Hohenheim, Univ., Diss., 2008.

A descoberta de novos alvos terapêuticos capazes de reverter o efeito tumoral imunossupressor sobre as células dendríticas (DCs) é de grande relevância clínica. Identificamos que monócitos de pacientes com leucemia linfóide crônica (LLC) têm alterações na sinalização de STAT6 induzida por IL-4 que previne a maturação fenotípico-funcional das DCs. Embora os monócitos dos pacientes apresentem alta expressão de IL-4R, a atividade de STAT6 está inibida devido aos elevados níveis de SOCS5. IL-10 reproduz esta desregulação de STAT6/SOCS5 nos monócitos de doadores saudáveis, causando diferenciação defeituosa das DCs. Isso indica que SOCS5 está envolvida nas alterações das DCs de pacientes. Ainda, encontramos que a inibição de STAT3 com pirimetamina, um composto em ensaio clínico para LLC, não afeta a maturação das DCs, diferentemente da inibição de STAT5 por JQ1. Isso mostra que STAT5 é importante para a maturação das DCs, e sugere que a JQ1, mas não a pirimetamina, pode causar imunossupressão. Já SOCS5 pode ser um novo potencial alvo para terapia do câncer. / Discovery of new targets to reverse tumor immunosuppression on dendritic cells (DCs) hold great therapeutic promise. Here, we identify that monocytes from chronic lymphocytic leukemia (CLL) patients have alteration in IL-4-induced STAT6 signaling that prevents DCs phenotypic and functional maturation. Although patients monocytes display high IL-4R expression, STAT6 activity is inhibited because of elevated SOCS5 levels. IL-10-treatment of healthy donors monocytes reproduces this altered mechanism (STAT6/SOCS5) and leads to a defective DC differentiation. These findings indicate that a high SOCS5 level is involved on CLL-DCs impaired function. Moreover, we find that pharmacologic inhibition of STAT3 by pyrimethamine, a clinical trial compound for CLL, does not affect LPS-induced DCs maturation while STAT5 inhibition by JQ1 prevents it. Our findings show that STAT5 is important for DCs maturation, and suggest that JQ1, but not pyrimetamine, can cause immunosuppression. Additionally, SOCS5 emerges as a new potential target for cancer treatment.

Submitted by Fabio Sobreira Campos da Costa (fabio.sobreira@ufpe.br) on 2017-07-12T15:39:14Z No. of bitstreams: 2 license_rdf: 811 bytes, checksum: e39d27027a6cc9cb039ad269a5db8e34 (MD5) Dissertação Juan Luiz Coelho da Silva.pdf: 2693101 bytes, checksum: b946d507d9f21698d6349e8ecf91e259 (MD5) / Made available in DSpace on 2017-07-12T15:39:14Z (GMT). No. of bitstreams: 2 license_rdf: 811 bytes, checksum: e39d27027a6cc9cb039ad269a5db8e34 (MD5) Dissertação Juan Luiz Coelho da Silva.pdf: 2693101 bytes, checksum: b946d507d9f21698d6349e8ecf91e259 (MD5) Previous issue date: 2016-03-11 / FACEPE / Algumas evidências destacam mutações no gene ASXL1 como um evento importante na evolução clínica de pacientes com neoplasias hematológicas, particularmente em leucemias mieloides agudas e síndrome mielodisplásicas. Contudo, seu impacto prognóstico em neoplasias mieloproliferativas (NMP) ainda é pouco explorado. Aqui, nós caracterizamos 208 pacientes com NMP cromossomo Filadélfia (Ph) negativo (policitemia vera, PV; trombocitemia essencial, TE; mielofibrose primária, MFP), de acordo com mutações no gene ASXL1, e correlacionamos esses achados com características clinico-laboratoriais desses pacientes. A pesquisa das mutações foi realizada por sequenciamento sanger, em que polimorfismos germinativos e mutações sinonímias foram excluídas das análises. Mutações no ASXL1 foram detectadas em 22/208 pacientes (10%), das quais quatro foram observadas em pacientes com PV (4/54; 7%), onze em pacientes com TE (11/123; 9%) e sete com MFP (7/31; 22%). As características clínicas e laboratoriais foram similares entre pacientes com ASXL1 mutado e não mutado. Quando as entidades foram avaliadas individualmente (PV, TE e MFP), observou-se associação entre mutações no ASXL1 e idade mais avançada em pacientes com TE (P = 0,049) e desenvolvimento de esplenomegalia em pacientes com MFP (P = 0,026). Com uma mediana de seguimento de 5,1 anos (IC95%: 4,5 a 7,3 anos), 136 pacientes (65%) desenvolveram algum tipo de manifestação clínica, sendo o desenvolvimento de complicações vasculares o mais frequente (n=54; 26%), seguido por esplenomegalia (n=47; 22%), eventos hemorrágicos (n=30; 14%) e trombose (n=21; 10%). Mutações no gene ASXL1 não foram associadas com o desenvolvimento das referidas manifestações. Dentro deste seguimento, apenas dois pacientes evoluíram para síndrome mielodisplásica e um para leucemia mieloide aguda, todos sem mutações no gene ASXL1. / Accumulating evidences report mutation in ASXL1 as an important predictor to clinical outcomes of patients with hematological malignancies, particularly acute myeloid leukemia and myelodysplastic syndrome. However, the prognostic impact in myeloproliferative neoplasm (MPN) remains underexplored. Here, we evaluated clinical and laboratory features of 208 Philadelphia negative MPN patients (polycythemia vera, PV; essential thrombocythemia, ET; primary myelofibrosis, PMF), according to mutations in ASXL1. Screening for ASXL1 mutations were performedby Sanger sequencing. Germline variations were excluded. ASXL1 mutations were detected in 22/208 patients (10%), of which four in PV patients (4/54-7%), 11 in ET patients (11/123-9%) and seven in PMF (7/31-22%). Baseline features were similar between ASXL1-mutated and non-mutated patients. Evaluated individually (PV, ET, PMF), we observed that ET patients harboring ASXL1 mutations were older (P = 0,049) than ASXL1 non-mutated patients. Similarly, PMF patients presented higher frequency of splenomegaly in ASXL1mutated group (P = 0,026). No other features were associated with ASXL1mutations. The median follow-up was 5,1 years (CI95%: 4,5-7,3 years). One hundred and thirty six patients (65%) developed some of the clinical common manifestations, which the most frequent was vascular complications (n=54; 26%), followed by splenomegaly (n=47; 22%), bleeding (n=30;14%) and thrombosis (n=21;10%). ASXL1 mutations were not associated with development of such events. In our cohort, only two patients have evolved for myelodysplastic syndrome and one for acute myeloid leukemia, all of them without mutations in ASXL1.

La SUMOylation est une modification post-traductionnelle qui gouverne divers processus cellulaires incluant immunité innée et défense antivirale. Des effecteurs de la synthèse d’IFN, de son signal de transduction ainsi que des facteurs de restriction sont modifiés par SUMO (Small Ubiquitin Modifier). Par ailleurs, certains virus exploitent la machinerie SUMO afin de contrecarrer les mécanismes de défense antivirale suggérant l’implication de SUMO dans l’interface virus et défense antivirale. A l’aide d’un modèle cellulaire exprimant les différents paralogues SUMO1, SUMO2 ou SUMO3 ou en diminuant l’expression de l’unique enzyme de conjugaison à SUMO, Ubc9, nous avons montré un effet différentiel de SUMO sur deux virus de la famille des Rhabdoviridae (virus de la stomatite vésiculaire (VSV) et le virus de la rage) et sur la réponse aux IFN alpha et IFN gamma. Le premier axe de recherche a permis de montrer que l’expression de SUMO inhibe la synthèse de l’IFN suite à l’infection par le VSV et le virus de la rage, rendant les cellules plus sensibles à l’infection par le virus de la rage. IRF3 est conjuguée à SUMO, ce qui corrèle avec l’inhibition de sa phosphorylation et l’inhibition de la synthèse d’IFN beta. En revanche, bien que la synthèse de l'IFN soit diminuée, l’expression de SUMO confère la résistance au VSV et inhibe sa transcription primaire. L’activité anti-VSV de SUMO est abolie par la déplétion de MxA. L’effet de SUMO est médié par sa capacité à augmenter l’oligomérisation et la stabilité de MxA. Par ailleurs, ce travail a permis d’identifier MxA comme nouvelle cible de la machinerie SUMO. MxA interagit avec SUMO de manière covalente sur la lysine K48 et de manière non covalente avec SUMO1. Le second axe de recherche a permis d’identifier SUMO comme un nouveau régulateur de la réponse aux IFN. La SUMOylation de STAT1 inhibe sa phosphorylation, régulant négativement le signal de transduction et par conséquent la transcription et la réponse biologique en réponse à l’IFN gamma. En revanche, l’expression de SUMO n’altère ni le signal de transduction ni la transcription en réponse à l’IFN alpha.Par ailleurs, dans les cellules exprimant SUMO3, l’IFN gamma et l’IFN alpha induisent la SUMOylation de PML par SUMO3 ce qui entraîne sa dégradation via le protéasome et inhibe les réponses biologiques médiées par PML. Ce travail a permis de montrer un rôle central de SUMO dans l’immunité intrinsèque et innée, médié par la SUMOylation de protéines cellulaires telles qu’IRF3, MxA, STAT1 ou PML. / SUMOylation modulates several cellular process including innate immunity and antiviral defense. Many key regulators involved in IFN induction, IFN signaling as well as various restriction factors are SUMOylated. Using cells stably expressing the different paralogs of SUMO; SUMO1, SUMO2 or SUMO3 and cells depleted of the only known SUMO conjugating enzyme, Ubc9, we show a differential effect on two viruses from Rhabdoviridae family (Vesicular Stomatitis Virus (VSV) and rabies virus) and on the response to IFN alpha and IFN gamma. First, we report that SUMO expression inhibits VSV- and rabies virus-induced IFN synthesis. Consequently, SUMO expression renders cells more sensitive to rabies virus infection. Overexpression of SUMO leads to IRF3 SUMOylation correlating rabies viral infection with both the inhibition of IRF3 activation and IFN beta synthesis. However, although SUMO inhibits VSV-induced IFN, SUMO confers resistance to VSV by inhibiting VSV primary transcription. Furthermore, the anti-VSV effect of SUMO is abolished in MxA depleted cells. Mechanistically, SUMO enhances MxA oligomerization resulting in the stabilization of the MxA protein. We also identified MxA as a new target of SUMO machinery. MxA interacts covalently with SUMO2/3 on lysine K48 and non-covalently with SUMO1. We then investigated the various roles of SUMO at different steps of the JAK/STAT pathway, including STAT activation, transcriptional response and IFN-induced biological effects, identifying SUMO as a new regulator of IFN response. The overexpression of SUMO leads to STAT1 SUMOylation and to a decrease in IFN-induced STAT1 phosphorylation resulting in an inhibition of IFN-gamma-induced transcription and biological responses. In contrast, SUMO expression does not alter IFN alpha signaling and transcriptional response. In addition, in SUMO3 expressing, IFN gamma;and IFN alpha induce SUMOylation of PML by SUMO3 inducing its degradation via the proteasome and inhibition of biological responses mediated by PML. Taken together our results show that SUMO plays a crucial role in innate and intrinsic immunity mediated by SUMOylated proteins such as IRF3, MxA, STAT1 or PML.

Ligand-activated signal transduction is a process critical to cell survival and function as it serves as a means of communication between the cells and their environment. Endocytosis is generally thought to down-regulate incoming signals by reducing the surface availability of receptors. However, increasing evidence in many systems suggests a notion which is referred to as the „signalling endosome" hypothesis - that endocytosis can also actively contribute to signalling apart from clearance of activated receptors and thereby attenuation of signalling. The functional aspect of signalling endosomes has been well-characterized in several pathways including RTK and TGF-β signalling. There are, however, various other signalling pathways where the active mechanism of endocytotic regulation is yet to be understood. In this study, we probe this aspect in the cytokine signalling system, where the receptors are known to internalize but the significance of such internalization and precise mechanism is unclear. My thesis aims to elucidate the function and molecular details of internalization of cytokine receptor using interleukin-4 receptor (IL-4R) signalling as a model. IL-4 and IL-13 ligands can induce assembly of three distinct complexes: IL4 induced IL-4Rα – IL-2Rγ (type I), IL-4 induced IL-4Rα – IL-13Rα1 (type II) or the IL-13 induced IL-13Rα1-IL-4Rα (type II). The formation of any of these complexes triggers signalling through the JAK/STAT pathway. However, models of how the oligomerization of the transmembrane receptors and activation takes place are very diverse and lack a clear molecular and biophysical understanding of the underlying receptor dynamics. Previous results of the lab had shown that the affinities between subunits are low, precluding complex formation at the plasma membrane at physiological concentrations. In addition, IL-4R subunits localize in to endosomal structures adjacent to the plasma membrane. It had already been shown that the shared IL-4R subunit IL-2Rγ is internalized by a specific, actin dependent, Rac1/Pak1 regulated endocytosis route in the IL-2 context. We could show that pharmacological suppression of this endocytosis pathway also prevented IL-4 induced JAK/STAT signalling, placing endocytosis upstream of signalling. Here I show using immuno-EM techniques that these endosomal structures are multivesicular bodies. Importantly, I could show that receptor subunits are highly enriched in the limiting membrane of these endosomes relative to the adjacent plasma membrane. Using quantitative loading assays I could furthermore demonstrate that this enrichment is achieved by constitutive internalization of receptors from the cell surface into cortical endosomes. The trafficking kinetics of the receptor subunits is independent of ligand occupancy. Pharmacological inhibition shows that receptors and ligand traffic via the previously identified Rac1/Pak1 pathway. Finally, Vav2 was identified as a candidate Guanine Exchange Factor (GEF) that may regulate Rac1 activity and thereby control the actin polymerization cascade driving IL-4R endocytosis. Immunoprecipitations showed that Vav2 interacts both with the cytoplasmic tail region of the receptors and the receptor associated 2 kinase JAK3. Vav2 may thus couple the receptor/JAK complexes to the Rac1/Pak1 mediated endocytosis route. Taken together, our results suggests that stable „signalling endosomes‟ adjacent to the plasma membrane act as enrichment centres, where ligand and receptor concentrations are locally increased by constitutive trafficking. The confined environment of the endosome then compensates for the weak affinities between the ligand and receptor and facilitates ligand-mediated receptor dimerization. Importantly, overexpression of both type II IL-4R subunits renders signal transduction resistant to endocytosis inhibition, strongly suggesting that the critical factor effecting signalling is sufficient concentration, which the endosomes facilitate achieving. The endosomes are thus dispensable as signalling scaffolds when the receptors are in sufficient concentration, where activated receptors could interact with downstream pathway components. Endocytosis thus provides a crucial means for the signalling process to overcome the thermodynamic hurdles for receptor oligomerization. In conclusion, our data propose a novel, purely thermodynamic role of endosomes in regulating cytokine receptor signalling not seen in any other signalling pathway.

The JAK-STAT pathway is an intracellular signaling pathway that is found to have crucial roles in hematopoiesis, immune response and the development of many other tissues in mammals. The pathway is conserved in Drosophila melanogaster, and is much simpler: there is only one Drosophila JAK (Hopscotch, Hop) and STAT (STAT92E) respectively, while there are at least 4 JAKs and 7 STATs in mammals. The pathway has been intensively studied in Drosophila, and has been implicated in many tissue development and cellular processes. In this work, I present several roles of JAK signaling in oogenesis.First, JAK signaling is required for cell differentiation within a specific lineage of follicle cells – stalk cells and polar cells. Unpaired (upd), which encodes the known ligand for the pathway, is expressed specifically in the polar cells in the developing egg. Reduced function of Upd or Hop results in fusions of egg chambers, which is primarily caused by improper formation of stalk cells, while general activation of the pathway in the egg chamber produces an extra number of stalk cells and sometimes eliminates polarfollicle cells. Based on the known function of the Notch pathway in oogenesis, we propose a model that Notch signaling determines a pool of precursors for the polar and stalk cells while JAK activity determines their specific fates within that pool.Second, JAK signaling is also involved in epithelial follicle cell differentiation. Consistent with the expression pattern of upd in the ovary, there is a gradient of JAK activity expanding from the poles, and this JAK activation gradient is both required and sufficient to suppress the main body follicle cell fate. Also, different levels of JAK activity are required and sufficient to determine both anterior and posterior terminal follicle cell fates. Consistent with these data is a model that a gradient of JAK activity triggered by Upd from the poles pre-patterns the epithelium into three domains and pre-determines sub-populations of terminal follicle cell fates prior to the EGFR activation, and cooperates with EGFR activity later to define posterior terminal follicle cell fates. This provides the first evidence for a morphogenic function of the JAK-STAT pathway in any organism.

The JAK/STAT pathway is a non-receptor tyrosine kinase signaling pathway that is well conserved and highly re-utilized in many mammalian and Drosophila developmental processes. Compared to dozens of ligands and receptors in mammalian JAK/STAT, Drosophila JAK/STAT pathway is simpler with one receptor and three ligands, Upd, Upd2 and Upd3, which have similar amino acid sequences. Previous literature shows that upd and upd2 exhibit the same dynamic striped expression pattern in embryos and have semi-redundant functions during embryogenesis. Do Upd and Upd3 also have redundant functions? To answer this question, the functions of Upd3 in Drosophila development were investigated in this dissertation. In addition, the coordinate expression mechanism of upd and upd3 in eye discs was also analyzed. To study the functions of Upd3 in development, the expression pattern of upd3 was examined and detected in larval eye discs, wing discs, haltere discs, lymph glands and adult ovaries with in situ hybridization to upd3 mRNA and an upd3 reporter line. Consistent with the expression pattern, the loss of function mutants of upd3 exhibit small eyes, outstretched wings, downward extended halteres and reduced circulating blood cell concentration, demonstrating the roles of Upd3 in these tissues’ development. However, functions of Upd3 in other aspects of immune response were not detected. To investigate the mechanism of the coordinate expression of upd and upd3, the genetic and molecular relationship of upd, upd3 and os was dissected. The os alleles, oso, oss and os1, are a group of classical alleles which display outstretched wings, small eyes, or both, respectively. The genetic complementation tests of upd, upd3 and os showed that both upd and upd3 failed to complement os while upd complemented upd3, suggesting functions of both upd and upd3 are affected in os alleles. Consistent with the genetic tests, the expression of upd and upd3 in eye discs is lost in os allele. Molecularly, putative enhancer regions are deleted at the 5’ end of upd3 in os alleles. Hence, a transcriptional co-regulation model of upd and upd3 is proposed in which upd and upd3 share a common cis-regulatory region, lesions of which cause the os phenotype.

The purpose of the study was to analyze the relationship between the state and religious communities in Sweden, using historical and contemporary background and Resource mobilization theory.  Results and conclusions show that Sweden is by law a Christian state. The Swedish state's definition of religion is narrow, and is reflected in legal texts. Furthermore, state subsidies to religious communities are distributed, administered and controlled by the communities themselves through political posts. The state and some religious organizations have, according to Resource mobilization theory, an unequal exchange relationship in which the religious communities get credibility and contributions in exchange for social work and confessional alternatives in public service. The religious organization´s most important resources in terms of money, legitimacy and political affairs come from the state. In this way, the state gives selected religious organizations the opportunity for political influence, while excluding others, according to Resource mobilization theory.

Our lab recently described a role for JAK-STAT signaling in the maintenance of quiescence during the murine hair cycle. Research into signaling pathways and cytokines/growth factors involved in the mammalian hair cycle has not focused extensively on the JAK-STAT pathway. In this thesis, I investigated the upstream effector(s) and downstream mechanisms of JAK-STAT signaling in the HFSC during telogen, using a variety of methods, including murine conditional mutants of the JAK-STAT pathway, pharmacological and immunological techniques. The mechanism through which OSM exerts this effect is via JAK-STAT5 signaling downstream of the OSM receptor, which is antagonized by pharmacological JAK inhibition. Conditional epidermal ablation of OSMR or STAT5 during early- and mid-telogen (P42 – P60) shortens the telogen phase significantly, and inhibition of macrophages by way of neutralizing antibodies, small molecule inhibitors, and genetic ablation (with Csf1r-CreER::R26-iDTR mice) during telogen also promotes hair growth. Single-cell RNA sequencing of dermal immune cells across murine telogen identified a distinct subset of TREM2+ macrophages that are enriched for OSM, and gene-set analysis suggests these “trichophages” are similar to the microglia of the central nervous system. I show that this distinct subset of TREM2+ macrophages predominate during early- and mid-telogen, where they produce Oncostatin M (OSM), which is sufficient to maintain quiescence of hair follicle stem cells (HFSCs). Proliferation of HFSCs and hair growth is associated with depletion of this subset of TREM2+ macrophages. Interestingly, macrophage markers and OSM were found to be upregulated in the balding scalp of males with androgenetic alopecia, suggesting that this mechanism is physiologically relevant in the control of human hair cycling.

Immune cells must accurately interpret environmental signals to make robust cell fate decisions and control inflammatory signalling. Many signals (e.g. Tumor Necrosis Factor alpha (TNFα) or interferon gamma (IFNγ)) converge on just a few key signalling systems such as Nuclear Factor kappa B (NF-κB) or Signal Transducers and Activators of Transcription (STAT), which exhibit complex activation dynamics that control patterns of downstream gene expression. Often, seemingly identical cells show heterogeneous or random behaviour to a common stimulus. Therefore, a key question is how can immune cells coordinate inflammatory signalling in the presence of this noise. The NF-kB system dynamics were studied in response to rapidly changing inflammatory signals. It was shown that pulsed TNFa cytokine stimulations induced digital single-cell NF-kB responses, with only a fraction of cells able to respond to repeated pulses. These responses appeared to be reproducible in individual cells, but heterogeneous in the population. Mathematical models of the NF-kB signalling network suggested that single-cell responses were governed through a refractory state potentially encoded via 'extrinsic' noise in the levels of signalling molecules related to the TNFa signal transduction pathway. Such signal processing enabled robust and reproducible single cell responses and maintained acute tissue-level signalling, with fewer cells responding to shorter pulsing intervals. The NF-kB system is involved in effector cytokine propagation in response to pathogen infection. It was shown that in macrophages, the dose of TLR4 stimulation (mimicking the pathogen infection) was encoded in graded (yet heterogeneous) NF-kB dynamics in single cells. This resulted in analogue inflammatory gene expression patterns in the population. However, individual cells substantially differed in their ability to encode TLR4 signal and to regulate TNFa expression, which was explained by extrinsic noise in the NF-kB system. Quantitative mathematical modelling showed that tissue-level environment modulates heterogeneous single-cell TNFa outputs; by effectively removing it from circulation. This may determine the interaction distance between tissue-resident immune cells to enable propagation of cellular inflammation. Heterogeneity of single cell macrophage signalling was also observed in NF-kB and STAT1 system responses to a range of IFN stimulation doses. Although each system showed substantial variability between cells, their responses were surprisingly well correlated in individual cells. It was however apparent (based on gene expression studies) that individual cells may not be able to precisely discriminate different IFNg doses. Overall, this work suggests that heterogeneity in the NF-kB (and other) regulatory networks might be a part of an inherent design motif in the inflammatory response, which enables robust control of the tissue-level inflammatory response by preventing homogeneous and thus potentially harmful activation.