Inflammasome Activation of Dermal Fibroblasts by an
Endogenous
Peptide Activate Tissue Resident Immune Cells
Carol M Artett1, Sihem Sassi-Gaha1, James D Thacker2
1Department
of Microbiology and Immunology, Drexel University College of Medicine, Philadelphia, PA 19129
2TherimuneX Pharmaceuticals, Inc., Doylestown, PA 18902
Abstract
Results
We challenged 42 Swiss Webster mice with a lethal dose of S.
typhimurium (5 x103 cfu/mouse) intraperitoneally; and 21 received a μM dose
of the natural pDAG (isolated from goat serum) one day prior to the challenge.
The mortality of the mice was monitored daily; natural pDAG conferred
protection in the treated mice (Fig 2A). Eighty percent of the treated mice
survived compared to 10% that did not (P <0.0001 – pooled data from 2
studies). As pDAG induced IL-6 (Fig 3), we measured acute phase protein
production in rabbits. We examined the production of antibodies in rabbits
challenged with M. tuberculosis (Freund’s Complete Adjuvant, FCA) with or
without 15 g of purified pDAG. Six New Zealand White female rabbits
received 0.25 ml FCA, however three also received 15 g natural pDAG.
Blood was drawn one day prior to the inoculation and on days 3, 5, 7, 10, 12,
14, and 17. Serum was analyzed for total immunoglobulin and M. tuberculosis
specific IgM antibodies by ELISA. Natural pDAG induced higher IgM levels
than FCA alone (Fig 2B).
100
pDAG+FCA
90
1.4
pDAG
IgM Titer, AU
Control
60
ATP
IL-33
IL-18
K+ efflux
acALY18
oligomerization
Active
NALP3
PYD
CARD
NF-B
pro-IL-33
pro-IL-18
20
0.4
10
0.2
0
1
2
3
4
5
6
7
8
9
0
10
0
Day
3
pro-IL-1
ASC
Pro-caspase 1
Inflammasome
Proteolytic cleavage
p50 p65
Nucleus
NF-B
p50 p65
NF-B responsive
Gene transcription
Figure 1. Pathway of inflammasome activation.
10
12
14
17
acALY-18 induced fibroblast secretion of
IL-6 and IL-8
IL-1β orchestrates IL-6 and IL-8 secretion (6), and because acALY-18
(synthetic peptide for natural pDAG) induced IL-1β, we measured IL-6 and IL-8
secretion from fibroblasts treated with acALY-18/lipofectamine. We found that
after 24 h, IL-6 in the culture media was 230 ng/ml (p < 0.01) and IL-8 was 100
ng/ml media (p < 0.001) significantly more than that which was observed in
lipofectamine treated fibroblasts.
Conditioned media from fibroblasts treated
with acALY-18, induced the expression of
inflammatory mediators in THP-1 cells
We measured the response of THP-1 cells to acALY-18/lipofectamine
after 24 hours. RNA was extracted and inflammasome transcripts; CASP1,
IL-18, IL-33, and IL-6 were quantified. All transcripts were then normalized to
β-actin (Fig 4A). We found that acALY-18 induced the expression of CASP1
1.2-fold, IL-1β 2.5-fold, and IL-33 1.6-fold (Fig 4A). However, when
conditioned media from acALY-18/lipofectamine stimulated cells was used to
culture the THP-1 cells, we found that the THP-1 cells differentiated into
macrophages and the expression of CASP1 was induced 2-fold, IL-18 3-fold,
IL-1β 7220-fold, and IL-33 1338-fold (Fig 4B). It is significant that the
expression of these transcripts was found to be exponentially higher with the
conditioned media than with THP-1/acALY-18 alone.
250
pDAG does not induce a cytokine storm
Protein
Media
Only
pDAG
0.04 ng/ml
pDAG
0.4 ng/ml
pDAG
4 ng/ml
LPS
100 ng/ml
IL-1β
-
-
-
+
+++
IL-2
-
-
-
+
+
IL-4
-
-
-
-
+
IL-5
-
-
-
-
-
IL-6
-
-
++
++++
++++
IL-7
-
-
-
-
-
IL-8
-
+
++
++++
++++
IL-10
-
-
-
-
++
IL-12p70
-
-
-
-
+
IL-13
-
-
-
-
+
IL-17
-
-
-
-
+
GM-CSF
-
-
-
+
-
INF-
-
-
-
-
+++
TNF-α
-
-
-
+
+++
G-CSF
-
-
-
+
++
MCP-1
-
-
+
++
++
MIP-1β
-
-
+
++
++++
Table
1.
Natural
pDAG
induces
pro-inflammatory
cytokines
from
human
leukocytes. Buffy coats were
isolated from fresh human blood
and cultured overnight with
natural pDAG or LPS. The
cytokine or chemokine profile
was determined using the BioPlex Protein Array System (BioRad, Hercules CA).
KEY:
++++  100 fold increase
+++ = 25 – 100 fold increase
++ = 5 – 25 fold increase
+ = 1 – 5 fold increase
- = no change from untreated
control buffy coat cells).
THP1
Relative Expression
200
Relative Expression
We have shown that an injection of a nM dose of natural pDAG provided a
protective benefit in a lethal bacterial challenge in the mouse. Therefore, we
wanted to determine the chemokine/cytokine profile induced by immune cells from
a freshly isolated human buffy coat. The buffy coat was incubated overnight in
RPMI supplemented with purified natural pDAG.
We determined if synthetic acALY-18 or natural pDAG were
contaminated by LPS by the following methods:
1. Premixing acALY-18 or natural pDAG with polymyxin B prior to
incubating with fibroblasts. We found that IL-6 and IL-8 mRNA
continued to be elevated with polymyxin B treated acALY-18 or
the natural pDAG isolate.
2. As LPS action requires serum proteins, we incubated acALY18 and natural pDAG in fibroblasts cultures in the absence of
FBS. We found that acALY-18 and natural pDAG were still able
to induce IL-6 and IL-8 mRNA in fibroblasts in the absence of
serum.
3. Boiled LPS is able to maintain its functionality. Therefore we
boiled acALY-18 and the natural pDAG product prior to adding
them to fibroblasts. IL-8 and IL-6 expression was found to be
ablated.
These findings confirm that LPS does not contaminate our acALY18 or natural pDAG preparations.
Methods
Transfection: Cells were plated one day prior to transfection at
80% confluence. Natural pDAG or the synthetic peptide to
natural pDAG (acALY-18) was diluted in Opti-MEM I medium to
yield a solution of 5 ug/ml. PLUS reagent (Invitrogen, Carlsbad
CA) and acALY-18 peptide were mixed and incubated 5 minutes.
Lipofectamine (Invitrogen) was added to PLUS-peptide mixture
and incubated for 30 minutes prior to use. 10 ul of PLUSpeptide/lipofectamine solution was added to the dish with the
culture media. Lipofectamine alone was included as a control.
Cells were incubated for 24 h at 37°C, 5% CO2.
Conditioned media: Fibroblasts were treated with peptide/
lipofectamine as described, overnight. Culture media was
removed and replaced with fresh media and the cells incubated
for 3 days. The fibroblast conditioned media was collected and
used to culture THP-1 cells for 3 days.
Real Time PCR: RNA was extracted using RNeasy kit (Qiagen).
IL-1 alpha, IL-1 beta, IL-6, IL-8, IL-18, IL-33, Caspase-1, MCP-1,
Collagen 1A1, and β-actin were measured by SYBR Green
Quantitative PCR. All message transcripts were normalized to
β-actin and control were normalized to 100.
Conclusions
10000
300
1000
Caspase-1
IB
7
Fig 2B. Natural pDAG acts like an Adjuvant.
All rabbits were treated with FCA, but 3 of the
rabbits were given 15 g purified natural pDAG.
Rabbits were bled on days, 3, 5, 7, 10, 12, 14,
and 17 and total immunoglobulin and IgM
specific M. tuberculosis antibodies were
measure in triplicate. No difference was
observed in total immunoglobulin between the
two groups; however, pDAG induced IgM
antibodies and by day 3 were significantly
greater (p = 0.012). This difference increased
with time: at day 5, p = 0.0004 and p < 0.0001 at
day 17.
Fig 2A. Survival curve of the S.
typhimurium challenged mice. 42 Swiss
Webster mice were challenged with a lethal
dose, 5 x 103 cfu/mouse of S. typhimurium
+/- 5 μg natural pDAG, 24 h prior to the
lethal inoculation. Mice were maintained and
monitored for 10 days. Mortality was
observed in the control group on day 4 but
not in the natural pDAG group until day 7. By
day 10, 80% of the natural pDAG group
were alive, compared to 10% in the
untreated group; p < 0.0001, pooled data
from 2 studies.
active
Proteolysis
Of IB
5
Day
IL-1
TLR
Inactive
NALP3
0.6
30
We therefore investigated the signaling of acALY-18 This
data allows us for the first time, to understand the mechanism
whereby pDAG is able to stimulate the innate immune system via
the inflammasome.
IB
p50 p65
1
0.8
The inflammasome has recently received attention due to
its role in the immune response under cellular stress (3). The
fundamental role of the inflammasome is to detect immune
danger signals (3). Signals leading to the activation of the
inflammasome are poorly understood; however, it is known that
the inflammasome is formed by a member of the NLRP (Nacht,
LRR and pyrin domain containing protein) family and ASC that
activates caspase-1 (CASP1). NLRP3 is the most extensively
studied inflammasome as it detects microbial products, cellular
stress and endogenous danger signals including extracellular
ATP (4), hypotonic stress, or toxins associated with cell injury.
The inflammasome modulates the activation of the proinflammatory cytokines, IL-1, IL-18, and IL-33.
Danger
Signals
1.2
40
Hamm et al (1). reported a serum fraction derived from the
goat (Capra hircus) that was effective as an adjunctive therapy
with standard antibiotics for treatment of suppurative lower
respiratory disease in horses. They reported that 86% of horses
treated with the serum fraction, in addition to standard antibiotics,
recovered within three weeks, whereas only 10% of horses
treated with antibiotic alone recovered (1). As no further analyses
was performed on the goat serum, we therefore sought to identify
the immunological compound mediating this effect. Indeed, we
determined that the compound 1-peptidyl-2,3-diacylglyceride
(pDAG) was the efficacious molecule (2). Studies in our
laboratory have revealed that pDAG mediates its activity by
activating the inflammasome, thereby conferring an advantage to
the recipient.
Cytosol
FCA
80
Introduction
Toxins
Figure 3. Inflammatory
Cytokine & Receptor rtPCR
Array
mRNA
expression from 84 proinflammatory
genes.
Fibroblasts treated with 3nM
acALY-18
showed
upregulated (red circles) or
down-regulated
(green
circles) genes > 2.0-fold.
Genes upregulated were
C8A, CCL2, CXCR4, IL1A,
IL1B, IL1RN, and IL-6.
Genes down regulated were
TLR2 and TLR6. Pooled
data from 3 independent
experiments.
1.6
70
acALY-18 is not contaminated with LPS
As the natural pDAG substrate in the mice challenged with a lethal
dose of S. typhimurium and dogs infected with parvovirus was given
subcutaneously and we demonstrated that diacylglycerol was a membrane
transport moiety (2), we therefore thought it was important to look at the
effects of intracellular acALY-18 on fibroblasts as a possible primary effector
cell. We treated fibroblasts with 3 nM concentration of acALY-18 plus
lipofectamine to aid in the transmembrane transport of the peptide. Cells
were harvested after 72 h and assayed using the Inflammatory Cytokine &
Receptor rt-PCR Array from SABiosciences (Frederick, MD). This array
examines 84 inflammatory genes and the results are presented as a scatter
plot (Fig 3). Key genes found to be up-regulated were C8A 6.3 fold;
CCL2/MCP1 2.2 fold; CXCR4 3.6 fold; IL1A 4.5 fold, IL1B 8.8 fold; IL1RN 9.3
fold; IL6 6 fold). Two genes were significantly down-regulated and these were
TLR2 100 fold and TLR6 18 fold).
1.8
50
Signal
acALY-18 upregulated innate immune
signaling genes in fibroblasts
Natural pDAG Protects Mice from Lethal
Bacteremia and acts as an Adjuvant in Rabbits
% Survival
Activation of innate immunity is an important strategy for host
defense as it is an early response to a pathogen derived danger
signal. Recently, we discovered a lipopeptide, 1-peptidyl-2,3diacylglyceride, that acts like a danger signaling molecule to induce
an innate immune response. The peptide moiety alone (acALY-18)
activates the inflammasome inducing the secretion of IL-1β, and
subsequently IL-6, IL-8, MIP-1α, and MCP-1 in fibroblasts and
keratinocytes. Subcutaneous administration of acALY-18 protected
mice from a lethal dose of S. typhimurium (survival: 80% treated vs.
10% untreated, P< 0.0001). We extended our initial observations
with acALY-18 to further determine its effect in fibroblasts and
subsequent effects on immune cells. Arrays confirmed the upregulation of IL-1β, MIP-1α, MCP-1, and IL-6; C8A, IL1-α, IL-1F8,
IL-1R2, IL-1RN were also increased. THP-1 cells responded to
acALY-18 fibroblast conditioned media with the up-regulation of
CASP1 (2-fold), IL-18 (2.6 fold), IL-33 (7.5 fold), (IL-1β 3600 fold),
and IL-6 (2.6 fold). The up-regulation of IL-1β and IL-33 was greater
than that observed in acALY-18 treated THP-1 cells (2.5 fold and
1.6 fold, respectively), suggesting that the fibroblast response to
acALY-18 can mediate immune cell activation. Additional studies
are further investigating the activation of the immune response by
acALY-18. acALY-18 could directly translate to a novel therapeutic
to enhance non-specific innate immune signaling against pathogens
resistant to antibiotics.
Results
150
100
THP1 +
acALY-18
THP1 + Fib. cond.
Media
100
10
THP1 + acALY-18 Fib.
Cond. Media
50
1
0
CASP1
IL-18
IL-1beta
IL-33
Fig 4A. acALY-18/lipofectamine induced the
expression of CASP1, IL-1β, and IL-33 in
THP-1 . THP-1 cells were treated with acALY18/lipofectamine for 24 h. RNA was harvested
and transcripts measured and normalized to βactin. The response of THP-1 cells to acALY18 was modest.
CASP1
IL-18
IL-1beta
IL-33
Fig 4B. Conditioned media from acALY18/lipofectamine
treated
fibroblasts
induced the expression of CASP1, IL-1β,
and IL-33 in THP-1 cells. THP-1 cells were
treated with conditioned media from
fibroblasts
treated
with
acALY18/lipofectamine for 72 h or conditioned
media from fibroblasts not treated with
acALY-18. RNA was harvested and
transcripts measured and normalized to βactin. We found that the expression of all
these transcripts were exponentially greater
than that observed in THP-1 cells treated
with acALY-18/lipofectamine (Fig 2A).
1. Natural pDAG confers protection against lethal bacteremia in
mice
2. Natural pDAG acts like an adjuvant inducing specific IgM
antibodies
3. acALY-18 (synthetic peptide of natural pDAG) induces IL-6
and IL-8 secretion from fibroblasts
4. Natural pDAG does not induce a cytokine storm but
upregulates the expression of specific cytokines
5. acALY-18 upregulates the expression of some immune
signaling genes in fibroblasts
6. THP-1 cells respond to acALY-18 in a similar manner as
fibroblasts, however acALY-18 fibroblast conditioned media
exponentially induced the expression of cytokines
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