Apolipoprotein E and Periostin Are Potential Biomarkers of Nasal Mucosal Inflammation. A Parallel Approach of In Vitro and In Vivo Secretomes

Abstract

No previously suggested biomarkers of nasal mucosal inflammation have been practically applied in clinical fields, and nasal epithelium-derived secreted proteins as biomarkers have not specifically been investigated. The goal of this study was to identify secreted proteins that dynamically change during the differentiation from basal cells to fully differentiated cells and examine whether nasal epithelium-derived proteins can be used as biomarkers of nasal mucosal inflammation, such as chronic rhinosinusitis. To achieve this goal, we analyzed two secretomes using the isobaric tag for relative and absolute quantification technique. From in vitro secretomes, we identified the proteins altered in apical secretions of primary human nasal epithelial cells according to the degree of differentiation; from in vivo secretomes, we identified the increased proteins in nasal lavage fluids obtained from patients 2 weeks after endoscopic sinus surgery for chronic sinusitis. We then used a parallel approach to identify specific biomarkers of nasal mucosal inflammation; first, we selected apolipoprotein E as a nasal epithelial cell-derived biomarker through screening proteins that were upregulated in both in vitro and in vivo secretomes, and verified highly secreted apolipoprotein E in nasal lavage fluids of the patients by Western blotting. Next, we selected periostin as an inflammatory mediator-inducible biomarker from in vivo secretomes, the secretion of which was not induced under in vitro culture conditions. We demonstrated that those two nasal epithelium-derived proteins are possible biomarkers of nasal mucosal inflammation.

Keywords: air–liquid interface culture; mucosal secretions; nasal epithelium; secreted proteins; secretome.

Figure 1.

Changes in primary human nasal epithelial cell (HNEC) composition during differentiation. (A) Scheme of the HNEC differentiation during air–liquid interface (ALI) culture (e.g., ALI culture for 15 d [ALI 15D]). (B) HNECs in different stages of differentiation were analyzed by immunofluorescence staining of cytospin slides with antibodies against specific marker proteins; ciliated cells are shown as acetylated-α-tubulin (Ac-α-tubulin)–positive cells (yellow); secretory cells are shown as MUC5AC-positive cells (green); and basal cells are shown as p63-positive cells (red). (C) Percentages of ciliated, secretory, intermediate (subtracted the ciliated, secretory, and basal cell portions from total cell numbers), and basal cells during differentiation of HNECs are presented as bar graphs. Results are the means ± SEM; n = 4; ***P < 0.001 versus ALI 2D by one-way ANOVA with Bonferroni post hoc correction. MUC5AC = mucin 5AC, oligomeric mucus/gel-forming.

Figure 2.

Proteomic characterization of secretomes from HNECs using isobaric tag for relative and absolute quantification (iTRAQ). (A) A heat map showing the relative abundance of 3,506 proteins identified in the iTRAQ-ALI-4Ds dataset. The color key indicates the relative abundance of each protein (−3 to 3) across eight samples from the iTRAQ-ALI-4Ds dataset. (B–D) Volcano plots demonstrating fold changes in protein abundance between ALI 2D and ALI 6D (B), ALI 2D and ALI 10D (C), and ALI 2D and ALI 15D (D). The x-axis represents the log2 ratio, and the y-axis represents significant differences (−log10 of P value, n = 2). A total of 269 proteins were changed by at least twofold (P < 0.01, n = 2): ALI 2D versus ALI 6D (37 proteins), ALI 2D versus ALI 10D (112 proteins), and ALI 2D versus ALI 15D (120 proteins). (E–G) Temporal profiles of eight mucin family members. Tethered mucins MUC1, MUC4, and MUC16 (E); gel-forming mucins MUC5A and MUC5B (F); minor mucins MUC18, MUC20, and MUC21 (G). Numbers in parenthesis are the total numbers of identified peptide sequences (Σ# PSMs) for the protein. **P < 0.01 by one-way ANOVA with Bonferroni post hoc correction.

Figure 3.

Characterization of proteins in HNEC secretomes. (A) Significant biological pathways enriched among 3,506 proteins identified by iTRAQ are represented as bar graphs. (B) Pie graph showing cellular localization of the HNEC secretomes classified by the top 10 cellular compartment in functional annotation with gene ontology analysis (GO). GnRH = gonadotropin-releasing hormone; HIF-1 = hypoxia-inducible factor 1; SNARE = SNAP receptor; TCA cycle = tricarboxylic acid cycle; VEGF = vascular endothelial growth factor. ErbB = erythroblastic leukemia viral oncogene homolog.

Figure 4.

Characterization of secretomes from patients after endoscopic sinus surgery. (A) Heat map showing the relative abundance of 660 proteins identified in the iTRAQ-H9 dataset. The color key indicates the relative abundance of each protein (−3 to 3) across nine samples from the iTRAQ-H9 dataset. (B) Venn diagram showing overlapping proteins (492) between HNEC secretomes (in vitro) and patient secretomes (in vivo). (C) Significant biological pathways enriched among 660 secretome proteins are represented as bar graphs. ECM = extracellular matrix.

Figure 5.

Comparison between in vitro and in vivo secretomes. (A) Heat map showing four clusters of in vitro secretome proteins based on their temporal profiles: cluster A (84 proteins secreted highly at ALI 2D); cluster B (246 proteins at ALI 6D); cluster C (664 proteins at ALI 10D); and cluster D (76 proteins at ALI 15D). (B) Heat map showing 86 in vivo secretome proteins with high abundance in patient group (FC > 2, P < 0.05). (C) Temporal profiles of three apolipoproteins (APOs): APOE; clusterin (CLUS; also known as APOJ); and prolow-density lipoprotein receptor–related protein 1 (LRP1, also known as ApoE receptor). (D) Relative quantities of APOs and periostin (POSTN) in in vivo secretome samples from nasal lavage (NAL) fluid of patients and that of control subjects are presented as bar graphs. Numbers in parenthesis are the Σ# PSMs for the protein. Results are the means ± SEM; *P < 0.05, **P < 0.01, and ***P < 0.001 versus control group. (E) Amount of ApoE in in vitro secretome samples isolated from HNECs cultures during differentiation were analyzed by Western blotting (upper panel). Gels were stained with Coomassie blue (lower panel). (F) Amount of POSTN and ApoE in NAL fluid secretomes was analyzed by Western blotting (upper panel). Gels were stained with Coomassie blue (lower panel). FC = fold change.

Figure 6.

Expression of APOE and POSTN in the nasal mucosa. Nasal mucosal sections from a patient with allergic rhinitis (AR) stained with antibodies against APOE (A, middle panel) or POSTN (B, middle panel), or without primary antibodies (negative control, top panels of A and B). Arrowheads indicate the APOE immunoreactive signals. Nasal mucosal sections from a patient without AR (Non AR, bottom panels) stained with antibodies against APOE (A, bottom panel) or POSTN (B, bottom panel) as control. Scale bars: 50 μm.