Bone marrow and Lymphoid tissues

The lymphoid tissues are a part of the circulatory system and the immune system, which plays an essential role in the defense against exogenous pathogens, removal of interstitial fluids as well as production and development of lymphocytes. The lymphoid tissue is usually classified into primary and secondary lymphoid tissue. The former consists of the bone marrow and thymus gland, and the latter includes the tonsil, lymph node and spleen. In the pig transcriptomic analysis, bone marrow is analyzed separately from the other lymphoid tissues (represented by thymus, tonsil, lymph node and spleen grouped together) and used for quantitative RNA measurements, representing the lymphoid system.

The gene classification strategy highlights genes with an elevated level of expression in one or a group of tissues compared to all other tissues. 248 genes are classified as bone marrow elevated out of which 16 genes are highlighted as bone marrow enriched. Based on the expression in lymphoid tissues 1360 genes are classified as elevated compared to other tissue types, out of which 185 genes are highlighted as tissue enriched in lymphoid tissues.

The function of the bone marrow and lymphoid tissues are similar between the pig and human. Histological image of the pig tissues used in the analysis can be found in the pig tissue dictionary.

Bone marrow

The bone marrow is a kind of semi-solid tissue composed of many types of cells and connective tissue. It can be found in some spongy or cancellous portions of bones. The bone marrow is the hematopoietic organ in the body and its main function is to maintain constant levels of the different blood cell types in the bloodstream, such as producing erythrocytes, leukocytes and thrombocytes. The bone marrow samples in this study were analyzed in full, i.e. including adipose tissue, erythrocytes and possibly bone residues from the sampling extraction.

Gene expression in bone marrow 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, 15211 genes are detected above cut of (1NX) in pig bone marrow. The tissue distribution category highlights 25 genes only detected in bone marrow while 248 genes are classified as bone marrow elevated compared to other tissues. Table 1 shows the overlap for the bone marrow elevated genes and tissue distribution category.

Figure 1, (A) The distribution of all genes across the five categories based on transcript specificity in bone marrow as well as in all other tissues. (B) The distribution of all genes across the six categories, based on transcript detection (NX≥1) in bone marrow 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 bone marrow.

Bone marrow enriched expression

PRTN3 encodes a serine protease, which can degrade proteins like elastin, fibronectin and laminin. It may play a role in neutrophil transendothelial migration. ELANE encodes an elastases that belongs to the subfamily of serine proteases, it can hydrolyze many proteins in addition to elastin and may play a role in degenerative and inflammatory diseases through proteolysis of collagen-IV and elastin. Myeloperoxidase (MPO ) is a heme protein synthesized in the process of myeloid differentiation. It constitutes the main component of eosinophils in neutrophils and be responsible for producing hypohalous acids central to the microbicidal activity of neutrophils. AZU1encodes a preprotein that can be processed into a mature azurophil granule antibiotic protein, with monocyte chemotactic and antimicrobial activity. Azurophil granules are specialized lysosomes of the neutrophil, they contain at least 10 proteins implicated in the killing of microorganisms. Eosinophil peroxidase (EPX) is mainly expressed in eosinophils, it mediates tyrosine nitration of secondary granule proteins in mature resting eosinophils, playing a role in inducing bacterial fragmentation and lysis.

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

Lymphoid tissue

Thymus is a primary lymphoid tissue where the T-cells mature and thus plays a vital role in immunity. The porcine tonsils are lymph-epithelial organs located in the openings of the digestive and respiratory tracts, which act as the first line of defense of the immune system against ingested or inhaled foreign pathogens. The lymph structure of porcine tonsil resembles that of lymphoid nodes. They are composed of lymphoid follicles with germinal centers that are abundant in B-lymphocytes. Lymph nodes are small bean-shaped structures with dense connective tissue that are white in color and spread along the lymph vessels that form the ganglion chain. Lymph nodes are the main sites of lymphocytes including B cells and T cells. The spleen can be found on the left side of the abdominal cavity, it plays an essential role in immune function since it acts primarily as a blood filter for antigen recognition. Spleen also removes old blood cells and serves as a reservoir for blood cell production, such as erythrocytes and platelets.

Lymphoid tissues in the transcriptomic analysis is represented by the highest expression value of the grouped lymphoid tissues; thymus, tonsil, lymph node and spleen. Gene expression in lymphoid tissues 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, 16617 genes are detected above cut of (1NX) in pig lymphoid tissues. The tissue distribution category highlights 60 genes only detected in lymphoid tissues while 1360 genes are classified as lymphoid tissues elevated compared to other tissues. Table 3 shows the overlap for the lymphoid tissues elevated genes and tissue distribution category.

Figure 2, (A) The distribution of all genes across the five categories based on transcript specificity in lymphoid tissues as well as in all other tissues. (B) The distribution of all genes across the six categories, based on transcript detection (NX≥1) in lymphoid tissues 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 lymphoid tissues

Lymphoid tissue enriched expression

In the list of genes classified as lymphoid tissue enriched, most genes with highest tissue specificity score are due to the unique expression profile in thymus, for example PSMB11, RAG1 and DNTT. Further down the list of genes we can also find spleen specific genes, such as SLC11A1 or more generally expressed lymphoid enrched genes, CD27.

Interleukins (for example IL24) and Toll-like receptors (for example TLR8) are represented in the list of lymphoid tissue enriched genes.

The representation of local lymphoid follicles in other tissue types, is obvious when studying certain examples (CD4, CD3D and IL22RA2), where the highest expression is observed in lymphoid tissues followed by a specific but much lower expression in tissues of the respiratory system and gastrointestinal tract - all with local lymphoid follicles.

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

Expression variation within lymphoid tissues

The expression variation category enables further details related to expression heterogeneity within the tissues that include grouped samples. Although many cell types and functions are shared between the 4 tissues grouped into the lymphoid tissues, the within tissue variation categorization resulted in 3143 genes highlighted as variable.

Gene expression in lymphoid tissues and bone marrow compared to other tissues

In order to illustrate the relation of bone marrow and lymphoid 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 3. An interactive network plot of the tissue enriched and group enriched genes connected to their respective enriched tissues (grey circles). Black circles shows lymphoid tissues and bone marrow.. 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 3 tissues, but the resulting lists show the complete set of group enriched genes in the particular tissue.

Relevant references

Pig as a model for immunology research Pabst R. (2020)