* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
Download Multiple Functions of the New Cytokine
Survey
Document related concepts
Peptide synthesis wikipedia , lookup
Gene therapy of the human retina wikipedia , lookup
Polyclonal B cell response wikipedia , lookup
Secreted frizzled-related protein 1 wikipedia , lookup
Paracrine signalling wikipedia , lookup
Signal transduction wikipedia , lookup
Two-hybrid screening wikipedia , lookup
Endogenous retrovirus wikipedia , lookup
Monoclonal antibody wikipedia , lookup
Ribosomally synthesized and post-translationally modified peptides wikipedia , lookup
Expression vector wikipedia , lookup
Transcript
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, * 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 Pharmaceuticals 2016, 9, 41; doi:10.3390/ph9030041 www.mdpi.com/journal/pharmaceuticals Pharmaceuticals2016, 2016,9,9,41 41 Pharmaceuticals of13 12 22of 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. Pharmaceuticals 2016, 9, 41 Pharmaceuticals 2016, 9, 41 3 of 13 3 of 12 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]. Pharmaceuticals 2016, 9, 41 Pharmaceuticals 2016, 9, 41 4 of 13 4 of 12 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 Pharmaceuticals 2016, 9, 41 Pharmaceuticals 2016, 9, 41 5 of 13 5 of 12 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 6 of 13 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 7 of 13 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. References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. Friend, S.L.; Hosier, S.; Nelson, A.; Foxworthe, D.; Williams, D.E.; Farr, A. A thymic stromal cell line supports in vitro development of surface IgM+ B cells and produces a novel growth factor affecting B and T lineage cells. Exp. Hematol. 1994, 22, 321–328. [PubMed] Quentmeier, H.; Drexler, H.G.; Fleckenstein, D.; Zaborski, M.; Armstrong, A.; Sims, J.E.; Lyman, S.D. Cloning of human thymic stromal lymphopoietin (TSLP) and signaling mechanisms leading to proliferation. Leukemia 2001, 15, 1286–1292. [CrossRef] [PubMed] Reche, P.A.; Soumelis, V.; Gorman, D.M.; Clifford, T.; Liu, M.; Travis, M.; Zurawski, S.M.; Johnston, J.; Liu, Y.J.; Spits, H.; et al. Human thymic stromal lymphopoietin preferentially stimulates myeloid cells. J. Immunol. 2001, 167, 336–343. [CrossRef] [PubMed] Ziegler, S.F.; Roan, F.; Bell, B.D.; Stoklasek, T.A.; Kitajima, M.; Han, H. The biology of thymic stromal lymphopoietin (TSLP). Adv. Pharmacol. 2013, 66, 129–155. [PubMed] Bjerkan, L.; Schreurs, O.; Engen, S.A.; Jahnsen, F.L.; Baekkevold, E.S.; Blix, I.J.; Schenck, K. The short form of TSLP is constitutively translated in human keratinocytes and has characteristics of an antimicrobial peptide. Mucosal Immunol. 2015, 8, 49–56. [CrossRef] [PubMed] Fornasa, G.; Tsilingiri, K.; Caprioli, F.; Botti, F.; Mapelli, M.; Meller, S.; Kislat, A.; Homey, B.; Di Sabatino, A.; Sonzogni, A.; et al. Dichotomy of short and long thymic stromal lymphopoietin isoforms in inflammatory disorders of the bowel and skin. J. Allergy Clin. Immunol. 2015, 136, 413–422. [CrossRef] [PubMed] Sonesson, A.; Kasetty, G.; Olin, A.I.; Malmsten, M.; Mörgelin, M.; Sørensen, O.E.; Schmidtchen, A. Thymic stromal lymphopoietin exerts antimicrobial activities. Exp. Dermatol. 2011, 20, 1004–1010. [CrossRef] [PubMed] Petersen, T.N.; Brunak, S.; von Heijne, G.; Nielsen, H. SignalP 4.0: Discriminating signal peptides from transmembrane regions. Nat. Methods 2011, 8, 785–786. [CrossRef] [PubMed] Arnold, K.; Bordoli, L.; Kopp, J.; Schwede, T. The SWISS-MODEL workspace: A web-based environment for protein structure homology modelling. Bioinformatics 2006, 22, 195–201. [CrossRef] [PubMed] Harada, M.; Hirota, T.; Jodo, A.I.; Doi, S.; Kameda, M.; Fujita, K.; Miyatake, A.; Enomoto, T.; Noguchi, E.; Yoshihara, S.; et al. Functional analysis of the thymic stromal lymphopoietin variants in human bronchial epithelial cells. Am. J. Respir. Cell Mol. Biol. 2009, 40, 368–374. [CrossRef] [PubMed] Pharmaceuticals 2016, 9, 41 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 11 of 13 Rothenberg, M.E.; Spergel, J.M.; Sherrill, J.D.; Annaiah, K.; Martin, L.J.; Cianferoni, A.; Gober, L.; Kim, C.; Glessner, J.; Frackelton, E.; et al. Common variants at 5q22 associate with pediatric eosinophilic esophagitis. Nat. Genet. 2010, 42, 289–291. [CrossRef] [PubMed] Mjösberg, J.; Bernink, J.; Golebski, K.; Karrich, J.J.; Peters, C.P.; Blom, B.; te Velde, A.A.; Fokkens, W.J.; van Drunen, C.M.; Spits, H. The transcription factor GATA3 is essential for the function of human type 2 innate lymphoid cells. Immunity 2012, 37, 649–659. [CrossRef] [PubMed] Melum, G.R.; Farkas, L.; Scheel, C.; Van Dieren, B.; Gran, E.; Liu, Y.J.; Johansen, F.E.; Jahnsen, F.L.; Baekkevold, E.S. A thymic stromal lymphopoietin-responsive dendritic cell subset mediates allergic responses in the upper airway mucosa. J. Allergy Clin. Immunol. 2014, 134, 613–621. [CrossRef] [PubMed] Soumelis, V.; Reche, P.A.; Kanzler, H.; Yuan, W.; Edward, G.; Homey, B.; Gilliet, M.; Ho, S.; Antonenko, S.; Lauerma, A.; et al. Human epithelial cells trigger dendritic cell mediated allergic inflammation by producing TSLP. Nat. Immunol. 2002, 3, 673–680. [CrossRef] [PubMed] Kinoshita, H.; Takai, T.; Le, T.A.; Kamijo, S.; Wang, X.L.; Ushio, H.; Hara, M.; Kawasaki, J.; Vu, A.T.; Ogawa, T.; et al. Cytokine milieu modulates release of thymic stromal lymphopoietin from human keratinocytes stimulated with double-stranded RNA. J. Allergy Clin. Immunol. 2009, 123, 179–186. [CrossRef] [PubMed] Kato, A.; Favoreto, S.; Avila, P.C.; Schleimer, R.P. TLR3- and Th2 cytokine-dependent production of thymic stromal lymphopoietin in human airway epithelial cells. J. Immunol. 2007, 179, 1080–1087. [CrossRef] [PubMed] Fontenot, D.; He, H.; Hanabuchi, S.; Nehete, P.N.; Zhang, M.; Chang, M.; Nehete, B.; Wang, Y.H.; Wang, Y.H.; Ma, Z.M.; et al. TSLP production by epithelial cells exposed to immunodeficiency virus triggers DC-mediated mucosal infection of CD4+ T cells. Proc. Natl. Acad. Sci. USA 2009, 106, 16776–16781. [CrossRef] [PubMed] Allakhverdi, Z.; Comeau, M.R.; Jessup, H.K.; Yoon, B.R.; Brewer, A.; Chartier, S.; Paquette, N.; Ziegler, S.F.; Sarfati, M.; Delespesse, G. Thymic stromal lymphopoietin is released by human epithelial cells in response to microbes, trauma, or inflammation and potently activates mast cells. J. Exp. Med. 2007, 204, 253–258. [CrossRef] [PubMed] Zhou, B.; Comeau, M.R.; De Smedt, T.; Liggitt, H.D.; Dahl, M.E.; Lewis, D.B.; Gyarmati, D.; Aye, T.; Campbell, D.J.; Ziegler, S.F. Thymic stromal lymphopoietin as a key initiator of allergic airway inflammation in mice. Nat. Immunol. 2005, 6, 1047–1053. [CrossRef] [PubMed] Headley, M.B.; Zhou, B.; Shih, W.X.; Aye, T.; Comeau, M.R.; Ziegler, S.F. TSLP conditions the lung immune environment for the generation of pathogenic innate and antigen-specific adaptive immune responses. J. Immunol. 2009, 182, 1641–1647. [CrossRef] [PubMed] Yoo, J.; Omori, M.; Gyarmati, D.; Zhou, B.; Aye, T.; Brewer, A.; Comeau, M.R.; Campbell, D.J.; Ziegler, S.F. Spontaneous atopic dermatitis in mice expressing an inducible thymic stromal lymphopoietin transgene specifically in the skin. J. Exp. Med. 2005, 202, 541–549. [CrossRef] [PubMed] Bergot, A.S.; Monnet, N.; Le Tran, S.; Mittal, D.; Al-Kouba, J.; Steptoe, R.J.; Grimbaldeston, M.A.; Frazer, I.H.; Wells, J.W. HPV16 E7 expression in skin induces TSLP secretion, type 2 ILC infiltration and atopic dermatitis-like lesions. Immunol. Cell Biol. 2015, 93, 540–547. [CrossRef] [PubMed] Mahmutovic-Persson, I.; Akbarshahi, H.; Bartlett, N.W.; Glanville, N.; Johnston, S.L.; Brandelius, A.; Uller, L. Inhaled dsRNA and rhinovirus evoke neutrophilic exacerbation and lung expression of thymic stromal lymphopoietin in allergic mice with established experimental asthma. Allergy 2014, 69, 348–358. [CrossRef] [PubMed] Li, M.; Messaddeq, N.; Teletin, M.; Pasquali, J.L.; Metzger, D.; Chambon, P. Retinoid X receptor ablation in adult mouse keratinocytes generates an atopic dermatitis triggered by thymic stromal lymphopoietin. Proc. Natl. Acad. Sci. USA 2005, 102, 14795–14800. [CrossRef] [PubMed] Lay, M.K.; Céspedes, P.F.; Palavecino, C.E.; León, M.A.; Díaz, R.A.; Salazar, F.J.; Méndez, G.P.; Bueno, S.M.; Kalergis, A.M. Human metapneumovirus infection activates the TSLP pathway that drives excessive pulmonary inflammation and viral replication in mice. Eur. J. Immunol. 2015, 45, 1680–1695. [CrossRef] [PubMed] He, R.; Geha, R.S. Thymic stromal lymphopoietin. Ann. N. Y. Acad. Sci. 2010, 1183, 13–24. [CrossRef] [PubMed] Pharmaceuticals 2016, 9, 41 27. 28. 29. 30. 31. 32. 33. 34. 35. 36. 37. 38. 39. 40. 41. 42. 43. 44. 45. 46. 12 of 13 Nagata, Y.; Kamijuku, H.; Taniguchi, M.; Ziegler, S.; Seino, K. Differential role of thymic stromal lymphopoietin in the induction of airway hyperreactivity and Th2 immune response in antigen-induced asthma with respect to natural killer T cell function. Int. Arch. Allergy Immunol. 2007, 144, 305–314. [CrossRef] [PubMed] Rochman, I.; Watanabe, N.; Arima, K.; Liu, Y.J.; Leonard, W.J. Cutting edge: Direct action of thymic stromal lymphopoietin on activated human CD4+ T cells. J. Immunol. 2007, 178, 6720–6724. [CrossRef] [PubMed] Wong, C.K.; Hu, S.; Cheung, P.F.; Lam, C.W. Thymic stromal lymphopoietin induces chemotactic and prosurvival effects in eosinophils: Implications in allergic inflammation. Am. J. Respir. Cell Mol. Biol. 2010, 43, 305–315. [CrossRef] [PubMed] Ziegler, S.F.; Artis, D. Sensing the outside world: TSLP regulates barrier immunity. Nat. Immunol. 2010, 11, 289–293. [CrossRef] [PubMed] Reardon, C.; Lechmann, M.; Brüstle, A.; Gareau, M.G.; Shuman, N.; Philpott, D.; Ziegler, S.F.; Mak, T.W. Thymic stromal lymphopoetin-induced expression of the endogenous inhibitory enzyme SLPI mediates recovery from colonic inflammation. Immunity 2011, 35, 223–235. [CrossRef] [PubMed] Siracusa, M.C.; Saenz, S.A.; Hill, D.A.; Kim, B.S.; Headley, M.B.; Doering, T.A.; Wherry, E.J.; Jessup, H.K.; Siegel, L.A.; Kambayashi, T.; et al. TSLP promotes interleukin-3-independent basophil haematopoiesis and type 2 inflammation. Nature 2011, 477, 229–233. [CrossRef] [PubMed] Arima, K.; Watanabe, N.; Hanabuchi, S.; Chang, M.; Sun, S.C.; Liu, Y.J. Distinct signal codes generate dendritic cell functional plasticity. Sci. Signal. 2010, 3, ra4. [CrossRef] [PubMed] Roan, F.; Bell, B.D.; Stoklasek, T.A.; Kitajima, M.; Han, H.; Ziegler, S.F. The multiple facets of thymic stromal lymphopoietin (TSLP) during allergic inflammation and beyond. J. Leukoc. Biol. 2012, 91, 877–886. [CrossRef] [PubMed] Ito, T.; Wang, Y.H.; Duramad, O.; Hori, T.; Delespesse, G.J.; Watanabe, N.; Qin, F.X.; Yao, Z.; Cao, W.; Liu, Y.J. TSLP-activated dendritic cells induce an inflammatory T helper type 2 cell response through OX40 ligand. J. Exp. Med. 2005, 202, 1213–1223. [CrossRef] [PubMed] Rimoldi, M.; Chieppa, M.; Salucci, V.; Avogadri, F.; Sonzogni, A.; Sampietro, G.M.; Nespoli, A.; Viale, G.; Allavena, P.; Rescigno, M. Intestinal immune homeostasis is regulated by the crosstalk between epithelial cells and dendritic cells. Nat. Immunol. 2005, 6, 507–505. [CrossRef] [PubMed] Iliev, I.D.; Spadoni, I.; Mileti, E.; Matteoli, G.; Sonzogni, A.; Sampietro, G.M.; Foschi, D.; Caprioli, F.; Viale, G.; Rescigno, M. Human intestinal epithelial cells promote the differentiation of tolerogenic dendritic cells. Gut 2009, 58, 1481–1489. [CrossRef] [PubMed] Wang, G. Improved methods for classification, prediction, and design of antimicrobial peptides. Methods Mol. Biol. 2015, 1268, 43–66. [PubMed] Brogden, K.A. Antimicrobial peptides: Pore formers or metabolic inhibitors in bacteria? Nat. Rev. Microbiol. 2005, 3, 238–250. [CrossRef] [PubMed] Powers, J.P.; Hancock, R.E. The relationship between peptide structure and antibacterial activity. Peptides 2003, 24, 1681–1691. [CrossRef] [PubMed] Zasloff, M. Antimicrobial peptides of multicellular organisms. Nature 2002, 415, 389–395. [CrossRef] [PubMed] Schittek, B.; Hipfel, R.; Sauer, B.; Bauer, J.; Kalbacher, H.; Stevanovic, S.; Schirle, M.; Schroeder, K.; Blin, N.; Meier, F.; et al. Dermcidin: A novel human antibiotic peptide secreted by sweat glands. Nat. Immunol. 2001, 2, 1133–1137. [CrossRef] [PubMed] Diamond, G.; Beckloff, N.; Weinberg, A.; Kisich, K.O. The roles of antimicrobial peptides in innate host defense. Curr. Pharm. Des. 2009, 15, 2377–2392. [CrossRef] [PubMed] Guani-Guerra, E.; Santos-Mendoza, T.; Lugo-Reyes, S.O.; Teran, L.M. Antimicrobial peptides: General overview and clinical implications in human health and disease. Clin. Immunol. 2010, 135, 1–11. [CrossRef] [PubMed] Glaser, R.; Harder, J.; Lange, H.; Bartels, J.; Christophers, E.; Schroder, J.M. Antimicrobial psoriasin (S100A7) protects human skin from Escherichia coli infection. Nat. Immunol. 2005, 6, 57–64. [CrossRef] [PubMed] Sorensen, O.E.; Follin, P.; Johnsen, A.H.; Calafat, J.; Tjabringa, G.S.; Hiemstra, P.S.; Borregaard, N. Human cathelicidin, hCAP-18, is processed to the antimicrobial peptide LL-37 by extracellular cleavage with proteinase 3. Blood 2001, 97, 3951–3959. [CrossRef] [PubMed] Pharmaceuticals 2016, 9, 41 47. 48. 49. 50. 51. 13 of 13 Nordahl, E.A.; Rydengard, V.; Nyberg, P.; Nitsche, D.P.; Morgelin, M.; Malmsten, M.; Björck, L.; Schmidtchen, A. Activation of the complement system generates antibacterial peptides. Proc. Natl. Acad. Sci. USA 2004, 101, 16879–16884. [CrossRef] [PubMed] Papareddy, P.; Rydengard, V.; Pasupuleti, M.; Walse, B.; Morgelin, M.; Chalupka, A.; Malmsten, M.; Schmidtchen, A. Proteolysis of human thrombin generates novel host defense peptides. PLoS Pathog. 2010, 6, e1000857. [CrossRef] [PubMed] Papareddy, P.; Kalle, M.; Kasetty, G.; Mörgelin, M.; Rydengård, V.; Albiger, B.; Lundqvist, K.; Malmsten, M.; Schmidtchen, A. C-terminal peptides of tissue factor pathway inhibitor are novel host defense molecules. J. Biol. Chem. 2010, 285, 28387–28398. [CrossRef] [PubMed] Baker, B.S. The role of microorganisms in atopic dermatitis. Clin. Exp. Immunol. 2006, 144, 1–9. [CrossRef] [PubMed] Schroeder, B.O.; Wu, Z.; Nuding, S.; Groscurth, S.; Marcinowski, M.; Beisner, J.; Buchner, J.; Schaller, M.; Stange, E.F.; Wehkamp, J. Reduction of disulphide bonds unmasks potent antimicrobial activity of human β-defensin 1. Nature 2011, 469, 419–423. [CrossRef] [PubMed] © 2016 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC-BY) license (http://creativecommons.org/licenses/by/4.0/).