RESUMO
Myddosome is an oligomeric complex required for the transmission of inflammatory signals from TLR/IL1Rs and consists of MyD88 and IRAK family kinases. However, the molecular basis for the self-assemble of Myddosome proteins and regulation of intracellular signaling remains poorly understood. Here, we identify OTUD5 acts as an essential regulator for MyD88 oligomerization and Myddosome formation. OTUD5 directly interacts with MyD88 and cleaves its K11-linked polyubiquitin chains at Lys95, Lys231 and Lys250. This polyubiquitin cleavage enhances MyD88 oligomerization after LPS stimulation, which subsequently promotes the recruitment of downstream IRAK4 and IRAK2 to form Myddosome and the activation of NF-κB and MAPK signaling and production of inflammatory cytokines. Consistently, Otud5-deficient mice are less susceptible to LPS- and CLP-induced sepsis. Taken together, our findings reveal a positive regulatory role of OTUD5 in MyD88 oligomerization and Myddosome formation, which provides new sights into the treatment of inflammatory diseases.
Assuntos
Inflamação , Fator 88 de Diferenciação Mieloide , Animais , Humanos , Camundongos , Células HEK293 , Inflamação/metabolismo , Inflamação/patologia , Quinases Associadas a Receptores de Interleucina-1/metabolismo , Quinases Associadas a Receptores de Interleucina-1/genética , Lipopolissacarídeos/farmacologia , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fator 88 de Diferenciação Mieloide/metabolismo , Transdução de SinaisRESUMO
Fungal infections have emerged as a major concern among immunocompromised patients, causing approximately 2 million deaths each year worldwide. However, the regulatory mechanisms underlying antifungal immunity remain elusive and require further investigation. The E3 ligase Trim26 belongs to the tripartite motif (Trim) protein family, which is involved in various biological processes, including cell proliferation, antiviral innate immunity, and inflammatory responses. Herein, we report that Trim26 exerts protective antifungal immune functions after fungal infection. Trim26-deficient mice are more susceptible to fungemia than their wild-type counterparts. Mechanistically, Trim26 restricts inflammatory neutrophils infiltration and limits proinflammatory cytokine production, which can attenuate kidney fungal load and renal damage during Candida infection. Trim26-deficient neutrophils showed higher proinflammatory cytokine expression and impaired fungicidal activity. We further demonstrated that excessive neutrophils infiltration in the kidney was because of the increased production of chemokines CXCL1 and CXCL2, which are mainly synthesized in the macrophages or dendritic cells of Trim26-deficient mice after Candida albicans infections. Together, our study findings unraveled the vital role of Trim26 in regulating antifungal immunity through the regulation of inflammatory neutrophils infiltration and proinflammatory cytokine and chemokine expression during candidiasis.
Assuntos
Candidíase , Neutrófilos , Animais , Camundongos , Antifúngicos , Candida albicans , Candidíase/metabolismo , Candidíase/microbiologia , Citocinas/metabolismo , Infiltração de Neutrófilos , Proteínas com Motivo Tripartido , Ubiquitina-Proteína LigasesRESUMO
The enzyme cyclic GMP-AMP synthase (cGAS) is a key sensor for detecting misplaced double-stranded DNA (dsDNA) of genomic, mitochondrial, and microbial origin. It synthesizes 2'3'-cGAMP, which in turn activates the stimulator of interferon genes pathway, leading to the initiation of innate immune responses. Here, we identified Listerin as a negative regulator of cGAS-mediated innate immune response. We found that Listerin interacts with cGAS on endosomes and promotes its K63-linked ubiquitination through recruitment of the E3 ligase TRIM27. The polyubiquitinated cGAS is then recognized by the endosomal sorting complexes required for transport machinery and sorted into endosomes for degradation. Listerin deficiency enhances the innate antiviral response to herpes simplex virus 1 infection. Genetic deletion of Listerin also deteriorates the neuroinflammation and the ALS disease progress in an ALS mice model; overexpression of Listerin can robustly ameliorate disease progression in ALS mice. Thus, our work uncovers a mechanism for cGAS regulation and suggests that Listerin may be a promising therapeutic target for ALS disease.
Assuntos
Esclerose Lateral Amiotrófica , Ubiquitina-Proteína Ligases , Animais , Camundongos , Esclerose Lateral Amiotrófica/tratamento farmacológico , Esclerose Lateral Amiotrófica/imunologia , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Imunidade Inata/genética , Nucleotidiltransferases/metabolismo , Proteólise , Transdução de Sinais/fisiologia , Modelos Animais de Doenças , Ubiquitina-Proteína Ligases/antagonistas & inibidores , Ubiquitina-Proteína Ligases/imunologia , Ubiquitina-Proteína Ligases/metabolismoRESUMO
STING (stimulator of interferon genes) exerts protective cellular responses to viral infection via induction of interferon production and autophagy. Here, we report the role of STING in modulating the immune responses toward fungal infection. Upon Candida albicans stimulation, STING transited alongside the endoplasmic reticulum (ER) to the phagosomes. In phagosomes, STING directly bound with Src via the N-terminal 18 amino acids of STING, and this binding prevented Src from recruiting and phosphorylating Syk. Consistently, Syk-associated signaling and production of pro-inflammatory cytokines and chemokines were increased in mouse BMDCs (bone-marrow-derived dendritic cells) lacking STING with fungal treatment. STING deficiency improved anti-fungal immunity in systemic C. albicans infection. Importantly, administration of the N-terminal 18-aa (amino acid) peptide of STING improved host outcomes in disseminated fungal infection. Overall, our study identifies a previously unrecognized function of STING in negatively regulating anti-fungal immune responses and offers a potential therapeutic strategy for controlling C. albicans infection.
Assuntos
Nucleotídeos , Transdução de Sinais , Animais , Camundongos , Citocinas/metabolismo , Imunidade Inata , Interferons/metabolismo , Nucleotídeos/metabolismo , Fagossomos/metabolismo , Fagossomos/microbiologiaRESUMO
Objective To express and purify the dormancy survival regulator Rv2628 of Mycobacterium tuberculosis, so as to prepare and identify its rabbit polyclonal antibody. Methods Using the genome of Mycobacterium tuberculosis H37Rv strain as a template, the Rv2628 gene was amplified to construct a recombinant expression plasmid which was transformed into an Escherichia coli protein expression strain and induced expression by IPTG. The target protein was purified using Ni-NTA chromatography column, and the rabbit polyclonal antibody was obtained by immunizing New Zealand white rabbits with purified Rv2628 protein. The specificity of polyclonal antibodies was verified by Western blot analysis and indirect ELISA, respectively. Results The PET-30A-RV2628 recombinant carrier was successfully constructed. After induction by IPTG, the RV2628 protein was mainly expressed in the form of inclusion. The high-purity Rv2628 protein was obtained by Ni-NTA column purification. Rabbit anti-Rv2628 polyclonal antibody was obtained after immunizing the rabbit. The antibody had good antigen binding properties and the antibody titer reached 1:1 093 500. Conclusion The high-purity Rv2628 protein and high-titer rabbit anti-Rv2628 polyclonal antibody were successfully prepared.
Assuntos
Mycobacterium tuberculosis , Coelhos , Animais , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/metabolismo , Isopropiltiogalactosídeo , Anticorpos , Ensaio de Imunoadsorção Enzimática , Western Blotting , Especificidade de AnticorposRESUMO
The receptor tyrosine kinase EPHB2 (EPH receptor B2) is highly expressed in many human cancer types, especially in gastrointestinal cancers, such as colorectal cancer. Several coding mutations of the EPHB2 gene have been identified in many cancer types, suggesting that EPHB2 plays a critical role in carcinogenesis. However, the exact functional mechanism of EPHB2 in carcinogenesis remains unknown. In this study, we find that EPHB2 is required for TNF-induced signaling activation and proinflammatory cytokine production in colorectal epithelial cells. Mechanistically, after TNF stimulation, EPHB2 is ubiquitinated by its E3 ligase RNF186. Then, ubiquitinated EPHB2 recruits and further phosphorylates TAB2 at nine tyrosine sites, which is a critical step for the binding between TAB2 and TAK1. Due to defects in TNF signaling in RNF186-knockout colorectal epithelial cells, the phenotype of colitis-propelled colorectal cancer model in RNF186-knockout mice is significantly reduced compared with that in wild-type control mice. Moreover, we find that a genetic mutation in EPHB2 identified in a family with colorectal cancer is a gain-of-function mutation that promoted TNF signaling activation compared with wild-type EPHB2. We provide evidence that the EPHB2-RNF186-TAB2-TAK1 signaling cascade plays an essential role in TNF-mediated signal transduction in colorectal epithelial cells and the carcinogenesis of colorectal cancer, which may provide potential targets for the treatment of colorectal cancer.
Assuntos
Neoplasias Colorretais , Receptor EphA1 , Animais , Humanos , Camundongos , Carcinogênese , Neoplasias Colorretais/genética , Citocinas , Células Epiteliais/metabolismo , Receptor EphA1/metabolismo , Transdução de Sinais , Tirosina , Ubiquitina/metabolismo , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo , Receptor EphB2RESUMO
Fungal infections cause ~1.5 million deaths each year worldwide, and the mortality rate of disseminated candidiasis currently exceeds that of breast cancer and malaria. The major reasons for the high mortality of candidiasis are the limited number of antifungal drugs and the emergence of drug-resistant species. Therefore, a better understanding of antifungal host defense mechanisms is crucial for the development of effective preventive and therapeutic strategies. Here, we report that DOCK2 (dedicator of cytokinesis 2) promotes indispensable antifungal innate immune signaling and proinflammatory gene expression in macrophages. DOCK2-deficient macrophages exhibit decreased RAC GTPase (Rac family small GTPase) activation and ROS (reactive oxygen species) production, which in turn attenuates the killing of intracellular fungi and the activation of downstream signaling pathways. Mechanistically, after fungal stimulation, activated SYK (spleen-associated tyrosine kinase) phosphorylates DOCK2 at tyrosine 985 and 1405, which promotes the recruitment and activation of RAC GTPases and then increases ROS production and downstream signaling activation. Importantly, nanoparticle-mediated delivery of in vitro transcribed (IVT) Rac1 mRNA promotes the activity of Rac1 and helps to eliminate fungal infection in vivo. Taken together, this study not only identifies a critical role of DOCK2 in antifungal immunity via regulation of RAC GTPase activity but also provides proof of concept for the treatment of invasive fungal infections by using IVT mRNA.
Assuntos
Candidíase , Proteínas Ativadoras de GTPase , Fatores de Troca do Nucleotídeo Guanina , Imunidade Inata , Proteínas rac de Ligação ao GTP , Animais , Candidíase/imunologia , Proteínas Ativadoras de GTPase/metabolismo , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Humanos , Camundongos , Camundongos Endogâmicos C57BL , RNA Mensageiro , Espécies Reativas de Oxigênio/metabolismo , Proteínas rac de Ligação ao GTP/metabolismoRESUMO
Therapeutic blockade of the immune checkpoint proteins programmed cell death protein 1 (PD-1) and cytotoxic T lymphocyte antigen 4 (CTLA4) has transformed cancer treatment. However, the overall response rate to these treatments is low, suggesting that immune checkpoint activation is not the only mechanism leading to dysfunctional anti-tumour immunity. Here we show that butyrophilin-like protein 2 (BTNL2) is a potent suppressor of the anti-tumour immune response. Antibody-mediated blockade of BTNL2 attenuates tumour progression in multiple in vivo murine tumour models, resulting in prolonged survival of tumour-bearing mice. Mechanistically, BTNL2 interacts with local γδ T cell populations to promote IL-17A production in the tumour microenvironment. Inhibition of BTNL2 reduces the number of tumour-infiltrating IL-17A-producing γδ T cells and myeloid-derived suppressor cells, while facilitating cytotoxic CD8+ T cell accumulation. Furthermore, we find high BTNL2 expression in several human tumour samples from highly prevalent cancer types, which negatively correlates with overall patient survival. Thus, our results suggest that BTNL2 is a negative regulator of anti-tumour immunity and a potential target for cancer immunotherapy.
Assuntos
Butirofilinas/genética , Butirofilinas/metabolismo , Interleucina-17/metabolismo , Linfócitos T/metabolismo , Evasão Tumoral/fisiologia , Animais , Linfócitos T CD8-Positivos/imunologia , Antígeno CTLA-4 , Feminino , Expressão Gênica , Células HEK293 , Humanos , Imunoterapia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neoplasias , Linfócitos T Citotóxicos/imunologia , Microambiente TumoralRESUMO
It is well known that exosomes could serve as anti-microbial immune factors in animals. However, despite growing evidences have shown that the homeostasis of the hemolymph microbiota was vital for immune regulation in crustaceans, the relationship between exosomes and hemolymph microbiota homeostasis during pathogenic bacteria infection has not been addressed. Here, we reported that exosomes released from Vibrio parahaemolyticus-infected mud crabs (Scylla paramamosain) could help to maintain the homeostasis of hemolymph microbiota and have a protective effect on the mortality of the host during the infection process. We further confirmed that miR-224 was densely packaged in these exosomes, resulting in the suppression of HSP70 and disruption of the HSP70-TRAF6 complex, then the released TRAF6 further interacted with Ecsit to regulate the production of mitochondrial ROS (mROS) and the expression of Anti-lipopolysaccharide factors (ALFs) in recipient hemocytes, which eventually affected hemolymph microbiota homeostasis in response to the pathogenic bacteria infection in mud crab. To the best of our knowledge, this is the first document that reports the role of exosome in the hemolymph microbiota homeostasis modulation during pathogen infection, which reveals the crosstalk between exosomal miRNAs and innate immune response in crustaceans.
Assuntos
Proteínas de Artrópodes/metabolismo , Braquiúros/imunologia , Exossomos/genética , Hemolinfa/imunologia , Imunidade Inata/imunologia , MicroRNAs/genética , Vibrioses/imunologia , Animais , Proteínas de Artrópodes/genética , Braquiúros/microbiologia , Perfilação da Expressão Gênica , Hemócitos/imunologia , Hemócitos/metabolismo , Hemócitos/microbiologia , Hemolinfa/metabolismo , Hemolinfa/microbiologia , Homeostase , Microbiota , Filogenia , Vibrioses/microbiologia , Vibrio parahaemolyticus/fisiologiaRESUMO
The successive emergences and accelerating spread of novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) lineages and evolved resistance to some ongoing clinical therapeutics increase the risks associated with the coronavirus disease 2019 (COVID-19) pandemic. An urgent intervention for broadly effective therapies to limit the morbidity and mortality of COVID-19 and future transmission events from SARS-related coronaviruses (SARSr-CoVs) is needed. Here, we isolate and humanize an angiotensin-converting enzyme-2 (ACE2)-blocking monoclonal antibody (MAb), named h11B11, which exhibits potent inhibitory activity against SARS-CoV and circulating global SARS-CoV-2 lineages. When administered therapeutically or prophylactically in the hACE2 mouse model, h11B11 alleviates and prevents SARS-CoV-2 replication and virus-induced pathological syndromes. No significant changes in blood pressure and hematology chemistry toxicology were observed after injections of multiple high dosages of h11B11 in cynomolgus monkeys. Analysis of the structures of the h11B11/ACE2 and receptor-binding domain (RBD)/ACE2 complexes shows hindrance and epitope competition of the MAb and RBD for the receptor. Together, these results suggest h11B11 as a potential therapeutic countermeasure against SARS-CoV, SARS-CoV-2, and escape variants.
Assuntos
Enzima de Conversão de Angiotensina 2/efeitos dos fármacos , Enzima de Conversão de Angiotensina 2/imunologia , Anticorpos Neutralizantes/administração & dosagem , Tratamento Farmacológico da COVID-19 , SARS-CoV-2/efeitos dos fármacos , Animais , Anticorpos Monoclonais/administração & dosagem , Anticorpos Monoclonais/imunologia , Anticorpos Neutralizantes/imunologia , COVID-19/imunologia , COVID-19/mortalidade , COVID-19/virologia , Chlorocebus aethiops , Modelos Animais de Doenças , Epitopos , Feminino , Células HEK293 , Haplorrinos , Humanos , Macaca fascicularis , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Pandemias , SARS-CoV-2/imunologia , SARS-CoV-2/isolamento & purificação , Células Vero , Ativação ViralRESUMO
Opportunistic fungal infections have become one of the leading causes of death among immunocompromised patients, resulting in an estimated 1.5 million deaths each year worldwide. The molecular mechanisms that promote host defense against fungal infections remain elusive. Here, we find that Myosin IF (MYO1F), an unconventional myosin, promotes the expression of genes that are critical for antifungal innate immune signaling and proinflammatory responses. Mechanistically, MYO1F is required for dectin-induced α-tubulin acetylation, acting as an adaptor that recruits both the adaptor AP2A1 and α-tubulin N-acetyltransferase 1 to α-tubulin; in turn, these events control the membrane-to-cytoplasm trafficking of spleen tyrosine kinase and caspase recruitment domain-containing protein 9 Myo1f-deficient mice are more susceptible than their wild-type counterparts to the lethal sequelae of systemic infection with Candida albicans Notably, administration of Sirt2 deacetylase inhibitors, namely AGK2, AK-1, or AK-7, significantly increases the dectin-induced expression of proinflammatory genes in mouse bone marrow-derived macrophages and microglia, thereby protecting mice from both systemic and central nervous system C. albicans infections. AGK2 also promotes proinflammatory gene expression in human peripheral blood mononuclear cells after Dectin stimulation. Taken together, our findings describe a key role for MYO1F in promoting antifungal immunity by regulating the acetylation of α-tubulin and microtubules, and our findings suggest that Sirt2 deacetylase inhibitors may be developed as potential drugs for the treatment of fungal infections.
Assuntos
Candida albicans/fisiologia , Candidíase/imunologia , Imunidade Inata/imunologia , Leucócitos Mononucleares/imunologia , Microtúbulos/imunologia , Miosina Tipo I/metabolismo , Miosina Tipo I/fisiologia , Acetilação , Animais , Antifúngicos/farmacologia , Candidíase/tratamento farmacológico , Candidíase/metabolismo , Candidíase/microbiologia , Humanos , Leucócitos Mononucleares/efeitos dos fármacos , Leucócitos Mononucleares/metabolismo , Leucócitos Mononucleares/microbiologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microtúbulos/efeitos dos fármacos , Microtúbulos/metabolismo , Microtúbulos/microbiologia , Miosina Tipo I/genética , Transdução de SinaisRESUMO
IL-17A plays an essential role in the pathogenesis of many autoimmune diseases, including psoriasis and multiple sclerosis. Act1 is a critical adaptor in the IL-17A signaling pathway. In this study, we report that an anti-sense long noncoding RNA, TRAF3IP2-AS1, regulates Act1 expression and IL-17A signaling by recruiting SRSF10, which downregulates the expression of IRF1, a transcriptional factor of Act1. Interestingly, we found that a psoriasis-susceptible variant of TRAF3IP2-AS1 A4165G (rs13210247) is a gain-of-function mutant. Furthermore, we identified a mouse gene E130307A14-Rik that is homologous to TRAF3IP2-AS1 and has a similar ability to regulate Act1 expression and IL-17A signaling. Importantly, treatment with lentiviruses expressing E130307A14-Rik or SRSF10 yielded therapeutic effects in mouse models of psoriasis and experimental autoimmune encephalomyelitis. These findings suggest that TRAF3IP2-AS1 and/or SRSF10 may represent attractive therapeutic targets in the treatment of IL-17-related autoimmune diseases, such as psoriasis and multiple sclerosis.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas de Ciclo Celular/metabolismo , Encefalomielite Autoimune Experimental/metabolismo , Fator Regulador 1 de Interferon/metabolismo , Interleucina-17/metabolismo , Psoríase/metabolismo , RNA Longo não Codificante/metabolismo , RNA/metabolismo , Proteínas Repressoras/metabolismo , Fatores de Processamento de Serina-Arginina/metabolismo , Transdução de Sinais/genética , Animais , Proteínas de Ciclo Celular/genética , Técnicas de Inativação de Genes , Células HaCaT , Células HeLa , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , RNA/genética , RNA Longo não Codificante/genética , Proteínas Repressoras/genética , Fatores de Processamento de Serina-Arginina/genética , TransfecçãoRESUMO
Invasive fungal infections have become a leading cause of death among immunocompromised patients, leading to around 1.5 million deaths per year globally. The molecular mechanisms by which hosts defend themselves against fungal infection remain largely unclear, which impedes the development of antifungal drugs and other treatment options. In this article, we show that the tyrosine kinase receptor EPH receptor B2 (EPHB2), together with dectin-1, recognizes ß-glucan and activates downstream signaling pathways. Mechanistically, we found that EPHB2 is a kinase for Syk and is required for Syk phosphorylation and activation after dectin-1 ligand stimulation, whereas dectin-1 is critical for the recruitment of Syk. Ephb2-deficient mice are susceptible to Candida albicans-induced fungemia model, which also supports the role of EPHB2 in antifungal immunity. Overall, we provide evidence that EPHB2 is a coreceptor for the recognition of dectin-1 ligands and plays an essential role in antifungal immunity by phosphorylating Syk.
Assuntos
Candida albicans/fisiologia , Candidíase/imunologia , Receptor EphB2/metabolismo , Animais , Modelos Animais de Doenças , Interações Hospedeiro-Patógeno , Humanos , Lectinas Tipo C/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fosforilação , Receptor EphB2/genética , Transdução de Sinais , Quinase Syk/metabolismo , Células THP-1RESUMO
Although genome-wide association studies have identified the gene RNF186 encoding an E3 ubiquitin-protein ligase as conferring susceptibility to ulcerative colitis, the exact function of this protein remains unclear. In the present study, we demonstrate an important role for RNF186 in macroautophagy/autophagy activation in colonic epithelial cells and intestinal homeostasis. Mechanistically, RNF186 acts as an E3 ubiquitin-protein ligase for EPHB2 and regulates the ubiquitination of EPHB2. Upon stimulation by ligand EFNB1 (ephrin B1), EPHB2 is ubiquitinated by RNF186 at Lys892, and further recruits MAP1LC3B for autophagy. Compared to control mice, rnf186-/- and ephb2-/- mice have a more severe phenotype in the DSS-induced colitis model, which is due to a defect in autophagy in colon epithelial cells. More importantly, treatment with ephrin-B1-Fc recombinant protein effectively relieves DSS-induced mouse colitis, which suggests that ephrin-B1-Fc may be a potential therapy for human inflammatory bowel diseases.Abbreviations: ACTB: actin beta; ATG5: autophagy related 5; ATG16L1: autophagy related 16 like 1; ATP: adenosine triphosphate; Cas9: CRISPR associated protein 9; CD: Crohn disease; CQ: chloroquine; Csf2: colony stimulating factor 2; Cxcl1: c-x-c motif chemokine ligand 1; DMSO: dimethyl sulfoxide; DSS: dextran sodium sulfate; EFNB1: ephrin B1; EPHB2: EPH receptor B2; EPHB3: EPH receptor B3; EPHB2K788R: lysine 788 mutated to arginine in EPHB2; EPHB2K892R: lysine 892 mutated to arginine in EPHB2; ER: endoplasmic reticulum; FITC: fluorescein isothiocyanate; GFP: green fluorescent protein; GWAS: genome-wide association studies; HRP: horseradish peroxidase; HSPA5/BiP: heat shock protein family A (Hsp70) member 5; IBD: inflammatory bowel diseases; Il1b: interleukin 1 beta; Il6: interleukin 6; IRGM:immunity related GTPase M; i.p.: intraperitoneally; IPP: inorganic pyrophosphatase; KD: knockdown; KO: knockout; MAP1LC3B: microtubule associated protein 1 light chain 3 beta; MTOR: mechanistic target of rapamycin kinase; NOD2: nucleotide binding oligomerization domain containing 2; PI3K: phosphoinositide 3-kinase; PtdIns3K: class III phosphatidylinositol 3-kinase; RNF186: ring finger protein 186; RNF186A64T: alanine 64 mutated to threonine in RNF186; RNF186R179X: arginine 179 mutated to X in RNF186; RPS6: ribosomal protein S6; Tnf: tumor necrosis factor; SQSTM1: sequestosome 1; Ub: ubiquitin; UBE2D2: ubiquitin conjugating enzyme E2 D2; UBE2H: ubiquitin conjugating enzyme E2 H; UBE2K: ubiquitin conjugating enzyme E2 K; UBE2N: ubiquitin conjugating enzyme E2 N; UC: ulcerative colitis; ULK1:unc-51 like autophagy activating kinase 1; WT: wild type.
Assuntos
Autofagia , Efrina-B1 , Ubiquitina-Proteína Ligases , Animais , Autofagia/genética , Classe III de Fosfatidilinositol 3-Quinases/metabolismo , Colo , Células Epiteliais/metabolismo , Estudo de Associação Genômica Ampla , Homeostase , Camundongos , Receptor EphB2 , Ubiquitina-Proteína Ligases/metabolismoRESUMO
The TAGAP gene locus has been linked to several infectious diseases or autoimmune diseases, including candidemia and multiple sclerosis. While previous studies have described a role of TAGAP in T cells, much less is known about its function in other cell types. Here we report that TAGAP is required for Dectin-induced anti-fungal signaling and proinflammatory cytokine production in myeloid cells. Following stimulation with Dectin ligands, TAGAP is phosphorylated by EPHB2 at tyrosine 310, which bridges proximal Dectin-induced EPHB2 activity to downstream CARD9-mediated signaling pathways. During Candida albicans infection, mice lacking TAGAP mount defective immune responses, impaired Th17 cell differentiation, and higher fungal burden. Similarly, in experimental autoimmune encephalomyelitis model of multiple sclerosis, TAGAP deficient mice develop significantly attenuated disease. In summary, we report that TAGAP plays an important role in linking Dectin-induced signaling to the promotion of effective T helper cell immune responses, during both anti-fungal host defense and autoimmunity.
Assuntos
Antifúngicos/imunologia , Candidíase/imunologia , Diferenciação Celular , Proteínas Ativadoras de GTPase/química , Proteínas Ativadoras de GTPase/metabolismo , Receptor EphB2/metabolismo , Transdução de Sinais/efeitos dos fármacos , Células Th17/metabolismo , Animais , Antifúngicos/farmacologia , Proteínas Adaptadoras de Sinalização CARD/metabolismo , Citocinas/metabolismo , Modelos Animais de Doenças , Encefalomielite Autoimune Experimental/imunologia , Encefalomielite Autoimune Experimental/microbiologia , Feminino , Proteínas Ativadoras de GTPase/genética , Humanos , Lectinas Tipo C/metabolismo , Masculino , Camundongos Knockout , Esclerose Múltipla/complicações , Esclerose Múltipla/imunologia , Fosforilação , Receptor EphB2/imunologia , Receptores Imunológicos , Receptores de Reconhecimento de Padrão/metabolismo , Células Th17/imunologiaRESUMO
Serine proteases (SPs) are important in various immune responses, including prophenoloxidase (proPO) activation, antimicrobial peptides (AMPs) synthesis, and hemolymph coagulation in invertebrates. In this study, SP3 and SP5 of mud crab (Scylla paramamosain) were studied. SP3 and SP5 were expressed in all examined tissues (mainly in hemocytes), and are associated with the immune responses of mud crab to Vibrio parahemolyticus and Staphylococcus aureus, as well as interacted with TRAF6, and are involved in the activation of anti-lipopolysaccharide factors (ALFs) probably through the TLR/NF-κB pathway. Depletion of SP3 inhibited the expression of ALF1, ALF2, ALF3, and ALF6, while knockdown of SP5 significantly decreased ALF5, and ALF6. Furthermore, both SP5 and TRAF6 regulated the PO activity in the hemolymph of mud crab. Overexpression assay showed that both SP3 and SP5 could enhance the promoter activities of ALFs in mud crab. Taken together, the results of this study indicate that SP3 and SP5 might play important roles in the immune system of mud crab against pathogen invasion.
Assuntos
Regulação da Expressão Gênica/imunologia , Imunidade Inata/genética , Penaeidae/genética , Penaeidae/imunologia , Serina Proteases/genética , Serina Proteases/imunologia , Sequência de Aminoácidos , Animais , Peptídeos Catiônicos Antimicrobianos , Proteínas de Artrópodes/química , Proteínas de Artrópodes/genética , Proteínas de Artrópodes/imunologia , Catecol Oxidase/genética , Catecol Oxidase/metabolismo , Precursores Enzimáticos/genética , Precursores Enzimáticos/metabolismo , Perfilação da Expressão Gênica , Filogenia , Alinhamento de Sequência , Serina Proteases/química , Staphylococcus aureus/fisiologia , Fator 6 Associado a Receptor de TNF/genética , Fator 6 Associado a Receptor de TNF/metabolismo , Vibrio parahaemolyticus/fisiologiaRESUMO
Tolls and Toll-like receptors (TLRs) were the first pattern recognition receptors (PRRs) identified to play key roles in host innate immunity. However, relatively little is known about other types of Toll-like receptors in Scylla paramamosain, although a Toll-like receptor (SpToll1) has recently been cloned. In this study, we cloned and characterized another novel Toll-like receptor 2 (SpToll2) from S. paramamosain. The full-length cDNA of SpToll2 is 3391 bp with a 2646 bp open reading frame (ORF) encoding a putative protein of 881 amino acids, and predicted to contain six extracellular leucine-rich repeat (LRR) domains, a transmembrane domain and an intracellular Toll/IL-1 receptor (TIR) domain. Phylogenetic analysis revealed that SpToll2 clustered with Drosophila Toll1, and shared high homology with PtToll4. Real-time qPCR analysis showed that SpToll2 was widely expressed in all tissues tested, with the highest level found in hemocytes and hepatopancreas while the lowest in heart and muscle. The transcript levels of both SpToll1 and SpToll2 in mud crabs hemocytes was induced following challenge with Vibrio parahaemolyticus, Staphylococcus aureus, Polyinosinic: polycytidylic acid (Poly I:C) and white spot syndrome virus (WSSV). In addition, recombinant SpToll1-LRR and SpToll2-LRR proteins could bind to V. parahaemolyticus, S. aureus, Escherichia coli, and Beta Streptococcus. In order to study the signaling pathway of AMPs' expression in mud crab, RNA interference were used to test the expression of SpAMPs after the challenges with V. parahaemolyticus or S. aureus. The data suggested that SpToll1and SpToll2 could regulate the transcripts of several AMPs and four immune related mediators (SpMyD88, SpTube, SpPelle and SpTRAF6) at different scale. While silencing of SpToll1 post pathogens challenge attenuated the expression of SpHistin, SpALF1 and SpALF5 in mud crab's hemocytes, depletion of SpToll2 post pathogens challenge inhibited the expression of SpALF1-6, SpGRP, SpArasin and SpHyastastin. Furthermore, the results of overexpression assay also showed SpToll1 and SpToll2 could enhance the promoter activities of SpALFs in mud crab. Taken together, these results indicated that SpToll1 and SpToll2 might play important roles in host defense against pathogen invasions in S. paramamosain.
Assuntos
Peptídeos Catiônicos Antimicrobianos/genética , Proteínas de Artrópodes/genética , Braquiúros/genética , Receptor 1 Toll-Like/genética , Receptor 2 Toll-Like/genética , Animais , Peptídeos Catiônicos Antimicrobianos/metabolismo , Proteínas de Artrópodes/metabolismo , Braquiúros/microbiologia , Linhagem Celular , Clonagem Molecular , Perfilação da Expressão Gênica , Hemócitos/metabolismo , Hemócitos/microbiologia , Hepatopâncreas/metabolismo , Hepatopâncreas/microbiologia , Interações Hospedeiro-Patógeno , Filogenia , Staphylococcus aureus/fisiologia , Receptor 1 Toll-Like/classificação , Receptor 1 Toll-Like/metabolismo , Receptor 2 Toll-Like/classificação , Receptor 2 Toll-Like/metabolismo , Vibrio parahaemolyticus/fisiologiaRESUMO
As pattern recognition receptors, C-type lectins (CTLs) play important roles in immune system of crustaceans through identifying and binding to the conservative pathogen-associated molecular patterns (PAMPs) on pathogen surfaces. In this study, a new CTL, SpCTL-B, was identified from the hemocytes of mud crab Scylla paramamosain. The full-length of SpCTL-B cDNA was 1278 bp with an open reading frame (ORF) of 348 bp. The predicted SpCTL-B protein contains a single carbohydrate-recognition domain (CRD). SpCTL-B transcripts were distributed in all examined tissues with the highest levels in hepatopancreas. After challenged with Vibrio parahaemolyticus, LPS, polyI:C and white spot syndrome virus (WSSV), the mRNA levels of SpCTL-B in hemocytes and hepatopancreas were up-regulated. The recombinant SpCTL-B (rSpCTL-B) purified by Ni-affinity chromatography showed stronger binding activities with Staphylococcus aureus, ß-hemolytic Streptococcus, Escherichia coli, Aeromonas hydrophila, Vibrio alginolyticus than those with V. parahaemolyticus and Saccharomyces cerevisiae. rSpCTL-B exhibited a broad spectrum of microorganism-agglutination activities against Gram-positive bacteria (S. aureus, ß-hemolytic Streptococcus) and Gram-negative bacteria (E. coli, V. parahaemolyticus, A. hydrophila, V. alginolyticus) in a Ca2+-dependent manner. The agglutination activities of rSpCTL-B could be inhibited by D-mannose and LPS, but not by d-fructose and galactose. The antimicrobial assay showed that rSpCTL-B exhibited the growth inhibition against all examined gram-positive bacteria and gram-negative bacteria. When SpCTL-B was silenced by RNAi, the bacterial clearance ability in mud crab was decreased and the transcript levels of five antimicrobial peptides (AMPs) (SpCrustin, SpHistin, SpALF4 (anti-lipopolysaccharide factor), SpALF5 and SpALF6) were significantly decreased in hemocytes. In our study, knockdown of SpCTL-B could down-regulate the expression of SpSTAT at mRNA transcriptional level and protein translational level in mud crab. Meantime, the phagocytosis rate and the expression of three phagocytosis related genes were declined after RNAi of SpCTL-B in hemocytes in mud crab. Collectively, our results suggest that SpCTL-B might play its roles as a pattern recognition receptor (PRR) in immune response towards pathogens infection through influencing the expression of AMPs and the phagocytosis of hemocytes in mud crab S. paramamosain.
Assuntos
Proteínas de Artrópodes/metabolismo , Bactérias/imunologia , Infecções Bacterianas/imunologia , Braquiúros/imunologia , Hemócitos/fisiologia , Lectinas Tipo C/metabolismo , Receptores de Reconhecimento de Padrão/metabolismo , Animais , Peptídeos Catiônicos Antimicrobianos/genética , Peptídeos Catiônicos Antimicrobianos/metabolismo , Proteínas de Artrópodes/genética , Carga Bacteriana/genética , Cálcio/metabolismo , Clonagem Molecular , Regulação da Expressão Gênica , Lectinas Tipo C/genética , Moléculas com Motivos Associados a Patógenos/metabolismo , Fagocitose , Ligação Proteica , RNA Interferente Pequeno/genética , Receptores de Reconhecimento de Padrão/genéticaRESUMO
As a member of the immunoglobulin superfamily, Down syndrome cell adhesion molecule (Dscam) could function in the innate immunity of invertebrates. Recently, it is shown that arthropod Dscams play similar functions as antibodies in the adaptive immune system. Dscam could produce thousands of isoforms by alternative splicing and specifically bind to various pathogens. In the present study, we cloned the first Dscam from mud crab Scylla paramamosain (SpDscam), with full-length cDNA 7363 bp containing an open reading frame (ORF) of 6069bp and encoding 2022 amino acids, which had typical domain architecture as other arthropods, i.e., 10 immunoglobulin domains (Ig), 6 fibronectin type 3 domains (FN III), transmembrane and cytoplasmic tail. Quantitative real-time PCR revealed that SpDscam was highly expressed in brain, skin, muscle, intestine and hepatopancreas, but weakly expressed in hemolymph, heart and gill. SpDscam had three alternative splicing regions, located at the N-terminal of Ig2 and Ig3 as well as on the whole Ig7. In these regions, 32, 41 and 14 exons were detected, together with the two exon types of transmembrane domain, indicating SpDscam could potentially encode at least 36,736 unique isoforms. SpDscam induced by Vibrio parahaemolyticus challenge had strong binding ability to V. parahaemolyticus. Further, SpDscam induced by V. parahaemolyticus possessed a clearance of V. parahaemolyticus in S. paramamosain. Collectively, the results indicated SpDscam was a hypervariable pattern-recognition receptor (PRR) by alternative splicing in innate immunity system of mud crab S. paramamosain.
Assuntos
Processamento Alternativo , Braquiúros/genética , Braquiúros/imunologia , Moléculas de Adesão Celular/genética , Moléculas de Adesão Celular/imunologia , Regulação da Expressão Gênica/imunologia , Imunidade Inata/genética , Filogenia , Sequência de Aminoácidos , Animais , Proteínas de Artrópodes/química , Proteínas de Artrópodes/genética , Proteínas de Artrópodes/imunologia , Sequência de Bases , Moléculas de Adesão Celular/química , Perfilação da Expressão Gênica , Alinhamento de Sequência , Vibrio parahaemolyticus/fisiologiaRESUMO
Tumor necrosis factor receptor-associated factor 6 (TRAF6) is a key cytoplasm signal adaptor that mediates signals activated by tumor necrosis factor receptor (TNFR) superfamily and the Interleukin-1 receptor/Toll-like receptor (IL-1/TLR) superfamily. The full-length 2492 bp TRAF6 (Sp-TRAF6) from Scylla paramamosain contains 1800 bp of open reading frame (ORF) encoding 598 amino acids, including an N-terminal RING-type zinc finger, two TRAF-type zinc fingers and a conserved C-terminal meprin and TRAF homology (MATH) domain. Multiple alignment analysis shows that the putative amino acid sequence of Sp-TRAf6 has highest identity of 88% with Pt-TRAF6 from Portunus trituberculatus, while the similarity of Sp-TRAF6 with other crustacean sequences was 54-55%. RT-PCR analysis indicated that Sp-TRAF6 transcripts were predominantly expressed in the hepatopancreas and stomach, whereas it was barely detected in the heart and hemocytes in our study. Moreover, Sp-TRAF6 transcripts were significantly up-regulated after Vibrio parahemolyticus and LPS challenges. RNA interference assay was carried out used by siRNA to investigate the genes expression patterns regulated by Sp-TRAF6. The qRT-PCR results showed that silencing Sp-TRAF6 gene could inhibit SpALF1, SpALF2, SpALF5 and SpALF6 expression in hemocytes, while inhibit SpALF1, SpALF3, SpALF4, SpALF5 and SpALF6 expression in hepatopancreas. Taken together, the acute-phase response to immune challenges and the inhibition of SpALFs gene expression indicate that Sp-TRAF6 plays an important role in host defense against pathogen invasions via regulation of ALF gene expression in S. paramamosain.