Transcript 1-peptidyl-2,3-diacylglyceride (PDAG

Characterization of a Unique, Naturally-occurring Immunomodulatory
Lipopeptide: 1-peptidyl-2,3-diacylglyceride (PDAG)
Mitali Purohit*, Carol Artlett*, Sihem Sassi Gaha*, James D.
1*
Thacker ,
Richard F. Rest*
*Department of Microbiology and Immunology, Drexel University College of Medicine
1TherimuneX Pharmaceuticals, Inc., Doylestown, PA
To control disease caused by emerging antibiotic-resistant
pathogens, there is a constant quest to develop novel therapeutic
agents and adjuncts to antimicrobials. We have pursued a broadspectrum strategy that involves boosting the innate immune system to
combat a broad range of infectious agents. In the search of such
immunomodulatory molecules, we screened the low-molecular weight
inflammatory proteome of mammalian plasma and serum and identified
a unique lipopeptide – 1-peptidyl-2,3-diacylglyceride (PDAG). PDAG
stimulates the innate immune response resulting in activation of tissue
resident immune cells, recruitment of phagocytic cells, and clearance of
pathogens. Serum-derived PDAG stimulates significant release from
whole human blood of proinflammatory cytokines IL-6, IL-8, MCP-1,
MIP-1α and MIP-1β. The cytokine profile induced by PDAG differs from
that induced by LPS, indicating a lack of endotoxin contamination of the
natural product.
In addition, PDAG stimulates isolated human
macrophages and THP-1 macrophage-like cells to release IL-6 and IL8 in a dose and time dependent manner. Interestingly, PDAG also
stimulates cultured human fibroblasts to secrete IL-6. We determined
whether PDAG activity was due to the entire molecule, PDAG peptide
or DAG. Neither DAG nor synthetic PDAG peptide alone induce
cytokine release from whole blood. PDAG is an exciting new and
unique immunoregulatory molecule that has a plethora of possible
clinical uses. [This work is funded by Drexel University College of
Medicine.]
INTRODUCTION
PDAG IS HOMOLOGOUS TO TRPC1
The PDAG peptide sequence is homologous with an extra-cellular loop
of TRPC-1, a transmembrane Ca2+ channel protein.
Diverse cells and mediators are involved in innate immunity.
We wanted to investigate the effects of PDAG on fibroblasts
and keratinocytes which are excellent sources of cytokines and
chemokines during inflammatory response to infections.
PDAG
Untreated
PDAG INDUCES CYTOKINE RELEASE
FROM HUMAN FIBROBLASTS
Figure 1: Putative domain structure and topology of TRPC1. Percent identity
between TRPC1 and at least one other member of the TRP family is
indicated. The red circle indicates the PDAG peptide sequence. Wes, et al.
Proc. Natl. Acad. Sci. USA Vol. 92, pp. 9652-9656
8
7
6
5
mRNA 4
(relative
3
level)
2
1
0
Control
MIP-1a
PDAG INDUCES CYTOKINE & CHEMOKINE
RELEASE FROM HUMAN BLOOD
The evolution and rapid spread of resistant microorganisms are
significant problems in nosocomial infections and are of increasing
importance in community acquired infections. There is an urgent need
for alternatives to antimicrobials that can either have direct
microbicidal activity or that can exert their effects by stimulating the
innate immune system. Immunomodulatory peptides, such as the
defensins, stimulate a number of effects on innate immune cells,
including monocytes, macrophages, and neutrophils to act as
chemoattractants, promote histamine release from mast cells, and
enhance phagocytosis at the site of infection.
IL-1a
PDAG
IL-1b
IL-8
IL-33
IL-18
700
mRNA (relative level)
ABSTRACT
PDAG PROTECTS IN A MOUSE
PERITONITIS MODEL
600
500
400
Control
Figure 6: Swiss Webster mice were challenged with Salmonella
typhimurium (5x103 cfu/mouse) by intraperitoneal injection. Mice
were treated with natural PDAG in normal saline (1.5 mg /mouse;
n=15) or saline alone (n=15) by subcutaneous injection a day before
bacterial challenge. Mortality was monitored daily for ten days. By
day 10, all mice in the control group were dead where as there was
20% death in the PDAG treated group. Administration of PDAG did
not cause a cytokine storm. Lymphopenia, neutrophilia, and
monocytosis were observed in the infected control mice, whereas
circulating neutrophils and monocytes in PDAG treated mice were at
or near normal levels. The bacterial load in the spleens of the mice
was 50% less in PDAG treated mice on day 1 (data not shown).
PDAG
SUMMARY
300
200
100
0
MCP-1
NFkB
NALP-3
Caspase-1
IL-6
Figure 4: Normal human fibroblasts were cultured in DMEM with 10% FBS at
37oC, with or without natural PDAG. RNA was purified from treated and control
fibroblasts, and assayed for the indicated cytokines or signaling molecules by
quantitative RT PCR. Values are normalized to -actin expression.
In our investigation of the low-molecular weight inflammatory
proteome in mammalian plasma and serum we identified a new
immunoactive lipopeptide with the general structure :
PDAG
Dialyze Caprine serum
Reduce dialysate volume by lyophilization
Extract lyophilized fraction with chloroform/ methanol and
remove solids
Prepare purified PDAG by preparative reversed phase HPLC.
PDAG samples are >99% chromatographically pure.
Determine PDAG composition by mass spectrometry and
sequence the PDAG peptide
PDAG INDUCES IL-6 AND IL-8
RELEASE FROM MACROPHAGES
1200
1000
800
600
400
200
0
relative m-RNA level
ISOLATION OF NATURAL PDAG
Figure 2: Fresh heparinized human blood was diluted five-fold with
RPMI 1640 medium and incubated with dilutions of purified natural
PDAG for 13 or 24 hrs. After incubation, samples were centrifuged and
plasma was assayed using the Excelarray Human Inflammation kit, or
Chemotaxis kit (Thermo Fisher Scientific). PDAG induced a dose and
time dependent release of cytokines.
Amount of Cytokine
(pg)
wherein X1 is a linear peptide and X2 and X3 are arachidonic and
stearic acid respectively. The peptide is covalently linked to the
diacylglycerol by an ester bond at the C-terminus of the peptide. We
hypothesize that 1-peptidyl-2,3-diacylglycerol, “PDAG”, activates the
innate immune cascade, activates tissue resident immune cells at the
site of injury, recruits professional phagocytic cells to local tissue,
clears pathogens, and abates disease progression.
PDAG INDUCES CYTOKINE RELEASE
FROM HUMAN KERATINOCYTES
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120
100
80
60
40
20
0
Control
PDAG
IL-6
IL-8
Cytokines
Figure 3: PMA-differentiated THP-1 cells in RPMI, 10% HI FBS and 50
µM beta-mercaptoethanol were treated with 1:100 natural PDAG for 24
hrs. Supernatants were assayed using the Excelarray Human
Inflammation kit. LPS is used as a positive control. Similar results were
seen with human monocyte derived macrophages.
Delayed cell
migration and
recruitment
Modified from NATURE BIOTECHNOLOGY VOLUME 25 NUMBER 4 APRIL 2007
FUTURE STUDIES
• Determine whether PDAG stimulates neutrophils,
monocytes or macrophages to express antibacterial
activities in vitro.
IL-6
Control
LPS 5ng/ml
PDAG (1:100)
Controlled induction of
cytokines and
chemokines
Cytokines
IL-8
Figure 5: PDAG treatment of keratinocytes induces increased expression of IL-6
and IL-8 as determined by RT-PCR. Values are normalized to β-actin.
• Determine the cellular origin of PDAG and conditions
for its production or release.
• Determine what pathways are activated by PDAG in
non-immune cells.
• Determine if PDAG induces an antimicrobial effect in
vivo.
FUNDING: This research has been funded by Drexel University
College of Medicine in a cooperative agreement with TherimuneX
Pharmaceuticals, Inc.