Increased Serum Cold-Inducible RNA-Binding Protein Levels in Psoriasis

Abstract

Background

Psoriasis is a chronic inflammatory skin disorder typified by well-demarcated erythematous plaques with scales. While considered an immune-driven condition, its underlying molecular triggers remain insufficiently defined. Cold-inducible RNA-binding protein (CIRP), a stress-response protein, has recently been recognized as a damage-associated molecular pattern that can stimulate immune responses.

Objective

This study aimed to explore the potential association between circulating CIRP levels and the clinical as well as histological characteristics of psoriasis.

Methods

Serum CIRP concentrations were analyzed in 67 individuals diagnosed with psoriasis and 20 healthy controls. Relationships between CIRP expression and various clinical and histological indices were also examined.

Results

Patients with psoriasis exhibited significantly elevated serum CIRP levels compared to healthy individuals. Although correlations were observed between CIRP and certain clinical and histological indicators, CIRP levels did not significantly differ based on disease severity (Psoriasis Area and Severity Index score), joint involvement, or nail changes.

Conclusion

Our findings support the notion that CIRP may be involved in the immunopathogenesis of psoriasis and could be considered a prospective target for therapeutic modulation.

INTRODUCTION

Psoriasis is a long-standing inflammatory skin disorder marked by sharply defined, scaly, erythematous plaques and is predominantly mediated by T cells¹,². Cytokines including tumor necrosis factor (TNF)-α, interleukin (IL)-17A, and IL-22, secreted by activated T cells, are known to promote keratinocyte overgrowth and altered differentiation, culminating in characteristic psoriatic lesions².

Recent insights suggest that innate immune activation through pattern recognition receptors in response to endogenous damage-associated molecular patterns (DAMPs) or exogenous pathogen-associated molecular patterns (PAMPs) may serve as a trigger for psoriatic inflammation³. These innate responses—primarily involving keratinocytes and plasmacytoid dendritic cells—can subsequently drive adaptive immune activation².

Cold-inducible RNA-binding protein (CIRP) belongs to the family of cold shock proteins responding to cold stress⁴. Extracellular CIRP was recently demonstrated to act as a DAMPs that leads to inflammatory responses⁴,⁵. Furthermore, previous studies suggest that CIRP is responsible for inflammation in macrophages, neutrophil, lymphocytes and dendritic cells, and acts through TLR4-MD2.

Recently, DAMPs and cytokines have been emphasized in the pathogenesis of psoriasis². However, the role of CIRP in psoriasis is poorly understood. We investigated the clinical significance of serum CIRP levels and psoriasis.

MATERIALS AND METHODS

Study population

A total of 67 individuals diagnosed with psoriasis (ages 11–80, average age 40.3 years) and 20 age- and sex-matched healthy individuals without psoriasis were enrolled. Clinical information, including age, sex, disease duration, severity, presence of psoriatic arthritis, nail involvement, family history, comorbidities, aggravating factors, and treatment history, was extracted from medical records. Disease severity was assessed using the Psoriasis Area and Severity Index (PASI). Histological parameters—including rete ridge elongation, lymphocytic infiltration around dermal vessels, capillary dilation in the papillary dermis, parakeratosis, Munro’s microabscesses, and Kogoj pustules—were graded on a 3-point scale (0: absent, 1: mild-to-moderate, 2: marked) by 2 independent dermatologists. Patients with systemic conditions known to alter CIRP levels (e.g., autoimmune disorders, infections, malignancies) were excluded. Informed consent was obtained, and the study was approved by the Institutional Review Board of Chungnam National University Hospital (CNUH-IRB-2021-01-015).

Sample collection and processing

Peripheral venous blood was drawn under aseptic conditions. The samples were centrifuged at approximately 1,000 g (3,000 rpm) for 15 minutes to isolate serum, which was subsequently stored at −20°C or −80°C until further analysis.

Immunohistochemical analysis

Skin biopsy specimens from both psoriasis patients and healthy controls were fixed in 10% formalin, embedded in paraffin, and sectioned into 4 μm slices. After deparaffinization in xylene and rehydration through an ethanol gradient, sections were incubated overnight at 4°C with a primary CIRP antibody (dilution 1:100; Abcam, Cambridge, UK). Following incubation with a secondary antibody for 30 minutes at ambient temperature, diaminobenzidine staining was performed, and hematoxylin was used for counterstaining.

Serum CIRP quantification via enzyme-linked immunosorbent assay (ELISA)

Circulating CIRP concentrations were measured using a commercial ELISA kit (Cusabio Biotech, Wuhan, China), following the manufacturer’s instructions.

Cell culture

Human skin tissues were collected with written informed consent from donors, following approval by the Institutional Review Board of Chungnam National University Hospital (IRB No. 2016-07-009). Primary human epidermal keratinocytes were isolated and cultured as previously described⁶. Cells were maintained in keratinocyte serum-free medium (Life Technologies Corporation, Grand Island, NY, USA) supplemented with bovine pituitary extract and recombinant human epidermal growth factor.

Production of recombinant adenovirus

Total RNA was extracted from cultured human keratinocytes using the Easy-Blue RNA extraction kit (Intron, Daejeon, Korea), and 2 μg of RNA was reverse transcribed using M-MLV reverse transcriptase (ELPIS Biotech, Daejeon, Korea). The resulting cDNA was amplified using primers specific for human CIRP, and the PCR product was subcloned into the pENTR/CMV-Flag vector containing attL recombination sites (Invitrogen, Carlsbad, CA, USA). Recombinant adenovirus expressing CIRP was generated using the ViraPower adenoviral expression system (Invitrogen) via site-specific recombination with a Gateway destination vector using LR Clonase. The final adenoviral construct was transfected into 293A cells with Lipofectamine 2000 (Invitrogen). Cells were harvested when ~80% cytopathic effect was observed, and viral particles were collected for use in subsequent experiments.

Western blot

Cells were lysed using Proprep solution (Intron), and total protein concentrations were determined with a BCA protein assay kit (Pierce Biotechnology, Rockford, IL, USA). Equal amounts of protein were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred to nitrocellulose membranes, and probed with the appropriate primary antibodies. After incubation with horseradish peroxidase-conjugated secondary antibodies, the protein bands were visualized using enhanced chemiluminescence detection reagents (Intron).

ELISA

Keratinocytes were transduced with recombinant adenoviruses expressing CIRP or LacZ control for 12 hours. Cells were replenished with fresh medium and then further treated with 1 mM poly(I:C) for 1 day. Secreted cytokines were measured using an ELISA kit (R&D Systems, Minneapolis, MN, USA), following the manufacturer’s instructions. Culture medium was collected and secreted TNF-α, IL-8 and IL-1β were determined using commercial ELISA kits purchased from R&D Systems.

Statistical analysis

Data were analyzed using IBM SPSS Statistics software (version 20.0; IBM Corp., Armonk, NY, USA). Comparisons of CIRP serum levels between groups were made using the Mann-Whitney U test. Associations with clinical variables (e.g., PASI score, psoriatic arthritis, nail involvement) were also assessed using this test. The Kruskal-Wallis test was employed to evaluate differences in CIRP levels across disease severity groups. Statistical significance was defined as a p-value <0.05.

RESULTS

Patient characteristics

Serum samples from 67 individuals with psoriasis and 20 healthy participants were collected. The average disease duration was 12.9±10.5 years. The mean PASI score was 16.6±10.6, mostly in severe psoriasis. Of the total patients, 9% had a family history of psoriasis in second-degree relatives, including parents and siblings. Nail involvement was present in 19.4% of patients. Similarly, psoriatic arthritis was observed in 16.4%. The patients were treated with systemic immunosuppressant (cyclosporine, methotrexate) or ultraviolet phototherapy with topical application. A summary of patient demographics and clinical characteristics is provided in Table 1.

Table 1. characteristic of patients with psoriasis.

Characteristics		Values
All patients		67 (100.0)
Age (yr)		40.3±15.6
Sex
	Male	45 (67.2)
	Female	22 (32.8)
Duration (yr)		12.9±10.5
PASI		16.6±10.6
Psoriatic arthritis		11 (16.4)
Nail psoriasis		13 (19.4)
Family history		6 (9.0)
Comorbidity
	DM	6 (9.0)
	HTN	8 (12.0)
	Hyperlipidemia	4 (6.0)
	Other	Ankylosing spondylitis 2
		Ulcerative colitis 1
		Epilepsy 1
		Interstitial lung disease 1
		Cutaneous squamous cell carcinoma 1
		Gout 1
		Myocardial infarction 1
Treatment
	Cyclosporin	35 (52.2)
	MTX	22 (32.8)
	Phototherapy	38 (56.7)

Values are presented as number (%) or mean ± standard deviation.

PASI: Psoriasis Area and Severity Index, DM: diabetes mellitus, HTN: hypertension, MTX: methotrexate.

CIRP expression in psoriatic skin

Immunohistochemical staining showed elevated nuclear expression of CIRP in epidermal keratinocytes within psoriatic lesions. CIRP was mainly expressed in the nuclei of epidermal keratinocytes in psoriasis skin specimens, but was minimally expressed in normal skin (Fig. 1).

Fig. 1. Expression of CIRP in normal and psoriasis skin tissues. Paraffin-embedded normal skin and psoriasis lesions were immunohistochemically stained with anti-CIRP (original magnification, ×100).

CIRP: cold-inducible RNA-binding protein.

Serum CIRP levels in psoriasis patients

Analysis using ELISA revealed that patients with psoriasis had significantly higher levels of circulating CIRP compared to healthy controls (28.3±16.6 pg/ml vs. 18.4±4.1 pg/ml, p=0.00009) (Fig. 2A). However, when stratified by disease severity—PASI <10, PASI 10–19.9, and PASI ≥20—no significant differences in serum CIRP levels were observed among groups (Fig. 2B).

Fig. 2. Serum CIRP levels in psoriasis patients and healthy controls. (A) Serum CIRP level was significantly higher in psoriasis patients than in healthy controls (p=0.00009, ^**p<0.01). (B) There was no correlation with severity of psoriasis according to PASI. (C, D) CIRP level was not related to the presence or absence of psoriatic arthritis or nail psoriasis.

CIRP: cold-inducible RNA-binding protein, PASI: Psoriasis Area and Severity Index.

Associations between serum CIRP levels and clinical and histological features

CIRP levels did not differ significantly with the presence or absence of psoriatic arthritis. The mean serum CIRP levels were 40.1±26.83 pg/ml vs. 25.0±11.43 pg/ml in those with psoriatic arthritis and those without psoriatic arthritis, respectively (p=0.096) (Fig. 2C). Additionally, there was no significant difference in serum CIRP levels between those with and without nail psoriasis (29.1±19.2 pg/ml vs. 27.8±16.09 pg/ml, p=0.833) (Fig. 2D). There was no correlation between the stage of the following histological findings characteristic of psoriasis (uniform rete ridge elongation, perivascular lymphocyte infiltration, dilated capillaries in papillary dermis, parakeratosis, Munro’s microabscess, and Kogoj spongiform pustules) and the CIRP level.

CIRP’s role in inflammatory response in keratinocytes

Treatment of poly(I:C) in a time-dependent manner and intracellular CIRP was increased at 6 hours after treatment. Secreted CIRP was significantly increased in a time-dependent manner. To determine the effect of CIRP on inflammation, we transduced an adenovirus overexpressing CIRP into keratinocytes. CIRP was detected in both intracellular lysates and supernatants by overexpression of CIRP (Fig. 3A and B). Treatment of poly(I:C) after overexpression of CIRP was done. Overexpression of CIRP was significantly increased secretion of inflammatory cytokines including TNF-α, IL-8 and IL-1β (Fig. 3C). These data indicated that CIRP has a synergistic effect on poly(I:C)-induced inflammation in keratinocytes. To further assess the psoriasis-specific relevance of CIRP, we evaluated the expression of additional markers such as S100A7, S100A8, DEFB4, IL-17, and CCL20 following CIRP overexpression. While S100A7, A8, DEFB4, and IL-17 showed mild, non-significant increases, CCL20 was significantly upregulated (Supplementary Fig. 1).

Fig. 3. CIRP expression and its pro-inflammatory effect in keratinocytes. (A) Keratinocytes were treated with poly(I:C) (1 μg/ml) for the indicated time periods, and CIRP expression was analyzed in intracellular lysates and culture supernatants by western blot. (B) Keratinocytes were transduced with CIRP-overexpressing adenovirus, and CIRP levels were assessed in intracellular and supernatant fractions by western blot. (C) After CIRP overexpression, keratinocytes were further stimulated with poly(I:C) (1 μg/ml) for 24 hours. The secretion of TNF-α, IL-8, and IL-1β was measured by enzyme-linked immunosorbent assay (p=0.0014, ^**p<0.01).

CIRP: cold-inducible RNA-binding protein, TNF: tumor necrosis factor, IL: interleukin, SN: supernatant, pIC: poly(I:C).

DISCUSSION

Recent studies have emphasized the role of CIRP as an inflammatory mediator in autoimmune and inflammatory diseases⁴,⁵. In this study, we demonstrated that serum levels of CIRP were significantly elevated in patients with psoriasis compared to healthy controls. Furthermore, immunohistochemical analysis revealed increased expression of CIRP in the nuclei of epidermal keratinocytes within psoriatic lesions, suggesting a potential role of CIRP in the pathogenesis of psoriasis.

Although serum CIRP levels were elevated in psoriasis patients overall, there was no significant correlation between CIRP levels and disease severity as measured by PASI scores, or with specific clinical features such as psoriatic arthritis or nail involvement. Similar to the PASI analysis, no significant association was observed between CIRP levels and Body Surface Area involvement (data not shown). This suggests that CIRP elevation may reflect a general pro-inflammatory state in psoriasis rather than being a marker of disease extent or particular manifestations.

CIRP, a member of the cold shock protein family, has been recently identified as a DAMP molecule capable of inducing inflammatory responses via TLR4-MD2 signaling pathways⁷,⁸. Recent studies have demonstrated that CIRP acts as a key DAMP molecule in various autoimmune and inflammatory diseases by activating the TLR4-MD2 complex and triggering nuclear factor-κB signaling, which leads to the production of pro-inflammatory cytokines⁷. This mechanism has been implicated in conditions such as rheumatoid arthritis, sepsis, and pulmonary fibrosis⁴,⁹,¹⁰.

A recent study examined CIRP expression in psoriasis and reported elevated CIRP mRNA and protein levels in lesional skin compared to non-lesional and control skin¹¹. While this study provided important evidence for the local upregulation of CIRP in psoriatic lesions, our findings extend these observations by evaluating circulating serum CIRP levels and further exploring its functional role in keratinocytes. This provides novel insight into the systemic and mechanistic relevance of CIRP in psoriasis pathogenesis.

While previous studies have implicated CIRP in various inflammatory diseases through its action on immune cells such as macrophages, neutrophils, lymphocytes, and dendritic cells⁵, its involvement in psoriasis had not been clearly elucidated. Our findings provide novel insights into the potential contribution of CIRP to psoriasis-associated inflammation.

The in vitro experiments further supported the pro-inflammatory role of CIRP in keratinocytes. Overexpression of CIRP in keratinocytes synergistically enhanced poly(I:C)-induced secretion of key inflammatory cytokines, including TNF-α, IL-8, and IL-1β. These cytokines are well-known mediators in the psoriatic inflammatory cascade¹², driving keratinocyte hyperproliferation and immune cell recruitment. Therefore, our data suggest that CIRP may amplify innate immune responses in keratinocytes, contributing to the chronic inflammatory environment characteristic of psoriasis.

Given the increasing recognition of DAMPs in the initiation and perpetuation of psoriatic inflammation³, CIRP could represent a novel target for therapeutic intervention. Modulating CIRP expression or blocking its interaction with TLR4 may offer new avenues to attenuate inflammation in psoriasis. However, further studies are warranted to elucidate the precise mechanisms by which CIRP influences keratinocyte behavior and immune activation in vivo.

A limitation of this study is that all patients had severe disease state of psoriatic lesion, which was represented by the PASI score over 10. In this study, there was no statistical significance between the expression level of CIRP and disease severity. Longitudinal studies are needed to assess whether CIRP levels fluctuate with disease activity or treatment response.

In conclusion, our findings highlight the potential role of CIRP as a mediator of inflammation in psoriasis. Elevated serum CIRP levels and its pro-inflammatory effects on keratinocytes suggest that CIRP may contribute to disease pathogenesis. Further research into CIRP-targeted strategies could pave the way for innovative treatments in psoriasis management.

SUPPLEMENTARY MATERIAL

Supplementary Fig. 1

Psoriasis-related cytokine expression following CIRP overexpression in keratinocytes.