Slit2 Modulates the Inflammatory Phenotype of Orbit-Infiltrating Fibrocytes in Graves' Disease

Abstract

Human CD34⁺ fibrocytes, circulating monocyte lineage progenitor cells, have recently been implicated in thyroid-associated ophthalmopathy (TAO), the ocular manifestation of Graves' disease (GD). Fibrocytes express constitutive MHC class II (MHC-2) and, surprisingly, thyroglobulin (Tg) and functional thyrotropin (TSH) receptor (TSHR). Underlying expression of these thyroid proteins is the autoimmune regulator protein (AIRE). Fibrocytes respond robustly to TSH and thyroid-stimulating Igs by generating extremely high levels of inflammatory cytokines, such as IL-6. In TAO, they appear to infiltrate the orbit, where they transition to CD34⁺ orbital fibroblasts (OF). There, they coexist with CD34^- OF as a mixed fibroblast population (GD-OF). In contrast to fibrocytes, GD-OF express vanishingly low levels of MHC-2, Tg, TSHR, and AIRE. Further, the amplitude of IL-6 induction by TSH in GD-OF is substantially lower. The molecular basis for this divergence between fibrocytes and CD34⁺ OF remains uncertain. In this article, we report that Slit2, an axon guidance glycoprotein, is constitutively expressed by the CD34^- OF subset of GD-OF. Culture conditioned medium (CM) generated by incubating with GD-OF and CD34^- OF substantially reduces levels of MHC-2, Tg, TSHR, and AIRE in fibrocytes. Expression can be restored by specifically depleting CM of Slit2. The effects of CD34^- OF CM are mimicked by recombinant human Slit2. TSH induces Slit2 levels in GD-OF by enhancing both Slit2 gene transcription and mRNA stability. These findings suggest that Slit2 represents a TSH-inducible factor within the TAO orbit that can modulate the inflammatory phenotype of CD34⁺ OF and therefore may determine the activity and severity of the disease.

Figure 1

Higher levels of (A) autoimmune regulator protein (AIRE), (B) MHC Class II (MHC-2) (C) thyroglobulin (Tg), (D) thyrotropin receptor (TSHR) in fibrocytes than orbital fibroblasts from patients with Graves’ disease (GD-OF). Confluent cell layers were harvested and (left panels) RNA isolated, reverse-transcribed, and subjected to real-time PCR and (right panels) stained with the labeled antibodies for flow cytometry as described in the Methods section. mRNA data were normalized to their respective GAPDH levels and are expressed as the mean ± SD of triplicate determinations. MFI data: AIRE, fibrocytes, 813.35 ± 30.18 vs GD-OF, 223.35 ± 31.2 (4-fold); MHC-2, fibrocytes, 490.56 ± 13.35 vs GD-OF, 18.73 ± 17.82 (11,797-fold); Tg, fibrocytes, 965.97 ± 21.85 vs GD-OF, 14.35 ± 15.84 67-fold); TSHR, fibrocytes, 193.58 ± 24.2 vs GD-OF, 26.92 ± 18.82 (7-fold). Data from flow analysis denote fluorescence intensity compared with isotype controls. *** P<0.001. A total of three experiments were performed.

Figure 2

(A) Expression pattern of AIRE, MHC-2, Tg, and TSHR in parental (mixed) GD-OF and pure CD34⁺ OF and CD34⁻ OF subsets. Parental GD-OF strains were subjected to sham cytometric sorting or sorted into CD34⁺ OF and CD34⁻ OF subsets and cultured for 3 d. (B) Conditioned media were generated by incubating GD-OF , CD34⁺ OF , and CD34⁻ OF in DMEM for 48 h. These media were then used to cover fibrocytes for 3 d. Monolayers were harvested, RNA extracted, reverse-transcribed and subjected to real-time PCR. Data are expressed as the mean ± SD of 3 independent replicates. Values were normalized to their respective GAPDH levels and are expressed as mean ± SD of triplicate determinations. *** p<0.001; ** p<0.01. Experiments were performed three times.

Figure 3

(A) Depleting medium conditioned by GD-OF of Slit2 restores fibrocyte gene expression. (A) Conditioned media (CM) from GD-OF and CD34⁻ OF were incubated with uncoated beads or beads coated with anti-Slit2 (100 µg), anti-HSP47 (100 µg), or isotype IgG (100 µg) as described in Methods. Fibrocyte monolayers were incubated with these media for 5 d. (B) Knocking down Slit2 with specific siRNA enhances gene expression in GD-OF. GD-OF were transfected with either scrambled (control) siRNA (3µg) or Slit2-specific siRNA (3µg) and incubated 3 d. RNA was extracted, reversed transcribed, and cDNAs subjected to real-time PCR for the targets indicated. Values were normalized to their respective GAPDH levels and expressed as mean ± SD of triplicate determinations. *** p<0.001, ** p<0.01; * p<0.05. Experiments were performed three times.

Figure 4

Slit2 is preferentially expressed in GD-OF compared to fibrocytes. It is a TSH-inducible protein as a consequence of increased gene transcription and mRNA stability. (A). Three strains of confluent fibrocytes and GD-OF remained untreated or were treated with bTSH (5mIU/ml) for 6 h, RNA extracted, reversed transcribed, and cDNA subjected to real-time PCR for Slit2. (B) Fibrocytes and GD-OF were treated with bTSH (5mIU/ml) for the graded intervals indicated along the abscissa. Medium was collected and subjected to a specific Slit2 ELISA as described in Methods. (C) A 2970 bp fragment of the human Slit2 gene promoter was cloned as described in Methods and transfected into fibrocytes and GD-OF. Cell layers were assayed for luciferase activity. (D) Confluent GD-OF were untreated or treated with bTSH for 2 h and subjected to a ChiP transcription assay as detailed in Methods. (E) GD-OF were pre-treated with bTSH for 2 h. All cultures were treated at time “0” with DRB (20 µg/mL) without or in the continued presence of bTSH for the intervals indicated along the abscissa. RNA was extracted, reverse transcribed, and subjected to real-time quantitative PCR for Slit2. Values were normalized to their respective GAPDH levels. Slit2 ELISA, luciferase assay, and ChiP results were normalized to the respective protein levels. Data were expressed as the mean ± SD of triplicate determinations. *** p<0.001,** p<0.01. Experiments were performed three times.

Figure 5

rhSlit2 attenuates expression of AIRE, MHC-2, Tg and TSHR in fibrocytes. (A) Cultured PBMCs were treated with rhSlit2 (50ng/mL) for 7–9 d, fibrocyte monolayers were harvested, stained with the antibodies indicated and subjected to flow cytometric analysis. (B) Fibrocytes were untreated or treated with rhSlit2 and then some were treated with bTSH for 6 h, monolayers harvested, RNA was extracted, reverse-transcribed, and subjected to real-time PCR for the targets indicated. Values were normalized to their respective GAPDH levels and β-actin levels and are expressed as the mean ± SD of triplicate determinations. ** p<0.01. Experiments were performed three times.

Figure 6

Theoretical model for the modulatory role of Slit2 on intra-orbital pathogenesis of TAO. When CD34⁻ OF-derived Slit2 predominates, the inflammatory phenotype of CD34⁺ OF is down regulated and the patient with GD either fails to manifest TAO or the disease is mild (Left panel). When CD34⁺ OF dominates the fibroblast population and the impact of Slit2 is inadequate, TAO is severe (Right panel).