Analysis of the skin microbiome in health and disease

Humans have evolved facing a continuous exposure to infectious agents, and it
is likely that our genetic make-up has been shaped by selective pressures from
external microorganisms and our commensal flora of 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 (refs 1-3). Atopic dermatitis is a disease characterized by
inflammation and itch, affecting up to 15% of all children. 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. Psoriasis is also a common
inflammatory skin disease affecting 2% of the adult population. A major genetic
association was found with HLA-Cw6, and streptococci have been implicated in
disease development. Recent findings by our lab have elucidated a major part of
the genetic basis (>60%) when we found that polymorphisms in antimicrobial
proteins (beta-defensins) and skin barrier proteins (LCE3C/B) were associated
with the disease. Collectively, these data have prompted us to investigate the
role of microorganisms in psoriasis and atopic dermatitis.
Recent investigations have started to make an inventory of skin and gut
microbiome by high-throughput approaches such as microarrays or by conventional
sequencing approaches (refs 4-5). Both have their drawbacks, as microarrays
are restricted towards known sequences, and clone-by-clone sequencing yields
incomplete coverage. The deep-sequencing technology as will be used in this
project overcomes these problems, and allows unbiased identification of
virtually all skin-inhabiting organisms. Our aim is to establish causal
relationships between microbiome and human skin disease. This would not only
shed a completely different light on the pathogenesis, but also provide means
to investigate a different approach to prevention and therapy which would
entail the normalization of the bacterial skin, throat and gut flora, e.g. by
use of natural products like food or probiotics.

1. Palmer CN, Irvine AD, Terron-Kwiatkowski A, Zhao Y, Liao H, Lee SP, Goudie
DR, Sandilands A, Campbell LE, Smith FJ, O'Regan GM, Watson RM, Cecil JE, Bale
SJ,Compton JG, DiGiovanna JJ, Fleckman P, Lewis-Jones S, Arseculeratne G,
SergeantA, Munro CS, El Houate B, McElreavey K, Halkjaer LB, Bisgaard H,
Mukhopadhyay S, McLean WH Common loss-of-function variants of the epidermal
barrier protein filaggrin are a major predisposing factor for atopic
dermatitis. Nat Genet. 2006 38:441-6
2. Hollox EJ, Huffmeier U, Zeeuwen PL, Palla R, Lascorz J, Rodijk-Olthuis D,
van de Kerkhof PC, Traupe H, de Jongh G, den Heijer M, Reis A, Armour JA,
Schalkwijk J. Psoriasis is associated with increased beta-defensin genomic copy
number. Nat Genet. 2008 40:23-5
3. Rafael de Cid1*, Eva Riveira-Munoz1*, Patrick L.J.M. Zeeuwen2*, Jason
Robarge3* (*shared first authorship), Wilson Liao, Emma N. Dannhauser, Emiliano
Giardina, Philip E. Stuart, Rajan Nair, Cynthia Helms, Georgia Escaramís, Ester
Ballana, Gemma Martín-Ezquerra, Martin den Heijer, Marijke Kamsteeg, Irma
Joosten, Evan E. Eichler, Conxi Lázaro, Ramón M. Pujol, Lluís Armengol, Gonçalo
Abecasis, James T. Elder, Giuseppe Novelli, John A.L. Armour, Pui Kwok, Anne
Bowcock, Joost Schalkwijk, and Xavier Estivill. Deletion of the late cornified
envelope (LCE) 3B and 3C genes as a susceptibility factor for psoriasis. Nature
Genet 2009, 41:211-5
4. Gao Z, Tseng CH, Strober BE, Pei Z, Blaser MJ (2008). Substantial
alterations of the cutaneous bacterial biota in psoriatic lesions. PLoS ONE 3:
e2719.
5. Grice EA, Kong HH, Conlan S, Deming CB, Davis J, Young AC, Bouffard GG,
Blakesley RW, Murray PR, Green ED, Turner ML, Segre JA (2009). Topographical
and temporal diversity of the human skin microbiome. Science 324: 1190-1192.

The major aim is to apply deep sequencing technology to obtain a detailed
unbiased overview of the bacterial microbiome of human skin. This will be
performed in healthy individuals, psoriasis patients and atopic dermatitis
patients. Furthermore, we will study the dynamics of skin microbiome
composition in regressing lesions (patients) and following superficial injury
(tapestripping of healthy individuals). A second aim is to collect throat swaps
and stool samples from these individuals for sequencing of the corresponding
microbiomes. Bioinformatics will be used to defect patterns of microbiome
composition that correlate with disease or disease activity.

STUDY DESIGN

Methodological considerations
This is a pilot study on the microbiome of relatively small groups of
individuals. These analyses 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).

Experimental groups:

Group 1. normal skin of healthy controls (n=10)
Group 2. tape stripped skin of healthy volunteers (see below for tapestripping)
(n=10)
Group 3. lesional and non-lesional skin of un treated atopic dermatitis
patients (n=10)
Group 4. lesional and non-lesional skin of untreated plaque psoriasis patients
(n=10)
Group 5. lesional and non-lesional skin of untreated psoriasis inversa patients
(n=10)
Group 6. lesional skin and non-lesional skin, throat swabs and stool of plaque
psoriasis patients during therapy with systemic medication (n=10); as controls
we will study samples of normal skin, throat swabs and stools of healthy
controls (n=10)

Before the start of the actual study, optimization of skin sampling and
bacterial DNA extraction is performed, which requires a small number of
volunteers:

Group 7. optimization studies: controls (n=5) and psoriasis patients (n=5)


Analysis of skin microbiome

groups 1,3,4,5 and 7
Volunteers will be recruited from our list or via our outpatient department. An
anamnesis will be taken by a clinician who will be part of the project team. In
this way we will recruit individuals for experimental groups 1 to 5 and 7.
We will start by optimizing our sampling and DNA extraction procedure on skin
of healthy volunteers and psoriasis patients (group 7). We will compare skin
sampling by scraping with a scalpel and standard swabs. The best method (DNA
yield, microbial diversity) will be selected for further study.
DNA from skin samples of groups 1,3,4 and 5 will be extracted and amplified
with universal 16S RNA primers and subjected to deep sequencing using the Roche
454 technology. Bioinformatic analysis will be performed in collaboration with
NIZO (The Dutch Dairy Institute, Ede). In this way we will obtain a
comprehensive analysis of the relative quantities of various bacterial taxa in
the samples, under steady state conditions (normal skin, lesional psoriasis and
atopic dermatitis skin).
Volunteers will receive for sample collection in one visit a ¤20 fee and travel
expenses.



group 2: tape-stripped skin
In this group we will subject normal skin to superficial injury by repeated
application of adhesive tape on skin, which removes one layer of stratum
corneum with its constituent microorganisms. This can be done 20-30 times
before hitting the first viable cell layer which is where the sterile
compartment of the epidermis is likely to start. This procedure creates a
superficial injury whilst the normal skin flora is largely removed. The
epidermis will recover within a week by increasing cell proliferation and
altered differentiation. We will sample control skin at day 0 and day 14;
tape-stripped skin is samples at day 0, day 1, day 2, day 4, day 7 and day 14.
Bacterial DNA extraction and analysis is performed as described above.
Volunteers will receive a ¤180 fee and travel expenses.

group 6: patients during therapy
Individuals from group 6 are patients treated by biologicals (anti-TNF). These
are patients that receive regular therapy and will be asked to participate in
the following study: During a 3 month period following the start of the
treatment they will be asked to provide stool samples, throat swabs and skin
swabs/scrapings during regular control visits in our clinic (4 times). They
will receive a fee of ¤160 for participation. The samples will be stored for
further analysis (deep sequencing of the microbiome). In parallel, control
skin, throat swabs and stools from healthy volunteers will be analysed at two
time points (3 month interval). They will receive a fee of ¤80 and
reimbursement of travel expenses.
The analyses of these samples will teach us if the skin microbiome of lesional
skin normalizes under therapy, and if changes precede or follow clinical
improvement. It also allows us to correlate the skin changes with changes of
the microbiome in other locations (throat and gut).

No significant risks or burden. No benefit.