Edith Darcissac

Intralesional Regulatory T-Cell Suppressive Function during Human Acute and Chronic Cutaneous Leishmaniasis Due to Leishmania guyanensis

ABSTRACT The levels of regulatory T cells (Treg cells), analyzed by Foxp3 mRNA expression, were determined in lesions from patients with acute cutaneous leishmaniasis (ACL) and chronic cutaneous leishmaniasis (CCL). We demonstrated that Treg cells preferentially accumulate in lesions from ACL patients during the early phase of infection (lesion duration of less than 1 month). In addition, levels of Foxp3 mRNA transcripts were significantly higher in specimens from patients with CCL than in those from patients with ACL, suggesting a critical role of intralesional Treg cells in CCL. Intralesional Treg cells from both ACL and CCL patients were shown to have suppressive functions in vitro, since they inhibited the gamma interferon (IFN-γ) produced by CD4+ CD25− T cells purified from peripheral blood mononuclear cells from the same patient in response to Leishmania guyanensis stimulation. Intralesional 2,3-indoleamine dioxygenase (IDO) mRNA expression was associated with that of Foxp3, suggesting a role for IDO in the suppressive activity of intralesional Treg cells. In addition, a role, albeit minor, of interleukin-10 (IL-10) was also demonstrated, since neutralization of IL-10 produced by intralesional T cells increased IFN-γ production by effector cells in an in vitro suppressive assay. These results confirm the role of intralesional Treg cells in the immunopathogenesis of human Leishmania infection, particularly in CCL patients.

Interleukin-16 (IL-16) Inhibits Human Immunodeficiency Virus Replication in Cells from Infected Subjects, and Serum IL-16 Levels Drop with Disease Progression

The role of recombinant interleukin-16 (rIL-16) in regulating human immunodeficiency virus type 1 (HIV-1) replication in endogenously infected cells has been investigated. Cultures of CD8 cell-depleted mitogen-activated lymphocytes from 22 of 26 HIV-1-infected subjects presented variable levels of secreted p24 antigen. The presence of rIL-16 throughout the 14-day culture period dramatically inhibited p24 release into the culture supernatants. This effect was found to be mediated through inhibition of viral transcription but to be independent of the induced levels of other cytokines or chemokines known to regulate viral replication. Analysis of serum samples from HIV-1-infected subjects over a period of 8 years showed maintained or even increased IL-16 levels during the whole asymptomatic phase and a significant drop on progression to disease. These results strongly support a potential therapeutic value of rIL-16 in HIV-1 infection and the use of serum IL-16 levels to monitor disease progression.

Macrophage stimulation with Murabutide, an HIV-suppressive muramyl peptide derivative, selectively activates extracellular signal-regulated kinases 1 and 2, C / EBPβ and STAT1: role of CD14 and Toll-like receptors 2 and 4

The smallest unit of bacterial peptidoglycans known to be endowed with biological activities is muramyl dipeptide (MDP). A clinically acceptable synthetic derivative of MDP, namely murabutide (MB), has been found to present interesting pharmacological properties and to suppress HIV‐1 replication in monocyte‐derived macrophages (MDM). We have addressed the signaling events activated in MDM following stimulation with either MB or the potent immunostimulant LPS. We also examined whether signaling by muramyl peptides involves the use of cell surface receptors, including CD14 and Toll‐like receptor 2 (TLR2) or TLR4 that are known to be signal‐transducing receptors for other bacterial cell wall components. We demonstrate that, unlike LPS, the safe immunomodulator MB selectively activates extracellular signal‐regulated kinases (Erk) 1 / 2, in the absence of detectable Jun N‐terminal kinase (JNK) or p38 mitogen‐activated kinase activation. Furthermore, STAT1 activation but weakor no activation of STAT3 or STAT5 respectively, could be detected in MB‐stimulated MDM. Using MonoMac6 cells, we observed high C / EBPβ and AP‐1 but weaker and transient NF‐κB activation by MB.Moreover, the truncated form of C / EBPβ, known to repress HIV‐1 transcription, was detected in extracts from MB‐treated THP‐1 cells. Surprisingly, neither MB nor MDP were able to transduce signals via CD14 and TLR2 or 4. These findings present major differences in the early cell activation process between LPS and muramyl peptides, and strongly argue for the implication of co‐receptors other than TLR2 and TLR4 in mediating the signaling events induced by defined subunits of bacterial peptidoglycans.

Immunopharmacological activities and clinical development of muramyl peptides with particular emphasis on murabutide

Certain immunopharmacological activities of muramyl peptides have been associated with inflammatory and undesirable side-effects typically observed following the administration of the prototype molecule muramyl dipeptide. This activity is now demonstrated not to be linked to a direct activation of inflammatory processes in endothelial cells. Neither MDP nor other structural derivatives were able to induce inflammatory cytokines release or E-selectin gene expression in cultured human umbilical vein endothelial cells. However, oral administration of muramyl peptides has been reported to induce certain biological effects, including the downregulation of anamnestic, antigen-specific IgE responses, which are not observed following parenteral administration. We elaborate on these findings and extend them to show the efficacy of a new muramyl peptide in suppressing polyclonally induced serum IgE levels in anti-IgD-treated mice. The comparative effects of muramyl peptides, selected for clinical development, on the induction of cytokines in human whole blood are then presented at the level of mRNA accumulation and protein secretion. Moreover, the cytokine profile induced in vitro and in vivo by the combination of the safe immunostimulant, Murabutide, with interferon-alpha is examined. This combination reveals a selective and beneficial synergistic activity and induces anti-inflammatory cytokines in the absence of synergistic toxicity. The potential and the implications for the use of a therapeutic combination of an immunostimulant with a cytokine are discussed.

The Synthetic Immunomodulator Murabutide Controls Human Immunodeficiency Virus Type 1 Replication at Multiple Levels in Macrophages and Dendritic Cells

ABSTRACT Macrophages and dendritic cells are known to play an important role in the establishment and persistence of human immunodeficiency virus (HIV) infection. Besides antiretroviral therapy, several immune-based interventions are being evaluated with the aim of achieving better control of virus replication in reservoir cells. Murabutide is a safe synthetic immunomodulator presenting a capacity to enhance nonspecific resistance against viral infections and to target cells of the reticuloendothelial system. In this study, we have examined the ability of Murabutide to control HIV type 1 (HIV-1) replication in acutely infected monocyte-derived macrophages (MDMs) and dendritic cells (MDDCs). Highly significant suppression of viral replication was consistently observed in Murabutide-treated cultures of both cell types. Murabutide did not affect virus entry, reverse transcriptase activity, or early proviral DNA formation in the cytoplasm of infected cells. However, treated MDMs and MDDCs showed a dramatic reduction in nuclear viral two-long terminal repeat circular form and viral mRNA transcripts. This HIV-1-suppressive activity was not mediated by inhibiting cellular DNA synthesis or by activating p38 mitogen-activated protein kinase. Furthermore, Murabutide-stimulated cells expressed reduced CD4 and CCR5 receptors and secreted high levels of β-chemokines, although neutralization of the released chemokines did not alter the HIV-1-suppressive activity of Murabutide. These results provide evidence that a clinically acceptable immunomodulator can activate multiple effector pathways in macrophages and in dendritic cells, rendering them nonpermissive for HIV-1 replication.

Variations in serum IL-7 and 90K/Mac-2 binding protein (Mac−2 BP) levels analysed in cohorts of HIV-1 patients and correlated with clinical changes following antiretroviral therapy

Serum levels of interleukin‐7 (IL‐7), a non‐redundant cytokine that plays a crucial role in lymphopoiesis, are known to be elevated in HIV‐1‐infected subjects. To examine further the association between levels of IL‐7, CD4+ cell counts and viraemia, we analysed these parameters in a large cohort of HIV‐1 patients along with serum levels of 90K, a marker of disease severity but with no established involvement in lymphopoiesis. While IL‐7 levels were only found to correlate with CD4+ cell counts, 90K levels presented strong correlations with both CD4+ cell numbers and with plasma viral loads (VLs). These correlations were maintained in patients naive to treatment with antiretrovirals (n = 38) but were abolished when the analysis was restricted to the group receiving highly active antiretroviral therapy (HAART, n = 82). Moreover, although 90K levels were significantly reduced in patients on HAART, IL‐7 levels continued to be elevated despite successful treatment. The influence of HAART on the variations in these serum parameters was further assessed in a longitudinal study on 32 subjects. The HAART‐induced decrease in VLs and increase in CD4+ counts were found to correlate with a reduced serum level of 90K and IL‐7, respectively. Nevertheless, following a median period of 33 months of immunological and virological successful HAART, serum levels of IL‐7 continued to be significantly elevated compared with those detected in healthy controls. These findings suggest that immunotherapy with IL‐7, aimed to replenish T‐cell stock in HAART‐treated subjects, may have a limited impact on the process of immune reconstitution.

Recombinant interleukin‐16 selectively modulates surface receptor expression and cytokine release in macrophages and dendritic cells

Interleukin‐16 (IL‐16), a natural ligand for the CD4 receptor, has been found to modulate T‐lymphocyte function and to inhibit human immunodeficiency virus type 1 (HIV‐1) replication. Antigen‐presenting cells (APC), including macrophages and dendritic cells, are known to express functional surface CD4 molecules, to be susceptible to HIV‐1 infection and to play a critical role in different immune processes. Therefore, we evaluated the ability of recombinant IL‐16 (rIL‐16) to regulate receptor expression and cytokine release in monocyte‐derived macrophages (MDM) and monocyte‐derived dendritic cells (MDDC). Recombinant IL‐16 was found to up‐regulate CD25 and CD80 but to down‐regulate CD4 and CD86 surface expression in MDM cultures. However, no change could be observed on the level of CD4, CD80 and CD86 expression in IL‐16‐stimulated MDDC, although a significant up‐regulation of CD25 and CD83 was consistently detected. Furthermore, the level of gene expression of the chemokine receptors CCR5 and CXCR4 was significantly reduced in rIL‐16‐treated MDM and costimulation with IL‐2 did not modify the activity of the recombinant cytokine. The effects on chemokine receptor gene expression were less evident in MDDC and only a transient down‐regulation of weak intensity could be detected following stimulation with rIL‐16. Analysis of supernatants from rIL‐16‐stimulatedcultures revealed a different profile of released cytokines/chemokines among the two cell populations studied. These findings establish an important role for IL‐16 in modulating the activity of APC and may have relevance regarding the protection of reservoir cells against HIV‐1 infection.

Selective Regulation of Human Immunodeficiency Virus-Infected CD4+ Lymphocytes by a Synthetic Immunomodulator Leads to Potent Virus Suppression In Vitro and in hu-PBL-SCID Mice

ABSTRACT We have previously observed that the synthetic immunomodulator Murabutide inhibits human immunodeficiency virus type 1 (HIV-1) replication at multiple levels in macrophages and dendritic cells. The present study was designed to profile the activity of Murabutide on CD8-depleted phytohemagglutinin-activated lymphocytes from HIV-1-infected subjects and on the outcome of HIV-1 infection in severe combined immunodeficiency mice reconstituted with human peripheral blood leukocytes (hu-PBL-SCID mice). Maintaining cultures of CD8-depleted blasts from 36 patients in the presence of Murabutide produced dramatically reduced levels of viral p24 protein in the supernatants. This activity correlated with reduced viral transcripts and proviral DNA, was evident in cultures harboring R5, X4-R5, or X4 HIV-1 isolates, was not linked to inhibition of cellular DNA synthesis, and did not correlate with β-chemokine release. Moreover, c-myc mRNA expression was down-regulated in Murabutide-treated cells, suggesting potential interference of the immunomodulator with the nuclear transport of viral preintegration complexes. On the other hand, daily treatment of HIV-1-infected hu-PBL-SCID mice with Murabutide significantly reduced the viral loads in plasma and the proviral DNA content in human peritoneal cells. These results are the first to demonstrate that a clinically acceptable synthetic immunomodulator with an ability to enhance the hosts nonspecific immune defense mechanisms against infections can directly regulate cellular factors in infected lymphocytes, leading to controlled HIV-1 replication.

The transcriptional response of human macrophages to murabutide reflects a spectrum of biological effects for the synthetic immunomodulator.

The synthetic immunomodulator murabutide (MB) presents multiple biological activities with minimal toxicity in animals and in man. Although MB is known to target cells of the reticuloendothelial system and to regulate cytokine synthesis, the molecular mechanisms underlying several of its biological effects are still largely unknown. In an effort to define cellular factors implicated in the immunomodulatory and HIV‐suppressive activities of MB, we have undertaken profiling the regulated expression of genes in human monocyte‐derived macrophages (MDM) following a 6‐h stimulation with this synthetic glycopeptide. Oligonucleotide microarray analysis was performed on RNA samples of differentiated MDM from four separate donors, using probe sets corresponding to 1081 genes. We have identified, in a reproducible fashion, the enhanced expression of 40 genes and the inhibition of 16 others in MB‐treated MDM. These regulated genes belonged to different families of immune mediators or their receptors, transcription factors and kinases, matrix proteins and their inhibitors, ion channels and transporters, and proteins involved in cell metabolic pathways. Additional verification of the regulated expression of selected genes was carried out using Northern blots or the quantification of released proteins in MDM cultures. The profile of MB‐regulated genes in MDM provides a molecular basis for some of its previously reported biological activities, and reveals new set of genes targeted by the immunomodulator suggesting potential application in novel therapeutic indications.

Bioecological Drivers of Rabies Virus Circulation in a Neotropical Bat Community

Introduction In addition to the commonly accepted importance of the vampire bat in the maintenance and transmission of the rabies virus (RABV) in South America, RABV infection of other species is widely evidenced, challenging their role in the viral cycle. Methodology / Principles findings To identify the bioecological drivers of RABV circulation in neotropical bat communities, we conducted a molecular and serological survey on almost 1,000 bats from 30 species, and a 4-year longitudinal survey in two colonies of vampire bats in French Guiana. RABV was molecularly detected in a common vampire and in a frugivorous bat. The sequences corresponded to haematophagous bat-related strains and were close to viruses circulating in the Brazilian Amazon region. Species’ seroprevalence ranged from 0 to 20%, and the risk of seropositivity was higher in bats with a haematophagous diet, living in monospecific colonies and in dense forests. The longitudinal survey showed substantial temporal fluctuations, with individual waves of seroconversions and waning immunity. The high prevalences observed in bat communities, in most habitats and in species that do not share the same microhabitats and bioecological patterns, the temporal variations, and a rather short period of detectable antibodies as observed in recaptured vampires suggest (i) frequent exposure of animals, (ii) an ability of the infected host to control and eliminate the virus, (iii) more relaxed modes of exposure between bats than the commonly assumed infection via direct contact with saliva of infected animals, all of which should be further investigated. Conclusions / significance We hypothesize that RABV circulation in French Guiana is mainly maintained in the pristine forest habitats that may provide sufficient food resources to allow vampire bats, the main prevalent species, to survive and RABV to be propagated. However, on the forest edge and in disturbed areas, human activities may induce more insidious effects such as defaunation. One of the ecological consequences is the disappearance of resources for tertiary or secondary consumers. Populations of vampires may then shift to alternative resources such as cattle, domestic animals and humans. Therefore, a good forest status, allowing both a dilution effect in highly rich bat communities and the maintenance of large populations of medium-sized and large mammals used as prey by vampires, should prevent their migration to anthropized areas.