Fig 1.
Activated epithelial cells cause lymphocytic salivary gland infiltration.
A) Lymph node positive staining control for CD45 immunostaining. B) Low resolution immunostaining for CD45, denoting all leukocytes, in WT and A20-/- mice at 30 weeks of age. C) High resolution of boxed area in B showing foci of CD45+ cells. D) Quantification of proportion of CD45+ cells in WT and A20-/- mice submandibular salivary glands mice at 10, 20 and 30 weeks of age. E) Quantification of foci, defined as > 50 CD45+ cells together, per 4mm2 of submandibular SG tissue. n = 6 biological replicates for all groups, apart from 10 week time point, where n = 1 and 3 for WT and A20-/- respectively. *** = p < 0.001, Two Way ANOVA. F) Lymph node positive staining control for CD3 T cell immunostaining. G) Low resolution microscopy of CD3 immunostaining, denoting T cells, in WT and A20-/- mice at 30 weeks. H) High resolution of boxed area in F. I) Quantification of proportion of T cells in WT and A20-/- mice at submandibular salivary glands at 10, 20 and 30 weeks of age. J) Lymph node positive staining control for B220 immunostaining. K) Low resolution immunostaining images of B220, denoting B cells, in WT and A20-/- mice at 30 weeks of age. L) High resolution of boxed area in J. M) Quantification of proportion of B cells in WT and A20-/- mice submandibular glands at 10, 20 and 30 weeks of age. For all quantification data n numbers are as panel E. *** = p < 0.001.
Fig 2.
Activated epithelial cells cause leukocyte striated duct invasion and local increased cytokine production.
A) Leukocytic (CD45+) invasion of a striated duct in A20-/- mice at 30 weeks of age. B) CD3+ T cell invasion of a striated duct in serial section to A. C) B220+ B cell invasion of a striated duct in serial section to A. Second panels in A-C are high resolution images of inset boxes. D) Quantification of invaded striated ducts frequency per 4 mm2 of glandular tissue. *** = p< 0.001, Two Way ANOVA. E) Relative expression of the pro-inflammatory cytokines (IFNα, IFNɣ, TNFα and IL-6) and pSS-associated chemokines (CXCL10, CXCL13) in A20-/- mice at 20 and 30 weeks of age. n ≥ 5 per group. * p < 0.05, ** p < 0.01, *** p < 0.001, student’s t-test.
Fig 3.
Activated epithelial cells lead to reduction in both volume of saliva produced and Mucin 10 present in saliva.
A) Whole stimulated saliva produced by A20-/- mice and WT littermate controls. n = 6 mice per group per time point Two Way ANOVA testing showed significant difference at the 30 week time point. ** p < 0.01 B) Area under curve analysis of saliva production in A. Student’s t-test was performed for statistical analysis, ** p < 0.01. C) Coomassie and Periodic Acid Shift stained gel of whole stimulated saliva from WT and A20-/- mice. Position of bands representing Mucins 10 is shown. Each lane represents saliva from a separate biological replicate. D) Quantification amount of Mucin 10 in saliva, relative to total protein. Each data point represents a separate biological replicate. *** p < 0.001, student’s t-test.