RESUMO
IL-1R integrates signals from IL-1α and IL-1ß, and it is widely expressed across tissues and immune cell types. While the expression pattern and function of IL-1R within the innate immune system is well studied, its role in adaptive immunity, particularly within the CD8 T cell compartment, remains underexplored. Here, we show that CD8 T cells dynamically upregulate IL-1R1 levels during priming by APCs, which correlates with their proliferation status and the acquisition of an effector phenotype. Notably, this IL-1 sensitivity persists in memory CD8 T cells of both mice and humans, influencing effector cytokine production upon TCR reactivation. Furthermore, our study highlights that antiviral effector and tissue-resident CD8 T cell responses against influenza A virus infection become impaired in the absence of IL-1 signaling. Altogether, these data support the exploitation of IL-1 activity in the context of T cell vaccination strategies and warrant consideration of the impact of clinical IL-1 inhibition on the rollout of T cell immunity.
Assuntos
Linfócitos T CD8-Positivos , Influenza Humana , Humanos , Animais , Camundongos , Imunidade Adaptativa , Interleucina-1 , Antivirais , Camundongos Endogâmicos C57BLRESUMO
Human respiratory syncytial virus (RSV) is a major cause of lower respiratory tract disease, especially in young children and the elderly. The fusion protein (F) exists in a pre- and postfusion conformation and is the main target of RSV-neutralizing antibodies. Highly potent RSV-neutralizing antibodies typically bind sites that are unique to the prefusion conformation of F. In this study we screened a single-domain antibody (VHH) library derived from a llama immunized with prefusion-stabilized F and identified a prefusion F-specific VHH that can neutralize RSV A at subnanomolar concentrations. Structural analysis revealed that this VHH primarily binds to antigenic site I while also making contacts with residues in antigenic site III and IV. This new VHH reveals a previously underappreciated membrane-proximal region sensitive for neutralization.ImportanceRSV is an important respiratory pathogen. This study describes a prefusion F-specific VHH that primarily binds to antigenic site I of RSV F. This is the first time that a prefusion F-specific antibody that binds this site is reported. In general, antibodies that bind to site I are poorly neutralizing, whereas the VHH described here neutralizes RSV A at subnanomolar concentrations. Our findings contribute to insights into the RSV F antigenic map.
RESUMO
Lipopolysaccharides (LPS) can lead to a lethal endotoxemia, which is a systemic inflammatory response syndrome (SIRS) characterized by a systemic release of cytokines, such as TNF. Endotoxemia is studied intensely, as a model system of Gram-negative infections. LPS- and TNF-induced SIRS involve a strong induction of interferon-stimulated genes (ISGs), some of which cause cell death in the intestinal epithelium cells (IECs). It is well known that glucocorticoids (GCs) protect against endotoxemia. By applying numerous mutant mouse lines, our data support a model whereby GCs, via their glucocorticoid receptor (GR), apply two key mechanisms to control endotoxemia, (i) at the level of suppression of TNF production in a GR monomer-dependent way in macrophages and (ii) at the level of inhibition of TNFR1-induced ISG gene expression and necroptotic cell death mediators in IECs in a GR dimer-dependent way. Our data add new important insights to the understanding of the role of TNF in endotoxemia and the two separate key roles of GCs in suppressing TNF production and activity.
Assuntos
Endotoxemia , Lipopolissacarídeos , Animais , Citocinas , Endotoxemia/induzido quimicamente , Endotoxemia/genética , Glucocorticoides , Inflamação/genética , Lipopolissacarídeos/toxicidade , Camundongos , Fator de Necrose Tumoral alfa/genéticaRESUMO
Glucocorticoid resistance (GCR) is defined as an unresponsiveness to the therapeutic effects, including the antiinflammatory ones of glucocorticoids (GCs) and their receptor, the glucocorticoid receptor (GR). It is a problem in the management of inflammatory diseases and can be congenital as well as acquired. The strong proinflammatory cytokine TNF-alpha (TNF) induces an acute form of GCR, not only in mice, but also in several cell lines: e.g., in the hepatoma cell line BWTG3, as evidenced by impaired Dexamethasone (Dex)-stimulated direct GR-dependent gene up- and down-regulation. We report that TNF has a significant and broad impact on this transcriptional performance of GR, but no impact on nuclear translocation, dimerization, or DNA binding capacity of GR. Proteome-wide proximity-mapping (BioID), however, revealed that the GR interactome was strongly modulated by TNF. One GR cofactor that interacted significantly less with the receptor under GCR conditions is p300. NFκB activation and p300 knockdown both reduced direct transcriptional output of GR whereas p300 overexpression and NFκB inhibition reverted TNF-induced GCR, which is in support of a cofactor reshuffle model. This hypothesis was supported by FRET studies. This mechanism of GCR opens avenues for therapeutic interventions in GCR diseases.
Assuntos
Resistência a Medicamentos/genética , Proteína p300 Associada a E1A/metabolismo , Glucocorticoides/farmacologia , Inflamação/tratamento farmacológico , Receptores de Glucocorticoides/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Células A549 , Animais , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Dexametasona/farmacologia , Dexametasona/uso terapêutico , Regulação para Baixo/efeitos dos fármacos , Regulação para Baixo/imunologia , Proteína p300 Associada a E1A/genética , Feminino , Técnicas de Silenciamento de Genes , Glucocorticoides/uso terapêutico , Células HEK293 , Humanos , Inflamação/imunologia , Camundongos , NF-kappa B/metabolismo , Mapeamento de Interação de Proteínas , Mapas de Interação de Proteínas/efeitos dos fármacos , Mapas de Interação de Proteínas/imunologia , RNA Interferente Pequeno/metabolismo , RNA-Seq , Receptores de Glucocorticoides/imunologia , Regulação para Cima/efeitos dos fármacos , Regulação para Cima/imunologiaRESUMO
OBJECTIVES: Sepsis causes very high mortality and morbidity rates and remains one of the biggest medical challenges. This study investigates whether plasma levels of both matrix metalloproteinase 8 and tumor necrosis factor receptor 1 are associated with sepsis severity and also investigates the therapeutic applicability of simultaneous inhibition of the two molecules in sepsis. DESIGN: Observational human pilot study-prospective controlled animal study. SETTING: University hospital and research laboratory. SUBJECTS: Sepsis patients and C57BL/6 mice deficient for matrix metalloproteinase 8 and/or tumor necrosis factor receptor 1. INTERVENTION: Plasma and whole blood RNA were collected from 13 sepsis patients for 7 consecutive days and within 24 hours of admission to ICU. Matrix metalloproteinase 8 and tumor necrosis factor receptor 1 plasma and expression levels were determined in these patients. Mice deficient for both matrix metalloproteinase 8 and tumor necrosis factor receptor 1 were generated and subjected to endotoxemia and cecal ligation and puncture. Additionally, a bispecific Nanobody that simultaneously blocks matrix metalloproteinase 8 and tumor necrosis factor receptor 1 was created. MEASUREMENTS AND MAIN RESULTS: Plasma levels of matrix metalloproteinase 8 and tumor necrosis factor receptor 1 were positively correlated with the Sequential Organ Failure Assessment score (r, 0.51 and 0.58) and interleukin 6 levels (r, 0.59 and 0.52) in 13 sepsis patients. Combined elimination of tumor necrosis factor receptor 1 and matrix metalloproteinase 8 in double knockout mice resulted in superior survival in endotoxemia and CLP compared with single knockouts and wild-type mice. Cotreatment with our bispecific Nanobody in CLP resulted in improved survival rates (28% vs 19%) compared with untreated mice. CONCLUSIONS: Inhibition of matrix metalloproteinase 8 and tumor necrosis factor receptor 1 might have therapeutic potential to treat sepsis and proof-of-principle was provided as therapeutics that inhibit both tumor necrosis factor receptor 1 and matrix metalloproteinase 8 are effective in CLP.
Assuntos
Inflamação/fisiopatologia , Metaloproteinase 8 da Matriz/fisiologia , Inibidores de Metaloproteinases de Matriz/farmacologia , Receptores Tipo I de Fatores de Necrose Tumoral/antagonistas & inibidores , Sepse/fisiopatologia , Animais , Interleucina-6/sangue , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Projetos Piloto , Estudos Prospectivos , Receptores Tipo I de Fatores de Necrose Tumoral/fisiologiaRESUMO
Divergence of alternative splicing represents one of the major driving forces to shape phenotypic diversity during evolution. However, the extent to which these divergences could be explained by the evolving cis-regulatory versus trans-acting factors remains unresolved. To globally investigate the relative contributions of the two factors for the first time in mammals, we measured splicing difference between C57BL/6J and SPRET/EiJ mouse strains and allele-specific splicing pattern in their F1 hybrid. Out of 11,818 alternative splicing events expressed in the cultured fibroblast cells, we identified 796 with significant difference between the parental strains. After integrating allele-specific data from F1 hybrid, we demonstrated that these events could be predominately attributed to cis-regulatory variants, including those residing at and beyond canonical splicing sites. Contrary to previous observations in Drosophila, such predominant contribution was consistently observed across different types of alternative splicing. Further analysis of liver tissues from the same mouse strains and reanalysis of published datasets on other strains showed similar trends, implying in general the predominant contribution of cis-regulatory changes in the evolution of mouse alternative splicing.
Assuntos
Processamento Alternativo , RNA Mensageiro/metabolismo , Elementos Reguladores de Transcrição , Análise de Sequência de RNA/métodos , Animais , Células Cultivadas , Evolução Molecular , Fibroblastos/metabolismo , Células HEK293 , Humanos , Fígado/metabolismo , Camundongos , Células NIH 3T3RESUMO
Current research on new therapeutic strategies for sepsis uses different animal models, such as the lipopolysaccharide-induced endotoxemia model and the cecal ligation and puncture (CLP) peritonitis model. By using genetic and pharmacologic inhibition of the type I interferon (IFN) receptor (IFNAR1), we show that type I IFN signaling plays a detrimental role in these sepsis models. Mortality after CLP was reduced even when type I IFN responses were blocked after the onset of sepsis. Our findings reveal that type I IFNs play an important detrimental role during sepsis by negatively regulating neutrophil recruitment. Reduced neutrophil influx likely occurs via the induction of the CXC motif chemokine 1. Moreover, human white blood cells exposed to heat-killed Pseudomonas aeruginosa secrete IFN-ß and stimulate type I IFN signaling. We provide data that support pharmacologic inhibition of type I IFN signaling as a novel therapeutic treatment in severe sepsis.
Assuntos
Bacteriemia/imunologia , Endotoxemia/imunologia , Interferon Tipo I/antagonistas & inibidores , Receptor de Interferon alfa e beta/antagonistas & inibidores , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/imunologia , Análise de Variância , Animais , Anticorpos Monoclonais/farmacologia , Bacteriemia/tratamento farmacológico , Bacteriemia/metabolismo , Ceco/lesões , Ceco/cirurgia , Endotoxemia/tratamento farmacológico , Endotoxemia/metabolismo , Feminino , Humanos , Interferon Tipo I/imunologia , Interferon Tipo I/metabolismo , Estimativa de Kaplan-Meier , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neutrófilos/imunologia , Neutrófilos/metabolismo , Receptor de Interferon alfa e beta/imunologia , Receptor de Interferon alfa e beta/metabolismoRESUMO
IMPORTANCE: Human metapneumovirus (hMPV) is an important respiratory pathogen for which no licensed antivirals or vaccines exist. Single-domain antibodies represent promising antiviral biologics that can be easily produced and formatted. We describe the isolation and detailed characterization of two hMPV-neutralizing single-domain antibodies that are directed against the fusion protein F. One of these single-domain antibodies broadly neutralizes hMPV A and B strains, can prevent proteolytic maturation of F, and binds to an epitope in the F trimer interface. This suggests that hMPV pre-F undergoes trimer opening or "breathing" on infectious virions, exposing a vulnerable site for neutralizing antibodies. Finally, we show that this single-domain antibody, fused to a human IgG1 Fc, can protect cotton rats against hMPV replication, an important finding for potential future clinical applications.
Assuntos
Metapneumovirus , Anticorpos de Domínio Único , Humanos , Metapneumovirus/genética , Metapneumovirus/metabolismo , Anticorpos Antivirais , Anticorpos Neutralizantes , Epitopos , Proteínas Virais de Fusão/metabolismoRESUMO
Cell death coordinates repair programs following pathogen attack and tissue injury. However, aberrant cell death can interfere with such programs and cause organ failure. Cellular FLICE-like inhibitory protein (cFLIP) is a crucial regulator of cell death and a substrate of Caspase-8. However, the physiological role of cFLIP cleavage by Caspase-8 remains elusive. Here, we found an essential role for cFLIP cleavage in restraining cell death in different pathophysiological scenarios. Mice expressing a cleavage-resistant cFLIP mutant, CflipD377A, exhibited increased sensitivity to severe acute respiratory syndrome coronavirus (SARS-CoV)-induced lethality, impaired skin wound healing, and increased tissue damage caused by Sharpin deficiency. In vitro, abrogation of cFLIP cleavage sensitizes cells to tumor necrosis factor(TNF)-induced necroptosis and apoptosis by favoring complex-II formation. Mechanistically, the cell death-sensitizing effect of the D377A mutation depends on glutamine-469. These results reveal a crucial role for cFLIP cleavage in controlling the amplitude of cell death responses occurring upon tissue stress to ensure the execution of repair programs.
Assuntos
Apoptose , Viroses , Animais , Camundongos , Caspase 8/genética , Pele/metabolismo , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Migratory dendritic cells expressing CD103 are the targets for mucosal vaccines. These belong to either of two lineage-restricted subsets, cDC1 or cDC2 cells, which have been linked to priming of functionally distinct CD4 T cells. However, recent studies have identified plasticity in cDC2 cells with overlapping functions with cDC1 cells, while the converse has not been reported. We genetically engineered a vaccine adjuvant platform that targeted the cholera toxin A1 (CTA1) ADP-ribosylating enzyme to CD103+ cDC1 and cDC2 cells using a single-chain antibody (scFv) to CD103. Unexpectedly, intranasal immunization with the CTA1-svFcCD103 adjuvant modified cDC1 cells to effectively prime Th17 cells, a function previously limited to cDC2 cells. In fact, cDC2 cells were dispensible, while cDC1 cells, lacking in Batf3-/- mice, were critical. Following intranasal immunizations isolated cDC1 cells from mLN exclusively promoted Rorgt+ T cells and IL-17, IL-21, and IL-22 production. Strong CD8 T cell responses through antigen cross presentation by cDC1 cells were also observed. Single-cell RNAseq analysis revealed upregulation of Th17-promoting gene signatures in sorted cDC1 cells. Gene expression in isolated cDC2 cells was largely unaffected. Our finding represents a major shift of paradigm as we have documented functional plasticity in cDC1 cells.
Assuntos
Influenza Humana , Infecções por Orthomyxoviridae , Difosfato de Adenosina/metabolismo , Adjuvantes Imunológicos , Animais , Toxina da Cólera/metabolismo , Células Dendríticas , Humanos , Influenza Humana/metabolismo , Camundongos , Infecções por Orthomyxoviridae/metabolismo , Células Th17RESUMO
Neuraminidase of influenza A and B viruses plays a critical role in the virus life cycle and is an important target of the host immune system. Here, we highlight the current understanding of influenza neuraminidase structure, function, antigenicity, immunogenicity, and immune protective potential. Neuraminidase inhibiting antibodies have been recognized as correlates of protection against disease caused by natural or experimental influenza A virus infection in humans. In the past years, we have witnessed an increasing interest in the use of influenza neuraminidase to improve the protective potential of currently used influenza vaccines. A number of well-characterized influenza neuraminidase-specific monoclonal antibodies have been described recently, most of which can protect in experimental challenge models by inhibiting the neuraminidase activity or by Fc receptor-dependent mechanisms. The relative instability of the neuraminidase poses a challenge for protein-based antigen design. We critically review the different solutions that have been proposed to solve this problem, ranging from the inclusion of stabilizing heterologous tetramerizing zippers to the introduction of inter-protomer stabilizing mutations. Computationally engineered neuraminidase antigens have been generated that offer broad, within subtype protection in animal challenge models. We also provide an overview of modern vaccine technology platforms that are compatible with the induction of robust neuraminidase-specific immune responses. In the near future, we will likely see the implementation of influenza vaccines that confront the influenza virus with a double punch: targeting both the hemagglutinin and the neuraminidase.
Assuntos
Vacinas contra Influenza/imunologia , Influenza Humana/prevenção & controle , Neuraminidase/imunologia , Proteínas Virais/imunologia , Anticorpos Antivirais/sangue , Anticorpos Antivirais/imunologia , Deriva e Deslocamento Antigênicos , Antígenos Virais/imunologia , Antígenos Virais/ultraestrutura , Domínio Catalítico/genética , Domínio Catalítico/imunologia , Proteção Cruzada , Evolução Molecular , Humanos , Imunogenicidade da Vacina , Vacinas contra Influenza/administração & dosagem , Vacinas contra Influenza/genética , Influenza Humana/imunologia , Influenza Humana/virologia , Alphainfluenzavirus/enzimologia , Alphainfluenzavirus/genética , Alphainfluenzavirus/imunologia , Betainfluenzavirus/enzimologia , Betainfluenzavirus/genética , Betainfluenzavirus/imunologia , Mutação , Nanopartículas , Neuraminidase/administração & dosagem , Neuraminidase/genética , Neuraminidase/ultraestrutura , Vacinas Sintéticas/administração & dosagem , Vacinas Sintéticas/genética , Vacinas Sintéticas/imunologia , Vacinas Sintéticas/ultraestrutura , Proteínas Virais/administração & dosagem , Proteínas Virais/genética , Proteínas Virais/ultraestruturaRESUMO
The ectodomain of matrix protein 2 (M2e) of influenza A viruses is a universal influenza A vaccine candidate. Here, we report potential evasion strategies of influenza A viruses under in vivo passive anti-M2e IgG immune selection pressure in severe combined immune-deficient (SCID) mice. A/Puerto Rico/8/34-infected SCID mice were treated with the M2e-specific mouse IgG monoclonal antibodies (MAbs) MAb 65 (IgG2a) or MAb 37 (IgG1), which recognize amino acids 5 to 15 in M2e, or with MAb 148 (IgG1), which binds to the invariant N terminus of M2e. Treatment of challenged SCID mice with any of these MAbs significantly prolonged survival compared to isotype control IgG treatment. Furthermore, M2e-specific IgG2a protected significantly better than IgG1, and even resulted in virus clearance in some of the SCID mice. Deep sequencing analysis of viral RNA isolated at different time points after treatment revealed that the sequence variation in M2e was limited to P10H/L and/or I11T in anti-M2e MAb-treated mice. Remarkably, in half of the samples isolated from moribund MAb 37-treated mice and in all MAb 148-treated mice, virus was isolated with a wild-type M2 sequence but with nonsynonymous mutations in the polymerases and/or the hemagglutinin genes. Some of these mutations were associated with delayed M2 and other viral gene expression and with increased resistance to anti-M2e MAb treatment of SCID mice. Treatment with M2e-specific MAbs thus selects for viruses with limited variation in M2e. Importantly, influenza A viruses may also undergo an alternative escape route by acquiring mutations that result in delayed wild-type M2 expression. IMPORTANCE Broadly protective influenza vaccine candidates may have a higher barrier to immune evasion compared to conventional influenza vaccines. We used Illumina MiSeq deep sequence analysis to study the mutational patterns in A/Puerto Rico/8/34 viruses that evolve in chronically infected SCID mice that were treated with different M2e-specific MAbs. We show that under these circumstances, viruses emerged in vivo with mutations in M2e that were limited to positions 10 and 11. Moreover, we discovered an alternative route for anti-M2e antibody immune escape, in which a virus is selected with wild-type M2e but with mutations in other gene segments that result in delayed M2 and other viral protein expression. Delayed expression of the viral antigen that is targeted by a protective antibody thus represents an influenza virus immune escape mechanism that does not involve epitope alterations.
Assuntos
Anticorpos Antivirais/uso terapêutico , Imunoglobulina G/uso terapêutico , Vírus da Influenza A/genética , Vírus da Influenza A/imunologia , Mutação , Proteínas da Matriz Viral/genética , Proteínas da Matriz Viral/imunologia , Animais , Sequenciamento de Nucleotídeos em Larga Escala , Evasão da Resposta Imune , Camundongos Endogâmicos BALB C , Camundongos SCID , Proteínas da Matriz Viral/classificaçãoRESUMO
Viruses are the most common cause of acute respiratory tract infections (ARTI). Human metapneumovirus (hMPV) frequently causes viral pneumonia which can become life-threatening if the virus spreads to the lungs. Even though hMPV was only isolated in 2001, this negative-stranded RNA virus has probably been circulating in the human population for many decades. Interestingly, almost all adults have serologic evidence of hMPV infection. A well-established host immune response is evoked when hMPV infection occurs. However, the virus has evolved to circumvent and even exploit the host immune response. Further, infection with hMPV induces a weak memory response, and re-infections during life are common. In this review, we provide a comprehensive overview of the different cell types involved in the immune response in order to better understand the immunopathology induced by hMPV. Such knowledge may contribute to the development of vaccines and therapeutics directed against hMPV.
Assuntos
Imunidade Celular , Metapneumovirus/imunologia , Infecções por Paramyxoviridae/imunologia , Infecções Respiratórias/imunologia , Humanos , Evasão da Resposta Imune , Imunidade Inata , Pulmão/imunologia , Pulmão/patologia , Pulmão/virologia , Metapneumovirus/patogenicidade , Metapneumovirus/fisiologia , Infecções por Paramyxoviridae/patologia , Infecções por Paramyxoviridae/virologia , Infecções Respiratórias/patologia , Infecções Respiratórias/virologia , Replicação ViralRESUMO
Sera of camelid species contain a special kind of antibody that consists only of heavy chains. The variable antigen binding domain of these heavy chain antibodies can be expressed as a separate entity, called a single domain antibody that is characterized by its small size, high solubility and oftentimes exceptional stability. Because of this, most single domain antibodies fold correctly when expressed in the reducing environment of the cytoplasm, and thereby retain their antigen binding specificity. Single domain antibodies can thus be used to target a broad range of intracellular proteins. Such intracellular single domain antibodies are also known as intrabodies, and have proven to be highly useful tools for basic research by allowing visualization, disruption and even targeted degradation of intracellular proteins. Furthermore, intrabodies can be used to uncover prospective new therapeutic targets and have the potential to be applied in therapeutic settings in the future. In this review we provide a brief overview of recent advances in the field of intracellular single domain antibodies, focusing on their use as research tools and potential therapeutic applications. Special attention is given to the available methods that allow delivery of single domain antibodies into cells.
Assuntos
Espaço Intracelular/imunologia , Anticorpos de Domínio Único/imunologia , Animais , Sistemas de Liberação de Medicamentos , Humanos , Anticorpos de Domínio Único/químicaRESUMO
mRNA-lipoplex vaccines are currently being explored in phase II clinical trials for the treatment of patients with advanced solid tumors. Mechanistically, these mRNA-lipoplex vaccines are characterized by the induction of type I interferon (IFN) centered innate responses. Earlier studies have identified type I IFNs as major regulators of the T cell response instigated by mRNA-lipoplex vaccines. However, stimulatory or, in contrast, profound inhibitory effects of type I IFNs were described depending on the study. In this mouse study, we demonstrated that the opposing roles of type I IFN signaling on the magnitude of the vaccine-evoked T cell responses is dependent on the route of mRNA-lipoplex administration and is regulated at the level of the T cells rather than indirectly through modulation of dendritic cell function. This study helps to understand the double-edged sword character of type I IFN induction upon mRNA-based vaccine treatment and may contribute to a more rational design of mRNA vaccination regimens.
RESUMO
The cytokine TNF drives inflammatory diseases, e.g., Crohn's disease. In a mouse model of TNF-induced systemic inflammatory response syndrome (SIRS), severe impact on intestinal epithelial cells (IECs) is observed. Zinc confers complete protection in this model. We found that zinc no longer protects in animals which lack glucocorticoids (GCs), or express mutant versions of their receptor GR in IECs, nor in mice which lack gut microbiota. RNA-seq studies in IECs showed that zinc caused reduction in expression of constitutive (STAT1-induced) interferon-stimulated response (ISRE) genes and interferon regulatory factor (IRF) genes. Since some of these genes are involved in TNF-induced cell death in intestinal crypt Paneth cells, and since zinc has direct effects on the composition of the gut microbiota (such as several Staphylococcus species) and on TNF-induced Paneth cell death, we postulate a new zinc-related anti-inflammatory mechanism. Zinc modulates the gut microbiota, causing less induction of ISRE/IRF genes in crypt cells, less TNF-induced necroptosis in Paneth cells, and less fatal evasion of gut bacteria into the system.
Assuntos
Interferons , Zinco , Animais , Morte Celular , Mucosa Intestinal , Camundongos , Celulas de PanethRESUMO
Sepsis in humans and experimental animals is characterized by an acute inflammatory response. glucocorticoids (GCs) are widely used for the treatment of many inflammatory disorders, yet their effectiveness in sepsis is debatable. One of the major anti-inflammatory proteins induced by GCs is glucocorticoid-induced leucine zipper (GILZ, coded by the TSC22D3 gene). We found that TSC22D3 mRNA expression is downregulated in white blood cells of human sepsis patients. Interestingly, transgenic GILZ-overexpressing mice (GILZ-tg) showed better survival rates in the cecal ligation and puncture (CLP) model of mouse sepsis. To our surprise, GILZ had only mild anti-inflammatory effects in this model, as the systemic proinflammatory response was not significantly reduced in GILZ-tg mice compared with control mice. During CLP, we observed reduced bacterial counts in blood of GILZ-tg mice compared with control mice. We found increased expression of Tsc22d3 mRNA specifically in peritoneal exudate cells in the CLP model, as well as increased capacity for bacterial phagocytosis of CD45 GILZ-tg cells compared with CD45 GILZ-wt cells. Hence, we believe that the protective effects of GILZ in the CLP model can be linked to a more efficient phagocytosis.
Assuntos
Peritonite/metabolismo , Peritonite/prevenção & controle , Sepse/metabolismo , Sepse/prevenção & controle , Fatores de Transcrição/metabolismo , Animais , Ceco/lesões , Humanos , Interleucina-6/sangue , Antígenos Comuns de Leucócito/metabolismo , Ligadura/efeitos adversos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Peritonite/sangue , Peritonite/etiologia , Fagocitose/genética , Fagocitose/fisiologia , Punções/efeitos adversos , Sepse/etiologia , Fatores de Transcrição/genéticaRESUMO
Lower respiratory tract infections, such as infections caused by influenza A viruses, are a constant threat for public health. Antivirals are indispensable to control disease caused by epidemic as well as pandemic influenza A. We developed a novel anti-influenza A virus approach based on an engineered single-domain antibody (VHH) construct that can selectively recruit innate immune cells to the sites of virus replication. This protective construct comprises two VHHs. One VHH binds with nanomolar affinity to the conserved influenza A matrix protein 2 (M2) ectodomain (M2e). Co-crystal structure analysis revealed that the complementarity determining regions 2 and 3 of this VHH embrace M2e. The second selected VHH specifically binds to the mouse Fcγ Receptor IV (FcγRIV) and was genetically fused to the M2e-specific VHH, which resulted in a bi-specific VHH-based construct that could be efficiently expressed in Pichia pastoris. In the presence of M2 expressing or influenza A virus-infected target cells, this single domain antibody construct selectively activated the mouse FcγRIV. Moreover, intranasal delivery of this bispecific FcγRIV-engaging VHH construct protected wild type but not FcγRIV-/- mice against challenge with an H3N2 influenza virus. These results provide proof of concept that VHHs directed against a surface exposed viral antigen can be readily armed with effector functions that trigger protective antiviral activity beyond direct virus neutralization.
Assuntos
Anticorpos Antivirais/imunologia , Vírus da Influenza A/imunologia , Influenza Humana/imunologia , Influenza Humana/metabolismo , Influenza Humana/virologia , Receptores de IgG/metabolismo , Anticorpos de Domínio Único/imunologia , Proteínas da Matriz Viral/imunologia , Sequência de Aminoácidos , Animais , Anticorpos Biespecíficos/química , Anticorpos Biespecíficos/imunologia , Anticorpos Antivirais/química , Linhagem Celular , Humanos , Camundongos , Modelos Moleculares , Peptídeos/química , Peptídeos/imunologia , Conformação Proteica , Receptores de IgG/química , Anticorpos de Domínio Único/química , Relação Estrutura-Atividade , Proteínas da Matriz Viral/químicaRESUMO
Since their discovery in the 1990s, single-domain antibodies (VHHs), also known as Nanobodies®, have changed the landscape of affinity reagents. The outstanding solubility, stability, and specificity of VHHs, as well as their small size, ease of production and formatting flexibility favor VHHs over conventional antibody formats for many applications. The exceptional ease by which it is possible to fuse VHHs with different molecular modules has been particularly explored in the context of viral infections. In this review, we focus on VHH formats that have been developed to combat viruses including influenza viruses, human immunodeficiency virus-1 (HIV-1), and human respiratory syncytial virus (RSV). Such formats may significantly increase the affinity, half-life, breadth of protection of an antiviral VHH and reduce the risk of viral escape. In addition, VHHs can be equipped with effector functions, for example to guide components of the immune system with high precision to sites of viral infection.
RESUMO
TNF is an important mediator in numerous inflammatory diseases, e.g., in inflammatory bowel diseases (IBDs). In IBD, acute increases in TNF production can lead to disease flares. Glucocorticoids (GCs), which are steroids that bind and activate the glucocorticoid receptor (GR), are able to protect animals and humans against acute TNF-induced inflammatory symptoms. Mice with a poor transcriptional response of GR dimer-dependent target genes were studied in a model of TNF-induced lethal inflammation. In contrast to the GRWT/WT mice, these GRdim/dim mice displayed a substantial increase in TNF sensitivity and a lack of protection by the GC dexamethasone (DEX). Unchallenged GRdim/dim mice had a strong IFN-stimulated gene (ISG) signature, along with STAT1 upregulation and phosphorylation. This ISG signature was gut specific and, based on our studies with antibiotics, depended on the gut microbiota. GR dimers directly bound to short DNA sequences in the STAT1 promoter known as inverted repeat negative GRE (IR-nGRE) elements. Poor control of STAT1 in GRdim/dim mice led to failure to repress ISG genes, resulting in excessive necroptosis induction by TNF. Our findings support a critical interplay among gut microbiota, IFNs, necroptosis, and GR in both the basal response to acute inflammatory challenges and pharmacological intervention by GCs.