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The Journal of Immunology, 2006, 177: 1240-1249.
Copyright © 2006 by The American Association of Immunologists

Wolbachia Endosymbiotic Bacteria of Brugia malayi Mediate Macrophage Tolerance to TLR- and CD40-Specific Stimuli in a MyD88/TLR2-Dependent Manner1

Joseph D. Turner*, R. Stuart Langley*, Kelly L. Johnston*, Gill Egerton*, Samuel Wanji{dagger} and Mark J. Taylor2,*

* Filariasis Research Laboratory, Molecular and Biochemical Parasitology, Liverpool School of Tropical Medicine, Liverpool, United Kingdom; and {dagger} Research Foundation in Tropical Diseases and Environment, Buea, Cameroon


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Lymphatic filarial nematodes are able to down-regulate parasite-specific and nonspecific responses of lymphocytes and APC. Lymphatic filariae are reliant on Wolbachia endosymbiotic bacteria for development and survival. We tested the hypothesis that repeated exposure to Wolbachia endosymbionts would drive macrophage tolerance in vitro and in vivo. We pre-exposed murine peritoneal-elicited macrophages to soluble extracts of Brugia malayi female worms (BMFE) before restimulating with BMFE or TLR agonists. BMFE tolerized macrophages (in terms of IFN-beta, IL-1beta, IL-6, IL-12p40, and TNF-{alpha} inflammatory cytokine production) in a dose-dependent manner toward self, LPS, MyD88-dependent TLR2 or TLR9 ligands (peptidoglycan, triacyl lipopeptide, CpG DNA) and the MyD88-independent/TRIF-dependent TLR3 ligand, polyinosinic-polycytidylic acid. This was accompanied with down-regulation in surface expression of TLR4 and up-regulation of CD14, CD40, and TLR2. BMFE tolerance extended to CD40 activation in vitro and systemic inflammation following lethal challenge in an in vivo model of endotoxin shock. The mechanism of BMFE-mediated macrophage tolerance was dependent on MyD88 and TLR2 but not TLR4. Evidence that desensitization was driven by Wolbachia-specific ligands was determined by use of extracts from Wolbachia-depleted B. malayi, aposymbiotic filarial species, and a cell line stably infected with Wolbachia pipientis. Our data promote a role for Wolbachia in contributing toward the dysregulated and tolerized immunological phenotype that accompanies the majority of human filarial infections.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Both Brugia malayi and Wuchereria bancrofti, filarial nematode parasites of the lymphatic and blood systems, cause a spectrum of acute and chronic inflammatory mediated pathologies in humans (1). In the majority of asymptomatic infections, the phenotype of human filariasis is permissive and chronic coincident with a hyporesponsive immune state (2). Hyporesponsiveness extends to both lymphocyte and APC functions and multiple mechanisms of dysregulation have been identified in humans and model systems, including parasite-mediated apoptosis of dendritic cells (DC)3 (3) and T cells (4), down-regulation of MHC class I and class II on filarial exposed DC (5), the generation of T cell suppressing alternatively activated macrophages (6) and the development of regulatory T cell subsets (7).

Most recently, defective monocyte responses toward TLR ligands have been described in filariasis patients (8), highlighting a novel form of filarial-driven immune dysregulation with important implications for the readiness of the innate immune system to respond appropriately toward pathogens, vaccines, or allergens. Effective regulation of innate inflammation is important to prevent collateral damage to host tissues and chronic inflammatory disease. This regulation is reflected by the tolerized state of innate immune cells to reactivation by stimulatory ligands. The phenomenon has mostly been studied in macrophages tolerized to LPS (9), although other stimulatory ligands produce a similar lack of responsiveness. Indeed, induction of tolerance to one ligand can impart tolerance to challenge with other ligands, a concept referred to as cross-tolerance or heterotolerance (10, 11). A complex system of regulatory events occurs following activation of immune cells, which can include down-regulation of receptors, blockade of signaling pathways, and the induction of suppressive cytokines (12).

Filarial nematodes are dependent upon the endosymbiotic bacteria Wolbachia for development, fertility, and survival (13). On release from the worm, the endosymbionts or their products induce innate inflammatory responses of macrophages (14) and neutrophils (15). In addition, a recombinant form of the major surface protein of nematode Wolbachia (WSP) activates macrophages and DC via signaling through TLR2 and TLR4 (16). In this study, we tested the hypothesis that Wolbachia bacteria of B. malayi would induce homotolerance and heterotolerance toward multiple TLR ligands in macrophages and drive the tolerization of innate immunity in filarial infection.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Parasite preparations

B. malayi parasites were obtained from TRS Laboratories. B. malayi adults were isolated from the peritonea of infected Mongolian jirds (Meriones unguicalatus). For Wolbachia-depleted B. malayi, infected jirds were treated with 2.5 mg/ml tetracycline in drinking water for 6 wk before parasite isolation. Adult Acanthocheilonema viteae were isolated from infected jirds maintained at the University of Liverpool (Liverpool, U.K.). Adult Loa loa were recovered from s.c. tissues of a 2-year-old drill (Mandrillus leucophaeus) born in captivity, 7 mo after s.c. inoculation with 200 infective larvae in the inguinal region. Adult worms were soaked three times for 5 min in sterile RPMI 1640 containing 50 U/ml penicillin/50 µg/ml streptomycin solution (Invitrogen Life Technologies) and rinsed in sterile Dulbecco’s PBS (Sigma-Aldrich) before being mechanically minced. Worm material was subsequently sonicated on ice five times for 15-s pulses with 30-s intervals using a Vibracell sonicator (Jencons). Soluble material from sonicated worms was extracted into Dulbecco’s PBS during an overnight incubation/agitation at 4°C. Insoluble material was removed by centrifugation at 13,000 rpm for 30 min at 4°C. Protein concentrations of soluble extracts were determined by the Bradford method (Bio-Rad). All procedures were conducted under sterile conditions. Filarial worm extracts were assessed for endotoxin levels by kinetic-chromogenic limulus amebocyte lysate assay by a commercial endotoxin testing service (European Endotoxin Testing Service; Cambrex). Extracts containing <0.05 endotoxin units (~5 pg/ml Escherichia coli LPS) per 100 µg of protein were used in this study.

Generation of Wolbachia pipientis-infected C6/36 cell line

Aa23 cells, naturally infected with W. pipientis strain wAlbB, were grown as described previously (17). C6/36 cells (European Collection of Cell Cultures) were grown as recommended in L15 Leibovitz medium containing 2 mM L-glutamine, 1% nonessential amino acids, 2% tryptose phosphate broth (Sigma-Aldrich), and 5% FCS. Subculture of W. pipientis from Aa23 into C6/36 cells was conducted as previously described (18) with modifications. Briefly, Aa23 cells were grown to confluence without medium replacement. Supernatant (5 ml) was harvested and filtered through a 0.8-µm filter to remove remaining whole cells. Medium was discarded from confluent C6/36 cells and replaced with Aa23 inoculum. Cultures were incubated with gentle shaking at room temperature for 1 h before adding 25 ml of medium and incubating at 26°C. Infection was monitored by an immunofluorescent Ab test as previously described (15), and when stable infection was observed the cells were maintained using the same protocol as uninfected C6/36 cells. Cell extracts of infected and uninfected C6/36 cells were prepared by pelleting cells at 2000 rpm, washing twice in sterile PBS before sonicating, and extracting soluble material as described for parasite extracts.

Cell culture

Peritoneal macrophages were prepared from 6- to 10-wk-old C3H/HeN and BALB/c female mice and 6- to 8-wk-old C57BL/6 and C57BL/6 MyD88, TLR2, or TLR4–/– male mice. All animals were maintained at the University of Liverpool. MyD88, TLR2, and TLR4–/– mice were a gift from Prof. S. Akira (Osaka University, Osaka, Japan). Experimental procedures were approved by the Home Office (London, U.K.). Macrophages were elicited by 1-ml i.p. injection of 2% thioglycolate solution (BD Biosciences). Cells were harvested 4 days postinjection and macrophages were enriched by overnight adherence to plastic tissue culture flasks at 37°C/5% CO2 in 10% FCS (Perbio) and 50 U/ml penicillin/50 µg/ml streptomycin solution. Adherent cells were removed by mechanical scraping, resuspended in RPMI 1640 containing 5% FCS and 50 U/ml penicillin/50 µg/ml streptomycin solution, and seeded onto flat-bottom 12-well or 96-well plates (Corning) at a density of 1 x 106 viable cells/ml. Adherent cells were typically >95% CD14 positive as assessed by flow cytometry. Cells were stimulated with indicated doses of control stimuli, parasite extracts, or cell extracts for up to 48 h. Recombinant murine TGF-beta1, anti-mouse TGF-beta1, and matching mouse IgG1 isotype Abs (all R&D Systems) were added to macrophages during initial stimulation at the stated doses. Supernatants were removed and cells were washed twice in tissue culture medium for 1.5-h intervals. Macrophages were restimulated with indicated doses of Brugia malayi female worm extract (BMFE), Staphylococcus aureus peptidoglycan (Fluka), ultra-pure E. coli LPS (serotype O111:B4), PAM3CSK4 (palmitoyl-3-cysteine-serine-lysine-4), unmethylated CpG motif containing oligodeoxynucleotide (ODN) 1826, and polyinosinic-polycytidylic (poly(I:C)) acid, all purchased from Autogen Bioclear. CD40L-expressing J558 mouse plasmacytoma cells (a gift from Dr. P. Lane, University of Birmingham, Birmingham, U.K.) and the parental J558L cell line (European Collection of Cell Cultures) were maintained in 10% FCS and IMDM (Sigma-Aldrich) and cocultured with BMFE-exposed macrophages at a ratio of 1:1 in the presence of 0.5 µg of IFN-{gamma} (R&D Systems). Macrophage viability was assessed in situ by colorimetric assessment of 10% Alamar blue metabolic reduction following the manufacturer’s instructions (Serotec) or postculture by microscopic assessment of 0.2% trypan blue exclusion.

In vivo induction of LPS tolerance

Eight- to 10-wk-old C3H/HeN mice were inoculated with 0.2-ml volumes of PBS, 20 mg/kg BMFE, or 3 µg/kg E. coli LPS (O111:B4; Sigma-Aldrich) by the i.p. route. After 18 h, mice were tail bled before being challenged with 5 µg/kg LPS in combination with 1 g/kg D-galactosamine (Sigma-Aldrich). Mice were tail bled 1.5 h following challenge and terminally bled following euthanasia after 4 h.

Flow cytometry

Monoclonal Abs used for cell surface receptor staining were: rat anti-mouse TLR2-FITC (clone 6C2), CD40-FITC (clone HM40-3), TLR4-PE (clone MTS510), and CD14-allophycocyanin (clone Sa2-8) along with appropriate isotype controls IgG2a-PE, IgG2a-FITC, and IgG2a-allophycocyanin (all Abs supplied by eBioscience). Adherent cells were removed, washed in fresh tissue culture medium, and centrifuged for 5 min at 1500 rpm. Cells were resuspended in 100 µl of FACS buffer (PBS containing 0.5% FCS, 2 mM EDTA) and incubated for 15 min with anti-CD16/CD32 (eBioscience) to block nonspecific binding to Fc{gamma}R. Abs were added at recommended concentrations and incubated for 45 min in the dark at room temperature. Following incubation with Abs, cells were washed two times with 1 ml of FACS buffer and centrifuged for 5 min at 1500 rpm. Cells were resuspended in FACS buffer to a final volume of 200 µl. Data acquisition was performed on a FACSVantage flow cytometer (BD Biosciences) and analyzed with WinMDI version 2.8 (<http://facs.scripps.edu/software.html>). Live cells were gated based on their forward and side scatter characteristics.

Quantitative PCR

Genomic DNA was extracted from individual male and female B. malayi worms using QIAamp DNA extraction kits (Qiagen) and amplified immediately. A minimum of 11 worms was sampled per group. Quantitative PCR of WSP was undertaken as previously described (19). In brief, fluorescent dsDNA (DNA-intercalating dye Sybr green, Quantitect; Qiagen) incorporation into amplified WSP product was determined by a DNA Engine Opticon thermal cycler and related software (MJ Research). Degenerate primers for wsp used were forward primer 5'-TGTTGGT(AG)TTGGT(GC)TTGGTG-3' and reverse primer 5'-AACCAAA(AG)TAGCGAGC(CT) CCA3'. Quantification was calculated by reference to a linear standard curve of log 10 diluted cloned wsp.

Immunoassay

Macrophage supernatants were diluted 1/2 to 1/5 and levels of IFN-beta, TGF-beta1, and TNF-{alpha} were determined by ELISA following the manufacturer’s instructions (BioSource International and R&D Systems). Levels of IL-1beta, IL-6, and IL-12p40 in serum or macrophage culture supernatant diluted 1/2 were measured simultaneously using a bead-based fluorometric multiplex assay (Bio-Rad) and measured on a Luminex 100 analyzer.

Statistics

Significant differences in cytokine levels were assessed using Student’s t test.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
BMFE induce a state of homotolerance in murine macrophages a dose- and time-dependent manner

We investigated whether BMFE could render peritoneal-elicited macrophages refractory to challenge stimulation, in terms of their ability to produce proinflammatory cytokines. There was a clear dose-dependent effect of 24-h pretreatment with BMFE on the production of IL-6, IL-12p40, or TNF-{alpha} to a secondary stimulation with BMFE (Fig. 1A). This was significant when cells were exposed to greater than or equal to 50 µg/ml BMFE. There were no effects of BMFE pre-exposure on macrophage viability or total cell number as determined by trypan blue exclusion or Alamar blue reduction (data not shown). Similar effects were observed when a microfilarial B. malayi extract preparation was used (data not shown). Subsequently, we investigated whether the initial length of macrophage exposure to BMFE was important in the induction of BMFE homotolerance (Fig. 1B). We found that as little as a 1.5-h preincubation with 200 µg/ml BMFE was sufficient to induce a significant reduction in the secretion of IL-12p40 and TNF-{alpha}, but an incubation length of ≥18 h was required to fully inhibit (to unstimulated control cell levels) IL-12p40 or TNF-{alpha} production. The kinetics of IL-6 desensitization were delayed in comparison to IL-12p40 or TNF-{alpha} with a preincubation length of 3 h required before significant reduction to restimulation was observed. However, by preincubating macrophages for 18 h, IL-6 secretion in response to BMFE restimulation was also completely prevented.


Figure 1
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  		FIGURE 1. BMFE homotolerance in C3H/HeN peritoneal-elicited macrophages. A, Triplicate macrophage cultures were exposed to 2.5–200 µg/ml doses of BMFE or a sham dose of PBS for 24 h before being washed twice in tissue culture medium and restimulated with a 200 µg/ml dose of BMFE for 20 h. Data illustrated are the mean level ± SEM of IL-6, IL-12p40, or TNF-{alpha} production plotted as percentages of the PBS pre-exposed/BMFE-challenged positive control (mean levels ± SEM are IL-6 = 1810.95 ± 487.95 pg/ml, IL-12p40 = 57.95 ± 6.64 pg/ml, and TNF-{alpha} = 910.10 ± 214.50 pg/ml). Data are representative of three separate experiments. Similar results were obtained using peritoneal macrophages from BALB/c and C57BL/6 mice (data not shown). B, Triplicate macrophage cultures were preincubated with 200 µg/ml BMFE or PBS for periods between 1.5 and 48 h before stimulus was removed, cells washed twice and restimulated with a 200-µg dose of BMFE for 24 h. Data illustrated are the mean level ± SEM of IL-6, IL-12p40, or TNF-{alpha} plotted as percentages of PBS preincubated controls per time point. Data are representative of two separate experiments. Significant reductions in cytokine production (*, p < 0.05 and **, p < 0.01) compared with the PBS pretreated positive control are indicated.

 
BMFE mediates heterotolerance to LPS in vitro and in vivo

A number of bacterial and viral ligands can replace LPS in the ability to tolerize macrophages to LPS challenge. We therefore investigated whether the tolerizing effect of BMFE on proinflammatory cytokine production to itself could extend to LPS stimulation (Fig. 2A). We found that IL-12p40 and TNF-{alpha} production in response to E. coli LPS was significantly reduced if macrophages were pre-exposed with ≥100 µg/ml BMFE. Levels of LPS-induced IL-1beta and IL-6 secretions were also significantly reduced when macrophages were pretreated with 200 µg/ml BMFE. The degree of desensitization induced by BMFE pretreatment of macrophages, even at high dose, was not as profound as when LPS itself was used as an initial stimulus. Because BMFE showed the potential to mediate in vitro macrophage heterotolerance toward LPS stimulation at the level of proinflammatory cytokine production, we tested its capacity to reduce inflammatory cytokine production in vivo, using a low-dose murine model of endotoxin shock. We found that prior inoculation with 20 mg/kg BMFE significantly reduced systemic TNF-{alpha} levels 1.5 h following the LD50 challenge dose of E. coli LPS and also levels of circulating IL-1beta 4 h postchallenge with LPS (Fig. 2B). BMFE mediated a marginal reduction in levels of IL-6 1.5 h following LPS challenge (Fig. 2B). As with in vitro findings, although BMFE-mediated LPS tolerance, in terms of inhibiting proinflammatory cytokine release, this model was relatively less effective (and delayed with respect to effects on IL-1beta release) compared with animals that had received a sublethal inoculation of LPS before challenge.


Figure 2
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  		FIGURE 2. BMFE mediated LPS tolerance. A, C3H/HeN-elicited peritoneal macrophages were pretreated with PBS, LPS at 0.01 µg/ml or doses of BMFE between 2.5 and 200 µg/ml for 24 h before being washed twice and restimulated with E. coli LPS at 0.01 µg/ml for 20 h. Data are the mean IL-1beta, IL-6, IL-12p40, or TNF-{alpha} production ± SEM plotted as percentages of the PBS-pretreated/LPS-challenged positive control. Mean levels ± SEM are IL-1beta = 44.05 ± 8.58 pg/ml, IL-6 = 953.96 ± 106.60 pg/ml, IL-12p40 = 413.37 ± 27.59 pg/ml, and TNF-{alpha} = 3704.43 ± 740.55 pg/ml. Significant reductions in cytokine production (*, p < 0.05 and **, p < 0.01) compared with the positive control are indicated. Data are representative of three separate experiments. Similar effects were observed with peritoneal macrophages from BALB/c and C57BL/6 mice (data not shown). B, C3H/HeN mice were inoculated with PBS, sublethal injections of LPS or 20 mg/kg BMFE i.p. and challenged with a lethal dose of LPS in combination with D-galactosamine after 18 h. Circulating levels of IL-1beta, IL-6, and TNF-{alpha} were measured at 1.5 and 4 h postchallenge. Data are mean levels of cytokine ± SEM measured from nine animals per group. Significant reductions in cytokine production (*, p < 0.05 and **, p < 0.01) compared with PBS-inoculated/LPS-challenged animals are indicated.

 
BMFE-mediated heterotolerance affects multiple TLR responses and TLR-independent stimulation of murine macrophages

Proinflammatory gene expression by LPS is critically dependent on signaling via MyD88 (20). This MyD88 dependency is shared by a number of other TLRs including TLR2 (21, 22) and TLR9 (23). However, at least three other adaptor proteins are necessary for LPS-mediated proinflammatory cytokine production, including Toll/IL-1R (TIR) domain-containing adaptor-inducing IFN-beta (TRIF) (24). Thus BMFE-mediated LPS tolerance, at the level of cytokine expression, could target TLR signals proceeding exclusively via MyD88, MyD88-independent TLR pathways, or combinations of TLR signaling pathways. To test this possibility, we examined the ability of BMFE to desensitize TLR2- and TLR9-specific signaling and TLR3 signaling, which uses TRIF but not MyD88 (25) and results in the activation of both proinflammatory cytokines and IFN-beta (23, 26). Pretreatment of macrophages with ≥50 µg/ml BMFE significantly reduced TNF-{alpha} output to the TLR2 ligand, S. aureus peptidoglycan, and also a synthetic lipopeptide agonist of TLR2, PAM3CSK4 (Fig. 3A). TLR9 signaling in response to an unmethylated CpG motif containing ODN was also reduced, at the level of TNF-{alpha}, following pre-exposure to the same concentrations of BMFE. The TLR cross-tolerizing effect of BMFE exposure was transient because pre-exposed macrophages totally recovered (in terms of the ability to secrete TNF-{alpha} in response to lipopeptide) if left in culture for 48 h following the removal of the BMFE stimulus (Fig. 3B). To investigate an effect of BMFE pretreatment on the TRIF-dependent pathway, we stimulated cells with dsRNA poly(I:C), a TLR3 ligand. Both IFN-beta and TNF-{alpha} production to poly(I:C) was impaired following BMFE exposure (Fig. 3C). Thus, BMFE cross-desensitization extended to numerous TLR responses including TLR responses that were independent of MyD88 signaling. The ability of BMFE to cross-desensitize macrophages to TLR-independent stimulation was explored using a mouse plasmacytoma cell line, J558, stably transfected with murine CD40L. Coculture of primary macrophages and CD40L expressing J558, in the presence of IFN-{gamma}, led to potent activation in terms of proinflammatory cytokine release. Activation was dependent on CD40-CD40L interaction as coculture of parental J558 cells (lacking CD40L) with primary macrophages or addition of IFN-{gamma} alone did not result in significant cytokine production (Fig. 3D). When macrophages were pre-exposed to BMFE, CD40-specific cytokine release was significantly reduced with IL-12p40 production most severely affected (Fig. 3D). Therefore, prior exposure to BMFE induced disruption of macrophage CD40-dependent responses in addition to TLR-specific activation.


Figure 3
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  		FIGURE 3. BMFE-mediated heterotolerance in C3H/HeN-elicited peritoneal macrophages. A, Triplicate cultures were exposed to PBS, BMFE at doses of 2.5–200 µg/ml, or 5 µg/ml peptidoglycan (PG), 0.1 µg/ml lipopeptide (PAM3CSK4), or 5 µM CpG ODN 1826 controls for 24 h before being washed twice and restimulated with 5 µg/ml peptidoglycan, 0.1 µg/ml PAM3CSK4, or 5 µM ODN 1826 for 20 h. Data are mean TNF-{alpha} production ± SEM plotted as percentages of the PBS pretreated/peptidoglycan, PAM3CSK4, or ODN 1826-challenged positive control. Mean TNF-{alpha} levels ± SEM are peptidoglycan 1010.70 ± 89.25 pg/ml; PAM3CSK4 8924.78 ± 256.64 pg/ml; and ODN 1826 919.06 ± 162.93 pg/ml. B, Triplicate cultures were exposed to PBS, 200 µg/ml BMFE, or 0.1 µg/ml PAM3CSK4 for 24 h, washed twice, and challenged for a 20-h period with 0.1 µg/ml PAM3CSK4 immediately after washing or following a 24- or 48-h recovery period in 5% FCS/RPMI 1640 medium. Data are mean TNF-{alpha} production ± SEM plotted as percentages of the PBS-pretreated/PAM3CSK4-challenged positive control and are representative of two separate experiments. Similar results were observed using macrophages derived from BALB/c mice (data not shown). C, Triplicate cultures were preincubated with PBS, 100–200 µg/ml BMFE, or 40 µg/ml dsRNA poly(I:C) for 24 h, washed twice, and challenged with 40 µg/ml poly(I:C) for 20 h. Data are mean IFN-beta or TNF-{alpha} production ± SEM plotted as percentages of the PBS-pretreated/poly(I:C)-challenged positive control (mean levels ± SEM are IFN-beta = 4322.82 ± 170.72 pg/ml and TNF-{alpha} = 1153.91 ± 148.37 pg/ml). Data are representative of three separate experiments. D, Triplicate cultures were preincubated with 200 µg/ml BMFE for 24 h, washed twice, and cocultured with J558 cells expressing CD40L or parental J558 in the presence of 0.5 µg/ml recombinant IFN-{gamma} for 20 h. Data are mean IL-6, IL-12p40, or TNF-{alpha} production ± SEM plotted as percentages of the PBS pretreated/J558CD40L + IFN-{gamma}-challenged positive control. Mean levels ± SEM are IL-6 = 772.83 ± 123.45 pg/ml, IL-12p40 = 4143.74 ± 622.52 pg/ml, and TNF-{alpha} = 5534.04 ± 359.87 pg/ml. Data are representative of three separate experiments. In A, C, and D significant reductions in cytokine production (*, p < 0.05 and **, p < 0.01) compared with the positive control are indicated.

 
BMFE-induced heterotolerance is associated with down-regulation of surface-expressed TLR4 but up-regulation of TLR2, CD14, and CD40

Reduced expression of TLR4 on the surface of macrophages following LPS exposure has been proposed as a mechanism of homotolerance toward LPS challenge (27). We investigated whether reductions in surface receptors could explain BMFE-mediated tolerance to both TLR- and CD40-specific stimuli (Fig. 4). After a 24-h incubation with 200 µg/ml BMFE, surface expression of CD14 had increased, indicating that impaired expression of this facet of the LPS receptor complex was not responsible for reduced responsiveness toward LPS following BMFE pre-exposure. Likewise, LPS stimulation led to notable increases in CD14. LPS pre-exposure led to reductions in surface expression of TLR4, almost to background levels, supporting a previous finding that down-regulation of this member of the LPS receptor complex is associated with LPS homotolerance. When peritoneal macrophages were exposed to BMFE, we also observed down-regulation of surface expressed TLR4, albeit to a lesser degree compared with the effects of LPS. Interestingly, the TLR2 ligand, PAM3CSK4 was also able to induce notable reductions in surface TLR4 molecules, suggesting that the LPS cross-tolerizing effects of this TLR2 ligand might proceed via a shared mechanism as LPS homotolerance. Contrastingly, both TLR2 and CD40 expression were up-regulated on BMFE, LPS, and PAM3CSK4 exposed macrophages at 24 h, indicating that impaired inflammatory responses proceeding via either of these receptors following BMFE exposure was not due to a reduction in receptor surface expression. No modulatory effects on MHC class II expression were observed in BMFE-tolerized macrophages (data not shown).


Figure 4
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  		FIGURE 4. Changes in peritoneal-elicited macrophage surface markers following BMFE exposure. A total of 1 x 106 C57BL/6 macrophages was treated with PBS, 200 µg/ml BMFE, 0.01 µg/ml LPS, or 0.1 µg/ml PAM3CSK4 for 24 h before being analyzed for surface expression of CD14, TLR4, TLR2, or CD40 by FACS. Isotype controls (dashed line histogram) for each label are plotted. Differences between isotype and specific Ab mean fluorescence intensity values are calculated as fold differences (ratio of specific Ab mean fluorescence intensity:isotype Ab mean fluorescence intensity) and shown on horizontal bar. Data are representative of three independent experiments.

 
BMFE exposure induces an increase in macrophage TGF-beta1 production, but TGF-beta1 is not responsible for BMFE-associated heterotolerance

The anti-inflammatory cytokines IL-10 and TGF-beta1 are produced by macrophages in response to inflammatory stimuli such as LPS. There is evidence to suggest that both IL-10 and TGF-beta1 production can subsequently inhibit inflammatory responses to LPS challenge via an autocrine feedback loop. We therefore investigated whether there was any evidence to suggest BMFE-mediated heterotolerance proceeded via IL-10 or TGF-beta1. In a time course of 1.5–48 h exposure to BMFE, IL-10 production by macrophages was at background levels, whereas TGF-beta1 production was significantly enhanced compared with levels in unstimulated controls (Fig. 5A). To ascertain whether BMFE-mediated TGF-beta1 was responsible for heterotolerance to LPS, we preincubated macrophages with BMFE or recombinant TGF-beta1 in the presence of neutralizing anti-TGF-beta1 Ab for 24 h before challenging with LPS. Neutralizing Ab treatment successfully reduced available TGF-beta1 in BMFE-stimulated macrophage supernatants to below unstimulated control levels as assessed by ELISA (Fig. 5B). Furthermore, neutralizing Ab successfully blocked the inhibitory effects of recombinant TGF-beta1 on TNF-{alpha} production following LPS stimulation (Fig. 5C). Despite the obvious efficacy of TGF-beta1 Ab treatment, TGF-beta1 neutralization did not significantly reverse BMFE-induced LPS heterotolerance (Fig. 5C). Therefore, although BMFE enhances macrophage TGF-beta1 production, TGF-beta1 is not a mechanism of BMFE-mediated heterotolerance.


Figure 5
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  		FIGURE 5. BMFE-induced TGF-beta1 production does not mediate LPS tolerance of peritoneal-elicited macrophages via an autocrine feedback loop. A, Triplicate C3H/HeN macrophage cultures were treated with 200 µg/ml BMFE or sham treated with PBS for periods between 1.5 and 48 h. Mean cytokine concentrations ± SEM are representative of two independent experiments. Significant increases in TGF-beta1 (*, p < 0.05) compared with PBS controls are indicated. B, Triplicate C57BL/6 macrophage cultures were sham-treated with PBS or treated with 200 µg/ml BMFE for 24 h in the presence of 1 µg/ml anti-TGF-beta1 or matching isotype Ab control. Data represent mean TGF-beta1 production ± SEM. C, Triplicate C57BL/6 macrophage cultures were pretreated with 200 µg/ml BMFE, 10 ng/ml recombinant TGF-beta1, or sham treat ed with PBS for 24 h. Indicated cultures were treated in the presence of 1 µg/ml anti-TGF-beta1 or matching isotype Ab control. Cultures were washed twice in tissue culture medium and restimulated with LPS at 0.01 µg/ml for 20 h. Data represent mean ± SEM TNF-{alpha} production ± SEM plotted as percentages of the PBS pretreated/LPS-challenged positive control (typical values 3704.43 ± 740.55 pg/ml). Data plotted are representative of three independent experiments.

 
Soluble factors derived from Wolbachia endosymbionts are required to induce BMFE homotolerance and heterotolerance

B. malayi are stably infected with the endosymbiotic bacteria, Wolbachia. Previous studies by our laboratory have demonstrated that the innate inflammatory potential of B. malayi is dependent on the presence of Wolbachia (14). To investigate the role of Wolbachia in mediating homotolerance and heterotolerance to TLR- and CD40-specific stimuli, we used extracts of B. malayi adult worms treated with tetracycline antibiotic in vivo to reduce Wolbachia loads and extracts from the related filariae, Loa loa (L. loa female extract (LLFE) and L. loa male extract (LLME)) and Acanthocheilonema viteae (A. viteae female extract (AVFE)), which do not contain Wolbachia endosymbionts (28, 29). The median total number of Wolbachia per individual parasite, determined by quantitative PCR, was 7.79 x 106 and 0.68 x 106 in female and male adult B. malayi, respectively. Following recovery of treated B. malayi adult worms, tetracycline had reduced Wolbachia numbers on average by >15-fold in female and ~2-fold in male B. malayi compared with untreated worms (Fig. 6A). Macrophages were exposed to soluble extracts derived from drug-treated and control male and female worms and their ability to respond to challenge doses of BMFE was assessed. The degree of BMFE homotolerance, at the level of TNF-{alpha} production, was related to the amount of Wolbachia available in the parasite tissues (Fig. 6B). Significant BMFE homotolerance was only observed if macrophages were pre-exposed to control female extract, BMFE. Extracts from tetracycline-treated female worms (BMFEtet), did not induce homotolerance. In addition, only a marginal degree of homotolerance was observed when cells were pretreated with B. malayi male worm extract (BMME), derived from male adult worms that contain >10-fold less Wolbachia compared with female worms. This marginal homotolerance was not observed if macrophages were pre-exposed to extract derived from tetracycline-treated male B. malayi (BMMEtet) (Fig. 6B). Soluble extracts derived from Wolbachia aposymbiotic filarial species also did not have the capacity to induce macrophage tolerance toward BMFE (Fig. 6B). Similar to these findings, filarial extract-mediated macrophage heterotolerance toward TLR2-, TLR4-, and CD40-specific stimuli was dependent on the presence of normal levels of Wolbachia in parasite tissues. Extracts from tetracycline-treated female BMFE or extracts from Wolbachia-free filariae did not significantly affect subsequent responsiveness, in terms of TNF-{alpha} production, toward bacterial LPS, peptidoglycan, or PAM3CSK4 nor was responsiveness following CD40 ligation significantly reduced if macrophages were pre-exposed to Wolbachia-depleted or Wolbachia-free filarial extracts (Fig. 6C). The dependence of Wolbachia products in mediating macrophage tolerance was further substantiated using the insect cell line C6/36 stably transfected with W. pipientis derived from the mosquito Aedes albopictus. Soluble cell extracts from transfected but not parental C6/36 cells could mediate significant heterotolerance toward LPS and lipopeptide, at the level of TNF-{alpha} production (Fig. 6D).


Figure 6
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  		FIGURE 6. Soluble Wolbachia products are responsible for BMFE-mediated homotolerance and heterotolerance in C3H/HeN peritoneal-elicited macrophages. A, WSP gene copy number was determined by quantitative PCR in adult male (BMME) or female (BMFE) worms and corresponding worms following drug treatment in vivo with tetracycline (tet) antibiotic. B, Triplicate macrophage cultures were pretreated with 200 µg/ml BMFE, B. malayi male extract (BMME) the corresponding extracts from tetracycline-treated B. malayi (BMFEtet, BMMEtet), L. loa female and male extracts (LLFE, LLME), or A. viteae female extract (AVFE) for 24 h. Cells were washed twice and challenged with 200 µg/ml BMFE for 20 h. Data are mean TNF-{alpha} production ± SEM illustrated as a percentage of the PBS- pretreated/BMFE-challenged positive control (typical values are 910.10 ± 214.50 pg/ml). Similar results were obtained using macrophages derived from C57BL/6 mice (data not shown). Data are representative of three independent experiments. C, Triplicate macrophage cultures were stimulated with 200 µg/ml BMFE, BMFEtet, LLFE, or AVFE, washed twice, and challenged with 0.01 µg/ml LPS, 5 µg/ml peptidoglycan, and 0.1 µg/ml PAM3CSK4 or cocultured with J558 CD40L cells with 0.5 µg/ml IFN-{gamma} for 20 h. Data are mean TNF-{alpha} production ± SEM illustrated as a percentage of the PBS-pretreated/LPS, peptidoglycan (PG), PAM3CSK4, or J558CD40L + IFN-{gamma}-challenged positive control (typical values are LPS = 3704.43 ± 740.55 pg/ml, peptidoglycan = 1010.70 ± 89.25 pg/ml, PAM3CSK4 = 8924.78 ± 256.64 pg/ml, and J558CD40L+IFN-{gamma} = 5534.04 ± 359.87 pg/ml). Similar results were obtained using macrophages derived from C57BL/6 mice (data not shown). Data are representative of three separate experiments. D, Triplicate cultures were preincubated with 200 µg/ml soluble extract from C6/36 cells infected with W. pipientis (C6/36Wp) or uninfected C6/36 for 24 h, washed twice, and challenged with either 0.01 µg/ml LPS or 0.1 µg/ml PAM3CSK4 for 20 h. Data are mean TNF-{alpha} production ± SEM illustrated as a percentage of the PBS-pretreated/LPS or PAM3CSK4-positive control (typical values are shown in Figs. 2 and 3). Similar effects were observed using macrophages derived from C57BL/6 mice (data not shown). Data are representative of three independent experiments.

 
BMFE-mediated heterotolerance is abrogated in the absence of MyD88 and TLR2 but is unaffected in the absence of TLR4

The innate inflammatory activity of filarial Wolbachia or Wolbachia-containing extracts has been associated with TLR4 signaling (14, 30). Furthermore, recombinant WSP signals using both TLR2 and TLR4 (16). As these receptors use the adaptor protein MyD88 (either exclusively, in the case of TLR2, or in combination with other TIR-containing adaptor molecules, in the case of TLR4), we investigated the extent to which Wolbachia–MyD88 signaling was involved in the induction of the tolerized macrophage phenotype using MyD88 knockout mice. Following BMFE pre-exposure, peritoneal macrophages deficient in MyD88 were completely unaffected in terms of their ability to respond to the TLR3 stimulus, poly(I:C), at the level of IFN-beta production (Fig. 7A). Furthermore, in the absence of MyD88, macrophages could not be tolerized to CD40-specific stimulation, and produced maximal levels of IL-6, IL-12p40, and TNF-{alpha} (Fig. 7B). Therefore, the TLR3 and CD40 cross-tolerizing effects of Wolbachia products within BMFE are totally dependent on MyD88 signaling processes. To determine whether either of the MyD88-dependent receptors TLR2 or TLR4 are required for BMFE-related heterotolerance, we used TLR2 or TLR4 knockout mice. After 24 h pre-exposure to BMFE, TLR4–/– macrophages were rendered refractory to PAM3CSK4 challenge to the same extent as wild-type macrophages at the level of TNF-{alpha} secretion (Fig. 8A). A similar degree of heterotolerance to PAM3CSK4 or CpG DNA was observed in macrophages from TLR4 mutant mice (C3H/HeJ) compared with wild-type C3H/HeN-derived macrophages (data not shown). Contrastingly, BMFE pre-exposure had no effect on the ability of TLR2–/– macrophages to respond to LPS, as judged by comparison of TNF-{alpha} release with wild-type macrophages (Fig. 8B). Thus the heterotolerizing effects of BMFE are dependent on TLR2 but not TLR4 signaling.


Figure 7
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  		FIGURE 7. Wolbachia-mediated macrophage tolerance proceeds via MyD88. A, C57BL/6 MyD88–/– macrophages or wild-type controls were pretreated with 100–200 µg/ml BMFE or 40 µg poly(I:C) for 24 h, washed twice, and challenged with 40 µg/ml poly(I:C) for 20 h. Data are mean IFN-beta production ± SEM expressed as a percentage of the PBS-pretreated/poly(I:C)-challenged positive control (typical values are shown in Fig. 3). B, C57BL/6 MyD88–/– macrophages or wild-type controls were pretreated with 100–200 µg/ml BMFE for 24 h and washed twice before being cocultured with J558 cells expressing CD40L or parental cells in the presence of 0.5 µg/ml IFN-{gamma} for 20 h. Data are mean IL-6, IL-12p40, or TNF-{alpha} production ± SEM expressed as a percentage of the PBS- pretreated/J558CD40L + IFN-{gamma}-challenged positive control (typical values are IL-6 = 772.83 ± 123.45 pg/ml, IL-12p40 = 4143.74 ± 622.52 pg/ml, and TNF-{alpha} = 5534.04 ± 359.87 pg/ml). Data are representative of three independent experiments.

 

Figure 8
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  		FIGURE 8. Wolbachia-mediated tolerance proceeds via TLR2 but not TLR4 signaling in peritoneal-elicited macrophages. A, C57BL/6 wild-type or TLR4–/– mice were pretreated with 200 µg/ml BMFE or 0.1 µg/ml PAM3CSK4 for 24 h before being washed twice in tissue culture medium and restimulated with PAM3CSK4 for 20 h. Data are mean TNF-{alpha} production ± SEM plotted as a percentage of the PBS-pretreated/PAM3CSK4-challenged positive control (typical value is 8924.78 ± 256.64 pg/ml). B, C57BL/6 wild-type or TLR2–/– mice were pretreated with 200 µg/ml BMFE or 0.01 µg/ml LPS for 24 h before being washed twice in tissue culture medium and restimulated with LPS for 20 h. Data are mean TNF-{alpha} production ± SEM plotted as a percentage of the PBS-pretreated/LPS-challenged positive control (typical value 3704.43 ± 740.55 pg/ml). Data plotted are representative of two independent experiments.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
Previous studies by our group and others have determined that the inflammatory potential of filariae is due to the presence of an endosymbiont bacterium, Wolbachia. Crucially, a role for TLR recognition of Wolbachia ligands has been demonstrated in vitro (16) and in in vivo models of filarial inflammatory pathogenesis (14, 30). Wolbachia has also been implicated in propagating innate inflammatory responses in human filariasis (31). We therefore hypothesized that Wolbachia-TLR interactions would subsequently induce negative regulation of inflammatory processes in order that collateral damage to host tissues would be limited during chronic infection. More specifically, we postulated that repeated TLR-dependent signaling by a Wolbachia product or products during chronic infection could lead to a state of refractoriness similar to the phenomenon of endotoxin tolerance, where repeated LPS challenge leads to impaired TLR4 signaling processes in monocytic cells (9). The data presented in this study provide evidence to support this hypothesis. BMFE could mediate a complete homotolerant state in murine peritoneal macrophages in terms of proinflammatory cytokine production toward a secondary stimulus and a degree of heterotolerance toward multiple TLR ligands, a process that could be replicated in vivo using a low dose model of endotoxin shock. BMFE could also mediate tolerance to a TLR-independent stimulus, CD40L. The ability of BMFE to render peritoneal macrophages tolerant to TLR- and CD40-specific ligands was dependent on the quantity of Wolbachia bacteria contained within parasite tissues and thus available as components of the soluble preparation. Wolbachia-depleted B. malayi extract was unable to induce significant homotolerance or heterotolerance, and Wolbachia-negative filarial extracts also were deficient in mediating macrophage tolerance. Although a secreted product of A. viteae, ES-62, has previously been shown to mediate in vitro LPS heterotolerance (32), a soluble preparation of A. viteae used in this study was unable to induce a significant degree of homotolerance or heterotolerance, suggesting ES-62 is not a component of soluble extract-induced tolerance.

The finding that TLR3- and CD40-specific heterotolerance is dependent on MyD88 signaling supports a role for Wolbachia-TLR interactions in inducing negative regulation of these pathways. Both TLR4 and more recently TLR2 have been implicated in Wolbachia signaling (14, 16, 30). Our data strongly indicates that for the mediation of macrophage tolerance, Wolbachia TLR4-dependent interaction is dispensable, whereas TLR2-dependent signaling is essential. This result raises the possibility that distinct Wolbachia ligands are present within BMFE and that these ligands differ in their ability to induce negative regulation of inflammatory gene activation via their affinities for individual TLRs. Conversely, a single Wolbachia factor may provoke macrophage activation via both TLR2 and TLR4 but subsequent negative regulation occurs exclusively as a result of TLR2 signaling. The relative importance of TLR2 vs TLR4 Wolbachia signaling in macrophage activation and the Wolbachia molecules involved in BMFE-mediated activation and tolerance are the current focus of our laboratory.

The potential mechanisms underpinning TLR-associated macrophage tolerance are numerous, with multiple negative regulatory molecules targeting the TLR pathway at distinct points following activation. One such mechanism, which has been implicated in LPS macrophage tolerance, is the down-regulation of surface expressed TLR4, observed between 1 and 24 h following priming doses of LPS (27). Our data confirm this LPS effect on TLR4. We could also detect a subtle reduction in surface TLR4 when macrophages were exposed to Wolbachia TLR ligands, promoting a role for Wolbachia exposure in the down-regulation of TLR4 and subsequent hyporesponsiveness to LPS challenge, in terms of proinflammatory cytokine release. Reduced TLR4 surface expression as a mechanism of LPS tolerance was previously thought to be confined to the effects of LPS pre-exposure (33). However, in this study we identified that the synthetic lipopeptide, PAM3CSK4, could also mediate TLR4 down-regulation at the plasma membrane, to a similar extent as LPS. Thus, our data indicate that TLR2-conferred LPS heterotolerance as well as LPS homotolerance may proceed by down-regulating surface expression of TLR4.

Although we could associate prior macrophage exposure to Wolbachia molecules with modulatory effects on TLR4 expression, there was no such evidence to support similar effects on TLR2 and CD40 surface expression. In fact both of these receptors were increased at the plasma membrane of tolerized macrophages following Wolbachia exposure. Therefore, the tolerizing effects of Wolbachia TLR ligands extend further than down-regulation of surface receptors. The breadth of Wolbachia-mediated macrophage tolerance, extending to TRIF- and CD40-dependent responses as well as MyD88-dependent responses, may indicate that endosymbiont molecules are capable of activating multiple regulatory responses with separate mechanisms affecting distinct activation signaling pathways. For instance, several different mechanisms negatively regulate MyD88-specific responses by directly targeting upstream signaling events such as the MyD88 molecule itself (variant MyD88; MyD88s, TIR-containing orphan receptor ST2L) or facets of the IL-1R-associated kinase–MyD88 complex (suppressor of cytokine signaling 1 (SOCS1) or Toll-interacting protein (TOLLIP)) (12). Other mechanisms target downstream events that are shared between signaling pathways. Wolbachia TLR signaling may up-regulate a negative regulator or regulators that target molecules shared between the MyD88, TRIF, and CD40 pathways. The effects of Wolbachia exposure on these signaling cascades are currently under investigation.

Equivocal evidence exists for a role of the regulatory cytokines, IL-10 and TGF-beta1 in mediating LPS tolerance in vitro via an autocrine feedback loop (34, 35). However, TGF-beta1 has recently been demonstrated to negatively effect MyD88-dependent inflammatory gene activation by mediating ubiquitination of MyD88 (36). We could not establish a role for either of these cytokines in mediating Wolbachia-driven macrophage tolerance. BMFE-stimulated cells produced measurable TGF-beta1 but not IL-10 after 24 h. Blocking available TGF-beta1 by neutralizing Ab, administered during BMFE preincubation could not reverse the tolerization of macrophages to LPS challenge. Thus other mechanisms are responsible for the multifaceted tolerizing effects of Wolbachia, most likely involving disruption of TLR and CD40 signaling pathways.

Our data imply that prior exposure to TLR-reactive Wolbachia ligands can alter macrophage responsiveness. Considering the longevity of filarial infection (a minimum estimated reproductive lifespan of 4–6 years (37)) and the sustained production and turnover of microfilariae from patent infections, filariasis patients will be continually and systemically exposed to Wolbachia, as reflected in the identification of elevated anti-Wolbachia Ab responses in filariasis patients (38). In addition, release of Wolbachia within uterine contents and death of developing larvae or adult parasites through natural or immune-mediated attrition will further enhance the exposure to Wolbachia and their TLR ligands within the lymphatic and blood system. There is therefore ample opportunity for Wolbachia to interact with cells of the monocyte/macrophage cell lineage, an interaction that may well increase with chronicity and multiplicity of infection. Wolbachia-mediated negative regulation of macrophage inflammatory responses may have a direct effect on the susceptibility toward other microbial infections, an important implication in view of the evidence that recurrent secondary bacterial infections are a feature of chronic lymphoedema pathology (elephantiasis) (39). Because macrophages also serve as APCs, Wolbachia-mediated tolerance may also extend to a dysregulated APC–lymphocyte interface with concomitant effects on the generation of acquired immune responses. Certainly, the effects on CD40-CD40L coreceptor interaction, with profound attenuated IL-12p40 responses following Wolbachia exposure, may indicate that Wolbachia-driven macrophage tolerance extends to APC processes. In this light, we are currently investigating the effects of Wolbachia priming on subsequent DC maturation and polarization.

The effects of Wolbachia-TLR interactions on the development or modulation of the adaptive immune response to filarial infection, are yet to be determined. Certainly, the aposymbiotic filarial species, L. loa and A. viteae, are capable of modulating lymphocyte responses (40, 41), and various nematode-derived products with immunoregulatory activities have been identified (42). We have determined that Wolbachia mediates a novel form of filarial tolerance to innate inflammatory stimuli. Thus Wolbachia may contribute an additional level of immune cell regulation in the face of chronic filarial infection. Future studies should address whether a breakdown in negative regulation of Wolbachia-induced inflammation predisposes individuals to develop clinical inflammatory-mediated pathologies.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was funded by The Wellcome Trust and by a European Commission Grant ICA4-2002-10051 from The European Union. M.J.T. holds a Senior Wellcome Fellowship in Basic Biomedical Science. Back

2 Address correspondence and reprint requests Dr. Mark J. Taylor, Filariasis Research Laboratory, Molecular and Biochemical Parasitology, Liverpool School of Tropical Medicine, Pembroke Place, L3 5QA Liverpool, U.K. E-mail address: mark.taylor{at}liv.ac.uk Back

3 Abbreviations used in this paper: DC, dendritic cell; BMFE, Brugia malayi female worm extract; WSP, Wolbachia surface protein; ODN, oligodeoxynucleotide; TIR, Toll/IL-1R; TRIF, TIR domain-containing adaptor-inducing IFN-beta. Back

Received for publication December 5, 2005. Accepted for publication April 20, 2006.


   References
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
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