PAMPs, DAMPs and our evolving understanding of Sepsis and …...•RR >20, PCO2

Carl J. Hauser MD, FACS, FCCM

PAMPs, DAMPs and our evolving understanding

of Sepsis and SIRS

Gulf War Subcommittee

Disclosures / Competing interests

FUNDING• NIH • DoD (CDMRP) • CIMIT • No commercial funding

RAC-GWVI Meeting November 1-2, 2010 Presentation 3 - Hauser

BURNSSURGERY

OTHERSPANCREATITIS

INFECTION SIRS TRAUMA

≥ 2 of the following:

•Temp >38°C, <36°C

•Pulse >90

•RR >20, PCO2 <32

•WBC >12,000, <4000 or >10% bands

Systemic Inflammatory Response Syndrome (SIRS)

Inflammatory response to illness of any source

1/3 of all hospitalized patients – More than half of all ICU patients– Nearly all SICU patients– Morbidity and mortality 2° organ

failure • Lung (ALI / ARDS) > liver/kidney

Burden of SIRS


Inflammation can reflectInfection or ‘Sterile SIRS’

Hemoperitoneum vsbacterial peritonitis

Aspiration vs bacterial pneumonia

Clinical concepts of SIRS inadequate

“““STUFFSTUFFSTUFF”””

SIRSSIRS DEATH

Infection

Trauma

Fractures

Shock

CytokinesCytokines

??

??

??

??

ALIALI

‘DANGER’ molecules

Mechanistic understanding of SIRS

WRONG!


‘Danger’ molecules

SIRSSIRS DEATH

Infection

TRAUMA activate innate immunity Fractures

Shock

??

??

ALIALIinnate innate immunityimmunity

Redundant cytokine cascade

PRRPRR

PRRPRR

Inflammatory program

Pathogens → PAMPs → PRRInjury → DAMPs → PRR

??

Recognized by Pattern Recognition Receptors (PRR)

In the setting of infections

PAMPsPAMPsTLR / GPCRTLR / GPCR

In non-infective conditions

Ancient (invertebrates, multi-celled)PMN, Mφ, DC, NKC

No clonal expansionPRR on germ-line (TLRs, GPCRs)

multi-functional

Immediate response to danger motifs

Rapid responses in trauma, sepsis

Innate immunity


Exogenous infective motifs (LPS, FPs, bacterial sugars, ‘CpG’ DNA, dsRNA, flagellin…)

Bind PRRs immune activationCytokines etc

Symptomatic infective SIRS (“sepsis”)↑ NO· release → vasodilatation↑ PMN-EC interactions → capillary leak

PAMPs

Non-infective motifs ? Endogenous products of tissue injury

? Intracellular motifs released by mechanical injury

? Membrane motifs changed by toxins? New motifs 2’ to metabolic, I/R stress

Bind PRRs immune activationCytokines etc

?? …symptomatic non-infective SIRS

?? DAMPs…


Putative DAMP PRRHMGB-1 TLR4S-100 RAGEHSP 30/60 TLR4B7-H3 TREM

Few knownSignal through PRR’s like PAMPs

Intracellular DAMPs

Mitochondria as DAMPs…why are clinical sepsis and SIRS so

often indistinguishable?Mitochondria were saprophytic

bacteriaBecame endo-symbiontsEvolved into organelles

?‘Septic’ response to MT?


13 ‘endogenous’ peptides begin with n-formyl-met

? Do they activate FP receptors‘Bacteria-like’ DNA

Unmethylated ‘CpG’ repeats? Do they activate TLR-9

Do mitochondria contain DAMPs?

Does mechanical tissue injury cause

circulation of mitochondrial debris ?

(MTD)


Fluo

resc

ence

(nor

m)

1000

100

10

10 10 20 30 40

Cycles

Fluo

resc

ence

(nor

m)

1000

100

10

10 10 20 30 40

Cycles

PatientsVolunteers

Cyt B

Cyt C-III PatientsVolunteers

Fluo

resc

ence

(nor

m)

1000

100

10

10 10 20 30 40

NADH PatientsVolunteers

0

10

20

30

40 PatientsVolunteers

Ct v

alue

s (c

ycle

s)

Cyt B Cyt C-III NADH

* * *

a c

b d Cycles

mtDNA circulates after blunt trauma

Zhang, Hauser , Nature 2010

Do shock / ischemia-reperfusion injury

result in circulation of MTD ?


Plasma mtDNA in rat HS

mtD

NA

(µg/

ml)

Naïve 3h 1d 3d 7d

A

0.00

0.05

0.10

0.15

0.20

0.25

*

*

*

*

Zhang, Hauser; Shock 2010

Volunteer Plasma Patient Plasma Reaming Fluid

Ct c

ount

(cyc

les)

0

10

20

30

40Plasma Cyto B in Femur Fx

**

*P<0.01 vs vol plasma (ANOVA)

mtDNA appears in plasma of FFx patients

214 -fold increase

Hauser, J Ortho Trauma 2010


Do MTD activate inflammatory cell

signaling ?

mtFPs activate [Ca2+]i (via FPR1)

c d0 50 100 150 200 250

0

40

80

120

Ca2+

[Ca2+

] i(n

M)

Seconds

MTD + Anti-FPR1

MTD

500

0Anti-FPR1

+-

Cal

cium

Influ

x (A

UC

) (nM

·s)MTD

*

No MTD

Zhang, Hauser , Nature 2010


0

200

400

600

0 2min 5min 10min

p-p3

8 (A

rbitr

ary

unit)

p-p38p38

**

mtDNA (μg/ml) 5 10ODN - + - + p-p38

p38

mtDNA (μg/ml) 5 10ODN - + - + p-p38

p38

mtDNA activates p38 via TLR9

TLR9 blocked by CQ, ODNsZhang, Hauser , Nature 2010

Does MTD activate inflammatory cell

phenotypes ?


0

100

200

300

400

500

MTDLPS

MTD (µg/ml) LPS (µg/ml)0 10 20 40 100 200 400 1 10

*

*4hr

0

1

2

3

4

5

MTDLPS

**

0 10 20 40 100 200 400 1 10MTD (µg/ml) LPS (µg/ml)

24hr

***

***

IL-8

(pg/

ml)

IL-8

(ng/

ml)

MTD activates cytokine production

Zhang, Hauser Nature 2010

mtDNA activates PMN / EC interactions


Do mitochondrial DAMPs activate innate

immunity in vivo?

0

500

1000

1500

2000

Lung

MM

P-8

(uni

ts)

*

Naïve Media MTD0

1

2

3

4

5

6

Naïve Media MTD

BA

LF %

PM

N

*

MTD → PMN attack on lung

MMP-8 in lung PMN in BALF



MTD causes ALI

Naïve Media MTDNaïve Media MTD

0

10

20

30

40

50

Sham 3h 6h 24h

BA

LF T

NFα

(pg/

ml) *

0

50

100

150

200

Sham 3h 6h 24h

BA

LF IL

-6 (p

g/m

l)

*

0

50

100

150

BA

LF a

lbum

in (µ

g/m

l)

Naïve Media MTD

*

0.0

0.5

1.0

1.5

2.0

2.5

Lung

wet

/dry

wei

ght R

atio

Naïve Media MTD

*A B

C D

Albumin Wet/Dry

TNF-α IL-6


Sham i.v. MT (=5% liver injury) at 6h

MTD causes ALI



Media MTD

Airway

Airway

MTD ALI is oxidant-related

4-HNE stainsZhang, Hauser Nature 2010

Nature editorial March 4, 2010

Evolutionary conservation of PAMPs and DAMPs in bacteria and mitochondria cause many similarities between sepsis and SIRS


BURNS

OTHERSPANCREATITIS

Innate immune cells

TISSUE TRAUMA

So what is ‘septic’ SIRS?

SURGERY

INFECTION

PAMPs from infection cause SIRS

PAMPs

Bystander Organ Injury

OTHERS

PANCREATITIS

INFECTION

What is non-infectious SIRS?

necrosis

Injury causes SIRS without infectionSIRS then predisposes to infection2° Sepsis perpetuates SIRS →MOF→death

BURNS SURGICAL WOUNDS

FRACTURESCrush injury

DAM

Ps

DAMPs

DA

MPs

DA

MPsInnate

immune cells Bystander

Organinjury


1) Remove PAMPs (bio-markers)Antibiotic Tx Drainage, source control

2) Rx SIRS after source controlTarget PRR, signal cascadesSteroids, aPC, anti-cytokine Tx(All dangerous w/o source control)

Treatment of infective SIRS

1) Remove DAMPs (bio-markers)Debride / drain sourcesAvoid antibiotics

2) Prevent / treat SIRS earlyTarget DAMPs and PRRInterrupt inflammatory signalingSafe w/o infection (but ??healing)

Treatment of endogenous SIRS


Hauser LabKiyoshi ItagakiQin ZhangMustafa RaoofTolga Sursal

Junger LabYu Chen

Yuka Sumi

LondonKarim Brohi

Acknowledgements


PAMPs, DAMPs and our evolving understanding of Sepsis and …...•RR >20, PCO2

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