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Molecular Mechanisms > Infection control by innate immune system > Flashcards

Infection control by innate immune system Flashcards

1
Q

what are the key features of innate immune cells?

A
  • No memory
  • Fast acting – depend on PRRs
  • Inherited receptors (pattern recognition) - germline-encoded with less diversity
  • Often concentrated within barrier tissues – skin, mucosal tissue (lung, gut)
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2
Q

what are the 2 roles of innate immune cells?

A
  1. recognise pathogens via APCs
  2. Engulf and destroy pathogens
    - sentinel cells sound alarm in response to pathogen
    - granulocytes and phagocytes
    - clear infection
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3
Q

how do innate cells recognise pathogens?

A

via PRRs on surface of leukocyte recognising and binding PAMPs on pathogen surface

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4
Q

what happens when a PRR binds to a PAMP?

A

Binding initiates IC signalling pathway via enzymes, kinases, TFs

Causes cellular immune responses:
- Pathogen killing
- Antigen presentation – activate adaptive immunity
- Gene expression – TFs are activated in nucleus
- Leads to cytokine and chemokine production

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5
Q

what sorts of cytokines can be produced by immune cells in response to pathogens?

A

Interferons: switch on antiviral responses

Growth factors: Boost immune cell numbers

Interleukins: Direct immune responses

Chemokines: co-ordinate cell movement

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6
Q

what are Toll-like receptors (TLRs)?

A
  • important for antibacterial immunity
  • both intracellular and extracellular
  • TLR signals converge on MyD88 adaptor protein, which is a scaffold which helps initiate intracellular signalling cascade
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7
Q

what happens when MyD88 is lost or mutated?

A

Mutations in MyD88 can’t activate immune responses via TLRs - wipes out ability for PAMP recognition

Patients lacking MYD88 die early due to early and overwhelming bacterial infection

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8
Q

what kinds of PAMPs do endosomal TLRs recognise?

A

Endosomal TLRs recognise intracellular pathogenic nucleic acids
e.g. TLR3 recognises viral dsRNA

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9
Q

what are C-type lectin receptors (CLRs)?

A
  • important for anti-fungal family
  • bigger family than TLR
  • many signals converge on intracellular signalling pathway using CARD9
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10
Q

what is an example of a CLR?

A

Dectin 1 and dectin 2 – recognise carbohydrate in fungal cell wall called beta-glucan

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11
Q

what happens to CARD9-deficient patients?

A

Deficiency in CARD9 means patients cannot deal with fungal infections

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12
Q

do PRRs integrate against a pathogen? why?

A

Lots of different PAMPs in a pathogen e.g. proteins, carbohydrates
- When pathogen encounters macrophage, it engages multiple PRRs at the same time
- Complex IC signalling which overlap and cross-regulate each other – can amplify or downregulate signals
- many PRRs activate the same TF, NF-kB which is a master regulator of immune responses

Vaccine which affects TLRs should be researched to see how it affects CLRs

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13
Q

what is the role of NK-kB?

A

transcription factor which drives expression of cytokines and chemokines - master regulator of immune responses

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14
Q

what is an example of PRR co-operation?

A
  • a dormant, non-metabolically resting fungal spore weakly activates TLR2, so no downstream immune response via NF-kB = avoids unnecessary immune response
  • a germinating, potentially invasive fungal spore undergoes a shape change to expose other PAMPs such as beta-glucan
  • dectin 1 can now recognise beta-glucan and several PRRs are engaged
  • dectin 1 amplifies TLR2 signal - synergistic
  • NF-kB is activated = inflammation to clear spore
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15
Q

what are tissue-resident macrophages (TRMs)?

A
  • these are found in all tissues and initiate immune responses
  • organs have specialised TRMs e.g. microglia in brain
  • arise during development and embryogenesis - not replenished by bone marrow
  • important for immune surveillance
  • can quickly recognise pathogen and recruit leukocytes
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16
Q

where do TRMs develop from?

A

TRMs arise from embryonic precursors:
- Yolk sac produces TRMs
- E.g. microglia come from yolk sac, enter developing brain and stay there in situ – not replenished by monocytes from bone marrow – microglia are highly long lived since birth
- Other TRMs develop from foetal liver e.g. Kupffer cells (liver TRMs), heart macrophages, lung macrophages
- Some TRMs can be produced by bone marrow – splenic and intestinal macrophages have more turnover from bone marrow

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17
Q

where are monocyte-derived macrophages developed?

A

from the bone marrow

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18
Q

would a bone marrow transplant be useful to replenish TRMs?

A

no, as these arise from yolk sac during developmet

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19
Q

what are cytokines?

A

Signal through specific
receptors
- Pro-inflammatory (e.g. TNFa, IFNg)
- Anti-inflammatory (e.g. IL-10)
- Help ‘direct’ an immune response

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20
Q

what are chemokines?

A

Chemokines are small proteins which bind GPCRs
- Coordinate immune cell migration
- Two cysteine residues in its structure
- If side by side = CCL, if separated by a residue = CXCL
- One chemokine can bind multiple chemokine receptors

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21
Q

how do chemokines direct leukocyte movement?

A

TRM releases chemokines – high concentration near macrophage
- Chemokines diffuse into tissue – gradient of chemokine expression over distance
- Inflammatory immune cells sense chemokine and move towards to gradient until it reaches site of highest concentration, at the site of infection

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22
Q

what is the role of chemotaxis?

A

to bring inflammatory cells from the blood into tissues
- cause immune cells to slow down in circulation and extravasate
- chemokines indicate that immune cells need t slow down

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23
Q

what is the process of chemotaxis?

A
  1. chemokines indicate immune cell to slow down
  2. interaction between leukocyte and endothelial cells to tether and slow down
  3. rolling adhesion
  4. tight binding
  5. diapedesis
  6. migration
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24
Q

what is rolling adhesion in chemotaxis?

A

Rolling adhesion: weak binding between carbohydrates on leukocyte and selectins on endothelial cell – tethers and slows down leukocyte
- Leads to activation of endothelium to express other adhering proteins

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25
Q

what is tight binding in chemotaxis?

A

Tight-binding: holds leukocyte in place via integrins on leukocyte and ICAM on endothelial cells

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26
Q

what is diapedesis in chemotaxis?

A

immune cell squeezes between endothelium and enters tissue

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27
Q

what happens if a leukocyte is deficient in adhesion molecules?

A

inflammation which has no immune cells, so no pus
- Immune cells can’t get to site to fight infection

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28
Q

what immune cells need to be recruited by chemotaxis?

A

neutrophils
monocytes

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29
Q

what are neutrophils?

A
  • Short-lived (~2 days in blood)
  • Made in bone marrow via growth factors inducing haematopoeisis
  • Numerous
  • Quickly infiltrate infected tissue via chemokines
  • Granules
  • Highly phagocytic
  • Oxidative killing
  • 3-lobed nucleus
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30
Q

what are monocytes?

A
  • Short-lived (~2 days in blood)
  • Made in bone marrow via growth factors
  • Numerous
  • Quickly infiltrate infected tissue via chemokines
  • No granules
  • Less phagocytosis and killing
  • Differentiate into inflammatory macrophages once in tissues
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31
Q

what is neutropenia?

A

deficiency in neutrophils:
- patients at high risk for bacterial/fungal infections

32
Q

how do neutrophils induce killing?

A

via granular products

via NETosis

33
Q

what is contained in neutrophil granules?

A

toxic molecules:
primary
- Myeloperoxidase – oxidative killing
- Cathepsins
- Defensins – breakdown cell wall
- Lysozyme
- Elastase

Specific (secondary) – sequester nutrients from pathogen
- Lactoferrin
- Collagenase

  • Gelatinase (tertiary)
  • Gelatinase
  • MMP9
34
Q

how are large fungi killed by neutrophils?

A
  • some fungi form long filamentous hyphae - big so can’t phagocytose
  • neutrophils use NETosis - extracellular killing mechanism
35
Q

what is NETosis?

A

Specialised cell death pathway
- Neutrophils turn themselves inside-out - breakdown nuclear membrane, decondense chromatin and release as net
- NETs are sticky and can stop pathogens moving
- NETs also full of toxic molecules and enzymes; damage cell walls and membranes to burst pathogen cells - trap and kill larger pathogens

36
Q

what are inflammatory macrophages?

A
  • Monocyte-derived
  • They are NOT the same as tissue- resident macrophages
  • Make more cytokines and chemokines to amplify the immune response and boost chemotaxis
  • Tend to express more PRRs and be better at antigen presentation - interact with T cells better
  • Good at directing type of immune responses to pathogen
37
Q

how is inflammation resolved?

A

After infection clearance, many inflammatory macrophages (IM) apoptose, which are cleared by TRMs
- IMs can persist in tissue and have a TRM-like phenotype (can still be distinguished)
– the history of infection a person has changes how they can fight future infections

38
Q

what are the two types of inducible macrophages?

A

M1 - inflammatory

M2 - anti-inflammatory

39
Q

what are M1 inflammatory macrophages?

A

Induced to differentiate via Th1 cells releasing TNFa and IFNy
- Strong antimicrobial properties
- Toxic enzymes e.g. NADPH oxidase = respiratory burst, nitric oxide synthase = nitrogen-based killing
- use of arginine to generate NO
- Good at APC
- Blocked TCA cycle – increased metabolite production via glycolysis

40
Q

what are M2 anti-inflammatory macrophages?

A

Induced to differentiate by Th2 releasing IL-4 and IL-13
- Tissue remodelling
- Wound healing
- Metabolism is changed – use aerobic mitochondrial respiration and TCA cycle
- use fatty acids
- produce anti-inflammatory cytokines
- promote angiogenesis and ECM repair

41
Q

in tissues, how prevalent are M1 and M2 macrophages?

A

In a tissue there is a mix of both – highly plastic
– macrophages can switch based on cytokines and chemokines thatare present
- also depends on which PRRs are being engaged to be specific to PAMPs on the pathogen which can be altered
-Reality of immune system is more complex and plastic

42
Q

what amino acid dictates the function of macrophages?

A

arginine

43
Q

how do M1s and M2s use arginine differently?

A

Use arginine differently:
- M1 convert arginine using inducible nitric oxide synthase into nitric oxide for killing

  • M2 convert arginine via arginase-1 to ornithine and urea which are important for wound healing, toxic to parasites

Macrophages can express both enzymes – ratio of enzymes determines which phenotype as they compete for arginine

44
Q

how does the mitochondrial metabolism change between M1s and M2s?

A

M2 use oxidative phosphorylation

M1 use glycolysis

45
Q

what is the general immune response to pathogen being recognised?

A

Bacteria in tissues recognised by TRM via PRRs:
- Dendritic cells act as APC
- TRMs make chemokines
- Recruits monocytes and neutrophils via chemotaxis
- Monocytes differentiate to M1/M2 macrophages which die or take on TRM-like phenotype
- Neutrophils kill bacteria via oxidative killing, NETosis
- Clear infection

46
Q

why is it important to study fungal diseases?

A
  • fungi are persistent - 25% of world population have mucosal/skin fungal infection which can be recurring
  • they can be lethal e.g. cryptococcis - same number of people die from fungi as TB
  • high mortality rate
  • poorer diagnosis treatments available
  • affects immunocompromised
47
Q

why are diagnosis and treatment of fungi limited?

A
  • no vaccines for fungi
  • Fungi often in our microbiome – only become problematic when immune system gets damaged, so hard to vaccinate against
  • only 3 classes of antifungal drugs
  • increasing resistance

We are closest relatives to fungi:
- They are similar to our cells
- Drugs need to target things unique to fungus, but difficult because they use a lot of the same enzymes that we use = toxcitiy to own cells

48
Q

why is anti-fungal drug resistance on the rise?

A

Candida auris in asia is different to candida auris in europe
- It is independently arising
- Why? This fungus grows at higher temperatures to 42C, so climate change may influence this – fungus adapts to warmer temps around the world
- Candida auris is already resistant to 2 of the 3 antifungal drug classes – limited treatment
- Colonises skin easily – easy transmission on surfaces or between people

49
Q

why is anti-fungal drug development limited?

A
  • limited pathogen-specific targets e.g. cell wall
  • slow to develop - enhanced awareness needed
  • patient compliance - may not take full treatment
  • expensive, especially in developing countries
50
Q

what is Candida albicans?

A

yeast commensal in gut
- Not usually harmful – normal commensal flora
- Can cause thrush and life-threatening infection
- Can escape gut into the bloodstream and enter liver and kidneys, spleen and brain – 50% mortality when entering these tissues
- Abdominal surgery – fungus can easily leave
- Colonise IV catheters and enter bloodstream via skin

51
Q

what is Aspergillus?

A

black mould
- Produces spores (conidia) – can be breathed in commonly and not usually cause problems as immune system deals with it
- Patients with damaged lungs e.g. COPD, cystic fibrosis – it can cause problems
- Can exacerbate asthma
- Can cause large fungal structures in lung – lung bleeding and disease
- Can enter brain from lung, 90% mortality when in brain

52
Q

what is Cryptococcus?

A

Yeast causing meningitis in Africa
Risk factor is HIV – reduction of CD4 T cells, so susceptible to this yeast

53
Q

what is Mucor?

A

rare fungal infection
- Linked with COVID19 and steroid use
- It is angiogenic – invade blood vessels
- Deforming infection in sinuses behind eye and nose – people can lose part of their face

54
Q

how are TRMs affected by cryptococcus?

A

Fungus forms holes opening in the brain – immune response inactive as cryptococcus secrete molecules to dampen response and aid its own growth

55
Q

what is Corynespora cassiicola?

A
  • Most commonly a pathogen of plants/grasses
  • Temperature sensitive – not good in warm temps – infection didn’t enter blood, stayed in colder tissues
  • Cases of human infection incredibly rare
  • forms long filaments and spores
  • patient in this study had recurring infections with anti-fungals having no effect
56
Q

what are the consequences of a dectin 1 biallelic deletion mutation?

A

both copies of dectin 1 gene affected
- dectin 1 expression significantly reduced on patient monocytes as the there is a deleterious mutation
- protein not transcribed properly so is truncated and can’t reach surface

57
Q

what is the structure of dectin 1?

A
  • has carbohydrate recognition domain to bind beta-glucan on fungal cell wall
  • transmembrane region
  • cytoplasmic tail for signalling
58
Q

how can dectin 1 be tested for recognition of a fungus?

A

To test if dectin 1 recognises fungus of interest, make soluble dectin1 and use as staining tool
- Take head of carbohydrate recognition site and fuse to Fc region of antibody
- use dectin-1 like an antibody for flow cytometry and fluorescent microscopy
- Fluorescence = dectin1 binding
- Add Fc-dectin1 to fungus and see if it binds

59
Q

Does dectin 1 recognise C. cassiicola?

A

dectin 1 is a receptor for this fungus, but recognises it at specific points
- recognises ends and middle of fungal branches, where beta-glucans is concentrated

60
Q

how can a patient’s cells be tested or an immune response against a fungus?

A

Patient PBMC sample taken in tissue culture dish
- Add fungus
- Take media and measure cytokines IL-1B and TNFa
- compare to healthy volunteer fungal infection
- patient makes reduced cytokines in response to the fungus compared to healthy control

61
Q

how can it be proved that dectin 1 deficiency leads to susceptibility to the fungus?

A

mouse model:
- reproduce in mouse gives confidence that this gene mutation causes susceptibility

62
Q

what is the challenge of using a mouse model to recapitulate C. cassiicola infection? how was this overcome?

A

mouse has fur - difficult to access and recapitulate skin infection
- adding the fungus to skin doesn’t cause infection
- injection of fungus into bloodstream didn’t work due to higher temperature, so fungus gets killed
- need to infect cooler tissues such as ear or tail, but these lack subcutaneous tissue

they infected footpad
- Inject into fatty paw pad
- Can measure swelling using callipers to measure inflammation and infection control
- Infect one foot, use other foot as control

63
Q

how is inflammation resolution affected by dectin 1 deficiency in mouse paw pad?

A

Swelling in WT after a week, but inflammation reduces after a month – immune response at first, then infection cleared and resolved

In dectin 1 K/O mouse, there is long-lasting swelling in foot – problem with immune response
- slices of infected tissue showed granuloma inflammation - lots of neutrophils and monocytes persisting at the site as infection isn’t being cleared
- chronic inflammation and fibrosis

64
Q

did dectin 1 deficient mice make inflammatory cytokines?

A

Measured cytokines in footpad
- Dectin 1 K/O had less cytokines IL-1B and TNFa compared to WT control – need dectin 1 to activate expression of cytokines in response to fungus

65
Q

are immune cells present at infection site in dectin 1 deficient mice?

A

Yes, but no cytokines are being produced, so are non-functional
- chemotaxis is fine
- immune cell killing is not effective due to lack of IL-1B and TNFa

66
Q

what happens to mice which lack TNFa and IL-1B and their receptors when infected with the fungus?

A

IL-1B is important early on – increased swelling when lacking IL-1B

TNFa important in later responses – increased swelling when lacking TNFa

Both cytokines are important to protect against infection

67
Q

what is TNFa?

A

classic inflammatory cytokine
- activates other immune cells
- Activates expression of antimicrobial enzymes and proteins – nitric oxide synthase
- Drives the classic/M1
activation pathway
- Causes fever (systemic effect)

68
Q

what is IL-1B?

A

Made by inflammasomes
- Act as a chemokine; helps recruit inflammatory cells to site of infection
- Boost neutrophil and macrophage killing pathways

69
Q

how is TNFa produced?

A

Dectin 1 activation leads to CARD9 activation which activates NF-KB
- TNFa is transcribed via NF-KB

70
Q

how is IL-1B produced?

A

signal 1: inflammasome priming
- PRR recognition leads to transcription of pro-IL-1B (inactive precursor)
- also transcribes pro-caspase 1

signal 2: upregulation of inflammasome due to cell damage/high cholesterol
- pro-caspase 1 forms inflammasome complex with NLRP3, ASC (scaffold proteins)
- form donut-shape protein complex which only forms with signal 2
- pro-caspase 1 cleaved to form active caspase 1
- caspase proteolytic inflammosome processes pro-IL-1B to form active IL-1B

both signals needed!

71
Q

how is IL-1B secreted?

A

mature IL-1B is secreted via gasdermin D pore (expressed via signal 1) – forms pore in membrane for IL-1B to leave

72
Q

why is IL-1B production tightly controlled?

A

IL-1B is highly reactive - can cause psoriasis

73
Q

what happens to macrophages following the release of IL-1B?

A

Full activation of pathway causes many pores in macrophage membrane so undergoes pyroptosis (apoptosis due to many pores)
- IL-1B is a classic signature of pyroptosis
- Lots of IL-1B to activate lots of innate immunity and inflammation – potent
- lots of levels of control needed

74
Q

how are TNFa and IL-1B important for C. cassiicola infection?

A

if macrophage lacks these cytokines, fungus cannot be killed

if macrophage has either TNFa or IL-1B, there is poor killing

if both cytokines are present, killing of fungus is much more efficient

75
Q

what is the overall process of dectin 1 recognition of C. cassiicola in WT and deficient macrophages?

A

WT:
- Dectin1 recognises beta glucan at specific points
- Signals via CARD9 to make TNFa and IL-1B for fungal killing via macrophages

K/O:
- Absence of dectin1 – no recognition of beta glucan
- No CARD9 activation, no cytokine production – no fungal killing – overwhelming fungal infection

76
Q

what happens when CARD9 is deficient?

A

CARD9 is downstream of multiple PRRs:
- Need to use CARD9 K/O to see how this would affect the infection
- CARD9-defifcient patients have more extreme phenotype – other receptors must be involved
- CARD9 is crucial for protection, dectin 1 loss was more treatable – patient with CARD9 deficiency died from the same fungus, while the dectin1 patient survived

Molecular Mechanisms (6 decks)

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