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Expression of IL-10-triggered STAT3-dependent IL-4Rais required for induction of arginase 1 in visceralleishmaniasis

Arunima Biswas1, Arijit Bhattacharya2, Susanta Kar1

and Pijush K. Das1

1 Molecular Cell Biology Laboratory, Infectious Diseases and Immunology Division,

Indian Institute of Chemical Biology, Kolkata, India2 Department of Biotechnology, Presidency College, Kolkata, India

Although enhanced macrophage-specific arginase activity is directly related to increased

parasite burden in cutaneous leishmaniasis (CL), the regulation and precise role of arg-

inase in the disease outcome of visceral leishmaniasis (VL) has yet to be explored. As in

CL, BALB/c mice infected with Leishmania donovani showed increased levels of arginase in

acute infection. Arginase 1 is the major isoform associated with infection and while the

IL-4-induced arginase pathway is operative in CL, IL-10 plays a crucial role in modulating

arginase activity in VL, although a synergism with IL-4 is required. IL-10, in combination

with IL-4, regulated both in vivo and ex vivo arginase 1 induction in a STAT6 and C/EBPb-

dependent fashion. Further investigation toward the cause of such synergism suggests

that induction of a STAT3-dependent IL-10-mediated cascade in VL triggers the expression

and surface localization of the IL-4 receptor alpha (IL-4Ra) which, in turn, enhances IL-4

responsiveness toward STAT6 and C/EBPb-dependent signaling for arginase 1. This couldalso offer a mechanistic explanation for the fact that, in spite of the low level of IL-4 in VL,

enhanced IL-4-Ra expression by IL-10 might markedly amplify IL-4-mediated arginase 1

signaling and provide a possible mechanism for synergistic induction of arginase 1.

Keywords: Arginase 1 . IL-4 . IL-10 . IL-4 receptor a . Visceral leishmaniasis

Introduction

Arginase is classically considered as a rate-limiting enzyme of the

urea cycle in liver, but has been found to be present in a number

of organs and tissues where the urea cycle is not operative. To

date, two distinct isoforms of arginase have been identified in

mammals. They are encoded by different genes differing in their

cellular localization as well as their mode of regulation: type 1

arginase, a cytosolic enzyme expressed at high levels in liver, and

type 2 arginase, a mitochondrial enzyme found in several tissues

in addition to liver [1]. A growing body of evidence suggests that

arginase induction is correlated with parasite-specific immuno-

suppression of host, thereby facilitating pathogen survival and

growth inside the hostile environment of phagocytic cells.

Macrophages were found to upregulate arginase 1 expression

upon activation by Th2 cytokines and shown to have a

detrimental role in parasite infection by limiting the Th1-

dependent parasite clearance [2]. Interestingly, cAMP and TGF-

b are known to induce arginase 1 induction in macrophages [3].

In Chagas disease, induction of the arginase pathway could be

used by Trypanosoma cruzi to spread inside host [4]. Arginase

activity was triggered in macrophages from mice infected with the

helminth Schistosoma mansoni and was associated with an

increase in concentration of circulating L-ornithine-derived poly-

amines [5]. Intracellular pathogens like Salmonella enterica and

Mycobacterium tuberculosis also utilize host arginase for their ownCorrespondence: Dr. Pijush K. Dase-mail: [email protected]

& 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.eji-journal.eu

DOI 10.1002/eji.201040940 Eur. J. Immunol. 2011. 41: 9921003Arunima Biswas et al.92


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survival within the macrophages [6, 7]. Moreover, in bacteria like

Helicobacter pyroli, arginase plays a major role in its survival as

Helicobacter arginase impairs host T-cell function and allows the

bacteria to efficiently compete with its host in the mucous layer

[8]. Induction of host arginase seems to be important for growth

of trypanosomatidae including Leishmania major in susceptible

host. Murine infection by L. major showed arginase 1-dependentdisease progression and arginase inhibition by the specific

inhibitor, No-hydroxy L-arginine (NOHA), controlled parasite

growth in vitro. The treatment of L. major-infected mice with

Th2 cytokines (IL-4 and IL-10), which are inducers of arginase 1,

led to a proportional increase in the number of intracellular

amastigotes, supporting the idea that host arginase activity may

be involved in promoting parasite proliferation [9]. In contrast to

the self-healing cutaneous leishmaniasis (CL) caused by L. major,

visceral leishmaniasis (VL) is a debilitating and fatal infection

caused by L. donovani and is associated with fever, cachexia,

hepatosplenomegaly, anemia and blood cytopenia. The non-

healing parasite infection is attributed to the expansion of CD41

Th2 cells, characterized by the production of IL-4, IL-10 and IL-13

[10, 11]. However, the regulation of immune responses is

complex and Th2 dominance does not fully explain the non-

healing or reactivated forms of the disease [11, 12].

Upon transmission to the mammalian macrophage, the

intracellular Leishmania parasites are either killed or hosted

depending on the balance of the two inducible enzymes, indu-

cible nitric oxide synthase (iNOS) and arginase. These two

enzymes compete for a common substrate, L-arginine, and are

competitively regulated by cytokines secreted by Th1 and Th2

cells [13]. Although the Km of arginase is in the millimolar range

and that of iNOS in micromolar range, the arginase Vmax at body

pH is 1000 times greater than that of NOS, indicating that similarrates of substrate usage occur for both enzymes at a low arginine

concentration [14]. Though the role of arginase 1 is established

in the case of L. major infection [15] and the roles of Th2 cyto-

kines are well documented in modulating arginase activity in

infection [9], the intricate mechanisms behind the activation of

host arginase are not well documented in diseased models.

Earlier studies indicated that IL-4 either alone or in combination

with IL-10 induced arginase activities, which were further

increased by L. major infection [16]. Reports also suggested that

IL-4 controlled arginase levels in a STAT6-dependent manner,

which was independent of LPS/IFN-g [17]. Moreover, induction

of arginase I transcription by IL-4 requires a composite

DNA response element for STAT6 and C/EBP [18].

Although arginase induction plays a crucial role in the disease

propagation in CL [15], its role in VL is yet to be elucidated. In

the present study, we tried to elucidate the role of arginase in the

disease progression of experimental VL. Since, Th2 cytokines

have been strongly linked with the exacerbation of VL and

promote survival of the parasite by downregulating host-medi-

ated oxidative and inflammatory pathways, we further investi-

gated the distinct role of IL-10 and IL-4 in regulating arginase

activity in VL and wanted to decipher the intricate molecular

mechanisms behind their action.

Results

Arginase in VL and its regulation by Th2 cytokines

Although the induction of arginase in the establishment of CL is

well documented, there are no reports suggesting the role of

arginase in L. donovani-infected mice. We, therefore, first checkedwhether induction of arginase is associated with disease

progression in VL. In L. major-infected mice, arginase activity

increased with a maximum of 5.2-fold induction after 4 wk of

infection compared within activity at 0 wk in footpad homo-

genates (Fig. 1A). Interestingly, similar to L. major infection,

arginase activity in splenocytes of L. donovani-infected mice

increased significantly after the 2nd wk of infection (3.8-fold),

with a maximum of 6.5- and 5.2-fold increase after 4 and 6 wk

respectively over 0 wk infected control (Fig. 1B). Furthermore,

administration of NOHA, the specific inhibitor of arginase, could

significantly suppress (78.1% reduction after 6 wk of infection)

the spleen parasite burden, suggesting thereby that arginase

induction may be directly associated with disease progression in

VL (Fig. 1C).

Since infection with Leishmania parasites is known to induce a

Th2 response, which plays a crucial role in arginase induction, we,

therefore, examined the status of Th2 cytokines in the course of L.

donovani and L. major infection in BALB/c mice and their effect on

arginase induction. Analysis of draining lymph node cells from the

foot pad of L. major-infected mice showed IL-4 to be the major

cytokine that was enhanced rapidly after 2 wk of infection (4.6-

fold increase compared to uninfected mice, po0.001) and main-

tained at 9.9- and 8.4-fold increase after 4 and 6 wk of infection.

However, IL-10 increased moderately during the course of infec-

tion (2.5- and 2.2-fold increase after 4 and 6 wk, respectively,po0.01) (Fig. 1D). In CL, IL-4 seemed to be the major cytokine

controlling the arginase activity as anti-IL-4 Ab administration

after 2 wk of infection decreased enzyme activity by 78.2% after

4 wk, whereas administration of anti-IL-10 Ab could not exert any

appreciable effect (Fig. 1A). In contrast, ELISA studies with

splenocytes of L. donovani-infected mice showed a significant

increase in IL-10 production at 2 wk of infection (3.4-fold,

po0.001) with a maximum of 9.6- and 7.2-fold increase after 4

and 6 wk of infection, respectively, compared to uninfected

control (Fig. 1E). IL-4 production, on the other hand, was at much

lower level (2.1- and 1.9-fold after 4 and 6 wk of infection).

As IL-10 was found to be the major Th2 cytokine in VL and IL-

4 happens to be the prime modulator of arginase activity in CL,

we wanted to determine the role of these cytokines on the

induction of arginase in VL. Similar to L. major infection,

increased arginase activity in L. donovani infection could be

markedly reversed by administration of anti-IL-4 Ab (72.5 and

67.6% reduction after 4 and 6 wk of infection, respectively).

Interestingly, unlike L. major infection, anti-IL-10 Ab adminis-

tration could also reduce the increased arginase activity in L.

donovani-infected mice almost to the similar extent as that of

anti-IL-4 Ab (65.1 and 61.3% decrease after 4 and

6 wk of infection, po0.001) (Fig. 1B). Furthermore, combined

Eur. J. Immunol. 2011. 41: 9921003 Immunity to infection 993



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administration of both these anti-cytokine Abs could cause almost

complete reduction of arginase activity. Among the two existing

isoforms of arginase, the expression of arginase 1 was signifi-

cantly increased both at the mRNA (4.8-fold) and protein (3.3-

fold) level in L. donovani-infected mice as revealed by RT-PCR

(Fig. 1F) and Western blot analysis (Fig. 1G) respectively, after

4 wk of infection, whereas the expression of arginase 2 remained

almost unaltered in splenocyte homogenate.

Since arginase is known to suppress IL-12/IFN-g production,

we assessed the levels of these cytokines after treatment with

NOHA and also after treatment with anti-IL-4 and anti-IL-10

mAbs in infected mouse splenocytes. Arginase inhibition by

NOHA could elevate IFN-g and IL-12 production with a maximum

of 4.9- and 6.1-fold respectively, after 4 wk of infection

(Fig. 1H). Anti-IL-4 and anti-IL-10 Abs could also increase IFN-g

(3.56- and 2.67-fold, respectively) and IL-12 (4.5- and 3.5-fold,

respectively) levels at 4 wk after infection, whereas the values for

combined treatment with both anti-cytokine Abs were much

higher (5.8- and 7.2-fold increase for IFN-g and IL-12, respec-

tively) (Fig. 1H). Since iNOS competes with arginase for the same

Figure 1. Regulation of arginase by Th2 cytokines in leishmaniasis. Arginase activities were measured at the indicated times after infection infootpad lysates of (A) L. major-infected mice and in (B) splenocyte lysates ofL. donovani-infected mice treated with anti-cytokine mAbs as describedMaterials and methods. (C) Spleen parasite burden was determined in L. donovani-infected mice treated with NOHA i.p. five times a wk, starting 2 wkafter infection. Cytokine production by (D) DLN cells isolated from L. major-infected mice and stimulated with L. major-promastigotes and(E) splenocytes isolated from L. donovani-infected mice and stimulated with SLA (50 mg/mL). Arginase 1 and 2 (F) mRNA and (G) protein levels in L.donovani-infected mouse splenocytes determined at the indicated times after infection. (H) Cytokine protein and (I) iNOS mRNA (graph) andprotein (Western blot) production by splenocytes isolated from NOHA- and anti-cytokine mAb-treated L. donovani-infected mice at the indicatedtimes after infection. (FI) GAPDH and b-actin were used as internal controls for mRNA and protein, respectively. (I) Bands on the blot wereanalyzed densitometrically and fold changes are indicated. (J) Splenocytes were isolated from NOHA- and anti-cytokine mAb-treated L. donovani-infected mice 4 wk after infection and incubated with SLA (50mg/mL) and supernatant NO levels were measured by the Griess method. The dataare representative of three independent experiments and are expressed as mean 1 SD, n53. po0.001, po0.0001; Students t-test.

Eur. J. Immunol. 2011. 41: 9921003Arunima Biswas et al.94



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substrate L-arginine, we checked whether arginase inhibition with

NOHA or treatment with anti-cytokine mAbs could enhance

expression of iNOS in infected mice. Administration of NOHA as

well as anti-IL-4 and anti-IL-10 mAbs could markedly increase the

expression of iNOS both at the mRNA (5.5-fold for NOHA and

6.11-fold for combined anti-cytokine Abs) (Fig. 1I) and protein

(4.1-fold for NOHA and 4.7-fold for combined anti-cytokine Abs)levels (Fig. 1I, inset). Nitrite generation was also significantly

increased in NOHA-treated (4.2 mM to 17.11 mM) and combined

anti-cytokine Ab-treated (4.219.4 mM) mice splenocytes (Fig.

1J). These results indicate that, in a murine model of VL, IL-10

might play a major role in modulating arginase activity in addi-

tion to IL-4 and that arginase 1 is the major isoform associated

with infection. Moreover, arginase inhibition could markedly

increase Th1 response as well as the expression of iNOS and the

generation of nitrite in infected mice.

Role of Th2 cytokines on arginase activity

To ascertain the roles of IL-4 and IL-10 in the ex vivo activation ofarginase following L. donovani infection, peritoneal macrophages

were cultured in splenocyte supernatants from either PBS-treated

or L. donovani-infected mice in the presence or absence of mAbs

against various cytokines. Peritoneal macrophages cultured in

splenocyte supernatants from PBS-treated mice showed very little

arginase activity (62.3 mU/mg), which was considerably

increased (4.6-fold, po0.001) when cultured in supernatants of

Figure 2. Effect of IL-4 and IL-10 on arginase activity in VL. (A) Supernatants from cultured splenocytes isolated from mice infectedwith L. donovani-infected for 4 wk (splenocyte supernatant) were added to peritoneal macrophages (5105 cells/mL) isolated from BALB/cmice and cultured either alone or in the presence of anti-cytokine mAbs as indicated and arginase activity was measured. (B) RAW 264.7 cells(5105cells/mL) were treated with recombinant IL-4 and/or IL-10 (10 ng/mL) for 24 h and arginase activity was evaluated. (C, D) RAW 264.7 cellswere transfected with arginase luciferase reporter plasmid (31/3810) followed by incubation for 24 h with (C) splenocyte supernatant fromL. donovani-infected (4 wk) mice either alone or in the presence of anti-cytokine mAbs or (D) IL-4 and/or IL-10 and luciferase activity determined.The cultures were set in triplicate and the data are representative of four individual experiments. The error bar represents the mean 1 SD, n54.po0.001, po0.0001; Students t-test.




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splenocytes obtained from infected mice (Fig. 2A). Similar to the

in vivo situation, anti-IL-4 and anti-IL-10 Abs could markedly

reduce arginase activity (78.1 and 68.4% reduction, respectively)

in peritoneal macrophages when added to the splenocyte

supernatants of infected mice. Combined administration of both

anti-cytokine Abs almost completely reduced the arginase

activity. To further ascertain the individual roles of IL-4 and IL-10 in regulating the arginase activity, RAW 264.7 cells were

treated with recombinant cytokines for 8 h. Arginase activity

increased appreciably (4.7-fold, po0.001) in recombinant IL-4-

treated macrophages whereas no induction of the enzyme activity

was observed in recombinant IL-10-treated macrophages

(p50.07) (Fig. 2B). Interestingly, co-administration of both

these cytokines showed a marked increase of arginase activity

(7.5-fold, po0.0001) (Fig. 2B). These results along with our

previous observations suggest that although IL-10 could not

induce arginase activity individually, it had a pronounced

synergistic effect on the induction of arginase by IL-4.

Arginase 1 expression in murine macrophages is controlled by

an enhancer that is present 3 kb upstream of the basal promoter

containing STAT6 and C/EBPb-binding sites [18]. These consti-

tute the IL-4 responsive elements. To ascertain the effect of Th2

cytokines on arginase 1 promoter activity, RAW cells were

transfected with an arginase 1 promoter construct containing IL-4

response elements (31/3810). The transfected cells were cultured

in splenocyte supernatants obtained from infected mice in the

presence or absence of anti-IL-4 and anti-IL-10 mAb. The luci-

ferase activity of the arginase promoter increased by 21.4-fold

(po0.0001) when macrophages were cultured with splenocyte

supernatants from infected mice as compared with macrophages

cultured in splenocyte supernatants from PBS-treated mice.

Addition of either anti-IL-4 or anti-IL-10 Ab to the splenocyte

supernatant significantly decreased the arginase promoter activ-

ity by 79.1 and 69.5%, respectively (Fig. 2C). Combined

administration of both anti-cytokine antibodies could almost

completely inhibit arginase promoter activity. Arginase 1

promoter activity in RAW264.7 cells remained unaffected byrecombinant IL-10 treatment but was markedly induced by

recombinant IL-4 either alone or in combination with IL-10 (14.4-

and 26.3-fold, respectively, po0.0001, Fig. 2D). These results

suggest that similar to enzyme activity, IL-10 could only induce

arginase promoter activity in synergy with IL-4 in VL.

Role of IL-10 in the regulation of STAT6 and C/EBPbbinding to the arginase 1 promoter in VL

Although IL-10 is the major Th2 cytokine in VL and plays a

synergistic role with IL-4 in inducing arginase activity in the in vivo

as well as the ex vivo situation, individually it has no role in

augmenting enzyme nor promoter activity in an in vitro situation.

We, therefore, wanted to observe whether IL-10 has any role in

regulating the IL-4 response elements of the arginase 1 promoter

in vivo. The transcription factors STAT6 and C/EBPb play a major

role in the induction of arginase 1 transcription by binding to

composite DNA response elements in the arginase 1 promoter

region [19]. DNA-binding analysis in the nuclear extracts prepared

from splenocytes of infected mice revealed that L. donovani

infection resulted in markedly increased STAT6 and C/EBPb

binding (2.8 and 2.4-fold, respectively) after 4 wk of infection

Figure 3. Effect of Th2 cytokines on binding of STAT6 and C/EBPb. L. donovani-infected mice were administered with anti-IL-4 mAb and/or anti-IL-10 mAb starting 2 wk after infection. Splenocytes were isolated 4 wk after infection. EMSA of (A) STAT6 and (B) C/EBPb were performed usingsplenocyte nuclear extracts. Bands were analyzed densitometrically and fold changes are indicated. The results are representative of threeseparate experiments.




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(Fig. 3A and B). Administration of anti-IL-4 Ab could reduce

STAT6 binding by 62.9% (Fig. 3A) and C/EBPb binding by 61.8%

(Fig. 3B). Combined administration of anti-IL-10 and anti-IL-4 Abs

could reduce STAT6 binding by 88.1%, and C/EBPb binding by

85.1%. It was indeed interesting to observe that anti-IL-10 Ab

administration could also reduce STAT6 binding by 58.5% (Fig.

3A) and C/EBPb binding by 57.1 % (Fig. 3B) in L. donovani-infected mice, although these transcription factors are mainly

induced by IL-4 and no previous report has indicated the

involvement of IL-10 in their regulation. These results suggest

that in the diseased condition both IL-4 and IL-10 may be involved

in regulating the transcription factors required for arginase 1

expression.

IL-10-induced IL-4Ra expression modulates arginaseactivity following infection

Having found that IL-10 might have a role in the activation of

STAT6 and C/EBPb in VL, we next investigated the mechanism by

which these transcription factors are activated in L. donovani-

infected mice. IL-10 is known to induce a number of genes in

macrophages including the gene for IL-4Ra [19, 20], which might

be responsible for synergistic induction of arginase-1 expression

by IL-4 and IL-10. We, therefore, wanted to assess whether

L. donovani infection could modulate the expression of IL-4Ra in

an in vivo situation. A time-course analysis after infection

revealed that the expression of IL-4Ra was significantly increased

at both the mRNA and protein level in infected mice, which was

maximal after 4 wk of infection (6.3- and 4.2-fold at mRNA and

protein levels respectively, po0.0001) (Fig. 4A and B). Admin-

istration of anti-IL-10 Ab significantly decreased IL-4Raexpression in infected mice (75.1% in mRNA and 62.1% in

protein levels) and this was further enhanced by co-administra-

tion of anti-IL-4 and anti-IL-10 Ab (88.4 and 76.5% reduction

respectively, in mRNA and protein levels). On the other

hand, the expression of IL-4Ra was moderately inhibited by

administration of anti-IL-4 Ab alone (21.1% reduction in mRNA

level and 19.2% in protein levels, po0.01) (Fig. 4C and D).

We further examined the surface localization of IL-4Ra by

immunofluorescence labeling and two-color flow cytometric

analysis. Based on surface expression of CD-11b, macrophages of

splenocytes were gated by anti-CD-11b-FITC and the level

of IL-4Ra protein expression was indicated by the mean fluores-

cence intensity of anti-IL-4Ra-PE staining (Fig. 4E). The percen-

tage of macrophages having IL-4Ra expression was significantly

increased in infected mice (30.573.1 compared to 6.070.59% in

control mice, po0.0001). When anti-IL-4 Ab was administered in

infected mice, IL-4Ra expression decreased moderately

(24.272.4% compared to 30.573.1% in infected mice). Inter-

estingly, administration of anti-IL-10 Ab alone could significantly

reduce the IL-4Ra expression (16.871.6 compared to 30.573.1%

in infected mice, po0.001). Combined dose of both antibodies

could further downregulate IL-4Ra surface expression

(10.271.0% compared to 30.573.1% in infected mice). These

results indicate that in VL, IL-10 might somehow be involved in

enhancing IL-4Ra expression. Furthermore, to ascertain whether

IL-4Ra expression could directly modulate arginase activity, we

administered anti-IL-4Ra-Ab in infected mice and monitored the

enzyme activity. Arginase activity was significantly attenuated

(65.7% reduction) after 4 wk of infection when administered with

anti-IL-4Ra-Ab (Fig. 4F). Western blot analysis also showed thatanti-IL-4Ra-Ab administration could markedly reduce the expres-

sion of arginase 1 (62.1% reduction) at the protein level (Fig. 4G).

Collectively, these results suggest that IL-10-mediated IL-4Ra

expression may be necessary for arginase 1 induction in VL.

Role of STAT3 in IL-10-mediated IL-4Ra expression

Since IL-10 might have a role in regulating expression of IL-4Ra in

VL, we were interested to assess the status of IL-10-regulated

transcription factors following infection. Though STAT3 is reported

to be the major transcription factor involved in IL-10 signaling and

mice lacking STAT3 in macrophages have a strikingly similar

phenotype as IL-10-deficient mice [20], other transcription factors

like Sp1 and Sp3 are also regulated by IL-10. We, therefore,

checked the nuclear translocation and DNA binding of IL-10-

regulated transcription factors following infection in vivo. DNA

binding of Sp1/Sp3 was not induced in infected mice, whereas

STAT3 binding increased significantly after 4 wk of infection

(Fig. 5A). In vivo EMSA analysis revealed that specific complex

formation with the oligonucleotide containing a STAT3 site

increased 3.2-fold using nuclear extracts from infected compared

to uninfected mice and administration of anti-IL-10 Ab could

significantly reduce the enhanced binding (Fig. 5B). In contrast,

administration of anti-IL-4 Ab had neither any significant effect onSTAT3 DNA binding when administered alone nor any additive

effect when administered in combination with anti-IL-10 (Fig. 5B).

These results suggest that the activation and nuclear translocation

of STAT3 in infected mice might be regulated by IL-10.

To further correlate IL-10-mediated STAT3 induction with IL-

4Ra expression and arginase 1 induction following infection, an

siRNA-mediated knock-down system was adopted in RAW 264.7

cells for the inhibition of STAT3. Silencing of STAT3 significantly

suppressed IL-4Ra expression at both the mRNA (78.2% reduc-

tion) and protein levels (77.4% reduction) in macrophages

cultured in splenocyte supernatants obtained from infected mice

(Fig. 5C and E). Arginase 1 activity and expression were also

found to be significantly reduced at both the mRNA and protein

levels in STAT3 siRNA-transfected macrophages when cultured in

splenocyte supernatants obtained from infected mice (66.1%

reduction in activity and 58.1 and 52.2% reduction in mRNA and

protein level expression, respectively) (Fig. 5DF). The efficacy of

siRNA on STAT3 expression was evaluated by western blot

analysis. Expression of STAT3 was observed to be significantly

reduced in cells expressing STAT3-specific siRNA compared to

cells expressing control siRNA (Fig. 5E). These results suggest

that IL-4Ra expression and arginase 1 induction may be modu-

lated by STAT3-mediated IL-10 signaling in VL.




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Figure 4. Effect of Th2 cytokines on IL-4Ra expression. IL-4Ra (A) mRNA and (B) protein expression in L. donovani-infected (4 wk) mousesplenocytes at indicated times after infection. GAPDH and b-actin were used as internal controls for RNA and protein, respectively. IL-4Ra(C) mRNA and (D) protein expression in splenocytes isolated from L. donovani-infected (4 wk) mice treated with anti-IL-4 and/or anti-IL-10 mAbstarting at 2 wk after infection. (E) Splenocytes were isolated 4 wk after infection from L. donovani-infected, anti-cytokine mAb-treated mice andanalyzed by flow cytometry gating on CD11b and IL-4Ra was tagged with anti-IL-4Ra-PE antibody. Arginase 1 (F) protein expression and (G) activitywas determined in splenocytes isolated from L. donovani-infected mice treated with anti-IL-4Ra mAb starting at 2 wk after infection. The data arerepresentative of three independent experiments. Error bars represent mean 1 SD, n53. po0.01, po0.001, po0.0001 versus as indicated(A and C); po0.001 versus infected control (G).




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Discussion

Arginase, a competitor of iNOS for the substrate arginine, is

known to help microbes avoid the NO-dependent killing in

macrophages during infection with intracellular pathogens like

Toxoplasma gondii, M. tuberculosis and L. major [21]. In a mouse

model of CL, arginase 1 is induced during disease development

[16] and is mediated by the balance between IL-4 and IL-12.

Moreover, inhibition of arginase 1 delays disease outcome in

susceptible mice [16]. In the present study, we have demon-

strated that BALB/c mice infected with L. donovani had increased

levels of arginase 1 in acute infection and were associated with

increased IL-10 synthesis. The pathway that works toward the

induction of arginase 1 in VL is unique and different from the

Figure 5. Effect of IL-10-regulated transcription factors in IL-4Ra-mediated arginase expression. (A) Splenocytes were isolated from mice infectedwith L. donovani (4 wk) and EMSA for STAT3 and Sp1/Sp3 were performed using the splenocyte nuclear extracts. (B) Splenocyte nuclear extractsprepared from L. donovani-infected mice treated with anti-cytokine mAbs were used for EMSA of STAT3. Cold competitor oligonucleotides wereused for specific binding. (A, B) Bands were analyzed densitometrically and fold changes are indicated. (CE) RAW 264.7 cells were transfected(24 h) with control and STAT3 siRNA and cultured for 24 h in supernatant of cultured splenocytes obtained from control or 4-w k infected mice and

IL-4Ra and arginase 1 (C, D) mRNA and (E) protein expression was determined. (E) STAT3 siRNA specificity was determined by Western blotting inwhole cell extracts from RAW 264.7 cells expressing either STAT3 or control siRNAs. (F) RAW 264.7 cells were transfected with STAT3 siRNA andcultured for 24 h in supernatant of cultured splenocytes obtained from control or 4-wk infected mice and arginase activity determined. Results arerepresentative of three individual experiments. Error bars represent mean 1 SD, n53. po0.001; Students t-test




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IL-4-induced STAT6 and C/EBPb-dependent arginase 1 pathway

that may be operative in CL. In L. donovani infection, arginase 1

may be induced by a STAT3-dependent IL-10-mediated cascade,

which may trigger the expression and surface localization of

IL-4Ra to induce the IL-4-mediated signaling for arginase 1. As

far as Leishmania arginase is concerned, the lack of this enzyme

as in arg/

L. major or in arg/

L. mexicana showed impairedinfectivity resulting in delayed onset of lesion development,

attenuated pathology, and low parasite burden [22]. Results also

suggest that parasite-encoded arginase of L. major subverts

macrophage microbicidal activity by diverting arginine away

from iNOS [23]. L-arginine depletion and arginase-1-induced

polyamine production favors the growth of L. major, and

parasite-encoded arginase seems to partially control parasite

replication and disease manifestation in macrophages [22].

However, reports on the role of L. donovani arginase in disease

progression are scanty.

It has been reported that during progressive infection in

hamsters with L. donovani, there is low expression of NOS2 but

high splenic arginase I mRNA expression and increased

arginase activity as well as its downstream products, polyamines

[24]. Increased arginase activity and polyamine synthesis were

also evident in an in vitro model of infected hamster

macrophages [24]. A report also suggests that arginase is

involved in L. infantum parasite survival in the host and the

failure to control the progression of splenic infection in mice may

be related to the actions of IL-10 attenuating the cytotoxic

response via NO downregulation [25]. Pathogenesis and failure

to check the proliferation of the intracellular parasites in leish-

maniasis has been ascribed to polarized Th2 response [25], but

the precise mechanism resulting in the inability to

control disease progression is not very well documented.The Th1/Th2 paradigm to intracellular infection is largely

based on investigations using L. major and the roles of IL-12 and

IL-4 in driving Th1 and Th2 cell development for resistance and

susceptibility respectively are well established [26]. Since

previous studies correlated high levels of IL-4 with increased

arginase activities and increased parasite burden in L. major-

infected BALB/c mice [16], we aimed to study the status

of host arginase in L. donovani-infected mice and the cellular

mechanism underlying its modulation. Studies suggested that

IL-4, though increased slightly in visceral infection by

L. donovani compared to cutaneous infection by L. major, does

not promote chronic disease progression and indeed may play a

protective role. Some reports also pointed toward chronic disease

progression by IL-4 in VL, but the results are not conclusive about

the exact role of IL-4 in VL. Unlike L. major infection, where IL-4

plays a major role in disease outcome, IL-10 is the major Th2

cytokine that drives the disease progression in L. donovani

infection and serves as an immunosuppressive factor in VL, which

renders macrophages unresponsive to activation signals by

downregulation of TNF-a and NO [27].

Our studies indicated a significant increase in IL-10 levels in

VL and administration of anti-IL-10 Ab markedly abrogated

arginase induction in L. donovani-infected mice. Earlier studies in

CL indicated IL-4 and IL-13 to be the major cytokines driving

increased arginase activity in macrophages [28]. However, a

synergistic increase of arginase by IL-4 and IL-10 has also been

reported [29]. In the present study, increased arginase activity in

VL was found to be associated with upregulation of arginase 1

enzyme at both the mRNA and protein level. Though mitochon-

drial arginase 2 expression could restrict macrophage NOproduction in Helicobacter pylori infection [30], we have excluded

the role of arginase 2 in increasing arginase activity as arginase 2

was not induced in L. donovani-infected mice.

One of the interesting observations was that, although the IL-4

level is low in VL, the induction of arginase was as high as that in

CL. From our in vivo and ex vivo studies, it seemed that the

increased IL-10 level might have a role in the induction of argi-

nase following infection byL. donovani. But IL-10 alone could not

induce arginase activity as observed in the in vitro setup

of RAW 264.7 macrophages. IL-10 could only induce in synergy

with IL-4 and, interestingly, that synergistic increase was much

higher than IL-4 treatment alone. We, therefore, were interested

to address the question as to how IL-10 might be participating in

the arginase-mediated disease progression in synergy with IL-4.

The arginase 1 promoter was extensively studied and the

presence of an enhancer located 3 kb upstream of its transcription

start site, which is responsive to signals delivered by IL-4 [19],

was confirmed. IL-4 and IL-13 are by far the most potent signals

to regulate the arginase 1 enhancer when factors like STAT6, C/

EBPb and P.U1 form complex in the correct temporal order to

initiate its action.

Since distinct mechanisms have been reported for the regu-

lation of arginase 1 expression in different types of infection,

which might be STAT6-dependent or STAT6-independent, as is

the case for Mycobacterium where it is dependent on C/EBPb andMyD88 [21], we wanted to know the detailed molecular

mechanisms underlying the activation of arginase 1 following L.

donovani infection. It was interesting to note that IL-10 could

control the arginase 1 enhancer region in a STAT6 and C/EBPb-

dependent fashion in L. donovani-infected mice. However, the

mechanisms behind such activation were not clear as

STAT6 and C/EBPb are well-known IL-4-induced transcription

factors [19]. IL-10 has been reported to induce a highly restricted

number of genes in resting macrophages [15] including

IL-4Ra. IL-4Ra-deficient mice with L. major infection showed

strikingly delayed disease progression [31] and synergistic

induction of arginase 1 by IL-4 and IL-10 was associated with the

upregulation of IL-4Ra expression [20]. Interestingly, our data

indicated that IL-10 could markedly induce IL-4Ra expression

following L. donovani infection, which might enhance IL-4

responsiveness in vivo followed by STAT6 and C/EBPb-mediated

arginase induction. This could also offer a mechanistic

explanation for the fact that in spite of very low levels of

IL-4 in VL enhanced IL-4Ra expression by IL-10 might markedly

amplify IL-4-mediated arginase 1 signaling and could provide a

possible mechanism of synergistic induction of arginase 1.

The importance of IL-4Ra was further validated by the use of

anti-IL-4Ra Ab, which could reduce arginase activity and




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expression in L. donovani-infected mice. The IL-10 receptor

normally activates STAT3 and loss of this transcription factor

mimics the loss of IL-10 itself [20, 32]. STAT3 is also known to

play major roles in infection with Salmonella and Toxoplasma.

Rapid and sustained activation of STAT3 was observed in hosts

after cell invasion byT. gondii and Salmonella typhi [33, 34]. Our

data further indicated that among the different transcriptionfactors that function in an IL-10-dependent manner, STAT3 was

the most potent one, which was activated during L. donovani

infection and, in turn, regulated IL-4Ra expression and arginase

activation. This led us to suggest that in VL, IL-10-mediated

STAT3-dependent expression of IL-4Ra recruited IL-4, which in

turn activated STAT6/C/EBPb-mediated arginase 1 expression.

In conclusion, we speculate that induction of arginase 1 by an

IL-10-mediated STAT3-dependent pathway activating IL-4Ra

expression could be a potential mechanism by which the patho-

gen L. donovani escapes the host immune response and this could

not only be relevant for leishmaniasis but also have serious

implications for diseases in which the host defense depends on

cell-mediated immune responses.

Materials and methods

Reagents

All antibodies were from Santa Cruz Biotechnology and BD

Pharmingen. All other chemicals were from Sigma unless

otherwise indicated.

Cell culture and infections

L. donovani promastigotes (MHOM/IN/1983/AG83) were grown

as described previously [35]. The murine macrophage cell line

RAW 264.7 was maintained as described before [36]. For VL,

female BALB/c mice (2025g) were injected with 107L. donovani

promastigotes via the tail vein. Visceral infections were assessed

weekly in terms of spleen parasite burdens, expressed as

Leishman-Donovan units (LDU) as described earlier [36]. In

separate experiments mice were administered with 100 mg of the

arginase inhibitor No-hydroxy-L-arginine (NOHA) in 100mL of

PBS, intraperitoneally five times a wk, starting after 2 wk of

infection and continued throughout the course of infection. For

CL, mice were injected with 2106 L. major LV39 parasites

subcutaneously into the footpad. To study the effect of cytokines

on arginase activity, infected mice were administered with anti-

IL-4 mAb (2.5mg/kg body weight, i.p. and twice a wk) and anti-

IL-10 mAb (2.0mg/kg, i.p., twice a wk) either alone or in

combination starting at 2 wk of infection and continued up to

6 wk. Anti-IL-4Ra mAb (2.5 mg/kg body weight, i.p. and twice a

wk) was administered separately. The investigation conforms to

the Guide for the Care and Use of Laboratory Animals published

by US National Institutes of Health (NIH Publication No. 8523

revised 1996) and with the approval of the Institutional Animal

Care and Use Committee.

Splenocyte culture

Splenocytes from BALB/c mice were isolated from differentgroups of mice as described previously [37]. The cells (5 106/

mL) were stimulated with 20 mg/mL soluble leishmanial antigen

(SLA) for 48h. Splenocyte supernatant were obtained by

centrifuging the culture plates and kept at 201C until further

use. SLA was prepared as previously described [38].

Real-time PCR

Total RNA was isolated from splenocytes using the RNeasy

Minikit (Qiagen). One microgram of RNA was used as a template

for cDNA synthesis and quantitative Real time PCR (ABI 7500

Fast Real Time PCR system; Applied Biosystems) was performed

using Taqman Fast Universal PCR Master Mix (Applied Biosys-

tems) as described earlier [39].

Cytokine analysis by ELISA

The levels of cytokines in splenocytes from L. donovani-infected

mice were measured as described previously [35] using a

sandwich ELISA kit (BD Biosciences). For L. major-infected mice,

draining lymph nodes were harvested and 5106 cells/mL was

restimulated with 1106 cells/mL L. major promastigotes. The

level of cytokines in culture supernatants was determinedaccording to the manufacturers protocols.

Arginase activity

Arginase activity was measured according to Corraliza et al. [40].

Spleen tissue ($100mg) from mice was crushed and resuspended

50 mM Tris-HCl buffer, pH 7.5, followed by homogenization. After

addition of 0.1% Triton X-100, the cell lysates were incubated at 251C

for 10 min with shaking. Macrophages were lysed in buffer contain-

ing 0.1% Triton X-100, 100mg/mL pepstatin, 100mg/mL aprotinin

and 100mg/mL antipain. To the lysed cells, 10mM MnCl2, pH

7.4 mM, 50mM Tris-HCl were added to activate the enzyme by

heating for 10 min at 561C. Arginine hydrolysis was carried out by

incubating 25mL of the activated lysate with 25 mM L-arginine (pH

9.7) for 60 min at371C and the reaction was stopped with 400mL o f a

mixture of H2SO4, H3PO4 and H2O (1:3:7, v/v). The urea formed was

measured at 540nm after the addition of a-nitrosopropiophenone

(dissolved in 100% ethanol) and subsequent heating at 1001C for

45 min. For L. major-infected mice, footpads were homogenized and

arginase activity was measured using 510 mL of the homogenates.

One unit of enzyme activity is defined as the amount of enzyme that

catalyzed the formation of 1mmol of urea/min.




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Quantification of NO

For determination of NO generation in different groups of

infected BALB/c mice after 4 wk of infection, splenocytes

(2106/mL) were isolated and stimulated with or without

50mg/mL SLA for 48 h before performing nitrite assay by the

Greiss reaction as described previously [35].

EMSA

Nuclear extracts from splenocytes were isolated and used for

EMSA as described earlier [36]. For the preparation of

radiolabeled probes representing standard consensus sequences

of various transcription factors, the following oligonucleotides

were used: STAT-6: 50-GTA TTT CCC AGA AAA GGA AC-30;

STAT-3: 50-GAT CCT TCT GGG AAT TCC TAG ATC-30, C/EBP:

50-TGC AGA TTG CGC AAT CTG CA-3 0 and Sp1/Sp3: 50-ATT CGA

TCG GGG CGG GGC GAG C-30. The DNAprotein complex was

electrophoresed and analyzed by autoradiography.

Immunoblot analysis

Immunoblot analyses in splenocyte lysates were performed as

described earlier [41].

Transient transfection and reporter assay

Transfections were carried out in 2 106 cells with the arginase 1

promoter construct (31/3810, a kind gift from Dr. PeterJ. Murray, Department of Infectious Diseases, St. Jude Childrens

Research Hospital, Memphis, TN, USA) [42] in serum-free

medium using Lipofectamine (Invitrogen) according to the

manufacturers instructions. Luciferase activity was determined

as described earlier [36]. For siRNA transfection, cells

were transfected with 1 mg of STAT3 siRNA or control siRNA

according to the manufacturers instructions (Santa Cruz

Biotechnology).

Flow cytometry

Fluorochrome-conjugated mAb against CD11b and IL-4Ra

were obtained from BD Pharmingen. The splenocytes

were washed and Fc receptors were blocked with 5% FCS,

Fcg IgG and 0.5% BSA in PBS for 30min. The cells were

stained for surface markers with monoclonal PE and FITC-

conjugated antibodies directed against mouse IL-4Ra and

CD-11b, respectively, at 41C for 30 min in dark. After

staining, cells were centrifuged and resuspended in PBS.

At least 105 events were acquired on a FACS canto (BD

Biosciences) for subsequent analysis using FACS DIVA software

(BD Biosciences).

Statistical analysis

Experiments were performed at least three times each. Macro-

phage cultures were performed in triplicate and the animal

experiments were carried out with 56 mice per group. Students

t-test was used to evaluate the significance of the differences

between the means of the control and the experimental groups.

Acknowledgements: This work was supported by the

Department of Science and Technology and the Network

Project (NWP 0038) grant of the Council of Scientific and

Industrial Research, Government of India. We thank Dr. Anindita

Bhattacharya for critically analyzing the manuscript.

Conflict of interest: The authors declare no financial or

commercial conflict of interest.

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Abbreviations: CL: cutaneous leishmaniasis IL-4Ra: IL-4 receptor alpha

iNOS: inducible nitric oxide synthase NOHA: No-hydroxy-L-arginine

SLA: soluble leishmanial antigen VL: visceral leishmaniasis

Full correspondence: Dr. Pijush K. Das, Molecular Cell Biology

Laboratory, Indian Institute of Chemical Biology, 4 Raja S.C. Mullick

Road, Kolkata 700032, India

Fax: 1033-2473-5197

e-mail: [email protected]

Received: 11/8/2010

Revised: 16/12/2010

Accepted: 20/1/2011

Accepted article online: 31/1/2011



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