Introduction

Cold-inducible RNA-binding protein (CIRP) is a universal stress-response protein, and its expression may be regulated by hypoxia, ultraviolet radiation, glucose starvation, and oxygen radicals in addition to cold shock [1]. Intracellular CIRP may play an important role in anti-apoptotic processes and cell protection in response to stress through stabilizing mRNAs and facilitating their translation [2]. Concurrently, secreted extracellular CIRP (circulating CIRP) participates in the proinflammatory process via Toll-like receptor 4/myeloid differentiation 2 (TLR4/MD2)-mediated and triggering receptor expressed on myeloid cell‑1 (TREM-1)-mediated intracellular inflammatory signaling [3]. To date, roles of circulating CIRP in various inflammatory diseases such as rheumatoid arthritis, ulcerative colitis, sepsis, acute lung injury, and cardiac ischemia have been reported [4]. Antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV) is a representative small-vessel vasculitis with histopathological features exhibiting necrotizing vasculitis affecting mainly small-sized vessels including capillaries, adjacent arterioles, and venules. In addition, AAV may occasionally initiate inflammatory processes in medium-sized arteries [5, 6]. It can be assumed that circulating CIRP may be associated with the activity, function, and damage status of AAV, given that TLR4 and TREM‑1 have been reported to be involved in the pathogenesis of AAV [7, 8]. However, there have been no studies on the clinical role of circulating CIRP in patients with AAV to date. Hence, this study investigated whether circulating CIRP could be a biomarker to reflect the current activity, function, and damage status in patients with microscopic polyangiitis (MPA) and granulomatosis with polyangiitis (GPA).

Materials and methods

Study population

This study selected 65 microscopic polyangiitis (MPA) and granulomatosis with polyangiitis (GPA) patients who met the inclusion criteria described below from the Severance Hospital ANCA-Associated Vasculitides (SHAVE) cohort (a prospective observational cohort of patients with MPA, GPA, and (eosinophilic granulomatosis with polyangiitis) EGPA). The inclusion criteria were as follows: i) the first classification of MPA and GPA at the Division of Rheumatology, Department of Internal Medicine, Yonsei University College of Medicine, and Severance Hospital; ii) fulfilment of both the 2007 European Medicines Agency algorithms for AAV and polyarteritis nodosa, and the 2012 revised International Chapel Hill Consensus Conference Nomenclature of Vasculitides [5, 6]; iii) reclassified as having MPA and GPA according to the 2022 American College of Rheumatology (ACR)/European Alliance of Associations for Rheumatology (EULAR) classification criteria for MPA and GPA [9, 10]; iv) well-documented medical records regarding clinical and laboratory data at diagnosis including AAV subtype, ANCA positivity, AAV-specific indices, the systemic items belonging to Birmingham Vasculitis Activity Score (BVAS), and routine laboratory results; v) absence of serious medical conditions such as malignancies, infectious diseases requiring hospitalization, and systemic and autoimmune diseases other than AAV; and vi) no exposure to glucocorticoids more than 20 mg/day or immunosuppressive drugs for AAV treatment within the first year prior to AAV diagnosis. This study was approved by the Institutional Review Board (IRB) of Severance Hospital (4-2016-0901) and, when required, written informed consent was obtained from patients at the time of blood sampling. The IRB waived the need for written informed consent when it had been previously obtained at entry into the SHAVE cohort.

AAV-specific indices and blood collection

AAV-specific indices include BVAS version 3 [11], five-factor score (FFS) [12], the Korean version of the Short-Form 36-Item Health Survey (SF-36) physical component summary (PCS) and mental component summary (MCS) [13], and the vasculitis damage index (VDI) [14]. Whole blood was obtained from patients who provided consent on the same day that the assessments of AAV-specific indices were completed. Serum was immediately isolated from whole blood and stored at −80 °C. In this study, AAV-specific indices and serum stored at AAV diagnosis were used.

Clinical and laboratory data

Clinical and laboratory data were collected as described in Table 1. The titers of myeloperoxidase (MPO)-ANCA and proteinase 3 (PR3)-ANCA were measured using an immunoassay, the novel anchor-coated highly sensitive (hs) Phadia ELiA (ThermoFisher Scientific/Phadia, Freiburg, Germany) and human native antigens, on the Phadia250 analyzer. An immunoassay for MPO-ANCA and PR3-ANCA is recommended as the first screening method for ANCA [15]; however, perinuclear (P)-ANCA or cytoplasmic (C)-ANCA positivity was also accepted as ANCA positivity according to the 2022 ACR/EULAR classification criteria for MPA and GPA [9, 10].

Table 1 Characteristics of patients (N = 65)
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High activity of AAV

The highest tertile of BVAS was defined as high activity of AAV.

Measurement of circulating CIRP

Circulating CIRP was quantified in stored sera using enzyme-linked immunosorbent assay kits (MBL, Nagoya, Japan) according to the manufacturer’s instructions.

Statistical analyses

All statistical analyses were performed using IBM SPSS Statistics for Windows, version 26 (IBM Corp., Armonk, NY, USA). Continuous variables were expressed as medians with interquartile ranges, whereas categorical variables were expressed as numbers (percentages). The correlation coefficient (r) between the two variables was obtained using Pearson correlation analysis. The significant area under the curve (AUC) was confirmed and the optimal cut-off was extrapolated by performing a receiver operator characteristic (ROC) curve analysis. The value of circulating CIRP with the maximized sum of sensitivity and specificity for the lower limit of the highest tertile of BVAS was selected as its cut-off for high activity of AAV. The relative risk (RR) of the cut-off for high activity of AAV was analyzed using contingency tables and the chi-square test. P-values ≤ 0.05 were considered statistically significant.

Results

Characteristics

The median age of the study subjects was 65.0 years and 53.8% were women. Among the 65 patients, 39 and 26 had MPA and GPA, respectively. MPO-ANCA (or P‑ANCA) and PR3-ANCA (or C‑ANCA) were positive in 47 and 13 patients, respectively. The median BVAS, FFS, SF-36 PCS, MCS, and VDI scores were 12.0, 2.0, 47.5, 50.3, and 3.0, respectively. The most common systemic item defined by the form of BVAS was pulmonary manifestation (66.2%), followed by renal manifestation (61.5%). Median erythrocyte sedimentation rate (ESR), C‑reactive protein (CRP) level, and circulating CIRP were 57.0 mm/h, 7.0 mg/L, and 6.4 ng/mL, respectively (Table 1).

Correlations

Circulating CIRP was significantly correlated with BVAS (r = 0.256, P = 0.039). However, circulating CIRP was not correlated with other AAV-specific indices or acute-phase reactants, including ESR and CRP (Fig. 1).

Fig. 1
figure 1

Correlation analyses of circulating CIRP with the current BVAS, ESR, and CRP. CIRP cold-inducible RNA-binding protein, BVAS Birmingham Vasculitis Activity Score, ESR erythrocyte sedimentation rate, CRP C-reactive protein

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Cut-off and relative risk

Using the ROC curve, the cut-off of circulating CIRP for high activity of AAV was 6.16 ng/mL; the sensitivity and specificity were 75.0 and 53.7%, respectively. When patients were divided into two groups according to circulating CIRP ≥ 6.16 ng/mL, 37 of the 65 patients were assigned to the group with circulating CIRP ≥ 6.16 ng/mL. High activity of AAV was identified more frequently in patients with circulating CIRP ≥ 6.16 ng/mL than in those with circulating CIRP < 6.16 ng/mL (48.6% vs. 21.4%, P = 0.024). In addition, patients with circulating CIRP ≥ 6.16 ng/mL exhibited a significantly higher risk for high activity of AAV than those with circulating CIRP < 6.16 ng/mL (RR 3.474, 95% confidence interval 1.146, 10.533; Fig. 2).

Fig. 2
figure 2

ROC curve and relative risk. a ROC curve of circulating CIRP based on high activity of AAV (BVAS ≥ 16), b Relative risk of the cut-off of CIRP for high activity of AAV (BVAS ≥ 16). ROC receiver operator characteristic, CIRP cold-inducible RNA-binding protein, AAV antineutrophil cytoplasmic antibody-associated vasculitis, BVAS Birmingham vasculitis activity score

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Discussion

This study is the first to demonstrate that circulating CIRP could reflect the current BVAS and predict high activity of AAV in patients with MPA and GPA. However, circulating CIRP was not significantly correlated with other AAV-specific indices or acute-phase reactants, whereas BVAS was significantly correlated with SF-36 PCS (r = −0.340, P = 0.006), SF-36 MCS (r = −0.344, P = 0.05), ESR (r = 0.472, P < 0.001), and CRP (r = 0.342, P = 0.005). These results suggest that circulating CIRP reflects BVAS through rather complex and diverse mechanisms.

This study suggests several possibilities given several shared roles in the inflammatory processes between circulating CIRP and AAV pathophysiology, although the direct mechanism by which circulating CIRP is correlated with BVAS remains uncertain. The first shared mechanism is the upregulated production of various proinflammatory cytokines and chemokines [16]. Circulating CIRP was reported to promote the production of tumor necrosis factor‑α, interleukin-1β, interleukin‑6, and C‑X‑C motif chemokine ligand 2, which initiate and aggravate the inflammatory response and recruit neutrophils into inflamed tissues [17]. These cytokines may play an important role in not only ANCA formation but also in neutrophil priming in AAV pathogenesis [16]. The second shared mechanism is neutrophil extracellular trap (NET) formation [18]. Circulating CIRP has been reported to induce neutrophil extracellular trap (NET) formation in patients with sepsis and acute pancreatitis [19, 20]. Subsequently, circulating CIRP-induced NET formation might deteriorate phagocytic clearance of apoptotic cells by macrophages [21], leading to a vicious circle. The third shared mechanism is endothelial dysfunction [16]. It was reported that circulating CIRP can increase the formation of reactive oxygen species and caspase‑1 activation via nucleotide-binding domain-like receptor protein 3 (NLRP3) inflammasome activation [17, 22]. Based on these shared mechanisms, it was concluded that circulating CIRP has a reasonable background supporting its clinical significance as a biomarker to reflect the activity of MPA and GPA.

This study performed subgroup analyses according to the subtype of AAV, ANCA type, and systemic manifestations based on the items of BVAS, and obtained several interesting findings. First, regarding the subtype of AAV, MPA patients exhibited a significantly higher median circulating CIRP than GPA patients (7.6 ng/mL vs. 5.4 ng/mL, P = 0.001). Second, regarding AAV type, circulating CIRP was not significantly correlated with MPO-ANCA or PR3-ANCA titers (r = 0.183 and r = −0.082, respectively). In addition, no significant differences in circulating CIRP were noted based on the presence of MPO-ANCA (or P‑ANCA) or PR3-ANCA (or C‑ANCA) positivity. Third, regarding systemic manifestations based on BVAS, circulating CIRP exhibited a significant correlation with the total score of renal manifestation (r = 0.264, P = 0.033). Concurrently, MPA patients showed renal manifestation more frequently than GPA patients (84.6% vs. 26.9%, P < 0.001). Therefore, it was concluded that the subgroup analyses elucidated that circulating CIRP serves as a more significant biomarker in MPA patients through a higher frequency of renal manifestations.

This study has several limitations. Firstly, the size of this study was not large enough to provide potentially reliable information on the clinical significance of CIRP in MPA and GPA patients. Secondly, the results of this study could not be verified in a validation cohort consisting of patients from other hospitals because there is no additional AAV cohort other than our cohort for which clinical data of BVAS, FFS, and SF-36 and blood samples of Korean patients who have been newly diagnosed with AAV are immediately available for use. Thirdly, this study could not provide evidence supporting the hypothetical mechanisms of the correlation between circulating CIRP and the current BVAS. Lastly, the absence of paired results for the same patients was also a critical limitation. However, this study has clinical implications in that it is the first pilot study to demonstrate the possibility that circulating CIRP could reflect the current activity of MPA and GPA. A prospective future study with more patients, in particular by including patients from other hospitals, will provide serial, dynamic, and reliable data regarding the clinical potential of circulating CIRP in MPA and GPA.

Conclusion

This study suggests the clinical potential of circulating CIRP as a biomarker for reflecting the current BVAS and predicting high activity of AAV in patients with MPA and GPA.