The long term effects of coal tar treatment on the microbiome and DNA methylome in atopic dermatitis skin.

Atopic dermatitis: clinical features and therapy
Humans have evolved facing a continuous exposure to infectious agents, and it
is likely that our genetic make-up has been shaped by the selective pressure
from external microorganisms and our commensal flora of the skin and gut.
Recent data on two major inflammatory skin diseases, psoriasis and atopic
dermatitis (*eczema*) have indicated that the chemical and physical barrier of
the skin, which protects us against infection, plays a crucial role in the
development of these diseases [1-3]. Atopic dermatitis is a disease
characterized by inflammation and itch, affecting up to 15% of children in the
Western society. Skin of these patients has a poor antimicrobial defense as
demonstrated by several labs including our own. Skin of atopic dermatitis
patients is often infected and nearly always colonized by Staphylococcus
aureus. Recently, it was found that mutations in filaggrin, a gene involved in
skin barrier function, explains a large part of the heritability of this
disease.
Patients with atopic dermatitis are often treated with topical corticosteroids,
systemic immunosuppressants or UV light therapy. Most of these treatments show
short remission times, or even rebound effects. In our day care practice
patients are often treated with coal tar, an ancient topical therapy of (until
recently) unknown mechanism. Recently, our group found coal tar to activate the
aryl hydrocarbon receptor (AhR), thereby unraveling (a part of) the working
mechanism. Activation of AhR by polycyclic hydrocarbons, which are present in
large amounts in coal tar, appears to (a) enhance epidermal differentiation
leading to restored epidermal barrier function thereby attenuating allergen
exposure, and (b) dampens the inflammatory cues of the T helper (Th)2 response
[4]. Treatment of patients with coal tar shows a favorable remission time in
comparison to other topical therapies, like corticosteroid application. A
scientific explanation for this has not been supplied thus far.

Metagenomics
Metagenomics is the study of genetic material recovered directly from
environmental samples or individuals. While traditional microbiology and
microbial genome sequencing and genomics rely on clonal cultures, early
environmental gene sequencing cloned specific genes (often the 16S rRNA gene)
to produce a profile of diversity in a natural sample. Such work revealed that
the vast majority of microbial biodiversity has been missed by
cultivation-based methods. These studies have used "shotgun" Sanger sequencing
or massively parallel pyrosequencing to get largely unbiased samples of all
genes from all the members of the sampled communities (so-called microbiomes).
Recent investigations have provided an inventory of the skin and gut microbiome
by current next generation sequencing (NGS) technologies allowing an unbiased
identification of virtually all skin-inhabiting bacteria [5, 6]. Since a
correlation between skin microbiome and atopic dermatitis has been reported
[7], restoring the normal skin microbiome could hold a therapeutic effect in
itself.

Epigenetics
Epigenetics is the study of heritable changes in gene activity that are not
caused by changes in the DNA sequence; it also can be used to describe the
study of stable, long-term alterations in the transcriptional potential of a
cell that are not necessarily heritable. The term also refers to the changes
themselves: functionally relevant changes to the genome that do not involve a
change in the nucleotide sequence. Examples of mechanisms that produce such
changes are DNA methylation and histone modification, each of which alters how
genes are expressed without altering the underlying DNA sequence. Epigentic
changes are likely to be involved in cellular differentiation of normal
epidermal morphogenesis. Recent studies have indicated that diseases (such as
psoriasis) can induce epigenetic changes in skin tissue that may contribute to
maintenance of disease [8]. Also, recent pilot studies have shown distinct
tissue-specific patterns of DNA methylation associated with atopic dermatitis
[9].

1. Palmer, C.N., et al., Common loss-of-function variants of the epidermal
barrier protein filaggrin are a major predisposing factor for atopic
dermatitis. Nat Genet, 2006. 38(4): p. 441-6.
2. Hollox, E.J., et al., Psoriasis is associated with increased beta-defensin
genomic copy number. Nat Genet, 2008. 40(1): p. 23-5.
3. de Cid, R., et al., Deletion of the late cornified envelope LCE3B and LCE3C
genes as a susceptibility factor for psoriasis. Nat Genet, 2009. 41(2): p.
211-5.
4. van den Bogaard, E.H., et al., Coal tar induces AHR-dependent skin barrier
repair in atopic dermatitis. J Clin Invest, 2013. 123(2): p. 917-27.
5. Gao, Z., et al., Substantial alterations of the cutaneous bacterial biota in
psoriatic lesions. PLoS One, 2008. 3(7): p. e2719.
6. Grice, E.A., et al., Topographical and temporal diversity of the human skin
microbiome. Science, 2009. 324(5931): p. 1190-2.
7. Kong, H.H., et al., Temporal shifts in the skin microbiome associated with
disease flares and treatment in children with atopic dermatitis. Genome Res,
2012. 22(5): p. 850-9.
8. Roberson, E.D., et al., A subset of methylated CpG sites differentiate
psoriatic from normal skin. J Invest Dermatol, 2012. 132(3 Pt 1): p. 583-92.
9. Rodriguez, E., et al., An Integrated Epigenetic and Transcriptomic Analysis
Reveals Distinct Tissue-Specific Patterns of DNA Methylation Associated with
Atopic Dermatitis. J Invest Dermatol, 2014.

We hypothesize that the long lasting therapeutic effect of coal tar could be
mediated by effects on the skin microbiome and/or effects on cellular memory by
epigenetic changes. In atopic dermatitis patients, coal tar therapy could
modify the skin microbiome in such a way that colonization by S.aureus is
reduced and that a *normal* skin microbiome is established (largely consisting
of S.epidermidis and P.acnes, no detectable S.aureus). In addition, coal tar
therapy could induce epigenetic changes in epidermal (stem) cells or
lymphocytes, that would render the skin resistant to disease for a prolonged
period, thereby explaining the remission time.

*
Our study aims to address these issues in:
(A) a short term study to address the effect of coal tar on the skin microbiome
of healthy volunteers
(B) a long term study of the microbiome of atopic dermatitis patients that are
treated with regular coal tar therapy in a day care setting
(C) a pilot study to investigate if coal tar application on lesional atopic
dermatitis skin causes gross epigenetic changes in the skin cells

A. EFFECT OF COAL TAR ON THE MICROBIOME OF HEALTHY VOLUNTEERS
Our goal is to study the effect of coal tar on the skin microbiome in a group
of healthy volunteers (n=10). Three areas (4 x 4 cm, divided in 4 quadrants) on
the lower back will be treated as follows:

1. control without treatment
2. lanette/vaseline followed by zinc-oxide paste(base ointment)
3. coal tar solution (LCD) 10% in lanette/vaseline followed by coal tar (pix
lithantracis) 5% in zinc oxide paste

Treatment will be stopped at day 7. Skin scrape samples (non-invasive) will be
taken before, during and after treatment at day 0, 2, 7 and 14, from the 4
quadrants respectively (to avoid repeated sampling from the same area).
Microbial DNA will be extracted from these skin scrape samples and analyzed.
All healthy volunteers will be reimbursed for their travel costs, based on
public transportation, and receive ¤50 for participation in the study.

Methodological considerations
This is an explorative study on the microbiome of a small group of healthy
individuals. The microbiome analysis will generate large amounts of
experimental data (>10.000 datapoints per sample, comparable to microarray
studies), creating the problem of family wise errors. For this reason the
analysis will not primarily focus on the quantitative comparison of individual
taxa but rather on the shifts of the microbial communities within samples (at
higher taxonomical aggregation levels, from genus to phylum).

Analysis of the skin microbiome
Microbial DNA from skin samples will be extracted and amplified using the
universal 16S rRNA primers and subjected to deep sequencing technology. Because
of the rapidly evolution of sequencing techniques, the exact method of deep
sequencing is to be determined. Bioinformatics analysis will be performed in
collaboration with NIZO (The Dutch Dairy Institute, Ede) and CMBI (Centre for
Molecular BioInformatics, Nijmegen). We have previously performed such studies
with success (see CMO 1243; this has resulted in a publication by Zeeuwen et al
2012, (Genome Biol. 2012 Nov 15;13(11):R101)

B. EFFECT OF COAL TAR ON SKIN MICROBIOME OF AD PATIENTS
We aim to analyze the skin microbiome of adult patients with atopic dermatitis
before, during and after treatment with coal tar (n=10). In addition we will
analyze the effect of long term coal tar treatment on the skin microbiome. This
is a regular AD treatment in the day care centre of our department. Patients
are enrolled for coal tar treatment via the out-patient department and will be
asked to participate in the following study protocol. Patients that are already
on coal tar treatment are excluded. Patients receiving therapy with indifferent
ointments or corticosteroids are eligible. Two areas of lesional AD skin, the
right and left inner elbow (ca 5 x 8 cm) are selected for the experiment. These
two areas will be divided in 4 quadrants for sampling at the first 4 timepoints.

Area #1: lanette/vaseline followed by zinc-oxide paste(control treatment, base
ointment)
Area #2: coal tar solution (LCD) 10% in lanette/vaseline followed by coal tar
(pix lithantracis) 5% in zinc-oxide paste

Five patients will have coal tar on the right arm and base ointment on the
left, and 5 patients vice versa. Except for the arm with the base ointment, the
patients receive the standard regular skin care. Patients will receive the
regular instruction by the nurse practictioner how to apply the ointments and
bandages at home. Patients will visit the day care clinic, 4 times during the
first 2 weeks. Thereafter, the visit frequency is reduced, as assessed by the
dermatologist. Third week: the experimental treatment is stopped and patients
are only treated by their regular coal tar therapy, at a frequency determined
by the medical staff.
Skin scrapes are taken at baseline (t=0) and at the 3 following visits to the
day care clinic (end of week 2). When patients have finished the therapy at the
day care clinic (on average between 4 and 12 weeks treatment), a skin scrape
will be taken from the experimental area #2 (= exit measurement). The patients
will be asked to continue participation in our study by bi-monthly visits and
skin sampling of experimental area #2. Patients are asked to contact us when
they feel that their eczema exacerbates. We will then take skin scrapes again
from area #2.
Patients that participate in our study will be reimbursed for unscheduled
travel costs based on public transportation. Apart from travel costs, patients
that volunteer to participate will receive ¤50 when the exit measurement is
done. Patients that participate in the follow-up study will receive an
additional ¤50.

Methodological considerations
The design is constrained by various practical, ethical and financial reasons.
We have opted for the simplest protocol possible, in order not to interfere
with daily practice at the day care centre, and to minimize the burden of the
patient. Nevertheless we feel that we will extract sufficient useful
information from these investigations.
The microbiome analysis will generate large amounts of experimental data
(>10.000 datapoints per sample, comparable to microarray studies), creating the
problem of family wise errors. For this reason the analysis will not primarily
focus on the quantitative comparison of individual taxa but rather on the
shifts of the microbial communities between samples (at higher taxonomical
aggregation levels, from genus to phylum).

Analysis of the skin microbiome
Microbial DNA from skin samples will be extracted and amplified using the
universal 16S rRNA primers and subjected to deep sequencing technology. Because
of the rapidly evolution of sequencing techniques, the exact method of deep
sequencing is to be determined. Bioinformatics analysis will be performed in
collaboration with NIZO (The Dutch Dairy Institute, Ede) and CMBI (Centre for
Molecular BioInformatics, Nijmegen). We have previously performed such studies
with success (see CMO 1243; this has resulted in a publication by Zeeuwen et al
(Genome Biol. 2012 Nov 15;13(11):R101).

C. EFFECT OF COAL TAR ON SKIN DNA METHYLOME
In a pilot study (3 patients) we want to investigate if gross epigenetic
changes can be observed in atopic dermatitis skin, following coal tar
treatment. When such changes are observed, a larger study with appropriate
controls will be designed. This will then include larger sample numbers and
other therapeutic modalities. In view of the high costs associated with this
kind of research, we think it is justifiable to perform an uncontrolled pilot
study first to see if there is any effect.
The study will be performed in 3 AD patients that will be treated with coal tar
solution (LCD) 10% in lanette/vaseline followed by coal tar (pix lithantracis)
5% in zinc-oxide paste. We will take three 4-mm full thickness skin biopsies
from lesional skin, before and after topical treatment for 7 days (6 biopsies
in total).
Patients will be recruited via our list of volunteers, our day care centre or
out-patient department. Volunteers will receive a ¤15,- fee per biopsy and
additional travel costs will be reimbursed, based on public transportation.
Biopsies will be processed for DNA extraction and subsequent methylome analysis.

Analysis of the skin DNA methylome
DNA methylation status from skin biopsies will be analyzed in collaboration
with the Department of Molecular Biology (Radboud Institute of Molecular Life
Sciences, RIMLS, Nijmegen). Because of the rapid evolution of methylome
analysis techniques, the exact method of analysis will be chosen in a later
state. Bioinformatics analysis will be performed in collaboration with the CMBI
(Centre for Molecular BioInformatics, Nijmegen).

Collection of skin scrapes is not invasive and poses a limited burden on the
participants. Full thickness skin biopsies usually heal well, with a minor
scar. Individuals that have a tendency to develop hypertrophic scars or keloids
are excluded. None of the participants have direct benefits of the proposed
studies.
For patients that participate in study B there is the following burden:
normally they would be completely treated with coal tar, but here one arm will
be treated with base ointment without coal tar for 2 weeks. From week 2 onwards
they will receive the regular coal tar therapy.