MODULATING TRIM21 TO AMELIORATE DISEASE SYMPTOMS IN PATIENTS WITH AUTOIMMUNE DISEASESPHILIP ALLEN DY

SS

0.1-4.8% population (3)

40-50 years old

F:M Ratio9:1

pSS vs sSS

Manifestations? -Glandular

-Extraglandular

Diagnosis? Treatment?

AUTOIMMUNE DISEASES

Sjögren’s Syndrome (SS):

Is an inflammatory disease that can affect

many parts of the body but most often

affects the tear and saliva glands.

Dry eyes and dry mouth are the hallmark

of the disease(1).

SS mostly affects women with a ratio of

9:1 to men.

SS can exist either as a primary disorder

(pSS) or in patients with other

rheumatologic disorders such as SLE and

RA.

The patho-aetiology of SS is still unknown.

(2)Adapted from Kallen, 2014. Sjogrens Syndrome Effects on the Body Image. [Online] Available at: http://autoimmunesupport.com/category/sjogrens-syndrome/ [Accessed 19 March 2015].

(2)

SLE

14.6 – 68% per 100,000 people (US)

Symptoms vary

15-45 years old

F:M9:1

Diagnosis?

Treatment? Belimumab?

Systemic Lupus Erythematosus (SLE):

Is a chronic disease causing

inflammation such as pain and

swelling.

SLE affects many organs of the body.

Similar to SS, SLE affects women more

than men.

Branded as a ‘great imitator’ (4) due to

its wide range of symptoms that

mimics other diseases.

(5) Adapted from The Lupus Effect, (n.d.). Symptoms. [Online] Available at: http://thelupuseffect.com/about-lupus/symptoms/ [Accessed 19 March 2015].

(5)

PROJECT AIMS

Objectives:

To validate compounds that regulate the activity

of Trim21

To identify miRs whose expression is altered in

patients with autoimmune conditions compared

to healthy controls

Hypothesis:

The treatment of drug compounds that will

essentially increase the activity of Trim21

leading to the increase of its ability to reduce

type I IFN production thus reducing

inflammatory response

Differential expression of microRNAs may

play a role in the in the pathogenesis and

progression of pSS

(6) Adapted from Jefferies, C., Wynne, C. & Higgs, R. Antiviral TRIMs: friend or foe in autoimmune and autoinflammatory disease? Nat Rev Immunol 2011; 11: 617-25.

(6)

LUCIFERASE REPORTER ASSAY

Luciferase Reporter Assay:

Useful method for studying gene expression.

Uses two reporter enzymes, Firefly and

Renilla.

Both of these enzymes are situated within

specific light emitting plasmids.

The amount of light given off directly

correlates to the activity of the system.

The Renilla enzyme acts as an internal

control and the Firefly enzyme correlates to

the effect of the specific experimental

conditions.(7) Adapted from Ní Gabhann, J. (n.d.). Luciferase Reporter Assay Plate Setup Protocol.

(7)

LUCIFERASE ASSAY PROTOCOL

1x105 cells/ml seeded in a 96-

well plate

Add Luciferase Assay Mix Reagent or Renilla Mix

Read Luminescence

Plasmid DNA added to each well

Transfection Mix

A

B

C

TESTING INDIVIDUAL COMPONENTS OF VC

Individual components of VC: CD, PEG, and

NaOH.

Positive control: TRIF which drives IFN- β

promoter activity or Trim21 which is

inhibiting IFN- promoter activity.β

Negative control: EV

Therefore, TRIF+TRIM21 together provides

our threshold level.

A

B

A

B

DRUG COMPOUNDS @ 50NM CONCENTRATION

Single concentration at 0.05 M with different diluents including VC itself, PBS, and media based of μprevious results.

Further analysis resulted in the conclusion in mycoplasma infection = false interpretation of experimental data.

DRUG COMPOUNDS DOSE RESPONSE

3A compounds did not yield promising results since the drug compounds does not seem to be inhibiting IFN- promoter activity.β

On the other hand, the treatment with 4A compounds show a significant increase in the inhibition of IFN- promoter activity (p<0.05).β

A

B

C

A

B

C

DRUG COMPOUNDS DOSE RESPONSE

Increased concentration of our drug compounds for both 3A and 4A.

However, no inhibition of IFN- promoter activity exists.β

Also, due to miscalculations, only one VC was employed at 25 M.μ

A

B

C

A

B

C

THERMAL DENATURATION ASSAY

Differential Scanning Fluorimetry (DSF):

Used as as screening method for Trim21 and IRF3 proteins.

Measures the thermal stability of a target protein.

Binding of low Mw ligands can increase the thermal stability of a

protein.

The thermal stability change is measured a fluorescent dye, Sypro Orange (8).

The shifting of the curve decides the binding of the ligands.

A shift towards the left = destabilisation of the protein whereas a shift to the right = binding of ligand to protein.

2-degree shift to the right is significant.

THERMAL SHIFT ASSAYS

Determination of the optimal concentration for our proteins: Trim21 and IRF3 as well as the optimal concentration for DMSO, in which the compounds were solubilised in.

DMSO serves as our reference control.

THERMAL SHIFT ASSAY OF 3A AND 4A COMPOUNDS With the DMSO control in yellow, both

3A and 4A compounds with Trim21 protein have shifted to the right greater than 2-degrees.

In contrast, both 3A and 4A compounds with IRF3 protein have shifted to the left to our DMSO control.

PATHWAY OF MICRORNA

RNA Pol II

Exportin-5

Dicer

miRNA gene

Drosha-DGCR8 Pre-miRNA

Nucleus

Cytoplasm

miRISC

miRNA/miRISC

Target mRNA

miRISC

Target Degradation

(9) Adapted from Pilson, Q. (2015). Profiling microRNA in Primary Sjögren’s Syndrome Related Dry Eyes.

miRs are small, non-coding RNA molecule (~22nucleotides).

miRs have been shown to regulate inflammation

Trim21 (Ro52) functions in the ubiquitination process.

Trim21: common target of circulating autoantibodies in AID.

Trim21 has been identified as a key negative regulator of

inflammation.

(10) Adapted from Oke, V. & Wahren-Herlenius, M. The immunobiology of Ro52 (TRIM21) in autoimmunity: a critical review. J Autoimmun 2012; 39: 77-82.

BIOINFORMATICS

MIRS OF INTEREST

Two upregulated and two downregulated miRs were picked for further analysis and their respective gene targets were searched.

PRIMER OPTIMISATION

Primers for miRs of interest and their predicted targets were

optimised at three different annealing temperatures.

Optimal annealing temperatures were picked based on their

corresponding amplicon size.

Mw ladder: 1kb DNA ladder visualised by SyproRed staining

on a 1% Agarose gel.

QPCR ANALYSIS OF PBMCS OF PSS PATIENTS VS HEALTHY CONTROLS

A B

miR-30e was found to be overexpressed in pSS versus healthy controls from screening.

Real-time analysis confirms it is overexpressed in pSS patients.

SOCS1 is a predicted target of miR-30e that is involved in cytokine signal transduction.

SOCS1 gene expression in pSS patients vs healthy controls showed decreased

expression, as hoped.

n = 6 n = 8

A B

miR-98 was found to be underexpressed in pSS versus healthy controls from screening.

Real-time investigation demonstrates it is underexpressed in pSS patients.

IL10 is a predicted target of miR-98 that plays a vital role in inflammatory and immune

reactions.

IL10 gene expression in pSS patients vs healthy controls confirmed increased

expression, but not deemed to be statistically significant (p<0.05).

n = 6 n = 2

A B C

miR-194 was found to be underxpressed in pSS versus healthy controls from screening.

Real-time analysis confirms it is underexpressed in pSS patients.

CXCL3 is a predicted target of miR-194 that regulates monocyte migration and adhesion.

JAK3 is another predicted target of miR-194 that functions in signal transduction.

Both predicted targets should be upregulated. CXCL3 was found to be overexpressed as

expected but JAK3 showed underexpression in pSS patients.

n = 8 n = 8 n = 2

miR-125a was found to be underexpressed in pSS versus healthy controls from screening.

Real-time investigation did not agree with initial bioinformatic analysis as miR-125a was

found to be overexpressed in pSS patients.

No primers for miR-125a targets were ordered on time.

n = 8

SUMMARY Initial preliminary results of drug compounds look promising.

4A compounds: significant increase in the inhibition of TRIF driven IFN- promoter βactivity.

3A compounds: modifications in the structure of 3A compound could potentially increase the activity of Trim21.

Thermal melts

In our case, the expected results were achieved following 3A and 4A compounds with a positive shift when bound to Trim21 and a negative shift when bound to IRF3.

miRs of interest shows promising results.

Three miRs showed differential expression in SS vs SLE patients targeting Trim21.

Extensive analysis is required to further validate the legitimacy of the miRs and specifically, their gene targets in expression in pSS patients (more samples required).

Finally, further investigation of miRs could potentially lead to the development of personalised microRNA-base therapies as novel therapeutics.

ACKNOWLEDGEMENTS

SUPERVISORS

• Prof. Caroline Jefferies

• Prof. Conor Murphy

• Dr. Joan Ní Gabhann

• Dr. Qistina Pilson

• Dr. Siobhán Smith

DIT KEVIN ST.• Dr. Sara Lynch

• Dr. Claire Wynne

MOLECULAR IMMUNOLOGY RESEARCH GROUP, RCSI

• Dr. Joan Ní Gabhann

• Dr. Jay Chandanshive

• Dr. Marian Brennan

• Shane O’Grady

• Leah Arkins

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Immunol 2011; 11: 617-25.

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2012; 39: 77-82.