Evaluation of Degradable Itaconate Biopolymers as Therapeutic Tools to Regulate Pathological Fibrosis

Introduction: Fibrosis is broadly defined as the thickening or scarring of tissue as a result of tissue damage. Fibrotic conditions present a significant health challenge; an estimated 10% of the developed world population is impacted by fibrosis. Roughly 18% of global deaths can be attributed to pathological fibrotic conditions. Fibrosis can become pathological due to a dysfunction in the innate-immunity driven macrophage-fibroblast axis, posing a significant clinical challenge Within this abnormal axis, the chronically inflammatory macrophage cells (i.e., classically activated macrophages, LPS activation; M[LPS]) establish an excessive and continual inflammatory state leading to recruitment and differentiation of myofibroblast cells, which in turn excessively secrete extracellular matrix. Fibroblasts are not just passive downstream effector cells in this axis; they are able to increase macrophage recruitment and modulate their activity.
The metabolite itaconate (IA) has been demonstrated to have anti-fibrotic effects. IA intrinsically regulates multiple macrophage activation states; IA acts as a reversible brake system for macrophage inflammatory function wherein it dampens generation of succinate-mediated reactive oxygen production, inflammasome-mediated maturation of IL1β, and pro-inflammatory gene transcription. IA has been demonstrated to block monocyte polarization into M[IL4] like macrophages. Lastly, IA limits fibroblast proliferation rate and myofibroblast activity. Our lab has developed degradable IA-based biopolymers that can release IA at high sustained local doses necessary for metabolite-based regulation, and previously validated their ability to recapitulate the release of inflammatory cytokines in M[LPS] macrophages. In this work, we examined if IA polymers can be used to regulate the macrophage-fibroblast axis relevant to fibrotic micro-environments.

Materials and

Methods: IA polymers were synthesized in a one-pot polycondensation reaction of dimethyl IA and a 1,12-dodecanediol at a 1:1 ratio. This reaction is catalyzed with stannous octoate (0.01 mol/mol ester), and linear chain formation is forced via addition of 4-methoxyphenol (0.5 weight % of all reactants). To assess degradation-based regulation, IA polymers were degraded in distilled water at a concentration of 0.1g/ml for 1-60 days, and the supernatant was used for further analysis. Degradation products and relative concentrations were characterized by HPLC-MS. Degradation product conditioned media was assessed for cytotoxicity through serial dilution and subsequent viability staining (propidium iodide (PI), Carboxyfluorescein diacetate- succinimidyl ester (CFDA-SE), and Hoechst 33342) on bone marrow derived macrophages (BMDMs) and quantified as % cell death. Regulation of macrophage phenotype was assessed with monoculture of murine BMDM (C57BL6) macrophage cells generated through 7-day differentiation with M-CSF and stimulated for 24 hours with either LPS (100ng/ml) or LPS + IFNg (100ng/ml & 20ng/ml). Regulation of fibroblast activity was assessed with monoculture of murine NIH3T3 or human dermal fibroblast cells that have been stimulated for 24 hours with TGFβ (20ng/ml). Optimal polymer-conditioned media dilutions were determined using reactive nitrogen/oxygen production (RNS/ROS) assays (Griess assay), and fibroblast proliferation assays. Relative gene expression of M[LPS]/M[LPS+IFNg] effector pathway genes (e.g., Il1β, Il6, Hmox1, Nos2) and fibroblast activation markers (e.g., αSma, Fap, Col1α1) were examined and compared against untreated controls. ELISA was used to assess pro-inflammatory cytokine release (e.g., IL1β, IL6, TNFα) from M[LPS]/M[LPS+IFNg]. Lastly, αSMA staining was used to assess fibroblast activation.

Results, Conclusions, and Discussions: IA polymers degrade in water to release monomers (i.e., IA and diol) and long-lived stable oligomeric groups. As degradation time increased from 1-60 days, the relative concentrations of IA and IA-diol oligomers increased. We expect these oligomeric groups increase membrane permeability due to the addition of the long-chain alcohol and in turn increase regulatory capacity. Serial dilutions of polymer degradation conditioned media dilutions were characterized through live-dead staining, Greiss reagent assays, and proliferation assays to appreciate temporal and cell specific regulatory capacity of the IA & IA-oligmer degradation products. Polymer conditioned media demonstrated dilution dependent cytotoxicity and regulation of RNS/ROS; five times diluted (5X) 10-day degraded polymer conditioned media was confirmed to have minimal cytotoxicity and regulation of RNS/ROS production or reduction in proliferation rate maintained that was significantce compared to untreated controls (Figure 1). Therefore, 5X 10-day degraded polymer conditioned media was used for further analysis.
Treatment of M[LPS]/M[LPS+IFNg] cells with polymer conditioned media presented significant regulation of pro-inflammatory cytokine release (IL1β, IL6, TNFα) compared to untreated controls. Significant genetic downregulation of pro-inflammatory cytokine genes (Il1β, Il6) and ROS genes (Nos2), and upregulation of the antioxidant Hmox1 was also noted compared to untreated controls. This suggest that our IA polymers can strongly regulate macrophage inflammatory activity and phenotype present in a fibrotic micro-environment. Treatment of fibroblast and myofibroblasts (Tgfb+) with polymer conditioned media presented significant downregulation of fibroblast activation markers (αSma, Fap, Col1α1, Fn1) and strong reduction in proliferation rate. Relative expression of αSMA was decreased compared to control populations. This suggest that our IA polymers are also able to regulate fibrotic fibroblast activity.

Conclusion: The presented modulation of macrophage phenotype and fibroblast activity following exposure to IA polymer breakdown products without impacting the viability of cells in the local micro-environment suggests the potential of these polymer materials to be used an anti-fibrotic agent that specifically target the abnormal macrophage-fibroblast axis present in pathological fibrosis. Future investigations will look to expand on material characterization to specific applications and structures to enable localized delivery of IA, relying on manipulation of polymer properties to extend degradation baseddegradation-based payload delivery.

Acknowledgements (Optional):