Impacto de la variabilidad genética del mycobacterium tuberculosis en la respuesta innata del epitelio bronquial humano

En trabajos previos de nuestro laboratorio hemos demostrado que la cepa M de Mycobacterium tuberculosis (Mtb) multirresistente a drogas prevalente en Argentina, ha montado diversos mecanismos para evadir la respuesta inmune innata y adaptativa del hospedador. Lo que indicaría que dicha cepa podría haber desarrollado mecanismos de evasión para permanecer invisible a los mecanismos de eliminación bacteriana del hospedador, lo cual explicaría su éxito epidemiológico. El rol del epitelio bronquial humano en el contexto de la infección por Mtb aún resulta desconocido así como también su interacción con los neutrófilos (PMN) en estas mismas condiciones. En este trabajo de tesis, nos propusimos evaluar si las cepas de Mtb M y la de referencia en el laboratorio, H37Rv son capaces de invadir y replicar dentro de la línea celular del epitelio bronquial humano Calu-6 y si el epitelio infectado con las mismas podría alterar las respuestas funcionales de los PMN, unas de las primeras células de la inmunidad innata en llegar al pulmón.Facultad de Ciencias Exacta

Dimensionality Reduction of Longitudinal 'Omics Data using Modern Tensor Factorization

Precision medicine is a clinical approach for disease prevention, detection and treatment, which considers each individual's genetic background, environment and lifestyle. The development of this tailored avenue has been driven by the increased availability of omics methods, large cohorts of temporal samples, and their integration with clinical data. Despite the immense progression, existing computational methods for data analysis fail to provide appropriate solutions for this complex, high-dimensional and longitudinal data. In this work we have developed a new method termed TCAM, a dimensionality reduction technique for multi-way data, that overcomes major limitations when doing trajectory analysis of longitudinal omics data. Using real-world data, we show that TCAM outperforms traditional methods, as well as state-of-the-art tensor-based approaches for longitudinal microbiome data analysis. Moreover, we demonstrate the versatility of TCAM by applying it to several different omics datasets, and the applicability of it as a drop-in replacement within straightforward ML tasks

Role of neutrophils in CVB3 infection and viral myocarditis

Coxsackievirus B3 (CVB3) is a globally prevalent enterovirus of the Picornaviridae family that is frequently associated with viral myocarditis (VM). Neutrophils, as first responders, may be key cells in determining viral disease outcomes; however, neutrophils have been poorly studied with respect to viral infection. Although neutrophils have been ascribed a relevant role in early cardiac inflammation, their precise role in CVB3 infection has not yet been evaluated. In this study, we aimed to determine if the interaction between human neutrophils and CVB3 could lead to viral replication and/or modulation of neutrophil survival and biological functions, and whether neutrophil depletion in a murine model has a beneficial or harmful effect on CVB3 infection. Our results show that CVB3 interacted with but did not replicate in human neutrophils. Neutrophils recognized CVB3 mainly through endosomal TLR-8, and infection triggered NFκB activation. Virus internalization resulted in increased cell survival, up-regulation of CD11b, enhanced adhesion to fibrinogen and fibronectin, and the secretion of IL-6, IL-1β, TNF-α, and IL-8. Supernatants from infected neutrophils exerted chemotactic activity partly mediated by IL-8. The infected neutrophils released myeloperoxidase and triggered neutrophil extracellular trap formation in the presence of TNF-α. In mice infected with CVB3, viral RNA was detected in neutrophils as well as in mononuclear cells. After neutrophil depletion, mice showed reduced VM reflected by a reduction in viral titers, cell exudates, and CCL-2 mRNA levels, as well as the abrogation of reactive cardiomyocyte hypertrophy. Our results indicate that neutrophils have relevant direct and indirect roles in the pathogenesis of CVB3-induced VM.Instituto de Biotecnología y Biología MolecularFacultad de Ciencias Exacta

Role of neutrophils in CVB3 infection and viral myocarditis

Coxsackievirus B3 (CVB3) is a globally prevalent enterovirus of the Picornaviridae family that is frequently associated with viral myocarditis (VM). Neutrophils, as first responders, may be key cells in determining viral disease outcomes; however, neutrophils have been poorly studied with respect to viral infection. Although neutrophils have been ascribed a relevant role in early cardiac inflammation, their precise role in CVB3 infection has not yet been evaluated. In this study, we aimed to determine if the interaction between human neutrophils and CVB3 could lead to viral replication and/or modulation of neutrophil survival and biological functions, and whether neutrophil depletion in a murine model has a beneficial or harmful effect on CVB3 infection. Our results show that CVB3 interacted with but did not replicate in human neutrophils. Neutrophils recognized CVB3 mainly through endosomal TLR-8, and infection triggered NFκB activation. Virus internalization resulted in increased cell survival, up-regulation of CD11b, enhanced adhesion to fibrinogen and fibronectin, and the secretion of IL-6, IL-1β, TNF-α, and IL-8. Supernatants from infected neutrophils exerted chemotactic activity partly mediated by IL-8. The infected neutrophils released myeloperoxidase and triggered neutrophil extracellular trap formation in the presence of TNF-α. In mice infected with CVB3, viral RNA was detected in neutrophils as well as in mononuclear cells. After neutrophil depletion, mice showed reduced VM reflected by a reduction in viral titers, cell exudates, and CCL-2 mRNA levels, as well as the abrogation of reactive cardiomyocyte hypertrophy. Our results indicate that neutrophils have relevant direct and indirect roles in the pathogenesis of CVB3-induced VM.Fil: Rivadeneyra, Leonardo. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; ArgentinaFil: Charó, Nancy Lorena. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; ArgentinaFil: Kviatcovsky, Denise. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; ArgentinaFil: de la Barrera, Silvia Susana. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; ArgentinaFil: Gomez, Ricardo Martin. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Biotecnología y Biología Molecular. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Instituto de Biotecnología y Biología Molecular; ArgentinaFil: Schattner, Mirta Ana. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; Argentin

Mycobacterium tuberculosis Multidrug-Resistant Strain M Induces Low IL-8 and Inhibits TNF- α Secretion by Bronchial Epithelial Cells Altering Neutrophil Effector Functions

M strain, the most prevalent multidrug-resistant strain of Mycobacterium tuberculosis (Mtb) in Argentina, has mounted mechanisms to evade innate immune response. The role of human bronchial epithelium in Mtb infection remains unknown as well as its crosstalk with neutrophils (PMN). In this work, we evaluate whether M and H37Rv strains invade and replicate within bronchial epithelial cell line Calu-6 and how conditioned media (CM) derived from infected cells alter PMN responses. We demonstrated that M infects and survives within Calu-6 without promoting death. CM from M-infected Calu-6 (M-CM) did not attract PMN in correlation with its low IL-8 content compared to H37Rv-CM. Also, PMN activation and ROS production in response to irradiated H37Rv were impaired after treatment with M-CM due to the lack of TNF-α. Interestingly, M-CM increased H37Rv replication in PMN which would allow the spreading of mycobacteria upon PMN death and sustain IL-8 release. Thus, our results indicate that even at low invasion/replication rate within Calu-6, M induces the secretion of factors altering the crosstalk between these nonphagocytic cells and PMN, representing an evasion mechanism developed by M strain to persist in the host. These data provide new insights on the role of bronchial epithelium upon M infection.Fil: Kviatcovsky, Denise. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; ArgentinaFil: Rivadeneyra, Leonardo. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; ArgentinaFil: Balboa, Luciana. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; ArgentinaFil: Yokobori, Noemí. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; ArgentinaFil: López, Beatriz. Dirección Nacional de Institutos de Investigación. Administración Nacional de Laboratorios e Institutos de Salud. Instituto Nacional de Enfermedades Infecciosas; ArgentinaFil: Ritacco, Gloria Viviana. Dirección Nacional de Institutos de Investigación. Administración Nacional de Laboratorios e Institutos de Salud. Instituto Nacional de Enfermedades Infecciosas; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Schattner, Mirta Ana. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; ArgentinaFil: Sasiain, María del Carmen. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; ArgentinaFil: de la Barrera, Silvia Susana. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; Argentin

Diverging biological roles among human monocyte subsets in the context of tuberculosis infection

Circulating monocytes (Mo) play an essential role in the host immune response to chronic infections. We previously demonstrated that CD16pos Mo were expanded in TB (tuberculosis) patients, correlated with disease severity and were refractory to dendritic cell differentiation. In the present study, we investigated whether human Mo subsets (CD16neg and CD16pos) differed in their ability to influence the early inflammatory response against Mycobacterium tuberculosis. We first evaluated the capacity of the Mo subsets to migrate and engage a microbicidal response in vitro. Accordingly, CD16neg Mo were more prone to migrate in response to different mycobacteria-derived gradients, were more resistant to M. tuberculosis intracellular growth and produced higher reactive oxygen species than their CD16pos counterpart. To assess further the functional dichotomy among the human Mo subsets, we carried out an in vivo analysis by adapting a hybrid mouse model (SCID/Beige, where SCID is severe combined immunodeficient) to transfer each Mo subset, track their migratory fate during M. tuberculosis infection, and determine their impact on the host immune response. In M. tuberculosis-infected mice, the adoptively transferred CD16neg Mo displayed a higher lung migration index, induced a stronger pulmonary infiltration of murine leucocytes expressing pro- and anti-inflammatory cytokines, and significantly decreased the bacterial burden, in comparison with CD16pos Mo. Collectively, our results indicate that human Mo subsets display divergent biological roles in the context of M. tuberculosis infection, a scenario in which CD16neg Mo may contribute to the anti-mycobacterial immune response, whereas CD16pos Mo might promote microbial resilience, shedding light on a key aspect of the physiopathology of TB disease.Fil: Balboa, Luciana. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; ArgentinaFil: Barrios Payan, Jorge. Instituto Nacional de Ciencias Médicas y Nutrición "Salvador Zubirán"; MéxicoFil: González Domínguez, Erika. Instituto Politécnico Nacional; MéxicoFil: Lastrucci, Claire. Institut de Pharmacologie et de Biologie Structurale; FranciaFil: Lugo Villarino, Geanncarlo. Institut de Pharmacologie et de Biologie Structurale; FranciaFil: Mata Espinoza, Dulce. Instituto Nacional de Ciencias Médicas y Nutrición "Salvador Zubirán"; MéxicoFil: Schierloh, Luis Pablo. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; ArgentinaFil: Kviatcovsky, Denise. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; ArgentinaFil: Neyrolles, Olivier. Institut de Pharmacologie et de Biologie Structurale; FranciaFil: Maridonneau Parini, Isabelle. Institut de Pharmacologie et de Biologie Structurale; FranciaFil: Sánchez Torres, Carmen. Instituto Politécnico Nacional; MéxicoFil: Sasiain, María del Carmen. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; ArgentinaFil: Hernández Pando, Rogelio. Instituto Nacional de Ciencias Médicas y Nutrición "Salvador Zubirán"; Méxic

Effect of the BTK inhibitor ibrutinib on macrophage- and γδ T cell-mediated response against Mycobacterium tuberculosis

.The Bruton’s tyrosine kinase (BTK) inhibitor ibrutinib is approved by the Food and Drug Administration for its use as first-line treatment in chronic lymphocytic leukemia (CLL). Despite its efficacy, patients treated with ibrutinib rarely achieve complete responses and usually remain under treatment until progression.Fil: Colado, Ana. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; ArgentinaFil: Genoula, Melanie. Centre National de la Recherche Scientifique; Francia. International Associated Laboratory; Francia. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; ArgentinaFil: Cougoule, Céline. Université Paul Sabatier; Francia. International Associated Laboratory; Francia. Centre National de la Recherche Scientifique; FranciaFil: Marin Franco, Jose Luis. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; Argentina. Centre National de la Recherche Scientifique; Francia. International Associated Laboratory; FranciaFil: Almejún, María Belén. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; Argentina. Academia Nacional de Medicina de Buenos Aires; ArgentinaFil: Risnik, Denise Mariel. Academia Nacional de Medicina de Buenos Aires; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; ArgentinaFil: Kviatcovsky, Denise. International Associated Laboratory; Francia. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; Argentina. Centre National de la Recherche Scientifique; FranciaFil: Podaza, Enrique Arturo. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; Argentina. Academia Nacional de Medicina de Buenos Aires; ArgentinaFil: Elías, Esteban Enrique. Academia Nacional de Medicina de Buenos Aires; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; ArgentinaFil: Fuentes, Federico. Academia Nacional de Medicina de Buenos Aires; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; ArgentinaFil: Maridonneau Parini, Isabelle. Centre National de la Recherche Scientifique; Francia. International Associated Laboratory; Francia. Université Paul Sabatier; FranciaFil: Bezares, Fernando R.. Gobierno de la Ciudad de Buenos Aires. Hospital General de Agudos "Dr. Teodoro Álvarez"; ArgentinaFil: Fernandez Grecco, Horacio. Sanatorio Julio Méndez. Departamento de Hematología; ArgentinaFil: Cabrejo, María del Rosario. Sanatorio Julio Méndez. Departamento de Hematología; ArgentinaFil: Jancic, Carolina Cristina. Universidad de Buenos Aires. Facultad de Medicina; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; ArgentinaFil: Sasiain, María del Carmen. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; Argentina. International Associated Laboratory; Francia. Centre National de la Recherche Scientifique; FranciaFil: Giordano, Mirta Nilda. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; Argentina. Universidad de Buenos Aires. Facultad de Medicina; ArgentinaFil: Gamberale, Romina. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; Argentina. Universidad de Buenos Aires. Facultad de Medicina; ArgentinaFil: Balboa, Luciana. Universidad de Buenos Aires. Facultad de Medicina; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; ArgentinaFil: Borge, Mercedes. Universidad de Buenos Aires. Facultad de Medicina; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; Argentin

M-stimulated CD8⁺ T cells express low levels of lytic molecules.

<p>Intracellular expression of perforin, granzyme and granulysin in control or <i>Mtb-</i>stimulated CD8⁺ T cells from 15 PPD⁺ N (<b>A and B</b>) and 7 PPD^NEG N (<b>C</b>). Results are expressed as % of Perforin⁺, of Granzyme B⁺ or of Granulysin⁺ in the CD3⁺CD8⁺ T lymphocyte gate. <b>A</b> Representative dot plots; <b>B and C</b> graphs showing %Perforin⁺/CD8, %Granzyme B⁺/CD8 and %Granulysin⁺/CD8 (median and percentiles 25–75). Statistical differences: PBMC+<i>Mtb</i> vs. control PBMC * = p<0.05, between strains: § = p<0.05. <b>D.</b> PBMC from 5 PPD⁺ N were cultured alone (Control Ly) or with <i>Mtb</i> strains (H37Rv-Ly, M-Ly and 410-Ly) for 6 d and then co-cultured for further 4 h with non-stimulated (Ly+control-Mac) or <i>Mtb</i>-stimulated (Ly+<i>Mtb</i>-Mac) autologous macrophages. Cells were surface stained for CD3 and CD8 and intracellular perforin expression and evaluated by FACS. %Perforin⁺/CD8, %Granzyme B⁺/CD8 and %Granulysin⁺/CD8 was then determined and results are expressed as (median and percentiles 25–75). Statistical differences Ly+Mac+<i>Mtb</i> vs Ly+Mac-C: * p<0.05.</p

IL-2 enhances CD107 expression in CD8⁺ T cells.

<p><b>A.</b> PBMC from 12 PPD⁺ N were cultured for 5 d alone or with <i>Mtb</i> strains H37Rv, M or 410 in the presence or not of IL-2 (100 U/ml). Then intracellular expression of perforin, granzyme B and granulysin and surface expression of CD107 were determined on CD8⁺ T cells. Results are expressed as % of positive cells in the CD3⁺CD8⁺ T cells gate and individual data are shown (Black dots = PPD+ N; white dots = PPD^NEG N). Statistical differences: PBMC+IL-2 vs. control PBMC+IL-2 * = p<0.05, between strains: § = p<0.05. B. PBMC from 10 PPD⁺N were cultured for 5 d with <i>Mtb</i> strains with the addition or not of different amounts of IL-2. Then the % perforin⁺ and %CD107⁺ cells were determined in CD3⁺CD8⁺ T cells and dose response curves are shown. Statistical differences PBMC+IL-2 vs. non treated cells: * = p<0.05. <b>C.</b> PBMC from 5 healthy donors were cultured for 5 d with <i>Mtb</i> strains in the presence or not of neutralizing anti-IL-2. Then the % perforin⁺ and %CD107⁺ cells were determined in CD3⁺CD8⁺ T cells. Results are expressed as mean and 25–75 percentiles. Statistical differences PBMC+a-IL-2 vs. untreated cells: * = p<0.05.</p

M strain induces low expression of CD69 in CD8⁺ T cells.

<p><b>A and B,</b> Surface CD69 expression on CD8⁺ T cells from 18 h-cultured PBMC in 15 PPD⁺ N (A) and 7 PPD^NEG N (B). <b>C and D,</b> Surface CD25 expression in CD8⁺ T cells from 5 days-cultured PBMC in 13 PPD⁺N (C) and 7 PPD^NEG N (D). Results are expressed as %CD69⁺ and %CD25⁺ cells in the CD8⁺CD3⁺ T cells gate. Statistical differences: PBMC+<i>Mtb</i> strains vs. control PBMC * = p<0.05, among strains: § = p<0.05. <b>E.</b> PBMC from PPD⁺ N (n = 3) were cultured for 18 h alone (C) or with strains H37Rv, M or 410 with or without anti-CD3 and/or anti-CD28. Representative dot plots show the % of CD69⁺ cells in the CD3⁺CD8⁺ lymphocyte gate. <b>F and G.</b> PBMC from 6 PPD⁺ N were cultured alone or with <i>Mtb</i> strains and the %CD69⁺ (F) and %CD25⁺ (G) cells within the CD3⁺CD8⁺ lymphocyte gate was determined at different time points. Statistical differences, PBMC+<i>Mtb</i> strains vs. control PBMC: * = p<0.05, between strains: § = p<0.05.</p