PT  - JOURNAL ARTICLE
AU  - Firl, Christina
AU  - Clancy, Robert
AU  - Buyon, Jill
TI  - 1302 Fetal cardiac and neonatal dermal fibroblast phenotypes and cytokine reactivity following macrophage crosstalk in the pathogenesis of neonatal lupus
AID  - 10.1136/lupus-2023-lupus21century.90
DP  - 2024 May 01
TA  - Lupus Science & Medicine
PG  - A106--A108
VI  - 11
IP  - Suppl 2
4099  - http://lupus.bmj.com/content/11/Suppl_2/A106.short
4100  - http://lupus.bmj.com/content/11/Suppl_2/A106.full
SO  - Lupus Sci Med2024 May 01; 11
AB  - Background/Purpose Driven by fetal exposure to maternal anti-SSA/Ro autoantibodies, neonatal lupus (NL) typically involves in utero permanent cardiac and/or transient post-natal cutaneous manifestations. Considering the contribution of fibroblasts to NL pathogenesis, this explored similarities and differences between fetal cardiac fibroblasts and neonatal dermal fibroblasts in the context of maternal anti-SSA/Ro- propagated injury and cytokine influence.Methods In an in vitro model of anti-SSA/Ro-initiated scarring, human cardiac fetal fibroblasts or neonatal dermal fibroblasts were co-cultured with supernatants (sup) of THP-1 macrophages transfected with DOTAP or hY3 (noncoding ssRNA binding Ro60 and TLR 7/8 agonist), IFNα, or activated TGF-β1 for 24–72 hours. Relative fibroblast heterogeneity was confirmed via expression of S100 Calcium-Binding Protein A4 (S100A4) and/or myofibroblast marker alpha-Smooth Muscle Actin (α-SMA). Functional fibroblast readouts—cell migration and wound closure—were assessed by a ‘scratch’ assay, wherein the gap made by a scratch across a surface of confluent cells was recorded at 0 and 24 hours.Results Immunofluorescence after 24, 48, and 72 hours of treatment (DOTAP sup, hY3 sup, or IFNα or TGF-β1) established a time-course of S100A4 and α-SMA expression in cardiac and dermal fibroblasts. 24 hours following addition of hY3 sup or IFNα, cardiac fibroblasts (figure 1) and dermal fibroblasts (figure 2) almost exclusively expressed S100A4. At 48 hours, both were increasingly α-SMA-positive, primarily through the emergence of a dual-positive (α-SMA+ S100A4+) subpopulation. At 72 hours, α-SMA surpassed S100A4 as the predominant marker expressed in cardiac fibroblasts cultured with hY3 sup or IFNα. By contrast, in dermal fibroblasts, α-SMA expression declined between 48 and 72 hours, and S100A4 expression all but disappeared by 72 hours post- treatment with hY3 sup or IFNα. The addition of neutralizing antibodies to IFNα inhibited the expression of S100A4 and α-SMA in cardiac and dermal fibroblasts exposed to hY3 sup, validating type I Interferon as the dominant cytokine. TGF-β1 consistently produced α-SMA+ myofibroblasts in cardiac and dermal fibroblasts across all time points, without inducing S100A4. Turning to function, addition of hY3 sup or IFNα resulted in increased (%) wound closure after 24 hours in cardiac (83%, 86% respectively) and dermal (81%, 77% respectively) fibroblasts (figure 3). Co-treatment with an S100A4 inhibitor, Niclosamide, mitigated the wound closure in fibroblasts cultured with hY3 sup or IFNα after 24 hours for cardiac (24%, 27% respectively) and dermal fibroblasts (22%, 28% respectively).Conclusion These results suggest the initial macrophage-fibroblast injury in NL, mediated by anti-SSA/Ro, is driven by secreted type I IFN with subsequent S100A4 expression in cardiac and dermal fibroblasts, and associated increase in cell migration. While α-SMA+ scarring myofibroblasts increase in cardiac fibroblasts over time, dermal fibroblasts appeared to recover from this scarring phenotype perhaps contributing to the more transient and non-fibrotic phenotype of cutaneous NL. These results provide insights into the pathogenesis of different manifestations of NL and potential new targets for therapy.Abstract 1302 Figure 1 Human fetal cardiac fibroblasts treated with DOTAP mac sup (1:5 dilution), hY3 mac sup (1:5), IFNα (2000U/mL), or activated TGF-β1 (2 ng/mL) for 24–72 hours. N=3Abstract 1302 Figure 2 Human neonatal dermal fibroblasts treated with DOTAP mac sup (1:5 dilution), hY3 mac sup (1:5), IFNα (2000U/mL), or TGF-β1 (2 ng/mL) for 24–72 hours. N=2Abstract 1302 Figure 3 Human neonatal dermal fibroblasts (A) and fetal cardiac fibroblasts (B) were treated with DOTAP mac sup (1:5 dilution), hY3 mac sup (1:5), TGF-β1 (2 ng/mL), IFNα (2000 U/mL) separately and in combination with Niclosamide (150 nM) for 24 hours. Scratch was made at t=0 and wound closure (%area) was assessed at t=24 hours (N=2).