Mechanisms of pain in autoimmunity : the role of antibodies

Chronic pain in autoimmune diseases, like rheumatoid arthritis (RA), is a common and life- changing problem for many patients. Treatment is usually aimed at reducing inflammation and preserving the function of affected tissues. Chronic pain, however, often persists despite optimal disease control. Autoimmune pain arises from multiple mechanisms with a wide range of characteristics that differs between individuals. For effective management of the pain, it is essential to understand these mechanisms. One of the hallmarks in the pathogenesis in most autoimmune diseases is the presence of autoantibodies. In RA, several types of antibodies are well characterized, but little is known about their interaction with the sensory system. Thus, the aim of this thesis is explore mechanisms involved in pain signaling, specifically the role of disease-relevant antibodies as inducers of pain. In Paper I and II, we investigate the effect of anti-citrullinated protein antibodies (ACPA) on pain behavior and interaction with immune cells. When injected into mice, both polyclonal human ACPA or murinized monoclonal ACPA induces spontaneous and evoked pain-like behavior in the absence of inflammation. Additionally, the antibodies induce trabecular bone loss measured with micro-CT. The antibodies localize to joint and bone marrow, binding osteoclasts and its precursors. Using cultures of mice and human osteoclasts, we show that ACPA bind structures on the cells, causing proliferation and release of the chemokine CXCL1/IL-8. The effect of the release is increased bone resorption and activation of sensory neurons, causing pain-like behavior, which can be reversed by treating the mice with the CXCR1/2 blocker reparixin. In Paper III, we demonstrate that mice injected with antibodies specific to the cartilage protein collagen type II (anti-CII mAbs) displays pronounced mechanical hypersensitivity and reduction in locomotion at time points when visual, histological and molecular indications of inflammation were completely absent. Further, this effect was not mediated by the activation of complement factors or by changes in the cartilage structure. Instead our data point to a direct action of anti-CII mAb/collagen immune complexes on the sensory neurons through neuronally expressed Fc-gamma receptor IIb (FcγRIIb), causing increased inward currents, intracellular Ca2+ levels, and calcitonin-gene related peptide (CGRP) release. Importantly, the nociceptive properties of anti-CII mAbs were lost when the Fc-FcγR interaction was disrupted in vivo. In summary, we have described two novel mechanisms of how disease-relevant antibodies can activate sensory neurons, causing pain-like behavior. These results deepen the understanding of pain mechanisms in autoimmune disease and potentially to new ways of treating the pain component in patients

Autoantibodies to citrullinated proteins induce joint pain independent of inflammation via a chemokine-dependent mechanism

OBJECTIVE: An 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. METHODS: Antibodies 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. RESULTS: Mice 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. CONCLUSIONS: The 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

Differential ACPA Binding to Nuclear Antigens Reveals a PAD-Independent Pathway and a Distinct Subset of Acetylation Cross-Reactive Autoantibodies in Rheumatoid Arthritis

Rheumatoid arthritis (RA) associated anti-citrullinated protein autoantibodies (ACPA) target a wide range of modified proteins. Citrullination occurs during physiological processes such as apoptosis, yet little is known about the interaction of ACPA with nuclear antigens or apoptotic cells. Since uncleared apoptotic cells and neutrophil extracellular trap (NET) products have been postulated to be central sources of autoantigen and immunostimulation in autoimmune disease, we sought to characterize the anti-nuclear and anti-neutrophil reactivities of ACFA. Serology showed that a subset of anti-CCP2 seropositive RA patients had high reactivity to full-length citrullinated histones. In contrast, seronegative RA patients displayed elevated IgG reactivity to native histone compared to controls, but no citrulline-specific reactivity. Screening of 10 single B-cell derived monoclonal AGFA from RA patients revealed that four ACPA exhibited strong binding to apoptotic cells and three of these had anti-nuclear (ANA) autoantibody reactivity. Modified histones were confirmed to be the primary targets of this anti-nuclear ACPA subset following immunoprecipitation from apoptotic cell lysates. Monoclonal ACPA were also screened for reactivities against stimulated murine and human neutrophils, and all the nuclear-reactive monoclonal ACPA bound to NETs. Intriguingly, one ACPA mAb displayed a contrasting cytoplasmic perinuclear neutrophil binding and may represent a different NET-reactive ACPA subset. Notably, studies of CRISPR-Cas9 PAD4 KO cells and cells from PAD KO mice showed that the cytoplasmic NET-binding was fully dependent on PAD4, whilst nuclear- and histone-mediated NEI reactivity was largely PAD-independent. Our further analysis revealed that the nuclear binding could be explained by consensus-motif driven ACPA cross-reactivity to acetylated histones. Specific acetylated histone peptides targeted by the monoclonal antibodies were identified and the anti-modified protein autoantibody (AMPA) profile of the ACPA was found to correlate with the functional activity of the antibodies. In conclusion, when investigating monoclonal ACPA, we could group ACPA into distinct subsets based on their nuclear binding-patterns and acetylation-mediated binding to apoptotic cells, neutrophils, and NETs. Differential anti-modified protein reactivities of RA-autoantibody subsets could have an important functional impact and provide insights in RA pathogenesis

Spinal actions of lipoxin A4 and 17(R)-resolvin D1 attenuate inflammation-induced mechanical hypersensitivity and spinal TNF release.

Lipoxins and resolvins have anti-inflammatory and pro-resolving actions and accumulating evidence indicates that these lipid mediators also attenuate pain-like behavior in a number of experimental models of inflammation and tissue injury-induced pain. The present study was undertaken to assess if spinal administration of lipoxin A4 (LXA4) or 17 (R)-resolvin D1 (17(R)-RvD1) attenuates mechanical hypersensitivity in the carrageenan model of peripheral inflammation in the rat. Given the emerging role of spinal cytokines in the generation and maintenance of inflammatory pain we measured cytokine levels in the cerebrospinal fluid (CSF) after LXA4 or 17(R)-RvD1 administration, and the ability of these lipid metabolites to prevent stimuli-induced release of cytokines from cultured primary spinal astrocytes. We found that intrathecal bolus injection of LXA4 and17(R)-RvD1 attenuated inflammation-induced mechanical hypersensitivity without reducing the local inflammation. Furthermore, both LXA4 and 17(R)-RvD1 reduced carrageenan-induced tumor necrosis factor (TNF) release in the CSF, while only 17(R)-RvD1attenuated LPS and IFN-γ-induced TNF release in astrocyte cell culture. In conclusion, this study demonstrates that lipoxins and resolvins potently suppress inflammation-induced mechanical hypersensitivity, possibly by attenuating cytokine release from spinal astrocytes. The inhibitory effect of lipoxins and resolvins on spinal nociceptive processing puts them in an intriguing position in the search for novel pain therapeutics

Pain-like behavior in the collagen antibody-induced arthritis model is regulated by lysophosphatidic acid and activation of satellite glia cells.

Inflammatory and neuropathic-like components underlie rheumatoid arthritis (RA)-associated pain and lysophosphatidic acid (LPA) is linked to both joint inflammation in RA patients and to neuropathic pain. Thus, we investigated a role for LPA signalling using the collagen antibody-induced arthritis (CAIA) model. Pain-like behavior during the inflammatory phase and the late, neuropathic-like phase of CAIA was reversed by a neutralizing antibody generated against LPA and by an LPA1/3 receptor inhibitor, but joint inflammation was not affected. Autotaxin, an LPA synthesizing enzyme was upregulated in dorsal root ganglia (DRG) neurons during both CAIA phases, but not in joints or spinal cord. Late-phase pronociceptive neurochemical changes in the DRG were blocked in Lpar1 receptor deficient mice and reversed by LPA neutralization. In vitro and in vivo studies indicated that LPA regulates pain-like behavior via the LPA1 receptor on satellite glia cells (SGCs), which is expressed by both human and mouse SGCs in the DRG. Furthermore, CAIA-induced SGC activity is reversed by phospholipid neutralization and blocked in Lpar1 deficient mice. Our findings suggest that the regulation of CAIA-induced pain-like behavior by LPA signalling is a peripheral event, associated with the DRGs and involving increased pronociceptive activity of SGCs, which in turn act on sensory neurons

17 (R)-RvD1 attenuate IFN-γ and LPS induced TNF induction in rat primary astrocytes.

<p>Bar graphs showing levels of TNF in media from primary rat astrocyte cultures (A) following stimulation with IFN-γ (1000 U/ml, 24 h), LPS (2 µg/ml, 24 h) or the combined effect of IFN-γ (1000 U/ml, 4 h) followed by LPS (2 µg/ml, 20 h). Pretreatment with 17(R)-RvD1 (500 nM, 30 min) significantly reduced IFN-γ (B) and LPS-induced (C)-TNF release. No significant effect was seen after pretreatment with LXA4 (500 nM, 30 min). Each bar represents mean ± S.E.M, * represents a significant difference at p<0.05 as compared to PBS (A) or indicated in the figure (B-D).</p

FPR2/ALX mRNA is present in the rat spinal cord and cultured spinal astrocytes.

<p>Bar graphs showing the expression of (A) FPR2/ALX mRNA in the rat ipsilateral spinal cord plotted versus time following carrageenan injection to the hind paw, presented as a percent of mRNA levels in control (naïve) rat spinal cord. Each bar represents the mean ± S.E.M, n= 6-10. * represents a significant difference at p<0.05 as compared with naïve spinal cord. FPR2/ALX mRNA is expressed in the rat (B) and human astrocytes (C). GPR32 mRNA is expressed in human astrocytes (C). mRNA levels are expressed as relative units and each bar represents the mean ± S.E.M for three repeats. The immunohistochemical images show the expression of FPR2/ALX (D), the astrocyte marker GFAP (E) and the colocalization of FPR2/ALX and GFAP (F) in naïve rat lumbar spinal cord.</p

Intrathecal injection of LXA4 and 17(R)-RvD1 does not change carrageenan-induced inflammation.

<p>Bar graphs show that the increased paw size measured 4 h following carrageenan injection was not altered by pretreatment with LXA4 (A) or 17(R)-RvD1 (B) as compared to pretreatment with vehicle. All data are presented as mean ± S.E.M, * represents a significant difference at p<0.05 as compared with i.t. vehicle.</p

17(R)-RvD1 reduced TNF-induced ERK activation in human primary astrocytes.

<p>Bar graphs and representative western blots showing MAPK phosphorylation levels in control and TNF (50 ng/ml) stimulated cells. In astrocytes, 17(R)-RvD1 (265 nM) inhibited TNF-induced ERK (A), but not p38 (B) or JNK (C) phosphorylation. Each bar represents the mean ± S.E.M for three repeats. * represents p<0.05 for comparisons indicated in the figure.</p