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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. Using a single-cell transcriptome analysis we observed that the expression of GSDMD and inflammasome-related genes were increased in neutrophils from COVID-19 patients. Furthermore, high expression of GSDMD was found associated with NETs structures in the lung tissue of COVID-19 patients. The activation of GSDMD in neutrophils requires live SARS-CoV-2 and occurs after neutrophil infection via ACE2 receptors and serine protease TMPRSS2. In a mouse model of SARS-CoV-2 infection, the treatment with GSDMD inhibitor (disulfiram) reduced NETs release and organ damage. These results demonstrated that GSDMD-dependent NETosis plays a critical role in COVID-19 immunopathology, and suggests that GSDMD inhibitors, can be useful to COVID-19 treatment.In BriefHere, we showed that the activation of the Gasdermin-D (GSDMD) pathway in neutrophils controls NET release during COVID-19. The inhibition of GSDMD with disulfiram, abrogated NET formation reducing lung inflammation and tissue damage. These findings suggest GSDMD as a target for improving the COVID-19 therapy.

A tuberculose (Tb) é uma doença infecciosa crônica ocasionada pelo bacilo Mycobacterium tuberculosis (Mtb). No espaço alveolar, o bacilo entra em contato com células do sistema imune inato como as células dendríticas (DCs), assim como células epiteliais (AEC). Na Tb, o fator de transcrição HIF-1? (Fator Induzido por Hipóxia 1 alfa) se encontra elevado em macrófagos infectados e células epiteliais alveolares adjacentes. Por isso, o objetivo deste trabalho foi investigar o papel de HIF-1? na resposta pró- inflamatória de AEC do tipo II (AEC-II) e na modulação da função de DCs em contato com AEC-II durante a infecção por Mtb. Células MLE-15 foram infectadas com Mtb H37Rv (MOI10) para avaliação da permissividade à infecção (microscopia eletrônica), crescimento dos bacilos (CFU) e ativação através da análise de citocinas (ELISA), nitrito (ensaio de Griess), TLR2, TLR9 e HIF-1? (qPCR e/ou Western blotting). A modulação DCs derivadas da medula óssea (BMDCs) por AEC-II, foi analisada de forma direta (contato) ou indireta (meio condicionado - CM) de MLE-15 não infectadas (CM - NIC) ou infectadas (CM - IC). Foram determinadas citocinas (ELISA), nitrito (Ensaio de Griess), expressão de HIF-1?, enzimas glicolíticas, moléculas co-estimuladoras e CCR7 (citometria de fluxo). Ensaio de migração foi realizando em câmera de boyden. A proliferação de células T CD4+ naive em co-cultura com BMDCs e a manutenção da Th17 foram avaliadas por citometria de fluxo. Eventualmente, BMDCs foram previamente infectadas com Mtb (MOI2) e, em seguida, estimuladas com CM-NIC ou CM-IC. Células MLE-15 mostraram-se permissivas à infecção, não conseguindo controlar o crescimento dos bacilos. A infecção induziu aumento da produção de IL-6, NO2-,CCL5, S100A9 e IFN- ?. Apesar do acúmulo inicial de HIF-1?, a expressão gênica caiu com o passar do tempo. A expressão gênica de TLR2 e TLR9 também estava aumentada. A regulação positiva10 de HIF-1? em células epiteliais induziu uma redução de IL-6 e NO, sem, no entanto, interferir significativamente no número de CFU. BMDCs estimuladas com CM - IC mostraram maior produção de IL-1?, IL-12, IL-6 e IL-10, maior expressão gênica de GLUT1 e HK2, além do acúmulo inicial de HIF-1?, que foi degradado em 24 horas, acompanhado de baixa expressão de iNOS. A expressão MHC-II, CD80, CD86 e CCR7 estava aumentada em BMDCs submetidas ao CM - IC, enquanto a indução do acúmulo de HIF-1? através do seu estabilizador, DMOG, foi capaz de reverter negativamente essa resposta. A maior maturação de BMDCs ocasionou maior proliferação de células TCD4+ naive, desfavorecendo a indução de células T CD4+ IFN?+. Entretanto, as citocinas produzidas favorecerem a manutenção de células TCD4+ produtoras de IL-17. No entanto, o fenótipo de maior maturação foi perdido em BMDCs infectadas estimuladas com CM - IC, aliado à baixa produção de TNF e alta produção de IL-10. Em conclusão, HIF-1? mostrou uma função anti-inflamatória, reduzindo a produção de moléculas pró- inflamatórias por AEC-II e regulando negativamente a maturação e a migração de DCs. Além disso, apesar de AEC-II infectadas por Mtb favorecerem a maturação e migração de DCs, o Mtb é capaz de subverter essa resposta Tuberculosis (Tb) is a chronic infectious disease caused by the bacillus Mycobacterium tuberculosis (Mtb). In the alveolar space, the bacillus contacts cells from the immune system, such as dendritic cells (DCs), as well as alveolar epithelial cells (AEC). In Tb, the transcription factor HIF-1? (Hypoxia-inducible factor 1- alpha) is accumulated in infected macrophages and adjacent alveolar epithelial cells. Therefore, the aim of this work was to investigate the role of HIF-1? in the proinflammatory response of type II AEC (AEC-II) and modulation of DCs function upon contact with AEC-II during Mtb infection. MLE-15 cells were infected with Mtb H37Rv (MOI10) to evaluate the permissiveness to infection (electron microscopy), killing ability (CFU) and cell activation through cytokine analysis (ELISA), nitrite (Griess assay), TLR2, TLR9 and HIF-1? (qPCR and / or Western blotting). Bone marrow derived DCs (BMDCs) modulation by AEC-II, were analyzed directly (contact) or indirectly (conditioned medium - CM) of uninfected (CM-NIC) or infected (CMIC) MLE-15. We determined cytokines (ELISA), nitrite (Griess Assay), HIF-1? expression, glycolytic enzymes, co-stimulatory molecules and CCR7 (flow cytometry). Chemotaxie assay was performed on Boyden camera. Proliferation of naive CD4 + T cells in co-culture with BMDCs and maintenance of Th17 were assessed by flow cytometry. Eventually, BMDCs were previously infected with Mtb (MOI2) and then stimulated with CM-NIC or CM-IC. MLE-15 cells were permissive to infection, failing to control the bacilli growth. Infection induced increased production of IL-6, NO2-, CCL5, S100A9 and IFN-?. Despite the initial accumulation of HIF-1?, gene expression dropped over time. The gene expression of TLR2 and TLR9 was also increased. Positive regulation of HIF-1? in epithelial cells induced a reduction of IL-6 and NO, but did not significantly interfere with the number of CFU. BMDCs stimulated with CM-IC showed higher production of IL-1?, IL-12, IL-6, and IL-10, greater GLUT1 and HK2 gene expression, in addition to the initial12 accumulation of HIF-1?, which was degraded within 24 hours, accompanied by low iNOS expression. The expression of MHC-II, CD80, CD86 and CCR7 was increased in BMDCs undergoing CM-IC, while the induction of HIF-1? accumulation through its stabilizer, DMOG, was able to negatively revert this response. The higher maturation of BMDCs resulted in a greater proliferation of naive CD4 + T cells, but hampered induction of CD4 + IFN? + T cells. However, cytokines produced favor the maintenance of IL-17 producing CD4 + cells. The phenotype of higher maturation was lost in Mtb-infected BMDC, accompanied by low TNF production and high IL-10 production. In conclusion, HIF-1? showed an anti-inflammatory function, reducing the production of proinflammatory molecules by AEC-II and negatively regulating the maturation and migration of DCs. In addition, although Mtb-infected AEC-II favor maturation and migration of DCs, Mtbinfection of DCs is capable of subverting this response

Campylobacter spp. were detected - using culture, ELISA, PCR, and qPCR - among children (0–36 months) 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.