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Drug-resistant Pseudomonas aeruginosa (PA) poses an emerging threat to human health with urgent need for alternative therapeutic approaches. Here, we deciphered the B cell and antibody response to the virulence-associated type III secretion system (T3SS) in a cohort of patients chronically infected with PA. Single-cell analytics revealed a diverse B cell receptor repertoire directed against the T3SS needle-tip protein PcrV, enabling the production of monoclonal antibodies (mAbs) abrogating T3SS-mediated cytotoxicity. Mechanistic studies involving cryoelectron microscopy identified a surface-exposed C-terminal PcrV epitope as the target of highly neutralizing mAbs with broad activity against drug-resistant PA isolates. These anti-PcrV mAbs were as effective as treatment with conventional antibiotics in vivo. Our study reveals that chronically infected patients represent a source of neutralizing antibodies, which can be exploited as therapeutics against PA.
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Anticuerpos Antibacterianos , Anticuerpos Neutralizantes , Infecciones por Pseudomonas , Pseudomonas aeruginosa , Humanos , Anticuerpos Antibacterianos/farmacología , Microscopía por Crioelectrón , Inmunoglobulinas/metabolismo , Pseudomonas aeruginosa/efectos de los fármacos , Pseudomonas aeruginosa/fisiología , Infecciones por Pseudomonas/tratamiento farmacológicoRESUMEN
The novel coronavirus pandemic, first reported in December 2019, was caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). SARS-CoV-2 infection leads to a strong immune response and activation of antigen-presenting cells, which can elicit acute respiratory distress syndrome (ARDS) characterized by the rapid onset of widespread inflammation, the so-called cytokine storm. In response to viral infections, monocytes are recruited into the lung and subsequently differentiate into dendritic cells (DCs). DCs are critical players in the development of the acute lung inflammation that causes ARDS. Here we focus on the interaction of a specific SARS-CoV-2 open reading frame protein, ORF8, with DCs. We show that ORF8 binds to DCs, causes a pre-maturation of differentiating DCs, and induces the secretion of multiple proinflammatory cytokines by these cells. In addition, we identified DC-SIGN as a possible interaction partner of ORF8 on DCs. Blockade of ORF8 leads to reduced production of IL-1ß, IL-6, IL-12p70, TNF-α, MCP-1 (also named CCL2), and IL-10 by DCs. Therefore, a neutralizing antibody blocking the ORF8-mediated cytokine and chemokine response could be an improved therapeutical strategy against severe SARS-CoV-2.
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Background: Symptoms lasting longer than 12 weeks after severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) infection are called post-coronavirus disease (COVID) syndrome (PCS). The identification of new biomarkers that predict the occurrence or course of PCS in terms of a post-viral syndrome is vital. T-cell dysfunction, cytokine imbalance, and impaired autoimmunity have been reported in PCS. Nevertheless, there is still a lack of conclusive information on the underlying mechanisms due to, among other things, a lack of controlled study designs. Methods: Here, we conducted a prospective, controlled study to characterize the humoral and cellular immune response in unvaccinated patients with and without PCS following SARS-CoV-2 infection over 7â¯months and unexposed donors. Results: Patients with PCS showed as early as 6â¯weeks and 7â¯months after symptom onset significantly increased frequencies of SARS-CoV-2-specific CD4+ and CD8+ T-cells secreting IFNγ, TNF, and expressing CD40L, as well as plasmacytoid dendritic cells (pDC) with an activated phenotype. Remarkably, the immunosuppressive counterparts type 1 regulatory T-cells (TR1: CD49b/LAG-3+) and IL-4 were more abundant in PCS+. Conclusion: This work describes immunological alterations between inflammation and immunosuppression in COVID-19 convalescents with and without PCS, which may provide potential directions for future epidemiological investigations and targeted treatments.
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Durable cell-mediated immune responses require efficient innate immune signaling and the release of pro-inflammatory cytokines. How precisely mRNA vaccines trigger innate immune cells for shaping antigen specific adaptive immunity remains unknown. Here, we show that SARS-CoV-2 mRNA vaccination primes human monocyte-derived macrophages for activation of the NLRP3 inflammasome. Spike protein exposed macrophages undergo NLRP3-driven pyroptotic cell death and subsequently secrete mature interleukin-1ß. These effects depend on activation of spleen tyrosine kinase (SYK) coupled to C-type lectin receptors. Using autologous cocultures, we show that SYK and NLRP3 orchestrate macrophage-driven activation of effector memory T cells. Furthermore, vaccination-induced macrophage priming can be enhanced with repetitive antigen exposure providing a rationale for prime-boost concepts to augment innate immune signaling in SARS-CoV-2 vaccination. Collectively, these findings identify SYK as a regulatory node capable of differentiating between primed and unprimed macrophages, which modulate spike protein-specific T cell responses.
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COVID-19 , Proteína con Dominio Pirina 3 de la Familia NLR , COVID-19/prevención & control , Vacunas contra la COVID-19 , Humanos , Inmunidad Innata , Inflamasomas/metabolismo , Interleucina-1beta , Péptidos y Proteínas de Señalización Intracelular/genética , Proteínas Tirosina Quinasas/metabolismo , ARN Mensajero/genética , SARS-CoV-2 , Glicoproteína de la Espiga del Coronavirus/genética , Quinasa Syk , VacunaciónRESUMEN
Metastatic small cell lung cancer (SCLC) is not curable. While SCLC is initially sensitive to chemotherapy, remissions are short-lived. The relapse is induced by chemotherapy-selected tumor stem cells, which express the AC133 epitope of the CD133 stem cell marker. We studied the effectiveness of AC133-specific CAR T cells post-chemotherapy using human primary SCLC and an orthotopic xenograft mouse model. AC133-specific CAR T cells migrated to SCLC tumor lesions, reduced the tumor burden, and prolonged survival in a humanized orthotopic SCLC model, but were not able to entirely eliminate tumors. We identified CD73 and PD-L1 as immune-escape mechanisms and combined PD-1-inhibition and CD73-inhibition with CAR T cell treatment. This triple-immunotherapy induced cures in 25% of the mice, without signs of graft-versus-host disease or bone marrow failure. AC133+ cancer stem cells and PD-L1+CD73+ myeloid cells were detectable in primary human SCLC tissues, suggesting that patients may benefit from the triple-immunotherapy. We conclude that the combination of AC133-specific CAR T cells, anti-PD-1-antibody and CD73-inhibitor specifically eliminates chemo-resistant tumor stem cells, overcomes SCLC-mediated T cell inhibition, and might induce long-term complete remission in an otherwise incurable disease.
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Neoplasias Pulmonares , Carcinoma Pulmonar de Células Pequeñas , Animales , Antígeno B7-H1 , Línea Celular Tumoral , Humanos , Inmunoterapia Adoptiva , Neoplasias Pulmonares/patología , Ratones , Recurrencia Local de Neoplasia , Carcinoma Pulmonar de Células Pequeñas/patología , Carcinoma Pulmonar de Células Pequeñas/terapiaRESUMEN
This article has been retracted: please see Elsevier Policy on Article Withdrawal (https://www.elsevier.com/about/our-business/policies/article-withdrawal). This article has been retracted at the request of the Editor-in-Chief. Following the publication of the above article, the Editor was notified that an error occurred in which all images were published with incorrect versions. The Editor has taken the decision that the manuscript is no longer acceptable in its current form, nor with a corrigendum, as the extensive changes to the figures and publication would lead to ambiguity for our readers. We have therefore made the decision to retract this manuscript from Cancer Letters with the possibility of resubmission and republication of the manuscript in its corrected form after peer review.
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Infections with viral pathogens are widespread and can cause a variety of different diseases. In-depth knowledge about viral triggers initiating an immune response is necessary to decipher viral pathogenesis. Inflammasomes, as part of the innate immune system, can be activated by viral pathogens. However, viral structural components responsible for inflammasome activation remain largely unknown. Here we analyzed glycoproteins derived from SARS-CoV-1/2, HCMV and HCV, required for viral entry and fusion, as potential triggers of NLRP3 inflammasome activation and pyroptosis in THP-1 macrophages. All tested glycoproteins were able to potently induce NLRP3 inflammasome activation, indicated by ASC-SPECK formation and secretion of cleaved IL-1ß. Lytic cell death via gasdermin D (GSDMD), pore formation, and pyroptosis are required for IL-1ß release. As a hallmark of pyroptosis, we were able to detect cleavage of GSDMD and, correspondingly, cell death in THP-1 macrophages. CRISPR-Cas9 knockout of NLRP3 and GSDMD in THP-1 macrophages confirmed and strongly support the evidence that viral glycoproteins can act as innate immunity triggers. With our study, we decipher key mechanisms of viral pathogenesis by showing that viral glycoproteins potently induce innate immune responses. These insights could be beneficial in vaccine development and provide new impulses for the investigation of vaccine-induced innate immunity.
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Inmunidad Innata/inmunología , Inflamasomas/inmunología , Macrófagos/inmunología , Proteína con Dominio Pirina 3 de la Familia NLR/inmunología , Proteínas del Envoltorio Viral/inmunología , Proteínas Virales de Fusión/inmunología , Línea Celular Tumoral , Citomegalovirus/inmunología , Hepacivirus/inmunología , Humanos , Interleucina-1beta/biosíntesis , Interleucina-1beta/inmunología , Piroptosis/inmunología , Coronavirus Relacionado al Síndrome Respiratorio Agudo Severo/inmunología , SARS-CoV-2/inmunología , Células THP-1RESUMEN
This article has been retracted: please see Elsevier Policy on Article Withdrawal (https://www.elsevier.com/about/our-business/policies/article-withdrawal). This article has been retracted at the request of the Editor-in-Chief. Following the publication of the above article, the Editor was notified that an error occurred in which all images were published with incorrect versions. The Editor has taken the decision that the manuscript is no longer acceptable in its current form, nor with a corrigendum, as the extensive changes to the figures and publication would lead to ambiguity for our readers. We have therefore made the decision to retract this manuscript from Cancer Letters with the possibility of resubmission and republication of the manuscript in its corrected form after peer review.
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5'-Nucleotidasa/genética , Antígeno AC133/genética , Antígeno B7-H1/genética , Carcinoma Pulmonar de Células Pequeñas/terapia , 5'-Nucleotidasa/antagonistas & inhibidores , Antígeno AC133/inmunología , Animales , Anticuerpos Antiidiotipos/farmacología , Antígeno B7-H1/antagonistas & inhibidores , Línea Celular Tumoral , Femenino , Xenoinjertos , Humanos , Inmunoterapia Adoptiva/tendencias , Masculino , Ratones , Metástasis de la Neoplasia , Células Madre Neoplásicas/efectos de los fármacos , Células Madre Neoplásicas/inmunología , Células Madre Neoplásicas/patología , Receptor de Muerte Celular Programada 1/antagonistas & inhibidores , Receptor de Muerte Celular Programada 1/genética , Receptores Quiméricos de Antígenos/genética , Receptores Quiméricos de Antígenos/inmunología , Receptores Quiméricos de Antígenos/uso terapéutico , Carcinoma Pulmonar de Células Pequeñas/genética , Carcinoma Pulmonar de Células Pequeñas/inmunología , Carcinoma Pulmonar de Células Pequeñas/patología , Linfocitos T/inmunología , Carga TumoralRESUMEN
Innate immunity triggers responsible for viral control or hyperinflammation in COVID-19 are largely unknown. Here we show that the SARS-CoV-2 spike protein (S-protein) primes inflammasome formation and release of mature interleukin-1ß (IL-1ß) in macrophages derived from COVID-19 patients but not in macrophages from healthy SARS-CoV-2 naïve individuals. Furthermore, longitudinal analyses reveal robust S-protein-driven inflammasome activation in macrophages isolated from convalescent COVID-19 patients, which correlates with distinct epigenetic and gene expression signatures suggesting innate immune memory after recovery from COVID-19. Importantly, we show that S-protein-driven IL-1ß secretion from patient-derived macrophages requires non-specific monocyte pre-activation in vivo to trigger NLRP3-inflammasome signaling. Our findings reveal that SARS-CoV-2 infection causes profound and long-lived reprogramming of macrophages resulting in augmented immunogenicity of the SARS-CoV-2 S-protein, a major vaccine antigen and potent driver of adaptive and innate immune signaling.
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COVID-19 , Glicoproteína de la Espiga del Coronavirus , Humanos , Inmunidad Innata , Inflamasomas , Interleucina-1beta , Macrófagos , Proteína con Dominio Pirina 3 de la Familia NLR/genética , SARS-CoV-2RESUMEN
The ongoing SARS-CoV-2 pandemic stresses the need for effective antiviral drugs that can quickly be applied in order to reduce morbidity, mortality, and ideally viral transmission. By repurposing of broadly active antiviral drugs and compounds that are known to inhibit viral replication of related viruses, several advances could be made in the development of treatment strategies against COVID-19. The nucleoside analog remdesivir, which is known for its potent in vitro activity against Ebolavirus and other RNA viruses, was recently shown to reduce the time to recovery in patients with severe COVID-19. It is to date the only approved antiviral for treating COVID-19. Here, we provide a mechanism and evidence-based comparative review of remdesivir and other repurposed drugs with proven in vitro activity against SARS-CoV-2.
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Adenosina Monofosfato/análogos & derivados , Alanina/análogos & derivados , Antivirales/uso terapéutico , Tratamiento Farmacológico de COVID-19 , Reposicionamiento de Medicamentos , SARS-CoV-2/efectos de los fármacos , Adenosina Monofosfato/farmacología , Adenosina Monofosfato/uso terapéutico , Alanina/farmacología , Alanina/uso terapéutico , Amidas/farmacología , Amidas/uso terapéutico , Antivirales/farmacología , Benzamidinas , Reposicionamiento de Medicamentos/métodos , Ésteres/farmacología , Ésteres/uso terapéutico , Guanidinas/farmacología , Guanidinas/uso terapéutico , Guanina/farmacología , Guanina/uso terapéutico , Humanos , Indoles/farmacología , Indoles/uso terapéutico , Lopinavir/farmacología , Lopinavir/uso terapéutico , Inhibidores de Proteasas/farmacología , Inhibidores de Proteasas/uso terapéutico , Pirazinas/farmacología , Pirazinas/uso terapéutico , Ribavirina/farmacología , Ribavirina/uso terapéutico , Ritonavir/farmacología , Ritonavir/uso terapéutico , SARS-CoV-2/fisiología , Internalización del Virus/efectos de los fármacos , Replicación Viral/efectos de los fármacosAsunto(s)
Adenosina Monofosfato/análogos & derivados , Alanina/análogos & derivados , Antivirales/sangre , COVID-19/sangre , Fallo Renal Crónico/sangre , Diálisis Renal/tendencias , Adenosina Monofosfato/administración & dosificación , Adenosina Monofosfato/sangre , Adenosina Monofosfato/farmacocinética , Anciano , Alanina/administración & dosificación , Alanina/sangre , Alanina/farmacocinética , Antivirales/administración & dosificación , Antivirales/farmacocinética , COVID-19/complicaciones , Humanos , Infusiones Intravenosas/métodos , Fallo Renal Crónico/complicaciones , Fallo Renal Crónico/tratamiento farmacológico , Masculino , Tratamiento Farmacológico de COVID-19RESUMEN
The prevalence of bacterial pathogens being resistant to antibiotic treatment is increasing worldwide, leading to a severe global health challenge. Simultaneously, the development and approval of new antibiotics stagnated in the past decades, leading to an urgent need for novel approaches to avoid the spread of untreatable bacterial infections in the future. We developed a highly comprehensive screening platform based on quantification of pathogen driven host-cell death to detect new anti-virulence drugs targeting Pseudomonas aeruginosa (Pa) and Salmonella enterica serovar Typhimurium (ST), both known for their emerging antibiotic resistance. By screening over 10,000 small molecules we could identify several substances showing promising effects on Pa and ST pathogenicity in our in vitro infection model. Importantly, we could detect compounds potently inhibiting bacteria induced killing of host cells and one novel comipound with impact on the function of the type 3 secretion system (T3SS) of ST. Thus, we provide proof of concept data of rapid and feasible medium- to high-throughput drug screening assays targeting virulence mechanisms of two major Gram-negative pathogens.
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Acute myeloid leukemia (AML) initiating and sustaining cells maintain high cell-surface similarity with their cells-of-origin, i.e., hematopoietic stem and progenitor cells (HSPCs), and identification of truly distinguishing leukemia-private antigens has remained elusive to date. To nonetheless utilize surface antigen-directed immunotherapy in AML, we here propose targeting both, healthy and malignant human HSPC, by chimeric antigen receptor (CAR) T-cells with specificity against CD117, the cognate receptor for stem cell factor. This approach should spare most mature hematopoietic cells and would require CAR T termination followed by subsequent transplantation of healthy HSPCs to rescue hematopoiesis. We successfully generated anti-CD117 CAR T-cells from healthy donors and AML patients. Anti-CD117 CAR T-cells efficiently targeted healthy and leukemic CD117-positive cells in vitro. In mice xenografted with healthy human hematopoiesis, they eliminated CD117-expressing, but not CD117-negative human cells. Importantly, in mice xenografted with primary human CD117-positive AML, they eradicated disease in a therapeutic setting. Administration of ATG in combination with rituximab, which binds to the co-expressed CAR T-cell transduction/selection marker RQR8, led to CAR T-cell depletion. Thus, we here provide the first proof of concept for the generation and preclinical efficacy of CAR T-cells directed against CD117-expressing human hematopoietic cells.
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Inmunoterapia Adoptiva , Proteínas Proto-Oncogénicas c-kit/antagonistas & inhibidores , Receptores de Antígenos de Linfocitos T/inmunología , Receptores Quiméricos de Antígenos/inmunología , Linfocitos T/inmunología , Linfocitos T/metabolismo , Animales , Biomarcadores , Biopsia , Médula Ósea/metabolismo , Médula Ósea/patología , Línea Celular Tumoral , Modelos Animales de Enfermedad , Expresión Génica , Neoplasias Hematológicas/diagnóstico , Neoplasias Hematológicas/inmunología , Neoplasias Hematológicas/metabolismo , Neoplasias Hematológicas/terapia , Células Madre Hematopoyéticas/inmunología , Células Madre Hematopoyéticas/metabolismo , Humanos , Inmunofenotipificación , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/inmunología , Leucemia Mieloide Aguda/patología , Leucemia Mieloide Aguda/terapia , Depleción Linfocítica , Ratones , Proteínas Proto-Oncogénicas c-kit/genética , Proteínas Proto-Oncogénicas c-kit/metabolismo , Receptores de Antígenos de Linfocitos T/genética , Receptores Quiméricos de Antígenos/genética , Resultado del Tratamiento , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Recently, an immunodeficiency syndrome caused by guanine-adenine-thymine-adenine 2 (GATA2) deficiency has been described. The syndrome is characterized by (i) typical onset in early adulthood, (ii) profound peripheral blood cytopenias of monocytes, B lymphocytes, and NK cells, (iii) distinct susceptibility to disseminated non-tuberculous mycobacterial (NTM) and other opportunistic infections (particularly human papillomavirus), and (iv) a high risk of developing hematologic malignancies (myelodysplastic syndromes (MDS); acute myeloid leukemias (AML)). Considerable clinical heterogeneity exists among patients with GATA2 deficiency, but once infectious symptoms occur or MDS/AML arises, survival declines significantly. Allogeneic hematopoietic cell transplantation (HCT) currently provides the only curative treatment option for both MDS/AML and dysfunctional immunity with life-threatening opportunistic infections. Strategies regarding timing of allogeneic HCT, antimicrobial prophylaxis and treatment, intensity of the preparative regimen, and optimal donor and graft source have not been clearly defined due to the rarity of the disease. Here, we provide a comprehensive analysis of the available literature and published case reports on the use of allogeneic HCT in patients with GATA2 deficiency. In addition, a case of a young woman with GATA2 deficiency, who developed an immune reconstitution inflammatory syndrome in her mycobacterial skin lesions post allogeneic HCT is presented and illustrates distinct problems encountered in this disease context.
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Factor de Transcripción GATA2/deficiencia , Trasplante de Células Madre Hematopoyéticas , Síndrome Inflamatorio de Reconstitución Inmune/etiología , Síndromes de Inmunodeficiencia/terapia , Infecciones por Mycobacterium no Tuberculosas/etiología , Adolescente , Adulto , Aloinjertos , Niño , Preescolar , Susceptibilidad a Enfermedades , Femenino , Humanos , Síndromes de Inmunodeficiencia/genética , Masculino , Persona de Mediana Edad , Infecciones por Mycobacterium no Tuberculosas/inmunología , Infecciones por Mycobacterium no Tuberculosas/patología , Mycobacterium abscessus/aislamiento & purificación , Síndromes Mielodisplásicos/etiología , Trasplante de Células Madre de Sangre Periférica , Embolia Pulmonar/etiología , Úlcera Cutánea/etiología , Verrugas/etiología , Adulto JovenRESUMEN
Acid sphingomyelinase (ASM) is a key enzyme in sphingolipid metabolism that converts sphingomyelin to ceramide, thereby modulating membrane structures and signal transduction. Bacterial pathogens can manipulate ASM activity and function, and use host sphingolipids during multiple steps of their infection process. An increase in ceramides upon infection results in the formation of ceramide-enriched membrane platforms that serve to cluster receptor molecules and organize intracellular signaling molecules, thus facilitating bacterial uptake. In this review, we focus on how extracellular bacterial pathogens target ASM and modulate membrane properties and signaling pathways to gain entry into eukaryotic cells or induce cell death. We describe how intracellular pathogens interfere with the intralysosomal functions of ASM to favor replication and survival. In addition, bacteria utilize their own sphingomyelinases as virulence factors to modulate sphingolipid metabolism. The potential of ASM as a target for treating bacterial infections is also discussed.
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Infecciones Bacterianas/enzimología , Infecciones Bacterianas/metabolismo , Esfingolípidos/metabolismo , Esfingomielina Fosfodiesterasa/metabolismo , Animales , HumanosRESUMEN
The Gram-negative extracellular bacterium Neisseria meningitidis is one of the most common aetiological agents of bacterial meningitis affecting predominantly young children worldwide. This bacterium is normally a quiescent coloniser of the upper respiratory tract, but in some individuals it enters the blood stream and causes invasive diseases, such as septicaemia and meningitis. Interactions of N. meningitidis with human endothelial cells are crucially involved in pathogencitiy, and great efforts have been made to understand these molecular interactions. The aim of this review article is to provide an overview of the interactions of meningococcal virulence factors with host endothelial cells at the blood-cerebrospinal fluid barrier.
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Barrera Hematoencefálica/microbiología , Células Endoteliales/microbiología , Neisseria meningitidis/patogenicidad , Factores de Virulencia/metabolismo , Líquido Cefalorraquídeo/microbiología , Humanos , Infecciones Meningocócicas/microbiología , Neisseria meningitidis/metabolismo , Sistema Respiratorio/microbiologíaRESUMEN
The interaction with brain endothelial cells is central to the pathogenicity of Neisseria meningitidis infections. Here, we show that N. meningitidis causes transient activation of acid sphingomyelinase (ASM) followed by ceramide release in brain endothelial cells. In response to N. meningitidis infection, ASM and ceramide are displayed at the outer leaflet of the cell membrane and condense into large membrane platforms which also concentrate the ErbB2 receptor. The outer membrane protein Opc and phosphatidylcholine-specific phospholipase C that is activated upon binding of the pathogen to heparan sulfate proteoglycans, are required for N. meningitidis-mediated ASM activation. Pharmacologic or genetic ablation of ASM abrogated meningococcal internalization without affecting bacterial adherence. In accordance, the restricted invasiveness of a defined set of pathogenic isolates of the ST-11/ST-8 clonal complex into brain endothelial cells directly correlated with their restricted ability to induce ASM and ceramide release. In conclusion, ASM activation and ceramide release are essential for internalization of Opc-expressing meningococci into brain endothelial cells, and this segregates with invasiveness of N. meningitidis strains.