Multiple Functions of the New Cytokine-Based Antimicrobial Peptide Thymic Stromal Lymphopoietin (TSLP)

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.

Full text

pharmaceuticals
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 ,*
1Department of Oral Biology, Dental Faculty, University of Oslo, PB 1052 Blindern, N-0316 Oslo, Norway;
louise.bjerkan@medisin.uio.no
2Division of Dermatology and Venereology, Department of Clinical Sciences Lund, Lund University, BMC,
Tornavägen 10, SE-22184 Lund, Sweden; andreas.sonesson@med.lu.se
3Dermatology and Venereology, Skane University Hospital, Lasarettsgatan 15, SE-22185 Lund, Sweden
*Correspondence: karl.schenck@odont.uio.no; 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
Pharmaceuticals 2016,9, 41; doi:10.3390/ph9030041 www.mdpi.com/journal/pharmaceuticals

Pharmaceuticals 2016,9, 41 2 of 13
of the immune system, describe their qualities as an AMP, and outline some of the mechanisms behind
their antimicrobial effects.
2. TSLP Variants
Three transcript variants of human TSLP are annotated in the RefSeq (Reference Sequence)
database (National Center for Biotechnology Information, National Library of Medicine, Bethesda, MD,
USA), two long and one shorter variant, but only one of the long (variant 1) and the short (variant 2)
variants give rise to coding RNA. Variant 1 consists of four exons and variant 2 is two 5
1
exons shorter
but contains an alternate 5
1
exon compared to variant 1 (Figure 1A). The relevance of distinguishing
between TSLP variants in mice is currently uncertain as a murine short TSLP variant has not been
described or annotated in RefSeq so far.
The long form (lfTSLP) encodes a 159 amino acid (aa) protein. The short form (sfTSLP) encodes
a sequence that is identical in the C terminal region of long TSLP and consists of 63 aa (UniProt
entry: G3XAM8) and/or a 60 aa (UniProt entry: Q96AU7) (Figure 1B). UniProt has two entries for
the short isoform because there are two potential methionine start codons, separated by two amino
acids. A putative signal sequence is identified in the long TSLP isoform, with a predicted cleavage
site after the threonine residue at amino acid 28, leaving a mature lfTSLP protein of 131 amino
acids [
2
]. The calculated molecular weight (MW) of lfTSLP with, or without, the signal sequence is
18.1 kDa and 15 kDa, respectively, but in Western blotting the apparent MW is 23 kDa, probably due to
post-transcriptional modifications (PTM) [5].
The N-terminal sequence of sfTSLP also contains a potential N-terminal signal sequence of 20 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 5.2 kDa (63 aa) or 4.8 kDa (60 aa), respectively. In Western blotting, the observed MW lies at
9 kDa, probably due to PTM [
5
]. The PTM might be glycosylation because two potential sites for
N-linked glycosylation are present in the long isoform and one potential site is seen in the short isoform
(Figure 1B). sfTSLP is predicted to consist of two
α
-helices (Figure 1C,D; [
9
]). As yet, onlya few studies
have examined the expression of sfTSLP [5,6,10–13].
Pharmaceuticals 2016, 9, 41 2 of 12
human TSLP and their expression in the human body, summarize their activity on different elements
of the immune system, describe their qualities as an AMP, and outline some of the mechanisms
behind their antimicrobial effects.
2. TSLP Variants
Three transcript variants of human TSLP are annotated in the RefSeq (Reference Sequence)
database (National Center for Biotechnology Information, National Library of Medicine, Bethesda,
MD, USA), two long and one shorter variant, but only one of the long (variant 1) and the short (variant
2) variants give rise to coding RNA. Variant 1 consists of four exons and variant 2 is two 5′ exons
shorter but contains an alternate 5′ exon compared to variant 1 (Figure 1A). The relevance of
distinguishing between TSLP variants in mice is currently uncertain as a murine short TSLP variant has
not been described or annotated in RefSeq so far.
The long form (lfTSLP) encodes a 159 amino acid (aa) protein. The short form (sfTSLP) encodes
a sequence that is identical in the C terminal region of long TSLP and consists of 63 aa (UniProt entry:
G3XAM8) and/or a 60 aa (UniProt entry: Q96AU7) (Figure 1B). UniProt has two entries for the short
isoform because there are two potential methionine start codons, separated by two amino acids. A
putative signal sequence is identified in the long TSLP isoform, with a predicted cleavage site after
the threonine residue at amino acid 28, leaving a mature lfTSLP protein of 131 amino acids [2]. The
calculated molecular weight (MW) of lfTSLP with, or without, the signal sequence is 18.1 kDa and 15
kDa, respectively, but in Western blotting the apparent MW is 23 kDa, probably due to post-
transcriptional modifications (PTM) [5].
The N-terminal sequence of sfTSLP also contains a potential N-terminal signal sequence of 20 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 5.2 kDa (63 aa) or 4.8 kDa (60 aa), respectively. In Western blotting, the observed MW lies at 9
kDa, probably due to PTM [5]. The PTM might be glycosylation because two potential sites for N-
linked glycosylation are present in the long isoform and one potential site is seen in the short isoform
(Figure 1B). sfTSLP is predicted to consist of two α-helices (Figure 1C,D; [9]). As yet, onlya few studies
have examined the expression of sfTSLP [5,6,10–13].
Figure 1. Cont.
Figure 1. Cont.

Pharmaceuticals 2016,9, 41 3 of 13
Pharmaceuticals 2016, 9, 41 3 of 12
Figure 1. TSLP transcript variants and protein isoforms. (A) Graphics showing the TSLP gene (green),
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 and protein variants, respectively,
are indicated (NCBI). (B) Amino acid sequence of human TSLP isoforms. The putative signal
sequences of the TSLP isoforms are marked in blue, and the mature protein in black. N-linked
glycosylation sites are marked green, and methionine start codons are marked red. Bold black
characters indicate the position of MKK34. (C) JNet secondary structure prediction of lfTSLP based
on the amino acid sequence. Helices are marked as red tubes, and sheets are marked as green arrows.
JNETCONF: The confidence estimate for the prediction, high values indicate high confidence.
Modified from the web-based application Jpred (The Barton Group, School of Life Sciences, University
of Dundee, UK). (D) Three-dimensional structure (Swiss-model, [9]) of lfTSLP (left) and sfTSLP (right).
3. Expression and Regulation of TSLP Variants
Use of variant-specific reagents is necessary to study the expression of the two human TSLP
variants separately. At the mRNA level, this differentiation can be obtained by the use of variant-
specific primers, which are constructed based on unique mRNA sequences in the two transcript
variants. Detection of variant-specific protein expression, however, requires an indirect approach. As
there is a total overlap of sfTSLP with the lfTSLP amino acid C-terminal sequence, antibodies raised
against sfTSLP epitopes will recognize both variants and the production of sfTSLP-specific antibodies
is, therefore, not possible. On the other hand, it is possible to generate antibodies specific for lfTSLP,
either by immunization with peptide sequences that lie within the specific lfTSLP sequence, or by
retrieving monoclonals that recognize such sequences. Thus, distinguishing between sfTSLP and
lfTSLP has to be overcome by comparing the combined content of lfTSLP and sfTSLP in samples,
using one antibody that recognizes both forms, and another antibody that binds to the unique
sequence of lfTSLP (a long-specific anti-TSLP antibody). This approach has been used both for
Western blotting and immunohistology [5,6] (Figure 2).
The expression pattern of the two human isoforms is dependent on both tissue localization and
disease state. Most of the TSLP literature of the last two decades has only focused on lfTSLP, as the
translation of sfTSLP only recently has been documented [5]. The expression of TSLP has largely been
associated with inflammatory conditions by which it was found to be upregulated. We now know
that this was due to increased expression of lfTSLP [5,6]. In vivo, lfTSLP is upregulated in conditions
such as atopic dermatitis, asthma, ulcerative colitis, and smokeless tobacco-exposed oral mucosa,
while it is absent in healthy tissues (Figure 2) [5,6,10,14]. In vitro studies of cultured dermal and oral
keratinocytes exposed to pro-inflammatory factors, such as interferon γ (IFN-γ), tumor necrosis
factor α (TNF-α) in combination with interleukin 1β (IL-1-β), and polyriboinosinic:polyribocytidylic
acid (poly(I:C)), show upregulation of lfTSLP mRNA and protein [5,15]. The intestinal epithelial cell
line Caco-2, challenged with Salmonella typhimurium, shows upregulation of both mRNA and protein
lfTSLP expression [6]. Finally, TH2 cytokines were found to be potent inducers of TSLP in human
bronchial epithelial cells [16]. In normal nasal mucosa cultured in the presence of the inflammatory
TH2 cytokines; IL-4, IL-13, and TNF-α, lfTSLP mRNA 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
Figure 1.
TSLP transcript variants and protein isoforms. (
A
) Graphics showing the TSLP gene (green),
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 and protein variants, respectively, are
indicated (NCBI). (
B
) Amino acid sequence of human TSLP isoforms. The putative signal sequences
of the TSLP isoforms are marked in blue, and the mature protein in black. N-linked glycosylation
sites are marked green, and methionine start codons are marked red. Bold black characters indicate
the position of MKK34. (
C
) JNet secondary structure prediction of lfTSLP based on the amino acid
sequence. Helices are marked as red tubes, and sheets are marked as green arrows. JNETCONF:
The confidence estimate for the prediction, high values indicate high confidence. Modified from the
web-based application Jpred (The Barton Group, School of Life Sciences, University of Dundee, UK).
(D) Three-dimensional structure (Swiss-model, [9]) of lfTSLP (left) and sfTSLP (right).
3. Expression and Regulation of TSLP Variants
Use of variant-specific reagents is necessary to study the expression of the two human
TSLP variants separately. At the mRNA level, this differentiation can be obtained by the use of
variant-specific primers, which are constructed based on unique mRNA sequences in the two transcript
variants. Detection of variant-specific protein expression, however, requires an indirect approach.
As there is a total overlap of sfTSLP with the lfTSLP amino acid C-terminal sequence, antibodies raised
against sfTSLP epitopes will recognize both variants and the production of sfTSLP-specific antibodies
is, therefore, not possible. On the other hand, it is possible to generate antibodies specific for lfTSLP,
either by immunization with peptide sequences that lie within the specific lfTSLP sequence, or by
retrieving monoclonals that recognize such sequences. Thus, distinguishing between sfTSLP and
lfTSLP has to be overcome by comparing the combined content of lfTSLP and sfTSLP in samples, using
one antibody that recognizes both forms, and another antibody that binds to the unique sequence of
lfTSLP (a long-specific anti-TSLP antibody). This approach has been used both for Western blotting
and immunohistology [5,6] (Figure 2).
The expression pattern of the two human isoforms is dependent on both tissue localization and
disease state. Most of the TSLP literature of the last two decades has only focused on lfTSLP, as the
translation of sfTSLP only recently has been documented [
5
]. The expression of TSLP has largely been
associated with inflammatory conditions by which it was found to be upregulated. We now know
that this was due to increased expression of lfTSLP [
5
,
6
].
In vivo
, lfTSLP is upregulated in conditions
such as atopic dermatitis, asthma, ulcerative colitis, and smokeless tobacco-exposed oral mucosa,
while it is absent in healthy tissues (Figure 2) [
5
,
6
,
10
,
14
].
In vitro
studies of cultured dermal and
oral keratinocytes exposed to pro-inflammatory factors, such as interferon
γ
(IFN-
γ
), tumor necrosis
factor
α
(TNF-
α
) in combination with interleukin 1
β
(IL-1-
β
), and polyriboinosinic:polyribocytidylic
acid (poly(I:C)), show upregulation of lfTSLP mRNA and protein [
5
,
15
]. The intestinal epithelial cell
line Caco-2, challenged with Salmonella typhimurium, shows upregulation of both mRNA and protein
lfTSLP expression [
6
]. Finally, T
H
2 cytokines were found to be potent inducers of TSLP in human
bronchial epithelial cells [
16
]. In normal nasal mucosa cultured in the presence of the inflammatory
T
H
2 cytokines; IL-4, IL-13, and TNF-
α
, lfTSLP mRNA 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
].

Pharmaceuticals 2016,9, 41 4 of 13
Although not emphasized in these studies, the increased TSLP expression detected is presumably due
to lfTSLP.
Pharmaceuticals 2016, 9, 41 4 of 12
[18]. Although not emphasized in these studies, the increased TSLP expression detected is
presumably due to lfTSLP.
Figure 2. Immunohistochemical (IHC) staining and in situ hybridization (ISH) of sections of oral
mucosa (A–C), skin (D–F), salivary gland (G–I), and smokeless tobacco (“snus”; J,K) for TSLP
variants. Left column: IHC staining with anti-TSLP antibody recognizing both lfTSLP and sfTSLP
(brown color). Middle column: IHC staining with anti-TSLP antibody recognizing lfTSLP only. As no
specific staining is detected in (B,E,H), this means that the staining in (A,D,G) represents sfTSLP. In
oral mucosa exposed to smokeless tobacco, lfTSLP is seen (K). Right column: ISH staining by use of
sfTSLP-specific probe (blue color) which confirms strong expression of sfTSLP in oral 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
epidermis, salivary glands, and gut epithelial cells (Figure 2) [5,6]. Under inflammatory conditions,
sfTSLP appears to be downregulated as observed in lesional biopsy material from atopic dermatitis
(AD) and in the intestine of patients with Crohn’s disease [6]. Exposure to S. typhimurium also
downregulates sfTSLP mRNA and protein expression in Caco-2 cells [6]. To this date, sfTSLP protein
expression has only been identified in the gut, skin, oral epithelium and salivary glands [5,6].
The divergent expression pattern for the two translated TSLP variants is consistent with the
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
Figure 2.
Immunohistochemical (IHC) staining and in situ hybridization (ISH) of sections of oral
mucosa (
A
–
C
), skin (
D
–
F
), salivary gland (
G
–
I
), and smokeless tobacco (“snus”;
J
,
K
) for TSLP variants.
Left column: IHC staining with anti-TSLP antibody recognizing both lfTSLP and sfTSLP (brown color).
Middle column: IHC staining with anti-TSLP antibody recognizing lfTSLP only. As no specific staining
is detected in (
B
,
E
,
H
), this means that the staining in (
A
,
D
,
G
) represents sfTSLP. In oral mucosa exposed
to smokeless tobacco, lfTSLP is seen (
K
). Right column: ISH staining by use of sfTSLP-specific probe
(blue color) which confirms strong expression of sfTSLP in oral 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 epidermis, salivary
glands, and gut epithelial cells (Figure 2) [
5
,
6
]. Under inflammatory conditions, sfTSLP appears to
be downregulated as observed in lesional biopsy material from atopic dermatitis (AD) and in the
intestine of patients with Crohn’s disease [
6
]. Exposure to S. typhimurium also downregulates sfTSLP
mRNA and protein expression in Caco-2 cells [
6
]. To this date, sfTSLP protein expression has only
been identified in the gut, skin, oral epithelium and salivary glands [5,6].
The divergent expression pattern for the two translated TSLP variants is consistent with the
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

Pharmaceuticals 2016,9, 41 5 of 13
upstream from the lfTSLP under steady-state conditions is relatively inert in most of the cell lines
present in the UCSC database. Thus, under steady-state conditions, sfTSLP represents the homeostatic
form of TSLP. In inflammation, lfTSLP is up- and sfTSLP is downregulated.
The expression and regulation pattern of TSLP in mice overlaps to a large extend that of
human lfTSLP. A role of TSLP in human allergic diseases is well supported by a variety of mouse
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 regulated
by retinoid X receptors (RXR) [
24
]. In the latter study, keratinocyte-specific ablation of RXRs resulted
in upregulation of TSLP and development of AD-like skin inflammation. Further, the phenotype of
mice lacking TSLP signaling (tslpr(
´
/
´
)) and challenged with human metapneumovirus (hMPV)
show reduced lung infection and hMPV replication [
25
]. These mice displayed a decreased number of
neutrophils, as well a reduction in levels of thymus and activation-regulated chemokine/CCL17, IL-5,
IL-13, and TNF-αin the airways upon hMPV infection compared to WT mice.
4. Human TSLP Variants and Immunoregulation
4.1. Long-Form TSLP (lfTSLP)
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-7R
α
)
and the TSLP receptor chain (TSLPR) [
2
,
26
]. The functional receptor for lfTSLP is expressed on both
hematopoietic and non-hematopoietic cell lineages including DCs, T cells, B cells, natural killer cells,
monocytes, basophils, eosinophils, and epithelial cells [
3
,
18
,
19
,
27
–
32
]. Activation of the TSLP receptor
has been shown to signal through multiple signal transducer and activator of transcription (STAT)
proteins, including STAT 1, 3, 4, 5, 6, and Janus kinase (JAK) 1 and 2 in peripheral blood-derived
CD11c+DCs (Figure 3) [5,33,34].
Pharmaceuticals 2016, 9, 41 5 of 12
upstream from the lfTSLP under steady-state conditions is relatively inert in most of the cell lines
present in the UCSC database. Thus, under steady-state conditions, sfTSLP represents the
homeostatic form of TSLP. In inflammation, lfTSLP is up- and sfTSLP is downregulated.
The expression and regulation pattern of TSLP in mice overlaps to a large extend that of human
lfTSLP. A role of TSLP in human allergic diseases is well supported by a variety of mouse 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
regulated by retinoid X receptors (RXR) [24]. In the latter study, keratinocyte-specific ablation of
RXRs resulted in upregulation of TSLP and development of AD-like skin inflammation. Further, the
phenotype of mice lacking TSLP signaling (tslpr(−/−)) and challenged with human metapneumovirus
(hMPV) show reduced lung infection and hMPV replication [25]. These mice displayed a decreased
number of neutrophils, as well a reduction in levels of thymus and activation-regulated
chemokine/CCL17, IL-5, IL-13, and TNF-α in the airways upon hMPV infection compared to WT
mice.
4. Human TSLP Variants and Immunoregulation
4.1. Long-Form TSLP (lfTSLP)
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) and the TSLP receptor chain (TSLPR) [2,26]. The functional receptor for lfTSLP is expressed on
both hematopoietic and non-hematopoietic cell lineages including DCs, T cells, B cells, natural killer
cells, monocytes, basophils, eosinophils, and epithelial cells [3,18,19,27–32]. Activation of the TSLP
receptor has been shown to signal through multiple signal transducer and activator of transcription
(STAT) proteins, including STAT 1, 3, 4, 5, 6, and Janus kinase (JAK) 1 and 2 in peripheral blood-
derived CD11c+ DCs (Figure 3) [5,33,34].
Figure 3. STAT5 phosphorylation in response to lfTSLP, 60 aa sfTSLP, 63 aa sfTSLP, or lfTSLP
combined with sfTSLP in blood-derived CD1c myeloid DCs incubated with poly(I:C) for 24 h, and
then treated with sfTSLP or/and lfTSLP for 15 min. Phosphorylation of STAT5 was assessed by flow
cytometry. From [5].
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 lfTSLP-
stimulated DCs with allogeneic CD4+ T cells results in the generation of inflammatory Th2 cells
producing classical Th2 cytokines including IL-4, IL-5, IL-13, but in contrast to conventional Th2 cells,
these cells also produce TNF-α and not IL-10 [14]. This inflammatory Th2 phenotype is induced
Figure 3.
STAT5 phosphorylation in response to lfTSLP, 60 aa sfTSLP, 63 aa sfTSLP, or lfTSLP combined
with sfTSLP in blood-derived CD1c myeloid DCs incubated with poly(I:C) for 24 h, and then treated
with sfTSLP or/and lfTSLP for 15 min. Phosphorylation of STAT5 was assessed by flow cytometry.
From [5].
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
lfTSLP-stimulated DCs with allogeneic CD4
+
T cells results in the generation of inflammatory Th2 cells
producing classical Th2 cytokines including IL-4, IL-5, IL-13, but in contrast to conventional Th2 cells,
these cells also produce TNF-
α
and not IL-10 [
14
]. This inflammatory Th2 phenotype is induced

Pharmaceuticals 2016,9, 41 6 of 13
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
is highly expressed in acute and chronic AD lesions [
14
]. In allergic rhinitis, TSLP treatment of CD1c
+
DCs potently augments allergen-specific TH2 memory responses [13].
In contrast to its role in inflammation, TSLP has also been suggested to have homeostatic,
tolerogenic functions [
36
,
37
]. It was, however, at that time unknown that the sfTSLP peptide is
also translated, and that this peptide has an inhibiting effect on DCs [
5
]. After a re-evaluation of
earlier 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)
sfTSLP is constitutively expressed by several types of epithelial cells, as described above. sfTSLP
appears to act on DCs on which it inhibits cytokine secretion [
6
]. sfTSLP does not bind to the TSLPR
because it is not capable to block binding of lfTSLP to this receptor (Figure 3) [
5
,
6
]. The specific
receptor for sfTSLP is currently unknown. sfTSLP induces phosphorylation of p38
α
, extracellular
signal-regulated kinase 1/2, and Lyn, but has no effect on STAT5 phosphorylation (Figure 3) [
5
,
6
]. Very
little else is yet known about the immunoregulatory action of sfTSLP. As sfTSLP can be downregulated
by inflammation, this might contribute to an aggravation of local infection in view of its antimicrobial
activity (see below).
5. Human TSLP Variants as Antimicrobial Peptides
AMPs can be classified in a variety of approaches [
38
], fitting into one of four major structural
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 [
39
–
41
]. However, dermcidin, an AMP secreted by sweat glands [
42
], is often classified based
on its anionicity.
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
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
required for antimicrobial activity at physiological conditions, such a positive net charge of +11
and a theoretical isoelectric point (pI) of 9.63 (calculated by using the Protparam tool; Swiss Institute
of Bioinformatics, Lausanne, Switzerland). Furthermore, analysis of the hydrophobic moment (
µ
H)
revealed a region, likely in the C-terminal part of TSLP, with conspicuous amphiphathic properties
(Figure 4) (calculated by using the European Molecular Biology Open Software Suite (EMBOSS);
The Sanger Centre, Wellcome Trust Genome Campus, Hinxton, Cambridge, UK).
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
is highly expressed in acute and chronic AD lesions [14]. In allergic rhinitis, TSLP treatment of CD1c+
DCs potently augments allergen-specific TH2 memory responses [13].
In contrast to its role in inflammation, TSLP has also been suggested to have homeostatic,
tolerogenic functions [36,37]. It was, however, at that time unknown that the sfTSLP peptide is also
translated, and that this peptide has an inhibiting effect on DCs [5]. After a re-evaluation of earlier
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)
sfTSLP is constitutively expressed by several types of epithelial cells, as described above. sfTSLP
appears to act on DCs on which it inhibits cytokine secretion [6]. sfTSLP does not bind to the TSLPR
because it is not capable to block binding of lfTSLP to this receptor (Figure 3) [5,6]. The specific
receptor for sfTSLP is currently unknown. sfTSLP induces phosphorylation of p38α, extracellular
signal-regulated kinase 1/2, and Lyn, but has no effect on STAT5 phosphorylation (Figure 3) [5,6].
Very little else is yet known about the immunoregulatory action of sfTSLP. As sfTSLP can be
downregulated by inflammation, this might contribute to an aggravation of local infection in view of
its antimicrobial activity (see below).
5. Human TSLP Variants as Antimicrobial Peptides
AMPs can be classified in a variety of approaches [38], fitting into one of four major structural
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 [39–41]. However, dermcidin, an AMP secreted by sweat glands [42], is often
classified based on its anionicity.
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
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
required for antimicrobial activity at physiological conditions, such a positive net charge of +11 and
a theoretical isoelectric point (pI) of 9.63 (calculated by using the Protparam tool; Swiss Institute of
Bioinformatics, Lausanne, Switzerland). Furthermore, analysis of the hydrophobic moment (μH)
revealed a region, likely in the C-terminal part of TSLP, with conspicuous amphiphathic properties
(Figure 4) (calculated by using the European Molecular Biology Open Software Suite (EMBOSS); The
Sanger Centre, Wellcome Trust Genome Campus, Hinxton, Cambridge, UK).
Figure 4. Plot of hydrophobic moment (μH) for the mature lfTSLP (131 amino acids).
Our data displayed antimicrobial activity of TSLP against both bacteria and fungi [5,7] (Figure
5A,B). To further investigate the antimicrobial properties of TSLP and which regions of the molecule
exhibited the antimicrobial effects, overlapping 20-mer peptides were synthesized [7]. The
experiments showed that the antimicrobial effect preferentially was located in regions of the C-
terminal part of TSLP [7]. When a 34 aa long synthetic peptide (MKK34; Figure 1B) spanning the C-
Figure 4. Plot of hydrophobic moment (µH) for the mature lfTSLP (131 amino acids).
Our data displayed antimicrobial activity of TSLP against both bacteria and fungi [
5
,
7
]
(Figure 5A,B). To further investigate the antimicrobial properties of TSLP and which regions of
the molecule exhibited the antimicrobial effects, overlapping 20-mer peptides were synthesized [
7
].

Pharmaceuticals 2016,9, 41 7 of 13
The experiments showed that the antimicrobial effect preferentially was located in regions of the
C-terminal part of TSLP [
7
]. When a 34 aa long synthetic peptide (MKK34; Figure 1B) spanning the
C-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
predicted regions that could display
α
-helical conformation. A helical wheel projection of MKK34 is
visualized in Figure 6.
Pharmaceuticals 2016, 9, 41 7 of 12
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
predicted regions that could display α-helical conformation. A helical wheel projection of MKK34 is
visualized in Figure 6.
Figure 5. Antimicrobial activity of short and long forms of thymic stromal lymphopoietin (sfTSLP
and lfTSLP). (A) lfTSLP exhibited a larger zone of inhibition of growth of Escherichia coli ATCC 25922
in comparison with LL-37: (a) control; (b) 10 μM LL-37; and (c) 10μM TSLP. Mean values and standard
deviations (n = 4). (B) In a viable count assay, indicated bacterial (Escherichia coli, Pseudomonas
aeruginosa, Staphylococcus aureus, and Staphylococcus epidermidis) and fungal isolates (Candida albicans
and Candida parapsilosis) were subjected to 2 μM of TSLP. The number of cfu was registered. (C)
Suspensions of the indicated bacterial and fungal species were treated for 2 h with 60 amino acid (aa)
sfTSLP, and 63 aa sfTSLP or lfTSLP peptide at a concentration of 1.35 mM before being plated on agar.
Colony-forming units per ml were determined after incubation overnight. The values were
normalized to the levels obtained without the addition of test peptides (broken line). (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).
Figure 5.
Antimicrobial activity of short and long forms of thymic stromal lymphopoietin (sfTSLP
and lfTSLP). (
A
) lfTSLP exhibited a larger zone of inhibition of growth of Escherichia coli ATCC
25922 in comparison with LL-37: (a) control; (b) 10
µ
M LL-37; and (c) 10
µ
M TSLP. Mean values
and standard deviations (n = 4). (
B
) In a viable count assay, indicated bacterial (Escherichia coli,
Pseudomonas aeruginosa, Staphylococcus aureus, and Staphylococcus epidermidis) and fungal isolates
(Candida albicans and Candida parapsilosis) were subjected to 2
µ
M of TSLP. The number of cfu was
registered. (
C
) Suspensions of the indicated bacterial and fungal species were treated for 2 h with
60 amino acid (aa) sfTSLP, and 63 aa sfTSLP or lfTSLP peptide at a concentration of 1.35 mM before
being plated on agar. Colony-forming units per ml were determined after incubation overnight.
The values were normalized to the levels obtained without the addition of test peptides (broken line).
(
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 8 of 13
Pharmaceuticals 2016, 9, 41 8 of 12
Figure 6. Helical structure of MKK34. A helical wheel projection was constructed using the amino
acid sequence of MKK34.
The findings that the main antimicrobial activity of TSLP is located in its C-terminal part is
particularly relevant since both MKK34 and sfTSLP are found in this region, and sfTSLP is translated
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
fungi, stronger than the well-characterized AMP LL-37 [5,7] (Figure 5B,C). sfTSLP exerted potent
antimicrobial activity against all the tested species, including Streptococcus mitis, Escherichia coli,
Enterococcus faecalis, Bacillus cereus, Staphylococcus epidermidis, and Candida albicans [5] (Figure 5C).
Moreover, addition of polyclonal anti-TSLP antibody to sfTSLP before it was incubated with S. mitis,
reduced the antimicrobial activity by about half, showing that the reduction in colony-forming units
per mL was specifically due to the action of sfTSLP [5]. Dose-response curves using S. mitis showed
that the effect of sfTSLP was stronger than that of LL-37 (Figure 5D). Furthermore, the susceptibility
of isolates of Staphylococcus aureus, S. epidermidis, E. coli, and Pseudomonas aeruginosa to MKK34 was
tested in antimicrobial assays [7]. The Gram-positive isolates were generally less susceptible to
MKK34 in comparison to Gram-negative bacteria [7].
AMPs are reported to possess different antibacterial spectrums. The well-characterized AMP
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 and TSLP-derived peptides, such as MKK34 and sfSTLP, exert broad antimicrobial activity on
Gram-negative bacteria, Gram-positive bacteria, as well as fungi, that are of importance in host
defense [5,7]. Moreover, MKK34 may be a contributor to the in vivo resistance of human skin to
Gram-negative bacterial colonization and infection and hypothetically support the maintenance of
preferentially Gram-positive bacterial (S. epidermidis) colonization at the human skin.
Several classical and recently discovered AMPs, such as LL-37 and AMPs derived from 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
atopic eczema. Moreover, AD skin is frequently colonized by S. aureus and characterized by a chronic
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 this,
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.
When analyzed by SDS-PAGE, the incubation products revealed degradation of TSLP by both HLE
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
Figure 6.
Helical structure of MKK34. A helical wheel projection was constructed using the amino acid
sequence of MKK34.
The findings that the main antimicrobial activity of TSLP is located in its C-terminal part is
particularly relevant since both MKK34 and sfTSLP are found in this region, and sfTSLP is translated
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
fungi, stronger than the well-characterized AMP LL-37 [
5
,
7
] (Figure 5B,C). sfTSLP exerted potent
antimicrobial activity against all the tested species, including Streptococcus mitis, Escherichia coli,
Enterococcus faecalis, Bacillus cereus, Staphylococcus epidermidis, and Candida albicans [
5
] (Figure 5C).
Moreover, addition of polyclonal anti-TSLP antibody to sfTSLP before it was incubated with S. mitis,
reduced the antimicrobial activity by about half, showing that the reduction in colony-forming units
per mL was specifically due to the action of sfTSLP [
5
]. Dose-response curves using S. mitis showed
that the effect of sfTSLP was stronger than that of LL-37 (Figure 5D). Furthermore, the susceptibility of
isolates of Staphylococcus aureus,S. epidermidis,E. coli, and Pseudomonas aeruginosa to MKK34 was tested
in antimicrobial assays [
7
]. The Gram-positive isolates were generally less susceptible to MKK34 in
comparison to Gram-negative bacteria [7].
AMPs are reported to possess different antibacterial spectrums. The well-characterized AMP
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 and TSLP-derived peptides, such as MKK34 and sfSTLP, exert broad antimicrobial activity
on Gram-negative bacteria, Gram-positive bacteria, as well as fungi, that are of importance in host
defense [
5
,
7
]. Moreover, MKK34 may be a contributor to the
in vivo
resistance of human skin to
Gram-negative bacterial colonization and infection and hypothetically support the maintenance of
preferentially Gram-positive bacterial (S. epidermidis) colonization at the human skin.
Several classical and recently discovered AMPs, such as LL-37 and AMPs derived from
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
atopic eczema. Moreover, AD skin is frequently colonized by S. aureus and characterized by a chronic
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 this,
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.
When analyzed by SDS-PAGE, the incubation products revealed degradation of TSLP by both HLE

Pharmaceuticals 2016,9, 41 9 of 13
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
from the C-terminal part and comprising 42 amino acids (Thr88-Lys129), encompassing the previously
characterized synthetic MKK34 peptide. When the V8 degradation products were tested, the results
showed similar antibacterial activity of the degradation product against the Gram-negative bacteria
E. coli as the holoprotein TSLP [7].
Pharmaceuticals 2016, 9, 41 9 of 12
from the C-terminal part and comprising 42 amino acids (Thr88-Lys129), encompassing the
previously characterized synthetic MKK34 peptide. When the V8 degradation products were tested,
the results showed similar antibacterial activity of the degradation product against the Gram-
negative bacteria E. coli as the holoprotein TSLP [7].
Figure 7. Enzymatic digestion of TSLP. (A) TSLP was digested with S. aureus V8 proteinase, and
cleavage products were visualized by Western blot analysis using polyclonal antibodies against
human TSLP. Products produced by V8 cleavage of TSLP revealed a major immunoreactive protein
fragment at about 16 kDa. (B) TSLP and LL-37 were incubated with and without human neutrophil
(leukocyte) elastase (HLE), S. aureus V8 proteinase or Pseudomonas aeruginosa elastase (PAE) and
analyzed under non-reducing conditions by SDS-PAGE. From ref. [7].
AMPs are known to exert their effects by different mechanisms: some are membrane-active and
others are not [43]. To investigate if MKK34 exerted membrane active properties, we performed
liposome leakage models and fluorescence studies on peptide-treated bacteria. This showed that
MKK34 exerted membrane-penetrating effects on bacterial membranes of E. coli, as well as on
liposomes [7]. Moreover, electron microscopy analysis revealed severe membrane damage of
MKK34-treated bacteria (Figure 8).
Figure 8. Electron microscopy analysis. Staphylococcus aureus and Pseudomonas aeruginosa were
incubated with 30 μM of MKK34 and LL-37 for 2 h at 37 °C and visualized by negative staining. Scale
bar 1 μm. Control: buffer control. From [7], image courtesy of Matthias Mörgelin, Lund University,
Lund, Sweden.
The redox state of AMPs can be a determining factor for their activity: reduction of disulphide-
bridges in human beta-defensin-1 (hBD-1) vastly potentiates its antimicrobial effect and free cysteines
in the carboxy terminus seem important for the bactericidal effect [51]. Of the three disulphide
bridges in lfTSLP (Cys34-Cys110, Cys69-Cys75, and Cys90-Cys137; [2]), one is present in 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.
Figure 7.
Enzymatic digestion of TSLP. (
A
) TSLP was digested with S. aureus V8 proteinase, and
cleavage products were visualized by Western blot analysis using polyclonal antibodies against human
TSLP. Products produced by V8 cleavage of TSLP revealed a major immunoreactive protein fragment
at about 16 kDa. (
B
) TSLP and LL-37 were incubated with and without human neutrophil (leukocyte)
elastase (HLE), S. aureus V8 proteinase or Pseudomonas aeruginosa elastase (PAE) and analyzed under
non-reducing conditions by SDS-PAGE. From ref. [7].
AMPs are known to exert their effects by different mechanisms: some are membrane-active and
others are not [
43
]. To investigate if MKK34 exerted membrane active properties, we performed
liposome leakage models and fluorescence studies on peptide-treated bacteria. This showed that
MKK34 exerted membrane-penetrating effects on bacterial membranes of E. coli, as well as on
liposomes [
7
]. Moreover, electron microscopy analysis revealed severe membrane damage of
MKK34-treated bacteria (Figure 8).
Pharmaceuticals 2016, 9, 41 9 of 12
from the C-terminal part and comprising 42 amino acids (Thr88-Lys129), encompassing the
previously characterized synthetic MKK34 peptide. When the V8 degradation products were tested,
the results showed similar antibacterial activity of the degradation product against the Gram-
negative bacteria E. coli as the holoprotein TSLP [7].
Figure 7. Enzymatic digestion of TSLP. (A) TSLP was digested with S. aureus V8 proteinase, and
cleavage products were visualized by Western blot analysis using polyclonal antibodies against
human TSLP. Products produced by V8 cleavage of TSLP revealed a major immunoreactive protein
fragment at about 16 kDa. (B) TSLP and LL-37 were incubated with and without human neutrophil
(leukocyte) elastase (HLE), S. aureus V8 proteinase or Pseudomonas aeruginosa elastase (PAE) and
analyzed under non-reducing conditions by SDS-PAGE. From ref. [7].
AMPs are known to exert their effects by different mechanisms: some are membrane-active and
others are not [43]. To investigate if MKK34 exerted membrane active properties, we performed
liposome leakage models and fluorescence studies on peptide-treated bacteria. This showed that
MKK34 exerted membrane-penetrating effects on bacterial membranes of E. coli, as well as on
liposomes [7]. Moreover, electron microscopy analysis revealed severe membrane damage of
MKK34-treated bacteria (Figure 8).
Figure 8. Electron microscopy analysis. Staphylococcus aureus and Pseudomonas aeruginosa were
incubated with 30 μM of MKK34 and LL-37 for 2 h at 37 °C and visualized by negative staining. Scale
bar 1 μm. Control: buffer control. From [7], image courtesy of Matthias Mörgelin, Lund University,
Lund, Sweden.
The redox state of AMPs can be a determining factor for their activity: reduction of disulphide-
bridges in human beta-defensin-1 (hBD-1) vastly potentiates its antimicrobial effect and free cysteines
in the carboxy terminus seem important for the bactericidal effect [51]. Of the three disulphide
bridges in lfTSLP (Cys34-Cys110, Cys69-Cys75, and Cys90-Cys137; [2]), one is present in 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.
Figure 8.
Electron microscopy analysis. Staphylococcus aureus and Pseudomonas aeruginosa were
incubated with 30
µ
M of MKK34 and LL-37 for 2 h at 37
˝
C and visualized by negative staining. Scale
bar 1
µ
m. Control: buffer control. From [
7
], image courtesy of Matthias Mörgelin, Lund University,
Lund, Sweden.
The redox state of AMPs can be a determining factor for their activity: reduction of
disulphide-bridges in human beta-defensin-1 (hBD-1) vastly potentiates its antimicrobial effect and
free cysteines in the carboxy terminus seem important for the bactericidal effect [
51
]. Of the three
disulphide bridges in lfTSLP (Cys34-Cys110, Cys69-Cys75, and Cys90-Cys137; [
2
]), one is present in

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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