Biliverdin reductase-A attenuated GMH-induced inflammatory response in the spleen by inhibiting toll-like receptor-4 through eNOS/NO pathway

Background: Germinal matrix hemorrhage (GMH) is a common neurologic event with high morbidity and mortality in preterm infants. Spleen has been reported to play a critical role in inflammatory responses by regulating peripheral immune cells which contributes to secondary brain injury. Methods: The current study investigated the mechanistic role of biliverdin reductase-A (BLVRA) in the splenic response and brain damage in neonates following a collagenase GMH model. Neurological outcomes and splenic weights were assessed. Neutrophil production and infiltration were quantitated in the spleen and brain, respectively. Western blot was performed in both splenic and brain tissues to measure protein levels of toll-like receptor 4 and proinflammatory cytokines. Results: BLVRA treatment alleviated GMH-induced developmental delay and attenuated splenic atrophy at 1 and 3 days after GMH. Quantification analysis showed that spleen-stored peripheral immune cells mobilized into circulation and infiltrated in the brain following GMH, which was abrogated by BLVRA administration, resulting in reduced splenic inflammatory response. Furthermore, we showed that regulation of eNOS/NO signaling by BLVRA stimulation blunted toll-like receptor-4 (TLR4) signal. The eNOS-generated NO, in part, translocated BLVRA into the nucleus, where BLVRA inhibited TLR4 expression. Conclusion: We revealed a BLVRA-dependent signaling pathway in modulating the splenic inflammation in response to GMH via the eNOS/NO/TLR4 pathway.