Metabolic signature and proteasome activity controls synovial migration of CDC42hiCD14+ cells in rheumatoid arthritis

Objective Activation of Rho-GTPases in macrophages causes inflammation and severe arthritis in mice. In this study, we explore if Rho-GTPases define the joint destination of pathogenic leukocytes in rheumatoid arthritis (RA) and how JAK inhibition mitigates these effects. Methods CD14+ cells of 136 RA patients were characterized by RNA-sequencing, and cytokine measurement to identify biological processes and transcriptional regulators specific for CDC42 hiCD14+ cells, which were summarized in a metabolic signature. Effect of hypoxia, and IFN-γ signaling on the metabolic signature of CD14+ cells was assessed experimentally. To investigate its connection with joint inflammation, the signature was translated into the single cell characteristics of CDC42 hi synovial tissue macrophages. Sensitivity of the metabolic signature to the RA disease activity and treatment effect was assessed experimentally and clinically. Results CDC42 hiCD14+ cells carried the metabolic signature of genes functional in the oxidative phosphorylation and proteasome-dependent cell remodeling, which correlated with the cytokine-rich migratory phenotype and antigen presenting capacity of these cells. Integration of CDC42 hi CD14+ and synovial macrophages marked with the metabolic signature revealed the important role of the interferon-rich environment and immunoproteasome expression in homeostasis of these pathogenic macrophages. The CDC42 hiCD14+ cells were targeted by JAK-inhibitors and responded with downregulation of immunoproteasome and MHC-II molecules, which disintegrated the immunological synapse, reduced cytokine production and alleviated arthritis. Conclusion This study shows that the CDC42-related metabolic signature identifies the antigen-presenting CD14+ cells that migrate to joints to coordinate autoimmunity. Accumulation of CDC42 hiCD14+ cells disclose patients perceptive to JAKi treatment. ### Competing Interest Statement The authors have declared no competing interest. ### Funding Statement This work has been funded by grants from the Swedish Research Council (MB, 2017-03025 and 2017-495 00359), the Swedish Association against Rheumatism (MB, R-566961, R-751351 and R-860371), the 496 King Gustaf V:s 80-year Foundation (MB, FAI-2018-0519 and FAI-2020-0653), the Regional 497 agreement on medical training and clinical research between the Western Gotaland county council and 498 the University of Gothenburg (MB, ALFGBG-717681, ALFGBG-965623; RP, ALFGBG-965012, 499 ALFGBG-926621), the University of Gothenburg. The authors declare that the funding sources have 500 no role in study design; in the collection, analysis, and interpretation of data; in the writing of the 501 report; and in the decision to submit the paper for publication. ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: The study was approved by the Swedish Ethical Review Authority (659-2011) and done in accordance with the Declaration of Helsinki. The trial is registered at [ClinicalTrials.gov][1] (ID [NCT03449589][2]) I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable. Yes All data produced in the present study are available upon reasonable request to the authors [1]: http://ClinicalTrials.gov [2]: /lookup/external-ref?link_type=CLINTRIALGOV&access_num=NCT03449589&atom=%2Fmedrxiv%2Fearly%2F2023%2F06%2F16%2F2023.06.15.23291416.atom