Identification and functional analysis of anti-citrullinated protein antibodies in rheumatoid arthritis

Rheumatoid arthritis (RA) is a complex autoimmune disease and typically manifested by joint inflammation and bone erosion with approximately 0.5% of the global population affected. To date, it is believed that genetic predisposition (e.g. HLA-DRB1 alleles) and environment (e.g. cigarette smoking) are involved as risk factors for the development of RA. A hallmark of RA preceding the disease onset is the emergence of autoantibodies, including rheumatoid factors (RFs) and anticitrullinated protein antibodies (ACPAs). Being the most specific (>90%) and sensitive (>60%) autoantibodies in RA, ACPAs have been included in the clinical criteria for the classification of RA. The function of ACPAs in RA is still unclear. Although patients with ACPA positivity are associated with more severe arthritis and in vitro studies have shown certain pathogenic effects of ACPAs, the in vivo evidence remains lacking. On the other hand, extensive but common Nglycosylation in the variable domain of ACPAs (90%) has been unveiled, questioning if these Nglycans serve a functional role.

In Study I, we expressed several monoclonal ACPAs derived from RA patients and identified their specificities using a panel of citrullinated peptides. We found one of the ACPAs, clone E4, could protect against collagen antibody induced arthritis in mice. The protection is joint-specific and depending on the interaction between E4 in complex with citrullinated alpha-enolase and FCGR2B on activated macrophages, enhancing the IL-10 secretion and supressing osteoclastogenesis by macrophages. In Study II, we focused on the variable domain glycans (VDGs) in ACPAs by employing crystallography, glycobiology and functional B cell assay. We showed that 1) VDGs are positioned in the vicinity of the paratope with an impact on the antigen-binding; 2) VDGs could enhance B cell activation, and 3) VDG-expressing B cell receptors stay longer on the cell surface. In Study III, we investigated the two most significant arthritis QTLs in inbred rats, Ncf1 and Clec4b, and showed that Ncf1 and Clec4b together modulate the severity of arthritis in rats and their expression on neutrophils modulate the production of reactive oxygen species by neutrophils.

Taken together, the findings revealed a protective, rather than pathogenic effect of certain ACPAs in RA and elucidated the unique properties of VDGs in ACPAs and their functional impact on autoreactive B cells.

List of scientific papers

I. Yibo He, Changrong Ge, Àlex Moreno-Giró, Bingze Xu, Christian M. Beusch, Katalin Sandor, Jie Su, Lei Cheng, Erik Lönnblom, Christina Lundqvist, Linda M. Slot, Dongmei Tong, Vilma Urbonaviciute, Bibo Liang, Taotao Li, Gonzalo Fernandez Lahore, Mike Aoun, Vivianne Malmström, Theo Rispens, Patrik Ernfors, Camilla I. Svensson, Hans Ulrich Scherer, René E. M. Toes, Inger Gjertsson, Olov Ekwall, Roman A. Zubarev & Rikard Holmdahl. A subset of antibodies targeting citrullinated proteins confers protection from rheumatoid arthritis. Nature Communications. 14, 691 (2023).
https://doi.org/10.1038/s41467-023-36257-x

II. Theresa Kissel, Changrong Ge, Lise Hafkenscheid, Joanneke C. Kwekkeboom, Linda M. Slot, Marco Cavallari, Yibo He, Karin A. van Schie, Rochelle D. Vergroesen, Arieke S.B. Kampstra, Sanne Reijm, Gerrie Stoeken-Rijsbergen, Carolien Koeleman, Lennard M. Voortman, Laura H. Heitman, Bingze Xu, Ger J.M. Pruijn, Manfred Wuhrer, Theo Rispens, Tom W.J. Huizinga, Hans Ulrich Scherer, Michael Reth, Rikard Holmdahl, Rene E.M. Toes. Surface Ig variable domain glycosylation affects autoantigen binding and acts as threshold for human autoreactive B cell activation. Science Advances. 8, eabm1759 (2022).
https://doi.org/10.1126/sciadv.abm1759

III. Mike Aoun, Xiaojie Cai, Bingze Xu, Gonzalo Fernandez Lahore, Michael Yi Bonner, Yibo He, Liselotte Bäckdahl and Rikard Holmdahl. Glycan Activation of Clec4b Induces Reactive Oxygen Species Protecting against Neutrophilia and Arthritis. Antioxidants. 11(1): 12 (2022).
https://doi.org/10.3390/antiox11010012