Correspondence: Vinod Singh, Department of Microbiology, Barkatullah University, Bhopal, Madhya Pradesh, India. Tel.: +755 242 9331; fax: +755 267 7729; e-mail: email@example.com
The leishmaniases are a group of diseases caused by protozoan parasites of the genus Leishmania. Various Leishmania species can cause human infection, producing a spectrum of clinical manifestations. It is estimated that 350 million people are at risk, with a global yearly incidence of 1–1.5 million for cutaneous and 500 000 for visceral leishmaniasis (VL). VL is a major cause of morbidity and mortality in East Africa and the Indian subcontinent. Coinfection with HIV enhances the risk of the disease. The only control measure currently available in India is case detection and treatment with antimonial drugs, which are expensive, not always available and cannot be self-administered. Newer drugs like oral miltefosine have not become widely available. Vector and reservoir control is difficult due to the elusive nature of the vector and the diversity of the animal reservoir. A detailed knowledge of immune response to the parasite would help in designing prophylactic and therapeutic strategies against this infection.
Tropical medicine has captured the interest of generations of medical students and researchers living in countries where these diseases are endemic. Some of the world's most important health problems including malaria, leishmaniasis, trypanosomiasis and schistosomiasis are caused by parasites. Millions of people are at risk of infections, disease and death from parasitic infections (Bittencourt & Barral-Netto, 1995). Parasites belonging to the genus Leishmania are among the most diverse of human pathogens, both in terms of geographical distribution and in the variety of clinical syndromes caused by them (reviewed by Melby, 2002). Leishmania protozoa are obligate intracellular parasites and over 20 species and subspecies of Leishmania infect humans, each causing a different spectrum of signs and symptoms. These range from simple, self-healing skin ulcers caused by Leishmania major and other dermotropic species, more severe chronic mucocutaneous infections caused by Leishmania braziliensis to severe, life-threatening visceral disease caused by the Leishmania donovani complex, including Leishmania infantum/chagasi (reviewed by Alexander et al., 1999).
Within the mammalian host, Leishmania resides as amastigotes in phagocytic cells such as macrophages, dendritic cells (DCs) and neutrophils. The clinical manifestations of leishmaniasis depend not only upon the species of parasite infecting the host, but the general health and genetic constitution of the infected individual (reviewed by Bogdan et al., 1996). Leishmaniases often represent zoonotic infections of stray and domestic dogs, rodents, hyraxes or sloth with variable penetration to humans. There are at least 30 species of Leishmania, of which 12 named and several unnamed species infect humans (Lainson & Shaw, 1987). Currently, c. 12 million people are infected worldwide in c. 88 tropical/subtropical countries (16 developed and 72 developing countries) and c. 2 million new infections are reported annually (WHO report, 1998). In the past decade, the number of cases in endemic areas has increased sharply. In addition, leishmaniasis is spreading to several nonendemic areas of the world due to coinfections with HIV (reviewed by Alvar et al., 1997).
Cure in all forms of leishmaniasis is affected through cellular immune response capable of activating host macrophages to eliminate the parasite. Although leishmanial infections induce strong humoral responses, antibodies appear to play no role in protection and in fact are associated with nonhealing forms of leishmaniasis. Because there are many areas where different species and different forms of the disease overlap, detailed knowledge of the immune response and pathogenesis is extremely important to develop vaccines for the various forms of leishmaniasis. Not only do organisms of this genus have the ability to withstand, inhibit or circumvent the microbicidal activity of host macrophages, but under the appropriate circumstances, they can subvert the induction of both innate and adaptive immune responses. Early classical experiments established that T-cell-deficient mice rapidly succumb to disease following inoculation with any one of several species of Leishmania, and that transfer of normal T cells confers resistance to the animals. The CD4+ subset of T cells is crucial for resistance, whereas CD8+ T cells seem to participate more in the generation of immune memory than as effector cells involved in parasite elimination (reviewed by Awasthi et al., 2004).
However, recent studies have suggested that CD8+ T cells may also be involved in the clearance of primary infection. Cytokines form a complex network of synergistic and antagonistic interactions, which not only induce but also control immune responses. Thus, it is universally accepted that the nature of the T-cell response is one of the crucial factors controlling experimental and human leishmaniasis; however, there are marked differences in the immune responses observed in experimental and human leishmaniasis. The mechanisms leading to the differential expression of cytokines and ultimately to the division of T-helper cells responses are not entirely clear. The present review provides an insight into the immune mechanisms associated with leishmanial infection.
Leishmaniasis: what is known?
The observations that only a small proportion of individuals develop active disease in endemic areas and successfully cured patients seldom become reinfected suggest that vaccination against leishmaniasis is feasible. Although several vaccination strategies have been tested in experimental leishmaniasis and a number of vaccine trials have been initiated, the focus is on cutaneous leishmaniasis (CL), caused by L. major. Relatively fewer efforts have been focused on visceral leishmaniasis (VL), which include the use of attenuated or killed parasites, crude antigen fractions, purified L. donovani membrane proteins and DNA vaccines. These strategies have had variable degrees of success against parasite challenge with L. donovani in experimental models, and very few of these molecules have reached phase I trials.
The control of leishmanial infection is mediated by a Th1-type immune response, and experimental studies in murine models of CL have established a clear-cut dichotomy between Th1-mediated protection and Th2-mediated disease susceptibility (reviewed by Sacks & Noben-Trauth, 2002). Although, in human CL, disease is associated with a Th2 cytokine profile and immunity with a Th1 profile, immune response defining disease vs. protection is not so well established in VL (reviewed by Reed & Scott, 1993; reviewed by Miralles et al., 1994). Gamma-interferon (IFN-γ) and interleukin-12 (IL-12), the signature cytokines for Th1 responses, are decreased during acute VL. These responses persist at high levels after successful treatment and are accompanied by high IL-10 levels (reviewed by Awasthi et al., 2004; reviewed by Saha et al., 2006). Recently, IL-10 has been suggested to play a role in counterbalancing the exacerbated polarized response that may develop following a cure by various workers (Kemp et al., 1999; Belkaid et al., 2001; reviewed by Trinchieri, 2007).
Earlier investigators have used the Th1 and Th2 paradigm as a strategy for the selection of an antigen in vaccine development against leishmaniasis. Thus, leishmanial antigens that predominantly stimulate Th1 responses in patient cells or mice infected with the parasite have been accepted as ‘potential protective antigens’ and therefore promising vaccine candidates. Conversely, antigens that predominantly stimulate a Th2 response from these cells have been regarded as of lesser interest as vaccine candidates because they are likely to be associated with pathology. However, it has been observed that several leishmanial antigens that induce a Th1 response during infection are not necessarily protective in vivo. Hence, it may not be appropriate to use the stimulation of Th1 responses as readouts for antigen selection in vaccine development against leishmaniasis (reviewed by Campos-Neto, 2002).
Immune response in experimental and human CL
In human and experimental leishmaniasis, immunity is predominantly mediated by T lymphocytes. T cells play a major role in generating specific and memory T-cell responses to intracellular parasitic infections and these have been extensively characterized in Leishmania infection. Th1 and Th2 cells can be distinguished by the cytokines they secrete: Th1 cells secrete activators of cell-mediated immunity such as IFN-γ, while Th2 cells secrete cytokines such as IL-4, which promote antibody responses. The Th1/Th2 paradigm of resistance/susceptibility to intracellular infection is largely based on investigations using L. major. Most strains of mice (C57BL/6, C3H, CBA) develop a self-limiting cutaneous disease when infected with L. major. In these mice, resolution of infection is mediated by Th1 cells that produce IFN-γ. IFN-γ induces the production of nitric oxide (NO) in phagocytic cells that harbor L. major (principally macrophages), which leads to destruction of the parasite. T-cell differentiation either to Th1- or Th2-type effector cells depends chiefly on the priming during differentiation. IL-4 induces Th2 whereas IL-12 induces Th1 differentiation (Fig. 1). Therefore, infection with L. major in these strains of mice resembles self-limiting CL in humans (reviewed by Scott, 1998).
Upon infection with L. major, mice of the resistant phenotype clearly develop a dominant Th1 phenotype of immune response to the parasite antigens. By contrast, BALB/c mice develop a typical Th2 response. Several systems have been used to correlate resistance or susceptibility with Th1/Th2 responses. Perhaps the most compelling one is that involving mice genetically deficient in either IFN-γ or IL-4, the phenotypic surrogates of the Th1 and Th2 CD4+ T-cell responses, respectively (reviewed by Rogers et al., 2002). Targeted disruption of the IFN-γ gene in C57BL/6 mice causes these animals, which are otherwise resistant to infection with L. major, to become highly susceptible to these organisms (Wang et al., 1994). Moreover, IL-4-transgenic resistant C57BL/6 mice expressing low levels of this cytokine fail to clear the infection. In addition, targeted disruption of the IL-4 gene in BALB/c mice causes these animals, which are otherwise susceptible to infection with L. major, to become highly resistant to these organisms (Kopf et al., 1996).
Nonhealing BALB/c mice infected with L. major have been shown to contain transcripts of IL-4 in their draining lymph node cells, in marked contrast to C57BL/6 mice that expressed transcript for IFN-γ but not IL-4. Kinetic analysis has shown sustained expression of IL-4 mRNA in infected BALB/c mice with a significant elevation of serum immunoglobulin E (IgE) levels that was not observed in C57BL/6. Leishmania-specific CD4+ T cells could passively transfer resistance or exacerbation of disease to immunodeficient or sublethally irradiated naive hosts depending on Th1 or Th2 cytokine characteristics (Scott et al., 1990). The nature of immune response generated against Leishmania depends on the type of leishmanial antigen recognized by the T cells.
The identification of the precise role of type 2 responses remains more elusive, and characterization of Th2 cytokine functions is an area of continuing speculation and investigation. Studies on the role of the Th2 ‘archetypal’ cytokine, IL-4, has provided some contradictory observations (reviewed by Alexander et al., 1999). This may arise, in part, due to different parasite strains or species being examined or different tissue sites (footpad, ear or base of the tail) being infected. Also, redundancy in IL-4 function via the compensatory activity of other cytokines such as IL-13, which shares many of the properties of IL-4, may obscure otherwise significant activity. Protective roles for IL-4 and IL-13 have also been described during L. major and L. donovani infections but not during Leishmania mexicana or Leishmania amazonensis infections (reviewed by Alexander et al., 2000).
During early infection with L. major, both resistant and susceptible hosts have been shown to exhibit mixed Th1/Th2 responses of CD4+ cell population with IL-2, IL-4 and IL-13 production, while IFN-γ transcripts were variable in different strains of mice. Strikingly, IL-4 production in infected mice was similar to fully developed Th2 clones in all the strains of mice analyzed. Administration of antibody to CD4 or IL-4 led to healing of infection, suggesting that an IL-4-producing CD4+ population plays a critical role in disease progression during the early stages of infection. The IL-4 induction in Leishmania infection was shown to be dependent on other T-cell factors such as IL-2. Administration of anti-IL-2 or anti-IL-2 receptor antibody ameliorated the L. major infection, indicating that IL-2 may also be a susceptibility factor for leishmaniasis (Heinzel et al., 1993a, b). This was confirmed by a report showing that IL-2 induced IL-4 production in CD4+ T cells (Ben-Sasson et al., 1990). It has also been demonstrated that IL-4-deficient mice raised from BALB/c embryonic stem cells remained susceptible to L. major infection (Noben-Trauth et al., 1996) and some workers have shown that, depending on the phase of response and the type of APCs, IL-4 could promote a Th1 response (Biedermann et al., 2001). In situations where IL-4 deficiency has not significantly inhibited disease progression, other cytokines such as IL-13 and IL-2 could be fulfilling the role. It is now recognized that the significance of the type 2 responses to the outcome of infection with leishmania is largely dependent on the strain or species of parasite studied.
Susceptibility and resistance to Leishmania infection in the mouse model are also associated with the emergence of a unique subset of T cells, namely the T regulatory cells (Treg) and with the levels of the cytokine, IL-10 (Belkaid et al., 2002). IL-10−/− BALB/c mice were relatively resistant to infection, indicating that endogenous IL-10 plays an important role in allowing disease progression in IL-10-sufficient mice. In this model, one of the mechanisms of IL-10 induction was the triggering of Fc receptor signaling on macrophages by IgG antibody-coated L. major amastigotes. Although IL-10 was originally listed as a Th2 cytokine, it has recently been shown to have suppressive or regulatory roles in autoimmune disease, host vs. graft rejection and parasitic infections (Reed et al, 1994). Treg cells (CD4+CD25+) suppress the activity of effector T-cell populations (CD4+CD25−) specific for self-antigens as well as foreign invaders such as leishmania parasites through the production of IL-10. Interestingly, during infection of C57BL/6 mice with L. major, CD4+CD25+ T cells accumulate in the leishmanial skin lesions, and these cells produce IL-10 upon in vitro stimulation with parasite antigens (Belkaid et al., 2002). IL-10 is also a potent inhibitor of IFN-γ production and has been shown to be a key cytokine that favors the persistence of the parasites in skin lesions (Belkaid et al., 2001). Therefore, Treg cells and IL-10 are important regulators of resistance/susceptibility to leishmaniasis.
In another model of cutaneous infection with the Friedlin strain of L. major, a Th1-mediated mechanism has been shown to result in clinical cure in resistant C57BL/6 mice. Although a small number of parasites persist, sterile cure is achieved only if IL-10 is neutralized (Belkaid et al., 2001). In this model, IL-10 is produced mostly by CD4+ T cells. However, all of the IL-10-producing CD4+ cells at the site of infection, and half of those in the draining lymph nodes, also produce IFN-γ (Belkaid et al., 2001). Other studies have shown that during the chronic phase of L. major infection, approximately half of the CD4+ T cells at the site of infection are antigen-specific CD25+Foxp3+ Treg cells, which inhibit the response of CD25−-effector T cells via both IL-10-dependent and -independent mechanisms (Belkaid et al., 2002; Suffia et al., 2006). These Treg cells produce most of the IL-10 responsible for the maintenance of chronic infection, whereas CD25− T cells produce most of the IFN-γ (Belkaid et al., 2002).
IL-10 has also been shown to function in a Th1 cell-polarized manner in a model of nonhealing L. major in conventionally resistant C57BL/6 mice (Chatelain et al., 1999). IL-10 prevents clinical cure in this model and thereby reflects the conditions underlying nonhealing forms of clinical disease. Accumulating evidence from other mouse models of nonhealing or disseminating forms of leishmaniasis have reinforced pathogenetic mechanisms that take into account the presence of parasite-driven Th1 responses that are suppressed either in magnitude or function by IL-10. IL-10 is crucial for suppressing the healing response in mice with cutaneous lesions caused by L. mexicana and in preventing the clearance of L. donovani from the liver and spleen (Padigel et al., 2003). Even in the L. major BALB/c infection model, Th2 cell immune polarization appears to be superimposed on IL-10-mediated suppressive pathways to account for the hypersusceptibility of this mouse strain, as BALB/c IL-4Rα-deficient mice are not fully resistant until IL-10 function is also impaired (Noben-Trauth et al., 2003).
Recently, the source of IL-10 in C57BL/6 mice infected intradermally with a clinical isolate of L. major (NIH/Sd) has been characterized by Anderson et al. (2007). They showed that these mice produced heavily infected, nonhealing lesions, even in the presence of a vigorous Th1 response that is reminiscent of clinical leishmaniasis. In L. major NIH/Sd mice, IL-10 produced by T cells but not by innate cells was required for the suppression of the healing response (Fig. 2a). Most of the IL-10 was produced by innate cells at the lesion site, in contrast to the majority of IL-10 in the draining lymph nodes that was produced by T cells, including both CD25+Foxp3+ Treg cells and CD4+CD25−Foxp3− T cells. A majority of the latter cells also produced IFN-γ (Anderson et al., 2007). Further, adoptive transfer experiments using these two IL-10 producing cell subsets showed that only the IL-10 produced by the CD4+CD25−Foxp3− T cells suppressed the healing response, whereas IL-10 from the CD25+Foxp3+ Treg cells was ineffective (Fig. 2b) (Anderson et al., 2007). However, depletion of the Treg cells resulted in an increased parasite burden, suggesting that these cells promoted host resistance against this strain of L. major, possibly by suppressing a deleterious Th2 response and/or by decreasing IL-10 production by the Th1 cells (Fig. 2c).
Belkaid (2003) has shown that IL-10 plays an essential role in L. major persistence in genetically resistant C57BL/6 mice after spontaneous healing of their lesions. They have demonstrated that a sterile cure was achieved in IL-10−/− mice but not in IL-10-sufficient mice. This requirement for IL-10 in establishing latency was determined in mice infected either by intradermal injection of L. major metacyclic promastigotes or by exposure of the skin to infected sandflies that mimic natural infection. Most importantly, IL-10-sufficient C57BL/6 mice treated transiently during the chronic phase with anti-IL-10 receptor antibodies achieved a sterile cure, suggesting that IL-10 was actively involved in preventing complete parasite elimination even in the presence of a Th1 response.
A role for Treg in the pathogenesis of Leishmania infection is not restricted to resistant strains. In susceptible BALB/c mice, cells that suppress L. major protective immunity have been shown to belong to an IL-4- and IL-10-producing population of cells with a regulatory T-cell phenotype that also inhibited colitis (Xu et al., 2003). In this susceptible strain, the removal of Treg transiently exacerbated the Th2 response but eventually led to a better control of the infection. Thus, the outcome of chronic infection by L. major was tightly controlled by the equilibrium between Treg and effector T cells. Recently, Suffia et al. (2005) have shown that the αE chain (CD103) of the αEβ7 plays a critical role in the retention of Treg and suppression of effector T cells.
IL-12 and IFN-γ are the protective cytokines based on their ability to influence Th1 development in vitro in various systems (reviewed by Awasthi et al, 2004). Normally, resistant mice depleted of IL-12 by genetic means or antibody neutralization become susceptible to L. major, while BALB/c mice treated with IL-12 develop a Th1 response and resistance (Heinzel et al., 1993a, b; Sypek et al., 1993; Mattner et al., 1996). Macrophages make very little IL-12 in response to Leishmania and infected macrophages show a decreased ability to make IL-12 in response to various stimuli (Carrera et al., 1996). Several reports have shown that DCs are the source of IL-12 during early infection (Gorak et al, 1998; von Stebut et al., 1998; Konecny et al., 1999; Marovich et al., 2000; Quinones et al., 2000). Furthermore, the stimulation of IL-12 production by DCs probably requires more than one signal, and there are several host components that could contribute to the IL-12 response (Snijders et al., 1998). For example, CD40–CD40L interactions enhance IL-12 production, and mice lacking this pathway are susceptible to CL. Other interactions between T cells and DCs may also contribute to IL-12 production in leishmaniasis. While IL-12 is the essential cytokine in the development of Th1 responses in leishmaniasis, under certain circumstances, other cytokines, such as IL-1α, migration-inhibitory factor (MIF), type 1 IFNs, IL-18 and tumor necrosis factor (TNF), also contribute to the development of resistance (reviewed by Scott, 2003).
Furthermore, IL-12 has been shown to be critical in promoting a Th1 response; IL-12 holds promise as an immunomodulatory agent. Indeed, IL-12 has been shown to be an efficacious adjuvant in a vaccine against leishmaniasis (reviewed by Sacks & Noben-Trauth, 2002; Awasthi et al., 2004). BALB/c mice vaccinated with Leishmania antigens alone and then challenged with live parasites developed a Th2 response and were unable to resolve their lesions or control parasite replication. In contrast, mice immunized with Leishmania antigens and IL-12 were protected from infection and developed a Th1 response (reviewed by Jones et al., 1998). Similar protection was obtained in vaccination studies using a recombinant LACK antigen (a defined Leishmania antigen-Leishmania homolog of the receptor for activated C kinase), together with IL-12 as an adjuvant (Mougneau et al., 1995).
Endogenous IL-12 has been shown to play a critical role in leishmaniasis. The capacity of exogenous IL-12 to heal infected BALB/c mice correlated with the ability of IL-12 to suppress IL-4 transcription and protein production. It is now known that transcription factor T-bet regulates Th1, while GATA-3 regulates Th2 development of naive T cells. These factors reciprocally regulate each other's expression depending on the priming condition (reviewed by Agnello et al., 2003). These transcription factors possibly play an important regulatory role both in resistance or susceptibility for Leishmania infection (Martins et al., 2005).
However, in addition to IL-12 and IL-4, several other cytokines have marked effects on infection with L. major in mice. For instance, TNF-α is critical for the resolution of an L. major infection because infection with the parasite in TNF-α knockout mice is fatal (Wilhelm et al., 2001). Among the many ways in which TNF-α may play a role, the most obvious is its ability to enhance macrophage activation, NO production and thus parasite clearance. Similar to IL-12 and TNF-α, IFN-α/β is also produced by antigen-presenting cells. IFN-α/β (also known as type 1 IFN) can induce cell activation, including activation of macrophages to produce NO, which kills L. major (reviewed by Bogdan et al., 2000). As a result, treating mice infected with L. major with a neutralizing anti-IFN-α/β was detrimental for the course of infection.
Taken as a whole, these observations suggest that several cytokines produced by antigen-presenting cells (IL-12, TNF-α and IFN-α/β) can promote the development of a protective Th1/IFN-γ response to L. major infection. There are also cytokines (which again can be produced by antigen-presenting cells) that promote the development of a Th2 response to infection with L. major in mice. Transforming growth factor-β (TGF-β) can inhibit the production of IFN-γ and can ‘deactivate’ macrophages, making them more permissive to infection with Leishmania (reviewed by Barral-Netto et al., 1992). IL-6 has been proposed to favor the development of Th2 responses. However, when IL-6-deficient mice on a susceptible BALB/c background were infected with L. major, the course of infection was not different from control animals. The absence of IL-6 led to down-regulation of both Th1 (IL-12)- and Th2-associated (IL-4, IL-10 and IL-13) cytokines. Thus, in mice infected with L. major, IL-6 may promote the development of both Th1 and Th2 responses (Rogers et al., 2002).
Studies on human CL have also contributed to the current knowledge about the disease. Lesions induced by parasites transmitted by needle injection and sandflies have been studied in healthy volunteers. A clear dichotomy in the T-cell response to invading Leishmania parasites, as is seen in mice, has not been demonstrated in humans (Fig. 1). The cytokine response of peripheral T cells in patients with CL revealed mixed Th1 and Th2 immunity (Ajdary et al., 2000; Bottrel et al., 2001). Recently, more groups have studied local immune responses in the skin, because they might be most relevant for the outcome of infection. In localized CL (L. braziliensis or L. major), Th1 cells predominate over Th2 cells. IL-4 was detected only in cases of diffuse MCL (reviewed by Gaafar et al., 1995; Bourreau et al., 2003). Other investigators have clearly detected IL-13 and IL-4 in the skin after initial lesion development, suggesting that Th2 cytokines play an immunoregulatory role in early infections (reviewed by von Stebut & Udey, 2004). However, cure of the infection was regularly associated with the production of IFN-γ only, while IL-10 was present in persisting lesions. In addition, treatment of nonhealing CL with IFN-γ resulted in rapid and complete resolution of lesions (Kolde et al., 1996).
Patients with cutaneous and mucosal leishmaniasis due to L. braziliensis infection have a strong T-cell response, characterized by a high lymphocyte proliferative response to Leishmania antigens and IFN-γ production (Follador et al., 2002). Delayed-type hypersensitivity (DTH) to the leishmania antigen is also present in these patients and the intradermal skin test is usually used for the diagnosis of cutaneous and mucosal leishmaniasis. Although IFN-γ is produced during L. braziliensis infection, it is not clear why these patients develop disease. It is possible that these patients may have some abnormalities at the site of the lesion. Immunological studies of cells from tissues of leishmaniasis patients have shown an immune response pattern similar to that of their peripheral blood mononuclear cells (PBMCs), with evidence of a Th0 or Th1/Th2 mixed cytokine pattern. Some differences were found in the cytokine profile when patients with lesions of short duration were compared with those with old lesions (reviewed by Ribeiro-de-Jesus et al., 1998).
Patients with chronic lesions show a strong expression of proinflammatory cytokines such as TNF-α. Because the cytokines secreted in the early phases of infection in the experimental models of leishmaniasis are important to determine the progression or control of the infection, it may be expected that alterations during early infections in humans may account for parasite multiplication and clinical outcome of the disease (reviewed by Ribeiro-de-Jesus et al., 1998). In fact, in early infection (<60 days of disease) patients with CL showed a decrease in IFN-γ production and an increase in IL-10 production when compared with patients who had the disease for more than 2 months (Almeida et al, 1995). More recently, it was reported that absent or low IFN-γ production may occur in up to 50% of the patients with early CL (reviewed by Ribeiro-de-Jesus et al., 1998).
Several lines of evidence show that IFN-γ and TNF-α are important for the control of leishmaniasis. In human VL and DCL, there is evidence that the absence of IFN-γ allows parasite multiplication and progression from infection to disease. It is also expected that T-cell responses and monocyte functions are important in the control of L. braziliensis infection. In fact, there is evidence of a Th1 type of response in subjects with subclinical L. braziliensis infection and in subjects with self-healing CL (reviewed by Ribeiro-de-Jesus et al., 1998).
Immune response in experimental and human VL
Studies of infections with the visceralizing Leishmania species, L. donovani and L. infantum/chagasi have underscored the fact that host responses to these parasites differ significantly from L. major infection. In rodent models, the Th1/Th2 paradigm is important in determining the outcome of murine L. major infection (Miralles et al., 1994). This dichotomy is not as influential during murine L. donovani and L. chagasi disease, in which curative type 1 responses are instead suppressed by IL-10 and TGF-β. Leishmania chagasi directly affects its local environment by activating latent TGF-β, and both L. donovani and L. chagasi suppress host macrophage responses to IFN-γ (reviewed by McMahon-Pratt & Alexander, 2004). There are localized immune responses in the liver and spleens of infected animals, which lead to apparent tissue ‘tropism’ and unique patterns of localized growth or cure of parasite infection (reviewed by Ribeiro-de-Jesus et al., 1998; Wilson et al., 2005). Studies of L. donovani infection in inbred strains of mice have shown that a major susceptibility gene Lsh influences the disease outcome of leishmanial infection (reviewed by Garg & Dube, 2006). Protective immunity against L. donovani, as with species causing CL, is dependent on an IL-12-driven type 1 response and IFN-γ production, which results in the induction of parasite killing by macrophages primarily via the production of reactive nitrogen and oxygen intermediates. A disease-promoting role for IL-4 and the Th2 response in VL, however, is more difficult to identify. For example, the differential production of Th1 and Th2 cytokines does not control the rate of cure of murine VL (reviewed by Bogdan et al., 1993).
As in human CL, no constant association between Th1 responses and resistance to disease with predominance of cells that produce IFN-γ has been identified in human VL (Louzir et al., 1998; Antonelli et al., 2004; Khalil et al., 2005). The levels of IFN-γ and IL-4 are elevated during active disease and decline significantly after cure. In active human visceral disease, PBMCs exhibit a poor proliferative response to parasite antigens and fail to generate IFN-γin vitro (Haldar et al., 1983). This lack of IFN-γ production by PBMCs seems to predict progression of the infection into fulminant VL (Carvalho et al., 1985, 1989). Although patients are unresponsive to a leishmanin skin test during this phase, a good response is seen 6–12 months after successful treatment (Costa et al., 1999; reviewed by Reiner & Locksley et al., 1995). In contrast, lymphocytes from patients cured of disease by therapy or subclinical acquisition of a positive skin test reactivity to leishmanial antigens demonstrate a vigorous proliferative response and readily release IFN-γ, IL-2 and IL-12 on stimulation with parasite antigens in vitro (Carvalho et al., 1994). Thus, both spontaneous and drug-induced healing is thought to be followed by strong protective immunity (Cillari et al., 1995). The absence of lymphocyte proliferation and IFN-γ production upon leishmanial antigen stimulation have been used as markers of clinical evolution (Khalil et al., 2005). They have also been used as readouts for protection in the development and assessment of candidate vaccine antigens (reviewed by Handman, 2001).
The prevalence of serological conversion in endemic areas of VL has been found to be higher than the prevalence of disease. This suggests that many infected individuals can control the disease. The strongest evidence that IFN-γ is involved in the control of Leishmania infection comes from a longitudinal study evaluating lymphoproliferative response and IFN-γ production by PBMCs of children living in an endemic area of VL. It was seen that L. chagasi-infected children whose Leishmania antigen-stimulated PBMCs produced IFN-γ could control their infection, while children who had poor IFN-γ production progressed to disease (Bacellar et al., 1991; Carvalho et al., 1992). Although it is likely that several mechanisms may participate in the inability of lymphocytes from VL patients to produce this cytokine, one important point is related to the imbalance of cytokines produced in response to leishmania antigens, i.e. high production of IL-4 and IL-10 and low IL-2 and IFN-γ production (Khalil et al., 2005).
Exogenous IL-12 was shown to induce IFN-γ production in VL-infected mice and by PBMCs from patients. It was also involved in regulating the host response to chemotherapy. In VL patients, IL-12 enhances Th1 responses and restores lymphocyte proliferative responses, IFN-γ production and cytotoxic responses (Ghalib et al., 1995; Bacellar et al., 1996). IL-12 also decreases spontaneous or antigen-induced PBMC apoptosis in VL patients. IL-12 used in combination with leishmania antigen restores proliferation of PBMC from VL patients more strongly than the use of anti-IL-4 or anti-IL-10 monoclonal antibodies, or even of both monoclonals combined (reviewed by Barral-Netto et al., 1998). Although lymphocytes from patients with VL have a strong expression of mRNA for IL-4 and sera from VL patients have high IL-4 levels, there is no evidence that IL-4 is involved in the down-regulation of the Th1 type of response in human leishmaniasis (reviewed by Alexander et al., 2000; Wilson et al., 2005). It has been shown that in vitro addition of mAb against IL-4 did not restore the lymphocyte proliferative response or IFN-γ production in L. chagasi-stimulated PBMC from VL patients. IL-4 also did not suppress lymphocyte proliferative response or IFN-γ production in subjects cured of leishmaniasis (reviewed by Ribeiro-de-Jesus et al., 1998). Thus, the prominent role of IL-4 as the leading Th2 cytokine in murine leishmaniasis was not consistently seen in human leishmaniasis.
IL-10 seems to represent the main macrophage-deactivating cytokine in contrast to IFN-γ, being present in many different clinical presentations of human leishmaniasis. IL-10 blunts several immunological responses mediated by lymphocytes from leishmania-infected individuals (Ghalib et al., 1993). Patients with VL have increased expression of mRNA for IL-10 in bone marrow and lymph node cells and high levels of IL-10 in L. chagasi-stimulated PBMC supernatants (D'Oliveira et al., 1997). Moreover, the addition of monoclonal antibodies to anti-IL-10 restores the lymphocyte proliferative response and IFN-γ production in PBMC from VL patients (Hailu et al., 2005).
The fact that IL-10 abrogates the effect of IL-12 in inducing IFN-γ production in L. chagasi-stimulated PBMCs of VL patients strongly suggests that IL-10 is the major cytokine involved in the progression of leishmania infection to visceral disease (Bacellar et al., 1996). IL-10 has been shown to block Th1 activation and consequently a cytotoxic response by down-regulating IL-12 and IFN-γ production. Additionally, because IL-10 also inhibits macrophage activation (reviewed by Ribeiro-de-Jesus et al., 1998), it decreases the ability of these cells to kill Leishmania. Bone marrow and lymph node cells from Sudanese individuals with acute VL have been shown to simultaneously express IL-10 and IFN-γ transcripts and IL-10 decreased after resolution of disease (Ghalib et al., 1993; Karp et al., 1993). Leishmania-specific T cells recovered from cured VL patients in Sudan have been found to express IFN-γ, IL-4 or both IFN-γ and IL-10 (Kemp et al., 1999).
In humans, measurements of cytokines in culture supernatants of Leishmania antigen-activated PBMCs and T-cell clones have helped in determining whether Th1 and Th2 immune responses are stimulated. Studies of tissue cytokine mRNA expression have revealed a role for IL-10 in downregulating CD4+ T-cell responses and the involvement of IL-10 in the disease pathology of L. donovani infections. However, active VL also finds a correlation with enhanced induction of IFN-γ, IL-2, IL-10 and IL-4. After cure, levels of IFN-γ, IL-4 and IL-10 persist, suggesting a coexistence of Thl and Th2 in Kala azar patients as well as in cured individuals (Caldas et al., 2005). Because IL-10 usually exhibits human macrophage-deactivating properties, high levels of IL-10 may represent a necessary counterbalance to an extremely polarized immune response, limiting tissue damage (reviewed by Bogdan et al., 1993; Trinchieri, 2001).
Other Th2 cytokines like IL-13 (Babaloo et al., 2001) and TGF-β (Gantt et al., 2003) have been reported to be produced in VL, although their biological role in modulating the Leishmania-specific immune responses is not well defined. Studies on regulatory T cells (CD4+CD25+), which function through TGF-β and IL-10 production, could help to understand the role of TGF-β. However, it is evident that parasite survival is favored by the conversion of latent TGF-β of the host to active TGF-β by some parasite-derived factors, which help to create its immediate microenvironment to its own survival advantage (Gantt et al., 2003).
Taken together, the above findings do not provide very clear-cut evidence for the existence of a Th1/Th2 dichotomy in the T-cell response in human leishmaniasis. Probably, the outcome of the infection is determined by the balance between the two parasite-specific T-cell populations. While infections with L. major result in self-healing and development of long-lasting immunity unless the patient is immunocompromised, infections with L. donovani either result in subclinical infection with consequent development of protective immunity or in clinical disease with fatal outcome if not treated. It is not clear whether host factors such as a pre-existing cytokine environment or genetic background, parasite factors such as virulence and inoculum size or the combination of host/parasite factors play a role in the development of these two types of clinical manifestation. Other less-explored cytokines may also prove important in the immunoregulation of human leishmaniasis. Future strategies for vaccination or immunotherapy must take into account such findings, which do not always parallel mouse studies. Thus, even in humans it is difficult to demarcate the responses leading either to visceral disease or to protective immunity with L. donovani. These studies suggest and provide a basis for screening for potential vaccine candidates.
Th1/Th2 paradigm in vaccine development?
The immunology surrounding Leishmania infection is complicated both from the standpoint of the host response to a given Leishmania species and the fact that different species can elicit very different responses. Particularly difficult from a vaccine development standpoint is the fact that it is not entirely understood what constitutes a protective response in humans. The animal models currently available are perhaps somewhat, but not entirely, predictive of how effective a vaccine candidate will be in humans. In general, the lack of easy assays to define the potency of a vaccine candidate makes progress in this area difficult. Many antigens with the potential for protective immunization have been discovered through fundamental research rather than a direct search for vaccine candidates (Table 1).
Table 1. Summary of vaccination studies in human and experimental models for visceral leishmaniasis (VL)
Form of vaccine
Stage of parasite/Leishmania strain
Evaluated in parasite–host system
107 amastigotes/L. donovani
Killed promastigote L. donovani+glucan
Autoclaved L. donovani+BCG
L. donovani/hamster and langur
108 formaldehyde killed promastigotes (UR6)
Autoclaved L. major+BCG
L. donovani/hamster/langur/human (clinical trial) L. infantum/dog
Killed promastigote of L. infantum+FCA
Merthiolated promastigote of L. brazilensis+BCG
L. chagasi/dog (phases I–III)
Fraction of L. donovani+β-1,3-glucan
FML+FIA/BCG/saponin/IL-12 QS 21/Quil A/aluminum hydroxide/Riedel De Haen
During the past decade, several investigators have used the Th1/Th2 paradigm to design strategies for antigen discovery/selection in vaccine development against leishmaniasis. Thus, leishmanial antigens that predominantly stimulate Th1 responses in patient cells or spleen or lymph node cells from mice infected with L. major have commonly been accepted as ‘potential protective antigens’ and therefore promising vaccine candidates. Conversely, antigens that predominantly stimulate a Th2 response from these cells have been regarded as of lesser interest because they are likely to be associated with pathology (reviewed by Campos-Neto et al., 1995). Paradoxically, leishmanial antigens against which a Th1 response is developed during infection may not necessarily be protective antigens. For example, lymph node cells of BALB/c mice chronically infected with L. major, upon stimulation with the Ldp23 antigen, produce high levels of IFN-γ and undetectable amounts of IL-4, a typical Th1 response (reviewed by Campos-Neto, 2005).
Immunization of BALB/c mice with Ldp23 in combination with adjuvants that preferentially induce Th1 responses, such as IL-12 and monophosphoryl lipid A plus squalene (MPL-SE), despite stimulating a strong antigen-specific Th1 response in the absence of any detectable Th2 response, did not result in protection (reviewed by Campos-Neto, 2002). This lack of correlation with protection was also observed with eight other leishmanial antigens that have been discovered and selected on the basis of the Th1/Th2 paradigm (reviewed by Campos-Neto, 2002). In contrast, the LACK antigen has been shown to stimulate a strong Th2 response that could be detected in lymph node and spleen cells soon after infection of BALB/c mice with L. major. The sera of these animals contain high titers of IgG1 anti-LACK antibodies. In spite of this, LACK confers substantial protection in BALB/c mice if administered in conjunction with adjuvants that stimulate Th1 responses (Julia et al., 1996) (Fig. 3).
More recently, an L. mexicana cysteine protease antigen named CPB2.8 was shown to be a potent Th2-inducing molecule during experimental leishmaniasis. However, as in the case of LACK, it conferred significant protection when administered with Th1-modulating adjuvants (Pollock et al., 2003). The LmSTI1 (a Leishmania homolog of a yeast stress-inducible protein 1) antigen stimulated mixed Th1/Th2 responses in lymph node cells of BALB/c mice infected with L. major and the sera of these animals contained high titers of IgE, IgG1 and IgG2a anti-LmSTI1 antibodies (Webb et al., 1996). As is the case for LACK, LmSTI1 is readily recognized by the mouse lymph node and spleen cells soon after infection. Moreover, LmSTI1 induces excellent protection in BALB/c mice and in monkeys if used with IL-12 (Campos-Neto et al, 2001) or MPL-SE (Skeiky et al., 2002) as an adjuvant. Interestingly, these two antigens (LACK and LmSTI1) do not share sequence similarities and yet are equally involved in stimulating primarily a Th2 response during the infectious process caused by L. major in BALB/c mice.
Therefore, it seems that the biased Th2 response is not dependent on a particular molecular characteristic of leishmanial antigens. Infection of mice with an avirulent mutant phosphoglycan-deficient L. major (lpg2 parasites) has been shown to result in minor disease with prolonged survival of the parasite in the animals. For reasons not yet clearly understood, lymph node cells from mice infected with this mutant parasite have been shown to produce minimal levels of IL-4 and IL-10 after in vitro stimulation with L. major soluble antigens in contrast to mice infected with wild-type parasites. However, the IFN-γ production by mice infected with this mutant was only slightly lower than in mice infected with wild-type L. major. Not surprisingly, vaccination of BALB/c mice with lpg2 parasites conferred excellent protection against challenge with virulent organisms (Uzonna et al., 2004).
Thus, the protection conferred by lpg2 parasites is basically not associated with enhanced IFN-γ production in response to leishmanial antigens but with a dramatic suppression of IL-4 and IL-10 responses to the same antigens. These studies indicate that a Th1 response alone should not be used as a readout for antigen selection in vaccine development against leishmaniasis. However, they by no means challenge the concept that a Th1 response is essential for protection against leishmaniasis. Indeed, as mentioned above, inducing a Th1 immune response to LACK before infection results in protection (Julia et al., 1996). Conversely, immunization of BALB/c mice with LmSTI1 formulated with alum, an adjuvant that polarizes the immune response to Th2 phenotype, results in no protection, in clear contrast to immunization with the IL-12 adjuvant (reviewed by Reed et al., 2003).
Conclusions and further possibilities
To understand the nature of human infection with these parasites and to develop better chemotherapeutic and vaccine strategies, further in-depth studies focused on the immune modulation in subclinical and asymptomatic individuals are required. As there is tremendous ecological and genetic diversity among the different human populations exposed to the parasite, a conclusive understanding of the parameters of resistance vs. control in humans is difficult. Moreover, the immunological data available are still scarce. There is also an urgent need for a better experimental model mimicking human infections. It is evident that there is a marked occurrence of both Th1 and Th2 components of CMI response during VL as documented through the detection of serum and tissue cytokines. Recent reports suggest the involvement of immune cells other than the Th1 andTh2 subsets of CD4+ T cells. Among these, CD8+ cells, macrophages and NK cells play major roles. In addition, recent experimental data obtained with studies on CD4+CD25+ Treg cells point to a probable regulatory function of these cells in maintaining the immune homeostasis in human leishmaniasis. In contrast to the earlier ideas that antagonistic functions of IFN-γ and IL-4 determine the outcome of protection or pathogenesis of the disease, recent studies emphasize the importance of the balance of the two regulatory cytokines IL-12 and IL-10, critical for the regulation of the immune modulation during infection, pathogenesis and chemotherapy.
Macrophages are proposed primary host cells for Leishmania but the role of these cells has not been well characterized either in disease prevention or in progression independent of T cell. The effector functions of macrophages for Leishmania have always been described in a T-dependent manner. The fate of infected macrophages in pre-T-cell phase is not well known. Because T cells come later during infection, it is possible that the parasite modulates its host in terms of signaling or antigen presentation for its own benefit and induces factors that provide a disease-progressive environment and prime T cells for Th2 differentiation. It is also possible that parasites start modulating the macrophages at the time of entry and later on modulated parasitized macrophages interact with T cells and may induce IL-4 and disease-inducing factors from T cells that help in disease progression and parasite survival in a susceptible host. The above discussion suggests the delayed or later role of T cells that may be a part of the same series that starts from the macrophages. It is now known that IL-10 plays a role in disease progression but whether with IL-4 or before the IL-4 phase is not known. It is also known that Leishmania-parasitized macrophages produce IL-10 but not IL-4, suggesting the role of IL-10 before IL-4 in disease progression or in the susceptibility of the host. This suggests the crucial role of IL-10 in disease initiation independent of T cells and in disease progression later on in combination with IL-4.
Thus, the Th1/Th2 paradigm of resistance/susceptibility is an oversimplification of a far more complicated network of regulatory/counter-regulatory interactions. These will differ according to the Leishmania species being studied, the host organism used and the tissue site examined. Studies using these organisms are providing fascinating new insights into the basic immunological mechanisms controlling the outcome of infectious diseases in general, which will aid the future rational development of appropriate strategies for immune intervention or vaccination.