A polarized cell system amenable to subcellular resolution imaging of influenza virus infection.

The life cycle of influenza A viruses (IAV), and notably intracellular trafficking of the viral genome, depends on multiple interactions with the cellular cytoskeleton and endomembrane system. A limitation of the conventional cellular models used for mechanistic study and subcellular imaging of IAV infection is that they are cultured in two dimensions (2D) under non-polarizing conditions, and therefore they do not recapitulate the intracellular organization of the polarized respiratory epithelial cells naturally targeted by IAVs. To overcome this limitation, we developed an IAV-infection assay in a 3D cell culture system which allows imaging along the baso-lateral axis of polarized cells, with subcellular resolution. Here we describe a protocol to grow polarized monolayers of Caco2-TC7 cells on static Cytodex-3 microcarrier beads, infect them with IAV, and subsequently perform immunostaining and confocal imaging, or electron microscopy, on polarized IAV-infected cells. This method can be extended to other pathogens that infect human polarized epithelial cells.

SARS-CoV-2 infection induces the dedifferentiation of multiciliated cells and impairs mucociliary clearance.

Understanding how SARS-CoV-2 spreads within the respiratory tract is important to define the parameters controlling the severity of COVID-19. Here we examine the functional and structural consequences of SARS-CoV-2 infection in a reconstructed human bronchial epithelium model. SARS-CoV-2 replication causes a transient decrease in epithelial barrier function and disruption of tight junctions, though viral particle crossing remains limited. Rather, SARS-CoV-2 replication leads to a rapid loss of the ciliary layer, characterized at the ultrastructural level by axoneme loss and misorientation of remaining basal bodies. Downregulation of the master regulator of ciliogenesis Foxj1 occurs prior to extensive cilia loss, implicating this transcription factor in the dedifferentiation of ciliated cells. Motile cilia function is compromised by SARS-CoV-2 infection, as measured in a mucociliary clearance assay. Epithelial defense mechanisms, including basal cell mobilization and interferon-lambda induction, ramp up only after the initiation of cilia damage. Analysis of SARS-CoV-2 infection in Syrian hamsters further demonstrates the loss of motile cilia in vivo. This study identifies cilia damage as a pathogenic mechanism that could facilitate SARS-CoV-2 spread to the deeper lung parenchyma.

A simple, inexpensive and multi-scale 3-D fluorescent test sample for optical sectioning microscopies.

Fluorescence standards allow for quality control and for the comparison of data sets across instruments and laboratories in applications of quantitative fluorescence. For example, users of microscopy core facilities can expect a homogenous and time-invariant illumination and an uniform detection sensitivity, which are prerequisites for imaging analysis, tracking or fluorimetric pH or Ca2+ -concentration measurements. Similarly, confirming the three-dimensional (3-D) resolution of optical sectioning microscopes calls for a regular calibration with a standardized point source. The test samples required for such measurements are typically different ones, they are often expensive and they depend much on the very microscope technique used. Similarly, the ever-increasing choice among microscope techniques and geometries increases the demand for comparison across instruments. Here, we advocate and demonstrate the multiple uses of a surprisingly versatile and simple 3-D test sample that can complement existing and much more expensive calibration samples: commercial tissue paper labeled with a fluorescent highlighter pen. We provide relevant sample characteristics and show examples ranging from the sub-µm to cm scale, acquired on epifluorescence, confocal, image scanning, two-photon (2P) and light-sheet microscopes.

The zoonotic pathogen Leptospira interrogans mitigates environmental stress through cyclic-di-GMP-controlled biofilm production.

The zoonotic bacterium Leptospira interrogans is the aetiological agent of leptospirosis, a re-emerging infectious disease that is a growing public health concern. Most human cases of leptospirosis result from environmental infection. Biofilm formation and its contribution to the persistence of virulent leptospires in the environment or in the host have scarcely been addressed. Here, we examined spatial and time-domain changes in biofilm production by L. interrogans. Our observations showed that biofilm formation in L. interrogans is a highly dynamic process and leads to a polarized architecture. We notably found that the biofilm matrix is composed of extracellular DNA, which enhances the biofilm's cohesiveness. By studying L. interrogans mutants with defective diguanylate cyclase and phosphodiesterase genes, we show that biofilm production is regulated by intracellular levels of bis-(3'-5')-cyclic dimeric guanosine monophosphate (c-di-GMP) and underpins the bacterium's ability to withstand a wide variety of simulated environmental stresses. Our present results show how the c-di-GMP pathway regulates biofilm formation by L. interrogans, provide insights into the environmental persistence of L. interrogans and, more generally, highlight leptospirosis as an environment-borne threat to human health.

Hepatic stellate cell hypertrophy is associated with metabolic liver fibrosis.

Hepatic fibrosis is a major consequence of chronic liver disease such as non-alcoholic steatohepatitis which is undergoing a dramatic evolution given the obesity progression worldwide, and has no treatment to date. Hepatic stellate cells (HSCs) play a key role in the fibrosis process, because in chronic liver damage, they transdifferentiate from a "quiescent" to an "activated" phenotype responsible for most the collagen deposition in liver tissue. Here, using a diet-induced liver fibrosis murine model (choline-deficient amino acid-defined, high fat diet), we characterized a specific population of HSCs organized as clusters presenting simultaneously hypertrophy of retinoid droplets, quiescent and activated HSC markers. We showed that hypertrophied HSCs co-localized with fibrosis areas in space and time. Importantly, we reported the existence of this phenotype and its association with collagen deposition in three other mouse fibrosis models, including CCl4-induced fibrosis model. Moreover, we have also shown its relevance in human liver fibrosis associated with different etiologies (obesity, non-alcoholic steatohepatitis, viral hepatitis C and alcoholism). In particular, we have demonstrated a significant positive correlation between the stage of liver fibrosis and HSC hypertrophy in a cohort of obese patients with hepatic fibrosis. These results lead us to conclude that hypertrophied HSCs are closely associated with hepatic fibrosis in a metabolic disease context and may represent a new marker of metabolic liver disease progression.

Insights into amebiasis using a human 3D-intestinal model.

Entamoeba histolytica is the causative agent of amebiasis, an infectious disease targeting the intestine and the liver in humans. Two types of intestinal infection are caused by this parasite: silent infection, which occurs in the majority of cases, and invasive disease, which affects 10% of infected persons. To understand the intestinal pathogenic process, several in vitro models, such as cell cultures, human tissue explants or human intestine xenografts in mice, have been employed. Nevertheless, our knowledge on the early steps of amebic intestinal infection and the molecules involved during human-parasite interaction is scarce, in part due to limitations in the experimental settings. In the present work, we took advantage of tissue engineering approaches to build a three-dimensional (3D)-intestinal model that is able to replicate the general characteristics of the human colon. This system consists of an epithelial layer that develops tight and adherens junctions, a mucus layer and a lamina propria-like compartment made up of collagen containing macrophages and fibroblast. By means of microscopy imaging, omics assays and the evaluation of immune responses, we show a very dynamic interaction between E. histolytica and the 3D-intestinal model. Our data highlight the importance of several virulence markers occurring in patients or in experimental models, but they also demonstrate the involvement of under described molecules and regulatory factors in the amoebic invasive process.

USF1 defect drives p53 degradation during Helicobacter pylori infection and accelerates gastric carcinogenesis.

OBJECTIVE: Helicobacter pylori (Hp) is a major risk factor for gastric cancer (GC). Hp promotes DNA damage and proteasomal degradation of p53, the guardian of genome stability. Hp reduces the expression of the transcription factor USF1 shown to stabilise p53 in response to genotoxic stress. We investigated whether Hp-mediated USF1 deregulation impacts p53-response and consequently genetic instability. We also explored in vivo the role of USF1 in gastric carcinogenesis. DESIGN: Human gastric epithelial cell lines were infected with Hp7.13, exposed or not to a DNA-damaging agent camptothecin (CPT), to mimic a genetic instability context. We quantified the expression of USF1, p53 and their target genes, we determined their subcellular localisation by immunofluorescence and examined USF1/p53 interaction. Usf1-/- and INS-GAS mice were used to strengthen the findings in vivo and patient data examined for clinical relevance. RESULTS: In vivo we revealed the dominant role of USF1 in protecting gastric cells against Hp-induced carcinogenesis and its impact on p53 levels. In vitro, Hp delocalises USF1 into foci close to cell membranes. Hp prevents USF1/p53 nuclear built up and relocates these complexes in the cytoplasm, thereby impairing their transcriptional function. Hp also inhibits CPT-induced USF1/p53 nuclear complexes, exacerbating CPT-dependent DNA damaging effects. CONCLUSION: Our data reveal that the depletion of USF1 and its de-localisation in the vicinity of cell membranes are essential events associated to the genotoxic activity of Hp infection, thus promoting gastric carcinogenesis. These findings are also of clinical relevance, supporting USF1 expression as a potential marker of GC susceptibility.

DNA Hypermethylation Downregulates Telomerase Reverse Transcriptase (TERT) during H. pylori-Induced Chronic Inflammation.

Helicobacter pylori infection causes chronic gastritis and is the major risk factor of gastric cancer. H. pylori induces a chronic inflammation-producing reactive oxygen species (ROS) which is a source of chromosome instabilities and contributes to the development of malignancy. H. pylori also promotes DNA hypermethylation, known to dysregulate essential genes that maintain genetic stability. The maintenance of telomere length by telomerase is essential for chromosome integrity. Telomerase reverse transcriptase (TERT) is the catalytic component of telomerase activity and an important target during host-pathogen interaction. We aimed to investigate the consequences of H. pylori on the regulation of TERT gene expression and telomerase activity. In vitro, hTERT mRNA levels and telomerase activity were analysed in H. pylori-infected human gastric epithelial cells. In addition, C57BL/6 and INS-GAS mice were used to investigate the influence of H. pylori-induced inflammation on TERT levels. Our data demonstrated that, in vitro, H. pylori inhibits TERT gene expression and decreases the telomerase activity. The exposure of cells to lycopene, an antioxidant compound, restores TERT levels in infected cells, indicating that ROS are implicated in this downregulation. In vivo, fewer TERT-positive cells are observed in gastric tissues of infected mice compared to uninfected, more predominantly in the vicinity of large aggregates of lymphocytes, suggesting an inflammation-mediated regulation. Furthermore, H. pylori appears to downregulate TERT gene expression through DNA hypermethylation as shown by the restoration of TERT transcript levels in cells treated with 5'-azacytidine, an inhibitor of DNA methylation. This was confirmed in infected mice, by PCR-methylation assay of the TERT gene promoter. Our data unraveled a novel way for H. pylori to promote genome instabilities through the inhibition of TERT levels and telomerase activity. This mechanism could play an important role in the early steps of gastric carcinogenesis.

Effect of Helicobacter pylori Infection on GATA-5 and TFF1 Regulation, Comparison Between Pediatric and Adult Patients.

BACKGROUND: GATA factors, which constitute a family of transcription regulatory proteins, participate in gastrointestinal development. Trefoil factor 1 (TFF1) plays a crucial role in mucosal defense and healing, and evidence suggests that GATA-5 mediated its regulation. Gastric cancer is a multiple-step process triggered by Helicobacter pylori and is characterized by accumulation of molecular and epigenetic alteration. The aim of this study was to evaluate the effect of H. pylori infection on the regulation of GATA-5 and TFF1 in vitro and in vivo. RESULTS: Infected cells exhibited upregulation of GATA-5 and TFF1 after 48 h. An increase in GATA-5 and TFF1 mRNA levels was also found in mice samples after 6 and 12 months of infection, respectively. In human samples, we found an association between H. pylori infection and GATA-5 upregulation. In fact, among H. pylori-infected patients, hypermethylation was observed in 45.5% of pediatric samples, in 62.6% of chronic gastritis samples, and in 63% of gastric cancer samples. Regarding TFF1, the expression levels were similar in pediatrics and adults patients, and were independent of H. pylori infection, and the expression of these factors was downregulated in gastric cancer samples. GATA-5 promoter methylation was associated with a decrease in TFF1 mRNA levels. CONCLUSIONS: Our results suggest that the upregulation of GATA-5 and TFF1 observed in vitro and in vivo may be correlated with a protective effect of the mucosa in response to infection. The epigenetic inactivation of GATA-5 observed in human biopsies from infected patients may suggest that this alteration is an early event occurring in association with H. pylori infection.

Penetration of the Human Pulmonary Epithelium by Aspergillus fumigatus Hyphae.

Background: The airway epithelium is the first barrier interacting with Aspergillus fumigatus conidia after their inhalation, suggesting that this structure functions as point of entry of this fungus to initiate pulmonary aspergillosis. Methods: To study epithelial entry by A fumigatus, primary human reconstituted pseudostratified epithelium cultured in air-liquid interface as well as bronchial epithelial cell monolayers were infected with conidia. Results: Under these experimental conditions, we found that A fumigatus hyphae traversed the bronchial epithelium through a mechanism involving the recruitment of actin, which formed a tunnel that allows hyphae to enter the cells without disturbing their integrity. Conclusions: These findings describe a new mechanism by which A fumigatus hyphae penetrate the airway epithelial barrier and can infect its human host.

Fluorescence imaging host pathogen interactions: fifteen years benefit of hindsight .

We consider in review current state-of-the-art fluorescence microscopy for investigating the host-pathogen interface. Our perspective is honed from years with literally thousands of microbiologists using the variety of imaging technologies available within our dedicated BSL2/BSL3 optical imaging research service facilities at the Institut Pasteur Paris founded from scratch in 2001. During fifteen years learning from the success and failures of introducing different fluorescence imaging technologies, methods, and technical development strategies we provide here a synopsis review of our experience to date and a synthesis of how we see the future in perspective for fluorescence imaging at the host-pathogen interface.

Helicobacter pylori targets mitochondrial import and components of mitochondrial DNA replication machinery through an alternative VacA-dependent and a VacA-independent mechanisms.

Targeting mitochondria is a powerful strategy for pathogens to subvert cell physiology and establish infection. Helicobacter pylori is a bacterial pathogen associated with gastric cancer development that is known to target mitochondria directly and exclusively through its pro-apoptotic and vacuolating cytotoxin VacA. By in vitro infection of gastric epithelial cells with wild-type and VacA-deficient H. pylori strains, treatment of cells with purified VacA proteins and infection of a mouse model, we show that H. pylori deregulates mitochondria by two novel mechanisms, both rather associated with host cell survival. First, early upon infection VacA induces transient increase of mitochondrial translocases and a dramatic accumulation of the mitochondrial DNA replication and maintenance factors POLG and TFAM. These events occur when VacA is not detected intracellularly, therefore do not require the direct interaction of the cytotoxin with the organelle, and are independent of the toxin vacuolating activity. In vivo, these alterations coincide with the evolution of gastric lesions towards severity. Second, H. pylori also induces VacA-independent alteration of mitochondrial replication and import components, suggesting the involvement of additional H. pylori activities in mitochondria-mediated effects. These data unveil two novel mitochondrial effectors in H. pylori-host interaction with links on gastric pathogenesis.

Circulating mitochondrial DNA level, a noninvasive biomarker for the early detection of gastric cancer.

BACKGROUND: Gastric cancer represents a major health burden worldwide and is often diagnosed at an advanced stage. Biomarkers for screening and prevention of gastric cancer are missing. Changes in peripheral blood mitochondrial DNA (mtDNA) have emerged as a potential preventive/diagnosis biomarker for cancer risk. We aimed to determine whether peripheral leukocytes mtDNA levels are associated with stages of the gastric carcinogenesis cascade. METHODS: We measured mtDNA by quantitative real-time PCR assay in peripheral leukocytes of 28 patients with non-atrophic gastritis (NAG), 74 patients with gastric cancer, and 48 matched asymptomatic controls. In parallel, the serologic level of IL8 was determined. RESULTS: Mean mtDNA level was higher in patients with gastric cancer (P = 0.0095) than in controls, with values >8.46 significantly associated with gastric cancer (OR, 3.93). Three ranges of mtDNA values were identified: interval I, <2.0; interval II, 2.0-20; and interval III, >20. Interval I included mainly NAG cases, and few gastric cancer samples and interval III corresponded almost exclusively to patients with gastric cancer. All controls fell in interval II, together with some NAG and gastric cancer cases. IL8 levels were significantly higher in patients with gastric cancer (P < 0.05), with levels >50 pg/mL observed exclusively in patients with gastric cancer, allowing to distinguish them within interval II. We validated mtDNA results in a second cohort of patients, confirming that mtDNA was significantly higher in gastric cancer than in patients with preneoplasia. CONCLUSIONS: Circulating levels of mtDNA and IL8 constitute a potential biomarker for the early detection of gastric cancer. IMPACT: Our findings lead us to propose a new noninvasive method to detect patients with gastric cancer risk.

Initial formation of IGROV1 ovarian cancer multicellular aggregates involves vitronectin.

Ovarian cancer progression is frequently associated with the development of malignant ascites. Multicellular aggregates of carcinoma cells (spheroids) found within ascites are thought to be able to promote peritoneal carcinomatosis. We have previously demonstrated the involvement of the vitronectin/alphav integrin adhesive system in the dissemination of ovarian cancer cells and continue to investigate the influence of these molecules by studying their role(s) in spheroid behavior. The aim of this study was to generate ovarian cancer multicellular aggregates and to focus on the role of vitronectin and alphav integrins in their initiation. IGROV1 cancer cells cultured in the absence of adhesive substratum formed multicellular aggregates comparable to spheroids. After 21 days, a fraction of the cells within clusters remained viable and proliferated recurrently. Within the multicellular aggregates, vitronectin and alphav integrins were co-localized at intercellular sites, suggesting their involvement in cell-cell interactions. Initial formation of IGROV1 aggregates was inhibited using anti-vitronectin and anti-alphav integrin blocking antibodies or the cyclic peptide cRGDfV. Vitronectin expression persisted during cluster disaggregation on fibronectin. These results demonstrate the ability of IGROV1 cells to generate multicellular aggregates and point to a contributory role for the vitronectin/alphav integrin system in the initial step of this process. These events could represent a prerequisite for further dissemination.

Mesothelial vitronectin stimulates migration of ovarian cancer cells.

Ovarian carcinomas, the most fatal gynaecological malignancies, are associated with poor prognosis predominantly because of a high recurrence rate. Ovarian cancer cells spread widely throughout the abdominal cavity leading to peritoneal metastasis. The influence of the mesothelial microenvironment on the biological mechanisms leading to cancer cell colonization of the mesothelium is poorly understood. This study aims to investigate whether mesothelial secretions affect the migration of ovarian cancer cells and focuses on the role of the adhesive molecule Vn (vitronectin) and its integrin receptors. An in vitro co-culture model indicated that clusters of IGROV1 and SKOV3 cells adhere to MeT-5A mesothelial cells preferentially at intercellular sites, invade the mesothelial monolayer and alter the integrity of the mesothelium. In addition, mesothelial CM (cell-conditioned medium) induces migration of IGROV1 and SKOV3 cells in Boyden chambers and wound healing assays. Furthermore, blocking molecules directed against vitronectin or its alphav integrin receptor decrease mesothelial-CM-induced migration by approximately 40% and 60-70% for IGROV1 and SKOV3 ovarian cancer cells, respectively, in Boyden chamber assays. Wound healing assays that allow cell migration to be measured over 24 h periods demonstrated that blocking molecules prevent the migration of IGROV1 and SKOV3 cells. Vitronectin is present in CM MeT-5A (mesothelial conditioned medium) and in metastatic peritoneal tissue sections. The expression of vitronectin at the periphery of mesothelial cells and within ovarian cancer cell clusters suggests a potential role for this molecule during intraperitoneal implantation of ovarian cancer cells. Vitronectin could represent a target for the development of anti-adhesive strategies to impede ovarian cancer dissemination.

Vitronectin and its receptors partly mediate adhesion of ovarian cancer cells to peritoneal mesothelium in vitro.

Epithelial ovarian cancer cells metastasize by implanting onto the peritoneal mesothelial surface of the abdominal cavity. Adhesive molecules that lead to this implantation remain unclear. The aim of our study was to focus on the role of vitronectin (Vn) and its receptors, alpha(v) integrins and urokinase plasminogen activator receptor (uPAR), in the interactions of ovarian adenocarcinoma cells (IGROV1 and SKOV3 cell lines) with mesothelial cells (MeT-5A cell line and primary cultures). For all cell lines, immunofluorescence staining disclosed the presence of Vn over the whole cell surface and in thin continuous deposits underlining the cell periphery. Recruitment of Vn receptors to cell-cell contact sites was also revealed. We developed two distinct methods for the evaluation of in vitro cell-cell adhesion using cocultures of the tumor and mesothelial cells. Both adhesion assays revealed a strong ability of ovarian cancer cells to adhere preferentially to mesothelial intercellular junctions. Adhesion of ovarian carcinoma cells to mesothelial cells was significantly inhibited using anti-Vn-, -alpha(v)-integrin- and -uPAR-blocking antibodies or cyclic peptide cRGDfV. These results evidence the ability of ovarian carcinoma cells to bind to peritoneal mesothelium in vitro and strongly suggest that Vn and its receptors contribute to this crucial event.