Biology subjects

Sellin, M. E.

Publications and source records attributed to Sellin, M. E..

5 recordsLinked to original sources

bioRxiv2023

A Two-Step Activation Mechanism Enables Mast Cells to Differentiate their Response between Extracellular and Invasive Enterobacterial Infection

Mast cells (MCs) localize to mucosal tissues and contribute to innate immune defenses against infection. How MCs sense, differentiate between, and respond to bacterial pathogens remains a topic of ongoing debate. Using the prototype enteropathogen Salmonella Typhimurium (S.Tm) and other closely related enterobacteria, we here demonstrate that MCs can regulate their cytokine secretion response to distinguish between extracellular and invasive bacterial infection. Tissue-invasive S.Tm and MCs colocalize in the Salmonella-infected mouse gut. Toll-like Receptor 4 (TLR4) sensing of extracellular S.Tm, or pure LPS, causes a slow and modest induction of MC cytokine transcripts and proteins, including IL-6, IL-13, and TNF. By contrast, type-III-secretion-system-1 (TTSS-1)-dependent S.Tm invasion of both mouse and human MCs triggers rapid and potent inflammatory gene expression and >100-fold elevated cytokine secretion. The S.Tm TTSS-1 effectors SopB, SopE, and SopE2 here elicit a second activation signal, including Akt phosphorylation downstream of effector translocation, which combines with TLR activation to promote the full-blown MC response. Supernatants from S.Tm-infected MCs boost macrophage survival and maturation from bone-marrow progenitors. Taken together, this study shows that MCs can differentiate between extracellular and host-cell invasive enterobacteria via a two-step activation mechanism and tune their inflammatory output accordingly.

immunology↗

bioRxiv2023

Intraluminal neutrophils limit epithelium damage by reducing pathogen assault on intestinal epithelial cells during Salmonella gut infection

Recruitment of neutrophils into the gut epithelium is a cardinal feature of intestinal inflammation in response to enteric infections. Previous work using the model pathogen Salmonella Typhimurium (S. Tm) established that invasion of intestinal epithelial cells by S.Tm leads to recruitment of neutrophils into the gut lumen, where they can reduce pathogen loads transiently. Notably, a fraction of the pathogen population can survive this defense, re-grow to high density, and continue triggering enteropathy. However, the functions of intraluminal neutrophils in the defense against enteric pathogens and their effects on preventing or aggravating epithelial damage are still not fully understood. Here, we address this question via neutrophil depletion in different mouse models of Salmonella colitis, which differ in their degree of enteropathy. In an antibiotic pre-treated mouse model, neutrophil depletion by an anti-Ly6G antibody exacerbated epithelial damage. This could be linked to compromised neutrophil-mediated elimination and reduced physical blocking of the gut-luminal S.Tm population such that the pathogen density remained high near the epithelial surface throughout the infection. The removal of luminal S. Tm by gentamicin, an antibiotic restricted to the gut lumen, reversed the effect of neutrophil depletion on epithelial cell loss. Strikingly, when using germ-free mice and an S. Tm ssaV mutant capable of epithelium invasion, but attenuated for survival and growth within host tissues, neutrophil depletion caused exacerbated immune activation of the gut mucosa and a complete destruction of the epithelial barrier. Together, our data indicate that intraluminal neutrophils are central for maintaining epithelial barrier integrity during acute Salmonella-induced gut inflammation, by limiting the sustained pathogen assault on the epithelium in a critical window of the infection. Highlights{circ} After the first wave of mucosal invasion (day 1 p.i.), S. Tm maintains the assault from the lumen, triggering the continued expulsion of epithelial cells in antibiotic pre-treated mice. {circ}Neutrophil recruitment into the gut lumen is essential to limit this continued Salmonella attack on the epithelium. {circ}In antibiotic pre-treated SPF mice, neutrophil depletion exacerbates S. Tm invasion, causing excessive epithelial cell loss, which compromises epithelial barrier integrity at later time points (day 2-3 p.i.). {circ}In germ-free mice, neutrophil depletion exacerbates epithelial responses and epithelial barrier destruction even more strongly than in streptomycin pre-treated SPF mice. {circ}Gentamicin treatment and ssaV mutant infections indicate that neutrophils prevent epithelial damage by eliminating and physically blocking gut-luminal pathogens.

immunology↗

bioRxiv2022

High-efficiency transfection of Acanthamoeba castellanii using a cationic polymer

The free-living amoeba Acanthamoeba castellanii is an ecologically, clinically, and evolutionarily important microorganism. A. castellanii amoebae are directly pathogenic to humans and serve as reservoirs for bacterial pathogens (e.g., Legionella pneumophila) but also regulate the proliferation of other microorganisms in the soil. Despite their importance, no reliable genetic system has been developed, hampering the use of A. castellanii and related species as model organisms. Transfecting A. castellanii with plasmids is possible with commercial kits, but it is expensive, inefficient, and vulnerable to product discontinuation. In this contribution, we present a method for efficient transfection of A. castellanii with readily available and inexpensive polyethylenimines. We systematically explore the methods parameters, obtaining up to 100-fold higher efficiency than currently used protocols. The method presented here provides a robust step towards a complete genetic toolbox for A. castellanii, hence expanding its use as a model organism.

microbiology↗

bioRxiv2022

Gasdermin D is the only Gasdermin that provides non-redundant protection against acute Salmonella gut infection

Gasdermins (GSDMs) share a common functional domain structure and are best known for their capacity to form membrane pores. These pores are hallmarks of a specific form of cell death called pyroptosis and mediate the secretion of pro-inflammatory cytokines such as interleukin 1{beta} (IL1{beta}) and interleukin 18 (IL18). Thereby, Gasdermins have been implicated in various immune responses against cancer and infectious diseases such as acute Salmonella Typhimurium (S.Tm) gut infection. However, to date, we lack a comprehensive functional assessment of the different Gasdermins (GSDMA-E) during S.Tm infection in vivo. Here, we have performed littermate-controlled oral S.Tm infections to investigate the impact of all murine Gasdermins. While GSDMA, -C and -E appear dispensable, we show that GSDMD (i) restricts S.Tm loads in the gut tissue and systemic organs, (ii) controls gut inflammation kinetics, and (iii) prevents epithelium disruption by 72h of the infection. Full protection requires GSDMD expression by both bone-marrow-derived lamina propria cells and intestinal epithelial cells (IECs). In vivo experiments, 3D- and 2D-enteroid infections further show that infected IEC extrusion proceeds also without GSDMD, but that GSDMD controls the permeabilization and morphology of the extruding cells and affects extrusion kinetics. As such, this work identifies a non-redundant multipronged role of GSDMD in mucosal tissue defence against a common enteric pathogen. HIGHLIGHTSO_LIGasdermin D restricts Salmonella Typhimurium (S.Tm) translocation across the gut tissue, controls gut inflammation kinetics, and prevents epithelium disruption by 72h of the infection. C_LIO_LIGasdermins A, C and E appear dispensable for protection against acute S.Tm gut infection. C_LIO_LIGasdermin D in bone-marrow-derived lamina propria cells and intestinal epithelial cells complement each other to suppress gut tissue S.Tm loads. C_LIO_LIGasdermin D is not required for extrusion of infected intestinal epithelial cells but drives their permeabilization and affects qualitative features of the extrusion process. C_LI

immunology↗

bioRxiv2021

High Definition DIC Imaging Uncovers Transient Stages of Pathogen Infection Cycles on the Surface of Human Adult Stem Cell-Derived Intestinal Epithelium

Interactions between individual pathogenic microbes and host tissues involve fast and dynamic processes that ultimately impact the outcome of infection. Using live-cell microscopy, these dynamics can be visualized to study e.g. microbe motility, binding and invasion of host cells, and intra-host-cell survival. Such methodology typically employs confocal imaging of fluorescent tags in tumor-derived cell line infections on glass. This allows high-definition imaging, but poorly reflects the host tissues physiological architecture and may result in artifacts. We developed a method for live-cell imaging of microbial infection dynamics on human adult stem cell-derived intestinal epithelial cell (IEC) layers. These IEC monolayers are grown in alumina membrane chambers, optimized for physiological cell arrangement and fast, but gentle, differential interference contrast (DIC) imaging. This allows sub-second visualization of both microbial and epithelial surface ultrastructure at high resolution without using fluorescent reporters. We employed this technology to probe the behavior of two model pathogens, Salmonella enterica Typhimurium (Salmonella) and Giardia intestinalis (Giardia), at the intestinal epithelial surface. Our results reveal pathogen-specific swimming patterns on the epithelium, showing that Salmonella adheres to the IEC surface for prolonged periods before host-cell invasion, while Giardia uses circular swimming with intermittent attachments to scout for stable adhesion sites. This method even permits tracking of individual Giardia flagella, demonstrating that active flagellar beating and attachment to the IEC surface are not mutually exclusive. Thereby, this work describes a powerful, generalizable, and relatively inexpensive approach to study dynamic pathogen interactions with IEC surfaces at high resolution and under near-native conditions. ImportanceKnowledge of dynamic niche-specific interactions between single microbes and host cells is essential to understand infectious disease progression. However, advances in this field have been hampered by the inherent conflict between the technical requirements for high resolution live-cell imaging on one hand, and conditions that best mimic physiological infection niche parameters on the other. Towards bridging this divide, we present methodology for differential interference contrast (DIC) imaging of pathogen interactions at the apical surface of enteroid-derived intestinal epithelia, providing both high spatial and temporal resolution. This alleviates the need for fluorescent reporters in live-cell imaging and provides dynamic information about microbe interactions with a non-transformed, confluent, polarized and microvilliated human gut epithelium. Using this methodology, we uncover previously unrecognized stages of Salmonella and Giardia infection cycles at the epithelial surface.

microbiology↗