Inflammasome priming in salivary gland epithelial cells – A potential disease model of salivary gland inflammation in primary Sjögren’s Syndrome (pSS) By Aden Lau, Susan Lester, Mariea Bosco, Dr Eugene Roscioli, Dr Peter Zalewski, A/Prof. Maureen Rischmueller Rheumatology Department, TQEH

Disclosure We have no commercial relationship in the past 12 months

Introduction Primary Sjögren’s Syndrome (pSS) is characterized by chronic inflammation in the lacrimal and salivary gland About 2/3 of pSS patients have anti-nuclear autoantibodies (ANAs) anti-Ro/La, which target the autoantigen YRNA ribonucleoprotein (YRNP) Anti-Ro/La are associated with disease severity Patients with Ro/La tend to have more severe disease

pSS disease model based on current knowledge Focus on epithelial cells => Starts with Cell Death => Nuclear autoantigens (e.g. YRNP) are exposed => Antibodies generated against them => forming immune complexes => APC => Activate Type I IFN which is an innate immune mechanism that promotes inflammation => Prolonged inflammation kills epithelial cells => Self-perpetuating inflammation => The fact immune complexes from adaptive immunity can activate innate immunity is fascinating and we wonder how pSS disease model based on current knowledge Nocturne, G. & Mariette, X. (2013) Advances in understanding the pathogenesis of primary Sjögren’s syndrome Nat. Rev. Rheumatol. doi:10.1038/nrrheum.2013.110

Inflammasome-mediated inflammation pathway in immune cells That’s how we came to be interested in this innate immune machinery called inflammasome => There are a number of inflammasomes => activation of inflammasome requires two signals in immune cells (e.g. macrophages) => First, inflammasome primed through TLRs upon detection of PAMPs => now relevant genes are upregulated and inflammasome is ready => it requires a specific signal for each inflammasome for activation => families of inflammasomes, each has unique activation mechanism NLRP3: activated by ext. ATP, extensively studied in inflammatory diseases, mutation related to auto-inflammatory syndrome => expected to be activated at the beginning but instead AIM2: is more interesting, activated by int. dsDNA End results of any inflammasome activation is caspase 1 -> release of IL-1b Prolonged inflammation leads to cell death that can be apoptotic or pyroptotic These pathways are well understood in immune cells but we don’t know about epithelial cells Inflammasome-mediated inflammation pathway in immune cells

Elevated serum IL-18 levels in Ro/La+ pSS Our department has found…=> this implies that Ro/La immune complexes have a role in the inflammasome pathway Elevated serum IL-18 levels in Ro/La+ pSS Lester, S., et al. (2013). "Epistasis with HLA DR3 implicates the P2X7 receptor in the pathogenesis of primary Sjogren's syndrome." Arthritis Res Ther 15(4): R71.

Hypothesis Salivary gland epithelial cells (SGECs) possess necessary inflammasome apparatus for actively participating in salivary gland inflammation in pSS

Aim Our study aims to set up a salivary gland epithelial cell (SGEC) model for inflammasome activation by TLR agonists in a way that serves as a disease model for inflammasome activation by Ro/La immune complexes Because we don’t know anything about inflammasome in epithelial cell so we use this cell model as a starting point

Methods Readout: Expression of inflammasomes and inflammatory cytokine A253 Salivary gland epithelial cell line TLR agonists : LPS (TLR4) Poly(I:C) (TLR3) Imiquimod (TLR7) CL264 (TLR7) 6-hour Treatment Repeated twice Readout: Expression of inflammasomes and inflammatory cytokine qPCR Western Blot

Key Findings: Poly(I:C), a TLR3 agonist and a dsRNA analogue, primed the AIM2 inflammasome in A253 salivary gland epithelial cells NLRP3 was interesting because of other inflammatory disease studies

AIM2 is primed when treated with poly(I:C) (qPCR) Normalized to housekeeping genes X-Y axes LPS

TLR3 agonist primed AIM2 in Salivary Gland Epithelial Cells TLR agonists Confirmed at the protein level -> these are western blot results X: Each lane represents a TLR agonist treatment Y: Tested these proteins in response to treatments TL3 => AIM2, 1st time in human epithelial cells

AIM2 is expressed in acini and ducts in salivary gland biopsies Ro/La - pSS 100m In addition to cell model Red arrows black arrows… Apparently ducts and acini express AIM2 in Ro/La + Quantitative analysis is being done and we’ll see if AIM2 is differentially expressed in Ro/La+ patients compared to ctrl Ro/La + pSS 100m

FL IL-1beta levels increased in pSS ducts Our collaborators have found increased IL-1beta in pSS compared to sicca in our patients Our group is doing co-localization studies on AIM2 and IL-1beta => strong evidence for AIM2 being responsible for salivary gland inflammation To sum up, we have multiple lines of evidence supporting the hypothesis that AIM2 inflammasome is activated in pSS salivary gland inflammation A systematic evaluation on these data is being done Gilboa-Geffen, A., et al. (2011). "Activation of the alternative NFkappaB pathway improves disease symptoms in a model of Sjogren's syndrome." PLoS One 6(12): e28727

Active Caspase-1 is present in pSS ductal cells Apart from the AIM2 protein, active caspase-1

Summary TLR3 agonist (Poly I:C, dsRNA) induces AIM2 and IL-1 in SGEC line Inflammasome machineries (AIM2,caspase-1, IL-1) exist in SGECs Before I finish, lets take a look at what our findings have added to the big picture Given the resemblance of TLR3 stimulation to viral infection, this finding supports the proposed viral trigger of pSS Support out hypothesis of SGECs playing an active role in salivary gland inflammation It would be interesting to look at the role of Ro/La IC in inflammasome activation Ro/La Immune complexes  AIM2 activation?

Acknowledgement A/Prof. Maureen Rischmueller Susan Lester Dr Eugene Roscioli Mariea Bosco Dr Peter Zalewski Dr Hai Tran Melissa Bubicich

Thank you!

AIM2 inflammasome in other inflammatory diseases AIM2 is upregulated in B+P+ CRS patients Jardeleza, C., et al. (2013). "Inflammasome gene expression alterations in Staphylococcus aureus biofilm-associated chronic rhinosinusitis.“ Rhinology 51(4): 315-322. AIM2 is a key inflammatory mediator upon S. Aureus infection in other sites (e.g. CNS infection) Hanamsagar, R., et al. (2014). "Critical role for the AIM2 inflammasome during acute CNS bacterial infection." J Neurochem 129(4): 704-711.

TLR expression on SGEC and BAFF induction by Poly I:C Ittah, M., et al. (2008). "Viruses induce high expression of BAFF by salivary gland epithelial cells through TLR- and type-I IFN-dependent and -independent pathways.“ Eur J Immunol 38(4): 1058-1064.

Emerging evidence that epithelial cells in pathogen detection and inflammatory signalling TLR 1,3,7,9 are expressed on the apical surface of human airway epithelial cells Ioannidis, I., et al. (2013). "Toll-like receptor expression and induction of type I and type III interferons in primary airway epithelial cells." J Virol 87(6): 3261-3270.