RNA-Seq and CyTOF immuno-profiling of regenerating lacrimal glands identifies a novel subset of cells expressing muscle-related proteins

<div><p>The purpose of the present studies was to use CyTOF and RNA-Seq technologies to identify cells and genes involved in lacrimal gland repair that could be targeted to treat diseases of lacrimal gland dysfunction. Lacrimal glands of female BALB/c mice were experimentally injured by intra-glandular injection of interleukin 1 alpha (IL-1α). The lacrimal glands were harvested at various time points following injury (1 to 14 days) and used to either prepare single cell suspensions for CyTOF immuno-phenotyping analyses or to extract RNA for gene expression studies using RNA-Seq. CyTOF immuno-phenotyping identified monocytes and neutrophils as the major infiltrating populations 1 and 2 days post injury. Clustering of significantly differentially expressed genes identified 13 distinct molecular signatures: 3 associated with immune/inflammatory processes included genes up-regulated at days 1–2 and 3 associated with reparative processes with genes up-regulated primarily between days 4 and 5. Finally, clustering identified 65 genes which were specifically up-regulated 2 days post injury which was enriched for muscle specific genes. The expression of select muscle-related proteins was confirmed by immunohistochemistry which identified a subset of cells expressing these proteins. Double staining experiments showed that these cells are distinct from the myoepithelial cells. We conclude that experimentally induced injury to the lacrimal gland leads to massive infiltration by neutrophils and monocytes which resolved after 3 days. RNAseq and immunohistochemistry identified a group of cells, other than myoepithelial cells, that express muscle-related proteins that could play an important role in lacrimal gland repair.</p></div

Differential expression of collagen chains by day.

<p>Differential expression of collagen chains by day.</p

Double-staining of β-taxilin and desmin proteins.

<p>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.</p

Differential expression of matrix modifying enzymes by day.

<p>Differential expression of matrix modifying enzymes by day.</p

Histological analysis of IL-1α injected lacrimal glands.

<p>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.</p

Quantification of immune cell populations.

<p>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.</p

Venn diagram of overlap in significant gene regulation for each day.

<p>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.</p

Mass cytometry (CyTOF immunophenotyping) antibody panel.

<p>Mass cytometry (CyTOF immunophenotyping) antibody panel.</p

Expression pattern of significantly differentially expressed genes by day.

<p>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.</p

Representative molecular signatures of lacrimal gland inflammation and repair.

<p>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.</p