Differential Immune Modulatory Differential Immune Modulatory Activity of P. Aeruginosa Activity of P. Aeruginosa Quorum-sensing Signal Quorum-sensing Signal

MoleculesMolecules

Presented by Inderdeep S. Presented by Inderdeep S. AtwalAtwal

Background InformationBackground Information• Pseudomonas aeruginosa is a gram-Pseudomonas aeruginosa is a gram-

negative bacterianegative bacteria• Capable of causing disease in plants, Capable of causing disease in plants,

animals and immunocompromised animals and immunocompromised humanshumans

• Has the ability to colonize a wide variety Has the ability to colonize a wide variety of tissues in the body and is capable of of tissues in the body and is capable of causing extensive tissue damagecausing extensive tissue damage

• This ability to cause damage is a direct This ability to cause damage is a direct result of its quorum sensing result of its quorum sensing

P. Aeruginosa Quorum-sensing P. Aeruginosa Quorum-sensing signal moleculessignal molecules

QSSM and immune responseQSSM and immune response

• Early immunological experiments Early immunological experiments showed that 3-oxo-C12-HSL were showed that 3-oxo-C12-HSL were shown to suppress interleukin-12(IL-shown to suppress interleukin-12(IL-12) and tumor necrosis factor 12) and tumor necrosis factor alpha(TNF-alpha(TNF-άά) ) secretion by LPS secretion by LPS stimulated macrophages and stimulated macrophages and suppresses T-cell proliferation. suppresses T-cell proliferation.

• In contrast T-helper 2 (Th2)-In contrast T-helper 2 (Th2)-dependent antibody secretion was dependent antibody secretion was enhanced by 3-oxo-C12-HSL at low enhanced by 3-oxo-C12-HSL at low micromolar concentrations.micromolar concentrations.

Subverting the Immune Subverting the Immune SystemSystem• The observations led to a hypothesis The observations led to a hypothesis

that the QSSM is a subversive system that the QSSM is a subversive system

• The QSSM could steer T-cell The QSSM could steer T-cell responses away from a host-responses away from a host-protective T-helper 1(TH1) protective T-helper 1(TH1) phenotype, to possibly promote phenotype, to possibly promote pathogen survivalpathogen survival

Experimental BasisExperimental Basis

• The goal of this study was to study The goal of this study was to study PQS, a chemically distinct QSSM from PQS, a chemically distinct QSSM from 3-oxo-C12-HSL3-oxo-C12-HSL

• They were especially looking to see if They were especially looking to see if this QSSM was capable of modulating this QSSM was capable of modulating immune responses in a similar immune responses in a similar mannermanner

Results-PBMC proliferation and Results-PBMC proliferation and IL-2 secretion following IL-2 secretion following stimulation with ConAstimulation with ConA• Intially screened PQS, 3-oxo-C12-HSL, and Intially screened PQS, 3-oxo-C12-HSL, and

3-oxo-C6-HSL in a mitogen-driven T-cell 3-oxo-C6-HSL in a mitogen-driven T-cell proliferation.proliferation.

• C4-HSL and C6-HSL were shown to have no C4-HSL and C6-HSL were shown to have no activity in a previous studyactivity in a previous study

• They found that PQS and 3-oxo-C12-HSL They found that PQS and 3-oxo-C12-HSL inhibited cell proliferation in a dose inhibited cell proliferation in a dose dependent manner when peripheral blood dependent manner when peripheral blood cells isolated and stimulated with ConAcells isolated and stimulated with ConA

• PQS was shown to be the more potent PQS was shown to be the more potent antiproliferative molecule in the assayantiproliferative molecule in the assay

ContinuedContinued

• Concurrent MTS assay showed that Concurrent MTS assay showed that the immune-suppressive window for the immune-suppressive window for 3-oxo-C12-HSL and PQS was evident 3-oxo-C12-HSL and PQS was evident in the absence of cytotoxicityin the absence of cytotoxicity

Effect of P. Aeruginosa QSSM on hPBMC Effect of P. Aeruginosa QSSM on hPBMC proliferation viability and IL-2 secretionproliferation viability and IL-2 secretion

Effect of P. Aeruginosa QSSM on Effect of P. Aeruginosa QSSM on hPBMC proliferation viability-hPBMC proliferation viability-stimulated by ConAstimulated 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 PBMC proliferation and IL-2 secretion following stimulation with anti-CD3/anti-following stimulation with anti-CD3/anti-CD28 antibodiesCD28 antibodies• Specific stimulation of T cells by the engagement Specific stimulation of T cells by the engagement

of the T cell receptor CD3 complex with specific of the T cell receptor CD3 complex with specific antibodies requires a further antibody coligation antibodies requires a further antibody coligation of CD28, a coreceptor of T cell activationof CD28, a coreceptor of T cell activation

• The CD28 pathway provides intracellular The CD28 pathway provides intracellular coactivation signals which are required for the coactivation signals which are required for the production of cytokines, such as IL-2 and gamma production of cytokines, such as IL-2 and gamma interferon to drive T-cell proliferation. interferon to drive T-cell proliferation.

• Using this fact, the group further studyed 3-oxo-Using this fact, the group further studyed 3-oxo-C12-HSL and PQSC12-HSL and PQS

• Both QSSMs consistently inhibited T-cell Both QSSMs consistently inhibited T-cell proliferation when the cells were cross-linked with proliferation when the cells were cross-linked with anti-CD3 and anti-CD28 antibodiesanti-CD3 and anti-CD28 antibodies

Effect of P. Aeruginosa QSSM on hPBMC Effect of P. Aeruginosa QSSM on hPBMC proliferation and IL-2 secretion following proliferation and IL-2 secretion following stimulation by anti-CD3/anti-CD28 stimulation by anti-CD3/anti-CD28 antibodiesantibodies

PQS and 3-oxo-C12-HSL inhibited cell proliferation induced by anti-CD3/anti-CD28 antibodies.

Effect of P. Aeruginosa QSSM on hPBMC Effect of P. Aeruginosa QSSM on hPBMC proliferation and IL-2 secretion following proliferation and IL-2 secretion following stimulation by anti-CD3/anti-CD28 stimulation by anti-CD3/anti-CD28 antibodiesantibodies

Only 3-oxo-C12-HSL inhibited IL-2 release. PQS actually showed a slight induction of IL-2 release.

LPS-stimulated TNF-LPS-stimulated TNF-άά secretion from secretion from hPBMChPBMC

• LPS driven TNF-LPS driven TNF-άά secretion assay, 3-secretion assay, 3-oxo-C12-HSL at 50 oxo-C12-HSL at 50 µM and above µM and above suppressed secretionsuppressed secretion

• PQS significantly promoted secretion PQS significantly promoted secretion above 25 µM above 25 µM

Effect of P. Aeruginosa QSSM on Effect of P. Aeruginosa QSSM on hPBMC TNF-hPBMC TNF-άά , stimulated by E. Coli , stimulated by E. Coli LPS of hPBMC.LPS of hPBMC.

ConclusionsConclusions• The experiment showed that QSSM, 3-oxo-The experiment showed that QSSM, 3-oxo-

C12-HSL and PQS are able to regulate C12-HSL and PQS are able to regulate several cascades on mammalian immune several cascades on mammalian immune response in vitro.response in vitro.

• PQS and 3-oxo-C12-HSL significantly PQS and 3-oxo-C12-HSL significantly reduced the ability of lymphocytes to reduced the ability of lymphocytes to respond to ConA.respond to ConA.

• The antiproliferative activity of PQS occurred The antiproliferative activity of PQS occurred without any effect on cell viability, while 3-without any effect on cell viability, while 3-oxo-C12-HSL suppressed proliferation before oxo-C12-HSL suppressed proliferation before cell viability.—this effect is what they term cell viability.—this effect is what they term as immune-suppressive windowas immune-suppressive window

Conclusion ContinuedConclusion Continued• Attempting to study specifically the two compounds Attempting to study specifically the two compounds

affects on T-cell stimulation, a more specific T cell affects on T-cell stimulation, a more specific T cell assay was used with anti-CD3 antibody and anti-assay was used with anti-CD3 antibody and anti-CD28 antibody to drive T-cell proliferationCD28 antibody to drive T-cell proliferation

• PQS was more potent than 3-oxo-C12-HSL in PQS was more potent than 3-oxo-C12-HSL in suppressing T-cell proliferationsuppressing T-cell proliferation

• With respect to IL-2 production in response to T cell With respect to IL-2 production in response to T cell activation, 3-oxo-C12-HSL inhibited the release of activation, 3-oxo-C12-HSL inhibited the release of this cytokine when T cells were stimulatedthis cytokine when T cells were stimulated

• Suggesting that 3-oxo-C12-HSL is acting upstream Suggesting that 3-oxo-C12-HSL is acting upstream of IL-2 secretion while PQS is preventing of IL-2 secretion while PQS is preventing proliferation by acting downstream of IL-2proliferation by acting downstream of IL-2

• TNF-TNF-άά secretion was assessed in assays where LPS secretion was assessed in assays where LPS was used to drive was used to drive TNF-TNF-άά secretion from hPBMC— secretion from hPBMC—showing that 3-oxo.. Plays a suppresive role and showing that 3-oxo.. Plays a suppresive role and PQS playing a stimulatoryPQS playing a stimulatory

What comes of this What comes of this research?research?• The production of a dual wave of The production of a dual wave of

immune modulants in compromised immune modulants in compromised patients, in combination with other patients, in combination with other immunologically confounding immunologically confounding virulence factors, may confer an virulence factors, may confer an advantage for the bacteriaadvantage for the bacteria

• Further studies need to be made to Further studies need to be made to elaborate the actual mechanisms elaborate the actual mechanisms behind the subversion system.behind the subversion system.