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Deficiency of Receptor-Interacting Protein Kinase 3 Reduces Lung Injury and Inflammation in Neonatal Sepsis
PEDIATRIC SURGERY die by necrosis and apoptosis. Receptor-interacting protein kinase 3 (RIPK3) is a key protein in regulating a newly identified death mechanism, programmed necrosis or necroptosis. We hypothesized that deficiency of RIPK3 would be beneficial in neonatal sepsis.
Recently Discovered Oligosaccharide Inhibits Toll-Like Receptor 4 and Prevents Intestinal Stem Cell Apoptosis in Experimental Necrotizing Enterocolitis Laura Y Martin, MD, Chhinder P Sodhi, PhD, Hongpeng Jia, MD, Yukihiro Yamaguchi, PhD, Misty Good, MD, Peter Wipf, Jungeun Sung, Peng Lu, PhD, Diego F Nino, MD, PhD, David J Hackam, MD, FACS Johns Hopkins Hospital, Baltimore, MD
METHODS: Sepsis was induced in neonatal (age 5 to 7 days) C57BL6 (WT) and RIPK3 knockout (KO) mice by intraperitoneal injection of adult cecal slurry (0.9 mg/g, lethal dose 100 in 20 hours). Ten hours after cecal slurry injection, mice were sacrificed and tissues were collected for various analyses. Sham mice received intraperitoneal 0.9% saline injections.
INTRODUCTION: Necrotizing enterocolitis (NEC) is a severe inflammatory disease of the newborn resulting in toll-like receptor 4 (TLR4) activation and apoptosis of intestinal stem cells (ISCs). Breast milk is protective against NEC through unknown mechanisms. We recently identified a novel human milk oligosaccharide (HMO) analogue, C34, which inhibits TLR4 and prevents NEC. We hypothesized that C34 protects againsts NEC by blocking TLR4-mediated activation of p53 upregulated modulator of apoptosis (PUMA) and preventing stem cell apoptosis.
RESULTS: Serum cytokines interleukin (IL)-6 and IL-1b increased by 483- and 42-fold, respectively, 10 hours after sepsis in WT mice, compared with sham (Table). These cytokines were reduced by 66.7% and 48.8%, respectively, in septic RIPK3-KO mice. Lung IL-1b protein levels were also reduced by 44.3% in septic RIPK3KO mice, compared with septic WT mice. The lung mRNA levels of neutrophil chemoattractant MIP-2 in WT mice was increased 83-fold after sepsis, but a significant reduction was observed in RIPK3-KO mice. Furthermore, lung myeloperoxidase (MPO) activity was reduced in septic RIPK3-KO mice by 26.4%. Resultant lung tissue injury was attenuated in septic RIPK3-KO mice, as judged by maintained alveolar space, thin alveolar septae, and reduced cellular infiltration on hematoxylin and eosin histologic staining.
METHODS: Experimental NEC was induced and outcomes were evaluated in wild-type and PUMA knock-out mice with and without the addition of C34. Additionally, the effects of C34 on lipopolysaccharide (LPS)-induced apoptosis were evaluated in NF-kB luciferase reporter mice. Finally, enteroids cultured from ISCs of neonatal wild type (C57Bl/6) and Lgr5 green fluorescent protein (Lgr5+GFP+) mice were treated with LPS C34. Stem cell proliferation, apoptosis, differentiation, and NF-kB and PUMA expression were evaluated using TUNEL staining, immunohistochemistry, and reverse transcription polymerase chain reaction (RT-PCR). Morphometric analysis was performed using live cell imaging.
Table. Variable WT sham WT sepsis KO sham KO sepsis Serum IL-6, ng/mL 0.09 0.01 46.0 9.1* 0.10 0.01 15.3 1.0*y Serum IL-1b, pg/mL 8.9 4.8 384.5 33.5* Nondetectable 196.9 59.2*y Lung IL-1b, pg/mg 7.0 0.7 50.3 7.2* 5.5 0.3 28.0 2.3*y Lung MIP-2 mRNA, 1.0 0.3 83.8 49.4* 0.25 0.03 25.2 2.7y fold Lung MPO activity, 0.93 0.09 1.4 0.2* 0.66 0.09 1.03 0.06y U/mg
RESULTS: In wild-type mice, experimental NEC disrupted mucosal architecture, increased apoptosis, and decreased stem cells. PUMA knock-out mice and wild-type mice co-treated with C34 did not demonstrate these effects. C34 blocked LPS-induced NFkB activation in reporter mice, as measured by luciferase expression. LPS-treated enteroids demonstrated increased apoptosis, fewer stem cells, and poor morphology, which correlated with increased PUMA and NF-kB expression by RT-PCR. Treatment with C34 decreased apoptosis, preserved morphology and stem cell numbers, and decreased PUMA and NF-kB expression.
Mean SEM, n¼4-8/group; 1-way ANOVA. *p<0.05 vs WT sham. y p<0.05 vs WT sepsis.
CONCLUSIONS: RIPK3 deficiency is protective against systemic and lung inflammation, lung neutrophil infiltration, and lung injury in murine neonatal sepsis. Inhibition of RIP-mediated necroptosis may have therapeutic potential in neonatal sepsis.
CONCLUSIONS: TLR4 inhibitor C34 prevents NEC-associated ISC apoptosis in a PUMA-dependent manner.
Early vs Delayed Gastroschisis Closure: Outcomes Across US Children’s Hospitals Dani O Gonzalez, MD*, Jennifer N Cooper, PhD, Shawn D ST Peter, MD, FACS, Peter C Minneci, MD, FACS, Katherine J Deans, MD, FACS Nationwide Children’s Hospital Columbus, OH, Icahn School of Medicine at Mount Sinai, New York, NY, Children’s Mercy Hospital and Clinics, Kansas City, MO
Deficiency of Receptor-Interacting Protein Kinase 3 Reduces Lung Injury and Inflammation in Neonatal Sepsis Laura W Hansen, MD, Weng-Lang Yang, PhD, Alexandra C Bolognese, MD, Jose M Prince, MD, FACS, Jeffrey M Nicastro, MD, FACS, FCCM, Gene F Coppa, MD, FACS, Ping Wang, MD, FACS Hofstra Northwell School of Medicine, The Feinstein Institute for Medical Research, Manhasset, NY
INTRODUCTION: Different management strategies exist for gastroschisis, including timing of closure. In all patients with gastroschisis, it is not known whether there are national differences in length of stay (LOS) and time to enteral autonomy associated with early vs delayed closure.
INTRODUCTION: Neonates are highly susceptible to sepsis, with increased mortality rates. During sepsis, massive numbers of cells
S86
http://dx.doi.org/10.1016/j.jamcollsurg.2016.06.168 ISSN 1072-7515/16
Recently Discovered Oligosaccharide Inhibits Toll-Like Receptor 4 and Prevents Intestinal Stem Cell Apoptosis in Experimental Necrotizing Enterocolitis Laura Y Martin, MD, Chhinder P Sodhi, PhD, Hongpeng Jia, MD, Yukihiro Yamaguchi, PhD, Misty Good, MD, Peter Wipf, Jungeun Sung, Peng Lu, PhD, Diego F Nino, MD, PhD, David J Hackam, MD, FACS Johns Hopkins Hospital, Baltimore, MD
METHODS: Sepsis was induced in neonatal (age 5 to 7 days) C57BL6 (WT) and RIPK3 knockout (KO) mice by intraperitoneal injection of adult cecal slurry (0.9 mg/g, lethal dose 100 in 20 hours). Ten hours after cecal slurry injection, mice were sacrificed and tissues were collected for various analyses. Sham mice received intraperitoneal 0.9% saline injections.
INTRODUCTION: Necrotizing enterocolitis (NEC) is a severe inflammatory disease of the newborn resulting in toll-like receptor 4 (TLR4) activation and apoptosis of intestinal stem cells (ISCs). Breast milk is protective against NEC through unknown mechanisms. We recently identified a novel human milk oligosaccharide (HMO) analogue, C34, which inhibits TLR4 and prevents NEC. We hypothesized that C34 protects againsts NEC by blocking TLR4-mediated activation of p53 upregulated modulator of apoptosis (PUMA) and preventing stem cell apoptosis.
RESULTS: Serum cytokines interleukin (IL)-6 and IL-1b increased by 483- and 42-fold, respectively, 10 hours after sepsis in WT mice, compared with sham (Table). These cytokines were reduced by 66.7% and 48.8%, respectively, in septic RIPK3-KO mice. Lung IL-1b protein levels were also reduced by 44.3% in septic RIPK3KO mice, compared with septic WT mice. The lung mRNA levels of neutrophil chemoattractant MIP-2 in WT mice was increased 83-fold after sepsis, but a significant reduction was observed in RIPK3-KO mice. Furthermore, lung myeloperoxidase (MPO) activity was reduced in septic RIPK3-KO mice by 26.4%. Resultant lung tissue injury was attenuated in septic RIPK3-KO mice, as judged by maintained alveolar space, thin alveolar septae, and reduced cellular infiltration on hematoxylin and eosin histologic staining.
METHODS: Experimental NEC was induced and outcomes were evaluated in wild-type and PUMA knock-out mice with and without the addition of C34. Additionally, the effects of C34 on lipopolysaccharide (LPS)-induced apoptosis were evaluated in NF-kB luciferase reporter mice. Finally, enteroids cultured from ISCs of neonatal wild type (C57Bl/6) and Lgr5 green fluorescent protein (Lgr5+GFP+) mice were treated with LPS C34. Stem cell proliferation, apoptosis, differentiation, and NF-kB and PUMA expression were evaluated using TUNEL staining, immunohistochemistry, and reverse transcription polymerase chain reaction (RT-PCR). Morphometric analysis was performed using live cell imaging.
Table. Variable WT sham WT sepsis KO sham KO sepsis Serum IL-6, ng/mL 0.09 0.01 46.0 9.1* 0.10 0.01 15.3 1.0*y Serum IL-1b, pg/mL 8.9 4.8 384.5 33.5* Nondetectable 196.9 59.2*y Lung IL-1b, pg/mg 7.0 0.7 50.3 7.2* 5.5 0.3 28.0 2.3*y Lung MIP-2 mRNA, 1.0 0.3 83.8 49.4* 0.25 0.03 25.2 2.7y fold Lung MPO activity, 0.93 0.09 1.4 0.2* 0.66 0.09 1.03 0.06y U/mg
RESULTS: In wild-type mice, experimental NEC disrupted mucosal architecture, increased apoptosis, and decreased stem cells. PUMA knock-out mice and wild-type mice co-treated with C34 did not demonstrate these effects. C34 blocked LPS-induced NFkB activation in reporter mice, as measured by luciferase expression. LPS-treated enteroids demonstrated increased apoptosis, fewer stem cells, and poor morphology, which correlated with increased PUMA and NF-kB expression by RT-PCR. Treatment with C34 decreased apoptosis, preserved morphology and stem cell numbers, and decreased PUMA and NF-kB expression.
Mean SEM, n¼4-8/group; 1-way ANOVA. *p<0.05 vs WT sham. y p<0.05 vs WT sepsis.
CONCLUSIONS: RIPK3 deficiency is protective against systemic and lung inflammation, lung neutrophil infiltration, and lung injury in murine neonatal sepsis. Inhibition of RIP-mediated necroptosis may have therapeutic potential in neonatal sepsis.
CONCLUSIONS: TLR4 inhibitor C34 prevents NEC-associated ISC apoptosis in a PUMA-dependent manner.
Early vs Delayed Gastroschisis Closure: Outcomes Across US Children’s Hospitals Dani O Gonzalez, MD*, Jennifer N Cooper, PhD, Shawn D ST Peter, MD, FACS, Peter C Minneci, MD, FACS, Katherine J Deans, MD, FACS Nationwide Children’s Hospital Columbus, OH, Icahn School of Medicine at Mount Sinai, New York, NY, Children’s Mercy Hospital and Clinics, Kansas City, MO
Deficiency of Receptor-Interacting Protein Kinase 3 Reduces Lung Injury and Inflammation in Neonatal Sepsis Laura W Hansen, MD, Weng-Lang Yang, PhD, Alexandra C Bolognese, MD, Jose M Prince, MD, FACS, Jeffrey M Nicastro, MD, FACS, FCCM, Gene F Coppa, MD, FACS, Ping Wang, MD, FACS Hofstra Northwell School of Medicine, The Feinstein Institute for Medical Research, Manhasset, NY
INTRODUCTION: Different management strategies exist for gastroschisis, including timing of closure. In all patients with gastroschisis, it is not known whether there are national differences in length of stay (LOS) and time to enteral autonomy associated with early vs delayed closure.
INTRODUCTION: Neonates are highly susceptible to sepsis, with increased mortality rates. During sepsis, massive numbers of cells
S86
http://dx.doi.org/10.1016/j.jamcollsurg.2016.06.168 ISSN 1072-7515/16