LD downregulates LPS- and TNFα-stimulated CD40 expression on DCs

Because available reports have provided contradictory results regarding regulation of CD40 expression by LD in DCs [14–18], we first clarified whether LD upregulates or downregulates CD40 expression in DCs. For this purpose, we infected bone marrow-derived DCs (BMDCs), established from BALB/c mice, with LD promastigotes (extracellular form; LDPm) or amastigotes (intracellular form; LDAm) for varying times (6 h, 12 h or 24 h), stimulated with LPS for 24 h and assessed CD40 expression via flow cytometry. Relative to untreated BMDCs, BMDCs treated with LPS displayed more CD40 expression (Fig 1A and 1B, S1A and S1B Fig). In contrast, BMDC infection with LDPm or LDAm resulted in a temporal reduction in LPS-stimulated CD40 expression, with maximum inhibition occurring at 24 h postinfection (Fig 1A and 1B, S1A and S1B Fig). The LPS-stimulated CD40 expression was also found to be lower on splenic DCs (sDCs) derived from LD-infected mice than those derived from uninfected mice (Fig 1C). Further, to verify whether LD inhibits CD40 expression induced by other DC maturation stimuli, we analyzed the effect of LD infection on tumor necrosis factor alpha (TNFα)-stimulated CD40 expression. For these experiments, we infected BMDCs with LDPm for 24 h because our aforementioned observations (Fig 1A, S1A Fig) demonstrated maximum downregulation of LPS-induced CD40 expression at 24 h post LDPm infection. Subsequently, we stimulated these LDPm-infected BMDCs with TNFα for 24 h and analyzed CD40 expression on BMDCs via flow cytometry. We found that BMDC infection with LDPm for 24 h also inhibited the CD40 upregulation induced by TNFα (Fig 1D, S1C Fig). Thus, regardless of the forms of LD parasite (i.e., extracellular or intracellular form), LD inhibits LPS- and TNFα-stimulated CD40 expression in DCs.

Fig 1

LD inhibits LPS- and TNFα-stimulated CD40 upregulation on DCs.

(A and B) Expression of CD40 on BMDCs left uninfected (DC) or infected with LD promastigotes (LDPm; A) or amastigotes (LDAm; B) for indicated times and then stimulated with LPS for 24 h; evaluated by flow cytometry. Isotype represents immunostaining with an isotype-matched control antibody. (C) Splenocytes from uninfected (Uninf) and LD-infected (Inf) mice were cultured (at 45 days postinfection) in the presence or absence of LPS for 24 h. CD40 expression on DCs (CD11c⁺ gated cells outlined in the middle panels) was analyzed by flow cytometry. (D) Flow cytometry analysis of CD40 expression by BMDCs left uninfected or infected for 24 h with LDPm and then stimulated with TNFα for 24 h. In all experiments, LD strain AG83 was used. Data are representative of three independent experiments. For (A), (B) and (D), the relative mean fluorescence intensity (MFI) values of CD40 expression pooled from three independent experiments are shown in S1 Fig.

Runx proteins promote CD40 upregulation on DCs

Before determining how LD suppressed LPS- or TNFα-stimulated CD40 expression in DCs, we sought to identify a previously unrecognized mechanism associated with transcriptional regulation of CD40 by LPS and TNFα. While examining the mouse CD40 promoter sequence with the TFBIND program, we found two potential Runx-binding sites (R1: ^-489TGTGGT^-484 and R2: ^-464TGCGGT^-459; base positions are relative to the transcription initiation site) (Fig 2A). Accordingly, we tested whether LPS and TNFα induce the binding of Runx proteins to the CD40 promoter in DCs. Electrophoretic mobility shift assay (EMSA) using mouse CD40 promoter-specific Pr1 and Pr2 probes, which contained R1 and R2 sites, respectively (Fig 2A), demonstrated an increased binding of nuclear proteins to each of these probes upon stimulation of BMDCs and sDCs with LPS or TNFα (Fig 2B, S2 and S3 Figs). This nuclear protein binding, however, was blocked by mutation of the Runx-binding sequences (Fig 2C). Antibody-mediated supershift analysis subsequently affirmed the binding of Runx1 and Runx3 to Pr1 and Pr2 probes in LPS- or TNFα-treated DCs (Fig 2D). Correspondingly, chromatin immunoprecipitation (ChIP) assays demonstrated an enhanced recruitment of Runx1 and Runx3 to the CD40 promoter following LPS or TNFα stimulation (Fig 2E, S4 Fig). In addition, in vivo footprint analysis showed protected (i.e., weaker) bands within R1 and R2 sites of the mouse CD40 promoter in LPS- and TNFα-treated BMDCs but not in untreated BMDCs (Fig 2F), which is indicative of the occupancy of both R1 and R2 sites by Runx proteins following LPS or TNFα stimulation. Confocal microscopic studies further demonstrated that BMDC stimulation with LPS or TNFα augmented nuclear translocation of Runx1 and Runx3 (Fig 2G, S5 Fig). Thus, LPS and TNFα trigger the nuclear translocation and subsequent binding of Runx proteins to the CD40 promoter in DCs. Together these data establish the CD40 promoter as a direct target for Runx in DCs.

Fig 2

LPS and TNFα induce nuclear localization and binding of Runx proteins to the CD40 promoter.

(A) Schematic presentation of mouse CD40 promoter indicating the location of putative Runx-binding sites (R1 and R2) and ChIP primers (P1 and P2), and details of oligonucleotide probes used for EMSA. Mouse CD40 promoter-specific Pr1 and Pr2 probes contain putative wild-type Runx-binding sites, and Mut-Pr1 and Mut-Pr2 probes contain mutations (in italics) at Runx-binding sites. Base positions are relative to the transcription start site. (B and C) EMSA of nuclear extracts of BMDCs treated with LPS or TNFα for indicated times; assessed with indicated probes. Numbers below lanes in (B) represent densitometry [normalized to OCT-1 binding (control)] relative to that of untreated BMDCs (0 h). The densitometry results pooled from three independent experiments for (B) are shown in S2 Fig. (D) Supershift EMSA [with immunoglobulin G (IgG; control) or antibody to (α-) Runx1, Runx2 or Runx3] of nuclear extracts of BMDCs treated with LPS or TNFα for 0.5 h; assayed with Pr1 or Pr2 probes. (E) Binding of Runx proteins to -578/-373 region of the CD40 promoter in BMDCs treated with LPS or TNFα (time, above lanes); assessed via ChIP using the primers shown in (A) and indicated antibodies (left margin). Amplification of mouse GAPDH promoter (S4 Fig) and chromatin immunoprecipitated by rabbit IgG were used as negative controls, and input DNA (2%) as an internal control. Numbers below lanes represent densitometry, normalized to input DNA and presented relative to that of untreated BMDCs (0 h). (F) In vivo footprint analysis of mouse CD40 promoter showing DMS-protected bands (indicated by open circles) at R1 and R2 sites in LPS- or TNFα-treated but not in untreated (UT) BMDCs. (G) Translocation of Runx1 or Runx3 (red) into the nuclei (blue; Hoechst staining) in BMDCs left untreated (UT) or treated with LPS or TNFα for 0.5 h; assessed by confocal microscopy. Pink color (merge) shows nuclear translocation of Runx1 or Runx3. Scale bar, 10 μm. Confocal microscopy images of DCs immunostained with isotype control antibodies are given in S5 Fig. Data are representative of three (B-E) or two (F and G) independent experiments.

Next, we examined the role of each of the two Runx-binding sites (R1 and R2) in regulating mouse CD40 promoter activity. We transfected a mouse DC cell line JAWSII, which endogenously expresses Runx1 and Runx3 (S6A Fig), with the wild-type CD40 promoter-luciferase plasmid or with similar plasmid harboring mutations in either R1or R2 site or both R1 and R2 sites (S6B Fig). We then stimulated JAWSII cells with LPS and measured the CD40 promoter activity via luciferase reporter assay. In response to LPS, the activity of wild-type CD40 promoter was strongly enhanced (S6C Fig, left panel). However, mutation of R1 or R2 site alone drastically reduced LPS-stimulated CD40 promoter activity, with a more pronounced inhibition observed when both sites were mutated (S6C Fig, left panel). We obtained similar results with TNFα stimulation (S6C Fig, right panel). Therefore, both R1 and R2 sites serve as critical determinants of CD40 promoter activity in DCs.

To determine whether Runx proteins were required for LPS- or TNFα-stimulated upregulation of CD40 expression in DCs, we silenced Runx1 and/or Runx3 expression by small interfering RNAs (siRNAs; Fig 3A). Downregulation of Runx1 or Runx3 expression highly attenuated LPS-stimulated CD40 expression at both mRNA and protein levels (Fig 3B and 3C, S7A Fig). Combined Runx1 and Runx3 silencing, however, had no added effect (Fig 3B and 3C, S7A Fig). Similar to our siRNA data, overexpression of Runx dominant negative mutant (Runx DN), which is known to block the transcriptional activity of both Runx1 and Runx3 [19], largely impaired LPS-stimulated CD40 upregulation on BMDCs (Fig 3D). The latter finding ruled out the possibility that the downregulation of LPS-stimulated CD40 expression occurred due to off-target effects of Runx1- and Runx3-specific siRNAs. Accordingly, we used these Runx1- and Runx3-specific siRNAs for subsequent experiments. We further noted that similar to LPS stimulation, TNFα-induced CD40 mRNA and protein expression was also reduced following Runx1 or Runx3 silencing and was completely blocked when Runx1 and Runx3 were silenced together (Fig 3E and 3F, S7B Fig). Thus, both Runx1 and Runx3 are required for LPS- and TNFα-driven CD40 upregulation in DCs.

Fig 3

Both LPS and TNFα require Runx proteins to upregulate CD40 expression in DCs.

(A) Immunoblot analysis of Runx1 and Runx3 expression in lysates of BMDCs left untransfected (- siRNA) or transfected with control (Ctrl) siRNA, Runx1 siRNA, Runx3 siRNA or Runx1 and Runx3 siRNAs (Runx1+3 siRNA). β-actin serves as a loading control. Data are representative of three independent experiments. (B) Real-time PCR quantification of CD40 mRNA in BMDCs transfected with siRNAs as in (A) and then cultured with (+) or without (-) LPS for 12 h. Results were normalized to the expression of ACTB mRNA (encoding β-actin) and are presented as fold change relative to untransfected BMDCs cultured without LPS. Data are a compilation of three independent experiments (n = 2 in each experiment). Each symbol represents data of individual replicate. Error bars represent SD. (C) Flow cytometry analysis of CD40 expression on BMDCs transfected with indicated siRNAs (left margin) and cultured with (+) or without (-) LPS for 24 h. Data are representative of three independent experiments. (D) BMDCs were transfected with GFP-tagged Runx dominant negative vector (Runx DN vec) or control vector (Ctrl vec) and then cultured with (+) or without (-) LPS for 24 h. CD40 expression on gated GFP⁺ DCs (i.e., transfected DC population) was analyzed via flow cytometry. Top panel [untransfected BMDCs (- vec) cultured without LPS] shows the gating strategy used for flow cytometry analysis. BMDCs were first gated based on forward and side scatter, and then further gated based on GFP expression. Data are representative of two separate experiments. (E) Real-time PCR analysis of CD40 mRNA expression in BMDCs transfected with siRNAs as in (A), then stimulated for 12 h with (+) or without (-) TNFα. Results were normalized to the expression of ACTB mRNA (encoding β-actin) and are presented as fold change relative to untransfected BMDCs cultured without TNFα. Data are a compilation of three independent experiments (n = 2 in each experiment). Each symbol represents data of individual replicate. Error bars represent SD. (F) Flow cytometry analysis of CD40 expression on BMDCs transfected with indicated siRNAs (left margin) and cultured with (+) or without (-) TNFα for 24 h. Data are representative of three independent experiments. For (C) and (F), the relative MFI values of CD40 expression pooled from three independent experiments are presented in S7 Fig. *p < 0.001, **p < 0.01; NS, not significant.

Phosphatidylinositol 3-kinase (PI3K)-Akt signaling controls Runx-mediated CD40 expression in DCs

We then attempted to identify the upstream signaling events controlling Runx-mediated CD40 expression in DCs. In this regard, one potential regulator could be PI3K, because it associates with toll-like receptor 4 (TLR4; LPS receptor) and is required for LPS-induced CD40 expression in DCs [9,20]. The role for PI3K in TNFα-stimulated CD40 expression, however, remains unclear, although it is known to interact with the TNF receptor-1 (TNFR1) in other cell types [21]. Therefore, we initially determined the role for PI3K in LPS- and TNFα-induced upregulation of CD40 expression in DCs. While LPS and TNFα upregulated CD40 expression in untreated or control (dimethylsulfoxide; DMSO)-treated BMDCs, these effects of LPS and TNFα were substantially reduced in BMDCs treated (for 1 h) with the PI3K inhibitors wortmannin (Wort) or Ly294002 (LY) (Fig 4A, S8A Fig). Furthermore, pretreatment of BMDCs with Wort or LY inhibited LPS- or TNFα-stimulated nuclear translocation and binding of Runx proteins to the CD40 promoter without affecting Runx protein expression (Figs 4B, S8B and S9). Thus, PI3K is necessary for promoting nuclear translocation and binding of Runx proteins to the CD40 promoter, and subsequent upregulation of CD40 expression in DCs following LPS or TNFα treatment.

Fig 4

LPS and TNFα promote Runx-mediated CD40 upregulation on DCs via the PI3K-Akt pathway.

(A) Flow cytometry analysis of CD40 expression on BMDCs left untreated (UT) or treated with dimethylsulfoxide (DMSO, 0.1%; control treatment), Wort or LY for 1 h and then stimulated for 24 h with (+) or without (-) LPS (left panels) or TNFα (right panels). The relative MFI data for CD40 expression pooled from three independent experiments is presented in S8A Fig. (B) BMDCs were either left untreated or treated with DMSO, Wort or LY (for 1 h) and then stimulated with LPS or TNFα for 0.5 h or left unstimulated (US). The binding of nuclear Runx1 and Runx3 to the CD40 promoter was assessed via EMSA (probes as in Fig 2), and the expression of Runx1 and Runx3 in DC lysates was determined by immunoblot analysis. The OCT-1 binding in EMSA and β-actin expression in immunoblot analysis served as internal controls. Numbers below lanes indicate densitometry quantification of Runx1 and Runx3 binding to the CD40 promoter (EMSA panels; normalized to OCT-1 binding), and the levels of Runx1 and Runx3 proteins [immunoblot (IB) analysis panels; normalized to β-actin]; presented relative to that of untreated BMDCs that had been left unstimulated. S8B Fig shows densitometry results pooled from three independent experiments. (C and D) Immunoprecipitation (IP; antibodies, above lanes) of lysates of BMDCs treated with LPS (C) or TNFα (D) for indicated times, followed by immunoblot analysis with antibodies to various PI3K isoforms (C and D), TLR4 (C) or TNFR1 (D). WCL, whole-cell lysate (no immunoprecipitation). (E) Immunoblot analysis of p110β/PI3K and β-actin in lysates of BMDCs left untransfected or transfected with control siRNA or PI3K p110β-specific siRNA (p110β siRNA). (F and G) EMSA (probes as in Fig 2) assessing the binding of nuclear Runx1 and Runx3 to the CD40 promoter (F), and flow cytometry analysis measuring CD40 expression (G) in BMDCs transfected with siRNAs as in (E) and then cultured with or without LPS or TNFα for 0.5 h (F) or 24 h (G). (H) Immunoblot analysis of total and phosphorylated (p-) Akt in BMDCs transfected with indicated siRNAs, then cultured with or without LPS (upper panel) or TNFα (lower panel) for 0.3 h. (I) EMSA of nuclear extracts of BMDCs left untreated or treated with Akt inhibitor XIII (AI-XIII) for 1 h and then cultured with or without LPS or TNFα for 0.5 h, analyzed with probes as in Fig 2. (J) Analyzing (by flow cytometry) the effect of AI-XIII treatment (for 1 h) on CD40 expression by BMDCs treated with LPS (left panel) or TNFα (right panel) for 24 h. All data are representative of three independent experiments.

We then determined the type of PI3K complexes that were induced by LPS and TNFα, and mediated Runx binding to the CD40 promoter. Coimmunoprecipitation analyses showed that upon LPS stimulation, TLR4 interacted with the PI3K regulatory subunit p85α, and catalytic subunit p110β but not p110α or p110δ (Fig 4C). The same PI3K complex consisting of p85α and p110β subunits also interacted with TNFR1 following TNFα stimulation (Fig 4D). Next, to assess the potential role of PI3K complex p85α-p110β in Runx-mediated regulation of CD40 expression in DCs, we silenced p110β expression by siRNA (Fig 4E). Whereas LPS or TNFα stimulated Runx binding to the CD40 promoter and upregulated CD40 expression in untransfected and control siRNA-transfected BMDCs, these effects of LPS or TNFα were blocked in p110β-silenced BMDCs (Fig 4F and 4G). Therefore, the PI3K complex p85α-p110β is necessary for LPS- and TNFα-stimulated CD40 upregulation on DCs via Runx. Notably, the PI3K complex p85α-p110β mediated the above-mentioned effects through its downstream effector Akt. For example, silencing of PI3K p110β blocked the ability of LPS and TNFα to induce Akt phosphorylation (Figs 4H and S10). Furthermore, pretreatment of BMDCs with Akt inhibitor XIII (AI-XIII) largely inhibited LPS- and TNFα-stimulated Runx binding to the CD40 promoter and subsequent CD40 upregulation (Fig 4I and 4J). Collectively, our results showed a critical role for the PI3K-Akt pathway in Runx-mediated CD40 upregulation on DCs.

LD inhibits LPS- and TNFα-induced Runx binding to the CD40 promoter by suppressing PI3K-Akt pathway

Having shown that the PI3K-Akt-Runx pathway is required for LPS- and TNFα-induced CD40 upregulation, we investigated the relevance of this signaling pathway in LD-mediated suppression of CD40 expression in DCs. Initially, we determined whether LD infection influences Runx binding to the CD40 promoter. For these experiments, we infected BMDCs with LDPm for 12 or 24 h prior to LPS or TNFα treatment, because at these time point of LD infection we observed pronounced inhibition of CD40 expression on DCs (Figs 1A and 1B, S1A and S1B). Although LPS or TNFα efficiently triggered Runx binding to the CD40 promoter in uninfected BMDCs, these effects of LPS or TNFα were suppressed in BMDCs infected with LDPm (Figs 5A and S11, upper panels). Of note, the intracellular expression of Runx1 and Runx3, and the surface expression of TLR4, TNFR1 and TNFR2 were not affected by LD infection (Figs 5A, 5B and S11, lower panels). These results ruled out the possibility that LD inhibited Runx binding to the CD40 promoter by downregulating the expression of Runx proteins, or TLR4 and TNF receptors. On further investigation it became clear that LDPm actually suppressed the nuclear translocation of Runx1 and Runx3 despite LPS and TNFα stimulation, which eventually resulted in less Runx binding to the CD40 promoter in DCs (Figs 5A, 5C and S11, upper panels). Similar to LDPm, LDAm also inhibited LPS- or TNFα-stimulated binding of Runx proteins to the CD40 promoter (Fig 5D). In addition, sDCs derived from LD-infected mice showed reduced Runx binding to the CD40 promoter despite LPS stimulation as compared to those derived from uninfected mice (Fig 5E). These observations indicate that LD exhibits an inhibitory effect on nuclear translocation and subsequent binding of Runx proteins to the CD40 promoter in DCs.

Fig 5

LD attenuates LPS- and TNFα-induced Runx binding to the CD40 promoter by inhibiting PI3K-Akt pathway.

(A) EMSA (probes as in Fig 2) evaluating the binding of Runx1 and Runx3 to the CD40 promoter (top), and immunoblot analysis showing Runx1 and Runx3 expression (bottom) in BMDCs left uninfected (Uninf) or infected with LDPm for indicated times and then stimulated with (+) LPS (left panels) or TNFα (right panels) for 0.5 h or left unstimulated (-). Numbers below lanes represent densitometry (as in Fig 4B); presented relative to uninfected BMDCs that had been left unstimulated. A compilation of densitometry results from three independent experiments is shown in S11 Fig. (B) Flow cytometry analysis of TLR4, TNFR1 and TNFR2 expression on BMDCs left uninfected or infected with LDPm for indicated times. (C) Confocal microscopy of translocation of Runx1 or Runx3 (red) into the nuclei (blue; Hoechst staining) in BMDCs left uninfected or infected with LDPm for 24 h and then stimulated with LPS or TNFα for 0.5 h or left unstimulated (US). Pink color (merge) shows nuclear translocation of Runx1 or Runx3. Scale bar, 10 μm. (D and E) Nuclear lysates were prepared from BMDCs left uninfected or infected with LDAm for 24 h and then kept untreated (-) or treated (+) for 0.5 h with LPS or TNFα (D). In some experiments (E), nuclear lysates were prepared from sDCs that were derived from uninfected (Uninf mice) or LD-infected mice (Inf mice; isolated on day 45 postinfection) and then treated with LPS for 0.5 h. Lysates were subjected to EMSA (probes as in Fig 2) to detect the binding of Runx1 and Runx3 to the CD40 promoter. Numbers below lanes indicate relative densitometry quantification of Runx1 and Runx3 binding to the CD40 promoter (normalized to OCT-1 binding). (F and G) Immunoblot analysis of total and phosphorylated Akt in lysates of BMDCs infected with LDPm (F) or LDAm (G) for 24 h or left uninfected and then treated with LPS (left panels) or TNFα (right panels) for 0.3 h. Below lanes, densitometry, normalized to total Akt and presented relative to uninfected BMDCs given control treatment (neither LPS nor TNFα). In all experiments, LD strain AG83 was used. Data are representative of three (A, B, and D-G) or two (C) independent experiments.

Because the PI3K-Akt pathway was found critical for Runx binding to the CD40 promoter, it seemed likely that LD attenuated Runx binding to the CD40 promoter by suppressing PI3K-Akt signaling. To confirm this possibility, we analyzed the effect of LD infection on LPS- and TNFα-induced Akt phosphorylation. We found that BMDC infection with LDPm or LDAm attenuated LPS- and TNFα-induced Akt phosphorylation (Fig 5F and 5G). Collectively, these data indicate that LD suppresses LPS- and TNFα-induced activation of the PI3K-Akt pathway and subsequent nuclear translocation and binding of Runx proteins to the CD40 promoter, leading to the downregulation of CD40 expression on DCs.

LD inhibits PI3K-Akt-Runx-CD40 axis via Src homology phosphotyrosine phosphatase-1 (SHP-1)

Next, we examined the mechanism by which LD suppressed PI3K-Akt-Runx-CD40 axis in DCs. Previous reports showed that the PI3K-Akt signaling is negatively regulated by SHP-1 and that the activity of SHP-1 is induced in macrophages following LD infection [22,23]. Therefore, we wondered whether SHP-1 was involved in LD-mediated suppression of CD40 expression in DCs. Notably, the role of SHP-1 in the regulation of Runx activity and CD40 expression remains unknown. Initially, we determined whether LD induces SHP-1 activation in DCs. Kinetic analyses showed that SHP-1 activation, as measured by phosphorylation of SHP-1 at Tyr536 and Tyr564 [24,25], was augmented at 6 h after LDPm infection and continued up to 24 h postinfection (Figs 6A and S12A). Like LDPm, LDAm also triggered SHP-1 activation in DCs (Fig 6B). This SHP-1 induction by LD, however, was not affected by LPS or TNFα treatment (Fig 6C). We further found that silencing of SHP-1 by siRNA blocked the inhibitory effect of LDPm on LPS- and TNFα-induced Akt phosphorylation, binding of Runx proteins to the CD40 promoter, and upregulation of CD40 mRNA and protein expression by DCs (Figs 6D–6H, S12B and S12C). However, SHP-1 silencing did not affect the intracellular expression of Runx1 and Runx3, and the surface expression of TLR4, TNFR1 and TNFR2 (Figs 6F, S12B and S13). Therefore, silencing of SHP-1 restored LPS- or TNFα-mediated CD40 upregulation on LD-infected DCs by reinstating Runx binding to the CD40 promoter and not by enhancing the expression of Runx proteins, or TLR4 and TNF receptors. Similar to SHP-1 silencing by siRNA, overexpression of catalytically inactive SHP-1 dominant negative mutant (SHP-1 DN; SHP-1 carrying mutation at cysteine 453 (C453S); [26]) also prevented LD-induced inhibition of LPS-stimulated CD40 expression in DCs (Fig 6I and 6J). This data negates the possibility that the blockade of LD-induced inhibition of CD40 expression upon SHP-1 silencing occurred due to off-target effect of SHP-1-specific siRNA. Our findings thus revealed a previously unidentified inhibitory role for SHP-1 in the regulation of CD40 expression. Together these results indicate that LD inhibits LPS- and TNFα-stimulated CD40 upregulation on DCs by suppressing the PI3K-Akt-Runx pathway through SHP-1.

Fig 6

SHP-1 mediates LD-induced inhibition of CD40 expression in DCs.

(A and B) Immunoblot analysis of total SHP-1 or SHP-1 phosphorylated at Tyr536 or Tyr564 in lysates of BMDCs infected for 0–24 h with LDPm (A) or LDAm (B). Numbers below lanes represent densitometry, normalized to total SHP-1 and presented relative to that of uninfected BMDCs (0 h). Data are representative of three independent experiments. (C) SHP-1 phosphorylation at Tyr536 or Tyr564, determined by immunoblot, in BMDCs left uninfected or infected with LDPm for 24 h and then treated with LPS (left panels) or TNFα (right panels) for indicated times. The expression of total SHP-1 serves as a loading control. Below lanes, densitometry results as in (A), presented relative to uninfected BMDCs treated with LPS or TNFα for 0 h. Data are representative of three independent experiments. (D) BMDCs were left untransfected or transfected with control siRNA or SHP-1-specific siRNA. Cell lysates were immunoblotted for SHP-1 or β-actin. Data are representative of three independent experiments. (E and F) Analysis of Akt phosphorylation (E; determined by immunoblot analysis), the binding of nuclear Runx1 and Runx3 to the CD40 promoter (F; assessed by EMSA using probes as in Fig 2) and the expression of Runx1 and Runx3 (F; analyzed by immunoblot analysis) in BMDCs transfected with indicated siRNAs and then infected with LDPm for 24 h or left uninfected, and treated with or without LPS (left panels) or TNFα (right panels) for 0.3 h (E) or 0.5 h (F). Below lanes, densitometry (as in Figs 4B and 5F), presented relative to control BMDCs (BMDCs left untransfected and uninfected, and given no LPS or TNFα treatment). Data are representative of three independent experiments. (G) Real-time PCR analysis of CD40 mRNA expression in BMDCs transfected with indicated siRNAs and then infected with LDPm for 24 h and cultured with or without LPS (left panel) or TNFα (right panel) for 12 h. Results were normalized to the expression of ACTB mRNA (encoding β-actin) and are presented as fold change relative to control BMDCs (BMDCs left untransfected and uninfected, and cultured without LPS or TNFα). Data are a compilation of three independent experiments (n = 2 in each experiment). Each symbol represents data of individual replicate. Error bars represent SD. (H) Flow cytometry analysis of CD40 expression by BMDCs transfected with indicated siRNAs, then infected with LDPm for 24 h and stimulated with LPS (left panels) or TNFα (right panels) for 24 h. Data are representative of three independent experiments. (I) Immunoblot analysis of SHP-1 and β-actin (loading control) in BMDCs left untransfected (- vec) or transfected with control vector (Ctrl vec) or SHP-1 DN vector (SHP-1 DN vec). Data are representative of two independent experiments. (J) Flow cytometry analysis of CD40 expression by BMDCs transfected as in (I), then infected with LDPm for 24 h and cultured with (+) or without (-) LPS for 24 h. Data are representative of two independent experiments. In all experiments, LD strain AG83 was used. The pooled densitometry results for (A) and (F), and the relative MFI data of CD40 expression for (H) from three independent experiments are shown in S12 Fig. *p < 0.001; NS, not significant.

Sodium antimony gluconate (SAG)-induced DC antileishmanial response relies on Runx-mediated CD40 upregulation

Given that Runx proteins are necessary for CD40 upregulation on DCs (Fig 3) and that CD40 expressed by DCs contributes to host protection [7], we speculated a role for Runx in regulating antileishmanial immune responses mediated by DCs. To test this hypothesis, we focused on SAG (the first-line drug against VL [27]) because it upregulates CD40 expression on DCs [28]. Initially, we evaluated the role of Runx in SAG-induced CD40 expression in DCs. EMSA and supershift analyses showed that the treatment of BMDCs and sDCs with SAG led to increased Runx1 and Runx3 binding to the CD40 promoter (Figs 7A–7C and S14A). Previously, we have shown that SAG activates PI3K-Akt signaling in DCs [28]. In fact, phosphorylation of Akt was triggered within 0.25 h after SAG treatment, remained sustained up to1 h, and then started decreasing at 3 h (Fig 7D). Our data here further suggest that SAG requires the PI3K complex p85α-p110β and its downstream effector Akt to induce Runx binding to the CD40 promoter (Fig 7E–7G). Indeed, silencing of PI3K p110β by siRNA or pretreatment with Akt inhibitor AI-XIII substantially reduced the binding of Runx proteins to the CD40 promoter despite SAG treatment (Fig 7E and 7G). The PI3K-Akt pathway promoted SAG-induced binding of Runx proteins to the CD40 promoter by enhancing their (i.e., Runx1 and Runx3) nuclear translocation. For example, BMDC treatment with SAG considerably increased the nuclear translocation of Runx1 and Runx3 proteins (Fig 7H). This SAG-induced effect, however, was inhibited when we pretreated BMDCs with PI3K inhibitors Wort or LY (Fig 7H). Further analyses depicted that silencing of Runx1 or Runx3 decreased SAG-stimulated CD40 mRNA and protein expression in BMDCs. In addition, combined silencing of Runx1 and Runx3 robustly inhibited the upregulation of CD40 mRNA and protein expression induced by SAG (Figs 7I, 7J and S14B). Interestingly, SAG, unlike LPS or TNFα (Fig 1), upregulated CD40 expression on DCs even after LD infection (Figs 8A and S15A). This finding correlates with the observations that SAG effectively blocked LD-induced SHP-1 activation (Fig 8B), whereas LPS and TNFα did not (Fig 6C). Consequently, SAG continued to drive Akt phosphorylation (Fig 8B), which in turn augmented the nuclear translocation and the binding of Runx proteins to the CD40 promoter (Figs 8C and Fig 8D; S15B, left two panels) and thereby increased CD40 expression on BMDCs despite LD infection (Figs 8A and S15A). However, neither SAG treatment nor LD infection had any effect on Runx1 and Runx3 expression in BMDCs (Figs 8D and S15B, right two panels). In addition, we did not detect any significant change in both percentage of LD-infected DCs and intracellular parasite number following SAG treatment for 0.3 h or 1 h (S16 Fig). The latter finding indicated that the inhibition of LD-induced SHP1 activation, which occurred as early as 0.3 h or 1 h after SAG treatment (Fig 8B), was not an outcome of SAG-mediated clearance of intracellular LD parasites. Instead, SAG, being a potent inhibitor of SHP-1 [29–36], directly inhibited LD-induced SHP-1 activation at the above-mentioned time points. This conclusion is supported by a report [29] demonstrating that SAG forms stable complexes with SHP-1 and that SAG directly targets the catalytic domain of SHP-1 to inhibit its activity. Although our results (Figs 8B and S16) showed a direct inhibitory effect of SAG on LD-induced SHP-1 activation at early time points, a prolonged SAG exposure might be needed to continuously suppress LD-induced SHP-1 activation. In that case, the inhibition of LD-induced SHP-1 activation may partly be contributed by SAG-mediated reduction of intracellular parasite load. Nevertheless, our findings demonstrated that SAG promotes CD40 upregulation on LD-infected DCs via Runx by directly or indirectly inhibiting LD-induced SHP-1 activation.

Fig 7

SAG induces CD40 upregulation on DCs via the PI3K-Akt-Runx pathway.

(A and B) EMSA of nuclear extracts of BMDCs (A) or sDCs (B) treated with SAG for indicated times, assessed with probes as in Fig 2. Numbers below lanes in (A) represent densitometry (normalized to OCT-1 binding), presented relative to untreated BMDCs (0 h). Data are representative of three independent experiments. (C) Supershift EMSA (antibodies and probes are indicated above and below lanes, respectively) analyzing the binding of nuclear Runx1, Runx2 and Runx3 to the CD40 promoter in BMDCs treated with SAG for 0.5 h. Data are representative of three independent experiments. (D) Immunoblot analysis of total and phosphorylated Akt in lysates of BMDCs treated with SAG for indicated times. Numbers below lanes represent densitometry, normalized to total Akt and presented relative to untreated BMDCs (0 h). Data are representative of two independent experiments. (E) EMSA of nuclear extracts of BMDCs left untransfected or transfected with control siRNA or PI3K p110β-specific siRNA (p110β siRNA) and then treated with or without SAG for 0.5 h, assessed with probes as in Fig 2. Below lanes, densitometry as in (A); presented relative to untransfected BMDCs not treated with SAG. Data are representative of three independent experiments. (F) Immunoblot analysis of total and phosphorylated Akt in lysates of BMDCs transfected with siRNAs as in (E) and then treated with SAG for 0.3 h. Below lanes, densitometry (as in Fig 5F), presented relative to untransfected BMDCs given no SAG treatment. Data are representative of three independent experiments. (G) EMSA (probes as in Fig 2) of nuclear extracts of BMDCs left untreated or treated with AI-XIII for 1 h and then cultured with or without SAG for 0.5 h. Below lanes, densitometry as in (A), presented relative to untreated BMDCs cultured without SAG. Data are representative of three independent experiments. (H) Confocal microscopy of the translocation of Runx1 or Runx3 (red) into the nuclei (blue; Hoechst staining) in BMDCs left untreated (UT) or treated with DMSO, Wort or LY for 1 h, then cultured with or without SAG for 0.5 h. Pink color (merge) shows nuclear translocation of Runx1 or Runx3. Scale bar, 10 μm. Data are representative of two independent experiments. (I) Real-time PCR analysis of CD40 mRNA expression in BMDCs left untransfected (- siRNA) or transfected with control siRNA or Runx1- and/or Runx3-specific siRNA, then cultured for 12 h with or without SAG. Results were normalized to the expression of ACTB mRNA (encoding β-actin) and are presented as fold change relative to untransfected BMDCs cultured without SAG. Data are compilation of three independent experiments (n = 2 in each experiment). Each symbol represents data of individual replicate. Error bars represent SD. (J) Analyzing (by flow cytometry) the effect of Runx1 and/or Runx3 silencing on CD40 expression by BMDCs treated for 24 h with or without SAG. Data are representative of three independent experiments. The densitometry results for (A), and the relative MFI of CD40 expression for (J) pooled from three independent experiments are shown in S14 Fig. *p < 0.001, **p < 0.01; NS, not significant.

Fig 8

SAG-mediated SHP-1 inhibition allows PI3K-Akt-Runx signaling to upregulate CD40 expression on LD-infected DCs.

(A) CD40 expression profile on BMDCs left uninfected or infected with LDPm for 24 h, then treated with or without SAG for 24 h, assessed by flow cytometry. Data are representative of three independent experiments. (B) Immunoblot analysis of total and phosphorylated SHP-1 and Akt in lysates of BMDCs infected with LDPm for 24 h or left uninfected and then treated with SAG for indicated times. Number below lanes indicate densitometry of phosphorylated SHP-1 (normalized to total SHP-1) or phosphorylated Akt (normalized to total Akt), presented relative to uninfected BMDCs treated with SAG for 0 h. Data are representative of three independent experiments. (C) Confocal microscopy of the translocation of Runx1 or Runx3 (red) into the nuclei (blue; Hoechst staining) in BMDCs left uninfected or infected with LDPm for 24 h, then cultured with or without SAG for 0.5 h. Pink color (merge) shows nuclear translocation of Runx1 or Runx3. Scale bar, 10 μm. Data are representative of two independent experiments. (D) EMSA (with probes as in Fig 2) assessing the binding of nuclear Runx1 and Runx3 to the CD40 promoter (upper panel) and immunoblot analysis depicting Runx1 and Runx3 expression (lower panel) in BMDCs left uninfected or infected with LDPm for 24 h, then cultured for 0.5 h with or without SAG. Numbers below lanes indicate densitometry quantification of Runx1 and Runx3 binding to the CD40 promoter (EMSA panels; normalized to OCT-1 binding), and the levels of Runx1 and Runx3 proteins [immunoblot (IB) analysis panels; normalized to β-actin]; presented relative to uninfected BMDCs cultured without SAG. Data are representative of three independent experiments. For all experiments LD strain AG83 was used. The relative MFI of CD40 expression for (A) and the densitometry results for (D) pooled from three independent experiments are shown in S15 Fig.

Next, we tested whether Runx proteins, by promoting CD40 expression, contributed to SAG-induced antileishmanial response of DCs in vivo. To assess this, we silenced (or not) Runx1 and Runx3 expression in DCs and then treated with SAG. In some cases, we overexpressed CD40 in Runx1- and Runx3-silenced DCs prior to SAG treatment (S17 Fig). Afterward, we adoptively transferred these DCs into LD-infected syngeneic mice on the days 15, 25, 35 and 45 postinfection. On day 48 postinfection, we analyzed liver and spleen weights, splenic and liver parasite load, and the frequencies of splenic CD4⁺ and CD8⁺ T cells producing IFNγ or IL-10 (Fig 9A–9C and Fig 10). Upon transferring SAG-treated DCs, but not PBS-treated DCs, the liver and spleen weights and parasite load were drastically reduced in LD-infected mice. These effects of SAG-treated DCs were largely compromised after Runx1 and Runx3 silencing. However, forced CD40 expression in SAG-treated Runx1- and Runx3-silenced DCs improved the efficacy of these cells to reduce liver and spleen weights and parasite burden in LD-infected mice (Figs 9B, 9C and S18). We further observed that upon transfer of SAG-treated DCs, but not PBS-treated DCs, the frequencies of IFNγ-producing CD4⁺ and CD8⁺ T cells were increased and IL-10-producing CD4⁺ and CD8⁺ T were reduced in spleens of LD-infected mice. Silencing of Runx1 and Runx3 in SAG-treated DCs, however, reversed these effects. Strikingly, forced CD40 expression restored the ability of SAG-stimulated Runx1- and Runx3-silenced DCs to increase IFNγ-producing and reduce IL-10-producing CD4⁺ or CD8⁺ T cell populations to an extent similar to that observed in case of SAG-treated DCs (Figs 10 and S19). Collectively, these data suggest that Runx proteins play critical roles in mediating SAG-induced antileishmanial response of DCs by promoting CD40 expression and thereby augmenting type-1 T cell immunity.

Fig 9

Runx-mediated CD40 expression is required for clearance of LD infection in mice by SAG-treated DCs.

(A) Experimental scheme for adoptive transfer experiments. BALB/c BMDCs were treated with PBS or SAG for 24 h. Alternatively, BALB/c BMDCs were transfected with control siRNA, or with Runx1 and Runx3 siRNAs alone or together with an empty vector (Vec) or CD40-expressing vector (CD40) prior to SAG treatment (see S17 Fig showing the level of CD40 expression on these DCs). After such treatment, DCs were transferred i.v. into LD-infected (Inf) BALB/c mice on days 15, 25, 35 and 45 postinfection. In some sets of the experiment, age-matched uninfected (Uninf) mice and LD-infected BALB/c mice were left without any DC transfer (no DC transfer). On day 48 postinfection, spleens and livers were isolated for analysis. (B and C) Spleen and liver weights (B), and splenic and liver parasite burden [measured by stamp-smear method and presented as Leishman-Donovan Unit (LDU); (C)] in mice treated as in (A); assessed on day 48 postinfection. Each symbol represents an individual mouse. Data are compilation of two separate experiments (n = 3 mice per group in each experiment). The horizontal bars represent the mean. The representative pictures of spleens and livers from these mice are shown in S18 Fig. In all experiments, LD strain AG83 was used.*p < 0.001, **p < 0.01; NS, not significant.

Fig 10

SAG-induced Runx-dependent CD40 upregulation on DCs promotes type-1 T cell response in LD-infected mice.

Runx1- and Runx3-silenced BMDCs (with or without CD40 overexpression), and control-silenced BMDCs were treated with SAG and then adoptively transferred into LD-infected mice. Alternatively, age-matched uninfected and LD-infected mice were left without any DC transfer (details given in Fig 9 legend). (A and B) Frequency of IFNγ- and IL-10-producing CD4⁺ (A) or CD8⁺ (B) T cells in splenocytes isolated from these mice, assessed by flow cytometry (representative data of n = 6). Numbers indicate the percentage of cells in the respective quadrants. Gating strategy used for flow cytometry analysis is described in S19 Fig. (C) Graphs displaying cumulative data of the percentage of IFNγ- and IL-10-producing CD4⁺ (upper panels) or CD8⁺ (lower panels) T cells from two independent experiments (n = 3 mice per group in each experiment). Each symbol represents an individual mouse; horizontal bars represent the mean. In all experiments, LD strain AG83 was used. *p < 0.001, **p < 0.01, ***p < 0.05; NS, not significant.

Persistent SHP-1 activation by antimony-resistant LD impairs SAG-induced CD40 upregulation on DCs

Up to now, we demonstrated the ability of LD to regulate CD40 expression on DCs using normal LD [i.e., antimony-sensitive LD (Sb^SLD)] strain AG83, and lastly showed that this Sb^SLD strain failed to inhibit SAG-induced CD40 expression on DCs (Fig 8A). In contrast, our previous study [28], coupled with the data presented here (Figs 11A, 11B and S20A), showed that the antimony-resistant LD (Sb^RLD) strain GE1F8R effectively inhibited SAG-stimulated CD40 expression by DCs at both mRNA and protein levels. Since LD required SHP-1 to regulate PI3K-Akt-Runx pathway-mediated CD40 expression in DCs (Fig 6), we determined the involvement of these signaling elements in the modulation of CD40 expression by Sb^RLD. Initially, we compared the ability of Sb^RLD and Sb^SLD to induce SHP-1 activation (measured by SHP-1 phosphorylation at Tyr536 and Tyr564 as described above) in DCs following SAG treatment. While Sb^SLD strain AG83 could not induce SHP-1 phosphorylation in the presence of SAG, Sb^RLD strain GE1F8R continued to drive SHP-1 phosphorylation in BMDCs despite SAG treatment (Fig 11C). A previous study [22] has reported that SHP-1, upon phosphorylation, prevents the activation of PI3K-Akt pathway by physically interacting with the p85α subunit of PI3K complex. Consistent with this report, our results showed that in response to both AG83 (Sb^SLD) and GE1F8R (Sb^RLD) infection, SHP-1 physically interacted with the PI3K complex p85α-p110β (Fig 11D). The interaction between SHP-1 and the PI3K complex p85α-p110β, however, was prevented in AG83 (Sb^SLD)-infected DCs upon SAG treatment (Fig 11D). On the contrary, SHP-1 constantly interacted with the PI3K complex p85α-p110β in GE1F8R (Sb^RLD)-infected DCs despite SAG treatment (Fig 11D). Consequently, SAG-induced Akt phosphorylation (Fig 11E), the nuclear translocation and the binding of Runx proteins to the CD40 promoter (Figs 11F, 11G and S20B, left two panels), and the upregulation of CD40 mRNA and protein expression (Figs 11A, 11B and S20A) were largely impaired in GE1F8R (Sb^RLD)-infected BMDCs. In contrast, these SAG-induced events were readily detected in BMDCs infected with AG83 (Sb^SLD) (Figs 11A, 11B, 11E–11G, S20A and S20B). Notably, the intracellular expression of Runx1 and Runx3 remained unchanged despite Sb^RLD or Sb^SLD infection or SAG treatment (Figs 11G and S20B, right two panels). Thus, Sb^RLD, but not Sb^SLD, suppresses SAG-induced PI3K-Akt-Runx signaling and subsequent CD40 upregulation on DCs by persistently activating SHP-1.

Fig 11

Sb^RLD prevents SAG-induced Runx-dependent CD40 upregulation on DCs by persistently activating SHP-1.

(A) Real-time PCR analysis of CD40 mRNA expression in BMDCs left uninfected or infected for 24 h with promastigotes of Sb^RLD strain GE1F8R or Sb^SLD strain AG83, then cultured with or without SAG for 12 h. Results were normalized to the expression of ACTB mRNA (encoding β-actin) and presented as fold change relative to uninfected BMDCs cultured without SAG. Data are a compilation of three independent experiments (n = 2 in each experiment). Each symbol represents data of individual replicate. Error bars represent SD. (B) Flow cytometry analysis of CD40 expression on BMDCs infected as in (A) and then cultured with or without SAG for 24 h. Data are representative of three independent experiments. (C) Immunoblot analysis of total and phosphorylated SHP-1 in lysates of BMDCs left uninfected or infected with promastigotes of Sb^RLD strain GE1F8R or Sb^SLD strain AG83 for 24 h and then treated with SAG for indicated times. Number below lanes indicate densitometry of phosphorylated SHP-1 (normalized to total SHP-1), presented relative to uninfected BMDCs treated with SAG for 0 h. Data are representative of three independent experiments. (D) Immunoprecipitation (IP) and immunoblot (IB) analysis of the interaction of SHP-1 with the PI3K complex p85α-p110β in BMDCs infected with promastigotes of Sb^RLD strain GE1F8R or Sb^SLD strain AG83 for 24 h and then treated with SAG for indicated times. IgG represents immunoglobulin G (IP control), and WCL is whole-cell lysate (no IP). Data are representative of two independent experiments. (E) Immunoblot analysis of total and phosphorylated Akt in lysates of BMDCs left uninfected or infected with promastigotes of Sb^RLD strain GE1F8R or Sb^SLD strain AG83 for 24 h and then treated with SAG for indicated times. Number below lanes indicate densitometry of phosphorylated Akt (normalized to total Akt), presented relative to uninfected BMDCs treated with SAG for 0 h. Data are representative of three independent experiments. (F) Confocal microscopy of the translocation of Runx1 or Runx3 (red) into the nuclei (blue; Hoechst staining) in BMDCs left uninfected or infected with promastigotes of Sb^RLD strain GE1F8R or Sb^SLD strain AG83 for 24 h and then treated with (+) or without (-) SAG for 0.5 h. Pink color (merge) shows nuclear translocation of Runx1 or Runx3. Scale bar, 10 μm. Data are representative of two independent experiments. (G) EMSA (with probes as in Fig 2) of the binding of nuclear Runx1 and Runx3 to the CD40 promoter (upper panel), and immunoblot analysis of Runx1 and Runx3 expression (lower panel) in BMDCs left uninfected or infected with promastigotes of Sb^RLD strain GE1F8R or Sb^SLD strain AG83 for 24 h and then cultured for 0.5 h with or without SAG. Below lane, densitometry (as in Fig 8D); presented relative to uninfected BMDCs cultured without SAG. Data are representative of three independent experiments. The relative MFI data of CD40 expression for (B), and densitometry result for (G) pooled from three independent experiments are shown in S20 Fig. *p < 0.001, **p < 0.01; NS, not significant.

To extrapolate our findings in human DCs, we further verified the role of Runx in the regulation of SAG-induced CD40 expression by Sb^SLD and Sb^RLD in human monocyte-derived DCs (HuMoDCs). Initially, we tested whether Runx proteins regulate SAG-induced CD40 expression in HuMoDCs. Computational analysis using the TFBIND program identified two putative Runx-binding sites (R3: ^-456TGTGGC^-451 and R4: ^-433GGTGGT^-428; base positions are relative to the transcription initiation site) in the human CD40 promoter (Fig 12A). Subsequent analyses involving EMSA, supershift EMSA and ChIP assays confirmed the binding of Runx1 and Runx3 to both R3 and R4 sites in CD40 promoter following stimulation of HuMoDCs with SAG (Fig 12B–12E). Furthermore, silencing of Runx1 and/or Runx3 expression by siRNA greatly inhibited SAG-induced CD40 upregulation on HuMoDCs (Fig 12F and 12G). These results suggest that Runx1 and Runx3 are required for SAG-induced CD40 expression by HuMoDCs. Importantly, GE1F8R (Sb^RLD), unlike AG83 (Sb^SLD), impaired SAG-induced Runx binding to the CD40 promoter and upregulation of CD40 expression on HuMoDCs (Fig 12H and 12I). Collectively, our data demonstrate that Sb^RLD, but not Sb^SLD, suppresses SAG-induced CD40 expression on DCs by preventing Runx binding to the CD40 promoter. Sb^RLD imparts this inhibitory effect on Runx and the upstream PI3K-Akt signaling pathway by prolonging SHP-1 activation even in the presence of SAG. Overall, our findings indicate the active involvement of Runx proteins in differential regulation of SAG-stimulated CD40 expression by Sb^RLD and Sb^SLD in DCs.

Fig 12

Sb^RLD inhibits SAG-induced CD40 upregulation on HuMoDCs by impairing Runx binding to the CD40 promoter.

(A) A schematic of human CD40 promoter showing the location of putative Runx-binding sites (R3 and R4) and ChIP primers (P3 and P4), and details of oligonucleotide probes used for EMSA. Human CD40 promoter-specific Pr3 and Pr4 probes contain putative wild-type Runx-binding sites, and Mut-Pr3 and Mut-Pr4 probes contain mutations (in italics) at Runx-binding sites. Base positions are relative to the transcription start site. (B and C) EMSA of nuclear extracts of HuMoDCs treated with SAG for indicated times; assayed with indicated probes. OCT-1 binding (in B) serves as an internal control. Numbers below lanes in (B), densitometry readings (as in Fig 2B); presented relative to untreated HuMoDCs (0 h). (D) Supershift EMSA (antibodies and probes are indicated above and below lanes, respectively) to assess the binding of nuclear Runx1, Runx2 and Runx3 to the CD40 promoter in HuMoDCs treated with SAG for 0.5 h. (E) ChIP assay [with the primers shown in (A) and antibodies indicated at left margin] to determine the binding of Runx proteins to -480/-276 region of the CD40 promoter in HuMoDCs treated with SAG (time, above lanes). Amplification of human GAPDH promoter and chromatin immunoprecipitated by rabbit IgG were used as negative controls, and input DNA (2%) as an internal control. Numbers below lanes represent densitometry, normalized to input DNA and presented relative to that of untreated HuMoDCs (0 h). huCD40, human CD40; huGAPDH, human GAPDH. (F) Immunoblot analysis of Runx1 and Runx3 expression in HuMoDCs left untransfected, or transfected with control siRNA, Runx1 siRNA, Runx3 siRNA or Runx1 and Runx3 siRNAs (Runx1+3 siRNA). β-actin serves as a loading control. (G) Flow cytometry analysis of CD40 expression on HuMoDCs transfected with indicated siRNAs and then cultured with or without SAG for 24 h. (H) EMSA of nuclear extracts of HuMoDCs left uninfected or infected with promastigotes of Sb^RLD strain GE1F8R or Sb^SLD strain AG83 for 24 h and then cultured for 0.5 h with (+) or without (-) SAG; assayed with indicated probes. OCT-1 binding serves as an internal control. Numbers below lanes represent densitometry readings (as in Fig 2B); presented relative to control HuMoDCs (HuMoDCs left uninfected and cultured without SAG). (I) HuMoDCs were either left uninfected or infected with promastigotes of Sb^SLD strain AG83 or Sb^RLD strain GE1F8R for 24 h and then treated (for 24 h) with or without SAG. CD40 expression on HuMoDCs was assessed by flow cytometry. All data are representative of two independent experiments.

Ethics statement

The use of LD for this study was approved by the Biosafety Committee of Institute of Microbial Technology (#IMTECH/IBSC/2015/15 and CSIR-IMTECH/IBSC/2018/22). All animal studies were approved by the Institutional Animal Ethics Committee of the Institute of Microbial Technology (#IAEC/16/10) and performed according to the National Regulatory Guidelines issued by CPSEA (Committee for the Purpose of Supervision of Experiments on Animals), Govt. of India. For HuMoDC preparation, the buffy coats of healthy donors were obtained from the Postgraduate Institute of Medical Education and Research, Chandigarh, India, with approval of the Institutional Ethics Committees of Institute of Microbial Technology [IEC (May 2020) #1] and Postgraduate Institute of Medical Education and Research (PGI/IEC/2020/EIC000344). Informed consents were obtained from all blood donors.

Reagents

The following antibodies were used for immunoblot analysis: anti-Runx1 (sc-28679), anti-Runx3 (sc-376543), anti-PI3K p110β (sc-376412), anti-PI3K p110δ (sc-7176), anti-TLR4 (sc-293072), anti-β-actin (sc-47778), HRP-conjugated anti-rabbit IgG (sc-2004) and HRP-conjugated anti-mouse IgG (sc-2005; all from Santa Cruz Biotechnology); anti-phospho-SHP-1 (Tyr536, ab51171; Abcam); and anti-PI3K p85α (4292), anti-PI3K p110α (4255), anti-phospho-Akt (Ser473; 9271), anti-Akt (9272), anti-phospho-SHP-1 (Tyr564; 8849), anti-SHP-1 (3759) and anti-TNFR1 (13377; all from Cell Signaling Technologies). For EMSA, ChIP and immunoprecipitation assays, the antibodies and reagents used were as follows: anti-Runx1 (sc-28679 X), anti-Runx2 (sc-10758 X), rabbit IgG (sc-2027), mouse IgG (sc-2025) and protein A/G PLUS-Agarose beads (sc-2003; all from Santa Cruz Biotechnology); anti-Runx3 (39301; Active Motif); Armenian hamster IgG (400901, Biolegend); and anti-TLR4 (ab22048) and anti-TNFR1 (ab106099; both from Abcam). For confocal microscopy following antibodies were used: anti-Runx1 (sc-28679), anti-Runx3 (sc-376543), and respective isotype control antibodies [rabbit IgG (sc-2027) for anti-Runx1, and mouse IgG (sc-2025) for anti-Runx3] were obtained from Santa Cruz Biotechnology. Alexa Fluor 568 (AF)-conjugated goat anti-rabbit IgG (A-11011) and goat anti-mouse IgG (A-11004) secondary antibodies were procured from Thermo Fisher Scientific. For flow cytometry analysis, following antibodies were obtained from Biolegend: APC-conjugated anti-mouse CD11c (117310), APC-conjugated anti-mouse CD3 (100235), FITC-conjugated anti-mouse CD8 (100705), PE-conjugated anti-mouse IL-10 (505007), PE-conjugated anti-mouse CD40 (124610), PE-conjugated anti-mouse TNFR1 (113003), PE-conjugated anti-human CD40 (313006), PE-conjugated anti-mouse TNFR2 (113405), PE-conjugate Armenian hamster IgG (400907), PE-conjugated rat IgG2a,κ (400507), PE-conjugated mouse IgG1,κ (400111), APC-conjugated Armenian hamster IgG (400911) and APC-conjugated rat IgG2b,κ (400611). Other antibodies such as FITC-conjugated anti-mouse CD4 (11-0041-82), PE-conjugated anti-mouse IFNγ (12-7311-83) and PE-conjugated anti-mouse TLR4 (12-9041-80) were procured from eBioscience (Thermo Fisher Scientific); and FITC-conjugated rat IgG (sc-2340) and PE-conjugated rat IgG (sc-516648) were from Santa Cruz Biotechnology. The Akt inhibitor XIII (AI-XIII; 124030) was from Merck Life Science. Recombinant mouse TNFα, granulocyte-macrophage colony-stimulating factor (GM-CSF) and IL-4 were from Peprotech. Recombinant human GM-CSF and IL-4, and anti-mouse CD11c magnetic beads were from Miltenyi Biotec. The ON-TARGETplus non-targeting control pool siRNAs and SMARTpool siRNAs targeting mRNA encoding mouse Runx1, Runx3, PI3K p110β and SHP-1; and human Runx1 and Runx3 were from Dharmacon. Notably, the same siRNAs were previously used by other groups [51–54]. LPS (E. coli O111:B4), PI3K inhibitors Wortmannin (Wort; W3144) and Ly294002 (LY; L9908), and all other reagents were procured from Sigma-Aldrich unless stated otherwise.

Animals

BALB/c mice and golden hamsters (Mesocricetus auratus) were maintained and bred under pathogen-free conditions at the Institute of Microbial Technology animal facility.

Parasite culture

The LD strain AG83 [MHOM/IN/83/AG83; American Type Culture Collection (ATCC PRA-413)], provided by Dr. Nahid Ali (CSIR-Indian Institute of Chemical Biology, India), was maintained in male or female golden hamsters between 4–6 wks of age as described [55]. Amastigotes were isolated from spleens of infected hamsters as described [56]. Subsequently, amastigotes were transformed into promastigotes and maintained as described [57]. This LD strain AG83 was also used as Sb^SLD [28] in some experiments. For some comparative analyses with Sb^SLD strain AG83, the Sb^RLD strain GE1F8R (MHOM/IN/89/GE1) [28] was used.

DC preparation and treatment

BMDCs and sDCs were prepared from male or female BALB/c mice between 8–12 wks of age as described [58]. In some experiments, HuMoDCs, either prepared from human buffy coats as described [58] upon approval by the Ethical Committees mentioned above or purchased from Lonza (CC-2701), were used. DCs (5 x 10⁶/well) were then treated with LPS (1 μg/ml) or TNFα (100 ng/ml) for specified times in complete RPMI 1640 medium (10% FBS, penicillin/streptomycin, L-glutamine and 2-mercaptoethanol). Alternatively, DCs were treated with clinical grade of SAG (40 μg/ml; a gift from Albert David Ltd., Kolkata, India) for specified times as described [28]. The dose of SAG mentioned here represents the concentration of Sb^V. For blocking PI3K or Akt activation, DCs were treated with PI3K inhibitors Wort (200 nM) or LY (50 μM) or Akt inhibitor AI-XIII (5 μM) for 1 h prior to LPS, TNFα or SAG stimulation. In some other experiments, DCs (2.5 x 10⁵) were transfected with 500 ng green fluorescent protein (GFP)-tagged Runx DN or empty vector (a gift from Dr. Vanja Lazarevic, National Cancer Institute, NIH, USA; [19]) using the TransIT-2020 transfection reagent (Mirus) before LPS stimulation.

DC infection with LD

DCs (5 x 10⁶/well) were infected in vitro with amastigotes or promastigotes (stationary phase) of LD (i.e., Sb^SLD) at parasite to DC ratio (multiplicity of infection; MOI) of 10:1 for indicated times in RPMI 1640 complete medium. Subsequently, DCs were washed, resuspended in RPMI 1640 complete medium and stimulated with LPS, TNFα or SAG for specified times. In some cases, DCs (5 x 10⁵ cells in 500 μl RPMI 1640 complete medium) were adhered on 18 mm x 18 mm cover slips, infected with promastigotes of LD (i.e., Sb^SLD) for 24 h (parasite to DC ratio of 10:1) and then treated with SAG for indicated times. The number of intracellular parasites in DCs was determined via Giemsa staining. For some experiments, DCs were transfected with empty vector (pCMV) or SHP-1 DN-expressing vector (pCMV-SHP-1 DN (C453S); a gift from Dr. Yuping Lai, East China Normal University, Shanghai, China [59]) using the TransIT-2020 transfection reagent (Mirus) prior to LD infection. In other experiments, DCs were infected with promastigotes of Sb^RLD at parasite to DC ratio of 10:1 for 24 h in RPMI 1640 complete medium and then stimulated with SAG.

Quantitative RT-PCR

The cDNA synthesis and quantitative RT-PCR were carried out using the SuperScript III Platinum SYBR Green one-step qRT-PCR kit (Invitrogen) and the following primers: mouse CD40, forward 5'-GCTATGGGGCTGCTTGTTGA-3' and reverse 5' -ATGGGTGGCATTGGGTCTTC-3' [60]; and mouse ACTB (encoding β-actin), forward 5′-GCTCTGGCTCCTAGCACCAT-3′ and reverse 5′-GCCACCGATCCACACCGCGT-3′ [61]. Gene expression was measured by the change in threshold method (ΔΔC_T) and normalized to the ACTB mRNA expression.

In vivo footprinting

Dimethylsulfate (DMS) treatment of DCs and naked DNA, ligation-mediated PCR and in vivo footprint analyses were done as described [62]. Details of primers used for footprint analyses are mentioned in S1 Table.

EMSA

Nuclear extracts were prepared as described [63]. EMSA was performed as described [64] using various P³²-labeled DNA probes (S1 Table) specific for mouse or human CD40 promoter. An OCT-1 probe, 5'-TGTCGAATGCAAATCACTAGAA-3', was used as control. Supershift analysis was performed as described [65] using 4 μg of rabbit IgG, anti-Runx1, anti-Runx2 or anti-Runx3. Bands were visualized using a phosphoimager (Fujifilm FLA-9000).

ChIP

ChIP assay was performed using the ChIP-IT kit (Active Motif) and antibodies anti-Runx1, anti-Runx2, anti-Runx3 or rabbit IgG. After immunoprecipitation, followed by DNA extraction, PCR was performed to amplify -578/-373 region of mouse CD40 promoter using primers: P1, 5'-GCTGTCCCCTACTCGTAGGAATTTCCTTCT-3' and P2, 5'-GTTACCCCACCCCACCCACAATACCCC-3'. For a negative control, mouse glyceraldehyde-3-phosphate dehydrogenase (GAPDH) promoter was amplified by using primers: 5'-CACCCTGGCATTTTCTTCCA-3' and 5'-GACCCAGAGACCTGAATGCTG-3' [28]. Alternatively, PCR was carried out to amplify -480/-276 region of human CD40 promoter using primers: P3, 5'-TGAAACGCCTCCCCATAC-3' and P4, 5'- CTGCGACCGGAGAGAG-3'. In that case, amplification of human GAPDH promoter using primers 5'-GCCTGAGCAGTCCGGTGT-3' and 5'-GATCGGTGCTGGTTCCCA-3' [66] served as a negative control.

Immunoprecipitation and immunoblot analysis

Crosslinking of proteins in DCs was performed using dimethyl 3,3-dithiopropionimidate dihydrochloride (Sigma-Aldrich) as described [58]. DCs were subsequently lysed with the cell lysis buffer (Cell Signaling Technology). Immunoprecipitation and immunoblot analysis were performed as described [58].

Densitometry analysis

Densitometry analysis was performed using Scion Image software (Scion Corporation).

Reporter assay

The pGL3-luciferase reporter plasmid containing wild-type mouse CD40 promoter fragment (-505 to +22 region, represented here as p505-luc and previously described as D9 [8]) was provided by Prof. Herman Waldmann (University of Oxford, UK). The mutant CD40 promoter-luciferase constructs p505-MutR1-luc, p505-MutR2-luc and p505-Mut(R1+R2)-luc, in which the Runx-binding sites R1 (^-489TGTGGT^-484), R2 (^-464TGCGGT^-459) or both R1 and R2 were substituted with GAATTC sequence, were created by Mutagenex Inc. JAWSII cells (American Type Culture Collection; 5 x 10⁵/well) were transfected with either of the above-mentioned reporter constructs (900 ng) together with a control renilla luciferase reporter pRL-CMV (100 ng) using the TransIT-2020 transfection reagent (Mirus). After 24 h, cells were either left untreated or treated for 24 h with LPS or TNFα. Luciferase activity was assayed with the dual-luciferase reporter assay kit (Promega).

RNA-mediated interference

DCs were transfected with 60 nM siRNA using Lipofectamine RNAiMAX reagent (Invitrogen).

Confocal microscopy

Immunostaining and confocal microscopy were performed as described [67]. Briefly, BMDCs (2.5 x 10⁵) were adhered to coverslips (12 mm) for 6 h and stimulated with LPS, TNFα or SAG for 0.5 h. In some experiments, DCs were treated with PI3K inhibitors Wort or LY for 1 h, or infected with promastigotes of normal LD (AG83; Sb^SLD strain) or antimony-resistant LD (GE1F8R; Sb^RLD strain) for 24 h prior to LPS, TNFα or SAG stimulation as specified. Cells were washed with phosphate buffered saline (PBS), fixed for 5 min in 4% paraformaldehyde, permeabilized with 0.1% Triton X-100 for 5 min and then incubated with 3% BSA for 1 h at room temperature. Subsequently, cells were incubated with anti-Runx1, anti-Runx3 or corresponding isotype control antibody (all 1: 500 dilutions) for 1 h at room temperature. After washing, cells were further incubated for 1 h at room temperature with Alexa Fluor 568-conjugated goat anti-rabbit IgG or goat anti-mouse IgG (1: 1000 dilution). For staining of nuclei, Hoechst (Thermo Fisher Scientific) was used. After mounting, images were acquired with a NIKON A1R Laser scanning confocal microscope. Images were processed using NIS-Elements version 4.13.01 and Adobe Photoshop software.

Assessment of CD40 expression on sDCs derived from LD-infected mice

BALB/c mice between 4–6 wks of age were injected i.v. with stationary phase LDPm (2 x 10⁷/mouse) or left uninfected. After 45 days, splenocytes were prepared from these LD-infected mice or age-matched uninfected mice and stimulated with LPS for 24 h. The level of CD40 expression on sDCs was measured via flow cytometry after gating on CD11c⁺ population.

DC transfer experiments

BALB/c BMDCs were transfected or not with Runx1- and Runx3-specific siRNAs or with control siRNA and then stimulated with SAG for 24 h. In some experiments, prior to SAG stimulation, Runx1 and Runx3 siRNA-treated DCs were transfected with 2 μg empty vector or CD40-expressing vector (pcDNA3-CD40) using the TransIT-2020 transfection reagent (Mirus). DCs were then injected i.v. into LD-infected BALB/c mice (on days 15, 25, 35 and 45 postinfection). On day 48 postinfection, spleens and livers were isolated to measure their weights and parasite burden, and the frequencies of splenic CD4⁺ and CD8⁺ T cells producing IFNγ or IL-10. The spleen and liver parasite burden was measured by stamp-smear method and is presented as Leishman-Donovan Unit (LDU) [68]. For detection of IFNγ and IL-10 within CD4⁺ and CD8⁺ T cells, the RBC-depleted splenocytes were first surface-stained with anti-CD3-APC together with anti-CD4-FITC, anti-CD8-FITC or rat IgG-FITC (isotype control). Splenocytes were then subjected to intracellular staining using a Fixation/Permeabilization Buffer kit (eBioscience; 88-8823-88), and PE-labeled anti-mouse IFNγ, anti-mouse IL-10 or rat IgG (isotype control). The percentage of IFNγ- or IL-10-producing CD4⁺ and CD8⁺ cells in the CD3-gated cell population was determined via flow cytometry. The mouse CD40 cDNA used in this experiment was obtained from Prof. David Wagner (University of Colorado, USA) and cloned into a pcDNA3 plasmid (Invitrogen) using BamHI and XhoI sites.

Flow cytometry

Flow cytometry was performed with a C6 Accuri flow cytometer (BD Biosciences). Data were analyzed with FlowJo software (Tree Star).

Statistical analysis

One-way ANOVA (SigmaPlot 11.0 program) was used for statistical analyses. A “p” value < 0.05 was considered significant.