Human macrophage mechanosensitivity is modulated by lipopolysaccharide stimulation

Background: Macrophages can sense and phenotypically adapt to their mechanical environment. Macrophages express a variety of mechano-sensitive ion channels (MSC), which have been demonstrated to mediate macrophage activation upon mechanical stimulation.¹ Here we present evidence that human macrophage mechanosensitivity is in turn modulated by immunogenic stimuli, suggesting a complementary mechanism. Lipopolysaccharide (LPS), the main membrane component of gram-negative bacteria, represents a potent macrophage-activator, which is commonly used for in vitro research.² Our data indicates that LPS stimulation increases macrophage mechanosensitivity. This hitherto unknown modulation of macrophage susceptibility to mechanical cues could provide an important regulatory mechanism with respect to inflammatory- and mechano-(co-)stimulation, and could serve as a potential therapeutic target.

Methods: Human macrophages were isolated from healthy donor blood samples by high-density gradient centrifugation after consent was obtained. MSC activity in macrophages was studied by single-cell patch clamp recordings applying defined membrane pressure using a high-speed-pressure clamp (ALA Scientific), as previously published.³ In order to molecularly identify the underlying ion channels, MSC activity was further modulated by selective ion channel agonists and antagonists, as well as in knockdown experiments. In multicellular preparations of human macrophages, changes in cytosolic [Ca2+] served as a surrogate readout for MSC activity. [Ca2+] dynamics were visualized by fluorescent imaging of macrophages loaded with the Ca2+-sensitive dye Fluo-4-AM, applying MSC agonists. For all experimental conditions, we assessed effects of LPS stimulation (100 ng/mL, 37°C) to examine its modulatory role on MSC activity as compared to untreated control cells. The study is conducted in accordance with the Declaration of Helsinki and has been approved by the ethics committee of the University of Freiburg.

Results and Conclusions: In our pilot study, we demonstrate MSC activity in human blood-derived macrophages by patch-clamp recordings and [Ca2+] imaging. We observe a considerable variability in mean MSC current amplitudes between donors. LPS stimulation has been studied in macrophages with a low baseline MSC activity and led to higher MSC current amplitudes. This could be explained by either different macrophage subpopulations or by a varying degree of macrophage pre-activation. Hence, our findings indicate a potential immuno-regulatory mechanism of macrophage susceptibility to mechanical cues. This mechanism could well be of importance in clinical contexts where inflammatory stimulation goes along with shear stress mediated mechano-stimulation, such as in cardiopulmonary bypass surgery.

References:
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3.    Simon-Chica, A. et al. Piezo1 stretch-activated channel activity differs between murine bone marrow-derived and cardiac tissue-resident macrophages. J. Physiol. 1–20 (2024) doi:10.1113/JP284805.