Background/Purpose: Localized scleroderma (LS) is an inflammation-driven fibrotic disease, presenting clinically as
linear bands, patches, or regions of skin and underlying connective tissues with initial thickening of skin followed by dermal atrophy and pigmentation changes.  LS can lead to long-term complications for pediatric patients such as joint contractures and limb-length discrepancies. Fibrosis in scleroderma arises from activated fibroblasts producing excess collagen. Previous studies have shown increased interleukin-6 (IL-6) production in scleroderma patient tissues, which activates fibroblasts via the JAK/STAT3 signaling pathway. Interleukin-12 (IL-12) has also been shown to be elevated in some patients, and signals through the JAK/STAT4 pathway.  IL-12 has been associated with extracellular matrix regulation and degradation. We hypothesized that increased IL-6 and IL-12 signaling will increase fibroblast activity in vitro in scratch assays of healthy control primary fibroblasts.

Methods: Fibroblast cultures from five healthy control 4mm skin biopsies and one affected (LS) biopsy were stimulated with vehicle, IL-6, IL-12, and IL-6 + IL-12 (3 biological replicates per culture and stimulation condition n=72). Scratch assays were performed by inducing scratches with a p200 pipette tip and observed at regular intervals for 48 hours. Images were obtained at 3 locations for each scratch (n=216) at 4 timepoints (n=864 total measurements). Scratch width was measured digitally, and percent gap closure and rate of gap closure were calculated for each condition.

Results: Gap closure time was reduced in all three treatment groups (IL-6, IL-12, IL-6 + IL-12) compared to control. The IL-6 group showed the highest gap closure rate (mean: 4.31%/hr) which was significant (p< 0.05) compared with control (mean: 3.14%/hr). IL-6 + IL-12 group also showed a significant intermediate rate (4.15%/hr), and the IL-12 group (3.69%/hr) showed the smallest increase. All treated groups showed increased stimulation of fibroblast wound healing activity, but there was no additive effect or difference in stimulation of LS primary fibroblasts over healthy control.

Conclusion: IL-6 had the most significant effect on fibroblast activity, while IL-12 suggested a less robust response. IL-12 stimulation conditions were selected based on prior literature and may need further optimization for stimulation concentration, although it is notable that IL-6 did not provide an overriding effect for the lower IL-12 stimulation, which was consistent across cell lines and replicates. 

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/interleukin-6-and-interleukin-12-stimulation-increases-fibroblast-migration-in-localized-scleroderma-and-healthy-control-primary-fibroblasts/