IL‐17A is not a treatment target in progressive vitiligo
First published: 07 May 2019
EU Clinical Trials Register (EudraCT number: 2015‐003552‐48).
Funding information:
Novartis Pharmaceuticals provided funding for this study. The research activities of R. Speeckaert and N. van Geel are supported by the Scientific Research Foundation‐Flanders (Krediet aan Navorsers: 1504718N and FWO Senior Clinical Investigator: 1831512N, respectively).
Abstract
Multiple reports confirm elevated circulating IL‐17 levels and increased numbers of Th17 lymphocytes in patients with non‐segmental vitiligo. Additionally, melanocyte damaging characteristics have been ascribed to IL‐17. A single‐arm pilot study using secukinumab in active non‐segmental vitiligo was conducted. The large majority of patients developed additional skin depigmentations limiting further enrollment. Overall, laboratory analysis revealed no change in secreted chemokines or Th subsets. Th17 lymphocytes correlated with Th2, Th9, and Th22 cells while an inverse link with Th1 cells and serum sCD25 levels was observed. In contrast, Th17.1 cells correlated positively with Th1 lymphocytes. Confirmatory results were found in an independent group of patients with vitiligo showing a significant increase in Th17.1 and Th1 lymphocytes in progressive vitiligo patients compared to healthy controls, which was not found for Th17 cells. These results do not support a direct pathogenic role of IL‐17 or Th17 cells in vitiligo. Nonetheless, a delicate Th17/Th17.1/Th1 balance seems evident which changes markedly according to disease activity. This may offer new treatment options by interfering with cytokines that drive differentiation of Th17 cells toward Th1.
Significance
Several studies reported increased IL‐17 concentrations and elevated Th17 lymphocytes in patients with vitiligo, suggesting that IL‐17 might be an attractive treatment target. Nonetheless, anti‐interleukin 17A treatment resulted in no clinical or laboratory improvements in progressive vitiligo patients. Th17 lymphocytes were not increased in progressive vitiligo or linked to biomarkers of disease activity which was confirmed in an independent larger patient group. Th17.1 cells may explain the findings that pointed to an activated Th17 pathway in previous studies.
Important progress has been made to unravel the pathogenesis of vitiligo. Nonetheless, treatment options remain currently limited. Increasing evidence documents the important role of the CD8CXCR3‐CXCL9/10‐IFNγ axis in vitiligo (Rashighi & Harris, 2015). However, this pathway is crucial for skin immunosurveillance and blocking certain key factors may carry a potential risk of important long‐term side effects such as skin cancer (Blechman et al., 2017). A different approach which interferes with cell lineage polarization rather than cell function could be an interesting option.
Intriguingly, elevated values of IL‐17 and Th17 cells have been repeatedly documented in the blood and skin of patients with vitiligo by independent research groups (Singh et al., 2016). A link between IL‐17 levels and the extent of body surface involvement has been reported. Variable results were published regarding their association with disease activity (Abdallah et al., 2018; Aly et al., 2017; Basak, Adiloglu, Ceyhan, Tas, & Akkaya, 2009; Bhardwaj, Rani, Srivastava, Kumar, & Parsad, 2017; Elela, Hegazy, Fawzy, Rashed, & Rasheed, 2013).
Several in vitro experiments suggested a pathological role of IL‐17 in vitiligo. Melanocytes exposed to IL‐17 exhibited morphological shrinking and inhibition of melanogenesis. IL‐17 increases mitochondrial dysfunction and reactive oxygen species and induces autophagy (Zhou et al., 2018). After exposure to IL‐17, keratinocytes and fibroblasts release the pro‐inflammatory cytokines IL‐1β, IL‐6, and TNF‐α. Therefore, IL‐17 has been proposed as an initiating factor in the development of vitiligo (Kotobuki et al., 2012). IL‐17 may potentiate IL‐1β‐induced skin inflammation via NOD‐like receptor family, pyrin‐domain containing‐1 (NLRP1) and NLRP3 (Cho, Suh, Lee, Kang, & Woo, 2012; Zwicker et al., 2017). In a mouse model of vitiligo, an increased number of retinoic acid receptor‐related orphan receptor gamma (RORγT) T cells were found with clear increased expression of IL‐17 (Eby et al., 2014). Additionally, a clinical study found that successful treatment with NB‐UVB lead to a marked decrease in IL‐17 in vitiligo suggesting that therapeutic targeting of the IL‐17 axis could be an interesting option in vitiligo (Hegazy, Fawzy, Gawdat, Samir, & Rashed, 2014). However, a case of new onset vitiligo was reported in a patient receiving secukinumab (Méry‐Bossard et al., 2017).
In order to investigate the role of the Th17 pathway in vitiligo and the potential of IL‐17 inhibition, we conducted a pilot trial to determine whether secukinumab could be a promising therapy in active vitiligo. The primary objective of this study was to investigate the capacity of a targeted anti‐IL‐17A treatment with secukinumab to induce repigmentation in vitiligo patients with active disease. The secondary objective was to determine whether the disease could be stabilized and whether the quality of life and the impact of the disease on the daily life of patients with vitiligo could be improved by this treatment.
The goal of this study was to include 10 patients with active non‐segmental vitiligo with moderate to extensive vitiligo (body surface area ≥3%). Active vitiligo was defined as clear evidence of progression using digital pictures for follow‐up (maximum time between pictures = 7 months) and/or disease progression during the last 6 months (as reported by the patient) or at least one clinical activity sign (hypopigmented areas, vitiligo lesions with blurred borders, or confetti‐like depigmentations). The patients were required to exhibit vitiligo on the hands and/or face and have a Fitzpatrick 3–6 skin type and an impact score [= impact of vitiligo on daily life scored by patients on scale from 0 to 10] >8 or a Dermatology Life Quality Index (DLQI) >10. Patients with additional autoimmune diseases (except thyroid disease) were excluded. Other treatments (including topical therapy) were not allowed during the study period. This study was approved by the ethical committee of Ghent University Hospital. The trial was registered in the EU Clinical Trials Register (EudraCT number: 2015‐003552‐48).
Secukinumab 300 mg was administered at baseline, weeks 1, 2, 3, 4, and subsequently every 4 weeks. The total treatment time was 7 months and the total follow‐up time 9 months.
Photographic documentation was performed at each visit using UV photography. The area of involvement was calculated with the Vitiligo Extent Score (VES), and changes were assessed with the VESplus (van Geel et al., 2016). Blood was drawn at baseline, 2 weeks, and after 1, 2, 3, 4, 6, 8, and 9 months.
For biomarker monitoring, serum CXCL9 and CXCL10 were measured using ELISA (R&D Systems) while sCD25, sCD27, BAFF, and CCL20 (R&D Systems) were investigated using Luminex (R&D Systems). Flow cytometry was carried out for samples at baseline, 2 weeks, 1 month, and 2 months. Before freezing, peripheral blood mononuclear cells were suspended in 1 ml of freezing medium (50% FCS, 40% RPMI 1640, 10% DMSO) at a concentration of 1 × 107/ml and pipetted into 1 ml cryovials. The tubes were transferred into a prechilled (4°C) controlled‐rate freezing container and placed into a −80°C freezer. After 24 hr, all samples were transferred into a liquid nitrogen tank. For analysis, cryotubes were continuously moved in a 37°C water bath, and as soon as the samples were completely thawed, they were pipetted into a 15‐ml tube containing RPMI medium. The cells were washed two times at room temperature. Cell recovery and viability were determined by live/dead staining (Aqua, Thermo Fischer Scientific).
Given the interesting laboratory results of the pilot trial, an independent patient group was recruited to investigate T helper subsets with a focus on Th17.1 lymphocytes. A total of 77 patients with non‐segmental vitiligo and 30 healthy controls were enrolled in this study including 48 patients with active vitiligo during the last 6 months and 29 patients without disease activity in the last 6 months (as reported by the patient and verified by follow‐up pictures for patients in follow‐up). The affected body surface area (BSA) was measured using the VES. All patients signed written informed consent, and this study was approved by the local ethics committee.
Peripheral blood mononuclear cells were labeled with CD3‐Vio‐Blue, CD8‐APC‐Vio770, anti‐CCR10‐PE, CD183 (CXCR3)‐VioBright, CD194 (CCR4)‐PE‐Vio770, and CD196 (CCR6)‐APC (all from Miltenyi) and analyzed on an LSR II flow cytometer (BD Biosciences). The frequency of CD4+ CXCR3+ CCR4− CCR6− Th1 cells, CD4+ CCR4+ CCR6− Th2 cells, CD4+ CCR4− CCR6+ Th9 cells, CD4+ CCR4+ CCR6+ Th17, CD4+ CXCR3+ CCR4− CCR6+ Th17/1 cells, CD4+ CCR4+ CCR6+ CCR10+ Th22 cells, and CD8+ CXCR3+ cells was determined using FlowJo (Treestar). The gating strategy was performed as recommended by Myltenyi Biotec (“Flow cytometry of Th subsets,” February 2015). All percentages of Th subsets were calculated as percentage of CD4 cells.
For continuous variables, the Mann–Whitney U test was performed in case 2 independent groups were considered. If multiple groups were present, the Kruskal–Wallis test was conducted. To assess the relation between two continuous variables, Pearson’s correlation was done. In particular circumstances (= to assess a non‐linear relation), Spearman’s correlation was performed. In case of paired samples, Wilcoxon analysis was carried out. Statistical analysis was done using SPSS 25 (SPSS Inc.) and Medcalc 18.5 (Medcalc Software).
Eight patients were enrolled and treated with secukinumab. During the follow‐up period, 7/8 patients showed signs of further depigmentation while two patients showed limited repigmentation (Table 1, Figure S1). In two patients, both the patient and physician decided to stop the study (after 4 months [6/10 injections] and 6 months [9/10 injections], respectively) because of disease progression that was considered to be handled better with conventional treatment. As the primary endpoint (repigmentation) and secondary endpoint (stop of progression) were not reached, further recruitment was prematurely halted at eight patients (initial goal = 10 patients). Patient satisfaction of the treatment dropped during the follow‐up period (mean: 9 vs. 7.25), and in none of the patients, clear clinical efficacy was noted. No significant adverse events were observed.
| Patient number | Age | Disease duration (years) | Percentage of affected body surface area (%) | Ethnic origin | Associated autoimmune disease | Fitzpatrick skin type | Timing of progression (months) | Timing of repigmentation (months) | Total change in affected BSA (%) |
|---|---|---|---|---|---|---|---|---|---|
| 1 | 62 | 18 | 9.92 | Caucasian | No | III | 2–7 | 7–9 | +0.33a |
| 2 | 30 | 19 | 7.12 | Caucasian | No | III | 1–6 | / | +0.19 |
| 3 | 46 | 38 | 27.45 | Caucasian | No | III | 2–9 | / | +0.60 |
| 4 | 70 | 11 | 50.77 | Asian | No | IV | 3–6 | / | +1.30 |
| 5 | 59 | 16 | 9.83 | Indian | No | V | 4 | / | +0.21 |
| 6 | 42 | 12 | 14.36 | Indian | Thyroid | V | 1–3 | 2 | +0.34 |
| 7 | 46 | 11 | 12.36 | Caucasian | Thyroid | III | 3–9 | 1–3 | +2.00 |
| 8 | 47 | 8 | 47.56 | Caucasian | No | III | / | 6–8 | −0.08 |
- a Areas that progressed during treatment (0–7 months) repigmented again after stop of secukinumab (7–9 months).
In general, CXCL9, CXCL10, sCD25, sCD27, and BAFF showed no clear changes over time. CCL20, an IL‐17‐induced chemokine, demonstrated the most marked decrease although no significance was reached. Also, no obvious differences were noted in the percentage of CD3+ CD8+ lymphocytes, Th1, Th2, Th9, Th17, Th17.1, Th22 subsets, and the Th1/Th7 ratio. In contrast, CD3+ CD8+ CXCR3+ cells tended to increase toward month 3 (p = NS). Remarkably, the evolution of CD3+ CD8+ CXCR3+ cells at 4 and 8 weeks versus baseline showed a more negative trend in patients with high baseline Th17 levels (p = 0.015 and p = 0.021, respectively) suggesting that baseline Th17 levels influence the response to IL‐17 inhibition in vitiligo. One patient with high baseline Th17 lymphocytes and a decrease in CD8+ CXCR3+ cells after 4 and 8 weeks also displayed a significant decrease of both CXCL10 and CXCL9 during follow‐up (p = 0.010 and p < 0.001, respectively). This was however not in correspondence with the clinical evolution as a progression of 0.60% BSA was observed in this patient during the study period. Nonetheless, this laboratory finding suggests that the immunological response in patients with vitiligo might differ according to the status of the Th17 pathway.
Baseline Th17 cells were inversely correlated with sCD25 at baseline (ρ = −0.881, p = 0.004) which is surprising as sCD25 is a biomarker for active disease in vitiligo (Speeckaert, Lambert, & van Geel, 2016). Additionally, in a multiple regression model the percentage of Th17 cells was negatively linked to the percentage of Th1 cells, while positively linked with Th2, Th9, and Th22 lymphocytes. These findings do not support previous reports stating that the Th17 pathway is correlated with active disease. In contrast, Th17.1 lymphocytes were positively correlated with Th1 cells (p < 0.001). This result led us to investigate more closely the Th17.1 subset in a larger patient group which had not yet been extensively performed in vitiligo.
Additional analyses were performed to confirm the immunophenotyping results of the clinical trial pointing to a different role of Th17 cells compared Th17.1 lymphocytes. A total of 77 patients with non‐segmental vitiligo and 30 healthy controls were additionally recruited including 48 patients with active vitiligo and 29 patients with stable disease. Forty‐eight of seventy‐seven (62.3%) had active vitiligo in the last 6 months and the median affected BSA was 1.56% (IQR: 0.64–6.13). Forty‐two of seventy‐seven (54.5%) received topical treatment and 2/77 (2.6%) UVB treatment in the period before blood sampling. Marked differences in T helper subset percentages were noted in vitiligo patients with important changes depending on disease activity. In progressive vitiligo patients, Th1 cells were increased (p = 0.039) and Th2 cells decreased (p = NS). In patients with stable disease, significantly elevated Th9, Th17, and Th22 lymphocytes were observed (Figure 1a–f). A clearly decreased Th1/Th17 ratio was seen in stable vitiligo compared to the active counterpart (p = 0.016). This points to a Th1 predominance in the active process of vitiligo (Figure 1g–i).
Figure 1
T helper subsets according to disease activity (a–i). Th1 cells were significantly increased in active vitiligo patients (a), whereas Th9, Th17, and Th22 cells were only increased in stable vitiligo patients (c, e, d). Th17.1 cells were elevated both in active and stable vitiligo patients (f). The total lineage of Th1/Th17.1/Th17 was elevated in patients with vitiligo (g) although the Th1/Th17 ratio was increased in active compared to stable vitiligo (h). The Th17.1/Th17 ratio was increased in patients with vitiligo (i) indicating that Th17.1 cells may contribute in patients with vitiligo for a more important part to the production of IL‐17 compared to healthy controls
In contrast, Th17.1 cells were significantly increased in both active and stable vitiligo patients compared to healthy controls (p = 0.015 and p = 0.015, respectively). This result indicates that Th17.1 cells are the only subset of the Th17 pathway elevated in active disease. Th17.1 cells correlated with both Th1 (ρ = 0.284, p = 0.012) and Th17 lymphocytes (ρ = 0.437, p < 0.001). Interestingly, the link between Th17.1 and Th1 was only present in patients with relatively low Th1 cells (Figure 2f).
Figure 2
Correlation analysis between T helper subsets in patients with vitiligo. Th1 cells were inversely correlated with Th2, Th17, and Th9 lymphocytes (a–c). Th9 cells were inversely correlated with the Th1/Th17 ratio and the Th1/Th17.1 ratio (d, e), which corresponds to the immune environment associated with Th9 cells (including high levels of IL‐9 and TGF‐β) which blocks the differentiation of Th17 cells into Th17.1 and Th1 cells. In a relatively low Th1‐skewed milieu, Th1 and Th17.1 cells were positively correlated although this curve flattens in patients with very high numbers of Th1 cells suggesting that Th17.1 might differentiate toward Th1 cells in a strongly Th1‐dominated environment (f). In contrast, Th17.1 cells continue to increase in a Th17 dominant environment (g)
A multiple regression model showed independent contributions of Th2, Th9, and Th22 subsets to predict the amount of Th17 cells. These results are in line with the clinical observation of decreased disease activity in patients with more Th17 cells.
Overall, no relation between T helper subsets and the extent of vitiligo was observed. Nonetheless, in patients with active vitiligo both Th17 cells (r = 0.355, p = 0.016) and Th22 cells (r = 0.400, p = 0.006) showed a positive correlation with the percentage of affected BSA.
Cytotoxic lymphocytes tended to be higher in patients with active vitiligo, and CD3+ CD8+ CXCR3+ cells were increased in both stable and active vitiligo patients versus healthy controls (p < 0.001). In a multivariate regression model including Th1, Th2, Th9, Th17, and Th22 as independent variables, only Th1 cells were independently linked with the percentage of CD3+ CD8+ cells (p < 0.001).
In this study, a clinical trial using secukinumab in active non‐segmental vitiligo patients was conducted. Unfortunately, we observed progression in 7/8 patients without clear signs of repigmentation leading to premature stop of further enrollment. Similarly, immunophenotyping of these patients did not reveal an overall decrease of the best validated biomarkers in vitiligo such as CXCL9/10, sCD25/27, Th1 cells, or cytotoxic cells (Rashighi et al., 2014; Speeckaert et al., 2016; Wang et al., 2016). Moreover, Th17 lymphocytes were inversely correlated with markers of disease activity. These findings render an important contributing role of IL‐17 in the progression of vitiligo unlikely.
The results were confirmed in an independent larger patient group showing that the Th1/Th17.1/Th17 balance is shifted toward Th1 differentiation instead of Th17 in active non‐segmental vitiligo. Increased percentages of Th17.1 lymphocytes were found which likely contribute to the high circulating IL‐17 levels previously reported in progressive vitiligo. Th17.1 cells represent an intermediate phenotype with both Th1 and Th17 characteristics. Th17.1 cells produce both IL‐17 and IFN‐γ besides other cytokines such as GM‐CSF. An important plasticity of Th17 cells has been documented in other autoimmune diseases such as arthritis (Nistala et al., 2010). Th17 cells are able to differentiate into Th17.1 cells in particular immune environments (e.g., high IL‐12, IL‐23, and low TGF‐β concentrations). Subsequently, these Th17.1 cells carry the capacity to polarize into Th1 cells. Interestingly, the correlation between Th17.1 cells and Th1 lymphocytes was only present if the number of Th1 cells was low (Figure 2f), whereas highly elevated Th1 values did not induce a further increase in Th17.1 lymphocytes. This substantiates the theory that in a skewed Th1 environment, Th17.1 cells are converted into Th1 lymphocytes. Blocking differentiation of Th17 into Th1 cells might therefore represent a promising therapeutic option in vitiligo.
These data demonstrate that Th17 lymphocytes and IL‐17 do not play a direct major role in the immune‐mediated cytotoxic responses leading to melanocyte destruction in vitiligo. However, therapeutic interventions that target IL‐17 may not inhibit Th17/1 lymphocytes and pathogenic ex‐Th17 cells (Basdeo et al., 2017). This may have also important implications for other disorders as vitiligo can be considered as the best in vivo human model to study cytotoxic responses and immunosurveillance of the skin. In this regard, these experiments support the evidence that IL‐17 inhibition does not substantially hamper immune‐mediated cytotoxicity. Theoretically, this might lead to decreased long‐term side effects in disorders such as psoriasis, psoriasis arthritis, or hidradenitis suppurativa compared to treatments that affect the Th1 pathway.
Limitations of this study were the inclusion of patients with a rather limited body surface involvement. This may have implications on the observed results as Th17 cells were linked with disease extent. Regarding the pilot trial using IL‐17 inhibition, laboratory analyses (e.g., patient 3) showing decreased CD8+CXCR3+ percentages and lower CXCL10/9 levels indicate that in some patients, beneficial clinical results might be observed in larger studies. Another limitation is that the determination of T‐cell subsets based on chemokine receptor staining may not be a direct representation of their cytokine production. However, this approach was chosen as our main goal was to differentiate Th17 from Th17.1 lymphocytes.
In conclusion, these experiments point to the delicate Th1/Th17.1/Th17 balance in non‐segmental vitiligo. While IL‐17 inhibition did not demonstrate convincing results in active vitiligo, targeted modulation that interacts with the differentiation of Th17 toward Th17/1 and Th1 may be a new and promising approach.
ACKNOWLEDGEMENT
Novartis Pharmaceuticals provided funding for this study. They were involved in the design and protocol of the study and reviewed the final version of the manuscript before submission.
CONFLICT OF INTEREST
None declared.
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