Notch Ligand Delta-like 4 (Dll4) Induces Epigenetic Mechanism in Regulatory T Cell Function During Pulmonary Viral Infection

Respiratory Syncytial Virus (RSV) infects more than 90% of children under two years of age. It is the number one leading cause of hospitalizations for infants and young children. Inpatients suffer from RSV-induced moderate to severe bronchiolitis of the lower respiratory tract. RSV pathology that includes mucus hypersecretion, airway obstruction, bronchiolitis, eosinophilia is driven by pathogenic immune responses. Activated CD4 T cells include T helper cells type 1 (Th1), Th2, Th17, and Treg. Th2 and Th17 responses are pathogenic in that they trigger peribronchioalveolar lymphocytic aggregates, augment mucus production and reduce viral clearance. Regulatory T cells (Treg)-defined as Foxp3+ CD4 T cells-can be further induced by infection to mitigate RSV immunopathology including pathogenic Th2 responses. Understanding how Treg cells are regulated may reveal potential targets to therapeutically alleviate RSV-induced immunopathology. A recent study demonstrated that Notch ligand-Delta-like 4 (Dll4) was up-regulated to attenuate RSV pathology, and intracellular Notch signaling perturbs Treg cell stability in autoimmune disease. But the role of Notch ligand Dll4 in Treg cells in pulmonary infection is still elusive. In this dissertation, we will demonstrate 1) The role of Dll4 in Treg and 2) How Dll4 regulates Treg during RSV infection. The current dissertation demonstrates that Dll4 was up-regulated by RSV infection especially on CD11b+ pulmonary dendritic cells (DC). Using systemic Dll4 neutralization and a Dll4 knockdown DC transfer model, we found that Dll4 enhanced Foxp3 expression and reduced RSV immunopathology. Dll4 promoted peripheral Treg cell differentiation, harnessed Treg cell effector function, and stabilized Treg from Th17-like identities in vitro and in vivo during RSV infection. Using an epigenetic enzyme PCR array, we found that Dll4 and Notch signaling enhanced the expression of a novel histone methyltransferase-SET and MYND domain containing protein 3 (SMYD3) in vitro and in vivo during RSV infection. SMYD3 catalyzed Dll4-facilitated histone 3 lysine 4 tri-methylation (H3K4me3) around the Foxp3 loci in induced Treg cells, and SMYD3 differentially regulated Dll4-stimulated genes discovered by RNA-seq. Consistent with Dll4 activation, SMYD3 sustained Treg cells promoted anti-inflammatory IL-10 expression and prevented IL-17A production from T cells to mitigate pathogenic cytokines and mucus production. Collectively, the studies in this dissertation suggest that Notch and its ligand Dll4 augment immunomodulatory Treg cell programs to ameliorate RSV immunopathology in part through an epigenetic mechanism-SMYD3. The results presented herein lay the foundation for future therapeutics targeting pulmonary and other mucosal inflammatory diseases.PHDMolecular & Cellular PathologyUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttps://deepblue.lib.umich.edu/bitstream/2027.42/144081/1/tingha_1.pd