OC 46.3 - Why do peptidyl arginine deiminase (PAD) 4 deficient mice have improved survival following cecal ligation & puncture (CLP)? Implications for neutrophil extracellular traps (NETs) in immune homeostasis

Monday, June 24, 2024

15:15 – 15:30 ICT

Room: 111 A-C

Presenting Author(s)

Veronica Bochenek, BA

Research Technician
Children's Hospital of Philadelphia
Philadelphia, Pennsylvania, United States

Co-Author(s)

Anh Ngo, PhD

Research Post Doc Fellow
Children's Hospital of Philadelphia
Philadelphia, Pennsylvania, United States
SM

Samir Maarouf

Student
University of Pennsylvania
Philadelphia, Pennsylvania, United States
EM

Ethan Mack

Clinical Pathology Resident
University of Pennsylvania
Philadelphia, Pennsylvania, United States
SY

Santosh Yadav

Research Associate
Children's Hospital of Philadelphia
Philadelphia, Pennsylvania, United States
AS

Abigail Skidmore

Student
Lafayette College
Philadelphia, Pennsylvania, United States
NL

Nate Levine

Researcher
University of Pennsylvania
Philadelphia, Pennsylvania, United States
AK

Anna Kowalska

Researcher
Children's Hospital of Philadelphia
Philadelphia, Pennsylvania, United States
Mortimer Poncz, MD

Professor of Pediatrics
Children's Hospital of Philadelphia
Philadelphia, Pennsylvania, United States
Kandace Gollomp

Attending Physician
Children's Hospital of Philadelphia
Philadelphia, Pennsylvania, United States

Background: NETs are webs of decondensed chromatin that capture bacteria. However, mice deficient in PAD4 (Padi4-/-), a crucial mediator of NET release (NETosis), have been found to have improved survival in the CLP model of polymicrobial sepsis, leading some to conclude that NETs do more harm than good. As NETosis is a highly-conserved immune function in multicellular organisms, we alternatively propose that improved outcomes are due to alterations in the inflammatory milieu and the gastrointestinal (GI) microbiome in Padi4-/- mice.

Aims: To characterize the effect of PAD4 deficiency on murine immune function.

Methods: Wildtype (WT) and Padi4-/- mice received peritoneal injections of cecal slurry (CS) from WT or Padi4-/- donors. Plasma, tissue samples, and blood counts were obtained at baseline and 24h post-injection, and a sub-cohort was monitored for survival. Histology and flow cytometry were used to quantify neutrophil tissue infiltration. Proteomic analysis was performed on plasma samples using the Olink platform. 16S bacterial sequencing was used to characterize GI microbiota.

Results: At baseline, Padi4-/- mice had elevated circulating neutrophils, higher neutrophil CXCR4 levels, increased tissue neutrophil infiltration most prominently in the jejunal villi, and higher plasma levels of proinflammatory cytokines including TNFα, IL5, and IL6, and lower IL23r. 16S sequencing showed differences in GI microbiome composition in Padi4-/- animals (Table 1). Both WT and Padi4-/- mice that received CS from WT had more severe sepsis and higher mortality compared to WT and Padi4-/- animals that received CS from Padi4-/- donors (Fig.1).

Conclusion(s): PAD4 expression influences baseline inflammation, neutrophil mobilization, and GI microbiome composition. Results of CS experiments suggest that published reports of improved survival of Padi4-/- mice following CLP were related to altered inflammatory signaling and reduced pathogenicity of the autologous GI inoculum. The basis for differences in inflammation and GI microbiota in Padi4-/- mice merits further study.