Innate Immunity Reprogramming in Sepsis

Mohamed Elgazzar, PhD

Assistant Professor

Internal Medicine

East Tennessee State UniversityEast Tennessee State University

When Toll-like receptors (TLRs) sense a threat they signal innate cells such as neutrophils and macrophages to initiate the acute phase of inflammation

If the threat is limited, the inflammatory response resolves within hours

If the threat is severe, the acute phase is replaced by cell reprogramming that sustains a chronic inflammatory phase

Background

Sepsis represents an uncontrolled immune response to exposure to microbes and microbial products, such as during a traumatic injury

Reflects dysregulation of temporal sequence that normally protects against threats

Develops into two contrasting phenotypes: SIRS & CARS SIRS is induced by bacterial infection or non-infectious

causes such as trauma or major surgery

Sepsis

During SIRS- hyperinflammation characterized by excessive production of inflammatory mediators “cytokine storm,” damage to the vasculature, and hypotension, and if not reated early can result in vascular shock, organ dysfunction and death

During CARS- hypoinflammation and immunosuppression characterized by down-regulation of inflammatory mediators due to tolerance of neutrophils and macrophages to bacterial toxins, significant apoptosis of lymphocytes and dendritic cells, and persistent primary and secondary infection

Pathophysiology

acute phase chronic phase

(immunoactivation) (immunosuppression)

Infla

mm

atio

n in

de

x - anti-inflammatory cytokines-T-cell apoptosi-reduced antigen presentation-expansion of MDSCs

- proinflammatory cytokines- decreased bacterial clearance

SIRS MARS CARS

1 to 5 6 to

Time course changes in sepsis (days)

Sepsis phenotypesSepsis phenotypes

Phenotypes of severe inflammation & sepsis

baseline(infection/injury)

activated (poised promoters)

hyperinflammatory phase(cytokine storm)

silencedhypoinflammatory phase

resolving(reversal of gene reprogramming)

outcomesmortality or survival

severe

threat

severe

threat

mild

threat

mild

threat

Mortality rates are higher in humans and animals with chronic sepsis

Treatment modalities targeting the hyperinflammatory phase (SIRS) were often effective in animal models but failed in human clinical trials;

Reason: a delay between the onset of sepsis and the delivery of anti-inflammatory therapy when most patients enter the immunosuppressive (chronic) phase

Clinical Significance

Tolerance or hyporesponsiveness of innate cells to stimulation by bacterial toxins sustains immunosuppression and chronic infections

We detected this phenotype in in vitro cell model of sepsis and in septic patients

There is:

1. an epigenetic component that silences transcription of inflammatory genes,

2. a microRNA (miRNA) component that represses translation of these genes, and

3. a cellular component manifested by disruption of myeloid cell development and expansion of MDSCs, and

Mechanism

Induction of endotoxin tolerance in THP-1 human monocyte cell model

Responsive Tolerant

0 1 2 4time in LPS (h)

RNAProtein

1st LPS 2nd LPS

020406080

100120140

0 2 4 6 8 10 12rela

tive

expr

essi

on (

fold

)

TNF

El Gazzar et al (2007); J Biol Chem

p65:p50

S10

activated

H3p50:p50

K9

basal

me p

p65:RelB

p50:p50

K9 RelB

silenced

?me

Modules of proinflammatory gene transcription silencing (the epigenetic component)

Chromatin remodeling is a dynamic process in sepsis

McCall & El Gazzar (2010); J Innate Immun

Conclusions

NF-kB transcription factors, and DNA and histone based epigenetic processes cooperatively interact to silence proinflammatory gene expression during the systemic hyperinflammatory phase

Do interactions between epigenetic signals and transcription factors contribute to chromatin remodeling?

Although we can reverse the epigenetic-mediated transcription silencing of inflammatory genes, we cannot recover protein levels

This Suggests an additional layer of (translational) repression

MicroRNAs (miRNAs) are small (~22 nucleotide-long) non-coding RNAs that have emerged as key posttranscriptional regulators of gene expression

In mammals, miRNAs are predicted to control ~30% of all protein-coding genes

By base pairing to complementary AU-rich sequences in the 3`UTR region of the target mRNA, miRNAs mediate mRNA degradation or translational repression

miRNA sequences and their predicted target genes can be analyzed using a number of prediction algorithms such as miRBase (http://microrna.sanger.ac.uk) and micoRNA targets (http://www.microrna.org)

MicroRNA-dependent translation repression in sepsis

microRNA biogenesis

LPS

NF-kB

TLR4

AAAAAA 3`5`GpppAREORF

repression

3’UTR5’UTR

CDE

213

-miR-125b at 94-115-miR-579 at 489-502-miR-221 at 591-613-miR-181a at 487-507

-ARE= 34-nt at 462-495-CDE= 15-nt at 570-585

mi-RISC

miR-221miR-579miR-125b

Ago2

TIAR TTP

AUF1

Model of translation repression of TNF in sepsis

(The microRNA component)

El Gazzar et al (2010); J Biol Chem

Model of translation repression by microRNAs

RBM4AAAAA

Ago2

translationarrest

RBM4

RBM4AAAAA

Ago2cap

p-body

cytoplasm

and/or

RBM4

Tolerant

MKP-1

RBM4RBM4

RBM4

p

AAAAAeIF4A/4G

translation

Responsive

cytoplasm

nucleus

Ago2

mRNA degradation

AAAAA

p

p

We discovered the epigenetic and microRNA codes that sustain chronic sepsis, by repressing proinflammatory gene expression

We can reverse the epigenetic and miRNA-based gene repression program

This is clinically significant because reversing gene repression correlates with resolution of sepsis and survival: patients who survive late sepsis exhibit innate cell competency and inflammatory gene activation

Conclusions …

Inflammation-induced reprogramming (i.e., during SIRS) of innate cells may underlie the development of the hyporesponsive/immunosuppessive state

Evidence supports expansion of bone marrow progenitor cell populations during inflammation

We hypothesize that the initial hyperinflammatory (acute) phase of sepsis induces reprogramming of innate cell differentiation and/or maturation which may sustain immunosuppression and the chronic sepsis phenotype.

Hypothesis

Sham (n=20)CLP (n=20)

CLP + vehicle control (n=25)CLP + CD34+ cells (n=30)

Days post CLP

% s

urv

ival

0 2 4 6 8 10 12 14 16 18 20 22 24 26 280

20

40

60

80

100

Adoptive transfer of CD34+ hematopoietic progenitors

improves late sepsis survival

Adoptive transfer of CD34+ hematopoietic progenitors

improves late sepsis survival

Brudecki et al (2011); Infect Immunity

Circulating levels of proinflammatory cytokinesCirculating levels of proinflammatory cytokines

0

200

400

600

800

1000

1200

1400

Sham CLP CLP +

CD34

IL-6

(p

g/m

l)

days 14-16(chronic phase)

0

200

400

600

800

Sham CLP CLP +

CD34

TN

F

(p

g/m

l)

**

0

200

400

600

800

1000

1200

1400

Sham CLP CLP +

CD34

IL-6

(p

g/m

l)

**

days 2-4 (acute phase)

Resolved inflammationResolved inflammation

0

200

400

600

800

Sham CLP CLP +CD34

TN

F(

pg

/ml)

Peritoneal macrophages from chronically septic mice

reconstituted with CD34+ cells have normal immune rsponse

Peritoneal macrophages from chronically septic mice

reconstituted with CD34+ cells have normal immune rsponse

days 14-16

*

0

1000

2000

3000

4000

Sham CLP CLP +

CD34

IL-6

(p

g/m

l)

days 2-4

0

1000

2000

3000

4000

Sham CLP CLP +

CD34

IL-6

(p

g/m

l)

Ex vivo stimulationEx vivo stimulation

0

500

1000

1500

2000

Sham CLP CLP +

CD34

TN

F(

pg

/ml)

*

0

500

1000

1500

2000

Sham CLP CLP +

CD34

TN

F(

pg

/ml)

Peritoneum Blood

Bacterial load

days 2-4

days 14-16

CF

U/1

ml

CLP

CLP +

CD34

0

200

400

600

CF

U/1

ml

CLP

CLP +

CD34

0

200

400

600

CLP

CLP +

CD34

CF

U/m

ou

se

0

1.0 108

2.0 108

3.0 108

4.0 108

CLP

CLP +

CD34

*p=0.001

CF

U/m

ou

se

0

1.0 108

2.0 108

3.0 108

4.0 108

CD34+ cells enhance bacterial clearance

in chronically septic mice

CD34+ cells enhance bacterial clearance

in chronically septic mice

Macrophages Neutrophils

Phagocytic activity

days 14-16

B

days 2-4

*

0

20

40

60

80

100

120

CLP CLP +

CD34

mea

n flu

ores

cenc

e (5

85 n

m)

0

20

40

60

80

100

120

CLP CLP +

CD34

mea

n flu

ores

ence

(5

85 n

m)

0

20

40

60

80

100

120

CLP CLP +

CD34

mea

n flu

ores

cenc

e (5

85 n

m)

mea

n flu

ores

ence

(5

85 n

m) *

0

20

40

60

80

100

120

CLP CLP +

CD34

A

Fluorescein-conjugated E. coli emission (585 nm)

coun

tsDay

s 2-

4D

ays

14-1

6

CLP

100 101 102 103 104

CLP + CD34

100 101 102 103 104100 101 102 103 104

CLP + CD34

CLP

10 10 10 10 100 1 2 3 4

CLP + CD34

100 101 102 103 104

CLP + CD34

100 101 102 103 104

CLP

100 101 102 103 104

CLP

100 101 102 103 104

CD34+ cells improve bacterial phagocytic activity

of innate cells in chronically septic mice

CD34+ cells improve bacterial phagocytic activity

of innate cells in chronically septic mice

CD34+ cell-derivatives home to sites of inflammationCD34+ cell-derivatives home to sites of inflammation

Day 2

Day 5

bone marrow SpleenPeritoneum

The initial hyperinflammatory (acute) phase of sepsis reprograms innate cell differentiation and/or maturation to initiate and sustain immunosuppression and chronic inflammation

These processes may be linked to inflammation-driven myelopoiesis

Conclusions

MDSCs expand in BM, spleen, lymph nodes in nearly all inflammatory conditions

They are a mixed population that includes progenitors of macrophages, plymorphonuclear and dendritic cells

In mouse, they are phenotyped as GR1+ CD11b+ myeloid cells. In human, they are CD33+ CD11b+ cells

They are potently immunosuppressive, affecting innate and adaptive immunity

In tumor-bearing animals and human, their elimination improve anti-tumor immunity

Myeloid-derived suppressor cells (MDSCs) underlie chronic sepsis pathogenesis

Dramatic expansion of Gr1+ CD11b+ MDSCs cells in late sepsis

day 0 day 3 day 6 day 12A

C

CD11b-PE

Gr1

-FIT

C10 10 10 10 100 1 2 3 41

01

01

01

01

00

12

34

10 10 10 10 100 1 2 3 410

10

10

10

10

01

23

4

10 10 10 10 100 1 2 3 410

10

10

10

10

01

23

4

10 10 10 10 100 1 2 3 410

10

10

10

10

01

23

4

BB

**

0

20

40

60

80

100

0 3 6 12

Days post CLP

Gr1

+ C

D11

b+ c

ells

(%

) day 3 day 6

MDSCs can enhance or attenuate the systemic inflammatory response

saline (n=20)MDSCs from D3 (n=30)MDSCs from D12 (n=35)

Days post CLP

% s

urv

iva

l

0 2 4 6 8 10 12 14 16 18 20 22

0

20

40

60

80

100

MDSCs from chronically septic mice lose differentiation potential

CD11b-PE

F4/

80-A

PC

CD11b-PE

F4/

80-A

PC

MHC II-FITCC

D11

c+-P

EMHC II-FITC

CD

11c-

PE

Day 12

CD11b-PE

F4/

80-A

PC

CD11b-PE

F4/

80-A

PC

MHC II-FITC

CD

11c+

-PE

MHC II-FITC

CD

11c-

PE

Day3

B

CD11b-PE

Grr

1-F

ITC

A

10 10 10 10 100 1 2 3 410

10

10

10

10

01

23

4

10 10 10 10 100 1 2 3 410

10

10

10

10

01

23

4

10 10 10 10 100 1 2 3 410

10

10

10

10

01

23

4

10 10 10 10 100 1 2 3 410

10

10

10

10

01

23

4

10 10 10 10 100 1 2 3 410

10

10

10

10

01

23

4

10 10 10 10 100 1 2 3 410

10

10

10

10

01

23

4

10 10 10 10 100 1 2 3 410

10

10

10

10

01

23

4

10 10 10 10 100 1 2 3 410

10

10

10

10

01

23

4

10 10 10 10 100 1 2 3 410

10

10

10

10

01

23

4

Total MDSCs CD31+-enriched MDSCs

LT-HSC ST-HSC MPP CMP GMPGranulocyte

MonocyteMP

GP

normalnormalnormal

LT-HSC ST-HSC MPP CMP GMPGranulocyte

MonocyteMP

GP

GMPImmature

granulocyte

Immaturemonocyte

MP

GP

septicseptic

Pathway of hematopoietic stem cell differentiation

and development of innate cell repertoire

Pathway of hematopoietic stem cell differentiation

and development of innate cell repertoire

MiRNAs disrupt myeloid cell repertoire during sepsisMiRNAs disrupt myeloid cell repertoire during sepsis

MPP CMP GMP

miR-21+

miR-181+

miR-21+

miR-181+

miR-21+

miR-181+normal

Gfi1+ Gfi1+

Immature MDSC

miR-21b+++

miR-181+++granulocyte

dendritic cell

monocyte

MPP CMP

miR-21+++

miR-181+++

miR-21+++

miR-181+++sepsis

granulocyte

dendritic cell

monocyte

Fig. 9. Depicts disruption of myeloid cell repertoire by miR-21 and miR-181b

The initial hyperinflammatory (acute) phase of sepsis reprograms innate cell differentiation and/or maturation to initiate and sustain immunosuppression and chronic inflammation

Expansion of Gr1+ CD11b+ myeloid-derived suppressor cells (MDSCs) may underlie the immunosuppression in chronic sepsis

MDSC expansion in sepsis is a programmed response to inflammation, regardless of its sources

microRNAs are likely to play a role in this sepsis-induced innate immunity cell reprogramming and MDSC expansion

Conclusions and directions…

LAB COLLABORATORS

Laura Brudecki Charles McCall, MD

Research Assistant Wake Forest University

Jessica Jordan (PhD student) Benjamin Garcia, PhD Keeley Haggard (undergrad.) Princeton University

Donald Feruson, PhD

ETSU

Acknowledgement