Role of interferon regulatory factor 5 (IRF5) in the regulation of t helper cytokines

Amar Singh, Ashwinder Kaur (2021) Role of interferon regulatory factor 5 (IRF5) in the regulation of t helper cytokines. PhD thesis, University of Nottingham.

[img] PDF (Thesis - as examined) - Repository staff only - Requires a PDF viewer such as GSview, Xpdf or Adobe Acrobat Reader
Available under Licence Creative Commons Attribution.
Download (5MB)


Interferon regulatory factor 5 (IRF5) has a diverse role in the induction of pro-inflammatory cytokines and chemokines downstream to various signalling pathways contributing to the pathogenesis of various autoimmune and inflammatory diseases. Using Irf5 knockout mice under different experimental settings, studies have reported that IRF5 plays a crucial role in T helper (Th) polarization. Previous studies have shown that IRF5 has a potential role in regulating Th2 cytokine production, particularly Interleukin-13 (IL-13). Sequence analysis human Il13 promoter showed the presence of the IFN sensitive response element (ISRE) motif, the region that IRFs can recognize through their helix turn helix nucleotide domain. As a transcription factor, IRF5 has been shown to recognize and bound to this region to regulate the expression of its targeted genes. Based on these findings, we were intrigued to examine the transcriptional role of IRF5 in regulating the expression of human IL-13 and assess the modulation of IRF5 in regulating the expression of Th1 and Th2 associated cytokines.

Two of the predominant transcriptionally active IRF5 isoforms, IRF5 variant 4 (IRF5v4) and variant 5 (IRF5v5) were used in this study. To evaluate the binding of the IRF5 to Il13/ISRE region, DNA pulldown assay was performed using protein lysates extracted from HEK 293T cells transfected with either IRF5v4 or IRF5v5 in the presence or absence of the Toll-like receptor (TLR) adaptor protein, Myeloid differentiation primary response 88 (MyD88) expression. The ectopic MyD88 was included to mediate activation of IRF5 spliced isoforms. The transactivation of Il13 promoter activities by IRF5 spliced isoforms were examined through dual luciferase reporter assay. To do so, HEK 293T cells were transiently transfected with varying concentrations and combinations of IRF5v4 or IRF5v5 in the presence or absence of MyD88. The human IL13 promoter driven by firefly luciferase reporter vector and Renilla luciferase control vector were included for dual luciferase reporter activities. To evaluate the modulation of IRF5 spliced isoforms in regulating Th1 and Th2 cytokines, stably expressing IRF5 isoforms were generated in Jurkat cells, a human T cell line. Cells were stimulated with a combination of Phorbol-12-myristate 13-acetate (PMA) and Ionomycin for evaluating cytokines expression by reverse transcription polymerase chain reaction (RT-PCR) and secretion by enzyme-linked immunosorbent assay (ELISA).

Based on the DNA pulldown assay, our findings showed that both activated IRF5 spliced isoforms can bind to the oligonucleotide corresponding to Il13/ISRE. In luciferase reporter assay, our results showed that both IRF5v4 and IRF5v5 can transactivate Il13 promoter activation, however, their activities were suppressed when higher MyD88 concentrations were tested. By using semi-quantitative RT-PCR, we demonstrated that stable expression of IRF5v4 and IRF5v5 in Jurkat cells modulated Th1 and Th2 associated cytokines in response to co-stimulation of PMA and Ionomycin. Upon stimulation, overexpressed IRF5v5 showed upregulation of IFN and downregulation of IL-4 and IL-13 gene expression. On the other hand, IRF5v4 showed induction of IL-13 but inhibition of IL-4 gene expression. In protein level, IRF5v4 had a diminished level of IFNγ secretion. Interestingly, both IRF5v4 and IRF5v5 were shown to augment IL-2 expression in gene and protein levels. Moreover, IRF5v4 and IRF5v5 were able to secrete moderate amounts of IL-10 in comparison to control cells. In conclusion, our findings provided useful insight into IRF5 spliced isoforms’ involvement in inducing human Il13 promoter activity as well as the differential regulation of the expression of Th1 and Th2 cytokines in the human T cell line.

Item Type: Thesis (University of Nottingham only) (PhD)
Supervisors: Fang, Chee Mun
Keywords: IRF5; IL-13 promoter analysis; Th1/Th2 cytokines; immune system; transcription factor
Subjects: R Medicine > RS Pharmacy and materia medica
Faculties/Schools: University of Nottingham, Malaysia > Faculty of Science and Engineering — Science > School of Pharmacy
Item ID: 65826
Date Deposited: 04 Aug 2021 04:43
Last Modified: 03 Aug 2023 04:30

Actions (Archive Staff Only)

Edit View Edit View