Exploring the Role of Medicinal Plant-Based Immunomodulators for Effective Therapy of Leishmaniasis

Leishmaniasis is a pestilent affliction that importunately needs better therapeutics necessitated by the absence of effective vaccine, emergence as HIV co-infection, and the dread of debilitating chemotherapy. The Leishmania parasites incapacitate host macrophages by preventing the formation of phagolysosomes, impeding antigen presentation to T cells, leading to suppression of cell-mediated immunity. An ideal approach to cure leishmaniasis includes administration of antileishmanial compounds that can concomitantly establish an effective Th1 response via restoration of requisite signaling between macrophages and T cells, for subsequent activation of macrophages to eliminate intracellular amastigotes. Plants have provided an opulent treasure of biomolecules that have fueled the discovery of antileishmanial drugs. Modulation of immune functions using medicinal plants and their products has emerged as an effective therapeutic strategy. Herein, we review the plant extracts and natural products that have resulted in therapeutic polarization of host immunity to cure leishmaniasis. These immunostimulatory phytochemicals as source of potential antileishmanials may provide new strategies to combat leishmaniasis, alone or as adjunct modality.


INTRODUCTION
Leishmaniasis is a neglected; usually poverty-associated complex vector-borne disease that is caused by more than 21 species of parasites belonging to the order Kinetoplastida and family Trypanosomatidae. The disease manifests into different clinical indexes depending on the parasite tropism and ranges from selfhealing cutaneous lesions, to malign mucocutaneous leishmaniasis and fatal visceral manifestations. The disease is endemic in 98 countries, where 10-12 million people are afflicted worldwide with 1.5-2.5 million new cases, death toll of 70,000 and 350 million at risk of developing the infection (1)(2)(3). Since Leishmania parasites reside and multiply within the parasitophorous vacuoles of macrophages, failure of host immunity to contain the infection results in immunosuppression. Thus, the host becomes susceptible to various secondary infections including HIV (4,5).
The antiquated therapeutic modalities for leishmaniasis are crippled because of variable efficacy, drug resistance, and pronounced side effects. Even the known antileishmanial drugs such as amphotericin B (AmB), sodium stibogluconate (SSG), and miltefosine exert their antileishmanial effect via host immunomodulation [(6) and references therein]. Modulation of the host immune response via generation of antileishmanial vaccine would certainly be a propitious step in leishmaniasis control, but is impeded by the digenetic life cycle of Leishmania, and antigenic diversity among different Leishmania species, making prospects of a cross-protective vaccine a distant future (7,8). Thus, in the absence of any vaccine, a quintessential approach to control leishmaniasis shall be based on discovery of drugs from alternative sources that can directly kill the parasite as well as activate sentinels of immune system for clearance of the pathogen. Herein, we further elaborate the mechanisms employed by Leishmania parasites to evade host immune responses, lacunae in current chemotherapy and discuss potential role of natural immunomodulators in antileishmanial therapy.

MODULATION OF NEUTROPHIL FUNCTIONS
Within 24 h of Leishmania infection, neutrophils are recruited to the site of infection, serving as early and transitory host to Leishmania promastigotes (9). Although exact mechanisms underlying the recruitment of neutrophils remain unclear, the role of both parasites or vector-derived molecules is speculated (9,10). The role of neutrophils in Leishmania infection is variable, Leishmania species specific, and also depends on the host genetics (11)(12)(13)(14). Although neutrophils are responsible for early containment of different Leishmania species (15)(16)(17)(18), they play equally pivotal role in harboring the parasites till they reach their evolutionary destined host cells, i.e., macrophages. Leishmania promastigotes deviously modulate neutrophil phagocytic functions in more than one way. Internalized Leishmania promastigotes block the production of CXC-chemokine interferon gamma (IFN-γ) inducible protein-10, which results in decreased recruitment and activation of natural killer (NK) cells and Th1 cells (10). Leishmania donovani promastigotes have been shown to induce NETs (fibrous traps of DNA, histones, and proteins) in which they get trapped but escape their microbicidal activity by aid of lipophosphogylcan (LPG) (19). Leishmania parasites also extend the life span of neutrophils and delay their apoptosis by various mechanisms (20,21). Since ingestion of apoptotic neutrophils by macrophages does not trigger macrophage microbicidal defenses, it creates safe passage for stealth entry of Leishmania parasites. The theory that neutrophils act as Trojan horses is well perceived in case of L. donovani (10,22), but there is also evidence that Leishmania parasites escape neutrophils before infecting the macrophages (23), a Trojan rabbit strategy where viable promastigotes hide in the shadow of apoptotic neutrophils (9).

ENTRY INTO MACROPHAGES
In their quest for survival, Leishmania parasites face the arduous challenge to gain entry inside the macrophages and silence their impeccable defenses. The parasites express a wide array of ligands on their surfaces, which interact with a variety of macrophage receptors. Some of the key receptors that mediate promastigote-macrophage binding include the receptors for complement, fibronectin, mannose-fucose, and other sugars [(24) and references therein]. Interestingly, the foremost ligands employed by parasite for its phagocytic uptake are not encoded by the parasite itself; instead, parasite gets ingested into the macrophages via opsonin-dependent pathways. The sharp-witted Leishmania parasites not only circumvent complement-mediated lysis but also modulate the complement system for their active uptake inside the macrophages. C3bi and C3b are two major complement system components that bind to promastigote surface (25)(26)(27)(28) and facilitate their intracellular uptake via CR3 and CR1, respectively. The uptake via CR3 is more advantageous since internalization via these receptors does not result in oxidative burst and also suppresses the secretion of IL-12 and other pro-inflammatory signals, thus hampering the initiation of cell-mediated immunity (CMI) (29,30).

ESTABLISHING INFECTION IN MACROPHAGES
Once inside the macrophage phagosomes, promastigotes create an intracellular niche for their survival by silencing the macrophages through multifarious schemes. Predominantly, Leishmania parasites retard phagosome maturation, delay enodosome-phagosome fusion, inhibit hydrolytic enzymes in phagolysosomes, prevent generation of reactive-nitrogen and -oxygen species, suppress antigen presentation, and repress pro-inflammatory cytokine production. LPG present on the surface of Leishmania parasites retards phagosome maturation by inducing Cdc42-and Rac1 (Rho family GTPases, F-actin regulators)-dependent F-actin accumulation, which also involves inhibition of PKCα leading to impaired recruitment of LAMP-1 and rab7 (31)(32)(33)(34). It has been reported earlier that incorporation of LPG selectively into one of the leaflets of lipid bilayers of phagosome membrane alters its biophysical properties making it less fusogenic [(31) and references therein]. LPG impairs the acquisition of vesicular proton-ATPase, which is involved in acidification of phagolysosomes (35). Leishmania parasites either decimate or suppress expression of major histocompatibility complex (36)(37)(38). Leishmania parasites also interfere with protein expression of their host cells (39), suppress the secretion of pro-inflammatory cytokines (IFN-γ, TNF-α, IL-6, IL-12), and induce secretion of anti-inflammatory cytokines (IL-4, IL-10, and TGF-β) via modulation of host cell signaling (40)(41)(42).
Thus, establishment of Leishmania infection involves complex in-depth interactions between a vast repertoire of immunostimulating and immunosuppressive molecules that finally determine a species-dependent outcome of infection.

THERAPEUTIC MODALITIES
The pentavalent antimonials, SSG and Meglumine Antimoniate (MA), have been employed in treatment of visceral leishmaniasis (VL) and cutaneous leishmaniasis (CL) for more than 60 years. The use of SSG and MA as first-line treatment for VL has been already abandoned after failure rates of~65% in endemic regions of Bihar, India. Antimonials show variable efficacy against CL and VL, and accompanying severe side effects have demerited their use. The second line of drug AmB is now employed to treat the antimony-resistant patients but the need of hospitalization, prolonged duration of treatment, and infusion-related side effects are drawbacks. These constraints have now been overcome by lipid formulations of AmB that prevent tissue retention, thereby reducing the toxicity and promoting preferential uptake by reticuloendothelial cells that harbor the parasites. However, cost of liposomal AmB is a serious limitation and frequent noxious effects and drug resistance associated with pentamidine has also led to its withdrawal. Paromomycin has been registered for use in India against VL but is oto-and nephrotoxic. Miltefosine, the first oral drug, is of limited use because of its teratogenicity (43)(44)(45).

NATURAL IMMUNOMODULATORS: ROLE IN ANTILEISHMANIAL THERAPY
The drug discovery against leishmaniasis has been more reliant on therapeutic switching rather than discovery of novel drugs. Since elevation of host immunity is critical in parallel to the drug-mediated killing of Leishmania parasites, the antileishmanial arsenal may be benefited by antileishmanials that can prong a bifurcated attack, i.e., elimination of the parasites as well as restoration of CMI. The potential of immunomodulators in treating experimental leishmaniasis gained momentum with the discovery of antileishmanial activity of imiquimod (46,47), an agonist for toll-like receptor 7, which is present on macrophages and dendritic cells (DC) and promotes the development of Th1 immune response [(48) and references therein]. Several other synthetic compounds such as S 2 complex (an organic complex of copper chloride, ascorbic acid, and nicotinamide) (49), acetyl salicylic acid (50), and immunomodulatory peptide from cystatin (51) have been demonstrated to possess dual immune-modulating and antileishmanial activities.
Various herbal formulations and plant secondary metabolites such as flavonoids, isoflavonoids, saponins, alkaloids, sesquiterpenes, polysaccharides, tannins, indoles, and glucans are known to be immunomodulatory in different diseases (52,53). Among natural resources, plants have been most extensively explored for bioactive leishmanicidal and immunomodulatory compounds. Plant extracts contain a plethora of biomolecules that can naturally kill Leishmania parasites and also exert immunostimulatory properties, on otherwise depressed immune system during the diseased state, as has been extensively reviewed in Table 1.
In brief, the studies examining immunomodulatory effect of bioactive plant extracts or compounds have reported skewing of Frontiers in Immunology | Immunotherapies and Vaccines  has also been demonstrated (74). IL-12 is the central cytokine produced by DC, NK, and T cells, which activates macrophages to produce IFN-γ and TNF-α. Different plant secondary metabolites and extracts have induced IL-12 up-regulation (Table 1), indicating the worthy potential of natural resources. Macrophages also produce IL-18, which in synergism with IL-12 stimulates IFNγ production and aids in parasite clearance (80,81). Pelargonium sidoides extracts have been shown to increase mRNA levels of IL-18 in Leishmania major infection (73).
In parallel to stimulation of IL-12 and other pro-inflammatory cytokines, expression levels of IL-4, IL-10, and TGF-β have also been widely investigated ( Table 1). IL-4, IL-10, and TGF-β inhibit the production of IFN-γ from macrophages. IL-4 is known to potently inhibit macrophage activation, but IL-10 plays a cardinal role in progression of both CL and VL. In both, murine and human VL, despite the production of adequate amounts of IFN-γ, the hosts are unable to mount an effective CMI response, and this host inefficiency is attributed to increased levels of IL-10. Kane and Mosser (82) demonstrated that host-derived IgG present on Leishmania amastigotes ligates to Fcγ receptors on inflammatory macrophages and modulates them to secrete IL-10 in high amounts in CL. The levels of these Th2 cytokines have been observed to decline along with successful recuperation of CMI after treatment with immunomodulatory extracts and molecules of natural origin ( Table 1).
It can thus be well established that natural immunomodulators can skew the Th1-Th2 balance but pro-and anti-inflammatory cytokines play diverse and inter-regulatory roles. As also supported by Couper et al. (83), understanding the dynamics of Th1-Th2 paradigm has changed over the years and it is conceived that both Th1 and Th2 cells can mediate inflammation as well as aid parasite clearance. For instance, Néris et al. (57) reported that Licarin A, treated L. major-infected macrophages exhibit decline in IL-6 as well as IL-10 levels. IL-6 is a characteristic pro-inflammatory cytokine, which also negatively regulates Th1 differentiation and promotes CD4 + Th2 differentiation mediated by IL-4 (84). However, as the study presented only the in vitro data, and also the proor anti-immunopotentiating effect can be dose dependent (85) further in vivo studies may throw proper light on mechanism of action of Licarin A.

CONCLUSION
The use of herbal preparations to modulate the immune response to cure or avert diseases has been described in traditional systems of Indian, Unani, and Chinese medicine. The natural substances with dual, antileishmanial, and immunomodulatory properties have been carefully evaluated, however, it should be noted that human leishmaniasis varies in immunological pattern from murine leishmaniasis. Thus, the natural immunomodulators need to be evaluated more systematically and specifically in terms of dosage, biodistribution, kinetics, and interactions with other drugs and their putative role in other co-infections should also be examined.
Nonetheless, the concept of using natural immunomodulators to treat parasitic infections including leishmaniasis holds mighty potential in achieving the control of this disease. These natural immunomodulatory molecules can serve as scaffolds for synthesis and discovery of new immunodrugs. Also, the use of natural immunomodulators in synergy with existing drugs may involve the functional manipulation of multiple molecular targets leading to improved therapeutic efficacy and reduced toxicity.