ArticlePDF Available

Abstract and Figures

The role of microbial dysbiosis in scalp disease has been recently hypothesized. However, little information is available with regards to the association between microbial population on the scalp and hair diseases related to hair growth. Here we investigated bacterial communities in healthy and Alopecia areata (AA) subjects. The analysis of bacterial distribution at the genus level highlighted an increase of Propionibacterium in AA subjects alongside a general decrease of Staphylococcus. Analysis of log Relative abundance of main bacterial species inhabiting the scalp showed a significant increase of Propionibacterium acnes in AA subjects compared to control ones. AA scalp condition is also associated with a significant decrease of Staphylococcus epidermidis relative abundance. No significant changes were found for Staphylococcus aureus. Therefore, data from sequencing profiling of the bacterial population strongly support a different microbial composition of the different area surrounded hair follicle from the epidermis to hypodermis, highlighting differences between normal and AA affected the scalp. Our results highlight, for the first time, the presence of a microbial shift on the scalp of patients suffering from AA and gives the basis for a larger and more complete study of microbial population involvement in hair disorders.
Content may be subject to copyright.
RESEARCH ARTICLE
Scalp bacterial shift in Alopecia areata
Daniela Pinto
1,2,3
, Elisabetta Sorbellini
2,3
, Barbara Marzani
1,2,3
, Mariangela Rucco
3
,
Giammaria Giuliani
1,2
, Fabio RinaldiID
1,2,3
*
1Giuliani SpA, Milan, Italy, 2Human Advanced Microbiome Project-HMAP, Milan, Italy, 3International Hair
Research Foundation (IHRF), Milan, Italy
*fabio.rinaldi@studiorinaldi.com
Abstract
The role of microbial dysbiosis in scalp disease has been recently hypothesized. However,
little information is available with regards to the association between microbial population on
the scalp and hair diseases related to hair growth. Here we investigated bacterial communi-
ties in healthy and Alopecia areata (AA) subjects. The analysis of bacterial distribution at the
genus level highlighted an increase of Propionibacterium in AA subjects alongside a general
decrease of Staphylococcus. Analysis of log Relative abundance of main bacterial species
inhabiting the scalp showed a significant increase of Propionibacterium acnes in AA sub-
jects compared to control ones. AA scalp condition is also associated with a significant
decrease of Staphylococcus epidermidis relative abundance. No significant changes were
found for Staphylococcus aureus. Therefore, data from sequencing profiling of the bacterial
population strongly support a different microbial composition of the different area sur-
rounded hair follicle from the epidermis to hypodermis, highlighting differences between nor-
mal and AA affected the scalp. Our results highlight, for the first time, the presence of a
microbial shift on the scalp of patients suffering from AA and gives the basis for a larger and
more complete study of microbial population involvement in hair disorders.
Introduction
Alopecia areata (AA) is the second most common type of hair loss disorder for human beings.
It occurs in the form of a non-scarring alopecia which affects the scalp and, eventually, the
entire body [1]. An incidence higher than 2% has been reported for AA, with a lifetime risk of
1.7% both in men and women [2].
For subjects affected by AA, the catagen phase is either extremely short or doesn’t occur at
all, and in turn proceeds rapidly to telogen phase. From a clinical point of view, this led to sin-
gle or several annular or patchy bald lesions usually on the scalp [3,4]. These lesions can extend
to the entire scalp (Alopecia totalis) or to the entire pilar area of the body (Alopecia
universalis).
The management of AA still remains a challenge and is mainly aimed at containing it.
Among treatments currently available [5], in 2012, the British Association of Dermatologists
recommended two main treatments with a C grade of recommendation: i) topical and intrale-
sional corticosteroid (limited patchy hair loss); ii) immunotherapy (extensive patchy hair loss
and Alopecia totalis/universalis) [6].
PLOS ONE | https://doi.org/10.1371/journal.pone.0215206 April 11, 2019 1 / 11
a1111111111
a1111111111
a1111111111
a1111111111
a1111111111
OPEN ACCESS
Citation: Pinto D, Sorbellini E, Marzani B, Rucco M,
Giuliani G, Rinaldi F (2019) Scalp bacterial shift in
Alopecia areata. PLoS ONE 14(4): e0215206.
https://doi.org/10.1371/journal.pone.0215206
Editor: Brenda A. Wilson, University of Illinois at
Urbana-Champaign, UNITED STATES
Received: July 6, 2018
Accepted: March 28, 2019
Published: April 11, 2019
Copyright: ©2019 Pinto et al. This is an open
access article distributed under the terms of the
Creative Commons Attribution License, which
permits unrestricted use, distribution, and
reproduction in any medium, provided the original
author and source are credited.
Data Availability Statement: All relevant data are
within the paper.
Funding: Financial assistance received from by
Giuliani SpA. The funder provided support in the
form of salaries for authors [DP, BM and FR], but
did not have any additional role in the study design,
data collection and analysis, decision to publish, or
preparation of the manuscript. The specific roles of
these authors are articulated in the ‘author
contributions’ section.
Competing interests: DP, BM are employed by
Giuliani S.p.A.; FR serves as consultant for Giuliani
S.p.A; GG is part of Board of Directors of Giuliani S.
Causes behind AA are not yet fully understood, and there have been debates dating back to
the beginning of the 1800s. Many associations have been proposed by researchers over the
years [7]. However, clinical evidence and association with other immune disorders [8] under-
line the role of immunity and inflammation in the early development of AA [911]. Interest-
ingly, authors [11] reported the efficacy of PRP (Platelet-rich plasma) on AA as a potent anti-
inflammatory agent by suppressing cytokine release and limiting local tissue inflammation
[11].
Other comimon (common?) recognized offenders are hormonal imbalance, psychological
stress, genetic tendencies, other local skin disorders and also nutritional deficiencies [5]. More
recently, some authors reported evidence of the link between the gut microbiome and AA
[12,13] but little information is currently available as regards microbial communities on the
scalp [14,15]. Due to its unique features, the scalp is expected to harbor a specific microbiome,
which is expected to play a peculiar role in scalp conditions related to hair growth [16].
In this work, we present data on bacterial communities in healthy and AA subjects, on a
sample of Italian population. Our results highlight, for the first time, the presence of a signifi-
cative bacterial disequilibrium on the scalp of AA subjects compared to healthy population;
this disequilibrium also extends in the subepidermal compartments of the scalp.
Material and methods
Subjects recruitment
Fifteen healthy and AA subjects, respectively (20–60 years old; 40% male) were recruited from
a private Italian dermatological clinic (Milan, Italy).
All subjects have been enrolled under dermatological control. AA subjects have been previ-
ously evaluated about their disease history and by means of clinical examinations. Subjects
have been enrolled in control population after clinical examinations and in absence of any his-
tory of dermatological or scalp disorders.
All enrolled subjects had to meet the following criteria: i) no antibiotics in the 30 days lead-
ing up to the sampling; ii) no probiotics in the last 15 days; iii) the last shampoo was performed
48h before sampling; iv) no pregnancy or lactation; v) suffering from other dermatological dis-
eases; vi) no anti-tumor, immunosuppressant or radiation therapy in the last 3 months; vii) no
topical or hormonal therapy on the scalp in the last 3 months.
The study was approved by the Ethical Independent Committee for Clinical, not pharmaco-
logical investigation in Genoa (Italy) and in accordance with the ethical standards of the 1964
Declaration of Helsinki. All of the volunteers signed the informed consent.
Swab sample collection
The scalp surface has been sampled by means of swab procedure according to previously
reported methods [17,18] with minor modifications. Sterile cotton swabs were soaked for at
least 30s in ST solution (NaCl 0.15 M and 0.1% Tween 20) before sampling. A comb was used
to separate hair fibers and collect samples from a total area of 16cm
2
from a different area of
the scalp. After collection, the head of each swab was cut and stored in ST solution. Samples
from the same subjects were collected together and stored at 4˚C until DNA extraction. Sterile
cotton swabs placed in ST solution have been used as negative controls.
Biopsy samples collection
A total of 4 female subjects (two control and two AA, respectively) were also sampled for the
microbial community in the subepidermal compartments of the scalp. A 4-mm punch biopsy
Scalp bacterial shift in Alopecia areata
PLOS ONE | https://doi.org/10.1371/journal.pone.0215206 April 11, 2019 2 / 11
p.A. This does not alter our adherence to PLOS
ONE policies on sharing data and materials.
specimen was collected from each subject. In AA subjects, the specimen was obtained from a
well-developed lesion. The sampled area was disinfected prior to the surgery to avoid contami-
nation from surface bacteria. Epidermis, dermis and hypodermis were aseptically separated
and stored in Allprotect medium (Qiagen) according to manufacturer conditions until DNA
extraction.
Bacterial DNA extraction
Bacterial DNA from scalp swabs was extracted by mean of QIAamp UCP Pathogen Mini Kit
(Qiagen, Milan, Italy) according to manufacturer protocol, with minor modifications [19].
The DNeasy Tissue kit (Qiagen, Milan, Italy) was used for DNA extraction from biopsy speci-
mens. Extracted DNA was finally suspended in DNAse free water and quantified by the
QIAexpert system (Qiagen, Milan, Italy) before qRT-PCR and sequencing.
High throughput 16S amplicon generation, sequencing and analysis
DNA samples extracted from scalp swabs were amplified for the variable region V3-V4 using the
universal prokaryotic primers: 341 F CTGNCAGCMGCCGCGGTAA [20,21] and 806bR GGACTA
CNVGGGTWTCTAAT [2224] utilizing a modified dual-indexed adapter-linked single step pro-
tocol. Library preparation and Illumina MiSeq V3-V4 sequencing were carried out at StarSEQ
GmbH, Mainz, Germany, according to the method of Caporaso et al. [25] and Kozich et al., [26]
with minor modifications. Amplicons were generated using a high fidelity polymerase (AccuS-
tart II PCR ToughMix, Quantabio, Beverly, MA). The amplicons were then normalized to equi-
molar concentrations using SequalPrep Plate Normalization Kit (ThermoFisher Scientific,
Monza, Italy) and the final concentration of the library was determined using a fluorometric kit
(Qubit, Life technologies, Carlsbard, CA, USA). Libraries were mixed with Illumina-generated
PhiX control libraries and denatured using fresh NaOH. Runs were performed using Real-Time
Analysis software (RTA) v. 1.16.18 and 1.17.22, MiSeq Control Software (MCS) v. 2.0.5 and
2.1.13, varying amounts of a PhiX genomic library control, and varying cluster densities. Four
sequencing runs were performed with RTA v. 1.18.54, MCS v. 2.6, a target of 25% PhiX, and
600–700 k/mm2 cluster densities according to Illumina specifications for sequencing of low
diversity libraries. We used 25% PhiX to balance the runs and use 600 bp V3 chemistry for
sequencing. Basecalls from Illumina High Throughput Sequencing (HTS) machines were con-
verted to fastQ files using bcl2fastq (Illumina) software, v2.20.0.42 and quality control carried
out by mean of, v0.11.5. bcl2fastq (Illumina) software, v2.20.0.422. Quality control of fastq reads
was carried out using FastQC v0.11.5. The quality trimming of primers and adaptors was carried
out using Cutadapt, v. 1.14 [27] and Sickle v. 1.33 [28] toolkits, respectively.
Paired-end reads were assembled using Pandaseq v. 2.11[29] using a threshold of 0.9 and a
minimum overlap region length of 50. Clustering was carried out using closed-reference OTU
picking and de novo OUT picking protocol of QIIME v1.9 [25] at 97% identity.
Greengenes database v13_8 was used as a reference for bacterial taxonomic assignment
[30]. Amplicon reads were also analyzed as regards alpha diversity by mean of Shannon index,
using QIIME v1.9.
Bacteria quantification by qRT-PCR
Relative abundance of bacterial DNA of main bacterial species on the scalp was assessed by
means of real-time quantitative PCR (RT qPCR). Microbial PCR assay kit (Qiagen, Milan, Italy)
with gene-specific primers and TaqMan MGB probe targeting Propionibacterium acnes,Staphy-
lococcus epidermidis and Staphylococcus aureus 16S rRNA gene, respectively, were used. Gen-
bank accession numbers of 16S rRNA gene sequences for P. acnes, S. aureus and S. epidermidis
Scalp bacterial shift in Alopecia areata
PLOS ONE | https://doi.org/10.1371/journal.pone.0215206 April 11, 2019 3 / 11
were ADJL01000005.1, ACOT01000039.1 and ACJC01000191.1, respectively. Samples were
mixed with 12.5μL of Microbial qPCR Mastermix, 1 μL of Microbial DNA qPCR Assay, 5ng of
genomic DNA sample and Microbial-DNA-free water up to a final volume of 25 μL.
Nine separate PCR reactions are prepared for each sample, including Positive PCR Control,
No Template Control, and Microbial DNA Positive Control, as well as the Microbial DNA
qPCR Assay. Pan-bacteria (Genebank accession number HQ640630.1) assays that detect a
broad range of bacterial species are included to serve as positive controls for the presence of
bacterial DNA. Assays for human GAPDH and HBB1 (Genebank accession numbers
NT_009759.16 and NT_009237.18, respectively) have been included to determine proper sam-
ple collection and used to assess the presence of human genomic DNA in the sample and,
eventually, subtracted from calculation. Thermal cycling conditions used were as follows; 95˚C
for 10 min, 40 cycles of 95˚C for 15 sec, 60˚C for 2 min. PCR reactions were performed in
duplicate using an MX3000p PCR machine (Stratagene, La Jolla, CA). Amplification-curve
plotting and calculation of cycle threshold (Ct) values were performed using MX3000p soft-
ware (v.3; Stratagene) and data were further processed by Excel. ΔΔCt method [31] was used
to calculate bacterial load of each swab sample. Obtained values have been used for calculation
of Bacterial Load-Fold Change (AA/Healthy subjects). Data is finally expressed as Log of the
relative abundance of each sample versus the control group.
Statistical analysis
Data is expressed as log Relative abundance (RA) ±SEM for qRT-PCR analysis. Results were
checked for normal distribution using D’Agostino & Pearson normality test before further
analyses. Statistically significant differences on bacterial community between healthy and AA
group were determined using Wilcoxon test (p 0.05). All the comparisons were performed
pairwise for each group. Analyses were performed with GraphPad Prism 7.0 (GraphPad Soft-
ware, Inc., San Diego, CA). P–values equal to or less than 0.05 were considered significant.
Results
Microbiota profiling of the scalp in AA subjects
The human scalp’s bacterial composition of Control (n = 15) and AA (N = 15) subjects have
been analyzed by IlluminaSeq (Fig 1). We obtaining about 585,219 and 544,578 high quality
reads for the total V3-V4 sequences from control and AA subjects, respectively. About 56.3%
of sequences from the control group were assigned to Actinobacteria phylum and 35.2% to Fir-
micutes. As regards, AA group Actinobacteria were around 57.4% and Firmicutes decreased to
29.2%. The analysis of bacterial distribution at the genus level, interestingly, highlighted an
increase of Propionibacterium from 45.6% to 55.1% in AA subjects. Alongside data showed a
general decrease of Staphylococcus from 32.6% to 27.4% (Fig 1A). Therefore, the percentage of
other less abundant bacteria genus was similar (around 5%) both in control and AA subjects.
Alpha-diversity (Shannon diversity index) was significantly higher (p 0.001) in AA subjects
than in the control group (Fig 1B).
Microbial shift of the scalp surface in AA subjects
As previously reported by other authors [14,15], P.acnes,S.epidermidis and S.aureus are the
three major microbial species found on the scalp.
Relative abundance of predominant bacteria on scalps both of control and AA subjects has
been analyzed by mean of RT q-PCR. Primers and TaqMan MGB probe specific for 16S region
of P.acnes,S.epidermidis and S.aureus were used.
Scalp bacterial shift in Alopecia areata
PLOS ONE | https://doi.org/10.1371/journal.pone.0215206 April 11, 2019 4 / 11
Pan bacteria specific targets designed to detect the broadest possible collection of bacteria
involved in human biology were used as control. Student’s test analysis of log Relative abun-
dance comparing control and AA subjects showed a significant (p<0.01) increase of P.acnes
(from 1.6 to 1.8 log RA) in AA subjects compared to control ones (Fig 2A). AA scalp condition
is also associated with a significant (p<0.05) decrease of S.epidermidis relative abundance
(from 1.4 to 1.01 log RA) (Fig 2B) while no significant changes were found for S.aureus
(Fig 2C).
Microbial shift due to AA is also clear as regards the proportion of bacterial populations
analyzed. The ratio P.acnes/ S.epidermidis is significantly higher (p<0.05) in AA subjects
(mean ratio = 2.1±0.3) compared to control subjects (mean ratio = 1.3±0.1) (Fig 2D). Addi-
tionally, the P.acnes/ S.aureus ratio was also significantly higher (p<0.01) in AA subjects
(mean ratio = 1.4±0.1 vs mean ratio = 1.2±0.1) (Fig 2E). No significative differences were
found in the ratio S.epidermidis / S.aureus (Fig 2F).
Fig 1. Bacterial profiling in control and AA subjects. (A)% of bacteria at genus level in the control and AA groups.
Results are presented as the percentage (%) of total sequences, (p0.05). (B) Shannon diversity index for bacterial
population observed in control and AA subjects (p0.05).
https://doi.org/10.1371/journal.pone.0215206.g001
Fig 2. Relative abundance of main bacterial species on the scalp of AA andcontrol subjects by RT qPCR. Box and
Whisker comparing the log relative abundance of P.acnes,S.epidermidis and S.aureus collected by swabbing the scalp.
(A) Log Relative abundance of P.acnes in Control and AA subjects. (B) Log Relative abundance of S.epidermidis in
Control and AA subjects. (C) Log Relative abundance of S. aureus in Control and AA subjects. Ratios P. acnes/ S.
epidermidis (D), P. acnes/ S. aureus (E) and S. epidermidis / S. aureus (F) in Control and AA subjects. Values are
presented as mean +/- SEM, in duplicate. Box-and-Whiskers plot showing median with 25th to 75th percentile. The
center line of each box represents the median; data falling outside the whiskers range are plotted as outliers of the data.
https://doi.org/10.1371/journal.pone.0215206.g002
Scalp bacterial shift in Alopecia areata
PLOS ONE | https://doi.org/10.1371/journal.pone.0215206 April 11, 2019 5 / 11
AA alteration of bacterial distribution in the subepidermal compartments
of the scalp
Two bioptic samples were collected respectively from control and AA subjects and divided in
the main subepidermal compartments. Extracted genomic DNAs were analyzed by Illumina-
Seq and analyzed for bacterial distribution.
Similar proportions of Firmicutes (24.6% vs 27.6%) and Proteobacteria (16.2% vs 16.9%)
were reported in epidermis of both control and AA subjects (Fig 3) while a higher proportion
of Actinobacteria (33.3% vs 22.4%) and Bacteroidetes (20.1% vs 9.9%) were found in AA sub-
jects compared to control (Fig 3). Bacterial community in dermis shifted to a lower proportion
of Actinobacteria (6.1% vs 11.3%) in AA subjects while Proteobacteria (14.9% vs 8.1%) and
Bacteroidetes (14.2% vs 4.0%) increased compared to control (Fig 3). Also hypodermis showed
a peculiar bacterial distribution which results, also in this case affected by scalp condition. AA
subjects showed a significative higher proportion of Proteobacteria,Bacteroidetes and espe-
cially Firmicutes than control subjects (Fig 3). In general less variability was observed for bacte-
rial communities in AA subjects and this may reflect in a compromised healthiness of the
scalp.
Most interesting, the analysis at species level of bioptic samples highlighted the presence of
Prevotella copri in both AA samples, in all analyzed compartments.
Akkermansia muciniphila was also found (less than 1.5% of total population) in AA sub-
compartments of the scalp, in particular in the hypodermis.
Discussion
In this study, we reported, for the first time, the relationship between microbial shift on the
scalp and hair growth disorder, in particular, Alopecia areata. We conducted analysis by mean
of qRT-PCR and 16S sequencing.
A diversified and abundant microbial community host the skin [32] and this symbiotic rela-
tionship results, most of the time, as beneficial for both the host and microbial community
[3335]. Bacteria mainly belong to Corynebacteriaceae, Propionibacteriae, and
Fig 3. Bacterial profiling of scalp biopsy samples from control and AA subjects. % of bacteria at phylum level in the
control and AA groups in the epidermis, dermis and hypodermis. Results are presented as the percentage (%) of total
sequences.
https://doi.org/10.1371/journal.pone.0215206.g003
Scalp bacterial shift in Alopecia areata
PLOS ONE | https://doi.org/10.1371/journal.pone.0215206 April 11, 2019 6 / 11
Staphylococcaceae [3639] and are differently distributed according to the physiochemical
properties of each skin site they host [39,40]. Many scientific published evidence reported the
strict correlation between microbial disequilibrium and skin conditions [4145]. Little is still
reported with regards to the microbiome inhabiting the scalp and hair growth disorders
[14,15,46]. Clavaud and collaborators [15] and, more recently, Xu et al. [14] reported, the
implication of microorganisms in the development of dandruff. Characterization of scalp bac-
terial species involved in hair disorders such as Alopecia androgenetica, Alopecia areata, and
Lichen Planopilaris has been poorly investigated and, only recently, the piece bit of evidence
has been reported [16].
We focused our attention on bacterial population of the scalp of healthy and AA subjects
looking at main bacterial species on the scalp [15] (P.acnes,S.aureus, and S.epidermidis) and
at their reciprocal balancing. We quantified their relative abundance by means of accurate
gene-specific primers and probe targeting 16S region, by RT qPCR. Our results are concurrent
with Wang’s work [46] highlighting the reciprocal inhibition exerted by bacteria, each other,
on the scalp (Propionibacterium vs Staphylococcus and vice-versa). AA subjects showed an
increase in P.acnes and a decrease of Staphylococcus, especially S.epidermidis, suggesting the
role of Propionibacterium/Staphylococcus balancing in AA. A role of P.acnes with hair casts
and Alopecia has previously been hypothesized by Wang and collaborators [46] even though
not deeply investigated. P.acnes is able to synthesize many enzymes involved in the metabo-
lism of porphyrins that, once activated, may contribute to oxidation and follicular inflamma-
tion. Therefore, a speculation about the role of the hypoxic condition of the follicular region
may be speculated in AA and this may encourage P.acnes overgrowth. A role of hypoxia has
been reported in the progression of other skin condition such as psoriasis [47] and atopic der-
matitis [48]. The presence of A.muciniphila, a strictly anaerobic bacteria, around the hair folli-
cle in analyzed AA subjects may be suggestive of a hypoxic ecosystem in which this bacteria
can find favorable growth conditions.
Data from IlluniaSeq profiling also suggested a higher diversity of bacterial species inhabit-
ing the scalp of AA subjects. These results are in line with previous work [15] on other scalp
conditions. On the basis of the present and previous results, a link with a higher susceptibility
of an unhealthy scalp to be colonized by microorganisms could be postulated but further anal-
ysis are needed to understanding the reason behind this high variety.
Beyond the superficial relationship between the microorganism with skin, microbes can
also communicate with cells of the subepidermal compartments [49] and are involved also in
deep immunological response [5054]. As reported by Nakatsuji et al., [49] high interpersonal
variability was observed as regards epidermal and subepidermal microbial population. In this
study, data from sequencing profiling of the bacterial population strongly support a different
microbial composition of different area surrounded hair follicle from the epidermis to hypo-
dermis, highlighting differences between normal and AA affected scalp. We can hypothesize
the role of this different microbial composition in AA symptoms and manifestations.
Microbial changing at different subepidermal compartment may be linked to an autoim-
mune component of the pathology as to skin barrier skin disruption, as previously shown for
other skin disorders [55].
Most interesting, the analysis at species level of bioptic samples highlighted the peculiar
presence of P.copri and A.muciniphila in both AA samples, in all analyzed compartments.
These findings are very intriguing. The finding of Prevotella copri as one of the most abundant
bacteria in subepidermal compartments of AA scalp may be linked to the autoimmune compo-
nent of this hair condition. For example, P.copri has been found as relevant in the pathogene-
sis of rheumatoid arthritis [56], another chronic inflammatory autoimmune disorder that can
affect other parts of the body including the skin. Therefore the identification of A.muciniphila
Scalp bacterial shift in Alopecia areata
PLOS ONE | https://doi.org/10.1371/journal.pone.0215206 April 11, 2019 7 / 11
in the subepidermal compartments of the scalp of AA subjects could open to new therapeutic
approaches in the management of AA. The link between A.muciniphila and skin disease has
been yet discussed as it has been considered a gut signature of psoriasis [57].
The present work reported data from an initial pilot study. Future studies should be aimed
at better investigate both the role of microbial community shifts and hypoxia in hair scalp dis-
eases. Also the study of additional factors such as inclusion of samples from non-lesional sites
in AA and non-AA subjects and from other baldness disease besides AA and the role should
be considered.
Conclusions
Our study highlighted, for the first time, the presence of a microbial shift on the scalp of
patients suffering from AA and gives the basis for a larger and more complete study of micro-
bial population involvement in hair disorders. Therefore, the reported findings as the availabil-
ity of sophisticated and quick methods to evaluate the microbial composition of the scalp open
to new therapeutic approaches in the management of hair disorders.
Larger studies are still needed for a more precise identification of bacterial community on
the scalp as for the analysis of fungal component in AA subjects but the results of the present
work permit to asses, for the first time, the involvement of microbial changing in hair disorder,
in particular AA, also in the subepidermal compartments of the scalp.
Author Contributions
Conceptualization: Elisabetta Sorbellini, Barbara Marzani, Giammaria Giuliani, Fabio
Rinaldi.
Data curation: Daniela Pinto, Mariangela Rucco.
Formal analysis: Daniela Pinto.
Investigation: Daniela Pinto, Elisabetta Sorbellini, Mariangela Rucco, Fabio Rinaldi.
Methodology: Daniela Pinto, Fabio Rinaldi.
Supervision: Elisabetta Sorbellini, Fabio Rinaldi.
Writing – original draft: Daniela Pinto, Elisabetta Sorbellini, Barbara Marzani, Fabio Rinaldi.
Writing – review & editing: Daniela Pinto, Elisabetta Sorbellini, Barbara Marzani, Fabio
Rinaldi.
References
1. Odom RB, Davidsohn IJ, William D, Henry JB, Berger TG. Clinical diagnosis by laboratory methods. In:
Elston, Dirk M. (Ed.), Andrews’ Diseases of the Skin: Clinical Dermatology. Saunders Elsevier. 2006.
2. Dawber R. Alopecia areata. Monogr Dermatol. 1989; 2:89–102.
3. Tan E, Tay YK, Goh CL, Chin Giam Y. The pattern and profile of alopecia areata in Singapore—a study
of 219 Asians. Int J Dermatol. 2002 Nov; 41(11):748–53. PMID: 12452996
4. Camacho F. Alopecia areata. Clinical characteristics and dermatopathology. In: Trichology: Diseases of
the Pilosebaceous Follicle. Aula Medical Group S. A, Madrid; 1997. pp. 440–471.
5. Syed SA, Sandeep S. Alopecia areata: A review. Journal of the Saudi Society of Dermatology & Der-
matologic Surgery. 2013 Jul; 17(2):37–45.
6. Messenger AG, McKillop J, Farrant P, McDonagh AJ, Sladden M. British Association of Dermatologists’
guidelines for the management of alopecia areata 2012. Br J Dermatol. 2012 May; 166(5):916–26.
https://doi.org/10.1111/j.1365-2133.2012.10955.x PMID: 22524397
7. McElwee KJ, Gilhar A, Tobin DJ, Ramot Y, Sundberg JP, Nakamura M, et al. What causes alopecia
areata? Exp Dermaol. 2013; 22(9):609–626.
Scalp bacterial shift in Alopecia areata
PLOS ONE | https://doi.org/10.1371/journal.pone.0215206 April 11, 2019 8 / 11
8. McDonagh AJ, Tazi-Ahnini R.Epidemiology and genetics of alopecia areata. Clin Exp Dermatol. 2002;
27, 405–409. PMID: 12190641
9. Hordinsky M, Ericson M. Autoimmunity: alopecia areata. J Investig Dermatol SympProc. 2004 Jan; 9
(1):73–8. Review https://doi.org/10.1111/j.1087-0024.2004.00835.x PMID: 14870990
10. Brenner W, Diem E, Gschnait F. Coincidence of vitiligo, alopecia areata, onychodystrophy, localized
scleroderma and lichen planus. Dermatologica. 1979; 159(4):356–60. PMID: 478074
11. Trink A, Sorbellini E, Bezzola P, Rodella L, Rezzani R, Ramot Y, et al. A randomized, double-blind, pla-
cebo- and active-controlled, half-head study to evaluate the effects of platelet-rich plasma on alopecia
areata. Br J Dermatol.2013 Sep; 169(3):690–4. https://doi.org/10.1111/bjd.12397 PMID: 23607773
12. Borde A, Åstrand A. Alopecia areata and the gut-the link opens up for novel therapeutic interventions.
Expert Opin Ther Targets. 2018 Jun; 22(6):503–511. https://doi.org/10.1080/14728222.2018.1481504
PMID: 29808708
13. Rebello D, Wang E, Yen E, Lio PA, Kelly CR. Hair Growth in Two Alopecia Patients after Fecal Micro-
biota Transplant. ACG Case Rep J. 2017 Sep 13; 4:e107. https://doi.org/10.14309/crj.2017.107 PMID:
28932754
14. Xu Z, Wang Z, Yuan C, Liu X, Yang F, Wang T et al. Dandruff is associated with the conjoined interac-
tions between host and microorganisms. Scientific Reports. 2016; 6:24877. https://doi.org/10.1038/
srep24877 PMID: 27172459
15. Clavaud C, Jourdain R, Bar-Hen A, Tichit M, Bouchier C, Pouradier F, et al. Dandruff is associated with
disequilibrium in the proportion of the major bacterial and fungal populations colonizing the scalp. PLoS
One. 8(3):e58203. Erratum in: PLoS One 2013;8(10). https://doi.org/10.1371/journal.pone.0058203
PMID: 23483996
16. Rinaldi F, Pinto D, Marzani B, Rucco M, Giuliani G, Sorbellini E. Human microbiome: What’s new in
scalp diseases. J Transl Sci. 2018 Apr; Volume 4(6): 1–4.
17. Grice EA, Kong HH, Conlan S, Deming CB, Davis J, Young AC, et al. Topographical and Temporal
Diversity of the Human Skin Microbiome. Science (New York, NY). 2009; 324(5931):1190–1192.
https://doi.org/10.1126/science.1171700 PMID: 19478181
18. Paulino LC, Tseng CH, Strober BE, Blaser MJ. Molecular analysis of fungal microbiota in samples from
healthy human skin and psoriatic lesions. J Clin Microbiol. 2006 Aug; 44(8):2933–41. https://doi.org/10.
1128/JCM.00785-06 PMID: 16891514
19. Gao Z, Perez-Perez GI, Chen Y, Blaser MJ. Quantitation of Major Human Cutaneous Bacterial and
Fungal Populations. Journal of Clinical Microbiology. 2010; 48(10):3575–3581. https://doi.org/10.1128/
JCM.00597-10 PMID: 20702672
20. Klindworth A, Pruesse E, Schweer T, Peplies J, Quast C, Horn M, et al. Evaluation of general 16S ribo-
somal RNA gene PCR primers for classical and next-generation sequencing-based diversity studies.
Nucleic Acids Res. 2013 Jan; 7; 41(1):e1. https://doi.org/10.1093/nar/gks808 PMID: 22933715
21. Takahashi S, Tomita J, Nishioka K, Hisada T, Nishijima M. Development of a Prokaryotic Universal
Primer for Simultaneous Analysis of Bacteria and Archaea Using Next-Generation Sequencing. Bourt-
zis K, ed. PLoS ONE. 2014; 9(8):e105592. https://doi.org/10.1371/journal.pone.0105592 PMID:
25144201
22. Apprill A, McNally S, Parsons R, Weber L. Minor revision to V4 region SSU rRNA 806R gene primer
greatly increases detection of SAR11 bacterioplankton. Aquat Microb Ecol. 2015; 75:129–137.
23. Parada AE, Needham DM, Fuhrman JA. Every base matters: assessing small subunit rRNA primers for
marine microbiomes with mock communities, time series and global field samples. Environ Microbiol.
2016 May; 18(5):1403–14. https://doi.org/10.1111/1462-2920.13023 PMID: 26271760
24. Walters W, Hyde ER, Berg-Lyons D, Ackermann G, Humphrey G, Parada A. Improved Bacterial 16S
rRNA Gene (V4 and V4-5) and Fungal Internal Transcribed Spacer Marker Gene Primers for Microbial
Community Surveys. 2016. 1(1), e00009–15.
25. Caporaso JG, Lauber CL, Walters WA, Berg-Lyons D, Lozupone CA, Turnbaugh PJ, et al. Global pat-
terns of 16S rRNA diversity at a depth of millions of sequences per sample. Proc Natl Acad Sci U S A.
2011; 108(Suppl 1):4516–22.
26. Kozich JJ, Westcott SL, Baxter NT, Highlander SK, Schloss PD. Development of a dual-index sequenc-
ing strategy and curation pipeline for analyzing amplicon sequence data on the MiSeq Illumina sequenc-
ing platform. Appl Environ Microbiol. 2013 Sep; 79(17):5112–5120. https://doi.org/10.1128/AEM.
01043-13 PMID: 23793624
27. Martin M. Cutadapt removes adapter sequences from high-throughput sequencing reads. EMBnet. J.
2011; 17:10–12.
28. Joshi NA, Fass JN. Sickle: A sliding-window, adaptive, quality-based trimming tool for FastQ files (Ver-
sion 1.33) [Software]. 2011.
Scalp bacterial shift in Alopecia areata
PLOS ONE | https://doi.org/10.1371/journal.pone.0215206 April 11, 2019 9 / 11
29. Masella AP, Bartram AK, Truszkowski JM, Brown DG, Neufeld JD. PANDAseq: paired-end assembler
for illumina sequences. BMC Bioinformatics. 2012 Feb; 14:13–31.
30. Desantis TZ, Hugenholtz P, Larsen N, Rojas M, Brodie EL, Keller K, et al. Greengenes, a chimera-
checked 16S rRNA gene database and workbench compatible with ARB. Appl. Environ. Microbiol.
2006; 72: 5069–5072. https://doi.org/10.1128/AEM.03006-05 PMID: 16820507
31. Vigetti D, Viola M, Karousou E, Rizzi M, Moretto P, Genasetti A, et al. Hyaluronan-CD44-ERK1/2 regu-
late human aortic smooth muscle cell motility during aging. J Biol Chem 2008; 283:4448–58. https://doi.
org/10.1074/jbc.M709051200 PMID: 18077444
32. Findley K, Grice EA. The Skin Microbiome: A Focus on Pathogens and Their Association with Skin Dis-
ease. Miller V, ed. PLoS Pathogens. 2014; 10(11):e1004436.
33. Noble WC. Staphylococci on the skin. In The Skin Microflora and Microbial Skin Disease; Noble, W.C.,
Ed.; Cambridge University Press: London, UK, 2004; pp. 135–152.
34. Katsuyama M, Ichikawa H, Ogawa S, Ikezawa Z. A novel method to control the balance of skin micro-
flora. Part 1. Attack on biofilm of Staphylococcus aureus without antibiotics. J Dermatol Sci. 2005 Jun;
38(3):197–205. Epub 2005 Mar 2. Erratum in: J Dermatol Sci. 2005 Sep; 39(3):196. Masako, Kat-
suyama [corrected to Katsuyama, Masako]; Hideyuki, Ichikawa [corrected to Ichikawa, Hideyuki]; Shi-
geyuki, Ogawa [corrected to Ogawa, Shigeyuki]; Zenro, Ikezawa [corrected to Ikezawa, Zenro].
15927813. https://doi.org/10.1016/j.jdermsci.2005.01.006 PMID: 15927813
35. Lambers H, Piessens S, Bloem A, Pronk H, Finkel P. Natural skin surface pH is on average below 5,
which is beneficial for its resident flora. Int J Cosmet Sci. 2006 Oct; 28(5):359–70. https://doi.org/10.
1111/j.1467-2494.2006.00344.x PMID: 18489300
36. Dethlefsen L, McFall-Ngai M, Relman DA. An ecological and evolutionaryb perspective on human-
microbe mutualism and disease. Nature. 2007 Oct 18; 449(7164):811–8. Review. https://doi.org/10.
1038/nature06245 PMID: 17943117
37. Grice EA, Kong HH, Renaud G, Young AC; NISC Comparative Sequencing Program, Bouffard GG, Bla-
kesley RW, Wolfsberg TG, Turner ML, Segre JA. A diversity profile of the human skin microbiota.
Genome Res. 2008 Jul; 18(7):1043–50. https://doi.org/10.1101/gr.075549.107 PMID: 18502944
38. Reid G, Younes JA, Van der Mei HC, Gloor GB, Knight R, Busscher HJ. Microbiota restoration: natural
and supplemented recovery of human microbial communities. Nat Rev Microbiol. 2011 Jan; 9(1):27–
38. https://doi.org/10.1038/nrmicro2473 PMID: 21113182
39. Human Microbiome Project Consortium. Structure, function and diversity of the healthy human micro-
biome. Nature. 2012 Jun 13; 486(7402):207–14. https://doi.org/10.1038/nature11234 PMID: 22699609
40. Kong HH. Skin microbiome: genomics-based insights into the diversity and role of skin microbes.
Trends Mol Med. 2011 Jun; 17(6):320–8. https://doi.org/10.1016/j.molmed.2011.01.013 PMID:
21376666
41. Cogen AL, Nizet V, Gallo RL (2009). Skin microbiota: A source of disease or defence? Br J Dermatol
158, 442–455.
42. Brogden NK, Mehalick L, Fischer CL, Wertz PW, Brogden KA. The emerging role of peptides and lipids
as antimicrobial epidermal barriers and modulators of local inflammation. Skin Pharmacol Physiol.
2012; 25(4):167–81. https://doi.org/10.1159/000337927 PMID: 22538862
43. Zeeuwen PL, Kleerebezem M, Timmerman HM, Schalkwijk J. Microbiome and skindiseases. Curr Opin
Allergy Clin Immunol. 2013 Oct; 13(5):514–20. https://doi.org/10.1097/ACI.0b013e328364ebeb PMID:
23974680
44. Belkaid Y, Hand TW. Role of the microbiota in immunity and inflammation. Cell. 2014 Mar 27; 157
(1):121–41. https://doi.org/10.1016/j.cell.2014.03.011 Review. PMID: 24679531
45. Wang L, Clavaud C, Bar-Hen A, Cui M, Gao J, Liu Y, et al. Characterization of the major bacterial-fungal
populations colonizing dandruff scalps in Shanghai, China, shows microbial disequilibrium. Exp Derma-
tol. 2015 May; 24(5):398–400. https://doi.org/10.1111/exd.12684 PMID: 25739873
46. Wang E, Lee JS-S, Hee TH. Is Propionibacterium Acnes Associated with Hair Casts and Alopecia?
International Journal of Trichology. 2012; 4(2):93–97. https://doi.org/10.4103/0974-7753.96907 PMID:
23180917
47. Rosenberger C, Solovan C, Rosenberger AD, Jinping L, Treudler R, Frei U, et al. Upregulation of hyp-
oxia-inducible factors in normal and psoriatic skin. J Invest Dermatol. 2007 Oct; 127(10):2445–52.
https://doi.org/10.1038/sj.jid.5700874 PMID: 17495954
48. Manresa MC, Taylor CT. Hypoxia Inducible Factor (HIF) Hydroxylases as Regulators of Intestinal Epi-
thelial Barrier Function. Cell Mo Gastroenterol Hepatol. 2017 Feb 20; 3(3):303–315.
49. Nakatsuji T, Chiang HI, Jiang SB, Nagarajan H, Zengler K, Gallo RL. The microbiome extends to sub-
epidermal compartments of normal skin. Nat Commun. 2013; 4:1431. https://doi.org/10.1038/
ncomms2441 PMID: 23385576
Scalp bacterial shift in Alopecia areata
PLOS ONE | https://doi.org/10.1371/journal.pone.0215206 April 11, 2019 10 / 11
50. Yuki T, Yoshida H, Akazawa Y, Komiya A, Sugiyama Y, Inoue S. Activation of TLR2 enhances tight
junction barrier in epidermal keratinocytes. J Immunol. 2011 Sep 15; 187(6):3230–7. 7. https://doi.org/
10.4049/jimmunol.1100058 PMID: 21841130
51. Lai Y, Di Nardo A, Nakatsuji T, Leichtle A, Yang Y, Cogen AL, et al. Commensal bacteria regulate
TLR3-dependent inflammation following skin injury. Nature medicine. 2009; 15(12):1377–1382. https://
doi.org/10.1038/nm.2062 PMID: 19966777
52. Lai Y, Cogen AL, Radek KA, Park HJ, Macleod DT, Leichtle A, et al. Activation of TLR2 by a small mole-
cule produced by Staphylococcus epidermidis increases antimicrobial defense against bacterial skin
infections. J Invest Dermatol. 2010 Sep; 130(9):2211–21. https://doi.org/10.1038/jid.2010.123 PMID:
20463690
53. Wanke I, Steffen H, Christ C, Krismer B, Go
¨tz F, Peschel A, et al. Skin commensals amplify the innate
immune response to pathogens by activation of distinct signaling pathways. J Invest Dermatol. 2011
Feb; 131(2):382–90. 10. https://doi.org/10.1038/jid.2010.328 PMID: 21048787
54. Naik S, Bouladoux N, Wilhelm C, Molloy MJ, Salcedo R, Kastenmuller W, et al. Compartmentalized
Control of Skin Immunity by Resident Commensals. Science. 2012 Aug; 337(6098):1115–9. https://doi.
org/10.1126/science.1225152 PMID: 22837383
55. De Benedetto A, Kubo A, Beck LA. Skin barrier disruption: a requirement for allergen sensitization? J
Invest Dermatol. 2012; 132:949–963. https://doi.org/10.1038/jid.2011.435 PMID: 22217737
56. Pianta A, Arvikar S, Strle K, Drouin EE, Wang Q, Costello CE, et al. Evidence of the Immune Relevance
of Prevotella copri, a Gut Microbe, in Patients With Rheumatoid Arthritis. Arthritis Rheumatol. 2017
May; 69(5):964–975. https://doi.org/10.1002/art.40003 PMID: 27863183
57. Tan L, Zhao S, Zhu W, Wu L, Li J, Shen M, Lei L, et al. The Akkermansia muciniphila is a gut microbiota
signature in psoriasis. Exp Dermatol. 2018 Feb; 27(2):144–149. https://doi.org/10.1111/exd.13463
PMID: 29130553
Scalp bacterial shift in Alopecia areata
PLOS ONE | https://doi.org/10.1371/journal.pone.0215206 April 11, 2019 11 / 11
... Actinobacteria <1% 20%-30% [2,3,7,29,58,[101][102][103] Actinomycetota <1% 20%-30% ...
... Different immune mechanisms are recognised as important players in microbiota management and upon microbial infection. Microbial recognition is made by pattern recognition receptors, including toll-like receptors (TLRs), which are differentially distributed in the HF, [58,59] and nucleotide-binding and oligomerisation domain (NOD)-like receptors, present, amongst others, in keratinocytes. [60] This recognition is imperative in the activation of an immune response to fight infections and for the development of tolerance to commensal microbes. ...
... In HF diseases, such as AGA and AA, overexpression of these receptors is observed and correlates with dysbiosis. [58,59] Therefore, understanding whether dysbiosis or the change in TLR expression is the primary stimulus, is crucial for comprehension of the pathobiology. Further, grasping how the microbiota exploit these receptors is important for finding potential targets and advance the treatment of inflammatory diseases where activation of TLRs elicits an inappropriate immune response. ...
Article
The microbiome of human hair follicles (HFs) has emerged as an important player in different HF and skin pathologies, yet awaits in‐depth exploration. This raises questions regarding the tightly linked interactions between host environment, nutrient dependency of host‐associated microbes, microbial metabolism, microbe‐microbe interactions and host immunity. The use of simple model systems facilitates addressing generally important questions and testing overarching, therapeutically relevant principles that likely transcend obvious interspecies differences. Here, we evaluate the potential of the freshwater polyp Hydra, to dissect fundamental principles of microbiome regulation by the host, that is the human HF. In particular, we focus on therapeutically targetable host‐microbiome interactions, such as nutrient dependency, microbial interactions and host defence. Offering a new lens into the study of HF – microbiota interactions, we argue that general principles of how Hydra manages its microbiota can inform the development of novel, microbiome‐targeting therapeutic interventions in human skin disease. Microbial management in Hydra exhibits parallels to the human hair follicle. Therefore, these organisms provide an instructive model to study microbial regulation and interactions with the host that can be applied to study dysbiosis in hair follicle and hair follicle‐associated diseases research, by complete/targeted manipulation of the Hydra microbiome.
... Due to its unique features, the scalp is expected to harbor a specific microbiome, which is expected to play a peculiar role in scalp conditions related to hair growth [7]. Several researchers studied the scalp microbiome profile regarding AA and they suggested the notion that an imbalance between skin microbes that maintain skin homeostasis causes inflammation [8][9][10][11][12]. Several microbes such as Cutibacterium acnes, Staphylococcus, and Malassezia were suggested to be implicated in AA, but it still needs to be clarified using various cohorts of different populations. ...
... The severity of AA was evaluated based on the extent of scalp hair loss, progression of disease, and response of treatment, and was divided into mild AA (n = 26) and severe AA (n = 7) ( Table 1). The scalp samples were obtained by means of swab procedure according to previously reported methods [8][9][10] with minor modifications. Sterile cotton swabs were soaked for at least 30 s in solution (0.9% NaCl and 0.1% Tween 20). ...
... Several scientific published evidence have reported the strict correlation between microbial disequilibrium and skin conditions [4,5,[7][8][9][10]. Given this growing body of literature, it is becoming increasingly clear that the modulation of the microbiota may be a novel and important adjunct modality. ...
Article
Full-text available
Little is known about the scalp bacterial composition of alopecia areata (AA) patients. The aim of this study was to investigate the differences in the scalp microbiome of AA patients according to their prognosis, in addition to healthy controls. A total of 33 AA patients and 12 healthy controls (HC) were included in this study. The microbiomes were characterized by sequencing 16S rRNA genes on the Illumina MiSeq platform. The scalp microbiome was more diverse in AA patients compared to HC, but not significantly different according to the severity of AA. Nevertheless, the higher proportion of Corynebacterium species and the lower proportion of Staphylococcus caprae among the Staphylococcus species were noticed in severe AA patients compared to HC or mild AA. The higher ratio of Cutibacterium species to S. caprae was noticed in severe AA. We highlight the potential predictive role of scalp microbiome profiling to a worse prognosis of patients with alopecia areata.
... The role of the microbiome has also been reported in hair growth disorders such as non-cicatricial and cicatricial alopecia [39][40][41][49][50][51]. ...
... In our first study [40], preliminary results obtained from relatively small-sized samples highlighted, for the first time, the presence of microbial dysbiosis on the scalp of subjects affected by hair growth disorders such as AA, Lichen planopilaris, and, to a lesser extent, androgenetic alopecia. ...
Article
Full-text available
The continuous research advances in the microbiome field is changing clinicians’ points of view about the involvement of the microbiome in human health and disease, including autoimmune diseases such as alopecia areata (AA). Both gut and cutaneous dysbiosis have been considered to play roles in alopecia areata. A new approach is currently possible owing also to the use of omic techniques for studying the role of the microbiome in the disease by the deep understanding of microorganisms involved in the dysbiosis as well as of the pathways involved. These findings suggest the possibility to adopt a topical approach using either cosmetics or medical devices, to modulate or control, for example, the growth of overexpressed species using specific bacteriocins or postbiotics or with pH control. This will favour at the same time the growth of beneficial bacteria which, in turn, can impact positively both the structure of the scalp ecosystem on the host’s response to internal and external offenders. This approach, together with a “systemic” one, via oral supplementation, diet, or faecal transplantation, makes a reliable translation of microbiome research in clinical practice and should be taken into consideration every time alopecia areata is considered by a clinician.
... Many patients have no evident cause of scalp pruritus which is called pruritus of undetermined origin, itch or pruritus of unknown origin, or idiopathic itch/pruritus (Weisshaar and Dalgard 2009;Elizabeth et al. 2018). The therapy for this kind of patients is still a challenge to dermatologists (Elizabeth et al. 2018 In the past 10 years, the relationships between the dysbiosis of microbiota and skin diseases on scalp, such as dandruff, dermatitis and hair growth diseases, have brought attention (Clavaud et al. 2013;Park et al. 2017;Paulino 2017;Grimshaw et al. 2019;Pinto et al. 2019;Carmona-Cruz et al. 2022;Woo et al. 2022). However, few studies have investigated the microbiota on simplex scalp pruritus, though the scalp normal microbiota is speculated to be an important factor involved in the pathogenesis of scalp pruritus (Rattanakaemakorn and Suchonwanit 2019). ...
Article
Full-text available
Scalp pruritus is a common skin problem that remains therapeutic challenge. The relationships between the dysbiosis of microbiota and skin diseases have caught attention recently. However, there are few reports about microbiota on itchy scalp. This study investigated scalp microbial characteristics of subjects with mild scalp pruritus of undetermined origin and preliminarily screened physiological factors and bacteria potentially related to pruritus. The pruritus severity of 17 qualified females was evaluated by Visual Analogue Scale (VAS). Microbiota collection was done at both itchy (n = 20) and non-itchy sites (n = 27) at occiput and crown of the same subject and Illumina sequencing was performed at the V3–V4 hypervariable regions of 16S rRNA. The corresponding sebum content, hydration, pH, trans-epidermal water loss, erythema index and porphyrin numbers were also measured by skin tester. We identified 3044 amplicon sequence variants from 821 genera. The itchy and non-itchy sites had different microbiota structures (p = 0.045, by multivariate analysis of variance), while there were large inter- and intra-individual variations. Both sites had Staphylococcus, Cutibacterium and Lawsonella as predominant genera, which were not significantly related to pruritus. The use of three genera Lactobacillus, Morganella and Pseudomonas, could well distinguish non-itchy from itchy groups, whereas different composition patterns existed inside each group. Our investigation indicated that though the bacterial community structure on itchy scalp was individual specific, there was difference between itchy and non-itchy sites. The study provides new insights into microbiota profiling on itchy scalp, which will help microbiota-targeted therapeutic experiment or products design for scalp pruritus.
... There is a striking acquisition of ectopic immune-competence (most easily represented by upregulated MHC-I expression) in the cycling part of the growing HF (24), as well as other features of immunological dysregulation (25,26). Dysregulated redox balance (27,28), and microbial dysregulation (29,30) may also feature in AA pathogenesis. While these and numerous other factors have been reported in AA over the decades (31), the classical features of active disease include the brisk infiltration of immune cells in a classic 'Swarm of Bees'-like pattern (32), and the production of autoantibodies to HF-associated proteins (33)(34)(35). ...
Article
Full-text available
Alopecia areata (AA) is a chronic, multifactorial, polygenic, and heterogeneous disorder affecting growing hair follicles in susceptible individuals, which results in a non-scarring and reversible hair loss with a highly unpredictable course. Despite very considerable research effort, the nature of the precipitating factor(s) responsible for initiating AA in any given hair follicle remains unclear, due largely to significant gaps in our knowledge of the precise sequence of the etiopathogenic events in this dermatosis. However, disease-related changes in the immune-competence of the lower growing hair follicle, together with an active immune response (humoral and cellular) to hair follicle-associated antigens, are key associated phenomena. Confirmation of the hair follicle antigen(s) implicated in AA disease onset has remained stubbornly elusive. While it may be considered somewhat philosophical by some, it is also unclear whether immune-mediated hair loss in AA results from a) an ectopic (i.e., in an abnormal location) immune response to native (unmodified) self-antigens expressed by the healthy hair follicle, b) a normal immune response against modified self-antigens (or neoantigens), or c) a normal immune response against self-antigens (modified/non-modified) that were not previously visible to the immune system (because they were conformationally-hidden or sequestered) but become exposed and presentable in an MHC-I/-II molecule-restricted manner. While some candidate hair follicle antigen target(s) in AA are beginning to emerge, with a potential role for trichohyalin, it is not yet clear whether this represents the initial and immunodominant antigenic focus in AA or is simply one of an expanding repertoire of exposed hair follicle tissue damage-associated antigens that are secondary to the disease. Confirmation of autoantigen identity is essential for our understanding of AA etiopathogenesis, and consequently for developing a more informed therapeutic strategy. Major strides have been made in autoantigen discovery in other autoimmune conditions. In particular, some of these conditions may provide insights into how post-translational modifications (e.g., citrullination, deamidation, etc.) of hair follicle-restricted proteins may increase their antigenicity and so help drive the anti-hair follicle immune attack in AA.
... Symbiosis of Corynebateriaceae, Propionibacteriaceae, Staphylococcaceae, and Malassezia is related to a healthy scalp, while dysbiosis can cause pathological conditions. Pinto et al. found microbial shifts in individuals with AA exhibiting overcolonization with C. acnes along with a reduced S. epidermidis abundance, however, it has not been determined if these differences are cause or consequence of the disease (Pinto et al., 2019). The role of CMV in triggering AA was suggested after finding DNA sequences in biopsies of AA, but subsequent studies did not confirm this fact (Offidani et al., 2000). ...
Article
Full-text available
The human skin harbors a wide variety of microbes that, together with their genetic information and host interactions, form the human skin microbiome. The role of the human microbiome in the development of various diseases has lately gained interest. According to several studies, changes in the cutaneous microbiota are involved in the pathophysiology of several dermatoses. A better delineation of the human microbiome and its interactions with the innate and adaptive immune systems could lead to a better understanding of these diseases, as well as the opportunity to achieve new therapeutic modalities. The present review centers on the most recent knowledge on skin microbiome and its participation in the pathogenesis of several skin disorders: atopic and seborrheic dermatitis, alopecia areata, psoriasis and acne.
... There is an increasing interest in the role of HF dysbiosis in various hair diseases, such as AA and AGA, whereas very little is as yet known about the HF microbiome in LPP and FFA (Constantinou et al., 2021a(Constantinou et al., , 2021bGaber et al., 2015;Lousada et al., 2021;Pinto et al., 2019;Polak-Witka et al., 2020). Because Staphylococcus aureus overcolonization is thought to be one of the main pathogenic factors in the neutrophilic PCA, folliculitis decalvans (Chiarini et al., 2008;Otberg et al., 2008), it is important to characterize the effect of bacterial colonization and HF dysbiosis also on LPP and FFA. ...
Article
Full-text available
Lichen planopilaris (LPP) and frontal fibrosing alopecia (FFA) are primary, lymphocytic cicatricial hair loss disorders. These model epithelial stem cell diseases are thought to result from a CD8+ T cell-dominated immune attack on the hair follicle’s stem cell niche (bulge) after the latter has lost its immune privilege for as yet unknown reasons. This induces both apoptosis and pathological epithelial-mesenchymal transition (EMT) in epithelial stem cells, thus depletes the bulge, causes fibrosis, and ultimately abrogates hair follicle’s capacity to regenerate. Here, we synthesize recent progress in LPP and FFA pathobiology research, integrate our limited current understanding of the roles that genetic, hormonal, environmental and other factors may play, and define major open questions. We propose that LPP and FFA share a common initial pathobiology, which then bifurcates into two distinct clinical phenotypes, with macrophages possibly playing a key role in phenotype determination. As particularly promising translational research avenues towards direly needed progress in the management of these disfiguring, deeply distressful cicatricial alopecia variants, we advocate to focus on the development of bulge immune privilege and epithelial stem cell protectants such as, for example, topically effective, hair follicle-penetrating and immunoinhibitory preparations that contain tacrolimus, PPARg and/or cannabinoid receptor-1 agonists.
Article
Introduction: Alopecia areata (AA) is a non-scarring, hair loss disorder and a common autoimmune-mediated disease with an estimated lifetime risk of about 2%. To date, the treatment of AA is mainly based on suppression or stimulation of the immune response. Genomics and transcriptomics studies generated important insights into the underlying pathophysiology, enabled discovery of molecular disease signatures, which were used in some of the recent clinical trials to monitor drug response and substantiated the consideration of new therapeutic modalities for the treatment of AA such as abatacept, dupilumab, ustekinumab and Janus Kinase (JAK) inhibitors. Areas covered: In this review, genomics and transcriptomics studies in AA are discussed in detail with particular emphasis on their past and prospective translational impacts. Microbiome studies are also briefly introduced. Expert opinion: The generation of large datasets using the new high-throughput technologies has revolutionized medical research and AA has also benefited from the wave of omics studies. However, the limitations associated with JAK inhibitors and clinical heterogeneity in AA patients underscore the necessity for continuing omics research in AA for discovery of novel therapeutic modalities and development of clinical tools for precision medicine.
Article
While popular belief harbors little doubt that perceived stress can cause hair loss and premature graying, the scientific evidence for this is arguably much thinner. Here, we investigate whether these phenomena are real, and show that the cyclic growth and pigmentation of the hair follicle (HF) provides a tractable model system for dissecting how perceived stress modulates aspects of human physiology. Local production of stress-associated neurohormones and neurotrophins coalesces with neurotransmitters and neuropeptides released from HF-associated sensory and autonomic nerve endings, forming a complex local stress-response system that regulates perifollicular neurogenic inflammation, interacts with the HF microbiome and controls mitochondrial function. This local system integrates into the central stress response systems, allowing the study of systemic stress responses affecting organ function by quantifying stress mediator content of hair. Focusing on selected mediators in this “brain-HF axis” under stress conditions, we distill general principles of HF dysfunction induced by perceived stress.
Article
The human microbiome encompasses the microorganisms that live in and on the body. During the prenatal and infantile periods, foundations for the cutaneous and gut microbiomes are being established and refined concurrently with the development of immune function. Herein, we review the relevance of the microbiome to 5 conditions commonly encountered in pediatric dermatology: acne, alopecia areata, atopic dermatitis, psoriasis, and seborrheic dermatitis. Understanding the role microbes play in these conditions may establish the groundwork for future therapeutic interventions.
Article
Full-text available
Nowadays, the study of human microbiome represents a novel diagnostic and therapeutic approach to treat many human conditions, also including that strictly related to skin and scalp. The findings we included in the present work represent just an overview of a larger pioneer study on the involvement of changing of the microbiome in scalp diseases, especially that related to hair growth. Even just preliminary, our results strongly highlighted, for the first time, the role exerted by unbalancing on the normal resident microbial community in hair growth-related conditions.
Article
Full-text available
Clostridium difficile infections can be life-threatening but are increasingly being treated successfully with fecal microbiota transplantation (FMT). We report two patients with alopecia universalis who developed subsequent hair regrowth after FMT for treatment of recurrent C. difficile infections. Gut microbiota may have immunomodulatory effects in autoimmune conditions such as alopecia areata, and further study may elucidate disease mechanisms and lead to alternative treatment options for these patients for whom treatment options are currently limited.
Article
Full-text available
Human health is dependent upon the ability of the body to extract nutrients, fluids and oxygen from the external environment while at the same time, maintaining a state of internal sterility. Therefore, the cell layers that cover the surface areas of the body such as the lung, skin and gastrointestinal mucosa provide vital semi-permeable barriers which allow the transport of essential nutrients, fluid and waste products while at the same time keeping the internal compartments free of microbial organisms. These epithelial surfaces are highly specialized and differ in their anatomical structure depending on their location to provide appropriate and effective site-specific barrier function. Given this important role, it is not surprising that significant disease is often associated with alterations in epithelial barrier function. Examples of such diseases include inflammatory bowel disease (IBD)¹, chronic obstructive pulmonary disease (COPD)² and atopic dermatitis (AD)³. These chronic inflammatory disorders are often characterized by diminished tissue oxygen levels (hypoxia). Hypoxia triggers an adaptive transcriptional response governed by hypoxia inducible factors (HIFs), which are repressed by a family of oxygen-sensing HIF-hydroxylases. Here, we review recent evidence suggesting that pharmacologic hydroxylase inhibition may be of therapeutic benefit in IBD through the promotion of intestinal epithelial barrier function through both HIF-dependent and HIF-independent mechanisms.
Article
Full-text available
Dandruff is an unpleasant scalp disorder common to human populations. In this study, we systematically investigated the intra- and inter-associations among dandruff, physiological conditions such as sebum of the scalp, host demographics such as gender, age and the region of the scalp, and the microorganisms on the scalp. We found that the physiological conditions were highly relevant to the host age and varied in different regions of the same scalp. The sebum quantity and water content were negatively correlated with the formation of dandruff and had significant relationships with the two dominant but reciprocally inhibited bacteria on the scalp (Propionibacterium and Staphylococcus). The dominant fungus (Malassezia species) displayed contrary roles in its contribution to the healthy scalp micro-environment. Bacteria and fungi didn’t show a close association with each other, but the intramembers were tightly linked. Bacteria had a stronger relationship with the severity of dandruff than fungi. Our results indicated that the severity of dandruff was closely associated with the interactions between the host and microorganisms. This study suggests that adjusting the balance of the bacteria on the scalp, particularly by enhancing Propionibacterium and suppressing Staphylococcus, might be a potential solution to lessen dandruff.
Article
Full-text available
We continue to uncover a wealth of information connecting microbes in important ways to human and environmental ecology. As our scientific knowledge and technical abilities improve, the tools used for microbiome surveys can be modified to improve the accuracy of our techniques, ensuring that we can continue to identify groundbreaking connections between microbes and the ecosystems they populate, from ice caps to the human body. It is important to confirm that modifications to these tools do not cause new, detrimental biases that would inhibit the field rather than continue to move it forward. We therefore demonstrated that two recently modified primer pairs that target taxonomically discriminatory regions of bacterial and fungal genomic DNA do not introduce new biases when used on a variety of sample types, from soil to human skin. This confirms the utility of these primers for maintaining currently recommended microbiome research techniques as the state of the art.
Article
Introduction: This review aims to raise the potential of the modern society’s impact on gut integrity often leading to increased intestinal permeability, as a cause or driver of Alopecia Areata (AA) in genetically susceptible people. With the increasing rate of T cell-driven autoimmunity, we hypothesize that there is a common root cause of these diseases that originates from chronic inflammation, and that the gut is the most commonly exposed area with our modern lifestyle. Areas covered: We will discuss the complexity in the induction of AA and its potential link to increased intestinal permeability. Our main focus will be on the gut microbiome and mechanisms involved in the interplay with the immune system that may lead to local and/or peripheral inflammation and finally, tissue destruction. Expert opinion: We have seen a link between AA and a dysfunctional gastrointestinal system which raised the hypothesis that an underlying intestinal inflammation drives the priming and dysregulation of immune cells that lead to hair follicle destruction. While it is still important to resolve local inflammation and restore the IP around the hair follicles, we believe that the root cause needs to be eradicated by long-term interventions to extinguish the fire driving the disease.
Article
Objective: Prevotella copri, an intestinal microbe, may over-expand in stool samples of patients with new-onset rheumatoid arthritis (NORA), but it is not yet clear whether the organism has immune relevance in RA pathogenesis. Methods: HLA-DR-presented peptides (T cell epitopes) from P. copri were sought directly from patients' synovial tissue or peripheral blood mononuclear cells (PBMC) using tandem mass spectrometry, followed by testing the antigenicity of peptides or their source proteins using samples from RA patients or comparison groups. T cell reactivity was determined by ELISpot assays; antibody responses were measured by ELISA, and cytokine/chemokine determinations were made by Luminex. 16S rDNA of P. copri was sought in serum and synovial fluid samples using nested PCR. Results: In PBMC, we identified an HLA-DR-presented peptide from a 27-kD protein of P. copri (Pc-p27), which stimulated Th1 responses in 42% of NORA patients. In both NORA and chronic RA patients, one subgroup had IgA antibody responses to Pc-p27 or the whole organism, which correlated with Th17 cytokine responses and frequent anti-citrullinated protein antibodies (ACPA). The other subgroup had IgG P. copri antibodies, which were associated with Prevotella DNA in synovial fluid, P. copri-specific Th1 responses, and less frequent ACPA. In contrast, P. copri antibody responses were rarely found in patients with other rheumatic diseases or in healthy controls. Conclusion: Subgroups of RA patients have differential IgG or IgA immune reactivity with P. copri, which appears to be specific for this disease. These observations provide evidence that P. copri is immune-relevant in RA pathogenesis. This article is protected by copyright. All rights reserved.
Article
Microbial community analysis via high-throughput sequencing of amplified 16S rRNA genes is an essential microbiology tool. We found the popular primer pair 515F (515F-C) and 806R greatly underestimated (e.g. SAR11) or overestimated (e.g. Gammaproteobacteria) common marine taxa. We evaluated marine samples and mock communities (containing 11 or 27 marine 16S clones), showing alternative primers 515F-Y (5'- GTGYCAGCMGCCGCGGTAA) and 926R (5'- CCGYCAATTYMTTTRAGTTT) yield more accurate estimates of mock community abundances, produce longer amplicons that can differentiate taxa unresolvable with 515F-C/806R, and amplify eukaryotic 18S rRNA. Mock communities amplified with 515F-Y/926R yielded closer observed community composition vs. expected (r(2) =0.95) compared to 515F-Y/806R (r(2) ∼0.5). Unexpectedly, biases with 515F-Y/806R against SAR11 in field samples (∼4-10-fold) were stronger than in mock communities (∼2-fold). Correcting a mismatch to Thaumarchaea in the 515F-C increased their apparent abundance in field samples, but not as much as using 926R rather than 806R. With plankton samples rich in eukaryotic DNA (>1μm size fraction), 18S sequences averaged ∼17% of all sequences. A single mismatch can strongly bias amplification, but even perfectly matched primers can exhibit preferential amplification. We show that beyond in silico predictions, testing with mock communities and field samples is important in primer selection. This article is protected by copyright. All rights reserved.