Acta Tropica 98 (2006) 305–310 Rapid communication Osteopontin is involved in Th1-mediated immunity against Plasmodium falciparum infection in a holoendemic malaria region in Vietnam Yoshimasa Maeno a,∗ , Shusuke Nakazawa b , Le Duc Dao c , Nguyen Van Tuan c , Nguyen Duc Giang c , Truong Van Hanh c , Koki Taniguchi a a b Department of Virology and Parasitology, Fujita Health University School of Medicine, 1-98 Kutsukake, Toyoake, Aichi 470-1192, Japan Department of Protozoology, Institute of Tropical Medicine, Nagasaki University, 1-12-4 Sakamoto, Nagasaki, Nagasaki 852-8523, Japan c Molecular Biology Department, National Institute of Malariology, Parasitology and Entomology, BC 10200 Tu-Liem, Hanoi, Vietnam Received 28 March 2005; received in revised form 16 January 2006; accepted May 2006 Available online 12 June 2006 Abstract We examined the role of osteopontin (OPN) in immunity against Plasmodium falciparum infection We measured the mRNA levels for OPN and several cytokines in RNA preparations extracted from dried blood on filter paper obtained from falciparum malaria patients in Vietnam Expression of OPN mRNA was detected in 134 of 161 patients The expression of both interleukin-12 p40 and interferon-␥ mRNAs in the group positive for OPN mRNA was significantly higher than that in the group negative for OPN mRNA The level of parasitemia in the OPN mRNA-positive group was much lower than that in the negative one These results suggest that OPN might suppress multiplication of the parasites through T helper cells-mediated immune responses © 2006 Elsevier B.V All rights reserved Keywords: Osteopontin; Plasmodium falciparum; IL-12; IFN-␥; mRNA; Vietnam Malaria remains a major cause of mortality worldwide; however, neither the immunity to infection of hosts nor the pathogenesis of the disease is well understood Humans living in endemic areas appear to be able to control parasitemia through an interferon-␥ (IFN-␥) response as a means of protective immunity (Winkler et al., 1998) Studies on murine malaria have suggested that immune responses in mice against nonlethal murine blood-stage malaria Plasmodium chabaudi chabaudi comprise the production of interleukin (IL)-12, tumor ∗ Corresponding author Tel.: +81 562 93 2486; fax: +81 562 93 4008 E-mail address: ymaeno@fujita-hu.ac.jp (Y Maeno) 0001-706X/$ – see front matter © 2006 Elsevier B.V All rights reserved doi:10.1016/j.actatropica.2006.05.001 necrosis factor-␣ (TNF-␣), and IFN-␥ (Jacobs et al., 1996; De Souza et al., 1997; Stevenson et al., 1995; Su and Stevenson, 2002) These cytokines induced by malaria infection facilitate the modulation of parasitemia in the host Osteopontin (OPN) is a sialated phosphoprotein, an extracellular matrix protein, produced by activated macrophages, T cells, and natural killer cells, and is implicated in a variety of functions, such as the adhesion and migration of T cells and macrophages (Denhardt and Guo, 1993; Giachelli et al., 1998; Kawashima et al., 1999; O’Regan et al., 2000; O’Regan and Berman, 2000; Senger et al., 1988) Recent studies demonstrated that OPN was involved in the host response against infectious pathogens (Ashkar et al., 2000; Nau 306 Y Maeno et al / Acta Tropica 98 (2006) 305–310 et al., 1999; O’Regan and Berman, 2000) The immune response to Listeria monocytogenes or Mycobacterium bovis in osteopontin knockout (OPN-KO) mice is defective, because of the deficiency of the IL-12 and IFN␥ dependent T helper (Th) cells responses (Ashkar et al., 2000; Nau et al., 1999) These findings suggested that OPN might polarize the Th1 related cytokine response To our knowledge, however, no reports have been presented the data on an aspect of OPN in malaria infection In the present study, we examined whether or not infection by Plasmodium falciparum induces OPN production Next, we evaluated whether OPN is involved in immunity against malaria infection This study was conducted with volunteers, who were diagnosed as having falciparum malaria by examining Giemsa-stained thick blood films, living in Phu Rieng Village, Binh Phouc Province, Vietnam Malaria is transmitted perennially in this area One hundred sixty-one volunteers aged 10 months–72 years (mean ± standard deviation: 20 ± 14 years) were enrolled in this study The sex ratio of the participants was 1.21: 88 men and 73 women Thirty-five of the 161 volunteers were infected with P vivax simultaneously Treatment was done according to a Vietnamese policy The volunteers were mostly asymptomatic, but 24% of them showed a high body temperature (≥39 ◦ C) Thirty-two Giemsastained blood smear-negative healthy Vietnamese (with a past history of exposure to Plasmodium species) and 20 healthy Japanese (never exposed to Plasmodium species) volunteers were also enrolled as controls The study was reviewed and approved by the ethical committee in Fujita Health University School of Medicine and in Institute of Tropical Medicine, Nagasaki University in Japan and National institute of Malariology, Parasitology and Entomology in Vietnam as a Japan-Vietnam Collaborative Research Program All volunteers except infant patients provided informed consent; in infant patients, consent was obtained from a close relative Blood samples were obtained by means of finger pricking and spotted onto chromatography-grade filter papers (ET31CHR; Whatman, Maidstone, UK) Each blood-spotted filter paper was dried in the air immediately, put into a sealed plastic bag, and stored at room temperature (as lower possible) until reverse transcription-PCR (RT-PCR) analysis In this study area, blood sampling by venipuncture is problematic because of the reluctance of people, the need for the electricity and medical facilities, and transportation system of the samples and medical equipments Therefore, we carried out mRNA analysis in the dried blood for this study that has been found to be satisfactory for the immunological examination in the field-based studies (Maeno et al., 2003) The target cytokines in this study were IL-12 p40, IFN-␥, IL-4, and IL-10 P falciparum asexual stagespecific ribosomal RNA (A18S rRNA) and human actin were used as an indicator of the parasite density and an internal control, respectively Extraction of total RNA and reverse transcription for first-strand synthesis of cDNA from each dried spot were carried out as previously described (Maeno et al., 2003) The cDNA was subjected to PCR using specific oligonucleotide primers to amplify cDNAs encoding -actin, cytokines (IL-12 p40, IFN-␥, IL-4, and IL-10), OPN, and A18S rRNA PCRs for -actin, IL-12 p40, IFN-␥, IL-4, and IL-10 mRNA were carried out using Primer set kits (Maxim Biotech Inc., South San Francisco, CA) according to the manufacturer’s instructions For OPN and A18S rRNA, reaction mixtures (25 l) comprised 0.8 g of cDNA as a template, 0.5 M each primer, 200 M dNTP, 0.625 units of Taq DNA polymerase (QIAGEN, Tokyo, Japan), and × PCR buffer (containing 1.5 mM MgCl2 ) Target cDNA was amplified using a PCR protocol consisting of a denaturation step (94 ◦ C, min) followed by 35 cycles (94 ◦ C, min; optimal annealing temperature [OPN: 54 ◦ C, A18S rRNA: 68 ◦ C], 30 s; and 72 ◦ C, min), and a final extension step (72 ◦ C, min) The primer sequences for OPN were -TTG CAG TGA TTT GCT TTT GC-3 (sense) and -GCC ACA GCA TCT GGG TAT TT-3 (antisense) The primer sequences for A18S rRNA were previously described (Niederwieser et al., 2000) A GeneAmp PCR System 2400 (PerkinElmer, Norwalk, CT) was used for all the PCRs The PCR products were separated by electrophoresis on a 1.5% agarose gel and stained with ethidium bromide The densities of the DNA bands were determined with Lane & Spot Analyzer software (Atto, Tokyo, Japan) Expression of results for A18S rRNA was standardized with g total RNA in a dried blood spot and for the target mRNAs of cytokines and OPN were standardized by quantification of -actin mRNA used as an internal control Statistical evaluation was performed with Mann– Whitney test (two-tailed) All analyses were performed using SPSS software (SPSS Japan, Tokyo, Japan), a value of p < 0.05 being considered significant The RT-PCR product of OPN mRNA was detected in 134 of 161 specimens, the average expression level of OPN mRNA being 0.43 (Table 1) The positive ratio and the expression level of OPN mRNA were similar between a single infection with P falciparum and a mixed infection with P falciparum and P vivax OPN mRNA was not detected in any blood from healthy Y Maeno et al / Acta Tropica 98 (2006) 305–310 307 Table Positive rate and level of OPN mRNA expression in participants infected with P falciparum Category Number of patients Number of OPN mRNA-positive casesa Expression level of OPN mRNAb Falciparum malaria P falciparum single infection P falciparum and P vivax mixed infection Healthy Vietnamese Healthy Japanese 161 126 35 32 20 134 (83%) 105 (83) 29 (83) (0) (0) 0.43 0.44 0.43 0.00 0.00 Sex Female Male 73 88 59 (81) 75 (88) 0.54 ± 0.08 0.36 ± 0.05 Age ≤10 11–20 21–30 31≤ 54 40 35 32 46 (85) 32 (80) 28 (80) 28 (88) 0.64 0.37 0.30 0.35 a b ± ± ± ± ± ± ± ± ± 0.04 0.07 0.07 0.00 0.00 0.10 0.07 0.08 0.07 The measurement of OPN mRNA in dried blood spots was carried out by semi-quantitative RT-PCR Data were standardized by quantification of -actin mRNA used as an internal control, and were expressed as means ± S.E.M Vietnamese and Japanese controls The positive ratio and expression level of OPN mRNA were not different between sexes or among generations although there was a tendency that these ratio and level were high in people under 10 years and over 31 years of age (Table 1) To elucidate a relationship between OPN and pathologic change, we evaluated whether there was a difference in the expression levels of A18S rRNA and in body temperature due to the expression of OPN mRNA or not First, we compared the expression levels of A18S rRNA between the OPN mRNA-positive and -negative groups in the single infection with P falciparum and the mixed infection with P falciparum and P vivax, respectively In the single infection group, the expression level of A18S rRNA in OPN mRNA-positive group was significantly lower than that in negative group (Fig 1a) The similar result in the single infection group was observed in the mixed infection group The expression level of A18S rRNA in both groups, moreover, was inversely correlated to the level of OPN mRNA expression (Fig 1b) Next, we examined the relationship between the expression levels of A18S rRNA and body temperature in both the OPN mRNA-positive and -negative groups (Fig 1c and d) We divided the specimen into two groups with a border value of body temperature 39 ◦ C In the group with body temperature lower than 39 ◦ C, the level of A18S rRNA expression of OPN mRNA-positive group in P falciparum single infection was significantly lower than that in negative one, in particular (Fig 1c) The similar results was observed in the mixed infection group, but the difference was not significant In both infection groups with body temperature higher than 39 ◦ C, the level of A18S rRNA expression in OPN mRNA-positive group was lower than that in OPN mRNA-negative group, but not significant (Fig 1d) Recent studies suggested that OPN is involved at an early time point in an establishment of Th1 immunity by upregulating the production of IL-12 (Ashkar et al., 2000; Nau et al., 1999) We evaluated the roles of OPN in the Th1 and Th2 immune responses to P falciparum infection In the single infection with P falciparum, the expression levels of mRNA for IL-12 p40 and IFN-␥ in the OPN mRNA-negative group were significantly lower than that in the positive group (Fig 2a and b) Similar results for the expression levels of mRNA for IL-12 p40 and IFN-␥ were obtained in the mixed infection group, but expression of IL-12 p40 mRNA did not show a significant difference between the OPN mRNApositive and -negative groups (Fig 2a and b) It has been shown that the serum level of IL-12 is elevated from an early period of Plasmodium infection (Sam and Stevenson, 1999a,b; Su and Stevenson, 2000) The presence of IL-12 affects the initiation and regulation of IFN-␥ production, and macrophage effector functions (Sam and Stevenson, 1999a,b) In fact, IL-12 p40 gene deficient mice developed significantly higher parasitemia during acute P c chabaudi infection and those mice exhibited both impaired IFN-␥ and nitric oxide responses by spleen cells (Bastos et al., 2002) On human falciparum malaria cases, plasma levels of IL-12 were lower in Gabonese children with severe malaria than with mild malaria, but the basal level of IL-12 in the same children after recovery was similar to that with mild malaria (Luty et al., 2000) Similarly, the plasma 308 Y Maeno et al / Acta Tropica 98 (2006) 305–310 Fig (a–d) Expression level of P falciparum asexual stage-specific ribosomal RNA (A18S rRNA) in dried blood with or without OPN mRNA The measurement of target RNA expression was carried out by RT-PCR Data are standardized with g total RNA in a dried blood spot for A18S rRNA The horizontal line within the box stands for the median value; the box includes the 25th and 75th percentiles and each bar represents less than 10th or more than 90th percentiles The marks, and ×, indicate a loser (1.5–3 interquartile range) and an extreme value (more than three interquartile range), respectively * Significant difference between the OPN mRNA-positive and -negative groups (P < 0.01; Mann–Whitney test) Fig Expression levels of mRNA for IL-12 p40 (a), IFN-␥ (b), IL-4 (c), and IL-10 (d) in dried blood with or without OPN mRNA The measurement of target mRNA expression was carried out by semi-quantitative RT-PCR Data are standardized by quantification of -actin mRNA used as an internal control The horizontal line within the box stands for the median value; the box includes the 25th and 75th percentiles and each bar represents less than 10th or more than 90th percentiles The marks, and ×, indicates a loser (1.5–3 interquartile range) and an extreme value (more than three interquartile range), respectively The statistically significant difference between the OPN mRNA-positive and -negative groups is indicated (* P < 0.01, ** P < 0.05; Mann–Whitney test) Y Maeno et al / Acta Tropica 98 (2006) 305–310 level of IL-12 in Papua New Guineans infected with P falciparum was strongly inversely correlated with parasitemia (Boutlis et al., 2003) On the other hand, the role of OPN in infectious diseases has been demonstrated in infection by L monocytogenes, herpes simplex virus-type (HSV-1), and M bovis (Ashkar et al., 2000; Nau et al., 1999) IL-12 and IFN-␥ dependent Th1 responses to L monocytogenes or M bovis bacillus Calmette-Gu´erin are defective in OPN gene deficient mice (Ashkar et al., 2000; Nau et al., 1999) Taken these evidences and our findings into consideration, OPN may play a role in activation of Th1-mediated immune responses in the both P falciparum single and mixed infections We analyzed kinetics of expression levels of mRNA for OPN, IL-12 p40, and IFN-␥ of 16 people with P falciparum single infection who were under the followup survey for weeks; from 1-week before detection of parasites and treated until two weeks after treatment The expression levels of mRNA for OPN, IL-12 p40, and IFN-␥ before parasite detection was low When the parasite was detected, the level of those expressions increased and those mRNAs decreased immediately after a treatment (Fig 3) On Th2-related cytokine analysis, the expression levels of mRNA for IL-4 and IL-10 in the OPN mRNAnegative group of P falciparum single infection was found to be significantly higher than that in the positive group (Fig 2c and d) Similar results were observed in the mixed infection group (Fig 2c and d) The role Fig Kinetics of the expression levels of mRNA for OPN, IL-12 p40, and IFN-␥ in dried blood from 16 patients with P falciparum single infection The measurement of target mRNA expression was carried out by semi-quantitative RT-PCR Data are standardized by quantification of -actin mRNA used as an internal control Each point was expressed as the mean of 16 patients 309 of OPN in Th2 immune responses was demonstrated in HSV-1 or L monocytogenes infection Staege et al (2001) indicated that IL-4 downregulated OPN production as one of the means of deactivation of macrophage functions in L monocytogenes infection Ashkar et al (2000) showed that OPN-KO mice showed an increased IL-10 production in HSV-1 or L monocytogenes infection while WT mice showed a decreased production In this study, however, some people in OPN mRNApositive group of both single and mixed infection groups showed higher expression level of mRNAs for IL-4 or IL10 The cases with the higher expression of IL-4 mRNA showed lower expression of mRNAs for IL-12 p40 and IL-10 In contrast, the cases with the higher expression of IL-10 mRNA showed lower expression of IL-4 mRNA but expression of IL-12 p40 mRNA was high (Fig 2c and d) von der Weid et al (1994) demonstrated that IL-4 deficient mice infected with P chabaudi showed a decrease in the expression of mRNA for Th2-associated cytokines, such as IL-5 and IL-6, but not IL-10 IL10 is a potent inhibitor of Th2-cell functions, and its expression within the CD4 subset during malaria parasite clearance definitely distinguishes its regulation from that of IL-4 (de Waal Malefyt et al., 1993; Winkler et al., 1998) Recent in vitro and in vivo studies, furthermore, have shown that an increase in IFN-␥ and IL-10 coproducing CD4+ cells was found in falciparum malaria patients (Ramharter et al., 2003; Winkler et al., 1998) The co-production of these cytokines has been found to be particularly induced by IL-12 (Windhagen et al., 1996) At least IL-4 and IL-10 appeared to be antagonistic toward OPN in the present study It remains to be clarified whether or not OPN regulates the pathway of these Th2 cytokines The behavior of IL-4 and IL-10 may not be act harmoniously to parasites Although OPN is secreted from activated T cells, macrophages, and natural killer cells in pathogen infections, we did not detect the expression of OPN mRNA in the blood samples from 27 malaria-infected people Inhibition of OPN mRNA expression by Th2 related cytokines, especially IL-4, is conceivable, as shown in Fig Kumaratilake and Ferrante (1992) have also shown that IL-4 suppressed macrophage activity toward malaria parasites The present study suggests that the expression of OPN may lead a Th1-dominant immune responses rather than a Th2-dominant through the induction of IL-12 and may be involved in the control of symptom and suppression of parasitemia, as reported that OPN is an important factor for pathogen control in L monocytogenes and M bovis BCG infection (Ashkar et al., 2000; Nau et al., 1999) 310 Y Maeno et al / Acta Tropica 98 (2006) 305–310 Acknowledgements This work was partially supported by a Grant-inAid for Japan-Vietnam 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