A role for fungal {beta}-glucans and their receptor Dectin-1 in the induction of autoimmune arthritis in genetically susceptible mice

Abstract

A combination of genetic and environmental factors can cause autoimmune disease in animals. SKG mice, which are genetically prone to develop autoimmune arthritis, fail to develop the disease under a microbially clean condition, despite active thymic production of arthritogenic autoimmune T cells and their persistence in the periphery. However, in the clean environment, a single intraperitoneal injection of zymosan, a crude fungal beta-glucan, or purified beta-glucans such as curdlan and laminarin can trigger severe chronic arthritis in SKG mice, but only transient arthritis in normal mice. Blockade of Dectin-1, a major beta-glucan receptor, can prevent SKG arthritis triggered by beta-glucans, which strongly activate dendritic cells in vitro in a Dectin-1-dependent but Toll-like receptor-independent manner. Furthermore, antibiotic treatment against fungi can prevent SKG arthritis in an arthritis-prone microbial environment. Multiple injections of polyinosinic-polycytidylic acid double-stranded RNA also elicit mild arthritis in SKG mice. Thus, specific microbes, including fungi and viruses, may evoke autoimmune arthritis such as rheumatoid arthritis by stimulating innate immunity in individuals who harbor potentially arthritogenic autoimmune T cells as a result of genetic anomalies or variations.

Figure 1.

Chemical or biological compounds trigger arthritis in SKG mice. (A) Arthritis scores of 6-mo-old SKG mice maintained in the indicated environment. SPF-born nonarthritic SKG mice were foster nursed to non-SPF BALB/c mice at 7 d of age or transferred to the non-SPF environment at 8 wk of age. (B) 5 × 10⁷ splenocytes, 10⁸ thymocytes, or 5 × 10⁶ BM cells prepared from each nonarthritic SPF SKG mouse were adoptively transferred to individual 6-wk-old nude or SCID mice. BM cells were T cell depleted by treatment with anti–Thy-1, anti-CD4, and anti-CD8 antibodies and rabbit complement before transfer. Arthritis scores 6 mo after transfer are shown. (C) Arthritis scores of SPF SKG mice 5 mo after administration of indicated compounds. SKG mice between 8 and 12 wk of age maintained in the SPF environment were treated as follows: i.p. injection of 2 mg zymosan, 1 μg LPS, 200 μg ConA, 500 μl pristane, 1 mg CP, or 150 μl PBS; i.v. administered with 400 ng PTX on days 0 and 2; i.p. 150 μg poly(I:C) or 150 μl PBS three times per week for 17 wk, 10 nmol CpG or non-CpG once per week for 4 wk; and i.v. with 0.3 mg agonistic anti-CD40 or control rat IgG on days 0, 7, and 14.

Figure 2.

Silent fungal infections under non-SPF trigger arthritis in SKG mice. (A) PCRs of fungal 18SrDNA or mouse Rag 1 gene from esophagus (Es), lung (Lg), liver (Lv), or foot including ankle joint (Ft) of BALB/c or SKG mice in non-SPF. (B) Lungs of BALB/c or SKG mice in non-SPF or SPF were assessed for fungi by real-time quantitative PCR. (C–F) Methenamine silver (C and E) or hematoxylin and eosin (D and F) stainings of lungs from SKG mice (C and D) or BALB/c (E and F) of non-SPF. Arrows indicate argyrophilic fungal cysts. (G) Male SKG mice of 10 wk of age in the non-SPF facility were treated with four cycles of four daily i.p. injections of 75 μg amphotericin B (arrows) at 10-d intervals. (H) SKG mice in the SPF facility received the same protocol of antifungal treatment 10 d after i.p. administration of 2 mg zymosan (open inverted triangle). (I) Concentration of serum β-glucan in non-SPF or SPF 12-wk-old SKG mice.

Figure 3.

Purified β-glucans trigger arthritis in SKG mice. (A and B) Arthritis score and incidence of SKG mice or BALB/c in the SPF environment after a single i.p. injection of indicated amounts of (A) curdlan or (B) laminarin. Vertical bars represent the means ± SD. Arthritis scores are significantly different (P < 0.05) between SKG and BALB/c mice treated with 3 mg curdlan during 2–17 wk; between SKG mice treated with 3 and 10 mg curdlan during 4–7 wk; between SKG and BALB/c mice treated with 30 mg laminarin during 3–17 wk; and between SKG mice treated with 3 and 30 mg laminarin during 4–17 wk. (C–H) Hematoxylin and eosin staining of ankle joints. Arthritic joints of SPF SKG mice 20 wk after treatment with 2 mg zymosan (C), 30 mg laminarin (D), or 3 mg curdlan (E). (F and G) An arthritic (F, 8 wk after treatment) or cured (G, 20 wk) ankle joint from BALB/c mice with transient arthritis triggered by 3 mg curdlan. (H) An ankle joint of 40-wk-old nonarthritic SKG mice in SPF. (I) Arthritis scores of BALB/c or SKG donor mice 9 wk after administration of 2 mg zymosan. Nude mice were transferred with 5 × 10⁷ splenocytes from arthritic BALB/c or SKG mice and assessed for arthritis 12 wk after transfer.

Figure 4.

Purified β-glucans activate BM-DCs. (A) The expression of CD86 on CD11c⁺ BM-DCs cultured with laminarin, curdlan, or zymosan at indicated concentrations. (B) The expression of indicated surface markers on CD11c⁺ BM-DCs cultured with 1 μg/ml LPS, 10 mg/ml laminarin, 100 μg/ml curdlan, or 100 μg/ml zymosan. Percentages of positive cells are also shown. (C) The expression of CD86 on TLR4-, TLR2-, or MyD88-deficient or wild-type CD11c⁺ BM-DCs cultured with 10 mg/ml laminarin (LAM), 100 μg/ml curdlan (CDL), 10 μg/ml LPS, or 10 μg/ml Pam₃CSK₄. (D) BM-DCs were incubated with or without 100 μg/ml of laminarin for 1 h before the culturing with 10 μg/ml of curdlan or zymosan (ZYM), and assessed for CD86 expression. (E) The expression of Dectin-1 on unstimulated DCs or DCs activated by 1 μg/ml LPS, 10 mg/ml laminarin, 100 μg/ml curdlan, or 100 μg/ml zymosan. (F) BM-DCs were incubated with 10 μg/ml of 2A11 anti–Dectin-1 mAb or isotype-control IgG for 1 h before culture with indicated β-glucans or cultured with β-glucans alone. The expression of CD86 on CD11c⁺ BM-DCs 24 h after the stimulation is shown. Shaded histograms indicate unstimulated DCs in A, C, D, and F or staining controls in B and E. Ordinate indicates cell number. A representative result of three independent experiments is shown in A–F.

Figure 5.

Production of TNF-α by β-glucan–stimulated DCs. (A) BM-DCs were incubated with 10 mg/ml laminarin, 100 μg/ml curdlan, 100 μg/ml zymosan, or 1 μg/ml LPS from day 5 for 24 h and the amount of TNF-α was assessed by ELISA. (B) TNF-α production by BM-DCs that were incubated with 10 μg/ml of 2A11 mAb or control IgG2b for 1 h before culture with 100 μg/ml curdlan or 100 μg/ml zymosan. (C) TNF-α production by MyD88-deficient or wild-type BM-DCs that were cultured with 100 μg/ml curdlan, 100 μg/ml zymosan, or 10 μg/ml LPS. Vertical bars represent the means ± SD of triplicates.

Figure 6.

Dectin-1 as a major receptor for β-glucans in triggering SKG arthritis by β-glucans. (A) 0.5 mg 2A11 anti–Dectin-1 mAb or rat IgG were administered i.v. into SPF SKG mice two or three times, 2 wk apart (closed triangle), before or after i.p. injection of indicated amounts of laminarin, curdlan, or zymosan (open triangle). Vertical bars represent the means ± SD. Asterisks indicate significant differences (P < 0.05) at the indicated time points. (B) SKG mice were treated i.v. with 0.5 mg 2A11 or rat IgG, and the expression of CD11b and surface-bound rat IgG or Dectin-1 on peripheral blood was analyzed 1 wk later. (C) The synovium from arthritic SKG mice treated with 30 mg laminarin was digested for 1 h; resulting single cell suspensions were analyzed for the expression of indicated surface markers. Shaded histograms indicate staining controls. A representative result of three independent experiments is shown in B and C.