Saudi Cultural Missions Theses & Dissertations
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Item Restricted Investigating the Mechanobiology of Macrophages: Implications for Inflammatory Bowel Disease(The university of Sheffield, 2024) Alaklobie, Mubarak; Erdmann, KaiMacrophages are essential cells of the innate immune system, playing a key role in regulating inflammation, tissue repair, and homeostasis. Their behaviour is tightly controlled by various signalling pathways, including mechanical forces that influence their shape, movement, and function. This process, known as mechanotransduction, allows cells to sense and respond to mechanical signals from their environment, converting these signals into biochemical responses that regulate cellular behaviour. Dysregulation of macrophage functions can lead to chronic inflammatory diseases and cancer. Recent studies have shown that mechanical cues, such as extracellular matrix (ECM) stiffness, fluid flow, cell crowding, and topography, modulate macrophage behaviour in various physiological and pathological contexts. However, the effect of ECM stiffness at relevant physiological levels, particularly in inflammation and fibrosis, has not been fully understood. Previous studies have often relied on single or limited marker approaches, which may not capture the full complexity of macrophage polarization. To address this gap, we conducted a series of experiments aimed at characterizing THP-1 and bone marrow-derived macrophage (BMDM) protocols to ensure proper validation and reproducibility for our study. We then adapted ECM stiffness values, mimicking the conditions seen in inflammatory bowel disease (IBD), representing both normal and inflamed-fibrotic tissue. Experiments were conducted to assess macrophage polarization states in response to varying stiffness levels. Our results reveal that increasing ECM stiffness promotes the expression of YAP and IL-6 in M1 macrophages, driving a shift towards a pro-inflammatory phenotype. In contrast, M2 macrophages exhibited elevated levels of the anti-inflammatory markers CD163 and IL-10, reflecting an adaptive response to softer ECM conditions. Interestingly, M0 macrophages, which are considered to be non-polarized, adopted a hybrid phenotype, expressing both YAP and CD163, underscoring the inherent plasticity of macrophages when subjected to mechanical stress. In primary BMDMs, stiff ECM conditions induced also mixed phenotypes with favoured M1 polarization, as shown by a significant overlap with established M1 gene expression signatures, further emphasizing the role of ECM stiffness in driving pro-inflammatory responses. These findings challenge the traditional binary M1/M2 polarization model, suggesting that macrophage responses to mechanical cues are nuanced and context dependent. In the second part of this thesis, we investigated the mechanical regulation of the Poly(C)-binding protein 1 (PCBP1) in macrophages and its role in macrophage polarisation. PCBP1 is a multifunctional RNA-binding protein that plays a crucial role in regulating mRNA stability, splicing, and translation. It is also involved in iron metabolism, acting as an iron chaperone, and is involved in DNA damage repair. Our experiments demonstrate that ECM stiffness and cell density regulate PCBP1 subcellular localization in macrophages. In stiff ECM and low-density environments, PCBP1 localises mainly to the nucleus, while in soft ECM and high cell density, it remained cytoplasmic. PCBP1 knockdown increased CD163 expression, suggesting it modulates M2 polarization. Finally, we demonstrate a possible role of PCBP1 in ECM stiffness dependent DNA damage repair, suggesting a novel mechanism of mechanoprotection.14 0Item Restricted Investigating Asymmetric Dimethylarginine (ADMA) and Calcium-sensing receptor (CaSR) signalling in the macrophage(University of Glasgow, 2024-04) Alanazi, Sarah; Leiper, JamesMacrophages play a significant role in maintaining physiological homeostasis and orchestrating immune responses. In vascular diseases like atherosclerosis, they actively participate in plaque formation and progression by accumulating lipids and modulating inflammatory processes. A G-protein coupled receptor (GPCR) known as the calcium-sensing receptor (CaSR) is activated by a plethora of ligands and couples to distinct signalling cascades. Initially recognised for its role in regulating calcium (Ca2+) levels in calciotropic tissues, the CaSR has since been revealed to exhibit tissue/cell-specific effects. In macrophages, CaSR expression is confirmed where it influences immunological processes. Our research group demonstrated that asymmetric dimethylarginine (ADMA), an endogenous nitric oxide synthase (NOS) inhibitor, acts as a CaSR ligand in adipocytes, exerting NOS- independent effects. Based on this, it was hypothesised that a similar signalling pathway operates in macrophages, modulating inflammation and can be targeted in disease. In this study, immunocytochemistry confirmed the expression of both CaSR and the ADMA-metabolising enzyme, dimethylarginine dimethylaminohydorlase-2 (DDAH-2), in the RAW264.7 macrophage cell line used in this investigation, indicating their functional presence within these cells. Furthermore, their mRNA expression was unaffected by the pro-inflammatory stimuli, lipopolysaccharides (LPS). Next, the immunological impact of the DDAH-2/ADMA metabolic pathway in inflammation was explored using RNA-sequencing of LPS-stimulated murine macrophages with a macrophage-specific DDAH-2 knockout. This revealed that DDAH-2 plays a crucial role in modulating several genes associated with the LPS- response, exerting an anti-inflammatory effect. Specifically, DDAH-2 attenuates LPS-stimulated effects in macrophages, including antigen presentation, phagocytosis and chemotaxis. Additionally, DDAH-2 was found to influence the expression of a number of CaSR target genes that were previously identified in the literature. Furthermore, qPCR investigations on the role of ADMA in the expression of selected genes that displayed differential expression upon DDAH-2 deletion showed that ADMA regulates Il17a expression, highlighting a pro-inflammatory ii effect of ADMA in these cells. This study further revealed that ADMA does not influence the expression of any of the other selected genes for validation, including those recognsised as CaSR targets. This demonstrates that DDAH-2 has ADMA-independent effects in activated macrophages. This thesis also delved into the impact of CaSR on macrophage function, revealing its pro-inflammatory role. Specifically, in non-stimulated macrophages, activation of CaSR by its positive allosteric modulator (PAM), cinacalcet, induced IL-6 expression. ADMA treatment in these cells did not elicit a similar response, indicating the absence of ADMA- CaSR crosstalk in IL-6 regulation. This study further demonstrated the pro- inflammatory effect of ADMA in non-stimulated macrophages where ADMA induced a dose-dependent induction of IL-1β and TNF-α which was independent of CaSR signalling. By developing a novel macrophage-specific CaSR knockout mouse model, this thesis validated the utility of this model for future investigations into macrophage CaSR. This validation was achieved through confirmed significant deletion of CaSR and the viability of these mice. Furthermore, this work demonstrated that macrophage CaSR deficiency resulted in the upregulation of TNF-α in non- stimulated macrophages, highlighting CaSR’s role in regulating both pro- and anti- inflammatory effects in macrophages. Additionally, the absence of the CaSR in macrophages resulted in an age- and sex-dependent reduction in weight gain observed in female mice, an observation that requires exploration in future studies. Taken together, this thesis demonstrates DDAH-2, ADMA, and CaSR as regulators of the macrophage response. Nevertheless, it did not reveal a signalling crosstalk between ADMA and CaSR within this cell type. Future research should focus on elucidating the mechanisms underlying the observed effects, where they can be explored as potential drug targets against pathologies where macrophages are implicated.20 0Item Restricted Macrophage Function in Processing of Excess Dietary Fat(Saudi Digital Library, 2023-06) Bajonaid, Amal; Van Dyke, ThomasBackground: Chronic low-grade metabolic inflammation is one of the hallmarks of metabolic diseases. The risk of periodontitis is approximately threefold higher in diabetic individuals. To examine this relationship, we used a strain of transgenic mice overexpressing the receptor for Resolvin E1 (ERV1tg) that have a reduced inflammatory phenotype depicted by higher resolving (M2) to inflammatory macrophage (M1) ratio and are resistant to periodontitis and compared them to wild type (WT). Objective: investigate the role of resolving macrophages in processing of excess dietary fat in ERV1 tg mice and determine the difference that leads to sexual dimorphism in energy utilization by metabolic organs. Materials and Methods: 6-week-old ERV1tg and WT male and female mice were fed HFD for 16 weeks, 5 mice per group. Differences in expression of uncoupling protein 1 (UCP-1), peroxisome proliferator-activated receptor γ coactivator 1 (PGC-1⍺), nuclear respiratory factor 1 (NRF-1), sirtuin1 (SIRT1) and estrogen-related receptor alpha (ERRα) in visceral adipose tissue (VAT), liver and brown adipose tissue (BAT) were assessed using real-time quantitative PCR and western blot. mRNA expression was normalized to peptidylprolyl isomerase A (PPIA) as a reference gene. Protein expression was normalized to Actin-B. to investigate the impact of RvE1 on cholesterol uptake by macrophages and efflux to HDL, we used RAW264.7 murine macrophage cell line and performed in vitro cholesterol uptake and efflux assay and investigated the impact of varying concentrations of RvE1 on cholesterol uptake and efflux to HDL. Statistical analysis: One-way ANOVA with ⍺=0.05. Results: Expression of PGC1-α mRNA in VAT was significantly higher in ERV1 tg female mice (4.4-fold ± 0.66) compared to ERV1 tg male mice (0.65-fold ± 0.12, p <0.0001). Relative NRF- 1 mRNA expression in VAT was significantly higher in ERV1 tg female mice (1.6-fold ± 0.12) compared to ERV1 tg male mice (0.65 ± 0.13, p < 0.0001). ERV1 tg female mice showed significantly higher expression of Sirt-1 mRNA in VAT (3.1-fold ± 0.26) compared to ERV1 tg male mice (1.7-fold ± 0.29, p < 0.0001). NRF-1 mRNA expression in the liver was significantly higher in ERV1 tg female mice (2.5-fold ± 0.21) compared to ERV1 tg male mice (2.0-fold ± 0.59, p = 0.0002). Relative Sirt-1 mRNA expression in BAT was significantly higher in ERV1 tg female mice (2.2-fold ± 0.12) compared to WT female (0.5-fold ± 0.08, p < 0.0001), and ERV1 tg male mice (1.3-fold ± 0.29) compared to WT male mice (0.47-fold ± 0.09, p < 0.0001). Conclusions: This study provides direct evidence for molecular targets of RvE1-mediated regulation of obesity that display sexual dimorphism suggesting that female ERV1 tg mice have higher metabolic flexibility through increased mitochondrial function to adapt to excess energy.22 0