IL-1α, IL-6, and GM-CSF Are Downstream Mediators of IL-17A that Promote Asymmetric Stem Cell Self-Renewal in Human Keratinocytes

IL-17A, a key cytokine in the inflammation of psoriasis, is upregulated in psoriatic skin and stimulates transcriptional activity correlating with the psoriasis transcriptome (Chiricozzi et al., 2011Chiricozzi A. Guttman-Yassky E. Suárez-Fariñas M. Nograles K.E. Tian S. Cardinale I. et al.Integrative responses to IL-17 and TNF-α in human keratinocytes account for key inflammatory pathogenic circuits in psoriasis.J Invest Dermatol. 2011; 131: 677-687Abstract Full Text Full Text PDF PubMed Scopus (404) Google Scholar). Asymmetric stem cell (SC) self-renewal (ASR) is increased in psoriasis. IL-17A increases ASR in human keratinocytes (KCs), and ASR divisions are IL-17A‒dependent in the imiquimod-induced psoriasis-like mouse model (Charruyer et al., 2017Charruyer A. Fong S. Vitcov G.G. Sklar S. Tabernik L. Taneja M. et al.Brief report: interleukin-17A-dependent asymmetric stem cell divisions are increased in human psoriasis: a mechanism underlying benign hyperproliferation.Stem Cells. 2017; 35: 2001-2007Crossref PubMed Scopus (9) Google Scholar). Epidermal maintenance requires a balance of SC self-renewal and differentiation (Figure 1a). ASR divisions promote stratification by producing one basal SC and one (suprabasal) non-SC (a committed transit-amplifying progenitor) in mice (Lechler and Fuchs, 2005Lechler T. Fuchs E. Asymmetric cell divisions promote stratification and differentiation of mammalian skin.Nature. 2005; 437: 275-280Crossref PubMed Scopus (726) Google Scholar; Niessen et al., 2013Niessen M.T. Scott J. Zielinski J.G. Vorhagen S. Sotiropoulou P.A. Blanpain C. et al.aPKCλ controls epidermal homeostasis and stem cell fate through regulation of division orientation.J Cell Biol. 2013; 202: 887-900Crossref PubMed Scopus (51) Google Scholar; Williams et al., 2011Williams S.E. Beronja S. Pasolli H.A. Fuchs E. Asymmetric cell divisions promote Notch-dependent epidermal differentiation.Nature. 2011; 470: 353-358Crossref PubMed Scopus (302) Google Scholar) and humans (Charruyer et al., 2017Charruyer A. Fong S. Vitcov G.G. Sklar S. Tabernik L. Taneja M. et al.Brief report: interleukin-17A-dependent asymmetric stem cell divisions are increased in human psoriasis: a mechanism underlying benign hyperproliferation.Stem Cells. 2017; 35: 2001-2007Crossref PubMed Scopus (9) Google Scholar). Symmetric SC divisions lead to two SCs or two differentiated committed progenitor cells. ASR can be identified by asymmetric segregation of fate-determinant proteins such as Numb (Figure 1b). We hypothesized that IL-17A‒induced KC cytokines play a role in the increased ASR of psoriasis. We examined psoriasis-related cytokines produced by KCs after IL-17A treatment and then determined their effects on ASR. IL-17A increases KC production of IL-1α, IL-6, IL-8, and GM-CSF protein. Whereas there are robust studies of IL-17A‒induced KC gene expression (e.g., Chiricozzi et al., 2011Chiricozzi A. Guttman-Yassky E. Suárez-Fariñas M. Nograles K.E. Tian S. Cardinale I. et al.Integrative responses to IL-17 and TNF-α in human keratinocytes account for key inflammatory pathogenic circuits in psoriasis.J Invest Dermatol. 2011; 131: 677-687Abstract Full Text Full Text PDF PubMed Scopus (404) Google Scholar), this is not the case for protein expression. IL-17A increases IL-6 and IL-8, but results for IL-1α and IL-1β in KCs, HaCaT cells, and bronchial epithelial cells are inconsistent (Albanesi et al., 2000Albanesi C. Scarponi C. Cavani A. Federici M. Nasorri F. Girolomoni G. Interleukin-17 is produced by both Th1 and Th2 lymphocytes, and modulates interferon-gamma- and interleukin-4-induced activation of human keratinocytes.J Invest Dermatol. 2000; 115: 81-87Abstract Full Text Full Text PDF PubMed Scopus (241) Google Scholar; Laan et al., 2001Laan M. Lötvall J. Chung K.F. Lindén A. IL-17-induced cytokine release in human bronchial epithelial cells in vitro: role of mitogen-activated protein (MAP) kinases.Br J Pharmacol. 2001; 133: 200-206Crossref PubMed Scopus (167) Google Scholar; Tan et al., 2017Tan Q. Yang H. Liu E.M. Wang H. Establishing a role for interleukin-17 in atopic dermatitis-related skin inflammation.J Cutan Med Surg. 2017; 21: 308-315Crossref PubMed Scopus (19) Google Scholar; Teunissen et al., 1998Teunissen M.B. Koomen C.W. de Waal Malefyt R. Wierenga E.A. Bos J.D. Interleukin-17 and interferon-γ synergize in the enhancement of proinflammatory cytokine production by human keratinocytes.J Invest Dermatol. 1998; 111: 645-649Abstract Full Text Full Text PDF PubMed Scopus (470) Google Scholar). We investigated psoriasis-related cytokines produced by human KCs in response to IL-17A by ELISA. Neonatal skin samples were obtained with written informed parental consent as well as approval from the University of California, San Francisco Committee on Human Research. All studies abided by the rules of the Internal Review Board and the tenets of the Declaration of Helsinki. IL-17A significantly increased IL-6, IL-8, and GM-CSF. The increase in IL-1α approached significance (P = 0.06) (Figure 1c). No significant difference was detected in IL-1β, IL-2, IL-4, IL-10, IFN-γ, or TNF-α. TNF-α was not produced by IL-17A‒treated KCs and in previous studies, was not associated with hyperproliferation (Takahashi et al., 2009Takahashi H. Tsuji H. Hashimoto Y. Ishida-Yamamoto A. Iizuka H. Cell proliferation and cytokine induction by tnf-α of psoriatic keratinocytes are not different from normal keratinocytes in vitro.Indian J Dermatol. 2009; 54: 237-239Crossref PubMed Scopus (12) Google Scholar). In psoriasis, TNF-α may work by indirect activation of T helper type 17 responses through the activation of myeloid dendritic cells (Chiricozzi et al., 2011Chiricozzi A. Guttman-Yassky E. Suárez-Fariñas M. Nograles K.E. Tian S. Cardinale I. et al.Integrative responses to IL-17 and TNF-α in human keratinocytes account for key inflammatory pathogenic circuits in psoriasis.J Invest Dermatol. 2011; 131: 677-687Abstract Full Text Full Text PDF PubMed Scopus (404) Google Scholar), explaining why this key psoriasis cytokine did not increase ASR. IL-1α, IL-6, and GM-CSF but not IL-8 increase ASR divisions. Next, we analyzed the effect of IL-1α, IL-6, GM-CSF, and IL-8 on ASR using immunofluorescence staining in vitro. IL-6, IL-1α, and GM-CSF significantly increased ASR (51.2 ± 1.1%, 48.4 ± 4.0%, and 47.8 ± 2.7%, respectively) in IL-17A–treated KCs versus vehicle-treated cells (37.3 ± 0.8%, 36.4 ± 3.8%, and 34.2 ± 1.0%, respectively) (Figure 1d and e and Supplementary Figure S1). The increased ASR observed with IL-1α, IL-6, and GM-CSF treatment correlated with decreased symmetric differentiation and no change in symmetric self-renewal divisions. No significant change in division type was detected with TNF-α or IL-8 treatment. IL-1α, IL-6, and GM-CSF antibodies reduce IL-17A‒induced ASR. Next, we showed that IL-1α, IL-6, and GM-CSF antibodies inhibited IL-17A‒induced ASR divisions in a dose-dependent manner (Figure 2 and Supplementary Figure S1). IL-1α antibody significantly inhibited IL-17A‒induced ASR at ≥0.1 μg/ml, IL-6 antibody at ≥0.05 μg/ml, and GM-CSF antibody at ≥0.05 μg/ml (Figure 2a–c and Supplementary Figure S2a–c). The most effective doses of IL-1α, IL-6, and GM-CSF antibodies inhibited 77.3 ± 8%, 54.3 ± 24.3%, and 59.3 ± 11.8% of the IL-17A‒induced ASR, respectively, resulting in ASR levels not significantly different from those of the vehicle-treated KCs (Figure 2a–c and e). Next, we tested whether there was an additive effect of the antibodies (Figure 2d and Supplementary Figure S2d). GM-CSF+IL6, IL1α+IL6, and GM-CSF+IL1α antibodies inhibited 76.7 ± 6.2%, 57.7 ± 24.9%, and 65.4 ± 19% of IL-17A‒induced ASR divisions, respectively (Figure 2e). No additive effect on IL-17A‒induced ASR divisions was detected (Figure 2d and e). This may be because individual cytokine antibodies tested resulted in ASR levels not significantly different from those of the vehicle-treated KCs. It is also possible that we missed untested IL-17A‒induced mediators that affect ASR. Cytokines that we found to produce ASR divisions may arise less or more predominantly from KCs than from other psoriasis tissues. However, our studies show that autocrine and paracrine secretion of psoriasis cytokines may play a significant role in SC activation in psoriasis. Combination treatment resulted in a level of ASR not significantly different from that of the vehicle, suggesting that we have identified some of the key players in SC activation in psoriasis. The concept of cytokine regulation of SC divisional behavior is evolving. ASR divisions were increased in murine hyperproliferative inflammatory bowel disease (Bu et al., 2016Bu P. Wang L. Chen K.Y. Srinivasan T. Murthy P.K.L. Tung K.L. et al.A miR-34a-numb feedforward loop triggered by inflammation regulates asymmetric stem cell division in intestine and colon cancer.Cell Stem Cell. 2016; 18: 189-202Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar) and IL-22‒treated intestinal SCs (Zwarycz et al., 2019Zwarycz B. Gracz A.D. Rivera K.R. Williamson I.A. Samsa L.A. Starmer J. et al.IL22 inhibits epithelial stem cell expansion in an ileal organoid model.Cell Mol Gastroenterol Hepatol. 2019; 7: 1-17Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar). Thus, there is mounting evidence that cytokines can regulate SC self-renewal and/or differentiation and specifically ASR in psoriasis and other inflammatory diseases. Notably, the mediators of ASR we identified—IL-1a, IL-6, and GM-CSF—all activate NF-kB, a transcription factor upregulated in psoriasis (Ebner et al., 2003Ebner K. Bandion A. Binder B.R. de Martin R. Schmid J.A. GMCSF activates NF-kappaB via direct interaction of the GMCSF receptor with IkappaB kinase beta.Blood. 2003; 102: 192-199Crossref PubMed Scopus (55) Google Scholar; Wang et al., 2003Wang L. Walia B. Evans J. Gewirtz A.T. Merlin D. Sitaraman S.V. IL-6 induces NF-kappa B activation in the intestinal epithelia.J Immunol. 2003; 171: 3194-3201Crossref PubMed Scopus (155) Google Scholar). Perturbation of NF-kB signaling in various studies resulted in either epidermal hyperplasia or hypoplasia (Lizzul et al., 2005Lizzul P.F. Aphale A. Malaviya R. Sun Y. Masud S. Dombrovskiy V. et al.Differential expression of phosphorylated NF-kappaB/RelA in normal and psoriatic epidermis and downregulation of NF-kappaB in response to treatment with etanercept.J Invest Dermatol. 2005; 124: 1275-1283Abstract Full Text Full Text PDF PubMed Scopus (157) Google Scholar). In addition, NF-kB activation is required for ASR in neural SCs (Zhang et al., 2012Zhang Y. Liu J. Yao S. Li F. Xin L. Lai M. et al.Nuclear factor kappa B signaling initiates early differentiation of neural stem cells.Stem Cells. 2012; 30: 510-524Crossref PubMed Scopus (74) Google Scholar). Future studies will be needed to determine a possible role for NF-kB in the increased ASR resulting from IL-17A treatment. ASR is responsible for epidermal stratification. mInsc and LGN and aPKC are required to establish planar polarity during ASR (Dainichi et al., 2016Dainichi T. Hayden M.S. Park S.G. Oh H. Seeley J.J. Grinberg-Bleyer Y. et al.PDK1 is a regulator of epidermal differentiation that activates and organizes asymmetric cell division.Cell Rep. 2016; 15: 1615-1623Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar; Williams et al., 2011Williams S.E. Beronja S. Pasolli H.A. Fuchs E. Asymmetric cell divisions promote Notch-dependent epidermal differentiation.Nature. 2011; 470: 353-358Crossref PubMed Scopus (302) Google Scholar). Genetic manipulations in mice that increase ASR (mInsc overexpression) produced a thicker epidermis (Williams et al., 2011Williams S.E. Beronja S. Pasolli H.A. Fuchs E. Asymmetric cell divisions promote Notch-dependent epidermal differentiation.Nature. 2011; 470: 353-358Crossref PubMed Scopus (302) Google Scholar), and the manipulations that decreased ASR (Lgn deletion or knockout of Pdk1) produced a thinner epidermis (Dainichi et al., 2016Dainichi T. Hayden M.S. Park S.G. Oh H. Seeley J.J. Grinberg-Bleyer Y. et al.PDK1 is a regulator of epidermal differentiation that activates and organizes asymmetric cell division.Cell Rep. 2016; 15: 1615-1623Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar; Williams et al., 2011Williams S.E. Beronja S. Pasolli H.A. Fuchs E. Asymmetric cell divisions promote Notch-dependent epidermal differentiation.Nature. 2011; 470: 353-358Crossref PubMed Scopus (302) Google Scholar). Our work suggests that ASR divisions are important to the epidermal hyperplasia evoked by IL-17A (Charruyer et al., 2017Charruyer A. Fong S. Vitcov G.G. Sklar S. Tabernik L. Taneja M. et al.Brief report: interleukin-17A-dependent asymmetric stem cell divisions are increased in human psoriasis: a mechanism underlying benign hyperproliferation.Stem Cells. 2017; 35: 2001-2007Crossref PubMed Scopus (9) Google Scholar) and in psoriasis. It will be important to dissect molecular mechanisms underlying increased ASR in psoriasis to identify new disease targets that can be used to specifically combat cutaneous manifestations. Datasets related to this article can be found at https://doi.org/10.6084/m9.figshare.12241745, hosted at Figshare Data repository. Hang Li: http://orcid.org/0000-0003-1843-8329 Alex Charruyer: http://orcid.org/0000-0003-4655-4781 Tracy Weisenberger: http://orcid.org/0000-0002-5849-2103 Ayman Khalifa: http://orcid.org/0000-0001-5828-0816 Robert Nguyen: http://orcid.org/0000-0002-6186-3243 Ruby Ghadially: http://orcid.org/0000-0002-2880-7987 The authors state no conflict of interest. This study was supported by a Novartis Investigator-Initiated grant (Novartis Pharmaceuticals, AIN457AUSNCO2T) and a Merit Review Award from the Veterans Health Administration Office of Research and Development (BX000794). Conceptualization: HL, AC, RG; Data Curation: HL, AC; Formal Analysis: HL, AC, RN, RG; Funding Acquisition: RG; Investigation: HL, TW, AK, RN; Methodology: AC, RG; Project Administration: AC, RG; Resources: RG; Supervision: AC, RG; Validation: HL, AC, RG; Visualization: HL, AC, RN, RG; Writing - Original Draft Preparation: HL, AC, RN, RG; Writing - Review and Editing: AC, RG.