Pathophysiology Of Inflammatory Skin Diseases Research Articles

Maximizing the Utility of Transcriptomics Data in Inflammatory Skin Diseases.

Inflammatory skin diseases are induced by disorders of the host defense system of the skin, which is composed of a barrier, innate and acquired immunity, as well as the cutaneous microbiome. These disorders are characterized by recurrent cutaneous lesions and intense itch, which seriously affecting life quality of people across all ages and ethnicities. To elucidate molecular factors for typical inflammatory skin diseases (such as psoriasis and atopic dermatitis), transcriptomic profiling assays have been largely performed. Additionally, single-cell RNA sequencing (scRNA-seq) as well as spatial transcriptomic profiling have revealed multiple potential translational targets and offered guides to improve diagnosis and treatment strategies for inflammatory skin diseases. High-throughput transcriptomics data has shown unprecedented power to disclose the complex pathophysiology of inflammatory skin diseases. Here, we will summarize discoveries from transcriptomics data and discuss how to maximize the transcriptomics data to propel the development of diagnostic biomarkers and therapeutic targets in inflammatory skin diseases.

Recapitulating T cell infiltration in 3D psoriatic skin models for patient-specific drug testing

Drug screening studies for inflammatory skin diseases are currently performed using model systems that only partially recapitulate human diseased skin. Here, we developed a new strategy to incorporate T cells into human 3D skin constructs (HSCs), which enabled us to closely monitor and quantitate T cell responses. We found that the epidermis promotes the activation and infiltration of T cells into the skin, and provides a directional cue for their selective migration towards the epidermis. We established a psoriatic HSC (pHSC) by incorporating polarized Th1/Th17 cells or CCR6+CLA+ T cells derived from psoriasis patients into the constructs. These pHSCs showed a psoriatic epidermal phenotype and characteristic cytokine profiles, and responded to various classes of psoriasis drugs, highlighting the potential utility of our model as a drug screening platform. Taken together, we developed an advanced immunocompetent 3D skin model to investigate epidermal-T cell interactions and to understand the pathophysiology of inflammatory skin diseases in a human-relevant and patient-specific context.

Oral electrophilic nitro-fatty acids inhibit cutaneous immune responses

Abstract Allergic contact dermatitis (ACD) is a common inflammatory skin disease affecting as many as 15–20% of the population. ACD results from a delayed-type hypersensitivity reaction occurring in the skin upon re-exposure to the offending hapten and mediated by the activation of hapten-specific T cells. An increasing body of literature demonstrates that electrophilic nitro-fatty acids (NO2-FAs) exert potent anti-inflammatory actions by modulating metabolic and inflammatory pathways. Therefore, we evaluated the potential regulatory effects of NO2-FAs on ACD by a well-established model of contact hypersensitivity in mice utilizing 2,4-dinitrofluorobenzene (DNFB) as the hapten. Contrary to our hypothesis, topical pretreatment of nitro-oleic acid (OA-NO2) prior to DNFB application potentiated ACD. Given that ACD is mediated by adaptive immunity, topical administration of OA-NO2 may have an adverse effect on keratinocytes thereby limiting OA-NO2’s potential to systemically suppress the adaptive immune response. Therefore, we next compared the effect of oral OA-NO2 on ACD. In contrast to topical administration, oral OA-NO2 dose-dependently suppressed ACD. Moreover, oral OA-NO2 downregulated the production of cutaneous inflammatory cytokines. Interestingly, in vitro experiments demonstrate that OA-NO2 leads to a dose-dependent inhibition of keratinocyte proliferation and expression of IL-1β, TNF-α, and IL-6. Overall, these data emphasize the complexity of the cutaneous microenvironment. A better understanding of the immunoregulatory effects of NO2-FAs in the skin microenvironment will contribute to therapeutics and to our understanding of the pathophysiology of inflammatory skin diseases, such as ACD and psoriasis.

Higher Expression of Toll-like Receptors 3, 7, 8, and 9 in Pityriasis Rosea.

BackgroundPityriasis rosea (PR) is a common papulosquamous skin disease in which an infective agent may be implicated. Toll-like receptors (TLRs) play an important role in immune responses and in the pathophysiology of inflammatory skin diseases. Our aim was to determine the possible roles of TLRs 3, 7, 8, and 9 in the pathogenesis of PR.MethodsTwenty-four PR patients and 24 healthy individuals (as controls) were included in this case control study. All recruits were subjected to routine laboratory investigations. Biopsies were obtained from one active PR lesion and from healthy skin of controls for the detection of TLR 3, 7, 8, and 9 gene expression using real-time polymerase chain reaction.ResultsThis study included 24 patients (8 females and 16 males) with active PR lesions, with a mean age of 28.62 years. Twenty four healthy age- and sex-matched individuals were included as controls (8 females and 16 males, with a mean age of 30.83 years). The results of the routine laboratory tests revealed no significant differences between both groups. Significantly elevated expression of all studied TLRs were detected in PR patients relative to healthy controls (p < .001).ConclusionsTLRs 3, 7, 8, and 9 might be involved in the pathogenesis of PR.

From morphology to biochemical state - intravital multiphoton fluorescence lifetime imaging of inflamed human skin.

The application of multiphoton microscopy in the field of biomedical research and advanced diagnostics promises unique insights into the pathophysiology of inflammatory skin diseases. In the present study, we combined multiphoton-based intravital tomography (MPT) and fluorescence lifetime imaging (MPT-FLIM) within the scope of a clinical trial of atopic dermatitis with the aim of providing personalised data on the aetiopathology of inflammation in a non-invasive manner at patients’ bedsides. These ‘optical biopsies’ generated via MPT were morphologically analysed and aligned with classical skin histology. Because of its subcellular resolution, MPT provided evidence of a redistribution of mitochondria in keratinocytes, indicating an altered cellular metabolism. Two independent morphometric algorithms reliably showed an even distribution in healthy skin and a perinuclear accumulation in inflamed skin. Moreover, using MPT-FLIM, detection of the onset and progression of inflammatory processes could be achieved. In conclusion, the change in the distribution of mitochondria upon inflammation and the verification of an altered cellular metabolism facilitate a better understanding of inflammatory skin diseases and may permit early diagnosis and therapy.

Expression of IL-33 in the epidermis: The mechanism of induction by IL-17

BackgroundInterleukin (IL)-33 is a dual functional, IL-1 family member cytokine, whose exact roles in inflammatory skin diseases are still unknown. IL-17A is a key cytokine in the pathogenesis of psoriasis. ObjectivesWe investigated if IL-17A could induce IL-33 in epidermal keratinocytes, and the signaling mechanisms involved. MethodsIL-33 levels were evaluated by RT-PCR and western blot in human keratinocytes following IL-17A simulation. IL-33 immunohistochemical staining of psoriatic skin samples was also performed and compared with that of control tissues. The role of signaling pathways downstream of IL-17A was investigated using small molecule inhibitors of EGFR, ERK, p38, and JAK. Adenovirus vector expressing dominant negative STAT1 was also utilized. ResultsIL-33 and its receptor, ST2L, were expressed in the psoriatic epidermis, and the associated infiltrating cells. IL-17A induced IL-33 expression at mRNA and protein levels in a time- and concentration-dependent manner. IL-17A caused phosphorylation of EGFR, ERK, p38, and STAT1. IL-17A-induced IL-33 expression was blocked by the addition of EGFR, ERK, p38, and JAK inhibitors, and dominant negative STAT1-expressing adenovirus vector. ConclusionIL-17A induced IL-33 in NHEKs through EGFR, ERK, p38, and JAK/STAT1 pathways, which were necessary for the induction of IL-33. IL-33, induced by IL-17A in epidermal keratinocytes, may be involved in the pathophysiology of inflammatory skin diseases, including psoriasis.

Human Beta-Defensin 3 Induces Maturation of Human Langerhans Cell–Like Dendritic Cells: An Antimicrobial Peptide that Functions as an Endogenous Adjuvant

Human beta defensins (hBDs) are antimicrobial peptides that play an important role in innate immune responses at epithelial barriers such as the skin. However, the role that hBDs play in initiating cellular immune responses that contribute to antigen-specific adaptive immunity is not well understood. Here we show that one member of the hBD family, hBD3, can induce maturation and T helper type 1 (Th1) skewing function in human Langerhans cell-like DCs (LC-DCs). Specifically, hBD3 potently induces phenotypic maturation of LC-DCs, including increased expression of CCR7 which mediates functional chemotactic responses to CCL19 and CCL21. HBD3-stimulated LC-DCs induce strong proliferation and IFN-γ secretion by naïve human T cells. HBD3 also induces phenotypic maturation of primary human skin-migratory dendritic cells derived from human skin explants. These results suggest an important role for hBD3 in inducing DC activation, migration, and polarization. Thus hBD3 contributes to the integration of innate and adaptive immune responses in the skin and may be a useful adjuvant for skin immunization and an important factor in the pathophysiology of inflammatory skin diseases.

Co-culture of healthy human keratinocytes and T-cells promotes keratinocyte chemokine production and RORγt-positive IL-17 producing T-cell populations

Both keratinocytes and T-cells are crucial players in cutaneous immune responses. We hypothesized that direct interactions between keratinocytes and T-cell subsets could shape the nature or strength of the local immune response. We investigated direct interactions between keratinocytes and T-cell subsets, focused on keratinocyte chemokine production and T-cell phenotype and cytokine production. A newly developed in vitro serum free co-culture model using primary keratinocytes and T-cells subsets from healthy human donors was used. Keratinocyte chemokine production was analyzed with luminex, T-cell phenotype and cytokine production were analyzed with flow cytometry. Our data show that upon co-culture with CD4(pos) or CD8(pos) T-cells primary human keratinocytes increased production of functionally active chemokines CCL2, CCL20 and CXCL10 and that regulatory T-cells did not regulate keratinocyte chemokine production. Next to that, we found that keratinocytes skewed CD4(pos) and CD8(pos) T-cell populations toward an IL-17(pos) CCR6(pos) RORγt(pos) phenotype in a cell-cell contact independent manner, and that Treg were able to decrease the absolute number of IL-17 producing T-cells in keratinocyte/T-cell co-cultures. Correspondingly, freshly isolated skin-derived T-cell populations contained relatively high percentages of IL-17(pos) cells. We provide evidence that keratinocyte/T-cell communication may regulate leukocyte influx in the skin, and that keratinocytes enrich T-cell populations for Th17/Tc17 cells. Accumulation of Th17/Tc17 cells, but not chemokine production, appears under the control of regulatory T-cells. Dysregulation of these processes may well contribute to the pathophysiology of inflammatory skin diseases.

Epidermal Proteases in the Pathogenesis of Rosacea

A number of different proteases and their inhibitors have a role in skin physiology and in the pathophysiology of inflammatory skin diseases. Proteases are important in the desquamation process and orderly regulation of the skin's barrier function. On the basis of the catalytic domain, proteases are classified into aspartate-, cysteine-, glutamate-, metallo-, serine-, and threonine proteases. Particularly, serine proteases (SPs) contribute to epidermal permeability barrier homeostasis, as acute barrier disruption increases SP activity in skin and inhibition by topical SP inhibitors accelerated recovery of barrier function after acute abrogation. In rosacea, increased levels of the vasoactive and inflammatory host-defense peptide cathelicidin LL-37 and its proteolytic peptide fragments were found, which were explained by an abnormal production of tryptic activity originating from kallikrein-related peptidase (KLK) 5. It is therefore possible that also other proteases, even from microbial or parasite origin, have a role in rosacea by forming alternate angiogenic and proinflammatory cathelicidin peptides. Further, the regulation of protease activity, in particular KLK-5 activity, might have a role in rosacea. This review briefly summarizes our current knowledge about keratinocyte-derived proteases and protease inhibitors, which might have a role in the pathophysiology of rosacea.

Galanin-Like Peptides Exert Potent Vasoactive Functions In Vivo

The cutaneous vasculature plays a key role in the pathophysiology of inflammatory skin diseases. The vascular activity is under the control of the peripheral nervous system that includes locally released neuropeptides. Recently, we detected receptors for the neuropeptide galanin in association with dermal blood vessels, suggesting a role of the galanin-peptide-family in the regulation of the cutaneous microvasculature. Therefore, we have investigated galanin and galanin-like peptide (GALP), a neuropeptide previously only considered to be involved in metabolism and reproduction in the central nervous system, for vaso-modulatory activity in the murine skin in vivo. Picomole amounts of intradermally injected galanin and GALP decreased cutaneous blood flow and inhibited inflammatory edema formation. Both the full-length GALP (1-60) and the putative smaller proteolytic fragment GALP (3-32) showed similar effects. These activities are most likely mediated by galanin receptors galanin receptor subtype 2 (GalR2) and/or galanin receptor subtype 3 (GalR3), because reverse transcription-PCR analysis of murine skin revealed messenger RNA (mRNA) expression of GalR2 and GalR3 but not of galanin receptor subtype 1. The lack of galanin receptor mRNAs in endothelial and smooth muscle cells indicates a neuronal localization of these receptors around the vessels. These results indicate functional activity of GALP in the periphery in vivo and suggest a potential role as an inflammatory modulator.

Cytokine and Anti-Cytokine Therapies for Psoriasis and Atopic Dermatitis

Since the discovery of cytokines as key mediators in inflammation, targeting the cytokine network has represented a promising therapeutic approach. Psoriasis and atopic dermatitis, as T cell-mediated diseases with a strong cytokine component and a high unmet medical need, have moved into the focus of experimental therapies. Whereas pro-inflammatory cytokines such as tumor necrosis factor (TNF)-alpha are overexpressed in both diseases, a type 1 cytokine pattern predominates in psoriasis and a type 2 cytokine pattern is of pathophysiological importance at least in the initial stages of atopic dermatitis. Strategies for intervention into the cytokine network have included antagonism of pro-inflammatory cytokines (e.g. TNFalpha, interleukin [IL]-1, IL-8, IL-12, IL-18, IL-23) with neutralizing antibodies and soluble receptors, application of recombinant cytokines (e.g. IL-4, IL-10, IL-11, interferon [IFN]-gamma) to shift the cytokine balance, and administration of small molecules to modulate cytokine expression or signaling. Results from the clinic have led to novel therapeutic options as well as a better understanding of the pathophysiology of inflammatory skin diseases. This review highlights the various therapeutic strategies, results from the clinic (that are in some cases preliminary), and insights that can be drawn from the more advanced clinical studies and the use of approved cytokine-directed therapies.

Transgenic mice over-expressing a serine protease in the skin: evidence of interferon gamma-independent MHC II expression by epidermal keratinocytes.

Stratum corneum chymotryptic enzyme (SCCE; also known as kallikrein 7) is a serine protease that is preferentially expressed in cornifying epithelia and possibly involved in the desquamation process. We have recently described transgenic mice over-expressing human SCCE in the epidermis showing increased epidermal thickness, hyperkeratosis, and an apparent dermal inflammation with pruritus. This suggests that SCCE may be involved in the pathophysiology of inflammatory skin diseases. We therefore carried out a further characterization of the skin changes observed in scce-transgenic mice. An increase in number of dermal cells was verified by stereological measurements showing a more than twofold increase of the volume fraction of dermis occupied by cell nuclei. In some, but not all, animals the number of dermal mast cells was increased. The dermal cell infiltrate was shown to consist mainly of macrophages and granulocytes. The number of epidermal and dermal T-lymphocytes was not increased. Dermal changes were found in transgenic animals before the age they became pruritic. No increase in interferon-gamma expression could be detected in the skin of transgenic animals. In spite of this, keratinocytes of adult transgenic mice were found to express MHC II antigen. We suggest that increased expression and/or activity of epidermal SCCE may lead to skin changes that contribute to development and maintenance of inflammatory skin diseases.

Epidermal Overexpression of Stratum Corneum Chymotryptic Enzyme in Mice: A Model for Chronic Itchy Dermatitis

Identification of tissue-specific mechanisms involved in the pathophysiology of inflammatory skin diseases could offer new possibilities to develop effective therapies with fewer systemic effects. The serine protease stratum corneum chymotryptic enzyme is preferentially expressed in cornifying epithelia. We have previously reported on increased expression of the stratum corneum chymotryptic enzyme in psoriasis. Here is reported an increased epidermal expression of stratum corneum chymotryptic enzyme also found in chronic lesions of atopic dermatitis. Transgenic mice expressing human stratum corneum chymotryptic enzyme in suprabasal epidermal keratinocytes were found to develop pathologic skin changes with increased epidermal thickness, hyperkeratosis, dermal inflammation, and severe pruritus. The results suggest that stratum corneum chymotryptic enzyme may be involved in the pathogenesis of inflammatory skin diseases, and that stratum corneum chymotryptic enzyme and related enzymes should be evaluated as potential targets for new therapies.

12-Hydroxyeicosatetraenoic acid (12-HETE) is a chemotactic stimulus for epidermal cells

Several observations point to an important role of 12HETE and LTB4 in cutaneous physiology and in the pathophysiology of inflammatory skin diseases, in particular psoriasis [13]. Normal epidermal cells possess a high capacity for the transformation of arachidonic acid (AA) into 12-HETE by 12-1ipoxygenase. It is the main eicosanoid which is generated in epidermal homogenates besides PGE2 and P G D 2 [9, 14]. In contrast, the 5lipoxygenase product LTB4 is formed mainly by leukocytes [4] which infiltrate skin during inflammation. In psoriasis, highly elevated concentrations of 12-HETE are found in lesional skin [2, 8]. In addition, 12-HETE and LTB4 are chemoattractants for PMNLs which could contribute to the infiltration of PMNLs in psoriatic skin and other cutaneous inflammatory reactions [3, 6]. Although the metabolic processes which lead to the formation of 12-HETE in skin are well understood, the exact function of this AA-metabolite in cutaneous biology is not clear. Growth stimulation of keratinocytes by 12-HETE was reported by Kragballe and Fallon [11], but has not been confirmed by other groups [7, 12]. Here we report a novel aspect of 12-HETE and LTB 4 in cutaneous biology. We detected a chemotactic response of epidermal cells towards 12-Sand 12-R-HETE as well as LTB4. The response of the cells towards 12-HETE was in the range of the KD of a high affinity 12-HETE receptor described recently by our group [7]. Eicosanoids (12-S-HETE, 12-R-HETE, LTB4) were supplied by Paesel (Frankfurt/Main, FRG) as free acids in ethanol. The ethanol was evaporated under nitrogen immediately before use, and the eicosanoids were dissolved in serum-free DMEM to the required concentration. EGF was purchased from Sebak (Aidenbach, FRG). Fibroblast-conditioned medium (FCM) was pre-