Substrate Elasticity Exerts Functional Effects on Primary Microglia

Abstract

Microglia-the brain's primary immune cells-exert a tightly regulated cascade of proand anti-inflammatory effects upon brain pathology, either promoting regeneration or neurodegeneration. Therefore, harnessing microglia emerges as a potential therapeutic concept in neurological research. Recent studies suggest that-besides being affected by chemokines and cytokines-various cell entities in the brain relevantly respond to the mechanical properties of their microenvironment. For example, we lately reported considerable effects of elasticity on neural stem cells, regarding quiescence and differentiation potential. However, the effects of elasticity on microglia remain to be explored.Under the hypothesis that the elasticity of the microenvironment affects key characteristics and functions of microglia, we established an in vitro model of primary rat microglia grown in a polydimethylsiloxane (PDMS) elastomer-based cell culture system. This way, we simulated the brain's physiological elasticity range and compared it to supraphysiological stiffer PDMS controls. We assessed functional parameters of microglia under "resting" conditions, as well as when polarized towards a pro-inflammatory phenotype (M1) by lipopolysaccharide (LPS), or an anti-inflammatory phenotype (M2) by interleukin-4 (IL-4). Microglia viability was unimpaired on soft substrates, but we found various significant effects with a more than twofold increase in microglia proliferation on soft substrate elasticities mimicking the brain (relative to PDMS controls). Furthermore, soft substrates promoted the expression of the activation marker vimentin in microglia. Moreover, the M2-marker CD206 was upregulated in parallel to an increase in the secretion of Insulin-Like Growth Factor-1 (IGF-1). The upregulation of CD206 was abolished by blockage of stretch-dependent chloride channels. Our data suggest that the cultivation of microglia on substrates of brain-like elasticity promotes a basic anti-inflammatory activation state via stretch-dependent chloride

Two-Photon Polymerization of 2.5D and 3D Microstructures Fostering a Ramified Resting Phenotype in Primary Microglia

“…When it comes to the effect of stiffness on the morphology of microglia however, the literature shows some contradiction. In a study performed by Blaschke et al, rat primary microglia were shown to a have less round and more polar morphology and express more anti-inflammatory cytokines when cultured on a soft polydimethylsiloxane (PDMS) substrate (Young’s modulus = 0.6 kPa) versus a stiffer one (Young’s modulus = 1.2 MPa) ( Blaschke et al, 2020 ). On the other hand, Dudiki et al showed in their study that when culturing murine primary microglia on fibronectin-coated hyaluronic acid-based hydrogels of 60 and 600 Pa Young’s moduli, the bipolarization of the cells increased by approximately 3 times on the stiffer hydrogel ( Dudiki et al, 2020 ).…”

“…Cells have the ability to sense the mechanical strength of their surrounding environment. Studies performed on macrophages have shown the various and inconsistent effects of substrate stiffness on the morphology and cytokine expression of these cells ( Sridharan et al, 2019 ; Blaschke et al, 2020 ; Chen et al, 2020 ; Dudiki et al, 2020 ). While some results show the polarization of microglia on softer substrates ( Blaschke et al, 2020 ), others show the opposite ( Dudiki et al, 2020 ).…”

Multiscale Mechanobiology in Brain Physiology and Diseases

“…Indeed, changes in matrix stiffness were found to induce a morphological adaptation of microgliocytes. It was reported that soft substrates enhanced microglia polarization and increased their proliferation ( Blaschke et al, 2020 ). Similar to other cell types, microglial cells were observed to migrate towards the stiffer zone of a mechanical gradient ( Bollmann et al, 2015 ).…”

Mechanosensing and the Hippo Pathway in Microglia: A Potential Link to Alzheimer’s Disease Pathogenesis?

“…For example, during retinal development, microglia migrate through a stiffness gradient, and subsequently change their shape from ramified to bipolar-rod-like, a process which helps establish the vascular architecture of the developing retina [44]. In the context of microglia, soft PDMS-based elastic substrates (which vary between 1 and 0.6 kPa) have been reported to increase in vitro microglial proliferation and anti-inflammatory characteristics [45], whereas stiffer polyacrylamide/poly-D-lysine-based substrates (10-30 kPa) increase microglial pro-inflammatory characteristics [46]. It has therefore been suggested that brain tissue stiffness changes may be associated with the development of neurodegenerative pathologies; additionally, it has been suggested that microglia may act as mechanosensing cells in the pathogenesis of AD, or indeed other neurodegenerative diseases with neuroinflammatory components.…”