Mechanosensing in macrophages in health and disease

Student

Principle Investigator

Scientific interest within the context of the graduate college:

Our lab is interested in analyzing immune cells in the tissue context, using state-of-the-art, functional intravital microscopy and histocytometry approaches.^1,2,3 In addition to reacting to stimuli from hematopoietic and non-hematopoietic cells, for example via direct cell-cell contacts or via soluble mediators, immune cells are exposed to a variety of other stimuli present in the tissue, among them mechanical cues. The significance of those stimuli for immune cells has not been explored in detail. Recently, a role for the mechanosensor Piezo1 in shaping myeloid cell activation has been identified.⁴ Here, we aim to understand how mechanical cues in barrier tissues affect the function of myeloid cells. We will focus the lamina propria of the intestine, a tissue that in the healthy organism is constantly subjected to contractions (peristalsis). We hypothesize that this mechanical stimulation impacts on the phenotype of macrophages in the lamina propria. As alterations in peristalsis appear in certain situations, such as parasitic infections, we aim to test to what extent mechanosensing in macrophages affects their function under those conditions.

Project description:

To monitor mechanosensing in vivo, we aim to use a reporter system, which allows for the detection of Ca²⁺ flux, induced by the mechanosensor Piezo1, in intestinal macrophages by intravital microscopy.⁵ The objectives for this project are:

Aim 1: Determine the effect of mechanical stimuli on the phenotype of macrophages in vitro. Myeloid cells will be cultured and various, defined mechanical stimuli will be applied in a specialized chamber. The phenotypes of the cells will be compared by flow cytometry.

Aim 2: Define the role of the Ca²⁺ channel Piezo1 in myeloid mechanosensing and establish a reporter system for the quantification of Piezo1-mediated Ca²⁺ flux in vitro and in vivo. The impact of Piezo1 stimulation on cytoplasmic Ca²⁺ concentration will be assessed using specific inhibitors/activators in vitro. To quantify Piezo1-induced Ca²⁺ flux in the cytoplasm in vivo, we plan to use mice carrying a genetically encoded cytoplasmic calcium sensor in macrophages. We will relate the sensor signals to mechanical stimuli in vitro. Subsequently, we aim to employ this system to locate and quantify mechanically induced Ca²⁺ flux in myeloid cells by intravital FRET-FLIM microscopy,⁶ and correlate this signal to tissue dynamics, to map the pressure conditions in vivo.

Aim 3: Determine to what extent Piezo1-mediated mechanosensing in myeloid cells of the lamina propria contributes to the intestinal immune response in vivo. The phenotype of intestinal macrophages will be compared by flow cytometry and multiplexed histology in mice with a deficiency of Piezo1 in myeloid cells to wildtype mice. Initial analyses will be performed under homeostatic conditions. Subsequently, we will challenge the mice by infecting them with intestinal parasites, which are known to mechanically impact on the tissue, either by attaching to the intestinal wall or by inducing altered gut peristalsis.

References

1. Pascual-Reguant A, Kohler R, Mothes R, Bauherr S, Uecker R, Holzwarth K, Kotsch K, Seidl M, Philipsen L, Müller W, Hernández D, Romagnani C, Niesner R, Hauser AE. Phenotypic and spatial characteristics of human Innate Lymphoid Cells revealed by highly multiplexed histology. Nat Commun. 2021. Accepted for publication.
2. Stefanowski J, Lang A, Rauch A, Aulich L, Kohler M, Fiedler AF, Buttgereit F, Schmidt-Bleek K, Duda GN, Gaber T, Niesner RA, Hauser AE. Spatial Distribution of Macrophages During Callus Formation and Maturation Reveals Close Crosstalk Between Macrophages and Newly Forming Vessels. Front Immunol. 2019;10: 2588. doi: 10.3389/fimmu.2019.02588.
3. Holzwarth K, Kohler R, Philipsen L, Tokoyoda K, Ladyhina V, Wahlby C, Niesner RA, Hauser AE. Multiplexed fluorescence microscopy reveals heterogeneity among stromal cells in mouse bone marrow sections. Cytometry A. 2018;93: 876-888. doi: 10.1002/cyto.a.23526.
4. Solis AG, Bielecki P, Steach HR, Sharma L, Harman CCD, Yun S, de Zoete MR, Warnock JN, To SDF, York AG, Mack M, Schwartz MA, Dela Cruz CS, Palm NW, Jackson J, Flavell RA. Mechanosensation of cyclical force by PIEZO1 is essential for innate immunity. Nature. 2019; 573: 69-74. doi: 10.1038/s41586-019-1485-8.
5. Lindquist RL, Bayat-Sarmadi J, Leben R, Niesner R, Hauser AE. NAD(P)H Oxidase Activity in the Small Intestine Is Predominantly Found in Enterocytes, Not Professional Phagocytes. Int J Mol Sciences. 2018; 19:1365. doi: 10.3390/ijms19051365.
6. Ulbricht C, Leben R, Rakhymzhan A, Kirchhoff F, Nitschke L, Radbruch H, Niesner RA, Hauser AE. Intravital quantification of absolute cytoplasmic B cell calcium reveals dynamic signaling across B cell differentiation stages. Elife. 2020; 10:e56020. doi: 10.7554/eLife.56020.