Peter Helding Kvist

TPA induction leads to a Th17-like response in transgenic K14/VEGF mice: a novel in vivo screening model of psoriasis

Psoriasis is a common chronic inflammatory skin disease, characterized by epidermal hyperplasia, immune cell infiltration, increased dermal angiogenesis and local up-regulation of a variety of inflammatory mediators. Psoriasis is thought to be driven primarily by CD4(+) T cells with a T(h)1 and/or T(h)17 phenotype. Transgenic keratin 14 (K14)/vascular endothelial growth factor (VEGF) mice have previously been reported to develop a psoriasis-like phenotype. The aim of this study was to further characterize the model for validation as an in vivo screening model of psoriasis. Inflammation was induced in the ear skin with five topical applications of 12-O-tetradecanoyl phorbol-13-acetate (TPA) and a significantly increased inflammation was found in TPA-induced K14/VEGF transgenic animals compared with wild-type mice. The amount of VEGF in the ear tissue was significantly elevated resulting in increased dermal angiogenesis. Furthermore, intense epidermal hyperplasia, CD3(+) infiltration and significantly increased amounts of (TNF) tumor necrosis factor alpha, IL-1 beta, IL-6, IL-12/23p40, IL-12p70, IL-22 and IL-17 were detected in the inflamed ear skin. This cytokine profile strongly suggests a T(h)17-mediated inflammation. All findings were a result of induced over-expression of VEGF. Topical treatment with betamethasone-17-valerate (BMS) significantly reduced ear skin inflammation and epidermal hyperplasia and also decreased the CD3(+) infiltration. In conclusion, the TPA-induced phenotype in K14/VEGF animals displayed several features of psoriasis, including a T(h)17 cytokine profile and a chronic-like progression, and can be used as an in vivo screening model of psoriasis.

Regulatory T Cells Control VEGF-Dependent Skin Inflammation

Transgenic mice expressing vascular endothelial growth factor (VEGF) under the keratin 14 promoter have been described to develop a psoriasis-like inflammation characterized by increased angiogenesis, acanthosis, and immune cell infiltration. We have recently shown that applying 12-O-tetradecanoylphorbol-13-acetate (TPA) in these mice induces a severe and long-lasting skin inflammation with a Th17 cell signature. Here, we aimed to study the function of CD4(+) T cells using this model. Lymphocytes isolated from inflamed ears showed a significantly higher number of activated T cells, in contrast to the primarily naive lymphocytes isolated from blood. In addition, there was an increase in regulatory T cells (CD4(+)CD25(+)CD127(-/low)) within the skin. To clarify the function of CD4(+) cells, we depleted CD4(+) T cells using antibody. CD4 depletion resulted in augmented ear thickness and proinflammatory cytokine levels, indicating that CD4(+) T cells have a suppressive rather than a proinflammatory function in this model. Subsequently, sorted regulatory CD4(+)CD25(+) T cells were transferred to naive K14/VEGF transgenic mice before TPA challenge. CD4(+)CD25(+) T-cell transfer significantly reduced ear thickness and proinflammatory cytokine production compared to controls. This shows that a persistent skin inflammation with similarities to psoriasis can be controlled by a single injection of few regulatory T cells.

Comparison of the effects of vitamin D products in a psoriasis plaque test and a murine psoriasis xenograft model

The aim of the present study was to compare the effects of Daivobet® and calcipotriol on clinical score and biomarker responses in a modified version of the Scholtz-Dumas psoriasis plaque assay. Furthermore, it was the aim to compare the effects of calcipotriol and betamethasone in the murine psoriasis xenograft model. Twenty four patients with psoriasis were treated topically once daily for three weeks, whereas the grafted mice were treated for four weeks. Clinical responses were scored twice weekly and biopsies were taken at the end of each study to analyse for skin biomarkers by histology and immunohistochemistry. The results clearly demonstrate effects on both clinical signs and biomarkers. In the patient study the total clinical score was reduced significantly with both Daivobet® and calcipotriol. Both treatments reduced epidermal thickness, Ki-67 and cytokeratin 16 expression. T cell infiltration was significantly reduced by Daivobet® but only marginally by calcipotriol. Both treatments showed strong effects on the epidermal psoriatic phenotype.Results from the xenograft model essentially showed the same results. However differences were observed when investigating subtypes of T cells.The study demonstrates the feasibility of obtaining robust biomarker data in the psoriasis plaque test that correlate well with those obtained in other clinical studies. Furthermore, the biomarker data from the plaque test correlate with biopsy data from the grafted mice.

Recent Advances in Continuous Glucose Monitoring: Biocompatibility of Glucose Sensors for Implantation in Subcutis

Tight glycemic control slows or prevents the development of short- and long-term complications of diabetes mellitus. Continuous glucose measurements provide improved glycemic control and potentially prevent these diabetic complications. Glucose sensors, especially implantable devices, offer an alternative to classical self-monitored blood glucose levels and have shown promising glucose-sensing properties. However, the ultimate goal of implementing the glucose sensor as the glucose-sensing part of a closed loop system (artificial pancreas) is still years ahead because of malfunctions of the implanted sensor. The malfunction is partly a consequence of the subcutaneous inflammatory reaction caused by the implanted sensor. In order to improve sensor measurements and thereby close the loop, it is crucial to understand what happens at the tissue-sensor interface.

Induced keratinocyte hyper-proliferation in α2β1 integrin transgenic mice results in systemic immune cell activation

alpha2beta1 integrins are normally confined to the proliferating basal layers of the epidermis. However, during wound healing and in psoriasis, these integrins are expressed on keratinocytes in suprabasal layers correlating with a less differentiated phenotype. Transgenic mice expressing alpha2beta1 integrins under the involucrine promoter have previously been demonstrated, to various degrees, spontaneously develop a skin disorder resembling psoriasis. Herein, we show that a mild epidermal wounding induces a uniform acanthosis together with an influx of immune cells. The disease initiates as a normal wound healing process and is completely restored in wildtype mice by day 14. However, in the integrin transgenic mice a chronic inflammation develops, a process that can be compared to the Koebner phenomenon in psoriatic patients. In this study, we have followed the integrin transgenic mice for five weeks, where substantial keratinocyte hyper-proliferation, inflammatory infiltration and high cytokine levels within the skin can still be observed. In addition, draining lymph nodes were dramatically increased in size and contained highly activated T cells, as well as APCs secreting large amounts of pro-inflammatory cytokines. Furthermore, the systemic immune response was affected with increased spleen size, elevated cytokine levels in the serum and altered lymphocyte trafficking patterns, very much resembling what is seen in psoriasis patients. Finally, CD4(+) T cell depletion was not able to affect the onset or progression of skin inflammation. This suggests that altered keratinocyte differentiation and proliferation can drive a skin inflammation and cause chronic immune cell activation both at a local and systemic level.

A murine model of epicutaneous protein sensitization is useful to study efficacies of topical drugs in atopic dermatitis

We studied the suitability of our murine model for the treatment trials of atopic dermatitis (AD). In this model topical application of ovalbumin (OVA) together with bacterial superantigen, staphylococcal enterotoxin B (SEB) induces a cutaneous disease resembling AD. Injured mouse skin was treated with three different drugs: a class III corticosteroid, a calcineurin inhibitor and a type 4 phosphodiesterase inhibitor. One-week treatment with corticosteroid and phosphodiesterase inhibitor remarkably decreased both epidermal and dermal thickness, whereas the calcineurin inhibitor affected only the epidermal thickness. All investigated drugs reduced the infiltration of eosinophils and mast cells onto OVA/SEB sensitized skin areas, whereas CD4+ and CD8+ T cells as well as CD11c+ dendritic cells variously diminished after corticosteroid and calcineurin inhibitor treatments. Cutaneous expression of interleukin -4, -13, -10 and interferon-gamma also decreased differently depending on drug type. Interestingly, the calcineurin inhibitor and phosphodiesterase inhibitor increased total IgE antibodies and decreased SEB-specific IgG2a antibodies in OVA/SEB sensitized mice. All these drugs can ameliorate cutaneous inflammation, although the degree of recovery depends on the type of the drug. In summary, our results show that this mouse model can be used to test new topical treatments for AD.

Effect of Subcutaneous Glucose Sensor Implantation on Skin mRNA Expression in Pigs

BACKGROUND Tight glycemic control has the potential to reduce long- and short-term effects of diabetes mellitus. New and improved glucose sensors for short-term implantation in the subcutis offer an alternative to the classical self-monitored blood glucose concentration in the management of diabetes. The use of glucose sensors has an advantage over the latter due to its capacity to obtain continuous glucose measurements. However, instability of in vivo glucose sensor measurements has been reported, and this bioinstability is likely to be influenced by the inflammatory reaction to the implanted sensor. Gene expression analysis is now performed in an existing porcine model of subcutaneous glucose sensor implantation to investigate the time course of inflammation from a new perspective. METHODS Tissue surrounding glucose sensors was sampled to different time points (2 h, 24 h, 3 days, and 7 days) after implantation in the subcutis of pigs. From the tissue RNA was extracted, cDNA was synthesized, and real-time reverse transcription-polymerase chain reaction (RT-PCR) was performed for the quantification of immunoregulatory genes. RESULTS Genes coding for adhesion molecules, chemokines, cytokines, CD markers, and antigen presentation molecules were differentially expressed over time. Most of the investigated genes were significantly up-regulated 24 h and 7 days after implantation. CONCLUSIONS The present study demonstrated that the technology for targeted multiple-gene expression by real-time RT-PCR is useful in the evaluation of the immune response to subcutaneously implanted glucose sensors and that the expression levels also seemed to correspond to the histomorphological observations over time.

CD4+ T cell depletion changes the cytokine environment from a TH1/TH2 response to a TC17-like response in a murine model of atopic dermatitis.

Atopic dermatitis (AD) is a common inflammatory skin disease often associated with co-morbidities including allergic hypersensitivity. We have studied induced AD-like disease in NC/Nga mice using the hapten FITC. Following FITC-treatment the NC/Nga mice develop AD-like skin lesions in regard to the histopathological and immunological changes. Consistent with AD in humans the number of CD4(+) T cells within the blood and draining lymph nodes increases considerably. To evaluate the contribution of T(H) cells on disease development we examined the effect of CD4 depletion. Following CD4 depletion the mice still develop AD-like skin lesions characterized by e.g. increased epidermal proliferation, hyperkeratosis and cellular infiltrate, however, the underlying immunological mechanisms change. CD4 depletion results in increased IL-17A and IL-22 production, which traditionally are associated with T(H)17 cells. Using confocal microscopy, we demonstrate that epidermal CD8(+) cells are positive for IL-17A, indicating that these cells are T(C)17 cells, the cytotoxic T cell counterpart to T(H)17 cells. In conclusion, we show that NC/Nga mice develop AD-like disease following CD4 depletion. This is mirrored by an increased production of IL-17A, which we suggest are produced by T(C)17 cells. These findings support that CD8(+) T cells can play a role in AD.