A SYSTEMS BIOLOGY APPROACH TO OCULAR INFLAMMATION

Ocular inflammation is a chronic, progressive and degenerative inflammation of
any or all compartments of the eye including the uveal tract (uveitis and
age-related macular degeneration), the cornea and orbita and cause an immediate
threat to vision. The pathogenesis of ocular inflammation is poorly understood.
Current treatment strategies include various immunosuppressive medications but
fail to prevent or stop the decline in visual function, and usually induce many
adverse effects that affect quality of life, as immunosuppressant therapy is
often life-long. Hence, further research to understand pathogenesis and improve
prognosis for these patients is highly justified. Systems biology is an
interdisciplinary approach that systematically describes the complex
interactions between all the parts in a biological system, with a view to
elucidating new biological rules capable of predicting the behavior of the
biological system. To this aim, data are collected from all the components of a
biological system, analyzed and integrated in order to generate a mathematical
model that describes or predicts the response of the system to individual
perturbations.

Systems biology is an interdisciplinary approach that systematically describes
the complex interactions between all the parts in a biological system, with a
view to elucidating new biological rules capable of predicting the behavior of
the biological system. To this aim, data are collected from all the components
of a biological system, analyzed and integrated in order to generate a
mathematical model that describes or predicts the response of the system to
individual perturbations. To delineate these networks acquisition of high
throughput analyses of distinct layers (e.g. mRNA, microRNA, methylation
status, proteome) and various cellular subsets (e.g. B cells, T cells,
dendritic cells, NK cells) that constitute the network is obligatory. Due to
the integration of all the biological information using high throughput
technology, molecular signatures that typify different forms and disease
activity of intraocular inflammation will be identified and provide novel
therapeutic targets.

We aim to employ an *omics*-driven systems biology approach on patients with
ocular inflammation in order to identify the molecular pathways underlying the
pathogenesis and develop clinically useful markers to predict disease outcome
and treatment responses. Decipher the molecular pathways that underlie
intraocular inflammation.

1. To investigate the frequency, function and phenotype of circulating immune
cells; T cells, B cells, plasmacytoid dendritic cells, monocytes, and NK cells.
Plasma and serum will be collected to measure the circulating cytokines using
Luminex and the immune cells will be isolated. The surface markers and the
intracellular cytokine production of these cells will be analyzed by flow
cytometry. The T cells, pDCs and monocytes will be cultured and after
stimulation the supernatants will be analyzed by Luminex, the gene expression
of the cells will be investigated by qPCR.
2. To investigate the transcriptome and epigenome of circulating immune cells
by RNA sequencing miRNA profiling and genome-wide methylation of cell subsets.
3. To identify, using high throughput technology, molecular signatures that
typify different forms and disease activity of intraocular inflammation.

This study is designed as an observational cross-sectional study for the
duration of 4 years. For the present study we will recruit patients with
uveitis, corneal disease and age-related macular degeneration at the outbound
department of ophthalmology. We estimate (on the basis of the current flow of
new patients that we will be able to include 630 patients with uveitis
(anterior uveitis (n=60), intermediate uveitis (n=60) posterior uveitis (n=60),
panuveitis (n=60) and scleritis (n=60)), age-related macular degeneration
(n=60), retinal dystrophy (n=60), multifocal choroidal retinitis (n=60),
inflammatory corneal disease (n=40), orbital inflammatory diseases and lymphoma
(n=60), and strabismus (n=30). From this cohort and for the purpose of the
current study proposal, patients with ocular inflammation will be asked for a
blood sample (ranging between 11mL and 81mL total blood, dependent on age). A
venipuncture is performed to obtain a certain amount of blood dependent on age:
- Age of 0-6: a total of 11 mL blood; 6 mL EDTA blood for plasma samples and 5
mL clotting blood for collection of serum or 9 mL Lithium Heparin or Sodium
Heparin blood and 2 mL clotting blood.
- Age of 7-15: a total of 29 mL blood; 27 mL Lithium Heparin or Sodium Heparin
blood and 2 mL clotting blood.
- Age of 16 and above: a total of 81 mL blood; 70 mL Lithium Heparin or Sodium
Heparin blood for investigating the circulating immune cells using flow
cytometry, luminex and qPCR, 6 mL EDTA blood for collection of plasma and 5 mL
clotting blood for collection of serum.

Blood drawn from the study patients enters the Radstake lab after which various
immune cell subsets will be carefully isolated (B, T and NK cells, myeloid DCs,
plasmacytoid DCs and monocytes) for the determination of the transcriptome (RNA
sequencing) and epigenome (miRNA profiling and genome-wide methylation).
Simultaneously, these cell subsets will be phenotypically analyzed employing
extensive flow cytometry protocols. Besides the regulation at the cellular
level, we will study the circulatome by exploiting the Luminex core facility
(UDAIR) present in our lab. UDAIR is optimized to measure ~ 150 cytokines /
chemokines / growth factors in small sample volumes (50 ul). In addition, we
will study the level of oxidative stress markers and the presence of
inflammatory lipids at the fentomolar level in collaboration with our
metabolomics facility. Both UDAIR and the metabolomics facility enable us to
study the circulatome and merge this data with what we see on the cellular
level thereby identifying causative pathways / circulating markers of
relevance.

The risks of a venepuncture are generally considered to be very low. It is
possible that a haematoma develops at the venepuncture site. The risks of
topical mydriatics are considered low. After administration, the patient may
experience a blurry vision and a sensation of photophobia. Topical mydriatrics
will not be given to patients who are pregnant or are breastfeeding or have a
high(er) chance of developing glaucoma. Flare measurements are conducted during
the regular ophthalmic examination. The side effects are nil. Patients can have
short-term complaints of afterimages.