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Suppression of Molecular Inflammatory Pathways by Toll-Like Receptor 7, 8, and 9 Antagonists in a Model of IL-23-Induced Skin Inflammation

Suppression of Molecular Inflammatory Pathways by Toll-Like Receptor 7, 8, and 9 Antagonists in a... Psoriasis is a complex inflammatory disease resulting from the activation of T helper (Th) 1 and Th17 cells. Recent evidence suggests that abnormal activation of Toll-like receptors (TLRs) 7, 8 and 9 contributes to the initiation and maintenance of psoriasis. We have evaluated the effects of TLR antagonists on the gene expression profile in an IL-23-induced skin inflammation model in mice. Psoriasis-like skin lesions were induced in C57BL/6 mice by intradermal injection of IL-23 in the dorsum. Two TLR antagonists were compared: IMO-3100, an antagonist of TLRs 7 and 9, and IMO-8400, an antagonist of TLRs 7, 8 and 9, both of which previously have been shown to reduce epidermal hyperplasia in this model. Skin gene expression profiles of IL-23-induced inflammation were compared with or without TLR antagonist treatment. IL-23 injection resulted in alteration of 5100 gene probes (fold change ≥ 2, FDR < 0.05) including IL-17 pathways that are up-regulated in psoriasis vulgaris. Targeting TLRs 7, 8 and 9 with IMO-8400 resulted in modulation of more than 2300 mRNAs while targeting TLRs 7 and 9 with IMO-3100 resulted in modulation of more than 1900 mRNAs. Both agents strongly decreased IL-17A expression (>12-fold reduction), normalized IL-17 induced genes such as beta-defensin and CXCL1, and normalized aberrant expression of keratin 16 (indicating epidermal hyperplasia). These results suggest that IL-23-driven inflammation in mouse skin may be dependent on signaling mediated by TLRs 7, 8, and 9 and that these receptors represent novel therapeutic targets in psoriasis vulgaris and other diseases with similar pathophysiology. Citation: Suárez-Fariñas M, Arbeit R, Jiang W, Ortenzio FS, Sullivan T, et al. (2013) Suppression of Molecular Inflammatory Pathways by Toll-Like Receptor 7, 8, and 9 Antagonists in a Model of IL-23-Induced Skin Inflammation. PLoS ONE 8(12): e84634. doi:10.1371/journal.pone.0084634 Editor: Deyu Fang, Northwestern University Feinberg School of Medicine, United States of America Received September 4, 2013; Accepted November 16, 2013; Published December 27, 2013 Copyright: © 2013 Suárez-Fariñas et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: Funding for this study was provided by an unrestricted grant given to Dr. James Krueger by IDERA Pharmaceuticals, Inc. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript, however, they played a partial role in conception of the study and conducted the in vivo model on which this study is based. Competing interests: The authors have read the journal's policy and have the following conflicts: Robert Arbeit, Weiwen Jiang and Tim Sullivan are employees of Idera Pharmaceuticals, Inc. James G. Krueger has fulfilled the role of advisory to Idera Pharmaceuticals, Inc. This does not alter the authors’ adherence to all the PLOS ONE policies on sharing data and materials. * E-mail: kruegej@rockefeller.edu psoriasis. This cytokine milieu further activates keratinocytes Introduction and other resident cutaneous cells and induces abnormal Psoriasis is a chronic inflammatory disease of the skin, expression of antimicrobial peptides and other defensin genes characterized by keratinocyte hyperplasia, dermal leukocyte [6]. infiltration and dermal vascular enhancement [1]. It affects The critical role played by the IL23/Th17 axis in psoriasis has approximately 2% of the population and almost 90% of been highlighted in recent studies [7],[8]. IL-23 is produced by individuals suffer from the most common form known as plaque antigen presenting cells such as DCs, and in addition to driving psoriasis [2]. Immune cell infiltrates within psoriatic lesions differentiation of naïve CD4+ T cell precursors towards the predominantly consist of CD3+ Th1, Th17 cells and CD11c+ Th17 phenotype [9], IL-23 also stimulates survival and dendritic cells (DCs) [3], [4], [5]. The cytokines produced by expansion of Th17 populations [10]. In turn, IL-17 produced by these cells, such as tumor necrosis factor-α (TNFα), interferon- Th17 cells exerts direct regulatory control over the expression γ (IFNγ), IL-17, IL-22, IL-23, IL-12 and IL-1β, create an of defensins, S100 family proteins, and LL-37 [11],[12], all of inflammatory cascade, contributing to the pathogenesis of which contribute to innate immune responses within skin. PLOS ONE | www.plosone.org 1 December 2013 | Volume 8 | Issue 12 | e84634 TLR7, 8, and 9 Antagonists in IL-23 Mouse Model Lesional (LS) skin from humans exhibits higher expression of scores in psoriasis patients [25]. TLRs are transmembrane IL-23 in keratinocytes and dermal tissue in comparison to non- receptors that recognize pathogen-associated molecular lesional patterns (PAMPs) and mediate innate immune defense against pathogens. TLRs 3, 7, 8 and 9 are all located in the endosome, (NL) and normal skin [13],[14]. The high efficacy of however, TLR3, TLR7 and TLR8 bind RNA while TLR9 binds antibodies that target IL-23 and IL-17 further substantiates the integral role these cytokines play in psoriasis [15]. Studies DNA containing unmethylated CG dinucleotides [26]. The performed in mice reveal IL-23-mediated inflammation to be expression and activity of TLRs 7, 8 and 9 is regulated by highly dependent upon production of IL-17 [16]. Cutaneous interactions between these receptors. Although TLR8 has been IL-23 injections in mice result in epidermal hyperplasia and deemed nonfunctional in mice, recent evidence suggests it exerts regulatory control over other TLRs [27]. TLRs are mainly parakeratosis, somewhat reminiscent of the human psoriasis expressed on immune cells such as antigen presenting cells, phenotype [17]. These observed changes make the IL-23 with TLR7 and 9 on plasmacytoid DCs (pDCs) and B cells, and treated mouse a useful model for human skin inflammation. TLR8 on myeloid DCs (mDCs). A positive feedback loop of Although morphological similarities are readily visible, the inflammation is created when these pDCs and mDCs are extent to which there is genomic overlap between human activated by immune complexes consisting of self-nucleic acids psoriasis and the IL-23 treated mouse model remains to be and LL-37 (cathelicidin), an antimicrobial peptide elucidated. overexpressed in psoriatic lesions [28], [29]. Interactions Other mouse models with phenotypes that appear somewhat between TLRs on DCs and immune complexes induces analogous to human psoriasis have been analyzed on a production of type I IFN and facilitates T cell autoreactivity, genomic level. A recent study performed novel transcriptomics- ultimately contributing to lesional tissue changes [30]. It based comparisons between human psoriasis and five different appears that TLR7- and TLR9-signaling stimulates IL-23 psoriasiform mouse models [18]. Four transgenic models, K14- secretion by DCs [31], [32], consequently up-regulating IL-17 AREG, K5-STAT3C, K5-TGFβ1 and K5-Tie2, were investigated production [33]. A TLR7/8 agonist used to treat skin in addition to an imiquimod (IMQ)-induced model. The K14- abnormalities such as cancerous lesions, IMQ, has been AREG and K5-STAT3C both manifested inflammatory shown to exacerbate psoriasis in patients [34]. A significant phenotypes via disruption of keratinocyte homeostasis, in turn role for TLRs in psoriasis pathogenesis [35] is further causing increased cytokine release and a profound supported by the finding that IL-23/IL-17 dependent features of inflammatory response. Overexpression of human growth clinical psoriasis were induced by topical application of IMQ factor amphiregulin and a constitutive activation of a signaling [23]. component, Stat3, are the inciting events responsible for the In this study, we used a genome wide expression profile K14-AREG and K5-STAT3C, respectively [19], [20]. The K5- analysis to characterize the IL-23-induced model of skin Tie2 model, a result of a tyrosine kinase overexpression within inflammation. Comparison of global gene expression patterns basal keratinocytes, and the K5-TGFβ1 model, caused by as well as individual pathway analysis allowed us to determine overexpression of a latent form of transforming growth factor how closely the IL-23-induced mouse model resembled the beta 1, both initiate inflammation via keratinocyte human psoriasis phenotype. For completeness, five previously dysregulation, in conjunction with other mechanisms such as analyzed mouse models [18] were included in comparison with perturbance of the basement membrane and angiogenesis the IL-23-induced mouse model and human psoriasis. [21], [22]. IMQ, an agonist of TLRs 7 and 8, causes T cell Furthermore, treatment responses to two different TLR infiltration and keratinocyte and vascular hyperplasia upon antagonists were evaluated in the IL-23-induced mouse model. topical application [23]. For comparison, a human psoriasis transcriptome was extrapolated from differences in gene Results expression between psoriatic LS and normal skin using whole- genome microarray analysis. Transcriptomes for each mouse model were acquired in similar fashion, using naïve mouse skin Significant up-regulation of inflammatory cytokines in IL-23-induced inflammatory mouse model as a control to compare against each inflammatory phenotype. Global correspondence of gene expression between human Genetic changes associated with the IL-23-induced mouse psoriasis and all five mouse models was deemed significant phenotype were elucidated by comparison of full-thickness [18]. Further determination of ‘genomic fidelity’ between mouse IL-23 injected skin with naïve mouse skin using mouse4302 inflammatory models and human psoriasis will aid in the Affymetrix gene array. As expected, IL-17A mRNA was up- identification of representative models of clinical psoriasis. regulated by 13 fold in the diseased model, in addition to other Reliable animal models for the human psoriasis phenotype are IL-17-regulated genes in keratinocytes such as CXCL1 by 120- valuable as the genetic and immunological underpinnings of fold, lipocalin (LCN2) by 53-fold, S100A8 by 36-fold and both the disease have not yet been fully delineated and the search defensin (Defb4) and S100A9 by 30-fold. Innate cytokines were for novel and improved treatments continues. also highly induced in the inflammatory model including IL-6, Potential therapeutic value has recently been demonstrated which was up-regulated by 95-fold, and members of the IL-1 by a study showing that antagonism of TLRs 7, 8 and 9 family, namely, IL1-F5, F6, F8, F9 and IL1β, which increased reduced IL-23-induced epidermal hyperplasia and IL-17 by 55-fold. Keratin 16 mRNA increased by approximately 12- expression [24]. Additionally, data collected from a Phase 2 fold, which may directly correlate with the observed epidermal clinical trial has shown a TLR 7 and 9 antagonist reduced PASI hyperplasia in the IL-23 mouse model. Surprisingly, interferon-γ PLOS ONE | www.plosone.org 2 December 2013 | Volume 8 | Issue 12 | e84634 TLR7, 8, and 9 Antagonists in IL-23 Mouse Model mRNA exhibited an increase of almost 20-fold in addition to Comparative analysis of 6 inflammatory mouse models reveals differential expression of inflammatory downstream interferon-response genes: CXCL11 by 29-fold; pathways CXCL9 by 21-fold; and STAT1 by 4.7-fold. Using predefined We applied the previously described method of gene rank cut-off values of fold-change (FCH)>2 and false-discovery rate overlap analysis to five additional mouse models to evaluate (FDR)<0.05, we identified a total of 2346 up and 2762 down- their correspondence with the human psoriasis phenotype as regulated probe-sets, which encoded 1726 and 1775 unique represented by MAD3. In agreement with previously described genes, respectively. This profile of differentially regulated results [18], we found the K5-Tie2, IMQ, K14-AREG, K5-Stat3C genes, which we will herein refer to as the IL-23-induced and K5-TGFbeta1 mouse models all shared expression mouse transcriptome, can be found in Table S1. patterns with human psoriasis. Although the K14-AREG mouse was the closest of the transgenic models to the human Significant correspondence between IL-23-induced phenotype, side by side comparison revealed the IL-23 mouse mouse model of inflammation and human psoriasis resembled the expression patterns in human psoriasis with In order to compare how well the IL-23-induced model greatest fidelity overall (Figure S1). Various core inflammatory represented human psoriasis, analysis was performed using pathways that are represented in the overall gene set were two previously published human psoriasis transcriptomes. One analyzed using GSEA (Figure 2). The of the transcriptomes, MAD3, is meta-analysis-derived and Gudjonsson(LSvsNormal) showed slightly exaggerated based on differences in gene expression between LS and NL expression of IL-17 driven pathways in comparison to the skin across three independent profiling studies in humans [36]. MAD3, which had overall greater correlation with the The other human transcriptome, which will herein be referred to expression profiles of all mouse models. The IL-23 mouse model best matched the attenuated Th2 profile seen in human as Gudjonsson(LSvsNormal), is based on differences in gene psoriasis, whereas the amphiregulin model showed a slightly expression between psoriasis lesions and normal healthy more robust expression of Th2 pathways. Interestingly, the control skin [37], and served as the representative human IL-17 and TNF axes were well-represented in the IL-23 and psoriasis transcriptome in a previous comparative analyses amphiregulin models, however, the IL-23 model displayed with five mouse models [18]. Analysis of concordance between stronger representation of IFNα induced genes, in better human and the IL-23-induced mouse model revealed 25% of accord with human phenotype. Additionally, genes regulated by differentially expressed genes (DEGs) in the IL-23 IL-22 in keratinocytes were also more correctly represented transcriptome were present in MAD-3, compared to 15% in the within the IL-23 model. Overall, the IL-23 model best reflected Gudjonsson(LSvsNormal) (Figure 1A). As choice of cut-offs the cytokine-mediated processes found in human psoriasis, can influence the intersections between studies’ DEGs [38], we although it clearly does not encompass the full psoriasis used previously described methods [18] to compare human genotype. and mouse transcriptomes using ranked gene lists. The 5000 most strongly up-regulated and 5000 most strongly down- Treatment with TLR antagonists regulates IL-23- regulated genes were identified in the IL-23-induced mouse induced gene expression transcriptome, and then ranked by estimated FCH between LS Immunological pathway modifications by two different TLR and normal. For each rank k, the top k murine genes were antagonists were measured in the IL-23-induced mouse model, identified and the overlap between them and the murine which was chosen for its histological [24] and molecular orthologs of the human transcriptomes was determined. There resemblance to human psoriasis. Skin lesions were induced in was statistically significant overlap of top k up- and down- mice by intradermal injection of IL-23 into the dorsum, later regulated transcripts for the IL-23 mouse and both human followed by subcutaneous injections of either IMO-3100 (TLR7/9 antagonist) or IMO-8400 (TLR7/8/9 antagonist), distal transcriptomes (Figure 1B, 1C). Comparison between the to the IL-23 injection site. Principal component analysis based human transcriptomes themselves showed high degree of on the acquired microarray data illustrates that both TLR overlap, with psoriasis-increased ranked gene lists exhibiting antagonists exert significant effects on gene expression greater overlap than psoriasis-decreased (Figure 1D). Global patterns exhibited by the IL-23-induced mouse model. similarity between the IL-23-induced mouse model and human Expression patterns exhibited by the IMO-3100 and the psoriasis was further demonstrated with gene-set enrichment IMO-8400 treated mice exhibited a shift towards the expression analysis (GSEA). Using a previously described method [38], profile of naïve mice, with slightly more profound effect seen in ten previously published human psoriasis transcriptomes were the IMO-8400 treated cohort (Figure 3A). Analysis of gene treated like individual gene sets in order to quantify the extent overlap was accomplished by comparison of the naïve mouse to which up- and down-regulated genes correlated with ordered gene expression profile with that of the IL-23-induced DEGs from the IL-23-induced mouse (Table 1). Excluding the phenotype both pre- and post-treatment with both TLR methylation in psoriasis transcriptome, normalized enrichment antagonists. Using a cut-off level of FDR<0.05 and FCH>2, it scores (NES) spanned 2.04 - 2.41 for up and -2.77 to -1.88 for was found that IMO-3100 modulated 26% of the IL-23- down-regulated DEGs in the IL-23-induced LS skin, indicating regulated genes, while the additive effect of TLR8 antagonism significant enrichment of human psoriasis gene sets in the in IMO-8400 was associated with 36% alteration of IL-23 genes IL-23-induced phenotype. (Figure 3B). Ingenuity pathway analysis revealed that the PLOS ONE | www.plosone.org 3 December 2013 | Volume 8 | Issue 12 | e84634 TLR7, 8, and 9 Antagonists in IL-23 Mouse Model Figure 1. Murine IL-23 induced model of inflammation corresponds to the human psoriasis phenotype: Statistically significant overlap of DEGs and ranked gene lists. A. Venn diagrams illustrate relative overlap of orthologous DEGs between human psoriasis and the IL-23-induced mouse model. There are 10% more common DEGs between the IL-23 mouse model and MAD3 compared to that with Gudjonsson(LSvsNormal). B, C. Overlap between top k up- (red lines) and k down-regulated (dark blues lines) genes in the IL-23 transcriptome and the murine orthologs of the human MAD3 and Gudjonsson(LSvsNormal) transcriptomes, respectively, was estimated for k=1,…5000.. D. As a reference, the overlap between both human transcriptomes was analyzed in similar fashion. Statistically significant overlap is seen for all three depictions of ranked gene overlap, as the light blue regions represent degree of overlap expected under the null hypothesis of random overlap. doi: 10.1371/journal.pone.0084634.g001 PLOS ONE | www.plosone.org 4 December 2013 | Volume 8 | Issue 12 | e84634 TLR7, 8, and 9 Antagonists in IL-23 Mouse Model Suppression of inflammatory pathways by TLR Table 1. GSEA analysis for the IL23-induced phenotype in antagonism mice versus psoriasis transcriptomes in human. Fold changes in inflammatory genes highly implicated in psoriasis pathogenesis determined by microarray are displayed for the IL-23-induced mouse model as well as treatment groups Psoriasis in Table 2. Treatment with each TLR antagonist significantly Up Down Transcriptomes Reference 1 2 3 suppressed many of the inflammatory genes, with substantial Size ES NES Size ES NES CS reduction in IL-17A mRNA (>12-fold) as well as β-defensin, Gudjonsson ’09 425 0.71 2.41 214 -0.58 -2.43 0.65 [52] CXCL1, CXCL2, CXCL3, LCN2, and other IL-17 pathway (LSvsNL) molecules, including the IL-21 receptor and IL-12Rβ1. Gudjonsson ’10 482 0.70 2.43 336 -0.59 -2.54 0.64 [37] Additionally, IL-6, an up-stream regulator of Th17 development, (LSvsNormal) was reduced by 98-fold along with IFNγ, which decreased by 8- MAD5 579 0.68 2.37 390 -0.58 -2.57 0.63 [36] fold and 11.5-fold with IMO-3100 and IMO-8400, respectively. MAD3 899 0.66 2.33 608 -0.59 -2.70 0.63 [36] Yao’08 820 0.68 2.40 730 -0.55 -2.59 0.62 [40] IFNγ pathway genes like CXCL9 and IL-12Rβ1 were also Suárez-Fariñas ’10 500 0.67 2.34 633 -0.52 -2.43 0.60 [38] reduced. IL1 decreased 6-fold with IMO-3100 and 12-fold with NGS (Jabbari/ IMO-8400 and decline in NFkB mRNA was observed. Several 895 0.62 2.15 748 -0.58 -2.77 0.60 [53] SF’12) cytokine receptors that signal though CD132 and JAK3 were Suárez-Fariñas + 1362 0.58 2.04 949 -0.53 -2.54 0.55 [39] down-regulated. The results of GSEA analysis, which Zhou ‘03 220 0.68 2.27 344 -0.42 -1.88 0.55 [54] evaluated global treatment effects for the entire set of genes, Methylation in yielded similar results. Down-regulation of genes Psoriasis 379 -0.65 -2.22 457 0.34 1.55 -0.49 [55] overexpressed in keratinocytes cultured with IL-17, IFNγ and (LSvsNormal) IL1 as well as the JAK-Stat pathway and IL-23, IL-12 and IL-27 Size indicates the number of genes in each transcriptome canonical pathways following IMO-8400 treatment (Table 3). ES = enrichment score Interestingly, the type I diabetes pathway was found to be NES = normalized enrichment score down-regulated following treatment as well. CS = connectivity core, calculated as ½(ES(Up)-ES(Down)) doi: 10.1371/journal.pone.0084634.t001 Discussion While several different inflammatory models in mouse skin additional antagonism of TLR8 with IMO-8400 was linked to have shown some features that are consistent with human several canonical pathways including immune cell trafficking, psoriasis, it is clear that not all features of disease are inflammatory response and antimicrobial response pathways. represented within the available models. At present, psoriasis Upstream regulators were largely inflammatory in nature, is best defined by the array of genes that are dysregulated in including gene networks involved in both innate and adaptive diseased tissue, identified by comparison of LS to NL tissue immune responses as well as IFNγ signaling and components across multiple studies and with a meta-analysis of different of the IL-17 pathway such as IL-21. A nearly inverse pattern of studies consistently showing greater than 1000 genes, gene expression for IL-23-injected mice and naïve mice is collectively defining the psoriasis transcriptome [36]. The study portrayed by the heatmap in Figure 3C. The disparity in gene with the largest number of samples, Suárez-Fariñas et al. [39], expression between disease and naïve state is visibly has detected more than 4000 genes that are dysregulated by reconciled to some extent by treatment with TLR antagonists, criteria of greater than 2 FCH and a FDR <0.05. In this study, evidenced by post-treatment expression profiles bearing we first sought to determine the extent by which the IL-23 greater resemblance to the naïve rather than the inflammatory model reflects molecular and inflammatory pathways expressed in human psoriasis and secondly, to determine how state (Figure 3C). TLR antagonist treatment resulted in an this model relates to other inflammatory models where average FCH of 1.94 towards recovery for genes altered as a transcriptome profiles have been made available. Within this result of IL-23-induced inflammation, with a 53.21% context, the key inflammatory pathways in psoriasis, such as improvement seen with administration of IMO-8400. Overall, IL-23 stimulated activation of IL-17 and downstream genes, are 52% and 39% of genes in the IL-23-induced phenotype well represented. Also, the IL-23 model produces the least decreased by >50% in the IMO-8400 and IMO-3100 groups, amount of Th2 activation, which in conjunction with Th17, Th22 respectively. Interestingly, genes shared between the IL-23- and IFNγ are the defining elements of inflammation in mouse and the MAD3 human psoriasis transcriptomes psoriasis. High expression of IFNα related genes in LS skin has experienced 10 points higher improvement compared with further implicated an important role for Type I IFNs in genes unique to the mouse model (62.26% vs. 51.6%) as seen pathogenesis of psoriasis [40], [41]. in Figure 3D, upper panel. A FCH towards recovery of 2.32 TLR antagonism may represent a strategy for regulating the was observed for human and mouse orthologous genes vs. complex inflammatory environment in skin caused by psoriasis. 1.89 for purely mouse genes (Figure 3D, lower panel). A previously published study by Jiang et al., examined a TLR7, Collectively, these results indicate TLR7, 8, and 9 antagonism 8, and 9 antagonist in the IL-23 mouse model, focusing on partially resolves IL-23-induced inflammation. psoriasis related cytokines as measured by RT-PCR [24]. The PLOS ONE | www.plosone.org 5 December 2013 | Volume 8 | Issue 12 | e84634 TLR7, 8, and 9 Antagonists in IL-23 Mouse Model Figure 2. Pathways enriched in human psoriasis and inflammatory mouse model transcriptomes with GSEA. Degree of enrichment of key inflammatory pathways implicated in psoriasis pathogenesis compared in human and murine transcriptomes is portrayed by the bubbles representing normalized enrichment score (NES) and false discovery rate (FDR) values. Six inflammatory mouse model transcriptomes; IL-23-induced (IL-23), K14-AREG (AREG for both ear and tail), K5-Stat3c (Stat3), K5Tie2 (Tie-2), K5- TGFβ1 (TGFβ) and IMQ as well as two human psoriasis transcriptomes; MAD3 and Gudjonsson(LSvsNormal), were queried with sets of cytokine-treated keratinocyte, monocyte, fibroblast, inflammatory DC and reconstituted human epidermis (RHE) pathways. doi: 10.1371/journal.pone.0084634.g002 PLOS ONE | www.plosone.org 6 December 2013 | Volume 8 | Issue 12 | e84634 TLR7, 8, and 9 Antagonists in IL-23 Mouse Model Figure 3. IL-23-induced gene expression profile shifts towards recovery with TLR antagonism. A. Differences in gene expression in naïve, IL-23, IL-23+IMO-3100 and IL-23+IMO-8400 treated mice were analyzed using principal component analysis. Partial normalization of gene expression patterns in the IL-23-induced mouse model was seen following treatment with TLR antagonists, with IMO-8400 exerting a slightly greater effect. B. IMO-8400 modulated 10% more of the IL-23 altered genes compared to IMO-3100, indicating additive effects of TLR8 antagonism in addition to TLR7 and 9. C. Shifts in gene expression profiles for IL-23 treated mice towards naïve mice following treatment with TLR antagonists are displayed in heatmap. Of the genes that shifted towards recovery with TLR antagonism, a greater proportion consisted of shared human and mouse genes rather than those that were unique murine genes. doi: 10.1371/journal.pone.0084634.g003 PLOS ONE | www.plosone.org 7 December 2013 | Volume 8 | Issue 12 | e84634 TLR7, 8, and 9 Antagonists in IL-23 Mouse Model Table 2. Selected list of murine genes orthologous to Table 2 (continued). inflammatory genes implicated in psoriasis pathogenesis that are regulated by TLR antagonists. Fold Fold Fold ChangeIL-23 ChangeIL-23 ChangeIL-23 Fold Fold Fold Symbol Description +Saline +IMO-3100 +IMO-8400 ChangeIL-23 ChangeIL-23 ChangeIL-23 chemokine (C-X-C 2 2 Cxcr3 3.04 -1.80 -1.77 Symbol Description +Saline +IMO-3100 +IMO-8400 motif) receptor 3 Defb3 defensin beta 3 1012.96 -20.76 -16.62 interleukin 1 family, 2 2 Il1f5 2.46 1.01 1.08 chemokine (C-X-C member 5 (delta) Cxcl1 120.8 -18.62 -7.54 motif) ligand 1 FDR is not statistically significant chemokine (C-X-C FCH does not reach significance threshold Cxcl1 109.08 -15.37 -6.50 motif) ligand 1 doi: 10.1371/journal.pone.0084634.t002 Il6 interleukin 6 94.80 -98.77 -98.23 Il1b interleukin 1 beta 55.03 -5.94 -12.11 Table 3. GSEA analysis for the effect of IMO-8400 over Lcn2 lipocalin 2 53.35 -5.83 -3.87 canonical pathways (C2 collection) and psoriasis related chemokine (C-X-C Cxcl1 47.74 -13.51 -7.05 pathways (a selection of the significant pathways is motif) ligand 1 presented). S100 calcium binding S100a8 protein A8 (calgranulin 36.88 -3.33 -2.11 A) MolSigDb C2 collection N ES NES FDR Defb4 defensin beta 4 30.65 -20.58 -29.92 -4 IL-23 Pathway (PID) 31 -0.84 -2.57 <10 S100 calcium binding -4 Cytokine Receptor Interaction (KEGG) 191 -0.61 -2.54 <10 1 2 S100a9 protein A9 (calgranulin 30.44 -2.18 -1.50 -4 Cell Cycle (Reactome) 344 -0.55 -2.46 <10 B) -4 IL-12 Pathway (PID) 56 -0.68 -2.35 <10 chemokine (C-X-C Cxcl11 29.81 -29.61 -28.68 Graft Versus Host Disease (KEGG) 23 -0.80 -2.24 4.94E-05 motif) ligand 11 IL-27 Pathway (PID) 23 -0.77 -2.23 4.09E-05 Il24 interleukin 24 27.08 -27.08 -27.08 Type I Diabetes Mellitus (KEGG) 24 -0.75 -2.17 1.01E-04 interleukin 1 family, 2 2 Il1f6 23.85 -1.10 -1.12 Natural Killer Cell Mediated Cytotoxicity (KEGG) 93 -0.58 -2.15 3.23E-04 member 6 Allograft Rejection (KEGG) 22 -0.76 -2.12 5.30E-04 chemokine (C-X-C Cxcl9 21.68 -16.00 -20.66 Chemokine Signaling Pathway (KEGG) 161 -0.50 -2.04 0.0017 motif) ligand 9 JAK-STAT Signaling Pathway (KEGG) 113 -0.52 -2.02 0.0019 Ifng interferon gamma 19.77 -8.45 -11.57 Interferon Signaling (Reactome) 117 -0.51 -2.00 0.0023 Tbx21 T-box 21 (T-bet) 13.66 -2.45 -1.76 Toll Endogenous Pathway (PID) 23 -0.70 -1.99 0.0029 chemokine (C-X-C Cxcl10 13.08 -2.50 -2.98 Toll-Like Receptor Signaling Pathway (KEGG) 81 -0.53 -1.96 0.0046 motif) ligand 10 STAT3 Pathway (ST) 11 -0.81 -1.90 0.0076 Il17a interleukin 17A 12.89 -12.84 -12.55 IL-6 Pathway (PID) 44 -0.53 -1.76 0.0293 Krt16 keratin 16 11.92 -3.23 -2.07 Psoriasis-related gene sets tumor necrosis factor Genes down-regulated after 2 weeks of IL-17 Tnfsf10 (ligand) superfamily, 8.69 -5.44 -7.95 -4 675 -0.57 -2.70 <10 antagonist Ixekinumab member 10 -4 Up-regulated by IFNγin Normal Skin (JID, 2012) 722 -0.53 -2.54 <10 chemokine (C-X-C Cxcl2 8.05 -5.10 -7.44 -4 KC IL-17 Up 41 -0.72 -2.36 <10 motif) ligand 2 -4 RHE IFNγ Up 178 -0.56 -2.33 <10 chemokine (C-C motif) Ccr2 7.26 -13.83 -13.84 -4 Fibroblasts IL-17 Up 37 -0.73 -2.29 <10 receptor 2 -4 Additive IL-17 & IL-22 KC 19 -0.74 -2.02 3.51x10 interleukin 1 family, 2 2 Il1f8 6.97 1.04 -1.04 -4 Synergistic IL-17 & IL-22 KC 26 -0.67 -2.00 4.05 x10 member 8 -4 KC IFNα Up 24 -0.69 -1.98 6.19 x10 chemokine (C-C motif) Ccr2 6.41 -4.97 -6.35 Additive IL-17 & TNFα in KC 165 -0.43 -1.76 0.0062 receptor 2 Synergistic IL-17 & TNFα in KC 128 -0.38 -1.51 0.0386 signal transducer and KC TNF Up 460 -0.31 -1.42 0.0633 Stat1 activator of 4.69 -1.87 -3.60 KC IFNγUp 872 -0.28 -1.33 0.0957 transcription 1 N = number of genes detected in each pathway interleukin 1 family, 2 2 Il1f9 3.81 -1.36 -1.21 doi: 10.1371/journal.pone.0084634.t003 member 9 interleukin 1 family, 2 2 Il1f5 3.53 -1.06 1.02 member 5 (delta) transcripts identified in that paper confirmed many of the Il21r interleukin 21 receptor 3.06 -2.47 -2.85 cytokines that were detected in this study using gene array analysis and thus additional confirmation was rendered PLOS ONE | www.plosone.org 8 December 2013 | Volume 8 | Issue 12 | e84634 TLR7, 8, and 9 Antagonists in IL-23 Mouse Model unnecessary. The data presented here extends the analysis targeting TLR7, 8, and 9 impacts a broader array of IL-23- initiated by Jiang et al., by measuring the entire IL-23 mouse induced inflammation pathways than does targeting TLR7 and transcriptome, including the regulation of many genes under 9. This may also translate into future trials where targeting cytokine-driven pathways. Use of gene arrays has permitted a TLR7, 8, and 9 may be more efficacious in treating psoriasis. deeper analysis of the inflammatory pathways that may be Additionally, although results of the clinical trial of the TLR7/9 regulated by TLRs. An earlier study that indicated that TLR7 antagonist are still being analyzed, transcriptomic data from the may be involved in psoriasis pathogenesis made the IL-23 model herein provides biomarker pathways that may be observation that application of IMQ could induce psoriatic analyzed in psoriasis patients undergoing trials with TLR lesions at sites of inflammation. The mechanism of this was antagonists. The shift in disease-associated gene expression of IL-17 and suggested to be through activation of TLR7 on pDCs, leading to increased production of IFNs, with downstream effects on IL-23 towards normal levels in the IL-23 mouse following several inflammatory pathways regulated by IFN-induced treatment with TLR antagonists suggests a potential role for genes [42]. pDCs also express TLR9, which although differs in TLRs in the psoriatic inflammatory cascade. It appears that which ligands it binds, shares a similar mode of endosomal TLR-regulated innate immune pathways may be an important facet of the cutaneous immune system in normal individuals transport and signaling pathway with TLR7 [43]. Hence, if [35]. Additionally, the involvement of various immune cell types activated, TLR9 might play a similar pathogenic role in psoriatic in TLR signaling and the potential utility of TLR antagonists in inflammation. In contrast, TLR8 is expressed mainly by mDCs [44], which are the dominant cell population in psoriasis lesions cutaneous inflammatory diseases further necessitates that [45], and where activation of this TLR would be predicted to greater efforts be made in understanding the roles that these activate NFkB responsive pathways [46], which may include endosomic receptors fulfill within skin. IL-23 production from DCs. Evidence that TLR7, 8 and 9 may participate in psoriasis pathogenesis is also suggested by the Materials and Methods ability of LL-37-RNA and DNA complexes to activate pDCs and mDCs. Normally, the interaction between TLRs with Animals endocytosed viral nucleic acids results in activation of mDCs All protocols were approved by the Idera Institutional Animal and pDCs. In psoriasis, self-DNA and -RNA may be bound by Care and Use Committee. Female C57BL/6 mice, age of 6 LL-37, conferring protection against extracellular degradation weeks, were purchased from The Jackson Laboratory (Bar and consequently allowing access to endosomal TLRs. Harbor, ME). Mice were housed at the Idera Pharmaceuticals, Complexed DNA and LL-37 activates pDCs via TLR9, resulting Inc. animal facility for 1 week before initiating the study. All in IFNα secretion [29]. Alternatively, self-RNA and cathelicidin protocols were approved by the Idera Institutional Animal Care complexes are also able to directly stimulate pDCs by binding and Use Committee (n=5 per group). to TLR7 and can also trigger mDCs through activation of TLR8 [28]. Induction of disease Another consideration is that of the role of TLRs in Induction of lesions on dorsal skin was achieved by daily keratinocytes. In addition to producing elevated levels of intradermal injection of recombinant murine IL-23 (3 µg, cathelicidin, keratinocytes in psoriatic LS skin have been found eBioscience, San Diego, CA) from day 1 to 4 in 100μl PBS. to express significantly higher levels of TLR9 mRNA in IL-23-treated mice were injected subcutaneously at a distal site comparison to NL psoriatic skin or that of atopic dermatitis. with 15 mg/kg of each antagonist in 100μl PBS or, with 100μl Additionally, when cultured with LL-37, keratinocytes further PBS on day 4, 5 and 6 (n=5 per group). All mice were increased expression of TLR9 mRNA in vitro [47]. Activation of euthanized on day 7 and skin samples at the IL-23 injection TLR3, 4, 5, and 9 in keratinocytes with various PAMPs has led site were collected for evaluation. to nuclear translocation of subunit p65 of NFkB in vitro [46]. The observed TLR activation in keratinocytes and subsequent Synthesis and purification of TLR antagonists triggering of NFkB, offers a potential mechanism by which The antagonist oligonucleotides IMO-3100 and IMO-8400 keratinocytes may participate in IL-17 and TNF regulated were synthesized and purified as described earlier [49], [50] inflammatory pathways. Keratinocytes respond to IL-17 with an and contained < 0.075 EU/mg of endotoxin measured by the up-regulation of neutrophil-attracting chemokines as well as Limulus assay (Bio-Whittaker, Walkersville, MD). CCL20, which interacts with CCR6+ cells including mDCs and Th17 cells that subsequently may become part of the lesional Microarray Hybridization environ [48]. Although the exact role of TLRs in keratinocytes is not yet fully understood, further study is clearly warranted. Skin biopsies were stored in RNA Later at –20°C until used. Recently, the TLR7 and 9 antagonist used in this study, Skin total RNA was isolated using RNeasy Mini kit (Qiagen, IMO-3100, was tested in a Phase 2 psoriasis treatment trial Valencia, CA) by a modified protocol. Briefly, 20mg of skin [25]. Although prior examination of a TLR7, 8, and 9 antagonist samples were homogenized in 700μl QIAzol Lysis reagent has been conducted in the IL-23 mouse model [24], no study (Qiagen), followed by 140μl chloroform (Sigma, St. Louis, MO) prior to this has allowed for characterization of how gene and aqueous phase was collected after centrifugation at 12000 circuits may be differentially affected by TLR7 and 9 vs. TLR7, ×g for 15 minutes at 4°C. Absolute ethanol was added to the 8, and 9 antagonism. Results presented herein suggest aqueous phase at 1.5 volume, mixed and loaded on to RNeasy PLOS ONE | www.plosone.org 9 December 2013 | Volume 8 | Issue 12 | e84634 TLR7, 8, and 9 Antagonists in IL-23 Mouse Model Mini spin columns. Total RNA was purified according to the Supporting Information manufacture’s suggestion and later hybridized to GeneChip mouse4302 (Affymetrix, Santa Clara, CA). Raw data have Figure S1. Overlap of ranked gene sets between mouse been deposited in NCBI’s Gene Expression Omnibus and are models and MAD3. Ranked gene overlap analysis was accessible through accession number GSE50400. performed for 5 previously published mouse models and the IL-23 mouse model, using MAD3 as the human psoriasis Statistical analysis reference transcriptome. The red and dark blue lines in each Affymetrix (Santa Clara, CA) CEL files were scanned using figure respectively represent overlap between top and bottom software packages Harshlight [51] and arrayQualityMetrics ranked human orthologs from the murine model transcripts with from R/Bioconductor (www.bioconductor.org). Expression MAD3. Light blue regions represent overlap as predicted under values (in log -scale) were obtained using the GCRMA the null hypothesis. Results correlated with those previously algorithm. Genes with expression higher than 2 in at least 3 described [18], except that the IL-23 model included in our samples and standard deviation of 0.1 were included in the analysis exhibited slightly superior overlap of up-regulated statistical analysis. To identify DEGs moderated t-tests were genes with human, compared to the K14-AREG and K5-TGFβ1 used in the limma package framework. Resultant P-values models, which otherwise overlap best with psoriasis vulgaris. were adjusted for multiple hypotheses using the Benjamini– With respect to down-regulated transcripts, the K14-AREG and Hochberg procedure, which controls the FDR. The cutoffs used K5-Tie-2 models are roughly comparable to the IL-23 model. to determine DEGs were FDR<0.05 and FCH>2. Annotation, (TIF) including orthologs between human and mouse, were retrieved using R’s package biomart. Table S1. The IL-23-induced mouse model transcriptome. The overlap between the top k genes in the murine (XLSX) transcriptome and their orthologs on the published human transcriptome was determined for k=1,…,5000. For each rank Author Contributions k, the top k murine genes were identified and the overlap between them and the mouse orthologs of the human Conceived and designed the experiments: JGK WJ TS RA. transcriptomes was determined. Confidence Interval for the null Performed the experiments: WJ. Analyzed the data: MSF JGK hypothesis of random overlap between human and mouse was RA. Contributed reagents/materials/analysis tools: TS MSF estimated via simulations. For each rank k (k=1,…K) , k genes JGK RA WJ. 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PLOS ONE | www.plosone.org 12 December 2013 | Volume 8 | Issue 12 | e84634 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png PLoS ONE Pubmed Central

Suppression of Molecular Inflammatory Pathways by Toll-Like Receptor 7, 8, and 9 Antagonists in a Model of IL-23-Induced Skin Inflammation

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© 2013 Suárez-Fariñas et al
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Abstract

Psoriasis is a complex inflammatory disease resulting from the activation of T helper (Th) 1 and Th17 cells. Recent evidence suggests that abnormal activation of Toll-like receptors (TLRs) 7, 8 and 9 contributes to the initiation and maintenance of psoriasis. We have evaluated the effects of TLR antagonists on the gene expression profile in an IL-23-induced skin inflammation model in mice. Psoriasis-like skin lesions were induced in C57BL/6 mice by intradermal injection of IL-23 in the dorsum. Two TLR antagonists were compared: IMO-3100, an antagonist of TLRs 7 and 9, and IMO-8400, an antagonist of TLRs 7, 8 and 9, both of which previously have been shown to reduce epidermal hyperplasia in this model. Skin gene expression profiles of IL-23-induced inflammation were compared with or without TLR antagonist treatment. IL-23 injection resulted in alteration of 5100 gene probes (fold change ≥ 2, FDR < 0.05) including IL-17 pathways that are up-regulated in psoriasis vulgaris. Targeting TLRs 7, 8 and 9 with IMO-8400 resulted in modulation of more than 2300 mRNAs while targeting TLRs 7 and 9 with IMO-3100 resulted in modulation of more than 1900 mRNAs. Both agents strongly decreased IL-17A expression (>12-fold reduction), normalized IL-17 induced genes such as beta-defensin and CXCL1, and normalized aberrant expression of keratin 16 (indicating epidermal hyperplasia). These results suggest that IL-23-driven inflammation in mouse skin may be dependent on signaling mediated by TLRs 7, 8, and 9 and that these receptors represent novel therapeutic targets in psoriasis vulgaris and other diseases with similar pathophysiology. Citation: Suárez-Fariñas M, Arbeit R, Jiang W, Ortenzio FS, Sullivan T, et al. (2013) Suppression of Molecular Inflammatory Pathways by Toll-Like Receptor 7, 8, and 9 Antagonists in a Model of IL-23-Induced Skin Inflammation. PLoS ONE 8(12): e84634. doi:10.1371/journal.pone.0084634 Editor: Deyu Fang, Northwestern University Feinberg School of Medicine, United States of America Received September 4, 2013; Accepted November 16, 2013; Published December 27, 2013 Copyright: © 2013 Suárez-Fariñas et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: Funding for this study was provided by an unrestricted grant given to Dr. James Krueger by IDERA Pharmaceuticals, Inc. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript, however, they played a partial role in conception of the study and conducted the in vivo model on which this study is based. Competing interests: The authors have read the journal's policy and have the following conflicts: Robert Arbeit, Weiwen Jiang and Tim Sullivan are employees of Idera Pharmaceuticals, Inc. James G. Krueger has fulfilled the role of advisory to Idera Pharmaceuticals, Inc. This does not alter the authors’ adherence to all the PLOS ONE policies on sharing data and materials. * E-mail: kruegej@rockefeller.edu psoriasis. This cytokine milieu further activates keratinocytes Introduction and other resident cutaneous cells and induces abnormal Psoriasis is a chronic inflammatory disease of the skin, expression of antimicrobial peptides and other defensin genes characterized by keratinocyte hyperplasia, dermal leukocyte [6]. infiltration and dermal vascular enhancement [1]. It affects The critical role played by the IL23/Th17 axis in psoriasis has approximately 2% of the population and almost 90% of been highlighted in recent studies [7],[8]. IL-23 is produced by individuals suffer from the most common form known as plaque antigen presenting cells such as DCs, and in addition to driving psoriasis [2]. Immune cell infiltrates within psoriatic lesions differentiation of naïve CD4+ T cell precursors towards the predominantly consist of CD3+ Th1, Th17 cells and CD11c+ Th17 phenotype [9], IL-23 also stimulates survival and dendritic cells (DCs) [3], [4], [5]. The cytokines produced by expansion of Th17 populations [10]. In turn, IL-17 produced by these cells, such as tumor necrosis factor-α (TNFα), interferon- Th17 cells exerts direct regulatory control over the expression γ (IFNγ), IL-17, IL-22, IL-23, IL-12 and IL-1β, create an of defensins, S100 family proteins, and LL-37 [11],[12], all of inflammatory cascade, contributing to the pathogenesis of which contribute to innate immune responses within skin. PLOS ONE | www.plosone.org 1 December 2013 | Volume 8 | Issue 12 | e84634 TLR7, 8, and 9 Antagonists in IL-23 Mouse Model Lesional (LS) skin from humans exhibits higher expression of scores in psoriasis patients [25]. TLRs are transmembrane IL-23 in keratinocytes and dermal tissue in comparison to non- receptors that recognize pathogen-associated molecular lesional patterns (PAMPs) and mediate innate immune defense against pathogens. TLRs 3, 7, 8 and 9 are all located in the endosome, (NL) and normal skin [13],[14]. The high efficacy of however, TLR3, TLR7 and TLR8 bind RNA while TLR9 binds antibodies that target IL-23 and IL-17 further substantiates the integral role these cytokines play in psoriasis [15]. Studies DNA containing unmethylated CG dinucleotides [26]. The performed in mice reveal IL-23-mediated inflammation to be expression and activity of TLRs 7, 8 and 9 is regulated by highly dependent upon production of IL-17 [16]. Cutaneous interactions between these receptors. Although TLR8 has been IL-23 injections in mice result in epidermal hyperplasia and deemed nonfunctional in mice, recent evidence suggests it exerts regulatory control over other TLRs [27]. TLRs are mainly parakeratosis, somewhat reminiscent of the human psoriasis expressed on immune cells such as antigen presenting cells, phenotype [17]. These observed changes make the IL-23 with TLR7 and 9 on plasmacytoid DCs (pDCs) and B cells, and treated mouse a useful model for human skin inflammation. TLR8 on myeloid DCs (mDCs). A positive feedback loop of Although morphological similarities are readily visible, the inflammation is created when these pDCs and mDCs are extent to which there is genomic overlap between human activated by immune complexes consisting of self-nucleic acids psoriasis and the IL-23 treated mouse model remains to be and LL-37 (cathelicidin), an antimicrobial peptide elucidated. overexpressed in psoriatic lesions [28], [29]. Interactions Other mouse models with phenotypes that appear somewhat between TLRs on DCs and immune complexes induces analogous to human psoriasis have been analyzed on a production of type I IFN and facilitates T cell autoreactivity, genomic level. A recent study performed novel transcriptomics- ultimately contributing to lesional tissue changes [30]. It based comparisons between human psoriasis and five different appears that TLR7- and TLR9-signaling stimulates IL-23 psoriasiform mouse models [18]. Four transgenic models, K14- secretion by DCs [31], [32], consequently up-regulating IL-17 AREG, K5-STAT3C, K5-TGFβ1 and K5-Tie2, were investigated production [33]. A TLR7/8 agonist used to treat skin in addition to an imiquimod (IMQ)-induced model. The K14- abnormalities such as cancerous lesions, IMQ, has been AREG and K5-STAT3C both manifested inflammatory shown to exacerbate psoriasis in patients [34]. A significant phenotypes via disruption of keratinocyte homeostasis, in turn role for TLRs in psoriasis pathogenesis [35] is further causing increased cytokine release and a profound supported by the finding that IL-23/IL-17 dependent features of inflammatory response. Overexpression of human growth clinical psoriasis were induced by topical application of IMQ factor amphiregulin and a constitutive activation of a signaling [23]. component, Stat3, are the inciting events responsible for the In this study, we used a genome wide expression profile K14-AREG and K5-STAT3C, respectively [19], [20]. The K5- analysis to characterize the IL-23-induced model of skin Tie2 model, a result of a tyrosine kinase overexpression within inflammation. Comparison of global gene expression patterns basal keratinocytes, and the K5-TGFβ1 model, caused by as well as individual pathway analysis allowed us to determine overexpression of a latent form of transforming growth factor how closely the IL-23-induced mouse model resembled the beta 1, both initiate inflammation via keratinocyte human psoriasis phenotype. For completeness, five previously dysregulation, in conjunction with other mechanisms such as analyzed mouse models [18] were included in comparison with perturbance of the basement membrane and angiogenesis the IL-23-induced mouse model and human psoriasis. [21], [22]. IMQ, an agonist of TLRs 7 and 8, causes T cell Furthermore, treatment responses to two different TLR infiltration and keratinocyte and vascular hyperplasia upon antagonists were evaluated in the IL-23-induced mouse model. topical application [23]. For comparison, a human psoriasis transcriptome was extrapolated from differences in gene Results expression between psoriatic LS and normal skin using whole- genome microarray analysis. Transcriptomes for each mouse model were acquired in similar fashion, using naïve mouse skin Significant up-regulation of inflammatory cytokines in IL-23-induced inflammatory mouse model as a control to compare against each inflammatory phenotype. Global correspondence of gene expression between human Genetic changes associated with the IL-23-induced mouse psoriasis and all five mouse models was deemed significant phenotype were elucidated by comparison of full-thickness [18]. Further determination of ‘genomic fidelity’ between mouse IL-23 injected skin with naïve mouse skin using mouse4302 inflammatory models and human psoriasis will aid in the Affymetrix gene array. As expected, IL-17A mRNA was up- identification of representative models of clinical psoriasis. regulated by 13 fold in the diseased model, in addition to other Reliable animal models for the human psoriasis phenotype are IL-17-regulated genes in keratinocytes such as CXCL1 by 120- valuable as the genetic and immunological underpinnings of fold, lipocalin (LCN2) by 53-fold, S100A8 by 36-fold and both the disease have not yet been fully delineated and the search defensin (Defb4) and S100A9 by 30-fold. Innate cytokines were for novel and improved treatments continues. also highly induced in the inflammatory model including IL-6, Potential therapeutic value has recently been demonstrated which was up-regulated by 95-fold, and members of the IL-1 by a study showing that antagonism of TLRs 7, 8 and 9 family, namely, IL1-F5, F6, F8, F9 and IL1β, which increased reduced IL-23-induced epidermal hyperplasia and IL-17 by 55-fold. Keratin 16 mRNA increased by approximately 12- expression [24]. Additionally, data collected from a Phase 2 fold, which may directly correlate with the observed epidermal clinical trial has shown a TLR 7 and 9 antagonist reduced PASI hyperplasia in the IL-23 mouse model. Surprisingly, interferon-γ PLOS ONE | www.plosone.org 2 December 2013 | Volume 8 | Issue 12 | e84634 TLR7, 8, and 9 Antagonists in IL-23 Mouse Model mRNA exhibited an increase of almost 20-fold in addition to Comparative analysis of 6 inflammatory mouse models reveals differential expression of inflammatory downstream interferon-response genes: CXCL11 by 29-fold; pathways CXCL9 by 21-fold; and STAT1 by 4.7-fold. Using predefined We applied the previously described method of gene rank cut-off values of fold-change (FCH)>2 and false-discovery rate overlap analysis to five additional mouse models to evaluate (FDR)<0.05, we identified a total of 2346 up and 2762 down- their correspondence with the human psoriasis phenotype as regulated probe-sets, which encoded 1726 and 1775 unique represented by MAD3. In agreement with previously described genes, respectively. This profile of differentially regulated results [18], we found the K5-Tie2, IMQ, K14-AREG, K5-Stat3C genes, which we will herein refer to as the IL-23-induced and K5-TGFbeta1 mouse models all shared expression mouse transcriptome, can be found in Table S1. patterns with human psoriasis. Although the K14-AREG mouse was the closest of the transgenic models to the human Significant correspondence between IL-23-induced phenotype, side by side comparison revealed the IL-23 mouse mouse model of inflammation and human psoriasis resembled the expression patterns in human psoriasis with In order to compare how well the IL-23-induced model greatest fidelity overall (Figure S1). Various core inflammatory represented human psoriasis, analysis was performed using pathways that are represented in the overall gene set were two previously published human psoriasis transcriptomes. One analyzed using GSEA (Figure 2). The of the transcriptomes, MAD3, is meta-analysis-derived and Gudjonsson(LSvsNormal) showed slightly exaggerated based on differences in gene expression between LS and NL expression of IL-17 driven pathways in comparison to the skin across three independent profiling studies in humans [36]. MAD3, which had overall greater correlation with the The other human transcriptome, which will herein be referred to expression profiles of all mouse models. The IL-23 mouse model best matched the attenuated Th2 profile seen in human as Gudjonsson(LSvsNormal), is based on differences in gene psoriasis, whereas the amphiregulin model showed a slightly expression between psoriasis lesions and normal healthy more robust expression of Th2 pathways. Interestingly, the control skin [37], and served as the representative human IL-17 and TNF axes were well-represented in the IL-23 and psoriasis transcriptome in a previous comparative analyses amphiregulin models, however, the IL-23 model displayed with five mouse models [18]. Analysis of concordance between stronger representation of IFNα induced genes, in better human and the IL-23-induced mouse model revealed 25% of accord with human phenotype. Additionally, genes regulated by differentially expressed genes (DEGs) in the IL-23 IL-22 in keratinocytes were also more correctly represented transcriptome were present in MAD-3, compared to 15% in the within the IL-23 model. Overall, the IL-23 model best reflected Gudjonsson(LSvsNormal) (Figure 1A). As choice of cut-offs the cytokine-mediated processes found in human psoriasis, can influence the intersections between studies’ DEGs [38], we although it clearly does not encompass the full psoriasis used previously described methods [18] to compare human genotype. and mouse transcriptomes using ranked gene lists. The 5000 most strongly up-regulated and 5000 most strongly down- Treatment with TLR antagonists regulates IL-23- regulated genes were identified in the IL-23-induced mouse induced gene expression transcriptome, and then ranked by estimated FCH between LS Immunological pathway modifications by two different TLR and normal. For each rank k, the top k murine genes were antagonists were measured in the IL-23-induced mouse model, identified and the overlap between them and the murine which was chosen for its histological [24] and molecular orthologs of the human transcriptomes was determined. There resemblance to human psoriasis. Skin lesions were induced in was statistically significant overlap of top k up- and down- mice by intradermal injection of IL-23 into the dorsum, later regulated transcripts for the IL-23 mouse and both human followed by subcutaneous injections of either IMO-3100 (TLR7/9 antagonist) or IMO-8400 (TLR7/8/9 antagonist), distal transcriptomes (Figure 1B, 1C). Comparison between the to the IL-23 injection site. Principal component analysis based human transcriptomes themselves showed high degree of on the acquired microarray data illustrates that both TLR overlap, with psoriasis-increased ranked gene lists exhibiting antagonists exert significant effects on gene expression greater overlap than psoriasis-decreased (Figure 1D). Global patterns exhibited by the IL-23-induced mouse model. similarity between the IL-23-induced mouse model and human Expression patterns exhibited by the IMO-3100 and the psoriasis was further demonstrated with gene-set enrichment IMO-8400 treated mice exhibited a shift towards the expression analysis (GSEA). Using a previously described method [38], profile of naïve mice, with slightly more profound effect seen in ten previously published human psoriasis transcriptomes were the IMO-8400 treated cohort (Figure 3A). Analysis of gene treated like individual gene sets in order to quantify the extent overlap was accomplished by comparison of the naïve mouse to which up- and down-regulated genes correlated with ordered gene expression profile with that of the IL-23-induced DEGs from the IL-23-induced mouse (Table 1). Excluding the phenotype both pre- and post-treatment with both TLR methylation in psoriasis transcriptome, normalized enrichment antagonists. Using a cut-off level of FDR<0.05 and FCH>2, it scores (NES) spanned 2.04 - 2.41 for up and -2.77 to -1.88 for was found that IMO-3100 modulated 26% of the IL-23- down-regulated DEGs in the IL-23-induced LS skin, indicating regulated genes, while the additive effect of TLR8 antagonism significant enrichment of human psoriasis gene sets in the in IMO-8400 was associated with 36% alteration of IL-23 genes IL-23-induced phenotype. (Figure 3B). Ingenuity pathway analysis revealed that the PLOS ONE | www.plosone.org 3 December 2013 | Volume 8 | Issue 12 | e84634 TLR7, 8, and 9 Antagonists in IL-23 Mouse Model Figure 1. Murine IL-23 induced model of inflammation corresponds to the human psoriasis phenotype: Statistically significant overlap of DEGs and ranked gene lists. A. Venn diagrams illustrate relative overlap of orthologous DEGs between human psoriasis and the IL-23-induced mouse model. There are 10% more common DEGs between the IL-23 mouse model and MAD3 compared to that with Gudjonsson(LSvsNormal). B, C. Overlap between top k up- (red lines) and k down-regulated (dark blues lines) genes in the IL-23 transcriptome and the murine orthologs of the human MAD3 and Gudjonsson(LSvsNormal) transcriptomes, respectively, was estimated for k=1,…5000.. D. As a reference, the overlap between both human transcriptomes was analyzed in similar fashion. Statistically significant overlap is seen for all three depictions of ranked gene overlap, as the light blue regions represent degree of overlap expected under the null hypothesis of random overlap. doi: 10.1371/journal.pone.0084634.g001 PLOS ONE | www.plosone.org 4 December 2013 | Volume 8 | Issue 12 | e84634 TLR7, 8, and 9 Antagonists in IL-23 Mouse Model Suppression of inflammatory pathways by TLR Table 1. GSEA analysis for the IL23-induced phenotype in antagonism mice versus psoriasis transcriptomes in human. Fold changes in inflammatory genes highly implicated in psoriasis pathogenesis determined by microarray are displayed for the IL-23-induced mouse model as well as treatment groups Psoriasis in Table 2. Treatment with each TLR antagonist significantly Up Down Transcriptomes Reference 1 2 3 suppressed many of the inflammatory genes, with substantial Size ES NES Size ES NES CS reduction in IL-17A mRNA (>12-fold) as well as β-defensin, Gudjonsson ’09 425 0.71 2.41 214 -0.58 -2.43 0.65 [52] CXCL1, CXCL2, CXCL3, LCN2, and other IL-17 pathway (LSvsNL) molecules, including the IL-21 receptor and IL-12Rβ1. Gudjonsson ’10 482 0.70 2.43 336 -0.59 -2.54 0.64 [37] Additionally, IL-6, an up-stream regulator of Th17 development, (LSvsNormal) was reduced by 98-fold along with IFNγ, which decreased by 8- MAD5 579 0.68 2.37 390 -0.58 -2.57 0.63 [36] fold and 11.5-fold with IMO-3100 and IMO-8400, respectively. MAD3 899 0.66 2.33 608 -0.59 -2.70 0.63 [36] Yao’08 820 0.68 2.40 730 -0.55 -2.59 0.62 [40] IFNγ pathway genes like CXCL9 and IL-12Rβ1 were also Suárez-Fariñas ’10 500 0.67 2.34 633 -0.52 -2.43 0.60 [38] reduced. IL1 decreased 6-fold with IMO-3100 and 12-fold with NGS (Jabbari/ IMO-8400 and decline in NFkB mRNA was observed. Several 895 0.62 2.15 748 -0.58 -2.77 0.60 [53] SF’12) cytokine receptors that signal though CD132 and JAK3 were Suárez-Fariñas + 1362 0.58 2.04 949 -0.53 -2.54 0.55 [39] down-regulated. The results of GSEA analysis, which Zhou ‘03 220 0.68 2.27 344 -0.42 -1.88 0.55 [54] evaluated global treatment effects for the entire set of genes, Methylation in yielded similar results. Down-regulation of genes Psoriasis 379 -0.65 -2.22 457 0.34 1.55 -0.49 [55] overexpressed in keratinocytes cultured with IL-17, IFNγ and (LSvsNormal) IL1 as well as the JAK-Stat pathway and IL-23, IL-12 and IL-27 Size indicates the number of genes in each transcriptome canonical pathways following IMO-8400 treatment (Table 3). ES = enrichment score Interestingly, the type I diabetes pathway was found to be NES = normalized enrichment score down-regulated following treatment as well. CS = connectivity core, calculated as ½(ES(Up)-ES(Down)) doi: 10.1371/journal.pone.0084634.t001 Discussion While several different inflammatory models in mouse skin additional antagonism of TLR8 with IMO-8400 was linked to have shown some features that are consistent with human several canonical pathways including immune cell trafficking, psoriasis, it is clear that not all features of disease are inflammatory response and antimicrobial response pathways. represented within the available models. At present, psoriasis Upstream regulators were largely inflammatory in nature, is best defined by the array of genes that are dysregulated in including gene networks involved in both innate and adaptive diseased tissue, identified by comparison of LS to NL tissue immune responses as well as IFNγ signaling and components across multiple studies and with a meta-analysis of different of the IL-17 pathway such as IL-21. A nearly inverse pattern of studies consistently showing greater than 1000 genes, gene expression for IL-23-injected mice and naïve mice is collectively defining the psoriasis transcriptome [36]. The study portrayed by the heatmap in Figure 3C. The disparity in gene with the largest number of samples, Suárez-Fariñas et al. [39], expression between disease and naïve state is visibly has detected more than 4000 genes that are dysregulated by reconciled to some extent by treatment with TLR antagonists, criteria of greater than 2 FCH and a FDR <0.05. In this study, evidenced by post-treatment expression profiles bearing we first sought to determine the extent by which the IL-23 greater resemblance to the naïve rather than the inflammatory model reflects molecular and inflammatory pathways expressed in human psoriasis and secondly, to determine how state (Figure 3C). TLR antagonist treatment resulted in an this model relates to other inflammatory models where average FCH of 1.94 towards recovery for genes altered as a transcriptome profiles have been made available. Within this result of IL-23-induced inflammation, with a 53.21% context, the key inflammatory pathways in psoriasis, such as improvement seen with administration of IMO-8400. Overall, IL-23 stimulated activation of IL-17 and downstream genes, are 52% and 39% of genes in the IL-23-induced phenotype well represented. Also, the IL-23 model produces the least decreased by >50% in the IMO-8400 and IMO-3100 groups, amount of Th2 activation, which in conjunction with Th17, Th22 respectively. Interestingly, genes shared between the IL-23- and IFNγ are the defining elements of inflammation in mouse and the MAD3 human psoriasis transcriptomes psoriasis. High expression of IFNα related genes in LS skin has experienced 10 points higher improvement compared with further implicated an important role for Type I IFNs in genes unique to the mouse model (62.26% vs. 51.6%) as seen pathogenesis of psoriasis [40], [41]. in Figure 3D, upper panel. A FCH towards recovery of 2.32 TLR antagonism may represent a strategy for regulating the was observed for human and mouse orthologous genes vs. complex inflammatory environment in skin caused by psoriasis. 1.89 for purely mouse genes (Figure 3D, lower panel). A previously published study by Jiang et al., examined a TLR7, Collectively, these results indicate TLR7, 8, and 9 antagonism 8, and 9 antagonist in the IL-23 mouse model, focusing on partially resolves IL-23-induced inflammation. psoriasis related cytokines as measured by RT-PCR [24]. The PLOS ONE | www.plosone.org 5 December 2013 | Volume 8 | Issue 12 | e84634 TLR7, 8, and 9 Antagonists in IL-23 Mouse Model Figure 2. Pathways enriched in human psoriasis and inflammatory mouse model transcriptomes with GSEA. Degree of enrichment of key inflammatory pathways implicated in psoriasis pathogenesis compared in human and murine transcriptomes is portrayed by the bubbles representing normalized enrichment score (NES) and false discovery rate (FDR) values. Six inflammatory mouse model transcriptomes; IL-23-induced (IL-23), K14-AREG (AREG for both ear and tail), K5-Stat3c (Stat3), K5Tie2 (Tie-2), K5- TGFβ1 (TGFβ) and IMQ as well as two human psoriasis transcriptomes; MAD3 and Gudjonsson(LSvsNormal), were queried with sets of cytokine-treated keratinocyte, monocyte, fibroblast, inflammatory DC and reconstituted human epidermis (RHE) pathways. doi: 10.1371/journal.pone.0084634.g002 PLOS ONE | www.plosone.org 6 December 2013 | Volume 8 | Issue 12 | e84634 TLR7, 8, and 9 Antagonists in IL-23 Mouse Model Figure 3. IL-23-induced gene expression profile shifts towards recovery with TLR antagonism. A. Differences in gene expression in naïve, IL-23, IL-23+IMO-3100 and IL-23+IMO-8400 treated mice were analyzed using principal component analysis. Partial normalization of gene expression patterns in the IL-23-induced mouse model was seen following treatment with TLR antagonists, with IMO-8400 exerting a slightly greater effect. B. IMO-8400 modulated 10% more of the IL-23 altered genes compared to IMO-3100, indicating additive effects of TLR8 antagonism in addition to TLR7 and 9. C. Shifts in gene expression profiles for IL-23 treated mice towards naïve mice following treatment with TLR antagonists are displayed in heatmap. Of the genes that shifted towards recovery with TLR antagonism, a greater proportion consisted of shared human and mouse genes rather than those that were unique murine genes. doi: 10.1371/journal.pone.0084634.g003 PLOS ONE | www.plosone.org 7 December 2013 | Volume 8 | Issue 12 | e84634 TLR7, 8, and 9 Antagonists in IL-23 Mouse Model Table 2. Selected list of murine genes orthologous to Table 2 (continued). inflammatory genes implicated in psoriasis pathogenesis that are regulated by TLR antagonists. Fold Fold Fold ChangeIL-23 ChangeIL-23 ChangeIL-23 Fold Fold Fold Symbol Description +Saline +IMO-3100 +IMO-8400 ChangeIL-23 ChangeIL-23 ChangeIL-23 chemokine (C-X-C 2 2 Cxcr3 3.04 -1.80 -1.77 Symbol Description +Saline +IMO-3100 +IMO-8400 motif) receptor 3 Defb3 defensin beta 3 1012.96 -20.76 -16.62 interleukin 1 family, 2 2 Il1f5 2.46 1.01 1.08 chemokine (C-X-C member 5 (delta) Cxcl1 120.8 -18.62 -7.54 motif) ligand 1 FDR is not statistically significant chemokine (C-X-C FCH does not reach significance threshold Cxcl1 109.08 -15.37 -6.50 motif) ligand 1 doi: 10.1371/journal.pone.0084634.t002 Il6 interleukin 6 94.80 -98.77 -98.23 Il1b interleukin 1 beta 55.03 -5.94 -12.11 Table 3. GSEA analysis for the effect of IMO-8400 over Lcn2 lipocalin 2 53.35 -5.83 -3.87 canonical pathways (C2 collection) and psoriasis related chemokine (C-X-C Cxcl1 47.74 -13.51 -7.05 pathways (a selection of the significant pathways is motif) ligand 1 presented). S100 calcium binding S100a8 protein A8 (calgranulin 36.88 -3.33 -2.11 A) MolSigDb C2 collection N ES NES FDR Defb4 defensin beta 4 30.65 -20.58 -29.92 -4 IL-23 Pathway (PID) 31 -0.84 -2.57 <10 S100 calcium binding -4 Cytokine Receptor Interaction (KEGG) 191 -0.61 -2.54 <10 1 2 S100a9 protein A9 (calgranulin 30.44 -2.18 -1.50 -4 Cell Cycle (Reactome) 344 -0.55 -2.46 <10 B) -4 IL-12 Pathway (PID) 56 -0.68 -2.35 <10 chemokine (C-X-C Cxcl11 29.81 -29.61 -28.68 Graft Versus Host Disease (KEGG) 23 -0.80 -2.24 4.94E-05 motif) ligand 11 IL-27 Pathway (PID) 23 -0.77 -2.23 4.09E-05 Il24 interleukin 24 27.08 -27.08 -27.08 Type I Diabetes Mellitus (KEGG) 24 -0.75 -2.17 1.01E-04 interleukin 1 family, 2 2 Il1f6 23.85 -1.10 -1.12 Natural Killer Cell Mediated Cytotoxicity (KEGG) 93 -0.58 -2.15 3.23E-04 member 6 Allograft Rejection (KEGG) 22 -0.76 -2.12 5.30E-04 chemokine (C-X-C Cxcl9 21.68 -16.00 -20.66 Chemokine Signaling Pathway (KEGG) 161 -0.50 -2.04 0.0017 motif) ligand 9 JAK-STAT Signaling Pathway (KEGG) 113 -0.52 -2.02 0.0019 Ifng interferon gamma 19.77 -8.45 -11.57 Interferon Signaling (Reactome) 117 -0.51 -2.00 0.0023 Tbx21 T-box 21 (T-bet) 13.66 -2.45 -1.76 Toll Endogenous Pathway (PID) 23 -0.70 -1.99 0.0029 chemokine (C-X-C Cxcl10 13.08 -2.50 -2.98 Toll-Like Receptor Signaling Pathway (KEGG) 81 -0.53 -1.96 0.0046 motif) ligand 10 STAT3 Pathway (ST) 11 -0.81 -1.90 0.0076 Il17a interleukin 17A 12.89 -12.84 -12.55 IL-6 Pathway (PID) 44 -0.53 -1.76 0.0293 Krt16 keratin 16 11.92 -3.23 -2.07 Psoriasis-related gene sets tumor necrosis factor Genes down-regulated after 2 weeks of IL-17 Tnfsf10 (ligand) superfamily, 8.69 -5.44 -7.95 -4 675 -0.57 -2.70 <10 antagonist Ixekinumab member 10 -4 Up-regulated by IFNγin Normal Skin (JID, 2012) 722 -0.53 -2.54 <10 chemokine (C-X-C Cxcl2 8.05 -5.10 -7.44 -4 KC IL-17 Up 41 -0.72 -2.36 <10 motif) ligand 2 -4 RHE IFNγ Up 178 -0.56 -2.33 <10 chemokine (C-C motif) Ccr2 7.26 -13.83 -13.84 -4 Fibroblasts IL-17 Up 37 -0.73 -2.29 <10 receptor 2 -4 Additive IL-17 & IL-22 KC 19 -0.74 -2.02 3.51x10 interleukin 1 family, 2 2 Il1f8 6.97 1.04 -1.04 -4 Synergistic IL-17 & IL-22 KC 26 -0.67 -2.00 4.05 x10 member 8 -4 KC IFNα Up 24 -0.69 -1.98 6.19 x10 chemokine (C-C motif) Ccr2 6.41 -4.97 -6.35 Additive IL-17 & TNFα in KC 165 -0.43 -1.76 0.0062 receptor 2 Synergistic IL-17 & TNFα in KC 128 -0.38 -1.51 0.0386 signal transducer and KC TNF Up 460 -0.31 -1.42 0.0633 Stat1 activator of 4.69 -1.87 -3.60 KC IFNγUp 872 -0.28 -1.33 0.0957 transcription 1 N = number of genes detected in each pathway interleukin 1 family, 2 2 Il1f9 3.81 -1.36 -1.21 doi: 10.1371/journal.pone.0084634.t003 member 9 interleukin 1 family, 2 2 Il1f5 3.53 -1.06 1.02 member 5 (delta) transcripts identified in that paper confirmed many of the Il21r interleukin 21 receptor 3.06 -2.47 -2.85 cytokines that were detected in this study using gene array analysis and thus additional confirmation was rendered PLOS ONE | www.plosone.org 8 December 2013 | Volume 8 | Issue 12 | e84634 TLR7, 8, and 9 Antagonists in IL-23 Mouse Model unnecessary. The data presented here extends the analysis targeting TLR7, 8, and 9 impacts a broader array of IL-23- initiated by Jiang et al., by measuring the entire IL-23 mouse induced inflammation pathways than does targeting TLR7 and transcriptome, including the regulation of many genes under 9. This may also translate into future trials where targeting cytokine-driven pathways. Use of gene arrays has permitted a TLR7, 8, and 9 may be more efficacious in treating psoriasis. deeper analysis of the inflammatory pathways that may be Additionally, although results of the clinical trial of the TLR7/9 regulated by TLRs. An earlier study that indicated that TLR7 antagonist are still being analyzed, transcriptomic data from the may be involved in psoriasis pathogenesis made the IL-23 model herein provides biomarker pathways that may be observation that application of IMQ could induce psoriatic analyzed in psoriasis patients undergoing trials with TLR lesions at sites of inflammation. The mechanism of this was antagonists. The shift in disease-associated gene expression of IL-17 and suggested to be through activation of TLR7 on pDCs, leading to increased production of IFNs, with downstream effects on IL-23 towards normal levels in the IL-23 mouse following several inflammatory pathways regulated by IFN-induced treatment with TLR antagonists suggests a potential role for genes [42]. pDCs also express TLR9, which although differs in TLRs in the psoriatic inflammatory cascade. It appears that which ligands it binds, shares a similar mode of endosomal TLR-regulated innate immune pathways may be an important facet of the cutaneous immune system in normal individuals transport and signaling pathway with TLR7 [43]. Hence, if [35]. Additionally, the involvement of various immune cell types activated, TLR9 might play a similar pathogenic role in psoriatic in TLR signaling and the potential utility of TLR antagonists in inflammation. In contrast, TLR8 is expressed mainly by mDCs [44], which are the dominant cell population in psoriasis lesions cutaneous inflammatory diseases further necessitates that [45], and where activation of this TLR would be predicted to greater efforts be made in understanding the roles that these activate NFkB responsive pathways [46], which may include endosomic receptors fulfill within skin. IL-23 production from DCs. Evidence that TLR7, 8 and 9 may participate in psoriasis pathogenesis is also suggested by the Materials and Methods ability of LL-37-RNA and DNA complexes to activate pDCs and mDCs. Normally, the interaction between TLRs with Animals endocytosed viral nucleic acids results in activation of mDCs All protocols were approved by the Idera Institutional Animal and pDCs. In psoriasis, self-DNA and -RNA may be bound by Care and Use Committee. Female C57BL/6 mice, age of 6 LL-37, conferring protection against extracellular degradation weeks, were purchased from The Jackson Laboratory (Bar and consequently allowing access to endosomal TLRs. Harbor, ME). Mice were housed at the Idera Pharmaceuticals, Complexed DNA and LL-37 activates pDCs via TLR9, resulting Inc. animal facility for 1 week before initiating the study. All in IFNα secretion [29]. Alternatively, self-RNA and cathelicidin protocols were approved by the Idera Institutional Animal Care complexes are also able to directly stimulate pDCs by binding and Use Committee (n=5 per group). to TLR7 and can also trigger mDCs through activation of TLR8 [28]. Induction of disease Another consideration is that of the role of TLRs in Induction of lesions on dorsal skin was achieved by daily keratinocytes. In addition to producing elevated levels of intradermal injection of recombinant murine IL-23 (3 µg, cathelicidin, keratinocytes in psoriatic LS skin have been found eBioscience, San Diego, CA) from day 1 to 4 in 100μl PBS. to express significantly higher levels of TLR9 mRNA in IL-23-treated mice were injected subcutaneously at a distal site comparison to NL psoriatic skin or that of atopic dermatitis. with 15 mg/kg of each antagonist in 100μl PBS or, with 100μl Additionally, when cultured with LL-37, keratinocytes further PBS on day 4, 5 and 6 (n=5 per group). All mice were increased expression of TLR9 mRNA in vitro [47]. Activation of euthanized on day 7 and skin samples at the IL-23 injection TLR3, 4, 5, and 9 in keratinocytes with various PAMPs has led site were collected for evaluation. to nuclear translocation of subunit p65 of NFkB in vitro [46]. The observed TLR activation in keratinocytes and subsequent Synthesis and purification of TLR antagonists triggering of NFkB, offers a potential mechanism by which The antagonist oligonucleotides IMO-3100 and IMO-8400 keratinocytes may participate in IL-17 and TNF regulated were synthesized and purified as described earlier [49], [50] inflammatory pathways. Keratinocytes respond to IL-17 with an and contained < 0.075 EU/mg of endotoxin measured by the up-regulation of neutrophil-attracting chemokines as well as Limulus assay (Bio-Whittaker, Walkersville, MD). CCL20, which interacts with CCR6+ cells including mDCs and Th17 cells that subsequently may become part of the lesional Microarray Hybridization environ [48]. Although the exact role of TLRs in keratinocytes is not yet fully understood, further study is clearly warranted. Skin biopsies were stored in RNA Later at –20°C until used. Recently, the TLR7 and 9 antagonist used in this study, Skin total RNA was isolated using RNeasy Mini kit (Qiagen, IMO-3100, was tested in a Phase 2 psoriasis treatment trial Valencia, CA) by a modified protocol. Briefly, 20mg of skin [25]. Although prior examination of a TLR7, 8, and 9 antagonist samples were homogenized in 700μl QIAzol Lysis reagent has been conducted in the IL-23 mouse model [24], no study (Qiagen), followed by 140μl chloroform (Sigma, St. Louis, MO) prior to this has allowed for characterization of how gene and aqueous phase was collected after centrifugation at 12000 circuits may be differentially affected by TLR7 and 9 vs. TLR7, ×g for 15 minutes at 4°C. Absolute ethanol was added to the 8, and 9 antagonism. Results presented herein suggest aqueous phase at 1.5 volume, mixed and loaded on to RNeasy PLOS ONE | www.plosone.org 9 December 2013 | Volume 8 | Issue 12 | e84634 TLR7, 8, and 9 Antagonists in IL-23 Mouse Model Mini spin columns. Total RNA was purified according to the Supporting Information manufacture’s suggestion and later hybridized to GeneChip mouse4302 (Affymetrix, Santa Clara, CA). Raw data have Figure S1. Overlap of ranked gene sets between mouse been deposited in NCBI’s Gene Expression Omnibus and are models and MAD3. Ranked gene overlap analysis was accessible through accession number GSE50400. performed for 5 previously published mouse models and the IL-23 mouse model, using MAD3 as the human psoriasis Statistical analysis reference transcriptome. The red and dark blue lines in each Affymetrix (Santa Clara, CA) CEL files were scanned using figure respectively represent overlap between top and bottom software packages Harshlight [51] and arrayQualityMetrics ranked human orthologs from the murine model transcripts with from R/Bioconductor (www.bioconductor.org). Expression MAD3. Light blue regions represent overlap as predicted under values (in log -scale) were obtained using the GCRMA the null hypothesis. Results correlated with those previously algorithm. Genes with expression higher than 2 in at least 3 described [18], except that the IL-23 model included in our samples and standard deviation of 0.1 were included in the analysis exhibited slightly superior overlap of up-regulated statistical analysis. To identify DEGs moderated t-tests were genes with human, compared to the K14-AREG and K5-TGFβ1 used in the limma package framework. Resultant P-values models, which otherwise overlap best with psoriasis vulgaris. were adjusted for multiple hypotheses using the Benjamini– With respect to down-regulated transcripts, the K14-AREG and Hochberg procedure, which controls the FDR. The cutoffs used K5-Tie-2 models are roughly comparable to the IL-23 model. to determine DEGs were FDR<0.05 and FCH>2. Annotation, (TIF) including orthologs between human and mouse, were retrieved using R’s package biomart. Table S1. The IL-23-induced mouse model transcriptome. The overlap between the top k genes in the murine (XLSX) transcriptome and their orthologs on the published human transcriptome was determined for k=1,…,5000. For each rank Author Contributions k, the top k murine genes were identified and the overlap between them and the mouse orthologs of the human Conceived and designed the experiments: JGK WJ TS RA. transcriptomes was determined. Confidence Interval for the null Performed the experiments: WJ. Analyzed the data: MSF JGK hypothesis of random overlap between human and mouse was RA. Contributed reagents/materials/analysis tools: TS MSF estimated via simulations. For each rank k (k=1,…K) , k genes JGK RA WJ. 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