ATR-FTIR evaluation of structural and functional changes on murine macrophage cells upon activation and suppression by immunotherapeutic oligodeoxynucleotides


Thesis Type: Postgraduate

Institution Of The Thesis: Orta Doğu Teknik Üniversitesi, Faculty of Arts and Sciences, Department of Biology, Turkey

Approval Date: 2015

Student: EMRULLA SPAHİU

Consultant: MAYDA GÜRSEL

Abstract:

In the present study, ATR-FTIR was used to examine the structural and functional changes on murine macrophage cells upon activation and suppression by immunotherapeutic ODNs. Macrophage cells play a key role in innate and adaptive immunity. Their possession of high flexibility helps them to successfully respond to environmental signals. In this context, they can impressively alter their physiology in relation to their function and these changes have large molecular signals in the infrared spectral region that can be traced with FTIR spectroscopy. LPS and CpG ODN are used as well-known stimulants of macrophages to produce the powerful pro-inflammatory agents such as cytokines, chemokines and highly specific Ig\\\'s. CpG ODN is potentially used in many immunotherape utic therapies, but is found to cause allergy, toxic shock and death. The need for a suppressive agent led to the innovation of suppressive ODN, whose mechanism is not elucidated. In the present study, murine macrophage-like RAW 264.7 cells are treated with PBS medium, CpG ODN, LPS, suppressive ODNA151, and K3 Flip-ODN in four different time scales, to monitor the changes on the course of time. Homogenous /μL cell samples were prepared and FTIR spectra were recorded in triplicates. In total n=5 was performed. The spectra recorded underwent smoothing, baseline correction and normalization steps, and quantitative analysis was performed. One way-ANOVA with Dunnet’s test for multiple comparisons and Kruskal–Wallis with Dunn\\\'s test for multiple comparisons was used for the analysis of normally and non-normally distributed data, respectively. All results were expressed as mean SEM. The results clearly indicate that CpG-induced macrophage cells show prominent features in th eir IR spectra for their activation. The time period till 2h of stimulation seems to be the period of extensive RNA synthesis, and as it ceases, DNA synthesis starts from the 6th hour of stimulation upon both CpG ODN and LPS stimulation, except LPS at 6 hours, due to the property of LPS being an anti-proliferative agent. It is also found that stimulation of macrophage cells with CpG ODN and LPS decreases protein content. This is explained by the production of numerous oxidants, causing oxidative stress and in intention of protecting the cell; proteasomes start the degradation of oxidized proteins. The membrane fluidity increases during activation with CpG ODN and LPS, as a result of decreased unsaturated lipid content, increased lipid peroxidation and decreased cholesterol content. It was also noted that these changes may have altered the signal transduction processes, ion permeability and many other primary functions of cell membranes could be altered.