1. PRR lecture, Minerva edition
(some graphics and other items removed for copyright and clarity, will be explained in lecture)
Prof Ian Sabroe

2. Covering How we detect microorganisms and viruses
The need to detect and respond to molecular ‘patterns’
Receptors on the cell surface and cell membranes: TLRs
Receptors inside the cell: NLRs, RLRs

3. The problem
Mr J was well until two days ago. Suddenly he has fever, increased breathlessness, sputum production.
What will determine if he lives or dies?

4. Immunisation
Development of immunological memory is powerful and protective but takes weeks…
Vaccination/exposure, antigen presentation to T cells, first production of IgM, establishment of high specificity IgG, production of large amounts of IgGs from expanded B cells…
If pneumonia, you’re already dead

5. To handle pathogens we need a rapid response
‘Innate’ immunity: present since birth, no requirement for memory (except an evolutionary one)
Neutropaenic sepsis after chemotherapy/BMT

6. Handling of pathogens Bacteria, fungi: phagocytosis and killing
Viruses: cellular shut-down, self-sacrifice, cellular resistance

7. The problem
Immunity can take a long time but infections arrive quickly and unpredictably
We live in continual relationship with pathogens and commensals. How many times might you have died today?
Infection is often associated with injury, and damage and danger are important concepts

8. More problems
Adaptive immunological memory is highly specific, cross-reacting or self-targeting specificity is a problem.
There are a LOT of bacterial species and viruses out there. How do we tell bad from good at a moment’s notice?
Only vertebrates have an adaptive system; there must be other effective immune systems.

9. Charles Janeway
Incredibly for the most ancient part of our immune system, innate immunity and PRR concepts first proposed by Charlie Janeway, 1989
Self-non-self discrimination by recognition of unchanging patterns of microbes

10. What is pattern recognition?

11. Patterns
Limited characteristics
Gram +ve/-ve
dsRNA
CpG motifs

12. History of the most famous PRRs

13. Drosophila Toll

14. C3H/HeJ: mammals have Toll as well
Can’t see LPS/endotoxin
Can’t respond to Gram- negative bacteria
Science, 1998

15. The PRR family Secreted and circulating PRRs
Cell-associated PRRs (more traditional receptors)

16. Secreted and circulating PRRs
Antimicrobial peptides secreted in lining fluids, from epithelia, and phagocytes.
Defensins, cathelicidin
Lectins and collectins: carbohydrate-containing proteins that bind carbohydrates or lipids in microbe walls. Activate complement, improve phagocytosis.
Mannose binding lectin (deficiency syndromes), surfactant proteins A and D

18. Secreted and circulating PRRs (2)
Pentraxins. Proteins like CRP, which have some antimicrobial actions, can react with the C protein of pneumococci, activate complement, and promote phagocytosis.

19. Cell-associated PRRs
Receptors that are present on the cell membrane or in the cytosol of the cells.
Recognise a broad range of molecular patterns.
TLRs are the main family

21. TLRs TLR Ligand (exogenous) Ligand (endogenous) TLR1/2
Gram positive lipopeptides
TLR3
Double-stranded RNA
mRNA
TLR4
LPS, pneumolysin, viral proteins
Heat shock proteins, HMGB1, hyaluronan, fibrinogen
TLR5
Flagellin
TLR2/6
TLR7
Single-stranded RNA
RNA?
TLR8
TLR9
CpG DNA
DNA, mitochondrial DNA? (context may matter)

22. The special case of TLR4 pg amounts of LPS can signal
As little as 10 molecules/cell
Polymorphisms in TLR4 affect endotoxin responsiveness, associated with human plagues etc?

23. What do TLRs do?

24. Other membrane-bound PRRs
Family of receptors that may participate in pathogen recognition and particularly in pathogen phagocytosis:
Mannose receptor on macrophages (fungi).
Dectin-1. Widespread on phagocytes, helps recognise beta glucans in fungal walls.
Scavenger receptors on macrophages.

25. Not all pathogens are extracellular
Viruses multiply in the cytoplasm of cells
Bacteria such as Salmonella burst out from the phagolysosome and multiply in the cytoplasm of macrophages

26. NLRs
Named after first members of the family, NODs, Nod-like receptors.
Rapidly expanding family of another 22 human proteins that detect intracellular microbial pathogens.
Detection of peptidoglycan, muramyl dipeptide, etc.
Best known are NOD1, NOD2, NLRP3

27. NOD2 Widespread expression
Recognises muramyl dipeptide (MDP), a breakdown product of peptidoglycan
Activates inflammatory signalling pathways
Non-functioning mutations: Crohn’s disease
Hyperfunctioning mutations: Blau syndrome (rare, chronic granulomatous inflammation of skin, eyes and joints)

28. RLRs
Named after first members of the family: Rig-I. Rig- like helicases = RLRs
Best known are RIG-I and MDA5, who’s roles are to detect intracellular double-stranded viral RNA and DNA.
They couple effectively to activation of interferon production, enabling an antiviral response
NOD2 can also activate anti-viral signalling

31. So far…
Recognition of microbes and viruses depends on seeing ancient, conserved features of them: pattern recognition.
Families of receptors exist to detect these in fluids, cell surfaces and compartments, and intracellularly
Obvious roles in pathogen responses
Additional roles in homeostasis and damage recognition

32. Homeostasis
Blood neutrophil numbers may be dependent upon TLR4 signalling, independent of LPS in homeostasis.
Induction of endotoxin tolerance in the newborn gut.
Maturation of the normal immune system.
Maintaining a balance with commensal organisms.

33. Polly Matzinger Bunny girl, waitress, jazz musician, dog trainer.
And rather a good scientist.
Damage hypothesis - hydrophobicity, dynamic interactions.

34. Damage recognition
TLRs also adapted to recognise a range of endogenous damage molecules, which may share characteristics of hydrophobicity.
Appearance of host molecules in unfamiliar contexts can activate TLRs.
TLR signalling by cellular damage products activates immunity to initiate tissue repair and perhaps enhance local antimicrobial signalling.

36. PRRs in adaptive immunity
Activation of TLRs and other PRRs drives cytokine production by antigen-presenting cells that can increase the likelihood of successful T cell activation.
TLR4 agonists already used as vaccine adjuvants.
TLR7/8/9 adjuvants in development.

38. PRRs and disease Recognition of host molecules in autoimmune disease
Failure to recognise pathogens or increased inflammatory responses
Atherosclerosis, arthritis, COPD, etc.

39. Translation to therapy
Enhance TLR signalling: improve immunity, adjuvants
Inhibit TLR signalling: sepsis syndromes
Modify adaptive immune response: bias Th and Treg responses

40. Summary
Recognition of molecular patterns is key to survival and successful establishment of and engagement with commensal microbiome
PRRs are highly conserved systems of immunity, evolved to recognise unchanging patterns