PIGIMMUNITY: a systems biology approach to boost innate immunity in the pig – PIGIMMUNITY

Production of recombinant flagellin
The recombinant flagellin FliC?174-400 came from S. enterica serovar Typhimurium FliC and was produced with a histidine tag

Primary culture of pig airway epithelial cells (AECs)
Epithelial cells were dissected from the bronchi 1-month-old pigs by enzymatic digestion (0.1% proteases mix and 0.01% desoxyribonuclease I) during 24h at 4°C. The enzymatic digestion was stopped by the addition of a 10% volume of foetal bovine serum and cells were recovered and seeded onto 0,4 µm semi-permeable membranes (Greiner bio-one, Les Ulis, France) treated with collagen type 1. Cells were cultured until confluency and then cultured in an air-liquid-interface (ALI) Differentiation of the epithelium was evaluated by measurement of the transepithelial electrical resistance and determination of the presence of cilia and mucus cells using Anti- ß –Tubuline-Cy3 and Anti-MUC5AC antibodies. Tight junctions were stained with purified monoclonal mouse anti-human ZO-1 antibodies. An anti-cytokeratin antibody was used to determine if the epithelium was contaminated with cells from other origin

Development of an immortalized porcine bronchial epithelial cell line
Differentiated pig airway epithelial cells were shipped to InSCREENex GmbH to develop an immortalized cell line. Differentiation of the cell line into an airway epithelium was evaluated as above described

Ca2+ imaging of AECs
Ca2+ imaging was performed in air-liquid interface (ALI) cultures of airway epithelial cells (AECs). Cells were loaded with fura-2/AM (5 µM; Invitrogen) for 60 min.

Gene expression analysis
To monitor the TLR5 inflammatory response airway epithelial cells (AECs) were stimulated in the apical side with different concentrations of flagellin, or inhibtors depending on the experimental design. RNA was extracted and gene expression evaluated by Biomark HD RT-qPCR or RNA-seq.

Confocal imaging
P65 nuclear translocation was imaged to determine the activation of the NFkB pathway.

Task 1.1: Determination of primary airway epithelial cell (AEC) response to flagellin
The objective of this task was to understand the mechanisms used by AECs to process TLR5 signaling in response to flagellin
Works and results achieved:
We examined the dose-dependent activation of TLR5 signaling. We observed an ultrasensitive NF-?B response (Hill coefficient >1) to flagellin with an EC50 of 13.68 ng/ml and a maximum at 100 ng/ml. No activation was observed for P38. Maximum P65 activation correlated with the maximum expression of pro-inflammatory cytokines. RNA-seq analysis identified a total of 106 differentially expressed genes (DEGs) in AECs stimulated with flagellinMost of the DEGs were genes encoding pro-inflammatory cytokines and genes involved in the NF-?B pathway. Bioinformatic analysis showed that the DEGs were enriched in genes harboring NF-?B motifs, but not P38.
Inhibition of the sarco-/ER Ca2+ ATPase with thapsigargin lead to an increase in expression of CXCL2, CXCL8, CCL20 and NFKBIA genes, compared to the non-pre-treated AECs in response to flagellin. However, when we measured the cytosolic Ca2+ dynamics in AECs loaded with the Ca2+ dye fura-2, we found that flagellin stimulation did not lead to a Ca2+ spike.
Task 1.2. Evaluation of the effect of flagellin pre-stimulation in the response to a second inflammatory stimulus
Pretreatment of AECs with a low flagellin input (1 ng/ml) did not have any effect on the AECs response to a second flagellin or LPS stimulus. However, a high flagellin pre-treatment significantly decreased the cells response to a second flagellin stimulus (no effect on LPS). Evaluation of NFkB nuclear translocation showed that this effect is maintained up to 1 week after flagellin pre-treatment
Cells pre-treated with flagellin had a 7-fold increase in histamine-induced Ca2+ release. This data suggest that flagellin may have an effect on calcium homeostasis, which can affect the intensity of the immune response.

Determination of the role of NF-?B and Ca2+signalling during the flagellin pre-stimulation period.
Each of this signalling pathways will be selectively inhibited during pre-stimulation to determine whether cell response to a second stimulus is restored to values similar to those of non-pre-stimulated controls. Gene expression using the Biomark HD Fluidigm and NFkB translocation will be measured

Determination effect of flagellin on airway epithelial cells (AECs) response to Actinobacillus pleuropneumoniae (App)
Here we aim to better understand the effect of flagellin pre-treatment on App infection. First, AECs will be pre-treated with flagellin and infected with App. The immune response will be evaluated by Biomark HD Fluidigm and confocal imaging. Then, we will evaluate the role of flagellin in the pig lungs in vivo. For that, 8-10-week old pigs will be inoculated with flagellin and changes in the gene expression and immune cell population will be evaluated. We will later determine the in vivo protective effect of flagellin against App. Pigs will be inoculated with a flagellin concentration that produces an increased immune response in the lungs and then infected with App. Clinical assessment, haematological and histological parameters, bacterial charge and pig survival will be evaluated as bona fide clinical parameter to evaluate the relevance of flagellin immune stimulation in App infection

Mathematical modelling of TLR5 signalling
Data collected in tasks 1 and 2 will be used for parameter estimation and model refinement. The model will produce new hypotheses and insights into TLR5 signalling that will inform experimental design in a reciprocal manner. Parameter sensitivity analyses will be used to identify sensitive/robust parameters as well as signalling behaviour. This will produce new insights on how levels and duration of stimuli control the immune response and will enable identification of novel potential molecular targets to improve immunity.

Manuscripts submitted or accepted in peer-reviewed international journals
1. Airway administration of flagellin regulates the inflammatory response to Pseudomonas aeruginosa. Raquel López-Gálvez, Isabelle Fleurot, Pablo Chamero, Sascha Trapp, Michel Olivier, Claire Chevaleyre, Céline Barc, Mickael Riou, Christelle Rossignol, Antoine Guillon, Mustapha Si-Tahar, Tobias May, Pascal Barbry, Andrea Bähr, Nikolai Klymiuk, Jean-Claude Sirard, Ignacio Caballero. American Journal of Respiratory Cell and Molecular Biology. 2021. ACCEPTED
2. TLR5 signalling is hyper-responsive in porcine cystic fibrosis airways epithelium. Isabelle Fleurot, Raquel López-Gálvez, Pascal Barbry, Antoine Guillon, Mustapha Si-Tahar, Andrea Bähr, Nikolai Klymiuk, Jean-Claude Sirard, Ignacio Caballero. Journal of Cystic Fibrosis. 2021 SUBMITTED

Others:
Development of porcine bronchial epithelial cell line. In order to develop some of the experiments of the project, we have developed a bronchial epithelial cell line that retains the features of primary bronchial epithelial cells. This cell line will be available to the scientific community

The pig industry is facing an increasing number of challenges, in the perspective of a sustainable and safe food production. By 2050, the world population is expected to increase to more than 9 billion people and the demand for animal protein will at least double. Increased food production gains have been achieved largely by livestock intensification, with intensive farms becoming more dependable on antibiotics for disease prevention and treatments. This has led to the emergence of new antibiotic-resistance bacteria and, subsequently, increased therapeutic failure. An example is the increased trend in antibiotic resistance of Actinobacillus pleuropneumoniae (A.pp.), the causative agent of porcine pleuropneumonia. New legislation and programs have been set up to reduce antibiotic use and the apparition of antibiotic resistances. However, there is the risk of increase disease incidence, with great economic costs.
Stimulation of innate immunity has been identified as a promising prophylactic and/or therapeutic strategy to combat infectious diseases due to their ability to activate host immune mechanisms with a broad spectrum of protection. In this regard, flagellin, an agonist of Toll-like receptor 5 (TLR5), receptor of the innate immune system, emerges as a possible alternative strategy to increase resistance to both respiratory and intestinal diseases. This protection seem to be linked to a pro-inflammatory activity of flagellin that promotes the recruitment and maturation of immune cells, therefore preparing the mucosal environment for the arriving bacteria. At the same time, flagellin pre-treatment seems to reduce tissue damage, promoting full resolution of the process. Understanding this process will serve to develop better and safer strategies that combine high level of protection to infection whilst avoiding exacerbated inflammation, which will lead to a reduction in the use of antibiotics.
PIGIMMUNITY proposes a systems biology approach to determine the factors that control flagellin-induced modulation of the immune system and resistance to disease. Single cell studies, mathematical modelling and in vivo evaluation of the protective effect of flagellin will be combined to improve our understanding of the mechanisms underlying flagellin protection against pathogens. The project will generate comprehensive knowledge on the mechanisms that regulate pig innate immunity, more specifically TLR5 signalling. It will identify new pathways and molecular targets that enhances pig immunity and resistance to disease. This approach will serve to develop better and safer therapies that combines high level of protection to infection whilst avoiding exacerbated inflammation, improving production yields and pig industry competitiveness. It will also help to decrease the use of antibiotics, and, therefore, its environmental residues, contributing to the reduction of the development of antibiotic resistances.