TWEAK mediates inflammation in experimental atopic dermatitis and psoriasis

Atopic dermatitis (AD) and psoriasis are driven by alternate type 2 and type 17 immune responses, but some proteins might be critical to both diseases. Here we show that a deficiency of the TNF superfamily molecule TWEAK (TNFSF12) in mice results in defective maintenance of AD-specific T helper type 2 (Th2) and psoriasis-specific Th17 cells in the skin, and impaired expression of disease-characteristic chemokines and cytokines, such as CCL17 and TSLP in AD, and CCL20 and IL-19 in psoriasis. The TWEAK receptor, Fn14, is upregulated in keratinocytes and dermal fibroblasts, and TWEAK induces these cytokines and chemokines alone and in synergy with the signature T helper cytokines of either disease, IL-13 and IL-17. Furthermore, subcutaneous injection of recombinant TWEAK into naive mice induces cutaneous inflammation with histological and molecular signs of both diseases. TWEAK is therefore a critical contributor to skin inflammation and a possible therapeutic target in AD and psoriasis.

The only thing that they might want to consider in the discussion is that TWEAK induces noncanonical NF-κB and sensitisation to TNF induced killing. The authors themselves comment on the fact that this may be keratinocyte driven. The phenotypes they see are very reminiscent of the keratinocyte deficient TRAF2 mice eLIfe Etemadi et al 2015 (which have constitutive non-canonical NF-κB and are sensitive to TNF killing). If the authors have a few more words left in the discussion it might be worth making the comparison.
Reviewer #2 (Remarks to the Author): Thank you for this interesting work looking at the role of TWEAK in the important skin diseases AD and psoriasis. The work is novel and original but I have some questions and concerns, outlined below: (1) Your paper argues strongly that TWEAK plays a role in both AD and psoriasis, however the data may also be interpreted as showing a role in non-specific skin inflammation. It would be helpful for you to consider this possibility and if you still feel the data support specific roles in both AD and psoriasis please explain more fully how the one pathway can 'drive' what are usually mutually exclusive skin diseases in human.
(2) Your introduction and discussion raise the role of TWEAK in systemic inflammation; is there any evidence of extracutaneous involvement in the mouse models?
(3) The title is rather bold and should perhaps be re-worded to reflect the evidence presented of TWEAK maintaining/mediating inflammation (rather than 'driving') and also the fact that the data are from mouse models of AD and psoriasis.
(4) The abstract is unclear where it lists the inflammatory mediators in each disease without clearly stating which is for which disease. It is also not clear why the final statement reports 'TWEAK is a ubiquitous regulator of skin inflammation ...' this seems to be over-stating your data.
(5) The results of s/c injection of TWEAK are said to produce 'localised skin inflammation' but the distribution of inflammation is not shown in the results.
(6) What was the rationale for choosing to study the 5 chemokines (page 9, line 203)?
(7) Page 10, line 251, the data show that TWEAK plays a role in TSLP and IL-19 production but not that it is *critical*.
(8) The gene set enrichment analysis results are presented for the first time in the discussion. It would be helpful to expand this analysis (there is a considerable amount of publically available data on genetic variation and gene expression in AD and psoriasis) if possible, and this could provide support for the authors' hypothesis of a role for TWEAK in these diseases.
(9) Is there any evidence of increased or deceased apoptosis occurring within the skin of the mouse models as may be predicted from the knowledge of TWEAK?
(10) Why is there only 1 control for many of the key experiments? (Fig 1 a, b,g,h,Fig 2 a,b,g,h,Fig 4 d,f,g,h) The analyses would be more convincing if a greater number of controls could be included.
(10) In suppl fig 1, the Fn14 appears to be green (not red/brown as stated) and the lines indicating skin surface (panel B) and dermoepidermal junction (panel C) do not appear to be in the correct place.

Reviewer #1
We appreciate the positive comments of the reviewer.
1. The only thing that they might want to consider in the discussion is that TWEAK induces non-canonical NF-κB and sensitisation to TNF induced killing…The phenotypes they see are very reminiscent of the keratinocyte deficient TRAF2 mice eLIfe Etemadi et al 2015 (which have constitutive non-canonical NF-κB and are sensitive to TNF killing). If the authors have a few more words left in the discussion it might be worth making the comparison.
We have included a comment on non-canonical NFB signaling and apoptosis in the discussion and referenced the Etemadi paper (page 16).

Reviewer #2
Again, we are grateful for the enthusiasm from reviewer 2.

Your paper argues strongly that TWEAK plays a role in both AD and psoriasis, however the data may also be interpreted as showing a role in non-specific skin inflammation. It would be helpful for you to consider this possibility and if you still feel the data support specific roles in both AD and psoriasis please explain more fully how the one pathway can 'drive' what are usually mutually exclusive skin diseases in human.
To address the reviewers comment, we have added to the discussion of the manuscript to explain more fully why we think TWEAK can be active and play an important role in what are considered two different diseases (page 13/14). We should point out there is a precedent for this in a related molecule, TNF, that is active in diseases as diverse as psoriasis, rheumatoid arthritis, and inflammatory bowel disease. The notion that AD and psoriasis are mutually exclusive diseases in humans stems in an immunological standpoint from data showing they are driven by separate lineages of T cells and other immune cell types that contribute Th2 vs. Th17 cytokines respectively. Although these drive the phenotype of each disease, this does not argue against the involvement of common factors that would co-operate with these individual T cell populations or cytokines. Our data clearly demonstrate that TWEAK is important for disease in established models of AD and psoriasis that display the Th2 (IL-13) or Th17 (IL-17) phenotypes, but in either case TWEAK and its receptor are upregulated at the same time. The rationale for the importance of these molecules is then that they are not restricted to a particular lineage of cells and do not control development of these lineages of cells, and they do not rely on these cells or their cytokines for being produced or upregulated. TWEAK alone cannot promote complete AD or psoriasis phenotypes but it can promote chemokines common to both AD and psoriasis from keratinocytes and fibroblasts, as well as characteristic cytokines of both diseases such as TSLP and IL-19. As importantly, TWEAK can synergize with the disease-specific cytokines IL-13 and IL-17 in these activities, further explaining why this one pathway can be important for pathology in two diseases thought to be fundamentally different from one another. In terms of the reviewers reference to non-specific skin inflammation, we are not sure what is meant by this statement, but it is certainly possible that TWEAK will contribute to other skin pathologies and this is something that should be investigated in the future.

Your introduction and discussion raise the role of TWEAK in systemic inflammation; is there any evidence of extracutaneous involvement in the mouse models?
Both experimental models described induce robust and localized skin pathology on the rostral back of the animals. Nevertheless, we and others have seen a systemic immune response in both HDM and IMQ models. The HDM model induces adenopathy of the draining (axillary) lymph nodes and abundant IgE serum levels (Kawakami, Methods Mol Biol. 2015;1220:497-502). Similarly, the IMQ model leads to lymphadenopathy, splenomegaly and blood neutrophilia at the later stages of disease (Vinter, Exp Dermatol. 2016Dec 11. doi: 10.1111). While we have not focused our study on systemic effects, we did observe diminished adenopathy and splenomegaly in animals deficient for TWEAK or treated with TWEAK-neutralizing reagents in the respective models. It remains to be seen if TWEAK produced in the skin can itself become systemic and then directly affect immune responses in other organs, and this would be interesting to study in the future.
3. The title is rather bold and should perhaps be re-worded to reflect the evidence presented of TWEAK maintaining/mediating inflammation (rather than 'driving') and also the fact that the data are from mouse models of AD and psoriasis.
As requested we have altered the title of the manuscript.

The abstract is unclear where it lists the inflammatory mediators in each disease without clearly stating which is for which disease. It is also not clear why the final statement reports 'TWEAK is a ubiquitous regulator of skin inflammation ...' this seems to be over-stating your data.
We have modified the abstract to further clarify the distinct immunological mechanisms of AD and psoriasis and state the respective cytokines involved. To prevent overstatement of the study's implication, we changed the wording in the final sentence from "ubiquitous regulator" to "critical contributor".

The results of s/c injection of TWEAK are said to produce 'localised skin inflammation' but the distribution of inflammation is not shown in the results.
We have clarified in the text that injection of rTWEAK into the rostral regions of the animals back induced a clinically defined dermatitis only in the region of injection (page 8)

What was the rationale for choosing to study the 5 chemokines (page 9, line 203)?
We chose to focus on CCL2, 5 and 7 as they are involved in the influx and maintenance of monocytes/macrophages, eosinophils, and T cells common to both diseases, and CCL17 and CCL20, which are believed to be restricted to distinct populations associated with one or the other disease, eosinophils and Th2 cells for CCL17, and Th17 cells for CCL20. This is clarified in the text on page 9. 7. Page 10, line 251, the data show that TWEAK plays a role in TSLP and IL-19 production but not that it is *critical*.
We have altered the text to read "played a role in" rather than "was critical for" (page 11).
8. The gene set enrichment analysis results are presented for the first time in the discussion. It would be helpful to expand this analysis (there is a considerable amount of publically available data on genetic variation and gene expression in AD and psoriasis) if possible, and this could provide support for the authors' hypothesis of a role for TWEAK in these diseases.
We included the gene set enrichment analysis in the paper as we thought it was a nice addition to the publications showing upregulation of Fn14 and TWEAK at the protein level in skin or serum samples from patients with AD or psoriasis. We believe the protein data is more important than the genetic analysis, but as the reviewer thinks that both are valuable to our conclusions and hypotheses regarding the human diseases, we have moved our analysis to the end of the results section while at the same time quoting the more conventional studies of protein expression in AD and psoriasis patients (page 11/12).

9.
Is there any evidence of increased or deceased apoptosis occurring within the skin of the mouse models as may be predicted from the knowledge of TWEAK?
While TWEAK alone is only a weak pro-apoptotic stimulus, it can sensitize cells to other mediators, such as TNF. As remarked by reviewer 1, a recent study by Emetadi et al described a spontaneous psoriasis-like disease in animals with a deficiency in Traf2 that involved keratinocyte cell death and was partially dependent on TNF, and in line with the reviewers request we now discuss the possible involvement of TWEAK (page 16). While our experiments did not specifically focus on apoptosis, we did not observe excessive apoptotic features within the skin of our experimental animals. Furthermore, treatment of keratinocytes or dermal fibroblasts with rTWEAK alone or with IL-13 or IL-17 did not elicit overt cellular death. However, at present we cannot make a definitive statement about TWEAK and apoptosis in the skin.

10.
Why is there only 1 control for many of the key experiments? (Fig 1 a,b,g,h, Fig 2  a,b,g,h, Fig 4 d,f,g,h) The analyses would be more convincing if a greater number of controls could be included.
We have now combined independent experiments in Figure 1a We apologize for the errors. The figure legend has been changed accordingly and the lines for skin surface and epidermal-dermal junction redrawn.