Osteoarthritis treatment via intra-articular delivery of indoleamine 2,3-dioxygenase galectin-3 fusion protein

Abstract

Purpose: Inflammation caused by acute injury or post-traumatic OA directly impacts joint dysfunction and OA pain-related behaviors. Therefore, controlling inflammation can be pivotal in improving OA symptomology. Current treatments to improve OA symptomology, including intra-articular injections of steroids and other anti-inflammatory drugs, fail to provide long term pain relief due to rapid joint clearance. As such, strategies to anchor anti-inflammatory therapeutics to joint tissues can improve bioavailability and enhance joint residence time. Here, we developed an indoleamine 2,3-dioxygenase (IDO) galectin-3 (Gal3) fusion protein, termed IDO-Gal3. IDO can polarize immune cells via catalysis of tryptophan into kynurenines, ultimately reprogramming the local environment into an anti-inflammatory state. Gal3, a carbohydrate binding protein, can bind to lubricin at the surface of articular cartilage, and therefore serves to extend IDO’s joint residence time and anti-inflammatory effects. Methods: Gal3 joint retention was assessed using 16 male Lewis rats. All rats underwent medial meniscus transection (MMT) surgery on the right hind limb. After 8 weeks, 50 μl of Nano-Glo Luciferase (NL) or Nano-Glo Luciferase Galectin-3 fusion (NL-Gal3) was injected into both knees. NL and NL-Gal3 joint retention was quantified via IVIS on 1, 2, 4, 8, 12, 16, 20, 24, and 28 days post-injection. Joint tissue distribution was assessed after euthanasia on day 28. IDO-Gal3’s effects on pain-related behaviors and intra-articular inflammation was assessed using an additional 16 male Lewis rats. Rats underwent baseline von Frey and gait behavioral testing. Three component ground reaction force data, spatiotemporal characteristics of footprint patterns, and foot strike toe off events were simultaneously collected using the Gait Analysis Instrumentation and Technology Optimized for Rodents (GAITOR) suite. All rats received MMT surgery to their right hind limb. One rat was lost one week after surgery reducing the n to 15. Von Frey testing was conducted weekly after surgery and gait data was collected on weeks 3, 5, and 7 post-surgery. At 8 weeks post-surgery, rats received a 30 μl saline (n=7) or IDO-Gal3 (n=8) injection to their operated limb. Gait and von Frey data were collected weekly until euthanasia at 12 weeks. Immediately following euthanasia, magnetic capture of IL-6, MCP-1, and CTX-II was performed on operated and contralateral limbs. Briefly, magnetic capture uses superparamagnetic beads to capture biomarkers in the joint and assay ex vivo. After magnetic capture, operated and contralateral knees were sectioned for histology and stained with toluidine blue or hematoxylin and eosin. A 2-way ANOVA was used to compare intra-articular biomarkers levels in operated and contralateral knees of IDO-Gal3 and saline injected knees. Results: NL-Gal3 was retained in the joint out to 28 days, whereas NL alone cleared with 7 days. NL and NL-Gal3 cleared from operated and contralateral limbs at similar rates (Figure 1A). 50% paw withdrawal threshold in the operated limb decreased after MMT surgery, indicating heightened sensitivity to touch. After intra-articular IDO-Gal3 injection, the 50% paw withdrawal threshold reached our maximum testing range, suggesting decreased limb sensitivity. After saline injection, rats also showed decreased limb sensitivity, but to a lesser extent than IDO-Gal3 (Figure 1B). At 4 weeks after injection and 12 weeks post-surgery intra-articular levels of IL-6 were significantly lower in IDO-Gal3 treated knees compared to other controls (p=0.02) (Figure 1C). Conclusions: The half-life of NL in the joint was less than a day after injection. Gal3 functionalization increased NL joint retention to at least 4 weeks, and promoted NL accumulation in cartilage and menisci. High Gal3-NL cartilage accumulation is likely due to Gal3 binding lubricin at the articular cartilage surface. Moreover, these data demonstrate disease does not alter joint retention for these treatments. IDO fused to Gal3 was evaluated for its effects on pain-related behaviors and intra-articular inflammation. After saline injection, von Frey data showed most rats returned to the “normal” sensitivity range for Lewis rats (25-35 g of force), while after IDO-Gal3 injection, most rats returned to and stayed at baseline sensitivity level within a week of injection. Here, IDO quickly decreases limb sensitivity and maintains pain reversal for at least 4 weeks post-injection. Decreased limb sensitivity could be caused by immune modulation of molecular markers associated with OA. Intra-articular levels of IL-6 in the operated limb were significantly reduced in IDO-Gal3 treated rats. Intra-articular levels of CTX-II and MCP-1 were both suppressed in operated limbs for IDO-Gal3 treated rats. Thus, IDO’s immunosuppressive effects may improve OA symptomology and prognosis.