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Scalp bacterial shift in Alopecia areata

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  • Giuliani, Milan, Italy
  • Rinaldi & Associated, Milano, Italy

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.
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Scalp bacterial shift in Alopecia areata
Daniela Pinto
, Elisabetta Sorbellini
, Barbara Marzani
, Mariangela Rucco
Giammaria Giuliani
, Fabio RinaldiID
1Giuliani SpA, Milan, Italy, 2Human Advanced Microbiome Project-HMAP, Milan, Italy, 3International Hair
Research Foundation (IHRF), Milan, Italy
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.
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
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 | April 11, 2019 1 / 11
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.
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
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
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 | 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
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 | 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.
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 | 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).
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.
Scalp bacterial shift in Alopecia areata
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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
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.
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
Scalp bacterial shift in Alopecia areata
PLOS ONE | 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 | 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.
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
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
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Scalp bacterial shift in Alopecia areata
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... Several epidemiological studies have indirectly reported correlations between scalp bacteria and dermatosis ( Pinto et al . 2019 ;Filaire et al. 2020 ) . Pinto et al . ( 2019 ) and Filaire et al . ( 2020 ) compared bacterial community structures on the human scalp between healthy individuals and patients with alopecia areata and male pattern alopecia and suggested that the bacterial community structure on the scalp is a causal factor of scalp dermatosis ( Pinto et ...
... Several epidemiological studies have indirectly reported correlations between scalp bacteria and dermatosis ( Pinto et al . 2019 ;Filaire et al. 2020 ) . Pinto et al . ( 2019 ) and Filaire et al . ( 2020 ) compared bacterial community structures on the human scalp between healthy individuals and patients with alopecia areata and male pattern alopecia and suggested that the bacterial community structure on the scalp is a causal factor of scalp dermatosis ( Pinto et al. 2019 ;Filaire et al . 2020 ) . However, th ...
... ( 2019 ) and Filaire et al . ( 2020 ) compared bacterial community structures on the human scalp between healthy individuals and patients with alopecia areata and male pattern alopecia and suggested that the bacterial community structure on the scalp is a causal factor of scalp dermatosis ( Pinto et al. 2019 ;Filaire et al . 2020 ) . ...
Scalp bacteria on the human scalp and scalp hair comprise distinct community structures for sites and individuals. To evaluate their effect on human keratinocyte cellular activity, including that of the hair follicular keratinocytes, the expression of several longevity genes was examined using HaCaT cells. A screening system that uses enhanced green fluorescent protein (EGFP) fluorescence was established to identify scalp bacteria that enhance silent mating type information regulation 2 homolog-1 (SIRT1) promoter activity in transformed HaCaT cells (SIRT1p-EGFP). The results of quantitative polymerase chain reaction (qPCR) revealed that several predominant scalp bacteria enhanced (Cutibacterium acnes and Pseudomonas lini) and repressed (Staphylococcus epidermidis) the expressions of SIRT1 and telomerase reverse transcriptase (TERT) genes in HaCaT cells. These results suggest that the predominant scalp bacteria are related to the health of the scalp and hair, including repair of the damaged scalp and hair growth, by regulating gene expression in keratinocytes.
... We suggest that the role of C. acnes in our model of heatkilled GMNL-653 shampoo treatment might be contrary and different from that in the dandruff scalp. Other studies also provided evidence that the role of C. acnes is correlated with hair growth disorder [44][45][46][47]. For example, elevated C. acnes in the middle and lower compartments of hair follicles are associated with elevated immune response in patients with alopecia areata [45,46]. ...
... For example, elevated C. acnes in the middle and lower compartments of hair follicles are associated with elevated immune response in patients with alopecia areata [45,46]. A significant increase in C. acnes and a decrease in S. epidermidis were also observed in patients with alopecia areata [44] and androgenetic alopecia [47] compared with those in control. Further investigation of the potential role of C. acnes in the regulation of scalp hair growth under heatkilled GMNL-653 treatment using an in vitro culture of human hair follicles or in mouse models is necessary. ...
... These findings suggest that heat-killed L. paracasei GMNL-653 has the potential to improve scalp condition and enhance hair growth. Several studies reported that an increase in C. acnes abundance is strongly associated with hair growth disorders, such as alopecia areata and androgenetic alopecia [44,46,47]. Our results also showed that the use of heat-killed GMNL-653 shampoo could decrease the abundance of C. acnes in scalp (Fig. 8D). ...
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Background The equilibrium of the scalp microbiome is important for maintaining healthy scalp conditions, including sebum secretion, dandruff, and hair growth. Many different strategies to improve scalp health have been reported; however, the effect of postbiotics, such as heat-killed probiotics, on scalp health remains unclear. We examined the beneficial effects of heat-killed probiotics consisting of Lacticaseibacillus paracasei, GMNL-653, on scalp health. Results Heat-killed GMNL-653 could co-aggregate with scalp commensal fungi, Malassezia furfur, in vitro, and the GMNL-653-derived lipoteichoic acid inhibited the biofilm formation of M. furfur on Hs68 fibroblast cells. The mRNA of hair follicle growth factors, including insulin-like growth factor-1 receptor (IGF-1R), vascular endothelial growth factor, IGF-1, and keratinocyte growth factor was up-regulated in skin-related human cell lines Hs68 and HaCaT after treatment with heat-killed GMNL-653. For clinical observations, we recruited 22 volunteer participants to use the shampoo containing the heat-killed GMNL-653 for 5 months and subsequently measured their scalp conditions, including sebum secretion, dandruff formation, and hair growth. We applied polymerase chain reaction (PCR) to detect the scalp microbiota of M. restricta, M. globosa, Cutibacterium acnes, and Staphylococcus epidermidis. A decrease in dandruff and oil secretion and an increase in hair growth in the human scalp were observed after the use of heat-killed GMNL-653-containing shampoo. The increased abundance of M. globosa and the decreased abundance of M. restricta and C. acnes were also observed. We further found that accumulated L. paracasei abundance was positively correlated with M. globosa abundance and negatively correlated with C. acnes abundance. S. epidermidis and C. acnes abundance was negatively correlated with M. globosa abundance and positively correlated with M. restricta. Meanwhile, M. globosa and M. restricta abundances were negatively associated with each other. C. acnes and S. epidermidis abundances were statistically positively correlated with sebum secretion and dandruff, respectively, in our shampoo clinical trial. Conclusion Our study provides a new strategy for human scalp health care using the heat-killed probiotics GMNL-653-containing shampoo. The mechanism may be correlated with the microbiota shift.
... Typically, these methods include skin swabs, pore and tape strips, and cyanoacrylate gel biopsies [10][11][12]. Although these may suffice to characterise the resident skin microbiome communities, they fail to probe the deeper regions of the HF epithelium, completely miss the HF mesenchyme ( Figure S1), and do not robustly distinguish between the HF and skin microbiomes [13,14]. ...
... Due to the low biomass of the samples, each compartment was pooled from five HFs per donor. Unlike previous studies [11,12,19], our method yielded enough DNA to omit a PCR amplification step. 16S rRNA gene (V1-V2) sequencing was performed at the University of Kiel in a pair-end modality on a Illumina NextSeq 500 platform rendering 2×150 bp pair-end sequences. ...
... Comparably, in the middle and upper HF regions 35.2% and 36.1% of the reads assigned to Proteobacteria and 24.3% and 23.6% to Actinobacteria, respectively. Similarly, analysis at genera level demonstrated a predominance of Cutibacterium, Streptococcus and Corynebacterium in all regions of the HF, as previously described by others [10][11][12] (Figs. 2 and 3). However, while previous works reported these genera as the most abundant in the HF, comprising between 60 and 90% of the total sequences [11,12,14], LCM-based 16 S rRNA sequencing revealed a lower abundance of these microbes in all three HF regions (Fig. 3). ...
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Objective: Human hair follicles (HFs) are populated by a rich and diverse microbiome, traditionally evaluated by methods that inadvertently sample the skin microbiome and/or miss microbiota located in deeper HF regions. Thereby, these methods capture the human HF microbiome in a skewed and incomplete manner. This pilot study aimed to use laser-capture microdissection of human scalp HFs, coupled with 16S rRNA gene sequencing to sample the HF microbiome and overcome these methodological limitations. Results: HFs were laser-capture microdissected (LCM) into three anatomically distinct regions. All main known core HF bacterial colonisers, including Cutibacterium, Corynebacterium and Staphylococcus, were identified, in all three HF regions. Interestingly, region-specific variations in α-diversity and microbial abundance of the core microbiome genera and Reyranella were identified, suggestive of variations in microbiologically relevant microenvironment characteristics. This pilot study therefore shows that LCM-coupled with metagenomics is a powerful tool for analysing the microbiome of defined biological niches. Refining and complementing this method with broader metagenomic techniques will facilitate the mapping of dysbiotic events associated with HF diseases and targeted therapeutic interventions.
... Suzuki et al. reported that Cutibacterium, Corynebacterium, and Staphylococcus are the most abundant genera on the scalp surface, and scalp dysbiosis occurs in patients with AGA (Suzuki et al., 2021). The scalp condition of patients with AA was associated with more abundant Cutibacterium acne and fewer Staphylococcus epidermis compared to that of healthy controls (Pinto et al., 2019). Additionally, C. acnes is associated with immune response gene expression in hair follicles and can be involved in AGA pathogenesis and hair miniaturization (Ho et al., 2019). ...
... Similar to the results of Suzuki et al. (2021) and Pinto et al. (2019), in the scalp microbiome of our cohort, the alpha diversity of AGA+ showed an increasing trend compared with that of CON, especially for the Chao indices (bacterial richness) in women. The high Chao index of AGA+ could be derived to the significant increase in the number of non-skin inhabitant bacteria. ...
... Frontiers in Microbiology 08 decreased Staphylococcus (Pinto et al., 2019;Won et al., 2022) in the AA group. In particular, Won et al. noted that Corynebacterium and Staphylococcus spp. ...
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Androgenetic alopecia (AGA) is a non-scarring and progressive form of hair loss occurring in both men and women. Although genetic predisposition and sex steroid hormones are the main causes, many factors remain unknown, and various extrinsic factors can negatively affect the lifespan of hair. We investigated skin–gut axis microorganisms as potential exogenous factors causing AGA, through comparative analyses of the scalp and gut microbiome in individuals with and without AGA in a Korean cohort. Using 16S rRNA gene sequencing, we characterized the scalp and gut microbiomes of 141 individuals divided into groups by sex and presence of AGA. Alpha diversity indices in the scalp microbiome were generally higher in individuals with AGA than in healthy controls. These indices showed a strong negative correlation with scalp-inhabitant bacteria (Cutibacterium and Staphylococcus), indicating that the appearance of non-inhabitant bacteria increases as hair loss progresses. No significant differences in diversity were observed between the gut microbiomes. However, bacterial functional differences, such as bile acid synthesis and bacterial invasion of epithelial cells, which are related to intestinal homeostasis, were observed. The networks of the scalp and gut microbiome were more complex and denser with higher values of the network topology statistic coefficient values (i.e., transitivity, density, and degree centrality) and more unique associations in individuals with AGA than in healthy controls. Our findings reveal a link between skin–gut microorganisms and AGA, indicating the former’s potential involvement in the latter’s development. Additionally, these results provide evidence for the development of cosmetics and therapeutics using microorganisms and metabolites involved in AGA.
... These initial observations have allowed us to hypothesize that there may be menopause of the HF independent of the clinical menopause in women, probably due to the change in the metabolism of the HF because of several individual factors such as changes in estrogen receptor responses, genetics, and last but not least, the microbiota. Indeed, the role of the microbiota in the pathophysiology of the HF is now scientifically established [60][61][62][63][64]. ...
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This article explores the link between menopause and changes in the hair follicle (HF) lifecycle, focusing on hormonal and metabolic dynamics. During menopause, hormonal fluctuations and aging can impact the HF, leading to phenomena such as thinning, loss of volume, and changes in hair texture. These changes are primarily attributed to a decrease in estrogen levels. However, not all women experience significant hair changes during menopause, and the extent of transformations can vary considerably from person to person, influenced by genetic factors, stress, diet, and other elements. Furthermore, menopause mirrors the aging process, affecting metabolism and blood flow to the HFs, influencing the availability of vital nutrients. The article also discusses the key role of energy metabolism in the HF lifecycle and the effect of hormones, particularly estrogens, on metabolic efficiency. The concept of a possible “menopause” clinically independent of menopause is introduced, related to changes in HF metabolism, emphasizing the importance of individual factors such as estrogen receptor responses, genetics, and last but not least, the microbiota in determining these dynamics.
... murinus) overgrowth, impairs biotin biosynthesis by the intestinal microbiota and the intestinal metabolic functions, leading to alopecia. It was found that the lack of biotin in the diet of dysbiotic mice treated with antibiotics led to a systemic biotin deficiency, causing the development of alopecia [47]. ...
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Human skin is a habitat to a variety of microbes that, along with their host genetic material, make up microbiome of the human skin. The composition of the microbiota in the gut and skin is influenced by many factors, such as life stage, nutrition, lifestyle and gender. Recently, there is more and more discussions about the increasing role of the microbiome in the development of other diseases. According to many studies, any changes in the skin microbiota are associated with the development of several dermatoses. Better understanding of the human microbiome and its interactions with the immune system could help us understand many diseases as well as could have an impact on the development of some new therapeutic methods. In this article, the current knowledge on the skin microbiome and its influence on the development of alopecia areata will be discussed. Alopecia areata (AA) is caused by an autoimmune process that destroys the hair follicles. The exact pathogenesis is unknown, but the triggering factors include: immune disorders, environmental exposures, genetic predisposition, and possibly the microbiome.
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Alopecia areata (AA) is an autoimmune disease that has a complex underlying immunopathogenesis characterized by nonscarring hair loss ranging from small bald patches to complete loss of scalp, face, and/or body hair. Although the etiopathogenesis of AA has not yet been fully characterized, immune privilege collapse at the hair follicle (HF) followed by T-cell receptor recognition of exposed HF autoantigens by autoreactive cytotoxic CD8 ⁺ T cells is now understood to play a central role. Few treatment options are available, with the Janus kinase (JAK) 1/2 inhibitor baricitinib (2022) and the selective JAK3/tyrosine kinase expressed in hepatocellular carcinoma (TEC) inhibitor ritlecitinib (2023) being the only US Food and Drug Administration–approved systemic medications thus far for severe AA. Several other treatments are used off-label with limited efficacy and/or suboptimal safety and tolerability. With an increased understanding of the T-cell–mediated autoimmune and inflammatory pathogenesis of AA, additional therapeutic pathways beyond JAK inhibition are currently under investigation for the development of AA therapies. This narrative review presents a detailed overview about the role of T cells and T-cell–signaling pathways in the pathogenesis of AA, with a focus on those pathways targeted by drugs in clinical development for the treatment of AA. A detailed summary of new drugs targeting these pathways with expert commentary on future directions for AA drug development and the importance of targeting multiple T-cell–signaling pathways is also provided in this review.
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In this current review, research spanning the last decade (such as transcriptomic studies, phenotypic observations, and confirmed comorbidities) has been synthesized into an updated etiology of hair loss and applied to the new cosmeceutical paradigm of hair rejuvenation. The major etiological components in scalps with hair loss are denoted as the ‘big eight strikes’, which include the following: androgens, prostaglandins, overactive aerobic metabolism of glucose, bacterial or fungal over-colonization, inflammation, fibrosis, metabolism or circulation problems, and malnutrition. The relevance of the ‘big eight’ to nine categories of hair loss is explained. In cases of androgenetic alopecia or female pattern hair loss, both elevated DHT and increased frequency of androgen receptors lead to problems with the metabolism of glucose (sugar), redox imbalance, disruption to the electron transport chain, and PPAR-γ overactivity (the latter is unique to androgenetic alopecia, where the reverse occurs in other types of hair loss). These etiological factors and others from ‘the big eight’ are the focal point of our hypothetical narrative of the attenuative mechanisms of commercial cosmeceutical hair serums. We conclude that cosmeceuticals with the potential to improve all eight strikes (according to published in vitro or clinical data) utilize bioactive peptides and plant compounds that are either flavonoids (isoflavones, procyanidins, flavanols, and flavonols) or sterols/triterpenes. It is noteworthy that many therapeutic interventions are generic to the multiple types of hair loss. Lastly, suggestions are made on how scalp and hair health can be improved by following the cosmeceutical approach.
Alopecia areata (AA) is a dermatological disease that causes nonscarring hair loss. It can occur at any age and has an unpredictable and variable evolution in individuals. The aim of this review is to provide an update on the novel therapies currently being used, as well as upcoming therapeutic options in the treatment of AA.
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Alopecia areata (AA) ist eine chronische, multifaktorielle, polygene und heterogene Erkrankung, die wachsende Haarfollikel bei anfälligen Personen betrifft und zu einem nicht vernarbenden und reversiblen Haarausfall mit höchst unvorhersehbarem Verlauf führt. Trotz sehr erheblichem Forschungsaufwand bleibt die Art des/der auslösenden Faktors/en, der/die für die Initiierung von AA in einem bestimmten Haarfollikel verantwortlich ist (sind), unklar, was weitgehend auf signifikante Wissenslücken bezüglich der genauen Abfolge der ätiopathogenen Ereignisse bei dieser Dermatose zurückzuführen ist. Jedoch sind krankheitsbedingte Veränderungen in der Immunkompetenz im unteren Bereich des wachsenden Haarfollikels zusammen mit einer aktiven Immunantwort (humoral und zellulär) auf Haarfollikel-assoziierte Antigene wichtige Schlüsselphänomene. Die genaue Identität des/der Haarfollikel-Antigens/e, das/die mit dem Ausbruch der AA-Krankheit in Verbindung steht/stehen, blieb schwer zu fassen. Während es von einigen als eher philosophische Frage angesehen werden mag, ist auch unklar, ob immunvermittelter Haarausfall bei AA die Folge von a) einer ektopischen (d.h. an einer abnormalen Stelle) Immunantwort auf native (unmodifizierte), vom gesunden Haarfollikel exprimierte Selbst-Antigene ist oder von b) einer normalen Immunantwort gegen modifizierte Selbst-Antigene (oder Neoantigene) oder von c) einer normalen Immunantwort gegen Selbst-Antigene (modifiziert/nicht modifiziert), die zuvor für das Immunsystem nicht sichtbar waren (weil sie konformationell versteckt oder sequestriert waren), aber auf eine MHC-I/II-Molekül-abhängige Weise exponiert und präsentierbar werden. Während sich einige potenzielle Haarfollikel-Zielantigene bei AA abzuzeichnen beginnen, mit einer potenziellen Rolle von Trichohyalin, ist noch unklar, ob dies den anfänglichen und immundominanten antigenen Fokus bei AA darstellt oder ob es nur eines aus einem wachsenden Repertoire von Antigenen ist, die mit den exponierten Gewebeschäden im Haarfollikel assoziiert sind und sekundär zur Krankheit beitragen. Die Bestätigung der Autoantigenidentität ist für unser Verständnis der AA-Ätiopathogenese und folglich für die Entwicklung einer fundierteren therapeutischen Strategie von großer Bedeutung. Große Fortschritte sind bei der Entdeckung von Autoantigenen im Zusammenhang mit anderen Autoimmunerkrankungen erzielt worden. Insbesondere können einige dieser Erkrankungen darüber Aufschluss geben, wie posttranslationale Modifikationen (z.B. Citrullinierung, Desamidierung usw.) von Haarfollikel-spezifischen Proteinen ihre Antigenität erhöhen und so dazu beitragen können, den Anti-Haarfollikel-Immunangriff bei AA voranzutreiben.
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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.
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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.
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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.
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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.
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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.
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.
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.
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.