Human Perifollicular Macrophages Undergo Apoptosis, Express Wnt Ligands, and Switch their Polarization during Catagen

Abstract

In human and rodent skin, the hair follicle (HF) mesenchyme (connective tissue sheath [CTS]), is densely populated with macrophages (Bertolini et al., 2013Bertolini M. Meyer K.C. Slominski R. Kobayashi K. Ludwig R.J. Paus R. The immune system of mouse vibrissae follicles: cellular composition and indications of immune privilege.Exp Dermatol. 2013; 22: 593-598Crossref PubMed Scopus (11) Google Scholar, Christoph et al., 2000Christoph T. Müller-Röver S. Audring H. Tobin D.J. Hermes B. Cotsarelis G. et al.The human hair follicle immune system: cellular composition and immune privilege.Br J Dermatol. 2000; 142: 862-873Crossref PubMed Scopus (235) Google Scholar, Paus et al., 1998Paus R. Van Der Veen C. Eichmüller S. Kopp T. Hagen E. Müller-Röver S. et al.Generation and cyclic remodeling of the hair follicle immune system in mice.J Invest Dermatol. 1998; 111: 7-18Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar). Notably, the number of these perifollicular macrophages (pMΦ) fluctuates greatly during the murine hair cycle, being the highest during hair growth (anagen), decreasing in regression (catagen), and reaching the lowest count around resting (telogen) HFs (Paus et al., 1998Paus R. Van Der Veen C. Eichmüller S. Kopp T. Hagen E. Müller-Röver S. et al.Generation and cyclic remodeling of the hair follicle immune system in mice.J Invest Dermatol. 1998; 111: 7-18Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar). In rat skin, macrophage-like cells show similar hair cycle-associated fluctuations, along with a switch toward a fibroblast growth factor-5+ phenotype that promotes catagen (Suzuki et al., 1998Suzuki S. Kato T. Takimoto H. Masui S. Oshima H. Ozawa K. et al.Localization of rat FGF-5 protein in skin macrophage-like cells and FGF-5S protein in hair follicle: possible involvement of two Fgf-5 gene products in hair growth cycle regulation.J Invest Dermatol. 1998; 111: 963-972Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar). Only later was it demonstrated that pMΦ can regulate HF cycling, while apoptotic pMΦ around murine telogen HFs secrete Wnt signals that can activate quiescent epithelial HF stem cells inducing anagen (Castellana et al., 2014Castellana D. Paus R. Perez-Moreno M. Macrophages contribute to the cyclic activation of adult hair follicle stem cells.PLOS Biol. 2014; 12: e1002002Crossref PubMed Scopus (57) Google Scholar). However, the role of pMΦ in human HF biology remains obscure, despite abnormalities in their number and activation status being recognized in inflammatory hair diseases (Harries et al., 2013Harries M.J. Meyer K. Chaudhry I. E Kloepper J. Poblet E. Griffiths C.E. et al.Lichen planopilaris is characterized by immune privilege collapse of the hair follicle’s epithelial stem cell niche.J Pathol. 2013; 231: 236-247Crossref PubMed Scopus (94) Google Scholar) (Supplementary Text S1a). As a basis for hypothesis building, we asked what happens to the pMΦ number, distribution and polarization during catagen development of healthy organ cultured anagen scalp HFs (Langan et al., 2015Langan E.A. Philpott M.P. Kloepper J.E. Paus R. Human hair follicle organ culture: theory, application and perspectives.Exp Dermatol. 2015; 24: 903-911Crossref PubMed Scopus (56) Google Scholar), as this is clinically the most relevant for hair loss disorders (Paus and Cotsarelis, 1999Paus R. Cotsarelis G. The biology of hair follicles.N Engl J Med. 1999; 341: 491-497Crossref PubMed Scopus (802) Google Scholar) and can be followed ex vivo. To investigate, human HFs were obtained with ethics committee approval, institutional approval and informed, written patient consent. These were organ-cultured for 1 to 6 days collecting anagen, early-, mid- and late HFs for comparison (Kloepper et al., 2010Kloepper J.E. Sugawara K. Al-Nuaimi Y. Gáspár E. van Beek N. Paus R. Methods in hair research: how to objectively distinguish between anagen and catagen in human hair follicle organ culture.Exp Dermatol. 2010; 19: 305-312Crossref PubMed Scopus (78) Google Scholar). HF sections were analyzed by immunofluorescence microscopy for the macrophage marker CD68 (Barros et al., 2013Barros M.H.M. Hauck F. Dreyer J.H. Kempkes B. Niedobitek G. Macrophage polarisation: an immunohistochemical approach for identifying M1 and M2 macrophages.PLOS ONE. 2013; 8: e80908Crossref PubMed Scopus (377) Google Scholar) (for methods, see Supplementary Text S2). Consistent with previous research (Christoph et al., 2000Christoph T. Müller-Röver S. Audring H. Tobin D.J. Hermes B. Cotsarelis G. et al.The human hair follicle immune system: cellular composition and immune privilege.Br J Dermatol. 2000; 142: 862-873Crossref PubMed Scopus (235) Google Scholar), pMΦ predominantly localized to proximal CTS around the proliferative hair bulb as well as the central CTS, with more than half of all CD68+ pMΦ detected in the peribulbar CTS of anagen VI HFs (Figure 1a and b, Supplementary Figure S1a). The number of pMΦ decreased progressively to the infundibulum and was maintained in all the hair cycle stages (Figure 1a and b, Supplementary Figure S1). Immuno-electron microscopy confirmed that these CD68+ cells showed the ultrastructural characteristics of macrophages (Arismendi-Morillo et al., 2010Arismendi-Morillo G. Castellano-Ramírez A. Medina Z. Ultrastructural characterization of macrophage-like mononuclear leukocytes in human astrocytic tumors.Ultrastruct Patho. 2010; 34: 321-326Crossref PubMed Scopus (3) Google Scholar) (Supplementary Figure S2). When human HFs entered catagen, the pMΦ number rapidly decreased to reach a minimum in late catagen (Figure 1a and c), along with a 1.5-fold decrease in CD68 transcript levels (Figure 1d). The fact that freshly isolated, non-cultured HFs in (anagen/catagen/telogen) also showed this cycle-dependent decline in CD68+ pMΦs (Supplementary Figure S3) excludes the possibility that this observation represents an organ culture artifact. Next, we asked whether the catagen-associated decline in human pMΦs resulted from apoptosis, as in mice (Castellana et al., 2014Castellana D. Paus R. Perez-Moreno M. Macrophages contribute to the cyclic activation of adult hair follicle stem cells.PLOS Biol. 2014; 12: e1002002Crossref PubMed Scopus (57) Google Scholar). Indeed, as the HFs progressed through catagen, the number of apoptotic macrophages (CD68+/TUNEL+ immunofluorescence) increased significantly (Figure 1e and f). Therefore apoptosis, rather than emigration from the CTS, is likely responsible for this decline. As murine pMΦ are known to secrete Wnts, we asked whether they do so in the CTS of human HFs, thus contributing to anagen maintenance. Both WNT10a and 7b, which is also produced by murine pfMΦ (Castellana et al., 2014Castellana D. Paus R. Perez-Moreno M. Macrophages contribute to the cyclic activation of adult hair follicle stem cells.PLOS Biol. 2014; 12: e1002002Crossref PubMed Scopus (57) Google Scholar), were highly expressed by macrophages in anagen with protein levels decreasing as they enter catagen (Figure 2a and b). Regarding RNA, in situ hybridization (Supplementary Text S2) demonstrated that LEF1 mRNA, a down-stream Wnt mediator (Supplementary Figure S1), and WLS mRNA, found in Wnt-secreting cells (Supplementary Figure S2), were highly expressed around the anagen bulb. However, the WNT10a mRNA was low in the CTS, being restricted to the hair matrix tips (Supplementary Figure S4). AXIN2 mRNA was also expressed in low levels in the CTS during anagen (Supplementary Figure S4). During catagen, AXIN2 and LEF1 were expressed in the CTS, while WLS and WNT10a were almost absent. These data suggest that the human pMΦ niche is Wnt active and suggests that macropahges secrete Wnt ligands. Given the critical role of Wnt signaling in hair growth control (Geyfman et al., 2015Geyfman M. Plikus M.V. Treffeisen E. Andersen B. Paus R. Resting no more: re-defining telogen, the maintenance stage of the hair growth cycle.Biol Rev. 2015; 90: 1179-1196Crossref PubMed Scopus (89) Google Scholar, Schneider et al., 2009Schneider M.R. Schmidt-Ullrich R. Paus R. The hair follicle as a dynamic miniorgan.Curr Biol. 2009; 19: R132-R142Abstract Full Text Full Text PDF PubMed Scopus (634) Google Scholar), this raises the question whether pMΦ are one important source of WNTs (Castellana et al., 2014Castellana D. Paus R. Perez-Moreno M. Macrophages contribute to the cyclic activation of adult hair follicle stem cells.PLOS Biol. 2014; 12: e1002002Crossref PubMed Scopus (57) Google Scholar) (Supplementary Text S1b). As macrophages exist in different phenotypes, pMΦ polarization was explored using the dual immunofluorescence of CD68 with the M1 marker CD86 or RAGE, which is the receptor for advance glycation end-products (RAGE being upregulated by an M1 phenotype [Jin et al., 2015Jin X. Yao T. Zhou Z.Z. Zhu J. Zhang S. Hu W. et al.Advanced glycation end products enhance macrophages polarization into M1 phenotype through activating RAGE/NF- κ B pathway.Biomed Res. 2015; 2015732450Google Scholar]). Although the CD86 expression was low (Figure 2c) (< 5% of pMΦ), RAGE was highly expressed in anagen in 25% of the pMΦ, decreasing though catagen (Figure 2d) to 10% by late catagen. Interestingly, advance glycation end-products are produced in proliferative tissues such as tumors (Supplementary Figure S4). As RAGE is upregulated and stimulated by high levels of advance glycation end-products (Jin et al., 2015Jin X. Yao T. Zhou Z.Z. Zhu J. Zhang S. Hu W. et al.Advanced glycation end products enhance macrophages polarization into M1 phenotype through activating RAGE/NF- κ B pathway.Biomed Res. 2015; 2015732450Google Scholar), RAGE+ pMΦ might bind to and remove advance glycation end-products from the HF microenvironment during proliferative anagen, consistent with a previous hypothesis that increased advance glycation end-products levels detected by pMΦs may promote catagen induction (Supplementary Figure S4). Conversely, an M2 phenotype (CD206/CD163) (Martinez and Gordon, 2014Martinez F.O. Gordon S. The M1 and M2 paradigm of macrophage activation: time for reassessment.F1000Prime Rep. 2014; 6: 13Crossref PubMed Scopus (2907) Google Scholar) was least present in anagen and early/mid catagen but rapidly increased to 50% (CD206) and 30% (CD163) (Figure 2e and f) of all the macrophages in late catagen, representing a distinct hair cycle associated phenotypic switch. As an M2 phenotype is linked with tissue remodeling (S6), this switch may be important for the extensive catagen-associated HF remodeling. An exception to this was the high levels of CD206 around the anagen bulge region (40% of pMΦ), (Supplementary Figure S1b) where they may contribute to immune privilege (Supplementary Text 1e). Limiting causal investigation, the selective depletion of pMΦs by clodronate (Castellana et al., 2014Castellana D. Paus R. Perez-Moreno M. Macrophages contribute to the cyclic activation of adult hair follicle stem cells.PLOS Biol. 2014; 12: e1002002Crossref PubMed Scopus (57) Google Scholar) failed repeatedly in our ex vivo human model, potentially because of insufficient tissue penetration by these large liposomes (Supplementary Figure S3, Supplementary Figure S4, Supplementary Table S1, and Supplementary Text S1c) Taken together, our pilot study demonstrates that pMΦ dramatically change in number, activity and phenotype during the anagen-catagen transformations of human scalp HFs and suggest that these cells may engage in both Wnt secretion and AGE recognition, particularly in the bulb in human HFs, a region that changes extensively during the anagen-catagen switch (Kloepper et al., 2010Kloepper J.E. Sugawara K. Al-Nuaimi Y. Gáspár E. van Beek N. Paus R. Methods in hair research: how to objectively distinguish between anagen and catagen in human hair follicle organ culture.Exp Dermatol. 2010; 19: 305-312Crossref PubMed Scopus (78) Google Scholar, Oh et al., 2016Oh J.W. Kloepper J. Langan E.A. Kim Y. Yeo J. Kim M.J.M. et al.A guide to studying human hair follicle cycling in vivo.J Invest Dermatol. Elsevier, Inc. 2016; 136: 34-44Abstract Full Text Full Text PDF PubMed Scopus (167) Google Scholar). Therefore, pMΦ deserve to be systematically explored as therapeutic intervention targets in human hair growth control. Research data that is necessary to interpret the information presented here will be made available to any researcher, with minimal reuse restrictions. All the tissue was obtained with written and informed patient consent adhering to the ‘Declaration of Helsinki Principles’ with ethical and institutional approval from the University of Manchester. All samples, slides and biological material were tracked and stored according the ‘Human Tissue Act’ guidelines’ in an HTA licensed freezer. Jonathan Alan Hardman: https://orcid.org/0000-0002-1653-7908 Ferhan Muneeb: https://orcid.org/0000-0002-2564-9792 Jenny Pople: https://orcid.org/0000-0002-3560-8994 Ranjit Bhogal: https://orcid.org/0000-0003-4835-4871 Asim Shahmalak: https://orcid.org/0000-0003-1411-3666 Ralf Paus: https://orcid.org/0000-0002-3492-9358 This work was funded by Unilever in the form of a research grant awarded to RP. All other authors state no conflict of interest. We would like to thank R Marotta/IIT for support with IM-TEM and acknowledge Kim Mace for reviewing the manuscript. This work was supported by grants from Unilever, UK, and the NIHR Manchester BRC (BRC-1215-20007) “Inflammatory Hair Diseases” program. Conceptualization: JAH, RP, JP, RB; Data Curation: JAH, FM; Formal Analysis: JAH, FM; Funding Acquisition: RP, RB; Project Administration: JAH, RP; Tissue acquisition: AS; Writing – Original Draft Preparation: JAH, RP; Writing – review and editing: JAH, JP, FM. The human hair follicle (HF) is a mini-organ containing a vast array of cells, including stem, epithelial, mesenchymal and immune cells. Interestingly, the human HF has a unique immune profile owing to the fact that two of its key cell populations are immune privilege (IP), specifically the rapidly proliferating matrix keratinocytes of the bulb and the epithelial stem cell niche, the bulge (Bertolini et al., 2013Bertolini M. Meyer K.C. Slominski R. Kobayashi K. Ludwig R.J. Paus R. The immune system of mouse vibrissae follicles: cellular composition and indications of immune privilege.Exp Dermatol. 2013; 22: 593-598Crossref PubMed Scopus (11) Google Scholar, Paus et al., 2005Paus R. Nickoloff B.J. Ito T. A “hairy” privilege.Trends Immunol. 2005; 26: 32-40Abstract Full Text Full Text PDF PubMed Scopus (248) Google Scholar, Purba et al., 2017Purba T.S. Brunken L. Peake M. Shahmalak A. Chaves A. Poblet E. et al.Characterisation of cell cycle arrest and terminal differentiation in a maximally proliferative human epithelial tissue: lessons from the human hair follicle matrix.Eur J Cell Biol. 2017; 96: 632-641Crossref PubMed Scopus (26) Google Scholar, Purba et al., 2014Purba T.S. Haslam I.S. Poblet E. Jiménez F. Gandarillas A. Izeta A. et al.Human epithelial hair follicle stem cells and their progeny: current state of knowledge, the widening gap in translational research and future challenges.BioEssays. 2014; 36: 513-525Crossref PubMed Scopus (98) Google Scholar). It is when HF IP is lost that inflammatory hair diseases, such as alopecia areata and lichen planopilaris, develop (Bertolini et al., 2016Bertolini M. Pretzlaff M. Sulk M. Bähr M. Gherardini J. Uchida Y. et al.Vasoactive intestinal peptide, whose receptor-mediated signalling may be defective in alopecia areata, provides protection from hair follicle immune privilege collapse.Br J Dermatol. 2016; 175: 531-541Crossref PubMed Scopus (27) Google Scholar, Harries et al., 2013Harries M.J. Meyer K. Chaudhry I. E Kloepper J. Poblet E. Griffiths C.E. et al.Lichen planopilaris is characterized by immune privilege collapse of the hair follicle’s epithelial stem cell niche.J Pathol. 2013; 231: 236-247Crossref PubMed Scopus (94) Google Scholar; Imanishi et al., 2017; Strazzulla et al., 2018Strazzulla L.C. Wang E.H.C. Avila L. Lo Sicco K. Brinster N. Christiano A.M. et al.Alopecia areata.J Am Acad Dermatol. 2018; 78: 1-12Abstract Full Text Full Text PDF PubMed Scopus (213) Google Scholar). Despite growing evidence that the HF’s immune microenvironment may have an important regulatory role in regulating hair growth (Castellana et al., 2014Castellana D. Paus R. Perez-Moreno M. Macrophages contribute to the cyclic activation of adult hair follicle stem cells.PLOS Biol. 2014; 12: e1002002Crossref PubMed Scopus (57) Google Scholar, Kloepper et al., 2013Kloepper J.E. Kawai K. Bertolini M. Kanekura T. Paus R. Loss of γδ T cells Results in Hair Cycling Defects.J Invest Dermatol. 2013; 133: 1666-1669Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar, Maurer et al., 1995Maurer M. Paus R. Czarnetzki B.M. Mast cells as modulators of hair follicle cycling.Exp Dermatol. 1995; 4: 266-271Crossref PubMed Scopus (59) Google Scholar, Peters et al., 2017Peters E.M.J. Müller Y. Snaga W. Fliege H. Reißhauer A. Schmidt-Rose T. et al.Hair and stress: A pilot study of hair and cytokine balance alteration in healthy young women under major exam stress.PLOS ONE. 2017; 12: e0175904Crossref PubMed Scopus (25) Google Scholar), in addition to their immune function, work so far has tended to focus on mouse HFs. Specifically in rodent skin, the HF mesenchyme (connective tissue sheath [CTS]), is densely populated with macrophages (MΦ) (Bertolini et al., 2013Bertolini M. Meyer K.C. Slominski R. Kobayashi K. Ludwig R.J. Paus R. The immune system of mouse vibrissae follicles: cellular composition and indications of immune privilege.Exp Dermatol. 2013; 22: 593-598Crossref PubMed Scopus (11) Google Scholar, Christoph et al., 2000Christoph T. Müller-Röver S. Audring H. Tobin D.J. Hermes B. Cotsarelis G. et al.The human hair follicle immune system: cellular composition and immune privilege.Br J Dermatol. 2000; 142: 862-873Crossref PubMed Scopus (235) Google Scholar, Paus et al., 1998Paus R. Van Der Veen C. Eichmüller S. Kopp T. Hagen E. Müller-Röver S. et al.Generation and cyclic remodeling of the hair follicle immune system in mice.J Invest Dermatol. 1998; 111: 7-18Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar), which fluctuates greatly during the hair cycle, being the highest during active hair growth (anagen), showing a sharp decline during regression (catagen) and reaching the lowest count in telogen (Paus et al., 1998Paus R. Van Der Veen C. Eichmüller S. Kopp T. Hagen E. Müller-Röver S. et al.Generation and cyclic remodeling of the hair follicle immune system in mice.J Invest Dermatol. 1998; 111: 7-18Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar). In rats, macrophage-like cells show a similar pattern, and these cells have a fibroblast growth factor-5+ phenotype that might promote catagen (Suzuki et al., 1998Suzuki S. Kato T. Takimoto H. Masui S. Oshima H. Ozawa K. et al.Localization of rat FGF-5 protein in skin macrophage-like cells and FGF-5S protein in hair follicle: possible involvement of two Fgf-5 gene products in hair growth cycle regulation.J Invest Dermatol. 1998; 111: 963-972Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar). Only much later was it demonstrated that pMΦ can profoundly regulate HF cycling, while apoptotic perifollicular macrophages (pMΦ) around murine telogen HFs transcribe and secrete Wnt signals, which can activate quiescent epithelial HF stem cells and induce anagen (Castellana et al., 2014Castellana D. Paus R. Perez-Moreno M. Macrophages contribute to the cyclic activation of adult hair follicle stem cells.PLOS Biol. 2014; 12: e1002002Crossref PubMed Scopus (57) Google Scholar). However, the characteristics and functional role of pMΦ in human HF biology remain obscure, even though abnormalities in their number and activation status are recognized in some inflammatory hair diseases (Harries et al., 2013Harries M.J. Meyer K. Chaudhry I. E Kloepper J. Poblet E. Griffiths C.E. et al.Lichen planopilaris is characterized by immune privilege collapse of the hair follicle’s epithelial stem cell niche.J Pathol. 2013; 231: 236-247Crossref PubMed Scopus (94) Google Scholar). Despite this link, human studies stalled in the early 2000s, and although they were able to demonstrate distinct spatial changes in the macrophage population, being the highest in the proximal HF (Christoph et al., 2000Christoph T. Müller-Röver S. Audring H. Tobin D.J. Hermes B. Cotsarelis G. et al.The human hair follicle immune system: cellular composition and immune privilege.Br J Dermatol. 2000; 142: 862-873Crossref PubMed Scopus (235) Google Scholar), the hair cycle-dependent changes were not investigated. Moreover, to our knowledge, no study has investigated the macrophage phenotypes during the human hair cycle. With the profound differences between the activation state of macrophages, M1 traditionally thought to be inflammatory and M2 anti-inflammatory, and the function of each population with respect to cytokine and chemokine production (Arora et al., 2017), this article will investigate the human follicular macrophage immune-microenvironment with respect to the spatial, temporal and activation state of perifollicular macrophages to better understand whether they are important for HF biology and hair cycle control in humans. As a first step towards exploring this role, we asked what happens to pMΦ in healthy human scalp HFs when these undergo spontaneous catagen development ex vivo (Langan et al., 2015Langan E.A. Philpott M.P. Kloepper J.E. Paus R. Human hair follicle organ culture: theory, application and perspectives.Exp Dermatol. 2015; 24: 903-911Crossref PubMed Scopus (56) Google Scholar). We focused on the anagen VI-catagen switch because this represents the clinically most relevant HF transformation, since premature catagen development underlies the vast majority of hair loss cases seen in clinical practice (Paus and Cotsarelis, 1999Paus R. Cotsarelis G. The biology of hair follicles.N Engl J Med. 1999; 341: 491-497Crossref PubMed Scopus (802) Google Scholar). As a basis for hypothesis-building, in this pilot study we investigated whether the number, localization and/or polarization phenotype (i.e., “inflammatory” M1 versus repair-associated M2 macrophages, (Murray, 2017Murray P.J. Macrophage polarization.Annu Rev Physiol. 2017; 79: 541-566Crossref PubMed Scopus (1336) Google Scholar, Saha et al., 2017Saha S. Shalova I.N. Biswas S.K. Metabolic regulation of macrophage phenotype and function.Immunol Rev. 2017; 280: 102-111Crossref PubMed Scopus (113) Google Scholar)) of human pMΦs changes during the anagen-catagen transformation, and whether pMΦ undergo apoptosis during this time. In mice, pMΦ have been shown to secrete products even when they are undergoing apoptosis (Castellana et al., 2014Castellana D. Paus R. Perez-Moreno M. Macrophages contribute to the cyclic activation of adult hair follicle stem cells.PLOS Biol. 2014; 12: e1002002Crossref PubMed Scopus (57) Google Scholar). This has shown to be essential for anagen initiation. As macrophages are known to secrete many products to their local environment, it may be that they secrete products necessary for anagen maintenance, such as the Wnts. Moreover, a switch in phenotype between anagen and catagen may represent a secretory phenotype change that is important for catagen initiation. In addition to this, secretory products from these macrophages may be important in scar-less remodeling of the dermis as the lower thirds involute (Kasuya et al., 2018Kasuya A. Ito T. Tokura Y. M2 macrophages promote wound-induced hair neogenesis.J Dermatol Sci. 2018; 91: 250-255Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar). Moreover, M2c macrophages have been shown to be potent producers of MMP9 with macrophages also being demonstrated to have abilities to prevent fibrotic tissue, which may both be essential for regression during catagen (Adhyatmika et al., 2015Adhyatmika A. Putri K.S.S. Beljaars L. Melgert B.N. The elusive antifibrotic macrophage.Front Med. 2015; 2: 81Crossref PubMed Scopus (45) Google Scholar, Chistiakov et al., 2018Chistiakov D.A. Myasoedova V.A. Revin V.V. Orekhov A.N. Bobryshev Y.V. The impact of interferon-regulatory factors to macrophage differentiation and polarization into M1 and M2.Immunobiology. 2018; 223: 101-111Crossref PubMed Scopus (156) Google Scholar). Together this highlights multiple ways that these pMΦ influence the HF. In our experiments, it has not been possible to selectively ablate macrophages. This limitation prevents us from conclusively demonstrating how important pMΦ are to the HF. While we endeavored to deplete the macrophage population using clodronate, commonly used to selectively deplete MΦ by apoptosis induction in mice in vivo (Castellana et al., 2014Castellana D. Paus R. Perez-Moreno M. Macrophages contribute to the cyclic activation of adult hair follicle stem cells.PLOS Biol. 2014; 12: e1002002Crossref PubMed Scopus (57) Google Scholar, Lehenkari et al., 2002Lehenkari P.P. Kellinsalmi M. Näpänkangas J.P. Ylitalo K.V. Mönkkönen J. Rogers M.J. et al.Further insight into mechanism of action of clodronate: inhibition of mitochondrial ADP/ATP translocase by a nonhydrolyzable, adenine-containing metabolite.Mol Pharmacol. 2002; 61: 1255-1262Crossref PubMed Scopus (274) Google Scholar), this repeatedly failed to deplete the pMΦ in human CTS ex vivo, despite the pre-activation of macrophage phagocytic activity with PEA and IFN organ-culture (for details, see Supplementary Figure S4, Supplementary Table 1). Since targeted macrophage ablation may not be possible in this ex vivo model system, the information that can be gained may be limited. Next, we asked whether pMΦ polarization changes throughout the length of the CTS during HF cycling (bulb-infundibulum). While the protein expression of RAGE, CD163 and CD86 showed only marginal differences between any of the examined CTS regions, CD206 was markedly upregulated around the bulge region in anagen, being expressed on 40% of the pMΦs (Supplementary Figure S1b). However, by late catagen, CD206+ pMΦs also became prominent in the peribulbar and suprabulbar region. This invites the hypothesis that CD206+ pMΦs are involved in the catagen-associated remodeling of the regressing HF epithelium during late catagen. As the stem cell-containing bulge region is a region of immune privilege (Ito et al., 2008Ito T. Ito N. Saatoff M. Hashizume H. Fukamizu H. Nickoloff B.J. et al.Maintenance of hair follicle immune privilege is linked to prevention of NK cell attack.J Invest Dermatol. 2008; 128: 1196-1206Abstract Full Text Full Text PDF PubMed Scopus (191) Google Scholar), high levels of TGFβ may promote an anti-inflammatory phenotype in this area. These data show a clear phenotypic switch in the human pMΦ from a prevalent M1 phenotype in anagen VI HFs reflecting a protective function of pMΦ from advance glycation end-products accumulation in this proliferative tissue (Adhyatmika et al., 2015Adhyatmika A. Putri K.S.S. Beljaars L. Melgert B.N. The elusive antifibrotic macrophage.Front Med. 2015; 2: 81Crossref PubMed Scopus (45) Google Scholar, Chistiakov et al., 2018Chistiakov D.A. Myasoedova V.A. Revin V.V. Orekhov A.N. Bobryshev Y.V. The impact of interferon-regulatory factors to macrophage differentiation and polarization into M1 and M2.Immunobiology. 2018; 223: 101-111Crossref PubMed Scopus (156) Google Scholar). As excessive advance glycation end-products accumulation is associated with tissue pathologies, including diabetes, inflammation, kidney fibrosis, and cancer, RAGE expression here may act as a stress response system to maintain long-term human hair growth (Bucciarelli et al., 2002Bucciarelli L.G. Wendt T. Rong L. Lalla E. Hofmann M.A. Goova M.T. et al.RAGE is a multiligand receptor of the immunoglobulin superfamily: implications for homeostasis and chronic disease.Cell Mol Life Sci. 2002; 59: 1117-1128Crossref PubMed Scopus (263) Google Scholar, Lee et al., 2018Lee E.J. Kang M.K. Kim D.Y. Kim Y.H. Oh H. Kang Y.H. Chrysin inhibits advanced glycation end products-induced kidney fibrosis in renal mesangial cells and diabetic kidneys.Nutrients. 2018; 10: 882Crossref Scopus (30) Google Scholar, Mallipattu and Uribarri, 2014Mallipattu S.K. Uribarri J. Advanced glycation end product accumulation: A new enemy to target in chronic kidney disease?.Curr Opin Nephrol Hypertens. 2014; 23: 547-554Crossref PubMed Scopus (53) Google Scholar, Ramasamy et al., 2011Ramasamy R. Yan S.F. Schmidt A.M. Receptor for AGE (RAGE): signalling mechanisms in the pathogenesis of diabetes and its complications.Ann N Y Acad Sci. 2011; 1243: 88-102Crossref PubMed Scopus (328) Google Scholar). In catagen, this switches to a predominant M2 phenotype by late catagen, when pMΦ may facilitate tissue remodeling, for example, in phagocytosis of the thickened, excess basement membrane around the involuted HF epithelium (Parakkal, 1970Parakkal P.F. Morphogenesis of the hair follicle during catagen.Z Zellforsch Mikrosk Anat. 1970; 107: 174-186Crossref PubMed Scopus (39) Google Scholar). Excitingly, these data also suggest there may be differences, as their role in catagen may exceed that shown in mice (Mesa et al., 2015Mesa K.R. Rompolas P. Zito G. Myung P. Sun T.Y. Brown S. et al.Niche-induced cell death and epithelial phagocytosis regulate hair follicle stem cell pool.Nature. 2015; 522: 94-97Crossref PubMed Scopus (87) Google Scholar). Discarded occipital scalp hair follicles (HFs) from hair transplant surgery were obtained from either the Farjo Medical Centre (Manchester, UK) or the Crown Cosma Clinic (Manchester, UK). All tissue was obtained with written and informed patient consent adhering to the ‘Declaration of Helsinki Principles’ with ethical and institutional approval from the University of Manchester. Al