Innate immune defences & inflammation 2:The induced response

Question 1

Cells of the innate & adaptive immune system

Answer 1

The discovery of innate lymphoid cells (ILCs) is blurring the traditional boundaries between innate and adaptive immune systems. Invariant natural killer T cells, some B cells at epithelial barriers (B1 cells) and gamma delta T cells have innate qualities whereas NK cells may adapt after their first encounter with a pathogen due to innate immune memory. T and B cells also have innate immune receptors such as Toll-Like receptors (TLR).

Question 2

Functions of neutrophils

Answer 2

1. Phagocytosis
2. They can use reactive oxygen and nitrogen species when they engulf and phagocytose a pathogen to destroy it
3. They also produce lots of soluble molecules like antimicrobial peptides

Question 3

Functions of Macrophages

Answer 3

1. They also do phagocytosis
2. They are inflammatory mediators
3. They do Antigen presentation
4. can release inflammatory cytokines to generate more inflammation and recruit more cells to the sited of infection as well as complement proteins that activate part fo the complement cascade
5. hey can use reactive oxygen and nitrogen species when they engulf and phagocytose a pathogen to destroy it

Question 4

Functions of Dendritic Cells

Answer 4

They come in 2 types;

1. Plasmacytoid dendritic cells of lymphoid origin and they are very good at producing interferons that antiviral cytokine.

2. Myeloid dendritic cells and are very good at antigen presentation and also they produce lots of pro-inflammatory cytokines.

Question 5

Functions of the Natural Killer cells

Answer 5

• They are very good at lysing virally infected cells, they can release POERFORIN nad GRANZYME.
• Perforin can form pores in the membrane of pathogens and granzyme can then go in to induce apoptosis.

Question 6

Phagocyte recruitment

Answer 6

In order to get the innate immune cells to the site of infection, it will be achieved via phagocytic recruitment.

Tissue residence cells are gonna be releasing cytokines and chemokines and some of those cytokines like Tumor Necrotic Factor (TNF) can affect the vascular endothelium and cause it to become more permeable. It also leads to the upregulation of the adhesion molecules eg (ICAM-1 and VCAM-1).

The adhesion molecules can then bind to integrins on leucocytes moving through the blood. integrins are any of a class of animal transmembrane proteins involved in the adhesion of cells to each other and to their substrate.

the leucocytes start to bind to the adhesion molecules through their integrins, they start to roll and then arrest.

because the vascular endothelium has become more permeable, they can now move through to the site of infection in the tissue.

Question 7

Phagocytosis

Answer 7

• Once the innate immune cells are inside the cell, they will phagocytose foreign materials that they come across.
• This tends to be performed by neutrophils, dendritic cells and macrophages.
• macrophages are monocytes in the blood and differentiate into macrophages in the tissue
• These cells will ingest any foreign things that they come across
• in the image, you can see a macrophage in purple how it’s put out all its pseudopodia (tentacles) sampling the environment and coming across the green e-coli. they are going to pull them into the membrane and engulf them.

HELPS AVAILABLE

• to help with the action of phagocytosis, we have opsonins.
• the complement component ((C3b) is a good molecule that can bind to pathogens to aid opsonisation
• we also have collectins like (mannose-binding lectin) as well as antibodies

What all of these are going to do is that they are going to engage with receptors on the phagocytic cells like the complement receptors, the FC receptors for antibody, mannose receptor, and also Scavenger receptors that can bind to lipids that can recognise, viruses, bacteria as well as apoptotic cells.

Question 8

Receptor mediated phagocytosis

Answer 8

• so the white cells will bind to the opsonins which are highlighting the molecules to be phagocytosed
• they can then invaginate their membrane and take them into an endosome
• that will then fuse with lysosomes which have a special environment that is going to break down that in infection.
• when they fuse together we call this the phagolysosome

Question 9

Antimicrobial mechanisms of phagocytes

Answer 9

• Inside the phagolysosome is an environment that is very bacteriocidal, you’ve got an acidic environment with a low pH , lots of nitrogen and oxygen-derived products that are going to break down any pathogens they come across.
• You’ve also got the presence of lots of antimicrobial peptides like the defencins, the cathelicidin, LL37.
• we also have lysozyme that breaks down peptidoglycans on gram-positive bacteria.
• also in neutrophils, there are LACTOFERRIN and Lactoferrn requested ion which is very important for bacteria cell growth as it stops them from continuing to grow.

Question 10

Neutrophil Extracellular Traps (NETs)

Answer 10

Neutrophils can form nets, almost like a fishing net. when they get activated, they undergo a special form of cell death which is called ‘NETosis’.

When they do this, they burst and can release their chromatin and they spread it out trapping microorganisms and they hold them there ready to be phagocytosed by cells that come along.

Chromatin is a substance within a chromosome consisting of DNA and protein.

in the image, the net from the neutrophil that has lysed/died, holding in the net is trapped the orangy coloured SHIGELA and along comes these other sort of yellow like cells that will come along and phagocytose the pathogen.

Question 11

Pattern recognition receptors (PRRs)

Answer 11

As well as phagocytosing any pathogen that they come across, the innate immune cells also have various families of pattern recognition receptors that they use to recognise antigens.

There are 5 families that they use to induce inflammation but also to upregulate things like MHC involved in antigen presentation.

1. C type lectin receptors (CLRs)

2. Toll-like receptors (TLRs)

3. NOD-like receptors (NLRs)

4. Rig-I like receptors (RLRs)

5. Cytosolic DNA sensors (CDS)

• some of these receptors are found on the cell surface, i..e CLR’s and some of the TLR’s are found on the cell surface.
• some of the TLR’s are then found in the endosome and the rest of the families are found in the cytosol. because it’s inevitable that some infections will get into the cytoplasm of the cell. particularly viruses like to replicate in the cytoplasm of the host cell. but also things that are taken up by phagocytosis can sometimes escape or leak out of the endosome and get into the cytoplasmic compartments.
• These receptors are very clever, they don’t recognise a specific antigen as you would get with antibody but they recognise conserve structures that are found across several different pathogens
• they recognise broad ranges of pathogen rather than specific infections
• what they are recognising is. PAMPS.

Question 12

Pattern-associated molecular patterns (PAMPs)

Answer 12

PAMPs - Microbes evolve rapidly, so innate immunity must focus on highly conserved and essential components of microbes (cell wall structures; nucleic acids).

these are things that pathogens will always need to survive even when they evolve because they’re good at evolving. but PAMPS are things that they must always have and these are what the immune system recognises.

-some of the PAMPS may also be referred to as MAMPS (microbial associated molecular patterns). this is because they recognise those broad structures across microbes and some of those microbes like gut bacteria are not always pathogenic.

DAMPs – Damage associated molecular patterns; these are your own host molecules released from, dead and dying cells. particularly necrotic cells. this is important when you have tissue damage or inflammation that the innate immune system can stimulate an inflammatory response to induce wound healing.

-Apart from pamps, the innate immune system can also recognise DAMPS.

Question 13

Microbe vs Man

Answer 13

Random mutations that occur in microbes play a significant role in their ability to survive and adapt due to the speed at which they replicate.

In 3 hours, single bacteria could divide so much. every time that cells divide, you can have random mutations can occur.

Meanwhile it takes humans 20-40 yrs to replicate.

Question 14

C type lectin receptors (CLRs)

A type of pattern recognition receptor

Answer 14

• CLRs are expressed by most cell type that phagocytoses glycoproteins and microbes for antigen presentation to T lymphocyte.
• CLRs bind to carbohydrates in a calcium-dependent manner
• Type I CLRs assist with antigen uptake by phagocytes
• Type II CLRs are involved in fungal recognition
• Soluble CLRs include MBL (mannose-binding lectin) that binds carbohydrates on pathogen surfaces. MBL can activate compliment and also induce phagocytosis by acting as an opsonin.

Question 15

Toll-like receptors

Answer 15

• The toll-like receptors form transmembrane receptors so they go through the membrane once and on the outside they have this region that has Leucine-rich repeat which forms solenoid shape which then engages with the PAMPS or DAMPS.
• when activated, you get a conformational change in the TIR domain which is a toll interleukin one receptor homology domain and this is shared between all of the toll-like receptors but it gets its name because its also shared by the IL-1 receptor family.

Question 16

TLRs form functional hetero/homodimers

Answer 16

Toll like receptors come together in pairs so that they can bring two-tier domains into close proximity to allow them to trigger a signalling cascade.

• they either come together as two of the same type of toll-like receptor which is homodimer or two different ones can come together forming a heterodimer and this can change the signalling that goes on downstream.
• it can also change what ligands they can bind to so TLR2 does this, it binds to either TLR 1 or TLR2.

Question 17

Cellular location of TLRs

Answer 17

There are 10 human toll-like receptors

1. TLR 2 and 6 recognise - lipopeptides
2. TLR 2 and 1 recognise lipopeptides
3. TLR 5 - recognises flagella which are found on the flagellum of some bacteria
4. TLR 4 binds to LPS found on the coat of gram-negative bacteria.
   - The rest of the toll-like receptors are found within the endosome and they all recognise nucleic acid structures.
   - TL10 10 exists, it newly found. located predominantly in the endosome and it recognises double-stranded RNA’s in competition with TLR 3.
   - TLR 7 and 8 recognise single-stranded RNA and TRL 9 recognises CPG motifs within DNA.

Question 18

TLRs recognise exogenous and endogenous ligands

Answer 18

The TLR in the extracellular space will mainly recognise bacterial products whereas the intracellular (endosomal) ones mainly recognise viral products.

Although there is an exception because DNA is also found in bacteria.

As well as picking things up in pathogenic cells, TLR also pick up things from the host cells, they are shown in the image.

-In Lupus, there is complement deficiency and too many autoantibodies not being removed, those immune complexes from the system. the immune complexes that form in lupus have their antibodies bound to our own DNA and RNA. They can be taken up by immune cells and in the endolysosome, they can then trigger the activation of toll-like receptors to induce interferon which is classic of lupus.

Question 19

TLR signalling cascade

Answer 19

• when you activate the toll-like receptors, they will trigger signalling pathways that are complex but you don’t need to learn them.
• you need to learn that at the top of the cascade, they use 4 different adaptor proteins (MyD88/MAL, TRIF/TRAM). in this picture we are looking at TLR 4 because it uses all the adaptive proteins. However, not all TLRs engage all of the,
• depending on which adaptor molecules they engage, can fire tune the signalling downstream.
• most of them are gonna engage with MyD88 and that drives pathways down to NF-Kb and AP1 which is gonna produce things like IL-1, IL-6 and tnf (the proinflammatory cytokines)

if a TLR engages with. TRIF, it can drive downstream activation of transcription factors that drive the production of Type 1 interferons like interferon alpha and beta.

the other things that TLR do as well as driving the production of inflammation is;

1. They do produce a little bit of anti inflammatory cytokines but generally, its a pro inflammatory event that is produced.

2. they also lead to the upregulation of important molecules like MHC molecules and con stimulatory molecules involved in antigen presentation in activating the adaptive immune system.

3. they can also release antimicrobial peptides at the site of infection and complement components to help drive the complement cascade.

Question 20

TLR adaptor proteins

Answer 20

• All of the TLR’s apart from TLR 3 uses Myd88.
• TLR 2 and 4 use MAL
• TLR 3 only uses TRIF

Question 21

MyD88 gain of function mutation

Answer 21

what happens if you have a mutation in Myd88?

if you take a gain of function mutation in myd88, this has been found to be associated in 90% of patients with Waldenström macroglobulinemia which is a rare type of non-Hodgkin lymphoma.

• so what is happening is that you have too much signalling from myd88 leading to cell growth and cell survival
• lymphoma cells are proliferating in the bone marrow causing neutropenia, thrombocytopenia
• B cells are then driving large amounts of IgM to be produced and that causes a lot of problems

Question 22

A life without MyD88?

Answer 22

• we can actually live without myDD8.
• children found deficient in My88 were suffering from life-threatening pyogenic infections
• you would think that if you didn’t have the TLRs functioning well, you would be susceptible to more infections than the ones found.
• so people released that there has to be some sort of compensatory mechanisms.

Question 23

Are TLRs important?

Answer 23

-if you can survive and combat most infections without using most of those TLR’s , what happens if you dont’t have TLR3?

-TLR 3 is the only one of the 10 TLR’s that has been linked to immunodeficiency

• If you are deficient for TLR3, you are more susceptible to herpes simplex encephalitis. this is an inflammation of the brain due to an infection with HSV-1.
• HSV-1 is a double strande DNA virus.

when viruses replicate within the cell in a cytoplasm, they will produce double-stranded RNA as a replication intermediate and this can be recognised by TLR 3.

-other components of the TLR 3 signalling cascade, if you have defects or missing parts of that signalling cascade, those people are also susceptible to Herpes simplex (HSE).

Question 24

TLRs in disease

Answer 24

• HIV is a single-stranded RNA virus that can drive activation of inflammation through TLR8.
• In sepsis, bacteria activate TLR 2 and 4 and that is detrimental because they cause too much activation and you can end up with multi-organ failure
• you then have diseases like TB that can activate TLR 2 and 4 but also sterile inflammatory conditions
• in lupus, your own autoantibodies binding your own DNA and RNA triggering unwanted inflammation through the endosomal toll-like receptors.
• In Alzheimer’s disease, the amyloid-beta is thought to activate inflammation through TLR 2 and 4 that can lead to neuronal cell death.
• Atherosclerosis- oxidised lipids that form in as plaques can drive inflammatory signalling through TLR 2 and 4 and that inflammation can lead to destabilisation of the plaque and they can rupture and break off causing problems.

*There has been lots of work going on in the pharmaceutical industry to drive inflammation.

i. e a TLR 7 ligand can be used for genital warts and skin cancer because driving that inflammation can help turn on the body’s defences to remove the cancer cells.
- TLR antagonists are also being looked at to be used in like autoimmune diseases and sepsis.

Question 25

Nod-like receptors (NLRs)

Answer 25

**The meaning of the new terminology of NLR = Nucleotide-binding Leucine-Rich.** Nod-like receptors (NLRs) is their old name. **-instead of being in the cell surface of the endosome, these receptors are only found in the cytoplasm.** -they are another type of pattern recognition receptors **_they come as 2 types,_** 1. NLRCs (card domain) 2. NLRPs (pyrin domain) depending on which signalling domain they use to send the signal transduction. The NLRCs have a card domain and the NLRP have a pyrin domain.

Question 26

NLRCs

Answer 26

***_Two examples:_*** 1. NLRC1 (NOD1) 2. NLRC2 (NOD2) They have a leucine rich domain that can bind to **peptidoglycan** (PAMPA) which is present on the cell membrane of most bacteria and they use that card domain to send a signal which will go down to NFK-B and drive cytokine production.

Question 27

NOD1 and NOD2

Answer 27

There are other types of the the NLRC receptors but we re only gonna talk about the first 2. ***•NOD1 and NOD2 detect similar yet distinct peptides of peptidoglycan*** * NOD1 binds **_γ-glutamyl diaminopimelic acid (iE-DAP)_** (Mainly Gramm-ve Bacteria) * NOD2 binds **_muramyl dipeptide_** (both Gram+ve and Gram-ve bacteria)

Question 28

NOD2 gain of function mutation

Answer 28

linked to early-onset **_sarcoidosis_** . This is an autoimmune inflammatory disease where granulomas develop in the different organs of the body. Gain-of-function mutation: A mutation that confers new or enhanced activity on a protein.

Question 29

NOD2 loss of function mutation

Answer 29

is associated with susceptibility to **_Crohn’s disease,_** a chronic intestinal inflammatory disorder. Because you are not defending yourself with a NOD receptor against pathogenic bacteria slipping through from the tissues of the gut, you end up with a large amount of inflammation. Loss-of-function mutations, which are more common, result in reduced or abolished protein function.

Question 30

NLRPs

Answer 30

There are different members of them but we are only gonna talk about 1. ***_-The best characterised is NLRP3 aka (NALP3)_*** - NALP3 comes together with 2 other molecules; ASC and Procaspace 1 to form this high order structure which is called the **_inflammasome_**. the inflammasome has been termed a sensor of cell danger. - it is not fully understood how it's activated but we know that cellular stresses can cause the formation of the inflammasome. things like; ***K+ efflux, ATP release, reactive oxygen species production and lysosomal damage that can come through ingesting crystal during phagocytosis.*** ***_Inflammasome activation is essential for IL-1 and IL-18 secretion._***

Question 31

The Inflammasome senses danger

Answer 31

***Activated by cellular infection or cell stress.*** ***-it many different diseases, you can get crystals that form in the body that gets taken up by cells and that drives the activation and the formation of the NALP3 and inflamasome.*** ***-this was first recognised in GOUT (a painful condition of the joint), caused by the formation of uric acid crystals.*** ***-monocytes, macrophages and other cells will try and get rid of the crystals and that will then damage the phagolysosome and trigger activation of the NALP3 inflammasome.*** ***-this was also found in people who have ingested asbestos, people with alzeimers, etc.***

Question 32

Frustrated phagocytosis

Answer 32

-It's any form of cellular stress and this also happens in people who have had hip replacements. they have artificial hips that have sometimes metal or ceramic that grind over each other over the years. The little fragments break off, they are very small but compared to the size of the phagocytes, they are very large. Being game for anything, the phagocytes come along and decide that the wear particle shouldn't be there and its gonna get rid of it. obv the ceramic is much larger than the ecoli that the macrophages will normally ingest and dispose of. also, its made of ceramic and the macrophages will not be able to break them down. the WBC gets stuck in the process of trying to phagocytose the ceramic and that puts the cell under stress which leads to inflammasome activation and production of IL-1 and IL-18. what then happens with this patient is they get inflamed hip and have to have a precision surgery to have the tissue removed.

Question 33

Inflammsome cleavage of pro-IL-1 and pro-IL-18

Answer 33

***The activation of the inflammasome is important for the activation of IL1 and IL8 secretion.*** _What's the production of IL1 and IL18 by the inflammasome doing?_ - You will get activation of either the **IL1 receptor family or the toll-like receptors to lead to NF-KB activation** driving that **transcription and translation of IL-1B** (the pro-inflammatory cytokine). - but it's not made in its finished form. its made as **pro-IL1,** and that then needs to be cleaved by **caspase 1** which is released from the **inflammasome complex.** the caspase 1 is in a **pro form** and it's only during the **inflammasome activation** that it becomes active and become **the IL1.** that then makes the mature and activate form of **IL1** which can be released from the cell. - The same process goes for **IL18,** it also requires the exact same process of cleavage. - There are other inflammasomes that could also lead to the maturation of IL1 but only learn about this one for now.

Question 34

Gain of function mutations in **NLRP3**

Answer 34

This leads to diseases where people produce too much IL1 and collectively these diseases are called the **Cryopyrin-Associated Periodic Syndromes (CAPS)** **_This disease has 2 types_** **_1. Muckle wells syndrome (Prevalence unknown)_** Can occur spontaneously or be triggered by cold, heat, fatigue, or other stresses. Symptoms of fever, rash, arthralgia, conjunctivitis, uveitis, sensorineural deafness, and potentially life-threatening amyloidosis. ***this one is more serious.*** **_2. Familial cold autoinflammatory syndrome (1: 1000000)_** Triggered by exposure to cold Symptoms of fever urticarial rash with headache, arthralgia, and sometimes conjunctivitis \*because people who have these diseases have too much IL1, they both suffer from fever, because IL1 is a good inducer of fever. its very good at protecting from viruses. they also get rashes, painful joints, conjunctivitis etc. -cold tend to trigger the activation of their NALP3 inflammasome. Both conditions can be treated with biological therapy, an injection of **_Anakinra (IL-1RA)_**. this is a biological version of IL1 receptor antagonist which is being naturally made in the body which blocks the action of IL1.

Question 35

RIG-I-like receptors (RLRs)

Answer 35

There are 2 types of these receptors. ***1. RIG-I*** ***2. MDA5*** As well as the NOD receptors, these are found in the cytoplasm and are sensors of **_cytoplasmic RNA,_** it could be an RNA virus or it could be released as an intermediate of viral replication in the cytoplasm. They will signal to produce proinflammatory cytokines but they are also good inducers of large amounts of type 1 interferon. That antiviral cytokine. **_RIG-1_** -RIG-I binds to single-stranded RNA containing a 5 prime triphosphate, although we humans also have RNA with a 5 prime triphosphate, but our RNA is being released into the cytoplasm to go off to the ribosome to make protein is capped and doesn't get recognised by RGI- I. **_MDA5_** - MDA5 instead prefers to bind to long double-stranded RNA, it's very important for recognising picornaviruses. - some mutations have been recently discovered in some people and they are generally related to interferon diseases. - Mutations are rare but some have been identified in people with ***systemic lupus erythematosus and Aicardi–Goutières syndrome*** ( an inflammatory encephalopathy that mainly affects the brain but some other issues too).

Question 36

Cytosolic DNA sensors

Answer 36

***A few cytosolic DNA sensors have recently been discovered but we are only gonna talk about STING and C GAS which have been related to a disease that has been discovered.*** - when you get a viral infection, if the virus is a double-stranded DNA virus, the double-stranded DNA will bind to C GAS and that then activates the C GAS to convert ATP and GTP into cyclic GAMP an that acts as a ligand for the molecule STING. - STING then sends a signal to produce TYPE 1 interferon, the interferon-alpha and beta. some bacteria can also activate this pathway because they release from them some cyclic dinucleotides like cyclic-di-GMP or cyclic-di-AMP and they can also activate STING. - A gain of function mutation in sting has been discovered and this constitutively sends a signal to produce TYPE 1 interferon. so anybody with this mutation called SAVI (Stimulator of interferon genes (STING)-associated vasculopathy with onset in infancy). - These are young children that have too much interferon and these abnormal inflammation is causing damage to the skin , particularly the blood vessels of the lungs and other areas of the body.

Question 37

Acute Phase Response

Answer 37

Collectively, all the innate cell receptors, as well as the soluble molecules of the immune system, are gonna come together to cause an acute phase response. so you've got lots of these proteins produced as part of the acute phase response, the soluble molecules that are gonna help with those initial stages of the infection, so things like the complement components, a mannose-binding lectin that drives the complement cascade, CRP that can drive complement as well as acting as an opsonin. you've also got increased amyloid protein being produced and this is really ramped up by cytokines being released on activation of pattern recognition receptor-like IL6 and it triggers their production from the liver. many of the acute phase proteins are gonna end up helping with opsonisation and phagocytosis or driving complement activation. You can use this response to measure whether someone has inflammation. i.e you could measure CRP in the blood, but you can also measure the erythrocyte sedimentation rate. this becomes raised if somebody has an inflammation because the serum becomes more viscous (thick) due to all the extra proteins being made and released. -it doesn/'t has to be an infection before you could measure these things, it could also be autoimmunity cos many of the pattern recognition receptors get activated in autoimmune diseases.