RESUMO
Hypertension is accompanied by dysbiosis and a decrease in the relative abundance of short-chain fatty acid (SCFA)-producing bacteria. However, there is no report to examine the role of C. butyricum in blood pressure regulation. We hypothesized that a decrease in the relative abundance of SCFA-producing bacteria in the gut was the cause of spontaneously hypertensive rats (SHR)-induced hypertension. C. butyricum and captopril were used to treat adult SHR for six weeks. C. butyricum modulated SHR-induced dysbiosis and significantly reduced systolic blood pressure (SBP) in SHR (p < 0.01). A 16S rRNA analysis determined changes in the relative abundance of the mainly SCFA-producing bacteria Akkermansia muciniphila, Lactobacillus amylovorus, and Agthobacter rectalis, which increased significantly. Total SCFAs, and particularly butyrate concentrations, in the SHR cecum and plasma were reduced (p < 0.05), while C. butyricum prevented this effect. Likewise, we supplemented SHR with butyrate for six weeks. We analyzed the flora composition, cecum SCFA concentration, and inflammatory response. The results showed that butyrate prevented SHR-induced hypertension and inflammation, and the decline of cecum SCFA concentrations (p < 0.05). This research revealed that increasing cecum butyrate concentrations by probiotics, or direct butyrate supplementation, prevented the adverse effects of SHR on intestinal flora, vascular, and blood pressure.
Assuntos
Clostridium butyricum , Hipertensão , Ratos , Animais , Pressão Sanguínea/fisiologia , Ratos Endogâmicos SHR , Disbiose/complicações , RNA Ribossômico 16S , Ácidos Graxos Voláteis , Butiratos/análiseRESUMO
Legionella pneumophila widely exists in natural and artificial water environments, which enables it to infect people. L. pneumophila infection causes Legionnaires' disease (LD), which is a significant but relatively uncommon respiratory infection. Approximately 90% of LD is caused by L. pneumophila serogroup 1 (Lp1). Meteorological conditions may affect the infectivity and virulence of Lp1, but the exact relationship between them is still unclear. In this study, we evaluated the virulence of Lp1 by screening of total 156 Lp1 strains isolated from cooling tower water in different regions of China by detecting their abilities to activate NF-κB signaling pathway in vitro. In addition, we screened the distribution of some selected virulence genes in these strains. The virulence, virulence gene distribution, and the meteorological factors were analyzed. We found that both the virulence and the distribution of virulence genes had a certain regional and meteorological correlation. Although the loss of several virulence genes showed significant effects on the virulence of Lp1 strains, the distribution of virulence genes had very limited effects on the virulence of Lp1. IMPORTANCE LD is likely to be underrecognized in many countries. Due to the widespread existence of L. pneumophila in natural and artificial water environments and to the lack of cross-protection against different strains, L. pneumophila is a potentially serious threat to human health. Therefore, effective monitoring of the virulence of L. pneumophila in the water environment is very important to prevent and control the prevalence of LD. Understanding the virulence of L. pneumophila can not only help us to predict the risk of possible outbreaks in advance but can also enable more targeted clinical treatment. This study highlights the importance of understanding the epidemiology and ecology of L. pneumophila isolated from public facilities in terms of public health and biology. Due to the potential for water sources to harbor and disseminate L. pneumophila and to the fact that geographical conditions influence the virulence of L. pneumophila, timely and accurate L. pneumophila virulence surveillance is urgently needed.
Assuntos
Legionella pneumophila , Doença dos Legionários , China/epidemiologia , Surtos de Doenças , Ecologia , Humanos , Doença dos Legionários/epidemiologia , Microbiologia da ÁguaRESUMO
Influenza virus matrix 1 protein (M1) is highly conserved and plays essential roles at many stages of virus life cycle. Here, we used a yeast two-hybrid system to identify the host protein SLD5, a component of the GINS complex, which is essential for the initiation of DNA replication in eukaryotic cells, as a new M1 interacting protein. M1 from several different influenza virus strains all interacted with SLD5. Overexpression of SLD5 suppressed influenza virus replication. Transient, stable, or inducible expression of M1 induced host cell cycle blockade at G0/G1 phase. Moreover, SLD5 partially rescued M1 expression- or influenza virus infection-induced G0/G1 phase accumulation in cell lines and primary mouse embryonic fibroblasts. Importantly, SLD5 transgenic mice exhibited higher resistance and improved lung epithelial regeneration after virus infection compared with wild-type mice. Therefore, influenza virus M1 blocks host cell cycle process by interacting with SLD5. Our finding reveals the multifunctional nature of M1 and provides new insight for understanding influenza virus-host interaction.
Assuntos
Ciclo Celular/genética , Proteínas Cromossômicas não Histona/genética , Interações Hospedeiro-Patógeno/genética , Vírus da Influenza A Subtipo H1N1/genética , Infecções por Orthomyxoviridae/genética , Proteínas da Matriz Viral/genética , Células A549 , Animais , Proteínas Cromossômicas não Histona/metabolismo , Cães , Fibroblastos/metabolismo , Fibroblastos/virologia , Regulação da Expressão Gênica , Células HEK293 , Humanos , Vírus da Influenza A Subtipo H1N1/crescimento & desenvolvimento , Vírus da Influenza A Subtipo H1N1/metabolismo , Pulmão/metabolismo , Pulmão/virologia , Células Madin Darby de Rim Canino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Infecções por Orthomyxoviridae/metabolismo , Infecções por Orthomyxoviridae/patologia , Infecções por Orthomyxoviridae/virologia , Cultura Primária de Células , Ligação Proteica , Transdução de Sinais , Técnicas do Sistema de Duplo-Híbrido , Proteínas da Matriz Viral/metabolismo , Replicação Viral/genéticaRESUMO
NK-cells have traditionally been viewed as innate effector lymphocytes that serve as a first line of defence against a range of viruses and tumours. More recently, the importance of NK-cell immunoregulatory functions has been highlighted. NK-cells can inhibit antiviral T-cell responses, and also play an important role in controlling harmful T-cell activity in autoimmunity and transplantation settings. Moreover, immunopathological effects of NK-cells during infection have been reported. Nevertheless, the phenotype and function of NK-cells in the thymus during influenza virus infection is not understood. In the present study, we demonstrated that influenza A virus (IAV) infection in mice led to severe thymic atrophy caused by increased thymic T-cell apoptosis and suppressed proliferation. We found that NK-cells played a critical role in this phenotype. IFN-c production by NK-cells was a contributing factor for thymic atrophy during IAV infection. Taken together, our data indicate that NK-cells are involved in the thymic atrophy associated with IAV infection.
Assuntos
Vírus da Influenza A Subtipo H1N1/fisiologia , Influenza Humana/virologia , Células Matadoras Naturais/imunologia , Timo/imunologia , Animais , Atrofia/imunologia , Atrofia/virologia , Feminino , Humanos , Influenza Humana/imunologia , Influenza Humana/patologia , Interferon gama/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Timo/patologiaRESUMO
Natural killer (NK) cells play an important role in protective immunity against viral infection and tumor progression, but they also contribute to rejection of bone marrow grafts via contact-dependent cytotoxicity. Ligation of activating NK receptors with their ligands expressed on target cells induces receptor clustering and actin reorganization at the interface and triggers polarized movement of lytic granules to the contact site. Although activation of the small GTPase Rac has been implicated in NK cell-mediated cytotoxicity, its precise role and the upstream regulator remain elusive. Here, we show that DOCK2, an atypical guanine nucleotide exchange factor for Rac, plays a key role in NK cell-mediated cytotoxicity. We found that although DOCK2 deficiency in NK cells did not affect conjugate formation with target cells, DOCK2-deficienct NK cells failed to effectively kill leukemia cells in vitro and major histocompatibility complex class I-deficient bone marrow cells in vivo, regardless of the sorts of activating receptors. In DOCK2-deficient NK cells, NKG2D-mediated Rac activation was almost completely lost, resulting in a severe defect in the lytic synapse formation. Similar results were obtained when the Rac guanine nucleotide exchange factor activity of DOCK2 was selectively abrogated. These results indicate that DOCK2-Rac axis controls NK cell-mediated cytotoxicity through the lytic synapse formation.
Assuntos
Citotoxicidade Imunológica , Proteínas Ativadoras de GTPase/metabolismo , Sinapses Imunológicas/metabolismo , Células Matadoras Naturais/citologia , Células Matadoras Naturais/imunologia , Proteínas rac de Ligação ao GTP/metabolismo , Animais , Transplante de Medula Óssea , Membrana Celular/metabolismo , Citocinas/biossíntese , Ativação Enzimática , Proteínas Ativadoras de GTPase/deficiência , Fatores de Troca do Nucleotídeo Guanina , Antígenos de Histocompatibilidade Classe I/imunologia , Células Matadoras Naturais/enzimologia , Camundongos , Camundongos Endogâmicos C57BL , Fosfatidilinositol 3-Quinases/metabolismoRESUMO
To migrate efficiently through the interstitium, dendritic cells (DCs) constantly adapt their shape to the given structure of the extracellular matrix and follow the path of least resistance. It is known that this amoeboid migration of DCs requires Cdc42, yet the upstream regulators critical for localization and activation of Cdc42 remain to be determined. Mutations of DOCK8, a member of the atypical guanine nucleotide exchange factor family, causes combined immunodeficiency in humans. In the present study, we show that DOCK8 is a Cdc42-specific guanine nucleotide exchange factor that is critical for interstitial DC migration. By generating the knockout mice, we found that in the absence of DOCK8, DCs failed to accumulate in the lymph node parenchyma for T-cell priming. Although DOCK8-deficient DCs migrated normally on 2-dimensional surfaces, DOCK8 was required for DCs to crawl within 3-dimensional fibrillar networks and to transmigrate through the subcapsular sinus floor. This function of DOCK8 depended on the DHR-2 domain mediating Cdc42 activation. DOCK8 deficiency did not affect global Cdc42 activity. However, Cdc42 activation at the leading edge membrane was impaired in DOCK8-deficient DCs, resulting in a severe defect in amoeboid polarization and migration. Therefore, DOCK8 regulates interstitial DC migration by controlling Cdc42 activity spatially.
Assuntos
Imunidade Adaptativa/genética , Movimento Celular/genética , Células Dendríticas/fisiologia , Fatores de Troca do Nucleotídeo Guanina/fisiologia , Proteína cdc42 de Ligação ao GTP/metabolismo , Imunidade Adaptativa/imunologia , Animais , Técnicas de Cultura de Células , Movimento Celular/imunologia , Células Cultivadas , Células Dendríticas/metabolismo , Feminino , Fatores de Troca do Nucleotídeo Guanina/genética , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Células HEK293 , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos KnockoutRESUMO
Aquaporin-1 (AQP-1) is a water channel protein highly expressed in the vascular endothelial cells of proliferating tissues including malignant cancers. Given that in APC ubiquitinated peptides are effectively introduced into proteasomes from which CD8 epitopes are excised, we fused ubiquitin with AQP-1 (pUB-AQP-1) to produce a DNA vaccine. In C57BL/6J mice immunized with pUB-AQP-1, the growth of B16F10 melanoma was profoundly inhibited. The antitumor effect of the pUB-AQP-1 DNA vaccine was largely mediated by CD8 T cells, which secrete IFN-γ, perforin, and granzyme-B in the presence of APCs transfected with pUB-AQP-1. AQP-1-specific CD8 T cells possessed cytotoxic activity both in vivo and in vitro. After tumor challenge, the microvessel density decreased and the ratio of total blood vessel area to tumor area was significantly reduced as compared with control mice, resulting in a dramatic suppression of tumor growth. The immunization effect was completely abrogated in immunoproteasome-deficient mice. Strikingly this pUB-AQP-1 DNA vaccine was also effective against Colon 26 colon tumors (BALB/c) and MBT/2 bladder tumors (C3H/HeN). Thus, this ubiquitin-conjugated DNA immunization-targeting tumor vasculature is a valid and promising antitumor therapy. This vaccine works across the barriers of tumor species and MHC class I differences in host mice.
Assuntos
Aquaporina 1/imunologia , Vacinas Anticâncer/farmacologia , Neoplasias Experimentais/terapia , Linfócitos T Citotóxicos/imunologia , Vacinas de DNA/farmacologia , Animais , Western Blotting , Vacinas Anticâncer/imunologia , Feminino , Imuno-Histoquímica , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C3H , Camundongos Endogâmicos C57BL , Neoplasias Experimentais/irrigação sanguínea , Neoplasias Experimentais/imunologia , Neovascularização Patológica/terapia , Complexo de Endopeptidases do Proteassoma/imunologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transfecção , Ubiquitina/imunologia , Vacinas de DNA/imunologiaRESUMO
The spine is the most common site of bone metastases, as 20%-40% of cancer patients suffer from spinal metastases. Treatments for spinal metastases are scarce and palliative, primarily aiming at relieving bone pain and preserving neurological function. The bioactive agents-mediated therapies are the most effective modalities for treating spinal metastases because they achieve systematic and specific tumor regression. However, the clinical applications of some bioactive agents are limited due to the lack of targeting capabilities, severe side effects, and vulnerability of drug resistance. Fortunately, advanced biomaterials have been developed as excipients to enhance these treatments, including chemotherapy, phototherapy, magnetic hyperthermia therapy, and combination therapy, by improving tumor targeting and enabling sustaining and stimuli-responsive release of various therapeutic agents. Herein, the review summarizes the development of biomaterials-mediated bioactive agents for enhanced treatments of spinal metastases and predicts future research trends.
Assuntos
Neoplasias da Coluna Vertebral , Humanos , Neoplasias da Coluna Vertebral/tratamento farmacológico , Neoplasias da Coluna Vertebral/secundário , Materiais Biocompatíveis/uso terapêutico , FototerapiaRESUMO
RNA virus infection usually triggers a range of host immune responses, including the induction of proinflammatory cytokines, interferons, and interferon-stimulated genes (ISGs). Here, we report that UBL7, a ubiquitin-like protein, is upregulated during RNA virus infection and induced by type I interferon as an ISG. UBL7-deficient mice exhibit increased susceptibility to viral infection due to attenuated antiviral innate immunity. UBL7 enhances innate immune response to viral infection by promoting the K27-linked polyubiquitination of MAVS. UBL7 interacts with TRIM21, an E3 ubiquitin ligase of MAVS, and promotes the combination of TRIM21 with MAVS in a dose-dependent manner, facilitating the K27-linked polyubiquitination of MAVS and recruiting of TBK1 to enhance the IFN signaling pathway. Moreover, UBL7 has a broad-spectrum antiviral function as an immunomodulatory adaptor protein. Therefore, UBL7 positively regulates innate antiviral signaling and promotes positive feedback to enhance and amplify the antiviral response.
Assuntos
Interferon Tipo I , Infecções por Vírus de RNA , Viroses , Animais , Camundongos , Antivirais , Imunidade Inata , Interferon Tipo I/metabolismo , Ubiquitina/metabolismoRESUMO
BACKGROUND: FAT4 (FAT Atypical Cadherin 4) is a member of the cadherin-associated protein family, which has been shown to function as a tumor suppressor by inhibiting proliferation and metastasis. The Wnt/ß-catenin pathway activation is highly associated with PD-L1-associated tumor immune escape. Here, we report the mechanism by which FAT4 overexpression regulates anti-tumor immunity in cervical cancer by inhibiting PD-L1 N-glycosylation and cell membrane localization in a ß-catenin-dependent manner. METHODS: FAT4 expression was first detected in cervical cancer tissues and cell lines. Cell proliferation, clone formation, and immunofluorescence were used to determine the tumor suppressive impact of FAT4 overexpression in vitro, and the findings were confirmed in immunodeficient and immunocomplete mice xenografts. Through functional and mechanistic experiments in vivo and in vitro, we investigated how FAT4 overexpression affects the antitumor immunity via the ß-catenin/STT3/PD-L1 axis. RESULTS: FAT4 is downregulated in cervical cancer tissues and cell lines. We determined that FAT4 binds to ß-catenin and antagonizes its nuclear localization, promotes phosphorylation and degradation of ß-catenin by the degradation complexes (AXIN1, APC, GSK3ß, CK1). FAT4 overexpression decreases programmed death-ligand 1 (PD-L1) mRNA expression at the transcriptional level, and causes aberrant glycosylation of PD-L1 via STT3A at the post-translational modifications (PTMs) level, leading to its endoplasmic reticulum (ER) accumulation and polyubiquitination-dependent degradation. We found that FAT4 overexpression promotes aberrant PD-L1 glycosylation and degradation in a ß-catenin-dependent manner, thereby increasing cytotoxic T lymphocyte (CTL) activity in immunoreactive mouse models. CONCLUSIONS: These findings address the basis of Wnt/ß-catenin pathway activation in cervical cancer and provide combination immunotherapy options for targeting the FAT4/ß-catenin/STT3/PD-L1 axis. Schematic cartoons showing the antitumor immunity mechanism of FAT4. (left) when Wnts bind to their receptors, which are made up of Frizzled proteins and LRP5/6, the cytoplasmic protein DVL is activated, inducing the aggregation of degradation complexes (AXIN, GSK3ß, CK1, APC) to the receptor. Subsequently, stable ß-catenin translocates into the nucleus and binds to TCF/LEF and TCF7L2 transcription factors, leading to target genes transcription. The catalytically active subunit of oligosaccharyltransferase, STT3A, enhances PD-L1 glycosylation, and N-glycosylated PD-L1 translocates to the cell membrane via the ER-to-Golgi pathway, resulting in immune evasion. (Right) FAT4 exerts antitumor immunity mainly through following mechanisms: (i) FAT4 binds to ß-catenin and antagonizes its nuclear localization, promotes phosphorylation and degradation of ß-catenin by the degradation complexes (AXIN1, APC, GSK3ß, CK1); (ii) FAT4 inhibits PD-L1 and STT3A transcription in a ß-catenin-dependent manner and induces aberrant PD-L1 glycosylation and ubiquitination-dependent degradation; (iii) Promotes activation of cytotoxic T lymphocytes (CTL) and infiltration into the tumor microenvironment.
Assuntos
Antígeno B7-H1 , Neoplasias do Colo do Útero , beta Catenina , Animais , Feminino , Humanos , Camundongos , Antígeno B7-H1/genética , beta Catenina/metabolismo , Caderinas , Glicogênio Sintase Quinase 3 beta/genética , Microambiente Tumoral , Proteínas Supressoras de Tumor , Neoplasias do Colo do Útero/genéticaRESUMO
Mounting data suggest that environmental pollution due to airborne fine particles (AFPs) increases the occurrence and severity of respiratory virus infection in humans. However, it is unclear whether and how interactions with AFPs alter viral infection and distribution. We report synergetic effects between various AFPs and the H1N1 virus, regulated by physicochemical properties of the AFPs. Unlike infection caused by virus alone, AFPs facilitated the internalization of virus through a receptor-independent pathway. Moreover, AFPs promoted the budding and dispersal of progeny virions, likely mediated by lipid rafts in the host plasma membrane. Infected animal models demonstrated that AFPs favored penetration of the H1N1 virus into the distal lung, and its translocation into extrapulmonary organs including the liver, spleen, and kidney, thus causing severe local and systemic disorders. Our findings revealed a key role of AFPs in driving viral infection throughout the respiratory tract and beyond. These insights entail stronger air quality management and air pollution reduction policies.
Assuntos
Poluição do Ar , Vírus da Influenza A Subtipo H1N1 , Animais , Humanos , Pulmão , Proteínas de Transporte , Modelos AnimaisRESUMO
Despite advances in cervical cancer screening and human papilloma virus (HPV) vaccines, cervical cancer remains a global health burden. The standard treatment of cervical cancer includes surgery, radiation therapy, and chemotherapy. Radiotherapy (RT) is the primary treatment for advanced-stage disease. However, due to radioresistance, most patients in the advanced stage have an adverse outcome. Recent studies have shown that long noncoding RNAs (lncRNAs) participate in the regulation of cancer radiosensitivity by regulating DNA damage repair, apoptosis, cancer stem cells (CSCs), and epithelial-mesenchymal transition (EMT). In this review, we summarize the molecular mechanisms of long noncoding RNAs in cervical cancer and radiosensitivity, hoping to provide a theoretical basis and a new molecular target for the cervical cancer RT in the clinic.
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Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causal pathogen of the ongoing global pandemic of coronavirus disease 2019 (COVID-19). Loss of smell and taste are symptoms of COVID-19, and may be related to cilia dysfunction. Here, we found that the SARS-CoV-2 ORF10 increases the overall E3 ligase activity of the CUL2ZYG11B complex by interacting with ZYG11B. Enhanced CUL2ZYG11B activity by ORF10 causes increased ubiquitination and subsequent proteasome-mediated degradation of an intraflagellar transport (IFT) complex B protein, IFT46, thereby impairing both cilia biogenesis and maintenance. Further, we show that exposure of the respiratory tract of hACE2 mice to SARS-CoV-2 or SARS-CoV-2 ORF10 alone results in cilia-dysfunction-related phenotypes, and the ORF10 expression in primary human nasal epithelial cells (HNECs) also caused a rapid loss of the ciliary layer. Our study demonstrates how SARS-CoV-2 ORF10 hijacks CUL2ZYG11B to eliminate IFT46 and leads to cilia dysfunction, thereby offering a powerful etiopathological explanation for how SARS-CoV-2 causes multiple cilia-dysfunction-related symptoms specific to COVID-19.
Assuntos
Cílios , SARS-CoV-2 , Ubiquitina-Proteína Ligases , Animais , Células Cultivadas , Cílios/metabolismo , Cílios/patologia , Proteínas do Citoesqueleto , Células Epiteliais/metabolismo , Células Epiteliais/virologia , Humanos , Camundongos , SARS-CoV-2/patogenicidade , Olfato , Ubiquitina-Proteína Ligases/metabolismoRESUMO
When developing malaria vaccines, the most crucial step is to elucidate the mechanisms involved in protective immunity against the parasites. We found that CD8(+) T cells contribute to protective immunity against infection with blood-stage parasites of Plasmodium yoelii. Infection of C57BL/6 mice with P. yoelii 17XL was lethal, while all mice infected with a low-virulence strain of the parasite 17XNL acquired complete resistance against re-infection with P. yoelii 17XL. However, the host mice transferred with CD8(+) T cells from mice primed only with P. yoelii 17XNL failed to acquire protective immunity. On the other hand, the irradiated host mice were completely resistant to P. yoelii 17XL infection, showing no grade of parasitemia when adoptively transferred with CD8(+) T cells from immune mice that survived infection with both P. yoelii XNL and, subsequently, P. yoelii 17XL. These protective CD8(+) T cells from immune WT mice had the potential to generate IFN-gamma, perforin (PFN) and granzyme B. When mice deficient in IFN-gamma were used as donor mice for CD8(+) T cells, protective immunity in the host mice was fully abrogated, and the immunity was profoundly attenuated in PFN-deficient mice. Thus, CD8(+) T cells producing IFN-gamma and PFN appear to be involved in protective immunity against infection with blood-stage malaria.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Malária/imunologia , Parasitemia/imunologia , Plasmodium yoelii/imunologia , Transferência Adotiva , Animais , Linfócitos T CD8-Positivos/metabolismo , Linfócitos T CD8-Positivos/transplante , Convalescença , Proteínas de Ligação a DNA/deficiência , Proteínas de Ligação a DNA/genética , Feminino , Granzimas/biossíntese , Interferon gama/biossíntese , Interferon gama/deficiência , Interferon gama/genética , Macrófagos/imunologia , Malária/prevenção & controle , Vacinas Antimaláricas/imunologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Parasitemia/prevenção & controle , Plasmodium yoelii/patogenicidade , Proteínas Citotóxicas Formadoras de Poros/biossíntese , Proteínas Citotóxicas Formadoras de Poros/deficiência , Proteínas Citotóxicas Formadoras de Poros/genética , Quimera por Radiação , VirulênciaRESUMO
Gut microorganisms participate in many physiological processes. In particular, Clostridium butyricum can modulate gut microorganisms and treat diseases. The colonization and persistence of strains in the gut contribute to beneficial effects, and the colonization by C. butyricum in the gut is currently unknown. We investigated the total intestinal contents of C. butyricum at 12 h, 24 h, 48 h, and four and six days using real-time reverse transcription-PCR, after oral administration of C. butyricum to rats for seven consecutive days. We assessed the bacterial community structure using Illumina MiSeq sequencing. The results showed that C. butyricum was mainly colonized in the colon. The total content of C. butyricum in the gut increased significantly at 12 h after administration. Exogenous C. butyricum could still be detected in the gut six days after administration. Administration of C. butyricum significantly enhanced gut microbial diversity. The relative abundance of short-chain fatty acid-producing bacterial genera was shown to be higher than that of the control group, and treatment with C. butyricum elevated Firmicutes and diminished Bacteroidetes phyla compared with to the control group. These findings laid the foundation for the study of probiotic colonization capacity and the improvement of microflora for the prevention of gut diseases.
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MicroRNAs (miRNAs) regulate gene expression and thereby influence cell development and function. Numerous studies have shown the significant roles of miRNAs in regulating immune cells including natural killer (NK) cells. However, little is known about the role of miRNAs in NK cells with aging. We previously demonstrated that the aged C57BL/6 mice have significantly decreased proportion of mature (CD27- CD11b+ ) NK cells compared with young mice, indicating impaired maturation of NK cells with aging. Here, we performed deep sequencing of CD27+ NK cells from young and aged mice. Profiling of the miRNome (global miRNA expression levels) revealed that 49 miRNAs displayed a twofold or greater difference in expression between young and aged NK cells. Among these, 30 miRNAs were upregulated and 19 miRNAs were downregulated in the aged NK cells. We found that the expression level of miR-l8la-5p was increased with the maturation of NK cells, and significantly decreased in NK cells from the aged mice. Knockdown of miR-181a-5p inhibited NK cell development in vitro and in vivo. Furthermore, miR-181a-5p is highly conserved in mice and human. MiR-181a-5p promoted the production of IFN-γ and cytotoxicity in stimulated NK cells from both mice and human. Importantly, miR-181a-5p level markedly decreased in NK cells from PBMC of elderly people. Thus, our results demonstrated that the miRNAs profiles in NK cells change with aging, the decreased level of miR-181a-5p contributes to the defective NK cell development and function with aging. This opens new strategies to preserve or restore NK cell function in the elderly.
Assuntos
Envelhecimento/genética , Envelhecimento/imunologia , Células Matadoras Naturais/imunologia , MicroRNAs/metabolismo , Animais , Células Cultivadas , Citotoxicidade Imunológica , Regulação da Expressão Gênica , Humanos , Células K562 , Camundongos , Camundongos Endogâmicos C57BL , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas c-bcl-2/metabolismoRESUMO
Proteasome-mediated proteolysis is responsible for the generation of immunogenic epitopes presented by MHC class I molecules, which activate antigen-specific CD8+ T cells. Immunoproteasomes, defined by the presence of the three catalytic subunits LMP2, MECL-1, and LMP7, have been hypothesized to optimize MHC class I antigen processing. In this study, we demonstrate that the infection of mice with a protozoan parasite, Toxoplasma gondii, induced the expression of LMP7 mRNA in APC and increased the capacity of APC to induce the production of IFN-gamma by antigen-specific CD8+ T cells. In vitro infection of a DC cell line with T. gondii also induced the expression of LMP7 and resulted in enhanced proteasome proteolytic activity. Finally, mice lacking LMP7 were highly susceptible to infection with T. gondii and showed a reduced number of functional CD8+ T cells. These results demonstrate that proteasomes containing LMP7 play an indispensable role in the survival of mice infected with T. gondii, presumably due to the efficient generation of CTL epitopes required for the functional development of CD8+ T cells.
Assuntos
Complexos Multienzimáticos/genética , Toxoplasma/imunologia , Toxoplasmose/genética , Toxoplasmose/imunologia , Animais , Western Blotting , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Linfócitos T CD8-Positivos/parasitologia , Células Cultivadas , Cisteína Endopeptidases/biossíntese , Cisteína Endopeptidases/genética , Cisteína Endopeptidases/metabolismo , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Células Dendríticas/parasitologia , Indução Enzimática , Expressão Gênica , Predisposição Genética para Doença , Interações Hospedeiro-Patógeno , Imunidade Inata/genética , Imunidade Inata/imunologia , Interferon gama/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Complexos Multienzimáticos/biossíntese , Complexos Multienzimáticos/metabolismo , Complexo de Endopeptidases do Proteassoma , Interferência de RNA , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Tempo , Toxoplasma/fisiologia , Toxoplasmose/parasitologiaRESUMO
Cytotoxic CD8(+) T cells are particularly important to the development of protective immunity against the intracellular protozoan parasite, Trypanosoma cruzi, the etiological agent of Chagas disease. We have developed a new effective strategy of genetic immunization by activating CD8(+) T cells through the ubiquitin-fusion degradation (UFD) pathway. We constructed expression plasmids encoding the amastigote surface protein-2 (ASP-2) of T. cruzi. To induce the UFD pathway, a chimeric gene encoding ubiquitin fused to ASP-2 (pUB-ASP-2) was constructed. Mice immunized with pUB-ASP-2 presented lower parasitemia and longer survival period, compared with mice immunized with pASP-2 alone. Depletion of CD8(+) T cells abolished protection against T. cruzi in mice immunized with pUB-ASP-2 while depletion of CD4(+) T cells did not influence the effective immunity. Mice deficient in LMP2 or LMP7, subunits of immunoproteasomes, were not able to develop protective immunity induced. These results suggest that ubiquitin-fused antigens expressed in antigen-presenting cells were effectively degraded via the UFD pathway, and subsequently activated CD8(+) T cells. Consequently, immunization with pUB-ASP-2 was able to induce potent protective immunity against infection of T. cruzi.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Doença de Chagas/prevenção & controle , Neuraminidase/imunologia , Complexo de Endopeptidases do Proteassoma/metabolismo , Vacinas Protozoárias/imunologia , Trypanosoma cruzi/imunologia , Vacinas de DNA/imunologia , Sequência de Aminoácidos , Animais , Antígenos/imunologia , Antígenos/metabolismo , Doença de Chagas/imunologia , Cisteína Endopeptidases/genética , Feminino , Ativação Linfocitária , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Dados de Sequência Molecular , Complexos Multienzimáticos/genética , Neuraminidase/genética , Neuraminidase/metabolismo , Complexo de Endopeptidases do Proteassoma/genética , Vacinas Protozoárias/genética , Vacinas Protozoárias/metabolismo , Ubiquitina/metabolismo , Vacinação , Vacinas de DNA/genética , Vacinas de DNA/metabolismoRESUMO
Acquired immunity against infection with Trypanosoma cruzi is dependent on CD8(+)T cells. Here, to develop a vaccine strategy taking advantage of activated CD8(+)T cells, we constructed a DNA vaccine, designated pGFP-TSA1, encoding a fusion protein linking GFP to a single CTL epitope of TSA1, a leading candidate for vaccine against T. cruzi. C57BL/6 mice vaccinated with this plasmid showed suppressed parasitemia and prolonged survival. Vaccination with pGFP-TSA1 enhanced epitope-specific cytotoxicity and IFN-gamma secretion by CD8(+)T cells. Furthermore, the depletion of CD8(+)T cells prior to challenge infection with T. cruzi completely abolished this protection, indicating that CD8(+)T cells are the principal effector T cells involved. When mice deficient in the proteasome activator PA28alpha/beta or the immunoproteasome subunits LMP2 and LMP7 were used, the protective immunity against infection was profoundly attenuated. Our findings clearly demonstrate that vaccination with pGFP-TSA1 successfully induces protection dependent on CD8(+)T cell activation, in which immunoproteasomes play a crucial role. It is noteworthy to document that physical binding of the epitope and GFP is required for induction of this protection, since mice vaccinated with pTSA1-IRES-GFP failed to acquire resistance, probably because the epitope and GFP are separately expressed in the antigen-presenting cells.
Assuntos
Doença de Chagas/prevenção & controle , Cisteína Endopeptidases/imunologia , Vacinas Protozoárias/administração & dosagem , Trypanosoma cruzi/imunologia , Vacinação , Animais , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Epitopos de Linfócito T/imunologia , Epitopos de Linfócito T/metabolismo , Feminino , Proteínas de Fluorescência Verde/metabolismo , Esquemas de Imunização , Injeções Intradérmicas , Interferon gama/biossíntese , Ativação Linfocitária , Complexo Principal de Histocompatibilidade/imunologia , Proteínas de Membrana/genética , Proteínas de Membrana/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Complexos Multienzimáticos/imunologia , Plasmídeos/imunologia , Complexo de Endopeptidases do Proteassoma/imunologia , Subpopulações de Linfócitos T , Linfócitos T Citotóxicos , Vacinas de DNA/administração & dosagemRESUMO
We have developed a DNA vaccine encoding a fusion protein of ubiquitin (Ub) and target proteins at the N-terminus for effective induction of antigen-specific CD8(+) T cells. A series of expression plasmids encoding a model antigen, ovalbumin (OVA), fused with mutated Ub, was constructed. Western blotting analyses using COS7 cells transfected with these plasmids revealed that there were three types of amino acid causing different binding capacities between Ub and OVA. Natural Ub with a C-terminal glycine readily dissociated from OVA; on the other hand, artificially mutated Ub, the C-terminal amino acid of which had been exchanged to valine or arginine, stably united with the polypeptide, while Ub with a C-terminal alanine partially dissociated. The ability of DNA vaccination to induce OVA-specific CD8(+) T cells closely correlated with the stability of Ub fusion to OVA. Our strategy could be used to optimize the effect of genetic vaccines on the induction of CD8(+) T cells.