Mohammad, Salaheddin
(2022)
The influence of stromal fibroblast on antigen-presenting cell
function and alteration of their biology.
PhD thesis, University of Nottingham.
Abstract
Dendritic cells (DC) and macrophages (Mɸ) are antigen presenting cells (APC) that orchestrate
immune activation, immune and tissue homeostasis, and disease pathology. In the case of
Mɸ that act in tissue repair and wound healing, it is important that local inflammation is
tightly controlled. Disorder in inflammatory processes leads to impaired regeneration,
excessive remodelling or immune suppression. In contrast, the major functions of DCs include
priming and tolerization of immune responses, disruption of which has major impact on
responses to infectious disease, malignancy, or can result in autoimmunity. Whilst a sizeable
body of research has studied DCs and Mɸ, there has been relatively little attention paid to
the role of stromal cells that share their tissue microenvironment.
In vitro, human laboratory models of Mɸ and DCs were differentiated from monocytes under
the influence of cytokines; IL-4 and GM-CSF for DCs, or GM-CSF or M-CSF to differentiate GM-
Mɸ or M-Mɸ, respectively. These two major polarized Mɸ populations exhibited unique
cytokine profiles, ie GM-Mɸ expressed a pro-inflammatory cytokine profile including IL-12
and IL-23, whilst M-Mɸ exhibited elevated IL-10 with minimal IL-12.
Initial studies demonstrated the impact of fibroblasts (FB) on Mɸ in conventional planar co-
culture and showed the presence of FB affected their cytokine secretion profile. However,
further attempts to establish a more physiological model with 3D-co-culture of these cells in
spheroids were promising to improve this system. In planar coculture, FB increased IL-23
secretion by GM-Mɸ but this was not observed in the spheroid model. Secretion of IL-10 by
M-Mɸ was decreased in both planar and spheroid co-cultures in the presence of dermal
fibroblasts. We explored the impact of ionising radiation (IR) delivered as a single dose.
Irradiation (6Gy) altered the FB-mediated effect on Mɸ. In mono-culture, IR significantly
increased IL-12 secretion by GM-Mɸ (p<0.001), suppressed IL-10 by GM-Mɸ and M-Mɸ
(p<0.001 and p<0.0001), and decreased IL-23 secretion by Mɸ (p<0.001 and p<0.01). In
contrast, irradiated co-cultures showed increased IL-12 expression by GM-Mɸ (p<0.00001),
and suppressed IL-10 by M-Mɸ (p<0.001). This highlighted the potential for complex
interactions between APC and stromal cells. Glucocorticoids (GCs) display both
immunosuppressive and anti-inflammatory features, allowing them to be used for treatment
in various immune-mediated inflammatory disorders, we investigated the effect of Dex
administration. Dex in acute and prolonged periods of culture suppressed cytokine secretion
by the Mɸ. We explored that Dex at high concentration and treated with TGF-β significantly
(p<0.01 and p<0.001) decreased IL-23 and IL-10 by GM-Mɸ. In contrast, Dex at low
concentration and treated with TGF-β at low concentration significantly (p<0.0001) reduced
IL-23 by M-Mɸ.
Recently, it has been shown that ECM can convey specific signals to cells. We therefore
explored the hypothesis that FB-derived ECM regulates macrophage behaviour. We
developed a model of Mɸ differentiation to investigate the influence of FB-derived ECM
components on their differentiation and function.
Fibroblast cell lines and primary fibroblasts from breast cancer patients were cultured on
plastic for an extended 10-day period to deposit ECM. Subsequently, monocytes were
cultured on decellularized ECM in the presence of differentiating cytokines. Interestingly, the
presence of ECM from BJ6 cell line fibroblast suppresses CD169 and CD86 on monocyte
(CD14+)(p<0.05 and p<0.001). BJ6-ECM down-regulates CD169 on GM-Mɸ (p<0.05), whereas
BJ6-ECM down-regulates CD169 and CD86 on M-Mɸ (p<0.05 and p<0.001). Tumour-
associated FB upregulates CD204 and PD-1 on M-Mɸ. Whilst there was also evidence of
impact on cytokine secretion by Mɸ this was less clear. Taken together, this evidence suggests
that phenotypic consequence of Mɸ regulate by FB-derived-ECM-and does represent the
physiological situation and the potential promising to provide outlook on future experimental
implications that may lead to the design of novel co-culture experiments. Further planned
studies of this system (including sequencing the matrisome deposited by FB, and expression
profiling of the resulting macrophages) have the potential to reveal key ECM proteins
responsible for the development of the tumour-associated Mɸ phenotype
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