Inlammatory response of a human meniscal in vitro model studied by proteomics

Abstract

Purpose: There is likely an inflammatory phenotype of OA where pro-inflammatory cytokines contribute to the pathogenesis. Degenerative meniscus lesions have been associated with both OA etiology and its progression. Thus, we sought to establish a human meniscus in vitro model to explore the meniscal tissue’s response to cytokine treatment where we used a discovery proteomics approach to determine and follow the release of matrix proteins. Methods: One lateral meniscus was harvested within 40 hours postmortem from a human donor (male, age 79) without known chronic joint disease. The procedure was approved by the local ethics committee at Lund University. The meniscus was visually inspected to be macroscopically intact. We then obtained 12 slices (1mm wide) cut radially from the meniscal body. These slices were further divided in two by the inner 1/3 (inner zone) or the peripheral 2/3 (outer zone). The explants were either left untreated (controls, n=6+6) or with interleukin-1ß (IL-1ß) (n=6+6) at 5 ng/ml, and cultured in serum-free DMEM media (supplemented with ITS and BSA) for 21 days. Medium changes were carried out every 3 days, and used medium was collected and stored at -20°C for analysis. The glycosaminoglycan (GAG) release was quantified by the 1,9-dimethylmethylene blue (DMMB) assay for all samples and viability was checked using AlamarBlue at day 0,6,12 and 21. Tissue explants were frozen (-20°C) at the end of the 21 day culture. Liquid chromatography-mass spectrometry (LC-MS) runs using a Q-Exactive orbitrap were performed for medium samples at all the time points for the identification and quantification of proteins using pooled samples of the six replicates for each zone and treatment. Proteome Discoverer 2.2 software was used for the discovery analysis and the quantification was made at the MS1 level using label-free quantification with feature detection. The total treatment effect was estimated by summing up all the time points and calculating the fold change. Results: Explant viability was mainly stable during the whole cultivation period and that IL-1ß had moderate effect on glycosaminoglycan release with a significant increased release only at day 6 and 9 (see Figure 1). The proteomics data, however, revealed a distinct elevation in the levels of multiple cytokines and proteins related to inflammation in response to IL-1ß. The LC-MS experiment from different samples (n=28) resulted in a total of 479 proteins identified in at least two samples at a protein confidence <0.01. The principal component analysis (PCA) plot for the four sample groups are shown in Figure 2, illustrating a clear separation between control and IL-1ß treated groups, and also a time dependent response in all groups. The cumulative release until day 21 was used to pick up overall treatment effects such as up-regulation of several cytokines and proteins involved in the inflammatory response, including C-X-C motif chemokines, interleukins, and complement components. We found differences in outer vs inner zones, particularly the more cartilage specific proteins such as collagen II and aggrecan were prominent in the inner avascular zone, which could be expected as the different zones contain different cell populations. Interestingly we also found release of MMP-13 only in the inner zone. Proteins with an increased release upon IL-1 treatment are listed in Table 1 for both inner and outer zones with a major overlap seen. Conclusions: This pilot proteomics approach allowed us to unambiguously identify and quantify multiple proteins in the media of human meniscus explant cultures upon IL-1ß exposure that may mimic the early events in the meniscus phenotype of OA development. We followed the quantitative release of more than 200 proteins including cytokines, proteases as well as other extracellular proteins throughout the 21-day culture period. Several of the upregulated proteins belonged to the cytokine family.View Large Image Figure ViewerDownload Hi-res image Download (PPT)View Large Image Figure ViewerDownload Hi-res image Download (PPT)