Cytokines and visceral leishmaniasis: a comparison of plasma cytokine profiles between the clinical forms of visceral leishmaniasis.

It is not well established whether cytokine production differs in response to different clinical forms of visceral leishmaniasis (VL). In this work, we performed a cross-sectional study to investigate the plasma levels of cytokines [interferon (IFN)-γ, tumour necrosis factor (TNF)-α, interleukin (IL)-2, IL-4, IL-10 and IL-12] involved in the pathogenesis of VL in 80 subjects from VL endemic areas, including subjects with active VL, subjects with asymptomatic infection, subjects with cured VL and uninfected controls. The patients were recruited by sampling from a referral hospital and by random selection from a population-based cohort study. The results showed significant differences in the plasma concentration of all cytokines between the groups (p < 0.05). Patients with the active disease had higher plasma levels of IL-10, IL-4, INF-γ and TNF-α relative to the other groups and they produced more IL-12 than asymptomatic and cured subjects. Only the IL-2 concentration was higher in the asymptomatic and cured subjects relative to the patients with active disease (p < 0.05). Our results suggest that these cytokines can be used as markers in epidemiological studies conducted in endemic areas to distinguish between different clinical forms of VL. However, their usefulness should be confirmed in investigations conducted in other endemic areas.

online | memorias.ioc.fiocruz.br Mem Inst Oswaldo Cruz, Rio de Janeiro,Vol. 107(6): 735-739, September 2012 Visceral leishmaniasis (VL), or kala-azar, is a serious public health problem with worldwide distribution and is among the seven priority endemic diseases in the world. In Latin America, this disease has been described in at least 12 countries. Approximately 90% of the cases occur in Brazil and the disease is endemic in the state of Maranhão (MA) (Michalick & Genaro 2005, Maia-Elkhoury et al. 2008, MS/SVS 2009, WHO 2009.
Infection with Leishmania chagasi, the etiological agent of VL, is characterised by a broad spectrum of clinical manifestations that range from asymptomatic infection to discrete (oligosymptomatic), moderate and severe manifestations, which if not treated can result in death. These clinical forms reflect the balance between the multiplication of the parasite in mononuclear phagocytes, the host immune response and the degenerative conditions resulting from the infection (Medeiros et al. 1998, Peruhype-Magalhães et al. 2005, MS/SVS 2009. The factors that determine the development of VL have not been completely identified, but the specific cellular immune response to Leishmania seems to play a key role in the control of infection (Holaday 2000, Bacelar & Carvalho 2005, Michalick & Genaro 2005. In experimental mouse models using Leishmania major, protection against the disease is attributed to a T helper (Th)1 immune response that is characterised by the production susceptibility to the disease is attributed to a Th2 type response with the production of IL-4 and IL-10 (Park et al. 2000, Belkaid et al. 2002, Stetson et al. 2002, Goto & Prianti 2009). However, this dichotomy is not evident in mice infected with Leishmania donovani (Melby et al. 2001, Peruhype-Magalhães et al. 2005. In humans, VL is considered to be the prototype of immune dysfunction, compromising both cellular and humoral immunity. The immunological mechanism involved in the infection and the relationship between the immune system and the clinical forms of the disease have been studied by evaluating cellular profiles and cytokine production in bone marrow, spleen and lymph node aspirates, as well as in peripheral blood mononuclear cells and in plasma samples from patients with different clinical forms of VL. Analysis of plasma samples is a suitable alternative to the other methods because it is simpler and less invasive (Hailu et al. 2004, Peruhype-Magalhães et al. 2005, Khoshdel et al. 2009 are higher in patients with the active disease relative to asymptomatic individuals and are also higher in asymptomatic individuals compared to uninfected individuals (Ansari et al. 2006, Khoshdel et al. 2009). However, the basal levels of these cytokines have been detected in asymptomatic cases from Brazil, a finding that requires Financial support: FAPEMA further investigation (Peruhype-Magalhães et al. 2006). A significant reduction in these cytokines, particularly reliable marker of the cured disease, has been observed in patients after treatment of VL (Caldas et al. 2005, Ansari et al. 2006. Knowledge of the mechanisms involved in the regulation of cytokine production during the various clinical forms of VL infection and after treatment may contribute to a better understanding of the immunological phenomena that occur in the disease. Therefore, the objective of the present study was to compare the plasma cytokine IL-4, IL-10 and IL-12] between patients with active VL, asymptomatic individuals, individuals with a history of the disease and uninfected (healthy) controls from endemic areas in the state of MA.

PATIENTS, MATERIALS AND METHODS
A cross-sectional study of subjects from VL endemic areas in MA was conducted between June 2008-2009. The sample consisted of 80 subjects divided into four groups (n = 20): group 1, subjects with active VL (patients), group 2, subjects with asymptomatic infection, group 3, subjects with a history of VL (cured), and group 4, subjects not infected with L. chagasi (control).
Patients in group 1 were recruited from a referral hospital and the other three groups (asymptomatic infection, history of VL and control) were randomly selected from a cohort study conducted on the island of São Luís, MA, in 2008. That population-based study involved 365 subjects (relatives and neighbours of 50 cases).
The following clinical and laboratory criteria were used to define the four groups: (i) active VL, subjects presenting clinical and laboratory signs of the disease (MS/SVS 2006) and a positive bone marrow aspirate who were at the beginning of treatment, (ii) asymptomatic, subjects showing no clinical manifestations of the disease, but had a positive Montenegro skin test and anti-Leishmania antibodies [enzyme-linked immunosorbent assay (ELISA)], (iii) post-treatment of VL/cured, subjects with confirmation of the disease by a positive bone marrow aspirate (myelogram) who were considered to be clinically cured after treatment (12 months after the end of treatment), and (iv) uninfected (healthy) controls, subjects without clinical manifestations of the disease who had a negative Montenegro skin test and no anti-Leishmania antibodies (ELISA). Cytokines were measured by a sandwich ELISA using commercial kits according to the manufacturer's instructions (EBioscience). Absorbance was read at 450 nm using a spectrophotometer and the results are expressed in pg/ mL based on a standard curve. The sensitivity of the test pg/mL for IL-4 and IL-10.
The statistical analysis was performed using Graph-Pad Prism software, version 5.0 (GraphPad Software, Inc, 2007). Because the cytokine levels did not show a normal distribution, nonparametric tests were used. The Kruskal-Wallis test was first applied to compare three or more independent samples and the Dunn's post-test was used to compare the mean ranks. Differences were con- The study was approved by the Human Research Ethical Committee of the University Hospital, Federal University of Maranhão (protocol 33104-1295/2006).
A significant difference (p < 0.001) in cytokine production was observed between the four groups. A com-tive disease relative to those in the other groups (Figure). However, the plasma IL-2 levels were higher in the asymptomatic and cured subjects than in the subjects with the active disease (p < 0.05). The plasma IL-12 levels were higher in the group with the active disease relative to the asymptomatic and cured subjects (p < 0.05), but no significant difference in IL-12 levels was observed when the active disease group was compared to the uninfected controls (Figure).  (60) 18 (90) 0 (0) 1 (5) 1 (5) 32 (32) 9 (11.3) 6 (7.5) 33 (41.2)

DISCUSSION
Although the majority of the study population were females, most of the subjects in the active VL group were males. Previous studies have indicated that hormonal changes can influence susceptibility to VL, as the disease has the highest prevalence in adolescents after puberty and adult men (Jerônimo et al. 2004). It has been experimentally shown that testosterone increases replication of the parasite (Liu et al. 2006).
In our study, the majority of the subjects were older than 15 years. The second largest demographic population consisted of subjects who were younger than five years old; this young demographic was highly represented in the group with a history of the disease. In contrast to the present findings, national statistical data reveal a predominance of VL in children up to 10 years old (58%).
The plasma concentrations of most of the analysed er in the subjects with active VL relative to the other groups, demonstrating an exacerbation of the immune response in these patients; this elevation in cytokine levels is in agreement with results from other studies (Hailu et al. 2004, Peruhype-Magalhães et al. 2005, Kurkjian et al. 2006, Khoshdel et al. 2009). Here, a mixed cytokine profile (Th1 and Th2) was detected in the patients with the active disease. This mixed profile has also been reported by other investigators (Goto & Lindoso 2004, Peruhype-Magalhães et al. 2005, Goto & Prianti 2009). The increase in inflammatory cytokines could be associated with the physiopathological alterations seen during the active disease, whereas the production of IL-10 has been suggested to be important for the survival and persistence of the parasite inside macrophages (Peruhype-Magalhães et al. 2006. detected in the patients with the active disease, which is in agreement with results from other studies (Hailu et is likely produced in lymphoid organs where Leishmania proliferates (Goto & Prianti 2009). However, few studies support this hypothesis because it is difficult to obtain tissue samples from patients with the active disease. Several investigators, however, have detected the expression of 1993), lymph nodes (Ghalib et al. 1993) and spleen (Nylen -ceral leishmaniasis (VL), cured subjects (CUR), asymptomatic subjects (AS) and uninfected controls (CON). Results are expressed as pg/mL. The horizontal line indicates the median. p value was obtained by the Kruskal-Wallis test. The letters (a, b, c, d) indicate significant differences (p < 0.05) for VL, CUR, AS and CON, respectively, obtained by Dunn's post-test. IL-10 in the group with active disease indicates a lack of counter-regulation by these cytokines and that IL-10 is the main cytokine involved in the establishment and progression of infection (Caldas et al. 2005, Peruhype-Magalhães et al. 2005, Ansari et al. 2006). In addition, IL-10 protects against the side effects of an exaggerated inflammatory response, playing an important role in the regulation of the inflammatory response (Trinchieri 2007).
The concentration of IL-4, a cytokine that generally plays an immunosuppressive role (Miralles et al. 1994), was higher in patients with the active disease. However, its role in the induction of Th2 responses is has not been resolved. In vitro, when IL-4 is added after cell stimulaet al. 2001), whereas the addition of IL-4 to cells before al. 2000, Biedermann et al. 2001, Peruhype-Magalhães et al. 2005. A synergism between IL-4 and IL-10 has been reported in the literature. High concentrations of these cytokines have been shown to promote the deactivation of macrophage leishmanicidal activity and, consequently, favour the multiplication of the parasite and the development of the disease and are accompanied by the expression of Th2 (Peruhype-Magalhães et al. 2005). In the present study, high levels of IL-4 and IL-10 were observed in patients with the active disease.
Another cytokine found at high concentrations in pa-100% of these subjects. This cytokine plays an important role in the progression of infectious diseases because it induces the excessive production of nitric oxide. In addi-ease, such as fever, anorexia, weight loss, increased energy expenditure and cutaneous and mucosal pallor, and mediates the polyclonal activation of B cells (Engwerda et al. 2004). In contrast, Ansari et al. (2006) found minimal levels of this cytokine during the active disease in Indian patients with VL. In experimental models using L. donovani during the active disease. In addition, this cytokine has been reported to be a reliable marker of VL that has been cured ( Barral-Netto et al. 1991), which explains the detec-Here, the concentration of IL-12 was higher in patients with the active disease relative to asymptomatic and cured subjects, but not when compared to healthy controls. IL-12, which is produced by cells of the mononuclear phago-duction because it interacts with natural killer cells and T cells. IL-12 also plays a key role in the initiation and maintenance of Th1 responses and the subsequent production sera of patients with VL from Iran (Khoshdel et al. 2009), southeast Ethiopia (Hailu et al. 2005) and Brazil (Caldas et al. 2005) have shown significantly higher levels of IL-12 in patients with the active disease relative to those in asymptomatic and healthy subjects.
In the present study, lower IL-2 levels were observed in patients with active VL relative to asymptomatic and cured role in the immune response and contributes to parasite death. Its absence may indicate a defect in the immune response (Murray et al. 1993, Bacellar & Carvalho 2005. Basal levels of IL-2 were detected in patients with active VL from India (Ansari et al. 2006) and Brazil (Peruhype-Magalhães et al. 2005), suggesting the absence of effective lymphocyte activation during the active disease.
A comparison of cured and asymptomatic subjects showed no significant difference in the serum levels of the cytokines assayed, which is in agreement with studies conducted in Iran (Khoshdel et al. 2009) and Brazil (Peruhype-Magalhães et al. 2005). These findings indicate that, after the infection is cured, the immune response is effective in controlling the disease, followed by the establishment of protective mechanisms similar to those that occur in asymptomatic individuals (Peruhype-Magalhães et al. 2005).
In conclusion, the production of Th1 and Th2 cytokines is increased in patients with VL during the active disease. Cured subjects have a cytokine profile similar to that seen in subjects with asymptomatic infection, that cytokine levels should be evaluated in combination with the clinical and epidemiological characteristics of VL. Taken together, our results suggest that these cytokines can be used as markers in epidemiological studies conducted in endemic areas to distinguish between the different clinical forms of VL. However, their usefulness should be confirmed in investigations conducted in other endemic areas.