Modulation of osteoclasts and their myeloid precursors by specialised pro-resolving mediators in health and rheumatoid arthritis

Riedlová, Patrícia (2024) Modulation of osteoclasts and their myeloid precursors by specialised pro-resolving mediators in health and rheumatoid arthritis. PhD thesis, University of Glasgow.

Abstract

Rheumatoid arthritis (RA) is an autoimmune chronic inflammatory disease, which is associated with pathological degradation of bone/cartilage when boneresorbing osteoclasts dominate over bone-forming osteoblasts. Recent data have shown that novel specialised pro-resolving lipid mediators (SPMs)(e.g., RvE1, RvD1) have the capacity to modulate inflammation, reduce pain and swelling, and assist in cartilage repair, and bone remodelling. However, although prior studies have demonstrated the effect of RvE1 and RvD1 on osteoclast inhibition, this research was limited to murine models and cell lines, and the ability of SPMs to modulate primary human cells in this context has not been investigated.

This thesis therefore investigates modulation of differentiation (osteoclastogenesis) and function (osteolysis) of primary human CD14+ monocytes and pre-dendritic cells by pro-resolving lipid mediators (e.g., RvE1, RvD1, 17- HDHA, and MaR1). Notably, these studies have been done in both a “steady-state” and “TNF-driven” setting to understand the capability of SPMs to function in a pseudo-inflammatory context. The generated results indicate that exposure of CD14+ monocytes to particular SPMs can lead to inhibition of osteoclastogenesis. However, the “pathological” and pseudo-inflammatory context is particularly relevant, as although RvE1 can lead to osteoclast inhibition in cells derived from healthy donors exposed to TNF, it has no impact in cells derived from individuals with RA. In contrast, the reverse is true for RvD1, where it is able to inhibit osteoclastogenesis of CD14+ monocytes from individuals with RA exposed to TNF, but not healthy controls. Mechanistic studies undertaken to examine the cellular pathways associated with this inhibition, revealed that in both the healthy and RA context RvE1 and RvD1 exposure was associated with lower superoxide production, indicative of mitochondrial modulation.

Given the capacity of SPMs to interact with multiple receptors, further studies were performed to understand the cellular requirements for SPM-driven modulation of the observed cell fate decisions. Thus, the expression of SPM receptors present on osteoclasts and osteoclast precursors was examined. Initial analysis was based on RNAseq datasets for healthy monocytes and monocytes from patients with different rheumatic diseases, as well as healthy osteoclasts differentiated from either monocytes or dendritic cell precursors. Transcriptional profiling showed that RvE1 and RvD1 SPM receptors, namely LTB4R, CMKLR1, and FPR2 were expressed in both, monocytes and osteoclasts. Subsequent evaluation at the protein level, revealed that LTB4R was significantly more expressed in healthy monocytes and osteoclasts compared to RA cells, while the levels of CMKLR1 and FPR2 were comparable. Higher expression of LTB4R receptor in healthy cells supports the inhibitory actions of RvE1 on osteoclastogenesis in healthy but not in RA osteoclasts.

Having identified a potential mechanism of SPM-driven cellular changes through mitochondrial modulation, studies were undertaken to evaluate, whether there are metabolic differences in RA patient and healthy donor monocytes and monocyte-derived osteoclasts, which could explain the differences observed in their response to SPMs. Initial results showed a significant increase in glucose uptake, fatty acid uptake, mitochondrial membrane potential, and increase in mitochondrial mass/number of mitochondria in RA monocytes compared to healthy. Interestingly, no differences were observed in the metabolic enzymes and ATP production. For a better understanding of whether this difference was maintained throughout the differentiation into macrophages and osteoclasts, the metabolic state of these cells was further tested. While glucose and fatty acid uptake was significantly different in RA macrophages and osteoclasts, the size/number of mitochondria and the membrane potential was no longer significantly different when compared to healthy cells.

In conclusion, work in this thesis for the first time demonstrates the ability of RvE1 and RvD1 to inhibit osteoclasts differentiated from primary human cells in health and RA, suggesting a promising role for SPMs and their pathways to modulate the disease. Moreover, these studies have revealed that RA monocytes have a higher glucose and fatty acid uptake, compared to healthy monocytes, which was retained in differentiated macrophages and osteoclasts. These findings suggest overall increased metabolic activity in RA myeloid compartment, which could lead to higher bone resorption and inflammation in RA. Further research is now needed to better understand the drivers of this altered state and to determine the signalling pathways that could potentially be therapeutically targeted to reduce bone resorption and inflammation.

• Item Type: Thesis (PhD)
• Qualification Level: Doctoral
• Additional Information: Supported by funding from the European Union's Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie Grant (agreement No. 812890).
• Colleges/Schools: College of Medical Veterinary and Life Sciences > School of Infection & Immunity
• Supervisor's Name: Goodyear, Professor Carl
• Date of Award: 2024
• Depositing User: Theses Team
• Unique ID: glathesis:2024-84382
• Copyright: Copyright of this thesis is held by the author.
• Date Deposited: 24 Jun 2024 13:25
• Last Modified: 25 Jun 2024 07:30
• URI: https://theses.gla.ac.uk/id/eprint/84382