RESUMO
Rearrangement of the actin cytoskeleton is critical for cytotoxic and immunoregulatory functions as well as migration of natural killer (NK) cells. However, dynamic reorganization of actin is a complex process, which remains largely unknown. Here, we investigated the role of the protein Cereblon (CRBN), an E3 ubiquitin ligase complex co-receptor and the primary target of the immunomodulatory drugs, in NK cells. We observed that CRBN partially colocalizes with F-actin in chemokine-treated NK cells and is recruited to the immunological synapse, thus suggesting a role for this protein in cytoskeleton reorganization. Accordingly, silencing of CRBN in NK cells results in a reduced cytotoxicity that correlates with a defect in conjugate and lytic synapse formation. Moreover, CRBN depletion significantly impairs the ability of NK cells to migrate and reduces the enhancing effect of lenalidomide on NK cell migration. Finally, we provided evidence that CRBN is required for activation of the small GTPase Rac1, a critical mediator of cytoskeleton dynamics. Indeed, in CRBN-depleted NK cells, chemokine-mediated or target cell-mediated Rac1 activation is significantly reduced. Altogether our data identify a critical role for CRBN in regulating NK cell functions and suggest that this protein may mediate the stimulatory effect of lenalidomide on NK cells.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/imunologia , Movimento Celular/imunologia , Citotoxicidade Imunológica/imunologia , Células Matadoras Naturais/imunologia , Ubiquitina-Proteína Ligases/imunologia , Proteínas rac1 de Ligação ao GTP/imunologia , Movimento Celular/efeitos dos fármacos , Citotoxicidade Imunológica/efeitos dos fármacos , Humanos , Agentes de Imunomodulação/farmacologia , Células Matadoras Naturais/efeitos dos fármacos , Lenalidomida/farmacologiaRESUMO
Sarcoidosis is an enigmatic multisystem disease characterized by the development and accumulation of granulomas: a compact collection of macrophages that have differentiated into epithelioid cells and which are associated with T helper (Th)1 and Th17 cells. Although no single causative factor has been shown to underlie sarcoidosis in humans, its etiology has been related to microbial, environmental, and genetic factors. We examine how these factors play a role in sarcoidosis pathogenesis. Specifically, we propose that dysfunction of mTOR, Rac1, and autophagy-related pathways not only hampers pathogen or nonorganic particle clearance but also participates in T cell and macrophage dysfunction, driving granuloma formation. This concept opens new avenues for potentially treating sarcoidosis and may serve as a blueprint for other granulomatous disorders.
Assuntos
Autofagia , Sarcoidose , Serina-Treonina Quinases TOR , Proteínas rac1 de Ligação ao GTP , Autofagia/genética , Humanos , Macrófagos/imunologia , Macrófagos/patologia , Sarcoidose/genética , Sarcoidose/imunologia , Serina-Treonina Quinases TOR/imunologia , Células Th1/imunologia , Células Th1/patologia , Células Th17/imunologia , Proteínas rac1 de Ligação ao GTP/imunologiaRESUMO
Pathogenic intestinal bacteria lead to significant disease in humans. Here we investigated the role of the multifunctional protein, Apurinic/apyrimidinic endonuclease 1 (APE1), in regulating the internalization of bacteria into the intestinal epithelium. Intestinal tumor-cell lines and primary human epithelial cells were infected with Salmonella enterica serovar Typhimurium or adherent-invasive Escherichia coli. The effects of APE1 inhibition on bacterial internalization, the regulation of Rho GTPase Rac1 as well as the epithelial cell barrier function were assessed. Increased numbers of bacteria were present in APE1-deficient colonic tumor cell lines and primary epithelial cells. Activation of Rac1 was augmented following infection but negatively regulated by APE1. Pharmacological inhibition of Rac1 reversed the increase in intracellular bacteria in APE1-deficient cells whereas overexpression of constitutively active Rac1 augmented the numbers in APE1-competent cells. Enhanced numbers of intracellular bacteria resulted in the loss of barrier function and a delay in its recovery. Our data demonstrate that APE1 inhibits the internalization of invasive bacteria into human intestinal epithelial cells through its ability to negatively regulate Rac1. This activity also protects epithelial cell barrier function.
Assuntos
Colo , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/imunologia , Células Epiteliais , Infecções por Escherichia coli , Escherichia coli/imunologia , Mucosa Intestinal , Infecções por Salmonella , Salmonella typhimurium/imunologia , Proteínas rac1 de Ligação ao GTP/imunologia , Colo/imunologia , Colo/microbiologia , Colo/patologia , Células Epiteliais/imunologia , Células Epiteliais/microbiologia , Células Epiteliais/patologia , Infecções por Escherichia coli/imunologia , Infecções por Escherichia coli/patologia , Células HT29 , Humanos , Mucosa Intestinal/imunologia , Mucosa Intestinal/microbiologia , Mucosa Intestinal/patologia , Infecções por Salmonella/imunologia , Infecções por Salmonella/patologiaRESUMO
Growing evidence indicate that large antigen-containing particles induce potent T cell-dependent high-affinity antibody responses. These responses require large particle internalization after recognition by the B cell receptor (BCR) on B cells. However, the molecular mechanisms governing BCR-mediated internalization remain unclear. Here we use a high-throughput quantitative image analysis approach to discriminate between B cell particle binding and internalization. We systematically show, using small molecule inhibitors, that human B cells require a SYK-dependent IgM-BCR signaling transduction via PI3K to efficiently internalize large anti-IgM-coated particles. IgM-BCR-mediated activation of PI3K involves both the adaptor protein NCK and the co-receptor CD19. Interestingly, we here reveal a strong NCK-dependence without profound requirement of the co-receptor CD19 in B cell responses to large particles. Furthermore, we demonstrate that the IgM-BCR/NCK signaling event facilitates RAC1 activation to promote actin cytoskeleton remodeling necessary for particle engulfment. Thus, we establish NCK/PI3K/RAC1 as an attractive IgM-BCR signaling axis for biological intervention to prevent undesired antibody responses to large particles.
Assuntos
Apresentação de Antígeno/imunologia , Linfócitos B/imunologia , Ativação Linfocitária/imunologia , Fagocitose/imunologia , Transdução de Sinais/imunologia , Proteínas Adaptadoras de Transdução de Sinal/imunologia , Linfócitos B/metabolismo , Humanos , Imunoglobulina M/imunologia , Proteínas Oncogênicas/imunologia , Fosfatidilinositol 3-Quinases/imunologia , Receptores de Antígenos de Linfócitos B/imunologia , Proteínas rac1 de Ligação ao GTP/imunologiaRESUMO
ß2-integrins promote neutrophil recruitment to infected tissues and are crucial for host defense. Neutrophil recruitment is defective in leukocyte adhesion deficiency type-1 (LAD1), a condition caused by mutations in the CD18 (ß2-integrin) gene. Using a model of Citrobacter rodentium (CR)-induced colitis, we show that CD18-/- mice display increased intestinal damage and systemic bacterial burden, compared to littermate controls, ultimately succumbing to infection. This phenotype is not attributed to defective neutrophil recruitment, as it is shared by CXCR2-/- mice that survive CR infection. CR-infected CD18-/- mice feature prominent upregulation of IL-17 and downregulation of IL-22. Exogenous IL-22 administration, but not endogenous IL-17 neutralization, protects CD18-/- mice from lethal colitis. ß2-integrin expression on macrophages is mechanistically linked to Rac1/ROS-mediated induction of noncanonical-NLRP3 (nucleotide-binding domain, leucine-rich-containing family, pyrin domain-containing-3) inflammasome-dependent IL-1ß production, which promotes ILC3-derived IL-22. Therefore, ß2-integrins are required for protective IL-1ß-dependent IL-22 responses in colitis, and the identified mechanism may underlie the association of human LAD1 with colitis.
Assuntos
Antígenos CD18/genética , Citrobacter rodentium/patogenicidade , Colite/genética , Infecções por Enterobacteriaceae/genética , Interleucinas/genética , Macrófagos/imunologia , Animais , Antígenos CD18/deficiência , Antígenos CD18/imunologia , Citrobacter rodentium/imunologia , Colite/imunologia , Colite/microbiologia , Colite/mortalidade , Infecções por Enterobacteriaceae/imunologia , Infecções por Enterobacteriaceae/microbiologia , Infecções por Enterobacteriaceae/mortalidade , Feminino , Regulação da Expressão Gênica/imunologia , Inflamassomos/imunologia , Inflamassomos/metabolismo , Interleucina-17/genética , Interleucina-17/imunologia , Interleucina-1beta/genética , Interleucina-1beta/imunologia , Interleucinas/imunologia , Intestinos/imunologia , Intestinos/microbiologia , Intestinos/patologia , Macrófagos/microbiologia , Macrófagos/patologia , Masculino , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/imunologia , Camundongos , Camundongos Knockout , Proteína 3 que Contém Domínio de Pirina da Família NLR/genética , Proteína 3 que Contém Domínio de Pirina da Família NLR/imunologia , Neuropeptídeos/genética , Neuropeptídeos/imunologia , Infiltração de Neutrófilos , Neutrófilos/imunologia , Neutrófilos/microbiologia , Neutrófilos/patologia , Espécies Reativas de Oxigênio/imunologia , Espécies Reativas de Oxigênio/metabolismo , Análise de Sobrevida , Proteínas rac1 de Ligação ao GTP/genética , Proteínas rac1 de Ligação ao GTP/imunologia , Interleucina 22RESUMO
BACKGROUND/AIMS: Chronic inflammation contributes to the development of type 2 diabetes mellitus by targeting the insulin receptor substrate protein-1 (IRS-1) signaling pathway. Previous studies showed that Leukemia related protein 16 (LRP16) reduced insulin stimulated glucose uptake in adipocytes by impairing the IRS-1 signaling pathway. We explored the mechanism by which LRP16 promotes the inflammatory response. METHODS: We screened LRP16 induced proteins in the lipopolysaccharide (LPS)-stimulated inflammatory response using liquid chromatography-mass spectrometry (LC-MS) and analyzed the potential biological functions of these proteins using online bioinformatics tools. mRNA expression and protein expression of target genes were measured by real time PCR and Western blot, respectively. RESULTS: A total of 390 differentially expressed proteins were identified. The mitogen-activated protein kinase (MAPK) signaling pathway was the primary activated pathway in LRP16-expressing cells. Overexpression of LRP16 activated ERK1/2 and Rac1, which are two key players related to the MAPK signaling pathway. Furthermore, knock down of endogenous LRP16 by RNA interference (RNAi) reduced Rac1 expression, ERK activation, and inflammatory cytokine expression in human adipocytes stimulated by LPS. The stimulatory effect of LRP16 was diminished by suppressing Rac1 expression and treating the cells with the ERK specific inhibitor, PD98059. CONCLUSION: These findings revealed the functions of LRP16 in promoting the inflammatory response through activating the Rac1-MAPK1/ERK pathway in human adipocytes.
Assuntos
Inflamação/imunologia , Lipopolissacarídeos/imunologia , Sistema de Sinalização das MAP Quinases , Proteínas de Neoplasias/imunologia , Transdução de Sinais , Proteínas rac1 de Ligação ao GTP/imunologia , Adipócitos , Hidrolases de Éster Carboxílico , Linhagem Celular , Diabetes Mellitus Tipo 2/imunologia , HumanosRESUMO
Endosomal traffic of TCR and signaling molecules regulates immunological synapse formation and T cell activation. We recently showed that Rab11 endosomes regulate the subcellular localization of the tyrosine kinase Lck and of the GTPase Rac1 and control their functions in TCR signaling and actin cytoskeleton remodeling. HIV-1 infection of T cells alters their endosomal traffic, activation capacity, and actin cytoskeleton organization. The viral protein Nef is pivotal for these modifications. We hypothesized that HIV-1 Nef could jointly alter Lck and Rac1 endosomal traffic and concomitantly modulate their functions. In this study, we show that HIV-1 infection of human T cells sequesters both Lck and Rac1 in a pericentrosomal compartment in an Nef-dependent manner. Strikingly, the Nef-induced Lck compartment contains signaling-competent forms (phosphorylated on key Tyr residues) of Lck and some of its downstream effectors, TCRζ, ZAP70, SLP76, and Vav1, avoiding the proximal LAT adaptor. Importantly, Nef-induced concentration of signaling molecules was concomitant with the upregulation of several early and late T cell activation genes. Moreover, preventing the concentration of the Nef-induced Lck compartment by depleting the Rab11 effector FIP3 counteracted Nef-induced gene expression upregulation. In addition, Nef extensively sequesters Rac1 and downregulates Rac1-dependent actin cytoskeleton remodeling, thus reducing T cell spreading. Therefore, by modifying their endosomal traffic, Nef hijacks signaling and actin cytoskeleton regulators to dually modulate their functional outputs. Our data shed new light into the molecular mechanisms that modify T cell physiology during HIV-1 infection.
Assuntos
Linfócitos T CD4-Positivos/virologia , Infecções por HIV/imunologia , Proteína Tirosina Quinase p56(lck) Linfócito-Específica/metabolismo , Produtos do Gene nef do Vírus da Imunodeficiência Humana/imunologia , Proteínas rac1 de Ligação ao GTP/metabolismo , Citoesqueleto de Actina/imunologia , Citoesqueleto de Actina/metabolismo , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/metabolismo , Endossomos/imunologia , Endossomos/metabolismo , Endossomos/virologia , Infecções por HIV/metabolismo , Humanos , Proteína Tirosina Quinase p56(lck) Linfócito-Específica/imunologia , Transporte Proteico/imunologia , Transdução de Sinais/imunologia , Proteínas rac1 de Ligação ao GTP/imunologiaRESUMO
Bacterial pathogens are associated with severe infections (e.g., sepsis) and exacerbation of debilitating conditions such as chronic obstructive pulmonary disease (COPD). The interactions of bacterial pathogens with macrophages, a key component of innate immunity and host defense, are not clearly understood and continue to be intensively studied. Having previously demonstrated a role of Wnt5A signaling in phagocytosis, we proceeded to decipher the connection of Wnt5A signaling with infection by pathogenic bacteria, namely Pseudomonas aeruginosa (PA) and Streptococcus pneumoniae (SP), which are related with the progression of COPD and sepsis. We found that during the initial hours of infection with PA and SP, there is decrease in the steady state levels of the Wnt5A protein in macrophages. Suppression of Wnt5A signaling, moreover, impairs macrophage clearance of the bacterial infection both in vitro and in vivo. Activation of Wnt5A signaling, on the other hand, enhances clearance of the infection. Macrophage-mediated containment of bacterial infection in our study is dependant on Wnt5A-induced Rac1/Disheveled activation and cytochalasin D inhibitable actin assembly, which is associated with ULK1 kinase activity and LC3BII accumulation. Our experimental findings are consistent with Wnt5A signaling-dependent induction of autophagic killing (xenophagy) of PA and SP, as further substantiated by transmission electron microscopy. Overall, our study unveils the prevalence of a Wnt5A-Rac1-Disheveled-mediated actin-associated autophagy circuit as an important component of innate immunity in host macrophages that may turn out crucial for restricting infection by leading bacterial pathogens.
Assuntos
Proteínas Desgrenhadas/imunologia , Interações Hospedeiro-Patógeno/imunologia , Neuropeptídeos/imunologia , Infecções Pneumocócicas/imunologia , Infecções por Pseudomonas/imunologia , Proteína Wnt-5a/imunologia , Proteínas rac1 de Ligação ao GTP/imunologia , Animais , Linhagem Celular , Macrófagos/imunologia , Camundongos Endogâmicos BALB C , Peritonite/imunologia , Pseudomonas aeruginosa , Infecções Respiratórias/imunologia , Streptococcus pneumoniaeRESUMO
Various physiologically active effects of polymerized polyphenols have been reported. In this study, we synthesized a polymerized polyphenol (mL2a-pCA) by polymerizing caffeic acid using mutant Agaricus brasiliensis laccase and analyzed its physiological activity and mechanism of action. We found that mL2a-pCA induced morphological changes and the production of cytokines and chemokines in C3H/HeN mouse-derived resident peritoneal macrophages in vitro. The mechanisms of action of polymerized polyphenols on in vitro mouse resident peritoneal cells have not been characterized in detail previously. Herein, we report that the mL2a-pCA-induced production of interleukin-6 (IL-6) and monocyte chemotactic protein-1 (MCP-1) in C3H/HeN mouse-derived resident peritoneal cells was inhibited by treatment with the Rac1 inhibitor NSC23766 trihydrochloride. In addition, we found that mL2a-pCA activated the phosphorylation Rac1. Taken together, the results show that mL2a-pCA induced macrophage activation via Rac1 phosphorylation-dependent pathways.
Assuntos
Lacase/química , Ativação de Macrófagos/imunologia , Macrófagos/imunologia , Macrófagos/patologia , Neuropeptídeos/imunologia , Polifenóis/administração & dosagem , Polifenóis/química , Proteínas rac1 de Ligação ao GTP/imunologia , Animais , Células Cultivadas , Relação Dose-Resposta a Droga , Ativação Enzimática , Ativação de Macrófagos/efeitos dos fármacos , Macrófagos/efeitos dos fármacos , Masculino , Camundongos , Camundongos Endogâmicos C3H , Fosforilação/efeitos dos fármacosRESUMO
The endogenously activated rheumatoid arthritis (RA) synovial fibroblasts (RSFs) are likely to be the key to curing the disease. RSFs express Tolllike receptors (TLRs) rendering them prone to activation by exogenous and endogenous TLR ligands, resulting in the production of chemokines and cytokines Germline deletion of tumor necrosis factorαinduced protein8 like 2 (TIPE2, also known as TNFAIP8L2) results in fatal inflammation and hypersensitivity to TLR and T cell receptor stimulation. The present study demonstrates an inverse association between TIPE2 and cytokine gene expression in RSFs following lipopolysaccharide (LPS) stimulation. Enhanced TIPE2 expression decreased Rasrelated C3 botulinum toxin substrate (Rac) activation and interferon regulatory factor 3 phosphorylation, and phosphoinositide 3kinase and Rac inhibition significantly diminished LPSinduced cytokine gene expression in RSFs. In conclusion, the findings of the present study demonstrate that TIPE2 serves a negative role in activating the Rac signaling pathway and in the initiation of the immune response by decreasing the activity of proinflammatory cytokines. These results may be useful in designing novel strategies for the prevention and treatment of RA.
Assuntos
Artrite Experimental/genética , Fibroblastos/imunologia , Fator Regulador 3 de Interferon/genética , Peptídeos e Proteínas de Sinalização Intracelular/genética , Sinoviócitos/imunologia , Proteínas rac1 de Ligação ao GTP/genética , Animais , Artrite Experimental/induzido quimicamente , Artrite Experimental/imunologia , Artrite Experimental/patologia , Linhagem Celular , Fibroblastos/patologia , Regulação da Expressão Gênica , Fator Regulador 3 de Interferon/imunologia , Interferon gama/genética , Interferon gama/imunologia , Interleucina-1/genética , Interleucina-1/imunologia , Interleucina-6/genética , Interleucina-6/imunologia , Peptídeos e Proteínas de Sinalização Intracelular/imunologia , Lipopolissacarídeos , Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositol 3-Quinases/imunologia , Fosforilação , Ratos , Transdução de Sinais , Membrana Sinovial/imunologia , Membrana Sinovial/patologia , Sinoviócitos/patologia , Receptores Toll-Like/genética , Receptores Toll-Like/imunologia , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/imunologia , Proteínas rac1 de Ligação ao GTP/imunologiaRESUMO
Enhanced turnover of subchondral trabecular bone is a hallmark of rheumatoid arthritis (RA) and it results from an imbalance between bone resorption and bone formation activities. To investigate the formation and activation of osteoclasts which mediate bone resorption, a Fas-deficient MRL/lpr mouse model which spontaneously develops autoimmune arthritis and exhibits decreased bone mass was studied. Various assays were performed on subchondral trabecular bone of the temporomandibular joint (TMJ) from MRL/lpr mice and MRL+/+ mice. Initially, greater osteoclast production was observed in vitro from bone marrow macrophages obtained from MRL/lpr mice due to enhanced phosphorylation of NF-κB, as well as Akt and MAPK, to receptor activator of nuclear factor-κB ligand (RANKL). Expression of sphingosine 1-phosphate receptor 1 (S1P1) was also significantly upregulated in the condylar cartilage. S1P1 was found to be required for S1P-induced migration of osteoclast precursor cells and downstream signaling via Rac1. When SN50, a synthetic NF-κB-inhibitory peptide, was applied to the MRL/lpr mice, subchondral trabecular bone loss was reduced and both production of osteoclastogenesis markers and sphingosine kinase (Sphk) 1/S1P1 signaling were reduced. Thus, the present results suggest that Fas/S1P1 signaling via activation of NF-κB in osteoclast precursor cells is a key factor in the pathogenesis of RA in the TMJ.
Assuntos
Artrite Reumatoide/imunologia , Reabsorção Óssea/imunologia , NF-kappa B/imunologia , Osteoclastos/efeitos dos fármacos , Receptores de Lisoesfingolipídeo/imunologia , Articulação Temporomandibular/imunologia , Receptor fas/imunologia , Animais , Artrite Reumatoide/tratamento farmacológico , Artrite Reumatoide/genética , Artrite Reumatoide/patologia , Autoimunidade , Células da Medula Óssea/efeitos dos fármacos , Células da Medula Óssea/imunologia , Células da Medula Óssea/patologia , Reabsorção Óssea/genética , Reabsorção Óssea/patologia , Reabsorção Óssea/prevenção & controle , Diferenciação Celular , Modelos Animais de Doenças , Feminino , Regulação da Expressão Gênica , Lisofosfolipídeos/imunologia , Macrófagos/efeitos dos fármacos , Macrófagos/imunologia , Macrófagos/patologia , Camundongos , Camundongos Endogâmicos MRL lpr , Proteínas Quinases Ativadas por Mitógeno/genética , Proteínas Quinases Ativadas por Mitógeno/imunologia , NF-kappa B/antagonistas & inibidores , NF-kappa B/genética , Neuropeptídeos/genética , Neuropeptídeos/imunologia , Osteoclastos/imunologia , Osteoclastos/patologia , Osteogênese/efeitos dos fármacos , Osteogênese/imunologia , Peptídeos/farmacologia , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Fosfotransferases (Aceptor do Grupo Álcool)/imunologia , Cultura Primária de Células , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/imunologia , Ligante RANK/genética , Ligante RANK/imunologia , Receptores de Lisoesfingolipídeo/genética , Transdução de Sinais , Esfingosina/análogos & derivados , Esfingosina/imunologia , Articulação Temporomandibular/efeitos dos fármacos , Articulação Temporomandibular/patologia , Receptor fas/genética , Proteínas rac1 de Ligação ao GTP/genética , Proteínas rac1 de Ligação ao GTP/imunologiaRESUMO
Lamellipodia are sheet-like cell protrusions driven by actin polymerization mainly through Rac1, a GTPase molecular switch. In Fcγ receptor-mediated phagocytosis of IgG-opsonized erythrocytes (IgG-Es), Rac1 activation is required for lamellipodial extension along the surface of IgG-Es. However, the significance of Rac1 deactivation in phagosome formation is poorly understood. Our live-cell imaging and electron microscopy revealed that RAW264 macrophages expressing a constitutively active Rac1 mutant showed defects in phagocytic cup formation, while lamellipodia were formed around IgG-Es. Because activated Rac1 reduced the phosphorylation levels of myosin light chains, failure of the cup formation is probably due to inhibition of actin/myosin II contractility. Reversible photo-manipulation of the Rac1 switch in macrophages fed with IgG-Es could phenocopy two lamellipodial motilities: outward-extension and cup-constriction by Rac1 ON and OFF, respectively. In conjunction with fluorescence resonance energy transfer imaging of Rac1 activity, we provide a novel mechanistic model of phagosome formation spatiotemporally controlled by Rac1 switching within a phagocytic cup.
Assuntos
Macrófagos/imunologia , Modelos Imunológicos , Neuropeptídeos/imunologia , Fagossomos/imunologia , Receptores de IgG/imunologia , Proteínas rac1 de Ligação ao GTP/imunologia , Animais , Camundongos , Fosforilação/imunologia , Células RAW 264.7RESUMO
OBJECTIVES: Inappropriate activation of neutrophils plays a pathological role in antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV). The aim of this study was to investigate the functions of semaphorin 4D (SEMA4D) in regulation of neutrophil activation, and its involvement in AAV pathogenesis. METHODS: Serum levels of soluble SEMA4D were evaluated by ELISA. Blood cell-surface expression of membrane SEMA4D was evaluated by flow cytometry. To determine the functional interactions between neutrophil membrane SEMA4D and endothelial plexin B2, wild-type and SEMA4D-/- mice neutrophils were cultured with an endothelial cell line (MS1) stained with SYTOX green, and subjected to neutrophil extracellular trap (NET) formation assays. The efficacy of treating human neutrophils with recombinant plexin B2 was assessed by measuring the kinetic oxidative burst and NET formation assays. RESULTS: Serum levels of soluble SEMA4D were elevated in patients with AAV and correlated with disease activity scores. Cell-surface expression of SEMA4D was downregulated in neutrophils from patients with AAV, a consequence of proteolytic cleavage of membrane SEMA4D. Soluble SEMA4D exerted pro-inflammatory effects on endothelial cells. Membranous SEMA4D on neutrophils bound to plexin B2 on endothelial cells, and this interaction decreased NET formation. Recombinant plexin B2 suppressed neutrophil Rac1 activation through SEMA4D's intracellular domain, and inhibited pathogen-induced or ANCA-induced oxidative burst and NET formation. CONCLUSIONS: Neutrophil surface SEMA4D functions as a negative regulator of neutrophil activation. Proteolytic cleavage of SEMA4D as observed in patients with AAV may amplify neutrophil-mediated inflammatory responses. SEMA4D is a promising biomarker and potential therapeutic target for AAV.
Assuntos
Vasculite Associada a Anticorpo Anticitoplasma de Neutrófilos/imunologia , Antígenos CD/imunologia , Células Endoteliais/imunologia , Armadilhas Extracelulares/imunologia , Proteínas do Tecido Nervoso/imunologia , Neutrófilos/imunologia , Semaforinas/imunologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Antígenos CD/genética , Ensaio de Imunoadsorção Enzimática , Armadilhas Extracelulares/efeitos dos fármacos , Feminino , Citometria de Fluxo , Humanos , Masculino , Camundongos , Camundongos Knockout , Pessoa de Meia-Idade , Proteínas do Tecido Nervoso/farmacologia , Neutrófilos/efeitos dos fármacos , Espécies Reativas de Oxigênio/imunologia , Explosão Respiratória/efeitos dos fármacos , Semaforinas/genética , Proteínas rac1 de Ligação ao GTP/efeitos dos fármacos , Proteínas rac1 de Ligação ao GTP/imunologiaRESUMO
BACKGROUND/AIMS: Regulatory T (Treg) cells have been associated with neuroprotection by inhibiting microglial activation in animal models of Parkinson's disease (PD), a progressive neurodegenerative disease characterized by dopaminergic neuronal loss in the nigrostriatal system. Herein, we show that Treg cells directly protect dopaminergic neurons against 1-methyl-4-phenylpyridinium (MPP+) neurotoxicity via an interaction between the two transmembrane proteins CD47 and signal regulatory protein α (SIRPA). METHODS: Primary ventral mesencephalic (VM) cells or VM neurons were pretreated with Treg cells before MPP+ treatment. Transwell co-culture of Treg cells and VM neurons was used to assess the effects of the Treg cytokines transforming growth factor (TGF)-ß1 and interleukin (IL)-10 on dopaminergic neurons. Live cell imaging system detected a dynamic contact of Treg cells with VM neurons that were stained with CD47 and SIRPA, respectively. Dopaminergic neuronal loss, which was assessed by the number of tyrosine hydroxylase (TH)-immunoreactive cells, was examined after silencing CD47 in Treg cells or silencing SIRPA in VM neurons. RESULTS: Treg cells prevented MPP+-induced dopaminergic neuronal loss and glial inflammatory responses. TGF-ß1 and IL-10 secreted from Treg cells did not significantly prevent MPP+-induced dopaminergic neuronal loss in transwell co-culture of Treg cells and VM neurons. CD47 and SIRPA were expressed by Treg cells and VM neurons, respectively. CD47-labeled Treg cells dynamically contacted with SIRPA-labeled VM neurons. Silencing CD47 gene in Treg cells impaired the ability of Treg cells to protect dopaminergic neurons against MPP+ toxicity. Similarly, SIRPA knockdown in VM neurons reduced the ability of Treg cell neuroprotection. Rac1/Akt signaling pathway in VM neurons was activated by CD47-SIRPA interaction between Treg cells and the neurons. Inhibiting Rac1/Akt signaling in VM neurons compromised Treg cell neuroprotection. CONCLUSION: Treg cells protect dopaminergic neurons against MPP+ neurotoxicity by a cell-to-cell contact mechanism underlying CD47-SIRPA interaction and Rac1/Akt activation.
Assuntos
1-Metil-4-fenilpiridínio/toxicidade , Antígeno CD47/genética , Neurônios Dopaminérgicos/efeitos dos fármacos , Receptores Imunológicos/genética , Linfócitos T Reguladores/efeitos dos fármacos , Animais , Antígeno CD47/imunologia , Comunicação Celular , Morte Celular/efeitos dos fármacos , Técnicas de Cocultura , Cultura em Câmaras de Difusão , Neurônios Dopaminérgicos/citologia , Neurônios Dopaminérgicos/imunologia , Embrião de Mamíferos , Feminino , Expressão Gênica , Interleucina-10/farmacologia , Mesencéfalo/citologia , Mesencéfalo/efeitos dos fármacos , Mesencéfalo/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Neuropeptídeos/genética , Neuropeptídeos/imunologia , Cultura Primária de Células , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/imunologia , Receptores Imunológicos/imunologia , Transdução de Sinais , Baço/citologia , Baço/efeitos dos fármacos , Baço/imunologia , Linfócitos T Reguladores/citologia , Linfócitos T Reguladores/imunologia , Fator de Crescimento Transformador beta1/farmacologia , Proteínas rac1 de Ligação ao GTP/genética , Proteínas rac1 de Ligação ao GTP/imunologiaRESUMO
Neutrophil extravasation is a critical step of the innate immune system's response to inflammation. This multistep process is tightly regulated by adhesion and signaling molecules in the endothelium and neutrophils. Activation of the ß2 integrin LFA-1 is critical for adhesion of leukocytes to postcapillary venules. This step requires coordinated activation of signaling pathways in chemokine-stimulated neutrophils, including GTPase activation and cytoskeletal remodeling, leading to conformational changes in LFA-1. Hematopoietic cell-specific lyn substrate 1 (HS1) is a cortactin-related and leukocyte-specific actin-binding protein (ABP) that regulates several processes in various immune cells. It has been shown in vitro that HS1 is important for neutrophil chemotaxis and transendothelial migration of NK cells, but its role in neutrophil extravasation in vivo has not been investigated yet. Intravital microscopy of CXCL1-stimulated cremaster venules revealed an increased rolling velocity and reduced neutrophil adhesion and transmigration in HS1 knockout (KO) mice. CXCL1-induced rapid neutrophil arrest in vivo and adhesion under flow conditions in vitro were also reduced significantly. Whereas random motility of neutrophils was unaffected, chemotaxis toward a CXCL1 gradient was reduced in the absence of HS1. Further analysis of the underlying mechanisms demonstrated that HS1 controls CXCL1-induced activation of the small GTPases Ras-related C3 botulinum toxin substrate 1 (Rac1) and Ras-related protein 1 (Rap1), thus supporting LFA-1-mediated neutrophil adhesion. Importantly, with the use of Rac1 KO neutrophils, we could show that Rac1 acts upstream of Rap1. Our results establish HS1 as an important regulator of proper Rac1 and Rap1 activation and neutrophil extravasation.
Assuntos
Fator Estimulador de Colônias de Granulócitos/imunologia , Antígeno-1 Associado à Função Linfocitária/imunologia , Neuropeptídeos/imunologia , Neutrófilos/imunologia , Peritonite/imunologia , Proteínas rac1 de Ligação ao GTP/imunologia , Proteínas rap1 de Ligação ao GTP/imunologia , Músculos Abdominais/irrigação sanguínea , Músculos Abdominais/citologia , Músculos Abdominais/imunologia , Animais , Adesão Celular/efeitos dos fármacos , Quimiocina CXCL1/genética , Quimiocina CXCL1/imunologia , Quimiocina CXCL1/farmacologia , Quimiotaxia/efeitos dos fármacos , Fator Estimulador de Colônias de Granulócitos/deficiência , Fator Estimulador de Colônias de Granulócitos/genética , Imunidade Inata , Microscopia Intravital , Antígeno-1 Associado à Função Linfocitária/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neuropeptídeos/genética , Infiltração de Neutrófilos/efeitos dos fármacos , Neutrófilos/efeitos dos fármacos , Neutrófilos/patologia , Peritonite/genética , Peritonite/patologia , Cultura Primária de Células , Proteínas rac1 de Ligação ao GTP/genética , Proteínas rap1 de Ligação ao GTP/genéticaRESUMO
In myelodysplastic syndromes (MDS), functional defects of neutrophils result in high mortality because of infections; however, the molecular basis remains unclear. We recently found that miR-34a and miR-155 were significantly increased in MDS neutrophils. To clarify the effects of the aberrant microRNA expression on neutrophil functions, we introduced miR-34a, miR-155, or control microRNA into neutrophil-like differentiated HL60 cells. Ectopically introduced miR-34a and miR-155 significantly attenuated migration toward chemoattractants fMLF and IL-8, but enhanced degranulation. To clarify the mechanisms for inhibition of migration, we studied the effects of miR-34a and miR-155 on the migration-regulating Rho family members, Cdc42 and Rac1. The introduced miR-34a and miR-155 decreased the fMLF-induced active form of Cdc42 to 29.0 ± 15.9 and 39.7 ± 4.8% of that in the control cells, respectively, although Cdc42 protein levels were not altered. miR-34a decreased a Cdc42-specific guanine nucleotide exchange factor (GEF), dedicator of cytokinesis (DOCK) 8, whereas miR-155 reduced another Cdc42-specific GEF, FYVE, RhoGEF, and PH domain-containing (FGD) 4. The knockdown of DOCK8 and FGD4 by small interfering RNA suppressed Cdc42 activation and fMLF/IL-8-induced migration. miR-155, but not miR-34a, decreased Rac1 protein, and introduction of Rac1 small interfering RNA attenuated Rac1 activation and migration. Neutrophils from patients showed significant attenuation in migration compared with healthy cells, and protein levels of DOCK8, FGD4, and Rac1 were well correlated with migration toward fMLF (r = 0.642, 0.686, and 0.436, respectively) and IL-8 (r = 0.778, 0.659, and 0.606, respectively). Our results indicated that reduction of DOCK8, FGD4, and Rac1 contributes to impaired neutrophil migration in MDS.
Assuntos
Quimiotaxia de Leucócito , MicroRNAs/imunologia , Síndromes Mielodisplásicas/imunologia , Ativação de Neutrófilo , Neutrófilos/fisiologia , Idoso , Idoso de 80 Anos ou mais , Proliferação de Células , Quimiotaxia/imunologia , Feminino , Fatores de Troca do Nucleotídeo Guanina/deficiência , Fatores de Troca do Nucleotídeo Guanina/genética , Fatores de Troca do Nucleotídeo Guanina/imunologia , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Células HL-60 , Humanos , Interleucina-8/imunologia , Masculino , MicroRNAs/genética , MicroRNAs/fisiologia , Proteínas dos Microfilamentos/deficiência , Proteínas dos Microfilamentos/genética , Proteínas dos Microfilamentos/metabolismo , Pessoa de Meia-Idade , Neutrófilos/imunologia , RNA Interferente Pequeno , Proteína cdc42 de Ligação ao GTP/genética , Proteína cdc42 de Ligação ao GTP/imunologia , Proteínas rac1 de Ligação ao GTP/genética , Proteínas rac1 de Ligação ao GTP/imunologiaRESUMO
BACKGROUND/AIMS: Lipopolysaccharide (LPS) is a potent activator of vascular smooth muscle cells (VSMCs) proliferation, but the underlying mechanism remains unknown. In this study, we knocked down Toll-like receptor 4 (TLR4) and Ras-related C3 botulinum toxin substrate 1 (Rac1) expression using small interfering RNA (siRNA) in order to investigate the effects and possible mechanisms of LPS-induced VSMCs proliferation. METHODS: VSMCs proliferation was monitored by 5-ethynyl-2'-deoxyuridine staining, and Rac1 activity was measured via Glutathione S-transferase pull-down assay. mRNAs encoding proliferating cell nuclear antigen (PCNA), smooth muscle 22α (SM22α), myosin heavy chain (MYH) and transient receptor potential channel 1 (TRPC1) were detected by qRT-PCR. The expression of total Akt, p-Akt (308), p-Akt (473), SM22α, MYH and TRPC1 protein was analysed by Western blot. RESULTS: Treatment with TLR4 siRNA (siTLR4) or Rac1 siRNA (siRac1) significantly decreased LPS-induced VSMCs proliferation. Moreover, LPS-induced activation of Rac1 through TLR4 was observed. Western blot analysis revealed that transfection with siTLR4 or siRac1 inhibited LPS-induced Akt phosphorylation. We discovered that LPS stimulated VSMCs proliferation via phenotypic modulation and that this effect was partially inhibited by pre-treatment with siTLR4 or siRac1. Further, TLR4 and Rac1 are involved in LPS-induced activation of TRPC1. CONCLUSION: This study suggests that LPS exerts an effect on VSMCs proliferation and that the TLR4/Rac1/Akt signalling pathway mediates this effect.
Assuntos
Proliferação de Células , Lipopolissacarídeos/imunologia , Músculo Liso Vascular/citologia , Proteínas Proto-Oncogênicas c-akt/imunologia , Transdução de Sinais , Receptor 4 Toll-Like/imunologia , Proteínas rac1 de Ligação ao GTP/imunologia , Animais , Células Cultivadas , Feminino , Masculino , Músculo Liso Vascular/imunologia , Ratos Sprague-DawleyRESUMO
The activity of Rac in leukocytes is essential for immunity. However, its role in NK cell-mediated anti-microbial signaling remains unclear. In this study, we investigated the role of Rac in NK cell mediated anti-cryptococcal killing. We found thatCryptococcus neoformansindependently activates both Rac and SFK pathways in NK cells, and unlike in tumor killing,Cryptococcusinitiated a novel Rac â PI3K â Erk cytotoxicity cascade. Remarkably, Rac was not required for conjugate formation, despite its essential role in NK cytotoxicity againstC. neoformans Taken together, our data show that, unlike observations with tumor cells, NK cells use a novel Rac cytotoxicity pathway in conjunction with SFK, to killC. neoformans.
Assuntos
Classe Ia de Fosfatidilinositol 3-Quinase/imunologia , Cryptococcus neoformans/fisiologia , Citotoxicidade Imunológica , Células Matadoras Naturais/imunologia , Proteínas rac de Ligação ao GTP/imunologia , Proteínas rac1 de Ligação ao GTP/imunologia , Quinases da Família src/imunologia , Linhagem Celular Tumoral , Classe Ia de Fosfatidilinositol 3-Quinase/genética , Regulação da Expressão Gênica , Interações Hospedeiro-Patógeno , Humanos , Células Matadoras Naturais/efeitos dos fármacos , Células Matadoras Naturais/microbiologia , Proteína Quinase 1 Ativada por Mitógeno/genética , Proteína Quinase 1 Ativada por Mitógeno/imunologia , Proteína Quinase 3 Ativada por Mitógeno/genética , Proteína Quinase 3 Ativada por Mitógeno/imunologia , Fosforilação/efeitos dos fármacos , Cultura Primária de Células , Pironas/farmacologia , Quinolinas/farmacologia , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Transdução de Sinais , Proteínas rac de Ligação ao GTP/antagonistas & inibidores , Proteínas rac de Ligação ao GTP/genética , Proteínas rac1 de Ligação ao GTP/antagonistas & inibidores , Proteínas rac1 de Ligação ao GTP/genética , Quinases da Família src/genética , Proteína RAC2 de Ligação ao GTPRESUMO
Generation of reactive oxygen species (ROS) during infection is an immediate host defense leading to microbial killing. APE1 is a multifunctional protein induced by ROS and after induction, protects against ROS-mediated DNA damage. Rac1 and NAPDH oxidase (Nox1) are important contributors of ROS generation following infection and associated with gastrointestinal epithelial injury. The purpose of this study was to determine if APE1 regulates the function of Rac1 and Nox1 during oxidative stress. Gastric or colonic epithelial cells (wild-type or with suppressed APE1) were infected with Helicobacter pylori or Salmonella enterica and assessed for Rac1 and NADPH oxidase-dependent superoxide production. Rac1 and APE1 interactions were measured by co-immunoprecipitation, confocal microscopy and proximity ligation assay (PLA) in cell lines or in biopsy specimens. Significantly greater levels of ROS were produced by APE1-deficient human gastric and colonic cell lines and primary gastric epithelial cells compared to control cells after infection with either gastric or enteric pathogens. H. pylori activated Rac1 and Nox1 in all cell types, but activation was higher in APE1 suppressed cells. APE1 overexpression decreased H. pylori-induced ROS generation, Rac1 activation, and Nox1 expression. We determined that the effects of APE1 were mediated through its N-terminal lysine residues interacting with Rac1, leading to inhibition of Nox1 expression and ROS generation. APE1 is a negative regulator of oxidative stress in the gastrointestinal epithelium during bacterial infection by modulating Rac1 and Nox1. Our results implicate APE1 in novel molecular interactions that regulate early stress responses elicited by microbial infections.
Assuntos
DNA Liase (Sítios Apurínicos ou Apirimidínicos)/metabolismo , Mucosa Gástrica/imunologia , Infecções por Helicobacter/imunologia , Mucosa Intestinal/imunologia , Infecções por Salmonella/imunologia , Proteínas rac1 de Ligação ao GTP/metabolismo , Western Blotting , Linhagem Celular , Imunofluorescência , Mucosa Gástrica/metabolismo , Mucosa Gástrica/microbiologia , Infecções por Helicobacter/metabolismo , Humanos , Imunoprecipitação , Mucosa Intestinal/metabolismo , Mucosa Intestinal/microbiologia , Microscopia Confocal , Espécies Reativas de Oxigênio/imunologia , Espécies Reativas de Oxigênio/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Infecções por Salmonella/metabolismo , Proteínas rac1 de Ligação ao GTP/imunologiaRESUMO
OBJECTIVE: To prepare rabbit polyclonal antibody (pAb) against Ras-related C3 botulinum toxin substrate 1 (Rac1) and identify the functions of the antibody. METHODS: The Rac1 fragment was amplified from cDNA of human cervical cancer HeLa cells, and then inserted into the vector pET-32a to construct the recombinant plasmid pET-32a-Rac1. The recombinant protein Rac1 was fused with His-tag and the fusion protein His-Rac1 was expressed in E.coli expression system. Thereafter, His-tagged Rac1 was purified to immunize rabbits as immunogen. The titer and specificity of rabbit polyclonal antibody against Rac1 were identified by ELISA, Western blotting and immunohistochemistry, respectively. RESULTS: The fusion protein His-Rac1 was successfully expressed in E.coli. The pAb could specifically detect Rac1 in human HeLa cells and MCF-7 cells in Western blotting. Moreover, the pAb could specifically recognized Rac1 protein in human kidney tissues. CONCLUSION: The pAb of human Rac1 protein was successfully prepared, which could specifically recognize Rac1 protein in several natural samples.