Establishment of an imiquimod-induced psoriasis-like in vitro model in CaCl2-differentiated human keratinocytes: morphological, biochemical, and molecular approaches

Keskin S., Çakır M., Uyanikgil Y., Çakır Ş., Açıkgöz E.

16th National and 2nd International Congress of Histology and Embryology (NICHE2024), Sakarya, Türkiye, 26 - 28 Eylül 2024, ss.1

  • Yayın Türü: Bildiri / Özet Bildiri
  • Basıldığı Şehir: Sakarya
  • Basıldığı Ülke: Türkiye
  • Sayfa Sayıları: ss.1
  • Van Yüzüncü Yıl Üniversitesi Adresli: Evet

Özet

 

Introduction: Psoriasis is a chronic inflammatory skin disease involving complex immune cell and keratinocyte interactions, with no definitive treatment due to its complex pathogenesis. The development of anti-psoriatic drugs is lengthy, costly, and marked by high clinical phase failure rates, underscoring the need for in vitro models with high predictive accuracy. Existing models inadequately reflect keratinocyte properties, highlighting the potential of new models simulating keratinocyte differentiation and psoriatic inflammation to enhance drug efficacy and reduce time and cost in clinical trials.

Objective: The aim of the present study was to investigate the mechanisms of imiquimod (IMQ)-induced inflammation and cell-cell interactions in CaCl2-differentiated human keratinocytes (HaCaT) using morphological, biochemical, and molecular methods in a modified in vitro psoriasis-like model.

Material &Method: To establish an in vitro psoriasis-like model, HaCaT cells were first differentiated with 2 mM CaCl2 for 48 h and then stimulated with 25 μM IMQ for 24 h to induce psoriasis-like inflammation. The experimental groups were divided into four categories: Ca-free control, 2 mM CaCl2, 25 μM IMQ, and 2 mM CaCl2 + 25 μM IMQ. Cell morphology in all groups was evaluated using an inverted microscope. Hematoxylin and eosin (H&E) staining was used to examine cell morphology, while immunofluorescence (IF) staining for cytokeratin 17 (CK17) and proliferating cell nuclear antigen (PCNA) was performed to assess proliferation. Actin and prohibitin IF staining was conducted to analyze intercellular connections. Psoriasis-associated inflammatory cytokines were measured using ELISA, and the expression of Toll-like receptors (TLRs), NOD-like receptor protein 3 (NLRP3) inflammasome, angiogenic, hypoxia, and transforming growth factor (TGF)-β/Smads genes was analyzed using qRT-PCR.

Results: IMQ-stimulated HaCaT cells exhibited morphological features and structural changes similar to psoriatic keratinocytes. IF staining revealed that the expression levels of CK17, PCNA, actin, prohibitin, and pro-inflammatory cytokines were significantly elevated in the IMQ group compared to the control. Additionally, angiogenic, hypoxia, and TGF-β/Smads gene expressions were markedly increased in the IMQ group, primarily through the activation of the TLR and NLRP3 inflammasome pathways, compared to other groups.

Conclusion: The findings demonstrate that the IMQ-induced in vitro psoriasis-like model in CaCl2-differentiated HaCaT cells successfully replicated the inflammatory responses observed in psoriatic tissue at the microscopic, molecular, and biochemical levels in human keratinocytes. Moreover, the ability of this in vitro model to mimic psoriatic inflammation offers a practical and valuable tool for evaluating the antipsoriatic efficacy of new drug candidates.

 

Keywords: Psoriasis, CaCl2, Imiquimod, Keratinocyte, Inflammation, In Vitro Model

 

Acknowledgement: This research was conducted as part of the doctoral project numbered TDK-2022-10132 and was supported by the VAN YYU Scientific Research Projects Unit.