Chemokine Receptor CCR2-Expressing Inflammatory Monocytes Contribute to the Exacerbated Inflammatory Response Associated with Sepsis

CRITICAL CARE CONCLUSIONS: Deficiency of CIRP results in less renal injury after I/R by attenuating inflammation and oxidative stress. Furthermore, blockade of CIRP shows a protective effect, indicating CIRP as a target in the treatment of renal I/R.

A Novel Cold Shock Protein Induces Renal Injury after Ischemia-Reperfusion Cindy Cen, MD, Weng-Lang Yang, PhD, Hao-Ting Yen, Jeffrey M Nicastro, MD, FACS, Gene F Coppa, MD, FACS, Ping Wang, MD Hofstra North Shore-LIJ School of Medicine, Manhasset, NY; The Feinstein Institute for Medical Research, Manhasset, NY

BRCA1 as a Novel Biomarker of Beta1-Blockade in Sepsis Irada Ibrahim-Zada, MD, PhD, Alejandro Lencinas, PhD, Peter M Rhee, MD, FACS, Irina Maskaykina, Randall S Friese, MD, FACS University of Arizona Tucson, AZ

INTRODUCTION: Renal ischemia-reperfusion (I/R) injury, commonly caused by major surgery and shock, leads to acute kidney injury and is associated with high morbidity and mortality. Cold-inducible RNA-binding protein (CIRP), a cold shock protein, has been recently identified as a damage-associated molecular pattern (DAMP). We hypothesized that CIRP exacerbates severity of injury in renal I/R.

INTRODUCTION: Previously, we established a survival advantage of beta 1-blockade in an endotoxemic animal model. We hypothesized that inflammatory response genes can predict survival benefits of continuous infusion of esmolol in sepsis. Our aim was to identify molecular mechanisms and prognostic biomarker of the survival benefits of esmolol treatment in sepsis.

METHODS: Renal ischemia was induced in 8-week-old male C57BL/6 wild-type (WT) mice and CIRP knockout (KO) mice via bilateral clamping of renal pedicles for 30 minutes, followed by reperfusion for 24 hours and harvest of blood and renal tissue for analysis. Anti-CIRP antibody or nonimmunized IgG was injected intravenously (10 mg/kg body weight) at the time of reperfusion.

METHODS: An Affymetrix microarray study in C57BL/6 mice injected with lipopolysaccharide (LPS 12.5 mg/kg) and treated with either esmolol or saline (control) was performed. Regulatory gene motifs were identified using Transcription Factor database. Three-step gene validation was performed: 1) in silico: 30 human blood samples (sepsis vs controls); 2) in vivo: by qRT-PCR/IHC in blood/tissues (heart, liver, lungs, kidneys, and spleen) at 48 hours post-LPS in the mice model (esmolol vs saline) using antiBRCA1 primary antibody; and 3) in vitro: in SK-HEP1 liver cells at 48 hours post-LPS (esmolol vs control).

RESULTS: After renal I/R, CIRP KO mice demonstrated reductions of blood urea nitrogen (BUN) and creatinine of 53% and 60%, respectively, compared with that in WT mice (Table). Serum interleukin-6 levels were significantly reduced by 70% in KO mice. Levels of oxidatively modified protein markers, nitrotyrosine and 4hydroxynonenal, and inflammatory mediator cyclo-oxygenase-2 were also significantly decreased in the kidneys of the KO mice compared with WT mice after I/R (Table). Renal caspase-3 activity was decreased in KO mice, which corresponded to the reduction of apoptotic cells determined by TUNEL assay. Injection of neutralizing anti-CIRP antibody into WT mice led to an 82% reduction in BUN compared with nonimmunized IgG after renal I/R (p<0.05).

Variable

Wild type, Wild type, ischemia/ control reperfusion

Knockout, control

Knockout, ischemia/ reperfusion

Blood urea nitrogen, mg/dL 19
2 .0

160
17*

20
1.8

75
21*y

0.2
0.04

1.3
0.3*

0.35
0.04

0.5
0.08*,y

160
22*

ND

48
5.4*,y

Creatinine, mg/dL

Serum IL-6, pg/mL 8.9
8.9

1.3
0.1*

0.86
0.06 0.96
0.05y

4-Hydroxynonenal, fold 1.0
0.01

1.2
0.1

0.87
0.06

0.9
0.05y

Cyclo-oxygenase-2 mRNA, fold

1.0
0.09

11
2.5*

1.9
0.4

5.1
1.3*,y

Caspase-3 activity, fold

1.0
0.2

3.3
0.8*

1.0
0.1

0.9
0.3y

Nitrotyrosine, fold

1.0
0.02

RESULTS: A microarray analysis revealed 10 candidate genes: 6 shared a common motif for the BRCA1 gene. An 18- and 24fold decrease in BRCA1 expression was observed at 48 hours in the esmolol group in heart and liver, respectively. BRCA1 expression had a 2,000-fold decrease in esmolol group in SK-HEP1 cells (p<0.05). Immunostaining of BRCA1 protein in lungs and kidneys illustrated a significant reduction in the density of staining at 48 hours in experimental group. CONCLUSIONS: Our results demonstrate that esmolol down-regulates BRCA1, leading to the inhibition of transcription of NF-kB and TNF complexes and promoting cell survival in sepsis. BRCA1 has a novel role as a key transcriptional regulator of inflammatory response associated with survival during beta-blockade in sepsis. Chemokine Receptor CCR2-Expressing Inflammatory Monocytes Contribute to the Exacerbated Inflammatory Response Associated with Sepsis Zoltan H Nemeth, MD, PhD, Jaroslaw W Bilaniuk, MD, FACS, Louis T Di Fazio, MD, FACS, Samantha R Paglinco, Rolando H Rolandelli, MD, FACS Morristown Medical Center, Morristown, NJ

Mean
SEM, n¼5-7/group, 1-way ANOVA. *p<0.05 vs respective control. y p<0.05 vs WT I/R. ND, nondetectable.

ª 2015 by the American College of Surgeons Published by Elsevier Inc.

INTRODUCTION: It is well documented that the chemokine receptor CCR2 induces neutrophil activation and tissue infiltration in systemic inflammation. There is little known, however, about the function of CCR2-expressing monocytes in septic immune

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http://dx.doi.org/10.1016/j.jamcollsurg.2015.07.081 ISSN 1072-7515/15

Vol. 221, No. 4S1, October 2015

responses. Therefore, we set out to elucidate the functions of CCR2-expressing monocytes using cecal ligation and puncture (CLP)-induced sepsis.

Scientific Forum Abstracts

Table. Markers of Inflammation, Organ Dysfunction, and Shock Marker

METHODS: We used CCR2 Depleter (DTR) mice that express a functional diphtheria toxin receptor under the control of CCR2 promoter. In these mice, CCR2-expressing monocytes and macrophages are selectively eliminated with diphtheria toxin treatment. Although the control mice receive the same treatment, their CCR2 positive cells remain intact. The immunologic status of these septic animals was assessed by measuring cytokine levels and bacterial counts from blood, peritoneal lavage fluid, and vital organs 6 and 16 hours after CLP in CCR2 DTR and control (CO) mice. Survival and various markers of tissue damage were assessed as well. RESULTS: The CCR2 DTR mice had higher survival rates than CO animals. This was associated with better bacterial clearance and lower pro-inflammatory cytokine and chemokine levels in the blood and peritoneal lavage. In addition, septic CCR2 DTR animals showed milder lung and kidney inflammation and neutrophil infiltration. Furthermore, mice lacking CCR2 receptorexpressing cells showed less severe apoptosis in the thymus and down-regulated inflammation in the heart, lung, and kidney. CONCLUSIONS: CCR2 DTR mice were protected against septic organ injury, indicating that the chemokine receptor CCR2 is involved in the tissue damaging immune over-activation and neutrophil infiltration of vital organs. Clinical Validation of a Murine Model of Sepsis: Defining Thresholds for Deterioration Anthony J Lewis, MD, Christopher W Seymour, MD, Du Yuan, Matthew R Rosengart, MD, MPH, FACS University of Pittsburgh, Pittsburgh, PA INTRODUCTION: Murine models of sepsis administer, and therefore test, interventions at fixed time intervals after the insult. This ignores the inherent variability in magnitude and temporality of the host response and lacks clinical relevance to human trials using biochemical and physiologic inclusion and exclusion criteria. We previously characterized the variable murine response to cecal ligation and puncture (CLP) using wireless telemetry technology. We now seek to define and validate criteria for acute deterioration to generate a murine model more relevant to the conduct of human trials. METHODS: C57BL/6 mice aged 8 to 12 weeks underwent 1-cm ligation/double 21-gauge puncture CLP, during which time an HD-X11 wireless telemetry monitor (DSI) was implanted. Deterioration criteria for heart rate and core temperature were defined using a population-based analysis. Mice meeting criteria for deterioration were sacrificed, paired with a mouse that had not met deterioration threshold. We analyzed blood gases to characterize shock, ELISA of interleukin (IL)-6, IL-10, tumor necrosis factor (TNF)-a to characterize inflammation, and Cystatin C as a marker of acute kidney injury (AKI).

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IL-6, Q&/mL TNF-a, Q&/mL IL-10, Q&/mL Cystatin C, ng/mL pH Base excess HC03-, mmol/L

Mice meeting criteria

Mice not meeting criteria

p Value

28,646 55.4 1,549 700 6.95 14.4 17.7

12,239 18.9 462 581 7.02 11 20.0

0.04 0.03 0.04 <0.05 0.02 0.02 <0.05

RESULTS: A 10% reduction in both heart rate and temperature defined threshold. Mice reached deterioration thresholds from 339 to 529 minutes post-procedure. Mice meeting deterioration criteria had significantly worse shock, systemic inflammation, and AKI (Table) when compared with mice that had not reached the deterioration threshold. CONCLUSIONS: The defined experimental thresholds identify mice that are acutely deteriorating, and therefore may serve as a cohort more relevant to trials of human disease. These criteria will aid in standardization of planned trials for therapeutic intervention in sepsis. Cold-Inducible RNA-Binding Protein Release During Sepsis Causes Acute Lung Injury Alexandra Cerutti, MD, Weng-Lang Yang, PhD, Jeffrey M Nicastro, MD, FACS, Gene F Coppa, MD, FACS, Ping Wang, MD Hofstra North Shore-LIJ School of Medicine, Manhasset, NY, The Feinstein Institute for Medical Research, Manhasset, NY INTRODUCTION: Cold-inducible RNA-binding protein (CIRP) is a novel inflammatory mediator that stimulates the release of proinflammatory cytokines from macrophages. In sepsis, the integrity of vascular endothelium is damaged, causing acute lung injury. We hypothesized that extracellular CIRP contributes to acute lung injury in sepsis through endothelial cell (EC) activation. METHODS: C57BL/6 mice (20-25g) were subjected to sepsis by cecal ligation and puncture (CLP). Another set of mice received intravenous injection of recombinant murine CIRP (rmCIRP, 5 mg/kg BW) or PBS (Vehicle). At 5 hours after CLP or injection, lungs were harvested for analysis. RESULTS: Protein levels of CIRP in the lungs were increased 2fold at 5 hours after CLP. After rmCIRP injection in healthy mice, lungs extravasated significantly more Evans-blue dye (EBD) compared with Vehicle (Table). In addition, the lungs of rmCIRP-treated mice had significantly higher levels of EC activation markers E-selectin and ICAM-1. Levels of proinflammatory cytokines tumor necrosis factor (TNF)-a and interleukin (IL)-1b were also increased in the rmCIRP-treated mice. On lung histology, rmCIRP-treated mice had significant edema and neutrophil