Publications by authors named "Benoit Stijlemans"

67 Publications

Macrophage miR-210 induction and metabolic reprogramming in response to pathogen interaction boost life-threatening inflammation.

Sci Adv 2021 May 7;7(19). Epub 2021 May 7.

Laboratory of Tumor Inflammation and Angiogenesis, CCB, VIB, Leuven, Belgium.

Unbalanced immune responses to pathogens can be life-threatening although the underlying regulatory mechanisms remain unknown. Here, we show a hypoxia-inducible factor 1α-dependent microRNA (miR)-210 up-regulation in monocytes and macrophages upon pathogen interaction. MiR-210 knockout in the hematopoietic lineage or in monocytes/macrophages mitigated the symptoms of endotoxemia, bacteremia, sepsis, and parasitosis, limiting the cytokine storm, organ damage/dysfunction, pathogen spreading, and lethality. Similarly, pharmacologic miR-210 inhibition improved the survival of septic mice. Mechanistically, miR-210 induction in activated macrophages supported a switch toward a proinflammatory state by lessening mitochondria respiration in favor of glycolysis, partly achieved by downmodulating the iron-sulfur cluster assembly enzyme ISCU. In humans, augmented miR-210 levels in circulating monocytes correlated with the incidence of sepsis, while serum levels of monocyte/macrophage-derived miR-210 were associated with sepsis mortality. Together, our data identify miR-210 as a fine-tuning regulator of macrophage metabolism and inflammatory responses, suggesting miR-210-based therapeutic and diagnostic strategies.
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http://dx.doi.org/10.1126/sciadv.abf0466DOI Listing
May 2021

Immunogenicity Risk Profile of Nanobodies.

Front Immunol 2021 9;12:632687. Epub 2021 Mar 9.

In vivo Cellular and Molecular Imaging Laboratory (ICMI), Vrije Universiteit Brussel, Brussels, Belgium.

Nanobodies (Nbs), the variable domains of camelid heavy chain-only antibodies, are a promising class of therapeutics or imaging reagents entering the clinic. They possess unique characteristics, including a minimal size, providing fast pharmacokinetics, high-target specificity, and an affinity in the (sub-)nanomolar range in conjunction with an easy selection and production, which allow them to outperform conventional antibodies for imaging and radiotherapeutic purposes. As for all protein theranostics, extended safety assessment and investigation of their possible immunogenicity in particular are required. In this study, we assessed the immunogenicity risk profile of two Nbs that are in phase II clinical trials: a first Nb against Human Epidermal growth factor Receptor 2 (HER2) for PET imaging of breast cancer and a second Nb with specificity to the Macrophage Mannose Receptor (MMR) for PET imaging of tumor-associated macrophages. For the anti-HER2 Nb, we show that only one out of 20 patients had a low amount of pre-existing anti-drug antibodies (ADAs), which only marginally increased 3 months after administering the Nb, and without negative effects of safety and pharmacokinetics. Further immunogenicity assessment assays showed that both non-humanized Nbs were taken up by human dendritic cells but exhibited no or only a marginal capacity to activate dendritic cells or to induce T cell proliferation. From our data, we conclude that monomeric Nbs present a low immunogenicity risk profile, which is encouraging for their future development toward potential clinical applications.

One Sentence Summary: Nanobodies, the recombinant single domain affinity reagents derived from heavy chain-only antibodies in camelids, are proven to possess a low immunogenicity risk profile, which will facilitate a growing number of Nanobodies to enter the clinic for therapeutic or diagnostic applications.
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http://dx.doi.org/10.3389/fimmu.2021.632687DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7985456PMC
March 2021

A Critical Blimp-1-Dependent IL-10 Regulatory Pathway in T Cells Protects From a Lethal Pro-inflammatory Cytokine Storm During Acute Experimental Infection.

Front Immunol 2020 4;11:1085. Epub 2020 Jun 4.

Research Unit of Cellular and Molecular Immunology, Vrije Universiteit Brussel (VUB), Brussels, Belgium.

In many infectious diseases, the immune response operates as a double-edged sword. While required for protective immunity, infection-induced inflammation can be detrimental if it is not properly controlled, causing collateral body damage and potentially leading to death. It is in this context that the potent anti-inflammatory cytokine interleukin-10 (IL-10) is required to dampen the pro-inflammatory immune response that hallmarks trypanosomosis. Effective control of this infection requires not just the action of antibodies specific for the parasite's variable surface glycoprotein (VSG) coat antigens, but also a pro-inflammatory immune response mediated mainly by IFNγ, TNF, and NO. However, strict control of inflammation is mandatory, as IL-10-deficient mice succumb from an unrestrained cytokine storm within 10 days of a infection. The relevant cellular source of IL-10 and the associated molecular mechanisms implicated in its trypanosomosis associated production are poorly understood. Using an IL-10 reporter mouse strain (Vert-X), we demonstrate here that NK cells, CD8 T cells and CD4 T cells as well as B cells and plasma cells constitute potential cellular sources of IL-10 within the spleen and liver during acute infection. The IL-10 wave follows peak pro-inflammatory cytokine production, which accompanied the control of peak parasitemia. Similar results were observed following conventional experimental needle infection and physiological infections via -infected tsetse flies. Our results show that conditional T cell-specific ablation of the IL-10 regulating gene (encoding for the Blimp-1 transcription factor), leads to an uncontrolled trypanosome-induced pro-inflammatory syndrome like the one observed in infected IL-10-deficient mice. This result indicates that the biological role of IL-10-derived from non-T cells, including NK cells, is of minor importance when considering host survival. The cytokine IL-27 that is also considered to be an IL-10 regulator, did not affect IL-10 production during infection. Together, these data suggest that activates a Blimp-1-dependent IL-10 regulatory pathway in T cells that acts as a critical anti-inflammatory rheostat, mandatory for host survival during the acute phase of parasitemia.
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http://dx.doi.org/10.3389/fimmu.2020.01085DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7325990PMC
March 2021

A New Family of Diverse Skin Peptides from the Microhylid Frog Genus .

Molecules 2020 Feb 18;25(4). Epub 2020 Feb 18.

Amphibian Evolution Lab, Biology Department, Vrije Universiteit Brussel, Pleinlaan 2, 1050 Elsene, Belgium.

A wide range of frogs produce skin poisons composed of bioactive peptides for defence against pathogens, parasites and predators. While several frog families have been thoroughly screened for skin-secreted peptides, others, like the Microhylidae, have remained mostly unexplored. Previous studies of microhylids found no evidence of peptide secretion, suggesting that this defence adaptation was evolutionarily lost. We conducted transcriptome analyses of the skins of and two African microhylid species long suspected to be poisonous. Our analyses reveal 17 evolutionary related transcripts that diversified from to those of cytolytic peptides found in other frog families. The 19 peptides predicted to be processed from these transcripts, named phrynomantins, show a striking structural diversity that is distinct from any previously identified frog skin peptide. Functional analyses of five phrynomantins confirm the loss of a cytolytic function and the absence of insecticidal or proinflammatory activity, suggesting that they represent an evolutionary transition to a new, yet unknown function. Our study shows that peptides have been retained in the defence poison of at least one microhylid lineage and encourages research on similarly understudied taxa to further elucidate the diversity and evolution of skin defence molecules.
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http://dx.doi.org/10.3390/molecules25040912DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7070584PMC
February 2020

Development of a recombinase polymerase amplification lateral flow assay for the detection of active Trypanosoma evansi infections.

PLoS Negl Trop Dis 2020 02 18;14(2):e0008044. Epub 2020 Feb 18.

Research Unit for Cellular and Molecular Immunology (CMIM), Vrije Universiteit Brussel (VUB), Brussels, Belgium.

Background: Animal trypanosomosis caused by Trypanosoma evansi is known as "surra" and is a widespread neglected tropical disease affecting wild and domestic animals mainly in South America, the Middle East, North Africa and Asia. An essential necessity for T. evansi infection control is the availability of reliable and sensitive diagnostic tools. While DNA-based PCR detection techniques meet these criteria, most of them require well-trained and experienced users as well as a laboratory environment allowing correct protocol execution. As an alternative, we developed a recombinase polymerase amplification (RPA) test for Type A T. evansi. The technology uses an isothermal nucleic acid amplification approach that is simple, fast, cost-effective and is suitable for use in minimally equipped laboratories and even field settings.

Methodology/principle Findings: An RPA assay targeting the T. evansi RoTat1.2 VSG gene was designed for the DNA-based detection of T. evansi. Comparing post-amplification visualization by agarose gel electrophoresis and a lateral flow (LF) format reveals that the latter displays a higher sensitivity. The RPA-LF assay is specific for RoTat1.2-expressing strains of T. evansi as it does not detect the genomic DNA of other trypanosomatids. Finally, experimental mouse infection trials demonstrate that the T. evansi specific RPA-LF can be employed as a test-of-cure tool.

Conclusions/significance: Compared to other DNA-based parasite detection methods (such as PCR and LAMP), the T. evansi RPA-LF (TevRPA-LF) described in this paper is an interesting alternative because of its simple read-out (user-friendly), short execution time (15 minutes), experimental sensitivity of 100 fg purified genomic T. evansi DNA, and ability to be carried out at a moderate, constant temperature (39°C). Therefore, the TevRPA-LF is an interesting tool for the detection of active T. evansi infections.
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http://dx.doi.org/10.1371/journal.pntd.0008044DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7048301PMC
February 2020

Hepatocyte-derived IL-10 plays a crucial role in attenuating pathogenicity during the chronic phase of T. congolense infection.

PLoS Pathog 2020 02 3;16(2):e1008170. Epub 2020 Feb 3.

Lab of Cellular and Molecular Immunology, Vrije Universiteit Brussel (VUB), Brussels, Belgium.

Bovine African Trypanosomosis is an infectious parasitic disease affecting livestock productivity and thereby impairing the economic development of Sub-Saharan Africa. The most important trypanosome species implicated is T. congolense, causing anemia as most important pathological feature. Using murine models, it was shown that due to the parasite's efficient immune evasion mechanisms, including (i) antigenic variation of the variable surface glycoprotein (VSG) coat, (ii) induction of polyclonal B cell activation, (iii) loss of B cell memory and (iv) T cell mediated immunosuppression, disease prevention through vaccination has so far been impossible. In trypanotolerant models a strong, early pro-inflammatory immune response involving IFN-γ, TNF and NO, combined with a strong humoral anti-VSG response, ensures early parasitemia control. This potent protective inflammatory response is counterbalanced by the production of the anti-inflammatory cytokine IL-10, which in turn prevents early death of the host from uncontrolled hyper-inflammation-mediated immunopathologies. Though at this stage different hematopoietic cells, such as NK cells, T cells and B cells as well as myeloid cells (i.e. alternatively activated myeloid cells (M2) or Ly6c- monocytes), were found to produce IL-10, the contribution of non-hematopoietic cells as potential IL-10 source during experimental T. congolense infection has not been addressed. Here, we report for the first time that during the chronic stage of T. congolense infection non-hematopoietic cells constitute an important source of IL-10. Our data shows that hepatocyte-derived IL-10 is mandatory for host survival and is crucial for the control of trypanosomosis-induced inflammation and associated immunopathologies such as anemia, hepatosplenomegaly and excessive tissue injury.
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http://dx.doi.org/10.1371/journal.ppat.1008170DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7018099PMC
February 2020

Identification of Nanobodies against the Acute Myeloid Leukemia Marker CD33.

Int J Mol Sci 2020 Jan 2;21(1). Epub 2020 Jan 2.

Laboratory of Cellular and Molecular Immunology, Vrije Universiteit Brussel, 1050 Brussels, Belgium.

Nanobodies (Nbs) are the smallest antigen-binding, single domain fragments derived from heavy-chain-only antibodies from Camelidae. Among the several advantages over conventional monoclonal antibodies, their small size (12-15 kDa) allows them to extravasate rapidly, to show improved tissue penetration, and to clear rapidly from blood, which are important characteristics for cancer imaging and targeted radiotherapy. Herein, we identified Nbs against CD33, a marker for acute myeloid leukemia (AML). A total of 12 Nbs were generated against recombinant CD33 protein, out of which six bound natively CD33 protein, expressed on the surface of acute myeloid leukemia THP-1 cells. The equilibrium dissociation constants (K) of these six Nbs and CD33 range from 4 to 270 nM, and their melting temperature (Tm) varies between 52.67 and 67.80 °C. None of these Nbs showed leukemogenicity activity in vitro. The selected six candidates were radiolabeled with Tc, and their biodistribution was evaluated in THP-1-tumor-bearing mice. The imaging results demonstrated the fast tumor-targeting capacity of the Nbs in vivo. Among the anti-CD33 Nbs, Nb_7 showed the highest tumor uptake (2.53 ± 0.69 % injected activity per gram (IA/g), with low background signal, except in the kidneys and bladder. Overall, Nb_7 exhibits the best characteristics to be used as an anti-CD33 targeting vehicle for future diagnostic or therapeutic applications.
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http://dx.doi.org/10.3390/ijms21010310DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6981622PMC
January 2020

Dehydrin ERD14 activates glutathione transferase Phi9 in Arabidopsis thaliana under osmotic stress.

Biochim Biophys Acta Gen Subj 2020 03 20;1864(3):129506. Epub 2019 Dec 20.

VIB-VUB Center for Structural Biology (CSB), Vlaams Instituut voor Biotechnologie (VIB), Brussels, Belgium; Structural Biology Brussels (SBB), Vrije Universiteit Brussel (VUB), 1050 Brussels, Belgium; Institute of Enzymology, Research Centre for Natural Sciences of the Hungarian Academy of Sciences, Budapest, Hungary. Electronic address:

Background: Fully intrinsically disordered plant dehydrin ERD14 can protect enzymes via its chaperone-like activity, but it was not formally linked with enzymes of the plant redox system yet. This is of particular interest, as the level of HO in Arabidopsis plants increases during osmotic stress, which can be counteracted by overexpression of ERD14.

Methods: The proteomic mass-spectrometry analysis of stressed plants was performed to find the candidates affected by ERD14. With cross-linking, microscale thermophoresis, and active-site titration kinetics, the interaction and influence of ERD14 on the function of two target proteins: glutathione transferase Phi9 and catalase was examined.

Results: Under osmotic stress, redox enzymes, specifically the glutathione transferase Phi enzymes, are upregulated. Using microscale thermophoresis, we showed that ERD14 directly interacts with GSTF9 with a K of ~25 μM. ERD14 activates the inactive GSTF9 molecules, protects GSTF9 from oxidation, and can also increases the activity of the enzyme. Aside from GSTF9, we found that ERD14 can also interact with catalase, an important cellular HO scavenging enzyme, with a K of ~0.13 μM, and protects it from dehydration-induced loss of activity.

Conclusions: We propose that fully intrinsically disordered dehydrin ERD14 might protect and even activate redox enzymes, helping plants to survive oxidative stress under dehydration conditions.

General Significance: ERD14 has a direct effect on the activity of redox enzymes.
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http://dx.doi.org/10.1016/j.bbagen.2019.129506DOI Listing
March 2020

The Trypanosomal Transferrin Receptor of Trypanosoma Brucei-A Review.

Trop Med Infect Dis 2019 Oct 1;4(4). Epub 2019 Oct 1.

Laboratory of Cellular and Molecular Interactions (CMIM), Vrije Universiteit Brussels, Brussels, 1050 Ixelles, Belgium.

Iron is an essential element for life. Its uptake and utility requires a careful balancing with its toxic capacity, with mammals evolving a safe and bio-viable means of its transport and storage. This transport and storage is also utilized as part of the iron-sequestration arsenal employed by the mammalian hosts' 'nutritional immunity' against parasites. Interestingly, a key element of iron transport, i.e., serum transferrin (Tf), is an essential growth factor for parasitic haemo-protozoans of the genus . These are major mammalian parasites causing the diseases human African trypanosomosis (HAT) and animal trypanosomosis (AT). Using components of their well-characterized immune evasion system, bloodstream parasites adapt and scavenge for the mammalian host serum transferrin within their broad host range. The expression site associated genes (ESAG6 and 7) are utilized to construct a heterodimeric serum Tf binding complex which, within its niche in the flagellar pocket, and coupled to the trypanosomes' fast endocytic rate, allows receptor-mediated acquisition of essential iron from their environment. This review summarizes current knowledge of the trypanosomal transferrin receptor (TfR), with emphasis on the structure and function of the receptor, both in physiological conditions as well as in conditions where the iron supply to parasites is being limited. Potential applications using current knowledge of the parasite receptor are also briefly discussed, primarily focused on potential therapeutic interventions.
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http://dx.doi.org/10.3390/tropicalmed4040126DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6958415PMC
October 2019

Neutrophils enhance early Trypanosoma brucei infection onset.

Sci Rep 2018 07 25;8(1):11203. Epub 2018 Jul 25.

Unit of Veterinary Protozoology, Department of Biomedical Sciences, Institute of Tropical Medicine Antwerp (ITM), Antwerp, Belgium.

In this study, Trypanosoma brucei was naturally transmitted to mice through the bites of infected Glossina morsitans tsetse flies. Neutrophils were recruited rapidly to the bite site, whereas monocytes were attracted more gradually. Expression of inflammatory cytokines (il1b, il6), il10 and neutrophil chemokines (cxcl1, cxcl5) was transiently up-regulated at the site of parasite inoculation. Then, a second influx of neutrophils occurred that coincided with the previously described parasite retention and expansion in the ear dermis. Congenital and experimental neutropenia models, combined with bioluminescent imaging, indicate that neutrophils do not significantly contribute to dermal parasite control and elicit higher systemic parasitemia levels during the infection onset. Engulfment of parasites by neutrophils in the skin was rarely observed and was restricted to parasites with reduced motility/viability, whereas live parasites escaped phagocytosis. To our knowledge, this study represents the first description of a trypanosome infection promoting role of early innate immunological reactions following an infective tsetse fly bite. Our data indicate that the trypanosome is not hindered in its early development and benefits from the host innate responses with the neutrophils being important regulators of the early infection, as already demonstrated for the sand fly transmitted Leishmania parasite.
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http://dx.doi.org/10.1038/s41598-018-29527-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6060092PMC
July 2018

The anuran skin peptide bradykinin mediates its own absorption across epithelial barriers of the digestive tract.

Peptides 2018 05 20;103:84-89. Epub 2018 Mar 20.

Amphibian Evolution Lab, Biology Department, Vrije Universiteit Brussel, Pleinlaan 2, 1050 Elsene, Belgium. Electronic address:

When faced with a potential predator, a wide range of frog species secrete a mixture of peptide toxins from their skin to defend themselves. We have recently shown that antimicrobial peptides (AMPs) in a frog's defensive poison enhance the uptake of these peptides across epithelia, thereby speeding up the process of predator intoxication. This study provides evidence that bradykinin, a widespread peptide toxin in anurans (frogs), is capable to pass through epithelial barriers independent of this delivery system. We quantified bradykinin peptides secreted by Bombina orientalis during acute stress, and found that at biologically relevant concentrations, bradykinin passage across model epithelia occurs even in the absence of AMPs. Monitoring of transepithelial electric resistance showed that bradykinin treatment caused a subtle yet prolonged reduction in barrier function, indicating that the peptide itself is capable to increase the permeability of epithelia. Yet, bradykinin does not cause cells to leak lactate dehydrogenase, suggesting that it does not damage cell membranes. Moreover, imaging of bradykinin-treated monolayers shows no endocytosis of fluorescent propidium iodide, indicating that the peptide does not perforate cell membranes at smaller scale and therefore is unlikely to cross epithelia via a transcellular passage. Together, these observations suggest that bradykinin, unlike other amphibian neuropeptide toxins, mediates its own passage across mucosal barriers, possibly through a paracellular route. This "self-administering" property, combined with the fact that bradykinins can potently disturb multiple physiological processes, could explain why these peptides are one of the most widespread antipredator peptides in the defensive secretions of frogs.
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http://dx.doi.org/10.1016/j.peptides.2018.03.010DOI Listing
May 2018

African Trypanosomiasis-Associated Anemia: The Contribution of the Interplay between Parasites and the Mononuclear Phagocyte System.

Front Immunol 2018 15;9:218. Epub 2018 Feb 15.

Laboratory of Cellular and Molecular Immunology, Vrije Universiteit Brussel (VUB), Brussels, Belgium.

African trypanosomosis (AT) is a chronically debilitating parasitic disease of medical and economic importance for the development of sub-Saharan Africa. The trypanosomes that cause this disease are extracellular protozoan parasites that have developed efficient immune escape mechanisms to manipulate the entire host immune response to allow parasite survival and transmission. During the early stage of infection, a profound pro-inflammatory type 1 activation of the mononuclear phagocyte system (MPS), involving classically activated macrophages (i.e., M1), is required for initial parasite control. Yet, the persistence of this M1-type MPS activation in trypanosusceptible animals causes immunopathology with anemia as the most prominent pathological feature. By contrast, in trypanotolerant animals, there is an induction of IL-10 that promotes the induction of alternatively activated macrophages (M2) and collectively dampens tissue damage. A comparative gene expression analysis between M1 and M2 cells identified galectin-3 (Gal-3) and macrophage migration inhibitory factor (MIF) as novel M1-promoting factors, possibly acting synergistically and in concert with TNF-α during anemia development. While Gal-3 enhances erythrophagocytosis, MIF promotes both myeloid cell recruitment and iron retention within the MPS, thereby depriving iron for erythropoiesis. Hence, the enhanced erythrophagocytosis and suppressed erythropoiesis lead to anemia. Moreover, a thorough investigation using MIF-deficient mice revealed that the underlying mechanisms in AT-associated anemia development in trypanosusceptible and tolerant animals are quite distinct. In trypanosusceptible animals, anemia resembles anemia of inflammation, while in trypanotolerant animals' hemodilution, mainly caused by hepatosplenomegaly, is an additional factor contributing to anemia. In this review, we give an overview of how trypanosome- and host-derived factors can contribute to trypanosomosis-associated anemia development with a focus on the MPS system. Finally, we will discuss potential intervention strategies to alleviate AT-associated anemia that might also have therapeutic potential.
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http://dx.doi.org/10.3389/fimmu.2018.00218DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5818406PMC
April 2019

Novel half-life extended anti-MIF nanobodies protect against endotoxic shock.

FASEB J 2018 06 25;32(6):3411-3422. Epub 2018 Jan 25.

Laboratory of Cellular and Molecular Immunology, Vrije Universiteit Brussels, Brussels, Belgium.

Sepsis-leading to septic shock-is the leading cause of death in intensive care units. The systemic inflammatory response to infection, which is initiated by activated myeloid cells, plays a key role in the lethal outcome. Macrophage migration inhibitory factor (MIF) is an upstream immunoregulatory mediator, released by myeloid cells, that underlies a common genetic susceptibility to different infections and septic shock. Accordingly, strategies that are aimed at inhibiting the action of MIF have therapeutic potential. Here, we report the isolation and characterization of tailorable, small, affinity-matured nanobodies (Nbs; single-domain antigen-binding fragments derived from camelid heavy-chain Abs) directed against MIF. Of importance, these bioengineered Nbs bind both human and mouse MIFs with nanomolar affinity. NbE5 and NbE10 inhibit key MIF functions that can exacerbate septic shock, such as the tautomerase activity of MIF (by blocking catalytic pocket residues that are critical for MIF's conformation and receptor binding), the TNF-inducing potential, and the ability of MIF to antagonize glucocorticoid action. A lead NbE10, tailored to be a multivalent, half-life extended construct (NbE10-NbAlb8-NbE10), attenuated lethality in murine endotoxemia when administered via single injection, either prophylactically or therapeutically. Hence, Nbs, with their structural and pharmacologic advantages over currently available inhibitors, may be an effective, novel approach to interfere with the action of MIF in septic shock and other conditions of inflammatory end-organ damage.-Sparkes, A., De Baetselier, P., Brys, L., Cabrito, I., Sterckx, Y. G.-J., Schoonooghe, S., Muyldermans, S., Raes, G., Bucala, R., Vanlandschoot, P., Van Ginderachter, J. A., Stijlemans, B. Novel half-life extended anti-MIF nanobodies protect against endotoxic shock.
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http://dx.doi.org/10.1096/fj.201701189RDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6137717PMC
June 2018

MIF inhibition interferes with the inflammatory and T cell-stimulatory capacity of NOD macrophages and delays autoimmune diabetes onset.

PLoS One 2017 2;12(11):e0187455. Epub 2017 Nov 2.

Laboratory of Clinical and Experimental Endocrinology (CEE), Department of Chronic Diseases, Metabolism and Ageing (CHROMETA), KU Leuven, Leuven, Belgium.

Macrophages contribute in the initiation and progression of insulitis during type 1 diabetes (T1D). However, the mechanisms governing their recruitment into the islets as well as the manner of retention and activation are incompletely understood. Here, we investigated a role for macrophage migration inhibitory factor (MIF) and its transmembrane receptor, CD74, in the progression of T1D. Our data indicated elevated MIF concentrations especially in long-standing T1D patients and mice. Additionally, NOD mice featured increased MIF gene expression and CD74+ leukocyte frequencies in the pancreas. We identified F4/80+ macrophages as the main immune cells in the pancreas expressing CD74 and showed that MIF antagonism of NOD macrophages prevented their activation-induced cytokine production. The physiological importance was highlighted by the fact that inhibition of MIF delayed the onset of autoimmune diabetes in two different diabetogenic T cell transfer models. Mechanistically, macrophages pre-conditioned with the MIF inhibitor featured a refractory capacity to trigger T cell activation by keeping them in a naïve state. This study underlines a possible role for MIF/CD74 signaling pathways in promoting macrophage-mediated inflammation in T1D. As therapies directed at the MIF/CD74 pathway are in clinical development, new opportunities may be proposed for arresting T1D progression.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0187455PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5667746PMC
December 2017

Nanobodies As Tools to Understand, Diagnose, and Treat African Trypanosomiasis.

Front Immunol 2017 30;8:724. Epub 2017 Jun 30.

Laboratory of Cellular and Molecular Immunology, Vrije Universiteit Brussel (VUB), Brussels, Belgium.

African trypanosomes are strictly extracellular protozoan parasites that cause diseases in humans and livestock and significantly affect the economic development of sub-Saharan Africa. Due to an elaborate and efficient (vector)-parasite-host interplay, required to complete their life cycle/transmission, trypanosomes have evolved efficient immune escape mechanisms that manipulate the entire host immune response. So far, not a single field applicable vaccine exists, and chemotherapy is the only strategy available to treat the disease. Current therapies, however, exhibit high drug toxicity and an increased drug resistance is being reported. In addition, diagnosis is often hampered due to the inadequacy of current diagnostic procedures. In the context of tackling the shortcomings of current treatment and diagnostic approaches, nanobodies (Nbs, derived from the heavy chain-only antibodies of camels and llamas) might represent unmet advantages compared to conventional tools. Indeed, the combination of their small size, high stability, high affinity, and specificity for their target and tailorability represents a unique advantage, which is reflected by their broad use in basic and clinical research to date. In this article, we will review and discuss (i) diagnostic and therapeutic applications of Nbs that are being evaluated in the context of African trypanosomiasis, (ii) summarize new strategies that are being developed to optimize their potency for advancing their use, and (iii) document on unexpected properties of Nbs, such as inherent trypanolytic activities, that besides opening new therapeutic avenues, might offer new insight in hidden biological activities of conventional antibodies.
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http://dx.doi.org/10.3389/fimmu.2017.00724DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5492476PMC
June 2017

African Trypanosomes Undermine Humoral Responses and Vaccine Development: Link with Inflammatory Responses?

Front Immunol 2017 24;8:582. Epub 2017 May 24.

Laboratory of Cellular and Molecular Immunology, Vrije Universiteit Brussel (VUB), Brussels, Belgium.

African trypanosomosis is a debilitating disease of great medical and socioeconomical importance. It is caused by strictly extracellular protozoan parasites capable of infecting all vertebrate classes including human, livestock, and game animals. To survive within their mammalian host, trypanosomes have evolved efficient immune escape mechanisms and manipulate the entire host immune response, including the humoral response. This report provides an overview of how trypanosomes initially trigger and subsequently undermine the development of an effective host antibody response. Indeed, results available to date obtained in both natural and experimental infection models show that trypanosomes impair homeostatic B-cell lymphopoiesis, B-cell maturation and survival and B-cell memory development. Data on B-cell dysfunctioning in correlation with parasite virulence and trypanosome-mediated inflammation will be discussed, as well as the impact of trypanosomosis on heterologous vaccine efficacy and diagnosis. Therefore, new strategies aiming at enhancing vaccination efficacy could benefit from a combination of (i) early parasite diagnosis, (ii) anti-trypanosome (drugs) treatment, and (iii) anti-inflammatory treatment that collectively might allow B-cell recovery and improve vaccination.
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http://dx.doi.org/10.3389/fimmu.2017.00582DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5442186PMC
May 2017

Reprint of: The non-mammalian MIF superfamily.

Immunobiology 2017 06 26;222(6):858-867. Epub 2017 May 26.

Laboratory of Cellular and Molecular Immunology, Vrije Universiteit Brussel (VUB), Brussels, Belgium; Myeloid Cell Immunology Lab, VIB-UGent Center for Inflammation Research, Ghent, Belgium. Electronic address:

Macrophage migration inhibitory factor (MIF) was first described as a cytokine 50 years ago, and emerged in mammals as a pleiotropic protein with pro-inflammatory, chemotactic, and growth-promoting activities. In addition, MIF has gained substantial attention as a pivotal upstream mediator of innate and adaptive immune responses and with pathologic roles in several diseases. Of less importance in mammals is an intrinsic but non-physiologic enzymatic activity that points to MIF's evolution from an ancient defense molecule. Therefore, it is not surprising that mif-like genes also have been found across a range of different organisms including bacteria, plants, protozoa, helminths, molluscs, arthropods, fish, amphibians and birds. While Genebank analysis identifying mif-like genes across species is extensive, contained herein is an overview of the non-mammalian MIF-like proteins that have been most well studied experimentally. For many of these organisms, MIF contributes to an innate defense system or plays a role in development. For parasitic organisms however, MIF appears to function as a virulence factor aiding in the establishment or persistence of infection by modulating the host immune response. Consequently, a combined targeting of both parasitic and host MIF could lead to more effective treatment strategies for parasitic diseases of socioeconomic importance.
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http://dx.doi.org/10.1016/j.imbio.2017.05.004DOI Listing
June 2017

The non-mammalian MIF superfamily.

Immunobiology 2017 03 12;222(3):473-482. Epub 2016 Oct 12.

Laboratory of Cellular and Molecular Immunology, Vrije Universiteit Brussel (VUB), Brussels, Belgium; Myeloid Cell Immunology Lab, VIB Inflammation Research Center, Gent, Belgium. Electronic address:

Macrophage migration inhibitory factor (MIF) was first described as a cytokine 50 years ago, and emerged in mammals as a pleiotropic protein with pro-inflammatory, chemotactic, and growth-promoting activities. In addition, MIF has gained substantial attention as a pivotal upstream mediator of innate and adaptive immune responses and with pathologic roles in several diseases. Of less importance in mammals is an intrinsic but non-physiologic enzymatic activity that points to MIF's evolution from an ancient defense molecule. Therefore, it is not surprising that mif-like genes also have been found across a range of different organisms including bacteria, plants, ‎protozoa, helminths, molluscs, arthropods, fish, amphibians and birds. While Genebank analysis identifying mif-like genes across species is extensive, contained herein is an overview of the non-mammalian MIF-like proteins that have been most well studied experimentally. For many of these organisms, MIF contributes to an innate defense system or plays a role in development. For parasitic organisms however, MIF appears to function as a virulence factor aiding in the establishment or persistence of infection by modulating the host immune response. Consequently, a combined targeting of both parasitic and host MIF could lead to more effective treatment strategies for parasitic diseases of socioeconomic importance.
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http://dx.doi.org/10.1016/j.imbio.2016.10.006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5293613PMC
March 2017

MIF-Mediated Hemodilution Promotes Pathogenic Anemia in Experimental African Trypanosomosis.

PLoS Pathog 2016 09 15;12(9):e1005862. Epub 2016 Sep 15.

Laboratory of Cellular and Molecular Immunology, Vrije Universiteit Brussel (VUB), Brussels, Belgium.

Animal African trypanosomosis is a major threat to the economic development and human health in sub-Saharan Africa. Trypanosoma congolense infections represent the major constraint in livestock production, with anemia as the major pathogenic lethal feature. The mechanisms underlying anemia development are ill defined, which hampers the development of an effective therapy. Here, the contribution of the erythropoietic and erythrophagocytic potential as well as of hemodilution to the development of T. congolense-induced anemia were addressed in a mouse model of low virulence relevant for bovine trypanosomosis. We show that in infected mice, splenic extramedullary erythropoiesis could compensate for the chronic low-grade type I inflammation-induced phagocytosis of senescent red blood cells (RBCs) in spleen and liver myeloid cells, as well as for the impaired maturation of RBCs occurring in the bone marrow and spleen. Rather, anemia resulted from hemodilution. Our data also suggest that the heme catabolism subsequent to sustained erythrophagocytosis resulted in iron accumulation in tissue and hyperbilirubinemia. Moreover, hypoalbuminemia, potentially resulting from hemodilution and liver injury in infected mice, impaired the elimination of toxic circulating molecules like bilirubin. Hemodilutional thrombocytopenia also coincided with impaired coagulation. Combined, these effects could elicit multiple organ failure and uncontrolled bleeding thus reduce the survival of infected mice. MIF (macrophage migrating inhibitory factor), a potential pathogenic molecule in African trypanosomosis, was found herein to promote erythrophagocytosis, to block extramedullary erythropoiesis and RBC maturation, and to trigger hemodilution. Hence, these data prompt considering MIF as a potential target for treatment of natural bovine trypanosomosis.
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http://dx.doi.org/10.1371/journal.ppat.1005862DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5025191PMC
September 2016

Immune Evasion Strategies of Trypanosoma brucei within the Mammalian Host: Progression to Pathogenicity.

Front Immunol 2016 24;7:233. Epub 2016 Jun 24.

Laboratory of Cellular and Molecular Immunology, Vrije Universiteit Brussel (VUB), Brussels, Belgium; Department of Structural Biology, VIB, Brussels, Belgium.

The diseases caused by African trypanosomes (AT) are of both medical and veterinary importance and have adversely influenced the economic development of sub-Saharan Africa. Moreover, so far not a single field applicable vaccine exists, and chemotherapy is the only strategy available to treat the disease. These strictly extracellular protozoan parasites are confronted with different arms of the host's immune response (cellular as well as humoral) and via an elaborate and efficient (vector)-parasite-host interplay they have evolved efficient immune escape mechanisms to evade/manipulate the entire host immune response. This is of importance, since these parasites need to survive sufficiently long in their mammalian/vector host in order to complete their life cycle/transmission. Here, we will give an overview of the different mechanisms AT (i.e. T. brucei as a model organism) employ, comprising both tsetse fly saliva and parasite-derived components to modulate host innate immune responses thereby sculpturing an environment that allows survival and development within the mammalian host.
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http://dx.doi.org/10.3389/fimmu.2016.00233DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4919330PMC
July 2016

Molecular Imaging with Kupffer Cell-Targeting Nanobodies for Diagnosis and Prognosis in Mouse Models of Liver Pathogenesis.

Mol Imaging Biol 2017 02;19(1):49-58

Laboratory of Cellular and Molecular Immunology, Vrije Universiteit Brussel, Pleinlaan 2, Building E, 8th floor, Brussels, Belgium.

Purpose: Kupffer cells (KCs), the liver resident macrophages, are important mediators of tissue homeostasis and pathogen clearance. However, depending on the inflammatory stimuli, KCs have been involved in divergent hepato-protective or hepato-destructive immune responses. The versatility of KCs in response to environmental triggers, in combination with the specific biomarkers they express, make these macrophages attractive in vivo targets for non-invasive monitoring of liver inflammation or pathogenicity. This study aims to determine whether V-set and Ig domain-containing 4 (Vsig4) and C-type lectin domain family (Clec) 4, member F (Clec4F) can be used as imaging biomarkers for non-invasive monitoring of KCs during distinct liver inflammation models.

Procedure: Flow cytometry (FACS), immuno-histochemistry (IHC), and single-photon emission computed tomography (SPECT) with Tc-99m labeled anti-Vsig4 or anti-Clec4F nanobodies (Nbs) was performed to evaluate in mice KC dynamics in concanavalin A (ConA)-induced hepatitis and in non-alcoholic steatohepatitis induced via methionine choline deficiency (MCD).

Results: In homeostatic mice, Nbs targeting Clec4F were found to accumulate and co-localize with Vsig4-targeting Nbs only in the liver. Upon induction of acute hepatitis using ConA, down-regulation of the in vivo Nb imaging signal was observed, reflecting reduction in KC numbers as confirmed by FACS and IHC. On the other hand, induction of steatohepatitis resulted in higher signals in the liver corresponding to higher density of KCs. The Nb-imaging signals returned to normal levels after resolution of the investigated liver diseases.

Conclusions: Anti-Clec4F and anti-Vsig4 Nbs targeting KCs as molecular imaging biomarkers could allow non-invasive monitoring/staging of liver pathogenesis.
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http://dx.doi.org/10.1007/s11307-016-0976-3DOI Listing
February 2017

An Anti-proteome Nanobody Library Approach Yields a Specific Immunoassay for Trypanosoma congolense Diagnosis Targeting Glycosomal Aldolase.

PLoS Negl Trop Dis 2016 Feb 2;10(2):e0004420. Epub 2016 Feb 2.

Laboratory of Cellular and Molecular Immunology (CMIM), Vrije Universiteit Brussel, Brussels, Belgium.

Background: Infectious diseases pose a severe worldwide threat to human and livestock health. While early diagnosis could enable prompt preventive interventions, the majority of diseases are found in rural settings where basic laboratory facilities are scarce. Under such field conditions, point-of-care immunoassays provide an appropriate solution for rapid and reliable diagnosis. The limiting steps in the development of the assay are the identification of a suitable target antigen and the selection of appropriate high affinity capture and detection antibodies. To meet these challenges, we describe the development of a Nanobody (Nb)-based antigen detection assay generated from a Nb library directed against the soluble proteome of an infectious agent. In this study, Trypanosoma congolense was chosen as a model system.

Methodology/principal Findings: An alpaca was vaccinated with whole-parasite soluble proteome to generate a Nb library from which the most potent T. congolense specific Nb sandwich immunoassay (Nb474H-Nb474B) was selected. First, the Nb474-homologous sandwich ELISA (Nb474-ELISA) was shown to detect experimental infections with high Positive Predictive Value (98%), Sensitivity (87%) and Specificity (94%). Second, it was demonstrated under experimental conditions that the assay serves as test-of-cure after Berenil treatment. Finally, this assay allowed target antigen identification. The latter was independently purified through immuno-capturing from (i) T. congolense soluble proteome, (ii) T. congolense secretome preparation and (iii) sera of T. congolense infected mice. Subsequent mass spectrometry analysis identified the target as T. congolense glycosomal aldolase.

Conclusions/significance: The results show that glycosomal aldolase is a candidate biomarker for active T. congolense infections. In addition, and by proof-of-principle, the data demonstrate that the Nb strategy devised here offers a unique approach to both diagnostic development and target discovery that could be widely applied to other infectious diseases.
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http://dx.doi.org/10.1371/journal.pntd.0004420DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4737498PMC
February 2016

The Possible Role of Staphylococcus epidermidis LPxTG Surface Protein SesC in Biofilm Formation.

PLoS One 2016 22;11(1):e0146704. Epub 2016 Jan 22.

KU Leuven-University of Leuven, Department of Microbiology and Immunology, Laboratory of Clinical Bacteriology and Mycology, B-3000, Leuven, Belgium.

Staphylococcus epidermidis is the most common cause of device-associated infections. It has been shown that active and passive immunization in an animal model against protein SesC significantly reduces S. epidermidis biofilm-associated infections. In order to elucidate its role, knock-out of sesC or isolation of S. epidermidis sesC-negative mutants were attempted, however, without success. As an alternative strategy, sesC was introduced into Staphylococcus aureus 8325-4 and its isogenic icaADBC and srtA mutants, into the clinical methicillin-sensitive S. aureus isolate MSSA4 and the MRSA S. aureus isolate BH1CC, which all lack sesC. Transformation of these strains with sesC i) changed the biofilm phenotype of strains 8325-4 and MSSA4 from PIA-dependent to proteinaceous even though PIA synthesis was not affected, ii) converted the non-biofilm-forming strain 8325-4 ica::tet to a proteinaceous biofilm-forming strain, iii) impaired PIA-dependent biofilm formation by 8325-4 srtA::tet, iv) had no impact on protein-mediated biofilm formation of BH1CC and v) increased in vivo catheter and organ colonization by strain 8325-4. Furthermore, treatment with anti-SesC antibodies significantly reduced in vitro biofilm formation and in vivo colonization by these transformants expressing sesC. These findings strongly suggest that SesC is involved in S. epidermidis attachment to and subsequent biofilm formation on a substrate.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0146704PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4723045PMC
July 2016

M-CSF and GM-CSF Receptor Signaling Differentially Regulate Monocyte Maturation and Macrophage Polarization in the Tumor Microenvironment.

Cancer Res 2016 Jan 16;76(1):35-42. Epub 2015 Nov 16.

Laboratory of Myeloid Cell Immunology, VIB, Brussels, Belgium. Laboratory of Cellular and Molecular Immunology, Vrije Universiteit Brussel, Brussels, Belgium.

Tumors contain a heterogeneous myeloid fraction comprised of discrete MHC-II(hi) and MHC-II(lo) tumor-associated macrophage (TAM) subpopulations that originate from Ly6C(hi) monocytes. However, the mechanisms regulating the abundance and phenotype of distinct TAM subsets remain unknown. Here, we investigated the role of macrophage colony-stimulating factor (M-CSF) in TAM differentiation and polarization in different mouse tumor models. We demonstrate that treatment of tumor-bearing mice with a blocking anti-M-CSFR monoclonal antibody resulted in a reduction of mature TAMs due to impaired recruitment, extravasation, proliferation, and maturation of their Ly6C(hi) monocytic precursors. M-CSFR signaling blockade shifted the MHC-II(lo)/MHC-II(hi) TAM balance in favor of the latter as observed by the preferential differentiation of Ly6C(hi) monocytes into MHC-II(hi) TAMs. In addition, the genetic and functional signatures of MHC-II(lo) TAMs were downregulated upon M-CSFR blockade, indicating that M-CSFR signaling shapes the MHC-II(lo) TAM phenotype. Conversely, granulocyte macrophage (GM)-CSFR had no effect on the mononuclear tumor infiltrate or relative abundance of TAM subsets. However, GM-CSFR signaling played an important role in fine-tuning the MHC-II(hi) phenotype. Overall, our data uncover the multifaceted and opposing roles of M-CSFR and GM-CSFR signaling in governing the phenotype of macrophage subsets in tumors, and provide new insight into the mechanism of action underlying M-CSFR blockade.
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http://dx.doi.org/10.1158/0008-5472.CAN-15-0869DOI Listing
January 2016

E-cadherin expression in macrophages dampens their inflammatory responsiveness in vitro, but does not modulate M2-regulated pathologies in vivo.

Sci Rep 2015 Jul 31;5:12599. Epub 2015 Jul 31.

1] Laboratory of Myeloid Cell Immunology, VIB, Brussels, Belgium [2] Laboratory of Cellular and Molecular Immunology, Vrije Universiteit Brussel, Brussels, Belgium.

IL-4/IL-13-induced alternatively activated macrophages (M(IL-4/IL-13), AAMs or M2) are known to express E-cadherin, enabling them to engage in heterotypic cellular interactions and IL-4-driven macrophage fusion in vitro. Here we show that E-cadherin overexpression in Raw 264.7 macrophages inhibits their inflammatory response to LPS stimulation, as demonstrated by a reduced secretion of inflammatory mediators like interleukin (IL)-6, tumor necrosis factor (TNF) and nitric oxide (NO). To study the function of E-cadherin in M(IL-4/IL-13) macrophages in vivo, we generated macrophage-specific E-cadherin-deficient C57BL/6 mice. Using this new tool, we analyzed immunological parameters during two typical AAM-associated Th2-driven diseases and assessed Th2-associated granuloma formation. Although E-cadherin is strongly induced in AAMs during Taenia crassiceps helminth infections and allergic airway inflammation, its deletion in macrophages does not affect the course of both Th2 cytokine-driven diseases. Moreover, macrophage E-cadherin expression is largely redundant for granuloma formation around Schistosoma mansoni ova. Overall, we conclude that E-cadherin is a valuable AAM marker which suppresses the inflammatory response when overexpressed. Yet E-cadherin deletion in macrophages does not affect M(LPS+IFNγ) and M(IL-4) polarization in vitro, nor in vivo macrophage function, at least in the conditions tested.
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http://dx.doi.org/10.1038/srep12599DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4521155PMC
July 2015

Iron Homeostasis and Trypanosoma brucei Associated Immunopathogenicity Development: A Battle/Quest for Iron.

Biomed Res Int 2015 18;2015:819389. Epub 2015 May 18.

Laboratory of Cellular and Molecular Immunology, Vrije Universiteit Brussel (VUB), 1050 Brussels, Belgium ; Department of Myeloid Cell Immunology, Vlaams Instituut voor Biotechnologie (VIB), 1050 Brussels, Belgium.

African trypanosomosis is a chronic debilitating disease affecting the health and economic well-being of developing countries. The immune response during African trypanosome infection consisting of a strong proinflammatory M1-type activation of the myeloid phagocyte system (MYPS) results in iron deprivation for these extracellular parasites. Yet, the persistence of M1-type MYPS activation causes the development of anemia (anemia of chronic disease, ACD) as a most prominent pathological parameter in the mammalian host, due to enhanced erythrophagocytosis and retention of iron within the MYPS thereby depriving iron for erythropoiesis. In this review we give an overview of how parasites acquire iron from the host and how iron modulation of the host MYPS affects trypanosomosis-associated anemia development. Finally, we also discuss different strategies at the level of both the host and the parasite that can/might be used to modulate iron availability during African trypanosome infections.
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http://dx.doi.org/10.1155/2015/819389DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4450282PMC
March 2016

NK-, NKT- and CD8-Derived IFNγ Drives Myeloid Cell Activation and Erythrophagocytosis, Resulting in Trypanosomosis-Associated Acute Anemia.

PLoS Pathog 2015 Jun 12;11(6):e1004964. Epub 2015 Jun 12.

Laboratory of Cellular and Molecular Immunology, Vrije Universiteit Brussel, Brussels, Belgium; Department of Structural Biology, Vlaams Instituut voor Biotechnologie (VIB), Brussels, Belgium; Faculty of Health Sciences (IDM, Division Immunology), University of Cape Town, Cape Town, South Africa.

African trypanosomes are the causative agents of Human African Trypanosomosis (HAT/Sleeping Sickness) and Animal African Trypanosomosis (AAT/Nagana). A common hallmark of African trypanosome infections is inflammation. In murine trypanosomosis, the onset of inflammation occurs rapidly after infection and is manifested by an influx of myeloid cells in both liver and spleen, accompanied by a burst of serum pro-inflammatory cytokines. Within 48 hours after reaching peak parasitemia, acute anemia develops and the percentage of red blood cells drops by 50%. Using a newly developed in vivo erythrophagocytosis assay, we recently demonstrated that activated cells of the myeloid phagocytic system display enhanced erythrophagocytosis causing acute anemia. Here, we aimed to elucidate the mechanism and immune pathway behind this phenomenon in a murine model for trypanosomosis. Results indicate that IFNγ plays a crucial role in the recruitment and activation of erythrophagocytic myeloid cells, as mice lacking the IFNγ receptor were partially protected against trypanosomosis-associated inflammation and acute anemia. NK and NKT cells were the earliest source of IFNγ during T. b. brucei infection. Later in infection, CD8+ and to a lesser extent CD4+ T cells become the main IFNγ producers. Cell depletion and transfer experiments indicated that during infection the absence of NK, NKT and CD8+ T cells, but not CD4+ T cells, resulted in a reduced anemic phenotype similar to trypanosome infected IFNγR-/- mice. Collectively, this study shows that NK, NKT and CD8+ T cell-derived IFNγ is a critical mediator in trypanosomosis-associated pathology, driving enhanced erythrophagocytosis by myeloid phagocytic cells and the induction of acute inflammation-associated anemia.
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http://dx.doi.org/10.1371/journal.ppat.1004964DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4466398PMC
June 2015

Development of a pHrodo-based assay for the assessment of in vitro and in vivo erythrophagocytosis during experimental trypanosomosis.

PLoS Negl Trop Dis 2015 Mar 5;9(3):e0003561. Epub 2015 Mar 5.

Laboratory of Cellular and Molecular Immunology, Vrije Universiteit Brussel, Brussels, Belgium; Department of Structural Biology, Vlaams Instituut voor Biotechnologie (VIB), Brussels, Belgium.

Extracellular trypanosomes can cause a wide range of diseases and pathological complications in a broad range of mammalian hosts. One common feature of trypanosomosis is the occurrence of anemia, caused by an imbalance between erythropoiesis and red blood cell clearance of aging erythrocytes. In murine models for T. brucei trypanosomosis, anemia is marked by a very sudden non-hemolytic loss of RBCs during the first-peak parasitemia control, followed by a short recovery phase and the subsequent gradual occurrence of an ever-increasing level of anemia. Using a newly developed quantitative pHrodo based in vitro erythrophagocytosis assay, combined with FACS-based ex vivo and in vivo results, we show that activated liver monocytic cells and neutrophils as well as activated splenic macrophages are the main cells involved in the occurrence of the early-stage acute anemia. In addition, we show that trypanosomosis itself leads to a rapid alteration of RBC membrane stability, priming the cells for accelerated phagocytosis.
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http://dx.doi.org/10.1371/journal.pntd.0003561DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4352936PMC
March 2015

Macrophage dynamics are regulated by local macrophage proliferation and monocyte recruitment in injured pancreas.

Eur J Immunol 2015 May 19;45(5):1482-93. Epub 2015 Mar 19.

Myeloid Cell Immunology Laboratory, VIB, Brussels, Belgium.

Pancreas injury by partial duct ligation (PDL) activates a healing response, encompassing β-cell neogenesis and proliferation. Macrophages (MΦs) were recently shown to promote β-cell proliferation after PDL, but they remain poorly characterized. We assessed myeloid cell diversity and the factors driving myeloid cell dynamics following acute pancreas injury by PDL. In naive and sham-operated pancreas, the myeloid cell compartment consisted mainly of two distinct tissue-resident MΦ types, designated MHC-II(lo) and MHC-II(hi) MΦs, the latter being predominant. MHC-II(lo) and MHC-II(hi) pancreas MΦs differed at the molecular level, with MHC-II(lo) MΦs being more M2-activated. After PDL, there was an early surge of Ly6C(hi) monocyte infiltration in the pancreas, followed by a transient MHC-II(lo) MΦ peak and ultimately a restoration of the MHC-II(hi) MΦ-dominated steady-state equilibrium. These intricate MΦ dynamics in PDL pancreas depended on monocyte recruitment by C-C chemokine receptor 2 and macrophage-colony stimulating factor receptor as well as on macrophage-colony stimulating factor receptor-dependent local MΦ proliferation. Functionally, MHC-II(lo) MΦs were more angiogenic. We further demonstrated that, at least in C-C chemokine receptor 2-KO mice, tissue MΦs, rather than Ly6C(hi) monocyte-derived MΦs, contributed to β-cell proliferation. Together, our study fully characterizes the MΦ subsets in the pancreas and clarifies the complex dynamics of MΦs after PDL injury.
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http://dx.doi.org/10.1002/eji.201445013DOI Listing
May 2015

Generation of a nanobody targeting the paraflagellar rod protein of trypanosomes.

PLoS One 2014 31;9(12):e115893. Epub 2014 Dec 31.

Cellular and Molecular Immunology, Vrije Universiteit Brussel, Pleinlaan 2, B-1050 Brussels, Belgium; Structural Biology Research Center, VIB (Flanders Institute for Biotechnology), Pleinlaan 2, B-1050 Brussels, Belgium.

Trypanosomes are protozoan parasites that cause diseases in humans and livestock for which no vaccines are available. Disease eradication requires sensitive diagnostic tools and efficient treatment strategies. Immunodiagnostics based on antigen detection are preferable to antibody detection because the latter cannot differentiate between active infection and cure. Classical monoclonal antibodies are inaccessible to cryptic epitopes (based on their size-150 kDa), costly to produce and require cold chain maintenance, a condition that is difficult to achieve in trypanosomiasis endemic regions, which are mostly rural. Nanobodies are recombinant, heat-stable, small-sized (15 kDa), antigen-specific, single-domain, variable fragments derived from heavy chain-only antibodies in camelids. Because of numerous advantages over classical antibodies, we investigated the use of nanobodies for the targeting of trypanosome-specific antigens and diagnostic potential. An alpaca was immunized using lysates of Trypanosoma evansi. Using phage display and bio-panning techniques, a cross-reactive nanobody (Nb392) targeting all trypanosome species and isolates tested was selected. Imunoblotting, immunofluorescence microscopy, immunoprecipitation and mass spectrometry assays were combined to identify the target recognized. Nb392 targets paraflagellar rod protein (PFR1) of T. evansi, T. brucei, T. congolense and T. vivax. Two different RNAi mutants with defective PFR assembly (PFR2RNAi and KIF9BRNAi) were used to confirm its specificity. In conclusion, using a complex protein mixture for alpaca immunization, we generated a highly specific nanobody (Nb392) that targets a conserved trypanosome protein, i.e., PFR1 in the flagella of trypanosomes. Nb392 is an excellent marker for the PFR and can be useful in the diagnosis of trypanosomiasis. In addition, as demonstrated, Nb392 can be a useful research or PFR protein isolation tool.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0115893PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4281110PMC
October 2015