Processing human skin samples for single-cell assays

Saluzzo, Simona; Gail, Laura-Marie; Neuwirth, Teresa; Pandey, Ram Vinay; Farlik, Matthias; Stary, Georg

doi:10.1016/j.xpro.2022.101470

Cited by 4 publications

(1 citation statement)

References 11 publications

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“…Single leucocyte suspensions were prepared from skin via enzymatic digestion and from whole blood using a Ficoll-Paque gradient, as previously described in detail. 18 For sc-RNA-seq using the 10X Genomics platform (10X Genomics, Pleasanton, CA, USA), CD45 + live cells were sorted into phosphate-buffered saline (PBS)/10% fetal calf serum for library preparation with the Chromium™ Single Cell 5′ Library & Gel Bead Kit v2 (10X Genomics) according to the manufacturer’s instructions. Briefly, sorted cells were partitioned into single-cell GEMs (Gel beads in EMulsion droplets) for cDNA synthesis and subsequently amplified and prepared for sequencing.…”

Section: Methodsmentioning

confidence: 99%

Diverse macrophage populations contribute to distinct manifestations of human cutaneous graft-versus-host disease

Strobl,

Gail,

Krecu

et al. 2023

British Journal of Dermatology

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Background Graft-versus-host disease (GvHD) is a major life-threatening complication of allogeneic haematopoietic stem cell transplantation (HSCT), limiting the broad application of HSCT for haematological malignancies. Cutaneous GvHD is described as a post-transplant inflammatory reaction by skin-infiltrating donor T cells and remaining recipient tissue-resident memory T cells. Despite the major influence of lymphocytes on GvHD pathogenesis, the complex role of mononuclear phagocytes (MNPs) in tissues affected by GvHD is increasingly appreciated. Objectives To characterize the identity, origin and functions of MNPs in patients with acute cutaneous GvHD. Methods Using single-cell RNA sequencing and multiplex tissue immunofluorescence, we identified an increased abundance of MNPs in skin and blood from 36 patients with acute cutaneous GvHD. In cases of sex-mismatched transplantation, we used expression of X-linked genes to detect rapid tissue adaptation of newly recruited donor MNPs resulting in similar transcriptional states of host- and donor-derived macrophages within GvHD skin lesions. Results We showed that cutaneous GvHD lesions harbour expanded CD163+ tissue-resident macrophage populations with anti-inflammatory and tissue-remodelling properties including interleukin-10 cytokine production. Cell–cell interaction analyses revealed putative signalling to strengthen regulatory T-cell responses. Notably, macrophage polarization in chronic cutaneous GvHD types was proinflammatory and drastically differed from acute GvHD, supporting the notion of distinct cellular players in different clinical GvHD subtypes. Conclusions Overall, our data reveal a surprisingly dynamic role of MNPs after HSCT. Specific and time-resolved targeting to repolarize this cell subset may present a promising therapeutic strategy in combatting GvHD skin inflammation.

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Section: Methodsmentioning

confidence: 99%

Diverse macrophage populations contribute to distinct manifestations of human cutaneous graft-versus-host disease

Strobl,

Gail,

Krecu

et al. 2023

British Journal of Dermatology

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The Human Skin Cell Atlas: Mapping the Organ at Unprecedented Depth

Farlik,

Weninger

2023

Journal of Investigative Dermatology

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Th17-associated cytokines IL-17 and IL-23 in inflamed skin of Darier disease patients as potential therapeutic targets

Ettinger,

Burner,

Sharma

et al. 2023

Nat Commun

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Darier disease (DD) is a rare, inherited multi-organ disorder associated with mutations in the ATP2A2 gene. DD patients often have skin involvement characterized by malodorous, inflamed skin and recurrent, severe infections. Therapeutic options are limited and inadequate for the long-term management of this chronic disease. The aim of this study was to characterize the cutaneous immune infiltrate in DD skin lesions in detail and to identify new therapeutic targets. Using gene and protein expression profiling assays including scRNA sequencing, we demonstrate enhanced expression of Th17-related genes and cytokines and increased numbers of Th17 cells in six DD patients. We provide evidence that targeting the IL-17/IL-23 axis in a case series of three DD patients with monoclonal antibodies is efficacious with significant clinical improvement. As DD is a chronic, relapsing disease, our findings might pave the way toward additional options for the long-term management of skin inflammation in patients with DD.

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Processing human skin samples for single-cell assays

Cited by 4 publications

References 11 publications

Diverse macrophage populations contribute to distinct manifestations of human cutaneous graft-versus-host disease

Diverse macrophage populations contribute to distinct manifestations of human cutaneous graft-versus-host disease

The Human Skin Cell Atlas: Mapping the Organ at Unprecedented Depth

Th17-associated cytokines IL-17 and IL-23 in inflamed skin of Darier disease patients as potential therapeutic targets

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