Suppression of wear-particle-induced pro-inflammatory cytokine and chemokine production in macrophages via NF-κB decoy oligodeoxynucleotide: a preliminary report

Abstract

Total joint replacement (TJR) is very cost-effective surgery for end-stage arthritis. One important goal is to decrease the revision rate, mainly because TJR has been extended to younger patients. Continuous production of ultra-high molecular weight polyethylene (UHMWPE) wear particles induces macrophage infiltration and chronic inflammation, which can lead to periprosthetic osteolysis. Targeting individual pro-inflammatory cytokines directly has not reversed the osteolytic process in clinical trials, owing to compensatory up-regulation of other pro-inflammatory factors. It is hypothesized that targeting the important transcription factor NF-κB could mitigate the inflammatory response to wear particles, potentially diminishing osteolysis. In the current study, NF-κB activity in mouse RAW 264.7 and human THP1 macrophage cell lines, as well as primary mouse and human macrophages, was suppressed via competitive binding with double strand decoy oligodeoxynucleotide (ODN) containing an NF-κB binding element. It was found that macrophage exposure to UHMWPE particles induced multiple pro-inflammatory cytokine and chemokine expression, including TNF-α, MCP1, MIP1α and others. Importantly, the decoy ODN significantly suppressed the induced cytokine and chemokine expression in both murine and human macrophages, and resulted in suppression of macrophage recruitment. The strategic use of decoy NF-κB ODN, delivered locally, could potentially diminish particle-induced periprosthetic osteolysis.

Keywords: Macrophage; NF-κB decoy oligodeoxynucleotide; Periprosthetic osteolysis; Wear particles.

Fig.1. Naked NF-κB decoy ODN suppressed LPS induced TNF-α expression in mouse macrophages

a) TNF-α production by RAW 264.7 cells exposed to 1μg/ml LPS followed by 0.5 μM ODN delivery using 3 different transducing agents: a cationic polymer (C32-122), Lipidoid, or Lipofectamine 2000 (Invitrogen). The expression of TNF-α was detected by ELISA. “Alone= no transfection agent was used. TNF-α has no difference; b) Total lactate dehydrogenase activity (an indicator of viable cell number) when cultures of RAW 264.7 cells are exposed to transducing agents with or without ODN. Total lactate dehydrogenase activity and therefore live cell viability is decreased when the ODN is delivered using a transduction agent; c) upper panel, illustration of reporter luciferase gene driven by NF-κB response elements; lower panel, effect of LPS (1 μg/ml) on the RAW264.7 NF-κB luciferase reporter cell clone; d) upper panel, RAW264.7 NF-κB luciferase reporter cells in response to PE particles with decoy ODN (NF-κB ODN) or scrambled ODN (S-ODN) for 24h, the result was read by IVIS imaging system; lower panel, quantification result of luciferase activity. *p< .05, ** p< .01, *** p<.005

Fig.2. NF-κB decoy ODN suppressed pro-inflammatory cytokines mRNA expression in primary mouse macrophages exposed to UHMWPE and/or LPS

Characterization of mouse bone marrow derived macrophage (a). Primary mouse macrophages cultured for one week were stained for macrophage surface marker including F4/80 (APC) and CD11b (Cy7PE) and analyzed by flow cytometry. Effect of NF-κB decoy ODN (0.5μM) on TNF-α, MCP1, IL-1β, and IL-6 expression by mouse bone marrow-derived macrophages stimulated with UHMWPE and LPS (1 μg/ml) for 6, 12, 24 and 48 h (b-e). The RNA samples were collected at indicated time points and converted into complementary DNA. Quantitative PCR analysis was performed by using the Taq-Man system as described in the Materials and Methods. *p< .05, ** p< .01, *** p<.005.

Fig.3. NF-κB decoy ODN suppressed pro-inflammatory cytokines mRNA expression in human THP1 macrophage cells exposed to UHMWPE and/or LPS

Effect of NF-κB decoy ODN (0.5μM) on TNF-α, MCP1, IL-1β, and IL-6 expression by human THP1 macrophages stimulated with UHMWPE and LPS (1 μg/ml) for 6, 12, 24 and 48 h (a-d). The RNA samples were collected at indicated time points and converted into complementary DNA. Quantitative PCR analysis was performed by using the Taq-Man system as described in the Materials and Methods. *p< .05, ** p< .01,*** p<.005..

Fig.4. NF-κB decoy ODN suppressed TNF-α and MCP1 cytokine expression in primary mouse macrophages and THP1 cells exposed to UHMWPE and/or LPS

Effect of NF-κB decoy ODN (0.5μM) on TNF-α and MCP1 production by mouse bone marrow-derived macrophages (a, b) and THP1 cells (c, d) stimulated with UHMWPE and LPS (1 μg/ml) for 6, 12, 24 and 48 h. The supernatant samples were collected at indicated time points and analyzed by magnetic beads cytokine assay system. *p< .05, ** p< .01.

Fig.5. NF-κB decoy ODN suppressed THP1 cell migration attracted by the conditioned media from cells exposed to UHMWPE and/or LPS

The supernatant were collected from THP1 cells stimulated with ODNs, UHMWPE, and LPS (0.1 μg/ml) for 24 (a) and 48 h (b). The migration assay was performed using a ChemoTx® Disposable Chemotaxis System. The migrated cell number was quantified by detecting the double strand DNA concentration using pico-green detection dye. *p< .05, ** p< .01.