Transcript Atm

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unpublished data
How DNA deletions are scored in vivo
Mouse strain: pun mouse (C57BL/6J-pun/pun)
 dilute gray fur color
 pink eyes
 pun mutation
70 kb
pun
Exons
1-5
6-18
homologous
6-18
19-23
 recombination (in embryonic life)
HR leads to a deletion of
exons 6-18
wild type p
Exons
1-5
6-18
19-23
70 kb deletion at the pun locus results in pigmented spots on
the fur
 Fur spot assay
reverted premelanocytes
expand clonally to form a fur
spot
DNA deletion spot
pun reversion results in pigmented spots on the fur and
retinal pigment epithelium (RPE)
DNA deletion spots on the fur of pun mice
The eye and the Retinal
Pigment Epithelium (RPE)
RPE
neural retina
RPE
choroid
optic nerve
DNA deletion spots on the RPE
1 eye spot = 1 deletion event in the RPE
1-cell spot
4-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
34-cells spot
Distribution of spots,%
Eye spot frequency in Atm deficient mice
Number of spots/RPE
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
Difference in the frequency of genetic instability Harvard UCLA
Semi-conventionalized Atm-/- mice in a non-sterile facility have
increased DNA deletions compared to Atm-/- mice in a sterile
environment
p<0.01
*
p<0.05
*
Ribosomoal intergenic spacer analysis (RISA) shows that mice in
different facilities have different spectra of 18S rRNA
Markers
Semi-conventialized
mice
“Conventional”
mice
Mice in a sterile
environment
ISA results show that antibiotic treatment followed by re-inoculation wit
fecal samples from donor mice reconstitute the intestinal flora
Markers
Semi-conventialized
After 4 weeks of antibiotic
Donor mice
mice
treatment
Conventionalized mice
Older Atm-/- mice
0.06
non-treated
hOGG1-treated
Olive Tail Moment
0.05
p= 0.053
0.04
0.03
0.02
0.01
0
80th Percentile % Tail DNA
RF
2.5
CF
Sterile
Non-sterile
CCF
non-treated
hOGG1-treated
*
2
1.5
1
0.5
0
RF
CF
Sterile
Non-sterile
CCF
Older wildtype mice
0.16
**
0.14
Olive Tail Moment
0.12
0.1
non-treated
0.08
0.06
0.04
0.02
0
wt RF
wt CF
hOGG1-treated
Micronucleus Assay
*
12
*
MN/ 1000 erythrocytes
10
8
6
4
2
0
Atm-/- RF
Atm-/- CF
wt RF
* Indicates p<0.05 compared to wt control and as indicated
wt CF
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 nonsterile 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
Micronucleus
Micronuclei per 1000 RBCs
Comet Assay
120
100
80
60
40
0
Lbj
20
PBS
% difference in Olive Tail
Moment
140
12
10
8
*
6
PBS
Lbj
4
2
0
0
2
5
Weeks During Treatment
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 : IL1beta, 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
PCoA of Bray-Curtis difference between gut communities (all data)
Each data point is a bacterial community from the gut of one mouse
Sterile
Conventional
DLAM-Conventional
Semi-conventional
Restricted
ATM-/ATM +/ATM +/+
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
5
Helicobacteraceae
4.5
4
Relative abundance, %
•
•
3.5
3
2.5
2
1.5
1
0.5
0
restriced,
restriced, older
younger ATM-/ATM-/-
restriced, older DLAM, younger
wt
ATM-/-
DLAM, older
ATM-/-
DLAM, older wt
Conventional,
younger ATM-/-
Comparing bacterial communities with PCA of unifrac
score, a phylogenetic similarity metric
Restricted
Sterile
Semi-conventional
Conventional
DLAM Conventional
Unweighted unifrac
(presence/absence of taxa)
Weighted unifrac
(considers relative abundance of taxa)
Identification of bacteria that causeor suppress genetic instability
and lymphoma in mice
Candidate protective bacteria that are
statistically (P < 0.000) more abundant
in RM than CM
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 causative bacteria that are statistically
(P < 0.000) more abundant
in CM than RM
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 Reliene
Irene Maier
Lynn Yamamoto
Angeline Tilly
Jared Liu
David Berry
Alexander Loi
Mike Davoren
Yelena Rivina