Introduction: Newborn neonatal infants that require intensive care often exhibit unstable patterns of breathing and require oxygen support for several weeks. These infants are at risk of late onset, nosocomial, infection. Toll-like receptors (TLR) and their down downstream signalling pathways are important mediators of the innate immune system and upon activation by, damage associated molecular patterns or pathogen associated molecular patterns, promote inflammation. TLRs have a critical role in facilitating immune-adrenal crosstalk during inflammation and may modulate adrenal physiology under common clinical conditions of oxygen dysregulation and infection.

Objective: To quantify TLR expression in the adrenal gland of male and female rat pups which have been exposed to chronic oxygen dysregulation and acute gram-positive protein challenge.

Methods: Ethical approval was obtained locally by University College Cork and project authorisation was issued by Health Products Regulatory Authority Ireland AE19130/P100,/P163 and experiments performed under authorisation in accordance with Irish and European directive 2010/63/EU. Sprague Dawley rat pups and their dam were exposed either to room air or intermittent hypoxia (FiO₂ 8%)-hyperoxia (FiO₂ 30%)—normoxia (FiO₂ 21%)- (IHH) 24- hour protocol from postnatal day (PND) 3-12 and acutely challenged the animals with either sterile saline i.p. or mixture of 3mg/kg of lipoteichoic acid and 5mg/kg peptidoglycan i.p. on PND 13. Adrenal glands were dissected following rapid decapitation and either snap frozen in dry ice and stored at -80C and later used for TLR mRNA analysis of TLR-1, -2, -4, -9, MYD88, NOD2 and NFκB or cryoprotected and frozen in cooled isopentane and later used for histological assessment.

Results: TLR-4, TLR-9, MYD88, NOD2 and NFκB mRNA expression were increased in IHH exposed animals compared to Sham animals (P≤0.02; n=8 per group). IHH exposure interacted with LTA&PGN administration to reveal a higher expression of TLR-2 mRNA (gas x drug, P=0.01) and NFκB mRNA (gas x drug, P=0.05), (n=8 per group). GAPDH mRNA was utilised as the housekeeper for the mRNA analysis performed on the adrenal gland.

Discussion and Conclusions: We have previously reported that in PND13 rat pups LTA&PGN administration increased plasma cytokine and cortisol concentrations. Interestingly, Ilβ, IL-10, Leptin and Gro/KC were increased many fold when animals were pre-exposed to oxygen dysregulation. In this further analysis we sought to investigate the expression of TLR expression and some of its downstream signalling molecules. We found that TLR-2 was robustly increased with gram- positive bacterial proteins only when the animals were pre-exposed to oxygen dysregulation. This indicates that in early life pre-exposure to oxygen dysregulation may act to prime the body to generate a heightened inflammatory response upon infection.