TLR2 dimer‐specific ligands selectively activate protein kinase C isoforms in Leishmania infection

Abstract

Of the thirteen Toll‐like receptors (TLRs) in mice, TLR2 has a unique ability of forming heterodimers with TLR1 and TLR6. Such associations lead to selective cellular signalling and cellular responses such as cytokine expression. One of the signalling intermediates is protein kinase C (PKC); of which, eight isoforms are expressed in macrophages. Leishmania—a protozoan parasite that resides and replicates in macrophages—selectively modulates PKC‐α, PKC‐β, PKC‐δ and PKC‐ζ isoforms in macrophages. As TLR2 plays significant roles in Leishmania infection, we examined whether these PKC isoforms play selective roles in TLR2 signalling and TLR2‐induced anti‐leishmanial functions. We observed that the TLR2 ligands—Pam₃CSK₄ (TLR1/2), PGN (TLR2/2) and FSL (TLR2/6)—differentially phosphorylated and translocated PKC‐α, PKC‐β, PKC‐δ and PKC‐ζ isoforms to cell membrane in uninfected and L. major‐infected macrophages. The PKC isoform‐specific inhibitors differentially altered IL‐10 and IL‐12 expression, Th1 and Th2 responses and anti‐leishmanial effects in macrophages and in BALB/c mice. While PKC isoforms’ inhibitors had insignificant effects on the Pam3CSK4‐induced anti‐leishmanial functions, PGN‐induced pro‐leishmanial effects were enhanced by PKC‐(α + β) inhibitors, whereas PKC‐(δ + ζ) inhibitors enhanced the anti‐leishmanial effects of FSL. These results indicated that the ligand‐induced TLR2 dimerization triggered differential dose‐dependent and kinetic profiles of PKC isoform activation and that selective targeting of PKC isoforms using their respective inhibitors in combination significantly modulated TLR2‐induced anti‐leishmanial functions. To the best of our knowledge, this is the first demonstration of TLR2 dimer signalling through PKC isoforms and TLR2‐induced PKC isoform‐targeted anti‐leishmanial therapy.

TLR2 dimer‐specific ligands selectively induce PKC isoforms activation. L. major impairs PKC phosphorylation and membrane translocation in macrophages. TLR2‐ligand + PKC inhibitor co‐treatments alter parasite load and Th subset response.

Abbreviations

FSL

fibroblast‐stimulating lipopeptide

PGN

proteoglycans

P3C

Pam₃CSK₄

PKC

protein kinase C

TLR

Toll‐like receptor

INTRODUCTION

Toll‐like receptors (TLRs) act as innate immune sensors that recognize pathogen‐associated molecular patterns (PAMPs) and are therefore termed pathogen recognition receptors (PRRs) [1]. TLRs can be broadly divided based on their location—either on cell surface or in endosomal compartments [2]. Among the cell surface TLRs, TLR2 forms homodimers or forms heterodimers with TLR1 or TLR6 [2]. These TLR2 dimers recruit adaptor proteins at the membrane to form signalosome complex [2, 3] and signal through Myd88‐dependent pathway [2, 4], leading to production of various cytokines [5, 6, 7, 8]. Although different kinases such as IRAK1‐IRAK4 and the kinases that activate NF‐κB are shown to be involved in TLR2 signalling, little is known about the other kinases involved in TLR2 signalling [9, 10]. As TLRs are involved in pathogen recognition and subsequent immune response modulation, we tested the activity of TLR2 ligands in Leishmania‐infected macrophages and susceptible BALB/c mice.

Leishmania is a protozoan parasite that resides and replicates within mammalian macrophages and inflicts a complex of diseases called Leishmaniases in 88 countries. TLR2 is shown to play important roles in Leishmania infection [11, 12, 13]. We previously showed that the TLR1/2 ligand, Pam3CSK4, enhanced ERK‐1/2 phosphorylation and IL‐10 production, whereas TLR2/6 ligand, BPP‐cys‐MPEG, increased p38MAPK phosphorylation and IL‐12 production [12]. Leishmania alters many kinases and phosphatases in macrophages to engineer its survival within the host [14]. One of these kinases is represented by the isoforms of protein kinase C (PKC). Protein kinase C is a serine/threonine kinase that regulates diverse cellular responses including immune responses [15, 16, 17]. Based on cofactor requirements for their activation, protein kinase C (PKC) isoforms are divided into three classes—classical PKC isoforms (α, βI, βII, γ—using calcium and DAG as cofactors for activation), novel PKC isoforms (δ, ε, η, θ—using only DAG, but not calcium, for activation) and atypical PKC isoforms (ζ,λ/ι—independent of both calcium and DAG) [18]. According to their cofactor requirements, the primary structures of these PKC isoforms show the presence or deletion of Ca⁺⁺‐binding sites and DAG‐binding sites [18]. PKC isoforms are shown to regulate immune cell signalling, and their inhibition is shown to alter immune cell effector functions in Leishmania infection [19]. Although the roles of TLR2 and PKC are known in Leishmania infection, whether PKC isoforms participate in TLR1/2 and TLR2/6 signalling in uninfected and Leishmania‐infected macrophages and, if they do, whether TLR2 signalling through PKC isoforms affects Leishmania infection in a susceptible host remain to be examined.

Herein, using three different TLR2 ligands—Pam₃CSK₄ (TLR2/1), PGN (TLR2/2) and FSL (TLR2/6)—we demonstrated that different TLR2 dimer‐specific ligands resulted in differential cytokine production (IL‐10 and IL‐12) through selective activation of protein kinase C isoforms. Treatment of BALB/c‐derived macrophages with these ligands resulted in different time‐ and dose‐dependent phosphorylation and membrane translocation patterns among the PKC isoforms. Subsequently, using different PKC inhibitors, we observed that this differential PKC isoform activation led to generation of differential IL‐10 and IL‐12 production. Treatment of BALB/c mice, a susceptible host, with FSL significantly reduced L. major infection. TLR2 ligands in the presence of PKC‐(α + β) inhibitors significantly reduced the infection, and the anti‐leishmanial effect was accompanied by increased IFN‐γ production. Thus, the data presented here show for the first time that PKC isoforms selectively participate in TLR1/2 and TLR2/6 signalling and subsequent effector functions and that the PKC‐(α + β) inhibitors significantly enhance the pro‐leishmanial effect of PGN, whereas PKC‐(δ + ζ) inhibitors significantly augment the anti‐leishmanial effect of TLR2/6 in a susceptible host.

MATERIALS AND METHODS

Reagents

PKC‐α/β myristoylated pseudosubstrate peptide inhibitor, PKC‐β inhibitor and PKC‐ζ myristoylated pseudosubstrate peptide inhibitors were from Calbiochem (San Diego, CA). Cell‐permeable myristoylated translocation peptide inhibitor for PKC‐δ (V1‐1; Myr‐SFNSYELGSL‐OH) was synthesized from GenPro Biotech (New Delhi, India). Abs specific for p‐PKC‐βII, p‐PKC‐δ, p‐PKC‐ζ (Cell Signaling Technology, Danvers, MA), p‐PKC‐α, PKC‐α, p‐PKC‐βI, PKC‐βI, PKC‐βII, PKC‐δ, PKC‐ζ (Santa Cruz Biotechnology, Santa Cruz, CA) and Pan‐Ras (Pierce, Rockford, IL) were derived. Anti‐cytokine antibodies (IL‐10, IL‐12) and standard cytokines for ELISA were from BD Biosciences (San Diego, CA). PAM3CSK4, PGN and FSL were procured from Invivogen (San Diego, CA).

Mice and parasite

BALB/c mice were originally procured from the Jackson Laboratories (Bar Harbor, ME) and were bred and maintained in the National Center for Cell Science's experimental animal facility. All animal handling protocols were approved by the Institute's Animal Care and Use Committee. L. major (strain MHOM/Su73/5ASKH) was maintained in vitro in RPMI‐1640 medium with 10% FCS (Life Technologies, BRL, Grand Island, NY), and the virulence was maintained through passaging in BALB/c mice.

Peritoneal macrophages were harvested from BALB/c mice after five days of thioglycolate (3% thioglycolate, 2 ml) injection. Macrophages were cultured in RPMI‐1640 with 10% FCS in RPMI‐1640. After 6 h, non‐adherent cells were washed out and macrophages were incubated at 37° temperature with 5% CO₂ in humidified atmosphere for 24 h before stimulation.

L. major infection of macrophages

Thioglycolate‐elicited BALB/c‐derived peritoneal macrophages were cultured in RPMI‐1640 supplemented with 10% FCS and were infected with stationary‐phase L. major promastigotes at a 1:10 (macrophage:parasite) ratio for 6 h, following which the extracellular parasites were washed out and the PKC inhibitors were added. 2 h later, TLR ligands were added to the culture without removing the inhibitors. Treated and untreated macrophages were washed, fixed with methanol and stained with Giemsa. Amastigotes were counted in 6‐h and 72‐h infected macrophages [20, 21].

PKC isoform inhibitor and treatment

PKC‐α/β inhibitor is myristoylated pseudosubstrate peptide (PKC 20‐28, myristoyl‐FARKGALRQ‐OH), which contains pseudosubstrate peptide region of PKC‐α and PKC‐βI isoforms, and its N‐terminus is myristoylated to provide membrane permeability [22]. Cell‐permeable PKC‐βII inhibitor is chemically an anilino‐monoindolylmaleimide compound, which specifically inhibits PKC‐β isoform [22, 23]. PKC‐ζ inhibitor is cell‐permeable, pseudosubstrate peptide inhibitor of PKC‐ζ and acts as a competitive inhibitor [24]. Cell‐permeable PKC‐δ inhibitor (V1‐1; Myr‐SFNSYELGSL‐OH) is a myristoylated pseudosubstrate inhibitor, which contains pseudosubstrate peptide sequence from PKC‐δ isoform [25, 26]. The doses of PKC inhibitors were PKC‐α, 15 μm; PKC‐β, 7 nm; PKC‐δ, 10 μm; and PKC‐ζ, 15 μm.

Subcellular fractionation

Cells were washed twice with ice‐chilled PBS and were scraped in 1 ml PBS after treatment with the PKC isoform inhibitors and TLR ligands for indicated time periods. After washing, cells were centrifuged and resuspended in lysis buffer (20 mm Tris [pH 7.5], 150 mm NaCl, 10% glycerol, 1 mm EDTA, 1 mm EGTA, protease inhibitor mixture, and phosphate inhibitor mixture) without nonidet P‐40. The cell suspension was sonicated using sonicator (Vibra‐Cell, New Town, CT; five times for 10 s each). Sonicated samples were centrifuged at 1000 × g for 10 min at 4° to remove nuclei, granules and unbroken cells. Supernatants were collected and ultracentrifuged (TLA‐100 rotor; Beckman Coulter) at 100 000 × g at 4° for 30 min to get membrane fractions. After centrifugation, membrane fraction‐containing pellet was collected, washed and resuspended in lysis buffer containing 1% nonidet P‐40 [22].

L. major infection to mice

BALB/c mice were inoculated with 2 × 10⁶ stationary‐phase L. major parasites in the left footpad. After three days of infection, TLR2 ligands (P3C‐2 μg, PGN‐10 μg and FSL‐5 μg) and PKC inhibitors (5 mg/kg body weight) were injected. 35 days post‐infection, mice were killed and lymph nodes were collected for evaluating parasite load and FACS assay.

Macrophage–T‐cell co‐culture

BALB/c‐derived peritoneal macrophages were infected with L. major promastigotes for 6 h at a 1:10 ratio followed by washing of extracellular parasites. The infected macrophages were co‐cultured with the CD4⁺ T cells at the ratio of 3:1 (T cells: macrophages) isolated from the lymph node of L. major‐infected, PKC inhibitor and TLR2 ligand‐treated mice. 72 h after incubation, macrophages were washed and fixed with methanol. The cells were stained with Giemsa stain, and intracellular amastigotes were counted under a light microscope (E‐600; Nikon).

Western blotting

BALB/c peritoneal macrophages after treatment with indicated doses of isoform‐specific PKC inhibitors and TLR ligands for indicated time periods were washed twice with ice‐chilled PBS and lysed with cell lysis buffer (20 mm Tris [pH 7.5], 150 mm NaCl, 10% glycerol, 1 mm EDTA, 1mM EGTA, 1% nonidet P‐40 and protease inhibitor mixture; Roche Applied Science, Mannheim, Germany) and phosphatase inhibitor mixture (Pierce) for 10 min at 4°. The cell lysates were centrifuged at 18 000 × g for 15 min. The supernatant was collected, and amount of protein in cell lysates was estimated using the Bradford reagent assay (Bio‐Rad, Hercules, CA, USA). Equal amount of proteins were loaded and run on SDS‐PAGE. The resolved proteins on the gel were transferred to PVDF membrane (Millipore, Burlington, MA, USA). The membrane was blocked with 5% non‐fat milk in TBST (25 mm Tris [pH 7.6], 137 mm NaCl and 0·2% Tween‐20) for 75 min, washed and incubated with primary antibody at 4° overnight and HRP‐conjugated secondary antibody for one and half hour. Immunoreactive bands appeared upon treatment with luminol reagent (Santa Cruz Biotechnology, Dallas, TX, USA) were developed on the X‐ray films.

Cytokine ELISA

Macrophages were treated with indicated concentrations of PKC inhibitors for 2 h in serum‐free medium followed by TLR ligand treatment for 48 h. Cell‐free supernatants from the control and treated samples were collected and examined for IL‐10 and IL‐12 expression by ELISA. ELISA plates were coated with capture antibody for IL‐10 (1 μg/ml) and IL‐12 (2 μg/ml) at 4° for 12 h. Plates were washed thrice with wash buffer (0·05% Tween‐20 in 1× PBS) and incubated with blocking solution (1% BSA in PBS) for 2 h (for IL‐12) at room temperature or 4 h (for IL‐10) at 4°. After washing, culture supernatants (100 μl/well) and standards were added to the ELISA plates and incubated overnight at 4°. After washing, biotin‐conjugated detection antibody was added to the ELISA plates. After 2 h (for IL‐12) or 8 h (for IL‐10), plates were washed and incubated with peroxidase‐conjugated streptavidin (Roche Applied Science) for 30 min. The plates were washed and developed by adding 100 μl TMB substrate. Chromogenic reaction was stopped with 50 μl of 1N H₂SO₄ solution, and the plates were read at 450 nm.

FACS assay

Lymphocytes from the draining lymph nodes of treated and control mice were isolated and treated with PMA (20 ng/ml) and ionomycin (1 μg/ml) for 6 h followed by addition of Brefeldin (Golgi plug) for 2 h. For multi‐colour FACS analyses, after blocking with anti‐CD16/anti‐CD32 Fc blocking antibody (553140, BD Pharmingen, San Diego, CA), cells were stained with anti‐CD4 PB (558107, BD Pharmingen, San Diego, CA) for 45 min at 4° in dark and washed twice with FACS buffer (1× PBS, 10 mm HEPES buffer, and 3% FCS). Intracellular staining with anti‐t‐bet AF647 (644804, BioLegend, San Diego, CA), anti‐GATA3 eF660 (50‐9966‐42, eBioscience, San Diego, CA), anti‐IL‐4 PE (554434, BD Pharmingen, San Diego, CA) and anti‐IFN‐γ PE (554412, BD Pharmingen, San Diego, CA) was performed using a Cytofix/Cytoperm Plus Kit with GolgiPlug (BD Pharmingen) as per the manufacturer's instruction.

Statistical analysis

All statistical analysis was performed using SigmaPlot v10.0. Error bar indicates mean ± SEM from two independent set of experiments. One‐way ANOVA (Holm–Sidak method) was used to test statistical significance between two experimental groups. The values P < 0·05 (*), P < 0·01 (**) and P < 0·001 (***) were considered to be statistically significant.

RESULTS

Activation of PKC isoforms is differentially modulated by TLR2 ligands

PKC has been widely implicated in signalling pathways. We previously showed that different TLR2 dimers induced different signalling pathways leading to the generation of two counteractive pro‐ and anti‐inflammatory cytokines—IL‐12 and IL‐10—respectively [12]. In Leishmania‐infected macrophages, the activation of PKC‐α and PKC‐β was impaired, while activation of PKC‐δ and PKC‐ζ isoforms was enhanced [22]. However, the role of TLR2 in PKC isoform activation and, in particular, in Leishmania‐infected macrophages remains unknown. So, we examined the activation of PKC‐α, PKC‐β, PKC‐δ and PKC‐ζ isoforms in different TLR2 dimer‐induced signalling.

We treated the BALB/c‐derived elicited peritoneal macrophages with different TLR dimer‐specific ligands and followed the phosphorylation of these four PKC isoforms as a function of ligand doses and time, as indicated, after the TLR ligand treatment. The treatments generated a pattern of PKC isoform phosphorylation, as compared to the expression of the respective PKC isoforms (Figure 1a). The TLR1/2‐specific ligand Pam3CSK4 (P3C) induced highest phosphorylation of both PKC‐α/βI and PKC‐βII compared with total respective isoforms at a medium dose of 50 ng/ml (Figure 1b). For PKC‐βI and PKC‐ζ, the highest phosphorylation was achieved at the highest dose (100 ng/ml). After enhanced phosphorylation at the lowest P3C dose, PKC‐δ phosphorylation gradually decreased with increasing doses of the ligand, whereas PKC‐ζ phosphorylation increased with the same treatment, showing reciprocity in the activation between PKC‐δ and PKC‐ζ. During PGN stimulation, PKC‐βII and PKC‐ζ showed highest phosphorylation at a medium dose of 2·5 µg/ml. PKC‐βI phosphorylation gradually increased at medium and higher doses of PGN. Conversely, p‐PKC‐δ substantially changed during PGN stimulation. p‐PKC‐α was maximally activated at low dose of PGN. FSL, a TLR2/6 ligand, stimulation did not show any significant changes in PKC‐βI, PKC‐δ and PKC‐ζ phosphorylation (Figure 1b). PKC‐βII showed maximum phosphorylation at high dose of FSL, but PKC‐α phosphorylation gradually increased with the increase in doses. These data indicate that as a function of doses of the respective ligands, TLR2 dimers induce selective phosphorylation of PKC‐α, PKC‐β, PKC‐δ and PKC‐ζ isoforms. Given that Leishmania expresses lipophosphoglycan (LPG) and possibly ligands for other TLRs, too, it is possible that during Leishmania infection, differential engagement of TLR2 dimers may significantly modulate the fate of the infection. It is indeed plausible that pathogens expressing ligands for different TLR2 dimers may induce selective host PKC phosphorylation.

FIGURE 1.

Activation of PKC isoforms is differentially modulated by TLR2 ligands. PKC isoform phosphorylation induced by different TLR ligands. (a) BALB/c‐derived peritoneal macrophages (4 × 10⁶/well) were plated and allowed for 48‐h resting. After 2 h of serum starvation, cells were treated with indicated dose of Pam₃CSK₄ (TLR1/2 ligand), PGN (TLR2 ligand) and FSL (TLR2/6 ligand) for 15 min or left untreated. Cells were lysed, cell lysate was clarified by centrifugation, and equal amount of protein was loaded to check the PKC isoform phosphorylation by Western blotting. The antibodies used and the detected PKC isoform's molecular weight are shown in the parenthesis. (b) Densitometric units were quantified from the above gel images and plotted as bar graphs

PKC isoform phosphorylation shows differential kinetics following TLR2 ligand treatment

Next, we selected a single dose for each TLR ligand based on the dose where phosphorylation of most PKC isoforms was altered and also based on the titrations performed in various other assays. We followed the kinetics of PKC‐α, PKC‐β, PKC‐δ and PKC‐ζ isoform phosphorylation from 3 min to 60 min after the treatment of macrophages with each of the ligand single dose, as chosen from the previous dose‐dependent PKC phosphorylation experiment. PKC phosphorylation attained the peak at a particular dose of TLR2 ligands (Figure 2, lower panel). In case of P3C treatment (Figure 2, left panel), PKC‐α phosphorylation gradually increased till 30 min, whereas maximum PKC‐βI phosphorylation was achieved 15 min after the ligand treatment and diminished at later time‐points. The remaining PKC isoforms have shown almost constant phosphorylation. With PGN treatment, PKC‐α phosphorylation peaked after 15 min (Figure 2, middle panel), whereas PKC‐βII phosphorylation peaked 30 min after stimulation, but the other PKC isoforms showed almost unchanged phosphorylation at all studied time‐points after PGN treatment. Following FSL stimulation, PKC‐ζ showed highest phosphorylation at 3 min (Figure 2, right panel) and p‐PKC‐βII levels increased till 15 min (Figure 2, right panel) but PKC‐α, PKC‐βI and PKC‐δ phosphorylation remained unaltered. FSL treatment did not affect PKC‐δ phosphorylation when compared to the ratio of phospho‐PKC‐δ to total PKC‐δ (Figure 2, right panel). These observations indicate differential kinetics of the PKC isoform phosphorylation following TLR activation. These observations imply that the differential TLR2 dimer stimulation may selectively mobilize cofactor availability to different PKC isoforms and also determine the translocation of PKC isoforms to membranes wherein the active PKC isoforms perform their functions.

FIGURE 2.

PKC isoform phosphorylation shows differential kinetics following TLR2 ligand treatment. Kinetics of phosphorylation and membrane translocation pattern of PKC isoforms by TLR2 ligands. The single dose of the TLR ligands was chosen by optimum responses in various reported assays. Thioglycolate‐elicited macrophages were seeded at density of 4 × 10⁶ cells per well and rested for 48 h. After 2 h of serum starvation, cells were treated with indicated doses of Pam₃CSK₄, PGN and FSL for indicated periods of time. After cell lysis, equal amount of protein was loaded in SDS‐PAGE, immunoblotted on PVDF and developed on X‐ray film for analysing the kinetics of PKC isoform phosphorylation. Densitometric analysis of phosphorylation patterns is shown by line curves (top panel)

PKC isoforms show differential membrane translocation in TLR ligand‐treated macrophages

Activated PKC isoforms translocate to the membrane for triggering downstream signalling cascades. So, we studied the PKC isoform translocation to macrophage membrane after TLR2 ligand treatment. We observed membrane translocation of PKC‐α, PKC‐βI, PKC‐βII, PKC‐δ and PKC‐ζ at 5 min and 10 min after P3C treatment (Figure 3, left panel). By contrast, PGN treatment failed to translocate PKC‐βI, PKC‐δ and PKC‐ζ to membrane although PKC‐α and PKC‐βII translocated between 3 min and 15 min after PGN treatment (Figure 3, middle panel). In case of FSL treatment, we observed significantly higher translocation of PKC‐α, PKC‐βII, PKC‐δ and PKC‐ζ, but not of PKC‐βI, at all time‐points. The data suggest that P3C, PGN and FSL induce differential membrane translocation of PKC isoforms. As PKC isoforms play significant roles in the activation of downstream signalling events [22], it is plausible that the TLR2 dimers use the spatiotemporal activation of PKC isoforms as a principle to regulate the kinetics of PKC‐regulated macrophage functions.

FIGURE 3.

PKC isoforms show differential membrane translocation following TLR ligand treatment of macrophages. TLR‐induced membrane translocation of PKC isoforms. BALB/c‐derived peritoneal macrophages were treated with indicated doses of TLR ligands, and membrane fractions were collected by ultracentrifugation and dissolved in NP‐40 containing buffer. After estimation of the protein, equal amount of the protein was loaded in the SDS‐PAGE and immunoblotted to identify the membrane translocation of PKC isoforms. The line graphs represent the densitometric units of PKC isoforms translocated to membrane

Leishmania infection alters TLR2‐induced PKC activation in macrophages

Leishmania differentially modulates TLR2 association with TLR1 and TLR6 to establish its infection within the host [12]. We observed that PKC activation is differentially affected by different TLR2 dimers. Therefore, we have compared the phosphorylation and membrane translocation patterns of PKC isoforms in TLR2‐treated L. major‐infected (IM) and uninfected macrophages (UIM) (Figure 4a). FSL treatment significantly enhanced PKC‐α phosphorylation in IM as compared to UIM. P3C treatment increased phosphorylation of PKC‐βII and PKC‐ζ in both UIM and IM. PGN and FSL increased phosphorylation of PKC‐δ in UIM but decreased in IM (Figure 4a). As these ligands exhibit specificity towards TLR1‐TLR2, TLR2‐TLR2 or TLR2‐TLR6 dimers, it is possible that the responsiveness of macrophages depends on the relative expression of these TLRs and their preferred association during an infection.

FIGURE 4.

Leishmania infection alters TLR2‐induced PKC activation in macrophages. Phosphorylation of PKC isoforms in uninfected and infected macrophages upon treatment with TLR2 ligands. (a) BALB/c‐derived peritoneal macrophages were infected with L. major parasites at a ratio of 1:10 (10 parasites per macrophage). Both uninfected and infected macrophages were treated with Pam₃CSK₄ (50 ng/ml), PGN (5 μg/ml) and FSL (50 ng/ml) for 15 min and lysed. Equal amount of protein was used to perform the Western blotting, and immunoblotting was done to check phosphorylation pattern of PKC isoforms. The graphs represent the densitometric units (right). (b) Membrane fraction of both uninfected and infected macrophages after treatment with TLR2 ligands was isolated and run on SDS‐PAGE to examine the translocation of PKC isoforms. Pan‐Ras was used as positive control for membrane fraction. Densitometric analysis was done, and densitometric values are plotted as bar graphs (right)

High membrane translocation of PKC‐α was observed in UIM but not in IM upon P3C treatment. IM showed increased membrane translocation of PKC‐α and PKC‐βI upon FSL treatment (Figure 4b). L. major infection significantly reduced the P3C‐induced membrane translocation of all PKC isoforms. L. major infection also depleted the P3C‐ and FSL‐induced translocation of PKC‐δ and PKC‐ζ (Figure 4b). The above result indicates that Leishmania parasite selectively altered the TLR2 ligand‐induced membrane translocation of PKC isoforms in stimulated macrophages.

TLR2 dimers selectively regulate IL‐10 and IL‐12 in the presence of PKC‐α and PKC‐β inhibitors

TLR2 hetero‐ or homodimerization regulates Leishmania clearance or persistence. So, we next evaluated the impact of PKC isoforms in regulating anti‐inflammatory cytokine IL‐10 and pro‐inflammatory cytokine IL‐12 after treatment with P3C (25, 50 and 100 ng/ml), PGN (1, 2·5 and 5 μg/ml) and FSL (25, 50 and 100 ng/ml). BALB/c‐derived peritoneal macrophages were treated with the indicated doses of PKC‐α and PKC‐β inhibitors 2 h before treatment with TLR ligands. We observed reduced IL‐10, but increased IL‐12, expression upon treatment with PKC‐α inhibitor and P3C treatment (Figure 5). However, PKC‐β inhibitor and P3C treatment increased the expression of both IL‐10 and IL‐12 in macrophages. PGN treatment after PKC‐α inhibition did not alter the expression of IL‐10 and IL‐12. PGN treatment significantly reduced IL‐12 levels with low doses of PKC‐α inhibitor. PKC‐β inhibitor and FSL‐treated macrophages showed very high expression of IL‐12 and reduced expression of IL‐10. PKC‐α and PKC‐β inhibition reduced PGN‐induced IL‐10 expression and IL‐12 expression (Figure 5). Collectively, the data suggest that differential expression of IL‐10 and IL‐12 by TLR ligands depends on the PKC‐α and PKC‐β isoform activation.

FIGURE 5.

TLR2 dimers differentially regulate IL‐10 and IL‐12 in the presence of PKC‐α and PKC‐β inhibitors. Effect of PKC‐α and PKC‐β inhibition on TLR2‐induced IL‐10 and IL‐12 in macrophages. (A) BALB/c‐derived peritoneal macrophages were seeded at density of 10⁶ cells per well and allowed for 48‐h resting. The cells were treated with indicated doses of specific PKC isoform inhibitor in serum‐free media for 2 h. Cells were further treated with TLR2 ligands 50 ng/ml P3C, 5 μg/ml PGN and 50 ng/ml FSL and incubated for another 48 h. Cell‐free supernatants were collected and estimated for IL‐10 and IL‐12 production by ELISA. The experiments were repeated twice, and data from one of the experiments are shown. The data represent mean ± SEM. The amount of IL‐10 and IL‐12 production was compared with untreated cells, and values P < 0·05 (*), P < 0·01 (**) and P < 0·001 (***) were considered to be statistically significant

TLR2 activation differentially regulates IL‐10 and IL‐12 expression through PKC‐δ and PKC‐ζ

We next examined the expression of IL‐10 and IL‐12 in macrophages treated with the indicated doses of TLR2 ligand PKC‐δ and PKC‐ζ inhibitors. We observed that PKC‐δ inhibitor and P3C‐treated macrophages showed inhibitor dose‐independent increase in IL‐10 and IL‐12 expression (Figure 6). PKC‐ζ inhibition allowed the secretion of P3C‐induced IL‐10 and inhibited the IL‐12 secretion in macrophages. IL‐10 expression remained unaltered in PKC‐δ inhibited, and PGN‐ or FSL‐treated macrophages. However, PGN and FSL increased IL‐10 and IL‐12 levels in macrophages treated with PKC‐δ inhibitor (Figure 6). FSL but not PGN increased IL‐10 and IL‐12 in PKC‐ζ inhibitor‐treated macrophages. The above data indicate that TLR ligands differentially modulate the IL‐10 and IL‐12 generation through PKC isoforms.

FIGURE 6.

TLR2 activation differentially regulates IL‐10 and IL‐12 expression through PKC‐δ and PKC‐ζ. Effect of PKC‐δ and PKC‐ζ inhibition on TLR2 ligands induced IL‐10 and IL‐12 in macrophages. BALB/c‐derived macrophages were subjected to PKC‐δ and PKC‐ζ inhibition and treated with the 50 ng/ml P3C, 5 μg/ml PGN and 50 ng/ml FSL for 48 h. Culture supernatants were used for quantification of IL‐10 and IL‐12 secretion by performing ELISA. The experiments were repeated twice, and data from one of the experiments are shown. The data represent mean ± SEM. The amount of IL‐10 and IL‐12 production was compared with untreated cells, and values P < 0·05 (*), P < 0·01 (**) and P < 0·001 (***) were considered to be statistically significant

PKC inhibition modulates in vitro parasite load following TLR2 ligand treatment

Anti‐ or pro‐parasitic roles of TLR2 ligands have been known, but how PKC isoforms affect these TLR2 functions remains largely unknown. So, we infected the BALB/c‐derived macrophages with L. major parasites and treated them with PKC‐α (15 μm), PKC‐β (7 nm), PKC‐ζ (15 μm) or PKC‐δ (10 μm) inhibitor for 2 h in serum‐free media, followed by treatment with P3C (50 ng/ml), PGN (5 μg/ml) and FSL (50 ng/ml) for 6 h and 72 h. We enumerated the parasite load and per cent infectivity. We observed that treatment with PKC isoform inhibitors reduced the amastigote count and per cent infectivity.

P3C treatment reduced parasite load in PKC‐δ‐ and PKC‐ζ‐inhibited macrophages implying that the anti‐leishmanial functions triggered by P3C may be inhibited by PKC‐δ and PKC‐ζ isoforms, which were indeed found to be inhibitory in CD40‐activated macrophages [22]. PGN alone increased parasite load but upon co‐treatment with PKC‐α or PKC‐β inhibitors, PGN significantly reduced parasite load. FSL alone—but not in combination with any PKC isoform inhibitor—reduced amastigote count. Per cent infectivity remained unaltered but amastigote count was hugely affected upon treatment with TLR2 ligands and PKC inhibitor (Figure 7). Thus, these data suggest that the pro‐parasitic and anti‐parasitic roles of PGN and FSL, respectively, can be selectively affected by the inhibitors of PKC isoforms.

FIGURE 7.

PKC inhibition modulates in vitro parasite load following TLR2 ligand treatment. Kinetics of parasite load in macrophages treated with PKC isoform inhibitor and TLR ligands. BALB/c‐derived peritoneal macrophages were plated at a density of 5 × 10⁴/well. After 48‐h rest, cells were infected with stationary‐phase L. major parasites at a ratio of 1:10 (10 parasites/ macrophage) for 6 h. The extracellular parasites were washed, and after 24‐h incubation, cells were treated with PKC‐α (15 μm), PKC‐β (7 nm), PKC‐ζ (15 μm) and PKC‐δ (10 μm) inhibitor for 2 h in serum‐free media followed by treatment with Pam₃CSk₄ (50 ng/ml), PGN (5 μg/ml) and FSL (50 ng/ml) for 6 h and 72 h. The cells were washed, fixed with chilled methanol and stained with Giemsa stain. The amastigotes were counted under a microscope: amastigote count in 100 infected macrophages (IM) (top panel) and per cent infectivity (middle panel) was calculated by dividing the number of infected cells by number of uninfected cells. Amastigote count in 100 macrophages (bottom panel) was calculated by multiplying the above two functions. The amastigote counts in PKC isoform inhibitor‐treated cells were compared with infection alone and values P < 0·05 (*), P < 0·01 (**) and P < 0·001 (***) were considered to be statistically significant

TLR2 ligands and PKC inhibitors differentially modulate parasite load and T cells in vivo

PKC isoforms play vital role in regulating Leishmania infection, and the in vitro data show the anti‐leishmanial role of TLR2‐PKC axis. To examine the in vivo effect of the proposed axis, we treated L. major‐infected and uninfected mice with TLR2 ligands and PKC inhibitors (5 mg/kg body weight) in combination or alone. We examined the T cells of popliteal lymph node of treated and untreated mice for anti‐leishmanial role. We first assessed parasite load in the draining popliteal lymph node. We observed that PGN significantly increased parasite load, which was further enhanced in the presence of PKC‐(α + β) inhibitors, whereas FSL reduced parasite load by itself and the anti‐leishmanial effect was further augmented by PKC‐(δ + ζ) inhibitor (Figure 8a). In susceptible BALB/c mice, PGN and FSL exerted opposite effects on parasite growth; in case of PGN, PKC‐(α + β) inhibitor enhanced parasite load suggesting that PKC‐(δ + ζ) perform pro‐leishmanial functions, whereas in case of FSL, PKC‐(δ + ζ) inhibitor significantly reduced parasite load suggesting that PKC‐(α + β) play anti‐leishmanial functions.

FIGURE 8.

PKC inhibitors and TLR2 ligands differentially modulate T‐cell population under in vivo. FSL along with PKC‐(α + β) inhibitor injection reduces parasite load in mice. Hind footpad of a BALB/c mouse was infected with 2 × 10⁶ L. major parasites. After three days of infection, PKC inhibitors and TLR2 ligands were injected into the footpads. 5 weeks after infection, lymph nodes from respective mice were isolated and crushed to make single‐cell suspension. (a) The supernatants were incubated at 37° in humidified water‐jacketed CO₂ incubator for 7 days and used for counting of parasite transformation from amastigote to promastigotes. The data shown are mean ± SEM from replicates. (b) 5 × 10⁴ macrophages/well were plated in chamber slides. After 24 h, cells were infected with stationary‐phase L. major parasites for 6 h. Extracellular parasites were washed, and cells were incubated for 36 h. T cells from the lymph nodes of the treated, untreated and naïve mice were isolated and co‐cultured at the ratio of 1:3 (3T cells per macrophage) for 48 h. Cells were washed, fixed and stained with Giemsa for counting the amastigotes in the macrophages. The graph represents the amastigote count per 100 infected macrophages and per cent infectivity calculated as described above. (c) The single‐cell suspensions from the lymph node were incubated with 1 μg/ml ionomycin and 20 ng/ml PMA for 6 h followed by treatment with Brefeldin for 2 h. The lymphocytes were stained (after FcR blockade) with fluorochrome‐conjugated antibodies specific to surface or intracellular molecules. The T cells were acquired in CD4⁺ Tbet⁺ IFN‐γ⁺ for Th1 cells and CD4⁺ GATA3⁺ IL‐4⁺ gates for Th1 and Th2 cells analysis, respectively. The figure represents the Th1 cells (left panel) and Th2 cells (right panel). The data shown are a representative figure from one of the two independent experiments

Th1 cells play anti‐leishmanial and Th2 cells play pro‐leishmanial roles. So, we performed flow cytometry analysis for investigating the CD4⁺Tbet⁺IFN‐γ⁺ Th1 cells and CD4⁺GATA3⁺IL‐4⁺ Th2 cells. We observed that P3C by itself did not affect Th1 or Th2 subsets, but with PKC‐(α + β) inhibitors, it increased both Th1 and Th2 numbers but PGN by itself increased Th1 cells. PKC‐(α + β) inhibitors reduced the PGN‐induced Th1 cell numbers but increased Th2 cells, suggesting that PGN‐induced Th1 response may depend on PKC‐(α + β) isoforms. FSL by itself enhanced both Th1 and Th2 cells but with (α + β) inhibitors substantially increased Th1 cells but reduced Th2 cell numbers; similar effects were observed with the PKC‐(δ + ζ) inhibitors but to a lesser extent (Figure 8b). These data suggest that FSL with PKC‐(δ + ζ) inhibitors may reduce Leishmania infection, as observed both in vitro (Figure 7) and in vivo (Figure 8a).

DISCUSSION

The data presented here reveal the selective roles of PKC isoforms in TLR signalling and the characteristics of the signalling affected by the parasite Leishmania that lives within mammalian macrophages causing the complex of diseases called Leishmaniases. First, TLR dimer‐specific ligands showed specificity in signalling through PKC isoforms. P3C increased phosphorylation of all PKC isoforms tested, whereas membrane translocation of only PKC‐α, PKC‐βII and PKC‐ζ was enhanced. By contrast, PGN increased both phosphorylation and membrane translocation of PKC‐α, PKC‐βII and PKC‐ζ isoforms. Uniquely, membrane translocation of PKC‐δ was unaltered by P3C, enhanced by PGN but reduced by FSL revealing a distinctive role for PKC‐δ in processing TLR ligand‐specific signalling in macrophages. Similarly, PKC‐βI phosphorylation was enhanced by P3C, unaltered by PGN but reduced by FSL whereas its membrane translocation was reciprocally regulated by PGN and FSL. Further specificity profile in TLR2 signalling was observed as FSL induced higher PKC‐ζ translocation despite its reduced phosphorylation. These observations clearly suggest that the specificity of TLR signalling depends on the ligand‐induced heterodimerization of TLR2 with either TLR1 or TLR6. The specificity is conferred, at least in part, by a combination of PKC isoform activation and membrane translocation. The differences in their activation and translocation kinetics may also signify as parameters of specificity, and in addition, the spatiotemporal regulation of the TLR ligands induced cellular responsiveness. Second, during L. major infection, the usage of PKC isoforms was altered, as PKC‐δ phosphorylation was reduced but that of PKC‐ζ was increased by all three TLR2 ligands. Considering that CD40 and TLR regulate each other's functions and that CD40‐induced PKC‐δ phosphorylation increased in Leishmania infection [22], it is possible that the membrane receptors may target PKC isoforms for crosstalk and final biological response modification. Third, membrane translocation of almost all PKC isoforms, excepting FSL‐induced PKC‐α and PKC‐βI, was almost undetectable in L. major‐infected macrophages. This observation clearly re‐emphasizes the previous observation that FSL working through TLR2‐TLR6 heterodimer enhances host‐protective immune response in L. major infection [12]. Fourth, PKC isoform inhibitors selectively influenced IL‐10 and IL‐12 production following treatment with different TLR ligands, while P3C‐induced IL‐10 and IL‐12 production is increased in the presence of inhibitors of all PKC isoforms, excepting IL‐12 in the presence of PKC‐ζ inhibitor. Uniquely, PKC‐δ inhibitor increased IL‐12 production induced by all TLR2 ligands. The observed differential sensitivity of the TLR ligands to PKC isoform‐specific inhibitors suggests that TLRs may cross talk through modulation of PKC isoforms or, alternatively stated, PKC isoforms may regulate each TLR ability to induce specific effector functions. However, this remains to be tested as an independent investigation. Fifth, uniquely, FSL alone reduced amastigote counts in macrophages verifying the previously implied functions of TLR2‐TLR6 heterodimers in Leishmania infection [12]. As befits parasitism, Leishmania significantly impairs the association between TLR2 and TLR6, although TLR2 expression is not significantly affected [12]. Finally, the anti‐leishmanial efficacy of TLR ligands in conjunction with different PKC inhibitors was worked out showing that FSL alone reduced parasite burden in mice but neither inhibitors appeared to significantly affect parasite load in vitro; FSL enhanced parasite clearance in BALB/c mice in the presence of PKC‐(δ + ζ) inhibitors. Therefore, these data indicate that PKC isoforms are differentially activated by TLR2 ligands and that L. major infection substantially affects this activation.

Dose‐dependent reciprocal regulation has been widely described for many cellular sensors. Mechanism of this reciprocal regulation lies behind the involvement of differential intracellular signalling cascade in the indicated condition [27] specified by the dose of ligands, period of treatments or subcellular location of an enzyme. These observations thus explain the TLR‐induced spatiotemporal regulation of PKC isoforms. We had previously shown that the ligand‐induced TLR dimerization plays an important role in cellular signalling [12]. The TLR1/TLR2 dimer‐specific ligand induced peak PKC‐α and PKC‐β phosphorylation at medium dose. Even a lower dose of a ligand activated a particular PKC isoform more profoundly. For example, P3C‐induced PKC‐δ phosphorylation is maximal at lower doses. These observations indicate the specificity in TLR2‐induced PKC isoform activation. The specificity of TLR2‐induced PKC isoform activation was also defined by the phosphorylation kinetic profiles. PKC isoforms attained highest phosphorylation at different time‐points with different TLR2 ligands. The specificity in the kinetics of PKC isoform activation might play important roles in TLR self‐regulatory property. Such self‐regulation can be attained through restriction of the access to regulators of a PKC isoform or by isoforms’ temporal separation; for example, PKC‐α and PKC‐β can be activated before the activation of PKC‐ζ isoform. Spatial relocalization of PKC isoforms is important to access their specific substrate for further continuation of the respective signalling pathway [28]. Although phosphorylation and membrane translocation are two hallmarks of PKC activation, the relationship between the two in a time frame is not fixed [28]. For example, following P3C treatment, p‐PKC‐δ migrates to the membrane but its phosphorylation does not change throughout the time course. PKC‐α is phosphorylated maximally 30 min after the P3C treatment, but it does not translocate to the membrane at the same time. The regulation becomes further complex as the sequence of phosphorylation of the serine and threonine residues may be related to translocation or post‐translational modification [29].

Parasites have evolved their strategies to exploit host machinery for their survival. In vitro count of parasites has indicated that P3C and PGN treatments, but not FSL, favour the infection. PKC‐ζ phosphorylation increased significantly in infected macrophages, as compared to uninfected macrophages, following P3C and PGN treatments, whereas FSL treatment enhanced PKC‐α and PKC‐βII phosphorylation in infected macrophages. While the unique activation of one PKC isoform reflects the specificity of TLR signalling through PKC isoforms, any redundancy in activation of a given PKC isoform suggests crosstalk between the TLR2 dimers. While PKC‐α and PKC‐β inhibitors enhanced parasite count and had higher IL‐10, but less IL‐12, secretion following P3C stimulation of L. major‐infected macrophages, PGN treatment showed significantly increased parasite count and higher IL‐10 secretion from PKC‐δ‐inhibited macrophages. Although FSL treatment reduced the parasite load, PKC‐β inhibition enhanced the amastigote count in macrophages.

It is known that the outcome of Leishmania infections depends on T cells [12]. IFN‐γ‐secreting Th1 cells eliminate the parasite, but IL‐4‐secreting Th2 cells enhance the parasite burden. Our observations here indicate that PKC isoforms of macrophages play vital roles in modulating the T‐cell response. As induction of Th1 response kills parasites, Th1 response is reduced, whereas Th2 response is upregulated during P3C treatment under PKC‐(α + β)‐inhibited condition. The same trend has been observed in PGN stimulation. As FSL treatment significantly reduced L. major infection, Th1 population was increased during PKC‐(α + β) inhibitor treatment. FSL treatment of L. major‐infected BALB/c mice enhanced both Th1 and Th2 cells but under PKC‐(α + β) inhibitor, FSL increased Th1 cells significantly more than the IL‐4‐secreting Th2 cells; however, PKC‐(δ + ζ) inhibitor had lesser effects on both subsets of Th cells.

Our observations also record some discrepancies between the PKC phosphorylation studies and translocation studies. In essence, not all PKC isoforms that were phosphorylated showed membrane translocation in infected macrophages. This observation may suggest that PKC isoform phosphorylation and membrane translocation may be uncoupled in L. major‐infected macrophages. It is plausible that in L. major‐infected macrophages, phosphorylation of all the serine and threonine residues that are required for translocation is impaired. Alternatively, some cofactors may be required for membrane translocation and these are sequestered away from the PKC isoforms. It is also possible that the PKC isoforms require some post‐translational or post‐activation modifications, which are impaired in L. major‐infected macrophages. However, determination of the exact mechanism of impaired translocation of PKC isoforms will require a series of independent investigations. In conclusion, while the molecular analyses of the role of PKC isoforms in TLR signalling specificity remain awaited, we demonstrate here that ligand‐induced selective TLR2 heterodimerization with either TLR1 or TLR6 triggers activation and membrane translocation of different PKC isoforms. As an immune evasion strategy, Leishmania modulates those responses to ensure its survival. The observations inscribed in this report provide a rationale how TLR2 ligands can in combination with PKC isoforms’ inhibitors be used for anti‐leishmanial therapy.

CONFLICT OF INTEREST

The authors declare no conflict of interest.

AUTHOR CONTRIBUTIONS

AM, SR, AP and NB performed experiments. JD, BS and AS conceptualized and designed the study, analysed the data and prepared the manuscript.

Mukherjee A, Roy S, Patidar A, Bodhale N, Dandapat J, Saha B, et al. TLR2 dimer‐specific ligands selectively activate protein kinase C isoforms in Leishmania infection. Immunology. 2021;164:318–331. 10.1111/imm.13373