Transcript Wk13-P.aerug.

Differential Immune Modulatory
Activity of P. Aeruginosa Quorumsensing Signal Molecules
Presented by Inderdeep S. Atwal
Background Information
• Pseudomonas aeruginosa is a gram-
negative bacteria
• Capable of causing disease in plants,
animals and immunocompromised humans
• Has the ability to colonize a wide variety
of tissues in the body and is capable of
causing extensive tissue damage
• This ability to cause damage is a direct
result of its quorum sensing
P. Aeruginosa Quorum-sensing
signal molecules
QSSM and immune response
• Early immunological experiments showed
that 3-oxo-C12-HSL were shown to
suppress interleukin-12(IL-12) and tumor
necrosis factor alpha(TNF-ά) secretion by
LPS stimulated macrophages and
suppresses T-cell proliferation.
• In contrast T-helper 2 (Th2)-dependent
antibody secretion was enhanced by 3oxo-C12-HSL at low micromolar
concentrations.
Subverting the Immune System
• The observations led to a hypothesis that
the QSSM is a subversive system
• The QSSM could steer T-cell responses
away from a host-protective T-helper
1(TH1) phenotype, to possibly promote
pathogen survival
Experimental Basis
• The goal of this study was to study PQS, a
chemically distinct QSSM from 3-oxo-C12HSL
• They were especially looking to see if this
QSSM was capable of modulating immune
responses in a similar manner
Results-PBMC proliferation and IL-2
secretion following stimulation with
ConA
• Intially screened PQS, 3-oxo-C12-HSL, and 3-oxo•
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C6-HSL in a mitogen-driven T-cell proliferation.
C4-HSL and C6-HSL were shown to have no
activity in a previous study
They found that PQS and 3-oxo-C12-HSL inhibited
cell proliferation in a dose dependent manner
when peripheral blood cells isolated and
stimulated with ConA
PQS was shown to be the more potent
antiproliferative molecule in the assay
Continued
• Concurrent MTS assay showed that the
immune-suppressive window for 3-oxoC12-HSL and PQS was evident in the
absence of cytotoxicity
Effect of P. Aeruginosa QSSM on hPBMC
proliferation viability and IL-2 secretion
Effect of P. Aeruginosa QSSM on hPBMC
proliferation viability-stimulated by ConA
The levels of IL-2
released from
ConA-stimulated
hPBMC in the
prescence of QSSM
revealed similar
patterns to those
for cellular
proliferation.
PBMC proliferation and IL-2 secretion following
stimulation with anti-CD3/anti-CD28 antibodies
• Specific stimulation of T cells by the engagement of the
T cell receptor CD3 complex with specific antibodies
requires a further antibody coligation of CD28, a
coreceptor of T cell activation
• The CD28 pathway provides intracellular coactivation
signals which are required for the production of
cytokines, such as IL-2 and gamma interferon to drive Tcell proliferation.
• Using this fact, the group further studyed 3-oxo-C12-HSL
and PQS
• Both QSSMs consistently inhibited T-cell proliferation
when the cells were cross-linked with anti-CD3 and antiCD28 antibodies
Effect of P. Aeruginosa QSSM on hPBMC
proliferation and IL-2 secretion following
stimulation by anti-CD3/anti-CD28 antibodies
PQS and 3oxo-C12HSL
inhibited cell
proliferation
induced by
antiCD3/antiCD28
antibodies.
Effect of P. Aeruginosa QSSM on hPBMC
proliferation and IL-2 secretion following
stimulation by anti-CD3/anti-CD28 antibodies
Only 3oxo-C12HSL
inhibited
IL-2
release.
PQS
actually
showed a
slight
induction
of IL-2
release.
LPS-stimulated TNF-ά secretion from
hPBMC
• LPS driven TNF-ά secretion assay, 3-oxoC12-HSL at 50 µM and above suppressed
secretion
• PQS significantly promoted secretion
above 25 µM
Effect of P. Aeruginosa QSSM on hPBMC
TNF-ά , stimulated by E. Coli LPS of hPBMC.
Conclusions
• The experiment showed that QSSM, 3-oxo-C12-
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HSL and PQS are able to regulate several
cascades on mammalian immune response in
vitro.
PQS and 3-oxo-C12-HSL significantly reduced
the ability of lymphocytes to respond to ConA.
The antiproliferative activity of PQS occurred
without any effect on cell viability, while 3-oxoC12-HSL suppressed proliferation before cell
viability.—this effect is what they term as
immune-suppressive window
Conclusion Continued
• Attempting to study specifically the two compounds
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affects on T-cell stimulation, a more specific T cell assay
was used with anti-CD3 antibody and anti-CD28
antibody to drive T-cell proliferation
PQS was more potent than 3-oxo-C12-HSL in
suppressing T-cell proliferation
With respect to IL-2 production in response to T cell
activation, 3-oxo-C12-HSL inhibited the release of this
cytokine when T cells were stimulated
Suggesting that 3-oxo-C12-HSL is acting upstream of IL2 secretion while PQS is preventing proliferation by
acting downstream of IL-2
TNF-ά secretion was assessed in assays where LPS
was used to drive TNF-ά secretion from hPBMC—
showing that 3-oxo.. Plays a suppresive role and PQS
playing a stimulatory
What comes of this research?
• The production of a dual wave of immune
modulants in compromised patients, in
combination with other immunologically
confounding virulence factors, may confer
an advantage for the bacteria
• Further studies need to be made to
elaborate the actual mechanisms behind
the subversion system.