Immunomodulation by chemotherapeutic agents against Leishmaniasis

International Immunopharmacology 11 (2011) 1668–1679

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Review

Immunomodulation by chemotherapeutic agents against Leishmaniasis Piu Saha, Debanjan Mukhopadhyay, Mitali Chatterjee ⁎ Dept. of Pharmacology, Institute of Post Graduate Medical Education and Research, 244 B, Acharya JC Bose Road, Kolkata-700 020, West Bengal, India.

a r t i c l e

i n f o

Article history: Received 2 August 2011 Accepted 3 August 2011 Available online 27 August 2011 Keywords: Leishmaniasis Anti-leishmanial Immunomodulation Chemotherapy

a b s t r a c t Leishmaniasis is caused by protozoan parasites of the genus Leishmania and causes a wide spectrum of clinical manifestations ranging from self-healing cutaneous lesions to the fatal visceral form. The use of pentavalent antimony, the mainstay of therapy of Leishmaniasis is now limited by its toxicity and alarming increase in unresponsiveness, especially in the Indian subcontinent. Furthermore, other anti-leishmanial drugs are unaffordable in many affected countries and as vaccination based approaches have not yet proved to be effective, chemotherapy remains the only alternative, emphasizing the need for identifying novel drug targets. In this review, we have described the different host immune signaling pathways that could be considered as potential drug targets for Leishmania chemotherapy. © 2011 Elsevier B.V. All rights reserved.

Contents 1. 2. 3. 4.

Leishmaniasis and clinical manifestations . . . . . . . . . . . . . . . . . . . . Disease immunopathogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . Chemotherapy of Leishmaniasis . . . . . . . . . . . . . . . . . . . . . . . . . Targeting of host immunity by anti-leishmanial drugs . . . . . . . . . . . . . . 4.1. Role of neutrophils. . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.2. Monocytes and macrophages . . . . . . . . . . . . . . . . . . . . . . . 4.3. Role of reactive oxygen species (ROS) and reactive nitrogen species (RNS) . 4.4. Role of DCs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.5. Lymphocytes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.6. Macrophage derived cytokines as a measure of immunomodulatory activity . 4.7. Effect on co-stimulatory molecules . . . . . . . . . . . . . . . . . . . . 5. Modulation of signaling events in Leishmania infection; role of chemotherapy . . . 5.1. Effect on expression of CD40 and MAPK signaling pathways . . . . . . . . 5.2. Toll like receptors and their responsiveness in Leishmania infection . . . . . 5.3. Leishmania infection and effect on JAK-STAT pathways . . . . . . . . . . . . 5.4. Modulation of NF-κB signaling pathways by Leishmania . . . . . . . . . . 5.5. Alterations of host cell kinases and phosphatases by Leishmania . . . . . . 6. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1. Leishmaniasis and clinical manifestations Leishmania spp. belonging to the order Kinetoplastida and family Trypanosomatidae are responsible for the disease Leishmaniasis which is spread through the sand fly. The unique digenetic life cycle of the parasite includes spindle shaped, flagellated procyclic promastigotes that follow⁎ Corresponding author. Tel.: + 91 33 2223 4135; fax: + 91 33 2223 4135. E-mail address: [email protected] (M. Chatterjee). 1567-5769/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.intimp.2011.08.002

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ing entry into the sandfly gut differentiate into non-dividing infective metacyclic cells; following a blood meal, they are taken up by circulating professional phagocytic cells like neutrophils and macrophages to eventually transform into rounded amastigotes [1]. Clinical manifestations depends on the Leishmania species involved and ranges from a life-threatening systemic infection (visceral, VL) to self limiting or chronic skin sores (cutaneous, CL), or dreaded metastatic complications that can cause facial disfigurement (mucosal, MCL). The clinical features of VL generally include prolonged and irregular fever,

P. Saha et al. / International Immunopharmacology 11 (2011) 1668–1679

often associated with rigor and chills, hepatosplenomegaly, lymphadenopathy, progressive anemia, weight loss and hypergammaglobulinemia (mainly IgG from polyclonal B cell activation) and concomitant hypoalbuminemia [2]. Even after recovery, African and Indian VL patients may present with a secondary form called Post Kala-azar Dermal Leishmaniasis [3].

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4. Targeting of host immunity by anti-leishmanial drugs Within the mammalian host, Leishmania reside as amastigotes in phagocytic cells that include neutrophils, macrophages and dendritic cells (DCs); therefore, an immunomodulatory compound could be potentially leishmanicidal by virtue of its potential to activate phagocytic cells.

2. Disease immunopathogenesis 4.1. Role of neutrophils Survival of the parasite within macrophages is crucial, which it ensures by deviously manipulating the macrophage related immune functions [4 and references therein]. The engulfed pathogen prevents formation of phagolysosomes, and thereby suppresses MHC-II mediated presentation of the parasite antigen to CD4 + T cells, eventually preventing macrophages from eliminating the phagocytosed pathogen [4]. Furthermore, the parasite prevents initiation of a respiratory burst, ensuring that the macrophage is a safe haven for the parasite. Therefore, it is only logical to conclude that activation of macrophages would be a de novo chemotherapeutic strategy for Leishmaniasis. The outcome of leishmanial infections is determined by two functionally distinct T-helper (Th) cell populations, Th1 (IFN-γ, IL-2) and Th2 (IL-4, IL-10 and IL-13). Generally, uncontrolled, non healing infections i.e. disease susceptibility is associated with Th2 proliferation and production of IL 4, 5 and 10 while in healing responses i.e. resistance to disease, there is expansion of IFN-γ producing CD4+ Th1 helper cells [5 and references therein]. This was confirmed when pretreatment of macrophages with IFN-γ and IL-12 induced resistance to L. major [6]. Accordingly, in experimental VL, key roles have been identified for IFN-γ and IL-12 in mediating expansion of protective Th1 cells while IL-4 and IL-10 mediate progression of infection following expansion of Th2 cells [5]. However, in human VL, the response is not a strictly polarized Th2 type, as measurement of splenic and bone marrow cytokine mRNA at disease presentation showed increased levels of both IL-10 and IFN-γ [7]. Similiarly at presentation, increased circulating levels of IL-10, IL-12 and IFN-γ, suggesting an initial mixed Th1- Th2 response have been reported, while disease resolution was associated with a simultaneous decrease in both IL-10 and IFN-γ, indicating that both Th1 and Th2 are components of the immune system during active disease and both regress with effective treatment [8]. Patients with VL usually demonstrate a negative skin test to Leishmania antigens and peripheral blood mononuclear cells fail to proliferate and produce IFN-γ when exposed to specific antigen in vitro, indicative of anergy; addition of anti-IL-10R antibody to T cells harvested from these patients, restored cytokine responses, corroborating a role for IL-10 in suppressing T-cell responses [9]; furthermore, cure was associated with a fall in mRNA levels of IL-10 [9,10]. Effective parasite elimination also requires restoration of macrophage function for production of toxic nitrogen and oxygen metabolites, necessary to kill resident amastigotes [11 and references therein].

The best characterized function of polymorphonuclear neutrophils (PMNs) is their preeminent role in phagocytosis and killing of invading microorganisms via generation of reactive oxygen species (ROS) and release of lytic enzymes. Following entry of Leishmania into the mammalian host, PMNs are recruited immediately to the site of infection within 24 h, implying that they possibly serve as host cells for Leishmania in the very early phase of infection [23,24]. Neutrophils being inherently short-lived undergo apoptosis [23], while Leishmania parasites are known to delay their apoptosis, possibly by interfering with production of ROS, which importantly facilitates their survival [25,26]. To trigger apoptosis, neutrophils utilize a mitogen activated protein kinase (MAPK) signaling pathway, p38 MAPK being a key player [27]. Recent data suggests a critical role for neutrophils in the early protective response against L. donovani, both as effector cells involved in parasite killing and for influencing development of a protective Th1 response [28]. Importantly, Leishmania parasites that enter macrophages via the uptake of infected, apoptotic PMNs then survive and multiply effectively [23]. The amount of TGF-β secreted by macrophages following uptake of infected PMNs was higher than after direct uptake of L. major promastigotes [23] indicating that uptake of infected, apoptotic PMNs creates a more effective anti-inflammatory milieu, beneficial for Leishmania survival. Therefore, as neutrophils harbor and transport Leishmania, targeting pathogens residing in neutrophils should be taken into consideration when designing novel antileishmanial compounds. Therefore, it is tempting to extrapolate that a compound capable of increasing phagocytic activity and generating an oxidative burst within Leishmania infected neutrophils would effectively eliminate parasites. Indeed, antimonials increase the phagocytic capacity of neutrophils along with increased production of superoxide [29]. Berberine chloride (Table 2) also promoted parasite elimination via enhancement of apoptosis in L. donovani infected neutrophils, subsequent to modulation of the MAP kinase pathways [30]. 4.2. Monocytes and macrophages To sustain infection, it is mandatory that Leishmania parasites establish themselves in macrophages, but considering the potent antimicrobial functions of macrophages, the subject of how Leishmania survive is a subject of intense research.

3. Chemotherapy of Leishmaniasis Currently, drugs used to treat Leishmaniasis are handicapped by emergence of strains resistant to conventional antimony, associated toxicities and high cost especially regarding lipid formulations of amphotericin B ([12], Table 1). A matter of concern is that Miltefosine, the only orally effective drug can potentially become ineffective, as studies with resistant amastigotes have shown the presence of mutant drug transporters [13]. Taken together, the current armamentarium of Leishmaniasis is limited and alternative therapies are strongly warranted. An area of growing interest is the potential of natural plant-derived products of diverse structural classes having anti-leishmanial properties [14–16] and includes naphthylisoquinoline alkaloids and synthetic analogs, Luteolin, Quassin, Curcumin, Artemisinin and several others [14,17–22].

(i) Phagocytosis: C3b is a complement protein that following binding to Leishmania surface glycoprotein gp63 increases parasite uptake into macrophages as gp63 cleverly converts C3b into iC3b, which then favors phagocytosis, yet prevents lytic clearance [31]. Antimonials [29], Pourouma guinensis [Oleanolic acid, 32] and Diphyllin isolated from Haplophyllum bucharicum Litv [33] influence the phagocytic activity of macrophages as do CpG oligodeoxynucleotide (CpG ODN) and miltefosine [34]. (ii) Acidification: Generally, following fusion of the phagosome with the endosomal compartment, a significant drop in pH ensues. However, Leishmania produce a surface acid phosphatase that inhibits the oxidative burst within macrophages, and additionally is an active proton pump keeps the intracellular pH close to

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Table 1 Currently available anti-leishmanial drugs: their mechanism of action on parasites, dosage, advantages and limitations. Drug

Mode(s) of action

Dosage (for VL)

advantages

Limitations

Pentavalent antimonials: Meglumine antimoniate (Glucantime) or sodium stibogluconate (Pentostam)

Activated within the amastigote/macrophage after conversion to the trivalent form. Shows direct parasiticidal activity by generation of ROS, depletion of thiols, modulation of bioenergetic pathways (glycolysis, fatty acid beta oxidation, inhibition of ADP phosphorylation, blocking of SH groups of amastigote proteins) and inhibition of topoisomerase II Complexes with 24-substituted sterols, such as ergosterol in the cell membrane, causing pores which alter ion balance, increase membrane permeability resulting in cell death; also acts as an inhibitor of ergosterol biosynthesis

20 mg/kg b.w., i.m., daily (600 mg total) for 30 days in India

Easily availability and low cost

Myalgia, pancreatitis, [13,130,131 cardiac arrhythmias, and ref. therein] hepatitis Acquired resistance

0.75–1.0 mg/kg for 15–20 infusions either daily on alternate days in India

Primary resistance is unknown

Need for prolonged hospitalization High cost, high fever with rigor, chills, hypokalemia, renal dysfunction High cost

Amphotericin B (polyene antibiotic)

Ref

[130,131 and ref. therein]

Lipid formulation of amphotericin -doB Ambisome/Abelcet/Amphotec

Highly effective, low toxicity

Paromomycin (aminoglycoside antibiotic), also known as aminosidine or monomycin

Effective, well tolerated Lack of efficacy in and relatively cheap, East Africa acts synergistically with antimonials

130 and ref. therein, 132]

Effective and safe

[130 and ref. therein]

Miltefosine (hexadecylphosph-ocholine)

Sitamaquine (8-aminoquinoline, originally WR6026)

Ambisome: 2.0 mg/ kg × 5 days, i.v. in India In bacteria, inhibits protein synthesis, but in 16 mg/kg × 21 days, i. Leishmania, the exact mechanism is not yet known. m.: 20 mg/ It is proposed to induce respiratory dysfunction in L. kg × 17 days, i.m. donovani promastigotes. It also promoted ribosomal subunit association of both cytoplasmic and mitochondrial forms, low Mg+ 2 which induced dissociation It interacts with the cell membrane of Leishmania by 100–150 mg for four weeks, p.o. in India modulation of cell surface receptors, inositol metabolism and phospholipase activation, cell death being mediated by apoptosis Unknown, possibly affects mitochondrial electron 1.75–2 mg/kg/day transport chain for 28 days in India.

neutral [35]. Tamoxifen similarly modulates the macrophage intravacuolar compartment by causing a rapid, long-lasting alkalinization [36].

4.3. Role of reactive oxygen species (ROS) and reactive nitrogen species (RNS) As nitric oxide (NO) is an effector molecule critical for elimination of intracellular Leishmania parasites, disease progression is ensured via enhancement of Th2 responses that causes deactivation of macrophages and decreased production of NO ([4], Fig. 1). Therefore, parasite removal should entail activation of infected macrophages by increased expression of inducible nitric oxide synthase (iNOS) to form NO [37]. During Leishmania infection, decreased expression or inactivation of iNOS may also be associated with increased activation of arginase as deprivation of L-Arginine impairs Leishmania majorspecific T-cell responses [38]. Following parasite engulfment by macrophages, NAD(P)H oxidases are initially activated, which transfer the reducing equivalents from NAD (P)H to molecular oxygen leading to formation of extremely reactive superoxide [11]. These then react with parasite membrane phospholipids leading to increased permeabilization as also react with the pathogen's macromolecules such as DNA leading to strand breaks; However, when the infection is sustained, macrophages are deactivated causing a decreased production of superoxide which is now beneficial for parasite survival (Fig. 1). Therefore, it is anticipated that if a similar pro-oxidant scenario is recreated by anti-leishmanial drugs, they can effectively eliminate the parasite [21,39]. Conventional anti-leishmanial drugs like antimonials [29,40], Miltefosine [41,42] and amphotericin B [43] increase generation of ROS and NO in Leishmania infected macrophages, as do other immunomodulatory compounds alone or in combination with sub-optimal doses of SAG e.g. a mononuclear

Little is known about its efficacy and toxicity

Vomiting and diarrhoea. nephrotoxic, teratogenic

[131]

[133]

diperoxovanadate compound K[VO(O2)2(H2O)]PV6 [44]. Similarly, an ethanolic extract of Tinospora sinensis [45], 18 Beta-glycyrrhetinic acid [46], tannins and related compounds [47], trinitroglycerin [48] also upregulated production of NO (Table 2). Interestingly, Cystatin along with IFN-γ, induced generation of NO but markedly reduced expression of iNOS at both mRNA and protein levels [49–51]. Other compounds that showed similar NO enhancing activity were Artemisinin [22], quassin [19], an aqueous extract of human placenta [52], eugenol-rich essential oil from Ocimum gratissimum [53], glycolipids along with other constituents from Desmodium gangeticum [54], a linalool-rich essential Oil from Croton cajucara [55], Kalanchoe pinnata [56,57] and Aloe vera leaf exudate [58,59]. A similar observation was evident following treatment with Chenopodium ambrosioides essential oils [60], Pyrazinamide [61], Himathantus sucuuba Latex (HsL) [62], quinovic acid glycosides and cadambine acid [63]. The scenario was similar with CpG-containing oligodeoxynucleotide, CpG-ODN [64], 2, 3, 7, 8-Tetrachlorodibenzo-Pdioxin [65] and Berberine chloride ([66], Table 2). However, some compounds exert their inhibitory effect on amastigotes independent of activation of NO and include (3S)-16, 17-Didehydrofalcarinol, an oxylipin [67], cyclosporin A [68], Nimodipine, a calcium channel blocker [69] and a supercritical fluid fraction obtained from Tabernaemontana catharinensis [70]. 4.4. Role of DCs The interaction of Leishmania parasites with DCs is complex, as depending upon the species of Leishmania, the DC subset and other exogenous stimuli involved, there can either be control of infection or disease progression, [71 and references therein]. The first study with murine skin DC implicated epidermal Langerhans cells as important cells for detection, uptake and transport of Leishmania to lymph nodes [72]. Dermal DCs efficiently incorporate parasites into vacuoles and

P. Saha et al. / International Immunopharmacology 11 (2011) 1668–1679

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Table 2 Antileishmanial compounds having immunomodulatory activity. Compound

Leishmania spp. IC50 in amastigotes (ex vivo)

In vivo

Mode(s) of action

Antimonials sodium antimony gluconate

L. donovani 1.38 μg/ml– 112.77 μg/ml

L. donovani, BALB/c mice, 20 mg/kg b.w. ×5 days, s.c. caused 85 % parasite inhibition.

Amphotericin B

L. donovani 0.033– 0.4 μM

Water-soluble amphotericin B (AmB)-arabinogalactan

L. donovani optimal dose, 2.0 μg/ml

Artemisinin, a sesquiterpene endoperoxide

L. donovani, 22 μM

L. infantum, hamsters, (1 mg/kg b.w., i.c. on days 25, 26 and 27 post-infection), reduced parasite load in liver and spleen by 88.8% and 87.2% on day 32 and on day 135, by 66.7% and 54% respectively L. donovani, hamsters; 6 mg/kg b.w., i.c. × 1, suppressed 99% parasitic burden in liver and spleen. BALB/c mice, 10–25 mg/kg b.w., p.o. reduced splenic weight and parasite burden by 82.6% and 86.0% respectively

[14,134,135] Induced ERK-1/2 and p38 MAPK phosphorylation, increased production of ROS, NO and TNF-α Increased phagocytic capacity of monocytes and neutrophils, enhanced generation of superoxide anion and production of TNF and NO Up regulated levels of IL-12, TNF-α and [43,136–139] expression of iNOS along with down regulated IL-10 and TGF-β production. It restored the impaired classical PKC and abrogated the atypical PKC

Artemisinin Artemether (i) Berberine chloride (quarternary isoquinoline alkaloid) (ii) 8 cyanodihydroberberine (iii) Tetrahydroberberine

L. major 3 × 10 (−5)M 3 X10 (-6)M (i) L. donovani 2.5 μM (i) L. donovani, hamsters, 50 and 100 mg/kg b.w., i.p. ×4 decreased parasite burden in liver by 48.5 and 61.1% respectively. 50 mg/kg b.w., i.p. ×5 days, repeated after 5 days reduced parasite no. from 1.67 × 109 to 0.163 × 109 (liver) and 1.55 × 109 to 0.097 × 109 (spleen) (i) L. donovani, L. braziliensis panamensis: hamsters , max. dose of 208 mg/kg b.w. (i.m.) twice daily ×4 caused 36% and 56% suppression (ii) L. braziliensis panamensis hamsters, max. dose of 208 mg/kg b.w., i.m. twice daily ×4 caused 54% and 46% suppression respectively. (iii) L. donovani, hamsters, max. dose of 416 mg/kg b.w. (i.m.), twice daily ×4, less toxic and more potent against L. donovani showed 50% suppression L. donovani L. donovani, BALB/c mice

Meglumine antimonate

CpG-containing oligodeoxynucleotide (CpG-ODN) incorporated in mannose-coated liposomes (i) Man-lip-CpG (ii) lip-CpG (iii) free CpG-ODN

Cystatin

Two recombinant barley cystatins (i) HvCPI5 (ii) HvCPI6

(i) 3.01 μg/ml, (ii) 4.05 μg/ml (iii) 4.61 μg/ml

L. donovani 5.2 μg/ml IFN-γ + Cystatin 4.3 μg/ml L. infantum (% reduction) (i) 0.1 μM and 1 μM: 35.9 ± 9.9 and 36.4 ± 2.2 respectively (ii) 0.1 μM and 1 μM: 31.9 ± 11.9 and 28.3± 7.8 respectively

(i) CpG-ODN, 2.5 mg/kg b.w./day, x 15, i.p. caused 100% parasite suppression (ii) 2.5 mg/kg b.w./day, ×15, i.p. caused 81% reduction (iii) 2.5 mg/kg b.w./day, ×15 i.p. caused 62 % reduction glc-lip- CpG, 2.5 mg/kg b.w./day ×15 days, i.p. caused 86 % reduction L .donovani, BALB/c mice, hamsters (i) 1 nM, single dose, i.p., free and liposomal forms + miltefosine, (2.5 mg/kg × 5 days, p.o.) caused 85% inhibition. (ii) In hamsters, 1 nM, single dose, i.p. + miltefosine (5 mg/kg b.w. ×5 days, p.o.) caused 81.7% inhibition. L. donovani, BALB/c mice, 20 mg/kg b.w./day ×4 caused marked suppression. 5 mg/kg b.w./day +104 U IFN-γ × 4 days i.v. eliminated all parasites 0.5 mg/kg b.w./day + IFN-γ (100 U/ml) × 4, i.v. decreased parasite burden

References

Increased TNF-α but had no effect on IFN-γ and production of NO

Restored NO and Th1 cytokines, IFN-γ and IL-2 production.

[21,22,140]

[39,66,141–143] Increased production of NO and expression of iNOS and IL-12p40 along with down regulation of IL-10, induced phosphorylation of p38 MAPK and decreased phosphorylation of ERK1/2.

Enhanced generation of NO, ROS and H2O2, reduced levels of IL-4, but increased levels of IFN-γ, IL-12 and iNOS.

[34,64]

Increased phagocytosis index and combination therapy involving free or liposomal CpG ODN with miltefosine increased TNF, IFN-γ and IL-2 cytokine levels and downregulated IL-4 and IL-5.

[49–51,144,145] Induced ERK1/2 phosphorylation and NF-kB DNA-binding activity, IFN-γmediated JAK-STAT activation, generation of NO from macrophages, increased levels of IL-12 and TNF-α and iNOS and decreased IL-10 secretion. Cystatin + IFN-γ induced TLR/MyD88 signaling

(continued on next page)

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Table 2 (continued) Compound

Leishmania spp. IC50 in amastigotes (ex vivo)

In vivo

Mode(s) of action

References

Desmodium gangeticum (Glycolipids and other constituents from (i) EtOH extract (ii) Aminoglucosyl glycerolipid (iii) Glycosphingolipid (cerebroside) Diperoxovanadate compd. K[VO(O2)2(H2O)] − PV6 (10 μM) + SAG in (i) Sb-sensitive (SbS-LD) (ii) Sb-resistant (SbR-LD)

L. donovani

L .donovani, hamsters, 250 mg/kg b.w. × 2, p.o. of EtOH extract decreased parasite burden by 41.2 ± 5.3%

Increased production of NO

[54,146]

Enhanced T-cell proliferation, IFN-γ, ROS, production of NO and reduced production of IL-10.

[44]

Modulated phagocytic activity of macrophages Decreased production of NO.

[33]

Increased production of NO

[53]

Diphyllin (3S)-16,17Didehydrofalcarinol, an Oxylipin isolated from, Tridax procumbens (i) MeOH crude extract (ii) Hexane extract (iii) Compound 1 Eugenol-rich essential oil from Ocimum gratissimum Glycyrrhizza glabra L. (Licorice) 18 Beta-glycyrrhetinic acid (GRA) Imiquimod

Kalanchoe pinnata (Crassulaceae) (Kp, synonimia Briophyllum pinnatum (i) Leafy aqueous extract (ii) Quercetin 3-O-α-Larabinopyranosyl (1→ 2) α-L-rhamnopyranoside, (iii) Free quercetin Flavonoids

Miltefosine

Momordica charantia (i) Aqueous extract (ii) Momordicatin Pourouma guinensis (i) Ursolic acid (ii) Oleanolic acid

(ii) 100 μg/ml: 72 ± 5% inhibition; (iii) 100 μg/ml: 53.4 ± 4.4% inhibition L. donovani killed by L. donovani, BALB/c mice; PV6 (0.5 μM, i.p.) alternative day × 4 weeks decreased parasite load by (i) 72% (i) 72.7 ± 7.44% (ii) 63.48% (ii) 49.9 ± 2.24% PV6 (0.5 μM) alternative day × 4 weeks + SAG 50 mg/kg b.w., i.m. twice weekly × 3 SbR-LD reduced hepatic and splenic parasite burden, 77.1 ± 5.86% and 79.2 ± 3.62% respectively Leishmania spp. 0.2 – μM L. mexicana

[67]

(iii) 0.48 μM

L. amazonensis 100 μg/ml

–

L. donovani 4.6 μg/ml

L. donovani, BALB/c mice, 50 mg/kg/b.w./day × 5, i.p. caused complete elimination

Reduced levels of IL-10 and IL-4, but [46] increased levels of IL-12, IFN-γ, TNF-α, and iNOS, induced NF-κB migration into the nucleus [147–150] – L. tropica, patients with CL, Imiquimod (5% cream) + Reduced neutrophil and macrophage Meglumine antimonite 20 mg/kg. b.w. weekly for 4 infiltration in the lesion, depleted CD1a + dendritic cell in epidermis and CD68 weeks. Reduced histiocytic cellular aggregation. + macrophages in dermis L. major, combination with Glucantime, decreased Decreased production of IFN-γ, IL-4 and parasitic load and thickness in footpad IL-10 Patients with CL, administered 7.5% Imiquimod every alternative day 20 days + 20 mg/kg b.w. Meglumine antimonate i.v, cure rate 100% [56,57,151–153] Suppressed antibody production and (i) L. amazonenesis, BALB/c mice, leafy extract, L. amazonenesis 500 developed delayed-type 8 mg/kg b.w, p.o. ×18 days suppressed parasite μg/ml caused 58% hypersensitivity and produced specific growth reduction antibodies. (i) 320 mg/kg b.w. , p.o. ×30 days suppressed parasitic burden L. amazonensis 8 (i) L. chagasi, BALB/c mice, 400 mg/kg b.w. p.o. (day Increased production of NO. −N100 μg/ml 1 to 29), decreased parasite burden in spleen and liver by 4 and 6 fold respectively (ii) L. amazonensis, BALB/c mice, 16 mg/kg b.w, p.o ×30 days reduced parasitic burden. [41,74,136,137,154] L. donovani 0.9–4.3 μM L. donovani, BALB/c mice, 20, 25 and 30 mg/kg b.w./ Stimulated T cells and macrophages, increased secretion of proday ×5, p.o. reduced parasite burden by 51%, 76%, inflammatory cytokines, including and 81% respectively. L. major 32–37 μM L. major, BALB/c mice 10 and 25 mg/kg b.w./day ×10, IFN-γ, and enhanced production of RNIs and ROIs as also expression of STAT p.o. decreased parasite burden by 87 and 98% receptors, induced PKC- and PI3Krespectively. dependent p38MAPK phosphorylation and production of CD40-induced IL-12; it also activated DCs. L. donovani, hamsters Inhibited superoxide dismutase (SOD) [155] (i) 300 mg/kg b.w. alt day ×15: 100% clearance activity (ii) 10 mg/kg b.w., alt day ×15: 100% clearance L .amazonenesis Inhibited phagocytic activity of [32] (i) 11 μg/mL macrophages. (ii) 27 μg/mL

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Table 2 (continued) Compound

Leishmania spp. IC50 in amastigotes (ex vivo)

In vivo

Mode(s) of action

Pyrazinamide

L. major 10.2 μg/ml

–

Quassin

L. donovani 25 μg/ml

–

Quinovic acid glycosides and cadambine acid isolated from Nauclea diderrichii. Tabernaemontana catharinensis (leaves) Superficial fluid of EtOH fraction Tamoxifen, an antioestrogen, a tri phenylethylene

L. infantum, 1.0 μM

–

Increased IL-12, TNF-α, and production [61] of NO. Increased expression of costimulatory molecules CD80, CD86, and MHC class II as also slightly increased IL-10 [19] Enhanced NO generation and iNOS expression both at a protein and mRNA level and up-regulated pro inflammatory cytokines such as TNF-α and IL-12. Induced NO production. [63]

L. amazonensis 100 μg/ ml. caused 88% inhibition L. amazonensis 11.1± 0.2 μM L.(V.) braziliensis 1.9 ± 0.2 μM L. (L.) chagasi 2.4 ± 0.3 μM

–

Inhibited production of NO and TGF-ß

[70]

L. braziliensis, BALB/c mice, 20 mg/kg b.w., i.p. ×15 days decreased parasite load by 99%

Induced rapid alkalinization of parasitophorous vacuoles.

[36,156,157]

L. chagasi, hamsters, 20 mg/kg b.w., i.p. ×15 days decreased parasite load by 95-98%.

Tannins (i) Proanthocyanidinins (ii) Hydrolyzable tannins

(i) L. donovani 0.7–8.0 nM (iii) Polyphenols and hydroxyl (ii) b 0.4–12.5 μg/ml L. major, L. donovani tannins 1–2 μM (iv) Sage phenolics (iii) L. major, L. donovani 1–250 μM (iv) Leishmania spp. 3.9–22.6 nm Terpenoids monoterpene L. amazonensis (i) Linalool-rich essential oil (i) 22.0 ng/ml from Croton cajucara (ii) Linalool (ii) 15.5 ng/ml 2,3,7,8-Tetrachlorodibenzo-PL. major, C57BL/6 mice, 40 μg/kg b.w., s.c. × 1, prior to dioxin (TCDD) infection (single dose) caused a 10 fold decrease in parasite burden. Thalidomide and glucantime L. major, BALB/c mice, thalidomide 30 mg/kg/b.w./day × 12 days (p.o.) + Glucantime 200 mg/kg/b.w./day × 12 days (i.p.) decreased parasite load (i) Tinospora sinensis Linn L. donovani (Ethanolic extract) (i) 29.83 ±3.4 μg/ml (ii) Hexane fraction (iii) Chloroform fraction (iv) Butanol fraction (v) Aqueous fraction Trinitroglycerine (TNG)

(ii) N 100 μg/ml (iii) N 10 μg/ml; (iv) 17.6 ± 4.1 μg/ml; (v) N100 μg/ml

Z-100, polysaccharide from L. amazonensis 13.0 Mycobacterium tuberculosis mg/L + meglumine antimoniate

References

L. donovani, hamsters (i) 500 mg/kg b.w./day × 5 days, p.o. inhibited by 76.2 ± 9.2%

[47,80,81,84,158,159] Activated macrophages and upregulated NO, TNF, IFN-γ, iNOS, IL-1, IL10, IL-12 and IL-18 expression.

Hydrolyzable tannins induced the release of NO, TNF and IFN.

Increased production of NO

[55]

Increased IL-2 production.

[65]

Up regulated IFN-γ and downregulated IL-10 production.

[79]

The ethanolic extract and butanol fraction enhanced ROS and NO production as also activated macrophages.

[45,160]

Increased NO production.

[48]

(iv) 250 mg/kg b.w., p.o. inhibited by 72.8 ± 4% L. major, BALB/c mice, 200 μg, s.c. × 15 days decreased parasite load in lesions (59% to 49%, in liver (98% to 64%), spleen (98% to 49%) and in lymph nodes (78% to 15%) L. amazonensis, BALB/c, 100 μg/kg/b.w. i.p. × 2 wks between the 6th and 8th wk was not antileishmanial; Z-100 + SAG (14/28 mg/kg b.w., i. l. × 2 wks between the 6th–8th wk caused 99% inhibition

are proposed to act as principal APCs in Leishmaniasis, while others suggest that lymph node resident DCs are initiators of the immune response [71]. Leishmania have cleverly devised several strategies to avoid DCs, as in humans, L. donovani blocks maturation of DC [73] and production of IL-12, essential for initiation of a protective immune response. Accordingly, Miltefosine in turn can activate DCs [74] as also does Pyrazinamide via increased secretion of proinflammatory molecules and enhanced expression of co-stimulatory molecules ([61], Table 2).

Upregulation of IFN-γ along with IL-10 [78] and IL-4. Both IgG1 and IgG2a were increased.

4.5. Lymphocytes T lymphocytes are generally responsible for intracellular pathogen elimination whereas B lymphocytes eliminate extracellular bacteria. In order to eliminate Leishmania, the macrophage needs to be activated by antigen specific T lymphocytes who by secreting IFN-γ, upregulate production of NO from macrophages. Both CD4 and CD8 cells are required for resolving the infection, along with a balance between Th1 and Th2, preferably a Th1 skewed response [5]. Therefore, essential prerequisites of an effective immunomodulatory, anti-leishmanial drug

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Fig. 1. Schematic representation of Leishmania induced signaling events within macrophages: The different targets (1–8) within the macrophages modulated by Leishmania are potential sites that can be targeted by the anti-leishmanial compounds. (1) Increased interaction of CD40/CD40L (2) Activation of MAPKs (3) Upregaulation of TLRs, IRAK-1, TRAF-6 and Myd88 (4) Activation of JAK-STAT pathways (5) Enhanced nuclear translocation of NF-κB (6) Down regulation of PKC ε,ξ (7) Upregulation of PKC α, β and enhanced maturation of phagolysosomes leading to increased assembly of NADPH oxidase complex and (8) Downregulation of PTPs which in turn would upregulate expression of pro-inflammatory mediators. IFN-γ R: IFN-γ receptor; IkB: Inhibitory kappa B; iNOS: Inducible nitric oxide synthase; IRAK-1: IL-1R- associated Kinase 1; JAK: Janus Kinase; MAPK: Mitogen Activated Protein Kinase; MD2: Myeloid Differentiation protein 2; MyD88: Myeloid Differentiation primary-response gene 88; NO: Nitric oxide; O2: Oxygen molecule; .O2-: Super oxide anion; PKC: Protein Kinase C; PTP: Protein Tyrosine Phosphatase; PV: Parasitophorous vaccules; STAT: Signal Transducer and Activator of Transcription; TIRAP: Toll-interleukin 1 receptor (TIR) domain containing adaptor protein; TRAF-6: TNF receptor-associated factor 6.

should be its potential to tilt the Th1-Th2 imbalance in favor of Th1. Furthermore in VL, T cell proliferation is impaired possibly due to loss of co-stimulatory molecule(s) [5], and so this too can be an additional target. Restoration of the lymphoproliferative capacity is achieved by Miltefosine [74], AmB in association with a suboptimum dose of stearylamine-bearing cationic liposomes [75], a mononuclear diperoxovanadate compound K [VO (O2)2(H2O)] (PV6) along with SAG [44] and a human placental extract ([52], Table 2). Although Leishmaniasis is characterized by the appearance of antileishmanial antibodies, B cells and antibodies are unimportant, as parasites tend to hide within the macrophage parasitophorus vacuole [5]. In experimental models, it has been observed that anti-leishmanial antibodies play a contributory role as IgG coated parasites upon ligation with Fc receptors on macrophages [1] or dendritic cells [76] induce increased production of IL-10, a key cytokine for disease persistence. Furthermore, this was corroborated by studies with B cell depleted animals that were found to be highly resistant to Leishmaniasis [77]. Liposomal Z-100 and Kalanchoe pinnata decreased IgG and its subclasses ([56,78], Table 2). 4.6. Macrophage derived cytokines as a measure of immunomodulatory activity

cytokines involved in the generation of NO and macrophage activation which are increased by antimonials [83], tannins and related compounds [47,80,81] as also sage phenolics [84]. Chemokines, a superfamily of low MW cytokines recruit distinct subsets of leukocytes and by activating them play an important role in Leishmaniasis. TNF-α and IL-1β together with MIP-1α (Macrophage inflammatory protein 1α) regulates transport of Leishmania from infected sites to lymph nodes [85 and references therein]. During Leishmaniasis, IFN-γ together with macrophage chemotactic protein 1 (MCP-1) eliminates L. major while conversely, IL-4 antagonizes production of MCP-1 [86]. Essential oil and extracts from Xylopia discreta induced differential production of MCP-1 in leishmaniasis [87]. IL-8 is another chemokine that controls the early infection of Leishmania via recruitment of neutrophils [88] and release of NO along with pro-inflammatory cytokines from macrophages [89]; SAG in fact induces IL-8 synthesis in patients with CL [83]. It has been shown that co-incubation of Leishmania parasites with PMNs inhibits the CXCchemokine and interferon-γ inducible protein-10 (IP-10), accounting for its Th1 inhibiting activity [88]. Furthermore, as IP-10 and CXCL-10 induce NK cells [85] it suggests that induction of chemokines within Leishmania infected cells could also be an effective strategy. 4.7. Effect on co-stimulatory molecules

The immunomodulatory potential of anti-leishmanial drugs has been established by measuring its influence on macrophage derived cytokines, mainly IFN-γ, IL-12, TNF-α and IL-10. Miltefosine [41,42,74], glucantime [79], and Amphotericin B [43] along with experimentally effective compounds such as 18 Beta-glycyrrhetinic acid [46], tannins [47,80,81], artemisinin [22], quassin [19], aqueous extract of human placenta [52], garlic [82], pyrazinamide [61], CpGcontaining oligodeoxynucleotide [64], Berberine chloride [66] and 2,3,7,8-Tetrachlorodibenzo-P-dioxin [65], collectively support the notion that upregulation of the Th1 response is an effective strategy for parasite elimination. IL-6 and IL-1β are potent pro-inflammatory

T cell mediated regulation of immune responses is intimately associated with co-stimulatory molecules present on APCs, as they can modulate the TCR-MHC interaction. Among them CD28, plays a pivotal role as their enhanced or diminished expression causes immune activation or anergy respectively [90] owing to their interaction with B7.1 (CD80) or B7.2 (CD86) present on monocyte/ macrophages and/or B cells. In PKDL, increased levels of circulating CD8 +28 - lymphocytes confers immune anergy, evidenced by their non proliferating nature which gets reversed following treatment [91]. The impaired expression of CD86 on monocytes as evidenced in

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PKDL was markedly increased following treatment with Miltefosine and SAG, the effect of Miltefosine being greater [92]. Pyrazinamide enhanced expression of CD80 and CD86 in Leishmania infected BALB/C mice [61] as did an aqueous extract of human placenta, evidenced by an increased expression of MHC molecules on APCs ([52], Table 2). 5. Modulation of signaling events in Leishmania infection; role of chemotherapy 5.1. Effect on expression of CD40 and MAPK signaling pathways An important co-stimulatory molecule that determines the outcome of macrophage-Leishmania interactions is CD40 as the CD40-CD40L interaction helps increase the Th1 immune response [93]. With regard to Leishmania infection, CD40 mediated MAPKs have been reported to promote parasite survival by modulating the expression of IL-10 and IL12 in macrophages [94]. MAPKs, a group of serine/theonine kinases are responsible for phosphorylation of cellular proteins which in turn triggers signals necessary for cell proliferation, differentiation and survival [95]. The CD40 of macrophages interacts with CD40-L of T cells and passes the signal onwards to produce IL-12 via p38MAPK and NF-kB (nuclear factor kB, Fig. 1). The released IL-12 then binds to IL-12 receptors present on macrophages, increases their production of IFN-γ, which then acts on infected macrophages to induce parasite killing. However, this CD40-CD40L interaction has been proposed to exert a dual effect, as when CD40 signaling is associated with depletion of cholesterol and TRAF-6, it causes activation of ERK1/2, higher levels of IL-10 follow along with decreased levels of IL-12p40. Conversely, if the CD40 signalosome is associated with normal levels of cholesterol and TRAF-2/3/5, it causes p38 MAPK activation which is accompanied with increased leishmanicidal IL-12 p40 and accompanying pro-inflammatory responses [93,96]. It has been proposed that Leishmania lipophosphoglycans stimulate the ERK pathway, which in turn inhibits macrophage production of IL-12 [94 and references therein]. It has also been demonstrated that parasites can modulate the TLR2-stimulated MAPK pathway by suppressing phosphorylation of p38 MAPK along with enhanced phosphorylation of ERK1/2 [97]. Nitric oxide, a crucial mediator for leishmanicidal activity, was found to be dependent on iNOS expression and was linked to the MAPKs signaling pathway ([42], Fig. 1). Ben-Othman et al., 2008 [98] showed that Leishmania initially activated but subsequently downregulated intracellular MAPKs and NF-kB signaling in macrophages. Taken together, as ERK and p38 MAP kinases differentially regulate induction of macrophage effector molecules which dictate the course of infection, one is tempted to propose that these kinases be considered as potential targets for development of novel strategies to combat Leishmaniasis, as demonstrated with Miltefosine [42]. The classical anti-leishmanial drug SAG modulates signaling pathways such that it induces an early wave of ROS-dependent parasite killing followed by a stronger late wave of NO-dependent parasite killing via phosphorylation of ERK1/2, and p38 MAPK [40]. The activation of ERK1/2 resulted in increased production of ROS while p38 MAPK activation increased release of TNF-alpha and NO [40]. Cystatin activated the ERK1/2 pathway in presence of IFN-γ and decreased iNOS induction in macrophages [51] while Berberine chloride, a potent anti-leishmanial compound exerted its leishmanicidal activity via increased IL-12 following enhanced phosphorylation of p38 MAPK, and was accompanied with a down regulation of ERK1/2 and decreased levels of IL-10 ([66], Table 2). 5.2. Toll like receptors and their responsiveness in Leishmania infection Toll like receptors (TLRs) have been identified as ancient receptors that are of critical importance for initiation of an efficient immune response [99]. Innate immunity coordinates the inflammatory response to pathogens, wherein the contribution of TLRs is widely recognized. These TLRs are located either on the plasma membrane or

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within the endosomal membrane of macrophages, DCs, NK cells as also T and B lymphocytes. Mammalian cells express up to 12 different TLRs [99] which share an intracellular domain, called Toll-IL-1R [99]; amongst them, some signal through the myeloid differentiation protein 88 (MyD88) [100] which ultimately leads to nuclear translocation of NF-κB and expression of proinflammatory cytokines that includes TNF-α, IL-12 along with iNOS, collectively causing host protection [101]. Following recognition of a pathogen-associated molecular pattern, MyD88 is then recruited to the TIR (toll-interleukin 1 receptor) [100], followed by additional recruitment of IL-1 receptor-associated-kinase-1 (IRAK-1) to the complex. IRAK-1 then gets phosphorylated by IRAK-4 as also undergoes autophosphorylation; subsequently, dissociation of IRAK-1 from MyD88 follows so that it can now interact with TRAF6, which finally activates various cascades, leading to activation of MAP kinase pathways, translocation of NF-κB to nucleus and secretion of proinflammatory cytokines [102]. As Leishmania infection is associated with inhibition of IRAK mediated signaling (Fig. 1), the control of Leishmania parasites in vivo requires the adaptor protein MyD88 [103] as genetically resistant C57BL/6 mice became susceptible to Leishmania in the absence of MyD88 ([104], Fig. 1). Furthermore, silencing of TLR2, TLR3, IRAK-1 and MyD88 expression by RNA interference also led to decrease production of NO and TNF-α by macrophages in response to L. donovani promastigotes [105]. Studies showed that TLR4 signaling can enhance the microbicidal activity of macrophages harboring parasites [103] and Bhattacharya et al., 2010 [106] have demonstrated that the leishmanicidal potential of Arabinosylated Lipoarabinomannan was mediated through upregulation of TLR2 signals corroborating its importance as an important chemotherapeutic strategy. 5.3. Leishmania infection and effect on JAK-STAT pathways Cytokines play a critical role in determining the nature of the host immune response in Leishmania infection as they trigger a signaling pathway through a cascade of intracytoplasmic proteins known as Janus Kinase and signal transducer and activator of transcriptions [STATs, 107]. The biological effects of IFN-γ are dependent upon activation of STAT1 transcription factors as ligation of IFN-γ with IFN-γ receptor (IFN-γ R) activates JAK1/JAK2 kinase which then phosphorylates STAT-1; the STAT1 then translocates to the nucleus and further enhances transcription of IFN-γ-induced genes (Fig. 1, [108]). The induction of iNOS by LPS and IFN-γ is primarily controlled by two regulatory regions present in the iNOS promoter that contains binding sequences for two transcription factors, NF-κB and IFN regulatory factor 1 [IRF-1, 109]. The synergistic role of IFN-γ in induction of iNOS is attributed to its ability to induce expression of STAT1 and IRF-1 transcriptional complexes that also bind to the IFN-γ activating sequence and IRF response element sequences respectively. It has been shown that IFN-γR−/− mice are highly susceptible to L. major infection corroborating that IFN-γR is essential for control of CL [110]. Leishmania infection has been shown to cause inhibition of the JAK2/STAT1 signaling cascade, as infected macrophages on stimulation with IFN-γ, showed defective phosphorylation of JAK1, JAK2, and STAT1 [111]. Both L. major and L. mexicana suppressed expression of IFN-γRα and IFN-γRβ, reduced levels of total JAK1 and JAK2, and downregulated IFN-γ-induced JAK1, JAK2, and STAT1 activation, the effects being more profound with L. mexicana than L. major (Fig. 1, [112]). Wadhone et al., 2009 [42] demonstrated that miltefosine effectively modulates host cell-dependent signaling pathways, restores responsiveness to IFN-γ via enhancement of IFN-γ receptors, IFN-γ induced STAT-1 phosphorylation and reduced activation of SHP-1 (the phosphatase implicated in down-regulation of STAT-1 phosphorylation). Another immunomodulatory anti-leishmanial cystatin, along with IFN-γ, modulated production of NO in macrophages that was partly dependent on activation of the JAK/STAT pathway [51].

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5.4. Modulation of NF-κB signaling pathways by Leishmania The NF-κB family includes five members of which p50, p65 (Rel A) and c-Rel, have been detected in macrophages, p50-p65 being the commonest [113]. In resting cells, NF-κB is retained in the cytoplasm complexed with its inhibitory subunit IκBα; following agonist stimulation, an enhanced serine phosphorylation of IκBα triggers its proteasomal degradation, resulting in subsequent activation of NF-κB; the MAPK signaling pathways have been identified as the upstream kinases that induce NF-κB activation via phosphorylation of its inhibitor, IκBα [114]. These signals induce IκB kinase (IKK) and after activation of the IKK complex, specific IκBα phosphorylation/degradation causes subsequent release of NF-κB and its translocation to the nucleus activates transcription of multiple κB-dependent genes, including iNOS and Th1 cytokines [115]. Therefore, preventing the degradation of IκB and its downstream events is a strategy used by L. donovani promastigotes to effectively shut down the NF-κB-dependent expression of proinflammatory cytokines, ultimately translating into lowered production of NO (Fig. 1, [116]). Conversely, 18 Beta-glycyrrhetinic acid mediated its anti-leishmanial activity by inducing nuclear migration of NF-κB in parasite-infected cells, concomitant with a diminished presence of IκB in the cytoplasm ([46], Table 2). Similarly, cystatin along with IFN-γ increased the activity of IKK leading to decreased IκBα levels and activation of NF-κB [51]. 5.5. Alterations of host cell kinases and phosphatases by Leishmania Protein kinase C (PKC) is a family of 10 isoenzymes involved in controlling the function of other proteins through phosphorylation of hydroxyl groups of their serine and threonine residues. PKCs play an important role in several signal transduction cascades and are activated by increased concentration of diacylglycerol (DAG) or Ca2+ [117]. During Leishmania infection, activation of PKC is inhibited and subsequent intracellular signaling, LPG being a key determinant [118] as also other glycosylinositol phospholipids [119,120]. Bhattacharyya et al., (2001) have reported that L. donovani infection selectively inhibited Ca2+-dependent PKC activity (PKC β) via IL-10 while Ca2+-independent PKC (PKC ε, ζ) activity was enhanced (Fig. 1, [121]). L. major is known to inhibit PKC-dependent c-fos and TNFα gene expression [122]. Infection of macrophages with L. donovani enhanced levels of intracellular ceramide which in turn downregulated classical PKC activity, upregulated Ca2+-independent atypical PKC-ζ expression [123]. Furthermore, PKC β is known to activate the assembly of NADPH oxidase subunit complex whereas Leishmania infection by causing downregulation of PKC β expression, inhibited the assembly of NADPH oxidase subunit complex and subsequently attenuated generation of ROS [Fig. 1, 93. It is also known that PKC isform α is responsible for F actin mediated phagolysomal maturation; once again, Leishmania by downregulating the expression of PKC α inhibited phagosomal maturation [93 and ref therein]. Furthermore, as Leishmania induced PKC ζ expression, which is known to inhibit the MAPKs, caused decreased production of proinflammatory mediators [Fig. 1, 124 and ref therein]. It has been seen that C–C chemokines, macrophage inflammatory protein (MIP-1 alpha) and macrophage chemoattractant protein (MCP-1) can restrict the parasitic burden via restoration of impaired PKC signaling and induction of free-radical generation in murine Leishmaniasis [125]. These chemokines restored Ca 2+-dependent PKC activity and inhibited Ca 2+-independent atypical PKC activity both in vivo and in vitro in L. donovani-infected macrophages [125]. Mukherjee et al., (2010) have demonstrated that amphotericin B can restore the impaired classical PKC and abrogate the atypical PKC pathways [43]. During Leishmania infection, activation of (phosphoinositide 3-kinase) PI3K signaling caused a down regulation of IL-12 [126]. SHP-1 Protein Tyrosine Phosphatase (PTP) is an important negative regulatory molecule of signaling pathways, related to the actions of interferons [127].

Macrophages infected with Leishmania in vitro have elevated SHP-1/PTP activity induced by gp 63, which led to colocalization of SHP-1 and JAK2, and thereby prevented IFN-γ induced tyrosine phosphorylation of JAK2 (Fig. 1, [128]). Leishmania induced PTPs are known to inhibit MAPKs leading to inhibition of nuclear translocation of NF-κB (Fig. 1, [93,124]). It has been shown that SAG induces ERK1/2 phosphorylation through activation of PI3K, protein kinase C, and Ras while p38 MAPK phosphorylation occurs through activation of PI3K and Akt [40]. 6. Conclusions The key pathogenic event in Leishmaniasis is harboring of the causative Leishmania parasite within phagolysosomes of macrophages. Therefore, to establish infection, Leishmania invariably develop mechanisms to neutralize the microbicidal machinery of macrophages. Hence, establishment of infection critically hinges on whether the balance tilts towards the host's ability to activate its armamentarium or the parasite's ability to escape or evade this host immune response. Macrophages are host cells for the parasite, but also importantly, sentinels of the immune system. The parasite interferes with the signaling system of the host, such that effector functions triggered by various cell surface receptors are either actively suppressed or are altered so as to result in immune suppression that will promote parasite survival. Therefore, our quest for anti-leishmanial drugs should focus on their direct parasiticidal and/or indirect immunomodulatory activity, achieved via restoration of impaired host signaling pathways. In this review, we have highlighted the participation of various immune cells, microbicidal molecules and altered signaling mechanisms in Leishmaniasis, together with the influence of anti-leishmanial drugs upon various immune cells like neutrophils, macrophages, DCs and lymphocytes. The different immune mechanisms impacted upon include increased generation of ROS and RNS, activation of co-stimulatory molecules and signaling pathways e.g. TLRs, MAPK, JAK-STAT, PKC, and translocation of NF-kB. Taken together, screening for compounds having the propensity to modulate the host defense signaling pathways alone or in combination with existing anti leishmanial drugs [129] may well prove to be an effective immunochemotherapeutic strategy in Leishmaniasis worthy of pharmacological consideration. Acknowledgements This work received financial assistance from the Indian Council of Medical Research (ICMR), Dept. of Science & Technology and Council of Scientific & Industrial Research CSIR, Govt. of India. PS and DM are recipients of a Senior Research Fellowship from CSIR and ICMR, Govt. of India respectively. References [1] Kane MM, Mosser DM. Leishmania parasites and their ploys to disrupt macrophage activation. Curr Opin Hematol 2000;7:26–31. [2] Berman JD. Human leishmaniasis: clinical, diagnostic, and chemotherapeutic developments in the last 10 years. Clin Infect Dis 1997;24:684–703 Review. [3] Ganguly S, Das NK, Barbhuiya JN, Chatterjee M. Post-kala-azar dermal leishmaniasis – an overview. Int J Dermatol 2010;49:921–31. [4] Naderer T, McConville MJ. The Leishmania–macrophage interaction: a metabolic perspective. Cell Microbiol 2008;10:301–8 Review. [5] Nylén S, Gautam S. Immunological perspectives of leishmaniasis. J Glob Infect Dis 2010;2:135–46. [6] Ota H, Takashima Y, Matsumoto Y, Hayashi Y, Matsumoto Y. Pretreatment of macrophages with the combination of IFN-gamma and IL-12 induces resistance to Leishmania major at the early phase of infection. J Vet Med Sci 2008;70:589–93. [7] Nylén S, Maurya R, Eidsmo L, Manandhar KD, Sundar S, Sacks D. Splenic accumulation of IL-10 mRNA in T cells distinct from CD4 + CD25+ (Foxp3) regulatory T cells in human visceral leishmaniasis. J Exp Med 2007;204:805–17. [8] Khoshdel A, Alborzi A, Rosouli M, Taheri E, Kiany S, Javadian MH. Increased levels of IL-10, IL-12, and IFN- in patients with visceral leishmaniasis. Braz J Infect Dis 2009;13:44–6.

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