LEE- Moleküler Biyoloji-Genetik ve Biyoteknoloji Lisansüstü Programı

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http://hdl.handle.net/11527/19400

Gözat

Konu "allerji ve immünoloji" ile LEE- Moleküler Biyoloji-Genetik ve Biyoteknoloji Lisansüstü Programı'a göz atma
  • Öge
    Molecular genetic assessment of immune modulators in eosinophils and t cells both in silico and in vitro utilizing photoacoustic imaging approaches
    (ITU Graduate School, 2025) Çelik, Zülal ; Çıracı Muğan, Ceren ; 521221117 ; Molecular Biology-Genetics and Biotechnology
    The immune system is a network of specialized cells and chemicals with distinct functions that have different roles in fighting infection. It protects the host from pathogenic invaders and provides memory of the infection to defend the host against future encounters. The ability of immune cells may extend beyond host defense to include growth, tissue homeostasis, and repair mechanisms. In recent decades, it has been demonstrated that the origin of inflammation determines the immune response and that the immune system plays a role in maintenance, renewal, and sterile inflammation. There are two kinds of responses to non-self: innate responses that can be developed regardless of exposure an adaptive responses that can develop more rapidly with repeated exposures. Anatomical and physiological barriers such as lysozyme in tears, mucosal secretions, gastric pH, and skin constitute the primary defense. When these barriers are compromised, innate immunity is activated to maintain tissue homeostasis and defend against infections, cancer, and toxins. It is rapid, non-specific. The cells of the Innate immunity are eosinophils, basophils, mast cells, monocytes, dendritic cells, neutrophils, and macrophages. They release chemokines and cytokines, remove pathogens by phagocytosis. Eosinophils are multifunctional granulocytes responding to mostly parasitic infections and allergies. They originate in the bone marrow from homeopoietic stem cells in response to IL-5 and GM-CSF. After maturation, they enter bloodstream and tissues via IL-5 and eotaxin signaling. On the other hand, the adaptive immune system includes B and T cells where. T cells mature in the thymus and B cells in the bone marrow, and produce antibodies. Upon maturation, they migrate to secondary lymphoid tissues to capture antigens and initiate responses primed by innate immune signals. T cells are categorized into naive, memory, and regulatory subsets. Upon activation, they produce IL-2 andthey differentiate into effector cells enhancing the clearance via cytokines and cytotoxic mediators. Pattern recognition receptors (PRRs) sense molecular patterns associated with pathogens, apoptotic cells, and senescent cells. They link innate and adaptive immunity by producing non-specific protective mediators. PRRs are grouped by domain homology into TLRs, NLRs, RLRs, and CLRs. They recognize PAMPs and DAMPs, distinguish sterile and non-sterile inflammation. Although associated with innate immunity, PRRs are also expressed by T and B cells, suggesting a functional overlap. NLRP3 is the most studied inflammasome-related PRR, first identified via mutations causing cryopyrin-associated syndromes. It contains NACHT, LRR, and PYD domains, enabling ASC recruitment and caspase-1 activation, leading to pro-IL-1β processing. NLRP3 is unique in being activated by diverse agonists including pathogens, toxins, environmental factors, and DAMPs. The use of advanced imaging techniques to visualize biological processes has become a key focus in this field. Photoacoustic (PA) imaging is a hybrid biomedical imaging technique based on the thermo-acoustic effect generated by laser stimulation. It integrates the benefits of optical and ultrasonic imaging. Consequently, PA imaging is applicable to both pre-clinical research and clinical medicine for the diagnosis of cancer, microcirculation irregularities, and evaluation of therapy efficacy. This thesis presents two-part exploration of innate and adaptive immunity. It begins with a closer look at eosinophil-like cells in the context of PRRs and continues with the functional impact of Montelukast on T cells using both molecular and PA imaging approaches. In the first chapter, the goal was to compare the basal expression of pattern recognition receptors (PRRs)—including both membrane-bound and cytosolic types—in human eosinophil-like cell lines (EoL-1 and HL-60) to human primary eosinophils (HPEs), using publicly available in silico databases. Given the limitations of working with primary eosinophils, determining cell lines that are most closely mimicking primary cells' receptor profiles was a necessary first step toward adopting the most suitable in vitro models. These findings were intended to support future mechanistic studies on eosinophil-driven immunity. The second chapter of this thesis, focuses on the how Montelukast (MNT), a leukotriene receptor antagonist commonly prescribed for asthma, influences inflammatory signaling in Jurkat T cells. To serve this purpose, T cell like Jurkat cells cells were treated with MNT for 48 hours, either alone or in combination with cell activator (C.A). Expression levels of several key immune-related genes and proteins, including CYSLTR1, NLRP3, ASC, IL-1β, IL-10, and signaling molecules such as Akt and ERK, were measured using molecular assays including RT-qPCR, immunoblotting, ELISA, and immunocytochemistry (ICC). In addition to molecular approaches, photoacoustic (PA) imaging was used to examine how MNT treatment affected Jurkat cells changed optical signal characteristics. This combined strategy widened our view on the molecular changes induced by Montelukast in T cells, while also testing the potential of PA imaging as a non-invasive approach for detecting drug-driven immune modulation. In the first chapter, basal expression of PRRs' mRNA levels of human eosinophils were analyzed in silico and comparative profiles of EoL-1 and HL-60 cell lines with HPEs were determined. The analysis showed that levels of TLR2 and NLRP3 mRNA were significantly higher in both cell lines than in HPE (p<0.01), whereas NLRP12, LGP2 and Dectin-1 receptors mRNA levels were higher in HPE than in both cell lines (p<0.01). In addition, TLR6, MDA5, NOD2, NLRP1, CLEC4A were detected to be expressed at higher basal levels in EoL-1 and/or HL-60 than in PE. Although TLR3, TLR8 and TLR9 expressions were not detected in PE, these receptors were expressed in cell lines. TLR5 expression was unique to HPE. MDL-1 (CLEC5A) levels were significantly higher in HL-60 cells as compared to HPE, while CLEC4A expression was higher in EoL-1 cells than in HPE. These data suggested that EoL-1 and HL-60 cells are suitable in vitro models for certain PRRs, however, studying these receptors in cell lines may be challenging, particularly for those such as LGP2, NLRP12 and Dectin-1, which have high expression levels in HPE. In the second chapter, effects of MNT on molecular inflammatory responses in Jurkat T cell line were evaluated at 48 hour post-stimulation. CYSLTR1 mRNA levels were significantly upregulated after 10-⁵ M MNT treatment when compared to control group (~2.5-fold, p < 0.0001). NLRP3 expression was also upregulated by C.A. treatment (p <0.01), and this response was further elevated in the MNT+ C.A. group as compared to control and only-C.A. (p < 0.01). These results showed that MNT may induce inflammation via NLRP3. When ASC adaptor protein was measured by immunoblotting, it was significantly downregulated in both C.A. and MNT+ C.A. groups (p <0.05). We then mesured IL-1β release by ELISA and showed that there was no significant difference between the treatment and control groups (ns). However, , intracellular IL-1β expression was detected to be increased in the C.A. group (p <0.001) and reached the highest level in the MNT+ C.A. group (p <0.01) by immunocytochemical analysis.. We also determine the signal transduction pathways that are potentially involved in the MNT responses. Initially, we investiagted AKT protein, which was decreased in the C.A. group (p <0.05) and no significant difference was observed in p-AKT level. However, the p-AKT/AKT ratio increased in the C.A. group (p <0.01) and this increase was maintained in the MNT+ C.A. group (ns). Phosphorylated ERK ½ (p-ERK1/2) level increased in the C.A. group (p < 0.05) and reached the highest levels in the MNT+ C.A. group (p < 0.01).. In addition we examine the anti-inflamatory response. For this, IL-10 production was analyzed and expectedly, IL10 was not significantly differentially expressed after treatment. As for the photoacoustic imaging results, PA signal amplitude was significantly decreased in the MNT-treated groups- both single treatment and in combination with C.A. when compared to the control and C.A. groups. The mean amplitude value was 2523.22 in the control group, 2283.59 in the C.A. group, 755.66 in the MNT group, and 1088.19 in the MNT+ C.A. group. This suggests that MNT effects on the cells was measured as a reduced photoacoustic signal. In conclusion, the first chapter provided an in silico comparison of PRR expression profiles between eosinophil-like cell lines and HPEs, presenting practical insights into selecting more reliable and suitable models for future in vitro studies on eosinophil-driven immune mechanisms. The second chapter investigated the effects of MNT on inflammatory signaling in Jurkat T cells, revealing changes in cytokine expression, inflammasome-related proteins, and intracellular signaling pathways. The inclusion of photoacoustic imaging enabled the detection of biophysical changes at the cellular level during treatment. By integrating computational, molecular, and imaging-based approaches, this study presents a multi-way exploration of immune responses. Future research may be extented to primary human T cells and other drugs that requires the determination of side effects for different therapeutic interventions.