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Leishmaniasis is an anthropozoonotic, vectorially transmitted disease, which is caused by different Leishmania species. It is estimated that 350 million people worldwide are at risk of acquiring the disease, which has an annual incidence of 2 million cases. Under the influence of characteristics of the vector, vertebrate host and parasite, leishmaniasis can appear in the cutaneous (localized, disseminated and diffuse), mucocutaneous and visceral forms. In all clinical manifestations, the immune response plays an important role, contributing to the clinical cure or disease progression. Components of innate and acquired immunity act dynamically attempting to control the infection, so the host can achieve clinical cure. Considering these aspects, this chapter describes the functions of some important elements in innate and acquired responses against Leishmania (i. e. chemokines, co stimulatory molecules, receptors, cytokines and cells) in the different clinical forms of leishmaniasis.
Keywords: leishmaniasis, innate immune response, cellular immune response
Leishmaniasis is an anthropozoonotic, vectorially transmitted disease, which is caused by different Leishmania species. It is estimated that 350 million people worldwide are at risk of acquiring the disease, which has an annual incidence of 2 million cases. Depending on some features of the parasite, vector and the vertebrate host, including immunological state, the development of the disease can happen under a spectrum of clinical forms (ROGERS et al., 2002). Localized cutaneous leishmaniasis is the most frequent outcome, being characterized by the presence of one or more ulcerated lesions which tend to self-healing. In rare cases, the lesions can be numerous due to multiple sand-fly bites or parasite dissemination by blood (BRASIL, 2007). In diffuse leishmaniasis, there are several popular or nodular lesions throughout the body surface that can persist indefinitely. The mucocutaneous form is the most aggressive, presenting infiltrative lesions, with ulceration and tissue destruction in the nasal cavity, pharynx and larynx (GONTIJO; CARVALHO, 2003).
The apperarance of different clinical manifestations is influenced by the host immune response. Thus, the presence of immune effector cells such as macrophages, natural killers cels, CD4+ and CD8+ T cells, cytokines, effector molecules and specific antibodies are critical components to the control of leishmaniasis (Birnbaum et al 2011, Liese et al, 2008; Oghumu et al, 2010). Considering these aspects, this chapter describes the functions of some important elements in innate and acquired responses against Leishmania in the different clinical forms of leishmaniasis.
2 INNATE IMMUNE RESPONSE
Innate responses develop after the initial sensing of invading microbes, leading to the production of effector molecules that contribute to contain initial infection and to mount the subsequent adaptive immune response (Liese et al, 2008; Faria et al, 2012).There is growing evidence that the innate immune response mechanisms are also important to the antiparasitic response and infection control (Faria et al, 2012, BirnBaum et al 2011). We will discuss the aspects of the innate immune response with more details below.
2.1 Contributing cells
Leishmania life cycle inside the host is dependent upon internalization by phagocytic cells either resident or recruited to the wound site (Faria et al, 2012). Leishmania spp. has been considered obligate intracellular pathogens of macrophages, but they also have adapted to live within different host cells than those previously described (Kaye et al, 2011, Ritter et al 2009).
Neutrophils rapidly infiltrate skin after Leishmania spp. infection, in cutaneous and visceral leishmaniasis, and are present in early lesions being the most immediate responders (Peters, 2009, Kaye et al 2011, Birnbaum et al 2011, Ritter, 2009). Both host protective and disease promoting roles for neutrophils have been reported (Peters, 2009; Nylen e Gautam, 2010). The protective role of neutrophils is associated with rapid recruitment to sites of tissue damage and pathogen entry, and the subsequent clearance of these recruited neutrophils by macrophage/monocyte populations (peters, 2009, Nyleen e Gautam, 2010). Neutrophil active kills promastigotes via reactive oxygen and reactive nitrogen species as wells as neutrophils extracellular traps (birbaum et al, 2011, Ritter, 2009; Nylen e Gautam, 2010). However, neutrophils are short-lived and undergo apoptosis, and when their corpses are phagocytosed by macrophages it allows silent entry of the parasites into macrophages, through direct ingestion of the parasite or through ingestion of parasites that hide outside the dead neutrophils (John, 2008; birbaum et al, 2011, Ritter, 2009). These apoptotic neutrophils at infection site may also suppress macrophages functions with the release of anti-inflammatory cytokines such as TGF-b and can cause immune mediated tissue pathology (John, 2008; Birbaum et al, 2011; kaye, 2011, Ritter 2009 , Nylen et gautam, 2010).
While the passage through neutrophils is believed to be transient, serving as a temporary safe hideaway, the parasites are able to establish productive infections in macrophages, where they differentiate to amastigotes and replicate inside the parasitophorous vacuole (Faria et al 2012, Nylen et gautam 2010). However, in human VL neutrophils have been reported to harbor parasites during active disease [9, 10] (Nylen et gautam 2010).
Leishmania amastigotes, the intracellular form of the parasite, are able to multiply within macrophages, dendritic cells (DC) and neutrophils (mureer, 2009). However, it is within mononuclear phagocytes that there is the best evidence for replication and long-term survival of Leishmania spp. (Kaye et al 2011). Resolution of infection with Leishmania is associated with presentation of Leishmania antigens by macrophages and dendritic cells (DCs) and priming of CD4+ and CD8+ T lymphocytes. Ultimately, induction of nitric oxide synthase (iNOS) and interferon-g (IFN-g) leads to macrophage nitric oxide (NO) production, reactive oxygen species (ROS), and parasite killing (Birnbaum et al 2011). The central irony of leishmaniasis is that the macrophage is both the principal immune effector cell charged with killing Leishmania amastigotes and also the principal site of parasite proliferation and dissemination (Birnbaum et al 2011).
A complex network of immune cells within the skin-dendritic cells, macrophages and Langerhans cells-have a prominent role in cutaneous leishmaniasis (Maurer, 2009; John, 2008). DCs not only play a key role in the development of a protective immune response to Leishmania, but also act as a host cell for the parasites (Carvalho, 2008). Resident dermal macrophages are also rapidly infected, and they become the dominant infected population after 24 hours allowing differentiation, growth of leishmania spp (Kaye et al 2011, Nylen e Gautam 2010). These antigen presenting cells engage pathogens and then likely travel along lymphatics to the nearest lymph node, where T cell responses are developed to control infection. In general, accumulation of protein antigen bearing DCs in lymph nodes was found to peak »24 h post inoculation (John, 2008, maurer, 2009).
Together with phagocytes, NK cells represent the first line of defense against pathogens by two principal mechanisms, cytolytic destruction of infected cells and secretion of pro-inflammatory cytokines. In patients, NK cell number and activity has mainly been associated with protection against or healing of disease. Patients with active leishmaniasis (cutaneous and visceral) have been reported to have a reduction in the frequency of peripheral NK cells (e.g. IFNÎ³, TNFÎ±) (Nyleen e gautam, 2010). The activation of NK cells in visceral and most likely also in cutaneous leishmaniasis results from the intimate interaction of NK cells with mDCs, which are triggered by Leishmania parasites for the production of IL-12 in a TLR9-dependent fashion (Liese,2008).
2.2 Effector molecules and Chemokines
Chemokines and chemokine receptors have been shown to play a crucial role in determining the outcome of leishmaniasis. Chemokines are chemotactic cytokines that coordinate recruitment of leukocytes involved in homeostasis as well as in innate and adaptive immune responses (Oghumu_2010). Infection with Leishmania induces the expression of a number of chemokine genes in the host. This could potentially be beneficial to the parasite through recruitment of host cells it can infect, survive in and proliferate. In addition to mediating cellular recruitment, chemokines can activate various cell populations, participate in cell mediated immunity and possess anti-leishmanial properties (Oghumu_2010).
Chemokines produced at the site of an infection are critical in determining the composition of infiltrating cells and defining the eventual outcome of the disease (Teixeira et al., 2006). Patients with visceral leishmaniasis show elevated concentrations of CXCL9 and CXCL10 in their serum during active infection and it has been suggested that these chemokines along with IFN-Î³ play an important immunopathogenic role in the disease (Oghumu_2010).
Regarding effector molecules, the key antileishmanial components in experimental cutaneous and visceral leishmaniasis are reactive nitrogen intermediates (NO and NO-derived metabolites) and reactive oxygen intermediates (O2- and subsequent metabolites) (Liese, 2012). While the production of NO is required for the leishmanicidal activity against L.major and L. braziliensis in the skin of infected mice, it is dispensable in the spleen and mildly important in the lymph node (Faria, 2012).
The entry of Leishmania parasite into host macrophages results in the onset of respiratory burst, characterized by the increased production of reactive oxygen species (ROS), like superoxide (O2 -) and hydrogen peroxide (H2O2), which is required for the killing of the parasites. These O2 - are generated by activities of a multi component enzyme complex i.e., nicotinamide adenine dinucleotide phosphate (NADPH)/NADH oxidase. Moreover, in later stages of infection, reactive nitrogen intermediates (RNI) viz. nitric oxide groups (NOx) are also produced by the activity of inducible nitric oxide (iNOS), which further contribute to innate immunity and parasitic elimination. However, in leishmanial infections, the microbicidal activities of macrophage are severely hampered, leading to the survival and proliferation of parasites inside the macrophages.
2.3 Toll-like receptors (TLRs)
The TLR family is highly relevant to immunity against Leishmania and other parasites, as they recognize pathogen-associated molecules and participate in innate responses to infections (Faria, 2012; birnbaum, 2011, singh, 2012). TLR activation induces innate responses in multiple ways, leading to the production of effector molecules such as nitric oxide, inflammatory cytokines, chemokines, and other anti-microbial products that can directly destroy the pathogens. A few Leishmania-derived molecules have been reported to activate TLRs, and the majority of the studies to date focused on the activation of TLR2, TLR4, and TLR9 (Maurer, 2009, Faria et al 2012, Singh, 2012)
Evidences indicate that TLR4 contributes most significantly to control the growth of Leishmania spp. in both phases of the immune response. The TLR4 has been found to be a strong regulator of inducible nitric oxide synthase (iNOS, a marker of innate immu-nity) leading to the death of parasites. In addition to TLR4, TLR2 and 9 have been detected in the skin of patients with cutaneous leishmaniasis (Singh, 2012)
Lipophosphoglycans (LPG) on the Leishmania cell surface have been implicated as agonists of TLR 2, 3, 6 and have also been associated with NK cell activation in L major infection (Birnbaum et al, 2011). Purified L. major lipophosphoglycan (LPG) induced the upregulation and stimulation of TLR2 on human NK cells, with additional enhancement of TNF-Î± and IFN-Î³ . LPGs of L. major, L. mexicana, L.aethiopica, and L. tropica were defined as TLR2 ligands in studies using murine macrophages, although the stimulation with L. tropica LPG was only marginal . More recently, it was shown that LPG stimulates cytokine production by human peripheral blood mononuclear cells via TLR2 as well . Those findings assign a protective role for TLR2 which seems required to mount an effective Th1 response (Faria, 2012).
2.4 Complement System
The complement system is a complex set of serum proteins forming a controlled sequence for the production of activated molecules. The role of the activated molecules is to increase inflammatory reactions mediated by antibodies. In addition, generation of the membrane attack complex C5b-C9 leads to the lysis of "unwanted" cells. The complement receptor system is directed against mediators generated by the host early on after parasite contact. In Leishmania infections, after transmission of metacyclic promastigotes into the dermis, the parasites interact with serum and activate complement in both the classical and the alternative pathways . Opsonization of Leishmania promastigotes with complement is very rapid and interestingly, lysis via the membrane attack complex (C5b-C9 complex) begins 60 s after serum contact . These results in efficient killing of »90% of all inoculated parasites within few minutes (Maurer, 2009).
2.5 Modullation of infection in innate immune response
Leishmania parasites are capable of utilizing different components of the host defense innate mechanisms to avoid their elimination from the host before an infection is established. Some of the parasites surface molecules are capable of activating the complement system, resulting in the binding of C3bi and C3b to the surface of the parasite. Leishmania parasites smartly use this opsonization to escape from the hostile environment by promoting phagocytosis via complement receptors in cells such as in macrophages, neutrophils and erythrocytes (Ritte, 2009; maurer, 2009; nyleen gautam, 2010). They can also entry macrophages utilizing engagement of non-triggering receptors such the phosphatidyl serine (PS) receptor. Leishmania can also evade effector mechanisms of the immune system by direct inhibit macrophage function by interfering with NFB transcription and IL-12 production, affecting macrophage phagosomal maturation and killing functions; they can down regulate MHC class II; promoting production of regulatory cytokines like IL-10 and TGFÎ² and Inhibit dendritic cell maturation and chemotaxis (nyleen gautam, 2010; Birnbaum, 2012, carvalho, 2008).
3 ACQUIRED IMMUNE RESPONSE
In human and experimental leishmaniasis, immunity is predominantly mediated by T lymphocytes. T cells play a major role in generating specific and memory T-cells responses to intracellular parasitic infection and these have been extensively characterized in leishmania infection . In addition, T lymphocytes play critical role in shaping the host immune response by secreting cytokines, which may act both synergistically and antagonistically through complex signaling pathways to direct both protective and non-protective immunities against intracellular parasites .
Although the immune response induced by infection with Leishmania has been the subject of many investigations, the mechanisms that underlie host resistance and pathogenesis in leishmaniasis are not entirely understood. During the late 80s and early 90s, the discovery of two distinct subpopulations of CD4+ T helper cells based on their cytokine production, Th1 and Th2 , finally explained resistance and susceptibility to L. major in the murine model. 
Early studies using mouse models of experimental cutaneous leishmaniasis (CL) have revealed a clear dichotomy between Th1-associated cytokines mediating protection and Th2-associated cytokines mediating susceptibility.[1,2,6,9]. Failure to mount an efficient anti-leishmania Th1 response was shown to cause progressive disease and absence of lesion resolution [7,9]. In resistant C57BL/6 mice, resolution of the disease is mediated as a consequence of INF-Î³ release by Th1 cells and upregulation of NO in macrophages the harbor parasites [8,9]. Conversely, persistence of lesions in BALB/c mice is due to Th2 -type CD4+ T cell differentiation and production of IL-4, which suppresses macrophage activation, resulting in parasite survival [7,9]. On the other hand, during VL, Th2 response and cytokines such as IL-4 and IL-13 seem to be necessary for immunity and efficient response to antileishmanial chemotherapy.[10,11]
In the murine model of L. major infection, the predominant CD4+ T cell subpopulation resulting from infection greatly influences the outcome of disease [12,13,14]. Interleukin -12 (IL-12) produced by macrophages and dendritics cells and interferon-gamma (INF-Î³) produced by natural killer cells (NK), and previously activated T cells, promote the development of Th1 cells, whereas IL-4 induces the development of Th2 cells. The Th1 subpopulation, important for induction of leishmaniasis resistance, produce INF-Î³ and tumour necrosis factor - alpha (TNF-Î±) which play an important role in cellular immune responses against intracellular pathogens by activating macrophages for intracellular killing of pathogens [15, 14]. On the other hand, Th2 cells produce IL-4, IL-5, IL-10, IL-13 and TGF-Î², and are associated with leishmaniasis susceptibility in L. major infection murine models [14,16,17,18].
Most data point to the fact that same or similar Th1 dependent mechanisms are involved in control of human disease. Self-healing forms of leishmaniasis and cure of VL is typically accompanied by parasites specific proliferation and INF-Î³ production. Human macrophages are activated to kill intracellular parasites by INF-Î³ and exougenous INF-Î³ can promote cure of human CL[19,23]. Though Th2 responses can act in favor of the parasite, polarized Th2 response has never been able to explain non-curative or visceralizing human disease. Th2 independent disease progression is also supported by studies on non-healing disease in Th1 phenotypic B6 mice[20,23]. In this context it can also be noted that in patients with VL the effect of INF-Î³ administration was limited[21,23] and in human LC, INF-Î³ production by CD4+ cells, alone, in response to leishmania antigens is not predictive of protection or disease development[22,23]. This indicates that other mechanisms acting in synergy with INF-Î³ or counteracting the effects of INF-Î³ as important. Thus, the Th1/Th2 dichotomy as an indicator of resistance and susceptibility might be a generalization and is far more complex than what we currently know and understand.[9,23]
Of particular interest in this context is the differentiation of NaÃ¯ve CD4+ Th cells into various effector lineages orchestrating different immune responses. NaÃ¯ve CD4+ Th cells can differentiate into IFN-Î³ producing Th1 cells; into Th2 cell secreting IL-4, IL-5, IL-13, and IL-10; or into the recently described Th17 cells. In addition, NaÃ¯ve CD4+ Th cells can differentiate into IL-10-secreting regulatory T cells like regulatory type 1 T cells, IL-10, and TGF-Î² producing Th3 cells or into Foxp3-expressing regulatory T cells . Some cytokines are described in the following section.
3.1 Th1 and Th2 Cytokines
The development of cell-mediated immune responses capable of controlling Leishmania infection and resolving disease usually requires CD4+ T cell-, IFN-g- and/or tumor necrosis factor (TNF)-dependent activation of macrophages. This leads to a (post)transcriptional upregulation of antimicrobial effector mechanisms, including the acidification of the phagolysosomes and the expression of inducible nitric oxide synthase. (Cumming, 2010 Bogdan et al 2008),
TNF-alpha is a key cytokine mediating T cell-mediated inflammation. It is involved in leukocyte recruitment by increasing expression of adhesion molecules on vascular endothelium and increasing angiogenesis. Although TNF-alpha promotes increased macrophage activation, and contributes to control of Leishmania parasites, deleterious consequences of excessive TNF-alpha production have been reported. The high levels of TNF-alpha and IFN-gamma secreted by mononuclear cells from these patients is positively correlated with lesion size and the use of drugs that down modulate production of TNF-alpha in combination with antimony increases the rate of healing and allows the cure of refractory cases of mucosal and cutaneous disease (Carvalho, 2008)
The major biological function of IFN-Î³ is to activate macrophages, inducing iNOS expression and NO production (Cumming, 2010; Liese, 2012), therefore enhancing the microbicidal activity of these cells to eliminate parasites and resolve Leishmania infection. The biological effects of IFN-Î³ are largely dependent upon the activation of STAT1 transcription factors. STAT1/IFN-Î³ signaling pathway induces the expression of Th1-associated transcription factor, T-bet. Both STAT1 and T-bet are required to mount an efficient Th1 response and as such, are indispensable for host defense against Leishmania infection in mice (Cummings_2010).
Known as a proinflammatory cytokine, IL-12 is a heterodimer composed of two subunits, p35 and p40 and is produced primarily by macrophages and dendritic cells (DCs) in response to microbial pathogens. IL-12 functions as the main physiological inducer of gamma interferon (IFN- Î³) by activated T cells and promotes Th1-type CD4+ T cell differentiation, and therefore is a key cytokine for the generation of protective immunity in response to Leishmania infection. The specific cellular effects of IL-12 are due to the activation of Janus kinase (JAK)-STAT pathways, primarily to the activation of the specific transcription factor, STAT4. In activated T cells and NK cells, STAT4 functions to induce IFN-Î³ production in response to IL-12 signaling (Cummings,2010, Liese, 2012).
The anti-immune and anti-inflammatory cytokine, IL-10 is produced by a variety of cells including T cells, monocytes, macrophages, DCs, and B cells. While many cells can produce IL-10, the main biological functions of IL-10 appear to be on macrophages and DCs. IL-10 functions to inhibit the production of proinflammatory cytokines IL-1, IL-6, IL-12, and tumor necrosis factor (TNF) by macrophages and DCs  and thus prevents the expansion of Th1-type cells required for protective immunity during Leishmania infection [14-16]. IL-10 also promotes activation, survival, and antibody production by B cells and the development of humoral immune responses which play a detrimental role in host defense against Leishmania infection by facilitating parasite entry into host cells . Studies demonstrated that IL-10 is a master cytokine in cutaneous and visceral leishmaniasis that is critical for the initial survival and long-term persistence of Leishmania parasites in both human and experimental models. Since IL-10 can act as an inhibitor of IFN-Î³ induced NO synthesis, it is likely that antagonistic effect of IL-10 is mediated by its ability to suppress production of NO, which is critical for elimination of parasites (Cummings_2010, Bogdan, 2008).
IL-4 is an important cytokine that has been shown to deactivate macrophages and to regulate the induction of Type-2 (Barata-Masini, 2007; Kamali-SARVESTANI, 2006). Furthermore, IL-4 inhibits the responsiveness of CD4+ T cells to IL-12, due to its down regulatory effects on the expression of the IL-12 receptor b2-subunit and also inhibits the deviation of CD4+ T cells towards Th1 cells by modulation of the regulatory function of the transcription factor T-bet (Barata e Kamali, ANO). Moreover, macrophage activation by IL-4 induces a pathway of arginine metabolism toward arginase with production of polyamines that enhance Leishmania growth (Kamal-SARVESTANI, 2006). Since IL-4 has been shown to suppress macrophages and Th1 cells and enhances Leishmania growth, it is conceivable that the host ability in production of this cytokine may determine the susceptibility to CL. This hypothesis is supported by recent report on the association of IL-4 gene polymorphisms with susceptibility to visceral leishmaniasis (Kamali-SARVESTANI, 2006).
3.2 Th17 responses in Leishmania infections
Similar to the Th1 and Th2 subsets, the Th17 subset is orchestrated by specific cytokines and transcription factors (González-García et al, 2009). The Th17 response has been studied since 1995, when it was found that T helper cells can produce IL-17 under stimulation with specific antigens (Yao et al, 1995). Nowadays, it is known that the production of Th17 specific cytokines is present in allergy and inflammatory diseases (Schmidt-Weber et al, 2007; Oukka, 2007). However, these inflammatory mediators can orchestrate protective responses to several agents, as it is shown in M. tuberculosis and T. cruzi infections (Torrado et al, 2010; Guedes et al, 2010).
The Th17 response is activated by a combination of the cytokines IL-6 and TGF-Î², and the transcription factors RORÎ³t, RORÎ± and Stat3 are essential for Th17 commitment (Kaufmann; Kuchroo, 2009; González-García et al, 2009). IL-6 plays an important role in the differentiation of the Th17 subset, since TGF- Î² can also induce Foxp3, a transcription factor required for the generation of regulatory T (Treg) cells, and the presence of IL-6 suppresses the induction of Foxp3 (Kaufmann; Kuchroo, 2009).
Th17 cells produce cytokines such as IL-17A, IL-17F, IL-22, IL-21 and IL-23, which promote Th17 responses functionality. The cytokine IL-27, on the other hand, is the main negative regulator of the Th17 response, despite its structure's similarity to IL-6 (González-García et al, 2009). Research over the role of these cytokines in many infections is under constant development. The research of the influence of Th17 cells in leishmaniasis is primordial to understand the mechanisms related to protective or damaging immune responses in this disease. In the next section, some features of these cytokines are described.
The IL-17 cytokines include a family of six members (IL-17A-F), of which at least two of them exhibit potent proinflammatory properties: IL-17A (also known as CTLA-8) and IL17-F, which seem to have similar functions. IL-17B and IL-17C are members of the family whose cellular sources are unknown at this point, and whose biology seems unrelated to IL-17A. IL-17D and IL-17E (alternative names: IL-27 and IL-25), in turn, are the two members of the IL-17 family with lowest homology to IL-17A. None of the last is produced by Th17 cells, and they exert a negative control on the Th17 subset development (González-García et al, 2009). In this chapter, we will refer to IL-17A as IL-17.
By signaling through the receptor IL-17RA, IL-17 can induce the production of different kinds of proteins, many of them related to inflammation, including chemokines (CXCL-1, CXCL-2, CXCL-8-10, CCL-2, CCL-20), cytokines (IL-6, TNFÎ±, G-CSF, GM-CSF), proteins of the acute phase response, tissue remodelling factors (MMP1, MMP3, MMP9, MMP13, TIMP2), and anti-microbial products (Î²-defensins, mucins, calgranulins) (González-García et al, 2009). The role of IL-17 in immune responses is being widely studied. It is known that IL-17 is a potent activator of neutrophils. Increased levels of this cytokine are responsible for neutrophil immigration, most likely via CXCL2, whereas IFN-Î³ is responsible for activating macrophages to kill intracellular pathogens (Kostka et al, 2009). IL-17 seems to have a role in the protective immunity against many bacterial and fungi infections, as in the case of Klebsiella pneumonieae, Mycobacterium tuberculosis, Candida albicans and Aspergillus fumigate infections (Happel et al, 2003; Matsuzaki; Umemura, 2007). IL-17 could also be defensive against some parasites, as in the infection with the protozoan Toxoplasma gondii (Kelly et al, 2005). Also, IL-17 production appears to be downstream of IL-1Î±Î² in several pathological conditions. DC derived IL-1 is important for efficient Th1 induction in leishmaniasis. (Kostka et al, 2009).
Whilst in some models IL-17 and IL-23 seem to have a protective role on the outcome of the infection, as in the case of extracellular pathogens (e.g., Klebsiella pneumonia bacteria, Toxoplasma gondii parasites and Cryptococcus neoformans fungi) (Khader; Gopal, 2010), in Schistosoma mansoni infections, increased levels of IL-23 and IL-17 are associated with disease exacerbation (Rutitzky; Lopes da Rosa; Stadecker, 2005).
As to leishmaniasis, Kostka et al (2009) reported that BALB/c mice produced increased levels of IL-17 after infection with L. major and that IL-17-deficient (IL-17-/-) BALB/c mice exhibited dramatically attenuated disease despite typical Th2 development. They also demonstrate that elevated levels of IL-17A in BALB/c mice were associated with increased production of IL-23, but not IL-6 and TGF-Î²1, by infected DC.
In humans, studies have shown that IL-17 is present at the initial phase of the immune response in the cutaneous forms of leishmaniasis (Bacellar et al. 2009; Kostka et al, 2009; Novoa et al, 2011), leading to the conclusion that this cytokine could be injurious for the disease resolution. On the other hand, Novoa et al (2011) observed an increase in IL-17 levels in individuals with subclinical ACL, in comparison to patients with active lesions, concluding that this cytokine presents a protective part in the immune response. Pitta et al (2009) have also shown that L. donovani, a visceral leishmaniasis agent, strongly induces IL-17 and IL-22 production in PBMCs of healthy individuals, suggesting that these cytokines can present a protective role in Leishmania infections.
Although IL-21 does not look like an essential factor for Th17 lineage commitment, it is able to induce IL-17 expression in collaboration with TGF-Î² even in the absence of IL-6. Furthermore, generation of Th17 cells is attenuated by blocking IL-21, and loss of its expression, or its receptor, results in defective Th17 differentiation. Similar to IL-6, IL-21 inhibits Foxp3 expression induced by TGF-Î². IL-21 is produced by Th17 cells under IL-6 induction and autocrinally induces its own synthesis and the expression of IL-23R to allow IL-23 responsiveness (González-García et al, 2009).
Furthermore, IL-21 has been recently proven to induce IL-10 production under stimulation with L. donovani antigens. It is also known to critically regulate Ig production, and could be a contributing factor to the high titers of anti-leishmanial Abs in VL patients (Ansari et al, 2011).
IL-22 is also produced by Th17 cells, and to a lesser extent by Th1 and NK cells, and is involved in immunity at the epithelium and mucosal surfaces (Korn; Bettelli; Oukka; Kuchroo, 2009; Pitta et al, 2009). The functional IL-22 receptor is expressed on hepatocytes, keratinocytes, and fibroblasts. IL-22 increases the production of proinflammatory molecules, such as the S-100A proteins and CXCL5. IL-17 and IL-22 synergistically increase the production of antimicrobial peptides, such as Î²-defensins, by epithelial cells (Kostka et al, 2009; Pitta et al, 2009).
Both IL-17 and IL-22 have been shown to increase protection against certain bacteria and fungal pathogens in experimental models (Pitta et al, 2009). As to protozoans, Pitta et al (2009) stated that IL-17 and IL-22 are the cytokines most strongly associated with protection in the visceral forms of leishmaniasis. These cytokines may contribute to protective immunity to L. donovani in several ways. Studies using animal models suggest that neutrophils could be involved in controlling the Leishmania infection through the generation of skin and liver granulomas that form around Leishmania at early stages of infection. Furthermore, IL-22 is involved in epithelial repair and liver protection in chronic infections. Both the increases in epithelial protective barrier function and the recruitment of inflammatory cells, including neutrophils, to the skin and liver, could contribute to protection against L. donovani (Pitta et al, 2009).
The function of IL-23 in promoting Th17 cell expansion or survival has been proposed. A recent report suggests that IL-23 maintains the Th17 phenotype without affecting proliferation or survival. On the other hand, IL-23 has been demonstrated to maintain the pathogenic Th17 functions compared with culture under TGF-Î² and IL-6, depending on IL-10 production by Th17 cells (González-García et al, 2009).
Recent studies have also implicate IL-23 and IL-17 in immunity against extracellular pathogens, as bacteria (Klebsiella pneumoniae), Toxoplasma gondii parasites and fungi (Cryptococcus neoformans). In Schistosoma mansoni infections, increased levels of IL-23 and IL-17 are associated with disease exacerbation (Khader; Gopal, 2010; Rutitzky; Lopes da Rosa; Stadecker, 2005). Kosksta et al (2009) suggests that DC-derived IL-23, in addition to IL-1Î² and IL-12p80, can contribute to disease susceptibility in BALB/c mice infected with Leishmania parasites.
IL-27 is one of the main negative regulators of Th17 development. This cytokine is structurally related to IL-6, but has many different actions. Research studies show a damaging role of IL-27 on IL-17 producer cells. These studies conclude that the absence of IL-27 signalling exacerbates chronic inflammation in correlation with increased number of Th17 cells. Moreover, IL-27 is able to promote IL-10 production, another negative player in the network of Th17 activity regulation (González-García et al, 2009).
Novoa (2009) reported a higher expression of mRNA for IL-27 ex vivo or in cultures stimulated with soluble Leishmania antigen in patients with active lesions compared to individuals with subclinical disease. Ansari et al (2011) also associated active visceral leishmaniasis with elevated levels of IL-27 in plasma and IL-27 mRNA in spleen.
IL-27 produced by macrophages, along with IL-21 from T cell sources, are suggested to be disease-promoting cytokines in visceral leishmaniasis by virtue of their roles in promoting the differentiation and expansion of Ag-specific, IL-10-producing T cells. The studies support the notion that IL-27 is a key instructional cytokine involved in regulating the balance between immunity and pathology in human visceral leishmaniasis (Ansari et al, 2011).
3.3 Antibodies and Leishmaniasis
The infection by Leishmania in humans is characterized by the appearance of anti-leishmanial antibodies in the serum of the patients. In respect of the humoral immune response, a successively high titer of specific antibodies can be observed in Localized ATL, Mucocutaneous Leishmaniasis (MCL) and Diffuse ATL. An exceptionally high titer of antibodies against Leishmania antigens can be detected in the most severe form of the disease, Visceral Leishmaniasis (VL), as a consequence of polyclonal activation of B cells as a result of the presence of large numbers of parasites in the bone marrow and spleen1.
To evaluate the humoral immune response on Leishmaniasis, works have shown the role of the immunoglobulins on immunopathological mechanisms which are involved in the resistance and/or pathogenesis of the infection2, 3, 4. In some studies the presence of antibodies against Leishmania braziliensis infection in the sera of infected patients is still unclear but it has been monitored and it is utilized for diagnosis and prognosis of ATL5, 6. Contrastingly, strong anti-leishmanial antibody titers are as well documented in VL7, 8.
However, it has been shown that the class IgG not only offers protection against this intracellular parasite, but indeed, it contributes to the progression of the infection. Previous analysis of Leishmania antigen-specific immunoglobulin isotypes and IgG subclasses in VL patients' sera has shown that elevated levels of IgG, IgM, IgE and IgG subclasses were lasting1. This is due to differential patterns of immunoglobulin isotypes observed during the disease progression. Drug resistance and cure were specific for antigens of Leishmania donovani. IgG subclass analysis has revealed expression of all the subclasses, with a prevalence of IgG1 during the disease9, nevertheless, some studies have shown the advantage of using specific subclass antibodies for the diagnosis of VL1,10,11.
Although studies have been evaluating the humoral immune response on ATL, the role of specific antibodies on the immunity against Leishmania is still not completely clear. On Cutaneous Leishmaniasis (CL) and Mucocutaneous Leishmaniasis (MCL), the cellular immunity and the prevalence of the isotypes IgG1, IgG2 and IgG3 have been associated with the Th1 response; on the other hand, the Th2 profile has been related to Diffuse Cutaneous Leishmaniasis (DCL), with the presence of IgG4. Studies lead the attention to the correlation of the subclasses of IgG with the clinical manifestations of ATL. Therefore, high levels of the isotypes IgG1, IgG2 and IgG3 and low levels or absence of the IgG4 isotype can be detected in the sera of patients with CL. In patients with MCL, there are high levels of IgG1 while the levels of IgG2, IgG3 and IgG4 are similar to the findings on the sera of patients with CL. The levels of IgG4 in patients with DL are highly elevated, as the level of IgG1 and IgG2 are similar to the patients with CL and MCL. Studies show that all specific isotypes anti-Leishmania, except for IgD, are detected in the sera of patients with ATL. There are high levels of IgE in patients with more time of disease evolution and high levels of IgA in patients with MCL4.
The intensity of the antibody response appears to reflect both the parasite load and the chronicity of the infection and it also can be observed high titers of antibodies in all clinical manifestations of ATL12. Studies with immunological and serological methods which are available to the research of antibodies in ATL, showed controversial results due to its low sensibility and specificity13, 14. However, studies have shown the advantages of using specific antibodies in the diagnosis of VL1, 10, 15.
4 IMMUNE REGULATION IN LEISHMANIASIS
To achieve cure in Leishmaniasis, the infected host must develop an immune response capable of eliminating the parasite, but harmless to itself. This balance is given by regulatory T cells, which exhibit two well-defined subpopulations: naturally occurring CD4+CD25+ Tregs, which originate in the thymus during ontogeny, and inducible Tregs, which develop in the periphery from conventional CD4+ T cells (Belkaid et al., 2006b). The first subpopulation of Tregs was firstly described as a population of CD4+ T cells that prevent the expansion of self-reactive lymphocytes and, therefore, autoimmune disease in mice (Sakaguchi et al., 1995). This population can be defined by their constitutive expression of the IL-2 receptor Î± chain (CD25), the cytotoxic T lymphocyte antigen (CTLA4), the TNF receptor family member GITR (glucocorticoid-induced TNF-receptor-related protein), and the a chain of the Î±ÎµÎ²7 integrin (CD103) (Belkaid, 2003). However, expression of these molecules is not specific to Tregs. In contrast, the forkhead/winged helix transcription factor Foxp3 is thought to program the development and function of Tregs and is specifically expressed in natural Tregs in mice, as well as in CD25- T cells with regulatory activity (Fontenot et al., 2003; Hori et al., 2003; Khattri et al., 2003).
Cells with regulatory functions have been frequently described in Leishmania infections, and the existence of concomitant immunity is discussed (Belkaid et al. 2002, 2006, 2007) This phenomenon consists in the long-term persistence of pathogens in a host that is also able to maintain strong resistance to reinfection. In the murine model of infection with L. major, CD4+CD25+ T cells accumulate in the dermis, where they suppress - by both interleukin-10-dependent and interleukin-10-independent mechanisms - the ability of CD4+CD25- effector T cells to eliminate the parasite from the site. The sterilizing immunity achieved in mice with impaired IL-10 activity is followed by the loss of immunity to reinfection, indicating that the equilibrium established between effector and regulatory T cells in sites of chronic infection might reflect both parasite and host survival strategies (Belkaid et al., 2002).
Regarding the experimental infection with Leishmania (Viannia) braziliensis, a Treg activity has also been related. CD4+CD25+ cells expressing GITR, CD103 and Foxp3 were detected throughout the duration of clinical disease both at the ear and in draining lymph nodes of infected mice. In both sites, they were capable of suppress CD4+CD25- proliferation. Interestingly, in the outcome of a reinfection, parasites were mainly detected in the LN draining the primary infection site where a high frequency of CD4+IFN-Î³+ T cells was also present. Thus, in this model, Tregs are present in healed mice but this population does not compromise an effective immune response upon reinfection with L. braziliensis (Falcão et al., 2012).
Suppression of T cell response is thought to be involved in the pathogenesis of human leishmaniasis. In patients with CL caused by L. braziliensis, a frequency of CD4+CD25+ cells was observed in the skin lesions, along with expression of CTLA-4 and GITR markers and secretion of IL-10 and TGF-Î². Moreover, CD4+CD25+ T cells in peripheral blood (PB) from the same patients exhibited higher levels of CTLA-4 than healthy individuals (Campanelli et al., 2006). Because CTLA-4 is highly expressed on Treg cells [Campanelli 13, 14], and because it is supposed that this molecule plays an important role in their suppressor function , it is possible that the suppressor activity of CD4+CD25+ T cells was increased in the patients with CL.
A similar immune regulation in human visceral leishmaniasis is observed. The presence of CD4+CD25+ in the bone marrow, one of the disease sites, and the production of IL- 10 by these lymphocytes may inhibit T cell activation in IL-10 dependent manner (Rai et al., 2012). In contrast, CD4+CD25+ lymphocytes did not accumulate in and were not a major source of IL-10 in the spleen, and their removal did not rescue antigen-specific interferon-Î³ responses. Thus, in different sites the regulation of immune response may be performed by different T cell subpopulations, once IL-10 is secreted in the spleen by CD25-Foxp3- T cells (Nylén et al., 2007).
It is also interesting to investigate whether there is an influence of mechanisms of immune regulation on the response to chemotherapy. The analysis of the frequency of CD25+ cells in PB from patients with active and cured CL showed a higher presence of cells expressing this marker after treatment. Thus, CD25+ T cell expansion, presented by patients, may be due the role of these cells in the modulation of an exacerbated response by effector T cells, and maintenance of a small number of parasites in the localized lesion as an antigenic stimulus to prevent reinfection (Almeida et al., 2010). Among all the data obtained so far, immune regulation seems to happen as a way to maintain a homeostatic environment to allow the achievement of clinical cure by the host and the parasite persistence. Nevertheless, conclusive role of Treg cells in suppression of immunity in patients and its consequences is yet to be well defined.
5 FINAL CONSIDERATIONS
A The balance between the innate and adaptive immune system and the parasite evasion mechanisms is critical for the decision if disease is observed and if (lifelong) immunity develops (Maurer, 2009).