Salivary ZG16B expression loss follows exocrine gland dysfunction related to oral chronic graft-versus-host disease

Abstract

Chronic graft-versus-host disease (cGVHD) targets include the oral mucosa and salivary glands after allogeneic hematopoietic stem cell transplant (HSCT). Without incisional biopsy, no diagnostic test exists to confirm oral cGVHD. Consequently, therapy is often withheld until severe manifestations develop. This proteomic study examined saliva and human salivary gland for a biomarker profile at first onset of oral cGVHD prior to initiation of topical steroid therapy. Whole saliva collected at onset of biopsy-proven oral GVHD was assessed using liquid chromatography-coupled tandem mass spectrometry with identification of 569 proteins, of which 77 significantly changed in abundance. ZG16B, a secretory lectin protein, was reduced 2-fold in oral cGVHD saliva (p <0.05), and significantly decreased in salivary gland secretory cells affected by cGVHD. Single-cell RNA-seq analysis of healthy MSG localized ZG16B expression to two discrete acinar cell populations. Reduced ZG16B expression may indicate specific cGVHD activity and possibly general salivary gland dysfunction.

Keywords: Pathophysiology; Proteomics; Transcriptomics.

Graphical abstract

Figure 1

Shotgun LC-MS/MS workflow and analysis Proteins extracted from eight saliva samples from two HVs, three post-transplanted patients without oral GVHD, and three post-transplanted patients with oral GVHD were proteolytically digested and labeled with isobaric iTRAQ tags. Peptides are then pooled at equal concentrations, fractioned using HPLC, and analyzed by mass spectrometer. Database search and bioinformatics procedures were used for protein identification, quantification, and selection of putative candidate biomarkers. Validation was performed by either WB analysis or immunofluorescence of two different cohorts of patients and five additional HVs.

Figure 2

MMP9, PIP, a1ACT, and Ezrin protein decreases in saliva from oral cGVHD patients (A–F) (A) Quantitative WB analysis of MMP9 (predicted MW of ∼92 kDa), ezrin (predicted MW of ∼81 kDa), PIP (predicted MW of ∼17 kDa), and calmodulin (predicted MW of ∼17 kDa) in the saliva samples from post-HSCT patients with (n = 12) or without (n = 12) oral cGVHD of cohort 1 was undertaken in 2 blots. Equal protein loading (10 μg/lane) and consistent electrotransfer of samples for the WB were confirmed by staining the entire nitrocellulose membrane using Revert Total Protein (TP) Stain immediately prior to blotting as shown by the representative proteins (range 50–90 kDa) visualized by the membrane stain in the bottom panel of (A). Full image for Revert staining of all membranes is shown in Figure S4. Densitometric analysis of (B) MMP9, (C) ezrin, and both glycosylated and nonglycosylated forms of (D) PIP and (E) calmodulin included the whole window shown and indicated no statistically significant differences between groups (p >0.05) calculated by Mann–Whitney test (unpaired, 2-tailed). Subgroup analysis of calmodulin expression (F) shows sex- and GVHD-related differences in expression that were not statistically significant (Kruskal–Wallis test with Dunn's correction for multiple comparisons). All values were normalized by total protein by lane and are plotted as mean ± SEM. Each datapoint represents one patient. See also Figures S1 and S5.

Figure 3

Decreased ZG16B expression detected in the saliva of post-HSCT patients with oral cGVHD compared with non-affected post-HSCT patients (A) WB for ZG16B (predicted MW of ∼22.7 kDa) in saliva samples from post-HSCT patients with (n = 12) or without (n = 12) oral cGVHD in 2 blots (top panel). TP staining (Revert, LI-COR) was used as normalization control as shown by the representative proteins (range 50–90 kDa) visualized in the bottom panel. Image for Revert staining of all membranes is shown in Figure S4 (B) Quantification of salivary ZG16B protein was performed in the whole area depicted in the blot. Values are plotted as mean ± SEM. Group-wise differences were calculated using Mann–Whitney test, unpaired, 2-tailed significance set at p≤0.05. (C) Quantitative data comparing level of ZG16B between male and female and patients receiving or not TBI in the conditioning regimen. Each square represents 1 patient. Bars indicate mean ± SD. There were no statistical differences between groups tested at a 95% confidence level. See also Figures S1 and S5.

Figure 4

Single-cell RNA-seq and Immunofluorescence analysis of HV's MSG confirms RNA and protein expression of ZG16B in serous (S-) and seromucous (SM-) acinar cells (A) Left, Experimental workflow of 10x single-cell RNAseq analysis of normal MSG. After dissociation, cells were captured in microfluidic oil droplets, lysed, sequenced, and analyzed. (B) Right, UMAP embedding of 16,289 high-quality cells that were clustered into 14 populations using the Seurat algorithm. (C) UMAP plots showing the expression of ZG16B (top), MUC7 (middle), and KRT19 across clusters. (D) Dot plots illustrate the expression of ZG16B and known epithelial markers. The color of each plot reflects the average expression level from low (yellow) to high (red), and the size of each dot reflects the percentage of positive cells for each gene. (E) STRING network analysis of genes shared between S-Acinar and SM-Acinar clusters shown in (C) The light blue lines represent database evidence; the purple lines represent experimental evidence; the yellow lines represent text mining evidence; and the black lines represent co-expression evidence. The red dotted line adds a new association between ZG16B and MUC7. (F) Representative immunofluorescence staining pattern of co-expression of ZG16B (green) and MUC7 (red) in S- and SM-acinar cells. DAPI (blue) indicates nucleated cells and KRT19 (Cyan) labels ductal cells. Magnification 40x, scale bar = 50μm. See also Tables S2 and S3 and Figure S3.

Figure 5

Onset of oral cGVHD is associated with loss of acinar-specific ZG16B and lymphocyte infiltration in minor salivary glands (A–C) IHC staining of ZG16B (red) in labial MSG of (A) healthy volunteers (n = 5), (B) post-HSCT patients without oral cGVHD (n = 5), and (C) post-HSCT patients with oral cGVHD (n = 5). DAPI (blue) indicates nucleated cells and aquaporin 5 (green) labels the apical membrane of acinar cells. Magnification 400x, scale bars = 50 μm. (D) Quantification of ZG16B staining was performed as described in STAR Methods. The mean and individual values are shown for n = 5 per group. A one-way ANOVA with Tukey's correction for multiple comparisons indicates significant (∗p ≤0.01) differences between the groups and specific differences between no oral cGVHD and oral cGVHD (p<05) and healthy and oral cGVHD. (E–G) IHC staining of CD45 to mark lymphocytes (red) and nuclei (blue) in labial MSG of (E) healthy volunteers (n = 5), (F) post-HSCT patients without oral cGVHD (n = 5), and (G) post-HSCT patients with oral cGVHD (n = 5). Magnification 400x, scale bars = 100 μm. (H) Quantification of CD45⁺ cells was performed as described in STAR Methods. The mean and individual values are shown for n = 4 to 5 per group. A one-way ANOVA with Tukey's correction for multiple comparisons indicates significant (∗p ≤0.01) differences between the groups and specific differences between no oral cGVHD and oral cGVHD (p<01) and healthy and oral cGVHD (p<0.05). (I–K) Representative images of Masson's trichrome-stained histological sections of (I) healthy volunteers (n = 5), (J) post-HSCT patients without oral cGVHD (n = 10), and (K) post-HSCT patients with oral cGVHD (n = 10) labial MSG from cohort 2 are shown. Blue staining marks collagen deposition. (L) Quantification of MSG fibrosis area adjusted by total area from Masson trichrome-stained sections shown as individual values with mean. A one-way ANOVA with Tukey's correction for multiple comparisons indicate no significant (∗p ≤0.05) differences between the groups Magnification 20x, scale bars = 200 μm. See also Figure S4.