Cystatin S western blot of cell homogenates.

<p>Lanes 1–3 are cell homogenates where cells were incubated with saliva, C = control. The centre lane in Day 4 contains a molecular weight standard, whilst day 8 and 16 have WMS added.</p

MUC5B western blot of cell homogenates.

<p>Lanes 1–3 are cell homogenates where cells were incubated with saliva, C = control (no saliva incubation).</p

IgA Binding to HT29 and HT29-MTX cells from matched control IgA alone and WMS over 20 minute incubation period.

<p>Significantly more IgA bound to HT29-MTX cells from WMS then from IgA, *P<0.01. Significantly more IgA also bound to HT29-MTX then to HT29-MTX cells from IgA alone, *P<0.001.</p

Concentration of IgA bound to equal protein levels of HT29 and HT29-MTX cell lines from UWMS at days 4, 8 and 16.

<p>Significantly more IgA is bound to HT29-MTX cells compared to HT29 cells at day 16 and also significantly more compared to the same cell line at day 4, where *P<0.05 Day 16 HT29-MTX compared to day 4 HT29-MTX, **P<0.01 day 16 HT29-MTX compared to Day 16 HT29.</p

SDS-PAGE gel and wsetern blots of cell homogenates.

<p>Panel A shows a gel stained with CBB and PAS of control cells where no saliva or IgA was bound. H indicates HT29 cells with the number indicating the time point, M indicates the HT29-MTX cells. Other lanes include the molecular weight standard, UWMS (W), PS (P), buccal cell homogenate (B) and TR146 cells (T). Boxes highlight mucus band of the MUC5AC. Panel B shows blots of the same samples, probing for MUC5AC and a cell loading control β-actin.</p

Concentration of IgA bound to equal protein levels of HT29 and HT29-MTX cell lines from PS at days 4, 8 and 16.

<p>There does appear to be an increase in IgA binding as the MUC5AC later increases on the HT29-MTX cells but this increase is not significant.</p

HT29 cell layers at day 4, 8 and 16 and HT29-MTX cells at days 4, 8 and 16, with melamine formaldehyde particles and TRITC 4 added, shows mucus layer on the HT29-MTX only and high lights cell membranes.

<p>HT29 cells at day 8 and 16 don’t show any mucus production like the day 4 time point. Arrows are described by labels alongside them, with double-ended arrows highlighting cell and mucus thickness.</p

Activated epithelial cells lead to reduction in both volume of saliva produced and Mucin 10 present in saliva.

<p>A) Whole stimulated saliva produced by A20^-/- mice and WT littermate controls. <i>n</i> = 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 <i>t</i>-test was performed for statistical analysis, ** <i>p</i> < 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. *** <i>p</i> < 0.001, student’s <i>t</i>-test.</p

Dysregulation of NF-kB in glandular epithelial cells results in Sjögren’s-like features

<div><p>The autoimmune disease primary Sjögren’s syndrome (pSS) is characterized by hypofunction of the salivary glands (SGs), the cause of which is not correlated to lymphocytic SG infiltration, as prevailing dogma often states. We knocked out the NF-κB proinflammatory pathway inhibitor A20 in keratin14⁺ epithelial cells, to investigate if immune activated epithelial cells are capable of initiating pSS SG hallmarks. We show that immune activated epithelial cells can cause T cell dominated leukocytic infiltration and immune foci development of the SGs, reflecting the early clinical picture. Infiltrating leukocytes invaded striated ducts, similar to early stage lymphoepithelial lesions observed clinically. Expression of proinflammatory cyto-/chemokines IFNɣ, TNFα, IL-6, CXCL10 and CXCL13 increased in A20^-/- SGs, and functionally both volume and mucin 10 content of whole stimulated saliva from A20^-/- mice was significantly reduced. Epithelial cells may therefore represent the initial trigger for pSS SG pathologies, as opposed to simple reactionaries to pre-existing stimuli.</p></div

Activated epithelial cells cause leukocyte striated duct invasion and local increased cytokine production.

<p>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. <i>n</i> ≥ 5 per group. * p < 0.05, ** p < 0.01, *** p < 0.001, student’s <i>t</i>-test.</p