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1.  MIF Contributes to Trypanosoma brucei Associated Immunopathogenicity Development 
PLoS Pathogens  2014;10(9):e1004414.
African trypanosomiasis is a chronic debilitating disease affecting the health and economic well-being of many people in developing countries. The pathogenicity associated with this disease involves a persistent inflammatory response, whereby M1-type myeloid cells, including Ly6Chigh inflammatory monocytes, are centrally implicated. A comparative gene analysis between trypanosusceptible and trypanotolerant animals identified MIF (macrophage migrating inhibitory factor) as an important pathogenic candidate molecule. Using MIF-deficient mice and anti-MIF antibody treated mice, we show that MIF mediates the pathogenic inflammatory immune response and increases the recruitment of inflammatory monocytes and neutrophils to contribute to liver injury in Trypanosoma brucei infected mice. Moreover, neutrophil-derived MIF contributed more significantly than monocyte-derived MIF to increased pathogenic liver TNF production and liver injury during trypanosome infection. MIF deficient animals also featured limited anemia, coinciding with increased iron bio-availability, improved erythropoiesis and reduced RBC clearance during the chronic phase of infection. Our data suggest that MIF promotes the most prominent pathological features of experimental trypanosome infections (i.e. anemia and liver injury), and prompt considering MIF as a novel target for treatment of trypanosomiasis-associated immunopathogenicity.
Author Summary
Uncontrolled inflammation is a major contributor to pathogenicity development during many chronic parasitic infections, including African trypanosome infections. Hence, therapies should aim at re-establishing the balance between pro- and anti-inflammatory responses to reduce tissue damage. Our experiments uncovered that macrophage migration inhibitory factor (MIF) plays a pivotal role in trypanosomiasis-associated pathogenicity development. Hereby, MIF-deficient and neutralizing anti-MIF antibody-treated wild type (WT) T. brucei-infected mice exhibited decreased inflammatory responses, reduced liver damage and anemia (i.e. the most prominent pathogenicity features) compared to WT control mice. The reduced tissue damage coincided with reduced infiltration of pathogenic monocytic cells and neutrophils, whereby neutrophil-derived MIF contributed more significantly than monocyte-derived MIF to tissue damage. MIF also promoted anemia development by suppressing red blood cell production and enhancing their clearance. The clinical significance of these findings follows from human genetic data indicating that low-expression (protective) MIF alleles are enriched in Africans. The current findings therefore offer promise for human translation and open the possibility of assessing MIF levels or MIF genotype as an indication of an individual's risk for severe trypanosomiasis. Furthermore, given the unmet medical need of African trypanosomiasis affecting millions of people, these findings highlight MIF as a potential new therapeutic target for treatment of trypanosomiasis-associated pathogenicity.
doi:10.1371/journal.ppat.1004414
PMCID: PMC4177988  PMID: 25255103
3.  Targeting of Human Antigen-Presenting Cell Subsets 
Journal of Virology  2013;87(20):11304-11308.
Antigen-presenting cells are a heterogeneous group of cells that are characterized by their functional specialization. Consequently, targeting specific antigen-presenting cell subsets offers opportunities to induce distinct T cell responses. Here we report on the generation and use of nanobodies (Nbs) to target lentivectors specifically to human lymph node-resident myeloid dendritic cells, demonstrating that Nbs represent a powerful tool to redirect lentivectors to human antigen-presenting cell subsets.
doi:10.1128/JVI.01498-13
PMCID: PMC3807283  PMID: 23864630
4.  Immunogenicity of targeted lentivectors 
Oncotarget  2014;5(3):704-715.
To increase the safety and possibly efficacy of HIV-1 derived lentivectors (LVs) as an anti-cancer vaccine, we recently developed the Nanobody (Nb) display technology to target LVs to antigen presenting cells (APCs). In this study, we extend these data with exclusive targeting of LVs to conventional dendritic cells (DCs), which are believed to be the main cross-presenting APCs for the induction of a TH1-conducted antitumor immune response. The immunogenicity of these DC-subtype targeted LVs was compared to that of broad tropism, general APC-targeted and non-infectious LVs. Intranodal immunization with ovalbumin encoding LVs induced proliferation of antigen specific CD4+ T cells, irrespective of the LVs' targeting ability. However, the cytokine secretion profile of the restimulated CD4+ T cells demonstrated that general APC targeting induced a similar TH1-profile as the broad tropism LVs while transduction of conventional DCs alone induced a similar and less potent TH1 profile as the non-infectious LVs. This observation contradicts the hypothesis that conventional DCs are the most important APCs and suggests that the activation of other APCs is also meaningful. Despite these differences, all targeted LVs were able to stimulate cytotoxic T lymphocytes, be it to a lesser extent than broad tropism LVs. Furthermore this induction was shown to be dependent on type I interferon for the targeted and non-infectious LVs, but not for broad tropism LVs. Finally we demonstrated that the APC-targeted LVs were as potent in therapy as broad tropism LVs and as such deliver on their promise as safer and efficacious LV-based vaccines.
PMCID: PMC3996667  PMID: 24519916
lentivector; targeting; antigen presenting cell; vaccine; antitumor immunotherapy
5.  Functional Relationship between Tumor-Associated Macrophages and Macrophage Colony-Stimulating Factor as Contributors to Cancer Progression 
The current review article describes the functional relationship between tumor-associated macrophages (TAM) as key cellular contributors to cancer malignancy on the one hand and macrophage-colony-stimulating factor (M-CSF or CSF-1) as an important molecular contributor on the other. We recapitulate the available data on expression of M-CSF and the M-CSF receptor (M-CSFR) in human tumor tissue as constituents of a stromal macrophage signature and on the limits of the predictive and prognostic value of plasma M-CSF levels. After providing an update on current insights into the nature of TAM heterogeneity at the level of M1/M2 phenotype and TAM subsets, we give an overview of experimental evidence, based on genetic, antibody-mediated, and pharmacological disruption of M-CSF/M-CSFR signaling, for the extent to which M-CSFR signaling can not only determine the TAM quantity, but can also contribute to shaping the phenotype and heterogeneity of TAM and other related tumor-infiltrating myeloid cells (TIM). Finally, we review the accumulating information on the – sometimes conflicting – effects blocking M-CSFR signaling may have on various aspects of cancer progression such as tumor growth, invasion, angiogenesis, metastasis, and resistance to therapy and we thereby discuss in how far these different effects actually reflect a contribution of TAM.
doi:10.3389/fimmu.2014.00489
PMCID: PMC4188035  PMID: 25339957
M-CSF; CSF-1; M-CSFR; CSF-1R; tumor-associated macrophages; M1; M2; cancer progression
6.  A Trypanosoma brucei Kinesin Heavy Chain Promotes Parasite Growth by Triggering Host Arginase Activity 
PLoS Pathogens  2013;9(10):e1003731.
Background
In order to promote infection, the blood-borne parasite Trypanosoma brucei releases factors that upregulate arginase expression and activity in myeloid cells.
Methodology/Principal findings
By screening a cDNA library of T. brucei with an antibody neutralizing the arginase-inducing activity of parasite released factors, we identified a Kinesin Heavy Chain isoform, termed TbKHC1, as responsible for this effect. Following interaction with mouse myeloid cells, natural or recombinant TbKHC1 triggered SIGN-R1 receptor-dependent induction of IL-10 production, resulting in arginase-1 activation concomitant with reduction of nitric oxide (NO) synthase activity. This TbKHC1 activity was IL-4Rα-independent and did not mirror M2 activation of myeloid cells. As compared to wild-type T. brucei, infection by TbKHC1 KO parasites was characterized by strongly reduced parasitaemia and prolonged host survival time. By treating infected mice with ornithine or with NO synthase inhibitor, we observed that during the first wave of parasitaemia the parasite growth-promoting effect of TbKHC1-mediated arginase activation resulted more from increased polyamine production than from reduction of NO synthesis. In late stage infection, TbKHC1-mediated reduction of NO synthesis appeared to contribute to liver damage linked to shortening of host survival time.
Conclusion
A kinesin heavy chain released by T. brucei induces IL-10 and arginase-1 through SIGN-R1 signaling in myeloid cells, which promotes early trypanosome growth and favors parasite settlement in the host. Moreover, in the late stage of infection, the inhibition of NO synthesis by TbKHC1 contributes to liver pathogenicity.
Author Summary
From the first invasive step onwards, African trypanosomes can efficiently undermine the protective immune response of their mammalian host to favor their survival within the host and successful transmission by its vector, the tsetse fly. Identifying the parasite factors affecting the protective immune response is thus critical to detail the immune evasion mechanisms of these organisms. We report here that during acute infection, a Trypanosoma brucei protein named Kinesin Heavy Chain 1 (TbKHC1) sustains the development of the first (most prominent) peak of parasitaemia in the blood and its control by the host. Mechanistically, TbKHC1 was found to interact with the SIGN-R1 molecule at the surface of immune cells. Hereby, TbKHC1 modulates arginine/NO metabolism in immune cells towards the production by the host of nutrients (polyamines) required for parasite growth via an IL-10-dependent induction of arginase 1 and down-regulation of iNOS activities. Consequently, IL-10/arginase 1 producing immune cells are impaired in their capacity to destroy the parasite, favouring parasite settlement. Moreover, in the late stage of infection, the inhibition of NO synthesis by TbKHC1 increases liver pathogenicity that contributes to compromised host survival. Thus, targeting TbKHC1 may benefit the host protective immunity against T. brucei parasite.
doi:10.1371/journal.ppat.1003731
PMCID: PMC3814429  PMID: 24204274
7.  Correction: Affinity Is an Important Determinant of the Anti-Trypanosome Activity of Nanobodies 
PLoS Neglected Tropical Diseases  2012;6(12):10.1371/annotation/95386e26-78b2-44b1-af86-0028ba783156.
doi:10.1371/annotation/95386e26-78b2-44b1-af86-0028ba783156
PMCID: PMC3553202
8.  Affinity Is an Important Determinant of the Anti-Trypanosome Activity of Nanobodies 
Background
The discovery of Nanobodies (Nbs) with a direct toxic activity against African trypanosomes is a recent advancement towards a new strategy against these extracellular parasites. The anti-trypanosomal activity relies on perturbing the highly active recycling of the Variant-specific Surface Glycoprotein (VSG) that occurs in the parasite's flagellar pocket.
Methodology/Principal Findings
Here we expand the existing panel of Nbs with anti-Trypanosoma brucei potential and identify four categories based on their epitope specificity. We modified the binding properties of previously identified Nanobodies Nb_An05 and Nb_An33 by site-directed mutagenesis in the paratope and found this to strongly affect trypanotoxicity despite retention of antigen-targeting properties. Affinity measurements for all identified anti-trypanosomal Nbs reveal a strong correlation between trypanotoxicity and affinity (KD), suggesting that it is a crucial determinant for this activity. Half maximal effective (50%) affinity of 57 nM was calculated from the non-linear dose-response curves. In line with these observations, Nb humanizing mutations only preserved the trypanotoxic activity if the KD remained unaffected.
Conclusions/Significance
This study reveals that the binding properties of Nanobodies need to be compatible with achieving an occupancy of >95% saturation of the parasite surface VSG in order to exert an anti-trypanosomal activity. As such, Nb-based approaches directed against the VSG target would require binding to an accessible, conserved epitope with high affinity.
Author Summary
Nanobodies, antigen binding fragments derived from a non-conventional class of antibodies in camelids, were previously shown to exert a direct activity against African trypanosomes without the need of a toxin. Their mode-of-action relies on interference with the highly active recycling of the Variant-specific Surface Glycoprotein (VSG) that occurs in the flagellar pocket of the parasite. By expanding the panel of anti-trypanosomal Nanobodies and by modification of their binding properties through site-directed mutagenesis, we have been able to show a strong correlation between their trypanotoxic activity and affinity for the cognate antigen. From these studies it was calculated that the parasite surface saturation needs to exceed 95% in order to achieve this anti-trypanosomal effect of Nanobodies, which can be considered as a critical cut-off value for future Nanobody-based or other small molecule drug approaches against the VSG target.
doi:10.1371/journal.pntd.0001902
PMCID: PMC3499403  PMID: 23166849
9.  Tsetse Salivary Gland Proteins 1 and 2 Are High Affinity Nucleic Acid Binding Proteins with Residual Nuclease Activity 
PLoS ONE  2012;7(10):e47233.
Analysis of the tsetse fly salivary gland EST database revealed the presence of a highly enriched cluster of putative endonuclease genes, including tsal1 and tsal2. Tsal proteins are the major components of tsetse fly (G. morsitans morsitans) saliva where they are present as monomers as well as high molecular weight complexes with other saliva proteins. We demonstrate that the recombinant tsetse salivary gland proteins 1&2 (Tsal1&2) display DNA/RNA non-specific, high affinity nucleic acid binding with KD values in the low nanomolar range and a non-exclusive preference for duplex. These Tsal proteins exert only a residual nuclease activity with a preference for dsDNA in a broad pH range. Knockdown of Tsal expression by in vivo RNA interference in the tsetse fly revealed a partially impaired blood digestion phenotype as evidenced by higher gut nucleic acid, hematin and protein contents.
doi:10.1371/journal.pone.0047233
PMCID: PMC3479092  PMID: 23110062
10.  Instruction of myeloid cells by the tumor microenvironment 
Oncoimmunology  2012;1(7):1135-1145.
The versatility and plasticity of myeloid cell polarization/differentiation has turned out to be crucial in health and disease, and has become the subject of intense investigation during the last years. On one hand, myeloid cells provide a critical contribution to tissue homeostasis and repair. On the other hand, myeloid cells not only play an important role as first line defense against pathogens but also they are involved in a broad array of inflammation-related diseases such as cancer. Recent studies show that macrophages can exist in different activation states within the same tumor, underlining their plasticity and heterogeneity. In this review, we will discuss recent evidence on how the tumor microenvironment, as it evolves, shapes the recruitment, function, polarization and differentiation of the myeloid cell compartment, leading to the selection of myeloid cells with immunosuppressive and angiogenic functions that facilitate tumor progression and dissemination.
doi:10.4161/onci.21566
PMCID: PMC3494626  PMID: 23170260
ER stress; exosomes; hypoxia; myeloid cell plasticity; myeloid cells; myeloid-derived suppressor cells; tumor-associated dendritic cells; tumor-associated macrophages; tumor-associated neutrophils
11.  Monocytes Contribute to Differential Immune Pressure on R5 versus X4 HIV through the Adipocytokine Visfatin/NAMPT 
PLoS ONE  2012;7(4):e35074.
Background
The immune system exerts a diversifying selection pressure on HIV through cellular, humoral and innate mechanisms. This pressure drives viral evolution throughout infection. A better understanding of the natural immune pressure on the virus during infection is warranted, given the clinical interest in eliciting and sustaining an immune response to HIV which can help to control the infection. We undertook to evaluate the potential of the novel HIV-induced, monocyte-derived factor visfatin to modulate viral infection, as part of the innate immune pressure on viral populations.
Results
We show that visfatin is capable of selectively inhibiting infection by R5 HIV strains in macrophages and resting PBMC in vitro, while at the same time remaining indifferent to or even favouring infection by X4 strains. Furthermore, visfatin exerts a direct effect on the relative fitness of R5 versus X4 infections in a viral competition setup. Direct interaction of visfatin with the CCR5 receptor is proposed as a putative mechanism for this differential effect. Possible in vivo relevance of visfatin induction is illustrated by its association with the dominance of CXCR4-using HIV in the plasma.
Conclusions
As an innate factor produced by monocytes, visfatin is capable of inhibiting infections by R5 but not X4 strains, reflecting a potential selective pressure against R5 viruses.
doi:10.1371/journal.pone.0035074
PMCID: PMC3320877  PMID: 22493731
12.  Expression and extracellular release of a functional anti-trypanosome Nanobody® in Sodalis glossinidius, a bacterial symbiont of the tsetse fly 
Background
Sodalis glossinidius, a gram-negative bacterial endosymbiont of the tsetse fly, has been proposed as a potential in vivo drug delivery vehicle to control trypanosome parasite development in the fly, an approach known as paratransgenesis. Despite this interest of S. glossinidius as a paratransgenic platform organism in tsetse flies, few potential effector molecules have been identified so far and to date none of these molecules have been successfully expressed in this bacterium.
Results
In this study, S. glossinidius was transformed to express a single domain antibody, (Nanobody®) Nb_An33, that efficiently targets conserved cryptic epitopes of the variant surface glycoprotein (VSG) of the parasite Trypanosoma brucei. Next, we analyzed the capability of two predicted secretion signals to direct the extracellular delivery of significant levels of active Nb_An33. We show that the pelB leader peptide was successful in directing the export of fully functional Nb_An33 to the periplasm of S. glossinidius resulting in significant levels of extracellular release. Finally, S. glossinidius expressing pelBNb_An33 exhibited no significant reduction in terms of fitness, determined by in vitro growth kinetics, compared to the wild-type strain.
Conclusions
These data are the first demonstration of the expression and extracellular release of functional trypanosome-interfering Nanobodies® in S. glossinidius. Furthermore, Sodalis strains that efficiently released the effector protein were not affected in their growth, suggesting that they may be competitive with endogenous microbiota in the midgut environment of the tsetse fly. Collectively, these data reinforce the notion for the potential of S. glossinidius to be developed into a paratransgenic platform organism.
doi:10.1186/1475-2859-11-23
PMCID: PMC3311065  PMID: 22335892
Sodalis glossinidius; Symbiont; Glossina; Paratransgenesis; Expression; Nanobody; Functional
13.  Do-it-yourself: construction of a custom cDNA macroarray platform with high sensitivity and linear range 
BMC Biotechnology  2011;11:97.
Background
Research involving gene expression profiling and clinical applications, such as diagnostics and prognostics, often require a DNA array platform that is flexibly customisable and cost-effective, but at the same time is highly sensitive and capable of accurately and reproducibly quantifying the transcriptional expression of a vast number of genes over the whole transcriptome dynamic range using low amounts of RNA sample. Hereto, a set of easy-to-implement practical optimisations to the design of cDNA-based nylon macroarrays as well as sample 33P-labeling, hybridisation protocols and phosphor screen image processing were analysed for macroarray performance.
Results
The here proposed custom macroarray platform had an absolute sensitivity as low as 50,000 transcripts and a linear range of over 5 log-orders. Its quality of identifying differentially expressed genes was at least comparable to commercially available microchips. Interestingly, the quantitative accuracy was found to correlate significantly with corresponding reversed transcriptase - quantitative PCR values, the gold standard gene expression measure (Pearson's correlation test p < 0.0001). Furthermore, the assay has low cost and input RNA requirements (0.5 μg and less) and has a sound reproducibility.
Conclusions
Results presented here, demonstrate for the first time that self-made cDNA-based nylon macroarrays can produce highly reliable gene expression data with high sensitivity and covering the entire mammalian dynamic range of mRNA abundances. Starting off from minimal amounts of unamplified total RNA per sample, a reasonable amount of samples can be assayed simultaneously for the quantitative expression of hundreds of genes in an easily customisable and cost-effective manner.
doi:10.1186/1472-6750-11-97
PMCID: PMC3217856  PMID: 22026914
14.  High Affinity Nanobodies against the Trypanosome brucei VSG Are Potent Trypanolytic Agents that Block Endocytosis 
PLoS Pathogens  2011;7(6):e1002072.
The African trypanosome Trypanosoma brucei, which persists within the bloodstream of the mammalian host, has evolved potent mechanisms for immune evasion. Specifically, antigenic variation of the variant-specific surface glycoprotein (VSG) and a highly active endocytosis and recycling of the surface coat efficiently delay killing mediated by anti-VSG antibodies. Consequently, conventional VSG-specific intact immunoglobulins are non-trypanocidal in the absence of complement. In sharp contrast, monovalent antigen-binding fragments, including 15 kDa nanobodies (Nb) derived from camelid heavy-chain antibodies (HCAbs) recognizing variant-specific VSG epitopes, efficiently lyse trypanosomes both in vitro and in vivo. This Nb-mediated lysis is preceded by very rapid immobilisation of the parasites, massive enlargement of the flagellar pocket and major blockade of endocytosis. This is accompanied by severe metabolic perturbations reflected by reduced intracellular ATP-levels and loss of mitochondrial membrane potential, culminating in cell death. Modification of anti-VSG Nbs through site-directed mutagenesis and by reconstitution into HCAbs, combined with unveiling of trypanolytic activity from intact immunoglobulins by papain proteolysis, demonstrates that the trypanolytic activity of Nbs and Fabs requires low molecular weight, monovalency and high affinity. We propose that the generation of low molecular weight VSG-specific trypanolytic nanobodies that impede endocytosis offers a new opportunity for developing novel trypanosomiasis therapeutics. In addition, these data suggest that the antigen-binding domain of an anti-microbial antibody harbours biological functionality that is latent in the intact immunoglobulin and is revealed only upon release of the antigen-binding fragment.
Author Summary
Haemoparasites, such as African trypanosomes, have developed potent immune evasion mechanisms to avoid antibody-mediated elimination. Consequently, trypanosome surface antigen-specific immunoglobulins in the absence of complement are non-trypanocidal. In contrast, certain monovalent nanobodies (Nb), monomeric antigen-binding domains derived from camelid Heavy-Chain Antibodies (HCAb) and which have a much lower molecular weight (15 kDa) than classical antibodies (150 kDa), efficiently lyse trypanosomes both in vitro and in vivo. This is surprising as classically immunoglobulin effector functions are mediated via the Fc-domain, which is absent from the Nb. We demonstrate that the Nb-mediated trypanolysis depends on the low molecular weight, monovalency and high affinity and is associated with loss of motility, a major block to endocytosis, energy depletion and cell death. Overall, targeting the parasite surface with low molecular weight, high affinity Nbs is sufficient to exert a direct therapeutic action. Therefore, the exploitation of Nbs against African trypanosomiasis represents a novel therapeutic strategy. Furthermore, demonstration that a high affinity antigen-binding Nb or Fab fragment lacking an effector domain (i.e., Fc-domain or an attached toxin) can exert a direct biological function, suggests that intact antibodies likely harbour latent functionality which only become revealed upon removal of the Fc-domain.
doi:10.1371/journal.ppat.1002072
PMCID: PMC3116811  PMID: 21698216
15.  Tip-DC Development during Parasitic Infection Is Regulated by IL-10 and Requires CCL2/CCR2, IFN-γ and MyD88 Signaling 
PLoS Pathogens  2010;6(8):e1001045.
The development of classically activated monocytic cells (M1) is a prerequisite for effective elimination of parasites, including African trypanosomes. However, persistent activation of M1 that produce pathogenic molecules such as TNF and NO contributes to the development of trypanosome infection-associated tissue injury including liver cell necrosis in experimental mouse models. Aiming to identify mechanisms involved in regulation of M1 activity, we have recently documented that during Trypanosoma brucei infection, CD11b+Ly6C+CD11c+ TNF and iNOS producing DCs (Tip-DCs) represent the major pathogenic M1 liver subpopulation. By using gene expression analyses, KO mice and cytokine neutralizing antibodies, we show here that the conversion of CD11b+Ly6C+ monocytic cells to pathogenic Tip-DCs in the liver of T. brucei infected mice consists of a three-step process including (i) a CCR2-dependent but CCR5- and Mif-independent step crucial for emigration of CD11b+Ly6C+ monocytic cells from the bone marrow but dispensable for their blood to liver migration; (ii) a differentiation step of liver CD11b+Ly6C+ monocytic cells to immature inflammatory DCs (CD11c+ but CD80/CD86/MHC-IIlow) which is IFN-γ and MyD88 signaling independent; and (iii) a maturation step of inflammatory DCs to functional (CD80/CD86/MHC-IIhigh) TNF and NO producing Tip-DCs which is IFN-γ and MyD88 signaling dependent. Moreover, IL-10 could limit CCR2-mediated egression of CD11b+Ly6C+ monocytic cells from the bone marrow by limiting Ccl2 expression by liver monocytic cells, as well as their differentiation and maturation to Tip-DCs in the liver, showing that IL-10 works at multiple levels to dampen Tip-DC mediated pathogenicity during T. brucei infection. A wide spectrum of liver diseases associates with alteration of monocyte recruitment, phenotype or function, which could be modulated by IL-10. Therefore, investigating the contribution of recruited monocytes to African trypanosome induced liver injury could potentially identify new targets to treat hepatic inflammation in general, and during parasite infection in particular.
Author Summary
Most infections are associated with host inflammatory responses that can result in multiple organ failure and death. It is therefore essential to understand the mechanisms balancing host immune response and tissue damage. Mouse models of African trypanosome infection represent valuable tools to study the mechanisms contributing to the inflammatory (pathogenic) or anti-inflammatory (anti-pathogenic) immune response. We recently identified TNF and NO producing DCs (Tip-DCs) as major contributors to liver pathogenicity in Trypanosoma brucei infected mice. Herein, the role of different chemokine and cytokines in the generation of Tip-DCs was investigated. Tip-DCs originated from bone marrow derived monocytes that egressed to the blood in a CCR2 chemokine receptor dependent manner. Then, monocytes extravasated to inflamed liver where IFN-γ and MyD88 signaling promoted their maturation to Tip-DCs. Both the egression of monocytes from bone marrow and their IFN-γ/MyD88 dependent maturation to Tip-DCs was counteracted by IL-10, hereby reducing liver pathogenicity. Liver injury, affecting millions of persons worldwide with often lethal consequences, frequently results from uncontrolled activation of recruited monocyte-derived cells that can be modulated by IL-10. Thus, the mechanisms regulating liver immunopathogenicity during parasitic infection identified herein could lead to new therapeutic policies in the field of hepatic inflammation.
doi:10.1371/journal.ppat.1001045
PMCID: PMC2920868  PMID: 20714353
16.  Transcriptome analysis of monocyte-HIV interactions 
Retrovirology  2010;7:53.
Background
During HIV infection and/or antiretroviral therapy (ART), monocytes and macrophages exhibit a wide range of dysfunctions which contribute significantly to HIV pathogenesis and therapy-associated complications. Nevertheless, the molecular components which contribute to these dysfunctions remain elusive. We therefore applied a parallel approach of genome-wide microarray analysis and focused gene expression profiling on monocytes from patients in different stages of HIV infection and/or ART to further characterise these dysfunctions.
Results
Processes involved in apoptosis, cell cycle, lipid metabolism, proteasome function, protein trafficking and transcriptional regulation were identified as areas of monocyte dysfunction during HIV infection. Individual genes potentially contributing to these monocyte dysfunctions included several novel factors. One of these is the adipocytokine NAMPT/visfatin, which we show to be capable of inhibiting HIV at an early step in its life cycle. Roughly half of all genes identified were restored to control levels under ART, while the others represented a persistent dysregulation. Additionally, several candidate biomarkers (in particular CCL1 and CYP2C19) for the development of the abacavir hypersensitivity reaction were suggested.
Conclusions
Previously described areas of monocyte dysfunction during HIV infection were confirmed, and novel themes were identified. Furthermore, individual genes associated with these dysfunctions and with ART-associated disorders were pinpointed. These genes form a useful basis for further functional studies concerning the contribution of monocytes/macrophages to HIV pathogenesis. One such gene, NAMPT/visfatin, represents a possible novel restriction factor for HIV.
doi:10.1186/1742-4690-7-53
PMCID: PMC2900222  PMID: 20546557
17.  Trypanosoma brucei Modifies the Tsetse Salivary Composition, Altering the Fly Feeding Behavior That Favors Parasite Transmission 
PLoS Pathogens  2010;6(6):e1000926.
Tsetse flies are the notorious transmitters of African trypanosomiasis, a disease caused by the Trypanosoma parasite that affects humans and livestock on the African continent. Metacyclic infection rates in natural tsetse populations with Trypanosoma brucei, including the two human-pathogenic subspecies, are very low, even in epidemic situations. Therefore, the infected fly/host contact frequency is a key determinant of the transmission dynamics. As an obligate blood feeder, tsetse flies rely on their complex salivary potion to inhibit host haemostatic reactions ensuring an efficient feeding. The results of this experimental study suggest that the parasite might promote its transmission through manipulation of the tsetse feeding behavior by modifying the saliva composition. Indeed, salivary gland Trypanosoma brucei-infected flies display a significantly prolonged feeding time, thereby enhancing the likelihood of infecting multiple hosts during the process of a single blood meal cycle. Comparison of the two major anti-haemostatic activities i.e. anti-platelet aggregation and anti-coagulation activity in these flies versus non-infected tsetse flies demonstrates a significant suppression of these activities as a result of the trypanosome-infection status. This effect was mainly related to the parasite-induced reduction in salivary gland gene transcription, resulting in a strong decrease in protein content and related biological activities. Additionally, the anti-thrombin activity and inhibition of thrombin-induced coagulation was even more severely hampered as a result of the trypanosome infection. Indeed, while naive tsetse saliva strongly inhibited human thrombin activity and thrombin-induced blood coagulation, saliva from T. brucei-infected flies showed a significantly enhanced thrombinase activity resulting in a far less potent anti-coagulation activity. These data clearly provide evidence for a trypanosome-mediated modification of the tsetse salivary composition that results in a drastically reduced anti-haemostatic potential and a hampered feeding performance which could lead to an increase of the vector/host contact and parasite transmission in field conditions.
Author Summary
Human African Trypanosomiasis, or sleeping sickness, is a devastating parasitic disease that is fatal if left untreated. Infections are acquired via the bite of an obligate blood feeding fly, the tsetse fly, that is exclusively present on the African continent. In this insect vector, the trypanosome parasite has a complex development ending in the salivary glands. In this experimental study we demonstrate that the Trypanosoma brucei parasites change the composition of the tsetse fly saliva making it less efficient to keep the blood fluid at the biting site in the mammalian host. This results in a more difficult blood feeding process and favors the fly biting activity on multiple hosts, thereby promoting the survival and circulation of the parasite within the natural host population. These findings give us a better understanding of how trypanosome infections in the human population can be maintained given the fact that only very few tsetse flies are actually carrying the parasite.
doi:10.1371/journal.ppat.1000926
PMCID: PMC2880569  PMID: 20532213
18.  Identification of a Tsetse Fly Salivary Protein with Dual Inhibitory Action on Human Platelet Aggregation 
PLoS ONE  2010;5(3):e9671.
Background
Tsetse flies (Glossina sp.), the African trypanosome vectors, rely on anti-hemostatic compounds for efficient blood feeding. Despite their medical importance, very few salivary proteins have been characterized and functionally annotated.
Methodology/Principal Findings
Here we report on the functional characterisation of a 5′nucleotidase-related (5′Nuc) saliva protein of the tsetse fly Glossina morsitans morsitans. This protein is encoded by a 1668 bp cDNA corresponding at the genomic level with a single-copy 4 kb gene that is exclusively transcribed in the tsetse salivary gland tissue. The encoded 5′Nuc protein is a soluble 65 kDa glycosylated compound of tsetse saliva with a dual anti-hemostatic action that relies on its combined apyrase activity and fibrinogen receptor (GPIIb/IIIa) antagonistic properties. Experimental evidence is based on the biochemical and functional characterization of recombinant protein and on the successful silencing of the 5′nuc translation in the salivary gland by RNA interference (RNAi). Refolding of a 5′Nuc/SUMO-fusion protein yielded an active apyrase enzyme with Km and Vmax values of 43±4 µM and 684±49 nmol Pi/min×mg for ATPase and 49±11 µM and 177±37 nmol Pi/min×mg for the ADPase activity. In addition, recombinant 5′Nuc was found to bind to GPIIb/IIIa with an apparent KD of 92±25 nM. Consistent with these features, 5′Nuc potently inhibited ADP-induced thrombocyte aggregation and even caused disaggregation of ADP-triggered human platelets. The importance of 5′Nuc for the tsetse fly hematophagy was further illustrated by specific RNAi that reduced the anti-thrombotic activities in saliva by approximately 50% resulting in a disturbed blood feeding process.
Conclusions/Significance
These data show that this 5′nucleotidase-related apyrase exhibits GPIIb/IIIa antagonistic properties and represents a key thromboregulatory compound of tsetse fly saliva.
doi:10.1371/journal.pone.0009671
PMCID: PMC2843633  PMID: 20351782
20.  The Role of B-cells and IgM Antibodies in Parasitemia, Anemia, and VSG Switching in Trypanosoma brucei–Infected Mice 
PLoS Pathogens  2008;4(8):e1000122.
African trypanosomes are extracellular parasitic protozoa, predominantly transmitted by the bite of the haematophagic tsetse fly. The main mechanism considered to mediate parasitemia control in a mammalian host is the continuous interaction between antibodies and the parasite surface, covered by variant-specific surface glycoproteins. Early experimental studies have shown that B-cell responses can be strongly protective but are limited by their VSG-specificity. We have used B-cell (µMT) and IgM-deficient (IgM−/−) mice to investigate the role of B-cells and IgM antibodies in parasitemia control and the in vivo induction of trypanosomiasis-associated anemia. These infection studies revealed that that the initial setting of peak levels of parasitemia in Trypanosoma brucei–infected µMT and IgM−/− mice occurred independent of the presence of B-cells. However, B-cells helped to periodically reduce circulating parasites levels and were required for long term survival, while IgM antibodies played only a limited role in this process. Infection-associated anemia, hypothesized to be mediated by B-cell responses, was induced during infection in µMT mice as well as in IgM−/− mice, and as such occurred independently from the infection-induced host antibody response. Antigenic variation, the main immune evasion mechanism of African trypanosomes, occurred independently from host antibody responses against the parasite's ever-changing antigenic glycoprotein coat. Collectively, these results demonstrated that in murine experimental T. brucei trypanosomiasis, B-cells were crucial for periodic peak parasitemia clearance, whereas parasite-induced IgM antibodies played only a limited role in the outcome of the infection.
Author Summary
African trypanosomiasis is a disease caused by different species of extracellular flagellated protozoan trypanosome parasites. Trypanosomes have developed a mechanism of regular antigenic variation of their variant-specific surface glycoprotein (VSG) coat which allows chronic infection. Replacement of this coat occurs at rapid regular time intervals, allowing the parasite to escape from an effective host antibody responses. So far, primary T-cell independent antibody responses have been described to constitute the main host defense mechanism, relying largely on IgM antibody induction. Using genetically engineered B lymphocyte- or IgM-deficient mouse strains, we show that lack of B-cells or IgM did not prevent infection-associated anemia. More importantly, we show that in the absence of IgM, parasitemia was controlled almost as well as in wild-type mice, with only slightly increased mortality. In addition, we show in vivo that antigenic variation is not affected by the lack of IgM.
doi:10.1371/journal.ppat.1000122
PMCID: PMC2483930  PMID: 18688274
21.  Macrophages, PPARs, and Cancer 
PPAR Research  2008;2008:169414.
Mononuclear phagocytes often function as control switches of the immune system, securing the balance between pro- and anti-inflammatory reactions. For this purpose and depending on the activating stimuli, these cells can develop into different subsets: proinflammatory classically activated (M1) or anti-inflammatory alternatively activated (M2) macrophages. The expression of the nuclear peroxisome proliferator-activated receptors (PPARs) is regulated by M1- or M2-inducing stimuli, and these receptors are generally considered to counteract inflammatory M1 macrophages, while actively promoting M2 activation. This is of importance in a tumor context, where M1 are important initiators of inflammation-driven cancers. As a consequence, PPAR agonists are potentially usefull for inhibiting the early phases of tumorigenesis through their antagonistic effect on M1. In more established tumors, the macrophage phenotype is more diverse, making it more difficult to predict the outcome of PPAR agonism. Overall, in our view current knowledge provides a sound basis for the clinical evaluation of PPAR ligands as chemopreventive agents in chronic inflammation-associated cancer development, while cautioning against the unthoughtful application of these agents as cancer therapeutics.
doi:10.1155/2008/169414
PMCID: PMC2443396  PMID: 18615187
22.  Tsetse Fly Saliva Accelerates the Onset of Trypanosoma brucei Infection in a Mouse Model Associated with a Reduced Host Inflammatory Response▿  
Infection and Immunity  2006;74(11):6324-6330.
Tsetse flies (Glossina sp.) are the vectors that transmit African trypanosomes, protozoan parasites that cause human sleeping sickness and veterinary infections in the African continent. These blood-feeding dipteran insects deposit saliva at the feeding site that enables the blood-feeding process. Here we demonstrate that tsetse fly saliva also accelerates the onset of a Trypanosoma brucei infection. This effect was associated with a reduced inflammatory reaction at the site of infection initiation (reflected by a decrease of interleukin-6 [IL-6] and IL-12 mRNA) as well as lower serum concentrations of the trypanocidal cytokine tumor necrosis factor. Variant-specific surface glycoprotein-specific antibody isotypes immunoglobulin M (IgM) and IgG2a, implicated in trypanosome clearance, were not suppressed. We propose that tsetse fly saliva accelerates the onset of trypanosome infection by inhibiting local and systemic inflammatory responses involved in parasite control.
doi:10.1128/IAI.01046-06
PMCID: PMC1695494  PMID: 16954393
23.  Ablation of NK Cell Function During Tumor Growth Favors Type 2-Associated Macrophages, Leading to Suppressed CTL Generation 
Several reports describe regulatory interactions between NK cells and CTLs. We addressed the issue of NK participation in the early anti-tumor defense by inoculating α-ASGM-1 treated mice with BW-Sp3 T lymphoma. Rejection of BW-Sp3 depends on strong CTL responses. Our results demonstrated that (i) NK cells are a prerequisite for efficient CTL generation and (ii) the absence of NK cells favors the outgrowth of alternatively activated macrophages that can suppress CTL restimulation. In vitro studies demonstrate that in splenic cultures from NK-deficient, tumor-bearing mice, the presence of alternatively activated macrophages correlates with a lack of Type 1 cytokines, while the production of Type 2 cytokines is promoted. Provision of the Type 1 cytokine, IFN-γ can boost overall CTL activity but does not revert the dominance of arginase producing adherent cells in the NK-deficient CTL cultures. The role of NK effector functions in the efficient switch of the immune system towards Type 1 activation was evaluated in cytotoxicity assays. The results indicate that the accessory function of NK can depend at least partially on their ability to preferentially engage arginase-producing cells, suggesting that NK/macrophage lytic interactions might be involved in the switch from Type 2 to Type 1-dependent immune responses.
doi:10.1080/10446670310001626580
PMCID: PMC2485417  PMID: 14768937
24.  Amastigote Load and Cell Surface Phenotype of Infected Cells from Lesions and Lymph Nodes of Susceptible and Resistant Mice Infected with Leishmania major  
Infection and Immunity  2003;71(5):2704-2715.
Cells of the dendritic cell (DC) lineage, by their unique ability to stimulate naive T cells, may be of crucial importance in the development of protective immune responses to Leishmania parasites. The aim of this study was to compare the impact of L. major infection on DCs in BALB/c (susceptible, developing Th2 responses), C57BL/6 (resistant, developing Th1 responses), and tumor necrosis factor (TNF)−/− C57BL/6 mice (susceptible, developing delayed and reduced Th1 responses). We analyzed by immunohistochemistry the phenotype of infected cells in vivo. Granulocytes (GR1+) and macrophages (CD11b+) appear as the mainly infected cells in primary lesions. In contrast, cells expressing CD11c, a DC specific marker, are the most frequently infected cells in draining lymph nodes of all mice tested. These infected CD11c+ cells harbored a particular morphology and cell surface phenotype in infected C57BL/6 and BALB/c mice. CD11c+ infected cells from C57BL/6 and TNF−/− C57BL/6 mice displayed a weak parasitic load and a dendritic morphology and frequently expressed CD11b or F4/80 myeloid differentiation markers. In contrast, some CD11c+ infected cells from BALB/c mice were multinucleated giant cells. Giant cells presented a dramatic accumulation of parasites and differentiation markers were not detectable at their surface. In all mice, lymph node CD11c+ infected cells expressed a low major histocompatibility complex II level and no detectable CD86 expression. Our results suggest that infected CD11c+ DC-like cells might constitute a reservoir of parasites in lymph nodes.
doi:10.1128/IAI.71.5.2704-2715.2003
PMCID: PMC153240  PMID: 12704145
25.  FIZZ1 and Ym as Tools to Discriminate between Differentially Activated Macrophages 
Developmental Immunology  2002;9(3):151-159.
Although it is well-established that macrophages can occur in distinct activation states, the molecular characteristics of differentially activated macrophages, and particularly those of alternatively activated macrophages (aaMφ), are still poorly unraveled. Recently, we demonstrated that the expression of FIZZ1 and Ym is induced in aaMφ as compared with classically activated macrophages (caMφ), elicited in vitro or developed in vivo during infection with Trypanosoma brucei brucei. In the present study, we analyzed the expression of FIZZ1 and Ym in caMφ and aaMφ elicited during Trypanosoma congolense infection and show that the use of FIZZ1 and Ym for the identification of aaMφ is not limited to T. b. brucei infection and is independent of the organ sources from which macrophages are obtained. We also demonstrate that FIZZ1 can be used to discriminate between different populations of aaMφ. Furthermore, we studied the effects of various stimuli, and combinations thereof, on the expression of FIZZ1 and Ym in macrophages from different mouse strains and demonstrate that regulation of the expression of FIZZ1 and Ym in macrophages is not dependent on the mouse strain. Finally, we show that these genes can be used to monitor the macrophage activation status without the need to obtain pure macrophage populations.
doi:10.1080/1044667031000137629
PMCID: PMC2276098  PMID: 12892049

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