Propionibacterium acnes Induces an IL-17 Response in Acne Vulgaris that Is Regulated by Vitamin A and Vitamin D

Abstract

Acne vulgaris is the most common skin disorder affecting millions of people worldwide and inflammation resulting from the immune response targeting Propionibacterium acnes has a significant role in its pathogenesis. In this study, we have demonstrated that P. acnes is a potent inducer of T helper 17 (Th17) and Th1, but not Th2 responses in human peripheral blood mononuclear cells (PBMCs). P. acnes stimulated expression of key Th17-related genes, including IL-17A, RORα, RORc, IL-17RA, and IL-17RC, and triggered IL-17 secretion from CD4(+), but not from CD8(+) T cells. Supernatants from P. acnes-stimulated PBMCs were sufficient to promote the differentiation of naive CD4(+)CD45RA T cells into Th17 cells. Furthermore, we found that the combination of IL-1β, IL-6, and transforming growth factor-β-neutralizing antibodies completely inhibited P. acnes-induced IL-17 production. Importantly, we showed that IL-17-expressing cells were present in skin biopsies from acne patients but not from normal donors. Finally, vitamin A (all-trans retinoic acid) and vitamin D (1,25-dihydroxyvitamin D3) inhibited P. acnes-induced Th17 differentiation. Together, our data demonstrate that IL-17 is induced by P. acnes and expressed in acne lesions and that both vitamin A and D could be effective tools to modulate Th17-mediated diseases such as acne.

Fig.1. P. acnes lab strain and clinical isolates stimulate production of IL-17 in human PBMCs

a) PBMCs were cultured (2–5 × 10⁶/ml) in the presence of P. acnes sonicate (2 µg/ml), live P. acnes (0.5 muliplicity of infection), M. tuberculosis (5 µg/ml), M. leprae (5 µg/ml), and Staphylococcus enterotoxin B (SEB 2 µg/ml) for seven days. b) PBMCs (2–5 × 10⁶/ml) were cultured either in the presence or absence of seven P. acnes clinical isolates (C1–C7). Levels of IL-17 accumulated in culture supernatants were measured using ELISA. Experiments were performed at least five times using PBMCs from five different donors with similar results. (** p ≤ 0.05, ***p ≤ 0.001)

Fig.2. P. acnes stimulate production of IL-17A and IFN-γbut not IL-4 in PBMCs

PBMCs were cultured (2–5 × 10⁶/ml) in the presence of P. acnes sonicate (2 µg/ml) or P. acnes clinical isolates for 7 days. a) Levels of IL-17, IL-4 and IFN-γaccumulated in culture supernatants were measured using ELISA. Experiments were performed at least five times using PBMCs from five different donors with similar results. b) PBMCs (2–5 × 10⁶/ml) were cultured either in the presence or absence of seven P. acnes clinical isolates (C1–C7). Levels of IL-17, IL-4, and IFN-γaccumulated in culture supernatants were measured using ELISA. Experiments were performed at least three times using PBMCs from three different donors with similar results. The overall group effect was statistically significant (p<0.001). c) Flow cytometry of PBMCs stimulated with P. acnes sonicate for 7 days. Intracellular cytokine staining for IFN-γ, IL-4 and IL-17 was performed on day 7. Plots are gated on CD4⁺ T cells. Each panel is representative of four independent experiments. d) Flow cytometry of peripheral blood stimulated with P. acnes sonicates for seven days. Intracellular cytokine staining for IFN-γand IL-17 was performed on day seven. Plots are gated on CD4⁺ and CD8⁺ T cells. Each panel is representative of three independent experiments. Data represent mean ± SD (** p ≤ 0.05, ***p ≤ 0.001)

Fig.3. Induction of IL-17, IL-17RA, IL-17RC, RORc and RORa mRNAs expression in PBMCs stimulated with P. acnes

PBMCs were cultured (2–5 × 10⁶/ml) with P. acnes sonicate (2 µg/ml). Real time PCR of IL-17, IL-17RA, IL-17RC, RORc and RORa mRNA expression was analyzed 24 hours following P. acnes stimulation. Gene expression was normalized to the housekeeping genes GAPDH and quantified by the comparative method 2^−ΔΔCT. Data are representative of four independent experiments. Data represent mean ± SD (***p ≤ 0.001)

Fig.4. Supernatants from PBMCs treated with P. acnes differentiate naïve CD4⁺T cells to IL-17 producing T cells

a) PBMCs (2–5 × 10⁶/ml) were stimulated overnight with P. acnes sonicate (2 µg/ml). Culture supernatants were then collected and used to stimulate naive CD4⁺CD45RA T cells for seven days in 96 well plates with plate bound anti-CD3 and soluble CD28. Cells were harvested and intracellular cytokine staining for IL-17 was performed. Each panel is representative of three independent experiments. b) PBMCs (2–5 × 10⁶/ml) were cultured with IL-17, IL-1β, IL-6, TGF-β, IL-23p19, IL-4 and IFN-γneutralizing antibodies for one hour followed by seven days of stimulation with P. acnes. IL-17 production was then measured using ELISA. Data are representative of four independent experiments. Data represent mean ± SD (** p ≤ 0.05, ***p ≤ 0.001)

Fig.5. Immunohistochemistry of IL-17 in acne lesion

Frozen skin sections were obtained from patients with acne lesion (a) and (b) and nomal control skin (c). Immunohistochemical staining was carried out using anti-human IL-17 antibody (a) and (c) and an isotype control (b). IL-17 expressing lymphoid cells (brown) can be seen scattered around the inflammation surrounding the pilosebaceous follicle (a). Data is representative of three independent experiments from three different lesions. Scale bars represent 75 Zm and 30 Zm respectively.

Fig.6. Effects of 1,25D3, 25D3 and ATRA on Th17 differentiation

Human PBMCs were stimulated with P. acnes sonicate (2 µg/ml) in the presence of 1, 25D3 (10⁻⁷ M), 25D3 (10⁻⁷ M) and ATRA (10⁻⁷ M). IL-17 (a), RORa (c), RORc (d), IL-17RA (e) and IL-17RC (f) mRNA expression was analyzed 24 hours following P. acnes stimulation. Gene expression was normalized to the housekeeping genes GAPDH and quantified by the comparative method 2^−ΔΔCT. Each panel is representative of three independent donors. b) PBMCs were cultured (2–5 × 10⁶/ml) with 1,25D3, 25D3 and ATRA for one hour followed by seven days of stimulation with P. acnes (sonicate or live), IL-17 production was then measured using ELISA. Data are representative of four independent experiments. Data represent mean ± SD (***p ≤ 0.001)