Homeostatic tissue responses in skin biopsies from NOMID patients with constitutive overproduction of IL-1ß.

The autoinflammatory disorder, Neonatal-onset Multisystem Inflammatory Disease (NOMID) is the most severe phenotype of disorders caused by mutations in CIAS1 that result in increased production and secretion of active IL-1ß. NOMID patients present with systemic and organ-specific inflammation of the skin, central nervous system and bone, and respond dramatically to treatment with IL-1 blocking agents. We compared the cellular infiltrates and transcriptome of skin biopsies from patients with NOMID (n = 14) before treatment (lesional (LS) and non-lesional (pre-NL) skin) and after treatment (post-NL) with the IL-1 blocker anakinra (recombinant IL-1 receptor antagonist, Kineret®, Swedish Orphan Biovitrum AB, SOBI), to normal skin (n = 5) to assess tissue responses in the context of untreated and treated disease. Abundant neutrophils distinguish LS skin from pre-NL and post-NL skin. CD11c(+) dermal dendritic cells and CD163(+) macrophages expressed activated caspase-1 and are a likely source of cutaneous IL-1 production. Treatment with anakinra led to the disappearance of neutrophils, but CD3(+) T cells and HLA-DR(+) cells remained elevated. Among the upregulated genes IL-6, IL-8, TNF, IL-17A, CCL20, and the neutrophil defensins DEFA1 and DEFA3 were differentially regulated in LS tissues (compared to normal skin). Important significantly downregulated pathways in LS skin included IL-1R/TLR signaling, type I and II cytokine receptor signaling, mitochondrial dysfunction, and antigen presentation. The differential expression and regulation of microRNAs and pathways involved in post-transcriptional modification were suggestive of epigenetic modification in the chronically inflamed tissue. Overall, the dysregulated genes and pathways suggest extensive "adaptive" mechanisms to control inflammation and maintain tissue homeostasis, likely triggered by chronic IL-1 release in the skin of patients with NOMID.

Lesional dendritic cells in patients with chronic atopic dermatitis and psoriasis exhibit parallel ability to activate T-cell subsets.

BACKGROUND: Atopic dermatitis (AD) and psoriasis represent polar immune diseases. AD is a T(H)2/T(H)22-dominant disease, whereas psoriasis is considered a T(H)1/T(H)17 disease. Local immune deviation is suggested to be regulated by dendritic cell (DC)-induced T-cell polarization and recruitment of specific T-cell subsets by chemokines. Although the role of chemokines is well documented, the actual contribution of DCs to activate polar T-cell subsets in human subjects is still a matter of speculation. OBJECTIVE: We sought to elucidate the significance of each cutaneous DC subset in disease-specific T-cell immune deviation. METHODS: We performed a comprehensive analysis of major cutaneous resident (Langerhans cells and blood dendritic cell antigen 1-positive dermal DCs) and inflammatory (inflammatory dendritic epidermal cells and blood dendritic cell antigen 1-negative dermal DCs) DC subsets directly isolated from the lesional skin of patients with AD and those with psoriasis. RESULTS: The ability of each DC subset to expand T(H)1, T(H)2, T(H)17, and T(H)22 subsets was similar between the 2 diseases, despite the association of both with accumulation of resident and inflammatory DCs. We also confirmed differential upregulation of chemokine expression in patients with AD (CCL17, CCL18, and CCL22) and psoriasis (CXCL1, IL-8, and CCL20). The expression of CCL17 and CCL22 was higher in Langerhans cells from patients with AD than from patients with psoriasis, whereas the opposite was observed for CXCL9 and CXCL10. CONCLUSION: Our results suggest that DC polarity does not directly drive differential T-cell subset responses. Alternatively, disease-specific chemokines might recruit specific memory T-cell subsets into the skin, which in turn might be activated and expanded by DCs at the site of inflammation, maintaining differential immune polarity in these diseases.

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-gamma 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-gamma 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.

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-gamma, 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-1beta, 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.

Amelioration of epidermal hyperplasia by TNF inhibition is associated with reduced Th17 responses.

Biological agents have dramatically improved treatment options for patients with severe psoriasis. Etanercept (tumor necrosis factor [TNF] receptor-immunoglobulin fusion protein) is an effective treatment for many psoriasis patients, and blockade of TNF is considered to be its primary action. However, in this clinical trial, we show that etanercept has early inhibitory effects on a newly appreciated type of T cells: T helper type 17 (Th17) cells. Etanercept reduced the inflammatory dendritic cell products that drive Th17 cell proliferation (interleukin [IL] 23), as well as Th17 cell products and downstream effector molecules (IL-17, IL-22, CC chemokine ligand 20, and beta-defensin 4). In contrast, Th1 cellular products and effector molecules (interferon gamma, lymphotoxin alpha, and myxovirus resistance 1) were reduced late in disease resolution. This study suggests a role for Th17 in addition to Th1 cells in the pathogenesis of psoriasis. Th17 cells may be particularly important in driving epidermal activation in psoriatic plaques, whereas Th1 cells must also be eliminated for final disease resolution.

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.

BACKGROUND: Alefacept (anti-CD2) biological therapy selectively targets effector memory T cells (Tem) in psoriasis vulgaris, a model Type 1 autoimmune disease. METHODS: Circulating leukocytes were phenotyped in patients receiving alefacept for moderate to severe psoriasis. RESULTS: In 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-gamma-producing) were also significantly reduced. CONCLUSION: Alefacept causes a selective reduction in circulating effector memory T cells (Tem) and relative preservation of central memory T cells (Tcm) in psoriasis.

Novel insight into the agonistic mechanism of alefacept in vivo: differentially expressed genes may serve as biomarkers of response in psoriasis patients.

Alefacept is an LFA3-Ig fusion protein that binds to CD2 and is thought to inhibit T cell activation by antagonism of CD2 signaling or by lysis of CD2(+) cells. Alefacept is potential future therapeutic for organ transplant recipients or graft-vs-host disease and is an approved therapeutic for psoriasis vulgaris, which is a T cell-mediated inflammatory disease. However, alefacept improves psoriasis in only approximately 50% of patients treated for 12 wk. We studied the immunologic effects of alefacept in a group of psoriasis patients during treatment. We found that T cells, especially CD8(+) T cells, were rapidly decreased in the peripheral circulation. Decreases in circulating T cells were not associated with induced apoptosis. Unexpectedly, in addition to suppression of inflammatory genes, we found a marked induction of mRNAs for STAT1, IL-8, and monokine induced by IFN-gamma during the first day of treatment in PBMC. We confirmed the agonistic effects of alefacept in PBMC in vitro, which were similar to CD3/CD28 ligation on T cells. These data establish that alefacept activates gene expression programs in leukocytes and suggest that its therapeutic action may be as a mixed agonist/antagonist. Furthermore, responding patients to alefacept treatment show unique patterns of gene modulation. Whereas alefacept down-regulated TCRs CD3D and CD2 in responders, nonresponders reveal a higher expression of T cell activation genes such as CD69 in pretreatment PBMC. These finding suggest a potential basis for categorizing responders vs nonresponders at an early time point in treatment or before treatment of a broad range of proinflammatory diseases. This study 1) establishes alefacept as a novel CD2 agonist molecule for induction of leukocyte activation genes (prior work proposed its mechanism as a CD2 antagonist) and 2) that differential activation of genes may categorize clinical responders to this agent, critical for cost-effective use of this drug.

Major differences in inflammatory dendritic cells and their products distinguish atopic dermatitis from psoriasis.

BACKGROUND: Atopic dermatitis (AD) and psoriasis represent contrasting poles of the T(H)1 versus T(H)2 paradigm. Both diseases have been associated with increased numbers of dendritic cells (DCs) in the skin, but the similarities and differences in DC populations need to be established. OBJECTIVE: We aimed to characterize the specific DC subsets, as well as chemokine and cytokine environment in chronic AD compared with psoriasis. METHODS: Skin biopsies were obtained from patients with acute exacerbation of chronic AD (n = 18), psoriasis (n = 15), and healthy volunteers (n = 15) for microarray analysis, RT-PCR, immunohistochemistry, and double-label immunofluorescence. RESULTS: Myeloid DCs upregulate CCL17 and CCL18 in AD, as opposed to TNF-alpha and inducible nitric oxide synthase (iNOS) in psoriasis. In our study, we identified cells phenotypically identical to the inflammatory dendritic epidermal cells in the dermis in both diseases, although to a lesser extent in psoriasis. We found substantially higher numbers of dermal CCL22 producing plasmacytoid DCs in AD. The thymic stromal lymphopoietin receptor showed significantly higher expression in AD, whereas the thymic stromal lymphopoietin ligand was upregulated more in psoriasis. CONCLUSION: There are major differences in myeloid and plasmacytoid subsets of cutaneous DCs and the chemokine/cytokine environment between AD and psoriasis. Distinct subsets within the CD11c(+) population may influence polarization through the production of regulatory mediators, including iNOS, TNF, CCL17, and CCL18. Plasmacytoid DCs may also influence T(H)2 polarization, having a more important role in AD than previously appreciated. CLINICAL IMPLICATIONS: Dermal inflammatory dendritic cells in AD and TNF and iNOS-producing DCs in psoriasis, and/or their regulatory products, may be potential targets for future therapeutic interventions.

Human basal cell carcinoma is associated with Foxp3+ T cells in a Th2 dominant microenvironment.

Basal cell carcinoma (BCC), the most common human cancer, undergoes spontaneous regression in certain circumstances, which is potentially immune-mediated. To understand the immune response surrounding BCCs, we characterized the genomic, protein, and cellular microenvironment associated with BCC in comparison to normal skin. Our results demonstrated the following: (1) CD4+ CD25+ Foxp3+ surround epithelial tumor aggregates; (2) Immature dendritic cells (DCs) were abundant in the tumor microenvironment; (3) BCC showed increased expression of IL-4, IL-10, and CCL22 and increased expression of interferon-associated genes (IFI27, IRF1, IRF7, and G1P2) and IL-12/23, gene indicating a Th2 dominant microenvironment. Our findings suggest a dynamic state within the immune microenvironment associated with BCC. The finding of phenotypic T regs, in conjunction with immature DCs and Th2 cytokines, suggests an attenuated state of immunity to human BCC. In contrast, abundant CD8+ T cells, an interferon signal, and IL-12/23 suggest partial host antitumor response. A better understanding of these opposing forces within the immune microenvironment may facilitate development of more potent immune-based treatment for BCC and other human carcinomas.

Increase in TNF-alpha and inducible nitric oxide synthase-expressing dendritic cells in psoriasis and reduction with efalizumab (anti-CD11a).

We find that CD11c(+) cells with many markers of dendritic cells (DCs) are a major cell type in the skin lesions of psoriasis. These CD11c(+) cells, which are evident in both epidermis and dermis, are the sites for the expression of two mediators of inflammation, inducible nitric oxide synthase (iNOS) and TNF-alpha in diseased skin. These cells express HLA-DR, CD40, and CD86, lack the Langerin and CD14 markers of Langerhans cells and monocytes, respectively, and to a significant extent express the DC maturation markers DC-LAMP and CD83. Treatment of psoriasis with efalizumab (anti-CD11a, Raptiva) strongly reduces infiltration by these DCs in patients responding to this agent. Disease activity after therapy was more related to DC infiltrates and iNOS mRNA levels than T cell infiltrates, and CD11c(+) cells responded more quickly to therapy than epidermal keratinocytes. Our results suggest that a type of DC, which resembles murine "Tip-DCs" that can accumulate during infection, has proinflammatory effects in psoriasis through nitric oxide and TNF-alpha production, and can be an important target for suppressive therapies.

Alefacept reduces infiltrating T cells, activated dendritic cells, and inflammatory genes in psoriasis vulgaris.

Psoriasis vulgaris, a skin disease that is considered to be the result of a type 1 autoimmune response, provides an opportunity for studying the changes that occur in a target-diseased tissue during innovative immunotherapies. To gain a more comprehensive picture of the response to an approved biological therapy, we studied alfacept, which is a CD2 binding fusion protein. We examined T cells, dendritic cells (DCs), and expression of a number of inflammatory genes. In 22 patients, 55% demonstrated a clear histological remission of the disease, with a 73% reduction in lesional lymphocytes and a 79% decrease in infiltrating CD8+ cells. Only histological responders showed marked reductions in the tissue expression of inflammatory genes IFN-gamma, signal transducer and activator of transcription 1, monokine induced by IFN-gamma, inducible NO synthase, IL-8, and IL-23 subunits. Parallel decreases in CD83+ and CD11c+ DCs also were measured by immunohistochemistry. Because we observed that alefacept binds primarily to T cells and not DCs, we suggest that T cells are the primary target for therapy, but that DCs and a spectrum of type 1 inflammatory genes are coordinately suppressed.