Plasma levels of interleukin-12 and interleukin-18 in Plasmodium falciparum malaria patients in South Darfur Mohammed I Abdul Fattah *, Mahmoud M Badr*, Yaser A Shahin **, *Department of Microbiology and Immunology,**Departement of Gastroentrology and Hepatology Faculty of Medicine , Benha University, Egypt. Abstract : The interaction between cytokines such as interleukin-12 ( IL-12 )and IL-18 plays an important role in malaria pathogenesis and outcome , modulating the immunoresponce in Plasmodium falciparum ( P. falciparum ) malaria. This work aimed at detecting the plasma levels of IL-12 and IL-18 in P. falciparum malaria patients and to correlate the production of these cytokines with the severity of the disease .this study was conducted on two groups : Group I , consisted of 80 patients with P. falciparum malaria infection they were classified into two groups according to disease severity , 30 patients with severe malaria and 50 patients with mild malaria .Group II,consisted of 30 healthy persons as a control group. The levels of IL-12 and IL-18 were higher (16.2±8.3 pg/ml and 2682.8 ± 332.0 pg/ml , respectively ) in patients with mild malaria than in patients with severe form of the disease (12.3±6.42 pg/ml and 1226± 267.2 pg/ml respectively ) A positive correlation was observed between IL-12 and IL-18.These findings suggest that the production of these cytokines may be coregulated and both have an immunoregulatory effect on the immune response to P. falciparum infection , so can be used as a reliable parameter to predict the progression of the disease and may be used as an immunotherapy against P.falciparum malaria infection , especially in the early phase of the disease. Further studies are needed to find out the relation between these cytokines and individual forms and complications of P. falciparum malaria infection . Introduction Malaria is caused by the infection of hemoprotozoan parasites belonging to the genus Plasmodium. There are four parasite species , namely, Plasmodium falciparum,P. vivax, P. ovale and P. malariae, responsible for human malaria infections. P. falciparum and P. vivax are the two most causative agents of severe malaria ; infection with P. falciparum causes the most severe disease and is responsible for more than 95% of malaria deaths worldwide [1]. The clinical symptoms and pathological consequences of human malaria are associated with the asexual blood stage of the parasite life cycle . The clinical symptoms range from light headache or mild fever to extremely severe, life-threatening complications such as cerebral malaria, sever anemia, renal failure, hypoglycaemia, circulatory collapse, repeated generalized convulsions and pulmonary edema [2]. Among these clinical complications, cerebral malaria and severe anemia are commonly observed in children, while pulmonary and renal disorders often occur in adults . Both parasite and host factors determine the severity and outcome of malaria infection [3]. There has been rather little work characterizing innate immune responses to malaria, assessing their role in protection or their potential to modulate adaptive responses. Innate immune mechanisms represent the first line of defense against invading 1 pathogens. For severe, acute infections such as malaria, the ability to mount an effective innate response may mean the difference between life and death [4] Pro-inflammatory cytokines ( eg. IL-12 and IL-18 ) , have been associated with protective cell-mediated immunity by their capacity to induce parasite killing by monocytes/ macrophages and neutrophils. Anti-inflammatory cytokines counteract the production and possible cytopathic effects of pro-inflammatory cytokines and may thus be associated with malaria susceptibility [5]. Human interleukin 12 (IL-12), also known as natural killer cell stimulatory factor (NKSF) and cytotoxic lymphocyte maturation factor (CLMFIL-12), is a major cytokine involved in the control of CD4_ Th1 responses and NK cells. IL-12 has been shown to be involved in protective immunity against malaria by regulating gamma interferon (IFN- γ ), tumor necrosis factor alpha (TNF- α ), and nitric oxide responses in experimental studies [6]. IL-18 is structurally related to IL-1β and is produced by monocytes and macrophages in response to microbial products such as lipopolysaccharides . Experimental studies have shown that IL-18 plays a dominant role in the IL-12-mediated IFN- γ induction in T cells . In mice deficient for the IL-18 gene, little or no IFN- γ is produced despite the presence of IL-12, which suggests that IL-18 is a key player in regulation of IFN- γ production [7]. Furthermore, IL-12 and IL-18 synergistically up regulate IFN- γ production of macrophages, T cells, and NK cells including the enhancement of NKand T-cell-mediated cytotoxic activities [8]. In vivo, TNF- α production is associated with parasite clearance and resolution of fever , but elevated levels of TNF- α has also been associated with cerebral malaria. A priori, one would expect a crucial role for IL-12—and possibly also IL- 18—in initiation of the inflammatory cytokine cascade [9] . Accordingly, associations have been reported between circulating levels of IL-12 and IL-18 and risk of severe P. falciparum malaria [1,4,8 ]. In a prospective epidemiological study, IL-12 production was positively associated with IFN- γ and TNF- α production and negatively associated with parasitemia [8] . Complications of severe anemia and cerebral malaria are the major cause of morbidity and mortality, but recent evidence suggests that the host’s immunologic response plays a relevant role in the pathophysiology of this disease in humans [10] The balance between Th1 and Th2 immune response and between pro-inflammatory and anti-inflammatory cytokines is important in determining the level of malaria parasitemia, disease outcome and rates of recovery , while the overproduction of both pro-inflammatory and anti-inflammatory cytokines can be responsible for disease severity and mortality [11]. The aim of this work was to detect the plasma levels of IL-12 and IL-18 in patients with malaria in South Darfur and to correlate the production of these cytokines with the severity of the disease . Subjects and Methods : This study was conducted on eighty patients with P. falciparum malaria during the period between July 2009 to february 2010 in Nyala Specialised Hospital , South 2 Darfur, Sudan. These patients constituted ( Group I ) ,Their age ranged between 2 to 35 years ( 16.43±5.7 ) ,they were 49 males and 31 females. According to the symptoms, physical signs , laboratory findings of malaria at the onset of the disease , hematological parameters, hyperparasitemia and evidence of neurological involvement , these patients were classified into two groups. Group I (A) _Severe malaria_ (complicated) : They were 30 patients , their age ranged between 2 to 39 years ( 19.6±6.1 ) , they were 19 males and 11 females. The inclusion criteria were established microscopically by the presence of the P. falciparum parasite , that was confirmed with P. falciparum rapid test and by the clinical and physical signs according to the WHO criteria: evidence of neurological compromission (prostration, lethargy), gastrointestinal symptoms, severe anemia (Ht<20%, Hb<6 g/dl), hyperparasitemia corresponding to >5 parasitised red cells/100 red blood cells or >5% parasitemia, hypoglycemia (serum glucose less than 40 mg/dl), acidosis with respiratory distress, oliguria, cardiovascular shock, jaundice and diffuse hemorrhages. Group I (B) _Mild malaria_ (uncomplicated) : They were 50 patients , their age ranged between 3 to 32 years ( 15.6±4.8 ) , they were 30 males and 20 females, their classification was established microscopically by the presence of <5 parasitised red cells/100 red blood cells or <5% parasitemia, with fever, headache and myalgia without any indication of severe malaria. Group II :Thirty healthy persons were also included in the study , their age ranged between 6 to 40 years ( 18± 7.4 ), they were 19 males and 11 females. All subjects enrolled as control group were negative at the thick-smear examination for P. falciparum, rapid test , without febrile episodes during the previous 6 months and without signs of anemia (Hb>10 g/dl). 1-Preparation of blood films and detection of P. falciparum : Thin and thick blood films were done to all subjects using finger – prick blood samples stained with standard Giemsa stain and examined by light microscopy. Diagnosis of P.falciparum infection was confirmed by dipstick test kits "ICT Malaria Pf" (ICT Diagnostics, Australia) containing: test cards, reagent A (lysing solution), capillary tubes coated with EDTA and product insert, describing test procedure. The ICT Malaria P.falciparum test was performed according to the test procedure described in the product insert briefly as follows: The test card was opened and the blood sample was taken by the capillary tube from the patient’s pricked finger, was put on the purple area of the sample pad of the dipstick strip located in the right hand side of the card. Once the purple pad was saturated with blood sample, one drop of reagent A was poured immediately above the purple pad (two drops below the blood spot and four drops on to a cleaning pad) located on the top of the left side of the card. After running up the lysed blood up to a limit line on the strip, the card was closed. The result could then be read through a viewing window 3-5 minutes after the color of blood has almost cleared. The test was considered positive when two lines were visible and negative when only a control line was observed. In case that no line or only the test line appeared, the result was considered to be invalid. 3 Venous blood ( 3 ml ) was collected from all subjects using the heparinised vaccutainer system , sera were separated immediately and frozen in -20◦C till the time of ELISA run . 2-Detection of IL-12 and IL-18 by ELISA : Interleukin -12 levels were determined by ELISA (NEOGEN corporation , Nandino Blvd. Lexington ,USA ) with a minimum detection level of 8 pg/ml. Briefly , coated ELISA plates with 96 wells . Standerd IL-12 and samples were added and incubated at room teprature for 2 h. Detection antibody was added and incubated at room temperature for 1 h. Avidin-HRP were added into each well and incubated for 30 minutes at room temperature . TMB Substrate was added and incubated for 15 minutes at room temperature then stop solution was added , and the plates were read in an ELISA reader at a wavelenghth of 450 nM. Interleukin -18 levels were determined by ELISA ( MBL international corporation Massachusetts , USA ) with a minimum detection level of 12.5 pg/ml. Briefly , coated ELISA plates with 96 wells . Standerd IL-18 and samples were added and incubated at room teprature for 1 h. Subsequently , peroxidase-conjugated anti – IL-18 was added and incubated at room temperature for 1 h. Peroxidase substrate solution was added into each well and incubated for 30 minutes at room temperature , then stop solution was added , and the plates were read in an ELISA reader at a wavelenghth of 450 nM. Results were tabulated and statistically analyzed using a statistical software (KyPlot, Kioshi Yoshioka, Japan 1999-2001v2) Results : Age and sex distribution in the studied groups are shown in ( table 1) . Laboratory findings of patients and control subjects are shown in ( table 2 ). In this study we found that there was a high significant difference between patients and controls in RBCs count , Hb. concentrations , Ht., MCHC, platelets conts and RBlG levels ( P<0.001 ) , while there was a significant difference in MCV, MCH and S.bilirubin ( P<0.01 ). We also found that there was a high significant difference between patients with severe malaria ( Group I – A ) and patients with mild malaria ( Group I-B ) in Hb concentrations , Ht , platelets counts , RBlG ,and S.bilirubin ( P<0.001 ) , and a significant difference in RBCs counts and MCV ( P<0.01 ), while there was no significant difference in Hb. levels and Ht ( P>0.05 ) . The correlation coefficients between IL-12 and IL-18 were r = 0.52 ( P<0.001 ) in Group I (A) and 0.41 ( P<0.05 )in group I (B) Table (1) : Age and sex distribution in the studied groups ; Males Females Total Age (years) 4 Group I - A 19 11 30 19.6 ± 6.1 Group I - B 30 20 50 15.6 ± 4.8 Group II 19 11 30 18.0 ± 7.4 Table (2): Laboratory parameters in subjects of the study Group I A Group II B RBCs 1.78±0.84 (Million/mm3) Hb 3.96±1.43 (G/dl.) Ht 11.96±4.2 (%) MCV 81.6±13.4 ( U3 ) MHC 28.1±4.18 ( yy ) MCHC 31.16±5.5 (%) 1 Platelets 150±90 ( mm3 ) RBl.G 36±4.8 (mg/dl) S.Bilirubin 3.2±0.63 ( mg/dl ) * HS : high significant Comparisons 3.82±0.91 4.11±0.66 Group I vs Group II t P Sig. 11.71 <0.001 HS 8.61±1.61 11.82±2.5 0.735 <0.001 HS 0.882 <0.001 HS 26.2±5.31 29.3±4.16 0.513 <0.001 HS 0,721 <0.001 HS 72.3±11.8 86.6±10.7 1.052 <0.01 S 2.461 <0.01 S 26.2±3.87 35.8±3.75 1.743 <0.01 S 0.46 >0.05 NS 32.08±4.6 38.22±3.1 1.271 <0.001 HS 0.269 >0.05 NS 238±170 318±99 6.41 <0.001 HS 8.72 <0.001 HS 86±7 105±11 8.64 <0.001 HS 15.36 <0.001 HS 0.7±0.12 0.5±0.41 1.34 <0.01 S 0.561 <0.001 HS * S : significant Group IA vs Group IB t P Sig. 8.54 <0.01 S * NS : non significant Table (3) : Plasma levels of IL-12 and IL-18 in the studied groups : Group I A IL-12 12.3±6.42 ( pg/ml ) 1226± IL-18 267.2 ( pg/ml ) * HS : high significant Group II B 16.2±8.3 7.8±2.8 Comparisons Group I vs Group II t P Sig. 2.703 <0.001 HS 2682.8 ± 110± 21.68 <0.001 332.0 104.1 * S : significant * NS : non significant HS Group IA vs Group IB t P Sig. 1.631 <0.01 S 9.361 <0.001 HS Discussion : Malaria remains one of the leading global health concerns with over 300 million clinical cases and more than 1 million deaths on annual basis1. Being a predominantly tropical disease, malaria is one of the top three killers among communicable diseases in Africa [11]. Cytokines are key mediators in the cellular program of innate and adaptive immune responses to P. falciparum, controlling both the induction and the regulation of important effector immune responses. A critical balance between early pro- and 5 anti-inflammatory cellular responses is crucial both for effectively controlling parasitemia and for preventing pathology [12]. In our study we found that plasma levels of IL-12 were elevated in patients with severe malaria ( 12.3±6.42 pg/ml ) and patients with mild malaria ( 16.2±8.2 pg/dl ) more than controls ( 7.8±2.8 pg/ml ) and we found a significant difference between patients with severe malaria ( Group I-A ) and patients with mild malaria ( Group I-B ) (p<0.01 ) and a high significant difference between malaria patients (Group I) and controls (Group II ) ( p<0.001 ). Also we found that the plasma levels of IL-18 were elevated in severe malaria patients ( 1226±267.2 pg/ml) and patients with mild malaria ( 2682±332.0 pg/ml) than in control group ( 110±104.1pg/ml ) , we found a high significant difference between patients with severe malaria and patients with mild malaria , also there were a high significant difference between malaria patients and controls (p<0.001 ) ( table 3). Our results are in consistent with Malaguarnera et al., 2002 [13] , who studied the plasma levels of IL-12 and IL-18 in 105 African children affected by mild and severe P. falciparum malaria from Ouagadougou , Burkina Faso and correlated its levels with disease severity and found that the levels of IL-18 and IL-12 were higher(25·7 ±7.6 and 17.1± 7·8 pg/ml, respectively) in children with mild malaria than in children with a severe form of the disease (21·5 ±10 pg/ml and 13·2 ± 5·5 pg/ml, respectively). A positive correlation was observed between IL-18 and IL-12. His findings suggested that the production of these two cytokines may be coregulated and both have an immunoregulatory effect on the immune response in P. falciparum infection. It was concluded that IL-18 may be involved in the regulation of IL-12 production , both could have a critical role in the adaptive immune response to malaria through induction of IFN- γ , which has a central role in the cell-mediated immune response against blood-stage infection, inducing phagocytosis and killing. Moreover IL-12 and IL-18 synergistically induce anti-CD3 stimulated T cells or anti CD40-stimulated B cells to differentiate into highly IFN- γ producing cells [14] . The molecular mechanism underlying the synergy between IL-18 and 12 may be explained in part by reciprocal modulation of cytokine receptor expression. Specifically, IL-18 has been demonstrated to up-regulate IL-12 R expression, while IL-12 has been shown to up-regulate expression of IL-18R [15] . Also , Chaisavaneeyakorn et al., 2003 [8], who compared plasmaIL-12 and IL-18 levels in six groups of children classified as: aparasitemic, asymptomatic, mild malaria, highdensity uncomplicated malaria (UC), moderate malarial anemia (MMA), or severe malarial anemia (SMA). IL-12 levels were significantly reduced in children with SMA (P < 0.05) but not in other groups compared to children in the aparasitemic control group. IL-18, was produced more frequently (70%) in children with UC (P _ 0.06) than in children in the aparasitemic control group (32%). However, in the SMA group the IL-18 response rate declined to 30%, which was similar to that in the aparasitemic control group, which showed a 32% response rate. This finding suggested that the IL-18 response may be impaired in children with SMA. The results from his study supported the hypothesis that impairment of IL-12 and/or IL-18 response may contribute to the development of severe malarial anemia in areas of holoendemicity for malaria [8]. 6 The finding that IL-12 was significantly lower in children with SMA was in agreement with rodent studies [16] and previous human studies involving Gabonese Children, [17], the lowest level of IL-12 was in children with SMA although children with MMA and UC also had lower levels of IL-12 [17]. These findings closely agree with the results from a study conducted in Burkina Faso, where the levels of IL-12 have been shown to be significantly reduced in children with severe disease compared to those with mild malaria [18]. Luty et al. [19] have suggested that malaria pigments may contribute to the suppression of IL-12 production. Using a rodent model, Xu et al. [20] have shown that IL-12 p40 gene expression is profoundly inhibited by Plasmodium berghei infection. They have further shown that IL-10 may play a role in this inhibition and that it may be regulated by transcriptional regulation of the IL-12 gene [20]. Since IL-12 is involved in directly activating protective immunity against both liver stage and blood stage parasites, a severe defect in the IL-12 response could directly affect protective immune pathways and contribute to anemia development. He found that IL-18 levels were lower or absent in healthy children but elevated following symptomatic P. falciparum infection as reported in a previous study [21]. Our results goes hand in hand with that obtained with Malaguarnera et al., 2002,[22], who studied, the levels of IL-12 in 73 African children, , with severe and mild P. falciparum malaria . The levels of IL-12 were found to be significantly elevated (21·6 ± 18·3 pg/ml) in patients who suffered less severely from the disease. In contrast, the levels of IL-12 were found to be lower (13·1 ± 7·11 pg/ml) in patients who suffered more severely from of the disease. He also established a critical role for IL-12 in the adaptive immune response to malaria, inducing development, proliferation and activity of Th1 cells . The outcome of the disease, such as susceptibility to severe anaemia and other aspects of malarial pathophysiology, could depend on the response of host macrophages to parasite products and, consequently, impaired IL-12 production [22]. Luty et al .[19] and Perkins et a.[17] demonstrated that the low levels of IL- 12 play a role in exacerbation of anaemia and other clinical complications, that provided evidence that high levels of IL-12 play an important role in the defense against P. falciparum infection and in protection against systemic damage induced by the presence of the parasite. Since IL-12 has an important role as the initiator of cell-mediated immunity, it could be used in therapy as a potent stimulator of the cell-mediated immune response against P. falciparum. Similar results were obtained by Musumeci et al., 2003 [11]. Different results were obtained by Nagamine et al.,2003 [23] , who studied serum levels of IL-18, IFN- γ and IgE in 96 patients with P.falciparum malaria in Bangkok , Thailand and stated that Il-18 were elevated in all groups of patients with P. falciparum malaria compared to normal levels . In addition , IL-18 levels were significantly higher in the severe group than in the uncomplicated group for samples obtained on admission.. Similar results were obtained by Kojima et al., 2004 [24], also he was found a significant correlation between IL-18 levels and the extent of parasitemia in the severe group . 7 It is well known that the peripheral parasitemia does not necessary reflect the total parasite burden in the host infected with P. falciparum and that the total burden of parasites may be much larger than the population that the circulating , as sequestration of peripheral RBC take place in various organs and tissues [24]. Therefore , the elevation of IL-18 levels and its correlation with the extent of parasitemia may be a simple reflection of immune response to protect from further severity . Patients with CM showed decreased levels of IL-18 production especialy in the late stage .The reason for such decrease in IL-18 production was not clear it was explained by the suppressor effect of nitric oxide on the action of caspase -1 that regulates IL-18 production by cleaving proIL-18 to induce mature IL-18, thereby inducing reduction of IL-18 levels . The impaired IL-18 response was suggested in African children with severe malarial anemia in areas of holoendemicity of malaria [24]. Conclusion We can conclude that IL-12 and IL-18 are upregulated during acute P.falciparum malaria infection , playing a rule in pathogenesis and defence against it . These cytokines may be used to detect the progression of the disease providing a useful prognostic parameters , also can be used as an immunotherapy to P.falciparum malaria infection especially in the early phase of the disease. Further studies are needed to find out the relation between these cytokines and individual forms and complications of P. falciparum malaria infection . References 1.Stevenson M., Su Z., Sam H., Mohan K., 2001 .Modulation of host responces to blood- stage malaria by interleukin-12 : from therapy to adjuvant activity: Microbes and Infection.3,49-59 2.Good M.,Kaslow D., Miller L., 1998. Pathwayes and strategies for developing a malaria blood-stage vaccine , Annu. Rev. Immunol. 16.57-87 3.Kwiatkowski D., 2000.Genetic susceptibility to malaria getting complex ,2000. Curr. Opin. Genet. Dev.320-324 4. Walther M., Woodruff J., Edele F., Jeffries D., Tongren J., King E., Andrews L., Bejon P., Sarah C. , De Souza B., Sinden R., Adrian V. S., and Eleanor M., 2006. Innate Immune Responses to Human Malaria: Heterogeneous Cytokine Responses to Blood-Stage Plasmodium falciparum Correlate with Parasitological and Clinical Outcomes : The Journal of Immunology, 177: 5736–5745. 5. 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Xu X., Sumita K., Feng C., Xiong X., Shen H., Maruyama S., Kanoh M., and Asano Y. 2001. Down-regulation of IL-12 p40 gene in Plasmodium bergheiinfected mice. J. Immunol. 167:235–241. 21. Torre D., M. Giola, F. Speranza, A. Matteelli, C. Basilico, and G. Biondi. 2001. Serum levels of interleukin-18 in patients with uncomplicated Plasmodium falciparum malaria. Eur. Cytokine Netw. 12:361–364. 22. Malaguarnera L, Imbesi R.M., Pignatelli S. , Malaguarnera J.and Musume C. , 2002. Increased levels of interleukin-12 in Plasmodium falciparum malaria: correlation with the severity of diseaseParasite Immunology,2002,24, 387–389 23.Nagamine Y.,Hayano M.,Kashiwamura S.,Okamura H.,Nakanishi K.,Krudsod S.,Wilairatana P.,Looareesuwan S.,Kojima S.,2003. Involvement of interleukin -18 in severe plasmodium falciparum malaria,transactions of the royal society of tropical medicine and hygien ,97,236-241 24.Kojima S.,Nagamine Y.,Hayano M.,Looareesuuwan S.,Nakanishi K.,2004.A potential role of interleukin 18 in severe Falciparum malaria . Acta Tropica 89 , 279-284 في بالزما الدم في مرضي المالريا18- واالنترلوكين12-تعيين مستوي االنترلوكين الخبيثة في جنوب دارفور **محمد إبراهيم عبد الفتاح *محمود محمد بدر* ياسر أحمد شاهين *قسم الميكروبيولوجيا والمناعة الطبية ** قسم أمراض الكبد والجهاز الهضمي –كلية طب بنها ) في مرضي18- و انترلوكين12- يهدف هذا البحث الي تعيين مستوي السيتوكينات ( انترلوكين .المالريا الخبيثة وربط مستواها بشدة المرض :وقد شملت هذه الدراسة مجموعتين من األفراد ) مريضا يعانون من المالريا الخبيثة80 ( المجموعة األولي: وقد تم تقسيمهم الي مجموعتين فرعيتين مريضا مصابون بمالريا خبيثة مضاعفة30 المجموعة األولي ( أ ) وتشمل . مريضا مصابون بمالريا خبيثة غير مضاعفة50 المجموعة األولي ( ب ) وتشمل . شخصا طبيعيا ال يعانون من المالريا وذلك كعينة ضابطه30 المجموعة الثانية وتشمل- 10 ولقد وجد أن مستوي السيتوكينات المعنية في البحث يزداد في مرضي المالريا الخبيثة أكثر من األشخاص الغير مصابين بالمالريا ويكون مستوي ارتفاعهما أكثر في المرضي الذين يعانون من المالريا الغير مضاعفة عن المرضي المصابون بالمالريا المضاعفة . ونستنتج من ذلك احتمالية وجود دور لهذه السيتوكينات في تحفيز المناعة للوقاية من مرض المالريا الخبيثة كما تؤثر في شدة المرض وحدوث المضاعفات ,مما يمكننا من االعتماد عليهما لتوقع شدة المرض وحدوث المضاعفات ويفتح المجال لالستعانة بهما كعالج لتقليل حدة المرض ومضاعفاته وخاصة في المراحل األولي لحدوثه . وننصح بإجراء المزيد من االبحاث حول عالقتهما بمراحل المرض و مضاعفاته كل علي حده. 11