Mechanism of Down-regulation of RNA Polymerase III-transcribed Non-coding RNA Genes in Macrophages by Leishmania*

The parasitic protozoan Leishmania invades mammalian macrophages to establish infection. We reported previously that Leishmania manipulates the expression of several non-coding RNA genes (e.g. Alu RNA, B1 RNA, and signal recognition particle RNA) in macrophages to favor the establishment of their infection in the phagolysosomes of these cells (Ueda, Y., and Chaudhuri, G. (2000) J. Biol. Chem. 275, 19428–19432; Misra, S., Tripathi, M. K., and Chaudhuri, G. (2005) J. Biol. Chem. 280, 29364–29373). We report here the mechanism of this down-regulation. We found that the non-coding RNA (ncRNA) genes that are repressed by Leishmania infection in macrophages contain a “B-box” in their promoters and thus require the polymerase III transcription factor TFIIIC for their expression. We also found that Leishmania promastigotes through their surface protease (leishmanolysin or gp63) activate the thrombin receptor PAR1 in the macrophages. This activation of PAR1 raised the cytosolic concentration of Ca2+ into the micromolar range, thereby activating the Ca2+-dependent protease μ-calpain. μ-Calpain then degraded TFIIIC110 to inhibit the expression of the selected ncRNA genes. Avirulent stocks of Leishmania not expressing surface gp63 failed to down-regulate ncRNAs in the exposed macrophages. Inhibition of PAR1 or calpain 1 in macrophages made them resistant to Leishmania infection. These data suggest that macrophage PAR1 and calpain 1 are potential drug targets against leishmaniasis.

leishmanial infection in the mononuclear phagocytes, mainly the macrophages, is determined by breaching the innate immune barrier of these cells. The research described here is based on the notion that Leishmania plays an active role in the violation of innate immunities of the macrophages to establish infection (4 -6).
Non-coding RNAs (ncRNAs) 2 perform their biological functions as RNA molecules. Small ncRNAs, which include mini-RNAs (e.g. Alu RNA, B2 RNA, H1 RNA, 7SL RNA, and vault RNA) and micro-RNAs, play many critical biological roles to collectively define the transcriptome and proteome of a cell (7)(8)(9)(10)(11)(12). We found that infection of macrophages with the parasitic protozoan Leishmania represses several specific ncRNA genes in macrophages to convert these cells into permissible hosts for the establishment of infection. Very interestingly, we found that the genes of the ncRNAs that are down-regulated by Leishmania in the infected macrophages are transcribed by RNA polymerase III and are dependent on the transcription factor TFIIIC, particularly its subunit TFIIIC110 (13,14).
Our results discussed here suggest that Leishmania activates the thrombin receptor, protease-activated receptor 1 (PAR1) (15)(16)(17) on the surface of the macrophages through its surface protease gp63 (18 -21). Gp63 is the major surface glycoprotein of Leishmania promastigotes (1, 18 -23). This is a zinc-dependent metalloprotease with a wide range of substrates, including casein, gelatin, albumin, hemoglobin, and fibrinogen (18 -21). Gp63 is thought to play important roles in parasite survival and modulation of the host response (18 -21). This metalloprotease is the major ectoprotease expressed by all pathogenic Leishmania and serves as a ligand for binding macrophage complement and fibronectin receptors (22,23). PAR1 belongs to a family of G-protein-coupled proteaseactivated receptors that were discovered as the receptor for the coagulation protease thrombin (EC 3.4.21.5) (15-17, 24 -27). Four PARs have now been identified. PAR1, PAR3, and PAR4 can all be activated by thrombin. PAR2 is activated by trypsin and by trypsin-like proteases but not by thrombin. Mammalian macrophages only express PAR1, PAR2, and PAR3; PAR4 was not detected in these cells (28). PAR1 is activated when thrombin binds to and cleaves its amino-terminal exodomain to unmask a new receptor amino terminus. This new amino terminus then serves as a tethered peptide ligand, binding intramolecularly to the body of the receptor to affect transmembrane signaling (24 -27). The irreversibility of the proteolytic activation mechanism of PAR1 stands in contrast to the reversible ligand binding that activates classical G-protein-coupled receptors and compels special mechanisms for desensitization and resensitization. In endothelial cells and fibroblasts, activated PAR1 rapidly internalizes and then sorts to lysosomes rather than recycling to the plasma membrane as do classical G-protein-coupled receptors (24 -27). This trafficking behavior is critical for termination of thrombin signaling. An intracellular pool of thrombin receptors refreshes the cell surface with new receptors, thereby maintaining thrombin responsiveness (15-17, 24 -27).
We show here that the activation of PAR1 induces the release of Ca 2ϩ in the cytoplasm of the macrophages, which in turn activates the calcium-dependent protease -calpain (29,30). Calpains are calcium ion-dependent proteases, and thus their activities are strictly regulated by the levels of free Ca 2ϩ ion in the cytosol of the cells (29,30). Ca 2ϩ ions are usually trapped inside membranous bodies like endoplasmic reticulum and are released from the endoplasmic reticulum after being induced by second messengers like inositol trisphosphate (31,32). Two major calpains, m-calpain and -calpain, are distinguished by their requirements of Ca 2ϩ concentrations for functional activation (29,30). Both consist of an 80-kDa large subunit (from the genes Capn1 and Capn2, respectively), each of which forms a heterodimer with a common 30-kDa small subunit (Capn4). These calpains (Capn1/Capn4 and Capn2/Capn4) differ in their calcium requirement for activation (ϳ50 M for -calpain and ϳ500 M for m-calpain) (29,30,33).
We report here that one of the subunits of human TFIIIC, TFIIIC110 (13, 34 -36), is the target of activated -calpain in the macrophages. We provide evidence that TFIIIC110 is responsible for the binding to the B-box-containing promoters in these cells. We show that -calpain degrades TFIIIC110, thus inhibiting the transcription of any ncRNA genes that require TFIIIC110, including 7SL RNA, in the Leishmaniainfected macrophages. We also provide evidence that PAR1 and calpain inhibitors are potential deterrents against the establishment of leishmanial infection of macrophages.

EXPERIMENTAL PROCEDURES
Materials-Thrombin, thrombin receptor-activating peptide 6 (SFFLRN; also known as TRAP6), azocasein, and calpain inhibitors calpeptin and calpastatin were purchased from Sigma. Primary antibodies for PAR1 (S-19) and CAPN1 and FITC-labeled secondary antibody were bought from Santa Cruz Biotechnology (Santa Cruz, CA). Antibodies against the human TFIIIC110 subunits were initially obtained from Prof. Robert White (Beatson Institute for Cancer Research, Glasgow, UK) and later on were procured from Sigma and Santa Cruz Biotechnology. The ORFs of mouse and human RNA polymerase III subunit D and TFIIIC110 cloned into pCMV6-Entry vector were procured from Origene (Rockville, MD). These plasmids when introduced inside the macrophages produced carboxyl-terminal Myc-and FLAG-tagged proteins upon selection of the cells with G418 (500 g/ml). Myc and FLAG antibodies were also procured from Origene. PAR1 antagonist SCH79797 was procured from Tocris Bioscience (Ellisville, MO).
Plasmids, Mutations, and Transfection-The development of plasmids pSUPER-7SL and pSUPER-7SLmut that contain three G to A mutations have been described before (4). The B-box sequence of these plasmids was mutated from GGCT-GGAGGAT to GACAGGAGGAT using reagents and protocols of the QuikChange site-directed mutagenesis kit from Stratagene (Agilent Technologies, Santa Clara, CA). The macrophages were transfected using Lipofectamine 2000 reagents and protocols from Invitrogen. Nucleotide sequences of primers used to amplify some of the polymerase III-transcribed genes and their flanks from human and mouse macrophages are given in supplemental Table 1S.
Leishmania and Macrophages-The promastigotes of Leishmania amazonensis (LV78, MPRO/BR/72/M1845, zymodeme MON-41) and Leishmania major (MHOM/IL/80/ Friedlin, zymodeme MON-103) were used in this study. The promastigotes were grown at 25°C in M199 medium with 10% heat-inactivated fetal bovine serum (4). Avirulent promastigotes were from a cloned laboratory stock of Leishmania that have been maintained in axenic culture medium for Ͼ10 years and have lost infectivity in cultured macrophages or in mice (4). The mouse macrophage cell line J774G8 and the human monocytic leukemia cell line THP1 were used in this study. These cells were grown in RPMI 1640 medium with 20% heat-inactivated (56°C, 30 min) fetal bovine serum at 37°C (4). THP1 cells were differentiated into macrophages with phorbol 12-myristate 13-acetate (10 ng/ml) for 24 h (4). The macrophages were incubated with the parasite cells at a macrophage/parasite ratio of 1:10 at 37°C for 4 h for exposure experiments (4). Primary macrophages were isolated from the bone marrow and peritoneal exudates of BALB/c mice following standard protocols (37,38). Macrophages were also derived from human blood monocytes (SeraCare Life Sciences, Gaithersburg, MD). Human blood neutrophils were also obtained from SeraCare Life Sciences. Infectivity of Leishmania promastigotes to the macrophages was determined as described before (4).
Activation of PAR1 with Thrombin and SFFLRN-Cultured macrophages were incubated at 37°C in growth medium without serum for 5 h before their treatment with thrombin (10 nM) or SFFLRN (100 M) for 0 -4 h before the assay or extraction of RNA and protein.
Quantitative Reverse Transcription (RT)-PCR-Total RNA was extracted from macrophages using TRIzol reagent (Invitrogen). For isolation of RNA from unexposed macrophages, an equivalent number of parasite cells were added after addition of TRIzol to the macrophages to account for contribution of Leishmania RNA. Extracted RNA (5 g) was treated with RQ1 DNase (Promega, Madison, WI) according to the supplier-provided protocols. The DNase-treated RNA (1 g) was reverse transcribed using iScript reverse transcriptase (Bio-Rad) in a total volume of 20 l. Experiments were also done in parallel with RNA samples without the treatment with reverse transcriptase to validate further that the amplification products were not coming from genomic DNA contaminants. One microliter of cDNA was used in real time PCR with a final volume of 25 l containing 10 pmol of each primer (see supplemental Table 2S) and 12.5 l of 2ϫ SYBR Green PCR Master Mix (Bio-Rad). All reactions were performed in triplicate in the iCycler iQ instrument (Bio-Rad). 18 S rRNA was used as an internal control. The mean value of the replicates for each sample was calculated and expressed as cycle threshold (C T ). The amount of gene expression was then calculated as the difference (⌬C T ) between the C T value of the sample for the target gene and the mean C T value of that sample for the endogenous control. Relative expression was calculated as the difference (⌬⌬C T ) between the ⌬C T values of the test sample and the control sample for each target gene. The relative quantification value was expressed and shown as 2 Ϫ⌬⌬CT (4,39,40).
Chromatin Immunoprecipitation (ChIP) Assay-A ChIP assay was performed as described (41). Briefly, cells were harvested, washed in phosphate-buffered saline, and resuspended to 1-2 ϫ 10 6 cells/ml in PBS. The cells were cross-linked with 1% formaldehyde, quenched with 125 mM glycine, washed twice with PBS, and lysed for 5 min by a 6-fold dilution in lysis buffer (50 mM Tris-HCl, pH 8, 10 mM EDTA, 1% SDS, protease inhibitor mixture, 1 mM PMSF). Lysate aliquots of 200 l were sonicated on ice into chromatin fragments with an average length of 500 bp. Paramagnetic beads (Dynabeads, Invitrogen) were washed twice in RIPA buffer (10 mM Tris-HCl, pH 7.5, 1 mM EDTA, 0.5 mM EGTA, 1% Triton X-100, 0.1% SDS, 0.1% sodium deoxycholate, 140 mM NaCl) and resuspended in 1 volume of RIPA buffer. Diluted chromatin (2 A 260 units in RIPA buffer) was added to antibody-bead complexes and immunoprecipitated overnight at 4°C. Precipitated immune complexes were washed five times, and crosslinks were reversed by incubating samples with 150 l of elution buffer (20 mM Tris-HCl, pH 7.5, 5 mM EDTA, 50 mM NaCl) containing 1% SDS and 50 g/ml proteinase K for 2 h at 68°C. DNA was recovered by capturing beads, the ChIP material was reincubated in 150 l of elution buffer/SDS/proteinase K for 5 min, and both supernatants were pooled. DNA was isolated using a QIAquick spin kit (Qiagen). Real time PCR was performed using primers described in supplemental Table 2S. Real time PCR data for antibody-bound fractions were compared with a 1:100 dilution of input DNA.
Western Blot Analysis-Whole cell extracts were prepared, protein concentration was determined using Bradford protein assays (Bio-Rad), and 25-50 g of protein was analyzed by 4 -12% SDS-PAGE and transferred to nitrocellulose membranes, which were then blocked with 5% nonfat milk in TBST buffer (100 mM Tris, pH 8.0, 1.5 M NaCl, 0.1% Tween 20) for 1 h. The membranes were incubated overnight with primary antibody in blocking buffer, then washed, and incubated with a horseradish peroxidase-conjugated secondary antibody for 1 h (39, 40). Immunoreac-tivity was detected by using the ECL detection system (Amersham Biosciences) (39,40).
Evaluation of Calcium Ion Levels-Standard fluorometric protocols were used (42). Briefly, macrophages in fully confluent 6-well plates were incubated with the calcium probe FURA2/AM (5 M) for 30 min at room temperature in RPMI 1640 medium. Cells were washed twice in Hanks' balanced salt solution with 0.25% BSA (w/v) and resuspended in Hanks' balanced salt solution-BSA containing 1 mM CaCl 2 and 1 mM MgCl 2 . Cell suspensions were incubated at 37°C for 5 min. These macrophages were exposed to Leishmania (1:10 in 150 l of RPMI 1640 medium with heat-inactivated fetal bovine serum at 37°C). Fluorescence was measured at 1 min, 5 min, 10 min, 15 min, 30 min, and 1 h at 510 nm in a Bio-Rad Versa Fluor spectrofluorometer.
Calpain Activity Assay-Calpain activity was measured using the reagent and protocol of the assay kit from Abcam (Cambridge, MA). Macrophages cultured in 6-well plates were exposed to Leishmania (1:10) for 4 h or treated with thrombin or SFFLRN post-serum starvation for 4 h. Control cells were not given any treatment or exposed to avirulent L. amazonensis promastigotes. Cells were harvested and resuspended in 100 l of extraction buffer. The samples were incubated on ice for 20 min and mixed several times by tapping during incubation. The protein concentration was determined using Bradford protein assays (Bio-Rad). Cell lysates (100 g of protein) were diluted with 85 l of extraction buffer and 10 l of 10ϫ reaction buffer, and 5 l of calpain substrate was added to each tube and incubated at 37°C for 1 h in the dark. Fluorescence was read using a 400-nm excitation filter and a 505-nm emission filter. Calpain activity is expressed as the difference in the rate of substrate hydrolysis in the presence or absence of calpain inhibitor. Chemical inhibition of calpains was accomplished with a mixture of calpastatin (1 M) and calpeptin (5 M).
Alkaline Phosphatase (AP)-PAR Cleavage Assay-We stably transfected macrophages with a PAR1-AP-pcDNA3 construct (17) for the surface expression of AP-PAR using Lipofectamine 2000 (Invitrogen). Fully confluent AP-PARexpressing J774G8 cells in a 6-well plate were exposed to Leishmania for 4 h. Another set of cells was serum-starved for 5 h before treating them with 10 nM thrombin or 100 M SFFLRN for 4 h. After 4 h, the medium was collected and briefly spun to pellet any floating cells. Surface alkaline phosphatase activity was measured in the medium using a Great EscAPe Fluorescent SEAP Assay kit (Clontech) (17).
Evaluation of Surface gp63 Activity with Azocasein as Substrate-Leishmania promastigotes were harvested, washed, diluted to 2 ϫ 10 8 /ml in Hanks' balanced salts solution containing 20 mg/ml azocasein at pH 7.4, and incubated at 25°C with constant agitation. After 0, 5, 10, 20, 30, 40, and 60 min, 0.5-ml aliquots were removed and mixed with an equal volume of 5% trichloroacetic acid on ice. The absorbance of each sample was determined at 366 nm after centrifugation. The rate of azocasein degradation per 10 8 promastigotes was determined. One unit of promastigote surface protease activity produces 1 mg/min acid-soluble azocasein peptides at pH 7.4 (44). The protease activity of gp63 was blocked using the pseudophosphorylated triaspartate peptide 3DMRPed (KKKKKFDFKKPFKLDGLDFKRNRK) (2 M for 30 min) as described (45).
siRNA Treatment-PAR1 siRNAs and corresponding control siRNAs were designed using the Block-IT RNAi designer software (Invitrogen) and purchased from Invitrogen. The nucleotide sequences of these siRNAs and respective control RNAs used in this study are given in supplemental Table 3S. Transfection of these siRNAs into the macrophages was done by lipofection using Lipofectamine 2000 following the supplier-provided protocols. Briefly, cells were transfected at ϳ50% confluence using 100 pmol of siRNA in 6-well plates, and whole-cell lysates were prepared 48 h after transfection. We isolated RNA from these cells using TRIzol reagents (Invitrogen). Knockdown of the expression of the target PAR1 mRNA by the experimental siRNA and the corresponding protein was verified by real time RT-PCR and immunoblot analysis, respectively (39,40).
Immunofluorescence Microscopy-Immunofluorescence staining and confocal analyses were performed as described (43). In brief, cells were cultured on glass coverslips in 24-well plates for 24 h, rinsed with ice-cold 1ϫ PBS, and fixed with 3.7% formaldehyde for 30 min. Thereafter, cells were washed three times with ice-cold PBS. The slides were blocked in PBS containing 5% normal goat serum and then were incubated with primary antibodies overnight at 4°C in the blocking buffer. Slides were then washed five times with PBS followed by incubation for 45 min with the FITC-conjugated secondary antibody. The slides were again washed three times with PBS, embedded in glycerol/PBS-based mounting medium, and examined using a fluorescence microscope (Nikon TE2000-E). Confocal images were obtained with a Nikon TE2000-UC1 laser scanning microscope (43).
Statistical Analysis-Each experiment was repeated at least three times. Results were expressed as means Ϯ S.E. Statistical analysis were performed using GraphPad Prism software (GraphPad Software, Inc., La Jolla, CA). p values were calculated using the two-sided Student's t test (paired or unpaired as appropriate) and analysis of variance test for significance. p values Ͻ0.05 and Ͻ0.01 were considered as significant.

Infection of Macrophages with Virulent Leishmania Promastigotes Decreases Levels of Selected ncRNAs in
Macrophages-An important observation we have made previously is the down-regulation of the 7SL RNA in macrophages that are infected (4 h) by Leishmania promastigotes and amastigotes (4). 7SL RNA is the RNA component of the signal recognition particle (46,47). We found that 7SL RNA was down-regulated in mouse as well as human macrophages during exposure to Leishmania (4). The down-regulation of 7SL RNA was at the transcriptional level as was revealed by nuclear run-on analysis (4). Avirulent stocks of Leishmania promastigotes failed to knock down the 7SL RNA levels in the macrophages (4). We now sought to understand the mechanism of how Leishmania induces the transcriptional inhibition of 7SL RNA in macrophages. Because 7SL RNA is transcribed by RNA polymerase III, we first explored whether any other RNA polymerase III-transcribed RNAs are affected in the macrophages by Leishmania. We found that Leishmania infection of macrophages not only down-regulates 7SL RNA gene expression but also decreases the levels of selected other ncRNA genes that are transcribed by RNA polymerase III (Fig. 1, A and  B). RNA polymerase III transcribes ncRNA genes from four different types of promoters (supplemental Fig. 1S) (see Refs. 34 and 35). Because 7SL RNA is transcribed by RNA polymerase III FIGURE 1. Inhibition of expression of several ncRNAs in macrophages exposed to Leishmania. A, real time RT-PCR data showing inhibition of the expressions of selected ncRNAs in L. amazonensis-exposed J774G8 cells. 18 S rRNA was used as an internal control, and results are expressed relative to 18 S rRNA. Results are mean Ϯ S.E. (error bars) (n ϭ 6). * indicates a statistically significant difference between the control and the experimental sets (p Ͻ 0.001). M, untreated J774G8 cells; MϩL, Leishmania-exposed J774G8 cells. B, real time RT-PCR data showing inhibition of the expressions of selected ncRNAs in L. major-exposed phorbol 12-myristate 13-acetate-differentiated THP1 (dTHP1) cells. 18 S rRNA was used as an internal control, and results are expressed relative to 18 S rRNA. Results are mean Ϯ S.E. (error bars) (n ϭ 6). * indicates a statistically significant difference between the control and the experimental sets (p Ͻ 0.001). C-F show the decrease in the levels of 7SL RNA, vault RNA (vRNA), and B2/Alu RNA in mouse peritoneal exudates cells (mPEC), mouse bone marrow-derived cells (mBMDC), and human peripheral blood monocyte-derived macrophages (hMDM) exposed to L. amazonensis promastigotes (C), L. amazonensis amastigotes (D), Leishmania bodies isolated from the lysate of L. major-infected human neutrophils (E), and L. donovani promastigotes (F), respectively. RNA levels in the unexposed and Leishmania-exposed macrophages were evaluated by real time RT-PCR using 18 S rRNA as an internal control. Results are mean Ϯ S.E. (error bars) (n ϭ 6). -Fold decrease indicates the ratio of the values from the unexposed and exposed macrophages. FEBRUARY 25, 2011 • VOLUME 286 • NUMBER 8 from a type 4 promoter (48), we initially asked whether the inhibition is specific for 7SL RNA or whether the transcription of the vault RNA gene, which is transcribed from a type 4 promoter (48,49), is also affected in macrophages by Leishmania infection. Our data suggest that both 7SL and vault RNA levels are significantly affected by Leishmania infection of macrophages (Fig. 1, A and B). 7SL RNA and vault RNA genes share only the internal promoter elements, which are the A-box and the B-box sequences (48,49). We thus concluded that Leishmania must be regulating the functions mediated by those elements. These elements are also shared by type 2 promoters of RNA polymerase III-transcribed genes (13,36). The type 1 promoter has the Abox in common (13,36).

Leishmania-induced ncRNA Manipulations in Macrophages
Thus, our next question was whether Leishmania induces the decrease of other ncRNAs expressed from the RNA polymerase III promoters that have A-box and/or B-box elements. Our RNA analysis data with selected type 2 and type 4 promoter-containing ncRNA genes show that the expression levels of these ncRNAs are inhibited by Leishmania infection of the macrophages (Fig. 1, A and B, and supplemental Fig.  1S). The type 2 and type 4 promoters tested require both internal A-box and B-box elements. The 5 S rRNA gene is expressed from a type 1 promoter that lacks a B-box element, and the expression level of this ncRNA was not affected in Leishmania-exposed macrophages (Fig. 1, A and B, and supplemental Fig. 1S). Type 3 promoters do not require any internal elements such as A-box and B-box. H1 RNA gene is expressed from a type 3 promoter, and its level in macrophages was not affected by Leishmania (Fig. 1, A and B, and supplemental Fig. 1S). We thus postulated that perhaps the factors binding to the B-box elements are the target for Leishmania action. We verified our ncRNA analysis data described in Fig. 1A with other Leishmania species like L. major and Leishmania donovani as well as with other mouse (MH-S, P388D1, and J774A1) and human (derived from U937 and THP1) macrophage cells (see Fig. 1). We also validated our observations with primary mouse peritoneal and bone marrow-derived macrophages as well as human blood monocytederived macrophages (Fig. 1, C-F). We also used BALB/c mouse tail base lesion-derived Leishmania amastigotes as well as human neutrophil-derived Leishmania bodies to achieve similar down-regulation of ncRNAs ( Fig. 1, C-F).
ncRNA Genes That Are Knocked Down in Macrophages during Leishmania Exposure Require TFIIIC110 for Their Transcription-The B-box-containing promoters of types 2 and 4 require the transcription factor TFIIIC (34,35). Recruitment of TFIIIC to the type 1 promoter does not involve the B-box element (34,35). Of the five subunits of TFIIIC, the TFIIIC110 subunit has been implicated as binding to the Bbox sequences of type 2 and type 4 RNA polymerase III promoters (13,14). We hypothesized that Leishmania inhibits the expression of ncRNA genes in the macrophages by preventing the recruitment of TFIIIC110 to the B-boxes of these promoters. To verify whether RNA polymerase III and TFIIIC110 are indeed recruited at the Leishmania-regulated ncRNA gene promoters in macrophages, we performed ChIP analysis as described above. We expressed either FLAGtagged RNA polymerase III subunit D or FLAG-tagged TFIIIC110 in the macrophages. Quantitative ChIP analysis with anti-FLAG monoclonal antibody revealed that the promoters of the Leishmania-regulated ncRNA genes indeed recruit TFIIIC110 and RNA polymerase III to their promoters, and the recruitment of these proteins is decreased during Leishmania infection of the macrophages (Fig. 2, A and B). We also verified the expected Leishmania-mediated decrease in binding of TFIIIC110 as well as RNA polymerase III to representative tRNA gene promoters in Leishmania-infected macrophages. We found similar inhibition of binding of TFIIIC110 and RNA polymerase III at the tRNA Leu and tRNA Arg gene promoters by quantitative ChIP analysis (supplemental Fig. 2S). Our study also shows that the type 1 and type 3 promoters of RNA polymerase III we tested do not bind to TFIIIC110 (Fig. 2B).
To evaluate further whether TFIIIC110 indeed binds to the B-box sequence at the 7SL RNA gene promoter in vivo, we developed a construct (Fig. 2C) with mutations at the B-box sequence and performed ChIP analysis for TFIIIC110 binding to this construct. We used a 7SL RNA gene construct with three G to A mutations for this purpose. These mutations helped us to design primers that will distinctly bind to the transcript from this construct but not that from the endogenous copies of the 7SL RNA genes. Although both TFIIIC110 and RNA polymerase III bind to the wild-type promoter DNA (Fig. 2D), neither of these factors bound the promoter containing the mutant B-box (Fig. 2D). We postulate that the recruitment of TFIIIC multimeric complex is initiated with the help of TFIIIC110 at the B-box-binding domain. Sequential binding of TFIIIB and RNA polymerase III then follows. For type 4 promoters, the external proximal and distal element binding factors may play additional roles in the recruitment of RNA polymerase III at these promoters (48). We also propose that TFIIIC110 is not required for the binding of TFIIIC at the type 1 promoters (e.g. 5 S rRNA) that lack B-box element (supplemental Fig. 1S).
Level of TFIIIC110 Is Decreased in Leishmania-infected Macrophages-Because TFIIIC110 is involved in the transcription of Leishmania-regulated ncRNA genes, we evaluated whether TFIIIC110 levels are decreased in Leishmaniainfected macrophages. We found that although the levels of TFIIIC110 mRNA remained unaltered the level of TFIIIC110 protein is decreased in Leishmania-exposed macrophages (Fig. 2, E and F). The decrease in the TFIIIC110 protein levels in the Leishmania-exposed macrophages were dose-and time-dependent (supplemental Fig. 3S). Neither the levels of mRNA (not shown) nor those of the protein of TFIIIC220, another subunit of TFIIIC, are decreased in Leishmania-infected macrophages (Fig. 2, E and F).
Calpain Is Activated for Leishmania-induced Degradation of TFIIIC110 in Macrophages-Because proteolytic degradation of TFIIIC110 in the macrophages could be a mechanism of a Leishmania-induced decrease in the TFIIIC110 levels, we sought to identify the protease involved in this process. We found that TFIIIC110 has a conserved PES sequence motif at the amino-terminal regions (supplemental Fig. 4S). PES sequences are often cleaved by calcium-dependent proteases of the calpain family (50). Moreover, RNA polymerase III-asso-ciated factors have been shown in other systems to be the targets of calpain proteases (51). We found that the levels of mRNA and protein of the 80-kDa regulatory subunit of -calpain, CAPN1, were increased due to Leishmania-macrophage interaction (Fig. 3A, B, and C). We also found a significant increase in calpain activity after exposure of the macrophages to Leishmania (Fig. 3D).
Leishmania exposure also significantly increased free calcium ion concentration (from 0.1-0.2 to 1.6 -3.0 M) in the cytoplasm of the macrophages (Fig. 4A). Because the increase in Ca 2ϩ levels in the Leishmania-exposed macrophages never reached mM levels, we propose that it is the -calpain that is activated during this process. Chemical inhibition of calpains with a mixture of calpastatin (1 M) and calpeptin (5 M) abrogated the actions of Leishmania on macrophage ncRNA gene expression (Fig. 4B). The calpain inhibitor mixture also inhibited Leishmania-induced cleavage of TFIIIC110 (Fig. 4,  C and D). These data strongly suggest the involvement of MϩL, Leishmania-exposed J774G8 cells. Real time PCR was used to measure the amount of DNA corresponding to each promoter fragment present in the IP samples. The relative binding level represents the amount of DNA in the FLAG IP minus the amount of DNA in the control IP. Control IPs were done using normal mouse IgG. Each IP was normalized to the amount of input DNA. Similar results were obtained with L. major promastigotes and with THP1-derived macrophages. Results are mean Ϯ S.E. (error bars) (n ϭ 6). * indicates a statistically significant difference between the control and the experimental sets (p Ͻ 0.001). C, nucleotide sequence of the 7SL RNA gene construct. The external (Activating Transcription Factor (ATF)/Cyclic AMP-Responsive Element (CRE) binding site and the TATA box) and the internal (A-box and B-box) elements are shown (48). The mutated nucleotides in the B-box are in positions ϩ51 (G to A) and ϩ53 (T to A). The three G residues that were mutated to A residues are also indicated. The transcription termination sequences are indicated. D, evaluation of the binding of RNA polymerase III and TFIIIC110 to the wild-type and the B-box-mutated 7SL RNA gene promoters. The binding of these factors to the exogenously introduced promoter construct was evaluated by quantitative ChIP analysis as described under "Experimental Procedures." Results shown are mean Ϯ S.E. (error bars) (n ϭ 6). The inhibition of binding of RNA polymerase III and TFIIIC110 to the B-box-mutated 7SL RNA gene promoter was highly significant (p Ͻ 0.0001). E, effect of L. amazonensis exposure of J774G8 macrophages on the TFIIIC220 (i) and TFIIIC110 (ii) protein levels. Western blot analysis was performed as described under "Experimental Procedures." ␤-Actin was used as a loading control. F, densitometric analysis of immunoblot data as in E from four independent experiments. Bands were developed using IR Dye 800-conjugated secondary antibody (LI-COR Biosciences) and visualized using the LI-COR Odyssey Infrared Imaging System. Quantitation and analysis of bands were performed using Odyssey software. ␤-Actin was used as a normalization control, and the results are expressed relative to the level of ␤-actin. Results are mean Ϯ S.E. (error bars) (n ϭ 4). * indicates that the changes observed in the levels of TFIIIC110 were statistically significant (p Ͻ 0.001). FEBRUARY 25, 2011 • VOLUME 286 • NUMBER 8 -calpain in the mediation of Leishmania-induced knockdown of 7SL RNA and other RNA polymerase III transcripts in macrophages. We could not evaluate the effect of siRNA-mediated knockdown of CAPN1 mRNA (which codes for the regulatory component of -calpain) on this process because the effect of knockdown on the mRNA and protein levels of CAPN1 was nullified by the exposure of the macrophages to Leishmania. The mechanism underlying this effect remains to be determined.

Leishmania-induced ncRNA Manipulations in Macrophages
Ability of Leishmania Promastigotes to Knock Down 7SL RNA in Macrophages Is Proportional to Surface Protease (gp63) Levels of Parasite Cells-Increased levels of intracellular Ca 2ϩ may be executed through the extracellular activation of any number of G-protein-coupled seven-transmembrane domain receptors (52, 53). PAR1, a G-protein-coupled recep-tor, is present on the macrophage cell surface in abundance (28). Because virulence of Leishmania promastigotes is often associated with the level of its surface protease gp63 (23, 54 -56), a potential activator of PAR1, we explored whether Leishmania gp63 is involved in 7SL RNA down-regulation in Leishmania-exposed macrophages. We reported previously that virulent promastigotes but not attenuated avirulent parasites FIGURE 3. Activation of calpain in Leishmania-exposed J774G8 macrophages. A, increase in the level of CAPN1 mRNA in L. amazonensis-and L. major-exposed J774G8 macrophages as revealed by real time RT-PCR analysis. Macrophages were treated for 4 h with zymosan (10 g/ml), avirulent L. amazonensis promastigotes (1:10 macrophage/Leishmania; Lama Avir), virulent strain of L. amazonensis (Lama Vir), or L. major (Lmaj Vir) (1:10 macrophage/Leishmania). ␤-Actin mRNA was used as a normalization control, and the results are expressed relative to the level of ␤-actin mRNA. Results are mean Ϯ S.E. (error bars) (n ϭ 6). * indicates a statistically significant difference between the control and the experimental sets (p Ͻ 0.001). B, increase in the level of CAPN1 protein in L. amazonensis-and L. major-exposed J774G8 cells as revealed by immunoblot analysis. M, untreated J774G8 cells; MϩL, Leishmania-exposed J774G8 cells. C, densitometric analysis of immunoblot data as in B from four independent experiments. Bands were developed using IR Dye 800conjugated secondary antibody (LI-COR Biosciences) and visualized using the LI-COR Odyssey Infrared Imaging System. Quantitation and analysis of bands were performed using Odyssey software. ␤-Actin was used as a normalization control, and the results are expressed relative to the level of ␤-actin. Results are mean Ϯ S.E. (error bars) (n ϭ 4). * indicates that the changes observed in the levels of CAPN1 were statistically significant (p Ͻ 0.001). D, increase in the calpain activity in L. amazonensis-and L. major-exposed J774G8 macrophages. Calpain activity is expressed as the difference in the rate of substrate hydrolysis in the presence or absence of calpain inhibitor as described under "Experimental Procedures." Macrophages were treated for 4 h with zymosan (10 g/ml), avirulent L. amazonensis promastigotes (1:10 macrophage/Leishmania; Lama Avir), virulent strain of L. amazonensis (Lama Vir), or L. major (Lmaj Vir) (1:10 macrophage/Leishmania). Results are mean Ϯ S.E. (error bars) (n ϭ 6). * indicates a statistically significant difference between the control and the experimental sets (p Ͻ 0.001). MϩL, Leishmania-exposed J774G8 cells; MϩLϩCI, Leishmania-exposed J774G8 cells treated with calpain inhibitors; vRNA, vault RNA. C, effect of calpain inhibitor mixture on L. amazonensis-induced degradation of TFIIIC110 in J774G8 cells. The level of TFIIIC110 was evaluated by Western blot analysis. ␤-Actin was used as a loading control. Leish, Leishmania; Calp-Inh, calpain inhibitors. D, densitometric analysis of immunoblot data as in C from four independent experiments. Bands were developed using IR Dye 800-conjugated secondary antibody (LI-COR Biosciences) and visualized using the LI-COR Odyssey Infrared Imaging System. Quantitation and analysis of bands were performed using Odyssey software. ␤-Actin was used as a normalization control, and the results are expressed relative to the level of ␤-actin. Results are mean Ϯ S.E. (error bars) (n ϭ 4). * indicates that the changes observed in the levels of TFIIIC110 were statistically significant (p Ͻ 0.001).
are able to inhibit 7SL RNA expression in macrophages (4). We therefore evaluated whether the level of gp63 on the surface of the virulent parasite cells is correlated with their ability to knock down 7SL RNA levels in the macrophages. We selected two cloned laboratory stocks of both the virulent and avirulent L. amazonensis promastigotes (4) for this study. Although the virulent strains are capable of establishing infection in cultured J774G8 cells and in BALB/c mice, the avirulent strains fail to do so. We evaluated the levels of surface protease gp63 protein (Fig. 5, A and B) as well as the surface protease activity (Fig. 5C) of these cells to verify that their virulence is correlated with gp63 content and activity. We found that the levels of gp63 are well correlated with the ability of the promastigotes of L. amazonensis to knock down 7SL RNA in J774G8 cells (Fig. 5D). Inhibition of the gp63 protease activity of these cells with a peptide inhibitor of gp63 (45) abrogated the ability of the parasite cells to knock down 7SL RNA in the macrophages (Fig. 5D). Macrophages treated with the inhibitor alone did not show any significant effect on 7SL RNA level in these cells. Interestingly, exposure of macrophages to L. amazonensis having little or no gp63 protein or protease activity on its cell surface increased the level of 7SL RNA in the macrophages (Fig. 5D). Similar increases in the levels of B2 RNA and vault RNA were also observed in the J774G8 cells exposed to L. amazonensis promastigotes lacking gp63 protease on their surface (supplemental Fig. 5S). The levels of ncRNAs like B2 and Alu RNA are known to be increased in stressed mammalian cells (57)(58)(59)(60)(61). Whether the phagocytosis of Leishmania promastigotes is imposing stress on the macrophages that increases the levels of these ncRNAs and whether gp63 protease activity is counteracting this mechanism are currently under study in our laboratory (see "Discussion").
Exposure of macrophages to virulent but not to the avirulent Leishmania promastigotes also led to a transient increase in the cytosolic Ca 2ϩ concentration in the cells (Fig. 5E) and increased the calpain activity ( Fig. 5F) as well as the degradation of TFIIIC110 in the treated macrophages (Fig. 5, G and  H). Inhibition of gp63 protease activity of Leishmania promastigotes abrogated their ability to elevate the Ca 2ϩ levels as well as calpain activity in the infected macrophages (Fig. 5, E  and F). Treatment of the virulent promastigotes with the gp63 inhibitor also nullified the effect of virulent promastigotes on macrophage TFIIIC110 levels (supplemental Fig. 6S). These data suggested a direct involvement of the surface protease of Leishmania promastigotes in the parasite-induced knockdown of 7SL RNA in the macrophages.
Activation of Thrombin Receptor PAR1 Also Decreases 7SL RNA Levels in Macrophages-To identify potential substrates for the gp63 protease on the surface of macrophages, we explored the cell surface receptors on the macrophages that are known to be activated by proteases. The thrombin receptor PAR1 is one such receptor (15)(16)(17). PAR1 is not only activated by thrombin but has also been shown to be activated by other proteases like the metalloprotease MMP1 (62). We found through immunofluorescence microscopy that PAR1 is abundantly expressed on human and mouse macrophages but is only poorly expressed on undifferentiated monocytes (Fig.   6A). We asked whether activation of PAR1 may play a role in reducing the level of 7SL RNA in the macrophages. We challenged J774G8 cells (3 ϫ 10 6 cells/ml) with purified bovine thrombin (Sigma; 10 nM) or the thrombin agonist peptide SFFLRN (Sigma; 100 M) for 4 h and measured 7SL RNA levels in these cells by real time RT-PCR. When compared with the control untreated cells, the level of 7SL RNA was 4 -5-fold less in the treated (for 4 h) cells (Fig. 6B). Activation of PAR1 also led to a transient increase in the cytosolic Ca 2ϩ concentration in the treated cells (Fig. 6C) and to an increase in the calpain activity (Fig. 6D) as well as the degradation of TFIIIC110 in the treated macrophages (Fig. 6, E and F). These data suggested that PAR1 activation in macrophages may mimic the action of virulent Leishmania promastigotes on these cells.
Leishmania Surface Protease gp63 Activates PAR1 on Macrophages-To directly evaluate the ability of Leishmania surface protease gp63 to cleave PAR1 on macrophages, we used a PAR1 cleavage reporter (17). This substrate, AP-PAR1, is a chimeric protein in which the carboxyl terminus of secreted alkaline phosphatase is joined to the amino-terminal exodomain of PAR1. The junction between alkaline phosphatase and PAR1 is amino-terminal to the thrombin cleavage site of PAR1 (17). Thus, alkaline phosphatase is tethered to the cell membrane by the amino-terminal exodomain of PAR1 and is released when this domain is cleaved by thrombin or other proteases. Soluble alkaline phosphatase activity in cell-conditioned medium provides an easily measured index of PAR1 cleavage (17). We stably transfected J774G8 cells with this construct and exposed the transfected cells to L. amazonensis to measure PAR1 cleavage. Bovine thrombin (Sigma) was used as a positive control for the PAR1 cleavage studies. Leishmania promastigotes with abundant levels of gp63 were able to cleave the AP-PAR1 from the macrophage cell surface, whereas avirulent cells lacking gp63 could not (Fig. 7A). The cleavage of AP-PAR1 induced by virulent parasite cells was largely attenuated by the inhibitor of gp63 (Fig.  7B), further suggesting a role for gp63 in this process. Our data also show that Leishmania amastigotes freshly isolated from infected macrophages or Leishmania bodies isolated from Leishmania-infected human neutrophils were also able to cleave AP-PAR1 from the recombinant J774G8 cells (supplemental Fig. 7S).

Knockdown of PAR1 in Macrophages Prevented Leishmania-induced Inhibition of 7SL RNA Synthesis-We used PAR1
Stealth siRNA or PAR1 antagonist SCH79797 (100 M) to knock down the function of PAR1 in J774G8 cells. We verified the knockdown of PAR1 mRNA and protein in the siRNA-treated J774G8 cells by quantitative PCR and Western blotting (Fig. 7, C, D, and E). Although virulent Leishmania promastigotes could down-regulate 7SL RNA gene expression in the macrophages treated with control siRNA, they failed to do so in macrophages treated with PAR1 siRNA or PAR1 antagonist (Fig. 7F). Interestingly, the level of 7SL was increased significantly in the PAR1 knocked-down macrophages upon Leishmania exposure (Fig. 7F). Similar results were obtained with B2 and vault RNAs (supplemental Fig. 8S). The mechanism of this increase is not known. These data further suggest FEBRUARY 25, 2011 • VOLUME 286 • NUMBER 8 FIGURE 5. Evaluation of role of promastigote surface protease gp63 on L. amazonensis-induced activation of calpain and degradation of TFIIIC110 in J774G8 cells. A, evaluation of the levels of gp63 protein in two each of the virulent and avirulent clones of L. amazonensis promastigotes. The level of gp63 was evaluated by Western blot analysis with the gp63 monoclonal antibody as described (18). ␣-Tubulin was used as a loading control. B, densitometric analysis of immunoblot data as in A from four independent experiments. Bands were developed using IR Dye 800-conjugated secondary antibody (LI-COR Biosciences) and visualized using the LI-COR Odyssey Infrared Imaging System. Quantitation and analysis of bands were performed using Odyssey software. ␤-Actin was used as a normalization control, and the results are expressed relative to the level of ␣-tubulin. Results are mean Ϯ S.E. (error bars) (n ϭ 4). * indicates that the changes observed in the levels of gp63 were statistically significant (p Ͻ 0.001). C, evaluation of the levels of surface gp63 protease activity in two each of the virulent and avirulent clones of L. amazonensis promastigotes. The level of gp63 protease was evaluated using azocasein as substrate as described (44). Results are mean Ϯ S.E. (error bars) (n ϭ 6). * indicates a statistically significant difference between the control and the experimental sets (p Ͻ 0.001). D, evaluation of the ability of the virulent and avirulent stocks of L. amazonensis promastigotes in knocking down the levels of 7SL RNA in J774G8 cells. Real time RT-PCR was done to evaluate the level of 7SL RNA. 18 S rRNA was used as an internal control. Results are mean Ϯ S.E. (error bars) (n ϭ 6). The decrease in the levels of 7SL RNA by the virulent promastigotes was statistically significant (p Ͻ 0.001). E, effect of pretreatment of the promastigotes with gp63 inhibitor (GP63In) 3DMRPed (2 M) on the ability of the virulent stocks of L. amazonensis to elevate the Ca 2ϩ levels in J774G8 cells. Avirulent stocks were used as controls. M, unexposed macrophages; MϩL, macrophages exposed to Leishmania promastigotes. Results are mean Ϯ S.E. (error bars) (n ϭ 6). * indicates that the inhibition in the levels of Ca 2ϩ in the virulent promastigote-exposed macrophages by gp63 inhibitor was statistically significant (p Ͻ 0.001). F, effect of pretreatment of the promastigotes with gp63 inhibitor 3DMRPed (2 M) on the ability of the virulent stocks of L. amazonensis to elevate calpain activity in J774G8 cells. Avirulent stocks were used as controls. M, unexposed macrophages; MϩL, macrophages exposed to Leishmania promastigotes. Lama, L. amazonensis; Lmaj, L. major; Vir, virulent; Avir, avirulent. Calpain activity is expressed as the difference in the rate of substrate hydrolysis in the presence or absence of calpain inhibitor as described under "Experimental Procedures." Results are mean Ϯ S.E. (error bars) (n ϭ 6). * indicates that the inhibition in the calpain activity in the virulent promastigote-exposed macrophages by gp63 inhibitor was statistically significant (p Ͻ 0.001). G, evaluation of the ability of the virulent and the avirulent stocks of L. amazonensis to degrade TFIIIC110 in J774G8 cells. The level of TFIIIC110 was evaluated by Western blot analysis. ␤-Actin was used as a loading control. H, densitometric analysis of immunoblot data as in G from four independent experiments. Bands were developed using IR Dye 800-conjugated secondary antibody (LI-COR Biosciences) and visualized using the LI-COR Odyssey Infrared Imaging System. Quantitation and analysis of bands were performed using Odyssey software. ␤-Actin was used as a normalization control, and the results are expressed relative to the level of ␤-actin. Results are mean Ϯ S.E. (error bars) (n ϭ 4). * indicates that the changes observed in the levels of TFIIIC110 were statistically significant (p Ͻ 0.001).

Leishmania-induced ncRNA Manipulations in Macrophages
a key role for PAR1 in Leishmania-induced down-regulation of 7SL RNA gene expression in macrophages.
Activation of PAR1 in Macrophages Favors Establishment of Leishmania Infection in These Cells-To evaluate whether macrophages with activated PAR1 are more hospitable to otherwise avirulent promastigotes of Leishmania, we treated J774G8 cells with the agonist peptide SFFLRN (100 M) and (error bars) (n ϭ 6). ** indicates that the decrease in the levels of 7SL RNA by the activation of PAR1 was statistically significant (p Ͻ 0.001). C, effect of activation of PAR1 on the cytosolic free Ca 2ϩ levels in J774G8 cells. PAR1 was activated either by thrombin or by the agonist peptide SFFLRN. Results are mean Ϯ S.E. (error bars) (n ϭ 6). ** indicates that the increase in the levels of Ca 2ϩ in the macrophages was statistically significant (p Ͻ 0.001). D, effect of activation of PAR1 on the calpain activity in J774G8 cells. PAR1 was activated either by thrombin or by the agonist peptide SFFLRN. Calpain activity is expressed as the difference in the rate of substrate hydrolysis in the presence or absence of calpain inhibitor as described under "Experimental Procedures." Results are mean Ϯ S.E. (error bars) (n ϭ 6). * indicates that the elevation of the calpain activity in the PAR1 activated macrophages was statistically significant (p Ͻ 0.001). E, evaluation of the effect of PAR1 activation on TFIIIC110 degradation in J774G8 cells. The level of TFIIIC110 was evaluated by Western blot analysis. ␤-Actin was used as a loading control. F, densitometric analysis of immunoblot data as in E from four independent experiments. Bands were developed using IR Dye 800-conjugated secondary antibody (LI-COR Biosciences) and visualized using the LI-COR Odyssey Infrared Imaging System. Quantitation and analysis of bands were performed using Odyssey software. ␤-Actin was used as a normalization control, and the results are expressed relative to the level of ␤-actin. Results are mean Ϯ S.E. (error bars) (n ϭ 4). * indicates that the changes observed in the levels of TFIIIC110 were statistically significant (p Ͻ 0.001). Exp/Con, experimental/control. (error bars) (n ϭ 6). * indicates that the inhibition in the AP release in the virulent promastigote-exposed macrophages by gp63 inhibitor was statistically significant (p Ͻ 0.001). C, knockdown of PAR1 mRNA levels in J774G8 cells by two different Stealth siRNAs. PAR1 mRNA levels were evaluated by real time RT-PCR. ␤-Actin mRNA was used as a normalization control, and the results are expressed relative to the level of ␤-actin mRNA. Results are mean Ϯ S.E. (error bars) (n ϭ 6). ** indicates that the knockdown of PAR1 mRNA levels was statistically significant (p Ͻ 0.001). D, knockdown of PAR1 protein level in J774G8 cells by Stealth siRNA-1. The level of PAR1 was evaluated by Western blot analysis. ␤-Actin was used as a loading control. A similar result was obtained with Stealth siRNA-2. E, densitometric analysis of immunoblot data as in D from four independent experiments. Bands were developed using IR Dye 800-conjugated secondary antibody (LI-COR Biosciences) and visualized using the LI-COR Odyssey Infrared Imaging System. Quantitation and analysis of bands were performed using Odyssey software. ␤-Actin was used as a normalization control, and the results are expressed relative to the level of ␤-actin. Results are mean Ϯ S.E. (error bars) (n ϭ 4). * indicates that the changes observed in the levels of PAR1 were statistically significant (p Ͻ 0.001). F, effect of PAR1 knockdown on the ability of the virulent stocks of L. amazonensis to decrease the level of 7SL RNA. PAR1 activity was knocked down either with Stealth siRNA (siRNA-1) or the PAR1 antagonist SCH79797 (100 M). Real time RT-PCR was done to evaluate the level of 7SL RNA. 18 S rRNA was used as an internal control. Results are mean Ϯ S.E. (error bars) (n ϭ 6). The changes in the levels of 7SL RNA by the knockdown of PAR1 were statistically significant (p Ͻ 0.001). challenged these cells with avirulent promastigotes of L. amazonensis. Avirulent Leishmania (laboratory stock AV2) failed to establish infection in the untreated macrophages but established infection in the macrophages treated with PAR1 agonist peptide (Fig. 8, A and B). These data further support a role for PAR1 in the establishment of leishmanial infection in macrophages.

PAR1 Antagonist or Calpain Inhibitors Prevent Virulent Leishmania from Establishing Infection in Cultured
Macrophages-We established that PAR1 and -calpain in macrophages are critical determinants for Leishmania promastigotes to down-regulate the expression of 7SL and other ncRNAs. Because the ability of Leishmania to inhibit the expression of 7SL RNA is important for their establishment of infection in macrophages (4), we evaluated whether the inhibition of PAR1 or calpain in macrophages will make the macrophages resistant to Leishmania infection. A monolayer of J774G8 macrophages was treated with or without the PAR1 antagonist SCH79797 (100 M) for 10 min or with calpain inhibitors calpastatin peptide (1 M) and calpeptin (5 M) for 30 min in the growth medium. These cells were then incu-bated with virulent L. amazonensis promastigotes (laboratory stock V1) in antibiotic-free growth medium (with or without the PAR1 antagonist or calpain inhibitors) at a macrophage/ parasite ratio of 1:10 continuously for 5 days. One hundred macrophages were examined microscopically for the number of infected macrophages and the total number of amastigotes inside those macrophages (4). Our data (Fig. 8, C and D) show that PAR1 antagonists or calpain inhibitors could inhibit the establishment of infection of L. amazonensis promastigotes inside cultured J774G8 cells. We repeated these experiments with the virulent promastigotes of L. major and obtained similar results. The drugs did not have any detectable toxic effects on the growth or morphology of the promastigotes of L. amazonensis at the test concentrations.

DISCUSSION
Intracellular parasites often manipulate host cells to make them more hospitable for the establishment of infection and propagation of the parasite inside them (4 -6, 63, 64). Recent evidence has shown that Leishmania follow this paradigm to infect mammalian macrophages (4,63,64). Clues to the mechanisms used by Leishmania to achieve its goal have mainly come from comparative studies between uninfected macrophages and macrophages with established Leishmania infection (4 -6, 63, 64). The data from these studies have provided valuable information about how Leishmania maintains infection inside the macrophages and proliferates without being killed. One of the major innovative aspects of our study is the finding that Leishmania manipulates the ncRNA metabolism of its host cells to immunocompromise them and thus establish infection in those cells. Our data show that parasite cells can knock down selected RNA polymerase III-transcribed ncRNAs in their host cells through the activation of PAR1 and -calpain. This leads to reduced RNA synthesis through the degradation of a subunit of an RNA polymerase III transcription factor subunit (TFIIIC110).
We have elaborated a mechanism that promotes Leishmania infection and parasite propagation using cultured macrophages, and we have verified our findings with mouse (BALB/c) peritoneal and bone marrow-derived macrophages as well as human blood monocyte-derived macrophages. One of our major findings from these studies is the involvement of Leishmania surface protease gp63 in the activation of macrophage PAR1. This protease is a major surface glycoprotein of the promastigote form of the parasite, which is the morphological form that exists inside the infected sandfly vector gut and is transmitted to the human host during the sandfly bite (65)(66)(67).
The promastigotes of all Leishmania species may not be the major form of the parasite that directly interacts with the macrophages during the initial stages of infection (68,69). Macrophages may encounter the promastigotes directly, or they may encounter membrane-coated Leishmania bodies derived from neutrophils or other macrophages (65-69). As mentioned above, we found that Leishmania bodies isolated from lysates from infected human macrophages or from infected human neutrophils were also capable of activating PAR1 on the macrophage cell surface. We thus hypothesize  6). The differences between treated and untreated were statistically significant (p Ͻ 0.001). C, effects of PAR1 antagonist and calpain inhibitors on the establishment of infection by virulent L. amazonensis in J774G8 cells. The macrophages were treated with PAR1 antagonist or calpain inhibitor mixture as described before. The treated or untreated macrophages were infected with virulent L. amazonensis (clone V1), and the establishment of infection was evaluated by measuring the percentage of macrophages infected (C) and the number of amastigotes per 100 macrophages (D) following standard protocols. Results are mean Ϯ S.E. (error bars) (n ϭ 6). ** indicates that the differences between treated and untreated were statistically significant (p Ͻ 0.001).
that as macrophages are the major host cells in the human body that allow survival and propagation of Leishmania whatever developmental stage of Leishmania these cells encounter their PAR1 is activated prior to infection.
Our study strongly suggests that replenishment of the macrophages with selected RNA polymerase III-transcribed ncRNAs and/or knockdown of PAR1 and -calpain may combat Leishmania infection. This research thus adds to our understanding of macrophage-Leishmania interactions and provides a basis for developing chemical regimens to prevent the establishment of leishmanial infection. The study will also serve as a paradigm for investigations with other pathogens of macrophages to evaluate whether they also exploit similar mechanisms to manipulate the immune functions of macrophages during their establishment of infection. A point to note is that the concentration of free Ca 2ϩ in the Leishmaniaexposed macrophages never reached a level to explain the relatively robust elevation of calpain activity in these cells. Although we do not have any evidence yet, we speculate that under in vivo conditions -calpain is activated at a lower concentration of Ca 2ϩ than what has been determined as required in vitro. We hypothesize that other yet to be determined changes induced by PAR1 activation may influence the activation of calpain to decrease the Ca 2ϩ concentration required for -calpain in vivo. More research is needed to understand this PAR1-activated increase in calpain activity in macrophages.
Another potentially important outcome of Leishmaniainduced knockdown of the activity of type 2 promoters of RNA polymerase III could be the targeted alleviation of ncRNA-mediated transcriptional arrest of protein-coding genes in macrophages. Recent studies have shown that stress induces the overexpression of ncRNAs like B2 and Alu RNAs in mammalian cells that can interact with RNA polymerase II to inhibit the transcription of protein-coding genes (58 -61). We recently found that during the phagocytosis of avirulent L. amazonensis promastigotes by mouse macrophages the level of B2 RNA is elevated more than 12-fold, and the transcription of several protein-coding genes in macrophages were significantly inhibited. 3 Importantly, the level of B2 RNA is down-regulated significantly when the macrophages engulf the gp63-coated virulent L. amazonensis promastigotes (see "Results"). We propose that one of the purposes of knocking down ncRNA gene expression in macrophages by Leishmania is to alleviate ncRNA-mediated transcriptional arrest in these cells so that the parasites can establish infection as well as proliferate.
Our data described here and elsewhere (4,70) along with other information in the literature (1-3, 5, 6) suggest the following working model of the steps involved in establishing Leishmania infection in macrophages (Fig. 9). (a) The virulent Leishmania promastigotes are anchored to the surface of macrophages to present their surface gp63 protease to the macrophage membrane PAR1. (b) gp63 binds to and proteolytically cleaves inactive PAR1 precursor protein to activate this receptor. (c) Through the activation of G-proteins, the Ca 2ϩ concentration inside the macrophages is increased, leading to the activation of -calpain. (d) Activated -calpain then degrades TFIIIC110. (e) The decrease in TFIIIC110 levels results in the inhibition of RNA polymerase III transcription of 7SL and other TFIIIC110-dependent ncRNA genes. From our previous data (4), we postulate that this decrease in 7SL RNA reduces the amount of signal recognition particles in the cell. The decrease in signal recognition particles, in turn, reduces the phagolysosomal enzymes, macrophage surface membrane receptors, and secretion of autoregulatory chemokines and cytokines from macrophages, thus helping Leishmania to establish infection in the macrophages (4). The Leishmania-induced decrease in the levels of other small ncRNAs, such as B2 RNA and Alu RNA, may also alleviate the stress-induced transcriptional arrest of protein-coding genes in macrophages, thus also helping to sustain the parasite inside the host cells.