Early-Life RSV-Infection Alters Local and Systemic Immune Cell Populations in Neonates through TSLP Production

Abstract

Respiratory syncytial virus (RSV), a ubiquitous human pathogen, infects nearly all children by the age of two. It produces severe lower respiratory tract disease, including bronchiolitis, and is characterized by excessive mucus production and immune-mediated lung damage. Severe RSV bronchiolitis in infancy is strongly correlated with development of recurrent wheezing later in childhood, and boys are twice as likely to be affected than girls. These findings suggest persistent immune system alterations well beyond the time of viral clearance, especially in boys. RSV has the ability to evade the type-1 immune response and prime toward a pathologic Th2/Th17 response. Immunopathology associated with severe RSV infection is believed to be the cause of significant airway diseases later in life. However, sex differences associated with these changes and/or if systemic alterations are occurring along with local lung alterations have not been previously defined. Our studies here show that early-life RSV infection leads to differential long-term effects based upon the sex of the neonate, leaving male mice prone to exacerbation upon secondary allergen exposure while overall protecting female mice. During initial viral infection, we observed better viral control in the female mice with correlative expression of interferon-β that was not observed in male mice. Additionally, we observed persistent immune alterations in male mice at 4 weeks post-infection. These alterations include Th2 and Th17-skewing, innate cytokine expression (Tslp and Il33), and infiltration of innate immune cells (DC and ILC2). Upon exposure to allergen, beginning at 4 weeks following early-life RSV-infection, male mice show severe allergic exacerbation while female mice appear to be protected; knockdown of TSLP signaling using TSLPR-/- mice abrogated allergen exacerbation in male mice. We also show that long-term systemic alterations are occurring following early-life RSV-infection. Bone marrow-derived dendritic cells (BMDC) isolated from early-life RSV-infected male mice at 4 weeks post-infection retained expression of maturation markers (Cd80/86, Ox40l). Chemokines/ cytokines associated with the inflammatory response during RSV-infection were also persistently expressed along with Kdm6b and Tslp. Knockdown of TSLP signaling using TSLPR-/- male mice abrogated this activated phenotype and led to enhanced T cell IFN-γ production. ATAC-seq data indicated differences in the chromatin landscape of WT and TSLPR-/- BMDC, showing more open regions of chromatin near anti-viral type-1 genes, Mid1, Spp1, and Cxcl11 in TSLPR-/- BMDC. qPCR showed increased expression of Mid1, Ifnb and Il12a. Further evaluation of BMDC showed KDM6 demethylases were upregulated upon RSV-infection. KDM6 Chemical inhibition (GSK J4) in BMDC decreased production of chemokines and cytokines associated with the inflammatory response during RSV-infection and decreased maturation marker (CD80/86, MHCII) expression, similar to the TSLPR-/- BMDC phenotype. RSV-infected BMDC treated with GSK J4 altered co-activation of T cell cytokine production. Airway sensitization of naïve mice with RSV-infected BMDCs exacerbate a live challenge with RSV infection but was inhibited when BMDCs were treated with GSK J4 prior to sensitization. Finally, in vivo treatment with GSK J4 during RSV-infection reduced immunopathology. Collectively, these studies highlight the importance of proper anti-viral immune responses to limit RSV-driven Th2 immunopathology and future disease by limiting persistent alterations of the immune system. These data validate neonatal sex-differences to RSV and indicate that sex of the neonate should be considered during RSV disease treatment. These studies identify TSLP as a promising therapeutic target, especially in boys hospitalized with severe RSV.