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

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

2. 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

3. P. Aeruginosa Quorum-sensing signal molecules

4. 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 3-oxo-C12-HSL at low micromolar concentrations.

5. 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

6. Experimental Basis • 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

7. 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. • 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

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

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

10. 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.

12. 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 T-cell 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 anti-CD28 antibodies

13. 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.

14. 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.

15. LPS driven TNF-άsecretion assay, 3-oxo-C12-HSL at 50 µM and above suppressed secretion PQS significantly promoted secretion above 25 µM LPS-stimulated TNF-ά secretion from hPBMC

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

17. 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. 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 Conclusions

18. 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 • 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 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

19. 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.