Actin polymerization as a key innate immune effector mechanism to control Salmonella infection.

Salmonellosis is one of the leading causes of food poisoning worldwide. Controlling bacterial burden is essential to surviving infection. Nucleotide-binding oligomerization domain-like receptors (NLRs), such as NLRC4, induce inflammasome effector functions and play a crucial role in controlling Salmonella infection. Inflammasome-dependent production of IL-1ß recruits additional immune cells to the site of infection, whereas inflammasome-mediated pyroptosis of macrophages releases bacteria for uptake by neutrophils. Neither of these functions is known to directly kill intracellular salmonellae within macrophages. The mechanism, therefore, governing how inflammasomes mediate intracellular bacterial-killing and clearance in host macrophages remains unknown. Here, we show that actin polymerization is required for NLRC4-dependent regulation of intracellular bacterial burden, inflammasome assembly, pyroptosis, and IL-1ß production. NLRC4-induced changes in actin polymerization are physically manifested as increased cellular stiffness, and leads to reduced bacterial uptake, production of antimicrobial molecules, and arrested cellular migration. These processes act in concert to limit bacterial replication in the cell and dissemination in tissues. We show, therefore, a functional link between innate immunity and actin turnover in macrophages that underpins a key host defense mechanism for the control of salmonellosis.

Biophotonic techniques for the study of malaria-infected red blood cells.

Investigation of the homeostasis of red blood cells upon infection by Plasmodium falciparum poses complex experimental challenges. Changes in red cell shape, volume, protein, and ion balance are difficult to quantify. In this article, we review a wide range of optical techniques for quantitative measurements of critical homeostatic parameters in malaria-infected red blood cells. Fluorescence lifetime imaging and tomographic phase microscopy, quantitative deconvolution microscopy, and X-ray microanalysis, are used to measure haemoglobin concentration, cell volume, and ion contents. Atomic force microscopy is briefly reviewed in the context of these optical methodologies. We also describe how optical tweezers and optical stretchers can be usefully applied to empower basic malaria research to yield diagnostic information on cell compliance changes upon malaria infection. The combined application of these techniques sheds new light on the detailed mechanisms of malaria infection providing potential for new diagnostic or therapeutic approaches.

The cavity-to-cavity migration of leukaemic cells through 3D honey-combed hydrogels with adjustable internal dimension and stiffness.

Whilst rigid, planar surfaces are often used to study cell migration, a physiological scenario requires three-dimensional (3D) scaffolds with tissue-like stiffness. This paper presents a method for fabricating periodic hydrogel scaffolds with a 3D honeycomb-like structure from colloidal crystal templates. The scaffolds, made of hydrogel-walled cavities interconnected by pores, have separately tuneable internal dimensions and adjustable gel stiffness down to that of soft tissues. In conjunction with confocal microscopy, these scaffolds were used to study the importance of cell compliance on invasive potential. Acute promyelocytic leukaemia (APL) cells were differentiated with all-trans retinoic acid (ATRA) and treated with paclitaxel. Their migration ability into the scaffolds' size-restricted pores, enabled by cell softening during ATRA differentiation, was significantly reduced by paclitaxel treatment, which interferes with cell shape recovery. These findings demonstrate the usability of the scaffolds for investigating factors that affect cell migration, and potentially other cell functions, in a realistic 3D tissue model.

The optical cell rotator.

The optical cell rotator (OCR) is a modified dual-beam laser trap for the holding and controlled rotation of suspended dielectric microparticles, such as cells. In contrast to optical tweezers, OCR uses two counter-propagating divergent laser beams, which are shaped and delivered by optical fibers. The rotation of a trapped specimen is carried out by the rotation of a dual-mode fiber, emitting an asymmetric laser beam. Experiments were performed on human erythrocytes, promyelocytic leukemia cells (HL60), and cell clusters (MCF-7). Since OCR permits the rotation of cells around an axis perpendicular to the optical axis of any microscope and is fully decoupled from imaging optics, it could be a suitable and expedient tool for tomographic microscopy.