Mild Traumatic Brain Injury Improves Murine Neutrophil Phagocytosis

CRITICAL CARE I: BASIC BIOLOGY F4/80 and CD11b (for macrophages), CD69 (early activation), and CD86 (M1 phenotype) and CD206 (M2 phenotype). ANOVA was used for analysis with significance of p<0.05.

Mild Traumatic Brain Injury Improves Murine Neutrophil Phagocytosis Elizabeth G King, MD, Evan L Chiswick, PhD, Terry Hsieh, BS, Daniel Remick, MD Boston University School of Medicine, Boston, MA

RESULTS: CLP did not affect WT peritoneal macrophage phenotype (CD11b -95.9%
1.7 vs 92.0
4.0) or M1 (CD86- 20.8%
4.8 vs 18.0
3.1) vs M2 phenotype (CD206- 38.5%
1.5 vs 38.2
8.8, p>0.05 for all groups). After CLP, iNKT-/- peritoneal macrophages displayed increased activation as compared to WT (CD69-61.2%
7.4 vs 41.7
4.1; p¼0.04). Peritoneal macrophages from iNKT-/- compared to WT displayed baseline elevated M1 characteristics (CD86- 39.1%
1.43 vs 20.8
4.8, p¼0.004), with no difference in M2 phenotype (CD206 -40.0
3.1 vs 38.5
2.6; p>0.05). After CLP this difference in M1/M2 phenotype was no longer evident as compared to WT (CD8622.35%
4.0 vs 18.0
3.1, p<0.05; CD206-51.7%
6.6 vs 38.2
8.8, p<0.05).

INTRODUCTION: Previous work demonstrated improved survival in mice subjected to mild traumatic brain injury (TBI) given Pseudomonas aeruginosa (PSA) pneumonia compared to non-TBI. Additionally, 4 hours after administration of PSA, bronchoalveolar lavage (BAL) fluid from TBI mice has significantly more neutrophils and fewer bacterial colony-forming units. This study examined the hypothesis that TBI improves neutrophil phagocytosis. METHODS: Adult female ICR mice underwent TBI induced by a weight drop model or sham TBI. After 48 hours, heat-killed Bodipy-labeled PSA (B-PSA) was administered intratracheally. Mice were sacrificed at 4 hours post-pneumonia for blood and BAL fluid. Flow cytometric analysis of BAL fluid and whole blood incubated with opsonized B-PSA was performed.

CONCLUSIONS: CLP does not change WT peritoneal macrophage M1 vs M2 phenotypes. However, iNKT deficient animals display increased peritoneal macrophage expression of M1 markers at baseline with normalization to WT M1/M2 levels after CLP. iNKT cells play an important role in modulating macrophage phenotype at baseline and affect macrophage activation after sepsis.

RESULTS: Peripheral blood of TBI mice demonstrated significantly increased neutrophil phagocytosis of B-PSA at both high (51
3% vs 61
3%, p¼0.03) and low (34
3% vs 53
9%, p¼0.04) bacterial concentrations. BAL fluid from TBI mice also had an increased percentage of B-PSA+ neutrophils, although this difference was not statistically significant. However, the subpopulation of BAL neutrophils with higher forward light scattering, correlating to neutrophil activation, had an increased percentage of phagocytosis (39%
2 vs 45%
2, p¼0.04).

The Anti-inflammatory Effect of 5-Aminoimidazole-4Carboxyamide Ribonucleoside (AICAR) in Sepsis Is Mediated by AMP-Activated Protein Kinase (AMPK)a2 in the Hypothalamus Nikhil Mulchandani, MD, Weng-Lang Yang, PhD, Fangming Zhang, MD, PhD, Jeffrey M Nicastro, MD, FACS, Gene F Coppa, MD, FACS, Ping Wang, MD Hofstra North Shore-LIJ School of Medicine, Manhasset, NY, Feinstein Institute for Medical Research, Manhasset, NY

CONCLUSIONS: The peripheral blood neutrophils and the more active subpopulation of neutrophils in the BAL fluid of TBI mice exhibit increased phagocytosis. These findings suggest that mild TBI may prime neutrophils to respond to a subsequent bacterial challenge. This is likely an important mechanism in the previously reported improved survival of TBI mice after PSA pneumonia.

INTRODUCTION: Acute lung injury is a common complication in patients with severe sepsis, leading to high mortality. AICAR is an activator of an energy-regulating enzyme, AMPK, and has been shown to attenuate inflammation. However, the specific isoform of AMPK responsible for AICAR’s action remains unclear. We hypothesized that AMPKa2, primarily located in the hypothalamus, is required to mediate AICAR’s activity in sepsis.

Peritoneal Macrophage Activation and Phenotype Changes After Polymicrobial Sepsis Is Dependent upon Invariant Natural Killer T (iNKT) Cells John S Young, MD, Chun-Shiang Chung, PhD, Whitney A Young, MD, William G Cioffi Jr, MD, FACS, Alfred Ayala, PhD, Daithi S Heffernan, MD, FRCSI Brown University, Providence, RI

METHODS: Adult male wild-type (WT) and AMPKa2-knockout (KO) C57BL/6 mice underwent intracerebroventricular (ICV) injection of 20 ng AICAR (n¼4-6/group) or vehicle (2 mL normal saline, n¼4-6/group), followed by cecal ligation and puncture (CLP) 30 min post-ICV. Blood and tissues were collected 20 h after CLP. Protein and mRNA expression were evaluated using ELISA and quality polymerase chain reaction, respectively.

INTRODUCTION: After sepsis, iNKT cells modulate peritoneal macrophage function, with peritoneal macrophages from iNKT-/- mice displaying altered bacterial clearance. Little, however, is known about the classically (M1) vs alternatively activated (M2) phenotypic characteristics of these iNKT-/- peritoneal macrophages. We hypothesized that peritoneal macrophage phenotype changes after polymicrobial sepsis would be dependent upon iNKT cells.

RESULTS: Treatment with AICAR by ICV injection significantly reduced AST levels elevated in septic WT mice (156.5
15.3 vs 230.9
19.2 U/L, p <0.05). However, AICAR’s effect on AST reduction was diminished in septic AMPKa2-KO mice (258.1
96.8 vs 334.9
24.0 U/L, p¼0.55). AICAR-treated WT mice had a significant decrease in levels of serum and lung cytokines,

METHODS: WT or iNKT -/- mice were subjected to cecal ligation and puncture (CLP); 24h later peritoneal lavage was harvested. Flow cytometry for cell surface expression was undertaken with

ª 2014 by the American College of Surgeons Published by Elsevier Inc.

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http://dx.doi.org/10.1016/j.jamcollsurg.2014.07.082 ISSN 1072-7515/14