Biology subjects

Wiarda, J. E.

Publications and source records attributed to Wiarda, J. E..

7 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↗

bioRxiv2025

Porcine epidemic diarrhea virus infection promotes Peyer's patch immune induction and epithelial defense via single-cell transcriptional reprogramming

Porcine epidemic diarrhea virus (PEDV) is an enteric coronavirus causing gastrointestinal disease in swine. To mitigate PEDV risks to swine health, agricultural economic losses, and global food security, a better understanding of host-pathogen interactions is required. Using single-cell RNA sequencing (scRNA-seq), we studied transcriptional cellular responses to PEDV infection in two anatomical compartments of intestinal jejunum: first-line barrier defenses of epithelia and underlying immune induction in Peyers patches. PEDV infection altered gene expression across all cell types and was associated with antiviral response pathways, indicating coordinated transcriptional reprogramming of diverse cell types. Signaling network inferences showed macrophages, dendritic cells, and non-resting B cells had increased signaling to T follicular helper cells associated with processes of T cell-dependent B cell activation during PEDV infection, indicating transcriptional promotion of immune induction in Peyers patches. Mature enterocytes were the primary targets for PEDV infection, and a 190-gene signature indicative of antiviral immune defense represented a conserved enterocyte response to PEDV infection, regardless of enterocyte stress state or infection status. The 190-gene signature was specific to the epithelial lineage and escalated with enterocyte maturation, indicating antiviral transcriptional reprogramming is mobilized across matured enterocytes of PEDV-infected intestinal segments. Results exemplify key roles of Peyers patches and epithelia as integral components for coordinated antiviral responsiveness in the intestine. Improved understandings of host-pathogen interactions during PEDV infection can identify indicators of PEDV protection versus susceptibility to target for future intervention strategies. IMPORTANCEPorcine epidemic diarrhea virus (PEDV) causes high death rates in young pigs and production losses in older animals, yet prevention and treatment options for PEDV remain limited. Understanding how PEDV infects pigs and how cells can control infection is crucial for developing better prevention and treatment strategies. Our work explores how intestinal cells are impacted by PEDV infection. We find all intestinal cells responded to infection despite diverse origins and functions. Processes promoting immune responses and epithelial barrier defenses against PEDV were initiated, culminating in a highly coordinated and conserved antiviral response. Results identify targets that may be useful in developing new PEDV prevention and control strategies that could positively impact animal health, agricultural economic prosperity, and global food security.

immunology↗

bioRxiv2025

United States PRRSV 1-4-4 L1C.5 isolate demonstrates similar pathogenicity to a historic Chinese highly pathogenic PRRSV

Porcine reproductive and respiratory syndrome virus (PRRSV) is a major economic and animal health burden on the United States swine industry due to morbidity- and mortality-associated losses affecting all stages of pig production. Currently, a large proportion of losses are attributed to a highly virulent PRRSV strain, PRRSV 1-4-4 L1C.5. To benchmark the virulence of PRRSV 1-4-4 L1C.5, a study was conducted to compare pathogenicity of this contemporary strain to a historical Chinese highly pathogenic PRRSV (HP-PRRSV) strain, JXwn06, that devastated the Chinese and other Asian swine industries since 2006, as well as a moderately virulent United States PRRSV strain, MN184, considered to be one of the most virulent PRRSV strains circulating in the United States in the early 2000s. Weaned pigs were inoculated with PRRSV strains L1C.5, JXwn06, MN184, or mock inoculum and necropsied at 2, 6, and 10 days post inoculation, or as needed due to severe disease. Clinical metrics, viral loads, cytokine concentrations, PRRSV-specific antibody concentrations, and pathology were compared between treatment groups to compare pathogenicity. Results indicate a high degree of similarity disease dynamics between L1C.5 and JXwn06 animals that diverged from MN184 and mock animals. Findings indicate L1C.5 and JXwn06 cause more severe morbidity and mortality in weaned pigs than MN184. Results may be applied to develop more effective strategies for mitigating PRRSV 1-4-4 L1C.5 outbreaks currently plaguing the United States swine industry.

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

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↗