Lisa C. Zaba

Psoriasis Vulgaris Lesions Contain Discrete Populations of Th1 and Th17 T Cells

The importance of T helper 17 (Th17) cells in inflammation and autoimmunity is now being appreciated. We analyzed psoriasis skin lesions and peripheral blood for the presence of IL-17-producing T cells. We localized Th17 cells predominantly to the dermis of psoriasis skin lesions, confirmed that IL-17 mRNA increased with disease activity, and demonstrated that IL-17 mRNA expression normalized with cyclosporine therapy. IL-22 mRNA expression mirrored IL-17 and both were downregulated in parallel with keratin 16. Th17 cells are a discrete population, separate from Th1 cells (which are also in psoriasis lesions), and Th2 cells. Our findings suggest that psoriasis is a mixed Th1 and Th17 inflammatory environment. Th17 cells may be proximal regulators of psoriatic skin inflammation, and warrant further attention as therapeutic targets.

Psoriasis is characterized by accumulation of immunostimulatory and Th1/Th17 cell-polarizing myeloid dendritic cells.

Myeloid dermal dendritic cells (DCs) accumulate in chronically inflamed tissues such as psoriasis. The importance of these cells for psoriasis pathogenesis is suggested by comparative T-cell and DC-cell counts, where DCs outnumber T cells. We have previously identified CD11c(+)-blood dendritic cell antigen (BDCA)-1(+) cells as the main resident dermal DC population found in normal skin. We now show that psoriatic lesional skin has two populations of dermal DCs: (1) CD11c(+)BDCA-1(+) cells, which are phenotypically similar to those contained in normal skin and (2) CD11c(+)BDCA-1(-) cells, which are phenotypically immature and produce inflammatory cytokines. Although BDCA-1(+) DCs are not increased in number in psoriatic lesional skin compared with normal skin, BDCA-1(-) DCs are increased 30-fold. For functional studies, we FACS-sorted psoriatic dermal single-cell suspensions to isolate these two cutaneous DC populations, and cultured them as stimulators in an allogeneic mixed leukocyte reaction. Both BDCA-1(+) and BDCA-1(-) myeloid dermal DC populations induced T-cell proliferation, and polarized T cells to become T helper 1 (Th1) and T helper 17 (Th17) cells. In addition, psoriatic dermal DCs induced a population of activated T cells that simultaneously produced IL-17 and IFN-gamma, which was not induced by normal skin dermal DCs. As psoriasis is believed to be a mixed Th17/Th1 disease, it is possible that induction of these IL-17(+)IFN-gamma(+) cells is pathogenic. These cytokines, the T cells that produce them, and the inducing inflammatory DCs may all be important new therapeutic targets in psoriasis.

Low Expression of the IL-23/Th17 Pathway in Atopic Dermatitis Compared to Psoriasis

The classical Th1/Th2 paradigm previously defining atopic dermatitis (AD) and psoriasis has recently been challenged with the discovery of Th17 T cells that synthesize IL-17 and IL-22. Although it is becoming evident that many Th1 diseases including psoriasis have a strong IL-17 signal, the importance of Th17 T cells in AD is still unclear. We examined and compared skin biopsies from AD and psoriasis patients by gene microarray, RT-PCR, immunohistochemistry, and immunofluorescence. We found a reduced genomic expression of IL-23, IL-17, and IFN-γ in AD compared with psoriasis. To define the effects of IL-17 and IL-22 on keratinocytes, we performed gene array studies with cytokine-treated keratinocytes. We found lipocalin 2 and numerous other innate defense genes to be selectively induced in keratinocytes by IL-17. IFN-γ had no effect on antimicrobial gene-expression in keratinocytes. In AD skin lesions, protein and mRNA expression of lipocalin 2 and other innate defense genes (hBD2, elafin, LL37) were reduced compared with psoriasis. Although AD has been framed by the Th1/Th2 paradigm as a Th2 polar disease, we present evidence that the IL-23/Th17 axis is largely absent, perhaps accounting for recurrent skin infections in this disease.

Normal human dermis contains distinct populations of CD11c+BDCA-1+ dendritic cells and CD163+FXIIIA+ macrophages

We used a panel of monoclonal antibodies to characterize DCs in the dermis of normal human skin. Staining for the CD11c integrin, which is abundant on many kinds of DCs, revealed cells in the upper dermis. These cells were positive for blood DC antigen-1 (BDCA-1; also known as CD1c), HLA-DR, and CD45, markers that are also expressed by circulating myeloid DCs. A small subset of CD11c+ dermal cells expressed DEC-205/CD205 and DC-lysosomal-associated membrane glycoprotein/CD208 (DC-LAMP/CD208), suggesting some differentiation or maturation. When BDCA-1+ cells were selected from collagenase digests of normal dermis, they proved to be strong stimulators for T cells in a mixed leukocyte reaction. A second major population of cells located throughout the dermis was positive for factor XIIIA (FXIIIA), but lacked CD11c and BDCA-1. They expressed the macrophage scavenger receptor CD163 and stained weakly for HLA-DR and CD45. Isolated CD163+ dermal cells were inactive in stimulating T cell proliferation, but in biopsies of tattoos, these cells were selectively laden with granular pigments. Plasmacytoid DCs were also present in the dermis, marked by CD123 and BDCA-2. In summary, the normal dermis contains typical immunostimulatory myeloid DCs identified by CD11c and BDCA-1, as well as an additional population of poorly stimulatory macrophages marked by CD163 and FXIIIA.

Effective treatment of psoriasis with etanercept is linked to suppression of IL-17 signaling, not immediate response TNF genes.

BACKGROUND TNF inhibitors have revolutionized the treatment of psoriasis vulgaris as well as psoriatic and rheumatoid arthritis and Crohn disease. Despite our understanding that these agents block TNF, their complex mechanism of action in disease resolution is still unclear. OBJECTIVE To analyze globally the genomic effects of TNF inhibition in patients with psoriasis, and to compare genomic profiles of patients who responded or did not respond to treatment. METHODS In a clinical trial using etanercept TNF inhibitor to treat psoriasis vulgaris (n = 15), Affymetrix gene arrays were used to analyze gene profiles in lesional skin at multiple time points during drug treatment (baseline and weeks 1, 2, 4, and 12) compared with nonlesional skin. Patients were stratified as responders (n = 11) or nonresponders (n = 4) on the basis of histologic disease resolution. Cluster analysis was used to define gene sets that were modulated with similar magnitude and velocity over time. RESULTS In responders, 4 clusters of downregulated genes and 3 clusters of upregulated genes were identified. Genes downmodulated most rapidly reflected direct inhibition of myeloid lineage immune genes. Upregulated genes included the stable dendritic cell population genes CD1c and CD207 (langerin). Comparison of responders and nonresponders revealed rapid downmodulation of innate IL-1beta and IL-8 sepsis cascade cytokines in both groups, but only responders downregulated IL-17 pathway genes to baseline levels. CONCLUSION Although both responders and nonresponders to etanercept inactivated sepsis cascade cytokines, response to etanercept is dependent on inactivation of myeloid dendritic cell genes and inactivation of the T(H)17 immune response.

Identification of Cellular Pathways of “Type 1,” Th17 T Cells, and TNF- and Inducible Nitric Oxide Synthase-Producing Dendritic Cells in Autoimmune Inflammation through Pharmacogenomic Study of Cyclosporine A in Psoriasis

Therapeutic modulation of psoriasis with targeted immunosuppressive agents defines inflammatory genes associated with disease activity and may be extrapolated to a wide range of autoimmune diseases. Cyclosporine A (CSA) is considered a “gold standard” therapy for moderate-to-severe psoriasis. We conducted a clinical trial with CSA and analyzed the treatment outcome in blood and skin of 11 responding patients. In the skin, as expected, CSA modulated genes from activated T cells and the “type 1” pathway (p40, IFN-γ, and STAT-1-regulated genes). However, CSA also modulated genes from the newly described Th17 pathway (IL-17, IL-22, and downstream genes S100A12, DEFB-2, IL-1β, SEPRINB3, LCN2, and CCL20). CSA also affected dendritic cells, reducing TNF and inducible NO synthase (products of inflammatory TNF- and inducible NO synthase-producing dendritic cells), CD83, and IL-23p19. We detected 220 early response genes (day 14 posttreatment) that were down-regulated by CSA. We classified >95% into proinflammatory or skin resident cells. More myeloid-derived than activated T cell genes were modulated by CSA (54 myeloid genes compared with 11 lymphocyte genes), supporting the hypothesis that myeloid derived genes contribute to pathogenic inflammation in psoriasis. In circulating mononuclear leukocytes, in stark contrast, no inflammatory gene activity was detected. Thus, we have constructed a genomic signature of successful treatment of psoriasis which may serve as a reference to guide development of other new therapies. In addition, these data also identify new gene targets for therapeutic modulation and may be applied to wide range of autoimmune diseases.

Evaluation of the Psoriasis Transcriptome across Different Studies by Gene Set Enrichment Analysis (GSEA)

Background Our objective was to develop a consistent molecular definition of psoriasis. There have been several published microarray studies of psoriasis, and we compared disease-related genes identified across these different studies of psoriasis with our own in order to establish a consensus. Methodology/Principal Findings We present a psoriasis transcriptome from a group of 15 patients enrolled in a clinical study, and assessed its biological validity using a set of important pathways known to be involved in psoriasis. We also identified a key set of cytokines that are now strongly implicated in driving disease-related pathology, but which are not detected well on gene array platforms and require more sensitive methods to measure mRNA levels in skin tissues. Comparison of our transcriptome with three other published lists of psoriasis genes showed apparent inconsistencies based on the number of overlapping genes. We extended the well-established approach of Gene Set Enrichment Analysis (GSEA) to compare a new study with these other published list of differentially expressed genes (DEG) in a more comprehensive manner. We applied our method to these three published psoriasis transcriptomes and found them to be in good agreement with our study. Conclusions/Significance Due to wide variability in clinical protocols, platform and sample handling, and subtle disease-related signals, intersection of published DEG lists was unable to establish consensus between studies. In order to leverage the power of multiple transcriptomes reported by several laboratories using different patients and protocols, more sophisticated methods like the extension of GSEA presented here, should be used in order to overcome the shortcomings of overlapping individual DEG approach.

Myeloid Dendritic Cells from Human Cutaneous Squamous Cell Carcinoma Are Poor Stimulators of T-Cell Proliferation

To determine the phenotype and function of myeloid dendritic cells (DCs) from human cutaneous squamous-cell carcinoma (SCC), we studied their surface marker expression and allo-stimulatory potential ex vivo. There were abundant CD11c(+) myeloid DCs, as well as TNF and inducible nitric oxide synthase (iNOS)-producing DCs, in and around SCC tumor nests. Although myeloid DCs from SCC, adjacent non-tumor-bearing skin, and normal skin, were phenotypically similar by flow cytometry, and there was a pronounced genomic signature of mature DCs in SCC, they showed different T-cell stimulatory potential in an allogeneic mixed leukocyte reaction. Myeloid DCs from SCC were less potent stimulators of allogeneic T-cell proliferation than DCs from non-tumor-bearing skin. Culture with a DC-maturing cytokine cocktail (IL-1beta, IL-6, TNF-alpha, and PGE(2)) enhanced stimulatory potential in DCs from non-tumor-bearing skin, whereas SCC-associated DCs remained poor stimulators of T-cell proliferation. The microenvironment associated with SCC showed expression of TGF-beta, IL-10, and VEGF-A, factors capable of suppressing the DC function. These findings indicate that CD11c(+)/HLA-DR(hi) DCs from SCC are mature, but are not potent stimulators of T-cell proliferation compared with phenotypically similar DCs isolated from non-tumor-bearing skin. Identification of mechanisms responsible for suppression of tumor-associated DCs may provide insight into the evasion of immunosurveillance by SCC.

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.

CARD14 Expression in Dermal Endothelial Cells in Psoriasis

Mutations in the caspase recruitment domain, family member 14 (CARD14) gene have recently been described in psoriasis patients, and explain the psoriasis susceptibility locus 2 (PSORS2). CARD14 is a scaffolding protein that regulates NF-κB activation, and psoriasis-associated CARD14 mutations lead to enhanced NF-κB signaling. CARD14 is expressed mainly in epidermal keratinocytes, but also in unidentified dermal cells. In this manuscript, the identity of the dermal cell types expressing CARD14, as well the potential functional consequence of overactive CARD14 in these dermal cell types, was determined. Using two-color immunofluorescence, dermal CARD14 did not co-localize with T-cells, dendritic cells, or macrophages. However, dermal CARD14 did highly co-localize with CD31+ endothelial cells (ECs). CARD14 was also expressed non-dermal endothelial cells, such as aortic endothelial cells, which may indicate a role of CARD14+ECs in the systemic inflammation and cardiovascular comorbidities associated with psoriasis. Additionally, phosphorylated NF-κB was found in psoriatic CARD14+ CD31+ ECs, demonstrating this pathway is active in dermal ECs in psoriasis. Transfection of dermal ECs with psoriasis-associated CARD14 mutations resulted in increased expression of several chemokines, including CXCL10, IL-8, and CCL2. These results provide preliminary evidence that CARD14 expression in ECs may contribute to psoriasis through increased expression of chemokines and facilitating recruitment of immune cells into skin.