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Patent Filarial Infection Modulates -Specific Type 1 Cytokine Responses in an IL-10-Dependent Manner in a Filaria/Malaria-Coinfected Population This information is current as of September 30, 2021. Simon Metenou, Benoit Dembélé, Siaka Konate, Housseini Dolo, Siaka Y. Coulibaly, Yaya I. Coulibaly, Abdallah A. Diallo, Lamine Soumaoro, Michel E. Coulibaly, Dramane Sanogo, Salif S. Doumbia, Marissa Wagner, Sekou F. Traoré, Amy Klion, Siddhartha Mahanty and Thomas B. Nutman Downloaded from J Immunol 2009; 183:916-924; Prepublished online 26 June 2009; doi: 10.4049/jimmunol.0900257 http://www.jimmunol.org/content/183/2/916 http://www.jimmunol.org/

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2009 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Patent Filarial Infection Modulates Malaria-Specific Type 1 Cytokine Responses in an IL-10-Dependent Manner in a Filaria/Malaria-Coinfected Population1,2

Simon Metenou,* Benoit Dembe´le´,† Siaka Konate,† Housseini Dolo,† Siaka Y. Coulibaly,† Yaya I. Coulibaly,† Abdallah A. Diallo,† Lamine Soumaoro,† Michel E. Coulibaly,† Dramane Sanogo,† Salif S. Doumbia,† Marissa Wagner,† Sekou F. Traore´,† Amy Klion,* Siddhartha Mahanty,* and Thomas B. Nutman3*

The effect of filarial infections on malaria-specific immune responses was investigated in Malian villages coendemic for filariasis (Fil) and malaria. Cytokines were measured from plasma and Ag-stimulated whole blood from individuals with Wuchereria Downloaded from .(19 ؍ and those without evidence of filarial infection (Fil؊; n (19 ؍ bancrofti and/or infections (Fil؉; n -Plasma levels of IL-10 (geometric mean [GM], 22.8 vs 10.4) were higher in Fil؉ compared with Fil؊, whereas levels of IFN inducible protein (IP)-10 were lower in Fil؉ (GM, 66.3 vs 110.0). Fil؉ had higher levels of spontaneously secreted IL-10 (GM, 59.3 vs 6.8 pg/ml) and lower levels of IL-2 (1.0 vs 1.2 pg/ml) than did Fil؊. Although there were no differences in levels of Staphylococcus ؍ aureus enterotoxin B-induced cytokines between the two groups, Fil؉ mounted lower IL-12p70 (GM, 1.11 vs 3.83 pg/ml; p responses following malaria (0.007 ؍ and IP-10 (GM, 29.43 vs 281.7 pg/ml; p ,(0.009 ؍ IFN-␥ (GM, 5.44 vs 23.41 pg/ml; p ,(0.007 http://www.jimmunol.org/ Ag (MalAg) stimulation compared with Fil؊. In contrast, Fil؉ individuals had a higher MalAg-specific IL-10 response (GM, 7318 (␤compared with those without filarial infection. Neutralizing Ab to IL-10 (but not to TGF (0.006 ؍ pg/ml vs 3029 pg/ml; p reversed the down-regulated MalAg-specific IFN-␥ and IP-10 (p < 0.001) responses in Fil؉. Together, these data demonstrate that filarial infections modulate the Plasmodium falciparum-specific IL-12p70/IFN-␥ secretion pathways known to play a key role in resistance to malaria and that they do so in an IL-10-dependent manner. The Journal of Immunology, 2009, 183: 916–924.

otwithstanding the marked reduction in reported cases of three causative agents of lymphatic filariasis (Wuchereria ban- Plasmodium falciparum (Pf),4 malaria remains crofti (Wb), malayi, and ), 33% live in parts N in 109 countries worldwide. An estimated 189–327 mil- of Africa (2) where malaria is highly endemic. Coinfection with by guest on September 30, 2021 lion cases of malaria occur annually, with 86% occurring in sub- malarial and filarial parasites is common (3–6), with documented Saharan Africa. In 2006 alone, malaria claimed ϳ1.1 million lives prevalences of concomitant malaria and Wb ranging from 0.3 to in Africa, 85% occurring in children below the age of 5 years (1). 0.4% in (3, 4) to 3.3% in parts of South America (5) and Among the ϳ129 million people worldwide infected by one of the 4.3% in Kenya (6). More recently, Mansonella perstans (Mp)/ malaria coinfections were reported in 18% of pregnant women in a filarial-endemic region of Uganda (7). *Laboratory of Parasitic Diseases, National Institute of Allergy and Infectious Dis- Because malaria and filarial infections each induce discrete par- † eases, National Institutes of Health, Bethesda, MD 20892; and Unit, Fac- asite-specific immune responses, it is likely that the immune re- ulty of Medicine, Pharmacy and Dentistry, University of Bamako, Bamako, Mali sponses directed at one parasite could (through bystander effects) Received for publication January 27, 2009. Accepted for publication May 15, 2009. alter the immune response to the other. More importantly, how- The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance ever, in those areas with intense Pf and Wb such as with 18 U.S.C. Section 1734 solely to indicate this fact. Mali or parts of , malaria/filaria coinfection 1 This work was supported by the Intramural Research Program of the Division of occurs at an early age (8), and modulation of the responses to Intramural Research, National Institute of Allergy and Infectious Diseases, National malaria infection by concomitant filarial infection could, theoret- Institutes of Health. ically at least, dramatically alter clinical outcome and/or severity 2 Because S. Metenou, A. Klion, S. Mahanty, and T. B. Nutman are government of malaria in nonimmune children. employees and this is a government work, the work is in the public domain in the United States. Notwithstanding any other agreements, the National Institutes of Human studies have generated conflicting results on the effect of Health reserves the right to provide the work to PubMed Central for display and use helminth infections on the acquisition and severity of malaria, in by the public, and PubMed Central may tag or modify the work consistent with its customary practices. You can establish rights outside of the United States subject to that helminth/malaria coinfection has been shown to both exacer- a government use license. bate (9–12) and (in other studies) modulate the severity of the 3 Address correspondence and reprint requests to Dr. Thomas B. Nutman, Laboratory disease in human malaria (13–16). Animal models of malaria/fi- of Parasitic Diseases, National Institute of Allergy and Infectious Diseases, 4 Center laria coinfection have also led to conflicting results. Using the Drive, Room B1-03, National Institutes of Health, Bethesda, MD 20892. E-mail ad- dress: [email protected] filarial parasite Litomosoides sigmodontis and the rodent malaria Plasmodium chabaudi chabaudi AS, mice with both infections 4 Abbreviations used in this paper: Pf, Plasmodium falciparum; AMA, apical mem- brane Ag; BmA, adult worm extract; Filϩ, filaria infected; FilϪ, filaria were shown to have more severe anemia and loss of body mass uninfected; FilAg, filaria Ag; GM, geometric mean; IP, IFN-inducible protein; than did mice infected with the malaria parasite alone (17). In a MalAg, malarial Ag; Mp, Mansonella perstans; MSP, merozoite surface protein; PE, phycoerythrin; PPD, purified protein derivative; SEB, Staphylococcus aureus entero- different murine model, pretreatment of mice with irradiated Bru- toxin B; Wb, . gia pahangi larvae prevented cerebral malaria (in a Plasmodium www.jimmunol.org/cgi/doi/10.4049/jimmunol.0900257 The Journal of Immunology 917 berghei model) but induced more severe anemia that led to death circulating filarial Ag (FilAg) (ELISA; TropBio) and/or detectable micro- (18). In a study of experimental coinfections in nonhuman pri- filariae (Wb or Mp) in peripheral blood samples drawn between 10 p.m. and 2 a.m. Microfilaremia was assessed by calibrated thick smear (total mates, monkeys with circulating microfilariae developed less se- Ϫ blood volume, 60 ␮l). Fil had a negative test for circulating FilAg, and no vere malarial disease when challenged with P. falciparum parasites microfilariae were seen on thick smear. Each Filϩ individual was matched (19) than did monkeys without microfilariae. by age with a FilϪ individual. That the cytokine milieu associated with filarial infections Malaria and filarial Ab should have a modulating effect on responses to coinfecting patho- gens has been inferred primarily by the bystander effects induced Filarial Ab levels were determined using a B. malayi adult worm extract by filarial infections on responses to administration (20, (BmA)-specific IgG and IgG4 ELISA as described previously (34). Ma- 21). These data notwithstanding, a few studies have examined the laria-specific IgG and IgG subclass Ab levels were determined using the recombinant Ag merozoite surface protein (MSP)-142 of the 3D7 strain immunologic interface between human malaria and filarial (or expressed in Escherichia coli and the apical membrane Ag (AMA)-1 of the other systemic helminth) infections. In a study performed in In- 3D7 strain expressed in Pichia pastoris (provided by Dr. C. Long, Labo- donesia in 1980, chronic malaria and brugian filariasis had oppos- ratory of Malaria and Vector Research (LMVR), NIAID, NIH) using a ing effects on the numbers of circulating CD4ϩ and CD8ϩ T cells multiplex assay with suspension array technology (Luminex). The suspen- sion array technology assay protocol used was modified from the technique (22). More recently in Ghana, it was found that cells from children published by Fouda and collaborators (35) and was performed using a from rural areas where malaria and helminth infections (Schisto- BioPlex platform and BioPlex Manager, version 5 (Bio-Rad Laboratories). soma hematobium, Trichiura trichiura, hookworm) were coen- The protocol modification included a second Ab step before the addition of demic produced higher levels of IL-10 in response to malaria- the PE conjugate. The secondary Ab included biotin-labeled goat anti- human IgG (Kirkegaard and Perry Laboratories), mouse anti-human IgG1 infected RBC (but not to LPS) compared with those from children Downloaded from (Calbiochem), mouse anti-human IgG2 (clone HP-6014), IgG3 (clone HP- in an urban setting without helminth infections (23). 6050), and IgG4 (clone HP-6023) (Sigma-Aldrich), R-PE-conjugated Ј In malaria-endemic regions of the world, adults are protected F(ab )2 fragment, donkey anti-mouse IgG (H and L chains) and R-PE- against severe malaria and high parasitemia by mechanisms that conjugated streptavidin (Jackson ImmunoResearch Laboratories). The pos- remain poorly understood. Protection has been attributed, in part, itive control Abs were rabbit anti-MSP142 and anti-AMA-1 antisera (pro- vided by Dr. C. Long, LMVR, NIAID, NIH). to the large repertoire of specific Ab acquired through repeated

exposure to the infection (24, 25). Cellular responses have also Whole blood culture http://www.jimmunol.org/ been shown to play an important role in protection from clinical Heparinized blood was collected from study subjects in the village and disease in malaria. Indeed, it has been shown that both IL-12 and transported at ambient temperature to the laboratory in Bamako, Mali, for IFN-␥ play important roles in mediating immunity to malarial dis- processing. Blood samples were diluted 1/1 in RPMI 1640 supplemented ease (26, 27). Early in acute infection, Pf induces high levels of the with penicillin/streptomycin (100 U/ml and 100 ␮g/ml, respectively), L- proinflammatory cytokines TNF-␣, IL-6, and IL-1␤, the presence glutamine (2 mM), and HEPES (10 mM) (all from Invitrogen). The malaria parasite extract (MalAg) was prepared using standard methods as described of which have generally been associated with severe malaria (28– by Metenou et al. (36). Briefly, P. falciparum-infected erythrocytes of the 30), although high plasma levels of TNF-␣ have also been asso- 3D7 strain (prepared by H. Zhou in C. Long’s laboratory at NIAID) were ciated with rapid parasite clearance and cure (30). Nevertheless, cultured in complete medium (RPMI 1640 containing, hypoxanthine, L- ϩ the severity of malarial disease is associated not so much with the glutamine, HEPES, 10% human type O serum, and gentamicin) and at by guest on September 30, 2021 ␣ 5% hematocrit erythrocyte suspension in an automated continuous culture absolute plasma levels of TNF- but rather with the ratio of system. Parasites were regularly screened for mycoplasma contamination TNF-␣ to IL-10, an important regulatory cytokine also present in and other microorganisms. At a parasitemia of ϳ10–20%, the mature asex- the plasma during malaria infection (31, 32). ual stages were purified by centrifugation on a 60% Percoll density gra- Because the immunologic hallmark of patent filarial infection is dient (36). This purification step resulted in preparations with 80–90% Ͼ a parasite Ag-specific, down-regulated immune response mediated parasitemia, consisting of 95% schizonts/mature trophozoites. These ␤ preparations of infected RBC were washed twice in sterile RPMI 1640 and largely by IL-10 (and TGF ), the current study was designed to freeze thawed two or three times to obtain parasite extract and used at a examine the influence of a filarial-induced regulatory environment final concentration of 104 infected RBC/ml. BmA was prepared as de- (IL-10 and TGF␤) on the immune response to malarial Ag scribed by Mitre et al. (37) and used at 10 ␮g/ml final concentration. The (MalAg) in an area coendemic for filarial infections (both Wb and diluted blood samples were left unstimulated or stimulated with a Pf sch- izont extract (MalAg), BmA, Staphylococcus aureus enterotoxin B (SEB; Mp) and Pf malaria. By examining both plasma and whole blood Toxin Technology), or a purified protein derivative (PPD; Statens Serum responses to MalAg, our data demonstrate that concurrent filarial Institut, Copenhagen, Denmark) for 24 h in a CO2 incubator at 37°C with ␥ ␤ infections modulate the malaria-specific IL-12p70/IFN- secretion 5% CO2. To assess the effect of the regulatory cytokines IL-10 and TGF , pathways known to play a key role in resistance to malarial par- blood samples stimulated with the same Ag were cultured in the presence ␮ ␤ asites and do so in an IL-10-dependent manner. of 20 g/ml neutralizing anti-human IL-10, anti-human TGF , a combi- nation of anti-IL-10 and anti-TGF␤,ora20␮g/ml control Ab of the same isotype (R&D Systems). The culture supernatants were collected and Materials and Methods stored at Ϫ70°C or in liquid nitrogen until assayed. Study population To assess the cellular source of IL-10, brefeldin A (Sigma-Aldrich) (20 ␮g/ml final concentration) was added to additional sets of Ag-stimulated The study was conducted in Tiene´gue´bougou and Bougoudiana, two vil- and unstimulated samples after 12 h of incubation. The cultures were in- lages situated ϳ105 km northeast of Bamako, Mali, in a malaria-endemic cubated for another 12 h, after which the samples were lysed, fixed, and area with seasonal transmission. Before the start of the study, screening of cryopreserved at Ϫ80oC until they were used. adults in the villages showed the prevalence of circulating filarial antigen- emia (TropBio) (33) to be 53% in Tiene´gue´bougou and 36% in Bougoudi- Cytokine analysis ana, respectively. The prevalence of Mp microfilariae by calibrated thick Cytokine analysis in supernatants and plasma was performed by suspen- smear examination was 62% in Tiene´gue´bougou and 63% in Bougoudiana. sion array technology in multiplex as described in (38). The assay was done The study was approved by the National Institute of Allergy and Infec- using Milliplex kits for human IL-2, IL-4, IL-6, IL-10, IL-12p70, IL-17A, tious Diseases (NIAID) Institutional Review Board and the Ethical Com- IFN-␥, TNF-␣, and IFN-inducible protein (IP)-10 (Millipore) according to mittee of the University of Mali (National Institutes of Health (NIH) Clini- the manufacturer’s protocol. calTrials.gov identifier NCT00471666). Informed consent was obtained from all participants. Flow cytometry Thirty-eight volunteers (19 filaria uninfected (FilϪ) and 19 filaria in- fected (Filϩ)) between 11 and 20 years of age were enrolled before the start Cryopreserved fixed cells were thawed, washed twice with PBS and 1% of the malaria transmission season. Filϩ was defined by a positive test for BSA, and washed twice more in permeabilization buffer (eBioscience). The 918 MALARIA-SPECIFIC RESPONSES REGULATED BY COINCIDENT FILARIAE

Table I. Study populationa

Filarial Status

Negative (n ϭ 19) Positive (n ϭ 19) p value

Gender (male/female) 15/4 14/5 NS Wb circulating Agϩ (n) 09 GM (U/ml) ͓95% CI͔ 0 320.4 ͓56.7–1810.0͔ Mp/microfilaremia (n)0 12 GM (mf/ml) ͓95% CI͔ 033͓18.0–60.0͔ WBC (ϫ103/␮l) GM ͓range͔ 7.0 ͓3.5–10.5͔ 7.8 ͓4.9–21.6͔ NS Hb (g/dL) GM ͓range͔ 11.6 ͓9.4–13.5͔ 12.24 ͓9.9–15.5͔ NS AMA-1 Ab GM MFI ͓95% CI͔ IgG 15,163 ͓10,353–22,207͔ 18,925 ͓15,019–23,846͔ NS IgG1 4,593 ͓2,587–8,156͔ 6,298 ͓4,032–9,840͔ NS IgG2 14.8 ͓3.3–66.0͔ 13.1 ͓3.7–46.1͔ NS IgG3 13.7 ͓2.5–75.2͔ 26.3 ͓4.3–159.8͔ NS IgG4 6.6 ͓1.9–23.4͔ 1.3 ͓0.7–2.3͔ 0.020 IgE 20.92 ͓4.2–104.3͔ 35.55 ͓7.8–162.6͔ NS MSP-1-specific Ab GM MFI ͓95% CI͔ Downloaded from IgG 17,056 ͓12,207–23,832͔ 17,790 ͓13,779–22,969͔ NS IgG1 1,977 ͓1,073–3,644͔ 1,326 ͓488.9–3,597.0͔ NS IgG2 24.27 ͓4.5–131.1͔ 10.9 ͓3.1–38.3͔ NS IgG3 4,270 ͓1,156–15,773͔ 4,270 ͓1,156–15,773͔ NS IgG4 6.5 ͓1.5–27.9͔ 5.5 ͓1.3–22.8͔ NS IgE 18.3 ͓3.9–85.6͔ 14.1 ͓3.3–60.3͔ NS

BmA-specific Ab http://www.jimmunol.org/ GM ͓95% CI͔ IgG (␮g/ml) 64.37 ͓40.4–102.6͔ 105.6 ͓66.5–167.7͔ NS IgG4 (ng/ml) 43.73 ͓5.5–348.7͔ 605.9 ͓110.8–3,313͔ 0.04

a CI, confidence interval; mf/ml, microfilariae per milliliter; WBC, white blood count; MFI, mean fluorescence intensity.

Ϫ cells were then stained with mouse anti-human CD3-allophycocyanin- viduals (105.9, as compared with 43.73 in Fil ; p ϭ 0.04) Cy5.5, mouse anti-human CD4-PE-Cy5, rat anti-human IL-10-allophyco- (Table I). cyanin (eBioscience), and mouse anti-human CD25-allophycocyanin-Cy7

(BD Pharmingen). Samples were acquired on a BD LSR II flow cytometer by guest on September 30, 2021 (BD Pharmingen) and analyzed using FlowJo (Tree Star). Cytokine levels in plasma and spontaneous cytokine secretion in Statistical analysis whole blood cultures ␥ The study subjects were matched for age and sex, the Wilcoxon signed Plasma levels of IFN- and IL-10 were determined in all sub- rank test was used for group comparisons, and the p values were corrected jects (Fig. 1A). The plasma levels of IL-10 (GM, 22.8 vs 10.4; for multiple comparisons using the Holm correction method. All analyses p Ͻ 0.004) were significantly higher in Filϩ compared with were performed using Prism version 5.0 (GraphPad Software). FilϪ, as was the ratio of IL-10 to IFN-␥ (GM, 13.4 vs 5.2; p ϭ 0.01). In contrast, plasma levels of IP-10 were significantly Results higher in the FilϪ compared with the Filϩ group (GM, 110.0 vs Characteristics of the study population and serologic responses 66.3; p Ͻ 0.02; data not shown). There were no significant The study was conducted in two adjacent Malian villages me- differences between the two groups in any of the other cytokines soendemic for malaria with a seasonal pattern of transmission measured in the plasma. Spontaneous production of IFN-␥, before the malaria season. Thirty-eight age-matched subjects IL-2, IL-4, IL-6, IL-10, IL-12p70, IL-17A, IP-10, and TNF-␣ (19 Filϩ and 19 FilϪ), a subset of patients participating in a was assessed in whole blood cultures. As shown in Fig. 1B, the larger longitudinal study of clinical malaria, were enrolled. The levels of spontaneously secreted IL-10 were significantly higher demographics and parasitologic status of the subjects are shown in Filϩ compared with FilϪ (GM, 59.3 vs 6.8; p ϭ 0.01). There in Table I. There were no significant differences in total leuko- were no differences in the levels of other spontaneously pro- cyte count or hemoglobin concentration between the two groups duced cytokines between the two groups. The ratio of sponta- (Table I). neous IL-10: IFN-␥ appeared to be higher in the Filϩ group but

Although MSP142-specific and AMA-1-specific IgG levels were did not reach statistical significance. also comparable between Filϩ and FilϪ subjects (Table I), analysis of the antimalarial IgG subclass responses revealed that geometric mean (GM) AMA-1-specific IgG4 levels were significantly higher MalAg-driven cytokines in FilϪ compared with Filϩ individuals (6.6 vs 1.3; p ϭ 0.02). No Malaria-specific induction of IL-12p70 (GM, 1.1 vs 3.8; p ϭ other differences in malaria-specific IgG isotype levels were iden- 0.005), IP-10 (29.4 vs 281.7; p ϭ 0.007), and IFN-␥ (5.4 vs tified between the two groups. 23.4; p ϭ 0.009) was significantly lower in Filϩ compared with FilAg (BmA)-specific IgG levels were comparable between FilϪ (Fig. 2). In contrast, Filϩ produced significantly more Filϩ and FilϪ, suggesting that exposure to filarial parasites was IL-10 in response to MalAg than did FilϪ (GM, 7318 vs 3029; equivalent between the two groups; however, the GM levels of p ϭ 0.006). The ratio of IL-10 to IFN-␥ was also significantly filaria-specific IgG4, known to be increased in active filarial in- higher in Filϩ (GM, 1186 vs 123.5; p ϭ 0.0004). There were no fection (33), were, as expected, significantly higher in Filϩ indi- differences in the levels of MalAg-induced IL-6, IL-17A, and The Journal of Immunology 919

FIGURE 1. A, Plasma cytokine levels in FilϪ (open triangles) and Filϩ (closed triangles) individuals. Each tri- angle represents an individual patient; the horizontal bar represents the GM for each group. B, Levels of cytokines produced spontaneously in whole blood cultures from FilϪ (open trian- gles) and Filϩ (filled triangles) indi- viduals. Each triangle represents an in- dividual patient; the horizontal bar represents the GM for each group. Downloaded from

TNF-␣ between the two groups. IL-2 and IL-4 were not detect- Because patent filarial infection has been associated with para- able in response to MalAg (Fig. 2) in whole blood cultures from site Ag-specific, down-regulated cytokine responses, filaria any of the subjects. (BmA)-specific induction of IFN-␥, IL-2, IL-4, IL-6, IL-10, IL- 12p70, IL-17A, IP-10, and TNF-␣ was assessed in both groups of ϩ Cytokine responses to SEB, PPD, and FilAg stimulation subjects (Fig. 3). As expected, Fil had diminished production of http://www.jimmunol.org/ Ϫ There were no differences between the groups in the levels of IL-2 ( p ϭ 0.01) and IP-10 ( p ϭ 0.04) compared with Fil . any of the cytokines produced in response to SEB stimulation, Surprisingly, there was also diminished production of IL-4 in suggesting that there was no intrinsic difference in the capacity BmA-stimulated whole blood cultures in Filϩ, perhaps reflect- of the cells from either group to respond to a global stimulus ing the contribution of cells other than T cells in the production (Fig. 3). In response to PPD (used here as a control Ag) stim- of these cytokines (37) or down-regulation by the filarial par- ulation, cytokine levels were similar between the two groups asites themselves (41). There were no differences in the levels except for those of IL-2 (which was significantly lower in Filϩ of IL-6, IL-17A, and TNF-␣ between the two groups. Although compared with FilϪ; p ϭ 0.02) and IL-10 (with Filϩ demon- more individuals in the FilϪ group produced detectable levels strating higher production; p ϭ 0.01). In fact, filarial infection of IFN-␥ than those in the Filϩ group, the two groups did not by guest on September 30, 2021 has been shown to be associated with down-regulation of IL-2 differ in their IFN-␥ response to BmA. BmA did not induce production and T cell proliferation, and the down-regulation detectable levels of IL-12p70 in any of the cultures under the was IL-10 mediated (39, 40). conditions tested.

FIGURE 2. Levels of malaria Ag- stimulated cytokines IL-10, IFN-␥, and IL-10:IFN-␥ ratio (A) and IL- 12p70, IP-10, TNF-␣, IL-17A, and IL-6 (B) produced in whole blood cul- tures from FilϪ (open triangles) and Filϩ (filled triangles) individuals. Each triangle represents the net cytokine production from an individual patient; the horizontal bar represents the geo- metric mean for each group. 920 MALARIA-SPECIFIC RESPONSES REGULATED BY COINCIDENT FILARIAE

FIGURE 3. Levels of cytokine pro- duced in response to SEB, PPD, and BmA (BMA) stimulation in whole blood cultures of FilϪ (hatched bars) and Filϩ; (filled bars) individuals. Each bar represents the geometric mean of the net production of each cy- .p Ͻ 0.05 ,ء .tokine Downloaded from

Effect of anti-IL-10, anti-TGF␤, and both together on isotype control Ab (Fig. 4A). Neutralizing anti-TGF␤ had a small http://www.jimmunol.org/ MalAg-induced IFN-␥, IL-12p70, and IP-10 but significant effect on the production of malaria-specific IP-10 To determine whether the decreased IL-12 and IP-10 response to but not on the other cytokines measured. Production of IL-12p70, ␥ MalAg in Filϩ was due to the observed increase in IL-10, whole IP-10, and IFN- in the presence of the combination of anti-IL-10 ␤ blood samples from Filϩ were cultured in the presence of neutral- and anti-TGF neutralizing Ab was comparable to that in the pres- izing anti-IL-10 Ab as well as in the presence of a neutralizing Ab ence of anti-IL-10 alone (Fig. 4B). ␤ to TGF (another known regulatory molecule) (Fig. 4A) and to a ϩ Ϫ combination of anti-IL-10 and anti-TGF␤ neutralizing Ab (Fig. CD4 CD25 T cells are the major source of T cell-derived IL-10 4B). The addition of neutralizing anti-IL-10 Ab induced a signif- by guest on September 30, 2021 icant increase in the production of malaria-specific IFN-␥ (49.6% To determine the source of IL-10, intracellular cytokine staining increase; p ϭ 0.02) and IP-10 (221.4% increase; p ϭ 0.0005), but coupled with cell surface staining was performed in cells cultured not IL-12p70, when compared with production in cultures with an with and without MalAg for 24 h in a small set of cultures. As can

FIGURE 4. Effect of neutralizing anti-IL-10 and anti-TGF␤ Ab on the levels of malaria-specific cytokine production in whole blood cultures of filaria-infected patients. Cytokines were measured in supernatants of whole blood cultures stimulated with malaria Ag in the presence or absence (isotype control) of neutralizing anti- IL-10 (A, top), anti-TGF␤ (A, bottom) Ab alone or in combination (B). Each line represents the net concentration of a given cytokine for an individual pa- tient in the presence or absence of the neutralizing Ab shown. The Journal of Immunology 921

FIGURE 5. Source of IL-10 pro- duction by CD4ϩ cells in MalAg-stim- ulated cultures. Shown are four repre- sentative scatter plots from two representative FilϪ (left panels) and two representative Filϩ (right panels)

subjects. The numbers in each quad- Downloaded from rant represent the percentage of total CD4ϩ cells for each individual. http://www.jimmunol.org/ by guest on September 30, 2021 be seen in Fig. 5 (and in additional data not shown), the frequency many of these same cytokines (IFN-␥, TNF-␣, IP-10, and IL- of CD4ϩCD25ϪIL-10ϩ cells was 3- to 6-fold higher than the fre- 12) (51). Current understanding of the pathogenesis of acute ϩ ϩ ϩ quency of CD4 CD25 IL-10 cells. malaria suggests that a failure to coordinate the up-regulation of anti-inflammatory responses (such as IL-10 and TGF␤) follow- Discussion ing the increase in inflammatory mediators leads to exuberant The regulatory networks induced during chronic helminth infec- overproduction of these proinflammatory cytokines and signif- tion (particularly schistosome and filarial infections) have been icant pathology (51, 52), including cerebral malaria (53), severe implicated in both the modulation of parasite-specific immune re- malarial anemia (54), and renal failure (55). sponses and responses to nonparasite Ag, the latter through by- Studies in humans and in animal models have failed to provide stander effects (so-called “spillover suppression”) (42). Not only an unequivocal conclusion about the role played by helminth in- does the helminth-induced attenuation of the immune response ex- fection on either susceptibility to malaria infection or the modu- tend to nonparasite soluble Ag but also to responses to orally and lation of pathology in malaria-associated disease. In some animal parenterally administered (20, 43, 44), other infectious models, helminth/malaria-coinfected animals have been protected diseases (e.g., Helicobacter pylori, Mycobacterium tuberculosis, against severe malaria (18), whereas other studies report an in- P. falciparum, and HIV) (45–47), and aeroallergens (48). The creased susceptibility to severe disease (17) or no effect on severe mechanisms underlying this modulation of host responses to by- disease whatsoever (56). More recently, Heligmosomoides polygy- stander Ag remain unknown, although IL-10 has been the cytokine rus-infected mice immunized with blood-stage parasites of P. most often implicated (39, 40, 49) in mediating these spillover chabaudi chabaudi effects. AS produced lower levels of malaria-specific ␥ Among the many infections purportedly influenced by con- Ab and malaria-specific IFN- but higher levels of IL-4, IL-13, ␤ comitant helminth infection, P. falciparum holds primacy in IL-10, and TGF (57), whereas others, using a similar model, that it is responsible for an estimated 286 million clinical cases showed that concurrent H. polygyrus had no effect on the devel- and 1.2 million deaths yearly, with 91% of the cases and deaths opment of cerebral malaria (56). occurring in Africa (1). The components of the immune re- Helminth/malaria coinfection studies in humans have also pro- sponse implicated as being the most effective in preventing ma- duced quite disparate results, with some studies showing more laria infection per se are proinflammatory mediators (IFN-␥ and severe malaria in the presence of helminth coinfections (9–12) and TNF-␣), and their efficacy appears to require induction early in others showing helminth-induced protection against malaria-asso- infection (50); however, serious pathology associated with ma- ciated disease (13–16). Despite the differences in conclusions of laria infection has also been associated with the production of each of these studies, all appear to be constant in their suggestion 922 MALARIA-SPECIFIC RESPONSES REGULATED BY COINCIDENT FILARIAE that the immune response to MalAg was influenced by the con- not, however, examine IL-10 production in monocytes, previously comitant helminth infection. shown to be an IL-10 source in filaria-infected individuals from There have been relatively few studies in humans examining India (39). the immune responses to MalAg in the context of a coinfecting Our data clearly demonstrate the modulating effects of helminth parasite. A recent study in Ghana where Pf malaria, chronic filarial infection on the Th1/proinflammatory responses intestinal helminths, and Schistosoma hematobium are coen- to MalAg in a filarial/Pf coendemic region of West Africa. Spe- demic found that children from a rural area with S. hematobium cifically, the presence of concomitant filarial infection modu- and produced higher levels of IL-10 in lates the Pf-specific production of IL-12p70, IFN-␥, and IP-10 response to malaria-infected erythrocytes compared with hel- in a manner dependent on IL-10. Although the clinical impact minth-free children from an urban setting (23). In the current of these immunologic changes remains to be determined, these study, we found that patent filarial infection was associated with data suggest that coinfections should be taken into consider- significantly higher IL-10 production in response to MalAg ation when designing vaccine strategies or other intervention stimulation as well as significantly lower levels of IL-12p70 and trials in areas where helminth infections coexist with other non- IFN-␥, and IP-10. IL-12p70, IFN-␥, and IP-10 have each been helminth infectious diseases. shown to play critical roles in mediating the outcome in malaria infection (26, 27, 58, 59). That the IL-12/IFN-␥ axis is impor- Acknowledgments tant in protection against malarial disease in human populations We thank National Institute of Allergy and Infectious Diseases intramural has been lent additional credence by two studies in which the editor Brenda Rae Marshall for assistance. early induction of IFN-␥ was associated with the prevention of Downloaded from clinical malaria (60, 61), as well as by studies of mice and Disclosures nonhuman primates (62, 63). Thus, the diminution of malaria- The authors have no financial conflict of interest. specific induction of IL-12/IFN-␥ by concomitant filarial infec- tion may have important implications for ongoing studies eval- References uating the clinical outcome of malaria. 1. World Health Organization. 2008. World Malaria Report 2008. Global Malaria

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