Abstract
Immune cells process a myriad of biochemical signals but their function and behavior are also determined by mechanical cues. Macrophages are no exception to this. Being present in all types of tissues, macrophages are exposed to environments of varying stiffness, which can be further altered under pathological conditions. While it is becoming increasingly clear that macrophages are mechanosensitive, it remains poorly understood how mechanical cues modulate their inflammatory response. Here we report that substrate stiffness influences the expression of pro-inflammatory genes and the formation of the NLRP3 inflammasome, leading to changes in the secreted protein levels of the cytokines IL-1β and IL-6. Using polyacrylamide hydrogels of tunable elastic moduli between 0.2 and 33.1 kPa, we found that bone marrow-derived macrophages adopted a less spread and rounder morphology on compliant compared to stiff substrates. Upon LPS priming, the expression levels of the gene encoding for TNF-α were higher on more compliant hydrogels. When additionally stimulating macrophages with the ionophore nigericin, we observed an enhanced formation of the NLRP3 inflammasome, increased levels of cell death, and higher secreted protein levels of IL-1β and IL-6 on compliant substrates. The upregulation of inflammasome formation on compliant substrates was not primarily attributed to the decreased cell spreading, since spatially confining cells on micropatterns led to a reduction of inflammasome-positive cells compared to well-spread cells. Finally, interfering with actomyosin contractility diminished the differences in inflammasome formation between compliant and stiff substrates. In summary, we show that substrate stiffness modulates the pro-inflammatory response of macrophages, that the NLRP3 inflammasome is one of the components affected by macrophage mechanosensing, and a role for actomyosin contractility in this mechanosensory response. Thus, our results contribute to a better understanding of how microenvironment stiffness affects macrophage behavior, which might be relevant in diseases where tissue stiffness is altered and might potentially provide a basis for new strategies to modulate inflammatory responses.
Highlights
Macrophages are innate immune cells responsible for engulfing microbes and cell debris and orchestrating inflammatory responses to maintain tissue homeostasis
After culturing bone marrow-derived macrophages (BMDMs) on the gels for 14–18 h, they were well adhered to the material in all conditions and their viability was above 85% (Figures 1B, 3E)
We explored the mechanosensitive response of macrophages by exposing BMDMs to substrate stiffness in the range of 0.2 to 33.1 kPa
Summary
Macrophages are innate immune cells responsible for engulfing microbes and cell debris and orchestrating inflammatory responses to maintain tissue homeostasis. Some studies suggest that stiffer substrates upregulate macrophage pro-inflammatory responses and polarize them towards an M1 phenotype (Blakney et al, 2012; Previtera and Sengupta, 2015; Okamoto et al, 2018; Hsieh et al, 2019; Sridharan et al, 2019), while others show that more compliant materials enhance their pro-inflammatory behavior (Adlerz et al, 2015; Scheraga et al, 2016; Gruber et al, 2018) Parameters such as the stiffness range, the adhesive ligand, the activation stimulus and the specific cell type used vary across the different studies and all these factors could influence the results. It is clear that further research is required to dissect how substrate stiffness modulates the behavior of macrophages and how this impacts on their ability to induce inflammatory responses
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