Toll-like receptor adaptor signaling molecule MyD88 on intervertebral disk homeostasis: in vitro, ex vivo studies

Abstract

MyD88 is an adapter protein that links toll-like receptors (TLRs) and Interleukin-1 receptors (IL-1Rs) with downstream signaling molecules. The MyD88 has been found to be an essential mediator in the development of osteoarthritis in articular cartilage. However, the role of the MyD88 pathway has yet to be elucidated in the intervertebral disk (IVD). Using in vitro techniques, we analyzed the effect of MyD88 pathway-specific inhibition on the potent inflammatory and catabolic mediator LPS and IL-1 in bovine and human nucleus pulposus (NP) cells by assessing matrix-degrading enzyme expression, including matrix metalloproteases (MMPs) and a disintegrin-like and metalloprotease with thrombospondin motifs (ADAMTS family). We also analyzed inhibition of MyD88 in the regulation of inducible nitric oxide synthase and TLR-2. Finally, we used an ex vivo organ culture model to assess the effects of MyD88 inhibitor (MyD88i) on catabolic factor-induced disk degeneration in mice lumbar disks. In bovine NP cells, MyD88i potently antagonizes LPS- or IL-1-mediated induction of cartilage-degrading enzyme production, including MMP-1, MMP-13, ADAMTS-4, and ADAMTS-5. MyD88i also attenuates the LPS- or IL-1-mediated induction of iNOS and TLR-2 gene expression. Our ex vivo findings reveal inhibition of MyD88 via counteraction of IL-1-mediated proteoglycan depletion. The findings from this study demonstrate the potent anti-inflammatory and anti-catabolic effects of inhibition of MyD88 pathway inhibition on IVD homeostasis, suggesting a potential therapeutic benefit of a MyD88i in degenerative disk disease in the future.

Fig. 1

Inhibition of MyD88 pathway counteracts LPS- and IL-1-induced catabolic effects in bovine NP cells. The monolayer of bovine NP cells was treated with MyD88i (100 μM), LPS (10 μg/mL), IL-1 (10 ng/mL) and combinations of either LPS and MyD88i or IL-1 and MyD88i. After stimulation for 24 h, cells were harvested and total RNA was extracted to perform real time qPCR targeting key cartilage degrading enzymes including MMP-13 (A), ADAMTS-4 (B), ADAMTS-5 (C) and the MyD88 pathway receptor, TLR-2 (D). Values are the mean and SD and are representative of 3 different donors in 3 separate experiments. A value of p<0.05 and p<0.01 indicate a significant and a highly significant difference in ANOVA, respectively.

Fig. 2

Inhibition of MyD88 pathway suppresses LPS- and IL-1-induced expression of MMP-13 in human NP cells. The monolayer of human NP cells was treated with MyD88i (100 μM), LPS (10 μg/mL), IL-1 (10 ng/mL) and combinations of either LPS and MyD88i or IL-1 and MyD88i for 24 h. After stimulation, cells were harvested and subjected to either total RNA extraction for real time qPCR (A) or preparation of cell lysates for western blotting (B) to assess altered level of a potent collagen-degrading enzyme MMP-13 using human specific anti-MMP-13 antibody. Values are the mean and SD and are representative of 3 different donors in 3 separate experiments. A value of p<0.05 and p<0.01 indicate a significant and a highly significant difference in ANOVA, respectively.

Fig. 3

The LPS- and IL-1-induced expression of matrix-degrading enzyme MMP-1 is significantly inhibited by MyD88 peptide inhibitor in human NP cells. The monolayer of human NP cells was treated with MyD88i (100 μM), LPS (10 μg/mL) and IL-1 (10 ng/mL) and combinations of either LPS and MyD88i or IL-1 and MyD88i for 24 h. A, After stimulation, cells were harvested and total RNA was extracted to perform real time qPCR targeting MMP-1 gene expression. B, Cell lysates were then prepared and analyzed by Western blotting with human specific anti-MMP-1 antibody. Values are the mean and SD and are representative of 3 different donors in 3 separate experiments. A value of p<0.05 and p<0.01 indicate a significant and a highly significant difference in ANOVA, respectively.

Fig. 4

Inhibition of MyD88 pathway suppresses LPS- and IL-1-induced expression of aggrecanase (ADMATS-4) and oxidative stress associated gene (iNOS) in human NP cells. Human NP cells in monolayer were treated with MyD88i (100 μM), LPS (10 μg/mL) or IL-1 (10 ng/mL) and combinations of either LPS and MyD88i or IL-1 and MyD88i for 24 h. After stimulation, cells were harvested and total RNA was extracted to perform real time qPCR to assess aggrecanase ADAMTS-4 (A) and oxidative stress associated gene iNOS (B). A value of p<0.05 and p<0.01 indicate a significant and a highly significant difference in ANOVA, respectively.

Fig. 5

MyD88 inhibitory peptide antagonizes LPS- and IL-1-induced protease activity in human NP cells. Human NP cells in monolayer were treated with MyD88i (100 μM), LPS (10 μg/mL) or IL-1 (10 ng/mL) and combinations of either LPS and MyD88i or IL-1 and MyD88i for 24 h. Gelatin zymography was performed by loading equal volumes of the conditioned media sample on polyacrylamide gel (A). Band images were digitally captured and the intensity of the bands (pixels/band) was obtained using the ImageJ densitometry analysis software in arbitrary optical density units (B). A value of p<0.01 indicates a highly significant difference in ANOVA.

Fig. 6

Histological assessments (original magnification: ×100). Lumbar spine disks of mouse were dissected after en bloc intradiskal pre-microinjection with MyD88i at the concentration of 150 μM per disk using 30 G needle (1.5 μL in volume). The MyD88i pre-injected disks were challenged with either IL-1 (100 ng/mL) or LPS (10 μg/mL) in DMEM/Ham's F-12 medium supplemented with 1% mini-ITS and further incubated for 6 days. Harvested disks were fixed, decalcified in EDTA and were paraffin embedded followed by serial disk sections of exactly 5-μm thickness to prepare slides. Safranin O–fast green staining was performed to assess general morphology and the loss of PG in disk ground substance.