Investigation of the role of endosomal Toll-like receptors in murine collagen-induced arthritis reveals a potential role for TLR7 in disease maintenance

Abstract

Introduction: Endosomal toll-like receptors (TLRs) have recently emerged as potential contributors to the inflammation observed in human and rodent models of rheumatoid arthritis (RA). This study aims to evaluate the role of endosomal TLRs and in particular TLR7 in the murine collagen induced arthritis (CIA) model.

Methods: CIA was induced by injection of collagen in complete Freund's adjuvant. To investigate the effect of endosomal TLRs in the CIA model, mianserin was administered daily from the day of disease onset. The specific role of TLR7 was examined by inducing CIA in TLR7-deficient mice. Disease progression was assessed by measuring clinical score, paw swelling, serum anti-collagen antibodies histological parameters, cytokine production and the percentage of T regulatory (Treg) cells.

Results: Therapeutic administration of mianserin to arthritic animals demonstrated a highly protective effect on paw swelling and joint destruction. TLR7-/- mice developed a mild arthritis, where the clinical score and paw swelling were significantly compromised in comparison to the control group. The amelioration of arthritis by mianserin and TLR7 deficiency both corresponded with a reduction in IL-17 responses, histological and clinical scores, and paw swelling.

Conclusions: These data highlight the potential role for endosomal TLRs in the maintenance of inflammation in RA and support the concept of a role for TLR7 in experimental arthritis models. This study also illustrates the potential benefit that may be afforded by therapeutically inhibiting the endosomal TLRs in RA.

Figure 1

Mianserin inhibits Toll-like receptor (TLR) 3, 7, and 9 signaling in murine macrophages. Murine bone marrow-derived macrophages were pre-incubated for 30 minutes with mianserin and then stimulated with (a) 100 ng/mL lipopolysaccharide (LPS), 1 μg/mL resiquimod (R-848), and 500 μM ODN M326 for 6 hours before measuring tumor necrosis factor (TNF) in the supernantants or (b) 20 μg/mL polyinosinic:polycytidylic acid (poly I:C) for 24 hours before measuring RANTES production in the supernantants. Data are shown as a percentage of the TLR ligand-only response at 30 μg/mL mianserin and as a representative graph showing the effect of a range of concentrations of mianserin. (c) Cell viability was measured by 3-[4,5 dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide (MTT) assay. **P < 0.01, ***P < 0.001. RANTES, Regulated upon Activation, Normal T-cell Expressed, and Secreted; unstim, unstimulated.

Figure 2

Mianserin inhibits disease progression, paw swelling, and interleukin-17 (IL-17) production in the collagen-induced arthritis (CIA) model of arthritis. DBA/1 CIA mice were therapeutically given an intraperitoneal injection of vehicle (phosphate-buffered saline,) or 10 mg/kg mianserin once a day for 7 days, starting on the day of disease onset. Mice were assessed for (a) clinical score (n = 20 mice per group) and (b) paw swelling on a daily basis (n = 15 to 17 mice per group). (c, d) Arthritis was scored histologically as described in Materials and methods for the vehicle control group (n = 18) and the mianserin-treated group (n = 17). (e) Mice were bled on day 7, and serum levels of IgG₁ and IgG_2a anti-collagen antibodies were measured (n = 20). Frames (a-e) show data pooled from three separate experiments. (f) On day 10 after disease onset, cells from draining inguinal lymph nodes were cultured and stimulated with bovine type II collagen (BCII) (50 mg/mL) or anti-CD3 monoclonal antibody (100 ng/mL) for 48 hours. Supernatants were analyzed for the production of interferon-gamma (IFNγ) and IL-17 by enzyme-linked immunosorbent assay. Each point represents an individual animal (n = 8). *P < 0.05, **P < 0.01, ***P < 0.001. unstim, unstimulated.

Figure 3

Suppression of arthritis in TLR7-deficient mice. TLR7^-/- (n = 12) and control C57Bl/6 (n = 14) mice were immunized with chicken collagen in complete Freund's adjuvant. Arthritis was assessed by (a) clinical score and (b) paw swelling on a daily basis from the first day of disease onset. Data are pooled from three independent experiments. (c) The number of paws affected and the disease incidence were recorded for each group from three separate experiments. (d) The first affected joint was collected after 10 days of disease onset and was fixed and stained with hematoxylin and eosin. (e) Arthritis was scored histologically for TLR7^-/- (n = 9) and control C57Bl/6 (n = 10) mice as described in Materials and methods and pooled from two independent experiments. *P < 0.05, **P < 0.01. TLR, Toll-like receptor; WT, wild-type.

Figure 4

Arthritis in TLR7^-/- mice is associated with decreased IL-17 levels and increased T_reg cells. (a) Mice from Figure 3 were bled on day 10, and serum levels of IgG₁ and IgG_2a/c anti-collagen antibodies were measured by enzyme-linked immunosorbent assay as described in Materials and methods. (b, c) Draining lymph node cells (DLNCs) were isolated 10 days after disease onset and stimulated with 100 ng/mL anti-CD3 monoclonal antibody or 50 μg/mL chicken collagen, and supernatants were collected for the measurement of IL-17 (b) and interferon-gamma (IFNγ) (c). (d) DLNCs or cells liberated from the affected joints (e) were stained with conjugated antibodies to CD4 and foxp3 to determine the percentage of T_reg cells. *P < 0.05, **P < 0.01. IL-17, interleukin-17; LN, lymph node; No stim, no stimulation; N.S., not significant; TLR, Toll-like receptor; T_reg, T regulatory; WT, wild-type.

Figure 5

TLR7^-/- mice have decreased cytokine expression in the paw tissue. RNA was isolated from whole affected paws of control C57Bl/6 wild-type (WT) (n = 4 or 5) or TLR7^-/- (n = 3 or 4) mice. Real-time polymerase chain reaction was performed to measure levels of tumor necrosis factor-alpha (TNFα), interleukin-1-beta (IL-1β), IL-6, IL-17, and interferon-gamma (IFNγ) using ABI primers. *P < 0.05. TLR, Toll-like receptor.