Respiratory system

In the pig transcriptomic analysis, both lung and upper respiratory system are sampled and used for quantitative RNA measurements. The gene classification strategy highlights genes with an elevated expression in one or a group of tissues compared to all other tissues.

The respiratory system is a gas exchange system that can be divided into two main parts: The upper respiratory system (including nasopharynx, olfactory epithelium, larynx and trachea) and the lung (bronchus and the lung). The system starts at the nostrils and then leads on to the two nasal passages, which are the channels for air through the nose and are coated with respiratory mucus membranes that help mucus move to the throat. Olfactory epithelium is a special epithelial tissue in the nasal cavity, which is responsible for directly detecting odors. The larynx is an organ located below the pharynx, involving in breathing, generating sounds and protecting the trachea from inhaling food. The trachea is the extension from larynx that branches into the two primary bronchi. The porcine trachea is notably longer and more cartilaginous than the human trachea. The bronchi continue to branch into narrower air conducting tubes throughout the lung, when finally small enough to not be supported by cartilage they are called bronchioles, which includes respiratory bronchioles delivering air to the alveoli (gas exchanging unit of the lung).

321 genes are classified as lung elevated out of which 10 genes are highlighted as lung enriched. Based on the expression in upper respiratory system 667 genes are classified as elevated compared to other tissue types, out of which 23 genes are highlighted as tissue enriched in upper respiratory system.

The function of the respiratory system is similar between the pig and human. Histological image of the pig tissues used in the analysis can be found in the pig tissue dictionary.

Lung

The lungs are the essential organs in the respiratory system. They are responsible for providing the blood circulatory system with oxygen, which will then be transported to all other organs in the body, and also helping remove carbon dioxide from the bloodstream. Like human, pigs also have two lungs, the left lung and the right lung, which are located within the thoracic cavity of the chest. They are also highly lobulated, with well-defined pulmonary lobules demarcated by interlobular septae, but notably, compared to human lungs, which have 5 lobes, the pig lungs have 6 lobes. In this study, lung tissue was sampled at the very tip of the left lung while bronchi samples were taken earlier in the respiratory branching system where clear cartilage supported bronchi was found.

Gene expression in lung is categorized based on two gene classification strategies, tissue detection and tissue specificity. Figure 1 summarizes the number of genes in respective category. In total, 16104 genes are detected above cut of (1NX) in pig lung. The tissue distribution category highlights 5 genes only detected in lung while 321 genes are classified as lung elevated compared to other tissues. Table 1 shows the overlap for the lung elevated genes and tissue distribution category.

Figure 1, (A) The distribution of all genes across the five categories based on transcript specificity in lung as well as in all other tissues. (B) The distribution of all genes across the six categories, based on transcript detection (NX≥1) in lung as well as in all other tissues. The combination of the two categories is shown in table 1.

Table 1, Number of genes in the subdivided categories of elevated expression and tissue distribution in lung

Lung enriched expression

The type II alveolar cells in the lung are responsible for the production of a special surfactant that plays an essential part in gaseous exchange between air and blood as well as reducing the surface tension of fluids that coat the lung. Like the lung of human, proteins associated with pulmonary surfactant are highly expressed in the lung. Typical examples of these proteins are SFTPB, SFTPC and SFTPD.

Table 2, The 10 genes with the highest level of enriched expression in lung. "mRNA (tissue)" shows the transcript level in lung as NX values. "Tissue specificity score (TS)" corresponds to the fold-change between the expression level in lung and the tissue with second highest expression level.

Upper respiratory system

The upper respiratory system is represented by 4 different tissue types in the analysis; nasopharynx, olfactory, trachea and larynx. All tissues include respiratory epithelium, while only larynx and trachea includes cartilage in the tissue, although bone or cartilage residues are possibly included in the olfactory and nasopharynx epithelium. The Nasopharynx was samples in the nose cavity while olfactory epithelium was sampled as far up dorsal in the cavity as possible.

Gene expression in upper respiratory system is categorized based on two gene classification strategies, tissue detection and tissue specificity. Figure 2 summarizes the number of genes in respective category. In total, 16894 genes are detected above cut of (1NX) in pig upper respiratory system. The tissue distribution category highlights 10 genes only detected in upper respiratory system while 667 genes are classified as upper respiratory system elevated compared to other tissues. Table 3 shows the overlap for the upper respiratory system elevated genes and tissue distribution category.

Figure 2, (A) The distribution of all genes across the five categories based on transcript specificity in upper respiratory system as well as in all other tissues. (B) The distribution of all genes across the six categories, based on transcript detection (NX≥1) in upper respiratory system as well as in all other tissues. The combination of the two categories is shown in table 3.

Table 3, Number of genes in the subdivided categories of elevated expression and tissue distribution in upper respiratory system

Upper respiratory system enriched expression

Example of genes with high expression enrichment in upper respiratory system are OBP expressed specifically in the nasopharynx epithelium, GLYATL3 only detected in the olfactory epithelium, and ENSSSCG00000007262 expressed in all of the upper respiratory system tissues.

Table 4, The 10 genes with the highest level of enriched expression in upper respiratory system. "mRNA (tissue)" shows the transcript level in upper respiratory system as NX values. "Tissue specificity score (TS)" corresponds to the fold-change between the expression level in upper respiratory system and the tissue with second highest expression level.

Expression comparison between different parts of the upper respiratory system

The expression variation category enables further classification based on expression heterogeneity with the selected tissues, which include several grouped samples. In this case, expression profile of olfactory epithelium, nasopharynx, larynx and trachea was compared to each other, resulting in 765 genes highlighted as variable in the upper respiratory system.

Gene expression in the respiratory system compared to other tissues

Since both lung and upper respiratory system includes cartilage, respiratory epithelium and different types of connective tissue, several genes are shared and classified as group enriched. The presence of ciliated cells in the respiratory epithelium also results in several genes shared between different tissues types, for example CFAP65 and IFN-DELTA-7 both examples of genes with a shared expression between tissues with ciliated cells.

In order to illustrate the relation of respiratory tissue to other tissue types, a network plot was generated, displaying the number of genes shared between different tissue types. Group enriched genes are defined as genes showing a 4-fold higher average level of mRNA expression in a group of 2-5 tissues, compared to all other tissues.

Figure 6. An interactive network plot of the tissue enriched and group enriched genes connected to their respective enriched tissues (grey circles). Black circles shows the upper respiratory tract and lung. Red nodes represent the number of tissue enriched genes and orange nodes represent the number of genes that are group enriched. The sizes of the red and orange nodes are related to the number of genes displayed within the node. Each node is clickable and results in a list of all enriched genes connected to the highlighted edges. The network is limited to group enriched genes in combinations of up to tissues, but the resulting lists show the complete set of group enriched genes in the particular tissue.

Relevant references

Further reading related to the nasal cavity in Bama minipigs, with a focus on lymphatic tissue Yang J et al. (2017).