Peripheral neuroimmune interactions in health and arthritis-induced pain

<p dir="ltr">Chronic joint pain is one of the leading reasons patients seek primary health care. Although advancements in anti-rheumatic drugs have significantly improved clinical outcomes for patients with rheumatoid arthritis (RA), persistent pain remains a substantial issue for many patients even after achieving clinical remission. Previous research using the collagen antibody-induced arthritis (CAIA) model demonstrated that it effectively recapitulates critical clinical features of RA, showing an inflammatory flare that resolves, while mechanical hypersensitivity, a pain-like behavior, persists beyond the flare resolution. This persistent pain has been linked to alterations in sensory neurons within the dorsal root ganglion (DRG) and the activation of surrounding satellite glial cells. Given the inflammatory nature of RA and the CAIA model, we hypothesized that peripheral neuroimmune interactions contribute to these observed changes. While the synovial immune environment is well characterized, immune heterogeneity in the DRG was less understood.</p><p dir="ltr">In Study I, we aimed to characterize the DRG neuroimmune landscape in relation to its vasculature and permeability. Immunohistology analysis revealed a greater density of macrophages and blood vessels in the DRG compared to adjacent nerve structures. Using intravenously injected fluorescent tracers, we confirmed that the DRG-blood barrier permits the entry of blood-derived molecules, primarily internalized by macrophages. We identified zonal permeability differences, correlating increased permeability with vessels expressing higher levels of PLVAP and lower levels of CLDN5. We demonstrated that caveolar endocytosis is responsible for this permeability. Single-cell transcriptomics identified two distinct DRG macrophage populations: CD163+ and CCR2+. Further experiments showed that only the CD163+ subset internalized intravenously injected tracers, indicating their specific role in blood surveillance within the DRG. Bone marrow chimera experiments revealed rapid turnover of CCR2+ macrophages from monocytes, while CD163+ macrophages exhibited significantly slower turnover, identifying them as long-lived, tissue-resident cells. This foundational study provided critical insights into the DRG immunovascular unit, paving the way for subsequent studies examining persistent pain.</p><p dir="ltr">In Study II, we utilized findings from the first study to investigate whether neuroimmune interactions contribute to persistent pain in the CAIA model. Single-cell transcriptomics with hashtagging enabled the creation of a temporal atlas of the DRG-joint axis during CAIA progression, revealing extensive joint remodeling during the inflammatory flare, characterized by neutrophil and monocyte infiltration, loss of long-lived, tissue-resident macrophage subtypes, and alterations in fibroblast architecture. Importantly, the macrophage-fibroblast landscape of the DRG remained altered after the resolution of the inflammatory flare. Interestingly, cellular changes within the DRG were minimal. In vivo depletion experiments demonstrated that inflammation and pain-like behaviors in the CAIA model are neutrophil-dependent. By profiling synovial CGRP+ nerve fiber sprouting, we identified that this sprouting occurs during inflammation resolution, secondary to neutrophil infiltration. Utilizing cell-cell interaction analysis on the DRG-joint atlas, we identified neutrophil-derived Osm and Sema4D as critical ligands driving neuronal remodeling. Examination of publicly available human RA datasets confirmed expression of these molecules by synovial neutrophils in RA patients. Thus, our findings conclusively showed that neutrophils, rather than macrophages, drive inflammation, persistent mechanical hypersensitivity, and nerve fiber sprouting in CAIA via OSM and SEMA4D.</p><p dir="ltr">Study III emerged as a technical advancement from Study II. Here, we leveraged DRG single-cell data to identify sensory neuron markers, subsequently developing a FACS-based method and gating strategy for their isolation. Microscopic validation confirmed neuronal enrichment based on size and morphology. This new protocol facilitates DRG neuron analysis via flow cytometry, eliminating the need for genetic reporter mice or retrograde dye injections.</p><p dir="ltr">In Study IV, we explored the anti-nociceptive properties of baricitinib, a JAK-STAT pathway inhibitor approved for RA treatment, noted clinically for superior pain alleviation compared to other RA medications. Baricitinib treatment reversed mechanical hypersensitivity in CAIA mice during the post-inflammatory phase and reduced CGRP+ nerve fiber sprouting. Expression analysis revealed JAK1 and STAT3 presence in both mouse and human sensory neurons, suggesting direct neuronal actions of baricitinib.</p><p dir="ltr">Collectively, this thesis delineates peripheral neuroimmune interactions in physiological and arthritic contexts, offering novel insights into the DRG macrophage-vascular unit, establishing tools for studying neuroimmune communication along the joint-DRG axis, and identifying novel therapeutic targets for nerve fiber sprouting and pain management. Additionally, it provides methodological improvements for transcriptomic studies of sensory neurons and elucidates previously unknown mechanisms of an existing RA therapeutic. Future research should aim to validate the translational potential of these findings in human RA.</p><h3>List of scientific papers</h3><p dir="ltr">I. CD163+ macrophages monitor enhanced permeability at the blood-dorsal root ganglion barrier. Harald Lund*, Matthew A. Hunt*, Zerina Kurtović, Katalin Sandor, Paul B. Kägy, Noah Fereydouni, Anais Julien, Christian Göritz, Elisa Vazquez-Liebanas, Maarja Andaloussi Mäe, Alexandra Jurczak, Jinming Han, Keying Zhu, Robert A. Harris, Jon Lampa, Jonas Heilskov Graversen, Anders Etzerodt, Lisbet Haglund, Tony L. Yaksh, and Camilla I. Svensson. J Exp Med, 2024, Vol. 221, No. 2. <a href="https://doi.org/10.1084/jem.20230675" rel="noreferrer" target="_blank">https://doi.org/10.1084/jem.20230675</a></p><p dir="ltr">II. Neutrophil-neuronal crosstalk drives arthritis-induced pain. Zerina Kurtović, Juan Antonio Vazquez Mora, Katalin Sandor, Alex Bersellini Farinotti, Nilesh Agalave, Sven David Arvidsson, Matthew A. Hunt, Nils Simon, Julia Dorothea Monika Döring, Alexandra Kuliszkiewicz, Lizeth Ponce Gomez, Sijing Ye, Giovanni Emmanuel López Delgado, Arisai Martinez Martinez, Eduardo Mendoza Sanchez, Khosiyat Makhmudova, Enriqueta Munoz Islas, Juan Miguel Jimenez Andrade, Harald Lund1#, Camilla I. Svensson#. [Manuscript]</p><p dir="ltr">III. A novel method to sort and enrich sensory neurons. Zerina Kurtović*, Juan Antonio Vazquez Mora*, Sven David Arvidsson*, Alex Bersellini Farinotti, Nils Simon, Sijing Ye, Michael Hagemann-Jensen, Harald Lund, Camilla Svensson. [Manuscript]</p><p dir="ltr">IV. Characterization of the antinociceptive effect of baricitinib in the collagen antibody-induced arthritis mouse model. Nils Simon*, Resti Rudjito*, Lydia Moll, Katalin Sandor, Juan Antonio Vazquez Mora, Zerina Kurtović, Alexandra Kuliszkiewicz, Carlos E. Morado Urbina, Sven David Arvidsson, Eduardo Mendoza-Sanchez, Giovanni E Lopez-Delgado, Qing Luo, Qiaolin Deng, Arisai Martínez Martínez, Jens Gammeltoft Gerwien, Paul Karila, Venkatesh Krishnan, Juan Miguel Jiménez-Andrade, Camilla I Svensson. Ann Rheum Dis, 2025, 84(3), 421-434. <a href="https://doi.org/10.1016/j.ard.2025.01.005" rel="noreferrer" target="_blank">https://doi.org/10.1016/j.ard.2025.01.005</a></p><p dir="ltr">*,# contributed equally</p>