Periodic Reporting for period 4 - AUTOCOMPLEMENT (The role of complement in the induction of autoimmunity against post-translationally modified proteins)
Reporting period: 2022-03-01 to 2023-08-31
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.
At the start of this 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.
We next analyzed if anti-PTM antibodies occur in major human autoimmune diseases. Here we focused on Rheumatoid Arthritis (RA), Systemic Lupus Erythematosus (SLE) and Auto-Immune Hepatitis (AIH). In all cohorts tested, we could readily identify anti-PTM autoantibodies targeting all 6 PTMs, but with clear differences in the frequencies and in the combinations of the anti-PTM antibodies. In each of the cohorts, the presence of anti-PTM antibodies was associated with disease severity and or response to therapy.
Combined the data clearly indicate: 4 of the 6 PTMs bind and activate complement directly, without prior binding of anti-PTM antibodies.
To study the impact on clearance we performed in vitro phagocytosis experiments, based on beads coated with PTM proteins and control proteins incubated with serum. We observed again that 4 of the 6 PTMs triggered complement activation and that this resulted in enhanced phagocytosis.
We have performed immunization experiments with PTM protein in the presence or absence of exogenous adjuvant and observed strong antibody responses against 4 of the 6 PTMs as well as a break of tolerance against the mouse carrier protein for some of the PTMs. Comparing the observed effects in different strains of mice revealed that the relative contribution of complement on the production of anti-PTM antibodies is limited, but complex and multifactorial.
In currently still ongoing experiments we analyze the impact of immunizing with PTM modified proteins on the development of not just autoimmunity but also on autoimmune diseases such as in this case the development of diabetes.
The results of these studies have been presented at several (international) conferences as well has have now been published in several peer-reviewed publications. Other manuscripts are still pending, and two PhD students will defend a thesis based on these results.
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.
We next analyzed if anti-PTM antibodies occur in major human autoimmune diseases. Here we focused on Rheumatoid Arthritis (RA), Systemic Lupus Erythematosus (SLE) and Auto-Immune Hepatitis (AIH). In all cohorts tested, we could readily identify anti-PTM autoantibodies targeting all 6 PTMs, but with clear differences in the frequencies and in the combinations of the anti-PTM antibodies. In each of the cohorts, the presence of anti-PTM antibodies was associated with disease severity and or response to therapy.
Combined the data clearly indicate: 4 of the 6 PTMs bind and activate complement directly, without prior binding of anti-PTM antibodies.
To study the impact on clearance we performed in vitro phagocytosis experiments, based on beads coated with PTM proteins and control proteins incubated with serum. We observed again that 4 of the 6 PTMs triggered complement activation and that this resulted in enhanced phagocytosis.
We have performed immunization experiments with PTM protein in the presence or absence of exogenous adjuvant and observed strong antibody responses against 4 of the 6 PTMs as well as a break of tolerance against the mouse carrier protein for some of the PTMs. Comparing the observed effects in different strains of mice revealed that the relative contribution of complement on the production of anti-PTM antibodies is limited, but complex and multifactorial.
In currently still ongoing experiments we analyze the impact of immunizing with PTM modified proteins on the development of not just autoimmunity but also on autoimmune diseases such as in this case the development of diabetes.
The results of these studies have been presented at several (international) conferences as well has have now been published in several peer-reviewed publications. Other manuscripts are still pending, and two PhD students will defend a thesis based on these results.
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.
Although the triggering of complement may not be the only factor driving the production of anti-PTM autoantibodies we did get to the realization that the PTM proteins do activate complement chronically.
For many chronic diseases, this ,ow level continuous triggering of complement may be a key driving factor for several complications, such as cardiovascular disease. Our future work will now be focusing on this key observation and we will try to develop inhibitors that specifically inhibit complement activation on PTM proteins.
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.
Although the triggering of complement may not be the only factor driving the production of anti-PTM autoantibodies we did get to the realization that the PTM proteins do activate complement chronically.
For many chronic diseases, this ,ow level continuous triggering of complement may be a key driving factor for several complications, such as cardiovascular disease. Our future work will now be focusing on this key observation and we will try to develop inhibitors that specifically inhibit complement activation on PTM proteins.