2019 Sc ie ntific Re tre a t F RI DAY, APRI L 26, 2019 DI SCOVE RY WORL D PAVI L I ON RE PROGRAMMI NG NE UT ROPHI L S T O CONT ROL T I SSUE I NJURY Benjamin Gantner, PhD Assistant Professor of Medicine, Division of Endocrinology T o g e the r, T a king o n Ca nc e r’ s T o ug he st Cha lle ng e s Medical College of Wisconsin
NE UT ROPHI L S SE ARCH F OR I NJURY AND I NF E CT I ON Damage-Associated Stimuli: ? -Signs of Injury -Mitochondrial DNA -Mitochondrial Peptides (Formylated) -Purinergic stimuli (ATP, etc.) Bacterial Stimuli: -Lipopolysaccharide (Gram Negatives) -Lipopeptides -Peptidoglycans
NE UT ROPHI L S F I ND T UMORS… Damage-Associated Stimuli: ? -Signs of Injury -Mitochondrial DNA -Mitochondrial Peptides (Formylated) -Purinergic stimuli (ATP, etc.) Bacterial Stimuli: -Lipopolysaccharide (Gram Negatives) -Lipopeptides -Peptidoglycans
I NT RAVI T AL I MAGI NG OF VE NT I L AT E D L UNGS Cove overslip Vacuum Lung ng Vacuum CANCE R COL L ABORAT I VE
SURGI CAL PRE PARAT I ON F OR L UNG I MAGI NG Cove overslip Vacuum Lung ng Vacuum CANCE R COL L ABORAT I VE
* Omiecinski, U.W. Seattle I NT RAVI T AL I MAGI NG OF VE NT I L AT E D L UNGS - Ne utro phils c ra wling in no rma l lung -5 minute s re a l time o r ~600 b re a ths Vascular Casts 40 second projection Albumin Neutrophil 100µm 100µm Unaligned Stabilized CANCE R COL L ABORAT I VE
I MAGI NG WI T H CE L L UL AR RE SOL UT I ON -Ne utro phils a dhe re to infla me d c a pilla rie s -5 minute s re a l time o r ~600 b re a ths ng crawling /min) 80 p < 0.000001 40 µm/m Neutrophil speed ( µ m/min) 40 p < 0.001 (µm Neutrophil c 30 city ( 20 veloci 10 0 Albumin 0 4 8 Hours a s after er A Aerosol L LPS Hours after aerosol LPS delivery Neutrophil 90 p < 0.02 100 p < 0.05 (µm) a (µm 2 ) 80 90 ess ( 80 100µm 100µm 70 Area ( Thicknes 70 60 60 50 ar A 50 40 Alveolar 40 30 Septal 30 20 20 10 10 0 0 Unaligned Stabilized Unstim LPS Unstim LPS CANCE R COL L ABORAT I VE
NI T RI C OXI DE SYNT HASE 1 PROMOT E S SE PT I C L E T HAL I T Y AND L UNG I NJURY NOS1 (Neuronal NOS) WT 100 nNOS ( n =42) Percent survival 80 NOS1 -/- (Neuronal NOS) P<0.0005 ( n =31) 60 NOS2 -/- (Inducible NOS) 40 ( n =40) iNOS NOS3 -/- 20 (Endothelial NOS) eNOS ( n =32) 0 0 24 48 72 96 Hours after LPS CANCE R COL L ABORAT I VE
NI T RI C OXI DE SYNT HASE 1 PROMOT E S SE PT I C L E T HAL I T Y AND L UNG I NJURY WT NOS1 -/- Untreated NOS1 (Neuronal NOS) LPS, 8h WT 100 nNOS ( n =42) Percent survival 80 NOS1 -/- (Neuronal NOS) P<0.0005 ( n =31) Lung Filtration Coefficient 60 0.07 NOS2 -/- (Inducible NOS) ml/min/cm H 2 O/g 40 0.06 ( n =40) WT iNOS 0.05 NOS3 -/- NOS1-/- 20 (Endothelial NOS) eNOS 0.04 ( n =32) ** 0 0.03 0 24 48 72 96 0.02 Hours after LPS 0.01 0 LPS: - + - + CANCE R COL L ABORAT I VE
NI T RI C OXI DE SYNT HASE 1 PROMOT E S SE PT I C L E T HAL I T Y AND L UNG I NJURY WT NOS1 -/- Untreated NOS1 (Neuronal NOS) LPS, 8h WT 100 nNOS ( n =42) Percent survival 80 NOS1 -/- (Neuronal NOS) P<0.0005 ( n =31) Lung Filtration Coefficient 60 0.07 NOS2 -/- (Inducible NOS) ml/min/cm H 2 O/g 40 0.06 ( n =40) WT iNOS 0.05 NOS3 -/- NOS1-/- 20 (Endothelial NOS) eNOS *Baig, et al, 2015, J Exp Med 0.04 ( n =32) ** 0 0.03 0 24 48 72 96 0.02 Hours after LPS 0.01 0 LPS: - + - + CANCE R COL L ABORAT I VE
I NACT I VAT I ON OF NOS1 L E ADS T O NON-I NJURI OUS SWARMI NG AND BL OCK S I NF L AMMAT ORY VASCUL AR I NJURY - 6 hours after LPS - 30mg/kg, i.p. - 5 minute time lapse Albumin Neutrophli 100µm WT NOS1 -/- CANCE R COL L ABORAT I VE
NOS1 DRI VE S I NJURY, NOT NE UT ROPHI L RE CRUI T ME NT Endot othel elial Neutrop ophil Alb lbumin in CANCE R COL L ABORAT I VE
NOS1 I S NOT RE QUI RE D F OR NE UT ROPHI L SE NT I NE L F UNCT I ON OR PHAGOCYT OSI S Endot othel elial Neutrop ophil Alb lbumin in Latex B Bea eads 5 min. r 5 rea eal time CANCE R COL L ABORAT I VE
RE PROGRAMMI NG NE UT ROPHI L S I N T UMORS? Damage-Associated Stimuli: -Signs of Injury -Mitochondrial DNA -Mitochondrial Peptides (Formylated) -Purinergic stimuli (ATP, etc.) Bacterial Stimuli: -Lipopolysaccharide (Gram Negatives) -Lipopeptides -Peptidoglycans
I f yo u wa nt to g o fa st, ACKNOWLEDGEMENTS g o a lo ne . • Mao Mao • Steve Vogel I f yo u wa nt to g o fa r, • Sofia Zaichick • Peter Toth • Saqib Baig • Richard Minshall g o to g e the r. Andre de Abreu Maricela Castellon • • • Farnaz Bakhshi • Suellen D`Arc dos – Afric a n Pro ve rb Santos Oliveira • Peter Hart • Rebecca Shilling • Jing Deng • John Christman • Marcelo Bonini • Chenxia He Support from: • Advancing a Healthier Wisconsin Endowment – Research and CANCE R COL L ABORAT I VE Education Program • Cancer Center Pilot Funding mc w.e du/ de pa rtme nts/ c a nc e r-c e nte r @ MCWCa nc e rCe nte r
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