Differential Immune Modulatory Activity of P. Aeruginosa Quorum- sensing Signal Molecules Presented by Inderdeep S. Atwal

Background Information Pseudomonas aeruginosa is a gram- negative bacteria Pseudomonas aeruginosa is a gram- negative bacteria Capable of causing disease in plants, animals and immunocompromised humans 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 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 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. 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 3- oxo-C12-HSL at low micromolar concentrations. In contrast T-helper 2 (Th2)-dependent antibody secretion was enhanced by 3- oxo-C12-HSL at low micromolar concentrations.

Subverting the Immune System The observations led to a hypothesis that the QSSM is a subversive 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 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-C12- HSL The goal of this study was to study PQS, a chemically distinct QSSM from 3-oxo-C12- HSL They were especially looking to see if this QSSM was capable of modulating immune responses in a similar manner 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- C6-HSL in a mitogen-driven T-cell proliferation. Intially screened PQS, 3-oxo-C12-HSL, and 3-oxo- C6-HSL in a mitogen-driven T-cell proliferation. C4-HSL and C6-HSL were shown to have no activity in a previous study 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 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 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-oxo- C12-HSL and PQS was evident in the absence of cytotoxicity Concurrent MTS assay showed that the immune-suppressive window for 3-oxo- C12-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 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 T- cell proliferation. The CD28 pathway provides intracellular coactivation signals which are required for the production of cytokines, such as IL-2 and gamma interferon to drive T- cell proliferation. Using this fact, the group further studyed 3-oxo-C12-HSL and PQS 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 anti- CD28 antibodies Both QSSMs consistently inhibited T-cell proliferation when the cells were cross-linked with anti-CD3 and anti- CD28 antibodies

Effect of P. Aeruginosa QSSM on hPBMC proliferation and IL-2 secretion following stimulation by anti-CD3/anti-CD28 antibodies PQS and 3- oxo-C12- HSL inhibited cell proliferation induced by anti- CD3/anti- CD28 antibodies.

Effect of P. Aeruginosa QSSM on hPBMC proliferation and IL-2 secretion following stimulation by anti-CD3/anti-CD28 antibodies Only 3- oxo-C12- HSL 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-oxo- C12-HSL at 50 µM and above suppressed secretion LPS driven TNF- ά secretion assay, 3-oxo- C12-HSL at 50 µM and above suppressed secretion PQS significantly promoted secretion above 25 µM 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- HSL and PQS are able to regulate several cascades on mammalian immune response in vitro. The experiment showed that QSSM, 3-oxo-C12- 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. 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-oxo- C12-HSL suppressed proliferation before cell viability.—this effect is what they term as immune-suppressive window The antiproliferative activity of PQS occurred without any effect on cell viability, while 3-oxo- C12-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 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 Attempting to study specifically the two compounds 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 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 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 IL- 2 secretion while PQS is preventing proliferation by acting downstream of IL-2 Suggesting that 3-oxo-C12-HSL is acting upstream of IL- 2 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 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 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. Further studies need to be made to elaborate the actual mechanisms behind the subversion system.