Identification of genes and tissue-specific proteins induced by Alquernat Immuplus

Objective

The aim of this trial is to evaluate the effect of natural immunostimulant pronutrients (Alquernat Immuplus) on the expression of specific genes related to the immune system in an in vitro culture of porcine alveolar macrophages.

Methodology

Procedure for obtaining specific tissue-proteins that have increased expression thanks to the use of immunostimulant pronutrients is based on the following stages:

• Development of two cellular cultures of alveolar macrophages: one is the control culture (without treatment), while the other culture is treated with a natural immunostimulant.
• Isolation of total RNA from treated and untreated alveolar macrophages.
• Construction of a complete transcriptomic library and RNA sequencing.
• Bioinformatic analysis: analysis of the differential expression between treated and untreated cultures, showed as volcano plot and heat map (Pictures 1 and 2).
• Data review: search in databases about the biological function of differentially expressed genes, to determine molecular basis of the beneficial effect of a natural immunostimulant on alveolar macrophages.

Picture 1. Volcano plot – Comparison between gene expression of control and treated cultures.

Volcano plot is a scatter diagram of -log10 (p) (on the vertical axis) as a function of M (on the horizontal axis). Genes that are expressed differently (statistically significant difference) and, therefore, with “fold change”, will tend to be found at the upper right and left sides of the chart.

Imagen 2. Heat map – Comparación entre la expresión génica del control y del inmunoestimulante natural.

In the case of the heat map, samples are grouped into control samples (right) and treated samples (left), and difference in expression is shown through a color code.

Results

Analysis revealed that treatment of alveolar macrophages with a natural immunostimulant causes the induction of the following groups of genes:

• Genes involved in production of cytokines, chemiokines and interferons

Induction of a chemokine ligand involved in the formation and function of lymphoid tissue at the mucosal level and the recruitment of helper and regulatory T cells. Expression of MASP2 peptidase related to complement and elimination of pathogens through these biochemical cascades of the innate immune system. Increased expression of regulatory protein ZBTB32 that participates in formation of an expansive nucleus of NK cells that fight viral infections.

• Genes involved in the cell cycle

Overexpression of genes involved in the cell cycle; pro-apoptotic genes and tumor suppressors. Phagocytosis of pathogens by pro-apototic cells achieves a significant reduction in viral or bacterial load. In addition, once lysates, these macrophages are an essential source of antigens that can stimulate T-specific cells.

• Genes related to formation of phagosomes and infiltration of macrophages

These mentioned structures participate in adhesion and degradation of extracellular matrices, and are essential for infiltration or extravasation of macrophages and dendritic cells.

• Genes participating in cellular signaling

Induction of expression of global transcription regulators and GPCRs (G protein-coupled receptors). These regulators and receptors respond to a variety of extracellular stimuli and, ultimately, give rise to specific cellular responses. This type of signaling is essential in macrophages for key processes such as differentiation/polarization, elimination of pathogens or activation/resolution of inflammatory response.

In addition, there are different genes whose expression is repressed in the presence of natural immunostimulatory pronutrients, such as:

• A gene that inhibits (and therefore, lower expression of this gene would have an inducer effect) signaling mediated by the binding of antigens to T and B cells.
• The gene that codes for the FLVCR2 receptor, whose overexpression correlates with a high viral load of PRRSV, and six transporters related to calcium metabolism. Previously published transcriptomic analyzes showed a clear differential expression of multiple genes involved in intracellular calcium homeostasis in pigs infected with highly pathogenic PRRSV strains.

Conclusions

As conclusions of this trial, it can be said that natural immunostimulants are able to:

1. Induce expression of genes related to the physiology of the specific and non-specific immune system.
2. Stimulate activity and response to both microbial and viral pathogens in porcine alveolar macrophages.
3. Increase innate immune response and promote chemotaxis, infiltration of macrophages in the intestine and presentation of antigens.
4. Modify gene expression to improve resistance to infection by highly pathogenic strains of PRRS virus.

All mentioned above is finally reflected at productive level as a better general condition of the animal, a better response to vaccination and a decrease in mortality on the farm.

This natural immune booster has been developed by Biovet S.A. and it is marketed under the name Alquernat Immuplus.