Jorge Geffner

Mouse bone marrow-derived mesenchymal stromal cells turn activated macrophages into a regulatory-like profile.

In recent years it has become clear that the therapeutic properties of bone marrow-derived mesenchymal stromal cells (MSC) are related not only to their ability to differentiate into different lineages but also to their capacity to suppress the immune response. We here studied the influence of MSC on macrophage function. Using mouse thioglycolate-elicited peritoneal macrophages (M) stimulated with LPS, we found that MSC markedly suppressed the production of the inflammatory cytokines TNF-α, IL-6, IL-12p70 and interferon-γ while increased the production of IL-10 and IL-12p40. Similar results were observed using supernatants from MSC suggesting that factor(s) constitutively released by MSC are involved. Supporting a role for PGE2 we observed that acetylsalicylic acid impaired the ability of MSC to inhibit the production of inflammatory cytokines and to stimulate the production of IL-10 by LPS-stimulated M. Moreover, we found that MSC constitutively produce PGE2 at levels able to inhibit the production of TNF-α and IL-6 by activated M. MSC also inhibited the up-regulation of CD86 and MHC class II in LPS-stimulated M impairing their ability to activate antigen-specific T CD4+ cells. On the other hand, they stimulated the uptake of apoptotic thymocytes by M. Of note, MSC turned M into cells highly susceptible to infection with the parasite Trypanosoma cruzi increasing more than 5-fold the rate of M infection. Using a model of inflammation triggered by s.c. implantation of glass cylinders, we found that MSC stimulated the recruitment of macrophages which showed a low expression of CD86 and the MHC class II molecule Iab and a high ability to produce IL-10 and IL-12p40, but not IL-12 p70. In summary, our results suggest that MSC switch M into a regulatory profile characterized by a low ability to produce inflammatory cytokines, a high ability to phagocyte apoptotic cells, and a marked increase in their susceptibility to infection by intracellular pathogens.

Tolerogenic signals delivered by dendritic cells to T cells through a galectin-1-driven immunoregulatory circuit involving interleukin 27 and interleukin 10

Despite their central function in orchestrating immunity, dendritic cells (DCs) can respond to inhibitory signals by becoming tolerogenic. Here we show that galectin-1, an endogenous glycan-binding protein, can endow DCs with tolerogenic potential. After exposure to galectin-1, DCs acquired an interleukin 27 (IL-27)-dependent regulatory function, promoted IL-10-mediated T cell tolerance and suppressed autoimmune neuroinflammation. Consistent with its regulatory function, galectin-1 had its highest expression on DCs exposed to tolerogenic stimuli and was most abundant from the peak through the resolution of autoimmune pathology. DCs lacking galectin-1 had greater immunogenic potential and an impaired ability to halt inflammatory disease. Our findings identify a tolerogenic circuit linking galectin-1 signaling, IL-27-producing DCs and IL-10-secreting T cells, which has broad therapeutic implications in immunopathology.

NADPH oxidase controls phagosomal pH and antigen cross-presentation in human dendritic cells

The phagocyte NADPH oxidase (NOX2) is critical for the bactericidal activity of phagocytic cells and plays a major role in innate immunity. We showed recently that NOX2 activity in mouse dendritic cells (DCs) prevents acidification of phagosomes, promoting antigen cross-presentation. In order to investigate the role of NOX2 in the regulation of the phagosomal pH in human DCs, we analyzed the production of reactive oxygen species (ROS) and the phagosomal/endosomal pH in monocyte-derived DCs and macrophages (M(diameter)s) from healthy donors or patients with chronic granulomatous disease (CGD). As expected, we found that human M(diameter)s acidify their phagosomes more efficiently than human DCs. Accordingly, the expression of the vacuolar proton ATPase (V-H(+)-ATPase) was higher in M(diameter)s than in DCs. Phagosomal ROS production, however, was also higher in M(diameter)s than in DCs, due to higher levels of gp91phox expression and recruitment to phagosomes. In contrast, in the absence of active NOX2, the phagosomal and endosomal pH decreased. Both in the presence of a NOX2 inhibitor and in DCs derived from patients with CGD, the cross-presentation of 2 model tumor antigens was impaired. We conclude that NOX2 activity participates in the regulation of the phagosomal and endosomal pH in human DCs, and is required for efficient antigen cross-presentation.

Control of dendritic cell differentiation by angiotensin II

Here we analyze the role of the angiotensinergic system in the differentiation of dendritic cells (DC). We found that human monocytes produce angiotensin II (AII) and express AT1 and AT2 receptors for AII. DC differentiated from human monocytes in the presence of AT1 receptor antagonists losartan or candesartan show very low levels of CD1a expression and poor endocytic and allostimulatory activities. By contrast, DC differentiation in the presence of either the AT2 receptor antagonist PD 123319 or exogenous AII results in the development of nonadherent cells with CD1a expression and endocytic and allostimulatory activities higher than control DC. Similar contrasting effects were observed in mouse DC obtained from bone marrow cultures supplemented with granulocyte‐monocyte colony‐stimulating factor. DC differentiated in the presence of the AT1 receptor antagonist losartan express lower levels of CD11c, CD40, and Ia and display a lower ability to endocyte horseradish peroxidase (HRP) and to induce antibody responses in vivo, compared with controls. By contrast, DC differentiation in the presence of either the AT2 receptor antagonist PD 123319 or exogenous AII results in cells with high levels of CD11c, CD40, and Ia, as well as high ability to endocyte HRP and to induce antibody responses in vivo. Our results support the notion that the differentiation of DC is regulated by AII.

Promotion of Neutrophil Apoptosis by TNF-α

We examined the ability of TNF-α to modulate human neutrophil apoptosis. Neutrophils cultured with TNF-α alone undergo a low but significant increase in the number of apoptotic cells. More interestingly, when neutrophils were pretreated with TNF-α for 1–2 min at 37°C and then were exposed to a variety of agents such as immobilized IgG, IgG-coated erythrocytes, complement-treated erythrocytes, zymosan, PMA, zymosan-activated serum, fMLP, Escherichia coli, and GM-CSF for 3 h at 37°C, a marked stimulation of apoptosis was observed. Similar results were obtained in neutrophils pretreated with TNF-α for 30 min, 1 h, 3 h, and 18 h. Dose-dependent studies showed that TNF-α enhances neutrophil apoptosis at concentrations ranging from 1 to 100 ng/ml. In contrast to the observations made in neutrophils pretreated with TNF-α, there was no stimulation of apoptosis when TNF-α was added to neutrophils previously activated by conventional agonists. Experiments performed to establish the mechanism through which TNF-α promotes neutrophil apoptosis showed that neither reactive oxygen intermediates nor the Fas/Fas ligand system appear to be involved. Our results suggest that TNF-α plays a critical role in the control of neutrophil survival by virtue of its ability to induce an apoptotic death program which could be triggered by a variety of conventional agonists.

Extracellular acidosis induces neutrophil activation by a mechanism dependent on activation of phosphatidylinositol 3-kinase/Akt and ERK pathways.

Inflammation in peripheral tissues is usually associated with the development of local acidosis; however, there are few studies aimed at analyzing the influence of acidosis on immune cells. We have shown previously that extracellular acidosis triggers human neutrophil activation, inducing a transient increase in intracellular Ca2+ concentration, a shape change response, the up-regulation of CD18 expression, and a delay of apoptosis. In this study, we analyzed the signaling pathways responsible for neutrophil activation. We found that acidosis triggers the phosphorylation of Akt (the main downstream target of PI3K) and ERK MAPK, but not that of p38 and JNK MAPK. No degradation of IκB was observed, supporting the hypothesis that NF-κB is not activated under acidosis. Inhibition of PI3K by wortmannin or LY294002 markedly decreased the shape change response and the induction of Ca2+ transients triggered by acidosis, whereas the inhibition of MEK by PD98059 or U0126 significantly inhibited the shape change response without affecting the induction of Ca2+ transients. We also found that acidosis not only induces a shape change response and the induction of Ca2+ transients in human neutrophils but also stimulates the endocytosis of FITC-OVA and FITC-dextran. Stimulation of endocytosis was partially prevented by inhibitors of PI3K and MEK. Together, our results support the notion that the stimulation of human neutrophils by extracellular acidosis is dependent on the activation of PI3K/Akt and ERK pathways. Of note, using mouse peritoneal neutrophils we observed that the enhancement of endocytosis induced by acidosis was associated with an improved ability to present extracellular Ags through a MHC class I-restricted pathway.

Bacterial DNA activates human neutrophils by a CpG-independent pathway

Bacterial DNA stimulates macrophages, monocytes, B lymphocytes, NK cells, and dendritic cells in a CpG‐dependent manner. In this work we demonstrate that bacterial DNA, but not mammalian DNA, induces human neutrophil activation as assessed by L‐selectin shedding, CD11b upregulation, and stimulation of cellular shape change, IL‐8 secretion, and cell migration. Induction of these responsesis not dependent on the presence of unmethylated CpG motifs, as neutrophil stimulatory properties were neither modified by CpG‐methylation of bacterial DNA nor reproduced by oligonucleotides bearing CpG motifs. We found that human neutrophils express Toll‐like receptor (TLR) 9 mRNA. However, as expected for a CpG‐independent mechanism, activation does not involve a TLR9‐dependent signaling pathway; neutrophil stimulation was not prevented by immobilization of bacterial DNA or by wortmannin or chloroquine, two agents that inhibit TLR9 signaling. Of note, both single‐stranded and double‐stranded DNA were able to induce activation, suggesting that neutrophils might be activated by bacterial DNA at inflammatory foci even in the absence of conditions required to induce DNA denaturation. Our findings provide the first evidence that neutrophils might be alerted to the presence of invading bacteria through recognition of its DNA via a novel mechanism not involving CpG motifs.

Spermatozoa capture HIV-1 through heparan sulfate and efficiently transmit the virus to dendritic cells.

Semen is the main vector for HIV-1 dissemination worldwide. It contains three major sources of infectious virus: free virions, infected leukocytes, and spermatozoa-associated virions. We focused on the interaction of HIV-1 with human spermatozoa and dendritic cells (DCs). We report that heparan sulfate is expressed in spermatozoa and plays an important role in the capture of HIV-1. Spermatozoa-attached virus is efficiently transmitted to DCs, macrophages, and T cells. Interaction of spermatozoa with DCs not only leads to the transmission of HIV-1 and the internalization of the spermatozoa but also results in the phenotypic maturation of DCs and the production of IL-10 but not IL-12p70. At low values of extracellular pH (∼6.5 pH units), similar to those found in the vaginal mucosa after sexual intercourse, the binding of HIV-1 to the spermatozoa and the consequent transmission of HIV-1 to DCs were strongly enhanced. Our observations support the notion that far from being a passive carrier, spermatozoa acting in concert with DCs might affect the early course of sexual transmission of HIV-1 infection.

Acidosis Improves Uptake of Antigens and MHC Class I-Restricted Presentation by Dendritic Cells

It is widely appreciated that inflammatory responses in peripheral tissues are usually associated to the development of acidic microenvironments. Despite this, there are few studies aimed to analyze the effect of extracellular pH on immune cell functions. We analyzed the impact of acidosis on the behavior of dendritic cells (DCs) derived from murine bone marrow. We found that extracellular acidosis (pH 6.5) markedly stimulated the uptake of FITC-OVA, FITC-dextran, and HRP by DCs. In fact, to reach similar levels of endocytosis, DCs cultured at pH 7.3 required concentrations of Ag in the extracellular medium almost 10-fold higher compared with DCs cultured at pH 6.5. Not only the endocytic capacity of DCs was up-regulated by extracellular acidosis, but also the expression of CD11c, MHC class II, CD40, and CD86 as well as the acquisition of extracellular Ags by DCs for MHC class I-restricted presentation. Importantly, DCs pulsed with Ag under acidosis showed an improved efficacy to induce both specific CD8+ CTLs and specific Ab responses in vivo. Our results suggest that extracellular acidosis improves the Ag-presenting capacity of DCs.

Extracellular DNA: A Major Proinflammatory Component of Pseudomonas aeruginosa Biofilms

We previously demonstrated that extracellular bacterial DNA activates neutrophils through a CpG- and TLR9-independent mechanism. Biofilms are microbial communities enclosed in a polymeric matrix that play a critical role in the pathogenesis of many infectious diseases. Because extracellular DNA is a key component of biofilms of different bacterial species, the aim of this study was to determine whether it plays a role in the ability of biofilms to induce human neutrophil activation. We found that degradation of matrix extracellular DNA with DNase I markedly reduced the capacity of Pseudomonas aeruginosa biofilms to induce the release of the neutrophil proinflammatory cytokines IL-8 and IL-1β (>75%); reduced the upregulation of neutrophil activation markers CD18, CD11b, and CD66b (p < 0.001); reduced the number of bacteria phagocytosed per neutrophil contacting the biofilm; and reduced the production of neutrophil extracellular traps. Consistent with these findings, we found that biofilms formed by the lasI rhlI P. aeruginosa mutant strain, exhibiting a very low content of matrix extracellular DNA, displayed a lower capacity to stimulate the release of proinflammatory cytokines by neutrophils, which was not decreased further by DNase I treatment. Together, our findings support that matrix extracellular DNA is a major proinflammatory component of P. aeruginosa biofilms.