Sensitivity of alveolar macrophages to substrate mechanical and adhesive properties

Abstract

In order to understand the sensitivity of alveolar macrophages (AMs) to substrate properties, we have developed a new model of macrophages cultured on substrates of increasing Young's modulus: (i) a monolayer of alveolar epithelial cells representing the supple (approximately 0.1 kPa) physiological substrate, (ii) polyacrylamide gels with two concentrations of bis-acrylamide representing low and high intermediate stiffness (respectively 40 kPa and 160 kPa) and, (iii) a highly rigid surface of plastic or glass (respectively 3 MPa and 70 MPa), the two latter being or not functionalized with type I-collagen. The macrophage response was studied through their shape (characterized by 3D-reconstructions of F-actin structure) and their cytoskeletal stiffness (estimated by transient twisting of magnetic RGD-coated beads and corrected for actual bead immersion). Macrophage shape dramatically changed from rounded to flattened as substrate stiffness increased from soft ((i) and (ii)) to rigid (iii) substrates, indicating a net sensitivity of alveolar macrophages to substrate stiffness but without generating F-actin stress fibers. Macrophage stiffness was also increased by large substrate stiffness increase but this increase was not due to an increase in internal tension assessed by the negligible effect of a F-actin depolymerizing drug (cytochalasine D) on bead twisting. The mechanical sensitivity of AMs could be partly explained by an idealized numerical model describing how low cell height enhances the substrate-stiffness-dependence of the apparent (measured) AM stiffness. Altogether, these results suggest that macrophages are able to probe their physical environment but the mechanosensitive mechanism behind appears quite different from tissue cells, since it occurs at no significant cell-scale prestress, shape changes through minimal actin remodeling and finally an AMs stiffness not affected by the loss in F-actin integrity.

Matrix Architecture Dictates Three-Dimensional Migration Modes of Human Macrophages: Differential Involvement of Proteases and Podosome-Like Structures

“…3 and 4, MDMs layered on the two 2D matrices were flat (Fig. 6A) as expected from previous data showing that cells flattened as the stiffness of the matrix increased (42,49,50). In addition, they formed podosomes characterized by the classical F-actin dots and matrix degradation was observed (Fig.…”

“…This behavior is typical of a solid-like material. More precisely, when G' is one order of magnitude higher than G" it relates to a "weak gel" material (42,43). As shown in Fig.…”

Dynamics of podosome stiffness revealed by atomic force microscopy

“…Podosome biophysical properties are likely to be coupled with their mechanosensing activity. However, investigation of podosome properties in macrophages plated on soft matrix is challenging as these cells tend to round up and form less podosomes (28). Although the exploration of podosome biomechanics is just beginning, here we show that AFM offers the opportunity to directly quantify biophysical processes in podosomes.…”

Traction patterns of tumor cells

“…Since our method relies on no further hypotheses and is based on the use of large samples, we have good confidence in our data. Other techniques which can be used are traction tests [13,25], micropipette experiments [4], AFM [13,14], or rheometry [35]. Gels were prepared on a silanated square coverglass 22mm x 22mm and covered with a circular coverglass (35mm diameter) functionalized with NaOH (0.1M), APTMS (10mn), and 0.5% glutaraldehyde (30mn).…”