Fig 2: Roles of MMP12 deficiency in oral keratinocytes. (a) TER (left) or FITC-dextran concentrations (right) of primary oral mucosal keratinocytes from human donors, n = 5. (b) TER (left) or FITC-dextran concentrations (right) of HOKs co-cultured with primary oral mucosal macrophages from human donors, n = 5. (c-d) Healthy MDMs were transduced with or without sgMMP12-lentivirus for 48 h prior to 12-hour co-culture with primary oral mucosal T cells from control or EOLP participants for activation. The activated macrophages were then co-cultured with HOKs for another 12 h. Schematic exhibiting the macrophage-keratinocyte co-culture system (c); TER (left) or FITC-dextran concentrations (right) of HOKs co-cultured with activated macrophages (d), n = 5. ** P < 0.01, *** P < 0.001 vs. corresponding control group; ###P < 0.001 vs. T cell-OLP sgCtrl group. NEOLP, non-erosive oral lichen planus; EOLP, erosive oral lichen planus; M, macrophage; K, keratinocyte; H, health. Data were expressed as means ± standard deviation. One-way ANOVA was used for statistical analysis.

Fig 3: MMP12 inhibitor prevents keratinocyte barrier from destruction. HOKs were co-cultured with primary health- or EOLP-derived oral mucosal macrophages with or without MMP12 inhibitor treatment for 12 h. Three MMP12 inhibitors, MMP408 (inhibitor-1), RXP470.1 (inhibitor-2), PF-00356231 hydrochloride (MMP12 inhibitor-3) were used. (a) Schematic graphic of HOKs and macrophages co-culture. (b) MMP12 activity of primary EOLP-derived oral mucosal macrophages with or without MMP12 inhibitor treatment. Recombinant MMP12 was used for a positive control. (c-f) TER (c) or FITC-dextran concentrations (d) of HOK monolayers, fibronectin concentrations in the supernatants of HOKs (e) and fibronectin expression in the cell membrane fraction of HOKs (f) were tested. *** P < 0.001 vs. corresponding control group; ###P < 0.001 vs. OLP-Ctrl group. Ctrl, control; FN, fibronectin; H, health. Data were expressed as means ± standard deviation. All experiments were carried out 5 times. One-way ANOVA was used for statistical analysis.

Fig 4: MMP12 degrades keratinocyte-produced fibronectin. (a) Elisa showing fibronectin concentrations in the culture media of HOKs treated with different doses of recombinant MMP12 for 12 h. (b) Western blot examinations of fibronectin expression in the membrane, cytosol or nuclear fraction of HOKs treated with different doses of recombinant MMP12 for 12 h. (c) TER (left) and FITC-dextran concentrations (right) in HOKs with or without FN1 knockout. (d) TER (left) and FITC-dextran concentrations (right) in FN1-overexpressed HOKs with or without recombinant MMP12 treatment for 12 h. (e-f) Elisa showing fibronectin concentrations in the oral mucosae (left) or epithelia (right) derived from control or EOLP donors. (g) Western blot tests of fibronectin expression in the membrane, cytosol or nuclear fraction of primary keratinocytes from control or EOLP donors. (h-i) Primary oral mucosal macrophages were enriched from healthy or EOLP donors, followed by 12-hour culture. The supernatants of macrophages were then added into HOKs media at a 50% final volumetric concentration for another 12 h. Schematic illustration of HOKs with media from macrophages (h); Elisa assessments of fibronectin concentrations in the supernatants of HOKs (i). (j-k) Primary oral mucosal macrophages were enriched from healthy or EOLP donors, followed by 12-hour culture. The supernatants of macrophage (1 ml) were treated with 20 µg MMP12 antibodies coupled to Pierce Protein A/G Magnetic Beads (ThermoFisher Scientific) for immunodepleting of MMP12, followed by mixture with HOKs media (v/v, 50%/50%) for another 12 h. Schematic illustration of HOKs with media from macrophages (j); western blot showing MMP12 levels in the macrophage media with or without MMP12 antibodies treatment (k); Elisa assessments of fibronectin concentrations in the supernatants of HOKs (l). *** P < 0.001 vs. corresponding control group; ###P < 0.001 vs. MMP or IgG group. NEOLP, non-erosive oral lichen planus; EOLP, erosive oral lichen planus; H, health; Ctrl, control; FN, fibronectin. Data were expressed as means ± standard deviation. All experiments were carried out 5 times. One-way ANOVA (a, d, e, f, l) and Student’s t test (c, i) were used for statistical analysis.

Fig 5: MMP12 expression in the human samples. (a) Real-time PCR analyses of MMP12 mRNA levels in oral mucosal macrophages from human donors, n = 10. (b) Western blot determinations of MMP12 protein levels in oral mucosal macrophages from human donors (left) and quantitative analyses (right), n = 5. (c-d) Saliva (c) or Serum (d) MMP12 concentrations of healthy or OLP donors measured by Elisa, n = 10. *** P < 0.001 vs. corresponding control group. NEOLP, non-erosive oral lichen planus; EOLP, erosive oral lichen planus. Data were expressed as means ± standard deviation. One-way ANOVA was used for statistical analysis.