Background
Pain from osteoarthritis (OA) is one of the top causes of disability worldwide, but effective treatments are lacking and limited by adverse effects. For e.g., an anti-nerve growth factor (NGF) monoclonal antibody (tanezumab) effectively modified pain, but also increased rates of rapidly progressive OA [
1]. Effective targets to modify OA-related pain without accelerating joint damage are urgently needed. Synovial inflammation (synovitis) is dominated by macrophage infiltration and strongly associated with worse pain and joint damage in knee OA patients [
2‐
5]. We and others suspect that synovial macrophages mediate pain outcomes, but the mechanisms controlling synovial macrophage activation in OA are not well understood.
Macrophages reside in healthy synovium and maintain tissue homeostasis through phagocytosis of extracellular matrix turnover products and efferocytosis of dead and dying cells [
6,
7]. During OA, peripheral macrophages are recruited to synovial tissue and activated [
8], contributing to the genesis of chronic OA-related pain through the release of nociceptive molecules including cytokines that act on sensory nerve fibres and via crosstalk with other cell types [
9]. Pro- (M1) or anti-inflammatory (M2) macrophage polarization can be stimulated
in vitro [
10], but exposure to disease-specific cues in the local joint microenvironment leads to a wider range of activation states [
11]. This complexity underlies the helpful and harmful roles played by macrophages during chronic inflammation and its resolution.
Synovial macrophages may be a key target for the treatment of OA-related pain [
6], but are also required for the maintenance of joint homeostasis. Supporting this catch-22 hypothesis, previous studies have shown that depletion of synovial macrophages (for e.g., with liposomal clodronate or genetic deletion) in animal models of OA leads to a paradoxical increase in inflammation and destruction of joint tissues [
12,
13]. Therefore, we hypothesize that inhibiting macrophage activation (instead of ablation) may be an effective strategy to improve pain outcomes while preserving the homeostatic functions of synovial macrophages during OA.
The mechanisms leading to synovial macrophage activation during OA development are not well understood. Interestingly, the signal transducer and activator of transcription (STAT) family plays key roles in regulating macrophage activation, polarization, and crosstalk in other diseases [
11,
14]. This suggests that STAT signaling may also be important for macrophage activation in OA, but the role of STAT signaling in OA macrophages in pain and synovial inflammation has not been described. Small molecule inhibitors of intra-cellular signaling mechanisms are routinely used to manage rheumatoid and psoriatic arthritis in the clinic [
15]. To test the role of STAT signaling in this study, we packaged highly selective small molecule STAT1 or STAT6 inhibitors in large multi-lamellar liposomes. After intra-articular injection, liposomes and their drug payload are phagocytosed by synovial macrophages, allowing us to selectively target macrophage activation. Our objectives were to identify major mechanisms of synovial macrophage activation and test the role of STAT signaling in macrophages on pain outcomes in experimental knee OA.
Discussion
Chronic knee pain is the most commonly reported symptom by patients suffering from OA, but existing treatments are limited by adverse effects [
31‐
33]. Depletion of macrophages can resolve experimental OA related pain [
34,
35], but this comes at the cost of increased inflammation and joint tissue damage [
12,
13]. This suggested to us that tuning macrophage activation may be more therapeutically effective than ablating macrophages completely. Using transcriptomics, we identified STAT signaling as a dominant intracellular mechanism associated with macrophage activation pathways in early-stage experimental knee OA. Strikingly, STAT6 inhibition (STAT6i) in synovial macrophages raised (improved) the threshold for mechanical sensitivity above baseline and control levels at the affected knee and distal hindpaw at all time points. This robust protection against mechanical pain sensitivity was achieved without worsening synovial or cartilage histopathology outcomes, and reduced synovial lining hyperplasia. In contrast, STAT1 inhibition (STAT1i) transiently lowered (worsened) knee withdrawal thresholds and increased hindpaw withdrawal thresholds, with no clear impact on synovial or cartilage histopathology aside from a trend toward improved cartilage damage at the 12-week endpoint. Lastly, we found that repeated intra-articular injection of clodronate liposomes to persistently deplete synovial macrophages prevented the development of mechanical pain sensitivity up to 12 weeks after OA induction. As expected, these benefits came at the cost of increased joint tissue damage including increased synovial fibrosis, vascularization, and perivascular edema. These results suggest that activated synovial macrophages mediate mechanical pain sensitivity in knee OA and that STAT6 may be a particularly important mechanism.
Macrophages are one of the most important synovial cell types in OA. Although studies have identified altered macrophage-related gene expression from whole synovial tissue [
36], our study is among the first to explore differential gene expression selectively within the synovial macrophage compartment during experimental knee OA development. In line with longitudinal gene expression studies in mouse models [
37], synovial macrophage gene expression followed a phasic pattern. Early-stage OA macrophage gene expression reflected changes in cellular metabolism, activation, motility, inflammation, and Wnt signaling, which transitioned to extracellular matrix remodeling after 12 weeks of OA development. Similar to transcriptomic analyses in an equine model of OA [
38], our gene set enrichment analyses suggested a major role for STAT signaling in mediating cell stress, metabolism, and angiogenesis, with major overlaps across MAPK, PI3K, and SMAD signaling.
A role for STAT signaling in OA cartilage has been described.
Latourte et al. demonstrated structural protection against joint destabilization-induced experimental knee OA in the mouse using prophylactic systemic inhibition of STAT3 [
39]. However, the effects of STAT inhibition on OA-related pain are not well-understood, and macrophage activation mechanisms are context-dependent [
40]. Given the complementary roles of STAT1 and STAT6 signaling in regulating macrophage activation, we chose highly selective inhibitors of STAT1 (fludarabine) and STAT6 (AS1517499) for targeted delivery to synovial macrophages via phagocytosis of drug-loaded liposomes.
The potential for dual roles played by macrophages in mediating OA outcomes has long been suspected, partly based on the well-described model of pro- (M1) and anti-inflammatory (M2) polarization. However, our data underscore the hazards of relying on the M1/M2 paradigm, and assuming that all inflammation is bad. For example, interleukin-4 (IL-4) is an anti-inflammatory cytokine and generally regarded as analgesic [
41]. IL-4 knock-out mice display increased hindpaw mechanical hypersensitivity and STAT6 is a mediator of IL-4 receptor signaling [
40,
42]. Surprisingly, we found that STAT6i robustly protected against the development of both distal and local pain, suggesting that STAT6 likely has alternative functions in OA synovial macrophages. Interestingly, Haraden
et al. reported that OA disease severity was correlated to synovial fluid levels of the M2-marker CD163 [
43], which aligns with our results and suggests that alternatively-activated macrophages may contribute to nociception. Additionally, atopic diseases characterised by STAT6 activation are associated with increased risk of OA [
44]. Based on the literature, we predicted that STAT1 would drive pro-inflammatory macrophages and nociception in OA. However, we only observed a brief benefit on distal pain, and worsening of local knee pain sensitivity with STAT1i. Thus, STAT1 signaling may only play a small role in mediating OA-related nociception.
Analgesic treatments are frequently associated with off-target effects. For example, NGF inhibition caused a rare but clinically-important increased risk of rapidly progressive OA [
1]. To ensure that macrophage-targeted treatments did not worsen joint tissue outcomes, we explored joint histopathology and crosstalk between treated synovial tissue and healthy primary articular chondrocytes in an
ex-vivo joint co-culture system [
16]. Importantly, no treatment led to any increase in articular cartilage damage. There were trends toward reduced cartilage damage with STAT1i and STAT6i, but we lacked statistical power to detect a small protective effect. We therefore cannot exclude the potential for a protective effect on joint damage, as Sun
et al. reported marked reduction in cartilage degeneration after depleting macrophages in obese mice prior to, and 1 week after, OA induction [
45].
We previously reported that synovial tissue from early-stage experimental knee OA co-cultured with healthy articular chondrocytes stimulates a transient anabolic response [
16]. In this study, we found that macrophage-depleted synovium from early-stage OA decreased
Col2a1 expression by healthy articular chondrocytes, suggesting that synovial macrophages control chondrocyte extracellular matrix gene expression through crosstalk mechanisms. Supporting this, synovial tissues treated with liposomal STAT1i caused increased sGAG secretion and expression of matrix (
Acan,
Col2a1, and
Prg4) and protease genes (
Adamts5 and
Mmp13), whereas STAT6i led to increased
Acan and
Ccl2, and decreased
Col2a1 gene expression. Overall, our
in vitro crosstalk experiments suggest that STAT1-mediated macrophage activation inhibits anabolic responses in chondrocytes, whereas STAT6-mediated macrophage activation may be more important for nociception with fewer effects on chondrocyte anabolism. Given these somewhat complementary findings, it may have been interesting to include a combined STAT1i-STAT6i treatment.
Synovial tissue function is key to maintaining joint homeostasis and we found that synovial macrophage depletion caused worse synovial vascularization, fibrosis, and perivascular edema, which was not seen with STAT1i or STAT6i. Similarly, other studies have found that transient and/or prophylactic depletion of macrophages resulted in worse synovitis, joint damage, and infiltration of T lymphocytes [
12,
13]. In those studies, depletion was performed at a single timepoint, whereas we used repeat dosing every 2 weeks to sustain macrophage depletion/suppression. Together with our findings, a clearer picture is emerging that macrophages are essential for maintaining joint homeostasis, while simultaneously playing pathological roles in nociception and engaging in crosstalk with other tissues.
Our study has limitations. Our surgically induced joint destabilization model of OA does not address other OA risk factors such as age, obesity, or female sex, and the effects of STAT inhibition should be confirmed in those settings. Although our study focused on two commonly used methods to assess mechanical pain sensitivity at the knee and hindpaw, other pain-related behavioural tests may have revealed different outcomes. We cannot rule out a role played by other phagocytes such as dendritic cells, mast cells, or neutrophils (rarely seen in OA), which may also have been targeted by liposomes. However, synovial macrophages are the dominant immune cells in healthy joints and are heavily recruited to the synovium of OA joints. Further studies will be required to determine whether different roles are played by tissue resident versus recruited (bone marrow-derived) synovial macrophages.
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