Elevated NK-cell transcriptional signature and dysbalance of resting and activated NK cells in atopic dermatitis.

BACKGROUND: Altered quantities, activity, and composition of natural killer (NK) cells in blood as well as expression changes of genes involved in NK-cell function in skin lesions of patients with atopic dermatitis (AD) were recently reported. OBJECTIVES: We sought to comprehensively analyze cutaneous NK-cell transcriptomic signatures in AD, and to examine changes under treatment. METHODS: We analyzed NK-cell signatures in skin transcriptome data from 57 patients with moderate to severe AD and 31 healthy controls. In addition, changes after 12 weeks of systemic treatment (dupilumab n = 21, cyclosporine n = 8) were analyzed. Deconvolution of leucocyte fractions was conducted. Immunofluorescence staining of NK cells was performed on paraffin-embedded skin sections. RESULTS: Immunofluorescence staining revealed a relatively high abundance of both NK cells and CD3+CD56+ cells in lesional as compared with nonlesional and healthy skin. Lesional and to a lesser extent nonlesional skin showed a strong upregulation of NK-cell markers together with a dysbalanced expression of inhibitory and activating receptors, which was not reverted under treatment. Digital cytometry showed a decrease in activated and an increase in resting NK cells in both lesional and nonlesional skin, which was reverted by both treatment with dupilumab and cyclosporine. The NK-cell transcriptomic signature remained upregulated after treatment, but there was a shift on the qualitative level, indicating a compositional change in NK-cell subsets toward CD56bright NK cells. CONCLUSIONS: Lesional AD skin shows a NK-cell dysregulation, which despite clinical improvement under systemic therapy was only partially reverted, and which may represent a yet underappreciated disease mechanism.

Controllers of cutaneous regulatory T cells: ultraviolet radiation and the skin microbiome.

For the maintenance of homeostasis termination of immune reactions is as equally important as their induction. In this scenario regulatory T cells (Treg) play an important role. Accordingly a variety of inflammatory diseases are caused by an impairment of Treg. Hence, it is important to identify triggers by which Treg can be induced and activated, respectively. For quite a long time it is known that ultraviolet radiation can induce Treg which inhibit cutaneous immune reactions including contact hypersensitivity. Since these Treg inhibit in an antigen-specific fashion they may harbor therapeutic potential. However similar Treg can be induced also by other triggers which include vitamin D and antimicrobial peptides. Recently it was discovered that the gut microbiome controls the development of Treg in the intestine. The same may apply for the skin. Short chain fatty acids, microbiota-derived bacterial fermentation products, appear to induce and to activate Treg in the skin. Topical application of short chain fatty acids was shown to inhibit contact hypersensitivity and to reduce inflammation in the murine imiquimod-induced psoriasis-like skin inflammation model. Together, these data indicate that induction and activation of Treg may be a potential therapeutic strategy to treat inflammatory diseases in the future.

Reevaluation of Lung Injury in TNF-Induced Shock: The Role of the Acid Sphingomyelinase.

Tumor necrosis factor (TNF) is a well-known mediator of sepsis. In many cases, sepsis results in multiple organ injury including the lung with acute respiratory distress syndrome (ARDS). More than 20-year-old studies have suggested that TNF may be directly responsible for organ injury during sepsis. However, these old studies are inconclusive, because they relied on human rather than conspecific TNF, which was contaminated with endotoxin in most studies. In this study, we characterized the direct effects of intravenous murine endotoxin-free TNF on cardiovascular functions and organ injury in mice with a particular focus on the lungs. Because of the relevance of the acid sphingomyelinase in sepsis, ARDS, and caspase-independent cell death, we also included acid sphingomyelinase-deficient (ASM-/-) mice. ASM-/- and wild-type (WT) mice received 50 µg endotoxin-free murine TNF intravenously alone or in combination with the pan-caspase inhibitor carbobenzoxy-valyl-alanyl-aspartyl-[O-methyl]-fluoromethylketone (zVAD) and were ventilated at low tidal volume while lung mechanics were followed. Blood pressure was stabilized by intra-arterial fluid support, and body temperature was kept at 37°C to delay lethal shock and to allow investigation of blood gases, lung histopathology, proinflammatory mediators, and microvascular permeability 6 hours after TNF application. Besides the lungs, also the kidneys and liver were examined. TNF elicited the release of inflammatory mediators and a high mortality rate, but failed to injure the lungs, kidneys, or liver of healthy mice significantly within 6 hours. Mortality in WT mice was most likely due to sepsis-like shock, as indicated by metabolic acidosis, high procalcitonin levels, and cardiovascular failure. ASM-/- mice were protected from TNF-induced hypotension and reflex tachycardia and also from mortality. In WT mice, intravenous exogenous TNF does not cause organ injury but induces a systemic inflammatory response with cardiovascular failure, in which the ASM plays a role.

Nrf2 establishes a glutathione-mediated gradient of UVB cytoprotection in the epidermis.

Ultraviolet (UV) B irradiation can severely damage the skin and even induce tumorigenesis. It exerts its effects by direct DNA modification and by formation of reactive oxygen species (ROS). We developed a strategy to genetically activate target gene expression of the transcription factor NF-E2-related factor 2 (Nrf2) in keratinocytes in vivo based on expression of a constitutively active Nrf2 mutant. Activation of Nrf2 target genes strongly reduced UVB cytotoxicity through enhancement of ROS detoxification. Remarkably, the protective effect was extended to neighboring cells. Using different combinations of genetically modified mice, we demonstrate that Nrf2 activates the production, recycling, and release of glutathione and cysteine by suprabasal keratinocytes, resulting in protection of basal cells in a paracrine, glutathione/cysteine-dependent manner. Most importantly, we found that endogenous Nrf2 controls selective protection of suprabasal keratinocytes from UVB-induced apoptosis through activation of cytoprotective genes. This finding explains the preferential UVB-induced apoptosis of basal cells, which is important for elimination of mutated stem cells as well as for preservation of skin integrity. Taken together, our results identify Nrf2 as a key regulator in the UV response of the skin.

Infrared A radiation promotes survival of human melanocytes carrying ultraviolet radiation-induced DNA damage.

The link between solar radiation and melanoma is still elusive. Although infrared radiation (IR) accounts for over 50% of terrestrial solar energy, its influence on human skin is not well explored. There is increasing evidence that IR influences the expression patterns of several molecules independently of heat. A previous in vivo study revealed that pretreatment with IR might promote the development of UVR-induced non-epithelial skin cancer and possibly of melanoma in mice. To expand on this, the aim of the present study was to evaluate the impact of IR on UVR-induced apoptosis and DNA repair in normal human epidermal melanocytes. The balance between these two effects is a key factor of malignant transformation. Human melanocytes were exposed to physiologic doses of IR and UVR. Compared to cells irradiated with UVR only, simultaneous exposure to IR significantly reduced the apoptotic rate. However, IR did not influence the repair of UVR-induced DNA damage. IR partly reversed the pro-apoptotic effects of UVR via modification of the expression and activity of proteins mainly of the extrinsic apoptotic pathway. In conclusion, IR enhances the survival of melanocytes carrying UVR-induced DNA damage and thereby might contribute to melanomagenesis.

Induction of regulatory T cells by a murine ß-defensin.

ß-Defensins are antimicrobial peptides of the innate immune system produced in the skin by various stimuli, including proinflammatory cytokines, bacterial infection, and exposure to UV radiation (UVR). In this study we demonstrate that the UVR-inducible antimicrobial peptide murine ß-defensin-14 (mBD-14) switches CD4(+)CD25(-) T cells into a regulatory phenotype by inducing the expression of specific markers like Foxp3 and CTLA-4. This is functionally relevant because mBD-14-treated T cells inhibit sensitization upon adoptive transfer into naive C57BL/6 mice. Accordingly, injection of mBD-14, comparable to UVR, suppresses the induction of contact hypersensitivity and induces Ag-specific regulatory T cells (Tregs). Further evidence for the ability of mBD-14 to induce Foxp3(+) T cells is provided using DEREG (depletion of Tregs) mice in which Foxp3-expressing cells can be depleted by injecting diphtheria toxin. mBD-14 does not suppress sensitization in IL-10 knockout mice, suggesting involvement of IL-10 in mBD-14-mediated immunosuppression. However, unlike UVR, mBD-14 does not appear to mediate its immunosuppressive effects by affecting dendritic cells. Accordingly, UVR-induced immunosuppression is not abrogated in mBD-14 knockout mice. Together, these data suggest that mBD-14, like UVR, has the capacity to induce Tregs but does not appear to play a major role in UVR-induced immunosuppression. Through this capacity, mBD-14 may protect the host from microbial attacks on the one hand, but tame T cell-driven reactions on the other hand, thereby enabling an antimicrobial defense without collateral damage by the adaptive immune system.

Role of fibroblasts in the pathogenesis of atopic dermatitis.

BACKGROUND: Atopic dermatitis (AD) is a common dermatosis that highly impairs a patient's quality of life. The recent discovery that epidermal barrier defects caused by an aberrant differentiation process of keratinocytes are comparably important to the well-characterized changes in immune response patterns attributed a crucial role to the keratinocytes. Fibroblasts are able to alter proliferation and differentiation of keratinocytes, but their role in AD is not yet fully understood. OBJECTIVE: We sought to determine the role of fibroblasts in skin proliferation and differentiation in patients with AD. METHODS: We used human 3-dimensional organotypic skin cultures consisting of atopic fibroblasts and healthy keratinocytes, as well as healthy fibroblasts and atopic keratinocytes, and compared them with their controls. The expression of differentiation markers in these organotypic cultures were analyzed by using immunohistology and quantitative RT-PCR. Furthermore, the fundamental role of fibroblast-secreted leukemia inhibitory factor was assessed by using small interfering RNA-mediated knockdown cultures. RESULTS: We observed that atopic fibroblasts influence the proliferation of keratinocytes and the terminal differentiation process, resulting in an in vivo-like morphology of AD. Subsequently, healthy fibroblasts were able to restore the structural deficits of the epidermis consisting of atopic keratinocytes. Partially, these effects were due to a reduced expression of the differentiation-associated cytokine leukemia inhibitory factor by atopic fibroblasts. CONCLUSION: These data demonstrate that fibroblasts and the modulation of fibroblast-derived factors might be new therapeutic targets for the alleviation of AD.

Ultraviolet radiation-induced upregulation of antimicrobial proteins in health and disease.

This article reviews recent data on the expression, regulation and activation of antimicrobial peptides (AMP) in human skin, and considers their potential protective and pro-inflammatory roles following upregulation by ultraviolet radiation (UVR). Antimicrobial peptides are small peptides that are key components of the innate immune system, originally identified by their vital role in protecting the body-environment interface from infection. However, it has now become clear that AMP have more extensive actions, including the provision of pivotal links with the adaptive immune system. Moreover, aberrant AMP expression may contribute to immuno-modulated inflammatory dermatoses including psoriasis, eczema and the photoaggravated condition lupus erythematosus. Recent work has demonstrated the direct upregulation of AMP in healthy skin by cutaneous UVR exposure. This may serve to protect the skin from risks imposed by both the biophysical barrier-compromise and the immunosuppression that are attributable to UVR exposure. Furthermore, it is observed that UVR provokes upregulation of AMP in an atypical manner in the photosensitivity disorder polymorphic light eruption. Dysregulated UVR responses of these pro-inflammatory proteins may play a role in the pathogenesis of certain immune-mediated diseases caused or aggravated by sunlight.

Mouse Models of Allergic Contact Dermatitis: Practical Aspects.

Allergic contact dermatitis is a frequently observed dermatosis, especially in industrialized countries. Regarded as a classical type IV immune reaction (delayed type), the process can be separated into two pathogenetic parts: the induction phase where sensitization takes place and the elicitation phase in which inflammation is induced upon re-exposure to the same antigen. A murine model was established decades ago, which reliably reproduces both phases. Epicutaneously applied low-molecular-weight sensitizers bind to proteins (haptens) and become full antigens, which results in sensitization. Subsequent administration of the same hapten onto ear skin causes a swelling response. This reaction is antigen specific because it cannot be induced in nonsensitized mice or in sensitized mice with a different hapten. This model was used to study the mechanisms involved in allergic contact dermatitis and also was intensively utilized to study immunologic mechanisms, including antigen presentation and development of T effector or regulatory T cells. The model's major merit is its antigen specificity. It is highly reproducible, reliable, and simple to perform. In this paper, the methods of this technique are described to help researchers successfully establish this widely used model in laboratories. Describing the complex pathomechanisms underlying the model is beyond the scope of this article.

Crosstalk between microbiome, regulatory T cells and HCA2 orchestrates the inflammatory response in a murine psoriasis model.

The organ-specific microbiome plays a crucial role in tissue homeostasis, among other things by inducing regulatory T cells (Treg). This applies also to the skin and in this setting short chain fatty acids (SCFA) are relevant. It was demonstrated that topical application of SCFA controls the inflammatory response in the psoriasis-like imiquimod (IMQ)-induced murine skin inflammation model. Since SCFA signal via HCA2, a G-protein coupled receptor, and HCA2 expression is reduced in human lesional psoriatic skin, we studied the effect of HCA2 in this model. HCA2 knock-out (HCA2-KO) mice reacted to IMQ with stronger inflammation, presumably due to an impaired function of Treg. Surprisingly, injection of Treg from HCA2-KO mice even enhanced the IMQ reaction, suggesting that in the absence of HCA2 Treg switch from a suppressive into a proinflammatory type. HCA2-KO mice differed in the composition of the skin microbiome from wild type mice. Co-housing reversed the exaggerated response to IMQ and prevented the alteration of Treg, implying that the microbiome dictates the outcome of the inflammatory reaction. The switch of Treg into a proinflammatory type in HCA2-KO mice could be a downstream phenomenon. This opens the opportunity to reduce the inflammatory tendency in psoriasis by altering the skin microbiome.

In vivo reprogramming of UV radiation-induced regulatory T-cell migration to inhibit the elicitation of contact hypersensitivity.

BACKGROUND: Regulatory T (Treg) cells induced by UV radiation (UVR) inhibit only the induction and not the elicitation of contact hypersensitivity (CHS) because they migrate into the lymph nodes but not the skin. The tissue-homing receptor expression and migratory behavior of Treg cells can be altered by means of in vitro coincubation with skin-derived antigen-presenting cells. On this in vitro treatment, Treg cells migrate into the skin and thus inhibit the elicitation of CHS. OBJECTIVE: We attempted to determine whether Treg cells can be induced by UVR in sensitized mice and manipulated entirely in vivo in such a way that they suppress the elicitation of immune responses. METHODS: Treg cells were induced by applying contact allergens onto UV-exposed skin in wild-type, langerin diphtheria toxin receptor knock-in, or depletion of Treg cell transgenic mice. RESULTS: UVR-induced Treg cells inhibit the elicitation of CHS in sensitized mice when stimulated by means of an antigen-specific boost through the skin. This requires cutaneous antigen-presenting cells that alter the migratory behavior of Treg cells and drive them out of the lymph nodes into the skin. CONCLUSIONS: The indication is that antigen-specific Treg cells can be induced in sensitized hosts and manipulated in such a way that they suppress the elicitation of specific immune reactions. Because this is achieved entirely in vivo without invasive interventions, our findings might have important implications for strategies aiming to induce and use Treg cells in a therapeutic setting.

Prevention of UV radiation-induced immunosuppression by IL-12 is dependent on DNA repair.

The immunostimulatory cytokine IL-12 is able to antagonize immunosuppression induced by solar/ultraviolet (UV) radiation via yet unknown mechanisms. IL-12 was recently found to induce deoxyribonucleic acid (DNA) repair. UV-induced DNA damage is an important molecular trigger for UV-mediated immunosuppression. Thus, we initiated studies into immune restoration by IL-12 to discern whether its effects are linked to DNA repair. IL-12 prevented both UV-induced suppression of the induction of contact hypersensitivity and the depletion of Langerhans cells, the primary APC of the skin, in wild-type but not in DNA repair-deficient mice. IL-12 did not prevent the development of UV-induced regulatory T cells in DNA repair-deficient mice. In contrast, IL-12 was able to break established UV-induced tolerance and inhibited the activity of regulatory T cells independent of DNA repair. These data identify a new mechanism by which IL-12 can restore immune responses and also demonstrate a link between DNA repair and the prevention of UV-induced immunosuppression by IL-12.

Infrared radiation does not enhance the frequency of ultraviolet radiation-induced skin tumors, but their growth behaviour in mice.

There is increasing concern about the interaction between infrared radiation (IR) and ultraviolet radiation (UVR) with regard to carcinogenesis because prolonged solar exposure is associated with an increased cumulative load not only of UVR but also of IR. We recently demonstrated that IR-pretreatment reduces UVR-induced apoptosis. As this might support the survival of UVR-damaged cells and thus carcinogenesis, we performed an in vivo photocarcinogenesis study. One group of mice were treated with IR prior to each UVR exposure; additional groups were treated with IR or UVR alone. IR alone did not induce skin cancer. UVR-induced tumor formation was not enhanced in IR-pretreated mice, but, in contrast, seemed to occur with delay. This correlated with a reduction of p53 mutated clones in the skin. However, once developed, tumors in IR-pretreated mice grew faster which was confirmed by their enhanced Ki-67 expression. The enhanced aggressiveness of tumors derived from IR-pretreated mice was associated with a higher prevalence of sarcomas than epithelial tumors. Hence, the impact of IR on UVR-induced carcinogenesis has to be interpreted with caution. Although IR may delay the onset of UVR-induced tumors, it might contribute to a worse outcome by shifting these tumors into a more aggressive phenotype.

Interleukin-12 suppresses ultraviolet radiation-induced apoptosis by inducing DNA repair.

Induction of apoptosis of keratinocytes by ultraviolet (UV) radiation is a protective phenomenon relevant in limiting the survival of cells with irreparable DNA damage. Changes in UV-induced apoptosis may therefore have significant impact on photocarcinogenesis. We have found that the immunomodulatory cytokine IL-12 suppresses UV-mediated apoptosis of keratinocytes both in vitro and in vivo. IL-12 caused a remarkable reduction in UV-specific DNA lesions which was due to induction of DNA repair. In accordance with this, IL-12 induced the expression of particular components of the nucleotide-excision repair complex. Our results show that cytokines can protect cells from apoptosis induced by DNA-damaging UV radiation by inducing DNA repair, and that nucleotide-excision repair can be manipulated by cytokines.

Induction of Regulatory T Cells and Correction of Cytokine Disbalance by Short-Chain Fatty Acids: Implications for Psoriasis Therapy.

Commensal microbes modulate the immune system in the colon through short-chain fatty acids, which induce regulatory T cells (Treg). Accordingly, the short-chain fatty acid sodium butyrate (SB) suppressed allergic contact dermatitis in mice through the activation of Treg. There is evidence that Treg exert the capacity to control inflammation in psoriasis. Thus, we were interested in studying the effect of SB in psoriasis, utilizing the imiquimod-induced psoriasis-like skin inflammation model. Topical application of imiquimod induced thickening of the skin, scales, and inflammation. This was associated with an upregulation of IL-17 and downregulation of IL-10 and FOXP3. Topically applied SB reduced imiquimod-induced inflammation and downregulated IL-17 and induced IL-10 and FOXP3 transcripts. The mitigating effect of SB was due to Treg because it was lost upon depletion of Treg in the depletion of regulatory T cell mice. Treg isolated from the blood of patients with psoriasis were reduced in their suppressive activity, which was normalized by SB. The fewer Treg numbers in the biopsies of psoriatic lesions as well as enhanced IL-17- and IL-6-expression levels and reduced IL-10- and FOXP3-expression levels were restored by SB. These data indicate that psoriasis is associated with an impairment of Treg and an altered cytokine milieu. Short-chain fatty acids appear to restore these alterations, thereby harboring a therapeutic potential for psoriasis.

Experimental extracorporeal photopheresis inhibits the sensitization and effector phases of contact hypersensitivity via two mechanisms: generation of IL-10 and induction of regulatory T cells.

Extracorporeal photopheresis (ECP) is used to treat immune-mediated diseases including transplant rejection and graft-vs-host-disease. An experimental murine model of ECP utilizing contact hypersensitivity (CHS) revealed that ECP inhibits the sensitization of CHS and induces regulatory T cells (Treg). In this study, we find that ECP inhibits not only the sensitization but also the effector phase of CHS, although Treg only inhibited sensitization. IL-10 was determined to be a critical component of the effector phase inhibition and also a driving force in developing Treg. Thus, we propose that the inhibition of the effector phase of CHS by ECP is a process that does not require Treg but may be mediated via enhanced IL-10 as suggested by the use of IL-10-deficient mice. This suggests that ECP has at least two mechanisms of action, one inhibiting the effector phase of CHS and one generating Treg, which in turn can inhibit CHS sensitization and is responsible for the transferable protection. Together, this may help explain the clinical benefits of ECP in prophylactic, acute, and therapeutic settings.

UV-B radiation induces the expression of antimicrobial peptides in human keratinocytes in vitro and in vivo.

BACKGROUND: Suppression of the adaptive immune system by UV radiation plays an important role in photocarcinogenesis. Exacerbation of skin infections has been proposed as a further consequence of UV-induced immunosuppression. Clinically bacterial infections are not a problem. For defense against bacteria, the innate immune response including the release of antimicrobial peptides is much more relevant than the adaptive immune response. Keratinocytes have the capacity to release antimicrobial peptides. OBJECTIVE: We asked whether UV radiation induces antimicrobial peptides in vitro and in vivo. METHODS: Antimicrobial peptide expression by normal human keratinocytes was measured by real-time PCR and fluorescence-activated cell sorting analysis. Biopsies taken from human volunteers and skin explants were studied with immunohistochemistry. RESULTS: Real-time PCR of normal human keratinocytes revealed a dose-dependent increase of human beta-defensin-2, -3, ribonuclease 7, and psoriasin (S100A7) after UV radiation. This was confirmed at the protein level by intracellular fluorescence-activated cell sorting and in vitro immunofluorescence analysis. Immunohistochemistry of biopsies taken from healthy volunteers exposed to different UV radiation doses revealed enhanced epidermal expression of antimicrobial peptides after UV exposure. This was also confirmed by exposing human skin explants to UV radiation. CONCLUSION: UV radiation exerts diverse effects on the immune system, suppressing the adaptive but inducing the innate immune response. This may explain why T-cell-mediated immune reactions are suppressed on UV exposure but not host defense reactions against bacterial attacks.

Correction to: The AHR represses nucleotide excision repair and apoptosis and contributes to UV-induced skin carcinogenesis.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

DNA repair and cytokine responses.

As sunscreens do not provide complete protection against solar/UV radiation, alternative protective strategies are necessary to cope with the increasing incidence of skin cancer. These strategies include the reduction of UVR-induced DNA damage by the topical application of bacterial DNA repair enzymes. Recent evidence suggests that nucleotide excision repair, the physiological repair system that is mostly responsible for the removal of UVR-mediated DNA damage, can be modulated by cytokines, including IL-12, IL-18, and alpha-melanocyte-stimulating hormone. The mechanisms involved and the biological as well as the potential therapeutic implications of these findings are discussed.Journal of Investigative Dermatology Symposium Proceedings (2009) 14, 63-66; doi:10.1038/jidsymp.2009.3.

Green tea phenol extracts reduce UVB-induced DNA damage in human cells via interleukin-12.

Green tea chemoprevention has been a focus of recent research, as a polyphenolic fraction from green tea (GTP) has been suggested to prevent UV radiation-induced skin cancer. Recently, it was demonstrated that GTP reduced the risk for skin cancer in a murine photocarcinogenesis model. This was accompanied by a reduction in UV-induced DNA damage. These effects appeared to be mediated via interleukin (IL)-12, which was previously shown to induce DNA repair. Therefore, we studied whether GTP induction of IL-12 and DNA repair could also be observed in human cells. KB cells and normal human keratinocytes were exposed to GTP 5 h before and after UVB. UVB-induced apoptosis was reduced in UVB-exposed cells treated with GTP. GTP induced the secretion of IL-12 in keratinocytes. The reduction in UV-induced cell death by GTP was almost completely reversed upon addition of an anti-IL-12-antibody, indicating that the reduction of UV-induced cell death by GTP is mediated via IL-12. The ability of IL-12 to reduce DNA damage and sunburn cells was confirmed in "human living skin equivalent" models. Hence the previously reported UV-protective effects of GTP appear to be mediated in human cells via IL-12, most likely through induction of DNA repair.