Protozoan parasites of the genus Leishmania are the causative agents of leishmaniasis, a spectrum of vector-borne neglected diseases affecting over 12 million people worldwide with growing geographical extension (1, 2). The clinical manifestations of leishmaniasis differ widely, depending principally on the causative species. An estimated 20 different Leishmania spp. cause the three main clinical disease manifestations: cutaneous, mucocutaneous, and visceral leishmaniasis (3). Among these, the visceral form is fatal if not treated. Most cutaneous leishmaniasis (CL) and visceral leishmaniasis (VL) patients develop long-term protective immunity after cure from the infection, indicating the feasibility of developing an effective prophylactic strategy against leishmaniasis (4, 5). However, no vaccine is currently available against human leishmaniasis.

Leishmania is a digenetic parasite, whose life cycle includes two hosts: the insect vector, and a vertebrate host. Following the bite of an infected sand fly, the obligate intracellular parasite Leishmania rapidly infects its host cells (2, 6). A better understanding of the spectrum of immune responses following infection with Leishmania parasites is required to develop more effective preventative approaches, as the host immune response is a key determinant of the outcome of leishmaniasis (7). Following inoculation, innate cells such as neutrophils, inflammatory monocytes, macrophages, dendritic cells (DCs), mast cells, and natural killer (NK) cells create either a permissive or hostile environment for the parasite through their effector functions, and subsequently modulate the adaptive immune responses towards host susceptibility or resistance. Although the adaptive branch of the immune response is crucial to control leishmaniasis, it has been extensively reviewed elsewhere and it is out of the scope of this review. Specifically, we focus on the innate immune responses in visceral leishmaniasis due to its significant mortality (7, 8).

Leishmania parasites have evolved highly successful strategies to evade the microbicidal activity of neutrophils, to prime infected macrophages towards an anti-inflammatory/alternative phenotype, and to undermine the Th1 polarizing functions of DCs, thereby attenuating the host protective adaptive immune responses (9–11). These altered immunological characteristics of the host cells upon infection are often induced by changes in the host metabolic pathways. Thus, there has been an emerging interest in profiling host metabolomics in the context of immune regulation, and especially to understand how metabolites and metabolic processes such as oxidative metabolism, glycolysis, and glutaminolysis can influence immune cell proliferation, differentiation and effector functions (12, 13). For instance, in pathogen-associated molecular pattern (PAMP)-activated neutrophils, mast cells, and DCs, glycolysis acts as a metabolic effector response that fuels reactive oxygen species (ROS) production, degranulation, and antigen presentation, respectively (13). Furthermore, changes in the metabolomic profile of infected immune cells can alter nutrient availability to the parasite, rendering the intracellular environment either permissive or hostile thus determining the anti-leishmanial activities (14). Leishmania parasites can also alter the metabolic pathways of the host cell to their advantage, for example by influencing nutrient uptake (15). It is hypothesized these metabolomic alterations can protect the parasite from elevated temperatures, low pH, and ROS in the parasitophorous vacuole (PV), leading to enhanced survival inside the host cell (15).

Increasing evidence points to a crucial role of innate immune cells in determining the outcome of the infection. This review particularly attempts to highlight the role of the innate immune cells, the metabolomic and epigenetic reprogramming, and immune regulation during VL, and discuss their implications with respect to the development of a vaccine against VL.

Monocytes, along with neutrophils, are the earliest immune cell populations to be recruited after infection of Leishmania (57) ( Figure 2 ). Recruitment of monocytes is modulated by pro-inflammatory cytokines and chemokines after infection with different Leishmania species. Monocyte subpopulations exist within a phenotypical spectrum ranging from long-lived patrolling monocytes (non-classical) that mainly monitor the vasculature as part of normal physiology (58) to short-lived inflammatory (classical) monocytes (iMOs, CD11b+ CX3CR1^lo Ly6C^hi CCR2+ CD115+) (59, 60). During the bloodmeal, Leishmania is injected along with sand fly salivary components, which modulate inflammatory responses that lead to CCR2-mediated chemotaxis of iMOs to the infection site (61), reviewed in (62, 63). iMOs play contradictory roles in different infections: while they mediate host defense against toxoplasmosis (64) and malaria (65), a detrimental role in VL was demonstrated (51). Monocytes can recognize Leishmania parasites via Toll-like receptors (TLR)s and produce pro- and anti-inflammatory cytokines that can play paradoxical roles during leishmaniasis (66–69) ( Figure 2 ). The expression of TLRs and the molecules on the parasite surface could also generate a diverse range of monocytic responses during infection with different Leishmania species. For instance, L. donovani infection of monocytes inhibits oxidative burst and antigen presentation and activates IL-10 expression via downregulation of TLR2 and TLR4 signaling (70, 71). In contrast, during L. tropica infection, monocytes showed higher expression of TLR9 in addition to TLR2/TLR4, and elevated TNF-α that resulted in a decrease of inducible nitric oxide synthase (72). Furthermore, L. braziliensis infection of human monocytes showed enhanced production of TNF-α and higher TNF-α/IL-10 ratios, and controlled parasite burdens more effectively compared to L. infantum infection (66). In addition to their role in pathogenesis, monocytes play an important role in granuloma formation, necessary for parasite clearance in murine models of L. donovani infection. However, an IL-17^-/- mouse model of L. donovani infection showed diminished monocyte and neutrophil infiltration resulting in smaller isolated granulomas in the liver and spleen, yet controlled parasite burden due to elevated IFN-γ production (73).

Studies of active VL patients in Brazil and India showed an increase of CD14⁺ monocytes, and elevated expression of IL-10, which affects macrophage polarization and the outcome of the disease (74, 75). Other studies with Indian VL patients reported an anti-inflammatory (non-classical) monocyte response in the peripheral blood characterized by reduced expression of TLR2 and TLR4, chemokine receptors and adhesion molecules, as well as impaired phagocytosis and oxidative burst, important for the elimination of Leishmania parasites (70, 76). These observations illustrate the critical role of monocytes in the early modulation of host immune responses and the clinical outcome of L. donovani infection. The divergent engagement of TLRs and associated immune responses following infection of monocytes with different Leishmania species suggests that targeting these interactions could be exploited in the development of effective anti-parasitic strategies.

Macrophages are considered the canonical hosts for Leishmania in the later stages of infection, following phagocytosis of Leishmania-infected apoptotic neutrophils ( Figure 3 ) (95). Neutrophils provide the chemical cues (MCP-1/CCL2, MIP-1α/CCL3, MIP-1β/CCL4 and MIP-2/CXCL2) that help recruit macrophages to the infection site (96). In addition, salivary components induce a significant influx of macrophages in a CCL2/MCP-1 dependent mechanism in L. chagasi infection (97). Macrophage response to infection is dependent on the interplay of a) the surface molecules on the Leishmania parasites, such as glycoprotein-63 (gp63), lipophosphoglycan (LPG) and glycosylphosphatidylinositol (GPI); and b) the receptors which recognize them, such as Toll-like receptors (TLRs) (98) fibronectin, mannose, complement and Fc receptors ( Figure 3 ) (99). Binding of Leishmania to complement and Fc receptors slows down phagosome maturation (100) and induces IL-10 production (99, 101, 102). TLRs recognize Leishmania surface gp63 and activate the NF-κB pathway in macrophages ( Figure 3 ) (103). Interestingly, Leishmania uses gp63 to negatively regulate ROS production (104) and is shown to be critical for Leishmania-mediated inhibition of NLRP3 inflammasome and attenuating IL-1b secretion (105, 106). Macrophages play a dual role in the infection, as they not only serve as permissive hosts, but are also as anti-Leishmania effector cells (107). These contrasting roles depend on the microenvironmental signals in the host tissue (108) in response to different Leishmania species and on the differential recognition of the parasites. Depending on the stimulus, infected macrophages can polarize towards a functional phenotype within the spectrum ranging from inflammatory macrophages (also called classically activated or M1) and anti-inflammatory macrophages (also called alternatively activated of M2, Figure 3 ). Macrophage polarization can determine the outcome of the disease (37, 109–111). As discussed in the previous section, iMOs usually mature into M1 macrophages, while anti-inflammatory monocytes are more likely to differentiate into M2 macrophages (59, 112). Some of the parasite factors, such as L. infantum-derived lipid mediators, can also induce M2 phenotype by preventing IFN-γ-mediated M1 polarization (113). Functionally, macrophages classified within the M1 and M2 spectrum show distinct activities. The relationship between the macrophage phenotype and the immune milieu during Leishmania infection is discussed in more detail elsewhere (107). It is important to note that the phenotypic spectrum of macrophages is quite dynamic, and this plasticity confers a wide range of distinct phenotypic characteristics. However, for the purpose of this review we refer to the pro-inflammatory and anti-inflammatory ends of the spectrum as M1 and M2, respectively, as has been previously established in Leishmania immunology.

Dendritic cells (DCs) are professional antigen-presenting cells (APCs), are ubiquitous in the peripheral tissues, and perform sentinel functions ( Figure 4 ). Several distinct subsets of DCs (plasmacytoid, conventional, monocyte derived and more) have been identified based on their functional specialization and expression of distinct markers (159). Similar to other phagocytic cells, DCs can take up antigens via Fc receptors, C-type lectin receptors (CLRs), and pattern recognition receptors such as TLRs ( Figure 4 ) (160). Recognition of Leishmania parasites by DCs is accomplished via TLR-2, -4, and -9 (161). TLR-9 has been identified as being responsible for DC activation and production of neutrophil chemo-attractants and IL-12 secretion in L. infantum infection in C57BL/6 mice (162). Following infection, DCs undergo maturation and express MHCII, CD80, CD86, CD40 and migrate to lymphoid tissues where they present antigens to naïve T cells ( Figure 4 ) (10). DCs are the main producers of IL-12, which is critical for polarization of naïve T cells into IFN-γ producing Th1 cells (163). L. donovani infected DCs start producing IL-12, IL-23 and IL-27 within 5 hours of infection, IL-12 being mostly produced in CD8α⁺ DC subsets (164). In the chronic stage of L. donovani infection, CD11c⁺ splenic DCs showed a functional impairment, correlated with reduced surface expression of MHC-II and impaired IL-12 production (165). Similarly, migration of splenic DC into marginal zones is regulated by the CCR7 ligands CCL19/CCL21, which also affect the ability of DCs to produce IL-12 (166).

Mast cells (MCs) have been shown to participate in the innate immune responses in CL and VL (8, 186). MCs participate in the initiation and orchestration of innate and adaptive defense to pathogens and in various inflammatory responses (187, 188). MCs are present in large numbers in the skin, predominantly in the superficial dermis, the site where Leishmania parasites are deposited after the bite of infected sand flies (189, 190).

Literature on MCs with respect to visceral infections of Leishmania is sparse. Much of our understanding of the role of MCs in Leishmania is derived from studies of CL. MC-derived TNF-α followed by neutrophil influx and MIP-1α/β release is required for the recruitment of macrophages during cutaneous granuloma formation, a hallmark of parasite-induced inflammatory responses (191). Indeed, MCs recruit effector cells of innate and adaptive immunity to sites of L. major infection and induce systemic protective dendritic cell (DC)-dependent adaptive immune responses that eventually control L. major infection. Differential MC infiltration patterns and responses have been reported depending on the mouse strains (8) and Leishmania species (186). For example, there was a significant uptake and killing of L. tropica by MCs compared to L. donovani. Interactions of MCs with both these Leishmania species ensues the release of MC extracellular traps (MCETs) analogous to NETs produced by neutrophils ( Figure 1 ). Although both L. donovani and L. tropica seem susceptible to MCETs, relatively higher number of viable promastigotes of L. donovani in comparison to those of L. tropica are observed indicating the relative resistance of L. donovani parasites. Thus, MCs play a very important role in early innate immune response to L. tropica and L. donovani and thus may play an important role in the success of vaccines against Leishmaniasis (186). Indeed, it has been hypothesized that MC-induced control of L. major infections is not only restricted to the induction of local inflammation, but that MC recruitment of pro-inflammatory cells (i.e. DCs) to sites of L. major inoculation ensures the development of protective, long-lasting memory responses against L. major (192), suggesting the relevance of MCs in vaccine immunity. Interestingly, vaccination with LdCen ^-/- parasites against virulent L. mexicana challenge resulted in reduced infiltration or absence of degranulated MCs, which correlated with protection (193). MCs may have a role in Leishmania vaccination, similar to that observed in BCG vaccination, where MCs phagocytize BCG, produce ROS, and release MCETs (194). More studies in experimental models and in humans are required to understand the immune mechanisms that determine MCs involvement in Leishmania vaccine-induced immunity. MCs-derived molecules have shown potent adjuvant activity in certain vaccines [Reviewed in (195, 196)]. In Toxoplasma gondii infection, the treatment with two different MCs-derived molecules, C48/80 or cromoglycate, can either increase or decrease the parasite burden by differentially favoring Th1 or Th2 responses, respectively (197). Similar results have also been observed in a Trypanosoma cruzi murine model, where cromoglycate administration led to an increase of parasitic burden (198). Administration of other MCs-derived molecules like tryptase, resulted in the cleavage of many Th2 cytokines, and consequent Th1 polarization (199). Similar studies in Leishmania exploring the role of MC-derived molecules as potential immunomodulators remain to be undertaken.

Natural killer (NK) cells, identified as CD3−CD56+ in humans, are characterized by their cytotoxic activity and cytokine production (200), and have been shown to participate in the immune response against Leishmania ( Figure 1 ). VL patients show three different NK subsets: CD56–CD161+, CD56+CD161–, and CD56+CD161+, with a loss of the CD56+CD161+ population in comparison to the healthy individuals (201). TLR-2 recognition of Leishmania LPG activates NK cells and induces the production of IFN-γ and TNF-α and the translocation of NF-κB to the nucleus. This activation seems to be more intense in response to metacyclic promastigotes, compared to procyclic promastigotes (202). IFN-γ production and NK cytotoxicity is also dependent on TLR-9 (203). Interestingly, the downregulation of STAT-1 related to the reduction in IFN-γ, TNF-α and TLR2 expression by NK cells allows the development of diffuse cutaneous leishmaniasis (DCL), a form of the disease characterized by the uncontrolled spread of the parasites (204). Moreover, lesion healing of DCL patients corelates with the increase of CD16+ CD56+ NK cells (205). Despite their important role, NK cells are not necessary for the establishment of an effective Th1 response against L. major (206), or L. tropica, whereas they are indispensable for the elimination of L. donovani amastigotes (207). A more detailed appraisal of the role of NK cells during leishmaniasis can be found in (208). Recent studies have identified NK cells to play a role in vaccine-mediated immunity. Laabs, et al. have shown that vaccination with live L. major combined with CpG DNA leads to DC and NK activation, as well as increased IFN-γ production by NK cells (209). Interestingly, NK cells have also been shown to acquire trained immunity, in the context of BCG vaccination. However, the exact epigenetic and metabolic reprogramming underlying this phenomenon remains to be fully elucidated (210, 211). Similar to other innate cell types, the role of trained immunity in NK cells needs to be further investigated in the context of Leishmania infection and vaccination.

There are few reports about the role of NK-T cells (NKTs) in leishmaniasis. NKTs are CD1d-restricted T cells with innate and adaptive properties, which react to a variety of stress proteins and glycolipids using either NK or T cell effector mechanisms (212). Glycoconjugates on the Leishmania surface are detected by CD1d+ NKT cells, which are protective against leishmaniasis in early stages of VL (213). CD8+ NKT cells are also protective, express IFN-γ and Killer cell immunoglobulin-like receptors (KIRs) and do not migrate towards the L. donovani infection site, whereas the CD4+ NKTs are found to be pathogenic as they migrate towards the infection site and express CD25, FoxP3 and IL-10 (214). In comparison to other immune cells, literature on the role of NK and NKT cells remains limited in VL. They may yet play important roles in protective immunity in anti-leishmania vaccines analogous to the limited studies in BCG vaccination that revealed potent trained immunity that contributed to protection.

The immune response to Leishmania infection is primarily initiated by innate immune cells which orchestrate the generation of protective innate and adaptive immunity against Leishmania parasites. Innate immune cells specialize in the clearance of invading pathogens. Leishmania parasites have evolved a range of evasion strategies to subvert normal innate cell function. While the role of the innate immune cells in host protection during Leishmania infection are being explored, important questions regarding their roles in shaping the protective immunity in a prophylactic vaccine setting remain to be investigated. Furthermore, chronic infections with Leishmania parasites have been shown to induce prominent changes in host metabolomic pathways that influence immune cell proliferation, differentiation, and effector functions. Current advances in metabolomics have made significant impact and present important implications in the management of VL. Metabolomic profiling during VL may reveal novel immune regulation networks that can be exploited for vaccine development. Additionally, while metabolic changes in different Leishmania species are well studied and could serve as biomarkers of virulence and disease progression, what would be a desirable metabolic profile for innate immune cells during vaccination remains to be investigated. In conclusion, innate immune cells are critical for the development protective immunity due to their profound role in shaping adaptive immunity. Furthermore, innate cells themselves may undergo epigenetic and metabolic changes that renders them to acquire trained immunity, an emerging concept that has been studied in various vaccines. Such studies of metabolomic and epigenetic changes of innate immune cells will benefit the development of efficacious vaccines against VL.

GV, TPF, PB, TO, and SG drafted the article. ARS and HLN conceived the theme of the article. HLN and SG reviewed the draft. All authors contributed to the article and approved the submitted version.

This work is supported by NIH/NIAID grants R21 AI130485 02, RO3 AI144253 01 (ARS), Global Health Innovative Technology Fund grants G2018-201 and G2019-213 (ARS, HLN) and by intramural funding from FDA.

Contributions by the FDA authors are an informal communication and represent their best judgment. These comments do not bind or obligate FDA.

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.

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