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. 2013 Apr 18;172(3):403–409. doi: 10.1111/cei.12074

Leishmania expressed lipophosphoglycan interacts with Toll-like receptor (TLR)-2 to decrease TLR-9 expression and reduce anti-leishmanial responses

S Srivastava 1, S P Pandey 1, M K Jha 1, H S Chandel 1, B Saha 1
PMCID: PMC3646439  PMID: 23600828

Abstract

Two different Toll-like receptors (TLRs) have been shown to play a role in host responses to Leishmania infection. TLR-2 is involved in parasite survival in macrophages upon activation by lipophosphoglycan (LPG), a virulence factor expressed by Leishmania. In contrast, activation of TLR-9 has been shown to promote a host-protective response. However, whether there is a relationship between the interaction of LPG and TLR-2, on one hand, with the effect of TLR-9, on the other hand, remains unknown. In this study, we report that in-vitro infection of macrophages with a L. major parasite with high expression levels of LPG results in decreased TLR-9 expression compared to infection with a L. major parasite with lower expression levels of LPG. Addition of anti-LPG as well as anti-TLR-2 antibodies prevents this reduction of TLR-9 expression. Also, the addition of purified LPG to macrophages results in a decrease of TLR-9 expression, which is shown to be mediated by transforming growth factor (TGF)-β and interleukin (IL)-10. Finally, in-vitro treatment of macrophages with anti-LPG and/or anti-TLR-2 antibodies before infection reduces the number of amastigotes in macrophages and co-treatment of mice with anti-TLR-2 antibodies and cytosine–phosphate–guanosine (CpG) reduces footpad swelling and parasite load in the draining lymph nodes, accompanied by an interferon (IFN)-γ-predominant T cell response. Thus, for the first time, we show how interactions between LPG and TLR-2 reduce anti-leishmanial responses via cytokine-mediated decrease of TLR-9 expression.

Keywords: Leishmania, LPG, TLR expression, TLR-2, TLR-9

Introduction

Leishmania major, a protozoan parasite that inflicts the disease cutaneous leishmaniasis, resides and replicates in macrophages. Befitting the principle of parasitism, Leishmania infection results in the deactivation of macrophages. This deactivation can result from various processes, such as suppression of oxidative burst by the Leishmania-expressed virulence factor lipophosphoglycan (LPG) 1,2 or by interleukin (IL)-10 3. IL-10 can act in an autocrine manner to inhibit macrophage activation 4. However, whether there is a causal association between LPG and IL-10 production is not known.

Natural killer (NK) cells express Toll-like receptor-2 (TLR-2), a receptor for LPG 5. TLR-2 is also expressed in macrophages, implying that the observed LPG-induced deactivation of macrophages can, possibly, result from an LPG–TLR-2 interaction. However, TLR-2-deficient mice on a genetically resistant C57BL/6 background and wild-type C57BL/6 mice were comparably resistant to L. braziliensis infection 6, but the mice deficient in myeloid differentiation primary response gene 88 (MyD88) – the adaptor molecule responsible for signalling from several TLRs – on the same background were susceptible to L. braziliensis infection, suggesting that more than one TLR is involved in resistance to Leishmania infection.

Another TLR that signals through MyD88 and also participates in the host-protective anti-leishmanial immune response is TLR-9. Host-protective anti-leishmanial immune response is elicited by using the TLR-9 ligand cytosine–phosphate–guanosine (CpG) in prophylactic mode 7–9. As TLR-9-deficient mice on a C57BL/6 background were transiently susceptible 10, the CpG motif containing L. major DNA was suggested to require TLR-9 for inducing a host-protective effect. TLR-9 has been shown to elicit an anti-leishmanial response through NK cells 11. Despite discrete reports on LPG-induced macrophage deactivation and the roles for TLR-2 and TLR-9 in anti-leishmanial prophylaxis, to our knowledge neither the relationship between the Leishmania-expressed LPG, TLR-2 and TLR-9 in anti-leishmanial immune response nor the anti-leishmanial efficacy of CpG in a therapeutic mode has ever been tested.

In this study, we first characterized the LPG expression levels on a virulent L. major strain and on a less virulent strain derived from the virulent strain. The virulence of the strains was expressed in terms of their ability to infect susceptible BALB/c mice and BALB/c mouse-derived peritoneal macrophages. We examined whether LPG was involved in the modulation of TLR-9 expression and function and whether TLR-2 would contribute to such modulation. We finally examined whether co-administration of CpG and anti-TLR-2 antibody could reduce infection in susceptible BALB/c mice. These experiments established a causal relationship between LPG, TLR-2 and TLR-9 in L. major infection in susceptible BALB/c mice.

Materials and methods

L. major strains, mice and infection with L. major parasite

The L. major strain (MHOM/Su73/5ASKH) was maintained in vitro in RPMI-1640 10% fetal calf serum (FCS); for maintenance of virulence, the parasite was passaged regularly through BALB/c mice by subcutaneous infection of the stationary-phase promastigotes (2 × 106/mouse). A less virulent parasite strain (HP), derived by continued in-vitro passage for more than 8 years, or a virulent parasite strain (LP) of L. major (MHOM/Su73/5ASKH) was used for testing the association between virulence, LPG expression and TLR-9 expression.

BALB/c mice (Jackson Laboratories, Bar Harbor, ME, USA) were bred and reared in the experimental animal facility of the National Centre for Cell Science, Pune, India. The animals were monitored regularly by resident veterinarians. Progress of the infection was studied weekly and the parasite load was assessed at the termination of the animals. All experimentations were in accordance with the animal use protocol approved by the Institutional Animal Care and Use Committee (IACUC) and the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA), the regulatory authorities for animal experimentation.

Thioglycolate-elicited peritoneal macrophages from BALB/c mice were cultured in vitro and infected with L. major promastigotes at a 1:10 ratio for 12 h, followed by washing of the extracellular parasites and termination of the cultures 72 h after infection. The amastigote numbers per 100 macrophages were determined after staining the cells with Giemsa stain, as described previously 4,12. BALB/c mice were infected by subcutaneous injection of stationary phase promastigotes (2 × 106); the progress of the infection was assessed weekly by measurement of footpad thickness using a digital micrometer (Mitutoyo, Kawasaki, Japan) and the parasite load in the draining lymph node was enumerated as described previously 12.

LPG expression on parasites

Parasites were fixed in 80% methanol and kept at 4°C for 20 min. For surface phenotyping, the following antibodies from Cedarlane Laboratories (Burlington, Ontario, Canada) were used: purified anti-LPG mouse IgM, rabbit anti-mouse IgM-phycoerythrin (PE) and isotype IgM. Samples were acquired on a fluorescence activated cell sorter (FACS)Vantage™ flow cytometer and analysed with CellQuest Pro Software (Becton Dickinson, Mountain View, CA, USA).

Reverse transcription–polymerase chain reaction (RT–PCR) for testing the expression of TLR-9, inducible nitric oxide synthase (iNOS) transforming growth factor (TGF)-β and IL-10

The BALB/c-derived peritoneal macrophages were infected with either 5ASKH/LP or 5ASKH/HP, as indicated, or treated with LPG (a kind gift from Professor Salvatore J. Turco, University of Kentucky, Lexington, KY, USA) for 24 or 36 h, followed by RNA extraction in Trizol (Life Technologies, Gaithersburg, MD, USA), reverse transcription using Moloney murine leukaemia virus (MMLV)-reverse transcriptase and PCR using the gene-specific primers, as described previously 4,12. In some experiments, flagellin, lipopolysaccharide (LPS), Pam3CSK4 and peptidoglycan (PGN; Invivogen, San Diego, CA, USA) were used to stimulate macrophages. The following primers: TLR-9 forward: 5′-ACTGAGCACCCCTGCTTCTA-3′, reverse: 5′-AGATTAGTCAGCGGCAGGAA-3′; TGF-β forward: 5′-GCAACAACGCCATCTATAGAG-3′, reverse: 5′-CCTGTATTCCGTCTCCTTGG-3′; IL-10 forward: 5′-CTGCTATGCTGCCTGCTCTT-3′, reverse: 5′-CTCTTCACCTGCTCCACTGC-3′; iNOS forward: 5′-AGCTCCTCCCAGGACCACAC-3′, reverse: 5′-ACGCTGAGTACCTCATTGGC-3′; glyceraldehyde 3-phosphate dehydrogenase (GAPDH) forward: 5′-GAGCCAAACGGTCATCATC-3′, reverse: 5′-CCTGCTTCACCACCTTCTTG-3′; and β-actin forward: 5′-GTCCCTGTATGCCTCTGGTC-3′, reverse: 5′-CAAGAAGGAAGGCTGGAAAAG-3 were obtained from GenoMechanix (Alachua, FL, USA). GAPDH and β-actin were used as the control housekeeping genes. The PCR conditions were standardized, as described previously 4,12. The expression levels of the above-mentioned genes were quantified using the Quantity-one Program (Bio-Rad, Hercules, CA, USA).

Treatment of mice with CpG and anti-TLR-2 antibodies

For the TLR-2 blocking experiment mice were injected subcutaneously with anti-TLR-2 antibody or IgG1 isotype antibody (80 mg/kg body weight; eBioscience, San Diego, CA, USA) before L. major infection. BALB/c mice were infected subcutaneously with the indicated parasite. Mice were treated subcutaneously with TLR ligands (CpG ODN1826: 10 μg/mouse) with anti-TLR-2 antibody (Imgenex, San Diego, CA, USA) on alternate days starting from the second day after infection to the seventh day. Mice were killed 5 weeks after L. major infection and the parasite load was assessed in the draining lymph node, as described 12. Cytokine production by the draining lymph node cells was assessed using the respective cytokine emnzyme-linked immunosorbent assay (ELISA) kits (BD PharMingen, San Jose, CA, USA), following the manufacturer's instructions.

Statistical analysis

The in-vitro cultures were performed in triplicate. The in-vivo experiments had a minimum of five mice per group. The error bars are presented as mean ± s.d. The statistical significance between the indicated experimental and control groups was deduced by using Student's t-test.

Results

Virulent or less virulent L. major parasites differ in LPG expression levels

As Leishmania-expressed lipophosphoglycan (LPG) is involved in the survival of the parasite in macrophages, LPG is considered as a virulence factor in Leishmania infection. It is reported that LPG interacts with TLR-2 5. However, whether LPG interfacing TLR has any possible implications in the regulation of L. major infection is not known. Therefore, we studied how LPG may interface TLR to regulate L. major infection.

First, we characterized the virulent (5ASKH/LP) and less virulent (5ASKH/HP) L. major parasites for their infection of BALB/c-derived thioglycolate-elicited peritoneal macrophages. It was observed that the 5ASKH/LP-infected macrophages had a very high level of infection, whereas 5ASKH/HP were almost eliminated (Fig. 1). One of the mechanisms by which Leishmania can be killed by the host is via iNOS induction 13. It was observed that 5ASKH/LP did not induce strong iNOS expression, while 5ASKH/HP induced extremely strong iNOS expression (Fig. 1b, upper panel), which is corroborated by nitric oxide (NO) production by these two different parasites (Fig. 1b, lower panel).

Fig. 1.

Fig. 1

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Lipophosphoglycan (LPG) expression levels and infectivity of a virulent (LP) Leishmania major strain (5ASKH) and a less virulent (HP) L. major parasite derived from the same virulent strain. (a) LP and HP differ in their infectivity. BALB/c-derived thioglycolate-elicited macrophages were infected with LP or HP promastigotes, as indicated, at a 1:10 ratio for 12 h followed by washing off the extracellular parasites. The slides were fixed, stained and evaluated for infection at the indicated time-points. Each culture was set in triplicate and the experiment was repeated more than three times. The data from one representative experiment are shown. The error bars represent mean ± standard deviation (s.d.). (b) 72 h after the culture with L. major infection (LP or HP) or without infection (UIM), RNA was extracted from these macrophages and reverse transcription–polymerase chain reaction (RT–PCR) was performed to assess inducible nitric oxide synthase (iNOS) expression (upper panel). Culture supernatant from the same culture was assessed for nitrite production using Griess reagent, as described earlier (lower panel). Each culture was set in triplicate and the experiment was repeated three times. The data from one representative experiment are shown. The error bars represent mean ± s.d. (*P < 0·001). (c) The HP and LP parasites were stained with fluorescein isothiocyanate (FITC)-conjugated anti-LPG antibody or a FITC-labelled isotype-matched antibody and were analysed on a flow cytometer. The experiment was repeated three times, of which one representative overlay is shown. (d) Macrophages were prestimulated with peptidoglycan (PGN) (10 μg/ml) or medium for 6 h, followed by infection with the less virulent strains (HP) or virulent strains (LP) of L. major. At different time-points, the macrophages were fixed, stained and assessed under a light microscope for parasite load. The data from one representative experiment are shown. The error bars represent mean ± s.d. (*P < 0·001).

Next, we tested the LPG expression profiles on these two parasites. It was observed that the virulent strain had far higher LPG expression levels than that expressed by the less virulent strain of L. major (Fig. 1c). Because LPG works through TLR-2, this observation suggests that TLR-2 stimulation helps the parasite to survive. To examine this plausible role of TLR-2 we pretreated macrophages with PGN, a TLR-2 ligand, at different time-points, followed by infection with the virulent or less virulent strain. It was observed that PGN prolonged the survival of the less virulent strain of the L. major parasite (Fig. 1d). These results show that the highly virulent L. major parasite had far higher levels of LPG expression than the less virulent L. major, that LPG helps parasite survival, and that TLR-2 may play a role in parasite survival.

LPG–TLR-2 interaction modulates TLR-9 expression in macrophages

Because TLR-9 deficiency promotes L. major infection, albeit transiently 10, as does LPG 2, which is reported to interact with TLR-2 5, we tested whether or not these two strains of L. major differ in their capacity to inhibit TLR-9 expression in macrophages. It was observed that 5ASKH/LP, but not 5ASKH/HP, inhibited TLR-9 expression in BALB/c-derived thioglycolate-elicited peritoneal macrophages (Fig. 2a,b). Corroborating this observation, anti-TLR-2 antibody or anti-LPG antibody prevented the 5ASKH/LP-induced down-regulation of TLR-9 expression in macrophages (Fig. 2,d). In addition other TLR-2 ligands, Pam3CSK4 and PGN, inhibited TLR-9 expression whereas the TLR-4 ligand, LPS, or the TLR-5 ligand, flagellin, did not impair TLR-9 expression (Fig. 2e). These observations suggest that LPG down-regulates TLR-9 expression possibly by interacting through TLR-2.

Fig. 2.

Fig. 2

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TLR9 expression is inhibited by LPG and TLR2. (a) BALB/c-derived macrophages were infected with LP or HP parasites (IM) or cultured uninfected (UIM) for the indicated time points, followed by RT-PCR for TLR9 expression. (b–d) BALB/c-derived macrophages were infected with LP promastigotes at a 1:10 ratio, as described above, in absence or presence of anti-TLR2 antibody (c) or anti-LPG (d) antibody or with corresponding isotype-matched antibodies (10 μg/ml). RNA was extracted from the macrophages, as indicated, and RT-PCR was performed to assess TLR9 expression. The panels on the right show the densitometric quantitation of TLR9 expression (*P < 0·001). (e) BALB/c-derived macrophages were treated with the indicated doses of Pam3CSK4 (P3C), PGN, LPS or Flagellin for 8 h, as indicated, followed by RNA extraction and RT-PCR to assess TLR9 expression; the densitometric quantitation was presented (right panels; medium to Pam3CSK4, P = 0·019; medium to PGN, P < 0·001; medium to LPS, P < 0·001; medium to flagellin, P = 0·002). (f) BALB/c-derived macrophages were treated with LPG for 24 h or 36 h, as indicated, followed by RNA extraction and RT-PCR to assess TLR9 and TGF-β expression (left panel); the densitometric quantitation was presented (right panels; *P < 0·01; **P < 0·001). The culture supernatants were assessed for IL-10 production by ELISA (the extreme right panel; **P < 0·001). (g) BALB/c-derived macrophages were treated with the indicated concentrations of the respective cytokines (rIL-10 or rTGF-β) for 12 h, followed by RT-PCR and densitometry, as described above, for the assessment of TLR9 expression. The experiments were repeated thrice and the error bars represent mean ± s.d. IM: infected macrophages; UIM: uninfected macrophages.

Next, we examined the mechanism of LPG-induced suppression of TLR-9 expression in macrophages. As TGF-β and IL-10 are found to promote L. major infection 14,15, albeit through different mechanisms 16, we examined if LPG induced these two cytokines. It was observed that in BALB/c-derived thioglycolate-elicited macrophages, LPG induced the expression of TGF-β (Fig. 2f, left and middle panel) and IL-10 (Fig. 2f, extreme right panel), both of which suppressed TLR-9 expression in a dose-dependent manner (Fig. 2g). All these observations suggest, for the first time, that LPG plays a significant role in inhibiting TLR-9 expression in macrophages and that TLR-2 plays a significant role in inhibiting TLR-9 expression.

TLR-2 blockade reduces L. major infection significantly

Because TLR-9 is reported to promote a host-protective immune response, but LPG is observed to suppress TLR-9 expression, we tested whether antibodies against TLR-2 or LPG would reduce L. major infection of BALB/c-derived peritoneal macrophages. It was observed that both anti-TLR-2 and anti-LPG antibodies reduced L. major infection significantly in macrophages (Fig. 3a).

Fig. 3.

Fig. 3

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Toll-like receptor (TLR)-2 blockade restores cytosine–phosphate–guanosine (CpG)-induced anti-leishmanial functions. (a) BALB/c-derived macrophages were infected with the LP promastigotes in the presence of anti-LPG antibody or anti-TLR-2 antibody or both; 72 h later, the numbers of amastigote were assessed under a light microscope, as described in Materials and methods. The error bars represent mean ± s.d. (*P < 0·01; **P < 0·005). (b,c) BALB/c mice were infected with 5ASKH-LP promastigotes and were treated with anti-TLR-2 antibody or CpG or both, as described in Materials and methods. Footpad swelling was measured every week (b, left panel; *P < 0·01; **P < 0·001), but the parasite load in the draining lymph node was estimated after killing the mice 5 weeks after infection (b, right panel; *P < 0·01; **P < 0·001; ***P < 0·0001). The draining lymph node cells were stimulated with crude soluble leishmanial antigens for 48 h, followed by enzyme-linked immunosorbent assay (ELISA) for interleukin (IL)-4 and interferon (IFN)-γ with the culture supernatants (*P < 0·01; **P < 0·001). The data shown are from one individual experiment that was repeated twice. The error bars represent mean ± standard deviation (s.d.).

Because TLR-2 blockade reduced L. major infection in vitro, we tested whether or not simultaneous treatment with anti-TLR-2 antibody and CpG would enhance reduction of the L. major parasite burden in BALB/c mice. It was observed that co-treatment of BALB/c mice with anti-TLR-2 antibody and CpG reduced L. major parasites significantly more than that reduced by CpG or anti-TLR-2 antibody alone (Fig. 3b). The reduction in parasite load was accompanied by an IFN-γ-predominant response (Fig. 3c). These observations suggest that co-targeting TLR-2 and TLR-9 enhances the anti-leishmanial function.

Discussion

LPG, a virulence factor in Leishmania 1, is shown to be important in Leishmania survival in macrophages because it suppresses oxidative bursts in macrophages 2. In accordance with these reports, we find that the less virulent L. major parasites express less LPG and induce higher iNOS expression and NO production than that induced by the high LPG-expressing virulent L. major parasites. Another possible mechanism of deactivation of macrophages by LPG is the induction of IL-10 and TGF-β. Both cytokines can deactivate macrophages, resulting in parasite survival 4,14. As the LPG–TLR-2 interaction takes place presumably before T cells are brought into anti-leishmanial defence, the LPG-induced IL-10 production from macrophages can influence the T cell response significantly. For example, we have shown previously that IL-10 can inhibit CD40-induced p38 mitogen-activated protein kinase (MAPK)-mediated IL-12 production from macrophages 4. Because the CD40–CD40-L interaction plays a crucial role in the host-protective anti-leishmanial immune response 4,12, this initial interaction between LPG and TLR-2 is a key strategy to deviate from or suppress the host-protective immune response.

LPG is not the only known parasite-derived molecule to alter the host immune response against the invading parasite. For example, dsRNA from Schistosoma mansoni eggs interacts with TLR-3 to establish pathogenesis through alterations in the T helper type 1 (Th1)/Th2 balance in this infection in mice 17, and the lipids derived from S. mansoni eggs are recognized by TLR-2, resulting in Th2-polarized (IL-10 producing) regulatory T cells (Tregs) 18. Similarly, Acanthocheilonema viteae secreted ES-62 and S. mansoni-derived glycan lacto-N-fucopentaose III (LNFPIII) work through TLR-4 to result in a polarized Th2 response 19,20. In the present study, we observed a TLR-2-dependent Th2 bias in Leishmania infection. It is possible that the LPG–TLR-2 interaction leads to the production of IL-10 and TGF-β, which results in inhibition of the host-protective Th1 cells and differentiation of Tregs, respectively 21,22. Tregs are shown to promote Leishmania infection 23. However, the roles played by TLR-2 in the inhibition of Th1 cell and enhancement of Treg differentiation needs to be investigated in detail.

Our data indicate a distinct role for TLR-2 in L. major infection. The efficacy of TLR-9 agonist treatment of a susceptible host increased after blocking TLR-2, suggesting that TLR-2 limits the host-protective functions of TLR-9. Our observations corroborate a previous report, showing that TLR-2-deficient mice had enhanced resistance to L. braziliensis infection, but MyD88-deficient mice were susceptible to the infection 6. In experimental Trypanosoma cruzi infection, the parasite load and mortality in wild-type or TLR-2-deficient mice on a C57BL/6 background were comparable, suggesting that TLR-2 might not play a role in T. cruzi infection 24. Similarly, the L. major parasite loads in TLR-2-deficient mice on a Leishmania-resistant C57BL/6 background were comparable to wild-type mice (data not shown). However, the addition of TLR-2 deficiency to TLR-9-deficient mice resulted in a higher parasite load and less survival compared to TLR-9 deficiency alone 24. Taken together, these observations suggest that in susceptible hosts, the inhibitory or suppressive roles of TLR-2 in protozoan infections are clearly visible, whereas on an already resistant background the enhanced resistance due to lifting of the inhibitory functions of TLR-2 is not expressly apparent. Thus, although these two protozoan parasites are related closely, their interactions with the host cells with different genetic make-up can result in differences in parasite load and T cell responses.

In conclusion, as anti-TLR-2 antibody prevented the LPG-modulated expression of TLR-9 and enhanced TLR-9-ligand-induced host protection significantly in a susceptible mouse strain, it is possible that TLR-2 modulates the anti-leishmanial immune response through altered expression of TLR-9. Although observed in the context of L. major infection, this regulatory role of TLR-2 appears to have broader implications in other infections.

Acknowledgments

The work is supported by the Department of Biotechnology, New Delhi (BT/PR/3288/BRB/10/966/2011).

Disclosures

None.

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