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Macrophage motility is driven by frontal-towing with a force magnitude dependent on substrate stiffness.

ABSTRACT: The ability of macrophages to properly migrate is crucial to their success as early responders during the innate immune response. Furthermore, improper regulation of macrophage migration is known to contribute to several pathologies. The signaling mechanisms underlying macrophage migration have been previously studied but to date the mechanical mechanism of macrophage migration has not been determined. In this study, we have created the first traction maps of motile primary human macrophages by observing their migration on compliant polyacrylamide gels. We find that the force generated by migrating macrophages is concentrated in the leading edge of the cell - so-called frontal towing - and that the magnitude of this force is dependent on the stiffness of the underlying matrix. With the aid of chemical inhibitors, we show that signaling through the RhoA kinase ROCK, myosin II, and PI3K is essential for proper macrophage force generation. Finally, we show that Rac activation by its GEF Vav1 is crucial for macrophage force generation while activation through its GEF Tiam1 is unnecessary.

SUBMITTER: Hind LE

PROVIDER: S-EPMC5102152 | biostudies-other | 2015 Apr

REPOSITORIES: biostudies-other

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Macrophage motility is driven by frontal-towing with a force magnitude dependent on substrate stiffness.

Hind Laurel E LE Dembo Micah M Hammer Daniel A DA

Integrative biology : quantitative biosciences from nano to macro 20150401 4

The ability of macrophages to properly migrate is crucial to their success as early responders during the innate immune response. Furthermore, improper regulation of macrophage migration is known to contribute to several pathologies. The signaling mechanisms underlying macrophage migration have been previously studied but to date the mechanical mechanism of macrophage migration has not been determined. In this study, we have created the first traction maps of motile primary human macrophages by ...[more]

PMID: 25768202

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Project description:Heterogeneous intercellular coupling plays a significant role in mechanical and electrical signal transmission in the heart. Although many studies have investigated the electrical signal conduction between myocytes and nonmyocytes within the heart muscle tissue, there are not many that have looked into the mechanical counterpart. This study aims to investigate the effect of substrate stiffness and the presence of cardiac myofibroblasts (CMFs) on mechanical force propagation across cardiomyocytes (CMs) and CMFs in healthy and heart-attack-mimicking matrix stiffness conditions. The contractile forces generated by the CMs and their propagation across the CMFs were measured using a bio-nanoindenter integrated with fluorescence microscopy for fast calcium imaging. Our results showed that softer substrates facilitated stronger and further signal transmission. Interestingly, the presence of the CMFs attenuated the signal propagation in a stiffness-dependent manner. Stiffer substrates with CMFs present attenuated the signal ∼24-32% more compared to soft substrates with CMFs, indicating a synergistic detrimental effect of increased matrix stiffness and increased CMF numbers after myocardial infarction on myocardial function. Furthermore, the beating pattern of the CMF movement at the CM-CMF boundary also depended on the substrate stiffness, thereby influencing the waveform of the propagation of CM-generated contractile forces. We performed computer simulations to further understand the occurrence of different force transmission patterns and showed that cell-matrix focal adhesions assembled at the CM-CMF interfaces, which differs depending on the substrates stiffness, play important roles in determining the efficiency and mechanism of signal transmission. In conclusion, in addition to substrate stiffness, the degree and type of cell-cell and cell-matrix interactions, affected by the substrate stiffness, influence mechanical signal conduction between myocytes and nonmyocytes in the heart muscle tissue.

Project description:Macrophages are innate immune cells that interact with complex extracellular matrix environments, which have varied stiffness, composition, and structure, and such interactions can lead to the modulation of cellular activity. Collagen is often used in the culture of immune cells, but the effects of substrate functionalization conditions are not typically considered. Here, we show that the solvent system used to attach collagen onto a hydrogel surface affects its surface distribution and organization, and this can modulate the responses of macrophages subsequently cultured on these surfaces in terms of their inflammatory activation and expression of adhesion and mechanosensitive molecules. Collagen was solubilized in either acetic acid (Col-AA) or N-(2-hydroxyethyl)piperazine-N'-ethanesulfonic acid (HEPES) (Col-HEP) solutions and conjugated onto soft and stiff polyacrylamide (PA) hydrogel surfaces. Bone marrow-derived macrophages cultured under standard conditions (pH 7.4) on the Col-HEP-derived surfaces exhibited stiffness-dependent inflammatory activation; in contrast, the macrophages cultured on Col-AA-derived surfaces expressed high levels of inflammatory cytokines and genes, irrespective of the hydrogel stiffness. Among the collagen receptors that were examined, leukocyte-associated immunoglobulin-like receptor-1 (LAIR-1) was the most highly expressed, and knockdown of the Lair-1 gene enhanced the secretion of inflammatory cytokines. We found that the collagen distribution was more homogeneous on Col-AA surfaces but formed aggregates on Col-HEP surfaces. The macrophages cultured on Col-AA PA hydrogels were more evenly spread, expressed higher levels of vinculin, and exerted higher traction forces compared to those of cells on Col-HEP. These macrophages on Col-AA also had higher nuclear-to-cytoplasmic ratios of yes-associated protein (YAP) and transcriptional co-activator with PDZ-binding motif (TAZ), key molecules that control inflammation and sense substrate stiffness. Our results highlight that seemingly slight variations in substrate deposition for immunobiology studies can alter critical immune responses, and this is important to elucidate in the broader context of immunomodulatory biomaterial design.

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Macrophage motility is driven by frontal-towing with a force magnitude dependent on substrate stiffness.

Publications

Macrophage motility is driven by frontal-towing with a force magnitude dependent on substrate stiffness.

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