IL-17–Secreting γδ T Cells Are Completely Dependent upon CCR6 for Homing to Inflamed Skin

James J. Campbell, Karen Ebsworth, Linda S. Ertl, Jeffrey P. McMahon, Dale Newland, Yu Wang, Shirley Liu, Zhenhua Miao, Ton Dang, Penglie Zhang, Israel F. Charo, Rajinder Singh and Thomas J. Schall
J Immunol November 1, 2017, 199 (9) 3129-3136; DOI: https://doi.org/10.4049/jimmunol.1700826

Abstract

mAbs that neutralize IL-17 or its receptor have proven efficacious in treating moderate-to-severe psoriasis, confirming IL-17 as an important driver of this disease. In mice, a rare population of T cells, γδT17 cells, appears to be a dominant source of IL-17 in experimental psoriasis. These cells traffic between lymph nodes and the skin, and are identified by their coexpression of the TCR variable regions γ4 and δ4. These cells are homologous to the Vγ9Vδ2 T cell population identified in human psoriatic plaques. In this study we report that a potent and specific small molecule antagonist of the CCR6 chemokine receptor, CCX2553, was efficacious in reducing multiple aspects of psoriasis in two different murine models of the disease. Administration of CCX2553 ameliorated skin inflammation in both the IL-23–induced ear swelling model and the topical imiquimod model, and significantly reduced the number of γδT17 cells in inflamed skin. γδT17 cells were greatly reduced in imiquimod-treated skin of CCR6−/− mice, but adoptively transferred wild-type (CCR6+/+) γδT17 cells homed normally to the skin of imiquimod-treated CCR6−/− mice. Our data suggest that γδT17 cells are completely dependent on CCR6 for homing to psoriasiform skin. Thus, CCR6 may constitute a novel target for a mechanistically distinct therapeutic approach to treating psoriasis.

Introduction

Psoriasis is a chronic, disfiguring, immune-mediated inflammatory skin disorder with many associated physical and psychological comorbidities (1). Although the pathogenesis of psoriasis is not fully understood, it is clear that the interplay between IL-17–secreting T cells and IL-23 is a central component of this complex disease (2). In murine models of psoriasis, the absence of the genes for IL-17 or IL-17RA greatly reduces inflammation (3–5), as does absence of RAG1 or RAG2 (5, 6). Interestingly, inflammation is essentially eliminated in mice that lack only γδ T cells in these disease models (7), but is not affected in mice specifically lacking the much more prevalent αβ T cells (8). Although γδ T cells comprise only ∼1% of the mature T cell pool, these data suggest that γδ T cells are critically important in the pathogenesis of psoriasis, but little is known of the mechanism whereby these cells are recruited to the affected skin.

Chemokines are potent regulators of leukocyte trafficking to sites of inflammation or injury. Earlier work had found that mice lacking the chemokine receptor CCR6 had reduced skin inflammation in the IL-23–injection model of psoriasis (9, 10), but the culprit cytokine-producing cells were not identified. More recently, CCR6 has been found to be expressed on γδ T cells in murine models of psoriasis CCR6 (11–13).

In this study, we tested the hypothesis that trafficking of pathogenic γδ T cells to psoriatic skin is critically dependent upon their expression of functional CCR6. We report that a small molecule antagonist of CCR6 effectively reduces psoriasiform inflammation in two different murine models, and specifically inhibits the trafficking of a rare population of γδ T cells to diseased skin. These data provide a more mechanistic understanding of the pathogenesis of psoriasis, and suggest a novel approach for treating this disease based on interruption of trafficking of a rare subset of T cells.

Materials and Methods

CCR6 antagonist CCX2553 (see Supplemental Table I). was discovered and synthesized according to procedures described by ChemoCentryx (U.S. Patent Application No. 15/353,889). For in vivo studies, mice were s.c. dosed with CCX2553 once daily (s.c., q.d. in N-methyl-2-pyrrolidone/Tween 80) at 90 mg/kg, unless stated otherwise, starting at day 0. Control compound SCH527123 (14) was also dosed s.c. every day at 30 mg/kg.

In some cases, mice were treated with function-blocking anti–IL-17RA Ab (R&D Systems, Minneapolis, MN) or isotype control dosed at 200 μg i.p. once daily using PBS as the vehicle.

All animal studies were approved by the ChemoCentryx Institutional Animal Care and Use Committee.

Mice

BALB/c and C57BL/6 (wild-type, WT, CD45.1-congenic and CCR6−/−) mice were purchased from the Jackson Laboratory (Bar Harbor, ME) and housed at the ChemoCentryx animal facility in accordance with guidelines described in the Guide and Use of Laboratory Animals of the National Research Council. All studies were approved by the ChemoCentryx Institutional Animal Care and Use Committee.

Imiquimod-induced psoriasiform dermatitis model

Psoriasiform dermatitis in BALB/c mice was induced as previously described (5). Briefly, 5% imiquimod (Fougera) was applied daily to the shaved and depilated backs of BALB/c mice for up to 4 d (note: shaved and depilated mice were rested for 3 d prior to start of imiquimod treatment). Control mice were treated with an application of Vaseline. For C57BL/6 mice (including the CCR6−/− strain), imiquimod was applied twice daily for up to 5 d. Erythema, desquamation, and skin thickening were scored independently on a scale from zero to four where 0 = no disease; 1 = slight disease; 2 = moderate disease; 3 = marked disease; 4 = very marked disease (5). Skin thickness was measured by taking an average of three measurements along the central axis of each mouse using a spring-loaded electronic caliper. The level of erythema was scored using a scoring table with red tints graded from one to four. The total Psoriasis Area and Severity Index (PASI) score (erythema plus scaling plus thickening) served as a measure of the severity of inflammation (scale 0–12). All scoring was done by the same investigator blinded to the treatment groups for each study.

Skin from the backs of mice was excised at the end of the study and processed for flow cytometry analysis, snap-frozen in liquid nitrogen for analysis of cytokine levels, or fixed in 10% neutral buffered formalin for histological analysis. Skin used for isolation of T cells was weighed after excision. In some studies, the inguinal lymph nodes were also collected at the end of study for analysis by flow cytometry.

IL-23–induced ear swelling model.

C57BL/6 mice were given and intradermal injections of rmIL-23 (500 μg) (Thermo Fisher Scientific, San Diego, CA) into the right ear every other day for 12 d. An intradermal injection of PBS was given into the left ear as a control. Ear swelling was measured by calipers. CCX2553 or anti–IL-17RA Ab was dosed from day 0 when given in prophylactic mode.

Adoptive transfer studies

CD45.1 congenic C57BL/6 mice (donor mice) were treated with 5% imiquimod (Fougera) on the ears and depilated back skin twice daily for 5 d. Cervical, axillary, and inguinal nodes were collected. Single-cell suspensions of lymph node cells in saline (7.5 × 106 in 200 μl) were injected into the tail vein of WT or CCR6−/− C57BL/6 mice). The injected cell population contained ∼6.5 × 106 T cells, of which 0.6% were γδ T cells. These recipient mice had been rested for 3 d since shaving and depilating the back skin, and were treated twice daily with imiquimod.

Cellular phenotyping of skin infiltrates

Excised skin was finely chopped with scissors then digested for 30 min at 37°C in 2 mg/ml collagenase A and 1 U/ml DNAse I (St. Louis, MO). The digestion was stopped by the addition of 8 ml of cold PBS with 10% FBS. Cells were then dislodged from skin debris by repeated pipetting through a 10 ml serological pipette, filtered through a 70 μM sieve, washed, and resuspended in FACS buffer (PBS with 10% FBS) for analysis.

mAbs were obtained from R&D Systems, BioLegend (San Diego, CA), or eBioscience (San Diego, CA). All flow cytometry protocols for skin-derived T cells used TCRVδ4 (GL2) in PE, CD90.2 (30-H12) in Per CP-Cy5.5, TCRVγ4 (UC310A6) in PE-Cy7, TCRβ (H57-597) in allophycocyanin-eFluor-780, TCRγδ (GL3) in BV-421 and LIVE/DEAD fixable Aqua stain (Molecular Probes, Eugene, OR). Some staining protocols also included CD45 (30-F11) in Alexa Fluor 488 and TCRVγ5 (536) in allophycocyanin.

Others included one of a panel of unconjugated monoclonals (or their isotype-matched controls) followed by an appropriate allophycocyanin-conjugated polyclonal (FAB)2 (Jackson ImmunoResearch, West Grove, PA). These unconjugated reagents included anti-CCR6 (140706) and anti-CXCR2 (242216), both rat IgG2as, and anti-CCR2 (475301), a rat IgG2b. CLA expression was assessed with a recombinant mouse E-selectin/human IgG1 Fc chimera (R&D Systems), followed by an allophycocyanin-conjugated polyclonal anti-human IgG. The isotype control for this E-selectin chimera involved incubating the chimera-stained cells in EDTA (prior to second-stage polyclonal) to remove Ca2+-dependent selectin-specific binding. After staining with unconjugated monoclonal and allophycocyanin polyclonal, the cells were blocked with 10% mouse, 10% hamster, and 10% rat sera (Jackson ImmunoResearch) prior to staining with direct conjugates. For the intracellular staining with allophycocyanin-conjugated anti-RORγt (B2D) or its rat IgG1 isotype control, the cells were fixed and permeabilized with the Fixation and Permeabilization Buffer Kit (R&D) after completion of the extracellular stain, and incubated following the kit instructions.

For staining cells from adoptive-transfer experiments, the protocol included CD45.2 (104) in Alexa Fluor 488 and CD45.1 (A20) in allophycocyanin.

Flow cytometry data were acquired with a FACSCanto II (BD Biosciences, San Jose, CA) cytometer, and analyzed using FlowJo v10.2 (FlowJo, Ashland, OR).

Cytokine protein analysis

Skin samples were snap-frozen and then homogenized in PBS containing protease inhibitors (Sigma-Aldrich, St. Louis, MO). Homogenates were analyzed for cytokine expression by Luminex multiplex assay (R&D Systems). Results were normalized to total protein determined by standard Bradford assay (Bio-Rad Laboratories, Hercules, CA).

Histology

Depilated, excised mouse skin was fixed in 10% neutral-buffered formalin (Sigma-Aldrich), dehydrated, and embedded in paraffin by conventional methods. Then 5 μm skin sections were cut from formalin-fixed paraffin-embedded blocks and transferred to glass slides. Sections were stained with H&E using standard procedures. Epidermal thickness was measured as the average of ≥80 measurements made along the length of representative H&E-stained skin sections using Photoshop CS4 software (Adobe).

Statistical analysis

Statistical significance was determined by Mann–Whitney calculation using GraphPad Prism 6.0 software.

Results

CCR6−/− mice are resistant to inflammation in the IL-23 ear skin injection model of psoriasis (9, 10). Based on this information, we tested the ability of CCX2553, a small molecule CCR6 antagonist, to ameliorate IL-23–induced ear swelling in BALB/c mice (Fig. 1A). CCX2553 was administered at 90 mg/kg once daily, which yielded trough plasma levels in excess of the IC90 for antagonism of murine CCR6 (see Fig. 1, Supplemental Table I). We found that CCX2553 (red circles) markedly and significantly reduced ear swelling in comparison with vehicle control (blue squares). In fact, the reduction in swelling caused by CCX2553 was comparable to that caused by an anti–IL-17RA function–blocking Ab (green inverted triangles). CCX2553 was also found to be effective in a therapeutic version of the IL-23 model (Supplemental Fig. 2).

FIGURE 1.
FIGURE 1.

Small molecule CCR6 antagonist, CCX2553, ameliorates inflammation in two well-known murine models of psoriasis. (A) Time course of BALB/c ear skin thickness after intracutaneous injection of PBS (black diamonds, five mice per group) or IL-23 (all other groups, 10 mice per group). (B) Time course of total PASI score on areas of depilated BALB/c skin treated with Vaseline (black diamonds, five mice per group) or imiquimod (all other groups, 10 mice per group). Data representative of at least five separate experiments. Blue squares: IL-23– (A) or imiquimod- (B) inflamed mice treated with vehicle. Red circles: IL-23– (A) or imiquimod- (B) inflamed mice treated with small molecule CCR6 antagonist CCX2553. Green inverted triangles: IL-23– (A) or imiquimod- (B) inflamed mice treated with anti–IL-17RA MAb. Purple empty circles (B only): imiquimod-inflamed mice treated with small molecule CXCR2 antagonist SCH527123. CCX2553 plasma levels were 418 ± 156 nM at trough after 12 d of dosing in (A) and 240 ± 40 nM at trough after 4 d of dosing in (B). SCH527123 plasma levels were 1094 ± 504 nM at trough after 4 d of dosing.

Although ear skin injection of IL-23 recapitulates some aspects of psoriasis and provides a useful murine model, imiquimod-induced lesions more closely resemble those of full-blown human psoriatic plaques (3–5). In this model, imiquimod, a TLR7/8 agonist, is topically applied once daily to a shaved and depilated area on the back of each mouse. The lesions are assessed by an evaluator blinded to treatment group. Redness, scaling, and total skin thickness together comprise a PASI score (5). In agreement with the IL-23 model, CCX2553 significantly reduced psoriasiform dermatitis in imiquimod-treated BALB/c mice (Fig. 1B). Treatment with CCX2553 decreased the PASI score to levels approaching those of anti–IL-17RA–treated mice (Fig. 1B). As CCX2553 possesses antagonism both for mouse CCR6 and CXCR2 (Supplemental Table I), we compared its activity to that of a selective CXCR2 antagonist, SCH527123 (14). SCH527123 had no appreciable effect on the PASI score (purple open circles), suggesting that the CCR6 antagonism displayed by CCX2553 is the biological activity relevant to our observations from the imiquimod model.

We repeated the assay and obtained imiquimod-treated skin for pathological evaluation. After PASI scoring, the mice were sacrificed and the skin was fixed for sectioning and H&E staining (Fig. 2). We evaluated the mean epidermal thickness across the entire section (Fig. 2A, 2B). Treatment with CCX2553 profoundly reduced the mean epidermal thickness (Fig. 2B), reaching levels comparable to those of anti–IL-17RA treatment. We used linear regression to compare epidermal thickness to total PASI score for each mouse, and found a very high correlation (R2 = 0.82, see Fig. 2C). Thus, this objective measurement of epidermal thickness validates the changes we observed in PASI scoring. Closer examination of histopathology sections from mice within the various treatment groups (Fig. 2D) demonstrates that imiquimod-treated skin showed additional hallmarks of psoriasis, including microhemorrhages in the dermis and increased scaling/exfoliation of the stratum corneum. These psoriatic changes were appreciably reduced in the skin from CCX2553 and anti–IL-17RA–treated mice (Fig. 2D, right panels).

FIGURE 2.
FIGURE 2.

H&E analysis of imiquimod-treated skin demonstrates CCX2553-induced reduction of epidermal thickness, correlating with a reduction in PASI score. Paraffin-embedded formalin-fixed dorsal skin from imiquimod-induced lesions on BALB/c mice at day 4 were sectioned and stained with H&E for histopathological examination. Images were obtained of each section, with the left and right sides corresponding to the flanks of the mouse and the area over the spine in the center. (A) Epidermal thickness was assessed at regular intervals across the section as shown. (B) Mean epidermal thickness in microns is shown for an experiment comprising 5 Vaseline-treated mice (black diamonds) or 10 imiquimod-treated mice dosed with vehicle (blue squares), CCX2553 (red circles), or anti–IL-17RA mAb (green inverted triangles). (C) Linear regression of total PASI score versus epidermal thickness for the experiment shown in (B). (D) Representative images of skin sections from one mouse of each experimental group analyzed in (B) and (C). Scale bar in left panel shows scale for all four images in (D). In addition to differences in epidermal thickness, note the microhemorrhages and stratum corneum scaling seen in the vehicle-treated imiquimod sample but not in the Vaseline-, CCX2553- or α–IL-17RA–treated samples.

We next assessed the production of psoriasis-related cytokines in the lesional skin of imiquimod-treated BALB/c mice (Fig. 3). Lesional skin from mice dosed daily with vehicle or CCX2553 was snap-frozen at the indicated time points, and later processed for Luminex assessment of cytokine protein levels. Psoriasis-associated proinflammatory cytokines IL-17A, IL-17F, and IL-23 were all elevated at various time points in the imiquimod-treated mice (blue bars) with respect to Vaseline control mice (black bars). In contrast, the levels of all three cytokines in CCX2553-treated mice remained near basal levels throughout the entire 4 d experiment (red bars).

FIGURE 3.
FIGURE 3.

Systemic treatment of mice with CCX2553 reduces IL-17A, IL-17F, and IL-23 protein levels within imiquimod-treated skin. Time course for cytokine protein production in skin of Vaseline-treated (black bars) or imiquimod-treated (blue or red bars) BALB/c mice dosed with vehicle (red bars) or CCX2553 (blue bars), as assessed by Luminex multiplex protein assay. Each bar represents the mean and SD of seven mice. Treatment of all 77 BALB/c mice was begun on the same day, and the mice sacrificed and skin snap-frozen on the indicated day (0.25 d indicating 6 h after start of treatment). The p value for the difference between vehicle- and CCX2553-dosed mice is shown only where significance was reached.

Thus, treatment with CCX2553 ameliorates imiquimod-induced psoriatic lesions at the level of total skin thickness, redness, scaling, epidermal thickness, and production of proinflammatory cytokines.

To better understand the anti-inflammatory effects of CCX2553, we analyzed the various T cell populations within the inflamed skin. Imiquimod-treated mice were dosed with vehicle, CCX2553, or anti–IL-17RA as shown in Figs. 1 and 2. On day 4, lesions were assessed for total PASI score by a blinded evaluator (Fig. 4A). The mice were sacrificed, and the inflamed skin removed, weighed, then minced and gently digested. Skin-derived cell preparations were stained for flow cytometry. T cell subpopulations within the gates shown in Fig. 4B were enumerated.

FIGURE 4.
FIGURE 4.

CCX2553-induced reduction in disease severity is accompanied by markedly reduced accumulation of γδT17 cells but not DETC or αβ T cells in imiquimod-treated skin. (A) Total PASI score on areas of depilated BALB/c skin treated with Vaseline (black diamonds, five mice per group) or imiquimod (all other groups, 10 mice per group) after 4 d of treatment. (B) Leukocytes were isolated from skin of the same mice whose PASI scores are shown in (A). Gating scheme used to identify T cell subsets isolated from skin. The gates used to define each T cell subset in (C–E) are indicated by arrows [Note: in addition to the TCRγδhi profile indicated, DETC cells were further identified by Vγ5 expression, which was present on essentially all TCRγδhi cells (data not shown)]. The number of cells found within each gate for each mouse was used to calculate the total number of cells per gram of skin (the skin was weighed immediately after removal from the sacrificed mouse). (C) γδT17 cells isolated per gram of skin. (D) DETC isolated per gram of skin. (E) Conventional αβ T cells isolated per gram of skin. Blue squares: imiquimod-inflamed mice treated with vehicle. Red circles: imiquimod-inflamed mice treated with small molecule CCR6 antagonist CCX2553. Green inverted triangles: imiquimod-inflamed mice treated with anti–IL-17RA MAb. This figure shows data from a single experiment, and is representative of a total of two experiments.

In agreement with previous studies (13, 15, 16), the majority of non-dendritic epithelial T cells (DETC) and γδ T cells expressed the variable chains Vγ4 and Vδ4 (Fig. 4B). Significantly, this Vγ4+Vδ4+ population, known as γδT17, was markedly reduced in response to CCX2553 or anti–IL-17RA (Fig. 4C). These treatments reduced the γδT17 population to near baseline levels. Accumulation of DETC cells in response to imiquimod was not affected by CCX2553 or anti–IL-17RA (Fig. 4D). Interestingly, skin-infiltrating conventional αβ T cells were not significantly increased by imiquimod, nor were they reduced by CCX2553 or anti–IL-17RA (Fig. 4E). Thus, we found that the CCX2553- and anti–IL-17RA–induced reduction in PASI score correlated with a decrease in γδT17 cells, but not with the other T cell subsets from skin.

We further characterized the γδ T cell population from imiquimod-treated skin (Fig. 5) using the same flow cytometry gates shown in Fig. 4B. We confirmed that the Vγ4/Vδ4 population does indeed meet the definition of γδT17 cells in our study, as demonstrated by its uniform expression of RORγt, the transcription factor controlling IL-17 production (Fig. 5, top left panel) (reviewed in Ref. 17). We also confirmed that these cells uniformly express both CCR2 and CCR6 at high levels (18) (Fig. 5, far right panels). Interestingly, γδT17 cells uniformly expressed extremely high levels of CLA, a well-known mediator of selective T cell homing into skin from the circulation (19). We did not observe RORγt, CLA, or CCR6 expression on DETC cells, and the combination of these three markers was rare on αβ T cells. CCR2 expression, however, was quite common on αβ T cells from imiquimod-treated skin (data not shown). Consistent with our finding that a selective CXCR2 antagonist did not affect the PASI score (Fig. 1B), CXCR2 was not expressed by γδT17 cells.

FIGURE 5.
FIGURE 5.

γδ T cells co-expressing Vγ4 and Vδ4 within imiquimod-treated skin uniformly express CLA, CCR6, and RORγt. Lymphocytes were isolated from BALB/c skin after 4 d of topical imiquimod treatment. Unconjugated mAbs or isotype-matched controls were used to stain for each of the Ags shown in this figure (except for RORγt), followed by an appropriate secondary polyclonal Ab conjugated to allophycocyanin. After blocking with mouse, rat, and hamster serum, cells were stained with the same set of directly conjugated MAbs shown in Fig. 3B. For the RORγt stain, cells were permeabilized and stained with allophycocyanin-conjugated αRORγt or isotype-matched controls after staining with the same set of directly conjugated Abs used in the other panels. γδT17 cells were gated as shown in Fig 3B, and histograms of specific staining for the indicated Ag (red) overlaid on its appropriate isotype-matched control (blue). As discussed in the manuscript, the absence of CXCR2 on γδT17 cells is relevant, as CCX2553 is a potent antagonist of both CCR6 and CXCR2.

We next asked whether the above findings could be validated by a genetic model of CCR6 deficiency. For the following set of studies, we used the C57BL/6 strain, as CCR6−/− mice are readily available on this background. C57BL/6 mice are more resistant to imiquimod than BALB/c mice (3). In our study, 5 d of twice-daily imiquimod application was required to yield PASI scores similar to those of BALB/c mice treated once daily for 4 d (data not shown). We therefore used the 5 d/twice a day treatment protocol for these studies.

Upon isolating T cells from the imiquimod-treated skin on day 5, we found that ∼1500 γδT17 cells accumulated per gram of skin in C57BL/6 mice (Fig. 6, left panel, blue squares), which was similar to the numbers seen in BALB/c skin (Fig. 4C). In contrast, only ∼50 γδT17 cells per g (a ∼30-fold reduction) were seen in CCR6−/− skin (Fig. 6, left panel, red circles). CCR6 deficiency did not detectably affect DETC or αβT cell populations in skin (Fig. 6, right panels).

FIGURE 6.
FIGURE 6.

CCR6-deficiency markedly reduces imiquimod-induced γδT17 cell accumulation in skin, whereas DETC and αβ T cell numbers in skin remain unaffected. Enumeration of cells isolated from imiquimod-treated C57BL/6 (blue squares) or CCR6-deficient C57BL/6 (red circles) skin on day 5. Numbers of T cells from each subset per gram of skin are shown. γδT17 cells (left panel), DETC (center panel), and αβ T cells (right panel) were identified using the gates shown in Fig. 4B. All data shown in Fig. 6 are from a single experiment with five mice per group and are representative of three separate experiments. The PASI scores were determined by a researcher who was blinded as to the genotype of the mice under evaluation, and are shown in Fig. 7B (open squares and open circles).

Interestingly, in parallel with the effects of CCX2553 treatment seen in the previous section, the reduced accumulation of γδT17 cells observed in CCR6−/− skin corresponded with reduced PASI scores (compare the second and forth columns in Fig. 7B). Untreated WT and CCR6−/− mice both possessed similar numbers of γδT17 cells, as represented within lymph nodes. However, these cells increased in number within the draining nodes only in the WT mice after topical imiquimod treatment, likely due to the absence of γδT17 cells in the skin of CCR6−/− mice that would have otherwise drained to the downstream node (Supplemental Fig. 1).

FIGURE 7.
FIGURE 7.

Adoptively transferred WT γδT17 cells home equally well to inflamed skin within both WT and CCR6−/− recipients, and restore full imiquimod-induced inflammatory skin response to CCR6−/− mice. Donor cells were obtained from the skin-draining lymph nodes of imiquimod-treated CD45.1 congenic C57BL/6 mice. (A) 7.5 × 106 of these CD45.1+ lymph node cells, a population containing 6.5 × 106 total T cells (of which <1% were γδ T cells), were transferred i.v. into the tail veins of WT or CCR6−/− mice on the C57BL/6 background. (B) Total PASI scores were evaluated for mice that received CD45.1 donor cells (solid squares and circles) and mice that did not receive donor cells (empty squares and circles) after 5 d of imiquimod treatment. (C) Donor-derived (CD45.1+) cells per gram of skin for γδT17 cells (left panel), DETC (center panel), and αβ T cells (right panel). (D) The ratio of donor-derived (CD45.1) to endogenous (CD45.2) cells was calculated for each gated T cell subpopulation isolated from imiquimod-treated skin. All data shown in this figure are from a single experiment with five mice per group, and are representative of two separate experiments. The PASI scores were determined by a researcher who was blinded as to the genotype of the mice under evaluation.

We next asked whether the absence of CCR6 was the sole reason for the selective reduction of γδT17 cells in the skin. It was possible that the CCR6−/− skin microenvironment lacked a secondary component necessary to attract these cells. We adoptively transferred WT lymph node cells from imiquimod-treated donors into CCR6−/− recipients. (Fig. 7A). As a control, we transferred donor cells into WT recipients in parallel. Donor-derived cells were tracked by their expression of CD45.1. Interestingly, we found that adoptive transfer of WT donor cells restored the PASI score of CCR6−/− animals back to the levels of WT mice (Fig. 7B, closed squares and circles).

Donor-derived (CD45.1) γδT17 cells accumulated in imiquimod-treated skin independently of whether the recipient mice lacked the CCR6 gene. Although donor γδT17 cells accumulated somewhat less efficiently in CCR6−/− skin, the difference was not significant (Fig. 7C, left panel). This demonstrates that the immunological microenvironment within imiquimod-treated CCR6−/− skin possesses all factors necessary to support the influx of γδT17 cells, but only when the γδT17 cells express functional CCR6. The contribution of donor-derived cells to the DETC compartment was negligible (Fig. 7C, center panel).

Donor-derived αβ T cells also accumulated equally well in skin whether or not the recipient expressed CCR6 (Fig. 7C, right panel). However, donor-derived γδT17 cells (Fig. 7C, left panel, 400–500 cells per g) were much more abundant than donor-derived αβ T cells (Fig. 7C, right panel, 50–60 cells per g) within the skin despite the fact that the transferred donor T cell population consisted of ∼97% αβ T cells (data not shown). This finding highlights the notion that imiquimod treatment induces a T cell migration environment that is dramatically selective for γδT17 cells over αβ T cells.

We calculated the ratio of donor-derived (CD45.1) to endogenous (CD45.2) cells within each T cell population from the skin of WT and CCR6−/− recipients (Fig. 7D). The γδT17 population in the skin of WT recipients contained only a very small component of donor-derived cells, but in marked contrast, donor-derived cells greatly outnumbered endogenous cells in CCR6−/− recipients. Donor-derived cells were only a small component of the DETC and αβ T cell populations, regardless of whether the recipient was WT or CCR6−/−. Taken together, the data in Fig. 7 suggest there is a specific role for CCR6 in selective trafficking of γδT17 cells, but not other types of T cells, to imiquimod-treated skin.

Discussion

Psoriasis remains a serious, and sometimes debilitating, skin disease for which there is a clear unmet clinical need. Surveys of 5604 psoriasis patients performed by the National Psoriasis Foundation between 2003 and 2011 indicate that more than half of the respondents were dissatisfied with their existing treatment options (20). Mild-to-moderate plaque psoriasis can sometimes be managed with topical therapy, but patients suffering from moderate-to-severe disease (involving more than 5–10% of body surface area) are candidates for systemic treatment. Newer biologics such as TNF-α and IL-17 inhibitors are more effective than older systemic treatments such as methotrexate, but are expensive and have immunosuppressive side effects (21). A more recently approved orally bioavailable phosphodiesterase-4 inhibitor, apremilast, is effective only in approximately one third of moderate-to-severe psoriasis patients (21), meaning there is still need for alternative treatment options.

In the current study, we used a novel small molecule antagonist of CCR6 to identify a rare population of T cells, γδT17 cells, which drive the pathogenesis of psoriasis in two well-characterized murine models. To quantify the degree of psoriasis, we used a modification of the PASI, which is a widely used clinical metric. Several lines of evidence support the conclusion that γδT17 cells are key pathogenic cells in psoriasis, and are recruited to the skin in a CCR6-dependent manner. First, the small molecule antagonist CCX2553 reduced the PASI score in both the imiquimod model of psoriasis, and in the IL-23–driven model. Second, administration of CCX2553 reduced the levels of IL-17A/F and IL-23 in the diseased skin, consistent with the therapeutic benefit afforded by blocking CCR6. Third, blocking CCR6 dramatically and specifically reduced the number of γδT17 cells, but not other T cells in the diseased skin. Finally, adoptively transferred WT γδT17 cells homed to the skin in CCR6-deficient recipients, and most importantly, restored the full inflammatory response to imiquimod. Taken together, these data provide strong support for the notion that CCR6-dependent trafficking of γδT17 cells is critical for the pathogenesis of psoriasis, and also provide a scientific rationale for developing orally available CCR6 antagonists for the treatment of psoriasis.

The TCR repertoire of γδ T cells is much less diverse than that of αβ T cells in humans and mice (reviewed in Ref. 22). In fact, there are several specialized γδ T cell subsets in which the entire population expresses only a single canonical TCR VγVδ combination (reviewed in Ref. 22). One well-known invariant γδ T cell type is the DETC, a self-renewing population found only in the epidermis, which expresses a TCR composed of Vγ5 and Vδ3 (23). Other subsets bearing canonical TCR combinations are found in the digestive and reproductive tracts (22). A γδ population bearing a new canonical TCR combination, Vγ4 and Vδ4, was recently recognized (13, 16). These cells appear to be highly relevant in psoriasis, as they are overrepresented in psoriatic skin and produce IL-17 (13). The latter property has led to naming this population γδT17. Unlike DETC, which are restricted to skin, these γδT17 cells have the ability to traffic between lymph nodes and skin via the peripheral blood and lymph (15, 18, 24, 25). This murine cell population appears to be homologous to a human γδ T subset that also secretes IL-17 (7). The human population is greatly enriched within psoriatic plaques, comprising a mean of ∼40% of all T cells in psoriasis skin, compared with a mean of only ∼15% in normal skin [12 psoriasis patients ranged 20–90% γδ T cells, 6 normal controls ranged from 2 to 30% (7)]. These human γδT17-like cells bear the canonical TCR combination of Vγ9 and Vγ2, and express CCR6 (26). Thus, in both human and murine models of psoriasis, a homologous population of γδT cells appears to drive the disease. There is thus a high likelihood that human psoriasis can be successfully treated by blocking CCR6, as we have found by blocking the entry of the γδT17 cell population in the murine psoriasis models.

A well-known mediator of skin-selective T cell homing, CLA (19), is uniformly expressed at extremely high levels on γδT17 cells. The combined expression of CLA and CCR6 was rare on the other T cell types from imiquimod-treated skin. The coexpression of CLA and CCR6 may therefore constitute a combinatorial homing “area code” (27, 28) for skin-selective trafficking of γδT17 cells from blood, by engaging their respective ligands (i.e., E-selectin and CCL20/MIP3α) on skin endothelium.

Ramírez-Valle et al. (18) report that γδT17 cells require the chemokine receptor CCR2 for efficient accumulation within the skin. We confirm that γδT17 cells express CCR2 (Fig. 5). CCX2553 does not affect CCR2 function (see Supplemental Table I), nor would CCR6 deficiency be likely to do so (CCR2 and CCR6 are on different mouse chromosomes). Our studies suggest, therefore, that CCR2 is not sufficient to overcome the effects of CCR6 deficiency. At this time, it is not clear what the respective contributions of these two receptors may be to this process.

A very recent publication reports that CCR6 is dispensable for the development of skin lesions in CCR6−/− mice (29). The techniques used by this research group differed from ours in important ways. First, Cochez et al. studied imiquimod treatment on ear skin, but we worked with the shaved, depilated skin of a mouse’s back. The back skin is generally considered better suited for measuring parakeratosis, epidermal thickening, acanthosis, and cellular infiltration (5, 30) (reviewed in Ref. 3). In addition, a portion of imiquimod-induced inflammation (i.e., the component not affected by α–IL-17RA) is known to be influenced by an excipient in the vehicle in which imiquimod is supplied (30) (reviewed in Ref. 3) (see Figs. 1–4). We hypothesize that the inflammation studied by Cochez et al. is more heavily influenced by the imiquimod-independent vehicle effect than was our model. The vehicle effect is likely independent of T cell infiltration (reviewed in Ref. 3).

In summary, we have demonstrated that CCX2553, a small molecule antagonist of chemokine receptor CCR6, can ameliorate psoriatic symptoms in mice, with an effectiveness similar to that of function-blocking Abs against IL-17 receptor. CCX2553 appears to function by preventing the accumulation of γδT17 cells in psoriatic skin, most likely by blocking the migration of γδT17 cells from the circulation. These studies provide a mechanistic basis for developing small molecule CCR6 antagonists as a novel therapeutic approach to treating psoriasis.

Disclosures

All authors are full-time employees of ChemoCentryx Inc.

Acknowledgments

We thank Chris Li for critical reading of the manuscript.

Footnotes

  • The online version of this article contains supplemental material.

  • Abbreviations used in this article:

    DETC
    dendritic epithelial T cell
    PASI
    Psoriasis Area and Severity Index
    WT
    wild type.

  • Received June 8, 2017.
  • Accepted September 1, 2017.

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