Spatiotemporal in vivo tracking of polyclonal human regulatory T cells (Tregs) reveals a role for innate immune cells in Treg transplant recruitment.

Regulatory T cells (Tregs) are emerging as a new cell-based therapy in solid organ transplantation. Adoptive transfer of Tregs has been shown preclinically to protect from graft rejection, and the safety of Treg therapy has been demonstrated in clinical trials. Despite these successes, the in vivo distribution and persistence of adoptively transferred Tregs remained elusive, which hampers clinical translation. Here we isolated human Tregs using a GMP-compatible protocol and lentivirally transduced them with the human sodium iodide symporter to render them traceable in vivo by radionuclide imaging. Engineered human Tregs were characterized for phenotype, survival, suppressive capacity, and reporter function. To study their trafficking behavior, they were subsequently administered to humanized mice with human skin transplants. Traceable Tregs were quantified in skin grafts by non-invasive nano-single-photon emission computed tomography (nanoSPECT)/computed tomography (CT) for up to 40 days, and the results were validated ex vivo. Using this approach, we demonstrated that Treg trafficking to skin grafts was regulated by the presence of recipient Gr-1+ innate immune cells. We demonstrated the utility of radionuclide reporter gene-afforded quantitative Treg in vivo tracking, addressing a fundamental need in Treg therapy development and offering a clinically compatible methodology for future Treg therapy imaging in humans.

Psoriatic skin inflammation induces a pre-diabetic phenotype via the endocrine actions of skin secretome.

OBJECTIVE: Psoriasis is a chronic inflammatory skin disease that is thought to affect ∼2% of the global population. Psoriasis has been associated with ∼30% increased risk of developing type 2 diabetes (T2D), with numerous studies reporting that psoriasis is an independent risk-factor for T2D, separate from underlying obesity. Separately, studies of skin-specific transgenic mice have reported altered whole-body glucose homeostasis in these models. These studies imply a direct role for skin inflammation and dysfunction in mediating the onset of T2D in psoriasis patients, potentially via the endocrine effects of the skin secretome on key metabolic tissues. We used a combination of in vivo and ex vivo mouse models and ex vivo human imiquimod (IMQ) models to investigate the effects of psoriasis-mediated changes in the skin secretome on whole-body metabolic function. METHODS: To induce psoriatic skin inflammation, mice were topically administered 75 mg of 5% IMQ cream (or Vaseline control) to a shaved dorsal region for 4 consecutive days. On day 5, mice were fasted for glucose and insulin tolerance testing, or sacrificed in the fed state with blood and tissues collected for analysis. To determine effects of the skin secretome, mouse skin was collected at day 5 from IMQ mice and cultured for 24 h. Conditioned media (CM) was collected and used 1:1 with fresh media to treat mouse explant subcutaneous adipose tissue (sAT) and isolated pancreatic islets. For human CM experiments, human skin was exposed to 5% IMQ cream for 20 min, ex vivo, to induce a psoriatic phenotype, then cultured for 24 h. CM was collected, combined 1:1 with fresh media and used to treat human sAT ex vivo. Markers of tissue inflammation and metabolic function were determined by qPCR. Beta cell function in isolated islets was measured by dynamic insulin secretion. Beta-cell proliferation was determined by measurement of Ki67 immunofluorescence histochemistry and BrDU uptake, whilst islet apoptosis was assessed by caspase 3/7 activity. All data is expressed as mean ± SEM. RESULTS: Topical treatment with IMQ induced a psoriatic-like phenotype in mouse skin, evidenced by thickening, erythema and inflammation of the skin. Topical IMQ treatment induced inflammation and signs of metabolic dysfunction in sub-cutaneous and epidydimal adipose tissue, liver, skeletal muscle and gut tissue. However, consistent with islet compensation and a pre-diabetic phenotype, IMQ mice displayed improved glucose tolerance, increased insulin and c-peptide response to glucose, and increased beta cell proliferation. Treatment of sAT with psoriatic mouse or human skin-CM replicated the in vivo phenotype, leading to increased inflammation and metabolic dysfunction in mouse and human sAT. Treatment of pancreatic islets with psoriatic mouse skin-CM induced increases in beta-proliferation and apoptosis, thus partially replicating the in vivo phenotype. CONCLUSIONS: Psoriasis-like skin inflammation induces a pre-diabetic phenotype, characterised by tissue inflammation and markers of metabolic dysfunction, together with islet compensation in mice. The in vivo phenotype is partially replicated by exposure of sAT and pancreatic islets to psoriatic-skin conditioned media. These results support the hypothesis that psoriatic skin inflammation, potentially via the endocrine actions of the skin secretome, may constitute a novel pathophysiological pathway mediating the development of T2D.

Can the skin make you fat? A role for the skin in regulating adipose tissue function and whole-body glucose and lipid homeostasis.

Prevalence of obesity and related complications such as type 2 diabetes (T2D) has increased dramatically in recent decades. Metabolic complications of obesity arise in part due to subcutaneous adipose tissue (SAT) dysfunction. However, it is currently unclear why some obese individuals develop insulin resistance and T2D and others do not. In this review, we discuss the role of the skin in regulating SAT function, and whether presence of inflammatory skin diseases such as psoriasis represent a novel risk mechanism mediating development of obesity-related complications.

An Atlas of Human Regulatory T Helper-like Cells Reveals Features of Th2-like Tregs that Support a Tumorigenic Environment.

Regulatory T cells (Tregs) play a pivotal role in maintaining immunological tolerance, but they can also play a detrimental role by preventing antitumor responses. Here, we characterized T helper (Th)-like Treg subsets to further delineate their biological function and tissue distribution, focusing on their possible contribution to disease states. RNA sequencing and functional assays revealed that Th2-like Tregs displayed higher viability and autocrine interleukin-2 (IL-2)-mediated activation than other subsets. Th2-like Tregs were preferentially found in tissues rather than circulation and exhibited the highest migratory capacity toward chemokines enriched at tumor sites. These cellular responses led us to hypothesize that this subset could play a role in maintaining a tumorigenic environment. Concurrently, Th2-like Tregs were enriched specifically in malignant tissues from patients with melanoma and colorectal cancer compared to healthy tissue. Overall, our results suggest that Th2-like Tregs may contribute to a tumorigenic environment due to their increased cell survival, higher migratory capacity, and selective T-effector suppressive ability.

Oxidative stress-associated senescence in dermal papilla cells of men with androgenetic alopecia.

Dermal papilla cells (DPCs) taken from male androgenetic alopecia (AGA) patients undergo premature senescence in vitro in association with the expression of p16(INK4a), suggesting that DPCs from balding scalp are more sensitive to environmental stress than nonbalding cells. As one of the major triggers of senescence in vitro stems from the cell "culture shock" owing to oxidative stress, we have further investigated the effects of oxidative stress on balding and occipital scalp DPCs. Patient-matched DPCs from balding and occipital scalp were cultured at atmospheric (21%) or physiologically normal (2%) O2. At 21% O2, DPCs showed flattened morphology and a significant reduction in mobility, population doubling, increased levels of reactive oxygen species and senescence-associated ß-Gal activity, and increased expression of p16(INK4a) and pRB. Balding DPCs secreted higher levels of the negative hair growth regulators transforming growth factor beta 1 and 2 in response to H2O2 but not cell culture-associated oxidative stress. Balding DPCs had higher levels of catalase and total glutathione but appear to be less able to handle oxidative stress compared with occipital DPCs. These in vitro findings suggest that there may be a role for oxidative stress in the pathogenesis of AGA both in relation to cell senescence and migration but also secretion of known hair follicle inhibitory factors.

Steroid synthesis by primary human keratinocytes; implications for skin disease.

Cortisol-based therapy is one of the most potent anti-inflammatory treatments available for skin conditions including psoriasis and atopic dermatitis. Previous studies have investigated the steroidogenic capabilities of keratinocytes, though none have demonstrated that these skin cells, which form up to 90% of the epidermis are able to synthesise cortisol. Here we demonstrate that primary human keratinocytes (PHK) express all the elements required for cortisol steroidogenesis and metabolise pregnenolone through each intermediate steroid to cortisol. We show that normal epidermis and cultured PHK express each of the enzymes (CYP11A1, CYP17A1, 3ßHSD1, CYP21 and CYP11B1) that are required for cortisol synthesis. These enzymes were shown to be metabolically active for cortisol synthesis since radiometric conversion assays traced the metabolism of [7-(3)H]-pregnenolone through each steroid intermediate to [7-(3)H]-cortisol in cultured PHK. Trilostane (a 3ßHSD1 inhibitor) and ketoconazole (a CYP17A1 inhibitor) blocked the metabolism of both pregnenolone and progesterone. Finally, we show that normal skin expresses two cholesterol transporters, steroidogenic acute regulatory protein (StAR), regarded as the rate-determining protein for steroid synthesis, and metastatic lymph node 64 (MLN64) whose function has been linked to cholesterol transport in steroidogenesis. The expression of StAR and MLN64 was aberrant in two skin disorders, psoriasis and atopic dermatitis, that are commonly treated with cortisol, suggesting dysregulation of epidermal steroid synthesis in these patients. Collectively these data show that PHK are capable of extra-adrenal cortisol synthesis, which could be a fundamental pathway in skin biology with implications in psoriasis and atopic dermatitis.