Does metabolic perturbation mediate the inflammatory phenotype of the obese OA synovial fibroblast?

Aims and Objectives
The overarching objective of this studentship is to investigate the relationship between cellular bioenergetics and the inflammatory phenotype of the OA synovial fibroblast.
The studentship will aim to

1. Determine whether obese OA and normal-weight OA synovial fibroblasts exhibit different metabotypes
2. Determine the relationship between the synovial fibroblast metabotype, resident immune cell populations and the inflammatory phenotype of the synovial fibroblast (including cytokine secretion and the expression of inflammation-associated snoRNAs) and the relationship to the inflammatory profile of joint synovial fluid
3. Examine whether modulation of cellular metabolism alters the inflammatory phenotype of the synovial fibroblast.

Materials &Methods
Patient tissues and patient-derived primary cells
1. Patients with end-stage OA of the knee and hip and hand. Ethics for the collection and use of synovial tissue from OA patients and at-risk cohorts is already in place at Birmingham (REC reference: 16/SS/0172) and the study is NIHR adopted (Dr Simon W jones). In total, the ethics allows for the collection of synovial tissue/fluid from patients with knee OA, hip OA, ankle OA, spinal OA, shoulder OA, elbow OA, hand OA and foot OA, with n=157 patients per group.
2. Frozen archived synovial joint tissues and joint fluid from different OA patient populations
3. Isolated cyroperserved primary cells (chondrocytes, synovial fibroblasts)
Methods
The student will be trained and become skilled in a wide array of molecular cell biology techniques, including but not limited to
Extraction of total RNA using Trizol reagent.
Quantification of extracted RNA and analysis of RNA integrity (Agilent Bioanalyser, Nanodrop, Qubit)
qRT-PCR
ELISA/Luminex technique for the measurement of pro-inflammatory cytokines
Isolation and culture of primary cells (synovial fibroblasts)
In vitro loss of function studies using RNAi (including siRNA and LNAs) and lipid transfections as well as electroporation
Seahorse assays to study cellular metabolism.
FACS analysis of synovial fibroblasts
Mitochondrial proteomic analyses

Study Design
Characterise the fluid metabolic signature of inflammatory OA and correlate this with obesity.
To determine whether global and local metabolic deviation is linked to the pathology of inflammatory OA, serum and synovial fluid from NW and obese OA patients will be processed and subject to 1D NMR spectroscopy. Metabolites will then be identified as previously described). The same fluids will also be analysed by Luminex to assess levels of pro and anti-inflammatory cytokines and chemotactic factors. Biomarker signatures able to stratify disease groups will be identified by PCA, OPLS-DA and PLS-R analysis and correlations made between clinical measurements indicative of systemic inflammation including CRP and hand involvement.

Characterise the metabolism in OA synovial fibroblasts from normal weight and obese individuals.
Primary synovial fibroblasts will be isolated by digestion of synovial tissue biopsies from NW and obese OA patients undergoing arthroplasty. To determine whether synovial fibroblast metabolic phenotype underpins the inflammatory phenotype observed in obese OA patients, metabolites will be extracted from cells and culture supernatants, subject to 1D NMR spectroscopy and metabolites identified as previously described. Culture supernatants will be analysed by Luminex to profile cytokines and chemotactic factors. Signatures able to stratify disease groups will be identified by PCA, OPLS-DA and PLS-R analysis and correlations made between clinical measurements indicative of systemic inflammation. Comparison of metabolites identified in Aim 1 will be made to elucidate the contribution of fibroblasts to the systemic phenotype of inflammatory OA. Key enzymes from metabolic pathways identified in metabolomic analysis will be validated by qPCR.