Gene Selenoprotein P Trypanosomiasis Through the IL-10 Inducible Pathogenicity Associated with African Alternatively Activated M

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 development of classically activated myeloid cells (M1), to a counterbalancing IL-10-dependent response associated with alternatively 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, respectively, 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 inflammation 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 Molećulaire, 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 program, 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 determined 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. Copyright 2008 Pageant Media Ltd. from Harlan and B&K Universal respectively. Heterozygous Sepp1 mice Liver glutamic pyruvic transaminase/alanine aminotransferase (ALT) lev- or Sepp1⌬240-361 mice that had been backcrossed 10 times with C57BL/6 els were measured in individual serum samples, using a commercial kit mice were mated, and female wild-type, Sepp1 KO, and Sepp1⌬240-361 (Boehringer Mannheim). offspring were selected for study by genotyping as reported (20, 21). Wild- type Sepp1 KO and Sepp1⌬240-361 mice were fed from the time of weaning Statistical analysis Torula yeast-based diet (22) supplemented with 1 mg selenium/kg as so- Results are presented as mean Ϯ SEM. All comparisons were tested for dium selenite (prepared by Harlan Sprague Dawley). Mice were kept under statistical signiﬁcance ( p Ͻ 0.05) using the unpaired t test from GraphPad ﬁlter barrier and used at 8–12 wk of age. As controls, noninfected age Prism 4.0 software. Studies were performed on 3–5 independent matched mice were used. experiments. T. congolense variant Ag type 13 (Tc13) (23) was provided by Dr. H. Tabel (University of Sakatchewan, Canada). Parasites stored at Ϫ80°C

http://classic.jimmunol.org Results were used to infect cyclophosphamide-treated (C57BL/6 ϫ BALB/C) mice bred in house by i.p. inoculation. At day 4 post infection, mice were bled, 1. M2-associated genes Ctss, F13a1, Sepp1, and Ngfb are and parasites were purified by diethyl-aminoethyl-cellulose chromatogra- up-regulated upon T. congolense infection in C57BL/6 mice but phy (24). C57BL/6 strains were then infected with 2000 purified parasites. not in relatively susceptible IL-10 KO C57BL/6 mice Parasitemia was monitored by tail blood puncture. Gadolinium chloride was injected through the tail vein at a dose of 5 mg/kg. Experiments were To envisage a potential role of M2 in the outcome of African performed in compliance with the relevant laws and institutional trypanosomiasis, the expression of 30 recently identified M2-as- ϩ Downloaded from guidelines. sociated genes (2) was addressed in CD11b MC fraction from the MC isolation liver of T. congolense-infected C57BL/6 mice that gradually ex- panded in the course of infection (Table I). Genes were considered Liver non-parenchymal cells were isolated as follows: animals were eu- ϩ significantly induced if the expression in CD11b myeloid cells thanized (CO2) and livers were perfused through the portal vein with 10 ml of 100 U/ml collagenase type III (Worthington Biochemical Corporation) from all infected mice tested (n Ն 5) was Ͼ3-fold higher than in in HBSS supplemented with 2 mM EDTA. Then, the liver was minced and all noninfected mice (n Ն 5). Twelve genes were found using incubated in 10 ml of 100 U/ml collagenase III (20 min, 37°C). The re- real time PCR to be preferentially induced in M2 expanding in ␮ sulting cell suspension was passed through a 100 m nylon mesh filter and the liver of T. congolense-infected C57BL/6 mice (Pla2g7, then centrifuged (300 g, 10 min, 4°C). After erythrocyte lysis, the pellet was resuspended in 10 ml of HBSS supplemented with 2 mM EDTA, 10% phospholipase A2, group VII; Psap, prosaposin; Sepp1, seleno- FCS, and overlayed on 10 ml of Lymphoprep (Lucron Bioproducts). After protein P, plasma, 1; Trem2, triggering receptor expressed on centrifugation (430 g, 25 min, 17°C), the layer of low-density cells at the MC 2; Ngfb, nerve growth factor ␤; F13a1, coagulation factor interface containing non-parenchymal cells was harvested. MCs were XIII, A1 subunit; Pmp22, peripheral myelin protein; Chi3l3/4, isolated by incubation of non-parenchymal cells with MACS CD11b Microbeads (Miltenyi Biotech; 10 ␮l/107 cells) in MACS buffer (Ca2ϩ/ Ym(1/2); Mrc1, mannose receptor C, type 1; Folr2, folate re- Mg2ϩ free HBSS, 2 mM EDTA, and 0.5% BSA) and passage through ceptor 2 (fetal); Lrg1, leucine-rich ␣-2-glycoprotein 1; and two consecutive positive LS selection columns (Miltenyi Biotech). The Ctss, cathepsin S). 6170 M2-ASSOCIATED GENES LIMIT SEVERITY OF PARASITE INFECTION

Table I. Number and percentage of CD11bϩ cells in the liver at Table III. Gene expression level of M2-associated genes Sepp1, Ngfb, different times post T. congolense infection in wild-type C57BL/6 micea F13a1, and Ctss in the liver of T. congolense-infected IL-10 KO C57BL/6 mice Day No. (ϫ106) % Day 9a Noninfected 1.1 Ϯ 0.26 30.1 Ϯ 4.2 Infected 7 5.2 Ϯ 1.1b 45.2 Ϯ 11.1 Wild-type Mice IL-10 KO Mice 9 6.1 Ϯ 1.8b 50.4 Ϯ 15.0 b 28 12.4 Ϯ 2.6b 55.3 Ϯ 12.1 Sepp1, selenoprotein P, plasma, 1 7.2 Ϯ 1.0 0.82 Ϯ 0.25 b 120 29.5 Ϯ 9.9b 40.2 Ϯ 9.0 Ngfb, nerve growth factor ␤ 5.0 Ϯ 0.78 1.8 Ϯ 0.21 F13a1, coagulation factor XIII, 4.2 Ϯ 0.63 0.49 Ϯ 0.30b a Data are shown as mean Ϯ SEM of three individual mice in each experimental A1 subunit group. Ctss, cathepsin S 11.9 Ϯ 1.49 0.78 Ϯ 0.54b b Higher ( p Ͻ 0.05) than in noninfected mice. a Relative gene expression levels as compared with noninfected wild-type mice (mean Ϯ SEM) were determined at day 9 post infection by reverse transcription followed by real time quantitative PCR and normalized with the house-keeping gene A kinetic of expression revealed that most of the above men- ϩ ribosomal protein S12 in CD11b MCs from wild-type and IL-10 KO mice. The tioned M2-associated genes were significantly induced from days analysis was performed only at day 9 post infection considering the survival of IL-10 21–28 post T. congolense infection (not shown), with the excep- KO mice (11 Ϯ 0.4 days). Gene expression levels in noninfected IL-10 KO mice were similar as in noninfected wild-type mice (not shown). tion of the genes coding for selenoprotein P (Sepp1), nerve growth b Lower ( p Ͻ 0.05) than in infected wild-type mice. factor ␤ (Ngfb), cathepsin S (Ctss), and coagulation factor XIIIa (F13a1) that appeared significantly induced as soon as the first peak of parasitemia was controlled (day 9 post infection) (Table II). Of note, the induction of the latter four genes coincided with including peroxynitrite, are formed during African trypanosomia- the time at which the IFN-␥ titer in the plasma of T. congolense- sis (29, 30). However, since ROS also cause liver injury (31), we infected C57BL/6 mice returned to normal values coupled to a hypothesized that an increased production of selenoprotein P in gradual increase of the IL-10 titer (18, 25). The early induction of liver MCs limits the pathogenicity of African trypanosomiasis by expression of these genes coinciding with the time of emergence of detoxifying ROS. IL-10 suggests their IL-10 dependence. To test this possibility, the course of T. congolense infection was To address the mechanisms by which the subset of M2 triggered compared in Sepp1 KO and wild-type C57BL/6 mice. Sepp1 KO in an IL-10 environment limit the pathogenicity and render the mice controlled the first peak of parasitemia less efficiently than host relatively resistant to the disease, the level of expression of the did wild-type animals, exhibiting higher parasite burden and im- M2-associated genes Ctss, F13a1, Sepp1, and Ngfb was deter- paired parasite clearance capacity (Fig. 1). Approximately 50% of mined in T. congolense-infected IL-10 KO C57BL/6 mice and Sepp1 KO mice died around 9 days post infection, i.e., 3 days after compared with the level of expression observed in wild-type the first peak of parasitemia was reached. Yet, Sepp1 KO mice that C57BL/6 mice. This analysis was performed at day 9 post infec- overcame the early wave of parasitemia died significantly earlier tion considering the short survival of IL-10 KO mice (Table III). than wild-type mice (around 95 days post infection vs 163 Ϯ 12

by guest on October 1, 2021. Copyright 2008 Pageant Media Ltd. The level of expression of all genes tested in infected IL-10 KO days post infection). Thus, in the absence of Sepp1 gene expres- mice returned to the level of noninfected wild-type mice. Thus, the sion, the host becomes more susceptible to T. congolense infection, induction of Ctss, F13a1, Sepp1, and Ngfb can be considered as in terms of parasite control ability and survival. IL-10 dependent in T. congolense-infected C57BL/6 animals. This Selenoprotein P has two domains with respect to selenium con- observation is highly suggestive for a role of the respective gene tent and functions. The N-terminal, selenium poor domain, encom- products in the IL-10-dependent control of disease symptoms in passing two potential redox motifs, was predicted to have enzy- African trypanosomiasis. matic (peroxidase) properties, while the C-terminal, selenium rich domain plays a role in selenium transport (21). To distinguish the 2. Sepp1 KO mice are more susceptible to T. congolense function of the selenoprotein P domains during trypanosome in- http://classic.jimmunol.org infection fection, C57BL/6 mice with a deletion of the C-terminal domain of ⌬ Sepp1 was selected to test whether the IL-10-induced, M2-associ- Sepp1 gene (Sepp1 240-361 mice) that have been recently gener- ated genes limit the pathogenicity and favor the resistance to T. ated (21) were used (Fig. 1). In contrast to Sepp1 KO mice, ⌬ congolense infection. Selenoprotein P, the gene product of Sepp1, Sepp1 240-361 mice controlled T. congolense parasitemia as efﬁ- is postulated to be an antioxidant molecule (26–28), and ROS, ciently and survived as long as wild-type animals, suggesting that the enzymatic activity of selenoprotein P, and not its selenium

Downloaded from transporter function, is important for African trypanosomiasis Table II. Gene expression level of M2-associated genes Sepp1, Ngfb, control. F13a1, and Ctss in the liver of T. congolense-infected wild-type KO C57BL/6 mice 3. Increased necrosis/apoptosis and oxidative stress occur in MCs from T. congolense-infected Sepp1 KO mice Wild-type Micea Pathogenic insults in African trypanosome-infected mice result in Day 9 3–4 Mo necrosis/apoptosis in the liver, including Kupffer cells (17). In Sepp1, selenoprotein P, plasma, 1 4.0 Ϯ 0.60 7.5 Ϯ 1.2 agreement, the shorter survival of T. congolense-infected Sepp1 Ngfb, nerve growth factor ␤ 4.1 Ϯ 0.55 8.0 Ϯ 2.0 KO C57BL/6 mice correlated with higher serum ALT values than F13a1, coagulation factor XIII, 5.6 Ϯ 0.61 7.8 Ϯ 1.6 in wild-type mice (Fig. 2A), indicating that hepatocytes from the A1 subunit former animals were more damaged. Moreover, the percentage of Ctss, cathepsin S 3.5 Ϯ 0.48 8.1 Ϯ 1.0 ϩ Ϫ liver CD11b MCs and of CD11b cells undergoing apoptosis, as a Relative gene expression levels as compared with noninfected wild-type mice evidenced in ﬂow cytometry by Annexin V staining, was higher in (mean Ϯ SEM) were determined at day 9 and 3–4 mo post infection by reverse transcription followed by real time quantitative PCR and normalized with the house- Sepp1 KO than in wild-type animals, both at the peak of para- keeping gene ribosomal protein S12. sitemia (day 7 post infection) and later on (day 90) during infection The Journal of Immunology 6171

FIGURE 1. (A) Parasitemia and (B) survival at different times post T. congolense infection of wild- type, Sepp1⌬240-361, and Sepp1 KO C57BL/6 mice (n ϭ 6). Beyond day 10 post infection, minor but similar waves of parasitemia were observed until the animals died in all experimental groups. -higher (p Ͻ 0.05) than in wild-type ,ء and Sepp1⌬240-361-infected mice.

(Fig. 2, B and C). The difference in apoptosis levels between in- P may play a role in reducing pathogenicity of T. congolense infected wild-type and Sepp1 KO mice correlated with an increased fection by protecting liver parenchymal and non-parenchymal cells accumulation of ROS within liver CD11bϩ MCs (Fig. 2, D and E). from apoptosis/necrosis via its N-terminal domain-mediated enzy- In contrast, T. congolense-infected Sepp1⌬240-361 mice had similar matic function. That the increased apoptosis/necrosis of liver MCs levels of ALT (Fig. 2A), of apoptotic CD11bϩ and CD11bϪ cells in Sepp1 KO mice affected the control of T. congolense growth (Fig. 2, B and C), and of ROS accumulation within CD11bϩ MCs was supported by the observation that treatment of wild-type mice at (Fig. 2, F and G) as infected wild-type mice. Thus, selenoprotein day 18 post infection with gadolinium chloride (32), which eliminates

FIGURE 2. A, ALT level at different times post T. congolense infection in the serum of wild-type, Sepp1⌬240-361, and Sepp1 KO C57BL/6 mice (n ϭ 5). B and C, Level of apoptosis at days 7 and 90 post T. congolense infection within CD11bϩ and CD11bϪ liver cells from wild- type, Sepp1⌬240-361, and Sepp1 KO by guest on October 1, 2021. Copyright 2008 Pageant Media Ltd. C57BL/6 mice. Percentage of Annexin Vϩ cells within gated CD11bϩ or CD11bϪ cells were determined by ﬂow (higher (p Ͻ 0.05 ,ء .(cytometry (n ϭ 9 than in wild-type- and Sepp1⌬240-361- infected mice. D–G, ROS production at days 7 and 90 post T. congolense infection within CD11bϩ liver cells from wild-type, Sepp1⌬240-361, and Sepp1

http://classic.jimmunol.org KO C57BL/6 mice. ROS production

was measured using H2DCFDA oxidation within gated CD11bϩ cells by ﬂow cytometry. Proﬁles are representative of one individual mouse of nine investigated in three independent experiments. For clarity, ROS production in

Downloaded from noninfected wild-type mice, which was similar to noninfected Sepp1 KO and Sepp1⌬240-361 mice, is not shown. D and E, Light line, noninfected Sepp1 KO mice; shaded line, infected wild- type mice; bold line, infected Sepp1 KO mice; F and G, Light line, noninfected Sepp1⌬240-361 mice; shaded line, infected wild-type mice; bold line, infected Sepp1⌬240-361 mice. 6172 M2-ASSOCIATED GENES LIMIT SEVERITY OF PARASITE INFECTION

FIGURE 3. A, Percentage of CD11bϩ cells determined by ﬂow cytometry in the liver of wild-type C57BL/6 mice treated or not treated (PBS) with gadolinium chloride at day 18 post T. congolense infection (n ϭ 5). Cells from nontreated mice were analyzed at the same time as cells from mice treated since 24 h with gadolinium chloride. B, parasitemia in wild-type C57BL/6 mice treated (2) or not treated (PBS) with gadolinium chloride at day 18 post T. congolense infection (n ϭ 5). Treated mice survived 22 Ϯ 1 days post infection vs 165 Ϯ 25 days post infection for not treated mice. #, lower (p Ͻ 0.05) than in control infected higher (p Ͻ 0.05) than in ,ء .mice control infected mice.

90% of liver MCs (Fig. 3) but only 20% of spleen MCs (not shown), Discussion abolished its ability to control the parasitemia and severely shortened The gene expression proﬁle of both human and mouse M2 is now Ϯ Ϯ Ͻ its survival (22 1 days vs 165 25 days in nontreated mice; p relatively well studied. Yet, the mechanisms and downstream ef- 0.01) (Fig. 3). fector molecules by which these cells contribute to protective (34, 4. Absence of Sepp1 does not affect cytokine environment 35) or detrimental (36, 37) type II immune response are poorly and expression of other M2-associated, characterized. In contrast, it is clearly established that IL-10 is a IL-10-dependent genes major and multifunctional regulator of innate and adaptive immu-

by guest on October 1, 2021. Copyright 2008 Pageant Media Ltd. nity. By controlling the immune response, IL-10 creates an envi- Cytokine levels in the plasma and the expression of Ngfb, ronment that favors the persistence of microbes and the develop- F13a1, and Ctss in MCs were measured during T. congolense ment of chronic diseases. However, IL-10 can protect the host infection in Sepp1 KO and Sepp1⌬240-361 mice and compared from the pathogenicity of diseases caused by exacerbated immune with wild-type C57BL/6 mice. The IFN-␥ and IL-10 titers were responses and, as such, avoids tissue or systemic lesions (38). similar in all experimental groups both at the peak of para- Consequently, IL-10 inducible genes represent novel and more sitemia (day 7 post infection) and later on (day 90) during in- specific therapeutic options to intervene in the host’s ability to fection (Fig. 4). Moreover, no significant difference in the production of TNF-␣ and NO, that are known to contribute to the mount a protective immune response during inflammatory/anti- inflammatory processes. In the present study, African trypanosome http://classic.jimmunol.org control of trypanosome growth (8, 33), was observed between infected Sepp1 KO, Sepp1⌬240-361, and wild-type mice (not infection was used as a model to identify IL-10-dependent, M2- shown). The expression in the liver MCs of Ngfb, F13a1, and associated genes that could contribute to protection of the host Ctss was similar in infected Sepp1 KO, Sepp1⌬240-361, and against the pathogenicity associated with the disease. wild-type mice at days 9 and 90 post infection (not shown). Limited tissue damages and long survival in a host relatively Together, these data suggest that the increased susceptibility of resistant to African trypanosome infection result from a balance T. congolense-infected Sepp1 KO mice is not reflected by a between (i) an inflammatory immune response that associates with Downloaded from change in the immune environment or in the expression of the the production of IFN-␥, TNF-␣, and NO as well as with the de- M2 genes potentially associated with lower severity of the dis- velopment of M1, and (ii) a counteracting anti-inflammatory ease tested. response that includes IL-10, the development of M2, and the

FIGURE 4. Plasma concentration of (A) IFN-␥ and (B) IL-10 in wild- type, Sepp1⌬240-361, and Sepp1 KO C57BL/6 mice at days 7 and 90 post T. congolense infection (n ϭ 5). or IL-10 (#) level higher (ء) ␥-IFN (p Ͻ 0.05) than in non infected mice (day 0 post infection). The Journal of Immunology 6173

expansion of Tregs. Both M2 (not shown) and Tregs (18) have Sepp1 KO mice, and Sepp1⌬240-361 mice-expressing selenoprotein been found to contribute to IL-10 production. Genetic polymor- P truncated of the selenium transporter domain but retaining the phism in host populations influences the progression both of nat- domain exerting anti-oxidant activity-in the resistant C57BL/6 ural (39–44) and experimental (45) African trypanosomiasis. background were infected with T. congolense. It has been shown Moreover, increased resistance is under the control of multiple that in Sepp1 KO or Sepp1⌬240-361 mice, severe neurological dys- genes that relate to the control of either the parasitemia or the function occurs in animals fed a selenium-deficient diet. Yet, feed- pathogenic effects of the parasite. These genes remain largely un- ing 0.25 mg selenium/kg largely prevents this phenotype in Sepp1 known (46–48). However, quantitative trait loci studies performed KO or Sepp1⌬240-361 mice (20–22, 73). In this context, it is im- in mice and cattle have revealed chromosomal regions linked to portant that in our experiments, Sepp1 KO and Sepp1⌬240-361 mice control of African trypanosomiasis. In particular in mice infected were supplied with 10 times the normal nutritional requirement, 1 with T. congolense, 5 quantitative trait loci Tir1 on chromosome mg selenium/kg, preventing pathological phenotype and selenium 17, Tir2 on chromosome 5 and Tir3a, Tir3b, and Tir3c on chro- depletion in most tissues including the liver. Our data show that in mosome 1 have been identified (49, 50), among which one, Tir3b, T. congolense-infected wild-type animals, the expression of Sepp1 maps close to the location of the IL-10 gene (51). mRNA gradually increased in liver CD11bϩ MCs after the clear- Herein, by comparing the expression of 12 M2 gene markers in ance of the first peak of parasitemia. Concomitantly, it was not MCs isolated from the liver of T. congolense-infected wild-type and affected in CD11bϪ cells. Of interest, the mRNA expression level IL-10 C57BL/6 KO mice, four genes (Sepp1, Ngfb, F13a1, and Ctss) of other selenoproteins tested (thioredoxin reductases 1–3 and glu- that are expressed in relatively resistant wild-type animals, but not in tathione peroxidases 1–4) in CD11bϪ cells and CD11bϩ MCs highly susceptible IL-10 KO mice, have been identified. Thus, Sepp1, from the liver was not up-regulated in T. congolense infected wild- Ngfb, F13a1, and Ctss can be considered as in vivo IL-10-dependent, type and Sepp1 KO mice (not shown). These data suggest that M2-associated genes, the respective gene products of which could selenoprotein P is the major selenoprotein induced in infected mice favor the control of the pathogenicity and hereby the resistance of and that none of the other selenoproteins tested compensates, in C57BL/6 mice to T. congolense infection. With the exception of the MCs, for the loss of selenoprotein P in Sepp1 KO mice. T. congolense gene product of Ctss, which contributes to Ag presentation and matrix infection is more pathogenic in Sepp1 KO mice. The increased par- degradation (52, 53), the other genes encode proteins controlling asitemia and reduced survival of these animals as compared with inflammatory/anti-inflammatory processes (54–58). wild-type mice correlate with increased liver injury, including apo- Despite producing higher levels of IL-10 than C57BL/6 mice, ptosis/necrosis of hepatocytes and non-parenchymal cells. Of note, BALB/C mice, that are highly susceptible to T. congolense infec- the increased susceptibility of Sepp1 KO mice to T. congolense in- tion and die within 10 Ϯ 0.3 days post infection, did not up-reg- fection does not associate with a change in cytokine environment or ulate the expression of Sepp1, Ngfb, F13a1, and Ctss in MCs (not in the expression of IL-10-dependent M2 genes associated with re- shown). It has been suggested that infected BALB/C mice are not sistance to the disease (Ngfb, F13a1, and Ctss). In addition, while all responsive to the effect of IL-10 (17). In fact, T. congolense-sus- IL-10 KO mice died after the first peak of parasitemia, only half of the ceptible BALB/c mice, that do not control the first peak of para- Sepp1 KO mice died at that moment, suggesting that the effect of sitemia, produce simultaneously IFN-␥ and IL-10 in the early stage of IL-10 on resistance to T. congolense is not restricted to the sole in-

by guest on October 1, 2021. Copyright 2008 Pageant Media Ltd. infection while resistant C57BL/6 mice change from a predominant duction of IL-10-dependent genes identified in the present study. IFN-␥ environment to a predominant IL-10 environment after the In contrast to Sepp1 KO mice, Sepp1⌬240-361 mice controlled T. control of the first peak of parasitemia (59). Thus, we cannot exclude congolense infection as efficiently as wild-type animals, suggest- that the mixed IFN-␥ and IL-10 response in BALB/C mice in the ing that the anti-oxidant activity of selenoprotein P is important to early stage of infection impairs the efficient activation of MCs, in- avoid tissue damage and ensure survival. In agreement, in T. con- cluding up-regulation of Sepp1, Ngfb, F13a1, and Ctss expression. golense-infected mice, liver MCs from Sepp1 KO mice, but not Selenoprotein P is the major transporter of selenium, a trace from Sepp1⌬240-361 mice, accumulate more ROS than wild-type element required for normal development (60, 61). This secreted mice. Although ROS can limit trypanosome growth (29, 30), par- glycoprotein is almost exclusively synthesized by the liver, but asitemia increases in Sepp1 KO-infected mice during the first, http://classic.jimmunol.org local production in virtually all tissues has been described (62– most aggressive wave of parasitemia. Since liver MCs were more 64), including M2 elicited in several infection and cancer models prone to undergo apoptosis/necrosis in the latter animals, and since (2). Selenoprotein P plasma level is depressed in a number of gadolinium chloride treatment, which eliminates 90% of liver MCs human pathologies including cirrohsis and Crohn’s disease (65, in wild-type mice, results in loss of parasite control and early death 66). Moreover, human and/or murine Sepp1 gene promoter activity of the host, selenoprotein P may contribute to trypanosomiasis is impaired by IFN-␥, TNF-␣, and IL-1␤ (67) while induced by control by preserving the integrity/viability of MCs. Yet, Sepp1

Downloaded from TGF-␤ (68) and IL-10 (2) suggesting a differential regulation of KO mice that survived the first peak of parasitemia succumbed selenoprotein P expression during type 1 or 2 inflammatory reac- earlier than wild-type and Sepp1⌬240-361 mice, suggesting that tions. In addition, selenoprotein P was proposed to function in MCs contribute to resistance to trypanosomiasis by more than their anti-oxidant defense, as a scavenger of peroxynitrite or as a phos- parasite clearance function. African trypanosome infection per- pholipid hydroperoxide thiol peroxidase (26, 28, 69). Accordingly, turbs the triglyceride metabolism (74) and the release of acute selenoprotein P expressed in human pancreatic cancer cells asso- phase proteins (25), two major functions of hepatocytes. Because ciates with resistance to chemotherapy by suppressing the induc- ALT level was increased in infected Sepp1 KO mice, a role for tion of ROS (70). Hereby, its pH-dependent heparin-binding prop- selenoprotein P and MCs in the protection of hepatocytes from erty suggests that selenoprotein P binds to host cell membranes in apoptosis/necrosis cannot be excluded. areas of inflammation that typically have low pH, preventing cell In summary, by analyzing the gene profile in M2-oriented MCs injury induced by ROS (70–72). in T. congolense-infected mice, we have identified IL-10-depen- Currently, a documented role for selenoprotein P in M2 and in dent genes that could prevent the pathogenicity and, thus, contrib- infectious diseases is lacking. In an attempt to validate our ap- ute to the resistance of the host to the disease. These genes code for proach to identify IL-10-dependent, M2-associated genes contrib- proteins potentially involved in the control of inflammatory pro- uting to the control of the severity of African trypanosomiasis, cesses. One of these genes coding for selenoprotein P was found 6174 M2-ASSOCIATED GENES LIMIT SEVERITY OF PARASITE INFECTION

essential to control the pathogenic effect of the parasite including 21. Hill, K. E., J. Zhou, L. M. Austin, A. K. Motley, A. J. Ham, G. E. Olson, excessive production of ROS as well as destruction of liver MCs J. F. Atkins, R. F. Gesteland, and R. F. Burk. 2007. The selenium-rich C-terminal domain of mouse selenoprotein P is necessary for the supply of selenium to brain and hepatocytes. In a more general context, M2 genes that con- and testis but not for the maintenance of whole body selenium. J. Biol. Chem. tribute to the limitation of severity of African trypanosome infec- 282: 10972–10980. 22. Hill, K. E., J. Zhou, W. J. McMahan, A. K. Motley, and R. F. Burk. 2004. tion may represent targets for therapeutic intervention aiming to Neurological dysfunction occurs in mice with targeted deletion of the selenopro- limit potentially harmful inflammatory responses. tein P gene. J. Nutr. 134: 157–161. 23. Otesile, E. B., M. Lee, and H. Tabel. 1991. 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