Explore the vast range of titles available.

ObjectivesRheumatoid arthritis (RA)-specific anti-citrullinated protein/peptide antibodies (ACPAs) appear before disease onset and are associated with bone destruction. We aimed to dissect the role of ACPAs in osteoclast (OC) activation and to identify key cellular mediators in this process.MethodsPolyclonal ACPA were isolated from the synovial fluid (SF) and peripheral blood of patients with RA. Monoclonal ACPAs were isolated from single SF B-cells of patients with RA. OCs were developed from blood cell precursors with or without ACPAs. We analysed expression of citrullinated targets and peptidylarginine deiminases (PAD) enzymes by immunohistochemistry and cell supernatants by cytometric bead array. The effect of an anti-interleukin (IL)-8 neutralising antibody and a pan-PAD inhibitor was tested in the OC cultures. Monoclonal ACPAs were injected into mice and bone structure was analysed by micro-CT before and after CXCR1/2 blocking with reparixin.ResultsProtein citrullination by PADs is essential for OC differentiation. Polyclonal ACPAs enhance OC differentiation through a PAD-dependent IL-8-mediated autocrine loop that is completely abolished by IL-8 neutralisation. Some, but not all, human monoclonal ACPAs derived from single SF B-cells of patients with RA and exhibiting distinct epitope specificities promote OC differentiation in cell cultures. Transfer of the monoclonal ACPAs into mice induced bone loss that was completely reversed by the IL-8 antagonist reparixin.ConclusionsWe provide novel insights into the key role of citrullination and PAD enzymes during OC differentiation and ACPA-induced OC activation. Our findings suggest that IL8-dependent OC activation may constitute an early event in the initiation of the joint specific inflammation in ACPA-positive RA.

Background In the murine K/BxN serum transfer rheumatoid arthritis (RA) model, tactile allodynia persists after resolution of inflammation in male and partially in female wild type (WT) mice, which is absent in Toll-like receptor (TLR)4 deficient animals. We assessed the role of TLR4 on allodynia, bone remodeling and afferent sprouting in this model of arthritis. Methods K/BxN sera were injected into male and female mice with conditional or stable TLR4 deletion and controls. Paw swelling was scored and allodynia assessed by von Frey filaments. At day 28, synovial neural fibers were visualized with confocal microscopy and bone density assayed with microCT. Microglial activity and TLR4 dimerization in spinal cords were examined by immunofluorescence and flow cytometry. Results In the synovium, K/BxN injected WT male and female mice showed robust increases in calcitonin gene related-peptide (CGRP + ), tyrosine hydroxylase (TH) + and GAP43 + nerve fibers. Trabecular bone density by microCT was significantly decreased in K/BxN WT female but not in WT male mice. The number of osteoclasts increased in both sexes of WT mice, but not in Tlr4 -/- K/BxN mice. We used conditional strains with Cre drivers for monocytes/osteoclasts (lysozyme M), microglia (Tmem119 and Cx3CR1), astrocytes (GFAP) and sensory neurons (advillin) for Tlr4 f/f disruption. All strains developed similar arthritis scores after K/BxN serum injection with the exception being the Tlr4 Tmem119 mice which showed a reduction. Both sexes of Tlr4 Lyz2 , Tlr4 Tmem119 and Tlr4 Cx3cr1 mice displayed a partial reversal of the chronic pain phenotype but not in Tlr4 Avi l , and Tlr4 Gfap mice. WT K/BxN male mice showed increases in spinal Iba1, but not GFAP, compared to Tlr4 -/- male mice. To determine whether spinal TLR4 was indeed activated in the K/BxN mice, flow cytometry of lumbar spinal cords of WT K/BxN male mice was performed and revealed that TLR4 in microglia cells (CD11b + /TMEM119 + ) demonstrated dimerization (e.g. activation) and a characteristic increase in lipid rafts. Conclusion These results demonstrated a complex chronic allodynia phenotype associated with TLR4 in microglia and monocytic cell lineages, and a parallel spinal TLR4 activation. However, TLR4 is dispensable for the development of peripheral nerve sprouting in this model.

ObjectiveAn interesting and so far unexplained feature of chronic pain in autoimmune disease is the frequent disconnect between pain and inflammation. This is illustrated well in rheumatoid arthritis (RA) where pain in joints (arthralgia) may precede joint inflammation and persist even after successful anti-inflammatory treatment. In the present study, we have addressed the possibility that autoantibodies against citrullinated proteins (ACPA), present in RA, may be directly responsible for the induction of pain, independent of inflammation.MethodsAntibodies purified from human patients with RA, healthy donors and murinised monoclonal ACPA were injected into mice. Pain-like behaviour was monitored for up to 28 days, and tissues were analysed for signs of pathology. Mouse osteoclasts were cultured and stimulated with antibodies, and supernatants analysed for release of factors. Mice were treated with CXCR1/2 (interleukin (IL) 8 receptor) antagonist reparixin.ResultsMice injected with either human or murinised ACPA developed long-lasting pronounced pain-like behaviour in the absence of inflammation, while non-ACPA IgG from patients with RA or control monoclonal IgG were without pronociceptive effect. This effect was coupled to ACPA-mediated activation of osteoclasts and release of the nociceptive chemokine CXCL1 (analogue to human IL-8). ACPA-induced pain-like behaviour was reversed with reparixin.ConclusionsThe data suggest that CXCL1/IL-8, released from osteoclasts in an autoantibody-dependent manner, produces pain by activating sensory neurons. The identification of this new pain pathway may open new avenues for pain treatment in RA and also in other painful diseases associated with autoantibody production and/or osteoclast activation.

Although the role of peripheral nerves in cancer progression has been appreciated, little is known regarding cancer/sensory nerve crosstalk and its contribution to bone metastasis and associated pain. In this study, we revealed that the cancer/sensory nerve crosstalk plays a crucial role in bone metastatic progression. We found that (i) periosteal sensory nerves expressing calcitonin gene–related peptide (CGRP) are enriched in mice with bone metastasis; (ii) cancer patients with bone metastasis have elevated CGRP serum levels; (iii) bone metastatic patient tumor samples express elevated calcitonin receptor-like receptor (CRLR, a CGRP receptor component); (iv) higher CRLR levels in cancer patients are negatively correlated with recurrence-free survival; (v) CGRP induces cancer cell proliferation through the CRLR/p38/HSP27 pathway; and (vi) blocking sensory neuron–derived CGRP reduces cancer cell proliferation in vitro and bone metastatic progression in vivo. This suggests that CGRP-expressing sensory nerves are involved in bone metastatic progression and that the CGRP/CRLR axis may serve as a potential therapeutic target for bone metastasis.

Background: Painful distal symmetrical polyneuropathy (DPN) is a frequent complication of type-2 diabetes mellitus (T2DM) that commonly presents as neuropathic pain and loss of skin nerve fibers. However, there are limited therapies to effectively treat DPN and many of the current animal models of T2DM-induced DPN do not appear to mirror the human disease. Thus, we validated a DPN mouse model induced by a cafeteria-style diet plus low-doses of streptozotocin (STZ). Methods: Female C57BL/6J mice were fed either standard (STD) diet or obesogenic cafeteria (CAF) diet for 32 weeks, starting at 8 weeks old. Eight weeks after starting diets, CAF or STD mice received either four low-doses of STZ or vehicle. Changes in body weight, blood glucose and insulin levels, as well as oral glucose- and insulin-tolerance tests (OGTT and ITT) were determined. The development of mechanical hypersensitivity of the hindpaws was determined using von Frey filaments. Moreover, the effect of the most common neuropathic pain drugs was evaluated on T2DM-induced mechanical allodynia. Finally, the density of PGP -9.5 + (a pan-neuronal marker) axons in the epidermis from the hindpaw glabrous skin was quantified. Results: At 22–24 weeks after STZ injections, CAF + STZ mice had significantly higher glucose and insulin levels compared to CAF + VEH, STD + STZ, and STD + VEH mice, and developed glucose tolerance and insulin resistance. Skin mechanical sensitivity was detected as early as 12 weeks post-STZ injections and it was significantly attenuated by intraperitoneal acute treatment with amitriptyline, gabapentin, tramadol, duloxetine, or carbamazepine but not by diclofenac. The density of PGP-9.5 + nerve fibers was reduced in CAF + STZ mice compared to other groups. Conclusion: This reverse translational study provides a painful DPN mouse model which may help in developing a better understanding of the factors that generate and maintain neuropathic pain and denervation of skin under T2DM and to identify mechanism-based new treatments.

Beta 2 adrenergic receptor (β2 AR) activation in the central and peripheral nervous system has been implicated in nociceptive processing in acute and chronic pain settings with anti-inflammatory and anti-allodynic effects of β2-AR mimetics reported in several pain states. In the current study, we examined the therapeutic efficacy of the β2-AR agonist clenbuterol in a rat model of persistent postsurgical hypersensitivity induced by disruption of descending noradrenergic signaling in rats with plantar incision. We used growth curve modeling of ipsilateral mechanical paw withdrawal thresholds following incision to examine effects of treatment on postoperative trajectories. Depletion of spinal noradrenergic neurons delayed recovery of hypersensitivity following incision evident as a flattened slope compared to non-depleted rats (-1.8 g/day with 95% CI -2.4 to -1.085, p < 0.0001). Chronic administration of clenbuterol reduced mechanical hypersensitivity evident as a greater initial intercept in noradrenergic depleted (6.2 g with 95% CI 1.6 to 10.8, p = 0.013) and non-depleted rats (5.4 g with 95% CI 1.2 to 9.6, p = 0.018) with plantar incision compared to vehicle treated rats. Despite a persistent reduction in mechanical hypersensitivity, clenbuterol did not alter the slope of recovery when modeled over several days (p = 0.053) or five weeks in depleted rats (p = 0.64). Systemic clenbuterol suppressed the enhanced microglial activation in depleted rats and reduced the density of macrophage at the site of incision. Direct spinal infusion of clenbuterol failed to reduce mechanical hypersensitivity in depleted rats with incision suggesting that beneficial effects of β2-AR stimulation in this model are largely peripherally mediated. Lastly, we examined β2-AR distribution in the spinal cord and skin using in-situ hybridization and IHC. These data add to our understanding of the role of β2-ARs in the nervous system on hypersensitivity after surgical incision and extend previously observed anti-inflammatory actions of β2-AR agonists to models of surgical injury.

Gestational diabetes mellitus (GDM) induces cardiovascular and metabolic disturbances in offspring. However, the effects of GDM in pain processing in offspring and whether male and female offspring are equally affected is not well known. Thus, we determined: i) whether GDM in mice affects offspring hindpaw mechanical sensitivity, capsaicin-induced spontaneous pain-like behaviors, and epidermal nerve fiber density (ENFD); and ii) whether there is sexual dimorphism in these parameters in offspring from GDM dams. GDM was induced in pregnant ICR mice via i.p. streptozotocin (STZ). Then, glucose levels from dams and offspring were determined. Male and female offspring 2-3 months of age were evaluated for: a) baseline mechanical sensitivity of the hind paw by using von Frey filaments; b) number of flinches and time spent guarding induced by intraplantar capsaicin (0.1%); and c) density of PGP-9.5 and CGRP axons in the epidermis from the hind paw glabrous skin. Prepartum levels of glucose in STZ-treated dams were significantly increased compared to vehicle-treated dams; however, GDM or vehicle offspring displayed normal and similar blood glucose levels. Male and female GDM offspring showed significantly greater mechanical sensitivity and capsaicin-induced pain behaviors compared to vehicle offspring. Male GDM offspring displayed a slightly more intense nociceptive phenotype in the capsaicin test. PGP-9.5 and CGRP ENFD in hind paw glabrous skin were greater in male and female GDM offspring versus their controls. Sexual dimorphism was generally not observed in GDM offspring in most of the studied parameters. These results suggest GDM induced greater pain-like behaviors in adult offspring regardless of sex along with an increased ENFD of PGP-9.5 and CGRP in the hind paw glabrous skin. We show that GDM peripheral neuropathy differs from diabetic peripheral neuropathy acquired in adulthood and set the foundation to further study this in human babies exposed to GDM.

Bone pain is a serious and debilitating symptom of multiple myeloma (MM) that impairs the quality of life of patients. The underlying mechanisms of the pain are unknown and understudied, and there is a need for immunocompetent preclinical models of myeloma‐induced bone pain. The aim of this study was to provide the first in‐depth behavioral characterization of an immunocompetent mouse model of MM presenting the clinical disease features: osteolytic bone disease and bone pain. We hypothesized that a widely used syngeneic model of MM, established by systemic inoculation of green fluorescent protein‐tagged myeloma cells (5TGM1‐GFP) in immunocompetent C57Bl/KaLwRijHsd (BKAL) mice, would present pain‐related behaviors. Disease phenotype was confirmed by splenomegaly, high serum paraprotein, and tumor infiltration in the bone marrow of the hind limbs; however, myeloma‐bearing mice did not present pain‐related behaviors or substantial bone disease. Thus, we investigated an alternative model in which 5TGM1‐GFP cells were directly inoculated into the intrafemoral medullary cavity. This localized myeloma model presented the hallmarks of the disease, including high serum paraprotein, tumor growth, and osteolytic bone lesions. Compared with control mice, myeloma‐bearing mice presented myeloma‐induced pain‐related behaviors, a phenotype that was reversed by systemic morphine treatment. Micro‐computed tomography analyses of the myeloma‐inoculated femurs showed bone disease in cortical and trabecular bone. Repeated systemic bisphosphonate treatment induced an amelioration of the nociceptive phenotype, but did not completely reverse it. Furthermore, intrafemorally injected mice presented a profound denervation of the myeloma‐bearing bones, a previously unknown feature of the disease. This study reports the intrafemoral inoculation of 5TGM1‐GFP cells as a robust immunocompetent model of myeloma‐induced bone pain, with consistent bone loss. Moreover, the data suggest that myeloma‐induced bone pain is caused by a combinatorial mechanism including osteolysis and bone marrow denervation. © 2019 The Authors. JBMR Plus published by Wiley Periodicals, Inc. on behalf of American Society for Bone and Mineral Research.

The adult K/BxN transgenic mouse develops spontaneous autoimmune arthritis with joint remodeling and profound bone loss. We report that both males and females display a severe sustained tactile allodynia which is reduced by gabapentin but not the potent cyclooxygenase inhibitor ketorolac. In dorsal horn, males and females show increased GFAP + astrocytic cells; however, only males demonstrate an increase in Iba1 + microglia. In dorsal root ganglia (DRG), there is an increase in CGRP + , TH + , and Iba1 + (macrophage) labeling, but no increase in ATF3 + cells. At the ankle there is increased CGRP + , TH + , and GAP-43 + fiber synovial innervation. Thus, based on the changes in dorsal horn, DRG and peripheral innervation, we suggest that the adult K/BxN transgenic arthritic mice display a neuropathic phenotype, an assertion consistent with the analgesic pharmacology seen in this animal. These results indicate the relevance of this model to our understanding of the nociceptive processing which underlies the chronic pain state that evolves secondary to persistent joint inflammation.

Type-1 diabetes mellitus (T1DM) is a chronic condition characterized by long-term hyperglycemia that results in several complications such as painful peripheral neuropathy, bone deterioration, and increased risk of bone fractures. Lithium, a first-line therapy for bipolar disorder, has become an attractive agent for attenuating peripheral neuropathy and menopause-induced bone loss. Therefore, our aim was to determine the effect of chronic lithium treatment on mechanical hypersensitivity and trabecular bone loss induced by T1DM in mice. T1DM was induced in male C57BL/6J mice by intraperitoneal injection of streptozotocin (STZ, 50 mg/kg/day, for 5 consecutive days). 12 weeks after T1DM-induction, mice received a daily intraperitoneal injection of vehicle, 30 or 60 mg/kg lithium (as LiCl) for 6 weeks. Throughout the treatment period, blood glucose levels and mechanical sensitivity were evaluated every 2 weeks. After lithium treatment, the femur and L5 vertebra were harvested for microcomputed tomography (microCT) analysis. T1DM mice showed significant hyperglycemia, mechanical hypersensitivity, and significant trabecular bone loss as compared with the control group. Chronic lithium treatment did not revert the hindpaw mechanical hypersensitivity nor hyperglycemia associated to T1DM induced by STZ. In contrast, microCT analysis revealed that lithium reverted, in a dose-dependent manner, the loss of trabecular bone associated to T1DM induced by STZ at both the distal femur and L5 vertebra. Lithium treatment by itself did not affect any trabecular bone parameter in non-diabetic mice.