Associations between glycan signature alterations and the cellular antigenic properties of passaged chondrocytes

Abstract

Background: Cartilage repair is a significant clinical challenge because of the limited intrinsic healing capacity. Current therapeutic strategies, such as cell transplantation therapy, aim to overcome this challenge by replacing damaged tissue with healthy cells. However, the long-term survival and functionality of transplanted cells remain major hurdles.

Objective: This study investigated the impact of chondrocyte passaging on glycan profiles and their antigenic properties. We hypothesized that alterations in glycan composition due to passaging may contribute to the enhanced ability to activate macrophages, thereby affecting the outcome of cell transplantation therapy.

Methods: Peritoneal macrophages and primary articular chondrocytes were isolated from C57BL/6 mice to establish direct and indirect coculture models. Macrophage activation was assessed by measuring the concentrations of IL-6 and nitric oxide in the culture supernatants or their gene expression. Glycome analysis of various glycoconjugates was performed by glycoblotting methods combined with the SALSA procedure for N-glycans and GSLs and the BEP method for O-glycans.

Results: Our results revealed that direct coculture of macrophages with passaged chondrocytes increased the production of proinflammatory cytokines, including IL-6 and NO, as the number of passages increased. With increasing passage number, the expression of GD3 substantially decreased, and the expression of GM3, especially GD1a, significantly increased. Coculturing passaged GM3S knockout chondrocytes with macrophages significantly suppressed IL-6 expression, implying reduced macrophage activation.

Conclusion: The observed activation of macrophages due to alterations in the glycan profile of chondrocytes provides a possible explanation for the antigenicity and immune rejection of transplanted cells.

Keywords: antigenicity; cellular transplantation; chondrocyte; glycome analysis; macrophage; passage culture.

Figure 1

Immunological behavior through coculture with macrophages and passaged cells. (A), Schematic overview of the increase in the macrophage immune response due to glycosylation with increasing passage number. (B), IL-6 levels (left panel) and nitric oxide (NO) levels (right panel) released into the medium at different passage numbers of chondrocytes in a macrophage coculture system (n = 4 mice). The data are presented as the means ± s.d.s; Kruskal−Wallis test. (C), IL-6-stained cells were mainly costained with F4/80-stained cells. Scale bars, 50 μm. P, passage; MΦ, macrophage; C, chondrocyte.

Figure 2

Fluctuations in N- and O-glycans with passaging. (A), Spectra of chondrocytes for each passage, annotated with confirmed N-glycan structures from MALDI-TOF MS. HM, high mannose; IS, internal standard. (B), Profiling of O-glycans. The glycan population detected included core substituents, and a trend toward the biosynthesis of branched O-glycans was observed. (C), Absolute value and relative levels of O-glycans in chondrocytes at each passage. P, passaged.

Figure 3

Evaluation of GSL-glycan profiles on chondrocyte per passage. (A), MALDI-TOF MS spectra showing GSL-glycans on chondrocytes after successive passages. IS, internal standard. (B), Expression profiles of ganglioside depending on passage number. (C), The total amount of GSL-glycans increased with each successive passage. SphinGOMAP (http://www.sphingomap.org/) online databases were used for the structural estimation of the GSL glycans. NS, not significant; P, passage.

Figure 4

Immunological behavior through coculture with macrophages and passaged cells. (A), Pathway of ganglioside synthesis showing the block in GM3 synthase (GM3S) null mice. The approved gene name for GM3S is ST3Gal-5. (B), mRNA expression of ST3Gal-5 in wild-type and GM3S-/- chondrocytes on passage 0 (n = 3 mice). Data are mean ± s.d.; unpaired t-test. (C), mRNA expression of il-6 in co-cultured with GM3S-/- chondrocytes and macrophage on passage 3 (n = 3 mice). Data are mean ± s.d.; unpaired t-test. P, passage; MΦ, macrophage; KO, knockout.