Mycobacterium tuberculosis infection modulates adipose tissue biology

Mycobacterium tuberculosis (Mtb) primarily resides in the lung but can also persist in extrapulmonary sites. Macrophages are considered the prime cellular habitat in all tissues. Here we demonstrate that Mtb resides inside adipocytes of fat tissue where it expresses stress-related genes. Moreover, perigonadal fat of Mtb-infected mice disseminated the infection when transferred to uninfected animals. Adipose tissue harbors leukocytes in addition to adipocytes and other cell types and we observed that Mtb infection induces changes in adipose tissue biology depending on stage of infection. Mice infected via aerosol showed infiltration of inducible nitric oxide synthase (iNOS) or arginase 1 (Arg1)-negative F4/80+ cells, despite recruitment of CD3+, CD4+ and CD8+ T cells. Gene expression analysis of adipose tissue of aerosol Mtb-infected mice provided evidence for upregulated expression of genes associated with T cells and NK cells at 28 days post-infection. Strikingly, IFN-γ-producing NK cells and Mtb-specific CD8+ T cells were identified in perigonadal fat, specifically CD8+CD44-CD69+ and CD8+CD44-CD103+ subpopulations. Gene expression analysis of these cells revealed that they expressed IFN-γ and the lectin-like receptor Klrg1 and down-regulated CD27 and CD62L, consistent with an effector phenotype of Mtb-specific CD8+ T cells. Sorted NK cells expressed higher abundance of Klrg1 upon infection, as well. Our results reveal the ability of Mtb to persist in adipose tissue in a stressed state, and that NK cells and Mtb-specific CD8+ T cells infiltrate infected adipose tissue where they produce IFN-γ and assume an effector phenotype. We conclude that adipose tissue is a potential niche for Mtb and that due to infection CD8+ T cells and NK cells are attracted to this tissue

Rewiring cellular metabolism via the AKT/mTOR pathway contributes to host defence against Mycobacterium tuberculosis in human and murine cells

Contains fulltext : 171426.pdf (publisher's version ) (Open Access)Cells in homeostasis metabolize glucose mainly through the tricarboxylic acid cycle and oxidative phosphorylation, while activated cells switch their basal metabolism to aerobic glycolysis. In this study, we examined whether metabolic reprogramming toward aerobic glycolysis is important for the host response to Mycobacterium tuberculosis (Mtb). Through transcriptional and metabolite analysis we show that Mtb induces a switch in host cellular metabolism toward aerobic glycolysis in human peripheral blood mononuclear cells (PBMCs). The metabolic switch is TLR2 dependent but NOD2 independent, and is mediated in part through activation of the AKT-mTOR (mammalian target of rapamycin) pathway. We show that pharmacological inhibition of the AKT/mTOR pathway inhibits cellular responses to Mtb both in vitro in human PBMCs, and in vivo in a model of murine tuberculosis. Our findings reveal a novel regulatory layer of host responses to Mtb that will aid understanding of host susceptibility to Mtb, and which may be exploited for host-directed therapy

Caveolin-1 protects B6129 mice against Helicobacter pylori gastritis.

Caveolin-1 (Cav1) is a scaffold protein and pathogen receptor in the mucosa of the gastrointestinal tract. Chronic infection of gastric epithelial cells by Helicobacter pylori (H. pylori) is a major risk factor for human gastric cancer (GC) where Cav1 is frequently down-regulated. However, the function of Cav1 in H. pylori infection and pathogenesis of GC remained unknown. We show here that Cav1-deficient mice, infected for 11 months with the CagA-delivery deficient H. pylori strain SS1, developed more severe gastritis and tissue damage, including loss of parietal cells and foveolar hyperplasia, and displayed lower colonisation of the gastric mucosa than wild-type B6129 littermates. Cav1-null mice showed enhanced infiltration of macrophages and B-cells and secretion of chemokines (RANTES) but had reduced levels of CD25+ regulatory T-cells. Cav1-deficient human GC cells (AGS), infected with the CagA-delivery proficient H. pylori strain G27, were more sensitive to CagA-related cytoskeletal stress morphologies ("humming bird") compared to AGS cells stably transfected with Cav1 (AGS/Cav1). Infection of AGS/Cav1 cells triggered the recruitment of p120 RhoGTPase-activating protein/deleted in liver cancer-1 (p120RhoGAP/DLC1) to Cav1 and counteracted CagA-induced cytoskeletal rearrangements. In human GC cell lines (MKN45, N87) and mouse stomach tissue, H. pylori down-regulated endogenous expression of Cav1 independently of CagA. Mechanistically, H. pylori activated sterol-responsive element-binding protein-1 (SREBP1) to repress transcription of the human Cav1 gene from sterol-responsive elements (SREs) in the proximal Cav1 promoter. These data suggested a protective role of Cav1 against H. pylori-induced inflammation and tissue damage. We propose that H. pylori exploits down-regulation of Cav1 to subvert the host's immune response and to promote signalling of its virulence factors in host cells

Effects of Helicobacter suis γ-glutamyl transpeptidase on lymphocytes: modulation by glutamine and glutathione supplementation and outer membrane vesicles as a putative delivery route of the enzyme

Helicobacter (H.) suis colonizes the stomach of the majority of pigs as well as a minority of humans worldwide. Infection causes chronic inflammation in the stomach of the host, however without an effective clearance of the bacteria. Currently, no information is available about possible mechanisms H. suis utilizes to interfere with the host immune response. This study describes the effect on various lymphocytes of the γ-glutamyl transpeptidase (GGT) from H. suis. Compared to whole cell lysate from wild-type H. suis, lysate from a H. suis ggt mutant strain showed a decrease of the capacity to inhibit Jurkat T cell proliferation. Incubation of Jurkat T cells with recombinantly expressed H. suis GGT resulted in an impaired proliferation, and cell death was shown to be involved. A similar but more pronounced inhibitory effect was also seen on primary murine CD4+ T cells, CD8+ T cells, and CD19+ B cells. Supplementation with known GGT substrates was able to modulate the observed effects. Glutamine restored normal proliferation of the cells, whereas supplementation with reduced glutathione strengthened the H. suis GGT-mediated inhibition of proliferation. H. suis GGT treatment abolished secretion of IL-4 and IL-17 by CD4+ T cells, without affecting secretion of IFN-γ. Finally, H. suis outer membrane vesicles (OMV) were identified as a possible delivery route of H. suis GGT to lymphocytes residing in the deeper mucosal layers. Thus far, this study is the first to report that the effects on lymphocytes of this enzyme, not only important for H. suis metabolism but also for that of other Helicobacter species, depend on the degradation of two specific substrates: glutamine and reduced glutatione. This will provide new insights into the pathogenic mechanisms of H. suis infection in particular and infection with gastric helicobacters in general

Efecto de los péptidos formilados en la regulación del receptor para el fragmento Fc de inmunologlobulina G de tipo I (FcyRI) en monocitos humanos

En un fenómeno inflamatorio se produce la migración unidireccional (quimiotaxis) de los polimorfonucleares neutrófilos hacia el foco inflamatorio, seguidos luego por los monocitos sanguíneos. Esta migración es la respuesta al gradiente de concentración de un agente quimiotáctico determinado, como el leucotrieno B4 o las quimioquinas. También existen productos como los péptidos formilados, que son quimiotácticos para monocitos y neutrófilos. Estos péptidos formilados son productos de clivaje de proteínas bacterianas o mitocondriales. Los receptores para la porción Fc de lgG (FcγRs) son capaces de interconectar la especificidad de los anticuerpos con las funciones efectoras de los monocitos y neutrófilos. Tres clases de FCyRshan sido identificados en los leucocitos humanos: FcγRI, FcγRII y FcγRIII. El FcγRI es un receptor de alta afinidad capaz de mediar Ia fagocitosis y Ia citotoxicidad celular dependiente de anticuerpos (ADCC). Este receptor se expresa constitutivamente en la superficie de los fagocitos mononucleares y su expresión es aumentada por citoquinas como el interferón-γ (IFN-γ)y la interleukina-10 (IL-10). En este trabajo, se ha demostrado que la preincubación de los monocitos humanos con el péptido formilado prototipo N-formil-metionil-leucil-fenilalanina (FMLP) fue capaz de inhibir la expresión del FcγRI inducida por IFN-γ e IL-10. Esta inhibición se correlacionó con la inhibición de funciones dependientes del FcγRI como la ADCC y Ia fagocitosis, pero el FMLP no afectó otras funciones mediadas por el IFN-γ o la IL-10. El mecanismo de acción del FMLP cuando fue agregado antes que las citoquinas, involucraría la activación de las Mitogen Activated Kinases (MAPKs) p38 y Erk1/2, pero no de la proteín kinasa C (pKC). A su vez, el FMLP no modificó ni la expresión de los receptores para IFN-γ o IL-1O ni la fosforilación del factor de transcripción STAT-1. Por otra parte, el FMLP también inhibió la expresión del FcγRI en monocitos humanos previamente activados con IFN-γ. Sin embargo, en este caso, el FMLP no modificó la expresión del FcγRI en células pretratadas con IL-10. Sobrenadantes obtenidos de monocitos tratados primero con IFN-γ y luego con FMLP, indujeron la disminución de la expresión del FcγRI en monocitos naive. Este efecto estaria mediado por serin y metaloproteasas. A su vez, sobrenadantes provenientes de neutrófilos tratados con FMLP también fueron capaces de inducir la disminución del FcγRI en monocitos naive. En un modelo murino de inflamación crónica, el FMLP indujo la disminución de la expresión de FcγRs en macrófagos activados con IFN-γ. En pacientes con tuberculosis pulmonar activa, el IFN-γ y la IL-1O indujeron el aumento en la expresión del FCγRI en los monocitos. Sin embargo, el FMLP no fue capaz de disminuir la expresión del FCγRI inducida por estas citoquinas. Además, sobrenadantes de neutrófilos tratados con FMLP provenientes de estos pacientes no alteraron la expresión del FCγRI en monocitos naive. Este trabajo contribuye a la comprensión global de los mecanismos mediante los cuales agentes conocidos como pro-inflamatorios pueden tener funciones anti-inflamatorias en determinadas circunstancias. Este efecto antiinflamatorio del FMLP funcionaría como mecanismo de atenuación de una respuesta inflamatoria exacerbada por parte del huésped. El hecho de que los monocitos de los pacientes con tuberculosis pulmonar activa no presenten estos mecanismos concuerda con esta interpretación

Effect of formyl peptides in the regulation of the type I receptor for the Fc portion of immunoglobulin G (FcyRI) in human monocytes

En un fenómeno inflamatorio se produce la migración unidireccional (quimiotaxis) de los polimorfonucleares neutrófilos hacia el foco inflamatorio, seguidos luego por los monocitos sanguíneos. Esta migración es la respuesta al gradiente de concentración de un agente quimiotáctico determinado, como el leucotrieno B4 o las quimioquinas. También existen productos como los péptidos formilados, que son quimiotácticos para monocitos y neutrófilos. Estos péptidos formilados son productos de clivaje de proteínas bacterianas o mitocondriales. Los receptores para la porción Fc de lgG (FcγRs) son capaces de interconectar la especificidad de los anticuerpos con las funciones efectoras de los monocitos y neutrófilos. Tres clases de FCyRshan sido identificados en los leucocitos humanos: FcγRI, FcγRII y FcγRIII. El FcγRI es un receptor de alta afinidad capaz de mediar Ia fagocitosis y Ia citotoxicidad celular dependiente de anticuerpos (ADCC). Este receptor se expresa constitutivamente en la superficie de los fagocitos mononucleares y su expresión es aumentada por citoquinas como el interferón-γ (IFN-γ)y la interleukina-10 (IL-10). En este trabajo, se ha demostrado que la preincubación de los monocitos humanos con el péptido formilado prototipo N-formil-metionil-leucil-fenilalanina (FMLP) fue capaz de inhibir la expresión del FcγRI inducida por IFN-γ e IL-10. Esta inhibición se correlacionó con la inhibición de funciones dependientes del FcγRI como la ADCC y Ia fagocitosis, pero el FMLP no afectó otras funciones mediadas por el IFN-γ o la IL-10. El mecanismo de acción del FMLP cuando fue agregado antes que las citoquinas, involucraría la activación de las Mitogen Activated Kinases (MAPKs) p38 y Erk1/2, pero no de la proteín kinasa C (pKC). A su vez, el FMLP no modificó ni la expresión de los receptores para IFN-γ o IL-1O ni la fosforilación del factor de transcripción STAT-1. Por otra parte, el FMLP también inhibió la expresión del FcγRI en monocitos humanos previamente activados con IFN-γ. Sin embargo, en este caso, el FMLP no modificó la expresión del FcγRI en células pretratadas con IL-10. Sobrenadantes obtenidos de monocitos tratados primero con IFN-γ y luego con FMLP, indujeron la disminución de la expresión del FcγRI en monocitos naive. Este efecto estaria mediado por serin y metaloproteasas. A su vez, sobrenadantes provenientes de neutrófilos tratados con FMLP también fueron capaces de inducir la disminución del FcγRI en monocitos naive. En un modelo murino de inflamación crónica, el FMLP indujo la disminución de la expresión de FcγRs en macrófagos activados con IFN-γ. En pacientes con tuberculosis pulmonar activa, el IFN-γ y la IL-1O indujeron el aumento en la expresión del FCγRI en los monocitos. Sin embargo, el FMLP no fue capaz de disminuir la expresión del FCγRI inducida por estas citoquinas. Además, sobrenadantes de neutrófilos tratados con FMLP provenientes de estos pacientes no alteraron la expresión del FCγRI en monocitos naive. Este trabajo contribuye a la comprensión global de los mecanismos mediante los cuales agentes conocidos como pro-inflamatorios pueden tener funciones anti-inflamatorias en determinadas circunstancias. Este efecto antiinflamatorio del FMLP funcionaría como mecanismo de atenuación de una respuesta inflamatoria exacerbada por parte del huésped. El hecho de que los monocitos de los pacientes con tuberculosis pulmonar activa no presenten estos mecanismos concuerda con esta interpretación.In the course of the inflammatory phenomenon, polymorphonuclear neutrophils migrate unidirectionally (chemotaxis) towards the inflammatory focus, followed by monocytes. This migration occurs in response to the concentration gradient of a chemotactic agent, such as leukotriene B4 or chemokines. Molecules such as formylpeptides are chemotactic for neutrophils and monocytes. These peptides are cleavage products of bacterial and mitochondrial proteins. The receptors for the Fc portion of IgG (FCγRs) act as a bridge between the specificity of antibodies and the effector functions of monocytes and neutrophils. Three classes of FcγRs have been identified on human leukocytes: FcγRI, FcγRII and FcγRIII. FcγRI is a high affinity receptor that mediates phagocytosis and antibody-dependent cell-mediated cytotoxicity (ADCC). This receptor is constitutively expressed on mononuclear phagocytes and its expression is enhanced by interferon-γ (IFN-γ) and interleukine-1O (IL-10). In this study, it has been demonstrated that pre-incubation of human monocytes with the prototypic formyl peptide N-formyI-methionyl-leucyl-phenylalanine (FMLP) inhibited the expression of FcγRI induced by IFN-γ or IL-10. This inhibition correlated with the inhibition of FcγRI-dependent functions such as ADCC and phagocytosis. However, FMLP did not affect other functions modulated by IFN-γ or IL-10. When FMLP was added to human monocytes before these cytokines, Mitogen Activated Kinases (MAPKs) p38 and Erk1/2, but not protein kinase C (pKC), were involved in the FMLP-mediated inhibition. FMLP altered neither the expression of IFN-γ/IL-10 receptors nor the phosphorylation of the transcription factor STAT1. On the other hand, FMLP also inhibited the expression of FcγRI in human monocytes that had been previously treated with IFN-γ. However, FMLP did not alter the expression of FcγRI in monocytes previously treated with IL-10. Supernatants obtained from monocytes treated first with IFN-γ and later with FMLP downregulated the expression of FcγRI on naive human monocytes. This effect was mediated by serine and metalloproteases. Moreover, this effect was also observed when using supernatants from FMLP-treated human neutrophils. In a murine model of chronic inflammation, FMLP downregulated the expression of FcγRd on IFN-γ-activated macrophages. In patients with active pulmonary tuberculosis, IFN-γ and IL-1O upregulated the expression of FcγRI. However, FMLP was not capable of downregulating the expression FcγRI induced by these cytokines. Besides, FMLP-treated neutrophils from these patients did not modify the expression of FcγRI on naive monocytes. This study contributes to the understanding of inflammatory phenomena. We have demonstrated that well-known pro-inflammatory molecules could behave, under certain circumstances, as anti-inflammatory agents. This antiinflammatory effect of FMLP would attenuate an exacerbated host immune response. The fact that monocytes from tuberculosis patients lack these antiinflammatory mechanisms is in agreement with this interpretation.Fil:Beigier Bompadre, Macarena. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina

Antigen Export during Liver Infection of the Malaria Parasite Augments Protective Immunity

Protective immunity against preerythrocytic malaria parasite infection is difficult to achieve. Intracellular Plasmodium parasites likely minimize antigen presentation by surface-expressed major histocompatibility complex class I (MHC-I) molecules on infected cells, yet they actively remodel their host cells by export of parasite factors. Whether exported liver-stage proteins constitute better candidates for MHC-I antigen presentation to CD8(+) T lymphocytes remains unknown. Here, we systematically characterized the contribution of protein export to the magnitude of antigen-specific T-cell responses against Plasmodium berghei liver-stage parasites in C57BL/6 mice. We generated transgenic sporozoites that secrete a truncated ovalbumin (OVA) surrogate antigen only in the presence of an amino-terminal protein export element. Immunization with live attenuated transgenic sporozoites revealed that antigen export was not critical for CD8(+) T-cell priming but enhanced CD8(+) T-cell proliferation in the liver. Upon transfer of antigen-specific CD8(+) T cells, liver-stage parasites secreting the target protein were eliminated more efficiently. We conclude that Plasmodium parasites strictly control protein export during liver infection to minimize immune recognition. Strategies that enhance the discharge of parasite proteins into infected hepatocytes could improve the efficacy of candidate preerythrocytic malaria vaccines. Importance: Vaccine development against Plasmodium parasites remains a priority in malaria research. The most advanced malaria subunit vaccine candidates contain Plasmodium surface proteins with important roles for parasite vital functions. A fundamental question is whether recognition by effector CD8(+) T cells is restricted to sporozoite surface antigens or extends to parasite proteins that are synthesized during the extensive parasite expansion phase in the liver. Using a surrogate model antigen, we found that a cytoplasmic antigen is able to induce robust protective CD8(+) T-cell responses, but protein export further enhances immunogenicity and protection. Our results show that a cytoplasmic localization does not exclude a protein's candidacy for malaria subunit vaccines and that protein secretion can enhance protective immunity

Hipótesis: una vía alternativa de regulación de procesos inflamatorios

La regulación de mecanismos inflamatorios es un evento crucial debido a que una alteración de los mismos, como sucede por ejemplo, en la sepsis, en enfermedades autoinmunes crónicas (artritis reumatoidea, lupus eritematoso) o en enfermedades infecciosas (tuberculosis, lepra), genera daños tisulares severos. Aunque hay un consenso general de que la regulación de procesos inflamatorios resulta de un balance entre vías proinflamatorias y antiinflamatorias, nosotros arribamos a la conclusión de que moléculas quimioatractantes / proinflamatorias como, por ejemplo, péptidos formilados bacterianos o complejos inmunes (CI), pueden también inducir, paradójicamente, potentes efectos antiinflamatorios. Así, demostramos que el péptido formilado prototipo N-formilmetionil-leucil-fenilalanina (FMLP), ejerce un drástico efecto antiinflamatorio, inhibiendo la secreción de factor de necrosis tumoral alfa (TNF-α) inducido por lipopolisacáridos, un potente inductor de la secreción de TNF-α. También determinamos que el FMLP y los CI inducen la disminución de la expresión de receptores para el fragmento Fc de IgG (FcγRII and FcγRIIIB) en neutrófilos humanos. Más aún, el FMLP inhibe la inducción de la expresión de los FcγRI por interferón gamma (IFN-γ) y los CI disminuyen la expresión de moléculas de clase II del complejo mayor de histocompatibilidad en monocitos humanos. Parte de esos efectos fueron mediados por la liberación de aspártico-, serino-, o metaloproteasas. Todos estos resultados nos permiten especular sobre un nuevo concepto en el cual la regulación de los procesos inflamatorios también puede llevarse a cabo por una vía alternativa, no convencional, en la cual un agente quimioatractante / proinflamatorio, bajo determinadas circunstancias, puede actuar como una molécula antiinflamatoria.Regulation of inflammation is a crucial event since its alteration, such as in sepsis and chronic autoimmune (i.e. rheumatoid arthritis, lupus erythematosus) or infectious diseases (i.e. tuberculosis, leprosy), determines severe tissue damage. Although there is a general consensus that regulation of inflammation results from a balance between proinflammatory and antiinflammatory pathways, we arrived at the conclusion that well known chemoattractants/proinflammatory molecules such as bacterial formyl peptides or immune complexes (IC), could induce, paradoxically, strong antiinflammatory effects. Thus, we demonstrated that N-formyl-methionyl-leucyl-phenylalanine (FMLP) exerted a drastic antiinflammatory effect, inhibiting the secretion of tumor necrosis alpha (TNF-α) induced by lipopolysaccharides, a potent TNF-α inducer. We also determined that in human neutrophils FMLP and IC induced the downregulation of receptors for the Fc portion of IgG (FcγRII and FcγRIIIB). Moreover, FMLP inhibited interferon gamma (IFN-γ)-induced FcγRI expression and IC downregulate class II molecules of the major histocompatibility complex on monocytes. Part of these effects were mediated by the release of aspartic-, serin-, or metalloproteases. All these results favor the postulation of a new concept on the regulation of inflammation carried out through an alternative and non conventional pathway, in which a chemoattractant/proinflammatory agent could, under certain circumstances, act as an antiinflammatory molecule.Fil: Isturiz, Martín Amadeo. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; ArgentinaFil: Beigier Bompadre, Macarena. No especifica;Fil: Barrionuevo, Paula. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; ArgentinaFil: Alves Rosa, Maria Fernanda. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Palermo, Marina Sandra. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; ArgentinaFil: Vulcano, Marisa. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentin

Usage of Murine T-cell Hybridoma Cells as Responder Cells Reveals Interference of Helicobacter Pylori with Human Dendritic cell-mediated Antigen Presentation

Direct effects of Helicobacter pylori (H. pylori) on human CD4+ T-cells hamper disentangling a possible bacterial-mediated interference with major histocompatibility complex class II (MHC-II)-dependent antigen presentation to these cells. To overcome this limitation, we employed a previously described assay, which enables assessing human antigen-processing cell function by using murine T-cell hybridoma cells restricted by human leukocyte antigen (HLA) alleles. HLA-DR1+ monocyte-derived dendritic cells were exposed to H. pylori and pulsed with the antigen 85B from Mycobacterium tuberculosis (M. tuberculosis). Interleukin-2 (IL-2) secretion by AG85Baa97-112- specific hybridoma cells was then evaluated as an integral reporter of cognate antigen presentation. This methodology enabled revealing of interference of H. pylori with the antigen-presenting capacity of human dendritic cells