Bacterial detection by NAIP/NLRC4 elicits prompt contractions of intestinal epithelial cell layers

The gut epithelium serves to maximize the surface for nutrient and fluid uptake, but at the same time must provide a tight barrier to pathogens and remove damaged intestinal epithelial cells (IECs) without jeopardizing barrier integrity. How the epithelium coordinates these tasks remains a question of significant interest. We used imaging and an optical flow analysis pipeline to study the dynamicity of untransformed murine and human intestinal epithelia, cultured atop flexible hydrogel supports. Infection with the pathogen Salmonella Typhimurium (S.Tm) within minutes elicited focal contractions with inward movements of up to ∼1,000 IECs. Genetics approaches and chimeric epithelial monolayers revealed contractions to be triggered by the NAIP/NLRC4 inflammasome, which sensed type-III secretion system and flagellar ligands upon bacterial invasion, converting the local tissue into a contraction epicenter. Execution of the response required swift sublytic Gasdermin D pore formation, ion fluxes, and the propagation of a myosin contraction pulse across the tissue. Importantly, focal contractions preceded, and could be uncoupled from, the death and expulsion of infected IECs. In both two-dimensional monolayers and three-dimensional enteroids, multiple infection-elicited contractions coalesced to produce shrinkage of the epithelium as a whole. Monolayers deficient for Caspase-1(-11) or Gasdermin D failed to elicit focal contractions but were still capable of infected IEC death and expulsion. Strikingly, these monolayers lost their integrity to a markedly higher extent than wild-type counterparts. We propose that prompt NAIP/NLRC4/Caspase-1/Gasdermin D/myosin–dependent contractions allow the epithelium to densify its cell packing in infected regions, thereby preventing tissue disintegration due to the subsequent IEC death and expulsion process.

Epithelial cells make up barriers that shield the interior of the body from the environment. In the homeostatic intestine, the surface of the epithelium is maximized to facilitate uptake of ingested nutrients, water, and electrolytes (1, 2). This large surface, however, makes the gut epithelium vulnerable to attack by pathogenic microorganisms. Pathogen onslaught and the ensuing inflammation causes death and loss of intestinal epithelial cells (IECs) (3, 4). This can be beneficial as damaged cells and intracellular pathogens are cleared from the mucosa. At the same time, cell loss may jeopardize epithelial integrity when insufficient IEC numbers remain to uphold the barrier. One way to prevent such an outcome would be to compact the epithelial layer in affected regions. It is well known that smooth muscle contraction results in shrinkage and compaction of the intestinal wall during inflammation (5), but whether and how the epithelium itself can alter its IEC packing upon infection appears less clear.As a system to sense mucosal intrusions, IECs express pattern recognition receptors (PRRs) [e.g., Toll-like receptors associated with cell membranes (6), and Nod-like receptors (NLRs) that form inflammasomes in the cytosol] (7–9). Epithelial recognition of pathogens through PRRs elicits a panel of countermeasures, including production of proinflammatory cytokines, chemokines, lipids (10–13), secretion of antimicrobial peptides (11, 14), and the death and expulsion of infected IECs into the lumen (12, 15, 16). An intricate cross-talk exists between IECs and immune cells residing in the underlying lamina propria (17). Epithelial defense signaling has in some cases also been shown to engage bystander epithelial cells surrounding a pathogen-infected IEC (18–20). Still, it remains poorly explored how PRR recognition of invading pathogens can instruct tissue-scale epithelial responses.Intestinal epithelial organoids (denoted “enteroids” when established from small intestinal crypts) provide a powerful experimental system to assess the behavior of untransformed epithelia in the absence of other mucosal cell types (21). Organoids can be grown as three-dimensional (3D) miniature organs within matrix domes (22, 23) or be disrupted to produce two-dimensional (2D) monolayers atop coated surfaces (24–27). By contrast to tumor cell lines, organoids maintain untransformed properties over time (28) and show reliable PRR expression patterns that mimic the intact gut epithelium (6, 9). Organoid models have for these reasons become attractive tools for physiological studies of gut infection (25, 29–32).In this work, we used time-lapse imaging to follow the tissue dynamicity of enteroid-derived mouse and human intestinal epithelia, placed atop pliable matrix supports. Upon infection with the prototypical enteropathogen Salmonella enterica serovar Typhimurium (S.Tm), we observed prompt and large epithelial contraction foci which preceded and could be uncoupled from IEC death and expulsion. Bacterial type-three secretion system (TTSS) and/or cytosolic flagellin triggered the epithelial NAIP/NLRC4 inflammasome, sublytic Gasdermin D pore formation, ion fluxes, and myosin-dependent contractions spreading from sensing events at focal epicentres. We show that this swift response allows the epithelium to increase its IEC packing at sites of infection, which may minimize the disruptive effects of subsequent cell death and expulsion.     

α-Lipoic Acid and Superoxide Dismutase in the Management of Chronic Neck Pain: A Prospective Randomized Study

Background and Objective

Since oxidative stress plays a pathogenetic role in chronic neck pain (CNP), we investigated whether a combination of α-lipoic acid (ALA) and superoxide dismutase (SOD) might improve pain control and the efficacy of physiotherapy (“multimodal therapy”) in patients with CNP.

Setting

This study was conducted in the Rehabilitation Unit of the Department of Surgical and Oncological Sciences at the University Policlinic in Palermo, Italy.

Design and Patients

This was a prospective, randomized, open study in outpatients.

Intervention

Patients randomly received either physiotherapy alone (group 2; n = 45) or a combination of ALA 600 mg and SOD 140 IU daily in addition to physiotherapy (group 1; n = 51), for 60 days. Pain was assessed by a visual analogue scale (VAS) and a modified Neck Pain Questionnaire (mNPQ). Treatment compliance and safety were also evaluated.

Results

Both groups experienced a significant reduction in the VAS and mNPQ scores after 1 month; however, while no further improvement was observed in group 2 at 60 days, group 1 showed a further VAS reduction (p < 0.001). In addition, in the mNPQ at 60 days, more patients in group 1 than in group 2 reported that their neck pain was improved (p < 0.01), and they showed greater compliance with prescribed physiotherapy (p = 0.048). No drug reaction was observed.

Conclusion

Use of ALA/SOD in combination with physiotherapy may be a useful approach to CNP, being antioxidants that act on nerve inflammation and disease progression.

Clinical Rehabilitation Impact

These preliminary observations suggest that some interesting goals (better pain control and physical wellbeing) can be achieved by multimodal therapy in CNP patients.     

DNA damage and repair following In vitro exposure to two different forms of titanium dioxide nanoparticles on trout erythrocyte

TiO₂ has been widely used to promote organic compounds degradation on waste aqueous solution, however, data on TiO₂ nanotoxicity to aquatic life are still limited. In this in vitro study, we compare the toxicity of two different families of TiO₂ nanoparticles on erythrocytes from Oncorhynchus mykiss trout. The crystal structure of the two TiO₂ nanoparticles was analyzed by XRD and the results indicated that one sample is composed of TiO₂ in the anatase crystal phase, while the other sample contains a mixture of both the anatase and the rutile forms of TiO₂ in a 2:8 ratio. Further characterization of the two families of TiO₂ nanoparticles was determined by SEM high resolution images and BET technique. The toxicity results indicate that both TiO₂ nanoparticles increase the hemolysis rate in a dose dependent way (1.6, 3.2, 4.8 μg mL^?1) but they do not influence superoxide anion production due to NADH addition measured by chemiluminescence. Moreover, TiO₂ nanoparticles (4.8 μg mL^?1) induce DNA damage and the entity of the damage is independent from the type of TiO₂ nanoparticles used. Modified comet assay (Endo III and Fpg) shows that TiO₂ oxidizes not only purine but also pyrimidine bases. In our experimental conditions, the exposure to TiO₂ nanoparticles does not affect the DNA repair system functionality. The data obtained contribute to better characterize the aqueous environmental risks linked to TiO₂ nanoparticles exposure. © 2011 Wiley Periodicals, Inc. Environ Toxicol 29: 117–127, 2014.     

Interleukin-1α, interleukin-1β and tumor necrosis factor-α genetic variants and risk of dementia in the very old: evidence from the “Monzino 80-plus” prospective study

The association among single nucleotide polymorphisms in inflammatory genes as interleukin-1 alpha (IL-1α), interleukin-1 beta (IL-1β) or tumor necrosis factor alpha (TNF-α) and dementia has been explored mostly in Alzheimer’s disease, while few data addressing their association with dementia in very old people are available. We performed a prospective, door-to-door population-based study of 80 years or older residents in eight municipalities of Varese province, Italy (the Monzino 80-plus study). No difference was found by a cross-sectional approach comparing IL-1α rs1800587, IL-1β rs3087258 and TNF-α rs1799724 genotypic and allelic frequencies between those affected and not affected by dementia. After a 5-year follow-up, the elderly carriers of T-allele of TNF-α rs1799724 were at an increased risk of dementia (p = 0.03). This association was no more significant adjusting for the apolipoprotein E epsilon-4 allele (APOE-ε4, p = 0.26), which was an independent predictor of dementia onset (p = 0.0002). In short, in this Italian population of oldest olds, dementia was associated to the APOE-ε4 allele only.