Non-TGFß profibrotic signaling in ulcerative colitis after in vivo experimental intestinal injury in humans.

Although impaired regeneration is important in many gastrointestinal diseases including ulcerative colitis (UC), the dynamics of mucosal regeneration in humans are poorly investigated. We have developed a model to study these processes in vivo in humans. Epithelial restitution (ER) and extracellular matrix (ECM) regulation after an experimental injury of the sigmoid colonic mucosa was assessed by repeated high-resolution endoscopic imaging, histological assessment, RNA sequencing, deconvolution analysis, and 16S rDNA sequencing of the injury niche microbiome of 19 patients with UC in remission and 20 control subjects. Human ER had a 48-h lag before induction of regenerative epithelial cells [wound-associated epithelial (WAE) and transit amplifying (TA) cells] along with the increase of fibroblast-derived stem cell growth factor gremlin 1 mRNA (GREM1). However, UC deconvolution data showed rapid induction of inflammatory fibroblasts and upregulation of major structural ECM collagen mRNAs along with tissue inhibitor of metalloproteinase 1 (TIMP1), suggesting increased profibrotic ECM deposition. No change was seen in transforming growth factor ß (TGFß) mRNA, whereas the profibrotic cytokines interleukin 13 (IL13) and IL11 were upregulated in UC, suggesting that human postinjury responses could be TGFß-independent. In conclusion, we found distinct regulatory layers of regeneration in the normal human colon and a potential targetable profibrotic dysregulation in UC that could lead to long-term end-organ failure, i.e., intestinal damage.NEW & NOTEWORTHY The study reveals the regulatory dynamics of epithelial regeneration and extracellular matrix remodeling after experimental injury of the human colon in vivo and shows that human intestinal regeneration is different from data obtained from animals. A lag phase in epithelial restitution is associated with induction of stromal cell-derived epithelial growth factors. Postinjury regeneration is transforming growth factor ß-independent, and we find a profibrotic response in patients with ulcerative colitis despite being in remission.

Stage-related increase in PIM2 expression in mycosis fungoides.

The oncogene PIM2 is upregulated in several malignancies but has never been investigated in mycosis fungoides (MF), the most common type of cutaneous T-cell lymphoma (CTCL). PIM2 is a well-known oncogene and is regulated by cell signaling pathways like the JAK/STAT- and NF-kB-pathway, key regulators in the pathogenesis of CTCL. The aim of this study was to examine the role of PIM2 in MF. PIM2 gene expression was measured in 81 formalin-fixed paraffin-embedded skin biopsies from patients with MF and 46 control biopsies from healthy skin (HS) and benign inflammatory skin disease (BID). Validation of PIM2 protein expression was performed on selected biopsies with immunohistochemical staining. We found a significant difference in gene expression levels between both early stage MF and HS (p < 0.0001), and BID (p < 0.0001). In addition, the PIM2 gene expression was higher in advanced-stage MF compared to early stage disease (p = 0.0001). No significant difference in gene expression levels was found between patients with and without disease progression. In conclusion, we found PIM2 expression is significantly increased in MF compared to controls, and in advanced-stage MF compared to early stage MF. These findings could potentially have diagnostic value in discriminating early stage MF from BID.

Keratinocytes Present Staphylococcus aureus Enterotoxins and Promote Malignant and Nonmalignant T Cell Proliferation in Cutaneous T-Cell Lymphoma.

Cutaneous T-cell lymphoma is characterized by malignant T cells proliferating in a unique tumor microenvironment dominated by keratinocytes (KCs). Skin colonization and infection by Staphylococcus aureus are a common cause of morbidity and are suspected of fueling disease activity. In this study, we show that expression of HLA-DRs, high-affinity receptors for staphylococcal enterotoxins (SEs), by KCs correlates with IFN-γ expression in the tumor microenvironment. Importantly, IFN-γ induces HLA-DR, SE binding, and SE presentation by KCs to malignant T cells from patients with Sézary syndrome and malignant and nonmalignant T-cell lines derived from patients with Sézary syndrome and mycosis fungoides. Likewise, preincubation of KCs with supernatant from patient-derived SE-producing S aureus triggers proliferation in malignant T cells and cytokine release (including IL-2), when cultured with nonmalignant T cells. This is inhibited by pretreatment with engineered bacteriophage S aureus-specific endolysins. Furthermore, alteration in the HLA-DR-binding sites of SE type A and small interfering RNA-mediated knockdown of Jak3 and IL-2Rγ block induction of malignant T-cell proliferation. In conclusion, we show that upon exposure to patient-derived S aureus and SE, KCs stimulate IL-2Rγ/Jak3-dependent proliferation of malignant and nonmalignant T cells in an environment with nonmalignant T cells. These findings suggest that KCs in the tumor microenvironment play a key role in S aureus-mediated disease activity in cutaneous T-cell lymphoma.

The junctional adhesion molecule-like protein (JAML) is important for the inflammatory response during contact hypersensitivity.

BACKGROUND: The junctional adhesion molecule-like protein (JAML) plays important roles in wound healing and activation of epidermal γδ T cells in mice. Whether JAML plays a role in contact hypersensitivity (CHS), the animal model of allergic contact dermatitis (ACD), is not known. METHODS: To examine the role of JAML in CHS, we used various mouse models of CHS in JAML knockout (KO) and wild-type (WT) mice. Furthermore, the expression of the JAML ligand coxsackievirus and adenovirus receptor (CXADR) on keratinocytes was accessed in vitro and in vivo. RESULTS: JAML KO mice had a diminished inflammatory response during both the sensitization and elicitation phase of CHS and had reduced numbers of CD8+ and CD4+ T cells in the epidermis. Furthermore, interferon γ (IFNγ), interleukin 1ß (IL-1ß) and CXCL10 production were significantly reduced in JAML KO mice during the elicitation phase. We found that CD8+ T cells express JAML and that JAML is essential for rapid flare-up responses to contact allergens. Finally, we show that keratinocytes up-regulate the JAML ligand CXADR following exposure to contact allergens. CONCLUSION: Our study is the first to show a central role of JAML in CHS and reveals a potential new target for the treatment of ACD in humans.

Imbalanced IL-1B and IL-18 Expression in Sézary Syndrome.

Sézary syndrome (SS) is a rare and aggressive type of cutaneous T-cell lymphoma, with an abnormal inflammatory response in affected skin. The cytokines IL-1B and IL-18, as key signaling molecules in the immune system, are produced in an inactive form and cleave to the active form by inflammasomes. In this study, we assessed the skin, serum, peripheral mononuclear blood cell (PBMC) and lymph-node samples of SS patients and control groups (healthy donors (HDs) and idiopathic erythroderma (IE) nodes) to investigate the inflammatory markers IL-1B and IL-18 at the protein and transcript expression levels, as potential markers of inflammasome activation. Our findings showed increased IL-1B and decreased IL-18 protein expression in the epidermis of SS patients; however, in the dermis layer, we detected increased IL-18 protein expression. In the lymph nodes of SS patients at advanced stages of the disease (N2/N3), we also detected an enhancement of IL-18 and a downregulation of IL-1B at the protein level. Moreover, the transcriptomic analysis of the SS and IE nodes confirmed the decreased expression of IL1B and NLRP3, whereas the pathway analysis indicated a further downregulation of IL1B-associated genes. Overall, the present findings showed compartmentalized expressions of IL-1B and IL-18 and provided the first evidence of their imbalance in patients with Sézary syndrome.

Endolysin Inhibits Skin Colonization by Patient-Derived Staphylococcus Aureus and Malignant T-Cell Activation in Cutaneous T-Cell Lymphoma.

Staphylococcus aureus is suspected to fuel disease activity in cutaneous T-cell lymphomas. In this study, we investigate the effect of a recombinant, antibacterial protein, endolysin (XZ.700), on S. aureus skin colonization and malignant T-cell activation. We show that endolysin strongly inhibits the proliferation of S. aureus isolated from cutaneous T-cell lymphoma skin and significantly decreases S. aureus bacterial cell counts in a dose-dependent manner. Likewise, ex vivo colonization of both healthy and lesional skin by S. aureus is profoundly inhibited by endolysin. Moreover, endolysin inhibits the patient-derived S. aureus induction of IFNγ and the IFNγ-inducible chemokine CXCL10 in healthy skin. Whereas patient-derived S. aureus stimulates activation and proliferation of malignant T cells in vitro through an indirect mechanism involving nonmalignant T cells, endolysin strongly inhibits the effects of S. aureus on activation (reduced CD25 and signal transducer and activator of transcription 5 phosphorylation) and proliferation (reduced Ki-67) of malignant T cells and cell lines in the presence of nonmalignant T cells. Taken together, we provide evidence that endolysin XZ.700 inhibits skin colonization, chemokine expression, and proliferation of pathogenic S. aureus and blocks their potential tumor-promoting effects on malignant T cells.

Malignant T cells induce skin barrier defects through cytokine-mediated JAK/STAT signaling in cutaneous T-cell lymphoma.

Cutaneous T-cell lymphoma (CTCL) is a devastating lymphoid malignancy characterized by the accumulation of malignant T cells in the dermis and epidermis. Skin lesions cause serious symptoms that hamper quality of life and are entry sites for bacterial infection, a major cause of morbidity and mortality in advanced diseases. The mechanism driving the pathological processes that compromise the skin barrier remains unknown. Here, we report increased transepidermal water loss and compromised expression of the skin barrier proteins filaggrin and filaggrin-2 in areas adjacent to TOX-positive T cells in CTCL skin lesions. Malignant T cells secrete mediators (including cytokines such as interleukin 13 [IL-13], IL-22, and oncostatin M) that activate STAT3 signaling and downregulate filaggrin and filaggrin-2 expression in human keratinocytes and reconstructed human epithelium. Consequently, the repression of filaggrins can be counteracted by a cocktail of antibodies targeting these cytokines/receptors, small interfering RNA-mediated knockdown of JAK1/STAT3, and JAK1 inhibitors. Notably, we show that treatment with a clinically approved JAK inhibitor, tofacitinib, increases filaggrin expression in lesional skin from patients with mycosis fungoides. Taken together, these findings indicate that malignant T cells secrete cytokines that induce skin barrier defects via a JAK1/STAT3-dependent mechanism. As clinical grade JAK inhibitors largely abrogate the negative effect of malignant T cells on skin barrier proteins, our findings suggest that such inhibitors provide novel treatment options for patients with CTCL with advanced disease and a compromised skin barrier.

Concomitant Inhibition of FASN and SREBP Provides a Promising Therapy for CTCL.

Cutaneous T cell lymphoma (CTCL) is a group of non-Hodgkin's primary cutaneous T cell lymphomas, with Mycosis Fungoides and Sézary syndrome (SS) being the two most common subtypes. Fatty acid synthase (FASN) is a crucial enzyme that catalyses the biosynthesis of fatty acids, which has been reported to play an oncogenic role in various malignancies but not in CTCL so far. Herein, we show that FASN is highly expressed in CTCL cell lines and in peripheral blood mononuclear cells (PBMCs) from CTCL patients, while it is not in PBMCs from healthy individuals. The inhibition of FASN in CTCL cell lines impairs cell viability, survival, and proliferation, but, interestingly, it also increases FASN expression. However, inhibiting sterol regulatory element binding protein (SREBP), a transcription factor that promotes the expression of FASN, partially reversed the upregulation of FASN induced by FASN inhibitors. Thus, the combination of FASN and SREBP inhibitors enhanced the effects on both CTCL cell lines and PBMCs from SS patients, where a valid inhibition on cell proliferation could be verified. Importantly, compared to non-malignant cells, primary malignant cells are more sensitive to the inhibition of FASN and SREBP, making the combination of FASN and SREBP inhibitors a promising novel therapeutic strategy in CTCL.

Macrophages Control the Bioavailability of Vitamin D and Vitamin D-Regulated T Cell Responses.

The active form of vitamin D3 (1,25(OH)2D3) has a great impact on T cell effector function. Thus, 1,25(OH)2D3 promotes T helper 2 (Th2) and regulatory T (Treg) cell function and concomitantly inhibits Th1 and Th17 cell function. Thus, it is believed that vitamin D exerts anti-inflammatory effects. However, vitamin D binding protein (DBP) strongly binds both 1,25(OH)2D3 and the precursor 25(OH)D3, leaving only a minor fraction of vitamin D in the free, bioavailable form. Accordingly, DBP in physiological concentrations would be expected to block the effect of vitamin D on T cells and dendritic cells. In the present study, we show that pro-inflammatory, monocyte-derived M1 macrophages express very high levels of the 25(OH)D-1α-hydroxylase CYP27B1 that enables them to convert 25(OH)D3 into 1,25(OH)2D3 even in the presence of physiological concentrations of DBP. Co-cultivation of M1 macrophages with T cells allows them to overcome the sequestering of 25(OH)D3 by DBP and to produce sufficient levels of 1,25(OH)2D3 to affect T cell effector function. This study suggests that in highly inflammatory conditions, M1 macrophages can produce sufficient levels of 1,25(OH)2D3 to modify T cell responses and thereby reduce T cell-mediated inflammation via a vitamin D-mediated negative feed-back loop.

Staphylococcus aureus Induces Signal Transducer and Activator of Transcription 5‒Dependent miR-155 Expression in Cutaneous T-Cell Lymphoma.

Staphylococcal enterotoxins are believed to fuel disease activity in cutaneous T-cell lymphoma. Recent data support this by showing that antibiotics inhibit malignant T cells in skin lesions in mycosis fungoides and Sézary syndrome, the most common forms of cutaneous T-cell lymphoma. Yet, it remains incompletely characterized how staphylococcal enterotoxins fuel disease activity. In this study, we show that staphylococcal enterotoxins induce the expression of the oncogenic microRNA miR-155 in primary malignant T cells. Thus, staphylococcal enterotoxins and Staphyloccocus aureus isolates from lesional skin of patients induce miR-155 expression at least partly through the IL-2Rg‒Jak‒signal transducer and activator of transcription 5 pathway, and the effect is augmented by the presence of nonmalignant T cells. Importantly, mycosis fungoides lesions harbor S. aureus, express Y-phosphorylated signal transducer and activator of transcription 5, and display enhanced miR-155 expression, when compared with nonlesional and healthy skin. Preliminary data show that aggressive antibiotic therapy is associated with decreased Y-phosphorylated signal transducer and activator of transcription 5 and miR-155 expression in lesional skin in two patients with Sézary syndrome. In conclusion, we show that S. aureus and its enterotoxins induce enhanced expression of oncogenic miR-155, providing mechanistic insight into the role of S. aureus in cutaneous T-cell lymphoma. Our findings support that environmental stimuli such as bacteria can fuel disease progression in cutaneous T-cell lymphoma.

JAK3 Is Expressed in the Nucleus of Malignant T Cells in Cutaneous T Cell Lymphoma (CTCL).

Perturbation in JAK-STAT signaling has been reported in the pathogenesis of cutaneous T cell lymphoma (CTCL). JAK3 is predominantly associated with the intra-cytoplasmic part of IL-2Rγc located in the plasma membrane of hematopoietic cells. Here we demonstrate that JAK3 is also ectopically expressed in the nucleus of malignant T cells. We detected nuclear JAK3 in various CTCL cell lines and primary malignant T cells from patients with Sézary syndrome, a leukemic variant of CTCL. Nuclear localization of JAK3 was independent of its kinase activity whereas STAT3 had a modest effect on nuclear JAK3 expression. Moreover, JAK3 nuclear localization was only weakly affected by blockage of nuclear export. An inhibitor of the nuclear export protein CRM1, Leptomycin B, induced an increased expression of SOCS3 in the nucleus, but only a weak increase in nuclear JAK3. Importantly, immunoprecipitation experiments indicated that JAK3 interacts with the nuclear protein POLR2A, the catalytic subunit of RNA Polymerase II. Kinase assays showed tyrosine phosphorylation of recombinant human Histone H3 by JAK3 in vitro-an effect which was blocked by the JAK inhibitor (Tofacitinib citrate). In conclusion, we provide the first evidence of nuclear localization of JAK3 in malignant T cells. Our findings suggest that JAK3 may have a cytokine-receptor independent function in the nucleus of malignant T cells, and thus a novel non-canonical role in CTCL.

Suppressed microRNA-195-5p expression in mycosis fungoides promotes tumor cell proliferation.

BACKGROUND: Several cancers, including mycosis fungoides (MF), have reported dysregulation of miR-195-5p. miR-195-5p plays a role in cell cycle regulation in several malignant diseases. OBJECTIVES: This study aimed to investigate: (a) the expression level of miR-195-5p in lesional MF skin biopsies and (b) the potential regulatory roles of miR-195-5p in MF. METHODS: Quantitative real-time polymerase chain reaction (RT-qPCR) was used to determine miR-195-5p expression in MF skin biopsies and cell lines. The effect of miR-195-5p and ADP-ribosylation factor-like protein 2 (ARL2) on cell cycle and apoptosis was measured by flow cytometry assays. Changes in ARL2 expression were determined by RT-qPCR and Western blotting (WB). RESULTS: We found lower expression levels of miR-195-5p in lesional skin from MF patients compared with non-lesional MF skin and skin from healthy volunteers. Additionally, miR-195-5p showed lower expression levels in the skin from patients with disease progression compared with patients with stable disease. In vitro studies showed that overexpression of miR-195-5p induced a cell cycle arrest in G0G1. Using microarray analysis, we identified several genes that were regulated after miR-195-5p overexpression. The most downregulated gene after miR-195-5p mimic transfection was ARL2. RT-qPCR and WB analyses confirmed downregulation of ARL2 following transfection with miR-195-5p mimic. Lastly, transfection with siRNA against ARL2 also induced a G0G1 arrest. CONCLUSION: Upregulation of miR-195-5p in MF inhibits cycle arrest by downregulation of ARL2. miR-195-5p may thus function as a tumor suppressor in MF and low miR-195-5p expression in lesional MF skin may promote disease progression.

MicroRNA-93 Targets p21 and Promotes Proliferation in Mycosis Fungoides T Cells.

BACKGROUND: Mycosis fungoides (MF), the most common form of cutaneous T-cell lymphoma (CTCL), is a lymphoproliferative disorder characterized by proliferation of malignant T cells in a chronic inflammatory environment in the skin. The nature of MF is still not fully understood, but aberrant microRNA (miR) expression and function seem to play an important role in the pathogenesis and disease progression and have been proposed as a putative disease marker. Recent studies have reported aberrant expression of miR-93 in situin MF lesions and linked dysregulated miR-93 expression to advanced stages of MF. However, the pathophysiological role of miR-93 in MF is unknown. OBJECTIVE: Here, we provide the first evidence that miR-93 targets the cell cycle regulator cyclin-dependent kinase inhibitor p21 and promotes growth of malignant T cells in MF. METHODS/RESULTS: Thus, inhibition of miR-93 in MF patient-derived malignant T-cell lines increases expression of p21 and inhibition of malignant proliferation. Notably, treatment with the histone deacetylase inhibitor Vorinostat (SAHA) reduces miR-93 expression and enhances p21 expression in the malignant T cells. Importantly, transfection with an miR-93 mimic partly blocks SAHA-induced p21 expression. CONCLUSIONS: we provide evidence that enhanced expression of the putative oncogenic miR, miR-93, represses the cell cycle inhibitor p21 and promotes proliferation of malignant T cells. Moreover, we demonstrate that SAHA triggers p21 expression - at least partly - through an inhibition of miR-93.

The Thioredoxin-Interacting Protein TXNIP Is a Putative Tumour Suppressor in Cutaneous T-Cell Lymphoma.

BACKGROUND: The thioredoxin-interacting protein (TXNIP) is involved in cellular metabolism and cell proliferation, and recently, deficient expression of TXNIP has been associated with progression and poor outcome for cancer patients. OBJECTIVES: To assess TXNIP expression and function in malignant T cells from cutaneous T-cell lymphoma (CTCL). METHODS: CTCL-derived malignant (MyLa2059, PB2B) and non-malignant (MyLa1850) cell lines were analysed by Western blotting and qPCR for TXNIP expression. Subsequently, the malignant CTCL cell lines were treated with GSK126 - an inhibitor of enhancer of zeste homolog 2 (EZH2) methyltransferase activity or assessed by bisulphite sequencing for TXNIP promoter methylation. Methylation was also assessed with the demethylating agent 5-azacytidine (5AZA). Finally, TXNIP was overexpressed in the malignant PB2B cell line via plasmid transduction, and the effect of TXNIP was further analysed by flow cytometry. RESULTS: We report on low expression of TXNIP protein in all cell lines representing different subtypes and stages of CTCL when compared to non-malignant T cells. Epigenetic silencing and other mechanisms were involved in the repression of TXNIP whereas forced expression of TXNIP strongly inhibited proliferation of malignant T cells. CONCLUSIONS: Epigenetic silencing and other as yet unknown mechanisms repress TXNIP expression in malignant T cells. As forced expression of TXNIP inhibits malignant proliferation, we propose that TXNIP is a putative tumour suppressor in CTCL.

MicroRNA-106b Regulates Expression of the Tumour Suppressors p21 and TXNIP and Promotes Tumour Cell Proliferation in Mycosis Fungoides.

A prognostic 3-miRNA classifier for early-stage mycosis fungoides has been developed recently, with miR-106b providing the strongest prognostic power. The aim of this study was to investigate the molecular function of miR-106b in mycosis fungoides disease progression. The cellular localization of miR-106b in mycosis fungoides skin biopsies was determined by in situ hybridization. The regulatory role of miR-106b was assessed by transient miR-106b inhibitor/mimic transfection of 2 mycosis fungoides derived cell lines, followed by quantitative real-time PCR (RT-qPCR), western blotting and a proliferation assay. MiR-106b was found to be expressed by dermal T-lymphocytes in mycosis fungoides skin lesions, and miR-106b expression increased with advancing mycosis fungoides stage. Transfection of miR-106b in 2 mycosis fungoides derived cell lines showed that miR-106b represses the tumour suppressors cyclin-dependent kinase inhibitor 1 (p21) and thioredoxin-interacting protein (TXNIP) and promotes mycosis fungoides tumour cell proliferation. In conclusion, these results substantiate that miR-106b has both a functional and prognostic role in progression of mycosis fungoides.

Staphylococcus aureus alpha-toxin inhibits CD8+ T cell-mediated killing of cancer cells in cutaneous T-cell lymphoma.

Staphylococcus aureus and its toxins have been linked to disease progression and mortality in advanced stages of cutaneous T-cell lymphoma (CTCL). CD8+ T cells play a crucial role in anti-cancer responses and high CD8+ T cell numbers in tumor lesions are associated with a favorable prognosis in CTCL. Here, we show that CD8+ T cells from both healthy donors and Sézary syndrome patients are highly susceptible to cell death induced by Staphylococcal alpha-toxin, whereas malignant T cells are not. Importantly, alpha-toxin almost completely blocks cytotoxic killing of CTCL tumor cells by peptide-specific CD8+ T cells, leading to their escape from induced cell death and continued proliferation. These findings suggest that alpha-toxin may favor the persistence of malignant CTCL cells in vivo by inhibiting CD8+ T cell cytotoxicity. Thus, we propose a novel mechanism by which colonization with Staphylococcus aureus may contribute to cancer immune evasion and disease progression in CTCL.

MicroRNAs in the Pathogenesis, Diagnosis, Prognosis and Targeted Treatment of Cutaneous T-Cell Lymphomas.

Cutaneous T-cell lymphoma (CTCL) represents a heterogeneous group of potentially devastating primary skin malignancies. Despite decades of intense research efforts, the pathogenesis is still not fully understood. In the early stages, both clinical and histopathological diagnosis is often difficult due to the ability of CTCL to masquerade as benign skin inflammatory dermatoses. Due to a lack of reliable biomarkers, it is also difficult to predict which patients will respond to therapy or progress towards severe recalcitrant disease. In this review, we discuss recent discoveries concerning dysregulated microRNA (miR) expression and putative pathological roles of oncogenic and tumor suppressive miRs in CTCL. We also focus on the interplay between miRs, histone deacetylase inhibitors, and oncogenic signaling pathways in malignant T cells as well as the impact of miRs in shaping the inflammatory tumor microenvironment. We highlight the potential use of miRs as diagnostic and prognostic markers, as well as their potential as therapeutic targets. Finally, we propose that the combined use of miR-modulating compounds with epigenetic drugs may provide a novel avenue for boosting the clinical efficacy of existing anti-cancer therapies in CTCL.

Staphylococcus aureus enterotoxins induce FOXP3 in neoplastic T cells in Sézary syndrome.

Sézary syndrome (SS) is a heterogeneous leukemic subtype of cutaneous T-cell lymphoma (CTCL) with generalized erythroderma, lymphadenopathy, and a poor prognosis. Advanced disease is invariably associated with severe immune dysregulation and the majority of patients die from infectious complications caused by microorganisms such as, Staphylococcus aureus, rather than from the lymphoma per se. Here, we examined if staphylococcal enterotoxins (SE) may shape the phenotype of malignant SS cells, including expression of the regulatory T-cell-associated marker FOXP3. Our studies with primary and cultured malignant cells show that SE induce expression of FOXP3 in malignant cells when exposed to nonmalignant cells. Mutations in the MHC class II binding domain of SE-A (SEA) largely block the effect indicating that the response relies at least in part on the MHC class II-mediated antigen presentation. Transwell experiments show that the effect is induced by soluble factors, partly blocked by anti-IL-2 antibody, and depends on STAT5 activation in malignant cells. Collectively, these findings show that SE stimulate nonmalignant cells to induce FOXP3 expression in malignant cells. Thus, differences in exposure to environmental factors, such as bacterial toxins may explain the heterogeneous FOXP3 expression in malignant cells in SS.

Ex vivo culture of lesional psoriasis skin for pharmacological testing.

BACKGROUND: Psoriasis is a chronic, inflammatory skin disorder resulting from a complex interplay between immune and skin cells via release of soluble mediators. While a lot is known about the molecular mechanisms behind psoriasis pathogenesis, there is still a need for preclinical research models that accuratelyreplicate the disease. OBJECTIVE: This study aimed to develop and characterize ex vivo culture of psoriasis skin as a model for pharmacological testing, where the immunological events of psoriasis can be followed. METHODS: Full thickness punch biopsies of lesional psoriasis skin were cultured in submerged conditions up to 144 h followingin situ T cell stimulation with rhIL-23 and anti-CD3 and anti-CD28 antibodies. The T cell mediated skin inflammation was assessed by gene and protein l analysis for a panel of inflammatory mediators. Tissue integrity and morphology were evaluated by histological analysis. RESULTS: T cell stimulation resulted in functional and psoriasis specificin situ activation of T cells. The expression levels of most of the proinflammatory mediators related to both immune and skin cells were comparable to these in freshly isolated tissue at 48 and 96 h of culture. Tissue integrity and morphology were sustained up to 96 h. Treatment with a corticosteroid reduced the expression of several pro-inflammatory cytokines and chemokines, whereas anti-IL-17A antibody treatment reduced the expression of the IL-17A downstream markers IL-8 and DEFB4. CONCLUSION: By preserving keyimmunopathological mechanisms of psoriasis, ex vivo culture of psoriasis skin can be used for the investigation of inflammatory processes of psoriasis and for preclinical drug discovery research.