Blocking the CTLA-4 and PD-1 pathways during pulmonary paracoccidioidomycosis improves immunity, reduces disease severity, and increases the survival of infected mice.

Immune checkpoint pathways, i.e., coinhibitory pathways expressed as feedback following immune activation, are crucial for controlling an excessive immune response. Cytotoxic T lymphocyte antigen-4 (CTLA-4) and programmed cell death protein-1 (PD-1) are the central classical checkpoint inhibitory (CPI) molecules used for the control of neoplasms and some infectious diseases, including some fungal infections. As the immunosuppression of severe paracoccidioidomycosis (PCM), a chronic granulomatous fungal disease, was shown to be associated with the expression of coinhibitory molecules, we hypothesized that the inhibition of CTLA-4 and PD-1 could have a beneficial effect on pulmonary PCM. To this end, C57BL/6 mice were infected with Paracoccidioides brasiliensis yeasts and treated with monoclonal antibodies (mAbs) α-CTLA-4, α-PD-1, control IgG, or PBS. We verified that blockade of CTLA-4 and PD-1 reduced the fungal load in the lungs and fungal dissemination to the liver and spleen and decreased the size of pulmonary lesions, resulting in increased survival of mice. Compared with PBS-treated infected mice, significantly increased levels of many pro- and anti-inflammatory cytokines were observed in the lungs of α-CTLA-4-treated mice, but a drastic reduction in the liver was observed following PD-1 blockade. In the lungs of α-CPI and IgG-treated mice, there were no changes in the frequency of inflammatory leukocytes, but a significant reduction in the total number of these cells was observed. Compared with PBS-treated controls, α-CPI- and IgG-treated mice exhibited reduced pulmonary infiltration of several myeloid cell subpopulations and decreased expression of costimulatory molecules. In addition, a decreased number of CD4+ and CD8+ T cells but sustained numbers of Th1, Th2, and Th17 T cells were detected. An expressive reduction in several Treg subpopulations and their maturation and suppressive molecules, in addition to reduced numbers of Treg, TCD4+, and TCD8+ cells expressing costimulatory and coinhibitory molecules of immunity, were also detected. The novel cellular and humoral profiles established in the lungs of α-CTLA-4 and α-PD-1-treated mice but not in control IgG-treated mice were more efficient at controlling fungal growth and dissemination without causing increased tissue pathology due to excessive inflammation. This is the first study demonstrating the efficacy of CPI blockade in the treatment of pulmonary PCM, and further studies combining the use of immunotherapy with antifungal drugs are encouraged.

Intranasal delivery of LaAg vaccine improves immunity of aged mice against visceral Leishmaniasis.

There are no approved vaccines yet for human visceral leishmaniasis (VL), the most severe form of the leishmaniasis clinical manifestations that is fatal in over 95 % of untreated cases. It is well-accepted that immunological changes during aging have deleterious impact on the efficacy of vaccines and response to infections. In this work, we compared the response of young and aged mice to intranasal vaccination with killed Leishmania amazonensis promastigote antigens (LaAg) that were then challenged with L. infantum infection, a species that causes visceral leishmaniasis. Intranasal vaccination with LaAg induced a similar reduction in parasitism and hepatosplenomegaly in both young and aged mice compared to their unvaccinated counterparts. Following infection, there was also a less prominent inflammatory profile particularly in the vaccinated aged group, with lower production of TNF-α and nitrite compared to the respective unvaccinated group. Interestingly, the LaAg intranasal vaccination promoted increased production of IFN-γ that was observed in both young- and aged vaccinated groups. Additionally, CD4+ and CD8+T cells from both vaccinated groups presented decreased expression of the inhibitory receptors PD-1 and KLRG1 compared to their unvaccinated controls. Interestingly, a strong positive correlation was observed between the expression of both inhibitory receptors PD-1 and KLRG1 and parasitism, which was more conspicuous in the unvaccinated-aged mice than in the others. Overall, this study helps define new strategies to improve vaccine effectiveness and provides a perspective for prophylactic alternatives against leishmaniasis.

Impact of an autophagy-inducing peptide on immunogenicity and protection efficacy of an adenovirus-vectored SARS-CoV-2 vaccine.

Because of continual generation of new variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), it is critical to design the next generation of vaccines to combat the threat posed by SARS-CoV-2 variants. We developed human adenovirus (HAd) vector-based vaccines (HAd-Spike/C5 and HAd-Spike) that express the whole Spike (S) protein of SARS-CoV-2 with or without autophagy-inducing peptide C5 (AIP-C5), respectively. Mice or golden Syrian hamsters immunized intranasally (i.n.) with HAd-Spike/C5 induced similar levels of S-specific humoral immune responses and significantly higher levels of S-specific cell-mediated immune (CMI) responses compared with HAd-Spike vaccinated groups. These results indicated that inclusion of AIP-C5 induced enhanced S-specific CMI responses and similar levels of virus-neutralizing titers against SARS-CoV-2 variants. To investigate the protection efficacy, golden Syrian hamsters immunized i.n. either with HAd-Spike/C5 or HAd-Spike were challenged with SARS-CoV-2. The lungs and nasal turbinates were collected 3, 5, 7, and 14 days post challenge. Significant reductions in morbidity, virus titers, and lung histopathological scores were observed in immunized groups compared with the mock- or empty vector-inoculated groups. Overall, slightly better protection was seen in the HAd-Spike/C5 group compared with the HAd-Spike group.

Lung Inflammation Induced by Inactivated SARS-CoV-2 in C57BL/6 Female Mice Is Controlled by Intranasal Instillation of Vitamin D.

The COVID-19 pandemic was triggered by the coronavirus SARS-CoV-2, whose peak occurred in the years 2020 and 2021. The main target of this virus is the lung, and the infection is associated with an accentuated inflammatory process involving mainly the innate arm of the immune system. Here, we described the induction of a pulmonary inflammatory process triggered by the intranasal (IN) instillation of UV-inactivated SARS-CoV-2 in C57BL/6 female mice, and then the evaluation of the ability of vitamin D (VitD) to control this process. The assays used to estimate the severity of lung involvement included the total and differential number of cells in the bronchoalveolar lavage fluid (BALF), histopathological analysis, quantification of T cell subsets, and inflammatory mediators by RT-PCR, cytokine quantification in lung homogenates, and flow cytometric analysis of cells recovered from lung parenchyma. The IN instillation of inactivated SARS-CoV-2 triggered a pulmonary inflammatory process, consisting of various cell types and mediators, resembling the typical inflammation found in transgenic mice infected with SARS-CoV-2. This inflammatory process was significantly decreased by the IN delivery of VitD, but not by its IP administration, suggesting that this hormone could have a therapeutic potential in COVID-19 if locally applied. To our knowledge, the local delivery of VitD to downmodulate lung inflammation in COVID-19 is an original proposition.

SARS-CoV-2 infection impacts carbon metabolism and depends on glutamine for replication in Syrian hamster astrocytes.

COVID-19 causes more than million deaths worldwide. Although much is understood about the immunopathogenesis of the lung disease, a lot remains to be known on the neurological impact of COVID-19. Here, we evaluated immunometabolic changes using astrocytes in vitro and dissected brain areas of SARS-CoV-2 infected Syrian hamsters. We show that SARS-CoV-2 alters proteins of carbon metabolism, glycolysis, and synaptic transmission, many of which are altered in neurological diseases. Real-time respirometry evidenced hyperactivation of glycolysis, further confirmed by metabolomics, with intense consumption of glucose, pyruvate, glutamine, and alpha ketoglutarate. Consistent with glutamine reduction, the blockade of glutaminolysis impaired viral replication and inflammatory response in vitro. SARS-CoV-2 was detected in vivo in hippocampus, cortex, and olfactory bulb of intranasally infected animals. Our data evidence an imbalance in important metabolic molecules and neurotransmitters in infected astrocytes. We suggest this may correlate with the neurological impairment observed during COVID-19, as memory loss, confusion, and cognitive impairment.

Avaliação dos níveis da citocina pró-inflamatória fator de necrose tumoral-α em pacientes com diabetes tipo 2 e sua relação com variações genéticas de enzimas antioxidantes e obesidade / Evaluation of proinflammatory cytokine tumor necrosis factor-α levels in obese vs nonobese patients with type 2 diabetes and their relationship to CAT C-262T polymorphism

Introdução: Diabetes tipo 2 (DM2) é um distúrbio multifatorial caracterizado pelo aumento dos níveis de radicais livres. Tanto o estresse oxidativo quanto a obesidade contribuem para um estado inflamatório da doença, principalmente pelo aumento da citocina TNF-α. Sabendo-se que a genética individual pode contribuir para o estresse oxidativo, o estudo avaliou o impacto das variações genéticas de enzimas antioxidantes C262T no gene CAT e polimorfismos nulos dos genes GSTM1 e GSTT1 nos níveis de TNF-α, assim como, avaliou se as variantes genéticas atuariam sinergicamente com a obesidade aumentando os níveis da citocina em diabéticos da Grande Vitória/ES, Brasil.Métodos: O polimorfismo no gene CAT foi avaliado pela técnica PCR/RFLP e nos genes GSTM1 e GSTT1 por PCR multiplex, em 56 pacientes, sendo 28 obesos e 28 não obesos. Níveis de TNF-α foram medidos pela técnica de ELISA sanduíche.Resultados: Frequências das variantes nulas de GSTM1 e GSTT1 foram 44,6% e 17,9%, respectivamente. As frequências genotípicas C262T-CAT foram 73,2%, 25% e 1,8% para homozigoto normal, heterozigoto e homozigoto polimórfico, respectivamente. Não houve associação entre genótipos polimórficos e aumento dos níveis de TNF-α, assim como, não foi demonstrado aumento significante da citocina quando avaliado o sinergismo entre obesidade e genética individual do paciente.Conclusão: Níveis de TNF-α não se elevam em diabéticos tipo 2 na presença dos polimorfismos nos genes CAT, GSTM1 e GSTT1, e a obesidade não atua no aumento dessa citocina na população estudada, separadamente ou em conjunto com a genética individual de variantes nos genes CAT, GSTM1 e GSTT1.

Introduction: Type 2 diabetes is a multifactorial disorder characterized by increased levels of free radicals. Both oxidative stress and obesity contribute to an inflammatory state of the disease, mainly by increasing the levels of the proinflammatory cytokine tumor necrosis factor-α (TNF-α). Considering that personal genetics may contribute to oxidative stress, this study assessed the impact of CAT C-262T polymorphism and GSTM1 and GSTT1 null polymorphisms on TNF-α levels in patients with type 2. diabetes. The study also evaluated whether the genetic variants act synergistically with obesity to increase TNF-α levels in patients with diabetes from Grande Vitória, Brazil.Methods: Fifty-six patients were included, of whom 28 were obese and 28 were nonobese. The CAT gene polymorphism was assessed using the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method, whereas GSTM1 and GSTT1 polymorphisms were assessed using multiplex PCR. TNF-α levels were measured using the sandwich ELISA technique.Results: Frequencies of GSTM1 and GSTT1 null polymorphisms were 44.6% and 17.9%, respectively. The genotype frequencies of CATC-262T polymorphism were 73.2%, 25.0%, and 1.8% for normal homozygote, heterozygote, and polymorphic homozygote, respectively. Polymorphic genotypes were not associated with increased TNF-α levels, and there was no significant increase in TNF-α levels when evaluating the synergism between obesity and personal genetics.Conclusion: The presence of CAT, GSTM1, and GSTT1 gene polymorphisms was not associated with increased TNF-α levels in patients with type 2 diabetes. Obesity alone or combined with personal genetics of CAT, GSTM1, and GSTT1gene polymorphisms did not promote increased TNF-α levels in the study population.

Pam3CSK4 adjuvant given intranasally boosts anti-Leishmania immunogenicity but not protective immune responses conferred by LaAg vaccine against visceral leishmaniasis.

The use of adjuvants in vaccine formulations is a well-established practice to improve immunogenicity and protective immunity against diseases. Previously, we have demonstrated the feasibility of intranasal vaccination with the antigen of killed Leishmania amazonensis promastigotes (LaAg) against experimental leishmaniasis. In this work, we sought to optimize the immunogenic effect and protective immunity against murine visceral leishmaniasis conferred by intranasal delivery of LaAg in combination with a synthetic TLR1/TLR2 agonist (Pam3CSK4). Intranasal vaccination with LaAg/PAM did not show toxicity or adverse effects, induced the increase of delayed-type hypersensitivity response and the production of inflammatory cytokines after parasite antigen recall. However, mice vaccinated with LaAg/PAM and challenged with Leishmania infantum presented significant reduction of parasite burden in both liver and spleen, similar to those vaccinated with LaAg. Although LaAg/PAM intranasal vaccination had induced higher frequencies of specific CD4+ and CD8+ T cells and increased levels of IgG2a antibody isotype in serum, both LaAg and LaAg/PAM groups presented similar levels of IL-4 and IFN-y and decreased production of IL-10 when compared to controls. Our results provide the first evidence of the feasibility of intranasal immunization with antigens of killed Leishmania in association with a TLR agonist, which may be explored for developing an effective and alternative strategy for vaccination against visceral leishmaniasis.