Listeria monocytogenes exploits efferocytosis to promote cell-to-cell spread

Efferocytosis, the process by which dying/dead cells are removed by phagocytosis, plays an important role in development, tissue homeostasis and innate immunity1. Efferocytosis is mediated, in part, by receptors that bind to exofacial phosphatidylserine (PS) on cells or cellular debris after loss of plasma membrane asymmetry. Here we show that a bacterial pathogen, Listeria monocytogenes (Lm), can exploit efferocytosis to promote cell-to-cell spread during infection. These bacteria can escape the phagosome in host cells using the pore-forming toxin Listeriolysin O (LLO) and two phospholipases C2. Expression of the cell surface protein ActA allows Lm to activate host actin regulatory factors and undergo actin-based motility in the cytosol, eventually leading to formation of actin-rich protrusions at the cell surface. We show that protrusion formation is associated with plasma membrane damage due to LLO’s pore-forming activity. LLO also promotes the release of bacteria-containing protrusions from the host cell, generating membrane-derived vesicles with exofacial PS. The PS-binding receptor TIM-4 contributes to efficient cell-to-cell spread by Lm in macrophages in vitro and growth of these bacteria is impaired in TIM-4−/− mice. Thus, Lm promotes its dissemination in a host by exploiting efferocytosis. Our study suggests that PS-targeted therapeutics may be useful in the fight against infections by Lm and other bacteria that utilize similar strategies of cell-to-cell spread during infection

Novel approaches : Tissue engineering and stem cells--In vitro modelling of the gut

In many intestinal diseases, the function of the epithelial lining is impaired. In this review, we describe the recent developments of in vitro intestinal stem cell cultures. When these stem cells are grown in 3D structures (organoids), they provide a model of the intestinal epithelium, which is closely similar to the growth and development of the in vivo gut. This model provides a new tool to study various diseases of malabsorption in functional detail and therapeutic applications, which could not be achieved with traditional cell lines. First, we describe the organization and function of the healthy small intestinal epithelium. Then, we discuss the establishment of organoid cultures and how these structures represent the healthy epithelium. Finally, we discuss organoid cultures as a tool for studying intrinsic properties of the epithelium, as a model for intestinal disease, and as a possible source for stem cell transplantations

Novel approaches: Tissue engineering and stem cells--In vitro modelling of the gut

In many intestinal diseases, the function of the epithelial lining is impaired. In this review, we describe the recent developments of in vitro intestinal stem cell cultures. When these stem cells are grown in 3D structures (organoids), they provide a model of the intestinal epithelium, which is closely similar to the growth and development of the in vivo gut. This model provides a new tool to study various diseases of malabsorption in functional detail and therapeutic applications, which could not be achieved with traditional cell lines. First, we describe the organization and function of the healthy small intestinal epithelium. Then, we discuss the establishment of organoid cultures and how these structures represent the healthy epithelium. Finally, we discuss organoid cultures as a tool for studying intrinsic properties of the epithelium, as a model for intestinal disease, and as a possible source for stem cell transplantations

DGAT2 partially compensates for lipid-induced ER stress in human DGAT1-deficient intestinal stem cells

Dietary lipids are taken up as FAs by the intestinal epithelium and converted by diacylglycerol acyltransferase (DGAT) enzymes into triglycerides, which are packaged in chylomicrons or stored in cytoplasmic lipid droplets (LDs). DGAT1-deficient patients suffer from vomiting, diarrhea, and protein losing enteropathy, illustrating the importance of this process to intestinal homeostasis. Previously, we have shown that DGAT1 deficiency causes decreased LD formation and resistance to unsaturated FA lipotoxicity in patient-derived intestinal organoids. However, LD formation was not completely abolished in patient-derived organoids, suggesting the presence of an alternative mechanism for LD formation. Here, we show an unexpected role for DGAT2 in lipid metabolism, as DGAT2 partially compensates for LD formation and lipotoxicity in DGAT1-deficient intestinal stem cells. Furthermore, we show that (un)saturated FA-induced lipotoxicity is mediated by ER stress. More importantly, we demonstrate that overexpression of DGAT2 fully compensates for the loss of DGAT1 in organoids, indicating that induced DGAT2 expression in patient cells may serve as a therapeutic target in the future

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 intrahost-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 tissue's 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 layers are grown in apical imaging chambers, optimized for physiological cell arrangement and fast, but gentle, differential interference contrast (DIC) imaging. This allows subsecond 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 serovar Typhimurium and Giardia intestinalis, at the intestinal epithelial surface. Our results reveal pathogen-specific swimming patterns on the epithelium and show that Salmonella lingers on the IEC surface for prolonged periods before host cell invasion, while Giardia uses circular swimming with intermittent attachments to scout for stable adhesion sites. The method even permits tracking of individual Giardia flagella, demonstrating that active flagellar beating and attachment to the IEC surface are not mutually exclusive. This work describes a generalizable and relatively inexpensive approach to resolving dynamic pathogen-IEC layer interactions, applicable even to genetically nontractable microorganisms. IMPORTANCE Knowledge 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 the one hand and conditions that best mimic physiological infection niche parameters on the other. Toward bridging this divide, we present a 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 nontransformed, 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

Trophozoite fitness dictates the intestinal epithelial cell response to Giardia intestinalis infection

Giardia intestinalis is a non-invasive, protozoan parasite infecting the upper small intestine of most mammals. Symptomatic infections cause the diarrhoeal disease giardiasis in humans and animals, but at least half of the infections are asymptomatic. However, the molecular underpinnings of these different outcomes of the infection are still poorly defined. Here, we studied the early transcriptional response to G. intestinalis trophozoites, the disease-causing life-cycle stage, in human enteroid-derived, 2-dimensional intestinal epithelial cell (IEC) monolayers. Trophozoites preconditioned in media that maximise parasite fitness triggered only neglectable inflammatory transcription in the IECs during the first hours of co-incubation. By sharp contrast, “non-fit” or lysed trophozoites induced a vigorous IEC transcriptional response, including high up-regulation of many inflammatory cytokines and chemokines. Furthermore, “fit” trophozoites could even suppress the stimulatory effect of lysed trophozoites in mixed infections, suggesting active G. intestinalis suppression of the IEC response. By dual-species RNA-sequencing, we defined the IEC and G. intestinalis gene expression programs associated with these differential outcomes of the infection. Taken together, our results inform on how G. intestinalis infection can lead to such highly variable effects on the host, and pinpoints trophozoite fitness as a key determinant of the IEC response to this common parasite. Author summary Diarrhoeal infectious diseases are still a major problem worldwide, each year killing nearly 800,000 children. These infections are caused by a variety of pathogenic bacteria, viruses, fungi and protozoa. The protozoan parasite Giardia intestinalis is the most common eukaryotic intestinal pathogen found in humans. In contrast to most bacteria and viruses that cause diarrhoea, Giardia parasites elicit a highly variable clinical picture and often do not cause pronounced inflammation in the intestine of infected patients. Here we show, by using human intestinal epithelial cells derived from adult intestinal stem cells, that “fit” Giardia parasites can actively suppress epithelial inflammatory signalling, while their “non-fit” counterparts instead induce inflammatory responses. These two faces of the parasite are associated with specific gene expression programs and may explain the earlier observed high variability in outcome of a Giardia infection, and its modulatory effect on infection by other intestinal pathogens

Enhanced Collagen Deposition in the Duodenum of Patients with Hyaline Fibromatosis Syndrome and Protein Losing Enteropathy

Hyaline fibromatosis syndrome (HFS), resulting from ANTXR2 mutations, is an ultra-rare disease that causes intestinal lymphangiectasia and protein-losing enteropathy (PLE). The mechanisms leading to the gastrointestinal phenotype in these patients are not well defined. We present two patients with congenital diarrhea, severe PLE and unique clinical features resulting from deleterious ANTXR2 mutations. Intestinal organoids were generated from one of the patients, along with CRISPR-Cas9 ANTXR2 knockout, and compared with organoids from two healthy controls. The ANTXR2-deficient organoids displayed normal growth and polarity, compared to controls. Using an anthrax-toxin assay we showed that the c.155C&gt;T mutation causes loss-of-function of ANTXR2 protein. An intrinsic defect of monolayer formation in patient-derived or ANTXR2(KO) organoids was not apparent, suggesting normal epithelial function. However, electron microscopy and second harmonic generation imaging showed abnormal collagen deposition in duodenal samples of these patients. Specifically, collagen VI, which is known to bind ANTXR2, was highly expressed in the duodenum of these patients. In conclusion, despite resistance to anthrax-toxin, epithelial cell function, and specifically monolayer formation, is intact in patients with HFS. Nevertheless, loss of ANTXR2-mediated signaling leads to collagen VI accumulation in the duodenum and abnormal extracellular matrix composition, which likely plays a role in development of PLE.De två första författarna delar förstaförfattarskapet.</p

Human Fetal TNF-α-Cytokine-Producing CD4 + Effector Memory T Cells Promote Intestinal Development and Mediate Inflammation Early in Life

Although the fetal immune system is considered tolerogenic, preterm infants can suffer from severe intestinal inflammation, including necrotizing enterocolitis (NEC). Here, we demonstrate that human fetal intestines predominantly contain tumor necrosis factor-α (TNF-α) + CD4 + CD69 + T effector memory (Tem) cells. Single-cell RNA sequencing of fetal intestinal CD4 + T cells showed a T helper 1 phenotype and expression of genes mediating epithelial growth and cell cycling. Organoid co-cultures revealed a dose-dependent, TNF-α-mediated effect of fetal intestinal CD4 + T cells on intestinal stem cell (ISC) development, in which low T cell numbers supported epithelial development, whereas high numbers abrogated ISC proliferation. CD4 + Tem cell frequencies were higher in inflamed intestines from preterm infants with NEC than in healthy infant intestines and showed enhanced TNF signaling. These findings reveal a distinct population of TNF-α-producing CD4 + T cells that promote mucosal development in fetal intestines but can also mediate inflammation upon preterm birth. The fetal immune system is considered anti-inflammatory; nonetheless, preterm infants are at risk for necrotizing enterocolitis (NEC), a severe intestinal inflammatory disease. Schreurs et al. demonstrate that fetal TNF-α + CD4 + T cells promote gut development early in life. However, in preterm babies these TNFα + CD4 + T cells can mediate intestinal inflammation, providing a potential mechanism for NEC