Hyperglycemia and Salivary Gland Dysfunction in the Non-obese Diabetic Mouse: Caveats for Preclinical Studies in Sjögren's Syndrome

Abstract

The Non-obese Diabetic (NOD) mouse model for type I diabetes also develops some features of Sjögren's syndrome (SS). Since the source of the mice and the environment exert a strong influence on diabetes, this study investigated SS development in NOD mice obtained from two vendors. Female NOD mice from The Jackson Laboratory (JAX) and Taconic Biosciences were monitored for blood glucose and pilocarpine-induced salivation. The gut microbiome was analyzed by 16S rRNA sequencing of stool DNA. At euthanasia, serum cytokines and sialoadenitis severity were evaluated. The onset of diabetes was significantly accelerated in JAX mice compared to Taconic mice. Although the gut microbiome between the two groups was distinct, both groups developed sialoadenitis. There was no correlation between the severity of sialoadenitis and reduced saliva production. Instead, salivary gland dysfunction was associated with hyperglycemia and elevation of serum IL1β, IL16, and CXCL13. Our data suggest that inflammatory pathways linked with hyperglycemia are confounding factors for salivary gland dysfunction in female NOD mice, and might not be representative of the mechanisms operative in SS patients. Considering that NOD mice have been used to test numerous experimental therapies for SS, caution needs to be exerted before advancing these therapeutics for human trials.

Figure 1

Salivary gland function in NOD mice from JAX and Taconic. Pilocarpine-induced saliva was measured at different time points in female NOD mice purchased from JAX (a) and Taconic (b). The filled circles represent mice with blood glucose >250 mg/dL. Statistical significance was analyzed by the Kruskal-Wallis test, and the adjusted p values were calculated using Dunn’s multiple comparison test. A p < 0.05 was considered significant. (c) The amount of saliva was compared between mice with blood glucose levels less than or greater than 250 mg/dL. Statistical significance was determined by two-tailed unpaired t-test at 95% confidence interval and a p < 0.05 was considered significant.

Figure 2

Hyperglycemia does not dictate the extent of sialoadenitis in NOD mice. (a) Representative H&E stained sections of submandibular glands from JAX and Taconic NOD mice euthanized at 20–24 weeks of age. For comparison, an image from an area without inflammation is also shown. (b) Taconic mice show significantly higher level of inflammation in their submandibular glands, compared to age-matched JAX mice. H&E stained sections of submandibular glands were quantified for the area fraction showing lymphocytic infiltration. Statistical significance was determined by a two-tailed unpaired t-test at 95% confidence level and a p < 0.05 considered significant. (c) The % area of inflammation in submandibular glands was compared between hyperglycemic and normoglycemic mice. Statistical significance was calculated by a two-tailed unpaired t-test and a p < 0.05 considered significant at 95% confidence interval. (d) Amount of saliva produced does not correlate with the level of inflammation in salivary glands of NOD mice. Pearson correlation test was used for analysis.

Figure 3

Serum cytokine and chemokine levels in NOD mice and their association with salivary gland dysfunction. (Top panel) A 33-plex assay was used to estimate serum cytokine and chemokine levels in NOD mice with blood glucose levels of >250 (JAX (n = 10), Taconic (n = 4), total n = 14) and <250 (JAX (n = 3), Taconic (n = 8), total n = 11) mg/dL. Only cytokines that showed statistical significance (p < 0.05) are shown. Statistical significance was determined using unpaired t-test for all cytokines except CXCL13 and IL10, where a two-tailed Mann Whitney test was performed. (Bottom Panel) Correlation between levels of serum cytokines and saliva amount in NOD mice from JAX (n = 13) and Taconic (n = 12). Sera collected at euthanasia (23–24 wks: JAX (n = 11), Taconic (n = 9); 20–21 wks: JAX (n = 2), Taconic (n = 3)) were used for this analysis. To compute correlation, two-tailed Spearman correlation test at 95% confidence interval was used.

Figure 4

Gene expression analysis in submandibular glands of Taconic mice that were either hyperglycemic (n = 6) or normoglycemic (n = 6). RNA isolated from submandibular glands of mice euthanized at 20–24 wks of age was used for expression analysis. The nCounter mouse Immunology panel (NanoString Technologies, Seattle, WA, USA) was used to analyze the expression of 561 genes. Differential expression analysis was performed by using the nSolver Analysis software (NanoString Technologies, Seattle, WA, USA). Benjamini-Yekutieli False Discovery Rate method was used to calculate the adjusted p values. Please note only 3 genes Nfkbia, Irak2, and C4a showed significant differential expression.