The gut microbiota and SES in preterm infants in the Chicago area - PowerPoint PPT Presentation

The gut microbiota and SES in preterm infants in the Chicago area Erika C. Claud, MD Professor Departments of Pediatrics Section of Neonatology Director of Neonatology Research The University of Chicago

Prematurity • What is full term? 37-40 weeks gestation • What is prematurity? < 37 weeks gestation • What is the limit of viability? 22-23 weeks gestation

Prematurity Premature, 23 weeks Full Term, 40 weeks

Smallest Survivor Birth weight 8.6 oz 12 oz.

Su Surv rviv ival al vs vs Birth Birth Weigh Weight for for EL ELBW W Infants Infants 100 100 80 80 (%) al (% 60 60 ival rviv Surv 40 40 Su 20 20 0 450 450 - - 500 500 501 501 - - 600 600 601 601 - - 700 700 701 701 - - 800 800 801 801 - - 900 900 901- 1000 901- 1000 Birth Birth Weigh Weight (g) (g) William Meadow, MD PhD Dept. of Pediatrics and MacLean Center for Clinical Medical Ethics The University of Chicago

Day of Day of Deat Death for for EL ELBW BW No Non- n-Survi urvivo vors rs 40 Non-Survivors (%) 30 20 10 0 1 2 3 4 5 7 10 14 21 Day of Death William Meadow, MD PhD Dept. of Pediatrics and MacLean Center for Clinical Medical Ethics The University of Chicago

Survival Su rvival as as a a function function of of birt birthweigh weight for all for all patients patients alive alive on on Da Day y 4 4 (n (n = = 249) 249) 100 100 80 80 ival al rviv 60 60 Surv % Su % 40 40 20 20 0 <500 <500 gms gms 5 0 1 - 6 2 5 5 0 1 - 6 2 5 626-750 626-750 7 5 1 - 8 7 5 7 5 1 - 8 7 5 8 7 6 - 1 0 0 0 8 7 6 - 1 0 0 0 Birthweight Birthwe ight (gms (gms) William Meadow, MD PhD Dept. of Pediatrics and MacLean Center for Clinical Medical Ethics The University of Chicago

Born too soon… The Preterm Infant - 12% of births Disproportionately account for: 40% of children who have cerebral palsy (CP) 25% of children with hearing impairment 35% of those with vision impairment.

The NICU NICU Delayed Feeding Antibiotics Breast H2 Milk vs. Blockers Formula Opioids Instrumentation

Like the canary in the coal mine—or asthmatics in air pollution studies—children born preterm may serve as a sentinel population owing to increased susceptibility to the sometimes modest effects of common toxicants, improving study power and decreasing necessary sample size.

Necrotizing Enterocolitis (NEC)- Inflammatory bowel necrosis that primarily afflicts premature infants after the initiation of enteral feeding. Risk Factors • Prematurity • Bacterial Colonization • Enteral Feeding • Hypoxia/Altered intestinal blood flow

NEC and Neurologic Outcome Neurodevelopmental and Growth Outcomes of Extremely Low Birth Weight Infants after NEC Hintz, et al Pediatrics 2005 Multicenter, retrospective analysis 1995-1998 Infants in NICHD Neonatal Network <1000gm 5553 ELBW entered into registry 2948 infants evaluated at 18 and 22 months 124 – surgically managed NEC 121 – Medically managed NEC

Neurodevelopmental Outcome associated with NEC SurgNEC PVL 14% vs. 7% BPD 57% vs. 43% CP 24% vs 15% Decreased growth all parameters Hintz, S. R. et al. Pediatrics 2005;115:696-703

Hypothesis: Enteral feeding results in colonization of the uniquely susceptible premature intestine with pathogenic bacteria, resulting in an exaggerated inflammatory response . FASEB 2001; 15: 1398-1403

Question : • Can microbiome analysis be used to identify the pathogenic bacteria associated with NEC? Methods: •20 patients – 10 with NEC 10 control • 4 sets of twins •Analysis of fecal samples prior to onset of NEC by 16S rRNA sequencing

Bacterial Diversity and NEC Wang et al ISME 3(8):944-54, 2009.

Bacterial Colonization and NEC Wang et al ISME 3(8):944-54, 2009.

Bacterial Colonization and NEC Wang et al ISME 3(8):944-54, 2009.

Shift in Microbiome Claud et al. Microbiome, 2013

Temporal progression of the preterm infant microbiota Claud et al. Microbiome, 2013

Modification of the early microbiome and NEC Absolute risk difference .04 NNT – 25 (death or NEC) 0.18 0.16 Absolute risk difference .02 NNT - 48 0.14 Risk of NEC 0.12 Absolute risk difference .03 0.1 NNT - 32 0.08 0.06 0.04 0.02 0 control probiotics prolonged abx <5 days abx control any breast milk AlFaleh K and Anabrees J The Cochrane Collaboration 2014; Cotten et al Pediatrics 2009 January; 123: 58-66; Meinzen-Derr et al J Perinatol 2009 January; 29 (1): 57-62

Prematurity Premature, 23 weeks Full Term, 40 weeks Host development coincides with microbiome development

Pediatrics 2006

Preterm Infant Microbiota ( M PI ) 3000 2500 Weight in grams 2000 1500 1000 M PI -H M PI -L 500 0 0 2 4 6 8 10 Week of life M PI -H M PI -L

Transfer of infant microbiome to germ free mice Germ free pregnant Litter Fecal lysate Daily weight Wean dam C57/Bl6 from infant 10 8 Weight in grams 3000 Weight in grams 6 2500 2000 4 1500 2 1000 500 0 0 0 10 20 30 0 2 4 6 8 10 Day of life Week of life

Inflammatory Response Related Networks M PI _L M PI _H

PCR validation of Microarray 20 Intestine Relative mRNA expression levels M PI -L M PI -H in M PI -L and M PI -H ileum 15 10 5 1 IL1 b TNF a PPAR g CXCL2 MCP-1 VCAM1 TLR5 FOXP3 IFN g IL6 LTA CXCL10 ICAM1 TLR4 TLR9

Cytokine Expression Serum Cytokine unit/ 0.1ml serum M PI -L M PI -H 2400 GF – SPF GermFree 1800 1200 600 0 IFN- g IL-1 b TNF a IL-4 IL-6 IL-10 IL-18 GM-CSF VEGF Ileum lysates 4000 3000 2000 1000 0 TNF a IFN- g VEGF GM-CSF IL-1 b IL-4 IL-6 IL-10 IL-18

NF- κ B Activation GF M PI -L M PI -H SPF pNFkB p65 60 40 20 0 GF M PI -L M PI -H SPF Labeling index for pNF k B p65 nuclear translocation

NF- κ B Dependent Cytokines GF M PI -L M PI -H SPF VCAM-1 Magnification 40x MCP-1

Inflammation and Prematurity BPD NEC PVL ROP

Neurodevelopmental Outcome in Preterm infants Neurodevelopmental Impairment at Age 2 Morbidities 1. Bronchopulmonary dysplasia 2. Necrotizing Enterocolitis 3. Intraventricular Hemorrhage 4. Periventricular Leukomalacia 5. Retinopathy of Prematurity 6. Sepsis

Regulation of cortex neuronal development by 11/16/17 gut microbiota. A. M PI -L M PI -H SPF GF 1 2 3 1 2 3 1 2 3 1 2 3 NeuN GAPDH B. M PI -L M PI -H SPF GF DAPI NeuN

Regulation of cortex myelination by gut microbiota. 11/16/17 A. M PI -L M PI -H SP GF F 1 2 3 1 2 3 1 2 3 1 2 3 MBP GAPDH B. M PI -L M PI -H SPF GF DAPI MBP

IGF1 • We have established that certain microbiota colonization normalized the growth in GF mice (M2). • Mutation(s) in the igf -1 gene or in the igf1r gene are found to be associated with severe body growth failure, microcephaly, and developmental delay. • In rodents, igf-1 gene disruption results in reduced brain size, CNS hypomyelination and loss of hippocampal granules and striatal parvalbumin-containing neurons. • GF mice have lower circulating IGF-1 comparing to SPF mice. • IGF1 crosses the blood brain barrier

Hypothesis • Microbial colonization can modulate brain development through regulation of IGF1.

Microbiome influences serum IGF-1 11/16/17 A B S e ru m IG F -1 4 w e e k s S e ru m IG F -1 2 w e e k s 5 0 0 5 0 0 4 0 0 4 0 0 IG F -1 (n g /m L ) IG F -1 (n g /m L ) 3 0 0 3 0 0 2 0 0 2 0 0 1 0 0 1 0 0 0 0 M P I -L G F M P I -H L F H - - G M P I M P I

Alterations in brain IGF-1 11/16/17 B ra in IG F -1 2 w e e k s B ra in IG F -1 4 w e e k s 8 0 8 0 A B 6 0 6 0 IG F -1 (n g /m l) IG F -1 (n g /m l) 4 0 4 0 2 0 2 0 0 0 G F M P I -L M P I -H M P I -L G F M P I -H C D I g f1 tra n s c rip ts 2 w e e k s I g f1 tra n s c rip ts 4 w e e k s Igf1 m R N A re la tiv e to G a pdh Igf1 m R N A re la tiv e to G a pdh 0 .0 0 5 0 .0 0 5 0 .0 0 4 0 .0 0 4 0 .0 0 3 0 .0 0 3 0 .0 0 2 0 .0 0 2 0 .0 0 1 0 .0 0 1 0 .0 0 0 0 .0 0 0 G F M P I -L M P I -H G F M P I -L M P I -H

Alterations in brain IGF1r and IGFBP3 11/16/17 I g f1 r tra n s c rip ts 2 w e e k s I g f1 r tra n s c rip ts 4 w e e k s E F Ig fr1 m R N A re la tiv e to G a pdh Ig fr1 m R N A re la tiv e to G a pdh 0 .0 0 8 0 .0 0 8 0 .0 0 6 0 .0 0 6 0 .0 0 4 0 .0 0 4 0 .0 0 2 0 .0 0 2 0 .0 0 0 0 .0 0 0 G F M P I -L M P I -H G F M P I -L M P I -H G H I g fb p 3 tra n s c rip ts 2 w e e k s 4 w k s ig fb p 3 tra n s c rip ts Igfbp3 m R N A re la tiv e to G a pdh Igfbp3 m R N A re la tiv e to G a pdh 0 .0 4 0 .0 4 0 .0 3 0 .0 3 0 .0 2 0 .0 2 0 .0 1 0 .0 1 0 .0 0 0 .0 0 G F M P I -L M P I -H G F M P I -L M P I -H

Effect of socioeconomic status on Neurodevelopment Patrianakos-Hoobler et al Dev Med Child Neurol. 2010 Apr;52(4):379-85 Microbiome?

The means by which poverty alters neurodevelopment are unknown. The microbiome is influenced by environment and in turn influences brain The microbiome as a potential mediator of socio-economic disparities in development. preterm infant neurodevelopmental trajectories from NICU discharge to school age We hypothesize that the microbiome is a biologic effector of the influence of SES and environment on neurodevelopment.