Purchase this article with an account.
Algis Grybauskas, Edward Wagner, Robert Andrew Burdi, Loyal Walker, Paul A. Knepper; TLR-4 Innate Immune Differential Response To Three Dietary Fatty Acids Challenged With Low Molecular Weight Hyaluronic Acid, a TLR-4 Ligand. Invest. Ophthalmol. Vis. Sci. 2012;53(14):6615.
Download citation file:
© ARVO (1962-2015); The Authors (2016-present)
Toll-like receptor 4 (TLR-4) has been implicated in an innate immune response which leads to inflammatory diseases such as primary open-angle glaucoma and inflammatory dementia, notably Alzheimer's Disease. This experiment was designed to determine whether three common dietary fatty acids - elaidic acid (EA), docosahexaenoic (DHA), and lauric acid (LA) - alter TLR-4 expression when challenged with a TLR-4 ligand, low molecular weight hyaluronic acid (LMW HA).
Naive PC-12 cells were grown in 10% FBS, 5% FCS in RPMI 1640 Medium at 37C, 5% CO2. Once cells reached confluency, they were removed and plated at 40,000 cells/well. Cells incubated overnight in complete media for 24 h and the next day were treated with 2, 20, 200 uM DHA, an omega-3 fatty acid (Sigma), 10, 100, 1000 10 uM LA, a saturated fatty acid (Sigma), or 20, 200, 2000 uM EA, a trans-monounsaturated fatty acid (Sigma). Cells were also treated with 0.2, 2.0, and 20 uM of 50 kDa HA (LMW HA; Sigma) or 1000 kDa high molecular weight HA (HMW HA; Hyalose). Experiments were conducted following cell treatment at 6 and 24 h. Also, 0.2uM LMW HA and LMW HA with TLR-4 neutralizing antibody (Santa Cruz) treated PC-12 cells were used to test TLR-4 activity. Cells were removed from plating and counted using a 0.1mm Fischer Scientific hemacytometer to determine cell viability. Western Blot analysis was used to measure TLR-4 receptor, CD44, and hyaluronidase-2 in cell lysates at 6 and at 24h. Integrated Optical Densitometry (IOD) was used to quantify WB results, and t-tests were used to evaluate cell viability data.
TLR-4 expression in the presence of EA along with LMW HA was robustly increased (2.5 fold) in comparison to PBS control using IOD. TLR-4 expression was observed to occur in a time-dependent manner. In comparison, DHA with LMW HA IOD measurements showed a slight increase (1.04 fold) to PBS control, and LA with LMW HA showed a slight decrease (1.11 fold) to PBS control. Cell viability counts using PC-12 cells treated with TLR-4 neutralizing antibody and LMW HA were significantly greater (2.1 fold, p<.0002) than cells treated with LMW HA alone.
The results of this study indicate that LMW HA activation of TLR-4 causes PC-12 cell death, and co-administration of TLR-4 neutralizing antibody blocked LMW HA effects. Cell viability of PC-12 cells, when challenged with LMW HA, was dependent on the type of fatty acid treatment. Notably, LMW HA in the presence of EA markedly upregulated TLR-4. These results suggest that dietary fatty acids could be an important factor in the progression of inflammatory neurodegenerative diseases.
This PDF is available to Subscribers Only