Exploring the cellular composition and intercellular interactions of respiratory mucosa is essential for understanding its defense mechanisms and developing targeted therapeutic strategies [27]. In this study, we performed scRNA-seq for porcine nasal mucosa, and generated a single-cell atlas comprising 17,201 cells, categorized into 30 clusters including nine epithelial cell types, four stromal cell types, and seven immune cell types. Subsequently, the distribution characteristics of the three most representative epithelial cells in porcine nasal mucosa were depicted. Our analysis showed that basal cells and ciliated cells are evenly distributed across the entire mucosal epithelial layer. Ciliated cells, mediating mucociliary clearance process, play a critical role in expelling particles and pathogens[28]. Basal cells function as tissue-specific stem cells in the airway, crucial for long-term self-renewal and vital in maintaining and repairing the respiratory epithelium [29, 30]. The strategic distribution of ciliated cells and basal cells likely contributes to reducing pathogen colonization and aid in homeostasis maintenance. In contrast, the distribution of goblet cells showed significant regional specificity, which predominating in the lower but scarce in the upper regions of the porcine nasal mucosa. Take into account goblet cells are responsible for mucus production, and mucus serves as a key component of the mucosal defense mechanism against microbial invasion [31, 32], this uneven distribution suggests a potential increased vulnerability to pathogen infection in the upper mucosal regions.
Although it is generally believed that domestic pigs share histological similarities with humans [33], existing researches have yet to effectively compare the cellular composition and transcriptional characteristics of porcine and human nasal mucosa. Our study revealed that both porcine and human nasal epithelia consist of basal cells, suprabasal cells, cycling basal cells, club cells, goblet cells, ciliated cells, ionocyte, serous cells and mucous cells. Moreover, the marker genes of these nasal epithelial cells are highly conserved between pigs and humans. These findings suggest that the functional roles of the nasal epithelium are likely similar across these two species. Duct cells, commonly found in the pancreas, were also identified in porcine nasal cavity, exhibiting robust energy metabolism activity. Given their role in secreting mucin and fluid/electrolytes, particularly HCO3− [34–36], the presence of these cells in the porcine nasal cavity may enhance the functional role of the mucosal barrier, meriting further investigation.
Pseudotime analysis, also known as trajectory inference, is used for inferring the differentiation trajectory of cells or the evolution process of cell subtypes [37, 38]. Our pseudotime analysis showed that the differentiation trajectories of nasal epithelial cells in pigs and humans are remarkably similar, both start with basal cells, progress to club cells, and then branch into either ciliated cells or goblet cells under the regulation of different transcription factors. The signaling pathways such as Notch, Wnt, and BMP/TGFβ play an important role in in nasal epithelium development [39]. Our results showed that the expression patterns of key transcription factors within these pathways (such as JAG1, TGFB1, BMP7 and WNT10A) are similar in both porcine and human nasal epithelial cells. These findings indicate a conserved development of nasal epithelial cells across pigs and humans.
Interactions between cellular populations are essential for the development of nasal mucosa and the maintenance of the mucosal barrier [40]. Our study identified that the ligand-receptor pairs between nasal epithelial cells in pigs and humans were mainly related to epithelial development, such as EGFR-TGFB1, EGFR-MIF, TGFBR3-TGFB1, NGFR-NTF3, EPHB4-EPHB1 and LRP1-MDK [41, 42]. However, the expression patterns of these receptors and ligands are species-specific. Taking the interaction between basal cells and cycling basal cells as an example, in pigs, the TGFBR3-TGFB1 and NGFR-NTF3 signaling pathways were specifically activated, whereas in humans, the EPHB4-EPHB1 and LRP1-MDK pathways were specifically activated. These findings suggest that these pathways may uniquely contribute to the development of nasal epithelial cells in each species. Notably, our research also highlighted the unique activation of the NECTIN1-NECTIN3 signaling pathway in pigs, which is known to play a crucial role in forming, maintaining, and modifying cellular junctions [43]. This specific activation implies its potential involvement in establishing early epithelial barriers in pigs.
The maintenance of the physical barrier of the nasal mucosa requires the involvement of cell–cell junctions, including tight junction, adherens junction and desmosomes [44, 45]. The observation of similar expression patterns of these junction molecules in porcine and human nasal epithelial cells suggested that the nasal epithelium of pigs and humans have similar physical barrier functions. Pattern recognition receptors (PRRs), which detect pathogens by recognizing pathogen-associated molecular patterns (PAMPs), play a vital role in host defense mechanisms [46]. Our research showed that pigs exhibit a significantly higher variety and expression levels of pattern recognition receptors compared to humans. Notably, TLR3, which targets double-stranded RNA from viruses [47, 48], and TLR4, activated by bacterial lipopolysaccharides [49, 50] were highly expressed in the porcine nasal epithelial cells. This suggests that porcine nasal mucosa may have stronger innate immune response to infection.
Pigs are susceptible to a variety of human respiratory pathogens. We found multiple human respiratory virus receptors exhibit similar expression patterns in porcine and human nasal epithelial cells, such as ANXA5, ITGB1, ITGA5 and EGFR. Among these, ANXA5 and EGFR have been identified as a co-receptor for the influenza virus [51, 52]. The similar expression patterns of these two receptors in both porcine and human nasal epithelial cells may explain why pigs and humans exhibit comparable susceptibility and pathogenic responses to influenza viruses. Notably, our results highlighted that ciliated cells exhibit the highest levels of viral receptor expression. Since ciliated cells are primary targets for RSV and SARS-COV-2 [53, 54], they are likely more susceptible to infections by various respiratory viruses. However, it is crucial to recognize that protein expression levels play a pivotal role in determining cellular functions. Consequently, it is essential to investigate the concordance between protein expression and transcription levels in greater detail.
In conclusion, our study substantially enhances understanding of the cellular composition and gene expression profiles in the nasal mucosa of domestic pigs. By conducting a comparative analysis of single-cell data from porcine and human nasal mucosa, we identified significant parallels between porcine and human nasal epithelial cells regarding cellular composition, differentiation trajectories, and transcription characteristics of cell-cell junction molecules and various respiratory virus receptors. These insights provide a strong foundation for using porcine nasal cavity as alternative models for studying human respiratory diseases.