Intestinal Microbiota: A Key Player in Longevity, Genomic Instability, and Lymphoma in Atm deficient mice

Robert H. Schiestl

Professor of Pathology, Environmental Health and Radiation Oncology

UCLA

Bacteria in our body

There are 10 fold more bacteria than human cells in our body, most of them contained in our intestines

Intestinal microbiota and inflammation

In 1995 Dr. Barry Marshall and Dr Robin Warren received the Nobel Price in Medicine for their discovery of H. pylori as a cause for stomach ulcers

Mammals w/o intestinal microbiota are immunodeficient

ATAXIA TELANGIECTASIA (AT)

Clinical manifestation:

Autosomal recessive disease (1 in 40.000-100.000 people affected)

Early-onset progressive cerebellar ataxia

High incidence of tumors (30% develop lymphoma or leukemia)

Growth retardation

Immunodeficiency

Biological markers: Chromosomal instability

Hypersensitivity to radiation

Imbalance in antioxidant levels and

antioxidative enzymes

Different median survival rates of Atm-/- mice

50% survival(months)

Background Animal facility Reference

2.2

129SvEv, Black Swiss, 129SvEv:Black Swiss

Not indicated [2]

2 Not indicated Not indicated [1]

4.25 129SvEv:Black Swiss Not indicated [20]

3.5 129SvEv:Black Swiss Not indicated [21]

6.4 129SvEv:C57BL/6J Not indicated [4]

10 129SvEv:C57BL/6J SPF [7]

5 129SvEv, 129SvJ:C57BL/65 Non-SPF [9]

4 129SvEv:C57BL/6J Non-SPF [10]

2.5 129SvEv Not indicated [8]

7 129SvEv:C57BL/6J Not indicated [5]

4 129SvEv Not indicated [22]

7.5 129SvEv Not indicated [6]

12.5 C57BL/6J SPF unpublished data

dilute gray fur color pink eyes pun mutation

Mouse strain: pun mouse (C57BL/6J-pun/pun)

Exons 1-5 6-18 6-18 19-23

70 kb

pun

wild type p

homologous recombination (in embryonic life)

Exons 1-5 6-18 19-23

HR leads to a deletion of exons 6-18

How DNA deletions are scored in vivo

70 kb deletion at the pun locus results in pigmented spots on the fur

DNA deletion spot

reverted premelanocytes expand clonally to form a fur spot

Fur spot assay

RPE

DNA deletion spots on the RPE

DNA deletion spots on the fur of pun mice

pun reversion results in pigmented spots on the fur and retinal pigment epithelium (RPE)

RPE

optic nerve

choroid

neural retina

The eye and the Retinal Pigment Epithelium (RPE)

1 eye spot = 1 deletion event in the RPE

1-cell spot 4-cells spot 34-cells spot

an eye spot is a group of pigmented cells next to each other or separated by no more than one unpigmented cell

a single cell deletion event can be detected among 50.000 RPE cells

Eye spot frequency in Atm deficient mice

Atm -/- mice have high number of eye spots as compared to wild type

8.1 ± 3.1 (n=28) vs 5.9 ± .9 (n=36) spots/eye, respectively; P=0.001

Dis

trib

utio

n of

spo

ts,%

Number of spots/RPE

Difference in the frequency of genetic instability Harvard - UCLA

p<0.05

p<0.01

*

*

Semi-conventionalized Atm-/- mice in a non-sterile facility have increased DNA deletions compared to Atm-/- mice in a sterile

environment

Markers

Ribosomoal intergenic spacer analysis (RISA) shows that mice in different facilities have different spectra of 18S rRNA

“Conventional” mice

Semi-conventialized mice

Mice in a sterile environment

Markers Semi-conventialized miceAfter 4 weeks of antibiotic

treatmentDonor mice Conventionalized mice

RISA results show that antibiotic treatment followed by re-inoculation with fecal samples from donor mice reconstitute the intestinal flora

Older Atm-/- mice

Older wildtype mice

* Indicates p<0.05 compared to wt control and as indicated

Micronucleus Assay

Semi-conventionalized mice in a non-sterile facility have a decreased median lifespan compared to mice housed in a sterile facility

Kaplan-Meier survival curve of Atm-/- mice housed in normal and sterile facilities. The survival curves of mice living in normal and sterile facilities are significantly different (p<0.05). n=34 and 31 for the sterile SPF facility and non-sterile SPF facility, respectively.

Lymphoma latency is shorter in semi-conventional mice in a non-sterile environment

Lymphoma latency is shorter in a non-sterile environment. The latency of lymphoma development in a non-sterile environment is significantly shorter than in a sterile environment (p<0.01). n=15 and 13 for the sterile SPF facility and non-sterile SPF facility, respectively

Lactobacillus johnsonii 456 treatment reduces DNA damage in ATM -/- mice

Changes in Lymphocytes in peripheral blood populations caused by Lactobacillus inoculation

Changes in Lymphocyte populations in spleen caused by Lactobacillus inoculation

When treated with LBJ, mice showed a marked decreased in T cell infiltration in the liver

•LBJ treatment decreases inflammatory cytokine levels in both blood and liver : IL-1beta, IL-12, and IFN-g

•LBJ treatment increases levels of IL-4, IL-10, and TGF-beta, which enable inflammatory control, especially in the liver.

•Inflammatory diseases and oxidative stress: Cancer, heart disease, neurological disease, arthritis and ageing etc.

„Restricted“ mice have very distinctive microbiota

ATM-/-ATM +/-ATM +/+Restricted

PCoA of Bray-Curtis difference between gut communities (all data)Each data point is a bacterial community from the gut of one mouse

SterileConventionalDLAM-ConventionalSemi-conventional

Group 1: Indicator phylotypes

• 32 indicator phylotypes for DLAM mice (both ATM-/- and wt). • Few genotype-specific phylotypes, consistent with the ANOVA result that the

genotype is less important. • Diversity of indicators, including some putative opportunstic pathogens, e.g. in

the Helicobacteraceae

Comparing bacterial communities with PCA of unifrac score, a phylogenetic similarity metric

Unweighted unifrac(presence/absence of taxa)

Weighted unifrac(considers relative abundance of taxa)

RestrictedSterileSemi-conventionalConventionalDLAM Conventional

Identification of bacteria that causeor suppress genetic instability and lymphoma in mice

in RM than CM in CM than RM

1. Lactobacillus johnsonii: 2, Clostridium polysaccharolyticum; 3, Clostridium populeti; 4, Eubacterium hadrum; 5, Clostridium oroticum; 6, Barnesiella intestinihominis; 7, Clostridium fimetarium; 8, Acetanaerobacterium elongatum; 9, Porphyromonadaceae bacterium C941; 10, Butyrivibrio crossotus; 11, Butyricimonas synergistica; 12, Clostridium chauvoei; 13, Lachnospiraceae bacterium DJF_VP30; 14, Porphyromonas sp. C1075; 15, Prevotella sp. oral clone CY006; 16, Rumen bacterium NK4A66; 17, Filifactor alocis; 18, Cyanobacterium sp. MS-B-20; 19, Clostridium tyrobutyricum; 20, Alistipes onderdonkii; 21, Barnesiella viscericola.

Candidate protective bacteria that are statistically (P < 0.000) more abundant

Candidate causative bacteria that are statistically (P < 0.000) more abundant

1, Dysgonomonas gadei; 2, Prevotellaceae bacterium P4P_62; 3, Belliella sp. MIM10; 4, Parabacteroides merdae; 5, Clostridium sp. AN-AS17; 6, Capnocytophaga ochracea; 7, Pedobacter koreensis; 8, Eubacterium sp. BU014; 9, Riemerella anatipestifer; 10, Helicobacter typhlonicus; 11, Petrimonas sulfuriphila; 12, Caminicella sporogenes; 13, Nubsella zeaxanthinifaciens; 14, Porphyromonas sp. MI10-1288x; 15, Sphingobacterium sp. NBRC 15338; 16, Proteiniphilum acetatigenes; 17, Parabacteroides goldsteinii; 18, Bacteroidetes bacterium P073B; 19, Porphyromonas catoniae; 20, Bacteroides nordii.

Who did the work?

Ramune RelieneIrene MaierLynn YamamotoAngeline TillyJared LiuDavid BerryAlexander LoiMike DavorenYelena Rivina