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Myeloid Adherent Cells Are Involved in Hair Loss in the Alopecia Areata Mouse Model

      Alopecia areata (AA), which is defined as an autoimmune hair loss disease, has a serious impact on the quality of life for patients with AA worldwide. In this study, to our knowledge, a previously unreported method of AA induction in C3H mice has been established and validated. Using this method, we showed that dermal injection of 1–3 million of a mixture of skin cells freshly isolated from AA-affected skin induces AA in more than 80% of healthy mice. Contrary to the previous protocol, the induction of AA by this approach does not need any surgical AA skin grafting, cell manipulation, or high number of activated T cells. We also showed that dermal injection of adherent myeloid cells (mainly CD11b+) in healthy mice is as potent as a mixture of none adherent CD3+ T cells and CD19+ B cells in the induction of AA. Interestingly, most of the mice (7 out of 8) that received non-adherent cells developed AA universalis, whereas most of the mice (5 out of 7) that received adherent cells developed patchy AA. Finally, we found a high number of stage-specific embryonic antigen-expressing cells whose expression in monocytes in an inflammatory disease causes the release of inflammatory cytokines, TNF-α and IL-1β, from these cells in AA-affected skin.

      Abbreviations:

      AA (alopecia areata), SSEA (stage-specific embryonic antigen)

      Introduction

      Alopecia areata (AA) is an autoimmune and inflammatory skin disease that causes sudden hair loss. According to the area of hair loss, patients with AA are defined as alopecia patchy (patchy hair loss), alopecia totalis (whole head hair loss), or alopecia universalis (total body hair loss). It is estimated that 0.1% to 0.2% of people are affected, with a lifetime risk of 2.1% (
      • Mirzoyev S.A.
      • Schrum A.G.
      • Davis M.D.P.
      • Torgerson R.R.
      Lifetime incidence risk of alopecia areata estimated at 2.1% by Rochester Epidemiology Project, 1990–2009.
      ).
      AA is an inflammatory skin disease in which T lymphocytes, including CD4+ and CD8+ T cells, usually infiltrate in the juxta-follicular area in the acute phase. In the chronic phase of AA, CD8+ T cells dominate around the hair bulbs (
      • Ito T.
      • Hashizume H.
      • Shimauchi T.
      • Funakoshi A.
      • Ito N.
      • Fukamizu H.
      • et al.
      CXCL10 produced from hair follicles induces Th1 and Tc1 cell infiltration in the acute phase of alopecia areata followed by sustained Tc1 accumulation in the chronic phase.
      ). Experimental evidence showed that injections of CD8+ cells from AA mice to non-AA mice could induce hair loss at local skin (
      • McElwee K.J.
      • Freyschmidt-Paul P.
      • Hoffmann R.
      • Kissling S.
      • Hummel S.
      • Vitacolonna M.
      • et al.
      Transfer of CD8(+) cells induces localized hair loss whereas CD4(+)/CD25(-) cells promote systemic alopecia areata and CD4(+)/CD25(+) cells blockade disease onset in the C3H/HeJ mouse model.
      ), whereas depletion of CD4+ or CD8+ T cells in the Dundee Experimental Bald Rat model could partially recover hair-growth (
      • McElwee K.J.
      • Spiers E.M.
      • Oliver R.F.
      In vivo depletion of CD8+ T cells restores hair growth in the DEBR model for alopecia areata.
      ,
      • McElwee K.J.
      • Spiers E.M.
      • Oliver R.F.
      Partial restoration of hair growth in the DEBR model for alopecia areata after in vivo depletion of CD4+ T cells.
      ). Despite a functional link between activated T lymphocytes and hair loss, the potential role of adherent immune cells in AA development is not clear.
      With the exception of highly controlled and regulated clinical trials for the treatment of AA, the use of humans for experimental investigation is impossible owing to obvious ethical issues. In the past, several animal models of AA have been developed, of which the most notable are the C3H/HeJ mouse model (
      • McElwee K.J.
      • Boggess D.
      • King LEJr
      • Sundberg J.P.
      Experimental induction of alopecia areata-like hair loss in C3H/HeJ mice using full-thickness skin grafts.
      ) and the Dundee Experimental Bald Rat model (
      • Michie H.J.
      • Jahoda C.A.
      • Oliver R.F.
      • Johnson B.E.
      The DEBR rat: an animal model of human alopecia areata.
      ). C3H/HeJ mice develop a spontaneous, complex polygenic AA-like hair loss with low incident rate (<1% in less than 6 months and around 20% by 12 months of age) (
      • Sundberg J.P.
      • Cordy W.R.
      • King Jr., L.E.
      Alopecia areata in aging C3H/HeJ mice.
      ). AA can be induced in C3H/HeJ mice by either skin grafting one piece of affected mouse skin to normal mice or dermal injection of 10 million non-cultured lymphocytes or cultured activated lymphocytes, which are obtained from the skin-derived lymph nodes of affected mice (
      • McElwee K.J.
      • Freyschmidt-Paul P.
      • Hoffmann R.
      • Kissling S.
      • Hummel S.
      • Vitacolonna M.
      • et al.
      Transfer of CD8(+) cells induces localized hair loss whereas CD4(+)/CD25(-) cells promote systemic alopecia areata and CD4(+)/CD25(+) cells blockade disease onset in the C3H/HeJ mouse model.
      ,
      • Silva K.A.
      • Sundberg J.P.
      Surgical methods for full-thickness skin grafts to induce alopecia areata in C3H/HeJ mice.
      ,
      • Wang E.H.C.
      • Khosravi-Maharlooei M.
      • Jalili R.B.
      • Yu R.
      • Ghahary A.
      • Shapiro J.
      • et al.
      Transfer of alopecia areata to C3H/HeJ mice using cultured lympho node-derived cells.
      ). However, these strategies require either an invasive surgery with the risk of graft-rejection and infection (
      • McElwee K.J.
      • Boggess D.
      • King LEJr
      • Sundberg J.P.
      Experimental induction of alopecia areata-like hair loss in C3H/HeJ mice using full-thickness skin grafts.
      ), a large number of cells from multiple donors sufficient only for a limited number of recipient mice (
      • McElwee K.J.
      • Freyschmidt-Paul P.
      • Hoffmann R.
      • Kissling S.
      • Hummel S.
      • Vitacolonna M.
      • et al.
      Transfer of CD8(+) cells induces localized hair loss whereas CD4(+)/CD25(-) cells promote systemic alopecia areata and CD4(+)/CD25(+) cells blockade disease onset in the C3H/HeJ mouse model.
      ), or the use of expensive CD3/CD28 beads and cytokine cocktails for lymphocyte expansion (
      • Wang E.H.C.
      • Khosravi-Maharlooei M.
      • Jalili R.B.
      • Yu R.
      • Ghahary A.
      • Shapiro J.
      • et al.
      Transfer of alopecia areata to C3H/HeJ mice using cultured lympho node-derived cells.
      ).
      In this study, to our knowledge, a previously unreported strategy has been established and validated for AA induction in C3H/HeJ mice and showed that myeloid cells isolated from AA-affected skin are as important as non-adherent lymphocytes in the induction of AA in healthy C3H mice. Evaluating the skin of these mice showed a high number of cells co-expressing stage-specific embryonic antigens (SSEAs) 1 and 3 in AA-affected skin. These SSEA-positive cells are derived from CD11b+ myeloid cells.

      Results

      Dermal Injection of a Mixture of Cells Isolated from AA-Affected Skin Induces AA in C3H/HeJ Mice

      The total number of cells isolated from AA-affected mouse skin was 74 ± 10 × 106 cells (ranged from 66 × 106 to 88 × 106 cells, n = 4). The result of FACS analysis showed the presence of CD3+ T cells (20%), CD19+ B cells (11.5%), and CD11b+ myeloid cells (16.1%) in a mixture of isolated skin cells (Figure 1a). With dermal injection of either 1 or 3 million of a mixture of skin cells isolated from AA-affected mouse skin to healthy C3H/HeJ mice, around 80% of mice developed AA (Figure 1b). As shown in Table 1, none of the mice that received either nothing (negative control) or 1 million of non-AA-affected skin cell mixture developed AA within 6 months post the injection of cells.
      Figure thumbnail gr1
      Figure 1Dermal injection of a mixture of cells isolated from AA-affected skin induces hair loss in healthy C3H/HeJ mice. (a) The percentage of CD3+, CD19+, and CD11b+ cells in a mixture of cells isolated from AA-affected skin evaluated by FACS analysis. (b) The clinical appearances of five mouse representatives that received either nothing (controls, top panels) or 1 million cells isolated from AA-affected skin (bottom panels). Note, the development of AA universalis is noticeable in mice that received a mixture of cells isolated from AA-affected skin. Left panels, abdominal skin of mice; right panels, dorsal skin of mice. AA, alopecia areata.
      Table 1AA Induction by Dermal Injection of Cells in C3H/HeJ Mice
      GroupNumber of cells mice receivedNumber of mice to be injectedNumber of mice that developed AAAA induction rate (%)
      Non-treated01000
      Skin cell mixture from normal mice1 × 1061000
      Skin cell mixture from AA-affected skin1 × 106393384.5
      Skin cell mixture from AA-affected skin3 × 1069777.8
      Activated T lymphocytes10 × 106421945.2
      Cultured suspension cells isolated from AA-affected skin3 × 10688100
      Cultured Adherent cells isolated from AA-affected skin3 × 1067685.7
      Abbreviation: AA, alopecia areata.

      Cultured Adherent Myeloid Cells Isolated from AA-Affected Skin Induces AA in C3H mice

      To address whether adherent myeloid cells are as important as lymphocytes in the induction of AA, a mixture of total cells isolated from AA-affected skin was cultured. After 24 hours, non-adherent cells were harvested and dermally injected into 8 healthy C3H/HeJ mice, while adherent cells were gently washed 3 times with PBS and remained in the culture for another 48 hours. Before cell injection, the harvested cells were subjected to FACS analysis and the result showed the presence of 14% CD3+ T cells, 9.7% CD19+ B cells, and 6.6% CD11b+ cells in the non-adherent cell mixture, whereas 10.5% CD11b+ cells and negligible number of CD3+ T cells (1.0%) and CD19+ B cells (0.6%) were present in the adherent cell mixture (Figure 2a). The results of AA induction showed that 8 out of 8 mice that received non-adherent cells developed AA, whereas 6 out of 7 mice that received adherent cells developed AA (Figure 2b). Interestingly, most of the mice (7 out of 8) that received non-adherent cells developed AA universalis, whereas most of the mice (5 out of 7) that received adherent cells developed patchy AA, and only one mouse that received adherent cells developed AA universalis (Figure 2b and Table 1).
      Figure thumbnail gr2
      Figure 2Alopecia universalis and patchy are induced by dermal injection of cultured non-adherent cells and adherent cells isolated from AA-affected mouse skin. (a) Percentage of CD3+, CD19+, and CD11b+ cells in cultured non-adherent and adherent cells isolated from AA-affected mouse skin. The mixture of cells was cultured for 24 hours, and non-adherent cells were harvested and analyzed by flow cytometry. Adherent cells were washed 3 times with PBS and kept in the culture for other 48 hours. Cells were then harvested by trypsin-EDTA and analyzed by flow cytometry. (b) The clinical appearances of AA universalis (top panel) and patchy (bottom panel) in five mouse representatives received 3 million of either non-adherent (top panel) or adherent cells (bottom panel) isolated from a mixture of AA-affected skin cells. Left panels, abdominal skin of mice; Right panels, dorsal skin of mice. AA, alopecia areata.

      Stage-Specific Embryonic Antigen (SSEA)-1 and SSEA-3 Positive Cells are Present in AA-Affected Skin

      As cells expressing SSEAs, markers for pluripotent stem cells (
      • Trusler O.
      • Huang Z.
      • Goodwin J.
      • Laslett A.L.
      Cell surface markers for the identification and study of human naive pluripotent stem cells.
      ), have been found in inflammatory tissues (
      • Galassi A.
      • Turatello L.
      • De Salvia A.
      • Neri M.
      • Turillazzi E.
      • La Russa R.
      • et al.
      Septic cardiomyopathy: the value of lactoferrin and CD15 as specific markers to corroborate a definitive diagnosis.
      ,
      • Kaminagakura E.
      • Bonan P.R.
      • Jorge J.
      • Almeida O.P.
      • Scully C.
      Characterization of inflammatory cells in oral paracoccidioidomycosis.
      ,
      • Kurose R.
      • Sawai T.
      • Oishi K.
      • Liu X.
      • Sasaki A.
      • Kurose A.
      • et al.
      Possibility of inhibiting arthritis and joint destruction by SSEA-3 positive cells derived from synovial tissue in rheumatoid arthritis.
      ) and activate monocytes which release inflammatory cytokines, TNF-α and IL-1β (
      • Lo S.K.
      • Golenbock D.T.
      • Sass P.M.
      • Maskati A.
      • Xu H.
      • Silverstein R.L.
      Engagement of the lewis X antigen (CD15) results in monocyte activation.
      ), here, we examined whether SSEA-positive cells are present in AA-affected skin. By using immunofluorescent staining for SSEA-3, we found the presence of SSEA-3 positive cells in AA-affected skin but not in the normal skin (Figure 3a, top panels). Most of the SSEA-3 positive cells also co-expressed SSEA-1 in AA-affected mouse skin (Figure 2b).
      Figure thumbnail gr3
      Figure 3SSEA-1 and SSEA-3 positive myeloid cells are present in AA-affected skin of C3H/HeJ mice and cultured adherent cells isolated from AA-affected skin of mice. (a) Detection of SSEA-3 positive cells in AA-affected skin as compared with that in non-AA-affected skin. (b) Double immunofluorescent staining with SSEA-1 and SSEA-3 antibodies in AA-affected skin. (c) Double immunofluorescent staining with CD11b and SSEA-1 antibodies in cultured adherent cells isolated from AA-affected skin of mice. DAPI (blue) was used as a nuclear counterstain. AA, alopecia areata; SSEA, stage-specific embryonic antigen. Bar = 50 μm.
      To examine the correlation between SSEA-positive cells and adherent CD11b+ myeloid cells, we performed double immunofluorescent staining for CD11b+ and SSEA-1 and showed that some of the CD11b+ cells, but not all, are also positive for SSEA-1. This suggests that, at least, some of the cells expressing SSEAs and CD11b+ might be directly or indirectly responsible for the induction of AA in C3H/HeJ mice.

      Discussion

      In this study, we have established and validated a simple model of AA induction by using dermal injection of a mixture of cells isolated from AA-affected skin without any cellular manipulation into C3H/HeJ. We also showed that adherent myeloid cells (CD11b, SSEAs positive) are as important as a mixture of non-adherent CD3+ T cells and CD19+ B cells in the induction of AA. Contrary to the previous protocol, the induction of AA in the same strain of mice does not need any surgical procedure for AA skin grafting (
      • McElwee K.J.
      • Boggess D.
      • King LEJr
      • Sundberg J.P.
      Experimental induction of alopecia areata-like hair loss in C3H/HeJ mice using full-thickness skin grafts.
      ,
      • Silva K.A.
      • Sundberg J.P.
      Surgical methods for full-thickness skin grafts to induce alopecia areata in C3H/HeJ mice.
      ) or higher number of activated T cells (
      • McElwee K.J.
      • Freyschmidt-Paul P.
      • Hoffmann R.
      • Kissling S.
      • Hummel S.
      • Vitacolonna M.
      • et al.
      Transfer of CD8(+) cells induces localized hair loss whereas CD4(+)/CD25(-) cells promote systemic alopecia areata and CD4(+)/CD25(+) cells blockade disease onset in the C3H/HeJ mouse model.
      ,
      • Wang E.H.C.
      • Khosravi-Maharlooei M.
      • Jalili R.B.
      • Yu R.
      • Ghahary A.
      • Shapiro J.
      • et al.
      Transfer of alopecia areata to C3H/HeJ mice using cultured lympho node-derived cells.
      ). Here, by using a simple procedure of isolating a mixture of total skin cells from AA-affected skin and dermally injecting them in healthy C3H mice, we showed that AA can be induced in around 80% of mice that received a mixture of cells isolated from AA-affected skin. For comparison, we have also used the previously reported protocol of AA induction in mice and found that 19 out of 42 (45.2%) mice that received dermally injected 10 million cultured activated T cells developed AA (Table 1). When using 10 times fewer cells (1 x 106 vs 1 x 107 cells) in our inducible AA model, 33 out of 39 mice (84.5%) that received isolated AA-affected skin cells developed AA (Table 1).
      In a series of experiments, we have evaluated the role of adherent myeloid cells (CD11b) in the AA induction and showed that cultured adherent cells isolated from AA-affected skin also induce AA with a similar success rate as that of non-adherent cells. This finding suggests that adherent cells have the capacity to activate naive T cells responsible for hair loss either directly at the site of injection or indirectly through migration to regional lymph nodes. This shows that dermally injected adherent cells causing patchy AA supports the idea that these cells might directly activate T cells at the site of injection. The result of FACS analysis revealed that the majority of adherent cells are positive for CD11b with a small number of T and B cells. Knowing that the association of SSEA-1 and SSEA-3 cells with monocytes in inflammatory diseases causes the release of inflammatory cytokines such as TNF-α and IL-1β (
      • Galassi A.
      • Turatello L.
      • De Salvia A.
      • Neri M.
      • Turillazzi E.
      • La Russa R.
      • et al.
      Septic cardiomyopathy: the value of lactoferrin and CD15 as specific markers to corroborate a definitive diagnosis.
      ,
      • Kaminagakura E.
      • Bonan P.R.
      • Jorge J.
      • Almeida O.P.
      • Scully C.
      Characterization of inflammatory cells in oral paracoccidioidomycosis.
      ,
      • Kurose R.
      • Sawai T.
      • Oishi K.
      • Liu X.
      • Sasaki A.
      • Kurose A.
      • et al.
      Possibility of inhibiting arthritis and joint destruction by SSEA-3 positive cells derived from synovial tissue in rheumatoid arthritis.
      ), we examined the presence of these cells in AA-affected skin. The result showed a high number of SSEA-1 and SSEA-3 cells in AA-affected skin (Figure 3), some of which were also positive for CD11b in the cultured adherent cells. However, it is not clear whether SSEA-positive adherent cells directly or indirectly activate T cells and monocytes in AA-affected skin or are co-expressed with CD11b without being involved in the development of AA. For this reason, more study is needed to investigate the potential role of SSEA-positive myeloid cells in the development of AA.
      In summary, here, we have established a reproducible and simple method to induce AA in C3H/HeJ mice and showed that adherent myeloid cells (CD11b, SSEAs) are as important as a mixture of non-adherent CD3+ T cells and CD19+ B cells in the induction of AA. Interestingly, alopecia universalis and patchy are induced in healthy C3H mice by dermally injected non-adherent and adherent cells isolated from AA-affected skin, respectively.

      Materials and Methods

      Animals

      Female C3H/HeJ mice aged nine weeks were purchased from The Jackson Laboratory (Bar Harbor, Marine). The University of British Columbia Animal Care Committee approved all experimental procedures. The methods were carried out in accordance with the approved guidelines. Mice were socially housed under a 12 hour light/dark cycle.

      Cell Isolation from AA-Affected Mouse Skin

      As a source of AA-affected skin, we first induced AA by using previously published protocol (
      • Wang E.H.C.
      • Khosravi-Maharlooei M.
      • Jalili R.B.
      • Yu R.
      • Ghahary A.
      • Shapiro J.
      • et al.
      Transfer of alopecia areata to C3H/HeJ mice using cultured lympho node-derived cells.
      ). Subsequently, AA was induced by using our established protocol. To isolate cells from mouse skin, about 80% of the skin from euthanized mice with AA universalis was collected, cut into small pieces, and washed 3 times in PBS with 3% antibiotic-antimycotic (Invitrogen Life Technologies, Burlington, ON, Canada). Minced skin was incubated in PBS with 1 mg per ml of collagenase V (Sigma-Aldrich, St. Louis, MO) at 37 °C for 90 minutes. After the neutralization of collagenase with equal volume of DMEM (Invitrogen Life Technologies) containing 10% fetal bovine serum (Invitrogen Life Technologies), the extract was filtered through a 70 μm cell strainer. Cells were pelleted by centrifugation (800 g, 10 minutes) and washed once with DMEM containing 10% fetal bovine serum. Skin cell mixture was either directly dermally injected into healthy C3H/HeJ mice or cultured in DMEM containing 2% fetal bovine serum.

      FACS Analysis

      For FACS analysis, the following antibodies were used: anti-mouse CD3e-PE (1:100, Invitrogen Life Technologies), anti-mouse CD19-APC (1:100, Invitrogen Life Technologies), and anti-mouse CD11b-PE (1:100, Invitrogen Life Technologies). Cells were stained with antibody for 1 hour at room temperature, washed 3 times, and analyzed by FACS (BD Acuuri C6, Mississauga, ON, Canada).

      AA Induction in C3H/HeJ Mice

      In this study, AA was induced by dermal injection of either 1 or 3 million cells obtained from a freshly isolated mixture of AA-affected skin cells. In another set of experiments, AA was induced by dermal injection of 3 million of either adherent or non-adherent cells.

      Fluorescent Staining

      Skin samples were fixed by 10% formalin and embedded in paraffin, and 5 μm sections were cut and used for immunofluorescent staining. Skin sections were deparaffinized, rehydrated, and treated with antigen retrieval buffer (10 mM sodium citrate, pH 6.0). The samples were then incubated with a blocking buffer (5% of bovine serum albumin in PBS) for 1 hour before incubation with the primary antibody overnight. For culture cells, after washing once with PBS, cells were fixed with 10% formalin for 30 minutes, washed by PBS, and blocked with blocking buffer. The following primary antibodies were used: SSEA-1 (MC480) (1:100, Cell Signalling Technologies, Whitby, ON, Canada), SSEA-3 (1:1000, R & D Biosystem, Minneapolis, MN), and CD11b (1:500, eBioscience, San Diego, CA).

      Data availability statement

      The data that support the findings of this study are available from the corresponding author, AG, on request.

      Conflict of Interest

      The authors state no conflict of interest.

      Acknowledgments

      This article is published as part of a supplement sponsored by the National Alopecia Areata Foundation .
      Funding for the Summit and publication of this supplement was provided by the National Alopecia Areata Foundation. This Summit was supported (in part) by the National Institute of Arthritis and Musculoskeletal and Skin Diseases under Award Number R13AR074890 . The opinions or views expressed in this professional supplement are those of the authors and do not necessarily reflect the official views, opinions or recommendations of the National Institutes of Health or the National Alopecia Areata Foundation. This study was supported by the Canadian Institutes of Health Research (Funding Reference Number: MOP-136945 ).

      Author Contributions

      Conceptualization: YL and AG; Data Curation: YL; Formal analysis: YL; Funding Acquisition: AG; Investigation: YL, RTK, GL, AG; Methodology: YL, RTK, GL; Project Administration: AG; Resources: AG; Supervision: AG; Validation: YL, AG; Writing-Original Draft Preparation: YL, AG; Writing-Review and Editing: RTK, AG.

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