1. 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 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