New Proteins Contributing to Immune Cell Infiltration and Pannus Formation of Synovial Membrane from Arthritis Diseases

Abstract

An inflamed synovial membrane plays a major role in joint destruction and is characterized by immune cells infiltration and fibroblast proliferation. This proteomic study considers the inflammatory process at the molecular level by analyzing synovial biopsies presenting a histological inflammatory continuum throughout different arthritis joint diseases. Knee synovial biopsies were obtained from osteoarthritis (OA; n = 9), chronic pyrophosphate arthropathy (CPPA; n = 7) or rheumatoid arthritis (RA; n = 8) patients. The histological inflammatory score was determined using a semi-quantitative scale based on synovial hyperplasia, lymphocytes, plasmocytes, neutrophils and macrophages infiltration. Proteomic analysis was performed by liquid chromatography-mass spectrometry (LC-MS/MS). Differentially expressed proteins were confirmed by immunohistochemistry. Out of the 1871 proteins identified and quantified by LC-MS/MS, 10 proteins (LAP3, MANF, LCP1, CTSZ, PTPRC, DNAJB11, EML4, SCARA5, EIF3K, C1orf123) were differentially expressed in the synovial membrane of at least one of the three disease groups (RA, OA and CPPA). Significant increased expression of the seven first proteins was detected in RA and correlated to the histological inflammatory score. Proteomics is therefore a powerful tool that provides a molecular pattern to the classical histology usually applied for synovitis characterization. Except for LCP1, CTSZ and PTPRC, all proteins have never been described in human synovitis.

Keywords: CTSZ; DNAJB11; EML4; LAP3; LCP1; MANF; PTPRC; inflammation; proteomics; synovial membrane.

Figure A1

Immunohistochemistry Illustration of Negative Controls Obtained for the 10 Highlighted Proteins.

Figure 1

Proteomic analysis by mass spectrometry of synovial membrane from OA, CPPA and RA patients. (A) Protein expressions significantly modulated in OA, CPPA or RA synovial biopsies using the multiple sample test with 1400 permutation-based FDR for statistical analysis. (B) Representation of protein expressions obtained by mass spectrometry (Log2 (LFQ)) for the three pathologies. One-way ANOVA test with a post hoc test of Tukey was applied on logarithmic values: * P < 0.05, ** P < 0.01 and *** P < 0.001. (C) Correlation coefficients between the 10 highlighted biomarkers calculated according to the parametric Pearson test. OA, osteoarthritis; CPPA, chronic pyrophosphate arthropathy; RA, rheumatoid arthritis. Gene name was used to abbreviate protein name.

Figure 2

Immunohistochemistry (IHC) quantification of the 10 highlighted proteins in synovial membrane from OA, CPPA and RA patients. (A) Representation of protein quantification (optical density values) obtained by QuPath after IHC. One-way ANOVA test (post hoc of Tukey) or Kruskal–Wallis test (post hoc test of Dunn’s) was applied depending on normal distribution: * P < 0.05 and ** P < 0.01 (B) Correlation coefficients between the 10 highlighted biomarkers calculated according to the non-parametric Spearman test. OA, osteoarthritis; CPPA, chronic pyrophosphate arthropathy; RA, rheumatoid arthritis.

Figure 3

Correlation between mass spectrometry (MS) and immunohistochemistry (IHC) quantification of the 10 highlighted proteins in synovial membrane from OA, CPPA and RA patients. Protein expression levels (Log2 (LFQ)) obtained by mass spectrometry were correlated to the percentage of positive cells obtained by IHC using the non-parametric Spearman test: * P < 0.05, ** P < 0.01 and *** P < 0.001.

Figure 4

Immunohistochemistry illustration of the 10 highlighted proteins obtained with paraffin-embedded biopsies from synovial membrane of OA, CPPA and RA patients. The histological inflammatory scoring (HIS) is also associated with each biopsy. Highly positive cells have an OD > 0.6 (red spot), moderately positive 0.4 > OD > 0.6 (orange spot), weakly positive 0.2 > OD > 0.4 (yellow spot) and negative cells < 0.2 (blue spot). OA, osteoarthritis; CPPA, chronic pyrophosphate arthropathy; RA, rheumatoid arthritis.