From Biology to Disease: Importance of Species-Specific Leishmania Antigens from the Subgenera Viannia (L. braziliensis) and Leishmania (L. amazonensis) in the Pathogenesis of American Cutaneous Leishmaniasis

Abstract

American cutaneous leishmaniasis (ACL) is one of the most complex parasitic diseases from a clinical-immunopathological point of view due to the great heterogeneity of Leishmania species responsible for the disease. Currently, fifteen Leishmania species of the subgenera Leishmania, Viannia and Mundinia may give rise to ACL in Latin America. In Brazil, seven species are associated to the disease, but L. (V.) braziliensis and L. (L.) amazonensis stand out for producing the broadest clinical-immunopathological spectrum: localized cutaneous leishmaniasis [LCL: DTH+/++], borderline disseminated cutaneous leishmaniasis [BDCL: DTH+/−], mucocutaneous or mucosal leishmaniasis [MCL/ML: DTH++++], and anergic diffuse cutaneous leishmaniasis [ADCL: DTH−]. Although human genetic profile plays important factor in the immunopathogenesis of ACL, it deserves to be highlighted the crucial role of species-specific antigens of L. (V.) braziliensis and L. (L.) amazonensis [lipophosphoglycans, phosphatidylserine, proteophosphoglycans, glycoprotein-63 and CD200 – a macrophage activation inhibitor molecule] in the modulation of T-cell immune response (CD4+/CD8+) that will define the infection evolution.

1. Introduction

American cutaneous leishmaniasis (ACL) is an infectious, non-contagious, chronic disease caused by fifteen well recognized species of protozoan parasites of the genus Leishmania Ross 1903, widely distributed throughout Latin America. The disease is characterized by involvement of the cutaneous and/or mucosal tissue, where leishmaniotic ulcer represents the most usual clinical manifestation of ACL. However, other types of cutaneous lesions can be seen, such as: papule, tubercle, nodule, infiltrated plaque and verrucous or keloid-like vegetating lesion, among others. The mucosal lesion can be installed in the nose, mouth or pharynx alone, or even simultaneously, however, it is more frequent in the nasal mucosa, giving it a granulomatous ulcerative aspect, which, depending on its depth, can reach the cartilaginous tissue causing perforation of the nasal septum [1, 2, 3, 4, 5, 6, 7, 8, 9, 10].

In Brazil, the leishmaniotic nature of the skin and/or mucosal lesions of ACL were only confirmed, for the first time, in 1909, by Lindenberg, who found amastigote forms of Leishmania in smears made from the skin and/or mucosal lesions of patients coming from the interior of the State of São Paulo [southeastern Brazil]. Nevertheless, it was Vianna who described the new parasite, Leishmania braziliensis Vianna 1911, the first Leishmania species recognized in the New World. From then on, the etiology of ACL referred to as “Bauru’s ulcer”, “brave wound” or “tapir nose” was finally characterized [11, 12].

Until the early 1970s, all clinical forms of ACL in Brazil were attributed to a single Leishmania species, Leishmania braziliensis [13]. However, research carried out in the Brazilian Amazon pointed to the presence of a new Leishmania species responsible for some cases of the “anergic diffuse cutaneous form” named Leishmania mexicana amazonensis Lainson & Shaw 1972 [=Leishmania (Leishmania) amazonensis Lainson & Shaw 1987] [14]. Later, as new Leishmania species were identified in association with ACL [15, 16], great heterogeneity was noticed among these parasites which motivated a new classification of these protozoan parasites of the genus Leishmania, whose taxonomic character of greater expression was the biological behavior of the parasite within the gut of the sandfly vectors, represented by the terms: “hypopylaria, peripylaria and suprapilaria” [14]. Thus, these authors proposed the taxonomic classification of all Leishmania species into two subgenera: Leishmania Saf’janova 1982, and Viannia Lainson & Shaw 1987, in which the species that were previously allocated within the “mexicana” complex [suprapylaria = with development from the midgut to the foregut] were then allocated within the subgenus Leishmania [the same name of the genus], while those that were previously allocated within the “braziliensis” complex [peripylaria = with development from the hindgut (pylorus) to the foregut] were allocated within the subgenus Viannia.

Currently, these two subgenera [Leishmania and Viannia] include all seven Leishmania species and a hybrid parasite responsible for ACL in Brazil, mainly in the Brazilian Amazon, such as: L. (V.) braziliensis, L. (V.) guyanensis, L. (V.) shawi, L. (V.) lainsoni, L. (V.) lindenbergi, L. (V.) naiffi, L. (L.) amazonensis and L. (V.) guyanensis/L. (V.) shawi [9, 10, 11, 12, 17, 18]. Nevertheless, it is important to note that, more recently, a new molecular approach of the V7V8 SSU rRNA, Hsp70, and gGAPDH genes enabled the description of a new subgenus named Mundinia Shaw, Camargo & Teixeira 2016 [19], where L. (M.) martiniquensis Desbois et al. 2014 is classified, an ACL agent in the Caribbean region (Table 1).

Viannia Lainson & Shaw 1987	Subgenera Leishmania Ross 1903	Mundinia Shaw et al. 2016
L. (V.) braziliensis* Vianna 1911	L. (L.) mexicana Biagi 1953	L. (M.) martiniquensis Desbois et al. 2014
L. (V.) peruviana Velez 1913	L. (L.) pifanoi Medina/Romero 1959
L. (V.) guyanensis* Floch 1954	L. (L.) amazonensis*Lainson/Shaw 1972
L. (V.) panamensis Lainson/Shaw 1972	L. (L.) garnhami Scorza et al. 1979
L. (V.) lainsoni* Silveira et al. 1987	L. (L.) venezuelensis Bonfante-Garrido 1980
L. (V.) naiffi* Lainson/Shaw 1989	L. (L.) waltoni Shaw et al. 2015
L. (V.) shawi* Lainson et al. 1989
L. (V.) lindenbergi* Silveira et al. 2002; L. (V.) utingensis‡ Braga et al. 2003

Table 1.

Taxonomic classification of Leishmania parasites responsible for the etiology of American cutaneous leishmaniasis [ACL].

*

Etiological agents of American cutaneous leishmaniasis [ACL] in Brazil.

‡

Unique record of human infection by molecular biology [ITS-1 PCR].

In Brazil, from an eco-epidemiological point of view, ACL is regarded as a primary zoonotic infection of wild mammals, such as rodents, marsupials, edentates and primates, whose transmission of the parasites between their mammalian hosts is carried out by the infected females of different species of sandfly vectors [Diptera: Psychodidae: Phlebotominae] [11, 12, 20, 21, 22, 23, 24]. In this country, the disease represents an important public health problem, mainly in the North, Northeast and Midwest regions, where ecological, climatic and socio-economic factors have contributed to the formation and maintenance of large foci of the disease [9, 10, 25, 26]. In the North region, that is, in the Brazilian Amazon, it deserves special attention not only due to its high casuistry, but also due to its great heterogeneity of Leishmania species, mainly in the State of Pará, where L. (V.) braziliensis and L. (L.) amazonensis have high pathogenic potential to give rise to one of the most complex parasitic diseases.

In this sense, it is worth mentioning that, together, L. (V.) braziliensis and L. (L.) amazonensis are responsible for the broadest clinical-immunopathological spectrum of ACL already known, consisting of four clinical forms: localized cutaneous leishmaniasis [LCL], the most common form placed at the center of this spectrum and supported by a well-balanced T-cell immune response, including the delayed-type hypersensitivity reaction (DTH), mainly in cases by L. (V.) braziliensis [DTH^+/++/CD4⁺ < CD8⁺/Th1 > Th2], but with a high prevalence rate [≥80%] suppression of the T-cell hypersensitivity response in cases by L. (L.) amazonensis [DTH^±/−/CD4⁺ < CD8⁺/Th1 > Th2]; mucocutaneous or mucosal leishmanaisis [MCL/ML], the extreme expression of the T-cell hypersensitivity response linked to a strong species-specific CD4⁺/Th1-type immune response against to infection [DTH⁺⁺⁺⁺/CD4⁺ > CD8⁺/Th1 > Th2]; anergic diffuse cutaneous leishmaniasis [ADCL], the extreme expression of the T-cell hyposensitivity response linked to a strong species-specific CD4⁺/Th2-type immune response against to infection [DTH⁻/CD4⁺ < CD8⁺/Th1 < Th2]; and borderline disseminated cutaneous leishmaniasis [BDCL], an intermediary form between the central LCL and the two polar MCL/ML and ADCL forms, which is distinguished by a partial or incomplete suppression of the T-cell immune response more evident in those cases due to L. (L.) amazonensis [DTH⁻/CD4⁺ < CD8⁺/Th1 ≥ Th2] than those due to L. (V.) braziliensis [DTH^±/CD4⁺ > CD8⁺/Th1 ≥ Th2] (Figure 1) [10, 18, 27, 28, 29, 30].

Figure 1.

Some clinical and immunopathological features of the ACL spectrum caused by L. (V.) braziliensis and L. (L.) amazonensis in Brazil. Anergic DCL = diffuse cutaneous leishmaniasis; BDCL = borderline disseminated cutaneous leishmaniasis; LCL = localized cutaneous leishmanaisis; ML = mucosal leishmaniasis; DTH = delayed-type hypersensitivity reaction; CD4 = lymphocyte TCD4; CD8 = lymphocyte TCD8; Th1 = Th1-type immune response; Th2 = Th2-type immune response; TLR = tool-like receptors 2, 4 and 9; TNF-α = tumor necrosis factor-alpha; IFN-у = interferon-gamma; IL-10 = interleukin-10; TGF-β = tumor growth factor-beta. (−) no reaction; (±) borderline reaction; (+) weak reaction; (++) moderate reaction; (+++) strong reaction; and (++++) exacerbated reaction.

That said, one could question the real significance of the taxonomic classification of the genus Leishmania proposed by Lainson & Shaw [14] based on a merely biological taxonomic character in relation to the pathogenesis of ACL. In fact, perhaps not even the authors themselves at that time realized how significant that taxonomic classification would have in relation to the pathogenesis of ACL. However, the immunopathogenic competence of those Leishmania species of the subgenera Leishmania and Viannia play in the pathogenesis of ACL is now better understood. In this way, it has been shown a clear dichotomy on the interaction between L. (L.) amazonensis and L. (V.) braziliensis with human T-cell immune response; while L. (L.) amazonensis shows a clear tendency to lead infection from the LCL, a persistent suppressed T-cell hypersensitivity response [DTH^±/−] clinical form in the center of the spectrum, towards the ADCL at the T-cell hyposensitivity pole [DTH⁻] and with a marked CD4⁺/Th2-type immune response, L. (V.) braziliensis shows an opposite trend from the well-balanced form of the T-cell hypersensitivity [DTH^+/++] in the center of the spectrum, leading infection towards the MCL/ML in the T-cell hypersensitivity pole [DTH⁺⁺⁺⁺] and with a prominent CD4⁺/Th1-type immune response [18, 27, 29, 30, 31, 32, 33]. More than that, there is now some revealing evidence regarding the importance of some species-specific Leishmania antigens of the subgenera Leishmania and Viannia, represented by a dense layer of glycoconjugate molecules also known as virulence factors, such as, lipophosphoglycans [LPG], glucoinositolphospholipids [GIPLs], proteophosphoglycans [PPGs], glycoprotein-63 [GP-63], phosphatidylserine [PS], and CD200 [macrophage activation inhibitor molecule], playing a crucial role in modulating T-cell immune response against infection, which will be the reason for a more complete approach later in this chapter [18, 34, 35, 36].

2. Clinical and immunopathological spectrum of ACL due to L. (V.) braziliensis and L. (L.) amazonensis

Firstly, what reasons would there be today to highlight the importance of L. (V.) braziliensis and L. (L.) amazonensis as the species of greatest medical interest regarding the clinical-immunopathological spectrum of ACL in Brazil, mainly in the Brazilian Amazon. Without a doubt, based on what has already been said above and also taking in account that L. (V.) braziliensis is historically the first Leishmania species described in the New World, where it shows the widest geographic distribution in Latin America, from Central America [where it has been identified as an agent of ACL in Honduras, Belize, Costa Rica, Ecuador, Guatemala, Mexico, Nicaragua, and Panama] to South America [where it is strongly associated to ACL, mainly in Brazil, but also in Colombia, Venezuela, Peru, Bolivia, Paraguay, and Argentina], it seems fair enough to regard L. (V.) braziliensis as the main Leishmania species in the context of ACL [10, 11, 12, 37].

On the other hand, alongside L. (V.) braziliensis in Brazil, there seems to be no doubt that L. (L.) amazonensis occupies the second position in the ranking of the most medically important Leishmania species in reason of its capacity to develop the ADCL, the extreme expression of T-cell hyposensitivity pole [DTH⁻], which although it occurs with low frequency is capable of reproducing severe [nodules and infiltrated plaques] and disseminated [sparing only the scalp] chronic and incurable skin lesions in patients with strong suppression of the T-cell immune response [CD4⁺ < CD8⁺/Th1 < Th2/IL-10 and TGF-β > TNF-α and IFN-γ] [18, 27, 29, 30, 38].

Based on what was said above, it does not seem inappropriate to assume that L. (V.) braziliensis and L. (L.) amazonensis are recognized as the two Leishmania species with the greatest pathogenic potential for reproducing the clinical-immunopathological spectrum of ACL in Brazil: LCL and BDCL by both Leishmania species, while MCL/ML by L. (V.) braziliensis alone, as well as ADCL by L. (L.) amazonensis alone, which, from now on, reinforces the crucial role of the species-specific antigens also known as virulence factors [LPGs, GIPLs, PPGs, GP-63, PS and CD200] of these Leishmania species in the orchestration of the clinical-immunopathological spectrum of ACL. In other words, while species-specific L. (V.) braziliensis-antigens modulate a strong pro-inflammatory response in MCL/ML with production of high levels of major pro-inflammatory cytokines [IFN-γ/TNF-α], species-specific L. (L.) amazonensis-antigens modulate a strong anti-inflammatory response in ADCL with production of high levels of major regulatory cytokines [IL-10/TGF-β], even though higher (P < 0.05) antigen-presenting cells densities [dendritic Langerhans cell (LC): CD1a– MHCII⁺, Langerin⁺, and dermal dendritic cell (dDC): CD11c–MHCII⁺, Langerin⁻ and MHCI⁺, Langerin⁺] have been shown in ADCL clinical form, in the T cell hyposensitivity pole [DTH⁻], compared to those in LCL clinical form, a well-balanced T-cell hypersensitivity form [DTH^+/++] in the ACL spectrum, evidencing a crucial role of these species-specific antigens of L. (L.) amazonensis and L. (V.) braziliensis in modulating T-cell immune response (CD4⁺/CD8⁺) against infection [18, 27, 29, 30, 39, 40, 41].

Nevertheless, one could question the non-inclusion of L. (V.) guyanensis, alongside L. (V.) braziliensis, among the species with pathogenic potential to cause MCL/ML in Brazil. In fact, recent evidence has shown L. (V.) guyanensis as a causative agent of MCL/ML in the Brazilian Amazon [42], principally in the north bank of the Amazon River in the State of Amazonas, which undoubtedly limits its importance as a causative agent of MCL/ML not only in the Brazilian territory, but in the Brazilian Amazon as well. In the State of Pará, for example, where MCL/ML patients are regularly present in the outpatient clinic of the “Ralph Lainson leishmaniasis laboratory” at the Evandro Chagas Institute [Science, Technology and Innovation Secretariat, Ministry of Health, Brazil] in the municipality of Ananindeua, whose demand for patients comes from all regions of the State of Pará and to a lesser extent from the States of Amapá, Maranhão, Tocantins and Mato Grosso, the only leishmanine parasite that has been identified from MCL/ML in the last forty years is L. (V.) braziliensis, which leaves no doubt as to its importance as the main agent of MCL/ML in Brazil [9, 10, 11, 12, 14, 18, 27, 43, 44].

2.1 Localized cutaneous leishmaniasis (LCL) due to L. (V.) braziliensis and L. (L.) amazonensis

In Brazil, LCL is, without a doubt, the most common clinical manifestation of ACL, having as causal agent any of the seven Leishmania species and the hybrid parasite [L. (V.) guyanensis/L. (V.) shawi] mentioned above, that is, six species of the subgenus Viannia, including the species of greatest medical interest like L. (V.) braziliensis, and one of the subgenus Leishmania, with L. (L.) amazonensis being the only species associated with ACL in Brazil. However, it is necessary to record that, although the medical importance of these two species is well recognized, the association of L. (V.) braziliensis with the disease is much greater than that of L. (L.) amazonensis, which is explained due to the higher anthropophilic potential of the main sandfly vectors of L. (V.) braziliensis, such as, Lutzomyia intermedia, Lut. whitmani, Lut. migonei, Psychodopygus wellcomei, Psy. complexus and Psy. davisi than that of Lut. flaviscutellata, the principal sandfly vector of L. (L.) amazonensis in Brazil [9, 10, 45, 46, 47, 48].

From a clinical point of view, however, it is important to say that, although the typical ulcerated skin lesion, with a raised and infiltrated edge and a granulomatous background, represents the major clinical sign most frequently observed in patients with LCL caused by L. (V.) braziliensis or any other species of the subgenus Viannia, especially after one to two months of disease progression (Figure 2A), the same cannot be said in relation to the skin lesion in patients with LCL caused by L. (L.) amazonensis as, in most cases, it is represented by a nodule or plaque with a highly infiltrated base and edge, with a thickened appearance, evidencing discrete ulceration or even an exulceration on the surface (Figure 2E, F), without that typical ulcerative character seen in the skin lesion caused by L. (V.) braziliensis. This clinical difference in the skin lesion of LCL between L. (V.) braziliensis and L. (L.) amazonensis clearly reflects distinct physiopathogenic mechanisms on the interaction of species-specific antigens of these Leishmania species with human T-cell immune response [CD4⁺/CD8⁺]. On the one hand, while L. (V.) braziliensis is able to induce a moderate to overt DTH^(+/++) in LCL patients, on the other, L. (L.) amazonensis interacts in contrary to this by suppressing the DTH^(±/−) in more than 80% of LCL patients, which might explain the greater severity of dermal necrosis and subsequent ulceration seen in the skin lesion induced by L. (V.) braziliensis, since dermal necrosis represents a defense mechanism in the elimination of the parasite strongly related to DTH response [49, 50]. On the contrary, in the skin lesion induced by L. (L.) amazonensis dermal necrosis is a more discrete phenomenon due to the suppression of DTH^(±/−), resulting in a heavy parasite load on the vacuolated macrophages and lesser intense ulcerative process compared to that seen in the skin lesion of LCL by L. (V.) braziliensis [6, 18, 27, 29, 51, 52, 53].

Figure 2.

Localized cutaneous leishmaniasis caused by L. (V.) braziliensis [A: Typical ulcerated skin lesion; B: Keloid skin lesion; C: Verrucous skin lesion; D: Ulcer-infiltrated skin lesion on the hand, with bone deformity (osteomyelitis)] and L. (L.) amazonensis [E and F: Skin lesions in the form of a highly infiltrated plaque, with a thick base and exulcerated surface]; G and H: Histological sections of skin lesions caused by L. (L.) amazonensis and L. (V.) braziliensis, respectively, stained for eosin & hematoxylin (HE), showing in (G) dermal cellular infiltrate predominantly of heavily parasitized vacuolated macrophages (arrow), in addition to lymphocytes and plasma cells; and in (H) epithelioid granulomatous reaction, with giant cells (arrow), and lymphoplasmacytic infiltrate, with few macrophages. Bars = 20 μm.

Another clinical feature that deserves to be highlighted here refers to the polymorphism of the skin lesions that can be seen in patients with LCL caused by L. (V.) braziliensis, where not only the typical ulcerated skin lesion can be seen but also papule-like lesions, tubercle, nodule, infiltrated plaque and verrucous or keloid vegetating lesion (Figure 2B–D), while in patients with LCL caused by L. (L.) amazonensis the skin lesions do not go beyond the nodule or infiltrated plaque with a highly infiltrated base and edge, with a thickened appearance, evidencing discrete ulceration or even an exulceration on the surface (Figure 2E, F). However, in general, these clinical features are not optimized by many authors in Brazil, perhaps due to the low frequency of patients with LCL caused by L. (L.) amazonensis outside the Brazilian Amazon or the lack of a specific diagnosis of the etiologic agent involved. The fact is that, in the State of Pará, in the Brazilian Amazon, a regular number (194) of patients with LCL caused by L. (L.) amazonensis have been observed in the last forty years in the outpatient clinic of the “Ralph Lainson leishmaniasis laboratory” at the Evandro Chagas Institute [Science, Technology and Innovation Secretariat, Ministry of Health, Brazil] (Silveira, personal observation), more precisely from the northeast of the State of Pará, which has allowed a significant gain regarding the clinical, histopathological and immunopathological understanding of LCL caused by L. (L.) amazonensis [18, 27, 29, 38, 51, 52, 53, 54, 55].

However, before the establishment of Leishmania-infection, either by L. (V.) braziliensis or L. (L.) amazonensis and the subsequent development of the skin lesion, that is, of the LCL itself, one cannot fail to remember the crucial role of those species-specific antigens of L. (V.) braziliensis and L. (L.) amazonensis play during the two major events in the development of infection: first, the event that determines Leishmania-infection, represented by the interaction parasite-macrophage or parasite-neutrophil-macrophage; and second, the event that determines the evolution of Leishmania-infection, represented by the interaction parasite-dermal dendritic cell and/or parasite-Langerhans dendritic cell with human T-cell immune response [CD4⁺/CD8⁺] [9, 10, 18, 29, 32, 33, 40, 56, 57]. The recognition of these two events markedly influenced by the species-specific antigens of L. (V.) braziliensis and L. (L.) amazonensis is of great interest for a better understanding of the clinical-immunopathological spectrum of ACL caused by these Leishmania species.

Thus, after the establishment of the infection in the macrophage, some individuals who show effective activation of the innate immune response via IFN-γ and TNF-α are able to neutralize the infection [asymptomatic], while the majority develop different degrees of susceptibility [symptomatic], which may give rise to the central LCL form of the clinical-immunopathological spectrum of ACL that, depending on the species-specific Leishmania-antigens involved, such as, L. (V.) braziliensis or L. (L.) amazonensis, may present different clinical, histopathological and immunopathological profiles of infection [29, 32, 58].

In LCL caused by L. (V.) braziliensis, it has already been seen above that the predominant skin lesion is the typical leishmaniotic ulcer, which can generally be recognized after one to two months of disease progression, revealing, in most cases, the epithelioid granuloma into the dermis, with the presence of giant cells, some necrosis areas, typical lymphoplasmocytic cellular infiltrate and scanty parasitized macrophages (Figure 2H) [59, 60]. From the immunopathological point of view, some parameters previously studied deserve to be highlighted here, such as: most patients [≥95%] present moderate to overt DTH^(+/++), accompanied by high levels of lymphocyte proliferation assay, while into the dermis of the skin lesion the cell immunohistochemical labeling with specific monoclonal antibodies has revealed higher (P < 0.05) CD8⁺ T-cells density compared to that of their CD4⁺ T-cells counterpart, although the CD4⁺/Th1-type immune response has been shown to outperform the CD4⁺/Th2-type one, in addition to higher (P < 0.05) cell densities expressing pro-inflammatory cytokines [TNF-α, IFN-γ] compared to those of regulatory ones [IL-10, TGF-β], as well as greater CD68⁺macrophage densities expressing Toll-like receptors 2 and 4 (TLR2, 4) compared to that of TLR9 were evidenced in the double immunohistochemical cell labeling (Figure 1) [9, 10, 18, 27, 29, 30, 53, 61, 62, 63].

On the other hand, in LCL caused by L. (L.) amazonensis it has also been seen that the main clinical feature of the skin lesion observed in this form of the disease is the nodule or plaque with a highly infiltrated base and edge, with a thickened appearance, evidencing discrete ulceration or even an exulceration on the surface, in which the monocyte cell infiltrate into the dermis stands out at the expense, mainly, of the non-activated vacuolated macrophage of the M2 phenotype, intensely parasitized, with few plasma cells and lymphocytes in the interstitium, characterizing a true vacuolated macrophage reaction into the dermis of patients with LCL caused by L. (L.) amazonensis (Figure 2G). It is interesting to note the harmony between these histopathological findings with those of immunopathological nature, so that, in most patients [≥80%] the delayed-type hypersensitivity reaction is absent [DTH⁻] not only after challenge with axenic promastigote antigens of L. (L.) amazonensis or L. (V.) braziliensis, but also with axenic amastigote antigen of L. (V.) lainsoni, as well as low lymphocyte proliferation rates and few areas with epithelioid granuloma and/or necrosis into the dermis of the skin lesion in these patients are found [10, 18, 27, 51, 52, 53, 64, 65].

In addition, regarding the immunopathological findings searched by immunohistochemical cell labeling with specific monoclonal antibodies, it seems notorious certain similarity with those in LCL caused by L. (V.) braziliensis, such as, a higher (P < 0.05) CD8⁺ T-cells density compared to that of their CD4⁺ T-cells counterpart, as well as a CD4⁺/Th1-type immune response over than that of CD4⁺/Th2-type one, although not as evident as that seen in LCL caused by L. (V.) braziliensis. In this way, it is worth mentioning the higher (P < 0.05) cell densities expressing some regulatory cytokines [IL-10, TGF-β] than that of pro-inflammatory one [TNF-α], but not than that of IFN-γ. It is quite possible that the significant expression of IFN-γ together with that of TLR2 [evidenced by double immunohistochemical cell labeling] has ensured the superiority of the CD4+/Th1-type immune response over the CD4+/Th2-type in LCL caused by L. (L.) amazonensis, although some findings seem to signal a persistent suppression of the T-cell immune response as evidenced by high prevalence [≥80%] of negative DTH⁽⁻⁾, as well as low rates of lymphocyte proliferation assay in these patients (Figure 1) [10, 18, 27, 29, 30, 32, 51, 53].

In summary, taking into account these clinical, histopathological and immunopathological features of LCL caused by L. (V.) braziliensis and L. (L.) amazonensis, it would be appropriate to say that: i) the ulcerated character of the skin lesion is more associated with L. (V.) braziliensis-infection [in L. (L.) amazonensis-infection, the skin lesion is less ulcerated, with a more infiltrated and thickened border], ii) where the presence of parasitized macrophages into the dermis is more scanty, iii) frequently [≥95% of cases] associated to positive DTH^(+/++) [unlike L. (L.) amazonensis-infection with negative DTH⁽⁻⁾ in ≥80% of cases] and high rates of lymphocyte proliferation assay, iv) with inflammatory response mainly represented by the epithelioid granuloma, giant cells, necrosis areas and consistent lymphoplasmacytic cell infiltrate [in L. (L.) amazonensis-infection, the inflammatory response is basically formed by non-activated vacuolated macrophages of the M2 phenotype, intensely parasitized, with few plasma cells and lymphocytes, characterizing a true vacuolated macrophage reaction], v) while the immunopathological scenario shows a significant protector role of CD8⁺ T-cells stands out, in greater density than their CD4⁺ T-cells counterpart, both in LCL by L. (V.) braziliensis and L. (L.) amazonensis, vi) the CD4⁺/Th1-type immune response over than that of its CD4⁺/Th2-type one, more evident in L. (V.) braziliensis-infection, vii) with higher cell densities of pro-inflammatory cytokines [IFN-γ, TNF-α] compared to those of regulatory ones [IL-10, TGF-β], in agreement with greater expressions of TLR2, 4 than that of TLR9 [which has shown higher interaction with L. (L.) amazonensis-infection, in which there has been demonstrated high prevalence [80%] of the T-cell immune response suppression].

2.2 Borderline disseminated cutaneous leishmaniasis (BDCL) due to L. (V.) braziliensis and L. (L.) amazonensis

The first point that must be clarified is why the term “borderline” is being used here to designate this form of the disease that is referred by most authors as “Disseminated cutaneous leishmaniasis” [66, 67, 68, 69, 70, 71, 72, 73]. In fact, this term was first used by Moriearty et al. [74] to designate a clinical case of ACL in a patient with long-lasting disseminated skin lesions, and atypical intradermal reaction to Leishmania-antigen [DTH⁻] from northeast Brazil [Três Braços municipality, Bahia State], where the species L. (V.) braziliensis predominates. Considering that this patient also did not respond to other intradermally tested antigens, his ACL clinical form was labeled by these authors as “Borderline cutaneous leishmaniasis” [74]. However, considering that this was a typical clinical case with long-lasting disseminated skin lesions, the first step would be to designate the case as “Borderline disseminated cutaneous leishmaniasis”. Anyway, considering that the term “borderline” was used by these authors in an attempt to characterize a probable patient’s T-cell immune response dysfunction against infection, a similar interpretation was made by the authors of this chapter to also characterize a partial or incomplete suppression of the T-cell immune response against Leishmania-infection [mainly evidenced by suppression of the delayed-type hypersensitivity reaction (DTH) and lymphocyte proliferation assay] in some individuals with long-lasting disseminated skin lesions caused by L. (V.) braziliensis or L. (L.) amazonensis, favoring the spread of infection and the consequent establishment of the clinical form that has been designated by the authors of this chapter as “borderline disseminated cutaneous leishmaniasis” [BDCL] [10, 18, 27, 28, 29].

The second point that deserves to be clarified refers to the physiopathogenic mechanisms involved in the process of the parasite dissemination, which, once again, seems to be strongly influenced by the species-specific antigens of L. (V.) braziliensis and L. (L.) amazonensis. In this way, what stands out the most is the dynamics of the parasite dissemination and its relationship with the partial or incomplete suppression of the patient’s T-cell immune response. In terms of BDCL caused by L. (V.) braziliensis, it has been observed that the parasite dissemination is relatively fast, so that in around three months, tens or even hundreds papular, acneiform or ulcerative skin lesions mainly can be counted, distributed in the vicinity of the primary skin lesion [spread by contiguity] or in different regions of the patient’s body [lymphohematogenous systemic spread] (Figure 3A–D). Furthermore, recent evidence has shown that L. (V.) braziliensis isolates from patients with BDCL from an endemic area in the municipality of Três Braços, Bahia State, in northeastern Brazil, were able to induce, in vitro, higher infectivity rates and greater expressions of TLR2, 4 than isolates from LCL patients, which reinforces the central idea of ​​this chapter on the crucial role of species-specific antigens of L. (V.) braziliensis and L. (L.) amazonensis, or even among intra-specific isolates of these Leishmania species in the pathogenesis of ACL. Another alternative would be that these L. (V.) braziliensis polymorphic variants would be producing lower systemic levels of TNF-α and IFN-γ in BDCL patients than those LCL ones, which may account for parasite dissemination due to the decreased ability to control parasite growth [18, 27, 29, 66, 68, 69, 71, 75, 76, 77].

Figure 3.

Borderline disseminated cutaneous leishmaniasis (BDCL) caused by L. (V.) braziliensis and L. (L.) amazonensis, respectively, with disseminated acneiform ulcerative skin lesions in A, B, C and D [L.(V.)b]; and infiltrated plaque or nodular (non-ulcerated) skin lesions in E, F, G, and H [L.(L.)a].

Regarding BDCL caused by L. (L.) amazonensis, however, the parasite dissemination is clearly slower, taking around a year for the first metastatic lesions to appear, mainly papules that progress to non-ulcerated nodules and/or infiltrated plaques [more than ten were never observed], also close to the primary skin lesion [spread by contiguity] or at greater distances [lymphohematogenous systemic spread] (Figure 3E–H). Therefore, the clinical-biological behavior of L. (L.) amazonensis-infection, mainly with regard to the parasite dissemination and its relationship with T-cell immune response, is quite different from that by L. (V.) braziliensis; whereas L. (V.) braziliensis-infection disseminates faster and exerts a more discrete suppression of the T-cell immune response [DTH^±], L. (L.) amazonensis-infection disseminates more slowly, but with a more intense suppression of the T-cell immune response [DTH⁻], which is reflected in the response to antimony therapy as patients with BDCL caused by L. (L.) amazonensis require double [four series] the dosage of antimony therapy used in the treatment of patients with BDCL caused by L. (V.) braziliensis [see that dosage below]. Additionally, it is interesting to note that, similarly to what has been shown in L. (V.) braziliensis isolates from BDCL patients, it has already been shown, also in vitro, greater infectivity rates of L. (L.) amazonensis isolates from BDCL than those from LCL, confirming our previous suspicious that intra-specific antigenic differences within L. (L.) amazonensis [or even within L. (V.) braziliensis] strains/races/populations from distinct geographical regions in Brazil may be influencing the pathogenesis of ACL [10, 18, 27, 28, 29, 38, 78].

As for the histopathological repercussion of these processes, the findings into the dermis of the skin lesions in BDCL patients do not differ much in qualitative terms from those already mentioned in LCL caused by L. (V.) braziliensis and L. (L.) amazonensis, however, it is necessary to emphasize a greater intensity of these findings that take part in these processes. For example, in BDCL caused by L. (V.) braziliensis what stands out is the presence of a more consistent lymphoplasmacytic cell infiltrate, accompanied by few to moderate parasitized macrophages, with few necrosis areas and epithelioid granuloma (Figure 4B). In BDCL caused by L. (L.) amazonensis, what also stands out is the exuberance of the vacuolated macrophage reaction [mainly from the M2 phenotype], intensely parasitized, accompanied by moderate lymphoplasmacytic cellular infiltrate, without necrosis areas, but in some cases with long-lasting evolution [more than a year] there may be found some epithelioid granulomas with giant cells (Figure 4A), which seem to represent a T-cell immune response reserve, since, as in LCL caused by this Leishmania species or by L. (V.) braziliensis, antimony therapy [12 mg/Sb^v/kg weight/22 days each series] has been used successfully at twice [four series] the dosage of that used in LCL [two series] [9, 10, 18, 27, 28, 29, 64].

Figure 4.

Histological sections of skin lesions stained for eosin & hematoxylin (HE) in borderline disseminated cutaneous leishmaniasis (BDCL) caused by L. (L.) amazonensis (A) and L. (V.) braziliensis (B), where can be seen in a typical vacuolated macrophage reaction, intensely parasitized (arrows), with few lymphocytes and plasma cells and, in B intense lymphoplasmacytic infiltrate with few macrophages. Bars = 20 μm.

In terms of the immunopathology of BDCL, it should be emphasized that during the parasite dissemination, which means a critical stage of the infection, both the DTH reaction and lymphocyte proliferation assay remain negative, clearly reflecting some degree of T-cell immune response suppression, mainly in those BDCL patients due to L. (L.) amazonensis whom present a more intense T-cell immune response suppression [DTH⁻] than those due to L. (V.) braziliensis whom show a more discrete T-cell immune suppression [DTH^±], which, recently, was also noted by higher (P < 0.05) cell densities expressing regulatory cytokines [IL-10, TGF-β] than those pro-inflammatory one [TNF-α] in BDCL caused by both L. (V.) braziliensis and L. (L.) amazonensis. Moreover, it is interesting to point out that regarding the CD4⁺/CD8⁺ T-cells expressions in BDCL, it was also recently recorded that in L. (V.) braziliensis-infection there was a higher (P < 0.05) CD4⁺ T-cells expression than that of their CD8⁺ T-cells counterpart, while in L. (L.) amazonensis-infection there was a lower (P < 0.05) CD4⁺ T-cells expression than that of their CD8⁺ T-cells counterpart, which suggests a crucial role of CD4⁺ T-cell immune response against infection, since in BDCL caused by L. (L.) amazonensis, where there was a more intense T-cell immune response suppression [DTH⁻], the CD4⁺ T-cells expression was lower (P < 0.05) than that of their CD8⁺ T-cells one, although the CD4+ Th1-type immune response is still slightly over than the CD4+ Th2-type immune response. Finally, looking at the expressions of TLR2, 4 and 9, it was seen once again that TLR2,4 had greater (P < 0.05) expressions in BDCL by L. (V.) braziliensis, where the T-cell immune response suppression had less impact [DTH^±], while TLR9 had greater (P < 0.05) expression in BDCL by L. (L.) amazonensis, where T-cell immune response suppression was more impactful [DTH⁻] (Figure 1) [9, 10, 18, 27, 28, 29, 30].

In summary, BDCL represents an intermediary form of ACL that can occupy the two places between the center (LCL) and the two polar forms (MCL/ML and ADCL) in the clinical-immunopathological spectrum of the disease, having L. (V.) braziliensis and L. (L.) amazonensis as the main etiological agents in Brazil, as well as there are some clinical and immunopathological features that differ BDCL between these two Leishmania species. The term “borderline” was introduced to characterize the clinical evidence of partial or incomplete T-cell immune response suppression against Leishmania-infection in BDCL, mainly in those patients infected by L. (L.) amazonensis who present greater suppression of T-cell immune response [DTH⁻] than those infected by L. (V.) braziliensis [DTH^±], which seems to be one of the main reasons for the spread of the parasite. However, in BDCL by L. (V.) braziliensis, the parasite spread is relatively rapid, requiring only three months to give rise to a hundred or more ulcerative skin lesions; in contrast, in BDCL by L. (L.) amazonensis, the parasite spread is slower, requiring around one year to produce some 5 to 10 infiltrated skin lesions. As for the histopathology, attention is drawn to the exuberance of the vacuolated macrophage reaction, mainly at the expense of the intensely parasitized M2 phenotype macrophage in the cellular infiltrate of BDCL by L. (L.) amazonensis, while in BDCL by L. (V.) braziliensis what stands out is the presence of the characteristic lymphoplasmacytic cell infiltrate with few parasitized macrophages. These histopathological findings seem to be in agreement with those immunopathological ones that revealed higher [P < 0.05] expression of regulatory [IL-10 and TGF-β] than pro-inflammatory [TNF-α] cytokines in BDCL by both L. (V.) braziliensis and L. (L.) amazonensis, but with higher [P < 0.05] expression of CD4⁺ T-cells than their CD8⁺ T-cells counterpart in BDCL caused by L. (V.) braziliensis, although the opposite was observed in BDCL caused by L. (L.) amazonensis [lower expression of CD4⁺ T-cells than their CD8⁺ T-cells counterpart], which reinforces a higher T-cell immune response suppression in BDCL by L. (L.) amazonensis. However, in spite of these observations, the partial or incomplete T-cell immune response suppression found in BDCL by L. (L.) amazonensis has been restored following successful treatment with four LCL-therapeutic antimony regimens [12 mg/Sb^v/kg weight/22 days each series with a 10-day interval between series], which suggest an evident supremacy of Th1 over Th2 immune response (Th1 ≥ Th2).

2.3 Mucocutaneous or mucosal leishmaniasis (MCL/ML) due to L. (V.) braziliensis

The first point that needs to be clarified concerns the possible similarities or differences between the both terms mucocutaneous and mucosal leishmaniasis. What is common to both is the involvement of nasobuccopharyngeal mucosal tissue, or just nasal [which is more frequent] or just buccopharyngeal, however, what distinguishes them is the dynamics of infection, since in some situations the infection may present simultaneous involvement of the skin and nasobuccopharyngeal mucosa tissue [mucocutaneous leishmaniasis - MCL] or only the nasobuccopharyngeal mucosa tissue [mucosal leishmaniasis - ML]. Thus, it seems clear that in the case of MCL, the involvement of the mucosal tissue is earlier (a few months after the appearance of the skin lesion) than in ML, in which the appearance of the mucosal lesion may be after years (average two to three years) of apparent healing (spontaneous or after irregular therapy) of the skin lesion. Nevertheless, it is necessary to say that in the Brazilian Amazon, especially in the State of Pará, the form more frequently observed is the ML, while the MCL form is more prevalent in the Brazilian northeast, especially in the State of Bahia, which suggests, once again, that intra-specific antigenic differences within L. (V.) braziliensis strains/races/populations from distinct geographical regions in Brazil may be influencing the pathogenesis of ACL [MCL/ML] [1, 2, 10, 18, 27, 43, 79, 80, 81, 82, 83].

Anyway, what needs to be emphasized is the extraordinary capacity of L. (V.) braziliensis to migrate, via lymphohematogenous, from a skin lesion to the nasobuccopharyngeal mucosa tissue, where it will develop simultaneously to the skin lesion(s) [in the case of MCL], or years, in general, after apparent spontaneous remission or the inadequate treatment-based healing of skin lesion [in the case of ML], an inflammatory process that begins with erythema and infiltration of the mucosal tissue, followed by granulation and ulceration of the cartilaginous tissue, giving an ulcerogranulomatous aspect to the mucosal lesion (Figure 5A–C). In advanced cases, usually with more than five years of evolution, the inflammatory process can promote uncomfortable conditions such as nasal obstruction, formation of crusts, epistaxis, rhinorrhea, pruritus and even perforation of the cartilaginous tissue of the nasal septum or palate. In order of frequency, mucosal lesions primarily occur in the nasal septum, palate, pharynx, and larynx, or may be simultaneous in some cases resulting in metastatic lesions of the nasobuccopharyngeal mucosal tissue [1, 2, 4, 6, 10, 18, 27, 43, 83, 84].

Figure 5.

Clinical aspects of mucocutaneous (MCL) or mucosal leishmaniasis (ML) caused by L. (V.) braziliensis in A, B and C, and in D a histological section of a mucosal lesion stained for eosin & hematoxylin [EH] showing a garnulomatous reaction with intense lymphoplasmacytic infiltrate. Bar = 20 μm.

Regarding the histopathology of MCL/ML, the most striking finding of this form of the disease is represented by the presence of the tuberculoid granuloma in the nasobuccopharyngeal mucosal tissue, which corroborates not only the remarkable DTH⁽⁺⁺⁺⁺⁾ and lymphocyte proliferation assay, but also the up-regulation of CD4⁺/Th1-type immune response observed in MCL/ML. In addition, other histopathological findings include abundant infiltration of lymphocytes and plasma cells, with scanty parasitized macrophages, followed by necrosis of the cartilaginous structures (Figure 5D) [31, 53, 59, 60].

In parallel with the histopathology of nasobuccopharyngeal mucosal lesions, it is important to note that some immunohistochemical studies have shown that MCL/ML is supported not only by the increased expression of CD4⁺ T-cells infiltrating the mucosal lesions of patients (as compared to corresponding CD8⁺ T-cells), as well as the increased expression of pro-inflammatory TNF-α and IFN-γ as compared to regulatory TGF-β and IL-10 cytokines and lysozyme stained activated macrophages (epithelioid cells), characterizing a typical CD4⁺/Th1-type immune response, which reinforces a prior hypothesis regarding the pathogenesis of MCL/ML caused by L. (V.) braziliensis that implies the activation of a persistent antigen-specific CD4⁺/Th1-type immune response at the lymph nodes, followed by the recruitment of CD4⁺/T-cells from the peripheral blood to the inflammatory infiltrate in the mucosal lesions. As such, those cells are preferentially primed to operate as cytokine Th1-producing cells (mainly IFN-γ and TNF-α). The strong T-cell hypersensitive response recorded in these patients can be attributed to a long-lasting antigen-specific CD4⁺/Th1 activation by species-specific L. (V.) braziliensis-antigens, resulting in the high production of IFN-γ and TNF-α cytokines. In addition, the moderate expression of TLR2, 4 should also be highlighted, however, higher than that of TLR9 in MCL, which reinforces the greater interaction of TLR2,4 with L. (V.) braziliensis-infection than TLR9 (Figure 1) [10, 18, 27, 29, 30, 31, 85]. TNF-α seems to be the major cytokine responsible for damaging the mucosal tissue [86, 87, 88, 89]. Corroborating these findings, it should also be noted recent findings on the gene expression of immune response in ACL by L. (V.) braziliensis, showing high levels of IFN-γ and TNF-α cytokines in older skin lesions containing detectable levels of parasite transcripts [90].

2.4 Anergic diffuse cutaneous leishmaniasis (ADCL) due to L. (L.) amazonensis

Anergic diffuse cutaneous leishmaniasis (ADCL) is regarded a rare clinical form of ACL caused by leishmanine parasites belonging to the subgenera Leishmania and Mundinia. In this way, although L. (L.) mexicana and L. (L.) amazonensis have been regarded as the major agents of ADCL in Latin America, L. (M.) martiniquensis and L. (L.) waltoni have also been incriminated, more recently, as etiologic agents of the disease in that region [12, 14, 44, 91, 92, 93].

In Brazil, principally in the Brazilian Amazon, L. (L.) amazonensis is regarded, so far, the only species with immunopathogenic competence to determine ADCL; that is, with the ability to deviate the T-cell immune response of the infected individual to the T-cell hyposensitivity pole [DTH⁻] of the clinical-immunopathological ACL spectrum, characterized by strong expression of the T-cell immune response of CD4⁺/Th2-type, with high production of well-known regulatory cytokines [IL-10 and TGF-β]. It is in this subverted or down-regulated immunopathological scenario, strongly influenced by the species-specific action of L. (L.) amazonensis-antigens, that the infection progresses to establish a clinical picture characterized by a diffuse infiltration of the skin, as well as by papular, tuberous, infiltrating and nodular skin lesions, which rarely ulcerate, distributed throughout the integument only sparing the scalp and the palmar (hand) and plantar (foot) regions (Figure 6A–C). Due to this persistent immunosuppressive character of CD4⁺/Th2-type immune response, most patients (mainly children under five years old) carry, for many years (sometimes four or more decades), a severe clinical picture, sometimes highly mutilating, leading to the chronification of the process since the drugs available (pentavalent antimony, amphotericin B, pentamidine and miltefosine) for the treatment are not able to change the immunogenetic status of the patients. In cases with advanced evolution (over thirty years), bone lesions may occur in the extremities (osteomyelitis) or involvement of the nasobuccopharyngeal mucosa due to the parasite dissemination by contiguity of lesions on the face [10, 18, 27, 29, 30, 43, 94, 95, 96, 97, 98, 99].

Figure 6.

Clinical aspects of anergic diffuse cutaneous leishmaniasis (ADCL) caused by L. (L.) amazonensis in A, B and C, and in D a histological section of a skin lesion stained for eosin & hematoxylin (HE) exhibiting diffuse vacuolated macrophage reaction, intensely parasitized (arrows), with few lymphocytes and plasma cells. Bar = 20 μm.

Based on the immunosuppressive character of the disease, it would not be difficult to expect that the major histopathological finding of the skin lesions would reveal a monocytic cellular infiltrate into the dermis predominantly composed by vacuolated macrophages with a M2 phenotype, intensely parasitized, in addition to a moderate presence of plasma cells and few lymphocytes (Figure 6D) [10, 18, 27, 28, 43, 52, 100, 101]. In general, this is the quasi-stigmatic histopathological picture of ADCL that together with the persistent suppressive CD4⁺/Th2-type immune response explain why the authors of this chapter use the term “anergic” to name this type of ACL with a prognosis that is still quite reserved.

From an immunopathological point of view, there is no doubt that the specific suppression of the T-cell immune response [CD4⁺/CD8⁺] provided by the species-specific L. (L.) amazonensis-antigens has wide repercussions on different fronts of the immune defense mechanisms against infection, evidenced by the strong suppression of the delayed-type hypersensitivity reaction (DTH⁻) and lymphocyte proliferation responses, as well as by the low production, in situ, of the main inflammatory cytokines [IFN-γ and TNF-α] and, in contrast, the high production of regulatory ones [IL-4, IL-10 and TGF-β], revealing the inability of the non-activated dermal macrophage, mainly the M2 phenotype, to contain the parasite multiplication and leading to very weak responses to conventional therapy regimens [antimony, pentamidine and amphotericin B]. Corroborating these findings, it is also worth mentioning that it is precisely in ADCL that the lowest expression of CD4⁺/CD8⁺ T-cells is shown among all ACL clinical forms, but, on the contrary, it is also in ADCL that the highest expression of Treg Foxp3⁺ regulatory cells is recorded, confirming the extremely suppressed profile of CD4⁺/Th2-type immune response in ADCL, which seems to be strongly associated to low transcripts of CD4⁺/Th1-type and cytotoxic CD8^+/T-cells and the highest expression of TLR9 in the clinical-immunopatholgical ACL spectrum [18, 27, 29, 30, 38, 51, 53, 64, 102, 103].

3. Importance of species-specific antigens of L. (V.) braziliensis and L. (L.) amazonensis in the pathogenesis of ACL

Leishmania parasites are well-known as obligate intracellular protozoan organisms because they carry out an essential phase of their reproduction within the mononuclear phagocytic cells, mainly the macrophage, in which the amastigote form of the parasite multiplies by binary division [14]. However, in order for the amastigote form of the parasite to resist the lethal action of a highly hostile environment inside the macrophage’s phagolysosome [highly acidic, oxidative, containing antimicrobial peptides, proteases and restrict nutrient access], the parasite makes use of a series of molecules on the surface of its plasma membrane, called glycoconjugates, in order to neutralize the lethal action of these hostile elements into the macrophage’s phagolysosome [104, 105]. This is a critical phase for the parasite that will result in its survival or death, so that, if overcome by the parasite, the infection will be definitively established in the vertebrate host [106, 107]. However, in addition to the protective effect that these glycoconjugate molecules exert on the survival of the parasite, it is also known that their action can play an important role in the virulence of the parasite, reason why these glycoconjugate molecules are recognized here as species-specific Leishmania-antigens of both subgenera Leishmania and Viannia, with pivotal role in the pathogenesis of the disease caused by these Leishmania parasites, represented here by L. (V.) braziliensis and L. (L.) amazonensis.

Among the previously mentioned glycoconjugate molecules [LPG, GIPLs, PPGs, GP-63, PS and CD200], LPG is undoubtedly one of most important species-specific Leishmania-antigens or, in other words, the major cell surface glycoconjugate of Leishmania parasites, which has the ability to protect the parasite against complement-mediated lysis, facilitating its attachment and entry into the macrophage, and inhibiting the phagolysosomal fusion and protein kinase C [108, 109, 110]. Furthermore, it seems to exert one of the broadest antigenic activity spectrum being able to induce not only a pro-inflammatory innate immune response, but also a suppressive one, depending on the subgenus of Leishmania species involved in the process, i.e., Leishmania or Viannia. In this sense, one cannot neglect the possibility that the species-specific antigens of these subgenera (Leishmania and Viannia) may be true messengers of glycoconjugate molecules formed from the contents of the sandfly’s digestive tract segment where the parasite reproduced; that is, that of the subgenus Leishmania in the midgut [suprapylaria], while that of the sugenus Viannia in the posterior intestine [peripylaria] [14]. It is possible that this may be influencing the immunobiological role of the glycoconjugate molecules that act as species-specific antigens for Leishmania species of the Leishmania (L. amazonensis) and Viannia (L. braziliensis) subgenera.

In this way, it has been shown that LPG exerts a pivotal role in modulating a cascade of activated inflammatory T-cell responses in experimental L. (V.) braziliensis-infections, including high expression of TNF-α, IL-1β, IL-6 and NO (such as those seen in ML). In addition, it should also been highlighted that L. (V.) braziliensis-LPG not only stimulates messenger RNA transcription, but also induces NF-kB translocation, resulting in NO expression as well as IFN-γ and TNF-α cytokines induction in ACL. That induction is mediated by linking L. (V.) braziliensis-LPG to TLR2 or TLR4, indicating the crucial role of those receptors in innate defenses, which was also evidenced in clinical samples of LCL caused by L. (V.) braziliensis [30, 34, 36]. On the other hand, LPG has also been shown to act as an important immunomodulatory factor activating TLR4 and promoting the preferential CD4⁺/Th2-type immune response in experimental L. (L.) amazonensis-infections, which is strongly related to ADCL [35]. Based on these results, the most emphatic impression left is that the PLG molecule seems to act in a bidirectional manner from an immunobiological point of view, depending on the subgenus, Leishmania [L. amazonensis] or Viannia [L. braziliensis], involved in the process, although, more recently, it has been shown that LPG of L. (L.) amazonensis isolates from different origins, that is, from an ADCL case (State of Bahia, Brazil), from a domestic dog with visceral leishmaniasis (State of Minas Gerais, Brazil) and from a wild rodent (Proechmys sp.) in the Brazilian Amazon, showed a significant pro-inflammatory response with expression of TNF-α and IL-6 cytokines [111].

Another interesting species-specific Leishmania-antigen is the PS molecule, a phospholipid commonly located on the inner face of the parasite plasma membrane with capacity for inhibiting the inflammatory response of Leishmania-infected macrophages (as a strategy to evade the host T-cell immune response). In that sense, higher PS exposure on the surface of late-stationary-phase promastigotes of L. (V.) braziliensis has been observed in patients with LCL as compared to patients with ML [112]. However, higher PS exposure has been noted in L. (L.) amazonensis isolates obtained from ADCL patients as compared to those from LCL patients [113]. Those findings represent consistent evidence that the variability of PS across different strains of Leishmania [L. amazonensis] and Viannia [L. braziliensis] subgenera can influence the development of different clinical forms of ACL.

Finally, it is worth mentioning the important role that the CD200 molecule plays in the pathogenesis of L. (L.) amazonensis-infection, which is capable of inhibiting the activation of the infected macrophage via interaction with TLR9; that is, the CD200-dependent iNOS inhibition allows parasite growth within the macrophage, increasing the virulence of L. (L.) amazonensis [114]. Those findings were recently confirmed clinically, with evident interactions of TLR2 with clinical forms associated with L. (V.) braziliensis, including LCL, BDCL, and ML, while TLR9 showed strong interactions with clinical forms associated with L. (L.) amazonensis (principally the down regulated ADCL form) [30].

4. Concluding remarks

Based on what was mentioned above, there seems to be no doubt about how important it is to know the biology of the parasites of the genus Leishmania in order to better understand their relationships not only with their primitive hosts, invertebrates [phlebotomine sand flies] and vertebrates [wild mammals], but also with man, who is merely an accidental host [14]. This knowledge today has gained a great advance, mainly helped by the development of molecular biology, which has allowed to deepen the knowledge of the biology of these parasites at the level of the molecules of their plasma membrane’ (glycoconjugates), allowing a better understanding of their more specific relationships with the human immune response, mainly the T-cell immune responses, CD4/Th1-type and CD4/Th2-type, whose modulation by these glycoconjugate molecules, referred to herein as species-specific antigens of Leishmania species of the Leishmania [L. amazonensis] and Viannia [L. braziliensis] subgenera, will be of the same greater importance regarding the modulation of the clinical-immunopathological spectrum of ACL in Brazil [10, 18, 27, 29, 30].

Acknowledgments

This work was supported by Evandro Chagas Institute (Secretary of Health and Environment Surveillance of Ministry of Health, Brazil); Tropical Medicine Nucleus (Federal University of Pará State, Brazil); and “Laboratório de Patologia de Moléstias Infecciosas (LIM-50), Hospital das Clínicas HCFMUSP, Faculdade de Medicina, Universidade de Sao Paulo, SP, BR, and Grant #2014/50315-0, São Paulo Research Foundation (FAPESP)”.

Conflict of interests

The author declares no conflict of interest.