Pediatric Multiple Organ Dysfunction Syndrome

The Inflammatory Response in the ICU: The Good, The Bad, and The Ugly Mark W. Hall, MD The Ohio State University College of Medicine Nationwide Children’s Hospital Columbus, OH

Disclosures • I have no conflicts of interest to disclose. • I will be talking about off-label use of medications during this talk.

My apologies in advance to United Artists. ©1966

Does this patient have an over- or under-active innate immune response?

In other words…..does this patient need an anti-inflammatory therapy or a pro-inflammatory therapy?

The Good and the Bad

Pro-inflammatory

PG

– – – – – –

IL-1β TNF IL-6 (?) IL-8 IL-17 Eicosanoids

Anti-inflammatory – – – – – –

IL-10 TGFβ IL-11 sTNF receptor IL-1ra IL-4

The Good and the Bad

Pro-inflammatory

PG

– – – – – –

IL-1β TNF IL-6 (?) IL-8 IL-17 Eicosanoids

• Yeah….but therapies targeting these guys have failed to consistently improve sepsis outcomes! – Anti-LPS – IL-1ra – Anti-TNFα* – Anti-bradykinin – Anti-PAF – Methylprednisolone/ dexamethasone

Immunologic balance • Systemic Inflammatory Response Syndrome (SIRS) – TNFα, IL-1β, IL-8, IFNγ

• Compensatory Antiinflammatory Response Syndrome (CARS) – IL-10, TGFβ

• Homeostasis: The patient is able to respond to a challenge but is not actively inflamed.

Monneret, Adv in Sepsis, 2005

The Bad

The Good The Ugly

Where can we look for markers of immunoparalysis?

What about the monocyte? 2. TNF 1.

3.

Activated Monocyte 1. Phagocytosis

2. Intracellular killing 3. Antigen presentation 4. Extracellular TNF production

TNF

4.

TNF

TNF

Immunoparalysis

TNF

HLA-DR expression < 30%

Deactivated Monocyte TNF Ex-vivo TNF

TNF TNF

HLA-DR Quantification simple flow cytometry

Monneret et al, Intensive Care Med, 2006

Monocyte HLA-DR expression < 30% has been associated with adverse outcomes from sepsis-induced MODS in adults and children.

July, 2006

• 86 adults with septic shock, sampled on Days 1-2 and 3-4. • Overall mortality 34% (61 survivors, 25 non-survivors)

Immunoparalysis

TNF

Deactivated Monocyte TNF Ex-vivo TNF

TNF

TNF Impaired ex-vivo LPS-induced TNF production

Immunoparalysis: Pittsburgh-Columbus Study • Immunoparalysis: Ex-vivo TNFα production < 200 pg/ml for more than 3 days • Combined data sets from children with MODS at Nationwide Children’s Hospital (30) and Children’s Hospital of Pittsburgh (40). Immune function monitored serially. • Immunoparalysis incidence: 24/70 (34%)

Immunoparalysis in the PICU • A state of prolonged immunoparalysis was associated with increased relative risks for the development of adverse outcomes in children with MODS:

Relative risks (RR) with 95%CI if immunoparalysis is present for more than 3 days: Outcome

RR with 95%CI

Nosocomial Sepsis

3.3 (1.8 – 6.0)

Death

5.8 (2.1 – 16)

Ex vivo TNF response (pg/ml)

*

1800 1400 1000 600

*

200 Day 1

Day 3

Day 7

Day 14

Pediatric MODS patients who developed nosocomial sepsis (■) had lower ex vivo TNFα production over time compared to those without nosocomial sepsis (▲).

Ex vivo TNF response (pg/ml)

1600

*

1400 1200 1000 800

*

*

600 400 200 0 Day 1

Day 3

Day 7

Day 14

Pediatric MODS patients who died (■) had lower ex vivo TNFα production over time compared to those who survived (▲).

Immunoparalysis in other ICU settings Immunoparalysis doesn’t just happen after sepsis!

In 36 children undergoing CPB, reduction in ex vivo TNFα response was associated with the development of SIRS/sepsis.

PICUFlu Sites in the LPS-stimulation Study

10000

p = 0.002

*

1000

ve vi Su r

D

d

100

ea d

Ex vivo TNF production (pg/ml)

The innate immune response and death

• Critically ill children who died (n = 6) had lower ex vivo LPS-induced TNFα production capacity than did children who survived (n = 71). • This remained true when controlling for age, PRISM, steroid use, and baseline immune compromise (p = 0.0005, multivariable regression).

Ex vivo TNF production (pg/ml)

Critical Trauma at NCH p = 0.003; ANOVA

2500 2000 1500

* control median and 25th-75th percentile

**

1000 500 0

1-2

3-4

5-6

Post-trauma Day n = 60, 13 developed nosocomial sepsis, 47 did not.

So what?

Evidence for role of immunomodulation • Döcke et al (Nature Medicine, 1997) – Survival in 6 of 9 adult patients with sepsisinduced immunoparalysis after treatment with IFN.

• Nierhaus et al (Intensive Care Medicine, 2003) – Normalization of monocyte function without increased systemic inflammation in 9 adult patients with severe sepsis and immunoparalysis after treatment with rhGM-CSF. Six patients survived.

GM-CSF in critically ill adults with SIRS n = 15

30%

n = 18

30%

Rosenbloom et al, Chest, 2005 Monocyte HLA-DR expression improved in GM-CSF treated group compared to placebo. Increased HLA-DR expression was associated with clearance of infection (p=0.02)

• Prospective, randomized, placebo-controlled, double-blind clinical trial

• Adults with severe sepsis/septic shock underwent immune monitoring. Those with monocyte HLA-DR expression < 8,000 molecules/cell for 2 days were randomized to get SQ GM-CSF or placebo for 8 days. • Monocyte HLA-DR expression, ex vivo LPS-induced TNFα production, cytokines, and outcomes were measured

n = 19 subjects per group.

• There was no increase in systemic IL-6 levels in the GM-CSF-treated group.

• There were no GM-CSF-related adverse events reported. • Patients in the GM-CSF arm were weaned from the ventilator sooner. Underpowered to address mortality.

The Good and The Bad?

Pro-inflammatory

PG

– – – – – –

IL-1β TNF IL-6 (?) IL-8 IL-17 Eicosanoids

Anti-inflammatory – – – – – –

IL-10 TGFβ IL-11 sTNF receptor IL-1ra IL-4

What about other aspects of immune function?

Cytopenias • Problem: Critical illness in the setting of leukopenia. – Cancer – Transplantation – Drug-induced – Sepsis-induced

Neutrophil Lymphocyte

ANC < 500/mm3: ↑ incidence of adverse outcomes in the setting of malignancy

ALC < 1000/mm3 or ↓CD4 count : ↑ incidence of adverse outcomes in the settings of HIV, malignancy, transplantation

Lymphocyte apoptosis in adult sepsis Hotchkiss, J Immunol, 2001

Lymphopenia is associated with negative outcomes in pediatric MODS (Felmet, JI, 2005) • Prolonged lymphopenia – Occurred only in patients with MODS (17/58 vs. 0/55 ) – Associated with secondary infection with OR 5.5 (95% CI 1.7-17)

• controlling for immune suppression and steroids – Associated with death OR 6.8 (95% CI 1.3-34)

• controlling for immune suppression, steroid use, and PRISM score

B cell depletion in spleens of children with MODS and lymphopenia

Summary: Restore Immune Balance • Neutropenia and lymphopenia contribute to adverse outcomes in the ICU and demand specific management: – Neutropenia: GCSF, antimicrobial prophylaxis, removal of contributory drugs, +/- WBC transfusion – Lymphopenia: antimicrobial prophylaxis, removal of contributory drugs

• Innate and adaptive immune suppression is common in critically ill children and we need to develop robust, standardized monitoring regimens. – Monocyte HLA-DR, Ex vivo TNFα production capacity

• Immunostimulatory therapies such as GM-CSF hold promise for the reversal of immunoparalysis and should be subject of prospective study.

Acknowledgements The Research Institute at Nationwide Children’s Hospital The Section of Pediatric Critical Care Medicine

Joseph A. Carcillo, MD

Mark D. Wewers, MD

NICHD NHLBI

CANTREAT study Lab protocol Mark W. Hall, MD Director, Immune Surveillance Laboratory The Research Institute at Nationwide Children’s Hospital Columbus, OH

Multicenter ex vivo stimulation TNFα quantification LPS-stimulation protocol development Site training and education Stimulation solution/kit manufacture - Quality control Batch-ship supernatants Ship stimulation reagents monthly

Stimulation procedure

CANTREAT sites

What you will need.... • Gloves, PPE (e.g. lab coat) • Blood tubes – 4 ml green top – 6 ml dark blue top

What you do.... • Collect blood – 4 ml green top – 6 ml dark blue top

Ex vivo stimulation (do in duplicate) • What you need: – Pipettes and tips – Stimulation tubes – Incubator

Ex vivo stimulation (do in duplicate) • What you need: – Microcentrifuge – Empty storage tubes

Ex vivo stimulation (do in duplicate) • 1. Within 30 minutes of collecting blood in the green top (sodium heparin) tube.... • 2. Pipette 50μl of whole blood into a labelled stimulation tube.

Ex vivo stimulation • 3. Incubate for exactly 4 hours at 37°C. • 4. Spin the tube in a microcentrifuge for 5 minutes at 1000 x g.

Ex vivo stimulation • 5. Carefully pipette the supernatant into a clean, labelled empty storage tube. • 6. Freeze at -80° C.

While you are waiting.... What you need.... • Centrifuge – 1000 x g – 4 and 6 ml tubes

What you do.... • Spin at 1000 x g for 5 minutes

What you will need.... • Pipettes and tips • Empty storage tubes

What you do.... • Pipette off plasma/serum in 500μl aliquots into labelled empty storage tubes. • Freeze at -80° C.

YES! NO! No

Each subject will have a sheet of preprinted labels that looks something like this.....

CANTREAT 01-001-1 Green Top tube

CANTREAT 01-001-1 Dk Blue Top tube

CANTREAT 01-001-1 LPS stim tube 1

CANTREAT 01-001-1 LPS stim tube 2

CANTREAT 01-001-1 Green Top plasma 1

CANTREAT 01-001-1 Green Top plasma 2

CANTREAT 01-001-1 Green Top plasma 3

CANTREAT 01-001-1 Green Top plasma 4

CANTREAT 01-001-1 Dk Blue Top serum 1

CANTREAT 01-001-1 Dk Blue Top serum 2

CANTREAT 01-001-1 Dk Blue Top serum 3

CANTREAT 01-001-1 Dk Blue Top serum 4

CANTREAT 01-001-1 LPS stim sup 1

CANTREAT 01-001-1 LPS stim sup 2

CANTREAT 01-001-1 Case Report Form

CANTREAT 01-001-2 Green Top tube

CANTREAT 01-001-2 Dk Blue Top tube

CANTREAT 01-001-2 LPS stim tube 1

CANTREAT 01-001-2 LPS stim tube 2

CANTREAT 01-001-2 Green Top plasma 1

CANTREAT 01-001-2 Green Top plasma 2

CANTREAT 01-001-2 Green Top plasma 3

CANTREAT 01-001-2 Green Top plasma 4

CANTREAT 01-001-2 Dk Blue Top serum 1

CANTREAT 01-001-2 Dk Blue Top serum 2

CANTREAT 01-001-2 Dk Blue Top serum 3

CANTREAT 01-001-2 Dk Blue Top serum 4

CANTREAT 01-001-2 LPS stim sup 1

CANTREAT 01-001-2 LPS stim sup 2

CANTREAT 01-001-2 Case Report Form

CANTREAT 01-001-3 Green Top tube

CANTREAT 01-001-3 Dk Blue Top tube

CANTREAT 01-001-3 LPS stim tube 1

CANTREAT 01-001-3 LPS stim tube 2

CANTREAT 01-001-3 Green Top plasma 1

CANTREAT 01-001-3 Green Top plasma 2

CANTREAT 01-001-3 Green Top plasma 3

CANTREAT 01-001-3 Green Top plasma 4

CANTREAT 01-001-3 Dk Blue Top serum 1

CANTREAT 01-001-3 Dk Blue Top serum 2

CANTREAT 01-001-3 Dk Blue Top serum 3

CANTREAT 01-001-3 Dk Blue Top serum 4

CANTREAT 01-001-3 LPS stim sup 1

CANTREAT 01-001-3 LPS stim sup 2

CANTREAT 01-001-3 Case Report Form

CANTREAT 01-001-4 Green Top tube

CANTREAT 01-001-4 Dk Blue Top tube

CANTREAT 01-001-4 LPS stim tube 1

CANTREAT 01-001-4 LPS stim tube 2

CANTREAT 01-001-4 Green Top plasma 1

CANTREAT 01-001-4 Green Top plasma 2

CANTREAT 01-001-4 Green Top plasma 3

CANTREAT 01-001-4 Green Top plasma 4

CANTREAT 01-001-4 Dk Blue Top serum 1

CANTREAT 01-001-4 Dk Blue Top serum 2

CANTREAT 01-001-4 Dk Blue Top serum 3

CANTREAT 01-001-4 Dk Blue Top serum 4

CANTREAT 01-001-4 LPS stim sup 1

CANTREAT 01-001-4 LPS stim sup 2

CANTREAT 01-001-4 Case Report Form

For one sampling time.... CANTREAT 01-001-1 Green Top tube

CANTREAT 01-001-1 Dk Blue Top tube

CANTREAT 01-001-1 LPS stim tube 1

CANTREAT 01-001-1 LPS stim tube 2

CANTREAT 01-001-1 Green Top plasma 1

CANTREAT 01-001-1 Green Top plasma 2

CANTREAT 01-001-1 Green Top plasma 3

CANTREAT 01-001-1 Green Top plasma 4

CANTREAT 01-001-1 Dk Blue Top serum 1

CANTREAT 01-001-1 Dk Blue Top serum 2

CANTREAT 01-001-1 Dk Blue Top serum 3

CANTREAT 01-001-1 Dk Blue Top serum 4

CANTREAT 01-001-1 LPS stim sup 1

CANTREAT 01-001-1 LPS stim sup 2

CANTREAT 01-001-1 Case Report Form

CANTREAT 01-001-2 Green Top tube

CANTREAT 01-001-2 Dk Blue Top tube

CANTREAT 01-001-2 Subject number LPS stim tube 1

CANTREAT CANTREAT 01-001-2 01-001-2 Green Top plasma 1 Site number Green Top plasma 2 CANTREAT 01-001-2 Dk Blue Top serum 1

CANTREAT 01-001-2 Dk Blue Top serum 2

CANTREAT CANTREAT 01-001-2 01-001-1 Green Top plasma 3 Green Top tube CANTREAT 01-001-2 Dk Blue Top serum 3

CANTREAT 01-001-2 LPS stim tube 2 CANTREAT 01-001-2 Sample number Green Top plasma 4 CANTREAT 01-001-2 Dk Blue Top serum 4

What you’ll get... • First shipment: – – – – – – –

24 stimulation tubes Green top tubes Dark Blue top tubes Empty storage tubes Labels SOP Incubation log

Save the boxes!

Stimulation tubes should be cool, not frozen. Let me know if otherwise!

What you’ll get... • Monthly shipments: – 24 stimulation tubes – Incubation log – Green/blue tubes, storage tubes periodically – If you need more.... just let me know!

Save the boxes!

Each quarter... • Ship your samples back to me: – Group samples from each subject into individual plastic bags. – Ship back in one of the monthly shipping containers on dry ice. (2.5 kg should do) – Overnight FedEx per Janet.....

Questions????