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This study introduces the nanobromhexine lipid particle (NBL) platform designed for effective pulmonary drug delivery. Inspired by respiratory virus transport mechanisms, NBL address challenges associated with mucus permeation and inflammation in pulmonary diseases. Composed of low molecular weight polyethylene glycol-coated lipid nanoparticles with bromhexine hydrochloride, NBL exhibit a size of 118 ± 24 nm, a neutral zeta potential, osmolarity of 358 ± 28 mOsmol/kg, and a pH of 6.5. Nebulizing without leakage and showing no toxicity to epithelial cells, NBL display mucoadhesive properties with a 60% mucin-binding efficiency. They effectively traverse the dense mucus layer of Calu-3 cultures in an air-liquid interface, as supported by a 55% decrease in MUC5AC density and a 29% increase in nanoparticles internalization compared to non-exposed cells. In assessing immunomodulatory effects, NBL treatment in SARS-CoV-2-infected lung cells leads to a 40-fold increase in anti-inflammatory MUC1 gene expression, a proportional reduction in pro-inflammatory IL-6 expression, and elevated anti-inflammatory IL-10 expression. These findings suggest a potential mechanism to regulate the excessive IL-6 expression triggered by virus infection. Therefore, the NBL platform demonstrates promising potential for efficient pulmonary drug delivery and immunomodulation, offering a novel approach to addressing mucus permeation and inflammation in pulmonary diseases.
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Pulmón , Nanopartículas , SARS-CoV-2 , Nanopartículas/administración & dosificación , Humanos , Pulmón/metabolismo , SARS-CoV-2/efectos de los fármacos , Sistemas de Liberación de Medicamentos , Inmunomodulación , Línea Celular , Mucina-1/metabolismo , COVID-19 , Lípidos/química , Lípidos/administración & dosificación , Moco/metabolismo , Polietilenglicoles/química , Células Epiteliales/metabolismo , Células Epiteliales/efectos de los fármacos , Tratamiento Farmacológico de COVID-19 , Mucina 5AC/metabolismo , LiposomasRESUMEN
AIM2 is an interferon-inducible HIN-200 protein family member and is well-documented for its roles in innate immune responses as a DNA sensor. Recent studies have highlighted AIM2's function on regulatory T cells (Treg) and follicular T cells (Tfh). However, its involvement in Th17 cell differentiation remains unclear. This study reveals that AIM2 promotes Th17 cell differentiation. AIM2 deficiency decreases IL-17A production and downregulates key Th17 associated proteins (RORγt, IL-1R1, IL-23R). AIM2 is located in the nucleus of Th17 cells, where it interacts with RORγt, enhancing its binding to the Il17a promoter. The absence of AIM2 hinders naive CD4 T cells from differentiating into functional Th17 cells and from inducing colitis in Rag1-/- mice. This study uncovers AIM2's role as a regulator of Th17 cell transcriptional programming, highlighting its potential as a therapeutic target for Th17 cell-mediated inflammatory diseases.
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In the present study, we show that SARS-CoV-2 can infect palatine tonsils, adenoids, and secretions in children without symptoms of COVID-19, with no history of recent upper airway infection. We studied 48 children undergoing tonsillectomy due to snoring/OSA or recurrent tonsillitis between October 2020 and September 2021. Nasal cytobrushes, nasal washes, and tonsillar tissue fragments obtained at surgery were tested by RT-qPCR, immunohistochemistry (IHC), flow cytometry, and neutralization assay. We detected the presence of SARS-CoV-2 in at least one specimen tested in 27% of patients. IHC revealed the presence of the viral nucleoprotein in epithelial surface and in lymphoid cells in both extrafollicular and follicular regions, in adenoids and palatine tonsils. Also, IHC for the SARS-CoV-2 non-structural protein NSP-16 indicated the presence of viral replication in 53.8% of the SARS-CoV-2-infected tissues. Flow cytometry showed that CD20+ B lymphocytes were the most infected phenotypes, followed by CD4+ lymphocytes and CD123 dendritic cells, CD8+ T lymphocytes, and CD14+ macrophages. Additionally, IF indicated that infected tonsillar tissues had increased expression of ACE2 and TMPRSS2. NGS sequencing demonstrated the presence of different SARS-CoV-2 variants in tonsils from different tissues. SARS-CoV-2 antigen detection was not restricted to tonsils but was also detected in nasal cells from the olfactory region. Palatine tonsils and adenoids are sites of prolonged RNA presence by SARS-CoV-2 in children, even without COVID-19 symptoms. IMPORTANCE This study shows that SRS-CoV-2 of different lineages can infect tonsils and adenoids in one quarter of children undergoing tonsillectomy. These findings bring advancement to the area of SARS-CoV-2 pathogenesis, by showing that tonsils may be sites of prolonged infection, even without evidence of recent COVID-19 symptoms. SARS-CoV-2 infection of B and T lymphocytes, macrophages, and dendritic cells may interfere with the mounting of immune responses in these secondary lymphoid organs. Moreover, the shedding of SARS-CoV-2 RNA in respiratory secretions from silently infected children raises concern about possible diagnostic confusion in the presence of symptoms of acute respiratory infections caused by other etiologies.
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The TIGIT+FOXP3+Treg subset (TIGIT+Tregs) exerts robust suppressive activity on cellular immunity and predisposes septic individuals to opportunistic infection. We hypothesized that TIGIT+Tregs could play an important role in intensifying the COVID-19 severity and hampering the defense against nosocomial infections during hospitalization. Herein we aimed to verify the association between the levels of the TIGIT+Tregs with the mechanical ventilation requirement, fatal outcome, and bacteremia during hospitalization. TIGIT+Tregs were immunophenotyped by flow cytometry from the peripheral blood of 72 unvaccinated hospitalized COVID-19 patients at admission from May 29th to August 6th, 2020. The patients were stratified during hospitalization according to their mechanical ventilation requirement and fatal outcome. COVID-19 resulted in a high prevalence of the TIGIT+Tregs at admission, which progressively increased in patients with mechanical ventilation needs and fatal outcomes. The prevalence of TIGIT+Tregs positively correlated with poor pulmonary function and higher plasma levels of LDH, HMGB1, FGL2, and TNF. The non-survivors presented higher plasma levels of IL-33, HMGB1, FGL2, IL-10, IL-6, and 5.54 times more bacteremia than survivors. Conclusions: The expansion of the TIGIT+Tregs in COVID-19 patients was associated with inflammation, lung dysfunction, bacteremia, and fatal outcome.
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Bacteriemia , COVID-19 , Infección Hospitalaria , Proteína HMGB1 , Humanos , Respiración Artificial , Linfocitos T Reguladores , Receptores Inmunológicos , FibrinógenoRESUMEN
BACKGROUND: The release of neutrophil extracellular traps (NETs) is associated with inflammation, coagulopathy, and organ damage found in severe cases of COVID-19. However, the molecular mechanisms underlying the release of NETs in COVID-19 remain unclear. OBJECTIVES: We aim to investigate the role of the Gasdermin-D (GSDMD) pathway on NETs release and the development of organ damage during COVID-19. METHODS: We performed a single-cell transcriptome analysis in public data of bronchoalveolar lavage. Then, we enrolled 63 hospitalized patients with moderate and severe COVID-19. We analyze in blood and lung tissue samples the expression of GSDMD, presence of NETs, and signaling pathways upstreaming. Furthermore, we analyzed the treatment with disulfiram in a mouse model of SARS-CoV-2 infection. RESULTS: We found that the SARS-CoV-2 virus directly activates the pore-forming protein GSDMD that triggers NET production and organ damage in COVID-19. Single-cell transcriptome analysis revealed that the expression of GSDMD and inflammasome-related genes were increased in COVID-19 patients. High expression of active GSDMD associated with NETs structures was found in the lung tissue of COVID-19 patients. Furthermore, we showed that activation of GSDMD in neutrophils requires active caspase1/4 and live SARS-CoV-2, which infects neutrophils. In a mouse model of SARS-CoV-2 infection, the treatment with disulfiram inhibited NETs release and reduced organ damage. CONCLUSION: These results demonstrated that GSDMD-dependent NETosis plays a critical role in COVID-19 immunopathology and suggests GSDMD as a novel potential target for improving the COVID-19 therapeutic strategy.
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Tratamiento Farmacológico de COVID-19 , Trampas Extracelulares , Animales , Disulfiram/metabolismo , Trampas Extracelulares/metabolismo , Ratones , Neutrófilos/metabolismo , SARS-CoV-2RESUMEN
The microbiota of the gut-lung axis affects local and far-reaching immune responses and might also trigger chronic and inflammatory diseases. We hypothesized that gut dysbiosis induced by obesity, which coexists in countries with a high tuberculosis burden, aggravates the host susceptibility and the pulmonary damage tolerance. To assess our hypothesis, we used a model of high-fat diet (HFD)-induced obesity, followed by infection of C57BL/6 mice with Mycobacterium tuberculosis. We showed that obesity increased the susceptibility, the pulmonary inflammation and IFN-γ levels in M. tuberculosis-infected mice. During the comorbidity obesity and tuberculosis, there is an increase of Bacteroidetes and Firmicutes in the lungs, and an increase of Firmicutes and butyrate in the feces. Depletion of gut microbiota by antibiotic treatment in the obese infected mice reduced the frequencies of CD4+IFN-γ+IL-17- cells and IFN-γ levels in the lungs, associated with an increase of Lactobacillus. Our findings reinforce the role of the gut-lung axis in chronic infections and suggest that the gut microbiota modulation may be a potential host-directed therapy as an adjuvant to treat TB in the context of IFN-γ-mediated immunopathology.
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Disbiosis/etiología , Disbiosis/microbiología , Interferón gamma/biosíntesis , Obesidad/complicaciones , Obesidad/microbiología , Neumonía/microbiología , Tuberculosis/complicaciones , Inmunidad Adaptativa , Animales , Carga Bacteriana , Susceptibilidad a Enfermedades , Disbiosis/inmunología , Trasplante de Microbiota Fecal , Heces/microbiología , Femenino , Leucocitos/metabolismo , Pulmón/inmunología , Pulmón/microbiología , Pulmón/patología , Ratones Endogámicos C57BL , Microbiota , Obesidad/inmunología , Neumonía/inmunología , Tuberculosis/inmunologíaRESUMEN
The innate response plays a crucial role in the protection against tuberculosis development. Moreover, the initial steps that drive the host-pathogen interaction following Mycobacterium tuberculosis infection are critical for the development of adaptive immune response. As alveolar MÏs, airway epithelial cells, and dendritic cells can sense the presence of M. tuberculosis and are the first infected cells. These cells secrete mediators, which generate inflammatory signals that drive the differentiation and activation of the T lymphocytes necessary to clear the infection. Throughout this review article, we addressed the interaction between epithelial cells and M. tuberculosis, as well as the interaction between dendritic cells and M. tuberculosis. The understanding of the mechanisms that modulate those interactions is critical to have a complete view of the onset of an infection and may be useful for the development of dendritic cell-based vaccine or immunotherapies.
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Inmunidad Adaptativa , Células Epiteliales Alveolares/inmunología , Células Dendríticas/inmunología , Interacciones Huésped-Patógeno/inmunología , Mycobacterium tuberculosis/fisiología , Tuberculosis Pulmonar/inmunología , Células Epiteliales Alveolares/patología , Células Dendríticas/patología , Humanos , Macrófagos Alveolares/inmunología , Macrófagos Alveolares/patología , Tuberculosis Pulmonar/patologíaRESUMEN
Tuberculosis kills more than 1 million people every year, and its control depends on the effective mechanisms of innate immunity, with or without induction of adaptive immune response. We investigated the interaction of type II alveolar epithelial cells (AEC-II) infected by Mycobacterium tuberculosis with dendritic cells (DCs). We hypothesized that the microenvironment generated by this interaction is critical for the early innate response against mycobacteria. We found that AEC-II infected by M. tuberculosis induced DC maturation, which was negatively regulated by HIF-1α-inducible NOS2 axis, and switched DC metabolism from an early and short peak of glycolysis to a low energetic status. However, the infection of DCs by M. tuberculosis up-regulated NOS2 expression and inhibited AEC-II-induced DC maturation. Our study demonstrated, for the first time, that HIF-1α-NOS2 axis plays a negative role in the maturation of DCs during M. tuberculosis infection. Such modulation might be useful for the exploitation of molecular targets to develop new therapeutic strategies against tuberculosis.
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Células Epiteliales Alveolares/inmunología , Células Dendríticas/inmunología , Subunidad alfa del Factor 1 Inducible por Hipoxia/inmunología , Mycobacterium tuberculosis/inmunología , Óxido Nítrico Sintasa de Tipo II/inmunología , Tuberculosis Pulmonar/inmunología , Células Epiteliales Alveolares/microbiología , Células Epiteliales Alveolares/patología , Animales , Células Dendríticas/microbiología , Células Dendríticas/patología , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , Ratones , Ratones Noqueados , Óxido Nítrico Sintasa de Tipo II/genética , Tuberculosis Pulmonar/genética , Tuberculosis Pulmonar/microbiologíaRESUMEN
Campylobacter spp. were detected - using culture, ELISA, PCR, and qPCR - among children (0-36months) with moderate to severe diarrhea in Northeastern Brazil. Our data showed that either the qPCR alone or PCR along with ELISA might be an alternative to culture to diagnose Campylobacter due to their enhanced sensitivity.