Elucidating the Complex Interrelationship on Early Interactions between Leishmania and Macrophages

Abstract

The host’s ability to eradicate or control infection caused by intracellular pathogens depends on early interactions between these microorganisms and host cells. These events are related to the organism’s nature and stage of development and host immune status. Pathogens are recognized by host cells, which respond to infection by either mounting an efficient response or becoming a replication niche. Early interactions between the protozoan Leishmania parasite and host cell receptors activate different signaling pathways that can result in microbe elimination or, alternatively, infection establishment and the migration of Leishmania infected cells to other host tissues. This chapter focuses on Leishmania-macrophage interaction via phagocytosis, which involves a range of parasite ligands characteristic of Leishmania species and parasite stage of development and diverse host cell receptors. We also discuss alternative Leishmania entry by cell invasion and review how Leishmania spp. survive and replicate within the phagocytic compartment they induce.

1. Introduction

Leishmaniasis is a wide-ranging group of diseases caused by different species of Leishmania parasites that result in a broad spectrum of clinical manifestations. Cutaneous leishmaniasis (CL) is characterized by skin and mucosal lesions. At the same time, Visceral leishmaniasis (VL) affects internal organs, such as the liver, spleen, and bone marrow, which can be fatal if left untreated [1]. The WHO has estimated that 30,000 new cases of VL and more than 1 million new cases of CL occur annually, with more than one billion people at risk of infection worldwide [2].

Clinical manifestations of Leishmaniasis are dependent on the infecting parasite species and host immune response [3]. The host’s ability to eradicate or control infection caused by intracellular pathogens depends on early interactions between these microorganisms and host cells. Firstly, pathogens are recognized by host cells, which either respond to infection by mounting an efficient response or becoming a replication niche. Early interactions between the protozoan Leishmania parasite and host cell receptors result in microbe elimination or infection establishment and the migration of Leishmania infected cells to other host tissues. The dissemination of infected cells containing Leishmania is crucial to parasite survival and infection in the vertebrate host [4].

Macrophages are crucial in the host response to Leishmania infection since these cells are considered the primary host cell for Leishmania parasites [5]. This chapter focuses on Leishmania–macrophage interaction via phagocytosis, which involves a range of parasite ligands characteristic of Leishmania species and parasite stage of development and diverse host cell receptors. We also discuss alternative Leishmania entry by cell invasion and how Leishmania parasites survive and replicate within the phagocytic compartment they induce and macrophage’s effector mechanisms and parasite killing.

2. General aspects of phagocytosis

Phagocytosis is a metabolism-dependent process involving the internalization of particulate material (>0.5 mm) by professional and non-professional phagocytes that can differentiate self from non-self, modified or damaged self-particles. It occurs in a series of distinct and complementary steps [6, 7]. Initially, when the phagocyte recognizes ligands of the particulate material by receptors on cellular membranes, occurs an increase in phosphatidyl bisphosphate (PIP2) levels, followed by a reduction in PIP2 mediated by the conversion of PIP2 to phosphatidyl triphosphate (PIP3) [8]. Next, PLCγ hydrolyzes PIP3 into diacylglycerol and inositol triphosphate (IP3) [9]. After the phagosome formation, maturation of this compartment by the acquisition of different proteins begins [10]. The parasite can promote changes in the kinetics of recruitment to the membrane and activation of these molecules, interfering with phagosome maturation and the microbicidal activity of macrophages [11].

The best-known receptors that induce the attachment and ingestion of different particles, opsonized or not, are those for the complement fractions (CR1 and CR3), those for the Fc region of antibodies (Fc RI, RII, and RIII), as well as the mannose and beta-glycan receptors involved in the recognition and phagocytosis of particles derived from yeast [12] or by circulating collectins and pentraxins [13]. In addition, microbial products defined by Medzhitov and Janeway in 1997 [14] as “pathogen-associated molecular patterns” (PAMPs) are recognized by pattern recognition receptors (PRRs), mainly the toll-like receptors (TLRs) [15] nod-like receptors [16] and dendritic cell receptors such as C-type lectins [17, 18]. The PRRs interplay between innate and adaptive immune responses by directly activating effector mechanisms and alerting the host organism to the presence of infectious agents, including the expression of a group of endogenous signals, such as inflammatory cytokines and chemokines [14, 19].

Like professional phagocytes, nonprofessional phagocytic cells have the machinery, cytoskeleton, and components for signal transduction necessary for phagocytosis [20]. Although intestinal epithelial cells and many nonprofessional cell lines phagocytose bacteria such as Shigella, Yersinia, and E. coli, the mechanisms involved in pathogen recognition by these nonprofessional phagocytes are less well-known than those by professional cells [21, 22]. The significant difference between the two types of phagocytes may be the presence of a broader spectrum and a more substantial number of receptors capable of performing phagocytosis on the membrane of professional cells [20, 23, 24, 25].

The molecular processes involved in the uptake of particulate material have been well studied using particles opsonized by immunoglobulins (Ig) class G (IgG) and cells expressing a receptor for the Fc region of IgGs. This interaction results in the clustering of ligand-associated receptors on the phagocytic cell surface. The signaling steps leading to IgG engulfment of opsonized particles are also well studied. These steps comprise the recruitment and activation of kinases, phosphorylation of the cytosolic portion of the receptor, and stimulation of GTPases of the Rac and Cdc42 families, which cooperate with phosphatidyl bisphosphate promoting the stabilization of WASP family proteins. In turn, the WASP protein activates the Arp2/3 complex, promoting actin filaments polymerization, followed by the emission of pseudopodia around the particle [26]. The phagocytosed agent will be internalized in a vesicle called a phagosome. The phagosome will then fuse with lysosomes, forming the phagolysosome. Phagocytosis depends on a complex network of vesicle trafficking pathways that interconnect most intracellular compartments linked to the membrane and actin cytoskeleton and requires the use of a large amount of plasma membrane for pseudopod extension around the target particle [27].

3. Phagosome biogenesis

By analogy with the flow of substances through the endocytic pathway, it is most likely that the ligands present on the surface of particles or microorganisms contribute to the determination of particle destiny within the cell [28, 29]. In addition, the particles and microorganisms’ composition present in vacuoles determines both the nature of the vacuolar contents [30, 31] and the ability of the organelles to fuse with other vesicles of the endocytic pathway [32, 33].

Newly formed phagosomes undergo a maturation process from the plasma membrane, which comprises a series of modifications, usually leading to the internalized particle’s degradation [6]. In the past, biochemical analyses have been performed on phagosomes isolated and purified at different time points after uptake by antibody-fixed and antibody-coated staphylococcus aureus present in J774 macrophages. These studies revealed that changes in the protein composition of phagosomes are similar to those already identified during endosome maturation or for compartments successively formed during the internalization of soluble components. Identical to the maturation process of endosome compartments, the protein content within phagosomes is partly recycled and sorted. These organelles, probably by fusion with pre-lysosomes, produce a final compartment that presents at the membrane lysosomal glycoproteins, mannose-6 phosphate receptors, and the ATP-dependent proton pump [29]. The maturation of phagosomes containing IgG-opsonized particles has already been well described for the recognition process. This process involves the remodeling of membranes, gaining and losing proteins, and lipid markers during their biogenesis. These steps comprise acquisition of Rab5 with the participation of Rab20 [34]; acquisition of proton pump V-ATPases, with the release of protons in the phagosomes and acidification of the intracellular medium [35]; conversion of the membrane of the initial phagosome into a late one, due to the recruitment of some proteins such as Mon1-Ccz1 by Rab5, which by the action of guanine exchange factor (GEF) recruit and activate Rab7. The formation of phagolysosomes culminates in the fusion between late phagosomes with lysosomes [36, 37].

4. Host cell and Leishmania interactions during phagocytosis - binding molecules and internalization process

The contact of promastigote forms of Leishmania spp. with different cell types, such as neutrophils, dendritic cells, and mainly macrophages, when entering the vertebrate host organism and some cells of the immune system will play an essential role in the development of the disease. The role of different cells, immune or not, at each stage of the infection remains controversial; however, it is possible to state that the parasites are mostly confined within macrophages in the chronic phase of Leishmaniasis. Regarding the cell invasion mechanism employed by the promastigote forms inoculated by the insect vector, it is believed that the main route of penetration into the host cell occurs by phagocytosis. Once phagocytosed, the parasite travels through the host cell endosomal pathway and not only survives but also replicates inside acidic parasitophorous vacuoles that fuse with host cell lysosomes [38, 39, 40].

The first studies showing the interaction of infective promastigotes with macrophages were carried out in the 70s [41] and focused on the observation of the interaction of the parasite with its host cell from the very first moments of interaction until its complete internalization, passing by all intermediate stages of parasite uptake. These studies carried out using scanning electron microscopy also showed, in a pioneering way, that the phagocytosis seems to start preferentially by the tip of the parasite’s flagellum. Although parasites could be found being phagocytosed by the cell body, the flagellum appears to be the preferred portion to trigger the internalization process. The promastigote flagellum is an anterior structure, extremely active and motile, towards which the parasite moves its body. Interestingly, and due to these morphological characteristics, there is an increasing debate proposing that the promastigote flagellum is, in fact, a sensory structure and that it is probably the first portion of the parasite to interact with host cells. When macrophages were treated with cytochalasin-D, a potent phagocytosis inhibitor, about 75% of the infection was blocked [42].

Given that 25% of the cells continued to be infected even with the drug treatment, these data showed the importance of phagocytosis as a way of invasion. Furthermore, they pointed to alternative routes of infection yet to be explored. As discussed below, we now know that these parasites can penetrate cells also through non-phagocytic pathways.

The internalization process of the Leishmania parasite is partially orchestrated like Fc receptor-dependent phagocytosis, particularly those steps involving the recognition of molecules on parasite surface by classical phagocytosis receptors or PRRs on the host cell membrane. Promastigotes have a dense glycocalyx on their surface, mainly consisting of lipophosphoglycan (LPG) molecules, which have long carbohydrate chains with repeating phosphoglycan units that are attached to the membrane by glycophosphatidylinositol (GPI) and glycoinositol phospholipid (GIPL) anchors [43]. The 63 kDa glycoprotein (gp63) is also abundant on the surface of amastigotes and exhibits proteolytic activity. Studies show that gp63 degrades immunoglobulins, complement factors, and lysosomal proteins. Thus, LPG and gp63 molecules protect the parasite from lysis by complement [44] and are involved in the interaction of the parasite with macrophages [45, 46, 47]. In addition to the direct interaction with parasite surface molecules such as LPG, gp63, and CR3 [48] the interaction of Leishmania with the macrophage can happen indirectly with receptors for complement fractions, such as CR1 and CR3, after opsonization of the parasite with C3b and C3bi, respectively [49, 50, 51, 52, 53]. Additionally, CR1 and CR3 act cooperatively with various receptors present on the macrophage surface, promoting phagocytosis of different Leishmania species [49, 52, 53, 54]. Complement receptors can interact with receptors for the Fc portion of Ig (FcR) when opsonized by IgGs [55, 56, 57]. Alternatively, the receptor for mannose and CR3 may act together to bind and internalize L. donovani [50, 58, 59]. In addition, the receptor for fibronectin [60, 61], as well as CR4 [48] and the receptor for C-reactive protein [62], may jointly participate in the phagocytosis of promastigotes of various Leishmania species, such as L. donovani, L. mexicana, and L. infantum.

In addition to classical phagocytosis receptors, PRRs also participate in the interaction of Leishmania with macrophages, being involved not in parasite internalization but rather in macrophage activation [63] and control of parasite proliferation [64] These Leishmania-macrophage interacting receptors have an influence on phagocytosis steps following ligand-receptor binding, such as actin polymerization [65] production of microbicidal molecules [66, 67], and the formation of the parasitophorous vacuole [68]. Scavenger Receptors (SRs) are PRRs that are part of a protein family with multiple transmembrane domains involved in receptor-mediated endocytosis of polyanionic ligands, including LDL (low-density lipoproteins) [69]. MARCO (Macrophage Receptor with Collagenous Structure), a specific scavenger receptor of the SR-A family, has a collagenous structure, and SRCR (Scavenger Receptor Cysteine-Rich) domain has also been implicated in infection of L. major by macrophages in vitro and in vivo [70] (Figure 1).

Figure 1.

Binding of Leishmania during parasite phagocytosis by macrophages. This image depicts the recognition step of the Leishmania internalization process by phagocytes. Before being internalized, promastigotes presenting several surface molecules, such as LPG, GPI, GIPL, and GP63, are recognized by host cell surface molecules, including CR1, CR3, CR4, Fc gamma receptors (FcγRs), fibronectin receptor, and the scavenger receptors, SRCR and MARCO. Some of these receptors, such as FcgRs and fibronectin, only recognize opsonized parasites. Alternatively, CR3 can bind to opsonized and non-opsonized Leishmania. For clarity, the opsonization process is not illustrated in this image.

5. A non-phagocytic route of invasion for Leishmania spp.

The fact that Leishmania spp. infect phagocytic cells created the perception that these parasites have a passive role in their internalization process, needing only to survive after being actively captured by the phagocyte. However, several groups have already described the infection of non-phagocytic cells in vivo and in vitro [71, 72, 73, 74]. Thus, as mentioned before, treatment with phagocytosis blocking agents did not abolish infection since a considerable percentage of parasites end up internalized in macrophages even in the presence of such blockers [41]. Taken together, these data pointed to something that remained unexplored until very recently: parasites of the genus Leishmania are capable of penetrating host cells through mechanisms that are independent of the host cell cytoskeleton, thus by non-phagocytic means. Recently, this mechanism was elucidated using non-phagocytic cells as host cells and promastigote forms of the parasite. It was clearly demonstrated that L. amazonensis promastigotes actively induce cell invasion without any cytoskeleton activity, therefore, by a mechanism distinct from phagocytosis [75]. Similar to what was observed for Trypanosoma cruzi [76], the infection involves calcium signaling, recruitment, and exocytosis of lysosomes engaged in the process of plasma membrane repair and lysosome-triggered endocytosis. Briefly, when damaged, eukaryotic cells respond to repair the plasma membrane in a sequential process that involves calcium-dependent exocytosis of lysosomes and endocytosis of plasma membrane lesions [77]. Leishmania parasites use this endocytic process to invade host cells in a cytoskeleton-independent manner. In the presence of inhibitory drugs, for example, this is likely the pathway that allows promastigotes to penetrate macrophages even if they cannot phagocytose them. The study of non-phagocytic invasion by Leishmania spp. and the role of non-phagocytic host cells in maintaining the pathogen’s life cycle is a neglected aspect of the biology of these organisms with great potential yet to be explored. It is possible, for example, that different routes of infection play different roles in the intracellular destination of parasites, impacting their replication rates and their abilities to evade the immune response and manipulate their host cells.

6. Establishment of Leishmania within host cell intracellular compartments

Phagolysosome biogenesis, also known as phagosome maturation, is a highly regulated membrane traffic process essential for pathogen intracellular fate, survival or intracellular death and degradation, and antigen processing presentation by professional phagocytes [78, 79]. This maturation process results from sequential fusion events between phagosomes and compartments of the endocytic pathway. Classical cell biology studies have revealed several aspects of the biogenesis of Leishmania-induced parasitophorous vacuoles [80, 81, 82, 83]. To date, the mechanisms that explain the differences in morphological characteristics of parasitophorous vacuoles have not been fully elucidated. However, above all, the relationship between these morphological differences and the course of infection needs further investigation.

For pathogens that live in intracellular compartments, the process of phagosome maturation does not necessarily follow the steps described for other particles. Once internalized, several microorganisms, including protozoa and bacteria, are adapted to live at least one phase of their life cycle inside host cells. It has been described at least three strategies adopted by different pathogens to evade the defense mechanisms developed by the host following infection. Some microorganisms induce the formation of vacuoles that do not acidify [84]. Other pathogens are phagocytosed and settle in acidified compartments from which they then escape to live in the cytoplasm of the host cell. The last group is formed by organisms adapted to survive in the phagolysosomes of the host cell, which is the case of Leishmania parasites [84].

After being recognized by receptors in the host cell surface, Leishmania parasites are internalized by macrophages and settle inside structures known as parasitophorous vacuoles. Once inside the parasitophorous vacuoles, the parasites transform into amastigote forms with a rounded shape without apparent flagellum. Recently, Batista et al. (2021) [85] have comprehensively revised several aspects of the dependence of host cell machinery on the biogenesis of Leishmania- and T. cruzi-induced parasitophorous vacuoles. It is well known that Leishmania-induced parasitophorous vacuoles are acidic compartments containing lysosomal enzymes that have access to soluble markers of the endocytic pathway [5, 86, 87]. Indeed, these compartments are bounded by a membrane enriched by late endosome and lysosome markers, including Rab 7, macrosialin, LAMP-1, LAMP-2, vacuolar ATPase, and MHC class II molecules [88, 89, 90] (Figure 2). These data support the hypothesis that Leishmania-induced parasitophorous vacuoles exhibit characteristics of phagolysosomes [83, 90]. In addition, several other biomolecules, including lipids, proteins, and sialoglycoproteins, are exchanged by parasites and host cells following contact. One example is the identification of endoplasmic reticulum (ER) markers in the early steps of Leishmania-induced parasitophorous vacuole formation, indicating the participation of ER during phagosome membrane formation [91, 92].

Figure 2.

Model of the parasitophorous vacuole composition induced by Leishmania. This image illustrates a representative parasitophorous vacuole induced by Leishmania in macrophages. These compartments generated by Leishmania interact with the endocytic pathway and endoplasmic reticulum compartments. These compartments acquire by fusion markers from the late endosome and lysosome vesicles, such as Rab 7, macrosialin, LAMP-1, LAMP-2, vacuolar ATPase, Nramp1, SLC38A9, and MHC class II molecules. In addition, these compartments also acquire some molecules from host cell membrane, the integral membrane protein CD36, and the secretory pathway, including calnexin, SEC22b, and syntaxin 5.

Despite similar characteristics among Leishmania-induced vacuoles, the kinetics of their formation [89, 93] and the morphology of these parasite-containing compartments vary depending on the species and growth stage of Leishmania. It has been shown that, after phagocytosis of promastigotes of [90] L. donovani orL. major, the parasites localize in transient phagosomes with little ability to fuse with late endosomes [89]. This delay probably favors promastigote differentiation into amastigote forms if parasites settled within an acidic lysosomal enzyme-rich parasitophorous vacuole induced by L. donovani. Another possible mechanism involved in maturation delay by L. donovani-induced parasitophorous vacuole is the upregulation of Rab5a, an early endosome protein, along with the recruitment of its effector protein EEA1 [94]. In contrast, Rab5a and EEA1 are early recruited to L. amazonensis parasitophorous vacuole and then rapidly exchanged by late endosome and lysosome markers [95, 96]. On the other hand, after internalization, amastigotes of L. donovani, L. amazonensis, and L. mexicana are found in compartments that rapidly fuse with late compartments of the endocytic pathway, an environment in which parasites seem to be resistant [93, 97]. Another difference found to exist among parasite-induced parasitophorous vacuoles regards to their size. Despite L. major, L. infantum, and L. braziliensis living in tight vacuoles containing only one parasite inside [83], parasites from the Mexican complex are phagocytosed and settle in tight vacuoles that increase in size and become large parasitophorous vacuoles with a high number of parasites [80, 98, 99] (Figure 3). The usual large size of these parasitophorous vacuoles seems to be dependent on host cell factors, including lysosomal traffic regulator LYST/Beige [100] CD36 receptor [101] and V-ATPase subunit d isoform 2 (ATP6V0d2) [102] as recently revised by Bahia et al., 2021 [85]. Interestingly, the large size is shown to be related to infection success because in host cells lacking CD36 receptors, parasitophorous vacuoles are small in size, and parasite multiplication is impaired [101].

Figure 3.

Model of the parasitophorous vacuole induced by different Leishmania species. Shown here a representative image of a tight parasitophorous vacuoles containing only one parasite inside induced by L. major and L. donovani, and a large parasitophorous vacuole with a high number of parasites induced by L. amazonensis and L. mexicana.

7. Macrophage effector mechanisms, parasite replication and infection amplification

Once internalized within the parasitophorous vacuoles, the amastigotes multiply by binary fission, facilitating infection amplification, and persistence in the mammalian host (Figure 4). It is commonly assumed that amastigotes are released after host cell burst, which could be occasioned by the burden imposed by the unrestrained replication of the parasite. However, this is still an unproven hypothesis. Thus, the process of infection amplification during leishmaniasis is still a black box. Therefore, our knowledge about the mechanisms that lead to infection amplification during Leishmania infection is, in fact, quite limited. However, some crucial clues come from important observations made from in vivo experiments depicting the very first moments of the infectious process using mice and promastigotes of Leishmania major naturally transmitted to the mammalian model through the infective bite of the insect vector [38]. These experiments showed that just after promastigote inoculation, neutrophils are the first immune cells to reach the site of infection and phagocytose the parasite. It has been also demonstrated that the parasite could modulate neutrophil viability, which may delay or accelerate the host cell death through apoptosis [103, 104]. The modulation of neutrophil viability helps the survival of the parasites until their final targets and the macrophages. These cells reach the site of infection being attracted by different chemotactic factors such as MIP 1β, which stimulates the phagocytosis of infected apoptotic bodies [104]. Together with the macrophage recruitment, the inhibition of IL-12 secretion and the high secretion of IL-10 and TGF-β generate an anti-inflammatory environment, favoring the parasite’s survival. This process of initial neutrophil invasion followed by phagocytosis of these infected and apoptotic cells by macrophages is known as the “Trojan horse” mechanism [105, 106]. From all this, at least one major conclusion can be made: macrophages do not need to be initially infected by the promastigotes inoculated by the vector. They can instead get infected by the ingestion of apoptotic cells or apoptotic cell bodies containing viable amastigotes previously internalized within another cell type. This would be an interesting strategy for the parasite because apoptotic mechanisms do not trigger inflammation, as it would lead to a silent infection of macrophages, favoring parasite replication.

Figure 4.

Human macrophages infected by L. amazonensis. Cells were infected in vitro using parasites expressing a red fluorescent protein, host cell cytoskeleton (F-actin - green) was labeled by Alexa 488-conjugated phalloidin (life technologies), and nuclei were labeled by DAPI (blue). The images show macrophages harboring L. amazonensis amastigote forms (red). Original images kindly provided by Prof. Jane Lima-Santos from Universidade Estadual de Santa Cruz, Bahia, Brazil.

As mentioned earlier, cell disruption with the release of amastigotes into the extracellular environment has never been demonstrated despite being often assumed to be a mechanism of amastigote spread. On the other hand, it is entirely plausible to hypothesize that the amplification of infection in leishmaniasis occurs by the ingestion of apoptotic bodies of dead infected macrophages by new macrophages. This is even more logical if we consider that the clearance of dead cells is one of the leading roles these phagocytes play in tissue remodeling. This lack of knowledge about whether amastigotes are released extracellularly is also reflected in the few studies approaching cell invasion by free amastigotes [107, 108, 109].

Still, it is necessary to emphasize that other mechanisms have also been proposed regarding the infection amplification process. Leishmania amastigotes can be transferred cell to cell, from macrophage to macrophage, as already observed in in vitro studies [110]. Furthermore, other studies have shown that amastigotes can also be released by exocytosis of the parasite after the fusion of the parasitophorous vacuole membrane with host cell plasma membrane, thus without cell rupture [72]. Hence, several possibilities explain the spread of amastigotes and the amplification of infection during leishmaniasis, which do not involve the rupture of the parasitized cell. It is entirely plausible to hypothesize that these silent and non-necrotic pathways of infection amplification, without cell leakage or release of free amastigotes in the extracellular environment (which could be quickly cleared by immune effectors, such as the complement system) have been evolutionarily selected and are responsible for the susceptibility of macrophages to the infection and the amazing ability of Leishmania spp. to survive within the cell type that was supposed to kill them.

The dissemination of infected cells containing Leishmania is critical to parasite survival and the establishment of infection in the vertebrate host. Thus, the ability of Leishmania-infected host cells to migrate may be necessary for lesion distribution on the host and the dissemination of disease. As the main host cells for Leishmania, macrophages are crucial for the establishment of infection. However, the role played by these cells in parasite homing to specific tissues and how parasites modulate macrophage function is still poorly understood. Previously published work has shown that infection with Leishmania modulates phagocyte functions associated with cell migration [111, 112, 113, 114]. Some of these studies have shown that infection with different Leishmania species reduces macrophage adhesion, which could facilitate parasite dissemination in vivo [113, 114, 115]. However, other authors have demonstrated that infection with Leishmania impairs the ability of macrophages to migrate [3, 111]. In contrast with previous work, it has been shown that the modulation induced by Leishmania could depend on parasite species [116]. Thus, the modulation of macrophage migration induced by Leishmania, as well as impacts on parasite dissemination, remains unelucidated.

In leishmaniasis, macrophages function as a replicative niche for Leishmania parasites and work as anti-leishmanial effector cells, as immunoregulators, and as permissive host cells for the long-term survival of persistent parasites [117]. Parasite recognition by macrophages and priming by cytokines, such as IFN-γ, lead to the activation of the macrophage’s microbicide machinery and production of reactive species, especially superoxide by the multimeric enzyme NADPH oxidase and nitric oxide (NO) by inducible nitric oxide synthase (iNOS) [118]. These critical molecules are known to be involved in the macrophage-mediated innate host defense against Leishmania parasites. Previously published studies have demonstrated that the killing of different Leishmania species (e.g., L. major, L. donovani, L. braziliensis) in vitro depended on ROS production. In addition, several studies in mouse models have provided convincing evidence for the crucial role of NO in Leishmania parasite killing [39, 117]. However, although IFN-γ has been recognized as the key activator of the leishmanicidal activity of human macrophages, the importance of both iNOS expression and NO production remains controversial in humans [119, 120, 121]. Thus, to survive within the hostile environment of macrophages, Leishmania parasites have evolved different strategies to circumvent the antimicrobial mechanisms developed by these cells.

8. Conclusions

Undoubtedly, Leishmania spp. parasites have evolved a series of adaptations to be captured by macrophages and, instead of succumbing after internalization, they not only survive in the endocytic pathway but also replicate within the intracellular compartment where several other parasites would meet death [122]. The mechanism involved in parasite survival within macrophages and their dissemination in the vertebrate host is still poorly understood. Thus, further studies to dissect the mechanisms involved in the early steps of Leishmania-macrophage interaction are critical for the full understanding of Leishmania pathogenesis.

Acknowledgments

This work was supported by grants from the Bahia State Research Support Foundation (FAPESB) and FAPEMIG; PSTV holds a grant (305235/2019-2) from National Council for Scientific and Technological Development (CNPq). PSTV and JPBMF are professors from PGPAT and PGBSMI financed by Higher Education Personnel Council–Brazil (CAPES)—Finance Code 001.

We thank Prof. Jane Lima-Santos for kindly providing the infected macrophage image.

Conflict of interest

The authors deny the existence of any conflict of interest.