IFN-γ enhances cell-mediated cytotoxicity against keratinocytes via JAK2/STAT1 in lichen planus

Abstract

Lichen planus (LP) is a chronic debilitating inflammatory disease of unknown etiology affecting the skin, nails, and mucosa with no current FDA-approved treatments. It is histologically characterized by dense infiltration of T cells and epidermal keratinocyte apoptosis. Using global transcriptomic profiling of patient skin samples, we demonstrate that LP is characterized by a type II interferon (IFN) inflammatory response. The type II IFN, IFN-γ, is demonstrated to prime keratinocytes and increase their susceptibility to CD8⁺ T cell-mediated cytotoxic responses through MHC class I induction in a coculture model. We show that this process is dependent on Janus kinase 2 (JAK2) and signal transducer and activator of transcription 1 (STAT1), but not JAK1 or STAT2 signaling. Last, using drug prediction algorithms, we identify JAK inhibitors as promising therapeutic agents in LP and demonstrate that the JAK1/2 inhibitor baricitinib fully protects keratinocytes against cell-mediated cytotoxic responses in vitro. In summary, this work elucidates the role and mechanisms of IFN-γ in LP pathogenesis and provides evidence for the therapeutic use of JAK inhibitors to limit cell-mediated cytotoxicity in patients with LP.

Fig. 1.. Transcriptomic profiling of LP lesions reflects an IFN-γ-dominant inflammation

(A) Principal components analysis of microarray data from LP (n=20), HLP (n=17) and healthy controls (n=24). (B) The Venn diagram displays the intersection (upper panel) and correlation (lower panel) of differentially expressed genes (FDR<0.1, FC>1.5 or <−1.5) across the LP and HLP transcriptomes. The number of up-regulated and down-regulated genes for each data set is reported in parentheses. (C) Graphs demonstrate the most highly enriched Gene Ontology categories in the transcriptomes of LP and HLP. (D, E) Expression level of type II IFN, IFNG, and IFNGR (D), and the IFN-response genes MX1, MX2, OAS1 and OASL (E) in our microarray data. (F) Further analysis and classification of differentially expressed genes in LP and HLP skin lesions to cytokine-induced genes. (G) QRT-PCR was employed to detect the IFNG, MX1, IL17A, and IL22 expression level in skin lesions of LP (n=22) and HLP (n=16) and normal controls (NC, n=23). One-way ANOVA. Data are presented as the mean ± SEM of measurements obtained in each sample. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Fig. 2.. IFN-γ increases keratinocyte susceptibility to cell-mediated cytotoxicity

(A) Keratinocytes from one donor were co-cultured with CD3/CD28 microbeads-activated PBMCs from a second donor (n=2) and cell death was evaluated by Annexin-V PI staining. The representative flow cytometry data (left panel) and statistical analysis (right panel) are shown.(B) TUNEL staining and statistical analysis of TUNEL-positive cells was used to detect keratinocyte cell death. DAPI was used for nuclear staining (scale bar, 100μm). (C) PBMCs were primed with IFN-γ and then treated with CD3/CD28 microbeads, and then added to unstimulated keratinocyte cultures. (D) CD3/CD28 microbeads-activated PBMCs were pre-incubated with anti-CD4, anti-CD8, or anti-Nkp44 blocking antibody for 1 h, then added to in vitro co-culture model, and keratinocyte death was evaluated by Annexin-V PI staining. Data were analyzed using one-way ANOVA followed by Dunnett’s posttest. Data are presented as the mean ± SD of measurements obtained in triplicate or quadruplicate experiments. *P < 0.05, **P < 0.01, ****p < 0.0001.

Fig. 3.. Cytotoxic responses to IFN-γ-primed keratinocytes are MHC class I dependent.

(A) The mRNA expression level of all MHC molecules from microarray data from LP (n=20), HLP (n=17), and healthy control skin (n=24). (B) Representative immunofluorescence staining of MHC class I and MHC class II in LP/HLP lesions (n=3) and controls (n=3). DAPI was used to show nuclear staining (Scale bar, 100μm). (C) QRT-PCR showed mRNA expression of MHC I and II molecules in keratinocytes stimulated with IFN-γ (10ng/ml). (D) IFN-γ-primed keratinocyte were pre-incubated with anti-MHC class I and II monoclonal antibody for 1 h, and then co-cultured with CD3/CD28 activated PBMCs. Statistical analysis was done using one-way ANOVA followed by Dunnett’s posttest. Data are presented as the mean ± SD of measurements obtained in triplicate or quadruplicate experiments. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Fig. 4.. IFN-γ signals through JAK2/STAT1 signaling in keratinocytes to induce MHC class I expression

(A) Induced modules network analysis of the differentially expressed genes shared by LP and HLP. Node size correlates with the number of connected nodes and edges. Nodes with 5 connections are marked larger. (B) Expression level of STAT1-6 in LP (n=20), HLP (n=17) and healthy control skin (n=24) in our microarray data. (C) Representative immunofluorescence staining of STAT1 and phosphorylated (p)-STAT1 in LP/HLP skin lesions and controls (four samples were stained and analyzed per group). DAPI was used for nuclear staining (scale bar, 100μm). (D) Keratinocytes were stimulated with IFN-γ (10 ng/ml) for indicated time. Western blot for JAK1, JAK2, p-JAK1, p-JAK2, STAT1, STAT2, p-STAT1, and p-STAT2 are shown. (E) mRNA expression of MHC I molecules in JAK1, JAK2, STAT1, and STAT2 KO cells after 24hr IFN-γ (10 ng/ml) stimulation. One-way ANOVA. Data are presented as the mean ± SD of measurements obtained in triplicate experiments. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Fig. 5.. Targeting JAK signaling protects keratinocytes from cell-mediated cytotoxicity

(A) JAK2 and STAT1 KO cells were primed with IFN-γ and co-cultured with CD3/CD28 activated PBMCs. Keratinocyte cell-death was evaluated for Annexin-V positivity by flow cytometry. (B) The representative flow cytometry data and analysis of keratinocyte cell-death in the co-culture model with or without baricitinib. (C) The mRNA expression of MHC I molecules in IFN-γ-induced keratinocytes with or without baricitinib for 24 h. One-way ANOVA. Data are presented as the mean ± SD of measurements obtained in triplicate or quadruplicate experiments. *P < 0.05, ***P < 0.001, ****P < 0.0001.

Fig. 6.. High IFN-γ responses characterizes diseases with prominent epidermal cell death.

(A) Representative TUNEL staining showing cell death in skin lesions of LP, CLE, psoriasis, and normal controls. DAPI was used for nuclear staining (scale bar, 100μm). (B) The mRNA expression of IFNG and MHC I molecules in chronic plaque psoriasis (PV) (n=12 or 7), LP (n=38 or 10), CLE (n=21 or 12) skin lesions, and healthy control skin (NC, n=11 or 15). One-way ANOVA. Data are presented as the mean ± SD of measurements obtained in each sample.(c) Representative immunofluorescence staining of MHC I and MHC II, STAT1 and p-STAT1 in PV and CLE skin lesions and normal controls. These staining were performed on more than 3 patient samples for each group. DAPI was used to highlight nuclear staining (scale bar, 100μm).