Protein engineering of the chemokine CCL20 prevents psoriasiform dermatitis in an IL-23-dependent murine model

Abstract

Psoriasis is a chronic inflammatory skin disease characterized by the infiltration of T cell and other immune cells to the skin in response to injury or autoantigens. Conventional, as well as unconventional, γδ T cells are recruited to the dermis and epidermis by CCL20 and other chemokines. Together with its receptor CCR6, CCL20 plays a critical role in the development of psoriasiform dermatitis in mouse models. We screened a panel of CCL20 variants designed to form dimers stabilized by intermolecular disulfide bonds. A single-atom substitution yielded a CCL20 variant (CCL20 S64C) that acted as a partial agonist for the chemokine receptor CCR6. CCL20 S64C bound CCR6 and induced intracellular calcium release, consistent with G-protein activation, but exhibited minimal chemotactic activity. Instead, CCL20 S64C inhibited CCR6-mediated T cell migration with nominal impact on other chemokine receptor signaling. When given in an IL-23-dependent mouse model for psoriasis, CCL20 S64C prevented psoriatic inflammation and the up-regulation of IL-17A and IL-22. Our results validate CCR6 as a tractable therapeutic target for psoriasis and demonstrate the value of CCL20 S64C as a lead compound.

Keywords: CCL20; Th17; X-ray; protein engineering; psoriasis.

Fig. 1.

Crystal structure of CCL20 S64C shows a CXC dimer conformation. (A) Previously reported X-ray crystal structures of CCL20 WT (PDB ID codes 2HCI and 1M8A) exhibited the dimeric arrangement common to most CXC chemokines instead of the typical CC dimer interface. (B) Hydrogen bonds linking backbone atoms of the β1 strands define the CXC-type dimer interface and form an intermolecular β-sheet centered at F23. (C) Strong electron density (pink mesh) confirms the formation of the engineered disulfide bond. The carboxyl terminus is stabilized by hydrogen bonds between the K52 and K57 sidechain of one subunit with the backbone carbonyl of M70 of the opposing subunit. (D) Two copies of the CCL20 NMR monomer structure (purple; PDB ID code 2JYO) superimposed on the CCL20 S64C structure (gray/cyan; PDB ID code 5UR7). (E) Canonical receptor binding epitopes (site 1) are structurally conserved between the monomer structure of CCL20 (purple; PDB ID code 2JYO) and dimeric structure of CCL20 S64C (cyan; PDB ID code 5UR7).

Fig. 2.

Biochemical characterization of CCL20 S64C activation of CCR6. (A) Binding of CCL20 proteins was observed by ¹²⁵I-CCL20 WT displacement from CCR6⁺ transfected COS-7 cells. The K_d values for CCL20 WT and S64C binding to CCR6 were calculated as 7.2 nM (n = 4) and 77.6 nM, respectively (n = 3). (B) Administration of CCL20 WT and S64C on CCR6⁺ Jurkat cells promoted intracellular calcium release with EC₅₀ values of 75.8 and 714.5 nM, respectively (n = 3). (C) Accumulation of ³H-IP₃ was determined by radioactive measurements on transfected CCR6⁺ COS-7 cells in response to CCL20 WT and S64C with resulting EC₅₀ values of 0.4 and 44.0 nM (n = 4). (D) Dose-dependent treatment of U2OS cells with CCL20 WT and S64C promoted β-arrestin-2 recruitment to CCR6 with EC₅₀ values of 1.2 nM (n = 5) and 72.2 nM (n = 4). (E) Treatment with CCL20 S64C reduced CCR6 cell surface expression less efficiently than CCL20 WT. *P < 0.05 vs. same WT group (n = 2). (F) Table, summary of experimental EC₅₀ values, corresponding logEC₅₀ ± SEM, and receptor internalization results. *P < 0.05 vs. CCL20 WT group.

Fig. 3.

CCL20 S64C blocks CCL20 WT-dependent cell migration and is a preventative therapeutic for IL-23–induced psoriasis. (A) Migration of CCR6⁺ Jurkat cells after 2-h incubation was measured by flow cytometry (n = 2). *P < 0.05 vs. equal concentration CCL20 WT. (B) Inhibition of CCR6⁺ Jurkat cell migration by CCL20 S64C in the presence of 30 nM CCL20 WT. ^#P < 0.07 vs. CCL20 WT positive control. (C) CCL20 S64C inhibition of CCL2 (1 nM)- or CXCL12 (10 nM)-dependent THP-1 cell migration (n = 2). (D) Time course of ear swelling measured as the difference in ear thickness from day 0. *P < 0.05 vs. CCL20 WT group (n = 6). Three mice were injected in both ears for each experiment. Similar results were obtained in two independent experiments; plot is representative of one independent experiment.

Fig. 4.

The CCL20 locked dimer blocks the recruitment of γδ-low–expressing T cells to the epidermis. (A) H&E staining after treatment for 6 d. (Scale bar: 100 µm.) Data are representative of at least three mice. Similar results were obtained in three independent experiments. (B) Mean epidermal thickness measured per mouse for each group (n = 3). Plotted values are averaged from two pooled mouse ears. Similar results were obtained in two independent experiments. (C) Representative flow cytometry results of epidermal cell suspensions from each mouse ear stained with mAbs against γδ-TCR. The numbers indicate the proportion of side-scatter^med γδ^low GDL T cells. Similar results were obtained in three independent experiments. (D) Total number of epidermal GDL T cells per mouse for each group (n = 3). Plotted values are averaged from two pooled mouse ears. Similar results were obtained in two independent experiments. All experimental data are expressed as mean ± SEM. *P < 0.05 vs. IL-23–alone groups for all experiments.