Anemo 2014 - Bianchi - Allarmine: come il sistema immunitario influenza il sanguinamento

1. Marco E. Bianchi
San Raffaele University & Scientific Institute
HMGB1
and epigenetics
Allarmine:
come il sistema immunitario
può influenzare il sanguinamento

2. I’m part-owner of HMGBiotech

3. Our organism recognizes pathogens thanks to Pathogen-Associated Molecular Patters,
PAMPs (Charles Janeway).
Pathogen-Associated Molecular Patterns

4. Immune cells sample their environment and expose antigens all of the time, but they
mount an immune resonse only to those antigens presented when PAMPs are present.
Thus, the immune system does not recognize an antigen as “foreign” or “non-self” as
opposed to “self”, but rather as “antigen which is present in a situation of danger” (i.e.
infection) as opposed to “antigen which is present in a situation of calm”
Antigen presenting cells
Antigens
T cells
Adjuvants
(PAMPs)
Polly Matzinger’s “Danger Theory”

5. Damaged cells are also evidence of danger, and they signal through
Damage-Associated Molecular Patters (DAMPs). HMGB1 is a DAMP.
Necrotic cells release Damage Associated Molecular Patterns

6. Since cells of the immune system see DAMPs and PAMPs with the same receptors,
the outcome is very similar.
You can have sepsis (or sepsis-like conditions like Multi-Organ Failure)
without pathogens –> “sterile inflammation”
Both trauma and pathogens can give “immune paralysis”
If you want a reason for this, in nature very often infection will follow trauma
(but not in our hospitals, fortunately)
Antibiotics will not reverse sepsis, at most they will protect against more infection
25 kDa
Message number 1: infection and trauma are seen as almost equal

7. HMGB1 is evolutionarily conserved from sponges onwards, 99% identical in all mammals.
Related proteins (Nhp6A/B) exist in yeast.
They chaperone DNA bending.
25 kDa
HMGB1 is a major component of chromatin, and a DAMP

8. HMGB1 is an ancient DAMP

9. HMGB1 is released by necrotic cells,
and retained by apoptotic cells
detergent necrotic apoptotic
Scaffidi & al
Nature 2002

10. All cells that die non-apoptotically release HMGB1
- Mechanical injury
- Excitotoxicity (hyperstimulation of neurons)
- Infection by viruses expressing anti-apoptotic proteins
- Death by alkylating agents
- Death by excessive ROS exposure
In fact, release of HMGB1
is now considered evidence against apoptotic death

11. HMGB1
DAPI
untreated apoptosis H2O2 hypoxia

12. Extracellular HMGB1 recruits and activates inflammatory cells
Andrassy et al, Circulation 2008
WT control WT-48h I/R WT-48h I/R + box A
Brain ischemia Muhammad & al, J Neuroscience 2008
Hepatitis Sitia et al, J Leukoc Biol 2007
Peritonitis Orlova et al, EMBO J 2007
Ischemia-Reperfusion of the heart
Andrassy et al, Circulation 2008

13. www.buckinstitute.org/TheScience/thegibson/documents/
(sulphonic,
irreversible)
sulphinic,
only reduced
enzymatically
Cysteine thiol chemistry
Disulfide bond
sulphenic,
usually
labile
SOH

14. The redox states of HMGB1 control its activities
Venereau et al., J Exp Med. 2012
OXIDATION
23 45 106

15. Fully reduced HMGB1 binds
2 molecules of SDF-1/CXCL12,
and wraps them almost completely
CXCL12 BoxB

16. PNAS
2010

17. Our initial model

18. And what about HMGB1 and coagulation?
Vessel damage causes
coagulation,
by exposure of collagen
and platelet adhesion and
activation

19. Activated platelets secrete HMGB1
Platelets contain a high amount of
HMGB1 in the cytosol.
Upon activation,
Platelets partially secrete HMGB1 in
microparticles and partially retain it on
their surface
resting activated

20. Neutrophils are activated by HMGB1
Recombinant HMGB1
activates neutrophils,
and so do activated
platelets and platelet-
derived microparticles

21. Blocking HMGB1 blocks neutrophil activation by platelets
Neutrophilactivation

22. Activated platelets produce ROS, which oxidize HMGB1,
and sulfone HMGB1 activates neutrophils

23. … and finally HMGB1-activated neutrophils produce NETs

24. Another instance where activated neutrophils cause thrombosis

25. Conclusions
A novel mechanism of coagulation
mediated by platelets and neutrophils,
and producing “white” thrombi
mainly composed of neutrophils
and NETs

26. Credits
Emilie Vénéreau
Mariagrazia Uguccioni
IRB Bellinzona
Daniel J. Antoine Ulf Andersson Angelo Manfredi
U. Liverpool Karolinska Institutet San Raffaele University
Kevin Tracey
North Shore University Hospital, NY
Maura Casalgrandi

27. It all holds together…al, Circulation 2008
NF-κB
CXCL12
Chemotaxis
CXCR4
NF-κB
RAGE
TLR4
Inflammation
cytokines/chemokines
HMGB1
secretion
more
CXCL12