Keratinocyte proline‐rich protein modulates immune and epidermal response in imiquimod‐induced psoriatic skin inflammation

Psoriasis is a persistent inflammatory skin disease thought to arise as a result of the infiltration of inflammatory cells and activation of keratinocytes. Recent advances in basic research and clinical experience revealed that the interleukin (IL)‐23/IL‐17 axis has been identified as a major immune pathway in psoriasis. However, it remains unclear how keratinocyte factors contribute to the pathology of psoriasis. Keratinocyte proline‐rich protein (KPRP) is a proline‐rich insoluble protein, which is present in the epidermis and is likely to be involved in the skin barrier function. Here, to investigate the potential roles of KPRP in psoriatic skin inflammation, Kprp‐modified mice were applied in the imiquimod (IMQ)‐induced skin inflammation model, which develops psoriasis‐like epidermal hyperplasia and cutaneous inflammation features. Then, heterozygous knockout (Kprp+/−) but not homozygous knockout (Kprp−/−) mice displayed attenuated skin erythema compared to control wild‐type mice. In addition, RNA sequencing, quantitative PCR and/or histological analysis detected changes in the expression of several molecules related to psoriatic inflammation or keratinocyte differentiation in Kprp+/− mice, but not Kprp−/− mice. Further analysis exhibited reduced IL‐17‐producing γδlow T cells and amplified epidermal hyperplasia in Kprp+/− mice, which were implied to be related to decreased expression of β‐defensins and increased expression of LPAR1 (Lysophosphatidic acid receptor 1), respectively. Thus, our results imply that KPRP has the potential as a therapeutic target in psoriatic skin inflammation.


| INTRODUC TI ON
Skin serves as a primary defence system and forms a barrier between the organism and the environment to prevent desiccation and entry of harmful substances. 1 In the epidermis, the stratum corneum, the outer layer of the epidermis, is filled with a large protein complex known as the cornified envelope (CE). 2 Involucrin, loricrin, filaggrin and small proline-rich proteins (SPRs) have been reported to be components of the CE.Filaggrin is well-known for its clinical importance in atopic dermatitis as well as with ichthyosis vulgaris. 3In terms of involucrin, envoplakin and periplakin, single knockout mice showed no obvious skin abnormalities.[6][7][8] Keratinocyte proline-rich protein (KPRP) is a proline-rich insoluble protein of approximately 75 kDa that is present in all layers of stratified squamous epithelium in the skin, tongue and oesophagus. 9ny proteins important for skin differentiation are encoded on human chromosome 1q21, constituting the epidermal differentiation complex.The human KPRP gene is located in this region.It has a proline-rich structure (approximately 19%), which is shared with the SPR family, the major proteins constituting CE. 10,11 Although it is not clear whether KPRP is a constituent protein of CE or a functional protein of some kind, the fact that it has so much in common with these CEassociated proteins suggests that it may be a protein of some form and likely to be involved in some way in the skin barrier.
We previously studied allelic copy number analysis of atopic dermatitis patients and found that KPRP expression was significantly downregulated in atopic dermatitis patients. 12The study further revealed that skin inflammation was exacerbated in Kprp+ /− mice by oxazolone and ovalbumin subcutaneous immunization with no remarkable skin phenotype under steady-state conditions.Given that KPRP is expressed in the epidermis and is regarded as a barrier-related molecule, here we planned to explore the role of KPRP in keratinocytes in psoriasis-like dermatitis using genetically modified mice.

| Mice
Kprp-deficient mice of C57BL/6 genetic background were generated and used as described in previous literature. 12Mice were 7-to 12-week-old for all experiments.All mice were maintained in a specific pathogen-free and conventional facility.All studies and procedures were approved by the Committee on Animal Experimentation of The University of Tokyo.

| Imiquimod (IMQ)-induced psoriasis mouse model
IMQ-induced skin inflammation model is one of the most widely used mouse models in preclinical psoriasis studies. 13,14Briefly, 8-to 12-week-old mice were shaved and depilated on the back skin 24 h before the treatment and then daily treated with either 62.5 mg of commercially available imiquimod cream (5%) (Beselna Cream; Mochida Pharmaceuticals, Tokyo, Japan) on the back skin and/or ears for 6 consecutive days (Days 0-6). 13The degree of dermatitis was evaluated using an erythema score, which was scored independently according to an objective scoring system called the Psoriasis Area and Severity Index on a scale from 0 to 4 (0, none; 1, slight; 2, moderate; 3, marked; and 4, very marked) as described previously. 14 terms of IMQ-induced psoriasis-like dermatitis in combination with topical LPA, we applied LPA simultaneously in addition to IMQ as described previously with minor modifications. 15,165% LPA (Oleoyl-L-α-LPA, Sigma-Aldrich) was prepared with a solvent mixture of ethanol, propylene glycol, water and polysorbate (4:4:1:1), 15 and imiquimod cream was applied topically after the 100 mL of vehicle (solvent) or LPA was applied.Six days after the start of the study, skin tissue from the back of the mice was collected and the back skin tissue was used for haematoxylin-eosin staining.

| Histological and immunohistochemical analyses
Mouse deparaffinized skin tissue sections were stained as described previously. 17,18The following antibodies were used: polyclonal rabbit anti-DEFB4A/BD-2 antibody (LS Bio, LS-C408333, Seattle, WA, USA).Tissues were subsequently stained with an avidin-biotin-peroxidase complex using a Vector ABC staining kit (Vector Laboratories, Burlingame, CA).Each section was examined independently by two investigators in a blinded manner.

| RNA isolation and quantitative reverse-transcription PCR analysis
Total RNA was extracted from pelleted cells or tissues and used for cDNA synthesis, and cDNA yields were measured by real-time PCR as described previously. 19Briefly, total RNA was extracted from skin tissue and cells using RNeasy Mini Kit (Qiagen, Crawley, UK). cDNA was prepared using a ReverTraAce qPCR RT Master Mix (Toyobo, Osaka, Japan).TaqMan assay, using primers/probes mix and TaqMan Universal PCR master mix (Thermo Fisher Scientific, MA, USA), was conducted for the evaluation of mRNA expression levels.Gapdh or GAPDH was used as an internal control to normalize the amounts of loaded cDNA.Relative fold differences were calculated by using the comparative ∆∆CT method.The sequences of the primers are described in Supplementary Table S1.

| Preparation of cells and flow cytometric analysis
Skin samples coated with imiquimod or control cream were incubated in 2 mg/mL Dispase (Gibco) in PBS at 37°C for 45 min to separate into the epidermis and dermis.Then, the dermis was minced and incubated in DMEM containing penicillin/streptomycin, HEPES buffer 66 μg/mL, collagenase type 2 (Worthington, Lakewood, NJ) 2 mg/mL, DNaseI 100 μg/mL, hyaluronidase 0.5 mg/ mL, 2% fetal bovine serum at 37°C for 60-90 min while rotating.Digestion enzymes were quenched by the addition of 5 mM EDTA and 10% fetal bovine serum, and digested ear tissue was mashed through a 70 μm nylon mesh.Single-cell suspensions were stained on ice with labelled antibodies and analysed on a FACSVerse flow cytometer (BD Biosciences, CA, USA).The following monoclonal antibodies were used for flow cytometry: TCRδ/γ (GL3; BioLegend), TCRβ (H57; BioLegend), CD45.2 (104; BioLegend) and Fixable Viability Dye eFluor 780 (eBioscience).These antibodies or dyes were widely used to identify γδ T cells in the tissue. 20Flow cytometry data were processed using FlowJo version 10.2 software (Tree Star, Inc., Ashland, OR).

| RNA sequencing
Total RNA was extracted from skin tissue using RNeasy Fibrous Tissue Mini Kit.RNA-Seq (RNA-Seq) was performed by DNA Chip Research Inc.The integrity and quantity of total RNA were measured using an Agilent 2100 Bioanalyzer RNA 6000 Nano Kit (Agilent Technologies, Santa Clara, CA).Total RNA obtained from each sample was subjected to sequencing library construction using the NEBNext Ultra Directional RNA Library Prep Kit for Illumina (New England Biolabs, MA) with the NEBNext rRNA Depletion Kit (Mouse) according to the manufacturer's protocols.The quality of the libraries was assessed with an Agilent2200 TapeStation High Sensitivity D1000 (Agilent Technologies).The equally pooled libraries of the samples were sequenced using the illumina NextSeq 500 system (Illumina, Inc., San Diego, CA) in 76-base-pair single-end reads.Sequencing adaptors, low-quality reads and bases were trimmed with Trimmomatic-0.32 tool. 21The sequence reads were aligned to the mouse reference genome (mm10) using Tophat 2.0.13 (bowtie2-2.2.3), 22    in the HEK293LTV producer cell line.Virus-containing supernatants were collected 24 h after transfection and directly used for infection.Cells were selected with 2 μg/mL puromycin (Sigma-Aldrich).

| IMQ-induced psoriasis-like dermatitis in combination with oral administration of LPAR1 antagonist
AM966 is known to be a highly selective LPA1 antagonist, which inhibited not only LPA-induced chemotaxis in vitro but also reduced lung injury and fibrosis induced by bleomycin in vivo. 24Then, based on the experimental conditions in this literature, 24 AM966 (Advanced ChemBlocks, Burlingame, CA) is administered orally to KPRP +/− and wild-type control mice at 30 mg/kg in 0.3% carboxymethylcellulose solution twice daily starting on Day 0. IMQ is applied externally to the right ear and back, and the right ear and back skin are collected.

| Statistical analysis
Data were analysed using GraphPad Prism 8 (GraphPad Software, San Diego, CA).Statistical analysis was carried out with one-way ANOVA with Dunnett's post hoc tests or two-way ANOVA followed by Bonferroni's post hoc test for multiple group comparisons and the two-tailed statistical analysis.Two-tailed, unpaired t-tests were performed when comparing only two groups.A value of p < 0.05 was considered statistically significant.

| Attenuated IMQ-induced psoriasis-like dermatitis in Kprp heterozygous mice
To confirm the role of KPRP in psoriasis-like dermatitis, we first applied IMQ cream to the back skin of wild-type, Kprp +/− or Kprp −/− mice and examined the clinical and histopathological characteristics.
Then, IMQ-induced psoriasis-like skin inflammation was evaluated by scoring skin erythema, as well as skin pathological changes.Time-series erythema scores had a significant decrease in Kprp +/− mice; however, no significant difference in Kprp −/− mice compared to wild-type mice on Days 5-6 (Figure 1A).These results indicate that KPRP is a clinical exacerbating factor for psoriatic dermatitis.
The lack of phenotype in KO mice suggests that a complementary mechanism may have worked, and a similar phenomenon was observed in another previous report on atopic dermatitis. 12Therefore, in this study, we planned to explore how KPRP contributes to psoriasiform dermatitis.

| Decreased expression of β -defensins in Kprp heterozygous mice
Considering the localized expression of KPRP to the upper granular layer of the epidermis, 12 we examined differences in gene expression profiles in the IMQ-treated epidermis by RNA-Seq between wild-type, Kprp +/− and Kprp −/− mice in IMQ-induced psoriasis-like dermatitis model.RNA was extracted from the epidermis on Day 2; then, each sample (300 μg total RNA) was pooled from three mice.
The heatmap was created regarding genes that have been reported to be expressed in keratinocytes and/or be involved in the pathogenesis of psoriasis.Several molecules related to psoriasis showed remarkable expression changes in heterozygous mice (Figure 1B).
In particular, given that the phenotype was observed only in the heterozygous mice in oxazolone and ovalbumin-induced skin inflammation 12 and our IMQ-induced skin inflammation (Figure 1A), we decided to focus on molecules known to be involved in psoriasis-like dermatitis and expressed in keratinocytes that showed little difference between wild-type and Kprp −/− but showed a decrease or increase only in Kprp +/− mice.Our RNA-Seq experiments revealed that the expression levels of Tnf, Il36g and Ccl20 in Kprp +/− were comparable to those in the wild-type (Figure 1B).Moreover, in terms of the IL-17 family, while IL-17A and IL-17F are products of activated lymphocytes but not keratinocytes, IL-17C are products of epidermal keratinocytes which drive feed-forward psoriatic skin inflammation. 25Although we therefore focused on IL-17C in RNA-Seq data, Il17c mRNA expressions were comparable between wild-type and Kprp +/− epidermis.On the contrary, some antimicrobial peptides and proteins (AMPs) were decreased only in samples from Kprp +/− , suggesting that these may contribute to IMQ-induced dermatitis in Kprp+/− mice (Figure 1B).Recent studies revealed that keratinocytes produce AMPs which activate immune cells and enhance the production of some of Th17 and Th1 cytokines in the molecular pathogenesis of psoriasis, 26 especially in pre-psoriatic skin. 27It is known that the expression of various AMPs is present mainly in the stratum corneum, to a lower extent also in the stratum granulosum, spinosum and stratum basale. 28In particular, AMPs such as DEFB4 and DEFB14 are produced by epithelial cells and they are reported to be increased in psoriasis lesions. 29Our RNA-Seq data showed decreased expression levels of Defb4 and Defb14 in Kprp +/− epidermis.To confirm the results obtained by RNA-Seq, additional confirmation by qPCR was performed.qPCR results showed the same trend as RNA-Seq data for multiple molecules.
In particular, expression levels of Defb4 and Defb14 decreased in Kprp +/− mice compared to those in wild-type or Kprp −/− mice (Figure 1C).Consistent with the results described above, further immunohistological examination revealed that staining signals of DEFB4 in epidermal cells were decreased in Kprp +/− mice compared to wild-type or Kprp −/− mice (Figure 1D).

| Increased expression of LPAR1 in Kprp heterozygous mice
Epidermal thickening is known as one of the clinical and histological features of psoriasis.Related to the increased epidermal thickening in IMQ-treated mice, we next focused on the lysophosphatidic acid (LPA) receptors in RNA-Seq data for the following reasons.LPA, a member of the lysophospholipid family of bioactive lipids, is known to bind to at least six G-protein coupled receptors (LPAR1-6) 30 and mediate diverse biological actions such as tissue repair and angiogenesis. 31,32ratinocytes express several LPA receptors such as lysophosphatidic acid receptor 1 (LPAR1) and LPAR5, 16,33 and LPA induces keratinocyte differentiation through the LPAR1/LPAR5 receptors. 16RNA-Seq analysis revealed that the expression of LPAR1 but not LPAR5 was increased in Kprp +/− mice (Figure 1B).To confirm the results observed by RNA-Seq, additional confirmation by qPCR analysis, using the back skin after IMQ application, also showed significantly increased Lpar1 expression in Kprp +/− mice compared to those in wild-type or Kprp −/− mice (Figure 1C).Incidentally, when naive back skin without IMQ application was analysed in the same way, heterozygous mice similarly showed a significant increase in Lpar1 mRNA expression compared to wild-type and homozygous mice (Figure S1).Given that a recent report showed the high expression of LPAR1 and LPAR5 in skin keratinocytes, 16 qPCR was also performed for Lpar5 to rule out complementary LPAR5 expression fluctuations and possible effects on LPA signals.Comparable Lpar5 expression in the skin was observed between wild-type and Kprp +/− mice (Figure 1C), showing that the upregulation of LPA1 was not a complementary phenomenon caused by the change in LPAR5 expression.

| KPRP transduction in immortalized human keratinocytes leads to increased DEFB4 and decreased LPAR1 expression levels
We next tried to confirm that KPRP controls upstream of gene expression of AMPs and LPAR1 in epidermal cells using gene-transduced HaCaT cells, a spontaneously immortalized keratinocyte cell line, carrying HaloTag-hKPRP or HaloTag-control.Results from qPCR analysis showed upregulated DEFB4 (the ortholog of mouse Defb4) and downregulated LPAR1 expression in HaCaT HaloTag-hKPRP cells compared to HaloTag-control cells (Figure 1E).These results indicated that expression of AMPs and LPAR1 is directly regulated by KPRP or indirectly regulated by any humoral factors in an autocrine/paracrine manner.

| Kprp heterozygous mice showed less IL-23/IL-17 axis and IL-17-producing cells in IMQ-treated skin
Mouse Defb4 and Defb14 are the human orthologs of human DEFB4 (β-defensin 2, hBD2) and DEFB103 (β-defensin 3, hBD3), respectively. 34Human β-defensins are highly expressed in skin lesions in psoriasis patients. 26Furthermore, TNF-α and IL-17A promote DEFB4 secretion in a synergetic manner, 35 and secreted DEFB4 was reported to act as a ligand for chemokine receptor 6, 36 which regulates T helper type 17 (Th17) cell migration to the inflamed psoriatic skin. 37Moreover, mouse DEFB14 stimulates Langerhans cells to produce IL-23, which is important for maintaining Th17 responses in psoriasis-like inflammation. 38Here, we used the IMQ-induced skin inflammation model, which recapitulates the hallmarks of human psoriasis, including infiltration of γδ T cells and Th17 cells. 13Given these known reports and our data showing reduced expression of β-defensins in IMQ-treated Kprp +/− mouse skin, we next examined the mRNA expression levels of psoriasis-related cytokines in imiquimod-induced psoriasiform skin lesions.Then, imiquimod-treated skin from Kprp +/− mice showed significantly reduced mRNA levels of Il17a and Il22, and a non-significant downward trend in those of Il23a (Figure 2A), indicating the pro-inflammatory effects of KPRP.On the contrary, in terms of Il1b, Il12b and Tnf, no difference was observed between wild-type and Kprp +/− (Figure 2A).
[41] Furthermore, regarding the IMQ-induced skin inflammation model, unconventional γδ T cells migrate into the skin and express cytokines such as IL-17A, which play critical roles in the development of psoriatic inflammation.In contrast to resident γδ hi T cells in mouse epidermis which have high levels of the γδ receptor but do not express significant levels of IL-17A, γδ low T cells in IMQ-treated mouse skin have low to intermediate levels of the γδ receptor and serve as the major producers of IL-17A in the psoriatic epidermis. 37,39,40,42,43en, we next prepared single-cell suspensions of the dermis from the skin of wild-type and Kprp +/− mice on Day 5. Detailed fractionation analysis further revealed that the percentage of γδ low T cells was decreased in Kprp +/− mice compared to wild-type control mice (Figure 2B,C).On the contrary, percentages of TCRβ-positive and γδ hi T cells in Kprp +/− mice were comparable to that in wild-type (Figure 2B,C).These results suggest that the reason why the expression levels of IL-17/IL-23 axis-related cytokines were decreased in the heterozygous mouse was due to the decrease of IL-17Aproducing γδ low T cells infiltrating the IMQ-treated skin.

| Kprp heterozygosity leads to epidermal hyperplasia in the psoriatic skin inflammation
Our RNA-Seq and qPCR analysis showed increased expression of Lpar1.LPA induces proliferation in both human and mouse keratinocytes. 15In addition, LPAR1 is known to regulate keratinocyte differentiation. 16These results and pieces of literature motivated us to further investigate the effects of differences in KPRP expression on epidermal cell hyperplasia.In particular, histological analysis was performed using mouse dorsal skin samples on Day 5. Histological examination showed that cell layers in the epidermis were increased in Kprp +/− mice (Figure 3A  Psoriasis is characterized by the abnormal proliferation/differentiation of keratinocytes. 44There are several reports of attempts to apply LPA externally to the animal skin.In particular, LPA promotes wound healing with neoepithelial thickness and induces histiocytemacrophage immigration in the rat skin. 45Moreover, topical application of LPA causes epidermal thickening by promoting keratinocyte proliferation without inflammation or erythema, 15 and repeated application of LPA has been reported to improve water transpiration in dry-skinned mice. 16In terms of LPA and psoriatic inflammation, LPA mediates imiquimod-induced psoriasis-like dermatitis by promoting keratinocyte proliferation through LPAR1. 46When we explored the effects of LPA on the IMQ-treated skin using wild-type mice, epidermal thickening by IMQ application was significantly enhanced by adding LPA compared to not adding it (Figure S2).Therefore, experiments using LPAR1-specific inhibitors in vivo were considered useful for confirming whether enhanced IMQ-induced epidermal thickening in Kprp heterozygous mice is due to the upregulation of LPAR1.
AM966 is known to be a highly selective LPA1 antagonist. 47AM966 inhibited LPA-induced intracellular calcium release and chemotaxis in vitro.In addition, blocking LPAR1 in vivo with AM966 attenuated bleomycin-induced lung injury and fibrosis 24 and gastrointestinal motility in mice. 48Then, we applied this antagonist to the IMQinduced skin inflammation model using wild-type and Kprp +/− mice.
The epidermal thickness of the skin on Day 5 from those Kprp +/− and wild-type mice under the AMM966 administration was comparable (Figure 3C,D), showing that one of the reasons for the thickening of the epidermis in Kprp +/− mice under psoriatic skin inflammation is increased expression of Lpar1 in the epidermis.

| DISCUSS ION
To the best of our knowledge, this is the first report suggesting that KPRP expression affects murine psoriatic skin inflammation.In Kprp heterozygous mice, but not in homozygously deleted mice, the expression of β-defensins in keratinocytes was decreased.In addition, the IL-23/IL-17 axis-related expression in the dermis was also decreased along with the number of IL-17-producing lymphocytes.
Apart from these observations, increased Lpar1 epidermal expression in Kprp heterozygous mice was implicated to lead to epidermal hyperplasia.Our results suggest that KPRP contributes to the pathogenesis of psoriatic dermatitis through multiple mechanisms.
In our study, the reason has not been elucidated why the phenotype is strongly observed in Kprp +/− mice but not in Kprp −/− mice.
A similar trend was also seen in our previous report on atopic skin inflammation. 12Several redundant mechanisms might exist to compensate for the loss of KPRP in keratinocytes.For our reference, cornified envelope molecules are known to maintain skin barrier function with compensatory mechanisms. 8Then, in our speculation, partial reduction of KPRP levels in Kprp +/− mice may not lead compensatory mechanism, which may work in Kprp −/− mice with increased additional factors to cover the function of KPRP.Human KPRP is known to closely interact with loricrin in both regulation of mRNA expression and localization in the skin. 12Furthermore, the proteomic analysis showed that KPRP directly or indirectly interacted with some components of the desmosome, especially with plakophilin 1. 12 Then, it may be possible that these interacting molecules act in a complementary manner.Further development of research on these intermolecular interactions is desired in the future.
The detailed mechanism of how changes in the expression level of KPRP are linked to changes in the expression of β-defensins and LPAR1 has not been clarified.Although gene transfer experiments using HaCaT suggest that changes in KPRP are directly related to the expression control, it might be also possible that KPRP affects the expression of some factor that acts on keratinocytes in an autocrine/paracrine manner.For example, the expression of β-defensins in keratinocytes is known to be upregulated by IL-1β. 49Given that IL-1β is slightly reduced in the data in Figure 1B, decreased IL-1β expression in Kprp +/− keratinocytes may affect expression levels of β-defensins.In terms of these discussions, our study has a limitation.
We here use mice with systemic gene deletion of Kprp, making it difficult to establish whether KPRP in keratinocytes really contributed to the phenotype.Strictly speaking, keratinocyte-specific knockout mice should be used to make a clear conclusion.However, given that KPRP is specifically expressed in the epidermis, we believe that the conditional mouse is not desperately needed.
The treatment with LPAR1 antagonist AMM966 results in dimin- In murine psoriasis inflammation, KPRP seems to work as a disease-exacerbating factor (Figure S1).If we think inflammation is more of a clinical problem than acanthosis, KPRP may need to be suppressed in psoriasis patients.On the contrary, in atopic dermatitis-like inflammation, KPRP behaves as a disease-relieving factor in some mouse models. 12Although these seem contradictory, these different skin diseases have different roles for keratinocytes in pathology.In fact, barrier abnormalities in keratinocytes are important in atopy, and the mechanism of exacerbation of atopic dermatitis in Kprp +/− mice is based on changes in barrier dysfunction. 12On the contrary, our data which can adequately align reads onto the location including splice sites in the genome sequence.The aligned reads were subjected to downstream analyses using StrandNGS 3.0 software (Agilent Technologies).The read counts allocated for each gene and transcript (RefSeq version 2013.4.1) were quantified using a Trimmed Mean of M-value (TMM) method.

F I G U R E 1
Attenuated psoriasis-like dermatitis in Kprp heterozygous mice and altered expression of β-defensins and LPAR1 by altered KPRP expression in keratinocytes.(A) (Upper panel) Time course of erythema scores in wild-type (WT), Kprp +/− or Kprp −/− mice.Representative data from one of two independent experiments are shown.(Lower panel) Representative images of imiquimod (IMQ)treated back skin of mice on Day 5. Bars = 50 μm.(B) Heatmap from RNA sequencing analysis depicting scaled expression [TMM (Trimmed Mean of M-value)] of selected genes expressed in keratinocytes and/or involved in the pathogenesis of psoriasis.RNA was extracted from the epidermis and pooled from three mice for each sample.(C) Quantitative real-time PCR analysis of Defb4, Defb14, Lpar1 and Lpar5 mRNA in WT, Kprp +/− and Kprp −/− mouse skin at Day 2 of IMQ treatment.Each dot represents one mouse (n = 3-7 mice for each group).(D) Representative images of immunohistochemical staining for DEFB4 in IMQ-induced psoriatic skin tissue after 6 days of IMQ treatment from wild-type, Kprp +/− and Kprp −/− mice.The results are representative of six mice.Bars = 50 μm.(E) Quantitative real-time PCR analysis of DEFB4 and LPAR1 mRNA in KPRP or control-transduced immortalized HaCaT cells (duplicate data).Each experiment was repeated at least twice.Error bars represent the standard error of the mean.*p < 0.05, **p < 0.01, ***p < 0.001.

F I G U R E 2
Kprp heterozygous mice showed less IL-23/IL-17-axis cytokines expression and IL-17-producing cells in imiquimod-treated skin.(A) Quantitative real-time PCR analysis for psoriasis-related cytokines in imiquimod-treated skin at Day 2 from wild-type (WT) and Kprp +/− mice (n = 6 mice for each group).(B) Dermal cell suspensions of imiquimod-treated WT and Kprp +/− mouse back skin at Day 4 were stained with antibodies specific for surface markers including CD45.2, TCRβ and TCRδ/γ, followed by flow cytometric analysis.Graphs show the percentages of TCRγδ low , TCRγδ hi and TCRβ-positive population in the CD45-positive dermal cells from WT and Kprp +/− mice (n = 8 and 6 mice, respectively).(C) Representative plots from flow cytometric analysis were shown.Flow cytometric analysis of TCRβ and TCRγδ expression by dermal cell suspensions pregated on CD45-positive and cell viability marker-negative cells from the indicated mice.Numbers show the fraction of cells in the indicated gate.Each experiment was repeated at least twice.Error bars represent the standard error of the mean.*p < 0.05.

F I G U R E 3
,B), indicating that KPRP negatively regulates epidermal hyperplasia.Enhanced epidermal hyperplasia under imiquimod-induced psoriatic skin in Kprp +/− mice is mediated via LPA-LPAR1 axis.(A, B) Histological evaluation of the imiquimod-treated back skin from wild-type (WT), Kprp +/− and Kprp −/− mice on Day 5. (A) Histological presentation stained with haematoxylin and eosin (H&E) of the skin from WT, Kprp +/− and Kprp −/− mice.Representative pictures from each group.Bars = 100 μm.(B) Numbers of epidermal cell layers were calculated per high-power field from back skin from WT, Kprp +/− and Kprp −/− mice (n = 6-8 mice in each group).(C, D) Histological evaluation of the imiquimod-treated back skin (Day 5) from WT and Kprp +/− mice, which were orally administered with vehicle or LPA1-antagonist (AM966) twice a day at the same time.(C) Histological presentation stained with H&E of the skin from WT and Kprp +/− mice.Representative pictures from each group.Bars = 100 μm.(D) Numbers of epidermal cell layers were calculated per high-power field from back skin from WT and Kprp +/− mice on Day 5 (n = 6 mice in each group).Error bars represent the standard error of the mean.***p < 0.001.

2 (
ished differences in the epidermal thickness between Kprp +/− and wild-type mice (Figure3C,D), suggesting that LPAR1 is involved in psoriatic skin inflammation, especially in enhanced epidermal hyperplasia in IMQ-treated Kprp +/− mouse skin.Needless to say, there should be the possibility that molecules other than LPAR1 are involved in the enhancement of acanthosis in heterozygous mice, and further investigation is required to verify this.Regarding psoriatic skin inflammation and LPAR1, LPAR1 antagonist was reported to alleviate IMQ-induced psoriasis-like symptoms in mice, and in particular, LPAR1 signalling is involved in cell cycle progression in keratinocytes.46Considering these results and publications, if it becomes possible to increase the expression of KPRP in the epidermis, combination with LPAR1 antagonist may effectively lead to the reduction of epidermal hypertrophy and the accompanying excess scale in the skin diseases such as psoriasis.Kprp +/− mice have less γδ low T cells in the IMQ-induced psoriatic skin inflammation.In patients with psoriasis, β-defensin DEFB4) is reported to be a responsive biomarker of IL-17A-driven skin pathology.50Moreover, nucleic acid complexes, such as AMPs including DEFB4 within the upper layer of the skin, stimulate plasmacytoid dendritic cells during the onset of psoriasis.Plasmacytoid DCs produce cytokines such as IFN-γ, TNF-α and IL-23 to interact with myeloid dendritic cells, leading to clonal expansion of Th17 cells which produce IL-17 and IL-22. 51These suggest the following: Reduced KPRP expression in keratinocytes might bring the reduced expression of antimicrobial peptides, thereby reducing the proliferation of IL-17-producing cells in the psoriatic skin inflammation.
on attenuated psoriatic skin inflammation in Kprp +/− mice are possibly due to decreased expression of humoral factors involved in the exacerbation of inflammation.Thus, KPRP in keratinocytes shows different roles for each skin disease, which should be important and noteworthy in considering disease-specific treatment strategies.In summary, here we showed the unique roles of KPRP in murine psoriatic skin inflammation.Although further studies are required to clarify the specific and detailed mechanism underlying these observations, our findings on KPRP and future analysis using clinical human samples can pave the way for a novel design for treating patients with psoriasis.AUTH O R CO NTR I B UTI O N SAiMatsuno conducted most of the experiments, analysed the results and wrote the manuscript.Hayakazu Sumida supervised and designed the study, analysed the results, wrote the manuscript and discussed the results.Hirofumi Nakanishi, Yoshifumi Ikeyama and Hiroto Katoh conducted experiments.Tsuyoshi Ishii and Ayumi Yoshizaki discussed the results.Issei Omori, Hinako Saito, Okuto Iwasawa and Ayaka Sugimori analysed and discussed the results.Shumpei Ishikawa and Shinichi Sato discussed the results, supervised experiments and revised the manuscript.