A tolerogenic role for Toll-like receptor 9 is revealed by B-cell interaction with DNA complexes expressed on apoptotic cells

Abstract

Intracellular protein complexes containing nucleic acids are common targets of autoantibodies in many autoimmune diseases. Central tolerance to these antigens is incomplete, yet nucleosomal DNA is expressed on the surface of cells dying by apoptosis. It is commonly believed that autoimmunity is prevented by the rapid uptake of apoptotic cells (ACs) by neighbors or professional phagocytes to which they deliver anti-inflammatory signals. Self-reactive, innate-like B cells contact and are selected by intracellular antigens expressed on ACs; however, how self-tolerance is maintained is not well understood. Here we report that IL-10 production by B cells, stimulated by contact with ACs, results from the engagement of Toll-like receptor 9 (TLR9) within the B cell after recognition of DNA-containing complexes on the surface of ACs. Until now, TLR9 ligation has been considered an inflammatory signal, but we have confirmed a hitherto unexpected immunoregulatory role by demonstrating the absence of the protective effect of ACs during experimental autoimmune encephalitis (EAE) in TLR9-deficient mice. Human circulating CD27(+) B cells also respond to DNA-bearing ACs, but not to DNase-treated cells, by secreting IL-10. Chronic autoimmune disease may arise if this tolerance mechanism is not reimposed after episodes of inflammation, or if the regulatory B-cell response is subverted.

Fig. 1.

Regulatory B cells from the MZB cell and B1a cell subsets recognize whole ACs via the BCRs and secrete IL-10. (A) IL-10–GFP reporter mice were injected with 20 × 10⁶ ACs, and MZB cells were harvested 1 wk later. IL-10 expression was determined by FACS analysis. (B) MZB cells were stimulated with ACs for up to 4 h in the absence of further stimulation and mRNA for IL-10 measured by quantitative PCR. (C) B cells from WT or transgenic B cells from mice specific for HEL (MD4) were separated into FOB and MZB cells. After stimulation for 72 h in the presence of OVA-specific transgenic T cells and OVA peptide with or without ACs, supernatants were collected and IL-10 was measured by ELISA. (D) MZB cells from MD4 and WT mice stimulated as in C, but with the cognate protein HEL added to some of the cultures. (E) Polyclonal MZB cells (HEL⁻) and HEL-specific MZB cells (HEL⁺) derived from the same SW_HEL mice were stimulated with the TLR4 ligand LPS or the TLR2 ligand PGN for 72 h with and without ACs, and IL-10 was measured. *Indicates a significant difference between IL-10 in stimulated B cells cocultured with ACs and HEL⁻ and HEL⁺ MZB cells. (F) Peritoneal-derived (PEC) CD5⁻ and CD5⁺ CD19 B cells and splenic MZB and FOB cells were stimulated as in C. (G) MZB cells were sorted into CD1d^hiCD5⁻ or CD1d^hiCD5⁺ve subsets and stimulated for 72 h as in C (OVA) or with various TLRs, including CpG (TLR9), R848 (TLR7), and PGN (TLR2), and IL-10 was measured after 72 h. Each figure is representative of at least three experiments performed with three mice per group. Error bars represent SEM. ***P ≤ 0.0004; **P ≤ 0.004; *P ≤ 0.04. The threshold for detection of IL-10 was 25 pg/mL

Fig. 2.

DNase treatment abolishes the capacity of AC to enhance IL-10 secretion and inhibits regulatory responses in vivo. (A) ACs expressed DNA-containing complexes, stained with Sytox (molecular probes, Invitrogen) on the cell surface, which was lost after treatment with DNase. (B) B cells were stimulated by OVA-specific T cells, OVA peptide, and ACs along with 50 μg/mL of DNase, and IL-10 was measured in supernatants after 72 h. (C) A single injection of vehicle (PBS) or ACs pretreated with DNase (DNase-AC) or untreated ACs (AC) was injected into mice at the time of immunization with type II collagen (CII) in complete Freund's adjuvant (CFA), and arthritis was assessed clinically. (D) Histology of H&E-stained sagittal sections through the knee joints of mice taken from Fig. 3C. Both the DNase-treated mice and the PBS-treated mice exhibited active synovitis associated with a fibrinopurulent exudate within the joint space, with less inflammation in the AC-treated mice. (E) Splenocytes from AC-treated mice (filled squares), DNase-treated mice (open squares), and WT mice (filled circles) obtained at the end of the experiment shown in Fig. 3C were restimulated in vitro with CII for 3 d, and IL-10 levels were measured. The CIA experiment is representative of two experiments with eight mice per group. The remaining figures are representative of at least three experiments performed with three mice per group. Error bars represent SEM. ***P ≤ 0.0004; **P ≤ 0.004; *P ≤ 0.04.

Fig. 3.

B-cell TLR9 signaling is required for the immunosuppressive response to ACs. (A) B cells were stimulated with the TLR ligands PGN (TLR2) and LPS (TLR4) alone or in the presence of ACs or ACs and chloroquine (AC Chloro) for 72 h, after which IL-10 in the supernatants was measured. (B) WT, MyD88-deficient (MyD88), and TLR9-deficient (TLR9) B cells were cocultured with ACs, OVA-specific T cells, and OVA peptide in vitro. After 72 h, IL-10 secretion was measured by ELISA. (C) PEC B1 cells were stimulated with the same TLR ligands as in Fig. 1A with and without AC for 72 h, and IL-10 was measured. (D) WT and TLR9-deficient mice were immunized with MOG/CFA, and EAE was scored. A single i.v. injection of ACs also was administered on day 0. Some TLR9-deficient mice also received an injection of 10 × 10⁶ CD19 B cells on day 0. The EAE experiment is representative of three experiments with five mice per group. The remaining figures are representative of at least three experiments performed with three mice per group. Error bars represent SEM. ***P ≤ 0.0004; **P ≤ 0.004; *P ≤ 0.04. The threshold for detection of IL-10 was 25 pg/mL.

Fig. 4.

Human CD27⁺ B cells respond to ACs by secreting IL-10. (A) Human B cells isolated from healthy volunteers were separated into CD27⁺ and CD27⁻ fractions and cultured in the presence of IL-4 for 3 d with AC (+AC) or without AC, after which IL-10 in the supernatants was measured. (B) B cells were stimulated with ACs and IL-4 in the presence of increasing concentrations of chloroquine for 3 d, and IL-10 secretion was measured by ELISA. A shows data collected from 14 healthy volunteer blood donors; B is representative of at least two separate experiments. Error bars represent SEM. ***P ≤ 0.0004; **P ≤ 0.004; *P ≤ 0.04. The threshold for detection of IL-10 was 25 pg/mL