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1.
Mol Cell ; 84(18): 3513-3529.e5, 2024 Sep 19.
Artigo em Inglês | MEDLINE | ID: mdl-39255795

RESUMO

Innate immunity serves as the primary defense against viral and microbial infections in humans. The precise influence of cellular metabolites, especially fatty acids, on antiviral innate immunity remains largely elusive. Here, through screening a metabolite library, palmitic acid (PA) has been identified as a key modulator of antiviral infections in human cells. Mechanistically, PA induces mitochondrial antiviral signaling protein (MAVS) palmitoylation, aggregation, and subsequent activation, thereby enhancing the innate immune response. The palmitoyl-transferase ZDHHC24 catalyzes MAVS palmitoylation, thereby boosting the TBK1-IRF3-interferon (IFN) pathway, particularly under conditions of PA stimulation or high-fat-diet-fed mouse models, leading to antiviral immune responses. Additionally, APT2 de-palmitoylates MAVS, thus inhibiting antiviral signaling, suggesting that its inhibitors, such as ML349, effectively reverse MAVS activation in response to antiviral infections. These findings underscore the critical role of PA in regulating antiviral innate immunity through MAVS palmitoylation and provide strategies for enhancing PA intake or targeting APT2 for combating viral infections.


Assuntos
Aciltransferases , Proteínas Adaptadoras de Transdução de Sinal , Imunidade Inata , Fator Regulador 3 de Interferon , Lipoilação , Ácido Palmítico , Transdução de Sinais , Imunidade Inata/efeitos dos fármacos , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/imunologia , Humanos , Animais , Ácido Palmítico/farmacologia , Camundongos , Células HEK293 , Fator Regulador 3 de Interferon/metabolismo , Fator Regulador 3 de Interferon/genética , Fator Regulador 3 de Interferon/imunologia , Aciltransferases/genética , Aciltransferases/imunologia , Aciltransferases/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Serina-Treonina Quinases/genética , Camundongos Endogâmicos C57BL , Antivirais/farmacologia , Proteínas de Neoplasias , Peptídeos e Proteínas de Sinalização Intracelular
2.
Braz J Med Biol Res ; 57: e13409, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38958367

RESUMO

Tuberculosis (TB), caused by Mycobacterium tuberculosis, remains the leading cause of mortality by a single infectious agent in the world. M. tuberculosis infection could also result in clinical chronic infection, known as latent TB infection (LTBI). Compared to the current limited treatment, several subunit vaccines showed immunotherapeutic effects and were included in clinical trials. In this study, a subunit vaccine of Ag85B with a novel mucosal adjuvant c-di-AMP (Ag85B:c-di-AMP) was delivered intranasally to a persistent M. tuberculosis H37Ra infection mouse model, which also presented the asymptomatic characteristics of LTBI. Compared with Ag85B immunization, Ag85B:c-di-AMP vaccination induced stronger humoral immune responses, significantly higher CD4+ T cells recruitment, enhanced Th1/Th2/Th17 profile response in the lung, decreased pathological lesions of the lung, and reduced M. tuberculosis load in mice. Taken together, Ag85B:c-di-AMP mucosal route immunization provided an immunotherapeutic effect on persistent M. tuberculosis H37Ra infection, and c-di-AMP, as a promising potential mucosal adjuvant, could be further used in therapeutic or prophylactic vaccine strategies for persistent M. tuberculosis infection as well as LTBI.


Assuntos
Adjuvantes Imunológicos , Modelos Animais de Doenças , Mycobacterium tuberculosis , Vacinas contra a Tuberculose , Animais , Adjuvantes Imunológicos/administração & dosagem , Vacinas contra a Tuberculose/imunologia , Vacinas contra a Tuberculose/administração & dosagem , Mycobacterium tuberculosis/imunologia , Camundongos , Feminino , Antígenos de Bactérias/imunologia , Aciltransferases/imunologia , Vacinas de Subunidades Antigênicas/imunologia , Vacinas de Subunidades Antigênicas/administração & dosagem , Proteínas de Bactérias/imunologia , Tuberculose/imunologia , Tuberculose/prevenção & controle , Tuberculose Latente/imunologia , Camundongos Endogâmicos BALB C , Administração Intranasal
3.
Int Immunopharmacol ; 139: 112811, 2024 Sep 30.
Artigo em Inglês | MEDLINE | ID: mdl-39068754

RESUMO

The eradication of tuberculosis remains a global challenge. Despite being the only licensed vaccine, Bacillus Calmette-Guérin (BCG) confers limited protective efficacy in adults and individuals with latent tuberculosis infections (LTBI). There is an urgent need to develop novel vaccines that can enhance the protective effect of BCG. Protein subunit vaccines have garnered significant research interest due to their safety and plasticity. Based on previous studies, we selected three antigens associated with LTBI (Rv2028c, Rv2029c, Rv3126c) and fused them with an immunodominant antigen Ag85A, resulting in the construction of a multistage protein subunit vaccine named A986. We evaluated the protective effect of recombinant protein A986 adjuvanted with MPL/QS21 as a booster vaccine for BCG against Mycobacterium tuberculosis (Mtb) infection in mice. The A986 + MPL/QS21 induced the secretion of antigen-specific Th1 (IL-2+, IFN-γ+ and TNF-α+) and Th17 (IL-17A+) cytokines in CD4+ and CD8+ T cells within the lung and spleen of mice, while also increased the frequency of central memory and effector memory T cells. Additionally, it also induced the enhanced production of IgG antibodies. Compared to BCG alone, A986 + MPL/QS21 boosting significantly augmented the proliferation of antigen-specific multifunctional T cells and effectively reduced bacterial load in infected mice. Taken together, A986 + MPL/QS21 formulation induced robust antigen-specific immune responses and provided enhanced protection against Mtb infection as a booster of BCG vaccine.


Assuntos
Antígenos de Bactérias , Vacina BCG , Citocinas , Mycobacterium tuberculosis , Tuberculose , Vacinas de Subunidades Antigênicas , Animais , Vacinas de Subunidades Antigênicas/imunologia , Mycobacterium tuberculosis/imunologia , Vacina BCG/imunologia , Antígenos de Bactérias/imunologia , Feminino , Tuberculose/prevenção & controle , Tuberculose/imunologia , Camundongos , Citocinas/metabolismo , Imunização Secundária , Modelos Animais de Doenças , Adjuvantes Imunológicos/administração & dosagem , Vacinas contra a Tuberculose/imunologia , Vacinas contra a Tuberculose/administração & dosagem , Anticorpos Antibacterianos/sangue , Anticorpos Antibacterianos/imunologia , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos BALB C , Aciltransferases/imunologia , Pulmão/imunologia , Pulmão/microbiologia , Pulmão/patologia , Proteínas de Bactérias/imunologia , Humanos
4.
Front Immunol ; 15: 1401626, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38868779

RESUMO

Zinc finger Asp-His-His-Cys motif-containing (zDHHC) proteins, known for their palmitoyltransferase (PAT) activity, play crucial roles in diverse cellular processes, including immune regulation. However, their non-palmitoyltransferase immunomodulatory functions and involvement in teleost immune responses remain underexplored. In this study, we systematically characterized the zDHHC family in the large yellow croaker (Larimichthys crocea), identifying 22 members. Phylogenetic analysis unveiled that each of the 22 LczDHHCs formed distinct clusters with their orthologues from other teleost species. Furthermore, all LczDHHCs exhibited a highly conserved DHHC domain, as confirmed by tertiary structure prediction. Notably, LczDHHC23 exhibited the most pronounced upregulation following Pseudomonas plecoglossicida (P. plecoglossicida) infection of macrophage/monocyte cells (MO/MΦ). Silencing LczDHHC23 led to heightened pro-inflammatory cytokine expression and diminished anti-inflammatory cytokine levels in MO/MΦ during infection, indicating its anti-inflammatory role. Functionally, LczDHHC23 facilitated M2-type macrophage polarization, as evidenced by a significant skewing of MO/MΦ towards the pro-inflammatory M1 phenotype upon LczDHHC23 knockdown, along with the inhibition of MO/MΦ necroptosis induced by P. plecoglossicida infection. These findings highlight the non-PAT immunomodulatory function of LczDHHC23 in teleost immune regulation, broadening our understanding of zDHHC proteins in host-pathogen interactions, suggesting LczDHHC23 as a potential therapeutic target for immune modulation in aquatic species.


Assuntos
Proteínas de Peixes , Macrófagos , Necroptose , Perciformes , Animais , Perciformes/imunologia , Macrófagos/imunologia , Macrófagos/metabolismo , Proteínas de Peixes/genética , Proteínas de Peixes/imunologia , Proteínas de Peixes/metabolismo , Necroptose/imunologia , Filogenia , Ativação de Macrófagos/imunologia , Doenças dos Peixes/imunologia , Doenças dos Peixes/microbiologia , Aciltransferases/genética , Aciltransferases/imunologia , Pseudomonas/fisiologia , Citocinas/metabolismo
5.
Vaccine ; 42(22): 125909, 2024 Sep 17.
Artigo em Inglês | MEDLINE | ID: mdl-38704256

RESUMO

Mucosal vaccines have the potential to elicit protective immune responses at the point of entry of respiratory pathogens, thus preventing even the initial seed infection. Unlike licensed injectable vaccines, mucosal vaccines comprising protein subunits are only in development. One of the primary challenges associated with mucosal vaccines has been identifying and characterizing safe yet effective mucosal adjuvants that can effectively prime multi-factorial mucosal immunity. In this study, we tested NanoSTING, a liposomal formulation of the endogenous activator of the stimulator of interferon genes (STING) pathway, cyclic guanosine adenosine monophosphate (cGAMP), as a mucosal adjuvant. We formulated a vaccine based on the H1 antigen (fusion protein of Ag85b and ESAT-6) adjuvanted with NanoSTING. Intranasal immunization of NanoSTING-H1 elicited a strong T-cell response in the lung of vaccinated animals characterized by (a) CXCR3+ KLRG1- lung resident T cells that are known to be essential for controlling bacterial infection, (b) IFNγ-secreting CD4+ T cells which is necessary for intracellular bactericidal activity, and (c) IL17-secreting CD4+ T cells that can confer protective immunity against multiple clinically relevant strains of Mtb. Upon challenge with aerosolized Mycobacterium tuberculosis Erdman strain, intranasal NanoSTING-H1 provides protection comparable to subcutaneous administration of the live attenuated Mycobacterium bovis vaccine strain Bacille-Calmette-Guérin (BCG). Our results indicate that NanoSTING adjuvanted protein vaccines can elicit a multi-factorial immune response that protects from infection by M. tuberculosis.


Assuntos
Administração Intranasal , Antígenos de Bactérias , Camundongos Endogâmicos C57BL , Mycobacterium tuberculosis , Nanopartículas , Vacinas contra a Tuberculose , Animais , Vacinas contra a Tuberculose/imunologia , Vacinas contra a Tuberculose/administração & dosagem , Mycobacterium tuberculosis/imunologia , Nanopartículas/administração & dosagem , Nanopartículas/química , Antígenos de Bactérias/imunologia , Antígenos de Bactérias/administração & dosagem , Camundongos , Adjuvantes Imunológicos/administração & dosagem , Feminino , Tuberculose/prevenção & controle , Tuberculose/imunologia , Pulmão/imunologia , Pulmão/microbiologia , Proteínas de Bactérias/imunologia , Aciltransferases/imunologia , Aciltransferases/genética , Linfócitos T CD4-Positivos/imunologia , Adjuvantes de Vacinas/administração & dosagem , Imunidade nas Mucosas/imunologia , Nanovacinas , Proteínas Recombinantes de Fusão
6.
Cytokine Growth Factor Rev ; 77: 30-38, 2024 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-38472042

RESUMO

Pyroptosis, a programmed cell death process, is vital for the immune response against microbial infections and internal danger signals. Recent studies have highlighted the importance of protein palmitoylation, a modification that involves attaching palmitate to cysteine residues, in regulating key proteins involved in pyroptosis. Palmitoylation of cGAS at residue C474 by ZDHHC18 affects its enzymatic activity and DNA binding ability. Similarly, ZDHHC9 promotes cGAS activity through palmitoylation at residues C404/405. NLRP3 palmitoylation at residue C844, mediated by ZDHHC12, impacts its stability and interactions with other proteins, crucial for activating the NLRP3 inflammasome and triggering inflammation. However, the role of ZDHHC5 in NLRP3 palmitoylation remains uncertain due to conflicting findings. Palmitoylation at C88/91 is essential for STING activation and induction of type I interferons. It modulates the formation of multimeric complexes and downstream signaling pathways. GSDMD palmitoylation at C191 is necessary for pore formation and membrane translocation, while GSDME palmitoylation at C407/408 is associated with drug-induced pyroptosis. Moreover, palmitoylation of NOD1 and NOD2 influences their membrane recruitment and immune signaling pathways in response to bacterial peptidoglycans, acting as upstream regulators of pyroptosis. This review summarizes the important roles for palmitoylation in regulating the function of key pyroptosis-related proteins, shedding light on the intricate mechanisms governing immune responses and inflammation.


Assuntos
Lipoilação , Proteína 3 que Contém Domínio de Pirina da Família NLR , Piroptose , Humanos , Animais , Proteína 3 que Contém Domínio de Pirina da Família NLR/imunologia , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Transdução de Sinais , Inflamassomos/imunologia , Proteínas de Membrana/imunologia , Proteínas de Membrana/metabolismo , Nucleotidiltransferases/metabolismo , Nucleotidiltransferases/imunologia , Inflamação/imunologia , Aciltransferases/imunologia
7.
J Leukoc Biol ; 115(6): 1053-1069, 2024 05 29.
Artigo em Inglês | MEDLINE | ID: mdl-38242866

RESUMO

Tuberculosis is one of the deadliest infectious diseases worldwide. Mycobacterium tuberculosis has developed strategies not only to evade host immunity but also to manipulate it for its survival. We investigated whether Mycobacterium tuberculosis exploited the immunogenicity of Ag85B, one of its major secretory proteins, to redirect host antituberculosis immunity to its advantage. We found that administration of Ag85B protein to mice vaccinated with Bacillus Calmette-Guérin impaired the protection elicited by vaccination, causing a more severe infection when mice were challenged with Mycobacterium tuberculosis. Ag85B administration reduced Bacillus Calmette-Guérin-induced CD4 T-cell activation and IFN-γ, CCL-4, and IL-22 production in response to Mycobacterium tuberculosis-infected cells. On the other hand, it promoted robust Ag85B-responsive IFN-γ-producing CD4 T cells, expansion of a subset of IFN-γ/IL-10-producing CD4+FOXP3+Treg cells, differential activation of IL-17/IL-22 responses, and activation of regulatory and exhaustion pathways, including programmed death ligand 1 expression on macrophages. All this resulted in impaired intracellular Mycobacterium tuberculosis growth control by systemic immunity, both before and after the Mycobacterium tuberculosis challenge. Interestingly, Mycobacterium tuberculosis infection itself generated Ag85B-reactive inflammatory immune cells incapable of clearing Mycobacterium tuberculosis in both unvaccinated and Bacillus Calmette-Guérin-vaccinated mice. Our data suggest that Mycobacterium tuberculosis can exploit the strong immunogenicity of Ag85B to promote its own survival and spread. Since Ag85B is normally secreted by replicating bacteria and is commonly found in the lungs of the Mycobacterium tuberculosis-infected host, our findings may advance the understanding on the mechanisms of Mycobacterium tuberculosis pathogenesis and immune evasion.


Assuntos
Aciltransferases , Antígenos de Bactérias , Vacina BCG , Proteínas de Bactérias , Camundongos Endogâmicos C57BL , Mycobacterium tuberculosis , Tuberculose , Animais , Camundongos , Aciltransferases/imunologia , Antígenos de Bactérias/imunologia , Proteínas de Bactérias/imunologia , Vacina BCG/imunologia , Linfócitos T CD4-Positivos/imunologia , Citocinas/metabolismo , Viabilidade Microbiana , Mycobacterium tuberculosis/imunologia , Tuberculose/imunologia , Tuberculose/microbiologia
8.
Nat Commun ; 12(1): 2750, 2021 05 12.
Artigo em Inglês | MEDLINE | ID: mdl-33980819

RESUMO

S-acylation is a reversible protein post-translational modification mediated by protein S-acyltransferases (PATs). How S-acylation regulates plant innate immunity is our main concern. Here, we show that the plant immune receptor P2K1 (DORN1, LecRK-I.9; extracellular ATP receptor) directly interacts with and phosphorylates Arabidopsis PAT5 and PAT9 to stimulate their S-acyltransferase activity. This leads, in a time-dependent manner, to greater S-acylation of P2K1, which dampens the immune response. pat5 and pat9 mutants have an elevated extracellular ATP-induced immune response, limited bacterial invasion, increased phosphorylation and decreased degradation of P2K1 during immune signaling. Mutation of S-acylated cysteine residues in P2K1 results in a similar phenotype. Our study reveals that S-acylation effects the temporal dynamics of P2K1 receptor activity, through autophosphorylation and protein degradation, suggesting an important role for this modification in regulating the ability of plants in respond to external stimuli.


Assuntos
Trifosfato de Adenosina/metabolismo , Proteínas de Arabidopsis/metabolismo , Arabidopsis/imunologia , Imunidade Vegetal , Proteínas Quinases/metabolismo , Acilação , Aciltransferases/genética , Aciltransferases/imunologia , Aciltransferases/metabolismo , Trifosfato de Adenosina/imunologia , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/imunologia , Mutação , Fosforilação , Plantas Geneticamente Modificadas , Ligação Proteica , Proteínas Quinases/genética , Proteínas Quinases/imunologia , Receptores de Reconhecimento de Padrão/genética , Receptores de Reconhecimento de Padrão/imunologia , Receptores de Reconhecimento de Padrão/metabolismo , Receptores Purinérgicos P2/genética , Receptores Purinérgicos P2/imunologia , Receptores Purinérgicos P2/metabolismo , Transdução de Sinais , Fatores de Tempo
9.
Cell Metab ; 32(6): 981-995.e7, 2020 12 01.
Artigo em Inglês | MEDLINE | ID: mdl-33264603

RESUMO

Mitochondria constantly adapt to the metabolic needs of a cell. This mitochondrial plasticity is critical to T cells, which modulate metabolism depending on antigen-driven signals and environment. We show here that de novo synthesis of the mitochondrial membrane-specific lipid cardiolipin maintains CD8+ T cell function. T cells deficient for the cardiolipin-synthesizing enzyme PTPMT1 had reduced cardiolipin and responded poorly to antigen because basal cardiolipin levels were required for activation. However, neither de novo cardiolipin synthesis, nor its Tafazzin-dependent remodeling, was needed for T cell activation. In contrast, PTPMT1-dependent cardiolipin synthesis was vital when mitochondrial fitness was required, most notably during memory T cell differentiation or nutrient stress. We also found CD8+ T cell defects in a small cohort of patients with Barth syndrome, where TAFAZZIN is mutated, and in a Tafazzin-deficient mouse model. Thus, the dynamic regulation of a single mitochondrial lipid is crucial for CD8+ T cell immunity.


Assuntos
Aciltransferases/imunologia , Síndrome de Barth/imunologia , Linfócitos T CD8-Positivos/imunologia , Cardiolipinas/imunologia , Mitocôndrias/imunologia , PTEN Fosfo-Hidrolase/imunologia , Animais , Síndrome de Barth/patologia , Linfócitos T CD8-Positivos/citologia , Células Cultivadas , Feminino , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout
10.
J Zhejiang Univ Sci B ; 21(11): 856-870, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33150770

RESUMO

The study and characterization of biomolecules involved in the interaction between mycobacteria and their hosts are crucial to determine their roles in the invasion process and provide basic knowledge about the biology and pathogenesis of disease. Promising new biomarkers for diagnosis and immunotherapy have emerged recently. Mycobacterium is an ancient pathogen that has developed complex strategies for its persistence in the host and environment, likely based on the complexity of the network of interactions between the molecules involved in infection. Several biomarkers have received recent attention in the process of developing rapid and reliable detection techniques for tuberculosis. Among the most widely investigated antigens are CFP-10 (10-kDa culture filtrate protein), ESAT-6 (6-kDa early secretory antigenic target), Ag85A, Ag85B, CFP-7, and PPE18. Some of these antigens have been proposed as biomarkers to assess the key elements of the response to infection of both the pathogen and host. The design of novel and accurate diagnostic methods is essential for the control of tuberculosis worldwide. Presently, the diagnostic methods are based on the identification of molecules in the humoral response in infected individuals. Therefore, these tests depend on the capacity of the host to develop an immune response, which usually is heterogeneous. In the last 20 years, special attention has been given to the design of multiantigenic diagnostic methods to improve the levels of sensitivity and specificity. In this review, we summarize the state of the art in the study and use of mycobacterium biomolecules with the potential to support novel tuberculosis control strategies.


Assuntos
Antígenos/química , Biomarcadores/química , Mycobacterium tuberculosis/imunologia , Tuberculose/diagnóstico , Tuberculose/imunologia , Aciltransferases/imunologia , Animais , Antígenos de Bactérias/imunologia , Proteínas de Bactérias/imunologia , Biomarcadores/metabolismo , Humanos , Sistema Imunitário , Incidência , Proteínas Recombinantes/química , Risco , Sensibilidade e Especificidade
11.
Front Immunol ; 11: 577815, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33117380

RESUMO

T cells recognizing epitopes on the surface of mycobacteria-infected macrophages can impart protection, but with associated risk for reactivation to lung pathology. We aimed to identify antibodies specific to such epitopes, which carry potentials for development toward novel therapeutic constructs. Since epitopes presented in the context of major histocompatibility complex alleles are rarely recognized by naturally produced antibodies, we used a phage display library for the identification of monoclonal human single domain antibody producing clones. The selected 2C clone displayed T cell receptor-like recognition of an HLA-A*0201 bound 199KLVANNTRL207 peptide from the Ag85B antigen, which is known to be an immunodominant epitope for human T cells. The specificity of the selected domain antibody was demonstrated by solid phase immunoassay and by immunofluorescent surface staining of peptide loaded cells of the T2 cell line. The antibody affinity binding was determined by biolayer interferometry. Our results validated the used technologies as suitable for the generation of antibodies against epitopes on the surface of Mycobacterium tuberculosis infected cells. The potential approaches forward the development of antibody in immunotherapy of tuberculosis have been outlined in the discussion.


Assuntos
Aciltransferases/imunologia , Antígenos de Bactérias/imunologia , Antituberculosos/farmacologia , Proteínas de Bactérias/imunologia , Antígenos HLA-A/imunologia , Epitopos Imunodominantes , Mycobacterium tuberculosis/imunologia , Anticorpos de Cadeia Única/farmacologia , Linfócitos T/imunologia , Tuberculose/prevenção & controle , Especificidade de Anticorpos , Antituberculosos/imunologia , Linhagem Celular Tumoral , Técnicas de Visualização da Superfície Celular , Ensaio de Imunoadsorção Enzimática , Imunofluorescência , Humanos , Anticorpos de Cadeia Única/genética , Anticorpos de Cadeia Única/imunologia , Tuberculose/imunologia , Tuberculose/microbiologia
12.
Life Sci ; 260: 118476, 2020 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-32971102

RESUMO

Hepatocellular carcinoma (HCC) is the sixth most common malignancy and has the third highest mortality rate among all tumors. Previous studies found that phosphatidylinositol glycan anchor biosynthesis class U (PIGU) was highly expressed in hepatocellular carcinoma (HCC), while the function of PIGU in HCC remains unknown. Here, we deeply investigated this issue. The expression levels of PIGU in HCC cells were measured by Western blotting. The functions of PIGU in HCC cells were assessed in vitro, followed by assessing the nuclear factor-kappa B (NF-κB) pathway-related protein levels. The xenograft mouse models were conducted to investigate the effects of PIGU in vivo. Moreover, the effects of PIGU downregulation on natural killer (NK)-92 cell-mediated cell killing were detected. The results showed that PIGU was highly expressed in HCC cells compared with normal liver cells. Functional studies showed that PIGU promoted viability, cell cycle progression, migration, and invasion and suppressed apoptosis in HCC cells. Mechanism studies indicated that PIGU silencing blocked the NF-κB pathway and the blockade of the NF-κB pathway reversed the effects of PIGU overexpression on HCC cell function, including cell viability, migration, invasion, and apoptosis. In vivo studies further verified the effects of PIGU on HCC cell function, and demonstrated that PIGU knockdown suppressed tumorigenesis. Additionally, we proved that PIGU downregulation significantly enhanced the sensitivity of HCC cells to NK-92 cell cytolysis. Collectively, PIGU may promote HCC progression through activating the NF-κB pathway and promoting immune escape, indicating that PIGU may serve as a promising therapeutic target for HCC treatment.


Assuntos
Aciltransferases/metabolismo , Carcinoma Hepatocelular/patologia , Neoplasias Hepáticas/patologia , NF-kappa B/metabolismo , Aciltransferases/genética , Aciltransferases/imunologia , Animais , Apoptose/genética , Carcinoma Hepatocelular/imunologia , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/mortalidade , Linhagem Celular Tumoral , Movimento Celular/genética , Proliferação de Células/genética , Regulação Neoplásica da Expressão Gênica , Técnicas de Silenciamento de Genes , Humanos , Neoplasias Hepáticas/imunologia , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/mortalidade , Masculino , Camundongos Nus , Evasão Tumoral , Ensaios Antitumorais Modelo de Xenoenxerto
13.
Proc Natl Acad Sci U S A ; 117(37): 22984-22991, 2020 09 15.
Artigo em Inglês | MEDLINE | ID: mdl-32868431

RESUMO

Immune evasion through membrane remodeling is a hallmark of Yersinia pestis pathogenesis. Yersinia remodels its membrane during its life cycle as it alternates between mammalian hosts (37 °C) and ambient (21 °C to 26 °C) temperatures of the arthropod transmission vector or external environment. This shift in growth temperature induces changes in number and length of acyl groups on the lipid A portion of lipopolysaccharide (LPS) for the enteric pathogens Yersinia pseudotuberculosis (Ypt) and Yersinia enterocolitica (Ye), as well as the causative agent of plague, Yersinia pestis (Yp). Addition of a C16 fatty acid (palmitate) to lipid A by the outer membrane acyltransferase enzyme PagP occurs in immunostimulatory Ypt and Ye strains, but not in immune-evasive Yp Analysis of Yp pagP gene sequences identified a single-nucleotide polymorphism that results in a premature stop in translation, yielding a truncated, nonfunctional enzyme. Upon repair of this polymorphism to the sequence present in Ypt and Ye, lipid A isolated from a Yp pagP+ strain synthesized two structures with the C16 fatty acids located in acyloxyacyl linkage at the 2' and 3' positions of the diglucosamine backbone. Structural modifications were confirmed by mass spectrometry and gas chromatography. With the genotypic restoration of PagP enzymatic activity in Yp, a significant increase in lipid A endotoxicity mediated through the MyD88 and TRIF/TRAM arms of the TLR4-signaling pathway was observed. Discovery and repair of an evolutionarily lost lipid A modifying enzyme provides evidence of lipid A as a crucial determinant in Yp infectivity, pathogenesis, and host innate immune evasion.


Assuntos
Aciltransferases/imunologia , Evasão da Resposta Imune/imunologia , Imunidade Inata/imunologia , Lipídeo A/imunologia , Yersinia pestis/imunologia , Animais , Evolução Biológica , Linhagem Celular , Linhagem Celular Tumoral , Células HEK293 , Humanos , Leucócitos Mononucleares/imunologia , Lipopolissacarídeos/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Polimorfismo de Nucleotídeo Único/imunologia , Células THP-1/imunologia , Células U937 , Yersinia pseudotuberculosis/imunologia
14.
J Immunol ; 205(2): 425-437, 2020 07 15.
Artigo em Inglês | MEDLINE | ID: mdl-32513849

RESUMO

The continuing emergence of viral pathogens and their rapid spread into heavily populated areas around the world underscore the urgency for development of highly effective vaccines to generate protective antiviral Ab responses. Many established and newly emerging viral pathogens, including HIV and Ebola viruses, are most prevalent in regions of the world in which Mycobacterium tuberculosis infection remains endemic and vaccination at birth with M. bovis bacille Calmette-Guérin (BCG) is widely used. We have investigated the potential for using CD4+ T cells arising in response to BCG as a source of help for driving Ab responses against viral vaccines. To test this approach, we designed vaccines comprised of protein immunogens fused to an immunodominant CD4+ T cell epitope of the secreted Ag 85B protein of BCG. Proof-of-concept experiments showed that the presence of BCG-specific Th cells in previously BCG-vaccinated mice had a dose-sparing effect for subsequent vaccination with fusion proteins containing the Ag 85B epitope and consistently induced isotype switching to the IgG2c subclass. Studies using an Ebola virus glycoprotein fused to the Ag 85B epitope showed that prior BCG vaccination promoted high-affinity IgG1 responses that neutralized viral infection. The design of fusion protein vaccines with the ability to recruit BCG-specific CD4+ Th cells may be a useful and broadly applicable approach to generating improved vaccines against a range of established and newly emergent viral pathogens.


Assuntos
Aciltransferases/imunologia , Antígenos de Bactérias/imunologia , Proteínas de Bactérias/imunologia , Linfócitos T CD4-Positivos/imunologia , Vacinas contra Ebola/imunologia , Ebolavirus/fisiologia , Doença pelo Vírus Ebola/imunologia , Mycobacterium bovis/imunologia , Proteínas do Envelope Viral/imunologia , Aciltransferases/genética , Animais , Anticorpos Antivirais/metabolismo , Formação de Anticorpos , Antígenos de Bactérias/genética , Proteínas de Bactérias/genética , Modelos Animais de Doenças , Vacinas contra Ebola/genética , Feminino , Humanos , Imunoglobulina G/sangue , Ativação Linfocitária , Camundongos , Camundongos Transgênicos , Proteínas Recombinantes de Fusão/genética , Proteínas do Envelope Viral/genética
15.
Z Naturforsch C J Biosci ; 75(9-10): 313-317, 2020 Sep 25.
Artigo em Inglês | MEDLINE | ID: mdl-32374296

RESUMO

The purpose of this study is to purify the LpxA protein of Chlamydia trachomatis (Ct) and prepare the polyclonal antibody against LpxA protein, so as to lay a foundation for studying the function of LpxA protein. The LpxA gene was amplified by PCR. The expression plasmid pET28a-LpxA was constructed by using pET28a as the vector. The fusion protein containing 6 histidine tag was induced by IPTG and purified by Ni2+ chromatography gel. The purified His-LpxA protein was used as an immunogen to immunize New Zealand rabbits subcutaneously through the back to prepare polyclonal antibody. Immunoblotting was used to detect the reaction between the antibody and His-LpxA. The determination of polyclonal antibody titer was detected by ELISA. The relative molecular weight of His-LpxA was 32.8 kDa, and it could be expressed in Escherichia coli. The purity of the purified protein was about 95%. After immunizing New Zealand rabbits, the antiserum was able to recognize the recombinant His-LpxA protein with a titer greater than 1:10240. In this study, LpxA protein was successfully purified and antiserum was prepared, which provided an experimental basis for studying the function of LpxA protein.


Assuntos
Aciltransferases/administração & dosagem , Aciltransferases/isolamento & purificação , Anticorpos Antibacterianos/sangue , Chlamydia trachomatis/imunologia , Aciltransferases/genética , Aciltransferases/imunologia , Animais , Chlamydia trachomatis/genética , Clonagem Molecular , Histidina/metabolismo , Imunização , Injeções Subcutâneas , Peso Molecular , Plasmídeos/genética , Coelhos , Proteínas Recombinantes/administração & dosagem , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/isolamento & purificação
16.
Front Immunol ; 11: 803, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32457748

RESUMO

Understanding the in vivo fate of vaccine antigens and adjuvants and their safety is crucial for the rational design of mucosal subunit vaccines. Prime and pull vaccination using the T helper 17-inducing adjuvant CAF01 administered parenterally and mucosally, respectively, has previously been suggested as a promising strategy to redirect immunity to mucosal tissues. Recently, we reported a promising tuberculosis (TB) vaccination strategy comprising of parenteral priming followed by intrapulmonary (i.pulmon.) mucosal pull immunization with the TB subunit vaccine candidate H56/CAF01, which resulted in the induction of lung-localized, H56-specific T cells and systemic as well as lung mucosal IgA responses. Here, we investigate the uptake of H56/CAF01 by mucosal and systemic innate myeloid cells, antigen-presenting cells (APCs), lung epithelial cells and endothelial cells in mice after parenteral prime combined with i.pulmon. pull immunization, and after parenteral or i.pulmon. prime immunization alone. We find that i.pulmon. pull immunization of mice with H56/CAF01, which are parenterally primed with H56/CAF01, substantially enhances vaccine uptake and presentation by pulmonary and splenic APCs, pulmonary endothelial cells and type I epithelial cells and induces stronger activation of dendritic cells in the lung-draining lymph nodes, compared with parenteral immunization alone, which suggests activation of both innate and memory responses. Using mass spectrometry imaging of lipid biomarkers, we further show that (i) airway mucosal immunization with H56/CAF01 neither induces apparent local tissue damage nor inflammation in the lungs, and (ii) the presence of CAF01 is accompanied by evidence of an altered phagocytic activity in alveolar macrophages, evident from co-localization of CAF01 with the biomarker bis(monoacylglycero)phosphate, which is expressed in the late endosomes and lysosomes of phagocytosing macrophages. Hence, our data demonstrate that innate myeloid responses differ after one and two immunizations, respectively, and the priming route and boosting route individually affect this outcome. These findings may have important implications for the design of mucosal vaccines intended for safe administration in the airways.


Assuntos
Aciltransferases/imunologia , Adjuvantes Imunológicos/administração & dosagem , Células Apresentadoras de Antígenos/imunologia , Antígenos de Bactérias/imunologia , Proteínas de Bactérias/imunologia , Imunidade Inata , Mycobacterium tuberculosis/imunologia , Células Mieloides/imunologia , Vacinas contra a Tuberculose/administração & dosagem , Tuberculose/prevenção & controle , Vacinação/métodos , Animais , Feminino , Imunidade nas Mucosas , Imunização Secundária , Memória Imunológica , Injeções Intramusculares , Pulmão/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Resultado do Tratamento , Tuberculose/imunologia , Tuberculose/microbiologia , Vacinas contra a Tuberculose/imunologia , Vacinas de Subunidades Antigênicas/administração & dosagem , Vacinas de Subunidades Antigênicas/imunologia
17.
Proc Natl Acad Sci U S A ; 117(3): 1280-1282, 2020 01 21.
Artigo em Inglês | MEDLINE | ID: mdl-31907319

RESUMO

Nucleic acid vaccines introduce the genetic materials encoding antigenic proteins into host cells. If these proteins are directed into the secretory pathway with a signal/leader sequence, they will be exposed to the host's glycosylation machinery, and, if their amino acid sequences contain consensus sequons for N-linked glycosylation, they may become glycosylated. The presence of host glycans on the proteins of microbial origin may prevent a strong protective immune response either through hindering access to key epitopes by lymphocytes or through altering immune responses by binding to immunoregulatory glycan-binding receptors on immune cells. Ag85A expressed by Mycobacterium tuberculosis (Mtb) is a bacterial surface protein that is commonly used in nucleic acid vaccines in multiple clinical trials. Here we show that, when Ag85A is expressed in mammalian cells, it is glycosylated, does not induce a strong humoral immune response in mice, and does not activate Ag85A-specific lymphocytes as highly as Ag85A natively expressed by the bacterium. Our study indicates that host glycosylation of the vaccine target can impede its antigenicity and immunogenicity. Glycosylation of the antigenic protein targets therefore must be carefully evaluated in designing nucleic acid vaccines.


Assuntos
Aciltransferases/imunologia , Antígenos de Bactérias/imunologia , Imunogenicidade da Vacina , Processamento de Proteína Pós-Traducional , Vacinas contra a Tuberculose/imunologia , Aciltransferases/genética , Aciltransferases/metabolismo , Animais , Antígenos de Bactérias/genética , Antígenos de Bactérias/metabolismo , Glicosilação , Células HEK293 , Humanos , Linfócitos/imunologia , Camundongos
18.
J Mol Med (Berl) ; 97(12): 1685-1694, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31786669

RESUMO

In an earlier study, a novel Sendai virus-vectored anti-tuberculosis vaccine encoding Ag85A and Ag85B (SeV85AB) was constructed and shown to elicit antigen-specific T cell responses and protection against Mycobacterium tuberculosis (Mtb) infection in a murine model. In this study, we evaluate whether the immune responses induced by this novel vaccine might be elevated by a recombinant DNA vaccine expressing the same antigen in a heterologous prime-boost vaccination strategy. The results showed that both SeV85AB prime-DNA boost (SeV85AB-DNA) and DNA prime-SeV85AB boost (DNA-SeV85AB) vaccination strategies significantly enhanced the antigen-specific T cell responses induced by the separate vaccines. The SeV85AB-DNA immunization regimen induced higher levels of recall T cell responses after Mtb infection and conferred better immune protection compared with DNA-SeV85AB or a single immunization. Collectively, our study lends strong evidence that a DNA vaccine boost might be included in a novel SeV85AB immunization strategy designed to enhance the immune protection against Mtb. KEY MESSAGES: A heterologous prime-boost regimen with a novel recombinant SeV85AB and a DNA vaccine increase the T cell responses above those from a single vaccine. The heterologous prime-boost regimen provided protection against Mtb infection. The DNA vaccine might be included in a novel SeV85AB immunization strategy designed to enhance the immune protection against Mtb.


Assuntos
Aciltransferases/imunologia , Antígenos de Bactérias/imunologia , Proteínas de Bactérias/imunologia , Mycobacterium tuberculosis/imunologia , Vacinas contra a Tuberculose/genética , Tuberculose/imunologia , Vacinas de DNA/imunologia , Aciltransferases/genética , Animais , Antígenos de Bactérias/genética , Proteínas de Bactérias/genética , Linfócitos T CD8-Positivos/imunologia , Feminino , Vetores Genéticos , Imunização Secundária , Interferon gama/metabolismo , Interleucina-2/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Mycobacterium tuberculosis/genética , Vírus Sendai/genética , Tuberculose/prevenção & controle , Vacinas contra a Tuberculose/imunologia , Fator de Necrose Tumoral alfa/metabolismo , Vacinação
19.
Front Immunol ; 10: 1349, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31293568

RESUMO

Liposomes have been long considered as a vaccine delivery system but this technology remains to be fully utilized. Here, we describe a novel liposome-based subunit vaccine formulation for tuberculosis (TB) based on phosphatidylserine encapsulating two prominent TB antigens, Ag85B, and ESAT-6. We show that the resulting liposomes (Lipo-AE) are stable upon storage and can be readily taken up by antigen presenting cells and that their antigenic cargo is delivered and processed within endosomal cell compartments. The Lipo-AE vaccine formulation combined with the PolyIC adjuvant induced a mixed Th1/Th17-Th2 immune response to Ag85B but only a weak response to ESAT-6. An immunization regimen based on systemic delivery followed by mucosal boost with Lipo-AE resulted in the accumulation of resident memory T cells in the lungs. Most importantly though, when Lipo-AE vaccine candidate was administered to BCG-immunized mice subsequently challenged with low dose aerosol Mycobacterium tuberculosis, we observed a significant reduction of the bacterial load in the lungs and spleen compared to BCG alone. We therefore conclude that the immunization with mycobacterial antigens delivered by phosphatidylserine based liposomes in combination with Poly:IC adjuvant may represent a novel BCG boosting vaccination strategy.


Assuntos
Aciltransferases/imunologia , Antígenos de Bactérias/imunologia , Vacina BCG/imunologia , Proteínas de Bactérias/imunologia , Lipossomos/imunologia , Tuberculose Pulmonar/prevenção & controle , Adjuvantes Imunológicos/administração & dosagem , Animais , Carga Bacteriana , Sistemas de Liberação de Medicamentos , Feminino , Humanos , Memória Imunológica/imunologia , Pulmão/microbiologia , Camundongos , Camundongos Endogâmicos C57BL , Mycobacterium tuberculosis/imunologia , Fosfatidilserinas/imunologia , Poli I-C/imunologia , Baço/microbiologia , Linfócitos T Auxiliares-Indutores/imunologia , Vacinação , Vacinas de Subunidades Antigênicas/imunologia
20.
J Infect Dis ; 220(8): 1355-1366, 2019 09 13.
Artigo em Inglês | MEDLINE | ID: mdl-31198944

RESUMO

BACKGROUND: The development of strategies to accelerate disease resolution and shorten antibiotic therapy is imperative in curbing the global tuberculosis epidemic. Therapeutic application of novel vaccines adjunct to antibiotics represents such a strategy. METHODS: By using a murine model of pulmonary tuberculosis (TB), we have investigated whether a single respiratory mucosal therapeutic delivery of a novel chimpanzee adenovirus-vectored vaccine expressing Ag85A (AdCh68Ag85A) accelerates TB disease control in conjunction with antibiotics and restricts pulmonary disease rebound after premature (nonsterilizing) antibiotic cessation. RESULTS: We find that immunotherapy via the respiratory mucosal, but not parenteral, route significantly accelerates pulmonary mycobacterial clearance, limits lung pathology, and restricts disease rebound after premature antibiotic cessation. We further show that vaccine-activated antigen-specific T cells, particularly CD8 T cells, in the lung play an important role in immunotherapeutic effects. CONCLUSIONS: Our results indicate that a single-dose respiratory mucosal immunotherapy with AdCh68Ag85A adjunct to antibiotic therapy has the potential to significantly accelerate disease control and shorten the duration of conventional treatment. Our study provides the proof of principle to support therapeutic applications of viral-vectored vaccines via the respiratory route.


Assuntos
Antituberculosos/uso terapêutico , Mycobacterium tuberculosis/imunologia , Vacinas contra a Tuberculose/administração & dosagem , Tuberculose Pulmonar/terapia , Vacinação/métodos , Aciltransferases/genética , Aciltransferases/imunologia , Adenoviridae/genética , Administração Intranasal , Animais , Antígenos de Bactérias/genética , Antígenos de Bactérias/imunologia , Terapia Combinada/métodos , Modelos Animais de Doenças , Feminino , Vetores Genéticos/genética , Humanos , Esquemas de Imunização , Injeções Intramusculares , Camundongos , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/isolamento & purificação , Mucosa Nasal , Pan troglodytes/virologia , Estudo de Prova de Conceito , Vacinas contra a Tuberculose/genética , Vacinas contra a Tuberculose/imunologia , Tuberculose Pulmonar/diagnóstico , Tuberculose Pulmonar/microbiologia , Vacinas Sintéticas/administração & dosagem , Vacinas Sintéticas/genética , Vacinas Sintéticas/imunologia
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