1. 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

2. Disclosure
We have no commercial relationship in the past 12 months

3. 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

4. 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: /nrrheum

5. 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

6. 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.

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

8. 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

9. 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

10. 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

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

12. 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

13. 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

14. 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

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

16. 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?

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

18. Thank you!

19. 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):
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):

20. 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):

21. 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):