RESUMEN
Chronic graft-versus-host disease (cGVHD) is an immune-mediated disorder characterized by chronic inflammation and fibrosis. Rho-associated coiled-coil-containing protein kinases (ROCKs) are key coordinators of tissue response to injury, regulating multiple functions, such as gene expression and cell migration, proliferation and survival. Relevant to cGVHD and autoimmunity, only the ROCK2 isoform drives a pro-inflammatory type 17 helper T (Th17) cell response. Moreover, ROCK2 inhibition shifts the Th17/regulatory T (Treg) cell balance toward Treg cells and restores immune homeostasis in animal models of autoimmunity and cGVHD. Furthermore, the selective inhibition of ROCK2 by belumosudil reduces fibrosis by downregulating both transforming growth factor-ß signaling and profibrotic gene expression, thereby impeding the creation of focal adhesions. Consistent with its anti-inflammatory and antifibrotic activities, belumosudil has demonstrated efficacy in clinical studies, resulting in an overall response rate of >70% in patients with cGVHD who failed 2 to 5 prior lines of systemic therapy. In summary, selective ROCK2 inhibition has emerged as a promising novel therapeutic approach for treating cGVHD and other immunologic diseases with unique mechanisms of action, targeting both immune imbalance and detrimental fibrotic responses.
Asunto(s)
Enfermedad Injerto contra Huésped/enzimología , Quinasas Asociadas a rho/inmunología , Acetamidas/farmacología , Animales , Enfermedad Crónica , Modelos Animales de Enfermedad , Fibrosis , Enfermedad Injerto contra Huésped/inmunología , Enfermedad Injerto contra Huésped/terapia , Humanos , Inmunomodulación , Modelos Inmunológicos , Inhibidores de Proteínas Quinasas/farmacología , Linfocitos T Reguladores/inmunología , Quinasas Asociadas a rho/antagonistas & inhibidoresRESUMEN
Duchenne Muscular Dystrophy (DMD) patients often suffer from both muscle wasting and osteoporosis. Our previous studies have revealed reduced regeneration potential in skeletal muscle and bone, concomitant with ectopic calcification of soft tissues in double knockout (dKO, dystrophin-/-; utrophin-/-) mice, a severe murine model for DMD. We found significant involvement of RhoA/ROCK (Rho-Associated Protein Kinase) signaling in mediating ectopic calcification of muscles in dKO mice. However, the cellular identity of these RhoA+ cells, and the role that RhoA plays in the chronic inflammation-associated pathologies has not been elucidated. Here, we report that CD68+ macrophages are highly prevalent at the sites of ectopic calcification of dKO mice, and that these macrophages highly express RhoA. Macrophages from dKO mice feature a shift towards a more pro-inflammatory M1 polarization and an increased expression of various senescence-associated secretory phenotype (SASP) factors that was reduced with the RhoA/ROCK inhibitor Y-27632. Further, systemic inhibition of RhoA activity in dKO mice led to reduced number of RhoA+/CD68+ cells, as well as a reduction in fibrosis and ectopic calcification. Together, these data revealed that RhoA signaling may be a key regulator of imbalanced mineralization in the dystrophic musculoskeletal system and consequently a therapeutic target for the treatment of DMD or other related muscle dystrophies.
Asunto(s)
Calcinosis/metabolismo , Macrófagos/metabolismo , Músculo Esquelético/metabolismo , Distrofia Muscular Animal/metabolismo , Miocardio/metabolismo , Quinasas Asociadas a rho/metabolismo , Proteína de Unión al GTP rhoA/metabolismo , Animales , Antígenos CD/metabolismo , Antígenos de Diferenciación Mielomonocítica/metabolismo , Calcinosis/inmunología , Calcinosis/patología , Senescencia Celular/genética , Senescencia Celular/inmunología , Modelos Animales de Enfermedad , Distrofina/genética , Macrófagos/inmunología , Ratones , Ratones Noqueados , Músculo Esquelético/inmunología , Músculo Esquelético/patología , Distrofia Muscular Animal/genética , Distrofia Muscular Animal/inmunología , Distrofia Muscular Animal/patología , Distrofia Muscular de Duchenne/genética , Distrofia Muscular de Duchenne/inmunología , Distrofia Muscular de Duchenne/metabolismo , Distrofia Muscular de Duchenne/patología , Miocardio/inmunología , Miocardio/patología , Utrofina/genética , Quinasas Asociadas a rho/inmunología , Proteína de Unión al GTP rhoA/inmunologíaRESUMEN
OBJECTIVE: To investigate the regulatory mechanism of manual acupuncture (MA) on microglial polarization-mediated neuroinflammation after traumatic brain injury (TBI), focusing on the RhoA/Rho-associated coiled coil-forming protein kinase (ROCK2) pathway. METHODS: Sprague Dawley (SD) rats were used to generate a TBI model using Feeney's freefall epidural impact method. MA was performed on half of the TBI model rats, while the others remained untreated. Acupuncture was administered at GV15, GV16, GV20, GV26, and LI4. At the end of the intervention, rat brain tissue samples were collected, and the microglial M1 polarization status was observed by immunofluorescence labeling of CD86, an M1 microglia-specific protein. RhoA/ROCK2 signaling components were detected by quantitative real-time polymerase chain reaction (qRT-PCR) and Western blotting. An enzyme-linked immunosorbent assay (ELISA) was used to detect the expression levels of inflammatory factors. RESULTS: Compared with normal rats, the CD86 expression density in the untreated TBI model rats was high and showed an aggregated expression pattern. The genes and proteins of the RhoA/ROCK2 signaling pathway were highly expressed, and inflammatory factors were significantly increased. The CD86 expression density in TBI rats after MA was reduced compared to that in untreated TBI rats and showed a scattered distribution. The expression of RhoA/ROCK2 signaling pathway genes and proteins was also significantly reduced, and inflammatory factors were decreased. CONCLUSION: These results show that MA may inhibit M1 polarization of microglia by regulating the RhoA/ROCK2 signaling pathway, thereby reducing neuroinflammation in TBI.
Asunto(s)
Terapia por Acupuntura , Lesiones Traumáticas del Encéfalo/inmunología , Lesiones Traumáticas del Encéfalo/terapia , Microglía/inmunología , Proteínas de Unión al GTP rho/inmunología , Quinasas Asociadas a rho/inmunología , Animales , Lesiones Traumáticas del Encéfalo/enzimología , Lesiones Traumáticas del Encéfalo/genética , Modelos Animales de Enfermedad , Humanos , Masculino , Ratas , Ratas Sprague-Dawley , Transducción de Señal , Proteínas de Unión al GTP rho/genética , Quinasas Asociadas a rho/genéticaRESUMEN
Oxymatrine (OMT) is an important quinoxaline alkaloid that has a wide range of pharmacological effects and has been shown to alleviate ulcerative colitis due to its profound anti-inflammatory effects. The RhoA/ROCK (Rho kinase) signaling pathway has been shown to be related to the pathogenesis of several autoimmune diseases; however, the specific mechanisms of RhoA/ROCK signaling in inflammatory bowel disease (IBD) remain elusive. Therefore, we sought to determine whether OMT could ameliorate acute intestinal inflammation by targeting the RhoA/ROCK signaling pathway. The potential therapeutic effect of OMT on acute intestinal inflammation and its impact on the RhoA/ROCK signaling pathway were assessed in six groups of mice treated with low, medium and high doses of OMT (25, 50 and 100 mg/kg, respectively), and an inhibitor of ROCK, Y-27632, as a positive control, after initiating dextran sodium sulfate (DSS)-induced acute intestinal inflammation. The model group and normal group were injected intraperitoneally with equal doses of PBS. Our results showed that OMT treatment could protect the integrity of the epithelial barrier, relieve oxidative stress, inhibit the expression of inflammatory mediators and pro-inflammatory cytokines, restrain the differentiation of Th17 cells and promote the differentiation of Treg cells via inhibition of the RhoA/ROCK pathway, thus providing therapeutic benefits for ulcerative colitis (UC). Therefore, inhibiting the RhoA/ROCK pathway might be a new approach that can be used in UC therapy, which deserves to be investigated further.
Asunto(s)
Alcaloides/farmacología , Antiinflamatorios/farmacología , Colitis Ulcerosa/tratamiento farmacológico , Quinolizinas/farmacología , Quinasas Asociadas a rho/genética , Proteína de Unión al GTP rhoA/genética , Amidas/farmacología , Animales , Diferenciación Celular , Proliferación Celular , Colitis Ulcerosa/inducido químicamente , Colitis Ulcerosa/genética , Colitis Ulcerosa/patología , Sulfato de Dextran , Modelos Animales de Enfermedad , Factores de Transcripción Forkhead/genética , Factores de Transcripción Forkhead/inmunología , Regulación de la Expresión Génica , Humanos , Interleucinas/genética , Interleucinas/inmunología , Masculino , Ratones , Ratones Endogámicos BALB C , Estrés Oxidativo , Piridinas/farmacología , Transducción de Señal , Linfocitos T Reguladores/efectos de los fármacos , Linfocitos T Reguladores/inmunología , Linfocitos T Reguladores/patología , Células Th17/efectos de los fármacos , Células Th17/inmunología , Células Th17/patología , Resultado del Tratamiento , Quinasas Asociadas a rho/antagonistas & inhibidores , Quinasas Asociadas a rho/inmunología , Proteína de Unión al GTP rhoA/antagonistas & inhibidores , Proteína de Unión al GTP rhoA/inmunologíaRESUMEN
Context: Atherosclerosis is a chronic inflammatory disease in which the plaques were built up inside of the artery. Interleukin-8 (IL-8, CXCL8) is an inflammatory factor, known to play an important role in the development of atherosclerosis. G31P is an antagonist of the IL-8 receptor, which plays roles in vascular smooth muscle cell (VSMC) proliferation and migration. Objective: This study is to investigate the therapeutic effect of G31P on atherosclerosis through a mouse model. Materials and methods: A mouse model of atherosclerosis was generated through feeding the ApoE-/- mice with high fat diet for 12 weeks. G31P was injected subcutaneously into the mice. The levels of keratinocyte chemoattractant (KC), CXCR2, TNF-α, and IFN-γ were analyzed through ELISA. The expressions of MMP-2, MMP-9, PCNA, and Mef2a in aortic tissues were detected through RT-qPCR. In A7r5 cells, the levels of p-ERK, ROCK1, and ROCK2 were analyzed by western blot. Intracellular calcium levels were measured through Fluo-3 AM assay. Results and disccussion: G31P suppressed the abnormal lipid profile and decreased the levels of KC, MMP-2, MMP-9, PCNA, and Mef2a in a mouse model of atherosclerosis. In addition, G31P also inhibited the expressions of p-ERK, ROCK1, ROCK2, and decreased the calcium concentrations in A7r5 cells. Conclusions: These findings indicate the potential therapeutic effects of G31P in suppressing the development of atherosclerosis by antagonizing the IL-8 receptor. G31P inhibits the proliferation and migration of VSMCs through regulating the Rho-kinase, ERK, and calcium-dependent pathways.
Asunto(s)
Aorta/inmunología , Aterosclerosis/tratamiento farmacológico , Interleucina-8/farmacología , Músculo Liso Vascular/inmunología , Miocitos del Músculo Liso/inmunología , Fragmentos de Péptidos/farmacología , Placa Aterosclerótica/tratamiento farmacológico , Animales , Aorta/patología , Aterosclerosis/genética , Aterosclerosis/inmunología , Aterosclerosis/patología , Citocinas/genética , Citocinas/inmunología , Modelos Animales de Enfermedad , Humanos , Metaloproteinasa 2 de la Matriz/genética , Metaloproteinasa 2 de la Matriz/inmunología , Metaloproteinasa 9 de la Matriz/genética , Metaloproteinasa 9 de la Matriz/inmunología , Ratones , Ratones Noqueados para ApoE , Músculo Liso Vascular/patología , Miocitos del Músculo Liso/patología , Placa Aterosclerótica/genética , Placa Aterosclerótica/inmunología , Placa Aterosclerótica/patología , Antígeno Nuclear de Célula en Proliferación/genética , Antígeno Nuclear de Célula en Proliferación/inmunología , Quinasas Asociadas a rho/genética , Quinasas Asociadas a rho/inmunologíaRESUMEN
Microvesicles (MVs) have been extensively identified in various biological fluids including bronchoalveolar lavage fluid (BALF), peripheral blood and ascitic fluids. Our previous study showed that MVs are responsible for acute lung injury, but the exact mechanism underlying MVs formation remains poorly understood. In the present study, we investigate the potential role of RhoA/Rock signaling in MVs generation and the biological activity of MVs in ventilator-induced lung injury (VILI). Our results revealed that high tide ventilation induced super MVs releasing into the lung and subsequently caused lung inflammation. Strikingly, intratracheal instillation of MVs that isolated from highly ventilated mice triggered significant lung inflammation in naive mice. The MVs production is strongly correlated with lung inflammation and the upregulation of RhoA, Rock and phospho-Limk (phosphorylation of Limk is the activated form). RhoA inhibitor decreased the expression of Rock and the phosphorylation of Limk, decreased MVs production and alleviated lung inflammation. Rock inhibitor also decreased the phosphorylation of Limk, decreased MVs production and alleviated lung inflammation. Our data demonstrated that the production of MVs requires RhoA/Rock signaling, and VILI might be potentially prevented by targeting RhoA/Rock signaling pathway.
Asunto(s)
ADP Ribosa Transferasas/uso terapéutico , Toxinas Botulínicas/uso terapéutico , Micropartículas Derivadas de Células/efectos de los fármacos , Lesión Pulmonar Inducida por Ventilación Mecánica/tratamiento farmacológico , Proteína de Unión al GTP rhoA/antagonistas & inhibidores , Amidas/uso terapéutico , Animales , Líquido del Lavado Bronquioalveolar/inmunología , Citocinas/inmunología , Pulmón/efectos de los fármacos , Pulmón/inmunología , Pulmón/patología , Ratones Endogámicos C57BL , Piridinas/uso terapéutico , Lesión Pulmonar Inducida por Ventilación Mecánica/inmunología , Lesión Pulmonar Inducida por Ventilación Mecánica/patología , Quinasas Asociadas a rho/antagonistas & inhibidores , Quinasas Asociadas a rho/inmunología , Proteína de Unión al GTP rhoA/inmunologíaRESUMEN
ARHGEF1 is a RhoA-specific guanine nucleotide exchange factor expressed in hematopoietic cells. We used whole-exome sequencing to identify compound heterozygous mutations in ARHGEF1, resulting in the loss of ARHGEF1 protein expression in 2 primary antibody-deficient siblings presenting with recurrent severe respiratory tract infections and bronchiectasis. Both ARHGEF1-deficient patients showed an abnormal B cell immunophenotype, with a deficiency in marginal zone and memory B cells and an increased frequency of transitional B cells. Furthermore, the patients' blood contained immature myeloid cells. Analysis of a mediastinal lymph node from one patient highlighted the small size of the germinal centers and an abnormally high plasma cell content. On the molecular level, T and B lymphocytes from both patients displayed low RhoA activity and low steady-state actin polymerization (even after stimulation of lysophospholipid receptors). As a consequence of disturbed regulation of the RhoA downstream target Rho-associated kinase I/II (ROCK), the patients' lymphocytes failed to efficiently restrain AKT phosphorylation. Enforced ARHGEF1 expression or drug-induced activation of RhoA in the patients' cells corrected the impaired actin polymerization and AKT regulation. Our results indicate that ARHGEF1 activity in human lymphocytes is involved in controlling actin cytoskeleton dynamics, restraining PI3K/AKT signaling, and confining B lymphocytes and myelocytes within their dedicated functional environment.
Asunto(s)
Linfocitos B , Enfermedades de Inmunodeficiencia Primaria , Transducción de Señal , Linfocitos B/inmunología , Linfocitos B/patología , Femenino , Centro Germinal/inmunología , Centro Germinal/patología , Humanos , Memoria Inmunológica/genética , Masculino , Fosfatidilinositol 3-Quinasas/genética , Fosfatidilinositol 3-Quinasas/inmunología , Enfermedades de Inmunodeficiencia Primaria/genética , Enfermedades de Inmunodeficiencia Primaria/inmunología , Enfermedades de Inmunodeficiencia Primaria/patología , Proteínas Proto-Oncogénicas c-akt/genética , Proteínas Proto-Oncogénicas c-akt/inmunología , Factores de Intercambio de Guanina Nucleótido Rho/deficiencia , Factores de Intercambio de Guanina Nucleótido Rho/inmunología , Hermanos , Transducción de Señal/genética , Transducción de Señal/inmunología , Linfocitos T/inmunología , Linfocitos T/patología , Quinasas Asociadas a rho/genética , Quinasas Asociadas a rho/inmunología , Proteína de Unión al GTP rhoA/genéticaRESUMEN
BACKGROUND: Numbers of mesenchymal stem cells (MSCs) are increased in the airways after allergen challenge. Ras homolog family member A (RhoA)/Rho-associated protein kinase 1 (ROCK) signaling is critical in determining the lineage fate of MSCs in tissue repair/remodeling. OBJECTIVES: We sought to investigate the role of RhoA/ROCK signaling in lineage commitment of MSCs during allergen-induced airway remodeling and delineate the underlying mechanisms. METHODS: Active RhoA expression in lung tissues of asthmatic patients and its role in cockroach allergen-induced airway inflammation and remodeling were investigated. RhoA/ROCK signaling-mediated MSC lineage commitment was assessed in an asthma mouse model by using MSC lineage tracing mice (nestin-Cre; ROSA26-EYFP). The role of RhoA/ROCK in MSC lineage commitment was also examined by using MSCs expressing constitutively active RhoA (RhoA-L63) or dominant negative RhoA (RhoA-N19). Downstream RhoA-regulated genes were identified by using the Stem Cell Signaling Array. RESULTS: Lung tissues from asthmatic mice showed increased expression of active RhoA when compared with those from control mice. Inhibition of RhoA/ROCK signaling with fasudil, a RhoA/ROCK inhibitor, reversed established cockroach allergen-induced airway inflammation and remodeling, as assessed based on greater collagen deposition/fibrosis. Furthermore, fasudil inhibited MSC differentiation into fibroblasts/myofibroblasts but promoted MSC differentiation into epithelial cells in asthmatic nestin-Cre; ROSA26-EYFP mice. Consistently, expression of RhoA-L63 facilitated differentiation of MSCs into fibroblasts/myofibroblasts, whereas expression of RhoA-19 switched the differentiation toward epithelial cells. The gene array identified the Wnt signaling effector lymphoid enhancer-binding factor 1 (Lef1) as the most upregulated gene in RhoA-L63-transfected MSCs. Knockdown of Lef1 induced MSC differentiation away from fibroblasts/myofibroblasts but toward epithelial cells. CONCLUSIONS: These findings uncover a previously unrecognized role of RhoA/ROCK signaling in MSC-involved airway repair/remodeling in the setting of asthma.
Asunto(s)
Remodelación de las Vías Aéreas (Respiratorias)/inmunología , Asma/metabolismo , Factor de Unión 1 al Potenciador Linfoide/metabolismo , Células Madre Mesenquimatosas/metabolismo , Quinasas Asociadas a rho/metabolismo , Proteína de Unión al GTP rhoA/metabolismo , Animales , Asma/inmunología , Asma/patología , Linaje de la Célula/inmunología , Factor de Unión 1 al Potenciador Linfoide/inmunología , Células Madre Mesenquimatosas/inmunología , Células Madre Mesenquimatosas/patología , Ratones , Ratones Endogámicos C57BL , Transducción de Señal/inmunología , Quinasas Asociadas a rho/inmunología , Proteína de Unión al GTP rhoA/inmunologíaRESUMEN
BACKGROUND: Eosinophil-associated RNases (EARs) are stored preformed in eosinophil cytoplasmic secretory granules and have a key role in eosinophil effector functions in host defence and inflammatory disorders. However, the secretion mechanisms of EARs are poorly understood. OBJECTIVE: Our study aimed to understand the involvement of cytoskeleton machinery in EAR secretion. METHODS: Fresh human and mouse eosinophils were stimulated with CCL11, and the secretion of enzymatically active EARs was detected using an RNase activity assay. The involvement of cytoskeletal elements or microtubules was probed using specific inhibitors. RESULTS: We found that dynamic polymerization of microtubules and cytoskeletal elements, such as Rho and Rac, is required for chemokine-mediated EAR secretion from human and mouse eosinophils. However, inhibition of ROCK (Rho-associated protein kinase) increased EAR secretion in human and mouse eosinophils even in the absence of chemokine stimulation, suggesting ROCK negatively regulates EAR secretion. CONCLUSIONS: Collectively, these data suggest a cytoskeleton-dependent mechanism of EAR secretion from eosinophils, findings that are pertinent to host defence, allergy and other eosinophil-associated diseases.
Asunto(s)
Proteína Catiónica del Eosinófilo/inmunología , Eosinófilos/inmunología , Proteínas de Unión al GTP rac/inmunología , Quinasas Asociadas a rho/inmunología , Animales , Quimiocina CCL11/genética , Quimiocina CCL11/inmunología , Proteína Catiónica del Eosinófilo/genética , Humanos , Ratones , Ratones Endogámicos BALB C , Ratones Transgénicos , Proteínas de Unión al GTP rac/genética , Quinasas Asociadas a rho/genéticaRESUMEN
Activation of T cell immune response is critical for the therapeutic efficacy of cancer immunotherapy. Current immunotherapies have shown remarkable clinical success against several cancers; however, significant responses remain restricted to a minority of patients. Here, we show a therapeutic strategy that combines enhancing the phagocytic activity of antigen-presenting cells with immunogenic cell death to trigger efficient antitumour immunity. Rho-kinase (ROCK) blockade increases cancer cell phagocytosis and induces antitumour immunity through enhancement of T cell priming by dendritic cells (DCs), leading to suppression of tumour growth in syngeneic tumour models. Combining ROCK blockade with immunogenic chemotherapy leads to increased DC maturation and synergistic CD8+ cytotoxic T cell priming and infiltration into tumours. This therapeutic strategy effectively suppresses tumour growth and improves overall survival in a genetic mouse mammary tumour virus/Neu tumour model. Collectively, these results suggest that boosting intrinsic cancer immunity using immunogenic killing and enhanced phagocytosis is a promising therapeutic strategy for cancer immunotherapy.
Asunto(s)
Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Inmunidad/efectos de los fármacos , Neoplasias Experimentales/tratamiento farmacológico , Inhibidores de Proteínas Quinasas/farmacología , Quinasas Asociadas a rho/antagonistas & inhibidores , Amidas/administración & dosificación , Amidas/farmacología , Animales , Linfocitos T CD8-positivos/efectos de los fármacos , Linfocitos T CD8-positivos/inmunología , Muerte Celular/efectos de los fármacos , Muerte Celular/inmunología , Línea Celular Tumoral , Células Cultivadas , Cisplatino/administración & dosificación , Células Dendríticas/efectos de los fármacos , Células Dendríticas/inmunología , Doxorrubicina/administración & dosificación , Humanos , Inmunidad/inmunología , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Transgénicos , Neoplasias Experimentales/inmunología , Neoplasias Experimentales/patología , Inhibidores de Proteínas Quinasas/administración & dosificación , Piridinas/administración & dosificación , Piridinas/farmacología , Linfocitos T Citotóxicos/efectos de los fármacos , Linfocitos T Citotóxicos/inmunología , Quinasas Asociadas a rho/inmunología , Quinasas Asociadas a rho/metabolismoRESUMEN
Dendritic cells (DC) are professional Antigen-Presenting Cells scattered throughout antigen-exposed tissues and draining lymph nodes, and survey the body for pathogens. Their ability to migrate through tissues, a 3D environment, is essential for an effective immune response. Upon infection, recognition of Pathogen-Associated Molecular Patterns (PAMP) by Toll-like receptors (TLR) triggers DC maturation. Mature DC (mDC) essentially use the protease-independent, ROCK-dependent amoeboid mode in vivo, or in collagen matrices in vitro. However, the mechanisms of 3D migration used by human immature DC (iDC) are still poorly characterized. Here, we reveal that human monocyte-derived DC are able to use two migration modes in 3D. In porous matrices of fibrillar collagen I, iDC adopted the amoeboid migration mode. In dense matrices of gelled collagen I or Matrigel, iDC used the protease-dependent, ROCK-independent mesenchymal migration mode. Upon TLR4 activation by LPS, mDC-LPS lose the capacity to form podosomes and degrade the matrix along with impaired mesenchymal migration. TLR2 activation by Pam3CSK4 resulted in DC maturation, podosome maintenance, and efficient mesenchymal migration. Under all these conditions, when DC used the mesenchymal mode in dense matrices, they formed 3D podosomes at the tip of cell protrusions. Using PGE2, known to disrupt podosomes in DC, we observed that the cells remained in an immature status and the mesenchymal migration mode was abolished. We also observed that, while CCL5 (attractant of iDC) enhanced both amoeboid and mesenchymal migration of iDC, CCL19 and CCL21 (attractants of mDC) only enhanced mDC-LPS amoeboid migration without triggering mesenchymal migration. Finally, we examined the migration of iDC in tumor cell spheroids, a tissue-like 3D environment. We observed that iDC infiltrated spheroids of tumor cells using both migration modes. Altogether, these results demonstrate that human DC adopt the mesenchymal mode to migrate in 3D dense environments, which relies on their capacity to form podosomes independent of their maturation status, paving the way of further investigations on in vivo DC migration in dense tissues and its regulation during infections.
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Movimiento Celular , Células Dendríticas/citología , Endopeptidasas/metabolismo , Podosomas/inmunología , Diferenciación Celular , Células Cultivadas , Quimiocinas/inmunología , Dendritas/inmunología , Células Dendríticas/enzimología , Humanos , Macrófagos/inmunología , Receptores Toll-Like/inmunología , Quinasas Asociadas a rho/inmunologíaRESUMEN
One of the main research areas in biology from the mid-1980s through the 1990s was the elucidation of signaling pathways governing cell responses. These studies brought, among other molecules, the small GTPase Rho to the epicenter. Rho signaling research has since expanded to all areas of biology and medicine. Here, we describe how Rho emerged as a key molecule governing cell morphogenesis and movement, how it was linked to actin reorganization, and how the study of Rho signaling has expanded from cultured cells to whole biological systems. We then give an overview of the current research status of Rho signaling in development, brain, cardiovascular system, immunity and cancer, and discuss the future directions of Rho signaling research, with emphasis on one Rho effector, ROCK*. *The Rho GTPase family. Rho family GTPases have now expanded to contain 20 members. Amino acid sequences of 20 Rho GTPases found in human were aligned and the phylogenetic tree was generated by ClustalW2 software (EMBL-EBI) based on NJ algorithm. The subfamilies of the Rho GTPases are highlighted by the circle and labeled on the right side. Rho cited in this review refers to the original members of Rho subfamily, RhoA, RhoB and RhoC, that are C3 substrates, and, unless specified, not to other members of the Rho subfamily such as Rac, Cdc42, and Rnd.
Asunto(s)
Encéfalo/inmunología , Sistema Cardiovascular/inmunología , Proteínas de Neoplasias/inmunología , Neoplasias/inmunología , Transducción de Señal/inmunología , Proteínas de Unión al GTP rho/inmunología , Quinasas Asociadas a rho/inmunología , Animales , Encéfalo/patología , Sistema Cardiovascular/patología , Historia del Siglo XX , Historia del Siglo XXI , Humanos , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/historia , Neoplasias/genética , Neoplasias/patología , Proteínas de Unión al GTP rho/genética , Proteínas de Unión al GTP rho/historia , Quinasas Asociadas a rho/genética , Quinasas Asociadas a rho/historiaRESUMEN
BACKGROUND/AIMS: The current study was designed to investigate the protective role of alkannin (ALK) on liver injury in diabetic C57BL/KsJ-db/db mice and explore its potential mechanisms. METHODS: An oral glucose tolerance test (OGTT) was performed. The levels of insulin, alanine aminotransferase (ALT), aspartate aminotransaminase (AST), total cholesterol (TC) and triglyceride (TG) were determined by commercial kits. The pro-inflammatory cytokines interleukin (IL)-1ß, IL-6 and tumour necrosis factor (TNF)-α were determined by ELISA. The levels of the ROCK/NF-κB pathway were determined by Western blotting. RESULTS: The contents of pro-inflammatory cytokines interleukin (IL)-1ß, IL-6 and tumour necrosis factor (TNF)-α were inhibited by ALK, metformin or fasudil in diabetic db/db mice. Further, Western blotting analysis showed that the expression of Rho, ROCK1, ROCK2, p-NF-κBp65, and p-IκBα was significantly reversed by ALK treatment. In human hepatic HepG2 cells, the hepatoprotective effects of ALK were further characterized. With response to palmitic acid-challenge, increased amounts of insulin, ALT, AST, TG, and TC were observed, whereas ALK pretreatment significantly inhibited their leakage in HepG2 cells without appreciable cytotoxic effects. The inflammation condition was recovered with ALK treatment as shown by changes of IL-1ß, IL-6 and TNF-α. Further, Western blotting analysis also suggested that ALK improves hepatic inflammation in a Rho-kinase pathway. CONCLUSION: The present study successfully investigated the role of Rho-kinase signalling in diabetic liver injury. ALK exhibited hepatoprotective effects in diabetic db/db mice, and it might act through improving hepatic inflammation through the Rho-kinase pathway.
Asunto(s)
Antiinflamatorios/uso terapéutico , Complicaciones de la Diabetes/tratamiento farmacológico , Inflamación/tratamiento farmacológico , Hepatopatías/tratamiento farmacológico , Hígado/efectos de los fármacos , Naftoquinonas/uso terapéutico , Animales , Supervivencia Celular/efectos de los fármacos , Citocinas/análisis , Citocinas/inmunología , Complicaciones de la Diabetes/sangre , Complicaciones de la Diabetes/inmunología , Complicaciones de la Diabetes/patología , Diabetes Mellitus/sangre , Diabetes Mellitus/tratamiento farmacológico , Diabetes Mellitus/inmunología , Diabetes Mellitus/patología , Células Hep G2 , Humanos , Inflamación/sangre , Inflamación/inmunología , Inflamación/patología , Hígado/inmunología , Hígado/patología , Hepatopatías/sangre , Hepatopatías/inmunología , Hepatopatías/patología , Ratones Endogámicos C57BL , Transducción de Señal/efectos de los fármacos , Quinasas Asociadas a rho/inmunologíaRESUMEN
Onconeural antibodies are associated with cancer and paraneoplastic encephalitis. While their pathogenic role is still largely unknown, their high diagnostic value is undisputed. In this study we describe the discovery of a novel target of autoimmunity in an index case of paraneoplastic encephalitis associated with urogenital cancer.A 75-year-old man with a history of invasive bladder carcinoma 6 years ago with multiple recurrences and a newly discovered renal cell carcinoma presented with seizures and progressive cognitive decline followed by super-refractory status epilepticus. Clinical and ancillary findings including brain biopsy suggested paraneoplastic encephalitis. Immunohistochemistry of the brain biopsy was used to characterize the inflammatory response. Indirect immunofluorescence assay (IFA) was used for autoantibody screening. The autoantigen was identified by histo-immunoprecipitation and mass spectrometry and was validated by expressing the recombinant antigen in HEK293 cells and neutralization tests. Sera from 125 control patients were screened using IFA to test for the novel autoantibodies.IFA analysis of serum revealed a novel autoantibody against brain tissue. An intracellular enzyme, Rho-associated protein kinase 2 (ROCK2), was identified as target-antigen. ROCK2 was expressed in affected brain tissue and archival bladder tumor samples of this patient. Brain histopathology revealed appositions of cytotoxic CD8+ T cells on ROCK2-positive neurons. ROCK2 antibodies were not found in the sera of 20 patients with bladder cancer and 17 with renal cancer, both without neurological symptoms, 49 healthy controls, and 39 patients with other antineuronal autoantibodies. In conclusion, novel onconeural antibodies targeting ROCK2 are associated with paraneoplastic encephalitis and should be screened for when paraneoplastic neurological syndromes, especially in patients with urogenital cancers, occur.
Asunto(s)
Autoanticuerpos/inmunología , Enfermedades Autoinmunes del Sistema Nervioso/enzimología , Encefalitis/enzimología , Encefalitis/inmunología , Síndromes Paraneoplásicos del Sistema Nervioso/enzimología , Síndromes Paraneoplásicos del Sistema Nervioso/inmunología , Quinasas Asociadas a rho/inmunología , Anciano , Autoanticuerpos/sangre , Enfermedades Autoinmunes del Sistema Nervioso/inmunología , Autoinmunidad , Encéfalo/enzimología , Encéfalo/inmunología , Carcinoma/inmunología , Células HEK293 , Humanos , Neoplasias Renales/inmunología , Masculino , Neoplasias de la Vejiga Urinaria/inmunologíaRESUMEN
The factors that govern T cell activation control the initiation and progression of adaptive immune responses. T cells recognize their cognate antigen on the surface of antigen-presenting cells (APCs) through the T cell receptor, which results in the formation of a contact region (immune synapse) between the two cells and the activation of the T cells. Activated T cells proliferate and differentiate into effector T cells that secrete cytokines, provide help to B cells, and kill target cells. We asked whether the actin cytoskeleton governs differences in signaling in effector T cells versus naïve (unstimulated) T cells. Using atomic force microscopy and quantitative confocal microscopy, we found that naïve T cells had a mechanically stiffer cortical cytoskeleton than that of effector cells, which resulted in naïve cells forming smaller immune synapses with APCs. This suggests that the cytoskeletal stiffness of the T cell before it undergoes antigen stimulation predicts its subsequent dynamic engagement with APCs and its activation potential. Cytoskeletal rigidity depended on the activity of the actin-severing enzyme cofilin through a pathway requiring the small guanosine triphosphatase RhoA and the kinases ROCK (Rho-activated kinase) and LIMK. These findings suggest that the baseline cytoskeletal state controls T cell responses and that the underlying pathway could be a therapeutic target for modulating adaptive immunity.
Asunto(s)
Citoesqueleto de Actina/inmunología , Inmunidad Adaptativa/inmunología , Receptores de Antígenos de Linfocitos T/inmunología , Transducción de Señal/inmunología , Citoesqueleto de Actina/metabolismo , Inmunidad Adaptativa/genética , Animales , Células Presentadoras de Antígenos/inmunología , Células Presentadoras de Antígenos/metabolismo , Western Blotting , Línea Celular Tumoral , Quinasas Lim/inmunología , Quinasas Lim/metabolismo , Activación de Linfocitos/inmunología , Ratones Endogámicos C57BL , Ratones Transgénicos , Microscopía de Fuerza Atómica , Microscopía Confocal , Receptores de Antígenos de Linfocitos T/genética , Receptores de Antígenos de Linfocitos T/metabolismo , Transducción de Señal/genética , Quinasas Asociadas a rho/inmunología , Quinasas Asociadas a rho/metabolismo , Proteína de Unión al GTP rhoA/inmunología , Proteína de Unión al GTP rhoA/metabolismoRESUMEN
INTRODUCTION: In the pathogenesis of sepsis-induced acute lung injury (ALI), the crosstalk between inflammation and coagulation plays a pivotal role. The aim of this study was to investigate the role of Rho kinase (ROCK) inhibitor in alleviating pulmonary inflammation and coagulation in lipopolysaccharide (LPS)-induced acute lung injury (ALI) models. METHODS: In the in vivo study, mice were randomized to four different groups: Control, Y-27632 (Y), LPS, and LPS+Y-27632 (LPS+Y). ALI was induced by intranasally administering LPS (10µg in 50µL PBS). Y-27632 (10mg/kg body weight,) was injected intraperitoneally at 18h and 1h before LPS challenge. Mice were euthanized at 3h or 8h post LPS challenge (N=8 per group). In the in vitro study, human pulmonary microvascular endothelial cells (HPMECs) were incubated with LPS alone (1µg/mL) or in combination with 10µM Y-27632 or 50µM BAY11-7082. Cells were pretreated with the inhibitors 30min before exposure to LPS. Three hours later, cells were isolated for subsequent analysis. RESULTS: The myeloperoxidase (MPO) activity and fibrinogen deposits in the lung tissue significantly decreased and the lung damage in ALI mouse was attenuated. Pretreatment with Y-27632 markedly reduced the LPS-induced expression of interleukins 1ß and 6, and the activation of nuclear factor (NF)-κB. Furthermore, ROCK inhibitor treatment antagonized the expression of tissue factor (TF) and plasminogen activator inhibitor (PAI)-1 in lung tissue and HPMECs. CONCLUSIONS: ROCK inhibition protects against the endotoxin-induced pulmonary inflammation and coagulation via NF-kappaB pathway modulation.
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Lesión Pulmonar Aguda/tratamiento farmacológico , Amidas/uso terapéutico , Coagulación Sanguínea/efectos de los fármacos , Pulmón/efectos de los fármacos , Nitrilos/uso terapéutico , Neumonía/prevención & control , Piridinas/uso terapéutico , Sulfonas/uso terapéutico , Quinasas Asociadas a rho/antagonistas & inhibidores , Lesión Pulmonar Aguda/sangre , Lesión Pulmonar Aguda/inmunología , Lesión Pulmonar Aguda/patología , Animales , Línea Celular , Humanos , Interleucinas/inmunología , Lipopolisacáridos/inmunología , Pulmón/inmunología , Pulmón/patología , Masculino , Ratones , Ratones Endogámicos C57BL , FN-kappa B/inmunología , Neumonía/sangre , Neumonía/inmunología , Neumonía/patología , Quinasas Asociadas a rho/inmunologíaRESUMEN
The mechanism of penicillin immediate hypersensitivity reactions has not been completely elucidated. These reactions are generally considered to be mediated by IgE, but penicillin-specific IgE could not be detected in most cases. This study demonstrated that penicillin was able to cause vascular hyperpermeability in a mouse model mimicking clinical symptoms of penicillin immediate hypersensitivity reactions. The first exposure to penicillin also induced immediate edema and exudative reactions in ears and lungs of mice in a dose-dependent manner. Vasodilation was noted in microvessels in ears. These reactions were unlikely to be immune-mediated reactions, because no penicillin-specific IgE was produced. Furthermore, penicillin treatment directly elicited rapid histamine release. Penicillin also led to F-actin reorganization in human umbilical vein endothelial cells and increased the permeability of the endothelial monolayer. Activation of the RhoA/ROCK signaling pathway was observed in ears and lungs of mice and in endothelial cells after treatment with penicillin. Both an anti-histamine agent and a ROCK inhibitor attenuated penicillin immediate hypersensitivity reactions in mice. This study presents a novel mechanism of penicillin immediate hypersensitivity reactions and suggests a potential preventive approach against these reactions.
Asunto(s)
Liberación de Histamina/inmunología , Hipersensibilidad Inmediata/inmunología , Penicilinas/inmunología , Quinasas Asociadas a rho/inmunología , Proteína de Unión al GTP rhoA/inmunología , Animales , Línea Celular Tumoral , Células Cultivadas , Oído/irrigación sanguínea , Células Endoteliales de la Vena Umbilical Humana/inmunología , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Humanos , Hipersensibilidad Inmediata/metabolismo , Pulmón/inmunología , Pulmón/metabolismo , Masculino , Ratones Endogámicos ICR , Microvasos/inmunología , Microvasos/metabolismo , Vasodilatación/inmunología , Quinasas Asociadas a rho/metabolismo , Proteína de Unión al GTP rhoA/metabolismoRESUMEN
CD1d-restricted invariant natural killer T (iNKT) cells are believed to play a key role in cancer immune surveillance, and are functionally deficient in patients with chronic myeloid leukaemia (CML). Herein, we have hypothesized that this defect might originate from BCR-ABL-dependent dysfunctions in myeloid dendritic cells (mDCs). Indeed, flow cytometry and confocal microscopy revealed that cell surface expression of CD1d was downregulated in CML mDCs, relative to healthy donor (HD) controls. The decreased cell surface display of CD1d could not be ascribed to defective mDC differentiation, as attested by normal expression of HLA-DR and the CD86 maturation marker. On the other hand, reduced membrane expression was not associated with decreased intracytoplasmic levels of CD1d or its mRNA transcripts, consistent with intracellular retention. In vitro treatment of CML mDCs with the Rho-associated protein kinase (ROCK) inhibitor Y-27632 partially restored both cell surface CD1d expression and CD1d-mediated antigen presentation, whereas it had no effect on HD mDCs. An inhibitor of BCR-ABL tyrosine kinase (TK), imatinib mesylate (IM), had no such activity. Similar recovery of CD1d expression occurred with fasudil, another ROCK inhibitor that is commonly used in clinical trials. Our data support the conclusion that BCR-ABL-dependent ROCK, but not TK, is involved in CD1d downregulation. We propose that ROCK, which is most likely activated by the DH/PH domain of BCR-ABL, mediates iNKT-cell immune subversion in CML patients by downregulating CD1d expression on CML mDCs. Our study reveals the ROCK-mDC axis as a new potential target to restore immune surveillance in patients with CML, offering new therapeutic perspectives for CML treatment. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.
Asunto(s)
Antígenos CD1d/inmunología , Inmunidad Innata , Leucemia Mielógena Crónica BCR-ABL Positiva/inmunología , Quinasas Asociadas a rho/inmunología , Amidas/farmacología , Diferenciación Celular , Células Dendríticas/enzimología , Células Dendríticas/inmunología , Inhibidores Enzimáticos/farmacología , Proteínas de Fusión bcr-abl/inmunología , Humanos , Mesilato de Imatinib/farmacología , Leucemia Mielógena Crónica BCR-ABL Positiva/tratamiento farmacológico , Leucemia Mielógena Crónica BCR-ABL Positiva/enzimología , Leucemia Mielógena Crónica BCR-ABL Positiva/patología , Células Mieloides/enzimología , Células Mieloides/inmunología , Células T Asesinas Naturales/enzimología , Células T Asesinas Naturales/inmunología , Inhibidores de Proteínas Quinasas/farmacología , Piridinas/farmacología , Quinasas Asociadas a rho/antagonistas & inhibidoresRESUMEN
Rho-associated kinase 2 (ROCK2) determines the balance between human T helper 17 (TH17) cells and regulatory T (Treg) cells. We investigated its role in the generation of T follicular helper (TFH) cells, which help to generate antibody-producing B cells under normal and autoimmune conditions. Inhibiting ROCK2 in normal human T cells or peripheral blood mononuclear cells from patients with active systemic lupus erythematosus (SLE) decreased the number and function of TFH cells induced by activation ex vivo. Moreover, inhibition of ROCK2 activity decreased the abundance of the transcriptional regulator Bcl6 (B cell lymphoma 6) and increased that of Blimp1 by reducing the binding of signal transducer and activator of transcription 3 (STAT3) and increasing that of STAT5 to the promoters of the genes Bcl6 and PRDM1, respectively. In the MRL/lpr murine model of SLE, oral administration of the selective ROCK2 inhibitor KD025 resulted in a twofold reduction in the numbers of TFH cells and antibody-producing plasma cells in the spleen, as well as a decrease in the size of splenic germinal centers, which are the sites of interaction between TFH cells and B cells. KD025-treated mice showed a substantial improvement in both histological and clinical scores compared to those of untreated mice and had reduced amounts of Bcl6 and phosphorylated STAT3, as well as increased STAT5 phosphorylation. Together, these data suggest that ROCK2 signaling plays a critical role in controlling the development of TFH cells induced by autoimmune conditions through reciprocal regulation of STAT3 and STAT5 activation.
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Lupus Eritematoso Sistémico/inmunología , Factor de Transcripción STAT3/inmunología , Factor de Transcripción STAT5/inmunología , Transducción de Señal/inmunología , Linfocitos T Colaboradores-Inductores/inmunología , Quinasas Asociadas a rho/inmunología , Adolescente , Adulto , Anciano , Animales , Modelos Animales de Enfermedad , Femenino , Compuestos Heterocíclicos de 4 o más Anillos , Humanos , Lupus Eritematoso Sistémico/genética , Lupus Eritematoso Sistémico/patología , Masculino , Ratones , Ratones Endogámicos MRL lpr , Persona de Mediana Edad , Células Plasmáticas/inmunología , Células Plasmáticas/patología , Factor 1 de Unión al Dominio 1 de Regulación Positiva/genética , Factor 1 de Unión al Dominio 1 de Regulación Positiva/inmunología , Proteínas Proto-Oncogénicas c-bcl-6/genética , Proteínas Proto-Oncogénicas c-bcl-6/inmunología , Factor de Transcripción STAT3/genética , Factor de Transcripción STAT5/genética , Transducción de Señal/genética , Linfocitos T Colaboradores-Inductores/patología , Quinasas Asociadas a rho/genéticaRESUMEN
Galectin-9 is a widely expressed protein that is involved in immune regulation and tumorpathogenesis and serves as a marker of a poor prognosis in various types of cancers. However, the clinical impact and the precise mechanism by which this protein contributes to colon tumor progression are unclear. In the present study, we detected the expression of galectin-9 and CD56 cells using immunohistochemistry. Spearman's rank correlation was used to clarify the association between galectin-9 expression and natural killer (NK) cell infiltration. The influence of galectin-9 on NK-92 cell migration was evaluated in vitro using transwell chemotaxis assays. The role of rh-galectin-9 in F-actin polarization in NK-92 cells was investigated using laser scanning confocal microscopy. We showed that galectin-9 was expressed in 101 (78.91%) colon tumor tissues and that was expressed at lower levels in these tissues than in para-tumor tissues. Low levels of galectin-9 expression were positively correlated with a poor histological grade and lymph node metastasis (P<0.05). A Kaplan-Meier method and Cox proportional hazards regression analysis showed that overall survival was longer in patients with high galectin-9 expression in an 8-year follow-up (P<0.05). Spearman's rank correlation indicated that there was a linear correlation between galectin-9 expression and CD56+ NK cell infiltration (R(2) = 0.658; P<0.0001). Galectin-9 stimulated migration in human NK-92 cells by affecting F-actin polarization through the Rho/ROCK1 signaling pathway. These results suggest that galectin-9 expression potentially represents a novel mechanism for tumors to escape immune surveillance in colon tumors.