1

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.

ART

RIPE (2 mo)u-IRIS IE (4 mo)

Blood draw

ART

ART

RIPE (1-2 mo) IE (4 mo)

Unm

aski

ng

TB-IR

ISCo

ntro

lG

roup

1: n

o M

TBCo

ntro

lgr

oup

2: M

TB

1-3 months

1-3 months

1-3 months

ART

ART + RIPE

Study design

Blood draw

Blood draw

1. Control HIV N= 58

2. MTB + HIV N= 31

3. Unmasking MTB IRIS

N= 2318 confirmed or probable TB IRIS• 11 pulmonary• 7 extra-pulmonary

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

Study participant characteristics

IL-8

pg/m

l

C-reactive protein

ng/m

l x 1

0-3

25

20

15

10

5

0

Β2-μglobulin

pg/m

l

IL-6

pg/m

l

HGF

pg/m

l

EGF

pg/m

l

CREB-regulated cytokines and acute-phase reactants in unmasking M. tuberculosis-associated IRIS

Benjamini Hochberg FDR < 0.10

G-protein-coupled

receptor(s)

Adenylate cyclase

cAMP

PKACREB

CyclooxygenasePTGS1/2

Prostaglandins

Aspirin

NSAID

COX-2 inhibitors

Hypothesis for unmasking MTB-IRIS pathogenesis

MyD88M. tuberculosis

HIV-1Other pathogens?

Innate effectorsCellular receptors

( e .g.: TLR, Mannose receptors, DC-SIGN, others)

PI3K, AktGSK3 MAPK

Inflammatory response

Pro-inflammatory genes

NF-kB

Adaptive effectors(e.g. TCR)

Gene expression patterns highlightsThreshold= 1.3 fold background expression; ANOVA p value cutoff= 0.05

Pattern 1 (total n= 80): Caspase 6, PKA1*, CD226, Heat Shock Protein B1, cMER

HIV TB u-IRIS

Pattern 2 (total n= 61): CDK7, BCL-10, MAPK-7, ERB-B2HIV TB u-IRIS

•Pattern 3 (total n=122): Adenylate cyclase 3*, prostaglanin E synthase, TNF superfamily, E2F4

HIV TB u-IRIS

Pattern 4 (total n= 157): CXCR4, IL-16, CD44, CD160, MAPK9 HIV TB u-IRIS

unmasking MTB IRIS

MTB (independent of IRIS)

*Upstream of CREB*Downstream of CREB

IL-16: A, Baier M, Serfling E, Kurth R. Proc Natl Acad Sci 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..

CREB family gene expression: heat map

* *

*

*

Gen

e E

xpre

ssio

n

HIV TB IRIS

3

4

5

6x 10

4 CREB1

HIV TB IRIS

0.5

1

1.5

2

x 104 CREB1

HIV TB IRIS

1000

2000

3000

4000

CREB5

HIV TB IRIS1200

1600

2000

2400CREB3L2

HIV TB IRIS200

300

400

500

CREBL1

HIV TB IRIS

400

500

600

CREBL2

HIV TB IRIS

300

400

500

CREBBP

HIV TB IRIS

3000

3500

4000

CREBBP

HIV TB IRIS

500

1000

1500

CREBZF

CREB family gene expression summary

Kruskall Wallis FDR < 0.10 Wilcoxon < 0.05 Wilcoxon < 0.10

TLR family gene expression: heat map

Gen

e E

xpre

ssio

n

HIV TB IRIS

200

600

1000

TLR10

HIV TB IRIS

1000

2000

3000

TLR8

HIV TB IRIS

500

1000

1500

TLR7

HIV TB IRIS500

1000

1500

2000

TLR6

HIV TB IRIS

400

800

1200

TLR5

HIV TB IRIS

500

1000

1500

TLR4

HIV TB IRIS

200

300

400

TLR2

HIV TB IRIS

1000

2000

3000

TLR1

*

*

*

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

Funding:NIH/NIAID grant RO1 AI069996 to L.A. The Philadelphia Foundation The Stengel-Miller familyAIDS funds from the Commonwealth of Pennsylvania and from the Commonwealth Universal Research Enhancement Program, Pennsylvania Department of HealthCancer Center Grant (P30 CA10815)Penn Center for AIDS Research (CFAR).

• The Wistar Institute, Philadelphia, PA, USA:• Noor Dawany • Andrew Kossenkov • Celia Chang • Louise Showe • Luis J. Montaner

• Department of Molecular Medicine and Haematology, University of the Witwatersrand, Johannesburg, South Africa:• Denise Lawrie• Deborah Glencross• Wendy Stevens

• Clinical HIV Research Unit, Department of Medicine, University of the Witwatersrand/Themba Lethu Clinic, Johannesburg, South Africa:• Study participants• Katerina Roussos• Prudence Ive • Francesca Conradie• Ian Sanne