Abstract
Purpose :
Mitochondrial dysfunction is a hallmark of many human diseases. Our laboratory has previously shown that hyperglycemia, as seen in diabetes, and salt-induced hyperosmolarity, as seen in dry eye disease, lead to mitochondrial dysfunction and metabolic abnormalities in corneal (CECs) and conjunctival epithelial cells. We have further found that the insulin-like binding protein-3 (IGFBP-3) plays a key role in mitigating these responses. This study sought to expand on our prior results and investigate the effects of inflammation on mitochondrial and metabolic homeostasis in CECs.
Methods :
Telomerase-immortalized human corneal epithelial cells were cultured in serum-free keratinocyte basal media. To establish an inflammatory environment, cells were treated with 50 ng/mL IL-1β for 24 or 48 hours. Metabolism was measured in real time using a Seahorse metabolic flux analyzer. Oxygen consumption rate (OCR), extracellular acidification rate (ECAR), and other metabolic parameters were quantified using a Seahorse XFp Cell Mito Stress Test kit. Total cell number per well was used for normalization. Protein expression for metabolic, mitochondrial and mitophagy-related proteins was assessed using western blot.
Results :
At 24 hours, IL-1β had no effect on basal levels of OCR or ECAR. Maximal and spare respiratory were also unchanged. At 48 hours, IL-1β decreased ECAR (P=0.035), while basal OCR was unchanged. This shifted cells towards a more respiratory phenotype. There was a corresponding decrease in spare respiratory capacity (P=0.023). Interestingly, there was a decrease in non-mitochondrial oxygen consumption at 24 hours in the IL-1β group (P=0.003) which was not evident at 48 hours. There were no changes in ATP-linked respiration at either time point. Consistent with the decrease in non-mitochondrial oxygen consumption, IGFBP-3 also showed a drop in intracellular expression levels at 24 hours. This was associated with an observed increased in BNIP3L/NIX. At 48 hours, IGFBP-3 levels were similar in both treated and control groups, BNIP3L/NIX trended downward, while both mTOR and COVI X were increasing. PINK1 was unchanged.
Conclusions :
These data confirm that inflammation triggers changes in metabolic and mitochondrial homeostasis in CECs. Further studies are needed to fully define the ability of CECs to respond and adapt to inflammatory stress.
This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.