Epithelial HMGB1 Delays Skin Wound Healing and Drives Tumor Initiation by Priming Neutrophils for NET Formation

•HMGB1 controls transition from pre-cancerous to cancerous lesions in skin•Epithelial HMGB1 promotes neutrophil recruitment and NET formation•TNF and RIPK1 activity mediate HMGB1-dependent NET formation and skin tumorigenesis•NET formation occurs in lesional and tumor-associated skin of RDEB patients Regenerative responses predispose tissues to tumor formation by largely unknown mechanisms. High-mobility group box 1 (HMGB1) is a danger-associated molecular pattern contributing to inflammatory pathologies. We show that HMGB1 derived from keratinocytes, but not myeloid cells, delays cutaneous wound healing and drives tumor formation. In wounds of mice lacking HMGB1 selectively in keratinocytes, a marked reduction in neutrophil extracellular trap (NET) formation is observed. Pharmacological targeting of HMGB1 or NETs prevents skin tumorigenesis and accelerates wound regeneration. HMGB1-dependent NET formation and skin tumorigenesis is orchestrated by tumor necrosis factor (TNF) and requires RIPK1 kinase activity. NETs are present in the microenvironment of keratinocyte-derived tumors in mice and lesional and tumor skin of patients suffering from recessive dystrophic epidermolysis bullosa, a disease in which skin blistering predisposes to tumorigenesis. We conclude that tumorigenicity of the wound microenvironment depends on epithelial-derived HMGB1 regulating NET formation, thereby establishing a mechanism linking reparative inflammation to tumor initiation. Regenerative responses predispose tissues to tumor formation by largely unknown mechanisms. High-mobility group box 1 (HMGB1) is a danger-associated molecular pattern contributing to inflammatory pathologies. We show that HMGB1 derived from keratinocytes, but not myeloid cells, delays cutaneous wound healing and drives tumor formation. In wounds of mice lacking HMGB1 selectively in keratinocytes, a marked reduction in neutrophil extracellular trap (NET) formation is observed. Pharmacological targeting of HMGB1 or NETs prevents skin tumorigenesis and accelerates wound regeneration. HMGB1-dependent NET formation and skin tumorigenesis is orchestrated by tumor necrosis factor (TNF) and requires RIPK1 kinase activity. NETs are present in the microenvironment of keratinocyte-derived tumors in mice and lesional and tumor skin of patients suffering from recessive dystrophic epidermolysis bullosa, a disease in which skin blistering predisposes to tumorigenesis. We conclude that tumorigenicity of the wound microenvironment depends on epithelial-derived HMGB1 regulating NET formation, thereby establishing a mechanism linking reparative inflammation to tumor initiation. Danger-associated molecular patterns (DAMPs) activate the immune system upon injury and have key roles in cancer by either promoting inflammation or by driving the cytotoxic anti-tumor host response (Hernandez et al., 2016Hernandez C. Huebener P. Schwabe R.F. Damage-associated molecular patterns in cancer: a double-edged sword.Oncogene. 2016; 35: 5931-5941Crossref PubMed Scopus (162) Google Scholar). However, the specific contribution of individual DAMPs in these processes are still enigmatic. High-mobility group box 1 (HMGB1) is a multifunctional protein that serves as a transcriptional regulator in the nucleus but upon secretion acts as a DAMP, potently activating inflammation (Scaffidi et al., 2002Scaffidi P. Misteli T. Bianchi M.E. Release of chromatin protein HMGB1 by necrotic cells triggers inflammation.Nature. 2002; 418: 191-195Crossref PubMed Scopus (3095) Google Scholar). HMGB1 performs its cytokine function by binding to various receptors, the best documented being RAGE (receptor for advanced glycation end products) and TLR-4 (Toll-like receptor 4) (Hori et al., 1995Hori O. Brett J. Slattery T. Cao R. Zhang J. Chen J.X. Nagashima M. Lundh E.R. Vijay S. Nitecki D. et al.The receptor for advanced glycation end products (RAGE) is a cellular binding site for amphoterin. Mediation of neurite outgrowth and co-expression of rage and amphoterin in the developing nervous system.J. Biol. Chem. 1995; 270: 25752-25761Crossref PubMed Scopus (973) Google Scholar, Yang et al., 2010Yang H. Hreggvidsdottir H.S. Palmblad K. Wang H. Ochani M. Li J. Lu B. Chavan S. Rosas-Ballina M. Al-Abed Y. et al.A critical cysteine is required for HMGB1 binding to Toll-like receptor 4 and activation of macrophage cytokine release.Proc. Natl. Acad. Sci. USA. 2010; 107: 11942-11947Crossref PubMed Scopus (543) Google Scholar). Increased levels of circulating HMGB1 have been shown to amplify the inflammatory responses that regulate tumor progression, invasion, and metastasis (Kang et al., 2013Kang R. Zhang Q. Zeh 3rd, H.J. Lotze M.T. Tang D. HMGB1 in cancer: good, bad, or both?.Clin. Cancer Res. 2013; 19: 4046-4057Crossref PubMed Scopus (288) Google Scholar), but the importance of HMGB1 in initiating neoplastic responses is largely unknown. In skin, we previously showed that flagellated bacteria can trigger TLR-5 activation in leukocytes, inducing the upregulation of cutaneous HMGB1 expression levels and the promotion of wound-induced tumorigenesis (Hoste et al., 2015Hoste E. Arwert E.N. Lal R. South A.P. Salas-Alanis J.C. Murrell D.F. Donati G. Watt F.M. Innate sensing of microbial products promotes wound-induced skin cancer.Nat. Commun. 2015; 6: 5932Crossref PubMed Scopus (68) Google Scholar). Although HMGB1 has previously been implicated in tissue regeneration (Huebener et al., 2015Huebener P. Pradere J.P. Hernandez C. Gwak G.Y. Caviglia J.M. Mu X. Loike J.D. Schwabe R.F. The HMGB1/RAGE axis triggers neutrophil-mediated injury amplification following necrosis.J. Clin. Invest. 2015; 125: 539-550Crossref PubMed Scopus (229) Google Scholar, Straino et al., 2008Straino S. Di Carlo A. Mangoni A. De Mori R. Guerra L. Maurelli R. Panacchia L. Di Giacomo F. Palumbo R. Di Campli C. et al.High-mobility group box 1 protein in human and murine skin: involvement in wound healing.J. Invest. Dermatol. 2008; 128: 1545-1553Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar, Tirone et al., 2018Tirone M. Tran N.L. Ceriotti C. Gorzanelli A. Canepari M. Bottinelli R. Raucci A. Di Maggio S. Santiago C. Mellado M. et al.High mobility group box 1 orchestrates tissue regeneration via CXCR4.J. Exp. Med. 2018; 215: 303-318Crossref PubMed Scopus (69) Google Scholar), the molecular mechanisms through which this DAMP mediates wound repair and neoplastic events in skin are still unknown. Chronic and acute wounding predisposes tissues to tumor formation (Arwert et al., 2012Arwert E.N. Hoste E. Watt F.M. Epithelial stem cells, wound healing and cancer.Nat. Rev. Cancer. 2012; 12: 170-180Crossref PubMed Scopus (281) Google Scholar). In skin, this association is remarkably clear in patients suffering from recessive dystrophic epidermolysis bullosa (RDEB), a rare genetic cutaneous blistering disorder. Skin of RDEB patients constantly undergoes injury and repair, predisposing the epidermis to squamous cell carcinoma (SCC) formation (Fine et al., 2009Fine J.D. Johnson L.B. Weiner M. Li K.P. Suchindran C. Epidermolysis bullosa and the risk of life-threatening cancers: the National EB Registry experience, 1986-2006.J. Am. Acad. Dermatol. 2009; 60: 203-211Abstract Full Text Full Text PDF PubMed Scopus (250) Google Scholar). We previously demonstrated that HMGB1 is abundantly secreted in SCC-associated skin of RDEB patients (Hoste et al., 2015Hoste E. Arwert E.N. Lal R. South A.P. Salas-Alanis J.C. Murrell D.F. Donati G. Watt F.M. Innate sensing of microbial products promotes wound-induced skin cancer.Nat. Commun. 2015; 6: 5932Crossref PubMed Scopus (68) Google Scholar). Moreover, levels of circulating HMGB1 have been shown to correlate with RDEB disease severity (Petrof et al., 2013Petrof G. Abdul-Wahab A. Proudfoot L. Pramanik R. Mellerio J.E. McGrath J.A. Serum levels of high mobility group box 1 correlate with disease severity in recessive dystrophic epidermolysis bullosa.Exp. Dermatol. 2013; 22: 433-435Crossref PubMed Scopus (23) Google Scholar). Neutrophils are the first immune cells recruited to wounded skin, where they can form neutrophil extracellular traps (NETs), enabling neutrophils to fight pathogens (Belaaouaj et al., 1998Belaaouaj A. McCarthy R. Baumann M. Gao Z. Ley T.J. Abraham S.N. Shapiro S.D. Mice lacking neutrophil elastase reveal impaired host defense against gram negative bacterial sepsis.Nat. Med. 1998; 4: 615-618Crossref PubMed Scopus (520) Google Scholar, Brinkmann et al., 2004Brinkmann V. Reichard U. Goosmann C. Fauler B. Uhlemann Y. Weiss D.S. Weinrauch Y. Zychlinsky A. Neutrophil extracellular traps kill bacteria.Science. 2004; 303: 1532-1535Crossref PubMed Scopus (4989) Google Scholar). NET formation is characterized by the release of neutrophil chromatin decorated with antimicrobial peptides in order to trap and potentially kill pathogens. Although being an important antimicrobial defense mechanism, NETs can also induce tissue damage, as is the case in diabetic conditions, where neutrophils are primed to form NETs, thereby delaying cutaneous wound healing (Menegazzo et al., 2015Menegazzo L. Ciciliot S. Poncina N. Mazzucato M. Persano M. Bonora B. Albiero M. Vigili de Kreutzenberg S. Avogaro A. Fadini G.P. NETosis is induced by high glucose and associated with type 2 diabetes.Acta Diabetol. 2015; 52: 497-503Crossref PubMed Scopus (107) Google Scholar, Wong et al., 2015Wong S.L. Demers M. Martinod K. Gallant M. Wang Y. Goldfine A.B. Kahn C.R. Wagner D.D. Diabetes primes neutrophils to undergo NETosis, which impairs wound healing.Nat. Med. 2015; 21: 815-819Crossref PubMed Scopus (433) Google Scholar). Since NETs have also been shown to impact on a plethora of diseases, including asthma and arthritis (Jorch and Kubes, 2017Jorch S.K. Kubes P. An emerging role for neutrophil extracellular traps in noninfectious disease.Nat. Med. 2017; 23: 279-287Crossref PubMed Scopus (426) Google Scholar, Khandpur et al., 2013Khandpur R. Carmona-Rivera C. Vivekanandan-Giri A. Gizinski A. Yalavarthi S. Knight J.S. Friday S. Li S. Patel R.M. Subramanian V. et al.NETs are a source of citrullinated autoantigens and stimulate inflammatory responses in rheumatoid arthritis.Sci. Transl. Med. 2013; 5: 178ra40Crossref PubMed Scopus (633) Google Scholar, Toussaint et al., 2017Toussaint M. Jackson D.J. Swieboda D. Guedán A. Tsourouktsoglou T.D. Ching Y.M. Radermecker C. Makrinioti H. Aniscenko J. Bartlett N.W. et al.Host DNA released by NETosis promotes rhinovirus-induced type-2 allergic asthma exacerbation.Nat. Med. 2017; 23: 681-691Crossref PubMed Scopus (139) Google Scholar), and have been observed in the microenvironment of various tumor types (Demers et al., 2016Demers M. Wong S.L. Martinod K. Gallant M. Cabral J.E. Wang Y. Wagner D.D. Priming of neutrophils toward NETosis promotes tumor growth.OncoImmunology. 2016; 5: e1134073Crossref PubMed Scopus (88) Google Scholar), inhibiting NET formation might be an interesting therapeutic strategy to treat disease. One important cytokine that has been implicated in neutrophil priming is tumor necrosis factor (TNF) (Yuo et al., 1991Yuo A. Kitagawa S. Kasahara T. Matsushima K. Saito M. Takaku F. Stimulation and priming of human neutrophils by interleukin-8: cooperation with tumor necrosis factor and colony-stimulating factors.Blood. 1991; 78: 2708-2714PubMed Google Scholar), which is also important for chemical- and wound-induced skin carcinogenesis (Hoste et al., 2015Hoste E. Arwert E.N. Lal R. South A.P. Salas-Alanis J.C. Murrell D.F. Donati G. Watt F.M. Innate sensing of microbial products promotes wound-induced skin cancer.Nat. Commun. 2015; 6: 5932Crossref PubMed Scopus (68) Google Scholar, Moore et al., 1999Moore R.J. Owens D.M. Stamp G. Arnott C. Burke F. East N. Holdsworth H. Turner L. Rollins B. Pasparakis M. et al.Mice deficient in tumor necrosis factor-alpha are resistant to skin carcinogenesis.Nat. Med. 1999; 5: 828-831Crossref PubMed Scopus (723) Google Scholar), implicating a role for TNF in mediating NET formation downstream of HMGB1 release. Here, we tested the hypothesis that a sole DAMP, namely HMGB1, impacts on pathological cutaneous remodeling and tumor initiation. We could demonstrate a crucial role for epithelial-derived HMGB1 in regenerative and neoplastic inflammation and show that keratinocyte-specific HMGB1 primes neutrophils for NET formation in full-thickness skin wounds, resulting in delayed cutaneous wound closure and pro-tumorigenic signaling via TNF and RIPK1 kinase signaling. These findings identify epithelial-derived HMGB1 as a crucial molecule mediating tumorigenicity of the wound microenvironment and suggest that HMGB1 neutralization or inhibition of NET formation may have potential as a therapeutic strategy to prevent skin cancer and accelerate cutaneous wound healing responses. In order to understand the in vivo function of HMGB1 as a DAMP in cutaneous regenerative and neoplastic responses, we selectively deleted HMGB1 in keratinocytes or monocytes/macrophages by crossing HMGB1 “floxed” mice (Huebener et al., 2015Huebener P. Pradere J.P. Hernandez C. Gwak G.Y. Caviglia J.M. Mu X. Loike J.D. Schwabe R.F. The HMGB1/RAGE axis triggers neutrophil-mediated injury amplification following necrosis.J. Clin. Invest. 2015; 125: 539-550Crossref PubMed Scopus (229) Google Scholar) to mice expressing keratin-5 Cre (Tarutani et al., 1997Tarutani M. Itami S. Okabe M. Ikawa M. Tezuka T. Yoshikawa K. Kinoshita T. Takeda J. Tissue-specific knockout of the mouse Pig-a gene reveals important roles for GPI-anchored proteins in skin development.Proc. Natl. Acad. Sci. USA. 1997; 94: 7400-7405Crossref PubMed Scopus (208) Google Scholar) or lysozyme M Cre (Clausen et al., 1999Clausen B.E. Burkhardt C. Reith W. Renkawitz R. Förster I. Conditional gene targeting in macrophages and granulocytes using LysMcre mice.Transgenic Res. 1999; 8: 265-277Crossref PubMed Scopus (1399) Google Scholar), respectively (Figures S1A and S1B). Neither keratinocyte-specific HMGB1 knockout (ΔKer HMGB1) mice nor myeloid-specific HMGB1 knockout (Δmyelo HMGB1) mice displayed overt phenotypes. However, when subjected to full-thickness skin wounding, healing rates were markedly accelerated in ΔKer HMGB1 mice relative to littermate HMGB1fl/fl (Cre negative) control mice (Figure 1A; Figure S1C). Interestingly, wound healing rates were indistinguishable between mice lacking HMGB1 in myeloid cells and control littermates (Figure S1D). The accelerated wound healing rates observed in ΔKer HMGB1 skin were not due to cell-autonomous keratinocyte effects, as primary keratinocytes in culture showed faster healing effects in scratch-wound assays in HMGB1-proficient versus HMGB1-deficient cultures (Figure S1E). HMGB1 is upregulated in various chronically inflamed conditions (Degryse et al., 2001Degryse B. Bonaldi T. Scaffidi P. Müller S. Resnati M. Sanvito F. Arrigoni G. Bianchi M.E. The high mobility group (HMG) boxes of the nuclear protein HMG1 induce chemotaxis and cytoskeleton reorganization in rat smooth muscle cells.J. Cell Biol. 2001; 152: 1197-1206Crossref PubMed Scopus (392) Google Scholar, Taniguchi et al., 2003Taniguchi N. Kawahara K. Yone K. Hashiguchi T. Yamakuchi M. Goto M. Inoue K. Yamada S. Ijiri K. Matsunaga S. et al.High mobility group box chromosomal protein 1 plays a role in the pathogenesis of rheumatoid arthritis as a novel cytokine.Arthritis Rheum. 2003; 48: 971-981Crossref PubMed Scopus (408) Google Scholar); therefore, we next investigated wound healing responses in transgenic mice expressing a constitutively active MAP-kinase kinase 1 (MEK1) in differentiating keratinocytes (InvEE mice). These mice exhibit chronic skin inflammation, allowing the study of wound healing responses in a pathological skin context (Hobbs et al., 2004Hobbs R.M. Silva-Vargas V. Groves R. Watt F.M. Expression of activated MEK1 in differentiating epidermal cells is sufficient to generate hyperproliferative and inflammatory skin lesions.J. Invest. Dermatol. 2004; 123: 503-515Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar). Upon skin wounding, InvEE mice lacking HMGB1 in keratinocytes closed their wounds markedly faster than HMGB1-proficient InvEE mice (Figure 1B; Figure S1F). In contrast, myeloid-cell-specific deletion of HMGB1 in InvEE mice did not result in altered wound closure rates (Figure S1G). Together, these data indicate that HMGB1 derived from keratinocytes, but not myeloid cells, plays an important role in reparative cutaneous inflammation. HMGB1 has a context-dependent role in either promoting tumorigenesis by triggering chronic inflammation or in protecting from tumor development by eliciting immunogenic cell death (Hernandez et al., 2016Hernandez C. Huebener P. Schwabe R.F. Damage-associated molecular patterns in cancer: a double-edged sword.Oncogene. 2016; 35: 5931-5941Crossref PubMed Scopus (162) Google Scholar, Kang et al., 2013Kang R. Zhang Q. Zeh 3rd, H.J. Lotze M.T. Tang D. HMGB1 in cancer: good, bad, or both?.Clin. Cancer Res. 2013; 19: 4046-4057Crossref PubMed Scopus (288) Google Scholar). To unravel the function of HMGB1 in cutaneous tumor initiation, we subjected InvEE mice lacking HMGB1 in either keratinocytes or in myeloid cells to wound-induced tumor formation. InvEE mice develop tumors at sites of full-thickness skin wounding with an incidence of 50% (Arwert et al., 2010Arwert E.N. Lal R. Quist S. Rosewell I. van Rooijen N. Watt F.M. Tumor formation initiated by nondividing epidermal cells via an inflammatory infiltrate.Proc. Natl. Acad. Sci. USA. 2010; 107: 19903-19908Crossref PubMed Scopus (58) Google Scholar). Strikingly, ΔKer HMGB1 mice were completely protected from wound-induced tumor formation (Figure 1C), while Δmyelo HMGB1 mice were equally sensitive to InvEE papillomagenesis (Figure S1H). Given that HMGB1 has important roles in the nucleus where it mediates the assembly of transcription factor complexes (Park et al., 2003Park J.S. Arcaroli J. Yum H.K. Yang H. Wang H. Yang K.Y. Choe K.H. Strassheim D. Pitts T.M. Tracey K.J. Abraham E. Activation of gene expression in human neutrophils by high mobility group box 1 protein.Am. J. Physiol. Cell Physiol. 2003; 284: C870-C879Crossref PubMed Scopus (367) Google Scholar, West et al., 2004West K.L. Castellini M.A. Duncan M.K. Bustin M. Chromosomal proteins HMGN3a and HMGN3b regulate the expression of glycine transporter 1.Mol. Cell. Biol. 2004; 24: 3747-3756Crossref PubMed Scopus (43) Google Scholar), we tested wound healing rates and skin tumorigenesis in the presence of Box A, an HMGB1 antagonist that prevents HMGB1 binding to its receptors (Kokkola et al., 2003Kokkola R. Li J. Sundberg E. Aveberger A.C. Palmblad K. Yang H. Tracey K.J. Andersson U. Harris H.E. Successful treatment of collagen-induced arthritis in mice and rats by targeting extracellular high mobility group box chromosomal protein 1 activity.Arthritis Rheum. 2003; 48: 2052-2058Crossref PubMed Scopus (262) Google Scholar). In agreement with the faster wound healing rates observed in ΔKer HMGB1 mice (Figure 1B), accelerated wound closure was also observed in InvEE mice that were intradermally injected with Box A at time of wounding and during early stages of repair (Figure 1D), and subsequent wound-induced tumor formation was strongly reduced upon Box A treatment (Figure 1E). Additionally, we intradermally injected mice with ethylpyruvate, an established inhibitor of HMGB1 secretion (Davé et al., 2009Davé S.H. Tilstra J.S. Matsuoka K. Li F. DeMarco R.A. Beer-Stolz D. Sepulveda A.R. Fink M.P. Lotze M.T. Plevy S.E. Ethyl pyruvate decreases HMGB1 release and ameliorates murine colitis.J. Leukoc. Biol. 2009; 86: 633-643Crossref PubMed Scopus (135) Google Scholar, Kim et al., 2016Kim Y.M. Park E.J. Kim J.H. Park S.W. Kim H.J. Chang K.C. Ethyl pyruvate inhibits the acetylation and release of HMGB1 via effects on SIRT1/STAT signaling in LPS-activated RAW264.7 cells and peritoneal macrophages.Int. Immunopharmacol. 2016; 41: 98-105Crossref PubMed Scopus (35) Google Scholar), and also observed a protective effect on InvEE papillomagenesis (Figure 1F). In order to identify the receptor through which HMGB1 mediates regenerative and tumorigenic responses in skin, we next injected InvEE mice with antagonistic agents against RAGE or TLR-4. Both TLR-4 as well as RAGE neutralization resulted in enhanced wound closure in early post-wounding stages (Figures S1I and S1J). In addition, treatment with a monoclonal TLR-4 blocking antibody at the time of wounding and during the early post-wounding stages completely protected InvEE mice from tumor development (Figure 1G). Treatment with a blocking peptide against RAGE also protected InvEE mice from wound-induced tumorigenesis, albeit to a lesser extent than treatment with a TLR-4 inhibiting antibody (Figure 1H). RNA sequencing of sorted live integrin-alpha 6-positive keratinocytes obtained from InvEE ΔKer HMGB1 and InvEE HMGB1-proficient mice indicated that HMGB1 is the sole cytokine that is differentially expressed in both genotypes (Figure 1I; Figure S1K; GEO: GSE138918). In order to confirm our observations in the InvEE model of wound-induced tumorigenesis in an independent skin tumor model, we next subjected ΔKer HMGB1 and control littermates to the two-stage chemically induced DMBA/TPA (7,12-dimethylbenz(a)anthracene and 12-O-tetradecanoylphorbol-13-acetate) skin carcinogenesis model. In this model, skin cancer is induced by exposure to a single dose of DMBA inducing oncogenic mutations in HRas (initiation), followed by the repeated topical application with the phorbol ester TPA (promotion) allowing mutagenized cells to expand and form benign papillomas (Abel et al., 2009Abel E.L. Angel J.M. Kiguchi K. DiGiovanni J. Multi-stage chemical carcinogenesis in mouse skin: fundamentals and applications.Nat. Protoc. 2009; 4: 1350-1362Crossref PubMed Scopus (337) Google Scholar). In agreement with the phenotype observed in wound-induced tumorigenesis, mice lacking HMGB1 in keratinocytes were also fully resistant to chemically induced tumor formation (Figures 1J and 1K). Together, these data prove that keratinocyte-derived HMGB1 is a key DAMP that delays regenerative wound healing responses and initiates neoplasia in chronically inflamed skin. HMGB1, in its function as a DAMP, has been characterized as a potent pro-inflammatory mediator (Scaffidi et al., 2002Scaffidi P. Misteli T. Bianchi M.E. Release of chromatin protein HMGB1 by necrotic cells triggers inflammation.Nature. 2002; 418: 191-195Crossref PubMed Scopus (3095) Google Scholar, Wang et al., 1999Wang H. Bloom O. Zhang M. Vishnubhakat J.M. Ombrellino M. Che J. Frazier A. Yang H. Ivanova S. Borovikova L. et al.HMG-1 as a late mediator of endotoxin lethality in mice.Science. 1999; 285: 248-251Crossref PubMed Scopus (2786) Google Scholar). To investigate whether the complete resistance of ΔKer HMGB1 mice to chemically induced skin tumor formation was due to reduced inflammation upon treatment with the pro-inflammatory phorbol ester TPA, we examined the response of ΔKer HMGB1 skin to topical TPA treatment. For this, ΔKer HMGB1 and control littermate mice were repetitively treated with TPA for a total of two to six applications, and skin was collected 24 h after the last treatment (Figure 2A). Surprisingly, ΔKer HMGB1 mice exhibited an exacerbated response to TPA relative to HMGB1fl/fl control mice. After two topical TPA treatments, a significantly higher increase in transepidermal water loss (TEWL) was observed in ΔKer HMGB1 compared to control mice. This response was even more pronounced in skin of ΔKer HMGB1 mice treated six times with TPA (Figure 2B), indicating a more severe skin barrier perturbation in the absence of keratinocyte-derived HMGB1. In agreement, an increase in epidermal thickness could be observed in skin of TPA-treated ΔKer HMGB1 mice relative to HMGB1fl/fl skin (Figures 2C and 2D), again indicating a more severe tissue response to the pro-inflammatory exposure to TPA in ΔKer HMGB1 skin relative to control skin. To further discriminate between the effect of HMGB1 deficiency in keratinocytes on DMBA-induced tumor formation versus TPA-mediated tumor promotion, we subjected ΔKer HMGB1 mice and control littermates to topical DMBA treatment twice a week for 15 weeks. ΔKer HMGB1 mice were able to mount a tumor-protective response to treatment with the carcinogen alone, as tumor formation was strongly delayed upon DMBA treatment (Figure 2E). However, in contrast to the DMBA/TPA model, to which ΔKer HMGB1 mice were fully resistant, tumors did arise in ΔKer HMGB1 mice treated with DMBA alone, albeit far later and to fewer extent than in control littermates (Figure 2E; Figure S2). In conclusion, given that ΔKer HMGB1 mice exhibit an enhanced sensitivity to epidermal barrier perturbation upon repetitive treatment with the phorbol ester TPA relative to their HMGB1fl/fl littermates, the tumor-protective effect of HMGB1 ablation in keratinocytes does not result from a reduced inflammatory response in these mice. However, our data indicate that, depending on the type of epithelial damage, keratinocyte-derived HMGB1 can either protect from epidermal barrier perturbation or delay epithelial repair. Neutrophils represent the first immune cell type that is recruited to sites of injury. Interestingly, genes encoding for proteins involved in neutrophil infiltration, such as the chemokines Cxcl-2 and Ccl5 (RANTES), were significantly downregulated in TPA-treated ΔKer HMGB1 mice relative to controls (Figure 3A). These findings are in agreement with the reduced neutrophil infiltration seen in livers of acetaminophen-intoxicated hepatocyte-specific HMGB1-deficient mice (Huebener et al., 2015Huebener P. Pradere J.P. Hernandez C. Gwak G.Y. Caviglia J.M. Mu X. Loike J.D. Schwabe R.F. The HMGB1/RAGE axis triggers neutrophil-mediated injury amplification following necrosis.J. Clin. Invest. 2015; 125: 539-550Crossref PubMed Scopus (229) Google Scholar). Therefore, we tested whether neutrophil infiltration is altered in wounds of mice lacking HMGB1 in keratinocytes. Indeed, as shown by flow cytometric analysis, the total amount of neutrophils (CD45+ CD11b+ Ly6-G+ cells) that infiltrated the skin at day 2 post-wounding was significantly lower in ΔKer HMGB1 compared to HMGB1fl/fl mice. In contrast, the total amount of macrophages (CD45+ CD11b+ CD64+ cells) present in these wounds did not differ between genotypes (Figures 3B and 3C). Neutrophils can form extracellular traps, termed NETs, which have been demonstrated to delay cutaneous wound healing responses (Wong et al., 2015Wong S.L. Demers M. Martinod K. Gallant M. Wang Y. Goldfine A.B. Kahn C.R. Wagner D.D. Diabetes primes neutrophils to undergo NETosis, which impairs wound healing.Nat. Med. 2015; 21: 815-819Crossref PubMed Scopus (433) Google Scholar). Given that we observed slower wound closure in HMGB1fl/fl relative to ΔKer HMGB1 mice, we analyzed whether the level of NET formation was reduced in ΔKer HMGB1 wounds. Western blot analysis of skin wound lysates showed prominent levels of citrullinated histone-3 (H3citr), an established marker for NET formation (Wong et al., 2015Wong S.L. Demers M. Martinod K. Gallant M. Wang Y. Goldfine A.B. Kahn C.R. Wagner D.D. Diabetes primes neutrophils to undergo NETosis, which impairs wound healing.Nat. Med. 2015; 21: 815-819Crossref PubMed Scopus (433) Google Scholar, Toussaint et al., 2017Toussaint M. Jackson D.J. Swieboda D. Guedán A. Tsourouktsoglou T.D. Ching Y.M. Radermecker C. Makrinioti H. Aniscenko J. Bartlett N.W. et al.Host DNA released by NETosis promotes rhinovirus-induced type-2 allergic asthma exacerbation.Nat. Med. 2017; 23: 681-691Crossref PubMed Scopus (139) Google Scholar), in HMGB1-proficient wounds, whereas H3citr levels are barely detectable in ΔKer HMGB1 wounds at day 2 post-wounding (Figure 3D). NETs are defined as neutrophilic extracellular DNA structures that colocalize with granule-derived proteins and histones (Konig and Andrade, 2016Konig M.F. Andrade F. A Critical Reappraisal of Neutrophil Extracellular Traps and NETosis Mimics Based on Differential Requirements for Protein Citrullination.Front. Immunol. 2016; 7: 461Crossref PubMed Scopus (121) Google Scholar, Boeltz et al., 2019Boeltz S. Amini P. Anders H.J. Andrade F. Bilyy R. Chatfield S. Cichon I. Clancy D.M. Desai J. Dumych T. et al.To NET or not to NET:current opinions and state of the science regarding the formation of neutrophil extracellular traps.Cell Death Differ. 2019; 26: 395-408Crossref PubMed Scopus (133) Google Scholar). We performed immunofluorescent staining of externalized DNA (DAPI) and assessed colocalization with neutrophil elastase (NE) and H3citr. Confocal microscopy of skin wounds of ΔKer HMGB1 and HMGB1fl/fl mice at early post-wounding stage verified that NET formation was markedly reduced in ΔKer HMGB1 wounds relative to control littermates (Figure 3E). NETs were also observed in the tumor stroma of wound-induced papillomas (Figure 3E) but were absent in unwounded ΔKer HMGB1 and HMGB1fl/fl mice. We also confirmed the reduced level of histone-3 citrullination in neutrophils in ΔKer HMGB1 skin wounds by flow cytometric quantification of live neutrophils (CD45+ CD11b+ Ly6G+ cells) that stained for citrullinated histone-3 in ΔKer HMGB1 and HMGB1fl/fl wounds at day 2 post-wounding (Figure 3F), although it should be stated that this might be due to the reduced neutrophil infiltration observed in ΔKer HMGB1 wounds. In conclusion, we could demonstrate that keratinocyte-derived HMGB1 drives neutrophil infiltration and NET formation in the early inflammatory response to full-thickness skin wounding, thereby providing in vivo relevance of HMGB1 acting as an upstream regulator of NET formation in skin reparative inflammation. The increased presence of NETs, delaying regenerative responses in skin, has recently bee