Pegylated Bisacycloxypropylcysteine, a Diacylated Lipopeptide Ligand of TLR6, Plays a Host-Protective Role against Experimental Leishmania major Infection This information is current as of September 25, 2021. Surya Prakash Pandey, Himanshu Singh Chandel, Sunit Srivastava, Sathishkumar Selvaraj, Mukesh Kumar Jha, Divanshu Shukla, Thomas Ebensen, Carlos A. Guzman and Bhaskar Saha

J Immunol 2014; 193:3632-3643; Prepublished online 5 Downloaded from September 2014; doi: 10.4049/jimmunol.1400672 http://www.jimmunol.org/content/193/7/3632 http://www.jimmunol.org/

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2014 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Pegylated Bisacycloxypropylcysteine, a Diacylated Lipopeptide Ligand of TLR6, Plays a Host-Protective Role against Experimental Leishmania major Infection

Surya Prakash Pandey,* Himanshu Singh Chandel,* Sunit Srivastava,* Sathishkumar Selvaraj,* Mukesh Kumar Jha,* Divanshu Shukla,* Thomas Ebensen,† Carlos A. Guzman,† and Bhaskar Saha*

TLRs recognize pathogen-expressed Ags and elicit host-protective immune response. Although TLR2 forms heterodimers with TLR1 or TLR6, recognizing different ligands, differences in the functions of these heterodimers remain unknown. In this study, we report that in Leishmania major-infected , the expression of TLR1 and TLR2, but not TLR6, increased; TLR2– TLR2 association increased, but TLR2–TLR6 association diminished. Lentivirus-expressed TLR1–short hairpin RNA (shRNA) Downloaded from or TLR2–shRNA administration reduced, but TLR6–shRNA increased L. major infection in BALB/c mice. Corroboratively,

Pam3CSK4 (TLR1–TLR2 ligand) and peptidoglycan (TLR2 ligand) increased L. major infection but reduced TLR9 expression, whereas pegylated bisacycloxypropylcysteine (BPPcysMPEG; TLR2–TLR6 ligand) reduced L. major number in L. major-infected macrophages, accompanied by increased TLR9 expression, higher IL-12 production, and inducible NO synthase expression. Whereas MyD88, Toll/IL-1R adaptor , and TNFR-a–associated factor 6 recruitments to TLR2 were not different in http://www.jimmunol.org/ Pam3CSK4-, peptidoglycan-, or BPPcysMPEG-treated macrophages, only BPPcysMPEG enhanced p38MAPK and activating transcription factor 2 activation. BPPcysMPEG conferred antileishmanial functions to L. major-infected BALB/c-derived T cells in a –T cell coculture and in BALB/c mice; the protection was TLR6 dependent and IL-12 dependent, and it was accompanied by reduced regulatory T cell number. BPPcysMPEG administration during the priming with fixed L. major protected BALB/c mice against challenge L. major infection; the protection was accompanied by low IL-4 and IL-10, but high IFN-g productions and reduced regulatory T cells. Thus, BPPcysMPEG, a novel diacylated lipopeptide ligand for TLR2–TLR6 hetero- dimer, induces IL-12–dependent, inducible NO synthase–dependent, T-reg–sensitive antileishmanial protection. The data reveal a novel dimerization partner-dependent duality in TLR2 function. The Journal of Immunology, 2014, 193: 3632–3643. by guest on September 25, 2021 oll-like 2 is a unique member among the TLRs, as mediated deactivation of macrophages (10). Lipophosphoglycan– it forms heterodimers with TLR1 or TLR6 and modulates TLR2 interaction diminishes the antileishmanial response by reduc- T downstream signaling pathways (1–3). TLR1–TLR2 and ing TLR9 expression (11). By contrast, TLR2-deficient mice on a re- TLR2–TLR6 heterodimers recognize triacylated or diacylated sistant C57BL/6 background and the wild-type controls are equally lipopeptides, respectively (4, 5). Following recognition of pathogens resistant to Leishmania braziliensis infection (12), suggesting that such as Leishmania, TLRs activate that, in TLR2 may not play a role in Leishmania infection. In contrast, as turn, elicits pathogen-specific adaptive immune response (6, 7). IL-10 and IL-12 play significant roles in L. major infection (13) and as Leishmania major, a protozoan parasite living as aflagellate amasti- stimulation of Leishmania donovani-infected human cell gotes within macrophages, causes cutaneous lesions in a susceptible line–derived macrophages with Pam3CSK4—a TLR1–TLR2 ligand— host such as a BALB/c mouse. Whereas recognition of Leishmania by produces more IL-10, but less IL-12 (14), TLR2 is implied in the a TLR may be followed by macrophage activation and parasite con- control of Leishmania infection. However, whether TLR2–TLR6 trol (8), the protozoan devises immune evasion strategies either by functions differ from TLR1–TLR2 functions has not been studied. indirect interference of the IFN-g–secreting host-protective Th1 cell In Brucella abortus infection, TLR6 is implicated in NF-kB–de- generation (9) or by direct interference through lipophosphoglycan- pendent IL-12 production (15). TLR6 deficiency results in higher Mycobacterium avium infection in lung (16) or in higher Aspergillus *National Centre for Cell Science, Ganeshkhind, Pune 411007, India; and †Department fumigatus burden in C57BL/6 mice (17). TLR6 and TLR9 synergize of Vaccinology and Applied Microbiology, Helmholtz Centre for Infection Research, to reduce the Influenza A infection (18). Thus, TLR6 has been Braunschweig, 38124 Braunschweig, Germany implicated in bacterial, fungal, and viral infections. Indeed, the My- Received for publication March 26, 2014. Accepted for publication July 30, 2014. coplasma fermentas-derived macrophage-activating lipopeptide of This work was supported in part by the Department of Biotechnology and Indian 2 kDa that signals through TLR2–TLR6 heterodimer exhibits sig- Council of Medical Research, Government of India, and the Helmholtz Association nificant pharmaceutical efficacy in tumor therapy (19) and allergic (Indo–German Science Centre for Infectious Diseases). airway inflammation (20). The synthetic derivative of macrophage- Address correspondence and reprint requests to Dr. Bhaskar Saha, National Centre for Cell Science, Ganeshkhind, Pune 411007, India. E-mail address: [email protected] activating lipopeptide of 2 kDa, pegylated bisacycloxypropylcysteine Abbreviations used in this article: ATF2, activating transcription factor 2; BPPcysMPEG, (BPPcysMPEG), has been shown to induce Th1-type response pegylated bisacycloxypropylcysteine; DC, ; iNOS, inducible NO syn- and prevent pollen-induced allergy (21). In contrast, TLR2–TLR6 thase; PGN, peptidoglycan; shRNA, short hairpin RNA; TIRAP, Toll/IL-1R adaptor heterodimer is shown to induce IL-12 (16), a known protein; TRAF6, TNFR-a–associated factor 6; Treg, regulatory T. for promoting IFN-g–predominated host-protective response in Copyright Ó 2014 by The American Association of Immunologists, Inc. 0022-1767/14/$16.00 L. major infection (22). In contrast, TLR2 was found to promote www.jimmunol.org/cgi/doi/10.4049/jimmunol.1400672 The Journal of Immunology 3633

L. major (11) and L. donovani (23) infections. Although TLR1– Moloney murine leukemia virus–reverse transcriptase, and was used for TLR2 and TLR2–TLR6 heterodimers are known for recognition of 25 ml reaction mixture (Invitrogen Life Technologies, Carlsbad, CA). Sam- triacylated lipopeptides or diacylated lipopeptides, respectively (4, ples were incubated at 37˚C for 1 h, followed by 10-min heat inactivation at 65˚C. cDNA from each sample was amplified with Taq DNA polymerase 5), whether they function differently is not known. (Invitrogen Life Technologies) in 25 ml reaction mix, under the following Antileishmanial host-protective T cell response is limited by conditions: 94˚C for 1 min, annealing for 1 min at a temperature between regulatory T (Treg) cells (24–28), which can be modulated by IL-2, 53 and 60˚C depending upon the -specific primers (Table I), and 72˚C IL-10, and CD40. In fact, TLR2 has been implicated in the regu- for 1 min for a total of 28–30 cycles. Each sample was amplified for mouse GAPDH or b-actin to ensure equal cDNA input, as described earlier (11). lation of Treg cells in different diseases (29, 30). Altogether, these Amplified PCR products were run in 1.2% agarose gel. The expression previous reports thus implied that TLR2 plays an important role levels of the above-mentioned were quantified using the Quantity in infection and can possibly be targeted for devising therapeutic One-4.6.1 (basic) software (Bio-Rad, Hercules, CA). or prophylactic strategies. Therefore, based on these findings, Real-time PCR was performed on the Eppendorf RealPlex mastercycler we examined the role of TLR1–TLR2–TLR6 system of TLRs in using 2XIQ SYBR Green supermix (5 ml; Bio-Rad), 10 ng cDNA as template, 2 ng forward primer, and 2 ng reverse primer, and the reactions L. major infection and tested whether BPPcysMPEG treatment were performed in thin-wall 0.2-ml strip tubes (Axygen, Union City, CA) of L. major-infected BALB/c mice can result in a TLR6-targeted for a total of 10 ml reaction mix. Relative quantitation was done using the novel therapy or prophylaxis. comparative threshold (DD CT) method. The mRNA expression levels of In this study, we report that BPPcysMPEG increased TLR9 ex- the target genes were normalized against those of GAPDH levels and expressed as relative fold change compared with untreated controls (32). pression in BALB/c-derived uninfected and L. major-infected mac- rophages and induced a higher IL-12:IL-10 ratio than that induced Immunoprecipitation and Western blotting by Pam3CSK4 and peptidoglycan (PGN). TLR1 short hairpin RNA For immunoprecipitation, cells were lysed in lysis buffer by incubation on ice Downloaded from (shRNA) or TLR2 shRNA reduced, but TLR6 shRNA aggravated for 1 h. Equal amounts of (500 mg) were incubated with 1 mganti– TLR-2 Ab for 8–10 h at 4˚C and pulled with protein A/G–agarose beads L. major infection in BALB/c mice. Pam3CSK4 and PGN augmented, whereas BPPcysMPEG reduced amastigote number in macrophages. (Pierce, Rockford, MI) at 4˚C for 3 h. Immunoprecipitates were washed twice in cold lysis buffer and once in cold TBST. Pellets were resuspended in BPPcysMPEG induced higher p38MAPK and activating transcrip- 20 ml23 SDS sample buffer and subjected to Western blot analysis (32). tion factor 2 (ATF2), whereas Pam3CSK4 and PGN induced more of For Western blotting, cells were treated with indicated reagents and lysed

ERK1/2 and ELK-1 activation. These contrasting effects of TLR2 with lysis buffer (20 mM Tris [pH 7.5], 150 mM NaCl, 10% glycerol, 1 mM http://www.jimmunol.org/ heterodimers with TLR1 or TLR6 reveal a TLR2 functional duality. EDTA, 1 mM EGTA, and 1% Nonidet P-40), protease inhibitor mixture (Roche Applied Science, Mannheim, Germany), and phosphatase inhibitor mixture (Pierce) by incubation on ice for 1 h. Lysates were centrifuged Materials and Methods (12,000 rpm, 15 min), and supernatants were quantified by bicinchoninic Parasites, mice, peritoneal macrophages, and reagents acid (Pierce, Rockford, Michigan). Equal amount of protein was loaded on SDS-PAGE, and resolved protein was transferred to polyvinylidene L. major (strain MHOM/Su73/5ASKH) was maintained in RPMI 1640 difluoride membrane (Millipore, Billerica, MA) and blocked with 5% with 10% FCS (Life Technologies-BRL, Grand Island, NY). The parasites nonfat dried milk in TBST (25 mM Tris [pH 7.6], 137 mM NaCl, and were passaged through BALB/c mice to maintain the virulence. C57BL/6, 0.2% Tween 20). Membranes were incubated with primary Ab at 4˚C inducible NO synthase (iNOS)–deficient (C57BL/6 background), BALB/c, overnight, washed with TBST, and incubated with HRP-conjugated sec- and IL-12–deficient (BALB/c background) mice, originally from The ondary Ab. Immunoreactive bands were visualized with the Luminol reagent by guest on September 25, 2021 Jackson Laboratory (Bar Harbor, ME), were maintained in the experi- (Santa Cruz Biotechnology, Santa Cruz, CA) (32). Densitometric analyses of mental animal facility of the National Centre for Cell Science. Animals bands were performed using Quantity One software. were used according to the Institute’s ethical animal use committee–ap- proved animal use protocol. FACS reagents and Treg cell analyses The elicited peritoneal macrophages were isolated as adherent cells Anti-CD3–PE-Cy7 (553062), anti-CD25–allophycocyanin-Cy7 (557658), from C57BL/6, iNOS-deficient, IL-12–deficient, and BALB/c mice 5 d after anti-GITR–PE (12-5874-82), and anti–IL-10–FITC (554466) Abs were pur- thioglycolate injection (3% thioglycolate, 2 ml; i.p.). Purity of macrophages chased from BD Biosciences (San Jose, CA); anti-CD127–allophycocyanin was tested routinely, as described earlier (31), and it was always between 97 (17-1271-82) Ab was purchased from eBioscience (San Diego, CA); and anti- and 98.5% (data not shown). Macrophages were cultured in RPMI 1640 CD4–PerCP-Cy5.5 (100434) and anti-Foxp3–Pacific Blue (126409) Abs were supplemented with 10% FCS. After 6 h, nonadherent cells were washed out purchased from BioLegend (San Diego, CA). For multicolor FACS analyses, and cells were incubated for 36 h at 37˚C containing 5% CO2 and humidified cells were stained (after blocking with 5% FCS) with fluorescently labeled atmosphere. Abs anti-CD3–PE-Cy7, anti-CD4–PE-Cy5, anti-CD25–allophycocyanin- Pam3CSK4, PGN, and CpG ODN1826 were purchased from Invivogen Cy7, anti-GITR–PE, and anti-CD127–allophycocyanin for 45 min at 4˚C (San Diego, CA). BPPcysMPEG was synthesized at the Helmholtz Centre in dark and washed twice with FACS buffer (13 PBS, 10 mM HEPES for Infection Research (Braunschweig, Germany). The primers for TLRs and buffer, and 3% FCS). Intracellular cytokine staining was performed using (Table I) were from Integrated DNA Technologies (San Diego, a Cytofix/Cytoperm-Plus Kit with GolgiPlug (555028; BD Pharmingen), as CA). Abs and standards for cytokine ELISA were from BD Pharmingen (San per the manufacturer’s instructions. Cells were acquired in the CD3+CD4+ Diego, CA). Immunoblotting Abs for p-IKKa/b, total IKKa,p-NF-kB, total CD127dimCD25+ gate by FACSCanto II flow cytometer (BD Biosciences) NF-kB (Cell Signaling, Danvers, MA), p-ATF2, total ATF2, p-ELK1, total and analyzed for expression of GITR and Foxp3 or Foxp3 and IL-10 using ELK1, p-p38MAPK, total p38MAPK, p-ERK1/2, total ERK1/2, TLR-1, BD FACSDiva software (version 5.2; BD Biosciences) (28). TLR-6, MyD88, Toll/IL-1R adaptor protein (TIRAP), TNFR-a–associated factor 6 (TRAF6), b-actin (Santa Cruz Biotechnology; Santa Cruz, CA), and Macrophage–T cell coculture TLR-2 (Imgenex, San Diego, CA) were obtained, as indicated. BALB/c-derived peritoneal macrophages from naive mice were infected L. major infection of macrophages with L. major promastigotes at a ratio of 1:10 for 6 h. The extracellular parasites were washed out, and macrophages were treated with the indi- C57BL/6, iNOS-deficient, IL-12–deficient, and BALB/c-derived peritoneal cated doses of BPPcysMPEG for 24 h. The popliteal was macrophages were infected with stationary phase L. major promastigotes at collected from the 21-d L. major-infected BALB/c mice. Infected macro- a ratio of 1:10 for 6 h, and extracellular parasites were washed out. Macro- phages were cocultured with lymph node CD4+ T cells at 1:3 ratio phages were treated with different doses of TLR ligands or left untreated for (macrophage:T cells). After 60 h, supernatants were collected for cytokine 60 h. Macrophages were fixed with chilled methanol and stained with Giemsa assays and cells for Treg analyses. For amastigote count, macrophages stain. Amastigotes were counted under the microscope, as reported earlier (11). were washed, fixed with chilled methanol, stained with Giemsa stain, and counted under a microscope (E-600; Nikon). RT-PCR and real-time PCR Production of lentiviral particles for TLR shRNA and control shRNA RNAwas extracted using TRI-Reagent (Sigma-Aldrich, St. Louis, MO). For cDNA synthesis, 2 mg total RNA from each sample was incubated with 0.6 TLR1 and TLR6 shRNA (GenBank accession number NM_030682, mg random primer, 0.1M DTT, 10 mM dNTPs, 40 U RNase out, and 200 U http://www.ncbi.nlm.nih.gov/nuccore/NM_030682, and NM_011604, 3634 ANTILEISHMANIAL FUNCTION OF BPPcysMPEG http://www.ncbi.nlm.nih.gov/nuccore/NM_011604) in pLKO.1 lentivirus DCs were treated with the indicated doses of BPPcysMPEG for 24 h. The vector; control shRNA in pLKO.1 lentivirus vector; TLR2 shRNA (Gen- popliteal lymph node was collected from the 21-d L. major-infected Bank accession number NM_011905, http://www.ncbi.nlm.nih.gov/nuccore/ BALB/c mice. Infected DCs were cocultured with lymph node CD4+ NM_011905) in pGIPZ lentivirus vector; and control shRNA in pGIPZ T cells at a 1:3 ratio (DC:T cells). After 60 h, supernatants were collected lentivirus vector were purchased from Open Biosystems (Huntsville, AL). and quantified for cytokine response. Cells were harvested and were used These shRNA were used for lentiviral production using Trans-Lentiviral for Treg analyses by flow cytometry (28). packaging system (replication-incompetent HIV-1–based lentivirus; Open Biosystems) in HEK293T cell line following the manufacturer’s protocol. In vivo L. major infection Briefly, 5.5 3 106 cells were seeded in 100-mm petriplates. Next day (day 1), Trans-Lentiviral packaging mix (28.5 mg) and TLR shRNA (9 mg) or BALB/c and IL-12–deficient mice were infected in the left footpads with 3 6 control shRNA (9 mg) plasmid DNA were mixed together. Transfection L. major promastigotes (s.c., 2 10 in 50 ml saline/mouse). Mice were was carried out in serum-free media using Arrest-In transfection reagent treated with BPPcysMPEG (1 mg/mouse) for alternating 3 d starting from (Open Biosystems) at a 1:5 DNA/transfection reagent ratio. After 6 h, the third day postinfection. In some experiments, 2 d prior to L. major 3 6 medium was replaced with complete growth medium (DMEM supplemented infection, mice were pretreated with 5 10 transduction units (in 50 ml HBSS; Life Technologies, Grand Island, NY) of TLR1 shRNA, TLR2 with 10% FCS), and cells were incubated in a CO2 incubator at 37˚C. Virus- containing supernatant was harvested after 48 and 72 h, filtered through a shRNA, TLR6 shRNA, or control shRNA expressing lentiviruses in the 0.45-mm filter (Millipore), and concentrated 100-fold (32). same footpad to be infected. Some mice were treated with BPPcysMPEG (1 mg/mouse) or CpG (10 mg/mouse) or both (s.c. into the footpad for 3 d In vitro lentiviral transduction alternatively starting from the third day postinfection). Seven mice were used in each group. The footpad swelling was scored using a digital mi- Peritoneal macrophages were transduced with lentivirally expressed TLR1 crometer (Mitituyo). Mice were sacrificed 5 wk after the infection; pop- shRNA (TLR1-Lv), TLR2 shRNA (TLR2-Lv), TLR6 shRNA (TLR6-Lv), liteal lymph nodes were collected for assessing parasite load (34) and for and control shRNA (control-Lv) particles (2 transduction units/cell) for 8 h

cytokine production by ELISA (32). Downloaded from in RPMI 1640 supplemented with FCS (0.5%) and polybrene (8 mg/ml) (Sigma-Aldrich). Cells were washed to remove residual viral particles. Testing the adjuvanticity of BPPcysMPEG in antileishmanial After 48-h transduction, uninfected and L. major-infected cells were immunization treated with BPPcysMPEG and CpG or left untreated for 48 h for amas- tigote count and ELISA (32). Three groups of mice, each containing 10 BALB/c mice, were immunized s.c. into the hind footpad with 1 3 106 formalin-fixed L. major. Fixation of Generation of monocyte–derived dendritic cells the parasites was confirmed by the failure of parasites to grow in vitro. Dendritic cells (DCs) were cultured from bone marrow progenitor cells Immunized mice were treated twice with either Pam3CSK4 (1mg/mouse) or http://www.jimmunol.org/ using a modified protocol of a previously described method (33). In brief, BPPcysMPEG (1 mg/mouse) into the same footpad, on alternate days, femoral cells were fractionated on a density gradient (Histopaque-1077; starting from the first day of immunization, and one group was left un- Sigma-Aldrich) to isolate mononuclear cells, which were incubated for treated as a control. After 5 wk, all immunized and control mice were 3 6 plastic adherence for 90 min. Nonadherent cells were washed out with 13 infected with 2 10 L. major promastigotes. Five weeks after the in- PBS. DC culture medium (RPMI 1640, 10% FCS, 100 U/ml penicillin, 100 fection, mice were sacrificed and popliteal lymph nodes were collected for mg/ml streptomycin, 50 mM 2-ME, 20 ng/ml GM-CSF, and 10 ng/ml IL-4) parasite enumeration (34), cytokine production assay, and Treg cell ana- was added to the adhered (1 3 106 cells/ml). Culture medium lyses (28). was replaced with fresh culture medium every 3 d. On day 6, dislodged cells were used as bone marrow–derived DCs. Purity of the DC was always Statistical analysis . 85% (data not shown) (27, 28, 33). The in vitro cultures were set in triplicates. We used 5–10 mice per group by guest on September 25, 2021 DC and T cell coculture for in vivo experiments, as indicated. Error bars signify SEM from one representative from multiple sets of experiments. Student t test was used to Bone marrow–derived DCs were infected with L. major promastigotes with assess the significance of the differences between the mean values for a ratio of 1:10 for 3 h. The extracellular parasites were washed out, and control and experimental groups.

Table I. The forward and reverse primers used in RT-PCR and real-time PCR

Gene Primer Sequence (59-39) Gene Primer Sequence (59-39)

RT-PCR Primers GAPDH F: GAGCCAAACGGGTCATCATC TLR5 F: CAACCAAACCAACGTCACCCTGTT R: CCTGCTTCACCACCTTCTTG R: TGGCTTCCTCTTCATCGCAGTCTT b-actin F: GTCCCTGTATGCCTCTGGTC TLR6 F: TTGTTTGCGCCCTGGCCTTAAT R: CAAGAAGGAAGGCTGGAAAAG R: TGTTCTTGGTGGCAGGTCTTTG iNOS F: CAGAGGACCCAGAGACAAGC TLR7 F: TGCAACTGTGATGCTGTGTGGT R: AAGACCAGAGGCAGCACATC R: TTTGACCTTTGTGTGCTCCTGG TLR1 F: CAATGTGGAAACAACGTGGA TLR8 F: TGCCAAACAACAGCACCCAA R: TGTAACTTTGGGGGAAGCTG R: TCAAAGACTCAGGCAACCCA TLR2 F: AAGAGGAAGCCCAAGAAAGC TLR9 F: ACTGAGCACCCCTGCTTCTA R: CGATGGAATCGATGATGTTG R: AGATTAGTCAGCGGCAGGAA TLR3 F: TCCTTGCGTTGCGAAGTGAA TLR11 F: CTGAAGGTGAAGGCTTGAGGTT R: TTGGGCGTTGTTCAAGAGGA R: CCTGTCATTTCCAATCCCAAA TLR4 F: ACCTGGCTGGTTTACACGTC TLR12 F: TTGAGCTGCAGGGCTTGATTGT R: CTGCCAGAGACATTGCAGAA R: GCTTGGTTAGGCCAGTGCTTGAAA TLR13 F: ACACAGTGAATGGTGCCGACTT R: AAAGTGGCTGCTGGTGAACACT Real-Time PCR Primers TLR1 F: CAATGTGGAAACAACGTGGA TLR9 F: ACTGAGCACCCCTGCTTCTA R: TGTAACTTTGGGGGAAGCTG R: AGATTAGTCAGCGGCAGGAA TLR2 F: AAGAGGAAGCCCAAGAAAGC iNOS F: GTGACACACAGCGCTACAA R: CGATGGAATCGATGATGTTG R: GAGCACAGCCACATTGATCT TLR6 F: TTGTTTGCGCCCTGGCCTTAAT GAPDH F: ATGGACTGTGGTCATGAGCC R: TGTTCTTGGTGGCAGGTCTTTG R: ATTGTCAGCAATGCATCCTG F, forward; R, reverse. The Journal of Immunology 3635

Results whether providing extraneous TLR2–TLR6-ligand BPPcysMPEG TLR1, TLR2, and TLR6 are differentially expressed on would restore TLR2–TLR6 association in the infected macrophages. L. major-infected macrophages It was observed that BPPcysMPEG enhanced the association be- tween TLR2 and TLR6 in both uninfected and L. major-infected Because several TLRs are implicated in the control of Leishmania macrophages (Fig. 1D). These results suggest that L. major selec- infection (11, 12, 14, 23, 35–39), we checked the expression of all tively augments TLR1 expression; the parasite enhances TLR1– TLRs in uninfected and L. major-infected macrophages by RT- TLR2 and TLR2–TLR2 association, but reduces TLR2–TLR6 as- PCR (Table I). We observed an increased expression of TLR1 and sociation. It is plausible that the parasite reduces the TLR2–TLR6 TLR11/TLR12, but decreased expression of TLR4 and TLR9 association as an immune evasion strategy. In that case, befitting the (Fig. 1A). Among these altered TLRs, TLR4 and TLR9 promote principle of parasitism, the reduced association between TLR2 and host-protective antileishmanial immune responses (23, 40–42) and TLR6 implies a host-protective role in L. major infection. TLR11 and TLR12 are not yet implicated in Leishmania infection. However, the role of TLR1 in Leishmania infection remained to TLR1 and TLR2 shRNA reduced L. major infection, but TLR6 be elucidated. As TLR1 functions as a heterodimer with TLR2, shRNA increased the infection which can also bind to TLR6, we first confirmed TLR1, TLR2, To test the plausible roles for TLR1, TLR2, and TLR6 in L. major and TLR6 expression in uninfected and L. major-infected mac- infection, the expression of each of these TLRs was silenced using rophages. The real-time PCR and Western blot data confirmed that respective shRNA, followed by L. major infection of the BALB/c TLR1 expression was increased in L. major infection (Fig. 1B). mice. We observed that TLR1 and TLR2 shRNA reduced the

Next, we tested the association between TLR1 and TLR2 and parasite load in macrophages (Fig. 2A); a part of the TLR2 Downloaded from between TLR2 and TLR6 in uninfected and L. major-infected shRNA-induced reduction in parasite load could be due to less macrophages. It was observed that TLR1–TLR2 and TLR2–TLR2 internalization (Fig. 2B). In BALB/c mice, TLR1 shRNA and associations were enhanced, but the TLR2–TLR6 association was TLR2 shRNA reduced parasite burden, whereas TLR6 shRNA- reduced in L. major-infected macrophages (Fig. 1C). Because the treated BALB/c mice increased the parasite load compared with ligands can promote heterodimerization of receptors, we tested that observed in control shRNA-treated mice (Fig. 2C). These http://www.jimmunol.org/

FIGURE 1. L. major infection differentially regulates the expression of TLR1, TLR2, and TLR6 and association of TLR1 or TLR6 with TLR2. BALB/c-derived thio- glycolate-elicited peritoneal macrophages were kept either uninfected (UIM) or infected with L. major promastigotes at 1:10 ratio for 72 h (IM). (A) The cells were lysed and the by guest on September 25, 2021 mRNA levels were analyzed for all TLRs by RT-PCR. Densitometric quantitation was performed using the Quan- tity One software (Bio-Rad), and the values for genes were normalized against b-actin (right panel)(A). (B) cDNA from the above experiments was used to check the expression of TLR1, TLR2, and TLR6 by real-time PCR. mRNA ex- pression levels of the target genes were normalized against GAPDH and expressed as relative fold change compared with uninfected controls. The cells from the above experi- ments were lysed, and proteins were used for Western blotting for TLR1, TLR2, and TLR6 (lower panel)(B). The densitometric quantitation was performed using the Quantity One software, and data are shown (lower right panel)(B). (C) Equal amount of protein was immuno- precipitated with anti-TLR2 Ab, and the association of TLR1, TLR2, and TLR6 was checked by immunoblotting using respective Abs. Densitometric values for TLR1, TLR2, and TLR6 expression were normalized against the values of b-actin (right panel)(C). (D) Macrophages were stim- ulated with BPPcysMPEG (100 ng/ml) for 15 min and lysed; equal amount of protein was immunoprecipitated with anti-TLR2; and the association of TLR6 was checked by immunoblotting. Data shown are densitometric unit for TLR6 (right panel)(D). From the same set of the experi- ment, equal amount of protein was resolved and blotted for b-actin to ensure the equal loading. The experiments were performed thrice. Results from one representative experi- ment are shown. The densitometric values represent mean 6 SEM. *p , 0.05, **p , 0.01, ***p , 0.001. D-Units, den- sitometric units. 3636 ANTILEISHMANIAL FUNCTION OF BPPcysMPEG observations suggest that TLR1 and TLR2 promote L. major in- protective cytokine (13, 22); iNOS (Fig. 2F), the enzyme that fection, whereas TLR6 plays an antileishmanial role. catalyzes the generation of NO, which kills Leishmania (43); and Because TLR2–TLR6 association was reduced in L. major-in- TLR9 (Fig. 2G), which is also reported to play a host-protective fected macrophages, but restored by its ligand BPPcysMPEG, role by inducing IL-12 (44). Thus, BPPcysMPEG is observed to we tested the effect of its ligand on parasite growth in BALB/c- enhance key host-protective functions in vitro. derived peritoneal macrophages and compared with the effects of BPPcysMPEG activates higher p38MAPK and ATF2 Pam3CSK4, a ligand for TLR1–TLR2 heterodimer, and PGN, a ligand for TLR2 on the infection. It was observed that Pam3CSK4 and Because Pam3CSK4, PGN, and BPPcysMPEG, the ligands for PGN increased the infection, but BPPcysMPEG reduced the in- TLR1, TLR2, and TLR6, respectively, induced differential ef- fection in a dose-dependent manner (Fig. 2D). Corroborating fector functions in macrophages, we examined the signaling in- with this observation, among these three ligands, BPPcysMPEG duced by these ligands. As TLR2 signals through the adaptor appeared to be the strongest inducer of IL-12 (Fig. 2E), a host- molecules MyD88, TIRAP, and TRAF6 (44), TLR2 was immu- Downloaded from http://www.jimmunol.org/ by guest on September 25, 2021

FIGURE 2. TLR1 and TLR2 increased, whereas TLR6 reduced L. major infection. BALB/c-derived thioglycolate-elicited peritoneal macrophages were transduced with lentivirally expressed TLR1, TLR2, TLR6 shRNA-Lv, or control shRNA-Lv (two transduction units per cell) for 48 h. (A) Untransduced or TLR-specific lentivirally transduced cells were infected with L. major for 6 h. Extracellular parasites were washed out, and parasite burden was assessed by Giemsa staining. RT-PCR was performed to check the inhibition of TLR1, TLR2, and TLR6 expression by respective TLR shRNA Lv or control shRNA Lv, transduced in macrophages (lower panel)(A). (B) Internalization of L. major parasites in peritoneal macrophages was checked 1 h postinfection. (C) BALB/c mice (n = 7) were injected s.c. with 5 3 106 transduction units (in 50 ml HBSS) TLR1, TLR2, TLR6 shRNA Lv, or control shRNA Lv into hind footpad and were infected s.c. with 2 3 106 L. major in same hind footpad 3 d after lentiviral treatment. Some mice left untreated were infected with L. major and were kept as controls. Footpad thickness was measured weekly to assess the severity of disease (upper panel). Five weeks after the infection, mice were sacrificed and parasites were enumerated from the popliteal lymph node cells (lower panel), as described earlier. The experiments were repeated twice. The error bars represent mean 6 SEM. (D and E) Thioglycolate-elicited BALB/c-derived macrophages were either left uninfected (UIM) or infected (IM) with L. major promastigotes at a ratio of 1:10 for 12 h, followed by washing out extracellular parasites and incubation for an additional 12 h. The cells were treated with the indicated doses of Pam3CSK4 (P3C), PGN, or BPPcysMPEG (BPP) for another 48 h. (D) Parasite burden was assessed by Giemsa staining. (E) IL-10 and IL-12 (p70) from the culture supernatants of uninfected and L. major-infected macrophages treated with the indicated doses of P3C, PGN, or BPP were assessed by ELISA. The significance tests were performed to compare the IL-12 productions induced by BPPcysMPEG and the other two ligands. (F and G) Uninfected and 64-h L. major-infected macrophages were treated with the indicated doses of P3C, PGN, or BPP for another 8 h, followed by assessment of iNOS expression by RT-PCR (F) and TLR9 expression by RT-PCR and real-time PCR (G). For real-time PCR, doses used were P3C (100 ng/ml), PGN (10 mg/ml), or BPP (100 ng/ml) (right panel)(G). The experiments were performed three times. The error bars show mean 6 SEM. *p , 0.05, **p , 0.01, ***p , 0.001. The Journal of Immunology 3637

noprecipitated from the Pam3CSK4-, PGN-, and BPPcysMPEG- assessed the mechanism of the host-protective function of stimulated macrophages, followed by immunoblotting for MyD88, BPPcysMPEG. TIRAP, and TRAF6. We observed that TLR2 recruited more BPPcysMPEG induces T cell–mediated Th1 response both MyD88 in response to PGN or BPPcysMPEG than to Pam3CSK4; only BPPcysMPEG drastically increased the TIRAP recruitment in vitro and in a susceptible host to TLR2, whereas Pam3CSK4 showed a tendency to increased re- As BPPcysMPEG showed antileishmanial effects in macrophages cruitment of TRAF6 to TLR2 (Fig. 3A). As signals from membrane in vitro, we tested its effect on the induction of T cell–mediated host receptors are taken to the nucleus via p38MAPK and ERK1/2 (45, protection in a macrophage–T cell coculture system. When L. major- 46) and transcription factors, it was possible that the observed infected macrophages were cocultured with the lymph node T cells differences between TLR2 ligands might occur due to differential from L. major-infected BALB/c mice, the amastigote numbers in- phosphorylation and activation of these signaling intermediates. creased in macrophages, but BPPcysMPEG treatment reduced the We observed that Pam3CSK4, PGN, and BPPcysMPEG also dif- parasite count (Fig. 4A). A lower dose of BPPcysMPEG increased fered in their ability to activate p38MAPK, ERK1/2, and transcrip- both IL-4 and IFN-g production, but IL-4 production was reduced, tion factors. Compared with Pam3CSK4 and PGN, BPPcysMPEG whereas IFN-g production increased, with the higher doses of activated more p38MAPK and ATF2 than ERK1/2 and ELK1 BPPcysMPEG (Fig. 4B); a resultant effect of these changes was (Fig. 3B). It was reported that the L. donovani-infected THP-1 cells, significantly enhanced IFN-g production (Fig. 4B, inset). These ob- a macrophage-like cell line, responded to Pam3CSK4 by increasing servations suggested that BPPcysMPEG enhanced the antileish- higher ERK1/2 and IL-10 production (14). Taken together, these manial effect even in presence of the disease-exacerbating T cells, observations suggest that BPPcysMPEG signals through p38MAPK which predominated in the draining lymph node of a L. major- Downloaded from and ATF2, whereas Pam3CSK4 and PGN signaled primarily through infected susceptible host. Therefore, we next examined the anti- ERK1/2 and ELK1. leishmanial effect of BPPcysMPEG treatment on L. major infection The expression of TLR1, TLR2, and TLR6; the associations in BALB/c mice, which received three BPPcysMPEG treatments between these three TLRs; and the effects of the respective TLR on alternate days starting from the third day postinfection. We ob- shRNAs and ligands on various effector functions, including the served a significant reduction in parasite load in the lymph node

L. major infection, identify TLR2–TLR6 and its ligand BPPcysMPEG of the BPPcysMPEG-treated BALB/c mice (Fig. 4C, inset). http://www.jimmunol.org/ as the host-protective receptor–ligand pair. Therefore, we further This antileishmanial effect was accompanied by heightened IFN-g by guest on September 25, 2021

FIGURE 3. BPPcysMPEG activates higher p38MAPK and ATF2. (A) Thioglycolate-elicited BALB/c-derived macrophages were treated with the in- dicated doses of Pam3CSK4, PGN, or BPP for 15 min and lysed, and an equal amount of protein was immunoprecipitated with anti-TLR2 Ab and blotted with Abs against MyD88, TIRAP, and TRAF6. Densitometric values were normalized against the values of b-actin (lower panel)(A). Densitometry shown is the collated data from two experiments. Error bars show mean 6 SEM. (B) Cell lysates were immunoblotted for p-p38MAPK, p-ERK1/2, p-IKKa/b, p-NF-kB, p-ATF2, p-ELK1 or total p38MAPK, ERK1/2, IKKa, NF-kB, ATF2, and ELK1. Densitometric values were normalized against the values of respective total proteins, as indicated (lower panel)(B). The data from two repeat experiments are shown in the densitometry. The ratios of p-p38/ERK1/2 or p-ATF2/p-ELK1 were calculated from the mean values of their respective lanes. 3638 ANTILEISHMANIAL FUNCTION OF BPPcysMPEG production, but lowered IL-4 production (Fig. 4D). An apparent and CpG (Fig. 5C). Whereas BPPcysMPEG and CpG increased difference in the profile of BPPcysMPEG-induced changes in IL-4 IFN-g production in these mice, TLR6shRNA reduced IFN-g and IFN-g productions in vitro (Fig. 4B) and in BALB/c mice production, but increased IL-4 production (Fig. 5D). These re- (Fig. 4D) was reconciled as the ratio of IFN-g to IL-4 (Fig. 4B, sults clearly suggested that the observed antileishmanial effects inset), which showed a drastic increase in IFN-g production com- of BPPcysMPEG were indeed due to TLR6 and that CpG and pared with IL-4. Another plausible explanation for the apparent BPPcysMPEG synergize to effect the observed antileishmanial discrepancy is more complex interaction between APCs and T functions. cells in vivo than that observed in defined macrophage–T cell co- BPPcysMPEG reduced Treg cells in cocultures with culture. Thus, the TLR2/TLR6 ligand, BPPcysMPEG, establishes macrophages or DCs host-protective T cells to exert its antileishmanial functions. Treg cells promote Leishmania survival and growth in a suscepti- TLR6 shRNA significantly abrogated the antileishmanial ble host, as the numbers of Treg cells increase in progressive in- effects of BPPcysMPEG fection (24–28), but depletion of Treg cells reduces the disease Because BPPcysMPEG, a TLR2/6 ligand, increased TLR9 ex- (26, 47). As BPPcysMPEG reduced L. major infection in BALB/c pression, we studied the synergism between TLR2/TLR6 and TLR9. mice (Fig. 4C), we examined whether BPPcysMPEG would de- BALB/c-derived thioglycolate-elicited macrophages were trans- crease the number of Treg cells. To test the hypothesis, we co- duced with TLR6 shRNA or control shRNA-expressing lentivirus, cultured the BPPcysMPEG-treated or untreated, L. major-infected followed by L. major infection and treatment with BPPcysMPEG and macrophages or bone marrow–derived DC with CD4+ T cells from

CpG, or left untreated as control. It was observed that TLR6 shRNA- the draining popliteal lymph nodes of 21-d L. major-infected Downloaded from treated cells reversed the antileishmanial response to BPPcysMPEG BALB/c mice. We observed that the BPPcysMPEG treatment or CpG alone or in combination (Fig. 5A). The TLR6 shRNA- reduced the number of CD3+CD4+CD127dimCD25+GITR+Foxp3+ transduced macrophages produced less IL-12, but higher IL-10 cells in both macrophage–CD4+ T cell and DC–CD4+ T cell cocul- in response to these ligands (Fig. 5B). Next, BALB/c mice were tures (Fig. 6) and reduced the number of IL-10+ cells in DC–CD4+ treated with lentivirally expressed TLR6 shRNA or control shRNA, T cell cocultures (Fig. 6B). The culture supernatants were collected +

followed by L. major infection and treatment with BPPcysMPEG from DC–CD4 T cell cocultures, and the levels of IL-12, IL-10, http://www.jimmunol.org/ or CpG, or were left untreated. It was observed that TLR6 shRNA IL-4, and IFN-g were assessed. We observed that a reduced significantly abrogated the antileishmanial effect of BPPcysMPEG number of Treg cells was associated with higher IL-12 and IFN-g, by guest on September 25, 2021

FIGURE 4. BPPcysMPEG elicits host-protective immune response. BALB/c-derived peritoneal macrophages were infected with L. major for 72 h. After washing out the extracellular parasites, macrophages were treated with the indicated doses of BPP and were cocultured with the CD4+ T cells isolated from the lymph nodes of L. major-infected (21-d–infected) and naive BALB/c mice at 1:3 ratio (MФ:T cells). (A) After 72-h infection, macrophages were washed, fixed, stained with Giemsa stain, and evaluated for the amastigotes per 100 macrophages. (B) IL-4 and IFN-g were assessed from culture supernatants, as described in Materials and Methods.(B, inset) Fold over change in cytokine IL-4 and IFN-g was determined from the values of BPP treated against the values of infected macrophages cocultured with infected T cells. Data shown are the representative of one experiment, repeated twice. Error bars show mean 6 SEM. (C) BALB/c mice were infected s.c. in hind footpad with 2 3 106 L. major promastigotes. Mice were treated with BPP (1 mg/mouse) into the L. major-infected footpad alternatively for 3 d starting from the third day postinfection or were injected with sterile PBS as control. Disease progression was scored weekly by evaluating the net footpad swelling (the thickness of the uninfected left footpad subtracted from the infected right footpad) by digital micrometer for 5 wk. (C, inset) Mice were sacrificed 5 wk after the infection, and parasite burden in lymph node cells was assessed. (D) Lymph node cells from mice, as indicated, in each group (n = 7) were pooled and stimulated with anti-CD3 (0.5 mg/ml) and anti-CD28 (2 mg/ml) for 60 h. Culture supernatants were assessed for the production of the cytokines IL-4 and IFN-g, as described in Materials and Methods. The experiment was repeated twice. Data shown are the representative of one experiment, and error bars show the mean 6 SEM. *p , 0.05, **p , 0.01. The Journal of Immunology 3639 Downloaded from http://www.jimmunol.org/ by guest on September 25, 2021 FIGURE 5. TLR6 shRNA significantly abrogated the antileishmanial effects of BPPcysMPEG. Thioglycolate-elicited BALB/c-derived mouse macro- phages were transduced with TLR6 shRNA (TLR6 Lv) or control shRNA (control Lv) lentiviral particles. Forty-eight hours later, lentivirally transduced cells were either left uninfected or infected with L. major at a ratio of 1:10, followed by treatment with BPP (50 ng/ml) and CpG (0.12 mM) for a total 72-h infection. (A) Cells were stained with Giemsa, and amastigotes were enumerated. The amastigote numbers counted in the control CpG or control shRNA- Lv were 565.5 6 27.5 and 684.5 6 19.5, respectively. (B) Culture supernatants collected were assessed for IL-10 and IL-12p70 by ELISA. Ratio between IL-12:IL-10 was determined from the respective treatments. The IL-12:IL-10 ratios in the control CpG- and control shRNA-Lv–treated cultures were 0.33 6 0.03 and 0.25 6 0.05, respectively. (C and D) TLR6 lentivirus reversed the CpG-induced antileishmanial immune response in susceptible BALB/c mice. Some groups of BALB/c mice were treated with TLR6 shRNA Lv, 2 d later infected with L. major, and treated with BPP (1 mg/mouse) and CpG (10 mg/ mouse) together or alone on alternating 3 d, starting from 2 d postinfection. (C) Parasite load was determined 5 wk after the infection. Number of parasites enumerated from control CpG- or control shRNA-Lv–treated mice were (69.75 6 9.4681) 3 106 and (88.333 6 13.4361) 3 106, respectively. (C, inset) Immunoblot analysis of TLR6 expression from footpad lesion (FP) and lymph node (LN) of L. major-infected BALB/c mice treated with control shRNA-Lv and TLR6 shRNA-Lv 5 wk after L. major infection. (D) Total lymph node cells from various mice groups were stimulated with anti-CD3 Ab (0.5 mg/ml) and anti-CD28 Ab (2 mg/ml) for 60 h. Culture supernatants from different groups were assessed for IL-4 and IFN-g production by ELISA. The values for IL-4: control CpG (478.5 6 33.5 pg/ml), control shRNA-Lv (913.0 6 8.0 pg/ml); the values for IFN-g: control CpG (384 6 12 pg/ml), control shRNA-Lv (313.5 6 90.5 pg/ml). The experiments were performed twice, and one representative data are shown. Error bars represent mean 6 SEM. *p , 0.05, **p , 0.01, ***p , 0.001. but reduced IL-10 and IL-4 levels (Fig. 6B, inset), suggesting that (Fig. 7C). Together these observations indicate that the anti- BPPcysMPEG-induced host protection was associated with de- leishmanial effect of BPPcysMPEG is partly iNOS mediated. To creased Treg cells and increased IL-12 induction. confirm the role of IL-12 in BPPcysMPEG-induced antileishmanial defense, we tested the effects of BPPcysMPEG treatment on BPPcysMPEG-induced host protection is IL-12 dependent parasite load, IL-4/IFN-g production, and Treg cell numbers in Because BPPcysMPEG increased the production of IL-12 that IL-12–deficient and wild-type BALB/c mice. We observed that played a host-protective role against Leishmania infection (13, 22), BPPcysMPEG treatment reduced the severity of the disease the uninfected and L. major-infected macrophages from IL-12– (Fig. 7D) and parasite load in BALB/c mice, but not in the IL-12– deficient or BALB/c mice were treated with BPPcysMPEG. It was deficient mice (Fig. 7E); the antileishmanial effect of BPPcysMPEG observed that BPPcysMPEG reduced the number of amastigotes was accompanied by high IFN-g, but less IL-4 productions in wild-type, but not IL-12–deficient macrophages (Fig. 7A), com- (Fig. 7E) and reduced Treg cell numbers (Fig. 7F) in BALB/c, but mensurating with its effect on iNOS expression (Fig. 7B). Cor- not in IL-12–deficient mice. These observations indicate that the roborating with these observations, the antileishmanial effect of BPPcysMPEG-induced antileishmanial defense is IL-12 depen- BPPcysMPEG was significantly reduced in iNOS-deficient mice dent, but Treg sensitive. 3640 ANTILEISHMANIAL FUNCTION OF BPPcysMPEG

FIGURE 6. BPPcysMPEG treatment depletes Treg cells in vitro. Thioglycolate-elicited peritoneal mouse macro- phages or bone marrow DCs derived from BALB/c mice, infected with L. major promastigotes, and treated with indicated doses of BPP for 24 h and stimulated with crude soluble leishmania Ags (CSA). CD4+ T cells were isolated from popliteal draining lymph nodes of L. major-infected BALB/c mice 21 d postinfection. CD4+ T cells were used for the coculture with macrophages or DCs from the above-described culture for 60 h. Upper panel of data represents the gating strategy for the FACS analyses. (A) +

Cocultured macrophage CD4 T cells were used to check Downloaded from the CD3+CD4+CD127dimCD25+GITR+Foxp3+ cells. (B) Cocultured DC CD4+ T cells were used to check Foxp3+ and IL-10+ cells in CD3+CD4+CD127dimCD25+GITR+ cells. (B, inset) Culture supernatants were collected from the cocultured DC CD4+ T cells and assayed for IL-10, IL- 12, IL-4, and IFN-g cytokines, as described in Materials and Methods. Fold over change at the level of cytokines http://www.jimmunol.org/ was determined from the values of BPP-treated DC CD4+ coculture against the untreated DC CD4+ T cells. by guest on September 25, 2021

BPPcysMPEG induces host-protective prophylactic these host-protective effects of BPPcysMPEG were accompanied by antileishmanial effects increased TLR9 expression, enhanced IL-12 production, and higher As BPPcysMPEG reduced parasite load, accompanied by an IFN- iNOS induction. Fourth, as a possible mechanism for its host- g–dominated response in in vitro and in BALB/c mice, we ex- protective effects, BPPcysMPEG induced leishmanicidal activities amined the adjuvanticity of BPPcysMPEG in establishing host- in T cells from the L. major-infected mice and reduced Treg cell protective antileishmanial memory response. The BPPcysMPEG numbers. These host-protective effects were TLR6 mediated, IL-12 treatment during priming with the fixed L. major promastigotes dependent, iNOS dependent, and Treg sensitive. Finally, targeting resulted in significantly reduced parasite load in these mice, TLR2–TLR6 led to significant reduction in parasite load in both compared with the untreated controls or unprimed controls, fol- therapeutic and prophylactic modes, accompanied by modulation lowing a challenge L. major infection (Fig. 8A). The protection was of T cell responses. Thus, the role for TLR2 in selective hetero- accompanied by high IFN-g, but low IL-4 (Fig. 8B). The protec- dimerizations with either TLR1 or TLR6 in L. major infection tion was associated with reduced Treg cells in the BPPcysMPEG- assumes significance, which was indicated by the strong anti- treated group (Fig. 8C). These observations together indicate that leishmanial effect of the TLR2–TLR6 ligand, BPPcysMPEG, but BPPcysMPEG possesses a strong adjuvanticity against L. major not by the ligand of the TLR1–TLR2 heterodimer. Altogether, to infection. our knowledge, this is the first demonstration of TLR2 functional duality, contingent upon its dimerization with either TLR1 or TLR6. The increased TLR1–TLR2 or TLR2–TLR2 association, but Discussion reduced TLR2–TLR6 association, implies selective targeting of This work documents several unique observations. First, the TLR2 partners by the parasite. It is possible that due to Leish- expression of the TLR2 associates, TLR1, TLR2, and TLR6, and mania-induced changes in membrane anisotropy of the infected their dimerizations is differentially modulated by L. major infec- macrophages (48), the interaction between the hydrophobic tion in macrophages. The reduced TLR2–TLR6 association in domains of these TLRs (49) is differentially affected, resulting in L. major-infected macrophages was restored by its synthetic li- altered associations in L. major-infected macrophages. In contrast, gand BPPcysMPEG. Second, TLR1 or TLR2 silencing reduced TLR1 or TLR2 silencing reduces, but TLR6 silencing increases, L. major infection, but TLR6 silencing increased the infection. L. major infection. It is possible that silencing of one TLR alters Conversely, the ligands Pam3CSK4 and PGN exacerbated the infec- the other two TLRs’ heterodimerizing propensity. Thus, the initial tion, but BPPcysMPEG reduced the infection in macrophages. Third, difference between TLR1–TLR2, TLR2–TLR2, and TLR2–TLR6 The Journal of Immunology 3641 Downloaded from

FIGURE 7. BPPcysMPEG-induced host protection against L. major infection is IL-12 dependent. Thioglycolate-elicited peritoneal macrophages derived from BALB/c or IL-12 knockout (IL-12 KO) mice were either infected with L. major promastigotes or left uninfected. (A) Twenty-four-hour–infected http://www.jimmunol.org/ macrophages were stimulated with BPP for another 48 h, cells were stained with Giemsa stain, and amastigotes were enumerated. (B) Macrophages were stimulated with indicated doses of BPP for 8 h. Cells were lysed for the synthesis of cDNA and were used for the amplification of iNOS by real-time PCR. Data shown represent the relative fold change between untreated and treated cells. (C) Macrophages derived from C57BL/6 or iNOS knockout (iNOS-KO) mice were infected with L. major promastigotes and were treated with the indicated doses of BPP. Seventy-two hours postinfection, amastigotes were counted. (D and E) BALB/c and IL-12 KO mice were infected with 2 3 106 L. major promastigotes. Two days postinfection, mice were treated with BPP (1 mg/mouse) alternatively for 3 d. (D) The footpad thickness was measured weekly. (E) Five weeks after the infection, mice were sacrificed and parasites were enumerated from the popliteal lymph node cells. Lymph node cells pooled from mice (n = 7) of each group were cultured into anti-CD3 Ab (0.5 mg/ ml)-precoated plate for 60 h. Supernatants were collected, and cytokine ELISA was performed for IFN-g and IL-4 (right panel). (F) Foxp3+ Treg cells were analyzed by FACS gated on CD3+CD4+CD127dimCD25+GITR+ cells; upper panel of data represents the gating strategy. Data shown are representative of by guest on September 25, 2021 one experiment, and the experiments were repeated twice. Error bars represent mean 6 SEM. *p , 0.05, **p , 0.01, ***p , 0.001. dimers can be induced either by alteration of the physical prop- this mode of antileishmanial function of BPPcysMPEG can be erties of the macrophage membrane or by alteration of the ratios attributed to innate immune mechanisms. among these three TLRs. Through a cascade of events, these Besides the innate immune mechanisms, BPPcysMPEG is also initial changes led to contrasting outcomes of L. major infection. shown to affect T cells in the coculture with macrophages and The cascade of events may be represented by the differential DCs. It is possible that the T cell–dependent antileishmanial func- signaling triggered by the ligand-induced homotypic or heterotypic tions of BPPcysMPEG build up from its innate immune effects. association of TLR2. For example, Pam3CSK4 and PGN trigger For example, the TLR2–TLR6–activated or TLR9-activated IL-12 primarily ERK1/2 and ELK1 activation, whereas BPPcysMPEG can set a Th1 bias, as BPPcysMPEG failed to exert its anti- induces p38MAPK phosphorylation, leading to activation of leishmanial effects in IL-12–deficient mice. In a different study, C-Jun (50) that can dimerize with the BPPcysMPEG-activated BPPcysMPEG, along with IFN-g, is shown to reduce the allergic ATF2 to induce inflammatory response (51, 52). In contrast, airway inflammation (55). Because the allergic manifestations in ERK1/2 activates ELK1 via AP-1, which binds to activated c-Fos the airway may indicate exaggerated Th2 response (21), the re- (53, 54). How these signaling pathways are reciprocally related to duction in the airway allergy by BPPcysMPEG implies possible the TLR1–TLR2– or TLR2–TLR6–induced contrasting outcomes— antagonism of Th2 differentiation. As Th2 responses exacerbate disease promotion or host protection—of L. major infection awaits L. major infection (56), it is possible that BPPcysMPEG exerts elucidation. therapeutic and prophylactic effects by facilitating Th1 response One mechanism of the host-protective antileishmanial function (57). The enhanced productions of IL-12 in macrophages in vitro of BPPcysMPEG is related to the enhanced expression of TLR9, and of IFN-g in BPPcysMPEG-treated mice indicate the Th1 IL-12, and iNOS. iNOS catalyzes the formation of NO (11, 43), response-facilitating role for TLR2/TLR6 heterodimers and thereby which kills Leishmania (11, 32). As TLR6 silencing significantly its host-protective role against L. major infection. compromised the antileishmanial function of BPPcysMPEG, the In contrast, one of the disease-promoting T cells in L. major in- failure of IL-12–deficient macrophages to reduce L. major infec- fection is Treg cells, as the reduction in parasite burden has been tion in response to BPPcysMPEG treatment suggests a significant correlated with reduced number of Treg cells (24–28). To explore role for autocrine IL-12 in TLR2/TLR6-dependent Leishmania the prophylactic role for TLR2–TLR6 heterodimer, we primed killing. Finally, as TLR9 recognizes L. major DNA and induces BALB/c mice with fixed L. major and BPPcysMPEG, followed IL-12 production (41), it is possible that the leishmanial DNA- by challenge infection with L. major. BPPcysMPEG, but not activated TLR9-induced IL-12 may exert antileishmanial func- Pam3CSK4, the ligand for the TLR1–TLR2 heterodimer, established tions in macrophages. As T cells were absent in all these assays, a host-protective antileishmanial immune response, accompanied 3642 ANTILEISHMANIAL FUNCTION OF BPPcysMPEG

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