TNF/iNOS/NO pathway mediates host susceptibility to endothelial-dependent circulatory failure and death induced by betacoronavirus infection.

Poor disease outcomes and lethality are directly related to endothelial dysfunction in betacoronavirus infections. Here, we investigated the mechanisms underlying the vascular dysfunction caused by the betacoronaviruses MHV-3 and SARS-CoV-2. Wild-type C57BL/6 (WT) and knockout mice for inducible nitric oxide synthase (iNOS-/-) or TNF receptor 1 (TNFR1-/-) were infected with MHV-3, and K18-hACE2 transgenic mice expressing human ACE2 were infected with SARS-CoV-2. Isometric tension was used to evaluate vascular function. Protein expression was determined by immunofluorescence. Tail-cuff plethysmography and Doppler were used to assess blood pressure and flow, respectively. Nitric oxide (NO) was quantified with the DAF probe. ELISA was used to assess cytokine production. Survival curves were estimated using Kaplan-Meier. MHV-3 infection reduced aortic and vena cava contractility, arterial blood pressure, and blood flow, resulting in death. Resistance mesenteric arteries showed increased contractility. The contractility of the aorta was normalized by removing the endothelium, inhibiting iNOS, genetically deleting iNOS, or scavenging NO. In the aorta, iNOS and phospho-NF-kB p65 subunit expression was enhanced, along with basal NO production. TNF production was increased in plasma and vascular tissue. Genetic deletion of TNFR1 prevented vascular changes triggered by MHV-3, and death. Basal NO production and iNOS expression were also increased by SARS-CoV-2. In conclusion, betacoronavirus induces an endothelium-dependent decrease in contractility in macro-arteries and veins, leading to circulatory failure and death via TNF/iNOS/NO. These data highlight the key role of the vascular endothelium and TNF in the pathogenesis and lethality of coronaviruses.

A Biosafety Level 2 Mouse Model for Studying Betacoronavirus-Induced Acute Lung Damage and Systemic Manifestations.

The emergence of life-threatening zoonotic diseases caused by betacoronaviruses, including the ongoing coronavirus disease 19 (COVID-19) pandemic, has highlighted the need for developing preclinical models mirroring respiratory and systemic pathophysiological manifestations seen in infected humans. Here, we showed that C57BL/6J wild-type mice intranasally inoculated with the murine betacoronavirus murine hepatitis coronavirus 3 (MHV-3) develop a robust inflammatory response leading to acute lung injuries, including alveolar edema, hemorrhage, and fibrin thrombi. Although such histopathological changes seemed to resolve as the infection advanced, they efficiently impaired respiratory function, as the infected mice displayed restricted lung distention and increased respiratory frequency and ventilation. Following respiratory manifestation, the MHV-3 infection became systemic, and a high virus burden could be detected in multiple organs along with morphological changes. The systemic manifestation of MHV-3 infection was also marked by a sharp drop in the number of circulating platelets and lymphocytes, besides the augmented concentration of the proinflammatory cytokines interleukin 1 beta (IL-1ß), IL-6, IL-12, gamma interferon (IFN-γ), and tumor necrosis factor (TNF), thereby mirroring some clinical features observed in moderate and severe cases of COVID-19. Importantly, both respiratory and systemic changes triggered by MHV-3 infection were greatly prevented by blocking TNF signaling, either via genetic or pharmacologic approaches. In line with this, TNF blockage also diminished the infection-mediated release of proinflammatory cytokines and virus replication of human epithelial lung cells infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Collectively, results show that MHV-3 respiratory infection leads to a large range of clinical manifestations in mice and may constitute an attractive, lower-cost, biosafety level 2 (BSL2) in vivo platform for evaluating the respiratory and multiorgan involvement of betacoronavirus infections. IMPORTANCE Mouse models have long been used as valuable in vivo platforms to investigate the pathogenesis of viral infections and effective countermeasures. The natural resistance of mice to the novel betacoronavirus SARS-CoV-2, the causative agent of COVID-19, has launched a race toward the characterization of SARS-CoV-2 infection in other animals (e.g., hamsters, cats, ferrets, bats, and monkeys), as well as adaptation of the mouse model, by modifying either the host or the virus. In the present study, we utilized a natural pathogen of mice, MHV, as a prototype to model betacoronavirus-induced acute lung injure and multiorgan involvement under biosafety level 2 conditions. We showed that C57BL/6J mice intranasally inoculated with MHV-3 develops severe disease, which includes acute lung damage and respiratory distress that precede systemic inflammation and death. Accordingly, the proposed animal model may provide a useful tool for studies regarding betacoronavirus respiratory infection and related diseases.

Corrigendum: Assessment of the accuracy of 11 different diagnostic tests for the detection of Schistosomiasis mansoni in individuals from a Brazilian area of low endemicity using latent class analysis.

[This corrects the article DOI: 10.3389/fmicb.2022.1048457.].

O Efeito da Vitamina D na infecção experimental pelo Schistosoma mansoni (Sambon, 1907) e na eficácia do tratamento com doses subcurativas de praziquantel em modelo murino

A vitamina D (vit.D), compreendida como um hormônio, é responsável pelos mecanismos de absorção do cálcio e fósforo, bem como desencadeia diversos eventos genomicos e não genomicos no organismo mediados por seus receptores VDRs presentes em uma ampla variedade de tecidos corporais como cérebro, coração, pele, intestino, gônadas, ossos, rins, paratireoides e células imunológicas, sendo essa última de interesse em nosso estudo. A vit.D apresenta importante papel imunoregulatório sobre linfócitos T e células APC, e sobre a diferenciação de células CD4+, promovendo uma alteração no perfil de resposta de Th1 para Th2, auxiliando no enfrentamento a determinados patógenos, bem como modulando o limiar de dano tissular potencial relacionado à resposta Th1. Além disso, participa da regulação da diferenciação das células precursoras em células mais especializadas no sistema monocítico-macrofágico. Estudos epidemiológicos evidenciam uma generalizada deficiência e insuficiência dessa vitamina em diversas regiões do mundo, especialmente na Ásia, África e América Latina o que pode predispor esses indivíduos a diversas doenças. Os continentes em destaque para hipovitaminose D apresentam também doenças relevantes de saúde pública, como é o caso da esquistossomose que afeta aproximadamente 240 milhões de pessoas ao ano, em 54 países, sendo o Brasil uma das regiões acometidas. O Praziquantel (PZQ), única droga disponível para o tratamento, não apresenta efeito contra formas jovens do parasito e possui eficácia reduzida em determinados grupos, pois depende da resposta imunológica do indivíduo para sua eficácia. Portanto, buscamos nesse trabalho compreender e avaliar o impacto da suplementação de 10.000UI de vit.D na esquistossomose e na eficácia do tratamento com subdoses de praziquantel. Estabelecemos um protocolo de suplementação de vitamina D que promoveu alterações séricas de seu metabolito ativo. Avaliamos o impacto dessa suplementação sobre o desenvolvimento e sobrevivência do S. mansoni, sobre níveis séricos de citocinas, anticorpos e na patologia hepática induzida pela infecção experimental e na eficácia do tratamento com doses subcurativas de Praziquantel. Nossos resultados mostraram que a suplementação com vitamina D não alterou a carga parasitaria, antes e após o tratamento, não afetou os níveis séricos das citocinas TNF- α, IL-4, IL-2 e imunoglobulinas IgG, IgG1. Porém, encontramos diferenças significativas nos níveis séricos das citocinas IL- 10, IL-17, IFN- e IL-6 e de IgG2c anti-SWAP e observamos um redução significativa na área do granuloma hepático, em animais suplementados em relação aos controles. Nossos resultados evidenciaram a ação da vitamina D sobre a resposta imunológica específica contra o parasito e seu possível impacto sobre a patologia hepática. Estudos ainda são necessários para melhor compreensão desses eventos.