LMP299Y1 Lecture Notes - Knockout Mouse, Myd88, Innate Immune System

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16 Dec 2013
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September 10, 2013
Mortality curves
Sharp drop and straight line 150 years ago equal likelihood of dying in twenties and sixties
Sixty percent of deaths due to infectious diseases
5 to ten thousand years ago mortality curves identical to 150 years ago suggests lack of medical
access or poor medical care
Infectious diseases have been with us a long time large impact on survival
Immune system effective at population level good enough so can allow for population to reach
reproductive years
How bacterial toxins cause disease, if they do and how the immune system detects bacteria/pathogen
and how it responds
Tests answer based
Host-microbe interactions
90% of cells are bacterial in body
Commensals in the gut
Difficult to grow in culture majority of commensal bacterial
When fighting pathogens, initiate inflammatory response if too dramatic, can damage host energy
intensive response
Do not want to be mounting response against commensal bacteria
Avoiding inflammation by commensals
Mucus layer acting as physical barrier so no interaction between microbes and epithelial cells and
initate inflammation
In small intestine absorbing nutrients in small intestine, one of the mechanisms to reduce
inflammation is to release anti-microbial peptides into lumen of the gut insert themselves in the
membrane of the microbes and disrupt the membrane and kills them close to the intestine and keep
them segregated away
PAPER Vaishnava, S. et al
Lectin = carbohydrate binding protein
Raising the question
Only bacteria cells have 16S rRNA probe; this probe can interact with all the microbes “UNIVERSAL”
Looked at mice lacking Myd88 what is the role of innate immune system in forming spatial
Physical separation dependent on myd88?
Why knockout myd88 but not say TLR4? MyD88 is almost universal so can make only one knockout
Suggesting Epithelial cells are sensing the microbe, and doing something to create the segregation zone
Results right side
Figure 1
Quantitating the wild-type mice in solid circles knockout in white circles
Looking at amount of fluorescence with increasing distance from villus
The graph in panel B show that there is a zone of segregation, where in the wild-type, low
fluorescence, but the graph also suggests another plateau
Panel C close to the mucosal layer in wild-type, there are icrobes present; whereas the other panels
suggest that there are none close to the mucosal layer
A hundredfold more microbes closer to mucosal surface between wildtype and knockout
In the lumen (panel C still) no difference between the numbers of the microbes
Luminal graph of panel C can show that this is not global, but rather localized to the mucosal area
What is the cell type that is important for this Myd88 effect? Knock out Myd88 only in epithelial cells
All the other cells still have Myd88
Figure 2
No zone of segregation in knockout mice
Myd88 in epithelial cells necessary for segregation zone
To determine whether Myd88 is sufficient to maintain the barrier, generate transgenic mice with Myd88
under epithelial promoter most of cells of mice lack Myd88 except for epithelial cells get zone back
in transgenic so sufficient expression in epithelial cells is all you need, is sufficient
How are the mice sensing the bacteria if get too close to epithelial cells? What the mice actually do to
promote the segregation?
A plausible mechanism… in paper
Grampositive one layer with peptidoglycan thick layer
Gram negative two layers with thin layer of peptidoglycan
Figure 2
Look for REgIII gamma with specific antibody
If overexpress Myd88 in transgenic, get increased staining of the reg three gamma
C only shows a correlation
Figure 3
Reg3 gamma knock out mice
In the knockout, the microbes are close to the villi
D is quantitative data
Only ten fold difference between the number of bacteria near mucosal layer between wildtype and reg
three gamma knockout
There could be other antimicrobial peptides under Myg88 regulation segregation is not only about
Reg3 gamma
Figure 4
If knockout Reg3 gamma, have more of an immune response??
Looking at cells that are expressing IgA secreted into lumen of intestine marker of inflammation
Abx is antibiotics
To ensure that the microbes are causing the IgA
Reg3 gamma sufficient in creating decrease in bacterial population close to mucosal figure 3D
Need to put the mice in the same cage
Mice eat each other’s feces