072 - Preterm murine neonatal neutrophils are distinct and more pro-inflammatory than adult murine neutrophils

Publication Number: 72.6241

Devashis Mukherjee, Case Western Reserve University School of Medicine, Cleveland, OH, United States; Asha Thomas, Case Western Reserve University, Cleveland, OH, United States; Sarah Cioffi, UH Rainbow Babies & Children's Hospital, Cleveland, OH, United States; Ernest R. Chan, Case Western Reserve University School of Medicine, Cleveland, OH, United States; Lalitha Nayak, Case Western Reserve University School of Medicine, Cleveland, OH, United States

Background: Preterm neonates are classically described as immune suppressed. Most research involves the adaptive immune system or monocytes/macrophages. Neutrophils are the most abundant immune cells, and neutropenia is a major risk factor for mortality in preterm sepsis. Neutrophils respond to endotoxin (lipopolysaccharide, LPS) by assembly of the NLRP3 inflammasome, leading to cleavage of gasdermin D (FL-GSDMD) to N-terminal (NT)-GSDMD, and pro-IL-1B to mature IL-1B. IL-1B is released into the circulation and is a key pro-inflammatory cytokine that is essential to the immune response.

Objective: To investigate murine neutrophil-specific GSDMD cleavage, IL-1B release, and transcriptomic changes from early neonatal life to adulthood.

Design/Methods: Bone marrow (BM) was dissected from postnatal day (P) – 4, P12, and 8-week-old mice and used for immunomagnetic isolation of neutrophils (BMNs). BMNs were plated in RPMI media, treated with E. coli LPS for 3 h, followed by nigericin for 30 mins to prime and activate the NLRP3 inflammasome. Activation of the NLRP3 inflammasome was measured by cleavage of FL-GSDMD to NT-GSDMD, and by IL-1B release into the supernatant. Supernatant was precipitated for protein using TCA method. Cell lysates and supernatants were resolved using gel electrophoresis, probed for GSDMD, IL-1B, and B-actin. Supernatant was used for IL-1B ELISA. Total RNA was extracted from BMNs for next-generation sequencing (Illumina). Differentially expressed genes (DEGs) were identified using a significanceof q-value < 0.05 and subjected to gene enrichment analysis.

Results: GSDMD cleavage is developmentally regulated with decreasing cleavage (NT-GSDMD) with increasing postnatal age (Fig.1A). IL-1B release into supernatant also decreased with postnatal age, both by immunoblotting (Fig.1A) and ELISA (Fig.1B). RNA-seq showed distinct signatures at 3 ages, with the adult transcriptome being uniquely different (Fig.2A, B). Gene ontology meta-analysis followed by selection for adult vs. P12 AND P4 DEGs showed that the top 5 processes involved immune signaling and response to external antigens (Fig.2C).

Conclusion(s): Our novel data show that release of the key pro-inflammatory cytokine, IL1B, in response to LPS increases in neutrophils with decreasing postnatal age, likely due to increased neutrophil-NLRP3 assembly, GSDMD cleavage, and the ability to release IL-1B extracellularly at an earlier age. Our data challenge the existing belief that the preterm immune system is immune-suppressed, and future mechanistic studies using our RNA-seq results will significantly advance the field of preterm neonatal sepsis.

Figure 1
Figure 1. (A) shows Western blots of cell lysate and supernatants from bone marrow neutrophils isolated from P5, P12, and adult (8-week) C57Bl/6 mice and treated with 500ng/mL of E. coli LPS for 3 hours to prime the NLRP3 inflammasome, and then 30 mins of nigericin 20uM to activate the NLRP3 assembly. Activation of the NLRP3 inflammasome leads to cleavage of full-length gasdermin D (FL-GSDMD) to N-terminal GSDMD (NT-GSDMD), which is increased at an earlier postnatal age. NT-GSDMD leads to pore formation and release of cleaved IL-1β into the supernatant, which is also increased at an earlier postnatal age as depicted by western blots of protein precipitated from the supernatant. (B) shows ELISA results from the supernatant, demonstrating increased IL-1β released by neutrophils at an earlier postnatal age. N=2 for each and hence statistical analysis was not performed.

Figure 2
Figure 2. Total RNA was isolated from bone marrow neutrophils from P5, P12, and adult (8-week-old) C57Bl/6 mice (n=7 for P5, P12, and n=6 for adult), used to prepare libraries and run on the NovaSeqX Illumina platform after RNA quantification. Differentially expressed genes were identified using a significance cutoff of q-value < 0.05, subjected to gene enrichment analysis, and cross-referenced using the Gene Ontology database. Additional pathway analysis was performed using iPathwayGuide (Advaita Bio). (A) shows a heatmap of the top 5000 DEGs across all ages, (B) shows the principal component analysis depicting the distinct neutrophil transcriptome at each age, and (C) shows the top 5 biological processes affected in the transcriptomic meta-analysis when selecting for DEGs significant between adult vs. P12 and adult vs. P4 ages.