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Infection and Immunity, October 2006, p. 5780-5789, Vol. 74, No. 10
0019-9567/06/$08.00+0     doi:10.1128/IAI.00678-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Interleukin-10 Anti-Inflammatory Response to Borrelia burgdorferi, the Agent of Lyme Disease: a Possible Role for Suppressors of Cytokine Signaling 1 and 3

Vida A. Dennis,1* Ayanna Jefferson,1 Shree R. Singh,2 Frédéric Ganapamo,3 and Mario T. Philipp1

Division of Bacteriology and Parasitology, Tulane National Primate Research Center, Tulane University Health Sciences Center, Covington, Louisiana,1 Department of Math and Science, Alabama State University, Montgomery, Alabama,2 Division of Immunology, Tulane National Primate Research Center, Tulane University Health Sciences Center, Covington, Louisiana3

Received 27 April 2006/ Returned for modification 30 May 2006/ Accepted 25 July 2006


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ABSTRACT
 
It has been established that interleukin-10 (IL-10) inhibits inflammatory cytokines produced by macrophages in response to Borrelia burgdorferi or its lipoproteins. The mechanism by which IL-10 exerts this anti-inflammatory effect is still unknown. Recent findings indicate that suppressors of cytokine signaling (SOCS) proteins are induced by cytokines and Toll-like receptor (TLR)-mediated stimuli, and in turn they can down-regulate cytokine and TLR signaling in macrophages. Because it is known that SOCS are induced by IL-10 and that B. burgdorferi and its lipoproteins most likely interact via TLR2 or the heterodimers TLR2/1 and/or TLR2/6, we hypothesized that SOCS are induced by IL-10 and B. burgdorferi and its lipoproteins in macrophages and that SOCS may mediate the inhibition by IL-10 of concomitantly elicited cytokines. We report here that mouse J774 macrophages incubated with IL-10 and added B. burgdorferi spirochetes (freeze-thawed, live, or sonicated) or lipidated outer surface protein A (L-OspA) augmented their SOCS1/SOCS3 mRNA and protein expression, with SOCS3 being more abundant. Pam3Cys, a synthetic lipopeptide, also induced SOCS1/SOCS3 expression under these conditions, but unlipidated OspA was ineffective. Neither endogenous IL-10 nor the translation inhibitor cycloheximide blocked SOCS1/SOCS3 induction by B. burgdorferi and its lipoproteins, indicating that the expression of other genes is not required. This temporally correlated with the IL-10-mediated inhibition of the inflammatory cytokines IL-1ß, IL-6, IL-12p40, IL-18, and tumor necrosis factor {alpha}. Our data are evidence to suggest that expression of SOCS is part of the mechanism of IL-10-mediated inhibition of inflammatory cytokines elicited by B. burgdorferi and its lipoproteins.


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INTRODUCTION
 
Lyme disease, caused by the spirochete Borrelia burgdorferi, is spread to humans and other mammals through the bite of infected Ixodes ticks (9). The spirochete can invade multiple organs (4, 59) and persist in them for a long time (47, 65). Spirochetal persistence in the tissues has been associated with severe pathology (14, 21, 65) and both acute and chronic inflammatory conditions (50, 59). Numerous studies have shown that B. burgdorferi and its lipoproteins can induce in a variety of cell types the release of proinflammatory cytokines, such as interleukin-1{alpha} (IL-1{alpha}) (10), IL-1ß (45), IL-6 (8, 23, 45, 54, 55, 64), IL-8 (10), IL-12 (30, 45, 58), tumor necrosis factor alpha (TNF-{alpha}) (8, 45, 53, 54, 55, 58, 64), gamma interferon (IFN-{gamma}) (22, 23, 24), IL-17 (35), granulocyte-macrophage colony-stimulating factor (GM-CSF) (67), and IL-18 (30). These cytokines may contribute to tissue inflammation and damage. Although inflammation is a critical response to tissue injury and is required for tissue repair and the clearance of infections, uncontrolled inflammation in itself may result in further tissue damage. The control of host responsiveness to B. burgdorferi and its lipoproteins is thus of paramount importance in order to protect against unrestrained inflammatory processes that may result in massive tissue destruction or potential organ dysfunction.

IL-10 is a multifunctional anti-inflammatory cytokine whose general effects are essentially targeted to limit the inflammatory response and prevent tissue damage. This is achieved by down-regulating the expression of inflammatory cytokines and chemokines and inhibiting effector functions of T cells and mononuclear phagocytes (20). B. burgdorferi and its lipoproteins are potent inducers of IL-10 in cells of the innate and acquired immune responses (22, 24, 27, 28, 45). More importantly, IL-10 has proved to be a key cytokine in regulating inflammatory responses in Lyme disease by controlling the production and function of various proinflammatory cytokines. We (22, 23, 26, 45) and others (8, 25, 31, 42, 51, 66) have reported on experiments in vitro showing that in response to B. burgdorferi and its lipoproteins, IL-10 dampens proinflammatory responses of cells that are involved in innate and acquired immunity. Additionally, we (F. Ganapamo, V. A. Dennis, and M. T. Philipp, unpublished data) as well as others (8) have observed that bone marrow-derived macrophages from C57BL/6J (C57) mice, which are Lyme disease resistant, produce higher levels of IL-10 (and lower levels of IL-6, IL-12, and TNF-{alpha}) than do macrophages from the disease-susceptible C3H/HeN (C3H) mice in response to B. burgdorferi or its lipoproteins. Therefore, the differential production of IL-10 and inflammatory cytokines by macrophages in C57 and C3H mice seemingly correlates with susceptibility and resistance to disease in the murine model of Lyme disease. Despite considerable research on the anti-inflammatory activity of IL-10 in Lyme disease, the molecular mechanism through which IL-10 exerts this effect remains largely undefined.

Suppressors of cytokine signaling (SOCS) proteins have been identified as negative feedback inhibitors for various cytokines (2, 39). To date, eight members (SOCS1 to SOCS7 and CIS) have been identified in this protein family, all sharing a central Src homology 2 (SH2) domain and a C-terminal conserved domain called the SOCS box (2, 40). SOCS inhibitory effects are derived from the direct interaction of SOCS proteins with cytokine receptors and/or Janus kinases (JAKs), thereby preventing recruitment of signal transducers and activators of transcription (STATs) to the signaling complex (48, 56). In addition, it was shown recently that SOCS induction and action can also be caused by a much broader variety of stimuli and might even act on signaling pathways distinct from JAK/STAT (32). In this regard, SOCS proteins can be induced by Toll-like receptor (TLR)-mediated stimuli and in turn can regulate TLR signaling in innate immune cells (7, 16, 18, 29, 37, 46, 60, 61).

SOCS1 and SOCS3 are the key physiological regulators of macrophages and play significant roles in the regulation of inflammation (40). SOCS3 in particular has been shown to be a major player in the IL-10-mediated inhibition of lipopolysaccharide (LPS)-induced proinflammatory actions in mouse J774 macrophages (5). Because SOCS1 and SOCS3 are induced by IL-10 (5, 12, 19) and because B. burgdorferi and its lipoproteins most likely interact with cells of the innate immune system via TLR2 or the heterodimers TLR2/1 and/or TLR2/6, we hypothesized that SOCS proteins are induced by IL-10 and B. burgdorferi and its lipoproteins in macrophages, and they may mediate the inhibition by IL-10 of concomitantly elicited cytokines.

To address this hypothesis, we first verified that cells of the mouse macrophage cell line J774 could be stimulated with B. burgdorferi spirochetes (freeze-thawed) or lipidated outer surface protein A (L-OspA) to produce proinflammatory cytokines, and that this effect could be inhibited with added recombinant IL-10. We then quantified SOCS1 and SOCS3 mRNA transcripts as a function of time poststimulation in the presence and absence of added recombinant IL-10 and examined expression of SOCS1 and SOCS3 proteins. SOCS1 and SOCS3 transcripts were also quantified as a function of stimulant dose. To ascertain whether the effects elicited by L-OspA could be extended to all bacterial lipoproteins, we stimulated macrophages with the synthetic lipohexapeptide tripalmitoyl-S-glyceryl-Cys-Ser-Lys4-OH (Pam3Cys). Finally, live spirochetes were also used as stimulants. The effect of B. burgdorferi and its lipoproteins was compared with that of LPS. Here we present the results of these studies.


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MATERIALS AND METHODS
 
Bacteria and lipoproteins. The JD1 strain of B. burgdorferi was used essentially throughout. The B31 strain was used in experiments utilizing live and sonicated spirochetes. Freeze-thawed B. burgdorferi spirochetes were prepared as previously described (23). Recombinant lipidated outer surface protein A (L-OspA) and unlipidated OspA (U-OspA) were kindly provided by GlaxoSmithKline Biologicals (Rixensart, Belgium). L-OspA and U-OspA preparations contained less than 0.25 endotoxin units (EU) per mg of protein, as assessed by Limulus amebocyte assay (Associates of Cape Cod, Woods Hole, Mass.).

Ab and reagents. Neutralizing antibody (Ab) to mouse IL-10, control isotype mouse immunoglobulin (IgG1), and mouse recombinant IL-10 (rIL-10) were from BD-PharMingen (San Diego, CA). Anti-SOCS1 Ab, anti-SOCS3 Ab, horseradish peroxidase-conjugated goat anti-rabbit IgG (Santa Cruz Biotechnology, Santa Cruz, CA), ß-actin (Novus Biological Inc., Littleton, CO), 12% Tris-HCl Ready Gel (Bio-Rad Life Science Research, Hercules, CA), and broad-range molecular weight standards (Bio-Rad) were used for standard Western blots. LPS from Escherichia coli strain 026:B6 and cycloheximide were from Sigma Chemical Company (St. Louis, MO). The lipohexapeptide tripalmitoyl-S-glyceryl-Cys-Ser-Lys4-OH (Pam3Cys) was obtained from Boehringer Mannheim (Indianapolis, IN).

Cell stimulation and culture conditions. The mouse J774 macrophage cell line was obtained from the American Type Culture Collection (Waldorf, MD). Cell culture medium consisted of Dulbecco's modified Eagle’s medium (Gibco-BRL, Life Technologies, Grand Island, NY), 10% heat-inactivated fetal bovine serum (endotoxin, <1.4 EU/ml; JRH Biosciences, Inc., Lenexa, KS), 1 mM HEPES (Sigma), 2 mM L-glutamine (Gibco), and 1 µg/ml antibiotic and antimycotic (Gibco) (complete medium). Cells (2 x 106/ml or 3 x 106/ml) were cultured in 24-well plates (Costar, Cambridge, MA) and incubated at 37°C in a humidified atmosphere with 5% CO2 for various periods of time, depending on the experimental procedure. Live spirochetes were incubated with cells in antibiotic-free medium. All cultures were subsequently centrifuged at 400 x g at 4°C for 10 min to collect cell-free supernatants or extract RNA from the cell pellet as described below. Supernatant and RNA samples were stored at –70°C until they were used.

To study the effect of exogenous IL-10 and B. burgdorferi stimulants on SOCS mRNA transcripts as well as cytokine mRNA transcript and production levels, macrophages were stimulated with rIL-10 (10 ng/ml) as well as L-OspA (1 µg/ml), freeze-thawed B. burgdorferi (1 x 107/ml), live B. burgdorferi spirochetes (macrophage-to-spirochete ratio of 1:10), B. burgdorferi sonicated spirochetes (1 x 107/ml), Pam3Cys (1 µg/ml), U-OspA (1 µg/ml), and LPS (1 µg/ml) in the presence or absence of rIL-10 (10 ng/ml).

For kinetics of SOCS mRNA expression, macrophages were stimulated with rIL-10 (10 ng/ml) as well as B. burgdorferi (1 x 107/ml), L-OspA, and LPS (1 µg/ml) in the presence or absence of rIL-10 (10 ng/ml). RNA was collected at 0, 30, and 120 min postincubation. For dose-response studies, cells were stimulated with various concentrations of rIL-10 (0.01, 0.1, 1, and 10 ng/ml), B. burgdorferi (1 x 104/ml to 1 x 107/ml), L-OspA, and LPS (0.001, 0.01, 0.1, and 1 µg/ml), or live spirochetes (cell-to-spirochete ratios of 1:1, 1:5, 1:10, and 1:20) and incubated for 24 h. SOCS expression was determined in these samples by reverse transcriptase PCR (RT-PCR).

To determine the effect of exogenous and endogenous IL-10 on SOCS transcript and cytokine production levels, cells were preincubated with rIL-10 (10 ng/ml) or with a neutralizing rat anti-mouse IL-10 Ab (25 µg/ml). Normal rat IgG1 Ab (25 µg/ml) was used as control. After 30 min of preincubation at 37°C, B. burgdorferi, L-OspA, and LPS were added to individual cultures to reach a final concentration of 1 µg/ml for L-OspA and LPS or 1 x 107/ml for B. burgdorferi. Cultures were incubated for an additional 2, 24, and 48 h as described above. In some experiments, cells were preincubated with L-OspA, B. burgdorferi, or LPS at similar concentrations prior to the addition of rIL-10 and incubated for an additional 24 h. The effect of cycloheximide (CHX; 1 µg/ml) on SOCS expression was determined by preincubating cells with CHX for 30 min prior to addition of stimulants for an additional 2 or 4 h. Supernatant and RNA samples were collected at the various time points and analyzed for cytokine production and for SOCS and cytokine mRNA transcripts levels, respectively.

Measurement of cytokine concentrations. Cytokine enzyme-linked immunosorbent assays (ELISAs) were performed as previously described (23). Concentrations of TNF-{alpha}, IL-6, IL-1ß, IL-12p40, and IL-18 cytokines were quantified in cell-free supernatants of macrophage cultures using Opti-EIA kits (BD-PharMingen) according to the manufacturer's instructions.

RT-PCR. Total RNA was isolated using an RNeasy Mini kit (QIAGEN Inc, Valencia, CA), which included DNase I digestion. A constant amount of target RNA was reverse transcribed using 100 U M-MLV Reverse Transcriptase (Invitrogen, Carlsbad, CA) at 42°C for 60 min in the presence of 50 µM random hexamers (Promega, Madison, WI). PCR was performed using primers previously described for mouse cytokines (43) and for SOCS1, SOCS2, and SOCS3 (19). PCR amplification protocols for cytokines (43) and SOCS (19) were essentially conducted as already described. First-strand synthesis containing each mRNA sample but no reverse transcriptase was performed to control for possible DNA contamination of mRNAs used as targets for PCR amplification. PCR-amplified fragments were fractionated by electrophoresis on agarose gels and were visualized by ethidium bromide staining. Cytokine PCR levels were normalized for the amount of mRNA encoding glyceraldehyde-3-phosphate dehydrogenase (GAPDH), the product of a house-keeping gene, detected in the same sample. Signals were semiquantified with 1D Image Analysis Software (Kodak Digital Science, Eastman Kodak Co., Rochester, NY). For some studies, the results are expressed in terms of fold increase over the mRNA levels of cells cultured with medium. Fold increases higher than 2 were considered upregulations of the investigated SOCS or cytokine gene.

Quantitative real-time PCR. Purified RNA (50 ng) obtained as described above was used as template in the quantitative PCR mix according to the manufacturer's standard protocol for QuantiTect primer assays for one-step PCR (QIAGEN). SOCS1 and SOCS3 QuantiTect primers were used (QIAGEN), and quantifications were made by means of SYBR green using ABI 7700 (Applied Biosystems, Foster City, CA). The specificity of the PCR was controlled by no-template controls. Specific cDNA was quantified by standard curves based on known amounts of product. Threshold values were normalized to the expression of GAPDH using QuantiTect primers (QIAGEN). Quantitative real-time PCR results are expressed as fold induction (normalized copy number of stimulated cells/normalized copy number of unstimulated cells).

Western blotting. J774 macrophages were stimulated with B. burgdorferi, L-OspA, or LPS in the presence or absence of rIL-10. Cells were washed and lysed for 30 min on ice in 250 µl of lysis buffer consisting of 50 mM Tris-HCl, pH 7.4; 1% Igepal; 0.25% sodium deoxycholate; 150 mM NaCL; 1 mM EDTA; 1 mM phenylmethylsulfonyl fluoride; 1 µg/ml each of aprotinin, leupeptin, and pepstatin; 1 mM Na3VO4; and 1 mM NaF. Lysates were cleared by centrifugation (14,000 x g, 10 min, 4°C); supernatants were collected, and protein determinations were made using the bicinchoninic acid protein kit (Pierce, Rockford, IL). Cell lysates at 25 µg were electrophoresed by sodium dodecyl sulfate-12% polyacrylamide gel electrophoresis and electrophoretically transferred to polyvinylidene difluoride membranes (GE Healthcare Life Sciences, Piscataway, NJ) in a buffer containing 25 mM Tris, 186 mM glycine, and 20% methanol. Membranes were blocked in phosphate-buffered saline-5% nonfat dry milk-0.1% Tween 20 for 1 h at room temperature, washed, and then incubated overnight at 4°C with agitation (on a rocker) with anti-SOCS1 or anti-SOCS3 antibody (1 µg/ml). After incubation, the membranes were washed and then incubated for 1 h at room temperature with horseradish peroxidase-conjugated goat anti-rabbit IgG (1:5,000). SOCS1 and SOCS3 immunoreactive protein bands were visualized using an Amersham enhanced chemiluminescence system (GE Healthcare Life Sciences). Anti-ß-actin Ab (1:5,000) was used as an internal control to verify protein loading and then visualized as described for SOCS1 and SOCS3.

Statistical analysis. All the data were analyzed using the two-tailed unpaired Student's t test wherever applicable. P < 0.05 was considered significant.


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RESULTS
 
Exogenous IL-10 down-modulates proinflammatory cytokines induced by B. burgdorferi spirochetes and L-OspA. In the present study, we first determined whether exogenous IL-10 added at the time of stimulation was able to affect the production of proinflammatory cytokines in cultured J774 mouse macrophages. We measured the concentration of IL-ß, IL-6, IL-12p40, IL-18, or TNF-{alpha} in the supernatants as a function of time, at 30 min, and at 2, 24, and 48 h after stimulation with freeze-thawed B. burgdorferi spirochetes (B. burgdorferi, 1 x 107/ml), L-OspA (1 µg/ml), or LPS (1 µg/ml). J774 macrophages were incubated with stimulants in the absence or presence of 10 ng/ml of rIL-10. All tested cytokines were below detection limits in the absence of stimulants or in the presence of IL-10 alone. In cultures with added stimulants, no cytokines were detected at 30 min, and only TNF-{alpha} production was observed at 2 h poststimulation. At 24 h poststimulation, cytokine concentrations were maximal for IL-1ß, IL-6, IL-12p40, and IL-18. In supernatants of cells that were equally stimulated but in the presence of 10 ng/ml of rIL-10, the concentrations of IL-1ß, IL-6, IL-18, and TNF-{alpha} were significantly reduced (P < 0.05 to P < 0.0000001) (Fig. 1A). Added IL-10 marginally (but not significantly) affected the level of IL-12p40 production as induced by all stimulants (data not shown). Exogenous IL-10 decreased IL-6, IL-12p40, and IL-1ß transcript levels but not those of IL-18 and TNF-{alpha} (Fig. 1B; data not shown). Our results indicate that IL-10 differentially affects the production and/or mRNA transcript levels of proinflammatory cytokines by J774 macrophages when these cells are stimulated with B. burgdorferi, L-OspA, or LPS.


Figure 1
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  		FIG. 1. Exogenous IL-10 inhibits proinflammatory cytokines at the protein (A) and mRNA (B) expression levels in J774 macrophages stimulated with L-OspA, freeze-thawed B. burgdorferi spirochetes (Bb), and LPS. Macrophages (3 x 106/ml) were incubated with L-OspA (1 µg/ml), B. burgdorferi (1 x 107/ml), or LPS (1 µg/ml) in the presence (black bars) or absence (white bars) of 10 ng/ml mouse recombinant IL-10 (rIL-10). At 2 h (TNF-{alpha}) and 24 h (IL-6, IL-12p40, IL-1ß, IL-18), mRNA and protein levels were determined by RT-PCR and cytokine-specific ELISAs, respectively. For RT-PCR, all values were normalized with respect to the mRNA levels of the "housekeeping" gene that codes for GAPDH. Results are presented as fold increase over the control (i.e., the level in unstimulated cells). Cytokine production levels are shown in picograms/milliliter. Asterisks indicate significant differences from cells incubated with L-OspA, B. burgdorferi, or LPS alone (P < 0.05 to P < 0.0000001). P values were calculated by use of the unpaired Student's t test. Each bar represents the means ± standard errors of duplicate cultures. Data are representative of two separate experiments.

SOCS1 and SOCS3 expression levels are upregulated in J774 macrophages in response to exogenous IL-10, B. burgdorferi, and L-OspA stimulation. To begin to address our hypothesis that SOCS may mediate IL-10 inhibition of inflammatory cytokines induced via TLR2 or the heterodimer TLR2/1 or TLR2/6, the TLRs that most likely interact with B. burgdorferi or its lipoproteins, we stimulated J774 macrophages with rIL-10 (10 ng/ml) as well as B. burgdorferi (1 x 107/ml), L-OspA (1 µg/ml), or LPS (1 µg/ml) both in the presence and absence of rIL-10 (10 ng/ml), and we quantified SOCS transcript levels as a function of time poststimulation. We focused initially on SOCS3, because this protein had been shown to be a major player in the IL-10-mediated inhibition of LPS-induced inflammatory cytokines in macrophages (5). RNA was collected from unstimulated and stimulated cells at 0, 30, and 120 min following incubation. As shown in Fig. 2A, the addition of IL-10 to macrophages induced a significant upregulation of SOCS3 mRNA transcript at 30 and 120 min (P < 0.05 to 0.005). Addition of rIL-10 to B. burgdorferi and L-OspA caused a significant increase (P < 0.01 to 0.03) in SOCS3 mRNA transcript levels at 30 min and a further increase at 120 min (P < 0.01 to 0.007). We also assessed SOCS1 and SOCS2 transcript levels, as induced by exposure to IL-10, alone or in combination with stimulants. No significant change in transcript levels was observed at 30 min poststimulation for SOCS1 (Fig. 2B) or SOCS2 (data not shown). At 120 min, the amount of SOCS1 transcript was weakly but not significantly enhanced after IL-10, B. burgdorferi, and L-OspA stimulation, yet IL-10 in combination with B. burgdorferi or L-OspA induced significant (P < 0.005) upregulation of SOCS1 at 120 min (Fig. 2B). SOCS2 transcript also was not induced by stimulants at 120 min (data not shown). Significant (P < 0.05 to 0.007) upregulation of SOCS (particularly SOCS3) expression levels in the presence of IL-10 was also observed when LPS was used as the stimulant (Fig. 2A and B). Finally, we determined SOCS1 and SOCS3 transcript levels after 24 h and 48 h poststimulation in the same conditions as those described above. Overall, SOCS3 mRNA levels were increased by one-and-a-half times the levels observed after a 2-h stimulation (data not shown). By quantitative real-time PCR, we further demonstrated, at 24-h poststimulation, a significant fold increase (P < 0.001) in SOCS3 mRNA induction levels in L-OspA- and IL-10-stimulated cells, which were further enhanced by costimulation of cells. SOCS1 was marginally upregulated in all cultures compared to upregulation of SOCS3 (Fig. 2C), corroborating the RT-PCR results. Quantitative real-time PCR studies were not conducted to assess the effects of stimulation with B. burgdorferi or LPS.


Figure 2
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  		FIG. 2. SOCS transcript levels in J774 macrophages as a function of time poststimulation. Macrophages were incubated with L-OspA, freeze-thawed B. burgdorferi (designated Bb or Bb), or LPS in the presence or absence of rIL-10 as described for Fig. 1. RNA was collected from all cultures at 0, 30, and 120 min and analyzed by RT-PCR to determine (A) SOCS3 and (B) SOCS1 mRNA transcript levels. (C) Quantitative real-time PCR was conducted at 24 h after stimulation of cells to determine SOCS1 and SOCS3 induction levels. For RT-PCR and quantitative real-time PCR, all values were normalized with respect to GAPDH mRNA levels. Results are presented as fold increase over the control (i.e., the level in unstimulated cells). The results shown are the means ± standard deviations of three separate experiments (A and B) as well as a representative of two separate experiments for SOCS3 and one for SOCS1 (C). Asterisks indicate significant differences (P < 0.05 to P < 0.001). (D) Macrophages were incubated with L-OspA, B. burgdorferi, or LPS in the presence or absence of rIL-10. After 24 h of incubation, cell lysates were prepared and SOCS3 protein levels were assessed by Western blotting. Results are representative of four separate experiments.

Immunoblotting experiments with J774 whole-cell extracts demonstrated that SOCS1 and SOCS3 mRNA expression induced by stimulation of the cells with B. burgdorferi, L-OspA, or LPS, both in the presence and absence of IL-10, resulted in subsequent translation of SOCS1 and SOCS3 proteins. SOCS3 proteins were markedly enhanced when stimulants were cultured with IL-10 (Fig. 2D, compare lanes 2 to 4 with 6 to 8). Similar results were obtained for SOCS1, except the expression levels were weak (data not shown). The results show that the enhanced SOCS1 and SOCS3 transcript levels observed in macrophages in response to stimulants with and without IL-10 could be functionally important, insofar as they result in a raised level of the corresponding proteins.

Minimal concentrations of stimulants and IL-10 required for induction of SOCS expression in macrophages. Macrophages were incubated with a range of concentrations of B. burgdorferi (1 x 104 to 1 x 107/ml), L-OspA (0.001, 0.01, 0.1, and 1 µg/ml), LPS (0.001, 0.01, 0.1, and 1 µg/ml), and rIL-10 (0.01, 0.1, 1, and 10 ng/ml); RNA was collected at 24 h (time of maximal SOCS expression), and RT-PCR was performed to assess both SOCS1 and SOCS3 transcript levels. Induction of SOCS1 transcript above the constitutive level that was observed in the presence of medium alone was clearly detectable at 106 spirochetes/ml and 0.1 µg/ml L-OspA, and it was barely detectable at 10 ng/ml rIL-10 (Fig. 3). Overall, induction of SOCS3 transcript was achieved at lower concentrations of stimulants and IL-10, with B. burgdorferi being effective at 105 spirochetes/ml, L-OspA at 0.01 µg/ml, and IL-10 at 1 ng/ml (Fig. 3). LPS was effective in inducing both the SOCS1 and SOCS3 transcripts, even at the lowest concentration employed in the study, 1 ng/ml (Fig. 3).


Figure 3
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  		FIG. 3. Analysis of the stimulant dose required to induce optimal expression of SOCS1 and SOCS3 mRNA transcripts. Macrophages (3 x 106/ml) were stimulated for 24 h in medium alone or with various concentrations of L-OspA, freze-thawed B. burgdorferi (Bb), LPS, and rIL-10. RNA was collected, and RT-PCR was performed for SOCS1 and SOCS3 mRNA transcripts. Concentrations in boldface induced the highest mRNA transcript levels for SOCS1 or SOCS3. Data are representative of two separate experiments.

Pam3Cys as well as sonicated and live B. burgdorferi spirochetes, but not U-OspA, induce SOCS1 and SOCS3 mRNA transcripts. To assess whether SOCS1 and SOCS3 mRNA transcripts would be induced in macrophages in response to any bacterial lipoprotein and not just L-OspA, we incubated macrophages with Pam3Cys, a synthetic lipopeptide that mimics the structure of the lipoprotein lipid moiety (1 µg/ml), or U-OspA (nonlipidated OspA, 1 µg/ml) in the presence or absence of rIL-10 (10 ng/ml). RNA samples were collected at 24 h for RT-PCR analysis. We also determined whether spirochetal antigens from sonicated spirochetes (1 x 107/ml) as well as live spirochetes in the presence or absence of rIL-10 (10 ng/ml) induced SOCS1 and SOCS3 expression in macrophages. Pam3Cys and sonicated spirochetes induced both SOCS1 and SOCS3 mRNA transcripts in macrophages (Fig. 4A). Concomitant incubation of rIL-10 with Pam3Cys or sonicated spirochetes further augmented SOCS1 and SOCS3 transcripts (Fig. 4A, lanes 3 and 4 versus lanes 7 and 8). Augmentation of SOCS1 and SOCS3 transcripts in macrophages correlated with the ability of IL-10 to down-regulate IL-6 (mRNA and protein levels) and IL-12p40 (mRNA levels) in the same cultures (Fig. 4A, lanes 3 and 4 versus lanes 7 and 8, and B). U-OspA had no effect on SOCS or cytokine induction levels either alone or combined with IL-10 (Fig. 4A and B).


Figure 4
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  		FIG. 4. Pam3Cys as well as sonicated and live B. burgdorferi (Bb or Bb) spirochetes, but not U-OspA, induce SOCS1 and SOCS3 mRNA transcripts. (A) Macrophages were preincubated with 1 µg/ml of Pam3Cys or U-OspA and 1 x 107/ml of sonicated spirochetes prior to addition of 10 ng/ml of rIL-10 for an additional 24 h. SOCS1, SOCS3, IL-6, and IL-12p40 mRNA levels were determined by RT-PCR. (B) IL-6 protein levels in these same cultures were determined by ELISA. Data are representative of three separate experiments. (C) Macrophages were stimulated with various cell:spirochete ratios for 24 h, after which RNA was collected and analyzed by RT-PCR for SOCS1 and SOCS3. (D) Macrophages were preincubated with a cell:spirochete ratio of 1:10 prior to addition of rIL-10 to determine SOCS1 and SOCS3 mRNA expression levels by RT-PCR. (E) IL-6 protein levels were determined by ELISA in these same cultures at 24 h. Data are representative of two separate experiments. Asterisks indicate significant differences (P < 0.0000001) from cells incubated with stimulant alone compared to costimulation with rIL-10.

For live spirochetes, we incubated J774 macrophages with various cell:spirochete ratios (1:1, 1:5, 1:10, and 1:20), and SOCS1 and SOCS3 mRNA transcripts were analyzed by RT-PCR after 24 h of stimulation. The results show that live spirochetes at all cell:spirochete ratios induced the expression of SOCS1 and SOCS3 in macrophages (Fig. 4C). Maximal SOCS upregulation was seen with cell:spirochete ratios of 1:5 and 1:10. Moreover, coexposure of macrophages to IL-10 and live spirochetes augmented SOCS, particularly SOCS3 expression (Fig. 4D), which was associated with down-regulation of IL-6 at the mRNA and protein levels (Fig. 4D and E). These data demonstrate that SOCS1 and SOCS3 induction can also be elicited by any bacterial lipoprotein. Further, they are evidence to suggest that the enhancement of SOCS expression may be elicited during infection with B. burgdorferi, either by spirochetal debris or by living spirochetes.

Pretreatment of macrophages with exogenous IL-10 before addition of L-OspA and B. burgdorferi does not affect the upregulation of SOCS1 and SOCS3 mRNA expression. Considering that SOCS3 in particular is induced by rIL-10 as early as 30 min poststimulation, we conducted experiments to determine whether prior treatment of macrophages with rIL-10, to permit the synthesis of SOCS3, before addition of L-OspA or B. burgdorferi would affect the augmentation of SOCS1 and SOCS3 mRNA transcript levels as observed by costimulation (Fig. 2A). Macrophages were pretreated with rIL-10 (10 ng/ml) at 37°C for 30 min prior to addition of L-OspA and B. burgdorferi for an additional 24-h incubation period. Figure 5A shows that SOCS1 and SOCS3 expression levels were augmented by pretreatment of macrophages with rIL-10 prior to addition of L-OspA or B. burgdorferi (lanes 6 and 7) compared with L-OspA or B. burgdorferi alone (lanes 2 and 3). SOCS1 and SOCS3 upregulation correlated with the down-modulation of IL-6 and IL-12p40 at the mRNA levels (Fig. 5A, lanes 2 and 3 versus lanes 6 and 7) and with IL-6 at the protein level (Fig. 5C). SOCS1 and SOCS3 mRNA expression levels also were augmented by pretreatment of macrophages with L-OspA or B. burgdorferi prior to addition of rIL-10 (data not shown). This study demonstrated a possible direct induction of SOCS1 and SOCS3 gene transcripts in macrophages by stimulants alone and by costimulation. Similar results were obtained for LPS, except pretreatment of cells with rIL-10 prior to LPS stimulation did not augment SOCS1 expression levels (Fig. 5A, lanes 4 and 8).


Figure 5
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  		FIG. 5. Direct induction of SOCS1 and SOCS3 mRNA transcript levels in macrophages. Macrophages (2 x 106/ml) were preincubated with (A) rIL-10 (10 ng/ml) or (B) anti-IL-10 Ab (25 µg/ml) or normal IgG1 (Cab; 25 µg/ml) as a control. After 30 min of preincubation at 37°C, L-OspA, freeze-thawed B. burgdorferi (Bb), and LPS were added to individual cultures to reach a final concentration of 1 µg/ml for L-OspA and LPS or 1 x 107/ml for B. burgdorferi. SOCS1, SOCS3, IL-6, and IL-12p40 mRNA transcript analyses were determined by RT-PCR after an additional 24 h of incubation of cells with stimulants. (C) IL-6 protein levels were determined in 24-h-cultured supernatants by ELISA. Asterisks indicate significant differences (P < 0.0000001) from cells incubated with stimulants alone compared with costimulation with rIL-10. (D) Macrophages were pretreated with 1 µg/ml of cycloheximide (CHX) as described above, followed by stimulation with L-OspA, B. burgdorferi, or LPS for an additional 4-h incubation period. SOCS1, SOCS3, and IL-6 transcripts were assessed by RT-PCR. All values were normalized with respect to GAPDH mRNA levels. Data are representative of three separate experiments (A, B, and C) and of two separate experiments (D).

SOCS1 and SOCS3 induction by B. burgdorferi or L-OspA is not dependent on endogenous IL-10. Since pretreatment with stimulants did not affect the upregulation of SOCS1 and SOCS3 transcripts levels, we next determined if endogenous IL-10 could be involved in this phenomenon. Both B. burgdorferi- and L-OspA-stimulated J774 macrophages produce IL-10 in the range of 100 to 500 pg/ml (data not shown). Although this concentration of IL-10 may be insufficient to stimulate SOCS1 or SOCS3 expression per se (Fig. 3), it was still important to neutralize IL-10 activity in these culture supernatants to ascertain whether this amount of cytokine could influence the stimulants' effect on the enhancement in the expression of SOCS1 and SOCS3 transcripts. Macrophages were preincubated with anti-IL-10 or a control antibody (25 µg/ml) at 37°C for 30 min prior to addition of B. burgdorferi or L-OspA for an additional 24 h. Cells with no antibodies added served as controls. The concentration of anti-IL-10 antibody used was sufficient to completely neutralize IL-10 activity in the cultures, as assessed by ELISA in a parallel experiment (data not shown) and as already described by us (24). Neutralizing antibody to IL-10 (or its isotype control) did not affect the B. burgdorferi- or L-OspA-induced SOCS1 or SOCS3 mRNA expression levels (Fig. 5B) or their IL-6 production levels (Fig. 5C). Anti-IL-10 antibody also did not affect SOCS1 and SOCS3 expression induced by LPS or its ability to stimulate the production of IL-6 (Fig. 5B and C). Similar results were obtained from samples stimulated for 2 or 48 h. The results demonstrate that endogenously produced IL-10 does not contribute to the enhancement of SOCS1 and SOCS3 expression by stimulants alone or by costimulation of macrophages.

De novo protein synthesis is not required for the enhancement of SOCS1 and SOCS3 expression induced by IL-10, B. burgdorferi, or L-OspA. Finally, we determined if other regulatory proteins could contribute to the induction of SOCS1 and SOCS3 by stimulants in macrophages, since both SOCS proteins are also induced by other cytokines, including IL-6, IL-12, and TNF-{alpha} (7, 32, 38). The latter are produced by B. burgdorferi- and L-OspA-stimulated J774 macrophages (Fig. 1). To determine whether the upregulation of SOCS1 and SOCS3 mRNA by stimulants required synthesis of other regulatory proteins, we pretreated macrophages with the protein synthesis inhibitor cycloheximide (CHX; 1 µg/ml, which completely blocks protein synthesis in J774 macrophages [41]) for 30 min, followed by stimulation with optimal concentrations of B. burgdorferi, L-OspA, LPS, or rIL-10 for an additional 2- or 4-h incubation period. SOCS1, SOCS3, and IL-6 mRNA analyses were evaluated by RT-PCR. SOCS3 and IL-6 mRNA accumulation was directly induced by CHX (Fig. 5D). This finding is of no surprise, since CHX is known to induce the stabilization of various mRNAs, including those of SOCS3 (60) and IL-6 (33). Triggering of cells with B. burgdorferi, L-OspA, or IL-10 induced SOCS1 and SOCS3 in the presence of CHX, indicating a direct SOCS induction without increased synthesis of a regulatory protein (Fig. 5D). Moreover, accumulation of IL-6 mRNA transcripts in macrophages in response to L-OspA and B. burgdorferi was not affected by CHX treatment (lanes 2 and 3 versus 7 and 8). Results for LPS were very similar to those obtained for L-OspA and B. burgdorferi (Fig. 5D, lane 4 versus lane 8). Our CHX results clearly show that SOCS1 and SOCS3 expression induced by stimulation of cells with B. burgdorferi, L-OspA, or IL-10 for 2 (data not shown) or 4 h was independent of de novo protein synthesis, suggesting the lack of involvement of one or more of these cytokines in mediating the expression of SOCS. CHX experiments were not conducted at 24 h, because at this time a significant loss of cell viability was observed in the cultures.


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DISCUSSION
 
SOCS proteins are induced by cytokines and TLR-mediated stimuli; in turn, they can prevent secretion of cytokines that underlie pathogenic mechanisms of inflammatory diseases (1, 2, 13) and regulate TLR signaling in innate immune cells (7, 16, 18, 29, 37, 46, 60). We hypothesized that SOCS are induced in macrophages by IL-10 and by B. burgdorferi and its lipoproteins and that they may mediate the inhibition by IL-10 of concomitantly elicited cytokines. Our results confirmed that (i) expression of both SOCS1 and SOCS3 is induced in macrophages in response to IL-10, as well as to B. burgdorferi and its lipoproteins, with SOCS3 being the more abundant; (ii) SOCS1 and SOCS3 mRNA transcript levels are induced by IL-10 as well as B. burgdorferi and its lipoproteins in the absence of de novo protein synthesis; and (iii) stimulation of cells with IL-10 together with B. burgdorferi and its lipoproteins additively affects SOCS1 and SOCS3 expression levels. This temporally correlates with the IL-10-mediated inhibition of expression of the proinflammatory cytokines IL-1ß, IL-6, IL-12p40, IL-18, and TNF-{alpha} at the protein and/or mRNA levels.

To our knowledge, our study is the first to demonstrate that B. burgdorferi can induce an upregulation of SOCS1 and SOCS3 expression in macrophages, a finding that has been similarly documented for other bacteria, such as Listeria monocytogenes (61), Mycobacterium bovis (34), Mycobacterium tuberculosis (49), Salmonella enterica (63), and Staphylococcus aureus (6), or bacterium-derived substances such as LPS (7, 15, 16, 60) and CpG-DNA (18). Our study verified that live B. burgdorferi organisms were as effective as freeze-thawed spirochetes, sonicated spirochetes, L-OspA, and the lipohexapeptide Pam3Cys in stimulating the expression of SOCS1 and SOCS3 in macrophages; this indicates that this phenomenon may be elicited in vivo both by live organisms and by residual spirochetal antigens left in the tissues. U-OspA did not induce an upregulation of SOCS1 and SOCS3 mRNA expression in cells, in agreement with the well-established fact that the immune-modulatory effect of lipoproteins is due to the lipid moiety.

The induction of SOCS1 and SOCS3 expression by B. burgdorferi and its lipoproteins in macrophages did not require endogenous IL-10 or synthesis of other regulatory proteins. Endogenous IL-10 was not involved in this phenomenon, most likely because the levels of IL-10 produced by B. burgdorferi and its lipoproteins, as demonstrated in this study, are insufficient to induce SOCS1 and SOCS3 expression in macrophages. The fact that B. burgdorferi or its lipoproteins can alone induce SOCS1 and SOCS3 expression indicates that the spirochete may also control inflammation via a mechanism independent from that involving IL-10, even though both involve SOCS. Several studies have revealed that SOCS expression as induced by TLR stimulation yielded functional proteins, as demonstrated by the ability of SOCS to inhibit inflammatory cytokine signaling in macrophages (17, 18, 34, 60, 61). Studies conducted by Dalpke et al. (17) showed that TLR triggering of mouse macrophages induced SOCS1/SOCS3, leading in turn to an inhibition of IFN-{gamma} activation of STAT1 and major histocompatibility complex class II upregulation. SOCS expression induced by LPS (60), CpG-DNA (18), M. bovis (34), S. enterica (63) or L. monocytogenes (61) was demonstrated to be functional by showing that it prevented IFN-{gamma}, IL-6, or GM-CSF signaling in macrophages. Recently, Mansell and coworkers (44) elegantly demonstrated that SOCS1 specifically regulates TLR2 and TLR4 signaling in macrophages via a mechanism mediated by Mal (MyD88 adapter-like protein), but not MyD88, to modulate proinflammatory responses. This study showed that SOCS1 induced by TLR2 and TLR4 stimuli interacts with Mal, resulting in the polyubiquitination and subsequent degradation of Mal. Removal of SOCS1 potentiated Mal-dependent phosphorylation and transactivation of NF-{kappa}B, resulting in an enhanced IL-6 and TNF-{alpha} proinflammatory response in mouse macrophages (44). It is likely that SOCS1 and SOCS3 proteins induced by B. burgdorferi and its lipoproteins may function in a fashion similar to that of mediators of a variety of signaling pathways to suppress macrophage activation and subsequently the inflammatory potential of these cells.

Our study also verified that costimulation of macrophages with IL-10 and B. burgdorferi and its lipoproteins additively augmented SOCS1 and SOCS3 expression. Augmentation of SOCS1 and SOCS3 by costimulation compared to stimulation with individual stimulants would suggest either reinforcement of a common signaling pathway or convergence of different pathways for optimal SOCS1 and SOCS3 expression. It is known that IL-10 induction of SOCS1 and SOCS3 expression in macrophages involves both JAK/STAT-dependent and -independent signaling pathways (5, 11, 36). Alternatively, stimulation of SOCS1 and SOCS3 expression by B. burgdorferi and its lipoproteins in macrophages is most likely dependent on a TLR signaling pathway. Recent reports demonstrated that TLR2 induction of SOCS1 or SOCS3 is dependent on a TLR pathway regulated by either MyD88 (3) or Mal (44) adapter proteins. Thus, a combination of signals (possibly via TLR and JAK/STAT pathways) would be required for optimal expression of SOCS1 and SOCS3 in macrophages, as observed in our study. The precise signaling pathways and transcription factors that mediate the induction of SOCS expression in macrophages by individual stimulants or by costimulation remain to be investigated.

Enhanced expression of SOCS1 and SOCS3 by costimulation temporally correlated with the ability of IL-10 to down-modulate production of several proinflammatory cytokines, including IL-6, IL-12p40, IL-1ß, IL-18, and TNF-{alpha}. Both SOCS1 and SOCS3 are known mediators of the IL-10 anti-inflammatory activity in cells (5, 19, 52). SOCS3, in particular, was shown to be essential for IL-10 inhibition of LPS-stimulated production of TNF-{alpha}, IL-6, GM-CSF, and nitric oxide (NO) in mouse macrophages (5). Recent studies also showed that SOCS3 mediated the IL-10 inhibition of LPS-induced inducible NO synthase protein and NO production in mouse macrophages (52). The same study observed that the SH2 domain, SOCS box, and both Tyr204 and Tyr221 were required for IL-10 inhibition of TNF-{alpha} expression in mouse macrophages. The upregulation of SOCS proteins in our study is evidence to suggest that expression of SOCS is part of the mechanism of IL-10-mediated inhibition of inflammatory cytokines elicited by B. burgdorferi and its lipoproteins. We are now focusing on RNA interference of SOCS1 and SOCS3 expression to determine their direct involvement in this phenomenon.

The results obtained in the present study may be of significance in the context of susceptibility and resistance to Lyme disease, as modeled in mice. The enhanced production of IL-10 and the lessened production of proinflammatory cytokines by bone marrow-derived macrophages of C57 (disease-resistant) mice compared to C3H (disease-susceptible) mice (Ganapamo, Dennis, and Philipp, unpublished) (8), coupled with the ability of IL-10 to control inflammation in vivo in C57 mice (8), are all indicative of macrophage deactivation during the early phase of the immune response in C57 mice. Although conjectural, it is likely that in C57 mice SOCS proteins are upregulated in macrophages by costimulation provided by endogenous IL-10 and live spirochetes; this might explain the diminished inflammation and disease severity that are observed in this mouse strain. In contrast, the inability of C3H mice to control the inflammatory response may be due to lack of SOCS induction by the low level of IL-10 produced by their macrophages and therefore the absence of the additional costimulatory signal needed for enhancement of SOCS, although one is provided by live spirochetes. Studies have shown the potential beneficial actions of SOCS proteins in inflammatory diseases (57, 62). In rheumatoid arthritis, where proinflammatory cytokines play an important role in disease pathogenesis, SOCS3 expression levels are elevated in joint tissues of patients (62). The use of socs3 gene therapy suppressed proliferation of synovial fibroblasts, as well as the production of IL-6, and significantly reduced the onset and progression of collagen- and antigen-induced arthritis in mice (57). Our findings of enhanced expression of SOCS1 and SOCS3 by costimulation with live spirochetes and IL-10 suggest that SOCS1 and SOCS3 are differentially expressed in C57 and C3H mice to account for the differences in their inflammatory and disease severity responses. Future studies will investigate if SOCS1 and SOCS3 (or their downstream mediators) are indeed differentially expressed in macrophages and tissues of C57 and C3H mice as well as the correlation of SOCS expression with inflammatory cytokine production. If we can demonstrate differential expression of SOCS in vivo, then the SOCS pathway may dictate susceptibility and resistance to Lyme disease, as modeled in mice, and perhaps also in humans.


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ACKNOWLEDGMENTS
 
This work was supported by grant UO1-CI000153 from the Centers for Disease Control and Prevention and by grant RR00164 from the National Center for Research Resources, National Institutes of Health.

Excellent secretarial help from Avery MacLean is gratefully acknowledged.


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FOOTNOTES
 
* Corresponding author. Mailing address: Division of Bacteriology and Parasitology, Tulane National Primate Research Center, 18703 Three Rivers Rd., Covington, LA 70433. Phone: (985) 871-6267. Fax: (985) 871-6390. E-mail: vida{at}tulane.edu. Back

Editor: D. L. Burns


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Infection and Immunity, October 2006, p. 5780-5789, Vol. 74, No. 10
0019-9567/06/$08.00+0     doi:10.1128/IAI.00678-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.



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