IMM250 – Lecture 4 -Innate immunity: Cellular and humoral mediators of defense
Nod-like receptors - cytosolic sensors of bacteria and danger
a. NLR Proteins : detects MAMPs and DAMPs.
b. NLR have two families:
i. NOD subfamily – detects peptidoglycan which some into the cytosol ; Nod 1 and
ii. NLRP subfamily – in the cytosol; detects DAMPs – high levels of ROS, and K+
NLRPs detect danger and form the “inflammasome”
a. NLRP3 is the sensor, and ASC is the adapter. NLRP3 gets activated behind the adapter.
There is a conformational change and then it binds to the adapter. Similar to TLR which
gets activated by the adapter.
b. NLRP3 Respond to high levels of ROS and K+ efflux.
c. N terminus - Pyrin domain | C terminus LRR domain
d. NLRP3 has a Pyrin domain, and so does ASC. ASC binds to NLRP3 through the Pyrin Pyrin
e. The domains in the end of the adapter and NLRP3 is important. it helps to bind the
proteins and form a complex. Similar to Pyrin domain of TLR.
f. Which is similar to domain of TLR it has a transmembrane domain, and a cytoplasmic
g. NOD domain – Nucloetide binding domain
The inflammasome activates Caspase 1 into an enzmatically active protein
a. Procaspase 1 has a Card domain, just as ASC protein.
b. So Pro-caspase I binds to the CARD domain ASC via the Card-Card and form a link.
c. Forms the complex within the cell, and auto activates the active form of pPro Caspase I,
Active Caspase 1 cleaves pro-IL-1β into an active cytokine
a. Caspase 1 is the active form and it can then cleave off its target the targets are the
pro- form of cytokines.
b. It cleaves the end of Pro –IL – I Beta allowing it to be an active cytokine. IL-1beta
c. IL-1beta is secreted from the cell and has lots of effects in deriving information.
1. Pathogen breaches barrier and then….
2. Releases MAMPs 7. Releases MAMPs
3. • Detected by TLR/Nod proteins 8. • Detected by TLR/Nod proteins
4. • Activates signal transduction 9. • Activates signal transduction
5. • Activates NFκB, IRF 10. • Activates NFκB, IRF
6. • Controls cytokine production 11. • Controls cytokine production
12. Summary of TLRs, NLRs and RLRs
a. TLRs i. Detect MAMPs (eg LPS)
ii. Signal transduction to NFκB and IRF
iii. Leads to cytokine and interferon production
i. Detect MAMPs – mainly nucleic acid from viruses
ii. Signal transduction to NFκB and IRF
iii. Leads to cytokine and
iv. interferon production
i. Nod proteins
1. Detect MAMPs(peptidoglycan)
ii. Signal transduction to NFκB
1. Leads to cytokine production
i. Detect DAMPs (ROS& K+)
ii. Signal transduction to inflammasome activation
iii. production of IL-1β (a cytokine - we will talk about this today)
13. Ok, the pathogen has been detected, now what….?
a. Innate immune mediators: both cellular and humoral (ie secreted)
i. Cells of the innate system
b. Killing mechanisms
i. Secreted mediators of the innate system
c. Killing and/or protective mechanisms
d. Pathogen breaches mucosal barrier releases MAMPS (detected by TLR), NON
proteins (detects peptidoglycan), RLR (detects acid mainly from viruses) activates
signal transduction responsible for cell MyD88 binding to piece of TLR to activate TF,
activates TF (NFkB master regulator, driving many reactions specially production of
cytokines), IRF (Pretty specific for interferons).
e. When cells re infected with pathogens like salmonella, the cells begin to di and on this
process they release endogenous DAMPs. High levels of ROS, K+ efflux. Sub family of
NLRP family detects DAMPs. It is within the sub family of NLR. It activates signal
transduction response. Then it forms the complex called inflammasome which
activates Caspase 1 to control the production and secretion of IL-1B.
f. TLR and RLR, NLR
i. TLR – MAMP ; LPS, Signal Transduction, NFkB, IRF, leads to cytokines and
ii. RLR – Viruses and, MAMPs (peptidoglycan) signal transduction NFkB, IRf,
leads to cytokines and interferons production
1. Non proteins : MAMPs – peptodiglycan
2. NLRP – DAMP -> signal transduction pathway leading to
inflammasome 14. General scheme of an innate immune response
a. Pathogen with MAMPs damages the epithelium to break through the epithelial barrier
b. Epithelial cells ‘activated’ upon contact with microorganism - PRRs are triggered, NFκB,
IRFs, inflammasome are activated Mediators of innate immune defense are produced by
activated epithelial cells These are cytokines and chemokines that call in cellular re-
c. Epithelial cells – which pathogens comes into contact releases MAMPs damages
Epethelium comes into the submucosal surface PRR gets triggered IRF,
Inflammsome, NFkB pathways turns on
d. Production of cytokines, and chemokines calls in for intercellular reinforcement.
e. This process calls for inflammation in the person
a. Heat - Swelling - Redness - Pain - Loss of Function
16. What are Cytokines and Chemokines?
a. Cytokine: a category of signaling molecules that, like hormones and neurotransmitters,
are used extensively in cellular communication; cytokines generally enhance the ability
of our cells to get rid of an invading pathogen - are messenger proteins that tell our
body how to get rid of invading pathogen
b. Chemokine: a family of small chemotactic cytokines that act to attract other cells to the
site where they are produced – class of cytokines, protein molecules, peptides (fes AA
chains), chemotactic attract different cells to the site where they are being produced.
c. Features of cytokines and chemokines:
d. A diverse collection of soluble proteins and peptides that modulate the behaviour of
cells at extremely small concentrations – produced in very very small amounts, but have
a huge impact on the cell.
e. Act both locally and systemically -they can have an effect on the site where they are
being produced, and they can come to the blood and have have systematic effect
f. Have many biological activities (pleiotropic effects) - they have many biological
acitivites, even though they are just one protein or one peptide. , they have many
g. Some examples - Cytokines: Interleukins (1-35 …!)or probably more, Tumor necrosis
factor alpha (TNFα), IL-1β
i. Chemokines: Interleukin 8 (also known as CXCL8) normal peptide models, found
in pretty infected cells , monocyte chemotactic peptide 1 (MCP1)
17. Cytokines and chemokines bring in “effector cells” First step: cellular re-enforcements must
escape from blood vessels – get the cellular mediators to the site of infection.
a. Cells that make up blood vessels (endothelial cells) underlying epithelium are
impermeable to cells in the blood stream and plasma
b. Epithelial cells once infected by salmonella produces cytokins and chemokines with a
gradient. High concenrtrations near the site close to the infection and lower mediators
spreading away. As these mediators are being produced they start have an effect on
other cells locally spread around it. These include cells which make up the blood vessels. c. Very close to the epithelial cells are blood vessels, they are actively absorbing stuff from
our food from the blood vessels. When mediators are being produced, they can act on
the blood vessels. cells which make up this blood vessles are called endothelial cells . It
starts to change the endothelial cells so the mediators from the blood vessel can start
coming to the site of infection. It prevents a formidable barrier against leakage of stuff
from our blood. Prevents other cells from moving across the barrier.
d. When The mediators are produced by epithelial cells they start to have an effect on the
i. Dilation – blood vessels become dilated. And blood starts rushing to the site.
Because the vessels are starting to get dilated.
ii. Endothelial cells lining the Blood vessels start getting gaps. – Permeability of
the blood vessels increases and plasma (liquid part of blood) starts to leak out of
the blood. Same time, the leukocytes in the blood start to stick to the
endothelial cells and tries to move across the endothelial cells and tries to move
to the site of inflammation, so they can kill the bacteria coming across the
18. What are the cell types brought to the site of infection by this process??
a. White blood cells (leukocytes)
i. Leukocytes of the innate system
ii. Leukocytes of the adaptive system - called “lymphocytes” (T and B cells)
19. Mechanism of Cell Migration - Tethering and rolling
a. If we want the leukocytes to come to the place of inflammation we have to bring them
from endothelial cells of the barrier.
b. The endothelial cels starts to change their own phenotype, because of the factors
excreted from the epithelial cells and start to get sticky for leukocytes.
c. Normally, endothelial cells leukocytes roll past them.
d. When the cytokines produced by the epithelail cells and starts to act on the endothelial
cells, they change their phenotype and start to produce adhesion molecules which catch
the leukocytes rolling past the endothelial cells.
e. Expresses molecules which are mediated by selectins. Which tether the leukocytes to
the surface of the endothelial cells.
20. Migration and diapedesis (also known as extravasation)
a. Firm adhesion flattens cell and allows for migration between the endothelial cells Innate
effector cells migrate towards site of infection by detecting and following a gradient of
chemokine produced by infected epithelial cell.
b. Cells have to get through the epithelial cells to the site of infection. This is called
diapedesis. They have to push their way through the epithelial cells.
c. Leukocytes are binding to the selectins on the endothelial cells d. The leukocytes starts to flatten out and starts pushing pseudopods between the barrier
of epithelail cells and tries to get through it.
e. The leukocytes then gets into the cell and tries to migrates to the site of infection, and
this is done via the gradient of the cytokines and chemokines. Lower  in the faraway
place. Higher  near the site.
f. They move to the infected site and try to clear it.
21. Cellular mediators of defense
22. Cells Of The Immune System
23. Stem cells are cells which can be anything they want to be. In this case, they are hemopoietic
stem cells: they have limited ability to diversify itself; pluripotent: they have the potential to be
many many dfferent cells.
a. Myeloid progenitor – most of the innate cells comes from this. To form cells within the
i. Macrophages, dendritic cell, monocyte, neutrophil,
ii. granulocytes: eosinophil, basophil mast cell.
b. Common lymphoid progenitor – T & B lymphocytes. Important for adaptive immune
c. NK cells: even though it is derived from the common lymphoid progenitor, it sits in
between innate and adaptive
i. Innate because: ability to detect infections and tumor cells and kills them 24.
a. I. Phagocytes - Monocyte/Macrophage – cells that eats stuff
b. “Big eaters”
c. Are called “monocytes” in the blood - roundish cells
d. When they reach the tissue they are called “macrophages” - flat with lots of protrusions
to seek out pathogens –lots of macrophages can be seen in the mucosal sites.roaming
around the mucosal sites, respiratory tract, and genital tract looking for pathogens. Start
to flatten out and have lots of protrusions to seek out pathogens.monocytes in their
blood, when they come to the tissue it is called macrophages.
e. Their job: eat pathogens, kill pathogens and help present chewed up pieces of pathogen
to T cells (ie “antigen presentation”), production of soluble mediators
25. “Resident” macrophages have different names
a. They all differentiate from blood monocytes
b. Liver - Kuppfer cells
c. Brain - microglial cells
d. Lung - avelolar macrophage
e. Bone – osteoclast
26. Phagocytosis 27. 28. Mechanisms by which phagocytes kill pathogens
a. Phagasomes quickly becomes acidified – so we have low pH around 3.5-4 in the
phagasomes. This could stop bacterial replication ( bacetriostatic) or could kill the
bacteria (bactericidal )
b. ROS – ros is delivered into the phagasome thorugh the respiratory burst pathway.
Because they are highyly toxic they can kill pathogens directly
c. Antimicrobial peptides – defensins and cationic proteins: they can form pores withi the
cell membrane of the bacteria and kill them directly, they are contained within the
lysozyme and the phagsome
d. Enzymes – Lysozyme
e. Competitors – lactoferrin – bacteria nee