Vitiligo is an autoimmune skin disease characterized by focal depigmentation due to melanocyte loss attributed to autoreactive T cells. Recent single cell studies highlight activation of IFNg pathways in both lesional and non-lesional skin and stressed keratinocytes with metabolic switch towards oxidative phosphorylation in lesional skin. In this integrative study we combine existing single cell datasets and put it into spatial context with 10x Visium spatial sequencing of 25 samples from non-, peri- and lesional vitiligo and healthy skin. We utilized bulk RNAseq to assess tissue responses to pan T cell stimulation and measure quantitatively deconvolution-derived cell types changes in health and vitiligo. Pigmented peri-lesional vitiligo was found to be a site of active IFNγ inflammation and oxidative phosphorylation, with IFNγ responses highly associated with Treg cell signal. In contrast, depigmented lesions displayed dormant IFNγ inflammation, that was revealed upon T cell stimulation. Besides IFNg responses, vitiligo epidermis reacted to T cell stimulation with local T cell proliferation. Lesional melanocytes had increased expression of the damage marker S100B and selenoamino acid metabolism, both previously proposed biomarkers of disease activity. Moreover, S100B protein was specifically upregulated in T cells-melanocytes colocalizations. The combined modality of the study revealed novel insights into spatially restricted disruptions of cellular environments in vitiligo.

Sex is an important biological variable in human biology with consequences for manifestations of various inflammatory diseases. Sex-associated difference is found in circulating T cell immunity as females are more prone to have autoimmune disease, but it is not clear whether tissue residing T cells are specific to sex. To investigate the effects of sex on the pool of resident T cells in human skin, the sex difference on skin T cell profiles were assessed. The effects of testosterone/estradiol on human CD8 T cell differentiation were also addressed. Both bulk and single RNA sequencing data from human skin from public repositories exhibited that female and male have distinct transcriptional signatures. Protein analyses revealed that female skin harbors around twice more cytotoxic and IFNγ producing CD8 resident memory T cells (Trm) in epidermis compared to that in males or testosterone-treated females. Furthermore, in vitro analyses displayed that testosterone hampers development of cytotoxic CD8 Trm from circulating T cells. These differences were not prominent in dermal skin compartment. Collectively, these results lead to regard sex as one of the variables to consider in the composition of epidermal skin T cells.

Cutaneous T cell lymphoma (CTCL) is a rare type of lymphoma primarily affecting the skin. The local immune response to CTCL include cytotoxic CD8+ T cells, macrophages, B cells, and as identified recently by our group, natural killer (NK) cells. In early stages of the disease the lymphoma cells are mainly localized in epidermis and the upper parts of dermis, but in later stages deeper parts of dermis are also engaged. We hypothesize that progression to advanced disease in part is the result of failed local immune responses, including perturbed spatial distribution of anti-lymphoma immune cells. Our aim is to investigate the distribution and properties of infiltrating immune cells in skin from CTCL patients with different clinical manifestations of the disease. Immunofluorescence and digital spatial profiling have been performed to compare cellular localization and protein expression in different skin substructures and tumor regions. Our preliminary results indicate that CD8+ T cells and NK cells localize mainly to the epidermis and papillary dermis in patch and plaque lesions, and in the reticular dermis in tumors. We also find more CD8+ T cells/mm2 in the perivascular infiltrates of tumors, compared to patches and plaque, possibly indicating a higher influx of CD8+T cells. With spatial profiling, we detect a tendency to an increased heterogeneity of protein expression in more advanced manifestations and several differentially expressed proteins in CD4+ lymphoma cells and infiltrating CD8+ T cells in patch, plaque and tumor, both in perivascular and dermal infiltrates. No significant differences were detected in the protein expression of lymphoma regions with high vs low density of infiltrating CD8+ T cells or the peripheral vs central regions of the lymphoma. In summary we identify altered spatial localization and protein expression of anti-lymphoma cells in different phenotypes of CTCL and different skin substructures.

Tissue-resident memory T (Trm) cells provide local protection against recurrent infections and are implicated in recurrent inflammatory skin diseases such as psoriasis. Once seeded, individual clones of Trm cells can persist in homeostatic skin up to a decade, despite the expected life-spans of memory T cells ranging between 150 days -2 years. In this study, we investigate local precursors capable of replenishing and therefore retaining site-specific T cell populations in steady state and disease affected skin. Employing single cell sequencing with T cell receptor (TCR) and CITEseq technology, we identified a subset of lower differentiated Trm cells marked by the expression of selectin CD62L in healthy skin, psoriasis lesions and cutaneous T cell lymphoma (CTCL). These lower differentiated CD62L+ Trm cells showed clonal sharing with highly enriched clones of CD69+ CD103+ CD49a+ cytotoxic T cells in the skin but were less abundant in the blood. Additionally, these had high proliferative potential and differentiated into cytotoxic CD103+CD49a+ Trm cells following activation. Taken together, our study reveals a source of local replenishment of Trm cells in healthy and diseased skin which could explain the longevity of localized disease memories.

Tissue-resident memory T cells (TRM) are seeded and persist for years at the site of inflammatory responses in non-lymphoid tissues. In human skin, enrichment of oligoclonal cytotoxic CD103+CD49a+ TRM in disequilibrium with circulating T cells indicates local renewal of epidermal T cells. Indeed, a subset of less differentiated skin resident CD62L+CD49a- CD103-CD69+ TRM cells was identified by single cell CITE-seq and T cell receptor sequencing. Resident CD62L+ CD69+ T cells showed clonal sharing with epidermal CD49a+ CD103+ CD69+ cytotoxic T cells. Following stimulation, CD62L+ CD69+ T cells displayed high proliferation potential and differentiated into cytotoxic CD103+ CD49a+ TRM cells. Furthermore, in cutaneous T cell lymphoma (CTCL), driven by one expanded clone of malignant CD8+ T cells, both lowly differentiated CD69+CD62L+ T cells and highly differentiated CD49a+CD103+ TRM cells phenotypes were detected in the malignant clone. In conclusion, we revealed a skin resident precursor population to CD49a+CD103+ TRM cells marked by the expression of CD69 and CD62L, capable of maintaining the local pool of skin resident memory T cells in health and skin diseases.

Vitiligo is an inflammatory disease that presents as well-demarcated, depigmented skin lesions. While different mechanisms are implicated in the development and maintenance of vitiligo lesions, immune-mediated destruction of melanocytes has been ascribed the major mechanism of depigmentation. We have previously observed enrichment of cytotoxic resident CD49a+ CD103+ T-cells in the skin of vitiligo patients and recent single cell data highlights crosstalk between CD8+ effector and Treg cells within the lesion, as well as a subclinical state of activation in the non-lesional skin. Here, T cell composition and how these cells interact in situ with melanocytes was assessed in lesional, peri-lesional, and non-lesional skin with Nanostring GeoMX spatial proteomics and 10x Genomics Visium spatial transcriptomic systems. We observed increased levels of S100B protein in the T cell and melanocyte colocalisations in vitiligo skin, previously proposed as a marker for disease activity. Spatial transcriptomic analysis corroborated these finding by revealing increased expression of S100 family genes in the basal epidermal layer of vitiligo lesion, combined with decreased expression of several collagen related genes. Pan- T cell activation in vitiligo skin activated IFNg pathway related genes and genes responsible for proteasome assembly proteins. While some of the genes were also upregulated in activated non-lesional and healthy skin, the levels of expression were several-fold higher in lesional vitiligo. Overall our study shows that the vitiligo skin is a site of quiet inflammation and melanocyte injury, with quiescent IFNg dominated pathophysiology that is induced following T cell activation.

Pathogenic memory T cells are implicated in the local relapse of allergic contact dermatitis (ACD), vitiligo and psoriasis. Here we investigate how resident T cells initiate relapses in response to the allergen Methylisothiazolinone (MI). MI–related ACD was sampled two months and two years after resolution of disease. T cells were activated in skin explants with MI or the pan T-cell agonist OKT-3. Epidermis was analysed by RNAseq, Nanostring and multiplex methods. Metalloproteinase 12 (MMP-12)–induction by ACD–related cytokines was assessed in primary keratinocytes and fibroblasts and a humanized xenograft mouse model was used. Ex vivo activation of T cells in resolved ACD induced chemokines and cytokines involved in immune cell recruitment and tissue remodeling, including the collagenase MMP-12. In response to cytokine stimulation, human primary fibroblasts and keratinocytes had the capacity to overexpress and release MMP-12. Local injections of MMP-12 and ACD–associated cytokines in vivo resulted in degradation of the Collagen IV–rich basement membrane separating the epidermis and dermis and a redistribution of T cells within the skin. Resident T cells in resolved ACD induce MMP-12–driven tissue remodeling that facilitates recruitment of local T cells. Our findings emphasize the potential for topical T cell eradication to promote deep remission of relapsing inflammatory dermatoses.

Le psoriasis est une maladie inflammatoire cutanée chronique. Dans les lésions après traitement, en rémission macroscopique, la signature moléculaire n’est cependant pas totalement normalisée. Les lymphocytes T mémoires cutanés (TRM) qui résident dans la peau en rémission du psoriasis produisent plus d’IL-17 et d’IL-22 que dans la peau des contrôles sains, et pourraient constituer l’origine cellulaire des anomalies transcriptomiques du psoriasis en rémission. L’impact direct de ces cellules sur le tissu cutané environnant reste cependant à étudier : l’activation des TRM est-elle suffisante pour entraîner une récidive locale du psoriasis ? Nous présentons ici la réponse tissulaire à l’activation lymphocytaire dans des biopsies de patients. Des biopsies cutanées ont été prélevées sur des contrôles sains, des patients avec psoriasis actif (n = 10) et en rémission (biothérapies n = 9, UVB n = 9 et PUVA n = 5). L’activation des lymphocytes Tétait obtenue par exposition des biopsies à un anticorps anti-CD3 (OKT3 1 μg/mL) comparé à un contrôle (isotype IgG2a 1 μg/mL). Après 16 heures d’incubation, une séparation enzymatique du derme et de l’épiderme était effectuée et l’ARN était extrait après homogénéisation des tissus par le TissueLyser (Qiagen). Des analyses transcriptomiques étaient effectuées par RT-PCR, Nanostring et séquençage d’ARN messager. La sécrétion cytokinique était analysée par Luminex et la microscopie était réalisée par imagerie confoncale (Zeiss LSM800). Dans tous les groupes (lésions actives, en rémission ou contrôles) et avec les trois méthodes transcriptomiques utilisées, une augmentation d’IFNγet des gènes kératinocytaires d’aval (CXCL10, CXCL9) était trouvée après activation lymphocytaire. Un signal IL17était aussi augmenté (DefB2, SPRR2), plus dans les lésions actives et en rémission sous biothérapie que dans la peau saine. Pour étudier l’intérêt clinique de l’équilibre entre ces deux réponses, la même expérience était réalisée sur une cohorte de 9 patients après traitement par UVB. Dans les biopsies de leur peau en rémission, une réponse en faveur d’IL-17 plutôt qu’IFNγ après activation lymphocytaire était corrélé à une récidive clinique plus précoce (p < 0,05). Ce model ex vivo d’activation lymphocytaire de biopsies, isolées de la circulation sanguine, permet une description précise des équilibres cytokiniques au sein de la peau humaine. Dans le psoriasis, la présence d’un déséquilibre en faveur de la production d’IL-17 après traitement par UVB semble être un facteur prédisposant à la récidive. La réponse cutanée à l’activation locale des lymphocytes T pourrait permettre une stratification des patients afin de prédire leur réponse aux traitements.

Chronic wounds represent a major and rising health and economic burden worldwide. There is a continued search toward more effective wound therapy. We found significantly reduced microRNA-132 (miR-132) expression in human diabetic ulcers compared with normal skin wounds and also in skin wounds of leptin receptor-deficient (db/db) diabetic mice compared with wild-type mice. Local replenishment of miR-132 in the wounds of db/db mice accelerated wound closure effectively, which was accompanied by increased proliferation of wound edge keratinocytes and reduced inflammation. The pro-healing effect of miR-132 was further supported by global transcriptome analysis, which showed that several inflammation-related signaling pathways (e.g., NF-kappa B, NOD-like receptor, toll-like receptor, and tumor necrosis factor signaling pathways) were the top ones regulated by miR-132 in vivo. Moreover, we topically applied liposome-formulated miR-132 mimics mixed with pluronic F-127 gel on human ex vivo skin wounds, which promoted re-epithelialization. Together, our study showed the therapeutic potential of miR-132 in chronic wounds, which warrants further evaluation in controlled clinical trials.

An increasing number of patients need reconstructive surgery after massive weight loss. The hypothesis was that surgical experience together with standardised management guidelines significantly decreases early complication rates after abdominoplasty for massive weight loss. The primary aim was to assess the 30-day complication rate after abdominoplasty following increased surgical training and experience. The secondary aim was to assess whether optimised management guidelines have an impact on the complication rate and patient safety.

Chronic wounds represent a major and rising health and economic burden worldwide.There is a continued search towards more effective wound therapy.We found significantly reduced microRNA-132 (miR-132) expression in human diabetic ulcers compared to normal skin wounds, and also in skin wounds of leptin receptor-deficient (db/db) diabetic mice compared to wild-type mice.Local replenishment of miR-132 in the wounds of db/db mice accelerated wound closure effectively, which was accompanied by increased proliferation of wound-edge keratinocytes and reduced inflammation.The pro-healing effect of miR-132 was further supported by global transcriptome analysis, which revealed that several inflammation-related signaling pathways, e.g.NF-kB, NOD-like receptor, Toll-like receptor, TNF signaling pathways, were the top ones regulated by miR-132 in vivo.Moreover, we applied topically liposome-formulated miR-132 mimics mixed with pluronic F-127 gel on human ex vivo skin wounds, which treatment promoted re-epithelialization. Together, our study demonstrated the therapeutic potential of miR-132 in chronic wound, which warrants further evaluation in controlled clinical trials.

Orexin A (OXA) is a novel peptide that appears to play a role in the regulation of food intake, arousal, and energy balance. The aim of this study was to study the effect of iv infusion of OXA on gastric emptying, appetite, leptin, ghrelin, and glucose metabolism in man ( six normal men) and the localization of OXA and orexin receptors (OXRs) 1 and 2 in the human gut. Gastric emptying was studied scintigraphically after ingestion of a Tc-99m-labeled omelet and iv infusion of OXA ( 10 pmol/ kg center dot min). Appetite ratings and blood samples were obtained at regular intervals. The immunohistochemical distribution of OXA and OXRs was examined using antibodies recognizing OXA, OX1R, and OX2R in human gastrointestinal tissue. OXA had no effect on lag phase or gastric half-emptying time. However, the gastric emptying rate was significantly slower without affecting appetite ratings. Plasma concentrations of insulin were increased by OXA, whereas plasma leptin decreased and ghrelin was unchanged. OXA immunoreactivity was observed in a subset of neurons and varicose nerve fibers in the mucosa, ganglia, and circular muscle layer and mucosal endocrine cells in the stomach and small intestine. OXA-immunoreactive cells in the islets of Langerhans contained insulin with a subset expressing OX2R. In conclusion, peripheral OXA seems to slightly affect the regulation of gastric emptying in humans without affecting appetite ratings. OXA decreased plasma levels of leptin, suggesting a possible interaction between leptin and OXA in the regulation of energy homeostasis.

Orexin A (OXA) increases food intake and inhibits fasting small bowel motility in rats. The aim of this study was to examine the effect of exogenous OXA and endogenous OXA on gastric emptying, acid secretion, glucose metabolism and distribution of orexin immunoreactivity in the stomach. Rats equipped with a gastric fistula were subjected to intravenous (IV) infusion of OXA or the selective orexin-1 receptor (OX1R) antagonist SB-334867-A during saline or pentagastrin infusion. Gastric emptying was studied with a liquid non-nutrient or nutrient, using 51Cr as radioactive marker. Gastric retention was measured after a 20-min infusion of OXA or SB-334867-A. Plasma concentrations of OXA, insulin, glucagon, glucose and gastrin were studied. Immunohistochemistry against OXA, OX1R and gastrin in gastric tissue was performed. OXA alone had no effect on either acid secretion or gastric emptying. SB-334867-A inhibited both basal and pentagastrin-induced gastric acid secretion and increased gastric retention of the liquid nutrient, but not PEG 4000. Plasma gastrin levels were unchanged by IV OXA or SB-334867-A. Plasma OXA levels decreased after intake of the nutrient meal and infusion of the OX1R antagonist. Only weak effects were seen on plasma glucose and insulin by OXA. Immunoreactivity to OXA and OX1R were found in the mucosa, myenteric cells bodies and varicose nerve fibers in ganglia and circular muscle of the stomach. In conclusion, endogenous OXA influences gastric emptying of a nutrient liquid and gastric acid secretion independent of gastrin. This indicates a role for endogenous OXA, not only in metabolic homeostasis, but also in the pre-absorptive processing of nutrients in the gut.

Ghrelin is a gut peptide that is secreted from the stomach and stimulates food intake. There are ghrelin receptors throughout the gut and intracerebroventricular ghrelin has been shown to increase gastric acid secretion. The aim of the present study was to examine the effects of peripherally administered ghrelin on gastric emptying of a non-nutrient and nutrient liquid, as well as, basal and pentagastrin-stimulated gastric acid secretion in awake rats. In addition, gastric contractility was studied in vitro. Rats equipped with a gastric fistula were subjected to an intravenous infusion of ghrelin (10–500 pmol kg− 1 min− 1) during saline or pentagastrin (90 pmol kg− 1 min− 1) infusion. After administration of polyethylene glycol (PEG) 4000 with 51Cr as radioactive marker, or a liquid nutrient with 51Cr, gastric retention was measured after a 20-min infusion of ghrelin (500 pmol kg− 1 min− 1). In vitro isometric contractions of segments of rat gastric fundus were studied (10− 9 to 10− 6 M). Ghrelin had no effect on basal acid secretion, but at 500 pmol kg− 1 min− 1 ghrelin significantly decreased pentagastrin-stimulated acid secretion. Ghrelin had no effect on gastric emptying of the nutrient liquid, but significantly increased gastric emptying of the non-nutrient liquid. Ghrelin contracted fundus muscle strips dose-dependently (pD2 of 6.93 ± 0.7). Ghrelin IV decreased plasma orexin A concentrations and increased plasma somatostatin concentrations. Plasma gastrin concentrations were unchanged during ghrelin infusion. Thus, ghrelin seems to not only effect food intake but also gastric motor and secretory function indicating a multifunctional role for ghrelin in energy homeostasis.

Orexin A (OXA) is found in the central nervous system (CNS) and in the gut. Peripheral administration of OXA to rats results in an inhibition of fasting motility. Plasma OXA increases during fasting and central administration of OXA increases food intake. The aim of the present study was to assess the pharmacokinetic profile of OXA and the effect of intravenously (IV) administered OXA on plasma concentrations of insulin and glucagon concentrations. Rats were given OXA IV (100 pmol kg−1 min−1) for time periods of 0, 10, 20, 30 min and for 10, 20, 30 min after ceasing a 30-min infusion. After each time period, rats were then sacrificed and blood obtained. OXA was also administered at increasing doses (0, 100, 300 and 500 pmol kg−1 min−1) for 30 min and blood was obtained. Plasma OXA, insulin and glucagon levels were measured using commercially available radioimmunoassay (RIA) kits. The plasma half-life of OXA was 27.1±9.5 min. Stepwise increasing infusion rates of OXA confirmed a linear concentration–time curve and thus first-order kinetics. Its volume of distribution indicated no binding to peripheral tissues. Plasma glucagon decreased during infusion of OXA, while insulin was unaffected. Plasma OXA was raised fourfold after food intake. Thus, OXA has a longer plasma half-life than many other peptides found in the gut. This needs to be taken into account when assessing effects of OXA on biological parameters after peripheral administration.

Orexin A (OXA)-positive neurons are found in the lateral hypothalamic area and the enteric nervous system. The aim of this study was to investigate the mechanism of OXA action on small bowel motility. Electrodes were implanted in the serosa of the rat small intestine for recordings of myoelectric activity during infusion of saline or OXA in naive rats, vagotomized rats, rats pretreated with guanethidine (3 mg/kg) or N(omega)-nitro-L-arginine (L-NNA; 1 mg/kg). Naive rats were given a bolus of the orexin receptor-1 (OX1R) antagonist (SB-334867-A; 10 mg/kg), and the effect of both OXA and SB-334867-A on fasting motility was studied. Double-label immunocytochemistry with primary antibodies against OXA, neuronal nitric oxide synthase (nNOS), and OX1R was performed. OXA induced a dose-dependent prolongation of the cycle length of the migrating myoelectric complex (MMC) and, in the higher doses, replaced the activity fronts with an irregular spiking pattern. Vagotomy or pretreatment with guanethidine failed to prevent the response to OXA. The OXA-induced effect on the MMC cycle length was completely inhibited by pretreatment with L-NNA (P < 0.05), as did SB-334867-A. The OX1R antagonist shortened the MMC cycle length from 14.1 (12.0-23.5) to 11.0 (9.5-14.7) min (P < 0.05) during control and treatment periods, respectively. Colocalization of OXA and nNOS was observed in myenteric neurons of the duodenum and nerve fibers in the circular muscle. Our results indicate that OXA inhibition of the MMC involves the OX1R and that activation of a L-arginine/NO pathway possibly originating from OX1R/nNOS-containing neurons in the myenteric plexus may mediate this effect. Endogenous OXA may have a physiological role in regulating the MMC.

reactive for NOS or VIP, whereas colocalization with calbindin was not observed.In functional experiments, galanin 1-16, the GAL-R1 preferring fragment, inhibited in a concentrationdependent manner (300 nM -3 I~M) the ascending excitatory reflex recorded in the oral compartment in response to gut wall distension (evoked by balloon inflation in the caudal compartment).The inhibitory effect of galanin 1-16 (max.60%) was antagonized by 1 p.M RWJ-54708, a selective GAL-R1 antagonist.Galanin 1-16 was ineffective when added in the imermediate or the caudal compartment up to 3 p,M.Conclusions: ChAT immunoreactive neurons labeling inhibitory GAL-R1 are cholinergic motor neurons responsible for the ascending excitatory reflex.GAL-R1 does not appear to be present on ascending interneurons and intrinsic primary afferent neurons.