Session Title: Genetics, Genomics and Proteomics I
Session Type: Abstract Submissions (ACR)
In this study we are analyzing leukocyte subtype responses from patients with rheumatoid arthritis (RA) to IL6 inhibition. A large contribution to RA immunopathogenesis is caused by the pleiotropic effects of interleukin 6 (IL6), which we are investigating directly on the biological active constituents of the cell, i.e. the proteins, using state-of-the-artmass spectrometry. This has enabled us to quantify changes in protein expression of thousands of proteins as a result of biologic treatment, and mapping of post-translational modifications such as citrullination, and phosphorylation.
Methods: 10 ACA positive RA patients fulfilling the ACR criteria are being enrolled prior to monotherapy IL6 inhibition and 4 months after compared to 10 healthy controls. PBMC were isolated and subdivided into CD14+, CD4+, CD8+, C19+, and CD56+cells using immunoaffinity Dynabeads. Each cell type was prepared for mass spectrometry analysis (Thermo Q Exactive Plus), by a filter-aided sample preparation (FASP) method. MS data was searched against a human isoform database derived from UNIPROT using the Matrixscience MASCOT, and MaxQuant search engines. Differentially expressed proteins were filtered by requiring 2 peptides pr. protein, ANOVA (P-value) cutoff 0.05, q-value (false discovery adjusted p-value using multiple hypothesis testing) cutoff 0.05, and a power of at least 80%. The method was validated using known expression responses to IL6 inhibition. While responses have been shown via array and RT-qPCR, we obtained a broader quantitative differential protein expression profile.
Results: The initial results provided the identification of 4258 different proteins obtained by combining 3 technical replicates of the 5 cell types. In brief, several pro-inflammatory proteins were down regulated, while anti-inflammatory proteins were up regulated. For example in CD14+cells, interferon-induced guanylate-binding protein 1 (belonging to the TRIFF pathway), and CXCL2 (also known as macrophage inflammatory protein 2-α) were relatively down regulated by 8.04 fold and 1.14*10^4 fold, respectively while TGFβ was up regulated by 16.8 fold. Myeloid differentiation primary response protein (MyD88) was relatively down regulated by 2.3 fold, which could be explained by IL6 inhibition. The down regulation of both the TRIFF pathway, and MyD88 indicates a down regulation of CD14 (monocyte differentiation antigen) dependent signaling, and hence a reduction in the NFκB signaling and TNFα production. Of note, we found that STAT6 was relatively down regulated by 4.7 fold, which has recently been suggested to correlate with decreased CD14 signaling in CD14-/- mice.
Our proteomics driven strategy enable detection and mapping of valuable information of pro-, and anti-inflammatory responses to IL6 inhibition treatment on the protein level. This detailed insight into in vivo individual cell responses in patients will improve diagnostic and treatment optimization and drive personalized medicine. Our method may reveal hidden mechanisms in treatment success or failure.
M. K. Meyer,
G. N. Andersen,
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ACR Meeting Abstracts - https://acrabstracts.org/abstract/cellular-responses-of-il6-inhibition-tocilizumab-in-rheumatoid-arthritis-using-high-accuracy-tandem-mass-spectrometry/