**4. Molecular aspects of the infection**

The first interactions between Leishmania and the host's immune response are closely linked to the evolution of the disease or protection against the protozoan, and the vector's saliva directly contributes to these interactions [48]. Sandfly saliva is composed of active molecules that cause an imbalance in homeostasis at the host site, and aid repast [49]. The saliva of these arthropods contains a vast repertoire of pharmacologically active molecules that hinder the host's hemostatic, inflammatory, and immunological responses [48, 49]. When sand fly saliva is injected into the host's skin, it induces infiltration of inflammatory cells [50] and antibody production [51–53]. These disturbances in tissue physiology may also favor the release of Leishmania parasites, as the key to the success of Leishmania parasitism is the ability to evade host immune responses [48]. In this setting, immune complexes are formed [53] in the early stages of exposure. In addition, sand fly saliva also modulates costimulatory molecules and cytokine release by antigen-presenting cells [54–56].

#### *Leishmaniasis: Molecular Aspects of Parasite Dimorphic Forms Life Cycle DOI: http://dx.doi.org/10.5772/intechopen.102370*

Several active compounds with pharmaceutical properties have already been isolated from the saliva of sand flies such as the anticoagulant compound of Lufaxine (Inhibitor of Factor Xa from *Lutzomyia longipalpis*). This recombinant protein has potent and specific anticoagulant activity against factor Xa, a serine protease that cleaves prothrombin to generate thrombin and is involved in both the extrinsic and intrinsic coagulation pathway [57], preventing the activation of receptor 2 activated by protease and thereby inhibiting inflammation and thrombosis in C57BL/6 mice [58].

The action of the LuloHya compound, which acts as a hyaluronidase [55], has also been reported, and when co-inoculated with the parasites provides a more successful infection by Leishmania [59–61]. The Lundep protein, on the other hand, acts as an endonuclease and helps in the survival of parasites by inhibiting neutrophil traps (NET) in addition to preventing the activation by contact of FXIIa in human plasma [56, 60].

One of the most studied salivary peptides is a potent vasodilator known as maxadilan (MAX). In addition to vasodilation, this compound can also act as an immunomodulator in the host. It can up-regulate cytokines associated with a type 2 response (IL-10, IL-6, and TGF-β) and down-regulate type 1 cytokines (IL-12p70 and TNF-α), NO, and CCR7. This increased parasite survival in the vertebrate host in the early stages of infection [55, 56]. Studies involving the inhibition of human complement by the saliva of the sand fly *Lutzomyia longipalpis* showed the existence of inhibitors of the classical pathway in this species. As the anti-complement compound Salo [62] and is also considered as a potential transmission-blocking vaccine candidate against leishmaniasis [63].

Pharmacologically active molecules such as Maxadilan in *L. longipalpis* or PP-1 PP-2A inhibitors [Protein phosphorylation and dephosphorylation reactions, mediated by protein kinases and PPs, respectively, trigger signal transduction events that control diverse cellular responses to internal and external signals [64, 65] present in the saliva of *P. papatasi*, probably evolved to facilitate blood-feeding. However, as with many other biomolecules, salivary factors also exhibit other activities. In this case, Leishmania parasites benefit from the immunomodulatory effects of certain salivary factors to facilitate their establishment in the hostile environment of vertebrate skin [66].

Taken together, these data indicate that saliva is an endless issue, and several factors remain to be defined and how blocking these molecules is an open field for alternative tools against transmission [48, 49, 67]. **Figure 3** briefly illustrates the main aspects of how the infection of leishmaniasis parasites occurs in the host.

Recognition of the parasite by the host's immune system cells is the key to triggering effective Leishmania-specific immunity [5]. However, the parasite can persist in the host's myeloid cells, evading, delaying, and manipulating the host's immunity to escape host resistance and ensure its transmission [5].

Neutrophils are the first to infiltrate infection sites, where they generate an inflammatory response that restricts the parasite and acts to protect the organism, fighting infection through a series of mechanisms, being considered important modulators of leishmaniasis [68]. They are responsible for the formation of weblike structures called neutrophil extracellular traps (NETs) that can capture and/or kill microorganisms [68]. However, for some species of Leishmania, neutrophils can act as carriers that facilitate the silent infection of macrophages [69–71]. The 'Trojan Horse' model is based on the silent transmission of the parasite from neutrophils to macrophages and dendritic cells when macrophages and cells phagocyte from apoptotic neutrophils that are contaminated by Leishmania [70]. This model is evidenced in the reported ability of some Leishmania species, such as *L. major* and *L. braziliensis* [72, 73], to induce neutrophil apoptosis.

#### **Figure 3.**

*Schematic representation of the leishmaniasis stages of infection. Parasite infection: Leishmania sp. enters through the lesion caused by the proboscis during the meal and infect local macrophages. Stimulated by compounds with vasodilating and anti-hemostatic properties present in the vector's saliva, an inflammatory reaction begins in the region where more immune cells are recruited to the site and can also be infected by protozoa in metacyclic form. Once phagocytosed, the protozoa become different in the amastigote form in the phagosome. Growth and survival of Leishmania sp.: Infected macrophages secrete anti-inflammatory and pro-inflammatory mediators, initiate immune response mechanisms, neutrophils release cytokines and reactive oxygen species–ROS in the region and monocytes, which differ into macrophages and dendritic cells, which become infected and migrate to other tissues. The increase is represented as blue arrows and decrease is represented as red arrows.*

Macrophages are the main effector population involved in parasite elimination [5]. However, macrophages are the main host cells where the parasites grow and divide. The parasites infect, multiply gradually, and finally destroy macrophages releasing large numbers of viable amastigotes in the region [74]. Once inside the macrophage, and depending on the Leishmania species, the parasites delay the formation and maturation of phagosomes, preventing phagosome acidification and

#### *Leishmaniasis: Molecular Aspects of Parasite Dimorphic Forms Life Cycle DOI: http://dx.doi.org/10.5772/intechopen.102370*

the action of proteases, while guaranteeing the nutrients necessary for its survival. Furthermore, the parasites modulate the cytokine secretion pattern and inhibit the generation of NO and ROS, while extending the survival of infected macrophages [5]. Genomic and transcriptomic analyzes have largely contributed to the understanding of the biology of Leishmania and revealed to us about the complex interactions that occur within the parasite–host-vector triangle, these interactions are responsible for the rapid activation and deactivation of various signaling pathways that lead to functions of macrophages [e.g., phagocytosis, chemokine secretion, and prostaglandin secretion] [75, 76]. Extracellular matrix interactions, metabolic changes, modulation of gene expression and several mechanisms that are still being studied have revealed how cell–cell interaction occurs and why leishmaniasis is such a complex disease as shown in **Figure 4**. The elimination of parasites by macrophages requires the preparation and development of an adaptive effector Th1 immunity driven by specific subtypes of dendritic cells [5].

Studies analyzing neutrophils infected by *L. major* parasites have shown that, when phagocytosed by cells in the skin tissue, they have the ability to inhibit the maturation and migration of dendritic cells, resulting in a delay in the development of adaptive immunity [72, 78, 79]. Dendritic cells are essential for the generation of a Th1-mediated immune response, fundamental for the control of leishmaniasis [80–82]. These parasites can act at different levels to inhibit dendritic cells, including modulation of the MAPK pathway, decreased antigen presentation capacity and IL-12 secretion, this inhibition being mediated by the activation of protein tyrosine phosphatase (PTPs) [83, 84]. In summary, the internalization of the opsonized protozoan by dendritic cells via FcγR (Fcγ receptor) promotes dendritic cell activation and IL-12 production. Furthermore, there is a down-regulation of costimulatory molecules, CD40 and CD86 after infection and gp63 cleaves the SNAREs protein (soluble NSF binding protein receptor), preventing the assembly of the NADPH oxidase complex [5]. An analysis of the gene expression of lesions with Cutaneous Leishmaniasis showed increased P27 [85] and decreased expression of the A2 gene [86]. IL-10 is important for the persistence of the parasite in the lesion, preventing its complete elimination from the lesion, despite the presence of a protective immune response [87]. Furthermore, circulating antibody is crucial for susceptibility to the development of tegumentary leishmaniasis [88] and a progressive increase in tissue IL-10 expression during infection suggests a role in susceptibility [89]. The amastigotes from the cutaneous leishmaniasis lesion are coated with IgG, and the internalization of opsonized amastigotes by macrophages induces the production of IL-10 and a consequent increase in the intracellular growth of the parasite [90].

Tissue damage is promoted by inappropriate epidermal signals driven by dendritic cells. Furthermore, studies indicate that nTregs are essential for the development and maintenance of persistent skin infection and reactivation of infections caused by the Leishmania parasite [91]. Understanding which dendritic cell populations are critical to triggering and achieving immunity to Leishmania and how parasites inhibit its activation and migration will help to improve a rational design of vaccines aimed at neutralizing the parasite's virulence factors, along with the use of the most appropriate adjuvants [5]. These recurrent injuries may result from the Koebner phenomenon [92] which refers to skin lesions appearing in lines of mechanical trauma, seen in some skin diseases such as psoriasis.

Antimicrobial peptides are innate immunity mechanisms that contribute to host defense. LL-37 is a peptide derived from human cathelicidin (CAP180, a multifunctional regulator of the innate and adaptive immune response, having a leishmanicidal activity, increasing phagocytosis in dendritic cells and macrophages, and acting as an activator or suppressor of the adaptive immune response depending on the concentration [91].

#### *Leishmaniasis - General Aspects of a Stigmatized Disease*

#### **Figure 4.**

*Diagram representing the major reports about modulation of internal reactions in macrophages infected by the parasite causing leishmaniasis. Leishmania sp. internalization and cell differentiation is successfully achieved, mediated by modulating the expression of genes linked to various cellular functions [12] and by the alteration of signaling events in the host cell, leading to increased production of autoinhibitory molecules such as TGF-beta and decreased induction of cytokines such as IL12 for protective immunity. The production of nitric oxide is also inhibited. furthermore, defective expression of major histocompatibility complex (MHC) genes silences subsequent macrophage-mediated T cell activation, resulting in abnormal immune responses [77]. SHP-1 downregulates JAK2, Erk1/Erk2 MAP, NF-B, IRF-1, and AP-1 kinases, thereby inhibiting IFN-inducible macrophage functions (e.g., nitric oxide, IL-12 production, and immunoproteasome formation), STAT1 degradation by the proteasome is dependent on PKC and other phosphatases (eg, phosphatase IP3 and calcineurin) and surface parasite molecules such as LPGs play a key role in altering several secondary pathways, for example, PKC, Ca+2 and phosphatidyl inositol), regulating important phagocyte functions such as NO and superoxide production [75]. The increase is represented as blue arrows and the decrease is represented as red arrows.*

#### *Leishmaniasis: Molecular Aspects of Parasite Dimorphic Forms Life Cycle DOI: http://dx.doi.org/10.5772/intechopen.102370*

Natural regulatory T cells rapidly accumulate in the dermis, where they suppress, both through IL-10 dependent and independent mechanisms, the capacity of CD4 + CD25 effector T cells to eliminate the parasite from the site [91]. One of the immunopathological consequences of active visceral leishmaniasis in humans is the suppression of T cell responses mainly to the Leishmania antigen [93]. The immune responses induced during visceral leishmaniasis in experimental data are markedly different from those induced in cutaneous leishmaniasis [94]. Furthermore, gene expression studies of tissues infected with visceral leishmaniasis reveal the modulation of the expression of genes P27, Ufm1 [85] and A2 [95]. A spectrum of clinical manifestations occurs in visceral leishmaniasis, ranging from asymptomatic or oligosymptomatic disease to progressive disease with severe manifestations such as hepatosplenomegaly, fever, pancytopenia, and hypergammaglobulinemia [96].

These particularities must have to be studied in order to permit the understanding of how different Leishmania species could promote different forms of the disease can generate such different immune responses [94].
