Transforming Growth Factor Beta Suppression of CD8+ T Cell Proliferation

Transforming Growth Factor Beta (TGF-beta) is highly suppressive to both CD4+ and CD8+ T cell proliferation and function. in tumor microenvironments, TGF-beta has been described as immune suppressive, particularly to CD8+ T cells, however, the molecular mechanism behind how TGF-beta signaling controls T cell growth is not fully understood. Here, we report that TGF-beta inhibits CD8+ T cell proliferation and reduces expression of the CARMA1/BCL10/MALT1 (CBM) signalosome complex in activated CD8+ T cells. the CBM signalosome is an essential scaffold that forms after T cell receptor (TCR) stimulation, leading to the activation of NF-kB and AP-1. This observed reduction in the CBM complex occurs simultaneously with a reduction in CD25 (IL-2Ralpha) expression. Together, the data suggest that TGF-beta inhibits antigen-stimulated CD8+ T cells by reducing CD25 expression and defines that the CBM complex may be one of the targets that mediates the effects of TGF-beta on CD25 expression

<i>In Vitro</i> Modelling of Cellular Processes in OM-BMDM Studies in Junbo Mice Reveal Defects in HIF and TGF-β Signalling

Chronic otitis media (OM) is a common cause of deafness in children. Two novel murine models of chronic OM, Junbo and Jeff, generated by chemical mutagenesis with ethyl nitrosurea (ENU), have been developed at MRC Harwell. Junbo heterozygote mice (Jbo/+) mice have a mutation in transcription factor Evi-1 and Jeff heterozygote mice (Jf/+) mice have a mutation in gene for Fbxo protein Fbxo11. Pathologic hypoxia has been characterized as a common feature of the inflamed middle ear in both models. HIF-VEGF pathways dysregulation has been indicated in both models previously. However, the mechanisms involved in pathogenesis of OM in these two models are yet to be elucidated. In this thesis I have described an in-vitro model system to study cellular processes in OM to study and identify pathways involved in OM pathogenesis. This model system involves culturing BMDM and exposing them to various treatments under standard defined conditions. Results from this thesis reveal that Evi-1 is expressed at comparable levels in both WT and Jbo/+ genotypes and that Evi-1A2288T mutation is a loss of function mutation which results in dysregulated expression of Smad responsive and hypoxia responsive genes such as Vegf and Glut-1 under prolonged hypoxic conditions. Although Jbo/+ BMDM are hyper-responsive to LPS in normoxia, as was indicated by higher Vegf and IL-6 levels, macrophage phagocytic function is potentially attenuated in Jbo/+ under hypoxic conditions as was indicated by lower levels of Tnf-α and IL-1β in Jbo/+ BMDM in LPS and hypoxia combination treatment. A perturbed resolution of IL-6 and IL-1β in hypoxia was also observed. Studies performed in this thesis revealed dysregulation in TGF-β pathway in both Jbo/+ and Jf/+ BMDM in normoxia which suggests a common role of TGF-β signalling in OM pathogenesis. Differential expression of cytokines was observed in Jbo/+ BMDM after prolonged treatment with TGF-β and hypoxia indicating a pro-angiogenic and pro-inflammatory phenotype. Levels of pro-inflammatory cytokines such as IL-1β, C5a, IL-17, IL-23, and Ccl5 were higher in Jbo/+ BMDM and levels of anti-inflammatory cytokines such as IL-1ra, IL-10, and IL-4 were lower in Jbo/+ BMDM after prolonged hypoxia and TGF-B treatment which further validates the role of dysregulated HIF and TGF-β signalling pathways in OM in Jbo/+ mice. The differential cytokine expression observed in Jbo/+ BMDM is favourable for Th17 cell differentiation and may indicate a role of cross-talk of innate immunity and cell mediated immunity in OM chronicity in hypoxia. To conclude, HIF signalling and TGF-β signalling in Jbo/+ BMDM is dysregulated. Pro-angiogenic function, impaired resolution of inflammation and predisposition to Th17 cell differentiation by Jbo/+ BMDM under hypoxia contribute to the prolonged and chronic inflammation in Jbo/+ mice and may provide some evidence of pathways that could be affected in OM patients

microRNA Expression Profile of Purified Alveolar Epithelial Type II Cells

Alveolar type II (ATII) cells are essential for the maintenance of the alveolar homeostasis. However, knowledge of the expression of the miRNAs and miRNA-regulated networks which control homeostasis and coordinate diverse functions of murine ATII cells is limited. Therefore, we asked how miRNAs expressed in ATII cells might contribute to the regulation of signaling pathways. We purified "untouched by antibodies" ATII cells using a flow cytometric sorting method with a highly autofluorescent population of lung cells. TaqMan® miRNA low-density arrays were performed on sorted cells and intersected with miRNA profiles of ATII cells isolated according to a previously published protocol. Of 293 miRNAs expressed in both ATII preparations, 111 showed equal abundances. The target mRNAs of bona fide ATII miRNAs were used for pathway enrichment analysis. This analysis identified nine signaling pathways with known functions in fibrosis and/or epithelial-to-mesenchymal transition (EMT). In particular, a subset of 19 miRNAs was found to target 21 components of the TGF-β signaling pathway. Three of these miRNAs (miR-16-5p, -17-5p and -30c-5p) were down-modulated by TGF-β1 stimulation in human A549 cells, and concomitant up-regulation of associated mRNA targets (BMPR2, JUN, RUNX2) was observed. These results suggest an important role for miRNAs in maintaining the homeostasis of the TGF-β signaling pathway in ATII cells under physiological conditions

Dissecting the cellular and mechanical changes driving keloid scarring

Here we exploit keloid scars, a fibroproliferative skin disorder with no known cause or cure, to investigate this feature of fibrosis. We show that ECM alignment is mimicked in vitro by culturing fibroblasts isolated from the keloid dermis, and therefore, keloid fibroblasts (KDF) are used to investigate the cellular and mechanical drivers of fibrotic matrix alignment. KDF develop a significantly aligned supracellular actin network, mediated by cell-cell adhesions, that enable the formation of aligned ECM. We reveal that autocrine interleukin-6 (IL- 6) in keloid fibroblasts is both necessary and sufficient to induce this phenotype. Downstream of IL-6, independent pharmacological inhibition of ERK, AKT and STAT3, prevent cell alignment, highlighting a role for these signalling pathways in this fibrotic phenotype. Upstream of IL-6, although our data were inconclusive, we propose a role for c-Jun in the constitutive production of IL-6 in keloid fibroblasts. Keloid fibroblasts also display aligned focal adhesions, which we hypothesise generate aligned traction forces that enable the anisotropic remodelling of the ECM. To examine patterns in tractions, traction force microscopy (TFM) was performed on KDF and NDF. No significant differences in traction alignment were found, highlighting that further optimisation may be required. Overall, the work presented in this thesis suggests a novel function for IL-6 in keloid pathogenesis, as IL-6 signalling has been shown to be vital in cell and ECM remodelling. These data suggest that therapeutic targeting of IL-6 may not only reduce inflammation, but al

Stem cell-based phenotypic screening and characterization of novel BMP mimetics

The identification of selective small molecule cytokine mimetics and signaling activators holds great promise for numerous applications in biomedicine as they overcome the typical drawbacks of physiological peptide- or protein-based ligands. Yet, the development of such modalities remains a challenging task in drug discovery. In this thesis, a phenotypic, target-agnostic, high-throughput screening assay is presented that probes Bone Morphogenetic Protein (BMP) signaling during mesodermal patterning of murine embryonic stem cells. This approach represents a novelty in BMP activator identification by harnessing embryonic development in vitro, hence potentially expanding the druggable space of BMP signaling activators. During mesoderm specification, BMP signaling can be temporally discriminated from Transforming Growth Factor- (TGF)-driven stages of cardiac differentiation. This selective and authentic orchestration of BMP signaling cues can be recapitulated for the discovery of genuine BMP activator- or potentiator chemotypes. Here, a robust hit validation workflow has been devised, including the orthogonal assessment of BMP activation during osteoblastogenesis as well as BMP-dependent reporter assays. Proof-of-concept is demonstrated from screening of nearly 7,000 chemically diverse compounds, yielding 2,3-disubstituted 4H-chromen-4-ones as a new potent BMP activating chemical modality. Chromenone 1 enhanced osteogenic differentiation and mineralization in vitro. Structure-activity relationship (SAR) studies with 29 different Chromenones revealed pharmacophoric features relevant to BMP activity and provided a valuable toolkit of active and structurally closely related inactive derivatives. Interestingly, mechanistic studies suggested that Chromenone 1 enhances canonical nuclear BMP-Smad signaling outputs through an unparalleled, kinase-independent, negative TGF-Smad feedback loop. This is in sharp contrast to the reported BMP sensitizer PD407824, revealing novel BMP potentiator biology as desired from a conceptionally new morphogenic and phenotypic drug discovery approach. An additional screening of 1,408 known bioactive compounds eventually furnished the triazolo[1,5 c]quinazoline CGS-15943 as a novel and unique BMP activating chemotype that exhibited highest selectivity among all profiled and characterized BMP modulators in the present work. Its capacity to amplify BMP signaling during embryogenesis could be further substantiated in vivo during zebrafish development (i.e., ventralization phenotypes). Target deconvolution studies unraveled novel targets of CGS-15943 with a pronounced potency for CK1- and --isoforms. Mechanistically, CGS-15943 amplifies osteogenic BMP signaling outputs through a dual targeting of CK1/ and PI3K (p110) isoforms by enhancing BMP signaling independent of a TGF-feedback. Together, the presented work underscores proof-of-concept for a novel phenotypic drug discovery strategy to identify unique BMP signaling modulators. Two new chemotypes ultimately expanded the druggable space of osteogenic BMP activators and sensitizers that may serve as valuable tools for various applications in (stem) cell biology and regenerative medicine. Notably, this work might spur future translation to even more complex 3D-gastruloid and organoid systems

Glucose metabolism in idiopathic pulmonary fibrosis

Idiopathic pulmonary fibrosis (IPF) is a life-threatening interstitial lung disease of unknown aetiology characterized by progressive scarring of the lung parenchyma. Histologically, the hallmark of the disease is the presence of interspersed fibroblastic foci in the lung, composed of contractile myofibroblasts synthesizing a dense collagen-rich matrix. Transforming growth factor-β1 (TGFβ1) has been recognized as a key cytokine in the pathophysiology of IPF and other fibrotic disorders. Highly proliferative cells, such as cancer cells, reprogram their glucose metabolism through the activation of the PI3K-AKT-mTOR axis towards enhanced glycolysis, a process known as aerobic glycolysis. In view of the high biosynthetic nature of myofibroblasts, this thesis aimed to (1) describe the changes in glucose metabolism that occur during the process of TGFβ1-induced fibroblast to myofibroblast differentiation, (2) examine whether these changes are regulated by the PI3K-AKT-mTOR axis, and (3) examine the relationship between glucose uptake and fibrogenesis in an experimental model of lung fibrosis. For the in vitro experiments, the metabolic profile of primary human lung fibroblasts was assessed by examining cellular glucose uptake, glycolytic flux and mitochondrial respiration. Furthermore, using highly selective and potent pharmacological inhibitors, the role of the PI3K-AKT-mTOR pathway in promoting changes in glucose metabolism during fibroblast differentiation was examined. For the in vivo experiments, position emission tomography-computed tomography scanning and autoradiography were performed in the murine bleomycin model of lung injury and fibrosis following administration of radioactive 18F-labeled fluoro-2-deoxyglucose. Taken together, the data presented in this thesis demonstrate that the metabolic phenotype of fibroblasts changes during TGFβ1-induced fibroblast differentiation and is regulated by mTOR, in a PI3K-AKT-independent manner. This metabolic switch may further explain the observation of increased glucose uptake in the fibrotic lesions in the bleomycin model of lung fibrosis. These findings support the notion that pharmacological targeting of glucose metabolism and/or the mTOR kinase may be beneficial in preventing myofibroblast differentiation in IPF