A small molecule CRTH2 antagonist inhibits FITC-induced allergic cutaneous inflammation

Abstract

A FITC-induced allergic contact hypersensitivity model was used to investigate the role that the prostaglandin D(2) receptor-chemoattractant receptor-homologous molecule expressed on T(h)2 cells (CRTH2) plays in modulating cutaneous inflammation. Our results show that inhibition of CRTH2, achieved via administration of a potent, small molecule antagonist, Compound A (Cmpd A), effectively blocked edema formation and greatly reduced the inflammatory infiltrate and skin pathology observed in drug vehicle-treated animals. Gene expression analysis revealed that Cmpd A administration down-regulated the transcription of a wide range of pro-inflammatory mediators. This correlated with decreases in cytokine and chemokine protein levels, notably IL-4, IL-1beta, tumor necrosis factor-alpha, transforming growth factor-beta, GRO-alpha, MIP-2 and thymic stromal lymphopoietin (TSLP) in FITC-challenged ears. The administration of an anti-TSLP-neutralizing antibody was only partially effective in lowering the FITC-induced inflammatory infiltrate and cytokine production compared with the CRTH2 antagonist. Taken together, these data suggest that blockade of CRTH2 inhibits multiple pathways leading to cutaneous inflammation in this model. This suggests that CRTH2 antagonism may be a viable route for therapeutic intervention in allergic skin diseases, such as atopic dermatitis.

Fig. 1.

The CRTH2 antagonist, Cmpd A, inhibits FITC-induced ear swelling in an allergic contact inflammation model. (A) One hour prior to ear challenge, the mice were dosed as follows, FITC/veh group—drug Veh delivered orally (p.o.), FITC/Cmpd A—10 mg kg⁻¹ Cmpd A delivered p.o. (Cmpd A is described in the Materials and methods, ref. 19), FITC/ramatroban—15 mg kg⁻¹ ramatroban p.o., FITC/Dex—Dex 5 mg kg⁻¹ delivered intra-peritoneally (i.p.) and FITC/BW868c—15 mg kg⁻¹ BW868c intravenously. Seven hours after challenge, the mice were dosed a second time. After 24 h, the thickness of both ears was measured using Digital Calipers. Ear edema was determined by subtracting the width of left ear (Veh treated) from the right ear (FITC treated) width. The thickness of the left ear (Veh challenged) after 24 h is almost identical to the untreated ear thickness. In this experiment, the percentage of ear inflammation was determined by comparing the ear edema from individual animals of the different treatment groups to the average change in right ear thickness of the FITC/veh group, which was set to 100%. (B) Histological analysis of H and E-stained right ear sections 24 h after FITC challenge. Inset is FITC/veh at high power (×100) showing eosinophils and neutrophils in the inflammatory infiltrate. (C) Kinetic analysis of FITC-induced ear swelling showing peak edema at 24 h and partial resolution at 48 h post-challenge. The Veh/veh group and FITC/Veh group were received with drug Veh, the FITC/Cmpd A cohort was administered 10 mg kg⁻¹ of Cmpd A p.o. and the FITC/Dex received 5 mg kg⁻¹ Dex i.p. All groups were treated 1 h prior to FITC challenge and 7 h post-ear challenge. The average change in ear thickness per group is shown ± SEM. (D) Cmpd A inhibits FITC-induced ear edema in a dose-dependent manner. Mice were treated with the described dose of Cmpd A or drug Veh 1 h prior to FITC ear challenge and 7 h after challenge. At 24 h, ear thickness was measured, and the change in thickness of the challenged (right) ear is shown ± SEM. For all experiments shown, a minimum of five animals were used per treatment group, and ***P < 0.001, **P < 0.01.

Fig. 2.

Heatmap of select genes down-regulated in FITC-challenged ears treated with Cmpd A at 4, 8 and 24 h. Total RNA was extracted and analyzed using Illumina BeadArrays. Levels of gene expression were investigated in four treatment groups at 4-, 8- and 24-h time points using Illumina BeadArrays. The groups included Veh-treated animals (Veh on both ears and oral administration of drug Veh alone, Veh/veh), challenged animals/drug Veh (right ear challenged with FITC, left ear with Veh; oral administration of drug Veh only, FITC/veh), challenged animals/Cmpd A treated (right ear challenged with FITC, left ear with Veh; oral administration of Cmpd A 10 mg kg⁻¹, FITC/Cmpd A) and challenged animals/Dex (right ear challenged with FITC, left ear with Veh; intra-peritoneal administration of Dex 5 mg kg⁻¹, FITC/Dex). The array data are representative of three independent experiments (biological replicates). The fold changes are noted on the respective color scales. In the clustergrams, genes are grouped into (A) cytokines, cytokine receptors, CC chemokines, CC chemokine receptors and CXC chemokines. (B) CD antigens, mucin cluster and adenosine pathway. (C) arachidonic acid pathway, signaling molecules and transcription factors. (D) Levels of S100 calcium-binding protein A9 (calgranulin B) (S100a9), as determined by RT–qPCR. The data were normalized relative to β-actin and are presented as fold increases or decreases between FITC + Veh/veh, FITC + Cmpd A/FITC and FITC + Dex/FITC-treated mice. The RT–qPCR is representative of two independent experiments (biological replicates) performed in duplicate.

Fig. 2.

Heatmap of select genes down-regulated in FITC-challenged ears treated with Cmpd A at 4, 8 and 24 h. Total RNA was extracted and analyzed using Illumina BeadArrays. Levels of gene expression were investigated in four treatment groups at 4-, 8- and 24-h time points using Illumina BeadArrays. The groups included Veh-treated animals (Veh on both ears and oral administration of drug Veh alone, Veh/veh), challenged animals/drug Veh (right ear challenged with FITC, left ear with Veh; oral administration of drug Veh only, FITC/veh), challenged animals/Cmpd A treated (right ear challenged with FITC, left ear with Veh; oral administration of Cmpd A 10 mg kg⁻¹, FITC/Cmpd A) and challenged animals/Dex (right ear challenged with FITC, left ear with Veh; intra-peritoneal administration of Dex 5 mg kg⁻¹, FITC/Dex). The array data are representative of three independent experiments (biological replicates). The fold changes are noted on the respective color scales. In the clustergrams, genes are grouped into (A) cytokines, cytokine receptors, CC chemokines, CC chemokine receptors and CXC chemokines. (B) CD antigens, mucin cluster and adenosine pathway. (C) arachidonic acid pathway, signaling molecules and transcription factors. (D) Levels of S100 calcium-binding protein A9 (calgranulin B) (S100a9), as determined by RT–qPCR. The data were normalized relative to β-actin and are presented as fold increases or decreases between FITC + Veh/veh, FITC + Cmpd A/FITC and FITC + Dex/FITC-treated mice. The RT–qPCR is representative of two independent experiments (biological replicates) performed in duplicate.

Fig. 3.

Oral administration of Cmpd A reduces pro-inflammatory cytokine levels in FITC-challenged ears. Protein lysates from ears were examined for cytokines by ELISA analysis. Right and left ears were harvested from mice at the indicated time points (4, 8, 24 and 48 h) for IL-4 (A); 8, 24 and 48 h for IFN-γ (B); and 24 h for TNF-α (C) and TGF-β (D). Each point shown represents the average cytokine value (±SEM) from a minimum of five mice, and each lysate was tested independently in duplicate or triplicate as described in the Materials and methods. Mice were treated in the following conditions in panels (A, B and D): Veh challenged on the right and left ear and drug Veh administered p.o. (Veh/veh), FITC challenged on the right ear, Veh on the left ear and drug Veh given p.o. (FITC/veh), FITC-challenged right ear, Veh-challenged left ear and Cmpd A (10 mg kg⁻¹) delivered p.o. (FITC/Cmpd A) and FITC-challenged right ear, Veh-challenged left ear and Dex (5 mg kg⁻¹) administered intra-peritoneally (FITC/Dex). In panel (C), one Cmpd A treatment group received 1 mg kg⁻¹ and another 0.1 mg kg⁻¹, as labeled. Values expressed as petagram per 50 μg of total protein, except for IL-4 which is expressed as petagram per 100 μg total protein. There were a minimum of five mice per treatment group. ***P < 0.001, **P < 0.01.

Fig. 4.

Cmpd A reduces MIP-2 and GRO-α production and the recruitment of neutrophils (GR-1⁺ cells) in FITC-challenged ears. Protein lysates from challenged ears isolated at 8 and 24 h were assayed by ELISA for MIP-2 (A) and GRO-α (B) protein levels. Values shown are the petagram of cytokine measured from 50 μg total protein, and the average of five mice per treatment group ± SEM is shown, as detailed in the Materials and methods. The treatment groups are Veh/veh, FITC/veh, FITC/Cmpd A 10 mg kg⁻¹ p.o. and FITC/Dex 5 mg kg⁻¹ intra-peritoneally (C) IHC analysis of ears isolated at 24 h post-FITC challenge and stained with an anti-GR-1 mAb to label neutrophils.

Fig. 5.

IL-1β and TSLP protein levels are reduced 4 h after FITC challenge by Cmpd A. Mice were treated with either drug Veh or Cmpd A (1 mg kg⁻¹) 1 h before FITC challenge to the right ear. The left ears were challenged with FITC Veh (acetone:dibutyl phthalate). After 4 h, the ears were harvested and the protein lysates from the right ears were assayed for (A) IL-1β (50 μg total protein) and (B) TSLP (100 μg total protein). As TSLP levels were high in the Veh/veh group, the left ears (Veh challenged) from mice in all the groups were assayed for TSLP protein levels (C). Additionally, six ears from untreated BALB/c mice were also examined for TSLP protein levels. A minimum of five animals were tested per condition except where noted above, and the results show the mean ± SEM. ***P < 0.001, *P < 0.05.

Fig. 6.

Anti-TSLP antibody treatment only partially inhibits allergic inflammation in contrast to CRTH2 antagonism. One hour prior to FITC challenge, mice received drug Veh p.o., drug Veh p.o. and 500 μg control antibody intravenously (i.v.), Cmpd A (1 mg kg⁻¹) p.o., Cmpd A (0.1 mg kg⁻¹) and control antibody i.v., anti-TSLP antibody (500 μg) i.v. and Cmpd A (0.1 mg kg⁻¹) p.o. and anti-TSLP antibody i.v. Seven hours after FITC challenge to the right ear, the appropriate groups received either drug Veh or Cmpd A p.o. (A) Twenty-four hours after challenge, ear thickness was determined and the change in thickness calculated. (B) Ear sections isolated 24 h after FITC challenge were stained with H and E. Protein lysates from the treated ears were assayed for IL-4 (C) and GRO-α (D). Fifty micrograms of total protein from lysates were analyzed by ELISA. The results show the mean cytokine levels ± SEM of a minimum of five mice per treatment group.

Fig. 7.

CRTH2 is expressed by basal epidermal cells in human skin. Human skin was analyzed by IHC for CRTH2 expression using an anti-human CRTH2 polyclonal antibody (rabbit polyclonal). Control staining with a polyclonal rabbit primary antibody is also shown. No reliable anti-mouse CRTH2 antibody is available, so CRTH2 expression in mouse skin was not examined.