Mycobacterium tuberculosis is a global health problem in part as a result of extensive cytotoxicity caused by the infection. Here, we show how M. tuberculosis causes caspase-1/NLRP3/gasdermin D-mediated pyroptosis of human monocytes and macrophages. A type VII secretion system (ESX-1) mediated, contact-induced plasma membrane damage response occurs during phagocytosis of bacteria. Alternatively, this can occur from the cytosolic side of the plasma membrane after phagosomal rupture in infected macrophages. This damage causes K+ efflux and activation of NLRP3-dependent IL-1β release and pyroptosis, facilitating the spread of bacteria to neighbouring cells. A dynamic interplay of pyroptosis with ESCRT-mediated plasma membrane repair also occurs. This dual plasma membrane damage seems to be a common mechanism for NLRP3 activators that function through lysosomal damage.

of cell mechanotransduction machinery [23] or controlling the geometry of in vitro neuronal networks. [24] Recently arrays of SU-8 nanopillars were employed to demonstrate that in contrast to previous measurements the highest cell traction forces are not generated at the cell periphery, but instead are associated with perinuclear adhesions. [25] In a different report an ultraflexible GaAs nanowire array was used as a nanomechanical biosensor to probe cell-induced forces by living cells with a resolution of 50 piconewton. [26] An attempt to control the geometry of in vitro cultivated neuronal cells revealed that the cytoskeleton dynamics at the axon shaft in primary hippocampal rat neurons were changed when cultivated on hard poly(dimethylsiloxane) nanopillar arrays, which in turn lead to enhanced formation of axon collateral branches. [27] An example of in vivo application is provided by Tang et al. who apply vertically oriented Au-TiO 2 nanowire arrays as artificial photoreceptors which restore functional and behavioral light sensitivity in blind mice. [28] Despite the range of applications, there is still much unknown about how cells are influenced by HARNs. High viability and reduced spreading area of adherent cells is often reported. [29] Arrays of nanowires have been shown to inhibit fibroblast migration with longer nanowires having a stronger effect, [30] while small patches of nanopillars inhibited neuronal cell migration. [31] Reduced spreading of cells on HARN arrays has been reported, [16,32] as well as altered expression of genes related to the cytoskeleton and cell adhesion. [33,34] It has also been demonstrated that the cell membrane is able to wrap tightly over HARNs while maintaining membrane integrity. [35][36][37] Careful investigations of the membrane integrity in cardiomyocyte-like HL-1 cells and Human embryonic kidney cells (HEK 293) on a variety of nanostructures by Dipalo et al. showed that vertical nanostructures can spontaneously penetrate the cellular membrane only in rare cases and under specific conditions. [35] Local membrane curvature can act as a biochemical signal for endocytic proteins, and HARNs which induced sites of positive membrane curvature were found to be hot spots for clathrin-mediated endocytosis (CME), as determined by preferential accumulation of CME-related proteins at these sites. [38,39] Nanopillars were also applied for controlled probing of nuclear mechanical properties. [40] In that report, the mechanism of nuclear deformation was linked to adhesive actin patches associating with the nanopillars, pulling the nucleus down, as well Surfaces decorated with high aspect ratio nanostructures are a promising tool to study cellular processes and design novel devices to control cellular behavior. However, little is known about the dynamics of cellular phenomenon such as adhesion, spreading, and migration on such surfaces. In particular, how these are influenced by the surface properties. In this work, fibroblast behavior is investigated on regular arrays of 1 µm high polym...

Surfaces decorated with high aspect ratio nanostructures are a promising tool to study cellular processes and design novel devices to control cellular behaviour, perform intracellular sensing or deliver effector molecules to cells in culture. However, little is known about the dynamics of cellular phenomenon such as adhesion, spreading and migration on such surfaces. In particular, how these are influenced by the surface properties. In this work, we investigate fibroblast behaviour on regular arrays of 1 micrometer high, polymer nanopillars with varying pillar to pillar distance (array pitch).NIH-3T3 fibroblasts spread on all arrays, and on contact with the substrate engulf nanopillars independently of the array pitch. As the cells start to spread, different behaviour is observed. On dense arrays which have the pitch equal or below 1 micrometer, cells are suspended on top of the nanopillars, making only sporadic contact with the glass support. Cells stay attached to the glass support and fully engulf nanopillars during spreading and migration on the sparse arrays which are characterized by a pitch of 2 micrometers and above. These alternate states have a profound effect on cell migration rates, which are strongly reduced on nanopillar sparse arrays. Dynamic actin puncta colocalize with nanopillars during cell spreading and migration. Strong membrane association with engulfed nanopillars might explain the reduced migration rates on sparse arrays. This work reveals several interesting phenomenon of dynamical cell behaviour on nanopillar arrays, and provides important perspectives on design and applications of high aspect ratio nanostructures.

AbstractMycobacterium tuberculosis (Mtb) is a major global health problem and causes extensive cytotoxicity in patient cells and tissues. Here we define an NLRP3, caspase-1 and gasdermin D-mediated pathway to pyroptosis in human monocytes following exposure to Mtb. We demonstrate an ESX-1 mediated, contact-induced plasma membrane (PM) damage response that occurs during phagocytosis or from the cytosolic side of the PM after phagosomal rupture in Mtb infected cells. This PM injury in turn causes K+ efflux and activation of NLRP3 dependent IL-1β release and pyroptosis, facilitating the spread of Mtb to neighbouring cells. Further we reveal a dynamic interplay of pyroptosis with ESCRT-mediated PM repair. Collectively, these findings reveal a novel mechanism for pyroptosis and spread of infection acting through dual PM disturbances both during and after phagocytosis. We also highlight dual PM damage as a common mechanism utilized by other NLRP3 activators that have previously been shown to act through lysosomal damage.Graphical abstract