Intestinal Microbiota: A Key Player in Longevity, Genomic Instability, and Lymphoma in Atm deficient...
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Transcript of Intestinal Microbiota: A Key Player in Longevity, Genomic Instability, and Lymphoma in Atm deficient...
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12.5 C57BL/6J SPF unpublished data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