Cholesterol-Ester Transfer Protein Alters M1 and M2 Macrophage Polarization and Worsens Experimental Elastase-Induced Pulmonary Emphysema

Cholesterol-ester transfer protein (CETP) plays a role in atherosclerosis, the inflammatory response to endotoxemia and in experimental and human sepsis. Functional alterations in lipoprotein (LP) metabolism and immune cell populations, including macrophages, occur during sepsis and may be related to comorbidities such as chronic obstructive pulmonary disease (COPD). Macrophages are significantly associated with pulmonary emphysema, and depending on the microenvironment, might exhibit an M1 or M2 phenotype. Macrophages derived from the peritoneum and bone marrow reveal CETP that contributes to its plasma concentration. Here, we evaluated the role of CETP in macrophage polarization and elastase-induced pulmonary emphysema (ELA) in human CETP-expressing transgenic (huCETP) (line 5203, C57BL6/J background) male mice and compared it to their wild type littermates. We showed that bone marrow-derived macrophages from huCETP mice reduce polarization toward the M1 phenotype, but with increased IL-10. Compared to WT, huCETP mice exposed to elastase showed worsened lung function with an increased mean linear intercept (Lm), reflecting airspace enlargement resulting from parenchymal destruction with increased expression of arginase-1 and IL-10, which are M2 markers. The cytokine profile revealed increased IL-6 in plasma and TNF, and IL-10 in bronchoalveolar lavage (BAL), corroborating with the lung immunohistochemistry in the huCETP-ELA group compared to WT-ELA. Elastase treatment in the huCETP group increased VLDL-C and reduced HDL-C. Elastase-induced pulmonary emphysema in huCETP mice promotes lung M2-like phenotype with a deleterious effect in experimental COPD, corroborating the in vitro result in which CETP promoted M2 macrophage polarization. Our results suggest that CETP is associated with inflammatory response and influences the role of macrophages in COPD

ROLE OF SIRTUIN 1 IN CD4+ T CELLS ACTIVATION AND DIFFERENTIATION IN A MURINE MODEL OF OBESITY AND TRANSPLANTATION

As histonas desacetilases (HDACs) removem grupos acetil de resíduos de lisina em diferentes proteínas, incluindo histonas. Sirtuinas são membros da classe III das HDACs e sirtuina 1 (Sirt1) desempenham um papel importante no metabolismo celular e regulação da resposta imune. Na obesidade, a expressão de Sirt1 está reduzida na maioria dos tecidos com alta atividade metabólica. Nos últimos anos, tornou-se evidente a contribuição de células T CD4&#43 na obesidade. No entanto, a contribuição da Sirt1 em células T na obesidade não foi totalmente investigada. Nossa hipótese foi que a Sirt1 no contexto da obesidade teria um papel importante na polarização dos linfócitos T CD4&#43, não somente por modificações epigenéticas, mas também por uma modulação metabólica da resposta imune, que culminaria em alterações no aceite de um transplante. Aqui, avaliamos o papel de Sirt1 na diferenciação e ativação de células T CD4+ em um modelo experimental de obesidade. Animais Sirt1&#43/&#43 e CD4-Sirt1-/- com oito semanas de idade foram submetidos à obesidade induzida por dieta (DIO) ou à dieta padrão por 12 semanas. Parâmetros morfológicos, bioquímicos, metabólicos, moleculares e de biologia celular foram avaliados durante e ao final do DIO. Após 12 semanas, os animais DIO tornaram-se obesos e inflamados e mostraram uma atividade reduzida de HDACs e uma expressão reduzida da expressão de Sirt1, Sirt3, CD36 e PGC-1 em células T CD4+ em linfonodos. Foram observadas alterações na expressão desses genes também em momentos diferentes no sangue periférico. Nos linfonodos observou-se também aumento da frequência de Th1, Th17 e redução de células Treg. As células T CD4&#43 também apresentaram um aumento nos marcadores relacionados à ativação ou exaustão, como KLRG-1 e PD-1 e uma mudança para um fenótipo de precursor de memória (MPEC). Observamos um aumento da captação de glicose nas células T CD4&#43 em animais DIO em comparação com os controles, especialmente em CD4-Sirt1-/-. Além disso, um aumento da massa mitocondrial e produção de superóxido mitocondrial e um perfil respiratório mitocondrial alterado (em termos de vazamento de prótons, capacidade respiratória máxima e de reserva) foram observados em células T CD4&#43 de animais obesos. A depleção condicional de Sirt1 em células T CD4&#43 aumentou a frequência de células Th1 e Th17, no contexto da obesidade, em comparação com os resultados obtidos em animais Sirt1&#43/&#43 em DIO. O perfil bioenergético das células T CD4+ e a captação de glicose também mostraram respostas metabólicas e demanda de glicose aumentadas nos animais DIO em comparação com os controles. Em relação aos resultados do transplante, não foram observadas diferenças entre Sirt1&#43/&#43 e CD4-Sirt1-/-, mas uma taxa de rejeição acelerada foi observada nos animais em DIO. Em conclusão, a deleção de Sirt1 nas células T CD4&#43 agrava o efeito da obesidade no perfil metabólico e funcional dessas células. Esses dados sugerem um papel protetor de Sirt1 nas células T CD4&#43 no contexto de distúrbios metabólicos.Histone deacetylases (HDACs) remove acetyl groups from lysine residues in different proteins, including histones. Sirtuins are members of class III HDACs and Sirtuin 1 (Sirt1) plays a role in cellular metabolism and immunological regulation. In obesity, the expression of Sirt1 is constitutively downregulated in most metabolic tissues. Recently, it has become evident the contribution of T cells to obesity. However, the importance of Sirt1 expression in T cells in the context of obesity has not been investigated. We hypothesized that Sirt1 in the context of obesity has an important role on CD4&#43 T cell polarization, not just from an epigenetic point of view, but by a metabolic modulation of the immune response and these modifications could also be involved in the progression of transplant rejection. Here, we evaluate the role of Sirt1 in the differentiation and activation of CD4&#43 T cells in an experimental model of obesity. Eight weeks old Sirt&#43/&#43 and CD4-Sirt1-/- animals were submitted to diet-induced obesity (DIO) or standard diet conditions for 12 weeks. Morphological, biochemical, metabolic, molecular and cell biology parameters were evaluated through and at the end of the DIO. After 12 weeks, DIO animals became obese and inflamed and showed a reduced activity of HDACs and a reduced expression of Sirt1, Sirt3, CD36 and PGC-1 expression in CD4&#43 T cells from lymph nodes, changes in the expression of these genes were observed also at different point times in peripheral blood. An increased frequency of Th1, Th17 and reduction of Treg cells in draining lymph nodes was also observed. CD4&#43 T cells also presented an increase in markers related to activation or exhaustion, such as KLRG-1 and PD-1 and a shift to a memory precursor phenotype (MPEC). We observed an increased glucose uptake in DIO animals compared to controls, especially in CD4-Sirt1-/- supporting increased glucose demand in T cells from DIO mice. In addition, an increased mitochondrial mass and mitochondrial superoxide production, an altered mitochondrial respiratory profile (in terms of proton leak, maximal and spare respiratory capacity) were observed in CD4&#43 T cells from obese animals. Conditional depletion of Sirt1 in CD4&#43 T cells increased the frequency of Th1 and Th17 cells, in the context of obesity, compared with the obtained results in Sirt1&#43/&#43 DIO animals. The bioenergetic profile of CD4&#43 T cells and glucose uptake also showed increased metabolic responses and glucose demand in DIO animals compared to controls. Regarding transplantation outcomes, no differences were observed between Sirt1+/+ and CD4-Sirt1-/- but an accelerated rejection rate was observed as a result of DIO. In conclusion, deletion of Sirt1 in CD4&#43 T cells aggravates the effect of obesity in the metabolic and functional profile of these cells. These data suggest a protective role of Sirt1 in CD4&#43 T cells in the context of metabolic disorders. All the procedures were evaluated and accepted by the ethical committees of the participant institutions and were performed according to the national and international regulations and guaranteeing the animal welfare. Ethics committee approval code: CEUA 909020031

Role of sirtuin 1 in CD4+ T cells activation and differentiationin a murine model of obesity and transplantation

Tesis Doctoral inédita cotutelada por la Universidade de Sâo Paulo y la Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Biología Molecular. Fecha de Lectura: 11-02-2022Esta tesis tiene embargado el acceso al texto completo hasta el 11-08-202

A Simple Proteomics-Based Approach to Identification of Immunodominant Antigens from a Complex Pathogen: Application to the CD4 T Cell Response against Human Herpesvirus 6B.

Most of humanity is chronically infected with human herpesvirus 6 (HHV-6), with viral replication controlled at least in part by a poorly characterized CD4 T cell response. Identification of viral epitopes recognized by CD4 T cells is complicated by the large size of the herpesvirus genome and a low frequency of circulating T cells responding to the virus. Here, we present an alternative to classical epitope mapping approaches used to identify major targets of the T cell response to a complex pathogen like HHV-6B. In the approach presented here, extracellular virus preparations or virus-infected cells are fractionated by SDS-PAGE, and eluted fractions are used as source of antigens to study cytokine responses in direct ex vivo T cell activation studies. Fractions inducing significant cytokine responses are analyzed by mass spectrometry to identify viral proteins, and a subset of peptides from these proteins corresponding to predicted HLA-DR binders is tested for IFN-γ production in seropositive donors with diverse HLA haplotypes. Ten HHV-6B viral proteins were identified as immunodominant antigens. The epitope-specific response to HHV-6B virus was complex and variable between individuals. We identified 107 peptides, each recognized by at least one donor, with each donor having a distinctive footprint. Fourteen peptides showed responses in the majority of donors. Responses to these epitopes were validated using in vitro expanded cells and naturally expressed viral proteins. Predicted peptide binding affinities for the eight HLA-DRB1 alleles investigated here correlated only modestly with the observed CD4 T cell responses. Overall, the response to the virus was dominated by peptides from the major capsid protein U57 and major antigenic protein U11, but responses to other proteins including glycoprotein H (U48) and tegument proteins U54 and U14 also were observed. These results provide a means to follow and potentially modulate the CD4 T-cell immune response to HHV-6B

Differences in IgE mediated basophil degranulation induced by proteic fractions from whole flea body extract in patients with papular urticaria by flea bite and healthy controls

Background Papular urticaria by flea bite (PUFB) is a chronic inflammatory disease in children. The aim of this study was to assess the functional activity of IgE to protein fractions from flea body extract, through basophil degranulation in PUFB patients and controls.Methods Basophil degranulation, measured by overexpression of CD63 surface molecules, was evaluated by flow cytometry in samples from patients and controls. Cell stimulation was performed with three fractions with different molecular weight from flea body extract using a Basotest® modified protocol. Mann–Whitney U-test was used for comparisons.Results Specific IgE from PUFB patients and healthy controls induced basophil degranulation to flea body extract with no significant differences between them (16.2 ± 3.1% vs 13.6 ± 2.8% p = 0.77). However, when flea extract was analyzed in fractions with proteins ranging different molecular weights, significant differences were observed on the response from patients compared with controls to <50 kD (14.9 ± 5.1% vs 9.7 ± 2.1% p = 0.0058) and 50–100 kD proteic fractions (8.3 ± 3.2% vs 2.8 ± 1.6% p = 0.0021).Conclusion In this study, was established that the differential response by IgE, in PUFB, depends from the molecular weight of the antigens contained in the flea extract. These antigens may be related to 30–35 kD proteins previously described as major allergens. Keywords: Basophil degranulation, CD63, Flea allergy, Papular urticaria, Basophil degranulation tes

Mapping of the CD4 T cell responses in PBMCs to HHV-6B proteins.

<p>A. IFN-γ ELISpot responses (SFU/10⁶ cells) by donors #037, 118 and 132 to pools 1–29. Error bars represent the standard deviation of the replicates. B. Heat maps summarizing the response of all donors (n = 5) to all pools tested (53). Positive responses were assessed by DFR2x and ER analyses (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0142871#sec002" target="_blank">Methods</a>). Pools for which positive responses were observed by DFR2x in at least 4 donors were selected for further analysis and are indicated by (+). C. IFN-γ ELISpot responses by different donors to individual peptides in pools 5, 11, 23 and 29. D. Heat-maps summarizing the IFN-γ responses to individual peptides present in the pools selected in B. Positive responses to individual peptides were assessed by ER and DFR2x analyses. Fourteen peptides that induced responses in multiple donors were selected for further validation, and are indicated by (+).</p

DRB1 haplotype and serologic status of donors used in this study.

<p>1. HHV-6 IgG titer measured by IFA</p><p>2. HCMV IgG status measured by ELISA</p><p>DRB1 haplotype and serologic status of donors used in this study.</p

Summary of HHV-6B proteins identified by LC/MS^E in gel-fractions that induced IFN-γ responses.

<p>1. Virus: extracellular HHV-6B virus; Cell lysate: lysate of HHV-6B-infected cells</p><p>Summary of HHV-6B proteins identified by LC/MS^E in gel-fractions that induced IFN-γ responses.</p