Development and optimization of real-time PCR assays to detect anti-microbial immune factors and their response to type I and II interferons.
Year: 2016
Type: dissertation
Abstract: Introduction: Tuberculosis is the leading cause of mortality in HIV-infected individuals worldwide.HIV-infected individuals have increased risk of developing active tuberculosis even at high CD4 T-cell counts and while undergoing effective antiretroviral therapy.The mechanisms that result in anti-TB immune dysfunction in HIV-infected people are not well understood.Previous studies have shown that a type II (IFN-γ) interferon-driven vitamin D-dependent antimicrobial pathway in monocytes and macrophages is important for the control of Mycobacterium tuberculosis (M.tb) growth in vitro.Notably, acute and chronic HIV infection are associated with high levels of type I interferons, specifically interferon alpha 2a (IFNα2a).Type I interferon has been shown to be upregulated during M. tb infection and has been found to be detrimental to M. tb control.We, therefore, hypothesised that HIV-1 infection induces an interferon-driven dysregulation of the vitamin D-dependent antimicrobial pathway in monocytes, which may predispose to tuberculosis susceptibility.The goal of this project is to lay the methodological i groundwork that will allow a comprehensive assessment of this hypothesis in a monocytic cell line and in primary human peripheral blood samples.Methods: In this study, a one-plate quantitative polymerase chain reaction (qPCR) protocol for the reliable detection of Cytochrome P450 family 27 subfamily B member 1 (CYP27b1), cytochrome P450 family 24 subfamily A member 1 (CYP24a1), Cathelicidin (CAMP), Beta-defensin (DEFB4), and Vitamin D receptor (VDR) by the LightCycler 480 (Roche) was optimised for the human monocyte monocytic cell line (THP-1).The assays were also used to evaluate the expression of these transcripts in primary human peripheral CD14+ monocytes isolated using immunomagnetic separation from healthy donors (n=3).Cells were stimulated with recombinant human interferon gamma (rhIFNγ) (2,000 IU/mL) and recombinant human interferon alpha (rhIFN2a) (2,500 IU/ mL) for 24 hours.The impact of Type I and Type II interferons on gene expression were calculated using the Pfaffl equation to compare expression of genes of interest (normalised to GAPDH) between stimulated and unstimulated conditions.Results: Quantitative PCR protocols were developed to reproducibly measure CAMP, VDR and CYP27b1 in THP-1 cells and primary human monocytes.Basal levels of these three transcripts were expressed at similar levels in THP-1 cells and primary monocytes relative to GAPDH, however, CYP27b2 was notably less expressed in CD14+ cells relative to THP-1 cells.DEFB4 and CYP24a1 could not be detected in THP-1 cells1s but were reproducibly detectable in primary human monocytes.Comparative analysis of THP-1s and healthy donor CD14+ monocytes after stimulation with type I and II interferon showed varying modulation of the genes of interest.In THP-1s IFNα2a had the expected inhibitory effect on the three detectable transcripts of interest (CAMP, CYP27b1 and VDR).Stimulation with IFNγ resulted in the unexpected down-regulation of CAMP and CYP27b1.In healthy donor CD14+ monocytes stimulated with IFNγ, CAMP, CYP24a1, CYP27b1 and DEFB4 were all significantly upregulated.IFNα2a resulted in a trend towards up-regulation of CAMP, CYP24a1, CYP27b1 and DEFB4.VDR expression was not modulated by either Type I or II interferon stimulation in healthy donor monocytes.
Author: Nathan Scott Kieswetter
Language: English
Subfield: Infectious Diseases
Field: Medicine
Domain: Health Sciences
Open Access status: green
OpenAlex ID: W2770767787