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Autoantigen TRIM21/Ro52 Is Expressed on the Surface of Antigen-Presenting Cells and Its Enhanced Expression in Sjögren's Synd

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Autoantigen TRIM21/Ro52 Is Expressed on the Surface of Antigen-Presenting Cells and Its Enhanced Expression in Sjögren's Synd Sjögren syndrome RMD Open: first published as 10.1136/rmdopen-2020-001184 on 15 June 2020. Downloaded from SHORT REPORT Autoantigen TRIM21/Ro52 is expressed on the surface of antigen-presenting cells and its enhanced expression in Sjögren’s syndrome is associated with B cell hyperactivity and type I interferon activity Maarten R Hillen ,1,2 Katia Urso,3 Emma Koppe,3 Ana Pinheiro Lopes,1,2 Sofie L M Blokland,1,2 Aridaman Pandit ,1,2 Tom Slocombe,3 André van Maurik,4 Joel A G van Roon,1,2 Timothy R D J Radstake1,3 To cite: Hillen MR, Urso K, Tripartite motif-containing protein 21 et al Key messages Koppe E, . Autoantigen (TRIM21)/Ro52 is an immunoglobulin TRIM21/Ro52 is expressed on the surface of antigen- receptor that together with Ro60 makes up What is already known on this subject ’ presenting cells and its Sjögren s syndrome-antigen A (SSA). ► TRIM21/Ro52 is a major autoantibody target in enhanced expression in A 60%–80% of patients with primary Sjög- pSS and regulates downstream signalling of type ’ Sjögren ssyndromeis ren’s syndrome (pSS) have anti-SSA autoanti- IIFNs. associated with B cell bodies and they form part of the pSS ► Apoptosis-associated surface expression of TRIM21 hyperactivity and type TRIM21 on non-immune cells is associated with enhanced Iinterferonactivity.RMD Open diagnosis. is a cytosolic Fc-receptor 2020;6:e001184. doi:10.1136/ that mediates neutralisation of opsonised binding of anti-Ro52 autoantibodies. rmdopen-2020-001184 viruses and other particles, acting as a last What this study adds http://rmdopen.bmj.com/ line of defence against viruses that are recog- ► TRIM21 ► Additional material is Enhanced expression of in type I IFN- published online only. To view nised by antibodies but still manage to pene- producing immune cells from patients with pSS is please visit the journal online trate the cell membrane.1 In addition, associated with an IFN signature and B cell (http://dx.doi.org/10.1136/rmdo TRIM21 regulates type I interferon (IFN) hyperactivity. pen-2020-001184). responses by mediating either stabilisation or ► TRIM21 is expressed on the cell surface of non- 12 MRH and KU contributed degradation of IFN regulatory factors. apoptotic antigen-presenting cells and its β equally. Uniquely, it has a very broad antibody specifi- expression is enhanced by IFN- exposure. Received 15 January 2020 city and recognises IgA, IgG and IgM antibo- How might this impact on clinical practice on September 24, 2021 by guest. Protected copyright. Revised 10 April 2020 dies with high affinity, allowing TRIM21 to be ► Elucidating the relationship between type I IFNs and Accepted 19 May 2020 effective against a large range of viruses.3 anti-SSA autoantibodies in pSS may aid Alternatively, TRIM21 can be triggered by identification of promising therapeutic avenues. immune complexes containing non-viral par- ticles such as cellular debris or nucleic acids,4 leading to rapid production of pro- inflammatory mediators including type expression in the major type I IFN- I IFNs.2 Interestingly, mice immunised with producing immune cells, plasmacytoid © Author(s) (or their TRIM21 employer(s)) 2020. Re-use develop anti-TRIM21 autoantibodies dendritic cells (pDCs) and monocytes, permitted under CC BY-NC. No and salivary gland inflammation, while trans- from patients with pSS. In addition, we commercial re-use. See rights fer of serum from anti-TRIM21-positive mice investigated TRIM21 expression in salivary and permissions. Published to naïve mice induces salivary gland gland tissue. For pDCs and salivary gland by BMJ. dysfunction,5 suggesting a direct link between tissues, RNA sequencing and analysis were For numbered affiliations see anti-TRIM21 autoantibodies and induction of performed separately as previously end of article. Sjögren’s-like symptoms. described.67For monocytes, TRIM21 TRIM21 Correspondence to Considering the relevance of as expression was assessed using quantitative Dr Joel AG van Roon; j.vanroo autoantigen in pSS and its role in regulating PCR (online supplementary methods) We n@umcutrecht.nl IFN signalling, we here investigated its observed increased mRNA expression of Hillen MR, et al. RMD Open 2020;6:e001184. doi:10.1136/rmdopen-2020-001184 1 RMD Open RMD Open: first published as 10.1136/rmdopen-2020-001184 on 15 June 2020. Downloaded from TRIM21 in purified pDCs from patients with pSS expression. After stimulation of PBMCs with IFN-β compared to both healthy controls and patients with for 24 hours, we observed a robust increase in non-Sjögren’s sicca (nSS), as previously described by TRIM21 mRNA expression (online supplementary fig our group.6 Similarly, pSS patients showed increased ure 4). In addition, IFN-β stimulation resulted in expression of TRIM21 in monocytes compared to enhanced surface expression of TRIM21 on mono- HC. Additionally, we observed that salivary glands cytes (figure 2A). To further verify that surface from patients with pSS expressed more TRIM21 com- expression of TRIM21 is enhanced by IFN-β,we pared to patients with nSS (figure 1A). TRIM21 used extracellular protein biotinylation. After stimu- expression was associated with systemic B cell hyper- lation, total PBMCs were lysed and the extracellular activity, positivity for autoantibodies against SSA and proteins were separated from the IC proteins and the expression of type I IFN-induced genes (ie, the evaluated using western blot. Corroborating the pre- IFN score) (online supplementary figure 1). We also vious results, TRIM21 expression was increased upon observed a significant difference in TRIM21 expres- IFN-β stimulation in both the intracellular and sion between SSB-positive and SBB-negative pSS extracellular fractions (figure 2B,C). patients in pDCs (p=0.047). No differences in The presence of anti-SSA antibodies is a hallmark TRIM21 expressed were observed between rheuma- of Sjögren’s syndrome and associated with increased toid factor-positive and rheumatoid factor-negative B cell hyperactivity. In addition, evidence suggests pSS patients in any of the compartments analysed that antibodies against TRIM21 directly contribute (not shown). to pSS immunopathology and symptoms.5 Interest- We next set out to confirm this enhanced expres- ingly, most anti-TRIM21 antibodies in pSS patients sion at the protein level in circulating cells in an recognise and block domains critical for the anti- independent cohort (online supplementary table 1, inflammatory effects of TRIM21,9 while its pro- online supplementary figure 2). However, intracellular inflammatory effects that lead to enhanced type (IC) staining by flow cytometry of both pDCs and I IFN production remain intact. This is corroborated monocytes against TRIM21 revealed no differences by the strong association between anti-SSA positivity between pSS and healthy controls (HC) donors (fig- and the presence of an IFN signature in patients with ure 1B,C). ImageStream analysis showed that TRIM21 pSS. Although we could not distinguish between posi- expression is located in the cytosol and in vesicles, tivity for anti-Ro52 and anti-Ro60 in the current consistent with literature8 (figure 1D,E). Strikingly, study, the observed association between increased we also observed expression of TRIM21 in non- TRIM21 expression and positivity for SSA and the permeabilised monocytes, suggesting surface expres- IFN score further suggests that TRIM21 overexpres- sion. pDCs also showed cell surface expression in sion perpetuates inflammation via intracellular path- http://rmdopen.bmj.com/ several donors. To verify that this apparent TRIM21 ways that enhance type-I IFN production. This is surface expression was not an artefact due to leakage in good agreement with studies that identify of cytosolic TRIM21, we excluded cells with TRIM21 as a central mediator in anti-viral immunity, a disrupted cell membrane from the analysis using where it drives production of type I IFN and other live/dead staining. When comparing surface expres- pro-inflammatory cytokines via ubiquitination of sion of TRIM21, pSS patients showed increased IFN regulatory factors and mediators in the nuclear expression on monocytes compared to healthy donors factor kappa-light-chain-enhancer of activated B cells (figure 1F,G). To investigate whether TRIM21 surface (NF-κB) pathway.2310 on September 24, 2021 by guest. Protected copyright. expression was also present on other immune cells, we Enhanced TRIM21 surface expression may also assessed CD11c-expressing classical dendritic cells drive inflammation in pSS. Surface expression of (cDCs) and T cells within the stained peripheral TRIM21 was hitherto only observed in non-immune blood mononuclear cells (PBMCs). cDCs showed cells that undergo apoptosis, where it was linked to a clear surface TRIM21 signal, while expression on enhanced binding of anti-SSA antibodies.11 As we T cells was very low or undetectable (online supple excluded dead and dying cells from our flow cytome- mentary figure 3). Consistent with the flow cytometry try analyses and corroborated our results in freshly data, ImageStream analysis of freshly isolated PBMCs isolated PBMCs, it is unlikely that the observed sur- resulted in a clear signal after surface staining for face expression represents apoptotic cells. Yet, TRIM21, which was mainly localised in vesicle-like TRIM21 surface expression may well facilitate bind- structures suggesting internalisation (online supple ing of anti-SSA antibodies and cognate SSA-reactive mentary figure 3). B cells, driving B cell activation and production of As the expression of TRIM21 was associated with anti-SSA autoantibodies, which is in-line with the the IFN score in the patients, we investigated whether association between increased TRIM21 expression typeIIFNswouldenhancesurfaceTRIM21 and anti-SSA positivity. 2 Hillen MR, et al. RMD Open 2020;6:e001184. doi:10.1136/rmdopen-2020-001184 Sjögren syndrome RMD Open: first published as 10.1136/rmdopen-2020-001184 on 15 June 2020. Downloaded from http://rmdopen.bmj.com/ Figure 1 Tripartite motif-containing protein 21 (TRIM21) is overexpressed on the cell surface of monocytes from patients with ’ primary Sjögren s syndrome (pSS).
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