Aram, Jehan Jalal
(2018)
Granulocyte-macrophage colony-stimulating factor: expression and regulation in human immune responses with relevance to multiple sclerosis.
PhD thesis, University of Nottingham.
Abstract
Background: Granulocyte-Macrophage Colony-Stimulating Factor (GM-CSF) is a haematopoietic growth factor and a pro-inflammatory cytokine produced by T cells and other immune cells. Recent evidence suggests that GM-CSF plays an important role in multiple sclerosis (MS) pathogenesis. Few recent studies have detected GM-CSF expression by immune cells in MS. In this thesis, the expression of GM-CSF and its receptor by different subtypes of peripheral blood mononuclear cells (PBMCs) in MS was investigated. In addition, GM-CSF regulation was studied in the above-mentioned cells in MS. Finally, GM-CSF neutralization was performed in a phase Ib clinical trial, and some immune-related effects were investigated.
Aims: To evaluate the expression of GM-CSF and its receptor by PBMC subsets in MS; to determine the key factors regulating their expression by PBMC subsets in MS; to detect the differentiation of helper T cells producing GM-CSF (Th-GM) in MS patients, and to detect the frequency of immune cells after GM-CSF neutralization in MS in vivo.
Subjects and Methods: Patients were mainly untreated relapsing-remitting MS (RRMS) during remission stage, and some were MS patients during a relapse. Healthy controls were also enrolled. All subjects consented to participation in the study before donating peripheral blood. PBMCs were isolated using Ficoll density gradient centrifugation. Flow cytometry and q-PCR were used to detect the expression of GM-CSF and its receptor. Multiplex bead assay was used to quantify GM-CSF with other pro-inflammatory and anti-inflammatory cytokines.
Results: The frequency of stimulated GM-CSF-expressing cells (helper T (Th), cytotoxic T (Tc), monocytes, NK cells, and B cells) is significantly higher in the mixed PBMC population of untreated RRMS patients when compared to healthy volunteers. The frequency of Th1 cells expressing GM-CSF was higher in MS patients than healthy controls. The expression of GM-CSF by isolated and stimulated NK cells was not different in MS patients and controls. PBMC culture supernatants were shown to contain significantly higher concentrations of IL-2, IL-12, IL-1β, and GM-CSF in MS patients than controls. Blocking IL-2 and IL-12 significantly reduced GM-CSF expression by Tc, NK, and B cells in MS patients, but not in healthy controls. MS patients during relapse had significantly higher frequency of Th-GM (CD3+CD8-IL-17-IFN-γ-IL-3+GM-CSF+) cells than healthy controls. EBV infected B cells expressed GM-CSF receptor in less frequency than non-infected B cells. In vivo GM-CSF neutralization in MS patients resulted in significant reduction in the frequency of CD8+ T cells and CD4+CD45RA+CD25++ (naïve) Tregs and an increase in CD4+CD35+foxp3 (total) Tregs.
Conclusions: Th1 (and Th in general), Tc, monocytes, NK and B cells are all high producers of GM-CSF in MS. IL-2 and IL-12 are the main regulators of GM-CSF expression by Tc, NK, and B cells in MS patients. GM-CSF and its receptor may not be major survival or proliferation factors for EBV infected B cells. The newly identified Th-GM cells were detected in higher frequency in MS patients during relapse, which may suggest a new source for GM-CSF production in MS. The recent safety, tolerability, and immune-related results of GM-CSF neutralization in MS are encouraging. Therefore, GM-CSF is a potential therapeutic target in MS.
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