Table 1.
Mass cytometry (CyTOF immunophenotyping) antibody panel.
Fig 1.
Histological analysis of IL-1α injected lacrimal glands.
Lacrimal gland tissue was excised 1, 2, 3, 4, and 7 days post IL-1α or saline (vehicle) injection and processed for hematoxylin and eosin staining. Scale bar represents 50 μm for all panels.
Fig 2.
Principal component and heatmap analyses of RNA-sequencing samples.
All samples were quality controlled to identify samples for exclusion from differential expression analysis. (A)Principal components analysis (PCA) was used to investigate replicate variability with 3 distinct clusters successfully identified. (B) Heatmap analysis was also used to cluster samples to identify samples for exclusion which identified 4 distinct clusters of samples. Samples (arrows in A, red circles in B) outside the defined clusters were excluded from downstream analyses.
Fig 3.
Expression pattern of significantly differentially expressed genes by day.
The significantly differentially expressed genes at days 1, 2, 4, and 5 were plotted to visualize the global expression of these genes across all time points (days 3, 7, 14 and 14S data not plotted based on the small number of genes). Day 4 and 5 significantly differentially expressed genes exclude day 1 and 2 expression for visualization purposes (very few genes up/down-regulated). Data points colored based on regulation at the day of interest; green = strongly up-regulated, red, up-regulated, orange = strongly down-regulated, blue = down-regulated.
Fig 4.
Venn diagram of overlap in significant gene regulation for each day.
A Venn diagram of the differentially expressed genes at days 1, 2, 3, 4, and 5 was created to identify the overlap in gene expression.
Fig 5.
Representative molecular signatures of lacrimal gland inflammation and repair.
Expression (log2[fold change]) pattern of clusters representative of the 8 consolidated clusters were plotted for days 1, 2, 3, 4, 5, and 7. Threshold for significant up/down-regulation (+/- 1 = log2[+/-2]) is indicated by dotted blue line.
Table 2.
Summary of identified molecular signatures.
Fig 6.
Quantification of immune cell populations.
The number of cells per thousand of analyzed cells were plotted by day for (A) CD45+ (immune cells) CD45- (non-immune cells), (B) cells with large population change, monocytes and neutrophils, and (C) cells with small population change, natural killer (NK), B, plasmacytoid dendritic (pDC), CD4+ and CD8+ T cells. Insert shows representative immunofluorescence staining of Ly-6G (neutrophils) 1 day post IL-1α injection.
Table 3.
Differential expression of collagen chains by day.
Table 4.
Differential expression of matrix modifying enzymes by day.
Table 5.
Differential expression of factors associated with the extracellular matrix and its maintenance by day.
Table 6.
Muscle-related genes from cluster type VIII.
Fig 7.
Immunohistochemical analysis of muscle related proteins in 2 day post injury lacrimal glands.
Lacrimal gland tissue from IL-1α injected mice were stained for muscle related proteins desmin, β-taxilin, ryanodine receptor 1 (Ryr-1), and sarcalumenin (Srl). Panels represent staining from 2 days post IL-1α injected lacrimal glands which were counterstained with DAPI to visualize cell nuclei. Scale bars represent 25 μm.
Fig 8.
Double-staining of β-taxilin and desmin proteins.
Lacrimal gland tissue from IL-1α injected mice were double-stained for muscle related proteins desmin and β-taxilin. Panels represent staining from 2 days post IL-1α injected lacrimal glands which were counterstained with DAPI to visualize cell nuclei. Arrows represent cells expressing both proteins; arrowheads represent cells expressing β-taxilin only; stars represent cells expressing desmin only; BV represents a blood vessel. Scale bars represent 25 μm.
Fig 9.
Double-staining of desmin and the myoepithelial cell marker, α-smooth muscle actin.
Lacrimal gland tissue from IL-1α and saline (vehicle) injected mice were double stained for desmin and α-smooth muscle actin (α-SMA) or desmin and collagen I (Col-I). Panels represent staining from 2 days post IL-1α injected lacrimal glands which were counterstained with DAPI to visualize cell nuclei. Scale bars represent 25 μm.