circRNA landscape in dorsal root ganglia from mice with collagen antibody-induced arthritis.

Circular RNAs are a novel class of RNA molecules that are covalently closed into a ring structure. They are an epigenetic regulatory mechanism, and their best-studied function is regulation of microRNA activity. As such circular RNAs may be involved in the switch from acute to chronic pain. They have previously been studied in the context of neuropathic pain models, but their importance in inflammation-induced chronic pain models is unexplored. Microarray analysis of dorsal root ganglia collected in the late phase of collagen antibody-induced arthritis (day 59) were used to elucidate the relevance of circular RNAs in the mechanical hypersensitivity caused by this model. 120 circular RNA genes were found to be significantly differentially regulated in female BALB/c mice with collagen antibody-induced arthritis. Six genes were chosen for RT-qPCR analysis in the late (day 54-60) as well as the inflammatory (day 11-12) phase of this model. This validated an increase in circNufip1 expression in the late phase of collagen antibody-induced arthritis. Additionally, it was found that circVps13 and circMicall1 are upregulated in the inflammatory phase. Interestingly, no changes were found in dorsal root ganglia from mice injected with Freund's Complete Adjuvant (day 3) nor mice with spared nerve injury (day 20), despite their similarities to inflammatory and late phase collagen antibody-induced arthritis, respectively. This study provides evidence that mild circular RNA changes occur in dorsal root ganglia of mice with collagen antibody-induced arthritis that are, bioinformatically, predicated to be involved in processes relevant to sensitization.

Insights into FcγR involvement in pain-like behavior induced by an RA-derived anti-modified protein autoantibody.

Joint pain is one of the most debilitating symptoms of rheumatoid arthritis (RA) and patients frequently rate improvements in pain management as their priority. RA is hallmarked by the presence of anti-modified protein autoantibodies (AMPA) against post-translationally modified citrullinated, carbamylated and acetylated proteins. It has been suggested that autoantibody-mediated processes represent distinct mechanisms contributing to pain in RA. In this study, we investigated the pronociceptive properties of monoclonal AMPA 1325:01B09 (B09 mAb) derived from the plasma cell of an RA patient. We found that B09 mAb induces pain-like behavior in mice that is not associated with any visual, histological or transcriptional signs of inflammation in the joints, and not alleviated by non-steroidal anti-inflammatory drugs (NSAIDs). Instead, we found that B09 mAb is retained in dorsal root ganglia (DRG) and alters the expression of several satellite glia cell (SGC), neuron and macrophage-related factors in DRGs. Using mice that lack activating FcγRs, we uncovered that FcγRs are critical for the development of B09-induced pain-like behavior, and partially drive the transcriptional changes in the DRGs. Finally, we observed that B09 mAb binds SGC in vitro and in combination with external stimuli like ATP enhances transcriptional changes and protein release of pronociceptive factors from SGCs. We propose that certain RA antibodies bind epitopes in the DRG, here on SGCs, form immune complexes and activate resident macrophages via FcγR cross-linking. Our work supports the growing notion that autoantibodies can alter nociceptor signaling via mechanisms that are at large independent of local inflammatory processes in the joint.

Antibody-induced pain-like behavior and bone erosion: links to subclinical inflammation, osteoclast activity, and acid-sensing ion channel 3-dependent sensitization.

ABSTRACT: Several bone conditions, eg, bone cancer, osteoporosis, and rheumatoid arthritis (RA), are associated with a risk of developing persistent pain. Increased osteoclast activity is often the hallmark of these bony pathologies and not only leads to bone remodeling but is also a source of pronociceptive factors that sensitize the bone-innervating nociceptors. Although historically bone loss in RA has been believed to be a consequence of inflammation, both bone erosion and pain can occur years before the symptom onset. Here, we have addressed the disconnection between inflammation, pain, and bone erosion by using a combination of 2 monoclonal antibodies isolated from B cells of patients with RA. We have found that mice injected with B02/B09 monoclonal antibodies (mAbs) developed a long-lasting mechanical hypersensitivity that was accompanied by bone erosion in the absence of joint edema or synovitis. Intriguingly, we have noted a lack of analgesic effect of naproxen and a moderate elevation of few inflammatory factors in the ankle joints suggesting that B02/B09-induced pain-like behavior does not depend on inflammatory processes. By contrast, we found that inhibiting osteoclast activity and acid-sensing ion channel 3 signaling prevented the development of B02/B09-mediated mechanical hypersensitivity. Moreover, we have identified secretory phospholipase A2 and lysophosphatidylcholine 16:0 as critical components of B02/B09-induced pain-like behavior and shown that treatment with a secretory phospholipase A2 inhibitor reversed B02/B09-induced mechanical hypersensitivity and bone erosion. Taken together, our study suggests a potential link between bone erosion and pain in a state of subclinical inflammation and offers a step forward in understanding the mechanisms of bone pain in diseases such as RA.

Sex- and cell-dependent contribution of peripheral high mobility group box 1 and TLR4 in arthritis-induced pain.

ABSTRACT: Spinal high mobility group box 1 protein (HMGB1) plays crucial roles in arthritis-induced pain; however, the involvement of peripheral HMGB1 has not been examined previously. In this study, we addressed the role of peripheral HMGB1 and explored if sex contributes differentially to nociception in arthritis. We found Hmgb1 expression to be elevated in the ankle joints of male and female mice subjected to collagen antibody-induced arthritis. Blocking the action of peripheral HMGB1, however, only reversed collagen antibody-induced arthritis-mediated hypersensitivity in males. Intra-articular injection of the toll-like receptor (TLR)4-activating, partially reduced disulfide, but not the fully reduced all-thiol, HMGB1 evoked mechanical hypersensitivity in both sexes. A sex-dependent temporal profile in expression of inflammatory factors in the ankle joint was observed in response to intra-articular injection of disulfide HMGB1, with male mice showing a delayed, yet longer-lasting increase in mRNA levels for several of the investigated factors. Intra-articular HMGB1 did not induce cellular infiltration in the ankle joint suggesting its action on tissue resident cells. To further explore possible sex differences in cellular involvement, we used the macrophage inhibitor, minocycline, and mice with specific TLR4 depletion in myeloid cells or nociceptors. We found that inhibition of resident macrophages attenuated HMGB1-induced pain-like behavior only in male mice. Interestingly, although the contribution of TLR4 on myeloid cells to nociception was minimal in females compared to males, TLR4 on nociceptors are important for HMGB1-induced pain in both sexes. Collectively, our work highlights sex- and cellular location-dependent roles of HMGB1 and TLR4 in peripheral pain mechanisms.

Sex-dependent role of microglia in disulfide high mobility group box 1 protein-mediated mechanical hypersensitivity.

ABSTRACT: High mobility group box 1 protein (HMGB1) is increasingly regarded as an important player in the spinal regulation of chronic pain. Although it has been reported that HMGB1 induces spinal glial activation in a Toll-like receptor (TLR)4-dependent fashion, the aspect of sexual dimorphisms has not been thoroughly addressed. Here, we examined whether the action of TLR4-activating, partially reduced disulfide HMGB1 on microglia induces nociceptive behaviors in a sex-dependent manner. We found disulfide HMGB1 to equally increase microglial Iba1 immunoreactivity in lumbar spinal dorsal horn in male and female mice, but evoke higher cytokine and chemokine expression in primary microglial culture derived from males compared to females. Interestingly, TLR4 ablation in myeloid-derived cells, which include microglia, only protected male mice from developing HMGB1-induced mechanical hypersensitivity. Spinal administration of the glial inhibitor, minocycline, with disulfide HMGB1 also prevented pain-like behavior in male mice. To further explore sex difference, we examined the global spinal protein expression using liquid chromatography-mass spectrometry and found several antinociceptive and anti-inflammatory proteins to be upregulated in only male mice subjected to minocycline. One of the proteins elevated, alpha-1-antitrypsin, partially protected males but not females from developing HMGB1-induced pain. Targeting downstream proteins of alpha-1-antitrypsin failed to produce robust sex differences in pain-like behavior, suggesting that several proteins identified by liquid chromatography-mass spectrometry are required to modulate the effects. Taken together, the current study highlights the importance of mapping sex dimorphisms in pain mechanisms and point to processes potentially involved in the spinal antinociceptive effect of microglial inhibition in male mice.

Paracrine Signaling by Extracellular Vesicles via Osteoblasts.

Extracellular vesicles (EVs), spherical bilayered proteolipids, behave as paracrine effectors since they are released from cells to deliver signals to other cells. They control a diverse range of biological processes by transferring proteins, lipids, and nucleic acids between cells and are secreted by a wide spectrum of cell types and are found in various biological fluids. EVs are formed at the plasma membrane or in endosomes and are heterogeneous in size and composition. Increasing understanding of the working mechanisms is promising for therapeutic and diagnostic opportunities. In this review, we will focus on the recent developments in this emerging field with special emphasis on the role of EVs in the bone microenvironment, with a central role for the osteoblasts in the communication with a diversity of cells, including bone metastases.