International Immunopharmacology 11 (2011) 1668–1679

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International Immunopharmacology

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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.

⁎ Corresponding author. Tel.: +91 33 2223 4135; faxE-mail address: ilatim@vsnl.net (M. Chatterjee).

1567-5769/$ – see front matter © 2011 Elsevier B.V. Adoi:10.1016/j.intimp.2011.08.002

a b s t r a c t

a r t i c l e i n f o

Article history:Received 2 August 2011Accepted 3 August 2011Available online 27 August 2011

Keywords:LeishmaniasisAnti-leishmanialImmunomodulationChemotherapy

Leishmaniasis is caused by protozoan parasites of the genus Leishmania and causes a wide spectrum of clinicalmanifestations ranging from self-healing cutaneous lesions to the fatal visceral form. The use of pentavalentantimony, the mainstay of therapy of Leishmaniasis is now limited by its toxicity and alarming increase inunresponsiveness, especially in the Indian subcontinent. Furthermore, other anti-leishmanial drugs areunaffordable in many affected countries and as vaccination based approaches have not yet proved to beeffective, chemotherapy remains the only alternative, emphasizing the need for identifying novel drugtargets. In this review, we have described the different host immune signaling pathways that could beconsidered as potential drug targets for Leishmania chemotherapy.

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© 2011 Elsevier B.V. All rights reserved.

Contents

1. Leishmaniasis and clinical manifestations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16682. Disease immunopathogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16693. Chemotherapy of Leishmaniasis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16694. Targeting of host immunity by anti-leishmanial drugs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1669

4.1. Role of neutrophils. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16694.2. Monocytes and macrophages . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16694.3. Role of reactive oxygen species (ROS) and reactive nitrogen species (RNS) . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16704.4. Role of DCs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16704.5. Lymphocytes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16734.6. Macrophage derived cytokines as a measure of immunomodulatory activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16744.7. Effect on co-stimulatory molecules . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1674

5. Modulation of signaling events in Leishmania infection; role of chemotherapy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16755.1. Effect on expression of CD40 and MAPK signaling pathways . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16755.2. Toll like receptors and their responsiveness in Leishmania infection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16755.3. Leishmania infection and effect on JAK-STAT pathways . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16755.4. Modulation of NF-κB signaling pathways by Leishmania . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16765.5. Alterations of host cell kinases and phosphatases by Leishmania . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1676

6. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1676Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1676References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1676

1. Leishmaniasis and clinical manifestations

Leishmania spp. belonging to the order Kinetoplastida and familyTrypanosomatidae are responsible for the disease Leishmaniasis which isspread through the sand fly. The unique digenetic life cycle of the parasiteincludes spindle shaped, flagellated procyclic promastigotes that follow-

ing entry into the sandfly gut differentiate into non-dividing infectivemetacyclic cells; following a blood meal, they are taken up by circulatingprofessional phagocytic cells like neutrophils and macrophages toeventually transform into rounded amastigotes [1].

Clinical manifestations depends on the Leishmania species involvedand ranges froma life-threatening systemic infection (visceral, VL) to selflimiting or chronic skin sores (cutaneous, CL), or dreaded metastaticcomplications that can cause facial disfigurement (mucosal, MCL). Theclinical features of VL generally include prolonged and irregular fever,

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often associated with rigor and chills, hepatosplenomegaly, lymphade-nopathy, progressive anemia,weight loss and hypergammaglobulinemia(mainly IgG from polyclonal B cell activation) and concomitanthypoalbuminemia [2]. Even after recovery, African and IndianVLpatientsmay present with a secondary form called Post Kala-azar DermalLeishmaniasis [3].

2. Disease immunopathogenesis

Survival of the parasite within macrophages is crucial, which itensures by deviously manipulating the macrophage related immunefunctions [4 and references therein]. The engulfed pathogen preventsformation of phagolysosomes, and thereby suppressesMHC-IImediatedpresentation of the parasite antigen to CD4+ T cells, eventuallypreventing macrophages from eliminating the phagocytosed pathogen[4]. Furthermore, the parasite prevents initiation of a respiratory burst,ensuring that themacrophage is a safe haven for the parasite. Therefore,it is only logical to conclude that activation of macrophages would be ade novo chemotherapeutic strategy for Leishmaniasis.

The outcome of leishmanial infections is determined by twofunctionally distinct T-helper (Th) cell populations, Th1 (IFN-γ, IL-2)and Th2 (IL-4, IL-10 and IL-13). Generally, uncontrolled, non healinginfections i.e. disease susceptibility is associated with Th2 proliferationandproduction of IL 4, 5 and 10while in healing responses i.e. resistanceto disease, there is expansion of IFN-γ producing CD4+Th1 helper cells[5 and references therein]. This was confirmed when pretreatment ofmacrophages with IFN-γ and IL-12 induced resistance to L. major [6].Accordingly, in experimentalVL, key roles have been identified for IFN-γand IL-12 in mediating expansion of protective Th1 cells while IL-4 andIL-10 mediate progression of infection following expansion of Th2 cells[5]. However, in human VL, the response is not a strictly polarized Th2type, as measurement of splenic and bone marrow cytokine mRNA atdisease presentation showed increased levels of both IL-10 and IFN-γ[7]. Similiarly at presentation, increased circulating levels of IL-10, IL-12and IFN-γ, suggesting an initial mixed Th1- Th2 response have beenreported, while disease resolution was associated with a simultaneousdecrease in both IL-10 and IFN-γ, indicating that both Th1 and Th2 arecomponents of the immune system during active disease and bothregress with effective treatment [8].

Patients with VL usually demonstrate a negative skin test toLeishmania antigens and peripheral blood mononuclear cells fail toproliferate and produce IFN-γ when exposed to specific antigen invitro, indicative of anergy; addition of anti-IL-10R antibody to Tcells 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 ofmacrophage function for production of toxic nitrogen and oxygenmetabolites, necessary to kill resident amastigotes [11 andreferences therein].

3. Chemotherapy of Leishmaniasis

Currently, drugs used to treat Leishmaniasis are handicapped byemergence of strains resistant to conventional antimony, associatedtoxicities and high cost especially regarding lipid formulations ofamphotericin B ([12], Table 1). A matter of concern is that Miltefosine,the only orally effective drug can potentially become ineffective, asstudies with resistant amastigotes have shown the presence ofmutant drug transporters [13]. Taken together, the current arma-mentarium of Leishmaniasis is limited and alternative therapies arestrongly warranted. An area of growing interest is the potential ofnatural plant-derived products of diverse structural classes havinganti-leishmanial properties [14–16] and includes naphthylisoquino-line alkaloids and synthetic analogs, Luteolin, Quassin, Curcumin,Artemisinin and several others [14,17–22].

4. Targeting of host immunity by anti-leishmanial drugs

Within the mammalian host, Leishmania reside as amastigotes inphagocytic cells that include neutrophils, macrophages and dendriticcells (DCs); therefore, an immunomodulatory compound could bepotentially leishmanicidal by virtue of its potential to activatephagocytic cells.

4.1. Role of neutrophils

The best characterized function of polymorphonuclear neutrophils(PMNs) is their preeminent role in phagocytosis and killing ofinvading microorganisms via generation of reactive oxygen species(ROS) and release of lytic enzymes. Following entry of Leishmania intothe mammalian host, PMNs are recruited immediately to the site ofinfection within 24 h, implying that they possibly serve as host cellsfor Leishmania in the very early phase of infection [23,24]. Neutrophilsbeing inherently short-lived undergo apoptosis [23], while Leishmaniaparasites are known to delay their apoptosis, possibly by interferingwith production of ROS, which importantly facilitates their survival[25,26]. To trigger apoptosis, neutrophils utilize a mitogen activatedprotein kinase (MAPK) signaling pathway, p38 MAPK being a keyplayer [27]. Recent data suggests a critical role for neutrophils in theearly protective response against L. donovani, both as effector cellsinvolved in parasite killing and for influencing development of aprotective Th1 response [28].

Importantly, Leishmania parasites that enter macrophages via theuptake of infected, apoptotic PMNs then survive and multiplyeffectively [23]. The amount of TGF-β secreted by macrophagesfollowing uptake of infected PMNswas higher than after direct uptakeof 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 harborand transport Leishmania, targeting pathogens residing in neutrophilsshould be taken into consideration when designing novel anti-leishmanial compounds.

Therefore, it is tempting to extrapolate that a compound capable ofincreasing phagocytic activity and generating an oxidative burst withinLeishmania infected neutrophils would effectively eliminate parasites.Indeed, antimonials increase the phagocytic capacity of neutrophilsalong with increased production of superoxide [29]. Berberine chloride(Table 2) also promoted parasite elimination via enhancement ofapoptosis in L. donovani infected neutrophils, subsequent tomodulationof the MAP kinase pathways [30].

4.2. Monocytes and macrophages

To sustain infection, it is mandatory that Leishmania parasitesestablish themselves in macrophages, but considering the potentantimicrobial functions of macrophages, the subject of how Leishmaniasurvive is a subject of intense research.

(i) Phagocytosis: C3b is a complement protein that followingbinding to Leishmania surface glycoprotein gp63 increasesparasite uptake into macrophages as gp63 cleverly convertsC3b into iC3b, which then favors phagocytosis, yet preventslytic clearance [31]. Antimonials [29], Pourouma guinensis[Oleanolic acid, 32] and Diphyllin isolated from Haplophyllumbucharicum Litv [33] influence the phagocytic activity ofmacrophages as do CpG oligodeoxynucleotide (CpG ODN)and miltefosine [34].

(ii) Acidification: Generally, following fusion of the phagosomewiththe endosomal compartment, a significant drop in pH ensues.However, Leishmania produce a surface acid phosphatase thatinhibits theoxidative burstwithinmacrophages, and additionallyis an active proton pump keeps the intracellular pH close to

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

Drug Mode(s) of action Dosage (for VL) advantages Limitations Ref

Pentavalent antimonials:Meglumine antimoniate(Glucantime) or sodiumstibogluconate (Pentostam)

Activated within the amastigote/macrophageafter conversion to the trivalent form.Shows direct parasiticidal activity bygeneration of ROS, depletion of thiols,modulation of bioenergetic pathways(glycolysis, fatty acid beta oxidation,inhibition of ADP phosphorylation,blocking of SH groups of amastigoteproteins) and inhibition oftopoisomerase II

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

Easily availability andlow cost

Myalgia, pancreatitis,cardiac arrhythmias,hepatitisAcquired resistance

[13,130,131and ref.therein]

Amphotericin B(polyene antibiotic)

Complexes with 24-substituted sterols, such asergosterol in the cell membrane, causing poreswhich alter ion balance, increase membranepermeability resulting in cell death; also actsas an inhibitor of ergosterol biosynthesis

0.75–1.0 mg/kg for15–20 infusionseither daily onalternate days inIndia

Primary resistance isunknown

Need for prolongedhospitalizationHigh cost, high feverwith rigor, chills,hypokalemia, renaldysfunction

[130,131and ref.therein]

Lipid formulation of amphotericinB Ambisome/Abelcet/Amphotec

-do- Ambisome: 2.0 mg/kg×5 days, i.v. inIndia

Highly effective, lowtoxicity

High cost [131]

Paromomycin (aminoglycosideantibiotic), also known asaminosidine or monomycin

In bacteria, inhibits protein synthesis, but inLeishmania, the exact mechanism is not yet known.It is proposed to induce respiratory dysfunction in L.donovani promastigotes. It also promoted ribosomalsubunit association of both cytoplasmic andmitochondrial forms, low Mg+2 which induceddissociation

16 mg/kg×21 days, i.m.: 20 mg/kg×17 days, i.m.

Effective, well toleratedand relatively cheap,acts synergistically withantimonials

Lack of efficacy inEast Africa

130 and ref.therein,132]

Miltefosine(hexadecylphosph-ocholine)

It interacts with the cell membrane of Leishmania bymodulation of cell surface receptors, inositolmetabolism and phospholipase activation, celldeath being mediated by apoptosis

100–150 mg for fourweeks, p.o. in India

Effective and safe Vomiting anddiarrhoea.nephrotoxic,teratogenic

[130 andref. therein]

Sitamaquine(8-aminoquinoline,originally WR6026)

Unknown, possibly affects mitochondrial electrontransport chain

1.75–2 mg/kg/dayfor 28 days in India.

Little is known about itsefficacy and toxicity

[133]

1670 P. Saha et al. / International Immunopharmacology 11 (2011) 1668–1679

neutral [35]. Tamoxifen similarly modulates the macrophageintravacuolar compartment by causing a rapid, long-lastingalkalinization [36].

4.3. Role of reactive oxygen species (ROS) and reactive nitrogen species(RNS)

As nitric oxide (NO) is an effector molecule critical for eliminationof intracellular Leishmania parasites, disease progression is ensuredvia enhancement of Th2 responses that causes deactivation ofmacrophages and decreased production of NO ([4], Fig. 1). Therefore,parasite removal should entail activation of infected macrophages byincreased expression of inducible nitric oxide synthase (iNOS) to formNO [37]. During Leishmania infection, decreased expression orinactivation of iNOS may also be associated with increased activationof arginase as deprivation of L-Arginine impairs Leishmania major-specific T-cell responses [38].

Following parasite engulfment by macrophages, NAD(P)H oxidasesare initially activated,which transfer the reducing equivalents fromNAD(P)H to molecular oxygen leading to formation of extremely reactivesuperoxide [11]. These then react with parasite membrane phospho-lipids leading to increased permeabilization as also react with thepathogen's macromolecules such as DNA leading to strand breaks;However, when the infection is sustained, macrophages are deactivatedcausing a decreased production of superoxide which is now beneficialfor parasite survival (Fig. 1). Therefore, it is anticipated that if a similarpro-oxidant scenario is recreated by anti-leishmanial drugs, they caneffectively eliminate the parasite [21,39]. Conventional anti-leishmanialdrugs like antimonials [29,40], Miltefosine [41,42] and amphotericin B[43] increase generation of ROS and NO in Leishmania infectedmacrophages, as do other immunomodulatory compounds alone or incombination with sub-optimal doses of SAG e.g. a mononuclear

diperoxovanadate compound K[VO(O2)2(H2O)]PV6 [44]. Similarly, anethanolic extract of Tinospora sinensis [45], 18 Beta-glycyrrhetinic acid[46], tannins and related compounds [47], trinitroglycerin [48] alsoupregulated production of NO (Table 2). Interestingly, Cystatin alongwith IFN-γ, induced generation of NO but markedly reduced expressionof iNOS at bothmRNA andprotein levels [49–51]. Other compounds thatshowed similar NO enhancing activity were Artemisinin [22], quassin[19], an aqueous extract of human placenta [52], eugenol-rich essentialoil from Ocimum gratissimum [53], glycolipids along with otherconstituents from Desmodium gangeticum [54], a linalool-rich essentialOil fromCroton cajucara [55],Kalanchoe pinnata [56,57] andAloe vera leafexudate [58,59]. A similar observation was evident following treatmentwith Chenopodium ambrosioides essential oils [60], Pyrazinamide [61],Himathantus sucuuba Latex (HsL) [62], quinovic acid glycosides andcadambine acid [63]. The scenario was similar with CpG-containingoligodeoxynucleotide, CpG-ODN [64], 2, 3, 7, 8-Tetrachlorodibenzo-P-dioxin [65] and Berberine chloride ([66], Table 2).

However, some compounds exert their inhibitory effect onamastigotes 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 supercriticalfluid fraction obtained from Tabernaemontana catharinensis [70].

4.4. Role of DCs

The interaction of Leishmania parasites with DCs is complex, asdepending upon the species of Leishmania, the DC subset and otherexogenous stimuli involved, there can either be control of infection ordisease progression, [71 and references therein]. The first study withmurine skin DC implicated epidermal Langerhans cells as importantcells for detection, uptake and transport of Leishmania to lymph nodes[72]. Dermal DCs efficiently incorporate parasites into vacuoles and

able 2ntileishmanial compounds having immunomodulatory activity.

Compound Leishmania spp.IC50 in amastigotes(ex vivo)

In vivo Mode(s) of action References

Antimonials sodiumantimony gluconate

L. donovani1.38 μg/ml–112.77 μg/ml

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

Induced ERK-1/2 and p38 MAPKphosphorylation, increased productionof ROS, NO and TNF-α

[14,134,135]

Meglumineantimonate

Increased phagocytic capacity ofmonocytes and neutrophils, enhancedgeneration of superoxide anion andproduction of TNF and NO

Amphotericin B L. donovani 0.033–0.4 μM

L. infantum, hamsters, (1 mg/kgb.w., i.c. on days 25, 26 and 27post-infection), reduced parasiteload in liver and spleen by 88.8%and 87.2% on day 32 and on day135, by 66.7% and 54%respectively

Up regulated levels of IL-12, TNF-α andexpression of iNOS along with downregulated IL-10 and TGF-β production.It restored the impaired classical PKCand abrogated the atypical PKC

[43,136–139]

Water-solubleamphotericin B(AmB)-arabinogalactan

L. donovani optimaldose, 2.0 μg/ml

L. donovani, hamsters; 6 mg/kgb.w., i.c.×1, suppressed 99%parasitic burden in liverand spleen.

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

Artemisinin, asesquiterpeneendoperoxide

L. donovani, 22 μM BALB/c mice, 10–25 mg/kg b.w., p.o.reduced splenic weight andparasite burden by 82.6% and86.0% respectively

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

[21,22,140]

ArtemisininArtemether

L. major 3×10 (−5)M3 X10 (-6)M

(i) Berberine chloride(quarternaryisoquinolinealkaloid)

(ii) 8 cyanodihydroberberine

(i) L. donovani 2.5 μM (i) L. donovani, hamsters, 50 and 100 mg/kgb.w., i.p. ×4 decreased parasite burden inliver by 48.5 and 61.1% respectively. 50 mg/kgb.w., i.p. ×5 days, repeated after 5 daysreduced parasite no. from 1.67×109 to0.163×109 (liver) and 1.55×109 to0.097×109 (spleen)

Increased production of NO andexpression of iNOS and IL-12p40 alongwith down regulation of IL-10, inducedphosphorylation of p38 MAPK anddecreased phosphorylation of ERK1/2.

[39,66,141–143]

(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. doseof 208 mg/kg b.w., i.m. twice daily ×4caused 54% and 46% suppression respectively.

(iii) Tetrahydroberberine

(iii) L. donovani, hamsters, max. dose of 416 mg/kgb.w. (i.m.), twice daily ×4, less toxic and morepotent against L. donovani showed 50% suppression

CpG-containingoligodeoxynucleotide(CpG-ODN) incorporatedin mannose-coatedliposomes

L. donovani L. donovani, BALB/c mice Enhanced generation of NO, ROS andH2O2, reduced levels of IL-4, butincreased levels of IFN-γ, IL-12 andiNOS.

[34,64]

(i) Man-lip-CpG (i) 3.01 μg/ml, (i) CpG-ODN, 2.5 mg/kg b.w./day, x 15, i.p.caused 100% parasite suppression(ii) lip-CpG (ii) 4.05 μg/ml(ii) 2.5 mg/kg b.w./day, ×15, i.p. caused 81%reduction

(iii) free CpG-ODN (iii) 4.61 μg/ml

(iii) 2.5 mg/kg b.w./day, ×15 i.p. caused 62 %reductionglc-lip- CpG, 2.5 mg/kg b.w./day ×15 days, i.p.caused 86 % reductionL .donovani, BALB/c mice, hamsters

Increased phagocytosis index andcombination therapy involving free orliposomal CpG ODN with miltefosineincreased TNF, IFN-γ and IL-2 cytokinelevels and downregulated IL-4 and IL-5.

(i) 1 nM, single dose, i.p., freeand liposomal forms +miltefosine, (2.5 mg/kg ×5 days, p.o.) caused 85% inhibition.(ii) In hamsters, 1 nM, singledose, i.p. + miltefosine (5 mg/kgb.w. ×5 days, p.o.) caused81.7% inhibition.

Cystatin L. donovani 5.2 μg/mlIFN-γ + Cystatin 4.3μg/ml

L. donovani, BALB/c mice, 20 mg/kgb.w./day ×4 caused marked suppression.5 mg/kg b.w./day +104 U IFN-γ ×4 days i.v. eliminated all parasites0.5 mg/kg b.w./day + IFN-γ (100 U/ml) ×4, i.v. decreased parasite burden

Induced ERK1/2 phosphorylation andNF-kB DNA-binding activity, IFN-γ-mediated JAK-STAT activation,generation of NO from macrophages,increased levels of IL-12 and TNF-α andiNOS and decreased IL-10 secretion.Cystatin + IFN-γ induced TLR/MyD88signaling

[49–51,144,145]

Two recombinantbarley cystatins

L. infantum(% reduction)

(i) HvCPI5 (i) 0.1 μM and 1 μM:35.9 ± 9.9 and 36.4 ±2.2 respectively

(ii) HvCPI6

(ii) 0.1 μM and 1 μM:31.9 ± 11.9 and 28.3±7.8 respectively

1671P. Saha et al. / International Immunopharmacology 11 (2011) 1668–1679

TA

(continued on next page)

Table 2 (continued)

Compound Leishmania spp.IC50 in amastigotes(ex vivo)

In vivo Mode(s) of action References

Desmodium gangeticum(Glycolipids and otherconstituents from

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

Increased production of NO [54,146]

(i) EtOH extract(ii) Aminoglucosylglycerolipid

(ii) 100 μg/ml: 72 ±5% inhibition;

(iii) Glycosphingolipid(cerebroside)

(iii) 100 μg/ml: 53.4 ±4.4% inhibition

Diperoxovanadatecompd. K[VO(O2)2(H2O)]−PV6 (10 μM) + SAG in

L. donovani killed by L. donovani, BALB/c mice; PV6 (0.5 μM, i.p.)alternative day×4 weeks decreasedparasite load by

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

[44]

(i) Sb-sensitive (SbS-LD) (i) 72% (i) 72.7 ± 7.44%(ii) Sb-resistant (SbR-LD) (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 splenicparasite burden, 77.1 ± 5.86% and 79.2 ±3.62% respectively

Diphyllin Leishmania spp. 0.2μM

– Modulated phagocytic activity ofmacrophages

[33]

(3S)-16,17-Didehydrofalcarinol, anOxylipin isolated from,Tridax procumbens

L. mexicana Decreased production of NO. [67]

(i) MeOH crude extract(ii) Hexane extract(iii) Compound 1

(iii) 0.48 μM

Eugenol-rich essentialoil from Ocimumgratissimum

L. amazonensis 100μg/ml

– Increased production of NO [53]

Glycyrrhizza glabra L.(Licorice) 18Beta-glycyrrhetinicacid (GRA)

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, butincreased levels of IL-12, IFN-γ, TNF-α,and iNOS, induced NF-κBmigration intothe nucleus

[46]

Imiquimod – L. tropica, patients with CL, Imiquimod (5% cream)+Meglumine antimonite 20 mg/kg. b.w. weekly for 4weeks. Reduced histiocytic cellular aggregation.

Reduced neutrophil and macrophageinfiltration in the lesion, depleted CD1a+ dendritic cell in epidermis and CD68+ macrophages in dermis

[147–150]

L. major, combination with Glucantime, decreasedparasitic load and thickness in footpad

Decreased production of IFN-γ, IL-4 andIL-10

Patients with CL,administered 7.5%Imiquimod everyalternative day 20days + 20 mg/kgb.w. Meglumineantimonate i.v,cure rate 100%

Kalanchoe pinnata(Crassulaceae)(Kp, synonimiaBriophyllum pinnatum

L. amazonenesis 500μg/ml caused 58%reduction

(i) L. amazonenesis, BALB/c mice, leafy extract,8 mg/kg b.w, p.o. ×18 days suppressed parasitegrowth

Suppressed antibody production anddeveloped delayed-typehypersensitivity and produced specificantibodies.

[56,57,151–153]

(i) Leafy aqueous extract (i) 320 mg/kg b.w. , p.o. ×30 days suppressedparasitic burden(ii) Quercetin 3-O-α-L-

arabinopyranosyl (1→ 2)α-L-rhamnopyranoside,

(i) L. chagasi, BALB/c mice, 400 mg/kg b.w. p.o. (day1 to 29), decreased parasite burden in spleen andliver by 4 and 6 fold respectively

Increased production of NO.

(iii) Free quercetin Flavonoids

L. amazonensis 8−N100 μg/ml

(ii) L. amazonensis, BALB/c mice, 16 mg/kg b.w, p.o×30 days reduced parasitic burden.

Miltefosine L. donovani 0.9–4.3 μM L. donovani, BALB/c mice, 20, 25 and 30 mg/kg b.w./day ×5, p.o. reduced parasite burden by 51%, 76%,and 81% respectively.

Stimulated T cells and macrophages,increased secretion of pro-inflammatory cytokines, includingIFN-γ, and enhanced production of RNIsand ROIs as also expression of STATreceptors, induced PKC- and PI3K-dependent p38MAPK phosphorylationand production of CD40-induced IL-12;it also activated DCs.

[41,74,136,137,154]

L. major 32–37 μM L. major, BALB/c mice 10 and 25mg/kg b.w./day ×10,p.o. decreased parasite burden by 87 and 98%respectively.

Momordica charantia L. donovani, hamsters Inhibited superoxide dismutase (SOD)activity

[155](i) Aqueous extract (i) 300 mg/kg b.w. alt day ×15: 100% clearance(ii) Momordicatin (ii) 10 mg/kg b.w., alt day ×15: 100% clearancePourouma guinensis L .amazonenesis Inhibited phagocytic activity of

macrophages.[32]

(i) Ursolic acid (i) 11 μg/mL(ii) Oleanolic acid (ii) 27 μg/mL

1672 P. Saha et al. / International Immunopharmacology 11 (2011) 1668–1679

Table 2 (continued)

Compound Leishmania spp.IC50 in amastigotes(ex vivo)

In vivo Mode(s) of action References

Pyrazinamide L. major 10.2 μg/ml – Increased IL-12, TNF-α, and productionof NO.

[61]

Increased expression of costimulatorymolecules CD80, CD86, andMHC class IIas also slightly increased IL-10

Quassin L. donovani 25 μg/ml – Enhanced NO generation and iNOSexpression both at a protein and mRNAlevel and up-regulated proinflammatory cytokines such as TNF-αand IL-12.

[19]

Quinovic acid glycosides andcadambine acid isolatedfrom Nauclea diderrichii.

L. infantum, 1.0 μM – Induced NO production. [63]

Tabernaemontana catharinensis(leaves) Superficial fluid ofEtOH fraction

L. amazonensis 100 μg/ml. caused 88%inhibition

– Inhibited production of NO and TGF-ß [70]

Tamoxifen, an antioestrogen, atri phenylethylene

L. amazonensis 11.1±0.2 μM

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

Induced rapid alkalinization ofparasitophorous vacuoles.

[36,156,157]

L.(V.) braziliensis 1.9 ±0.2 μML. (L.) chagasi 2.4 ±0.3 μM

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

Tannins Activated macrophages and up-regulated NO, TNF, IFN-γ, iNOS, IL-1, IL-10, IL-12 and IL-18 expression.

[47,80,81,84,158,159](i) Proanthocyanidinins (i) L. donovani 0.7–8.0

nM(ii) Hydrolyzable tannins(ii) b0.4–12.5 μg/mlL. major, L. donovani1–2 μM

(iii) Polyphenols and hydroxyltannins

(iii) L. major, L.donovani 1–250 μM

Hydrolyzable tannins induced therelease of NO, TNF and IFN.

(iv) Sage phenolics

(iv) Leishmania spp.3.9–22.6 nm

Terpenoids monoterpene L. amazonensis Increased production of NO [55](i) Linalool-rich essential oilfrom Croton cajucara

(i) 22.0 ng/ml

(ii) Linalool (ii) 15.5 ng/ml2,3,7,8-Tetrachlorodibenzo-P-dioxin (TCDD)

L. major, C57BL/6 mice, 40 μg/kg b.w., s.c.×1, prior toinfection (single dose) caused a 10 fold decrease inparasite burden.

Increased IL-2 production. [65]

Thalidomide and glucantime L. major, BALB/c mice, thalidomide 30mg/kg/b.w./day×12 days (p.o.) + Glucantime 200mg/kg/b.w./day×12 days (i.p.) decreased parasiteload

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

[79]

(i) Tinospora sinensis Linn(Ethanolic extract)

L. donovani(i) 29.83 ±3.4 μg/ml

L. donovani, hamsters(i) 500 mg/kg b.w./day×5 days, p.o. inhibited by76.2 ± 9.2%

The ethanolic extract and butanolfraction enhanced ROS and NOproduction as also activatedmacrophages.

[45,160]

(ii) Hexane fraction (ii) N100 μg/ml(iii) Chloroform fraction (iii) N10 μg/ml;(iv) Butanol fraction (iv) 17.6 ± 4.1 μg/ml; (iv) 250 mg/kg b.w., p.o. inhibited by 72.8 ± 4%(v) Aqueous fraction (v) N100 μg/mlTrinitroglycerine (TNG) L. major, BALB/c mice, 200 μg, s.c.×15 days

decreased parasite load in lesions (59% to 49%, inliver (98% to 64%), spleen (98% to 49%) and in lymphnodes (78% to 15%)

Increased NO production. [48]

Z-100, polysaccharide fromMycobacterium tuberculosis+ meglumine antimoniate

L. amazonensis 13.0mg/L

L. amazonensis, BALB/c, 100 μg/kg/b.w. i.p.× 2 wksbetween the 6th and 8th wk was notantileishmanial; Z-100 + SAG (14/28 mg/kg b.w., i.l.×2 wks between the 6th–8th wk caused 99%inhibition

Upregulation of IFN-γ along with IL-10and IL-4.

[78]

Both IgG1 and IgG2a were increased.

1673P. Saha et al. / International Immunopharmacology 11 (2011) 1668–1679

are proposed to act as principal APCs in Leishmaniasis, while otherssuggest that lymph node resident DCs are initiators of the immuneresponse [71].

Leishmania have cleverly devised several strategies to avoid DCs,as in humans, L. donovani blocks maturation of DC [73] andproduction of IL-12, essential for initiation of a protective immuneresponse. Accordingly, Miltefosine in turn can activate DCs [74] asalso does Pyrazinamide via increased secretion of proinflammatorymolecules and enhanced expression of co-stimulatory molecules([61], Table 2).

4.5. Lymphocytes

T lymphocytes are generally responsible for intracellular pathogenelimination whereas B lymphocytes eliminate extracellular bacteria. Inorder to eliminate Leishmania, the macrophage needs to be activated byantigen specific T lymphocytes who by secreting IFN-γ, upregulateproduction of NO from macrophages. Both CD4 and CD8 cells arerequired for resolving the infection, along with a balance between Th1and Th2, preferably a Th1 skewed response [5]. Therefore, essentialprerequisites of an effective immunomodulatory, anti-leishmanial drug

Fig. 1. Schematic representation of Leishmania induced signaling events within macrophages: The different targets (1–8) within the macrophages modulated by Leishmania arepotential 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-6andMyd88 (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 maturationof phagolysosomes leading to increased assembly of NADPH oxidase complex and (8) Downregulation of PTPs which in turn would upregulate expression of pro-inflammatorymediators. 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 ActivatedProtein 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.

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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 ofco-stimulatory molecule(s) [5], and so this too can be an additionaltarget. Restoration of the lymphoproliferative capacity is achieved byMiltefosine [74], AmB in association with a suboptimum dose ofstearylamine-bearing cationic liposomes [75], a mononuclear diperox-ovanadate compound K [VO (O2)2(H2O)] (PV6) along with SAG [44] anda human placental extract ([52], Table 2).

Although Leishmaniasis is characterized by the appearance of anti-leishmanial antibodies, B cells and antibodies are unimportant, asparasites tend to hide within the macrophage parasitophorus vacuole[5]. In experimental models, it has been observed that anti-leishmanialantibodies play a contributory role as IgG coated parasites upon ligationwith Fc receptors on macrophages [1] or dendritic cells [76] induceincreased production of IL-10, a key cytokine for disease persistence.Furthermore, this was corroborated by studies with B cell depletedanimals that were found to be highly resistant to Leishmaniasis [77].Liposomal Z-100 andKalanchoe pinnatadecreased IgG and its subclasses([56,78], Table 2).

4.6. Macrophage derived cytokines as a measure of immunomodulatoryactivity

The immunomodulatory potential of anti-leishmanial drugs hasbeen established by measuring its influence on macrophage derivedcytokines, mainly IFN-γ, IL-12, TNF-α and IL-10. Miltefosine[41,42,74], glucantime [79], and Amphotericin B [43] along withexperimentally effective compounds such as 18 Beta-glycyrrhetinicacid [46], tannins [47,80,81], artemisinin [22], quassin [19], aqueousextract of human placenta [52], garlic [82], pyrazinamide [61], CpG-containing oligodeoxynucleotide [64], Berberine chloride [66] and2,3,7,8-Tetrachlorodibenzo-P-dioxin [65], collectively support thenotion that upregulation of the Th1 response is an effective strategyfor parasite elimination. IL-6 and IL-1β are potent pro-inflammatory

cytokines involved in the generation of NO andmacrophage activationwhich are increased by antimonials [83], tannins and relatedcompounds [47,80,81] as also sage phenolics [84].

Chemokines, a superfamily of low MW cytokines recruit distinctsubsets of leukocytes and by activating them play an important role inLeishmaniasis. TNF-α and IL-1β together with MIP-1α (Macrophageinflammatory protein 1α) regulates transport of Leishmania frominfected sites to lymph nodes [85 and references therein]. DuringLeishmaniasis, IFN-γ together with macrophage chemotactic protein1 (MCP-1) eliminates L. major while conversely, IL-4 antagonizesproduction of MCP-1 [86]. Essential oil and extracts from Xylopiadiscreta induced differential production of MCP-1 in leishmaniasis[87]. IL-8 is another chemokine that controls the early infection ofLeishmania via recruitment of neutrophils [88] and release of NO alongwith pro-inflammatory cytokines from macrophages [89]; SAG in factinduces IL-8 synthesis in patients with CL [83]. It has been shown thatco-incubation of Leishmania parasites with PMNs inhibits the CXC-chemokine and interferon-γ inducible protein-10 (IP-10), accountingfor its Th1 inhibiting activity [88]. Furthermore, as IP-10 and CXCL-10induce NK cells [85] it suggests that induction of chemokines withinLeishmania infected cells could also be an effective strategy.

4.7. Effect on co-stimulatory molecules

T cell mediated regulation of immune responses is intimatelyassociated with co-stimulatory molecules present on APCs, as theycan modulate the TCR-MHC interaction. Among them CD28, plays apivotal role as their enhanced or diminished expression causesimmune activation or anergy respectively [90] owing to theirinteraction with B7.1 (CD80) or B7.2 (CD86) present on monocyte/macrophages and/or B cells. In PKDL, increased levels of circulatingCD8+28- lymphocytes confers immune anergy, evidenced by theirnon 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 Miltefosineand SAG, the effect of Miltefosine being greater [92]. Pyrazinamideenhanced expression of CD80 and CD86 in Leishmania infected BALB/Cmice [61] as did an aqueous extract of human placenta, evidenced byan increased expression of MHC molecules on APCs ([52], Table 2).

5. Modulation of signaling events in Leishmania infection; role ofchemotherapy

5.1. Effect on expression of CD40 and MAPK signaling pathways

An important co-stimulatory molecule that determines the outcomeof macrophage-Leishmania interactions is CD40 as the CD40-CD40Linteraction helps increase the Th1 immune response [93].With regard toLeishmania infection, CD40 mediated MAPKs have been reported topromote parasite survival by modulating the expression of IL-10 and IL-12 in macrophages [94]. MAPKs, a group of serine/theonine kinases areresponsible for phosphorylation of cellular proteins which in turntriggers signals necessary for cell proliferation, differentiation andsurvival [95]. The CD40 of macrophages interacts with CD40-L of T cellsand passes the signal onwards to produce IL-12 via p38MAPK andNF-kB(nuclear factor kB, Fig. 1). The released IL-12 thenbinds to IL-12 receptorspresent onmacrophages, increases their production of IFN-γ, which thenacts on infected macrophages to induce parasite killing. However, thisCD40-CD40L interaction has been proposed to exert a dual effect, aswhen CD40 signaling is associated with depletion of cholesterol andTRAF-6, it causes activation of ERK1/2, higher levels of IL-10 follow alongwith decreased levels of IL-12p40. Conversely, if the CD40 signalosome isassociated with normal levels of cholesterol and TRAF-2/3/5, it causesp38 MAPK activation which is accompanied with increased leishmani-cidal IL-12 p40 and accompanying pro-inflammatory responses [93,96].It has been proposed that Leishmania lipophosphoglycans stimulate theERK pathway, which in turn inhibitsmacrophage production of IL-12 [94and references therein]. It has also been demonstrated that parasites canmodulate the TLR2-stimulated MAPK pathway by suppressing phos-phorylation of p38 MAPK along with enhanced phosphorylation ofERK1/2 [97]. Nitric oxide, a crucial mediator for leishmanicidal activity,was found to be dependent on iNOS expression and was linked to theMAPKs signaling pathway ([42], Fig. 1). Ben-Othman et al., 2008 [98]showed that Leishmania initially activated but subsequently down-regulated intracellular MAPKs and NF-kB signaling in macrophages.Taken together, as ERK and p38 MAP kinases differentially regulateinduction of macrophage effector molecules which dictate the course ofinfection, one is tempted to propose that these kinases be considered aspotential targets for development of novel strategies to combatLeishmaniasis, as demonstrated with Miltefosine [42].

The classical anti-leishmanial drug SAG modulates signalingpathways such that it induces an early wave of ROS-dependentparasite killing followed by a stronger late wave of NO-dependentparasite killing via phosphorylation of ERK1/2, and p38 MAPK [40].The activation of ERK1/2 resulted in increased production of ROSwhile p38 MAPK activation increased release of TNF-alpha and NO[40]. Cystatin activated the ERK1/2 pathway in presence of IFN-γ anddecreased iNOS induction in macrophages [51] while Berberinechloride, a potent anti-leishmanial compound exerted its leishmani-cidal activity via increased IL-12 following enhanced phosphorylationof p38MAPK, andwas accompaniedwith a down regulation of ERK1/2and 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 receptorsthat are of critical importance for initiation of an efficient immuneresponse [99]. Innate immunity coordinates the inflammatoryresponse to pathogens, wherein the contribution of TLRs is widelyrecognized. These TLRs are located either on the plasmamembrane or

within the endosomal membrane of macrophages, DCs, NK cells asalso T and B lymphocytes. Mammalian cells express up to 12 differentTLRs [99] which share an intracellular domain, called Toll-IL-1R [99];amongst them, some signal through the myeloid differentiationprotein 88 (MyD88) [100] which ultimately leads to nucleartranslocation of NF-κB and expression of proinflammatory cytokinesthat includes TNF-α, IL-12 along with iNOS, collectively causing hostprotection [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 asalso undergoes autophosphorylation; subsequently, dissociation ofIRAK-1 from MyD88 follows so that it can now interact with TRAF6,which finally activates various cascades, leading to activation of MAPkinase pathways, translocation of NF-κB to nucleus and secretion ofproinflammatory cytokines [102]. As Leishmania infection is associatedwith inhibition of IRAK mediated signaling (Fig. 1), the control ofLeishmaniaparasites in vivo requires the adaptor proteinMyD88 [103] asgenetically resistant C57BL/6 mice became susceptible to Leishmania inthe absence of MyD88 ([104], Fig. 1). Furthermore, silencing of TLR2,TLR3, IRAK-1 and MyD88 expression by RNA interference also led todecrease production of NO and TNF-α bymacrophages in response to L.donovani promastigotes [105]. Studies showed that TLR4 signaling canenhance the microbicidal activity of macrophages harboring parasites[103] and Bhattacharya et al., 2010 [106] have demonstrated that theleishmanicidal potential of Arabinosylated Lipoarabinomannan wasmediated through upregulation of TLR2 signals corroborating itsimportance 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 hostimmune response in Leishmania infection as they trigger a signalingpathway through a cascade of intracytoplasmic proteins known asJanus Kinase and signal transducer and activator of transcriptions[STATs, 107]. The biological effects of IFN-γ are dependent uponactivation of STAT1 transcription factors as ligation of IFN-γ withIFN-γ receptor (IFN-γ R) activates JAK1/JAK2 kinase which thenphosphorylates STAT-1; the STAT1 then translocates to the nucleusand further enhances transcription of IFN-γ-induced genes (Fig. 1,[108]). The induction of iNOS by LPS and IFN-γ is primarily controlledby two regulatory regions present in the iNOS promoter that containsbinding sequences for two transcription factors, NF-κB and IFNregulatory factor 1 [IRF-1, 109]. The synergistic role of IFN-γ ininduction of iNOS is attributed to its ability to induce expression ofSTAT1 and IRF-1 transcriptional complexes that also bind to the IFN-γactivating 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 theJAK2/STAT1 signaling cascade, as infected macrophages on stimula-tion with IFN-γ, showed defective phosphorylation of JAK1, JAK2, andSTAT1 [111]. Both L. major and L. mexicana suppressed expression ofIFN-γRα and IFN-γRβ, reduced levels of total JAK1 and JAK2, anddownregulated IFN-γ-induced JAK1, JAK2, and STAT1 activation, theeffects being more profound with L. mexicana than L. major (Fig. 1,[112]).

Wadhone et al., 2009 [42] demonstrated that miltefosine effectivelymodulates host cell-dependent signaling pathways, restores respon-siveness to IFN-γ via enhancement of IFN-γ receptors, IFN-γ inducedSTAT-1 phosphorylation and reduced activation of SHP-1 (the phos-phatase implicated in down-regulation of STAT-1 phosphorylation).Another immunomodulatory anti-leishmanial cystatin, along withIFN-γ, modulated production of NO in macrophages that was partlydependent 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 thecommonest [113]. In resting cells, NF-κB is retained in the cytoplasmcomplexed with its inhibitory subunit IκBα; following agonist stimu-lation, an enhanced serine phosphorylation of IκBα triggers itsproteasomal degradation, resulting in subsequent activation of NF-κB;the MAPK signaling pathways have been identified as the upstreamkinases that induceNF-κB activation via phosphorylation of its inhibitor,IκBα [114]. These signals induce IκB kinase (IKK) and after activation ofthe IKK complex, specific IκBα phosphorylation/degradation causessubsequent release of NF-κB and its translocation to the nucleusactivates transcription of multiple κB-dependent genes, including iNOSand Th1 cytokines [115]. Therefore, preventing the degradation of IκBand its downstream events is a strategy used by L. donovanipromastigotes to effectively shut down the NF-κB-dependent expres-sion of proinflammatory cytokines, ultimately translating into loweredproduction of NO (Fig. 1, [116]). Conversely, 18 Beta-glycyrrhetinic acidmediated its anti-leishmanial activity by inducing nuclear migration ofNF-κB in parasite-infected cells, concomitant with a diminishedpresence of IκB in the cytoplasm ([46], Table 2). Similarly, cystatinalongwith IFN-γ increased the activity of IKK leading to decreased IκBαlevels 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 incontrolling the function of other proteins through phosphorylation ofhydroxyl groups of their serine and threonine residues. PKCs play animportant role in several signal transduction cascades and are activatedby increased concentration of diacylglycerol (DAG) or Ca2+ [117].During Leishmania infection, activation of PKC is inhibited andsubsequent intracellular signaling, LPG being a key determinant [118]as also other glycosylinositol phospholipids [119,120]. Bhattacharyya etal., (2001) have reported that L. donovani infection selectively inhibitedCa2+-dependentPKCactivity (PKCβ) via IL-10whileCa2+-independentPKC (PKC ε, ζ) activitywas enhanced (Fig. 1, [121]). L. major is known toinhibit PKC-dependent c-fos and TNFα gene expression [122]. Infectionof macrophages with L. donovani enhanced levels of intracellularceramide which in turn downregulated classical PKC activity, upregu-lated Ca2+-independent atypical PKC-ζ expression [123]. Furthermore,PKC β is known to activate the assembly of NADPH oxidase subunitcomplex whereas Leishmania infection by causing downregulation ofPKC β expression, inhibited the assembly of NADPH oxidase subunitcomplex and subsequently attenuated generation of ROS [Fig. 1, 93. It isalso known that PKC isform α is responsible for F actin mediatedphagolysomal maturation; once again, Leishmania by downregulatingthe expression of PKC α inhibited phagosomal maturation [93 and reftherein]. Furthermore, as Leishmania induced PKC ζ expression,which isknown to inhibit the MAPKs, caused decreased production of pro-inflammatory mediators [Fig. 1, 124 and ref therein].

It has been seen that C–C chemokines, macrophage inflammatoryprotein (MIP-1 alpha) and macrophage chemoattractant protein(MCP-1) can restrict the parasitic burden via restoration of impairedPKC signaling and induction of free-radical generation in murineLeishmaniasis [125]. These chemokines restored Ca2+-dependent PKCactivity and inhibited Ca2+-independent atypical PKC activity both invivo and in vitro in L. donovani-infected macrophages [125].Mukherjee et al., (2010) have demonstrated that amphotericin Bcan restore the impaired classical PKC and abrogate the atypical PKCpathways [43].

During Leishmania infection, activation of (phosphoinositide 3-kinase)PI3K signaling caused a down regulation of IL-12 [126]. SHP-1 ProteinTyrosine Phosphatase (PTP) is an important negative regulatorymoleculeof signaling pathways, related to the actions of interferons [127].

Macrophages infected with Leishmania in vitro have elevated SHP-1/PTPactivity 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 MAPKsleading to inhibition of nuclear translocation of NF-κB (Fig. 1, [93,124]). Ithas been shown that SAG induces ERK1/2 phosphorylation throughactivation of PI3K, protein kinase C, and Ras while p38 MAPKphosphorylation occurs through activation of PI3K and Akt [40].

6. Conclusions

The key pathogenic event in Leishmaniasis is harboring of thecausative Leishmaniaparasitewithin phagolysosomes ofmacrophages.Therefore, to establish infection, Leishmania invariably developmechanisms to neutralize themicrobicidalmachinery ofmacrophages.Hence, establishment of infection critically hinges on whether thebalance tilts towards the host's ability to activate its armamentariumor 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 thesignaling system of the host, such that effector functions triggered byvarious cell surface receptors are either actively suppressed or arealtered so as to result in immune suppression that will promoteparasite survival. Therefore, our quest for anti-leishmanial drugsshould focus on their direct parasiticidal and/or indirect immuno-modulatory activity, achieved via restoration of impaired hostsignaling pathways.

In this review, we have highlighted the participation of variousimmune cells, microbicidal molecules and altered signaling mechanismsin Leishmaniasis, together with the influence of anti-leishmanial drugsupon various immune cells like neutrophils, macrophages, DCs andlymphocytes. The different immunemechanisms impacted upon includeincreased generation of ROS and RNS, activation of co-stimulatorymolecules and signaling pathways e.g. TLRs, MAPK, JAK-STAT, PKC, andtranslocation of NF-kB. Taken together, screening for compounds havingthe propensity tomodulate the host defense signaling pathways alone orin combinationwith existing anti leishmanial drugs [129]maywell proveto be an effective immunochemotherapeutic strategy in Leishmaniasisworthy of pharmacological consideration.

Acknowledgements

This work received financial assistance from the Indian Council ofMedical Research (ICMR), Dept. of Science & Technology and Councilof Scientific & Industrial Research CSIR, Govt. of India. PS and DM arerecipients of a Senior Research Fellowship from CSIR and ICMR, Govt.of India respectively.

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