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.

Bacterial genotoxins induce T cell senescence.

Several types of pathogenic bacteria produce genotoxins that induce DNA damage in host cells. Accumulating evidence suggests that a central function of these genotoxins is to dysregulate the host's immune response, but the underlying mechanisms remain unclear. To address this issue, we investigated the effects of the most widely expressed bacterial genotoxin, the cytolethal distending toxin (CDT), on T cells-the key mediators of adaptive immunity. We show that CDT induces premature senescence in activated CD4 T cells in vitro and provide evidence suggesting that infection with genotoxin-producing bacteria promotes T cell senescence in vivo. Moreover, we demonstrate that genotoxin-induced senescent CD4 T cells assume a senescence-associated secretory phenotype (SASP) which, at least partly, is orchestrated by the ATM-p38 signaling axis. These findings provide insight into the immunomodulatory properties of bacterial genotoxins and uncover a putative link between bacterial infections and T cell senescence.

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.

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-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.

Cellular Interactions and Inflammation in the Pathogenesis of Cutaneous T-Cell Lymphoma.

Cutaneous T-cell lymphoma (CTCL) comprises a group of lymphoproliferative diseases characterized by the accumulation of malignant T cells in chronically inflamed skin lesions. In early stages, the disease presents as skin patches or plaques covering a limited area of the skin and normally follows an indolent course. However, in a subset of patients the cutaneous lesions develop into tumors and the malignant T cells may spread to the lymphatic system, blood and internal organs with fatal consequences. Despite intensive research, the mechanisms driving disease progression remain incompletely understood. While most studies have focused on cancer cell-intrinsic oncogenesis, such as genetic and epigenetic events driving malignant transformation and disease progression, an increasing body of evidence shows that the interplay between malignant T cells and non-malignant cells plays a crucial role. Here, we outline some of the emerging mechanisms by which tumor, stromal and epidermal interactions may contribute to the progression of CTCL with particular emphasis on the crosstalk between fibroblasts, keratinocytes and malignant T cells.

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.

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.

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.

Expression of the Voltage-Gated Potassium Channel Kv1.3 in Lesional Skin from Patients with Cutaneous T-Cell Lymphoma and Benign Dermatitis.

BACKGROUND: The voltage-gated potassium channel Kv1.3 (KCNA3) is expressed by effector memory T cells (TEM) and plays an important role in their activation and proliferation. Mycosis fungoides (MF), the most common subtype of cutaneous T-cell lymphoma (CTCL), was recently proposed to be a malignancy of skin-resident TEM. However, the expression of Kv1.3 in CTCL has not been investigated. OBJECTIVES: This study aims to examine the expression of Kv1.3 in situ and in vitro in CTCL. METHODS: The expression of Kv1.3 was examined by immunohistochemistry in skin lesions from 38 patients with MF, 4 patients with Sézary syndrome (SS), and 27 patients with benign dermatosis. In 4 malignant T-cell lines of CTCL (Myla2059, PB2B, SeAx, and Mac2a) and a non-malignant T-cell line (MyLa1850), the expression of Kv1.3 was determined by flow cytometry. The proliferation of those cell lines treated with various concentrations of Kv1.3 inhibitor ShK was measured by 3H-thymdine incorporation. RESULTS: Half of the MF patients (19/38) displayed partial Kv1.3 expression including 1 patient with moderate Kv1.3 positivity, while the other half (19/38) exhibited Kv1.3 negativity. An almost identical distribution was observed in patients with benign conditions, that is, 44.4% (12/27) were partially positive for Kv1.3 including 1 patient with moderate Kv1.3 positivity, while 55.6% (15/27) were Kv1.3 negative. In contrast, 3 in 4 SS patients displayed partial Kv1.3 positivity including 2 patients with weak staining and 1 with moderate staining, while 1 in 4 SS patients was Kv1.3 negative. In addition, all malignant T-cell lines, and a non-malignant T-cell line, displayed low Kv1.3 surface expression with a similar pattern. Whereas 2 cell lines (PB2B and Mac2a) were sensitive to Kv1.3 blockade, the other 2 (Myla2059 and SeAx) were completely resistant. CONCLUSIONS: We provide the first evidence of a heterogeneous Kv1.3 expression in situ in CTCL lesions.

The Escherichia coli protein toxin cytotoxic necrotizing factor 1 induces epithelial mesenchymal transition.

Some toxigenic bacteria produce protein toxins with carcinogenic signatures, which either directly damage DNA or stimulate signalling pathways related to cancer. So far, however, only a few of them have been proved to favour the induction or progression of cancer. In this work, we report that the Rho-activating Escherichia coli protein toxin, cytotoxic necrotising factor 1 (CNF1), induces epithelial to mesenchymal transition (EMT) in intestinal epithelial cells. EMT is a crucial step in malignant tumour conversion and invasiveness. In the case of CNF1, it occurs by up-regulation of the transcription factors ZEB1 and Snail1, delocalisation of E-cadherin and ß-catenin, activation of the serine/threonine kinase mTOR, accelerated wound healing, and invasion. However, our results highlight that nontransformed epithelial cells entail the presence of inflammatory factors, in addition to CNF1, to acquire a mesenchymal-like behaviour. All this suggests that the surrounding microenvironment, as well as the cell type, dramatically influences the CNF1 ability to promote carcinogenic traits.

Staphylococcal alpha-toxin tilts the balance between malignant and non-malignant CD4+ T cells in cutaneous T-cell lymphoma.

Staphylococcus aureus is implicated in disease progression in cutaneous T-cell lymphoma (CTCL). Here, we demonstrate that malignant T cell lines derived from CTCL patients as well as primary malignant CD4+ T cells from Sézary syndrome patients are considerably more resistant to alpha-toxin-induced cell death than their non-malignant counterparts. Thus, in a subset of Sézary syndrome patients the ratio between malignant and non-malignant CD4+ T cells increases significantly following exposure to alpha-toxin. Whereas toxin-induced cell death is ADAM10 dependent in healthy CD4+ T cells, resistance to alpha-toxin in malignant T cells involves both downregulation of ADAM10 as well as other resistance mechanisms. In conclusion, we provide first evidence that Staphylococcus aureus derived alpha-toxin can tilt the balance between malignant and non-malignant CD4+ T cells in CTCL patients. Consequently, alpha-toxin may promote disease progression through positive selection of malignant CD4+ T cells, identifying alpha-toxin as a putative drug target in CTCL.

Expression and function of Kv1.3 channel in malignant T cells in Sézary syndrome.

The voltage-gated potassium channel Kv1.3 (KCNA3) is expressed by a subset of chronically activated memory T cells and plays an important role in their activation and proliferation. Here, we show that primary malignant T cells isolated from patients with Sézary syndrome (SS) express Kv1.3 and are sensitive to potent Kv1.3 inhibitors ShK and Vm24, but not sensitive to a less potent inhibitor [N17A/F32T]-AnTx. Kv1.3 blockade inhibits CD3/CD28-induced proliferation and IL-9 expression by SS cells in a concentration-dependent manner. In parallel, CD3/CD28-mediated CD25 induction is inhibited, whereas Kv1.3 blockade has no effect on apoptosis or cell death as judged by Annexin V and PI staining. In conclusion, we provide the first evidence that malignant T cells in SS express functional Kv1.3 channels and that Kv1.3 blockade inhibits activation-induced proliferation as well as cytokine and cytokine receptor expression in malignant T cells, suggesting that Kv1.3 is a potential target for therapy in SS.

Antibiotics inhibit tumor and disease activity in cutaneous T-cell lymphoma.

It has been proposed that CD4 T-cell responses to Staphylococcus aureus (SA) can inadvertently enhance neoplastic progression in models of skin cancer and cutaneous T-cell lymphoma (CTCL). In this prospective study, we explored the effect of transient antibiotic treatment on tumor cells and disease activity in 8 patients with advanced-stage CTCL. All patients experienced significant decrease in clinical symptoms in response to aggressive, transient antibiotic treatment. In some patients, clinical improvements lasted for more than 8 months. In 6 of 8 patients, a malignant T-cell clone could be identified in lesional skin, and a significant decrease in the fraction of malignant T cells was observed following antibiotics but an otherwise unchanged treatment regimen. Immunohistochemistry, global messenger RNA expression, and cell-signaling pathway analysis indicated that transient aggressive antibiotic therapy was associated with decreased expression of interleukin-2 high-affinity receptors (CD25), STAT3 signaling, and cell proliferation in lesional skin. In conclusion, this study provides novel evidence suggesting that aggressive antibiotic treatment inhibits malignant T cells in lesional skin. Thus, we provide a novel rationale for treatment of SA in advanced CTCL.

Single-cell heterogeneity in Sézary syndrome.

Sézary syndrome (SS) is an aggressive leukemic variant of cutaneous T-cell lymphoma (CTCL) with a median life expectancy of less than 4 years. Although initial treatment responses are often good, the vast majority of patients with SS fail to respond to ongoing therapy. We hypothesize that malignant T cells are highly heterogeneous and harbor subpopulations of SS cells that are both sensitive and resistant to treatment. Here, we investigate the presence of single-cell heterogeneity and resistance to histone deacetylase inhibitors (HDACi) within primary malignant T cells from patients with SS. Using single-cell RNA sequencing and flow cytometry, we find that malignant T cells from all investigated patients with SS display a high degree of single-cell heterogeneity at both the mRNA and protein levels. We show that this heterogeneity divides the malignant cells into distinct subpopulations that can be isolated by their expression of different surface antigens. Finally, we show that treatment with HDACi (suberanilohydroxamic acid and romidepsin) selectively eliminates some subpopulations while leaving other subpopulations largely unaffected. In conclusion, we show that patients with SS display a high degree of single-cell heterogeneity within the malignant T-cell population, and that distinct subpopulations of malignant T cells carry HDACi resistance. Our data point to the importance of understanding the heterogeneous nature of malignant SS cells in each individual patient to design combinational and new therapies to counter drug resistance and treatment failure.