The Journal of Immunology Alternatively Activated Myeloid Cells Limit Pathogenicity Associated with African Trypanosomiasis through the IL-10 Inducible Gene Selenoprotein P1 Tom Bosschaerts,*† Martin Guilliams,*† Wim Noel,*† Michel He´rin,‡ Raymond F. Burk,§ Kristina E. Hill,§ Lea Brys,*† Geert Raes,*† Gholamreza Hassanzadeh Ghassabeh,*† Patrick De Baetselier,*† and Alain Beschin2*† Uncontrolled inflammation is a major cause of tissue injury/pathogenicity often resulting in death of a host infected with African trypanosomes. Thus, comparing the immune response in hosts that develop different degrees of disease severity represents a promising approach to discover processes contributing to trypanosomiasis control. It is known that limitation of pathogenicity requires a transition in the course of infection, from an IFN-␥-dependent response resulting in the devel- opment of classically activated myeloid cells (M1), to a counterbalancing IL-10-dependent response associated with alter- natively activated myeloid cells (M2). Herein, mechanisms and downstream effectors by which M2 contribute to lower the pathogenicity and the associated susceptibility to African trypanosomiasis have been explored. Gene expression analysis in IL-10 knockout and wild-type mice, that are susceptible and relatively resistant to Trypanosoma congolense infection, re- spectively, revealed a number of IL-10-inducible genes expressed by M2, including Sepp1 coding for selenoprotein P. Func- tional analyses confirm that selenoprotein P contributes to limit disease severity through anti-oxidant activity. Indeed, Sepp1 knockout mice, but not Sepp1⌬240-361 mice retaining the anti-oxidant motif but lacking the selenium transporter domain of selenoprotein P, exhibited increased tissue injury that associated with increased production of reactive oxygen species and increased apoptosis in the liver immune cells, reduced parasite clearance capacity of myeloid cells, and decreased survival. These data validate M2-associated molecules as functioning in reducing the impact of parasite infection on the host. The Journal of Immunology, 2008, 180: 6168–6175. he plasticity of macrophages and other CD11bϩ myeloid of type 2 cytokine-associated MCs (M2) exerting overlapping cells (MCs)3 in response to microenvironmental signals functions have been recognized (1). Indeed, IL-4/IL-13-activated results in a continuum of different activation forms. At MCs (designated M2a), that promote type 2 immune response by guest on October 1, 2021. Copyright 2008 Pageant Media Ltd. T one end of the spectrum, type 1 cytokine-associated MCs (M1)- and inflammation, share common characteristics with IL-10- induced by IFN-␥, TNF-␣, and microbial products like LPS rep- induced/deactivated MCs (M2c) and type II activated MCs resent the classical form of activation state and play a critical role (M2b) polarized via exposure to immune complexes and ligands in type 1 inflammation and in the fight against intracellular patho- of TLRs or IL-1R. M2c contribute to tissue remodeling whereas gens and tumors. At the other end of the spectrum, distinct subsets M2b induce type 2 immune responses and suppress type 1 in- flammation but promote type 2 inflammation. *Department of Molecular and Cellular Interactions and †Laboratory of Cellular In a recent investigation, we have identified a common gene and Molecular Immunology, Vrije Universiteit Brussel, Brussels; ‡Cell and Tissue signature for peritoneal and splenic M2, by gene expression anal- Laboratory, Unite´de Recherche en Physiologie Mole´culaire, Faculte´s Universi- http://classic.jimmunol.org ysis in MCs elicited in various pathologies including African taires Notre-Dame de la Paix, Namur, Belgium; and §Division of Gastroenterol- ogy, Hepatology, and Nutrition, Department of Medicine, Vanderbilt University trypanosomiasis, helminthiasis, and lymphoma progression (2). School of Medicine, Nashville, TN 37232 The wide-ranging M2 signature includes genes that were earlier Received for publication July 16, 2007. Accepted for publication February 20, 2008. documented as M2 markers (Arg1, Retnla, MglI, MglII, Mrc1, and The costs of publication of this article were defrayed in part by the payment of page Chi3l3/4) and genes that were not before associated with M2 charges. This article must therefore be hereby marked advertisement in accordance (Pla2g7, Psap, Sepp1, Pmp22, Trem2, Folr2, and Cdh1). Together with 18 U.S.C. Section 1734 solely to indicate this fact. Downloaded from with the gene expression profile of tumor-associated macrophages 1 This work, performed in frame of an Interuniversity Attraction Pole Program, was supported by grants from the Institute for Promotion of Innovation by Science (3) and nematode infection elicited macrophages (4) exhibiting an and Technology in Flanders and the Fund for Scientific Research Flanders, and by M2 phenotype, the common M2 gene signature constitutes a re- a grant from Institute for Promotion of Innovation by Science and Technology in Flanders for Generisch Basisonderzoek aan de Universiteiten. R.F.B. and K.E.H. sourceful tool to explore the functions of M2. In this regard, we are supported by National Institutes of Health Grant ES02497. have investigated the functional implication of a number of M2 2 Address correspondence and reprint requests to Dr. Alain Beschin, Department of signature genes in helminthiasis (5) and tumor progression (6). In Molecular and Cellular Interactions, Vrije Universiteit Brussel, Laboratory of Cellular the present work, we have examined the significance of M2-asso- and Molecular Immunology, Pleinlaan 2, 1050 Brussels, Belgium. E-mail address: [email protected] ciated genes in African trypanosomiasis. 3 Abbreviations used in this paper: MC, myeloid cell; ROS, reactive oxygen species; It has been documented that the heterogeneity of MCs affects the Sepp1, selenoprotein P; Sepp1⌬240-361, selenoprotein P truncated of amino acids 240– outcome of African trypanosomiasis (7). In both Trypanosoma 361; Ctss, cathepsin S; Ngfb, nerve growth factor ␤; F13a1, coagulation factor XIII, congolense- and Trypanosoma brucei-infected mice, the develop- A1 subunit; Treg, T regulatory cell; KO, knockout. ment of IFN-␥-dependent M1 in the acute stage of infection re- Copyright © 2008 by The American Association of Immunologists, Inc. 0022-1767/08/$2.00 stricts parasite growth by secreting molecules like TNF-␣, IL-6, www.jimmunol.org The Journal of Immunology 6169 and IL-12 as well as reactive oxygen and nitrogen species (8–14). CD11bϪ cell fraction was collected as flow-through during washing of This type 1 immune response is followed at later stage by the the columns with ice-cold MACS buffer. Purity of isolated MCs emergence of M2 and the expansion of Foxp3ϩCD4ϩ regulatory T checked by flow cytometry, on FACSCanto II using the FlowJo pro- gram, always exceeded 95%. cells (Tregs) in type 2 cytokine environment in T. congolense- but not in T. brucei-infected C57BL/6 mice. Moreover, the inability Cytokines and nitrite determination of T. brucei-infected mice to suppress the type 1 immune re- Blood collected by heart puncture on heparin (20 U/ml) was centrifuged sponse correlates with the increased pathogenicity of the dis- (10 000 g, 10 min) and stored at Ϫ80°C. Cytokines were quantified in the ease that results in tissue injury and early death of the host plasma using ELISAs for IFN-␥, IL-10 (Pharmingen), or TNF-␣ (R&D (15–18). Of note, in both T. congolense- and T. brucei-infected Systems) according to the manufacturer’s protocols. NO level was deter- mined by quantifying the accumulation of nitrate and nitrite in plasma as mice, IL-10 is able to avoid the inflammation mediated by in Ref. 13. IFN-␥, including overwhelming activation of M1, that results in destruction of the liver and uncontrolled parasite growth (13, Differential gene expression analysis 17, 18). Since 80% of these extracellular parasites have been Gene expression was analyzed by quantitative real time PCR using the suggested to be cleared from the circulation by liver MCs (19), conditions and primers described in Ref. 2. Results of the PCR analyses these data indicate that IL-10, by reducing the pathogenicity of were normalized against the house-keeping gene S12, in at least two the disease, protects the integrity of the liver and hereby its independent experiments involving at least five mice per condition. trypanosome clearance capacity. Reactive oxygen species (ROS) Considering the essential role of IL-10 in limiting the patho- ROS production was measured using H2DCFDA as a probe. Briefly, cells genicity and, thus, the susceptibility to the infection, the set of 6 ␮ (10 ) were incubated in serum-free DMEM containing 2 MH2DCFDA genes induced in M2 of C57BL/6 mice that are relatively re- (30 min, 37°C), washed twice with excess cold RPMI 1640, and analyzed sistant to T. congolense infection (2) provides a basis for ex- by flow cytometry. ploration of the processes that prevent disease severity in Af- Apoptosis rican trypanosomiasis. Herein, a number of genes that are ␮ 5 inducible by IL-10 in the liver of T. congolense-infected Cells were incubated with FITC-Annexin V (BD Biosciences; 4 l/10 cells, 15 min, room temperature) in binding buffer (10 mM HEPES, 140 C57BL/6 mice were identified. In addition, we demonstrated mM NaCl, and 2.5 mM CaCl2 (pH 7.4)). After washing with binding that one of these M2-associated genes, Sepp1 which codes for buffer, cells were analyzed by flow cytometry. selenoprotein P, is clearly involved in the limitation of the Flow cytometry pathogenicity associated with T. congolense infection and, thus, decreases the impact of the inflammatory immune response on After blocking FcR with 2.4G2 anti-CD16/32 Abs (BD Biosciences), cells 6 the host. were incubated with PE-anti-CD11b Abs (BD Biosciences; 1 ␮g/10 cells, 30 min, 4°C, in the dark). After washing with ice-cold RPMI 1640, cells were analyzed on FACSVantage station (BD Biosciences) Materials and Methods using CellQuest software. Mice, parasites, and infections Alanine aminotransferase (ALT) levels Female wild-type and IL-10 knockout (KO) C57BL/6 mice were purchased by guest on October 1, 2021.