Evidence for Regulated Interleukin-4 Expression in Chondrocyte-Scaffolds under In Vitro Inflammatory Conditions

Muhammad Farooq Rai,Michael F. G. Schmidt,Thomas Graeve,Sven Twardziok,Zhongjun Zhou

doi:10.1371/journal.pone.0025749

Muhammad Farooq Rai, Michael F. G. Schmidt + Show 3 more

Open Access

https://doi.org/10.1371/journal.pone.0025749

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Abstract

ObjectiveTo elucidate the anti-inflammatory and anabolic effects of regulated expression of IL-4 in chondrocyte-scaffolds under in vitro inflammatory conditions.MethodsMature articular chondrocytes from dogs (n = 3) were conditioned through transient transfection using pcDNA3.1.cIL-4 (constitutive) or pCOX-2.cIL-4 (cytokine-responsive) plasmids. Conditioned cells were seeded in alginate microspheres and rat-tail collagen type I matrix (CaReS®) to generate two types of tissue-engineered 3-dimensional scaffolds. Inflammatory arthritis was simulated in the packed chondrocytes through exogenous addition of recombinant canine (rc) IL-1β (100 ng/ml) plus rcTNFα (50 ng/ml) in culture media for 96 hours. Harvested cells and culture media were analyzed by various assays to monitor the anti-inflammatory and regenerative (anabolic) properties of cIL-4.ResultscIL-4 was expressed from COX-2 promoter exclusively on the addition of rcIL-1β and rcTNFα while its expression from CMV promoter was constitutive. The expressed cIL-4 downregulated the mRNA expression of IL-1β, TNFα, IL-6, iNOS and COX-2 in the cells and inhibited the production of NO and PGE2 in culture media. At the same time, it up-regulated the expression of IGF-1, IL-1ra, COL2a1 and aggrecan in conditioned chondrocytes in both scaffolds along with a diminished release of total collagen and sGAG into the culture media. An increased amount of cIL-4 protein was detected both in chondrocyte cell lysate and in concentrated culture media. Neutralizing anti-cIL-4 antibody assay confirmed that the anti-inflammatory and regenerative effects seen are exclusively driven by cIL-4. There was a restricted expression of IL-4 under COX-2 promoter possibly due to negative feedback loop while it was over-expressed under CMV promoter (undesirable). Furthermore, the anti-inflammatory /anabolic outcomes from both scaffolds were reproducible and the therapeutic effects of cIL-4 were both scaffold- and promoter-independent.ConclusionsRegulated expression of therapeutic candidate gene(s) coupled with suitable scaffold(s) could potentially serve as a useful tissue-engineering tool to devise future treatment strategies for osteoarthritis.

Highlights

Osteoarthritis (OA) is the most common musculoskeletal disorder worldwide
Analysis of pro-inflammatory cytokines Our results show that the expression of canine IL-4 (cIL-4) driven by both
Control experiments showed that non-transfected chondrocytes expressed high levels of both inducible nitric oxide synthase (iNOS) and COX-2 only after exogenous stimulation with canine recombinant interleukin-1 beta (IL-1b) and tumor necrosis factor alpha (TNFa)

Summary

Introduction

Osteoarthritis (OA) is the most common musculoskeletal disorder worldwide It is the major cause of morbidity in developed nations and has enormous social and economic consequences. Progressive loss of cartilage in OA results from an imbalance of anabolic and catabolic metabolisms [1,2] through a complex interaction of mechanical and biochemical factors [3,4,5]. Among the latter, a number of catabolic factors, including pro-inflammatory cytokines and proteases have been demonstrated to play major roles [1,6,7,8]. Pro-inflammatory mediators present in the joint could affect the transplanted chondrocytes, potentiating the need to suppress inflammation [13]

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