Homan Kentaro, Tokuhiro Taiki, Onodera Tomohiro, Hanamatsu Hisatoshi, Furukawa Jun-Ichi, Ebata Taku, Matsuoka Masatake, Kadoya Ken, Terkawi M Alaa, Iwasaki Norimasa
Department of Orthopaedic Surgery, Faculty of Medicine and Graduate School of Medicine, Hokkaido University, Sapporo, Japan.
Department of Advanced Medicine for Locomotor System, Faculty of Medicine and Graduate School of Medicine, Hokkaido University, Sapporo, Japan.
Front Immunol. 2024 Nov 25;15:1475473. doi: 10.3389/fimmu.2024.1475473. eCollection 2024.
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.
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.
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.
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.
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.
由于内在愈合能力有限，软骨修复是一项重大的临床挑战。当前的治疗策略，如细胞移植疗法，旨在通过用健康细胞替换受损组织来克服这一挑战。然而，移植细胞的长期存活和功能仍然是主要障碍。
本研究调查了软骨细胞传代对聚糖谱及其抗原特性的影响。我们假设传代导致的聚糖组成改变可能有助于增强激活巨噬细胞的能力，从而影响细胞移植治疗的结果。
从C57BL/6小鼠中分离出腹腔巨噬细胞和原代关节软骨细胞，建立直接和间接共培养模型。通过测量培养上清液中白细胞介素-6和一氧化氮的浓度或其基因表达来评估巨噬细胞的激活。通过糖印迹法结合用于N-聚糖和神经节苷脂的SALSA程序以及用于O-聚糖的BEP方法对各种糖缀合物进行糖组分析。
我们的结果表明，随着传代数的增加，巨噬细胞与传代软骨细胞的直接共培养增加了促炎细胞因子（包括白细胞介素-6和一氧化氮）的产生。随着传代数的增加，GD3的表达大幅下降，GM3（尤其是GD1a）的表达显著增加。将传代的GM3S基因敲除软骨细胞与巨噬细胞共培养可显著抑制白细胞介素-6的表达，这意味着巨噬细胞激活减少。
观察到的由于软骨细胞聚糖谱改变而导致的巨噬细胞激活为移植细胞的抗原性和免疫排斥提供了一种可能的解释。
