Systemic isotretinoin therapy normalizes exaggerated TLR-2-mediated innate immune responses in acne patients

Abstract

Retinoids are used in the treatment of inflammatory skin diseases and malignancies, but studies characterizing the in vivo actions of these drugs in humans are lacking. Isotretinoin is a pro-drug for all-trans retinoic acid, which can induce long-term remissions of acne; however, its complete mechanism of action is unknown. We hypothesized that isotretinoin induces remission of acne by normalizing the innate immune response to the commensal bacterium Propionibacterium acnes. Compared with normal subjects, peripheral blood monocytes from acne patients expressed significantly higher levels of Toll-like receptor 2 (TLR-2) and exhibited significantly greater induction of TLR-2 expression following P. acnes stimulation. Treatment of patients with isotretinoin significantly decreased monocyte TLR-2 expression and subsequent inflammatory cytokine response to P. acnes after 1 week of therapy. This effect was sustained 6 months following cessation of therapy, indicating that TLR-2 modulation may be involved in the durable therapeutic response to isotretinoin. This study demonstrates that isotretinoin exerts immunomodulatory effects in patients and sheds light on a potential mechanism for its long-term effects on acne. The modulation of TLR-2 expression on monocytes has important implications in other inflammatory disorders characterized by TLR-2 dysregulation.

Figure 1. Isotretinoin therapy down-regulates surface expression of TLR-2 on patients’ monocytes in vivo

Peripheral blood was taken from normal volunteers (Vols; n = 23) and acne patients at baseline and 1, 4, 8, and 20 weeks of isotretinoin therapy and six months after cessation of therapy (n = 25, 19, 19, 17, 10, and 8, respectively). Isolated monocytes were treated with P. acnes sonicate or no antigen (unstimulated) and analyzed by flow cytometry. A) Dot plots of monocytes from one representative patient are shown. B) Displayed is the mean MFI ± SEM of TLR2 expression for patients at baseline versus volunteers (top) as well as patients’ TLR-2 levels over the course of isotretinoin therapy (bottom). *P<0.05, **P<0.01, and ***P<0.001.

Figure 2. Acne patients’ monocytes express lower levels of TLR-4 compared to normal volunteers

Mean MFI ± SEM of TLR-4 is shown for patients at baseline versus volunteers (left) as well as patients’ TLR-4 levels over the course of isotretinoin therapy (right). *P<0.05, ** P<0.01, and ***P<0.001.

Figure 3. Isotretinoin therapy decreases inflammatory cytokine production by acne patients’ monocytes in response to P. acnes

Media supernatants from treated monocytes were analyzed for inflammatory cytokine concentrations using bead arrays. Basal cytokine concentrations in the serum (at 10% final concentration) were subtracted from levels found in the media supernatants to calculate the amounts produced de novo by the monocytes. Concentrations of cytokines ± SEM are displayed for unstimulated and P. acnes-treated monocytes from normal volunteers (Vols) (n = 22) and from patients at baseline (n = 25), 1 week (n = 19), 4 weeks (n = 19), 8 weeks (n = 17), and 20 weeks (n = 10) of isotretinoin therapy as well as six months after the cessation of therapy (n = 8). *P<0.05, **P<0.01, and ***P<0.001 compared with patients’ baseline. +P<0.05, ++P<0.01, and +++P<0.001 compared with normal volunteers.

Figure 4. Lymphocyte proliferative response to P.acnes is blunted in acne patients and is unaffected by isotretinoin

Lymphocytes from normal volunteers (Vols) (n = 11) and from acne patients at baseline (n = 14), 1 week (n = 13), 4 weeks (n = 11), 8 weeks (n = 10), and 20 weeks (n = 5) of isotretinoin therapy were treated with vehicle (no antigen), 1 μM P. acnes sonicate, or 1 μg/mL of each anti-CD3 and anti-CD28 antibodies for five days. For the last four hours of incubation, cells were pulsed with 1 μCi of ³H-thymidine, and samples were then analyzed by scintillation counting. Mean counts per minute (CPM) ± SEM are shown. +P<0.05 and ++P<0.01 compared with normal volunteers.

Figure 5. Acne patients have higher serum levels of IL-10 compared to normal subjects

Concentrations of cytokines in the serum of normal volunteers (Vols) (n = 13) and acne patients at baseline (n = 26), 1 week (n = 20), 4 weeks (n = 18), 8 weeks (n = 16), and 20 weeks (n = 9) of isotretinoin therapy as well as six months after the cessation of therapy (n = 8) were analyzed using bead arrays. Mean concentrations ± SEM are shown. +P<0.05 and ++P<0.01 compared with normal volunteers.