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. 2006 Oct;74(10):5780-9.
doi: 10.1128/IAI.00678-06.

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

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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 et al. Infect Immun. .
Free PMC article

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. Pam(3)Cys, 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-1beta, 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.

Figures

FIG. 1.
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 × 106/ml) were incubated with L-OspA (1 μg/ml), B. burgdorferi (1 × 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-α) and 24 h (IL-6, IL-12p40, IL-1β, 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.
FIG. 2.
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.
FIG. 3.
FIG. 3.
Analysis of the stimulant dose required to induce optimal expression of SOCS1 and SOCS3 mRNA transcripts. Macrophages (3 × 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.
FIG. 4.
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 × 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.
FIG. 5.
FIG. 5.
Direct induction of SOCS1 and SOCS3 mRNA transcript levels in macrophages. Macrophages (2 × 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 × 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).

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