Isolates with Antimony-Resistant but Not -Sensitive Phenotype Inhibit Sodium Antimony Gluconate-Induced Dendritic Cell Activation

Summary

The inability of sodium antimony gluconate (SAG)-unresponsive kala-azar patients to clear Leishmania donovani (LD) infection despite SAG therapy is partly due to an ill-defined immune-dysfunction. Since dendritic cells (DCs) typically initiate anti-leishmanial immunity, a role for DCs in aberrant LD clearance was investigated. Accordingly, regulation of SAG-induced activation of murine DCs following infection with LD isolates exhibiting two distinct phenotypes such as antimony-resistant (Sb^RLD) and antimony-sensitive (Sb^SLD) was compared in vitro. Unlike Sb^SLD, infection of DCs with Sb^RLD induced more IL-10 production and inhibited SAG-induced secretion of proinflammatory cytokines, up-regulation of co-stimulatory molecules and leishmanicidal effects. Sb^RLD inhibited these effects of SAG by blocking activation of PI3K/AKT and NF-κB pathways. In contrast, Sb^SLD failed to block activation of SAG (20 µg/ml)-induced PI3K/AKT pathway; which continued to stimulate NF-κB signaling, induce leishmanicidal effects and promote DC activation. Notably, prolonged incubation of DCs with Sb^SLD also inhibited SAG (20 µg/ml)-induced activation of PI3K/AKT and NF-κB pathways and leishmanicidal effects, which was restored by increasing the dose of SAG to 40 µg/ml. In contrast, Sb^RLD inhibited these SAG-induced events regardless of duration of DC exposure to Sb^RLD or dose of SAG. Interestingly, the inhibitory effects of isogenic Sb^SLD expressing ATP-binding cassette (ABC) transporter MRPA on SAG-induced leishmanicidal effects mimicked that of Sb^RLD to some extent, although antimony resistance in clinical LD isolates is known to be multifactorial. Furthermore, NF-κB was found to transcriptionally regulate expression of murine γglutamylcysteine synthetase heavy-chain (mγGCS_hc) gene, presumably an important regulator of antimony resistance. Importantly, Sb^RLD but not Sb^SLD blocked SAG-induced mγGCS expression in DCs by preventing NF-κB binding to the mγGCS_hc promoter. Our findings demonstrate that Sb^RLD but not Sb^SLD prevents SAG-induced DC activation by suppressing a PI3K-dependent NF-κB pathway and provide the evidence for differential host-pathogen interaction mediated by Sb^RLD and Sb^SLD.

Introduction

Kala-azar, caused by Leishmania donovani (LD), is regarded as the most severe form of leishmanial infection, which can be fatal in patients when left untreated. In the absence of an effective vaccine, treatment with pentavalent antimonial compounds such as sodium antimony gluconate (SAG) remains as the first-choice therapy for kala-azar. However, therapeutic utility of SAG is now jeopardized by the emergence of antimony-resistant strains of LD [1], which is becoming a major concern of the World Health Organization (www.who.int/infections-disease-report/2000).

Resistance to antimonial drugs, as observed in leishmanial infection, is marked by two independent “checkpoints”. The first is associated with the impaired biological reduction of the pentavalent antimony (Sb^V) prodrug to a toxic trivalent (Sb^III) form, although the site (macrophage (Mφ) and/or parasite) and mechanism of reduction (enzymatic or nonezymatic) are undefined. The second checkpoint involves a regulatory mechanism promoting reduced influx and/or enhanced efflux/sequestration of active drug that lowers its intracellular accumulation [2], [3]. Importantly, these two events are largely dependent on the intracellular level of thiol compounds such as glutathione (γglutamylcysteinylglycine, GSH) and parasite-specific trypanothione, which in turn are regulated by both host- and LD-γglutamylcysteine synthetase, a rate-limiting enzyme in glutathione biosynthesis [2]–[5]. Although the increased expression of γglutamylcysteine synthetase (γGCS) gene in antimony-resistant strains of LD is controversial [3]–[6], inhibition of γGCS by buthionine sulfoxamine (BSO) reverses Sb^III resistance in LD [7]. Therefore, γGCS expression contributes to antimony resistance in LD by regulating intracellular thiol level.

In addition to the mechanisms noted above, the unresponsiveness of kala-azar patients to treatment with SAG is also believed to be a consequence of skewed type-2 immune response that suppresses interferon (IFN) γ-mediated protective immunity [8]. Nonetheless, IFNγ production by T cells is reduced in non-responders compared to SAG-responders [8], [9]. The endogenous production of IFNγ and importantly, its principal inducer IL-12, determine the anti-leishmanial efficacy of SAG in a fully immunocompetent host infected with LD [10], [11]. Following LD infection, the early production of IL-12 is exclusively mediated by dendritic cells (DCs) [12]. This tempted us to speculate a possible involvement of DCs in regulating “SAG responsiveness” versus “unresponsiveness” in kala-azar patients.

DCs normally play a key role in initiating and regulating Leishmania-specific T cell reactivity [13], [14]. However, the T cell stimulatory capacity of DCs depends on their state of activation and maturation. In contrast to mature DCs, immature DCs exhibit a reduced capacity to stimulate T cells due to low expression of MHC and co-stimulatory molecules, and the lack of production of proinflammatory cytokines. Gene expression associated with the development, activation, maturation and antigen-presenting cell (APC) function of DCs is largely regulated by the transcription factor NF-κB [15]–[18]. For instance, inhibition of NF-κB activation suppresses DC maturation and APC function [19], [20]. NF-κB is a hetero- or homo-dimeric complex of structurally related proteins p50, p52, p65 (RelA), cRel and RelB. In resting cells, NF-κB is sequestered in the cytoplasm by the inhibitory proteins IκBα, IκBβ and IκBε [21]. However, cellular activation with wide range of stimuli such as LPS, TNFα and IL-1phosphorylates and thereby activates a multisubunit complex IκB kinase (IKK) consisting of IKKα/IKK1, IKKβ/IKK2 and IKKγ/NEMO [22]. Subsequently, activated IKK promotes downstream events, for example, phosphorylation followed by polyubiquitination and 26S proteasome-mediated degradation of IκB proteins [21]. NF-κB dimers then translocate to the nucleus, and bind to consensus sequences to induce gene transcription. Notably, the phosphatidylinositol 3-kinase (PI3K)/AKT pathway has been demonstrated in a variety of models to regulate NF-κB activation [20], [23], [24].

Studies demonstrated that stimulation with SAG induces the PI3K/AKT pathway and enhances production of proinflammatory cytokines and leishmanicidal effector molecules in Mφ [25]. Furthermore, SAG stimulates NF-κB activation in different cell types, such as CD4⁺ T cells and peripheral blood mononuclear cells [26]. Importantly, blockade of NF-κB activation is shown to impair γGCS expression in murine Mφ-like cell line [27]. However, direct role of NF-κB in transcriptional regulation of murine γGCS promoter is undefined. With this in mind, the current study was initiated to define the role of SAG in murine DC activation and its regulation by LD isolates with SAG-resistant (Sb^RLD) and SAG-sensitive (Sb^SLD) phenotype. We demonstrate that Sb^RLD but not Sb^SLD infection suppresses SAG-induced activation/maturation and γGCS expression of DCs by inhibiting NF-κB activation in a PI3K/AKT-dependent manner.

Results

Sb^SLD- and Sb^RLD-infected DCs respond differentially to SAG treatment

Although Mφs are regarded as a “primary target” for leishmanial infection, recent studies indicate DC infection with various Leishmania spp. including LD [28], [29]. Indeed, LD infection was observed in both immature bone marrow-derived DC (BMDC) (CD11c⁺CD8α^-) and splenic DC (sDC) (Figure 1A). To determine the leishmanicidal effect of SAG on intracellular Sb^RLD and Sb^SLD in DCs, BMDCs and sDCs were infected with GFP expressing promastigotes of Sb^SLD strain 2001 (GFP-2001) or Sb^RLD strain R5 (GFP-R5) for 3 hours, stimulated with SAG (20 µg/ml) for 24 hours, and the frequency of infected DCs measured via flow cytometry. A comparable level of DC infection was observed with both GFP-2001 and GFP-R5 (Figure 1B). However, the percentage of BMDCs or sDCs infected with GFP-2001 (Sb^SLD) was reduced by 5 to 9-fold following SAG treatment (Figure 1B). In marked contrast, SAG treatment failed to exhibit any significant effect on GFP-R5 (Sb^RLD) infection in DCs (Figure 1B). Furthermore, analyses via Giemsa staining demonstrated that intracellular amastigotes of other Sb^RLD strains exhibit similar resistance to the SAG-induced leishmanicidal effect in DCs. For instance, a significant reduction in both percentage of infected BMDCs and intracellular parasite number were observed in AG83 (Sb^SLD)- and to a lesser extent in 39 (Sb^RLD)-infected BMDCs after SAG treatment (10 and 20 µg/ml) for 24 and 48 hours (Figure S1). Titration of parasites demonstrated that parasite to DC ratio (multiplicity of infection; MOI) of 10∶1 was the optimum ratio for maximum LD infection in DCs (data not shown). Therefore, this MOI was used for all subsequent experiments unless otherwise stated.

Next, the regulation of SAG-induced activation and maturation of DCs by Sb^RLD and Sb^SLD was investigated. For this purpose, BMDCs and sDCs were infected with Sb^RLD and Sb^SLD promastigotes for 3 hours, washed and cultured with or without SAG (20 µg/ml) for an additional 48 hours. The activation and maturation of DCs were determined by analyzing MHC and co-stimulatory molecule expression and secretion of cytokines. Infection of BMDCs and sDCs with Sb^RLD strains 39 or GE1F8R induced more IL-10 production as compared with Sb^SLD strains AG83 or 2001 (Figures 1C and S2A). Interestingly, IL-10 secretion from Sb^RLD-infected DCs was not affected by SAG treatment (Figures 1C and S2A). In contrast, SAG treatment significantly inhibited IL-10 secretion by Sb^SLD-infected DCs (Figures 1C and S2A). Furthermore, SAG-stimulated secretion of proinflammatory cytokines such as IL-12p70 and TNFα from DCs were inhibited by Sb^RLD and not Sb^SLD infection (Figures 1D-E and S2B-C). Finally, SAG treatment up-regulated CD40, CD80, CD86, MHC-I (H2K^d) and MHC-II (IA^d) expression in BMDCs infected with Sb^SLD but not Sb^RLD (Figure 1F). Consistent with work by other groups [30], [31], co-stimulatory molecule and MHC expression of untreated BMDCs remained unaltered following Sb^RLD or Sb^SLD infection (Figure 1F). Together, these results demonstrate that SAG treatment protects DCs from Sb^SLD but not Sb^RLD infection. Moreover, stimulation with SAG fails to activate and mature Sb^RLD-infected DCs, while Sb^SLD-infected DCs are still capable of activation and maturation upon SAG treatment.

SAG treatment induces NF-κB activation in DCs

Since NF-κB is a key regulator of maturation and APC function of DCs, the effect of SAG treatment on NF-κB activation was investigated. BMDCs were treated with SAG for varying times and DNA binding activity of nuclear NF-κB was determined via electrophoretic mobility shift assay (EMSA). Relative to untreated BMDCs, a 23-fold increase in NF-κB DNA binding activity was initially observed by 0.3 hours, which persisted up to 1 hour after SAG treatment (Figure 2A). Notably, OCT-1 DNA binding was unaltered despite SAG treatment indicating that SAG-induced enhancement of nuclear DNA binding was NF-κB-specific (Figure 2A). Furthermore, supershift analysis using antibodies specific for each Rel family member demonstrated that SAG stimulation of BMDCs induced DNA binding of NF-κB complexes consisting of the p50, p65 and RelB subunits (Figure 2B). In contrast to SAG, BMDC treatment with varying concentrations (25 to 200 µg/ml) of sodium gluconate for 0.3 hours, or 200 µg/ml of sodium gluconate for various times failed to induce DNA binding activity of NF-κB (Figure 2C-D).

Consistent with the EMSA data, SAG treatment for 0.3, 0.5 and 1 hour induced degradation of IκB proteins in BMDCs (Figure 3A). Importantly, SAG-induced IκB degradation corresponded to enhanced IκBα phosphorylation (Figure 3B), which could be due to increased activity of upstream IKK complex. To test this hypothesis, BMDCs were stimulated with SAG for 0.3, 0.5 and 1 hour. IKK signalosome was immunoprecipitated from cytoplasmic extracts and kinase activity of the complex determined by measuring phosphorylation of an IκBα-GST substrate in vitro. BMDCs stimulated with SAG for 0.3, 0.5 and 1 hour exhibited approximately 3.6 to 4.7-fold increase in IKK activity compared to untreated BMDCs (Figure 3C). In comparison, the level of IKK1 and IKK2 proteins were similar in all BMDC extracts (Figure 3C). The SAG-induced NF-κB DNA binding, IκBα degradation and IKK activity were also observed in sDCs (Figure S3).

Of note, SAG treatment failed to activate the mitogen-activated protein kinase (MAPK) pathway in BMDCs. In contrast to LPS stimulation, phosphorylation of p38MAPK, ERK1/ERK2 and JNK was not detected in SAG-treated DCs (Figure 3D-F). Collectively, these findings demonstrate that stimulation with SAG induces activation of the IKK complex, phosphorylation and degradation of IκB proteins and downstream nuclear DNA binding of NF-κB in both BMDCs and sDCs, and that the induction of these events in DC is contributed by antimonial moiety of SAG. Furthermore, among different signaling pathways, which are known to regulate DC activation/maturation, NF-κB signaling is selectively induced by SAG treatment.

Sb^RLD but not Sb^SLD infection inhibits SAG-induced NF-κB activation in DCs

The effect(s) of Sb^RLD and Sb^SLD infection on SAG-stimulated NF-κB activation in DCs was determined. BMDCs were infected with either promastigotes or amastigotes of 39 (Sb^RLD) or 2001 (Sb^SLD) for varying times, stimulated with SAG for 0.3 hours and nuclear NF-κB DNA binding activity to H2K-specific probe measured via EMSA. SAG-induced NF-κB activity was completely inhibited in BMDCs upon infection with either 39 promastigotes (39Pm) or amastigotes (39Am) (Sb^RLD) at all times of LD infection analyzed (Figure 4A-B). In contrast, BMDC infection with 2001 promastigotes (2001Pm) or amastigotes (2001Am) (Sb^SLD) for up to 6 and 3 hours, respectively, failed to inhibit SAG-stimulated NF-κB DNA binding (Figure 4A-B). SAG-induced NF-κB DNA binding activity was also inhibited in 39Pm (Sb^RLD)-infected sDCs (Figure S4A). The ability of Sb^RLD to block SAG-stimulated NF-κB DNA binding in DCs was not LD strain specific. For example, BMDC infection with promastigotes of Sb^RLD strain GE1F8R (GE1F8RPm), unlike Sb^SLD strain AG83 (AG83Pm), completely prevented SAG-induced NF-κB DNA binding (Figure S4B).

Our finding that SAG-induced NF-κB activity is inhibited selectively in Sb^RLD-infected BMDCs was further confirmed by temporal analysis of IκB protein degradation and activation of IKK. BMDC infection with either 2001Pm or 2001Am (Sb^SLD) for up to 6 or 3 hours, respectively, had no significant effect on SAG-induced IκB degradation (Figure 4C-D). In contrast, IκB degradation stimulated by SAG was persistently inhibited by 39 (Sb^RLD) regardless of the duration of BMDC infection and form of parasite (Figure 4C-D). SAG-induced IκBα degradation was similarly inhibited in sDCs infected with 39Pm (Sb^RLD) and BMDCs infected with promastigotes of a different Sb^RLD strain (Figure S4C-D). Interestingly, inhibition of SAG-induced IκB degradation corresponded with a reduction in IκBα phosphorylation in BMDCs infected with Sb^RLD promastigotes (Figure 4C, E). Furthermore, SAG-induced IKK activation as determined by in vitro IKK activity or phosphorylation of IKK1 and IKK2 was inhibited in extracts prepared from BMDCs and sDCs infected with Sb^RLD but not Sb^SLD promastigotes (Figures 4F and S4E-F). BMDC infection with Sb^RLD and not Sb^SLD amastigotes also blocked SAG-stimulated IKK activity (Figure 4G). Additionally, pretreatment of BMDCs with parasite antigen(s) (Sb^RLDsAg) or culture supernatant (Sb^RLDs) of Sb^RLD inhibited SAG-induced NF-κB DNA binding activity, IκB degradation and IKK activity; whereas culture supernatant of Sb^SLD (Sb^SLDs) or Sb^SLD-derived antigen(s) (Sb^SLDsAg) did not (Figure 5).

As demonstrated in Figure 4A-D; SAG-induced NF-κB activation was inhibited in BMDCs infected at a MOI 10∶1 (promastigote or amastigote to DC) with Sb^SLD promastigotes or amastigotes for 24 or 6 hours, respectively, similar to Sb^RLD-infected BMDCs. Accordingly, we tested whether the increased intracellular parasite number at these time points of infection rendered 20 µg/ml of SAG insufficient to activate NF-κB. In fact, number of intracellular 2001 (Sb^SLD) or 39 (Sb^RLD) was significantly increased if BMDCs were infected with promastigotes for 24 hours rather than 6 hours (Figure S5A). Likewise, BMDCs infected with amastigotes of the above LD strains for 6 hours exhibited increased intracellular parasite number compared to BMDCs infected for 3 hours (Figure S5B). Notably, SAG (20 µg/ml)-induced NF-κB DNA binding and IκBα degradation were detected despite the presence of intracellular parasites in BMDCs infected with 2001Pm and 2001Am (Sb^SLD) for 6 and 3 hours, respectively (Figures 4A-D and S5A-B). Therefore, these two time points of BMDC infection were selected as a “reference” to analyze the basis of defective SAG-induced NF-κB activation in BMDCs infected with 2001Pm or 2001Am (Sb^SLD) for 24 or 6 hours, respectively. Initially, the association of increased intracellular parasite number with impairment of SAG-induced NF-κB activation was verified in both BMDCs infected with 2001Pm or 2001Am (Sb^SLD) for 24 or 6 hours, respectively. For this purpose, BMDCs were infected with 2001Pm (Sb^SLD) or 39Pm (Sb^RLD) for 24 hours at varying MOIs and stimulated with SAG (20 µg/ml) for 0.3 hours. Despite LD infection for fixed duration (24 hours), this approach established varying levels of intracellular parasite number, which was elevated in BMDCs infected at MOI 10∶1 compared to other MOIs (Figure S6A). BMDC infection with both 2001Pm (Sb^SLD) and 39Pm (Sb^RLD) at a MOI 10∶1 inhibited SAG-induced NF-κB DNA binding activity and IκBα degradation (Figure 6A-B). However, SAG-induced NF-κB DNA binding activity and IκBα degradation were observed only in 2001Pm (Sb^SLD)-infected BMDCs but not 39Pm (Sb^RLD)-infected BMDCs when BMDC infection was done at MOIs 2.5∶1 and 5∶1 (Figure 6A-B). Notably, at each of these MOIs both 2001Pm (Sb^SLD)-infected BMDCs and 39Pm (Sb^RLD)-infected BMDCs had comparable level of intracellular parasite number (Figure S6A). Using identical MOIs for BMDC infection, similar results were obtained when the intracellular parasite number and SAG-induced NF-κB activation were analyzed in BMDCs infected for 6 hours with 39Am (Sb^RLD) and 2001Am (Sb^SLD) (Figures 6C-D and S6B). These findings suggest that irrespective of the form of parasite, 2001 (Sb^SLD) and 39 (Sb^RLD) differentially regulate SAG-induced NF-κB signaling in DCs with low intracellular parasite number.

It is possible that with increased intracellular parasite number 2001(Sb^SLD), similar to 39 (Sb^RLD), developed the capacity to inhibit SAG-induced NF-κB activation and that occurred when BMDCs were infected at a MOI 10∶1 with 2001Pm or 2001Am (Sb^SLD) for 24 and 6 hours, respectively. However, this possibility was ruled out when NF-κB activation in response to 20 and 40 µg/ml of SAG treatment was analyzed in BMDCs infected for 6 and 24 hours with 2001Pm (Sb^SLD) or 39Pm (Sb^RLD) at a MOI of 10∶1. The effect of SAG (40 µg/ml) stimulation was also verified in BMDCs infected similarly with 2001Am or 39Am for 3 and 6 hours. Stimulation with both 20 and 40 µg/ml of SAG induced NF-κB DNA binding and IκBα degradation in BMDCs infected with 2001Pm (Sb^SLD) for 6 hours (Figure 6E-F). In contrast, BMDCs infected for 24 hours with 2001Pm (Sb^SLD) exhibited enhanced NF-κB DNA binding and IκBα degradation only when stimulated with 40 µg/ml of SAG (Figure 6E-F). Similarly, the inhibition of SAG-induced NF-κB DNA binding and IκBα degradation due to BMDC infection with 2001Am (Sb^SLD) for 6 hours was overcome by increasing the dose of SAG from 20 to 40 µg/ml (Figure 6G-H). On the contrary, 39Pm/39Am (Sb^RLD) continued to suppress NF-κB DNA binding and IκBα degradation at various durations of infection tested irrespective of dose of SAG used for stimulation (Figure 6E-H). Together, these results demonstrate that SAG-induced NF-κB signaling is impaired by Sb^RLD infection of DCs.

Sb^RLD inhibits SAG-induced NF-κB signaling, DC activation and leishmanicidal effects by suppressing the PI3K/AKT pathway in an IL-10-independent manner

Stimulation with SAG induces PI3K/AKT activation in Mφ [25]. Furthermore, PI3K/AKT regulates the NF-κB pathway in DCs via IKK [19], [20]. Therefore, the possibility that SAG-induced activation of NF-κB in DCs is PI3K/AKT-dependent was investigated. Initially, the effect of SAG stimulation on AKT activation was assessed by measuring phosphorylation of AKT. Compared to unstimulated BMDCs, SAG treatment induced a 5 to 7-fold increase in AKT phosphorylation in BMDCs (Figure 7A). Notably, SAG-induced AKT phosphorylation was not observed in BMDCs pretreated with PI3K inhibitors wortmannin (Wort) or Ly294002 (Ly) (Figure 7A). Next, the effect of PI3K inhibitors on SAG-induced NF-κB signaling was determined. Pretreatment with Wort or Ly effectively blocked SAG-induced IKK activity, IκBα degradation and nuclear NF-κB DNA binding in BMDCs (Figure 7B-D). Importantly, infection of BMDCs and sDCs with 39Pm (Sb^RLD) but not 2001Pm (Sb^SLD) for 3 hours inhibited SAG (20 µg/ml)-induced AKT phosphorylation (Figures 7E and S7). SAG (20 µg/ml)-induced AKT phosphorylation was also inhibited due to BMDCs infection for 1 and 3 hours with 39Am (Sb^RLD) but not 2001Am (Sb^SLD) (Figure 7F). Similar to 39Pm (Sb^RLD)-infected BMDCs, SAG (20 µg/ml)-induced AKT phosphorylation, however, was not observed if BMDCs were infected with 2001Pm (Sb^SLD) for 24 hours (Figure 7G). Interestingly, AKT phosphorylation was observed in these 2001Pm (Sb^SLD)-infected BMDCs but not 39Pm (Sb^RLD)-infected BMDCs upon stimulation with of 40 µg/ml of SAG (Figure 7G).

Since Sb^RLD infection stimulated high IL-10 secretion by DCs (Figures 1C and S2A), the possibility that Sb^RLD inhibited SAG-induced PI3K/AKT (Figures 7E-F and S7) and NF-κB pathways (Figures 4 and S4) in an IL-10-dependent manner was investigated by using a neutralizing αIL-10 Ab. Temporal analyses demonstrated that significant IL-10 production by BMDCs was initially detected after an infection for 12 hours with Sb^RLD but not Sb^SLD promastigotes (Figure 7H). Compared to Sb^SLD-infected BMDCs, IL-10 production was significantly increased in BMDCs infected with Sb^RLD for 24 and 48 hours (Figure 7H). Strikingly, SAG-induced AKT phosphorylation and NF-κB DNA binding activity were not restored in BMDCs infected with Sb^RLD for 3 and 24 hours despite αIL-10 Ab treatment (Figure 7I-J). However, αIL-10 Ab treatment effectively prevented inhibition of LPS-induced NF-κB DNA binding in BMDCs pretreated with IL-10 (Figure S8). Therefore, this finding ruled out the involvement of IL-10 in suppression of SAG-induced PI3K/AKT and NF-κB pathways in Sb^RLD-infected BMDCs.

Consistent with previous report [5], an overexpression of ATP-binding cassette (ABC) transporter MRPA (PGPA) was observed in Sb^RLD strains 39Pm and GE1F8RPm (Figure S9A). Whether MRPA plays any role in mediating the inhibitory effects of Sb^RLD on SAG-induced PI3K/AKT and NF-κB activation in DC was then investigated. Accordingly, SAG-induced AKT phosphorylation and NF-κB activation in BMDCs infected for 3 hours with 39Pm (Sb^RLD), 2001Pm (Sb^SLD) and isogenic 2001Pm expressing MRPA (2001Pm-MRPA) (Figure S9B) were compared. The latter was developed by transfecting 2001Pm (Sb^SLD) with a DNA construct expressing MRPA. A complete blockade of SAG-induced AKT phosphorylation and NF-κB DNA binding activity was observed in 39Pm (Sb^RLD)-infected BMDCs (Figure 8A-B). In contrast, SAG-induced AKT phosphorylation and NF-κB DNA binding activity were detected in 2001Pm (Sb^SLD)-infected BMDCs (Figure 8A-B). However, infection of BMDCs with 2001Pm-MRPA inhibited SAG-induced AKT phosphorylation and DNA binding activity of NF-κB in BMDCs, albeit partially (Figure 8A-B).

Next, a direct role for PI3K in Sb^RLD and Sb^SLD regulation of SAG-induced proinflammatory cytokine secretion and leishmanicidal effects in DCs was investigated using Wort or Ly. In contrast to 39Pm (Sb^RLD)-infected BMDCs, SAG-stimulated IL-12 and TNFα production were observed in both uninfected and 2001Pm (Sb^SLD)-infected BMDCs (Figure 9A-B). However, pretreatment of uninfected and 2001Pm (Sb^SLD)-infected BMDCs with Wort or Ly significantly inhibited SAG-induced secretion of IL-12 and TNFα (Figure 9A-B). Furthermore, PI3K inhibitors prevented SAG (20 µg/ml)-induced reduction of percentage of infected BMDCs and intracellular parasite number in BMDCs infected for 3 hours with 2001Pm (Sb^SLD) (Figure 9C-D). Importantly, BMDCs infected with 2001Pm (Sb^SLD) for 24 hours exhibited a significant reduction in both percentage of infected BMDCs and intracellular parasite number only when treated with 40 but not 20 µg/ml of SAG (Figure S10). Treatment of these 2001Pm (Sb^SLD)-infected BMDCs with Wort or Ly blocked the leishmanicidal effects of SAG (40 µg/ml) (Figure S10). In contrast to 2001Pm (Sb^SLD)-infected BMDCs, SAG-induced leishmanicidal effects were not observed in 39Pm (Sb^RLD)-infected BMDCs regardless of duration of infection and dose of SAG (Figures 9C-D and S10). In addition, BMDC infection for 3 hours with 2001Pm-MRPA, unlike 2001Pm (Sb^SLD), partly but significantly suppressed SAG-induced leishmanicidal effects (Figure 9E-F). Collectively, these data demonstrate that blockade of PI3K/AKT pathway by Sb^RLD impairs SAG-induced NF-κB signaling, DC activation and leishmanicidal function and that is IL-10-independent. Furthermore, these inhibitory effects of Sb^RLD are partly contributed by MRPA.

Inhibition of NF-κB activation by Sb^RLD suppresses SAG-stimulated murine γGCS heavy-chain gene expression in DC

Previous studies have demonstrated an association of antimony resistance of leishmanial parasite with γGCS heavy-chain (γGCS_hc) gene expression of host [4]. The latter encodes the catalytic subunit of γGCS [32]. In fact, a comparative analysis demonstrated that SAG-induced murine γGCS_hc (mγGCS_hc) expression was unaffected in 2001Pm (Sb^SLD)-infected BMDCs but selectively inhibited in 39Pm (Sb^RLD)-infected BMDCs (Figure 10A). The molecular basis for Sb^RLD-mediated suppression of mγGCS_hc expression in DC was then explored. Despite SAG stimulation, the inhibition of mγGCS_hc expression could be due to suppression of NF-κB activation in Sb^RLD-infected BMDC. To investigate this possibility, the regulatory role of NF-κB in mγGCS_hc promoter activity was initially ascertained. An approximately 1.0 kb DNA sequence upstream of the transcriptional start site of mγGCS_hc gene (Mus musculus chromosome 9 genomic contig, NT_039474.7; GI:149260095) was selected as the promoter region using Ensembl and UCSC browsers. The mγGCS_hc promoter was found to contain a putative NF-κB binding site ^-904GGGGAAACTT^-895 that differs from the consensus sequence GGGRNNYYCC at positions 7, 9 and 10 (Figure 10B). ChIP analysis demonstrated that SAG treatment of BMDCs induced NF-κB binding to −991/−673 region of mγGCS_hc promoter that includes the sequence ^-904GGGGAAACTT^-895 (Figure 10B-C). Furthermore, DNA binding of NF-κB complexes consisting of p50, RelB and p65 subunits specifically to the sequence

^-904GGGGAAACTT^-895 in SAG-treated BMDCs was confirmed via EMSA using mγGCS_hc probes containing wild-type sequence ^-904GGGGAAACTT^-895 (WT-mγGCS_hc probe) or mutant sequence ^-904CTCTAAGAAT^-895 (Mut-mγGCS_hc probe) (Figure 10D) and supershift analysis (Figure 10E).

Next, the role of NF-κB binding site ^-904GGGGAAACTT^-895 in regulation of promoter activity of mγGCS_hc gene was tested via luciferase reporter assay using p987-luc and Mut p987-luc, the reporter constructs of mγGCS_hc promoter fragment containing wild-type and mutant NF-κB binding site, respectively. Compared to control vector (pEGFP-C1) transfected cells, expression of NF-κB subunit p65 strongly induced luciferase activity of p987-luc (Figure 10F). This enhanced luciferase activity of p987-luc was completely blocked upon co-transfection with pEGFP-dominant negative IκBα (pEGFP-IκBαΔN) encoding the NF-κB-specific inhibitor, IκBαΔN (Figure 10F). The lack of luciferase activity of Mut p987-luc despite p65 expression (Figure 10F) further indicated that the sequence ^-904GGGGAAACTT^-895 is required for NF-κB-mediated transcriptional activation of mγGCS_hc gene. Interestingly, SAG-induced NF-κB DNA binding to WT-mγGCS_hc probe was inhibited in 39Pm (Sb^RLD)-infected BMDCs (Figure 10G). In contrast, SAG-induced NF-κB DNA binding to WT-mγGCS_hc probe was readily detected in 2001Pm (Sb^SLD)-infected BMDCs (Figure 10G). These findings suggest that Sb^RLD suppresses SAG-induced mγGCS_hc expression in DC by inhibiting NF-κB DNA binding to the mγGCS_hc promoter.

Discussion

Antimonial drugs activate innate effector cells to promote an anti-leishmanial effect [25]. However, regulation of antimonial drug-mediated immune activation by Sb^RLD and Sb^SLD is ill-defined. This is of particular interest in view of the lack of efficacy of antimonial compounds reported for SAG-unresponsive kala-azar patients. Recent studies indicated that DCs play a key role in regulating anti-leishmanial immune response [12]–[14], [33]. Accordingly, the role of SAG in activation of DCs, its regulation by Sb^RLD and Sb^SLD and the molecular mechanism involved therein were investigated. Here we provide evidence that Sb^RLD and Sb^SLD differentially regulate activation of DCs. Furthermore, SAG-induced signaling pathway associated with DC activation is selectively targeted by Sb^RLD infection.

In an agreement with an earlier report [28], both BMDCs and ex vivo sDCs were infected in vitro with LD promastigotes (Figure 1A). The “SAG-resistant” phenotype did not significantly affect the efficiency of LD infection, but did impact the susceptibility of LD to the leishmanicidal effects of SAG. In contrast, SAG treatment significantly impaired DC infectivity of Sb^SLD including reduction in both intracellular parasite number and percentage of infected DCs (Figures 1B and S1). The differential response of Sb^RLD and Sb^SLD towards SAG treatment was also noted in their ability to regulate activation and maturation of DCs. In contrast to Sb^SLD, Sb^RLD infection inhibited SAG-induced proinflammatory cytokine secretion and up-regulation of co-stimulatory molecule and MHC expression in DCs (Figures 1D-F and S2B-C). Noteworthy is that Sb^RLD induced increased IL-10 secretion by DCs compared to Sb^SLD (Figures 1C and S2A). This finding reinforces the inherent ability of Sb^RLD and Sb^SLD to differentially immunoregulate DC activation. Previous studies demonstrated that IL-10, a potent suppressor of anti-leishmanial immunity, minimizes responsiveness to SAG [34], [35]. Therefore, increased IL-10 production may play a critical role in disease pathogenesis in the host infected with Sb^RLD.

The second important finding arising from this study is that both promastigotes and amastigotes of Sb^RLD and Sb^SLD differentially regulate SAG-induced NF-κB activation in DCs. Indeed, Sb^RLD but not Sb^SLD infection blocks SAG-induced NF-κB signaling by suppressing IKK activation, and IκB protein phosphorylation and degradation (Figures 4 and S4). In this regard, it should be noted that SAG stimulation of uninfected DCs induced concomitant degradation of all three IκB proteins (Figure 3A), although degradation of IκBε generally occurs with delayed kinetics upon cellular activation [36], [37]. This finding, however, is consistent with a number of studies reporting rapid degradation of IκBβ and IκBε depending on the type of cell and the nature of the stimulation [19], [20], [36]–[39]. Nevertheless, the suppression of SAG-induced IκB protein degradation by Sb^RLD ultimately impaired nuclear NF-κB DNA binding activity (Figures 4 and S4). Surprisingly, BMDC infection at a MOI 10∶1 with Sb^SLD promastigotes or amastigotes for 24 or 6 hours, respectively, also inhibited SAG (20 µg/ml)-induced NF-κB activation (Figure 4). SAG (20 µg/ml)-induced NF-κB activation was restored in these BMDCs but not Sb^RLD-infected BMDCs upon lowering the MOIs (<10∶1) for BMDC infection, which established reduced levels of intracellular parasite number compared to MOI 10∶1(Figures 6A-D and S6). This finding suggests that intracellular parasite number plays a critical role for differential regulation of SAG-induced NF-κB activation by Sb^RLD and Sb^SLD. The early inhibition of NF-κB activation in Sb^SLD amastigote versus promastigote-infected BMDCs (Figure 4) is in agreement with the fact that DCs internalize amastigotes more efficiently than promastigotes [40]–[42]. However, Sb^RLD and Sb^SLD still retain their ability to differentially regulate SAG-induced NF-κB activation in BMDCs with high intracellular parasite number. This conclusion is supported by results demonstrating that despite stimulation with 40 µg/ml of SAG, NF-κB activation was blocked in BMDCs infected for above durations with Sb^RLD amastigotes or promastigotes at a MOI 10∶1 but readily observed in BMDCs infected similarly with Sb^SLD (Figure 6). Here, the SAG dose was increased from 20 to 40 µg/ml keeping in mind that SAG therapy requires multiple dosing schedules to ensure enough antimony accumulation in tissues of kala-azar patients [43]. Furthermore, equivalent and/or increased concentrations of SAG have previously been used by other groups [44], [45]. Under identical conditions, the recurrence of NF-κB activation in Sb^SLD-infected BMDCs by increasing the dose of SAG from 20 to 40 µg/ml (Figure 6) further suggested that 20 µg/ml of SAG was insufficient to activate NF-κB in these BMDCs due to high intracellular parasite number.

Noteworthy is that the inhibitory effect on NF-κB activation was not dependent on live Sb^RLD. For instance, parasite antigens derived from Sb^RLD (Sb^RLDsAg) and Sb^RLD culture supernatant (Sb^RLDs) but not the Sb^SLD-derived antigens (Sb^SLDsAg) or culture supernatant of Sb^SLD (Sb^SLDs) efficiently inhibited SAG-induced NF-κB activation (Figure 5). These findings suggest that the inhibition of NF-κB activation is specific for Sb^RLD/Sb^RLD-derived antigen(s)/factor(s) secreted by Sb^RLD. Strikingly, the inhibition of NF-κB activation correlated with suppression of SAG-induced DC activation by Sb^RLD infection (Figures 1, 4 and S2). Studies involving gene transfer of a modified IκBα recombinant into immature DC demonstrated that blockade of NF-κB activation alone prevents up-regulation of co-stimulatory molecule expression and production of proinflammatory cytokines [16], [18]. Based on these reports coupled with our own observations, we conclude that the Sb^RLD blocks SAG-induced NF-κB signaling to prevent DC activation and maturation.

Our results further suggest that Sb^RLD inhibits IKK and NF-κB activation by blocking SAG-induced PI3K/AKT signaling. SAG stimulation of DCs induced PI3K activation as measured by phosphorylation of AKT, a downstream signaling mediator of PI3K (Figures 7A and S7). Blockade of PI3K/AKT activation by Wort or Ly completely suppressed SAG-stimulated IKK activity and NF-κB signaling (Figure 7A-D), indicating a direct involvement of the PI3K/AKT pathway in NF-κB activation by SAG in DCs. Importantly, PI3K/AKT activation is negatively regulated by Src homology phosphotyrosine phosphatase (SHP)-1, which dephosphorylates PI3K [46]. Furthermore, SHP-1activity is inhibited by SAG [47]. Therefore, blockade of SHP-1activity by SAG may indirectly promote PI3K phosphorylation and activation of downstream AKT, IKK and NF-κB in DCs. Similar to uninfected DCs, SAG treatment induced PI3K/AKT activation in DCs infected with Sb^SLD promastigotes for 3 hours and Sb^SLD amastigotes for 1 and 3 hours (Figures 7E-F and S7). Importantly, PI3K inhibitors impaired SAG (20 µg/ml)-induced NF-κB pathway, DC activation and leishmanicidal effects in BMDCs infected with Sb^SLD promastigotes for 3 hours (Figures 9 and S11). These results suggest the inability of Sb^SLD to regulate SAG-induced PI3K/AKT and NF-κB pathways. Consequently, SAG continues to exhibit leishmanicidal effects in Sb^SLD-infected DCs. The inability of Sb^SLD to regulate SAG-induced leishmanicidal effects was maintained despite BMDC infection for 24 hours with Sb^SLD promastigotes. This was apparent when increased dose of SAG (40 µg/ml) was used for treatment (Figure S10). In fact, treatment with 40 µg/ml of SAG restored AKT phosphorylation and therefore exhibited leishmanicidal effects in a PI3K-dependent manner in these Sb^SLD-infected BMDCs (Figures 7G and S10). Interestingly, Sb^RLD infection mimicked the effects of the PI3K inhibitors in that all SAG-induced events as mentioned above were also blocked by Sb^RLD regardless of duration of infection, form of parasite and dose of SAG (Figures 7, 9, S10 and S11). One intriguing possibility is that IL-10 produced by Sb^RLD-infected DCs mediated Sb^RLD-induced suppression of PI3K/AKT and NF-κB pathways (Figures 1C, 4, 7, S2A and S7). This correlation can be made because IL-10 inhibits AKT activation and NF-κB pathway in DCs [19]. Furthermore, the inhibitory effects of IL-10 on DCs can be mediated in an autocrine manner [48]. However, this is unlikely since SAG-induced NF-κB activation was inhibited even in BMDCs infected with Sb^RLD for 1 and 3 hours, when IL-10 production was not detected (Figures 4 and 7H). Moreover, neutralization of IL-10 produced by BMDCs upon Sb^RLD infection for 24 hours failed to block Sb^RLD-induced inhibition of PI3K/AKT and NF-κB pathways (Figure 7H-J). Therefore, Sb^RLD infection of BMDCs for up to 24 hours inhibits SAG-induced PI3K/AKT and NF-κB pathways in an IL-10-independent manner and eventually impairs DC activation.

Another key finding is that the suppression of NF-κB activation by Sb^RLD but not Sb^SLD inhibits not only DC activation but also SAG-induced mγGCS_hc expression in DC (Figure 10). Importantly, regulation of host γGCS_hc expression and therefore host GSH level by Sb^RLD plays a key role for antimony resistance in LD infection [4]. Although regulation of γGCS_hc expression by NF-κB was shown in a murine Mφ-like cell line by blocking NF-κB activation [27], our observation establishes a direct role for NF-κB in mediating the promoter activity of the mγGCS_hc gene (Figure 10C-F). Interestingly, Sb^RLD blocked SAG-induced mγGCS_hc expression in DC by preventing NF-κB binding to the mγGCS_hc gene promoter (Figure 10G). This suggests a key role for NF-κB in Sb^RLD-mediated suppression of mγGCS_hc expression in DC. Our findings (Figures 10A and S12) are consistent with the recent work by Carter and colleagues demonstrating that Sb^RLD transcriptionally down-regulate host γGCS_hc expression and up-regulate their own γGCS_hc (LD-γGCS_hc) expression [4]. Whereas Sb^RLD-induced inhibition of host γGCS expression reduces host GSH level and impairs reduction of Sb^V to toxic Sb^III form; elevated expression of LD γGCS by Sb^RLD restores GSH level that promotes efflux of SAG and confers protection against oxidative stress [4].

The mechanism of antimony resistance in clinical LD isolates is unknown and may differ from laboratory-derived resistant parasites [3]. The true markers of clinical antimony resistance in LD isolates are still lacking [2]. A gene, PG1, is reported to confer antimony resistance in clinical isolates of LD [45], [49]. In addition, enhanced expression of several other genes including MRPA and proteophosphoglycans (PPG) was demonstrated in antimony-resistant compared to antimony-sensitive field isolates (Salotra P, Singh R, Nakhasi H. 2005. Clinical Microbiology and Infection. Vol 11, Suppl 2: 47) [5], [50]. As an initial effort to determine whether any Sb^RLD-specific factor(s) mediated the suppression of SAG-induced NF-κB signaling and leishmanicidal effects in DCs, the role for MRPA was investigated. Results obtained using 2001Pm and its isogenic strain expressing MRPA (2001Pm-MRPA) showed that the inhibitory effect of 2001Pm-MRPA on SAG-induced PI3K/AKT and NF-κB pathways and leishmanicidal activities mimics that of Sb^RLD to some extent (Figures 8 and 9). However, the real bearing of this observation in naturally occurring antimony-resistant LD isolates is still questionable and needs detailed investigation further. Moreover, the effects of SAG are likely to be inhibited in DCs by other Sb^RLD-specific factor(s) also. The relative contribution of these parasite-specific factors in Sb^RLD-mediated suppression of DC activation is currently under investigation. Furthermore, an association of antimony resistance with genetic variation among LD strains has been proposed [51]. Recent studies demonstrated that due to high genetic polymorphism, strain 39 is remarkably distinct not only from antimony-sensitive strain 2001 but also from other antimony-resistant clinical LD isolates exhibiting homology with antimony-sensitive parasites [51]. On the other hand, the antimony-sensitive strains 2001 and Dd8 exhibit significant genetic similarity [51]. The extreme genetic polymorphism might be a potential cause of antimony resistance in strain 39 [51]. These reports together with our findings emphasize the notion that antimony resistance of clinical LD isolates is “multifactorial” [3].

SAG unresponsiveness in kala-azar patients also entails a number of host-regulated events including dominance of a type-2 T cell response and altered host gene expression [3], [4], [8], [9]. Our findings demonstrate that SAG treatment induces PI3K-dependent NF-κB activation in DCs, which is blocked by Sb^RLD but not Sb^SLD infection. Dysregulation of SAG-induced NF-κB activation favors persistent survival of Sb^RLD in DCs despite SAG treatment by: 1) inhibiting NF-κB-dependent mγGCS expression, a key mediator of Sb^V reduction to Sb^III, and 2) preventing DC activation and maturation required for the initiation of the anti-leishmanial immune response. Notably, a heterogeneous response to SAG treatment may be observed in humans. This possibility is raised by a report demonstrating that SAG treatment of monocyte-derived DCs restores their capacity to respond to LPS in ∼60% of type 1diabetes patients [52]. Importantly, some variability in SAG responsiveness is also reported in kala-azar patients [53]. It is speculated that the genetic differences among individuals may influence the response following SAG therapy in the patients infected with same Leishmania species and living in the same endemic area [54]. Further studies are needed to define how genetic variation, if any, influences the outcome of SAG treatment in kala-azar patients.

Materials and Methods

Animals

BALB/c mice and golden hamsters (Mesocricetus auratus) were maintained and bred under pathogen-free conditions.

Ethics statement

Use of both mice and hamsters was approved by the Institutional Animal Ethics Committees of Institute of Microbial Technology and Indian Institute of Chemical Biology, India. All animal experimentations were performed according to the National Regulatory Guidelines issued by CPSEA (Committee for the Purpose of Supervision of Experiments on Animals), Ministry of Environment and Forest, Govt. of India.

Parasite cultures and preparation of soluble antigen from LD

Sb^RLD [GE1F8R (MHOM/IN/89/GE1), 39, R5] and Sb^SLD [AG83 (MHOM/IN/83/AG83), 2001] strains are gifts from Dr. Neeloo Singh (Central Drug Research Institute, India) and Dr. Shyam Sundar (Banaras Hindu University, India) and were maintained in golden hamsters as described [45], [55]–[57]. Amastigotes were obtained from spleens of infected hamsters as described [58]. Subsequently, amastigotes were transformed into promastigotes and maintained as described [59]. GFP-2001 and GFP-R5 are gifts from Dr. Neeloo Singh and were maintained in M199 complete medium (10% FBS, penicillin/streptomycin) with 720 µg/ml geneticin disulfate (G418) (Sigma, St. Louis, MO) [60]. Soluble antigens were prepared from Sb^SLD and Sb^RLD promastigotes (10⁹/ml) as described [59].

Transfection of 2001Pm

2001Pm (Sb^SLD) at mid log or stationary phase were washed twice with electroporation buffer (21 mM HEPES, pH 7.05; 137 mM NaCl; 5 mM KCl; 0.7 mM NaH₂PO₄; 6 mM glucose). The promastigotes were resuspended in ice-cold electroporation buffer to a final concentration of 10⁷/ml. An aliquot (400 µl) of parasite suspension was mixed with 35–40 µg of chilled pGEM 7ZF α-neo-α L. tarentolae MRPA or pGEM 7ZF α-neo-α DNA (kind gifts from Dr. Marc Ouellette, Laval University, Canada), transferred to a 2-mm gap cuvette and electroporated using BIO-RAD Gene-Pulser X cell instrument at 450 V and 500 µF (3.5 to 4 milli-seconds pulse time). After electroporation, parasites were immediately placed on ice for 10 minutes and cultured at 22°C for 24 hours in Schneider's insect medium with 1500 µg/ml paromomycin and 10 µM Biopterin. Subsequently, 40 µg/ml of G418 was added to the parasite culture. After 24 hours, 1 ml of this promastigote culture was transferred to a new flask containing 5 ml of fresh medium with 10 µM biopterin and 120 µg/ml G418. The culture was maintained for one month under drug pressure (once per week) to obtain the stable transfectants. The mRNA expression of MRPA in these stable transfectants was verified via RT-PCR.

DC preparation

BMDCs and sDCs were prepared from male or female BALB/c mice between 8–12 weeks of age as described [19]. Flow cytometric analyses indicated >85% and ∼90% purity of BMDCs and sDCs, respectively, based on CD11c expression.

DC infection with promastigotes or amastigotes of LD

DCs (5×10⁶/well) were infected in vitro at specified MOIs either with amastigotes of Sb^RLD or Sb^SLD; or promastigotes of stationary phase Sb^RLD, Sb^SLD or respective GFP-LD for indicated times in a 6-well plate in RPMI 1640 complete medium (10% FBS, penicillin/streptomycin, L-glutamine, sodium pyruvate, non-essential amino acids, 2-mercaptoethanol). Subsequently, DCs were washed, resuspended in RPMI 1640 complete medium and stimulated with SAG (10, 20 and 40 µg/ml) of clinical grade or sodium gluconate (25, 50, 100 and 200 µg/ml) for specified times. The doses of SAG mentioned here and for all experiments represent the concentration of Sb^V. DC infection with GFP-Sb^RLD or GFP-Sb^SLD was determined via flow cytometry. SAG containing 20 µg/ml of Sb^V, and 35 µg/ml of sodium gluconate had equivalent molar concentration. Sodium gluconate was purchased from Acros Organics, New Jersey, USA. SAG was obtained as kind gift from Albert David Ltd., Kolkata, India. For other experiments, DCs (2.5×10⁵/ml) were adhered on 22×22 mm cover slips, infected with promastigotes/amastigotes of Sb^SLD or Sb^RLD and treated with SAG for indicated times. The number of intracellular parasites in DCs was determined via Giemsa staining. In some experiments, DCs (5×10⁶/well) were infected with Sb^SLD or Sb^RLD promastigotes for 3 hours or left uninfected. Alternatively, infection of DCs (5×10⁶/well) with LD promastigotes was done for specified times. DCs were then treated or not with Wort (200 nM) or Ly (50 µM) (Cell Signaling Technology, Beverly, MA) 1 hour prior to SAG stimulation as described [19]. In some cases, DCs (5×10⁶/well) were infected with Sb^SLD or Sb^RLD promastigotes for 3 and 24 hours or left uninfected. Neutralizing αIL-10 monoclonal Ab (10 µg/ml)/isotype control rat IgG2_b Ab (10 µg/ml) (BD Biosciences, San Jose, CA) were added at 2^nd hour of BMDC infection. After infection, DCs were washed and stimulated with SAG for 0.3 hours.

DC pretreatment with soluble antigens/culture supernatant of LD or IL-10

DCs (5×10⁶/well) were pretreated for 3 hours with 50 µg/ml soluble antigens derived from Sb^RLD (Sb^RLDsAg) or Sb^SLD (Sb^SLDsAg) and stimulated with SAG for 0.3 hours in a 6-well plate. Alternatively, Sb^RLD or Sb^SLD promastigotes (10⁶/ml) were grown in M199 complete medium till the parasite concentration reached ≥10⁷/ml. The respective culture supernatants of Sb^RLD (Sb^RLDs) and Sb^SLD (Sb^SLDs) were then collected. Subsequently, DCs (5×10⁶/well) were cultured for 3 hours in RPMI 1640 complete medium containing Sb^RLDs or Sb^SLDs at specified complete medium to supernatant ratios and stimulated with SAG for 0.3 hours as above. In some experiments, BMDCs (5×10⁶/well) were pretreated with murine IL-10 (50 ng/ml) (PeproTech, Rocky Hill, NJ) for 24 hours as described [19], in the presence or absence of 10 µg/ml of αIL-10Ab/isotype control Ab and stimulated with LPS (500 ng/ml) for 0.5 hours.

EMSA and Western blot

Nuclear and cytoplasmic extracts were prepared from DC as described [61]. EMSA was performed as described [62] using ³²P-labeled DNA probes containing NF-κB binding sites derived from MHC-I H2K promoter:

5′-CAGGGCTGGGGATTCCCCATCTCCACAGTTTCACTTC-3′ [20]. In some experiments, DNA probes specific for murine γGCS heavy-chain (mγGCS_hc) promoter containing wild-type or mutant NF-κB binding sites as represented by WT-mγGCS_hc probe, ′-CGGTTCTGAAGGTGGGGAAACTTCTGAAGAAACTT-3′5and Mut-mγGCS_hc probe, 5′-CGGTTCTGAAGGTCTCTAAGAATCTGAAGAAACTT-3′ (NF-κB binding site is underlined and mutated bases are in italics) respectively, were used. A double stranded OCT-1 DNA probe, 5′-TGTCGAATGCAAATCACTAGAA-3′ was used as control. Supershift EMSAs were carried out as described [19] using following Abs: αp50, αRelB (Active Motif, CA); αp65 (Abcam plc. Cambridge, UK); αp52, αcRel, rabbit IgG (Santa Cruz Biotechnology, Santa Cruz, CA). Bands were visualized using a phosphoimager (Bio-Rad Molecular Imager FX, Hercules, CA).

Western blotting was carried out as described [20]. Blots were probed with Abs specific for: IκBα, IκBβ, IκBε, IKK1, IKK2 (Santa Cruz Biotechnology); pIκBα, pIKK1 (Ser180)/pIKK2 (Ser181), pAKT (Ser473), AKT, pSAPK/JNK (Thr183/Tyr185), SAPK/JNK, phospho-p38MAPK (Thr180/Tyr182), p38MAPK, pERK1/pERK2 (Thr202/Tyr204), ERK1/ERK2 (Cell Signaling Technology); β-actin (Sigma). Binding of secondary HRP-labeled goat-αrabbit (Santa Cruz Biotechnology) or goat-αmouse Abs (Sigma) was analyzed using SuperSignal^R West Pico or West Dura Chemiluminescent Substrate (Pierce, Rockland, IL).

IKK assay

IKK signalosome was immunoprecipitated from 700 µg of a whole DC lysate using Protein A/G agarose beads (Santa Cruz Biotechnology) and rabbit polyclonal αIKK1 Ab. In vitro kinase reaction was performed and kinase activity of immunoprecipitated IKK complex determined as described [20].

Flow cytometry

The following monoclonal antibodies used for flow cytometry were purchased from eBioscience (San Diego, CA): PE-αmouse CD11c, FITC-αmouse CD11b, FITC-αmouse CD40, FITC-αmouse CD86, FITC-αmouse CD80, FITC-αmouse H2K^d and FITC-αmouse IA^d. The fluorescence of stained cells was analyzed on a FACSCalibur (BD Biosciences) using Cell Quest Pro software.

Measurement of IL-12, TNFα and IL-10 secretions from DCs

DCs (1×10⁶/ml) were infected with promastigotes of Sb^RLD or Sb^SLD for 3 hours or left uninfected, washed and stimulated with SAG for additional 48 hours in a 24-well plate. Alternatively, DCs (1×10⁶/ml) were infected with promastigotes of Sb^RLD or Sb^SLD for varying times or left uninfected. The culture supernatants were analyzed for IL-12, TNFα and IL-10 productions in triplicate using ELISA kits (BD Biosciences) following the manufacturer's instructions.

Analysis of murine γGCS heavy-chain (mγGCS_hc) and LD γGCS heavy-chain (LD-γGCS_hc) expression in Sb^RLD- and Sb^SLD-infected BMDCs

BMDCs (5×10⁶/well) were infected with Sb^RLD or Sb^SLD for 3 hours or left uninfected, stimulated with SAG for 3 hours and RNA prepared using RNeasy minikit reagent (QIAGEN). The mRNA expression of mγGCS_hc, LD-γGCS_hc, murine glyceraldehyde-3-phosphate dehydrogenase (mGAPDH) and LD-tubulin genes were determined via reverse transcription-PCR using platinum quantitative RT-PCR Thermoscript One Step system kit (Invitrogen, Carlsbad, CA), and primers: LD-γGCS_hc 5′-TATCAAGTCTCGCTACGACT-3′, 5′-CGGAGTCCTTCAGAAGTT-3′; LD-tubulin 5′-ACATCACGAACTCGGTGTTT-3′, 5′-TTCGTCTTGATCGTCGCAAT-3′; mγGCS_hc 5′-AGAACAATCGCTTTAGGATCA-3′, 5′-AGAAGATGATCGATGCCTTC-3′; mGAPDH 5′-AGATTGTTGCCATCAACGAC 3′, 5′-ATGACAAGCTTCCCATTCTC-3′ [4].

Detection of MRPA expression in LD

RNA was isolated from LD promastigotes and mRNA expression of MRPA was detected by analyzing the amplification of 179 bp cDNA fragment of MRPA via reverse transcription-PCR using primers: 5′-GCGCAGCCGTTTGTGCTTGTGG-3′, 5′-TTGCCGTACGTCGCGATGGTGC-3′ [5]. mRNA expression of LD-tubulin was used as loading control.

Chromatin immunoprecipitation (ChIP)

BMDCs (5×10⁶/well) were stimulated with SAG for 0.3 hours or left untreated. ChIP was performed using ChIP-IT kit (Active Motif) following the manufacturer's instructions. After immunoprecipitation using rabbit IgG or NF-κB Abs such as αp50, αRelB and αp65, followed by DNA extraction; PCR was performed to amplify −991/−673 region of mγGCS_hc promoter using primers: P1, 5′-CCAGTTCCCAGAGCCTTCCG-3′ and P2, 5′-TTGTACGACTCCACATGGCATG-3′. For a negative control, mGAPDH promoter was amplified by using primers: 5′-CACCCTGGCATTTTCTTCCA-3 and 5′-GACCCAGAGACCTGAATGCTG-3′ [63].

Reporter assay

An approximately 1.0 kb (−987/+25) long 5′-flanking sequence of mγGCS_hc gene was amplified by PCR using murine genomic DNA (Promega, Madison, WI) and cloned into pGL3-Basic vector. Using this resulting construct, p987-luc, and a QuickChangeII PCR-based site-directed mutagenesis kit (Stratagene, Cedar Creek, TX); the construct (Mut p987-luc) containing a mutant NF-κB binding site, similar to that described in EMSA studies, in −987/+25 region of mγGCS_hc promoter was generated. Both constructs were confirmed by sequencing. NIH3T3 cells were transiently transfected with a DNA mixture containing p987-luc or Mut p987-luc (0.266 µg), pRL-CMV (0.200 µg), pEGFP-p65 (0.266 µg) and/or pEGFP-IκBαΔN (IκBα with amino acids 1–36 deleted) (0.266 µg) using lipofectamine LTX (Invitrogen). The latter two expression vectors are gifts from Dr. Johannes Schmid (Medical University of Vienna, Austria) and Dr. Susan Kandarian (Boston University, USA) respectively [64], [65]. The DNA amount in each transfection was kept constant. Cells were grown for 24 hours after transfection. The luciferase activity of the cell lysates was determined using Dual Luciferase Reporter Assay System (Promega) and GLOMAX luminometer (Promega) following the manufacturer's instructions. The level of luciferase activity was normalized to the level of Renilla luciferase activity.

Supporting Information

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