Periodic Reporting for period 3 - AUTOCOMPLEMENT (The role of complement in the induction of autoimmunity against post-translationally modified proteins) Reporting period: 2020-09-01 to 2022-02-28 Summary of the context and overall objectives of the project The ERC-Consolidator project entitled AUTOCOMPLEMENT is focused on the role of complement in the induction of antibody responses against post-translationally modified proteins. In many prevalent autoimmune diseases such as rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE) autoantibodies are used as diagnostic and prognostic tools. Several of these autoantibodies target proteins that have been post-translationally modified (PTM). Examples of such modifications are citrullination and carbamylation. The success of B cell-targeted therapies in many auto-antibody positive diseases suggests that B cell mediated auto-immunity is playing a direct pathogenic role. Despite the wealth of information on the clinical associations of these anti-PTM protein antibodies as biomarkers we have currently no insight into why these antibodies are formed. Immunization studies reveal that PTM proteins can induce antibody responses even in the absence of exogenous adjuvant. The reason why these PTM proteins have ‘autoadjuvant’ properties that lead to a breach of tolerance is currently unknown. In this proposal, I hypothesized that the breach of tolerance towards PTM proteins is mediated by complement factors that bind directly to these PTM. Complement could be involved in the autoadjuvant property of PTM proteins as next to killing pathogens complement can also boost adaptive immune responses. We are currently unravelling the importance of the complement–PTM protein interaction by answering these questions: 1) What is the physiological function of complement binding to PTM proteins? 2) Is the breach of tolerance towards PTM proteins influenced by complement? 3) Can the adjuvant function of PTM be used to increase vaccine efficacy and/or decrease autoreactivity? With AUTOCOMPLEMENT we will elucidate how PTM-reactive B cells receive ‘autoadjuvant’ signals. We envision that this insight will impact on patient care as we can now design strategies to either block unwanted ‘autoadjuvant’ signals to inhibit autoimmunity or to utilize ‘autoadjuvant’ signals to potentiate vaccination. Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far In this first part of the project we have focused on the production of a set of proteins that are modified by six different post-translational modifications (PTM) and on the biochemical characterization of the interaction of complement proteins with the PTM proteins. We have now successfully generated six PTM versions of two proteins to be used with human serum and also a mouse protein with all six modifications. The presence of the modifications has now been verified by biochemical methods and by mass-spectrometry. The modified proteins, and their controls have now been used to study the interaction with complement. Importantly, we could replicate the preliminary data used in the proposal, that carbamylated proteins, one of the PTM, indeed bind complement proteins. In addition, we observed that this is occurring for more, but not all, of the modifications. We have now confirmed that not only complement proteins from human serum are binding PTM proteins but that also mouse complement proteins can do so in a similar way. This is highly encouraging as it indicates that the mouse is indeed an appropriate animal model to use for the proposed studies. First immunization experiments in mice with PTM proteins have now been performed yielding interesting insight into the break of tolerance and the specificity of the anti-PTM response. The first large set of sera have now been used for the detection of the presence of anti-PTM protein antibodies. We could indeed detect autoantibodies that target these PTMs. Some individuals could harbor antibodies against several of the PTM’s while others only display reactivity against one PTM. Interestingly, in a subset of patients suffering from Rheumatoid Arthritis we observed no reactivity against any of the PTMs, as was also the case for the healthy controls. Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far) The research on Rheumatoid Arthritis (RA) has been largely focused on the presence of antibodies directed against one PTM, citrullination. We challenged this idea by showing that there are also antibody responses against another PTM, carbamylation. Our current data clearly expand this view indicating that a wide array of PTMs are recognized by antibodies present in the serum of RA patients. This has important implications for current efforts aimed to specifically down modulate anti-citrulline responses in RA. Now in this ERC project the main aim is to identify not just the clinical associations of the presence of the anti-PTM antibodies but the reason why antibodies against these PTM proteins are made. Our first large sets of data based on Mass Spec and ELISA clearly indicate that complement proteins are binding very prominently. This binding is in the absence of binding of antibodies, indicating a direct interaction.The notion that direct binding of complement to PTM protein endows the PTM proteins with an auto-adjuvant effect is the key element of the ERC grant. We expect to define this adjuvant effect at the molecular level in the human setting and experimentally in mice. We envisage that this knowledge can be used to inhibit unwanted immune responses against e.g. PTM proteins in autoimmunity, but may actually also be useful as an adjuvant for therapeutic vaccination protocols. Overview of AUTOCOMPLEMENT
