Dr. Trouw explains potential of Y-shaped biomarkers in Rheumatoid Arthritis.
Recently, Jing Shi and colleagues from the Leiden University Medical Center (LUMC, the Netherlands) found a new subset of autoantibodies which are involved in Rheumatoid Arthritis (RA), namely the Y-shaped proteins which tend to bind carbamylated proteins (anti-CarP antibodies). They published their findings in PNAS. I talked to Dr. Trouw, one of the authors, about this important hallmark.
Leendert Trouw started his life in Rotterdam, studied biology in Leiden, was a PhD fellow in the same city and completed his post-doc at Lund university in Malmö (Sweden). Currently, Dr. Trouw is an assistant-professor at the Department of Rheumatology at the LUMC. He and his colleagues focus on several important themes, including the role of autoantibodies in RA. The research group in Leiden managed to make numerous scientific publications about this subject, including the one about anti-CarP antibodies and more severe joint damage in RA.
How far are scientists in treating/understanding RA?
“Rheumatoid Arthritis (RA) is a very common disease, affecting around 1% of the Dutch population. It is an autoimmune disease, characterized by a chronic immune response of the body against its own tissue. In RA, this response primarily affects flexible (synovial) joints in hand and feet, resulting over time in massive deformities of the joints leading to pain and disability (right hand in picture below). Nowadays, there is a wide variety of therapeutic options, so the clinical picture is brighter. If patients can be treated soon enough, the current medication is sufficient to halt the disease process and hence prevent severe joint damage. In some early-treated individuals it even results in complete remission of the disease.”
What are important challenges in RA treatment?
“To make early treatment against RA possible, it is vital to predict who will develop RA in the next years and who will not. This is rather difficult. Several biomarkers in the blood can guide early identification of RA, of which autoantibodies Rheumatoid Factor (RF) and anti-citrullinated protein antibodies (ACPA) are important ones. They contribute to RA by worsening the joint destruction by activating nearby inflammatory cells. However, since not all patients have these self-molecule directed Y-shaped proteins in their blood, our lab tries to identify additional biomarkers which identify people at risk to develop RA, classify RA patients and predict more severe joint damage.”
How did your group discover the difference between anti-CarP antibodies and anti-citrullinated protein antibodies (ACPA)?
“Our lab searched for autoantibodies which are directed against homocitrulline containing proteins. Homocitrulline is a building block of protein, better known as amino acid. This amino acid is different from regular amino acids, because it is not encoded in the genome, which is the whole of genetic information in a cell. Instead, homocitrulline is generated via a chemical reaction on the amino acid Lysine. This process is called carbamylation of proteins and results in the generation of carbamylated proteins. The main target of the well-described autoantibody ACPA is a modified amino acid which shows a big resemblance with homocitrulline, namely citrulline (picture below).
After a challenging and important discussion with Dr. Tony Cerami, a world famous expert in protein chemistry, we started numerous experiments to answer the following question: Can the autoantibodies of interest actually distinguish between citrulline and homocitrulline or is the detection of carbamylated proteins just a very difficult way of measuring ACPA?
We found that antibodies can indeed detect the difference between citrulline or homocitrulline at a given position in a small protein. When testing patient samples we observed that several patients reacted to both citrullinated proteins and homocitrulline containing proteins. Interestingly, we also identified patients that only reacted to the carbamylated proteins. Therefore, we realized we had effectively identified a new antibody response in humans, namely the response of the anti-CarP antibody. This made us very excited.”
How do anti-CarP antibodies and ACPA distinguish between each other’s targets?
“There is a part of the ACPA and anti-CarP antibodies with a cross-reactive component, but a substantial group of autoantibodies only react with citrulline or with homocitrulline. Around 14% of the ACPA negative RA patients harbor these anti-CarP antibodies. This may be influenced by the modification itself, because homocitrulline is one CH2 group larger (picture above). Another reason for the distinction could be the different amino acids which surround the modified amino acids (R1 and R2 in picture above).”
What happened after the discovery of the anti-CarP antibody?
“A new antibody is not necessarily a useful antibody, so we studied the response of this antibody to understand the disease process underlying RA. We detected these anti-CarP antibodies in RA patients to know if this newly-discovered autoantibody could be clinically meaningful. These findings stimulated us to spent more time to investigate and describe the significance of the anti-CarP antibody. It was a pleasant journey, because we quickly learned more about this autoantibody response. We also discovered the associations with more severe joint damage in RA. Because these findings were very meaningful, we managed to publish it in PNAS.”
What is the reason carbamylated proteins and anti-CarP antibodies are prevalent in the joints?
“We are still performing experiments to answer this question. We now know that anti-CarP antibodies are present in almost 50% of the RA patients and in around 3% of the healthy controls. However, the percentages in other clinical conditions are much lower than in RA. Why this is especially happening in RA is still a mystery. Renal failure, smoking and chronic inflammation are all conditions associated with RA and with enhanced carbamylation, but why RA patients make these antibodies is unknown. Genetics of the RA population is probably involved in carbamylation or the production of anti-CarP antibodies. We are currently investigating this issue.”
How could anti-CarP antibodies increase the severity of RA?
“When anti-CarP antibodies attach to carbamylated proteins in the joint, several immune mechanisms could worsen the chronic inflammation, such as the enhanced complement activation, recruitment of immune cells and activation of these cells. These processes possibly contribute to more joint damage. However, there is currently no proof for this hypothesis. Animal studies are often useful to gain more insight about disease mechanisms. Therefore, these studies probably will probably help to elucidate the role of anti-CarP antibodies in RA.”
What is the clinical relevance of anti-CarP antibodies?
“In the present study, anti-CarP antibodies are associated with more severe damage in RA, especially in ACPA negative patients. Who knows, maybe one day anti-CarP antibodies will be included in EULAR/ACR classification criteria for RA, just like RF and ACPA. Possibly the presence of anti-CarP antibodies in the blood could influence RA treatment in the future. However, experimental studies will have to be done to investigate whether anti-CarP antibodies contribute to the development and severity of RA. If so, these antibodies not only classify RA, but also ‘predict’ who will develop RA and who will develop the more severe RA.”
Leendert Trouw and Jing Shi
antibodies, joints, rheumatoid arthritis, interview