Variety of endosomal TLRs and Interferons (IFN‐α, IFN‐β, IFN‐γ) expression profiles in patients with SLE, SSc and MCTD

A Paradowska‐Gorycka; A Wajda; B Stypinska; E Walczuk; E Rzeszotarska; M Walczyk; E Haladyj; K Romanowska‐Prochnicka; A Felis‐Giemza; A Lewandowska; M Olesińska

doi:10.1111/cei.13566

. 2021 Jan 17;204(1):49–63. doi: 10.1111/cei.13566

Variety of endosomal TLRs and Interferons (IFN‐α, IFN‐β, IFN‐γ) expression profiles in patients with SLE, SSc and MCTD

A Paradowska‐Gorycka ^1,^✉, A Wajda ¹, B Stypinska ¹, E Walczuk ¹, E Rzeszotarska ¹, M Walczyk ², E Haladyj ³, K Romanowska‐Prochnicka ^2,⁴, A Felis‐Giemza ², A Lewandowska ², M Olesińska ²

PMCID: PMC7944358 PMID: 33336388

Summary

We investigated Toll‐like receptor (TLR)‐3/‐7/‐8/‐9 and interferon (IFN)‐α/β/γ mRNA expression in whole blood and serum IFN‐α/β/γ levels in patients with mixed connective tissue disease (MCTD), systemic lupus erythematosus (SLE) and systemic sclerosis (SSc) and in healthy subjects to assess the association between the TLR–IFN expression and severity of and susceptibility to diseases, and identify potential biomarkers. Expression of the IFN‐γ, TLR‐3 and TLR‐8 was detected only in SLE patients. TLR‐7, IFN‐α and IFN‐β expression was highest in SLE, while TLR‐9 expression was highest in SSc patients. In SLE and MCTD patients a strong correlation was observed between TLR‐7 and IFN‐α expression and IFN‐β and IFN‐α expression. In MCTD patients, negative correlation between IFN‐α and TLR‐9 and TLR‐7 and TLR‐9 was revealed. TLR‐9 expression in anti‐U1‐70k‐negative, anti‐C negative and anti‐SmB‐negative MCTD patients was higher than in MCTD‐positive patients. We observed negative correlations between serum IFN‐α levels and TLR‐7 expression and C3 and C4 levels in SLE patients. In SLE patients we observed that with increased IFN‐γ, TLR‐3 and TLR‐8 expression increased the value of C3 and C4. Our results confirmed that the endosomal TLR–IFN pathway seems to be more important in SLE than in MCTD or SSc, and that IFN‐α and IFN‐β may be possible biomarkers for SLE.

Keywords: connective tissue disease, expression, interferon, pathogenesis, TLR

The summary of the article is: “We investigated TLR3/7/8/9 and IFN‐α/β/γ mRNA expression in whole blood and serum IFN‐α/β/γ levels in patients with mixed connective tissue disease (MCTD), systemic lupus erythematosus (SLE) and systemic sclerosis (SSc), and in healthy subjects to assess the association between the TLRs‐IFNs expression and severity of and susceptibility to diseases, and identify potential biomarkers. Expression of the IFN‐γ, TLR3, and TLR8 was detected only in SLE patients. Our study indicated that TLRs‐IFNs signaling pathway may be implicated in the pathogenesis of these diseases, but the underlying mechanism is distinct; probably, in each ACTDs, endosomal TLRs are activated on the different cell types and in different immune pathways.

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Introduction

Autoimmune connective tissue diseases (ACTDs) belong to a family of heterogeneous, multi‐system diseases where immune dysregulation leads to the production of numerous autoantibodies, irregular production of proinflammatory cytokines and, in consequence, multi‐organ damage [1, 2, 3]. Systemic lupus erythematosus (SLE), systemic sclerosis (SSc) and mixed connective tissue disease (MCTD) are members of the ACTD family, where various autoantibodies are present in more than 90% of patients [4, 5]. Although the precise causes of these diseases are complex and not well understood, several murine models, as well as human studies in lupus, have demonstrated a key role of endosomal Toll‐like receptors (TLRs) in the disease initiation, generation of autoantibodies and accumulation of clinical manifestations [3, 15]. Endosomal TLRs belong to the family of pattern recognition receptors (PRRs), and recognize bacterial and viral synthetic nucleic acids. TLR‐3 is specific for double‐stranded RNA (dsRNA), TLRs‐7 and ‐8 are activated by single‐stranded RNA (ssRNA) and TLR‐9 is specific for unmethylated CpG motifs within dsDNA (CpG DNA) [10, 16, 17, 18, 19, 20, 21, 22, 23]. Activation of the endosomal TLRs through self‐antigens on immune cells leads to the inappropriate production of type I interferons (IFN‐α, IFN‐β), type II IFN‐γ and up‐regulation of the IFN‐inducible genes, as well as unrestrained production of particular autoantibodies [13, 19, 22, 24, 25].

Type I IFN and IFN‐γ are pleiotropic cytokines important for immunity, inflammation regulation and induction of cell‐intrinsic anti‐microbial states, and act as a bridge between innate and adaptive immunity [13, 26, 27, 28]. In general, type I IFNs play a pathogenic role in ACTDs by supporting antigen presentation, dendritic cell (DC) maturation, lymphocyte differentiation and inducing chemokine and co‐stimulatory molecule expression [27, 28, 29, 30]. Furthermore, IFN‐γ increases antigen presentation, regulates macrophage activation and generates cytokines and inflammatory factors to support inflammation and inhibit differentiation of regulatory T (T_reg) cells [23, 31]. Type 1 IFN also possesses a protective role in ACTDs through the suppression of T helper type 17 (Th17) cell development, inhibition of inflammatory cytokine production and suppresses pathogenic cell proliferation [27].

Progress in the understanding of ACTDs pathogenesis is restricted by limited understanding of these diseases’ multiplicity. It seems that the TLR–IFN pathway could play a central role in the dysregulation of the immune system, which leads to different clinical manifestations in different patients. In the current study, we assess the endosomal TLRs, type I interferons and type II interferon expression in whole blood from patients with SLE, SSc and MCTD as well as in healthy subjects. Our aim was to (1) assess the association between the TLR–IFN pathway and clinical, as well as laboratory parameters, and (2) identify potential diagnostic/prognostic biomarkers for disease activity and its prognosis. This will not only improve understanding of the ACTDs pathogenesis but also may promote clearer classification and monitoring of the patients with various ACTDs.

Material and methods

Study population

Thirty‐two MCTD patients, 57 SLE patients, 31 SSc patients and 21 healthy blood donors participated in the present study. Peripheral blood samples were collected from all participants after signing informed written consent for the study. All ACTDs patients were diagnosed at the Department of Connective Tissue Diseases of the National Institute of Geriatrics, Rheumatology and Rehabilitation in Warsaw, Poland. Healthy subjects, matched to the patients by ethnicity, gender and age, were recruited from the Regional Center for Blood Donation and Blood Treatment in Warsaw, Poland. Autoantibodies to Sm, Ro, La, RibP, PCNA, CENPB, Scl‐70, Jo‐1, His and dsDNA were measured in sera using dot‐blot tests (recomLine ANA/ENA; Mikrogen Diagnostik, Neuried, Germany). The identification of anti‐nuclear antibodies (ANA) was performed by indirect immunofluorescence (IF) on human epithelial type 2 (Hep2) cell lines (Euroimmun Polska, Wroclaw, Poland), with a median titer of 1 : 5840 (range = 1 : 80–1 : 40 960). The presence of anti‐U1‐RNP was determined by electrochemiluminescence (ECLIA) using streptavidin‐coated paramagnetic beads (UNICAP100; Phadia, Uppsala, Sweden). The research protocol was approved by the Research Ethics Committee of the National Institute of Geriatrics, Rheumatology and Rehabilitation in Warsaw and research was conducted in accordance with the Declaration of Helsinki.

SLE patients

SLE patients met the American College of Rheumatology/Systemic Lupus International Collaborating Clinics (ACR/SLICC) 2012 classification criteria. Disease activity was assessed based on the SLE Disease Activity Index (SLEDAI) score; the damage index was assessed using the Systemic Lupus International Collaborating Clinics/American College of Rheumatology Damage Index (SLICC/ACR DI).

MCTD patients

MCTD patients met Alarcón‐Segovia and Villarreal classification criteria, which characterized strong specificity for MCTD. We excluded from the study patients with MCTD who met the classification criteria for two ACTDs at the time of blood sampling. Guidelines to estimate the MCTD patients’ disease phenotype have not yet been established. For the purpose of this study, we developed objective and subjective methods to evaluate the disease activity and damage status in patients with MCTD. To assess the MCTD clinical activity we used the activity index, which was used in our Institute’s SLEDAI index, and was named Mixed Connective Tissue Disease – Activity Index (MCTD‐AI). The MCTD‐AI scale contains the clinical and laboratory symptoms that indicate active disease. For each symptom that was found in the last 28 days, indicating the disease activity, the assigned number of points was calculated. The MCTD‐AI for a patient was the sum of all points, with a maximum of 52 (Supporting information, Table S1). MCTD‐Damage Index (DI) was developed based on the Systemic Lupus International Collaborating Clinics/American College of Rheumatology (SLICC/ACR) DI (Supporting information, Table S2). One point was scored for each symptom of damage that occurred in MCTD patients for at least 6 months; if the episode repeated, two points were scored.

SSc patients

SSc patients met ACR/European League Against Rheumatism (EULAR) 2013 classification criteria. Disease activity was measured by the European Scleroderma Research Group (EScSG) AI and DI.

RNA isolation and cDNA synthesis

Blood was collected in ethylenediamine tetraacetic acid (EDTA)‐coated vials (BD vacutainer; BD Biosciences, San Jose, CA, USA) and stored in aliquots at −80°C. Total RNA was extracted from 500 μl of whole blood using miRNA (A&A Biotechnology, Gydnia, Poland) and with Trizol reagent (Invitrogen, Carlsbad, CA, USA). The quantity and quality of isolated RNA were evaluated by Quawell Q5000 spectrophotometer. cDNA was prepared using high‐capacity cDNA reverse transcription using the RNase inhibitor kit (Life Technologies, Carlsbad, CA, USA), according to the manufacturer’s instructions.

Quantitative real‐time polymerase chain reaction (PCR)

For gene expression analysis, pre‐validated TaqMan Gene Expression Assays were used: IFN‐α (Hs00256882_s1), IFN‐β (Hs01077958_s1), IFN‐γ (Hs00989291_m1), TLR‐3 (Hs_01551078_m1), TLR‐7 (Hs_01933259_s1), TLR‐8 (Hs00152972_m1), TLR‐9 (Hs00152973_m1) and TaqMan Gene Expression Master Mix. All assays were guaranteed for their PCR efficiency of 100 ± 2%. Each sample was analyzed with two technical replications and the mean Ct value was taken for further analysis. A Ct value higher than 35 was taken as below quantification. Based on the literature we selected the most frequently used panel of genes in the analysis of gene expression in whole blood. Then, analysis of the six housekeeping genes candidates panel [glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH), b‐actin (ACTB), 60S acidic ribosomal protein P (RPL)0, RPL13, TATA‐box‐binding protein (TBP), importin‐8 (IPO8), succinate dehydrogenase (SDHA)] in the control group and groups of patients was conducted. The gene with the lowest variability and stable expression level between analyzed groups was selected. Finally, the relative expression was calculated by the ΔΔ Ct or ΔCt method (normalized to GAPDH as reference gene) using the Quant Studio 5 real‐time PCR System (Applied Biosystems, Foster City, CA, USA).

Measurement of IFN‐α, IFN‐β and IFN‐γ levels

IFN‐α, IFN‐β and IFN‐γ concentrations in serum of patients with SSc, SLE and MCTD and healthy subjects was measured by enzyme‐linked immunosorbent assay (ELISA), according to the manufacturer’s instructions (SunRed Biological Technology, Shanghai, China). For analysis we used 40 μl of serum. The ELISA standard ranges were 6–2000 pg/ml for IFN‐α, 8–2000 ng/l for IFN‐β and 2–600 ng/l for IFN‐γ.

Statistical analysis

Data normality was checked using the Shapiro–Wilk test. Statistical significance between healthy controls and SSc/SLE/MCTD was determined using the non‐parametric Mann–Whitney U‐test. A P‐value less than 0·05 was considered statistically significant. IFN‐α and IFN‐β protein levels were compared using the non‐parametric Kruskal–Wallis test and Dunn’s multiple comparison test. Correlation was determined by Spearman’s rank correlation coefficient. Receiver operator characteristic (ROC) curve analyses for gene expression in whole blood at baseline was performed and the area under the curve (AUC) was calculated. All calculations were performed using GraphPad Prism version 8.3.1.

Results

Study subjects’ characteristics

The demographic and clinical characteristics of all patients are presented in Table 1. The median age of SSc, MCTD and SLE patients was 60, 41 and 39 years, respectively. In patients with SSc the most common antibodies were anti‐Scl‐70, which were present in 54% of our SSc patients. In contrast, these antibodies were not present in SLE patients and in only 6% of MCTD patients. In SLE patients the most common antibodies were anti‐dsDNA, which were present in 56% of these patients. Anti‐dsDNA was not observed in patients with SSc, and observed only in 13% of MCTD patients. Moreover, anti‐U1‐RNP were observed in 90% of our MCTD patients. Anti‐U1‐RNP were present in 24% of SLE patients and in none of the SSc patients.

Table 1.

Characteristics of SSc, SLE and MCTD patients

	SSc, n = 1	SLE, n = 57	MCTD, n = 32
Age (years)
Median (min–max)	60 (28–87)	39·5 (21–74)	41 (19–56)
Gender
Female	25 (81%)	53 (93%)	24 (75%)
Male	6 (19%)	4 (7%)	8 (25%)
Disease duration (years)	2 (0·16–19)	1·2 (0, 1–29)	7 (0, 33–27)
Additional clinical characteristics	Number of patients with: lSSc: 12 dSSc:15 ILD: 16 Correct HRCT: 11 mRSS: 8 (2–32)	SLEDAI: 3 (0–12) SLICC: 0 (0–5) Number of patients with: Sjogren’s syndrome: 14 (28%) Arthritis: 34 (68%) Pulmonary fibrosis: 3 (6%) Facial erythema: 22 (44%) C3: 79·3 (36–162) C4:13·6 (3·1–102)	Damage index [median range (min–max)] MCTD‐DI: 2 (0–12) Number of patients with: Raynaud’s phenomenon 100% Arthritis 20 (62.5%) Sjogren’s syndrome: 5 (16%) Swollen hand and fingers: 12 (37.5%) PAH: 1 (3%) Facial erythema: 9 (28%) Pulmonary fibrosis 8 (2%) Myopathy 9 (28%)
ESR (mm/h)
Median (min–max)	11 (1–52)	32·5 (5–114)	17 (2–95)
CRP (mg/l)
Median (min–max)	4·5 (1–48)	8 (1–395)	7 (1–71)
Antibodies n positive (%)
	Anti‐dsDNA: 0 (0%)	Anti‐dsDNA: 28 (56%)	Anti‐dsDNA 4 (13%)
	Anti‐Jo‐1: 0 (0%)	Anti‐Jo: 0 (0%)	Anti‐Jo: 1 (3%)
	Anti‐Ro 60: 0 (0%)	Anti‐Ro: 23 (50%)	Anti‐Ro 60 5 (17%)
	Anti‐Ro 52: 4 (14%)		Anti‐Ro 52 7 (22%)
	Anti‐La: 0 (0%)	Anti‐La: 8 (17%)	Anti‐La: 1 (3%)
	Anti‐SmB: 0 (0%)	Anti‐Sm: 7 (15%)	Anti‐SmB: 9 (30%)
	Anti‐SmD: 0 (0%)		Anti‐SmD: 1 (4%)
	Anti‐Rib P: 1 (4%)	Anti‐Rib p: 4 (9%)	Anti‐Rib P: 3 (9%)
	Anti‐Scl‐70: 15 (54%)	Anti‐Scl‐70: 0 (0%)	Anti‐Scl‐70: 2 (6%)
	Anti‐His: 1 (4%)	Anti‐His: 14 (31%)	Anti‐His: 5 (16%)
	Anti‐CENP B: 7 (24%)	Anti‐CENP‐B: 2 (4%)	Anti‐CENP B: 2 (6%)
	Anti‐PCNA: 0 (0%)	Anti‐U1‐RNP: 11 (24%)	Anti‐U1‐RNP: 28 (90%)
	ACA: 3 (11%)		Anti‐U1‐70: 21 (75%)
	Anti‐CENP A: 7 (26%)	Anti‐CLIgM: 5 (11%)	Anti‐U1‐A: 22 (79%)
	Fibrillarin antibodies: 2 (8%)	Anti‐CLIgG: 10 (22%)	Anti‐U1‐C: 13 (46%)
			Anti‐PCNA: 2 (6%)
			RF: 3 (10%)

Medications, n (%):	Immunosuppressive drugs: Azathioprine: 4 (13%) Cyclophosphamide: 2 (7%) Other 15 (52%)	Immunosuppressive drugs: Cyclosporine: 3 (6%) Azathioprine: 12 (24%) Cyclophosphamide: 5 (10%)	Immunosuppressive drugs: Azathioprine: 2 (6%)
	Methotrexate: 8 (27%) Mycophenolate mofetil: 7 (23%)	Methotrexate: 5 (10%) Mycophenolate mofetil: 3 (6%)	Methotrexate: 7 (23%)
	Corticosteroids: 2 (7%)	Corticosteroids: 47 (94%)	Corticosteroids (Prednizone): 24 (77%)
	Vasodilators: 26 (84%) Sildenafil: 7 (23%) Prostacyclin analogue (Prostavasin): 10 (32%) Amlodipine or other Ca channel blocker: 25 (81%)	Anti‐malarics: Chloroquine: 26 (52%) Hydroxychloroquine: 21 (42%)	Anti‐malarics: Chloroquine: 16 (59%)

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lSSc = limited systemic sclerosis; dSSc = diffuse systemic sclerosis; ILD = interstitial lung disease; HRCT = high‐resolution computerized tomography; mRSS = modified Rodnan skin score; PAH = pulmonary artery hypertension; ESR = erythrocyte sedimentation rate; CRP = C‐reactive protein; anti‐RibP = anti‐protein ribosomal P; anti‐PCNA = antibodies directed against proliferating cell nuclear antigen; anti‐CENP B = anti‐centromere protein B; anti‐Scl‐70 = anti‐topoisomerase I; anti‐Jo‐1 = anti‐histidyl transfer RNA (t‐RNA) synthetase; anti‐dsDNA = anti‐double‐stranded DNA; ACA = anti‐centromere antibody; RF = rheumatoid factor; MCTD = mixed connective tissue disease.

Healthy subjects included in the present study [12 (60%) women and nine (40%) men, with a mean age of 31·7 ± 12·9 years] did not have a history of autoimmune and/or inflammatory disease at the time of sampling.

Differential expression of endosomal TLRs and type I and II interferons in ACTDs patients and healthy subjects

First, we investigated the mRNA expression levels of IFN‐α, IFN‐β, IFN‐γ and TLR‐3, TLR‐7 and TLR‐9 in whole blood in patients with SSc, SLE and MCTD as well as in healthy subjects. In the present study, we observed that the expression of IFN‐γ, TLR‐3 and TLR‐8 was detected only in patients with SLE, although at a very low level. In contrast, in the study group significant differences in the expression of IFN‐α, IFN‐β, TLR‐7 and TLR‐9 were observed (Fig. 1).

Fig. 1 — Expression of interferon (IFN)‐α (a), IFN‐β (b), Toll‐like receptor (TLR)‐7 (c) and TLR‐9 (d) mRNA in whole blood in healthy controls (HC), systemic sclerosis (SSc) patients, systemic lupus erythematosus (SLE) patients and mixed connective tissue disease (MCTD) patients (in HC, expression was taken as 1). *Significance at *P <* 0·05 (comparison to HC by Mann–Whitney test).

IFN‐α expression was at the same level in healthy subjects and in SLE patients. In patients with SSc and MCTD, IFN‐α mRNA levels were significantly lower than in healthy subjects (P = 0·01 and P = 0·03, respectively). In the case of IFN‐β the highest level of expression was observed in patients with SLE (half‐times higher than in healthy subjects). Patients with SSc were characterized by an almost two‐times lower mRNA level of IFN‐β compared to healthy subjects whereas, in MCTD, the IFN‐β mRNA level was comparable to that observed in healthy subjects. Patients with SSc were characterized significantly by a 2·5‐times higher TLR‐9 expression compared to levels in healthy controls (P = 0·03). The expression of TLR‐9 was detected in SLE and MCTD patients as well as in healthy subjects at very low levels. The relative expression of TLR‐7 was lower in all ACTD patients in comparison to healthy subjects (in the case of SSc and MCTD the level was statistically significant: P = 0·02 and P = 0·005, respectively).

Positive correlation between TLR‐7 and IFNs and negative correlation between TLR‐9 and TLR‐7

Correlation analysis was used to find relationships between the examined gene expression. In healthy subjects and in patients with SLE and MCTD we observed a positive correlation between IFN‐α mRNA and IFN‐β (r = 0·72, P < 0·001; r = 0·8, P < 0·001; r = 0·66, P < 0·001, respectively) and between IFN‐α and TLR‐7 (r = 0·86, P < 0·001; r = 0·81, P < 0·001; r = 0·57, P < 0·001, respectively) (Fig. 2). These correlations were not observed in SSc patients. Additionally, significant correlations between TLR‐7 and IFN‐β mRNA levels were observed in healthy subjects (r = 0·82, P < 0·001) and MCTD patients (r = 0·5, P = 0·007).

Fig. 2 — Correlation of analyzed gene expression in healthy subjects (a–c), mixed connective tissue disease (MCTD) patients (d–f), systemic lupus erythematosus (SLE) patients (g–i) and systemic sclerosis (SSc) patients (j–l).

In MCTD patients, a strong negative correlation between IFN‐α and TLR‐9 (r = −0·63, P = 0·002) and TLR‐7 and TLR‐9 (r = −0·66, P = 0·001) was revealed (Supporting information, Fig. S5A).

TLR‐3, TLR‐8 and IFN‐γ expression, as mentioned above, were observed only in SLE patients. A very strong correlation was observed between TLR‐8 and IFN‐γ (r = 0·87, P < 0·0001), IFN‐γ and TLR‐3 (r = 0·89, P < 0·0001) and TLR‐8 and TLR‐3 (r = 0·88, P < 0·0001). Furthermore, a high correlation was observed between IFN‐γ and TLR‐9 (r = 0·64, P < 0·0001) and TLR‐8 and TLR‐9 (r = 0·66, P < 0·0001) (Supporting information, Fig. S5B).

IFN‐α, IFN‐β and IFN‐γ serum levels in ACTDs patients and healthy subjects

In the present study, we also determined the IFN‐α, IFN‐β and IFN‐γ levels in serum. As shown in Supporting information, Table S3 and Fig. 3, the highest level of IFN‐α was observed in patients with MCTD (1827 pg/ml) and the lowest in healthy subjects (672·3 pg/ml).

Furthermore, IFN‐β serum levels were highest in patients with SSc (1150 pg/ml) and lowest in healthy subjects (190 pg/ml). In both measured cytokine levels the differences between analyzed groups were not statistically significant. The serum concentration of IFN‐γ was similar in all analyzed groups, including healthy controls.

Excluding SSc patients, we did not find a significant correlation between analyzed cytokine concentrations in serum. In SSc patients, a tendency of increasing concentrations of IFN‐α and IFN‐β in serum was noted (r = 0·54, P = 0·078) (Supporting information, Fig. S1).

Increased TLR‐9 expression in anti‐U1‐C and anti‐SmB‐negative MCTD patients

Regarding the importance of the signaling pathway in the pathogenesis of inflammatory diseases, we also analyzed whether examined TLR–IFN mRNA expression as well as IFN serum levels may have had an impact on the ACTDs phenotype.

No significant correlations were revealed between DI and selected gene mRNA expression and serum IFN levels in MCTD patients. The highest correlation (r = 0·38) was noted between DI and IFN‐α levels in serum; however, the result was not statistically significant (Supporting information, Fig. S2). Also, no correlation was found between mRNA levels of analyzed genes and C‐reactive protein (CRP) values. However, a higher level of TLR‐9 expression and IFN‐β serum concentration with a lower CRP value was observed (r = −0·36, P = 0·08 and r = −0·31, P = 0·23, respectively Fig. 4).

Fig. 4 — Expression of interferon (IFN)‐α, IFN‐β, Toll‐like receptor (TLR‐7) and TLR‐9 mRNA level normalized to reference gene and IFN‐α, IFN‐β serum levels in mixed connective tissue disease (MCTD) patients with different autoantibody profiles. *Significance at P < 0·05 (comparison to HC by Mann–Whitney test).

Next, we compared expression levels of selected gene and serum IFN levels with autoantibody profiles in patients with MCTD.

Anti‐U1‐70k‐negative versus anti‐U1‐70k‐positive

Significantly, we observed that anti‐U1‐70k‐negative MCTD patients revealed approximately three times lower IFN‐α serum levels (P = 0·005) and approximately three times higher IFN‐β serum levels (P = 0·65) compared to the anti‐U1‐70k‐positive MCTD patients. mRNA levels of IFN‐α, IFN‐β and TLR‐7 were similar in anti‐U1‐70k‐positive and ‐negative MCTD patients (P = 0·97, P = 0·79 and P > 0·99, respectively). TLR‐9 expression did not differ between anti‐U1‐70k‐positive and ‐negative MCTD patients (P = 0·91). However, TLR‐9 expression in anti‐U1‐70k‐negative MCTD patients was two times higher than in anti‐U1‐70k‐positive MCTD patients.

Anti‐U1‐A‐negative versus anti‐U1‐A‐positive

We did not find significant differences in gene expression and IFN serum levels between MCTD patients with positive and negative anti‐U1‐A antibodies.

Anti‐U1‐C‐negative versus anti‐U1‐C‐positive

MCTD patients negative to anti‐U1‐C had a significantly higher level of TLR‐9 expression when compared with anti‐U1‐C‐positive MCTD patients (P = 0·013). In contrast, in anti‐U1‐C‐positive MCTD patients, mRNA levels of IFN‐α, IFN‐β and TLR‐7 were elevated compared to anti‐U1‐C‐negative MCTD patients; however, the differences were not statistically significant (P = 0·11, P = 0·61 and P = 0·46, respectively). Serum IFN‐α levels were similar in anti‐U1‐C‐negative and anti‐C‐positive MCTD patients. Furthermore, in the case of serum IFN‐β levels, anti‐U1‐C‐positive MCTD patients were characterized by higher serum levels of this cytokine (median levels were 628 pg/ml) than anti‐U1‐C‐negative MCTD patients where median levels of IFN‐β were below the level of detection (P = 0·03).

Anti‐SmB‐negative versus anti‐SmB‐positive

In the case of anti‐SmB antibody, anti‐SmB‐negative MCTD patients were characterized by an approximately two times higher level of TLR‐9 expression than anti‐SmB‐positive MCTD patients, although the difference was not statistically significant (P = 0·52). mRNA levels of IFN‐α, IFN‐β and TLR‐7, as well as serum IFN‐α and IFN‐β levels, were similar in anti‐SmB‐positive and ‐negative MCTD patients (P = 0·84, P = 0·25, P = 0·85, P = 0·68 and P = 0·38, respectively).

Association between TLR‐7 expression and low levels of C3 and C4 (TLR‐7 is a biomarker of SLE disease activity) in patients with SLE

When we compared the examined genes with the SLE phenotype we found a negative average, a significant correlation between expression of TLR‐3/TLR‐8/TLR‐9/IFN‐γ and mean value of CRP (r = −0·40 and P = 0·01; r = −0·46 and P = 0·004; r = −0·36 and P = 0·02; r = −0·40 and P = 0·01, respectively; Supporting information, Fig. S3). Furthermore, we found no correlation between mRNA levels of IFN‐α, IFN‐β and TLR‐7 as well as serum IFN‐α and IFN‐β levels and mean values of CRP and autoantibody profiles in our SLE patients. In this study, we observed that serum IFN‐α levels have shown an average correlation SELENA_SLEDAI score (r = 0·38, P = 0·08; Supporting information, Fig. S4).

We also observed that SLE patients had low levels of C3 (mean value = 79·3) and C4 (mean value = 13·6). We observed a high and significant negative correlation between serum IFN‐α level as well as TLR‐7 expression and C3/C4 levels in SLE patients (Fig. 5). The higher the TLR‐7 expression level, the lower the C3 and C4 concentration.

Fig. 5 — Correlation between serum interferon (IFN)‐α levels (a) and Toll‐like receptor (TLR‐7) mRNA levels normalized to the reference gene in whole blood (b) of systemic lupus erythematosus (SLE) patients and C3 levels and between serum IFN‐α levels (c) and TLR7 mRNA levels normalized to the reference gene in whole blood (d) of SLE patients and C4 levels.

Although the mRNA levels of IFN‐γ, TLR‐3 and TLR‐8 in whole blood of SLE patients were very low, we observed a tendency towards increased expression with increased values of C3 and C4 parameters (Supporting information, Fig. S5).

Anti‐U1‐RNP antibody in MCTD and SLE patients

Anti‐U1‐RNP‐positive SLE patients revealed a significantly higher expression of IFN‐α, IFN‐β and TLR‐7 than anti‐U1‐RNP‐positive MCTD patients (P = 0·042, P = 0·03 and P = 0·009, respectively). We did not observe significant differences in analyzed gene expression between anti‐U1‐RNP‐positive and anti‐U1‐RNP‐negative SLE patients. Most of the patients with MCTD were anti‐U1‐RNP‐positive (90%). In contrast to MCTD, anti‐U1‐RNP‐positive SLE patients were much more likely to retain immunoglobulin (Ig)M‐U1‐snRNP instead of IgG antibodies [34]. Furthermore, anti‐U1‐RNP antibodies in patients with SLE seem to differ from patients with MCTD in terms of antigen recognition [35]. Taken together, the presence of high titers of IgG‐autoantibodies against U1‐snRNP is highly specific for MCTD.

Endosomal TLR–IFN expression in whole blood is not associated with phenotype of SSc

As in many different AITDs, TLRs play an important role in SSc. We did not observe a correlation between endosomal TLRs and IFN‐α/‐β/‐γ expression and clinical parameters, such as the mean values of CRP and ESR and modified Rodnan skin score (mRSS) in SSc patients. Based on the presence/absence of ILD in high‐resolution computed tomography (HRCT) scoring, we did not observe significant differences in gene expression in SSc patients (Supporting information, Table S4). Based on capillaroscopy analysis, SSc patients did not differ in analyzed gene expression levels. However, in patients with an active pattern of capillaroscopic changes the highest average level of TLR‐9 mRNA was noted. Nevertheless, the difference is not statistically significant.

Next, we compared expression levels of selected genes and serum IFN levels with autoantibody profiles in SSc patients. Similar mRNA levels of IFN‐α, IFN‐β and TLR‐7 between anti‐Scl70‐positive and ‐negative SSc patients were observed (Fig. 6).

Fig. 6 — Expression of interferon (IFN)‐α, IFN‐β, TLR‐7 and TLR‐9 normalized to the reference gene as well as serum IFN‐α and IFN‐β levels in anti‐Scl70‐positive systemic sclerosis (SSc) patients and anti‐Scl70‐negative SSc patients. *Significance at P < 0·05 [comparison to healthy controls (HC) by Mann–Whitney test].

Although the median value of TLR‐9 expression was higher in anti‐Scl70‐positive SSc patients (median = 1·63) than in anti‐Scl70‐negative SSc patients (median = 0·23), the difference was not statistically significant (P = 0·57). Moreover, serum IFN‐α and IFN‐γ levels were also similar in anti‐Scl70‐positive SSc patients and anti‐Scl70‐negative SSc patients. In the case of serum IFN‐β levels, a significant difference was observed between anti‐Scl70‐positive SSc patients and anti‐Scl70‐negative SSc patients. Serum IFN‐β levels were significantly higher in anti‐Scl70‐positive SSc patients than in anti‐Scl70‐negative SSc patients (751·8 versus 30·6 pg/ml, P = 0·002).

Finally, SSc patients were divided into two major categories based on skin involvement, limited cutaneous (lcSSc) and diffuse cutaneous (dcSSc). Both groups of SSc patients did not differ in age and mean value of ESR. Furthermore, lcSSc patients characterized a significantly three times lower level of the mean CRP value in comparison to dcSSc patients (P = 0·02). In patients with lcSSc, a significant positive correlation was observed between mRSS and mean CRP value (r = 0·69, P = 0·03; Supporting information, Fig. S6A) but, in contrast, a negative correlation was observed between mRSS and IFN‐β expression (r = −0·69, P = 0·03; Supporting information, Fig. S6B). Serum IFN‐α levels were lower in dcSSc than in lcSSc patients, but the difference was not significant (1086 versus 1696 pg/ml, P = 0·37). Serum IFN‐β levels were almost two times higher in lcSSc patients than in dcSSc patients, but the difference was not significant (P = 0·20).

We also investigated IFN‐α, IFN‐β and TLR‐7 mRNA expression in whole blood in patients with lcSSc and dcSSc (Supporting information, Fig. S7). We observed that dcSSc patients were characterized by significantly higher TLR‐7 mRNA expression than lcSSc patients (P = 0·004). Furthermore, IFN‐α, IFN‐β mRNA expression did not differ between lcSSc and dcSSc patients.

Based on the ROC curve analysis, the diagnostic usefulness of the IFN‐α, IFN‐β and TLR‐7 mRNA expression in ‘differentiating’ dcSSc from lcSSc patients was determined. Comparison between dcSSc patients and lcSSc patients has shown that the AUC (Fig. 7b) was significant for TLR‐7 (AUC = 0·82, P = 0·005).

Fig. 7 — mRNA levels of interferon (IFN)‐α, IFN‐β and Toll‐like receptor (TLR‐7) normalized to the reference gene in whole blood in patients with limited cutaneous systemic sclerosis (lcSSc) and diffuse cutaneous (dc)SSc. *Significance at P < 0·05 [comparison to healthy controls (HC) by Mann–Whitney test]. (a) Schematic illustration of receiver operating characteristic (ROC) curve to evaluate the diagnostic potential of area under the curve (AUC) (b) in dcSSc patients and lcSSc patients.

IFN‐α and IFN‐β as possible prognostic biomarkers for SLE

Based on ROC curve analysis, the diagnostic usefulness of the relative levels of IFN‐α, IFN‐β and TLR‐7 mRNA expression in the diagnosis of SLE, MCTD and SSc patients, as well as ‘differentiating’ SLE from MCTD or SSc, was determined. Comparison between SSc patients and healthy subjects showed that the AUC (Fig. 8a–c,f) was significant for IFN‐α (AUC = 0·71, P = 0·01), IFN‐β (AUC = 0·68, P = 0·02) and TLR‐7 (AUC = 0·69, P = 0·02).

Comparison between MCTD patients and healthy subjects has shown that the AUC (Fig. 8d–e) was significant for IFN‐α (AUC = 0·68, P = 0·03) and TLR‐7 (AUC = 0·73, P = 0·005). For distinguishing SLE patients from SSc patients, the diagnostic potential of IFN‐α and IFN‐β was determined at AUC = 0·71 (Fig. 8g–h). Furthermore, for distinguishing SLE patients from MCTD patients the diagnostic potential of IFN‐α and IFN‐β was determined at AUC = 0·67 (Fig. 8i) and AUC = 0·65 (Fig. 8j), respectively.

Discussion

Although ACTDs differ in their clinical manifestations, they have a shared background of inflammatory cascades [16, 21]. Despite many years of scientific research, it has still not been possible to identify the specific cause of ACTDs. However, it has been confirmed that their pathogenesis is associated with genetic and immunological factors that lead to a breakdown of immune tolerance [2]. Clinical manifestations, the presence of antibodies and development of the adaptive immune response suggest that aberrant activation of endosomal TLRs leads to the production of IFNs and participates in the pathogenesis of autoimmune connective tissue diseases [16, 22, 24].

To our knowledge, the current investigation presents, for the first time, the association and correlation between endosomal TLRs, type I and type II IFN expression and MCTD disease presentation and activity. Additionally, our study is the first, to our knowledge, which has compared TLR‐3/‐7/‐8/‐9 and IFN‐α/‐β/‐γ expression profiles in whole blood between SLE, SSc and MCTD patients. The main findings of the present study are that (1) IFN‐γ, TLR‐3 and TLR‐8 expression was detected only in SLE patients; (2) TLR‐7 expression was higher in SLE, whereas TLR‐9 expression was higher in SSc patients; (3) serum IFN‐α levels were highest in MCTD patients, whereas the serum IFN‐β levels were highest in SSc patients; (4) TLR‐9 expression in anti‐U1‐70k‐negative, anti‐C‐negative and anti‐SmB‐negative MCTD patients was higher than in anti‐U1‐70k‐positive, anti‐C‐positive and anti‐SmB‐positive MCTD patients; (5) we observed a high negative correlation between serum IFN‐α and TLR‐7 expression and C3/C4 levels in SLE patients; and (6) in SLE patients, the increase of IFN‐γ, TLR‐3 and TLR‐8 expression increased the value of C3 and C4. Nevertheless, we are aware that the present study has some limitations. Our sample size was limited and heterogeneous, so some subgroups of patients were, in comparison, not big enough to illustrate statistical significance. Moreover, patients’ disease activity was mild to moderate. Additionally, serum IFNs levels were measured directly using classic immunoassays, which are not always sensitive enough to detect the low concentration of IFN‐α in serum.

For decades, there has been discussion concerning whether MCTD is a distinct disease or whether these patients should be classified as SLE, SSc or myositis. The findings of high TLR‐3/‐7/‐8 and IFN‐α/‐β/‐γ expression in whole blood in patients with SLE compared to SSc and MCTD patients, comparable levels of TLR‐3/‐7/‐8 and IFN‐α/‐β/‐γ mRNA levels between SSc and MCTD patients and higher TLR‐9 expression in SSc patients than in SLE and MCTD patients indicate that the TLR–IFN signaling pathway may be implicated in the pathogenesis of these diseases. Nevertheless, the underlying mechanism is distinct. It is probable that, in each of the ACTDs, endosomal TLRs are activated on the different cell types and in different immune pathways. Further studies are required to validate this observation, as the relationship between endosomal TLRs expression and their response on immune cells, and especially B cells, remains poorly understood.

In the current study, we observed that TLR‐3/‐8/‐9 and IFN‐α/‐β/‐γ expression was up‐regulated in patients with SLE, which is consistent with previous studies on PBMCs [12, 32, 33, 34, 35]. However, in contrast to earlier studies on PBMCs [12, 14, 34, 35], we did not find differences in TLR‐7 expression in whole blood between SLE patients and healthy subjects. Wang et al. [36] also did not detect increased TLR‐7 expression in SLE patients compared to healthy subjects However, the authors found that SLE patients having a TLR‐7 rs3853839G allele (73·68% of SLE patients) revealed higher TLR‐7 expression than healthy subjects [37, 38]. Similarly, Sakata et al. [13] have shown that TLR‐7 expression on DCs was similar in SLE patients and healthy subjects. Recent studies have also highlighted the significance of TLR‐7 in SLE pathogenesis, and that TLR‐7 signaling in B cells promoted SLE development [39, 40, 41, 42]. The role of TLR‐8 in the pathogenesis of SLE is less clear, although some results from mice showed that TLR‐8 over‐expression leads to a multi‐organ inflammatory response, autoimmunity and arthritis [43]. However, the mechanisms of endosomal TLR‐mediated autoimmunity in the pathogenesis of SLE are not clearly defined. Some studies present the association between endosomal TLR and IFN expression and SLE activity; however, the results are contradictory. Klonowska‐Szymczyk et al. [33] and Wong et al. [44] did not find a correlation between TLR‐3, TLR‐7 or TLR‐9 expression and SLE activity and/or autoantibody profiles. Sakata et al. [13] and Murayama et al. [45] reported that TLR‐7‐mediated IFN‐α production was positively correlated and TLR‐9‐mediated IFN‐α production was negatively correlated with SLE disease severity and activity. In the present study, we observed a high negative correlation between TLR‐7 expression and C3/C4 levels in SLE patients and, at the same time, a very high significant positive correlation between IFN‐γ, TLR‐3, TLR‐8, C3 and C4 concentrations. The determination serum complement C3 and C4 levels are commonly used as markers of disease activity in SLE [46]. Complement system activation leads to inflammatory responses and plays a key role in the pathogenesis of SLE [47]. Changes in endosomal TLR–IFN signaling pathways may be caused by genetic, epigenetic and environmental factors, providing various links to SLE pathology [37, 48, 49].

It has been well identified that TLRs and IFNs are involved in the pathogenesis of SSc; however, their effect on risk and disease activity has been controversial. We observed that in SSc patients TLR‐7 and IFN‐α/‐β were down‐regulated, TLR‐9 was up‐regulated and TLR‐3/‐8 and IFN‐γ were not detected. Assassi et al. [32], Tan et al. [50] and York et al. [51] presented increased expression of IFN‐inducible genes in SSc patients compared to healthy subjects. In contrast to our study, Vreca et al. [52] showed significant up‐regulation of TLR‐7 expression and significantly lower expression levels of the TLR‐9 in PBMCs of SSc patients. Moreover, they also observed that increased TLR‐7 expression correlated with disease activity, advanced disease and digital ulcers. Furthermore, Ah Kioon et al. [10] have shown that TLR‐8 expression increased in PBMCs of SSc patients, which is in contrast to our study on whole blood. They also observed TLR‐8 expression on plasmacytoid DCs (pDCs) isolated from SSc patients, but not on pDCs isolated from SLE and healthy subjects. Assassi et al. [32] did not observe an association between IFN score and SSc disease duration, activity or clinical manifestations. In the case of SSc, the lack of a standardized disease activity method and the complex nature of the disease, which are seen as clinical and pathological features, significantly hindered the interpretation of results and led to discrepancies between studies.

Elevated serum IFN‐α levels are linked with SLE severity and activity [53, 54, 55, 56, 57, 58, 59, 60, 61]. Nevertheless, some evidence has shown that SLE patients with clinically inactive disease also have increased serum IFN‐α levels [1, 55, 56, 62, 63, 64]. The presence of IFN is extremely variable both within and between ACTDs. However, the IFN signature is present in the sera of approximately half of ACTD patients. In the present study, we observed that the serum IFN‐α levels were highest in MCTD patients, while serum IFN‐β levels were highest in patients with SSc. Fang et al. [65] observed that poly I:C exerts anti‐fibrotic effects that are mediated by IFN‐β. Furthermore, Azuma et al. [66] demonstrated that pulmonary fibrosis induced by bleomycin in mice was attenuated by IFN‐β treatment. Although evidence has linked the inflammatory response with profibrotic events in SSc patients, the roles of IFN are still unclear. It is also essential to note that available clinical evidence has shown that SSc may occur in patients after IFN‐β or IFN‐α therapy of hepatitis C or chronic myelogenous leukemia, respectively [67]. Moreover, treatment with anifrolumab (an anti‐IFNAR monoclonal antibody) in patients with SSc leads to TGF‐β signaling suppression, which suggests that type I IFNs are pathogenic for SSc. However, further studies are required to validate these observations.

In conclusion, our study indicates that the TLR–IFN signaling pathway may be implicated in the pathogenesis of these diseases, but the underlying mechanism is distinct. Probably, in each ACTD, endosomal TLRs are activated on different cell types and in different immune pathways. The endosomal TLR–IFN pathway seem to be more important in SLE than in MCTD or SSc, which should be confirmed in a larger patient sample. Further studies are required to validate this observation, because the relationship between endosomal TLRs expression and their response on immune cells remains poorly understood.

Disclosures

The authors declare that they have no conflicts of interest.

Author contributions

A. P. G. conceived, designed and performed experiments, analyzed and interpreted of data and wrote the manuscript. A. W. performed data analysis and wrote the manuscript, B. S., E. W., A. W. and E. R. performed experiments. M. W., E. H., K. R. P., A. F. G., A. L. and M. O. were involved in the classification of patients, clinical check of patients and treatment control. All authors read and approved the final version.

Supporting information

Table S1. Scale of MCTD activity (MCTD‐AI).

Table S2. Scale of MCTD damage (MCTD‐DI).

Table S3. IFN‐α, IFN‐β and IFN‐γ serum levels (median and range) in healthy subjects (HC), SSc, SLE and MCTD patients.

Table S4. Analyzed gene expression and capilaroscopy evaluation.

Fig. S1. Correlation between IFN‐α and IFN‐β serum level in SSc patients.

Fig. S2. Correlation between concentration of IFN‐α in serum and Damage index in MCTD patients.

Fig. S3. Correlation between CRP and IFN‐γ, TLR8, TLR3 and TLR9 in SLE patients.

Fig. S4. Correlation between IFN‐α levels in serum and SELENA_SLEDAI score in SLE patients.

Fig. S5. A‐Correlation of TLR7 and IFN‐α expression with TLR9 expression in MCTD patients. B‐ Correlations between TLR9, IFN‐γ and TLR8 in SLE patients.

Fig. S6. Correlation between mRSS and CRP in lcSSc patients (A) and IFN‐β mRNA expression in lcSSc patients (B).

Click here for additional data file.^{(713.5KB, doc)}

Acknowledgements

The technical assistance of Wieslawa Frankowska is gratefully acknowledged. We are also grateful to all the SLE, SSc and MCTD patients and healthy subjects whose co‐operation made this study possible. This work was supported by a grant from the Polpharma Scientific Foundation.

Data Availability Statement

Data available on request from the authors.

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