Nicholas Gulati

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.

Common clonal origin of central and resident memory T cells following skin immunization

Central memory T (TCM) cells in lymph nodes (LNs) and resident memory T (TRM) cells in peripheral tissues have distinct roles in protective immunity. Both are generated after primary infections, but their clonal origins have been unclear. To address this question, we immunized mice through the skin with a protein antigen, a chemical hapten, or a non-replicating poxvirus. We then analyzed antigen-activated T cells from different tissues using high-throughput sequencing (HTS) of the gene encoding the T cell receptor (TCR) β-chain (Trb, also known as Tcrb) using CDR3 sequences to simultaneously track thousands of unique T cells. For every abundant TRM cell clone generated in the skin, an abundant TCM cell clone bearing the identical TCR was present in the LNs. Thus, antigen-reactive skin TRM and LN TCM cell clones were derived from a common naive T cell precursor after skin immunization, generating overlapping TCR repertoires. Although they bore the same TCR, TRM cells mediated rapid contact hypersensitivity responses, whereas TCM cells mediated delayed and attenuated responses. Studies in human subjects confirmed the generation of skin TRM cells in allergic contact dermatitis. Thus, immunization through skin simultaneously generates skin TRM and LN TCM cells in similar numbers from the same naive T cells.

IL-17 Induces an Expanded Range of Downstream Genes in Reconstituted Human Epidermis Model

Background IL-17 is the defining cytokine of the Th17, Tc17, and γδ T cell populations that plays a critical role in mediating inflammation and autoimmunity. Psoriasis vulgaris is an inflammatory skin disease mediated by Th1 and Th17 cytokines with relevant contributions of IFN-γ, TNF-α, and IL-17. Despite the pivotal role IL-17 plays in psoriasis, and in contrast to the other key mediators involved in the psoriasis cytokine cascade that are capable of inducing broad effects on keratinocytes, IL-17 was demonstrated to regulate the expression of a limited number of genes in monolayer keratinocytes cultured in vitro. Methodology/Principal Findings Given the clinical efficacy of anti-IL-17 agents is associated with an impressive reduction in a large set of inflammatory genes, we sought a full-thickness skin model that more closely resemble in vivo epidermal architecture. Using a reconstructed human epidermis (RHE), IL-17 was able to upregulate 419 gene probes and downregulate 216 gene probes. As possible explanation for the increased gene induction in the RHE model is that C/CAAT-enhancer-binding proteins (C/EBP) -β, the transcription factor regulating IL-17-responsive genes, is expressed preferentially in differentiated keratinocytes. Conclusions/Significance The genes identified in IL-17-treated RHE are likely relevant to the IL-17 effects in psoriasis, since ixekizumab (anti-IL-17A agent) strongly suppressed the “RHE” genes in psoriasis patients treated in vivo with this IL-17 antagonist.

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.

TREM-1 as a Potential Therapeutic Target in Psoriasis

Our group recently described a population of antigen presenting cells that appear to be critical in psoriasis pathogenesis, termed inflammatory myeloid dendritic cells (CD11c+ BDCA1−). Triggering receptor expressed on myeloid cells type-1 (TREM-1) Signaling was a major canonical pathway in the published transcriptome of these cells. TREM-1 is a member of the immunoglobulin superfamily, active through the DAP12 signaling pathway, with an unknown ligand. Activation through TREM-1 induces inflammatory cytokines including IL-8, MCP/CCL2 and TNF. We now show that TREM-1 was expressed in the skin of healthy and psoriatic patients, and there was increased soluble TREM-1 in the circulation of psoriasis patients. In psoriasis lesions, TREM-1 was co-localized with dendritic cells as well as CD31+ endothelial cells. TREM-1 expression was reduced with successful NB-UVB, etanercept and anti-IL-17 treatments. An in vitro model of PGN-activated monocytes as inflammatory myeloid DCs was developed to study TREM-1 blockade, and treatment with a TREM-1 blocking chimera decreased allogeneic Th17 activation as well as IL-17 production. Furthermore, TREM-1 blockade of ex vivo psoriatic dendritic cells in an alloMLR also showed a decrease in IL-17. Together, these data suggest that the TREM-1 signaling pathway may be a previously unidentified therapeutic target to prevent the effects of inflammatory myeloid DCs in psoriasis.

Comparative genomic profiling of synovium versus skin lesions in psoriatic arthritis.

To our knowledge, there is no broad genomic analysis comparing skin and synovium in psoriatic arthritis (PsA). Also, there is little understanding of the relative levels of cytokines and chemokines in skin and synovium. The purpose of this study was to better define inflammatory pathways in paired lesional skin and affected synovial tissue in patients with PsA.

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 Expression Profiling of the Leading Edge of Cutaneous Squamous Cell Carcinoma: IL-24-Driven MMP-7

The precise mechanisms governing invasion at the leading edge of SCC and its subsequent metastasis are not fully understood. We aimed to define the cancer related molecular changes that distinguish non-invasive tumor from invasive SCC. To this end, we combined laser capture microdissection with cDNA microarray analysis. We defined invasion-associated genes as those differentially regulated only in invasive SCC nests, but not in actinic keratosis or in situ SCC, compared to normal epidermis. There were 383 up- and 354 down-regulated genes in the “invasion set.” SCC invasion was characterized by aberrant expression of various proteolytic molecules. We noted increased expression of MMP7 and IL-24 in invasive SCC. IL-24 induced the expression of MMP7 in SCC cells in culture. In addition, blocking of MMP7 by a specific antibody significantly delayed the migration of SCC cells in culture. These results suggest a possible contribution of IL-24 to SCC invasion via enhancing focal expression of MMP7, though IL-24 has been suggested to have anti-tumor growth effects in other cancer types. Identification of regional molecular changes that regulate cancer invasion may facilitate the development of new targeted treatments for aggressive cancer.

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.

Deficient cutaneous antibacterial competence in cutaneous T-cell lymphomas: role of Th2-mediated biased Th17-function

Purpose: Primary cutaneous T-cell lymphomas (CTCL) are neoplastic disorders of skin-homing T cells. Affected skin areas show morphologic similarities with alterations in other T-cell–mediated dermatoses. Furthermore, as in atopic dermatitis but in contrast with psoriasis, patients with CTCL are frequently afflicted by cutaneous bacterial infections that support the survival of lymphoma cells. Our aim was to investigate the mechanisms of elevated susceptibility to cutaneous infections in patients with CTCL. Experimental Design: Skin samples from CTCL, psoriasis, and atopic dermatitis patients were used to illuminate the antibacterial competence status and the presence of its modulating cytokines. For substantiation of findings, 3-dimensional epidermis models, isolated and in vitro generated Th-subpopulations, were applied. Parameters were analyzed via qPCR and IHC. Results: CTCL lesions compared with psoriatic lesions presented an impaired upregulation of antibacterial proteins (ABPs), with levels even below those in atopic dermatitis. This was associated with a relative deficiency of the ABP-inducing cytokine IL-17 and a strong presence of the ABP-downregulating cytokine IL-13. The simultaneous presence of the Th17-cell cytokine IL-26 indicated that IL-17 deficiency in CTCL lesions results from functional deviation of Th17 cells. Accordingly, IL-17 but not IL-26 production by Th17 cells in vitro was inhibited by IL-4Rα ligand. Levels of other ABP inducers were comparable between CTCL and psoriasis lesions. The same was true about IL-22/TNF-α targets, including the keratinocyte hyper-regeneration marker K16 and the matrix-degrading enzyme MMP1. Conclusion: Our results suggest that the cutaneous bacterial infections in CTCL are caused by impaired ABP induction as consequence of Th2-mediated biased Th17-cell function. Clin Cancer Res; 20(21); 5507–16. ©2014 AACR.