Mary Sullivan-Whalen

Th17 cells and activated dendritic cells are increased in vitiligo lesions.

Background Vitiligo is a common skin disorder, characterized by progressive skin de-pigmentation due to the loss of cutaneous melanocytes. The exact cause of melanocyte loss remains unclear, but a large number of observations have pointed to the important role of cellular immunity in vitiligo pathogenesis. Methodology/Principal Findings In this study, we characterized T cell and inflammation-related dermal dendritic cell (DC) subsets in pigmented non-lesional, leading edge and depigmented lesional vitiligo skin. By immunohistochemistry staining, we observed enhanced populations of CD11c+ myeloid dermal DCs and CD207+ Langerhans cells in leading edge vitiligo biopsies. DC-LAMP+ and CD1c+ sub-populations of dermal DCs expanded significantly in leading edge and lesional vitiligo skin. We also detected elevated tissue mRNA levels of IL-17A in leading edge skin biopsies of vitiligo patients, as well as IL-17A positive T cells by immunohistochemistry and immunofluorescence. Langerhans cells with activated inflammasomes were also noted in lesional vitiligo skin, along with increased IL-1ß mRNA, which suggest the potential of Langerhans cells to drive Th17 activation in vitiligo. Conclusions/Significance These studies provided direct tissue evidence that implicates active Th17 cells in vitiligo skin lesions. We characterized new cellular immune elements, in the active margins of vitiligo lesions (e.g. populations of epidermal and dermal dendritic cells subsets), which could potentially drive the inflammatory responses.

Severe atopic dermatitis is characterized by selective expansion of circulating TH2/TC2 and TH22/TC22, but not TH17/TC17, cells within the skin-homing T-cell population

BACKGROUND Past studies of blood T-cell phenotyping in patients with atopic dermatitis (AD) have provided controversial results and were mostly performed before the identification of TH9, TH17, and TH22 T-cell populations in human subjects. OBJECTIVE We sought to quantify TH1, TH2, TH9, TH17, and TH22 T-cell populations and corresponding CD8(+) T-cell subsets in both cutaneous lymphocyte antigen (CLA)-positive and CLA(-) T-cell subsets in patients with AD and control subjects. METHODS We studied 42 adults with severe AD (mean SCORAD score, 65) and 25 healthy subjects using an 11-color flow cytometric antibody panel. Frequencies of IFN-γ-, IL-22-, IL-13-, IL-17-, and IL-9-producing CD4(+) and CD8(+) T cells were compared in CLA(-) and CLA(+) populations. RESULTS We measured increased TH2/TC2/IL-13(+) and TH22/TC22/IL-22(+) populations (P < .1) in patients with severe AD versus control subjects, with significant differences in CLA(+) T-cell numbers (P < .01). A significantly lower frequency of CLA(+) IFN-γ-producing cells was observed in patients with AD, with no significant differences in CLA(-) T-cell numbers. The CLA(+) TH1/TH2 and TC1/TC2 ratio was highly imbalanced in patients with AD (10 vs 3 [P = .005] and 19 vs 7 [P < .001], respectively). Positive correlations were found between frequencies of IL-13- and IL-22-producing CD4(+) and CD8(+) T cells (r = 0.5 and 0.8, respectively; P < .0001), and frequencies of IL-13-producing CLA(+) cells were also correlated with IgE levels and SCORAD scores. Patients with AD with skin infections had higher CD4(+) IL-22(+) and IL-17(+) cell frequencies, which were highly significant among CLA(-) cells (IL-22: 3.7 vs 1.7 [P < .001] and IL-17: 1.7 vs 0.6 [P < .001]), with less significant effects among CLA(+) T cells (IL-22: 11 vs 7.5, P = .04). CONCLUSIONS Severe AD is accompanied by expansion of skin-homing TH2/TC2 and TH22/TC22 subsets with lower TH1/TC1 frequencies. These data create a critical basis for studying alterations in immune activation in adults and pediatric patients with AD.

Cyclosporine in patients with atopic dermatitis modulates activated inflammatory pathways and reverses epidermal pathology.

BACKGROUND Atopic dermatitis (AD) is the most common inflammatory disease. Evolving disease models link changes in epidermal growth and differentiation to T(H)2/T(H)22 cytokine activation. However, these models have not been tested by in vivo suppression of T-cell cytokines. Cyclosporine (CsA) is an immunosuppressant that is highly effective for severe disease, but its mechanism in AD skin lesions has not been studied. OBJECTIVE We sought to establish the ability of a systemic immunosuppressant to modulate immune and epidermal alterations that form the pathogenic disease phenotype and to correlate changes with clinical improvement. METHODS CsAs effects on AD skin pathology were evaluated by using gene expression and immunohistochemistry studies in baseline, week 2, and week 12 lesional and nonlesional biopsy specimens from 19 patients treated with 5 mg/kg/d CsA for 12 weeks. RESULTS After 2 and 12 weeks of treatment, we observed significant reductions of 51% and 72%, respectively, in SCORAD scores. Clinical improvements were associated with significant gene expression changes in lesional but also nonlesional skin, particularly reductions in levels of T(H)2-, T(H)22-, and some T(H)17-related molecules (ie, IL-13, IL-22, CCL17, S100As, and elafin/peptidase inhibitor 3), and modulation of epidermal hyperplasia and differentiation measures. CONCLUSIONS This is the first study that establishes a relationship between cytokine activation and molecular epidermal alterations, as well as correlations between disease biomarkers in the skin and clinical improvement. The reversal of the molecular phenotype with CsA and the associated biomarkers can serve as a reference for the successful modulation of tissue inflammation with specific immune antagonists in future studies, contributing to the understanding of the specific cytokines involved in epidermal pathology.

Human Keratinocytes' Response to Injury Upregulates CCL20 and Other Genes Linking Innate and Adaptive Immunity

In the early stages of wound healing, keratinocytes become “activated” and release inflammatory molecules such as interleukin-1 and interleukin-8 that are linked to innate immune responses and neutrophil recruitment. It is unclear, however, whether keratinocytes release molecules linked to adaptive immune responses, e.g. CCL20, in their early state of activation without signals from infiltrating T cells. This study aims to isolate the immediate alterations in protective and inflammatory gene expression that occur in epidermal keratinocytes, with a particular focus on molecules associated with cell-mediated immunity. We used dispase-separated epidermis, followed by intercellular disassociation by trypsinization, as a model for epidermal injury. We obtained a pure population of keratinocytes using flow cytometry. As a control for uninjured epidermis, we performed laser capture microdissection on normal human skin. Sorted keratinocytes had an early burst of upregulated gene expression, which included CCL20, IL-15, IL-23A, IFN-κ, and several antimicrobial peptides. Our results provide insight into the potential role of keratinocytes as contributors to cell-mediated inflammation, and expand knowledge about gene modulation that occurs during early wound healing. Our findings may be relevant to cutaneous diseases such as psoriasis, where micro-injury can trigger the formation of psoriatic plaques at the site of trauma.

Efficacy and safety of ustekinumab treatment in adults with moderate-to-severe atopic dermatitis.

Atopic dermatitis (AD) is the most common inflammatory skin disease, but treatment options for moderate‐to‐severe disease are limited. Ustekinumab is an IL‐12/IL‐23p40 antagonist that suppresses Th1, Th17 and Th22 activation, commonly used for psoriasis patients. We sought to assess efficacy and safety of ustekinumab in patients with moderate‐to‐severe AD. In this phase II, double‐blind, placebo‐controlled study, 33 patients with moderate‐to‐severe AD were randomly assigned to either ustekinumab (n=16) or placebo (n=17), with subsequent crossover at 16 weeks, and last dose at 32 weeks. Background therapy with mild topical steroids was allowed to promote retention. Study endpoints included clinical (SCORAD50) and biopsy‐based measures of tissue structure and inflammation, using protein and gene expression studies. The ustekinumab group achieved higher SCORAD50 responses at 12, 16 (the primary endpoint) and 20 weeks compared to placebo, but the difference between groups was not significant. The AD molecular profile/transcriptome showed early robust gene modulation, with sustained further improvements until 32 weeks in the initial ustekinumab group. Distinct and more robust modulation of Th1, Th17 and Th22 but also Th2‐related AD genes was seen after 4 weeks of ustekinumab treatment (i.e. MMP12, IL‐22, IL‐13, IFN‐γ, elafin/PI3, CXCL1 and CCL17; P<.05). Epidermal responses (K16, terminal differentiation) showed faster (4 weeks) and long‐term regulation (32 weeks) from baseline in the ustekinumab group. No severe adverse events were observed. Ustekinumab had clear clinical and molecular effects, but clinical outcomes might have been obscured by a profound “placebo” effect, most likely due to background topical glucocorticosteroids and possibly insufficient dosing for AD.

A Single Intradermal Injection of IFN-γ Induces an Inflammatory State in Both Non-Lesional Psoriatic and Healthy Skin

Psoriasis is a chronic, debilitating, immune-mediated inflammatory skin disease. As IFN-γ is involved in many cellular processes, including activation of dendritic cells (DCs), antigen processing and presentation, cell adhesion and trafficking, and cytokine and chemokine production, IFN-γ-producing Th1 cells were proposed to be integral to the pathogenesis of psoriasis. Recently, IFN-γ was shown to enhance IL-23 and IL-1 production by DCs and subsequently induce Th17 cells, important contributors to the inflammatory cascade in psoriasis lesions. To determine if IFN-γ indeed induces the pathways expressed in psoriasis lesions, a single intradermal injection of IFN-γ was administered to an area of clinically normal, non-lesional skin of psoriasis patients and biopsies were collected 24 hours later. Although there were no visible changes in the skin, IFN-γ induced many molecular and histological features characteristic of psoriasis lesions. IFN-γ increased a number of differentially expressed genes in the skin, including many chemokines concomitant with an influx of T cells and inflammatory DCs. Furthermore, inflammatory DC products TNF, iNOS, IL-23, and TRAIL were present in IFN-γ-treated skin. Thus, IFN-γ, which is significantly elevated in non-lesional skin compared to healthy skin, appears to be a key pathogenic cytokine that can induce many features of the inflammatory cascade of psoriasis.

Alefacept (anti-CD2) causes a selective reduction in circulating effector memory T cells (Tem) and relative preservation of central memory T cells (Tcm) in psoriasis

BackgroundAlefacept (anti-CD2) biological therapy selectively targets effector memory T cells (Tem) in psoriasis vulgaris, a model Type 1 autoimmune disease.MethodsCirculating leukocytes were phenotyped in patients receiving alefacept for moderate to severe psoriasis.ResultsIn all patients, this treatment caused a preferential decrease in effector memory T cells (CCR7- CD45RA-) (mean 63% reduction) for both CD4+ and CD8+ Tem, while central memory T cells (Tcm) (CCR7+CD45RA-) were less affected, and naïve T cells (CCR7+CD45RA+) were relatively spared. Circulating CD8+ effector T cells and Type 1 T cells (IFN-γ-producing) were also significantly reduced.ConclusionAlefacept causes a selective reduction in circulating effector memory T cells (Tem) and relative preservation of central memory T cells (Tcm) in psoriasis.

Molecular Characterization of Human Skin Response to Diphencyprone at Peak and Resolution Phases: Therapeutic Insights

Diphencyprone (DPCP) is a hapten that induces delayed-type hypersensitivity (DTH) reactions. It is used as an immune modulating therapeutic, but its molecular effects in human skin are largely unknown. We studied cellular and molecular characteristics of a recall response to 0.04% DPCP at 3 day (peak) and 14 day (resolution) timepoints using immune markers, RT-PCR and gene array approaches. A peak response showed modulation of ~7,500 mRNA transcripts, with high expression of cytokines that define all major effector T-cell subsets. Concomitant increases in T-cell and CD11c+ dendritic cell (DC) infiltrates were measured. The resolution reaction was characterized by unexpectedly high levels of T-cells and mature (DC-LAMP+) DCs, but with marked decreases in expression of IL-2, IFNγ, and other T-cell derived cytokines. However, negative immune regulators such as IDO1 that were high in peak reactions, continued to have high expression in resolution reactions. In the resolution reaction, ~1,500 mRNA transcripts were significantly different from placebo-treated skin. These data suggest the response to DPCP evolves from an inflammatory/effector peak at day 3 to a more regulated immune response after 14 days. This model system could be useful for further dissection of mechanisms of immune activation or negative immune regulation in human skin.

Gene Profiling of Narrowband UVB–Induced Skin Injury Defines Cellular and Molecular Innate Immune Responses

The acute response of human skin to UVB radiation has not been fully characterized. We sought to define the cutaneous response at 24 hours following narrowband UVB (NB-UVB, 312-nm peak), a therapeutically relevant source of UVB, using transcriptional profiling, immunohistochemistry, and immunofluorescence. There were 1,522 unique differentially regulated genes, including upregulated genes encoding antimicrobial peptides (AMPs) (S100A7, S100A12, human beta-defensin 2, and elafin), as well as neutrophil and monocyte/dendritic cell (DC) chemoattractants (IL-8, CXCL1, CCL20, CCL2). Ingenuity pathway analysis demonstrated activation of innate defense and early adaptive immune pathways. Immunohistochemistry confirmed increased epidermal staining for AMPs (S100A7, S100A12, human beta-defensin 2, and elafin). Inflammatory myeloid CD11c(+)BDCA1(-) DCs were increased in irradiated skin, which were immature as shown by minimal colocalization with DC-LAMP, and coexpressed inflammatory markers tumor necrosis factor (TNF) and TNF-related apoptosis-inducing ligand in irradiated skin. There were increased BDCA3(+) DCs, a cross-presenting DC subtype with immunosuppressive functions, and these cells have not been previously characterized as part of the response to UVB. These results show that the acute response of human skin to erythemogenic doses of NB-UVB includes activation of innate defense mechanisms, as well as early infiltration of multiple subtypes of inflammatory DCs, which could serve as a link between innate and adaptive immunity.

Post-therapeutic relapse of psoriasis after CD11a blockade is associated with T cells and inflammatory myeloid DCs.

To understand the development of new psoriasis lesions, we studied a group of moderate-to-severe psoriasis patients who experienced a relapse after ceasing efalizumab (anti-CD11a, Raptiva, Genentech). There were increased CD3+ T cells, neutrophils, CD11c+ and CD83+ myeloid dendritic cells (DCs), but no increase in CD1c+ resident myeloid DCs. In relapsed lesions, there were many CD11c+CD1c−, inflammatory myeloid DCs identified by TNFSF10/TRAIL, TNF, and iNOS. CD11c+ cells in relapsed lesions co-expressed CD14 and CD16 in situ. Efalizumab induced an improvement in many psoriasis genes, and during relapse, the majority of these genes reversed back to a lesional state. Gene Set Enrichment Analysis (GSEA) of the transcriptome of relapsed tissue showed that many of the gene sets known to be present in psoriasis were also highly enriched in relapse. Hence, on ceasing efalizumab, T cells and myeloid cells rapidly enter the skin to cause classic psoriasis. Trial registration Clinicaltrials.gov NCT00115076