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MAPK-directed phosphatases preferentially regulate pro- and anti-inflammatory cytokines in experimental visceral leishmaniasis: involvement of distinct protein kinase C isoforms Susanta Kar,* Anindita Ukil,† Gunjan Sharma,* and Pijush K. Das*,1 *Molecular Cell Biology Laboratory, Infectious Diseases and Immunology Division, Indian Institute of Chemical Biology, Kolkata, India; and †Department of Biochemistry, Calcutta University, Kolkata, India RECEIVED SEPTEMBER 29, 2009; REVISED JANUARY 8, 2010; ACCEPTED JANUARY 19, 2010. DOI: 10.1189/jlb.0909644

ABSTRACT The role of phosphatases in the impairment of MAPK signaling, which is directly responsible for Leishmaniainduced macrophage dysfunction, is still poorly understood. Gene expression profiling revealed that Leishmania donovani infection markedly up-regulated the expression of three phosphatases: MKP1, MKP3, and PP2A. Inhibition of these phosphatases prior to infection points toward preferential induction of the Th2 response through deactivation of p38 by MKP1. On the other hand, MKP3 and PP2A might play significant roles in the inhibition of iNOS expression through deactivation of ERK1/2. Among various PKC isoforms, PKC␨ was associated with induction of MKP3 and PP2A in infected macrophages, whereas PKC␧ was correlated with MKP1 induction. Inhibition of phosphatases in L. donovani-infected BALB/c mice shifted the cytokine balance in favor of the host by inducing TNF-␣ and iNOS expression. This was validated by cystatin, an immunomodulator and curing agent for experimental visceral leishmaniasis, which showed that inhibition of MKPs and PP2A activity may be necessary for a favorable T cell response and suppression of organ parasite burden. This study, for the first time, suggests the possibility of the involvement of MAPK-directed phosphatases in the establishment of L. donovani infection. J. Leukoc. Biol. 88: 9–20; 2010.

INTRODUCTION Macrophages provide an interface between innate and adaptive immunity and play key regulatory and effector functions

Abbreviations: BMM⫽bone marrow-derived macrophage, bpV(phen)⫽ potassium bisperoxo (1,10-phenanthroline) oxovanadate (V), DUSP⫽dual specificity phosphatase, iNOS⫽inducible NO synthase, LDU⫽LeishmanDonovan unit(s), MBP⫽myelin basic protein, MKP⫽MAPK phosphatase, NP-40⫽Nonidet P-40, p⫽phospho, PKC⫽protein kinase C, pNPP⫽p-nitrophenyl phosphate, PP2A⫽protein phosphatase 2A, PTK⫽protein tyrosine kinase, PTP⫽protein tyrosine phosphatase, siRNA⫽small interfering RNA, SLA⫽soluble leishmanial antigen

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for many aspects of the immune response. Multiple intracellular signal transduction pathways stringently regulate the expression of macrophage accessory and effector functions, and among them, MAPK is the most ancient and evolutionarily conserved signaling cascades [1]. The activation of MAPK requires dual phosphorylation of serine/threonine and tyrosine residue in the TXY kinase activation motif, and deactivation occurs through the action of PTPs, serine/threonine phosphatases, or DUSPs [2]. The kinetoplastidae parasitic protozoa Leishmania donovani induces an immune-silencing mechanism for its intracellular survival inside the hostile environment of macrophages. Impairment of MAPK signaling is one of the major manipulative strategies through which Leishmania can promote numerous phagocyte dysfunctions, such as failure to respond to IFN-␥ and inhibition of iNOS gene expression [3]. Infection by Leishmania amazonensis amastigotes altered phosphorylation of ERK1/2 in response to LPS [4], and L. donovani infection caused inactivation of ERK1/2, which was accompanied by the inhibition of transcription factors elk-1 and c-fos [5]. In both studies, it was suggested that PTPs are responsible for the dephosphorylation of ERK1/2. On the other hand, for establishing infection, Leishmania inhibits CD40-triggered p38 MAPK signaling [6]. Induction of one of the PTPs, Src homology 2 domain-containing tyrosine phosphatase-1, has been shown to be vital for inhibition of NO generation, which is mediated by JAK2 and ERK1/2 inactivation [7]. However, the role of other phosphatases, such as serine/threonine phosphatases and DUSPs (also known as MKPs) in the disease progression of leishmanaiasis, is yet to be elucidated. The first DUSP, MKP1, was initially thought to dephosphorylate ERK1/2 only and later, shown to deactivate MAPK in the order of p38 ⬎⬎ JNK ⬎⬎ ERK1/2 [8]. A series of studies using MKP1 null mice or a MKP1⫺/⫺ macrophage cell line demonstrated that MKP1 is a pivotal feedback control regulator of 1. Correspondence: Molecular Cell Biology Laboratory, Indian Institute of Chemical Biology, 4 Raja S.C. Mullick Road, Kolkata 700032, India. E-mail: [email protected]

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the innate immune response and controls the threshold and magnitude of p38 activation in macrophages [9, 10]. Another DUSP, MKP3, also termed Pyst 1 or VH6, is predominantly localized in the cytoplasm, highly specific for ERK1/2 inactivation and not inducible by growth factor or stress [11, 12]. Prolonged activation of p-ERK1/2 is associated with reduced activity of MKP3 during cellular senescence and hyperoxia [13]. MKP3, however, is not related to the serine/threonine phosphatases but belongs to the PTP superfamily. PP2A, a type-2 serine/threonine phosphatase, is an important negative regulator of the ERK signaling pathway and is involved in the control of many cellular functions [14, 15]. The inability of macrophages infected with Mycobacterium avium to sustain MAPK activation and to produce high levels of TNF-␣ was, in part, a result of an increase in PP2A activity [15]. PKC, which plays a major regulatory role in the diverse cellular signaling, has also been implicated in the differential regulation of MAPK-directed phosphatases. For instance, PKC␧ has been shown to play a crucial role in the negative regulation of MAPK through induction of MKP1 [16]. On the other hand, PKC␨, an atypical calcium-independent PKC isoform, has been involved in the control of mitogenic signal transduction and survival by inhibition of PP2A [17]. Although a recent report suggested increased ceramide synthesis following L. donovani infection with simultaneous induction of PKC␨ and PP2A [18], no direct relationship between these two has yet been documented. In the present investigation, we set out to explore the possible signaling events involved in L. donovani-induced expression and activation of MKPs and serine-threonine phosphatases. Moreover, we also assessed the effect of both of these classes of phosphatases on perturbation of Th1/Th2 cytokine balance and iNOS gene expression in in vitro and in vivo situations of visceral leishmaniasis. Importance of this pathway has been attempted further to validate by cystatin, a natural cysteine protease inhibitor that can synergize with subthreshold concentration of IFN-␥ in curing experimental visceral leishmaniasis through induction of a favorable T cell response and upregulation of NO [19].

MATERIALS AND METHODS

Materials All antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA, USA) and Cell Signaling Technology (Beverly, MA, USA). All other chemicals were from Sigma-Aldrich (St. Louis, MO, USA), unless indicated otherwise.

Animals and parasite L. donovani strain AG83 (MHOM/IN/1983/AG83), isolated from an Indian patient with Kala azar [19], was maintained in inbred BALB/c mice by i.v. passage every 6 weeks. L. donovani promastigotes were obtained by allowing isolated splenic amastigotes to transform in parasite growth medium for 72 h at 22°C. The growth medium consisted of medium 199 (Invitrogen, Carlsbad, CA, USA), supplemented with 10% (v/v) FCS. SLA was prepared from promastigotes as described earlier [3]. Briefly, freeze-thawed cell suspension (5⫻109 cells/ml in 100 mM Tris-HCl, pH 8.0, containing 1 mM EDTA, 50 ␮g/ml leupeptin, and 1.6 mM PMSF) was sonicated for 5 ⫻ 45 s at 20 kilocycles/min in an ice bath. The contents were centrifuged at 10,000 g for 20 min, and the supernatant was dialyzed, filtered, and stored at ⫺80°C. It was used at a concentration of 20 ␮g/ml. Amastigotes were

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isolated from splenocytes of infected mice (4 weeks) and cultured in vitro at 37°C, pH 5.5, in M199 plus 10% FBS, which mimics the temperature and pH of the host macrophage phagolysosome.

Cell culture and infections BMMs were prepared, as described earlier [20], from the femurs and tibias of 6- to 8-week-old BALB/c mice. BM cells were suspended in RPMI 1640, supplemented with 100 U/ml penicillin and 100 ␮g/ml streptomycin, 10% FCS, 5% horse serum, and 20% L929-conditioned media, and were incubated at 5% CO2, 95% humidity, at 37°C. On the 4th day, cells were fed again with L cell-conditioned medium; on Day 6, cells were harvested, counted, and used for experiments. The murine macrophage cell line RAW 264.7 was maintained at 37°C/5% CO2 in RPMI 1640 (Invitrogen) supplemented with 10% FCS, penicillin (100 U/ml), and streptomycin (100 ␮g/ml). No cytotoxic or cytostatic effect was observed for bpV(phen) (Alexis Biochemicals, San Diego, CA, USA) or triptolide or okadaic acid at the concentrations used (data not shown), as assessed for cell viability using a MTT-based colorimetric assay kit (Roche Applied Science, Indianapolis, IN, USA). L. donovani promastigotes and amastigotes were also assessed for cell viability against various doses of bpV(phen) and okadaic acid using a MTT assay. For in vitro infection experiments, macrophages were infected with stationary-phase promastigotes at a ratio of 10 parasites/macrophage [19]. Infection was allowed to proceed for 4 h, unphagocytized parasites were removed, and cells were cultured for 20 h. For in vivo experiments, female BALB/c mice were injected via the tail vein with 107 L. donovani promastigotes. Cystatin (5 mg/kg/day), in combination with IFN-␥ (5⫻105 U/kg/day), was administered on the 14th day postinfection for 4 consecutive days. To study the inhibitor profile in vivo, infected mice were treated with bpV(phen) (2.5 ␮mol/30 g body weight, i.p. daily) or okadaic acid (100 ␮g/kg, orally once in a week) over a 4-week period, starting at the 14th day after infection, and infection was assessed by removing spleen from infected mice up to 6 weeks; parasite burdens were determined from Giemsa-stained impression smears. Spleen parasite burdens, expressed as LDU, were calculated as the number of amastigotes/ 1000 nucleated cells ⫻ spleen weight (in grams) [21]. The investigation conforms to the Guide for the Care and Use of Laboratory Animals, published by the U.S. National Institutes of Health (NIH Publication No. 8523, revised 1996), and with the approval of the Institutional Animal Care and Use Committee.

Real-time PCR Total RNA was isolated from splenocytes, BMMs, or RAW 264.7 cells using the RNeasy mini kit (Qiagen, Valencia, CA, USA) and treated with Dnase 1, as recommended by the manufacturer. RNA (1 ␮g) was used as template for cDNA synthesis using the SuperScript first strand synthesis system for the RT-PCR kit (Invitrogen). Quantitative real-time PCRs (ABI 7500 Fast realtime PCR system, Applied Biosystems, Foster City, CA, USA) were performed using TaqMan Fast Universal PCR master mix (Applied Biosystems) with the following PCR amplification conditions: 40 cycles of 95°C for 15 s and 60°C for 1 min. Relative quantitation was performed using the ⌬⌬ comparative threshold method; data for each sample were normalized to ␤-actin mRNA levels and expressed as a fold change compared with uninfected controls.

Cytokine analysis by ELISA The level of various cytokines in the single-cell suspension of spleen cells, BMMs, or macrophages was measured using a sandwich ELISA kit (Quantikine M, R&D Systems, Minneapolis, MN, USA). Before analysis, spleen cells were stimulated with 20 ␮g/ml SLA for 48 h. The assay was performed as per the detailed instructions of the manufacturer. The detection limit of these assays was ⬎5.1, ⬎2.5, ⬎4, and ⬎4.6 pg/ml for TNF-␣, IL12p70, IL-10, and TGF-␤, respectively.

PTP activity assay PTP activity was measured using the PTP assay kit (Sigma-Aldrich), according to the manufacturer’s instructions. Briefly, cells were lysed in lysis

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Kar et al. MAPK-directed phosphatases in leishmaniasis buffer (50 mM Hepes, pH 7.4, containing 0.5% Triton X-100, 10% glycerol, 1 mM benzamidine, 10 ␮g/ml aprotinin, 10 ␮g/ml leupeptin, and 2 ␮g/ml pepstatin A), incubated on ice for 30 min, and centrifuged at 10,000 g for 15 min at 4°C. PTP activity in clear supernatants was determined using a monophosphorylated phosphotyrosine peptide as substrate. Free inorganic phosphate was detected with malachite green (SigmaAldrich), and OD was taken at 620 nm.

Secreted acid phosphatase assay L. donovani promastigotes and amastigotes were cultured for 48 h in the presence of bpV(phen) (10 ␮M) or okadaic acid (10 ␮M). Culture supernatants were harvested, and acid phosphatase activity was assayed according to Gottlieb and Dwyer [22]. All enzyme assays were done in quadruplicate and were repeated on four different, independently generated samples.

Ex vivo phosphatase assay Isolated splenocytes were lysed in lysis buffer [20 mM Tris-HCl, pH 7.5, 5 mM EGTA, 2 mM EDTA, 1 mM DTT, 0.1% NP-40, 10% glycerol, 10 ␮g/ml leupeptin, 10 ␮g/ml aprotinin, and 34.4 ␮g/ml 4-(2-aminoethyl)-benzenesulfonyl fluoride], and whole cell lysates were prepared by centrifuging at 15,000 g for 30 min at 4°C. PP2A activity was determined by using the serine/threonine phosphatase assay system from Promega (Madison, WI, USA). Enzyme activity was assessed by the release of phosphate from a chemically synthesized ser/thr phosphopeptide in PP2A reaction buffer. The amount of phosphate released was measured by the absorbance of the molybdate-malachite green phosphate complex at 620 nm. To evaluate MKP1 and MKP3 activity specifically, 100 ␮g of the whole cell lysate was incubated for 2 h at 4°C with 2 ␮g anti-MKP1 or anti-MKP3 antibodies, respectively, and 100 ␮l protein A-Sepharose (Santa Cruz Biotechnology). Immune complexes were collected by centrifugation at 10,000 g for 5 min at 4°C, washed four times, and finally, resuspended in 100 ␮l lysis buffer. The assay was performed in 200 ␮l reaction mixture containing 50 mM Hepes (pH 7.5), 0.1% ␤-ME, 2 mM pNPP (Sigma-Aldrich), and 20 ␮l immunoprecipitates at 37°C for 30 min. Reactions were terminated by addition of 50 ␮l 200 mM NaOH, and absorbance was taken at 410 nm. Nonspecific hydrolysis of pNPP by lysates was assessed in nonimmune IgG immunoprecipitates and subtracted from the values obtained for enzyme immunoprecipitates. In control experiments, Western blots of aliquots of the MKP1/MKP3 immunoprecipitates were conducted to ensure that comparable amounts of MKP1 and MKP3 were precipitated from infected samples.

(30 ␮g) from each sample were resolved by 10% SDS-PAGE and then transferred to a nitrocellulose membrane (Millipore, Billerica, MA, USA). The membranes were blocked with 5% BSA in wash buffer (TBS/0.1% Tween 20) for 1 h at room temperature and probed with primary antibody overnight at a dilution recommended by the suppliers. Membranes were washed three times with wash buffer and then incubated with HRP-conjugated secondary antibody and detected by the ECL detection system (Amersham Biosciences, Arlington Heights, IL, USA), according to the manufacturer’s instruction.

In vitro ERK dephosphorylation assay The in vitro ERK dephosphorylation assay was according to Nyunoya et al. [13]. Whole cell protein was obtained by lysing the cells in lysis buffer [50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1% NP-40, and complete protease inhibitors (Roche Applied Science)] and centrifuging at 15,000 g for 10 min at 4°C. Immunoprecipitation was performed by incubation of cell lysate (500 ␮g/500 ␮l) with 5 ␮g respective antibodies (anti-MKP1, antiMKP3, or anti-PP2A) and rocked overnight at 4°C. Protein G plus-agarose (40 ␮l/sample; Santa Cruz Biotechnology) was added, and rocking was allowed to continue for another 1 h. The immunoprecipitates were washed twice with lysis buffer and once with phosphatase buffer (50 mM Tris-HCl, pH 7.5, 1 mM MgCl2, 0.1 mM EDTA, and 0.9 mg/ml BSA) in nonreducing conditions without ␤-ME. Immunoprecipitated MKP1, MKP3, or PP2A was incubated with 0.1 ␮g rp-ERK (Stratagene, La Jolla, CA, USA) at 37°C in phosphatase buffer and released from agarose by boiling in 2⫻ sample buffer. The specific phosphatase activity for p-ERK was measured by Western blotting using anti-p-ERK antibody. In control experiments, Western blots of the aliquots of MKP1/MKP3 immunoprecipitates were conducted to ensure that comparable amounts of MKP1 and MKP3 were precipitated from infected samples. Rabbit IgG was used as a negative control.

Preparation of cytosol and membrane fractions PKC translocation was determined according to Valledor et al. [16]. The cells were lysed by scraping in cold hypotonic buffer T10 (10 mM Tris-HCl, pH 7.5, 1 mM EGTA, 10 mM NaCl), supplemented with complete protease inhibitors (Roche Applied Sciences) and 1 mM sodium orthovanadate. The cell lysates were spun at 100,000 g for 30 min at 4°C, and the supernatants comprise the cytosol fraction. The pellets were resuspended in cold T10 buffer containing 1% Triton X-100, homogenized on ice, kept at 4°C for 1 h, and then centrifuged at 100,000 g for 30 min at 4°C. The supernatant comprised the membrane fraction.

PTK activity measurement

PKC activity assay

PTK activity was assayed in the splenocytes according to Olivier et al. [23]. Briefly, cell lysates were prepared by centrifuging the cells in lysis buffer (1 M Tris-HCl, pH 8.0, 3 M NaCl, 100% glycerol, 10% NP-40, 0.5 M NaF, 50 ␮M nitrophenyl guanidobenzoate, 5 ␮M aprotinin, and 5 ␮M leupeptin) at 15,000 g for 30 min at 4°C in a microfuge. The phosphorylation reaction was initiated by addition of kinase reaction mixture {25 ␮M ATP in 50 mM Hepes, pH 7.4, 40 mM MgCl2, 2.5 mg/ml synthetic substrate, poly (GluTyr) (4:1), and 5 ␮M [␥-32P] ATP} to a total volume of 100 ␮l. After 10 min incubation at 22°C, the reaction was arrested by spotting 50 ␮l of the reaction solution onto Whatman 3 MM (Clifton, NJ, USA) square paper, washed thoroughly with 10% TCA containing 10 ␮M sodium pyrophosphate treated with anhydrous ethanol, air-dried, and counted by liquid scintillation using a Packard Tri-Carb 2100TR scintillation counter (Perkin Elmer, Boston, MA, USA).

PKC␧ activity was measured according to Valledor et al. [16]. Briefly, PKC␧ immnoprecipitated (2 ␮g antibody/150 ␮g total protein, in a total volume of 300 ␮l) from subcellular fractions. Immunocomplexes were separated by addition of 75 ␮l 20% protein A-Sepharose, incubated 2 h at 4°C, and pelleted. The pellets were washed twice with radioimmunoprecipitation assay buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1% Triton X-100, 1% deoxycholate, 2 mM EDTA, 1 mM EGTA), supplemented with protease inhibitors and 1 mM sodium orthovanadate, once with prereaction buffer (50 mM ␤-glycerophosphate, 10 mM MgCl2, 20 mM Tris-HCl, pH 7.5, 50 mM NaCl, 1 mM DTT, protease inhibitors, 1 mM sodium orthovanadate), and then resuspended in reaction buffer (prereaction buffer supplemented with 100 ␮M ATP, 33 ␮M 1, 2-sn-dioleoylglycerol, and 40 ␮g/ml L-␣-phosphatidylserine). Kinase reaction was initiated by the addition of 5 ␮Ci [␥-32P] ATP and 1 ␮M [25Ser] PKC; the 25Ser-substituted peptide was obtained from the pseudosubstrate region of PKC (Calbiochem, San Diego, CA, USA) and incubated for 10 min at 30°C. Each sample was spotted on a phosphocellulose filter (Whatman 3 MM) and washed in 5% TCA with 10 mM sodium pyrophosphate, and radioactivity was counted by liquid scintillation. PKC␨ activity was measured in immunoprecipitates by measuring the incorporation of 32P into the synthetic peptide MBP (Upstate Biotechnology, Lake Placid, NY, USA), a specific PKC substrate, under the conditions described above. PKC activities in nonimmune IgG immunoprecipitates

Immunoblot analysis Cells were lysed in lysis buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1% NP-40, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS, 5 mM iodoacetamide, and 2 mM PMSF), and the protein concentrations in cleared supernatants were measured using a Bio-Rad protein assay (Bio-Rad, Hercules, CA, USA). The supernatants containing an equal amount of protein

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were subtracted from the values obtained for enzyme (PKC␨ or -␧) immunoprecipitates.

Statistical analysis Data were analyzed by one-way ANOVA to determine the statistical significance of intergroup comparisons using GraphPad Prism, Version 4.0 (GraphPad Software, San Diego, CA, USA). P values ⬍0.05 were considered statistically significant.

phages along with a significant increase of 5.5-fold of TNF-␣ synthesis (Fig. 1D). Analysis of iNOS mRNA expression showed a 5.6-fold increase by bpV(phen) pretreatment over that of a low level found in infected cells (Fig. 1E). Taken together, these results suggest that the induction of macrophage PTP activity in a Leishmania-infected condition may be associated with enhanced Th2 response and reduced iNOS expression.

Taqman analysis for identification of MAPK-directed phosphatases following infection

RESULTS

Effect of L. donovani-induced PTP activation To see the effect of L. donovani infection on MAPK activation, normal and infected BMMs were stimulated with LPS (10 ng/ ml) for various time periods. LPS induced strong phosphorylation of p38 and ERK1/2 in normal macrophages, whereas this was reduced significantly (2.5- and 2.3-fold for p38 and ERK1/2, respectively, at 30 min, P⬍0.01; Fig. 1A) in L. donovani-infected macrophages. This reduction was consistent with a rapid increase in macrophage PTP activity, which was maximal at 90 min (4.6-fold) and was stable, as examined up to 12 h after infection (Fig. 1B). To evaluate further the role of PTP activation on evasion of the MAPK pathway, BMMs were treated with bpV(phen), a potent inhibitor of PTP, prior to infection, and then activation of p38 and ERK1/2 was assessed. As depicted in Figure 1C, pretreatment of macrophages with 10 ␮M bpV(phen) markedly enhanced the phosphorylation of p38 and ERK1/2 in infected macrophages in a time-dependent manner with maximum induction at 1 h. bpV(phen) treatment in control cells resulted in little activation of p38 and ERK1/2 (Fig. 1C). We then evaluated the effect of PTP inhibition on cytokine balance and iNOS expression. Leishmania infection caused an increased level of IL-10 (480⫾50 pg/ml) and low level of TNF-␣ (30⫾4 pg/ml) production (Fig. 1D). However, pretreatment with the PTP inhibitor resulted in 77% reduction in IL-10 in infected macro-

As L. donovani infection caused induction of PTP activity, we therefore attempted to obtain a global view of the expression and regulation of MAPK-directed phosphatases in infected BMMs by taking advantage of quantitative real-time PCR. To do this, 12 MKPs and three Ser/Thr phosphatases were selected for detailed transcriptional analysis, as they were all well-characterized to regulate the activity of MAPKs. Taqman probes for all of the MAPK-directed phosphatases were obtained from Applied Biosystems (listed in Tables 1 and 2). As shown in Table 1, of all the phosphatases examined, infection of BMMs led to a rapid increase of MKP1 (2.6-fold), MKP3 (2.8-fold), and PP2A (2.9-fold) gene expression at 1 h and attained maximum level at 2 h (7.4-, 6.5-, and 6.2-fold for MKP1, MKP3, and PP2A, respectively). Immunoblot analysis revealed a time-dependent increase in the expression of all three phosphatases in infected macrophages (Fig. 2). However, the maximum induction was observed at 90 min. Further analysis by real-time PCR of these phosphatases also showed comparable values at 90 min with that of 2 h (data not shown). The mRNA levels for remaining enzymes did not change or were increased marginally. Although the expression levels of all three phosphatases peaked at 90 min, they remain elevated significantly, up to 12 h of infection as studied (Tables 1 and 2).

Figure 1. Effect of L. donovani infection on PTP activation. (A) The expression and phosphorylation of MAPKs were detected by Western blotting in LPS (10 ng/ml)-stimulated normal or L. donovani (Ld)-infected BMMs for various time periods. (B) Induction of macrophage PTP activity during L. donovani infection was measured using a PTP assay kit. Results are expressed as the relative increase (n-fold) over PTP activity in control cells and represent the mean ⫾ sd (n⫽3). (C) BMMs were treated with bpV(phen) (10 ␮M) for 1 h, and in another set, BMMs were treated with bpV(phen) for 1 h followed by infection. In both sets, the levels of p-MAPKs were then measured by Western blotting at various time periods as indicated. (D) Cytokine levels in the cell culture supernatant were measured by ELISA in bpV(phen)-treated normal and infected (24 h) macrophages. (E) Taqman analysis of iNOS gene expression in bpV(phen)-treated normal and infected macrophages. mRNA levels were normalized to ␤-actin and expressed as a fold change compared with control. Results are expressed as mean ⫾ sd (n⫽3). **, P ⬍ 0.01; ***, P ⬍ 0.001.



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Kar et al. MAPK-directed phosphatases in leishmaniasis

TABLE 1. Real-Time PCR for MKPs following Infectiona GenBankTM number NM_013642c NM_010090 NM_028207 NM_176933 NM_026268c NM_008748 NM_029352 NM_022019 NM_028099 NM_023173 NM_130447 NM_024438

Gene name

Common name

Assay IDb

1h

2h

4h

12 h

DUSP1 DUSP2 DUSP3 DUSP4 DUSP6 DUSP8 DUSP9 DUSP10 DUSP11 DUSP12 DUSP16 DUSP19

MKP-1 PAC-1 VHR MKP-2 MKP-3 hVH5 MKP-4 MKP-5 PIR1 YVH1 MKP-7 SKRP1

Mm01309843_g1 Mm00839675_g1 Mm00459217_m1 Mm00723763_m1 Mm00518185_m1 Mm01158982_g1 Mm01187306_g1 Mm00517678_m1 Mm01190370_m1 Mm00517772_m1 Mm00459938_m1 Mm01324861_m1

2.6 ⫾ 0.4 1.4 ⫾ 0.4 1.2 ⫾ 0.5 1.1 ⫾ 0.2 2.8 ⫾ 0.6 1.4 ⫾ 0.3 1.2 ⫾ 0.2 1.3 ⫾ 0.5 1.5 ⫾ 0.3 1.4 ⫾ 0.3 1.3 ⫾ 0.4 1.1 ⫾ 0.2

7.4 ⫾ 0.6 1.1 ⫾ 0.6 1.4 ⫾ 0.3 1.2 ⫾ 0.5 6.5 ⫾ 0.8 1.1 ⫾ 0.3 1.2 ⫾ 0.3 1.4 ⫾ 0.4 1.3 ⫾ 0.2 1.3 ⫾ 0.4 1.4 ⫾ 0.3 1.3 ⫾ 0.4

6.4 ⫾ 0.5 1.2 ⫾ 0.3 1.5 ⫾ 0.6 1.0 ⫾ 0.4 5.7 ⫾ 0.9 1.3 ⫾ 0.2 1.1 ⫾ 0.3 1.2 ⫾ 0.4 1.2 ⫾ 0.3 1.3 ⫾ 0.5 1.4 ⫾ 0.4 1.1 ⫾ 0.3

6.8 ⫾ 1.8 1.4 ⫾ 0.5 1.3 ⫾ 0.4 1.1 ⫾ 0.3 5.6 ⫾ 1.4 1.2 ⫾ 0.3 1.3 ⫾ 0.3 1.2 ⫾ 0.4 1.4 ⫾ 0.3 1.1 ⫾ 0.5 1.3 ⫾ 0.4 1.2 ⫾ 0.3

a This table depicts the expression profile of MKPs at various time-points (1, 2, 4, and 12 h) after infection, which were carried out by real-time PCR as described in Materials and Methods. Assays were performed at least three times, and values are the mean fold changes relative to uninfected controls ⫾ sd of the three assays. bAssay ID number of Taqman probes.cSignificantly up-regulated genes meeting a two-fold cut-off.

Effect of MAPK-directed phosphatases on Th1/Th2 balance and iNOS expression As specific phosphatases were up-regulated significantly in an infected condition, we assessed their effect on the modulation of Th cell polarization and iNOS gene expression. Triptolide (1 ␮M) and okadaic acid (10 ␮M) were used for specific inhibition of MKP1 and PP2A, respectively. The siRNA-mediated knockdown system was used for the inhibition of MKP3, as a specific inhibitor is not available. BMM is not suitable for siRNA transfection; we therefore used the murine macrophage cell line RAW 264.7 for our subsequent experiments, as the expression pattern of MAPK-directed phosphatases observed in BMMs (Table 1) was similar to that observed in RAW 264.7 cells (data not shown). Inhibition of MKP1 resulted in a substantial decrease of IL-10 (69%) and TGF-␤ (67%) with a concomitant increase of TNF-␣ (3.5-fold) and IL-12 (3.2-fold) in infected macrophages (Fig. 3B). In contrast, PP2A inhibition resulted in much less of an alteration of cytokine levels (33% and 30% decrease of IL-10 and TGF-␤, respectively, accompanied by a 1.8- and 1.6-fold increase of TNF-␣ and IL-12, respectively; Fig. 3B). In control cells, triptolide and okadaic acid treatment resulted in a little induction of IL-12 and TNF-␣ (P⬍0.05 for triptolide) with no alteration of IL-10 and TGF-␤ synthesis (Fig. 3, A and D). MKP3 inhibition also resulted in a comparable effect on cytokine levels with that of PP2A inhibition (Fig. 3E). However, as far as iNOS expression was concerned, PP2A and MKP3 inhibition caused a marked

increase of 3.3- and 3.0-fold, respectively, and MKP1 inhibition resulted in only an increase of 1.6-fold (Fig. 3, C and F). The efficacy and specificity of chemical inhibitors and siRNA were evaluated by measuring the specific phosphatase activity in immunoprecipitated samples obtained from control and L. donovani-infected macrophages (data not shown). The efficacy of siRNA on MKP3 expression was assessed by Western blotting. MKP3 was reduced significantly in cells expressing MKP3specific siRNA compared with cells expressing control siRNA (Fig. 3G). These results suggest that MKP1, MKP3, and PP2A regulate Th1/Th2 cytokine balance and iNOS mRNA expression differentially in L. donovani-infected macrophages with preferential induction of Th2 cytokines by MKP1 and iNOS by MKP3 and PP2A.

Effect of MAPK-directed phosphatases on p38 and ERK1/2 As L. donovani infection induced dephosphorylaton of ERK1/2 and p38 in LPS-activated macrophages, we wanted to identify the specific phosphatases responsible for infection-induced inactivation of both of these MAPKs. Whole cell lysates were obtained from infected RAW 264.7 cells, and MKP1, MKP3, and PP2A were immunoprecipitated and used in an in vitro ERK dephosphorylation assay. As shown in Figure 4A, PP2A and MKP3 from infected macrophages could dephosphorylate rERK substrate efficiently, whereas MKP1 had little activity. Immunoblot analysis of MKP1, MKP3, and PP2A in control

TABLE 2. Real-Time PCR for Serine-Threonine Phosphatases following Infectiona GenBankTM number NM_013636 NM_017374c NM_001033

Gene name

Common name

Assay IDb

1h

2h

4h

12 h

Ppp1cc Ppp2cb Ppm2c

PP1 PP2A PP2C

Mm00849631_s1 Mm00479551_g1 Mm01221849_m1

1.2 ⫾ 0.3 2.9 ⫾ 0.4 1.3 ⫾ 0.5

1.2 ⫾ 0.2 6.2 ⫾ 0.7 1.4 ⫾ 0.3

1.3 ⫾ 0.2 5.8 ⫾ 1.1 1.2 ⫾ 0.4

1.4 ⫾ 0.3 6.2 ⫾ 1.4 1.1 ⫾ 0.4

a This table depicts the expression profile serine-threonine phosphatases at various time-points (1, 2, 4, and 12 h) after infection, and values are the mean fold changes relative to uninfected controls ⫾ sd of the three assays. bAssay ID number of Taqman probes. cSignificantly up-regulated genes meeting a twofold cut-off.

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Figure 2. Expression of phosphatases in BMMs following infection. BMMs were infected with L. donovani promastigotes (cell:parasite ratio, 1:10) for various time periods as indicated. The expression of MKP1, MKP3, and PP2A was evaluated by immunoblot analysis. ***, P ⬍ 0.001.

experiments suggested that comparable amounts of phosphatases were immunoprecipitated from infected macrophages (Fig. 4B). Further, as depicted in Figure 4, C and D, inhibition of PP2A by okadaic acid and MKP3 by siRNA resulted in a marked induction of ERK phosphorylation in infected macro-

phages. As a recombinant substrate for p38 is not available, we exploited the pharmacological inhibition of MKP1 and PP2A and siRNA-mediated knockdown of MKP3 to examine the role of these phosphatases on the kinetics of p38 activation. When MKP1 was inhibited by the use of triptolide (1 ␮M), we found an increase of p38 phosphorylation, as studied up to 8 h after infection with a maximum level at 4 h (Fig. 4E). In contrast, no up-regulation of p38 phosphorylation was found in infected macrophages pretreated with okadaic acid (10 ␮M), inhibitor of PP2A, or MKP3 siRNA (Fig. 4, E and F). These studies suggest that in an infected condition, MKP1 may act as a negative regulator of p38, whereas MKP3 and PP2A activity contributes significantly to the sustained inactivation of ERK1/2.

Involvement of PKC pathway in infection-induced expression of phosphatases Impairment of PKC isotypes is one of the crucial adaptive strategies that helps in the successful propagation of the Leishmania parasite within the hostile environment of macrophages. We therefore wanted to investigate the specific PKC isoforms that might be affected by infection and explore the effect of the inhibition of PKC on the induction of MKP1, PP2A, and MKP3. Immunoblot analysis using eight PKC isoform (␣, ␤I,

Figure 3. Effect of phosphatases on cytokine response and iNOS expression. RAW 264.7 cells were treated with triptolide or okadaic acid for 1 h and infected with L. donovani promastigotes for 4 h, noningested promastigotes were removed, and macrophages were cultured for another 20 h. The levels of cytokine in control (A) and infected cells (B) were measured by ELISA. iNOS expression (C) was determined in control and infected cells by Taqman analysis. To determine the effect of MKP3 inhibition, macrophages were transfected (24 h) with MKP3 siRNA, infected as mentioned above. Cytokine levels were measured in control (D) and infected (E) cells and iNOS expression (F) by Taqman analysis. (G) The specificity of MKP3 siRNA was determined in whole cell extracts from macrophages expressing MKP3-targeting or control siRNAs by Western blotting using a specific antibody against MKP3. Results are expressed as mean ⫾ sd (n⫽4). *, P ⬍ 0.05; **, P ⬍ 0.01; ***, P ⬍ 0.001. ns, Not significant.



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Figure 4. Effect of phosphatases on inactivation of MAPK. (A) MKP1, MKP3, and PP2A were immunoprecipitated from normal and infected (2 h) macrophages. Immunoprecipitated samples were incubated with rp-ERK substrate. Activity of phosphatases was measured by Western blotting (WB) using anti-p-ERK antibody. The amount of individual phosphatases was measured by stripping the blot and reprobing with antibodies against MKP1, MKP3, or PP2A. These data are representative of three experiments. (B) Western blot analysis of immunoprecipitated (IP) MKP1, MKP3, and PP2A was carried out in control (C) and infected cells using respective antibodies. Rabbit IgG was used as a negative control. (C) Macrophages were treated with triptolide or okadaic acid for 1 h and then infected with L. donovani promastigotes for 2 h. Levels of p-ERK1/2 were measured by Western blotting. (D) Macrophages were transfected (24 h) with control or MKP3 siRNA, infected with L. donovani promastigotes for 2 h, and p-ERK1/2 levels were measured by Western blotting. (E) Macrophages were treated with the inhibitors as mentioned in C, followed by infection for various time periods, and p-p38 levels were measured. (F) Macrophages were transfected with MKP3 siRNA as mentioned in D, followed by infection for various time periods, and p-p38 levels were measured. *, P ⬍ 0.05; **, P ⬍ 0.01.

␤II, ␦, ␧, ␮, ␭, and ␨)-specific antibodies revealed that only PKC␧ (2.4-fold; P⬍0.01) and PKC␨ (2.3-fold; P⬍0.01) were up-regulated significantly in infected macrophages, whereas expression of PKC␤I and -␤II was markedly attenuated (Fig. 5A). Leishmania infection resulted in a rapid translocation of both of these isoforms from cytosol to membrane with

a peak time of 15 min and 30 min for PKC␧ and -␨, respectively, which remained at a significantly increased level, as examined up to 1 h (Fig. 5B). To further correlate membrane translocation with kinase activity, we performed an immunocomplex kinase assay. The ratio of activity of PKC␧ in a membrane-to-cytosolic fraction was 1.7 at 15 min postinfection (Fig.

Figure 5. Induction of PKC isoforms following L. donovani infection. (A) RAW 264.7 cells were infected with L. donovani promastigotes for 4 h, noningested promastigotes were removed, and macrophages were cultured for another 12 h. Immunoblot analysis for various PKC isoforms were performed in normal and infected macrophages using anti-PKC isoform antibodies. (B) Macrophages were infected with L. donovani promastigotes for various time periods. Cells were lysed, membrane and cytosolic fractions were obtained by subcellular fractionation, and proteins (30 ␮g) from each fraction were subjected to immunoblot analysis for PKC␨ and -␧ expression using respective antibodies. Immunoprecipitates were obtained from membrane and cytosolic fractions using anti-PKC␨ and -␧ antibodies. ␣-Tubulin and GAPDH were used as internal control for membrane and cytosol, respectively. Activities of PKC␨ (C) and PKC␧ (D) were assayed using MBP and Ser-PKC as substrates, respectively, at various time-points after infection. Ratios of enzyme activities in membrane-to-cytosolic fraction are depicted in the insets of C and D. *, P ⬍ 0.05; **, P ⬍ 0.01; ***, P ⬍ 0.001.

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5C), whereas it was 2.3 for PKC␨ at 30 min postinfection compared with 0.6 for uninfected control (Fig. 5D). This led us to study the involvement of both of these isoforms in the activation of MKP1, MKP3, and PP2A in infected macrophages. As depicted in Figure 6A, pretreatment of RAW 264.7 cells with a PKC␰-specific inhibitor peptide decreased the ability of immunoprecipitated MKP3 and PP2A to dephosphorylate ERK significantly (2.5- and 2.2-fold, respectively) compared with infected control. In addition, as depicted in Figure 6B, inhibition of PKC␨ by a specific inhibitor peptide resulted in a marked induction of ERK phosphorylation in infected macrophages. However, ERK-directed phosphatase activity of MKP3 and PP2A was not altered in cells treated with PKC␧-specific inhibitor peptide prior to infection (Fig. 6A). In contrast, inhibition of PKC␧ by a specific inhibitor peptide reduced MKP1 activity significantly in infected cells, which was demonstrated by a reciprocal, time-dependent increase in phosphorylation of p38 (maximum level found at 4 h; Fig. 6C). This was further consistent with a markedly attenuated expression of MKP1 (2.1fold reduction compared with infected controls) in cells treated with PKC␧ inhibitor peptide prior to infection (Fig. 6D). Taken together, these results suggest that Leishmania infection rapidly induced activation/translocation of PKC isoforms ␧ and ␨, which in turn, differentially regulated the expression of MKP1, MKP3, and PP2A in infected macrophages.

Effect of cystatin on PTP/PTK balance We have demonstrated earlier that cystatin, a natural cysteine protease inhibitor, could cure experimental visceral leishmaniasis along with a suboptimal dose of IFN-␥. This curative effect was associated with a strong up-regulation of NO and favorable T cell response [3]. As induction of Th2 response and iNOS inhibition in infected macrophages were associated with induction of PTP activity, we therefore examined the effect of cystatin plus IFN-␥ on the status of PTP/PTK balance in the infected macrophages. Cystatin plus IFN-␥ inhibited PTP activity significantly in a time-dependent manner (Fig. 7A) with a maximum inhibition (2.5-fold) at 8 h after infection. A decrease of PTP activity was consistent with a time-dependent activation of PTK with a maximum of 4.5-fold induction at 8 h

(Fig. 7B). Immunoblot assay revealed a time-dependent activation of ERK1/2 in infected macrophages treated with cystatin plus IFN-␥ with maximal phosphorylation at 6 h after treatment (Fig. 7C). However, no induction of p38 was found in infected, treated cells. Moreover, expression of all of the phosphatases studied was down-regulated significantly (2.1-, 2.3-, and 2.2-fold for MKP1, MKP3, and PP2A, respectively) in an infected macrophage treated with cystatin plus IFN-␥ (Fig. 7D). Taken together, these results suggest that polarization of a host-protective Th1 response and iNOS expression by cystatin plus IFN-␥ in infected macrophages might be associated with shifting the PTP/PTK balance in favor of host through activation of ERK1/2 and inhibition of MKP1, MKP3, and PP2A.

Modulation of MAPK-directed phosphatases in in vivo infection by cystatin To evaluate the role of phosphatases in the modulation of disease progression, MKP1, MKP3, and PP2A activities were assayed in the splenocytes of infected animals at various time periods after infection. Similar to the in vitro scenario, activities of all of the phosphatases were increased in infected animals. L. donovani infection caused a substantial increase of MKP activity in the adherent spleen cells with a maximum induction (4.7-fold for MKP1 and 4.3-fold for MKP3) at 2 weeks postinfection (Fig. 8A). PP2A activity, on the other hand, was maximal (4.6-fold) at 3 weeks after infection (Fig. 8B). Nonadherent cells did not show any significant activity (data not shown). To evaluate further whether inhibition of MKPs and PP2A can modulate the parasite persistence in vivo, infected mice were treated with bpV(phen) or okadaic acid over a 4-week period. As MKPs belong to the PTP superfamily, we used bpV(phen), a general PTP inhibitor, for in vivo inhibition of MKPs. bpV(phen) treatment reduced the spleen parasitic burden by 77%, whereas 57% reduction was observed in the case of okadaic acid treatment (Fig. 8C). bpV(phen) or okadaic acid was tested for their ability to kill parasites by inhibition of MTT reduction in promastigotes and freshly isolated amastigotes of L. donovani at a concentration range of 2.5–10 ␮M (Fig. 8, D and E). Cell viability of promastigotes was inhibited moderately (36%) by bpV-

Figure 6. Involvement of PKC isoforms on phosphatase activation. (A) Macrophages were treated with cell-permeative PKC␨ and PKC␧ inhibitor peptide (1 ␮M) for 1 h and then infected with L. donovani promastigotes for 2 h. ERK dephosphorylation assay was carried out with immunoprecipitated MKP3 and PP2A as described in the legend of Figure 4A. (B) Macrophages were treated with PKC␨ or PKC␧ inhibitor peptide (1 ␮M) for 1 h and then infected with L. donovani promastigotes for 2 h. Levels of p-ERK1/2 were measured by Western blotting. (C) Macrophages were treated with PKC inhibitors as mentioned in B, followed by infection for various time periods, and p-p38 levels were measured. (D) Macrophages were treated with PKC inhibitors, infected for 2 h, and the levels of MKP1 were measured by Western blotting.



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Figure 7. Effect of cystatin ⴙ IFN-␥ on PTP/PTK balance. (A) PTP activity was assayed in infected or cystatin (0.5 ␮M) ⫹ IFN-␥ (100 U/ml)-treated (2 h postinfection) RAW 264.7 cells, as described in the legend of Figure 1B. Results are expressed as the relative increase (n-fold) over PTP activity in control cells and represent the mean ⫾ sd (n⫽4). (B) PTK activity in infected and cystatin ⫹ IFN-␥treated (2 h postinfection) macrophages was assayed using poly(GluTyr) as substrate. (C) L. donovaniinfected (2 h) macrophages were treated with cystatin ⫹ IFN-␥ for 0⫺8 h, and the levels of p-p38 and p-ERK1/2 were measured by Western blotting. (D) L. donovani-infected (2 h) macrophages were treated with cystatin ⫹ IFN-␥ for 4 h, and the levels of MKP1, MKP3, and PP2A were measured by Western blotting. **, P ⬍ 0.01; ***, P ⬍ 0.001.

(phen) at a dose of 10 ␮M, whereas the inhibition of amastigote viability was less (8% reduction in cell viability). On the other hand, okadaic acid (10 ␮M) had little effect on the cell viability of promastigotes and amastigotes (8% and 6% reduction in cell viability, respectively; Fig. 8, D and E). The abundant, secreted acid phosphatase activity in L. donovani is contributed primarily by promastigotes (38 nmol/ min/107 cells), whereas in amastigotes, it is low (9 nmol/ min/107 cells; Fig. 8F). As the amount of secreted acid phosphatase is less in amastigotes, therefore, its contribution toward the total phosphatase pool may be negligible. bpV(phen) at a dose of 10 ␮M inhibited the secreted acid phosphatase activity of promastigotes moderately (29% inhibition), with no effect on that of amastigotes (Fig. 8F). Okadaic acid (10 ␮M) had no significant effect on acid phosphatase activity of promastigotes as well as amastigotes (9% and 7% inhibition, respectively; Fig. 8F). These results suggest that inhibition of host but not parasite phosphatases by bpV(phen) and okadaic acid might have a role for reduced parasite burden. bpV(phen) treatment resulted in a 70% reduction of IL-10 synthesis in spleen cells of infected mice (Fig. 8G) along with a 5.2-fold increase in TNF-␣ production (Fig. 8H) 4 weeks after infection. Okadaic acid treatment, on the other hand, resulted in a 45% reduction of IL-10 (Fig. 8G) along with a 2.8-fold increase in TNF-␣ production (Fig. 8H). Analysis of iNOS mRNA expression in infected mice treated with bpV(phen) or okadaic acid showed a sixfold and 3.8-fold increase, respectively (Fig. 8I), at 2 weeks post-treatment, suggesting that activation of

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MKPs and PP2A is accompanied by inhibition of iNOS and shifting the cytokine balance toward the Th2 mode. For assessing the correlation between phosphatase level and cystatin treatment, we observed that administration of a curative dose of cystatin (5 mg/kg/day) plus IFN-␥ (5⫻105 U/kg/ day) for 4 consecutive days 2 weeks after infection resulted in a 2.6-, 2.9-, and 3.1-fold decrease in MKP1, MKP3, and PP2A activity, respectively (Fig. 8, A and B). Cystatin ⫹ IFN-␥ treatment also resulted in increased levels of TNF-␣ (1100⫾100 pg/ml compared with 110⫾10 pg/ml in infected mice; Fig. 8H) and iNOS mRNA expression (7.5-fold compared with infected control; Fig. 8I), with a concomitant decrease in IL-10 (275⫾28 pg/ml compared with 1250⫾130 pg/ml in infected mice; Fig. 8G) synthesis. These results suggest that inhibition of MKPs and PP2A by cystatin in vivo may be associated with its curative effect against visceral leishmaniasis.

DISCUSSION The capacity of Leishmania to alter the host-signaling system leading to functional inhibition of macrophages has been shown to involve the evasion of MAPK signaling cascades [24]. Deactivation of these signaling systems may be modulated by phosphatases [5], which play a crucial role for the parasite’s survival as well as successful propagation of the disease, but the identity of the molecular player(s) mediating specific or general inhibition as well as the level at which they interfere Volume 88, July 2010


Figure 8. Role of phosphatases in the in vivo modulation of parasitemia and cytokine balance. L. donovani-infected mice were treated with cystatin ⫹ IFN-␥ as described in Materials and Methods. Spleen cells were isolated and lysed, and phosphatases were immunoprecipitated from whole cell lysates with respective antibodies. MKP1 and MKP3 activity (A) was assayed in immunoprecipitated samples by pNPP hydrolysis. (A1) Western blot analysis of immunoprecipitated MKP1 and MKP3 was conducted in splenocyte lysates from control and infected (2-week) mice using respective antibodies. Rabbit IgG was used as a negative control. PP2A activity (B) was measured by assay kit. Results are expressed as the relative increase (n-fold) over enzyme activity in control cells. (C) Spleen parasite burdens were determined in infected mice treated with cystatin ⫹ IFN-␥ or bpV(Phen) or okadaic acid and expressed as LDU ⫾ sd for six animals. bpV(Phen) or okadaic acid was given at 2 weeks after infection for up to 6 weeks. Cystatin ⫹ IFN-␥ was administered on the 14th day postinfection for 4 consecutive days. Cell viability was evaluated for log phase promastigotes (D) and freshly isolated amastigotes (E) in the presence of increasing doses of bpV(phen) and okadaic acid. (F) Promastigotes and amastigotes were grown in the presence of bpV(phen) (10 ␮M) or okadaic acid (10 ␮M) for 48 h. Secretory acid phosphatase activity was measured in culture supernatants and expressed as nmol/min/107 cells. ELISA was performed for the levels of IL-10 (G) and TNF-␣ (H), and real-time PCR analysis was performed for the expression of iNOS (I) in the splenocyte of infected mice as well as mice treated with cystatin ⫹ IFN-␥, bpV(Phen), and okadaic acid. *, P ⬍ 0.05; **, P ⬍ 0.01; ***, P ⬍ 0.001.

with inflammatory gene expression are poorly understood. Our main finding is that infection by L. donovani parasites results in significant up-regulation of specific MAPK-directed phosphatases such as MKP1, MKP3, and PP2A, which might be involved in preferential deactivation of p38 and ERK1/2. To our knowledge, this is the first study to examine global changes in MAPK-directed phosphatase gene expression following infection and their downstream effect on Th cell polarization as well as iNOS expression. Using pharmacologic inhibitors, we have demonstrated a definite role of these phosphatases in disease progression. We have validated our observation further by using cystatin, a curing agent for experimental visceral leishmaniasis, and showed that inhibition of MKPs and 18 Journal of Leukocyte Biology


PP2A activity in vivo by cystatin may be necessary for shifting cytokine balance toward a Th1 mode and subsequent elimination of organ parasite burden of infected mice. Taqman analysis revealed two MAPK-directed phosphatases—MKP1 and MKP3—to be induced significantly in L. donovani-infected macrophages along with a serine/threonine phosphatase—PP2A. A number of phosphatases have been linked to ERK1/2 inactivation, including the serine/ theronine phosphatase PP2A; tyrosine phosphatases, hematopoietic PTP and striatal-enriched phosphatase; and the family of dual-specificity phosphatases [13, 25–27]. Hydrogen peroxide-mediated oxidative stress was found to increase ERK activity via a decrease in phosphatase activity,

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including PP2A and PTP [27]. On the other hand, delayed inactivation of ERK was found to be mediated, at least in part, by MKPs. Moreover, a recent study suggests that sustained ERK activity in hyperoxia-exposed cells is a result of the down-regulation of MKP3 and PP2A [13]. Consistent with these observations, induction of MKP3 and PP2A was found in our studies to be the cause of prolonged inactivation of ERK1/2 in L. donovani-infected macrophages. MKP1, on the other hand, did not have a significant role in ERK inactivation; rather, inhibition of MKP1 by triptolide markedly induced the phosphorylation of p38. It may be mentioned in this regard that maximal MKP1 induction did not correlate with a substantial dephosphorylation of ERK1/2 in LPS-stimulated cells, and ectopic expression of MKP1 did not alter the LPS-triggered ERK activation significantly [28]. Several in vitro studies suggested that MKP1 might be an important negative-feedback regulator of macrophage function and inflammatory responses to TLR signals transduced via the p38 pathway [29, 30]. MKP1 has been shown to modulate the production of proinflammatory cytokines and negatively regulate systemic levels of effector and regulatory cytokines produced by activated macrophages, T and NK cells [29]. In the present study also, specific inhibition of MKP1 by triptolide attenuated the synthesis of IL-10 and TGF-␤ significantly in infected macrophages and enhanced the synthesis of proinflammatory cytokines such as TNF-␣ and IL-12. A decrease in the production of TGF-␤ and IL-10 was also observed in infected macrophages pretreated with okadaic acid, an inhibitor of PP2A, or MKP3 siRNA, suggesting a role of these phosphatases also in the induction of anti-inflammatory cytokines. It may be noteworthy that the inability of macrophages infected with Leishmania or Mycobacteria to sustain MAPK activation and to produce high levels of TNF-␣ was, in part, a result of an increase in PP2A activity [15, 18]. In the present study, MKP3- and PP2A-mediated deactivation of ERK1/2 resulted in a substantial decrease in iNOS mRNA expression, whereas MKP1 had little effect. MKP1, rather, has a more significant role to play in cytokine balance. Induction of these phosphatases following infection was found to be associated with activation of different PKC isoforms. Although induction of PKC␨ following infection was essential for the activation of MKP3 and PP2A in vitro, MKP1 activation was dependent on PKC␧ induction. This is not surprising, as the use of antisense oligonucleotides directed toward PKC␧ was found to prolong significantly the time course of ERK activity correlating with the inhibition of MKP1 expression [16]. On the other hand, an increased endogenous ceramide level following L. donovani infection induced impairment of the kinase/phosphatase balance and was also responsible for the induction of PKC␨ [18]. In the present study, we observed that the use of the PKC␰ translocation inhibitor peptide markedly reduced the ERK-directed activity of MKP3 and PP2A in infected macrophages, which suggests a possible link between PKC␰ induction with activation of MKP3 and PP2A, and PP2A is known to be regulated by phosphorylation and subsequent methylation of its catalytic subunit. It is therefore possible that PKC␰ phosphorylates the PP2A catalytic subunit to enhance

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its ERK-directed activity and thereby, attenuates iNOS expression and Th1 cytokine synthesis. PP2A and MKPs may be needed for disease progression, as the use of bpV(phen) for inhibition of MKPs resulted in 77% reduction of spleen parasite burden, whereas the use of okadaic acid for inhibition of PP2A caused 57% reduction. In addition, administration of bpV(phen) or okadaic acid enhanced iNOS gene expression in infected mice, which suggests further that NO synthesis in visceral leishmaniasis is modulated by MKPs and serine/threonine phosphatases. bpV(phen) pretreatment also resulted in a strong up-regulation of iNOS in control and L. donovani-infected BMMs. bpV(phen) is known to be a potent inducer of iNOS, and selective modulation of MAPK by bpV(phen) resulted in enhanced iNOS expression and NO generation in macrophages stimulated with IFN-␥ [31]. We have demonstrated earlier that cystatin, a natural cysteine protease inhibitor, acts as an immunomodulator in activating macrophages for the release of NO and Th1 cytokines, which play a central role in curing visceral leishmaniasis in an experimental animal model [3]. We have demonstrated that cystatin switches the PTP/PTK balance in infected macrophages toward PTK with a parallel activation of ERK and down-regulation of MKP1, MKP3, and PP2A expression. Moreover, suppression of organ parasite burden of infected mice by cystatin plus IFN-␥ is accompanied by in vivo inhibition of MKPs and PP2A, resulting in the shifting of cytokine balance toward a Th1 mode. This is more likely, as inhibition of MKPs and PP2A in vivo by pharmacologic inhibitors not only shifted the cytokine balance toward Th1 but also diminished parasite burden significantly. Promotion of the Th1 response over that of Th2 is associated with control of disease in case of cutaneous leishmaniasis. Although such clear polarization is not the case in visceral leishmaniasis, the Th1 response correlates with resistance to infection [32]. In this light, the modulation of proinflammatory over anti-inflammatory cytokines by PTP inhibitors might favor a better control over infection in vivo. Use of cystatin further strengthened our observation that induction of MAPK-directed phosphatases may be needed for propagation of the Leishmania parasite as well as elicitation of a disease-progressive immunological response. Results of the present study emphasize the important role that MAPK-directed phosphatases play in the establishment of Leishmania infection. Moreover, the study evaluated the differential contribution of PKC isoforms in the induction of MAPKdirected phosphatases following infection and highlighted the fact that cystatin plus IFN-␥-mediated inhibition of MKPs and PP2A activity in infected macrophages promoted positive signal transduction, leading to the up-regulation of effector macrophage functions. Overall, this study has shed light on a novel mechanism used by Leishmania to alter macrophage signaling, which favors its persistence within the host and propagation of the disease. Finally, a better understanding of this pathway will help in developing therapeutic agents having immunomodulatory capacity which favors control not only for nonhealing leishmaniasis but also for other chronic infectious diseases. Volume 88, July 2010


AUTHORSHIP S. K., A. U., and P. K. D. designed research; S. K. and A.U. performed research; S. K., G. S., and P. K. D. analyzed data; S. K., A. U., and P. K D. wrote the paper.

ACKNOWLEDGMENTS This work was supported by the Network Project (NWP 0038) and Supra Institutional Project (SIP007) grant of the Council of Scientific and Industrial Research, Government of India.

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KEY WORDS: macrophage 䡠 protein phosphatase 2A 䡠 inducible NO synthase

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