Female Sex workers and its linkage to HIV susceptibility in vitro A Dichotomous Relationship of TLR7 and TLR8 Responses in HIV Exposed Seronegative Female Sex workers and its linkage to HIV susceptibility in vitro B. UNIVERSITY OF NAIROBI Omange R. W. 1 , Su R.-C.1, Dil N.1, Apidi W.A.1, Meeme C.K.3, Tatari N.2 Maina A.K.4,7, Kimani. M.3,8, Kimani J. 1,3, Mesa C. 3, Ramdahin S.1, Meyers A.F.A1,6, Gounni S. A.2, Plummer F.A.1,2,3, Ball T. B1,2,3,6. 1 Departments of Medical Microbiology and 2 Immunology University of Manitoba, 3 Dept of Medical Microbiology-Univ. of Nairobi, Kenya, 4 Kenya Medical Research Institute, Nairobi-Kenya, 5 National Microbiology laboratory, Winnipeg, 6 Public Health Agency of Canada (PHAC), 7 ITROMID-JKUAT, Juja-Kenya, 8 Methodist University, Nairobi-Kenya. Introduction Identifying the correlates of innate protection against Human Immunodeficiency Virus is an important goal for development of effective anti-HIV therapies and vaccines. Not all exposures to HIV result in infection. A small group of female commercial sex-workers in Nairobi have remained HIV exposed but seronegative (HESN) in spite many years of high risk sex work. Hypothesis Recognition of ssRNA analogous to HIV’s genetic material by TLR8 in HESN PBMCs, results in higher cognate receptor expression, increased TLR8 signal transduction, higher and more balance cytokine responses and greater inhibition of HIV replication in vitro compared to susceptible (HIV-N) controls. Objectives To quantify and compare cytokine responses and cytokine milieu of PBMCs from HESN and HIV-N before and after stimulation with TLR4, 7 and 8 agonists. To compare TLR4, 7 and 8 expression before and after cognate ligand stimulation of PBMCs from HESN and HIV-N. To compare TLR7 and 8 signalling and activation of related pathways with cognate ligand stimulation of PBMCs from HESN and HIV-N. To compare susceptibility to HIV primary isolates in vitro, of TLR7 and TLR8 pre-treated PBMCs from HESN and controls Methods Figure 1: Experimental Layout Figure 2: TLR7 and TLR8 Signalling Results Figure 3: Higher Cytokine Responses to TLR8 and not TLR4 or TLR7 stimulation by HESN PBMCs Lower cytokine responses to both TLR4 and TLR7 stimulations, but higher TLR8 responses in HESN PBMCs. Figure 4: Cytokine Correlations before and after TLR7 and TLR8 stimulation The cytokine microenvironment of HESN PBMCs is more co-ordinately regulated that of susceptible controls Figure 5: Lower of TLR7 and higher TLR8 expression and signalling in PBMCs of HESN Figure 6: Lower expression of specific cytokine mRNA in HESN PBMCs with TLR7 stimulation . Lower expression CSF2, CSF3, IL-10 and IL-12A mRNA in PBMCs of HESN with TLR7 stimulation. Figure 7: HIV infection assays Pre-treatment of PBMCs with TLR7 ligand enhanced susceptibility to infection with primary HIV isolates, whereas stimulation of TLR8 reduced susceptibility of HIV targets cells in PBMCs, more so in HESN when compared to controls. Conclusion PBMCs of HESN were hypo-responsive to TLR4 and TLR7 stimulations, but hyper-responsive to TLR8 through HIV analogous ssRNA. The differences in outcomes of the in vitro HIV infection assays following the activation of TLR7 or TLR8 in PBMCs, was linked to the distinct cytokine profiles generated by either stimulation. Acknowledgements Special thanks to the participants of this study, IID and GHTP for funding my PhD studies, advisory committee, my supervisors Dr T.B. Ball and Dr F.A. Plummer, and members of Plummer/Ball lab. Further in formation For further information regarding this poster, contact the first author using this email umomange@cc.umanitoba.ca A. A. B. C. D. B. F. G. I. E. H. J. K. L. N. M. B. A. C. D. Unstimulated TLR4 TLR7 TLR8 IFN-γ IL-12p40 IL-1β IL-2 IL-6 IL-10 TNF-α MIP-1α IP-10 IFNα2   IFN-γ IL12p40 IL-1β IL-2 IL-6 IL-10 TNF-α MIP-1α IP-10   IFN-γ IL12p40 IL-1β IL-2 IL-6 IL-10 TNF-α MIP-1α IP-10   IFN-γ IL12p40 IL-1β IL-2 IL-6 IL-10 TNF-α MIP-1α IP-10   PBMC isolation TLR7 TLR8 PMA + Iono Media Cytokine quantification Luminex Array TLR Signaling Gene Analysis ELISA (HIV p24) HESN TLR4 HIV-N The ‘dichotomy’ in TLR responsiveness of the HESN PBMCs was linked to differential expression of TLR7 or TLR8 and activation of cognate TLR signalling pathways with ligation Stimulation Up-regulated (HESN) Down-regulated (HESN) TLR7 TLR7, MyD88, TAB1, JUN, NFκB complex and MAPKs pathway, CSF2, CSF3, TLR8 TLR8, c-FOS, Heat shock proteins, NFκB complex proteints and MAPKs pathway. IRAK2