Biology subjects

Hill, D. R.

Publications and source records attributed to Hill, D. R..

6 recordsLinked to original sources

bioRxiv2022

Neutrophils prime unique transcriptional responses in intestinal organoids during infection with nontyphoidal Salmonella enterica serovars

Nontyphoidal strains of Salmonella enterica are a major cause of foodborne illnesses and infection with these bacteria result in inflammatory gastroenteritis. Neutrophils are a dominant immune cell type found at the site of infection in Salmonella-infected individuals, but how they regulate infection outcome is not well understood. Here we used a co-culture model of primary human neutrophils and human intestinal organoids to probe the role of neutrophils during infection with two of the most prevalent Salmonella serovars: Salmonella enterica serovar Enteritidis and Typhimurium. Using a transcriptomics approach, we identified a dominant role for neutrophils in mounting differential immune responses including production of pro-inflammatory cytokines, chemokines, and antimicrobial peptides. We also identified specific gene sets that are induced by neutrophils in response to Enteritidis or Typhimurium infection. By comparing host responses to these serovars, we uncovered differential regulation of host metabolic pathways particularly induction of cholesterol biosynthetic pathways during Typhimurium infection and suppression of RNA metabolism during Enteritidis infection. Together these findings provide insight into the role of human neutrophils in modulating different host responses to pathogens that cause similar disease in humans. ImportanceNontyphoidal serovars of Salmonella enterica are known to induce robust neutrophil recruitment in the gut during early stages of infection, but the specific role of neutrophils in regulating infection outcome of different serovars is poorly understood. Due to differences in human infection progression compared to small animal models, characterizing the role of neutrophils during infection has been challenging. Here we used a co-culture model of human intestinal organoids with human primary neutrophils to study the role of neutrophils during infection of human intestinal epithelium. Using a transcriptomics approach, we define neutrophil-dependent reprogramming of the host response to Salmonella, establishing a clear role in amplifying pro-inflammatory gene expression. Additionally, the host response driven by neutrophils differed between two similar nontyphoidal Salmonella serovars. These findings highlight the importance of building more physiological infection models to replicate human infection conditions to study host responses specific to individual pathogens.

immunology↗

bioRxiv2022

Human neutrophils direct epithelial cell extrusion to enhance intestinal epithelial host defense 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 then seeded with primary human polymorphonuclear leukocytes (PMN-HIOs), specifically neutrophils and analyzed for bacterial growth and host cell survival. Surprisingly, PMNs did not affect luminal colonization of Salmonella, but their presence reduced intraepithelial bacterial burden. Adding PMNs to infected HIOs resulted in substantial accumulation of shed intestinal epithelial cells that could be blocked by Caspase-1 or Caspase-3 inhibition. Cleaved Caspase-3 was present in epithelial cells, but expression of the inflammasome adaptor, ASC, was only detected in PMNs. Caspase inhibition also increased bacterial burden in the epithelium of the PMN-HIO, suggesting PMNs enhance activation of cell death pathways in human intestinal epithelial cells as a protective response to infection. These data support a critical function for neutrophils beyond their antimicrobial role whereby they amplify cell death and extrusion of epithelial cells from the Salmonella-infected intestinal monolayer. Significance statementNeutrophils are early responders to Salmonella intestinal infection, but how they influence infection progression and outcome is unknown. Here we use a co-culture model of human intestinal organoids and human primary neutrophils to study the contribution of human neutrophils to Salmonella infection of the intestinal epithelium. We found that neutrophils markedly enhanced epithelial defenses, including enhancing cell extrusion to reduce intraepithelial burden of Salmonella and association with the epithelium, rather than directly killing Salmonella in the HIO lumen. These findings reveal a novel role for neutrophils in the gut beyond killing invading pathogens and illuminate how neutrophils can reprogram cells in the gut environment to enhance antimicrobial defenses.

microbiology↗

bioRxiv2020

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 infection 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 host responses to individual serovars have been difficult to perform due to reliance on poorly representative 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, reflecting 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 elevated levels of reactive oxygen species production in SE-infected HIOs compared to mock-infected HIOs. Collectively, these results suggest that the HIO model is well suited to reveal host transcriptional programming specific to individual Salmonella serovars, and that individual NTS may provoke unique host epithelial responses during intestinal stages of infection. Author SummarySalmonella enterica is the major causative agent of bacterial infections associated with contaminated food and water. Salmonella enterica consists of over 2500 serovars of which Typhimurium (STM), Enteritidis (SE) and Typhi (ST) are the three major serovars with medical relevance to humans. These serovars elicit distinctive immune responses and cause different diseases in humans, including self-limiting diarrhea, gastroenteritis and typhoid fever. Differences in the human host response to these serovars are likely to be a major contributing factor to distinct disease outcomes but are not well characterized, possibly due to the limitations of human-derived physiological infection models. Unlike immortalized epithelial cell culture models, human intestinal organoids (HIOs) are three-dimensional structures derived from embryonic stem cells that differentiate into intestinal mesenchymal and epithelial cells, mirroring key organizational aspects of the intestine. In this study, we used HIOs to monitor transcriptional changes during early stages of STM, SE and ST infection. Our comparative analysis showed that HIO inflammatory responses are the dominant response in all infections, but ST infection induces the weakest upregulation of inflammatory mediators relative to the other serovars. In addition, we identified several cellular processes, including cell cycle and mitochondrial functions, that were inversely regulated between STM and SE infection despite these serovars causing similar localized intestinal infection in humans. Our findings reinforce HIOs as an emerging model system to study Salmonella serovar infection, and provide global host transcriptional response profiles as a foundation for understanding human infection outcomes.

microbiology↗

bioRxiv2020

Salmonella enterica serovar Typhimurium SPI-1 and SPI-2 shape the transcriptional landscape of epithelial cells 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 serovar Typhimurium (STm). While interaction of STm with the mammalian host has been well studied in vitro in transformed epithelial cell lines or in the complex intestinal environment in vivo, few tractable models recapitulate key features of the intestinal epithelium. Human intestinal organoids (HIOs) contain a polarized epithelium with functionally differentiated cell subtypes, including enterocytes and goblet cells. HIOs contain luminal space that supports bacterial replication and are more amenable to experimental manipulation than animals while more reflective of physiological epithelial responses. Here we use the HIO model to define transcriptional responses of the host epithelium to STm 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 STm strongly stimulates pro-inflammatory 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, STm infection led to significant down-regulation of host genes associated with cell cycle and DNA repair, an effect that required SPI-1 and SPI-2 T3SS. The transcriptional profile of cell cycle-associated target genes implicates multiple miRNAs as likely mediators of STm-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 reveal host cell cycle as a potential target during STm intracellular infection. ImportanceSalmonella enterica serovar Typhimurium (STm) 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, evaluating physiological host responses driven by major virulence determinants of Salmonella have been difficult to date. Here we use the 3D human intestinal organoid model to define early responses to infection with wildtype STm and mutants defective in the SPI-1 or SPI-2 Type 3 secretion systems. Both secretion system mutants show defects in a mouse model of oral Salmonella infection but the specific contributions of each secretion system are less well understood. We show that STm upregulates pro-inflammatory pathways independently of either secretion system while downregulation of host cell cycle pathways is dependent 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.

microbiology↗

bioRxiv2020

Loss of RpoS results in attenuated Escherichia coli colonization of human intestinal organoids and a competitive disadvantage within the germ-free mouse intestine

Pluripotent stem-cell-derived human intestinal organoids (HIOs) are three-dimensional, multicellular structures that model a previously uncolonized, naive intestinal epithelium in an in vitro system. We recently demonstrated that microinjection of the non-pathogenic 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 investigate the bacterial response to colonization of the HIO lumen. In E. coli and other Gram-negative bacteria, adaptation to environmental stress is regulated by the general stress response sigma factor, RpoS. We generated an isogenic {triangleup}rpoS ECOR2 mutant to compare challenges faced by a bacterium during colonization of the HIO lumen relative to the germ-free mouse intestine, which is currently the best available system for studying the initial establishment of bacterial populations within the gut. We demonstrate that loss of RpoS significantly decreases the ability of ECOR2 to colonize HIOs, though it does not prevent colonization of germ-free mice. Rather, the {triangleup}rpoS ECOR2 exhibits a fitness defect in the germ-free mouse intestine only in the context of microbial competition. These results indicate that HIOs pose a differentially restrictive luminal environment to E. coli during colonization, thus increasing our understanding of the HIO model system as it pertains to studying the establishment of intestinal host-microbe symbioses. ImportanceTechnological advancements have and will continue to drive the adoption of organoid-based 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 differ from 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.

microbiology↗

bioRxiv2017

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 diZcult 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.

developmental biology↗