The miRNA Landscape of Lacrimal Glands in a Murine Model of Autoimmune Dacryoadenitis

Abstract

Purpose: To analyze the changes in the lacrimal gland (LG) miRNAome from male nonobese diabetic (NOD) mice with autoimmune dacryoadenitis compared with LG from healthy male BALB/c and dacryoadenitis-free female NOD mice.

Methods: LG from these mice were collected for small RNA sequencing to identify dysregulated miRNAs; hits were validated by RT-qPCR in male NOD and BALB/c LG. Dysregulation of validated species within immune cell-enriched cell fractions and epithelial-enriched cell fractions from LG was probed by RT-qPCR. Ingenuity pathway analysis identified putative miRNA targets, which were examined in publicly available mRNA-seq datasets. Western blotting and confocal imaging of immunofluorescence enabled validation of some molecular changes at the protein level.

Results: Male NOD LG exhibited 15 and 13 significantly up- and downregulated miRNAs, respectively. Dysregulated expression of 14 of these miRNAs (9 upregulated, 5 downregulated) was validated in male NOD versus BALB/c LG by RT-qPCR. Seven of the upregulated miRNAs were increased owing to their abundance in immune cell-enriched cell fractions, whereas four downregulated miRNAs were largely expressed in epithelial-enriched cell fractions. Ingenuity pathway analysis predicted the upregulation of IL-6 and IL-6-like pathways as an outcome of miRNA dysregulation. Increased expression of several genes in these pathways was confirmed by mRNA-seq analysis, whereas immunoblotting and immunofluorescence confirmed Ingenuity pathway analysis-predicted changes for IL-6Rα and gp130/IL-6st.

Conclusions: Male NOD mouse LG exhibit multiple dysregulated miRNAs owing to the presence of infiltrating immune cells, and decreased acinar cell content. The observed dysregulation may increase IL-6Rα and gp130/IL-6st on acini and IL-6Rα on specific lymphocytes, enhancing IL-6 and IL-6-like cytokine signaling.

Figure 1.

miRNA expression from sRNAseq of stimulated LG from NOD and BALB/c mice. (A) Boxplot of the number of unique miRNAs per group.^† Red dots indicate the mean, and the other dots indicate the individual data points. (Constraints: at least 1 read detected per each miRNA per sample.) (B) Venn diagram of common and distinct miRNAs in LG from male (M) NOD, M BALB/c and female (F) NOD mice. (Constraints: miRNAs are detected in at least 50% of the biological replicates of a group.) (C) Barplot of the most highly expressed miRNAs^†† in each strain. ^†One-way ANOVA with Tukey's-HSD for multiple correction. ***P_adj < 10⁻⁴, ****P_adj < 10^{−5 ††}Average DESeq2 normalized counts >10⁴; error bars = standard error of the mean.

Figure 2.

Dysregulated miRNAs in LG from male NOD mice. miRNA that are (A) upregulated or (B) downregulated in LG from 13-week diseased male NOD mice relative to LG of age-matched female NOD and male BALB/c mice. Data are plotted as Log10 normalized miRNA counts as calculated by DESeq2 for each group. N = 5 samples for male NOD and BALB/c and N = 3 samples for female NOD mice; n = 1 LG from each of 5 mice per sample. miRNAs were considered differentially expressed if they were upregulated or downregulated in the male NOD versus male BALB/c comparison as well as in the male NOD versus female NOD comparison, had a mean expression value of ≥1000, had a significant unadjusted P value in at least one of the two comparisons; and showed a >95% sequence similarity with human miRNA. (*P < 5 × 10⁻², **P < 10⁻³, ***P < 10⁻⁴, ****P < 10⁻⁵, adjusted P values - DESeq2.)

Figure 3.

RT-qPCR validation of miRNA changes in unstimulated LG. Barplots showing log2 fold change in miRNA that are either (A) upregulated or (B) downregulated in unstimulated LG from 13-week-old male NOD mice relative to unstimulated LG from age-matched male BALB/c mice. miRNA Ct values were normalized to the endogenous control, snord68; ΔCt values for a given miRNA were then normalized to average ΔCt of that miRNA's expression in the healthy control group (male BALB/c). (Data are plotted as mean ± SEM. n = 3 LG from 3 mice/strain. *q < 0.05, **q < 0.01, ***q < 0.001, ****q < 0.0001, two-way ANOVA with fdr controlled at q = 0.05 for multiple comparisons correction.)

Figure 4.

miRNA expression in immune-enriched (IEF) and epithelia-enriched (EEF) cell fractions from male NOD mouse LG. Barplots comparing log2 relative expression of miRNAs that are either (A) upregulated or (B) downregulated in IEF of 13-week male NOD mice relative to their expression in EEF. Ct values were normalized to the endogenous control, snord68; ΔCt values for IEF were then normalized to average ΔCt of the EEF. (n = 5 mice/group, data are plotted as mean log2 fold change ± SEM. *q < 0.05, **q < 0.01, ***q < 0.001, ****q < 0.0001. ANOVA with repeated measures and fdr (q) controlled at 0.05 for multiple comparisons correction.)

Figure 5.

miRNAs targeting IL-6 signaling are dysregulated in male NOD LG IEF. (A) Arrows indicate direction of miRNA change (Created in BioRender). (B) RT-qPCR confirmation of gene expression changes in gp130/IL-6st, an IL-6 coreceptor, in 13-week-old male NOD relative to age-matched male BALB/c mouse LG. n = 3 LG from 3 separate mice, data are plotted as mean fold change ± SEM, *P = 0.05, unpaired t test.

Figure 6.

Dysregulated miRNAs target IL-6-like cytokines and their downstream effectors in EEF. (A) IL-6–like cytokine signaling pathway with gene expression as predicted by IPA owing to the presence of dysregulated miRNA in EEF. Color-coded genes in orange are IPAs estimated predictions with color saturation proportional to the intensity of predicted upregulation. (B) Heatmap showing levels of gene expression in LG of NOD male mice before (pre-dacryoadenitis onset, DO) and after onset of dacryoadenitis (Post DO) as compared with LG of age and sex matched BALB/c. (Bulk RNA-seq data generated by Ohno Y et. al. and raw data obtained from ENA Accession PRJDB9749.)

Figure 7.

Protein expression of IL-6 receptor subunits is altered in male NOD LG. Western blots showing protein expression of (A) gp130/IL-6st and (B) IL-6Rα in LG lysates from 13-week-old male BALB/c and NOD mice.^† Representative images of 5-µm LG sections showing immunofluorescence labeling of the IL-6 receptor subunits (C) gp130/IL-6st and (D) IL6-Rα in LG from male NOD and BALB/c mice. In (C), arrows point to acini expressing gp130/IL-6st. In (D), arrows point to lymphocytes expressing IL-6Rα, whereas the arrowheads point to an acinar lumen showing enhanced accumulation of IL-6Rα. Bars, 10 µm. ^†n = 5 LG from 5 separate mice, data are plotted as total protein normalized mean signal intensity ± SEM, *P < 0.05, ***P < 0.001, unpaired Student's t test.