The influence of stromal fibroblast on antigen-presenting cell function and alteration of their biology

Mohammad, Salaheddin (2022) The influence of stromal fibroblast on antigen-presenting cell function and alteration of their biology. PhD thesis, University of Nottingham.

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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

Item Type: Thesis (University of Nottingham only) (PhD)
Supervisors: Jackson, Andrew
Patel, Poulam
Grabowska, Anna
Keywords: Adaptive immunity; Fibroblasts; CD4+ cells; CD8+ cells; Tumour cells; Antigen presenting cells; Dendritic cells
Subjects: QS-QZ Preclinical sciences (NLM Classification) > QW Microbiology. Immunology
Faculties/Schools: UK Campuses > Faculty of Medicine and Health Sciences > School of Medicine
Item ID: 69103
Depositing User: Mohammad, Salaheddin
Date Deposited: 08 Nov 2023 10:21
Last Modified: 08 Nov 2023 10:21

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