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

Avaliação da influência da obesidade na microbiota pulmonar em modelo murino de asma alérgica

Asthma is the most common inflammatory airway disease and its prevalence has increased worldwide. Obesity is also a risk factor for asthma and preliminary studies demonstrated that obese asthmatic subjects presented high levels of IL-17A and neutrophils in the lungs, demonstrating that they have a distinct phenotype from classic allergy. Evidence has been raised indicating that the gut microbiota play an important role setting different features of asthma. However, there are not studies showing the influence of obesity in a pulmonary microbiota. Therefore, the present study aimed to evaluate the influence of obesity on the pulmonary microbiota in a model of allergic asthma. Female BALB/c mice fed a high fat diet during 14 weeks, sensitized and challenged with Ovalbumin (OVA), were euthanized 24h and 48h after the last OVA challenge. Pulmonary microbiota was analyzed through Fluorescent in situ Hybridization (FISH) and quantitative Polymerase Chain Reaction (qPCR). Lung microbiota effect over dendritic cells was evaluated in coculture with bone marrow derived dendritic cells (BDMC). Both obesity and allergy increased Firmicutes/Bacteroidetes ratio in the gut. Obese and obese-allergic mice presented higher pulmonary IL-17A levels. Obesity increased Proteobacteria and diminished Firmicutes counting, associated with higher TLR-4 and reduced TLR-2 expression by pulmonary DCs. After allergy induction, these markers increased over time, obese allergic mice, which presented diminished Proteobacteria and increased Firmicutes number, compared to allergic group. Obese BMDC coculture with lung microbiota, showed reduced TLR-2/4 and CD80 expression. These data suggests obesity is able to modify pulmonary microbiota, before and after allergy induction. These changes promoted lung homeostasis disruption, causing the delay of immune response development and Th17 profile onset. This work provides a novel mechanism by which obesity worsens asthma. Hence, the better understanding of those interactions will contribute to the development of new treatment strategies suitable for that distinct asthma phenotype.A asma é a doença inflamatória crônica mais frequente das vias aéreas e sua prevalência aumentou em todo o mundo. A obesidade é um fator de risco para asma e estudos preliminares demonstraram que indivíduos obesos asmáticos apresentam níveis elevados de IL-17A e neutrófilos nos pulmões, demonstrando que eles apresentam um fenótipo distinto da alergia clássica. Evidências indicam que a microbiota intestinal desempenha um papel importante na determinação das diferentes características da asma. No entanto, não há estudos mostrando a influência da obesidade sobre a microbiota pulmonar. Portanto, o presente estudo teve como objetivo avaliar a influência da obesidade na microbiota pulmonar em modelo de asma alérgica. Camundongos BALB/c fêmeas alimentados com uma dieta hiperlipídica durante 14 semanas, sensibilizados e desafiados com Ovalbumina (OVA), foram eutanasiados 24h e 48h após o último desafio com OVA. A microbiota pulmonar foi analisada através das técnicas de Hibridização Fluorescente in situ (FISH) e pela Reação em Cadeia da Polimerase quantitativa (qPCR). O efeito da microbiota pulmonar em células dendríticas foi avaliado através de co-cultura com células dendríticas derivadas da medula óssea (BDMC). Tanto a obesidade como a asma, aumentaram a proporção de Firmicutes/Bacteroidetes no intestino. Obesos e obesos-alérgicos apresentaram maiores níveis de IL-17A nos pulmões. A obesidade aumentou a contagem de Proteobacteria e reduziu a de Firmicutes nos pulmões, associada à maior expressão de TLR-4 e à redução da expressão de TLR-2 em CDs pulmonares. Após a indução da alergia, esses marcadores aumentaram ao longo do tempo, em animais obesos alérgicos, os quais apresentaram menor número de Proteobacteria e maior Firmicutes, em comparação ao grupo alérgico. A co-cultura de BMDCs de obesos com a microbiota pulmonar reduziu a expressão de TLR-2/4 e CD80. Esses dados sugerem que a obesidade é capaz de modificar a microbiota pulmonar, antes e após a indução da alergia. Essas alterações promoveram a quebra da homeostasia pulmonar, causando o atraso da resposta imune e o desenvolvimento de uma resposta Th17. Este trabalho fornece um novo mecanismo no qual a obesidade piora a asma. Assim, a melhor compreensão dessas interações contribuirão para o desenvolvimento de novas estratégias de tratamento adequadas para este fenótipo distinto de asma

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

Presentation_1_Blocking the CTLA-4 and PD-1 pathways during pulmonary paracoccidioidomycosis improves immunity, reduces disease severity, and increases the survival of infected mice.pptx

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.</p