Keratinocyte-Specific CCN1 Modulation Driven Regulation of Keratinocyte Proliferation and Inflammation in Psoriasis

doi:10.21203/rs.3.rs-3640474/v1

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Keratinocyte-Specific CCN1 Modulation Driven Regulation of Keratinocyte Proliferation and Inflammation in Psoriasis

https://doi.org/10.21203/rs.3.rs-3640474/v1

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Background: Psoriasis is a chronic autoimmune disease characterized by the hyperproliferation of keratinocytes and inflammatory cell infiltration. CCN1 is a matricellular protein that plays a crucial role in cell proliferation, differentiation, and adhesion, which are accepted as psoriasis-relevant.

Objective: Our study evaluated the function of CCN1 in psoriasis treatment using keratinocyte-specific Ccn1 transgenic mice.

Methods and Results: We constructed keratinocyte tissue-specific Ccn1 transgenic mice and induced a psoriasis model with imiquimod (IMQ). The results exhibited that, compared to the psoriatic model group, the keratinocyte-specific ablation of Ccn1 ameliorated IMQ-induced psoriasis-like skin lesions in mice. Furthermore, the downregulation of PCNA⁺ cells, NF-kB P50⁺, F4/80⁺ macrophage, CD3⁺ T lymphocytes, and pSTAT3 was confirmed by immunohistochemical staining in the epidermis of psoriatic lesions. Keratinocyte-specific Ccn1 overexpression also increased inflammation in mice after exposure to imiquimod, exhibiting more pronounced red and scaly plaques, epidermal hyperproliferation, and abnormal differentiation of keratinocytes. Thus, targeting CCN1 could broadly improve psoriasis treatment outcomes.

Conclusion: Overall, epidermal-specific CCN1 exerts a key player in keratinocyte proliferation and immunoinflammation in the psoriasis setting. Our study yields valuable insights into the pathogenesis and potential treatment of the disease.

CCN1

Psoriasis

keratinocyte

hyperproliferation

Immunoinflammation

Psoriasis is a common, chronic immune-mediated inflammatory skin disease characterized by increased keratinocyte proliferation and inflammation [1]. There are at least 100 million psoriatic individuals worldwide, with a prevalence of 0.51%-11.43% according to geographic location and race [2]. A consensus has been reached that psoriasis patients have a higher risk of developing several comorbidities, including metabolic syndrome [3], cardiovascular disease [4], and mental illness [5].

Keratinocyte differentiation and hyperproliferation are taken as hallmarks of psoriasis pathogenesis [6]. Previous study of our research group has confirmed that activated keratinocytes lead to characteristic skin lesions of psoriasis, which are characterized by raised, well-demarcated, erythematous oval plaques [7] covered with silvery scales [8]. The presence of abnormal keratinocytes also triggers inflammation in the skin of psoriatic mice [9], highlighting the critical role of inflammation in the pathogenesis of psoriasis[10].

Accumulating evidence has indicated that cellular communication network factor 1 (CCN1) is involved in psoriasis [11]. According to the research findings by Wu et al. [12], the expression of CCN1 was significantly increased in skin lesions of psoriasis patients as compared with normal donors. CCN1, also called Cysteine-rich 61 (Cyr61), is a multifunctional protein that is secreted into the extracellular matrix (ECM) and partakes in cellular functions such as cell proliferation, migration, adhesion, angiogenesis, inflammation, and tumorigenesis through binding to various integrin receptors [12–16]. Psoriasis severity is exacerbated by elevated CCN1 levels [12]. In vitro investigations showed that CCN1 stimulated keratinocyte proliferation in a dose-dependent manner [17]. Cytochemokines, particularly IL-17, IL-8, and IL-1β, are the ones that cause inflammation and activate keratinocytes. Through multiple signal transduction pathways, CCN1 plays a pro-inflammatory role in the development of psoriasis and upregulates keratinocyte cytokine production [18]. This, in turn, promotes inflammatory responses, keratinocyte differentiation, and hyperproliferation [11, 18, 19]. Additionally, in IMQ-induced psoriasis-like mice, inhibiting CCN1 with a specific mAb remarkably reduced epidermal inflammation and ameliorated disease progression [12]. Furthermore, CCN1 is intimately related to keratinocyte activation in the pathogenesis of psoriasis [17]. The epidermal hyperplasia in the skin lesions of IL-23-induced psoriasis-like mice is reduced after cutting down Ccn1 expression [17]. Our earlier investigations have also confirmed that CCN1 is capable of stimulating the proliferation of keratinocytes, thereby facilitating wound healing [20]. Thus, CCN1 might be a possible target for ameliorating epidermal hyperplasia and inflammation in psoriasis. The in vitro and in vivo phenotypes of CCN1 are intricately linked to keratinocytes. Nonetheless, the dearth of advanced experimental evidence underscores the imperative for additional validation. To address this, we engineered transgenic mice expressing Ccn1 exclusively in keratinocytes, thereby furnishing elevated-level experimental substantiation for the involvement of CCN1 in psoriasis.

In this study, we aimed to elucidate the physiological function of CCN1 concerning epidermal hyperplasia and inflammation. First, CCN1 was discovered to promote the aberrant proliferation and differentiation of keratinocytes. Next, we found that CD3, F4/80, and NF-kB decreased after knocking out Ccn1 in mice, suggesting that the knockdown of Ccn1 may possess a significant function in the remission of inflammation. Furthermore, more severe psoriatic skin lesions were observed in Ccn1 overexpression mice. Thus, we concluded that CCN1 increased epidermal hyperplasia and inflammation, demonstrating that CCN1 might be a potential target for ameliorating psoriasis.

Animals

Ccn1 ^flox / ^flox and keratin14-Cre mice were purchased from Jiangsu GemPharmatech Co., Ltd. Ccn1^flox/^flox homozygous mice were crossed with keratin14-Cre mice to produce Ccn1^KO mice as previously described [21]. To prevent conceivable genetic background differences, the F4 generation was utilized for all experiments (Ccn1^flox/flox; Cre⁺ and Ccn1^flox/flox; Cre⁻), and Ccn1^flox/flox; Cre⁻ animals were used as controls.

Six to eight-week-old mice weighing 18–25 g were recruited for the present study. Mice were bred and housed under specific pathogen-free conditions, along with adequate food and water. Animal procedures were approved by the Animal Ethics Committee of Shanghai Skin Disease Hospital, School of Medicine, Tongji University (no. 2023 − 101 (Dong) Yu).

Model establishment and interventions

In brief, the hair on the dorsal area of mice was shaved (2 × 2 cm²). Mice were categorized into five groups: control group, psoriatic model group (IMQ group), psoriatic model, Ccn1 knockdown group (IMQ + Ccn1^KO group), and Ccn1 overexpression group (IMQ + Ccn1^OE group) (n = 3). To elicit a psoriasis-like phenotype in mouse skin, the psoriasis model was established with 62.5 mg of 5% IMQ cream on the ear and dorsal skin for 8 days.

Generation of CKO mice via the CRISPR/Cas9 system

We made Ccn1 conditional knockout mice via the CRISPR/Cas9 system. Firstly, two sgRNAs targeting the introns on both sides of the floxed region of Ccn1 were respectively constructed and transcribed in vitro. And the donor vector with the loxp fragment was designed and constructed in vitro. Then Cas9 mRNA, sgRNA, and donor would be co-injected into zygotes. Thereafter, the zygotes were transferred into the oviducts of pseudopregnant ICR females at 0.5 dpc. F0 mice were born after 19 ~ 21 days of transplantation, and all the offspring of ICR females (F0 mice) were identified by PCR and sequencing of tail DNA. And positive F0 mice were genotyped by the methods. Finally, F0 mice were crossed with C57BL/6J mice to build up heterozygous mice.

Generation of cas9-ki mice

We made Ccn1 knockin mice via the CRISPR/Cas9 system. Firstly, one sgRNA targeting the near sequence of the inserted site was constructed and transcribed in vitro. The donor vector with the inserted fragment was designed and constructed in vitro. Then Cas9 mRNA, sgRNA, and donor will be co-injected into zygotes. Thereafter, the zygotes were transferred into the oviducts of pseudopregnant ICR females at 0.5 dpc. F0 mice were born after 19 ~ 21 days of transplantation, and all the offspring of ICR females (F0 mice) were identified by PCR and sequencing of tail DNA. Finally, crossing positive F0 mice with wild-type mice to build up heterozygous mice.

Histological analysis and immunohistochemistry assay

Mice were sacrificed after 8 days. The psoriatic tissues were fixed in 10% formalin and embedded in the paraffin routinely for 48 hours. The sections (5µm) were stained with hematoxylin-eosin (HE) and immunohistochemistry (IHC) after being dewaxed and rinsed with PBS.

For immunostaining, tissue sections were incubated with anti-CD3 (Abcam, ab16669, diluted 1:150), anti-NF-κB P50 (Abcam, ab32360, diluted 1:18000), anti-F4/80 (Abcam, ab300421, diluted 1:5000), anti-PCNA (Abcam, ab29, diluted 1:20000), pstat3 (Abcam, ab32360, diluted 1:200). The HE and IHC staining of skin tissues were observed under a microscope. Images were taken under a digital slice scanner at a magnification of 20×, and the average number of positive cells in each group was calculated. The immunostaining content was quantified using ImageJ software.

Western Blotting assay

Western blotting experiments were performed as described earlier [22]. Briefly, the protein was extracted from the mice's back lesions of psoriasis and subjected to SDS-PAGE. The blocked membranes were incubated with the primary antibody overnight. After washing the membrane, incubated it into the CCN1antibody ^(Proteintech^{, 26689−1−AP, diluted at 1:10}0⁰⁾. The β-actin (Abcam; ab8227; diluted 1:2,000) was chosen as an internal reference.

RNA extraction and quantification

RNA isolation from skin tissue using TRIzol reagent (Takara) Isolation and quantification of total RNA by RT-PCR in accordance with the manufacturer's instructions. The mRNA expression levels of the examined genes were normalized and then determined using the 2^−ΔΔCt method. Primer sequences are shown in Table S1.

Statistical analysis

All statistical analyses were performed using GraphPad Prism 9.0 software and were presented as mean ± standard error of the mean (SEM). The results were analyzed using Student’s t-test or ANOVA. P < 0.05 was considered significant.

Elevated CCN1 levels in keratinocytes coincide with more severe skin lesions in IMQ-induced psoriatic mice

To further investigate the involvement of CCN1 in the development of psoriasis, we constructed keratinocyte tissue-specific Ccn1 transgenic mice and produced a psoriasis model with imiquimod (IMQ). To verify the knockout impact of Ccn1 in keratinocytes, RT-PCR and immunohistochemical assays were performed. The findings demonstrated a substantial drop in both mRNA and protein levels of CCN1 in the skin tissue of Ccn1^KO mice, while an elevation was found in the skin tissue of Ccn1^OE mice in comparison to Ccn1^fl/fl (wild-type, WT) mice (Fig. 1). These results confirmed that Ccn1 was effectively knocked out or overexpressed in keratinocytes, indicating the successful establishment of a keratinocyte tissue-specific Ccn1 transgenic mouse model.

The IMQ-induced psoriasis-like phenotype in mice was examined with ear thickness and PASI (the Psoriasis Area and Severity Index). Compared with the IMQ group, ear thickness and PASI were significantly reduced in the IMQ + Ccn1^KO and raised in the IMQ + Ccn1^OE groups (Fig. 2). The results showed that the downregulation of Ccn1 in keratinocytes led to an improvement in skin lesions observed in mice with IMQ-induced psoriasis-like symptoms. Conversely, the overexpression of CCN1 in keratinocytes was found to enhance the occurrence of scaly psoriatic plaques. These results provided evidence supporting a positive correlation between CCN1 expression and the advancement and progression of psoriasis.

CCN1 exacerbates the psoriasis-like pathological features in the epidermis of IMQ-induced psoriatic mice

We further studied the potential relevance of CCN1 to the psoriasis-like pathological features in the epidermis of psoriasis. Hematoxylin and eosin staining of skin sections revealed considerable changes in the thickness of the epidermis among the experimental groups. Consistent with the lesion phenotypic changes, Ccn1 keratinocyte-specific deletion resulted in a notable reduction in both ear and back epidermal thickness, while conversely, overexpression of CCN1 caused epidermal thickening (Fig. 3).

Proliferating Cell Nuclear Antigen (PCNA) is a marker for cell proliferation and is typically detected within the nuclei of cells [23]. The number of PCNA + cells in ear lesions acts as a measure of keratinocyte proliferation. Utilizing immunohistochemical analysis, we found knockdown of Ccn1 dramatically diminished the number of PCNA + cells (Fig. 4), which implies a reduction in epidermal cell proliferation following Ccn1 knockout. These results collectively corroborate our conclusion that CCN1 exacerbates psoriasis-like pathological characteristics in the epidermis of IMQ-induced psoriatic mice. Specifically, CCN1 plays a crucial role in epidermal thickening via keratinocyte proliferation.

CCN1 knockout attenuates inflammation and immune cell infiltration

The proinflammatory property of CCN1 was evaluated by its capacity to upregulate F4/80 and CD3.

The CD3 antigen is associated with the T-cell receptor and is a common T-cell marker [24]. Our observations showed that after Ccn1 removal, the epidermal and dermal superficial CD3 infiltration decreased and the number of CD3 + T cells reduced (Fig. 5). F4/80 antigen was utilized to observe macrophages [25]. Macrophages are recognized to have a vital role in the maintenance of skin inflammation in psoriasis [26]. Changes found in the infiltration of F4/80 + macrophages in IMQ-induced psoriatic mice were consistent with the alterations observed in CD3 + infiltration (Fig. 4). Our findings conclusively illustrated that the keratinocyte-specific knockout of Ccn1 exerted a beneficial effect in mitigating inflammation and diminishing the infiltration of immune cells in the psoriatic stromal area.

Activated NF-kB p50 and pSTAT3 are strongly implicated in inflammation and immunological response [26, 27]. We conducted an assessment of the expression of nuclear NF-kB p50 and pSTAT3 in IMQ-induced psoriatic mice with a keratinocyte-specific knockout of Ccn1 (Figs. 4 and 5). Our findings revealed an extensive reduction in the expression levels of both nuclear NF-kB p50 and pSTAT3. The downregulation of NF-kB p50, a pivotal transcription factor involved in the regulation of pro-inflammatory gene expression [27], indicated a potential mitigation of the inflammatory response. Furthermore, the reduction in pSTAT3 activation, a critical component in immune cell activation and cytokine generation [28], suggests a potential attenuation of immune cell-driven responses.

Collectively, our findings suggest that CCN1 in keratinocytes may be an essential mediator in triggering the inflammatory response in psoriasis. The observed reduction in CD3, F4/80, and NF-kB expression in the CCN1^KO mice support the idea that CCN1 is involved in the upregulation of immune cell infiltration and inflammatory signaling pathways.

Studies have revealed that the increase of CCN1 contributed to the disease phenotypes of psoriasis [17]. It has been shown that CCN1 is emerging as a critical regulator of keratinocyte proliferation and inflammation in psoriasis [18]. However, there is a paucity of high-level experimental evidence confirming the role of CCN1 in keratinocyte proliferation and inflammation. We applied transgenic mice with a keratinocyte-specific Ccn1 gene to build psoriasis models. The findings demonstrated that keratinocyte-specific ablation of Ccn1 ameliorated IMQ-induced psoriasis-like skin damage in mice compared to the psoriasis model group. Further studies indicated the downregulation of PCNA⁺ cells, NF-κB P50⁺, F4/80⁺ macrophages, CD3⁺ T lymphocytes, and pSTAT3 in the psoriatic epidermal lesions. Keratinocyte-specific overexpression of Ccn1 also increased inflammation in mice after exposure to IMQ. Consequently, epidermal-specific Ccn1 contributes significantly to keratinocyte proliferation and immune inflammation in psoriasis.

CCN1 is a key regulator in skin biology as it can alter the critical functions of epidermal keratinocytes and skin fibroblasts [29]. Recent studies [19, 29] have shown that CCN1 protein is highly expressed in the skin lesions of psoriasis patients, as well as in psoriatic mouse skin induced by IMQ or interleukin-23 (IL-23). The utilization of neutralizing antibodies to inhibit the function of CCN1 in the psoriasis-like mouse model yields substantial enhancements in the psoriasis-like skin phenotype including a notable reduction in epidermal thickness and infiltration of inflammatory cells in the dermis. Additionally, following exposure to IMQ, overexpression of keratinocyte-specific CCN1 also increased inflammation in mice, manifested as more pronounced red and squamous plaques, as well as excessive epidermal proliferation and abnormal differentiation of keratinocytes. Our data suggest that this is consistent with the previously reported results [17], indicating that CCN1 plays an important role in the pathological process of psoriasis.

During the onset of psoriasis, there are various cytokine imbalances and abnormal intracellular signal transduction, accompanied by excessive proliferation of keratinocytes as well as dysfunction of macrophages and T cells [6]. This ultimately leads to symptoms such as excessive keratinization of epidermal tissue, disappearance of granular layers, and epidermal microabscess. In severe cases, it can even lead to complications such as arthritis, lymph node inflammation, immune dysfunction, and cardiovascular system disease [32]. Excessive proliferation of keratinocytes is an important pathological feature of psoriasis [6]. PCNA is a proliferating cell nuclear antigen that can reflect the proliferation of keratinocytes. The results of this study showed that PCNA was strongly expressed in the basal layer, spinous cell layer, and granular layer in the model group and was much higher than that in the control group, which was consistent with literature reports [33]. Compared with the model group, the expression of PCNA in the Ccn1^KO group was reduced, and the number of PCNA-positive expression cells was drastically reduced. Collectively, these results imply that keratinocyte-specific Ccn1 knockout could effectively inhibit the proliferation of keratinocytes around the stromal area of psoriatic skin lesions.

Previous researches have shown that immunological staining can identify the activation and aggregation of macrophages in psoriatic skin lesions. Once activated, macrophages can independently trigger the formation of psoriatic-like skin lesions independently of CD4⁺ T cells [34]. Macrophages, as a key natural immune cell, may play an important role in promoting immune disorders, abnormal proliferation of keratinocytes and vascular endothelial cells, leading to the onset of psoriasis by secreting inflammatory mediators such as IL-20, TNF-a, prokinetic protein (PK) 2, and macrophage migration inhibitory factor (MIF) [35]. The exclusive indicator of mouse macrophages, F4/80, has been shown in our experimental findings to substantially decrease in the number when Ccn1 is ablated in keratinocytes [36]. This decline in dermal macrophage infiltration, accompanied by a decrease in the number of F4/80 positive expression cells, suggests that CCN1 may play an essential role in the immunomodulatory processes of psoriasis by influencing macrophage activation and aggregation.

NF-κB, as a key regulator of inflammation, cell proliferation, differentiation, and apoptosis, is also considered to be an important regulator in the pathogenesis of psoriasis [37]. Previous studies have demonstrated that NF-κB is overactivated during the progression of psoriasis. Its activity increases to promote the secretion of pro-inflammatory factors by keratinocytes, while inhibiting NF-κB has a protective effect on psoriasis keratinocytes [38]. NF-κB p50 is activated and translocated to the nucleus, stimulating the proliferation and transcription of inflammatory regulatory genes and facilitating a positive feedback loop in psoriasis [39]. In our current study, NF-κB p50 expression was increased in the model group, but upon Ccn1 knockout, a notable decrease in the number of NF-κB p50⁺ cells in the epidermal nucleus was observed. These findings suggest that CCN1 potentially contributes to the pathogenesis of psoriasis by modulating the expression and activity of NF-κB p50, consequently influencing the inflammatory response and cellular proliferation associated with psoriasis.

T lymphocyte-mediated immunity plays a crucial role in the development and maintenance of psoriasis, and studies have confirmed that the number of CD3⁺ T cells in psoriasis patients' skin lesions is increased, which is consistent with our results [40]. As a central regulator of inflammation and immune response, transcription factor STAT3 has become a key participant in the development and pathological mechanism of psoriasis. Activated STAT3 (STAT3 phosphorylation) has a series of biological activities that regulate cell proliferation, apoptosis, and differentiation [41]. The results of this study showed that the positive expression of pSTAT3 was significantly higher than that of normal skin tissue, which is consistent with previous reports [42]. After the deletion of Ccn1, a significant decline was seen in the expression of pSTAT3, along with the number of cells expressing pSTAT3 positively. The aforementioned findings indicate a potential correlation between the regulatory mechanism of CCN1 and CD3 + T cell immune responses and suggest its potential impact on the inflammatory response and cellular proliferation in psoriasis through the modulation of pSTAT3 expression and activity.

As a result, epidermal-specific CCN1 is crucial for the proliferation of keratinocytes and immune inflammation in psoriasis. However, the mechanism by which CCN1 promotes epidermal hyperplasia and local inflammatory response in psoriasis remains unclear. Exploring the relationship between CCN1 and psoriasis-related inflammatory signaling pathways may serve as a new direction for further research on the mechanism of psoriasis.

In summary, we used IMQ to induce keratinocyte tissue-specific CCN1 transgenic mice to build psoriasis models, further confirming that epidermal-specific CCN1 plays a key role in keratinocyte proliferation and immunoinflammation in the psoriatic environment. Therefore, our study provides valuable insights into the pathogenesis of psoriasis and potential therapeutic approaches. By precisely targeting the expression of CCN1 in psoriatic lesions, we can regulate its positive feedback pathway in the inflammatory microenvironment, which may be an effective strategy for the treatment of psoriasis.

Acknowledgments

The authors state no acknowledgments.

Conflict of Interest

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as potential conflicts of interest.

Author Contributions

TTX, MJZ, and QZ contributed equally. YZ and BL conceived and designed the study. YL, XYF, and YL performed experimental work. JKS analyzed and interpreted the data. MJZ and YZ prepared the original draft. XJD and LK collected the literature and references. YZ and BL reviewed and edited the manuscript. All authors have read and approved the final manuscript.

Funding

This study was supported by the National Key Research and Development Program of China (2018YFC1705305), the National Natural Science Foundation of China (81973860, 82174383) the Shanghai Science and Technology Committee (21Y21920101, 21Y21920102), the Shanghai Development Office of TCM (ZY(2021-2023)-0302, ZY(2021-2023)-0209-13), the Shanghai Clinical Key Specialty Construction Project (shslczdzk05001), the Shanghai Sailing Program (21YF1448100, 21YF1441500, 22YF1441300), the Shanghai Municipal Health Commission Health Industry Clinical Research Special Project (20234Y0075, 20234Y0269), the Shanghai Dermatology Research Center (2023ZZ02017), the Shanghai Dermatology Hospital demonstration research ward project (SHDC2023CRW009), and evidence-based dermatology base sponsored by State Administration of Traditional Chinese.

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Keratinocyte-Specific CCN1 Modulation Driven Regulation of Keratinocyte Proliferation and Inflammation in Psoriasis

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Abstract

Figures

Introduction

Methods

Animals

Model establishment and interventions

Generation of CKO mice via the CRISPR/Cas9 system

Generation of cas9-ki mice

Histological analysis and immunohistochemistry assay

Western Blotting assay

RNA extraction and quantification

Statistical analysis

Results

Elevated CCN1 levels in keratinocytes coincide with more severe skin lesions in IMQ-induced psoriatic mice

CCN1 exacerbates the psoriasis-like pathological features in the epidermis of IMQ-induced psoriatic mice

CCN1 knockout attenuates inflammation and immune cell infiltration

Discussion

Conclusion

Declarations

References

Additional Declarations

Supplementary Files

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