Evaluation of the Involvement of Coiled-coil α-helical Rod Protein 1 (CCHCR1) in Imiquimod-induced Psoriasis Models

Dear Editor:

Psoriasis is a chronic skin disease characterized by keratinocyte hyperproliferation. The heredity of psoriasis is thought to be non-Mendelian; that is, it is caused by the accumulation of multiple genes with relatively weak effects in an individual. The oldest and strongest evidence for a susceptibility locus is in the HLA region (PSORS1, OMIM#177900)¹. Additionally, linkage results from genome-wide scans place a major psoriasis locus on chromosome 6p21.3¹.

The study was approved by the ethical committee of the Juntendo University (the approved no. 2020293). The informed consent was waived.

The CCHCR1 gene within the major psoriasis susceptibility locus (PSORS1) is a plausible candidate gene for the risk effect². Three psoriasis-associated susceptibility alleles, HLAC*w6, CCHCR1*WWCC, and CDSN*5 (corneodesmosin), have been identified in PSORS1². Psoriasis vulgaris and guttate psoriasis are genetically indistinguishable for their association with PSORS1, and the CCHCR1*WWCC association is weaker than that for HLAC*w6 but stronger than that for CDSN*5³. Tiala et al.² generated transgenic mice overexpressing the psoriasis-associated risk allele CCHCR1*WWCC and reported that CCHCR1 acts as a negative regulator of keratinocyte proliferation after observing the wound healing process.

Moreover, a variant of CCHCR1 has been identified as one of the susceptibility genes for alopecia areata (AA). Oka et al.⁴ revealed that approximately 15% of cases of AA occur in individuals with a nonsynonymous variant of CCHCR¹. We therefore created CCHCR1 knockout (KO) mice (Fig. 1) and evaluated whether the water avoidance stress test induced an AA phenotype⁵.

Fig. 1. (A) The site of CCHCR1 gene knockout (KO). (B) The result of CCHCR1 KO mice by genotyping. (C) The examination of CCHCR1 expression by RT–PCR. CCHCR1 expression was decreased in CCHCR1 KO mice. (1D) The examination of CCHCR1 expression by western blot. CCHCR1 expression was abolished at the protein level. Adapted from the article of Zhao et al. (Biomedicines 2021;9:840)⁵. WT: wild type.

Topical imiquimod is often used to create a mouse psoriasis model. The antiviral and antitumour effects of imiquimod are achieved by activation of TLR7 and TLR8. Increased production of inflammatory cytokines and chemokines directly induces antiviral activity mediated by interferon (IFN)-α, resulting in an influx of immune cells. Given that CCHCR1 is a negative regulator of keratinocyte proliferation, it is expected that keratinocyte proliferation will be stronger in CCHCR1 KO mice after imiquimod application.

The backs of 6 wild-type mice (C57BL/6NCrl) and 6 CCHCR1 KO mice were shaved, and 5% imiquimod cream (Beselna, Mochida Pharmaceutical) was applied for 6 consecutive days. Tissues were collected on the 7th day and evaluated by HE staining, Ki67 staining, and RT–PCR of interleukin (IL)-17A, IL-23, K17, and IFN-α.

Scaling and erythema were similarly observed in both wild-type mice and CCHCR1 KO mice (Fig. 2A). The thickness of the epidermis and stratum corneum, disappearance of the granule layer, and extension of epidermal protrusions were similarly observed in both groups of mice (Fig. 2B). Ki67 staining of the basal layer was similar in both groups, suggesting almost the same level of keratinocyte proliferation (Fig. 2C).

Fig. 2. (A) Clinical photographs of wild-type mice and CCHCR1 KO mice in chronological order. In this study, we used 6 CCHCR1 KO mice and 6 wild-type mice (C57BL/6NCrl). (B) Erythema and scaling became more severe each day, as described in the severity score from H&E staining of imiquimod-treated mice. The thickness of the epidermis and stratum corneum, disappearance of the granule layer, and extension of epidermal protrusions were similarly observed in both wild-type mice and CCHCR1 KO mice. (C) Ki67 staining of the basal layer was similar in both groups, suggesting almost the same level of keratinocyte proliferation. Ki67-positive cell counts were not significantly different between the groups. (D) The expression of interleukin (IL)-17A, IL-23, K17, and interferon (IFN)-α was detected in both imiquimod (IMQ)-treated wild-type mice and CCHCR1 KO mice, and there were no significant differences between the groups.

Ki67-positive cell counts were not significantly different between the groups (Fig. 2C). Moreover, the expression levels of IL-17A, IL-23, K17, and IFN-α were not significantly different between the groups (Fig. 2D). Considering that CCHCR1 acts as a negative regulator of keratinocyte proliferation, the expression levels of IL-17A, IL-23, K17, and IFN-α should increase in imiquimod-treated CCHCR1 KO mice. However, there were no statistically significant differences between the groups in this study. As described by Asumalahti et al.³, the CCHCR1*WWCC association was weaker than that of HLAC*w6; these results suggest that CCHCR1 may not be indispensable in psoriasis pathophysiology, at least in imiquimod-induced psoriasis models. Additional experiments will be required to finalize the conclusion.

ACKNOWLEDGMENT

The authors are thankful for the helpful discussions and technical assistance from employees of the Laboratory of Biomedical Research Resources Center at Juntendo University.