Disease resistance of individuals exposed to less-hygienic conditions during early life has been called the hygiene hypothesis, first postulated by Strachan (1989). It proposes that individuals exposed to less-hygienic conditions early in life may have altered immune system development which lowers the incidence of illness later in life (Strachan, 1989). While it is well understood that particular conditions, such as poor water sanitation, lead to the development and propagation of diseases, low environmental exposure has been associated with increased resistance to human chronic illnesses, such as asthma and allergies (Illi et al., 2001; MacNeill et al., 2013; Huang et al., 2021). It has been theorized that this low dose of exposure results in a mild stimulation of the immune system, particularly the adaptive immune system (Su et al., 2013).

A growing number of publications indicate that microbe exposure may be a significant contributor to the development of the immune system (reviewed by Zheng et al., 2020). Ennamorati et al. (2020) identified the gut microbiome as a major presenter of novel antigens to the immune system that influences the generation of regulatory T cells by the thymus in neonatal mice. The authors described how this process may facilitate homeostasis between microbiota and the host immune system in the host to prevent intestinal inflammation later in life. Studies in the chicken also indicate that intestinal microbiota may regulate T-cell production in the thymus in early life (Cheng et al., 2021). Moreover, postnatal microbial colonization of the body may act as a major stimulus of immune tissue development and modulation of miRNA expression (Masotti, 2012), which contributes to “training” of the immune system (Gury-BenAri et al., 2016). This early training of the immune system and development of microbial memory in early life may allow individuals to overcome an illness more quickly, thus, allowing them to spare energy to partition toward growth. In addition, development of a more robust immune system should reduce antibiotic use and subsequently antimicrobial resistance.

As livestock producers have strived to increase sustainability and growth efficiency of pigs, the environment and hygienic conditions have significantly changed over time. During early commercialized swine production, swine were reared outdoors and exposed to soil, weather, and predators. The move to modern, indoor housing has allowed for decreased exposure to parasites, fecal material, predators, and changes in weather conditions, which greatly improved the overall welfare of swine. It has been observed that early-life exposure of pigs to less-hygienic conditions (i.e., topsoil exposure) has increased growth (Dourmad et al., 2009; Lebret et al., 2011; Wenner et al., 2013; Vo et al., 2017) while decreasing feed intake (Lebret et al., 2011; Wenner et al., 2013). One explanation for these findings is that host exposure to microbes from the environment may influence host physiology.

Initial studies of neonatal pigs exposed to topsoil indicate microbial communities (Vo et al., 2017; Wen et al., 2021) and the immune cell population (Tsai et al., 2016; Wen et al., 2021) are altered later in life in response to early-life topsoil exposure. Both Vo et al. (2017) and Wen et al. (2021) identified higher levels of Prevotella at days 13 and 12, respectively, in the gut of piglets exposed to topsoil. In mice, perturbation of Prevotella in the gut microbiome resulted in exacerbated intestinal inflammation (Iljazovic et al., 2021). Moreover, Vo et al. (2017), who used the same animals described in this study, identified a higher abundance of Prevotella at weaning (d 21) and at day 35 (mid-nursery). Vo et al. (2017) also identified higher levels of Bacteroidetes and Ruminococcaceae at day 13 and Clostridium and Clostridium cluster XI, Coprococcus, Campylobacter, Streptococcus, Mitsuokella, Ruminococcus, Dialister, and Sarcina during postweaning at the end of the nursery phase (d 56) in response to topsoil exposure. Interestingly, Clostridium species have been associated with colonic regulatory T-cell accumulation and improvement of the local and systemic immune responses (Atarashi et al., 2011). Although these studies demonstrate that the microbiome is altered in pigs exposed to topsoil, it is still unclear which specific microbes are directly impacting the animal’s physiology. Little is known about the molecular mechanisms responsible for impacting animal growth or the immune system in this model. Understanding how the immune system is programmed in response to microbes and the environment is an important step in the development of technologies (e.g., probiotics) that may enhance efficiency in livestock production, and provide basic science knowledge about host–microbiome interactions that are beneficial to improve human health.

The objective of this study was to identify changes in peripheral blood mononuclear cell (PBMC) gene expression and associated pathways in response to early-life topsoil exposure in piglets from a subset of piglets exposed to topsoil from previous studies (Tsai et al., 2016; Vo et al., 2017). Specifically, we sought to determine if there was evidence of early, enhanced, or altered immune system development based on mRNA and miRNA expression profiles using RNA-sequencing (RNA-seq) technology. Transcriptome profiling is a powerful tool to evaluate global changes in gene expression within a tissue. As miRNAs are well-established regulators of gene expression and may facilitate communication between the host and microbiome, we evaluated the relationship between miRNAs and known or predicted mRNA targets (Masotti, 2012; Yuan et al., 2018; Gebert and MacRae, 2019).

The objective of this study was to identify potential mechanisms controlling how the immune system is programmed in response to microbes and the environment. To understand how early-life exposure to less-hygienic conditions alters immune training, piglets were exposed to topsoil only from d 4-d 21 of life, which resulted in the modulation of gut microbiota and growth performance (Vo et al., 2017). To determine changes in immune signaling, PBMC mRNA and miRNA gene expression were analyzed over the first 56 days of life. The following passages provide potential explanations for the observed changes in PBMC gene expression in response to early-life topsoil exposure along with the potential limitations in the interpretation of this study.

Genes identified as DE indicate changes in the development of the immune system which under the parameters of this study may be in response to interactions with novel antigens or nutrient availability. This analysis identified DE genes associated with T-cell activation. The first two DE genes, PTPRJ and ITGB3, are known to be involved in the formation of the immune synapse or area of communication between a T cell and an antigen-presenting cell (APC). Both genes were upregulated at d 20 in the topsoil group (Supplementary Figure S1, Figure 1C) which may be in response to increased contact with microbes during the nursery phase from d 4 to d 21. Corroborating these results, Lin et al. (2004) found increased levels of PTPRJ in T cells upon activation in mice, indicating that this increase in the topsoil-exposed piglets may have elevated T-cell activation at d 20. Conversely, the lower levels of PTPRJ in naïve T cells in mice (Lin et al., 2004) indicate that the lower expression of PTPRJ in the control piglets at d 20, when the piglets were in higher hygienic conditions, results from lower antigen contact and probably higher naïve T cells.

Two of the identified DE genes makeup components of the T-cells receptor (TCR) complex, TRBV30 and CD3D, are crucial to antigen recognition and initiation of cellular signaling. The diversity of paired alpha and beta chains of the TCR repertoire confers broad immune coverage against pathogens. Recent studies using next-generation sequencing have characterized the TCR repertoire of individuals. The TCR repertoire is composed of a diverse combination of alpha and beta TCR chains (Attaf et al., 2015), which may provide the pathogen exposure history and immunological memory of a given individual (DeWitt et al., 2018). TRBV30 is one of the several genes encoding the V region of the variable domain of TCR beta chain which is responsible for recognizing specific peptides presented by APCs (Davis et al., 1995). The CD3D gene is associated with TCR chain activation after antigen recognition (Dave et al., 1997; Garcillán et al., 2021). Both TRBV30 and CD3D exhibited similar trends of expression over time in this study. In piglets exposed to topsoil, expression levels increased from d 11 to d 20 and then decreased at d 56 (Figure 1C). In control piglets, expression levels decreased from d 11 to d 20 and increased at d 56. The increased expression at d 20 followed by a decrease at d 56 in the topsoil group as well as the delayed peak in the control piglets may indicate earlier development of the immune system in topsoil-exposed piglets compared with the control. Early-life exposure to topsoil may have provided microbes or other substances that resulted in these differences in gene expression that allow for faster response to future exposure of these microbes. Once ingested by the piglets, microbes and other constituents of the topsoil would interact with Paneth cells and APCs that stimulate the immune system.

Other DE genes with known function in immune response (i.e., BMP8A, GPD2, PHKA1 and PHKA2) (Supplementary Figure S1) exhibited a similar pattern of expression, which may support the hypothesis that exposure to topsoil alters the timing or development of the immune system. Of particular interest, BMP8A is a transforming growth factor beta (TGF-β) superfamily member contained within a signaling pathway essential to activation and homeostasis of naïve CD4⁺ T cells (Martínez et al., 2015).

The ontology enrichment analyses identified pathways involved in T-cell activation, specifically those involved in the interaction between the T cell and the APC, including the formation of the interactive space between the two also known as the immunological synapse. The DE genes STX3, PTPRJ, ITGB3, ITGA3, MYH9, and TLN1 were clustered in several GO terms related to processes involved in many aspects of the immune synapse and downstream signaling. These genes function in vesicle-mediated transport to the plasma membrane (GO:0098876), protein transport within plasma membrane (GO:0099632), microtubule-based protein transport (GO:0099118), integrin-mediated signaling pathways (GO:0007229), positive regulation of peptidyl-tyrosine phosphorylation (GO:0050731), and positive regulation of cell-substrate adhesion (GO:0010811). After T-cell receptor stimulation, cytoskeleton reorganization (actin and microtubules) is needed to adapt the cell conformation and formation of the synapse architecture (Valitutti et al., 1995; Martín-Cófreces and Sánchez-Madrid, 2018). Traffic through the membrane is important to regulate the activation of T cells by balancing the cellular localization of a wide type of components as receptors, signaling molecules or cytokines (reviewed by Soares et al., 2013). STX3 has a putative role in the formation of the immune synapse of cytotoxic T cells as it is involved in protein trafficking involving vesicle fusion and exocytosis. STX3 co-localized with CD3 proteins which are components of TCR-CD3 complex on T-cell surfaces (Pattu et al., 2012). Two additional DE genes, PTPRJ and ITGB3, are known to be involved in the formation of the immune synapse and clustered in GO terms related to cell-substrate adhesion and regulation of tyrosine phosphorylation. The ITGB3 gene plays a role in the immune synapse by encoding a subunit of specific integrins. Other DE genes involved in these processes include ITGA3, MYH9, and TLN1 (Figure 1C, Supplementary Figure S1), all of which increase at d 20 in piglets exposed to topsoil suggesting that these genes support the formation of the immune synapse (Figure 1C).

An important finding in relation to the development of the immune system was the identification of mir-143 as DE in response to topsoil exposure over time (Figure 2C). MicroRNAs are important regulators of gene expression with important roles in mediating the function of the immune system (Ma et al., 2011; Zhang et al., 2018). Typically, each miRNA suppresses gene expression in tens to thousands of mRNA transcripts to coordinate the use of pathways within the transcriptome (Gebert and MacRae, 2019). Previous studies indicate that overexpression of mir-143 may enhance the conversion of cytotoxic T cells into memory cells by reprogramming cellular metabolism (Zhang et al., 2018). In addition, mir-143 was identified as a candidate regulator of a miRNA-target co-expression network in response to topsoil and a hub miRNA within the brown miRNA module. It was also negatively correlated with a group of mRNAs (brown mRNA module) only in the piglets exposed to topsoil during early life. Target mRNA genes within the miRNA:mRNA correlated modules containing mir-143 were enriched for biological processes and pathways such as response to lipopolysaccharide and positive regulation of B-cell receptor signaling pathway. Together, mir-143, the other hub miRNAs, and their co-expressed target genes in the brown module make up a miRNA-target network acting as a putative driver of the immune response in the animals exposed to topsoil in early life.

Analysis of mRNA-miRNA interactions also identified mir-29a-3p and mir-148–5p as potential regulators of gene expression only in topsoil-exposed pigs. Upon bacterial infection, mir-29a-3p has been identified to be downregulated in innate immune cells, such as natural killer and adaptive immune cells such as helper T cells and cytotoxic T cells (Ma et al., 2011). Targets of mir-29a-3p were enriched for regulation of mTOR signaling which has a role in the innate and adaptive immune system by regulating metabolism. As reviewed by Chi. (2012), mTOR signaling can act in different pathways affecting T-cell activation, differentiation, and immune homeostasis. The third interesting candidate miRNA-target network identified was driven by mir-148a-5p, which was negatively correlated to the royalblue module. The mRNA genes within the royalblue mRNA module were enriched for genes involved in response to type I interferon and interferon-gamma-mediated signaling pathway. Type I interferon is secreted by the innate immune cells after detection of the pathogen, but also can act in the adaptive immune response by stimulating antibody production of B cells or intensifying the T-cells function (Reviewed by Ivashkiv and Donlin, 2014).

Understanding the role hygienic environments have on host gene expression is important in our understanding of the development of the immune system. The findings of this study indicate that early-life exposure of piglets to topsoil impacts both mRNA and miRNA expression in PBMCs, particularly genes and miRNA involved in the development of T cells and their ability to interact with antigen-presenting cells. Key regulatory genes identified include: PTPRJ, ITGB3, TRBV30, CD3D, mir-143, mir-29, and mir-148a. While these results need to be interpreted with caution as they represent only a snapshot in time across a small window of time from a subset of peripheral immune cells, these results support the current hygiene hypothesis in that less-hygienic environments during early life contribute to the development of the immune system. However, additional studies are needed to characterize how topsoil exposure may impact gene expression within the gut and impact gut phenotypes critical to animal health.