Transcriptional signature primes human oral mucosa for rapid wound healing

Abstract

Oral mucosal wound healing has long been regarded as an ideal system of wound resolution. However, the intrinsic characteristics that mediate optimal healing at mucosal surfaces are poorly understood, particularly in humans. We present a unique comparative analysis between human oral and cutaneous wound healing using paired and sequential biopsies during the repair process. Using molecular profiling, we determined that wound-activated transcriptional networks are present at basal state in the oral mucosa, priming the epithelium for wound repair. We show that oral mucosal wound-related networks control epithelial cell differentiation and regulate inflammatory responses, highlighting fundamental global mechanisms of repair and inflammatory responses in humans. The paired comparative analysis allowed for the identification of differentially expressed SOX2 (sex-determining region Y-box 2) and PITX1 (paired-like homeodomain 1) transcriptional regulators in oral versus skin keratinocytes, conferring a unique identity to oral keratinocytes. We show that SOX2 and PITX1 transcriptional function has the potential to reprogram skin keratinocytes to increase cell migration and improve wound resolution in vivo. Our data provide insights into therapeutic targeting of chronic and nonhealing wounds based on greater understanding of the biology of healing in human mucosal and cutaneous environments.

Copyright © 2018 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.

Figures

Figure 1. Comparison of paired oral and skin wounds in human subjects.

(A) Schematic of spatiotemporal human biopsy sample collection; Clinicaltrials.gov #NCT01078467. Baseline biopsies were performed to create paired identical wounds in the oral mucosa and skin (day 1). Follow-up biopsies of the wound area(s) were collected at day 3 and day 6 of healing in two different groups. (B) Table showing the time-course of the clinical study and biopsy sampling, and representative H&E pictures of longitudinal sections of biopsies taken at day 1. Thirty healthy subjects were randomized in three groups (with 10 subjects on each group). SM: smooth muscle. See Methods section for more information. (C) Representative pictures and healing time course of oral wounds and skin wounds in group 1 (after 3 mm primary biopsy), and group 2 (after 5 mm secondary biopsy). Markings in millimeters. Error bars represent SDs. Number of samples for each group as follows: 3 mm wounds, day 1 n=29, day 3 n=30, day 6 n=20, day 9 n=9, day 13 n=9, day 15 n=9; 5mm wounds, day 1 n=11, day 3 n=20, day 9 n=21, day 13 n=21, day 15 n=21. (D) Graphs show healing rate of 3 mm wounds in group 1 and 5 mm wounds in group 2 as well as the contraction percentage immediately after 3 mm biopsy in group 1 and 5 mm biopsy in group 2. n.s.: not significant, *p<0.05. Error bars represent SDs. (E) Representative H&E pictures of oral and skin wounds at day 3 and 6. At day 3 there is almost complete epithelization of the wound, with migratory tongue growing on top of blood clot. In the skin, migratory epithelial tongue is growing under the scab that covers the wound. At day 6, oral wounds are completely resolved, while skin wounds are still undergoing re-epithelialization. Magnification of dotted box is shown on the right. In magnification, epithelium is marked with a dotted line.

Figure 2. Wound-activated transcriptional networks are present in the unwounded oral mucosa.

(A) Schematic representation of biopsy site in the mucosa of the cheek and posterior axillary region of the arm (left) and unsupervised clustering analysis of RNA-Seq gene expression data of the 24 paired samples at Day 1, 3 and 6. O: oral, S: skin. Numbers indicate matching subject. Paired oral and skin samples were chosen randomly from four subjects for each day (24 total samples from 12 individual subjects) and were a mix of males and females. (B) CIRCOS plot of the differential gene expression during wound healing (ANOVA). No significant differences were found on Oral Day 6 versus Day 1 (Day 6/1). Ribbon connectors indicate the same genes present in different datasets. Number of genes with differential expression in each comparison: Oral Day 3 versus Day 1 (Day 3/1): 410 genes, Skin Day 3 versus Day 1 (Day 3/1): 1473 genes, Skin Day 6 versus Day 1 (Day 6/1): 1836 genes. See fig. S1a for explanation on CIRCOS plot. (C) Volcano plot indicating differential gene expression between unwounded (Day 1) oral mucosa and skin. Magnification on right panel, highlights the some of the most significantly upregulated genes in the oral mucosa over the skin. (D) IPA analysis results showing Diseases and Functions terms found in upregulated genes in the oral mucosa over the skin. (E) Unsupervised hierarchical clustering using a psoriasis gene signature with the gene expression of oral mucosa and skin data at baseline (Day 1) and during wound healing (Day 3 and 6).

Figure 3. Oral keratinocytes show reduced differentiation during wound healing.

(A) CIRCOS plot summarizing cross-reference of transcripts upregulated during skin wound healing (Skin D3/D1, Skin D6/D1, D=day) with those upregulated in the oral mucosa with respect to the skin at basal conditions (Oral D1/Skin D1). Black line indicates genes upregulated in the unwounded oral mucosa that are upregulated during skin wound healing (250 unique genes; see Table S2). Right panel, graph indicates GO biological process terms enriched in Oral D1/Skin D1 dataset. Ribbon connectors indicate that the same genes are present in different datasets. Oral D1/Skin D1: 760 genes, Skin D3/D1: 971 genes, Skin D3/D1: 1089 genes. (B) Relative mRNA expression levels of keratinization and epidermal cell differentiation markers throughout the wound healing process. (C) Representative pictures of unwounded (D1) and wounded (D3 and D6) oral mucosa and skin stained to show expression of the basal marker keratin 5 (K5, red), and differentiation markers keratin 4 (K4) for the oral mucosa and keratin 10 (K10) for the skin (green).(D) Representative pictures of unwounded (D1) and wounded (D3) oral mucosa and skin stained to show expression of the basal marker cytokeratin 5 (K5, red), and activated epithelium keratin 6 (K6, green). (E) Representative pictures of unwounded (D1) and wounded (D3 and D6) oral mucosa and skin stained to show expression of the basal marker keratin 5 (K5, green), and proliferation marker PCNA (red). Quantification of number of cells positive for the proliferation marker PCNA per area (mm2). **p<0.01 oral vs skin day 6, no asterisk means not statistically different between oral and skin. Error bars represent SDs of three independent samples. Magnification of the dotted box is shown on the right of each picture. For D1 magnification shows the basal, unwounded expression of corresponding marker. For D3 and D6 magnification shows the migratory tongue or wound area, A, or an adjacent epithelial area to the wound, B.

Figure 4. Inflammatory pathways are more active and sustained in skin wounds that in oral wounds.

(A) IPA analysis results showing diseases and functions terms found in differentially regulated genes during the wound healing process included terms related to inflammatory processes. (B) CIRCOS plot showing the genes exclusively upregulated during skin wound healing (black line) and GO biological process terms enriched in this dataset. Ribbon connectors indicate that the same genes are present in different datasets. Oral Day 3/Day 1: 276 genes, Skin Day 3/Day 1: 971 genes. D=day. (C) Relative mRNA expression levels of interleukins, chemokines and other inflammatory regulators during wound healing. (D) Representative pictures of recruitment of immune cells during the wound healing process in the oral mucosa and the skin. Bottom panels show quantification of recruitment of specific immune cell types during the wound healing process in the oral mucosa and the skin. * p<0.05, ** p<0.01, no asterisk means not statistically different, comparisons between day 3 and day 6 vs day 1 oral or skin respectively. Error bars represent SDs of three independent samples. (E) Representative images of unwounded (D1) and wounded (D3) oral mucosa and skin stained to detect expression of the basal marker keratin 5 (K5, red), and the immune-modulators SLPI (green) and AnnexinA1 (ANXA1, magenta). Magnification of dotted box is shown on the right of each picture.

Figure 5. Transcriptional networks in oral keratinocytes contributing to rapid wound resolution.

(A) Relative mRNA expression levels in the wound healing dataset of genes consistently upregulated in oral mucosa and oral keratinocytes, presented by fold change (FC) of the unwounded oral mucosa with respect to the unwounded skin (O1/S1). (B) Protein levels of PAX9, PITX2, PITX1 and SOX2 in primary cultures of human oral (NOK) and skin (NHEK) keratinocytes. (C) Representative pictures of unwounded (D1) and wounded (D3 and D6) oral mucosa and skin stained to show expression of the basal marker keratin 5 (K5, red), and the indicated transcription factor (green). Magnification of the dotted box is shown on the right of each picture. For D1 magnification shows the basal, unwounded expression of corresponding marker. For D3 and D6 magnification shows the migratory tongue or wound area, A, or an adjacent epithelial area to the wound, B.

Figure 6. Knockdown of oral signature genes in primary oral keratinocytes.

(A) Protein level of SOX2, PITX1 and total protein (RPS14) after transfection with respective siRNAs in NOK cells. (B) IPA analysis of RNA-seq data from NOK cells treated with siRNAs for SOX2 and PITX1: Diseases and functions terms related to migration and cell movement found in differentially regulated genes. *p≤0.05 (C) Relative cell migration length of NOK cells transduced with siRNA for SOX2, PITX1 and siControl. Values were determined by 3 microscopic fields in n=3 per group. *p < 0.05 siSOX2 and siPITX1 versus control. Error bars represent SDs of three independent experiments.

Figure 7. Overexpression of SOX2 and PITX1 in primary skin keratinocytes.

(A) Representative pictures of NHEK cells transduced with indicated adenoviruses and stained to show overexpression of corresponding proteins. No expression of PITX1 or SOX2 was observed in non-transduced cells (not shown). Bar= 20μm. (B) GO biological process terms enriched in datasets of genes differentially regulated by PITX1 and SOX2 overexpression in NHEK cells. (C) Fold change of the expression levels of genes related to differentiation and response to biotic stimulus in NHEK cells transduced with PITX1 and SOX2, presented as log2 fold change (log FC) over GFP expression. (D) Migrating NHEK cells transduced with SOX2, PITX1 and GFP (control) by adenoviral delivery. Images were taken at 0 and 24hs after removal of silicone insert. Values were determined by counting the number of migrating cells at 24hs in 6 microscopic fields in n=3 per group. **p < 0.01, *p < 0.05 adSOX2 and adPITX1 versus control. Error bars represent SDs of three independent experiments.

Figure 8. Conditional overexpression of SOX2 contributed to the promotion of cutaneous wound healing.

(A) Schematic representation of the experimental design used for K14CreERTM/LSL-SOX2 mice. Mice are treated with vehicle or tamoxifen five consecutive days by topical application on dorsal skin (Day 1-Day 5). The wounds were created as a 6 mm full-thickness excisional dorsal skin wound by biopsy punch (Day 6). (B) Representative images of unwounded skin stained to show expression of SOX2 (red), and the basal marker keratin 5 (K5, green) in K14CreERTM/LSL-SOX2 mice treated with ethanol as vehicle or tamoxifen. Bar = 50 um. (C) Photographs of the wound areas after topical treatment with vehicle or tamoxifen in K14CreERTM/SOX2 mice at 1, 3, 5, 7, and 9 days after wounding. Bar=1mm. (D) Percent wound area at each time point relative to the original wound area in K14CreERTM/LSL-SOX2 mice treated with vehicle or tamoxifen. Quantification of the wound areas in n = 7 wounds per groups was performed using Image J software. *p < 0.05, **p < 0.01 tamoxifen versus vehicle at each day. Error bars represent SDs of seven wounds. (E) Representative H&E-stained section Day 1(un-wounded) and Day5 (during wound healing, wound edges including epithelial tongue). Skin sections were from K14CreERTM/LSL-SOX2 mice after treatment with tamoxifen or vehicle.