Restoration of IFNγR Subunit Assembly, IFNγ Signaling and Parasite Clearance in Leishmania donovani Infected Macrophages: Role of Membrane Cholesterol

Despite the presence of significant levels of systemic Interferon gamma (IFNγ), the host protective cytokine, Kala-azar patients display high parasite load with downregulated IFNγ signaling in Leishmania donovani (LD) infected macrophages (LD-MØs); the cause of such aberrant phenomenon is unknown. Here we reveal for the first time the mechanistic basis of impaired IFNγ signaling in parasitized murine macrophages. Our study clearly shows that in LD-MØs IFNγ receptor (IFNγR) expression and their ligand-affinity remained unaltered. The intracellular parasites did not pose any generalized defect in LD-MØs as IL-10 mediated signal transducer and activator of transcription 3 (STAT3) phosphorylation remained unaltered with respect to normal. Previously, we showed that LD-MØs are more fluid than normal MØs due to quenching of membrane cholesterol. The decreased rigidity in LD-MØs was not due to parasite derived lipophosphoglycan (LPG) because purified LPG failed to alter fluidity in normal MØs. IFNγR subunit 1 (IFNγR1) and subunit 2 (IFNγR2) colocalize in raft upon IFNγ stimulation of normal MØs, but this was absent in LD-MØs. Oddly enough, such association of IFNγR1 and IFNγR2 could be restored upon liposomal delivery of cholesterol as evident from the fluorescence resonance energy transfer (FRET) experiment and co-immunoprecipitation studies. Furthermore, liposomal cholesterol treatment together with IFNγ allowed reassociation of signaling assembly (phospho-JAK1, JAK2 and STAT1) in LD-MØs, appropriate signaling, and subsequent parasite killing. This effect was cholesterol specific because cholesterol analogue 4-cholestene-3-one failed to restore the response. The presence of cholesterol binding motifs [(L/V)-X1–5-Y-X1–5-(R/K)] in the transmembrane domain of IFNγR1 was also noted. The interaction of peptides representing this motif of IFNγR1 was studied with cholesterol-liposome and analogue-liposome with difference of two orders of magnitude in respective affinity (KD: 4.27×10−9 M versus 2.69×10−7 M). These observations reinforce the importance of cholesterol in the regulation of function of IFNγR1 proteins. This study clearly demonstrates that during its intracellular life-cycle LD perturbs IFNγR1 and IFNγR2 assembly and subsequent ligand driven signaling by quenching MØ membrane cholesterol.


Introduction
Visceral Leishmaniasis (VL), a potentially fatal visceralizing disease, afflicts millions of people worldwide and is caused by infection with Leishmania donovani (LD), an obligate-intracellular trypanosomatid protozoan. During the past decades, a large body of evidences supported the notion that the cytokine interferon gamma (IFNc) plays a decisive role in anti-leishmanial defense [1,2]. A primary defect that may lead to pathogenesis in VL is the failure to activate parasitized macrophages (MØs) to eliminate LD in response to IFNc [3]. Intriguingly, the presence of elevated levels of serum IFNc in human VL [4][5][6][7][8] and high expression of IFNc mRNA in lymphoid organs [9] do not reconcile with large parasite burden observed at the active stage of the disease. The remarkable predominance of the Th1 cytokine IFNc along with impaired MØ effector function indicates a MØ specific desensitization to the available IFNc stimulus. This was evident from several in vitro studies [3,10,11] showing gross inhibition of the IFNc signaling pathways in the LD infected MØs (LD-MØs), but the exact mechanism that triggers the inhibition remained unknown till date.
IFNc binds to specific cell surface receptor IFNcR which consists of two heterodimeric subunits, IFNcR1 (a, ligand binding subunit) and IFNcR2 (b, signal-transducing subunit). Signal transduction of IFNc is initiated by its binding to IFNcR1 and subsequent receptor subunit multimerization [12]. IFNcR1 colocalizes partly with the ganglioside GM1, a classical marker of specialized cholesterol-rich membrane microdomains termed lipid-rafts [13]. Subsequent evidences disclosed that membrane lipid-rafts are intimately involved in the process of IFNc mediated signal transduction. Remarkably, irrespective of various cell types used in different reports, disruption of the plasma membrane rafts by cholesterol depletion using methyl-b-cyclodextrin (mBCD) or cholesterol sequestration with filipin reversibly affected not only the generation of the IFNc inducible tyrosine phosphorylation of signal transducer and activator of transcription 1 (STAT1) but also productive transcriptional signaling [14][15][16]. This is congruent with the prevalent hypothesis of lipid raft involvement in IFNc mediated signaling [17]. Membrane proteins such as G protein coupled receptors, transporters, or ion channels have been shown to be localized or enriched in lipid rafts. The structure-activity analysis shows that many of the cholesterol effects are due to specific sterol-protein interactions as shown in case of a number of membrane bound receptors such as those for cholecystokinin (type B), oxytocin and nicotinic acetylcholine (nACh) [18]. Refined structure of nACh receptor (nAChr) has been shown to have internal sites capable of containing cholesterol, whose occupation bolsters the protein structure [19]. Both oxytocin and serotonin 1A receptors contain the strict cholesterol consensus motif (CCC), and in both the cases cholesterol incorporation lead to dramatic increase in agonist affinity [20,21].
Previous studies from our group have shown that during their intracellular life-cycle LD parasites disrupt the membrane rafts of MØs by quenching cholesterol from the membrane. This was associated with defective antigen presenting function, which could be corrected by liposomal delivery cholesterol [22,23]. We also showed that systemic administration of cholesterol through liposomal delivery in LD infected hamsters has strong therapeutic efficacy [23], in agreement with the clinical practice of using cholesterol either directly [24] or as an adjunct to treatment [25]. It may be recalled that Kala-azar patients show progressive decrease in serum cholesterol as a function of splenic parasite load [26]. Therefore we investigated the potential involvement of LD mediated manipulation of MØ membrane cholesterol in IFNc signaling modulation.
Herein we report for the first time that the earliest activation induced signaling event of IFNc receptor oligomerization is inhibited due to quenching of the MØ membrane cholesterol by the LD parasites. Restoration of the MØ membrane cholesterol by exogenous delivery not only reinstates the receptor subunit interaction but also restores subsequent downstream signaling as well as the IFNc mediated leishmanicidal property.

Defective IFNcR subunit multimerization in LD infected MØs
Previous reports revealed that Leishmania spp. infected MØs are refractory to exogenous IFNc mediated clearance of intracellular parasites due to defective IFNc signaling [3, 11, 27, and 28]. In this study, the molecular mechanism/s triggering the impairment of IFNc signaling in LD-MØs have been investigated.
To investigate the mechanistic basis of defective IFNc signaling in LD-MØs, BALB/c derived peritoneal MØs (PEC) and homologous cell-line of RAW 264.7 were used. While performing the preliminary experiments ( Figure S1-S3), RAW 264.7 cells yielded results comparable to the native system of PEC; therefore all the subsequent experiments were performed using these cells unless otherwise mentioned.
To address this possibility, we utilized the fluorescence resonance energy transfer (FRET) method, a powerful spectrofluorometric technique that has been used to determine molecular interactions with higher resolution [42]. We assessed FRET between cell surface IFNcR1 and IFNcR2 molecules that were fluorescently labeled with receptor specific antibodies tagged with DyLight488 or DyLight594 respectively. As shown in Figure 1A (panel a), the emission spectrum excited with 485 nm light showed reciprocal changes in donor emission peak (517 nm) versus acceptor emission (620 nm), indicating that IFNc treatment promoted energy transfer between IFNc-R1 and -R2 in normal cells. In marked contrast, stimulation of LD infected cells with IFNc could induce minimal or no FRET signal between IFNcR1 and IFNcR2 ( Figure 1A, panel b and c). The FRET efficiency was calculated in each of these conditions. As this was found to be low in infected cells (E = 0.166 in 4 hr and E = 0.042 in 12 hr LD infection) compared to that (E = 0.465) of normal IFNc stimulated condition, LD infection must have significantly inhibited ligand induced receptor association. Thus the results indicate prominent reduction in the ligand induced IFNcR1-IFNcR2 FRET efficiency, which is reduced by 64.6 % at 4 hr and 90.9 % at 12 hr post LD infection than the normal condition.
To confirm the observation, we coimmunoprecipitated IFNcR1 and IFNcR2 from ligand stimulated or unstimulated normal and infected MØs. The results ( Figure 1B) indicate that IFNc induced copurification of the IFNcR2 subunits with IFNcR1 in normal condition of the MØs. Conversely, infection for 4 hr markedly reduced the extent of IFNcR2 coprecipitation with IFNcR1 even in IFNc stimulated condition ( Figure 1B), while the level of coprecipitated IFNcR2 went below detection level with increasing time of infection. Taken together, the results in this section imply that LD infection inhibits IFNc induced receptor oligomerization in MØs.

Ligand induced IFNcR subunit partitioning in detergent resistant membrane fraction is lost in LD infected MØs
Association with lipid-raft membrane domains is known to regulate the oligomerization of various cell surface receptors [43][44][45]. It is also accepted that localization of the IFNcR complex within intact lipid raft microdomains governs their efficient signal transduction capability [14][15][16]. The aforementioned results suggested that LD infection inhibits IFNc induced receptor oligomerization in MØs, despite having unaltered initial ligandreceptor association. We therefore speculated that differential

Author Summary
The disease Visceral Leishmaniasis or Kala-azar is extending its base in the Indian subcontinent and elsewhere. The emergence of drug resistant cases is aggravating the problem further. The kala-azar patients do not respond to the host-protective cytokine IFNc at the active stage of the disease, the cause of which is unknown. This research is designed to understand how cell surface receptors for IFNc respond under parasitized condition. Our results clearly showed that the Leishmania parasites during their intracellular life-cycle make the host cell membrane fluid by quenching cholesterol from the membrane, which renders the IFNcR nonfunctional despite their physical presence on the cell surface. Upon supplementation of cholesterol in infected MØs, the infected cells regain responsiveness to IFNc coupled with intracellular parasite killing. Thus supplementation of cholesterol together with IFNc may be a new approach to treat drug unresponsive Kala-azar cases.
membrane partitioning behavior of the subunits might affect the IFNc-R1 and -R2 interaction. It was therefore necessary to examine whether LD infection of the host cells delocalizes the IFNcR chains from the lipid raft microdomains that in consequence might hinder efficient oligomerization of the receptor subunits upon IFNc mediated stimulation.
To study the abrogation of IFNc induced IFNcR subunits partitioning, if any, under parasitized conditions, MØ membrane extracts with 1% Triton X-100 were subjected to optiprep density gradient separation and examined by immunoblotting to analyze the distribution of IFNcR subunit proteins ( Figure 2). Cholesterolrich lipid raft domains were characterized by the enrichment of known raft marker protein flotillin1, while the fractions containing the non-raft portions were identified with the surrogate marker of transferrin receptor [46,22]. The success of the biochemical isolation process was confirmed by determining the lipid raft constituent glycosphingolipid GM1 in each fraction by dot blot. Careful analysis of the fractionation study revealed that the constitutive association of IFNcR1 with the detergent resistant lipid raft fractions was discernibly disoriented at steady-state of the early infected MØs where appreciable amount of IFNcR1 was present in the detergent soluble fractions. Whereas, with increasing severity of the LD infection, the IFNcR1 protein was nearly completely delocalized from the lipid raft fractions and were recovered from the detergent soluble higher density fractions ( Figure 2). On the contrary, the localization pattern of the IFNcR2 subunit showed a more or less diffused distribution throughout the density gradients of the steady state of the normal MØs. Upon IFNc mediated activation, the IFNcR2 subunit is recruited to the lipid raft fractions almost to entirety which is accompanied by Figure 1. IFNc induced IFNcR subunit association is impaired by LD infection. A, Fluorescence spectrometric analysis of IFNcR1-IFNcR2 FRET in uninfected and infected RAW 264.7 cells. Cells were treated with/without 100 U/ml of rIFNc for 3 minutes, washed with ice cold PBS, transferred to ice and then fixed immediately. Cell-surface IFNcR1 and IFNcR2 were then stained with respective mAb linked directly to fluorophores, Dylight488 (DL488) for anti-IFNcR1 and Dylight594 (DL594) for anti-IFNcR2. The fluorescence emission spectra in response to 485 nm excitation before (solid black) and after (solid grey) IFNc treatment are shown with hashed black line representing the single stained Dylight488 control. 'E' in the figures represents the FRET efficiency of IFNc treated samples, calculated as described in Materials and Methods. B, Immunoblot Analysis of IFNcR1 and IFNcR2 in immunoprecipitates of anti-IFNcR1. IFNcR1was immunoprecipitated with either specific or control immunoglobulin from uninfected and infected MØs stimulated with/without 100 U/ml of rIFNc for 3 minutes as described in Materials and Methods. Copurifying IFNcR2 was detected by immunoblotting with anti-IFNcR2 (lower gel). doi:10.1371/journal.ppat.1002229.g001 robust receptor subunit interaction, as indicated in the above mentioned energy transfer and coIP experiments ( Figure 1). Intriguingly, the IFNcR2 subunit distribution pattern was markedly altered from the very early hours of infection through the late hours, when bulk of the proteins were recovered from the non-raft fractions, denoting complete detergent solubilization. Nevertheless, at early hours of LD infection stimulation of the cells with IFNc could elicit IFNcR2 redistribution albeit to a lesser extent and this partial IFNcR2 recruitment to the raft domains was reflected in concomitant decline in the efficiency of receptor subunit interaction and signaling potency. Notably, at late hours of LD infection, despite having simultaneous presence of both IFNcR1 and IFNcR2 within the non-raft fractions, the IFNc mediated stimulation of the MØs failed to induce any detectable subunit interaction as evidenced by the absence of FRET or coIP positivity at 12 hr postinfection.
The results were further validated in situ by confocal colocalization study, which recapitulated the observations of the floatation studies for the receptor distribution patterns. Analysis of the colocalization pattern of the individual subunits of the IFNcR assembly with the GM1 rich microdomains was quantified and expressed in terms of the Pearson's correlation coefficient (PCC) values. The PCC values obtained by colocalization analysis of constitutively raft associated IFNcR1 proteins with GM1 at the steady state of the naïve MØs were considered as basal, and each experimental condition assessed thereafter was represented as the function of this optimal (100%) colocalization efficiency. The results in Figure 3, demonstrate that the percentage colocalization of IFNcR1 with GM1 molecules upon IFNc mediated activation of the normal MØs remain unchanged (102%), signifying preassociation of the IFNcR1 protein with the GM1 enriched domain. The results also reveal that, there is a considerable decrease in the percentage of colocalized IFNcR1 with GM1 in the macrophage membrane at 4 hour post LD infection (71%) than the basal condition. However, IFNc stimulation could elicit a minor increase in the colocalization pattern (86.9%) sufficient to elicit a partial activation of signaling cascade. With increasing time of infection at 12 hr post infection, we observed a sharp decline in the percentage of the colocalized IFNcR1 (32.7%) along with absolute failure of IFNc to induce any detectable increase in the colocalization pattern at this condition (34.2%).
Comparison of the percentage colocalization of the IFNcR2 with GM1 at the steady-state of the MØs showed a steep decline both in the early as well as the late hours of LD infected MØs than the uninfected control. In agreement with the floatation studies, IFNc activation could elicit a partial increase in the colocalization efficiency at the early hours of LD infection (62.5%), though absence of the same at late hours of infection denotes a conspicuous block in the IFNc triggered translocational behavior of IFNcR2.

Impaired IFNc signaling complex formation under parasitized condition
To investigate further, we stimulated the uninfected and LD infected MØs with biotinylated IFNc and isolated the lipid rafts. Total receptors and engaged receptors were immunoprecipitated using anti-IFNcR1 and streptavidin-agarose respectively. The activated forms of the signaling complex were determined by resolubilization of the immunoprecipitates of streptavidin-agarose, reprecipitation by p-Tyr antibody and immunoblotting with antibodies against distinct components of the IFNc signaling complex namely IFNcR1, JAK1, JAK2 and STAT1. As depicted in Figure 4, endogenous IFNcR1 distribution was less affected during early (4 th ) hours of infection, but with increasing severity of infection it was completely delocalized from the raft fractions. Most striking of these results is that, though we could recover some ligand engaged IFNcR1 from non-raft portions at 4 hr post infection, IFNcR2 was only detectibly coimmunoprecipitated from the raft fractions. Another significant finding of this experiment was that though endogenous IFNcR1 and engaged IFNcR1 could be immunoprecipitated from both raft and nonraft fractions after 3 minutes of IFNc stimulation from 4 hr LD infected macrophages, the phosphorylated (activated) IFNcR1 was only detected in the immunecomplexes from the raft fractions along with other activated signaling intermediates. It is worthy of note that at late hours of infection, though engaged IFNcR1 ( Figure 4) and IFNcR2 ( Figure 2) were simultaneously present in the non-raft fractions, they could not generate any productive interaction as evidenced by the complete absence of IFNc triggered CoIP and/or phosphorylation signal. These results denote critical importance of cholesterol rich microenvironment for proper IFNcR functioning. MØs left untreated or treated with 100 U/ml of rIFNc (3 minutes, 37uC) were immediately washed with ice-cold PBS and crude membranes were prepared which were then extracted with 1% Triton X-100 and subjected to optiprep density gradient centrifugation to isolate lipid rafts. Proteins from equal volume of aliquots of the collected fractions were separated by SDS-PAGE and analyzed by western blotting using specific Ab against IFNcR1, IFNcR2, Flotillin 1 and Transferrin receptor. To analyze the distribution of GM1, 2 ml of each fraction was dot blotted onto a nitrocellulose membrane and detected using CTxB-HRP. 'TfR' denotes transferrin receptor for this and the subsequent experiments. doi:10.1371/journal.ppat.1002229.g002 Cholesterol dependent modulation of IFNc mediated receptor subunit interaction in LD infected MØs involving differential raft partitioning of IFNcR subunits The involvement of rafts in early IFNc signaling events such as induction of STAT1 tyrosine phosphorylation has previously been studied using methyl-ß-cyclodextrin (mBCD) as well as filipin and nystatin [14][15][16], all of which perturb the raft integrity via cholesterol depletion or sequestration [47]. Extending and verifying our original observation of decreased MØ membrane cholesterol in LD infected in vivo model [23], our in vitro system of infected RAW 264.7 cells also showed reduction in membrane cholesterol as function of time ( Figure 5A), the kinetics of which exactly coincided with the time points of waning IFNc signaling. To clearly establish the role of cholesterol in IFNc mediated receptor assembly, we replenished the LD-MØs with exogenous cholesterol in liposomal form (CH-Liposome). Figure 5B shows that cholesterol supplementation induced relocalization of IFNcR1 within the raft domains regardless of the time points of investigation after LD infection. Interestingly, in cholesterol replete MØs, the engaged receptors were entirely recovered from the raft fractions ( Figure 6A). This cholesterol induced IFNcR1 relocalization was prominently associated with a marked increase in IFNcR2 interaction, which was readily detected from the immunecomplexes isolated by total IFNcR1 and/or engaged IFNcR1 in the IFNc stimulated condition ( Figure 6A). The restored pattern of IFNcR subunit multimerization was more conclusively verified in FRET experiments, where cholesterol supplementation reinstated the IFNc inducible receptor subunit interactions ( Figure 6B).
To establish unambiguously the specific role of cholesterol in influencing the earliest step of IFNcR signaling in reconstituted membrane rafts of LD-MØs, we treated the infected MØs with liposomes prepared with oxidized cholesterol analogue of 4cholestene-3-one (AN-Liposome). The results of coimmunoprecipitation ( Figure 6A) and FRET ( Figure 6B) analyses between IFNcR subunits in AN-Liposome treated infected MØs showed that treatment with AN-liposome failed to restore IFNcR translocations as also the subunit interactions by IFNc stimulation.
In order to investigate whether the enhanced IFNcR oligomerization in cholesterol-replete cells directly involves action of cholesterol rather than its indirect effect via increased bulk membrane rigidity, we treated LD-MØs with DPPC, a phospholipid previously shown to induce increased membrane rigidity in model membranes [48]. The data in Figure 5B, 6A reveal that in contrast to cholesterol supplemented cells, the DPPC treated cells, despite having comparable bulk membrane rigidity as indicated by the FA values ( Figure 6B, insets), failed to induce IFNc receptor interaction indicating specific requirement of cholesterol in IFNcR subunit assembly.
To corroborate the finding of cholesterol specific IFNcR subunit interaction, RAW 264.7 cells were treated with mBCD, which disrupts lipid rafts by specific depletion of cholesterol [49]. As shown in Figure 7A, stimulation of mBCD treated cells with IFNc failed to induce association of IFNcR2 subunit with engaged IFNcR1, which was found to be localized in the non-raft membrane fractions. Comparable to the LD infected macrophages, mBCD treatment completely abolished the activation of IFNc signaling components while cholesterol replenishment of these cells restored the subunit assembly ( Figure 7) and activation pattern of IFNc induction ( Figure 7A). In additional control experiments, conditions of mBCD treatments in RAW 264.7 cells were first determined to ensure unaffected cell viability and unaltered expression of surface IFNcR and components of STAT1 activation axis (data not shown).

LPG plays no role in delocalization of IFNcR from raft
LPG is a GPI anchored molecule secreted/shed by the parasites after internalization in MØs by phagocytosis [50,51]. It is known to increase the total membrane rigidity. Consistent with previous reports [52][53][54], LPG induced significantly higher bulk membrane rigidity upon incubation with MØs ( Figure 8A) and was incorporated substantially within GM1 enriched membrane domains. However, it could not induce IFNcR1 delocalization from the more buoyant lipid raft fractions as indicated by dot blot analyses of individual membrane fractions of optiprep gradient ( Figure 8B). This intactness of IFNcR1 localization in the LPG incubated MØs was also reflected in undiminished IFNc functionality in these cells as assessed by the gamma activated sequence driven luciferase (GAS-Luc) reporter system ( Figure 8C), a finding supported previously by Ray et al. [11]. Notably, pharmacological depletion of membrane cholesterol from the LPG-incubated MØs completely disoriented the IFNcR1 localization pattern and disrupted the membrane rafts as indicated by the even distribution of GM1 molecules along the isolated density gradients of the cell membrane ( Figure 8B). Importantly, repletion of cholesterol in the system of LPG-incubated mBCD-treated MØs restored both the localization and functionality pattern of IFNcR1 ( Figure 8B, 8C).
As the infection in vivo propagates through amastigotes which lack LPG [53], we asked the question if their role in IFNc signaling inhibition was different from that of promastigotes. The result depicted in Figure 9 demonstrates that for all the tested time points of amastigote infection, deactivation of the IFNc signaling takes place as early as 5 min post-IFNc stimulation. The result was consistent with previous report [55] of immediate deactivation of early IFNc signaling at the level of JAK2 phosphorylation in LD amastigote infected primary human MØ, post 10 min of rIFNc stimulation.
Collectively the above results strongly imply that membrane cholesterol is critically involved in the initiation of IFNc signaling, probably by direct modulation of IFNc receptor functionality. and insoluble lipid raft (R) fractions were isolated as described in figure 2. The fractions 2-4 were pooled to yield a total raft (R) compartment and fractions 7-9 were pooled to obtain the non-raft (NR) compartment. Engaged receptors and their signaling complexes were immunoprecipitated using Streptavidin-agarose beads. For comparison, the total amount of receptor was immunoprecipitated by adding anti-IFNcR1 Ab to the soluble non-raft and raft fractions. Immunoprecipitates and corresponding total cell lysates were subjected to SDS-PAGE and immunoblotted using specific Ab against IFNcR1 and IFNcR2. Activated forms of the signaling subunits were determined by dissociating the engaged signaling complexes pulled down by Streptavidin-agarose beads, by boiling in 1% SDS, followed by immunoprecipitation using an anti-pTyr Ab. Activated and phosphorylated proteins were analyzed by western blotting using Ab against IFNcR1, JAK2, JAK1 and STAT1. Equal loading was verified by immunoblotting of respective whole proteins in whole cells lysates of each experimental set. Str: Streptavidin. doi:10.1371/journal.ppat.1002229.g004 IFNcR1 peptide containing CRAC motif exhibits preferential association with cholesterol There are number of reports on the existence of a common cholesterol recognition/interaction amino acid consensus (CRAC) motif in various proteins [56] including several membrane proteins that directly interact with cholesterol [57]. Very well characterized CRAC motif conforms to the pattern (L/V)-X 1-5 -Y-X 1-5 -(R/K), in which X 1-5 represents between one to five residues of any amino acid [56,57]. As an initial effort to explore the possibility that the functionality of constitutively raft associated IFNcR1 protein is directly modulated by the interaction of cholesterol, we tried to detect the cholesterol recognition motif in it. Motif analysis of IFNcR1 indeed identified two CRAC sites within the IFNcR1 subunit protein ( Figure S4). Multiple sequence alignment showed it to be highly conserved because the domain is present in many of the homologous proteins in different species (Table S1) nullifying its chance occurrence in murine IFNcR1. As denoted by Jamin et al. the CRAC motif is typically located adjacent to a transmembrane ahelix that positions this sequence within the nonpolar core of the bilayer, where it can form contacts along the length of the cholesterol molecule [58]. Sequence analysis of IFNcR1 predicted the localization of the CRAC motif (2269 to 2280) within a transmembrane a-helical portion ( Figure S5). Accordingly, we designed a peptide reproducing the CRAC motif of IFNcR1 protein (2269 to 2280) to analyze the binding of the motif with cholesterol liposome. It should be noted that the tyrosine residue in this motif has been shown by mutational analysis to be critical for cholesterol binding in other proteins [58]. Therefore, another peptide was designed to introduce substitution at positions 275 and 277 to replace tyrosine, intended to disrupt/attenuate the binding of the peptide with cholesterol . We used surface plasmon resonance (SPR) to examine the binding of IFNcR1 peptides to liposomal formulation of cholesterol or its oxidized analogue 4-cholestene-3one. Binding of cholesterol to the Wt IFNcR1 peptide is indicated by the high association rate (k a = 2.69610 3 M 21 s 21 ) and affinity (K D = 4.27610 29 M) compared to those of the mutant (k a = 27610 0 M 21 s 21 , K D = 3.77610 27 M). The specificity of the cholesterol-IFNcR1 peptide interaction was also evident from the 100 fold lower affinity of the Wt peptide for the cholesterol analogue (Table 1 and Figure 10).

Enhancement of IFNc induced intracellular parasite killing upon liposomal delivery of cholesterol in parasitized MØ
We assessed whether cholesterol repletion could restore IFNc induced leishmanicidal effects of the infected MØs. The results depicted in Figure 11 reveal that IFNc stimulation of the cholesterol repleted MØs mediated effective restoration of the To ascertain the direct correlation of cholesterol mediated IFNc signaling with NO production and consequent leishmanicidal effect without involvement of any secondary mediator, L-NMMA was added to cholesterol treated cells. This indeed completely abrogated the enhanced NO production and amastigote killing potential of cholesterol replenished MØs in response to IFNc (Figure 11 D, D').
We finally assessed the generalized biological response of cholesterol promoted IFNc signal enhancement in MØs. Analysis of the IFNc induced transcriptional activity by GAS driven luciferase reporter activity in cholesterol-supplemented systems of CH-Liposome treated LD infected cells and mBCD treated cells demonstrated enhanced bioactivity of IFNc, as manifested by Figure 6. Analysis of raft association of steady-state and activated IFNcR signaling complex in LD infected and differentially treated MØs. A, Immunoblot analysis of immunoprecipitates of isolated raft (R) and non-raft (NR) fractions from LD infected MØs left untreated or treated with either Cholesterol-liposome (CH-Lipo) or 4-cholestene-3-one-liposome (AN-Lipo) or DPPC-liposome (DPPC-Lipo), and stimulated with 0.5 ng/ml of biotinylated IFNc (3 minutes, 37uC) at indicated timepoints postinfection. Either anti-IFNcR1 or streptavidin-agarose beads were used for precipitating immunocomplexes associated with total endogenous cell surface IFNcR1 or IFNc engaged IFNcR1 as described in details in Materials and Methods. Activated forms of the signaling subunits were determined by resolubilizing the engaged signaling complexes followed by immunoprecipitation using an anti-pTyr Ab. Activated and phosphorylated proteins were analyzed by western blotting using Ab against IFNcR1, JAK2, JAK1, and STAT1. Immunoblotting of respective whole proteins in whole cells lysates of each experimental set verified equal loading. B, Analysis of ligand inducible IFNcR subunit interaction in LD infected and differentially treated MØs. Fluorescence spectrometric assay of IFNcR1-IFNcR2 FRET in infected RAW 264.7 cells left untreated or treated with Cholesterol-liposome (CH-Lipo) or 4-cholestene-3-one-liposome (AN-Lipo) or DPPC-liposome (DPPC-Lipo). Cells were treated with/without 100 U/ml of rIFNc for 3 minutes at 37uC and washed and transferred to ice then fixed immediately. FRET analyses were done exactly as described in figure legend 1A. Insets Measurements of fluorescence anisotropy (FA) value similarly treated cells as in figure 6B. 'E' represents the FRET efficiency of IFNc treated samples, calculated as described in Materials and Methods. doi:10.1371/journal.ppat.1002229.g006 higher GAS-Luc expression. This suggests that efficient MØ activation could be achieved by cholesterol treatment of deactivated MØ systems.

Discussion
Our study reinforces the previous findings that LD-MØs are refractory to IFNc mediated signaling, as evident from the inability to clear intracellular parasites ( Figure S1). This was due to defective IFNc signaling as apparent from a number of functional assays such as defective downstream signaling such as JAK2 phosphorylation ( Figure S3A) and IFNc driven GAS-Luc activity ( Figure S3B) under parasitized condition. Interestingly enough, the defect is not due to non-availability of IFNcR on the cell surface ( Figure S2A) or inability of the ligand to bind to the cognate receptor as there was a comparable binding affinity (K D ) in terms of binding of 125 I-IFNc between normal and infected conditions ( Figure S2B). It was also evident that LD-MØ did not show any generalized defect as IL-10 mediated STAT3 phosphorylation ( Figure S3C) and internalization of 125 I-IFNc remained unaffected ( Figure S2C). subjected or not (Control) to cholesterol depletion using 5 mM mBCD for 30 minutes at 37uC, and to cholesterol reloading using Cholesterolliposomes (CH-Lipo) (repletion). Following these treatments, cells were stimulated or not with 0.5 ng/ml of biotinylated IFNc for 3 minutes, 37uC, before harvesting of cells. Total endogenous cell surface IFNcR1 and ligand engaged IFNcR1 were precipitated from pooled raft (R) (fractions 2-4) and non-raft (NR) compartments (fractions 7-9) by anti-IFNcR1 or streptavidin-agarose beads as described in Materials and Methods. The immune complexes were analyzed by Western blot using the appropriate antibodies against IFNcR1 and IFNcR2. Equal loading was verified by respective whole protein immunoblotting from corresponding whole cell lysates. A sequential immunoprecipitation with pTyr antibody experiment from the resolubilized immunecomplexes confirmed the activation status of the signaling subunits when blotted with antibodies against IFNcR1, JAK2, JAK1 and STAT1. B, FRET analysis of ligand induced IFNcR subunit interaction in mBCD treated and cholesterol repleted MØs. Fluorescence spectrometric assay of IFNcR1-IFNcR2 FRET in RAW 264.7 cells that were either left untreated (Control) or subjected to cholesterol depletion or cholesterol reloading of the depleted cells using Cholesterol-liposomes (CH-Lipo) (repletion). Cells were treated with/without 100 U/ml of rIFNc for 3 minutes, 37uC and washed with ice cold PBS and transferred to ice then fixed immediately. FRET analyses were done exactly as described in figure legend 1A. Insets  The ligand receptor interaction of IFNc-IFNcR1 is a sequential multistep process which involves the binding of bivalent IFNc to the extracellular domain of the cognate receptor subunit of IFNcR1 in a ratio of 1:2 [59,60]. This provides the binding scaffold for IFNcR2, the second subunit of the receptor complex [61,62]. The receptor subunit assembly is unlikely to occur randomly at the plasma membrane of the stimulated cells but is confined to specific cholesterol rich membrane microdomains termed rafts. This can be inferred from indirect evidences derived by the use of cholesterol quenching drugs, which show perturbation of IFNc signaling by depletion of membrane cholesterol [14][15][16].
The defective IFNc signaling in LD-MØs was not due to LPG because treatment of normal MØs with exogenous LPG showed neither decrease in fluorescence anisotropy (FA) nor altered IFNc mediated promoter activity ( Figure 8). The defective signaling induced by promastigote mediated infection could also be reproduced with LPG deficient amastigotes ( Figure 9); implying that the effect is independent of LPG despite the intercalation of LPG into membrane raft ( Figure 8B and ref [54]) and increased the total membrane rigidity ( Figure 8A and ref [54]).
From the fractionation study ( Figure 2) and confocal microscopy (Figure 3), it was evident that IFNcR1 is raft associated whereas IFNcR2 is largely non-raft associated. Upon ligand driven stimulation, IFNcR2 moves to the raft fractions in normal condition (Figure 2). During infection, restraint in IFNcR2 recruitment in raft fraction even in the presence of IFNc, suggests that the association of IFNc-R1 and -R2 in LD-MØ is defective. This observation was further corroborated from co-IP studies ( Figure 1B) and confirmed by FRET analysis ( Figure 1A). An elegant study showed that the diffusion coefficient of both GPI linked and native I-E k are dependent on cholesterol concentration [63]. Our study also showed that membrane cholesterol decreases in LD-MØ as a function of time ( Figure 5A). Our results thus support the notion that the decreasing membrane cholesterol due to the presence of intracellular parasites affects the stimulation dependent mobility of IFNcR2 into rafts as shown in the case of T cell receptors at the plasma membrane [64].
It was also observed that stimulation of normal MØs with IFNc causes clustering of the entire IFNc signaling complex in the membrane raft (Figure 4) which was absent under parasitized condition. The effect caused by the intracellular parasites was mimicked by quenching of cholesterol from the membrane by mBCD and restored by liposomal delivery of cholesterol, as evident from FRET analysis ( Figure 7B) and fractionation studies ( Figure 7A). This observation indicates that intracellular parasites may cause the above defects by quenching cholesterol from the membrane. The specificity of cholesterol in inducing effective IFNc inducible receptor interaction was assessed by supplementation of the LD-MØs with the oxidized cholesterol analogue 4cholestene-3-one ( Figure 5B, 6A, 6B), which is known not to induce lipid ordered domain or raft formation due to its head group oxidation and differential partitioning behavior compared to cholesterol [65,66,23]. The results imply that the oxidized cholesterol analogue could not induce any productive interaction between the receptor subunits of IFNcR1 and IFNcR2, substantiating an obligatory role of cholesterol in maintaining the functional integrity of IFNcR. The membrane packing of cholesterol is supported by a hydrogen bond between the cholesterol OH group and the amide bond of sphingolipids [49]; this interaction is presumably absent in the case of the oxidised analogue because of the lack of OH group.
To assess whether increasing the membrane rigidity could restore the propensity of IFNcR1 and IFNcR2 subunit interaction, we supplemented the cell with DPPC-liposomes ( Figure 5B, 6A, 6B) to deliver saturated phospholipids, which increased the membrane rigidity very efficiently. Intriguingly and paradoxically, the increased membrane viscosity resulting from the delivery of DPPC ( Figure 5, 6) or LPG (Figure 8), as evident from fluorescence anisotropy values, was insufficient to potentiate IFNc induced receptor subunit interaction. Collectively these results suggest a direct role of cholesterol in stabilizing the IFNcR interactions, rather than an indirect effect of bulk membrane rigidity to increase the chance interaction of the receptor subunits by random proximity.
Membrane proteins such as G protein coupled receptors, transporters, or ion channels have been shown to be localized or enriched in lipid rafts. The role of cholesterol in receptor RAW 264.7 cells were transfected with pCIS-CK negative control vector or IFNc driven pGAS reporter plasmid. 12 hrs after transfection, cells were either left untreated or treated with mBCD (5 mM, 30 min, 37uC) and /or treated with 10 mg/ml of LPG for 18 hours. Treated cells were then replenished or not with CH-Lipo. Subsequently treated cells were stimulated with or without 100 U/ml of rIFNc eight hours before final harvesting for luciferase activity determination in cell lysates. The data represents the relative luciferase activity normalized to per microgram of protein content. Data are representative of three independent experiments. doi:10.1371/journal.ppat.1002229.g008 conformational stabilization has been reported by several groups, where stability of the active conformation of the receptors depends on the presence of cholesterol with specific molecular interaction between protein and adjacent cholesterol, such as cholecystokinin (type B), oxytocin and nACh receptors [18]. nAChr is reportedly shown to be stabilized in its activated form by a direct action of cholesterol [19]. There are spectroscopic analyses showing that variation in cholesterol concentration can perturb protein secondary structure; this has been shown in photosystem II [67].
Because of nonavailability of pure IFNcR1 receptors (available in the form of chimera with Fc receptor, which itself contains cholesterol binding motif) we could not study the conformation and determine kinetic values of IFNcR1 in association with cholesterol-liposome/analogue-liposome.    Figure 10). Consistent with previous reports, the interaction is tyrosine dependent as the tyrosine substituted peptide failed to interact with cholesterol since the association rate was very slow and/or the affinity for cholesterol was 100 fold reduced. These observations, thus clearly denote that Leishmania parasites during their intracellular life cycle by modulating membrane cholesterol, exert significant effect on the IFNcR function with subsequent modification of IFNc mediated intracellular parasite killing.
These results thus represent the first evidence of interaction of cholesterol and cholesterol binding motif in IFNcR1. It corroborates all the previous reports [14][15][16] of cholesterol dependent IFNcR1 functional regulation. One would expect cholesterolliposome to show leishmanicidal effect in association with IFNc. Indeed this study clearly showed such effect coupled with NO generation in LD-MØs upon treatment with the above combination. The effect was cholesterol specific because the leishmanicidal effect was not observed with analogue liposome or with DPCCliposome. This observation lends credence to our previous reports showing strong therapeutic role of liposomal-cholesterol in infected hamster model [23].
Survival strategy of the parasite LD might as well include specific degradation and/or negative regulation of expression of protein molecules of the infected cells such as STAT1 [68] and IFNcR1 [69] -two important components of the IFNc signaling pathway. The lack of reproducibility of these results in our system is perhaps attributable to the difference in cell system or parasite strains used for infection.
The mechanism by which LD modulates membrane cholesterol is yet unknown. Given the fact that dynamic equilibrium between the pools of free membrane cholesterol and cytoplasmic droplets of cholesterol esters is very tightly controlled [70], diminished membrane cholesterol in LD-MØs, may result from overall decrease in total cellular cholesterol. It may be recalled that in VL patients a progressive decline in serum cholesterol as a function of splenic parasite load [26] is indicative of reduced cellular cholesterol since serum cholesterol and total cellular cholesterol are in dynamic equilibrium [71]. Interestingly, in L. major infected macrophages decrease in cellular cholesterol is causally related to reduced mRNA level of HMG-CoA reductase, the rate limiting enzyme of cholesterol biosynthetic pathway [72]. Alternatively, different reports [73,74] also suggest that LD may exploit host cholesterol for its entry and survival, interfere with cholesterol trafficking or may exchange host cholesterol for ergosterol, with biophysical characteristics grossly different than cholesterol. Thus, these contentions warrant further studies and are currently under our investigation.
We would like to emphasize here that we have specifically dealt with the earliest and critical most step of IFNc signaling occurring within seconds (180 secs) of receptor ligation and demonstrate that LD infection can efficiently impose a block at this step by specific disruption of the receptor oligomerization by lowering the macrophage membrane cholesterol level. It should be mentioned here that IFNc signaling amplitude depends on the sustained durability of the activation phenomenon and is very tightly regulated by multiple negative regulatory mechanisms which include dephosphorylation of the activated signaling intermediates by inducible activity of phosphoprotein phosphatases [75], several minutes after ligand-receptor interaction. Seminal study by Blanchette et al. show that this basic physiological phenomenon of signal duration restrain is efficiently utilized by the opportunistic parasite Leishmania spp., to attenuate the IFNc signaling amplitude [76] which in turn affect the overall IFNc bioactivity in LD infected macrophages which likely contribute in part to the inhibition of complete restoration of the IFNc signaling pattern in cholesterol treated LD infected macrophages ( Figure 11A, A', B, B', C, C', E).
Of note, constitutive SHP-1 preassociation with JAK2 in the steady-state of normal cells as reported by various groups [77,78] denotes an active repression of JAK2 autophosphorylation. Experimental verification ( Figure S6) denotes that in cholesterol depleted systems with segregated IFNc receptor subunits as in established LD infected macrophages, the SHP-1 species remained associated with the JAK2 proteins, preventing its autophosphorylation upon ligand stimulation, which was in sharp contrast to uninfected macrophages where the SHP-1 is released from JAK2 to induce its autophosphorylation within minutes (3 minutes) after ligand stimulation. Interestingly, cholesterol supplementation in LD infected cells reinstated the JAK2 activation profile and release of SHP-1 from JAK2 immediately upon IFNc stimulation (our unpublished observation, data not shown).
There is a report that Leishmania parasites deliver effector proteins into host cells by exosome-based secretion and can modulate host-cell function [79]. Thus it is not unlikely that uninfected MØs in the infected splenic environment may trap parasite derived antigen (s) and display similar defects in IFNc signaling like infected MØs. It is worthy of mention that the late established phase of VL is marked with increased production of IL-10 [80]. IL-10, which has a prominent IFNc desensitizing role acting directly at the level of STAT1 deactivation [81], might account for the chronic infection in the presence of high circulating IFNc in VL, providing an additional negative regulatory step on IFNc signaling.
The central point that evolved from this study is that intracellular LD quench membrane cholesterol; this in turn affects nitric oxide synthase inhibitor blocks IFNc induced leishmanicidal effect in CH-Lipo treated infected MØs. RAW 264.7 cells were infected with LD at m.o.i of 10, for 6 hours. MØs were then washed and either treated with CH-Lipo or left untreated as infected control. Subsequently, at indicated timepoints postinfection, MØs were stimulated with or without 100 U/ml of rIFNc for 24 hours in presence or absence of 5 mM L-NMMA. Cell free supernatant was harvested for NO measurement. Data of Nitrite concentration (mM) is representative of three independent experiments. The amastigote numbers of the parallel sets treated similarly was demonstrated in D'. E, Enhanced IFNc-bioactivity in CH-Lipo replenished MØs as determined by reporter assay. RAW 264.7 cells were transfected with pCIS-CK negative control vector or IFNc driven pGAS reporter plasmid. 12 hrs after transfection, cells were either left untreated or treated with mBCD (5 mM, 30 min, 37uC) or infected with LD. Treated cells were then replenished or not with CH-Lipo. Subsequently, mBCD treated cells with or without CH-Lipo reloading were stimulated with or without 100U/ml of rIFNc eight hours before final harvesting for luciferase activity determination in cell lysates. While the infected cells with or without CH-Lipo treatment were stimulated or not with 100 U/ml of rIFNc at indicated timepoints post infection. Eight hours later, luciferase activities in cell lysates were measured as described in Materials and Methods. The data represents the relative luciferase activity normalized to per microgram of protein content. Data is representative of three independent experiments. doi:10.1371/journal.ppat.1002229.g011 the lateral mobility of IFNcR2 into the raft, IFNcR1-R2 multimerization and subsequent downstream signaling in response to IFNc. This defect could be corrected by exogenous delivery of liposomal cholesterol leading to intracellular parasite clearance. Based on this study, it can be reasonably speculated that cholesterol may also influence the conformation of IFNcR1. Thus in VL, cholesterol-liposomal formulation of IFNc treatment may emerge as a promising therapeutic strategy both in drug-sensitive and -resistant cases as it is directed specifically towards host than the parasites.

Parasite maintenance
LD strain AG83 (MHOM/IN/1983/AG83), originally obtained from an Indian kala-azar patient [82], was maintained in Golden hamsters as described previously [83]. Promastigotes obtained after transforming amastigotes from the spleen of infected animals were maintained in culture in Medium 199 supplemented with 10% FCS at 22uC. The culture was replenished with fresh medium every 72 h.

Cell lines and preparation of peritoneal exudates cells
Murine MØ-like tumor cell, RAW 264.7 was obtained from American Type Culture Collection. Cells were maintained in complete medium at 37uC with 5% CO 2 in a humidified atmosphere.
BALB/c mice were obtained from the animal facility of the institute and were housed under conventional conditions, with food and water ad libitum. BALB/c mice were injected with 2 ml of 4% starch i.p. The peritoneal cells (PEC) were harvested 2 days after the starch injection and were rested for 24 h before any treatments on these cells. In the meanwhile, the nonadherent cells were washed out to generate pure adherent populations.

Infection of PECs and RAW 264.7
PEC and RAW 264.7 cells were allowed to adhere for 4 hr at 37uC in presence of a 5% CO 2 atmosphere, after which the nonadherent cells were removed by gentle washing with serumfree medium. The adherent MØs, after overnight incubation in complete medium, were challenged with LD promastigotes at a MØ to parasite ratio of 1:10 and incubated further for 6 hr at 37uC. After initial attachment of 6 hr, excess parasites were washed with serum-free medium, and this was considered as the initial most (0 hr) time point of infection and the infection was allowed to progress for the indicated time periods thereafter, followed by rIFNc treatment with the suboptimal 10 U/ml, optimal 100 U/ml and supraoptimal dose of 1000 U/ml. The cultures were terminated after indicated timepoints and treatments, by washing and immediate fixing with methanol, followed by staining with Giemsa stain and counted under a light microscope (Leica). The parasite load was expressed as the number of amastigotes per MØ and % infected MØs as described earlier [22]. In some experiments freshly prepared lesion derived LD amastigotes were used for infection at a multiplicity of infection (m.o.i) of 10. The dose of 100 U/ml of rIFNc was chosen for optimum cell-activation as no additional benefit was obtained for the higher dose tested.

Measurement of NO
Nitric oxide (NO) generation was monitored by using the Griess reaction as described previously [84], and the results were expressed in mM of nitrite accumulation. In some experiments, MØs were cultured in the presence of N G -monomethyl-L-arginine (L-NMMA) a competitive inhibitor of inducible NO synthase, added to a final concentration of 5 mM [85].

Flow cytometry
Membrane expression of IFNcR1 and IFNcR2 were investigated on resting RAW 264.7 cells with incubation of 1610 6 cells with anti-mouse IFNcR1 mAb or anti-mouse IFNcR2 mAb for 30 min at 4uC followed by two washes with cold PBS supplemented with 1% FCS and 0.05% sodium azide (PBSazide). For secondary labeling, cells were stained with biotinconjugated anti-hamster antibody for 30 min at 4uC and were washed twice with cold PBS-azide and were then labeled with PEconjugated Streptavidin. After the final wash with cold PBS-azide, cells were fixed with cold 2% paraformaldehyde in PBS. Membrane protein expression was analyzed with a FACSAria II flow cytometer (Becton Dickinson, Mountain View, CA).

Radioiodinations and binding assays
Radioiodination of rIFNc was performed as previously described [86]. rIFNc (5 mg at 0.5 mg/ml) was radioiodinated with 5 ml (500 mCi) of Na 125 I (17 mCi/mg, BRIT, India) in the presence of 25 ml of 0.15 M potassium buffer, pH 7.4, and 10 ml chloramine-T (5 mg/ml) for 2 minutes. After neutralization of the reaction with 10 ml each of sodium metabisulfite (10 mg/ml), potassium iodide (70 mg/ml), and BSA (20 mg/ml), the reaction mixture was chromatographed over a 10-ml Sephadex G-10 column equilibrated with a Tris/NaCl/BSA buffer (10 mM Tris-HCl, pH 7.4, 0.15 M NaCl, and 0.33 mg/ml BSA). Fractions of 500 to 600 ml were collected, and the fraction containing the greatest activity was used for receptor binding studies. The specific activity of 125 I-IFNc was 110 mCi/mg of protein. Anti-mouse IFNcR1 (10 mg) was radioiodinated by following the method essentially described in [87], with minor modifications. Briefly, 50 ml anti-mouse IFNcR1 was incubated with 25 ml (500 mCi) Na 125 I (17 mCi/mg, BRIT, India) in the presence of 125 ml of 0.15 M potassium buffer, pH 7.4, and 50 ml chloramine-T (5 mg/ ml) for 4 min. The neutralization of the reaction was done as above and readily sieved on a 10-ml Sephadex G-10 column. Fractions of 400 ml were collected, and the fraction containing the greatest activity was used for receptor binding studies. The specific activity of 125 I -labeled anti-IFNcR1 was in the range of 100-105 mCi/mg of protein.
Saturation binding assays were performed as previously described with brief modifications [10]. To enumerate receptor numbers, 125 I-IFNc was added to final concentrations of 5-800 pM to 2610 7 cells per ml of RPMI medium containing 2% fetal bovine serum in duplicates. After 180 min of incubation at 4uC, cells were washed thoroughly with ice cold PBS. The radioactivities associated with separated cell pellet and the supernatant were quantified in a scintillation counter (Packard) to determine free and bound 125 I-IFNc respectively. To determine nonspecific binding, the amount of 125 I-IFNc bound to the cells in the presence of a 200-fold excess of unlabeled ligand was determined. Specific binding was calculated by subtracting nonspecific binding from the total radioactivity bound. After correcting for nonspecific binding, data of 125 I-IFNc saturation binding experiments were fitted to a one-site binding hyperbola and analyzed with non-linear regression analysis to determine K D and Bmax values using GraphPad Prism 5.0 (GraphPad, San Diego, USA) to obtain parameters of equilibrium binding, where Bmax denotes the maximal density of receptor sites and K D denotes the radioligand equilibrium dissociation constant. In addition, 125 I-IFNc saturation binding data were displayed using Scatchard plots where the X-axis is specific binding (Bound) and the Y-axis is specific binding divided by free radioligand concentration (Bound/Free) [88].
Ligand dependent and independent internalization assay 125 I-labeled murine IFNc and 125 I-labeled anti-mouse IFNcR1 were used respectively to determine the ligand induced and constitutive bulk flow endocytosis mediated IFNcR internalization and were performed as previously described [11,89] with minor modifications. For internalization studies, 2610 6 cells were replenished with ice-cold RPMI containing 2% fetal calf serum. Parallel wells received either 125 I-labeled IFNc or 125 I-labeled antimouse IFNcR1 and were incubated for 180 minutes in 4uC, after which unbound radioactivity was removed with brief wash with ice cold PBS and incubations were continued in 37uC medium without ligand to allow internalization to occur. At intervals thereafter, cells were immediately added with ice cold PBS and transferred to ice, to stop internalization. Membrane-associated ligand or antibody was removed from the cells with low pH acid wash buffer (0.2 M acetic acid, 0.5 M NaC1, pH 2.5, [90]). Internalized ligand or antibody was determined by solubilization of the acid-treated cells in 1 M NaOH. Radioactivity was measured with a scintillation counter (Packard). The amount of radioactivity remaining with cells that had been kept at 4uC all along before acid treatment was taken to represent 'nonspecific' acid-resistant radioactivity and was subtracted from the values obtained from the 37uC incubations to provide an estimate of specific ligand internalization. The data were fitted to linear regression analysis program in GraphPad Prism 5.0 (GraphPad, San Diego, USA) to obtain the rate of internalization by following the methods of Liu et al. [91].

Cell lysis and western blotting from whole cell lysates
Cell lysis and western blotting were carried as described previously [92] with fewer modifications. In general, uninfected or LD infected RAW 264.7 cells were treated with or without 100 U/ml of rIFNc for 3 min, 37uC. After washing twice with icecold PBS, cells were lysed in 1X RIPA Buffer from Cell signaling technology, Danvers, MA (20 mM Tris-HCl pH 7.5, 150 mM NaCl, 1 mM Na 2 EDTA, 1 mM EGTA, 1% NP-40, 1% sodium deoxycholate, 2.5 mM sodium pyrophosphate, 1 mM betaglycerophosphate, 1 mM Na 3 VO 4 , 1 mg/ml leupeptin, with recommended addition of 1 mM PMSF immediately before use).
In some experiments, cells were treated with 10 ng/ml of rIL-10 for 15 min, 37uC [93]. Protein concentration was determined using the Micro BCA protein assay kit (Pierce Chemical Co. Rockland, IL). Proteins (20 mg/lane) were separated by SDS-PAGE on a 10% gel under reducing conditions and electrotransferred to nitrocellulose membranes (HYBOND ECL, Amersham, Piscataway, NJ) in a transfer buffer consisting of 20 mM Tris-HCl, 150 mM glycine, and 20% methanol. Membranes were incubated at 4uC overnight in blocking buffer (Western Blocker Solution, Sigma). Primary and secondary Abs were diluted as recommended by the manufacturer in blocking buffer and incubated with the membranes for 1 hr, at room temperature with five washes in between, with wash buffer (20 mM Tris, 500 mM NaCl, pH 7.4). Detection of HRP-conjugated Abs was performed using SuperSignal West Pico Chemiluminescent Substrate (Pierce, Rockland, IL).

Transfection and transient expression of luciferase reporter gene constructs
Interferon gamma driven luciferase reporter activity was essentially done as in ref. [94]. RAW 264.7 cells were transfected with the pGAS cis-reporter plasmids or the pCIS-CK negative control plasmid by electroporation [95] by Genepulser X cell (Bio-Rad, Hercules, CA). Cells from three electroporations were pooled to eliminate differences between individual transfections and the mixture was then equally divided among the wells of a 24-well cluster dish (Corning CoStar Corp., Cambridge, MA). They were next allowed to adhere to the substratum for 4 hr before the medium was changed. After 24 hr (which was needed to reduce the background levels of luminescence), the medium was again changed and were either infected with LD or left uninfected. After the initial attachment of the parasites with the cells for 6 hr, the cells were washed and the infection was allowed to progress for the indicated time periods. Thereafter, cultures were incubated for 8 hr with medium alone or medium that contained 100 U/ml IFNc. The cells were harvested in 300 ml of passive lysis buffer (Promega Biotech, Madison, WI), and 50 ml aliquots of the clarified extracts were used to assay luciferase activity using Luciferase assay kits/reagents from Promega Biotech according to the manufacturer's protocols. Luciferase activity was normalized to the levels of the protein content. The average is reported along with the standard deviation of the mean. The pGAS cis-reporter plasmid and the pCIS-CK negative control plasmid were purchased from Stratagene, La Jolla, CA.

Liposome preparation
Unilamellar liposomes were prepared from PC with either cholesterol or an analogue of cholesterol (4-cholesten-3-one) at a molar ratio of 1:1.5 as described in ref. 22,23. For liposome preparation with DPPC, we followed the methods of Giraud et al. [96]. DPPC (13.8 mg) was dissolved in a chloroform-methanol (9:1 v/v) mixture. The solvent was removed by evaporation under vacuum, followed by overnight drying of the resulting film under vacuum to remove the residual solvents. The dry film of DPPC was rehydrated in PBS, pH 7.4, then alternately heated in a water bath at 60uC and mixed via a vortex mixer at room temperature for 5 min to allow full hydration of the phospholipid and finally sonicated using an ultrasound 2 mm tip in microprobe sonicator. According to the conventional procedure, repetitive 30-sec cycles of sonication/ice cooling were performed. Samples were briefly centrifuged to remove titanium particles.

FRET analysis and DyLight labeling of primary antibodies
For fluorescence resonance experiments, anti-mouse IFNcR1 and anti-mouse IFNcR2 were conjugated to DyLight488 or DyLight594 (Pierce; N-hydroxy succinimide (NHS) ester-activated fluorescent dyes) respectively, according to the manufacturer's protocol. Naïve or infected MØs were stimulated with/without IFNc for 3 min, 37uC to induce FRET signals. The stimulation was terminated by immediate transfer of cells to ice and washing with ice-cold PBS. The cells were then fixed with 2% paraformaldehyde/PBS at 4uC. After washing the paraformaldehyde and quenching the excess formaldehyde for 15 min with 0.1% NaBH 4 in PBS, staining was followed with excess antibodies to respective IFNcR subunits at 4uC, for 1 hr. Following 3 washes with cold buffer, cells were resuspended in PBS. Spectroscopic analysis of cells in a quartz cuvette was done with to 485 nm excitation and recording the emission spectra from 500 nm to 700 nm with emission and excitation slit of 2.5 nm. FRET efficiency (E) was obtained from the measured fluorescence intensity of the donor (Dylight488-IFNcR1) at its maximal emission wavelength (519 nm) in the presence and absence of the acceptor (Dylight594-IFNcR2) using the equation with E (%) = (1 -I da /I d ), where I da and I d are the fluorescence intensities in the presence and absence of acceptor, respectively [97,98].

Co-Immunoprecipitation and immunoblotting
For co-immunoprecipitation, experimental cells, treated as indicated in the figure legends, were aspirated off the medium and washed with ice cold PBS, lysed in Cell Lysis Buffer (1X), Cell signaling technology (20 mM Tris pH 7.5, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, 2.5 mM sodium pyrophosphate, 1 mM b-glycerophosphate, 1 mM Na 3 VO 4 , 1 mg/ml Leupeptin with 1 mM PMSF added immediately prior to use). After 10 min on ice, lysates were collected by scraping, sonicated for 15 s (0.3 s bursts), centrifuged at 10, 000 r.p.m. for 10 min at 4uC and the supernatant was recovered. Total protein concentrations were determined using the Micro BCA protein assay kit (Pierce Chemical Co.) with BSA as standard. Lysates were pre-cleared using Protein A/G plus Agarose beads (Pierce Chemical Co.). 200 ml of precleared supernatants were incubated overnight at 4uC with specific primary Ab to IFNcR1 or control unrelated whole rabbit IgG, and the immune complexes were collected on 20 ml of 50% bead slurry Protein A/G Plus-Agarose (prewashed and preblocked with 2% BSA for 2 hour, at 4uC in 25 mM Tris, 150 mM NaCl; pH 7.2) for 2 h, at 4uC, with gentle shaking. Precipitates were washed 5 times with 500 ml of 1X Cell Lysis Buffer, kept on ice during washes and microcentrifuged for 60 seconds at 4uC at 2500 x g, each time. The pellet was resuspended with 20 ml of 2X Laemmli sample buffer [(Bio-Rad, Hercules, CA), 62.5 mM Tris-HCl, pH 6.8, 25% glycerol, 2% SDS, 0.01% Bromophenol Blue, 5% b-mercaptoethanol], vortexed for 30 seconds and boiled for 5 min before resolving in SDS-PAGE gels and probed with antibodies as detailed in the figure legends and detected as previously stated.

Analysis of membrane fractions on floatation gradients
Total crude cell membranes were isolated as described in ref. [99]. Cells were homogenized in 1 ml of buffer [10 mM Tris-HCl (pH 7.4), 1 mM EDTA, 200 mM sucrose] and protease inhibitor mix (Roche Diagnostics, Mannheim, Germany)]. The nuclei and cellular debris were removed by centrifugation at 900 x g for 10 min at 4uC. The resulting supernatant was centrifuged at 110,000 x g for 75 min at 4uC to obtain the crude membrane pellet. Purification method of detergent-soluble and -insoluble fractions was adapted from ref. [100], with very few modifications. The crude membrane pellet was solubilized in 1 ml of Lysis buffer (Caveolae/ Rafts Isolation Kit, Sigma) containing 1% Triton X-100 and was agitated end to end for 30 min at 4uC. The sample was mixed with OptiPrep and to a final OptiPrep concentration of 35%. Then the density gradient was made of 5 layers of OptiPrep with 35,30,25,20, and 0%. The volume of each layer was 2 ml except for 1 ml of 0% OptiPrep. Samples were centrifuged at 200,000 x g for 4 h at 4uC. After centrifugation, nine fractions of 1-ml from the top to bottom of the ultracentrifuge tube were collected. As described in manufacturer's instruction, caveolin-1 positive fractions were found in fractions 2-4 (25-35% OptiPrep) counting from the top (data not shown), which were also enriched in GM1 content. Ganglioside GM1 was visualized with an HRPconjugated cholera toxin B subunit (1:2000, Sigma) in dot blots with 2 ml sample from each gradient fraction. In some experiments, fractions 2-4 or fractions 7-9 were combined, and are referred to as Triton-insoluble glycolipid enriched membranes (rafts) or Triton-soluble (non-raft) fractions, respectively. Equal volumes (50 ml) of each fraction were analyzed directly by western blotting.

Confocal microscopy, image processing and quantitation of signal overlap on fixed cells
Experimental cells were grown on glass-coverslips and treated as indicated in figure legends and after final treatment, washed with cold wash buffer. Cells, preblocked in ice cold blocking buffer (1X PBS with 2 % FBS) for 30 minutes in 4uC were stained with anti-mouse IFNcR1 or anti-mouse IFNcR2 in blocking buffer diluted to a predetermined optimal concentration and incubated at 4uC in the dark. The reaction was stopped by adding ice cold wash buffer and washed three times. For staining with secondary Ab, biotin-conjugated Fc specific anti-IgG was added and stained as described above and followed by streptavidin-PE. The cells were counterstained with FITC-CTxB following the protocol described above. The cells were then fixed with 1% paraformaldehyde, mounted with 90% glycerol on a glass slide, and observed under a laser scanning microscope [Leica (Mannheim, Germany) TCS SP2 AOBS system] by using a 63 oil-immersion objective. Double-stained images were obtained by sequential scanning for each channel to eliminate the ''cross-talk'' of chromophores and to ensure reliable quantification of colocalization [101]. Images were acquired and processed for colocalization analysis in TIFF format. Colocalization was estimated using Pearson's correlation coefficient [102] analyzed by ImageJ software (rsbweb. nih.gov/ij/).

Immunoprecipitation and immunoblotting from purified raft and non-raft fractions
Total IFNcR1 or engaged IFNcR1 were immunoprecipitated from pooled 'rafts' fraction (2, 3 and 4) and the 'non-raft' fraction (7, 8 and 9) as described previously [103,104] with minor modifications of both. Cells were stimulated for 3 minutes at 37uC with biotinylated rIFN-c [Recombinant murine IFN-c was conjugated to EZ linked N-hydroxysulfosuccinimide-biotin (Pierce, Rockford, IL) according to the manufacturer's instruction. Cells were incubated with 0.5 ng/ml biotin-conjugated IFNc, which is the calculated approximate equivalence to 100 U/ml of bioactive unconjugated IFNc in our system and gave unaltered stimulation index in terms of STAT1 phosphorylation and pGAS-Luc activity in supportive experiments as with the unconjugated rIFNc (data not shown)]. Stimulation was terminated by immediate washing with ice cold PBS and harvesting cells in lysis buffers. To immunoprecipitate total amount of IFNcR1 from isolated raft and non-raft fractions, 0.5 ml of the pooled raft and non-raft fractions were added to 0.5 ml of cell lysis buffer, were pre-cleared, and the signaling complex was immunoprecipitated overnight at 4uC with anti-IFNcR1 (10 mg/ml) and the recovery of the pulled immunecomplex was done exactly as described before. Alternatively, engaged IFNcR1 were immunoprecipitated from pooled raft and non-raft fractions with streptavidin-agarose beads. Pooled 'rafts' fraction (2, 3 and 4) and the 'non-raft' fraction (7, 8 and 9) pre-cleared of non-specific binding proteins by incubation with 50 ml of agarose beads (prewashed and suspended to 50% in 1X cell lysis buffer). Pre-cleared lysates were incubated with streptavidin-agarose beads (50 ml of even suspension of 50% bead slurry ) for 2 hr at 4uC with gentle end over end shaking, washed using our immunoprecipitation protocol and procured from the beads and analyzed as before. Control experiments confirmed that no proteins bound to streptavidin-agarose beads if cells were left untreated with biotinylated IFNc (data not shown). Where indicated, the IFNc signaling complex pulled down by immobilized streptavidin-agarose was dissociated in 50 ml of PBS containing 1% SDS by boiling for 10 min and diluted 20-fold with lysis buffer before it was subjected to a second immunoprecipitation using 10 mg/ml of an anti-pTyr Ab. The washing, procurement and immunoblot analysis were done exactly as mentioned before.

Methyl b-cyclodextrin (mBCD) treatment of cells
For some experiments, cells were treated with mBCD (Sigma-Aldrich, St. Louis, MO) to disrupt lipid rafts. In the initial set of experiments, cells were treated with different concentration of mBCD ranging from 2 to 20 mM in the absence of serum for 30 min at 37uC, after which cell viability was assessed by trypan blue exclusion and the cell membrane cholesterol content and the cell-membrane fluidity were measured (data not shown). Only conditions that allowed for cell viability comparable to mock treatment were used for these studies. It was observed that RAW 264.7 cells maintained the ability to exclude vital dyes such as trypan blue after treatment with 5 mM mBCD though treatment with the same essentially caused an acute depletion of cholesterol content from the cell membrane almost equivalent to the conditions of prevailing in MØ with 12 hr LD infection.

Measurement of fluorescence anisotropy (FA)
The membrane fluorescence and lipid fluidity of cells were measured following the method described by Shinitzky and Inbar [105]. Briefly, the fluorescent probe DPH was dissolved in tetrahydrofuran at 2 mM concentration. To 10 ml of rapidly stirring PBS (pH 7.2), 2 mM DPH solution was added. For labeling, 10 6 cells were mixed with an equal volume of DPH in PBS (C f 1 mM) and incubated for 2 hr at 37uC. Thereafter the cells were washed thrice and resuspended in PBS. The DPH probe bound to the membrane of the cell was excited at 365 nm and the intensity of emission was recorded at 430 nm in a spectrofluorometer. The FA value was calculated using the equation: FA = [(I I -I H )/ (I I + 2I H )], where I I and I H are the fluorescent intensities oriented, respectively, parallel and perpendicular to the direction of polarization of the exciting light [106,22].

Measurement of cholesterol content
Sample for cholesterol estimation was essentially prepared following the methods described in ref [107]. The crude plasma membrane fraction was isolated as described above from the experimental cells and suspended in a minimum volume of PBS. An aliquot was used for protein measurement. The rest of the pellet was extracted with 2:1 methanol/chloroform, followed by 0.5 ml of chloroform and 0.5 ml of water. The methanol/ chloroform (lipid phase) layer was dried under vacuum. The dry lipid was suspended in 200 ml of 1X Reaction buffer provided in Amplex Red Cholesterol Assay Kit (Molecular Probes' Invitrogen) and membrane cholesterol quantitation was performed exactly according to manufacturer's instructions.

Interaction of purified LPG with MØs
A method modified from ref. [108] was employed. Lyophilized lipophosphoglycan (LPG), purified from LD (a kind gift from Prof SJ Turco, University of Kentucky, Lexington, USA), was solubilized in double-distilled water to 1 mM stock and frozen in 220uC as aliquots. Stock solution was diluted in PBS and added to the MØ cultures to a final concentration of 10 mg/ml and was incubated at 37uC in 5% CO 2 and high humidity for indicated time duration, in each experiment. The cells were washed three times in cell-culture medium with 5% FCS at room temperature to remove unbound LPG followed by washing three times in PBS at 4uC to terminate the experiment.

Peptide synthesis and purification
Sequence analysis of IFNcR1 protein was done with ScanProsite software (EXPASY TOOLS) to find out consensus motif for cholesterol binding and the peptides were designed accordingly. Peptide designed and synthesized from wild type IFNcR1 protein, reproducing cholesterol binding motif (CRAC motif, residues 2269 to 2280) was designated as Wt IFNcR1 peptide (269-VILVFAYWYTKK-). Point mutant IFNcR1 peptide (269-VILVFAAWATKK-) reproduces the tyrosine to alanine substituted form of Wt IFNcR1 peptide. The peptides were synthesized on Rink Amide MBHA resin using standard solid phase Fmoc chemistry [109] with a capping step with 5% acetic anhydride and 5% lutidine in DMF after each coupling using PS3 peptide synthesizer (Protein Technologies Inc, USA). Fmoc-amino acids were activated with HBTU in presence of HOBt and DIEA. Peptides were cleaved from the resin and sidechain protecting groups were removed by incubating with 94% TFA, 2.5% EDT, 1.5% thioanisole, 1.5% water, 0.5% TIS for 3 hrs at room temperature after which peptides were precipitated with ice-cold diethyl ether. Peptides were then purified by HPLC (Waters, USA) on a reverse phase mbondapak C-18 column using 0-80% acetonitrile in 0.01% TFA and molecular weight determined by MALDI-TOF/TOF analyzer (Applied Biosystem, USA).

Binding studies using SPR
Binding experiments were done by surface plasmon resonance (SPR) as described [110], on a BIAcore 3000 system (Biacore AB, Piscataway, NJ). L1 sensor chip (Biacore) with hydrophobically modified dextran layer was used to for SPR experiment to study binding of peptide to immobilized liposomes. Freshly prepared liposome were immobilized on L1 sensor chip up to response unit of 2000-2500 at flow rate of 5 ml/min in PBS (pH = 7.2). To remove any multilamellar structures from the lipid surface, we injected NaOH (50 ml, 10 mM) at a flow rate of 50 ml /min, which resulted in a stable baseline corresponding to the lipid monolayer linked to the chip surface. The negative control BSA was injected (25 ml, 0.1 mg/ml in PBS) to confirm complete coverage of the nonspecific binding sites. After complete association required dissociation time was given to wash loosely bound liposomes and to achieve stable base line. Kinetic experiments were performed with serial dilution of the peptides. Concentration of the peptides used were 0.02 nM, 0.2 nM, 2 nM, 20 nM, 200 nM in PBS (pH = 7.2). Peptides binding measured by observing the change in SPR angle as 90 ml of peptide analyte flowed over the immobilized liposome for 3 min at flow rate 30 ml/min. L1 sensor chip was regenerated using 200 ml of 20 mM CHAPS at flow rate of 50 ml/ min. Different dilutions of protein samples in buffer were injected at a flow rate of 30 ml/min. The response was monitored as a function of time (sensorgram) at 25uC. SPR experiments were conducted in duplicate at each concentration to confirm the bindings were repeatable. Multiconcentration data were globally fit, and residuals were calculated and used to assess the goodness of fit with BIAevaluation 4.1 software (Biacore, Piscataway, NJ).

Statistical variation and presentation
Each experiment was performed thrice, and representative data from one set of these experiments are presented; the interassay variation was within 10%. Two-tailed Student's t test was performed to ascertain the significance of the differences between the means of the control and the experimental groups. We considered values of P,0.05 to be statistically significant. P value ,0.001 were considered extremely significant (***), P value ranging between 0.001 to 0.01 were very significant (**), P value 0.01 to 0.05 as significant (*) and P value .0.05 were not significant (ns) (Student's t test). Error bars indicate mean 6 SD. Data was analyzed by using Prism 5.0 (GraphPad, San Diego, CA). Figure S1 Loss of IFNc response in MØs from very early time point of LD infection. A, Monolayers of freshly plated and adhered mPEC (A and B) and RAW 264.7 (D and E) were either left untreated or exposed to promastigotes of Leishmania donovani at a multiplicity of infection (m.o.i) 10:1. After initial attachment of 6 hr, excess parasites were washed and the infection was allowed to progress for the indicated time periods, followed by rIFNc treatment with suboptimal 10 U/ml, optimal 100 U/ml and supraoptimal 1000 U/ml doses for 24 h. The intracellular parasite number was expressed as amastigotes per MØ and percentage infected cells in the culture. C and F, Culture supernatants from MØs treated exactly as in A-E were harvested and assayed for Nitrite concentration (mM) by Griess reaction as described in Materials and Methods. The results shown are from one of three identical experiments which yielded similar results, and represent the mean 6 SD of triplicate determinations for each experimental group. (TIF) Figure S2 Unaltered endogenous cell surface IFNcR expression, ligand binding and ligand induced receptor internalization kinetics in LD infected MØs. A, Flow cytometric analysis of surface expression of IFNcR1 and IFNcR2 in RAW 264.7 cell that were left uninfected or infected with LD at a m.o.i of 10 for indicated time periods. Numbers in the plot indicate the mean fluorescence intensity. B, Non-linear regression analysis of the binding of IFNc to the IFNcR protein in normal and LD infected MØs. Uninfected and infected MØs were treated with increasing concentrations of trace labeled 125I-IFNc for 3 hr at 4uC in the presence or absence of 200 fold excess of cold IFNc. Scatchard plots (insets) and KD and Bmax values were obtained using the Graph Pad Prism program by analyzing data for specific IFNc binding. Data represents the mean of duplicate measurements for each concentration of IFNc. C, Rate of ligand induced IFNcR internalization in uninfected and infected RAW 264.7. Cells left uninfected or infected for 4 hr or 12 hr with LD were treated with 125 I-IFNc (100 U/ml) for 3 hr at 4uC followed by incubation at 37uC for various time intervals. At the end of incubation, cells were rinsed twice with ice cold PBS and then treated with a low pH buffer (pH 2.5) for 5 minutes at 4uC that removes surface-bound ligand. The acid-inaccessible internalized ligand is presented as a fraction of total cell-associated radioactivity prior to cell transfer to 37uC. For control, we tested the behavior of steady-state internalization of IFNcR by labeling them with 125 I-IFNcR1 antibody for each experimental set tested. The constitutive and inducible internalization rate was shown in inset and calculated by linear regression analysis in Graph Pad Prism. (TIF) Figure S3 Suppression of IFNc signaling initiation in LD infected MØs. A, The MØs were infected with/without LD promastigotes for indicated time periods as described earlier, followed by treatment with 100 U/ml of rIFNc for 3 min. Expressions of phosphorylated JAK2 and whole JAK2 were measured in whole cell lysates via western blot using the same membrane. Densitometric readings represent the ratio of intensity of phosphorylated JAK2 (pJAK2) to JAK2 protein expression per unit area and are represented as arbitrary units (au). Equal loading was verified by immunoblotting of cytoplasmic actin. B, RAW 264.7 cells were transfected with either pGAS-Luc or the pCIS-CK negative control plasmid. After 12 hr of transfection, cells were either left uninfected or infected with LD and at indicated timepoints postinfection cells were treated with 100 U/ml rIFNc for 8 hrs, before final harvestation. Cell lysates were prepared, and luciferase assay was done as described under Materials and Methods. Medium control denotes the transfected cells without any subsequent treatments. The values shown in all panels are the average of triplicate measurements in a single experiment and are expressed as relative light units (RLU) normalized to total protein content in each sample. C, Assessment of the unrelated JAK-STAT pathway by IL-10 sensitization of LD infected MØs. The MØs were left uninfected or infected with LD promastigotes for indicated time periods as described earlier, followed by treatment with 10 ng/ml of rIL-10 for 15 minutes. Expressions of phosphorylated STAT3 and STAT3 were measured in whole cell lysates via western blot using the same membrane. Equal loading was verified by immunoblotting of actin. (TIF) Figure S4 Scanprosite analysis of mouse IFNcR1 protein sequence (Accession: EDL03452.1 GI: 148671505) denoting the CRAC motifs. Two CRAC motifs in IFNcR1 protein sequence located within amino acid position 2269 to 2280 and 2393 to 2399 are highlighted in yellow. (TIF) Figure S5 Transmembrane helix prediction result of mouse IFNcR1 protein sequence (Accession: EDL03452.1 GI: 148671505). The predicted a-helical portion of IFNcR1 protein sequence is denoted as letter 'H' in red. (TIF) Figure S6 SHP-1 forms a constitutive complex with JAK2 in macrophages and the release of SHP-1 is IFNc inducible. The MØs were infected with/without LD promastigotes for 12 hrs as described earlier, followed by treatment with 100 U/ml of rIFNc for 3 minutes. Cells were then treated with/ without cell permeable crosslinker of DSS, as per manufacture's protocol (Pierce). After cell lysis, protein extracts was immunoprecipitated with anti-JAK2. To verify the identity of JAK2 and SHP-1, immunoprecipitates were separated on polyacrylamide-SDS gel, and immunoblotting was performed with anti-JAK2 antibody or anti-SHP-1 antibody. (TIF)

Supporting Information
Table S1 Identification of the cholesterol recognition / interacting amino acid consensus pattern. Alignment of amino acid sequences showing presence of CRAC motif conforming to -L/V-(X) 1-5 -Y-(X) 1-5 -R/K-in IFNcR1 proteins in different species. (DOC)