Topical Antibiotic Treatment Significantly Reduced Depigmentation in Mouse Model

The use of topical antibiotics on the skin of mice significantly reduced depigmentation in a mouse model of vitiligo, according to a report published in Pigment Cell and Melanoma Research.¹

These results may be indicative of the efficacy of short-term topical antibiotic use in reducing depigmentation in vitiligo, according to the review's authors.

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Background and Methods

Previous research showed that extended oral neomycin use encouraged the colonization of Bacteroides bacteria in the gut, linked to a potential delay in vitiligo development in FH mice.² Yet, converting these findings into practical treatments for patients remains a challenge, according to researchers.

Researchers purchased a breeder pair of mice of both sexes, aged 17-30 weeks. The antibiotic treatment involved shaving the dorsal flanks of the mice and applying either white petrolatum (control), Bacitracin zinc, or Neosporin. Treatments were applied in a series of 4 weeks on, 2 weeks off, 2 weeks on, totaling 6 weeks. The ventral side was neither shaved nor treated to serve as an internal control for depigmentation.

To assess depigmentation, mice were scanned weekly to monitor ventral depigmentation away from the application site. Depigmentation was measured by flatbed scanning under anesthesia, and the percentage of depigmentation was calculated using Adobe Photoshop software.

Skin samples were obtained from both dorsal flanks and from untreated ventral skin for immunostaining. Antibodies were used to detect various cell markers, including CD3 for total T cells, FoxP3 for regulatory T cells, CD8a for cytotoxic T cells, and antibodies specific to PMEL. Mucosa-associated innate T cells were also stained for using specific antibodies. Imaging was done using a microscope, and cell quantification was performed using software.

For cytokine analysis, skin cryosections were homogenized, and the inflammatory cytokine content of 40 different proteins was analyzed using a cytokine array. Protein concentrations were determined using a BCA Protein Assay Kit. Bacterial DNA was isolated from fecal and skin samples, and 16S rRNA gene sequencing was performed to analyze the microbiome composition.

Findings

The results showed that both Bacitracin and Vaseline led to significant ventral depigmentation over the 14-week assessment period, with no significant difference between the two. However, Neosporin application halted ventral depigmentation significantly.

Furthermore, dorsal depigmentation did not significantly differ between antibiotic and Vaseline-treated sides, though a trend toward reduced depigmentation was observed with Neosporin treatment. Representative mouse scans illustrated the visible difference in depigmentation among groups, with Neosporin-treated mice exhibiting no significant depigmentation.

Immunostaining for the pre-melanosomal marker Pmel revealed that Neosporin-treated samples exhibited a significantly greater density of melanocyte-containing hair follicles compared to control samples, supporting the preservation of pigmentation. Quantitative evaluation showed a significant loss of melanocytes in Vaseline- or Bacitracin-treated skin compared to Neosporin-treated skin, indicating that depigmentation was accompanied by melanocyte loss.

Microbial communities from feces and skin strips were characterized using 16S rRNA gene amplicon sequencing. While alpha diversity did not significantly differ between treatment groups in fecal samples, beta diversity analysis revealed significant differences. Neosporin treatment led to distinct changes in fecal microbial communities compared to Bacitracin and Vaseline treatments.

Immunostaining for MR1 in dorsal skin tissue showed a significant difference in the number of MR1+ cells among groups, with Vaseline-treated mice exhibiting a significantly greater number of MR1-expressing cells compared to Neosporin and Bacitracin-treated mice.

Immunostaining on skin tissue samples revealed a reduction in total T cells and CD8 T-cell numbers in Neosporin-treated skin compared to control samples. FoxP3+ T cells increased significantly in Neosporin-treated skin.

Neosporin-treated skin showed reduced abundance of anti-inflammatory IL-1ra compared to control skin, as well as reduced expression of CCL2, C5/C5a, and TIMP.

"Utilizing a short treatment regimen of antibiotics, we observed a reduction in depigmentation in the pmel-1 mouse model of vitiligo, accompanied by changes in the microbiome and altered cytokine responses," wrote authors Touni et al. "Interestingly, Neosporin-treated mice markedly limited, even halted depigmentation."

Conclusions

These findings are strongly suggestive of the pivotal role of short-term applications of antibiotics in delaying vitiligo depigmentation, according to authors.

Specifically, neomycin may be considered a novel therapeutic strategy and warrants further research.

Potential limitations of the study, as noted by researchers, may include the observed alignment of microbial abundance with changes in the fecal microbiome rather than the skin microbiome. This, they wrote, may be influenced by the fact that mice are more likely to ingest antibiotics applied topically. Therefore, the outcomes of the study could be due to the effects of orally consumed, diluted antibiotics rather than a direct effect on the skin microbiome.

"Taken together, these findings contribute to a broader understanding of the immune-microbiome interactions in vitiligo and support the development of targeted interventions for this autoimmune skin disorder," according to Touni et al. "The modulation of Treg abundance in the skin, along with the alterations to the microbiome and cutaneous inflammatory responses, provide new avenues for therapeutic approaches aimed at suppressing autoimmunity and ultimately restoring skin pigmentation in vitiligo."

References

1. Touni AA, Sohn R, Cosgrove C, et al. Topical antibiotics limit depigmentation in a mouse model of vitiligo. Pigment Cell Melanoma Res. March 4, 2024. Accessed May 7, 2024. doi:10.1111/pcmr.13164
2. Dellacecca ER, Cosgrove C, Mukhatayev Z, et al. Antibiotics drive microbial imbalance and vitiligo development in mice. J Invest Dermatol. 2020. 140(3), 676–687.e6