Skin infiltrating NK cells in cutaneous T-cell lymphoma are increased in number and display phenotypic alterations partially driven by the tumor.

Cutaneous T-cell lymphomas (CTCL) are characterized by focal infiltration of malignant T cell clones in solitary skin lesions. Many CTCL patients experience an indolent disease, but some progress to advanced disease with high fatality. We hypothesized that natural killer (NK) cells participate in local control of tumor growth in CTCL skin. Immunohistochemistry and flow cytometry analysis of the density, localization, phenotype and function of NK cells in twenty-nine fresh or formalin-fixed skin biopsies from twenty-four CTCL patients and twenty-three biopsies from twenty healthy controls highlighted higher numbers of CD56+CD3- NK cells in CTCL skin. A reduced fraction of CTCL skin NK cells expressed the maturation marker CD57, the cytotoxic protein granzyme B and the activation marker CD69, indicating reduced tumor-killing abilities of the NK cells. Retained expression of immune checkpoint proteins or inhibitory proteins including PD1, TIM3, LAG3, CD73 and NKG2A and the activating receptors CD16 and NKp46 indicated maintained effector functions. Indeed, the capacity of NK cells to produce anti-tumor acting IFNγ upon PMA+ionomycin stimulation was similar in cells from CTCL and healthy skin. Co-cultures of primary human NK cells or the NK cell line NKL with CTCL cells resulted in reduced levels of granzyme B and CD69, indicating that close cellular interactions with CTCL cells induced the impaired functional NK cell phenotype. In conclusion, increased numbers of NK cells in CTCL skin exhibit a partially impaired phenotype in terms of activity. Enhancing NK cell activity with NK cell activating cytokines such as IL-15 or immune checkpoint blockade therefore represents a potential immunotherapeutic approach in CTCL.

Graded expression of the chemokine receptor CX3CR1 marks differentiation states of human and murine T cells and enables cross-species interpretation.

T cells differentiate into functionally distinct states upon antigen encounter. These states are delineated by different cell surface markers for murine and human T cells, which hamper cross-species translation of T cell properties. We aimed to identify surface markers that reflect the graded nature of CD8+ T cell differentiation and delineate functionally comparable states in mice and humans. CITEseq analyses revealed that graded expression of CX3CR1, encoding the chemokine receptor CX3CR1, correlated with the CD8+ T cell differentiation gradient. CX3CR1 expression distinguished human and murine CD8+ and CD4+ T cell states, as defined by migratory and functional properties. Graded CX3CR1 expression, refined with CD62L, accurately captured the high-dimensional T cell differentiation continuum. Furthermore, the CX3CR1 expression gradient delineated states with comparable properties in humans and mice in steady state and on longitudinally tracked virus-specific CD8+ T cells in both species. Thus, graded CX3CR1 expression provides a strategy to translate the behavior of distinct T cell differentiation states across species.

Human skin-resident CD8+ T cells require RUNX2 and RUNX3 for induction of cytotoxicity and expression of the integrin CD49a.

The integrin CD49a marks highly cytotoxic epidermal-tissue-resident memory (TRM) cells, but their differentiation from circulating populations remains poorly defined. We demonstrate enrichment of RUNT family transcription-factor-binding motifs in human epidermal CD8+CD103+CD49a+ TRM cells, paralleled by high RUNX2 and RUNX3 protein expression. Sequencing of paired skin and blood samples revealed clonal overlap between epidermal CD8+CD103+CD49a+ TRM cells and circulating memory CD8+CD45RA-CD62L+ T cells. In vitro stimulation of circulating CD8+CD45RA-CD62L+ T cells with IL-15 and TGF-ß induced CD49a expression and cytotoxic transcriptional profiles in a RUNX2- and RUNX3-dependent manner. We therefore identified a reservoir of circulating cells with cytotoxic TRM potential. In melanoma patients, high RUNX2, but not RUNX3, transcription correlated with a cytotoxic CD8+CD103+CD49a+ TRM cell signature and improved patient survival. Together, our results indicate that combined RUNX2 and RUNX3 activity promotes the differentiation of cytotoxic CD8+CD103+CD49a+ TRM cells, providing immunosurveillance of infected and malignant cells.

IL-22 Downregulates Peptidylarginine Deiminase-1 in Human Keratinocytes: Adding Another Piece to the IL-22 Puzzle in Epidermal Barrier Formation.

Increased presence of IL-22+ cells in the skin is a characteristic finding in skin barrier defects, such as psoriasis and atopic dermatitis. However, mechanistic insight into effects of IL-22 on epidermal functioning is yet to be elucidated. One crucial step during epidermal differentiation is deimination or citrullination. Here, we show reduced levels of peptidylarginine deiminase 1, an enzyme that converts peptidylarginine into citrulline in lesional psoriatic skin. IL-22 signaling through the IL-22 receptor complex was found to suppress expression of peptidylarginine deiminase 1 in epidermal keratinocytes. Subsequently, total peptidylarginine deiminase activity and extent of protein deimination in keratinocytes treated with IL-22 were reduced together with a significant decrease in deimination of keratin 1 and FLG, both important for epidermal differentiation. Vitamin D and acitretin partly restored the peptidylarginine deiminase 1 defect caused by IL-22. Collectively, we show that IL-22 downregulates deimination, thus identifying a potential target for treatment of skin barrier defects.

Vancomycin-Loaded Microneedle Arrays against Methicillin-Resistant Staphylococcus Aureus Skin Infections.

Skin and soft tissue infections (SSTIs) caused by methicillin-resistant Staphylococcus aureus (MRSA) are a major healthcare burden, often treated with intravenous injection of the glycopeptide antibiotic vancomycin (VAN). However, low local drug concentration in the skin limits its treatment efficiency, while systemic exposure promotes the development of resistant bacterial strains. Topical administration of VAN on skin is ineffective as its high molecular weight prohibits transdermal penetration. In order to implement a local VAN delivery, microneedle (MN) arrays with a water-insoluble support layer for the controlled administration of VAN into the skin are developed. The utilization of such a support layer results in water-insoluble needle shafts surrounded by drug-loaded water-soluble tips with high drug encapsulation. The developed MN arrays can penetrate the dermal barriers of both porcine and fresh human skin. Permeation studies on porcine skin reveal that the majority of the delivered VAN is retained within the skin. It is shown that the VAN-MN array reduces MRSA growth both in vitro and ex vivo on skin. The developed VAN-MN arrays may be extended to several drugs and may facilitate localized treatment of MRSA-caused skin infections while minimizing adverse systemic effects.

Neutrophil Recruitment to Noninvasive MRSA at the Stratum Corneum of Human Skin Mediates Transient Colonization.

Staphylococcus aureus is a leading cause of skin and soft issue infection, but paradoxically, it also transiently, and often harmlessly, colonizes human skin. An obstacle to understanding this contradiction has been a shortage of in vivo models reproducing the unique structure and immunology of human skin. In this work, we developed a humanized model to study how healthy adult human skin responds to colonizing methicillin-resistant S. aureus (MRSA). We demonstrate the importance of the outer stratum corneum as the major site of bacterial colonization and how noninvasive MRSA adhesion to corneocytes induces a local inflammatory response in underlying skin layers. This signaling recruits neutrophils to the skin, where they control bacterial numbers, mediating transiency in colonization. This work highlights the spatiotemporal aspects of human skin colonization and demonstrates a subclinical inflammatory response to noninvasive MRSA that allows human skin to regulate the bacterial population at its outer surface.

A skewed pool of resident T cells triggers psoriasis-associated tissue responses in never-lesional skin from patients with psoriasis.

BACKGROUND: Resident T cells are implicated in the maintenance and recurrence of psoriatic lesions. Whether skin that has not yet experienced psoriasis in patients with established disease harbors pathogenic T cells is less investigated. OBJECTIVE: We sought to analyze the composition of resident T cells and T cell-driven tissue responses in skin never affected by psoriasis from patients with mild disease. METHODS: Never-lesional skin from patients with psoriasis (NLP) was collected from those with mild disease. T-cell profiles were assessed by using confocal imaging and flow cytometry. Tissue responses to T-cell stimulation were measured by using multiplex and NanoString technology. RESULTS: T-cell activation ex vivo triggered psoriasiform and type I interferon tissue responses in NLP psoriasis. Accordingly, keratinocytes from NLP responded to IFN-γ stimulation with myxovirus 1 (MX1) expression and IFN-α release. Additionally, CCR6-expressing resident T cells poised to produce IFN-γ and IL-17 were enriched in epidermis from NLP, whereas dermal tissue responses and T-cell compositions were similar to those in healthy skin. Finally, keratinocytes from NLP exposed to IL-17 and skin explants exposed to common fungal antigens responded with upregulation of the CCR6 ligand CCL20. CONCLUSION: Epidermal resident T cells capable of triggering psoriasiform tissue responses accumulate in epidermis from NLP. Our global analysis of NLP reveals that microbial interplay with genetically predisposed keratinocytes might shape the local pool of resident T cells.

Resident T Cells in Resolved Psoriasis Steer Tissue Responses that Stratify Clinical Outcome.

Psoriasis is driven by focal disruptions of the immune-homeostasis in human skin. Local relapse following cessation of therapy is common and unpredictable, which complicates clinical management of psoriasis. We have previously shown that pathogenic resident T cells accumulate in active and resolved psoriasis, but whether these cells drive psoriasiform tissue reactions is less clear. Here, we activated T cells within skin explants using the pan-T cell activating antibody OKT-3. To explore if T cells induced different tissue response patterns in healthy and psoriasis afflicted skin, transcriptomic analyses were performed with RNA-sequencing and Nanostring. Core tissue responses dominated by IFN-induced pathways were triggered regardless of the inflammatory status of the skin. In contrast, pathways induced by IL-17A, including Defensin beta 2 and keratinocyte differentiation markers, were activated in psoriasis samples. An integrated analysis of IL-17A and IFN-related responses revealed that IL-17 dominated tissue response correlated with early relapse following UVB treatment. Stratification of tissue responses to T cell activation in resolved lesions could potentially offer individualized prediction of disease relapse during long-term immunomodulatory treatment.

MicroRNA-132 with Therapeutic Potential in Chronic Wounds.

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-κB, 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.

CD49a Expression Defines Tissue-Resident CD8+ T Cells Poised for Cytotoxic Function in Human Skin.

Tissue-resident memory T (Trm) cells form a heterogeneous population that provides localized protection against pathogens. Here, we identify CD49a as a marker that differentiates CD8+ Trm cells on a compartmental and functional basis. In human skin epithelia, CD8+CD49a+ Trm cells produced interferon-γ, whereas CD8+CD49a- Trm cells produced interleukin-17 (IL-17). In addition, CD8+CD49a+ Trm cells from healthy skin rapidly induced the expression of the effector molecules perforin and granzyme B when stimulated with IL-15, thereby promoting a strong cytotoxic response. In skin from patients with vitiligo, where melanocytes are eradicated locally, CD8+CD49a+ Trm cells that constitutively expressed perforin and granzyme B accumulated both in the epidermis and dermis. Conversely, CD8+CD49a- Trm cells from psoriasis lesions predominantly generated IL-17 responses that promote local inflammation in this skin disease. Overall, CD49a expression delineates CD8+ Trm cell specialization in human epithelial barriers and correlates with the effector cell balance found in distinct inflammatory skin diseases.

Stimulatory effect of endogenous orexin A on gastric emptying and acid secretion independent of gastrin.

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.

Effect of peripherally administered ghrelin on gastric emptying and acid secretion in the rat.

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 (51)Cr, 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.

Long-lasting adverse effects of prenatal hypoxia on developing autonomic nervous system and cardiovascular parameters in rats.

To determine whether prenatal hypoxia increases the risk of developing cardiovascular disorders as an adult and, if so, the identity of the cell mechanisms involved in such dysfunction, we evaluated the sympathoadrenal system and central areas related to cardiovascular events during development and the cardiovascular parameters in adults. Pregnant rats were exposed to hypoxia (10% oxygen) from embryonic day (E) 5 to E20 and the offspring studied at 1, 3, 9 and 12 weeks of age for neurochemistry and at 12 weeks of age for cardiovascular analysis. In the 1-, 3- and 9-week-old offspring, the levels and utilization of catecholamines were reduced in sympathetic ganglia, in target organs, in adrenals and in the rostral part of the A2 cell group in the nucleus tractus solitarius, but were increased in the locus coeruleus. In the 12-week-old adult offspring, the lowered autonomic nervous activity was restricted to cardiac-related structures, i.e. the stellate ganglion, heart and adrenals. In adult rats, prenatal hypoxia did not affect the cardiac parameters under resting conditions but increased blood pressure and the variability of blood pressure and heart rate under stress conditions. The altered metabolic activity of the sympathoadrenal system and related central areas during development and at adulthood for most structures might be part of the potential mechanisms contributing to cardiovascular disorders in adults.