Characterization of the in vitro cytokine response of phagocytes to mycobacteria

Dissertação mestrado Ciências (ramo de conhecimento em Genética Molecular)Tuberculosis, caused by Mycobacterium tuberculosis, remains one of the main threats to mankind. Despite the intense research on the immune response to tuberculosis, major questions remain unsolved, one of which relates to the fact that the efficacy of the current vaccine, Mycobacterium Bovis BCG, is variable. However, no other experimental vaccines against tuberculosis developed in the last 100 years were proven to be better than BCG. Therefore BCG remains the only vaccine available to date to prevent tuberculosis. A possible explanation for the variability of BCG efficacy relies on the fact that the immune response triggered by BCG and M. tuberculosis is different. Nevertheless, M. tuberculosis-based experimental vaccines tend not to protect better than BCG. We therefore hypothesized that the basic protective response triggered by BCG must be appropriate, although it needs to be improved in order to confer higher and longer lasting protection. In this work, we performed a comparative study of M. tuberculosis versus BCG infection on dendritic cells (DCs) and macrophages to better understand how these pathogens interact with cells of the innate immune system and how that might translate into an effective, or not, T helper (Th) cell response. We found that macrophages and DCs respond differently to M. tuberculosis or BCG stimulation in what concerns cytokine expression. The expression of IL-12p40, TNF and IL-6 is significantly higher in DCs than in macrophages stimulated with either mycobacteria. Nevertheless, the expression of IL-12p35 and IL-10 was similar in both cell types. We also found that the same cell type respond differently to M. tuberculosis or BCG. M. tuberculosis-stimulated DCs induced higher levels of p40 and p19 monomers and of the bioactive cytokines IL-12 and IL-23, respectively. BCGstimulated DCs produced higher amounts of TNF. The amounts of IL-6 and IL-10 secreted upon stimulation of DCs with M. tuberculosis or BCG were similar. The differential expression of IL-12 and IL-23 might be correlated in part to a higher activation of the MAP kinase ERK1/2 in DCs in response to M. tuberculosis than to BCG. Although macrophages were in general poorly induced to produce cytokines, when compared to DCs, the threshold of ERK1/2 activation was higher in stimulated macrophages. A consequence of the differential activation of DCs was reflected on the distinct type of Th responses developed when M. tuberculosis- or BCG-infected DCs presented OVA peptide to TCRtransgenic CD4+ T cells. M. tuberculosis-infected DCs were able to induce the development of both Th1 and Th17 responses, whereas BCG-infected DCs presented a shift towards Th17 responses. These differences are of interest considering the importance of Th1/Th17 balance during vaccination. Further understanding the molecular mechanisms dictating this differential Th response will be used for the development of new vaccines.A tuberculose é causada pelo Mycobacterium tuberculosis e constitui um grave problema a nível mundial. Apesar de todo o esforço dedicado ao estudo desta infecção, questões como a variabilidade da eficácia da corrente vacina em uso, M. bovis BCG, permanecem inexplicáveis. No entanto, nos últimos 100 anos, não foi experimentalmente desenhada outra vacina mais eficiente do que o BCG. Sendo assim, a única vacina disponível até ao momento para combater a tuberculose é o BCG. Uma possivel explicação para a variabilidade da vacina consiste no facto de o M. tuberculosis e o M. bovis BCG desencadearem uma resposta imunológica diferente.Contudo, vacinas experimentais baseadas no M. tuberculosis não conferem maior protecção do que o BCG. A nossa hipótese, é que a resposta desencadeada pelo BCG deve ser adequada, embora necessite de ser melhorada de modo a conferir uma protecção maior e mais prolongada. Neste trabalho foram comparadas as infecções por M. tuberculosis versus BCG em células dendríticas (DCs) e em macrófagos, com os objectivos de compreender melhor a interacção entre estes agentes patogénicos e o sistema imunológico inato, e como é que isso se traduz, ou não, numa reposta T de ajuda (Th) eficaz. Os nossos resultados mostram que as respostas induzidas pelos macrófagos e pelas DCs estimulados por M. tuberculosis ou por BCG, em termos de expressão de citocinas, são diferentes. A expressão de IL-12p40, TNF e IL-6 induzida por ambas as micobactérias foi significativamente maior em DCs do que em macrófagos. Contudo, a expressão de IL-12p35 e IL-10 foi semelhante em ambos os tipos celulares. Observamos, também, que o mesmo tipo celular responde de modo diferente ao M. tuberculosis ou ao BCG. As DCs estimuladas pelo M. tuberculosis induziram níveis elevados de p40 e p19, bem como das respectivas citocinas bioactivas IL-12 e IL-23. As DCs estimuladas pelo BCG produziram elevados níveis de TNF. A produção de IL-6 e IL-10 foi semelhante quer nas DCs estimuladas pelo M. tuberculosis quer pelo BCG. A expressão diferencial de IL-12 e de IL-23 poderá estar correlacionada, em parte, com uma maior activação da MAP cinase ERK1/2 pelas DCs em resposta ao M. tuberculosis relactivamente ao BCG. Embora a indução de citocinas pelos macrófagos fosse menor do que pelas DCs, o threshold de activação do ERK1/2 induzido pelos macrófagos foi maior. A diferente activação induzida pelo M. tuberculosis ou BCG em DCs reflectiu-se no tipo de resposta Th diferenciada. Em DCs estimuladas com M. tuberculosis ou BCG, que apresentam o péptido OVA a células T CD4+ cujo TCR é transgénico, observamos que as DCs estimuladas com M. tuberculosis induziram o desenvolvimento de respostas Th1 e Th17, enquanto que DCs estimuladas com BCG induziram uma resposta Th17 superior à observada com M. tuberculosis. Considerando a importância do balanço Th1/Th17 durante a vaccinação, as diferenças observadas são de extrema relevância, dado que a compreensão dos mecanismos moleculares que ditam esta resposta diferencial consiste numa estratégia para o desenvolvimento de novas vacinas contra a tuberculose

Torulaspora delbrueckii phenotypic and metabolic profiling towards its biotechnological exploitation

Wine is a particularly complex beverage resulting from the combination of several factors, with yeasts being highlighted due to their fundamental role in its development. For many years, non-Saccharomyces yeasts were believed to be sources of spoilage and contamination, but this idea was challenged, and many of these yeasts are starting to be explored for their beneficial input to wine character. Among this group, Torulaspora delbrueckii is gaining relevance within the wine industry, owing to its low volatile acidity production, increased release of aromatic compounds and enhanced color intensity. In addition, this yeast was also attracting interest in other biotechnological areas, such as bread and beer fermentation. In this work, a set of 40 T. delbrueckii strains, of varied geographical and technological origins, was gathered in order to characterize the phenotypic behavior of this species, focusing on different parameters of biotechnological interest. The fermentative performance of the strains was also evaluated through individual fermentations in synthetic grape must with the isolates’ metabolic profile being assessed by HPLC. Data analysis revealed that T. delbrueckii growth is significantly affected by high temperature (37 °C) and ethanol concentrations (up to 18%), alongside 1.5 mM SO2, showing variable fermentative power and yields. Our computation models suggest that the technological origin of the strains seems to prevail over the geographical origin as regards the influence on yeast properties. The inter-strain variability and profile of the products through the fermentative processes reinforce the potential of T. delbrueckii from a biotechnological point of view.This work was supported by the UID/BIA/04050/2013 (POCI-01-0145-FEDER-007569) and UID/BIA/04050/2019 strategic programs and by the project PTDC/BIA-MIC/32059/2017, which is funded by national funds through the FCT-Fundação para a Ciência e Tecnologia and by the ERDFEuropean Regional Development Fund through the COMPETE2020–Programa Operacional Competitividade e Internacionalização (POCI) and Sistema de Apoio à Investigação Científica e Tecnológica (SAICT). FS-S and TF were supported by Fellowships (UI/BD/150873/2021 and 2021.04595.BD, respectively) from Fundação para a Ciência e Tecnologia, Portugal

Cellular and Molecular Characterization of Microglia:A Unique Immune Cell Population

Microglia are essential for the development and function of the adult brain. Microglia arise from erythro-myeloid precursors in the yolk sac and populate the brain rudiment early during development. Unlike monocytes that are constantly renewed from bone marrow hematopoietic stem cells throughout life, resident microglia in the healthy brain persist during adulthood via constant self-renewal. Their ontogeny, together with the absence of turnover from the periphery and the singular environment of the central nervous system, make microglia a unique cell population. Supporting this notion, recent genome-wide transcriptional studies revealed specific gene expression profiles clearly distinct from other brain and peripheral immune cells. Here, we highlight the breakthrough studies that, over the last decades, helped elucidate microglial cell identity, ontogeny, and function. We describe the main techniques that have been used for this task and outline the crucial milestones that have been achieved to reach our actual knowledge of microglia. Furthermore, we give an overview of the "microgliome" that is currently emerging thanks to the constant progress in the modern profiling techniques

The nature of the silicaphilic fluorescence of PDMPO

PDMPO (2-(4-pyridyl)-5-((4-(2-dimethylaminoethylaminocarbamoyl)methoxy)phenyl)oxazole), has unique silica specific fluorescence and is used in biology to understand biosilicification. This ‘silicaphilic’ fluorescence is not well understood nor is the response to local environmental variables like solvent and pH. We investigated PDMPO in a range of environments: using UV-vis and fluorescence spectroscopy supported by computational data, (SPARC, molecular dynamics simulations, density functional theory calculations), dynamic light scattering and zeta potential measurements to understand the PDMPO–silica interaction. From absorption data, PDMPO exhibited a pKa of 4.20 for PDMPOH22+ to PDMPOH+ . Fluorescence emission measurements revealed large shifts in excited state pKa* values with different behaviour when bound to silica (pKa* of 10.4). PDMPO bound to silica particles is located in the Stern layer with the dye exhibiting pH dependent depolarising motion. In aqueous solution, PDMPO showed strong chromaticity with correlation between the maximum emission wavelength for PDMPOH+* and dielectric constant (4.8–80). Additional chromatic effects were attributed to changes in solvent accessible surface area. Chromatic effects were also observed for silica bound dye which allow its use as a direct probe of bulk pH over a range far in excess of what is possible for the dye alone (3–5.2). The unique combination of chromaticity and excited state dynamics allows PDMPO to monitor pH from 3 to 13 while also reporting on surface environment opening a new frontier in the quantitative understanding of (bio)silicification

Mycobacterium tuberculosis strains are differentially recognized by TLRs with an impact on the immune response

Tuberculosis associates with a wide spectrum of disease outcomes. The Beijing (Bj) lineage of Mycobacterium tuberculosis (Mtb) is suggested to be more virulent than other Mtb lineages and prone to elicit non-protective immune responses. However, highly heterogeneous immune responses were reported upon infection of innate immune cells with Bj strains or stimulation with their glycolipids. Using both in vitro and in vivo mouse models of infection, we here report that the molecular mechanism for this heterogeneity may be related to distinct TLR activations. Among this Mtb lineage, we found strains that preferentially activate TLR2, and others that also activate TLR4. Recognition of Mtb strains by TLR4 resulted in a distinct cytokine profile in vitro and in vivo, with specific production of type I IFN. We also uncover a novel protective role for TLR4 activation in vivo. Thus, our findings contribute to the knowledge of the molecular basis underlying how host innate immune cells handle different Mtb strains, in particular the intricate host-pathogen interaction with strains of the Mtb Bj lineage.This work has been funded by Fundacao para a Ciencia e Tecnologia, Portugal. Project grants: PTDC/SAU-MII/101977/2008 and PTDC/BIA-BCM/102776/2008. Personal grants: SFRH/BD/35981/2007 to JC; SFRH/BPD/3306/2007 to AC; SFRH/BPD/77399/2011 to LMT; SFRH/BI/33456/2008 to CS; and SFRH/BPD/33959/2009 to NSO. MS is a Ciencia 2007 fellow. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript

Elucidating tumour-associated microglia/macrophage diversity along glioblastoma progression and under ACOD1 deficiency

In glioblastoma (GBM), tumour-associated microglia/macrophages (TAMs) represent the major cell type of the stromal compartment and contribute to tumour immune escape mechanisms. Thus, targeting TAMs is emerging as a promising strategy for immunotherapy. However, TAM heterogeneity and metabolic adaptation along GBM progression represent critical features for the design of effective TAM-targeted therapies. Here, we comprehensively study the cellular and molecular changes of TAMs in the GL261 GBM mouse model, combining single-cell RNA-sequencing with flow cytometry and immunohistological analyses along GBM progression and in the absence of Acod1 (also known as Irg1), a key gene involved in the metabolic reprogramming of macrophages towards an anti-inflammatory phenotype. Similarly to patients, we identify distinct TAM profiles, mainly based on their ontogeny, that reiterate the idea that microglia- and macrophage-like cells show key transcriptional differences and dynamically adapt along GBM stages. Notably, we uncover decreased antigen-presenting cell features and immune reactivity in TAMs along tumour progression that are instead enhanced in Acod1-deficient mice. Overall, our results provide insight into TAM heterogeneity and highlight a novel role for Acod1 in TAM adaptation during GBM progression.publishedVersio

Tell me if you prefer bovine or poultry sectors and I’ll tell you who you are: Characterization of Salmonella enterica subsp. enterica serovar Mbandaka in France

IntroductionIn north-western France, Salmonella enterica susp. enterica serovar Mbandaka (S. Mbandaka) is most frequently isolated from bovine and dairy samples. While this serovar most often results in asymptomatic carriage, for a number of years it has caused episodes of abortions, which have serious economic consequences for the sector. Interestingly, this serovar is also isolated from Gallus gallus in the same geographic zone. Despite its prevalence in bovines in north-western France, S. Mbandaka has not been broadly studied at the genomic level, and its prevalence and host adaptation are still not fully understood.MethodsIn this study, we analyzed the genomic diversity of 304 strains of S. Mbandaka isolated from the bovine and poultry sectors in this area over a period of 5 years. A phylogenetic analysis was carried out and two approaches were followed to identify conserved genes and mutations related to host associations. The first approach targeted the genes compiled in the MEGARESv2, Resfinder, VFDB and SPI databases. Plasmid and phage contents were also investigated. The second approach refers to an in-house algorithm developed for this study that computes sensitivity, specificity, and accuracy of accessory genes and core variants according to predefined genomes groups.Results and discussionAll the analyzed strains belong to the multi-locus sequence type profile ST413, and the phylogenomic analysis revealed main clustering by host (bovine and poultry), emphasizing the circulation of 12 different major clones, of which seven circulate in poultry and five in the bovine sector in France and a likely food production chain adaptation of these clones. All strains present resistance determinants including heavy metals and biocides that could explain the ability of this serovar to survive and persist in the environment, within herds, and in food processing plants. To explore the wild animal contribution to the spread of this serovar in north-western France, we retrieved S. Mbandaka genomes isolated from wild birds from EnteroBase and included them in the phylogenomic analysis together with our collection. Lastly, screening of accessory genes and major variants allowed us to identify conserved specific mutations characteristic of each major cluster. These mutations could be used to design useful probes for food safety surveillance

TLR2 deficiency by compromising p19 (IL-23) expression limits T helper 17 cell responses to Mycobacterium tuberculosis

The authors are grateful to Drs. Manuel Teixeira da Silva, Fernando Rodrigues, Margarida Correia-Neves and Paul S. Redford for critically reading this manuscript and thank the personnel at the ICVS animal house facility for excellent animal husbandry.CD4+ Th1 cells producing IFN-γ are of extreme importance in controlling infections by Mycobacterium tuberculosis both in mice and in men. In addition to IFN-γ-producing T cells, IL-17-producing T cells (Th17) have been observed during mycobacterial infections. Nevertheless, their contribution for the host immune response to mycobacteria as well as the signals triggering M. tuberculosis -specific Th17 cell differentiation and maintenance are not fully understood. We show that signaling via Toll-like receptor (TLR) 2 has a major impact on the regulation of p19 (IL-23) expression in response to M. tuberculosis and therefore on the establishment of Th17 cell responses to M. tuberculosis infection. Diminished Th17 responses in the lung of M. tuberculosis -infected TLR2-deficient animals were not caused by defective cell differentiation in the draining lymph node (LN) but rather by reduced maintenance at the site of infection. Consistent with the decreased numbers of Th17 cells in the lungs of infected TLR2-deficient animals, we observed reduced expression of CXCL9, CXCL10 and CXCL11, chemokines involved in recall responses to M. tuberculosis. Our data provides insights into the TLR2 role in infection with M. tuberculosis, with implications in pathophysiology of the disease and vaccine design.Fundação para a Ciência e Tecnologia, Portugal (Project Grants PTDC/SAU/70895/2006 to AGC and PTDC/BIA-BCM/102776/2008 to MS; and Personal Grants SRFH/BD/33034/2006 to MTC; SFRH/BPD/3306/2007 to AC; SFRH/BD/35981/2007 to JC; SFRH/BI/33456/2008 to CS and PTDC/SAU-MII/70895/2006 to DRP) and by the Health Service of Fundação Calouste Gulbenkian. MS is a Ciência 2007 Fellow

Challenging the Science Curriculum Paradigm: TeachingPrimary Children Atomic-Molecular Theory

Solutions to global issues demand the involvement of scientists, yet concern exists about retention rates in science as students pass through school into University. Young children are curious about science, yet are considered incapable of grappling with abstract and microscopic concepts such as atoms, sub-atomic particles, molecules and DNA. School curricula for primary (elementary) aged children reflect this by their limitation to examining only what phenomena are without providing any explanatory frameworks for how or why they occur. This research challenges the assumption that atomic-molecular theory is too difficult for young children, examining new ways of introducing atomic theory to 9 year olds and seeks to verify their efficacy in producing genuine learning in the participants. Early results in three cases in different schools indicate these novel methods fostered further interest in science, allowed diverse children to engage and learn aspects of atomic theory, and satisfied the children’s desire for intellectual challenge. Learning exceeded expectations as demonstrated in the post-interview findings. Learning was also remarkably robust, as demonstrated in two schools eight weeks after the intervention, and in one school, one year after their first exposure to ideas about atoms, elements and molecules