/metabolismo , Solubilidad , Difracción de Rayos XRESUMEN
The chemokine CXC ligand 13 protein (CXCL13) is reported to closely related to the disease activity and severity of systemic lupus erythematosus (SLE), moreover, the level of CXCL13 was markedly raised in kidney tissues of lupus nephritis (LN) patients. The aim of the present study was to explore whether the blockade of CXCL13 has therapeutic effects on murine LN. MRL/lpr mice received 50µg anti-CXCL13 neutralizing antibody or isotype IgG by intraperitoneal injection everyday for six weeks, and renal damage of each group was determined. Our results showed that the blockade of CXCL13 significantly reduced urine protein, serum creatinine, and dramatically attenuated renal pathology injury. Treatment with anti-CXCL13Ab also reduced serum anti-dsDNA level, renal immune complex deposition as well as inflammatory cytokines secretion. Meanwhile, Th17/Treg ratio in spleens of MRL/lpr mice was significantly decreased by the blocking of CXCL13. These findings suggested that CXCL13 may be a promising target for the therapy of LN.
Asunto(s)
Quimiocina CXCL13/antagonistas & inhibidores , Nefritis Lúpica/tratamiento farmacológico , Animales , Autoanticuerpos/sangre , Recuento de Linfocito CD4 , Quimiocina CXCL13/fisiología , Evaluación Preclínica de Medicamentos , Femenino , Riñón/efectos de los fármacos , Riñón/metabolismo , Nefritis Lúpica/sangre , Ratones Endogámicos MRL lpr , Linfocitos T Reguladores/inmunología , Células Th17/inmunologíaRESUMEN
We investigated the expression of -CXC chemokine ligand 13 (CXCL13) and its receptor -CXC chemokine receptor 5 (CXCR5) in 98 breast cancer (BC) patients with infiltrating duct carcinoma, out of which 56 were found lymph node metastasis (LNM) positive. Interestingly, co-expression of CXCL13 and CXCR5 showed a significant correlation with LNM. Since, epithelial to mesenchymal transition (EMT) is highly associated with metastasis we investigated EMT-inducing potential of CXCL13 in BC cell lines. In CXCL13-stimulated BC cells, expression of various mesenchymal markers (Vimentin, N-cadherin), EMT regulators (Snail, Slug), and matrix metalloproteinase-9 (MMP9) was increased, whereas the expression of epithelial marker E-cadherin was found to be decreased. In addition, expression of receptor activator of nuclear factor kappa-B ligand (RANKL), which is known to regulate MMP9 expression via Src activation, was also significantly increased after CXCL13 stimulation. Using specific protein kinase inhibitors, we confirmed that CXCL13 stimulated EMT and MMP9 expression via RANKL-Src axis in BC cell lines. To further validate this observation, we examined gene expression patterns in primary breast tumors and detected significantly higher expression of various mesenchymal markers and regulators in CXCL13-CXCR5 co-expressing patients. Therefore, this study showed the EMT-inducing potential of CXCL13 as well as demonstrated the prognostic value of CXCL13-CXCR5 co-expression in primary BC. Moreover, CXCL13-CXCR5-RANKL-Src axis may present a therapeutic target in LNM positive BC patients.
Asunto(s)
Neoplasias de la Mama/patología , Quimiocina CXCL13/metabolismo , Transición Epitelial-Mesenquimal , Metástasis Linfática/patología , Receptores CXCR5/metabolismo , Adulto , Anciano , Antígenos CD/biosíntesis , Biomarcadores de Tumor/metabolismo , Cadherinas/biosíntesis , Línea Celular Tumoral , Movimiento Celular , Quimiocina CXCL13/antagonistas & inhibidores , Quimiocina CXCL13/biosíntesis , Femenino , Furanos/farmacología , Humanos , Indoles/farmacología , Metaloproteinasa 9 de la Matriz/biosíntesis , Persona de Mediana Edad , Inhibidores de las Quinasa Fosfoinosítidos-3 , Piridinas/farmacología , Pirimidinas/farmacología , Ligando RANK/biosíntesis , Ligando RANK/genética , ARN Mensajero/biosíntesis , Receptores CXCR5/antagonistas & inhibidores , Receptores CXCR5/biosíntesis , Transducción de Señal , Factores de Transcripción de la Familia Snail , Sulfonamidas/farmacología , Factores de Transcripción/biosíntesis , Vimentina/biosíntesis , Familia-src Quinasas/antagonistas & inhibidoresRESUMEN
The chemokine CXCL13 has a key role in secondary lymphoid tissue orchestration and lymphoid neogenesis. Transgenic mice deficient in CXCL13 or its receptor CXCR5 have severely impaired lymph node development, lack peritoneal B-lymphocytes and are deficient in circulating antibodies to common bacterial antigens. However, total circulating numbers of B-lymphocytes are slightly elevated and humoral responses to T-dependent or blood-borne antigens are relatively normal. Lymphoid neogenesis is an aberrant process that occurs in chronically inflamed tissue and provides a microenvironment supportive of pathogenic B-cell survival and activation. Here, we describe the impact of therapeutic dosing of a CXCL13 antibody in a mouse model of arthritis, and detail the contribution CXCL13 makes to lymphoid follicle microenvironment, without affecting humoral immune responses.
Asunto(s)
Enfermedades Autoinmunes/inmunología , Quimiocina CXCL13/antagonistas & inhibidores , Modelos Animales de Enfermedad , Tejido Linfoide/inmunología , Animales , Quimiocina CXCL13/inmunología , Ratones , Ratones Transgénicos , Receptores CXCR5/inmunologíaRESUMEN
Lymphocytes that invade nonlymphoid tissues often organize into follicle-like structures known as tertiary lymphoid organs (TLOs). These structures resemble those found in spleen or lymph nodes, but their function is unknown. TLOs are recognized in many autoimmune diseases, including the NOD mouse model of type 1 diabetes. In some cases, TLOs have been associated with the B lymphocyte chemoattractant, CXCL13. Studies presented in this article show that CXCL13 is present in inflamed islets of NOD mice. Ab blockade of this chemokine unraveled B lymphocyte organization in islet TLOs, without reducing their proportion in the islets. These chaotic milieus contained B lymphocytes with the same distinct repertoire of B cell receptors as those found in mice with well-organized structures. Somatic hypermutation, associated with T-B interactions, was not impaired in these disorganized insulitis lesions. Finally, loss of B lymphocyte organization in islets did not provide disease protection. Thus, B lymphocytes infiltrating islets in NOD mice do not require the morphology of secondary lymphoid tissues to support their role in disease.