Infection begins when a female sand fly interacts with the skin host and injects metacyclic promastigotes into the dermis. The proportion of metacyclic promastigotes delivered by the female sand fly into the skin is responsible for the success of the infection (Giraud et al., 2019). Flies infected for more days contain more parasite burden and are able to deliver higher numbers of parasites into the skin and their infective dose is enriched by non-metacyclic promastigotes (low-quality dose) (Giraud et al., 2019). This fact influences the pathology caused by Leishmania mexicana leading to exacerbated cutaneous lesions, enriched by neutrophils, and associated with low parasite load in the skin of mammalians (Giraud et al., 2019). A high-quality dose containing low quantity of promastigotes (~100–1,000 parasites) but enriched by metacyclic promastigotes generates milder lesions with higher parasite load and is more effective in transmitting to other sand flies, showing the influence of parasite-vector relationship on the skin inflammatory response and on the final parasite load in the mammalian host (Giraud et al., 2019). Moreover, the traumatic changes caused by the contact of proboscides with skin and the disruption of the epidermis layers associated with the sand fly saliva induce the endothelial activation and neutrophil infiltration (Peters et al., 2008; Peters and Sacks, 2009). The inflammatory infiltrate generated creates a toxic environment from which promastigotes have to escape to survive. For this, promastigotes have to escape from extracellular toxic environment, enter the host cells, and convert into amastigote form. In this context, components in the sand fly saliva may help promastigotes in this issue. For example, saliva contains endonuclease, which digests Neutrophil Extracellular Traps (NETs) and inhibits blood coagulation, possibly allowing the local spread of promastigotes (Chagas et al., 2014).

Resident macrophages together with infiltrated neutrophils are the first cells to interact with Leishmania parasite upon infection. Neutrophils may exert a variety of effector mechanisms, such as phagocytosis, enzymes, and antimicrobial proteins release and NET formation (Kennedy and De Leo, 2009). Leishmania amazonensis promastigotes induce NET formation (Guimarães-Costa et al., 2009) and NETs were also demonstrated in active and chronic lesions of Tegumentary Leishmaniasis (TL) caused by Leishmania braziliensis, showing that even amastigotes may induce NET formation (Guimarães-Costa et al., 2009; Morgado et al., 2015). In in vitro system, NETs exerted restraint and toxic effects for L. amazonensis promastigotes (Guimarães-Costa et al., 2009), which could be reverted by the expression of Leishmania- 3'Nucleotidase/Nuclease activity digesting NETs and escaping killing by released NETs (Guimarães-Costa et al., 2014). In patients infected with L. braziliensis, ecto-nucleotidase activity is correlated with clinical manifestations: isolates from mucosal lesions presented higher ecto-nucleotidase activity than skin lesions (Leite et al., 2012). Furthermore, higher ecto-nucleotidase activity is also correlated with higher parasite load, and modulation of immune response by inhibiting dendritic cell activation and NO production (Leite et al., 2012). The interaction between NETs and macrophages favor the survival and persistence of parasites since it stimulates the M2 profile of macrophages, which are susceptible to L. amazonensis infection but unable to kill the parasite (Guimarães-Costa et al., 2017). Recently, L. panamensis resistant strains to miltefosine and meglumine antimoniate were described (Regli et al., 2018). These strains were able to modulate neutrophils stimulating the release of NETs and ROS production and survived better within neutrophils than the drug-susceptible strains (Regli et al., 2018). Altogether, these published data suggest that the induction of NET formation may lead to escape from extracellular effector immune mechanisms, facilitating the access of a permissive cell to parasite infection and persistence.

Still in the extracellular milieu, promastigotes have to survive from the effect of complement molecules. For this, LPG present in the parasite surface interferes with the complement cascade (Hermoso et al., 1991) and with the insertion of membrane attack complex (Puentes et al., 1990). In addition, the metallopeptidase GP63 cleaves the C3b molecule (Brittingham et al., 1995).

To enter host cells, Leishmania spp. uses manose-fucose (Akilov et al., 2007), Fc (Kima et al., 2000), fibronectin (Brittingham et al., 1999), and Toll-like receptors (Kropf et al., 2004), as well as CR1 and CR3 complement receptors (Blackwell et al., 1985; Wenzel et al., 2012). Inside the phagolysossoma, Leishmania spp have to escape from the acid environment, the action of enzymes and the microbicidal effects from oxygen and nitrogen radicals. In L. donovani experimental infection, the inhibition of phagolysosomes biogenesis was observed (Matheoud et al., 2013). As an effect of the metallopeptidase GP63 that mediates the cleavage of SNAREs, L. donovani infection prevented NADPH oxidase complex from assembling, altering the pH and phagosome degradative properties (Matheoud et al., 2013). As a result, antigen presentation via MHC-I was impaired, reducing the T cell activation (Matheoud et al., 2013).

During parasite-host macrophage interaction, Leishmania spp. infection may exert some impacts on signaling pathways leading to the inability of macrophages to kill intracellular parasites (Awasthi et al., 2003). For example, in experimental infection of BALB/c mice with Leishmania major, the CD40 signaling pathway is impaired (Awasthi et al., 2003). Thus, it results in the impairment of protein kinase C (PKCα, βI, βII, and ε) (Sudan et al., 2012) and the consequent reduction in p38MAPK phosphorylation and NOS2 expression, resulting in an impairment in killing Leishmania amastigotes (Awasthi et al., 2003). On the other hand, L. major infection enhances PKCδ, ζ, and λ isoforms, promoting ERK1/ERK2 phosphorilation, IL-10 production, and parasite growth (Sudan et al., 2012). These effects can be reverted using anisomycin, a p38MAPK activator, establishing a host-protective memory T cell response (Awasthi et al., 2003). Leishmania donovani infection differentially regulates small G-proteins: enhances N-Ras expression, whereas it inhibits K-Ras and H-Ras expression (Husein et al., 2018). It also increases extracellular signal–regulated kinase 1/2 phosphorylation and simultaneously decreases p38 phosphorylation, leading to the reduction of IL-12 and the increase of IL-10 expression (Husein et al., 2018). In macular PKDL patients, L. donovani infection decreased leucocyte rolling (L-selectin shedding) and induced up-regulation of the cellular signaling factors involved in pathogenesis (ERK1/2) as well as down regulated the signaling elements (p38 MAPK) involved in the Th1 response (Singh et al., 2018). The role of IL-10-induced immunosuppression in parasite persistence in visceral leishmaniasis and PKDL has already been described (Zijlstra, 2016; Lima et al., 2017; Bunn et al., 2018; Viana et al., 2019). Leishmania infantum subverts the host inflammatory response through the adenosine A2_A receptor by inducing CD4⁺FOXP3⁺ T cells and IL-10 expression impairing the development of Th1-type adaptive immunity and promoting the establishment of infection (Lima et al., 2017). In experimental infection with L. donovani, the absence of IL-10 resulted in the control of parasite replication, but also caused tissue damage and the rupture of splenic microarchitecture (Bunn et al., 2018). PKDL is characterized by an intermediate position between a Th2 and Th1 response: the Th2 response shows the presence and persistence of IL-10 in the skin that was already present during VL, while systemically the Th1 response that was induced after VL therapy persists with IFN-γ production (Zijlstra, 2016).

Although not observed in L. major infection (Späth et al., 2008), in L. donovani infection, the inhibition of nitric oxide production by the protein tyrosine phosphatase (SHP-1) in mice macrophages was demonstrated in vitro and in vivo (Forget et al., 2006). The authors demonstrated that Leishmania major was able to inhibit the activation of JAK2 and ERK1/2 and the transcription factors NF-kB and AP-1 since SHP-1 is a negative regulator of these transcription factors (Forget et al., 2006; Blanchette et al., 2008). In addition, infection of macrophages with L. major induces the expression of Monarch-1, a negative regulator of NF-kB activation (Fata et al., 2013). Leishmania infantum infection induces the activation of phosphatidylinositol 3-kinase/Akt and extracellular signal-regulated kinase 1/2 and promotes the cleavage of the nuclear factor-kB p65^RelA subunit by infected DCs (Neves et al., 2010). Therefore, Leishmania can inhibit microbicidal oxygen and nitrogen radicals, enabling them to escape from effector mechanisms upon phagocytosis. Some of these effects could be reversed in vitro using meglumine antimoniate, but nitric oxide production was only recovered using the drug associated with TNF-α (Muniz-Junqueira and de Paula-Coelho, 2008).

In human cutaneous leishmaniasis, the oxidative burst of monocytes is higher than that from healthy donors (Carneiro et al., 2016). However, NO alone is not sufficient to control the parasite load (Carneiro et al., 2016). This capacity to resist or interfere with NO production by macrophages depends on the Leishmania species involved (Table 1; Campos et al., 2008). Therein, L. braziliensis isolated from Mucosal Leishmaniasis patients showed the highest infection index and the lowest NO production in vitro by macrophages when compared to the infection by the other species of subgenus Viannia (Campos et al., 2008). In contrast, L. naiffi, which generally causes a benign and autoresolutive infection in humans, showed the lowest infection index and the highest NO production (Campos et al., 2008). In another study, promastigotes isolated from patients with mucosal leishmaniasis express more thiol-specific antioxidant protein, and were more resistant to nitric oxide and H₂O₂ than strains obtained from localized cutaneous leishmaniasis (Ávila et al., 2018). Leishmania amazonensis, which is capable of inducing severe diffuse cutaneous form cursing with high parasite load and inactivation of macrophages, also developed strategies to escape from oxidative effects from phagocytes. For example, L. amazonensis activates the NF-kB repressor complex p50/p50 that negatively regulates NOS2 expression interfering with NO production (Calegari-Silva et al., 2009). Leishmania donovani produces ornithine decarboxylase, which plays a role in the synthesis of tripanothione, the major reduced thiol responsible for the modulation of the immune response and pathogenesis in visceral leishmaniasis (Yadav et al., 2015). In this last study, the effects of a recombinant ornithine decarboxylase from L. donovani (r-LdODC) on the immune response of peripheral blood mononuclear cells of patients affected by visceral leishmaniasis were observed (Yadav et al., 2015). The r-LdODC induced the production of IL-10 from CD4 T cells and the reduced IL-12 and NO production (Yadav et al., 2015). The induction of proinflammatory mediators such as TNF-α and IL-6 through the transcription factor IRF-5 by L. donovani has also been demonstrated (Hammami et al., 2015). IRF-5 also limits CD8 T cell expansion and inhibits IL-12 expression, favoring the establishment of persistent infection (Hammami et al., 2015).

Leishmania spp can also interfere with immune cell-cell communication, inhibiting an efficient immune response. For example, L. major can suppress IL-2Rα expression by peripheral human T lymphocytes (Khodadadi et al., 2013). This suppression impacts the proliferation of T cells, stimulation of NK cells, and IFN-γ production, thus resulting in a failure of immune response. In this sense, L. infantum may induce MHC Class II^high DC to polarize CD4⁺ T Cells toward a non-protective T-bet⁺ IFN-g⁺IL-10⁺ phenotype, which is associated with chronicity and prolonged parasite persistence (Resende et al., 2013). Differences in the capability to modulate the effector response from monocytes by both species have also been demonstrated (Viana et al., 2018). Leishmania infantum infections lead to lower expression of TLR2, TLR9, HLA-DR and TNF-α, resulting in less control of parasite load, which may reflect upon the distinct clinical course observed in patients presenting visceral and tegumentary leishmaniasis (Viana et al., 2018).

Once the parasite converts into intracellular amastigotes, the next step is dissemination. A study comparing L. major—promastigotes and amastigotes infection of macrophages—showed that while infection with promastigotes induced inflammatory mediators, such as TNF-α and chemokines, amastigote infection was silent resulting in increased parasite load (Wenzel et al., 2012).

Sand fly saliva is composed of multiple components with a variety of functions and is able to immunomodulate macrophages (Lerner et al., 1991; Morris et al., 2001; Oliveira et al., 2008). Maxadilan, the most well-defined component from Lutzomyia longipalpis saliva, alone decreases TNF-α, IL-10 and increases IL-6, IL-8, and IL-12 production in LPS stimulated human macrophages (Costa et al., 2004). In addition, saliva from Lutzomyia longipalpis increases the anti-inflammatory mediator PGE2 and reduces the lipid leukotriene B4 (LTB4), favoring L. infantum infection in C57BL6 mice (Araújo-Santos et al., 2014). Leishmania spp. may also affect macrophage profile and functions. Infection with L. braziliensis leads to the decrease of BLT1 receptor, which recognizes LTB4 (Morato et al., 2014). LTB4-BLT1 recognition activates pro-inflammatory responses and stimulates the leishmanicidal activity of macrophages and neutrophils by inducing ROS secretion (Morato et al., 2014; Plagge and Laskay, 2017). Depending on the species, Leishmania can differently modulate macrophages. For example, infection with L. baziliensis promotes the infiltration of intermediate monocytes that express TNF and IL1β and enhance the inflammatory response in human patients (Passos et al., 2015; Santos et al., 2018). LPG from L. braziliensis was able to induce higher TNF-α, IL-6, IL-1β, and NO production by infected macrophages than LPG from L. infantum (Ibraim et al., 2013). In addition, L. braziliensis activates NF-kB, while no ERK activation by L. infantum LPG was demonstrated (Ibraim et al., 2013). In fact, the production of IL-1β by cutaneous leishmaniasis patients is associated with chronic inflammation and tissue damage (Novais et al., 2017). L. amazonensis induces HDAC1, which negatively regulates NOS2 expression contributing to the hyporeactive state of macrophages and to the replication of amastigotes (Calegari-Silva et al., 2018). Leishmania infantum is able to modulate host macrophage mitochondrial metabolism by hijacking the SIRT1-AMPK axis (Moreira et al., 2015). Parasites alter the macrophage metabolism toward aerobic glycolysis (Warburg effect) with a concomitant decrease of mitochondrial function, leading to the accumulation of intracellular ATP (Moreira et al., 2015). Consequently, an activation of AMPK is observed and leads to catabolic processes to restore intracellular energy and nutrients that can nourish the parasite (Moreira et al., 2015). Besides that, Leishmania parasites activate the PERK/eIF2α/ATF-4 pathway in cultured macrophages and infected human tissue protecting themselves from the harmful consequences of cellular stress, thus contributing to parasite survival and progression of the infection (Dias-Teixeira et al., 2017). Recently, the modulation of human macrophages by L. infantum—MicroRNA hsa-miR-346 demonstrated to play a role in regulating macrophage functions since several MHC- or interferon-associated genes are targets for this miRNA (Diotallevi et al., 2018).

Apoptosis is a process of cell death that maintains tissue homeostasis (Trahtemberg and Mevorach, 2017). Apoptotic cells present morphological and functional characteristics, such as chromatin condensation, pyknotic, and fragmented nuclei, apoptotic bodies formation, and phosphatidylserine (PS) exposition (Elmore, 2007; Trahtemberg and Mevorach, 2017). PS is recognized by regulatory receptors, such as TIM-3, which inhibits the inflammatory response (Kerr et al., 2018). As an escape mechanism, promastigotes may also inhibit the inflammatory process due to exposition of PS (Wanderley et al., 2009). Different from amastigotes whose entire population expresses PS, part of L. amazonensis promastigotes are PS⁺ and modulate inflammatory infiltrate, while PS⁻ promastigotes are able to successfully infect macrophages and survive intracellularly due to host phagocyte inactivation and reduction of NO production (Wanderley et al., 2009). Apoptotic parasites stimulate TGF-β production, silencing macrophages and favoring the survival of viable parasites (Aga et al., 2002; van Zandbergen et al., 2004). Therefore, efficient in vivo and in vitro infection occurred only when PS positive and negative promastigotes subpopulations were added simultaneously (Wanderley et al., 2009). Apoptotic-like Leishmania also uses host cell autophagy machinery (Crauwels et al., 2015). Therefore, T cell proliferation is inhibited favoring the overall population survival (Crauwels et al., 2015). Autophagy machinery plays a role during homeostasis by clearing apoptotic bodies in a process known as LC3-associated phagocytosis and promotes an anti-inflammatory response with production of IL-10 and TGF-β and hampering the production of proinflammatory cytokines, such as TNF-α, IL-1B, and IL-6 (Crauwels et al., 2015), generating a microenvironment favorable to Leishmania survival and replication.

Still at the beginning of infection, the infiltrating neutrophils rapidly respond to the infection and phagocytize the promastigotes and die generating apoptotic bodies that inhibit the response of macrophages (Afonso et al., 2008; Ribeiro-Gomes and Sacks, 2012). In established infection, apoptosis of host cells has been correlated with disease progression and increase of parasite load, suggesting the parasite can use the normal regulatory process to escape from effector mechanisms from leucocytes (Moreira et al., 2013). Leishmania major infection induces apoptosis of lymphocytes by the down-regulation of Bcl-2 and over-expression of p53 and caspase-3 (Moshrefi et al., 2017). However, how Leishmania spp may use this mechanism to evade immune response has yet to be clarified. In fact, apoptosis of inflammatory cells may occur as a result of cellular exhaustion (Liu et al., 2018). Cellular exhaustion was first described in chronic viral infection and manifested at the beginning of infection, being characterized by the expression of PD-1 receptor (Programmed Death 1) (Barber et al., 2006). PD-1 expression is induced by repeated antigenic stimulation of T and B lymphocytes (Eichbaum, 2011). The PD-ligand 1 (PD-L1) is constitutively expressed by B and T lymphocytes, macrophages and dendritic cells from spleen (Eichbaum, 2011). PD-1 activation induces apoptosis, down-regulates cellular proliferation and cytokine expression (Joshi et al., 2009). PD-1 and CTLA-4 expression by CD8 cells from peripheral blood and spleen were demonstrated in human VL (Gautam et al., 2014) and in L. donovani-infected mice (Murphy et al., 1998). The exhaustion pathway blockage with anti-CTLA-4 antibody increased IFN-γ and IL-4 expression in murine spleen and liver, as well as accelerated the development of hepatic granulomatous response associated with the reduction of parasite load (Joshi et al., 2009).

Esch et al. (2013) studied dogs with VL that showed CD4⁺PD-1⁺ and CD8⁺PD-1⁺ cells in peripheral blood. These cells presented reduced proliferation index, as wells as reduced IFN-γ expression. Blockage of the exhaustion pathway in vitro recovered the T cell effector functions, such as proliferation index and oxidative radical production. As a result, the parasite load was partially controlled by monocytes. Exhaustion of macrophages has also been demonstrated in L. donovani infection (Medina-Colorado et al., 2017). The blockage of PD-L2 was able to induce a reduction in arginase-1 and to control the parasite load; however, no alterations in IFN-γ nor NOS2 were demonstrated (Medina-Colorado et al., 2017). Despite the Th1 response induced and the IFN-γ expression detected during leishmaniasis, amastigotes are able to survive probably by using normal regulatory mechanisms developed by the immune system that prevents tissue damage and maintains the homeostasis. In this context (Kong et al., 2017), observed that IFN-γ enhanced L. donovani growth and induced the counter-regulatory molecules STAT3, IL-10, Arg1, Ido1, and Irg1 in splenic macrophages, which suggests that splenic macrophages in VL are conditioned to respond to macrophage activation signals with a counter-regulatory response, which is ineffective and even disease-promoting (Kong et al., 2017). M2 macrophages were also observed during Leishmania spp infection (Moreira et al., 2016; Hu et al., 2018; Kumar et al., 2018; Lee et al., 2018). The M2 profile has been described as a cell type that participates in the processes of cellularity reduction in the inflammatory infiltrate, tissue remodeling, and healing (Tomiotto-Pellissier et al., 2018). In this regard, it produces IL-10, TGF-b, and endothelium growth factors make a cell unable to express NOS2 and kill intracellular amastigotes, favoring Leishmania replication and persistence.

Recently, the capacity of Leishmania spp to modify its genome constitution in order to better adapt to different environments has been demonstrated for L. donovani, L. major, and L. tropica complexes (Prieto Barja et al., 2017; Bussotti et al., 2018). Aneuploidy has been described for Leishmania parasites and this phenomenon generates karyotypic fluctuations that are correlated with phenotypic variations, impacting the proliferative, and infectivity capacities (Prieto Barja et al., 2017). Leishmania clones containing beneficial haplotypes selected by the environment give rise to specific copy number variation profiles that could represent a fitness gain to new hostile environments (Bussotti et al., 2018). Therefore, Leishmania is able to change its chromosome and gene copy number to adapt to environmental changes (Prieto Barja et al., 2017; Bussotti et al., 2018). In this sense, the microenvironment generated by immune cells from the mammalian host or by anti-Leishmania drugs may constitute a bottle neck for the selection of better adapted clones, enabling the persistence of parasites. For example, the allopurinol-resistance of L. infatum strain was associated with chromosome and gene copy number variations (Yasur-Landau et al., 2018). The gene encoding the s-adenosylmethionine synthetase (METK) showed diminished copy numbers in allopurinol-resistance strains (Yasur-Landau et al., 2018). The deletion of the gene LinJ.36.2510 that encodes 24-sterol methyltransferase was associated to amphotericin B-resistance (Rastrojo et al., 2018). And whole genomic sequencing of L. mexicana strain showed an association between anphotericin B resistance and mutation of the sterol biosynthesis gene (Pountain et al., 2019). Moreover, a comparative proteomic analysis revealed the role of the iron superoxide dismutase in miltefosine resistance of L. donovani strains (Veronica et al., 2019). In another study, genomic analysis of L. infantum strains evidenced an association between deletion or mutations in LiMT and LiRos3 genes and miltefosine-resistance (Mondelaers et al., 2016). Finally, the increase of thiol levels was associated to antimony-resistance indicating that the redox metabolism has a role in the antimony-resistance of new world VL isolates (Magalhães et al., 2018).

Once the parasite is able to successfully establish growth and survival in the cells from mammalian host, clinical disease can be detected. However, a certain number of infected individuals are able to control parasite replication as well as the immune response, becoming permissive to parasite persistence. In this case they are considered infected but not sick. In this context, patients with subclinical infection with L. braziliensis may present more CD8⁺IFN-γ⁺ cells and less Cytotoxic CD8⁺ T cells than patients manifesting cutaneous leishmaniasis (Cardoso et al., 2015). However, even these subclinical patients maintain latent parasites that may reactivate in case of immunosuppression (Conceição-Silva et al., 2018). How do parasites persist in mammalian tissues? How does mammalian host contain parasite replication? Answering these questions is crucial to understand the role of both sides to survive without serious damage. Consequently, this knowledge may improve the design of new methods for diagnosis, the development of new drugs, as well as the way to construct a vaccine.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.