Inflammatory and Metabolic Mechanisms Activated in Macrophages Exposed to Metal Implant Wear and Corrosion Products

Abstract

Wear and corrosion products from CoCrMo implants have been associated with adverse local tissue reactions that can ultimately lead to implant failure. However, the underlying molecular mechanisms remain largely unknown. Once activated, macrophages (the predominant immune cells in the periprosthetic tissues) secrete pro inflammatory cytokines, including interleukin-1β (IL-1β), which promote and maintain a pro-inflammatory milieu. More specifically, previous results from our group have shown that, among metal ions released from CoCrMo implants (mainly Co²⁺ and Cr³⁺), only Cr³⁺ led to caspase 1 cleavage in bone marrow-derived macrophages (BMDM), suggesting IL-1β release involving the NLRP3 inflammasome. Additionally, Co²⁺ was shown to induce an increase in oxidative stress markers and a metabolic reprogramming away from oxidative phosphorylation (OXPHOS) towards glycolysis in macrophages. However, the specific mechanisms through which CoCrMo implant wear particles (mainly Cr₂O₃ and CoCrMo) and ions (mainly Co²⁺ and Cr³⁺) may induce the activation of the NLRP3 inflammasome and/or potential metabolic alterations contributing to IL-1β synthesis, and thereby to the overall periprosthetic inflammation, remain largely unknown. The first objective of this thesis focused on identifying mechanisms through which Cr₂O₃ particles, CoCrMo particles, Co²⁺, and Cr³⁺ lead to IL-1β release by macrophages. Results showed that IL-1β release induced by Cr₂O₃ particles and Cr³⁺ was both NLRP3 and caspase-1 dependent in BMDM. In contrast, IL-1β release induced by CoCrMo particles and Co²⁺ occurred independently of NLRP3, being caspase 1 independent in response to CoCrMo particles and only partially caspase 1 dependent in response to Co²⁺. Further analysis suggested that the NLRP3 inflammasome activation by Cr₂O₃ particles was cathepsin B dependent and mediated by lysosomal destabilization, whereas activation by Cr³⁺ was mediated by ROS. The NLRP3 independent IL-1β release induced by CoCrMo particles and Co²⁺ was caspase 8 dependent. The second objective of this thesis focused on determining the effects of CoCrMo particles and Co²⁺ on macrophage metabolism. Results showed that Co²⁺ induced a metabolic shift away from OXPHOS towards glycolysis in BMDM. While only Co²⁺ enhanced pentose phosphate ribose-5-phosphate and ribulose-5-phosphate levels, CoCrMo particles bolstered hexoses and pyruvate levels. Moreover, both CoCrMo particles and Co²⁺ induced a non classical alteration of the tricarboxylic acid cycle highlighted by citrate depletion on which the induced IL-1β release was dependent. The third objective of this thesis focused on determining if itaconate and its derivative, 4-octyl itaconate (4-OI), can modulate the metabolic shift induced by Co²⁺ in macrophages. Results showed that both itaconate and 4-OI significantly inhibited Co²⁺ induced IL-1β release by BMDM. Interestingly, only 4 OI decreased both the precursor and mature forms of IL-1β. While neither itaconate nor 4-OI prevented Co²⁺-induced OXPHOS inhibition, both agents prevented glycolytic flux increase. Lastly, only 4-OI moderately increased CD206 expression, suggesting a shift towards an anti inflammatory or reparative phenotype. Overall, the findings of this thesis revealed: 1. the diversity and specificity of the mechanisms by which different wear particles and metal ions from CoCrMo implants can induce IL-1β release in macrophages; 2. the differential effects of CoCrMo particles and Co²⁺ on the metabolic adaptation of macrophages to elicit their pro-inflammatory response; and 3. a promising role for itaconate and its derivative, 4-OI, in mitigating Co²⁺-induced inflammatory response in macrophages, highlighting the therapeutic potential of 4 OI in macrophage metabolic reprogramming to ultimately improve CoCrMo implant longevity.