Noninvasive Imaging Techniques for Monitoring Cellular Response to Treatment in Stable Vitiligo

TO THE EDITOR

Vitiligo, an immune-mediated melanocyte destruction disorder (Harris, 2018), is often treated with immunomodulators and phototherapy (Bae et al, 2017). Surgical interventions can achieve 45%–47% success with >90% repigmentation (Ju et al, 2021) in stable vitiligo, but responses are unpredictable (Rajaram et al, 2017). Quantifying vitiligo treatment response to grafting is challenging because of the limited sensitivity of the Vitiligo Area Scoring Index (Gan et al, 2017; Komen et al, 2015), which can only capture macroscopic changes after 6–12 months (Gao et al, 2022). Reflectance confocal microscopy (RCM) and multiphoton microscopy (MPM) are noninvasive techniques for cellular-level skin imaging without labeling (Lentsch et al, 2022; Tkaczyk, 2017). RCM has been used to measure vitiligo treatment responses (Ardigo et al, 2007). MPM, which detects cellular metabolic changes via two-photon excitation fluorescence from reduced NADH (Pouli et al, 2016), has identified keratinocyte metabolic changes associated with vitiligo disease persistence (Shiu et al, 2022). Although MPM can visualize NADH contrast, existing clinical devices have a submillimeter scale field of view. Combining MPM with RCM can overcome this limitation (Lentsch et al, 2022). This approach has not been applied to vitiligo, in which this combination approach may be particularly useful for measuring keratinocyte metabolism changes and melanocyte migration simultaneously.

We evaluated vitiligo punch grafting effectiveness using MPM and RCM imaging on 12 stable vitiligo lesions in 11 patients (Supplementary Materials and Methods) over 5 visits (baseline, 1, 3, 6, and 10 weeks after treatment). Patients who received treatment had stable lesions for over a year, were unresponsive to prior treatments, and had a 3-month treatment washout. Patients provided written informed consent to participate in the study, which was approved by the University of California, Irvine Institutional Review Board. Vivascope1500 (CaliberID) and MPTflex (JenLab GmbH) were used for in vivo RCM and MPM imaging, respectively. We acquired images as described previously (Lentsch et al, 2022; Shiu et al, 2022) (see Supplementary Materials and Methods for details). RCM was performed at weeks 3 and 6 for 8 of the 12 lesions and at week 10 for 11 of the 12 lesions owing to device availability and participant dropout. Consistency was maintained by imaging the same graft at each visit to correlate optical biomarkers of melanocyte migration and keratinocyte metabolic changes with treatment responses.

RCM measured melanocyte density (number of melanocytes per unit area) and distance from the graft site. Five of the 12 lesions showed clinical repigmentation by week 10. Activated melanocytes with bipolar and/or stellate dendrites in patients showing clinical repigmentation were seen as early as 3 weeks in 2 patients and 6 weeks in most patients (Figure 1a and c), their appearance preceded clinical evidence of repigmentation, and their density increased over time (statistical significance was not determined secondary to the sample size). Nonresponders lacked activated melanocytes, consistent with prior findings identifying melanocyte activation’s repigmentation role (Abdallah et al, 2003), and visualized melanocyte count was significantly higher in responders (mean = 4.8, SD = 2.6) versus in nonresponders (mean = 0, SD = 0). Melanocyte distance from the graft increased in most responsive patients (Figure 1b), suggesting melanocyte migration. Patients in whom melanocytes were imaged did not exhibit later disappearance of melanocytes, suggesting that the lack of response was not due to melanocyte destruction.

Figure 1. In vivo RCM imaging following vitiligo treatment enables melanocyte quantification.

(a) Melanocyte density in responders and nonresponders at different visits. Each data point represents number of activated melanocytes visualized in a lesion per unit area per visit. *P-value < .05. (b) Average distance per melanocyte from punch graft in treatment responders. (c) Blue circle depicts clinical area that was imaged over time and the corresponding RCM image. Enlarged views of the area of interest are depicted in large red squares. Activated melanocytes are indicated by white arrows. Scale bars = 200 μm.

MPM can capture keratinocyte pigmentation and melanocyte migration changes after punch grafting in vitiligo (Figure 2a–e), but the limited field of view precludes accurate measurement of melanocyte density or migration. MPM images obtained from different epidermal depths within the same graft throughout the treatment were used to quantitatively assess metabolic changes using mitochondrial clustering analysis (Pouli et al, 2016) (Supplementary Materials and Methods). Baseline imaging of the normal skin before treatment exhibits depth-dependent changes in mitochondrial clustering (β), reflecting differences in metabolism. Basal and parabasal keratinocytes have higher b values compared with keratinocytes in higher epidermal layers as keratinocytes transition from glycolysis to oxidative phosphorylation during cell differentiation, progressing from a more fragmented mitochondria phenotype in the basal layer to a more fused phenotype in higher epidermal layers (Supplementary Figure S1) (Pouli et al, 2016). β values indicated altered keratinocyte metabolism in vitiligo lesions compared with the perilesional skin for all patients at baseline. Comparing the same metric at different treatment visits, basal keratinocytes in responsive vitiligo skin reverted to a more glycolytic profile within 6 weeks. In contrast, basal keratinocytes in nonresponsive lesions remained metabolically distinct from the adjacent normal skin (Figure 2f). β variability across the responsive vitiligo epidermis aligned with the nonlesional skin by 6 weeks, remaining consistent after 10 weeks (Figure 2g). Metabolic profile differences between keratinocytes in the lesional and nonlesional skin were insignificant in responders at 6 weeks but persisted in nonresponders beyond 10 weeks after the treatment. These findings suggest that keratinocyte metabolism shifts may serve as a potential indicator of treatment response failure. β variability values were compared using a linear mixed-effects model generated in SAS JMP Pro 14 (Supplementary Materials and Methods).

Figure 2. In vivo MPM imaging after vitiligo treatment captures repigmentation and metabolic changes.

MPM images showing nonpigmented keratinocytes in lesional acceptor (a) and melanin caps in nonlesional donor skin (b); (c) Clinical image of the imaged graft (blue arrow) and adjacent imaged location (red arrows). (d, e) MPM images showing activated melanocytes (arrows) in graft (c) and migrating melanocytes in a hair follicle in adjacent location. (f) Clinical images of treated areas and spline fits of β values showing basal keratinocytes reverting to a more glycolytic profile within 6 weeks in responders, while maintaining a distinct profile from normal skin in nonresponders. (g) β variability values of keratinocytes in treated vitiligo aligning with nonlesional skin for responders by 6 weeks. *P-value < .05.

In this study, we used RCM and MPM to monitor vitiligo treatment response at a cellular level, identifying changes in melanocyte morphology and keratinocyte metabolism that correlate with an early response. Melanocyte activation emerges as an early treatment response indicator, observed as early as 3 weeks after treatment initiation, preceding clinical repigmentation. Absence of melanocyte activation in nonresponders suggests that treatment failure is a consequence of lack of melanocyte activation rather than immune-mediated melanocyte destruction. However, it is possible that a small immune infiltrate went unnoticed, either because of limitations in detecting low-level T-cell responses or the study’s small sample size.

Although these results highlight the potential of noninvasive optical microscopy in predicting tissue-level responses that align with therapeutic outcomes, we acknowledge that the combination of MPM and RCM is time-consuming, which may limit its clinical applicability. Promising advances such as the fast, large area multiphoton exoscope (Fast et al, 2020), which can simultaneously gauge local tissue metabolic changes and melanocyte activation, holds potential for capturing and understanding early cytologic treatment responses in vitiligo, a crucial aspect in determining why some lesions respond to treatment while others do not. Future studies using fast, large area multiphoton exoscope on a larger group of patients with vitiligo will help validate our current findings and further establish the utility of in vivo nonlinear optical microscopy for monitoring local cytological responses to therapy.

Abbreviations:

MPM

multiphoton microscopy

RCM

reflectance confocal microscopy

DATA AVAILABILITY STATEMENT

No large datasets were generated from these data. The data that support the findings of this study are available from the corresponding author (JS) upon reasonable request.