Our scientific interest lies in understanding how cells of the immune system interact with their surrounding tissues. While molecular and biochemical processes underlying immune cell interactions have long been the subject of research, comparatively little attention has been paid to other stimuli to which immune cells are exposed. Those include physical cues, for example mechanical stimulation. Besides blood and lymph, which serve as liquid transport media and generate shear stress, immune cells in healthy individuals are found to reside in many diverse tissues, which strongly differ in their physical properties. Those properties, impacting on force transmission, are determined by both, the types and behavior of cells that make up the tissues, as well as the structure of the extracellular matrix. Other factors, such as hydrostatic pressure or external forces, also impact mechanically on the tissue under physiological conditions. Examples include the pressure exerted on lungs by respiratory movements, or on the intestine during peristalsis. Thus, immune cells are constantly exposed to mechanical stimuli. How tissue-resident immune cells such as macrophages or innate lymphoid cells sense mechanical stimuli present in the tissues they reside in, how they integrate and react to them, and whether they can adapt to varying mechanical situations in different tissues is not known. Furthermore, inflammatory conditions can alter the physical conditions of tissues. For example, fibrosis profoundly increases the rigidity of tissues. We previously found that the localization of immune cells in fibrotic areas of the lung, was associated with an increased expression of mechanosensors.1 We have also previously shown that innate lymphoid cells localize to fibrovascular niches in the tissue, locations which are sensitive to pressure changes such as edema or fibrosis, due to their proximity to vessels.2 We hypothesize that in a healthy organism, physical properties of the various organs contribute to immune homeostasis by transducing tissue-specific mechanical signals to immune cells, and tissue-resident immune cells can sense and adapt to the specific mechanical conditions surrounding them. Conversely, changes in these properties in the course of pathologies may affect immune cell activation via distorted force transmission.
Introduction: Macrophages can process such stimuli with the help of the mechanosensor Piezo-1, a calcium (Ca2+) channel, and are stimulated by them to differentiate.3 In the first round of the “Re-thinking health” graduate school, we successfully established a system to monitor and quantify mechanical force sensing in macrophages in real-time, by employing advanced microscopy methods. Using a reporter mouse expressing a fluorescent Ca2+ biosensor4 in macrophages, we found that bone marrow-derived macrophages treated with a Piezo-1 agonist respond with an immediate increase in cytoplasmic Ca2+. A similar Ca2+ increase is observed when the cells are being mechanically stimulated, using force indentation microscopy. Within a cell culture dish, the Ca2+ signal was transmitted to other, neighboring macrophages that were not directly mechanically stimulated, suggesting the transmission of force-induced signals within immune cell networks. In the proposed project, we plan to take advantage of our established system to further investigate the impact of mechanostimulation on macrophages, and also extend it to innate lymphoid cells. We also plan to investigate mechanical properties of the microenvironments hosting the immune cells, comparing various tissues.
Aim 1: Determine how innate immune cells from various tissues differ in their mechanosensing capacity
WP1: We will isolate macrophages from various tissues of the Ca2+ reporter mice, stimulate single cells using atomic force microscopy and analyze their capacity to respond to various degrees of mechanostimulation by live cell fluorescence microscopy. The impact of mechanostimulation on the phenotype of those cells will be determined by flow cytometry. We will also assess the capacity of innate lymphoid cells to respond to mechanical stimuli.
Aim 2: Determine the physical properties of various tissues hosting macrophages
WP2: Using a combination of third harmonic generation microscopy and force indentation, we will assess the strain and force transmission as well as the degree of mechanostimulation within various tissues from mice expressing the Ca2+ biosensor in macrophages.
Aim 3: Determine the impact of the mechanical microenvironment on macrophage phenotypes
WP3: We will culture macrophages in hydrogel scaffolds with various physical properties. Those gels will be exposed to mechanical pressure, and mechanosensing will be monitored in macrophages by measuring Ca2+ influx. Furthermore, the impact of mechanical stimulation on the phenotype of the macrophages will be assessed by flow cytometry.