Monocyte Dll4 Contributes to Systemic Inflammation in People Living With HIV.

Abstract

Background: In people living with HIV (PLWH), persistent systemic inflammation drives comorbidities, including cardiovascular and cerebrovascular diseases. Monocyte-mediated inflammation, rather than T-cell activation, is a major contributor to chronic inflammation. However, the underlying mechanisms remain elusive. Here, we investigated the role of Dll4 (delta-like 4)-Notch signaling in monocyte-mediated inflammation.

Methods: In clinical specimens, we quantified monocyte mDll4 (membrane-bound Dll4) expression and plasma levels of exDll4 (extracellular Dll4). We also analyzed the association between circulating exDll4 and monocyte subsets in PLWH. In vitro, human primary monocytes, endothelial cells, and T cells were stimulated with lipopolysaccharide or TNF-α (tumor necrosis factor-α) to assess DLL4 mRNA and Dll4 protein expression and exDll4 secretion. RNA sequencing was performed after DLL4 silencing to determine downstream signaling pathways. In addition, molecular and functional studies examined the effects of Dll4-Notch1 signaling on interferon-related gene expression, monocyte adhesion, and transmigration.

Results: Both male and female PLWH exhibited significantly increased mDll4 expression, activation of Dll4-Notch1 signaling, and increased Notch-targeted inflammatory factors. Although monocyte mDll4 did not differ by sex in PLWH, plasma exDll4 was significantly elevated in males, but not females, compared with HIV-uninfected individuals. In male PLWH, plasma exDll4 levels paralleled monocyte mDll4 expression. Circulating exDll4 was positively associated with proinflammatory monocytes and negatively associated with classical monocytes in male PLWH. Mechanistically, lipopolysaccharide or TNF-α robustly increased DLL4 mRNA and protein expression in human primary monocytes, along with exDll4 secretion from monocytes, but not from endothelial cells or T cells. RNA sequencing after DLL4 silencing showed that Dll4-Notch signaling suppresses a broad anti-pathogen program regulated by interferon and NF-κB, as well as cell-mobility-related genes. At the molecular level, Dll4 activated Notch1, which upregulated Hes1, forming a heterotrimer with STATs (signal transducers and activators of transcription), and repressed interferon pathway-related gene expression. Functionally, loss of Dll4 significantly reduced monocyte adhesion and transmigration.

Conclusions: Proinflammatory cues upregulate Dll4 in monocytes and activate Dll4-Notch signaling, thereby suppressing antiviral responses and enhancing monocyte transmigration. Together, these effects sustain systemic inflammation in PLWH of both sexes. Our data identify monocyte-derived Dll4 as a promising biomarker and therapeutic target for systemic inflammation.