With the development and market introduction of biologics, much progress has been made in recent years in the systemic treatment of psoriasis. Currently, targeted therapy with antibodies against IL-17 or IL-23 exhibits high efficacy and allows complete or almost complete clinical clearance of psoriasis lesions in a high percentage of cases (Blauvelt et al., 2017; Langley et al., 2014; Papp et al., 2017; Reich et al., 2018). However, patients with limited body area involvement (i.e. mild forms of psoriasis) who represent the majority of psoriasis patients with up to 90% (Stern et al., 2004) have been neglected. Not much innovation has occurred in the past few years on topical treatment of psoriasis that is mainly prescribed to these patients. Steroids, vitamin D3 and vitamin A analogues are most commonly used as topical agents in such patients, but besides changes in their pharmaceutical formulation, they have not been developed further in recent years (de Korte et al., 2008; Krueger et al., 2001; Langley et al., 2011; Schaarschmidt et al., 2015). Short courses of dithranol (1,8-dihydroxy-9-anthracenone or anthralin) have been successfully used as intermittent topical treatment of psoriasis since 1916 (Nast et al., 2012; Sehgal et al., 2014; Vleuten et al., 1996). Despite its numerous disadvantages like brown staining and irritation of perilesional skin, dithranol has remained one of the most effective topical treatment modalities in psoriasis. Analogous to the most recent generation of biologics (including anti-IL-17 and anti-IL-23 antibodies), dithranol delivers PASI75 rates in 66–82.5% of patients with fast action and clearance of skin lesion within very few weeks (Kemény et al., 1990; Painsi et al., 2015b; Sehgal et al., 2014; van de Kerkhof, 2015). Although dithranol has been used for many years, its exact mechanism of action has remained largely unknown (Holstein et al., 2017; Kemény et al., 1990; Kucharekova et al., 2006; Sehgal et al., 2014).

With the aim of unraveling dithranol’s therapeutic mechanisms and to possibly uncover new targets for topical treatment of psoriasis, we conducted a clinical trial and employed several mouse models including the c-Jun/JunB knockout model (Zenz et al., 2005) and the imiquimod psoriasis model (van der Fits et al., 2009) in order to address this issue and elucidate dithranol's effects. In this study, we demonstrate that dithranol exerts its anti-psoriatic effects by directly targeting keratinocytes and their crosstalk with neutrophils, as well as disrupting the IL-36 inflammatory loop. Consistent with this finding, we observed that dithranol’s therapeutic activity was completely independent of its pro-inflammatory effect mainly on perilesional skin, thus overthrowing the long-believed paradigm that dithranol-induced irritation is crucial for its anti-psoriatic action and unraveling irritation merely as a bystander effect of treatment.

This study demonstrates that topical dithranol directly targets keratinocytes (in particular their differentiation regulators and AMPs), keratinocyte-neutrophil crosstalk and inhibits the IL-36 inflammatory loop in psoriasis, thus unraveling after over 100 years of use, the therapeutic mechanism of one of the most effective topical treatments of psoriasis. Dithranol significantly diminished mRNA expression of pro-psoriatic IL-1 family members (IL36A, IL36G and IL36RN) (Supplementary file 1 and Figure 4). At day 6 after start of dithranol treatment, PASI had decreased by 33%, but at week 2–3 we saw a reduction of 58%, an effect that was paralleled by reduced expression of IL-36-related genes in psoriasis patients. The therapeutic importance of reduction of IL-1 family members in human skin was substantiated by results generated in the keratinocyte-based c-Jun/JunB mouse psoriasis model (Zenz et al., 2005). Expression of IL36RN (murine Il1f5) and IL36B (Il1f8) was significantly reduced in dithranol-treated psoriatic c-Jun/JunB lesions compared to controls (Figure 4) and among the top differentially expressed genes in our human and mouse dataset were genes involved in keratinocyte and epidermal differentiation (Table 4, Table 7). Dithranol strongly reduced mRNA expression of the keratinocyte differentiation regulator involucrin (IVL) and members of the serpin family (SERPBINB7, SERBINB13) both in lesional skin of patients and c-Jun/JunB knockout mice (Figure 4). Moreover, dithranol downregulated expression of AMPs such as ß-defensins (DEFB4A and DEFB4B) produced by keratinocytes (Liu et al., 2002; Schröder and Harder, 1999) within 6 days and chemotactic factors for neutrophils (such as CXCL5 and CXCL8) (Albanesi et al., 2018; Table 2) and neutrophilic infiltration (as determined by MPO staining) within 2–3 weeks of treatment in human psoriatic skin (Figure 2).

Surprisingly, there were no significant changes in overall T cell numbers including CD4+ and CD8+ T cells, as well as FoxP3 positivity indicative for regulatory T cells in the skin in the early phase (within 6 days) during dithranol treatment. Reflecting dithranol’s primary effect on the epidermal compartment, the reduction of T cell numbers in the epidermis preceded that in the dermis. Whereas dithranol had decreased T cell counts in the epidermis at week 2–3, an effect on T cell numbers in the dermis was only evident at the follow-up visit, 4–6 weeks after the end of dithranol treatment (Figure 2), in agreement with previous reports favoring dithranol’s effect on keratinocytes (Holstein et al., 2017; Swinkels et al., 2002a; Vleuten et al., 1996; Yamamoto and Nishioka, 2003).

Vleuten et al., 1996 and Swinkels et al., 2002a applied immunohistochemistry to analyze differentiation and proliferation markers as well as T cell numbers in the skin and observed, as we did, a decrease in keratin 16, restoration of filaggrin and a decrease of Ki67 in the epidermis after 2 weeks of dithranol treatment. However, their data on the effect of dithranol on dermal T cell numbers at 4 weeks was controversial (Swinkels et al., 2002a; Vleuten et al., 1996). The group of Eberle recently tested the effects of dithranol using primary keratinocytes, a 3D psoriasis tissue model and some biopsy samples from psoriasis patients and reported on a reduction in Ki67 and keratin 16 positive cells in the epidermis using immunostaining and an inhibition of the antimicrobial peptide DEFB4 using qPCR analysis. However, based on their observations, they concluded that dithranol’s anti-psoriatic effects cannot be explained by direct effects on keratinocyte differentiation or cytokine expression (Holstein et al., 2017).

Our genome-wide expression analysis indicates that dithranol primarily targets keratinocytes and that this is crucial for response to treatment, considering that differentially regulated genes in histological responders compared to non-responders belonged to pathways like keratinocyte differentiation, cornification and keratin filament formation (Supplementary file 3). The importance of dithranol’s direct effect on keratinocytes has been further substantiated by our findings generated using the mouse-tail model, a simple in vivo model to analyze effects of topical preparations on keratinocyte differentiation and parakeratosis (Bosman et al., 1992; Sebök et al., 2000; Wu et al., 2015). Similar to previous studies (Bosman et al., 1992; Hofbauer et al., 1988; Sebök et al., 2000; Wrench and Britten, 1975), we observed a strong increase in orthokeratosis after dithranol application in the mouse-tail test, reflecting its keratinocyte differentiation-inducing activity (Figure 3—figure supplement 1). Our transcriptional analysis indicated that the effect of dithranol in the mouse-tail test was linked to a strong upregulation of keratinocyte differentiation markers and several AMPs, while the pro-psoriatic antimicrobial peptide Camp/LL37 was downregulated, as well as Cxcl5, a chemotactic factor for neutrophils (Figure 3—figure supplement 2), but not other pro-psoriatic AMPs (Fritz et al., 2017; Wang et al., 2018) such as Defb3, S100a8 or S100a9. Although the mouse-tail test has evidently limitations since the disturbed cell differentiation of this model only reflects one of many aspects of psoriasis, it supports the primary effect of dithranol on keratinocytes with induced induction of orthokeratosis.

In contrast to its therapeutic effect in both keratinocyte-driven psoriasis models, the c-Jun/JunB model and mouse-tail test, dithranol did aggravate psoriatic lesions in the imiquimod model that has been solely shown to be immunologically mediated and dependent on the IL-17/IL-23 axis (van der Fits et al., 2009). In the latter model, biologics such as etanercept and anti-IL-17a agents (Liu et al., 2018), topical steroids (Sun et al., 2014) and vitamin d3 analogues (Germán et al., 2019) but also UVB and PUVA (Shirsath et al., 2018) were shown to have a beneficial effect. In contrast, dithranol significantly enhanced overall macroscopic skin thickness, consistent with a slight increase in epidermal hyperplasia and worsened inflammation (as measured by the density of cellular infiltrate in the dermis) (Figure 3—figure supplement 3). Dithranol treatment of healthy murine skin led to similar effects upon irritation, as it increased epidermal thickness and cellular infiltrate of the skin (data not shown). However, the irritant effect of dithranol may remain without functional anti-psoriatic relevance in human psoriasis (as indicated by the clinical results depicted in Figure 1—figure supplement 2 and discussed below) but might be crucial in the agent's therapeutic action in alopecia areata (Nasimi et al., 2019; Ngwanya et al., 2017).

Together, these data unambiguously demonstrate that dithranol directly acts on keratinocytes, their crosstalk with neutrophils and IL-36 signaling, with AMPs being the potential link (Figure 5). This goes also in line with the observation that Langerhans cells as another type of immune cells showed a delayed response to dithranol, with no changes during treatment and an evident increase in numbers only later on (at the follow-up visit 4–6 weeks after end of treatment). These results are consistent with previous studies performed by Swinkels et al., showing that there was no significant change in T cells or Langerhans cells during twelve days of dithranol treatment (Swinkels et al., 2002a).

Figure 5

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Our findings on dithranol’s effect on members of the IL-1 family, being beneficial for its anti-psoriatic efficacy, are well in line with recent work on blockade of IL-36 pathway as a novel strategy for the treatment of pustular psoriasis (Bachelez et al., 2019) as well as plaque-type psoriasis (Benezeder and Wolf, 2019; Mahil et al., 2017). Notably, human keratinocytes express IL-1 family members (IL-36α, IL-36β, IL-36γ, IL-36Ra) and their receptor IL-36R (D'Erme et al., 2015; Foster et al., 2014; Johnston et al., 2017). Furthermore, normal human keratinocytes show increased expression of IL-1 group mRNA after treatment with psoriasis-associated cytokines (TNFα, IL-1α, IL-17,IL-22) (Johnston et al., 2011). Genome-wide association studies revealed that deficiency in interleukin-36 receptor antagonist due to IL36RN mutations was associated with generalized pustular psoriasis (GPP) (Marrakchi et al., 2011; Sugiura et al., 2013). Supporting this notion, blocking IL-36 receptor was effective in reducing epidermal hyperplasia and showed comparable effects to etanercept in a psoriatic skin xenotransplantation model (Blumberg et al., 2010). Furthermore, successful treatment of psoriasis with the anti-psoriatic standard treatment etanercept is accompanied by a decrease in IL36A, IL36G and IL36RN expression (Johnston et al., 2011). A recent clinical phase one study provided proof-of-concept for the efficacy of BI 655130, a monoclonal antibody against the interleukin-36 receptor in the treatment of generalized pustular psoriasis (Bachelez et al., 2019). Intriguingly, topical short-contact dithranol therapy is efficacious not only in plaque-type psoriasis, but under certain conditions (i.e. after stabilization of disease activity with bland emollients) reportedly also in pustular psoriasis (Farber and Nall, 1993; Gerritsen, 1999). In this neutrophilic-driven condition, in which one might expect that dithranol worsens a heavy inflammatory state, it may have beneficial capacity by targeting the IL-36 pathway and neutrophils, both playing a most crucial role in this type of psoriasis (Bachelez, 2018; Bachelez et al., 2019; Johnston et al., 2017; Marrakchi et al., 2011; Sugiura et al., 2013).

As outlined above, we observed a strong effect of dithranol on keratinocyte-neutrophil crosstalk. Importantly, early response to anti-IL-17a blockade, as one of the most effective biological treatments currently available, has been linked to inhibition of keratinocyte-neutrophil crosstalk (Reich et al., 2015). Similar to our observation, anti-IL-17a treatment with secukinumab significantly reduced epidermal hyperproliferation after 2 weeks, decreased mRNA expression of keratinocyte-derived chemotactic factors and led to a strong decrease in IL-17a positive neutrophils. Apparently, disrupting the crosstalk between keratinocytes and neutrophils may be a crucial early effect in the clinical efficacy of secukinumab in psoriasis (Reich et al., 2015). In addition, decreased expression of AMPs like ß-defensin and S100 proteins was observed after only 1 week of anti-IL-17a treatment with secukinumab (Krueger et al., 2019) and after 2 weeks of ixekizumab treatment (Krueger et al., 2012), well in line with our observed early effect of dithranol on AMPs. Krueger et al. concluded that clinical efficacy of anti-IL-17a treatment is closely linked to early inhibition of keratinocyte-derived products such as chemokines and AMPs. Together this suggests that dithranol’s direct action on keratinocytes at the molecular level may disrupt IL-17 pathway dysregulation (without directly blocking IL-17 or its receptor), leading in turn to similar downstream effects as treatment with anti-IL-17 antagonists.

Our study also negates the paradigm that dithranol-induced irritation is crucial for its anti-psoriatic action (Gerritsen, 1999; Kucharekova et al., 2005; Prins et al., 1998; van de Kerkhof, 1991; Wiegrebe and Müller, 1995). As depicted in Figure 1—figure supplement 2, there was no correlation between dithranol-induced perilesional as well as lesional erythema and its anti-psoriatic effect, indicating that dithranol’s irritation (Prins et al., 1998; Swinkels et al., 2002c; Swinkels et al., 2002b) is a treatment side-effect unrelated to its therapeutic mechanism. However, this irritant effect of dithranol may be crucial for its action in alopecia areata, a condition, in which it was shown to be greatly effective, leading to hair regrowth in a high percentage of cases (Ngwanya et al., 2017). Similar to other topical treatment options (Nasimi et al., 2019; Yoshimasu and Furukawa, 2016), an initial irritant reaction to dithranol is followed by regrowth of hair within weeks after treatment.

What our work does not answer, is how dithranol exactly acts at the molecular level. Cell culture studies have shown that dithranol targets mitochondria (McGill et al., 2005), alters cellular metabolism (Hollywood et al., 2015) and induces apoptosis in keratinocytes (George et al., 2013; McGill et al., 2005). Dithranol also inhibits human monocytes in vitro, inhibiting secretion of IL-6, IL-8 and TNFα (Mrowietz et al., 1992; Mrowietz et al., 1997). Its effects on neutrophils were also shown in vitro, where dithranol leads to an increase in superoxide generation and a decrease in leukotriene production in neutrophils (Kavanagh et al., 1996; Schröder, 1986). Moreover, the potential receptor of dithranol remains undefined. Anti-psoriatic effects of other topical treatment options have recently been linked to modulation of the aryl hydrocarbon receptor (AhR) (Smith et al., 2017) and AhR may play a role in pathogenesis of psoriasis (Di Meglio et al., 2014). However, it appears that dithranol does not act via modulation of AhR, as we did not observe differences in the response of skin to dithranol comparing AhR knockout mice to controls (data not shown). There is need for further studies investigating how dithranol exactly acts on the molecular level, which receptor it potentially binds to or inhibits or whether it acts through modulation of a specific transcription factor such as NF-kB or STATs. Another question is whether the effect of dithranol is specific compared to other topical treatments such as steroids and vitamin D3 analogues. There seem to be some overlapping mechanisms between dithranol and vitamin D3 analogues such as calcipotriol that has been shown to act on keratinocytes to repress the expression of IL-36α/γ, an effect mediated through keratinocytic vitamin D receptor (Germán et al., 2019). Moreover, similar to dithranol, calcipotriol decreased expression of AMPs such as ß-defensins in keratinocytes of psoriatic plaques (Peric et al., 2009). At the same time, calcipotriol normalized the proinflammatory cytokine milieu and decreased IL-17A, IL-17F and IL-8 transcript abundance in lesional psoriatic skin. Calcipotriol also directly targets Th17 cells (Fujiyama et al., 2016) and CD8+IL-17+ cells (Dyring-Andersen et al., 2015), whereas we found that dithranol only has delayed effects on T cells. Moreover, cathelicidin (LL37) expression was increased by calcipotriol (Peric et al., 2009), juxtaposing the results of dithranol treatment at least in the mouse-tail test of present study. That dithranol and vitamin D3 analogues may have similar basic mechanisms of action is also supported by the notion that depending on the concentration, both dithranol (Nasimi et al., 2019; Ngwanya et al., 2017) and calcipotriol (El Taieb et al., 2019; Fullerton et al., 1998; Molinelli et al., 2020) can be irritant to the skin and induce hair regrowth in alopecia areata. Compared to vitamin D3 analogues topical steroids have an even broader mechanisms of action in psoriasis linked to their anti-inflammatory, antiproliferative, vasoconstrictive (Uva et al., 2012) and immunomodulatory properties, in particular suppressing the IL-23/IL-17 axis, with IL-23 produced by dendritic cells/macrophages and IL-17 produced by Th17 cells/γδ T cells/innate lymphoid cells (Germán et al., 2019).

Another question that this study does not answer is, whether topical dithranol therapy has any effects on systemic psoriatic inflammation. However nowadays, treatment with dithranol is mainly administered in refractory, circumscribed psoriatic lesions in patients who do not have significant systemic inflammation how it may be otherwise the case in patients with moderate to severe forms of psoriasis.

Together our work opens up several avenues for novel topical (and potentially also systemic) treatment strategies in psoriasis. Not only targeting the IL-36 pathway, but also keratinocyte differentiation regulators (e.g. involucrin), keratinocyte-produced AMPs (ß-defensins like DEFB4A, DEFB4B, DEFB103A, S100 proteins like S100A7, S100A12), and neutrophils and their chemotactic factors (CXCL5 and CXCL8) or members of the serpin family (SERPINB7 and SERPINB13), are promising approaches. Such approaches may not only be helpful for chronic plaque-psoriasis, but also for pustular psoriasis, in which a vicious loop between AMPs such as cathelicidin (LL-37) and IL-36 signaling may drive psoriatic disease (Benezeder and Wolf, 2019; Furue et al., 2018; Li et al., 2014; Madonna et al., 2019; Ngo et al., 2018).

All microarray data has been deposited at the public repository Gene Expression Omnibus (GEO) (http://www.ncbi.nlm.nih.gov/geo/) with accession numbers GSE145126 and GSE145127.

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Acceptance summary:

The role of keratinocytes in the pathogenesis of psoriasis has gained a lot of interest recently and your work supplements the new insights from the therapeutic point of view. Dithranol by itself may still be a therapeutic niche, but the data regarding the mode of action can stimulate drug development along these lines.

Decision letter after peer review:

Thank you for submitting your article "Dithranol targets keratinocytes, their crosstalk with neutrophils and inhibits the IL-36 inflammatory loop in psoriasis" for consideration by eLife. Your article has been reviewed by three peer reviewers, one of whom is a member of our Board of Reviewing Editors, and the evaluation has been overseen by Jos van der Meer as the Senior Editor. The following individual involved in review of your submission has agreed to reveal their identity: Claus Johansen (Reviewer #3).

The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.

As the editors have judged that your manuscript is of interest, but as described below that additional work is required before it is published, we would like to draw your attention to changes in our revision policy that we have made in response to COVID-19 (https://elifesciences.org/articles/57162). First, because many researchers have temporarily lost access to the labs, we will give authors as much time as they need to submit revised manuscripts. We are also offering, if you choose, to post the manuscript to bioRxiv (if it is not already there) along with this decision letter and a formal designation that the manuscript is "in revision at eLife". Please let us know if you would like to pursue this option. (If your work is more suitable for medRxiv, you will need to post the preprint yourself, as the mechanisms for us to do so are still in development.)

Summary:

Topic of interest but in major parts confirmatory to already published work and in other parts not conclusive. Please provide clarity about the data on AMPs in this manuscript and in the manuscript submitted to the other journal.

Essential revisions:

Statistical analysis (see reviewer 2). Interpretation of data (specific for dithranol or bystander effects (see reviewers 1 and 2). Interpretation of data regarding the mouse models used as there seem a number of inconsistencies (see reviewers 2 and 3).

Interpretation of results in the light of the critique of all reviewers.

Reviewer #1:

The manuscript by Benezeder et al. addresses an interesting topic. The role of keratinocytes for psoriasis pathogenesis has been neglected for many years and the work presented here re-focuses on keratinocyte-mediated effects.

Interestingly, the authors used dithranol as a model, which is challenging since this topical compound shows beneficial therapeutic effects but also induces dose- and individual-depended skin irritation. Although the methods employed are well established and state-of-the-art there are some points that need clarification.

• Psoriasis is viewed as systemic disorder and to judge the overall beneficial effect of any topical therapy including dithranol it should be shown whether there are effects on systemic inflammation (e.g. vascular inflammation). The authors should mention this as limitation of their approach.

• The most commonly used topical therapies are corticosteroids alone or in a fixed combination with a vitamin D analogue. The authors are encouraged to use such treatment as a control in order to prove that the observed effects are specific for dithranol.

• The irritant potential of dithranol is helpful in cases of otherwise refractory alopecia areata. The authors should briefly mention this and discuss if their data may explain the effect as an outlook for further investigation.

The reviewer is puzzled by the fact that he was asked to review a manuscript of the same group with the same first author with significant similarity to data that are described in this manuscript for another journal particularly about the effect of dithranol on antimicrobial peptides.

The authors must make clear if there is an overlap of data and which of the data presented in the other manuscript are also contained in this one.

Reviewer #2:

Benezeder et al. present a paper on dithranol action on keratinocytes in psoriasis. The authors provide data on clinical outcome, reduction of epidermal thickness, cell proliferation and keratin 16 expression. All changes were observed at week 2-3 of treatment. By microarray analysis on day 6 and week 2-3 differentially expressed genes (DEGs) were identified, among these genes such encoding keratinocyte differentiation, skin barrier function and antimicrobial peptides (AMPs), but also IL-17 or IL-36. In addition, the authors used dithranol treatment in a c-Jun/JunB knockout mouse model. Again, microarray analysis was used to follow effects. The authors focused on genes belonging to keratinocyte function. Microarray data generated from the c-Jun/JunB knockout mouse model was compared to human microarray data. Dithranol was also applied to mouse tails. Here, they found increased thickening and orthokeratosis, increase in keratin 16 expression and AMPs. Finally, they used the imiquimod psoriasis-like mouse model where dithranol increased skin thickening. The authors conclude that the effects of dithranol are mainly mediated by keratinocytes and IL-36.

Although this is an interesting study, there are some major caveats and conflicting results, which make it difficult to accept this paper.

1) The main messages of this study – decrease of psoriatic epidermal thickening, reduction of Ki67 positivity, reduction of keratin 16 expression and AMPs by dithranol have been shown by previous reports from human skin (van de Kerkhof et al., 1996 and 2002; Eberle et al., 2017).

2) There is plenty of evidence demonstrating that psoriasis is an IL-17-driven immune disease. All treatments with a rapid clinical response will find a decrease in epidermal thickening, a normalization of keratinocyte differentiation and a decrease in immune cell infiltration as well as cytokine expression. From this perspective, one cannot conclude that the one or other treatment will work through keratinocytes and not influence the immune system. The changes in keratinocytes as observed in Figure 2 (epidermal thickening, Ki67 expression) seem to be significant on week 2 to 3. A similar pattern with a decrease in epidermal thickening, keratin 16 and Ki67 expression is visible at week 2 of treatment when using monoclonal antibodies e.g. directed against IL-17. Similarly, antimicrobial peptides are regulated at early time points by immunobiologics (several well-performed studies on this topic are published by Krueger and colleagues in JACI).

3) The mouse model used (c-Jun/JunB knockout) is somehow artificial and does not require T cells for developing epidermal changes. Therefore, the findings – although of interest – cannot be translated to the human situation.

4) The Major concern is that most of the findings are descriptive. The authors performed many microarray analyses but studies on functional mechanisms confirming their hypothesis are missing.

5) The increase in epidermal thickening, the changes in keratin 16 and AMPs found in the mouse tail experiment with dithranol contradict the results found in human psoriasis (Figure 4).

6) IL-36 cytokines are mainly expressed by keratinocytes (Wenzel et al., J Invest Dermatol 2015), but the authors describe a decrease at week 2 to 3. If dithranol targets primarily keratinocytes and inhibits IL-36, the effect should be visible at earlier time points.

Reviewer #3:

The manuscript by Benezeder et al. investigates the mode of action by which dithranol mediates its anti-psoriatic effects. Although dithranol has been used as topical treatment for psoriasis for many years the mechanism by which dithranol works is not that well described and therefore a better understanding of the mode of action of dithranol is needed and could pave the way for other topical drugs for psoriasis.

The manuscript is interesting and well-written and the data are convincing.

I have the following comments:

1) Although the data in the manuscript clearly shows that the early mode of action of dithranol in psoriasis treatment is on keratinocyte-derived molecules and subsequently neutrophil infiltration to the skin the manuscript does not reveal the molecular mechanism behind this. Does dithranol use a membrane bound receptor, an intracellular receptor or does it inhibit specific transcription factors (e.g. NF-κB, STAT or IkBzeta)? Have the authors investigated this or is there anything in the literature about this. The authors should discuss this more in the manuscript.

2) Surprisingly, dithranol does not have an anti-psoriatic effect in the imiquimod-induced psoriasis model. In contrast, dithranol has an anti-psoriatic effect in the c-Jun/JunB mouse model. Were the mouse strains used in the two models the same? Were the mice similar in age and did the authors use females or males and was this the same in the two models? The authors write that dithranol rather worsened the psoriatic lesions in the IMQ model. And interestingly the authors write that "Dithranol treatment of healthy murine skin led to similar effects". Could this be the same effect as has been described in perilesional skin? Is the inflammatory effect of dithranol on perilesional skin known? If so the authors could examine if this is also what is seen in skin of IMQ-treated mice. These issues could be discussed in more detail.

3) In the psoriasis patients treated with dithranol biopsies were taken from lesional skin over time. Did the authors take biopsies from the same "target" lesion throughout the study?

4) In Table 1 the authors should in the legend include that the numbers in the table are based on data from 15 psoriatic patients.

Summary:

Topic of interest but in major parts confirmatory to already published work and in other parts not conclusive. Please provide clarity about the data on AMPs in this manuscript and in the manuscript submitted to the other journal.

We analyzed biopsy samples from 15 psoriasis patients at several time points, i.e., before, during, after dithranol therapy and at a follow-up visits and from a well-matched control group (in a total more than 70 human biopsies) and used three mouse models (IMQ model, c-Jun/JunB model and mouse tail test) to substantiate our findings on the effects of dithranol in diseased psoriatic skin. We are first by using genome-wide expression analysis to investigate the effects of dithranol and by doing so we were able to thoroughly dissect the mechanisms of dithranol in diseased psoriatic skin. As pointed out, psoriatic response to dithranol was characterized by a rapid decrease in expression of keratinocyte differentiation regulators (e.g. involucrin, SERPINB7 and SERPINB13), anti-microbial peptides (e.g. ß-defensins like DEFB4A, DEFB4B, DEFB103A, S100 proteins like S100A7, S100A12), chemotactic factors for neutrophils (e.g. CXCL5, CXCL8) and neutrophilic infiltration and was followed with much delay by reduction in T cell infiltration. These findings are novel and haven't been reported previously.

We understand the reviewer’s concern that we recently submitted another manuscript about dithranol’s effects to a different journal. However, in the latter manuscript (Letter to the Editor) we report on dithranol's irritant effect (possibly playing a role in the therapy of alopecia areata) on healthy skin exclusively using healthy murine skin as well as xenografted healthy human skin. We want to point out that there is no overlap at all between the data and conclusions of the two manuscripts. However, we are happy to provide this letter on a confidential basis to eLife if the editors wish so.

Essential revisions:

Statistical analysis (see reviewer 2). Interpretation of data (specific for dithranol or bystander effects (see reviewers 1 and 2). Interpretation of data regarding the mouse models used as there seem a number of inconsistencies (see reviewers 2 and 3).

Interpretation of results in the light of the critique of all reviewers.

We have responded to all these comments appropriately (see below), including a revision of statistics.

Reviewer #1:

The manuscript by Benezeder et al. addresses an interesting topic. The role of keratinocytes for psoriasis pathogenesis has been neglected for many years and the work presented here re-focuses on keratinocyte-mediated effects. Interestingly, the authors used dithranol as a model, which is challenging since this topical compound shows beneficial therapeutic effects but also induces dose- and individual-depended skin irritation.

Although the methods employed are well established and state-of-the-art there are some points that need clarification.

• Psoriasis is viewed as systemic disorder and to judge the overall beneficial effect of any topical therapy including dithranol it should be shown whether there are effects on systemic inflammation (e.g. vascular inflammation). The authors should mention this as limitation of their approach.

We agree with the reviewer and have mentioned this limitation in the tenth paragraph of the Discussion.

• The most commonly used topical therapies are corticosteroids alone or in a fixed combination with a vitamin D analogue. The authors are encouraged to use such treatment as a control in order to prove that the observed effects are specific for dithranol.

Indeed, some downstream effects of dithranol seem to be overlapping with that of vitamin D3 analogues (more than with that of topical steroids), whereas other effects such as keratinocyte-neutrophil crosstalk are differential and seem to be specific for dithranol. We discuss this now in the Discussion and provide a couple of references on papers investigating the molecular effects of calcipotriol and topical steroids on human psoriasis.

• The irritant potential of dithranol is helpful in cases of otherwise refractory alopecia areata. The authors should briefly mention this and discuss if their data may explain the effect as an outlook for further investigation.

We entirely agree with the reviewer on the effect of dithranol in alopecia areata (see also comment above to the Editor) and have added a respective sentence to the eighth paragraph of the Discussion.

The reviewer is puzzled by the fact that he was asked to review a manuscript of the same group with the same first author with significant similarity to data that are described in this manuscript for another journal particularly about the effect of dithranol on antimicrobial peptides.

The authors must make clear if there is an overlap of data and which of the data presented in the other manuscript are also contained in this one.

Please see response above to the Editor's comments.

Reviewer #2:

[…] Although this is an interesting study, there are some major caveats and conflicting results, which make it difficult to accept this paper.

1) The main messages of this study – decrease of psoriatic epidermal thickening, reduction of Ki67 positivity, reduction of keratin 16 expression and AMPs by dithranol have been shown by previous reports from human skin (van de Kerkhof et al., 1996 and 2002; Eberle et al., 2017).

Please see response above to the Editor's comments. We would like to stress that we discuss the results of those previous studies by van de Kerkhof et al. and Eberle et al., 2017 now in more detail in our paper (Discussion, second paragraph). In addition, we wish to point out that we are the first to show dithranol’s effect on a transcriptional level in a large patient cohort with multiple time points of biopsy sampling and in two psoriasis mouse models. Moreover, our main conclusion is that inhibition of keratinocyte-neutrophil crosstalk as well as IL-36 pathway is crucial for its anti-psoriatic effect. These findings are entirely novel.

2) There is plenty of evidence demonstrating that psoriasis is an IL-17-driven immune disease. All treatments with a rapid clinical response will find a decrease in epidermal thickening, a normalization of keratinocyte differentiation and a decrease in immune cell infiltration as well as cytokine expression. From this perspective, one cannot conclude that the one or other treatment will work through keratinocytes and not influence the immune system. The changes in keratinocytes as observed in Figure 2 (epidermal thickening, Ki67 expression) seem to be significant on week 2 to 3. A similar pattern with a decrease in epidermal thickening, keratin 16 and Ki67 expression is visible at week 2 of treatment when using monoclonal antibodies e.g. directed against IL-17. Similarly, antimicrobial peptides are regulated at early time points by immunobiologics (several well-performed studies on this topic are published by Krueger and colleagues in JACI).

We agree with the reviewer, however, our data clearly indicate that dithranol treatment first acts on keratinocytes and later by delayed direct or indirect effects on the immune system. Indeed, certain biologics even of the latest generation have similar effects on the molecular level, which is fascinating, that a traditional topical agent like dithranol can show similar effects as a systemic antibody therapy. We believe that dithranol’s primary effect on keratinocytes is crucial for response to treatment, considering that differentially regulated genes in histological responders compared to non-responders belonged to pathways like keratinocyte differentiation, cornification and keratin filament formation (as mentioned in the third paragraph of the Discussion and Supplementary file 3).

We thank the reviewer for hinting us to the work of Krueger et al., 2012 and 2019). We have slightly modified the last sentence of the Results section and added those references in the seventh paragraph of the Discussion section, together with a couple of respective sentences.

3) The mouse model used (c-Jun/JunB knockout) is somehow artificial and does not require T cells for developing epidermal changes. Therefore, the findings – although of interest – cannot be translated to the human situation.

We entirely agree that findings from mouse models cannot be directly translated to the human situation. However, in addition to the imiquimod model, a solely immunologically mediated psoriasis model, we specifically have chosen the c-Jun/JunB model that is based on keratinocytes rather than on the immune system. We had mentioned this in the manuscript but to make our purpose clearer, we have now modified the wording (Discussion, first paragraph): "The therapeutic importance of reduction of IL-1 family members in human skin was substantiated by results generated in the keratinocyte-based c-Jun/JunB mouse psoriasis model."

4) The Major concern is that most of the findings are descriptive. The authors performed many microarray analyses but studies on functional mechanisms confirming their hypothesis are missing.

We would have loved to substantiate our findings on the functional level and have mentioned this shortage of the paper "What our work does not answer, is how dithranol exactly acts at the molecular level. Cell culture studies have shown that dithranol targets mitochondria.…". Anti-psoriatic effects of other topical treatment options have recently been linked to modulation of AhR (Smith et al., 2017) and AhR may play a role in pathogenesis of psoriasis (DiMeglio et al., 2014). Thus, we made attempts to identify the receptor of dithranol by using aryl hydrocarbon receptor (AhR) knockout mice in order to substantiate our findings on the functional level. However, it appears that dithranol does not act via modulation of AhR, as we did not observe differences in the response of skin to dithranol comparing AhR knockout mice to controls. Certainly, future studies are indeed needed to identify dithranol’s yet unknown receptor. This all has now been worded in the Discussion.

5) The increase in epidermal thickening, the changes in keratin 16 and AMPs found in the mouse tail experiment with dithranol contradict the results found in human psoriasis (Figure 4).

We agree with the reviewer, the effects of dithranol in the mouse-tail test seem to be contradictory. However, this test has limitations since its usefulness only refers to which degree an agent or treatment increases orthokeratosis as a measure of keratinocyte differentiation-inducing activity (Figure 3—figure supplement 1), as we have demonstrated it for dithranol. On the other hand, as the reviewer observed, besides upregulation of keratinocyte markers dithranol increased certain psoriasis-associated AMPs such as Defb3 and S100a8 and S100a9 but decreased the pro-psoriatic antimicrobial peptide Camp/LL37 as well as Cxcl5, a chemotactic factor for neutrophils (Figure 3—figure supplement 2). We now have mentioned this in the Discussion, together with a couple of new references and stressed the limitations of the mouse tail-test (third paragraph).

6) IL-36 cytokines are mainly expressed by keratinocytes (Wenzel et al., J Invest Dermatol 2015), but the authors describe a decrease at week 2 to 3. If dithranol targets primarily keratinocytes and inhibits IL-36, the effect should be visible at earlier time points.

We thank the reviewer for providing the reference of Wenzel et al. with regard to IL-36 expression in keratinocytes. Although we already saw a reduction of keratinocyte-related genes at day 6, a lot of these genes were only normalized at week 2-3, accompanied with a significantly diminished mRNA expression of pro-psoriatic IL-1 family members (IL36A, IL36G and IL36RN) (Supplementary file 1 and Figure 4). At day 6, PASI had decreased by 33%, but at week 2-3 we saw a reduction of 58%. Most likely, dithranol’s effect on members of the IL-36 family might be linked to its strong clinical effect at week 2-3. The therapeutic importance of reduction of IL-1 family members in human skin was substantiated by results generated in the keratinocyte-based c-Jun/JunB mouse psoriasis model, where IL-36 family genes were significantly reduced in dithranol-treated psoriatic c-Jun/JunB lesions compared to controls. We have added a respective sentence on IL-36 in the first paragraph of the Discussion.

Reviewer #3:

[…] I have the following comments:

1) Although the data in the manuscript clearly shows that the early mode of action of dithranol in psoriasis treatment is on keratinocyte-derived molecules and subsequently neutrophil infiltration to the skin the manuscript does not reveal the molecular mechanism behind this. Does dithranol use a membrane bound receptor, an intracellular receptor or does it inhibit specific transcription factors (e.g. NF-κB, STAT or IkBzeta)? Have the authors investigated this or is there anything in the literature about this. The authors should discuss this more in the manuscript.

We agree with the reviewer on that point and have discussed the limitations of our work now in more detail (Discussion, ninth paragraph). What indeed our work does not answer, is how dithranol exactly acts at the molecular level, which we also mention in the ninth paragraph of the Discussion, paragraph 1. Little is known in the literature about dithranol's potential receptor that remains yet to be determined. Please see also our response in this regard to comment 4 of reviewer 2 about the aryl hydrocarbon receptor (AhR).

2) Surprisingly, dithranol does not have an anti-psoriatic effect in the imiquimod-induced psoriasis model. In contrast, dithranol has an anti-psoriatic effect in the c-Jun/JunB mouse model. Were the mouse strains used in the two models the same? Were the mice similar in age and did the authors use females or males and was this the same in the two models? The authors write that dithranol rather worsened the psoriatic lesions in the IMQ model. And interestingly the authors write that "Dithranol treatment of healthy murine skin led to similar effects". Could this be the same effect as has been described in perilesional skin? Is the inflammatory effect of dithranol on perilesional skin known? If so the authors could examine if this is also what is seen in skin of IMQ-treated mice. These issues could be discussed in more detail.

As stated in the manuscript (see subsection “Animals”), we used 6-9 week-old mice with different strains. BALB/c mice are commonly used for the Imiquimod model, as we did; the c-Jun/JunB knockout mice of our study have a mixed background, as described by Zenz et al., 2005. However, it is highly unlikely that the different mouse strains played any role in the outcome of the experiments since the two psoriasis models are per se completely different. Immunological aspects are important in the imiquimod model and molecular alterations dominate the c-Jun/JunB model. Concerning the effect of dithranol on healthy murine skin we have added a couple of sentences on dithranol’s irritant effect that may be rather a side effect without functional relevance in human psoriasis (Discussion, fourth paragraph) but crucial for dithranol's action in alopecia areata (see response to the comment 3 of reviewer #1 above). We now have added a couple of respective sentences on alopecia areata in the Discussion.

3) In the psoriasis patients treated with dithranol biopsies were taken from lesional skin over time. Did the authors take biopsies from the same "target" lesion throughout the study?

We have now precisely outlined how marker lesions were defined, scored, treated and biopsy-sampled in the Materials and methods subsection under the new heading “Marker lesions and scoring”. This has also been illustrated in Figure 1A.

4) In Table 1 the authors should in the legend include that the numbers in the table are based on data from 15 psoriatic patients.

We thank the reviewer for mentioning this and have changed the heading of Table 1 accordingly.

Author details

1. Theresa Benezeder

   Department of Dermatology, Medical University of Graz, Graz, Austria

   Contribution

   Conceptualization, Data curation, Formal analysis, Validation, Investigation, Visualization, Methodology, Writing - original draft, Project administration

   Contributed equally with

   Clemens Painsi

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0001-6218-2792

2. Clemens Painsi

   State Hospital Klagenfurt, Klagenfurt am Wörthersee, Austria

   Contribution

   Conceptualization, Data curation, Investigation, Project administration

   Contributed equally with

   Theresa Benezeder

   Competing interests

   No competing interests declared

3. VijayKumar Patra

   Department of Dermatology, Medical University of Graz, Graz, Austria

   Contribution

   Visualization, Methodology

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-7161-7767

4. Saptaswa Dey

   Department of Dermatology, Medical University of Graz, Graz, Austria

   Contribution

   Methodology

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0001-7532-7858

5. Martin Holcmann

   Institute of Cancer Research, Department of Medicine I, Comprehensive Cancer Center, Medical University of Vienna, Vienna, Austria

   Contribution

   Resources, Methodology

   Competing interests

   No competing interests declared

6. Bernhard Lange-Asschenfeldt

   State Hospital Klagenfurt, Klagenfurt am Wörthersee, Austria

   Contribution

   Resources, Project administration

   Competing interests

   No competing interests declared

7. Maria Sibilia

   Institute of Cancer Research, Department of Medicine I, Comprehensive Cancer Center, Medical University of Vienna, Vienna, Austria

   Contribution

   Resources, Writing - review and editing

   Competing interests

   No competing interests declared

8. Peter Wolf

   Department of Dermatology, Medical University of Graz, Graz, Austria

   Contribution

   Conceptualization, Supervision, Funding acquisition, Writing - review and editing

   For correspondence

   peter.wolf@medunigraz.at

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0001-7777-9444

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