The dynamics of endothelial changes in vivo following standardized skin injury.

BACKGROUND: Vascular changes, that is, functional and cellular endothelial changes, are of essential importance in healing. Alongside these vascular changes, inflammatory factors and hypoxia may play an important role in recovery. OBJECTIVES: To investigate the dynamics of functional and cellular endothelial changes and hypoxia in vivo following standardized skin damage caused by tape stripping. METHODS: Vascular changes (endothelial cell proliferation, vascular network size, vessel diameter) and hypoxia-inducible factor-1α were examined immunohistochemically using a CD31/Ki67 double staining and HIF-1α single staining. Cutaneous perfusion was evaluated using the Twente Optical Perfusion Camera (TOPCam). RESULTS: The initial phase is seen to be dominated by endothelial cell proliferation, HIF-1α expression, and vasodilatation. Cutaneous perfusion intensity is particularly increased in the first 16 hours. The late phase of recovery (after 72 hours) is characterized by a peak of expansion of the vascular network and a second peak of endothelial cell proliferation and HIF-1α expression. CONCLUSION: Endothelial cell proliferation and HIF-1α expression appear to be (strongly) related, having maximum levels at 16 hours and 72 hours. Angiogenesis and HIF-1α expression are not continuous processes, but rather occur intermittently.

Response of the Endothelium to the Epicutaneous Application of Leukotriene B4.

BACKGROUND: Vascular changes, both endothelial and functional, are crucial events in inflammatory responses. OBJECTIVES: To investigate the dynamics of endothelial cell (EC) and functional changes during acute inflammation in an in vivo model of the skin using leukotriene B4. METHODS: EC proliferation, vascular network size, vessel diameter (VD), and hypoxia-inducible factor (HIF)-1α were studied by immunohistochemical CD31/Ki67 double staining and single staining of HIF-1α. Cutaneous perfusion (CP) was assessed using the Twente Optical Perfusion Camera. RESULTS: The initial phase illustrated an increase in VD, Ki67+ EC, and HIF-1α expression and late-phase vascular expansion. The HIF-1α and Ki67+ EC expression was limited. CP and VD were augmented after 24 h. CONCLUSION: The early phase of inflammation is characterized by EC proliferation and HIF-1α expression. Vascular expansion continues over time. CP and VD are seen in both phases of inflammation. Angiogenesis, vascular network formation, and perfusion are time-dependent processes which are mutually related during inflammation.

Balance of Treg versus T-effector cells during systemic treatment with adalimumab and topical treatment with calcipotriol-betamethasone dipropionate ointment.

Diminished suppressive capacity of regulatory T cells (Treg) has been demonstrated in blood and in lesional skin of psoriatic patients. Treatment with anti-TNFα restored the number and function of circulating Treg in psoriasis. We aimed to study Treg in the skin of psoriatic patients undergoing topical treatment with calcipotriol-betamethasone dipropionate (CBD) ointment (n = 12) or systemic treatment with anti-TNFα agent adalimumab (n = 10). Skin biopsies were collected from patients with chronic plaque psoriasis who responded to the above-mentioned treatments with a SUM-score improvement of at least 50% (at the end of treatment). Biopsies were processed for immunohistochemistry. As Treg function is associated with a numerical balance between Treg and effector T cells, Foxp3/CD4 ratios were calculated. It appeared that both treatments cause a significant decrease in the presence of Foxp3+ cells. However, in patients that were treated with CBD ointment, we observed lower Foxp3/CD4 ratios after 8 weeks of treatment compared to baseline (t = 0: 0.41 ± 0.08; t = 8: 0.22 ± 0.04, P = 0.033), whereas in patients who were treated with adalimumab we observed an increase of the Foxp3/CD4 ratios after 1.5 and 16 weeks of treatment compared to baseline (t = 0: 0.25 ± 0.04; t = 1.5: 0.32 ± 0.06; t = 16: 0.49 ± 0.10, P = 0.15). Based on Foxp3/CD4 ratios, we can conclude that adalimumab treated skin differs from CBD treated skin with regard to the anti-inflammatory/inflammatory balance. We suggest that, in contrast to CBD ointment, adalimumab favours local Treg function in the skin.

Cellular sources of IL-17 in psoriasis: a paradigm shift?

Psoriasis is a common chronic inflammatory skin disease that results from interplay between the immune system and the epithelium. In the light of very successful anticytokine therapies for psoriasis, the focus has been directed towards the adaptive immune system. Expression studies, genetic studies and treatments specifically targeting players of the IL-23/IL-17 pathway, point at an important role for IL-17 in the pathogenesis of psoriasis. IL-17 stimulates the keratinocytes to produce psoriasis-associated molecules, eventually leading to chronic skin inflammation. The current opinion is that IL-17 is mainly produced by T cells, so-called T-helper 17 (Th17) cells, in psoriasis. However, evidence is accumulating that cells of the innate immune system, like neutrophils, mast cells, γδ T cells and innate lymphoid cells are the main source of IL-17 in psoriasis, rather than T cells. The paradigm in this field of research is shifting. With this viewpoint article, we will address this novel concept by critically summarizing the current literature on this subject. In psoriatic arthritis and atherosclerosis, important conditions related to psoriasis, it was also found that the majority of IL-17 is associated with cells of the innate immune system. This new concept changes our view of IL-17. Blocking IL-17 with targeted treatments might be more far-reaching than previously thought; not only IL-17 production by T cells but also by innate immune cells is blocked. Furthermore, therapies specifically targeting IL-17 may not only improve psoriasis, but also comorbidity that is associated with the IL-17 pathway, hereby preventing serious complications on the long term.

Are newly discovered drivers of immune-mediated skin disorders expressed in normal skin regenerating from standardized surface injury?

BACKGROUND: In healthy skin, tape stripping induces a transient wave of histological changes resembling immune-mediated skin diseases, such as psoriasis. The response to surface trauma may harbor mechanisms which are also relevant to the development of Koebner-positive skin disorders. However, studies on newly discovered drivers of inflammation in regenerative skin are lacking. METHODS: The course of epidermal proliferation and keratinization as well as key representatives of innate and acquired immunity were studied during the first 72 h after tape stripping. RESULTS: Epidermal rupture rapidly activates various epidermal processes, which remain upregulated for 72 h. Elastase+ and IL-17+ cells dominate the acute phase and their numbers decrease rapidly thereafter. The number of T-Bet+ cells increases more gradually, reaching maximum levels several hours later when the other cell types decrease. CONCLUSIONS: This model permits investigations on the sequence of crucial inflammatory processes set off by cutaneous injury, which are presumed to play a role within the pathogenesis of immune-mediated skin diseases exhibiting the Koebner phenomenon.

Impairment in work and activities of daily life in patients with psoriasis: results of the prospective BioCAPTURE registry.

Background: Little is known about the extent of impairments in work and activities of daily life (ADL) in patients with psoriasis, and the influence of contextual factors such as disease-related characteristics and treatment. Therefore, this study aimed to assess these impairments in patients with psoriasis who started using biologicals/small molecule inhibitors.Methods: Using data from the prospective BioCAPTURE registry, we collected patient, disease, and treatment parameters, as well as work/ADL impairments at baseline, 6 and 12 months. Changes in impairment parameters and correlations between impairment and patient/disease characteristics were assessed using generalized estimating equations.Results: We included 194 patients in our analysis. After biological initiation, disease activity decreased significantly (PASI 11.2 at baseline versus 3.9 at 12 months, p < 0.001). Work-for-pay in this cohort was lower than in the Dutch general population (53% versus 67%, p = 0.01). In patients who had work-for-pay, presenteeism improved over time (5% at baseline versus 0% at 12 months, p = 0.04). Up to half of the patients reported impairments in ADL, which did not change over time. Associations between impairments and contextual factors varied, but all impairments were associated with worse mental/physical general functioning.Conclusion: Patients with psoriasis using biologicals are less likely to have work-for-pay. Treatment improves the work productivity of employed patients, but we were unable to detect changes in ADL performance.

Lymphocyte Isolation from Human Skin for Phenotypic Analysis and Ex Vivo Cell Culture.

Human skin has an important barrier function and contains various immune cells that contribute to tissue homeostasis and protection from pathogens. As the skin is relatively easy to access, it provides an ideal platform to study peripheral immune regulatory mechanisms. Immune resident cells in healthy skin conduct immunosurveillance, but also play an important role in the development of inflammatory skin disorders, such as psoriasis. Despite emerging insights, our understanding of the biology underlying various inflammatory skin diseases is still limited. There is a need for good quality (single) cell populations isolated from biopsied skin samples. So far, isolation procedures have been seriously hampered by a lack of obtaining a sufficient number of viable cells. Isolation and subsequent analysis have also been affected by the loss of immune cell lineage markers, due to the mechanical and chemical stress caused by the current dissociation procedures to obtain single cell suspension. Here, we describe a modified method to isolate T cells from both healthy and involved psoriatic human skin by combining mechanical skin dissociation using an automated tissue dissociator and collagenase treatment. This methodology preserves expression of most immune lineage markers such as CD4, CD8, Foxp3 and CD11c upon the preparation of single cell suspensions. Examples of successful CD4(+) T cell isolation and subsequent phenotypic and functional analysis are shown.

Targeting PKC in human T cells using sotrastaurin (AEB071) preserves regulatory T cells and prevents IL-17 production.

Regulatory T-cells (Treg) are crucial for immune homeostasis and prevention of immune pathology. Yet, Treg may lose Foxp3 and start secreting IL-17, dependent on environmental cues. Our previous data revealed that Treg from severe psoriasis patients are particularly prone to such conversion. The question of how to maintain Treg stability in the context of inflammation awaits immediate resolution. The pan-protein kinase C (PKC) inhibitor sotrastaurin has shown efficacy in clinical trials of psoriasis. Here, we show that sotrastaurin inhibited effector T-cell responses, whereas the regulatory response was enhanced. Sotrastaurin prevented TCR/CD28-induced T-cell activation and pro-inflammatory cytokine production, but preserved a stable Treg phenotype as evidenced by maintenance of suppressive capacity, high Foxp3 and CD25 expression, and lack of IL-17A and IFNγ production. Moreover, in both circulating and dermal psoriatic Treg, prone to rapid induction of IL-17, sotrastaurin enhanced Foxp3 expression and prevented IL-17A and IFNγ production even when stimulated in the presence of the helper T 17-enhancing cytokines IL-1ß or IL-23. Thus, pharmacological inhibition of PKC may serve as a powerful tool to concurrently inhibit effector T cells and to facilitate Treg, thereby showing therapeutic potential for the treatment of psoriasis.

In vivo induction of cutaneous inflammation results in the accumulation of extracellular trap-forming neutrophils expressing RORγt and IL-17.

Clinical trials successfully using antibodies targeting IL-17 in psoriasis support the importance of IL-17 in the pathophysiology of this disease. However, there is a debate concerning the source and dynamics of IL-17 production in inflamed skin. Here we characterized IL-17-producing immune cells over time, using two established in vivo models of human skin inflammation that share many histological features with psoriasis, i.e., leukotriene B4 application and tape-stripping. Both treatments revealed a clear influx of neutrophils and T cells. Staining for IL-17 revealed that the majority of IL-17 was expressed by neutrophils and mast cells, in both models. Neutrophils, but not mast cells, coexpressed the IL-17-associated transcription factor RORγt and were able to form extracellular traps. While the presence of mast cells remained steady during the skin inflammatory process, the presence of neutrophils was clearly dynamic in time. Therefore, it is attractive to hypothesize that IL-17+/RORγt+ neutrophils contribute to human skin inflammation in vivo and possibly to the pathogenesis of skin diseases such as psoriasis. Surprisingly, T cells represented a minority of the IL-17-expressing cell population. These observations challenge the classical opinion that IL-17 is predominantly associated with T cells in skin inflammation.

Humanized mouse model of skin inflammation is characterized by disturbed keratinocyte differentiation and influx of IL-17A producing T cells.

Humanized mouse models offer a challenging possibility to study human cell function in vivo. In the huPBL-SCID-huSkin allograft model human skin is transplanted onto immunodeficient mice and allowed to heal. Thereafter allogeneic human peripheral blood mononuclear cells are infused intra peritoneally to induce T cell mediated inflammation and microvessel destruction of the human skin. This model has great potential for in vivo study of human immune cells in (skin) inflammatory processes and for preclinical screening of systemically administered immunomodulating agents. Here we studied the inflammatory skin response of human keratinocytes and human T cells and the concomitant systemic human T cell response.As new findings in the inflamed human skin of the huPBL-SCID-huSkin model we here identified: 1. Parameters of dermal pathology that enable precise quantification of the local skin inflammatory response exemplified by acanthosis, increased expression of human ß-defensin-2, Elafin, K16, Ki67 and reduced expression of K10 by microscopy and immunohistochemistry. 2. Induction of human cytokines and chemokines using quantitative real-time PCR. 3. Influx of inflammation associated IL-17A-producing human CD4+ and CD8+ T cells as well as immunoregulatory CD4+Foxp3+ cells using immunohistochemistry and -fluorescence, suggesting that active immune regulation is taking place locally in the inflamed skin. 4. Systemic responses that revealed activated and proliferating human CD4+ and CD8+ T cells that acquired homing marker expression of CD62L and CLA. Finally, we demonstrated the value of the newly identified parameters by showing significant changes upon systemic treatment with the T cell inhibitory agents cyclosporine-A and rapamycin. In summary, here we equipped the huPBL-SCID-huSkin humanized mouse model with relevant tools not only to quantify the inflammatory dermal response, but also to monitor the peripheral immune status. This combined approach will gain our understanding of the dermal immunopathology in humans and benefit the development of novel therapeutics for controlling inflammatory skin diseases.

Cutaneous complication after BCG vaccination: case report and review of the literature.

The bacille Calmette-Guérin (BCG) vaccination protects against tuberculosis (TB)-related meningitis and disseminated tuberculosis. While severe complications after BCG vaccination are relatively rare, different cutaneous reactions have been reported. We report a patient with a 7-mm erythematous nodule at a distance of 4 cm from the BCG injection site. Histopathologically, a necrotizing granulomatous reaction pattern was seen in the dermis. Although complementary stainings did not detect acid fast bacilli, we suspected the lesion was caused by the attenuated strains of Mycobacterium bovis from the vaccine. This specific complication is called BCG-itis in the literature. After the excisional biopsy, the lesion disappeared and further treatment was not necessary. In defining the best treatment for this boy, we discovered a lack of knowledge on BCG-related lesions and their subsequent treatment options in the literature. We will list existing literature on this topic and demonstrate that treatment of BCG-related complications is poorly defined.