Protein macroarray screening identifies the hnRNP-DL protein as a novel autoantigen targeted in rheumatoid arthritis (RA).
Sera from 26 RA patients and 40 control subjects, including osteoarthritis (OA) patients (n = 20) and self-reported healthy blood donors (n = 20), were analysed on protein macroarrays[31]. The 20 most sensitive autoantigens only found in the RA group are listed in the Additional file 2.
We identified α-hnRNPDL with second highest intensity score. HnRNP-A2/B1 and hnRNP-D (AUF1), have already been described as autoantibody targets in RA[11, 19]. Structure of hnRNP-DL and sequence alignment with hnRNP-D is shown in Additional file 1: supplementary Fig. 1. One of two different hnRNP-DL clones, expressing the protein fragment from amino acid 81 to 420, revealed autoantibody reactivity in 20% of RA sera (Additional file 2). This hnRNP-DL fragment was termed hnRNP-DLmir (major immunogenic region). Isoform hnRNP-DL2 (amino acid 120–420) could not be detected by RA sera.
Autoantibodies against native and citrullinated hnRNP-DL are predominantly present in sera of systemic lupus erythematosus (SLE) and RA patients.
To verify the results from protein macroarray screening, hnRNP-DLmir was expressed in E. coli BL21(DE3)pLysS, purified and tested for reactivity in ELISA as native (DL) and citrullinated protein version (cit-DL), using 1010 sera obtained from Risk-RA cohort (n = 71), from early RA cohorts (LURA n = 106; EIRA n = 404), from an established RA cohort (Predict n = 127), control cohorts of other autoimmune diseases (n = 216) and from healthy controls (n = 86).The majority of α-DL was found in sera of patients with SLE (34%) and RA (6–21%) and in patients with psoriasis arthritis (15%), patients with MS (5%) and scleroderma (5%) as well as healthy controls (2%) (Fig. 1B; Additional file 1: supplementary Fig. 2). Interestingly, we obtained very different sensitivities within the four investigated RA cohorts whereby Risk-RA- (13%) and EIRA cohort (21%) showed the highest sensitivities. Cohorts under certain therapy or advanced disease duration showed lower values (LURA 8%/Predict 6%).
Since citrullinated antigens, among them hnRNP-A2/B1[18], are the most specific targets in RA, we analysed autoantibody responses against cit-DL, with the highest signaling and positivity found in the early and established RA cohorts (64–100%). With special focus on the seropositive and seronegative RA patients only α-cit-DL signals differ significantly within all investigated RA cohorts, not α-DL values (Fig. 1A/B; Additional file 1: supplementary Table 1). Although α-cit-DL signals of seronegative patients were lower than those of seropositive patients, they were still significantly higher than in other diseases in EIRA and Predict cohort (Additional file 1: supplementary Table 2).
Noticeable 58% of the SLE patients in comparison to other diseases were α-cit-DL positive (α-DL 18%), although 98% of the tested SLE sera were α-CCP2-negative. We determined the difference between the ELISA signals, to get a value that describes the relationship between α-cit-DL and α-DL. This value we named CNDL-Index (ΔDL), shown in Fig. 1C, with the highest values detected in the RA cohorts. In established RA (Predict) the highest CNDL-Index (sensitivity: 100%/72% vs. healthy controls/other diseases) and exclusively positive values were detected. In contrast, 11–20% of early RA patients (Risk-RA/EIRA/ LURA) had a negative CNDL-index, where α-DL was higher than α-cit-DL. Besides, only SLE patients and single exceptions in other diseases had a negative CNDL-index below − 0.1. In the early RA cohort EIRA the CNDL-Index correlated positively to α-cit-DL and there, exclusively in the seronegative EIRA negatively to α-DL response (Additional file 1: supplementary Fig. 3).
Figure 1. Distribution of ELISA signals of α-hnRNP-DLmir autoantibodies. reactivities were predominantly found in SLE and RA.
A-C, Prevalence of citrullinated α-hnRNP-DLmir (cit-DL) (A), α-hnRNP-DLmir (DL) (B) and the difference between cit-DL and DL signal (ΔDL) (C) in sera from Risk-RA patients (n = 71), early RA patients (LURA n = 106; EIRA n = 404), established RA patients (Predict n = 127), SLE patients (n = 89), other diseases (n = 127) and healthy controls (n = 86) determined by ELISA. The dotted lines mark the cutoff versus other diseases (except SLE) or healthy controls with 98% specificity each. OD, optical density; SLE, systemic lupus erythematosus
Anti-cit-DL and CN DL -Index correlated with parenchymal changes in lung/shared epitope and identified people at risk to develop RA.
Anti-DL autoantibodies were detectable in early RA. Therefore, we investigated aCCP2-positive healthy subjects with musculoskeletal symptoms, classified as Risk-RA cohort, differentiating between subjects developing arthritis during follow-up and those remaining healthy without arthritis diagnosis. Further we analysed α-DL autoantibody association with certain risk factors for RA. We plotted respectively α-cit-DL, α-DL and the CNDL-index in the LURA cohort with the parenchymal changes in the lung and in the EIRA cohort with the genetic risk factor shared epitope.
In the Risk-RA cohort α-cit-DL and CNDL-Index were significantly elevated in progressors (Fig. 2A), in the LURA cohort in patients with parenchymal lung changes (Fig. 2B) and in the EIRA in patients with shared epitope, particularly in those carrying two copies (Fig. 2C). No significant differences were found for α-DL antibodies.
Figure 2. Anti-citrullinated hnRNP-DLmir autoantibodies are detectable even before the onset and in early status of disease. A-C, Anti-citrullinated hnRNP-DLmir (cit-DL), α-hnRNP-DLmir (DL) and ∆ OD between cit-DL and DL (ΔDL) were measured by ELISA. A, In Risk-patients of arthritis the OD-levels of citDL and ΔDL before onset are significantly specific in the patient group where the arthritis has already been diagnosed compared to the group without diagnosis (n = 71; non Arthritis n = 34/Arthritis n = 37; MannWhitneyU; citDL mediannon Arthritis=0.19/medianArthritis=0.46; p = 0.0006; NC-index mediannon Arthritis=0.10/medianArthritis=0.38; p = 0.0003). B-C, Cit-DL and ΔDL are significantly associated with parenchymal changes in the lung of early RA patients of the LURA cohort (B; n = 106; no n = 48/PC n = 58; MannWhitneyU; citDL medianno=0.23/medianPC=0.53; p = 0.0340; NC-index medianno=0.16/medianPC=0.44; p = 0.0332) and with and shared epitopes of the early RA patients of the EIRA cohort (C; n = 404; no n = 112/SE n = 213/double SE n = 79; MannWhitneyU; citDL medianno=0,27/medianSE=0.36; p = 0.0003, medianno=0,27/mediandouble SE=0.54; p < 0.0001, medianSE=0,36/mediandouble SE=0,54; p = 0.0453;
NC-index medianno=0,11/medianSE=0.21; p < 0.0001, medianno=0,11/mediandouble SE=0.34; p < 0.0001, medianSE=0,21/mediandouble SE=0,34; p = 0.0061).
Mann-Whitney U test was performed for analysing significance of indicated groups (*p < 0.05, **p < 0.01, ***p < 0.001 ****p < 0.0001). OD, optical density; nm, nano meter; ns, not significant; PC, parenchymal changes in lung; SE, shared epitope
High α-DL autoantibody levels found in 6-month EULAR responders for MTX or Enbrel® treatment
We examined our biomarkers (α-cit-DL, α-DL and CNDL-index) with therapy data of the EIRA and Predict cohort. 192 MTX treated EIRA patients were analysed (Fig. 3A-C; Additional file 1: supplementary Fig. 4A-C). The ROC analysis of α-DL signals reached 12% sensitivity with 90% specificity, using the RA-specific cutoff level (OD 0,371) for detecting MTX response. ROC results got more significance for detecting MTX responses in the seronegative group (cutoff 0,371; 16% sensitivity, 94% specificity; Additional file 1: supplementary Table 6).
Because α-DL correlated negatively to the CNDL-Index in the seronegative group (Additional file 1: supplementary Fig. 3), we analysed MTX-treated EIRA patients with negative CNDL-index. 87% of these patients were responders. We reached sensitivities in a range of 15–33% (100/75% specificity) to detect MTX response (Additional file 1: supplementary Table 7).
In Predict cohort (Enbrel®-treatment) no CNDL-index/response association were found since all patients had equally high positive CNDL-index and none of them negative values. ROC analysis of α-cit-DL or CNDL-index showed no specific response cutoff. But with α-DL we identified 23% of the EIRA patients as MTX responder and in the seronegative group 25% (90% specificity). Among the established RA cohort (Predict) α-DL reached 13% sensitivity and even 25% within the seronegative group for the detection of Enbrel® response (100% specificity; Fig. 3D-F; Additional file 1: supplementary Table 8).
Figure 3. Diagnostic performance of α-hnRNP-DLmir (DL) for the detection of therapy response.
High baseline titer against α-hnRNP-DLmir (DL) is rather present in 6-month EULAR Responder RA patients who had received MTX or α-TNF inhibitor therapy (Enbrel®). A-C, α-DL were measured by ELISA in patient sera from the EIRA cohort treated with MTX (n = 192) with 161 EULAR Responder and 31 EULAR non-Responder among 6 months. Above these values ROC analyses were performed for detecting DAS28 therapy response. D-F, α-DL were measured by ELISA in patient sera from the Predict cohort treated with Enbrel® therapy with 6-month EULAR response data (n = 94, responder n = 63, non-Responder n = 31). Based on the signals, ROC analysis was performed for detecting DAS28 therapy response.
OD, optical density; nm, nano meter; vs., versus; RA, rheumatoid arthritis; MTX, Methotrexat; seropos., rheumatoid factor IgM and/or α-CCP2 positive patients; seroneg., rheumatoid factor IgM and α-CCP2 negative patients.
Anti-cit-DL and α-DL increase the serodiagnostic sensitivity in early RA.
All RA cohorts were analysed to determine diagnostic sensitivities of α-cit-DL and α-DL, in RF IgM/ α-CCP2-seropositive and -negative patients.
The calculated cutoff versus healthy controls (96% specificity), identified 80% of the subjects in the Risk-RA cohort, which are exclusively aCCP2-positive. In the LURA/ EIRA cohort 32/73% of the seronegative patients were identified. In the Predict cohort all patients could be identified with one of our biomarkers and α- DL response was on average the lowest (6%). In SLE 84% in total were detected (α-DL: 34%; α-cit-DL: 80 %). In other autoimmune diseases about half (48%) of the patients were detected in total with our biomarker set.
Using the cutoff versus other diseases (96% specificity), we detected 51% of the seronegative established RA patients and 8–17% of the early RA patients (Table 1).
Table 1
Sensitivity of α-(cit)hnRNP-DLmir (α-DL) autoantibodies in in sera from Risk-RA patients, early RA patients (LURA/ EIRA), established RA patients (Predict), SLE patients (n = 89), other diseases and healthy controls determined by ELISA. The sensitivity is indicated in percent with 98% specificity versus healthy controls (left of slash) and versus other diseases SLE (right of slash). Total DL is the combined reactivity and describes the percentage of patients reacting positively to at least one of the three biomarkers α-cit-DL, α-DL and ΔDL.
| | | | | | RF and/or CCP positive | | | RF and CCP negative | |
| cit-DL | DL | ΔDL | total DL | | cit-DL | DL | ΔDL | total DL | | cit-DL | DL | ΔDL | total DL |
RiskRA | n = 71 | | | | | n = 71 | | | | | n = 0 | | | |
% pos. | 70/ 28 | 13/ 6 | 68/ 34 | 80/ 39 | | 70/ 28 | 13/ 6 | 68/ 34 | 80/ 39 | | - | - | - | - |
LURA | n = 106 | | | | | n = 81 | | | | | n = 25 | | | |
% pos. | 64/ 38 | 8/4 | 64/ 40 | 69/ 42 | | 77/ 48 | 7/ 2 | 77/ 52 | 80/ 53 | | 24/ 4 | 12/ 8 | 24/ 0 | 32/ 8 |
EIRA | n = 404 | | | | | n = 202 | | | | | n = 202 | | | |
% pos. | 80/ 33 | 21/ 9 | 68/ 31 | 84/ 40 | | 94/ 59 | 17/ 5 | 89/ 59 | 96/ 63 | | 65/ 7 | 25/ 14 | 46/ 2 | 73/ 17 |
Predict | n = 127 | | | | | n = 86 | | | | | n = 41 | | | |
% pos. | 100/ 67 | 6/ 0 | 100/ 72 | 100/ 72 | | 100/ 77 | 6/ 0 | 100/ 83 | 100/ 83 | | 100/ 46 | 5/ 0 | 100/ 51 | 100/ 51 |
SLE | n = 89 | | | | | | | | | | | | | |
% pos. | 80/ 58 | 34/ 18 | 72/ 57 | 84/ 73 | | | | | | | | | | |
other | n = 127 | | | | | | | | | | | | | |
% pos. | 45/ 2 | 4/ 2 | 41/ 2 | 48/ 4 | | | | | | | | | | |
HC | n = 86 | | | | | | | | | | | | | |
% pos. | 2/ 0 | 2/ 0 | 2/ 0 | 5/ 0 | | | | | | | | | | |
RA: Rheumatoid Arthritis; SLE, Systemic Lupus Erythematosus |
Localization and expression of hnRNP-DL in different cell lines and synovial tissue.
Affinity-purified α-DL autoantibodies from RA patient sera were used for localization of hnRNP-DL in HeLa- and HEp-2 cells. Sparing the nucleoli in interphase cells, staining with the α-DL autoantibodies showed a nucleoplasmic staining with large speckles (Fig. 4A-a-b). However, the nucleoplasmic staining produced by α-hnRNP-D and α-hnRNP-A2/B1 antibodies was more homogeneous (Fig. 4A-e-f) and stained as well as α-DL autoantibodies discrete cytoplasmic foci when cells were stressed by arsenite (Fig. 4A-c, -e-f). Notably, the co-localization experiment showed α-DL antibodies stained a subset of cytoplasmic stress granules (Fig. 4A-c), independent of size and localization. hnRNP-D could be detected in nearly all granules (Fig. 4A-g, yellow), like the controls Ataxin2 and RCK/p54 (Fig. 4A-d/h).
Since previous studies demonstrated hnRNP-A2/B1 and hnRNP-D to be highly expressed in synovial tissue of RA patients and arthritic mice[19, 28, 32, 33], we analysed the expression of hnRNP-DL in the human joint. Specific rabbit antibodies recognizing hnRNP-DL 1 and − 2 expression were tested by immunohistochemistry in synovial tissue of RA and OA patients and from healthy controls (Fig. 4B). These analyses revealed hnRNP-DL to be highly expressed in RA tissue. Nuclear and cytoplasmic expression was seen in cells of RA synovial tissue, in contrast to the exclusive nuclear staining observed in OA and normal tissue (Fig. 4B, arrows).
We further investigated the expression of hnRNP-DL under inflammatory conditions in IL1α- and TNFα-stimulated HepG2-, as well as in IL6-stimulated HeLa cells by immunoblotting (Fig. 4C). TNFα and particularly IL1α upregulate, whereas IL6 downregulates the expression of hnRNP-DL and furthermore induces its degradation.
We further detected citrullinated proteins of the same molecular weight as of hnRNP-DL (Fig. 4D) in the synovial tissue.
Figure 4. Localisation and Expression of cytokine-regulated, stress granule protein hnRNP-DLmir in cells and synovial tissue. Anti-human hnRNP-DLmir antibodies detect stress granules in immunofluorescence microscopy. Staining with an affinity-purified α-human hnRNP-DLmir antibody was performed in HEp-2 (a) and HeLa cells (b). HeLa cells were treated with 0.5 mM sodium arsenite to induce stress granules and stained with affinity-purified α-human hnRNP-DLmir antibodies (c), mouse α-human ATXN2 antibodies 63 (d), mouse α-human hnRNP-A2/B1 antibodies (e) and α-human AUF1 peptide-specific rabbit serum 19 (f). Co-localization of AUF1 and stress granules/P-bodies. Staining of HEp-2 cells with α-RCK/p54 64 antibodies (g) and double staining of HEp-2 cells with affinity-purified α-human AUF1 (green) and α-RCK/p54 64 antibodies (red) (h). Merged sections are visible in yellow. B, Expression of JKTBP in synovial tissue from a patient with rheumatoid arthritis, a patient with osteoarthritis and a healthy subject, each in 20-fold and detail in 40-fold magnification. C, Influence of cytokines on hnRNP-DL expression determined by immunoblotting. Cellular extracts from unstimulated, IL1α- or TNFα-stimulated HeLa cells and from unstimulated and IL6-stimulated HepG2 cells were probed with α-hnRNP-DL1/2-peptide specific rabbit serum. D, Citrullination of hnRNP-DL1/2 in synovial tissue from a patient with rheumatoid arthritis was investigated with an α-deiminated arginine antibody and an α-hnRNP-DL antibody.
Anti-DL in animal models of RA and SLE with association to TLR7/9 and MyD88 - supports reference to clinical pain
Anti-citDL/α-DL autoantibodies, in baseline samples, are associated with pain VAS after 6 months of various treatments of EIRA patients; Additional file 1: supplementary Table 3–5).
Therefore, we wanted to study the production of α-DL autoantibodies in the context of TLR and MyD88-knock-out mice, known to be involved in pain pathway[34, 35]. Because hnRNP-DL is highly conserved in human and mouse (similarity 98,5%[36]; Additional file 1: supplementary Fig. 1), we analysed α-DL in sera of mouse models of RA and SLE (Table 2).
In zymosan-treated SKG-mice[37], α-DL autoantibodies were twice as frequent (50%) compared to the less severe arthritis model without zymosan induction (25%).
Interestingly, in the interleukin-1 receptor antagonist-deficient (IL-1Ra−/−)-mouse arthritis model we found high signals of α-DL autoantibodies in all mice tested.
MRL/lpr-mice produce antibodies against hnRNPs[38] and snRNPs[39] α-DL autoantibodies were detectable in 85%, while none of them were positive for the citrullinated protein version.
We analysed sera from TLR7-, TLR9- and TLR7/TLR9-double deficient lupus-prone MRL/lpr-mice. This investigation revealed that α-DL autoantibodies were TLR7/-9 dependent and only completely absent in the double deficient mice, while they remained detectable in about 50% of the single TLR7- or TLR9-knockout MRL/lpr-mice. MyD88 plays a central role in TLR-pathway[40]. We tested MyD88-deficient mice, which did not produce α-DL autoantibodies except two mice with very low titer. Further we tested knock-out mice of Toll interleukin-1 receptor 8 (TIR8, SIGIRR, IL1R8), a negative regulator of TLR-IL1-receptor family signaling. Genetic inactivation of this protein, which is associated with severe autoimmunity and high autoantibody production[41], increased prevalence of α-DL autoantibodies by 50%, with a three times higher mean level of ELISA signal intensity (Table 2).
Table 2
Frequency of autoantibodies against recombinant hnRNP-DLmir in sera from different RA and SLE mouse models.
Mouse model | Model of | Autoantigen(s) assayed | No. of sera tested | % positive | ‡Ratio Mean OD positive |
SKG (-/+ Zymosan) | RA | hnRNP-DLmir | 8/8 | 25/50 | 2,48/1,23 |
Balb/c (IL-1Ra−/−) | RA | hnRNP-DLmir | 36 | 100 | 7,89 |
MRL-lpr | SLE | hnRNP-DLmir† | 20 | 85 | 4,22 |
MRL-lpr (MyD88−/−) | SLE | hnRNP-DLmir† | 20 | 10 | 1,3 |
MRL-lpr (TLR9−/−) | SLE | hnRNP-DLmir | 4 | 50 | 2,42 |
MRL-lpr (TLR7−/−) | SLE | hnRNP-DLmir | 7 | 43 | 2,55 |
MRL-lpr (TLR7/9−/−) | SLE | hnRNP-DLmir | 7 | 0 | - |
C57BL/6 lpr | SLE | hnRNP-DLmir† | 12 | 33 | 2,46 |
C57BL/6 lpr (SIGIRR/TIR8−/−) | SLE | hnRNP-DLmir† | 12 | 83 | 6,83 |
C57BL/6 (-/+ R848) | TLR7/8 agonist | hnRNP-DLmir | 10/10 | 0/10 | -/1,39 |
RA: Rheumatoid Arthritis; SLE: Systemic Lupus Erythematosus; SIGIRR/ TIR8: Single Ig IL-1-related receptor/ Toll/interleukin-1 receptor 8; R848: SIGIRR TLR7/8 agonist; MyD88: myeloid differentiation primary response gene 88; TLR: Toll-like receptor. † Additionally citrullinated hnRNP-DLmir were tested. In no case citrullination of hnRNP-DLmir resulted in a higher signal compared to the native hnRNP-DLmir form (no additional reactivity). ‡ Ratio Mean OD positive reflects the level of the positive signals in each mouse model and was calculated as the quotient of the mean value of the positive signals and the diagnostic cutoff. |