Inhibition of TYK2 and JAK1 ameliorates imiquimod-induced psoriasis-like dermatitis by inhibiting IL-22 and the IL-23/IL-17 axis

Abstract

Psoriasis is a chronic autoimmune disease affecting the skin and characterized by aberrant keratinocyte proliferation and function. Immune cells infiltrate the skin and release proinflammatory cytokines that play important roles in psoriasis. The Th17 network, including IL-23 and IL-22, has recently emerged as a critical component in the pathogenesis of psoriasis. IL-22 and IL-23 signaling is dependent on the JAK family of protein tyrosine kinases, making JAK inhibition an appealing strategy for the treatment of psoriasis. In this study, we report the activity of SAR-20347, a small molecule inhibitor with specificity for JAK1 and tyrosine kinase 2 (TYK2) over other JAK family members. In cellular assays, SAR-20347 dose dependently (1 nM-10 μM) inhibited JAK1- and/or TYK2-dependent signaling from the IL-12/IL-23, IL-22, and IFN-α receptors. In vivo, TYK2 mutant mice or treatment of wild-type mice with SAR-20347 significantly reduced IL-12-induced IFN-γ production and IL-22-dependent serum amyloid A to similar extents, indicating that, in these models, SAR-20347 is probably acting through inhibition of TYK2. In an imiquimod-induced psoriasis model, the administration of SAR-20347 led to a striking decrease in disease pathology, including reduced activation of keratinocytes and proinflammatory cytokine levels compared with both TYK2 mutant mice and wild-type controls. Taken together, these data indicate that targeting both JAK1- and TYK2-mediated cytokine signaling is more effective than TYK2 inhibition alone in reducing psoriasis pathogenesis.

Figure 1. SAR-20347 inhibits TYK2- and JAK1-mediated IL-12 and IFN-α signaling

(A) Representative curve from the biochemical TR-FRET assay comparing TYK2, JAK1, JAK2, and JAK3 specificity. (B) PBMCs were incubated with the indicated concentration of SAR-20347 or DMSO and stimulated with 10 ng/mL human IL-12 for 24 hours. IFN-γ concentration in the supernatant was compared to control cells incubated with DMSO and IL-12. (C) HEK-BLUE IFN-α/β reporter cells were incubated with SAR-20347 and stimulated with 200 U IFN-α for 24 hours. SEAP levels were compared to control cells incubated with DMSO and IFN-α. X-axes indicate concentration of SAR-20347. Graphs represent mean ± standard deviation for three experiments.

Figure 2. SAR20347 alters in vitro development of mouse T helper subsets

A) Flow cytometric analysis of CD4+ cells cultured with 1 μM SAR-20347 or tofacitinib in the presence of Th1, Th2, Th17, or iTreg lineage-driving cytokines for 4 days. Cells were stimulated and intracellular staining for relevant cytokines was performed. Representative of 3-4 experiments. B) Graphs represent percent population under Th1, Th2, Th17, and iTreg skewing conditions. average ± standard deviation, * indicates t-test p < 0.05 compared to no inhibitor control. C) CD4+ T cell proliferation in anti-CD28/anti-TCR-coated plates after 4 day culture with inhibitor. Two-way ANOVA (concentration, treatment), * indicates p < 0.05 compared to 0 μM inhibitor control (DMSO).

Figure 3. TYK2 and JAK1 mediate IL-22 signaling in vitro and in vivo

(A) siRNA knockdown of JAKs in HT-29 cells. HT-29 cells were treated with TYK2, JAK1, or scrambled siRNA for 48 hours, and then incubated with 10 ng/mL human IL-22 for 15 minutes. pSTAT3 levels were assessed by flow cytometry and shown compared to cells treated with no siRNA and no IL-22. Gray- no siRNA, no IL-22, Red - scrambled siRNA + IL-22, Light Blue - JAK1 siRNA + IL-22, Green - TYK2 siRNA + IL-22, Dark Blue - JAK1 and TYK2 siRNA + IL-22. (B) Effect of SAR-20347 on IL-22 signaling. HT-29 cells were pretreated with SAR-20347 or DMSO for 20 minutes, and then incubated with 10 ng/mL human IL-22 for 15 minutes. pSTAT3 levels were assessed by flow cytometry and the change in mean fluorescence intensity compared to DMSO controls (100%) is plotted. Inset: representative histogram of data from flow cytometry. Gray - no IL-22 + vehicle, Red - IL-22 + vehicle, Green - 0.01 μM, Light Blue – 0.3 μM, Dark Blue – 1 μM SAR-20347. (C) SAA levels in TYK2 mutant mice following IL-22 treatment. TYK2 mutant (N=9) or wild type (WT; B10; N=9) mice were injected (i.p.) with 10 μg mouse IL-22, and serum SAA levels were assessed 6 hours post-injection. (D) SAA levels in mice treated with SAR2037. C57BL/6 mice were pretreated with vehicle (N=6) or 50 mg/kg SAR-20347 (N=6), injected with 10 μg mouse IL-22 or vehicle and PBS (N=4), and serum SAA levels were assessed 6 hours post-injection. (C-D) Graphs represent mean ± standard deviation. * indicates t-test p < 0.05 compared to WT.

Figure 4. SAR-20347 reduces imiquimod-induced inflammation and keratinocyte cell numbers

(A) Clinical scores for redness and scaling were assessed 6 days following the onset of imiquimod treatment. (B) H&E stained slides were scored (0-4) for disease severity. (C) Epidermal thickness was evaluated by counting the number of keratinocytes comprising the epidermis (calculated by a blinded observer for 4 fields per subject and averaged). (D) Cell proliferation was measured as percent Ki67 staining was calculated by measuring Ki67 staining area in the epidermis (above the dotted line) in imiquimod treated animals relative the Ki67 area in control cream treated animals (not shown). (E) Representative H&E and (F) Ki67 micrographs. For (A)-(F), WT animals treated with vehicle (N=6) or 50 mg/kg SAR-20347 (N=5), and TYK2 mutant mice treated with vehicle (N=6). All animals were treated (p.o.) twice daily and treated topically daily with imiquimod. Graphs represent mean ± standard deviation. * indicates t-test p < 0.05 compared to WT.

Figure 5. SAR-20347 reduces imiquimod-induced inflammation and antimicrobial peptide production

All animals were treated with imiquimod. WT animals received either 50 mg/kg SAR-20347 (N=5) or vehicle (N=6), and TYK2 mutant mice treated with vehicle only (N=6). Skin RNA was extracted after 6 days of imiquimod treatment, and expression of IL-22, IL-23, IL-17, S100A9, IL-6, Defensin β1, S100A8, IL-20, IL-10, IL-2, and TNF was determined by qPCR. Graphs represent mean 2^(-ΔΔCt) ± standard deviation. * indicates t-test p < 0.05 for ΔCt values compared to WT. # indicates t-test p < 0.05 for ΔCt values compared to TYK2 mutant.

Figure 6. SAR-20347 treatment and TYK2 mutant mice show reduced IL-17 and Vγ3

Back skin samples from mice treated with imiquimod (A-C at 10x, D-F at 20x magnification). (A) and (D) WT/vehicle mice, (B) and (E) WT/SAR-20347-treated mice, and (C) and (F) TYK2 mutant mice treated with vehicle. Micrographs of IL-17 (green), Vγ3 (red), and Hoechst (blue) staining. (G) and (H) signal intensity for each animal was calculated in Image J and averaged. Graphs represent mean ± standard deviation. * indicates t-test p < 0.05 compared to WT.

Figure 7. Proposed mechanism of action of SAR-20347 in ameliorating psoriasis-like symptoms

Activated dendritic cells (DCs) produce IL-23 and other proinflammatory cytokines, including IL-6. IL-23 induces γδ T cell activation causing them to secrete IL-17 and IL-22. IL-6 contributes to the differentiation and, along with IL-23, proliferation of Th17 cells, which in turn also produce IL-22 and IL-17. Together with γδ T cells, Th17 cells activate keratinocytes to secrete antimicrobial peptides and proinflammatory cytokines such as IL-20, enhancing the inflammatory reactions in the skin. Treatment with SAR-20347 acts at multiple levels by blocking activity of IL-23, IL-22, and IL-6, and blocking their pathogenic effects on keratinocytes.