Tuberculosis (TB) is a prevalent disease causing an estimated 1.6 million deaths and 10.6 million new cases annually. Discriminating TB disease from differential diagnoses can be complex,... Show moreTuberculosis (TB) is a prevalent disease causing an estimated 1.6 million deaths and 10.6 million new cases annually. Discriminating TB disease from differential diagnoses can be complex, particularly in the field. Increased levels of complement component C1q in serum have been identified as a specific and accessible biomarker for TB disease but the source of C1q in circulation has not been identified. Here, data and samples previously collected from human cohorts, a clinical trial and a non-human primate study were used to identify cells producing C1q in circulation. Cell subset frequencies were correlated with serum C1q levels and combined with single cell RNA sequencing and flow cytometry analyses. This identified monocytes as C1q producers in circulation, with a pronounced expression of C1q in classical and intermediate monocytes and variable expression in non-classical monocytes. Show less
ObjectiveAntibodies targeting post-translationally modified proteins, such as anti-carbamylated protein antibodies (anti-CarP antibodies) are present in sera of rheumatoid arthritis (RA) patients.... Show moreObjectiveAntibodies targeting post-translationally modified proteins, such as anti-carbamylated protein antibodies (anti-CarP antibodies) are present in sera of rheumatoid arthritis (RA) patients. These autoantibodies associate with increased risk of RA development and with severity of joint destruction. It is not known which proteins in the RA joint are recognised by anti-CarP antibodies. Therefore, we investigated the presence and identity of carbamylated proteins in the human (inflamed) joint.MethodsWe obtained synovium, cartilage and synovial fluid from RA joints. Cartilage and synovium were obtained from controls. Samples were processed and used for immunohistochemistry or mass-spectrometric analysis to investigate the presence of carbamylated proteins. Anti-CarP antibody reactivity towards identified carbamylated proteins was tested by ELISA.ResultsImmunohistochemistry showed extensive staining of RA and control synovial tissue. Whole proteome analyses of the joint tissues revealed a large number of carbamylated peptidyllysine residues. We identified many carbamylated proteins in cartilage and were also able to detect carbamylation in synovial tissue and synovial fluid. Carbamylation was not exclusive to the RA joint and was also present in the joints of controls. Anti-CarP antibodies in the sera of RA patients were able to recognise the identified carbamylated proteins.ConclusionWe conclude that numerous carbamylated proteins are present in the RA joint. These carbamylated proteins can be recognised by anti-CarP antibodies, substantiating the notion that anti-CarP antibodies may play a role in the pathogenesis of RA. Show less
Although quite some data is available on anti-CarP antibodies, several questions remain unanswered, including the reproducibility of the clinical data on anti-CarP antibodies, such as their... Show moreAlthough quite some data is available on anti-CarP antibodies, several questions remain unanswered, including the reproducibility of the clinical data on anti-CarP antibodies, such as their presence before disease onset and association with joint damage. It is also unknown whether these findings can be expanded to non-caucasian populations. Furthermore, it is unclear how anti-CarP antibodies are induced, what proteins they recognize, whether they are able to recognize multiple carbamylated proteins and what the characteristics of these autoantibodies are. Here, some of these questions will be answered. In this thesis, I will first discuss the clinical implications of the presence of anti-CarP antibodies compared to RA-specific autoantibodies in both RA patients other relevant conditions (chapters 2 till 7). This is followed by more detailed investigations into the characteristics of anti-CarP antibodies and their antigens (chapters 8 till 12). Show less
Objectives Over 50% of rheumatoid arthritis (RA) patients harbor a variety of Anti-Modified Protein Antibodies (AMPA) against different post-translationally modified (PTM) proteins, including anti... Show moreObjectives Over 50% of rheumatoid arthritis (RA) patients harbor a variety of Anti-Modified Protein Antibodies (AMPA) against different post-translationally modified (PTM) proteins, including anti-carbamylated protein (anti-CarP) antibodies. At present it is unknown how AMPA are generated and how autoreactive B cell responses against PTM proteins are induced. Here we studied whether PTM foreign antigens can breach B cell tolerance towards PTM self-proteins. Methods Serum reactivity towards five carbamylated proteins was determined for 160 RA-patients and 40 healthy individuals. Antibody cross-reactivity was studied by inhibition experiments. Mass spectrometry was performed to identify carbamylated self-proteins in human rheumatic joint tissue. Mice were immunized with carbamylated- or non-modified (auto)antigens and analyzed for autoantibody responses. Results We show that anti-CarP antibodies in RA are highly cross-reactive towards multiple carbamylated proteins, including modified self- as well as modified non-self proteins. Studies in mice show that anti-CarP antibody responses recognizing carbamylated self-proteins are not only induced by immunization with carbamylated self-proteins but also by immunization with carbamylated proteins of non-self origin. Similar to the data observed with sera from RA patients, the murine anti-CarP antibody response was, both at the monoclonal- and polyclonal level, highly cross-reactive towards multiple carbamylated proteins, including carbamylated self-proteins. Conclusions Self-reactive AMPA-responses can be induced by exposure to foreign proteins containing PTM. These data show how autoreactive B cell responses against PTM self-proteins can be induced by exposure to PTM foreign proteins and provide new insights on the breach of autoreactive B cell tolerance. Show less
