A. C. Bolognese1,2, W. Yang1,2,3, L. W. Hansen2, J. Nicastro2, G. F. Coppa2, P. Wang1,2,3 1Elmezzi Graduate School Of Molecular Medicine,Manhasset, NY, USA 2Hofstra Northwell School Of Medicine,Department Of Surgery,Manhasset, NY, USA 3The Feinstein Institute For Medical Research,Center For Immunology And Inflammation,Manhasset, NY, USA
Introduction: Neonatal sepsis represents a unique therapeutic challenge owing to an immature immune system with a hyperactive innate immune response. Necroptosis, a form of programmed cell death, has been identified as an important mechanism of inflammation-induced cell death. Receptor-interacting protein kinase 1 (RIPK1) plays a key role in mediating this process. We hypothesized that inhibition of necroptosis via RIPK1 would be protective in neonatal sepsis.
Methods: Sepsis was induced in C57BL/6 mouse pups (5-7d) by intraperitoneal injection of adult cecal slurry (CS, 0.175 µg/g body weight, LD100). At 1 h after CS injection, the RIPK1 inhibitor necrostatin 1 stable (Nec-1s, 10 µg/g body weight) or vehicle (5% DMSO in PBS) was administered via retroorbital injection. At 20 h after CS injection, blood and lung tissues were collected for ELISA, PCR, and histologic analysis. For the survival study, pups were monitored for 7 days.
Results: At 20 h after sepsis induction, pups that received the vehicle showed an increase in serum levels of proinflammatory cytokines IL-1β and IL-6 compared to sham (396.30 vs. 1.58 pg/ml and 41.43 vs. 0.32 ng/ml, respectively). With Nec-1s treatment, serum levels of IL-1β and IL-6 were decreased by 81% and 72%, respectively, compared to the vehicle (IL-1β: 74.26 pg/ml, range 0-344; IL-6:11.69 ng/ml, range 0-32). In the lungs, sepsis induction resulted in a 10-fold increase in IL-1β mRNA levels compared to sham, while Nec-1s treatment decreased these levels to just 4-fold (p<0.05). Expression of the neutrophil chemokines KC and MIP-2 was also increased in the lungs in sepsis (1105- and 516-fold compared to sham, respectively, p<0.05), while Nec-1s treatment reduced these levels by 89% and 77%, respectively, compared to vehicle (p<0.05). The above findings correlated with largely retained normal lung architecture observed on histology after Nec-1s treatment compared to vehicle (Figure). In addition, treatment with Nec-1s resulted in a 29% survival rate compared to no survival in the vehicle-treated septic pups (Figure).
Conclusion: Inhibition of RIPK1 by Nec-1s decreases systemic and pulmonary inflammation, reduces lung injury, and increases survival after cecal slurry injection in neonatal mice. Targeting the necroptosis pathway represents a promising therapeutic strategy for neonatal sepsis.
