RESUMEN
Severe influenza A virus (IAV) infections can result in hyper-inflammation, lung injury and acute respiratory distress syndrome1-5 (ARDS), for which there are no effective pharmacological therapies. Necroptosis is an attractive entry point for therapeutic intervention in ARDS and related inflammatory conditions because it drives pathogenic lung inflammation and lethality during severe IAV infection6-8 and can potentially be targeted by receptor interacting protein kinase 3 (RIPK3) inhibitors. Here we show that a newly developed RIPK3 inhibitor, UH15-38, potently and selectively blocked IAV-triggered necroptosis in alveolar epithelial cells in vivo. UH15-38 ameliorated lung inflammation and prevented mortality following infection with laboratory-adapted and pandemic strains of IAV, without compromising antiviral adaptive immune responses or impeding viral clearance. UH15-38 displayed robust therapeutic efficacy even when administered late in the course of infection, suggesting that RIPK3 blockade may provide clinical benefit in patients with IAV-driven ARDS and other hyper-inflammatory pathologies.
Asunto(s)
Lesión Pulmonar , Necroptosis , Infecciones por Orthomyxoviridae , Inhibidores de Proteínas Quinasas , Proteína Serina-Treonina Quinasas de Interacción con Receptores , Animales , Femenino , Humanos , Masculino , Ratones , Células Epiteliales Alveolares/patología , Células Epiteliales Alveolares/efectos de los fármacos , Células Epiteliales Alveolares/virología , Células Epiteliales Alveolares/metabolismo , Virus de la Influenza A/clasificación , Virus de la Influenza A/efectos de los fármacos , Virus de la Influenza A/inmunología , Virus de la Influenza A/patogenicidad , Lesión Pulmonar/complicaciones , Lesión Pulmonar/patología , Lesión Pulmonar/prevención & control , Lesión Pulmonar/virología , Ratones Endogámicos C57BL , Necroptosis/efectos de los fármacos , Infecciones por Orthomyxoviridae/complicaciones , Infecciones por Orthomyxoviridae/tratamiento farmacológico , Infecciones por Orthomyxoviridae/inmunología , Infecciones por Orthomyxoviridae/mortalidad , Infecciones por Orthomyxoviridae/virología , Inhibidores de Proteínas Quinasas/administración & dosificación , Inhibidores de Proteínas Quinasas/farmacología , Inhibidores de Proteínas Quinasas/uso terapéutico , Proteína Serina-Treonina Quinasas de Interacción con Receptores/metabolismo , Proteína Serina-Treonina Quinasas de Interacción con Receptores/antagonistas & inhibidores , Síndrome de Dificultad Respiratoria/complicaciones , Síndrome de Dificultad Respiratoria/patología , Síndrome de Dificultad Respiratoria/prevención & control , Síndrome de Dificultad Respiratoria/virologíaRESUMEN
Only a small proportion of patients with cancer show lasting responses to immune checkpoint blockade (ICB)-based monotherapies. The RNA-editing enzyme ADAR1 is an emerging determinant of resistance to ICB therapy and prevents ICB responsiveness by repressing immunogenic double-stranded RNAs (dsRNAs), such as those arising from the dysregulated expression of endogenous retroviral elements (EREs)1-4. These dsRNAs trigger an interferon-dependent antitumour response by activating A-form dsRNA (A-RNA)-sensing proteins such as MDA-5 and PKR5. Here we show that ADAR1 also prevents the accrual of endogenous Z-form dsRNA elements (Z-RNAs), which were enriched in the 3' untranslated regions of interferon-stimulated mRNAs. Depletion or mutation of ADAR1 resulted in Z-RNA accumulation and activation of the Z-RNA sensor ZBP1, which culminated in RIPK3-mediated necroptosis. As no clinically viable ADAR1 inhibitors currently exist, we searched for a compound that can override the requirement for ADAR1 inhibition and directly activate ZBP1. We identified a small molecule, the curaxin CBL0137, which potently activates ZBP1 by triggering Z-DNA formation in cells. CBL0137 induced ZBP1-dependent necroptosis in cancer-associated fibroblasts and reversed ICB unresponsiveness in mouse models of melanoma. Collectively, these results demonstrate that ADAR1 represses endogenous Z-RNAs and identifies ZBP1-mediated necroptosis as a new determinant of tumour immunogenicity masked by ADAR1. Therapeutic activation of ZBP1-induced necroptosis provides a readily translatable avenue for rekindling the immune responsiveness of ICB-resistant human cancers.
Asunto(s)
Adenosina Desaminasa , Necroptosis , Neoplasias , Proteínas de Unión al ARN , Regiones no Traducidas 3' , Adenosina Desaminasa/metabolismo , Animales , Fibroblastos Asociados al Cáncer , Carbazoles/farmacología , Humanos , Inmunoterapia/tendencias , Interferones/metabolismo , Melanoma , Ratones , Neoplasias/tratamiento farmacológico , Neoplasias/genética , Neoplasias/patología , ARN Bicatenario/inmunología , Proteínas de Unión al ARN/metabolismoRESUMEN
BACKGROUND: CD83 is known to regulate lymphocyte maturation, activation, homeostasis, and antibody response to immunization and infection. While CD83 has a major part in B cell function, its role in influenza A virus infection has not yet been investigated. METHODS: We investigated the role of CD83 using C57BL/6J wild type mice and CD83 knockout (KO) mice after intraperitoneal administration of the influenza A/WSN/1933 virus. We analyzed cells of the peritoneal cavity, splenocytes, and cells of the bone marrow with FACS to investigate CD83 expression and cell population change in response to the virus infection. ELISA was performed with sera and peritoneal cavity fluids to detect A/WSN/1933 virus-specific IgG and the subclasses of IgG. RESULTS: FACS analysis data showed a transient but distinct induction of CD83 expression in the peritoneal B cells of wild type mice. CD83 KO mice exhibited a delayed recovery of B cells in the bone marrow after influenza virus infection and overall, a smaller T cell population compared to wild type mice. The peritoneal cavity and serum of the wild type mice contained a high titer of IgG within 14 days after infection, whereas the CD83 KO mice had a very low titer of IgG. CONCLUSIONS: These results show the importance of CD83 in lymphocytes homeostasis and antibody production during influenza A virus infection.
Asunto(s)
Antígenos CD/genética , Antígenos CD/inmunología , Regulación de la Expresión Génica/inmunología , Inmunoglobulinas/genética , Inmunoglobulinas/inmunología , Virus de la Influenza A/inmunología , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/inmunología , Infecciones por Orthomyxoviridae/inmunología , Animales , Anticuerpos Antivirales/sangre , Formación de Anticuerpos , Linfocitos B/inmunología , Células de la Médula Ósea/inmunología , Femenino , Inmunoglobulina G/sangre , Inmunoglobulina G/clasificación , Activación de Linfocitos/inmunología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Cavidad Peritoneal/citología , Bazo/citología , Antígeno CD83RESUMEN
CpG-DNA activates the host immune system to resist bacterial infections. In this study, we examined the protective effect of CpG-DNA in mice against Escherichia coli (E. coli) K1 infection. Administration of CpG-DNA increased the survival of mice after E. coli K1 infection, which reduces the numbers of bacteria in the organs. Pre-injection of mice with CpG-DNA before E. coli K1 infection increased the levels of the complement C3 but not C3a and C3b. The survival of the mice after E. coli K1 infection was significantly decreased when the mice were pre-injected with the cobra venom factor (CVF) removing the complement compared to the non-CVF-treated mice group. It suggests that the complement has protective roles against E. coli K1 infection. In addition, the survival of complement-depleted mice was increased by CpG-DNA pre-administration before E. coli K1 infection. Therefore, we suggest that CpG-DNA enhances the anti-bacterial activity of the immune system by augmenting the levels of complement systems after E. coli K1 infection and triggering other factors as well. Further studies are required to investigate the functional roles of the CpG-DNA-induced complement regulation and other factors against urgent bacterial infection.
Asunto(s)
Antibacterianos/farmacología , Proteínas del Sistema Complemento/efectos de los fármacos , Proteínas del Sistema Complemento/inmunología , Factores Inmunológicos/farmacología , Oligodesoxirribonucleótidos/farmacología , Animales , Antibacterianos/administración & dosificación , Antibacterianos/química , Activación de Complemento/efectos de los fármacos , Activación de Complemento/inmunología , Escherichia coli/efectos de los fármacos , Escherichia coli/inmunología , Infecciones por Escherichia coli/tratamiento farmacológico , Infecciones por Escherichia coli/inmunología , Infecciones por Escherichia coli/microbiología , Factores Inmunológicos/administración & dosificación , Factores Inmunológicos/química , Infusiones Parenterales , Ratones , Oligodesoxirribonucleótidos/administración & dosificación , Oligodesoxirribonucleótidos/química , Fagocitosis/efectos de los fármacos , Fagocitosis/inmunologíaRESUMEN
BACKGROUND: Inbred mice have several advantages, including genetic similarity to humans, a well-established gene manipulation system, and strong tolerance to inbreeding. However, inbred mice derived from a limited genetic pool have a small genetic diversity. Thus, the development of new inbred strains from wild mice is needed to overcome this limitation. Hence, in this study, we used a new strain of inbred mice called KWM/Hym. We sequenced the Mx1 gene to elucidate the genetic diversities of KWM/Hym mice and observed the biological alterations of the Mx1 protein upon influenza A infection. RESULTS: The Mx1 gene in KWM/Hym mice had 2, 4, and 38 nucleotide substitutions compared to those in the Mx1 gene in A2G, CAST/EiJ, and Mus spretus mice, respectively. Moreover, the Mx1 protein in KWM/Hym mice had 2 and 25 amino acid substitutions compared to those in the Mx1 protein in CAST/EiJ and M. spretus mice, respectively. To elucidate the function of the Mx1 protein, we inoculated the influenza A virus (A/WSN/1933) in KWM/Hym mice. Nine days after infection, all infected KWM/Hym mice survived without any weight loss. Four days after infection, the lungs of the infected KWM/Hym mice showed mild alveolitis and loss of bronchiolar epithelium; however, the pulmonary viral titers of the infected KWM/Hym mice were significantly lower than that in the infected BALB/c mice (2.17 × plaque-forming units mL-1). CONCLUSIONS: Our results demonstrate that the KWM/Hym mice are resistant to influenza A virus infection. Further, these mice can be used as a model organism to understand the mechanism of influenza A virus susceptibility.
RESUMEN
Inflammation and cell death are closely linked arms of the host immune response to infection, which when carefully balanced ensure host survival. One example of this balance is the tightly regulated transition from TNFR1-associated pro-inflammatory complex I to pro-death complex II. By contrast, here we show that a TRIF-dependent complex containing FADD, RIPK1 and caspase-8 (that we have termed the TRIFosome) mediates cell death in response to Yersinia pseudotuberculosis and LPS. Furthermore, we show that constitutive binding between ZBP1 and RIPK1 is essential for the initiation of TRIFosome interactions, caspase-8-mediated cell death and inflammasome activation, thus positioning ZBP1 as an effector of cell death in the context of bacterial blockade of pro-inflammatory signaling. Additionally, our findings offer an alternative to the TNFR1-dependent model of complex II assembly, by demonstrating pro-death complex formation reliant on TRIF signaling.
Asunto(s)
Interleucina-1beta/metabolismo , Lipopolisacáridos/farmacología , Proteínas de Unión al ARN/metabolismo , Proteína Serina-Treonina Quinasas de Interacción con Receptores/metabolismo , Proteínas Adaptadoras del Transporte Vesicular/metabolismo , Animales , Caspasa 8/metabolismo , Muerte Celular/efectos de los fármacos , Ratones Endogámicos C57BL , Unión Proteica/efectos de los fármacos , Receptores Tipo I de Factores de Necrosis Tumoral/metabolismo , YersiniaRESUMEN
In humans, respiratory infections with influenza A viruses can be lethal, but it is unclear whether non-respiratory influenza A infections can be equally lethal. Intraperitoneal infection makes the abdominal and pelvic organs accessible to pathogens because of the circulation of peritoneal fluid throughout the pelvis and abdomen. We found that high-dose intraperitoneal infection in mice with influenza A viruses resulted in severe sclerosis and structural damage in the pancreas, disruption of ovarian follicles, and massive infiltration of immune cells in the uterus. The intraperitoneal infections also caused robust upregulation of proinflammatory mediators including IL-6, BLC, and MIG. In addition, low-dose intraperitoneal infection with one influenza strain provided cross-protection against subsequent intraperitoneal or intranasal challenge with another influenza strain. Our results suggest that low-dose, non-respiratory administration might provide a route for influenza vaccination. Furthermore, these results provide insight on the pathological role of influenza A viruses in high-risk patients, including women and diabetic individuals.
RESUMEN
Influenza A virus (IAV) activates ZBP1-initiated RIPK3-dependent parallel pathways of necroptosis and apoptosis in infected cells. Although mice deficient in both pathways fail to control IAV and succumb to lethal respiratory infection, RIPK3-mediated apoptosis by itself can limit IAV, without need for necroptosis. However, whether necroptosis, conventionally considered a fail-safe cell death mechanism to apoptosis, can restrict IAV-or indeed any virus-in the absence of apoptosis is not known. Here, we use mice selectively deficient in IAV-activated apoptosis to show that necroptosis drives robust antiviral immune responses and promotes effective virus clearance from infected lungs when apoptosis is absent. We also demonstrate that apoptosis and necroptosis are mutually exclusive fates in IAV-infected cells. Thus, necroptosis is an independent, "stand-alone" cell death mechanism that fully compensates for the absence of apoptosis in antiviral host defense.
Asunto(s)
Caspasa 8/genética , Interacciones Microbiota-Huesped/genética , Virus de la Influenza A/inmunología , Necroptosis/genética , Infecciones por Orthomyxoviridae/inmunología , Inmunidad Adaptativa , Animales , Apoptosis/genética , Apoptosis/inmunología , Caspasa 8/metabolismo , Femenino , Técnicas de Sustitución del Gen , Interacciones Microbiota-Huesped/inmunología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Necroptosis/inmunología , Infecciones por Orthomyxoviridae/virología , Proteínas de Unión al ARN/metabolismo , Proteína Serina-Treonina Quinasas de Interacción con Receptores/metabolismoRESUMEN
Intraperitoneal inoculation with live influenza A virus confers protection against intranasal infections in mice and ferrets. However, the responses of peritoneal cells to influenza A virus have not been investigated. Here we show that intraperitoneal inoculation with A/WSN/1933 (H1N1) virus induced virus-reactive IgG production in the peritoneal cavity in mice. The infection resulted in substantial but transient B cell and macrophage depletion along with massive neutrophil infiltration, but virus growth was not detected. Influenza A viruses bound to α-2,6-linked sialic acids of B cells and macrophages and induced apoptotic death of peritoneal cavity cells. However, re-infection with A/WSN/1933 virus did not have adverse effects on immune cells most likely because of the neutralizing antibodies produced in response to the first exposure. Infection of BALB/c mice with A/WSN/1933 induced cross-protection against an otherwise lethal intraperitoneal dose of A/Hongkong/4801/2014 (H3N2) virus. This information suggests that immunological responses in the peritoneal cavity can induce effective defense against future virus infection. Considering the unexpected potent immunoregulatory activity of the peritoneal cells against influenza viruses, we suggest that comparative studies on various immune reactions after infection through different routes may contribute to better selection of vaccination routes in development of efficacious influenza vaccines.
Asunto(s)
Protección Cruzada/inmunología , Virus de la Influenza A/inmunología , Infecciones por Orthomyxoviridae/inmunología , Peritoneo/inmunología , Administración Intranasal , Animales , Anticuerpos Neutralizantes/inmunología , Anticuerpos Antivirales/inmunología , Perros , Subtipo H1N1 del Virus de la Influenza A/inmunología , Subtipo H1N1 del Virus de la Influenza A/fisiología , Subtipo H3N2 del Virus de la Influenza A/inmunología , Subtipo H3N2 del Virus de la Influenza A/fisiología , Virus de la Influenza A/fisiología , Vacunas contra la Influenza/inmunología , Inyecciones Intraperitoneales , Células de Riñón Canino Madin Darby , Ratones , Ratones Endogámicos BALB C , Infecciones por Orthomyxoviridae/virología , Cavidad Peritoneal/citología , Cavidad Peritoneal/virología , Vacunación/métodosRESUMEN
The cell surface transmembrane 4 superfamily member 5 protein (TM4SF5) has been implicated in various human cancers. Immunization with a peptide vaccine targeting human TM4SF5 has been shown to exert prophylactic and therapeutic effects against the development of hepatocellular carcinoma and colon cancer in mouse models. In this study, we developed a novel monoclonal antibody (mEC2CF) targeting a cyclic epitope of TM4SF5 and evaluated its reactivity to TM4SF5 in colorectal cancer (CRC) cells and cancer tissues. The isotype of mEC2CF was IgG2a and the antibody specifically recognized the cyclic peptide, based on ELISA. The antibody recognized recombinant TM4SF5 overexpressed in 293F cells, irrespective of Nglycosidase F treatment. The antibody was internalized into the cytosol after binding to the surface of TM4SF5expressing CRC cells, suggesting that this antibody may be useful in therapeutics. In addition, we evaluated TM4SF5 expression in the tissues of patients with CRC patients to determine its prognostic significance. TM4SF5 expression was assessed by immunohistochemistry using mEC2CF and tissue microarray blocks of 204 primary CRC samples. The overall rate of TM4SF5 overexpression in the samples (immunohistochemical score >4) was 27.0% (55 of 204). The increased expression of TM4SF5 was significantly associated with a shorter survival rate (P=0.0014) and a worse diseasefree survival (P=0.0483) of patients with CRC. No association was observed between TM4SF5 expression and clinicopathological characteristics, apart from tumor depth of invasion (P=0.027). These results suggest that our novel antibody can be used to detect endogenous and recombinant TM4SF5, and that TM4SF5 may be a possible marker for the poor prognosis of patients with CRC.
Asunto(s)
Biomarcadores de Tumor/genética , Neoplasias Colorrectales/genética , Proteínas de la Membrana/genética , Pronóstico , Adulto , Anciano , Anciano de 80 o más Años , Animales , Anticuerpos Antiidiotipos/biosíntesis , Anticuerpos Antiidiotipos/farmacología , Anticuerpos Monoclonales/biosíntesis , Anticuerpos Monoclonales/farmacología , Biomarcadores de Tumor/antagonistas & inhibidores , Biomarcadores de Tumor/inmunología , Biomarcadores de Tumor/aislamiento & purificación , Línea Celular Tumoral , Proliferación Celular/genética , Neoplasias Colorrectales/inmunología , Neoplasias Colorrectales/patología , Modelos Animales de Enfermedad , Supervivencia sin Enfermedad , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Inmunoglobulina G/inmunología , Masculino , Proteínas de la Membrana/antagonistas & inhibidores , Proteínas de la Membrana/inmunología , Proteínas de la Membrana/aislamiento & purificación , Ratones , Persona de Mediana Edad , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
CpG-DNA activates various immune cells, contributing to the host defense against bacteria. Here, we examined the biological function of CpG-DNA in the production of bacteria-reactive antibodies. The administration of CpG-DNA increased survival in mice following infection with methicillin-resistant S. aureus and protected immune cell populations in the peritoneal cavity, bone marrow, and spleen. CpG-DNA injection likewise increased bacteria-reactive antibodies in the mouse peritoneal fluid and serum, which was dependent on TLR9. B cells isolated from the peritoneal cavity produced bacteria-reactive antibodies in vitro following CpG-DNA administration that enhanced the phagocytic activity of the peritoneal cells. The bacteria-reactive monoclonal antibody enhanced phagocytosis in vitro and protected mice after S. aureus infection. Therefore, we suggest that CpG-DNA enhances the antibacterial activity of the immune system by protecting immune cells and triggering the production of bacteria-reactive antibodies. Consequently, we believe that monoclonal antibodies could aid in the treatment of antibiotic-resistant bacterial infections.
Asunto(s)
Adyuvantes Inmunológicos/administración & dosificación , Staphylococcus aureus Resistente a Meticilina/inmunología , Oligodesoxirribonucleótidos/administración & dosificación , Infecciones Estafilocócicas/terapia , Animales , Anticuerpos Antibacterianos/sangre , Anticuerpos Antibacterianos/inmunología , Anticuerpos Antibacterianos/metabolismo , Anticuerpos Monoclonales/sangre , Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales/metabolismo , Formación de Anticuerpos/efectos de los fármacos , Linfocitos B/efectos de los fármacos , Linfocitos B/inmunología , Linfocitos B/metabolismo , Modelos Animales de Enfermedad , Femenino , Humanos , Inyecciones Intraperitoneales , Ratones , Ratones Endogámicos BALB C , Ratones Noqueados , Fagocitosis/efectos de los fármacos , Fagocitosis/inmunología , Infecciones Estafilocócicas/sangre , Infecciones Estafilocócicas/inmunología , Infecciones Estafilocócicas/microbiología , Receptor Toll-Like 9/genética , Receptor Toll-Like 9/inmunología , Receptor Toll-Like 9/metabolismo , Resultado del TratamientoRESUMEN
Transmembrane 4 superfamily member 5 protein (TM4SF5) is a potential therapeutic target for hepatocellular carcinoma (HCC) and colon cancer. In a previous study, we demonstrated the prophylactic and therapeutic effects of a TM4SF5-specific peptide vaccine and monoclonal antibody in HCC and colon cancer in a mouse model. Here, we designed a cyclic peptide targeting TM4SF5. Cyclic peptide-specific antibodies were produced in mice after immunization with a complex of the peptide, CpG-DNA, and liposomes. Intravenous injection of the CT-26 mouse colon cancer cell line into mice induced tumors in the lung. Immunization with the peptide vaccine improved the survival rate and reduced the growth of lung tumors. We established a monoclonal antibody specific to the cyclic TM4SF5-based peptide and humanized the antibody sequence by complementarity determining region-grafting. The humanized antibody was reactive to the cyclic peptide and TM4SF5 protein. Treatment of CT-26 cells with the humanized antibody reduced cell motility in vitro. Furthermore, direct injection of the humanized anti-TM4SF5 antibody in vivo reduced growth of lung tumors in mouse metastasis model. Therefore, we conclude that the immunization with the cyclic peptide vaccine and injection of the TM4SF5-specifc humanized antibody have an anti-metastatic effect against colon cancer in mice. Importantly, the humanized antibody may serve as a starting platf.