Investigating the Mechanobiology of Macrophages: Implications for Inflammatory Bowel Disease

Abstract

Macrophages are essential cells of the innate immune system, playing a key role in regulating inflammation, tissue repair, and homeostasis. Their behaviour is tightly controlled by various signalling pathways, including mechanical forces that influence their shape, movement, and function. This process, known as mechanotransduction, allows cells to sense and respond to mechanical signals from their environment, converting these signals into biochemical responses that regulate cellular behaviour. Dysregulation of macrophage functions can lead to chronic inflammatory diseases and cancer. Recent studies have shown that mechanical cues, such as extracellular matrix (ECM) stiffness, fluid flow, cell crowding, and topography, modulate macrophage behaviour in various physiological and pathological contexts. However, the effect of ECM stiffness at relevant physiological levels, particularly in inflammation and fibrosis, has not been fully understood. Previous studies have often relied on single or limited marker approaches, which may not capture the full complexity of macrophage polarization.

To address this gap, we conducted a series of experiments aimed at characterizing THP-1 and bone marrow-derived macrophage (BMDM) protocols to ensure proper validation and reproducibility for our study. We then adapted ECM stiffness values, mimicking the conditions seen in inflammatory bowel disease (IBD), representing both normal and inflamed-fibrotic tissue. Experiments were conducted to assess macrophage polarization states in response to varying stiffness levels. Our results reveal that increasing ECM stiffness promotes the expression of YAP and IL-6 in M1 macrophages, driving a shift towards a pro-inflammatory phenotype. In contrast, M2 macrophages exhibited elevated levels of the anti-inflammatory markers CD163 and IL-10, reflecting an adaptive response to softer ECM conditions. Interestingly, M0 macrophages, which are considered to be non-polarized, adopted a hybrid phenotype, expressing both YAP and CD163, underscoring the inherent plasticity of macrophages when subjected to mechanical stress. In primary BMDMs, stiff ECM conditions induced also mixed phenotypes with favoured M1 polarization, as shown by a significant overlap with established M1 gene expression signatures, further emphasizing the role of ECM stiffness in driving pro-inflammatory responses. These findings challenge the traditional binary M1/M2 polarization model, suggesting that macrophage responses to mechanical cues are nuanced and context dependent.

In the second part of this thesis, we investigated the mechanical regulation of the Poly(C)-binding protein 1 (PCBP1) in macrophages and its role in macrophage polarisation. PCBP1 is a multifunctional RNA-binding protein that plays a crucial role in regulating mRNA stability, splicing, and translation. It is also involved in iron metabolism, acting as an iron chaperone, and is involved in DNA damage repair. Our experiments demonstrate that ECM stiffness and cell density regulate PCBP1 subcellular localization in macrophages. In stiff ECM and low-density environments, PCBP1 localises mainly to the nucleus, while in soft ECM and high cell density, it remained cytoplasmic. PCBP1 knockdown increased CD163 expression, suggesting it modulates M2 polarization. Finally, we demonstrate a possible role of PCBP1 in ECM stiffness dependent DNA damage repair, suggesting a novel mechanism of mechanoprotection.