Epidermal RAF prevents allergic skin disease

The RAS pathway is central to epidermal homeostasis, and its activation in tumors or in Rasopathies correlates with hyperproliferation. Downstream of RAS, RAF kinases are actionable targets regulating keratinocyte turnover; however, chemical RAF inhibitors paradoxically activate the pathway, promoting epidermal proliferation. We generated mice with compound epidermis-restricted BRAF/RAF1 ablation. In these animals, transient barrier defects and production of chemokines and Th2-type cytokines by keratinocytes cause a disease akin to human atopic dermatitis, characterized by IgE responses and local and systemic inflammation. Mechanistically, BRAF and RAF1 operate independently to balance MAPK signaling: BRAF promotes ERK activation, while RAF1 dims stress kinase activation. In vivo, JNK inhibition prevents disease onset, while MEK/ERK inhibition in mice lacking epidermal RAF1 phenocopies it. These results support a primary role of keratinocytes in the pathogenesis of atopic dermatitis, and the animals lacking BRAF and RAF1 in the epidermis represent a useful model for this disease. DOI: http://dx.doi.org/10.7554/eLife.14012.001


Introduction
The largest organ of the body, the skin, allows exchange with the environment while shielding the organism from insults of mechanical, chemical, and infectious nature. In the skin, the epidermis acts as a mechanical barrier which prevents water loss and the entry of potentially harmful chemicals; in addition, through the interplay between keratinocytes in the epidermis and immune cells based mainly in the underlying dermis, the skin works as an immunological barrier actively defending the body from pathogens (Pasparakis et al., 2014;Nestle et al., 2009). Maintaining these barrier functions throughout life requires continuous regeneration of the epidermis and appropriately balanced immune responses. Dysregulation of the regenerative process can lead to a wide range of defects, from epidermal thickening to malignancies; and an imbalance in the skin's immune reactions can lead to recurrent infections, or inflammatory/allergic diseases such as psoriasis or atopic dermatitis.
The RAF/MEK/ERK signaling pathway plays an essential role in the epidermis. Downstream of the EGFR and RAS, it acts in hair follicle development and wound healing; its mild activation in Rasopathies, genetic diseases caused by activating mutations in the pathway, results in skin phenotypes ranging from thickening of palms and soles to the development of papillomas; and strong activation in keratinocytes results in tumorigenesis (Kern et al., 2011;Ratushny et al., 2012). In animal models, epidermis-restricted inducible activation of RAF or MEK causes massive cutaneous hyperplasia and reduced differentiation (Khavari and Rinn, 2007). Consistently, RAF1 (also known as CRAF) and BRAF are essential for the development and progression of RAS-induced tumors although they fulfil different roles, with RAF1 preventing keratinocyte differentiation (Ehrenreiter et al., 2009) and BRAF promoting proliferation through MEK/ERK activation . The requirement for MEK/ERK in epidermal proliferation has been independently assessed (Khavari and Rinn, 2007;Dumesic et al., 2009;. In line with their prominent roles in cell proliferation and tumorigenesis in a number of tissues, the components of the EGFR/RAS/RAF/MEK/ERK pathway are attractive targets for molecule-based therapy. Many inhibitory compounds have been developed that are currently in clinical trials or have reached the clinic. Reflecting the function of the pathway in skin, cutaneous toxicities are one of the main adverse effects of these therapies; they can be severe and lead to an interruption of the therapy or to its termination (Belum et al., 2013;Curry et al., 2014;Dy and Adjei, 2013). These adverse effects can be roughly classified as inflammatory reactions, elicited chiefly by blocking EGFR or MEK; and proliferative events, caused by multikinase inhibitors such as sorafenib or by more specific RAF inhibitors (vemurafenib, dabrafenib). Cutaneous inflammation induced by agents blocking EGFR (erlotinib or cetuximab) and by MEK inhibitors (selumetinib and trametinib) is characterized by papulopustular rash, pruritus, and suppurative folliculitis in !80% of the patients (Curry et al., 2014). The effects of erlotinib and cetuximab are on-target, as recently demonstrated by two studies showing similar inflammatory phenotypes in mice with epidermis-restricted EGFR ablation (Mascia et al., 2013;Lichtenberger et al., 2013).
Skin rashes and pruritus are also observed in patients treated with inhibitors targeting RAF. Up to half of the cutaneous reactions induced by these compounds, however, consist of anomalous epidermal proliferation events ranging from different forms of keratosis to the development of druginduced papillomas, keratoacanthomas, and squamous cell carcinomas (4-30% depending on the study and the inhibitors used) (Anforth et al., 2013). These side effects are mechanism-based and eLife digest The skin is the largest organ of the body and shields it from damage. It is also home to cells of the immune system, which protect the body from infections. To maintain its role as a barrier, the skin regenerates throughout life, constantly producing new cells to replace the old ones. If this process goes wrong and the skin regenerates too much, the skin may produce tumors. Likewise, disrupting the immune barrier can lead to autoimmune or allergic skin diseases such as psoriasis or atopic dermatitis (also known as eczema).
The outer layer of the skin is called the epidermis, and is made up of cells known as keratinocytes. A family of proteins called RAF plays an important role in controlling how keratinocytes behave. These proteins are part of a signaling pathway that controls the production of new cells and is disrupted in skin tumors. Therapies that inhibit RAF are effective treatments for these tumors but have side effects that can affect the skin so severely that the treatment must be interrupted.
Keratinocytes also play a role in the development of allergic skin diseases. However, it is not known whether they do so by triggering the disease themselves, or by responding to stimuli produced by immune cells.
Raguz et al. investigated what would happen if RAF proteins were removed from keratinocytes in the epidermis of mice. This caused the mice to develop an allergic disease similar to human atopic dermatitis. This was an unexpected effect and different from the side effects caused by drugs that inhibit RAF proteins. By analyzing the signaling pathway that RAF is part of, Raguz et al. discovered that removing RAF from the epidermis reduces the pathway's ability to prevent excessive stress signals being sent throughout the skin. Under these conditions, the keratinocytes bring about inflammation and allergy by activating the immune cells in the skin and elsewhere.
Overall, the results presented by Raguz et al. indicate that allergic dermatitis can be triggered by defects in keratinocytes rather than in immune cells. Furthermore, RAF in the epidermis appears to prevent allergic skin diseases. Future studies could use mice that lack RAF in their epidermis to further understand atopic dermatitis and to investigate the way in which drugs that target RAF can damage the skin. rely on the paradoxical activation of MEK/ERK promoted by the inhibitors in cultured cells, animal models, and patients (Samatar and Poulikakos, 2014;Holderfield et al., 2014). Consistent with this, combination treatment (RAF plus MEK inhibitors) reduces cutaneous toxicity (Flaherty et al., 2012;Mattei et al., 2013).
In contrast to RAF inhibitor treatment, inducible epidermis-restricted ablation of BRAF and RAF1 causes the rapid regression of RAS-driven tumors without apparent cutaneous toxicity . Here, we systematically test the effect of compound BRAF/RAF1 ablation in epidermis and show that it gives rise to a progressive disease strongly resembling human atopic dermatitis. Mechanistically, the disease relies on the combination of reduced ERK and increased stress kinase activation, leading to chemokine/cytokine overproduction and chronic, systemic inflammation.

Results
Epidermal BRAF and RAF1 are essential to prevent local and systemic inflammation BRAF and RAF1 were ablated in the epidermis by introducing the Krt5-Cre transgene (Tarutani et al., 1997) into a Braf f/f ; Raf1 f/f background  ( Figure 1A). The mice (heretofore referred to as D/Dep2) were born at Mendelian ratio but with their eyes open ( Figure 1B), probably as a result of the migration defects of RAF1-deficient keratinocytes (Ehrenreiter et al., 2005). With time, however, they started to show symptoms of a progressive skin disease. These included intense itching and scratching resulting in partial alopecia and self-inflicted wounds ( Figure 1B). Histological examination of the non-affected areas revealed thickening of the epidermis correlated with increased proliferation and expansion of both the basal and suprabasal keratinocytes but not with keratinocyte apoptosis ( Figure 1C and In line with the dermal inflammatory reaction, D/Dep2 epidermis was characterized by the robust expression of the keratinocyte activation marker K6, of the cell adhesion molecule ICAM1, and by the sporadic expression of MHC class II molecules ( Figure 1E), all upregulated in inflammatory conditions including atopic dermatitis (Freedberg et al., 2001;Fan et al., 2003;Caughman et al., 1992). Consistent with this, D/Dep2 epidermal lysates enriched in K5/K10-positive keratinocytes  contained increased amounts of the cytokine TSLP, associated with skin allergic disorders (Ziegler et al., 2013) (Figure 1F), as well as of other chemokines and cytokines, with CCL7, IL18, IL5 and IL13 levels significantly higher than controls ( Figure 1G).
Epidermal ablation of BRAF and RAF1 had profound systemic effects. The mice failed to thrive ( Figure 2A) and presented with enlarged spleens and lymph nodes. The splenomegaly could largely be attributed to increased numbers of Mac1+Gr1+ cells ( Figure 2B), a cell type found both in the spleen of a mouse model of FITC contact hypersensitivity/S. aureus infection and in the blood and skin infiltrates of atopic dermatitis patients (Skabytska et al., 2014). The lymph nodes contained elevated numbers of activated T, B, and dendritic cells ( Figure 2C). In the blood, we observed leukocytosis and increased amounts of the chemokines CCL2 and CCL7, as well as of GCSF; in addition, serum IgEs were elevated in D/Dep2 mice ( Figure 2D). Thus, the phenotype of adult D/Dep2 mice resembled a skin-specific allergic disease.
To determine whether the systemic phenotype was secondary to the severe skin inflammation observed in adult animals, we examined D/Dep2 mice at weaning (3 weeks of age), at which stage they did not groom more than control littermates nor showed any signs of skin rash, except a mild eyelid irritation (Figure 3-figure supplement 1A). Skin architecture and keratinocyte proliferation were not altered at this stage ( Figure 3-figure supplement 1A). In terms of dermal infiltrate, a 2fold increase in mast cells could already be observed, but these cells were not activated ( Figure 3A and  only a trend could be observed for others ( Figure 3B and Figure 3-figure supplement 1C). Systemically, enlarged lymph nodes contained increased amounts of activated T, B, and dendritic cells, while the spleen was normal both in size and cell composition ( Figure 3C and Figure 3-figure supplement 1D). In the blood, the number of monocytes and granulocytes was increased, as were the levels of CCL2, CCL7, and GCSF. However, no significant difference in serum IgEs could be observed at this point ( Figure 3D). The increase in T, B, and dendritic cells in lymph nodes and the granulocytosis could be traced as far back as 10 days of age, at which time point GCSF was the only cytokine elevated in the serum of D/Dep2 mice ( Figure 3E-F). Thus, ablation of both Braf and Raf1 in mouse epidermis does not directly disturb skin architecture but rather affects the cross-talk between keratinocytes and the innate and adaptive immune system.

Lack of epidermal BRAF and RAF1 causes transient inside-out barrier defects
Inflammatory skin conditions often involve impaired skin barrier function. D/Dep2 embryos performed normally in an outside-in dye penetration assay, indicating that stratum corneum permeability was not affected ( Figure 4A). We next monitored the weight of E18.5 embryos of different  immunoblotting and quantified by Image J. ACTB served as a loading control. The results were normalized by arbitrarily setting one of the F/F2 samples as 1 and plotted as mean ± SEM. Data represent mean ± SEM. p = 0.011, p1 = 0.001, p2 = 0.007, p3 = 0.001, p4 = 3.06E-6, p5 = 1.37E-4, p6 = 0.002, p7 = 0.049, p8 = 0.042, p9 = 0.046 and p10 = 0.001. DOI: 10.7554/eLife.14012.003 The following figure supplement is available for figure 1:  The body weight of D/Dep2 mice is significantly reduced compared to their littermates (n = 5). The data was analyzed by two-way analysis of variance (ANOVA) test. (B) Increased spleen/body weight ratio and increased numbers of splenic Mac1 + Gr1 + cells in adult D/Dep2 animals (n = 4-5). (C) Enlarged lymph nodes and and activated T, B, and dendritic cells in adult D/Dep2 (n = 4-8). T cells (CD4 + or CD8 + ) and B cells (B220 + ) activation was determined by costaining with CD69; activated dendritic cells were identified as CD11c + and MHC II hi or CD80 + . (D) Circulating blood cells and plasma levels of chemokines and IgE antibodies in adult mice (n = 6-8). Data represent mean ± SEM. p1 = 0.0002, p2 = 0.023, p3 = 0.002, p4 = 0.002, p5 = 9.83E-8, p6 = 6.31E-7, p7 = 2.91E-7, p8 = 0.004, p9 = 0.001, p10 = 0.008, p11 = 0.011, p12 = 0.046, p13 = 0.050, p14 = 3.65E-4 and p15 = 0.001. DOI: 10.7554/eLife.14012.005 genotypes for 9 hr after birth as a proxy for transepidermal water loss and thus for the barrier function of tight junctions. D/Dep2 embryos lost three-fold the weight of littermate controls ( Figure 4B). Molecularly, reduced expression of E-cadherin (CDH1), an adherens junction protein known to regulate tight junctions (Tunggal et al., 2005), and of the tight junction protein claudin 1 (CLDN1), which is crucial for the maintenance of the inside-outside barrier (Furuse et al., 2002) and has been found downregulated in atopic dermatitis patients (De Benedetto et al., 2011) was still evident at P3 in D/ Dep2 epidermis, while occludin expression was normal (OCLN) ( Figure 4C). The architecture of P3 D/Dep2 skin was indistinguishable from that of controls (Figure 4-figure supplement 1A); P3 epidermal lysates contained highly variable amounts of chemokines and cytokines, predominantly CCL2, IL6 and IL18. All were increased in the D/Dep2 epidermal lysates, albeit not significantly . The E-cadherin and claudin 1 downregulation was transient and was no longer detectable in 3 weeks old D/Dep2 mice ( Figure 4D). Single deletion of RAF1 (RAF1D/Dep) and BRAF (BRAFD/Dep) did not lead to increased body weight loss in E18.5 embryos; consistently,  Results are displayed as percentage of initial weight (n = 41 for F/F2 and n = 11 for D/Dep2). The data was analyzed by two-way analysis of variance (ANOVA) test. (C, D) Immunoblot analysis of CDH1, CLDN1 and OCLN expression in epidermal lysates of 3 days old (C, n = 3; quantification shown in the plot, performed as in Figure 1F) or 3 weeks old D/Dep2 animals. TUBA and GAPDH are shown as loading controls. Data represent mean ± SEM. p1 = 0.0001, p2 = 0.028, p3 = 0.020. DOI: 10.7554/eLife.14012.008 The following figure supplement is available for figure 4: To assess the relevance of the transient perinatal barrier defect of D/Dep2 mice, we deleted Braf and Raf1 in 3 weeks old mice using tamoxifen-inducible Krt5-Cre (Indra et al., 1999). These animals, termed D/Dep2 TX , showed conversion of both F to D alleles and strongly reduced expression of BRAF and RAF1 proteins in tail tissue ( Figure 5A). D/Dep2 TX mice developed a milder disease than the D/Dep2 mice, characterized by much slower kinetics (8 months between ablation and overt symptoms), moderate keratinocyte hyperproliferation (assessed as increased epidermal thickness) and activation (as determined by K6 and ICAM1 expression), as well as by a modest increase in activated mast cells and granulocytes in the dermis ( Figure 5B). At the systemic level, we observed mild splenomegaly with an increase in Mac1+Gr1+ cells ( Figure 5C) as well as enlarged lymph nodes containing activated lymphocytes and dendritic cells ( Figure 5D and Figure 5-figure supplement 1). Increased numbers of lymphocytes and granulocytes were found in the blood, while IgE levels were comparable to controls ( Figure 5E). Thus, circumventing the transient barrier defect of the D/Dep2 animals postponed and attenuated the clinical manifestation of the disease.

Increased JNK and reduced ERK activation in D/Dep2 epidermis and cells
To gain insight in the molecular correlates of the phenotype, we analyzed MAPK signaling in the epidermis. We observed decreased activation of ERK and increased activation of JNK, assessed by the phosphorylation of both MAPK and their respective downstream targets pRSK and pJUN, in nonlesional epidermis of adult D/Dep2 and D/Dep2 TX mice; in contrast, phosphorylation of the p38 target MAPKAPK2 was not affected ( Figure 6A and B). A more detailed analysis of 3 weeks old animals confirmed low ERK activation and increased phosphorylation of JNK (and, to a lower degree, p38) in D/Dep2 epidermal lysates ( Figure 6C). Consistent with this phosphorylation pattern, the levels of the dual specificity phosphatase DUSP1 and DUSP10, negative regulators of the stress kinases (Patterson et al., 2009), were low in the D/Dep2 lysates. BRAFDep lysates were characterized by reduced ERK phosphorylation only, while RAF1Dep lysates showed increased phosphorylation of all three MAPK. The expression of ICAM1 as marker of inflammation was detected only in the D/Dep2 lysates, leading to the hypothesis that the combination of high JNK, low ERK activation is at the basis of the inflammatory phenotype of D/Dep2 animals ( Figure 6C).

Inhibition of ERK activation induces inflammatory skin disease in RAF1Dep mice
We tested this hypothesis by treating 6 weeks old RAF1Dep animals with a MEK inhibitor (MEKi; trametinib, in clinical use; daily by gavage for 32 days) to decrease ERK activation and determine whether this would phenocopy the D/Dep2 disease. MEKi efficiently reduced ERK phosphorylation and slightly increased JNK phosphorylation in both control and RAF1Dep epidermal lysates, but increased p38 phosphorylation was only observed in the RAF1Dep ( Figure 7A). Consistently, MEKi reduced the expression of the phosphatases DUSP1 and 10 and of CLDN1 in RAF1Dep epidermis only ( Figure 7A). Within a month, the RAF1Dep animals developed an inflammatory skin disease characterized by K6 and ICAM1 expression in the epidermis, by the presence of activated dermal mast cells and by an increase in TSLP ( Figure 7B). In control animals, inhibitor treatment did not affect, or reduced chemo-or cytokine amounts; GMCSF, IL5, IL2 and CCL4 were reduced significantly, and a trend could be observed for IL4, IL17, IL27, and CXCL1. In contrast, a comparison between inhibitor-treated RAF1F/F and RAF1Dep lysates revealed a significant upregulation of CCL2, GMCSF, IL18, IL5, IL13, IL2, IL17 and CCL4 ( Figure 7C and Figure 7-figure supplement 1). This correlated with increased Mac1+Gr1+ cells in the spleen and activated T, B, and dendritic cells in the lymph nodes ( Figure 7D) as well as with increased circulating IgEs and granulocytosis ( Figure 7E). Thus, inhibiting ERK in the RAF1Dep animals recreates the immunological environment necessary to bring about a disease very similar to that observed in D/Dep2 mice. The inflammatory skin disease of D/Dep2 mice can be prevented by JNK inhibition but not by MyD88, TNF or caspase 1/11 ablation Collectively, the data above suggest that JNK activation was responsible for the inflammatory skin phenotype. To assess whether this was the case in vivo, we treated D/Dep2 animals with the specific peptide inhibitor D-JNKI1. D-JNKI1 efficiently blocked JUN phosphorylation, increased ERK and RSK phosphorylation ( Figure 8A), and prevented the development of the disease as determined by ICAM1 and TSLP expression in the epidermis as well as by eyelid inflammation and mast cells accumulation in the dermis ( Figure 8A-B). Consistent with the data in Figure 3-figure supplement 1B, K6 expression could not be observed in the interfollicular epidermis in any of the experimental groups (Figure 8-figure supplement 1A). Compared to untreated 3 weeks old animals, vehicle (TAT-peptide) alone had minor effects on the level of some chemokines and cytokines; the increase in IL17 and IL18 in TAT-peptide-treated D/Dep2 vs F/F2 animals became significant; the opposite was observed for CCL7, IL13 and IL4 (compare Figures 3B and 8C; and . Treatment with D-JNKI1 reduced chemo-and cytokines in epidermal lysates of D/Dep2 animals to levels indistinguishable from, or lower than (CCL2, CCL4, GMCSF and IL4), those of F/F2 controls. Exceptions were IL6, which was significantly reduced in D/Dep2, but not in F/F2 epidermal lysates; and IL5, which was not affected by the inhibitor in either genotype ( Figure 8C and Figure 8-figure supplement 1B). At the systemic level, D-JNKI1 normalized the numbers of activated B and dendritic cells in lymph nodes, while the numbers of activated T cells where reduced in both F/F2 and D/Dep2 lymph nodes ( Figure 8D). Thus, JNK activation and the resulting chemo-and cytokine accumulation in the epidermis are required for disease development.
To determine whether the increased JNK activation observed in the D/Dep2 animals was due to a specific signal, we mated them to MyD88 knockout animals (Adachi et al., 1998), to block both TLR (with the exception of TLR3) and IL1 signaling (Janssens and Beyaert, 2002). We also used TNFA knockout mice to prevent TNF signaling (Kuprash et al., 2005), and caspase 1/11 knockout mice to ablate IL1 and IL18 production (Smith et al., 1997). None of these knockouts altered the progression (onset around 2 months of age) or severity of the D/Dep2 skin disease (Figure 8-figure supplement 2). The Myd88 knockout, however, reduced the splenomegaly observed in the D/Dep2 mice. This specific phenotype is caused by an increase in Mac1+/Gr1+ splenocytes ( Figure 2B), and its selective rescue in the compound D/Dep2;MyD88 knockout animals is likely due to the crucial role of MyD88 in the generation of these cells (Arora et al., 2010;Delano et al., 2007).
Interfering with the JNK pathway decreases the production of proinflammatory molecules in primary D/Dep2 keratinocytes and RAF knockdown HaCat cells A concomitant increase in JNK activation and decrease in ERK activation is the basis of the skin disease of D/Dep2 animals. To test whether this molecular phenotype was cell-autonomous and to gain some insight in the molecular mechanisms underlying JNK activation, we established and analyzed primary keratinocytes cultures. D/Dep2 keratinocytes were "primed" for inflammation: they constitutively expressed ICAM1 ( Figure 9A) and, upon stimulation, significantly increased amounts of chemokines (CCL2, CCL7) and cytokines (IL6, IL18, TSLP, IL13, Figure 9B; IL4 was not detected under these conditions). In addition, D/Dep2 keratinocytes showed decreased ERK activation in response to EGF and a strong increase in pJNK when co-treated with proinflammatory stimuli ( Figure 9A). Treatment with D-JNKI1, a cell-penetrating, protease-resistant peptide that prevents JNK interaction with its JBD-dependent targets (Borsello and Forloni, 2007), reduced the expression of the inflammatory proteins ICAM1, TSLP, CCL2 and CCL7, but did not have any major effect on ERK activation  Figure 1F. ACTB is shown as a loading control. Phosphorylation is expressed as the ratio between the signals obtained obtained with the phosphospecific antibody and with the protein-specific antibody. In both cases, the data are normalized to one of the F/F2 samples, which was arbitrarily set as 1. Data are plotted as mean ± SEM. p1 = 2.73E-4, p2 = 4.15E-5, p3 = 0.042, p4 = 0.031, p5 = 0.001, p6 = 0.023, p7 = 0.038, p8 = 0.049, p9 = 0.010, p10 = 0.030, p11 = 0.033, p12 = 0.023 and p13 = 0.018. DOI: 10.7554/eLife.14012.012 and on the stress kinase phosphatases DUSP1 and DUSP10, expressed at lower levels in D/Dep2 keratinocytes ( Figure 9C-D).
To ensure that the effects observed in the D/Dep2 cells and tissue could be reproduced by acute ablation, we performed knockdown experiments in the human keratinocyte cell line HaCat. Concomitant silencing of BRAF and RAF1 (double knockdown, KD2) abolished basal ERK phosphorylation, decreased ERK activation and increased JNK activation by a combination of EGF and proinflammatory stimuli (Figure 9-figure supplement 1A). Constitutive ICAM1 expression was not observed in KD2 cells, but they expressed higher levels of this molecule when treated with TNFa. ICAM1 expression was reduced by D-JNKI1 in KD2 cells and increased by MEKi in RAF1KD cells (Figure 9-figure supplement 1B-C). CCL2 mRNA accumulation, which depends on concomitant stress kinase activation and ERK inhibition, was also elevated in a JNK-dependent manner in KD2 cells (Figure 9-figure supplement 1B); MEK inhibition strongly increased CCL2 mRNA in WT cells (Pastore et al., 2005) and even more so in RAF1KD cells (Figure 9-figure supplement 1C).
Mixed lineage kinase 3 (MLK3) activates the JNK pathway (Gallo and Johnson, 2002) and can function both as a positive regulator of ERK signaling, via kinase-independent mechanisms (Chadee and Kyriakis, 2004;Chadee et al., 2006) and as a negative regulator of ERK, via JNKdependent mechanisms (Shen et al., 2003). Importantly, MLK3 binds to JIP1 (Whitmarsh et al., 1998), and signal flow from this upstream kinase would be interrupted by D-JKNI1. We thus determined whether MLK3 was implicated in the imbalance in ERK/JNK signaling observed in D/Dep2 primary keratinocytes and in KD2 HaCat cells. MLK3 downregulation by two independent shRNAs strongly reduced both ERK and JNK phosphorylation in F/F2 cells treated with EGF and proinflammatory stimuli; however, in D/Dep2 cells, only JNK phosphorylation was reduced ( Figure 9E). Consistently, MLK3 downregulation reduced ICAM1 upregulation and the induction of Ccl2 and Tslp mRNA ( Figure 9F). Essentially the same results were obtained by downregulating MLK3 in WT and KD2 HaCat cells (Figure 9-figure supplement 1D). Thus, MLK3 is responsible for JNK activation and the induction of inflammatory molecules in D/Dep2 keratinocytes and HaCat cells.

Discussion BRAF and RAF1 prevent keratinocyte-driven allergic inflammation
Keratinocytes have long been known to contribute to the pathogenesis of skin inflammatory disorders, but whether they do so by reacting to stimuli produced by immune cells or by initiating the cascade leading to the disease is still a matter of debate. We describe an essential role of BRAF and RAF1 in keratinocytes in the control of allergic inflammation. Mice lacking BRAF/RAF1 in keratinocytes develop a disease clinically very similar to human atopic dermatitis (Bieber, 2010). It starts with a barrier defect accompanied by the reduced expression of crucial tight junction proteins and by a marginally increased expression of chemokines and cytokines, particularly CCL2, IL6 and IL18. It then progresses to a stage in which the skin appears unaffected, yet both local (mast cell infiltration, chemokine and cytokine production) and systemic (T and B cell activation, increased amounts of circulating leukocytes and chemokines) anomalies become evident while TSLP and IgE levels are only slightly increased. This is reminiscent of the early phase of atopic dermatitis in children, prior to IgE sensitization (Bieber, 2010). Unlike the majority of atopic dermatitis patients (De Benedetto et al., 2012), however, the D/Dep2 animals don't have a stratum corneum defect, which may help explain the lack of symptoms at this stage. Adult mice present with a full-fledged disease characterized by rich dermal infiltrates, increased cytokine production, clearly elevated IgEs and mast cell activation, in good correlation with the intense pruritus causing extensive scratching and self-inflicted wounds.   . Increased stress kinase signaling and JNK pathway-dependent cytokine and chemokine production by primary keratinocytes lacking BRAF and RAF1. (A) Reduced ERK phosphorylation and increased JNK/p38 activation in primary D/Dep2 keratinocytes stimulated with EGF and/or TNFa and IL1b for 15 min. (B) Increased cytokine and chemokine production in primary D/Dep2 keratinocytes treated with EGF, TNFa and IL1b for 24 hr. Cytokine and chemokine production was determined by multiplex analysis, except for TSLP which was quantified by ELISA. Data represent mean ± SEM of 3-5 biological replicates. (C-D) Cells were pretreated with D-JNKI1 inhibitors prior to stimulation with EGF, TNFa and IL1b for 15 min (C) or 24 hr (D). Data represent the mean ± SEM of technical replicates (n = 3). (E-F) Effect of shRNA-mediated Mlk3 silencing on ERK and JNK phosphorylation and ICAM1 expression (E; stimulation with EGF, TNFa and IL1b for 15 min) and on the expression of Ccl2 and Tslp mRNA (F; stimulation with EGF, TNFa and IL1b for 24 hr) by F/F2 and D/Dep2 keratinocytes. shRen, shRNA targeting Renilla, used as a control; sh1 and sh2, targeting Mlk3, binding sites nucleotide 2266-2285 and 2383-2402, respectively. The shRNAs were encoded by lentiviral vectors coexpressing GFP. GFP immunoblots are shown to confirm similar levels of infection in all samples. Data represent mean ± SEM of 4 biological replicates. Each keratinocyte culture represents a pool of three mice. Immunoblots are representative of three independent experiments. p1 = 0.041, p2 = 0.040, p3 = 1.89E-4, p4 = 0.018, p5 = 0.046, p6 = 0.020, p7 = 0.008, p8 = 0.016, p9 = 0.001, p10 = 0.018, p11 = 3.23E-4, p12 = 1.47E-4, p13 = 0.007, p14 = 0.03, p15 = 0.035, p16 = 0.023 and p17 = 0.046. DOI: 10.7554/eLife.14012.018 Figure 9 continued on next page The initial barrier defect contributes to full-fledged disease, since ablation of BRAF/RAF1 after the third week causes a less severe condition lacking IgE sensitization and mast cell activation, yet recapitulating the systemic symptoms of the disease. This could be likened to late-onset, IgE-sensitization-independent dermatitis (Bieber, 2010). Thus, BRAF and RAF1 act together to ensure the timely establishment of the inside-out barrier of the epidermis and to prevent allergic inflammation. The data support the hypothesis that the keratinocytes play a primary role in the pathogenesis of atopic dermatitis, and the D/Dep2 animals may be useful as a model for this disease (Figure 10). A set of chemokines and Th2 cytokines underlies D/Dep2 allergic inflammation In this respect, our experiments provide information about the minimal set of keratinocyte-derived factors required for the establishment of allergic, atopic dermatitis-like inflammation.
In the phase before IgE sensitization and mast cell activation, we detected significantly higher levels of TSLP, CCL2, CCL7, GMCSF, IL2, IL5, IL6, and IL13 in D/Dep2 epidermal lysates; most chemokines and cytokines in the D/Dep2 animals were sensitive to D-JNKI1 inhibitor treatment, which brought D/Dep2 and F/F2 epidermal lysates down to indistinguishable levels. Conversely, the set of cytokines (TSLP, IL5, IL13, IL18, borderline IL2 and IL17) observed in adult, diseased D/Dep2 mice was similar to that promoting symptomatic disease in RAF1Dep animals treated with trametinib, which showed an increase in TSLP, IL2, IL5, IL13, as well as in IL18. These data can be interpreted to mean that ERK signaling negatively regulates the production of allergy-promoting factors. However, it is important to note that trametinib treatment reduced IL2 and IL5 as well as GMCSF in the F/F2 epidermis, indicating that sensitivity to ERK inhibition must be seen in the context of altered cellautonomous signaling (increased JNK activation in RAF1Dep epidermis) and/or tissue milieu. Along the same lines, D/Dep2 keratinocytes treated with inflammatory stimuli were significantly more efficient than F/F2 in producing CCL2, CCL7, IL6, IL18, TSLP and IL13, but not IL2 or IL5, resembling, but not fully reproducing, the spectrum observed in epidermal lysates. At this stage, it is impossible to determine whether these discrepancies might be due to the presence of cell types other than keratinocytes in the epidermal lysates or whether the culture and stimulation conditions used did not recapitulate the situation in vivo.
Be that as it may, the range of chemokines and cytokines overrepresented in the D/Dep2 mice and in the trametinib-treated RAF1Dep mice is consistent with allergic inflammation. In this respect, strong expression of CCL2 has been reported in the basal keratinocytes of atopic dermatitis patients (Giustizieri et al., 2001), and expression of CCL7, which can attract basophils, eosinophils, mast cells and Th2 cells, can be induced by allergens in atopic skin (Ying et al., 1995).
TSLP, the signature cytokine of allergic disease, promotes the functions of these same cell types (Ziegler et al., 2013), and is responsible for the extreme itching accompanying atopic dermatitis (Wilson et al., 2013). High levels of TSLP (Liu, 2006), but also of IL5 and IL13 have been reported in the skin of atopic dermatitis patients, particularly in those with elevated IgEs (Jeong et al., 2003). In mice, it could be shown that the expression of TSLP (Liu, 2006), IL4, IL5 and IL13 induces atopic dermatitis as well as asthma (Lee and Flavell, 2004), and IL5 knockout mice exposed to allergens are less prone than wild-type to develop skin eosinophilia and epidermal thickening (Spergel et al., 1999). Finally, expression of IL13 in keratinocytes is sufficient to induce a disease mimicking atopic dermatitis (Zheng et al., 2009). Thus, these cytokines appear instrumental for the development of atopic dermatitis with IgE sensitization. IL18, on the other hand, can stimulate both Th1 and Th2 responses (Nakanishi et al., 2001), and appears to be causally involved in a different type of atopic dermatitis. IL18 is essential for the induction of dermatitis by the epidermis-restricted expression of caspase 1, the enzyme that generates active IL18 from its precursor. In addition, expression of IL18 in keratinocytes induces a late-onset dermatitis associated with mastocytosis, but independent of IgE production (Konishi et al., 2002). More recently, IL18 knockout mice were shown to be resistant to infection-associated atopic dermatitis, in a mouse model generated by perturbing the stratum corneum with detergent prior to topical application of S. aureus protein A (Terada et al., 2006). However, stratum corneum defects could not be observed in D/Dep2 mice, and caspase 1 ablation does not modify the course of the D/Dep2 disease. Thus, IL18 is not a determining factor in the context of the D/Dep2 dermatitis, which rather resembles a Th2/IgE-driven disease.
An imbalance in MAPK signaling underlies skin inflammation in D/Dep2 mice Ablation of BRAF and RAF1 in the epidermis has non-redundant functions converging on MAPK cascades. Decreased ERK and increased JNK activation are observed in D/Dep2 epidermis in vivo independently of the severity of the disease as well as in D/Dep2 keratinocytes and BRAF/RAF1 knockdown HaCat cells. Thus, the imbalance in MAPK signaling represents an intrinsic defect of cells and tissues lacking BRAF/RAF1. While BRAF ablation, as in many other tissues/organs, is responsible for reduced ERK activation (Desideri et al., 2015), RAF1 ablation correlates with a previously unnoticed increase in stress kinase activation. Consistent with this, we have observed a reduction in the expression of two DUSPs involved in the inactivation of stress kinases, which is strongest in the D/Dep2 epidermis. This is in line with the reported positive role of p38 in the induction of DUSP expression and of ERK in its attenuation at multiple levels (Caunt and Keyse, 2013;Taxman et al., 2011), which would predict that decreased ERK signaling would be needed to reduce DUSP expression and stabilize JNK phosphorylation when p38 is active. Importantly, both DUSPs have been implicated in the production of cytokines by immunocompetent cells and in murine models of inflammatory and autoimmune disease (Lang et al., 2006).
We could further identify MLK3 as the kinase responsible for JNK activation as well as for the expression of inflammatory molecules in primary D/Dep2 keratinocytes and BRAF/RAF1 knockdown HaCat cells costimulated with EGF, TNFa and IL1b (as a proxy for the alterations observed in the D/ Dep2 animals). The lack of basal JNK activation in unstimulated cells suggests a cross-talk at the level of BRAF/RAF1 and MLK3 rather than direct MLK3 activation by BRAF/RAF1 ablation. Such a crosstalk was previously reported to occur in the context of ERK signaling, which is positively regulated by MLK3 via the stabilization of the RAF dimer (Chadee and Kyriakis, 2004;Chadee et al., 2006). It is possible that in turn, BRAF and RAF1 would restrict the involvement of MLK3 in the JNK pathway, at the same time promoting ERK and reducing JNK activation in stimulated cells (Figure 10).
While the mechanistic details remain to be fully elucidated, the relevance of the imbalance in MAPK signaling for the development of the atopic dermatitis-like disease in D/Dep2 animals was shown in vivo in two complementary ways: 1) by preventing disease onset with the specific peptide inhibitor D-JNKI1; and 2) by inducing disease in healthy RAF1Dep animals with a MEK inhibitor (Figure 10). The second set of experiments also clearly shows that the function of BRAF and RAF1 in the epidermis is not redundant. Finally, the combined RAF knockout and inhibitor data help explain why in patients the cutaneous toxicity of RAF inhibitors, which increase ERK activation, is mostly related to hyperproliferation (Anforth et al., 2013), while inhibitors of ERK activation promote the onset or exacerbate the course of cutaneous inflammatory reactions involving stress kinase activation (Curry et al., 2014).

Materials and methods
Animal studies and inhibitor treatment All strains were maintained on a Sv/129 background. Krt5-CreBraf f/f ;Raf1 f/f mice were generated for this study by mating Krt5-Cre;Braf f/f :: Krt5-Cre;Raf1 f/f animals. These strains, the Krt5-CreER(TX);Braf f/f ;Raf1 f/f mice as well as their genotyping and the tamoxifen-induced deletion of the RAF alleles have been previously described Ehrenreiter et al., 2005). Inducible RAF deletion was performed at the age of three weeks.

FlowCytomix analyte assay and ELISA
Cytokines and chemokines were detected in cell supernatants, serum samples and epidermal tissue lysates using the Affymetrix eBioscience bead-based multiplex immunoassay. Data were analyzed with FlowCytomix Pro2.4 software. GCSF (R&D Systems, DY414), TSLP (R&D Systems, DY555) and IgEs (Bethyl Laboratories, E90-115) in serum samples were detected by ELISA according to the manufacturer's protocol.

Barrier function assays
Water loss assay and toluidine blue dye staining of embryos were carried out as described (Tunggal et al., 2005).

Lentiviral vectors, cloning and transduction procedure
Two independent shRNAs against mouse MLK3 (shRNA1, binding site 2266-2285 and shRNA2, binding site 2383-2402) were designed as 97-bp oligomers containing a 20bp targeting sequence embedded in a shRNAmir stem, amplified and cloned into Xho and EcoRI sites of the miRE lentiviral recipient vector pRRL.SFFV.GFP.miRE.PGK.Puro (SGEP) (Fellmann et al., 2013). The SGEP plasmid containing Renilla shRNA served as a control. Lentiviral vectors were transfected in 293T cells. Viral supernatants were collected after 24 and 48 hr and passed through a 0.45-mm filter (Sarstedt, Germany). Each fresh viral supernatant was used for primary keratinocyte spinfection (1500 g, 30 min). Primary keratinocyte cultures were harvested 72 hr after first transduction.

Statistical analysis
Histological samples from at least three animals per condition and genotype were analyzed by counting or measuring at least 3-5 microscopic field/section and analyzed by ImageJ software. Values are expressed as mean (±SEM). The number of biological replicates and, where applicable, technical replicates are indicated in the figure legends. p values were calculated using the two-tailed Student's t test, hetero-or homoskedastic as determined by a previous F-test of equality of variances or, when indicated, by two-way analysis of variance (ANOVA) test. A p value 0.05 is considered statistically significant. data or reagents; MB, Conception, design and supervision of the project, Analysis and interpretation of data, Drafting and revising the article Author ORCIDs Manuela Baccarini, http://orcid.org/0000-0002-3033-391X

Ethics
Animal experimentation: The study was performed in accordance with the National Austrian legislation (Law of Animal Experiments 2012 ("TVG-Tierversuchsgesetz"; Federal Law regulating the "Experimentation on living animals" BGBI. I Nr.114/2012) and the overriding EU and international legislation and codes of conduct. Animals were bred and housed in the MFPL facility, which follows the "Charter of Fundamental Rights of the European Union", the opinion of the "European Group on Ethics in Science", and the "Protocol on the Protection and Welfare of Animals". Care of mice is performed by licensed animal caretakers according to FELASA recommendations. Animal experiments involving genetic manipulations are governed by the "Gentechnikgetsetz" (GTG, 12.07.1994). All experiments described in the manuscript were authorized by the Austrian Ministry of Science, Research and Economy (GZ: BMWF-66.006/0030-II/3b/2013 and BMWF-66.006/0012-II/10b/2010).