Biology subjects

Loving, C. L.

Publications and source records attributed to Loving, C. L..

10 recordsLinked to original sources

bioRxiv2025

A single-cell immune atlas of primary and secondary lymphoid organs in pigs

Single-cell RNA sequencing (scRNA-seq) has revolutionized understandings of cellular identities and functions due to the ability to study transcriptome-wide gene expression within individual cells. Multi-tissue scRNA-seq atlases have generated holistic understandings of body-wide cell dynamics and serve as key foundational resources for further scientific studies across a variety of species. Pigs are a valuable biomedical model, and pork is an essential global food source, but minimal understanding of immune cell identities and functions across anatomical locations limits agricultural and health advancements in pigs. To address current limitations, we apply scRNA-seq to create an atlas of immune cells recovered from key immune tissues including primary lymphoid organs (bone marrow and thymus) and secondary lymphoid organs (lymph node and spleen). Thymus data was compared to a previously published scRNA-seq dataset of pig thymus and shared a general consensus while also identifying several new thymic cell populations. Comparison of spleen to a human splenic scRNA-seq dataset also revealed conserved features, including two subsets of innate lymphoid cells conserved between pigs and humans. Spatial reconstruction of lymph node structure from scRNA-seq data revealed follicular organization with similar cell type distributions and cell signaling interactions to those in human lymph nodes. To expand accessibility of the scRNA-seq atlas for biological query, we deploy an interactive application and demonstrate its use for non-computational exploration of diverse cell populations recovered from bone marrow. Overall, results expand current foundational understandings of immune cell identities and functions in pig lymphoid organs and demonstrate pig-to-human immune similarities to consider for future research applications. Materials associated with this work are made readily accessible for others to investigate individual queries requiring foundational knowledge pertaining to pig immunity.

immunology↗

bioRxiv2024

A single-cell atlas of transcriptome changes in the intestinal epithelium at the suckling-to-weaning transition

The suckling-to-weaning dietary transition is a key step in mammalian intestinal development. However, the relative contributions of genetically wired and nutritional factors in this maturation process remain to be elucidated. Moreover, the cellular diversity of the intestinal epithelium has been overlooked in this context. The aim of our study was to identify the transcriptome changes induced in each cell type of the intestinal epithelium at the onset of solid food ingestion. We compared the single-cell transcriptome of epithelial cells isolated from the caecum of age-matched littermate suckling rabbits ingesting or not solid food. Our dataset provides the first single-cell atlas of the rabbit intestinal epithelium and highlights the interest of the rabbit as a model for studying BEST4+ epithelial cells, which are absent in mice. Solid food ingestion induced extensive transcriptome changes in each epithelial cell type, with the most pronounced changes noted in absorptive and BEST4+ cells. Some of the effects of solid food introduction were common to most epithelial cell types, such as the upregulation of ALDH1A1, which encodes for a vitamin A processing enzyme. Solid food ingestion remodeled epithelial defenses systems, as observed by the increased expression of interferon-stimulated genes in mature absorptive and BEST4+ cells. Solid food also upregulated the gene expression of the immunoglobulin transporter PIGR, specifically in cells located at the base of epithelial crypts and in goblet cells. In addition, solid food triggered epithelial differentiation, which was associated with modification of the expression of genes involved in handling of amino acids, lipids and bile acids, as well as changes in hormone expression by enteroendocrine cells. These cell type-specific transcriptome modifications induced by solid food ingestion coincided with changes in microbiota composition and metabolic activity, which may contribute to epithelial maturation. Overall, our work provides a single-cell atlas of the transcriptome changes induced in the intestinal epithelium at the suckling-to-weaning transition.

physiology↗

bioRxiv2023

Metabolic and genomic characterization of two novel butyrate producing Lachnospiraceae isolated from swine feces

Commensal bacteria from the swine gut microbiome that can be isolated have numerous potential applications in the animal production industry, including mitigation of disease, improving performance, and promoting colonization resistance to human foodborne pathogens. Butyrate-producing bacteria are targets for next-generation probiotics and microbiome-engineering strategies because butyrate is a metabolite of central importance in large intestinal homeostasis and may augment colonization resistance to enteric pathogens. However, relatively few butyrate-producers from swine have been cultured and extensively characterized. Here, we describe the substrate utilization, metabolic profiles, and genomic features of two novel species that produce high concentration of butyrate in vitro, Roseburia sp. 831b and Petralouisia sp. 499, isolated from swine feces. The complete genomes illustrated versatility in carbon metabolism and unique carbohydrate-active enzymes not observed in other species of Roseburia and Petralouisia that encode a combination of glycosidic hydrolases and carbohydrate-binding modules involved in starch and pectin utilization. Roseburia sp. 831b fermented a broader range and more complex mono- and polysaccharides than Petralouisia sp. 499. Fecal and cecal metagenomes from eight-week-old pigs challenged with Salmonella revealed that Roseburia sp. 831b increased to detectable abundances in the swine hindgut in most animals at [~]63-70 days of age. Additionally, the abundance of Roseburia sp. 831b in fecal metagenomes correlated with fecal butyrate concentrations in the pigs fed a diet supplemented with a prebiotic resistant potato starch. Together, these findings highlight the probiotic potential and ecological niche in the swine gastrointestinal tract for two novel butyrate-producers. ImportanceAntibiotics have been important for swine production and management of enteric pathogens; however, the Veterinary Feed Directive limits the use of medically important in-feed antibiotics for production purposes. As a result, there is a need for alternatives to antibiotics. Butyrate-producing bacteria can improve colonization resistance to human pathogens within the swine gastrointestinal tract by reinforcing the intestinal barrier, increasing mucus production, and reducing local oxygen and pH levels. Here, we demonstrate the versatile substrate utilization and metabolic potential of two novel species isolated from swine that produce high butyrate concentrations in vitro. These findings will help develop strategies that increase the abundance of these species and other butyrate producers in the swine gut. Further, isolating and characterizing swine butyrate producers is necessary for controlled studies that provide a mechanistic understanding of how this functional group of bacteria promotes swine gut health and colonization resistance to bacteria of public health concern.

microbiology↗

bioRxiv2023

Conserved B cell signaling, activation, and differentiation in porcine jejunal and ileal Peyer's patches despite distinct immune landscapes

Peyers patches (PPs) are B cell-rich sites of immune induction in the intestine, yet B cell signaling, activation, and differentiation associated with PPs are poorly defined. Single-cell and spatial transcriptomics were completed to study B cells from porcine jejunum and ileum containing PPs. Intestinal locations had distinct immune landscapes, including more follicular B cells in ileum and increased MHC-II-encoding gene expression in jejunal, non-resting B cells. Despite distinct landscapes, conserved B cell dynamics were detected across intestinal locations. B cell signaling to CD4+ macrophages that are putative phagocytic, cytotoxic, effector cells and deduced routes of B cell activation/differentiation, including resting B cells migrating into follicles to replicate/divide or differentiate into antibody-secreting cells residing in intestinal crypts, were conserved across locations. A six-biomarker panel recapitulated transcriptomics results of B cell phenotypes, frequencies, and spatial locations via ex vivo/in situ staining of gene/protein targets. Findings convey conserved B cell signaling, activation, and differentiation, despite location-specific immune environments in jejunum and ileum containing PPs. Results establish a benchmark of B cell dynamics for understanding intestinal immune induction important to promoting gut and overall health.

immunology↗

bioRxiv2022

Phenotypic and genomic comparison of human outbreak and cattle-associated Shiga toxin-producing Escherichia coli O157:H7

Escherichia coli O157:H7 (O157)-adulterated food products, including beef and produce, are associated with disease outbreaks in humans. Although cattle feces are a source for the contamination, it is unclear if diverse O157 human-associated outbreak isolates expressing a specific virulence phenotype can colonize and shed in the feces of cattle at a quantitatively similar levels to non-outbreak isolates. It is also unclear if other phenotypes, such as biofilm, cell attachment, and toxin production, differentiate environmental O157 isolates from O157 isolates associated with human illness. Genomic profiling of O157 isolates acquired through routine surveillance can inform if the isolates encode virulence genes associated with human disease, but many genotype-phenotype relationships remain unclear for O157. Therefore, the objective of this study was to compare a diverse set of O157 isolates, with the intent of identifying potential genotypic differences that could inform phenotypes such as cattle colonization and fecal shedding, in vitro cell attachment, biofilm production, and Shiga toxin production. In addition, the relationship between phenotypes and potential for foodborne illness as it relates to genomic virulence traits was explored. No significant differences in cattle colonization and fecal shedding were detected for the tested isolates, despite broad genomic differences. In addition, the in vitro phenotypic differences noted in biofilm and cell attachment did not associate with one LSPA-6 lineage compared to another. Overall, no differences in cattle shedding were observed, yet variations in genotype and phenotype were identified indicating further work is warranted to better understand the relationship between O157 genome and virulence. ImportanceFoodborne illness has a major impact on the health and wellbeing of the global population, besides creating substantial financial hardships for industry. While many bacteria and viruses are implicated in foodborne illness, Escherichia coli serotype O157:H7 (O157) is a common food adulterant that can cause human disease and food recalls. Cattle feces are a significant source of food-adulterating O157. A greater understanding of O157 genetics and its relation to phenotype is needed to develop mitigation strategies to limit spread of O157 into the food chain. The goal of the research was to identify O157 genomic and phenotypic attributes of O157 associated with cattle colonization and fecal shedding along with other factors involved in environmental persistence and illness in humans. It was observed that variations in biofilm formation and in vitro cellular adherence did not associate with enhanced cattle colonization or fecal shedding, indicating that the processes involved in cattle colonization are complex and not well understood.

microbiology↗

bioRxiv2022

Route of oxytetracycline administration differentially impacts the growth and gut microbiome of pigs co-infected with Bordetella bronchiseptica and Pasteurella multocida

Along with judicious antibiotic use, there is great interest in how the dose regimen of an antibiotic affects the animal gut microbiota. This study evaluated the impact of experimental respiratory infection alone or respiratory infection followed by oxytetracycline (oxytet) treatment on the animals health and its fecal microbiome. Piglets of approximately three weeks-of-age were separated into four groups (n=20 per group). One group remained non-infected and administered non-medicated feed and the other three groups were infected with Bordetella bronchiseptica (day 0) and Pasteurella multocida (day 4), with one group receiving non- medicated feed and the remaining two groups receiving oxytetr starting on day 7 by injection or in-feed (day 7-14). Infection with B. bronchiseptica and P. multocida negatively impacted piglet growth and induced mild pneumonia. Infection alone had minimal effect on the fecal microbiota community. When oxytet was administered either by injection or in-feed to treat the respiratory infections, both routes had minimal effect on clearing B. bronchiseptica and P. multocida in the animal. However, both routes appeared to limit lung lesion severity, and injected oxytet reduced the negative impact of infection on weight gain. Both routes had limited impact on the animals overall gut microbiome, including relative abundances of bacterial taxa and antibiotic resistance genes tet32, tetW, and aph2. Overall, oxytet administered by either route did not clear the respiratory infection, but oxytet administration minimized the negative health impacts of infection and had minor impact on the pig gut microbiome. ImportanceEfforts to address antibiotic resistance calls for improved antibiotic stewardship, including considering antibiotic administration route. While our previous study found in-feed oxytet had greater impact on the gut microbiome of healthy piglets than injected oxytet, it remained unknown if oxytet treatments would have the same impact on the microbiota of infected piglets. We evaluated the impact of respiratory infection alone or respiratory infection followed by oxytet treatment on the animals health and their gut microbiome profile. Respiratory infection negatively affected piglets health, but infection alone had minimal impact on the gut community. When oxytet was administered either in-feed or by injection to treat the respiratory infection, neither route of administration led to the clearance of the respiratory pathogens. However, oxytet minimized the negative health impacts of infection, and had minor impact on the pig gut microbiome. These findings are informative for disease management in food animals while integrating antibiotic stewardship practices.

microbiology↗

bioRxiv2022

Porcine intestinal innate lymphoid cells and lymphocyte spatial context revealed through single-cell RNA sequencing

Intestinal lymphocytes are crucial members of the mucosal immune system with impact over outcomes of intestinal health versus dysbiosis. Resolving intestinal lymphocyte complexity and function is a challenge, as the intestine provides cellular snapshots of a diverse spectrum of immune states. In pigs, intestinal lymphocytes are poorly described relative to humans or traditional model species. Enhanced understanding of porcine intestinal lymphocytes will promote food security and improve utility of pigs as a biomedical model for intestinal research. Single-cell RNA sequencing (scRNA-seq) was performed to provide transcriptomic profiles of lymphocytes in porcine ileum, with 31,983 cells annotated into 26 cell types. Deeper interrogation revealed previously undescribed cells in porcine intestine, including SELLhi{gamma}{delta} T cells, group 1 and group 3 innate lymphoid cells (ILCs), and four subsets of B cells. Single-cell transcriptomes in ileum were compared to those in porcine blood, and subsets of activated lymphocytes were detected in ileum but not periphery. Comparison to scRNA-seq human and murine ileum data revealed a general consensus of ileal lymphocytes across species. Lymphocyte spatial context in porcine ileum was conferred through differential tissue dissection prior to scRNA-seq. Antibody-secreting cells, B cells, follicular CD4 {beta} T cells, and cycling T/ILCs were enriched in ileum with Peyers patches, while non-cycling{gamma}{delta} T, CD8 {beta} T, and group 1 ILCs were enriched in ileum without Peyers patches. scRNA-seq findings were leveraged to develop advanced toolsets for further identification of ILCs in porcine ileum via flow cytometry and in situ staining. Porcine ileal ILCs identified via scRNA-seq did not transcriptionally mirror peripheral porcine ILCs (corresponding to natural killer cells) but instead had gene signatures indicative of tissue- and activation-specific functions, indicating potentially similar roles to intestinal ILCs identified in humans. Overall, the data serve as a highly-resolved transcriptomic atlas of the porcine intestinal immune landscape and will be useful in further understanding intestinal immune cell function.

immunology↗

bioRxiv2021

Reference transcriptomes of porcine peripheral immune cells created through bulk and single-cell RNA sequencing

Pigs are a valuable human biomedical model and an important protein source supporting global food security. The transcriptomes of peripheral blood immune cells in pigs were defined at the bulk cell-type and single cell levels. First, eight cell types were isolated in bulk from peripheral blood mononuclear cells (PBMCs) by cell sorting, representing Myeloid, NK cells and specific populations of T and B cells. Transcriptomes for each bulk population of cells were generated by RNA-seq with 10,974 expressed genes detected. Pairwise comparisons between cell types revealed specific expression, while enrichment analysis identified 1,885 to 3,591 significantly enriched genes across all 8 cell types. Gene Ontology analysis for the top 25% of significantly enriched genes (SEG) showed high enrichment of biological processes related to the nature of each cell type. Comparison of gene expression indicated highly significant correlations between pig cells and corresponding human PBMC bulk RNA-seq data available in Haemopedia. Second, higher resolution of distinct cell populations was obtained by single-cell RNA-sequencing (scRNA-seq) of PBMC. Seven PBMC samples were partitioned and sequenced that produced 28,810 single cell transcriptomes distributed across 36 clusters and classified into 13 general cell types including plasmacytoid dendritic cells (DC), conventional DCs, monocytes, B cell, conventional CD4 and CD8 {beta} T cells, NK cells, and {gamma}{delta} T cells. Signature gene sets from the human Haemopedia data were assessed for relative enrichment in genes expressed in pig cells and integration of pig scRNA-seq with a public human scRNA-seq dataset provided further validation for similarity between human and pig data. The sorted porcine bulk RNAseq dataset informed classification of scRNA-seq PBMC populations; specifically, an integration of the datasets showed that the pig bulk RNAseq data helped define the CD4CD8 double-positive T cell populations in the scRNA-seq data. Overall, the data provides deep and well-validated transcriptomic data from sorted PBMC populations and the first single-cell transcriptomic data for porcine PBMCs. This resource will be invaluable for annotation of pig genes controlling immunogenetic traits as part of the porcine Functional Annotation of Animal Genomes (FAANG) project, as well as further study of, and development of new reagents for, porcine immunology.

immunology↗

bioRxiv2020

Intraepithelial T cells diverge by intestinal location as pigs age

T cells resident within the intestinal epithelium play a central role in barrier integrity and provide a first line of immune defense. Intraepithelial T cells (IETs) are among the earliest immune cells to populate and protect intestinal tissues, thereby giving them an important role in shaping gut health early in life. In pigs, IETs are poorly defined, and their maturation in young pigs has not been well studied. Given the importance of IETs in contributing to early life and long-term intestinal health through interactions with epithelial cells, the microbiota, and additional environmental factors, a deeper characterization of IETs in pigs is warranted. The objective of this study was to analyze age- and intestinal location-dependent changes in IETs across multiple sites of the small and large intestine in pigs between 4 and 8 weeks of age. IETs increased in abundance over time and belonged to both {gamma}{delta} and {beta} T cell lineages. Similar compositions of IETs were identified across intestinal sites in 4-week-old pigs, but compositions diverged between intestinal sites as pigs aged. CD2+CD8+ {gamma}{delta} T cells and CD4-CD8+ {beta} T cells comprised >78% of total IETs at all intestinal locations and ages examined. Greater percentages of {gamma}{delta} IETs were present in large intestine compared to small intestine in older pigs. Small intestinal tissues had greater percentages of CD2+CD8- {gamma}{delta} IETs, while CD2+CD8+ {gamma}{delta} IET percentages were greater in the large intestine. Percentages of CD4-CD8+ {beta} IETs increased over time across all intestinal sites. Moreover, percentages of CD27+ cells decreased in ileum and large intestine over time, indicating increased IET activation as pigs aged. Percentages of CD27+ cells were also higher in small intestine compared to large intestine at later timepoints. Results herein emphasize 4 to 8 weeks of age as a critical window of IET maturation and suggest strong associations between intestinal location and age with IET heterogeneity in pigs.

immunology↗

bioRxiv2018

Resistant potato starch fuels beneficial host-microbe interactions in the gut

Interactions between diet, the microbiota, and the host set the ecological conditions in the gut and have broad implications for health. Prebiotics are dietary compounds that may shift these conditions towards health by promoting the growth of beneficial microbes. Pigs fed a diet amended with 5% resistant potato starch (RPS) exhibited alterations associated with gut health relative to swine fed an unamended diet (CON). RPS intake increased abundances of anaerobic Clostridia in feces and several tissues, as well as intestinal concentrations of butyrate. Functional gene amplicons suggested bacteria similar to Anaerostipes hadrus were stimulated by RPS intake. The CON treatment exhibited increased abundances of several genera of Proteobacteria (which utilize respiratory metabolisms) in several location. RPS intake increased the abundance of regulatory T cells in the cecum, but not periphery, and cecal immune status alterations were indicative of enhanced mucosal defenses. A network analysis of host and microbial changes in the cecum revealed that regulatory T cells positively correlated with butyrate concentration, luminal IgA concentration, expression of IL-6 and DEF1B, and several mucosa-associated bacterial taxa. Thus, the administration of RPS modulated the microbiota and host response, improved cecal barrier function, promoted immunological tolerance, and reduced the niche for bacterial respiration.\n\nImportanceThe gut microbiota is central to host health. Many disease states and disorders appear to arise from interactions between the gut microbial community and host immune system. As a result, methods and interventions to support the growth and activity of beneficial gut microbes are being actively pursued. Feeding the gut microbiota with dietary compounds, known as prebiotics, is one of the most promising ways to support gut health. Here we describe a successful prebiotic intervention in weaning swine, a relevant translational model for human health. This work unites microbial and immunological data and demonstrates one way a prebiotic intervention can play out for the benefit of the host.

systems biology↗