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.

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.

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

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.

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.

Comparative analysis of the interaction of Helicobacter pylori with human dendritic cells, macrophages, and monocytes.

Helicobacter pylori may cause chronic gastritis, gastric cancer, or lymphoma. Myeloid antigen-presenting cells (APCs) are most likely involved in the induction and expression of the underlying inflammatory responses. To study the interaction of human APC subsets with H. pylori, we infected monocytes, monocyte-derived dendritic cells (DCs), and monocyte-derived (classically activated; M1) macrophages with H. pylori and analyzed phenotypic alterations, cytokine secretion, phagocytosis, and immunostimulation. Since we detected CD163(+) (alternatively activated; M2) macrophages in gastric biopsy specimens from H. pylori-positive patients, we also included monocyte-derived M2 macrophages in the study. Upon H. pylori infection, monocytes secreted interleukin-1ß (IL-1ß), IL-6, IL-10, and IL-12p40 (partially secreted as IL-23) but not IL-12p70. Infected DCs became activated, as shown by the enhanced expression of CD25, CD80, CD83, PDL-1, and CCR7, and secreted IL-1ß, IL-6, IL-10, IL-12p40, IL-12p70, and IL-23. However, infection led to significantly downregulated CD209 and suppressed the constitutive secretion of macrophage migration inhibitory factor (MIF). H. pylori-infected M1 macrophages upregulated CD14 and CD32, downregulated CD11b and HLA-DR, and secreted mainly IL-1ß, IL-6, IL-10, IL-12p40, and IL-23. Activation of DCs and M1 macrophages correlated with increased capacity to induce T-cell proliferation and decreased phagocytosis of dextran. M2 macrophages upregulated CD14 and CD206 and secreted IL-10 but produced less of the proinflammatory cytokines than M1 macrophages. Thus, H. pylori affects the functions of human APC subsets differently, which may influence the course and the outcome of H. pylori infection. The suppression of MIF in DCs constitutes a novel immune evasion mechanism exploited by H. pylori.

Modulation of the CD4+ T-cell response by Helicobacter pylori depends on known virulence factors and bacterial cholesterol and cholesterol α-glucoside content.

Helicobacter pylori blocks the proliferation of human CD4(+) T cells, facilitated by vacuolating exotoxin (VacA) and γ-glutamyl transpeptidase (GGT). H. pylori-triggered T-cell reactions in mice correlate with bacterial cholesterol and cholesterol α-glucoside content but their role in human cells is unclear. We characterized the effect of VacA, GGT, and cholesterol on T-helper 1, T-helper 2, T-regulatory and T-helper 17 associated cytokines and T-cell proliferation. VacA, GGT, and bacterial cholesterol content exhibited differential and synergistic inhibitory effects on the expression of activation markers CD25 and CD69 and on interleukin 2, interleukin 4, interleukin 10, and interferon γ production. These factors did not affect the H. pylori-mediated abrogation of transforming growth factor ß secretion or increased interleukin 6 production. Cholesterol α-glucosyltransferase-deficient bacteria exerted strongly reduced antiproliferative effects on primary human CD4(+) T cells. In conclusion, H. pylori shapes rather than suppresses human CD4(+) T-cell responses, and glucosylated cholesterol is a relevant bacterial component involved in this modulation.

Helicobacter pylori induces miR-155 in T cells in a cAMP-Foxp3-dependent manner.

Amongst the most severe clinical outcomes of life-long infections with Helicobacter pylori is the development of peptic ulcers and gastric adenocarcinoma--diseases often associated with an increase of regulatory T cells. Understanding H. pylori-driven regulation of T cells is therefore of crucial clinical importance. Several studies have defined mammalian microRNAs as key regulators of the immune system and of carcinogenic processes. Hence, we aimed here to identify H. pylori-regulated miRNAs, mainly in human T cells. MicroRNA profiling of non-infected and infected human T cells revealed H. pylori infection triggers miR-155 expression in vitro and in vivo. By using single and double H. pylori mutants and the corresponding purified enzymes, the bacterial vacuolating toxin A (VacA) and gamma-glutamyl transpeptidase (GGT) plus lipopolysaccharide (LPS) tested positive for their ability to regulate miR-155 and Foxp3 expression in human lymphocytes; the latter being considered as the master regulator and marker of regulatory T cells. RNAi-mediated knockdown (KD) of the Foxp3 transcription factor in T cells abolished miR-155 expression. Using adenylate cyclase inhibitors, the miR-155 induction cascade was shown to be dependent on the second messenger cyclic adenosine monophosphate (cAMP). Furthermore, we found that miR-155 directly targets the protein kinase A inhibitor alpha (PKIalpha) mRNA in its 3'UTR, indicative of a positive feedback mechanism on the cAMP pathway. Taken together, our study describes, in the context of an H. pylori infection, a direct link between Foxp3 and miR-155 in human T cells and highlights the significance of cAMP in this miR-155 induction cascade.

Immune complexes inhibit differentiation, maturation, and function of human monocyte-derived dendritic cells.

The interaction between immune complexes (IC) and the receptors for the Fc portion of IgG (FcgammaRs) triggers regulatory and effector functions in the immune system. In this study, we investigated the effects of IC on differentiation, maturation, and functions of human monocyte-derived dendritic cells (DC). When IC were added on day 0, DC generated on day 6 (IC-DC) showed lower levels of CD1a and increased expression of CD14, MHC class II, and the macrophage marker CD68, as compared with normally differentiated DC. The use of specific blocking FcgammaR mAbs indicated that the effect of IC was exerted mainly through their interaction with FcgammaRI and to a lesser extend with FcgammaRII. Immature IC-DC also expressed higher levels of CD83, CD86, and CD40 and the expression of these maturation markers was not further regulated by LPS. The apparent lack of maturation following TLR stimulation was associated with a decreased production of IL-12, normal secretion of IL-10 and CCL22, and increased production of CXCL8 and CCL2. IC-DC displayed low endocytic activity and a reduced ability to induce allogeneic T cell proliferation both at basal and LPS-stimulated conditions. Altogether, these data reveal that IC strongly affect DC differentiation and maturation. Skewing of DC function from Ag presentation to a proinflammatory phenotype by IC resembles the state of activation observed in DC obtained from patients with chronic inflammatory autoimmune disorders, such as systemic lupus erythematosus disease and arthritis. Therefore, the altered maturation of DC induced by IC may be involved in the pathogenesis of autoimmune diseases.

A novel function for galectin-1 at the crossroad of innate and adaptive immunity: galectin-1 regulates monocyte/macrophage physiology through a nonapoptotic ERK-dependent pathway.

Several environmental factors can differentially regulate monocyte and macrophage response patterns, resulting in the display of distinct functional phenotypes. Galectin-1, an endogenous lectin found at peripheral lymphoid organs and inflammatory sites, has shown immunoregulatory activity in vivo in experimental models of autoimmunity and cancer. Whereas compelling evidence has been accumulated regarding the effects of galectin-1 on T cell fate, limited information is available on how galectin-1 may impact other immune cell types. In the present study, we report a novel role for galectin-1 in the regulation of monocyte and macrophage physiology. Treatment with galectin-1 in vitro differentially regulates constitutive and inducible FcgammaRI expression on human monocytes and FcgammaRI-dependent phagocytosis. In addition, galectin-1 inhibits IFN-gamma-induced MHC class II (MHC-II) expression and MHC-II-dependent Ag presentation in a dose-dependent manner. These regulatory effects were also evident in mouse macrophages recruited in response to inflammatory stimuli following treatment with recombinant galectin-1 and further confirmed in galectin-1-deficient mice. Investigation of the mechanisms involved in these functions showed that galectin-1 does not affect survival of human monocytes, but rather influences FcgammaRI- and MHC-II-dependent functions through active mechanisms involving modulation of an ERK1/2-dependent pathway. Our results provide evidence of a novel unrecognized role for galectin-1 in the control of monocyte/macrophage physiology with potential implications at the crossroad of innate and adaptive immunity.

Increased susceptibility to apoptosis of CD56dimCD16+ NK cells induces the enrichment of IFN-gamma-producing CD56bright cells in tuberculous pleurisy.

Tuberculous pleuritis is a good model for the study of specific cells at the site of active Mycobacterium tuberculosis (Mtb) infection. We investigated the frequency and phenotype of NK cells in paired samples of peripheral blood and pleural fluid (PF) from patients with tuberculosis (TB) or parapneumonic infection. We demonstrated for the first time a reduction of NK cells in PF from TB with an enrichment in the CD56brightCD16- subset. In agreement, in PF NK cells we observed an increased expression of CD94, NKG2A, CD62L, and CCR7 molecules and lower expression of Bcl-2 and perforin. The activation markers CD69 and HLA-DR were also increased. The enrichment in the CD56bright subset was due to an increased susceptibility to apoptosis of CD56+CD16+ NK cells mediated by heat-labile and stable soluble factors present in tuberculous effusions and not in PF from other etiologies. Furthermore, in TB patients, Mtb-induced IFN-gamma production by PF NK cells was not dependent on the presence of CD3+, CD19+, and CD14+ cells, suggesting a direct interaction of CD56bright cells with Mtb and/or the involvement of other accessory cells present at the site of Mtb infection.

Galectin-3 and soluble fibrinogen act in concert to modulate neutrophil activation and survival: involvement of alternative MAPK pathways.

Galectin-3 (Gal-3), a member of a family of highly conserved carbohydrate-binding proteins, has recently emerged as a novel cellular modulator at inflammatory foci. Here we investigated the effects of Gal-3 on central effector functions of human neutrophils, including phagocytosis, exocytosis of secretory granules, and survival. We examined the effects of Gal-3 alone or in combination with soluble fibrinogen (sFbg), an extracellular mediator that plays a key role during the early phase of the inflammatory response through binding to integrin receptors. In addition we evaluated the intracellular signals triggered by these mediators in human neutrophils. Human neutrophils incubated with recombinant Gal-3 alone increased their phagocytic activity and CD66 surface expression. In contrast to the known antiapoptotic effect of Gal-3 on many cellular types, Gal-3 enhanced PMN apoptotic rate. Preincubation with Gal-3 primed neutrophils to the effects of sFbg, resulting in a synergistic action on degranulation. On the other hand, Gal-3 and sFbg had opposite effects on PMN survival, and the simultaneous action of both agonists partially counteracted the proapoptotic effects of Gal-3. In addition, although sFbg induced its effects through the activation of the ERKs, Gal-3 led to p38 phosphorylation. Disruption of this signaling pathway abrogated Gal-3-mediated modulation of neutrophil degranulation, phagocytosis, and apoptosis. Together, our results support the notion that Gal-3 and sFbg are two physiological mediators present at inflammatory sites that activate different components of the MAPK pathway and could be acting in concert to modulate the functionality and life span of neutrophils.

Hypothesis: an alternative pathway for the regulation of inflammation.

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-alpha) induced by lipopolysaccharides, a potent TNF-alpha inducer. We also determined that in human neutrophils FMLP and IC induced the downregulation of receptors for the Fc portion of IgG (FcgammaRII and FcgammaRIIIB). Moreover, FMLP inhibited interferon gamma (IFN-gamma)-induced FcgammaRI 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.

Hypotesis: an alternative pathway for the regulation of inflammation

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 ntiinflamatorios. 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.(AU)

Hypothesis: an alternative pathway for the regulation of inflammation.

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-alpha) induced by lipopolysaccharides, a potent TNF-alpha inducer. We also determined that in human neutrophils FMLP and IC induced the downregulation of receptors for the Fc portion of IgG (FcgammaRII and FcgammaRIIIB). Moreover, FMLP inhibited interferon gamma (IFN-gamma)-induced FcgammaRI 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.

Hypotesis: an alternative pathway for the regulation of inflammation

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 ntiinflamatorios. 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.