RESUMEN
Upon infection, mycobacteria, such as Mycobacterium tuberculosis (Mtb) and nontuberculous mycobacteria (NTM), are recognized by host innate immune cells, triggering a series of intracellular processes that promote mycobacterial killing. Mycobacteria, however, have developed multiple counter-strategies to persist and survive inside host cells. By manipulating host effector mechanisms, including phagosome maturation, vacuolar escape, autophagy, antigen presentation, and metabolic pathways, pathogenic mycobacteria are able to establish long-lasting infection. Counteracting these mycobacteria-induced host modifying mechanisms can be accomplished by host-directed therapeutic (HDT) strategies. HDTs offer several major advantages compared to conventional antibiotics: (a) HDTs can be effective against both drug-resistant and drug-susceptible bacteria, as well as potentially dormant mycobacteria; (b) HDTs are less likely to induce bacterial drug resistance; and (c) HDTs could synergize with, or shorten antibiotic treatment by targeting different pathways. In this review, we will explore host-pathogen interactions that have been identified for Mtb for which potential HDTs impacting both innate and adaptive immunity are available, and outline those worthy of future research. We will also discuss possibilities to target NTM infection by HDT, although current knowledge regarding host-pathogen interactions for NTM is limited compared to Mtb. Finally, we speculate that combinatorial HDT strategies can potentially synergize to achieve optimal mycobacterial host immune control.
Asunto(s)
Mycobacterium tuberculosis , Micobacterias no Tuberculosas , Antibacterianos/uso terapéutico , Autofagia , Interacciones Huésped-PatógenoRESUMEN
Mycobacterium tuberculosis (Mtb) is the causative pathogen of tuberculosis, the most lethal infectious disease resulting in 1.3 million deaths annually. Treatments against Mtb are increasingly impaired by the growing prevalence of antimicrobial drug resistance, which necessitates the development of new antibiotics or alternative therapeutic approaches. Upon infecting host cells, predominantly macrophages, Mtb becomes critically dependent on lipids as a source of nutrients. Additionally, Mtb produces numerous lipid-based virulence factors that contribute to the pathogen's ability to interfere with the host's immune responses and to create a lipid rich environment for itself. As lipids, lipid metabolism and manipulating host lipid metabolism play an important role for the virulence of Mtb, this review provides a state-of-the-art overview of mycobacterial lipid metabolism and concomitant role of host metabolism and host-pathogen interaction therein. While doing so, we will emphasize unexploited bacteria-directed and host-directed drug targets, and highlight potential synergistic drug combinations that hold promise for the development of new therapeutic interventions.
RESUMEN
Antibiotic resistance is a major problem, with methicillin-resistant Staphylococcus aureus (MRSA) being a prototypical example in surgical and community-acquired infections. S. aureus, like many pathogens, is immune evasive and able to multiply within host immune cells. Consequently, compounds that aid host immunity (e.g., by stimulating the host-mediated killing of pathogens) are appealing alternatives or adjuncts to classical antibiotics. Azithromycin is both an antibacterial and an immunomodulatory drug that accumulates in immune cells. We set out to improve the immunomodulatory properties of azithromycin by coupling the immune activators, nitric oxide and acetate, to its core structure. This new compound, designated CSY5669, enhanced the intracellular killing of MRSA by 45% ± 20% in monocyte-derived macrophages and by 55% ± 15% in peripheral blood leukocytes, compared with untreated controls. CSY5669-treated peripheral blood leukocytes produced fewer proinflammatory cytokines, while in both monocyte-derived macrophages and peripheral blood leukocytes, phagocytosis, ROS production, and degranulation were unaffected. In mice with MRSA pneumonia, CSY5669 treatment reduced inflammation, lung pathology and vascular leakage with doses as low as 0.01 µmol/kg p.o. CSY5669 had diminished direct in vitro antibacterial properties compared with azithromycin. Also, CSY5669 was immunomodulatory at concentrations well below 1% of the minimum inhibitory concentration, which would minimize selection for macrolide-resistant bacteria if it were to be used as a host-directed therapy. This study highlights the potential of CSY5669 as a possible adjunctive therapy in pneumonia caused by MRSA, as CSY5669 could enhance bacterial eradication while simultaneously limiting inflammation-associated pathology.
Asunto(s)
Staphylococcus aureus Resistente a Meticilina , Neumonía Estafilocócica , Profármacos , Infecciones Estafilocócicas , Animales , Antibacterianos/farmacología , Antibacterianos/uso terapéutico , Azitromicina/farmacología , Azitromicina/uso terapéutico , Citocinas , Inflamación/tratamiento farmacológico , Ratones , Pruebas de Sensibilidad Microbiana , Óxido Nítrico , Neumonía Estafilocócica/tratamiento farmacológico , Profármacos/uso terapéutico , Especies Reactivas de Oxígeno , Infecciones Estafilocócicas/tratamiento farmacológico , Staphylococcus aureusRESUMEN
Despite high levels of CXCR3 ligands in mechanically ventilated COVID-19 patients, BALF CD8 T cells were not enriched in CXCR3+ cells but rather CCR6+ , likely due to high CCL20 levels in BALF, and had very high PD-1 expression. In mechanically ventilated, but not ward, patients Th-1 immunity is impaired. â.
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Linfocitos T CD8-positivos/inmunología , COVID-19/inmunología , Quimiocina CCL20/inmunología , Pulmón/inmunología , Receptores CCR6/inmunología , Respiración Artificial , SARS-CoV-2/inmunología , Anciano , Anciano de 80 o más Años , Linfocitos T CD8-positivos/patología , COVID-19/patología , COVID-19/terapia , Femenino , Humanos , Pulmón/patología , Recuento de Linfocitos , Masculino , Persona de Mediana EdadRESUMEN
BACKGROUND: Knowledge of the pathophysiology of COVID-19 is almost exclusively derived from studies that examined the immune response in blood. We here aimed to analyse the pulmonary immune response during severe COVID-19 and to compare this with blood responses. METHODS: This was an observational study in patients with COVID-19 admitted to the intensive care unit (ICU). Mononuclear cells were purified from bronchoalveolar lavage fluid (BALF) and blood, and analysed by spectral flow cytometry; inflammatory mediators were measured in BALF and plasma. FINDINGS: Paired blood and BALF samples were obtained from 17 patients, four of whom died in the ICU. Macrophages and T cells were the most abundant cells in BALF, with a high percentage of T cells expressing the ƴδ T cell receptor. In the lungs, both CD4 and CD8 T cells were predominantly effector memory cells (87·3% and 83·8%, respectively), and these cells expressed higher levels of the exhaustion marker programmad death-1 than in peripheral blood. Prolonged ICU stay (>14 days) was associated with a reduced proportion of activated T cells in peripheral blood and even more so in BALF. T cell activation in blood, but not in BALF, was higher in fatal COVID-19 cases. Increased levels of inflammatory mediators were more pronounced in BALF than in plasma. INTERPRETATION: The bronchoalveolar immune response in COVID-19 has a unique local profile that strongly differs from the immune profile in peripheral blood. Fully elucidating COVID-19 pathophysiology will require investigation of the pulmonary immune response.
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COVID-19/inmunología , Inmunidad Celular/fisiología , Mediadores de Inflamación/metabolismo , Anciano , Líquido del Lavado Bronquioalveolar/química , Líquido del Lavado Bronquioalveolar/citología , COVID-19/sangre , COVID-19/patología , Cuidados Críticos , Enfermedad Crítica , Femenino , Citometría de Flujo , Humanos , Macrófagos/fisiología , Masculino , Persona de Mediana Edad , Linfocitos T/fisiologíaRESUMEN
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of the current coronavirus disease 2019 (COVID-19) pandemic. Understanding the immune response that provides specific immunity but may also lead to immunopathology is crucial for the design of potential preventive and therapeutic strategies. Here, we characterized and quantified SARS-CoV-2-specific immune responses in patients with different clinical courses. Compared to individuals with a mild clinical presentation, CD4+ T-cell responses were qualitatively impaired in critically ill patients. Strikingly, however, in these patients the specific IgG antibody response was remarkably strong. Furthermore, in these critically ill patients, a massive influx of circulating T cells into the lungs was observed, overwhelming the local T-cell compartment, and indicative of vascular leakage. The observed disparate T- and B-cell responses could be indicative of a deregulated immune response in critically ill COVID-19 patients.
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Anticuerpos Antivirales/inmunología , Linfocitos B/inmunología , Linfocitos T CD4-Positivos/inmunología , COVID-19/inmunología , Inmunoglobulina G/inmunología , SARS-CoV-2/inmunología , Adulto , Anciano , Linfocitos B/patología , Linfocitos T CD4-Positivos/patología , COVID-19/patología , Femenino , Humanos , Masculino , Persona de Mediana Edad , Índice de Severidad de la EnfermedadRESUMEN
Patients refractory to platelet transfusions because of alloimmunization require HLA-matched platelets, which is only possible if a large HLA-typed donor pool is available. However, even then, patients with broad immunization or rare haplotypes may not have suitable donors. In these patients, transfusions with platelets showing low HLA class I expression may be an alternative to fully HLA-matched transfusions. In this study, we quantified the proportion of donors with consistently low HLA-B8, -B12, and -B35 expression on platelets using human monoclonal antibodies specific for these antigens. Furthermore, as model for in vivo clearance, antibody-mediated internalization of these platelets by macrophages was investigated. The expression of HLA-B8, -B12, or -B35 on platelets was extremely variable between individuals (coefficients of variation, 41.4% to 73.6%). For HLA-B8, but not for HLA-B12 or -B35, this variation was in part explained by zygosity. The variation was most pronounced in, but not exclusive to, platelets. Expression within one donor was consistent over time. Remarkably, 32% of 113 HLA-B8, 34% of 98 HLA-B12, and 9% of 66 HLA-B35 donors showed platelet antigen expression that was not or only minimally above background. Antibody-mediated internalization of platelets by macrophages correlated with antibody opsonization and antigen expression and was absent in platelets with low or minimal HLA expression. In conclusion, our findings indicate that a substantial proportion of donors have platelets with consistently low expression of specific HLA class I antigens. These platelets may be used to treat refractory patients with antibodies directed against these particular antigens, despite HLA mismatches.
Asunto(s)
Plaquetas/inmunología , Antígenos HLA-B/metabolismo , Antígeno HLA-B35/metabolismo , Antígeno HLA-B8/metabolismo , Isoanticuerpos/inmunología , Macrófagos/metabolismo , Donantes de Tejidos , Plaquetas/metabolismo , Antígenos HLA-B/inmunología , Antígeno HLA-B35/inmunología , Antígeno HLA-B8/inmunología , Prueba de Histocompatibilidad , Humanos , Macrófagos/inmunología , Selección de Paciente , Transfusión de Plaquetas/normasRESUMEN
BACKGROUND: Storage of platelet concentrates (PCs) results in reduced recovery and survival of transfused platelets (PLTs). Upon storage PLTs develop storage lesion that can be monitored by several laboratory tests. However, correlation of these descriptive tests with corrected count increments (CCIs), a marker frequently used to establish the effectiveness of PLT transfusions, is limited or unknown. This study investigated to what extent a functional test or a combined in vitro rating score improves the correlation of laboratory tests with 1-hour CCI. STUDY DESIGN AND METHODS: PCs were analyzed using six different laboratory tests (n = 123) before transfusion in a prophylactic setting to 74 hematooncologic patients. Linear regression and Spearman correlation were used to determine associations between descriptive (either separately or combined in an in vitro rating score) or functional test results and 1-hour CCIs obtained after transfusion. RESULTS: CD62P expression (r = -0.45), annexin V binding (r = -0.36), the updated in vitro rating score (r = 0.50), and PLT responsiveness after thrombin receptor activator for peptide-6 (TRAP) (r = 0.43-0.57) or adenosine diphosphate stimulation (r = 0.11-0.51) significantly correlated to 1-hour CCIs obtained after transfusion, whereas lactate concentration, ThromboLUX score, and thromboelastography measurements did not. The strongest correlations were observed for in vitro rating score and PLT responsiveness after TRAP stimulation and these tests could explain 24 and 33% of the observed variation in 1-hour CCI, respectively. CONCLUSION: Combining descriptive markers in one in vitro rating score improved correlation to 1-hour CCI compared to the tests separately. Of all tests investigated, mean PLT responsiveness after TRAP stimulation showed the strongest clinical correlation and was best able to predict the 1-hour CCI.
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Plaquetas , Selectina-P/metabolismo , Transfusión de Plaquetas , Control de Calidad , Adulto , Anciano , Plaquetas/citología , Plaquetas/metabolismo , Conservación de la Sangre/métodos , Femenino , Humanos , Masculino , Persona de Mediana Edad , Recuento de PlaquetasRESUMEN
BACKGROUND: Platelet transfusions can induce alloimmunization against HLA antigens. The use of pathogen-reduced platelet concentrates (PCs) was suggested to reduce HLA alloimmunization and concomitant transfusion refractoriness. METHODS: This study investigated HLA alloimmunization in available samples from 448 hemato-oncological patients who were randomized for the Pathogen Reduction Evaluation and Predictive Analytical Rating Score (PREPAReS) trial to receive either untreated or pathogen-reduced PCs (Mirasol, Terumo BCT Inc.). Anti-HLA Class I and II antibodies were determined before the first platelet transfusion and weekly thereafter using multiplex assay with standard cutoffs to detect low- as well as high-level antibodies. RESULTS: When using the lower cutoff, in patients who were antibody negative at enrollment, 5.4% (n = 12) developed anti-HLA Class I antibodies after receiving untreated PCs, while this was significantly higher in patients receiving pathogen-reduced PCs, 12.8% (n = 29; p = 0.009, intention-to-treat [ITT] analysis). A similar but nonsignificant trend was observed in the per-protocol (PP) analysis (5.4% vs. 10.1%; p = 0.15). HLA class II antibody formation was similar between both types of PCs in the ITT analysis, while the PP analysis showed a trend toward lower immunization after receiving pathogen-reduced PCs. Multivariate analysis identified receiving pathogen-reduced platelets as an independent risk factor for HLA Class I alloimmunization (ITT: odds ratio [95% confidence interval] = 3.02 [1.42-6.51], PP: odds ratio [95% confidence interval] = 2.77 [1.00-5.40]), without affecting HLA Class II alloimmunization. When using the high cutoff value, the difference in HLA Class I alloimmunization between study arms remained significant in the ITT analysis and again was not significant in the PP analysis. CONCLUSION: Our data clearly indicate that Mirasol pathogen inactivation does not prevent HLA Class I or II alloimmunization after platelet transfusions.
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Antígenos HLA , Neoplasias Hematológicas , Inmunización , Isoanticuerpos , Transfusión de Plaquetas/efectos adversos , Reacción a la Transfusión , Anciano , Femenino , Antígenos HLA/sangre , Antígenos HLA/inmunología , Neoplasias Hematológicas/sangre , Neoplasias Hematológicas/inmunología , Neoplasias Hematológicas/terapia , Humanos , Isoanticuerpos/sangre , Isoanticuerpos/inmunología , Masculino , Persona de Mediana Edad , Estudios Prospectivos , Reacción a la Transfusión/sangre , Reacción a la Transfusión/inmunologíaRESUMEN
OBJECTIVES: Therapeutic antibodies can provoke an antidrug antibody (ADA) response, which can form soluble immune complexes with the drug in potentially high amounts. Nevertheless, ADA-associated adverse events are usually rare, although with notable exceptions including infliximab. The immune activating effects and the eventual fate of these 'anti-idiotype' complexes are poorly studied, hampering assessment of ADA-associated risk of adverse events. We investigated the in vitro formation and biological activities of ADA-drug anti-idiotype immune complexes using patient-derived monoclonal anti-infliximab antibodies. METHODS: Size distribution and conformation of ADA-drug complexes were characterised by size-exclusion chromatography and electron microscopy. Internalisation of and immune activation by complexes of defined size was visualised with flow imaging, whole blood cell assay and C4b/c ELISA. RESULTS: Size and conformation of immune complexes depended on the concentrations and ratio of drug and ADA; large complexes (>6 IgGs) formed only with high ADA titres. Macrophages efficiently internalised tetrameric and bigger complexes in vitro, but not dimers. Corroborating these results, ex vivo analysis of patient sera demonstrated only dimeric complexes in circulation.No activation of immune cells by anti-idiotype complexes was observed, and only very large complexes activated complement. Unlike Fc-linked hexamers, anti-idiotype hexamers did not activate complement, demonstrating that besides size, conformation governs immune complex potential for triggering effector functions. CONCLUSIONS: Anti-idiotype ADA-drug complexes generally have restricted immune activation capacity. Large, irregularly shaped complexes only form at high concentrations of both drug and ADA, as may be achieved during intravenous infusion of infliximab, explaining the rarity of serious ADA-associated adverse events.
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Anticuerpos/inmunología , Formación de Anticuerpos/efectos de los fármacos , Complejo Antígeno-Anticuerpo/inmunología , Antirreumáticos/inmunología , Infliximab/inmunología , Cromatografía en Gel , Ensayo de Inmunoadsorción Enzimática , Humanos , Suero/inmunologíaRESUMEN
HLA antibodies are associated with refractoriness to platelet transfusion, leading to rapid platelet clearance, sometimes coinciding with clinical side effects such as fever and chills. The presence of HLA antibodies is not always manifested by clinical symptoms. It is currently unclear why refractoriness to platelet transfusion is only observed in a subset of patients. Here, we utilized the availability of a unique panel of human monoclonal antibodies to study whether these were capable of activating platelets. Three out of eight human HLA-specific monoclonal antibodies induced activation of HLA-matched platelets from healthy donors as evidenced by enhanced α-granule release, aggregation, and αIIbb3 activation. The propensity of HLA monoclonal antibodies to activate platelets was independent of the HLA subtype to which they were directed, but was dependent on the recognized epitope. Activation was fully inhibited either by blocking FcγRIIa, or by blocking FcγRIIa-dependent signaling with Syk inhibitor IV. Furthermore, activation required the presence of the IgG-Fc part, as F(ab')2 fragments of HLA monoclonal antibodies were unable to induce platelet activation. Mixing experiments revealed that activation of platelets occurred in an intra-platelet dependent manner. Accordingly, a proportion of sera from refractory patients with HLA antibodies induced FcγRIIa-dependent platelet activation. Our data show that a subset of HLA antibodies is capable of crosslinking HLA and FcγRIIa thereby promoting platelet activation and enhancing these cells' phagocytosis by macrophages. Based on these findings we suggest that FcγRIIa-dependent platelet activation may contribute to the decreased platelet survival in platelet-transfusion-dependent patients with HLA antibodies.
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Anticuerpos Monoclonales/farmacología , Plaquetas/metabolismo , Antígenos HLA/metabolismo , Inmunoglobulina G/farmacología , Activación Plaquetaria/efectos de los fármacos , Receptores de IgG/metabolismo , Humanos , Fragmentos Fab de Inmunoglobulinas/farmacología , Fragmentos Fc de Inmunoglobulinas/farmacologíaRESUMEN
The emergence of antimicrobial resistance has created an urgent need for alternative treatments against bacterial pathogens. Here, we investigated kinase inhibitors as potential host-directed therapies (HDTs) against intracellular bacteria, specifically Salmonella Typhimurium (Stm) and Mycobacterium tuberculosis (Mtb). We screened 827 ATP-competitive kinase inhibitors with known target profiles from two Published Kinase Inhibitor Sets (PKIS1 and PKIS2) using intracellular infection models for Stm and Mtb, based on human cell lines and primary macrophages. Additionally, the in vivo safety and efficacy of the compounds were assessed using zebrafish embryo infection models. Our screen identified 11 hit compounds for Stm and 17 hit compounds for Mtb that were effective against intracellular bacteria and non-toxic for host cells. Further experiments were conducted to prioritize Stm hit compounds that were able to clear the intracellular infection in primary human macrophages. From these, two structurally related Stm hit compounds, GSK1379738A and GSK1379760A, exhibited significant activity against Stm in infected zebrafish embryos. In addition, we identified compounds that were active against intracellular Mtb, including morpholino-imidazo/triazolo-pyrimidinones that target PIK3CB, as well as 2-aminobenzimidazoles targeting ABL1. Overall, this study provided insights into kinase targets acting at the host-pathogen interface and identified several kinase inhibitors as potential HDTs.
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Macrófagos , Mycobacterium tuberculosis , Inhibidores de Proteínas Quinasas , Salmonella typhimurium , Pez Cebra , Animales , Humanos , Salmonella typhimurium/efectos de los fármacos , Mycobacterium tuberculosis/efectos de los fármacos , Mycobacterium tuberculosis/enzimología , Inhibidores de Proteínas Quinasas/farmacología , Macrófagos/microbiología , Macrófagos/efectos de los fármacos , Macrófagos/metabolismo , Infecciones por Salmonella/tratamiento farmacológico , Infecciones por Salmonella/microbiología , Antibacterianos/farmacología , Línea Celular , Embrión no Mamífero/efectos de los fármacos , Tuberculosis/tratamiento farmacológico , Tuberculosis/microbiologíaRESUMEN
Mycobacterium tuberculosis (Mtb) as well as nontuberculous mycobacteria are intracellular pathogens whose treatment is extensive and increasingly impaired due to the rise of mycobacterial drug resistance. The loss of antibiotic efficacy has raised interest in the identification of host-directed therapeutics (HDT) to develop novel treatment strategies for mycobacterial infections. In this study, we identified amiodarone as a potential HDT candidate that inhibited both intracellular Mtb and Mycobacterium avium in primary human macrophages without directly impairing bacterial growth, thereby confirming that amiodarone acts in a host-mediated manner. Moreover, amiodarone induced the formation of (auto)phagosomes and enhanced autophagic targeting of mycobacteria in macrophages. The induction of autophagy by amiodarone is likely due to enhanced transcriptional regulation, as the nuclear intensity of the transcription factor EB, the master regulator of autophagy and lysosomal biogenesis, was strongly increased. Furthermore, blocking lysosomal degradation with bafilomycin impaired the host-beneficial effect of amiodarone. Finally, amiodarone induced autophagy and reduced bacterial burden in a zebrafish embryo model of tuberculosis, thereby confirming the HDT activity of amiodarone in vivo. In conclusion, we have identified amiodarone as an autophagy-inducing antimycobacterial HDT that improves host control of mycobacterial infections. IMPORTANCE: Due to the global rise in antibiotic resistance, there is a strong need for alternative treatment strategies against intracellular bacterial infections, including Mycobacterium tuberculosis (Mtb) and non-tuberculous mycobacteria. Stimulating host defense mechanisms by host-directed therapy (HDT) is a promising approach for treating mycobacterial infections. This study identified amiodarone, an antiarrhythmic agent, as a potential HDT candidate that inhibits the survival of Mtb and Mycobacterium avium in primary human macrophages. The antimycobacterial effect of amiodarone was confirmed in an in vivo tuberculosis model based on Mycobacterium marinum infection of zebrafish embryos. Furthermore, amiodarone induced autophagy and inhibition of the autophagic flux effectively impaired the host-protective effect of amiodarone, supporting that activation of the host (auto)phagolysosomal pathway is essential for the mechanism of action of amiodarone. In conclusion, we have identified amiodarone as an autophagy-inducing HDT that improves host control of a wide range of mycobacteria.
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Amiodarona , Autofagia , Macrófagos , Mycobacterium tuberculosis , Tuberculosis , Pez Cebra , Amiodarona/farmacología , Autofagia/efectos de los fármacos , Animales , Pez Cebra/microbiología , Humanos , Macrófagos/microbiología , Macrófagos/inmunología , Macrófagos/efectos de los fármacos , Mycobacterium tuberculosis/efectos de los fármacos , Mycobacterium tuberculosis/crecimiento & desarrollo , Tuberculosis/tratamiento farmacológico , Tuberculosis/microbiología , Modelos Animales de Enfermedad , Mycobacterium avium/efectos de los fármacos , Lisosomas/efectos de los fármacos , Lisosomas/metabolismo , Lisosomas/microbiologíaRESUMEN
The increasing prevalence of antimicrobial-resistant Staphylococcus aureus strains, especially methicillin-resistant S. aureus (MRSA), poses a threat to successful antibiotic treatment. Unsuccessful attempts to develop a vaccine and rising resistance to last-resort antibiotics urge the need for alternative treatments. Host-directed therapy (HDT) targeting critical intracellular stages of S. aureus emerges as a promising alternative, potentially acting synergistically with antibiotics and reducing the risk of de novo drug resistance. We assessed 201 ATP-competitive kinase inhibitors from Published Kinase Inhibitor Sets (PKIS1 and PKIS2) against intracellular MRSA. Seventeen hit compounds were identified, of which the two most effective and well-tolerated hit compounds (i.e., GW633459A and GW296115X) were selected for further analysis. The compounds did not affect planktonic bacterial cultures, while they were active in a range of human cell lines of cervical, skin, lung, breast and monocyte origin, confirming their host-directed mechanisms. GW633459A, structurally related to lapatinib, exhibited an HDT effect on intracellular MRSA independently of its known human epidermal growth factor receptor (EGFR)/(HER) kinase family targets. GW296115X activated adenosine monophosphate-activated protein kinase (AMPK), thereby enhancing bacterial degradation via autophagy. Finally, GW296115X not only reduced MRSA growth in human cells but also improved the survival rates of MRSA-infected zebrafish embryos, highlighting its potential as HDT.
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Staphylococcus aureus Resistente a Meticilina , Infecciones Estafilocócicas , Humanos , Animales , Staphylococcus aureus , Pez Cebra , Antibacterianos/farmacología , Antibacterianos/uso terapéutico , Infecciones Estafilocócicas/microbiología , Pruebas de Sensibilidad MicrobianaRESUMEN
OBJECTIVES: Lymphopenia at hospital admission occurs in over one-third of patients with community-acquired pneumonia (CAP), yet its clinical relevance and pathophysiological implications remain underexplored. We evaluated outcomes and immune features of patients with lymphopenic CAP (L-CAP), a previously described immunophenotype characterized by admission lymphocyte count <0.724 × 109 cells/L. METHODS: Observational study in 149 patients admitted to a general ward for CAP. We measured 34 plasma biomarkers reflective of inflammation, endothelial cell responses, coagulation, and immune checkpoints. We characterized lymphocyte phenotypes in 29 patients using spectral flow cytometry. RESULTS: L-CAP occurred in 45 patients (30.2%) and was associated with prolonged time-to-clinical-stability (median 5 versus 3 days), also when we accounted for competing events for reaching clinical stability and adjusted for baseline covariates (subdistribution hazard ratio 0.63; 95% confidence interval 0.45-0.88). L-CAP patients demonstrated a proportional depletion of CD4 T follicular helper cells, CD4 T effector memory cells, naïve CD8 T cells and IgG+ B cells. Plasma biomarker analyses indicated increased activation of the cytokine network and the vascular endothelium in L-CAP. CONCLUSIONS: L-CAP patients have a protracted clinical recovery course and a more broadly dysregulated host response. These findings highlight the prognostic and pathophysiological relevance of admission lymphopenia in patients with CAP.
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Infecciones Comunitarias Adquiridas , Linfopenia , Neumonía , Humanos , Inflamación , HospitalizaciónRESUMEN
Ebola virus can trigger a release of pro-inflammatory cytokines with subsequent vascular leakage and impairment of clotting finally leading to multiorgan failure and shock after entering and infecting patients. Ebola virus is known to directly target endothelial cells and macrophages, even without infecting them, through direct interactions with viral proteins. These interactions affect cellular mechanics and immune processes, which are tightly linked to other key cellular functions such as metabolism. However, research regarding metabolic activity of these cells upon viral exposure remains limited, hampering our understanding of its pathophysiology and progression. Therefore, in the present study, an untargeted cellular metabolomic approach was performed to investigate the metabolic alterations of primary human endothelial cells and M1 and M2 macrophages upon exposure to Ebola virus-like particles (VLP). The results show that Ebola VLP led to metabolic changes among endothelial, M1, and M2 cells. Differential metabolite abundance and perturbed signaling pathway analysis further identified specific metabolic features, mainly in fatty acid-, steroid-, and amino acid-related metabolism pathways for all the three cell types, in a host cell specific manner. Taken together, this work characterized for the first time the metabolic alternations of endothelial cells and two primary human macrophage subtypes after Ebola VLP exposure, and identified the potential metabolites and pathways differentially affected, highlighting the important role of those host cells in disease development and progression. KEY MESSAGES: ⢠Ebola VLP can lead to metabolic alternations in endothelial cells and M1 and M2 macrophages. ⢠Differential abundance of metabolites, mainly including fatty acids and sterol lipids, was observed after Ebola VLP exposure. ⢠Multiple fatty acid-, steroid-, and amino acid-related metabolism pathways were observed perturbed.
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Ebolavirus , Fiebre Hemorrágica Ebola , Humanos , Ebolavirus/fisiología , Células Endoteliales , Transducción de Señal , AminoácidosRESUMEN
MicroRNA-155 (miR-155) plays a crucial role in regulating host inflammatory responses during bacterial infection. Previous studies have shown that constitutive miR-155 deficiency alleviates inflammation while having varying effects in different bacterial infection models. However, whether miR-155 in myeloid cells is involved in the regulation of inflammatory and antibacterial responses is largely elusive. Mice with myeloid cell specific miR-155 deficiency were generated to study the in vitro response of bone marrow-derived macrophages (BMDMs), alveolar macrophages (AMs) and peritoneal macrophages (PMs) to lipopolysaccharide (LPS), and the in vivo response after intranasal or intraperitoneal challenge with LPS or infection with Klebsiella (K.) pneumoniae via the airways. MiR-155-deficient macrophages released less inflammatory cytokines than control macrophages upon stimulation with LPS in vitro. However, the in vivo inflammatory cytokine response to LPS or K. pneumoniae was not affected by myeloid miR-155 deficiency. Moreover, bacterial outgrowth in the lungs was not altered in myeloid miR-155-deficient mice, but Klebsiella loads in the liver of these mice were significantly higher than in control mice. These data argue against a major role for myeloid miR-155 in host inflammatory responses during LPS-induced inflammation and K. pneumoniae-induced pneumosepsis but suggest that myeloid miR-155 contributes to host defense against Klebsiella infection in the liver.
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Infecciones por Klebsiella , MicroARNs , Animales , Ratones , Lipopolisacáridos , Klebsiella/genética , Inflamación , Klebsiella pneumoniae/fisiología , Citocinas , Infecciones por Klebsiella/microbiología , MicroARNs/genética , Ratones Endogámicos C57BLRESUMEN
Background: Community-acquired pneumonia (CAP) represents a major health burden worldwide. Dysregulation of the immune response plays an important role in adverse outcomes in patients with CAP. Methods: We analyzed peripheral blood mononuclear cells by 36-color spectral flow cytometry in adult patients hospitalized for CAP (n=40), matched control subjects (n=31), and patients hospitalized for COVID-19 (n=35). Results: We identified 86 immune cell metaclusters, 19 of which (22.1%) were differentially abundant in patients with CAP versus matched controls. The most notable differences involved classical monocyte metaclusters, which were more abundant in CAP and displayed phenotypic alterations reminiscent of immunosuppression, increased susceptibility to apoptosis, and enhanced expression of chemokine receptors. Expression profiles on classical monocytes, driven by CCR7 and CXCR5, divided patients with CAP into two clusters with a distinct inflammatory response and disease course. The peripheral immune response in patients with CAP was highly similar to that in patients with COVID-19, but increased CCR7 expression on classical monocytes was only present in CAP. Conclusion: CAP is associated with profound cellular changes in blood that mainly relate to classical monocytes and largely overlap with the immune response detected in COVID-19.
Asunto(s)
COVID-19 , Infecciones Comunitarias Adquiridas , Neumonía , Adulto , Humanos , Leucocitos Mononucleares , Receptores CCR7 , InmunidadRESUMEN
The Mycobacterium avium (Mav) complex accounts for more than 80% of all pulmonary diseases caused by non-tuberculous mycobacteria (NTM) infections, which have an alarming increase in prevalence and vary in different regions, currently reaching 0.3-9.8 per 100,000 individuals. Poor clinical outcomes, as a result of increasing microbial drug resistance and low treatment adherence due to drug-toxicities, emphasize the need for more effective treatments. Identification of more effective treatments, however, appears to be difficult, which may be due to the intracellular life of NTM and concomitant altered drug sensitivity that is not taken into account using traditional drug susceptibility testing screenings. We therefore developed human cell-based in vitro Mav infection models using the human MelJuSo cell line as well as primary human macrophages and a fluorescently labeled Mav strain. By testing a range of multiplicity of infection (MOI) and using flow cytometry and colony-forming unit (CFU) analysis, we found that an MOI of 10 was the most suitable for Mav infection in primary human macrophages, whereas an MOI of 50 was required to achieve similar results in MelJuSo cells. Moreover, by monitoring intracellular bacterial loads over time, the macrophages were shown to be capable of controlling the infection, while MelJuSo cells failed to do so. When comparing the MGIT system with the classical CFU counting assay to determine intracellular bacterial loads, MGIT appeared as a less labor-intensive, more precise, and more objective alternative. Next, using our macrophage Mav infection models, the drug efficacy of the first-line drug rifampicin and the more recently discovered bedaquiline on intracellular bacteria was compared to the activity on extracellular bacteria. The efficacy of the antibiotics inhibiting bacterial growth was significantly lower against intracellular bacteria compared to extracellular bacteria. This finding emphasizes the crucial role of the host cell during infection and drug susceptibility and highlights the usefulness of the models. Taken together, the human cell-based Mav infection models are reliable tools to determine the intracellular loads of Mav, which will enable researchers to investigate host-pathogen interactions and to evaluate the efficacy of (host-directed) therapeutic strategies against Mav.
Asunto(s)
Infección por Mycobacterium avium-intracellulare , Mycobacterium tuberculosis , Humanos , Pruebas de Sensibilidad Microbiana , Mycobacterium avium , Complejo Mycobacterium avium , Infección por Mycobacterium avium-intracellulare/epidemiología , Infección por Mycobacterium avium-intracellulare/microbiologíaRESUMEN
Platelet transfusions are a frequently administered therapy for especially hemato-oncological patients with thrombocytopenia. Next to their primary function in hemostasis, currently there is increased attention for the capacity of platelets to affect the function of various cells of the immune system. Here, we investigate the capacity of platelets to immuno-modulate monocyte-derived dendritic cells (moDC) as well as primary dendritic cells and effects on subsequent T cell responses. Platelets significantly inhibited pro-inflammatory (IL-12, IL-6, TNFα) and increased anti-inflammatory (IL-10) cytokine production of moDCs primed with toll-like receptor (TLR)-dependent and TLR-independent stimuli. Transwell assays and ultracentrifugation revealed that a soluble factor secreted by platelets, but not microvesicles, inhibited DC activation. Interestingly, platelet-derived soluble mediators also inhibited cytokine production by human ex vivo stimulated myeloid CD1c+ conventional DC2. Moreover, platelets and platelet-derived soluble mediators inhibited T cell priming and T helper differentiation toward an IFNγ+ Th1 phenotype by moDCs. Overall, these results show that platelets are able to inhibit the pro-inflammatory properties of DCs, and may even induce an anti-inflammatory DC phenotype, with decreased T cell priming capacity by the DC. The results of this study provide more insight in the potential role of platelets in immune modulation, especially in the context of platelet transfusions.