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Arunima Biswas et al.

DOI 10.1002/eji.201040940

Eur. J. Immunol. 2011. 41: 9921003

Expression of IL-10-triggered STAT3-dependent IL-4Ra is required for induction of arginase 1 in visceral leishmaniasis
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, India Department of Biotechnology, Presidency College, Kolkata, India

Although enhanced macrophage-specic arginase activity is directly related to increased parasite burden in cutaneous leishmaniasis (CL), the regulation and precise role of arginase 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/EBPbdependent 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 could also 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 identied 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

Correspondence: Dr. Pijush K. Das e-mail: pijushdas@iicb.res.in

arginase induction is correlated with parasite-specic immunosuppression 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 Th1dependent parasite clearance [2]. Interestingly, cAMP and TGFb 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 polyamines [5]. Intracellular pathogens like Salmonella enterica and Mycobacterium tuberculosis also utilize host arginase for their own

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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 efciently 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-dependent disease progression and arginase inhibition by the specic 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 nonhealing 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 nonhealing 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, inducible 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 similar rates 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 cytokines 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-mediated oxidative and inammatory pathways, we further investigated 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, rst checked whether 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 homogenates (Fig. 1A). Interestingly, similar to L. major infection, arginase activity in splenocytes of L. donovani-infected mice increased signicantly 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 specic inhibitor of arginase, could signicantly 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 (4.6fold increase compared to uninfected mice, p o 0.001) and maintained at 9.9- and 8.4-fold increase after 4 and 6 wk of infection. However, IL-10 increased moderately during the course of infection (2.5- and 2.2-fold increase after 4 and 6 wk, respectively, p o 0.01) (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 signicant increase in IL-10 production at 2 wk of infection (3.4-fold, p o 0.001) 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 IL4 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 administration 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, po 0.001) (Fig. 1B). Furthermore, combined
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Figure 1. Regulation of arginase by Th2 cytokines in leishmaniasis. Arginase activities were measured at the indicated times after infection in footpad lysates of (A) L. major-infected mice and in (B) splenocyte lysates of L. donovani-infected mice treated with anti-cytokine mAbs as described Materials and methods. (C) Spleen parasite burden was determined in L. donovani-infected mice treated with NOHA i.p. ve times a wk, starting 2 wk after 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) and protein (Western blot) production by splenocytes isolated from NOHA- and anti-cytokine mAb-treated L. donovani-infected mice at the indicated times after infection. (FI) GAPDH and b-actin were used as internal controls for mRNA and protein, respectively. (I) Bands on the blot were analyzed densitometrically and fold changes are indicated. (J) Splenocytes were isolated from NOHA- and anti-cytokine mAb-treated L. donovaniinfected mice 4 wk after infection and incubated with SLA (50 mg/mL) and supernatant NO levels were measured by the Griess method. The data are representative of three independent experiments and are expressed as mean 1 SD, n 5 3. p o 0.001, p o 0.0001; Students t-test.

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 signicantly increased both at the mRNA (4.8-fold) and protein (3.3fold) 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, respectively) (Fig. 1H). Since iNOS competes with arginase for the same

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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 signicantly 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 addition 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 of arginase 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 infected with L. donovani-infected for 4 wk (splenocyte supernatant) were added to peritoneal macrophages (5 105 cells/mL) isolated from BALB/c mice and cultured either alone or in the presence of anti-cytokine mAbs as indicated and arginase activity was measured. (B) RAW 264.7 cells (5 105 cells/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 cells were transfected with arginase luciferase reporter plasmid (31/3810) followed by incubation for 24 h with (C) splenocyte supernatant from L. 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, n 5 4. p o 0.001, p o 0.0001; 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 IL10 in regulating the arginase activity, RAW 264.7 cells were treated with recombinant cytokines for 8 h. Arginase activity increased appreciably (4.7-fold, po 0.001) in recombinant IL-4treated macrophages whereas no induction of the enzyme activity was observed in recombinant IL-10-treated macrophages (p 5 0.07) (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 constitute 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 luciferase 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 signicantly decreased the arginase promoter activity 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 by recombinant IL-10 treatment but was markedly induced by recombinant IL-4 either alone or in combination with IL-10 (14.4and 26.3-fold, respectively, po 0.0001, 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/EBPb binding 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-IL10 mAb starting 2 wk after infection. Splenocytes were isolated 4 wk after infection. EMSA of (A) STAT6 and (B) C/EBPb were performed using splenocyte nuclear extracts. Bands were analyzed densitometrically and fold changes are indicated. The results are representative of three separate 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. donovaniinfected 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.

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 signicantly attenuated (65.7% reduction) after 4 wk of infection when administered with anti-IL-4Ra-Ab (Fig. 4F). Western blot analysis also showed that anti-IL-4Ra-Ab administration could markedly reduce the expression 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 IL-10-induced IL-4Ra expression modulates arginase activity 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. donovaniinfected 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 signicantly 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, po 0.0001) (Fig. 4A and B). Administration of anti-IL-10 Ab signicantly decreased IL-4Ra expression in infected mice (75.1% in mRNA and 62.1% in protein levels) and this was further enhanced by co-administration 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, po 0.01) (Fig. 4C and D). We further examined the surface localization of IL-4Ra by immunouorescence labeling and two-color ow 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 uorescence intensity of anti-IL-4Ra-PE staining (Fig. 4E). The percentage of macrophages having IL-4Ra expression was signicantly increased in infected mice (30.573.1 compared to 6.070.59% in control mice, p o 0.0001). 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). Interestingly, administration of anti-IL-10 Ab alone could signicantly reduce the IL-4Ra expression (16.871.6 compared to 30.573.1% in infected mice, p o 0.001). Combined dose of both antibodies could further downregulate IL-4Ra surface expression (10.271.0% compared to 30.573.1% in infected mice). These
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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-decient 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-10regulated transcription factors following infection in vivo. DNA binding of Sp1/Sp3 was not induced in infected mice, whereas STAT3 binding increased signicantly after 4 wk of infection (Fig. 5A). In vivo EMSA analysis revealed that specic 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 signicantly reduce the enhanced binding (Fig. 5B). In contrast, administration of anti-IL-4 Ab had neither any signicant effect on STAT3 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 IL4Ra 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 signicantly suppressed IL-4Ra expression at both the mRNA (78.2% reduction) 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 signicantly 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 efcacy of siRNA on STAT3 expression was evaluated by western blot analysis. Expression of STAT3 was observed to be signicantly reduced in cells expressing STAT3-specic siRNA compared to cells expressing control siRNA (Fig. 5E). These results suggest that IL-4Ra expression and arginase 1 induction may be modulated 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) mouse splenocytes 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 mAb starting at 2 wk after infection. (E) Splenocytes were isolated 4 wk after infection from L. donovani-infected, anti-cytokine mAb-treated mice and analyzed by ow cytometry gating on CD11b and IL-4Ra was tagged with anti-IL-4Ra-PE antibody. Arginase 1 (F) protein expression and (G) activity was determined in splenocytes isolated from L. donovani-infected mice treated with anti-IL-4Ra mAb starting at 2 wk after infection. The data are representative of three independent experiments. Error bars represent mean 1 SD, n 5 3. p o 0.01, p o 0.001, p o 0.0001 versus as indicated (A and C); p o 0.001 versus infected control (G).

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Figure 5. Effect of IL-10-regulated transcription factors in IL-4Ra-mediated arginase expression. (A) Splenocytes were isolated from mice infected with L. donovani (4 wk) and EMSA for STAT3 and Sp1/Sp3 were performed using the splenocyte nuclear extracts. (B) Splenocyte nuclear extracts prepared from L. donovani-infected mice treated with anti-cytokine mAbs were used for EMSA of STAT3. Cold competitor oligonucleotides were used for specic 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-wk infected mice and IL-4Ra and arginase 1 (C, D) mRNA and (E) protein expression was determined. (E) STAT3 siRNA specicity was determined by Western blotting in whole cell extracts from RAW 264.7 cells expressing either STAT3 or control siRNAs. (F) RAW 264.7 cells were transfected with STAT3 siRNA and cultured for 24 h in supernatant of cultured splenocytes obtained from control or 4-wk infected mice and arginase activity determined. Results are representative of three individual experiments. Error bars represent mean 1 SD, n 5 3. p o 0.001; Students t-test

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 demonstrated 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

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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 impaired infectivity 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 leishmaniasis 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. majorinfected 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 signicant 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 mitochondrial arginase 2 expression could restrict macrophage NO production 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 arginase following infection by L. 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 conrmed. 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 regulation 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 and MyD88 [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/EBPbdependent 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-decient 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 by T. gondii and Salmonella typhi [33, 34]. Our data further indicated that among the different transcription factors 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 pathogen 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.

revised 1996) and with the approval of the Institutional Animal Care and Use Committee.

Splenocyte culture
Splenocytes from BALB/c mice were isolated from different groups of mice as described previously [37]. The cells (5 106/ mL) were stimulated with 20 mg/mL soluble leishmanial antigen (SLA) for 48 h. 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 Biosystems) as described earlier [39].

Materials and methods

Cytokine analysis by ELISA Reagents
All antibodies were from Santa Cruz Biotechnology and BD Pharmingen. All other chemicals were from Sigma unless otherwise indicated. 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 5 106 cells/mL was restimulated with 1 106 cells/mL L. major promastigotes. The level of cytokines in culture supernatants was determined according to the manufacturers protocols.

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 107 L. 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 100 mL of PBS, intraperitoneally ve times a wk, starting after 2 wk of infection and continued throughout the course of infection. For CL, mice were injected with 2 106 L. major LV39 parasites subcutaneously into the footpad. To study the effect of cytokines on arginase activity, infected mice were administered with antiIL-4 mAb (2.5 mg/kg body weight, i.p. and twice a wk) and antiIL-10 mAb (2.0 mg/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

Arginase activity
Arginase activity was measured according to Corraliza et al. [40]. Spleen tissue ($100 mg) 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 containing 0.1% Triton X-100, 100 mg/mL pepstatin, 100 mg/mL aprotinin and 100 mg/mL antipain. To the lysed cells, 10 mM MnCl2, pH 7.4 mM, 50 mM Tris-HCl were added to activate the enzyme by heating for 10 min at 561C. Arginine hydrolysis was carried out by incubating 25 mL of the activated lysate with 25 mM L-arginine (pH 9.7) for 60 min at 371C and the reaction was stopped with 400 mL of a mixture of H2SO4, H3PO4 and H2O (1:3:7, v/v). The urea formed was measured at 540 nm 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 dened as the amount of enzyme that catalyzed the formation of 1 mmol of urea/min.

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Quantication of NO
For determination of NO generation in different groups of infected BALB/c mice after 4 wk of infection, splenocytes (2 106/mL) were isolated and stimulated with or without 50 mg/mL SLA for 48 h before performing nitrite assay by the Greiss reaction as described previously [35].

Statistical analysis
Experiments were performed at least three times each. Macrophage 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 signicance of the differences between the means of the control and the experimental groups.

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-30 and Sp1/Sp3: 50 -ATT CGA TCG GGG CGG GGC GAG C-30 . The DNAprotein complex was electrophoresed and analyzed by autoradiography. Acknowledgements: This work was supported by the Department of Science and Technology and the Network Project (NWP 0038) grant of the Council of Scientic and Industrial Research, Government of India. We thank Dr. Anindita Bhattacharya for critically analyzing the manuscript. Conict of interest: The authors declare no nancial or commercial conict of interest.

Immunoblot analysis
Immunoblot analyses in splenocyte lysates were performed as described earlier [41].

References
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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. Peter J. 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).

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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: pijushdas@iicb.res.in Received: 11/8/2010 Revised: 16/12/2010 Accepted: 20/1/2011 Accepted article online: 31/1/2011

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