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. Author manuscript; available in PMC: 2013 Jun 15.
Published in final edited form as: J Immunol. 2012 May 11;188(12):5906–5914. doi: 10.4049/jimmunol.1103233

CIKS/Act1-mediated signaling by IL-17 cytokines in context: Implications for how a CIKS gene variant may predispose to psoriasis

Søren Ulrik Sønder *, Andrea Paun *, Hye-Lin Ha *, Peter F Johnson †, Ulrich Siebenlist *,§
PMCID: PMC3370149  NIHMSID: NIHMS371817  PMID: 22581863

Abstract

Psoriasis is a relapsing skin disease characterized by abnormal keratinocyte proliferation and differentiation and by an influx of inflammatory immune cells. Recently IL-17 cytokines have been strongly implicated as critical for the pathogenesis of this disease. IL-17A (a.k.a. IL-17) and IL-17F are the signature cytokine of Th17 cells, but are also produced by innate cells, including γδ T cells present in skin, while epithelial cells, including keratinocytes, may produce IL-17C. IL-17 cytokines signal via the adaptor protein CIKS/Act1. Psoriasis is a disease with a strong genetic predisposition and the gene encoding CIKS has recently been identified as a susceptibility locus. Unexpectedly, one predisposing gene variant features a mutation that impairs rather than enhances CIKS-mediated IL-17 cytokine signaling, counter to the predicted role for IL-17 cytokines in psoriatic inflammation. Here we demonstrate, however, that this mutant adaptor does not impair the IL-17-specific contributions to the genetic response if combined with TNFα, a cytokine also prominent in psoriatic inflammation. Interestingly, TNFα signals compensate IL-17 signaling defects imposed by this mutant adaptor even for genes that are not induced by TNFα alone, including the transcription factors C/EBPδ and IκBζ, which help regulate secondary gene expression in response to IL-17. Based on these findings we discuss a scenario in which the mutant adaptor may interfere with homeostatic maintenance of epithelial barriers, thereby potentially enabling the initiation of inflammatory responses to insults, while this same mutant adaptor would still be able to mediate IL-17-specific contributions to inflammation once TNFα is present.

Introduction

IL-17A (a.k.a. IL-17) is the signature cytokine of Th17 cells, a helper T cell subset that is critical for defense against fungi and extracellular bacteria, but that has also been linked with a number of inflammatory and autoimmune diseases. IL-17 is not only produced by Th17 cells during adaptive responses, but is also produced during innate responses, most notably by γδT cells and NKT cells. Recruitment of neutrophils to sites of inflammation is a major corollary of IL-17 expression (reviewed in (1–3)). IL-17(A) is a member of family of cytokines (IL-17A-F). IL-17F is structurally and functionally closely related to, and usually co-produced with IL-17 (4). IL-17C may functionally overlap with IL-17, but is produced primarily by epithelial cells, including keratinocytes (5, 6). These cytokines signal via heteromeric receptors composed of members of the IL-17 receptor family (IL-17RA-RE), with the IL-17RA chain likely common to all receptors (7). Intracellularly the receptors for IL-17A/F, IL-17C and IL-17E (IL-25), and probably all members of this cytokine family signal via an adaptor protein termed CIKS (8)(a.k.a Act1 (9), Traf3ip2) (10–13).

Psoriasis is a common chronic inflammatory skin disease affecting up to 2–3% of the population worldwide. This disease has a strong hereditary component, with multiple susceptibility gene loci, including genes encoding proteins involved in keratinocyte differentiation and function as well as proteins involved in inflammatory responses (14–17). Recently IL-17 cytokines have been implicated as important drivers of psoriatic inflammation in human patients and/or in mouse models of the disease (6, 18–21). Furthermore, humanized anti-IL-17 antibodies have proven efficacious in treatment of this disease in clinical trials (22). Recent comprehensive genome-wide association studies have also revealed the gene encoding the IL-17 cytokine signaling adaptor CIKS to be a strong susceptibility locus for psoriasis and psoriatic arthritis, identified with two single nucleotide polymorphisms (SNPs) (23–25). One SNP predicts an amino acid change in the N-terminal part of this protein, a mutation known to have functional consequences. We and others have previously shown that this amino acid change lies in region required for recruitment of the adaptor protein TRAF6 during IL-17 signaling, which in turn is essential for activation of the NF-κB transcription factor and other downstream effectors (26, 27). NF-κB activation by IL-17 is important for the overall response to IL-17, as shown by us in studies with primary IL-17-stimulated fibroblasts (26). The fact that this particular mutation in CIKS impairs TRAF6 recruitment in response to IL-17, yet is also associated with increased susceptibility to psoriasis (24) does pose a paradox, as much evidence suggests that IL-17 actively drives psoriatic inflammation (1, 13, 20, 21).

To explain how an impaired CIKS adaptor might increase the risk to develop psoriasis we hypothesize that IL-17 cytokines may have two distinct roles in the skin: One role may be to help maintain barrier functions, such as warding off bacteria under normal, non-inflammatory conditions, and a second role may be to help drive inflammation during psoriatic flare-ups. We therefore asked whether the CIKS adaptor mutant linked to psoriasis would impair the contributions of IL-17 cytokines in an inflammatory context that includes TNFα; TNFα has been strongly implicated in psoriasis and anti-TNFα treatments have proven fairly effective (28, 29). We demonstrate that the mutant CIKS adaptor does not impair the responses to the combined action of these two cytokines, while it does impair responses to IL-17 alone, as assessed not only with primary mouse embryo fibroblasts, but also with primary keratinocytes and primary dermal fibroblasts. We show that TNFα signaling appears to fully compensate for the inability of this CIKS mutant to recruit TRAF6. Most surprisingly, TNFα signals in concert with IL-17 compensate for the defects in the IL-17-only response even for genes that are induced exclusively by IL-17, but not TNFα. These genes include the transcription factors IκBζ and C/EBPδ, factors that are rapidly induced and that help shape the overall response to IL-17 signaling. We discuss how the CIKS mutant and the thus impaired response to IL-17 alone may promote the initiation of psoriatic inflammation, while this mutant adaptor would still be able to mediate IL-17-specific inflammatory signals in the presence of TNFα, a cytokine with which IL-17 can synergize in induced expression of many genes.

Materials and Methods

Cell culture and reagents

Primary mouse embryo fibroblast cultures (MEFs) were established from wild-type (WT) and CIKS deficient (KO) mice as described previously (10). Primary keratinocytes and dermal fibroblasts were isolated from one-day old CIKS KO exactly as published in a detailed protocol (30). Briefly, pups were euthanized and the skin carefully removed. The dermis and epidermis were separated manually following trypsin digestion. Keratinocytes were extracted from the epidermal layer and dermal fibroblasts were extracted from the dermal layer. Keratinocytes were grown under low Ca conditions and cultures of epidermal keratinocytes and dermal fibroblasts were analyzed by phase contrast microscopy to confirm typical cobblestone morphology of the former and typical more elongated spindle-like morphology of the latter cells. Cells were grown for a total of up to five days prior to stimulation (see below). Mice were bred and housed in National Institute of Allergy and Infectious Diseases facilities, and all experiments were done with approval of the National Institute of Allergy and Infectious Diseases Animal Care and Use Committee and in accordance with all relevant institutional guidelines. Immortalized NEMO deficient MEFs were kindly donated by Dr. Manolis Pasparakis. IκBζ deficient mice were kindly provided by Dr. Shizuo Akira. MEFs were generated from C/EBPβ and C/EBPδ deficient mice, which have been described previously (31, 32). C/EBPβδ doubly deficient MEFs were generated from doubly deficient mice generated by crossbreeding of C/EBPβ and C/EBPδ singly deficient mice. Recombinant IL-17 (100 ng/ml, R&D Systems) and/or TNFα (2 or 10 ng/ml, Peprotech) were used for stimulation of cells.

Plasmids and lentivirus

Full length human CIKS and the CIKSΔT6 mutant (lacking amino acids #10–25 (26)) were cloned into a Gateway Entry vector (Invitrogen) and subcloned into a lentiviral FLAG Tag vector by Gateway LR recombination using the manufacturer’s protocols to generate expression clones. To insure low-level constitutive expression, the standard CMV promoter in these vectors was replaced with the PolII promoter. Plasmid constructs were confirmed by sequencing. Lentivirus preparations used for transduction of wild-type and mutant CIKS proteins into CIKS-deficient primary cells were generated with the ViraPower Lentiviral Expression System (Invitrogen) following the manufacturer’s instructions. Cells were transduced with virus during an overnight incubation and 24 or 48 hours later cells were stimulated with cytokines. In parallel experiments, GFP expressing lentivirus transductions revealed efficiencies of at least 60% (keratinocytes, dermal fibroblasts) and up to 80% (MEFs). Approximately equal transduction efficiencies for matched samples were confirmed with Western blots for expression of transduced proteins in all experiments.

RNA isolation and Real Time PCR (RT-PCR)

RNA was isolated using the RNeasy RNA isolation kit (Qiagen) according to the manufacturer’s instructions. cDNA was synthesized using oligo-dT and Superscript III (Invitrogen). Expression of Iκbζ, Zc3h12a, Il-6, Cxcl1, Cxcl2, Cxcl5, S100a8, Saa3, Lcn2 and β-actin was quantified by Taqman qPCR using primers from Applied Biosystems. All results are expressed as 2−ΔΔCt, where ΔΔCt = (Cttarget−CtΔ-actin) for stimulated samples - (Cttarget−CtΔ-actin) for unstimulated controls. Data are shown as the mean ± SEM.

Western Blots, quantification of chemokines/cytokines and antibodies

Whole cell extracts were isolated, loaded on to 10% SDS-polyacrylamide gel, electrophoresed and transferred to PVDF (Millipore) membrane. The following antibodies were used for Western analysis: anti-FLAG (BioLegend); anti-OCT-1 anti-C/EBPβ, anti-C/EBPδ and anti-β-actin (Santa Cruz).

The concentrations of CXCL1, CXCL5 and IL-6 in cell supernatants were quantified using the FlowCytomix bead-based immunoassays (eBioscience).

Nuclear extract and DNA binding assay

Nuclear extracts were isolated from nearly confluent 15cm dishes using the Cellytic NuCLEAR Extraction kit from Sigma according to the manufactures instructions. The ability of C/EBPβ to bind DNA was measured using the TransAM C/EBP α/β kit from Active Motif according to the manufactures instructions.

Statistical analysis

All data were analyzed as paired samples using the Student’s t test to determine statistical significance.

Results

The response to IL-17 plus TNFα does not require CIKS to recruit TRAF6

We set out to explore why a mutant CIKS allele that is unable to fully mediate signaling by IL-17 cytokines might yet be associated with increased susceptibility to psoriasis, an inflammatory disease thought to critically involve IL-17. Since IL-17 is known to synergize with other inflammatory cytokines, notably TNFα, and since psoriatic inflammation also critically involves TNFα, we first examined the effect of the CIKS mutation on the signaling response to the combined action of both cytokines. To address this question we used primary, CIKS deficient mouse embryo fibroblasts (MEFs) that were reconstituted, via lentiviral transduction, with either wild-type (WT) CIKS or a mutant CIKS protein in which the region between amino acids 10–25 containing the TRAF6 binding site was deleted (CIKSΔT6); CIKS deficient (KO) cells were used a negative control. As shown previously by us and others, the CIKS mutant lacking the TRAF6 binding site was impaired in its ability to signal for activation of NF-κB in response to IL-17 and thus impaired in its ability to induce expression of TRAF6/NF-κB dependent genes (26, 27). Cells were stimulated with IL-17, TNFα or IL-17 + TNFα and gene expression was measured by real-time PCR after 1h and 6h. These time points were chosen so as to be able to survey both immediate-early genes, of which some are induced only transiently, as well as later-induced genes, some of which comprise a secondary response dependent on induced expression of immediate-early transcriptional factors. The results confirm that the interaction between TRAF6 and CIKS controls the IL-17-driven expression of a number of genes, as IL-17 stimulation of MEFs reconstituted with the CIKSΔT6 mutant resulted in a block or significantly reduced expression of all of the test genes shown when compared to stimulation of cells reconstituted with WT CIKS (Fig. 1A; Fig. 1B shows approximately equal expression of WT CIKS and CIKSΔT6 in transduced cells). As expected, CIKS KO MEFs showed no response to IL-17 and the induction of those genes responsive to TNFα alone was completely unaffected by the status of CIKS in these cells. Importantly, there were no significant differences in gene induction between MEFs reconstituted with WT CIKS or CIKSΔT6 after stimulation with both TNFα plus IL-17. This included genes for which the two cytokines together provided a very strong synergistic stimulus (such as Cxcl1, Cxcl5, and late induction of Saa3, Lcn2 and IL-6), but interestingly also genes that were primarily induced by IL-17 (with wild-type CIKS), but not by TNFα (Zc3h12a, Lcn2; additional genes below). We confirmed these results by measuring the protein levels of IL-6, CXCL1 and CXCL5 in supernatants after 6h or 24h of stimulation (Fig 1C). Together these results suggested that the response to the combined actions of TNFα and IL-17 was not impaired by the inability of the CIKS adaptor to recruit TRAF6 in response to IL-17. Such a result might be expected if the only role of IL-17 in this context was to stabilize TNFα-induced mRNAs as this function of IL-17 is independent of TRAF6 (33); Cxcl1, Cxcl2 and Cxcl5 are among the genes whose mRNAs are known to be stabilized by IL-17 (34, 35). However, that IL-17 must make other specific contributions beyond stabilizing mRNAs was revealed by the ability of TNFα to also compensate for the CIKSΔT6 defect in the induction of genes that responded only to IL-17, not TNFα. These findings suggested that the CIKSΔT6 mutant, which significantly impaired the genetic response to IL-17 alone, did not impair the combined response of TNFα + IL-17.

Figure 1.

Figure 1

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TRAF6 binding motif in CIKS is required for IL-17, but not IL-17 + TNFα signaling. CIKS deficient primary MEFs were reconstituted via lentiviral transduction with wild-type (WT) CIKS or the CIKSΔT6 mutant lacking amino acids 10–25. Non-transduced cells were used as a negative control (KO). A) Real-time PCR analyses for indicated genes with RNAs isolated from cells stimulated for 1h or 6h with IL-17, TNFα, IL-17 + TNFα or left unstimulated. Fold induction is in reference to unstimulated cells. B) Western blot analysis of cell lysate collected from a representative experiment used in (A) to show approximately equal expression levels of transduced FLAG tagged CIKS proteins, with β-actin serving as a loading control. C) Concentrations of proteins in supernatants of MEF cultures were measured after 6h (CXCL1) or 24h (IL-6 and CXCL5) of stimulation as indicated. Data are shown as the mean ± SEM for four independent experiments. *, p < 0.05; NS, not significant (p > 0.05); Student’s t-test.

IκBζ is an important component of the IL-17-induced response

To gain a more comprehensive answer to the question whether the CIKSΔT6-imposed defects in IL-17-induced, TRAF6-dependent signaling might be fully compensated by TNFα-initiated events, we began to focus on transcription factors that are induced by IL-17; such factors are involved in secondary gene induction and thus have an impact on the expression of many genes. If these factors were also fully induced by TNFα plus IL-17 in the presence of the CIKSΔT6 mutant, then it would be reasonable to suggest that the conclusions based on the more limited analyses shown in Fig. 1 are likely valid for the entire genetic response in cells carrying this mutant CIKS adaptor.

We first focused on the transcription factor IκBζ, because its IL-17-induced expression is significantly dependent on TRAF6/NF-κB (26) and because knockdown studies in human epithelial cell lines have shown a role for IκBζ in IL-17-induced expression of at least two genes, namely hBD and NGAL (36, 37). Most importantly, while this factor was induced by IL-17, peaking after about 1h, it was not induced by TNFα in these primary MEFs (Fig. 2A). Consequently induction of this factor and all secondary genes regulated by it should comprise part of the IL-17-specific contribution in the overall response to TNFα + IL-17 in these primary cells. To experimentally determine whether IκBζ is indeed functionally significant in the context of stimulation with both TNFα + IL-17 in our experimental system, we generated WT and IκBζ deficient primary MEFs and assessed gene induction at 1h and/or 6h after stimulation with IL-17, TNFα or IL-17 + TNFα; such an analysis has not been reported previously (Fig. 2B and C). As expected, loss of IκBζ had no impact on induction of various test genes by 1h, but led to a significant block in the IL-17 + TNFα induced synergistic expression of in particular IL-6 and Lcn2 by 6h, and there was a trend toward lower expression of Cxcl1 and Cxcl2 under these conditions.

Figure 2.

Figure 2

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IκBζ is rapidly induced by IL-17 and is essential for expression of a subset of genes induced at later times. A) Real-time PCR analyses of IκBζ expression in WT MEFs at indicated times following stimulation with IL-17, TNFα, IL-17 + TNFα. B, C) Real-time PCR analyses for indicated genes with RNAs isolated from WT and IκBζ deficient MEFs stimulated for 1h and 6h (B) or just 6h (C) with IL-17, TNFα, IL-17 + TNFα. Data are shown as the mean ± SEM for four independent experiments and fold induction is in reference to unstimulated cells; *, p < 0.05 Student’s t-test. D) Western analysis of WT and IκBζ deficient MEFs (KO) stimulated for 2h with IL-17, IL-17 + TNFα or cells left unstimulated. Cell lysates were analyzed for expression levels of C/EBPβ and C/EBPδ; β-Actin served as a loading control. Blot shown is representative of at least three independent experiments.

We also investigated the relevance of IκBζ in the regulation of C/EBPδ and C/EBPβ with our primary cells, as these C/EBPs have previously been suggested to play an important role in the response to IL-17 in certain fibroblasts cell lines and epithelial cells (38, 39). The induced expression of the C/EBPδ protein, which can be observed as early as 2 h after stimulation, was completely dependent on IκBζ in primary MEFs; on the other hand, induced expression of C/EBPβ was independent of this factor (Fig 2D). This is in line with previous work showing that C/ebpδ induction by LPS is dependent on IκBζ (40). C/EBPδ and C/EBPβ are the only inflammation-relevant C/EBPs expressed in fibroblasts. C/EBPβ is already present at basal levels prior to stimulation and the two protein bands represent the LAP and LAP* isoforms, both of which have transactivating activity and differ in size due to alternative in-frame translational initiation sites (41). Together these data highlight the importance of the IL-17-specific induction of IκBζ in the overall response to stimulation with both IL-17 + TNFα.

C/EBPs are induced and/or activated by IL-17 and important for the IL-17-specific response

As shown above and previously by others (39), the levels of both C/EBPδ and C/EBPβ were induced in response to IL-17, and in the case of C/EBPβ, this protein was already present at basal levels prior to stimulation. Both factors have previously been implicated in the genetic response to IL-17 signaling in cell lines as well (38, 39, 42). While C/EBPδ activity appears to be regulated exclusively via induced expression, C/EBPβ is a self-inhibited transcription factor that also requires post-translational modification for activation, specifically phosphorylation (41, 43). Beyond that, C/EBPβ expression levels were also increased above basal levels by both IL-17 and TNFα, and this was dependent on NF-κB/NEMO (similarly, induced expression of C/EBPδ was dependent on NF-κB/NEMO). Furthermore, nuclear levels of C/EBPβ rose in parallel with increased cellular levels (Fig. 3A, B); however, DNA binding activity of C/EBPβ appeared to be activated primarily by IL-17, not TNFα in these primary MEFs (Fig. 3C); this has also previously been suggested by analysis with a stromal cell line (39). These data further highlight unique contributions of IL-17 when compared to TNFα.

Figure 3.

Figure 3

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IL-17 induced activation of C/EBPs. A) Western blot analysis of C/EBPβ and C/EBPδ expression in whole cell lysates isolated from WT and NEMO deficient MEFs (KO) after 2h stimulation with cytokines as indicated. Control is unstimulated cells; β-actin served as a loading control. B) Western blot analysis for expression levels of C/EBPβ in nuclear extract isolated from WT MEFs subjected to stimulation with cytokines for 2h as shown. Oct1 served as loading control. All Western blots were repeated 3 times and representative blots are shown. C) C/EBPβ DNA binding assay with nuclear extracts used in (B). Data are shown as the mean ± SEM for five independent experiments and fold induction is in reference to unstimulated cells; *, p < 0.05 Student’s t-test.

To assess the contributions of C/EBPs in the response to IL-17 + TNFα we generated primary MEFs lacking C/EBPβ, C/EBPδ or both factors; we then stimulated these cells with IL-17, TNFα or IL-17 + TNFα to measure induced expression of various genes over time, an analysis with primary cells which extends prior, more limited studies with cell lines (38) (Fig. 4A). C/EBPβ appeared to have a partial role in the immediate-early response to IL-17 or IL-17 + TNFα, as loss of C/EBPβ alone (or loss of both C/EBPs) trended towards or caused a significant, albeit partial reduction in the induced expression of Iκbζ and Zc3h12a. As expected, loss of C/EBPδ alone had little or no effect on these immediate-early events, which are presumably mediated by IL-17-induced activation of basal C/EBPβ protein levels. C/EBPs appeared to have no role in the induced expression of Cxcl1, Cxcl2 and Cxcl5 (measured at 1 and 6 h of stimulation), but C/EBPs were critical for the IL-17 + TNFα induced expression of Lcn2, Saa3 and IL-6 after 6 h of stimulation, as loss of both C/EBPβ and C/EBPδ largely abrogated expression of these genes at that time; loss of C/EBPδ alone had little effect, while loss of C/EBPβ trended towards or resulted in a partial reduction in the case of Saa3 and IL-6, respectively. Therefore the functions of the two C/EBP factors in the induced expression of these three later-induced genes were largely, though not completely redundant. Together these data highlight the importance of IL-17-induced expression/activation of C/EBPs in the overall response to IL-17 + TNFα in our primary MEF cultures.

Figure 4.

Figure 4

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C/EBPs are essential for a subset of IL-17-induced genes. A) Real-time PCR analyses for indicated genes with RNAs isolated from WT, C/EBPβ deficient (βKO), C/EBPδ deficient (δKO) and C/EBPβδ doubly deficient (βδKO) MEFs stimulated for 1h or 6h with IL-17, TNFα, IL-17 + TNFα. Data are shown as the mean ± SEM of four independent experiments and fold induction is in reference to unstimulated cells; *, p < 0.05 Student’s t-test. B) Western blot analysis of cell lysate collected from a representative experiment shown in (A) after 2h of IL-17 stimulation to confirm absence or show expression levels of C/EBPβ and C/EBPδ; β-actin served as a loading control.

We also examined C/EBPβ and C/EBPδ levels in wild-type MEFs and the various C/EBP deficient MEFs before and after 2 h of stimulation with IL-17 (Fig. 4B). The data confirm the loss of one or the other or of both C/EBPs in the corresponding knockout MEFs; in addition they show an apparently higher level of induced C/EBPδ in the C/EBPβ knockout MEFs, which may represent a compensatory effect.

TNFα compensates for defective IL-17-specific induction/activation of Iκbζ and C/EBP in cells with CIKS lacking the TRAF6 recruitment domain

The results above indicate that the transcription factors IκBζ, C/EBPβ and C/EBPδ are important not only in the overall response to IL-17 alone, but also in the response to IL-17 + TNFα. Furthermore, the results show that the induced activity of these particular transcription factors is largely due to IL-17, not to TNFα. We thus wanted to determine whether the IL-17-induced expression of these factors was impaired in cells carrying the CIKSΔT6 mutant, and if so, whether TNFα would be able to compensate for this defect even though TNFα alone could not induce either IκBζ or C/EBPδ or further activate C/EBPβ (it was able to increase levels of C/EBPβ). If so, this would allow us to extrapolate findings in Fig. 1 to suggest that the CIKSΔT6 mutant does not appear to impair the overall response to IL-17 plus TNFα. Loss of the TRAF6 recruitment domain in CIKS significantly impaired IL-17-induced expression of Iκbζ (Fig. 5A) and abolished IL-17-induced expression of C/EBPδ (which is dependent on IκBζ (40)) (Fig. 5B). By contrast, cells carrying the CIKSΔT6 mutant showed no significant reductions in the IL-17 + TNFα induced expression of Iκbζ and C/EBPδ or the enhanced induction of C/EBPβ. Therefore one would not expect this mutant CIKS protein to impair downstream effects of these transcription factors in the context of IL-17 + TNFα stimulation, as already evidenced by those test genes in Fig. 1 for which induction was dependent on these early-induced transcription factors. Based on these novel findings, we thus conclude that the mutant CIKSΔT6 adaptor is capable of transmitting IL-17-specific signals that are not activated downstream of TNFα alone, signals that are important for the overall genetic response to the combined action of both cytokines; furthermore, TNFα signals can compensate for the inability of the mutant CIKSΔT6 adaptor to transmit TRAF6-dependent signals downstream of IL-17 alone, required to induce many of the IL-17 target genes.

Figure 5.

Figure 5

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Co-stimulation with TNFα compensates for lack of TRAF6 mediated IL-17 signaling in expression of critical IL-17-specific genes. CIKS deficient primary MEFs were reconstituted via lentiviral transduction with wild-type CIKS or the CIKSΔT6 mutant. Non transduced cells were used as a negative control (KO). Cells were stimulated with IL-17, TNFα, IL-17 + TNFα. A) Real-time PCR analysis for IκBζ with RNAs isolated from cells stimulated for 1h. Data are shown as the mean ± SEM for four independent experiments and fold induction is in reference to unstimulated cells; *, p < 0.05 Student’s t-test. B) Western blot analysis for expression of C/EBPβ, C/EBPδ and FLAG tagged CIKS in cell lysate collected after 2h stimulation, with β-actin serving as a loading control. The blot is representative of 3 independent experiments.

The response to IL-17 plus TNFα is not impaired in primary keratinocytes or primary dermal fibroblasts carrying the CIKSΔT6 mutant adaptor

Psoriasis is an inflammatory skin disease also characterized by aberrant keratinocyte functions and differentiation (14–17). Because keratinocytes are known targets of IL-17 cytokines and because these cells might be wired differently for signaling by these cytokines than MEFs, we desired to determine whether primary keratinocytes behaved similar to primary MEFs when carrying the CIKSΔT6 mutant adaptor. In addition to epidermal keratinocytes, we also wanted to investigate signaling in dermal fibroblasts, because these cells are likely targets of IL-17 cytokines as well, given that the dermis harbors γδ T cells, a major source of IL-17 cytokines in mouse psoriasis models (44). To carry out these investigations we isolated primary keratinocytes from the epidermis and fibroblast from the dermis of the skins of one-day old CIKS deficient mice and then reconstituted these cell types with either WT CIKS or the CIKSΔT6 mutant via lentiviral transduction, prior to stimulating these cells with cytokines. Primary keratinocytes carrying the CIKSΔT6 mutant showed significantly impaired expression of some key target genes in response to IL-17 alone when compared to cells carrying the WT CIKS protein; by contrast, there was no difference after stimulation with both TNFα and IL-17 (Fig. 6A, B). The target genes included Lcn2, which exhibited strong synergistic induction by these two cytokines, as well as Iκbζ, for which TNFα merely corrected for the partial defect imposed by CIKSΔT6 in the IL-17-only mediated response. We also tested for induction of target genes in dermal fibroblasts reconstituted with either the WT or the CIKSΔT6 adaptor and again found that none of the responses were impaired when both cytokines were present (Fig. 6C, D), while IL-17 only signaling was impaired (Fig. 6C). Therefore the conclusions we were able to draw from the more extensive analysis of primary MEFs also appear to hold true for primary keratinocytes and primary dermal fibroblasts. In all of these cells the CIKSΔT6 mutant only impaired responses to signaling by IL-17 cytokines alone, but did not impair responses to the combined action of IL-17 plus TNFα.

Figure 6.

Figure 6

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The CIKSΔT6 mutant only impairs IL-17− but not IL-17+TNFα-induced target gene expression in primary keratinocytes and primary dermal fibroblasts. CIKS KO primary epidermal keratinocytes (A, B) and primary dermal fibroblasts (C, D), reconstituted with WT CIKS, the CIKSΔT6 mutant or left untransduced (KO) were stimulated with IL-17, TNFα, IL-17 + TNFα or left unstimulated for 2h (A, C) or 24h (B, D) before harvesting cells and real-time PCR analyses for induced target gene expression, as indicated. Data are shown as the mean ± SEM for at least four independent experiments and fold induction is in reference to unstimulated cells; *, p < 0.05; NS, not significant; Students t-test.

Discussion

Here we have shown that a mutant CIKS protein lacking the N-terminal TRAF6 binding domain significantly impaired IL-17-induced expression of target genes, but had no impact on the response to IL-17 + TNFα. This was the case even though the genetic response to IL-17 + TNFα included genes that were induced by IL-17, but not TNFα; furthermore these genes were induced by IL-17 in manner dependent on the TRAF6 recruitment domain of CIKS. Importantly, among the proteins encoded by these latter genes were transcription factors such as IκBζ and C/EBPδ, which in turn are involved in the expression of later-induced genes in response to both IL-17 and IL-17 + TNFα. Therefore our results may be extrapolated to suggest that the overall genetic response to IL-17 + TNFα appears to be largely unaffected by loss of the TRAF6 recruitment domain in CIKS. Consequently such a mutant CIKS adaptor would not be expected to interfere with IL-17-mediated contributions in an inflammatory context, such as in psoriasis, where TNFα is also present and critical for disease pathogenesis (28, 29). In addition to IL-17(A), IL-17F and IL-17C may contribute to psoriasis as well (5, 6, 22, 45, 46), and the results described here for IL-17 signaling via the mutant CIKS adaptor are likely to apply to these cytokines as well.

While the bulk of these studies were conducted with primary mouse embryo fibroblasts, we also confirmed key findings with primary keratinocytes and primary dermal fibroblasts. Both cell types are targets of IL-17 cytokines in skin, and aberrant functions along with disturbed differentiation/proliferation of keratinocytes are prominent features of psoriatic lesions in both human patients and in mouse models of this disease (16, 19, 22, 47). Together our findings provide strong evidence for why a mutant CIKS adaptor unable to recruit TRAF6 should not impair inflammatory responses downstream of IL-17 cytokines in the context of psoriasis, contrary to expectations.

How might TNFα signals compensate for the lack of TRAF6 recruitment in mediating IL-17-specific responses? Recruitment of TRAF6 to CIKS is required for IL-17-induced NF-κB activation, which plays an important role in the overall genetic response to IL-17. TRAF6 may also be involved, via TAK1, in the activation of MAP kinases by IL-17, although little is presently known about how this cytokine activates various MAP kinases. While ERK activation appears to be largely independent of TRAF6, activation of JNK appears to be dependent (26, 27, 48). TNFα is a very potent activator of NF-κB (49) and this is likely an important mechanism by which TNFα compensates for CIKS-mutant dependent defects in IL-17 signaling. Since our data suggest that TNFα can fully compensate for impaired IL-17 signaling imposed by this CIKS mutant, TNFα must be able to compensate for all TRAF6-mediated activities, even though TNFα itself does not signal via TRAF6, but instead signals via TRAF2 and TRAF5 (49). The precise and full details of the compensatory mechanisms will require further study.

How might TNFα and IL-17 cytokines synergize to induce expression of certain target genes and why might this be preserved in the presence of the CIKSΔT6 mutant? One mechanism likely involves IL-17-induced mRNA stabilization, which is independent of TRAF6 (33, 50, 51). Transcription of a given gene might be strongly induced by TNFα, but only weakly by IL-17, but its mRNA might be very unstable unless stabilized by IL-17; in this scenario the two cytokines would synergize even in the presence of the CIKSΔT6 mutant. Another mechanism likely involves synergistic interactions of IL-17-induced transcription factors such IκBζ with transcription factors activated by TNFα. Since TNFα also corrects for the partial defect in IL-17-induced, CIKSΔT6-mediated expression of IκBζ, synergy would be preserved in the presence of this mutant adaptor.

Our results clearly show that the combined action of IL-17 and TNFα is not impaired by the inability of the CIKS mutant to recruit TRAF6, so IL-17 should continue to drive psoriatic inflammation in patients carrying this mutation. An important remaining question however is whether this CIKS mutant might in fact compromise epithelial skin barriers under homeostatic, non-inflammatory conditions, conditions in which TNFα is not expressed. If barrier functions were compromised this may lead to heightened responses to insults, for example by allowing pathogens or their products to more easily penetrate skin, thereby causing inflammation. In such an inflammatory context IL-17 cytokines would then once again be able to fully contribute to disease pathogenesis, because this particular mutant CIKS adaptor does not interfere with the responses to IL-17 + TNFα, as shown here. Some prior reports have indeed suggested a role for IL-17 in maintaining barrier functions under homeostatic conditions (1, 52, 53). While TNFα is presumably not expressed in homeostatic, non-inflammatory conditions, there is evidence that IL-17 cytokines might be produced, albeit at low levels. Dermal γδT cells are poised to produce large amounts of IL-17 under inflammatory conditions (47), but a recent report suggests that they already constitutively express low levels of both IL-17(A) and IL-17F (44). A similar situation may apply to IL-17C, which is reportedly produced by keratinocytes in a mouse psoriasis model (5, 6), but which might also be expressed at low levels under non-inflammatory conditions. Low levels of these IL-17 cytokines could be envisioned to help induce sufficient levels of anti-microbial peptides to keep pathogens and even commensals at bay; low levels of IL-17 cytokines may also to induce sufficient levels of chemokines to attract cell types with anti-microbial activity or with the ability to insulate skin from various other insults. It is these activities of IL-17 cytokines that we suspect of being compromised in host skin carrying a mutant CIKS adaptor lacking the ability to recruit TRAF6. To fully prove our hypothesis in vivo will require the generation of mice expressing the CIKSΔT6 mutant in place of the wild-type gene; such mutant mice could then be tested for impaired barrier functions as well as for their ability to still develop full psoriatic phenotypes in appropriate animal models of psoriasis.

It is important to note that the genome-wide association studies showed that the CIKS susceptibility allele carrying a mutation in the TRAF6 recruitment domain is often found in a homozygous state in patients, albeit not always (23–25). Whether such a mutant might have similar functional consequences in the heterozygous state will need to be addressed in future studies. It is of course possible that patients carrying just one such mutant allele could carry additional genomic changes that predispose to psoriasis.

In sum, our data provide a possible explanation for how a mutant CIKS allele impaired in TRAF6 recruitment may promote and sustain psoriasis, contrary to initial expectations. Such a mutant would be fully capable of transmitting potent IL-17-mediated signals in an inflammatory context with TNFα, as shown here. At the same time we conjecture that such a mutant CIKS adaptor might actually increase the risk of inflammation. We hypothesize that this mutant may impair low-level IL-17 signaling under homeostatic conditions, thereby weakening barrier functions; this in turn might increase entry of microbes or microbial products and thus lead to inflammation. Our work also suggests that treatment of psoriasis with a combination of drugs that suppress both IL-17 and TNFα signaling might be particularly beneficial, with doses that may be suboptimal if given individually.

Acknowledgements

We thank Dr. Manolis Pasparakis for the gift of NEMO deficient MEFs, Dr. Shizuo Akira for the gift of IκBζ deficient mice, Nancy Martin for generating C/EBPβδ double deficient MEFs, Angie Hackley and Karen Saylor for animal technical support, Dr. Estefania Claudio for assistance with animal experiments and Dr. Francesca Mascia for technical and material help during isolation and culturing of primary skin cells from newborn mice. We are grateful to Dr. Anthony S. Fauci for continued support.

This research was supported by the Intramural Research Program of NIAID, NIH, and in part by the Intramural Research Program of the NCI, Center for Cancer Research, NIH.

Abbreviations

CIKS

Connection to IκB Kinase and Stress-activated protein kinases

TRAF6

TNF receptor associated factor 6

MEF

Mouse embryo fibroblast

C/EBP

CCAAT/Enhancer binding protein

NEMO

NF-κB essential modulator

SNP

Small nucleotide polymorphism modulator

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