Substrate Elasticity Exerts Functional Effects on Primary Microglia.

Stefan J Blaschke,Christina Hoffmann,Gereon R Fink,Maria A Rueger,Seda Demir,Jella-Andrea Abraham,Anna König,Nils Hersch,Michael Schroeter,Daniel N Olschewski,Marco Hoffmann,Sabine U Vay,Rudolf Merkel,Monika Rabenstein,Bernd Hoffmann

doi:10.3389/fncel.2020.590500

Abstract

Microglia—the brain’s primary immune cells—exert a tightly regulated cascade of pro- and 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 two-fold 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 channels. The results highlight the significance of the omnipresent but mostly overlooked mechanobiological effects exerted on microglia and contribute to a better understanding of the complex spatial and temporal interactions between microglia, neural stem cells, and glia, in health and disease.

Highlights

As primary immune cells of the central nervous system (CNS), microglia mediate, and regulate neuroinflammation in health and disease (Ransohoff and El Khoury, 2015)
Stainings confirmed that morphologically different microglia still expressed both the characteristic constitutive microglial marker CD11b as well as Iba-1 as a marker for activated microglia, while they did not express GFAP, indicating that there was no contamination with astrocytes (Supplementary Figure 1)
As relevant viscoelastic alteration of neural tissue is induced by chronic inflammation (Streitberger et al, 2012; Millward et al, 2015), stroke (Freimann et al, 2013), or aging (Arbogast et al, 1997; Arani et al, 2015), further knowledge about elasticity-dependent microglia function will help to understand microglia action in disease better and possibly support directed therapeutic strategies

Summary

Introduction

As primary immune cells of the central nervous system (CNS), microglia mediate, and regulate neuroinflammation in health and disease (Ransohoff and El Khoury, 2015). A tightly controlled regulation of both initiation and termination of microglial activation is vital for healthy development (Lenz and Nelson, 2018) and homeostasis of the CNS (Walberer et al, 2014; Yin et al, 2017) Understanding this complex interplay might expedite microglia-directed therapies in the future (Kim et al, 2015). In parallel to the subdivision of activation states of macrophages in other organs, a pro-inflammatory (M1) and an anti-inflammatory (M2) phenotype in microglia have been proposed upon activation (Tang and Le, 2016) with transitional states in between (Vay et al, 2018) While this concept has been a subject of thorough investigation, more in-depth knowledge of influencing factors, transitional states, or state reversibility is needed to develop novel therapeutic options targeting the microglial response to pathology

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