Endogenous Retinoids in the Pathogenesis of Alopecia Areata · 2017. 2. 1. · Alopecia areata (AA) is an autoimmune, nonscarring, hair loss disease affecting up to 1.7% of humans

Endogenous Retinoids in the Pathogenesis ofAlopecia AreataF. Jason Duncan1,7, Kathleen A. Silva2,7, Charles J. Johnson1,7, Benjamin L. King2, Jin P. Szatkiewicz2,Sonya P. Kamdar2, David E. Ong3, Joseph L. Napoli4, Jinshan Wang4, Lloyd E. King Jr3, David A. Whiting5,Kevin J. McElwee6, John P. Sundberg2,3 and Helen B. Everts1

Alopecia areata (AA) is an autoimmune disease that attacks anagen hair follicles. Gene array in graft-induced C3H/HeJ mice revealed that genes involved in retinoic acid (RA) synthesis were increased, whereas RA degradation geneswere decreased in AA compared with sham controls. This was confirmed by immunohistochemistry in biopsiesfrom patients with AA and both mouse and rat AA models. RA levels were also increased in C3H/HeJ mice with AA.C3H/HeJ mice were fed a purified diet containing one of the four levels of dietary vitamin A or an unpurified diet2 weeks before grafting and disease progression followed. High vitamin A accelerated AA, whereas mice that werenot fed vitamin A had more severe disease by the end of the study. More hair follicles were in anagen in mice fedhigh vitamin A. Both the number and localization of granzyme B–positive cells were altered by vitamin A. IFNg wasalso the lowest and IL13 highest in mice fed high vitamin A. Other cytokines were reduced and chemokinesincreased as the disease progressed, but no additional effects of vitamin A were seen. Combined, these resultssuggest that vitamin A regulates both the hair cycle and immune response to alter the progression of AA.

Journal of Investigative Dermatology (2013) 133, 334–343; doi:10.1038/jid.2012.344; published online 27 September 2012

INTRODUCTIONAlopecia areata (AA) is an autoimmune, nonscarring, hair lossdisease affecting up to 1.7% of humans (Safavi et al., 1995).Current treatments are often ineffective in inducing prolongedremission (Tosti and Duque-Estrada, 2009; Harries et al.,2010). AA is characterized by a loss of hair follicle immuneprivilege, increased IFN gamma (IFNg) and other T helper 1(Th1) cytokines, and an increase in CD8þ T cells (McElweeet al., 1996; Gilhar et al., 2005; King et al., 2008). NK or NKT

cells and T regulatory (Tregs) cells may also be involved(McElwee et al., 2005; Ito et al., 2008; Petukhova et al., 2010).Studies suggest that AA is a complex polygenetic disease(Sundberg et al., 2004; Petukhova et al., 2010). Little is knownabout the environmental factors, such as diet, that impact thecourse of AA.

Several interactions between retinoids and immunity exist(Duriancik et al., 2010). Vitamin A deficiency impairs thedevelopment of cell-mediated immunity (Smith et al., 1987)and promotes Th1 responses while delaying Th2 development(Cantorna et al., 1994). Recently, additional T-cell subtypeswere appreciated as important in autoimmunity, includingTh17 and T Tregs cells, which are both regulated by vitamin Ato maintain gut immune tolerance (Stockinger et al., 2007;Sojka et al., 2008). Retinoic acid (RA) synthesis occurs indendritic cells (DCs) in the gut (Iwata et al., 2004), whichincreases FOXP3-positive Tregs (Coombes et al., 2007) andinhibits Th17 cell development (Mucida et al., 2007) via RAreceptor alpha (RARA) in vitro (Schambach et al., 2007).Inducing in vivo endogenous RA synthesis through activationof toll-like receptor 2 (Manicassamy et al., 2009) or PPARGagonist (Housley et al., 2009) inhibited Th17 cells andincreased FOXP3. Exogenous RA inhibited Th17 cells buthad no effect on FOXP3 (Xiao et al., 2008), suggesting that RAneeds to be induced in a precise location endogenously tomaintain gut immune tolerance. Similar to the gut, skin hasa major barrier function. DCs in the dermis have similarcharacteristics to those in the gut, including langerinexpression (Bursch et al., 2007). Mouse ear dermal DCs hadaldehyde dehydrogenase activity and induced FOXP3-positive

See related commentary on pg 285ORIGINAL ARTICLE

1Department of Nutrition, The Ohio State University, Columbus, Ohio, USA;2The Jackson Laboratory, Bar Harbor, Maine, USA; 3Vanderbilt UniversityMedical Center, Nashville, Tennessee, USA; 4University of California,Berkeley, Berkeley, California, USA; 5Baylor Hair Research and TreatmentCenter, Dallas, Texas, USA and 6University of British Columbia, Vancouver,British Columbia, Canada

Correspondence: Helen B. Everts, Department of Nutrition, The Ohio StateUniversity, Columbus, Ohio 43210, USA. E-mail: [email protected]

7These authors contributed equally to this work.

Received 7 February 2012; revised 11 July 2012; accepted 2 August 2012;published online 27 September 2012

Abbreviations: AA, alopecia areata; ALDH1A1, 2, 3, retinal dehydrogenase1,2, 3; CRABP2, cellular retinoic acid–binding protein II; CRABP2, cellularretinoic acid–binding protein II; DGAT1, diacylglycerol acyltransferase 1;DHRS9, dehydrogenase reductase SDR family member 9; GZMB, granzyme B;LRAT, lecithin:retinol acyltransferase; NKG2D, natural killer group 2D; RA,retinoic acid; Raldhs, retinal dehydrogenases; RARA, B, G, retinoic acidreceptor alpha, beta, gamma; RBP1 (formerly CRBP), cellular retinol–bindingprotein 1; Roldhs, retinol dehydrogenases; STRA6, stimulated by retinoic acid6; Th1, T helper 1; VAA, vitamin A adequate; VAD, vitamin A deficient; VAE,vitamin A excess; VAH, vitamin A high. Genes and RNA message expressionare italicized, whereas proteins are not. Proteins from mice, rats, and humansare all capital letters.

334 Journal of Investigative Dermatology (2013), Volume 133 & 2013 The Society for Investigative Dermatology

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Tregs in an RAR-dependent manner, although it was notconfirmed that these cells also expressed langerin or retinaldehydrogenase 2 (ALDH1A2, (Guilliams et al., 2010). Collec-tively, the results from these studies suggest that RA synthesiswithin the gut and skin DCs regulates immune tolerance.

Excess RA leads to alopecia (Ruzicka et al., 1992; Ries andHess, 1999; Shih et al., 2009), which may result from manyfactors including dysregulated immune function. Vitamin Adeficiency leads to follicular hyperkeratosis and rupture inhumans and rodents (Wolbach and Howe, 1925; Girard et al.,2006). In rodents, vitamin A deficiency also leads to a thin haircoat that is frequently seen but rarely reported (Anzano et al.,1979, unpublished observation; Everts and Berdanier, 2002).The results from these studies suggest that precise RA levelsare needed for optimal hair follicle function.

RA synthesis occurs locally in or near the cells where it willultimately be used. Precise spatial and temporal levels of RAin the skin are achieved by regulating several key steps incellular vitamin A metabolism: storage as retinyl esters, RAsynthesis, and RA degradation (Everts, 2012). In brief, vitaminA circulates as retinol bound to retinol-binding protein (RBP4).Retinol is transported into the cell via stimulation by RA 6(STRA6) and binds cellular retinol binding protein 1 (RBP1,aka CRBP). This bound retinol can either be esterified bylecithin:retinol acyltransferase (LRAT) for storage or reversiblyoxidized to retinal via retinol dehydrogenases, such asdehydrogenase reductase SDR family member 9 (DHRS9) orRDH10. Retinal is further oxidized to RA by retinaldehydrogenases 1–3 (ALDH1A1–3). RA is then sent to thenucleus with the assistance of cellular RA binding protein 2(CRABP2) to bind its RA receptors alpha, beta, and gamma(RARA, B, C) and activate the transcription of 500þ genes(Balmer and Blomhoff, 2002). When RBP1/CRBP is saturatedor absent, retinol can be esterified by acyl CoA: diacylglycerolacyltransferase 1 (DGAT1) or cleared by conversion to retinalby alcohol dehydrogenases 1–4. Retinal is then oxidized to RAvia ALDH1A1 and further metabolized by cytochrome P45026 family members (CYP26A1, B1, C1) with the assistance ofcellular RA binding protein 1 (CRABP1).

To better understand AA, transcriptome analysis of C3H/HeJmice with AA was performed. As retinoid metabolism was notpart of the network analysis software, the expression ofretinoid metabolism genes was examined with the hypothesisthat RA metabolism was not altered in AA. Transcripts codingfor proteins metabolizing retinoids were altered, which wasconfirmed in biopsies from AA patients and rodent AA models.High dietary vitamin A accelerated disease progression andnumbers of hair follicles in anagen. Lack of vitamin A resultedin a more severe disease. A few immune factors were alsoaltered by diet, suggesting that retinoids alter AA by regulatingboth the hair cycle and immune response.

RESULTSThe capacity for RA synthesis was increased in AAAnalysis of graft-induced AA transcripts revealed that theexpression of most genes involved in RA synthesiswas significantly increased (Figure 1a, Supplementary TablesS3–S6 online), whereas the expression of Rbp4 and RA

degradation genes was significantly decreased (Figure 1b) inAA compared with sham controls at 10, 15, and sometimes 20weeks after grafting. Only Rbp1 (Crbp1), Crabp2, and Aldh1a3transcripts were significantly increased and Stra6 significantlydecreased in mice with spontaneous AA compared with wild-type C3H/HeJ mice (Figure 1c).

Because differences in the hair cycle between the AA miceand controls were found (Supplementary Figure S2 online) andRA synthesis components changed during the hair cycle(Everts et al., 2007), these gene array results were confirmedusing immunohistochemistry (IHC) on human, DEBR rat, andC3H/HeJ mouse skin, with or without AA, using antibodiesagainst specific RA synthesis and degradation proteins tobetter control for hair cycle changes. RBP1/CRBP had thegreatest increase in immunoreactivity in biopsies of patientswith AA and both rodent models (Figure 2a–d andSupplementary Figure S3a and b online). RBP1/CRBP wasalso high in CBA/CaHN-Btkxnl/J, SWR/J, and A/J mice with AA,but not in C3H/HeOuJ mice with AA (SupplementaryFigure S4 online). DHRS9 and CRABP2 were also increasedin mice but only slightly increased in AA patients and DEBRrats (Figure 2e–h, q–t and Supplementary Figure S3c, d, g, andh online). ALDH1A1 and ALDH1A2 were increased inbiopsies from humans, but were not different in rodent models(Figure 2i–l and Supplementary Figure S6a–d online).ALDH1A3 was greatly increased in mice, absent from thepre-medulla in DEBR rats, and not different in biopsies fromhumans, although no pre-medulla was present in humansamples (Figure 2m–p and Supplementary Figure S3e,f online). Immunoreactivity of CYP26B1 was not differentbetween C3H/HeJ mice with AA or controls, but localizationchanged during the hair cycle (data not shown). DHRS9,ALDH1A1, and ALDH1A2 also localized to infiltratingimmune cells in biopsies from human patients with AA andC3H/HeJ mice (Figure 2f, h, j, l and Supplementary FigureS6a–d arrow online). In DEBR rats, immune cells expressedDHRS9 but not ALDH1A1 or ALDH1A2 (SupplementaryFigure S3d online, data not shown). ALDH1A2 colocalizedwith DC markers langerin and natural killer group 2D(NKG2D; Supplementary Figure S6e, f arrow online).

Retinoid levels were measured by liquid chromatography/mass spectometry (LC/MS/MS) and HPLC to confirm thisexpression pattern. RA levels were significantly greater(Po0.05), whereas retinol levels were lower (P¼ 0.061), inAA mice compared with controls (Supplementary Figure S7aand b online). There was no difference in retinyl ester levels(Supplementary Figure S7c online).

Dietary vitamin A altered the progression of AA

To determine whether dietary vitamin A altered AA, C3H/HeJ mice were fed one of the five diets, starting 2 weeksbefore receiving a graft from a mouse with AA, and theprogression of the disease was analyzed. Ventral hair losswas significantly increased at 13, 14, and 18 weeks postgrafting in mice fed high vitamin A (12 IU, VAH) comparedwith mice fed unpurified chow (control, Figure 3a). Therewas also a trend at 14 weeks with mice fed VAH diet havingmore hair loss than mice fed the vitamin A–deficient diet

FJ Duncan et al.Retinoid Metabolism in Alopecia Areata

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(VAD, P¼0.055). Fifteen weeks after grafting, mice fed VAHdiet had significantly more epidermal hyperplasia in ventralskin compared with mice fed VAD or adequate vitamin A (4IU, VAA), or control diets (Figure 3b). Mice fed excessvitamin A (28 IU, VAE) also had significantly more epider-mal hyperplasia in ventral skin than mice fed VAD diet. Thepercentage of hair follicles in mid-anagen through mid-catagen, when active disease is seen, was also greater inmice fed VAH and VAE diets compared with the control dietin both ventral and dorsal skin (Figure 3c). In the dorsal skin,these findings were also significantly greater than mice fedVAA diet. By contrast, at 20 weeks post grafting, mice fedVAD diet had significantly more lymphocytes and outer rootsheath hyperplasia in ventral skin compared with all otherdiets (Figure 3d and e). Follicular dystrophy was significantlygreater in mice fed VAD, VAA, and VAH diets comparedwith mice fed control diet (Figure 3f). Follicular dystrophywas also significantly greater in mice fed VAD comparedwith mice fed VAE diets. Similar results were seen in dorsal

skin, with mice fed VAD diet having significantly morefollicular dystrophy than mice fed VAH and VAE diets(Po0.05, data not shown). Mice fed VAA diet also hadmore follicular dystrophy than mice fed VAH diet (Po0.05,data not shown).

There were no differences in body weight between any ofthe groups (Supplementary Figure S8a online). Mice fed thecontrol diet ate more food than any of the other diets(Po0.001), whereas mice fed VAH diet ate significantly lessthan mice fed VAD, VAE, and control diets (Po0.05;Supplementary Figure S8b online). Both food intake and bodyweight increased with time as expected, yet neither wassignificantly different between the mice receiving the AAand sham grafts (Supplementary Figure S8a and b online).

Dietary vitamin A altered the number and localization ofgranzyme B (GZMB)–positive cells

To determine how dietary vitamin A altered activated immunecells in AA, IHC of GZMB was performed and positive cells

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Figure 1. Retinoid metabolism is altered in alopecia areata. Fold changes (FC) in (a) retinoic acid (RA) synthesis and (b) retinol degradation proteins determined

by microarray analysis between C3H/HeJ mice grafted with alopecia areata skin and sham controls 5, 10, 15, and 20 weeks after surgery, or (c) between

mice with spontaneous disease and unaffected mice. *Po0.05, qo0.05 all genes, **Po0.05, qo0.05 all genes except Lrat, Rara, and Cyp26b1, ***Po0.05,qo0.05 only Rbp4, Adh1, and Dgat1. (d) Diagram of retinoid metabolism with genes significantly altered highlighted. Red, FC4þ 3; pink, FC 0 to þ3;dark green, FCo� 2; light green, FC 0 to �2. AA, alopecia areata.


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counted in the bulb, superbular, isthmus, and infundibulum ofstaged hair follicles. GZMB was significantly greater in AAmice versus shams (Figure 4a, c, e and g). Further statisticalanalysis was performed with only AA mice with hair folliclesin mid-anagen through mid-catagen, when disease is seen.GZMB was greatest in mice fed VAE diet 15 weeks postgrafting in and around the isthmus (Figure 4d). GZMB peakedat 10 weeks in and around the isthmus for mice fed VAH diet,

yet was high at both 10 and 15 weeks for mice fed VAA andcontrol diets. In contrast, mice fed VAD diet had a differentlocalization pattern with significantly more positive cells inand around the bulb at 10 and 15 weeks than mice fed theother diets (Figure 4h). At 10 weeks, most of the positive cellswere in and around the bulb and superbulbar, but by week 15these mice had higher numbers of cells in and around theisthmus (Figure 4d, f and h).

Figure 2. Retinoic acid synthesis enzymes and binding proteins are increased in alopecia areata. Immunohistochemistry (IHC) was performed on (a, e, i, m, q)

C3H/HeJ mouse skin collected 10 weeks after grafting with sham controls, (b, f, j, n, r) alopecia areata, (c, g, k, o, s) biopsies from human patients with tinea

capitis controls, (d, h, l, p, t) or alopecia areata with antibodies against (a–d) cellular retinol–binding protein (CRBP/RBP)1, (e–h) dehydrogenase reductase

SDR family member 9 (DHRS9), (i–l) 2, 3, retinal dehydrogenase 1, 2, 3 (ALDH1A1), (m–p) ALDH1A3, (q–t) and cellular retinoic acid–binding protein II

(CRABP2). Bar¼ 50mm. Arrow, positive immune cells.




High vitamin A increased IL13 and reduced IFNcCytokines are differentially regulated in AA (Carroll et al.,2002; Deeths et al., 2006; Freyschmidt-Paul et al., 2006;McPhee et al., 2012). ELISAs were performed to determinewhether Th1, Th2, or Th17 cytokine protein levels weredifferentially regulated by diet in mice. There were a fewdiet differences. Mice fed VAH diet had significantly moreIL13 than control chow-fed mice, but there were nodifferences between the AA and sham controls (Figure 5a).Levels of IFNg varied at different times with different diets,with low IFNg seen in mice fed VAH diet at 15 weeks andmice fed VAE diet at 20 weeks (Figure 5b).

Cytokine production and PDL1 was reduced, whereas CXCL9and CCL5 was increased, in AA

Protein levels of IL4, IL10, IL22, and IFNg were elevated in AAmice compared with sham mice at 5 weeks, but this was onlysignificant for IL4 (Supplementary Figure S9a–c and f online).

All cytokines progressively decreased in the AA mice andincreased in the sham mice with time after grafting. By 20weeks post grafting, IL10, IFNg, IL17, IL21, and IL22 weresignificantly decreased in mice that received the AA graftcompared with sham controls (Supplementary Figure S9a–fonline). Two factors essential for tolerance, CTLA4 and PDL1(CD274), were also tested in the skin by ELISA and IHC,repectively. CTLA4 was significantly lower by 20 weeks, butnot significantly different between AA and sham mice(Supplementary Figure S9g online). In contrast, PDL1(CD274) was significantly lower in mice that received theAA graft versus sham graft 15 and 20 weeks post grafting(Supplementary Figure S9h online).

Quantitative real-time PCR analysis revealed that IFNgmRNA levels were significantly increased in AA mice 15weeks post grafting compared with sham controls, althoughno diet effect was seen (Supplementary Figure S10 online).

To better select additional immune factors to analyze, anELISArray (Qiagen, Frederick, MD) was performed on the basisof results from a gene microarray and quantitative real-timePCR arrays (McPhee et al., 2012, ELISArray data not shown).CCL5 (RANTES) and CXCL9 (MIG) were significantlyincreased in the skin of mice receiving AA grafts comparedwith sham controls at 10, 15, and 20 weeks post grafting(Supplementary Figure S11a and b online). Neither chemokinewas altered by dietary vitamin A. Data for the control-fed micewere previously published (McPhee et al., 2012).

DISCUSSIONThis report shows that the expression of retinoid synthesisenzymes and binding proteins is increased in human patientswith AA and in two rodent models. DCs and NKG2D-positivecells also contained RA synthesis enzymes. RA levels werealso increased in C3H/HeJ mice with AA. Feeding high levelsof dietary vitamin A combined with increased RA synthesisaccelerated the onset of AA. Yet, not feeding vitamin Aresulted in more severe disease by the end of the study. Thisduality of responses suggests that precise vitamin A levels areneeded. Analysis of immune factors showed that dietaryvitamin A altered GZMB, IL13, and IFNg, and providedadditional findings on AA pathogenesis. Regulation of the haircycle may have also contributed to the accelerated disease.

This report shows increased RA synthesis in AA. To ourknowledge, any studies of RA alterations in AA are previouslyunreported. RA levels were also increased in the skin-specific(Krt14-cre recombinase) Scd1tm2Ntam-null mice with cicatricialalopecia (Flowers et al., 2011). The expression of RA synthesiscomponents was also increased in patients and C57BL/6Jmice with cicatricial alopecia (Everts et al., in press). Thus,RA synthesis may be secondary to these diseases. Regardlessof how changes occurred, alteration in retinoid metabolismmay make patients with AA more sensitive to exogenousretinoids.

Vitamin A toxicity leads to alopecia (Ruzicka et al., 1992;Ries and Hess, 1999; Shih et al., 2009). In mice, excess retinoland all transRA (Dgat1tm2Far null mice) within the basalepidermis and outer root sheath (Krt14 cre) led toprogressive cyclical alopecia with accelerated telogen to

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Mice were fed purified diets containing 0, 4, 12, or 28 IU dietary vitamin A/g

diet, or a control chow diet, starting 2 weeks before grafting and were killed (b, c)

5, 10, 15, or (d, e, f) 20 weeks post grafting. Ventral hair loss (a) was measured

weekly, with 300 representing a full coat of hair. Data are shown as mean±SEM.

Hematoxylin and eosin (H&E) slides were scored by JP Sundberg on a scale of

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(c) anagen, (d) lymphocytes, (e) outer root sheath hyperplasia, and (f) follicular

dystrophy. n¼ 8–11. *Significantly different from chow-fed mice, Po0.05,different letters are significantly different, Po0.05. ORS, outer root sheath.



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Figure 4. Granzyme B (GZMB) is significantly increased in mice with alopecia areata (AA) and altered by vitamin A. Immunohistochemistry (IHC) was

performed and GZMB–positive cells enumerated in (a, b) the infundibulum, (c, d) isthmus, (e, f) suprabulbar, and (g, h) bulb. (a, c, e, g) Mann–Whitney tests were

conducted to compare mice with AA lesions with mice without lesions (n¼40–50). (b, d, f, h) Generalized linear model with the Poisson response function wasthen performed on mice that received the AA graft with hair follicles in mid-anagen through mid-catagen (n¼ 1–10). Data are shown as mean±SE. *Po0.05versus no lesion; different letters within a time point are significantly different, Po0.05; t5, t10, t15 significantly different from 5, 10, and 15 weeks, respectively,Po0.05.

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Figure 5. Alterations in protein levels of cytokines by high vitamin A during alopecia areata (AA) progression in C3H/HeJ mice. (a) Protein levels of cytokines

IL13 and (b) IFNg were determined by ELISA from dorsal lumbar skin. Data were natural log-(ln) transformed and analysis of variance performed using SPSS, v19.Data are shown as mean±SE. n¼ 63–65 for a, and n¼ 16–19 for b. Different letters are significantly different Po0.05.




anagen transition (Shih et al., 2009). This increased anageninduction and alopecia could be reduced in Dgat1tm2Far-nullmice by severely reducing dietary vitamin A intake. In thisstudy, a moderate 3-fold increase in dietary vitamin Aaccelerated AA onset, possibly by inducing anagen, thetarget of AA. This dietary vitamin A level is well within theamount consumed by Americans who take supplements (Parket al., 2008). Thus, one mechanism for the increased AA withhigh vitamin A may be to regulate the hair cycle.

Vitamin A may also directly regulate the immune response,as RA regulates numerous immune cells (Duriancik et al.,2010). Vitamin A increases Th2 and reduces Th1 cytokines(Hoag et al., 2002; Iwata et al., 2003). The current study foundthat high vitamin A reduced protein levels of the Th1 cytokineIFNg and increased Th2 cytokine IL13 over the control diet. Itis noteworthy that there are many differences between thesediets besides the vitamin A content. The reduction in IFNgmay be important, as it was shown to have a key role in theetiology of AA (Freyschmidt-Paul et al., 2005; Gilhar et al.,2005; Freyschmidt-Paul et al., 2006; Nakamura et al., 2008).Several studies reported increased skin IFNg mRNA levels,similar to the current finding (Carroll et al., 2002; McPheeet al., 2012). IFNg is regulated posttranscriptionally (Khabarand Young, 2007), yet few studies have measured IFNgprotein levels in AA. Serum IFNg protein levels wereincreased in patients with AA (Barahmani et al., 2009; Arcaet al., 2004). Within the skin of patients with long-standingAA, IFNg levels were reduced (Deeths et al., 2006). This isconsistent with the drop in skin IFNg found in the currentstudy. Collectively, the results from these studies suggest thatalthough IFNg is essential for AA onset skin IFNg levels maydrop as AA progresses. High vitamin A may accelerate AAtoward a chronic stage and associated reduced IFNg. Futurestudies should analyze IFNg protein levels in the serum,spleen, and/or lymph nodes.

Vitamin A has a large role in maintaining gut immuneprivilege (Duriancik et al., 2010). RA synthesized in DCsincreased FOXP3þ Tregs and reduced Th17 cells to maintainmucosal immune tolerance. Increased Th17 cytokine levelsare hallmarks of autoimmune disease (Pelidou et al., 2000;Nakae et al., 2003; Langrish et al., 2005). The current reportshows that ALDH1A2 localized to dermal DCs, but Th17 cellsprobably do not contribute to AA in C3H/HeJ mice as cytokinelevels were reduced, not elevated. Th17 cytokines levels mayhave been too reduced for vitamin A to have any furthereffect. IL10, produced by numerous cell types (reviewed inO’Garra et al., 2008), is a key immunosuppressive cytokine inTreg differentiation (Asseman and Powrie, 1998; Assemanet al., 1999). IL10 can also activate CD8þ T cells (Grouxet al., 1998), and IL10 therapy produced variable clinicaloutcomes (O’Garra et al., 2008). AA frequency is reduced inIL10-deficient mice (Freyschmidt-Paul et al., 2002), suggestingthat IL10 may activate CD8þ T cells and exacerbate thedisease. Vitamin A may regulate IL10, but results areconflicting (Cantorna et al., 1994; Stephensen et al., 2004;Maynard et al., 2009). In the current report, IL10 was highestat 10 weeks, and then became significantly reduced as AAprogressed, but vitamin A had no effects. As IL10 has many

roles, it may be immunosuppressive in sham mice butproinflammatory in AA mice. The reduction in IL10 at 20weeks in AA may point to a loss in function or a reduction inthe number of Tregs in AA. Other studies (Zoller et al., 2002;McElwee et al., 2005; Petukhova et al., 2010) provide indirectevidence that Tregs are important in AA, as IL2 and itsreceptor IL2A (CD25) were reduced in AA. IL2 is essentialfor the maintenance and survival of Treg cells (Fontenot et al.,2005). Overall, the results of this study suggest that althoughTh17 cells may not be involved in AA Tregs possibly are.There was no effect of dietary vitamin A on the immunefactors tested, but RA synthesis enzymes are localized to DCs.Future studies on AA should examine factors such as FOXP3and Treg function to further analyze the role(s) of vitamin A inTreg responses in AA.

Both CTLA4 and PD1 (CD279) and its ligand PDL1 (CD274)are essential for suppressing T-cell responses directly andindirectly via Treg activation and preventing autoimmunediseases (Fife and Bluestone, 2008). PDL1 is expressed inmany immune privileged sites. To our knowledge, a reductionin PDL1 in AA that affected mouse hair follicles is previouslyunreported. PDL1 reduction later in disease is consistent withcompromised immune tolerance (privilege) in AA. In contrast,although CTLA4 decreased with time post grafting in AAaffected mouse skin, there were no significant differencesbetween AA and sham mice. These results conflict with geneassociation studies in humans (Petukhova et al., 2010; Johnet al., 2011) and functional studies in C3H/HeJ mice (Carrollet al., 2002; McElwee et al., 2002; Sundberg et al., 2011)linking CTLA4 to the pathogenesis of AA. One potentialreason for this difference may be the site tested. Skin wasexamined in this study, but CTLA4 alterations may occur inthe spleen and/or lymph nodes and should be examined infuture studies in these sites. The PD1 pathway, a secondcostimulatory pathway, should be examined for its role(s) inthe pathogenesis of AA.

Increased GZMB expression occurs in numerous autoim-mune and skin diseases (Boivin et al., 2009; Afonina et al.,2010). GZMB was localized to the dermis, subcutis, and hairfollicle connective tissue sheath in some but not all studies onAA patients (Sato-Kawamura et al., 2003). Small numbers ofGZMB-positive cells were detected in the dermis of C3H/HeJmice and were increased by tachykinin 1 (substance P,(Siebenhaar et al., 2007). None of these studies reported theGZMB-positive cell localization within the hair follicle. In thecurrent study, GZMB-positive cells were detected around andwithin the hair follicle, as well as in the isthmus—the site ofhair follicle stem cells (Cotsarelis et al., 1990) and immuneprivilege collapse in AA (Meyer et al., 2008). Most studiesonly mention GZMB’s role in inducing apoptosis, and someargue against GZMB having a major role in killing follicularcells in AA. More recent studies reveal additional roles forGZMB in cleaving extracellular matrix proteins, autoantigens,and receptors NOTCH1 and FGFR1 (Boivin et al., 2009).These effects could negatively affect the hair follicle in AA byaltering both the stucture of the connective tissue sheath andsignaling within the hair follicle stem cells and dermal papilla.Breakdown of the extracellular matrix can cause cell death



because of loss of cell–matrix interactions, allow moreimmune cells access to follicles, and contribute to collapseof hair follicle immune privilege. Thus, altered immunoloca-lization of GZMB by vitamin A may cause different types ofcellular damage at different follicular sites.

The chemokines tested in this study increased consistentlywith recruitment of immune cells (Hayashi, 1982; Shimokawaet al., 1983; Rathanaswami et al., 1993; Flier et al., 2001). Theincrease in CCL5 and CXCL9 in C3H/HeJ mice is similar toreported increases in gene and protein expression (Kuwanoet al., 2007; Subramanya et al., 2010) in patients with AA. Thecolocalization of RA synthesis enzymes with NKG2D cellssuggests that AA may be altered by RA effects on these cells.RA upregulates several NKG2D ligands (Cerwenka et al.,2000; Jinushi et al., 2003; Poggi et al., 2009), which activateNK, NKT, and CD8þ T cells (Mistry and O’Callaghan, 2007),but whether and how vitamin A regulates NKG2D and itsligands is unknown.

Initial attempts to treat AA with retinoids gave promisingresults. Topical tretinoin (0.05%) alone (Much, 1976), orcombined with intralesional triamcinolone acetonide(Kubeyinje and Cmathur, 1997), was effective in somepatients. The RXR agonist, bexarotene, also induced hairregrowth in some patients (Talpur et al., 2009). None ofthese studies had more than 42 patients, nor were theycontrolled for spontaneous hair regrowth. In the currentstudy, high vitamin A accelerated the disease, arguingagainst pharmacologic retinoids as an effective treatment forAA. It is also possible that the patients who responded toretinoid treatment were marginally vitamin A deficient andthat treatment corrected this deficiency. AA waxes and wanes;therefore, the stage of disease at the time of treatment mayalso alter retinoid effectiveness. In addition, studies in the gutsuggest that exogenous retinoids do not alter Tregs (Xiao et al.,2008), whereas upregulating RA within DCs did (Housleyet al., 2009; Manicassamy et al., 2009), suggesting thatregulating DC RA production may be an effective treatment.This study also highlights the need for precise regulation of RAin AA patients with adequate dietary vitamin A, as excessvitamin A from physicians and commercial sources such asskin- and hair-care products (Arechalde and Saurat, 2000) anddietary supplements (Park et al., 2008) may be detrimental.

MATERIALS AND METHODSAll procedures were carried out with approval from The Jackson

Laboratory IACUC for the microarray study, The Ohio State Uni-

versity IACUC for the diet study, and both IACUC boards for the mice

used for retinoid analysis. The microarray study was previously

reported (McPhee et al., 2012). Tissue arrays were created from

archived tissues (Sundberg et al., 1994, 1995; McElwee et al., 1998b,

1999). Biopsies from human patients (n¼ 2) were obtained fromarchives at Vanderbilt University (Nashville, TN) and Baylor Hair

Research and Treatment Center (Dallas, TX, Supplementary Table S1

online). The Vanderbilt University Institutional Review Board

approved all human work. Rat paraffin blocks of skin with or without

AA (n¼ 3) were provided by Kevin McElwee at the University ofBritish Columbia. IHC and immunoflurescence were performed as

previously described (Everts et al., 2007; Duncan et al., 2009).

Primary antibodies against Langerin (eBiosciences, San Diego, CA),

NKG2D, GZMB, and PDL1 (CD274; R&D, Minneapolis, MN) were

purchased, whereas the remaning antibodies were prepared and

validated in Dr Ong’s laboratory (Everts et al., 2007) (Supplementary

Table S2 online).

In the second study, recipient mice (n¼ 10) were fed purifiedAIN93M diets containing either 0 (VAD), 4 (VAA), 12 (VAH), or 28

(VAE) IU retinyl acetate per g diet (Research Diets, New Brunswick,

NJ), or control unpurified diet (chow, Harlen Teklad 7912, Indiana-

polis, IN), starting 2 weeks before grafting according to McElwee et al.

(1998a). Body weight, food intake, and hair loss were measured (Tang

et al., 2004) weekly and samples collected 5, 10, 15, and 20 weeks

post grafting. Histology was scored by JPS (Supplementary Figure S1

online). ELISA kits were purchased from R&D Systems, whereas the

ELISA array was purchased from SABiosciences (Frederick, MD), and

analyses were performed according to the kit instructions. RNA was

isolated using the RNAeasy Fibrous tissue kit (Qiagen, Valencia, CA),

cDNA prepared using the High Capacity cDNA Archive Kit (Applied

Biosystems, Carlsbad, CA), and quantitative real-time PCR performed

using a Taqman kit (Applied Biosystems).

Additional AA and sham mice (n¼ 6) were obtained (JPS) andretinoids analyzed as previously described (Kane et al., 2008a, b).

Data were analyzed using SPSS, v19 (IBM; Armonk, NY) after

consultation with the OSU statistical consulting services. Histological

scores were analyzed using a generalized linear model with the

multinomial logistic response function at each time point. See the

figure legends for additional analyses.

See Supplementary Data online for more details.

CONFLICT OF INTERESTThe authors state no conflict of interest.

ACKNOWLEDGMENTSThis work was supported by grants from the National Alopecia AreataFoundation (to HBE, LEK, and JPS) and the National Institutes of Health(AR052009 to HBE, AR041943 to LEK, and AR056635 to JPS). We thank JeniSquiric and Steven Naber from the OSU statistical department for their guidance.

SUPPLEMENTARY MATERIAL

Supplementary material is linked to the online version of the paper at http://www.nature.com/jid

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Endogenous Retinoids in the Pathogenesis of Alopecia AreataIntroductionResultsDiscussionMaterials and Methods

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Endogenous Retinoids in the Pathogenesis of Alopecia Areata · 2017. 2. 1. · Alopecia areata (AA) is an autoimmune, nonscarring, hair loss disease affecting up to 1.7% of humans