Finding a treatment for X-linked

Recessive Ichthyosis

Sofia Miettinen

Supervisor – Edel O’Toole Experimental Pathology iBSc 2018/19

Centre for Cell Biology and Cutaneous Research, Blizard

Institute, Barts and The London School of Medicine and

Dentistry

Introduction:

X-linked recessive ichthyosis (XLRI) is a congenital skin condition belonging to the ichthyosis

family. XLRI presents a few weeks post birth with brown or grey scales across the body mainly

over the limbs and trunk, with extracutaneous features such as comma shaped corneal opacities,

undescended testicles, and neurological changes such as autism, attention deficit hyperactivity

disorder and epilepsy present in some cases (1).

90% of XLRI cases are caused by deletion of the steroid sulfatase (STS) gene (1). In the XLRI

epidermis, STS enzyme activity is lost completely from all epidermal layers. This results in

cholesterol sulfate build up in the epidermis, causing the delayed desquamation phenotype.

This delay in desquamation is seen as an increased thickness in the stratum corneum and is

seen clinically as scaling across the skin.

Currently there is no ideal treatment for XLRI. Current treatments aim to reduce the presence

and appearance of the scaling of the skin and reduce dryness but achieve this with varying

effects.

Aims:

Previous work by the O’Toole lab included RNA sequencing of XLRI patient’s skin samples.

The lab also created a 3D organotypic model of XLRI skin using STS gene knockout cells that

encapsulates the core features seen in affected patients’ skin.

Using this RNA sequencing data, we aim to:

1. Work out which genes could be targeted to overcome the deficiency in the STS gene

2. Find a new treatment to improve the epidermal profile

Methods:

1. Cell lines –

CRISPR STS gene knockout in immortalized keratinocyte cell lines (N/TERT) were used.

Keratinocytes with no STS gene knockout activity will hereafter be called CRISPR wild type

keratinocytes (CR-WT) and STS gene knockout keratinocytes will be called CRISPR STS

knockout 1 keratinocytes (CR-KO1).

2. Immunohistochemistry –

Five micron thick sections were used.

2.1 Deparaffinization and rehydration protocol –

Paraffin embedded, fixed tissue (4% PFA) slides deparaffinized and rehydrated prior to

staining. Deparaffinization achieved via two 5 minute xylene washes, and rehydration via

3 minutes each ethanol washes (100%, 100%, 90%, 70%).

2.2 Haematoxylin and Eosin staining –

Slides immersed in Haematoxylin for 3 minutes, washed with running water for 5 minutes,

immersed in 1% acid alcohol and rinsed with running water. Slides then immersed in Eosin

for 2 minutes, and dehydrated using 3-minute ethanol washes (70%, 80%, 90%, 100%,

100%) and two 5 minute xylene washes. Slides mounted using Immu-Mount (Thermo

Fisher Scientific, USA). Imaged using LSM510Meta inverted Confocal microscope.

2.3 Nile Red staining –

Drop of 0.05% Nile Red (Sigma-Aldrich, USA) mixed with acetone added to each slide.

Imaged using DM2000 Epifluorescence microscope.

2.4 Immunofluorescent staining –

Paraffin section staining preparation

Heat-induced epitope retrieval via boiling for 10 minutes in 95 degrees Celsius

sodium citrate buffer (10mM Sodium Citrate, 0.05% Tween20, pH 6.0). Slides

cooled and rinsed thoroughly with distilled water.

IF staining protocol

Slides blocked in IFF buffer (PBS, 1% w/v BSA, 2% v/v FBS) with goat serum at

room temperature for 1 hour. Primary antibodies diluted with IFF, table 1. Slides

incubated with 50 μL primary antibody overnight at 4 degrees Celsius in a wet

chamber.

Slides washed with PBS three times. 50 μL Alexa Flour 568-red anti-rabbit

secondary antibodies (Invitrogen, USA) diluted in IFF buffer 1:100 concentration

and 4',6-Diamidino-2-phenylindole (DAPI) 1:100 added to each sample for 1 hour

at room temperature. Slides washed with PBS three times and mounted with Immu-

Mount (Thermo Fisher Scientific, USA). Imaging via DM2000 Epifluorescence

microscope.

Table 1 - Primary antibody concentrations for Immunofluorescence (IF) and Western

Blotting (WB)

3. Ethical approval –

The use of anonymized human skin samples was conducted according to the Declaration of

Helsinki principles and approved by the appropriate local ethics committees.

4. Statistics –

Statistical analysis performed using GraphPad. An unpaired t test was used for relative

expression of JunB. ImageJ used for quantification of immunostaining and western blots.

JunB Abcam Rabbit 1:100 1:1000

GAPDH Abcam Rabbit 1:1000

Results:

UniProt was used to select all the transcription factors from an RNA sequencing list of

differentially expressed genes in skin from patients with XLRI with a deletion in the STS gene.

The activator protein 1 (AP-1) transcription factor subunits were downregulated and selected

for further analysis as they are important in the regulation of differentiation. AP-1 is a

transcription factor formed of Jun (c-Jun, JunB, JunD), Fos (c-Fos, Fra1, Fra2) and ATF

(ATF2, ATF3) families of subunits (2).

Luciferase reporter assay results showed statistically significant decreased AP-1 promotor

activity under calcium shift conditions for CR-KO1 compared to CR-WT (p <0.05), figure 1.A.

Western blot showed a decrease in JunB in the nuclear fraction of CR-KO1 compared to CR-

WT, figure 1.B. JunB expression in 3D organotypic models of XLRI skin grown from CR-

WT and CR-KO1 cell lines was significantly reduced in CR-KO1 cell lines compared to CR-

WT (p < 0.01), figure 1.C and 1.D.

As JunB expression was significantly decreased, DSigDB was used to select drug treatments

that upregulate AP-1 expression: celecoxib, cholecalciferol, and quercetin were selected (3).

Haematoxylin and Eosin staining, figure 2, showed that the three drug treatments all

decreased epidermal thickness.

GAPDH

JunB

1 0.4

D C B A

Figure 1. Comparison of XLRI 3D organotypic models in CRISPR knockout (CR-KO)

and wildtype (CR-WT)

(A). Activator protein 1 (AP-1) expression in CR-KO1 keratinocyte cell line compared to CR-WT

keratinocyte cell line measured with dual luciferase assay with *P0.05 (B). JunB expression was

analyzed by western blot. GAPDH was used as an internal control. (C). Representative images of

immunofluorescence (IF) staining of JunB (red) in CR-WT and CR-KO1 3D organotypics. DAPI

(blue) was used as a nuclear stain. (D). Intensity of JunB was measured using ImageJ, the average

intensity per cell was calculated and shown as a percentage of CR-WT organotypic. Data represents

meanSD with **P0.01

Figure 2 – H&E staining of drug treated CR-WT and CR-KO1 cell line 3D organotypic models Haematoxylin and eosin staining was performed on 3D organotypic models grown from CR-WT and CR-KO1

cell lines. Images were taken from the thickest portion of the epidermis. Scale bar = 200 μm.

XLRI patients have increased polar lipids throughout the epidermis, and so treatment aims to

decrease the polar lipids content. All three drug treatments decreased polar lipids in the CR-

KO1 cell lines compared to the CR-KO1 negative control, figures 3-5.

JunB protein expression was validated as significantly decreased in CR-KO1 cell lines. As seen

in figure 6, JunB expression was increased by similar amounts by all concentrations of drug

treatments in the CR-KO1 3D models compared to CR-KO1 negative control.

Figure 3 – Nile red staining of celecoxib treated CR-WT and CR-KO1 3D organotypic models

3D organotypic model cultures grown using CR-WT and CR-KO1 cell lines were treated with celecoxib 0.05

μM and 0.5 μM. Nile red was added to CR-WT and CR-KO1 negative controls (NC) and drug treated 3D

organotypic cultures. Red = polar lipids, green = non-polar lipids. Scale bar = 100 μm.

Figure 4 – Nile red staining of cholecalciferol treated CR-WT and CR-KO1 3D organotypic models

3D organotypic model cultures grown using CR-WT and CR-KO1 cell lines were treated with

cholecalciferol 0.01 μM and 0.1 μM. Nile red was added to CR-WT and CR-KO1 negative controls (NC)

and drug treated 3D organotypic cultures. Red = polar lipids, green = non-polar lipids. Scale bar = 100 μm.

Figure 5 – Nile red staining of quercetin treated CR-WT and CR-KO1 3D organotypic models

3D organotypic model cultures grown using CR-WT and CR-KO1 cell lines were treated with quercetin 0.05

μM and 0.5 μM. Nile red was added to CR-WT and CR-KO1 negative controls (NC) and drug treated 3D

organotypic cultures. Red = polar lipids, green = non-polar lipids. Scale bar = 100 μm.

Figure 6 – JunB expression in celecoxib, cholecalciferol and quercetin treated CR-WT and CR-KO1

3D organotypic models

3D organotypic model culture grown using CR-WT and CR-KO1 cell lines tissue was stained for JunB. Blue

staining = nucleus, red staining = JunB. Scale bar = 100 μm.

Conclusion:

With the AP-1 subunits forming a complex cascade that can be altered by numerous changes,

it makes identifying the cause of the epidermal changes in XLRI difficult. From our data so

far, it is speculated that decreased JunB activity could be causing the changes.

XLRI has limited treatment options so far, with current treatment not targeting specific

pathways. A further understanding of exact pathways affected by STS gene knockout could

open novel treatment options. This work so far has suggested some interesting pathways for

follow-up to improve the treatment of XLRI.

References:

(1) Oji V, Tadini G, Akiyama M, Blanchet Bardon C, Bodemer C, Bourrat E, et al.

Revised nomenclature and classification of inherited ichthyoses: Results of the First

Ichthyosis Consensus Conference in Sorèze 2009. J Am Acad Dermatol. 2010 Oct

1;63(4):607–41.

(2) van Dam H, Castellazzi M. Distinct roles of Jun : Fos and Jun : ATF dimers in

oncogenesis. Oncogene. 2001 Apr 8;20(19):2453–64.

(3) Yoo M, Shin J, Kim J, Ryall KA, Lee K, Lee S, et al. DSigDB: drug signatures

database for gene set analysis. Bioinformatics. 2015 Sep 15;31(18):3069–71.