Th17 and Th1 T-cell responses in giant cell arteritis

Abstract

Background: In giant cell arteritis (GCA), vasculitic damage of the aorta and its branches is combined with a syndrome of intense systemic inflammation. Therapeutically, glucocorticoids remain the gold standard because they promptly and effectively suppress acute manifestations; however, they fail to eradicate vessel wall infiltrates. The effects of glucocorticoids on the systemic and vascular components of GCA are not understood.

Methods and results: The immunoprofile of untreated and glucocorticoid-treated GCA was examined in peripheral blood and temporal artery biopsies with protein quantification assays, flow cytometry, quantitative real-time polymerase chain reaction, and immunohistochemistry. Plasma interferon-gamma and interleukin (IL)-17 and frequencies of interferon-gamma-producing and IL-17-producing T cells were markedly elevated before therapy. Glucocorticoid treatment suppressed the Th17 but not the Th1 arm in the blood and the vascular lesions. Analysis of monocytes/macrophages in the circulation and in temporal arteries revealed glucocorticoid-mediated suppression of Th17-promoting cytokines (IL-1beta, IL-6, and IL-23) but sparing of Th1-promoting cytokines (IL-12). In human artery-severe combined immunodeficiency mouse chimeras, in which patient-derived T cells cause inflammation of engrafted human temporal arteries, glucocorticoids were similarly selective in inhibiting Th17 cells and leaving Th1 cells unaffected.

Conclusions: Two pathogenic pathways mediated by Th17 and Th1 cells contribute to the systemic and vascular manifestations of GCA. IL-17-producing Th17 cells are sensitive to glucocorticoid-mediated suppression, but interferon-gamma-producing Th1 responses persist in treated patients. Targeting steroid-resistant Th1 responses will be necessary to resolve chronic smoldering vasculitis. Monitoring Th17 and Th1 frequencies can aid in assessing disease activity in GCA.

Figure 1. Circulating Th1 and Th17 cells are markedly expanded in GCA and differentially suppressed by Glucocorticoids

PBMC were isolated from untreated patients (No Tx, n=26) and GC-treated patients (Tx, n=34) or age-matched healthy controls (Con, n=34). Cells were stimulated with PMA/ionomycin in the presence of brefeldin A for 4 hours, stained with PE anti-CD3, PerCP anti-CD4, FITC anti-IFN-γ, and APC anti-IL-17 antibodies. Alternatively, PBMC were stained with FITC anti-CD3, PerCP anti-CD4, PE anti-Foxp3 antibodies, and analyzed by flow cytometry. Frequencies of IL-17⁺ Th17 (A) and IFN-γ⁺ Th1 cells (B) amongst CD4 T cells, and Foxp3⁺ CD4 Treg (C) amongst CD3 T cells are presented. Representative cytometric dot plots of Th17 and Th1 cells from a healthy individual (D), a GCA patient before therapy (E), and a GCA patient on treatment (F) are shown. Plasma concentrations of IL-17 (G) and IFN-γ (H) protein were measured in six GCA patients before and after initiation of GC therapy and six age-matched healthy controls with ELISArray kits. Results are presented as absorbance value at 450nm. ns: not significant.

Figure 2. Steroid sensitivity of tissue IL-17 and steroid resistance of tissue IFN-γ in temporal artery lesions

Total RNA was isolated from temporal arteries harvested before initiation of GC therapy (No Tx) (n=8) and from a second-side biopsy 3-9 months after initiation of therapy (Tx). Arteries without GCA served as controls (Con) (n=8). Transcripts of IL-17 (A), IFN-γ (B), and Foxp3 (C) mRNA in the tissues were quantified by real-time PCR. Paraffin-embedded tissue sections of temporal arteries were stained with anti-CD3 (E) or anti-IL-17 (F-H) antibodies; (D) IL-17⁺ cells in arteries harvested before (G) and on treatment (H) were quantified in 10 randomly selected high power fields. (I) IFN-γ staining showed IFN-γ⁺ cells in the inner adventitia of a temporal artery on treatment. The images presented are representative of six biopsy samples. (J) Purified rabbit IgG served as isotype control antibody. Magnification 200× in (E), (F) and (J); 400× in (H) and (I); 600× in (G).

Figure 3. Glucocorticoids suppress Th17-promoting cytokines and spare Th1-promoting cytokines in the circulation

The proinflammatory cytokines IL-1β (A), IL-6 (B) and IL-12 (C) were measured in plasma samples of GCA patients before (No Tx) and after initiation of GC therapy (Tx) (n=6), and age-matched controls (Con) (n=6) by ELISArrays. Data are presented as absorbance values at 450nm. (D-H) CD14⁺ monocytes were purified from PBMC of GCA patients prior to steroid therapy (n=8) and after steroid therapy (n=8), or GCA negative donors (n=8). Transcripts of IL-1β (D), IL-6 (E), IL-23p19 (F), IL-12p40 (G) and IL-12p35 (H) in cell extracts were quantified by qRT-PCR.

Figure 4. Effects of Glucocorticoids on the expression of Th17-promoting and Th1-promoting cytokines in inflamed temporal arteries

RNA was extracted from temporal artery biopsies collected as described in Figure 2 prior to steroid therapy (first side) (No Tx) and after steroid therapy (second side) (Tx). Arteries without GCA served as controls (Con). Transcripts of IL-1β (A), IL-6 (B), IL-23p19 (C), IL-12p40 (D) and IL-12p35 (E) were quantified by qRT-PCR.

Figure 5. Dexamethasone prevents Th17- but not Th1-mediated vessel wall inflammation in human artery-SCID chimeras

Normal human arteries were engrafted into SCID mice. One week later mice were injected with LPS or saline as control (Con). 24 hours later, T-cell lines established from the blood of GCA patients were adoptively transferred (10⁷ cells/mouse). Chimeras were treated with either dexamethasone (4 mg/kg) (Tx) or saline (No Tx), and temporal arteries were explanted. T-cell lines from the GCA patients had similar proportions of Th17, Th1, and Th17/Th1 cells; a representative cytometric dot plot is shown (A). Tissue extracts from explanted arteries were analyzed for IL-17 (B) and IFN-γ (C) by qRT-PCR. Data are presented as mean ± SEM from three independent experiments. Tissue sections from explanted arteries were stained with anti-IL-17 antibodies (D-F). IL-17⁺ T cells (dark brown) were quantified in a minimum of 12 high-powered fields for each arterial cross-section (G). Transcript levels of IL-6 (H) and MMP-9 (I) in the implants were measured by qRT-PCR as well. Magnification 600× in (D), (E) and (F).