Immunobiology of leishmaniasis

Transcription

1 Indian Journal of Experimental Biology Vol. 47, June 2009, pp Review Article Immunobiology of leishmaniasis Umakant Sharma & Sarman Singh* Division of Clinical Microbiology, Department of Laboratory Medicine, All India Institute of Medical Sciences, New Delhi , India Leishmaniasis is a parasitic disease caused by various species of Leishmania, a unicellular kinetoplastid protozoan flagellate. It manifests mainly in 3 clinical forms; visceral leishmaniasis (VL), cutaneous leishmaniasis (CL) and mucocutaneous leishmaniasis (MCL), of which VL is the most severe form of the disease. VL is lethal if untreated and spontaneous cure is extremely rare. Cutaneous leishmaniasis usually has milder course and often results into a self-healing of ulcers. Resolution of leishmanial infection is dependent on the coordinated interactions between components of cell mediated immune response, specifically the activation of targeted T-cell populations for appropriate cytokine production and activation of macrophages. In murine model, the development of Th1 response is associated with control of infection, and Th2 response is associated with disease progression. However, Thl and Th2 dichotomy in the human system is not as distinct as in mice and the murine model does not strictly apply to human leishmaniasis. This review focuses the dichotomy of immune response against various clinical forms of the disease. An in-depth knowledge of sequences involved in the immune response to the parasite would help in designing prophylactic and therapeutic strategies against leishmaniasis. Keywords: Immunobiology, Immunomodulation, Leishmaniasis, Macrophage, T-cell response Leishmaniasis is one of the most diverse and complex of all vector borne diseases. It is caused by an obligate intracellular protozoan parasite belonging to the genus Leishmania. The disease, in different clinical forms, affects approximately 12 million people worldwide, with an increasing incidence of million new cases diagnosed every year and 350 million people at risk. It manifests mainly in 3 clinical forms; visceral leishmaniasis (VL), cutaneous leishmaniasis (CL) and mucocutaneous leishmaniasis (MCL), of which VL is the most severe form of the disease, lethal if untreated, is caused by species of Leishmania donovani complex. A total of about 21 Leishmania spp. have been identified to be pathogenic to human. It is characterized by diversity and complexity, both in terms of geographical distribution and in the variety of clinical syndromes. These range from simple, self-healing skin ulcers to severe, lifethreatening visceral disease. The disease is endemic in 88 countries on five continents. Of the 88 countries, 22 are in the New World and 66 in the Old World. More than 90% of the CL cases occur in Iran, Afghanistan, Syria, Saudi Arabia, Brazil, and Peru. VL is endemic in the tropical and sub-tropical regions *Correspondent author Telephone: ; Fax: sarman_singh@yahoo.com of Africa, Asia, Southern Europe, South and Central America. India accounts for half of the total 500,000 VL or kala-azar infections that are recorded annually worldwide 1. In addition, leishmaniasis is spreading to several nonendemic areas of the world due to coinfection with human immunodeficiency virus (HIV) 2,3. The parasites have a digenetic life cycle and exist in two distinct morphologies, the promastigote in sand fly vector, and the amastigote in mammalian host. The motile flagellated promastigotes exist, multiply and develop extracellularly in the alimentary tract of the blood sucking female sand fly vectors and are transmitted during the blood meal into mammalian host. Inside the mammalian hosts they infect macrophages of the reticuloendothelial tissue and differentiate into nonmotile amastigotes and multiply as such in the phagolysosomal vacuoles 4. Depending on the parasite species they either elicit cutaneous leishmaniasis (e.g. L. major, L. tropica, L. mexicana, L. braziliensis), mucocutaneous leishmaniasis (e.g. L. braziliensis) or visceral leishmaniasis (e.g. L. donovani, L. infantum/chagasi) 5-7. 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 the

2 SHARMA & SINGH: IMMUNOBIOLOGY OF LEISHMANIASIS 413 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. Although most of the informations on the immunologic mechanisms upon infection and protection from the Leishmania parasites are accumulated from the studies in mice, some critical findings of murine CL have been confirmed in humans in recent years. However, the immune responses to VL and pathogenesis of disease in human deviates considerably from the murine model. This review provides an insight into the immune mechanisms associated with leishmanial infection. Primary host response against the parasite invasion In order to develop a successful parasitic relationship with its host, the Leishmania must evade both the innate and adaptive immune responses. When Leishmania first enters the human body, it is in the promastigote form. Promastigotes are engulfed by macrophages but are resistant to proteolysis and degradation in the phagosome. Within the mammalian host, Leishmania resides as amastigotes in phagocytic cells such as macrophages, dendritic cells (DCs) and neutrophils. The complement protein C3b is one of the most potent immune opsonins. C3b binds to foreign material and promotes its uptake via C3b receptors on phagocytic cells. C3b binds to Leishmania parasite which results in uptake by the macrophage. Leishmania has a special surface glycoprotein, called gp63, which converts C3b into ic3b 8. From the parasitic's standpoint this conversion would favor phagocytic clearance rather than lytic clearance as Leishmania is very resistant to degradation once phagocytosed. Therefore, this conversion is crucial for Leishmania's survival. After being engulfed, the Leishmania must endure harsh conditions inside the phagosome like the oxidative burst used by the macrophage to destroy foreign material inside the phagosome. Macrophage plays a primary role in the host defense and regulation of immune responses upon activation 9. This process consists of an attack by superoxide and hydroxyl radicals on the parasite. Leishmania produces acid phosphatases on its surface which inhibit this burst. In addition to the oxidative burst, macrophages often attempt to degrade parasites with acidic enzymes. This occurs when lysosomes fuse with the phagosome. The Leishmania resists this attack through a proton pump present on the surface and allows its intracellular ph to remain close to neutral. Also, the protozoan molecule lipophosphoglycan (LPG) plays an active role by inhibiting lysosomal enzymes. The parasites perform a complex hostparasite interaction inside the severe environment of the phagolysosomes and eventually evade this immune defense mechanism 10. Infection of macrophages with Leishmania results in impaired microbicidal machinery as evidenced by decreased responsiveness to the lipopolysaccharide required for induction of interleukin (IL)-1 production 11. By continuing to live inside the macrophage, Leishmania effectively avoids the humoral branch of the immune system. During each of the steps described, the parasite evades and at times manipulates the human immune system and thus, avoids digestion. Humoral responses in leishmaniasis Infection of Leishmania in human is characterized by the appearance of anti-leishmanial antibodies in the sera of the patients. In CL, usually they are present at low levels during the active phase of the disease 12. However, in some studies the presence of antibodies against L. braziliensis infection in the sera of infected patients has been critically monitored and utilized for the diagnosis and prognosis of the disease In contrast, strong anti-leishmanial antibody titers are well documented in VL 18,19. The role of elevated anti-leishmanial antibodies in kalaazar patients towards protection or pathogenesis is still unclear. Critical analysis of Leishmania antigenspecific immunoglobulin (Ig) isotypes revealed elevated levels of IgG, IgM, IgE and IgG subclasses during disease IgG not only fails to provide protection against this intracellular pathogen, but it actually contributes to disease progression. Passive administration of antileishmanial IgG resulted in larger lesions in BALB/c mice with greater amount of IL-10 production 26. This result can be correlated with the highly elevated titers of anti-leishmanial antibodies during the active phase of the disease and a consecutive fall in the antibody titer after a successful cure. The elevated antibody titers against promastigote or amastigote antigens, their fractions or recombinant antigens have been extensively exploited for specific serodiagnosis in last two decades.

3 414 INDIAN J EXP BIOL, JUNE 2009 Cell mediated immune responses 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, 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 27 (Fig. 1). Therefore, infection with L. major in these strains of mice resembles self-limiting CL in humans. 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. 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 28. Moreover, IL-4-transgenic resistant C57BL/6 mice expressing low levels of this cytokine fail to clear the Fig. 1 Th1 and Th 2 dichotomy in leishmaniasis. 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 29. 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. 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 ameliorate the L. major infection, indicating that IL-2 may also be a susceptibility factor for leishmaniasis 30,31. 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 (T reg ) and with the levels of the cytokine, IL IL-10 deficient 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. T reg 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 32. 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 33. Therefore, T reg cells and IL-10 are important regulators of resistance/susceptibility to leishmaniasis. 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 34. 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 hyper-susceptibility of this mouse strain, as BALB/c IL-4Rα-deficient mice are not fully resistant until IL-10 function is also impaired 35. It has been shown that IL-10 plays an essential role in L. major persistence in genetically

4 SHARMA & SINGH: IMMUNOBIOLOGY OF LEISHMANIASIS 415 Fig. 2 Healing and non-healing immunological responses in cutaneous leishmaniasis. resistant C57BL/6 mice after spontaneous healing of their lesions (Fig. 2). They have demonstrated that a sterile cure was achieved in IL-10 deficient mice but not in IL-10 sufficient mice. 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 36. A role for T reg 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 37. In this susceptible strain, the removal of T reg 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 T reg and effector T cells (Fig. 2). IL-12 and IFN-γ are the protective cytokines based on their ability to influence Th1 development in vitro in various systems 38. 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 become resistant 30,31,39,40. Macrophages make very little IL-12 in response to Leishmania and infected macrophages shows a decreased ability to make IL-12 in response to various stimuli 41. Several reports have shown that DCs are the source of IL-12 during early infection 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 47. For example, CD40 CD40L interactions enhance IL-12 production, and the mice lacking this

5 416 INDIAN J EXP BIOL, JUNE 2009 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 48. 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 49,38. 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 50. 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 51. These transcription factors possibly play an important regulatory role both in resistance or susceptibility for Leishmania infection 52. 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 53. Among the ways in which TNF-α and IFN-α/β may play a role, the most obvious is the ability to enhance macrophage activation, nitric oxide production and thus parasite clearance. These observations suggest that several cytokines produced by antigen-presenting cells (IL-12, TNF-α and TFN-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 (Fig. 1). 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 54. 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 55. The absence of IL-6 led to down-regulation of both Th1 (IL-12) and Th2 (IL-4, IL-10 and IL-13) associated cytokines. Thus, in mice infected with L. major, IL-6 may promote the development of both Th1 and Th2 responses. In human CL, a clear dichotomy in the T-cell response to invading Leishmania parasites, as is seen in mice, has not been demonstrated in humans. The cytokine response of peripheral T cells in patients with CL revealed mixed Th1 and Th2 immunity 56,57. In localized CL (L. braziliensis or L. major), Th1 cells predominate over Th2 cells. IL-4 was detected only in cases of diffuse MCL 58,59. 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 60. 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 non-healing CL with IFN-γ resulted in rapid and complete resolution of lesions 61. 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 62. 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. Patient with chronic lesions shows 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 63. Many evidence shows 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.

6 SHARMA & SINGH: IMMUNOBIOLOGY OF LEISHMANIASIS 417 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 64. This dichotomy is not as influential during murine L. donovani and L. chagasi disease, in which curative type1 responses are instead suppressed by IL-10 and TGF-β. L. 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 65. 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 66. Protective immunity against L. donovani, as with species causing CL, is dependent on an IL-12-driven type1 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. It is well documented that VL is characterized by suppression of CMI, which is proved from the unresponsiveness of the patients to the Leishmanin skin test (LST) or Montenegro test. This test measures a DTH reaction to an intradermal injection of leishmanial antigens 67. Containment of the disease following a successful treatment is associated with a strong cell mediated DTH response 68. The cell mediated immune suppression is also evident from blastogenesis assay of the peripheral blood mononuclear cells (PBMCs) (lymphoproliferation) from untreated VL patients from Brazil, Africa and India Reduction in proportion of the helper T cells and the immunosuppression 73 is rapidly reversible with effective chemotherapy. 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 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-γ in vitro 71. This lack of IFN-γ production by PBMCs seems to predict progression of the infection into fulminant VL 77,78. In contrast, lymphocytes from patients cured of disease demonstrate a vigorous proliferative response and readily release IFN-γ, IL-2 and IL-12 on stimulation with parasite antigens in vitro 79. Thus, both spontaneous and drug-induced healing is thought to be followed by strong protective immunity 80. IL-12, known as an natural killer (NK) cell stimulating factor, cytotoxic lymphocytes maturation factor, and a central immunoregulation of the initiation and maintenance of the Th1 response, plays an important role in the induction of IFN-γ production by T and NK cells 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 86,87. IL-12 also decreases spontaneous or antigen induced PBMC apoptosis in VL patients. IL-12 plays a counter-regulatory effect against IL-10 in Leishmania infection, is proved from the observation that addition of recombinant IL-12 or neutralizing anti-il-10 mab could restore the IFN-γ production as well as the lymphoproliferative response in the Leishmania lysate stimulated PBMCs of active VL patients. Conversely, neutralizing anti- IL-12 or ril10 inhibits the production of IFN-γ 86,87. 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 88. IL-4 is considered to be the signature cytokine of Th-2 response. 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 89,90. 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 63. Thus, the prominent role of IL-4 as the leading Th2 cytokine in murine leishmaniasis was not consistently seen in human leishmaniasis. 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,

7 418 INDIAN J EXP BIOL, JUNE 2009 which help to create its immediate microenvironment to its own survival advantage 91. IL-10 was initially characterized as a Th2 cytokine but later on it was proved to be a pleiotropic cytokine, secreted from different cell types including the macrophages 92. Experimental evidences indicate that IL-10 plays an important regulatory role in the progression of VL. 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 93. 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 94. Moreover, the addition of monoclonal antibodies to anti-il-10 restores the lymphocyte proliferative response and IFN-γ production in PBMC from VL patients 95. 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 87. 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 63, it decreases the ability of these cells to kill Leishmania. Studies of tissue cytokine mrna expression have revealed a role for IL-10 in down regulating 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 96. Besides its possible role in susceptibility and progressive immunopathology in VL infection, high levels of endogenous IL-10 correlate with in the progression of PKDL. Along with PBMCs, dermal keratinocytes of the African PKDL patients who eventually developed PKDL produced significantly higher amount of IL-10 than the cured VL patients who did not develop PKDL 97. 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 immune-compromised, 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 preexisting 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. Immune responses in Leishmania-HIV co-infection Among all types of leishmaniasis, VL is the most frequent potential opportunistic disease associated with HIV-1 since the mid 1980s. Although 90% of the reported cases are from southwestern Europe, incidences of co-infection are increasing in eastern Africa 98,99 and the Indian subcontinent 3. VL is found to promote the clinical progression of AIDS 100 and it was suggested that Leishmania parasites could be seen as potential co-factor in HIV-1 pathogenesis 101. Further, L. donovani promastigotes and LPG are proved to mediate CD4 + T cell activation induced HIV-1 replication. Leishmanial antigen induced TNFα produced in the culture is important for the replication 102,103. The mutual interdependence as the agents for co-infection between these two pathogens is also reported. Co-infection in THP-1 macrophage cell line increases the multiplication of L. donovani amastigotes in the macrophages. It was shown that killed HIV preparation abrogate the proliferative response as well as IFN-γ production from the L. donovani antigen induced PBMCs of healthy individuals. Moreover, anti-il-10 could not enhance IFN- γ production in HIV-VL co-infected patients as is generally found in only VL patients 104,105. Immune response in Post Kala-azar Dermal Leishmaniasis (PKDL) patients As a sequel to kala-azar, PKDL, appears in a dermatotropic form of L. donovani infection in >50 per cent patients in Sudan and per cent patients in India 106. The immunopathogenesis of the disease is still not well understood. Although patients of PKDL

8 SHARMA & SINGH: IMMUNOBIOLOGY OF LEISHMANIASIS 419 often bear the different forms of PKDL lesions for years and thus some workers consider these cases as reservoir forvisceral leishmaniasis 107,108, while others have found that VL and PKDL causing strains are different 109. There are limited attempts to reveal the underlying immune mechanisms for the appearance of the disease 108. Studies, which have so far been made, indicate that unlike VL there is positive cell mediated immune response against Leishmanial antigen in PKDL in terms of DTH (LST), lymphoproliferation and production of IFN-γ by the PBMCs 71,72. PKDL patients manifest an aggravation of the disease with time in form of nodular lesions or erythematous plaque formation. Again, PKDL patients with early infection show better CMI than the chronic patients. These indicate that there are different domains of PKDL patients according to the immunopathogenesis of the disease. Though there is generalized prominence of Th1 in PKDL, as found from IFN-γ production, high levels of IL-10 in the lesions as well as plasma of the patients 97 indicate the importance of Th2 response in chronic PKDL. Prevalence of the CD8 + cells over CD4 + cells in PKDL lesions of Indian patients 110,111 suggest persistence of parasites as CD8 + cells might block the IL-2 production locally and in turn inhibit the IFN-γ production 112 favoring disease. Th1/Th2 paradigm 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. 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 lymphnode 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 113. 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 114. Conclusion To understand the nature of human infection with these parasites and to develop better chemotherapeutic and vaccine strategies, further indepth 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. 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 diseaseinducing factors from T cells that help in disease progression and parasite survival in a susceptible host. 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 Leishmaniaparasitized macrophages produce IL-10 but not IL-4, suggesting the role of IL-10 before IL-4 in disease

9 420 INDIAN J EXP BIOL, JUNE 2009 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. Acknowledgement Research fellowship to US by Indian Council of Medical Research (ICMR), New Delhi is gratefully acknowledged. 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