Adriana Bonomo

Apoptotic mimicry by an obligate intracellular parasite downregulates macrophage microbicidal activity

Programmed cell death by apoptosis of unnecessary or potentially harmful cells is clearly beneficial to multicellular organisms. Proper functioning of such a program demands that the removal of dying cells proceed without an inflammatory reaction. Phosphatidylserine (PS) is one of the ligands displayed by apoptotic cells that participates in their noninflammatory removal when recognized by neighboring phagocytes. PS ligation induces the release of transforming growth factor-beta (TGF-beta), an antiinflammatory cytokine that mediates the suppression of macrophage-mediated inflammation. In Hydra vulgaris, an organism that stands at the base of metazoan evolution, the selective advantage provided by apoptosis lies in the fact that Hydra can survive recycling apoptotic cells by phagocytosis. In unicellular organisms, it has been proposed that altruistic death benefits clonal populations of yeasts and trypanosomatids. Now we show that advantageous features of the apoptotic process can operate without death as the necessary outcome. Leishmania spp are able to evade the killing activity of phagocytes and establish themselves as obligate intracellular parasites. Amastigotes, responsible for disease propagation, similar to apoptotic cells, inhibit macrophage activity by exposing PS. Exposed PS participates in amastigote internalization. Recognition of this moiety by macrophages induces TGF-beta secretion and IL-10 synthesis, inhibits NO production, and increases susceptibility to intracellular leishmanial growth.

Mimicry of Apoptotic Cells by Exposing Phosphatidylserine Participates in the Establishment of Amastigotes of Leishmania (L) amazonensis in Mammalian Hosts

Signaling through exposed phosphatidylserine (PS) is fundamental for the TGFβ1-dependent, noninflammatory phagocytosis of apoptotic cells. This same mechanism operates in the internalization of amastigotes of Leishmania (L) amazonensis (L(L)a) in a process quoted as apoptotic mimicry. Now we show that the host modulates PS exposure by the amastigotes and, as a consequence, BALB/c mice-derived amastigotes expose significantly more PS than those derived from C57BL/6 mice. Due to this difference in the density of surface PS molecules, the former are significantly more infective than the latter, both in vivo, in F1 (BALB/c × C57BL/6) mice, and in vitro, in thioglycollate-derived macrophages from this same mouse strain. PS exposure increases with progression of the lesion and reaches its maximum value in amastigotes obtained at the time point when the lesion in C57BL/6 mice begins to decrease in size and the lesions in BALB/c mice are still growing in size. Synthesis of active TGFβ1, induction of IL-10 message, and inhibition of NO synthesis correlate with the amount of surface PS displayed by viable (propidium iodide-negative) infective amastigote. Furthermore, we also show that, similar to what happens with apoptotic cells, amastigotes of L(L)a are internalized by macropinocytosis. This mechanism of internalization is consistent with the large phagolysosomes characteristic of L(L)a infection. The intensity of macrophage macropinocytic activity is dependent on the amount of surface PS displayed by the infecting amastigote.

Cooperative Activation of TLR2 and Bradykinin B2 Receptor Is Required for Induction of Type 1 Immunity in a Mouse Model of Subcutaneous Infection by Trypanosoma cruzi

We have previously reported that exogenous bradykinin activates immature dendritic cells (DCs) via the bradykinin B2 receptor (B2R), thereby stimulating adaptive immunity. In this study, we show that these premises are met in a model of s.c. infection by Trypanosoma cruzi, a protozoan that liberates kinins from kininogens through its major protease, cruzipain. Intensity of B2R-dependent paw edema evoked by trypomastigotes correlated with levels of IL-12 produced by CD11c+ dendritic cells isolated from draining lymph nodes. The IL-12 response induced by endogenously released kinins was vigorously increased in infected mice pretreated with inhibitors of angiotensin converting enzyme (ACE), a kinin-degrading metallopeptidase. Furthermore, these innate stimulatory effects were linked to B2R-dependent up-regulation of IFN-γ production by Ag-specific T cells. Strikingly, the trypomastigotes failed to up-regulate type 1 immunity in TLR2−/− mice, irrespective of ACE inhibitor treatment. Analysis of the dynamics of inflammation revealed that TLR2 triggering by glycosylphosphatidylinositol-anchored mucins induces plasma extravasation, thereby favoring peripheral accumulation of kininogens in sites of infection. Further downstream, the parasites generate high levels of innate kinin signals in peripheral tissues through the activity of cruzipain. The demonstration that the deficient type 1 immune responses of TLR2−/− mice are rescued upon s.c. injection of exogenous kininogens, along with trypomastigotes, supports the notion that generation of kinin “danger” signals is intensified through cooperative activation of TLR2 and B2R. In summary, we have described a s.c. infection model where type 1 immunity is vigorously up-regulated by bradykinin, an innate signal whose levels in peripheral tissues are controlled by an intricate interplay of TLR2, B2R, and ACE.

Soluble Factors Released by Toxoplasma gondii-Infected Astrocytes Down-Modulate Nitric Oxide Production by Gamma Interferon-Activated Microglia and Prevent Neuronal Degeneration

ABSTRACT The maintenance of a benign chronic Toxoplasma gondii infection is mainly dependent on the persistent presence of gamma interferon (IFN-γ) in the central nervous system (CNS). However, IFN-γ-activated microglia are paradoxically involved in parasitism control and in tissue damage during a broad range of CNS pathologies. In this way, nitric oxide (NO), the main toxic metabolite produced by IFN-γ-activated microglia, may cause neuronal injury during T. gondii infection. Despite the potential NO toxicity, neurodegeneration is not a common finding during chronic T. gondii infection. In this work, we describe a significant down-modulation of NO production by IFN-γ-activated microglia in the presence of conditioned medium of T. gondii-infected astrocytes (CMi). The inhibition of NO production was paralleled with recovery of neurite outgrowth when neurons were cocultured with IFN-γ-activated microglia in the presence of CMi. Moreover, the modulation of NO secretion and the neuroprotective effect were shown to be dependent on prostaglandin E2 (PGE2) production by T. gondii-infected astrocytes and autocrine secretion of interleukin-10 (IL-10) by microglia. These events were partially eliminated when infected astrocytes were treated with aspirin and cocultures were treated with anti-IL-10 neutralizing antibodies and RP-8-Br cyclic AMP (cAMP), a protein kinase A inhibitor. Further, the modulatory effects of CMi were mimicked by the presence of exogenous PGE2 and by forskolin, an adenylate cyclase activator. Altogether, these data point to a T. gondii-triggered regulatory mechanism involving PGE2 secretion by astrocytes and cAMP-dependent IL-10 secretion by microglia. This may reduce host tissue inflammation, thus avoiding neuron damage during an established Th1 protective immune response.

Apoptotic mimicry: an altruistic behavior in host/Leishmania interplay

Apoptosis is the most common phenotype observed when cells die through programmed cell death. The morphologic and biochemical changes that characterize apoptotic cells depend on the activation of a diverse set of genes. Apoptosis is essential for multicellular organisms since their development and homeostasis are dependent on extensive cell renewal. In fact, there is strong evidence for the correlation between the emergence of multicellular organisms and apoptosis during evolution. On the other hand, no obvious advantages can be envisaged for unicellular organisms to carry the complex machinery required for programmed cell death. However, accumulating evidence shows that free-living and parasitic protozoa as well as yeasts display apoptotic markers. This phenomenon has been related to altruistic behavior, when a subpopulation of protozoa or yeasts dies by apoptosis, with clear benefits for the entire population. Recently, phosphatidylserine (PS) exposure and its recognition by a specific receptor (PSR) were implicated in the infectivity of amastigote forms of Leishmania, an obligatory vertebrate intramacrophagic parasite, showing for the first time that unicellular organisms use apoptotic features for the establishment and/or maintenance of infection. Here we focus on PS exposure in the outer leaflet of the plasma membrane--an early hallmark of apoptosis--and how it modulates the inflammatory activity of phagocytic cells. We also discuss the possible mechanisms by which PS exposure can define Leishmania survival inside host cells and the evolutionary implications of apoptosis at the unicellular level.

Trypanosoma cruzi infection modulates intrathymic contents of extracellular matrix ligands and receptors and alters thymocyte migration

Several T cell abnormalities have been described in the course of acute Trypanosoma cruzi infection in mice, including severe effects on the thymus. In the present study, looking at the expression of extracellular matrix ligands in the thymus, we observed that deposits of fibronectin and laminin increased progressively during the course of infection, reaching a maximum at the peak of parasitemia and thymic atrophy. Concomitantly, membrane expression of fibronectin and laminin receptors (VLA‐4, VLA‐5 and VLA‐6) was also enhanced on thymocyte subsets of infected mice. These results correlated with changes in intrathymic thymocyte migration ability during the acute phase of infection, when a higher fibronectin‐dependent transmigratory activity of CD4+CD8+ thymocytes was observed. Strikingly, we detected higher frequency of immature and high VLA‐expressing CD4+CD8+ T cells in the peripheral lymphoid organs of infected mice at thepeak of parasitemia. These cells seemed to be thymus dependent, since significantly lower amounts of them were found in thymectomized mice, and some of them carry “prohibited” Vβ segments of the TCR. Our data suggest an imbalance in the intrathymic cell trafficking following acute T. cruzi infection, likely due to dysregulated extracellular matrix‐dependent interactions.

T Cells Induce Pre-Metastatic Osteolytic Disease and Help Bone Metastases Establishment in a Mouse Model of Metastatic Breast Cancer

Bone metastases, present in 70% of patients with metastatic breast cancer, lead to skeletal disease, fractures and intense pain, which are all believed to be mediated by tumor cells. Engraftment of tumor cells is supposed to be preceded by changes in the target tissue to create a permissive microenvironment, the pre-metastatic niche, for the establishment of the metastatic foci. In bone metastatic niche, metastatic cells stimulate bone consumption resulting in the release of growth factors that feed the tumor, establishing a vicious cycle between the bone remodeling system and the tumor itself. Yet, how the pre-metastatic niches arise in the bone tissue remains unclear. Here we show that tumor-specific T cells induce osteolytic bone disease before bone colonization. T cells pro-metastatic activity correlate with a pro-osteoclastogenic cytokine profile, including RANKL, a master regulator of osteoclastogenesis. In vivo inhibition of RANKL from tumor-specific T cells completely blocks bone loss and metastasis. Our results unveil an unexpected role for RANKL-derived from T cells in setting the pre-metastatic niche and promoting tumor spread. We believe this information can bring new possibilities for the development of prognostic and therapeutic tools based on modulation of T cell activity for prevention and treatment of bone metastasis.

Study of membrane potential in T lymphocytes subpopulations using flow cytometry.

BackgroundIon channels are involved in the control of membrane potential (ψ) in a variety of cells. The maintenance of ψ in human T lymphocytes is essential for T-cell activation and was suggested to depend mostly on the voltage-gated Kv1.3 channel. Blockage of Kv1.3 inhibits cytokine production and lymphocyte proliferation in vitro and suppresses immune response in vivo. T lymphocytes are a heterogeneous cell population and the expression of Kv1.3 varies among cell subsets. Oxonol diBA-C4-(3) was used to determine ψ by flow cytometry. The presence of distinct T cell subsets was evaluated by immunophenotyping techniques and the contribution of Kv1.3 channels for the maintenance of ψ was investigated using selective blockers.ResultsThe distribution of ψ in T lymphocytes varied among blood donors and did not always follow a unimodal pattern. T lymphocytes were divided into CD3+/CD45RO- and CD3+/CD45RO+ subsets, whose peak channel values of ψ were -58 ± 3.6 mV and -37 ± 4.1 mV, respectively. MgTX (specific inhibitor of Kv1.3 channels) had no significant effect in the ψ of CD3+/CD45RO- subsets but depolarized CD3+/CD45RO+ cells to -27 ± 5.1 mV.ConclusionCombination of optical methods for determination of ψ by flow cytometry with immuophenotyping techniques opens new possibilities for the study of ion channels in the biology of heterogeneous cell populations such as T lymphocyte subsets.

T-lymphocyte function from peripheral blood stem-cell donors is inhibited by activated granulocytes

BACKGROUND PBSC transplant provides 10 times more T cells than BMT However, the incidence and severity of acute GvHD is similar among recipients of both types of transplants. Studies in mouse models suggest that the similar clinical outcome in BMT and PBSCT is due to differences in the lymphokine profiles. METHODS PBMC, PBMC from G-CSF mobilized donors (G-PBMC)and BM mononuclear cells (BM-MC) were analyzed by flow cytometry and ELISA to detect gamma-IFN and IL-4 production. Hematoxylin and eosin staining was used to identify morphology and annexin/propidium-iodide was used for apoptosis assays. RESULTS We show decreased production of gamma-interferon (85%) and IL-4 (60%) in G-PBMC when compared with either PBMC or BM-MCT cells on ex vivo assays. Surprisingly, 85% of fresh G-PBMC is composed of low-density granulocytes (LDG), which undergo apoptosis after 48 h in culture. At this same time, gamma-IFN production from G-PBMC T cell was reverted. In vitro, G-CSF converts granulocytes into LDGs, able to inhibit T-cell function by H2O2 production, and not through immune-deviation towards a Th2-type phenotype. DISCUSSION We show that the estimated numbers of Th1 and Th2 cells infused in BMT and PBSCT do not differ significantly. These findings are discussed with reference to the relatively low incidence of acute GvHD in PBSCT shown in the literature. We suggest that these results might depend on the high number of granulocytes and progenitors infused. The potential use of granulocytes as immunosupressive short-term therapy is now being investigated by our group using a mouse experimental model.

Comparative analysis of splenic cell proliferation induced by interleukin 3 and by syngeneic accessory cells (syngeneic mixed leukocyte reaction): evidence that autoreactive T-cell functioning instructs hematopoietic phenomena.

Murine syngeneic mixed leukocyte reaction (SMLR) was studied under totally autologous culture conditions using syngeneic normal mouse serum in the culture. SMLR was detected in splenic, but not in lymph node, nonadherent responding cell populations (NWNAC). In the absence of stimulator, accessory cells (AC), IL3-containing fluids also induced splenic, but not lymph node, NWNAC growth. SMLR-derived supernatants contained IL3, but not IL2, activity, and production of this IL3 activity could be prevented by adding anti-CD4 mAbs to SMLR cultures. Precursor frequencies of both SMLR and IL3 splenic responses were very low and similar, and there was a synergism between IL3 and AC in induction of NWNAC growth. Growth of responding NWNAC was further enhanced by T-cell depletion with anti-Thy1 mAb and complement. Lack of T-cell proliferation in the SMLR was confirmed by BUdR and light protection experiments. Autoradiographs indicated that the same cell type grew in both SMLR and IL3-induced NWNAC cultures. Besides blast cells, cells with the appearance of immature monocytes with 3H-labeled nuclei were found in both kinds of culture. No labeled lymphocytes could be found. Both SMLR and IL3-induced NWNAC cultures contained expanded numbers of M-CSF-responsive monocyte precursors. On the other hand, SMLR- but not IL3-induced cultures contained expanded numbers of IL3-responsive, immature precursors capable of giving rise to large colonies of monocytic-like cells. Although IL2 could not be detected in SMLR supernatants, both cell growth and IL3 production could be blocked with anti-IL2 receptor and anti-IL2 mAbs. Exogenous IL2, on the other hand, enhanced both cell growth and IL3 production in the SMLR. These results indicate that, under totally autologous conditions, CD4+ autoreactive T-cells do not proliferate in the SMLR, but rather instruct the growth of splenic hematopoietic precursors capable of differentiating along the monocytic lineage. Autoreactive T-cell activation in the SMLR seems to involve minimal IL2 production, which is critically necessary for triggering IL3 production in a markedly amplified manner. These results suggest a link between normal regulation of hematopoiesis and MHC-restricted, autoreactive T-cell activation.