Role of CXCL10 and IL-18 as markers of repigmentation response in non-segmental vitiligo treated with narrowband-UVB phototherapy: a prospective cohort study

  • Lía Hojman
    Affiliations
    Servicio de Dermatología, Departamento de Cirugía, Facultad de Medicina Clínica Alemana de Santiago – Universidad del Desarrollo, Santiago, Chile

    Laboratorio de Regulación e Inmunología del Cáncer, Programa Disciplinario de Inmunología, Facultad de Medicina, Universidad de Chile, Santiago, Chile
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  • Raúl Cabrera
    Correspondence
    Corresponding author: Raúl Cabrera
    Affiliations
    Servicio de Dermatología, Departamento de Cirugía, Facultad de Medicina Clínica Alemana de Santiago – Universidad del Desarrollo, Santiago, Chile
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  • Claudio Karsulovic
    Affiliations
    Laboratorio de Regulación e Inmunología del Cáncer, Programa Disciplinario de Inmunología, Facultad de Medicina, Universidad de Chile, Santiago, Chile

    Departamento de Medicina Interna, Clínica Alemana de Santiago
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  • Fabian Tempio
    Affiliations
    Laboratorio de Regulación e Inmunología del Cáncer, Programa Disciplinario de Inmunología, Facultad de Medicina, Universidad de Chile, Santiago, Chile

    Instituto Milenio de Inmunología e Inmunoterapia, Facultad de Medicina, U de Chile, Santiago, Chile
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  • Claudio Perez
    Affiliations
    Laboratorio de Terapia Celular, Hospital Clínico de la Universidad de Chile, Santiago, Chile
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  • Mercedes López
    Affiliations
    Laboratorio de Regulación e Inmunología del Cáncer, Programa Disciplinario de Inmunología, Facultad de Medicina, Universidad de Chile, Santiago, Chile

    Instituto Milenio de Inmunología e Inmunoterapia, Facultad de Medicina, U de Chile, Santiago, Chile
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Published:January 13, 2021DOI:https://doi.org/10.1016/j.jid.2020.12.021

      TO THE EDITOR

      Vitiligo is a depigmenting disease caused by loss of melanocytes due to autoimmune cytotoxic activity and, as it was recently described, basal melanocyte detachment(
      • Boukhedouni N.
      • Martins C.
      • Darrigade A.-S.
      • Drullion C.
      • Rambert J.
      • Barrault C.
      • et al.
      Type-1 cytokines regulate matrix metalloprotease-9 production and E-cadherin disruption to promote melanocyte loss in vitiligo.
      ).
      IFN-γ plays an important role in CXCL10 secretion and participates in TCD8+ cell recruitment to affected skin through its receptor, CXCR3(
      • Wang X.X.
      • Wang Q.Q.
      • Wu J.Q.
      • Jiang M.
      • Chen L.
      • Zhang C.F.
      • et al.
      Increased expression of CXCR3 and its ligands in patients with vitiligo and CXCL10 as a potential clinical marker for vitiligo.
      ). CXCL10 is significantly elevated in more severe vitiligo (
      • Abdallah M.
      • El-Mofty M.
      • Anbar T.
      • Rasheed H.
      • Esmat S.
      • Al-Tawdy A.
      • et al.
      CXCL-10 and Interleukin-6 are reliable serum markers for vitiligo activity: A multicenter cross-sectional study.
      ,
      • Zhang L.
      • Chen S.
      • Kang Y.
      • Wang X.
      • Yan F.
      • Jiang M.
      • et al.
      Association of Clinical Markers With Disease Progression in Patients With Vitiligo From China.
      ) and is important in the maintenance of the depigmented phenotype in a mouse model of vitiligo(
      • Rashighi M.
      • Agarwal P.
      • Richmond J.M.
      • Harris T.H.
      • Dresser K.
      • Su M.W.
      • et al.
      CXCL10 is critical for the progression and maintenance of depigmentation in a mouse model of vitiligo.
      ). Cytokine IL-18 promotes IFN-γ induction and TCD8+ cell activity and is proposed to act through NLRP3 inflammosome pathway activation in vitiligo(
      • Tang L.
      • Zhou F.
      Inflammasomes in Common Immune-Related Skin Diseases.
      ). We recently demonstrated that non-segmental vitiligo patients have different response velocities during narrowband-UVB phototherapy(
      • Cabrera R.
      • Hojman L.
      • Recule F.
      • Sepulveda R.
      • Delgado I.
      Predictive Model for Response Rate to Narrowband Ultraviolet B Phototherapy in Vitiligo: A Retrospective Cohort Study of 579 Patients.
      ), nevertheless we do not know how these inflammatory cytokines and cytotoxic cells behave during treatment.
      After approval by the institutional review board, we recruited and obtained written informed consent for 23 patients from the Phototherapy Unit of Clinica Alemana de Santiago, between April 2016 and June 2018(supplementary table 1), and followed them during 48 thrice-weekly sessions of phototherapy. Every patient was prescribed with phototherapy as they were classified as having progressive disease (defined as appearance of new achromic patches or enlargement of previous ones in the three months preceding enrollment); also, all patients were held-off of other treatments during a minimum of one month before enrollment. Six patients were classified as slow responders versus seventeen fast responders (supplementary table 2). The first had significantly more time from diagnosis (supplementary table 2). Most patients improved VASI score after 24 sessions of phototherapy, with a mean reduction of 42%. Interestingly, mainly fast responders contribute to VASI reduction (Figure 1a). On the other hand, patients with less than 2 years of vitiligo diagnosis, classified as recent onset vitiligo, had significantly higher VASI improvement than longstanding vitiligo patients (Figure 1b). Time from diagnosis showed a positive correlation with VASI at baseline and negative correlation with VASI improvement at 24 sessions (Figures 1c and 1d).
      Figure thumbnail gr1
      Figure 1Association between VASI improvement, time from diagnosis, response rate and frequency of CD8+ T Lymphocytes in vitiligo patients during phototherapy. (a) Normalized VASI score (obtained by two different dermatologists using Wood’s Light and standardized photographs) reduction during phototherapy at baseline, 24 and 48 thrice-weekly sessions checkpoints, in fast and slow responders. (b) Normalized VASI score reduction at same checkpoints in longstanding and recent onset vitiligo. (c) Correlation between VASI score at baseline and months since diagnosis on each patient (n=23). (d) Correlation between VASI score at 24 sessions of narrowband -UVB phototherapy and months since diagnosis (n=23). (e) PBMC extracted at three checkpoints were analysed using Live/dead, anti-CD3, anti-CD4, anti-CD8 markers. Gating strategy is shown. (f) TCD8+ frequency during phototherapy in all vitiligo patients. (g) Correlation between TCD8+ Lymphocytes frequency and VASI improvement at 24 sessions. (h) Correlation between TCD8+ Lymphocytes and serum levels of CXCL10. **** p=0.0001, *** p=0.0007, ** p=0.002, * p=0,034. Mann-Whitney test and Spearman rank correlation test were used.
      Blood samples were taken at 24 and 48 sessions checkpoints and flow cytometry to select live CD3+CD4-CD8+ cells was performed (Figure 1e). Circulating TCD8+ cells decrease during phototherapy reaching a mean of 45% at 48 sessions (Figure 1f). Also, TCD8+ cells correlate indirectly with VASI improvement at 24 sessions and directly with CXCL10 (Figure 1g and 1h), showing that, when the patient shows a better response to narrowband-UVB phototherapy, circulating cytotoxic T cells and serum CXCL10 diminish. Considering this and knowing that CXCL10 and IL-18 participate in the inflammatory lymphocyte response at the skin; we aim to measure CXCL10 and IL-18 serum levels at baseline, 24, and 48 sessions of phototherapy. IL-18 decreased significantly at 24 and 48 sessions a mean of 45% (Figure 2a). When analysing IL-18 in subgroups, only fast responders decreased IL-18 after 24 sessions and 48 sessions (Figure 2b). CXCL10 on the other hand, diminishes significantly at 24 and 48 sessions, paralleling the behavior of IL-18 (Figure 2c). Fast responders, as in IL-18 measurements, showed lower CXCL10 levels after treatment with a mean of 42% at 24 sessions (Figure 2d). When divided into recent onset and longstanding vitiligo, the first had higher CXCL10 levels at baseline and are the only that diminish after phototherapy (Figure 2e), correlating positive and significantly with IL-18 after 24 sessions (Figure 2f). Finally, to clarify an IL-18 possible role in INF-γ production, we analysed CD3+CD4-CD8+INF-γ+ cell count showing a significant decrease at 48 sessions (Figure 2g). Also, when plotted against IL-18 levels, INF-γ+ cells correlate positively and significantly (Figure 2h).
      Figure thumbnail gr2
      Figure 2CXCL10, IL-18 and TCD8+INF- γ+ Lymphocytes levels in vitiligo patients at baseline and during phototherapy. (a)Serum IL-18 levels in vitiligo patients during phototherapy obtained by ELISA at three checkpoints. (b) IL-18 levels during phototherapy in fast and slow responders. (c) Serum CXCL10 levels in vitiligo patients during phototherapy obtained by ELISA at three checkpoints. (d) CXCL10 levels during phototherapy in fast and slow responders’ groups. (e) CXCL10 levels during phototherapy in longstanding and recent onset vitiligo diagnosis groups. (f) Correlation between CXCL10 and IL-18 levels at 24 sessions checkpoint (n=21). (g) Circulating INF- γ+ TCD8+ lymphocytes count at three checkpoints. (h) Correlation between INF- γ+ TCD8+ lymphocytes and IL-18 levels at baseline (n=16). **** p=0.0001 / p=0.0003 *** p=0.0008 / p=0.0007, ** p=0.003/p=0.002. Mann-Whitney test and Spearman rank correlation test were used.
      These results support a role of CXCL10 and IL-18 in cytotoxic activity in vitiligo. It has been described that the activation of the IFN-γ, CXCL10, and CXCR3 axis is important for triggering and maintaining vitiligo in a mouse model(
      • Frisoli M.L.
      • Essien K.
      • Harris J.E.
      Vitiligo: Mechanisms of Pathogenesis and Treatment.
      ); And in humans, CXCL10 has been reported both as a severity marker(
      • Abdallah M.
      • El-Mofty M.
      • Anbar T.
      • Rasheed H.
      • Esmat S.
      • Al-Tawdy A.
      • et al.
      CXCL-10 and Interleukin-6 are reliable serum markers for vitiligo activity: A multicenter cross-sectional study.
      ) and a potential therapeutic target (
      • Rodrigues M.
      • Ezzedine K.
      • Hamzavi I.
      • Pandya A.G.
      • Harris J.E.
      New discoveries in the pathogenesis and classification of vitiligo.
      ). In contrast, IL-18 has not been studied for vitiligo until now.
      Cytotoxic activity against melanocytes by TCD8+ cells causes its elimination in vitiligo lesions(
      • Frisoli M.L.
      • Essien K.
      • Harris J.E.
      Vitiligo: Mechanisms of Pathogenesis and Treatment.
      ) and the role of many cytokines has been demonstrated in its pathogenesis (IL-2, IL-6, IL-8, GM-CSF, TNF-α, IFN-γ, IL-4, IL-17, IL-10, IL-13, TGF-β1)(
      • Tembhre M.K.
      • Sharma V.K.
      • Sharma A.
      • Chattopadhyay P.
      • Gupta S.
      T helper and regulatory T cell cytokine profile in active, stable and narrow band ultraviolet B treated generalized vitiligo.
      ,
      • Yu H.S.
      • Chang K.L.
      • Yu C.L.
      • Li H.F.
      • Wu M.T.
      • Wu C.S.
      Alterations in IL-6, IL-8, GM-CSF, TNF-alpha, and IFN-gamma release by peripheral mononuclear cells in patients with active vitiligo.
      ).
      We published previously different repigmentation rates in vitiligo treated with narrowband-UVB. We found individuals that responded very rapidly to phototherapy (very fast and fast responders), others responded very slowly (slow and very slow responders) and a third group responded in between these two subgroups (average responders). Although the majority of the fast responders had facial vitiligo, some of them also had vitiligo only in the trunk and extremities(
      • Cabrera R.
      • Hojman L.
      • Recule F.
      • Sepulveda R.
      • Delgado I.
      Predictive Model for Response Rate to Narrowband Ultraviolet B Phototherapy in Vitiligo: A Retrospective Cohort Study of 579 Patients.
      ). To apply this velocity repigmentation rate classification, we got results confirming that fast responders -although they had higher baseline levels of CXCL10 and IL-18- under narrowband-UVB treatment can lower blood levels of these cytokines at a higher rate than slow responders.
      In our cohort, despite all patients have progressive vitiligo, those with longstanding disease have a lower rate of response to narrowband-UVB phototherapy. They also had a lower baseline concentration of CXCL10 and IL-18. These findings suggest that recent onset vitiligo patients have a better response to phototherapy than longstanding vitiligo and that CXCL10 and IL-18 can probably be used as biologic markers to predict and monitor the clinical response to narrowband-UVB phototherapy in non-segmental vitiligo patients. Further studies are needed to confirm this hypothesis.

      Conflict of interests:

      The authors have declared that no conflict of interest exists
      Funding
      None
      Data availability statement
      All data generated or analyzed during this study are included in this published article and its supplementary information.

      Author Contributions

      Conceptualization: LH, RC, ML, CK; Data Curation: LH, CK; Formal Analysis: LH, RC, ML, CK; Funding Acquisition: LH; Investigation: LH, ML, FT, CK, CP, RC; Resources: LH, ML; Supervision: RC, ML; Writing - Original Draft Preparation: LH, RC, CK; Writing - Review and Editing: LH, RC, CK

      Acknowledgments

      None

      Supplementary data

      Methods
      Study Population
      Inclusion criteria for patients is described in the Table S1.
      Clinical and immunological assessment
      Disease diagnosis was assessed independently by 2 dermatologists (R.C., L.H) using daylight and Woods light (VISIA system 4th gen, Canfield Industries, NJ, USA). VASI score was measured at 0, 24, and 48 sessions with standardized photographs (Canon camera EOS t3i, Tokyo, Japan). CXCL10 and IL-18 levels were measured using ELISA (ELISA kit, anti-CXCL10 and anti-IL-18, BD Biosciences, California, USA). Flow cytometry (BD FACSverse flow cytometer, BD Biosciences, California, USA) was used for measurement of TCD8+ cells (Live/dead, anti-CD3, anti-CD4, anti-CD8, BD Biosciences, California, USA). Cytokine levels and TCD8+ cells were measured at the same checkpoints as VASI. VASI improvement was calculated as ([Baseline VASI - VASI]/Baseline VASI]x100).
      We classified patients as Slow or Fast responders using a modification of our Predictive Model of response for vitiligo patients(
      • Cabrera R.
      • Hojman L.
      • Recule F.
      • Sepulveda R.
      • Delgado I.
      Predictive Model for Response Rate to Narrowband Ultraviolet B Phototherapy in Vitiligo: A Retrospective Cohort Study of 579 Patients.
      ): Patients that showed 10% or more repigmentation at 24 sessions and/or 25% or more repigmentation at 48 sessions were considered Fast responders. For less repigmentation, they were considered Slow responders.
      We divided patients into recent onset (<24 months from diagnosis) or longstanding vitiligo (> 24 months).
      Treatment
      Patients received narrowband-UVB phototherapy thrice a week using the standardized protocol previously published(
      • Mohammad T.F.
      • Al-Jamal M.
      • Hamzavi I.H.
      • Harris J.E.
      • Leone G.
      • Cabrera R.
      • et al.
      The Vitiligo Working Group recommendations for narrowband ultraviolet B light phototherapy treatment of vitiligo.
      ). All patients received 24 narrowband-UVB sessions and 10 (43.5%) patients received 48 narrowband-UVB sessions to achieve a better repigmentation.
      Statistical analysis
      We used GraphPad software and Stata 13.1 for data analysis. Continuous variables were expressed as means (SDs) and discrete variables as percentage distributions. Fisher exact and χ2 tests were used to compare qualitative data and T test and analysis of variance test for quantitative data. Regression models were constructed according to the distribution and nature of variables.
      Tabled 1Supplementary table 1: Inclusion and Exclusion Criteria for the study
      InclusionExclusion
      >14 years of agePersonal history of malignant melanoma
      Clinical or histological diagnosis of non-segmental vitiligoUntreated or currently under treatment cancer
      Without current of in the past 30 days vitiligo treatmentHistory of primary or secondary immunodeficiency
      Signed written informed consentSubjects with history photosensitive pathologies like porphyria, lupus, untreated skin cancer
      ANA titers >1/80
      Patients unable to stay in standing position
      History of claustrophobia
      Tabled 1Supplementary Table 2: Baseline demographic characteristics of participants
      CharacteristicAll patientsRecent onset vitiligo*Longstanding vitiligo*Slow respondersFast respondersp valuep value
      (n=23)(n=9)(n=14)(n=6)(n=17)
      Age, mean (SD), y35,5 (10,6)34,1 (11,2)37,5 (11,5)36,7 (13)35,5 (9,9).52.40
      Female, No (%)10 (43%)2 (22%)8 (57%)5 (83%)5 (29,4%).07.64
      Time since diagnosis, mean (SD), mo104,8 (141)9,6 (10,2)178 (151,6)224,6 (173,3)64,7 (106,2).0002.0008
      * Recent onset vitiligo: < 24 months, longstanding vitiligo: > 24 months

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