Lecture 3: Innate Sensing by the Immune System

Question 1

Why do we have response against virus with spike protein but not spike protein alone?

Answer 1

Virus has PAMPs along with spike protein antigen –> virus serves as adjuvant

Question 2

do we mount immune response against an adjuvant? why?

Answer 2

yes –> adjuvant acts as PAMP or DAMP to trigger immune response

Question 3

difference btwn PAMP/DAMP and epitope that T cells recognize

Answer 3

T cells recognize PEPTIDES that have been processed by DCs

PAMP is usually not a peptide

Question 4

role of epigenetics in innate immunity?

Answer 4

epigenetics allows for trained immunity by altering gene expression in a way that is long-lasting

Question 5

what is trained innate immunity?

Answer 5

due to epigenetic changes, the cell can react differently than the first exposure

Question 6

difference btwn trained innate immunity and adaptive memory

Answer 6

trained innate immunity is NOT specific

Question 7

what is sterile inflammation? example?

Answer 7

sterile inflammation = inflammation that occurs in the absence of microbial stimuli

ex. lesion on internal organ

Question 8

does sterile inflammation support Janeway’s hypothesis or Matzinger’s theory?

Answer 8

Matzinger’s theory (DAMPs)

Question 9

advantage of having lots of non-lymphoid tissue-resident cells?

Answer 9

centralized location

Question 10

disadvantage of having lots of non-lymphoid tissue-resident cells?

Answer 10

can have non-specific hyperinflammatory response leading to unnecessary tissue damage

Question 11

2 examples of how our immune system could have evolved from pre-existing program to ensure tissue development and/or proper tissue physiology

Answer 11

1. macrophages initially undergoing efferocytosis to clean up dead cells during development
2. type 2 immune response evolved for tissue repair, rather than immunity

Question 12

3 ways in which “form follows function” impacts the activation of our immune system

Answer 12

1. DC has dendrites to reach out into environment
2. PRRs on outside vs inside of cell depending on pathogen
3. MHC interacting with TCR for specificity

Question 13

describe the cells of gut villi

Answer 13

bottom of crypts has proliferating stem cells that migrate up villi to make the rest of the differentiated epithelial cells with specialized functions

Question 14

how do proliferation and differentiation change further up the villi?

Answer 14

proliferation decreases, differentiation increases

Question 15

describe “form follows function” in the context of gut villi

Answer 15

1. stem cells are protected in the crypt
2. easier to dispose of dead cells in lumen rather than crypt

Question 16

describe our skin cells

Answer 16

proliferating basal cells at the bottom rise up to differentiate

top cells are superficial skin layer –> cells die and lose everything except lipids to create a seal and make us waterproof

Question 17

what do we see when looking at the gut microscopically?

Answer 17

can see villi projections covered in dead cells, mucous, food, bacteria, fungi, viruses

Question 18

what do we see when looking at the gut histologically?

Answer 18

villi acts as sieve

Question 19

what does DAPI stain?

Answer 19

nuclei

Question 20

what does UEA1 label?

Answer 20

mucous

Question 21

what does DCLK label?

Answer 21

subset of cells that produce IL-25 to activate innate immune response

Question 22

all PRR signaling pathways converge at:

Answer 22

MyD88 and NFkB conserved signaling pathway

Question 23

describe the MyD88 and NFkB signaling pathway

Answer 23

MyD88 adaptor protein recruits other proteins to activate NFkB pro-inflammatory TF

Question 24

if all PRR pathways converge at MyD88 and NFkB, how can we differentiate btwn diff pathogens and allow specificity? (4)

Answer 24

1. location
2. Magnitude of response
3. Certain cell types mount response against certain PAMPs due to epigenetics
4. Other adaptor proteins involved that activate and recruit other TFs

Question 25

why doesn’t bacteria colonizing our gut just pierce thru and stimulate the immune system?

Answer 25

TLR signaling promotes a physical separation (“DMZ”) of the microbiota and the small intestinal surface

Question 26

how did they find the DMZ?

Answer 26

used 16S rDNA probe to stain all bacteria –> no bacteria found at DMZ

Question 27

What happened to villi in cells without MyD88 expression? Why?

Answer 27

no more DMZ –> bacteria directly interacting with villi

without MyD88, the signaling pathway cannot function so the cells cannot respond to bacteria

Question 28

What type of cells express MyD88?

Answer 28

most cells but mainly innate immune cells

Question 29

what did the experiment with cells lacking MyD88 indicate?

Answer 29

epithelial cells aren’t just a physical barrier but play a role in sensing and mounting a response

Question 30

describe the CreLox mouse model used

Answer 30

Mouse 1: MyD88 gene flanked by loxP on either side
Mouse 2: Villin promoter driving Cre expression

Cross the mice:
- intestinal epithelial cells will activate villin promoter and therefore activate Cre expression
- Cre recombinase will cleave at loxP sites and delete MyD88

Question 31

what happened to the DMZ in the mice with deleted MyD88 from CreLox system?

Answer 31

no more DMZ

Question 32

How did they look at MyD88 gain of function?

Answer 32

constitutively overexpress MyD88 in Villin-expressing/intestinal epithelium cells with MyD88 deleted in all other cells

Question 33

results of MyD88 gain of function experiment

Answer 33

DMZ is present

Question 34

therefore, what is the role of MyD88

Answer 34

MyD88 is necessary and sufficient to limit bacterial association with the small intestine surface, i.e. create DMZ

Question 35

RegIIIy in context of:
- Control, WT mice
- MyD88 KO
- MyD88 overexpression

Answer 35

• WT: RegIIIy expressed
• KO: no RegIIIy expression
• Overexpression: lots of RegIIIy expressed

Question 36

what does RegIIIy do?

Answer 36

bactericidal –> pokes holes in bacteria to kill it

Question 37

what happens in RegIIIy KO?

Answer 37

No more DMZ

Question 38

what cells produce RegIIIy?

Answer 38

less differentiated cells make RegIIIy to protect stem cells

Question 39

after all these experiments, what do we know about how the DMZ works?

Answer 39

DMZ is full of mucous, acting as a barrier and also holds RegIIIy bc the mucous is sticky

therefore, bacteria physically cannot reach epithelium and will be killed by RegIIIy

Question 40

what is the epithelial escalator?

Answer 40

increased differentiation of cells as they move out of intestinal crypts / rise to outer skin layers

Question 41

difference btwn gain of function and loss of function experiment –> why are they used?

Answer 41

use CreLox system to block gene

use transgene to induce gene

used to test if molecule or cell type is necessary and sufficient for a specific function

Question 42

in addition to RegIIIy in intestinal epithelial cells, how can you test whether TLR2 in CD4+ T cells is important for gut barrier integrity?

Answer 42

KO TLR in CD4+ T cells to see how this changes gut barrier

Question 43

are DAMPs and PAMPs sensed differently?

Answer 43

no –> both recognized by PRRs

Question 44

how do anti-microbial proteins like RegIIIy and goblet cells work together for host defense at the intestinal barrier?

Answer 44

mucous forms physical barrier as viscous substance that traps RegIIIy to prevent bacterial infiltration into the gut