Autoantibodies and lipids - novel contributors to pain in fibromyalgia?

Fibromyalgia (FM) is a chronic primary pain condition that affects 2-8% of the population, predominantly women (80%). It is characterized by pain that arises from musculoskeletal tissue, and skin. Other concomitant symptoms are usually fatigue, anxiety, depression, cognitive and sleep impairment. Although FM is non- destructive, it can significantly diminish the quality of life and contribute to high socio-economic costs. Albeit promising findings regarding changes in FM, its pathophysiology remains poorly understood, and efficient treatments are lacking. This thesis sought to investigate cellular and molecular contributors to pain in FM.

Study I explored whether autoimmunity could be a key player in FM pathogenesis. Immunoglobulins G (IgG) isolated from the serum of FM patients were injected intraperitoneally into mice, resulting in polymodal hypersensitivities, reduced skin innervation, decreased locomotion, and reduced grip strength. Conversely, IgG isolated from healthy controls (HC). IgG was sequestered into the dorsal root ganglia (DRG), and consequently bound and activated satellite glial cells (SGC) in vitro and in vivo. Anti-SGC IgG also recognized epitopes present in postmortem human dorsal root ganglia (hDRG).

Study II assessed anti-SGC IgG levels in individual serum or plasma samples from 2 FM cohorts (Sweden and Canada, n=30/35), and HC. Additionally, correlations of anti-SGC IgG and phenotypic characteristics were investigated. Our screening assay, consisting of primary cell cultures enriched for SGCs that were "live" incubated with serum or plasma, showed that FM IgG bound more frequently and strongly to in vitro murine SGCs, and postmortem human DRGs; predominantly in SGCs but also to some extent neuronal soma, compared to HC. The percentage of anti-SGC IgG from FM patients was significantly correlated with disease severity, in the form of VAS and FIQ scores, and to a lesser extent PPT. K-means clustering analysis based on VASaverage, VASmax and FIQ, split the patients into severe and mild FM groups. Importantly, the FM severe group presented increased frequency and binding intensity of FM IgG to SGCs, indicating a distinct subset of FM patients with increased IgG reactivity.

Study III used lipidomic analysis to investigate lipid changes, associated to disease severity, and anti-SGC IgG levels in serum from FM normal-weight patients (n=35), compared to HC (n=33). High-performance liquid chromatography (HPLC) coupled to high-resolution mass spectrometry (LC- HRMS) was performed. Patients were divided into mild and severe groups, according to K-means clustering based on VASaverage, VASmax and FIQ scores. IgG binding to SGCs was assessed as described in Study II. Lipid levels were adjusted for independent covariates, such as BMI, age, and week of MS acquisition using linear regression (LR). Additionally, the relationship between anti-SGC IgG levels and lipid levels was analyzed with LR, and lipid levels and pain scores, such as VAS, FIQ, PPT and CPM were analyzed with Spearman rank's correlation. Decreased levels of lysophosphatidylcholine (LPC) (n=10), lysophsphatidylethanolamine (LPE) (n=7), phosphatidylcholine (PC) (n=4) and triglycerides (TGs) (n=5) were observed in FM. In contrast, diglycerides (DGs) (n=3) were found to be upregulated. A number of LPC species, such as 19:0, 22:0, and 24:1 were upregulated in the severe FM group, compared to the mild, and the latter two positively correlated with anti-SGC IgG levels. TGs correlated positively with pain ratings and to a lesser extent anti-SGC IgG levels. Sphingomyelins (SMs) (n=6) displayed increased concentrations in the mild FM group, compared to HC, but decreased in the severe FM group. Additionally, SMs (n=8) negatively correlated with clinical scores, with only one SM species showing a significant correlation with anti-SGC IgG levels. Not only SMs, but also one DG, PCs (n=3) and TGs (n=5) were negatively associated with phenotypic characteristics.

Study IV measured Lysophosphatidic acid (LPA) in FM serum and cerebrospinal fluid (CSF). Autotaxin (ATX) levels from FM serum samples were also evaluated. For comparison, serum LPA levels were measured also in other painful conditions, such as osteoarthritis (OA), disc degeneration disc (DDD), and lumbar disc herniation (LDH). To investigate LPA and ATX levels, we employed LPA and ATX ELISA assays. We found a significant increase of LPA in the serum of FM patients, compared to HC. LPA levels correlated positively with conditioned pain modulation (CPM), and to a less extent VASmin. In CSF, no change of LPA levels nor ATX in the serum of FM patients was found. However, we observed a significant correlation between LPA and ATX levels in one FM cohort, substantiating the hypothesis of ATX being the responsible enzyme for the generation of LPA in FM. Interestingly, we found an increase of LPA in the serum of OA female patients, that correlated with VASpain and KOOS. Conversely, LPA levels were decreased in DDD, compared to HC. Our study highlights the presence of LPA in painful conditions, however in FM, a dual role is suggested, that requires further investigation.

Taken together, this thesis highlights the presence of important peripheral contributors to pain in FM, such as autoimmunity, and changes in lipid metabolism, as seen in the following lipid classes: TG, LPC, SM as well as LPA, which can contribute to the symptomatology present in FM patients' subsets. Therapies that can reduce IgG titers or address the altered lipid metabolism might provide relief to these patients. Therefore, further mechanistic and longitudinal studies are required to fully elucidate pain mechanisms of FM.

List of scientific papers

I. Passive transfer of fibromyalgia symptoms from patients to mice. Andreas Goebel*, Emerson Krock*, Clive Gentry, Mathilde R. Israel, Alexandra Jurczak, Carlos Morado Urbina, Katalin Sandor, Nisha Vastani, Margot Maurer, Ulku Cuhadar, Serena Sensi, Yuki Nomura, Joana Menezes, Azar Baharpoor, Louisa Brieskorn, Angelica Sandström, Jeanette Tour, Diana Kadetoff, Lisbet Haglund, Eva Kosek, Stuart Bevan, Camilla I. Svensson#, and David A. Andersson#. J Clin Invest. 2021 Jul 1;131(13):e144201. https://doi.org/10.1172/JCI144201

II. Fibromyalgia patients with elevated levels of anti-satellite glia cell immunoglobulin G antibodies present with more severe symptoms. Emerson Krock, Carlos E. Morado-Urbina, Joana Menezes, Matthew A. Hunt, Angelica Sandström, Diana Kadetoff, Jeanette Tour, Vivek Verma, Kim Kultima, Lisbet Haglund, Carolina B. Meloto, Luda Diatchenko, Eva Kosek, Camilla I. Svensson. Pain. 2023 Aug 1;164(8):1828-1840. https://doi.org/10.1097/j.pain.0000000000002881

III. Fibromyalgia patients have altered lipid concentrations associated with disease symptom severity and anti-satellite glial cell IgG antibodies. Jenny E. Jakobsson* , Joana Menezes* , Emerson Krock, Matthew A. Hunt, Henrik Carlsson, Aina Vaidade, Payam Emami Khoonsari, Nilesh M. Agalave, Angelica Sandström, Diana Kadetoff, Jeanette Tour, Ida Erngren, Asma Al-Grety, Eva Freyhult, Katalin Sandor, Eva Kosek, Camilla I. Svensson, Kim Kultima. [Manuscript]

IV. Comparative analysis of lysophosphatidic acid levels in fibromyalgia and other painful conditions. Joana Menezes, Jenny E. Jackobsson, Alex Bersellini, Farinotti, Emerson Krock, Matthew Hunt, Nils Simon, Sigita Venckute Larsson, Kim Kultima#, Eva Kosek#, Camilla I. Svensson#. [Manuscript]

*, # Contributed equally