Transcript PowerPoint Presentation - Slide 1

pDAG Stimulates the Inflammasome while Truncated pDAG
Downregulates Inflammasome Signaling
Alicia M. Holmgren1, Sihem Sassi-Gaha1, James D. Thacker2, Carol M. Artlett1
1Department
Abstract
The inflammatory peptide, 1-peptidyl-2,3-diacylglyceride (pDAG )
isolated from goat serum, was found to be more effective at
curing horses with respiratory disease than antibiotic therapy
alone. Previous experiments demonstrated that pDAG was
capable of stimulating the production and secretion of
proinflammatory cytokines including IL-6, IL-8, and MCP-1 in
macrophage. We examined the ability of pDAG peptide to
stimulate fibroblasts. Caspase-1, IL-1beta, IL-33, IL-6, IL-8,
MCP-1, and MIP-1alpha increased in a dose-dependent manner
in normal fibroblasts incubated with pDAG. Furthermore, we
found that caspase-1, IL-1 beta, IL-33, IL-6, and MCP-1
increased in a time dependent manner. Further analysis of the
signaling of pDAG suggested that it operates through the
inflammasome and implies that pDAG is working as a stimulant
of the innate immune response. We also analyzed a truncated
form of pDAG, which differed from pDAG peptide by the deletion
of an isoleucine at the C-terminus. Normal fibroblasts treated
with truncated peptide decreased proinflammatory cytokine
mRNA levels when compared to controls. To investigate the
truncated peptide response further, we analyzed fibroblasts
isolated from SSc patients that have elevated inflammasome
signaling. The truncated peptide reduced the mRNA transcript
levels of many proinflammatory cytokines, such as caspase-1,
MCP-1 and IL-33; suggesting that it inhibited inflammasome
signaling. This data allows us for the first time to (1) understand
the mechanism whereby pDAG is able to stimulate an innate
immune response via inflammasome signaling and (2) identify a
truncated form of the peptide that reduces aberrant
inflammasome activation in a fibrotic disease like SSc.
of Microbiology and Immunology, Drexel University College of Medicine, Philadelphia, PA 19129
2TherimuneX Pharmaceuticals, Inc., Doylestown, PA
Results
pDAG Increases Proinflammatory Cytokines in Normal Fibroblasts
Tissue Culture: Normal fibroblasts from a 16 year old individual
(GM06112, passage 15), from a 31 year old (GM00024, passage
10 and 12), and fibroblasts from SSc affected lesions, passage 8,
were cultured in growth media (Dulbecco’s Modified Eagle’s
Medium
supplemented
with
10%
FBS
and
1%
penicillin/streptomycin) at 37°C and 5% CO2.
Transfection: Cells were plated in 500uL of culture medium one
day prior to transfection at 50-80% confluence in a 24 well plate.
pDAG, truncated pDAG peptide, or no peptide (control) was diluted
in Opti-MEM I medium to yield a solution of 5 ug/mL. PLUS
reagent (Invitrogen) and pDAG were mixed and incubated 5
minutes. Lipofectamine (Invitrogen) was added to PLUS-pDAG
mixture and incubated for 30 minutes prior to use. 0.1 uL-10 uL of
pDAG/PLUS/lipofectamine solution was added to a well with 500uL
growth media. Incubate 1-3 days at 37°C and 5% CO2.
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 βactin and control was set to 100.
.
Conclusions
Figure 2: Fibroblasts stimulated with pDAG
increase some proinflammatory cytokines in
a time-dependent manner. RT-PCR results are
normalized to beta-actin and controls are set to
100. Most cytokines are expressed highest after
3 day incubation with pDAG peptide. IL-18
appears to peak earlier, around Day 2.
Introduction
Hamm et al. (1), reported a serum fraction derived from goat
(Capra hircus) that was effective as an adjunctive therapy with
standard antibiotics for the treatment of suppurative lower
respiratory disease in horses. TherimuneX Pharmaceuticals, Inc.
isolated and identified the immunomodulatory compound in the
goat serum fraction as 1-peptidyl-2,3-diacylglyceride (pDAG).
Initial studies with pDAG have indicated that it triggers the
release of proinflammatory cytokines in monocytes and
monocyte derived macrophage, such as MCP-1, MIP-1, IL-6,
IL-8, and IL-1. Fibroblasts are sentinel cells in skin structures.
We therefore extended our studies to normal dermal fibroblasts
and investigated the potential for pDAG stimulation of nonimmune cells.
The inflammasome is a newly identified inflammatory signaling
pathway that triggers the activation and release of the IL-1 family
of cytokines (IL-1, IL-18, and IL-33), important mediators of
inflammation and the innate immune response. Recent studies
from our lab have indicated that the inflammasome is activated in
Systemic Sclerosis (SSc). SSc is a fibrotic, TGF-beta driven
autoimmune disease, characterized by uncontrolled fibrosis of
the dermis and internal organs. During production of a synthetic
pDAG peptide a truncated pDAG peptide was developed and
tested for immunomodulatory activity. The truncated pDAG
peptide was found to down regulate inflammasome activation in
SSc fibroblasts.
Methods
Proinflammatory cytokines, IL-1α & β, IL-33, MCP-1, etc., were
increased in a time dependent manner upon fibroblast stimulation
with pDAG indicating stimulation of the inflammasome.
Truncated pDAG was not able to stimulate proinflammatory
cytokine production and even decreased Caspase-1 and Collagen
1A1 mRNA.
Truncated pDAG reduced mRNA levels of inflammasome related
cytokine below control in SSc fibroblasts.
Future Studies
Truncated pDAG Does Not Trigger
Proinflammatory Cytokines in
Normal Fibroblasts
Truncated pDAG Decreases
Proinflammatory Cytokines in
SSc Fibroblasts
Determine if pDAG activates the inflammasome using knock-out
mice (NALP3-/-, ASC-/-, MyD88-/-, and TRIF-/-).
Investigate the cytokine and chemokine profile in mice treated with
pDAG by Real Time PCR, immunofluorescence of skin and lung,
ELISA of serum, lung, and skin.
Flow Cytometry of skin cells for activation: fibroblasts,
keratinocytes, macrophages, DCs, mast cells, B cells, and T cells.
Western blot analysis of inflammasome components
Caspase-1 and NALP3.
including
Continue work with SSc fibroblasts and truncated pDAG by western
blots for inflammasome proteins and ELISA for secreted
proinflammatory cytokines and chemokines.
Identify the mechanism by which the truncated pDAG decreases
collagen production and expression of proinflammatory cytokines
and chemokines.
References
Figure 3: Truncated pDAG is unable to stimulate normal
fibroblasts. RT-PCR results are normalized to beta-actin and
controls set to 100.
Very high concentrations are needed to
increase cytokine production using truncated pDAG when
compared to pDAG peptide.
Collagen 1A1 and caspase-1
expression are dramatically decreased in response to truncated
pDAG.
Figure 1: Pathway of inflammasome activation.
Figure 4: Truncated pDAG decreases proinflammatory cytokines
in SSc fibroblasts. RT-PCR results are normalized to beta-actin and
controls are set to 100. Fibroblasts were derived from the fibrotic
lesions of SSc patients.
1. Hamm, D., et al. 2002. Equine Vet. J. 34(1): 71-5.
2. Kummer, J.A., et al. 2007. J. Histochem. Cytochem. 55(5):
443-52.
3. Martinon, F. 2009. J Leukoc. Biol. 83(3): 507-11.