Antibody activation of sensory neurons : exploring novel pain mechanisms in rheumatoid arthritis

Chronic pain is a worldwide major problem that presents several challenges due to lack of treatment efficacy and/or side effects associated with long-term usage of analgesics. Autoimmune diseases such as rheumatoid arthritis (RA) are often characterized by pain components, which generally poorly respond to drug treatment. In fact, RA patients suffer from persistent pain even if the active disease and inflammation is under medical control or in remission. Moreover, pain appears years before the onset of the active disease. This indicates that RA pain components might underlie additional unknown mechanisms rather than only the classical view of pain strictly correlating with inflammation. Of note, recent studies show that RA autoantibodies are present in RA patients up to 10 years before the onset of inflammation and most of the available treatment options in the clinics do not affect antibody titers. Therefore, the aim of this thesis is to investigate possible autoantibody actions that could represent the missing link explaining pain in RA in the pre- and post-inflammatory phases of the disease. In study I, we explored the role of RA-relevant autoantibodies in directly activating sensory neurons. Injection of anti-collagen type II (CII) antibodies (Abs) promoted pain-like behavior in mice in the absence of any visual, histological or molecular inflammation. This pain-like behavior was not dependent on complement activation or destabilization of cartilage structure. Instead, our data suggested a direct activation of CII-immune complexes (ICs) on sensory neurons via the activation of Fc gamma receptors (FcγRs). Indeed, we found expression of FcγRI and FcγRIIb proteins on peripheral neuronal terminals in mouse skin. In addition, CII-IC in vitro stimulation of cultured dorsal root ganglia (DRGs) neuronal cells promoted release of a calcitonin gene related peptide (CGRP), intracellular increase of calcium levels and membrane depolarization. Interestingly, CGRP release was prevented in cultures from FcRγ chain deficient mice (lacking activating FcγRI, III and IV, but still expressing inhibitory FcγRIIb). Accordingly, injection of anti-CII Abs failed to induce pain-like behavior in FcRγ chain deficient mice or when the Ab-FcγR interaction was altered. Instead, mice expressing activating FcγRs only on non-hematopoietic cells (including neurons), but not on hematopoietic cells, displayed similar pain thresholds to wild type mice when injected with anti-CII Abs. Altogether our data suggested a novel RA-associated pain mechanism of direct interaction between Abs and FcγRI present on sensory neurons that is independent of inflammatory functions of pathological Abs. Finally, we showed that human DRG neurons also express the activating FcγRIIIA making our data translational to clinics, possibly explaining pain in RA patients before the onset of the disease or even when it is under medical control or in remission. In study II, we investigated pain-associated pathological actions of human anticitrullinated proteins antibodies (ACPA) purified from RA-patients. Injection of human ACPA, but not non-ACPA or IgGs from healthy individuals, promoted pain-like behavior in mice in the absence of visual, histological and molecular inflammation. Furthermore, ACPA did not induce significant increase of intracellular calcium levels or membrane depolarization in cultured DRG neurons, suggesting that ACPA do not exert their nociceptive functions through a direct action of their Fab region on sensory neurons. However, ACPA bound to osteoclasts, inducing the release of the mouse interleukin-8 analogue CXCL1, which subsequentially sensitized neurons. In fact, a CXCL1 receptor antagonist or an osteoclasts inhibitor prevented ACPAinduced pain-like behavior. In conclusion, we provided evidence of novel nociceptive actions of human ACPA, offering new targets in IL-8 and osteoclasts for the pain treatment of the ACPA-positive subgroup of RA patients. In study III, we characterized B35, Neuro-2a (N2a) and F11 neuroblastoma cell lines, trying to find an alternative method to primary DRG cultures from rodents for pain-related in vitro experiments. We compared the cell lines subjected to two differentiation media to promote the acquisition of more neuronal-like features on parameters such as morphology, proliferation, metabolic activity, expression of neuronal markers and functional activity. While B35 showed the highest neuronallike morphological features, N2a the highest neuronal markers expression and F11 the highest neuronal excitability in functional assays, all the cell lines compared to primary DRG cultures only to some extent. Therefore, our findings indicated that neuroblastoma cell lines should be carefully selected by researchers for studying neuronal processes, as they do not represent a complete substitute of primary DRG cultures. In summary, this thesis addresses the crucial need of better understanding the underlying pain mechanisms in RA and provides novel insights that could potentially benefit the clinical therapeutic strategies, opening new avenues for the development of innovative pain-relief drugs

Cartilage-binding antibodies induce pain through immune complex-mediated activation of neurons

Rheumatoid arthritis-associated joint pain is frequently observed independent of disease activity, suggesting unidentified pain mechanisms. We demonstrate that antibodies binding to cartilage, specific for collagen type II (CII) or cartilage oligomeric matrix protein (COMP), elicit mechanical hypersensitivity in mice, uncoupled from visual, histological and molecular indications of inflammation. Cartilage antibody-induced pain-like behavior does not depend on complement activation or joint inflammation, but instead on tissue antigen recognition and local immune complex (IC) formation. smFISH and IHC suggest that neuronal Fcgr1 and Fcgr2b mRNA are transported to peripheral ends of primary afferents. CII-ICs directly activate cultured WT but not FcRγ chain-deficient DRG neurons. In line with this observation, CII-IC does not induce mechanical hypersensitivity in FcRγ chain-deficient mice. Furthermore, injection of CII antibodies does not generate pain-like behavior in FcRγ chain-deficient mice or mice lacking activating FcγRs in neurons. In summary, this study defines functional coupling between autoantibodies and pain transmission that may facilitate the development of new disease-relevant pain therapeutics

Flexible Organic Electronic Ion Pump Fabricated Using Inkjet Printing and Microfabrication for Precision In Vitro Delivery of Bupivacaine

The organic electronic ion pump (OEIP) is an on-demand electrophoretic drug delivery device, that via electronic to ionic signal conversion enables drug delivery without additional pressure or volume changes. The fundamental component of OEIPs is their polyelectrolyte membranes which are shaped into ionic channels that conduct and deliver ionic drugs, with high spatiotemporal resolution. The patterning of these membranes is essential in OEIP devices and is typically achieved using laborious micro processing techniques. Here, we report the development of an inkjet printable formulation of polyelectrolyte, based on a custom anionically functionalized hyperbranched polyglycerol (i-AHPG). This polyelectrolyte ink greatly simplifies the fabrication process, and is used in the production of free standing, OEIPs on flexible polyimide substrates. Both i-AHPG and the OEIP devices are characterized, exhibiting favorable iontronic characteristics of charge selectivity and ability to transport aromatic compounds. Further, the applicability of these technologies is demonstrated by transport and delivery of the pharmaceutical compound bupivacaine to dorsal root ganglion cells with high spatial precision and effective nerve-blocking, highlighting the applicability of these technologies for biomedical scenarios.Funding: Swedish Foundation for Strategic Research; Knut and Alice Wallenberg Foundation; Swedish Research Council; European Research Council [834677]; Swedish Government Strategic Research Area in Materials Science on Advanced Functional Materials at Linkoping University (Faculty Grant SFO-Mat-LiU) [2009-00971]; Vinnova</p

Electrophoretic Delivery of Clinically Approved Anesthetic Drug for Chronic Pain Therapy

Despite a range of available pain therapies, most patients report so-called “breakthrough pain.” Coupled with global issues like opioid abuse, there is a clear need for advanced therapies and technologies for safe and effective pain management. Here the authors demonstrate a candidate for such an advanced therapy: precise and fluid-flow-free electrophoretic delivery via organic electronic ion pumps (OEIPs) of the commonly used anesthetic drug bupivacaine. Bupivacaine is delivered to dorsal root ganglion (DRG) neurons in vitro. DRG neurons are a good proxy for pain studies as they are responsible for relaying ascending sensory signals from nociceptors (pain receptors) in the peripheral nervous system to the central nervous system. Capillary based OEIPs are used due to their probe-like and free-standing form factor, ideal for interfacing with cells. By delivering bupivacaine with the OEIP and recording dose versus response (Ca2+ imaging), it is observed that only cells close to the OEIP outlet (≤75 µm) are affected (“anaesthetized”) and at concentrations up to 10s of thousands of times lower than with bulk/bolus delivery. These results demonstrate the first effective OEIP deliveryof a clinically approved and widely used analgesic pharmaceutical, and thus are a major translational milestone for this technology.Funding agencies: This work was supported by the Swedish Foundation for Strategic Research, the Knut and Alice Wallenberg Foundation, the Swedish Research Council, the European Research Council (AdG 2018 Magnus Berggren, 834677 and CoG 2019 Camilla Svensson, 866075), and Vinnova. Additional support was provided by the Swedish Government Strategic Research Area in Materials Science on Advanced Functional Materials at Linköping University (Faculty Grant SFO-Mat-LiU no. 2009-00971).</p

UPEC kidney infection triggers neuro-immune communication leading to modulation of local renal inflammation by splenic IFNγ.

Bacterial infection results in a veritable cascade of host responses, both local and systemic. To study the initial stages of host-pathogen interaction in living tissue we use spatially-temporally controlled in vivo models. Using this approach, we show here that within 4 h of a uropathogenic Escherichia coli (UPEC) infection in the kidney, an IFNγ response is triggered in the spleen. This rapid infection-mediated inter-organ communication was found to be transmitted via nerve signalling. Bacterial expression of the toxin α-hemolysin directly and indirectly activated sensory neurons, which were identified in the basement membrane of renal tubules. Nerve activation was transmitted via the splenic nerve, inducing upregulation of IFNγ in the marginal zones of the spleen that led to increasing concentrations of IFNγ in the circulation. We found that IFNγ modulated the inflammatory signalling generated by renal epithelia cells in response to UPEC infection. This demonstrates a new concept in the host response to kidney infection; the role of nerves in sensing infection and rapidly triggering a systemic response which can modulate inflammation at the site of infection. The interplay between the nervous and immune systems is an exciting, developing field with the appealing prospect of non-pharmaceutical interventions. Our study identifies an important role for systemic neuro-immune communication in modulating inflammation during the very first hours of a local bacterial infection in vivo

Cell–cell interactions in joint pain: rheumatoid arthritis and osteoarthritis

Rheumatoid and osteoarthritis are chronic conditions generating joint pain for which better management is required. We argue that a better understanding of the cell-to-cell interactions occurring within the joint will enhance mechanistic understanding of joint pain and could lead to new therapeutic avenues being explored.L.A.P. was supported by the University of Cambridge BBSRC Doctoral Training Programme (BB/M011194/1). E.K. acknowledges support from a Canadian Institutes of Health Research Fellowship (201910MFE-430366-253212) and an International Association for the Study of Pain John J. Bonica Fellowship. C.I.S. acknowledges support from the Swedish Research Council (542-2013-8373) and Knut and Alice Wallenberg Foundation (2018-0161). E.St.J.S acknowledges support from Versus Arthritis (RG 21973)

Cartilage-binding antibodies induce pain through immune complex-mediated activation of neurons

Rheumatoid arthritis-associated joint pain is frequently observed independent of disease activity, suggesting unidentified pain mechanisms. We demonstrate that antibodies binding to cartilage, specific for collagen type II (CII) or cartilage oligomeric matrix protein (COMP), elicit mechanical hypersensitivity in mice, uncoupled from visual, histological and molecular indications of inflammation. Cartilage antibody-induced pain-like behavior does not depend on complement activation or joint inflammation, but instead on tissue antigen recognition and local immune complex (IC) formation. smFISH and IHC suggest that neuronal Fcgr1 and Fcgr2b mRNA are transported to peripheral ends of primary afferents. CII-ICs directly activate cultured WT but not FcR gamma chain-deficient DRG neurons. In line with this observation, CII-IC does not induce mechanical hypersensitivity in FcR gamma chain-deficient mice. Furthermore, injection of CII antibodies does not generate pain-like behavior in FcR gamma chain-deficient mice or mice lacking activating Fc gamma Rs in neurons. In summary, this study defines functional coupling between autoantibodies and pain transmission that may facilitate the development of new disease-relevant pain therapeutics