Inhibition of CD40-Induced N-Ras Activation Reduces Leishmania major Infection

CD40, a transmembrane costimulatory receptor expressed on APCs such as macrophages and dendritic cells, plays crucial roles in autoimmune and infectious diseases, transplant rejection, and tumor regression (1–4). Blockade of CD40–CD154 interaction prevents autoimmune diseases and transplant rejection but abrogates host protection against pathogens. CD40 induces activation of ERK-1/2–mediated anti-inflammatory IL-10 production and p38MAPK-mediated proinflammatory IL-12 production, depending on the strength of signaling (5). Leishmania major, a protozoan parasite that causes cutaneous leishmaniasis, switches CD40 signaling to ERK-1/2 with concomitant decrease in p38MAPK activation (5). CD40-induced IL-10 promotes Leishmania infection whereas CD40-induced IL-12 protects hosts from the infection. These observations demonstrated a functional duality for CD40. Because CD40 reciprocally signals through Raf-MEK-ERK and PI3K-MKK-p38MAPK pathways to trigger counteractive effects on L. major infection, and because both PI3K and Raf-1 have a Ras-binding domain (6), the L. major–induced switching of the signal implies that Ras isoforms, that is, H-Ras, K-Ras and N-Ras, may have differential involvements in L. major infection.

Although Ras is implicated in CD40 signaling, differential involvement of the Ras isoforms in CD40-induced antileishmanial functions remained to be demonstrated. A recent report showed that mice deficient in either H-Ras or N-Ras in T cells had transiently higher L. major infection than did wild-type C57BL/6 mice, suggesting the role of these Ras isoforms in Th1 response (7). However, the report had two major lapses. First, the mice were on a genetically resistant background and so limited the infection and perturbation of macrophages. Second, the authors did not study the effect of K-Ras deficiency, as K-Ras–deficient mice die embryonically (8). Therefore, we examined the differential involvement of Ras isoforms in L. major infection. Our results demonstrate that in L. major–infected macrophages, their expression and activation are differentially modulated. The reciprocal modulation of expression and activity of N-Ras and K-/H-Ras isoforms are associated with the outcome of L. major infection. N-Ras short hairpin RNA (shRNA) and a novel N-Ras–blocking peptide derived from Sos-1/2, the N-Ras–activating guanine nucleotide exchange factor (GEF), provided significant host protection against L. major infection. Thus, although Ras isoforms have so far been reported as structurally and functionally homologous, and conventionally associated with tumor transformation, we demonstrate, for the first time to our knowledge, that Ras isoforms do have unique functions in untransformed cells. The biological significance of our findings is exemplified by its application in a susceptible model of cutaneous leishmaniasis.

Western blot Abs for p-p38MAPK, p–ERK-1/2, ERK-1/2, β-actin, H-Ras, N-Ras, K-Ras, and Luminol reagent were from Santa Cruz Biotechnology. Western blot Ab for p38MAPK was from Cell Signaling Technologies (Danvers, MA). Anti-cytokine Abs (IL-12, IL-10, IFN-γ, and IL-4), cytokine standard for ELISA, and anti-CD40 Ab (NA/LE; clone 3/23) were from BD Biosciences (San Diego, CA). EZ-detect Ras activation kit and UltraLink immobilized protein A/G Plus was from Pierce Biotechnology (Rockford, IL). Pam₃CSK₄ was from InvivoGen (San Diego, CA) and purified anti-lipophosphoglycan (LPG) was from Cedarlane Laboratories (Burlington, ON, Canada). Anti-TLR2–blocking and FITC-conjugated TLR2 and isotype Abs were procured from eBioscience or Imgenex.

C57BL/6, BALB/c, and CD40-deficient (BALB/c background) mice (6–8 wk old), originally from The Jackson Laboratory (Bar Harbor, ME), were bred in the Institute’s experimental animal facility. The progress of infection was monitored weekly, and parasite load was assessed after euthanization. All experiments were in accordance with the animal use protocol approved by the Institutional Animal Care and Use Committee and the Committee for the Purpose of Control and Supervision of Experiments on Animals, the regulatory authorities for animal experimentation. P388D1, a macrophage-like cell line, was from the American Type Culture Collection and was cultured in RPMI 1640 containing penicillin (100 U/ml), streptomycin (100 μg/ml), 2-ME (50 μM), sodium pyruvate (1 μM), HEPES (20 μM), and 10% heat-inactivated FCS. L. major (strain MHOM/Su73/5ASKH) was maintained in vitro in RPMI 1640 with 10% FCS (Life Technologies/BRL, Grand Island, NY), and its virulence was maintained by passaging through BALB/c mice.

Mice were injected with 2 ml thioglycolate (3%, i.p.). Four days later, peritoneal exudate cells were harvested in sterile HBSS, centrifuged (1200 rpm, 8 min), and the pellet was resuspended in RPMI 1640 supplemented with 10% FCS. The cells in RPMI 1640 with 10% FCS were counted in a cell counting chamber, seeded in culture plates according to experimental requirements, and were cultured at 37°C in a humidified CO₂ incubator for 12 h. Nonadherent cells were removed by replacing the medium with fresh RPMI 1640 with FCS. The culture was maintained for 24 h.

Following the indicated treatments, macrophages were washed twice with ice-cold PBS and resuspended in an appropriate (∼200–300 μl) volume of ice-cold lysis buffer (1% Nonidet P-40, 25 mM Tris-HCl [pH 7.5], 150 mM NaCl, 5 mM MgCl₂, 5% glycerol, phosphatase inhibitor mixture [Pierce Biotechnology], and protease inhibitor mixture [Roche Applied Science, Mannheim, Germany]) for 30 min and kept for gentle rocking at 4°C. Lysate was centrifuged (12,000 rpm, 15 min; 4°C) and supernatant was collected. Protein was quantified using a BCA kit and an equal amount of protein was run on SDS-PAGE. Resolved proteins were blotted to a polyvinylidene difluoride membrane (Millipore, Bedford, MA), blocked with 5% non-fat dried milk in TBST (25 mM Tris [pH 7.60], 137 mM NaCl, and 0.2% Tween 20), incubated with primary Ab at 4°C overnight, washed with TBST, and probed with HRP-conjugated secondary Ab. Immunoreactive bands were visualized by chemiluminescent Luminol reagent (5, 9). Densitometric analyses of the bands were performed by using ImageJ software.

C57BL/6- or BALB/c-derived elicited macrophages were infected with stationary phase L. major promastigotes at a ratio of 1:10 for 6 h (5), followed by removal of extracellular parasites (>75% infection), transduction with lentivirally expressed shRNA or Sos peptides (4 h), and treatment with anti-CD40 Ab (3 μg/ml) for 62 h. The cells were fixed with chilled methanol for 5 min and stained with Giemsa. The amastigotes per 100 macrophages were counted under a Nikon Eclipse E600 microscope.

The expression of transcripts encoding H-Ras, N-Ras, and K-Ras was analyzed by real-time PCR using following primers: H-Ras (forward, 5′-GCCATCAACAACACCAAGTC-3′, reverse, 5′-AGCCAGGTCACACTTGTTG-3′), K-Ras (forward, 5′-CTTGGATATTCTCGACACAGC-3′, reverse, 5′-CCTCCCCAGTTCTCATGTAC-3′), and N-Ras (forward, 5′-AGAGACCAGTACATGAGGACAG-3′, reverse, 5′-CACACGCTTAATTTGCTCC-3′). RNA from macrophages was isolated using TRI Reagent (Sigma-Aldrich, St. Louis, MO) and reverse transcription was carried out using Moloney murine leukemia virus reverse transcriptase (Invitrogen Life Technologies, Carlsbad, CA) as described previously (5). The reactions were performed in thin-wall 0.2-ml strip tubes (Axygen, Union City, CA) and the volume of each reaction mixture was 10 μl. Reaction mixture contains 10 ng cDNA, 2 ng forward primer, 2 ng reverse primer, and 2× iQ SYBR Green Supermix (5 μl; Bio-Rad; Hercules, CA). Quantitative real-time PCR was performed on an Eppendorf realplex4 Mastercycler under the following conditions: 95°C for 2 min, 40 cycles of 95°C for 1 min, 60°C for 30 s, 72°C for 35 s. Reactions were performed in duplicates. Relative quantitation was done using the comparative threshold (∆∆Ct) method. mRNA expression levels of the target genes were normalized against those of GADPH levels and expressed as a relative fold change compared with untreated controls.

Ras isoform transcripts in the 72 h L. major–infected macrophages were analyzed by RT-PCR (9) using the primers: H-Ras (forward, 5′-ATGACAGAATACAAGCTTGTGGTGG-3′, reverse, 5′-TCAGGACAGCACACATTTGCAG-3′), K-Ras (forward, 5′-GTGGTGGTTGGAGCTGGT-3′, reverse, 5′-GTCCTTGACTTCTTCTTCTTC-3′), N-Ras (forward, 5′-ATGACTGAGTACAAACTGGTG-3′, reverse, 5′-TACACATCAGCACACAGGG-3′), and GAPDH (forward, 5′-GAGCCAAACGGGTCATCATC-3′, reverse, 5′-CCTGCTTCACCACCTTCTTG-3′).

Active Ras pull-down assay was carried out using an active Ras pull-down and detection kit from Thermo Fisher Scientific (Rockford, IL). Ten million macrophages were used for the active Ras pull-down assay. After stimulation, cells were gently rinsed with ice-cold PBS. The PBS was removed and 600 μl lysis buffer was added. Lysate was vortexed briefly and centrifuged at 16,000 × g for 15 min at 4°C. Supernatant was subjected to protein estimation using a BCA assay kit. For each sample, a spin cup was placed into a collection tube and one to two glutathione resin cubes were added per cup, followed by 80 μg GST-Raf1 Ras-binding domain. Five hundred to 700 μg protein was loaded onto the spin cup. The reaction mixture was incubated at 4°C for 2–3 h with gentle rocking. The spin cup with collection tube was centrifuged (6000 × g; 1 min). The spin cup was transferred to a new collection tube. The spin cup containing the resins was washed thrice with lysis buffer and transferred to a new collection tube. Fifty microliters 2× reducing Laemmli sample buffer was added to resin and vortexed. Samples were heated for 5–10 min at 95°C and centrifuged (6000 × g; 2 min), followed by SDS-PAGE and Western blotting (5, 9).

Cycloheximide (50 μM for 3h; Sigma-Aldrich)-treated BALB/c-derived elicited macrophages (2 × 10⁷) were labeled with 0.5 mCi [³H]palmitic acid (MP Biomedicals, Solon, OH) for 4 h. Cells were suspended in TES buffer and disrupted by 20 passages through a 30-gauge needle. Homogenate was centrifuged (2000 × g; 10 min). The supernatant was loaded on a 4–24% iodixanol (Sigma-Aldrich) gradient and centrifuged (46,000 rpm; 1.5 h) for isolating plasma membrane and Golgi fractions (10).

The culture supernatant obtained from macrophages and lymph node cells was stimulated as indicated and assayed for IL-12/IL-10 and IL-4/IFN-γ secretion, respectively, by ELISA. Briefly, ELISA plates were coated overnight at 4°C with purified Abs to IL-10 (2 μg/ml), IL-12 (2 μg/ml), IL-4 (1 μg/ml), or IFN-γ (2 μg/ml). Plates were washed thrice (0.05% Tween 20 in PBS) and blocked for 2 h with 1% BSA. Plates were incubated overnight with cytokine standards or culture supernatants. Plates were washed and incubated with respective biotin-conjugated detection Abs for 1 h at 25°C. Plates were washed and incubated with peroxidase-conjugated streptavidin (Roche Applied Science) for 30 min followed by washing and development with tetramethylbenzidine substrate (BD Pharmingen, San Diego, CA). Reaction was stopped by the addition of 1 N H₂SO₄, and absorbance was measured at 450 nm.

N-Ras shRNA, H-Ras shRNA, K-Ras shRNA, and control shRNA were purchased from Open Biosystems (Huntsville, AL). The shRNAs were packaged in a Trans-Lentiviral packaging system, which is a replication-incompetent HIV-1–based lentivirus using the HEK293T cell line according to the manufacturer’s protocol. Briefly, 5.5 × 10⁶ cells were plated in a 100-mm culture dish. Sixteen hours later, 28.5 μg Trans-Lentiviral packaging mix was added to 9 μg shRNA plasmid DNA. Transfection was carried out in serum-free medium using Arrest-In transfection reagent (Open Biosystems) at 1:5 DNA: transfection reagent ratio. After 6 h, medium was changed with DMEM–10% FCS and cells were incubated for 72 h. Supernatant was harvested after 48 and 72 h and filtered though a 0.45-μm filter. Virus was concentrated 100-fold and titrated according to the manufacturer’s protocol (9).

C57BL/6, BALB/c, and CD40-deficient mice (BALB/c background) were injected s.c. with 2 × 10⁶ L. major promastigotes (50 μl). The disease was scored by measuring footpad swelling by a digital micrometer (Mitituyo, Kawasaki, Japan). Mice were sacrificed 5 wk after L. major infection and the parasite burden in the draining lymph node was assessed by limiting dilution analyses, as described earlier (5).

Three days after L. major infection, mice were treated with Sos-derived peptides, control peptides, and scrambled peptides (i.p., 6 mg/kg body wt for 3 alternate days) in combination with anti-CD40 Ab (50 μg/mouse; clone 3/23; from G. Klaus, National Institute of Medical Research, London, U.K.). For in vivo experiments using lentivirus treatment, mice were injected with 5 × 10⁶ transduction units (in 50 μl HBSS; Life Technologies) of lentivirus-expressing N-Ras shRNA (GenBank; http://www.ncbi.nlm.nih.gov/genbank/, accession no. NM_010937) in pGIPZ lentivirus vector, control shRNA in pGIPZ lentivirus vector, H-Ras shRNA (GenBank accession no. NM_008284), K-Ras shRNA (GenBank accession no. NM_021284) in pLKO.1 lentivirus vector, and control shRNA in pLKO.1 lentivirus vector. Lentivirus particles were injected s.c. in the L. major–infected hind footpad 2 d prior to infection.

BALB/c-derived peritoneal macrophages were transduced with lentiviral particles expressing Ras isoform–specific shRNA, TLR2 shRNA (GenBank accession no. NM_011905), or control shRNA for 8 h in RPMI 1640 supplemented with 0.5% FCS and 8 μg/ml Polybrene (Sigma-Aldrich). Residual viral particles were washed off. Forty-eight hours later, cells were stimulated with anti-CD40 Ab (3 μg/ml) for the indicated periods and processed for immunoblot analysis.

BALB/c-derived peritoneal macrophages were transduced with TLR2 shRNA or control shRNA. After 48 h, cells were scraped out and incubated with anti-CD16/32 for 30 min. After washing with FACS buffer (PBS with 2% FCS), cells were incubated with FITC-conjugated monoclonal anti-TLR2 Ab for 1 h, washed twice with FACS buffer, and acquired on a FACS CyAn ADP analyzer flow cytometer and analyzed by Summit software (Beckman Coulter, Brea, CA).

Model building of N-Ras was performed using co-ordinates for human N-Ras bound to GDP, obtained from the Protein Data Bank (11). The Ramachandran plot shows that 88.6% of all residues are in the most favored regions and 0.7% are in the disallowed regions. The guiding force in the initial docking of the peptide to its binding site is the electrostatic long-range interactions. Docking was performed using AutoDock (12). This ensures the complementary patches of positive and negative charges especially for those present in loop regions to be readily identified. After the initial docking, energy minimization via 1000 steps using conjugate gradient followed by short-run molecular dynamics simulation was performed with Gromacs simulation package.

The experiments were performed three times and the data from one representative experiment are shown. The cultures in vitro were in triplicates and six to eight mice were used per group. Data are presented as means ± SEM. Comparison between the groups was performed by employing a Student t test.

Because we observed alterations in CD40 signaling in L. major–infected macrophages (5, 9), we tested whether the parasite alters the expression of Ras isoforms to manipulate CD40 signaling. In L. major–infected macrophages, N-Ras and H-Ras were highly expressed, whereas K-Ras expression was reduced (Fig. 1A). Next, we assessed the kinetics of CD40-induced Ras GTPase activation in uninfected and L. major–infected macrophages. We observed that Ras activation increased with all doses, but the intensity of Ras GTPase activation at higher doses was significantly less in L. major–infected macrophages (Fig. 1B). Because K-Ras and H-Ras were activated at higher doses of anti-CD40 Ab treatments, the reduced Ras GTPase activation in L. major–infected macrophages corroborated with less K-Ras and H-Ras activation but higher N-Ras activation in infected macrophages (Fig. 1C). Higher expression but less activation of H-Ras in L. major–infected macrophages was perhaps due to its altered palmitoylation-dependent membrane targeting or entrapment in Golgi in the infected macrophages (Fig. 1D). Because CD40 activation resulted in N-Ras–mediated ERK-1/2–dependent IL-10 production (S. Chakraborty, A. Srivastava, A. Nair, S.P. Pandey, and B. Saha, unpublished observation), these data suggest that Leishmania switches CD40 signaling toward ERK-1/2–dependent IL-10 production by increasing N-Ras expression and activation in the infected macrophages, unveiling a novel parasite-employed immune evasion strategy.

During L. major infection, the host macrophage–expressed TLR2 interacts with the Leishmania expressed LPG (13). Blockade of TLR2 or LPG reduced L. major parasite number in infected macrophages (13, 14). Therefore, we tested whether TLR2 silencing reversed the L. major–modulated Ras expression. We observed that TLR2 silencing significantly prevented the upregulation of N-Ras expression and downregulation of K-Ras expression in TLR2 shRNA-transduced macrophages (Fig. 2A), which caused silencing of TLR2 expression (Fig. 2A, right panel). Likewise, both anti-TLR2 and anti-LPG Abs reversed the L. major–modulated expression of Ras isoforms (Fig. 2B). Corroborating these observations, a TLR2 ligand, Pam₃CSK₄, increased N-Ras expression but reduced K-Ras expression, recapitulating the L. major–modulated Ras isoforms expression in BALB/c-derived elicited macrophages (Fig. 2C). Thus, altogether these data indicate that while infecting the macrophages, the parasite selectively alters the expression of Ras isoforms.

Because L. major differentially regulated the expression and activity of Ras isoforms, we argued that these isoforms would have differential effects on L. major infection. Indeed, H-Ras–deficient mice and N-Ras–deficient mice on a resistant C57BL/6 background were reported to have higher L. major parasite load, albeit transient, than did the wild-type controls (7). Because these mice were developed as deficient in these genes and because Ras isoforms were reported to have roles in embryonic development (8, 15), the developmental defects on macrophages remain unknown. Therefore, we silenced N-Ras, K-Ras, and H-Ras expression in C57BL/6-derived elicited macrophages by transducing respective shRNA expressed in a lentiviral vector. L. major infection in these mice showed that the macrophages transduced with N-Ras shRNA had a parasite load comparable to that of the infection control or shRNA controls, but the macrophage transduced with K-Ras or H-Ras had a higher parasite load than did the control macrophages (Fig. 3A). We did not observe any differences in internalization among these groups (Fig. 3B). In C57BL/6 mice, K-Ras shRNA in a lentivirus increased the infection whereas N-Ras shRNA reduced the lesion size and infection (Fig. 3C, 3D). Thus, Ras isoforms play differential roles in L. major infection. Because N-Ras promoted L. major infection, its inhibition might reinstate host-protective immune response.

Because reduced N-Ras expression or activation resulted in host protection, its role in Ag-specific immune response was examined. Therefore, we cloned, expressed, and purified L. major MAPK10 (M10) (16). Peritoneal macrophages from CD40^+/+ mice were treated with the lentivirally expressed N-Ras shRNA or control shRNA, pulsed with M10, and injected s.c. in the hind footpad of CD40-deficient (CD40^−/−) mice. Treatment of mice with N-Ras shRNA but not the control shRNA resulted in increased IFN-γ and decreased IL-4 production from the M10-treated lymph node cells (Fig. 4A) and higher M10-specific IgM but lower IgG1 and insignificant IgG2a production (Fig. 4B), indicating N-Ras regulation of Ag-specific immune response. Because N-Ras regulated Ag-specific and antileishmanial immune responses, we examined whether Ag-specific priming using the Ag-pulsed, N-Ras–silenced macrophages would have a host-protective immunoprophylactic effect. We observed that priming of CD40^−/− mice, which were more susceptible to Leishmania than were CD40^+/+ mice, with the N-Ras shRNA-treated, M10-pulsed macrophages resulted in significantly lower parasite burden (Fig. 4C) and higher M10-specific IFN-γ production (Fig. 4D) than for the CD40^−/− mice that received the control shRNA-treated, M10-pulsed macrophages, demonstrating a novel N-Ras isoform–targeted host-protective immunoprophylaxis.

Because L. major infection enhances CD40-induced ERK-1/2 phosphorylation (5, 9) through activation of Raf-1, a Ras-binding protein (17), and because N-Ras also enhances ERK-1/2–mediated production of IL-10, a proparasitic cytokine (5, 9), we tested whether N-Ras silencing would reduce L. major infection in susceptible BALB/c mice. Treatment of macrophages with lentivirally expressed N-Ras shRNA but not control shRNA reduced CD40-induced phosphorylation of ERK-1/2 but augmented phosphorylation p38MAPK (Fig. 5A, left panel), accompanied by enhanced antileishmanial effects of CD40 (Fig. 5A, right panel). Administration of anti-CD40 Ab along with N-Ras shRNA but not control shRNA significantly reduced N-Ras expression in footpad and the draining lymph node (Fig. 5B, inset), footpad swelling, parasite load, and lymph node weight in L. major–infected susceptible BALB/c mice (Fig. 5B, 5C). Although a low dose (1 μg/ml) anti-CD40 Ab treatment of macrophages in vitro increased N-Ras activation, leading to enhanced IL-10 production, the high-dose anti-CD40 Ab (50 μg/mouse for 3 d) treatment alone conferred some resistance. Compared to untreated infected mice, lymph node cells from L. major–infected, N-Ras shRNA with anti-CD40 Ab–treated mice secreted significantly less IL-4 but more IFN-γ, a cytokine responsible for L. major elimination (Fig. 5D). Because IL-10 inhibits IFN-γ–secreting Th1 cells (18), that the N-Ras shRNA regulated less IL-10 production might have resulted in higher IFN-γ production. Corroborating higher IFN-γ production in N-Ras–treated mice, we observed increased leishmanial Ag-specific IgG2a in the sera of infected mice treated with N-Ras shRNA and anti-CD40 compared with the control groups (Fig. 5E). Additionally, macrophages obtained from N-Ras shRNA-treated L. major–infected BALB/c mice showed CD40-induced enhanced p38MAPK activation and IL-12 production but reduced N-Ras expression, ERK-1/2 activation, and IL-10 production compared with untreated or control shRNA-treated L. major–infected mice (Fig. 5F). N-Ras shRNA administration did not reduce footpad swelling, parasite load, and lymph node weight in L. major–infected CD40^−/− mice (Fig. 5G), suggesting that the antileishmanial effects of silencing of N-Ras was lost in the absence of CD40. These observations indicate that lentivirally expressed N-Ras shRNA mediates antileishmanial effects by enhancing the Th1 response, which is responsible for eliminating L. major. Collectively, these observations suggest that the parasite-enhanced N-Ras expression was crucial for the infection in a susceptible host; reducing N-Ras expression by N-Ras shRNA reinstates the host-protective antileishmanial functions of CD40. Whereas the CD40-sufficient mice have both proparasitic and antiparasitic signaling and effector pathways, the CD40-deficient mice do not have any of these activated. Therefore, the parasite devised immune evasion strategy by suppressing the antiparasitic signaling pathway exhibited in the CD40-sufficient mice.

As Sos interacts with N-Ras and activates it (S. Chakraborty, A. Srivastava, A. Nair, S.P. Pandey, and B. Saha, unpublished observation), we performed computational modeling of N-Ras–Sos interaction. We designed two Sos-derived peptides (24-mer Sos peptide 1; 23-mer Sos peptide 2) and assessed whether they blocked the association between Sos and N-Ras, recapitulating the N-Ras shRNA effects. The computational docking of Sos-derived peptides on N-Ras was performed (Fig. 6A). The significant point of interaction in N-Ras–Sos peptide 1 (Sos-P1) is between Gly²–Asp³⁸ and Met⁹–Glu⁶³ and the interaction observed for Sos peptide 2 (Sos-P2) is between Ser⁷–Asp³⁸ and Met⁸–Arg⁴¹ (19, 20). We observed that both Sos-P1 and Sos-P2 inhibited CD40-induced N-Ras activation (Fig. 6B). Treatment of macrophages with Sos-P1 reduced CD40-induced ERK-1/2 activation and IL-10 production, but reciprocally enhanced p38MAPK activation and IL-12 production (Fig. 6C, 6D). Similar to N-Ras shRNA, Sos-P1 enhanced CD40-induced antileishmanial activity in L. major–infected macrophages (Fig. 6E). Administration of Sos-P1 but not the control and scrambled peptides significantly reduced footpad swelling and parasite load in L. major–infected susceptible BALB/c mice (Fig. 6F). The lymph node cells from L. major–infected Sos-P1– and anti-CD40–treated mice produced more IFN-γ and less IL-4 compared with the lymph node cells from untreated infected mice (Fig. 6G). Alternatively, Sos-P2–N-Ras interaction (Fig. 6H) resulted in CD40-induced less ERK-1/2, but higher p38MAPK, phosphorylation (Fig. 6H, right panel), less IL-10, but higher IL-12, production (Fig. 6I), and stronger antileishmanial function (Fig. 6J). However, the antileishmanial effects of N-Ras activation blockade using Sos-P1 or Sos-P2 were not observed in CD40^−/− mice (data not shown), suggesting that the antileishmanial effects of inactivation of N-Ras was lost in the absence of CD40. Thus, although these observations identify CD40-induced N-Ras activation as a crucial antileishmanial target (Fig. 7), its use as a novel Ras isoform–targeted therapy requires further detailed investigation.

Befitting the principle of parasitism, the protozoan parasite Leishmania modulates host macrophage signaling (21, 22) to ensure its survival. Instead of indiscriminate suppression of host cell signaling, the parasite reduces the signaling that eventuates in its elimination but enhances those that support parasite survival (5). Whereas such counteractive signaling pathways may be part of two antagonistic receptors such as CD28 and CD152, we established the single receptor plasticity whereby CD40 signals reciprocally through ERK-1/2 and p38MAPK that result in counteractive functions such as parasite survival or parasite elimination, respectively (5). We established a novel bimodular architecture of the CD40 signaling pathway (23) and showed that L. major switches CD40 signaling from the p38MAPK module to the ERK-1/2 module. Because of two transmodular feedback loops, activation of ERK-1/2 in one module reduces p38MAPK activation in the other module. Ras isoforms play important roles in the sustenance of this reciprocity and are therefore targeted by the parasite L. major.

Our findings revealed, for the first time to our knowledge, that as an expression of receptor plasticity, CD40 can differentially activate cellular Ras isoforms to modulate its reciprocal signaling outcome and thus maintain the cellular homeostasis. In contrast, as cell membrane of the L. major–infected macrophages becomes more detergent-soluble, CD40 starts signaling more through the TRAF6–syk signalosome complex (9). Syk activates Sos-1/2 that works as a GEF for N-Ras activation. Thus, the initial switch from the TRAF3-lyn signalosome to TRAF6-syk signalosome that results in predominant signaling through the N-Ras-Raf-MEK-ERK module accentuates the activity of the ERK-1/2 module leading to collapse of the CD40 signaling reciprocity. As a result, Leishmania perpetuates in the host.

In the present study, we reveal several novel facts about Leishmania-induced Ras modulation. First, N-Ras and H-Ras expression increased but K-Ras expression decreased in L. major–infected macrophages. Activity of N-Ras was increased but activities of H-Ras and K-Ras were reduced. The reduced H-Ras activity despite its enhanced expression was due to its entrapment in Golgi owing to less palmitoylation. Thus, Ras isoforms are differentially targeted by the parasite. Second, such regulation was effected through LPG–TLR2 interaction during the infection. Blockade of this interaction reversed the L. major–induced changes in Ras isoform expression whereas treatment of macrophages with a TLR2 ligand recapitulated the infection-induced changes in Ras isoforms’ expression. Therefore, it is possible that the initial skewing toward the Ras-Raf-MEK-ERK pathway through TLR2 adds advantage to subsequent CD40 signaling through the ERK-1/2 module. Owing to the transmodular feedback loop between ERK and lyn, the p38MAPK signaling module is inhibited further. Thus, the L. major–enhanced N-Ras expression and activation present a novel form of TLR–CD40 crosstalk targeted for devising a novel immune evasion strategy or for formulating a novel immunotherapeutic strategy. Third, N-Ras–redirected CD40 signaling through ERK-1/2 leads to the production of IL-10 that helps L. major survive in a susceptible host (5) by inhibiting secretion of type 1 cytokines (24). Silencing N-Ras or blocking N-Ras activation hampers IL-10 production and skews CD40 signaling toward p38MAPK activation, leading to IL-12 production, a host-protective Th1 response against Leishmania infection (25). Fourth, we show that regulation of N-Ras in macrophages can control Ag-specific immune responses. Blockade of N-Ras in the Ag-presenting macrophages resulted in IFN-γ–dominated response against the purified M10 Ag. Finally, we exemplify, for the first time to our knowledge, how Ras isoform–specific targeting leads to both immunotherapeutic and immunoprophylactic strategies. Treatment of L. major–infected susceptible BALB/c mice with lentivirally expressed N-Ras shRNA significantly reduced Leishmania infection by mounting a host-protective Th1 response. Finding the physical association between Sos-1/2 and N-Ras prompted us to model the interaction for the first time. Based on the result of the modeled interaction, we designed the peptide that inhibited N-Ras activation. Administration of the peptide resulted in Th1-accompanied protection of a susceptible host from L. major infection.

The observations described in this study thus provide a plausible mechanism of Ras-targeted survival strategy of L. major (Fig. 7). As the parasite-expressed LPG interacts with the macrophage-expressed TLR2 during infection, K-Ras expression is downregulated but N-Ras expression is upregulated. Leishmania also switches CD40 signaling to syk that activates Sos-1/2, which acts as a GEF for N-Ras. Thus, N-Ras is doubly affected, leading to inhibition of the p38MAPK pathway and collapsing of the reciprocity. Because adjustment of signal strength in the counteractive kinase modules (p38MAPK and ERK-1/2) is key to reinstatement of cellular homeostasis, the loss of reciprocity implies that the cell cannot re-establish antileishmanial functions. As a result, the parasite survives. To impart the host-protective functions, the N-Ras isoform is targeted. Such selective modulation of Ras isoforms suggests a possible mechanism for achieving signaling specificity, one of the most difficult and unsolved issues in biology. Thus, given the pivotal roles of CD40 in immune responses and multiple roles of Ras GTPases in various cellular processes, our findings not only set up a new framework for receptor responsiveness and cellular functions but also imply a huge potential in Ras isoform–targeted therapy in many diseases.

The authors have no financial conflicts of interest.