Human neutrophil IL1ß directs intestinal epithelial cell extrusion during Salmonella infection.

Infection of the human gut by Salmonella enterica Typhimurium (STM) results in a localized inflammatory disease that is not mimicked in murine infections. To determine mechanisms by which neutrophils, as early responders to bacterial challenge, direct inflammatory programming of human intestinal epithelium, we established a multi-component human intestinal organoid (HIO) model of STM infection. HIOs were micro-injected with STM and seeded with primary human polymorphonuclear leukocytes (PMN-HIOs). PMNs did not significantly alter luminal colonization of Salmonella, but their presence reduced intraepithelial bacterial burden. Adding PMNs to infected HIOs resulted in substantial accumulation of shed TUNEL+ epithelial cells that was driven by PMN Caspase-1 activity. Inhibition of Caspases-1, -3 or -4 abrogated epithelial cell death and extrusion in the infected PMN-HIOs but only Caspase-1 inhibition significantly increased bacterial burden in the PMN-HIO epithelium. Thus, PMNs promote cell death in human intestinal epithelial cells through multiple caspases as a protective response to infection. IL-1ß was necessary and sufficient to induce cell shedding in the infected HIOs. These data support a critical innate immune function for human neutrophils in amplifying cell death and extrusion of human epithelial cells from the Salmonella-infected intestinal monolayer.

Perfusion System for Modification of Luminal Contents of Human Intestinal Organoids and Realtime Imaging Analysis of Microbial Populations.

Intestinal organoids are 3D cell structures that replicate some aspects of organ function and are organized with a polarized epithelium facing a central lumen. To enable more applications, new technologies are needed to access the luminal cavity and apical cell surface of organoids. We developed a perfusion system utilizing a double-barrel glass capillary with a pressure-based pump to access and modify the luminal contents of a human intestinal organoid for extended periods of time while applying cyclic cellular strain. Cyclic injection and withdrawal of fluorescent FITC-Dextran coupled with real-time measurement of fluorescence intensity showed discrete changes of intensity correlating with perfusion cycles. The perfusion system was also used to modify the lumen of organoids injected with GFP-expressing E. coli. Due to the low concentration and fluorescence of the E. coli, a novel imaging analysis method utilizing bacteria enumeration and image flattening was developed to monitor E. coli within the organoid. Collectively, this work shows that a double-barrel perfusion system provides constant luminal access and allows regulation of luminal contents and luminal mixing.

Comparative transcriptional profiling of the early host response to infection by typhoidal and non-typhoidal Salmonella serovars in human intestinal organoids.

Salmonella enterica represents over 2500 serovars associated with a wide-ranging spectrum of disease; from self-limiting gastroenteritis to invasive infections caused by non-typhoidal serovars (NTS) and typhoidal serovars, respectively. Host factors strongly influence infection outcome as malnourished or immunocompromised individuals can develop invasive infections from NTS, however, comparative analyses of serovar-specific host responses have been constrained by reliance on limited model systems. Here we used human intestinal organoids (HIOs), a three-dimensional "gut-like" in vitro system derived from human embryonic stem cells, to elucidate similarities and differences in host responses to NTS and typhoidal serovars. HIOs discriminated between the two most prevalent NTS, Salmonella enterica serovar Typhimurium (STM) and Salmonella enterica serovar Enteritidis (SE), and typhoidal serovar Salmonella enterica serovar Typhi (ST) in epithelial cell invasion, replication and transcriptional responses. Pro-inflammatory signaling and cytokine output was reduced in ST-infected HIOs compared to NTS infections, consistent with early stages of NTS and typhoidal diseases. While we predicted that ST would induce a distinct transcriptional profile from the NTS strains, more nuanced expression profiles emerged. Notably, pathways involved in cell cycle, metabolism and mitochondrial functions were downregulated in STM-infected HIOs and upregulated in SE-infected HIOs. These results correlated with suppression of cellular proliferation and induction of host cell death in STM-infected HIOs and in contrast, elevated levels of reactive oxygen species production in SE-infected HIOs. Collectively, these results suggest that the HIO model is well suited to reveal host transcriptional programming specific to infection by individual Salmonella serovars, and that individual NTS may provoke unique host epithelial responses during intestinal stages of infection.

Salmonella enterica Serovar Typhimurium SPI-1 and SPI-2 Shape the Global Transcriptional Landscape in a Human Intestinal Organoid Model System.

The intestinal epithelium is a primary interface for engagement of the host response by foodborne pathogens, like Salmonella enterica Typhimurium. While the interaction of S Typhimurium with the mammalian host has been well studied in transformed epithelial cell lines or in the complex intestinal environment in vivo, few tractable models recapitulate key features of the intestine. Human intestinal organoids (HIOs) contain a polarized epithelium with functionally differentiated cell subtypes, including enterocytes and goblet cells and a supporting mesenchymal cell layer. HIOs contain luminal space that supports bacterial replication, are more amenable to experimental manipulation than animals and are more reflective of physiological host responses. Here, we use the HIO model to define host transcriptional responses to S Typhimurium infection, also determining host pathways dependent on Salmonella pathogenicity island-1 (SPI-1)- and -2 (SPI-2)-encoded type 3 secretion systems (T3SS). Consistent with prior findings, we find that S Typhimurium strongly stimulates proinflammatory gene expression. Infection-induced cytokine gene expression was rapid, transient, and largely independent of SPI-1 T3SS-mediated invasion, likely due to continued luminal stimulation. Notably, S Typhimurium infection led to significant downregulation of host genes associated with cell cycle and DNA repair, leading to a reduction in cellular proliferation, dependent on SPI-1 and SPI-2 T3SS. The transcriptional profile of cell cycle-associated target genes implicates multiple miRNAs as mediators of S Typhimurium-dependent cell cycle suppression. These findings from Salmonella-infected HIOs delineate common and distinct contributions of SPI-1 and SPI-2 T3SSs in inducing early host responses during enteric infection and reinforce host cell proliferation as a process targeted by SalmonellaIMPORTANCESalmonella enterica serovar Typhimurium (S Typhimurium) causes a significant health burden worldwide, yet host responses to initial stages of intestinal infection remain poorly understood. Due to differences in infection outcome between mice and humans, physiological human host responses driven by major virulence determinants of Salmonella have been more challenging to evaluate. Here, we use the three-dimensional human intestinal organoid model to define early responses to infection with wild-type S Typhimurium and mutants defective in the SPI-1 or SPI-2 type-3 secretion systems. While both secretion system mutants show defects in mouse models of oral Salmonella infection, the specific contributions of each secretion system are less well understood. We show that S Typhimurium upregulates proinflammatory pathways independently of either secretion system, while the downregulation of the host cell cycle pathways relies on both SPI-1 and SPI-2. These findings lay the groundwork for future studies investigating how SPI-1- and SPI-2-driven host responses affect infection outcome and show the potential of this model to study host-pathogen interactions with other serovars to understand how initial interactions with the intestinal epithelium may affect pathogenesis.

The Lumen of Human Intestinal Organoids Poses Greater Stress to Bacteria Compared to the Germ-Free Mouse Intestine: Escherichia coli Deficient in RpoS as a Colonization Probe.

Pluripotent stem-cell-derived human intestinal organoids (HIOs) are three-dimensional, multicellular structures that model a naive intestinal epithelium in an in vitro system. Several published reports have investigated the use of HIOs to study host-microbe interactions. We recently demonstrated that microinjection of the nonpathogenic Escherichia coli strain ECOR2 into HIOs induced morphological and functional maturation of the HIO epithelium, including increased secretion of mucins and cationic antimicrobial peptides. In the current work, we use ECOR2 as a biological probe to further characterize the environment present in the HIO lumen. We generated an isogenic mutant in the general stress response sigma factor RpoS and employed this mutant to compare challenges faced by a bacterium during colonization of the HIO lumen relative to the germ-free mouse intestine. We demonstrate that the loss of RpoS significantly decreases the ability of ECOR2 to colonize HIOs, although it does not prevent colonization of germ-free mice. These results indicate that the HIO lumen is a more restrictive environment to E. coli than the germ-free mouse intestine, thus increasing our understanding of the HIO model system as it pertains to studying the establishment of intestinal host-microbe symbioses.IMPORTANCE Technological advancements have driven and will continue to drive the adoption of organotypic systems for investigating host-microbe interactions within the human intestinal ecosystem. Using E. coli deficient in the RpoS-mediated general stress response, we demonstrate that the type or severity of microbial stressors within the HIO lumen is more restrictive than those of the in vivo environment of the germ-free mouse gut. This study provides important insight into the nature of the HIO microenvironment from a microbiological standpoint.

TET1 contributes to allergic airway inflammation and regulates interferon and aryl hydrocarbon receptor signaling pathways in bronchial epithelial cells.

Previous studies have suggested a role for Tet1 in the pathogenesis of childhood asthma. However, how Tet1 contributes to asthma remains unknown. Here we used mice deficient for Tet1 in a well-established model of allergic airway inflammation and demonstrated that loss of Tet1 increased disease severity including airway hyperresponsiveness and lung eosinophilia. Increased expression of Muc5ac, Il13, Il33, Il17a, Egfr, and Tff2 were observed in HDM-challenged Tet1-deficient mice compared to Tet1+/+ littermates. Further, transcriptomic analysis of lung RNA followed by pathway and protein network analysis showed that the IFN signaling pathway was significantly upregulated and the aryl hydrocarbon receptor (AhR) pathway was significantly downregulated in HDM-challenged Tet1-/- mice. This transcriptional regulation of the IFN and AhR pathways by Tet1 was also present in human bronchial epithelial cells at base line and following HDM challenges. Genes in these pathways were further associated with changes in DNA methylation, predicted binding of transcriptional factors with relevant functions in their promoters, and the presence of histone marks generated by histone enzymes that are known to interact with Tet1. Collectively, our data suggest that Tet1 inhibits HDM-induced allergic airway inflammation by direct regulation of the IFN and AhR pathways.

Bacterial colonization stimulates a complex physiological response in the immature human intestinal epithelium.

The human gastrointestinal tract is immature at birth, yet must adapt to dramatic changes such as oral nutrition and microbial colonization. The confluence of these factors can lead to severe inflammatory disease in premature infants; however, investigating complex environment-host interactions is difficult due to limited access to immature human tissue. Here, we demonstrate that the epithelium of human pluripotent stem-cell-derived human intestinal organoids is globally similar to the immature human epithelium and we utilize HIOs to investigate complex host-microbe interactions in this naive epithelium. Our findings demonstrate that the immature epithelium is intrinsically capable of establishing a stable host-microbe symbiosis. Microbial colonization leads to complex contact and hypoxia driven responses resulting in increased antimicrobial peptide production, maturation of the mucus layer, and improved barrier function. These studies lay the groundwork for an improved mechanistic understanding of how colonization influences development of the immature human intestine.

Nasal DNA methylation differentiates corticosteroid treatment response in pediatric asthma: A pilot study.

BACKGROUND: Treatment response to systemic corticosteroid in asthmatic children is heterogeneous and may be mediated by epigenetic mechanism(s). We aim to identify DNA methylation (DNAm) changes responsive to steroid, and DNAm biomarkers that distinguish treatment response. MATERIALS AND METHODS: We followed 33 children (ages 5-18) presenting to the Emergency Department (ED) for asthma exacerbation. Based on whether they met discharge criteria in ≤24 hours, participants were grouped into good and poor responders to steroid treatment. Nasal samples were collected upon presentation to the ED (T0) and 18-24 hours later (T1). Genome-wide DNAm was measured for both time points in 20 subjects, and compared between T0 and T1 in good and poor responders respectively. DNAm at T1 was also compared between two responder groups. DNAm of selected CpGs was verified in the complete cohort, and expression of associated genes was examined. Interactions between DNAm, common single nucleotide polymorphism (SNP) located at the CpG sites and treatment responses were assessed. RESULTS: Three CpGs located in the OTX2 promoter showed responder-specific DNAm changes from T0 to T1, in which DNAm decreased in good but not in poor responders. Good and poor responders showed differential DNAm at T1 in 127 CpGs without and 182 CpGs with common SNP co-localization. Negative correlations between DNAm and gene expression were observed at CpGs located within the LDHC promoter, suggesting an impact of DNAm on gene regulation. Interactions between SNPs, DNAm and treatment response were detected. CONCLUSION: Acute systemic steroid treatment modifies nasal DNAm in good responders. Nasal DNAm, dependent or independent of SNPs, can differentiate response to treatment in acute asthmatic children.