A subset of antibodies targeting citrullinated proteins confers protection from rheumatoid arthritis

Although elevated levels of anti-citrullinated protein antibodies (ACPAs) are a hallmark of rheumatoid arthritis (RA), the in vivo functions of these antibodies remain unclear. Here, we have expressed monoclonal ACPAs derived from patients with RA, and analyzed their functions in mice, as well as their specificities. None of the ACPAs showed arthritogenicity nor induced pain-associated behavior in mice. However, one of the antibodies, clone E4, protected mice from antibody-induced arthritis. E4 showed a binding pattern restricted to skin, macrophages and dendritic cells in lymphoid tissue, and cartilage derived from mouse and human arthritic joints. Proteomic analysis confirmed that E4 strongly binds to macrophages and certain RA synovial fluid proteins such as α-enolase. The protective effect of E4 was epitope-specific and dependent on the interaction between E4-citrullinated α-enolase immune complexes with FCGR2B on macrophages, resulting in increased IL-10 secretion and reduced osteoclastogenesis. These findings suggest that a subset of ACPAs have therapeutic potential in RA.


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The experiments using human samples in the present study focus on tissue staining or molecular analysis with small sample size (n<5), the samples were randomly selected based only on clinical criteria, the findings therefore do not apply to only one sex or gender.
RA patients visiting LUMC with anti-CCP2 positivity, none of them was treated with B-cell depleting therapies or biologicals; RA patients (female) with anti-CEP1 positivity at clinical visits due to a need for joint effusions, with age range 27-47y and disease duration 1-16y; The cartilage explant samples originate from patients with fractured femur head or RA patients; all RA patients fulfilled the EULAR/ACR criteria for the classification of RA. The human thymic tissues were obtained from two children (age 64d old female and 153d old male), diagnosed with ventricular septal defect (VSD), and underwent corrective cardiac surgery, further genetic conditions not applicable. Buffy coats from healthy donors were obtained from Karolinska University Hospital (Solna, Stockholm). Please refer to the materials & methods section in the manuscript for more details.
Participants were recruited in the clinic in voluntary basis. All participants gave written informed consent priorly. Samples were randomly selected based on the experimental purpose without bias.
Local ethics committee of the LUMC, The Netherlands; regional ethics committee at the University of Gothenburg, Sweden; regional ethics committee at Karolinska Institutet, Sweden.
Depending on the experimental design and model, for example, in CAIA model, n=4 or 5 is the typical minimal replicate number, and in EAE model, n=10 is the typical minimal replicate number. In experiments using human samples with multiple groups, n=3 is the minimal replicate number for statistical analysis and in cell experiments with multiple groups, at least three replicates were used, as we deemed this to be sufficient based on the design of the various controls included and low observed variability between samples. More details are specified in each experimental design in the manuscript.
No data exclusions in the present study.
All results were successfully replicated, more details are provided in the present manuscript text and figure legends. Experiments were repeated either exact, or similar setup.
All samples were randomly allocated into experimental groups. Mice were kept in mixed cages to avoid cage effect.
Animal disease model experiments were scored in a blinded manner. In vitro experiments were not blinded, as readout measurements were automated and purely quantitative, eliminating any subjective bias. As an occasion, mannual counting of osteoclast number was performed in a blinded manner.  Table 1 in the manuscript): E4, L1-L12, E4NG, E4m, ACC1, ACC4, M2139, E4 hIgG1 and M2139 hIgG1. These mAbs are produced in-house, therefore the commercial information is not applicable. Dilution for the used antibodies are specified in the manuscript where applicable.
All antibodies were purified by affinity chromatography, endotoxin removal, dialysis/buffer exchange and validated by specificity tests either through ELISA, IHC or Western Blot etc., detailed information could be referred to peer-reviewed publications indicated in the manuscript, the present data in the manuscript, or by the manufacturers' product page.
For ATDC5, authentication was carried out by European Collection of Cell Cultures (ECACC), operated by Public Health England. Cells were analyzed and tested following the criteria: 1) no microbial growth vs. positive control; 2) cell count (>2x10^6 cells/amp, viability (>70%) and confluency (confluent in 2 days); 3) free of Mycoplasma contamination tested by PCR using Mycoplasma-specific primers. For Expi293F, 1) cells are demonstrated that they could be recovered as healthy logarithmically growing cells within 4 to 7 days after thawing. Viability was >90%. 2) cells are negative for Mycoplasma and 3) sterile.
All cell lines were tested free of Mycoplasma contamination.
No commonly misidentified lines were used.