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East Tennessee State University
Innate Immunity Reprogramming in Sepsis
Mohamed Elgazzar, PhD
Assistant Professor
Internal Medicine
Background
 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
Sepsis
 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
Pathophysiology
 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
Sepsis phenotypes
SIRS
MARS
CARS
acute phase
chronic phase
(immunoactivation)
(immunosuppression)
Inflammation index
- anti-inflammatory cytokines
-T-cell apoptosi
-reduced antigen presentation
-expansion of MDSCs
- proinflammatory cytokines
- decreased bacterial clearance
1 to 5
6 to
Time course changes in sepsis (days)
Phenotypes of severe inflammation & sepsis
baseline
(infection/injury)
outcomes
mortality or survival
mild
threat
activated
(poised promoters)
resolving
hyperinflammatory phase
(reversal of gene
reprogramming)
(cytokine storm)
silenced
hypoinflammatory phase
severe
threat
Clinical Significance
 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
Mechanism


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
Induction of endotoxin tolerance
in THP-1 human monocyte cell model
1st LPS
2nd LPS
relative expression (fold)
Responsive
140
120
100
80
60
40
20
0
Tolerant
RNA
Protein
0
2
4
6
8
10 12
0
time in LPS (h)
1
2
4
TNFa
Modules of proinflammatory gene transcription silencing
(the epigenetic component)
K9
me
S10
p
activated
basal
H3
p50:p50
p65:p50
K9
me
RelB
?
silenced
p50:p50
p65:RelB
El Gazzar et al (2007); J Biol Chem
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
MicroRNA-dependent translation repression in sepsis
 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 biogenesis
Model of translation repression of TNFa in sepsis
(The microRNA component)
LPS
TLR4
NF-kB
-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
Ago2
2
AUF1
TTP miR-221
3
miR-125b
1
miR-579
AAAAAA 3`
5`Gppp
ORF
5’UTR
repression
ARE CDE
3’UTR
El Gazzar et al (2010); J Biol Chem
Model of translation repression by microRNAs
Tolerant
p
Responsive
RBM4
RBM4
cytoplasm
MKP-1
cytoplasm
nucleus
p
Ago2
Ago2
RBM4
RBM4
RBM4
AAAAA
cap
and/or
Ago2
RBM4
AAAAA
translation
arrest

eIF4A/4G
mRNA
degradation
AAAAA
RBM4
p
translation
AAAAA
Conclusions …
 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
Hypothesis
 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.
Adoptive transfer of CD34+ hematopoietic progenitors
improves late sepsis survival
Sham (n=20)
CLP (n=20)
CLP + vehicle control (n=25)
CLP + CD34+ cells (n=30)
100
% survival
80
60
40
20
0
0
2
4
6
8
10
12
14
16
18
20
22
24
26
28
Days post CLP
Brudecki et al (2011); Infect Immunity
Circulating levels of proinflammatory cytokines
days 2-4
(acute phase)
*
*
600
IL-6 (pg/ml)
TNFa (pg/ml)
800
400
200
0
Sham
CLP
1400
1200
*
*
1000
800
600
400
200
0
CLP +
Sham
CLP
CD34
CLP +
CD34
days 14-16
(chronic phase)
IL-6 (pg/ml)
TNFa (pg/ml)
800
600
400
200
0
Sham
CLP
1400
1200
1000
800
600
400
200
0
CLP +
CD34
Resolved inflammation
Sham
CLP
CLP +
CD34
Peritoneal macrophages from chronically septic mice
reconstituted with CD34+ cells have normal immune rsponse
days 2-4
4000
IL-6 (pg/ml)
TNFa (pg/ml)
2000
1500
1000
500
3000
2000
1000
0
0
Sham
CLP
Sham
CLP +
CLP
CLP +
CD34
CD34
days 14-16
4000
*
1500
1000
500
IL-6 (pg/ml)
TNFa (pg/ml)
2000
*
3000
2000
1000
0
0
Sham
CLP
CLP +
CD34
Ex vivo stimulation
Sham
CLP
CLP +
CD34
CD34+ cells enhance bacterial clearance
in chronically septic mice
Bacterial load
days 2-4
4.0
108
3.0
 108
2.0

1.0
108
Blood
600
CFU/1 ml
CFU/mouse
Peritoneum
108
400
200
0
0
4.0
108
3.0
 108
2.0
108
1.0
108
0
600
*p=0.001
CFU/1 ml
CFU/mouse
days 14-16
400
200
0
CD34+ cells improve bacterial phagocytic activity
of innate cells in chronically septic mice
B
Phagocytic activity
Macrophages
Neutrophils
100
80
60
40
20
CLP + CD34
CLP
80
60
40
20
0
100 101 102 103 104 100 101 102 103 104 100 101 102 103 104 100 101 102 103 104
CLP
100
(585 nm)
CLP + CD34
120
0
CLP
CLP + CD34
CLP +
CLP
CD34
Fluorescein-conjugated E. coli emission (585 nm)
CLP +
CD34
mean fluorescence
(585 nm)
days 14-16
120
*
100
80
60
40
20
0
120
100
(585 nm)
CLP
mean fluoresence
CLP + CD34
mean fluoresence
CLP
120
(585 nm)
100 101 102 103 104 100 101 102 103 104 100 101 102 103 104 100 101 102 103 104
mean fluorescence
days 2-4
counts
Days 14-16
Days 2-4
A
*
80
60
40
20
0
CLP
CLP +
CD34
CLP
CLP +
CD34
CD34+ cell-derivatives home to sites of inflammation
bone marrow
Day 2
Day 5
Peritoneum
Spleen
Conclusions
 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
Myeloid-derived suppressor cells (MDSCs) underlie chronic
sepsis pathogenesis
 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
100 101 102 103 104
day 3
100 101 102 103 104
day 6
100 101 102 103 104
100 101 102 103 104
day 0
100 101 102 103 104
Gr1-FITC
100 101 102 103 104
A
100 101 102 103 104
Dramatic expansion of Gr1+ CD11b+ MDSCs cells in late sepsis
day 12
100 101 102 103 104
CD11b-PE
Gr1+ CD11b+ cells (%)
B
C
*
100
*
80
60
40
20
0
0
3
6
12
Days post CLP
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)
100
% survival
80
60
40
20
0
0
2
4
6
8
10
12
14
Days post CLP
16
18
20
22
MDSCs from chronically septic mice lose differentiation potential
CD11b-PE
100 101 102 103 104
MHC II-FITC
CD11c-PE
100 101 102 103 104
MHC II-FITC
CD11c-PE
F4/80-APC
100 101 102 103 104
100 101 102 103 104
100 101 102 103 104
100 101 102 103 104
CD11b-PE
MHC II-FITC
CD11c+-PE
F4/80-APC
Day 12
100 101 102 103 104
100 101 102 103 104
CD11b-PE
100 101 102 103 104
CD11b-PE
CD11b-PE
F4/80-APC
100 101 102 103 104
100 101 102 103 104
100 101 102 103 104
100 101 102 103 104
CD11c+-PE
F4/80-APC
100 101 102 103 104
100 101 102 103 104
100 101 102 103 104
Grr1-FITC
Day3
CD31+-enriched MDSCs
Total MDSCs
B
100 101 102 103 104
A
100 101 102 103 104
MHC II-FITC
Pathway of hematopoietic stem cell differentiation
and development of innate cell repertoire
normal
LT-HSC
ST-HSC
MPP
CMP
ST-HSC
MPP
CMP
Monocyte
GP
Granulocyte
GMP
septic
LT-HSC
MP
MP
Monocyte
GP
Granulocyte
MP
Immature
monocyte
GP
Immature
granulocyte
GMP
GMP
MiRNAs disrupt myeloid cell repertoire during sepsis
Gfi1+
Gfi1+
miR-21+
miR-181+
miR-21+
miR-181+
monocyte
normal
miR-21+
miR-181+
granulocyte
MPP
CMP
GMP
dendritic cell
monocyte
miR-21+++
sepsis miR-181+++
miR-21+++
miR-181+++
miR-21b+++
miR-181+++
granulocyte
MPP
CMP
Immature
MDSC
dendritic cell
Fig. 9. Depicts disruption of myeloid cell repertoire by miR-21 and miR-181b
Conclusions and directions…
 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
Acknowledgement
LAB
 Laura Brudecki
Research Assistant
 Jessica Jordan (PhD student)
 Keeley Haggard (undergrad.)
COLLABORATORS
Charles McCall, MD
Wake Forest University
Benjamin Garcia, PhD
Princeton University
Donald Feruson, PhD
ETSU