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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 ATMp38 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.

In cutaneous T cell lymphomas (CTCL), miR-21 is aberrantly expressed in skin and peripheral blood and displays anti-apoptotic properties in malignant T cells. It is, however, unclear exactly which cells express miR-21 and what mechanisms regulate miR-21. Here, we demonstrate miR-21 expression in situ in both malignant and reactive lymphocytes as well as stromal cells. qRT-PCR analysis of 47 patients with mycosis fungoides (MF) and Sezary Syndrome (SS) confirmed an increased miR-21 expression that correlated with progressive disease. In cultured malignant T cells miR-21 expression was inhibited by Tofacitinib (CP-690550), a clinical-grade JAK3 inhibitor. Chromatin immunoprecipitation (ChIP) analysis showed direct binding of STAT5 to the miR-21 promoter. Cytokine starvation ex vivo triggered a decrease in miR-21 expression, whereas IL-2 induced an increased miR-21 expression in primary SS T cells and cultured cytokine-dependent SS cells (SeAx). siRNA-mediated depletion of STAT5 inhibited constitutive- and IL-2-induced miR-21 expression in cytokine-independent and dependent T cell lines, respectively. IL-15 and IL-2 were more potent than IL-21 in inducing miR-21 expression in the cytokine-dependent T cells. In conclusion, we provide first evidence that miR-21 is expressed in situ in CTCL skin lesions, induced by IL-2 and IL-15 cytokines, and is regulated by STAT5 in malignant T cells. Thus, our data provide novel evidence for a pathological role of IL-2Rg cytokines in promoting expression of the oncogenic miR-21 in CTCL.

Sustained inflammation is a key feature of mycosis fungoides (MF), the most common form of cutaneous T-cell lymphoma (CTCL). Resident IL9-producing T cells have been found in skin infections and certain inflammatory skin diseases, but their role in MF is currently unknown.We analyzed lesional skin from patients with MF for the expression of IL9 and its regulators. To determine which cells were producing IL9, high-throughput sequencing was used to identify malignant clones and Vb-specific antibodies were employed to visualize malignant cells in histologic preparations. To explore the mechanism of IL9 secretion, we knocked down STAT3/5 and IRF4 by siRNA transfection in CTCL cell lines receiving psoralen+UVA (PUVA) ± anti-IL9 antibody. To further examine the role of IL9 in tumor development, the EL-4 T-cell lymphoma model was used in C57BL/6 mice.Malignant and reactive T cells produce IL9 in lesional skin. Expression of the Th9 transcription factor IRF4 in malignant cells was heterogeneous, whereas reactive T cells expressed it uniformly. PUVA or UVB phototherapy diminished the frequencies of IL9- and IL9r-positive cells, as well as STAT3/5a and IRF4 expression in lesional skin. IL9 production was regulated by STAT3/5 and silencing of STAT5 or blockade of IL9 with neutralizing antibodies potentiated cell death after PUVA treatment in vitro IL9-depleted mice exhibited a reduction of tumor growth, higher frequencies of regulatory T cells, and activated CD4 and CD8 T lymphocytes.Our results suggest that IL9 and its regulators are promising new targets for therapy development in mycosis fungoides. Clin Cancer Res; 22(13); 3328-39. ©2016 AACR.

Aberrant activation of Janus kinase-3 (Jak3) and its key down-stream effectors, Signal Transducer and Activator of Transcription-3 (STAT3) and STAT5, is a key feature of malignant transformation in cutaneous T-cell lymphoma (CTCL). However, it remains only partially understood how Jak3/STAT activation promotes lymphomagenesis. Recently, non-coding microRNAs (miRNAs) have been implicated in the pathogenesis of this malignancy. Here, we show that (i) malignant T cells display a decreased expression of a tumor suppressor miRNA, miR-22, when compared to non-malignant T cells, (ii) STAT5 binds the promoter of the miR-22 host gene, and (iii) inhibition of Jak3, STAT3, and STAT5 triggers increased expression of pri-miR-22 and miR-22. Curcumin, a nutrient with anti-Jak3 activity and histone deacetylase inhibitors (HDACi) also trigger increased expression of pri-miR-22 and miR-22. Transfection of malignant T cells with recombinant miR-22 inhibits the expression of validated miR-22 targets including NCoA1, a transcriptional co-activator in others cancers, as well as HDAC6, MAX, MYCBP, PTEN, and CDK2, which have all been implicated in CTCL pathogenesis. In conclusion, we provide the first evidence that de-regulated Jak3/STAT3/STAT5 signalling in CTCL cells represses the expression of the gene encoding miR-22, a novel tumor suppressor miRNA.

Cutaneous T-cell lymphomas (CTCL) are characterized by the presence of chronically inflamed skin lesions containing malignant T cells. Early disease presents as limited skin patches or plaques and exhibits an indolent behavior. For many patients, the disease never progresses beyond this stage, but in approximately one third of patients, the disease becomes progressive, and the skin lesions start to expand and evolve. Eventually, overt tumors develop and the malignant T cells may disseminate to the blood, lymph nodes, bone marrow, and visceral organs, often with a fatal outcome. The transition from early indolent to progressive and advanced disease is accompanied by a significant shift in the nature of the tumor-associated inflammation. This shift does not appear to be an epiphenomenon but rather a critical step in disease progression. Emerging evidence supports that the malignant T cells take control of the inflammatory environment, suppressing cellular immunity and anti-tumor responses while promoting a chronic inflammatory milieu that fuels their own expansion. Here, we review the inflammatory changes associated with disease progression in CTCL and point to their wider relevance in other cancer contexts. We further define the term "malignant inflammation" as a pro-tumorigenic inflammatory environment orchestrated by the tumor cells and discuss some of the mechanisms driving the development of malignant inflammation in CTCL.

Dissertação de mestrado em Bioquímica Aplicada, Lymphoma is the most common blood cancer. The incidence of Cutaneous T Cell Lymphoma (CTCL) has been increasing and, in 2016, affected 6.4 per million persons. Symptoms can include dry skin, itching, a red rash, and enlarged lymph nodes. The cutaneous burden of CTCL rapidly decreases quality of life for patients. Therefore, a need for research directed at the cutaneous symptoms and skin involvement arises. Thus, the major aim of this project was to shed light on the effect of malignant T cells on keratinocytes, which can induce changes on the structure and function of the epidermis. This project was divided into three segments which allowed to associate modification of epidermis’ components to disease morbidity and other CTCL complications: i) identification of genes related to skin barrier function whose expression responds to malignant T cells; ii) identification of signalling pathways in malignant T cells that lead to the modulation of keratinocyte gene expression; and iii) identification of signal transduction pathways in keratinocytes responsible for the alteration in gene expression. In this thesis it is provided novel evidence that malignant T cells are able to modulate keratinocyte gene and protein expression, which takes the form of down- regulation of KRT1, KRT10, DSC1, DSG1, and FLG, whose proteins are all important for the maintenance of a normally functioning epidermal barrier. In contrast, no expression modulation was seen for KRT5, KRT14, DSC3, and DSG3. This difference hints to a possible inhibition of keratinocyte differentiation by malignant T cells. Regarding pathway activity in malignant T cells, the JNK pathway seems to be responsible for the effects seen on KRT1, KRT10, and FLG. Other pathways, p38, JAK3, and ERK, also appear to have a role in the downregulation of KRT1, DSC1, and FLG, respectively. In keratinocytes, the JAK3 pathway seems to play a major role in the down- regulation of KRT1, KRT10, DSC1, and FLG. In accordance, JAK3 substrates, STAT3 and STAT6, become phosphorylated in response to malignant T cell supernatant, suggesting that FLG expression is modulated by the JAK3/STAT3 pathway. Taken together, this thesis identified the mechanism by which CTCL T cells induce barrier disruption in the epidermis, which could help explain the symptoms reported by the patients. Furthermore, it identified new putative targets for treatment of the cutaneous burden of CTCL with the prospect to increase quality of life and decrease complications related to bacterial infections often seen in CTCL patients., Linfoma é o tipo mais comum de cancro sanguíneo. A incidência de Linfoma Cutâneo de Células T (CTCL) tem vindo a aumentar e, em 2016, afetava 6,4 pessoas por milhão. Os sintomas podem incluir pele seca, comichão, erupções cutâneas vermelhas, e nódulos linfáticos dilatados, sendo que estes sintomas diminuem a qualidade de vida dos pacientes. Assim, surge a necessidade de investigar o envolvimento da pele neste tipo de linfoma. Desta forma, o principal objetivo deste projeto passou pela descoberta do efeito das células T malignas nos queratinócitos, que pode subsequentemente induzir alterações na estrutura e função da epiderme. Para atingir este objetivo, o projeto foi dividido em três tarefas que permitiram a associação da modificação de componentes da epiderme à morbilidade da doença e outras complicações: i) identificação de genes relacionados com a função de barreira da pele cuja expressão reage a células T malignas; ii) identificação de vias de sinalização nas células T malignas que levam à modulação de expressão génica nos queratinócitos; e iii) identificação de vias de transdução de sinal nos queratinócitos responsáveis pela alteração da expressão de genes. Nesta tese são apresentadas novas evidências de que as células T malignas são capazes de modular a expressão de genes e proteínas nos queratinócitos, causando especificamente a diminuição da expressão de KRT1, KRT10, DSC1, DSG1, e FLG, cujas proteínas são importantes para a manutenção de uma barreira epidermal funcional. Em contraste, não foi observada qualquer modulação da expressão de KRT5, KRT14, DSC3, e DSG3. Esta diferença indica uma possível inibição da diferenciação dos queratinócitos pelas células T malignas. A respeito da atividade de vias de sinalização nas células T malignas, a via JNK parece ser responsável pelos efeitos na expressão de KRT1, KRT10, e FLG. Outras vias, como p38, JAK3, e ERK, também parecem ter um papel na diminuição da expressão de KRT1, DSC1, e FLG, respetivamente. Nos queratinócitos, a via JAK3 parece ter um papel importante na diminuição da expressão de KRT1, KRT10, DSC1, e FLG. Em concordância, os substratos de JAK3, STAT3 e STAT6, são fosforilados em resposta a sobrenadante de células T malignas, sugerindo que a expressão de FLG é modulada pela via JAK3/STAT3. Em suma, esta tese identificou o mecanismo pelo qual as células T malignas de CTCL induzem disrupção da barreira da epiderme, que poderá explicar os sintomas reportados pelos pacientes. Identificaram-se, ainda, novos alvos putativos para o tratamento do envolvimento cutâneo do CTCL com a perspetiva de aumentar a qualidade de vida e diminuir as complicações relacionadas com infeções bacterianas, frequentemente observadas em pacientes com CTCL.