Activation of the CREB-dependent pathway distinguishes M.tuberculosis-mediated unmasking immune reconstitution syndrome (IRIS) from chronic HIV and HIV-MTB. Dawany N., Kossenkov A., Chang C., Conradie F., Ive, P. Sanne I., Montaner L.J., Showe L., Azzoni L.
Study design Blood draw u-IRIS RIPE (2 mo) IE (4 mo) Unmasking TB-IRIS ART 1-3 months Blood draw ART Control Group 1: no MTB ART 1-3 months Blood draw ART + RIPE Control group 2: MTB RIPE (1-2 mo) IE (4 mo) ART 1-3 months
Study participant characteristics Unmasking MTB IRIS diagnostic criteria: ACTG Enrollment: • ARV regimen: 106 of 112 subjects: D4t + 3Tc + EFV, median time 45 days (IQR= [28-61]. 2 IRIS exceptions, ART = 111 and 140 days) • No significant differences in: • ART duration and regimen • Baseline CD4 count and VL • Pre-ART CD4
CREB-regulated cytokines and acute-phase reactants in unmasking M. tuberculosis-associated IRIS IL-6 C-reactive protein 25 20 15 pg/ml ng/ml x 10-3 10 5 0 HGF EGF Β2-μglobulin pg/ml pg/ml pg/ml IL-8 Benjamini Hochberg FDR < 0.10 pg/ml
Hypothesis for unmasking MTB-IRIS pathogenesis Inflammatory response Adaptive effectors (e.g. TCR) Pro-inflammatory genes Aspirin • NF-kB NSAID COX-2 inhibitors GSK3 PI3K, Akt MAPK Innate effectors Cellular receptors ( e .g.: TLR, Mannose receptors, DC-SIGN, others) M. tuberculosis HIV-1 Other pathogens? MyD88
Gene expression patterns highlights Threshold= 1.3 fold background expression; ANOVA p value cutoff= 0.05 unmasking MTB IRIS u-IRIS Pattern 1 (total n= 80): Caspase 6, PKA1*, CD226, Heat Shock Protein B1, cMER HIV TB HIV TB u-IRIS Pattern 2 (total n= 61): CDK7, BCL-10, MAPK-7, ERB-B2 TB u-IRIS • Pattern 3 (total n=122): Adenylatecyclase 3*, prostaglanin E synthase, TNF superfamily, E2F4 HIV MTB (independent of IRIS) HIV TB u-IRIS Pattern 4 (total n= 157): CXCR4, IL-16, CD44, CD160, MAPK9 *Upstream of CREB IL-16: A, Baier M, Serfling E, Kurth R. Proc NatlAcadSci U S A. 1999 Feb 16;96(4):1541-6. CXCR4: Cristillo AD, Highbarger HC, Dewar RL, Dimitrov DS, Golding H, Bierer BE. FASEB J. 2002 Mar;16(3):354-64. HSP: Billack B, Heck DE, Mariano TM, Gardner CR, Sur R, Laskin DL, Laskin JD. Am J Physiol Cell Physiol. 2002 Oct;283(4):C1267-77. CD44: Rothman BL, Kennure N, Kelley KA, Katz M, Aune TM. J Immunol. 1993 Dec 1;151(11):6036-42. TNF: Matt T. Acta Med Austriaca. 2002;29(3):77-9.; Spriggs DR, Deutsch S, Kufe DW, Immunol Ser. 1992;56:3-34.. *Downstream of CREB
CREB family gene expression: heat map
CREB family gene expression summary * * * * Kruskall Wallis FDR < 0.10 Wilcoxon < 0.05 Wilcoxon < 0.10
TLR family gene expression: heat map
TLR family gene expression summary * * * Kruskall Wallis FDR < 0.10 Wilcoxon < 0.05 Wilcoxon < 0.10
Conclusions • Unmasking MTB-IRIS is characterized by: • Increased expression of cytokines and acute-phase reactants regulated by the cAMP/CREB pathway • Increased gene expression of CREBL2>CREB5 (transcription activators) • Decreased gene expression of CREBZF (transcription repressor) • These result support a role for an activation of the CREB pathway(s) in the pathogenesis of IRIS • Differential expression of TLR genes (e.g. increased TLR5) suggests another potential activation pathway. • Limitations • Small number of subjects • Lack of CREB and TLR protein expression and activation assessments • Lack of a comparative paradoxical MTB IRIS group • Possible confounding effect of treatment in the MTB control group • Planned studies • Validation of gene array results in larger longitudinal cohorts • Assessment of CREB and TLR protein levels and activity
Acknowledgements • Clinical HIV Research Unit, Department of Medicine, University of the Witwatersrand/ThembaLethu Clinic, Johannesburg, South Africa: • Study participants • Katerina Roussos • Prudence Ive • Francesca Conradie • Ian Sanne • Department of Molecular Medicine and Haematology, University of the Witwatersrand, Johannesburg, South Africa: • Denise Lawrie • Deborah Glencross • Wendy Stevens • Funding: • NIH/NIAID grant RO1 AI069996 to L.A. • The Philadelphia Foundation The Stengel-Miller family • AIDS funds from the Commonwealth of Pennsylvania and from the Commonwealth Universal Research Enhancement Program, Pennsylvania Department of Health • Cancer Center Grant (P30 CA10815) • Penn Center for AIDS Research (CFAR). • The Wistar Institute, Philadelphia, PA, USA: • NoorDawany • Andrew Kossenkov • Celia Chang • Louise Showe • Luis J. Montaner
