Abstract
BackgroundTumor necrosis factor (TNF)-α inhibitors represented by Etanercept (a fusion protein containing soluble TNF receptor II (sTNFRII) and the Fc segment of human IgG1) play a pivotal role in Rheumatoid arthritis (RA) treatment. However, long-term use increases the risk of infection and tumors for their systemic inhibition of TNF-α, which disrupts the regular physiological function of this molecular. Mesenchymal stem cells (MSCs)-based delivery system provides new options for RA treatment with their “homing” and immune-regulation capacities, whereas inflammatory environment (especially TNF-α) is not conducive to MSCs' therapeutic effects by inducing apoptosis/autophagy. Here, we constructed a strain of sTNFRII-Fc-expressing MSCs (sTNFRII-MSC), aiming to offset the deficiency of those two interventions.MethodsConstructed sTNFRII-Fc lentiviral vector was used to infect human umbilical cord-derived MSCs, and sTNFRII-MSC stable cell line was generated by monoclonal cultivation. In vitro and vivo characteristics of sTNFRII-MSC were assessed by coculture assay and an acute inflammatory model in NOD/SCID mice. The sTNFRII-MSC were transplanted into CIA model, pathological and immunological indicators were detected to evaluate the therapeutic effects of sTNFRII-MSC. The distribution of sTNFRII-MSC was determined by immunofluorescence assay. Apoptosis and autophagy were analyzed by flow cytometry, western blot and immunofluorescence.ResultssTNFRII-Fc secreted by sTNFRII-MSC present biological activity both in vitro and vivo. sTNFRII-MSC transplantation effectively alleviates mice collagen-induced arthritis (CIA) via migrating to affected area, protecting articular cartilage destruction, modulating immune balance and sTNFRII-MSC showed prolonged internal retention via resisting apoptosis/autophagy induced by TNF-α.ConclusionsTNFRII-Fc modification protects MSCs against apoptosis/autophagy induced by TNF-α, in addition to releasing sTNFRII-Fc neutralizing TNF-α to block relevant immune-inflammation cascade, and thus exert better therapeutic effects in alleviating inflammatory arthritis.
Highlights
Tumor necrosis factor (TNF)-α inhibitors represented by Etanercept (a fusion protein containing soluble TNF receptor II and the Fc segment of human immunoglobulin G1 (IgG1)) play a pivotal role in Rheumatoid arthritis (RA) treatment
As for the mechanism underlying the better therapeutic effects granted by soluble tumor necrosis factor receptor (TNFR) II (sTNFRII)-Mesenchymal stem cells (MSCs), we focus on the apoptosis and autophagy of implanted MSCs induced by TNF-α. sTNFRII-MSC maintained longer in collagen-induced arthritis (CIA) mice than naïve MSCs which was related to the protective effect of sTNFRII-Fc modification against apoptosis and autophagy induced by TNF-α on MSCs
The expression cassette of sTNFRII-Fc was cloned into the lentiviral vector GV358 containing enhanced green fluorescent protein (EGFP) and puromycin resistance gene (Fig. 1a), and gel electrophoresis revealed that a characteristic band was shown at 1.54 kb (Additional fi1e 1: Fig. 1D) which consistent with our predictions
Summary
Tumor necrosis factor (TNF)-α inhibitors represented by Etanercept (a fusion protein containing soluble TNF receptor II (sTNFRII) and the Fc segment of human IgG1) play a pivotal role in Rheumatoid arthritis (RA) treatment. TNF-α is originally produced as a trimeric type II transmembrane protein, which can be cleaved to a soluble form by the metalloproteinase TNF-converting enzyme, and primarily secreted by lymphocytes, monocytes and macrophages [4, 5] This extracellular TNF-α binds to its two receptors, tumor necrosis factor receptor (TNFR) I (expressed on almost all mammalian cell type) and TNFR II (restricted to immune cells and endothelial cells) on the cell membrane, and eventually activates the transcription factor nuclear factor-κB to mediate different physiological processes [6]. The current therapeutic paradigm of global TNF-α blockade has several limitations, including adverse effects (serious infection), low rates of disease remission and generation of anti-drug antibodies [11, 12] These limitations were due to systemic delivery of those TNF-α inhibitors, which required higher dosage to treat affected arthritic joints in addition to generating immunogenicity and affecting the inherent physiological function of TNF-α within the hematopoietic lineage [13, 14]. There is an urgent need for a safe, effective and accessible carrier to deliver drugs to the affected areas, and reduce the occurrence of adverse reactions
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