Sjögren’s disease autoantigen TRIM21/Ro52 is exposed during lytic cell death, facilitates immune complex formation and activates macrophages

Sjögren’s disease (SjD) causes localised inflammation of the lacrimal and salivary glands (SGs), and autoantibody production against ubiquitously expressed intracellular proteins such as TRIM21/Ro52 and TROVE2/Ro60. TRIM21 has vital intracellular roles, as an Fc receptor and E3 ubiquitin ligase. It binds antibody Fc domains on opsonised pathogens, which have escaped extracellular immunity and entered cytosols. TRIM21 ubiquitinates these pathogenic targets, driving their proteasomal degradation. However, whilst the intracellular functions of TRIM21 are well-established, how and why TRIM21 becomes an autoantigen in SjD remains unclear. Previous studies suggest apoptosis promotes self-antigen release, triggering germinal centre (GC) reactions for autoantibody production. However, apoptosis is generally anti-inflammatory and thus more recently, lytic cell death has been proposed as a mechanism for driving inflammation in autoimmune diseases. Therefore, we first aimed to identify the location and regulation of TRIM21 expression in murine and human cells. We then tested whether TRIM21 is preferentially released during lytic forms of cell death (pyroptosis, necroptosis or necrosis), which may promote autoimmune responses upon concomitant release of inflammatory alarmins. We found that TRIM21 is expressed ubiquitously in cells and tissues and can be further upregulated following pathogenic (e.g. LPS and Poly I:C) or cytokine (e.g. IFN) stimulation. TRIM21 is released upon lytic cell death (pyroptosis/necroptosis) but not apoptosis. Instead, during apoptosis, TRIM21-GFP punctae localised within apoptotic blebs and were not released into the cell supernatant. Earlier research demonstrates that intracellular TRIM21 binds antibody Fc with very high affinity. However, whether extracellular TRIM21 could maintain its antibody-binding capacity was unknown. We analysed TRIM21-antibody binding by ELISA, immunoprecipitation (IP) and non-denaturing protein-complex separation (Native Blue-PAGE). We showed that TRIM21 forms immune complexes (ICs) with circulating antibodies via its PRYSPRY Fc-interacting domain. Importantly, these complexes were even larger with plasma antibodies from TRIM21/Ro52-seropositive SjD patients, where TRIM21-antibody interactions were mediated via both Fc- and F(ab’)2 domains. ICs are pathogenic in autoimmune diseases such as systemic lupus erythematosus (SLE), and may promote inflammation and antigen presentation. Thus we sought to explore the uptake and functional consequences of TRIM21-ICs in vitro. We fed TRIM21 and TRIM21-ICs of different sizes to macrophages. We showed that larger ICs are taken up more efficiently and facilitate stronger pro-inflammatory cytokine secretion, compared to soluble, free TRIM21. This was also validated by gene expression analyses, with macrophages showing inflammatory and metabolic transcriptional changes. Antigen presentation is an important mechanism for driving autoimmune, antigen-specific immune reactions. Therefore, we tested whether TRIM21 could drive antigen cross-presentation, by employing the OT-I ovalbumin (OVA) TCR-transgenic mouse system. We generated TRIM21-OVA ICs, which we fed to murine macrophages and then detected CD8+ T cell activation, as evidence for cross-presentation. TRIM21-OVA ICs were more efficiently cross-presented that OVA alone, suggesting that extracellular TRIM21 may be able to bind opsonised antigens and enhance their cross-presentation. We suggest such TRIM21-dependent mechanisms of inflammation and antigen presentation may perpetuate additional SG damage in SjD. This may drive cycles of further tissue destruction, TRIM21 antigen release, IC formation and cross-presentation. Therefore, TRIM21’s ability to bind antibody Fc and form ICs causes it to be inherently autoimmunogenic.