Browsing by Author "Pires, David"
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- A 3d cell culture model of the tuberculosis granuloma that can be applied for host genetic studies in the context of a multicellular immunologic response to infectionPublication . David, Susana; Mandal, Manoj; Anes, Elsa; Pires, DavidIntroduction: The granuloma is an inflammatory infiltrate of mononuclear cells. Some bacterial infections are characterized by the formation of granulomas as part of the immune response to contain the infection. Granuloma models have contributed valuable insights into the genetic basis of granuloma formation during infection. For example, IFNGR1 and IFNGR2 variants have been found to disrupt the immune response, resulting in impaired granuloma formation and increased susceptibility to diseases by Mycobacterium sp. More easily implemented comprehensive models would facilitate the study of the different immune mechanisms and help identify new diseaseassociated genes. Our objective is to generate an in vitro 3D cell culture model using human primary cells and microspheres to generate a stratified granuloma model for future use in genetic, immunological and drug discovery studies. Methods: A commercial system was used to encapsulate human peripheral blood mononuclear cells (PBMC) infected with GFP-expressing M. tuberculosis and maintained in culture for several weeks. The cellular constituents of these granulomas and their organization were characterized by fluorescence microscopy and flow cytometry as well as the viability of the cells and the extent of bacterial replication in factor of time. Results: The results demonstrate a ready recruitment of cells towards infected macrophages, leading to the formation of densely populated aggregates. These aggregates maintained cell viability for several weeks and displayed an enhanced control of bacterial replication compared to the more common monolayer infection models. Moreover, the capsules can be easily disrupted when required to isolate genetic material for further analysis. Conclusion: The proposed 3D model resembles some structural and cellular characteristics of the tuberculosis granuloma and maintains its stability beyond more common 2D models of infection. These preliminary results demonstrate that this model can be used to further explore the determinants of granuloma formation and host response to infection.
- Cell-to-cell transmission of HIV-1 and HIV-2 from infected macrophages and dendritic cells to CD4+ T lymphocytesPublication . Calado, Marta; Pires, David; Conceição, Carolina; Ferreira, Rita; Santos-Costa, Quirina; Anes, Elsa; Azevedo-Pereira, José MiguelMacrophages (Mø) and dendritic cells (DCs) are key players in human immunodeficiency virus (HIV) infection and pathogenesis. They are essential for the spread of HIV to CD4+ T lymphocytes (TCD4+) during acute infection. In addition, they constitute a persistently infected reservoir in which viral production is maintained for long periods of time during chronic infection. Defining how HIV interacts with these cells remains a critical area of research to elucidate the pathogenic mechanisms of acute spread and sustained chronic infection and transmission. To address this issue, we analyzed a panel of phenotypically distinct HIV-1 and HIV-2 primary isolates for the efficiency with which they are transferred from infected DCs or Mø to TCD4+. Our results show that infected Mø and DCs spread the virus to TCD4+ via cell-free viral particles in addition to other alternative pathways. We demonstrate that the production of infectious viral particles is induced by the co-culture of different cell populations, indicating that the contribution of cell signaling driven by cell-to-cell contact is a trigger for viral replication. The results obtained do not correlate with the phenotypic characteristics of the HIV isolates, namely their co-receptor usage, nor do we find significant differences between HIV-1 and HIV-2 in terms of cis- or trans-infection. The data presented here may help to further elucidate the cell-to-cell spread of HIV and its importance in HIV pathogenesis. Ultimately, this knowledge is critical for new therapeutic and vaccine approaches.
- CRISPRi-mediated characterization of novel anti-tuberculosis targets: mycobacterial peptidoglycan modifications promote beta-lactam resistance and intracellular survivalPublication . Silveiro, Cátia; Marques, Mariana; Olivença, Francisco; Pires, David; Mortinho, Diana; Nunes, Alexandra; Pimentel, Madalena; Anes, Elsa; Catalão, Maria JoãoThe lack of effective therapeutics against emerging multi-drug resistant strains of Mycobacterium tuberculosis (Mtb) prompts the identification of novel anti-tuberculosis targets. The essential nature of the peptidoglycan (PG) layer of the mycobacterial cell wall, which features several distinctive modifications, such as the N-glycolylation of muramic acid and the amidation of D-iso-glutamate, makes it a target of particular interest. To understand their role in susceptibility to beta-lactams and in the modulation of host-pathogen interactions, the genes encoding the enzymes responsible for these PG modifications (namH and murT/gatD, respectively) were silenced in the model organism Mycobacterium smegmatis using CRISPR interference (CRISPRi). Although beta-lactams are not included in TB-therapy, their combination with beta-lactamase inhibitors is a prospective strategy to treat MDR-TB. To uncover synergistic effects between the action of beta-lactams and the depletion of these PG modifications, knockdown mutants were also constructed in strains lacking the major beta-lactamase of M. smegmatis BlaS, PM965 (M. smegmatis ΔblaS1) and PM979 (M. smegmatis ΔblaS1 ΔnamH). The phenotyping assays affirmed the essentiality of the amidation of D-iso-glutamate to the survival of mycobacteria, as opposed to the N-glycolylation of muramic acid. The qRT-PCR assays confirmed the successful repression of the target genes, along with few polar effects and differential knockdown level depending on PAM strength and target site. Both PG modifications were found to contribute to beta-lactam resistance. While the amidation of D-iso-glutamate impacted cefotaxime and isoniazid resistance, the N-glycolylation of muramic acid substantially promoted resistance to the tested beta-lactams. Their simultaneous depletion provoked synergistic reductions in beta-lactam MICs. Moreover, the depletion of these PG modifications promoted a significantly faster bacilli killing by J774 macrophages. Whole-genome sequencing revealed that these PG modifications are highly conserved in a set of 172 clinical strains of Mtb, demonstrating their potential as therapeutic targets against TB. Our results support the development of new therapeutic agents targeting these distinctive mycobacterial PG modifications.
- Cystatin F depletion in Mycobacterium tuberculosis-infected macrophages improves cathepsin C/granzyme B-driven cytotoxic effects on HIV-infected cells during coinfectionPublication . Mandal, Manoj; Pires, David; Calado, Marta; Azevedo-Pereira, Jose Miguel; Anes, ElsaCystatin F (CstF) is a protease inhibitor of cysteine cathepsins, including those involved in activating the perforin/granzyme cytotoxic pathways. It is targeted at the endolysosomal pathway but can also be secreted to the extracellular milieu or endocytosed by bystander cells. CstF was shown to be significantly increased in tuberculous pleurisy, and during HIV coinfection, pleural fluids display high viral loads. In human macrophages, our previous results revealed a strong upregulation of CstF in phagocytes activated by interferon γ or after infection with Mycobacterium tuberculosis (Mtb). CstF manipulation using RNA silencing led to increased proteolytic activity of lysosomal cathepsins, improving Mtb intracellular killing. In the present work, we investigate the impact of CstF depletion in macrophages during the coinfection of Mtb-infected phagocytes with lymphocytes infected with HIV. The results indicate that decreasing the CstF released by phagocytes increases the major pro-granzyme convertase cathepsin C of cytotoxic immune cells from peripheral blood-derived lymphocytes. Consequently, an observed augmentation of the granzyme B cytolytic activity leads to a significant reduction in viral replication in HIV-infected CD4+ T-lymphocytes. Ultimately, this knowledge can be crucial for developing new therapeutic approaches to control both pathogens based on manipulating CstF.
- Development and characterisation of a 3D cell culture model of the tuberculosis granulomaPublication . Pires, David; David, Susana; Mandal, Manoj; Soderberg, Julia; Anes, ElsaTuberculosis (TB) is a disease caused by Mycobacterium tuberculosis (Mtb) that results in 1.6 million deaths yearly. The TB granuloma is the hallmark cellular structure of latent TB that contains the spread of infection1 . More comprehensive in vitro models of TB that better resemble the cellular and immunoregulatory complexity of the granuloma would facilitate the study of the interplay between the bacteria and the different immune system cells2 . We aim to generate an in vitro, 3D cell culture model of the TB granuloma that can be easily implemented using readily available commercial reagents and materials. A commercial encapsulation system based on sodium cellulose sulphate (NaCS) and Poly (diallyldimethylammonium chloride) (PDADMAC)3 was used to generate small capsules containing human peripheral blood mononuclear cells (PBMC) in the presence of GFPexpressing Mtb H37Rv and maintained in culture for several weeks. The 3D structure formed by the cells inside and outside the capsules was evaluated by fluorescence microscopy and flow cytometry to distinguish the different cell types, and how they are organised inside the sphere and to measure cell survival and bacteria replication. The results show that human PBMCs readily form 3D cellular aggregates around infected cells and that cells cultivated outside the capsules are attracted and surround the capsules in response to infection. The model could be maintained for several weeks before bacteria-induced cell necrosis. PBMC’s viability remained stable, with more than 80 % live cells following two weeks of culture. Moreover, adding an exterior layer of cells helped control bacterial replication, suggesting relevant communication between cells inside and outside the capsules to control the infection.
- Development of a new mRNA vaccine platform for tuberculosisPublication . Matarazzo, Laura; Taina‑González, Laura; Pinheiro, Ricardo; Pires, David; Fuente, Maria de la; Bettencourt, Paulo J. G.
- Development of a new mRNA vaccine platform for tuberculosisPublication . Matarazzo, Laura; Taina‑González, Laura; Pinheiro, Ricardo; Pires, David; de la Fuente, María; Bettencourt, Paulo J. G.Background Tuberculosis (TB), caused by Mycobacterium tuberculosis (M.tb), is the frst cause of death by an infectious disease worldwide, killed 1.6 million people in 2021. Bacillus Calmette-Guerin (BCG) is the only approved vaccine for TB to date. However, while BCG is efective in preventing severe forms in children, its efcacy in adults is inconsistent and it does not prevent transmission, highlighting the need for new vaccine development [1]. The recent success of COVID-19 vaccines raised the interest for mRNA-based vaccines, as they are efective, safe and easy to produce. This project aims to develop a new mRNA vaccine platform for TB, based on mRNA coding for antigenic peptides from BCG and M.tb identifed by immunopeptidomics [2], and formulated with a patented technology of lipid nanoemulsions (NE) (WO2019138139A1), adapted for efcient intracellular delivery of mRNA [3]. Materials and methods We tested diferent prototypes of NE-mRNA formulations, coding for EGFP, in vitro. Human alveolar basal epithelial cells (A549), human monocytic cells (THP-1), and primary human monocyte-derived macrophages, were transfected with NE-mRNA formulations. Transfection efciency was assessed by measuring the percentage of transfected cells, and the intensity of GFP fuorescence. The cytotoxicity of the formulations was evaluated using AlamarBlue, and by 7-AAD viability staining. Results In vitro preliminary data using EGFP-mRNA-NE formulations indicate that NE formulations can efciently deliver mRNA and induce expression of the encoded protein in diferent cell types, with low cytotoxicity. Conclusions The NE technology presented here is safe, stable, and can efciently deliver mRNA to various cell types. Selected NE formulations will be used as a carrier for a new vaccine candidate against TB, based on mRNA encoding relevant antigenic peptides. These will be tested in mice for safety, immunogenicity, efcacy and dose optimization in order to generate an efective and sustained humoral and cellular immune response against TB. The mRNA vaccines are rapid and relatively simple to produce. The vaccine platform described here could be adapted to develop vaccines against other infectious diseases, particularly to quickly respond to emerging pathogens.
- Development of chitosan particles loaded with siRNA for cystatin C to control intracellular drug-resistant mycobacterium tuberculosisPublication . Pires, David; Mandal, Manoj; Matos, Ana I.; Peres, Carina; Catalão, Maria João; Azevedo-Pereira, José Miguel; Satchi-Fainaro, Ronit; Florindo, Helena F.; Anes, ElsaThe golden age of antibiotics for tuberculosis (TB) is marked by its success in the 1950s of the last century. However, TB is not under control, and the rise in antibiotic resistance worldwide is a major threat to global health care. Understanding the complex interactions between TB bacilli and their host can inform the rational design of better TB therapeutics, including vaccines, new antibiotics, and host-directed therapies. We recently demonstrated that the modulation of cystatin C in human macrophages via RNA silencing improved the anti-mycobacterial immune responses to Mycobacterium tuberculosis infection. Available in vitro transfection methods are not suitable for the clinical translation of host-cell RNA silencing. To overcome this limitation, we developed different RNA delivery systems (DSs) that target human macrophages. Human peripheral blood-derived macrophages and THP1 cells are difficult to transfect using available methods. In this work, a new potential nanomedicine based on chitosan (CS-DS) was efficiently developed to carry a siRNA-targeting cystatin C to the infected macrophage models. Consequently, an effective impact on the intracellular survival/replication of TB bacilli, including drug-resistant clinical strains, was observed. Altogether, these results suggest the potential use of CS-DS in adjunctive therapy for TB in combination or not with antibiotics.
- ESAT-6 a major virulence factor of mycobacterium tuberculosisPublication . Anes, Elsa; Pires, David; Mandal, Manoj; Azevedo-Pereira, José MiguelMycobacterium tuberculosis (Mtb), the causative agent of human tuberculosis (TB), is one of the most successfully adapted human pathogens. Human-to-human transmission occurs at high rates through aerosols containing bacteria, but the pathogen evolved prior to the establishment of crowded populations. Mtb has developed a particular strategy to ensure persistence in the host until an opportunity for transmission arises. It has refined its lifestyle to obviate the need for virulence factors such as capsules, flagella, pili, or toxins to circumvent mucosal barriers. Instead, the pathogen uses host macrophages, where it establishes intracellular niches for its migration into the lung parenchyma and other tissues and for the induction of long-lived latency in granulomas. Finally, at the end of the infection cycle, Mtb induces necrotic cell death in macrophages to escape to the extracellular milieu and instructs a strong inflammatory response that is required for the progression from latency to disease and transmission. Common to all these events is ESAT-6, one of the major virulence factors secreted by the pathogen. This narrative review highlights the recent advances in understanding the role of ESAT-6 in hijacking macrophage function to establish successful infection and transmission and its use as a target for the development of diagnostic tools and vaccines.
- Ethambutol and meropenem/clavulanate synergy promotes enhanced extracellular and intracellular killing of Mycobacterium tuberculosisPublication . Olivença, Francisco; Pires, David; Silveiro, Cátia; Gama, Bianca; Holtreman, Frederico; Anes, Elsa; Catalao, Maria JoãoIncreasing evidence supports the repositioning of beta-lactams for tuberculosis (TB) therapy, but further research on their interaction with conventional anti-TB agents is still warranted. Moreover, the complex cell envelope of Mycobacterium tuberculosis (Mtb) may pose an additional obstacle to beta-lactam diffusion. In this context, we aimed to identify synergies between beta-lactams and anti-TB drugs ethambutol (EMB) and isoniazid (INH) by assessing antimicrobial effects, intracellular activity, and immune responses. Checkerboard assays with H37Rv and eight clinical isolates, including four drug-resistant strains, exposed that only treatments containing EMB and beta-lactams achieved synergistic effects. Meanwhile, the standard EMB and INH association failed to produce any synergy. In Mtb-infected THP-1 macrophages, combinations of EMB with increasing meropenem (MEM) concentrations consistently displayed superior killing activities over the individual antibiotics. Flow cytometry with BODIPY FL vancomycin, which binds directly to the peptidoglycan (PG), confirmed an increased exposure of this layer after co-treatment. This was reinforced by the high IL-1β secretion levels found in infected macrophages after incubation with MEM concentrations above 5 mg/L, indicating an exposure of the host innate response sensors to pathogen-associated molecular patterns in the PG. Our findings show that the proposed impaired access of beta-lactams to periplasmic transpeptidases is counteracted by concomitant administration with EMB. The efficiency of this combination may be attributed to the synchronized inhibition of arabinogalactan and PG synthesis, two key cell wall components. Given that beta-lactams exhibit a time-dependent bactericidal activity, a more effective pathogen recognition and killing prompted by this association may be highly beneficial to optimize TB regimens containing carbapenems.
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