Biology subjects

Petrie, R. J.

Publications and source records attributed to Petrie, R. J..

4 recordsLinked to original sources

bioRxiv2024

Effects of injury size on local and systemic immune cell dynamics in volumetric muscle loss

We took a systems approach to the analysis of macrophage phenotype in regenerative and fibrotic volumetric muscle loss outcomes in mice together with analysis of systemic inflammation and of other leukocytes in the muscle, spleen, and bone marrow. Macrophage dysfunction in the fibrotic group occurred as early as day 1, persisted to at least day 28, and was associated with increased numbers of leukocytes in the muscle and bone marrow, increased pro-inflammatory marker expression in splenic macrophages, and changes in the levels of pro-inflammatory cytokines in the blood. The most prominent differences were in muscle neutrophils, which were much more abundant in fibrotic outcomes compared to regenerative outcomes at day 1 after injury. However, neutrophil depletion had little to no effect on macrophage phenotype or on muscle repair outcomes. Together, these results suggest that the entire system of immune cell interactions must be considered to improve muscle repair outcomes.

bioengineering↗

bioRxiv2024

The cytoplasm of living cells can sustain transient and steady intracellular pressure gradients

Understanding the physical basis of cellular shape change in response to both internal and external mechanical stresses requires characterisation of cytoplasmic rheology. At subsecond time-scales and micron length-scales, cells behave as fluid-filled sponges in which shape changes necessitate intracellular fluid redistribution. However, whether these cytoplasmic poroelastic properties play an important role in cellular mechanical response over length- scales and time-scales relevant to cell physiology remains unclear. Here, we investigated whether and how a localised deformation of the cell surface gives rise to transient intracellular flows spanning several microns and lasting seconds. Next, we showed that pressure gradients induced in the cytoplasm can be sustained over several minutes. We found that stable pressure gradients can arise from the combination of cortical tension, cytoplasmic poroelasticity and water flows across the membrane. Overall our data indicate that intracellular cytosolic flows and pressure gradients may play a much greater role than currently appreciated, acting over time- and length-scales relevant to mechanotransduction and cell migration, signifying that poroelastic properties need to be accounted for in models of the cell. Significance statementUnderstanding how cells change shape dynamically under the influence of external and internal forces requires characterisation of the mechanical response of the cytoplasm, the viscous material that fills their interior. The cytoplasm consists of a porous solid phase bathed in a fluid, the cytosol. As the cytoplasm is incompressible, any cellular shape change necessitates redistribution of the cytosol within the cell and its flow rate sets the time-scale for deformation. How the cytoplasmic mechanical response affects cell physiology remains poorly understood. We show that the unique physical properties of the cytoplasm allow cells to sustain cellular-scale pressure gradients over minute time-scales. As a consequence, pressure-driven mechanisms may play a much greater role in cell physiology than currently appreciated.

biophysics↗

bioRxiv2021

Cytoplasmic pressure maintains epithelial integrity and inhibits cell motility

Cytoplasmic pressure, a function of actomyosin contractility and water flow, can regulate cellular morphology and dynamics. In mesenchymal cells, cytoplasmic pressure powers cell protrusion through physiological three-dimensional extracellular matrices. However, the role of intracellular pressure in epithelial cells is relatively unclear. Here we find that high cytoplasmic pressure is necessary to maintain barrier function, one of the hallmarks of epithelial homeostasis. Further, our data show that decreased cytoplasmic pressure facilitates lamellipodia formation during the epithelial to mesenchymal transition (EMT). Critically, activation of the actin nucleating protein Arp2/3 is required for the reduction in cytoplasmic pressure and lamellipodia formation in response to treatment with hepatocyte growth factor (HGF) to induce EMT. Thus, elevated cytoplasmic pressure functions to maintain epithelial tissue integrity, while reduced cytoplasmic pressure triggers lamellipodia formation and motility during HGF-dependent EMT.

cell biology↗

bioRxiv2020

Regulation of extracellular matrix assembly and structure by hybrid M1/M2 macrophages

Aberrant extracellular matrix (ECM) assembly surrounding implanted biomaterials is the hallmark of the foreign body response, in which implants become encapsulated in thick fibrous tissue that prevents their proper function. While macrophages are known regulators of fibroblast behavior, how their phenotype influences ECM assembly and the progression of the foreign body response is poorly understood. In this study, we used in vitro models with physiologically relevant macrophage phenotypes, as well as controlled release of macrophage-modulating cytokines from gelatin hydrogels implanted subcutaneously in vivo to investigate the role of macrophages in ECM assembly. Primary human macrophages were polarized to four distinct phenotypes, which have each been associated with fibrosis, including pro-inflammatory M1, pro-healing M2, and a hybrid M1/M2, generated by exposing macrophages to M1- and M2-promoting stimuli simultaneously. Additionally, macrophages were first polarized to M1 and then to M2 (M1[->]M2) to generate a phenotype typically observed during normal wound healing. Human dermal fibroblasts that were cultured in macrophage-conditioned media upregulated numerous genes involved in regulation of ECM assembly, especially in M2-conditioned media. Hybrid M1/M2 macrophage-conditioned media caused fibroblasts to produce a matrix with thicker and less aligned fibers, while M2 macrophage-conditioned media caused the formation of a more aligned matrix with thinner fibers. Gelatin methacrylate hydrogels containing interleukin-4 (IL4) and IL13-loaded poly(lactic-co-glycolic acid) (PLGA) microparticles were designed to promote the M2 phenotype in a murine subcutaneous in vivo model. NanoString multiplex gene expression analysis of hydrogel explants showed that hydrogels with and without drug caused markers of both M1 and M2 phenotypes to be highly expressed, but the release of IL4+IL13 promoted upregulation of M2 markers and genes associated with regulation of ECM assembly, such as Col5a1 and Col6a1. Biochemical analysis and second harmonic generation microscopy showed that the release of IL4+IL13 increased total sulfated glycosaminoglycan content and decreased fibril alignment, which is typically associated with less fibrotic tissue. Together, these results show that hybrid M1/M2 macrophages regulate ECM assembly, and that shifting the balance towards M2 may promote architectural and compositional changes in ECM with enhanced potential for downstream remodeling.

bioengineering↗