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.

A method to fabricate inexpensive and transparent nanowire impalement devices is invented based on CuO nanowire arrays grown by thermal oxidation. By employing a novel process the nanowires are transferred to a transparent, cell-compatible epoxy membrane. Cargo delivery and detailed cell-nanowire interaction studies are performed, revealing that the cell plasma membrane tightly wraps the nanowires, while cell membrane penetration is not observed. The presented device offers an efficient investigation platform for further optimization, leading towards a simple and versatile impalement delivery system.

Nanoscale topographies and chemical patterns can be used as synthetic cell interfaces with a range of applications including study and control of cellular processes. Herein, we describe the fabrication of high aspect ratio nanostructures using electron beam lithography in the epoxy-based polymer SU-8. We show how nanostructure geometry, position and fluorescent properties can be tuned, allowing flexible device design. Further, thiol-epoxide reactions were developed to give effective and specific modification of SU-8 surface chemistry. SU-8 nanostructures were made directly on glass cover slips, enabling the use of high resolution optical techniques such as live-cell confocal, total internal reflection and 3D structured illumination microscopy to investigate cell interactions with the nanostructures. Details of cell adherence and spreading, plasma membrane conformation and actin organization in response to high aspect ratio nanopillars and nanolines were investigated. The versatile structural and chemical properties combined with high resolution cell imaging capabilities of this system are an important step towards better understanding and controlling cell interactions with nanomaterials.

TLR8 is an endosomal sensor of RNA degradation products in human phagocytes, and is involved in the recognition of viral and bacterial pathogens. We previously showed that in human primary monocytes and monocyte derived macrophages, TLR8 senses entire Staphylococcus aureus and Streptococcus agalactiae (group B streptococcus , GBS), resulting in the activation of IRF5 and production of IFNβ, IL-12p70, and TNF. However, the quantitative and qualitative impact of TLR8 for the sensing of bacteria have remained unclear because selective inhibitors have been unavailable. Moreover, while we have shown that TLR2 activation attenuates TLR8-IRF5 signaling, the molecular mechanism of this crosstalk is unknown. We here used a recently developed chemical antagonist of TLR8 to determine its role in human primary monocytes challenged with S. aureus , GBS, Streptococcus pneumonia, Pseudomonas aeruginosa , and E. coli . The inhibitor completely blocked cytokine production in monocytes stimulated with TLR8-agonists, but not TLR2-, and TLR4-agonists. Upon challenge with S. aureus , GBS, and S. pneumonia , the TLR8 inhibitor almost eliminated the production of IL-1β and IL-12p70, and it strongly reduced the release of IL-6, TNF, and IL-10. With P. aeruginosa infection, the TLR8 inhibitor impaired the production of IL-12p70 and IL-1β, while with E. coli infection the inhibitor had less effect that varied depending on the strain and conditions. Signaling via TLR2, TLR4, or TLR5, but not TLR8, rapidly eliminated IRAK-1 detection by immunoblotting due to IRAK-1 modifications during activation. Silencing of IRAK-1 reduced the induction of IFNβ and TNF by TLR8 activation, suggesting that IRAK-1 is required for TLR8-IRF5 signaling. The TLR-induced modifications of IRAK-1 also correlated closely with attenuation of TLR8-IRF5 activation, suggesting that sequestration and/or modification of Myddosome components by cell surface TLRs limit the function of TLR8. Accordingly, inhibition of CD14- and TLR4-activation during E. coli challenge increased the activation of IRF5 and the production of IL-1β and IL-12p70. We conclude that TLR8 is a dominating sensor of several species of pyogenic bacteria in human monocytes, while some bacteria attenuate TLR8-signaling via cell surface TLR- activation. Taken together, TLR8 appears as a more important sensor in the antibacterial defense system than previously known.

TLR8 is the major endosomal sensor of degraded RNA in human monocytes and macrophages. It has been implicated in the sensing of viruses and more recently also bacteria. We previously identified a TLR8-IFN regulatory factor 5 (IRF5) signaling pathway that mediates IFNβ and interleukin-12 (IL-12) induction by Staphylococcus aureus and is antagonized by TLR2. The relative importance of TLR8 for the sensing of various bacterial species is however still unclear. We here compared the role of TLR8 and IRF5 for the sensing of Group B Streptococcus (GBS), S. aureus, and Escherichia coli in human primary monocytes and monocyte-derived macrophages (MDM). GBS induced stronger IFNβ and TNF production as well as IRF5 nuclear translocation compared to S. aureus grown to the stationary phase, while S. aureus in exponential growth appeared similarly potent to GBS. Cytokine induction in primary human monocytes by GBS was not dependent on hemolysins, and induction of IFNβ and IL-12 as well as IRF5 activation were reduced with TLR2 ligand costimulation. Heat inactivation of GBS reduced IRF5 and NF-kB translocation, while only the viable E. coli activated IRF5. The attenuated stimulation correlated with loss of bacterial RNA integrity. The E. coli-induced IRF5 translocation was not inhibited by TLR2 costimulation, suggesting that IRF5 was activated via a TLR8-independent mechanism. Gene silencing of MDM using siRNA revealed that GBS-induced IFNβ, IL-12-p35, and TNF production was dependent on TLR8 and IRF5. In contrast, cytokine induction by E. coli was TLR8 independent but still partly dependent on IRF5. We conclude that TLR8-IRF5 signaling is more important for the sensing of GBS than for stationary grown S. aureus in human primary monocytes and MDM, likely due to reduced resistance of GBS to phagosomal degradation and to a lower production of TLR2 activating lipoproteins. TLR8 does not sense viable E. coli, while IRF5 still contributes to E. coli-induced cytokine production, possibly via a cytosolic nucleic acid sensing mechanism.

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...

Abstract. Live cell arrays are an emerging tool that expand traditional 2D in vitro cell culture, increasing experimental precision and throughput. A patterned cell system was developed by combining the cell repellent properties of polyvinyl alcohol hydrogels with the cell adhesive properties of self-assembled films of dopamine (polydopamine). It was shown that polydopamine could be patterned onto polyvinyl alcohol hydrogels by microcontact printing, which in turn effectively patterned the growth of several cell types (HeLa, HEK293, HUVEC and PC3). The cells could be patterned at levels down to single-cell confinement, and it was demonstrated that cell patterns could be maintained for at least 3 weeks. Further, polydopamine could be used to modify poly(vinyl alcohol) in situ using a cell compatible deposition buffer (1 mg mL −1 dopamine in 25 mm tris with a physiological salt balance). The treatment switched the PVA hydrogel from cell repellent to cell adhesive. By pre-patterning one cell population and depositing polydopamine in situ, a second cell population could be seeded, and adhered to the newly switched surface. Patterned co-cultures of HeLa/HeLa and HeLa/HUVEC were thus realized through simple chemistry and could be studied over time. The combination of polyvinyl alcohol and polydopamine was shown to be an attractive route to versatile live cell arrays with minimal infrastructure requirements and low experimental complexity.PACS numbers: 87.17. Rt, 87.18.Gh, 87.80.Fe Keywords: Cell micropatterning, cell arrays, polydopamine, poly(vinyl alcohol) hydrogels, micropatterned co-cultures Patterned cell arrays on polydopamine-modified poly(vinyl alcohol) hydrogels.

Flat surfaces decorated with micro-and nanostructures are important tools in biomedical research used to control cellular shape, in studies of mechanotransduction, membrane mechanics, cell migration and cellular interactions with nanostructured surfaces. Existing methods to fabricate surface-bound nanostructures are typically limited either by resolution, aspect ratio or throughput. In this work, we explore electron beam lithography based structuring of the epoxy resist SU-8 on glass substrate. We focus on a systematic investigation of the process parameters and determine limits of the fabrication process, both in terms of spatial resolution, structure aspect ratio and fabrication throughput. The described approach is capable of producing high-aspect ratio, surface bound nanostructures with height ranging from 100 nm to 4000 nm and with in-plane resolution below 100 nm directly on a transparent substrate. Fabricated nanostructured surfaces can be integrated with common techniques for biomedical research, such as high numerical aperture optical microscopy. Furthermore, we show how the described approach can be used to make nanostructures with multiple heights on the same surface, something which is not readily achievable using alternative fabrication approaches. Our research paves an alternative way of manufacturing nanostructured surfaces with applications in life science research.