IL36 and IL37Cytokines, Mediators or Potential Modulators of Airway Infection and Inflammation?

Abstract

Respiratory tract infections caused by viruses or bacteria are among the most common human diseases worldwide. More specifically, respiratory syncytial virus (RSV) is a major cause of acute lower respiratory infection (ALRI), and this frequently results in hospitalisation in young children, particularly among those under the age of 5, and the elderly. Infection with RSV is also correlated with various airway respiratory diseases, such as bronchiolitis and asthma. In vivo testing suggests that levels of IL-36 alpha, IL-36 gamma, and IL-37 protein can be very high in nasopharyngeal aspirate (NPA) during RSV and rhinovirus (RV) infections. This work thus hypothesises that, during RSV and RV infections, the epithelial cells in the airways may express IL-36α, IL-36γ, and their respective receptors in a manner that plays a crucial pro-inflammatory role in RSV and RV infections. In addition, this work hypothesises the possibility of the further expression of IL-36Rα and IL-37 cytokines by airway epithelial cells (AECs) in response to RSV and RV infections. The main aim of this work, as described in this thesis, is thus to investigate whether RSV infection can trigger AECs to express IL-36 and IL-37 cytokines and their receptors.IL-36α, IL-36γ, and IL-37 proteins were measured in NPAs obtained from PCR positive RSV or RV infected infants under the age of 5. The results were classified by disease severity, patient age, and oxygen requirements during hospital admission based on both RSV and RV patient samples. Human adult nasal airway epithelial cells (HNAECs) and A549 and BEAS-2B cultured AEC cells were used to characterise the expression of IL-36α, IL-36γ, and IL-36Rα cytokines and the relevant IL-36 receptors (IL-1RL2 and IL-1Rap) following RSV infection. The expression of IL-37 cytokine and its receptors (IL-18Ra and IL-1R8) was also examined in HNAECs and A549 and BEAS-2B. This work then extended this investigation to determine whether other pro-inflammatory cytokines, including IL-1β, TNF-α, IL-17, IL-22, and IFNγ, might influence the expression of IL-36γ in A549 cells during RSV infections, or whether there is any combined effect of these cytokines on the expression of IL-36γ. LPS was also examined with respect to the expression of IL-36γ cytokines in infected or non-infected cells. The levels of IL-36α protein were significantly higher than those of IL-36γ protein in NPAs from RSV and RV infected infants, though IL-36α, IL-36γ, and IL-37 were all seen in significantly higher proportions in NPAs from RV infected infants than in those from infants with RSV. RSV infection, based on HNAECs and cultured bronchial epithelial cell lines (A549 and BEAS-2B), induces increased expression of IL-36α, IL-36γ, and IL-36Rα mRNA. In both HNAECs and cultured cell lines (A594 and BEAS-2B), IL-36γ was seen to be the most upregulated IL-36 cytokine. It is also worth nothing that the IL-36 protein requires ultracentrifugation to enable its detection within the supernatant of infected HNAECs and cultured cell lines (A594 and BEAS-2B), while xo-stimulation with ATP is required for optimal secretion of IL-36γ from RSV infected cells. There was, nevertheless, a significant expression of IL-37 in both HNAECS and cultured cell lines (A594 and BEAS-2B).With regard to IL-37 receptors, the results showed significantly increased IL-18R1 mRNA expression in both HNAECs and culture cell lines A549 and BEAS2B. In addition, there was a significant increase in the expression of IL-1R8 mRNA in infected A549 and BEAS-2B cells; however, this was not found in HNAECs. The stimulation of A549 cells with either IL-1β or TNF-α played a significant role in the expression of IL-36γ mRNA, though the results showed no effects from these cytokines with respect to the expression of IL-36γ mRNA in RSV infected cells. The combination of IL-1β and TNF-α with other cytokines such as IL-17, IL-22, IFNγ, IFNβ and IL-4 also did not affect the expression of IL-36γ cytokine by A549 cells. Overall, the expression of IL-36 cytokines, and particularly the IL-36γ cytokine, by AECs was found to significantly increase as a response to RSV infection. IL-37 cytokines and their receptors were also expressed by airway epithelial cells in response to RSV infection, with the exception of IL-1R8 in HNAECs. These findings support the idea of a regulatory role for cytokines in terms of limiting the immune response induced by RSV infection. IL-1β and TNF-α cytokines may thus increase the expression of IL-36γ cytokines by A549 cells, although they appear to have no effect when combined with other cytokines.