Borrelia burgdorferi RNA induces type I and III interferons via Toll-like receptor 7 and contributes to production of NF-κB-dependent cytokines

Abstract

Borrelia burgdorferi elicits a potent cytokine response through activation of multiple signaling receptors on innate immune cells. Spirochetal lipoproteins initiate expression of NF-κB-dependent cytokines primarily via TLR2, whereas type I interferon (IFN) production is induced through the endosomal receptors TLR7 and TLR9 in human dendritic cells and TLR8 in monocytes. We demonstrate that DNA and RNA are the B. burgdorferi components that initiate a type I IFN response by human peripheral blood mononuclear cells (PBMCs). IFN-α protein and transcripts for IRF7, MX1, and OAS1 were induced by endosomal delivery of B. burgdorferi DNA, RNA, or whole-cell lysate, but not by lysate that had been treated with DNase and RNase. Induction of IFN-α and IFN-λ1, a type III IFN, by B. burgdorferi RNA or live spirochetes required TLR7-dependent signaling and correlated with significantly enhanced transcription and expression of IRF7 but not IRF3. Induction of type I and type III IFNs by B. burgdorferi RNA could be completely abrogated by a TLR7 inhibitor, IRS661. In addition to type I and type III IFNs, B. burgdorferi RNA contributed to the production of the NF-κB-dependent cytokines, IFN-γ, interleukin-10 (IL-10), IL-1β, IL-6, and tumor necrosis factor alpha (TNF-α), by human PBMCs. Collectively, these data indicate that TLR7-dependent recognition of RNA is pivotal for IFN-α and IFN-λ1 production by human PBMCs, and that RNA-initiated signaling contributes to full potentiation of the cytokine response generated during B. burgdorferi infection.

FIG 1

B. burgdorferi nucleic acids induce a type I IFN response in human PBMCs. Human PBMCs (5 × 10⁶) were stimulated for 12 h with 5 × 10⁷ live B. burgdorferi (Bb) spirochetes, DOTAP-complexed B. burgdorferi DNA or RNA (1 μg/ml), or B. burgdorferi whole-cell lysate (1 μg/ml) with or without DOTAP added. (A) Transcriptional expression of MX1, OAS1, and IRF7 was measured by RT-PCR and normalized to transcript levels for GAPDH. Data are presented as the mean fold changes ± standard deviations (SD) relative to PBMCs incubated with medium alone. (B) Protein concentration of IFN-α in cell-free supernatants was measured by ELISA. Data shown are the means ± SD of values from two donors assessed in triplicate in two independent experiments and are representative of one additional donor. P < 0.05 (*), P < 0.01 (**), and P < 0.001 (***) relative to PBMCs incubated with medium alone. (C) B. burgdorferi B515 lysate was prepared by vortexing in the presence of glass beads until cellular breakage was verified via dark-field microscopy. Whole-cell lysate, or lysate that had been treated with RNase A or DNase I, was used to stimulate PBMCs. In order to confirm the presence of TLR2 ligands in the lysate preparations, volumes containing 10 μg total protein were resolved by 12.5% SDS-PAGE and immunoblotted with specific antibodies for OspC and OspA, spirochetal lipoproteins known to initiate TLR2-mediated signaling. B. burgdorferi B515 lysate prepared from a separate culture using a chemical lysis method was included as a positive control (far left lane).

FIG 2

B. burgdorferi RNA induces transcription of IRF7 in human PBMCs via TLR7-dependent signaling. Human PBMCs (5 × 10⁶) were cultured in the presence of medium, a control ODN (5.6 μM), or the TLR7 inhibitor IRS661 (5.6 μM) for 1 h before stimulation with live B. burgdorferi (5 × 10⁷), DOTAP-complexed B. burgdorferi RNA (1 μg/ml), or a synthetic TLR7 agonist, R837 (5 μg/ml). PBMC RNA was isolated 12 h after addition of stimuli, and real-time RT-PCR was used to measure transcriptional expression of signaling mediators. GAPDH-normalized values were used to calculate fold changes in transcript levels for IRF7 or IRF3 relative to PBMCs incubated with medium alone. Columns represent the means ± SD of results obtained using PBMCs from two donors assessed in triplicate in independent experiments. ***, P < 0.001 relative to PBMCs incubated with medium alone; NS, not significantly different.

FIG 3

B. burgdorferi RNA promotes expression of IRF7 human PBMCs. Human PBMCs (5 × 10⁶) were incubated for 12 h with 5 × 10⁷ live B. burgdorferi spirochetes, DOTAP-complexed B. burgdorferi RNA (1 μg/ml), or B. burgdorferi whole-cell lysate (1 μg/ml) added without DOTAP. PBMC lysates were resolved by 12.5% SDS-PAGE for Western immunoblotting with rabbit anti-human IRF7 (A) or IRF3 (B). Signals were quantified by densitometry and are expressed as ratios to GAPDH. Columns represent the means ± SD of results from three donors. Western blot images from a single representative donor are shown. ***, P < 0.001 relative to PBMCs incubated with medium alone. NS, not significantly different.

FIG 4

TLR7-dependent signaling by B. burgdorferi RNA elicits production of type I and type III IFNs by human PBMCs. Human PBMCs (5 × 10⁶) were cultured in the presence of medium, a control ODN (5.6 μM), or the TLR7 inhibitor IRS661 (5.6 μM) for 1 h prior to stimulation with 5 × 10⁷ live B. burgdorferi spirochetes, DOTAP-complexed B. burgdorferi RNA (1 μg/ml), or B. burgdorferi whole-cell lysate (1 μg/ml) added without DOTAP. R837 (5 μg/ml) and Pam2CSK4 (1 μg/ml) were used as positive controls for activation of TLR7 or TLR2, respectively. IFN-α (A) and IFN-λ1 (B) protein concentrations in culture supernatants were quantitated by ELISA. Data represent the means ± SD of values from three donors assessed in triplicate in three independent experiments. P < 0.05 (*), P < 0.01 (**), and P < 0.001 (***) relative to PBMCs incubated with medium alone, as determined by one-way ANOVA with Tukey-Kramer's posttest, with the exception of the positive-control R837, which was determined by nonparametric Mann-Whitney U test. NS, not significantly different.

FIG 5

B. burgdorferi RNA induces transcription of IFN-responsive genes in human PBMCs via TLR7. Human PBMCs (5 × 10⁶) were cultured in the presence of medium, a control ODN (5.6 μM), or the TLR7 inhibitor IRS661 (5.6 μM) for 1 h before stimulation with live B. burgdorferi (5 × 10⁷), the synthetic TLR7 agonist R837 (5 μg/ml), or DOTAP-complexed B. burgdorferi RNA (1 μg/ml) that had been treated with RNase A or left untreated. Total RNA was isolated 12 h after addition of stimuli, and transcriptional expression of IFN-responsive genes was measured by real-time RT-PCR. GAPDH-normalized values were used to calculate fold changes in transcript levels for MX1 (A) or OAS1 (B) relative to PBMCs incubated with medium alone. Columns represent the mean fold changes ± SD obtained using PBMCs from two donors assessed in independent experiments. ***, P < 0.001 relative to PBMCs incubated with medium alone. NS, not significantly different.

FIG 6

B. burgdorferi RNA contributes to the induction of NF-κB-dependent cytokines by human PBMCs. Human PBMCs (5 × 10⁶) were incubated for 12 h with 5 × 10⁷ live B. burgdorferi spirochetes, DOTAP-complexed B. burgdorferi RNA (1 μg/ml), or B. burgdorferi whole-cell lysate (1 μg/ml) added without DOTAP. R837 (5 μg/ml) and Pam2CSK4 (1 μg/ml) were used as controls for activation of TLR7 and TLR2, respectively. Protein concentrations of IFN-γ, IL-1β, TNF-α, IL-10, and IL-6 in cell-free culture supernatants were measured by cytometric bead array using a MACSQuant analyzer. Columns depict the mean values ± SD of results from three donors assessed in triplicate in three independent experiments. P < 0.05 (*), P < 0.01 (**), and P < 0.001 (***) relative to PBMCs incubated with medium alone, as determined by Mann-Whitney U test.

FIG 7

Proposed model of B. burgdorferi-induced cytokine production by human dendritic cells. Following phagocytosis of B. burgdorferi, spirochetal RNA is detected by TLR7 and initiates MyD88-dependent signaling that leads to activation of IRF7 and production of IFN-α and IFN-λ1. TLR7-MyD88 signaling contributes to the production of NF-κB-dependent cytokines, including IL-6, IL-1β, TNF-α, IFN-γ, and IL-10. Additional activation of NF-κB by B. burgdorferi occurs via TLR2-dependent sensing of spirochetal lipoproteins. The figure is based on Petzke et al. (11) and adapted to include data from the present study.