Expression of Toll-like receptor genes in rat spleen and hypothalamus under conditions of stress exposure and administration of rat defensin RatNP-3

Infectious diseases and immune disorders present a problem for mankind for centuries. To combat various diseases and infections, it is essential to gain understanding of how immune responses are triggered and modulated. In this regard, Toll-like receptors (TLRs) have become one of the main topics of biomedical research, since this family of proteins acts as one of the early determinants of immune response activation. Stress factors are among the most common causes of changes in the immune response which lead to rearranged response of blood leukocytes, altered hormonal levels and cytokine production. It has also been shown that TLR4 gene expression increases under stressful conditions. Endogenous damage-associated molecular factors (DAMPs, or alarmins) are known to be potential ligands of Toll-like receptors. One may assume that, TLR antagonists may be among the endogenous compounds whose concentration increases under unfavorable conditions, Their task is to prevent excessive expression of these receptors. The aim of the study was to determine how preventive administration of the endogenous antimicrobial peptide defensin RatNP-3 affects TLR3 and TLR4 gene expression in hypothalamus and spleen of rats after acute emotional and physical stress induced by forced swimming in cold water for two minutes. Three hours after the stress exposure to the rats, the spleen and hypothalamus were removed from the animals, RNA was isolated and a reverse transcription reaction was performed to obtain cDNA. Expression of TLR3 and TLR4 genes was assessed, with a reference to the housekeeping gene GAPDH (glyceraldehyde-3-phosphate dehydrogenase) expression, being determined by real-time PCR. Three hours after stress exposure, the expression of TLR3 and TLR4 genes was found to increase in hypothalamus and spleen. However, the pre-exposure intraperitoneal administration of defensin reduced the expression level of these genes only in splenocytes, but did not affect their expression in hypothalamus. These results indicate to the stress-induced systemic activation of Toll-like receptors in hypothalamic structures of the brain and spleen cells. Since the administration of endogenous defensins did not affect expression of TLR genes in hypothalamus, one may suggest that the regulatory action of neutrophilic defensins is not mediated via central regulatory mechanisms.