Page 47 - Annual report 2021-22

Annual Report 2021-22 |

Archana Singh

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Archana Singh’s lab is interested in melanocyte dysfunction mechanisms across various skin diseases.

Cutaneous wound healing is known to result from a temporally coordinated communication among
keratinocytes, fibroblasts and immune cells. However, the role of melanocytes in wound healing
remains largely unknown. To investigate and compare the kinetics of wound healing in pigmented vs.
non-pigmented skin in guinea pigs at histological and molecular (transcriptional) levels, partial-
thickness wounds of 1x1 cm were created on pigmented and non-pigmented skin on the same guinea
pig. The wounds were examined at five-time points post-injury- day 5, day 9, day 12, day 14 and day
32 and compared at histological and molecular levels. The histological evaluation of skin during wound
healing revealed an accelerated transition from inflammatory to proliferative phase in pigmented skin
on day9 compared to a relatively delayed neo-epidermis formation in the non-pigmented skin, which
was corroborated by morphometric analysis of the wounds. Transcriptional profiling of the markers
of inflammatory, anti-inflammatory cytokines, macrophages, neutrophils and T cell lineages revealed
distinct transcriptional upregulation of macrophage marker F4/80, cytotoxic T cell markers- IFN-
gamma and IL-2, besides showing significant upregulation of transcription factors associated with Th1,
Th17 and Treg lineage during wound healing in pigmented skin.
The infiltration of neutrophils, one of the most dominant cell types found in the inflammatory phase,
was evaluated by analyzing expression of two markers- myeloperoxidase (MPO) and elastase (ELANE).
Both the markers showed significant upregulation on day 5, after which their expression declined
significantly when compared to the non-pigmented skin. The expression of macrophages (F4/80 and
CD86) was also significantly upregulated on day 5 in the pigmented skin, after which it declined
significantly. As several lineages of T cells are known to shape the wound healing response, the
expression of markers of Th1/Th2/Th17/Treg lineages by analyzing the expression of lineage-specific
transcription factors- TBX21, GATA3, ROR- γT and FOXP3 respectively was checked. Comparison of
pigmented vs. non-pigmented skin revealed a significant upregulation of Th1, Treg and Th17 lineage
markers in the inflammatory phase.

The higher melanin content in the re-epithelialized neoepidermis in the pigmented skin compared to
its basal level (day 1), as revealed in the histological studies, was intriguing and prompted
investigations into the role of melanocytes and/or melanin content during wound healing. Using
scratch wound assay (on keratinocytes) to mimic re-epithelialization in the presence of melanocyte-
derived conditioned media treated with either tyrosine or vehicle control (condition media from
melanocytes with basal level of pigmentation). Analysis of wound closure showed a significantly higher
keratinocyte migration in the presence of conditioned media derived from melanocytes. Moreover,
there was a significantly faster migration in tyrosine-treated melanocytes. Thus, our results suggested
that presence of melanocytes and the melanin content within them positively influenced keratinocyte
migration. This study implies an important non-canonical role of melanocytes in the wound healing
process in a guinea pig model.
Healing of large wounds remains a significant clinical challenge. Skin grafting, one of the most utilized
strategies, suffers from susceptibility to infection, poor proliferation, low transplantation efficiency,