"Introduction: High IL-1β levels are often found in osteoarthritic (OA) joints. Moreover, IL-1β credibly induces post-traumatic OA. Since cell morphology is an important regulator of cell behavior, we investigated how acute vs. chronic IL-1β dynamically changes multifaceted aspects of cell morphology in healthy and OA chondrocytes in relation to phenotypic outcome.
Methods: Acute inflammation was mimicked by treating healthy bovine chondrocytes with IL-1β for 3 days, followed by media lacking IL-1β for 3 days. Chronic IL-1β was simulated by adding IL-1β to the media for 6 days. Unstimulated chondrocytes served as the control. An automated high-throughput method for quantitatively measuring a panel of shape descriptors and gene expression profiling (ddPCR) was used. Significant correlations and the correlation coefficient were calculated.
Results: Chronic IL-1β in human OA chondrocytes significantly decreased COL2A1, SOX9, and ACAN and increased IL-6 and IL-8 vs. control. At day 6 after acute inflammation, healthy bovine chondrocytes expressed significantly lower COL1A2 and higher IL-6, while chronic IL-1β significantly decreased COL2A1 and increased IL-6 and IL-8 vs. control and acutely-stimulated chondrocytes. IL-1β caused OA chondrocytes to become less wide, smaller, longer, slimmer, less round and more circular, consistent with a de-differentiated phenotype, while acute IL-1β caused healthy chondrocytes to increase in size, length, aspect ratio and become less round and solid. Chronic inflammation led to a decrease in area, length and aspect ratio, while the cells got more circular, round and solid. Significant correlations showed that both types of chondrocytes responded to IL-1β but behaved differently in their response. In OA chondrocytes, COL1A2 positively correlated with SOX9, while COL2A1 positively correlated with SOX9 and ACAN and negatively with IL-8. ACAN also negatively correlated with IL-8 expression. In OA chondrocytes, COL2A1, SOX9 and ACAN positively correlated with roundness, while SOX9 negatively correlated with the cell’s major axis. The living cell number negatively correlated with OA grade, circularity and solidity. In healthy chondrocytes, there were positive correlations between COL1A2 and COL2A1 and IL-6 and IL-8, which was in contrast to OA chondrocytes. In healthy chondrocytes, COL1A2 also negatively correlated with IL-8, while COL2A1 negatively correlated with both IL-6 and IL-8. COL2A1 positively correlated with area, major axis and aspect ratio and negatively with circularity, roundness and solidity. Opposite correlations were found with IL-6 and IL-8 gene expression and the aforementioned cell descriptors.
Conclusion: IL-1β significantly alters chondrocyte morphology and the effects can be correlated to changes in chondrogenic and inflammatory gene expression. While IL-1β led to less of a de-differentiated cell shape in healthy chondrocytes vs. OA chondrocytes, importantly, IL-1β caused early morphological effects as well as functional effects in non-diseased and previously healthy chondrocytes suggesting that IL-1β could promote the “healthier” parts of the cartilage tissue to become diseased and enhance progression towards full OA. Quantitative cell morphometry may be a useful biological fingerprint for describing chondrocyte phenotype under inflammatory attack and used to understand how inflammation or therapeutic targeting of inflammation regulates cell function and outcome."