IARS was highly expressed in psoriatic epidermis
To investigate the expression profile of IARS in healthy donor epidermis and psoriatic epidermis, immunofluorescence (IF) staining against IARS was performed in skin biopsies obtained from healthy donors (n=3) and psoriasis patients (n=6). Compared with normal epidermis, IARS has significantly been upregulated in psoriatic epidermis (Fig. 1A, B). IARS was expressed in all epidermal layers in lesional skin. In perilesional skin, it was only expressed in the suprabasal layers of the epidermis as shown in Figure 1a. We calculated the percentage of IARS positive cells at 1mm, 2mm, 3mm, 4mm, 5mm, and 6mm from the lesional area, which represented decreasing epidermal thickness. As shown in Figure 1b, the percentage of IARS positive cells gradually decreased parallel to reduced thickness. In order to further confirm the up regulation of IARS in psoriatic lesions, we detected its protein level in normal and psoriatic epidermis by Western blot. Compared with normal epidermis, the protein level of IARS was increased in the psoriatic epidermis (Fig. 1C). Meanwhile, normal human epidermal keratinocytes (NHEKs) and psoriasis lesional epidermal keratinocytes (PLEKs) were used to detect the expression of IARS at a transcriptional level by qRT-PCR. Compared with NHEKs, the mRNA level of IARS was significantly up regulated in PLEKs (Fig. 1D). We searched the GEO database, were two published datasets GSE13355[23] and GSE53552[28] showed that IARS expression was significantly increased in the lesional skin of psoriasis patients, which was reversed after blocking IL-17 signaling by brodalumab (Fig. 1E, F).
IMQ-induced psoriasis-like skin inflammation in mice was alleviated by topical mupirocin
Since IARS was highly expressed in psoriatic epidermis, we used the inhibitor of IARS, mupirocin (MUP), to further explore the role of IARS in psoriasis. To assess whether topical mupirocin could relief IMQ-induced psoriasis-like skin inflammation in vivo, the experimental mice were treated with 6 different methods: 1) blank control (vehicle-treated mice); 2) topical application of IMQ for 6 consecutive days (IMQ-treated mice); 3) topical application of MUP and IMQ for 6 consecutive days (MUP+IMQ-treated mice); 4) topical application of IMQ for 6 consecutive days, accompanied by MUP at the 3rd to 6th day (IMQ-6days&MUP-4days-treated mice); 5) topical application of IMQ for 6 consecutive days,, with MUP application at the 4th to 6th day(IMQ-6days&MUP-3days-treated mice); 6)topical application of MUP for 2 consecutive days, followed by IMQ for another 4 days (MUP-2days&IMQ-4days-treated mice)(Figure 2a). Compared with the vehicle-treated mice, typical psoriasis-like skin lesions were shown in the IMQ-treated mice. However, these lesions were significantly relieved in the MUP+IMQ-treated mice with reduced scales and erythema. In group 4 and 5, we found that decreased application of mupirocin resulted in exacerbation of psoriasis-like lesions. In addition, mice developed typical psoriasis-like skin lesions even when mupirocin was applied 2 days in advance. The PASI scores at the 6th day of MUP+IMQ-treated mice were significantly lower than that of the IMQ-treated mice (n=5, *P< 0.05, **P< 0.01) (Fig. 2C). Then, H&E (hematoxylin-eosin) staining was performed to evaluate epidermal thickness and histopathological changes. Compared with the IMQ-treated mice, the MUP+IMQ-treated mice showed an obvious decrease in the severity of psoriasiform skin changes (Fig. 2B). Moreover, the epidermal thickness was significantly reduced (n=5, *P< 0.05, **P< 0.01) (Fig. 2C). In addition, keratin 14(K14) and Ki67, as cell proliferation-related indicators, were detected using immunohistochemistry. Upon IMQ induction, K14-positive cells were located in all layers of the epidermis. However, the percentage of K14-positive cells was significantly decreased after application of mupirocin (Fig. 2D). Similar results of Ki67 positive cells were observed between these two groups (Fig. 2D). Furthermore, the ear thickness was significantly decreased after IARS-antibody injection (n=5, *P< 0.05, **P< 0.01) (Fig. 2E, F). Taken together, the phenotypic and histological features of IMQ-induced psoriasis-like skin inflammation were improved by topical mupirocin and IARS-antibody injection treatment.
Mupirocin inhibited epidermal IARS in IMQ-induced psoriasis-like mouse model with decreased expression of cytokines and chemokines in the IL-17 signaling pathway
To determine, whether mupirocin is able to decrease epidermal IARS and inflammatory cytokine- and chemokine expression, we compared the expression of IARS and some cytokine- and chemokine expression levels among vehicle-treated mice, MUP+IMQ treated mice and IMQ-treated mice. As shown in Figure 3a, IMQ-treated mice showed full-layer IARS expression in the epidermis, which was consistent with psoriatic lesions of patients. Furthermore, when treated with MUP and IMQ, IARS protein level was significantly decreased (Fig. 3A). Immunoblotting results showed similar expression differences among these 3 groups (Fig. 3B). To look for the possible signaling pathway, RNA sequencing (RNA-Seq) was performed with mouse epidermis. Differentially expressed genes (DEGs) were discovered by DESeq with the threshold of |log2 fold change| > 2 and adjusted P value<0.05. Then, all down-regulated DEGs between MUP+IMQ-treated and IMQ-treated were analyzed by Kyoto Encyclopedia of Genes and Genomes (KEGG). These DEGs were statistically enriched in IL-17 signaling pathways. Among them, the top 5 signaling pathways were the IL-17 signaling pathway, the cytokine-cytokine receptor interaction, pantothenate and CoA biosynthesis, amoebiasis and salmonella infection (Fig. 3C). It is well known that the IL-23/IL-17 immune axis is a key driver in the development of psoriasis. Here, 15 DEGs were enriched in the IL-17 signaling pathway, including IL-17 family (IL-17A, IL-17B and IL-17F), chemokines (CXCL1, CXCL2 and CXCL3), and S100 protein family members (S100A8 and S100A9), etc. (Fig. 3D). RNA-Seq results of mouse epidermis suggested that many cytokines (IL-17 family, IL-1β) and chemokines (CXCL1, CXCL2 and CXCL3) were reduced in MUP+IMQ-treated mice. To further analyze their expression at the protein level, Luminex Multi-Analyte Assay was applied to mouse epidermis. As shown in Figure 3E, chemokines (CXCL2), and S100 Protein family (S100A9) were highly expressed in the epidermis of IMQ-treated mice compared with vehicle-treated and MUP+IMQ-treated mice, which was consistent with the RNA-Seq results. IL-17 family cytokine (IL-17E), chemokines (CCL3, CCL19), and Serpin E1 were down regulated in MUP+IMQ-treated mice. Among them, the concentration of IL-17A was detected by ELISA detection kit for epidermal protein. Results showed that the IL17A concentration in the epidermis of IMQ-treated mice was significantly higher than that in the vehicle-treated- and MUP+IMQ-treated mice as shown in Figure 3D. All of these findings were consistent with the transcriptome expression.
Mupirocin decreased the inflammatory cell infiltration in IMQ-induced mouse model
The infiltration of inflammatory cells in skin and lymph nodes (LN) was analyzed by IF, IHC and flow cytometry. In the IMQ-treated mice group, the number of CD4+ T cells and CD8+ T cells were significantly increased in the skin and draining lymph nodes compared with vehicle-treated mice, which could be reversed by MUP treatment at the same time (Fig. 4A, B, F). As “Munro” micro abscess is a typical pathological feature of psoriasis[29], we studied the number of neutrophils in the skin and the LN. Neutrophil numbers increased in the IMQ-induced psoriasis-like skin inflammation, but decreased significantly after MUP application (Fig. 4C). Similar results were observed in LN (Fig. 4J). Flow cytometry results of the epidermis showed that compared with IMQ-treated group, the percentage of LCs in the vehicle-treated and MUP+IMQ-treated mice were increased (P<0.05, Fig. 4D-J). Through the participation of skin draining LN, the initial immune response will generate a self-sustaining inflammatory cycle to maintain inflammation. The flow cytometry of LN showed similar results. The proportion of CD3+CD4+, CD3+CD8+ cells (Fig. 4E), neutrophils (Fig. 4F), IL-17A secreting gdT cells (Fig. 4G), dendritic cells (Fig. 4H), macrophages (Fig. 4I) and in the IMQ-treated mice were significantly higher than that in the vehicle-treated and MUP+IMQ-treated mice. (P<0.05 and P<0.01, Fig. 4J).
Mupirocin inhibited the proliferation of PLEKs and promoted their apoptosis
As keratinocytes play an important role in the pathogenesis of psoriasis, we further explored the effect of IARS on keratinocytes. PLEKs were treated with several concentrations of mupirocin (0μM, 10μM and 50μM) for 48h. Then the effects on the proliferation and apoptosis activities of keratinocytes were studied. First, the cell proliferation of the mupirocin-treated keratinocytes was determined by EdU assay. Compared with the control group, the number of EdU-positive cells were significantly decreased after mupirocin treatment in a dose-dependent manner (P<0.05; P<0.01; Fig. 5A). Moreover, the cell density of mupirocin-treated PLEKs was also markedly reduced (P<0.05; P<0.01; Fig. 5B). Also, we found that mupirocin could induce the expression of Bax and cleaved-caspase3 in keratinocytes, especially at the concentration of 50μM (Fig. 5C). In addition, flow cytometry with Annexin V-FITC/PI double staining was performed to determine the percentage of apoptotic cells. When the concentration of mupirocin reached 50μM, a significant increase in the percentage of apoptotic cells in the mupirocin-treated PLEKs (P<0.05; Fig. 5D, E) was detected. Briefly, the results above suggest that mupirocin could inhibit the proliferation of PLEKs and promote their apoptosis.
Regulation of IARS in PLEKs and NHEKs
To investigate the regulatory mechanism of mupirocin on keratinocytes, PLEKs were treated with various concentrations of mupirocin (0-100μM) for 24h. As shown in Fig 6A, the expression of IARS was dramatically inhibited in MUP concentration-dependent way. Among these groups, the IF results of IARS showed similar results in PLEKs (P<0.01; Fig. 6E, F). Interestingly, phosphorylation of STAT3 was also inhibited when the concentration reached 75μM (Fig. 6A). As a result, we continued to use high-concentration of MUP,100μM, to treat PLEKs at different time point. The results of Western blot analysis showed that the expression of p-STAT3 was remarkably down-regulated from 30min until 120min (Fig. 6B). Thus, mupirocin was able to inhibit IARS in keratinocytes, maybe through STAT3 signaling pathway. In addition to the inhibitory effect of mupirocin, it is necessary to explore whether drugs including clobetasol and calcipotriol could have similar effects in IARS. Here, IARS expression was examined in PLEKs using Western blot following a 48h treatment with clobetasol (Clob, 1μM and 10μM) and calcipotriol (Cal, 20nM and 200nM). After calcipotriol treatment, the expression of IARS was decreased in a dose-dependent manner, concomitant with a slight decrease in STAT3 phosphorylation levels (Fig. 6C). In contrast, we did not observe significant changes of IARS or p-STAT3 levels when treated with clobetasol (Fig. 6C). Moreover, IL-17A (200ng/ml) combined with TNFα (10ng/ml), IL-23 (100ng/ml), and IL-6 (100ng/ml) could promote IARS expression and STAT3 phosphorylation in NHEKs (Fig. 6D).