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Abstract
Objectives There is increasing evidence that microRNAs (miRNAs) are deregulated in autoimmune and cardiovascular diseases. The present study aimed to identify if miRNAs are deregulated in giant cell arteritis (GCA), a vasculitis affecting large-sized and medium-sized arteries, and to determine if miRNA levels might allow to discriminate between patients with GCA and those without.
Methods 58 patients who had temporal artery biopsy (TAB) for suspected GCA were included in the study and divided into three groups: patients with TAB-positive GCA showing a transmural inflammation (n=27), patients with TAB-negative GCA (n=8) and TAB-negative non-GCA patients with a final diagnosis different from GCA (n=23). To identify candidate miRNAs deregulated in GCA, we profiled the expression of 1209 miRNAs in inflamed TABs and normal TABs. Selected miRNAs were then validated by real-time PCRs and in situ hybridisation (ISH).
Results MiR-146b-5p, -146a, -155, -150, -21 and -299-5p were significantly more expressed in inflamed TABs from patients with GCA. miRNAs were mainly deregulated at the tissue level because peripheral blood mononuclear cells and polymorphonuclear cells from the three groups of patients and age-matched healthy controls had similar levels of miRNAs. ISH showed that miR-21 was mainly expressed by cells in the medial and intimal layers of inflamed TABs. Patients with TAB-negative GCA had a miRNA profile similar to TAB-negative non-GCA patients.
Conclusions MiR-146b-5p, -146a, -21, -150, -155, -299-5p are overexpressed in the presence of inflammation in TABs from patients with GCA.
- Giant Cell Arteritis
- Inflammation
- Autoimmunity
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Introduction
Giant cell arteritis (GCA) is a large-vessel and medium-vessel vasculitis that affects individuals older than 50 years.1 ,2 Its aetiology is still unknown, but arteries from patients with GCA become infiltrated by immune cells, mainly CD4+ T lymphocytes and monocytes and in about half of the patients’ multinucleated giant cells, with increased production of several cytokines, growth factors, elastolytic and proteolytic enzymes, which leads to tissue remodelling.3 Intimal thickening may produce stenoses or occlusions resulting in ischaemic events. Patients with GCA mostly have elevated serum erythrocyte sedimentation rate (ESR) and C reactive protein (CRP), but no GCA-specific serological markers have been identified. The gold standard for the diagnosis of GCA remains a positive temporal artery biopsy (TAB) showing a transmural inflammatory infiltrate, although adventitial or periarterial small-vessel vasculitis can also be consistent with GCA.1 ,2 ,4 ,5 However, TAB is an invasive procedure. In addition, vascular inflammation may be segmental or spare the temporal arteries6 ,7; hence, a TAB lacking inflammatory cells does not completely rule out GCA.8 Therefore, the identification of diagnostic circulating biomarkers would be desirable. Glucocorticoids (GCs) are still the cornerstone of the treatment of GCA because they act quickly, and can prevent irreversible ischaemic events.2 However, flares occur in >50% of patients when the GC dose is tapered, while 86% of patients experience GC-related adverse events.9 ,10 Adjunctive or alternative therapies have been investigated with conflicting results, and no GC-sparing agents have yet gained consensus.2 ,4 A better understanding of the pathogenesis of GCA to identify new targets for therapy is, thus, needed.
MicroRNAs (miRNAs) are small, non-coding RNAs that suppress gene expression at post-transcriptional level by inhibiting translation and/or inducing mRNA degradation. A single miRNA can regulate the expression of multiple target mRNAs through sequence binding. Deregulation of miRNAs has been reported in cancer, autoimmune and cardiovascular diseases (CVDs) such as atherosclerosis and aortic aneurysms.11 ,12 Moreover, miRNAs can regulate innate and adaptive immunity.13 ,14
Our aims were to identify if miRNAs are deregulated in GCA and to determine if miRNA levels might allow to distinguish patients with GCA with a negative TAB from those without GCA.
Methods
Please see the online supplementary file for details on isolation of peripheral blood mononuclear cells (PBMCs) and polymorphonuclear cells (PMNCs), miRNA high throughput profiling in TABs, real-time PCR, heat map representation of miRNA expression, in situ hybridisation (ISH), treatment of PBMCs with dexamethasone in vitro and statistical analyses.
Patients
Fifty-eight consecutive patients who underwent a TAB at the Arcispedale Santa Maria Nuova-IRCCS (Reggio Emilia, Italy) for suspected GCA were enrolled. Patients were divided into three groups: patients with positive (inflamed) TABs diagnosed with GCA (n=27), patients with negative (non-inflamed) TABs diagnosed with GCA on the basis of clinical, laboratory findings and follow-up evaluation (n=8) and patients with negative TABs with a different diagnosis (n=23, non-GCA control patients). Analyses were conducted on three different cohorts of patients: a discovery cohort for the high throughput profiling of miRNAs in TABs (n=15), a validation cohort to confirm miRNA differential expression in TABs by real-time PCR (n=31) and a cohort of patients for analyses on circulating cells (n=25), which partly overlapped with the previous one. Table 1 shows the characteristics of all the investigated patients.
Characteristics of patients at the time of TAB
Only patients with TABs showing transmural inflammation, as defined by Cavazza et al,16 were selected for the present study. Final diagnoses for the non-GCA patients were: polymyalgia rheumatica (PMR, n=14), non-arteritic anterior ischaemic optic neuropathy (n=2), fibromyalgia (n=3), fever of unknown origin (n=1) and neuropathic pain (n=3). For analyses on PBMCs and PMNCs, 10 age-matched healthy subjects were included as unaffected controls. The study was approved by the Local Ethics Committee and informed consent was obtained from all subjects.
Results
MiRNAs deregulated in TABs from patients with GCA
To identify candidate miRNAs likely involved in GCA, miRNA expression was profiled with the mirBase v16 Multi-Species microRNA array in inflamed TABs from patients with GCA (n=7) versus negative TABs from patients with a final diagnosis different from GCA (n=8). Only 10/1209 analysed miRNA showed a significant differential expression greater than twofold between inflamed and non-inflamed TABs with a false discovery rate <10% (see online supplementary table S1). These miRNAs were chosen for further analyses.
A signature of miRNAs overexpressed in inflamed TABs from patients with GCA
To validate data obtained from the miRNA array, expression levels of the selected miRNAs were investigated by real-time PCRs in different TAB samples. The discovery cohort of patients was comparable with the validation cohort of patients for age, ESR and CRP levels and clinical parameters (data not shown). In addition to inflamed TABs from patients with GCA (n=14) and non-inflamed TABs from non-GCA patients (n=11), the validation cohort included non-inflamed TABs from patients with a diagnosis of GCA (n=6). MiR-146b-5p, -146a, -21, -155, -150 and -299-5p were confirmed to be significantly more expressed in positive TABs from patients with GCA (figure 1A). They respectively showed 14.5, 7.2, 5.0, 2.9, 5.1 and 3.4-fold greater expression in inflamed TABs compared with normal TABs from non-GCA patients. Expression of miR-146b-5p was the most promising from a diagnostic perspective because it was possible to set a cut-off (=0.44), which discriminated inflamed from normal TABs with 100% specificity and sensitivity (receiver operating characteristic (ROC) curve). On the contrary, miR-4328, -3652, -885-3p and -432 showed a similar expression in inflamed and non-inflamed TABs, and were expressed at lower levels (data not shown). Negative TABs from patients with GCA patients had a miRNA expression profile similar to negative TABs from non-GCA patients (figure 1A). Depicting the expression levels of miRNAs by a heat map revealed patient-specific profiles. In particular, TAB-positive GCA patients #2, #8, #9, #10, #13, #14 overexpressed all the miRNAs, whereas TAB-negative non-GCA patients #8, #9, #10, #11 underexpressed all of them (figure 1B).
MiRNAs overexpressed in inflamed temporal artery biopsies (TABs) from patients with giant cell arteritis (GCA). Expression of miRNAs was investigated by real-time PCR in inflammation-positive TABs from patients with GCA (TAB pos GCA, n=14) versus non-inflamed normal TABs from patients with GCA (TAB neg GCA, n=6) and non-GCA control patients (TAB neg CTR, n=11). Results are shown as normalised expression: 2−ΔCt (ΔCt=Ct miRNA target–Ct miR-191). (A) Scatter plot visualisation of miRNA expression levels in the groups of patients. Data were compared with the Mann–Whitney U test. **p<0.01; ns=not statistically significant. Lines indicate median expression level. The dotted line in the miR-146b-5p graph indicates the expression level, which discriminates between inflamed and non-inflamed TABs. (B) Heat map visualisation of the profile of miRNA expression levels in each patient. The median Ct values of the miRNAs in the inflamed TABs were: 21.7 (miR-146b-5p), 22.1 (miR-146a), 15.0 (miR-21), 20.7 (miR-150), 25.5 (miR-155), 27.6 (miR-299-5p), 21.8 (miR-191).
To determine if miRNA levels correlated with the levels of miRNA targets, expressions of some genes known to be targeted by the miRNAs were investigated in TABs (see online supplementary table S2 for a list of validated targets of miR-146b-5p, -146a, -21, -150, -155, -299-5p). We selected genes with a potential relevance for GCA and that have been mostly investigated in the literature. Interleukin-1 receptor-associated kinase 1 (IRAK1) and suppressor of cytokine signalling 1 (SOCS1) showed a higher expression in inflamed TABs, whereas tumour necrosis factor (TNF) receptor-associated factor 6 (TRAF6), programmed cell death 4 (PDCD4), angiotensin II receptor type 1 (AGTR1) and cyclin-dependent kinase inhibitor 1A (CDKN1A) showed a similar expression in inflamed and non-inflamed TABs (see online supplementary figure S1). Gene expression levels of the selected targets did not negatively correlate with miRNA expression levels (data not shown). Avian myeloblastosis viral oncogene homolog (MYB) was not expressed in TABs in our experimental conditions.
MiRNA deregulation mainly occurred at tissue level
To determine which cell types mostly expressed the selected miRNAs in inflamed TABs, miRNA expression levels were correlated with the gene expression levels of markers of immune cells (CD45, CD4, CD68 reflecting total leucocytes, T helper cells and macrophages) and vascular cells (ACTA2, alpha smooth muscle actin, Von Willebrand factor (VWF), FAP, fibroblast activation protein alpha as markers of smooth muscle cells, endothelial cells (ECs) and fibroblasts potentially involved in tissue remodeling17). As expected, inflamed TABs from patients with GCA had a higher expression of CD45, CD4, CD68 mRNAs compared with normal TABs. Normal TABs from patients with GCA had a similar expression of CD45, CD4, CD68 mRNAs compared with normal TABs from non-GCA patients. Moreover, inflamed TABs from patients with GCA showed a higher expression of FAP and a lower expression of ACTA2 mRNAs compared with normal TABs from non-GCA patients, indicating tissue remodelling and a similar expression of VWF (see online supplementary figure S2). Considering all samples (inflamed plus non-inflamed TABs), positive correlations emerged between miRNA and CD45, CD4, CD68, FAP expression levels (see online supplementary figures S3–S6), negative correlations emerged between miRNA and ACTA2 expression levels (see online supplementary figure S7), whereas no correlations were found between miRNA and VWF expression levels (see online supplementary figure S8). Considering only the group of inflamed TABs, miR-146b-5p and miR-21 showed an inverse correlation with CD45 gene expression, but they did not correlate with CD4 and CD68 gene expression (see online supplementary figures S3–S5).
To further determine, which cells expressed the miRNAs in TABs, ISH was performed on formalin-fixed paraffin embedded tissue sections using anti-miRNA probes. MiR-146b-5p and miR-21 were investigated being the most promising from a diagnostic and pathophysiological perspective. ISH did not allow to identify any positive cells for miR-146b-5p in TABs, although it detected positive cells in thyroid cancer biopsies used as technical controls (see online supplementary methods). On the contrary, in all the inflamed TABs (n=9), miR-21 was detected by ISH in spindle-shaped cells of the medial layer and stellate fibroblast-like cells of the intimal layer whereas leucocytes were not or only sporadically stained (figure 2). MiR-21 was not detected in normal TABs from patients with GCA (n=8) or without GCA (n=14) (figure 2) with the exception of one negative TAB from a patient with GCA showing some positive cells only in the intimal layer (data not shown). It is likely that the level of expression of miR-146b-5p in inflamed TABs did not exceed the threshold of sensitivity of the ISH, thus, resulted undetectable. Indeed, miR-21, detected by ISH, showed the highest expression level by real-time PCR among the deregulated miRNAs (figure 1A). To determine if the investigated miRNAs were also deregulated in circulating leucocytes, miRNA expression levels were analysed in PBMCs and PMNCs from patients. Age-matched healthy subjects were used as controls. MiRNAs were expressed at comparable levels by circulating PBMCs and PMNCs of patients who underwent TABs and healthy subjects (figure 3) with the exception of miR-155, which showed a lower expression level in PBMCs from patients with GCA and miR-21, which showed a higher expression level in PMNCs from patients with GCA compared with healthy subjects (figure 3). MiR-299-5p was not detected in PMNCs in our experimental conditions.
Localisation of miR-21-overexpressing cells in TABs. Expression of miR-21 was determined by in situ hybridisation on formalin-fixed paraffin embedded TAB sections. The appearance of a dark-blue precipitate indicated the presence of miR-21-positive cells. Nuclei were counter stained with Nuclear Fast Red. A scrambled probe was used as negative control to account for non-specific staining. Representative images are shown. Magnification: 200× if not indicated. CTR, control patient without GCA; GCA, giant cell arteritis; TAB, temporal artery biopsy.
MiRNA expression in circulating cells. Expression of miRNAs was investigated by real-time PCR in peripheral blood mononuclear cells and polymorphonuclear cells (PMNCs) from patients with giant cell arteritis (GCA) with temporal artery biopsies (TABs) positive and negative for infiltrating immune cells compared with patients who underwent TABs, but had a different disease and healthy age-matched subjects. Cohort of patients whose PBMCs were analysed: TAB pos GCA, n=11; TAB neg GCA, n=7; TAB neg CTR, n=7; healthy CTR, n=10. Cohort of patients whose PMNCs were analysed: TAB pos GCA, n=7; TAB neg GCA, n=5; TAB neg CTR, n=5; healthy CTR, n=10. Results are shown as normalised expression: 2−ΔCt (ΔCt=Ct miRNA target–Ct miR-191). Data were compared with the Mann–Whitney U test. *p<0.05, **p<0.01, ns=not statistically significant. CTR, control. Lines indicate median expression levels.
Effects of GCs on miRNA expression
To determine if miRNAs were modulated by GCs, we correlated miRNA expression levels in TABs, PBMCs and PMNCs with GC daily dose. Only in the group of inflamed TABs, a negative correlation was found between miR-146b-5p and -299-5p levels and GC daily dose (data not shown). No correlations were found between miRNA expression levels in PBMCs and PMNCs and GC daily dose (data not shown).
To determine if GCs could directly regulate miRNA expression, PBMCs were treated with dexamethasone in vitro for 4 h and 24 h with and without phorbol 12-myristate 13-acetate (PMA) plus ionomycin (to activate T cells) and lipopolysaccharide (LPS) (to activate monocytes). Treatment with dexamethasone alone did not modify miRNA expression. Treatment with PMA plus ionomycin as well as LPS alone increased expression of miR-155, but the addition of dexamethasone did not affect such induction (see online supplementary figure S9).
Discussion
We found that miR-146b-5p, -146a, -21, -150, -155 and -299-5p were overexpressed in inflamed TABs from patients with GCA, whereas they were expressed at lower levels in normal, non-inflamed TABs from both GCA and non-GCA patients. Therefore, miRNA deregulation correlated with inflammation and miRNA profiling did not allow to distinguish patients with GCA and negative TABs from non-GCA patients. The increased expression of miRNAs in inflamed TABs might reflect the presence of infiltrating immune cell subsets as well as derive from activated arterial cells.
MiRNAs deregulation in GCA mainly occurred at tissue level because PBMCs and PMNCs from patients who underwent TABs showed similar levels of miRNAs, comparable with those of age-matched healthy subjects, with the exception of a lower expression of miR-155 by PBMCs and a higher expression of miR-21 by PMNCs from patients with GCA. A decreased expression of miR-155 has also been documented in patients with other CVDs18 ,19 suggesting that miR-155 might be a circulating marker of CVDs. Neutrophils have been supposed to have a role in GCA progression20; therefore, the higher expression of miR-21 by PMNCs of patients with GCA might reflect the activation of innate immune responses similarly to what has been found in mice exposed to aerosolised LPS.21
Most of the miRNAs deregulated in GCA belong to the so-called Immuno-miRs22 and Inflamma-miRs.23 They are expressed by immune cells,24 and can fine-tune immunity.13 ,14 ,22 ,25 Therefore, they might regulate the balance between immune cell subsets and pro-inflammatory/anti-inflammatory networks in GCA. Online supplementary table S3 reports the activities of miR-146a, -21 and -155 in T cells, macrophages and dendritic cells (DCs) the main players in GCA pathogenesis. MiR-155 is primarily a pro-inflammatory miRNA, but can also inhibit macrophages and DC activation in late phases of inflammatory responses. MiR-21 can exert both pro-inflammatory and anti-inflammatory activities on T cells and DCs while favouring an anti-inflammatory phenotype of macrophages.26 MiR-146a has been mainly reported to dampen inflammation by negative feedback loops.13 ,14 ,22 (see online supplementary table S3). MiRNA induction in GCA might reflect an attempt to curb chronic inflammation.
Interestingly, the deregulated miRNAs might reflect the presence of Th17 (miR-155 and miR-21), Th1 (miR-155), Th2 (miR-21 and miR-146a) and regulatory T cells (Tregs) (miR-146a, -21, -155) in TABs. Th1 and Th17 cells have a proven role in the pathogenesis of GCA.27–30 A decreased frequency of circulating Tregs has been documented in patients with GCA and PMR,29 and FoxP3 positive cells30 and CD25 positive cells31 (potentially Tregs) have been detected in TABs from patients with GCA. Furthermore, such trio of miRNAs might reflect the presence of macrophages of M1 (miR-155) and M2 (miR-21 and miR-146a) phenotypes both identified in TABs.32 Finally, miR-146a, -21, -155 can be induced by activation of Toll Like Receptor (TLR)33 ,34 and Nuclear Factor-κB (NF-κB)35 suggesting that these pathways might promote the development of GCA. Indeed, activation of TLR4 can induce the transmural immune infiltrate in GCA,36 ,37 whereas, to our knowledge, the role of NF-κB in the pathogenesis of GCA is still unclear.
MiR-150 can modulate development and function of NK, iNKT and B cells.13 ,14 B cell deregulation has recently been reported in patients with GCA and PMR38 while the role of NK and iNKT cells in GCA has not yet been defined.
Some validated miRNA targets—IRAK1, TRAF6, PDCD4, SOCS1, AGTR1 and CDKN1A—did not show a decreased expression in TABs, meaning that they might not be targeted by the miRNAs in GCA. Indeed, miRNA effects are disease and context dependent. Furthermore, miRNA effects are timely regulated being different in early and late phases of immune responses and inflammation. Moreover, gene expression was analysed in whole TABs while miRNA effects can vary in different cell types and most of the studies that aimed at identifying miRNA targets have been conducted in cell cultures in vitro. Finally, miRNAs bind to target genes and inhibits their translation, but not always induce mRNA downregulation. Therefore, an analysis of protein expression would be necessary to further confirm the gene expression data. In the literature, it has reported an increased protein expression of AGTR1 and CDKN1A in TABs from patients with GCA,39 ,40 which supports that AGTR1 and CDKN1A are not effectively targeted by miR-155 and -299-5p.
GCA is associated with senescence, and immune and vascular ageing have been suggested as risk factors.41 Interestingly, miR-146a, -146b-5p, -21 and -155 can be induced by cellular senescence (eg, of ECs, fibroblasts and DCs) and inflammaging,23 a low-grade, chronic, systemic inflammation occurring in the elderly due to a dysfunctional activation of immune cells and the acquisition of the senescence-associated secretory phenotype. Patients subjected to TABs were age-matched; therefore, the increased expression of such miRNAs in patients with TAB-positive GCA might indicate an accelerated/premature cellular senescence (eg, of vascular cells), which might increase the risk for inflammation, and might be involved in disease pathogenesis.
Noteworthy, we found an overlap between GCA-associated miRNAs and CVD-associated miRNAs. Similarly to temporal arteries from patients with GCA, in abdominal aortic aneurysms42 and atherosclerotic plaques,43 a higher expression of miR-146b-5p, -146a, -21 and -155 has been found. MiR-146a, -155 and -21 can regulate vascular smooth muscle cells (VSMCs), ECs and adventitial fibroblasts (AFs)12 ,44 ,45 as summarised in online supplementary table S4, suggesting that such miRNAs might contribute to arterial remodelling in chronic inflammation. MiR-146a has diverging effects on VSMCs and ECs (pro-atherogenic and atheroprotective, respectively). MiR-155 can exert atheroprotective effects both on VSMCs and ECs. MiR-21 is the only miRNA overexpressed in GCA that has pro-atherogenic effects on VSMCs, ECs and AFs (see online supplementary table S4), thus emerging as most promising in terms of pathological relevance for GCA. Noteworthy, miR-21 overexpression can stimulate neointimal hyperplasia, and local delivery of oligonucleotides inhibiting miR-21 decreased neointima formation in preclinical models of atherosclerosis.46–48 In inflamed TABs from patients with GCA, miR-21 was mainly expressed by cells in the medial and intimal layers; thus, we speculate that targeting miR-21 might be useful to counteract intimal hyperplasia and arterial remodelling in GCA.
The main limitation of our study is that most patients were receiving GCs when analyses were performed. It is still not clear whether GCs might have affected miRNA expression. We found a negative correlation between miR-146b-5p and -299-5p levels in inflamed TABs and GC daily dose, suggesting that GCs might inhibit the expression of those miRNAs. In vitro treatment of PBMCs with dexamethasone did not modify miRNA levels, indicating that miRNA modulation by GCs, if it happens, might be an indirect effect or an effect on tissue cells. The effects of GCs on miRNA expression have been recently reviewed,49 but the findings are controversial because GC effects appear to be cell-type and disease-specific.
In conclusion, miR-146b-5p, 146a, -21, -150, -155, -299-5p were overexpressed in the presence of inflammation in TABs from patients with GCA. Further studies are required to understand the role of such miRNAs in the pathogenesis of GCA and their potential as new therapeutic targets.
Acknowledgments
We thank Dr Nicolò Pipitone (Unit of Rheumatology, Arcispedale Santa Maria Nuova-IRCCS, Reggio Emilia, Italy) for his assistance in revising the manuscript and Dr Ione Tamagnini and Letizia Marchi (Unit of Pathology, Arcispedale Santa Maria Nuova-IRCCS, Reggio Emilia, Italy) for assistance in ISH.
References
Supplementary materials
Supplementary Data
This web only file has been produced by the BMJ Publishing Group from an electronic file supplied by the author(s) and has not been edited for content.
- Data supplement 1 - Online supplement
Footnotes
Handling editor Tore K Kvien
Contributors SC designed and performed most of the experiments, interpreted data and wrote the manuscript. AZ and LB contributed to experimental design, data interpretation and manuscript editing. FM and GP recruited and followed up the patients. AB contributed to ISH. DN, EF and BC performed the miRNA array profiling. LC and AM performed the TABs. AC did the histological diagnosis. MP contributed to data interpretation. CS conceived the study and edited the manuscript. All authors read and approved the manuscript.
Funding This study was supported by the Arcispedale Santa Maria Nuova-IRCCS, Reggio Emilia, Italy, and by the Fondazione Umberto Veronesi, Milan, Italy, with a postdoctoral fellowship for SC (project title: Regulation of inflammation in chronic vascular diseases at the crossroad between rheumatology and cardiology).
Competing interests None declared.
Ethics approval The study was approved by the Local Ethics Committee (Reggio Emilia, Italy).
Provenance and peer review Not commissioned; externally peer reviewed.