Counterregulation of Th2 immunity by interleukin 12 reduces host defenses against Strongyloides venezuelensis infection

Microbes and Infection 11 (2009) 571e578 www.elsevier.com/locate/micinf

Original article

Counterregulation of Th2 immunity by interleukin 12 reduces host defenses against Strongyloides venezuelensis infection Eleuza R. Machado a, Daniela Carlos a, Elaine V. Lourenc¸o b, Carlos A. Sorgi a, E´rika V. Silva a, Simone G. Ramos c, Marlene T. Ueta d, David M. Aronoff e, Lu´cia H. Faccioli a,* a

Departamento de Ana´lises Clı´nicas, Toxicolo´gicas e Bromatolo´gicas, Faculdade de Cieˆncias Farmaceˆuticas de Ribeir~ao Preto, Universidade de S~ao Paulo (USP), Av. do Cafe´ s/n, Ribeir~ao Preto, S~ao Paulo 14040-903, Brazil b Departamento de Biologia Celular e Molecular e Bioagentes Patogeˆnicos, Faculdade de Medicina de Ribeir~ao Preto, Universidade de S~ao Paulo, Av. dos Bandeirantes 3900, Ribeir~ao Preto-SP, CEP 14049-900, Brazil c Departmento de Patologia, Faculdade de Medicina de Ribeir~ao Preto, Universidade de S~ao Paulo, Av. Bandeirantes 3900, 14049-900 Ribeir~ao Preto, SP, Brazil d Departamento de Parasitologia, Instituto de Biologia, Universidade Estadual de Campinas, Cidade Universita´ria Zeferino Vaz s/n. CEP 13083-970, Caixa Postal 6109, Campinas, SP, Brazil e Division of Infectious Diseases, Department of Internal Medicine, University of Michigan Health System, Ann Arbor, MI 48109, USA Received 22 October 2008; accepted 6 March 2009 Available online 1 April 2009

Abstract The aim of this study was to investigate the role of interleukin 12 (IL-12) during Strongyloides venezuelensis infection. IL-12/ and wildtype C57BL/6 mice were subcutaneously infected with 1500 larvae of S. venezuelensis. On days 7, 14, and 21 post-infection, we determined eosinophil and mononuclear cell numbers in the blood and broncoalveolar lavage fluid (BALF), Th2 cytokine secretion in the lung parenchyma, and serum antibody levels. The numbers of eggs in the feces and worm parasites in the duodena were also quantified. The eosinophil and mononuclear cell counts and the concentrations of IL-3, IL-5, IL-10, IL-13, and IgG1 and IgE antibodies increased significantly in infected IL-12/ and wild-type mice as compared with uninfected controls. However, the number of eosinophils and mononuclear cells in the blood and BALF and the Th2 cytokine levels in the lungs of infected IL-12/ mice were greater than in infected wild-type C57BL/6 mice. In addition, serum IgE and IgG1 levels were also significantly enhanced in the infected mice lacking IL-12. Meanwhile, parasite burden and fecal egg counts were significantly decreased in infected IL-12/ mice. Together, our results showed that the absence of IL-12 upregulates the Th2 immune response, which is important for control of S. venezuelensis infection. Ó 2009 Elsevier Masson SAS. All rights reserved. Keywords: S. venezuelensis; Cytokines; Eosinophils; Antibodies; IL-12/ mice

1. Introduction Strongyloides stercoralis is a nematode prevalent in tropical and subtropical regions [1]. Acute infection is characterized by local reactions at the site of larval entry, pulmonary symptoms mimicking bronchitis, and non-specific gastrointestinal symptoms such as diarrhea, constipation, anorexia, and abdominal pain [1]. Due to autoinfection, S. stercoralis can * Corresponding author. Tel.: þ55 016 3602 4303; fax: þ55 016 3602 4725. E-mail address: [email protected] (L.H. Faccioli). 1286-4579/$ - see front matter Ó 2009 Elsevier Masson SAS. All rights reserved. doi:10.1016/j.micinf.2009.03.005

persist in an individual for decades, resulting in a chronic condition [2]. Chronic infection is often asymptomatic, but when symptoms occur they are usually mild, episodic, and prolonged, including gastrointestinal symptoms (vomiting, diarrhea, borborygmus, and epigastric pain), weight loss, or cutaneous reactions (pruritus, urticaria, and larva currens) [1,3]. In patients with impaired immune systems, hyperinfection syndrome and disseminated strongyloidiasis may develop [1,4], and these have become more common due to the widespread use of immunosuppressive drugs like corticosteroids [4,5]. Hyperinfection syndrome is characterized by

572

E.R. Machado et al. / Microbes and Infection 11 (2009) 571e578

severe gastrointestinal inflammation and respiratory tract involvement, skin rashes, recurrent Gram-negative bacteremia, and meningitis [4]. In autoimmune patients and renal transplant recipients, S. stercoralis infection may be confused with the underlying disease, delaying diagnosis and increasing the risk of death [1,4]. S. stercoralis infection is acquired by skin exposure to feces or fecally contaminated soil that harbor filariform larvae. The parasite is also capable of reinfesting its host without leaving the body [1,6], a process known as autoinfection. Female parasites lay eggs in the intestinal mucosa that hatch into noninfective, rhabditiform larvae. These may develop into filariform larvae and penetrate the intestinal mucosa or perianal skin, causing long-term persistence of the infection [2]. In human hosts and mouse models, the immune response to Strongyloides spp. is characterized by intraepithelial and tissue increases in eosinophils [7e9], neutrophils [8] and mast cells [10]; production of Th2-type cytokines, such as interleukin 3 (IL-3), IL-4, and IL-5 [7,11]; and increases in IgA, IgE, IgG1, IgG4, and IgM serum levels [7,11,12]. During helminthic infection, the development of adequate host defense requires this robust Th2 immune response [11,13]. The Th1-type cytokine IL-12 is produced by macrophages, B lymphocytes and other antigen-presenting cells in response to bacterial products and intracellular parasites [14]. The production of IL-12 by phagocytes helps to drive a pattern of Th1 immune responses that is characterized by the production of IL-2 and gamma interferon (IFN-g) [15e17], as well as other mediators such as tumor necrosis factor alpha (TNF-a), granulocyte macrophage colony-stimulating factor (GM-CSF), M-CSF, and IL-3 [17,18]. Studies have demonstrated that T cells stimulated with IL-12 differentiate into CD4þ helper T cells, which produce high levels of IFN-g and low levels of IL4 [19]. IL-12 also inhibits the production of the Th2 cytokines IL-4 and IL-10 while encouraging the synthesis of IFN-g and IL-2 by allergen-stimulated T cells [20]. The role of IL-12 during helminthic infection is not well characterized but appears to antagonize the Th2 response. For example, mice infected with Nippostrongylus braziliensis and treated with recombinant IL-12 displayed a decrease in Th2 cytokines and increased IFN-g release [21]. This change in cytokine secretion was accompanied by an inhibition in IgE production [21]. In S. stercoralis-infected mice treated with recombinant IL-12, a switch from a Th2 to a Th1 response was also observed [22]. S. venezuelensis [23] is a nematode of the wild rodent that serves as a useful model organism for studying hosteparasite interactions, molecular aspects of infection, the efficacy of putative therapeutic agents, and the standardization of new immunological techniques in human strongyloidiasis (serving as a source of heterologous antigen) [24]. S. venezuelensis evokes similar innate and acquired immune responses in mice as S. stercoralis, but is safer for use in the laboratory than the latter organism. In the present study, we investigated the role of IL-12 during S. venezuelensis infection in mice. Our results demonstrate that IL-12 suppresses Th2-type protective immunity, increasing the survival and persistence of parasites in the intestine of an infected host.

2. Materials and methods 2.1. Animals Male C57BL/6 mice weighing 18e25 g and male Rattus norvergicus (Wistar) rats weighing 120e180 g were obtained from the animal facilities of the Universidade de S~ao Paulo na Faculdade de Cieˆncias Farmaceˆuticas de Ribeir~ao Preto. Male IL-12 deficient mice (IL-12/) weighing 18e25 g were kindly provided by Prof. Dr. Jo~ao Santana Silva of the Universidade de S~ao Paulo na Faculdade de Medicina de Ribeir~ao Preto. All experiments were approved by and conducted in accordance with the guidelines of the University of S~ao Paulo at Ribeir~ao Preto Animal Care Committee of the (Protocol no 19-2). All animals were maintained under standard laboratory conditions. 2.2. Parasites The strain of S. venezuelensis (Sv L2) was isolated from the wild rodent Bolomys lasiurus in April 1986 [25]. The strain was maintained in Wistar rats, routinely infected in the Labora´torio de Imunologia e Inflamac¸~ao das Parasitoses (LIIP) na Faculdade de Cieˆncias Farmaceˆuticas de Ribeir~ao Preto, S~ao Paulo, Brazil. 2.3. Infection of mice with S. venezuelensis strain Infective third-stage larvae (L3) of S. venezuelensis were obtained from charcoal cultures of infected rat feces. The cultures were stored at 28  C for 72 h. The infective larvae were collected and concentrated using a Baermann apparatus and then washed several times in PBS before being counted. The larval count was adjusted to 15,000 L3/mL of PBS for murine infection. The IL-12/ and wild-type C57BL/6 mice were each divided into two subsets: a control group of uninfected mice and an experimental group of infected mice. The IL-12/ and wild-type mice allocated to the experimental group were individually inoculated via subcutaneous (s.c.) abdominal injection with 100 mL of phosphate buffered saline (PBS) containing 1.5  103 S. venezuelensis L3. For each of two separate experiments, 15 mice were infected with Sv L2 while nine control animals remained uninfected. On post-infection days 7, 14, and 21, five infected mice and three uninfected mice were euthanized. The data presented in the figures represent the mean of values acquired from two independent experiments. 2.4. Collection of blood and bronchoalveolar lavage fluid (BALF) On post-infection days 7, 14, and 21, mice were anesthetized with 30 mg/kg of tribromoethanol (Acros Organics, Fairlawn, NJ, USA) by s.c. injection. Blood samples were then collected by cardiac puncture, followed by euthanasia with an overdose of tribromoethanol. The chest cavity of each animal was carefully opened to expose the trachea, which was then catheterized and infused with three 1-mL aliquots of sterile 0.5% PBS/sodium citrate. The BALF was collected and placed

E.R. Machado et al. / Microbes and Infection 11 (2009) 571e578

on ice. Total cell counts in the blood and BALF were then immediately performed in a Neubauer chamber. Differential counts were obtained using Rosenfeld-straining cytospin preparations as previously described [26]. The blood was then centrifuged and the serum collected and stored at 70  C.

573

extract at a concentration of 20 mg/mL (50 mL/well) and the ELISA was conducted according to the literature [4,26]. The results are reported as the mean absorbance of samples per group (SEM). 2.7. Measurement of cytokines in the lung homogenates

2.5. Alkaline parasite extracts Alkaline extracts were prepared as previously described by Machado et al. [26]. Briefly, 1 mL of 0.15 M of sodium hydroxide was added to approximately 1.2  105 Sv larvae, which were maintained under gentle agitation for 6 h at 4  C. Subsequently, 0.3 M of hydrochloric acid was added until a pH of 7.0 was reached. This preparation was then centrifuged at 12,400g for 30 min at 4  C. The protein content of the supernatant was 1.96 mg/mL, as detected by the Lowry method [28]. This antigenic extract was then used for determination of serum antibody levels. 2.6. Measurement of antibodies in sera Specific IgG1, IgG2a, and IgE serum levels were determined by enzyme-linked immunosorbent assay (ELISA) according to the manufacturer’s instructions (Technical Data Sheet; BD Pharmingen, San Diego, CA, USA). High-binding 96-well plates were coated with S. venezuelensis alkaline

Fig. 1. Eosinophil numbers in blood (A) and BALF (B). Cells were obtained from IL-12/ and wild-type C57BL/6 mice after infection with S. venezuelensis (Sv) larvae. A group of uninfected mice was used as a control. Eosinophils were enumerated and identified by Rosenfeld staining. Data are expressed as mean  SEM from two independent experiments with infected and uninfected mice (n ¼ 6e10). *Significant compared to uninfected mice, # Wild-type C57BL/6 þ Sv vs. IL-12/ þ Sv, *#P < 0.05.

To determine cytokine levels, the murine lungs were removed on post-infection days 7, 14, and 21. Tissue samples were homogenized (Ultra-Turrax T8; IKA-Werke, Staufen, Germany) in 1.5 mL of RPMI medium, centrifuged at 1500g, filtered, and stored at 70  C until analysis. Commercially-available ELISA kits were used to measure IL3, IL-4, IL-5, IL-10, and IL-13 levels in the lung tissue according to the manufacturer’s instructions (BD Pharmingen). Detection sensitivities were >10 pg/mL. 2.8. Egg and adult worm counts On post-infection days 7, 14, and 21, groups of Sv L2infected wild-type and IL-12/ mice were placed individually on clean, moist absorbent paper and allowed to defecate. The Cornell-McMaster egg-counting technique was then used

Fig. 2. Mononuclear cell numbers in blood (A) and BALF (B). Cells were obtained from IL-12/ and wild-type C57BL/6 mice after infection with S. venezuelensis (Sv) larvae. A group of uninfected mice was used as a control. Mononuclear cells were enumerated and identified by Rosenfeld staining. Data are expressed as mean  SEM from two independent experiments with infected and uninfected mice (n ¼ 6e10). *Significant compared to uninfected mice, #Wild-type C57BL/6 þ Sv vs. IL-12/ þ Sv, *#P < 0.05.

574

E.R. Machado et al. / Microbes and Infection 11 (2009) 571e578

to determine the eggs per gram of feces [25]. A parasitological exam was performed twice, and the mean of the two results was calculated. The mice were then euthanized with an overdose of tribromoethanol. To count the adult parasites, 10-cm duodenal sections were excised, placed on Petri dishes containing saline, longitudinally sectioned, and incubated for 2 h at 37  C as described [7]. The adult worms from the intestines, as well as the eggs from the feces, were counted under a light microscope at a magnification of 100. 2.9. Histology Duodena (10-cm sections) were removed on post-infection days 7, 14, and 21. Tissue samples were fixed in 10% formalin and embedded in paraffin. To count inflammatory cells and determine worm burden, 5-mm sections were stained with hematoxylin and eosin and analyzed in a blinded fashion. 2.10. Statistical analysis Results are expressed as mean  SEM. Statistical comparisons were analyzed by one-way analysis of variance (ANOVA) followed by a Tukey post-test using the Prism 4.0 statistical program (GraphPad Software, San Diego, CA). The Student t-test was used only in the analysis of parasite and egg numbers. Differences at P < 0.05 were considered statistically significant.

3. Results 3.1. IL-12/ mice infected with S. venezuelensis develop higher numbers of eosinophils in the blood and BALF To confirm the importance of IL-12 in regulating inflammatory reactions to S. venezuelensis, the cellular influx from the blood to the bronchoalveolar space of IL-12/ and wildtype C57BL/6 mice was analyzed daily from day 7 to day 21 post-infection. Fig. 1A and B demonstrate increases in the number of eosinophils in the blood and BALF of wild-type and IL-12/ mice in response to infection with Sv larvae. Yet, the increase in blood eosinophil count was significantly higher in infected IL-12/ mice, as compared with wild-type mice on days 7 and 14 while there was a significant drop at day 21 post-infection (Fig. 1A). The number of eosinophils was also significantly enhanced in the BALF of infected IL-12/ mice, reaching a peak on post-infection day 21 (Fig. 1B). These data show that the absence of IL-12 amplifies the eosinophil recruitment induced by S. venezuelensis infection, suggesting that IL-12 downregulates eosinophil trafficking during infection. Infection with S. venezuelensis also induced significant increases in the mononuclear cell count in the blood and BALF of both IL-12/ and wild-type infected mice, as compared with uninfected mice during all periods of infection

Fig. 3. Cytokine levels in lung parenchyma from IL-12/ and wild-type C57BL/6 mice infected with S. venezuelensis (Sv) larvae. IL-13 (A), IL-10 (B), IL-5 (C) and IL-3 (D) concentrations were measured in the lung homogenates by ELISA method. The data are expressed as mean  SEM of two independent experiments with infected and uninfected mice (n ¼ 5e7). *Significant compared to uninfected mice, #Wild-type C57BL/6 þ Sv vs. IL-12/ þ Sv, *#P < 0.05.

E.R. Machado et al. / Microbes and Infection 11 (2009) 571e578

(Fig. 2A, B). For both types of infected mice, mononuclear cell numbers peaked on post-infection day 14 in the blood and on day 21 in the BALF. In addition, there was significant enhance in mononuclear cell count in infected IL-12/ when compared to wild-type mice only on post-infection day 7. Note that no difference was seen on days 14 and 21 postinfection. 3.2. Absence of IL-12 during S. venezuelensis infection enhances Th2 cytokine generation in the lung parenchyma As compared with uninfected mice, S. venezuelensisinfected IL-12/ and wild-type C57BL/6 mice exhibited higher levels of the Th2-type cytokines IL-3, IL-5, IL-13, and IL-10 in the lung parenchyma during all periods of infection. Also, differences were observed between the two infected groups of mice for all cytokine types (Fig. 3). From days 7 to 21 post-infection, the levels of IL-13 and IL-10 were significantly higher in lungs of IL-12/ infected mice than in the wild-type animals (Fig. 3A, 3B). Differences in IL-5 levels were also noted in all observation periods, when infected IL12/ mice produced higher levels of IL-5 than wild-type mice. Also, we observed increased IL-3 levels not only in the earlier phase (day 7) but also in the last analyzed period (day 21 post-infection) in infected IL-12/ mice (Fig. 3C, D).

Fig. 4. Specific antibodies IgG1 (A) and IgE (B) in the serum of IL-12/ and wild-type C57BL/6 mice infected with S. venezuelensis (Sv) larvae. The antibody concentrations were detected by ELISA. The data are expressed as mean  SEM of two independent experiments (n ¼ 5e7). *Significant compared to uninfected mice, #Wild-type C57BL/6 þ Sv vs. IL-12/ þ Sv, *# P < 0.05.

575

3.3. IL-12/ mice infected with S. venezuelensis display higher IgG1 and IgE serum levels in comparison to wild-type mice As demonstrated by Machado et al. in 2005, we also found that S. venezuelensis infection induced higher IgG1 and IgE serum levels in IL-12/ and wild-type mice than in the uninfected controls. The absence of IL-12 resulted in higher levels of circulating IgG1 on days 7 and 14 post-infection as shown in Fig. 4A. In accordance, elevated levels of IgE were observed in the serum of infected IL-12/ compared to wildtype C57BL/6 mice in these same time points (Fig. 4B). 3.4. IL-12 deficiency is associated with decreased parasite burden and female worm fertility We recovered fewer S. venezuelensis adult parasites from the intestine of IL-12/ mice than from wild-type animals. In addition, the absence of IL-12 resulted in decreased numbers of fecal eggs on day 7 post-infection (Fig. 5). On days 14 and 21 post-infection, in both IL-12/ and wild-type mice, adult parasites in the intestine and eggs in the feces were rare or absent. As histopathological analyses of the duodena of infected mice demonstrated (Fig. 6), on day 7 post-infection,

Fig. 5. Number of adult worms (A) and fecal eggs (B) recovered from IL-12/ and wild-type C57BL/6 mice infected with S. venezuelensis (Sv) larvae. Data are expressed as mean  SEM from two independent experiments (n ¼ 5e7). # Wild-type C57BL/6 þ Sv vs. IL-12/ þ Sv, #P < 0.05.

576

E.R. Machado et al. / Microbes and Infection 11 (2009) 571e578

Fig. 6. Histopathology of duodena from IL-12/ and wild-type C57BL/6 mice after infection with S. venezuelensis (Sv) larvae. A group of uninfected animals was used as a control. Representative hematoxylin and eosin-stained duodenal sections from uninfected wild-type (A) and IL-12/ (E) mice, from infected wild-type mice (B, C, and D), and from infected IL-12/ mice (F, G, and H). The arrows indicate sections containing adult worms. Tissues were fixed in 10% formalin and embedded in paraffin, and 5-mm sections were stained with hematoxylin and eosin. Magnification of panels, 100.

E.R. Machado et al. / Microbes and Infection 11 (2009) 571e578

numerous worms were localized beneath the epithelial layer, especially in the infected wild-type C57BL/6 mice. The worm count decreased in both infected groups on post-infection day 14, and parasites were completely expelled from the intestine on day 21 post-infection. In both IL-12/ (Fig. 6F) and wildtype infected (Fig. 6B) mice, adult worms were accompanied by intense infiltration of inflammatory cells and eosinophils into the epithelium. Meanwhile, near the intestinal lumen, mononuclear cell, but eosinophil infiltration was predominant. These inflammatory reactions were more accentuated in IL12/ mice than in the wild-type mice (Fig. 6). 4. Discussion In the present study, we newly demonstrate that a lack of IL-12 during S. venezuelensis infection enhances the protective Th2 immune response, decreasing parasite burden and female worm fertility. These findings may reflect decreased larval survival upon passage through the lungs and duodenum due to accentuated levels of Th2 cytokines and higher eosinophil numbers. In previous studies, we reported that S. venezuelensis infection increased eosinophil and mononuclear cell numbers in the blood and BALF, accompanied by enhanced leukotriene levels and IL-5 and IL-3 production [7]. Our present data confirm this augmented IL-5 and IL-3 levels and also demonstrate an increase in IL-10 in both IL-12/ and wildtype infected mice. Interestingly, in the absence of IL-12, the levels of IL-3, IL-5 and IL-10 as well as eosinophil numbers were increased in the lung parenchyma and bronchoalveolar space, in sharp contrast with cytokine levels and cell counts in wild-type animals. It is well established that IL-3 and IL-5 are eosinophil differentiation and survival factors [27,28], and that IL-5 induces eosinophil release from the bone marrow [27]. Thus, the present data strongly suggest that higher eosinophil numbers in the blood and BALF of IL-12/ infected mice resulted from increased IL-3 and IL-5 levels during parasitic infection. Proof of this causal mechanism requires further investigation. Previous studies demonstrated a role for IL-5 in antiStrongyloides host defenses [7]. Both IL-5 and eosinophils play key roles in innate and adaptive immune responses to Strongyloides parasites [7,8,25]. The intraepithelial infiltration of eosinophils in the gastrointestinal tract was found to contribute to the elimination of adult S. venezuelensis nematodes in mice. Immunoglobulins, in coordination with IL-5 and eosinophils, also contribute to host defense during parasite infection [29]. In this context, we speculate that IL-12-null mice exhibit stronger eosinophil-mediated effector mechanisms, resulting in increased larval death during migration through the lungs. Such a decrease in larval numbers may result in markedly lower numbers of parasites in the duodena and eggs in the feces. We further postulate that the increase in eosinophil numbers in IL-12/ infected mice might be responsible for increased IL-13 levels observed in the lung tissue, since eosinophils are a main source of IL-13 [30]. Since IL-13 is an important growth factor for IgE-producing B lymphocytes, we also detected significant differences in

577

parasite-specific IgE levels between IL-12/ and wild-type mice. Moreover, parasite-specific IgG1 levels were only increased in the absence of IL-12 with exception on the day 21 post-infection. In spite of the slight increase in parasitespecific IgG1 and IgE in the IL-12/ mice, in comparison with wild-type animals, these differences in serum antibody levels may be biologically relevant, contributing to more efficient effector mechanisms in the infected host. A role for IL-12 as a down-modulator of the Th2 immune response during S. stercoralis infection has been suggested [29]. Here, we demonstrate for the first time, that IL-12 truly counterregulates the Th2 immune response during S. venezuelensis infection. These findings may contribute to the development of new treatments for patients with Strongyloides hyperinfection syndrome, for example, using targeted monoclonal antibodies against IL-12 to improve the Th2 immune response to better eliminate parasites. Acknowledgments This study was supported by grants from the Fundac¸~ao de Amparo a` Pesquisa do Estado de S~ao Paulo (FAPESP -Process: 02/12856-2) and Conselho Nacional de Desenvolvimento Cientifico e Tecnologico. We are grateful to Elaine Medeiros Floriano from the Laborato´rio de Patologia da Faculdade de Medicina de Ribeir~ao Preto, Universidade de S~ao Paulo for help in the formulation of histologic preparations. References [1] D.I. Grove, Human strongyloidiasis, Adv. Parasitol. 38 (1996) 251e309. [2] J.L. Guyomard, S. Chevrier, J.L. Bertholom, C. Guigen, J.F. Charlin, Finding of Strongyloides stercoralis infection, 25 years after leaving the endemic area, upon corticotherapy for ocular trauma, J. Fr. Ophtalmol. 30 (2007) e4. [3] P. Giavina-Bianchi, F.De S. Silva, M. Toledo-Barros, D. Birolini, J. Kalil, L.V. Rizzo, A rare intestinal manifestation in a patient with common variable immunodeficiency and strongyloidiasis, Int. Arch. Allergy Immunol. 140 (2006) 199e204. [4] R.S. Vadlamudi, D.S. Chi, G. Krishnaswamy, Intestinal strongyloidiasis and hyperinfection syndrome, Clin. Mol. Allergy 4 (2006) 8. [5] L. Einsiedel, D. Spelman, Strongyloides stercoralis: risks posed to immigrant patients in an Australian tertiary referral centre, Intern. Med. J. 36 (2006) 632e637. [6] F.J. Thompson, G.L. Barker, L. Hughes, C.P. Wilkes, J. Coghill, M.E. Viney, A microarray analysis of gene expression in the free-living stages of the parasitic nematode Strongyloides ratti, BMC Genomics 7 (2006) 157. [7] E.R. Machado, M.T. Ueta, E.V. Lourenc¸o, F.F. Anibal, C.A. Sorgi, E.G. Soares, M.C. Roque-Barreira, A.I. Medeiros, L.H. Faccioli, Leukotrienes play a role in the control of parasite burden in murine strongyloidiasis, J. Immunol. 175 (2005) 3892e3899. [8] A.M. Galioto, J.A. Hess, T.J. Nolan, G.A. Schad, J.J. Lee, D. Abraham, Role of eosinophils and neutrophils in innate and adaptive protective immunity to larval Strongyloides stercoralis in mice, Infect. Immun. 74 (2006) 5730e5738. [9] A. Mir, D. Benahmed, R. Igual, R. Borras, J.E. O’Connor, M.J. Moreno, S. Rull, Eosinophil-selective mediators in human strongyloidiasis, Parasite Immunol. 28 (2006) 397e400. [10] Y. Sasaki, T. Yoshimoto, H. Maruyama, T. Tegoshi, N. Ohta, N. Arizono, K. Nakanishi, IL-18 with IL-2 protects against Strongyloides

578

[11]

[12]

[13]

[14]

[15]

[16]

[17]

[18]

[19]

[20]

E.R. Machado et al. / Microbes and Infection 11 (2009) 571e578 venezuelensis infection by activating mucosal mast cell-dependent type 2 innate immunity, J. Exp. Med. 202 (2005) 607e616. D. Negr~ao-Correa, V. Pinho, D.G. Souza, A.T. Pereira, A. Fernandes, K. Scheuermann, A.L. Souza, M.M. Teixeira, Expression of IL-4 receptor on non-bone marrow-derived cells is necessary for the timely elimination of Strongyloides venezuelensis in mice, but not for intestinal IL-4 production, Int. J. Parasitol. 36 (2006) 1185e1195. M. Satoh, H. Toma, S. Kiyuna, Y. Shiroma, A. Kokaze, Y. Sato, Association of a sex-related difference of Strongyloides stercoralis-specific IgG4 antibody titer with the efficacy of treatment of strongyloidiasis, Am. J. Trop. Med. Hyg. 71 (2004) 107e111. D.M. Zaiss, L. Yang, P.R. Shah, J.J. Kobie, J.F. Urban, T.R. Mosmann, Amphiregulin, a Th2 cytokine enhancing resistance to nematodes, Science 314 (2006) 1746. C.L. Langrish, B.S. McKenzie, N.J. Wilson, R.W. Malefyt, R.A. Kastelein, D.J. Cua, IL-12 and IL-23: master regulators of innate and adaptive immunity, Immunol. Rev. 202 (2004) 96e105. B. Naume, M.K. Gately, B.B. Desai, A. Sundan, T. Espevik, Synergistic effects of interleukin 4 and interleukin 12 on NK cell proliferation, Cytokine 5 (1993) 38e46. R. Manetti, P. Parronchi, M.G. Giudizi, M.P. Piccinni, E. Maggi, G. Trinchieri, S. Romagnani, Natural killer cell stimulatory factor (interleukin 12 [IL-12]) induces T helper type 1 (Th1)-specific immune responses and inhibits the development of IL-4-producing Th cells, J. Exp. Med. 177 (1993) 1199e1204. M. Aste-Amezaga, A. D’Andrea, M. Kubin, G. Trinchieri, Cooperation of natural killer cell stimulatory factor/interleukin-12 with other stimuli in the induction of cytokines and cytotoxic cell-associated molecules in human T and NK cells, Cell. Immunol. 156 (1994) 480e492. F.D. Finkelman, T. Shea-Donohue, J. Goldhill, C.A. Sullivan, S.C. Morris, K.B. Madden, W.C. Gause, J.F. Urban, Cytokine regulation of host defense against parasitic gastrointestinal nematodes: lessons from studies with rodent models, Annu. Rev. Immunol. 15 (1997) 505e533. E. Schmitt, P. Hoehn, T. Germann, E. Rude, Differential effects of interleukin-12 on the development of naive mouse CD4 þ T cells, Eur. J. Immunol. 24 (1994) 343e347. J.D. Marshall, H. Secrist, R.H. DeKruyff, S.F. Wolf, D.T. Umetsu, IL-12 inhibits the production of IL-4 and IL-10 in allergen-specific human CD4þ T lymphocytes, J. Immunol. 155 (1995) 111e117.

[21] F.D. Finkelman, K.B. Madden, A.W. Cheever, I.M. Katona, S.C. Morris, M.K. Gately, B.R. Hubbard, W.C. Gause, J.F. Urban Jr., Effects of interleukin 12 on immune responses and host protection in mice infected with intestinal nematode parasites, J. Exp. Med. 179 (1994) 1563e1572. [22] H.L. Rotman, S. Schnyder-Candrian, P. Scott, T.J. Nolan, G.A. Schad, D. Abraham, IL-12 eliminates the Th2 dependent protective immune response of mice to larval Strongyloides stercoralis, Parasite Immunol. 19 (1997) 29e39. [23] E. Brumpt, Pre´cis de Parasitologie, Masson et Cie, Paris, 1934, pp. 1042e1045. [24] E.R. Machado, M.T. Ueta, M.R. Gonc¸alves-Pires, J.B. de Oliveira, L.H. Faccioli, J.M. Costa-Cruz, Diagnosis of human strongyloidiasis using particulate antigen of two strains of Strongyloides venezuelensis in indirect immunofluorescence antibody test, Exp. Parasitol. 99 (2001) 52e55. [25] E.R. Machado, M.T. Ueta, E.V. Lourenc¸o, F.F. Anibal, M.C. RoqueBarreira, L.H. Faccioli, Comparison of immune responses in mice infected with different strains of Strongyloides venezuelensis, Parasite Immunol. 29 (2007) 549e557. [26] E.R. Machado, M.T. Ueta, M.R.F. Gonc¸alves-Pires, J.B.A. Oliveira, L.H. Faccioli, J.M. Costa-Cruz, Strongyloides venezuelensis alkaline extract for the diagnosis of human strongyloidiasis by enzyme-linked immunosorbent assay, Mem. Inst. Oswaldo Cruz. 98 (2003) 849e851. [27] Y. Yamaguchi, Y. Hayashi, Y. Sugama, Y. Miura, T. Kasahara, S. Kitamura, M. Torisu, S. Mita, A. Tominaga, K. Takatsu, Highly purified murine interleukin 5 (IL-5) stimulates eosinophil function and prolongs in vitro survival. IL-5 as an eosinophil chemotactic factor, J. Exp. Med. 167 (1988) 1737e1742. [28] K. Kimura, C.H. Song, A. Rastogi, G. Dranoff, S.J. Galli, C.S. Lantz, Interleukin-3 and c-Kit/stem cell factor are required for normal eosinophil responses in mice infected with Strongyloides venezuelensis, Lab. Invest. 86 (2006) 987e996. [29] M. Korenaga, Y. Hitoshi, N. Yamaguchi, Y. Sato, K. Takatsu, I. Tada, The role of interleukin-5 in protective immunity to Strongyloides venezuelensis infection in mice, Immunology 72 (1991) 502e507. [30] D. Myrtek, M. Knoll, T. Matthiesen, S. Krause, J. Lohrmann, D. Schillinger, M. Idzko, J.C. Virchow, K. Friedrich, W. Luttmann, Expression of interleukin-13 receptor alpha 1-subunit on peripheral blood eosinophils is regulated by cytokines, Immunology 112 (2004) 597e604.