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Review
Multiple Functions of the New Cytokine-Based
Antimicrobial Peptide Thymic Stromal
Lymphopoietin (TSLP)
Louise Bjerkan 1,† , Andreas Sonesson 2,3 and Karl Schenck 1, *
1
2
3
*
†
Department of Oral Biology, Dental Faculty, University of Oslo, PB 1052 Blindern, N-0316 Oslo, Norway;
[email protected]
Division of Dermatology and Venereology, Department of Clinical Sciences Lund, Lund University, BMC,
Tornavägen 10, SE-22184 Lund, Sweden; [email protected]
Dermatology and Venereology, Skane University Hospital, Lasarettsgatan 15, SE-22185 Lund, Sweden
Correspondence: [email protected]; Tel.: + 47-2284-0360
Present address: K.G. Jebsen Centre for Research on Influenza Vaccines, N-0450 Oslo, Norway
Academic Editor: Guangshun Wang
Received: 26 May 2016; Accepted: 30 June 2016; Published: 5 July 2016
Abstract: Thymic stromal lymphopoietin (TSLP) is a pleiotropic cytokine, hitherto mostly known to
be involved in inflammatory responses and immunoregulation. The human tslp gene gives rise to
two transcription and translation variants: a long form (lfTSLP) that is induced by inflammation, and
a short, constitutively-expressed form (sfTSLP), that appears to be downregulated by inflammation.
The TSLP forms can be produced by a number of cell types, including epithelial and dendritic cells
(DCs). lfTSLP can activate mast cells, DCs, and T cells through binding to the lfTSLP receptor (TSLPR)
and has a pro-inflammatory function. In contrast, sfTSLP inhibits cytokine secretion of DCs, but the
receptor mediating this effect is unknown. Our recent studies have demonstrated that both forms
of TSLP display potent antimicrobial activity, exceeding that of many other known antimicrobial
peptides (AMPs), with sfTSLP having the strongest effect. The AMP activity is primarily mediated by
the C-terminal region of the protein and is localized within a 34-mer peptide (MKK34) that spans
the C-terminal α-helical region in TSLP. Fluorescent studies of peptide-treated bacteria, electron
microscopy, and liposome leakage models showed that MKK34 exerted membrane-disrupting effects
comparable to those of LL-37. Expression of TSLP in skin, oral mucosa, salivary glands, and intestine
is part of the defense barrier that aids in the control of both commensal and pathogenic microbes.
Keywords: TSLP; AMP; immunoregulation
1. Introduction
Thymic stromal lymphopoietin (TSLP) was first identified in the culture supernatant of a murine
thymic stromal cell line and was shown to support B-cell growth and development [1]. The human
homologue of TSLP was cloned and characterized in 2001 and showed only 43% amino acid sequence
identity with mouse TSLP [2]. Despite the low amino acid sequence homology, human and murine
TSLP are functionally similar [3]. Human TSLP was identified as a four-helix bundle cytokine
containing six conserved cysteine residues and multiple sites for N-linked glycosylation [2,4].
Two variants of human TSLP peptides are expressed. Most studies hitherto have been focused
on a long form of TSLP (lfTSLP), while translation of a short form (sfTSLP) has been reported only
recently [5]. lfTSLP is inducible and associated with inflammation, and sfTSLP is constitutively
expressed and has an inhibiting effect on dendritic cells (DCs) [5,6]. Lately, we have shown that both
TSLP forms also act as strong antimicrobial peptides (AMP) [5,7]. Here, we describe the variants of
human TSLP and their expression in the human body, summarize their activity on different elements
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human TSLP and their expression in the human body, summarize their activity on different elements
ofthe
theimmune
immunesystem,
system,
describe
their
qualities
an AMP,
and outline
some
of the mechanisms
of
describe
their
qualities
as anasAMP,
and outline
some of
the mechanisms
behind
behind
their antimicrobial
their
antimicrobial
effects. effects.
2.
TSLP Variants
Variants
2. TSLP
Three
Three transcript
transcript variants
variants of
of human
human TSLP
TSLP are
are annotated
annotated in
in the
the RefSeq
RefSeq (Reference
(Reference Sequence)
Sequence)
database
(National
Center
for
Biotechnology
Information,
National
Library
of
Medicine,
Bethesda,
MD,
database (National Center for Biotechnology Information, National Library of Medicine,
Bethesda,
USA),
two long
and one
variant,
but only
one one
of the
longlong
(variant
1) and
thethe
short
(variant
2)
MD, USA),
two long
and shorter
one shorter
variant,
but only
of the
(variant
1) and
short
(variant
1 exons shorter
variants
give
rise
to
coding
RNA.
Variant
1
consists
of
four
exons
and
variant
2
is
two
5
2) variants give rise to coding RNA. Variant 1 consists of four exons and variant 2 is two 5′ exons
1 exon compared to variant 1 (Figure 1A). The relevance of distinguishing
but
contains
alternate
shorter
but an
contains
an 5alternate
5′ exon compared to variant 1 (Figure 1A). The relevance of
between
TSLP between
variants TSLP
in mice
is currently
as uncertain
a murineas
short
TSLPshort
variant
hasvariant
not been
distinguishing
variants
in miceuncertain
is currently
a murine
TSLP
has
described
or
annotated
in
RefSeq
so
far.
not been described or annotated in RefSeq so far.
The
Thelong
longform
form(lfTSLP)
(lfTSLP)encodes
encodesaa159
159amino
aminoacid
acid(aa)
(aa)protein.
protein.The
Theshort
shortform
form(sfTSLP)
(sfTSLP)encodes
encodes
aa sequence that
that isisidentical
identicalininthe
the
terminal
region
of long
TSLP
consists
aa (UniProt
CC
terminal
region
of long
TSLP
and and
consists
of 63of
aa63
(UniProt
entry:
entry:
G3XAM8)
and/or
a
60
aa
(UniProt
entry:
Q96AU7)
(Figure
1B).
UniProt
has
two
entries
for
G3XAM8) and/or a 60 aa (UniProt entry: Q96AU7) (Figure 1B). UniProt has two entries for the short
the
shortbecause
isoform there
because
are two potential
methionine
start separated
codons, separated
by twoacids.
amino
isoform
arethere
two potential
methionine
start codons,
by two amino
A
acids.
A
putative
signal
sequence
is
identified
in
the
long
TSLP
isoform,
with
a
predicted
cleavage
putative signal sequence is identified in the long TSLP isoform, with a predicted cleavage site after
site
after the threonine
at amino
acid 28,
leaving
a mature
lfTSLP
protein
131 [2].
amino
the threonine
residue atresidue
amino acid
28, leaving
a mature
lfTSLP
protein
of 131
aminoof
acids
The
acids
[2]. The
calculated
molecular
weight
(MW)
with,
without,
the signal
is
calculated
molecular
weight
(MW) of
lfTSLP
with,oforlfTSLP
without,
the or
signal
sequence
is 18.1sequence
kDa and 15
18.1
kDa
and
15
kDa,
respectively,
but
in
Western
blotting
the
apparent
MW
is
23
kDa,
probably
due
to
kDa, respectively, but in Western blotting the apparent MW is 23 kDa, probably due to postpost-transcriptional
modifications
(PTM)
transcriptional modifications
(PTM)
[5]. [5].
The
TheN-terminal
N-terminalsequence
sequenceof
ofsfTSLP
sfTSLPalso
alsocontains
containsaapotential
potentialN-terminal
N-terminalsignal
signalsequence
sequenceof
of20
20aa
aa
(SignalP,
[8]).
The
MW
of
sfTSLP
with
or
without
signal
sequence
is
7.4
kDa
(63
aa)
or
7.1
kDa
(60
aa)
(SignalP, [8]). The MW of sfTSLP with or without signal sequence is 7.4 kDa (63 aa) or 7.1 kDa (60 aa)
and
respectively. In
InWestern
Westernblotting,
blotting,the
theobserved
observedMW
MWlies
liesatat9
and 5.2
5.2 kDa
kDa (63
(63 aa) or 4.8 kDa (60 aa), respectively.
9kDa,
kDa,probably
probablydue
duetotoPTM
PTM[5].
[5].The
The
PTM
might
be
glycosylation
because
two
potential
sites
PTM might be glycosylation because two potential sites forfor
NN-linked
glycosylationare
arepresent
presentininthe
thelong
longisoform
isoformand
andone
onepotential
potential site
site is seen in the short
linked glycosylation
short isoform
isoform
(Figure
(Figure1B).
1B).sfTSLP
sfTSLPisispredicted
predictedto
toconsist
consistof
oftwo
twoα-helices
α-helices(Figure
(Figure1C,D;
1C,D;[9]).
[9]).As
Asyet,
yet,onlya
onlyafew
fewstudies
studies
have
examined
the
expression
of
sfTSLP
[5,6,10–13].
have examined the expression of sfTSLP [5,6,10–13].
Figure
Figure1.1.Cont.
Cont.
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Figure
1. TSLP
transcriptvariants
variantsand
andprotein
protein isoforms.
isoforms. (A)
gene
(green),
Figure
1. TSLP
transcript
(A)Graphics
Graphicsshowing
showingthe
theTSLP
TSLP
gene
(green),
the
long
form,
and
short
form
transcripts
(blue),
and
the
protein
products
(red).
Long
(NM_033035.4,
the long form, and short form transcripts (blue), and the protein products (red). Long (NM_033035.4,
NP_149024.1)
and
short
(NM_138551.3,
NP_612561.2)transcript
transcriptand
andprotein
proteinvariants,
variants,respectively,
respectively,are
NP_149024.1)
and
short
(NM_138551.3,
NP_612561.2)
are
indicated
(NCBI).
(B)
Amino
acid
sequence
of
human
TSLP
isoforms.
The
putative
signal
indicated (NCBI). (B) Amino acid sequence of human TSLP isoforms. The putative signal sequences
sequences
of
the
TSLP
isoforms
are
marked
in
blue,
and
the
mature
protein
in
black.
N-linked
of the TSLP isoforms are marked in blue, and the mature protein in black. N-linked glycosylation
glycosylation sites are marked green, and methionine start codons are marked red. Bold black
sites
are marked green, and methionine start codons are marked red. Bold black characters indicate
characters indicate the position of MKK34. (C) JNet secondary structure prediction of lfTSLP based
the position of MKK34. (C) JNet secondary structure prediction of lfTSLP based on the amino acid
on the amino acid sequence. Helices are marked as red tubes, and sheets are marked as green arrows.
sequence. Helices are marked as red tubes, and sheets are marked as green arrows. JNETCONF:
JNETCONF: The confidence estimate for the prediction, high values indicate high confidence.
The confidence estimate for the prediction, high values indicate high confidence. Modified from the
Modified from the web-based application Jpred (The Barton Group, School of Life Sciences, University
web-based application Jpred (The Barton Group, School of Life Sciences, University of Dundee, UK).
of Dundee, UK). (D) Three-dimensional structure (Swiss-model, [9]) of lfTSLP (left) and sfTSLP (right).
(D) Three-dimensional structure (Swiss-model, [9]) of lfTSLP (left) and sfTSLP (right).
3. Expression and Regulation of TSLP Variants
3. Expression and Regulation of TSLP Variants
Use of variant-specific reagents is necessary to study the expression of the two human TSLP
Use
variant-specific
reagents
is this
necessary
to study
expression
of the
two
human
variantsofseparately.
At the mRNA
level,
differentiation
canthe
be obtained
by the
use of
variantTSLP
variants
separately.
At
the
mRNA
level,
this
differentiation
can
be
obtained
by
the
use of
specific primers, which are constructed based on unique mRNA sequences in the two transcript
variant-specific
primers,
which are constructed
based on unique
mRNA
sequences
in theapproach.
two transcript
variants. Detection
of variant-specific
protein expression,
however,
requires
an indirect
As
there is Detection
a total overlap
of sfTSLP with the
lfTSLP
amino acidhowever,
C-terminal
sequence,
raised
variants.
of variant-specific
protein
expression,
requires
an antibodies
indirect approach.
will
recognize
variantsamino
and the
production
of sfTSLP-specific
antibodies
As against
there issfTSLP
a total epitopes
overlap of
sfTSLP
withboth
the lfTSLP
acid
C-terminal
sequence, antibodies
raised
is, therefore,
possible.
the other
hand,
it is possible
toproduction
generate antibodies
specific forantibodies
lfTSLP,
against
sfTSLP not
epitopes
willOn
recognize
both
variants
and the
of sfTSLP-specific
by immunization
peptide
lie within
the specific
lfTSLPspecific
sequence,
by
is, either
therefore,
not possible. with
On the
other sequences
hand, it is that
possible
to generate
antibodies
foror
lfTSLP,
retrieving
monoclonals
that
recognize
such
sequences.
Thus,
distinguishing
between
sfTSLP
and
either by immunization with peptide sequences that lie within the specific lfTSLP sequence, or by
lfTSLP has
to be overcome
by comparing
combined Thus,
content
of lfTSLP and between
sfTSLP insfTSLP
samples,
retrieving
monoclonals
that recognize
suchthe
sequences.
distinguishing
and
using
one
antibody
that
recognizes
both
forms,
and
another
antibody
that
binds
to
the
unique
lfTSLP has to be overcome by comparing the combined content of lfTSLP and sfTSLP in samples,
using
lfTSLP (a long-specific anti-TSLP antibody). This approach has been used both for
onesequence
antibodyofthat
recognizes both forms, and another antibody that binds to the unique sequence of
Western blotting and immunohistology [5,6] (Figure 2).
lfTSLP (a long-specific anti-TSLP antibody). This approach has been used both for Western blotting
The expression pattern of the two human isoforms is dependent on both tissue localization and
and immunohistology [5,6] (Figure 2).
disease state. Most of the TSLP literature of the last two decades has only focused on lfTSLP, as the
The expression pattern of the two human isoforms is dependent on both tissue localization and
translation of sfTSLP only recently has been documented [5]. The expression of TSLP has largely been
disease state. Most of the TSLP literature of the last two decades has only focused on lfTSLP, as the
associated with inflammatory conditions by which it was found to be upregulated. We now know
translation
of sfTSLP
recently
has beenofdocumented
[5].vivo,
The lfTSLP
expression
of TSLP has
largely been
that this was
due toonly
increased
expression
lfTSLP [5,6]. In
is upregulated
in conditions
associated
with
inflammatory
conditions
by
which
it
was
found
to
be
upregulated.
We
know
such as atopic dermatitis, asthma, ulcerative colitis, and smokeless tobacco-exposed oralnow
mucosa,
that
this
was
due
to
increased
expression
of
lfTSLP
[5,6].
In
vivo,
lfTSLP
is
upregulated
in
conditions
while it is absent in healthy tissues (Figure 2) [5,6,10,14]. In vitro studies of cultured dermal and oral
such
as atopic dermatitis,
ulcerative factors,
colitis, such
and smokeless
tobacco-exposed
oralnecrosis
mucosa,
keratinocytes
exposed toasthma,
pro-inflammatory
as interferon
γ (IFN-γ), tumor
while
it
is
absent
in
healthy
tissues
(Figure
2)
[5,6,10,14].
In
vitro
studies
of
cultured
dermal
and
factor α (TNF-α) in combination with interleukin 1β (IL-1-β), and polyriboinosinic:polyribocytidylic
oral
keratinocytes
exposed
to pro-inflammatory
factors,
such
as interferon
(IFN-γ), epithelial
tumor necrosis
acid
(poly(I:C)), show
upregulation
of lfTSLP mRNA
and
protein
[5,15]. Theγintestinal
cell
factor
(TNF-α)
in combination
with interleukin
1β (IL-1-β),
and polyriboinosinic:polyribocytidylic
lineαCaco-2,
challenged
with Salmonella
typhimurium,
shows upregulation
of both mRNA and protein
lfTSLP
expression
Finally, TH2of
cytokines
were found
to be potent
of TSLPepithelial
in human
acid
(poly(I:C)),
show[6].
upregulation
lfTSLP mRNA
and protein
[5,15].inducers
The intestinal
cell
bronchial
epithelial
cells
[16].
In
normal
nasal
mucosa
cultured
in
the
presence
of
the
inflammatory
line Caco-2, challenged with Salmonella typhimurium, shows upregulation of both mRNA and protein
TH2 cytokines;
and
lfTSLP
mRNA
is upregulated
Increased
mRNA
and
lfTSLP
expressionIL-4,
[6]. IL-13,
Finally,
THTNF-α,
2 cytokines
were
found
to be potent [13].
inducers
of TSLP
in human
protein
TSLP
expression
were
detected
upon
exposure
of
immunodeficiency
virus
in
cervical
bronchial epithelial cells [16]. In normal nasal mucosa cultured in the presence of the inflammatory
cellsIL-4,
[17] and
exposure
to poly(I:C)
andmRNA
a cocktail
of IL-1 and TNF
in airway
epithelial
cells
THepithelial
2 cytokines;
IL-13,
and TNF-α,
lfTSLP
is upregulated
[13].
Increased
mRNA
and
protein TSLP expression were detected upon exposure of immunodeficiency virus in cervical epithelial
cells [17] and exposure to poly(I:C) and a cocktail of IL-1 and TNF in airway epithelial cells [18].
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Although not emphasized in these studies, the increased TSLP expression detected is presumably due
[18]. Although not emphasized in these studies, the increased TSLP expression detected is
to lfTSLP.
presumably due to lfTSLP.
Figure
2. Immunohistochemical(IHC)
(IHC) staining
staining and
(ISH)
of sections
of oral
Figure
2. Immunohistochemical
and ininsitu
situhybridization
hybridization
(ISH)
of sections
of oral
mucosa (A–C), skin (D–F), salivary gland (G–I), and smokeless tobacco (“snus”; J,K) for TSLP
mucosa (A–C), skin (D–F), salivary gland (G–I), and smokeless tobacco (“snus”; J,K) for TSLP variants.
variants. Left column: IHC staining with anti-TSLP antibody recognizing both lfTSLP and sfTSLP
Left column: IHC staining with anti-TSLP antibody recognizing both lfTSLP and sfTSLP (brown color).
(brown color). Middle column: IHC staining with anti-TSLP antibody recognizing lfTSLP only. As no
Middle column: IHC staining with anti-TSLP antibody recognizing lfTSLP only. As no specific staining
specific staining is detected in (B,E,H), this means that the staining in (A,D,G) represents sfTSLP. In
is detected
in (B,E,H),
thistomeans
that the
staining
in (A,D,G)
represents
sfTSLP.
Instaining
oral mucosa
oral mucosa
exposed
smokeless
tobacco,
lfTSLP
is seen (K).
Right column:
ISH
by useexposed
of
to smokeless
tobacco,
lfTSLP
is
seen
(K).
Right
column:
ISH
staining
by
use
of
sfTSLP-specific
sfTSLP-specific probe (blue color) which confirms strong expression of sfTSLP in oral mucosa andprobe
(bluesalivary
color) which
expression
of sfTSLP
in oral
gland, confirms
and weak strong
expression
in skin. Modified
from
[5]. mucosa and salivary gland, and weak
expression in skin. Modified from [5].
In contrast to lfTSLP, sfTSLP (mRNA and protein) is the predominant form of TSLP
constitutively expressed in healthy tissues, including clinically healthy oral epithelium, skin
In contrast to lfTSLP, sfTSLP (mRNA and protein) is the predominant form of TSLP constitutively
epidermis, salivary glands, and gut epithelial cells (Figure 2) [5,6]. Under inflammatory conditions,
expressed in healthy tissues, including clinically healthy oral epithelium, skin epidermis, salivary
sfTSLP appears to be downregulated as observed in lesional biopsy material from atopic dermatitis
glands,
and
gut
cells
[5,6].
Underdisease
inflammatory
conditions,
sfTSLP appears
(AD)
and
inepithelial
the intestine
of (Figure
patients 2)
with
Crohn’s
[6]. Exposure
to S. typhimurium
also to
be downregulated
as observed
lesional
material
from
(AD)protein
and in the
downregulates sfTSLP
mRNA in
and
protein biopsy
expression
in Caco-2
cellsatopic
[6]. Todermatitis
this date, sfTSLP
intestine
of patients
with
Crohn’s
disease
S. typhimurium
also downregulates
sfTSLP
expression
has only
been
identified
in the[6].
gut,Exposure
skin, oral to
epithelium
and salivary
glands [5,6].
The
divergent
expression
pattern
for
the
two
translated
TSLP
variants
is
consistent
with
the
mRNA and protein expression in Caco-2 cells [6]. To this date, sfTSLP protein expression has only
of the
human
reveals that
two variants
not alternatively spliced, but
been analysis
identified
in the
gut,TSLP
skin,locus
oral that
epithelium
andthe
salivary
glandsare
[5,6].
are derived
fromexpression
the activity pattern
of two separate,
promotorTSLP
regions
[6]. TheissfTSLP
promotor
The
divergent
for the putative
two translated
variants
consistent
with the
appears
to
exhibit
a
high
capacity
to
bind
a
number
of
different
transcription
factors,
while
the
region
analysis of the human TSLP locus that reveals that the two variants are not alternatively spliced,
but are derived from the activity of two separate, putative promotor regions [6]. The sfTSLP promotor
appears to exhibit a high capacity to bind a number of different transcription factors, while the region
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upstream
from
the
is relatively
relativelyinert
inertininmost
mostofofthe
the
cell
lines
upstream
from
thelfTSLP
lfTSLPunder
understeady-state
steady-state conditions
conditions is
cell
lines
present
in the
database.
Thus, under
conditions,
sfTSLP represents
the homeostatic
present
in UCSC
the UCSC
database.
Thus, steady-state
under steady-state
conditions,
sfTSLP represents
the
homeostatic
form
of TSLP. In inflammation,
lfTSLP
is up-isand
sfTSLP is downregulated.
form
of TSLP. In
inflammation,
lfTSLP is up- and
sfTSLP
downregulated.
The
expressionand
and regulation
of of
TSLP
in mice
overlaps
to a large
of human
The
expression
regulationpattern
pattern
TSLP
in mice
overlaps
to aextend
largethat
extend
that of
lfTSLP.
A roleAofrole
TSLP
human
allergicallergic
diseasesdiseases
is well supported
by a variety
mouse of
models
human
lfTSLP.
of in
TSLP
in human
is well supported
by aofvariety
mouse
[19–22]
and
increased
lung
tissue
expression
of
TSLP
has
been
detected
in
mice
challenged
with
models [19–22] and increased lung tissue expression of TSLP has been detected in mice challenged
with
dsRNA
[23].
In
the
steady
state,
TSLP
expression
in
the
skin
of
mice
appears
to
be
negatively
dsRNA [23]. In the steady state, TSLP expression in the skin of mice appears to be negatively regulated
retinoid(RXR)
X receptors
[24]. study,
In the keratinocyte-specific
latter study, keratinocyte-specific
ablation
of
by regulated
retinoid Xby
receptors
[24]. In(RXR)
the latter
ablation of RXRs
resulted
RXRs
resulted
in
upregulation
of
TSLP
and
development
of
AD-like
skin
inflammation.
Further,
the
in upregulation of TSLP and development of AD-like skin inflammation. Further, the phenotype of
phenotype of mice lacking TSLP signaling (tslpr(−/−)) and challenged with human metapneumovirus
mice lacking TSLP signaling (tslpr(´/´)) and challenged with human metapneumovirus (hMPV)
(hMPV) show reduced lung infection and hMPV replication [25]. These mice displayed a decreased
show reduced lung infection and hMPV replication [25]. These mice displayed a decreased number of
number of neutrophils, as well a reduction in levels of thymus and activation-regulated
neutrophils, as well a reduction in levels of thymus and activation-regulated chemokine/CCL17, IL-5,
chemokine/CCL17, IL-5, IL-13, and TNF-α in the airways upon hMPV infection compared to WT
IL-13, and TNF-α in the airways upon hMPV infection compared to WT mice.
mice.
4. Human TSLP Variants and Immunoregulation
4. Human TSLP Variants and Immunoregulation
4.1. Long-Form TSLP (lfTSLP)
4.1. Long-Form TSLP (lfTSLP)
lfTSLP is closely related to IL-7, with which it shares an overlapping, but not identical, biological
lfTSLP is closely related to IL-7, with which it shares an overlapping, but not identical, biological
profile, and binds to a heterodimeric receptor complex consisting of the IL-7 receptor α-chain (IL-7Rα)
profile, and binds to a heterodimeric receptor complex consisting of the IL-7 receptor α-chain (ILand
the TSLP receptor chain (TSLPR) [2,26]. The functional receptor for lfTSLP is expressed on both
7Rα) and the TSLP receptor chain (TSLPR) [2,26]. The functional receptor for lfTSLP is expressed on
hematopoietic
and non-hematopoietic
cell lineages
including
DCs,DCs,
T cells,
B cells,
natural
killer
cells,
both hematopoietic
and non-hematopoietic
cell lineages
including
T cells,
B cells,
natural
killer
monocytes,
basophils,
eosinophils,
and
epithelial
cells
[3,18,19,27–32].
Activation
of
the
TSLP
receptor
cells, monocytes, basophils, eosinophils, and epithelial cells [3,18,19,27–32]. Activation of the TSLP
hasreceptor
been shown
to signal
signal transducer
and activator
of transcription
(STAT)
has been
shownthrough
to signalmultiple
through multiple
signal transducer
and activator
of transcription
proteins,
including
STAT
1,
3,
4,
5,
6,
and
Janus
kinase
(JAK)
1
and
2
in
peripheral
blood-derived
(STAT) proteins, including STAT 1, 3, 4, 5, 6, and Janus kinase (JAK) 1 and 2 in peripheral blood+ DCs (Figure
CD11c
3)(Figure
[5,33,34].
derived
CD11c+ DCs
3) [5,33,34].
Figure
3. STAT5
phosphorylation
in response
to lfTSLP,
aa sfTSLP,
63 aa sfTSLP,
or combined
lfTSLP
Figure
3. STAT5
phosphorylation
in response
to lfTSLP,
60 aa 60
sfTSLP,
63 aa sfTSLP,
or lfTSLP
combined
with
sfTSLP
in
blood-derived
CD1c
myeloid
DCs
incubated
with
poly(I:C)
for
24
and
with sfTSLP in blood-derived CD1c myeloid DCs incubated with poly(I:C) for 24 h, and thenh,treated
then
treated
with
sfTSLP
or/and
lfTSLP
for
15
min.
Phosphorylation
of
STAT5
was
assessed
by
flow
with sfTSLP or/and lfTSLP for 15 min. Phosphorylation of STAT5 was assessed by flow cytometry.
cytometry.
From [5].
From
[5].
lfTSLP has an impact on several immune functions and has, as mentioned above, been associated
lfTSLP has an impact on several immune functions and has, as mentioned above, been associated
with immune disorders, such as allergic diseases and intestinal inflammation. Co-culture of lfTSLPwith
immuneDCs
disorders,
such asCD4
allergic
diseases
intestinal
inflammation.
Co-culture
+ T cells
stimulated
with allogeneic
results and
in the
generation
of inflammatory
Th2 cells of
+ T cells results in the generation of inflammatory Th2 cells
lfTSLP-stimulated
DCs
with
allogeneic
CD4
producing classical Th2 cytokines including IL-4, IL-5, IL-13, but in contrast to conventional Th2 cells,
producing
classical
Th2 cytokines
IL-4,[14].
IL-5,This
IL-13,
but in contrast
conventional
Th2 cells,
these cells
also produce
TNF-α including
and not IL-10
inflammatory
Th2tophenotype
is induced
these cells also produce TNF-α and not IL-10 [14]. This inflammatory Th2 phenotype is induced
Pharmaceuticals 2016, 9, 41
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Pharmaceuticals
2016, 9, 41
6 of 12
through
the upregulation
of OX-40 ligand expression on lfTSLP-treated DCs [14,35]. Accordingly,
in atopic dermatitis (AD), lfTSLP protein is not detectable in non-lesional skin in AD patients, while it
through the upregulation of OX-40 ligand expression on lfTSLP-treated DCs [14,35]. Accordingly, in
is highly expressed in acute and chronic AD lesions [14]. In allergic rhinitis, TSLP treatment of CD1c+
atopic dermatitis (AD), lfTSLP protein is not detectable in non-lesional skin in AD patients, while it
DCs potently augments allergen-specific TH 2 memory responses [13].
is highly expressed in acute and chronic AD lesions [14]. In allergic rhinitis, TSLP treatment of CD1c+
In contrast to its role in inflammation, TSLP has also been suggested to have homeostatic,
DCs potently augments allergen-specific TH2 memory responses [13].
tolerogenic
functions [36,37]. It was, however, at that time unknown that the sfTSLP peptide is
In contrast to its role in inflammation, TSLP has also been suggested to have homeostatic,
also
translated,
and that
this peptide
has an inhibiting
effect
on DCs
a re-evaluation
tolerogenic functions
[36,37].
It was, however,
at that time
unknown
that[5].
theAfter
sfTSLP
peptide is also of
earlier
resultsand
andthat
further
is now clear
that
responsible
for this effect
in the
translated,
this investigations,
peptide has an itinhibiting
effect
on sfTSLP
DCs [5].isAfter
a re-evaluation
of earlier
intestine
[6].
results and further investigations, it is now clear that sfTSLP is responsible for this effect in the
intestine [6].
4.2. Short-Form TSLP (sfTSLP)
4.2.
Short-Form
TSLP (sfTSLP)
sfTSLP
is constitutively
expressed by several types of epithelial cells, as described above. sfTSLP
appearssfTSLP
to act is
onconstitutively
DCs on which
it inhibits
secretion
[6]. sfTSLP
does
not bind
to the
TSLPR
expressed
by cytokine
several types
of epithelial
cells, as
described
above.
sfTSLP
because
it
is
not
capable
to
block
binding
of
lfTSLP
to
this
receptor
(Figure
3)
[5,6].
The
specific
appears to act on DCs on which it inhibits cytokine secretion [6]. sfTSLP does not bind to the TSLPR
receptor
forit sfTSLP
is currently
unknown.
induces
of [5,6].
p38α,The
extracellular
because
is not capable
to block
binding sfTSLP
of lfTSLP
to thisphosphorylation
receptor (Figure 3)
specific
signal-regulated
kinaseis1/2,
and Lyn,
but has no
effectinduces
on STAT5
phosphorylation
(Figure
3) [5,6]. Very
receptor for sfTSLP
currently
unknown.
sfTSLP
phosphorylation
of p38α,
extracellular
signal-regulated
kinase
1/2,the
and
Lyn, but has no effect
(Figure
3) [5,6].
little
else is yet known
about
immunoregulatory
actiononofSTAT5
sfTSLP.phosphorylation
As sfTSLP can be
downregulated
little else this
is yet
known
about to
theanimmunoregulatory
action
of sfTSLP.
can be
by Very
inflammation,
might
contribute
aggravation of local
infection
in viewAsofsfTSLP
its antimicrobial
downregulated
by inflammation, this might contribute to an aggravation of local infection in view of
activity
(see below).
its antimicrobial activity (see below).
5. Human TSLP Variants as Antimicrobial Peptides
5. Human TSLP Variants as Antimicrobial Peptides
AMPs can be classified in a variety of approaches [38], fitting into one of four major structural
can peptides
be classified
a variety
approaches
[38], fittingupon
into one
of four
major structural
classes:AMPs
(1) linear
thatinmay
adoptofα-helical
conformation
bacterial
binding;
(2) β-sheet
classes:
(1)
linear
peptides
that
may
adopt
α-helical
conformation
upon
bacterial
binding;
(2)
β-sheet
peptides; (3) extended peptides with over-representation of specific amino acid residues; or (4)
looped
peptides;
(3)
extended
peptides
with
over-representation
of
specific
amino
acid
residues;
or based
(4)
peptides [39–41]. However, dermcidin, an AMP secreted by sweat glands [42], is often classified
looped
peptides
[39–41].
However,
dermcidin,
an
AMP
secreted
by
sweat
glands
[42],
is
often
on its anionicity.
classified based on its anionicity.
A common characteristic of AMPs is the propensity to form helical structure [40,43]. It has been
A common characteristic of AMPs is the propensity to form helical structure [40,43]. It has been
previously reported that TSLP contains several predicted helical regions [3] (Figure 1C). Thus, both
previously reported that TSLP contains several predicted helical regions [3] (Figure 1C). Thus, both
sfTSLP and lfTSLP are cationic peptides with regions that could display α-helical conformation.
sfTSLP and lfTSLP are cationic peptides with regions that could display α-helical conformation.
Moreover, analysis of the mature lfTSLP (131 amino acids) reveals that it may harbor qualities
Moreover, analysis of the mature lfTSLP (131 amino acids) reveals that it may harbor qualities
required
forforantimicrobial
at physiological
physiologicalconditions,
conditions,
such
a positive
net charge
+11
required
antimicrobial activity
activity at
such
a positive
net charge
of +11 of
and
and
theoretical
isoelectric
point
of 9.63
(calculated
by using
the Protparam
Swiss
Institute
aa
theoretical
isoelectric
point
(pI)(pI)
of 9.63
(calculated
by using
the Protparam
tool;tool;
Swiss
Institute
of
of Bioinformatics,
Lausanne,
Switzerland).
Furthermore,
analysis
of
the
hydrophobic
moment
(µH)
Bioinformatics, Lausanne, Switzerland). Furthermore, analysis of the hydrophobic moment (μH)
revealed
a region,
likely
conspicuousamphiphathic
amphiphathicproperties
properties
revealed
a region,
likelyininthe
theC-terminal
C-terminalpart
part of
of TSLP,
TSLP, with
with conspicuous
(Figure
4) 4)(calculated
theEuropean
European
Molecular
Biology
Open
Software
Suite (EMBOSS);
(Figure
(calculatedby
by using
using the
Molecular
Biology
Open
Software
Suite (EMBOSS);
The
TheSanger
Sanger
Centre,
Wellcome
Trust
Genome
Campus,
Hinxton,
Cambridge,
UK).
Centre, Wellcome Trust Genome Campus, Hinxton, Cambridge, UK).
Figure
Plotofofhydrophobic
hydrophobicmoment
moment (µH)
(μH) for
for the
Figure
4. 4.Plot
the mature
maturelfTSLP
lfTSLP(131
(131amino
aminoacids).
acids).
Our data displayed antimicrobial activity of TSLP against both bacteria and fungi [5,7] (Figure
Our To
data
displayed
antimicrobial
activity
of TSLP
against
and
[5,7]
5A,B).
further
investigate
the antimicrobial
properties
of TSLP
and both
whichbacteria
regions of
the fungi
molecule
(Figure
5A,B).
To
further
investigate
the
antimicrobial
properties
of
TSLP
and
which
regions
exhibited the antimicrobial effects, overlapping 20-mer peptides were synthesized [7]. The of
theexperiments
molecule exhibited
effects,
overlapping
20-mer
synthesized
showed the
thatantimicrobial
the antimicrobial
effect
preferentially
was peptides
located inwere
regions
of the C-[7].
terminal part of TSLP [7]. When a 34 aa long synthetic peptide (MKK34; Figure 1B) spanning the C-
Pharmaceuticals 2016, 9, 41
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The experiments showed that the antimicrobial effect preferentially was located in regions of the
Pharmaceuticals
2016,
9, 41 [7]. When a 34 aa long synthetic peptide (MKK34; Figure 1B) spanning
7 of 12
C-terminal
part of
TSLP
the
C-terminal part of TSLP was tested for antimicrobial activities, it exerted potent antimicrobial activity,
terminal part of TSLP was tested for antimicrobial activities, it exerted potent antimicrobial activity,
both in the presence of human plasma and in physiological salt conditions [7]. MKK34 contains
both in the presence of human plasma and in physiological salt conditions [7]. MKK34 contains
predicted
regions
thatthat
could
display
helical
wheel
projection
of MKK34
is
predicted
regions
could
displayα-helical
α-helical conformation.
conformation. AA
helical
wheel
projection
of MKK34
is
visualized
in Figure
6. 6.
visualized
in Figure
5. Antimicrobial
activity
shortand
and long
long forms
stromal
lymphopoietin
(sfTSLP
FigureFigure
5. Antimicrobial
activity
ofofshort
formsofofthymic
thymic
stromal
lymphopoietin
(sfTSLP
and
lfTSLP).
(A)
lfTSLP
exhibited
a
larger
zone
of
inhibition
of
growth
of
Escherichia
coli
ATCC
and lfTSLP). (A) lfTSLP exhibited a larger zone of inhibition of growth of Escherichia25922
coli ATCC
in comparison with LL-37: (a) control; (b) 10 μM LL-37; and (c) 10μM TSLP. Mean values and standard
25922 in comparison with LL-37: (a) control; (b) 10 µM LL-37; and (c) 10µM TSLP. Mean values
deviations (n = 4). (B) In a viable count assay, indicated bacterial (Escherichia coli, Pseudomonas
and standard deviations (n = 4). (B) In a viable count assay, indicated bacterial (Escherichia coli,
aeruginosa, Staphylococcus aureus, and Staphylococcus epidermidis) and fungal isolates (Candida albicans
Pseudomonas
aeruginosa,
Staphylococcus
aureus,
and
epidermidis)
fungal(C)
isolates
and Candida
parapsilosis)
were subjected
to 2 μM
of Staphylococcus
TSLP. The number
of cfu wasand
registered.
(Candida
albicans of
and
to treated
2 µM of
The
number
cfu was
Suspensions
theCandida
indicatedparapsilosis)
bacterial and were
fungalsubjected
species were
for TSLP.
2 h with
60 amino
acidof(aa)
registered.
oflfTSLP
the indicated
and fungal
species
for
2 h with
sfTSLP,(C)
andSuspensions
63 aa sfTSLP or
peptide at bacterial
a concentration
of 1.35 mM
beforewere
beingtreated
plated on
agar.
Colony-forming
units per
were
determined
afterpeptide
incubation
overnight. The of
values
werebefore
60 amino
acid (aa) sfTSLP,
and ml
63 aa
sfTSLP
or lfTSLP
at a concentration
1.35 mM
the levels
obtained without
the addition
test peptides
(broken
line).incubation
(D) Suspensions
being normalized
plated ontoagar.
Colony-forming
units
per ml ofwere
determined
after
overnight.
of Streptococcus
mitis wereto
treated
with equimolar
concentrations
of 60 aa of
sfTSLP,
LL-37, or(broken
lfTSLP line).
The values
were normalized
the levels
obtained without
the addition
test peptides
and analyzed as in C. From: [7] (A,B) and [5] (C,D).
(D) Suspensions of Streptococcus mitis were treated with equimolar concentrations of 60 aa sfTSLP,
LL-37, or lfTSLP and analyzed as in C. From: [7] (A,B) and [5] (C,D).
Pharmaceuticals 2016, 9, 41
Pharmaceuticals 2016, 9, 41
8 of 13
8 of 12
Figure 6.
helical
wheel
projection
waswas
constructed
using
the amino
acid
6. Helical
Helicalstructure
structureofofMKK34.
MKK34.A A
helical
wheel
projection
constructed
using
the amino
sequence
of MKK34.
acid sequence
of MKK34.
The findings
findings that
that the
the main
main antimicrobial
antimicrobial activity
activity of
of TSLP
TSLP is
is located
located in
in its
its C-terminal
C-terminal part
part is
is
The
particularly relevant
relevant since
since both
both MKK34
MKK34 and
and sfTSLP
sfTSLP are
are found
found in
in this
this region,
region, and
and sfTSLP
sfTSLP is
is translated
translated
particularly
and
constitutivelyexpressed
in
normal
tissues
[5].
In
our
studies,
both
forms
of
TSLP
and
MKK34
and constitutivelyexpressed in normal tissues [5]. In our studies, both forms of TSLP and MKK34
were
found
to
have
antimicrobial
action
against
Gram-positive
and
Gram-negative
bacteria,
and
were found to have antimicrobial action against Gram-positive and Gram-negative bacteria, and
fungi, stronger
stronger than
than the
the well-characterized
well-characterized AMP
AMP LL-37
LL-37 [5,7]
[5,7] (Figure
(Figure 5B,C).
5B,C). sfTSLP
sfTSLP exerted
exerted potent
potent
fungi,
antimicrobial
activity
against
all
the
tested
species,
including
Streptococcus
mitis,
Escherichia
antimicrobial activity against all the tested species, including Streptococcus mitis, Escherichia coli,
coli,
Enterococcus faecalis,
faecalis, Bacillus
Bacillus cereus,
cereus, Staphylococcus
Staphylococcus epidermidis,
Enterococcus
epidermidis, and
and Candida
Candida albicans
albicans [5]
[5] (Figure
(Figure 5C).
5C).
Moreover, addition
addition of
of polyclonal
polyclonal anti-TSLP
anti-TSLP antibody
antibody to
to sfTSLP
sfTSLP before
before it
it was
Moreover,
was incubated
incubated with
with S.
S. mitis,
mitis,
reduced
the
antimicrobial
activity
by
about
half,
showing
that
the
reduction
in
colony-forming
units
reduced the antimicrobial activity by about half, showing that the reduction in colony-forming units
per mL
mL was
was specifically
specifically due
due to
to the
the action
action of
of sfTSLP
sfTSLP [5].
[5]. Dose-response
Dose-response curves
curves using
using S.
S. mitis
mitis showed
showed
per
that
the
effect
of
sfTSLP
was
stronger
than
that
of
LL-37
(Figure
5D).
Furthermore,
the
susceptibility
that the effect of sfTSLP was stronger than that of LL-37 (Figure 5D). Furthermore, the susceptibility of
of isolates
of Staphylococcus
aureus,
S. epidermidis,
E. and
coli,Pseudomonas
and Pseudomonas
aeruginosa
to MKK34
was
isolates
of Staphylococcus
aureus,
S. epidermidis,
E. coli,
aeruginosa
to MKK34
was tested
tested
in antimicrobial
assays
The Gram-positive
isolates
were less
generally
less susceptible
to
in
antimicrobial
assays [7].
The [7].
Gram-positive
isolates were
generally
susceptible
to MKK34 in
MKK34
in
comparison
to
Gram-negative
bacteria
[7].
comparison to Gram-negative bacteria [7].
AMPs are
are reported
reported to
to possess
possess different
different antibacterial
antibacterial spectrums.
spectrums. The
The well-characterized
well-characterized AMP
AMP
AMPs
LL-37
has
a
broad
spectrum
whereas
psoriasin
preferentially
kills
E.
coli
[44,45].
Considering
that
LL-37 has a broad spectrum whereas psoriasin preferentially kills E. coli [44,45]. Considering that
TSLP
is
released
in
response
to
microbial
stimulation
of
epithelial
cells
[18],
our
findings
suggest
that
TSLP is released in response to microbial stimulation of epithelial cells [18], our findings suggest
TSLPTSLP
and and
TSLP-derived
peptides,
suchsuch
as MKK34
andand
sfSTLP,
exert
broad
antimicrobial
activity
on
that
TSLP-derived
peptides,
as MKK34
sfSTLP,
exert
broad
antimicrobial
activity
Gram-negative
bacteria,
Gram-positive
bacteria,
as
well
as
fungi,
that
are
of
importance
in
host
on Gram-negative bacteria, Gram-positive bacteria, as well as fungi, that are of importance in host
defense [5,7].
[5,7]. Moreover,
vivo resistance
resistance of
of human
human skin
skin to
to
defense
Moreover, MKK34
MKK34 may
may be
be aa contributor
contributor to
to the
the in
in vivo
Gram-negative bacterial
bacterial colonization
colonization and
infection and
and hypothetically
hypothetically support
support the
the maintenance
maintenance of
of
Gram-negative
and infection
preferentially
Gram-positive
bacterial
(S.
epidermidis)
colonization
at
the
human
skin.
preferentially Gram-positive bacterial (S. epidermidis) colonization at the human skin.
Several classical
AMPs,
such
as LL-37
and AMPs
derived
from larger
Several
classical and
andrecently
recentlydiscovered
discovered
AMPs,
such
as LL-37
and AMPs
derived
from
proteins,
are
generated
by
proteolytic
processing
resulting
in
bioactive
fragments
that
exert
larger proteins, are generated by proteolytic processing resulting in bioactive fragments that exert
antimicrobial
effects
[46–49].
As
mentioned
before,
TSLP
is
highly
expressed
by
keratinocytes
in
antimicrobial effects [46–49]. As mentioned before, TSLP is highly expressed by keratinocytes in
atopic eczema.
eczema. Moreover,
AD skin
skin is
is frequently
frequently colonized
colonized by
by S.
S. aureus
aureus and
and characterized
characterized by
by aa chronic
chronic
atopic
Moreover, AD
inflammatory
infiltrate
[14,50].
Therefore,
it
is
tempting
to
speculate
that
AD
skin
cleavage
of
TSLP
inflammatory infiltrate [14,50]. Therefore, it is tempting to speculate that AD skin cleavage of TSLP
by
proteases
(both
endogenous
and
bacterial)
produces
small
antimicrobial
fragments.
To
test
by proteases (both endogenous and bacterial) produces small antimicrobial fragments. To test this,
this,
we
incubated
TSLP
in
the
presence
of
neutrophil
(leukocyte)
elastase
(HLE),
which
is
produced
by
we incubated TSLP in the presence of neutrophil (leukocyte) elastase (HLE), which is produced by
leukocytes
during
inflammation,
as
well
as
in
the
presence
of
different
bacterial
derived
proteases.
leukocytes during inflammation, as well as in the presence of different bacterial derived proteases.
When analyzed
analyzed by
by SDS-PAGE,
SDS-PAGE, the
the incubation
incubation products
products revealed
revealed degradation
degradation of
of TSLP
TSLP by
by both
both HLE
HLE
When
and the bacterial proteases (P. aeruginosa elastase, S. aureus V8) (Figure 7). The S. aureus V8 proteinase
degraded TSLP into three distinct fragments, the major peptide fragment (fragment I) being derived
Pharmaceuticals 2016, 9, 41
9 of 13
(P. aeruginosa elastase, S. aureus V8) (Figure 7). The S. aureus V8 proteinase
99 of
of 12
12
degraded TSLP into three distinct fragments, the major peptide fragment (fragment I) being derived
from
the
C-terminal
part
comprising
42
acids
encompassing
the
from the
theC-terminal
C-terminal
part
and
comprising
42 amino
amino
acids (Thr88-Lys129),
(Thr88-Lys129),
encompassing
the
part
andand
comprising
42 amino
acids (Thr88-Lys129),
encompassing
the previously
previously
synthetic
MKK34
peptide.
When
the
products
were
previously characterized
characterized
synthetic
MKK34
peptide.
When
the V8
V8 degradation
degradation
products
were
tested,
characterized
synthetic MKK34
peptide.
When
the V8
degradation
products were
tested,
the tested,
results
the
showed
similar
of
degradation
against
Gramthe results
results
showed
similar antibacterial
antibacterial
activity
of the
the
degradation
product
against the
thebacteria
Gramshowed
similar
antibacterial
activity
of theactivity
degradation
product
againstproduct
the Gram-negative
negative
bacteria
E.
the
TSLP
negative
bacteria
E. coli
coli as
as
the holoprotein
holoprotein
TSLP [7].
[7].
E.
coli as the
holoprotein
TSLP
[7].
and
the bacterial
proteases
Pharmaceuticals
2016,
41
Pharmaceuticals
2016, 9,
9,
41
Figure
7. Enzymatic
Enzymatic
digestion
of
TSLP.
Figure 7.
Enzymatic digestion
digestionof
of TSLP.
TSLP. (A)
(A) TSLP
TSLP was
was digested
digested with
with S.
S. aureus
aureus V8
V8 proteinase,
proteinase, and
and
cleavage
products
were
visualized
Western
analysis
polyclonal
antibodies
cleavage products
productswere
werevisualized
visualized
by
Western
blot
analysis
using
polyclonal
antibodies
against
cleavage
byby
Western
blotblot
analysis
usingusing
polyclonal
antibodies
againstagainst
human
human
TSLP.
produced
by
of
aa major
protein
humanProducts
TSLP. Products
Products
produced
by V8
V8 cleavage
cleavage
of TSLP
TSLP revealed
major immunoreactive
immunoreactive
protein
TSLP.
produced
by V8 cleavage
of TSLP revealed
arevealed
major immunoreactive
protein fragment
fragment
at
about
16
kDa.
(B)
were
with
without
human
fragment
atkDa.
about
16TSLP
kDa.and
(B) TSLP
TSLP and
and
LL-37
were incubated
incubated
with and
and
without
human neutrophil
neutrophil
at
about 16
(B)
LL-37
wereLL-37
incubated
with
and without
human
neutrophil
(leukocyte)
(leukocyte)
elastase
(HLE),
S.
Pseudomonas
aeruginosa
elastase
(PAE)
and
elastase
(HLE),
S. aureus
V8 proteinase
or proteinase
Pseudomonas
elastase
(PAE) and
analyzed
under
(leukocyte)
elastase
(HLE),
S. aureus
aureus V8
V8
proteinase or
oraeruginosa
Pseudomonas
aeruginosa
elastase
(PAE)
and
analyzed
non-reducing
conditions
by
From
non-reducing
conditions
by SDS-PAGE.
ref. [7].
analyzed under
under
non-reducing
conditionsFrom
by SDS-PAGE.
SDS-PAGE.
From ref.
ref. [7].
[7].
AMPs
AMPs are
are known
known to
to exert
exert their
their effects
effects by
by different
different mechanisms:
mechanisms: some
some are
are membrane-active
membrane-active and
and
AMPs
are
known
to
exert
their
effects
by
different
mechanisms:
some
are
membrane-active
and
others
others are
are not
not [43].
[43]. To
To
investigate if
if MKK34
MKK34 exerted
exerted membrane
membrane active
active properties,
properties, we
we performed
performed
others
are
not
[43].
To investigate
investigate
if
MKK34
exerted
membrane
active
properties,
we
performed
liposome
leakage
models
and
fluorescence
studies
on
peptide-treated
bacteria.
This
showed
liposome
leakage
models
and
fluorescence
studies
on
peptide-treated
bacteria.
This
showed
that
liposome leakage models and fluorescence studies on peptide-treated bacteria. This showed that
that
MKK34
MKK34 exerted
exerted membrane-penetrating
membrane-penetrating effects
effects
on
bacterial
membranes
of
E.
coli,
as
well
as on
on
MKK34
exerted
membrane-penetrating
effects on
on bacterial
bacterial membranes
membranes of
of E.
E. coli,
coli, as
as well
well as
as
on
liposomes
[7].
Moreover,
electron
microscopy
analysis
revealed
severe
membrane
damage
liposomes
[7].
Moreover,
electron
microscopy
analysis
revealed
severe
membrane
damage
of
liposomes [7]. Moreover, electron microscopy analysis revealed severe membrane damage of
of
MKK34-treated
bacteria
(Figure
8).
MKK34-treated
bacteria
(Figure
8).
MKK34-treated bacteria (Figure 8).
Figure
Electron microscopy
microscopy analysis.
analysis.
Staphylococcusaureus
aureus and
and Pseudomonas
Pseudomonas aeruginosa
aeruginosa were
were
Figure
8.
Figure 8.
8. Electron
Electron
microscopy
analysis. Staphylococcus
Staphylococcus
aureus
and
Pseudomonas
aeruginosa
were
˝
incubated
with
30
µM
of
MKK34
and
LL-37
for
hh at
at
37
C and
and visualized
visualized by
by
negative
staining.
Scale
incubated
incubated with
with 30
30 μM
μM of
of MKK34
MKK34 and
and LL-37
LL-37 for
for 222 h
at 37
37 °C
°C
and
visualized
by negative
negative staining.
staining. Scale
Scale
bar
µm.
Control:
buffer control.
control. From
From[7],
[7], image
imagecourtesy
courtesyof
ofMatthias
MatthiasMörgelin,
Mörgelin,Lund
LundUniversity,
University,
bar
bar 111 μm.
μm. Control:
Control: buffer
buffer
control.
From
[7],
image
courtesy
of
Matthias
Mörgelin,
Lund
University,
Lund,
Sweden.
Lund,
Lund, Sweden.
Sweden.
The
of
can
factor
their
activity:
disulphideredox
state
of AMPs
AMPs
can be
be aa determining
determining
factor for
for
theirfor
activity:
reduction
ofreduction
disulphideThe redox
redoxstate
state
of
AMPs
can
be a determining
factor
their reduction
activity: of
of
bridges
in
human
beta-defensin-1
(hBD-1)
vastly
potentiates
its
antimicrobial
effect
and
free
cysteines
bridges
in
human
beta-defensin-1
(hBD-1)
vastly
potentiates
its
antimicrobial
effect
and
free
cysteines
disulphide-bridges in human beta-defensin-1 (hBD-1) vastly potentiates its antimicrobial effect and
in
carboxy
seem
important
for
the
the
three
disulphide
in the
the
carboxyinterminus
terminus
seem
important
forimportant
the bactericidal
bactericidal
effect [51].
[51]. Of
Of
the [51].
threeOf
disulphide
free
cysteines
the carboxy
terminus
seem
for the effect
bactericidal
effect
the three
bridges
Cys69-Cys75,
and
[2]),
bridges in
in lfTSLP
lfTSLP
(Cys34-Cys110,
Cys69-Cys75,
and Cys90-Cys137;
Cys90-Cys137;
[2]), one
one is
is present
present
in sfTSLP
sfTSLP
disulphide
bridges(Cys34-Cys110,
in lfTSLP (Cys34-Cys110,
Cys69-Cys75,
and Cys90-Cys137;
[2]),
one is in
present
in
(Cys90-Cys137).
(Cys90-Cys137). It
It will
will be
be interesting
interesting to
to examine
examine whether
whether the
the redox
redox state
state of
of cysteine
cysteine residues
residues in
in
sfTSLP
sfTSLP can
can affect
affect its
its structure
structure and
and antimicrobial
antimicrobial properties.
properties. It
It also
also remains
remains to
to be
be confirmed
confirmed that
that
sfTSLP
sfTSLP occurs
occurs in
in sufficiently
sufficiently high
high concentrations
concentrations in
in mucosal
mucosal secretions
secretions and
and exfoliated
exfoliated skin
skin to
to be
be
effective
effective as
as an
an antimicrobial
antimicrobial agent.
agent.
Pharmaceuticals 2016, 9, 41
10 of 13
sfTSLP (Cys90-Cys137). It will be interesting to examine whether the redox state of cysteine residues in
sfTSLP can affect its structure and antimicrobial properties. It also remains to be confirmed that sfTSLP
occurs in sufficiently high concentrations in mucosal secretions and exfoliated skin to be effective as an
antimicrobial agent.
Whether murine TSLP exhibits antimicrobial properties has not yet been investigated.
6. Conclusions
Taken together, the latest studies on TSLP have shown (1) that human TSLP is translated in two
forms; (2) that the short form is constitutively expressed in a steady-state, while the long form is absent;
(3) that the long form is induced by inflammation, while the short form appears to be downregulated
by inflammation; (4) that both forms exhibit potent AMP activity, with sfTSLP exhibiting the strongest
activity; (5) that the AMP activity is primarily localized in the C-terminal part of both forms; and (6) that
the C-terminal part of TSLP a has penetrating effect on bacterial membranes.
As human sfTSLP is constitutively expressed at major barrier surfaces consisting of skin and
mucosa, it is expected to play an important role against infection and regulation of inflammation at
these sites. lfTSLP has a broader role in the defense against infection because it participates in the
regulation of various immune activities, but it also preserves its antimicrobial functions.
Acknowledgments: We thank Artur Schmidtchen, Division of Dermatology and Venereology, Department of
Clinical Sciences Lund, Lund University, for helpful comments.
Conflicts of Interest: The authors declare no conflict of interest.
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