Charlotte M. Bonefeld

Diagnostic microRNA profiling in cutaneous T-cell lymphoma (CTCL)

Cutaneous T-cell lymphomas (CTCLs) are the most frequent primary skin lymphomas. Nevertheless, diagnosis of early disease has proven difficult because of a clinical and histologic resemblance to benign inflammatory skin diseases. To address whether microRNA (miRNA) profiling can discriminate CTCL from benign inflammation, we studied miRNA expression levels in 198 patients with CTCL, peripheral T-cell lymphoma (PTL), and benign skin diseases (psoriasis and dermatitis). Using microarrays, we show that the most induced (miR-326, miR-663b, and miR-711) and repressed (miR-203 and miR-205) miRNAs distinguish CTCL from benign skin diseases with > 90% accuracy in a training set of 90 samples and a test set of 58 blinded samples. These miRNAs also distinguish malignant and benign lesions in an independent set of 50 patients with PTL and skin inflammation and in experimental human xenograft mouse models of psoriasis and CTCL. Quantitative (q)RT-PCR analysis of 103 patients with CTCL and benign skin disorders validates differential expression of 4 of the 5 miRNAs and confirms previous reports on miR-155 in CTCL. A qRT-PCR-based classifier consisting of miR-155, miR-203, and miR-205 distinguishes CTCL from benign disorders with high specificity and sensitivity, and with a classification accuracy of 95%, indicating that miRNAs have a high diagnostic potential in CTCL.

IL-23 and TH17-mediated inflammation in human allergic contact dermatitis

BACKGROUND IL-17-producing T(H) (T(H)17) cells are key mediators of chronic inflammation in mice. Recent studies have implicated T(H)17-mediated inflammation in the pathogenesis of human autoimmune diseases; however, the involvement of T(H)17 cells in allergic disorders remains largely elusive. OBJECTIVE To investigate T(H)17-mediated inflammation in human beings with allergic contact dermatitis; in particular, the innate response of keratinocytes to contact allergen, the induction of allergen-specific T(H)17 cells, and the presence of T(H)17-related effector cells in inflamed skin. METHODS Human keratinocytes were stimulated with nickel in vitro followed by measurements of IL-23 and IL-12 production by quantitative PCR and ELISA. Allergen-specific memory T cells from the blood of individuals with nickel allergy and healthy controls were identified and characterized by using a short-term ex vivo assay. Nickel patch test lesions and normal skin were analyzed for the expression of T(H)17-related cells and molecules by using immunohistochemistry. RESULTS Keratinocytes were found to produce IL-23, but no detectable IL-12, in a response to nickel stimulation. Memory T cells isolated from peripheral blood of individuals with nickel allergy, but not healthy controls, contained T(H)17 and T(H)1 cells proliferating in response to nickel-pulsed DCs. Inflamed skin of nickel-challenged allergic individuals contained infiltrating neutrophils and cells expressing IL-17, IL-22, CCR6, and IL-22R. CONCLUSION Our results demonstrate the involvement of T(H)17-mediated immunopathology in human allergic contact dermatitis, including both innate and adaptive immune responses to contact allergens.

Malignant cutaneous T-cell lymphoma cells express IL-17 utilizing the Jak3/Stat3 signaling pathway.

IL-17 is a proinflammatory cytokine that is crucial for the hosts protection against a range of extracellular pathogens. However, inappropriately regulated expression of IL-17 is associated with the development of inflammatory diseases and cancer. In cutaneous T-cell lymphoma (CTCL), malignant T cells gradually accumulate in skin lesions characterized by massive chronic inflammation, suggesting that IL-17 could be involved in the pathogenesis. In this study we show that IL-17 protein is present in 10 of 13 examined skin lesions but not in sera from 28 CTCL patients. Importantly, IL-17 expression is primarily observed in atypical lymphocytes with characteristic neoplastic cell morphology. In accordance, malignant T-cell lines from CTCL patients produce IL-17 and the synthesis is selectively increased by IL-2 receptor β chain cytokines. Small-molecule inhibitors or small interfering RNA against Jak3 and signal transducer and activator of transcription 3 (Stat3) reduce the production of IL-17, showing that the Jak3/Stat3 pathway promotes the expression of the cytokine. In summary, our findings indicate that the malignant T cells in CTCL lesions express IL-17 and that this expression is promoted by the Jak3/Stat3 pathway.

STAT5-mediated expression of oncogenic miR-155 in cutaneous T-cell lymphoma

The pathogenesis of cutaneous T-cell lymphoma (CTCL) remains elusive. Recent discoveries indicate that the oncogenic microRNA miR-155 is overexpressed in affected skin from CTCL patients. Here, we address what drives the expression of miR-155 and investigate its role in the pathogenesis of CTCL. We show that malignant T cells constitutively express high levels of miR-155 and its host gene BIC (B cell integration cluster). Using ChIP-seq, we identify BIC as a target of transcription factor STAT5, which is aberrantly activated in malignant T cells and induced by IL-2/IL-15 in non-malignant T cells. Incubation with JAK inhibitor or siRNA-mediated knockdown of STAT5 decreases BIC/miR-155 expression, whereas IL-2 and IL-15 increase their expression in cell lines and primary cells. In contrast, knockdown of STAT3 has no effect, and BIC is not a transcriptional target of STAT3, indicating that regulation of BIC/miR-155 expression by STAT5 is highly specific. Malignant proliferation is significantly inhibited by an antisense-miR-155 as well as by knockdown of STAT5 and BIC. In conclusion, we provide the first evidence that STAT5 drives expression of oncogenic BIC/miR-155 in cancer. Moreover, our data indicate that the STAT5/BIC/miR-155 pathway promotes proliferation of malignant T cells, and therefore is a putative target for therapy in CTCL.

Increased number and frequency of group 3 innate lymphoid cells in nonlesional psoriatic skin

Psoriasis is a common immune‐mediated inflammatory disease that affects the skin and joints. The interleukin (IL)‐23/IL‐17A axis and IL‐22 play key roles in the pathogenesis of psoriasis. IL‐23‐responsive innate lymphoid cells (ILCs) with a high capacity to produce IL‐17 and/or IL‐22 have recently been identified and associated with inflammatory bowel diseases. The occurrence and role of ILCs in human skin are poorly understood.

The effect of short-chain fatty acids on human monocyte-derived dendritic cells

The gut microbiota is essential for human health and plays an important role in the pathogenesis of several diseases. Short-chain fatty acids (SCFA), such as acetate, butyrate and propionate, are end-products of microbial fermentation of macronutrients that distribute systemically via the blood. The aim of this study was to investigate the transcriptional response of immature and LPS-matured human monocyte-derived DC to SCFA. Our data revealed distinct effects exerted by each individual SCFA on gene expression in human monocyte-derived DC, especially in the mature ones. Acetate only exerted negligible effects, while both butyrate and propionate strongly modulated gene expression in both immature and mature human monocyte-derived DC. An Ingenuity pathway analysis based on the differentially expressed genes suggested that propionate and butyrate modulate leukocyte trafficking, as SCFA strongly reduced the release of several pro-inflammatory chemokines including CCL3, CCL4, CCL5, CXCL9, CXCL10, and CXCL11. Additionally, butyrate and propionate inhibited the expression of lipopolysaccharide (LPS)-induced cytokines such as IL-6 and IL-12p40 showing a strong anti-inflammatory effect. This work illustrates that bacterial metabolites far from the site of their production can differentially modulate the inflammatory response and generally provides new insights into host-microbiome interactions.

Protein Kinase C (PKC)α and PKCθ Are the Major PKC Isotypes Involved in TCR Down-Regulation

It is well known that protein kinase C (PKC) plays an important role in regulation of TCR cell surface expression levels. However, eight different PKC isotypes are present in T cells, and to date the particular isotype(s) involved in TCR down-regulation remains to be identified. The aim of this study was to identify the PKC isotype(s) involved in TCR down-regulation and to elucidate the mechanism by which they induce TCR down-regulation. To accomplish this, we studied TCR down-regulation in the human T cell line Jurkat, in primary human T cells, or in the mouse T cell line DO11.10 in which we either overexpressed constitutive active or dominant-negative forms of various PKC isotypes. In addition, we studied TCR down-regulation in PKC knockout mice and by using small interfering RNA-mediated knockdown of specific PKC isotypes. We found that PKCα and PKCθ were the only PKC isotypes able to induce significant TCR down-regulation. Both isotypes mediated TCR down-regulation via the TCR recycling pathway that strictly depends on Ser126 and the di-leucine-based receptor-sorting motif of the CD3γ chain. Finally, we found that PKCθ was mainly implicated in down-regulation of directly engaged TCR, whereas PKCα was involved in down-regulation of nonengaged TCR.

Constitutive and Ligand-Induced TCR Degradation

Modulation of TCR expression levels is a central event during T cell development and activation, and it probably plays an important role in adjusting T cell responsiveness. Conflicting data have been published on down-regulation and degradation rates of the individual TCR subunits, and several divergent models for TCR down-regulation and degradation have been suggested. The aims of this study were to determine the rate constants for constitutive and ligand-induced TCR degradation and to determine whether the TCR subunits segregate or are processed as an intact unit during TCR down-regulation and degradation. We found that the TCR subunits in nonstimulated Jurkat cells were degraded with rate constants of ∼0.0011 min−1, resulting in a half-life of ∼10.5 h. Triggering of the TCR by anti-TCR Abs resulted in a 3-fold increase in the degradation rate constants to ∼0.0033 min−1, resulting in a half-life of ∼3.5 h. The subunits of the TCR complex were down-regulated from the cell surface and degraded with identical kinetics, and most likely remained associated during the passage throughout the endocytic pathway from the cell surface to the lysosomes. Similar results were obtained in studies of primary human Vβ8+ T cells stimulated with superantigen. Based on these results, the simplest model for TCR internalization, sorting, and degradation is proposed.

Enhanced sensitization and elicitation responses caused by mixtures of common fragrance allergens

Background. Perfumes are complex mixtures composed of many fragrance ingredients, many of which are known to be only weak allergens when tested individually. It is therefore surprising that fragrance contact allergy is one of the most common forms of contact allergy.

Elucidating the role of interleukin-17F in cutaneous T-cell lymphoma

Inappropriately regulated expression of interleukin (IL)-17A is associated with the development of inflammatory diseases and cancer. However, little is known about the role of other IL-17 family members in carcinogenesis. Here, we show that a set of malignant T-cell lines established from patients with cutaneous T-cell lymphoma (CTCL) spontaneously secrete IL-17F and that inhibitors of Janus kinases and Signal transducer and activator of transcription 3 are able to block that secretion. Other malignant T-cell lines produce IL-17A but not IL-17F. Upon activation, however, some of the malignant T-cell lines are able to coexpress IL-17A and IL-17F, leading to formation of IL-17A/F heterodimers. Clinically, we demonstrate that IL-17F messenger RNA expression is significantly increased in CTCL skin lesions compared with healthy donors and patients with chronic dermatitis. IL-17A expression is also increased and a significant number of patients express high levels of both IL-17A and IL-17F. Concomitantly, we observed that the expression of the IL-17 receptor is significantly increased in CTCL skin lesions compared with control subjects. Importantly, analysis of a historic cohort of 60 CTCL patients indicates that IL-17F expression is associated with progressive disease. These findings implicate IL-17F in the pathogenesis of CTCL and suggest that IL-17 cytokines and their receptors may serve as therapeutic targets.