Dendritic cell (DC) activation by pattern recognition receptors like Toll-like-receptors (TLRs) is crucial for cancer immunotherapies. Here, we demonstrate the effectiveness of the TLR7/8 agonist imiquimod (IMQ) in treating both local tumors and distant metastases. Administered orally, IMQ activates plasmacytoid DCs (pDCs) to produce systemic type I interferons (IFN-I) required for TLR7/8 upregulation in DCs and macrophages, sensitizing them to topical IMQ treatment, which is essential for therapeutic efficacy. The mechanism involves c-Jun/AP-1 mediating TLR7/8 signaling in IFN-I-primed DCs, upregulating the pDC-recruiting chemokine CCL2 and the anti-angiogenic cytokine interleukin-12, which suppresses VEGF-A production leading to tumor necrosis and regression. Combining topical and systemic IMQ or IFN-I generates a CD8+ T cell-dependent response at metastatic sites, reinforced by PD-1 blockade, leading to long-lasting memory. Analysis of cohorts of patients with melanoma demonstrates DC-specific TLR7/8 upregulation by IFN-I, supporting the translational potential of combining systemic IFN-I and topical IMQ to improve immunotherapy of topically accessible tumors.

Dendritic cell (DC) progenitors adapt their transcriptional program during development, generating different subsets. How chromatin modifications modulate these processes is unclear. Here, we investigate the impact of histone deacetylation on DCs by genetically deleting histone deacetylase 1 (HDAC1) or HDAC2 in hematopoietic progenitors and CD11c-expressing cells. While HDAC2 is not critical for DC development, HDAC1 deletion impairs pro-pDC and mature pDC generation and affects ESAM+cDC2 differentiation from tDCs and pre-cDC2s, whereas cDC1s are unchanged. HDAC1 knockdown in human hematopoietic cells also impairs cDC2 development, highlighting its crucial role across species. Multi-omics analyses reveal that HDAC1 controls expression, chromatin accessibility, and histone acetylation of the transcription factors IRF4, IRF8, and SPIB required for efficient development of cDC2 subsets. Without HDAC1, DCs switch immunologically, enhancing tumor surveillance through increased cDC1 maturation and interleukin-12 production, driving T helper 1-mediated immunity and CD8+ T cell recruitment. Our study reveals the importance of histone acetylation in DC development and anti-tumor immunity, suggesting DC-targeted therapeutic strategies for immuno-oncology.

AbstractThe hair follicle stem cell niche is an immune-privileged microenvironment, characterized by reduced antigen presentation, thus shielding against permanent immune-mediated tissue damage. In this study, we demonstrated the protective role of hair follicle-specific epidermal growth factor receptor (EGFR) against scarring hair follicle destruction. Mechanistically, disruption of EGFR signaling generated a cell-intrinsic hypersensitivity within the JAK-STAT1 pathway, which, synergistically with interferon gamma expressing CD8 T-cell and NK-cell-mediated inflammation, compromised the stem cell niche. Hair follicle-specific genetic depletion of either JAK1/2 or STAT1 or therapeutic inhibition of JAK1/2 ameliorated the inflammation, restored skin barrier function and activated the residual stem cells to resume hair growth in mouse models of epidermal and hair follicle-specific EGFR deletion. Skin biopsies from EGFR inhibitor-treated and cicatricial alopecia patients revealed an active JAK-STAT1 signaling signature along with upregulation of antigen presentation and downregulation of key components of the EGFR pathway. Our findings offer molecular insights and highlight a mechanism-based therapeutic strategy for addressing chronic folliculitis associated with EGFR-inhibitor anti-cancer therapy and cicatricial alopecia.

FAM3C/ILEI is an important cytokine for tumor progression and metastasis. However, its involvement in inflammation remains elusive. Here, we show that ILEI protein is highly expressed in psoriatic lesions. Inducible keratinocyte-specific ILEI overexpression in mice (K5-ILEIind ) recapitulates many aspects of psoriasis following TPA challenge, primarily manifested by impaired epidermal differentiation and increased neutrophil recruitment. Mechanistically, ILEI triggers Erk and Akt signaling, which then activates STAT3 via Ser727 phosphorylation. Keratinocyte-specific ILEI deletion ameliorates TPA-induced skin inflammation. A transcriptomic ILEI signature obtained from the K5-ILEIind model shows enrichment in several signaling pathways also found in psoriasis and identifies urokinase as a targetable enzyme to counteract ILEI activity. Pharmacological inhibition of urokinase in TPA-induced K5-ILEIind mice results in significant improvement of psoriasiform symptoms by reducing ILEI secretion. The ILEI signature distinguishes psoriasis from healthy skin with uPA ranking among the top "separator" genes. Our study identifies ILEI as a key driver in psoriasis, indicates the relevance of ILEI-regulated genes for disease manifestation, and shows the clinical impact of ILEI and urokinase as novel potential therapeutic targets in psoriasis.

The hair follicle stem cell niche is an immune-privileged microenvironment, characterised by suppressed antigen presentation, thus shielding against permanent immune-mediated tissue damage. In this study, we demonstrate the protective role of hair follicle-specific epidermal growth factor receptor (EGFR) from scarring hair follicle degeneration. Mechanistically, disruption of EGFR signalling generates a cell intrinsic hypersensitivity within the JAK-STAT1 pathway, compromising the immune privilege in the context of CD8 T cell and NK cell-mediated inflammation. Genetic depletion of either JAK1/2 or STAT1 or topical therapeutic inhibition of JAK1/2 restores the immune privilege and activates stem cells to resume hair growth in mouse models of epidermal and hair follicle specific EGFR deletion. Skin biopsies from EGFR inhibitor-treated and from EGFR-independent cicatricial alopecia patients indicate active STAT1 signalling within the hair follicles. Notably, a case study of folliculitis decalvans, characterised by progressive hair loss, scaling and perifollicular erythema, demonstrates successful treatment with systemic JAK1/2 inhibition. Our findings offer mechanistic insights and present a therapeutic strategy for addressing scarring hair follicle destruction associated with EGFR-inhibitor therapy and cicatricial alopecia.

Supplementary Video 1. Impact of calpain inhibition on the dynamics of KP64-induced rapid cell death of A427 cells

Supplementary Figure 1. Role of p53 status and mitochondrial membrane depolarization in KP46-induced cell death Supplementary Figure 2. Expression and localization changes of pro- and anti-apoptotic Bcl-2 family members in response to KP46 treatment Supplementary Figure 3. Impact or KP46 on integrin β1 expression and subcellular localization in cancer cells Supplementary Figure 4. Alteration in intracellular Ca2+ levels in HCT-116 cells upon KP46 treatment Supplementary Figure 5 Effect of extracellular calpain inhibition on KP46 sensitivity in A427 and HCT-116 cells

Legends to all supplementary figures and the video

FAM3C/ILEI is an important factor in epithelial-to-mesenchymal transition (EMT) induction, tumor progression and metastasis. Overexpressed in many cancers, elevated ILEI levels and secretion correlate with poor patient survival. Although ILEI's causative role in invasive tumor growth and metastasis has been demonstrated in several cellular tumor models, there are no available transgenic mice to study these effects in the context of a complex organism. Here, we describe the generation and initial characterization of a Tet-ON inducible Fam3c/ILEI transgenic mouse strain. We find that ubiquitous induction of ILEI overexpression (R26-ILEIind) at weaning age leads to a shortened lifespan, reduced body weight and microcytic hypochromic anemia. The anemia was reversible at a young age within a week upon withdrawal of ILEI induction. Vav1-driven overexpression of the ILEIind transgene in all hematopoietic cells (Vav-ILEIind) did not render mice anemic or lower overall fitness, demonstrating that no intrinsic mechanisms of erythroid development were dysregulated by ILEI and that hematopoietic ILEI hyperfunction did not contribute to death. Reduced serum iron levels of R26-ILEIind mice were indicative for a malfunction in iron uptake or homeostasis. Accordingly, the liver, the main organ of iron metabolism, was severely affected in moribund ILEI overexpressing mice: increased alanine transaminase and aspartate aminotransferase levels indicated liver dysfunction, the liver was reduced in size, showed increased apoptosis, reduced cellular iron content, and had a fibrotic phenotype. These data indicate that high ILEI expression in the liver might reduce hepatoprotection and induce liver fibrosis, which leads to liver dysfunction, disturbed iron metabolism and eventually to death. Overall, we show here that the novel Tet-ON inducible Fam3c/ILEI transgenic mouse strain allows tissue specific timely controlled overexpression of ILEI and thus, will serve as a versatile tool to model the effect of elevated ILEI expression in diverse tissue entities and disease conditions, including cancer.

The skin is the outermost barrier protecting the body from pathogenic invasion and environmental insults. Its breakdown initiates the start of skin inflammation. The epidermal growth factor (EGFR) on keratinocytes protects this barrier, and its dysfunction leads to atopic dermatitis-like skin disease. One of the initial cytokines expressed upon skin barrier breach and during atopic dermatitis is TSLP. Here, we describe the expression and secretion of TSLP during EGFR inhibition and present an ex-vivo model, which mimics the early events after barrier insult. Skin explants floated on culture medium at 32 °C released TSLP in parallel to the activation of the resident Langerhans cell network. We could further show the up-regulation and activation of the AP-1 family of transcription factors during atopic-like skin inflammation and its involvement in TSLP production from the skin explant cultures. Inhibition of the c-Jun N-terminal kinase pathway led to a dose-dependent blunting of TSLP release. These data indicate the involvement of AP-1 during the early stages of atopic-like skin inflammation and highlight a novel therapeutic approach by targeting it. Therefore, skin explant cultures mimic the early events during skin barrier immunity and provide a suitable model to test therapeutic intervention.

Dendritic cell (DC) development is orchestrated by lineage-determining transcription factors (TFs). Although, members of the activator-protein-1 (AP-1) family, including Batf3, have been implicated in conventional (c)DC specification, the role of Jun proteins is poorly understood. Here, we identified c-Jun and JunB as essential for cDC1 fate specification and function. In mice, Jun proteins regulate extrinsic and intrinsic pathways, which control CD8α cDC1 diversification, whereas CD103 cDC1 development is unaffected. The loss of c-Jun and JunB in DC progenitors diminishes the CD8α cDC1 pool and thus confers resistance to Listeria monocytogenes infection. Their absence in CD8α cDC1 results in impaired TLR triggering and antigen cross-presentation. Both TFs are required for the maintenance of the CD8α cDC1 subset and suppression of cDC2 identity on a transcriptional and phenotypic level. Taken together, these results demonstrate the essential role of c-Jun and JunB in CD8α cDC1 diversification, function, and maintenance of their identity.

Toll-like receptor (TLR) stimulation induces innate immune responses involved in many inflammatory disorders including psoriasis. Although activation of the AP-1 transcription factor complex is common in TLR signaling, the specific involvement and induced targets remain poorly understood. Here, we investigated the role of c-Jun/AP-1 protein in skin inflammation following TLR7 activation using human psoriatic skin, dendritic cells (DC), and genetically engineered mouse models. We show that c-Jun regulates CCL2 production in DCs leading to impaired recruitment of plasmacytoid DCs to inflamed skin after treatment with the TLR7/8 agonist Imiquimod. Furthermore, deletion of c-Jun in DCs or chemical blockade of JNK/c-Jun signaling ameliorates psoriasis-like skin inflammation by reducing IL-23 production in DCs. Importantly, the control of IL-23 and CCL2 by c-Jun is most pronounced in murine type-2 DCs. CCL2 and IL-23 expression co-localize with c-Jun in type-2/inflammatory DCs in human psoriatic skin and JNK-AP-1 inhibition reduces the expression of these targets in TLR7/8-stimulated human DCs. Therefore, c-Jun/AP-1 is a central driver of TLR7-induced immune responses by DCs and JNK/c-Jun a potential therapeutic target in psoriasis.

Despite the introduction of biologics, topical dithranol (anthralin) has remained one of the most effective anti-psoriatic agents. Serial biopsies from human psoriatic lesions and both the c-Jun/JunB and imiquimod psoriasis mouse model allowed us to study the therapeutic mechanism of this drug. Top differentially expressed genes in the early response to dithranol belonged to keratinocyte and epidermal differentiation pathways and IL-1 family members (i.e. IL36RN) but not elements of the IL-17/IL-23 axis. In human psoriatic response to dithranol, rapid decrease in expression of keratinocyte differentiation regulators (e.g. involucrin, SERPINB7 and SERPINB13), antimicrobial 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 was followed with much delay by reduction in T cell infiltration. Targeting keratinocytes rather than immune cells may be an alternative approach in particular for topical anti-psoriatic treatment, an area with high need for new drugs.

Epidermal growth factor receptor (EGFR) signaling controls skin development and homeostasis in mice and humans, and its deficiency causes severe skin inflammation, which might affect epidermal stem cell behavior. Here, we describe the inflammation-independent effects of EGFR deficiency during skin morphogenesis and in adult hair follicle stem cells. Expression and alternative splicing analysis of RNA sequencing data from interfollicular epidermis and outer root sheath indicate that EGFR controls genes involved in epidermal differentiation and also in centrosome function, DNA damage, cell cycle, and apoptosis. Genetic experiments employing p53 deletion in EGFR-deficient epidermis reveal that EGFR signaling exhibits p53-dependent functions in proliferative epidermal compartments, as well as p53-independent functions in differentiated hair shaft keratinocytes. Loss of EGFR leads to absence of LEF1 protein specifically in the innermost epithelial hair layers, resulting in disorganization of medulla cells. Thus, our results uncover important spatial and temporal features of cell-autonomous EGFR functions in the epidermis.

Mice deficient in epidermal growth factor receptor ( Egfr −/− mice) are growth retarded and exhibit severe bone defects that are poorly understood. Here we show that EGFR-deficient mice are osteopenic and display impaired endochondral and intramembranous ossification resulting in irregular mineralization of their bones. This phenotype is recapitulated in mice lacking EGFR exclusively in osteoblasts, but not in mice lacking EGFR in osteoclasts indicating that osteoblasts are responsible for the bone phenotype. Experiments are presented demonstrating that signaling via EGFR stimulates osteoblast proliferation and inhibits their differentiation by suppression of the IGF-1R/mTOR-pathway via ERK1/2-dependent up-regulation of IGFBP-3. Osteoblasts from Egfr −/− mice show increased levels of IGF-1R and hyperactivation of mTOR-pathway proteins, including enhanced phosphorylation of 4E-BP1 and S6. The same changes are also seen in Egfr −/− bones. Importantly, pharmacological inhibition of mTOR with rapamycin decreases osteoblasts differentiation as well as rescues the low bone mass phenotype of Egfr −/− fetuses. Our results demonstrate that suppression of the IGF-1R/mTOR-pathway by EGFR/ERK/IGFBP-3 signaling is necessary for balanced osteoblast maturation providing a mechanism for the skeletal phenotype observed in EGFR-deficient mice.

Colorectal cancer (CRC) is one of the most commonly diagnosed cancers and a major cause of cancer mortality worldwide. At late stage of the disease CRC often shows (multiple) metastatic lesions in the peritoneal cavity which cannot be efficiently targeted by systemic chemotherapy. This is one major factor contributing to poor prognosis. Oxaliplatin is one of the most commonly used systemic treatment options for advanced CRC. However, drug resistance - often due to insufficient drug delivery - is still hampering successful treatment. The anticancer activity of oxaliplatin includes besides DNA damage also a strong immunogenic component. Consequently, the aim of this study was to investigate the effect of bacterial ghosts (BGs) as adjuvant immunostimulant on oxaliplatin efficacy. BGs are empty envelopes of gram-negative bacteria with a distinct immune-stimulatory potential. Indeed, we were able to show that the combination of BGs with oxaliplatin treatment had strong synergistic anticancer activity against the CT26 allograft, resulting in prolonged survival and even a complete remission in this murine model of CRC carcinomatosis. This synergistic effect was based on an enhanced induction of immunogenic cell death and activation of an efficient T-cell response leading to long-term anti-tumor memory effects. Taken together, co-application of BGs strengthens the immunogenic component of the oxaliplatin anticancer response and thus represents a promising natural immune-adjuvant to chemotherapy in advanced CRC.

Imiquimod (IMQ) acts as a toll-like-receptor-7/8 agonist and its topical application is used as a model for psoriasiform skin inflammation. In mice and humans, skin inflammation is characterized by infiltration of several immune cells (Drobits et al., 2012Drobits B. Holcmann M. Amberg N. Swiecki M. Grundtner R. Hammer M. et al.Imiquimod clears tumors in mice independent of adaptive immunity by converting pDCs into tumor-killing effector cells.J Clin Invest. 2012; 122: 575-585Crossref PubMed Scopus (213) Google Scholar, Kalb et al., 2012Kalb M.L. Glaser A. Stary G. Koszik F. Stingl G. TRAIL(+) human plasmacytoid dendritic cells kill tumor cells in vitro: mechanisms of imiquimod- and IFN-alpha-mediated antitumor reactivity.J Immunol. 2012; 188: 1583-1591Crossref PubMed Scopus (81) Google Scholar, Palamara et al., 2004Palamara F. Meindl S. Holcmann M. Luhrs P. Stingl G. Sibilia M. Identification and characterization of pDC-like cells in normal mouse skin and melanomas treated with imiquimod.J Immunol. 2004; 173: 3051-3061Crossref PubMed Scopus (177) Google Scholar) and activation of the IL-23/IL-17/IL-22 axis (Riol-Blanco et al., 2014Riol-Blanco L. Ordovas-Montanes J. Perro M. Naval E. Thiriot A. Alvarez D. et al.Nociceptive sensory neurons drive interleukin-23-mediated psoriasiform skin inflammation.Nature. 2014; 510: 157-161Crossref PubMed Scopus (331) Google Scholar, van der Fits et al., 2009van der Fits L. Mourits S. Voerman J.S. Kant M. Boon L. Laman J.D. et al.Imiquimod-induced psoriasis-like skin inflammation in mice is mediated via the IL-23/IL-17 axis.J Immunol. 2009; 182: 5836-5845Crossref PubMed Scopus (1360) Google Scholar). Major histocompatibility complex-II upregulation on keratinocytes, hyperproliferation, parakeratosis, and increased vascularization are also observed recapitulating psoriatic hallmarks (Flutter and Nestle, 2013Flutter B. Nestle F.O. TLRs to cytokines: mechanistic insights from the imiquimod mouse model of psoriasis.Eur J Immunol. 2013; 43: 3138-3146Crossref PubMed Scopus (194) Google Scholar). Hair removal is essential before IMQ is applied to the murine skin. Different laboratories use various depilation strategies such as razor shaving, depilation cream, or wax stripping. Shaving does not affect hair follicles (HF), whereas wax stripping results in depletion of HF stem cells leading to anagen entry (Amberg et al., 2016Amberg N. Holcmann M. Stulnig G. Sibilia M. Effects of imiquimod on hair follicle stem cells and hair cycle progression.J Invest Dermatol. 2016; 136: 2140-2149Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar, Paus et al., 1994Paus R. Handjiski B. Eichmuller S. Czarnetzki B.M. Chemotherapy-induced alopecia in mice. Induction by cyclophosphamide, inhibition by cyclosporine A, and modulation by dexamethasone.Am J Pathol. 1994; 144: 719-734PubMed Google Scholar). The effects of depilation cream have not been carefully investigated yet. We tested if these three different depilation strategies affect the inflammatory response by applying IMQ for 7 consecutive days on the back skin of late telogen (65-day-old) C57BL/6 mice 1 day after hair removal. All IMQ-treated mice exhibited splenomegaly as previously reported, which was not seen in untreated or vehicle-treated controls (Palamara et al., 2004Palamara F. Meindl S. Holcmann M. Luhrs P. Stingl G. Sibilia M. Identification and characterization of pDC-like cells in normal mouse skin and melanomas treated with imiquimod.J Immunol. 2004; 173: 3051-3061Crossref PubMed Scopus (177) Google Scholar) (Supplementary Figure S1a online). As it is important to distinguish infundibulum (IF) thickening from rete ridges found in human psoriatic skin, we analyzed IF and interfollicular epidermis (IFE), because both compartments harbor stem cells that can replenish injured epidermis. All treatment groups responded to IMQ with a similar increase in IFE thickening (Figure 1a, Supplementary Figure S1b). The IF was already longer in creamed and waxed mice compared with shaved mice and increased further after IMQ application in shaved and creamed, but not in waxed mice (Figure 1b). These results demonstrate that waxing masks the effects of IMQ on the IF. We next investigated whether these distinct effects of hair removal methods were specific for IMQ or applied also to other triggers of skin inflammation such as rmIL-23 injection. Similar to IMQ treatment we found acanthosis of the IFE after intradermal rmIL-23 injection although waxing induced significantly less IFE thickening than shaving or creaming (Figure 1c, Supplementary Figure S1c). IF length was already increased by phosphate buffered saline injection in creamed and waxed compared with shaved mice (Figure 1d). Except for creamed mice, the IF increased further on rmIL-23 injection (Figure 1d). These results show that different hair removal methods affect skin morphology in two skin inflammation models and that the effects on IFE are less strong with rmIL-23 when compared with IMQ. We recently showed that telogen HFs from shaved mice treated with IMQ started to enter anagen (Amberg et al., 2016Amberg N. Holcmann M. Stulnig G. Sibilia M. Effects of imiquimod on hair follicle stem cells and hair cycle progression.J Invest Dermatol. 2016; 136: 2140-2149Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar). An analysis of HF stem cell activation by staining for the proliferation marker Ki67 or the M-phase marker PSer10H3 revealed that telogen HFs of shaved skin remained quiescent, whereas all HFs of creamed or waxed skin had proceeded to mid-anagen (Figure 1e, Supplementary Figure S2a online). Because creaming and waxing per se already induced anagen, there was no additional effect induced by IMQ (Supplementary Figures S1b and S2a). A significant increase in proliferation was observed in the IFE and IF of all IMQ-treated mice (Figure 1f and g, Supplementary Figure S2b and c). However, skin exposed to depilation cream showed the lowest increase in proliferation on IMQ treatment (Figure 1f and g). Different depilation methods also affected other psoriatic hallmarks like microvessel density after IMQ treatment, whereby the most pronounced vessel increase was observed in waxed mice as determined by staining with MECA-32 (Supplementary Figure S2d and e). FACS analysis showed that all groups responded to IMQ with elevated numbers of CD45+ cells (Figure 2a). In contrast to shaving and creaming, waxing did not increase major histocompatibility complex-II+ dendritic cells after IMQ treatment (Supplementary Figure S3a online). Surprisingly, dendritic epidermal T cells (TCRδhi) were significantly lower in waxed compared with shaved or creamed mice and did not decrease further after IMQ treatment (Figure 2b), showing that waxing alone leads to dendritic epidermal T-cell activation and emigration. In line, TCRδlo cells did not change in waxed mice after IMQ treatment, but were increased in shaved and creamed mice (Figure 2c). TCRβ+ cells were not affected (Supplementary Figure S3b). Depilation cream induced an increase in dermal CD45+ cells in untreated and IMQ-treated mice (Supplementary Figure S3c). Macrophages increased after IMQ treatment in shaved and waxed mice, but remained low in creamed mice (Figure 2d). Waxed mice showed reduced neutrophils and CD11b+/Ly6Chi monocytes after IMQ treatment, compared with shaved mice (Figure 2e and f). Mast cells were already elevated in waxed compared with shaved and creamed mice, but were less activated in waxed mice despite their numerical increase after IMQ treatment (Supplementary Figure S3d–f). The changes in the inflammatory infiltrate after rmIL-23 injection under different depilation regimens were similar to what was observed with IMQ (Figure 2g–l), except for TCRδlo cells, which were increased in waxed skin (Figure 2i). Moreover, phosphate buffered saline injection alone led to increased macrophages and monocytes in mice depilated by cream or wax, but not by shaving indicating that injection injury is per se an inducer of these cells in combination with the respective depilation method (Figure 2j and l). Furthermore, unlike in IMQ-treated skin, we did not observe differences in monocyte numbers between shaved or waxed IL-23-treated skin (Figure 2l). However, because of the small area of skin treated with IL-23, a separate analysis of epidermis and dermis was not possible, which might account for the differences compared with IMQ treatment. Expression analysis of cytokines typical for psoriasiform skin inflammation by quantitative real-time reverse transcription-PCR showed that IL-6 and Cxcl-1 were induced in IMQ-treated creamed or waxed skin. A similar trend could be observed for TNFα, IL-23p19, and IL-17a (Figure 2m–q). In summary, hair removal by waxing showed the most prominent differences compared with shaving after IMQ or IL-23 treatment, possibly caused by removal of the stratum corneum through waxing (Mohammed et al., 2012Mohammed D. Yang Q. Guy R.H. Matts P.J. Hadgraft J. Lane M.E. Comparison of gravimetric and spectroscopic approaches to quantify stratum corneum removed by tape-stripping.Eur J Pharm Biopharm. 2012; 82: 171-174Crossref PubMed Scopus (34) Google Scholar). This extra irritation likely alters skin homeostasis. Some of these differences were also observed using depilation cream. However, we cannot exclude that ingredients of the depilation cream are responsible for the observed effects. On the basis of our results, we suggest standardizing the depilation methods between different laboratories using IMQ to allow a better comparison of experiments. Our recommendation is to apply topical IMQ on razor-shaved back skin during late telogen. This condition exhibits the greatest differences not only in HF stem cell activation but also in immune cell infiltration and psoriatic hallmarks. C57BL/6 mice were purchased from Jackson Laboratories and kept in the animal facility of the Medical University of Vienna for several generations in accordance with institutional policies and federal guidelines. Animal experiments were approved by the Animal Experimental Ethics Committee of the Medical University of Vienna and the Austrian Federal Ministry of Science and Research. The authors state no conflict of interest. We would like to thank Karin Komposch and Caroline Stremnitzer for fruitful discussions and critical reading of the manuscript. We are grateful to Temenusha Baykusheva-Gentscheva, Ksenija Prpa, Iva Vokic, and Ramona Rica for help with histology, to Theresia Lengheimer and Martina Hammer for animal care, and to Rainer Zenz for help in preparing the figures and manuscript. This work was supported by the Austrian Science Fund (FWF) grants FWF-DK W1212 and the Austrian Federal Government’s GEN-AU program “Austromouse” (GZ 200.147/1-VI/1a/2006 and 820966). Download .pdf (.71 MB) Help with pdf files Supplementary Data

Background & Aims Inhibitors of the epidermal growth factor receptor (EGFR) are the first-line therapy for patients with metastatic colorectal tumors without RAS mutations. However, EGFR inhibitors are ineffective in these patients, and tumor level of EGFR does not associate with response to therapy. We screened human colorectal tumors for EGFR-positive myeloid cells and investigated their association with patient outcome. We also performed studies in mice to evaluate how EGFR expression in tumor cells and myeloid cells contributes to development of colitis-associated cancer and ApcMin-dependent intestinal tumorigenesis. Methods We performed immunohistochemical and immunofluorescent analyses of 116 colorectal tumor biopsies to determine levels of EGFR in tumor and stroma; we also collected information on tumor stage and patient features and outcomes. We used the Mann-Whitney U and Kruskal-Wallis tests to correlate tumor levels of EGFR with tumor stage, and the Kaplan-Meier method to estimate patients’ median survival time. We performed experiments in mice lacking EGFR in intestinal epithelial cells (Villin-Cre; Egfrf/f and Villin-CreERT2; Egfrf/f mice) or myeloid cells (LysM-Cre; Egfrf/f mice) on a mixed background. These mice were bred with ApcMin/+ mice; colitis-associated cancer and colitis were induced by administration of dextran sodium sulfate (DSS), with or without azoxymethane (AOM), respectively. Villin-CreERT2 was activated in developed tumors by administration of tamoxifen to mice. Littermates that expressed full-length EGFR were used as controls. Intestinal tissues were collected; severity of colitis, numbers and size of tumors, and intestinal barrier integrity were assessed by histologic, immunohistochemical, quantitative reverse transcription polymerase chain reaction, and flow cytometry analyses. Results We detected EGFR in myeloid cells in the stroma of human colorectal tumors; myeloid cell expression of EGFR associated with tumor metastasis and shorter patient survival time. Mice with deletion of EGFR from myeloid cells formed significantly fewer and smaller tumors than the respective EGFR-expressing controls in an ApcMin/+ background as well as after administration of AOM and DSS. Deletion of EGFR from intestinal epithelial cells did not affect tumor growth. Furthermore, tamoxifen-induced deletion of EGFR from epithelial cells of established intestinal tumors in mice given AOM and DSS did not reduce tumor size. EGFR signaling in myeloid cells promoted activation of STAT3 and expression of survivin in intestinal tumor cells. Mice with deletion of EGFR from myeloid cells developed more severe colitis after DSS administration, characterized by increased intestinal inflammation and intestinal barrier disruption, than control mice or mice with deletion of EGFR from intestinal epithelial cells. EGFR-deficient myeloid cells in the colon of DSS-treated LysM-Cre; Egfrf/f mice had reduced expression of interleukin 6 (IL6), and epithelial STAT3 activation was reduced compared with controls. Administration of recombinant IL6 to LysM-Cre; Egfrf/f mice given DSS protected them from weight loss and restored epithelial proliferation and STAT3 activation, compared with administration of DSS alone to these mice. Conclusions Increased expression of EGFR in myeloid cells from the colorectal tumor stroma associates with tumor progression and reduced survival time of patients with metastatic colorectal cancer. Deletion of EGFR from myeloid cells, but not intestinal epithelial cells, protects mice from colitis-induced intestinal cancer and ApcMin-dependent intestinal tumorigenesis. Myeloid cell expression of EGFR increases activation of STAT3 and expression of survivin in intestinal epithelial cells and expression of IL6 in colon tissues. These findings indicate that expression of EGFR by myeloid cells of the colorectal tumor stroma, rather than the cancer cells themselves, contributes to tumor development.