A Role for Natural Regulatory T Cells in the Pathogenesis of Experimental Cerebral Malaria
2007; Elsevier BV; Volume: 171; Issue: 2 Linguagem: Inglês
10.2353/ajpath.2007.061033
ISSN1525-2191
AutoresFiona H. Amante, Amanda C. Stanley, Louise M. Randall, Yonghong Zhou, Ashraful Haque, Karli M. McSweeney, Andrew P. Waters, Chris J. Janse, Michael F. Good, Geoffrey R. Hill, Christian Engwerda,
Tópico(s)Complement system in diseases
ResumoCerebral malaria (CM) is a serious complication of Plasmodium falciparum infection that is responsible for a significant number of deaths in children and nonimmune adults. A failure to control blood parasitemia and subsequent sequestration of parasites to brain microvasculature are thought to be key events in many CM cases. Here, we show for the first time, to our knowledge, that CD4+CD25+Foxp3+ natural regulatory T (Treg) cells contribute to pathogenesis by modulating immune responses in P. berghei ANKA (PbA)-infected mice. Depletion of Treg cells with anti-CD25 monoclonal antibody protected mice from experimental CM. The accumulation of parasites in the vasculature and brain was reduced in these animals, resulting in significantly lower parasite burdens compared with control animals. Mice lacking Treg cells had increased numbers of activated CD4+ and CD8+ T cells in the spleen and lymph nodes, but CD8+ T-cell recruitment to the brain was selectively reduced in these mice. Importantly, a non-Treg-cell source of interleukin-10 was critical in preventing experimental CM. Finally, we show that therapeutic administration of anti-CD25 monoclonal antibody, even when blood parasitemia is established, can prevent disease, confirming a critical and paradoxical role for Treg cells in experimental CM pathogenesis. Cerebral malaria (CM) is a serious complication of Plasmodium falciparum infection that is responsible for a significant number of deaths in children and nonimmune adults. A failure to control blood parasitemia and subsequent sequestration of parasites to brain microvasculature are thought to be key events in many CM cases. Here, we show for the first time, to our knowledge, that CD4+CD25+Foxp3+ natural regulatory T (Treg) cells contribute to pathogenesis by modulating immune responses in P. berghei ANKA (PbA)-infected mice. Depletion of Treg cells with anti-CD25 monoclonal antibody protected mice from experimental CM. The accumulation of parasites in the vasculature and brain was reduced in these animals, resulting in significantly lower parasite burdens compared with control animals. Mice lacking Treg cells had increased numbers of activated CD4+ and CD8+ T cells in the spleen and lymph nodes, but CD8+ T-cell recruitment to the brain was selectively reduced in these mice. Importantly, a non-Treg-cell source of interleukin-10 was critical in preventing experimental CM. Finally, we show that therapeutic administration of anti-CD25 monoclonal antibody, even when blood parasitemia is established, can prevent disease, confirming a critical and paradoxical role for Treg cells in experimental CM pathogenesis. Cerebral malaria (CM) is a major cause of death in people infected with Plasmodium falciparum, with most deaths occurring in young children in sub-Saharan Africa. An estimated 10 to 20% of children who develop CM die, and a significant proportion of survivors have permanent neurological damage.1Snow RW Trape JF Marsh K The past, present and future of childhood malaria mortality in Africa.Trends Parasitol. 2001; 17: 593-597Abstract Full Text Full Text PDF PubMed Scopus (213) Google Scholar, 2Mung'Ala-Odera V Snow RW Newton CR The burden of the neurocognitive impairment associated with Plasmodium falciparum malaria in sub-Saharan Africa.Am J Trop Med Hyg. 2004; 71: 64-70PubMed Google Scholar, 3Carter JA Ross AJ Neville BG Obiero E Katana K Mung'ala-Odera V Lees JA Newton CR Developmental impairments following severe falciparum malaria in children.Trop Med Int Health. 2005; 10: 3-10Crossref PubMed Scopus (112) Google Scholar CM can be associated with sequestration of parasitized red blood cells (pRBCs) in the brain microvasculature4MacPherson GG Warrell MJ White NJ Looareesuwan S Warrell DA Human cerebral malaria: a quantitative ultrastructural analysis of parasitized erythrocyte sequestration.Am J Pathol. 1985; 119: 385-401PubMed Google Scholar and secretion of toxic molecules by parasites,5Schofield L Hackett F Signal transduction in host cells by a glycosylphosphatidylinositol toxin of malaria parasites.J Exp Med. 1993; 177: 145-153Crossref PubMed Scopus (400) Google Scholar as well as inflammatory components of the host immune response, including secretion of cytokines6Clark IA Rockett KA The cytokine theory of human cerebral malaria.Parasitol Today. 1994; 10: 410-412Abstract Full Text PDF PubMed Scopus (163) Google Scholar and recruitment of activated leukocytes to the brain.7Patnaik JK Das BS Mishra SK Mohanty S Satpathy SK Mohanty D Vascular clogging, mononuclear cell margination, and enhanced vascular permeability in the pathogenesis of human cerebral malaria.Am J Trop Med Hyg. 1994; 51: 642-647PubMed Google Scholar, 8Clark IA Awburn MM Whitten RO Harper CG Liomba NG Molyneux ME Taylor TE Tissue distribution of migration inhibitory factor and inducible nitric oxide synthase in falciparum malaria and sepsis in African children.Malar J. 2003; 2: 6Crossref PubMed Google Scholar, 9Taylor TE Fu WJ Carr RA Whitten RO Mueller JG Fosiko NG Lewallen S Liomba NG Molyneux ME Differentiating the pathologies of cerebral malaria by postmortem parasite counts.Nat Med. 2004; 10: 143-145Crossref PubMed Scopus (514) Google Scholar An experimental model of CM (ECM) caused by infection of C57BL/6 and CBA mice with P. berghei ANKA (PbA) displays many features of human CM and has allowed the identification of several important factors in CM pathogenesis. Both CD4+ and CD8+ T cells contribute to the development of ECM,10Yañez DM Manning DD Cooley AJ Weidanz WP van der Heyde HC Participation of lymphocyte subpopulations in the pathogenesis of experimental murine cerebral malaria.J Immunol. 1996; 157: 1620-1624PubMed Google Scholar, 11Hermsen C van de Wiel T Mommers E Sauerwein R Eling W Depletion of CD4+ or CD8+ T-cells prevents Plasmodium berghei induced cerebral malaria in end-stage disease.Parasitology. 1997; 114: 7-12Crossref PubMed Scopus (89) Google Scholar, 12Belnoue E Kayibanda M Vigario AM Deschemin JC van Rooijen N Viguier M Snounou G Renia L On the pathogenic role of brain-sequestered αβ CD8+ T cells in experimental cerebral malaria.J Immunol. 2002; 169: 6369-6375PubMed Google Scholar, 13Nitcheu J Bonduelle O Combadiere C Tefit M Seilhean D Mazier D Combadiere B Perforin-dependent brain-infiltrating cytotoxic CD8+ T lymphocytes mediate experimental cerebral malaria pathogenesis.J Immunol. 2003; 170: 2221-2228PubMed Google Scholar and the spleen seems to be a key site for priming of PbA-specific T-cell responses.14Hermsen CC Mommers E van de Wiel T Sauerwein RW Eling WM Convulsions due to increased permeability of the blood-brain barrier in experimental cerebral malaria can be prevented by splenectomy or anti-T cell treatment.J Infect Dis. 1998; 178: 1225-1227Crossref PubMed Scopus (43) Google Scholar In addition, the proinflammatory cytokines interferon (IFN)-γ,15Grau GE Heremans H Piguet PF Pointaire P Lambert PH Billiau A Vassalli P Monoclonal antibody against interferon γ can prevent experimental cerebral malaria and its associated overproduction of tumor necrosis factor.Proc Natl Acad Sci USA. 1989; 86: 5572-5574Crossref PubMed Scopus (283) Google Scholar, 16Amani V Vigario AM Belnoue E Marussig M Fonseca L Mazier D Renia L Involvement of IFN-gγ receptor-medicated signaling in pathology and anti-malarial immunity induced by Plasmodium berghei infection.Eur J Immunol. 2000; 30: 1646-1655Crossref PubMed Scopus (152) Google Scholar tumor necrosis factor,17Grau GE Fajardo LF Piguet PF Allet B Lambert PH Vassalli P Tumor necrosis factor (cachectin) as an essential mediator in murine cerebral malaria.Science. 1987; 237: 1210-1212Crossref PubMed Scopus (616) Google Scholar and LTα,18Engwerda CR Mynott TL Sawhney S De Souza JB Bickle QD Kaye PM Locally up-regulated lymphotoxin α, not systemic tumor necrosis factor α, is the principle mediator of murine cerebral malaria.J Exp Med. 2002; 195: 1371-1377Crossref PubMed Scopus (211) Google Scholar as well as perforin,13Nitcheu J Bonduelle O Combadiere C Tefit M Seilhean D Mazier D Combadiere B Perforin-dependent brain-infiltrating cytotoxic CD8+ T lymphocytes mediate experimental cerebral malaria pathogenesis.J Immunol. 2003; 170: 2221-2228PubMed Google Scholar all seem to play a role in ECM pathogenesis.Although the risk factors that predispose individuals to develop CM remain largely unknown, high blood parasitemia is significantly correlated with increased risk of CM.19Molyneux ME Taylor TE Wirima JJ Borgstein A Clinical features and prognostic indicators in paediatric cerebral malaria: a study of 131 comatose Malawian children.Q J Med. 1989; 71: 441-459PubMed Google Scholar Effective immune responses to Plasmodium blood stages only emerge in people living in malaria-endemic regions after several years of repeated malaria infections.20Greenwood BM Bradley AK Greenwood AM Byass P Jammeh K Marsh K Tulloch S Oldfield FS Hayes R Mortality and morbidity from malaria among children in a rural area of The Gambia, West Africa.Trans R Soc Trop Med Hyg. 1987; 81: 478-486Abstract Full Text PDF PubMed Scopus (528) Google Scholar Antibodies against the surface of the merozoite lifecycle stage of P. falciparum and cell-mediated immunity are both thought to be required for protective immunity, but they may also contribute to pathology.21Good MF Xu H Wykes M Engwerda CR Development and regulation of cell-mediated immune responses to the blood stages of malaria: implications for vaccine research.Annu Rev Immunol. 2005; 23: 69-99Crossref PubMed Scopus (149) Google Scholar Recently, CD4+CD25+ regulatory T (Treg) cells were shown to be rapidly induced in vivo in humans following P. falciparum infection, and this was associated with a burst of transforming growth factor-β production, decreased parasite-specific immune responses, and higher rates of parasite growth.22Walther M Tongren JE Andrews L Korbel D King E Fletcher H Andersen RF Bejon P Thompson F Dunachie SJ Edele F de Souza JB Sinden RE Gilbert SC Riley EM Hill AV Upregulation of TGF-beta, FOXP3, and CD4+CD25+ regulatory T cells correlates with more rapid parasite growth in human malaria infection.Immunity. 2005; 23: 287-296Abstract Full Text Full Text PDF PubMed Scopus (291) Google Scholar Treg cells have also been shown to enhance P. yoelii infection in BALB/c mice.23Hisaeda H Maekawa Y Iwakawa D Okada H Himeno K Kishihara K Tsukumo S Yasutomo K Escape of malaria parasites from host immunity requires CD4+CD25+ regulatory T cells.Nat Med. 2004; 10: 29-30Crossref PubMed Scopus (290) Google Scholar Together, these reports support a detrimental role for Treg cells in controlling parasites during malaria infections, although their effect on CM pathogenesis is unknown.Naturally occurring CD25+CD4+ Treg cells, constituting 5 to 10% of peripheral CD4+ T cells in mice and humans, express the forkhead/winged helix transcription factor Foxp3.24Sakaguchi S Naturally arising CD4+ regulatory T cells for immunologic self-tolerance and negative control of immune responses.Annu Rev Immunol. 2004; 22: 531-562Crossref PubMed Scopus (2883) Google Scholar They are produced in the thymus as a distinct and functionally mature population, but there is also evidence that they are induced in the periphery.25Kretschmer K Apostolou I Hawiger D Khazaie K Nussenzweig MC von Boehmer H Inducing and expanding regulatory T cell populations by foreign antigen.Nat Immunol. 2005; 6: 1219-1227Crossref PubMed Scopus (1027) Google Scholar Treg cells play a critical role in the maintenance of immunological self-tolerance, as well as the control of immune responses to pathogens,26Belkaid Y Rouse BT Natural regulatory T cells in infectious disease.Nat Immunol. 2005; 6: 353-360Crossref PubMed Scopus (863) Google Scholar commensal microbes, and environmental antigens.24Sakaguchi S Naturally arising CD4+ regulatory T cells for immunologic self-tolerance and negative control of immune responses.Annu Rev Immunol. 2004; 22: 531-562Crossref PubMed Scopus (2883) Google Scholar Treg cells mediate their effects by direct cell contact27Thornton AM Shevach EM Suppressor effector function of CD4+CD25+ immunoregulatory T cells is antigen nonspecific.J Immunol. 2000; 164: 183-190PubMed Google Scholar or the secretion of anti-inflammatory cytokines such as interleukin (IL)-10 and transforming growth factor-β.28Powrie F Read S Mottet C Uhlig H Maloy K Control of immune pathology by regulatory T cells.Novartis Found Symp. 2003; 252: 92-114Crossref PubMed Google Scholar Here, we show that Treg cells play an important role in modulating the host immune response to PbA during the pathogenesis of ECM. This is one of the first examples of Treg cells contributing to a pathogenic process during an infectious disease.Materials and MethodsMiceFemale C57BL/6 and CBA/CaH mice 5 to 6 weeks of age were purchased from the Australian Resource Centre (Canning Vale, Perth, Western Australia) and maintained under conventional conditions. Female C57BL/6 mice deficient in IL-10 (originally obtained from Jackson Laboratories, Bar Harbor, ME) were bred and maintained in house. All animal procedures were approved and monitored by the Queensland Institute of Medical Research Animal Ethics Committee.Parasites and InfectionsP. berghei ANKA (PbA) was used in all experiments after one in vivo passage in mice. A transgenic PbA (231c1l) line expressing luciferase and green fluorescent protein under the control of the ef1-α promoter was used for experiments involving in vivo imaging.29Franke-Fayard B Janse CJ Cunha-Rodrigues M Ramesar J Buscher P Que I Lowik C Voshol PJ den Boer MA van Duinen SG Febbraio M Mota MM Waters AP Murine malaria parasite sequestration: CD36 is the major receptor, but cerebral pathology is unlinked to sequestration.Proc Natl Acad Sci USA. 2005; 102: 11468-11473Crossref PubMed Scopus (242) Google Scholar All mice were infected by injecting 105 pRBCs intravenously (i.v.) via the lateral tail vein. Blood parasitemia was monitored by examination of Diff-Quick (Lab Aids, Narrabeen, NSW, Australia)-stained thin blood smears obtained from tail bleeds. Anemia was estimated by measuring hemoglobin levels using a HemoCue Hb 201 analyzer according to the manufacturer's instructions (HemoCue AB, Angelholm, Sweden). For serum cytokine analysis, 100 μl of blood was collected via the lateral tail vein before infection and 5 days after PbA infection. Blood was allowed to clot, and serum was collected and stored at −70°C until required.Disease AssessmentMice were monitored twice daily after day 5 postinfection (p.i.), and clinical ECM evaluated. Clinical ECM scores were defined by the presentation of the following signs: ruffled fur, hunching, wobbly gait, limb paralysis, convulsions, and coma. Each sign was given a score of 1. Animals with severe ECM (accumulative scores ≥4) were sacrificed by CO2 asphyxiation according to ethics guidelines, and the day of death was deemed to be the following day.AntibodiesAllophycocyanin-conjugated anti-TCRβ chain, phycoerythrin (PE)-Cy5- or PE-conjugated anti-CD4, PE-conjugated anti-CD69, anti-CD25-biotin (7D4), PE-Cy5-conjugated anti-CD8, PE-conjugated anti-Ly6G, fluorescein isothiocyanate-conjugated anti-Ly6C, allophycocyanin-conjugated anti-B220, fluorescein isothiocyanate-conjugated anti-CD19, allophycocyanin-conjugated anti-CD11c, PE-Cy5-conjugated anti-CD11b, fluorescein isothiocyanate-conjugated anti-CD45.2, biotin-conjugated anti-NK1.1, anti-intercellular adhesion molecule (ICAM)-1, anti-vascular cell adhesion molecule (VCAM)-1 monoclonal antibodies (mAbs), and Alexa Fluor 488-conjugated streptavidin were purchased from Biolegend (San Diego, CA) or BD Biosciences (Franklin Lakes, NJ). PE-Cy5-conjugated α-galactosylceramide (αGalCer) mouse CD1d tetramers were a generous gift from Dale Godfrey and Daniel Pellicci (University of Melbourne, Melbourne, VIC, Australia). PE-labeled anti-mouse Foxp3 mAb was purchased from eBioscience (San Diego, CA). Anti-CD25 (PC61; rat IgG1) and isotype control mAb (MAC49; rat IgG1) were purified from culture supernatants by protein G column purification (Amersham, Uppsala, Sweden) followed by endotoxin removal (Mustang Membranes; Pall Life Sciences, East Hills, NY). Purified control rat IgG were also used in some experiments and purchased from Sigma-Aldrich (Castle Hill, NSW, Australia).Preparation of Tissue Mononuclear CellsSpleen cells were isolated by digesting tissue in collagenase type 4 (1 mg/ml; Worthington Biochemical Corp., Lakewood, NJ) and deoxyribonuclease I (0.5 mg/ml; Worthington Biochemical) at room temperature for 40 minutes. Splenocytes and lymph node cells (from superficial cervical, axillary, brachial, mesenteric, and inguinal lymph nodes) were isolated by passing tissue through a 100-μm sieve and washing twice with phosphate-buffered saline supplemented with 2% (v/v) fetal calf serum (wash buffer). Red blood cells were then lysed using red cell lysis buffer (Sigma-Aldrich), according to the manufacturer's instructions, underlaid with fetal calf serum, and centrifuged at 443 × g for 5 minutes. Cell pellets were washed once more with wash buffer and cells counted. Brain mononuclear cells were isolated by digesting tissue as described above before passing through a 100-μm sieve and washing twice with wash buffer. The cell pellet was resuspended in 33% (v/v) Percoll and centrifuged at 693 × g for 12 minutes at room temperature. Supernatant containing debris was removed, and the leukocyte pellet was washed once in wash buffer, depleted of red blood cells as described above, underlaid with fetal calf serum, and centrifuged at 443 × g for 5 minutes. Cell pellets were washed once more with wash buffer and cells counted. Peripheral blood leukocytes were prepared from heparinized blood that was depleted of red blood cells by three or four treatments with red cell lysis buffer (Sigma-Aldrich) according to the manufacturer's instructions.Flow Cytometric AnalysisFor the staining of cell surface antigens, cells were incubated with fluorochrome-conjugated or biotinylated mAbs on ice for 30 minutes followed by Alexa Fluor 488-streptavidin incubation for an additional 30 minutes. Intracellular staining for Foxp3 was performed on fixed/permeabilized cells using PE-labeled anti-mouse Foxp3 kit (eBioscience), according to the manufacturer's instructions. Data were acquired on a FACSCalibur flow cytometer and analyzed using Cell Quest Pro software (BD Biosciences). Cell populations in the spleen and brain were defined as follows: CD4+ T cells (CD4+TCR+), CD8+ T cells (CD8+TCR+), B cells (B220+CD19+), neutrophils (CD11b+Ly6G+), macrophages/monocytes (CD11b+Ly6C+), dendritic cells (DC; CD11chi), NK cells (NK1.1+TCR−), NK T cells (CD1d αGalCer tetramer+ NK1.1+), and microglia (CD45intermediate (int)CD11c+ or CD11b+). Cytokines in serum samples collected 5 days p.i. were quantified using the cytometric bead array (CBA) inflammatory kit (BD Biosciences) on a FACScan cytometer equipped with Cell Quest Pro and CBA software (BD Biosciences).CD25+ T-Cell DepletionCD25+ T-cell depletion was performed by intraperitoneal (i.p.) injection of 0.5 mg of anti-CD25 mAb (PC61) 1 day or 14 days before PbA infection. The efficacy of CD25 depletion was confirmed by fluorescence-activated cell sorting (FACS) analysis using anti-CD4, anti-CD25, and anti-Foxp3 antibodies.IFNγ ELISPOTCD4+ and CD8+ T cells were positively selected from RBC-depleted splenocytes using magnetic activated cell sorting according to protocols recommended by the manufacturer of the metallo-conjugated anti-CD4 and anti-CD8 antibodies and positive selection columns (Miltenyi Biotec, Bergisch Gladbach, Germany). Cells isolated by this procedure were greater than 98% pure as assessed by FACS. The IFNγ ELISPOT was performed as previously described.18Engwerda CR Mynott TL Sawhney S De Souza JB Bickle QD Kaye PM Locally up-regulated lymphotoxin α, not systemic tumor necrosis factor α, is the principle mediator of murine cerebral malaria.J Exp Med. 2002; 195: 1371-1377Crossref PubMed Scopus (211) Google ScholarIn Vivo Bioluminescence ImagingLuciferase-expressing PbA pRBCs were visualized by imaging whole bodies or dissected organs with an I-CCD photon-counting video camera and in vivo imaging system (IVIS 100; Xenogen, Alameda, CA). On day 5 p.i., when ECM symptoms were observed in infected control animals, mice were anesthetized with fluorothane and injected subcutaneously with 0.1 ml of 5 mg/ml d-luciferin firefly potassium salt (Xenogen). Images were then captured on the IVIS 100 according to the manufacturer's instructions. Parasites were visualized in the brain after removal from mice that had been perfused with 20 ml of saline via the heart. Bioluminescence generated by luciferase transgenic PbA in mice or brain tissue was measured according to the manufacturer's instructions using the same regions of measurement for all samples being compared. The unit of measurement was photons/second/cm2/steer radiant (p/sec/cm2/sr).ImmunohistochemistryICAM-1 and VCAM-1 staining was conducted on 6-μm acetone-fixed brain sections, and primary antibodies were detected with appropriate secondary detection reagents and horseradish peroxidase according to the manufacturer's instructions (Vector Laboratories, Peterborough, UK). Sections were dehydrated and mounted before microscopic examination. These sections were then used to count ICAM-1- and VCAM-1-positive vessels in 25 consecutive microscopic fields of view at a final magnification of 400×.Real-Time Reverse Transcriptase-Polymerase Chain ReactionTotal RNA was extracted from the spleen using TRIzol reagent (Invitrogen Life Technologies, Carlsbad, CA), and an RNeasy Mini Kit with on-column DNase digestion (Qiagen, Valencia, CA). RNA samples were reverse-transcribed into cDNA using the cDNA Archive Kit (Applied Biosystems, Foster City, CA) according to the manufacturer's instructions. The number of IFNγ and IL-10 cDNA molecules in each sample were calculated by using TaqMan gene expression assays (Applied Biosystems), and the number of HPRT (forward: 5′-GTTGGATACAGGCCAGACTTTGTTG-3′; reverse: 5′-GATTCAACCTTGCGCTCATCTTAGGC-3′) (housekeeping gene) cDNA molecules in each sample were calculated by real-time reverse transcription-polymerase chain reaction using Platinum SYBR Green Master Mix (Invitrogen Life Technologies). All reverse transcription-polymerase chain reactions were performed on a Corbett Research RG-3000 Rotor Gene (Corbett Life Sciences, Sydney, NSW, Australia). Standard curves were generated with known amounts of cDNA for each gene, and the number of cytokine molecules per 1000 HPRT molecules in each sample was calculated.Statistical AnalysisDifferences in survival of treatment groups were analyzed using the Kaplan-Meier log-rank test. Differences in parasitemia, cytokine levels, and bioluminescence were analyzed using either the Mann-Whitney U-test or the Student's t-test where indicated. For all statistical tests, P < 0.05 was considered significant.ResultsMice Depleted of CD4+CD25+Foxp3+ Treg Cells Do Not Develop ECMTo establish whether natural Treg cells contribute to the development of ECM, we used an anti-CD25 mAb (PC61) to deplete CD4+CD25+Foxp3+ Treg cells in C57BL/6 mice. Specific depletion of CD4+CD25+Foxp3+ Treg cells and no other major lymphocyte population in the spleen was confirmed by flow cytometry 24 hours after antibody administration (Figure 1, A and B). Furthermore, we confirmed that CD4+CD25+Foxp3+ Treg cells were depleted and had not simply down-regulated CD25 expression, as previously reported,30Kohm AP McMahon JS Podojil JR Begolka WS DeGutes M Kasprowicz DJ Ziegler SF Miller SD Cutting Edge: Anti-CD25 monoclonal antibody injection results in the functional inactivation, not depletion, of CD4+CD25+ T regulatory cells.J Immunol. 2006; 176: 3301-3305PubMed Google Scholar because there was no accumulation of CD4+CD25−Foxp3+ Treg cells in mice receiving anti-CD25 mAb (data not shown). We also demonstrated that CD4+CD25+Foxp3+ Treg cells were depleted by anti-CD25 mAb treatment in the lymph nodes, blood, and brain (see Supplemental Figure 1 at http://ajp.amjpathol.org). These cells remained at similar numbers in the spleen and lymph nodes up until the time that control animals died with ECM but increased over the course of infection in the blood and brain (see Supplemental Figure 1 at http://ajp.amjpathol.org).Ninety percent of mice treated with an isotype control antibody the day before PbA infection developed severe neurological signs of ECM between days 6 and 9 p.i. and were subsequently sacrificed (Figure 1C). In contrast, mice depleted of CD4+CD25+Foxp3+ Treg cells the day before infection showed a significant (P < 0.001) increase in survival (80% survival on day 18 p.i.) (Figure 1C) and a small but significant reduction (P < 0.05) in blood parasitemia at days 5, 6, and 7 p.i. compared with control-treated mice (Figure 1D). Clinical scores were also reduced in mice depleted of Treg cells (Figure 1E), and these animals developed no neurological signs of ECM and survived until 3 weeks after infection, when they developed hyperparasitemia (Figure 1D) and severe anemia (hemoglobin levels <20 g/L). Histological examination of the brain showed no cerebral hemorrhages in ECM-resistant mice that received anti-CD25 mAb, unlike in control PbA-infected animals (data not shown).To exclude the possibility that anti-CD25 mAb might directly affect emerging antiparasitic immune responses, including the generation of inducible Treg cells, we next depleted Treg cells 14 days before PbA infection, a time period that allows continued Treg-cell depletion31Oldenhove G de Heusch M Urbain-Vansanten G Urbain J Maliszewski C Leo O Moser M CD4+CD25+ regulatory T cells control T helper cell type 1 responses to foreign antigens induced by mature dendritic cells in vivo.J Exp Med. 2003; 198: 259-266Crossref PubMed Scopus (201) Google Scholar as well as clearance of antibody from the blood32Loughry A Fairchild S Athanasou N Edwards J Hall FC Inflammatory arthritis and dermatitis in thymectomized, CD25+ cell-depleted adult mice.Rheumatology (Oxford). 2005; 44: 299-308Crossref PubMed Scopus (15) Google Scholar (see Supplemental Figure 2 at http://ajp.amjpathol.org). Specific depletion of CD4+CD25+Foxp3+ Treg cells and no other major leukocyte population was confirmed by flow cytometry 13 days after anti-CD25 mAb administration and the day before infection (Figure 2, A and B), and we again demonstrated that CD4+CD25+Foxp3+ Treg cells were depleted in the lymph nodes, blood, and brain using this anti-CD25 mAb treatment regime (see Supplemental Figure 1 at http://ajp.amjpathol.org).Figure 2Anti-CD25 mAb treatment 14 days before PbA infection prevents ECM in C57BL/6 mice. Mice were injected i.p. with 0.5 mg of anti-CD25 mAb (open bars or open triangles) or 0.5 mg of control mAb (closed bars or closed circles) 14 days before infection with PbA. FACS analysis of splenic CD4+, CD8+, NK, NK T, and B cell numbers (A) as well as CD4+CD25+Foxp3+ T cells (B) on the day of infection are shown. Data represent the mean ± SEM of individual samples from three mice per treatment group. Survival (C), parasitemia (mean ± SEM) (D), and clinical disease (mean score ± SEM) (E) were monitored. The open box in C–E indicates the time when mice displayed ECM symptoms. Data are from one representative experiment of four performed. Statistical differences of *P < 0.05 are indicated.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Mice depleted of Treg cells 14 days before infection failed to develop ECM (Figure 2C), had significantly (P < 0.05) lower blood parasitemia on day 6 p.i. (Figure 2D), and had lower clinical scores (Figure 2E) compared with control mice. Again, these mice survived until the third week after infection with no signs of ECM and ultimately developed hyperparasitemia (Figure 2D) and severe anemia. Therefore, our data show that Treg-cell depletion 14 days before PbA infection results in the same outcome as Treg-cell depletion the day before infection and indicate a critical role for natural Treg cells, but not inducible Treg cells, in ECM pathogenesis.Prevention of Cerebral PbA Accumulation in the Absence of Treg CellsAlthough small reductions in blood parasitemia were observed in the absence of Treg cells (Figure 1, Figure 2), this may not reflect overall differences in parasite burden because of the ability of PbA to accumulate in vasculature and tissue. Therefore, to visualize the effect of Treg-cell depletion on parasite burden, we infected mice 14 days after anti-CD25 mAb treatment with a transgenic PbA line that constitutively expressed luciferase. Following injection of luciferin into mice at day 5 p.i., when ECM symptoms were present, parasites were observed as bioluminescence in extremities such as the tail, ears, nose, and foot pads, where blood vessels were close to the surface of the skin, as well as tissues such as the lungs and brain (Figure 3A). In this experiment, a small, but significant (P < 0.05), reduction in blood parasitemia was observed in mice depleted of Treg cells 14 days before infection, as determined by microscopic examination of blood smears (Figure 3B). However, when bioluminescence generated from parasites was measured, a much larger and highly significant reduction (P < 0.001) was observed in mice treated with anti-CD25 mAb compared with controls (Figure 3C). This indicated that parasite accumulation in tissue and vasculature was greatly reduced in mice depleted of Treg cells compared with controls. Furthermore, these data showed that parasite burdens in control animals were greatly underestimated, and differences in parasite burden between mice lacking
Referência(s)