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. 2008 Jan;118(1):205-16.
doi: 10.1172/JCI32639.

Stimulation of TLR2 and TLR4 differentially skews the balance of T cells in a mouse model of arthritis

Shahla Abdollahi-Roodsaz  1 , Leo A B Joosten, Marije I Koenders, Isabel Devesa, Mieke F Roelofs, Timothy R D J Radstake, Marleen Heuvelmans-Jacobs, Shizuo Akira, Martin J H Nicklin, Fátima Ribeiro-Dias, Wim B van den Berg
Affiliations

Stimulation of TLR2 and TLR4 differentially skews the balance of T cells in a mouse model of arthritis

Shahla Abdollahi-Roodsaz et al. J Clin Invest. 2008 Jan.

Abstract

TLRs may contribute to the progression of rheumatoid arthritis through recognition of microbial or host-derived ligands found in arthritic joints. Here, we show that TLR2 and TLR4, but not TLR9, are involved in the pathogenesis of autoimmune arthritis and play distinct roles in the regulation of T cells and cytokines. We investigated the involvement of TLR2, TLR4, and TLR9 in the progression of arthritis using IL-1 receptor antagonist-knockout (IL1rn-/-) mice, which spontaneously develop an autoimmune T cell-mediated arthritis. Spontaneous onset of arthritis was dependent on TLR activation by microbial flora, as germ-free mice did not develop arthritis. Clinical and histopathological evaluation of IL1rn-/-Tlr2-/- mice revealed more severe arthritis, characterized by reduced suppressive function of Tregs and substantially increased IFN-gamma production by T cells. IL1rn-/-Tlr4-/- mice were, in contrast, protected against severe arthritis and had markedly lower numbers of Th17 cells and a reduced capacity to produce IL-17. A lack of Tlr9 did not affect the progression of arthritis. While any therapeutic intervention targeting TLR2 still seems complicated, the strict position of TLR4 upstream of a number of pathogenic cytokines including IL-17 provides an interesting potential therapeutic target for rheumatoid arthritis.

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Figures

Figure 1
Figure 1. Effect of microbial status of IL1rn–/– mice on the development of arthritis and cytokine response to TCR and TLR stimulations.
The incidence (A) and severity (B) of arthritis are completely abolished in germ-free (GF) IL1rn–/– mice, and infection of GF mice with Lactobacillus (arrows) leads to the same disease expression as in non–germ-free (NGF) animals. Arthritis severity was scored on a scale from 0 to 2 for each paw; n ≥ 6 per group. (C and D) Splenic lymphocytes (2 × 105) were stimulated with plate-coated anti-CD3 (2 μg/ml), Pam3Cys (100 ng/ml), or LPS (200 ng/ml) for 72 hours. IL-1 (C) and IL-17 (D) were measured using Luminex multianalyte technology (see Methods). Data are expressed as mean ± SD and are representative of 2 independent experiments.
Figure 2
Figure 2. Effect of direct TLR2 and TLR4 stimulation on the development and pathogenesis of IL1rn–/– arthritis.
Treatment of 5-week-old IL1rn–/– mice with 1 i.p. injection of Pam3Cys (4 mg/kg body weight) or LPS (400 μg/kg body weight) increased the incidence (A) and the macroscopic severity score (B) of arthritis. (C) Histological analysis of the ankle joints at 13 weeks of age expressed as mean ± SEM on a scale from 0 to 3. All parameters were scored by 2 observers blinded to the experimental protocol; n = 9 mice per group. *P < 0.05 and **P < 0.01.
Figure 3
Figure 3. Higher severity and increased histopathology of IL1rn–/– arthritis caused by Tlr2 deficiency.
Percentage incidence (A) and severity score (B) of arthritis in IL1rn–/–Tlr2+/+ mice compared with IL1rn–/–Tlr2–/– littermates. Severity was scored macroscopically on a scale of 0–2 for each paw; n > 20 mice per group. (C) Histological assessment of the ankle joints at 15 weeks (mean ± SEM) on a scale between 0 and 3 for each parameter; n = 14 mice per group. (D) Representative histological images of ankle joints. Cell influx and chondrocyte death were scored on H&E-stained (top row), and cartilage and bone damage (arrows) were scored on safranin O–stained (bottom row) tissue sections. PG depletion is apparent from loss of red staining. Original magnification, ×50 for H&E and ×100 for safranin O staining. B, bone; C, cartilage; JS, joint space; S, synovium. *P < 0.05 and **P < 0.01.
Figure 4
Figure 4. Influence of Tlr2 deficiency on various Th cell subsets and the expression of related cytokines.
mRNA expression of T cell markers in inflamed synovium of the ankle joints (A) and spleens (B) of IL1rn–/–Tlr2–/– compared with IL1rn–/–Tlr2+/+ mice. Synovial biopsies were pooled from more than 5 joints of 15-week-old mice, and spleens were from 4 nonarthritic 6-week-old mice. mRNA expression was measured by quantitative PCR. Relative mRNA expression compared with the housekeeping gene GAPDH (2-dCt × 1,000) is shown on the y axis. IFN-γ (C) and IL-17 (D) production by spleen and lymph node cells upon 72 hours stimulation with anti-CD3 (0.5 μg/0.2 ml/well) and anti-CD28 (2 μg/ml), measured by Luminex; results are mean ± SEM from a representative experiment with n > 4 mice per group. Protein expression of Foxp3 (E) and CD25 (F) on CD4+-gated CD25+Foxp3+ cells from whole blood and spleen of 15-week-old mice after correction for isotype-matched IgG control; mean ± SEM of n = 3 from a representative experiment. (G and H) T cell suppression assay on splenic cells of IL1rn–/–Tlr2+/+ and IL1rn–/–Tlr2–/– mice. (G) CD4+CD25– Teffs (50,000 cells/well) were stimulated with 1 μg/ml anti-CD3, 2 μg/ml concanavalin A, or 60 IU/ml IL-6 (negative control) in the presence of 50,000/well irradiated CD4– cells as APCs. After 3 days, proliferation was measured by [3H]thymidine incorporation. (H) For suppression assays, cells were incubated with anti-CD3 and APCs in the presence of titrated numbers of Tregs (CD4+CD25+). Percent suppression was calculated relative to the cultures without Tregs. Measurements were performed in triplicate, and values shown are mean ± SEM of 4 mice per group. mRNA expression of TGF-β1 in noninflamed and inflamed ankle synovium (I) and spleen (J), measured by quantitative PCR. Relative mRNA expression compared with GAPDH is shown. *P < 0.05.
Figure 5
Figure 5. Lower severity and reduced histopathology of IL1rn–/– arthritis caused by Tlr4 deficiency.
Similar incidence (A) and reduced severity (B) of arthritis in IL1rn–/–Tlr4–/– as compared with IL1rn–/–Tlr4+/+ littermates during the first 15 weeks of age. Severity was scored on a scale of 0 to 2 for each paw; n > 20 mice per group. (C) Histological assessment of the ankle joints at week 15 of age. Data are mean ± SEM (scale 0–3) of 14 mice per group. mRNA expression of IL-23p19 (D) and IL-17A (E) in synovial biopsies of the ankle joints of 15-week-old mice selected according to the degree of inflammation. mRNA expression was measured by quantitative real-time PCR. Relative expression compared with GAPDH (2-dCt × 10,000) is shown. (F) Representative images of the ankle joints. Cell influx and chondrocyte death (arrows) were scored on H&E-stained sections (top row), and cartilage and bone damage (arrows) were scored on safranin O–stained tissue sections (bottom row). Original magnification, ×100 for H&E and ×200 for safranin O staining. *P < 0.05 and ***P < 0.001.
Figure 6
Figure 6. Decreased IL-17 in IL1rn–/–Tlr4–/– mice and IL-1–mediated effects of TLR4 activation on IL-23/IL-17 production.
(A–D and F) Spleens and lymph nodes from 5- to 6-week-old mice without arthritis were isolated and disrupted. CD3+ T lymphocytes were isolated using magnetic beads (MACS). (A and B) Th17 FACS analysis following stimulation with PMA (50 ng/ml), ionomycin (1 μg/ml), and brefeldin (1 μl/ml) for 5 hours ; n = 4. (C) IL-17 production by splenic and lymph node T cells upon stimulation with anti-CD3 (0.5 μg/0.2 ml/well) and anti-CD28 (2 μg/ml) for 72 hours; measured by Luminex; n ≥ 6. (D) TLR4 activation of bone marrow–derived DCs by 100 ng/ml LPS for 24 hours resulted in higher IL-23 production in IL1rn–/– cells compared with BALB/c WT. IL-23 was measured by ELISA; n = 10. (E) TLR4 (200 ng/ml LPS) plus anti-CD3 (1 μg/ml) activation of splenic T cells leads to higher IL-1–mediated IL-17 production in IL1rn–/– cells compared with WT cells, as measured by Luminex; n = 5. (F) Bone marrow–derived DCs from IL1rn–/–Tlr4–/– mice are not compromised in IL-23 production upon non-TLR4 stimulations. DCs were stimulated with 100 ng/ml LPS, 100 ng/ml Pam3Cys, or a cocktail of IL-1β (25 ng/ml), TNF-α (25 ng/ml), IL-6 (100 ng/ml), and PGE2 (1 μg/ml) for 24 hours; n = 4 per group. Data are mean ± SEM. NRS, normal rabbit serum. *P < 0.05, **P < 0.01, and ***P < 0.001.
Figure 7
Figure 7. TLR4-mediated stimulation of cytokine production by rheumatoid synovial fluid and in synovial biopsies of RA patients.
(A) HEK293-TLR4 cells were stimulated with PMA, IL-1β, TNF-α, and various TLR ligands as indicated in Methods. Mean IL-8 concentrations (Luminex assay) of triplicates are shown. (B) HEK293 and HEK293-TLR4 cells were stimulated with synovial fluid of RA patients in the presence of a TNF-α blocker (Enbrel; 100 ng/ml) or remained unstimulated (medium control). IL-8 was measured in culture supernatants after 24 hours. Stimulation index over medium control of each cell line is shown on the y axis; n = 7. Data are mean ± SEM. ***P < 0.001. (C) Synovial biopsies of RA patients were cultured ex vivo with or without TLR4 antagonist (10 μg/ml) for 24 hours. Experiments were performed in triplicate. The concentration of cytokines was measured using Luminex. Data are mean ± SEM from 7 RA patients.
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