Agneta Richter-Dahlfors

Toll-like Receptor 4 Resides in the Golgi Apparatus and Colocalizes with Internalized Lipopolysaccharide in Intestinal Epithelial Cells

Toll-like receptor (TLR) 4 is mainly found on cells of the myelopoietic lineage. It recognizes lipopolysaccharide (LPS) and mediates cellular activation and production of proinflammatory cytokines. Less is known about the distribution and role of TLR4 in epithelial cells that are continuously exposed to microbes and microbial products. Here we show that the murine small intestinal epithelial cell line m-ICcl2 is highly responsive to LPS and expresses both CD14 and TLR4. Transcription and surface membrane staining for CD14 were up-regulated upon LPS exposure. Surprisingly, TLR4 immunostaining revealed a strictly cytoplasmic paranuclear distribution. This paranuclear compartment could be identified as the Golgi apparatus. LPS added to the supernatant was internalized by m-ICcl2 cells and colocalized with TLR4. Continuous exposure to LPS led to a tolerant phenotype but did not alter TLR4 expression nor cellular distribution. Thus, intestinal epithelial cells might be able to provide the initial proinflammatory signal to attract professional immune cells to the side of infection. The cytoplasmic location of TLR4, which is identical to the final location of internalized LPS, further indicates an important role of cellular internalization and cytoplasmic traffic in the process of innate immune recognition.

Vesicle-Mediated Export and Assembly of Pore-Forming Oligomers of the Enterobacterial ClyA Cytotoxin

The ClyA protein is a pore-forming cytotoxin expressed by Escherichia coli and some other enterobacteria. It confers cytotoxic activity toward mammalian cells, but it has remained unknown how ClyA is surface exposed and exported from bacterial cells. Outer-membrane vesicles (OMVs) released from the bacteria were shown to contain ClyA protein. ClyA formed oligomeric pore assemblies in the OMVs, and the cytotoxic activity toward mammalian cells was considerably higher than that of ClyA protein purified from the bacterial periplasm. The redox status of ClyA correlated with its ability to form the oligomeric pore assemblies. In bacterial cells with a defective periplasmic disulphide oxidoreductase system, the ClyA protein was phenotypically expressed in a constitutive manner. The results define a vesicle-mediated transport mechanism in bacteria, and our findings show that the localization of proteins to OMVs directly may contribute to the activation and delivery of pathogenic effector proteins.

Nanoscale features influence epithelial cell morphology and cytokine production

Available, easy and fast fabrication methods of nanostructured surfaces, and the knowledge that cells in vivo interacts with nanometer-sized structures/objects, led us to study the impact of nanotopography on cell morphology and cytokine production. Uroepithelial cells were seeded on three different substrate types: two with defined nanometer topographies and a flat control, all three having identical surface chemistry. The nanostructured substrates contained hemispherical pillars or step edges, the latter in the form of parallel grooves and ridges. Qualitative and quantitative analysis of cell morphology and cytokine production were studied. Both quantities were significantly different between cells cultured on hemispherically structured surfaces compared to flat control surfaces. Cells cultured on hemispherically structured surfaces showed a decrease in IL-6 and IL-8 production and were less spread, less round and more stellate (larger dispersion). Only cell morphology differed between cells cultured on grooved surfaces and flat control surfaces. These findings suggest that epithelial cell morphology and cytokine production are dependent on the underlying nanotopography.

Alpha-haemolysin of uropathogenic E. coli induces Ca2+ oscillations in renal epithelial cells.

Pyelonephritis is one of the most common febrile diseases in children. If not treated appropriately, it causes irreversible renal damage and accounts for a large proportion of end stage renal failures. Renal scarring can occur in the absence of inflammatory cells, indicating that bacteria may have a direct signalling effect on renal cells. Intracellular calcium ([Ca2+]i) oscillations can protect cells from the cytotoxic effects of prolonged increases in intracellular calcium. However, no pathophysiologically relevant protein that induces such oscillations has been identified. Here we show that infection by uropathogenic Escherichia coli induces a constant, low-frequency oscillatory [Ca 2+]i response in target primary rat renal epithelial cells induced by the secreted RTX (repeats-in-toxin) toxin α-haemolysin. The response depends on calcium influx through L-type calcium channels as well as from internal stores gated by inositol triphosphate. Internal calcium oscillations induced by α-haemolysin in a renal epithelial cell line stimulated production of cytokines interleukin (IL)-6 and IL-8. Our findings indicate a novel role for α-haemolysin in pyelonephritis: as an inducer of an oscillating second messenger response in target cells, which fine-tunes gene expression during the inflammatory response.

Organic electronics for precise delivery of neurotransmitters to modulate mammalian sensory function

Significant advances have been made in the understanding of the pathophysiology, molecular targets and therapies for the treatment of a variety of nervous-system disorders. Particular therapies involve electrical sensing and stimulation of neural activity, and significant effort has therefore been devoted to the refinement of neural electrodes. However, direct electrical interfacing suffers from some inherent problems, such as the inability to discriminate amongst cell types. Thus, there is a need for novel devices to specifically interface nerve cells. Here, we demonstrate an organic electronic device capable of precisely delivering neurotransmitters in vitro and in vivo. In converting electronic addressing into delivery of neurotransmitters, the device mimics the nerve synapse. Using the peripheral auditory system, we show that out of a diverse population of cells, the device can selectively stimulate nerve cells responding to a specific neurotransmitter. This is achieved by precise electronic control of electrophoretic migration through a polymer film. This mechanism provides several sought-after features for regulation of cell signalling: exact dosage determination through electrochemical relationships, minimally disruptive delivery due to lack of fluid flow, and on-off switching. This technology has great potential as a therapeutic platform and could help accelerate the development of therapeutic strategies for nervous-system disorders.

Induction of innate immune responses by Escherichia coli and purified lipopolysaccharide correlate with organ‐ and cell‐specific expression of Toll‐like receptors within the human urinary tract

Mucosal epithelial linings function as physical barriers against microbes. In addition, they participate in the first line of host defence by production of a variety of proinflammatory mediators when exposed to microbes and microbial agents. Here, we use a human urinary tract infection model to demonstrate that organ‐ and cell‐specific innate responses induced by lipopolysaccharides (LPS) present on Gram‐negative bacteria correlates with the expression of Toll‐like receptor 4 (TLR4). The presence of TLR4 on human bladder epithelial cells enables them to rapidly respond to bacterial infections in vitro and in vivo. In contrast, TLR4 is not expressed on human proximal tubule cells isolated from the renal cortex, which may explain the cortical localization of bacteria in pyelonephritis. TLR4‐negative renal epithelial cells, A498, are non‐responsive to purified LPS, however, they respond to viable bacteria via a mannose‐sensitive attachment‐mediated pathway. To identify LPS components recognised by bladder epithelial cells, a bacterial lipid A mutant and LPS of different chemotypes were tested. Full interleukin 8 induction required hexa‐acylated lipid A and was decreased by between 50% and 70% in the presence of O‐antigen. Taken together, we propose that multiple independent pathways, which are organ‐ and cell‐specifically expressed, mediate bacterial recognition and determine the outcome of innate responses to infection.

Gastric Mucosal Recognition of Helicobacter pylori Is Independent of Toll-Like Receptor 4

Little is known about the interactions between Helicobacter pylori, which specializes in colonizing the mucin layer that covers the gastric mucosa, and primary gastric epithelial cells. The expression pattern of Toll-like receptors (TLRs) in primary gastric epithelial cells and cell lines was compared. Primary cells did not express TLR4, whereas all cell lines expressed a nonsignaling form of TLR4. Because other cells within the mucosa expressed TLR4, it was next investigated whether H. pylori can be recognized by TLR4--they cannot. Moreover, H. pylori infection of primary cells induced a regulated production of interleukin (IL)-6, IL-8, and tumor necrosis factor-alpha, whereas infection of cell lines only resulted in IL-8 production. The cytokine production in all cell types was strictly cag dependent. These findings indicate that, although the epithelium is important in directing the immune response against H. pylori infections, the response is independent of TLR4.

Structural requirements for TLR4-mediated LPS signalling: a biological role for LPS modifications

Cells of the mucosal lining are the first to encounter invading bacteria during infection, and as such, they have developed numerous ways of detecting microbial intruders. Recently, we showed that epithelial cells recognize lipopolysaccharide (LPS) through the CD14-Toll-like receptor (TLR)-4 complex. Here, we identify the substructures of LPS that are recognized by the TLR4 receptor complex. In contrast to lipid A, the O-antigen does not mediate an inflammatory response; rather it interferes with the lipid A recognition. An Escherichia coli strain genetically modified to express penta-acylated lipid A not only showed reduced immunogenicity, but was also found to inhibit pro-inflammatory signalling induced by wild-type E. coli (hexa-acylated lipid A) as well as LPS from other bacteria of the Enterobacteriaceae family. Furthermore, penta-acylated LPS from Pseudomonas aeruginosa acted as an antagonist to hexa-acylated E. coli LPS, as did E. coli, as shown by its inhibitory effect on IL-8 production in stimulated cells. Hypo-acylated lipid A, such as that of P. aeruginosa, is found in several species within the gut microflora as well as in several bacteria causing chronic infections. Thus, our results suggest that the composition of the microflora may be important in modulating pro-inflammatory signalling in epithelial cells under normal as well as pathologic conditions.

The biocompatibility and antibacterial properties of collagen-stabilized, photochemically prepared silver nanoparticles.

Spherical 3.5 nm diameter silver nanoparticles (AgNP) stabilized in type I collagen (AgNP@collagen) were prepared in minutes (5-15 min) at room temperature by a photochemical method initiated by UVA irradiation of a water-soluble non-toxic benzoin. This biocomposite was examined to evaluate its biocompatibility and its anti-bacterial properties and showed remarkable properties. Thus, while keratinocytes and fibroblasts were not affected by AgNP@collagen, it was bactericidal against Bacillus megaterium and E. coli but only bacteriostatic against S. epidermidis. In particular, the bactericidal properties displayed by AgNP@collagen were proven to be due to AgNP in AgNP@collagen, rather than to released silver ions, since equimolar concentrations of Ag are about four times less active than AgNP@collagen based on total Ag content. This new biocomposite was stable over a remarkable range of NaCl, phosphate, and 2-(N-morpholino)ethanesulfonic acid concentrations and for over one month at 4 °C. Circular dichroism studies show that the conformation of collagen in AgNP@collagen remains intact. Finally, we have compared the properties of AgNP@collagen with a similar biocomposite prepared using α-poly-L-Lysine and also with citrate stabilized AgNP; neither of these materials showed comparable biocompatibility, stability, or anti-bacterial activity.

Organic bioelectronics in nanomedicine

BACKGROUND Nanomedicine is a research area with potential to shape, direct, and change future medical treatments in a revolutionary manner over the next decades. While the common goal with other fields of biomedicine is to solve medical problems, this area embraces an increasing number of technology platforms as they become miniaturized. Organic electronics has over the past two decades developed into an exciting and thriving area of research. SCOPE OF REVIEW Today, the organic electronics field stands at the interface with biology. As the area of organic bioelectronics advances, it holds promise to make major contributions to nanomedicine. The progress made in this direction is the topic of this review. MAJOR CONCLUSIONS We describe the inherent features of conducting polymers, and explain the usefulness of these materials as active scaffolds in cell biology and tissue engineering. We also explain how the combined ionic and electronic conductive nature of the polymers is used to precisely control the delivery of signal substances. This unique feature is key in novel devices for chemical communication with cells and tissues. GENERAL SIGNIFICANCE This review highlights the results from the creative melting pot of interdisciplinary research in organic bioelectronics. This article is part of a Special Issue entitled Nanotechnologies - Emerging Applications in Biomedicine.