Leishmaniasis is a broad term that is used for a group of vector-borne diseases caused by species of protozoan parasites of the Leishmania genus of which 18 spp. are pathogenic to humans (1). The disease presents in five main clinical forms: visceral leishmaniasis (VL, or kala-azar), cutaneous leishmaniasis (CL), mucocutaneous leishmaniasis (MCL), diffuse cutaneous leishmaniasis (DCL) and post-kala-azar dermal leishmaniasis (PKDL) (2). All types of leishmaniases are transmitted to an animal or human reservoir through the bite of female infected phlebotomine sand flies, which infect a range of 70 animal species, including humans, rodents, and canids in their transmission cycle (3).

The World Health Organization (WHO) classifies leishmaniasis as a neglected tropical disease because it is directly linked to economically disadvantaged populations in tropical regions (2). A total of 700,000 to one million cases of leishmaniasis occur annually in 102 countries, areas or territories worldwide, with 20,000–30,000 deaths (3).

The high prevalence of this disease is directly influenced by the success of long host–parasite coevolutionary process in which parasites Leishmania have the ability to manipulate the vertebrate immune system in their favor, through the synthesis of parasites molecules, but also by vector saliva molecules, which are injected into the blood-feeding site during transmission (4).

The Leishmania parasites exhibit a biological digenetic life cycle with variable morphology that alternates between two main distinct developmental stages: the free-living flagellated promastigote form found in the midgut of phlebotomine sandfly vectors and the obligate intracellular aflagellated amastigotes in phagolysosomal vesicles of the vertebrate phagocytic cells, mainly into macrophages (5, 6).

During the blood feeding of the infected sandfly, which inoculates the host with metacyclic promastigotes and a large portion of the salivary content of the insect. Phlebotomine saliva is composed of pharmacologically active components with anti-hemostatic, chemotactic and immunomodulatory properties, that directly influence the parasite infection process modulating the local immune response (7). At the site of the bite occurs a rapid and intense neutrophil infiltration after inoculation, followed by monocytes/macrophages (8, 9).

Neutrophils primarily phagocytize most (80–90%) of the parasites and produce chemokines and cytokines that recruit and activate different cell types to regulate the development of the adaptive immune response during Leishmania sp. infection (8). Neutrophils are important components of the initial immune response against Leishmania parasites, even though there are currently contradictory findings on their role in the Leishmania infection.

Although the effective participation of neutrophils in the elimination of the parasite has been reported for L. braziliensis, L. amazonensis (10–15) and Leishmania donovani (16), collectively, most of these studies reported that the leishmanicidal action of neutrophils is clearly insufficient to control the establishment of infection and the development of the disease [reviewed in (17)].

Subversion of neutrophil killing functions by Leishmania is a strategy that allows parasite spreading in the host with a consequent infection evolution, transforming the primary protective role of neutrophils into a deleterious one. Neutrophils do not eliminate the parasite but act as “Trojan horses,” becoming late apoptotic and rapidly internalized by macrophages and dendritic cells, increasing the infectivity and persistence of the parasite (18, 19).

Macrophages play a dual role in Leishmania infection. These cells are responsible for the destruction of internalized parasites but also provide a safe place for Leishmania replication. Therefore, macrophages are key to disease progression and the success or failure of the infection depends on the interplay between infecting Leishmania species and the type and magnitude of the host's immune response. Both of these factors are closely related to the clinical forms of leishmaniasis (20, 21).

Macrophages are normally at rest as naïve macrophages (M0), but the microenvironment in which these cells are found provides different signals that activate them and lead to the development of functionally distinct macrophage's phenotype, toward “classically activated” (M1) or “alternatively activated” (M2) with different disease outcomes (22, 23). Therefore, the activation of M1 macrophages by Th1 lymphocyte subpopulation, which produces various cytokines, primarily interferon gamma (IFN-γ) and tumor necrosis factor-alpha (TNF-α) is crucial for the elimination of this intracellular pathogen via the triggering of an oxidative burst. The host cells increase the production of reactive oxygen species (ROS), including superoxide, hydrogen peroxide, and hydroxyl radicals, and nitric oxide (NO), which exhibit high microbicidal capacity (20, 22).

In contrast, the activation of Th2 lymphocytes, which produce IL-4 and IL-13 cytokines, induces the M2 profile caractherized by polyamine biosynthesis via activation of the enzyme arginase (arg) and production of urea and L-ornithine, which are beneficial for Leishmania intramacrophage growth favoring parasite survival in the infected macrophages and disease progression (22, 24).

Different Leishmania species trigger distinct immune responses (25). These responses are far beyond the classical Th1/Th2 paradigm (26) and increase the interest in understanding the role of M1 and M2 macrophages in the context of different Leishmania species infection. The immunomodulatory influence of the saliva of different leishmaniasis vectors should also be considered in the differential recruitment/activation of macrophages subtypes.

It is known how important macrophages are in the resistance or susceptibility to Leishmania infection, therefore we reviewed the impact of macrophage plasticity and M1 and M2 phenotypes on infection outcome. We also consider the role of vector saliva, which is a well-established immunomodulatory element in the Leishmania infection, in macrophage plasticity and phenotype.

Macrophages are phagocytic cells that are found in several tissues. In innate immunity, macrophages are responsible primarily for the control of pathogens and in adaptive immunity, this cell participates in the recognition, processing, and presentation of antigens to T cells (27). Macrophages interact with T and B cells via intercellular contact and the production of molecules and mediators to participate in the inflammatory response and tissue repair (27).

Two main macrophages phenotypes are known, M1 and M2 (28). The M1, or the classically activated macrophage, is a pro-inflammatory subtype that exhibits microbicidal properties (29). The M2, or the alternatively activated macrophage, is an anti-inflammatory/regulatory subtype that plays a role in the resolution of inflammation and tissue repair (30). The polarization of macrophages into M1 and M2 phenotypes is dependent on the signals provided by the microenvironment (28, 30, 31).

The designations M1 and M2 originated from Th1 and Th2 cytokine patterns and are associated with the change in macrophage phenotypes (32). Macrophage subtypes also differ in the production of cytokines, chemokines and other mediators and the expression of receptors, surface molecules, and transcription factors, which can act as specific markers to aid in the identification and function of these cells (27, 29, 32–34).

M1 macrophages are characterized by a high production of pro-inflammatory cytokines (TNF-α, IL-1β, IL-6, IL-12, IL-18, IL-23, and Type 1 IFN), high phagocytosis rate, and the production of reactive oxygen and nitrogen species and act to control intracellular pathogens. This macrophage subtype plays a role in tumor control, and it may be involved in autoimmune diseases and tissue damage (7, 27, 34–39).

M1 macrophages constitute the first line of defense against intracellular pathogens and induce the development of the Th1 response via IL-12 secretion (40, 41). The polarization of M1 macrophages may be primarily due to the presence of lipopolysaccharide (LPS), IFN-γ or TNF-α. Granulocyte-macrophage colony-stimulating factor (GM-CSF) may also result in the differentiation and maintenance of the M1 phenotype (41, 42). Ruan et al. (43) demonstrated that complement system activation is also related to M1 polarization.

M1 polarization activates transcription factors, such as AP1, STATs, NFκBp65 and IRFs, which lead to the expression of pro-inflammatory genes, costimulatory molecules and chemokines to attract various immune cells (Table 1) (7, 27, 34–39). IRF4 and IRF5 are involved in the polarization of M1 and M2 macrophages, but the role of IRFs in M2 polarization is not completely clear (44).

The alternatively activated macrophages, or M2, exhibit an anti-inflammatory/immunoregulatory phenotype that is related to tissue remodeling and repair, resistance to some parasites and the promotion of tumor growth (30, 45, 46). M2 was initially characterized by the expression of mannose receptor (CD206), but a range of markers and mediators produced by these cells were described, including important chemokines for the recruitment of different cells (Table 1) (37, 45, 47).

Different stimuli, such as IL-4/IL-13, IL-10, TGF-β, M-CSF, vitamin D3, and immunocomplexes, induce M2 macrophages (48, 49). Other cytokines, such as IL-21 and IL-33, may also act on macrophage polarization and maintenance to an M2 phenotype (50–52). Li et al. (52) demonstrated that IL-21 reduced the expression of CD86, iNOS, TLR-4, and IL-6 and TNF-α production via STAT3 phosphorylation. IL-33 amplifies IL-13-induced M2 polarization (50, 51).

The classification of M2 macrophages was recently expanded and subdivides these cells into four subtypes, M2a, M2b, M2c, and M2d, according to stimulus and function (23, 45). M2a macrophages are polarized by macrophage colony-stimulating factor (M-CSF) and IL-4 or IL-13. This subtype is characterized by arg-1, IL-10, and SOCS3 expression and produces CCL24, CCL17, and CCL22, which are responsible for the recruitment of eosinophils, basophils and Th2 cells. These cells are involved in allergic reactions, parasite death and encapsulation, the promotion of fibrogenesis, tissue repair and cell proliferation (53–55).

The M2b phenotype is induced by the combination of immunocomplexes with IL-1βRa/TLR ligands, apoptotic cells or LPS. These cells secrete a large amount of IL-10 and inflammatory mediators, such as TNF-α and IL-6, and express iNOS (30, 53). M2b macrophages secrete the CCL1 chemokine, which results in the infiltration of eosinophils, Th2 lymphocytes and regulatory T cells (Tregs) (56). Therefore, M2b macrophages act as an immunoregulator and trigger activation of the Th2 response (23, 53).

M2c macrophage polarization results from IL-10, TGF-β and glucocorticoids (38, 57, 58). This subtype produces IL-10, TGF-β, CXCL13, CCL16, and CCL18, which leads to the down-regulation of pro-inflammatory cytokines and an increase in the recruitment of eosinophils and naïve T cells. M2c cells express high levels of arg-1, CD163, CD206, scavenger receptors, TLR1, TLR8, FPR1, CCR2, and CCR5, and this subtype is involved in tissue regeneration and angiogenesis (53–55, 59).

The M2d macrophage phenotype is involved in the inhibition of the immune response and the promotion of angiogenesis (60). These cells are induced by IL-6, toll-like receptor (TLR) ligands and adenosine A_2A receptor agonists (60, 61). Stimulation with adenosine may result in the polarization of M1 macrophages to M2d (53, 61). This phenotype expresses high levels of IL-10, VEGF, and iNOS and secretes CCL5, CXCL10, and CXCL6 and low levels of IL-12 and TNF-α (53, 60–63).

Signals of the microenvironment are of great importance of the change in the polarization state of macrophages, and the programming from one phenotype to another is closely related to the activation of specific transcription factors and microRNAs (miRNAs) (64, 65). miRNAs are small molecules of non-coding RNAs which can act on gene expression at the post-transcriptional level (64), regulating some important transcription factors in the M1 and M2 phenotypes (66, 67).

Li et al. (67) reviewed the role of various miRNAs which participate in the regulation and polarization of macrophages in murine and human models. The miRNAs miRNA-9, miRNA-146a, miRNA-146b, miRNA-124, miRNA-181a, miRNA let-7c, miRNA-93, and miRNA-210 act to suppress the M1 and promote M2 phenotype. On the other hand, miRNA-27a, miRNA-130a, miRNA-130b, miRNA-155, miRNA-21, miRNA-19a-3p, miRNA-23a, miRNA-125a, miRNA-125b, miRNA-26a, miRNA-26b, and miRNA-720 act on transcription factors involved in the promotion of M1 phenotype and M2 suppression [reviewed in (67)].

Although we discussed above on the macrophage polarization of M0 to M1 or M2, it is known that macrophages have high plasticity and can be repolarized or reprogrammed under specific stimuli, in other words, M1 macrophages can differentiate into M2, and vice versa (40, 68). M2 macrophage may be repolarized more quickly than M1 macrophages, and this repolarization can occur through exposure of TLR ligands such as LPS and/or IFN-γ, or by expression of miRNAs such as miR-155 (69, 70). Van den Bossche et al. (71) showed that exposure of M1 macrophages to IL-4 is not able to reprogram the macrophages to M2 (71). However, the miRNAs can suppress the M1 phenotype and promote the polarization to M2 as commented above (67).

Among over 800 species of phlebotomines recorded, 98 are proven or suspected vectors of human leishmaniases; these include 42 Phlebotomus species in the Old World and 56 Lutzomyia species in the New World (all: Diptera: Psychodidae) (72). It is known that through the insect bite, vector saliva plays an important role in the establishment of Leishmania infection by increasing the infectivity of the parasite and modulating the host immune response (73, 74). Arthropod saliva contains anti-inflammatory, chemotactic and anti-hemostatic components that influence the course of parasite transmission to the host (7, 75, 76).

Most studies of the role of vector saliva in disease course were performed prior to the establishment of the M1 and M2 macrophage concept. Therefore, these works do not use this nomenclature. However, they investigated molecules that are involved in the plastic response of macrophages. These molecules are discussed below.

Some groups produced extracts, homogenates, sonicates and salivary lysates to elucidate the function of vector saliva in Leishmania transmission and infection (Table 2). We review the literature and assemble the results of different groups to provide an overview of the function of saliva.

Saliva contains molecules that induce a long-lasting erythema, which facilitates the obtaining of blood from capillaries in the host tissue (79). Vector saliva may facilitate cell recruitment via the promotion of vasodilation (79, 80, 92). The recruited cells include neutrophils, eosinophils and macrophages. Neutrophils may be attracted by the presence of several mediators, such as LTB₄, and the role of these cells as a “Trojan horse” favors the establishment of infection (80, 92). The role of eosinophils is controversial, and whether these cells promote or suppress the infection is not well known (80, 95).

Some studies observed that vector saliva increased macrophages recruitment to the site of infection because of the modulation of chemotactic factors, such as CCL2/MCP-1, CCR2 and PGE₂ (80, 92). Although some studies have shown the participation of lipid mediators such as PGE₂ and LTB₄ in cell migration (73, 92), the role of these lipid mediators acting in the polarization or recruitment of a specific profile of macrophages is uncertain, differing between cell types and models studied (28, 96–98).

Vector saliva plays an important immunomodulatory role and favors the M2 profile in different manners. Vector saliva induces IL-10 to promote a regulatory response (81, 89, 90), and it is related to the activation of a Th2 response via the increase in IL-4 and IL-6 synthesis (85, 87) Rohousov et al. (88). Besides that, the salivary components reducing the M1 related parameters, such as the pro-inflammatory cytokines IFN-γ and IL-12, iNOS and nitric oxide (NO) (85, 86, 88, 90).

However, some studies showed the action of saliva inducing M1 parameters. C57BL/6 mice immunized with plasmids encoding salivary proteins developed a Th1 response, resulting in protection against L. major infection, demonstrating that saliva may provide a protective effect and conferring characteristics for the development of a vaccine against Leishmania (90, 99). In a clinical study, was observed that individuals from an endemic area exposed to P. duboscqi bite presented high serum levels of INF-γ and decrease of IL-13, IL-5, directing a Th1 profile. Nonetheless, when PBMC of those individuals were exposed to P. duboscqi saliva, the most presented a mixed Th1/Th2 response, without a specific polarized profile (82).

In addition to complexes containing salivary components, the biological activity of pharmacological compounds isolated from vector saliva was examined. Adenosine and adenosine monophosphatase are active compounds found in P. papatasi saliva, and these factors inhibit the function of dendritic cells via a PGE₂/IL-10-dependent mechanism to promote a tolerogenic profile that is characterized by the induction of regulatory T cells, which is also related to M2 polarization (73).

Maxadilan (Max) is a vasodilator peptide isolated from the saliva of arthropod vectors, and it reduces CD80 expression, which is responsible for T cell activation, and CCR7, which is involved in the development of adaptive immunity. Max increases CD86 expression on a subpopulation of dendritic cells, which leads to a preferential Th2 type response. Max also promotes an increased production of IL-6, IL-10, and TGF-β, and reduction of the Th1 cytokines, IL-1β, IL-12p70, TNF-α, and IFN-γ (87, 93). Max treatment of L. major-infected peritoneal exudate cells increased parasite load because of Th2 polarization and decreased NO production (94). These results suggest that Max acts on M2 polarization, as demonstrated previously for total saliva.

Parasites also play an important role in vector saliva modulation because these pathogens secrete promastigote secretory gel (PSG) in the insect gut. The vector regurgitates PSG with the other salivary components at the moment of blood-feeding (77). Leishmania mexicana-PSG regurgitated by L. longipalpis exacerbates skin infection via an increase in the recruitment of macrophages to the site of infection (77). PSG also increases parasite load in vitro and in vivo via increasing arginase activity (77).

In more detail, Giraud et al. (78) demonstrated that PSG exacerbates the inflammatory phase of the early wound response (high levels of cytokines IL-1β, IL-6, IL-10, TNFα and chemokines CCL2, CCL3, CCL4, CXCL2), to induce insulin-like growth factor 1 (IGF1)-signaling and later IGF1-dependent expression of arg-1 in macrophages. As a result, the M2 macrophages promote the effective infection of the parasites in a PGS-dependent manner (78).

In this way, it is noteworthy that some works show that high levels of pro-inflammatory cytokines are induced by saliva components, leading to a Th1 profile, that can act in a host protective way. However, the most of studies inferred that the different salivary components are allied to the immunomodulatory capacity of the parasite, and these components are essential tools in the success of the infection, via the down-regulation of a pro-inflammatory response and reduction of macrophages M1, which are fundamental to parasite elimination and disease resolution. The saliva also up-regulates Th2-standard cytokines and regulatory molecules, which act in M2 polarization, facilitate the promastigote infection and increase the survival and proliferation of intracellular amastigotes (Figure 1).

The role of macrophage subsets in Leishmania infection was not investigated thoroughly. However, the fundamental role of these cells in the development of the lesion support an improved understanding of the M1 and M2 profiles as an important tool in the pathogenesis of leishmaniasis. In vitro and in vivo studies of the host response to Leishmania (Table 3) and therapeutic strategies for modulating key molecules that control cellular activity were performed. We discuss the studies of species that cause the visceral and cutaneous forms of the disease.

The collected works suggest that vector saliva plays an immunomodulatory effect on macrophages, which leads to the polarization of macrophages to an M2 phenotype. Therefore, vector saliva may contribute to an increase in the infectivity and persistence of the parasite during the initial periods of infection. Different Leishmania species exhibit virulence factors that can subvert the microbicidal mechanisms of the host to favor its proliferation because of the M2 polarization. However, M1 macrophages act during later periods of the infection and trigger an exacerbated immune response that leads to a worsening of the lesions, despite the role of these cells as powerful microbicidal agents. Therefore, a balance between the initial microbicidal response (M1) followed by a restorative response (M2) at later periods of time would provide the utmost benefit for the host. Further studies are necessary to fully elucidate the balance between these two main macrophages populations in the control of Leishmania infection.

FT-P study design, literature review, data collection and manuscript writing. BB, JA, MG, AC, and MM-S literature review, data collection and manuscript writing. IC-C and JB text correction and organization. WP study design, text correction and organization.