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High Ratios of Circulating Pro-Inflammatory Cytokines to Anti-Inflammatory IL-10 Correlate with Regional Brain Atrophy in Chronic Suppressed HIV Infection

KJ Kallianpur, M Sakoda, TM Umaki, PJ Norris, SM Keating, JD Barbour, LC Ndhlovu, D Chow, ES Nakasone, CM Shikuma

The authors report no conflicts of interest.

H a w a i ' i C e n t e r f o r A I D SU n i v e r s i t y o f H a w a i ' i - M a n o a

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Background

Cytokine levels increase in HIV infection and predict disease progression, mortality

Inflammatory cytokines may contribute to blood-brain barrier disruption

HIV-associated neurocognitive disorders (HAND)

Brain atrophy, microstructural white matter alterations, subcortical brain injury associated with high plasma MCP-11,2

1Ragin et al, 2006; 2Ragin et al, 2010

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Chronic HIV disease Inflammatory markers (IL-6, IL-8,

TNF-α,…) remain elevated after successful combination antiretroviral therapy (cART)

Plasma cytokines associated with memory performance in HIV1

1Correia et al, 2013

IL-10 inhibits monocyte synthesis of

pro-inflammatory cytokines (IL-1β, IL-6, IL-8, TNF-α…)

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Why consider ratios of cytokines to IL-10?

Balance between TNF-α and IL-10 may be important in HIV pathogenesis3

High TNF-α/IL-10 → HIV disease progression4

The balance between pro-inflammatory mediators and anti-inflammatory IL-10 may be important in neurological disease.

1Remarque et al, 2001; 2Dhabhar et al, 2009; 3Fauci et al, 1996; 4Stylianou et al, 1999

Alzheimer’s disease1: IL-1β/IL-10Major depression2 : IL-6/IL-10, low IL-10

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Objectives

i. volumes of brain regions

ii. neuropsychological test performance

To investigate relationships of plasma cytokine levels (concentrations as well as ratios over IL-10) to

in chronic suppressed HIV disease.

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Methods and study participants N=52: HIV+ subjects in HAHC-CVD†

study who underwent neuropsychological (NP) testing and brain magnetic resonance imaging (MRI)

Age ≥ 40 years

On cART for > 6 months prior to entry

Cytokine levels measured by multiplex Luminex assay

Cross-sectional assessment

†HIV Aging with HIV-Cardiovascular Disease

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Abbreviation CytokineIL-1β, IL-6, IL-8, IL-10 Interleukin-1β, etc.

TNF-α Tumor necrosis factor-αMCP-1 Monocyte chemotactic protein-1VEGF Vascular endothelial growth factor IFN-γ Interferon-γ

NT-proBNP N-terminal prohormone of brain natriuretic peptide

sE-Selectin Soluble E-SelectinsVCAM-1 Soluble vascular cell adhesion molecule-1

sICAM-1 Soluble intercellular cell adhesion molecule-1

MMP-9 Matrix metallopeptidase-9MPO Myeloperoxidase tPAI-1 Tissue plasminogen activator inhibitor-1 CRP C-reactive proteinSAA Serum amyloid ASAP Serum amyloid P

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T1-weighted MRI at 3.0T (3D turbo field echo sequence); processed with FreeSurfer

Volumes of 10 brain structures, and 3 composite gray and white matter volumes

MRI data acquisition and processing

Regional brain volumes assessedThalamus Corpus callosumCaudate nucleus Cortical gray matterPutamen Cerebral white matterGlobus pallidus Cerebellar gray matterHippocampus Cerebellar white matterAmygdala Total subcortical gray

matterNucleus accumbens

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Global & composite NP z-scoresNP z-score NP domain

NPZ_14 GlobalNPZpm Psychomotor speedNPZlrn_mem Learning and memory NPZef Executive function

NPZwm Working memory

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Data analysis

Multivariate linear regression effects of cytokines (concentrations

and ratios to IL-10) on regional brain volumes

controlled for age, nadir CD4 count, and intracranial volume

Pearson correlation NP z-scores and cytokine levels

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Variable Mean ± SD, or n (%)N 52Age (years) 50.8 ± 7.1 (40.0 – 70.0)Sex (% male) 44 (85%) Education (years) 14.4 ± 2.3 (8 – 19)Undetectable plasma HIV RNA (< 50 copies/mL) 43 (88%)

Current CD4 count (cells/mm3)465.9 ± 213.2 (33.0 – 955.0)

Nadir CD4 cell count (cells/mm3) 154.5 ± 132.8 (0 – 550.0) Ethnicity (Caucasian) 28 (54%)NPZ_14 [global] -0.07 ± 0.56NPZpm [psychomotor speed]  0.28 ± 0.62NPZlrn_mem [learning & memory] -0.26 ± 1.03NPZef [executive function]  0.15 ± 0.99NPZwm [working memory] -0.14 ± 0.78

Results: Patient characteristics

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Brain region (volume, mm3)

Cytokine [ ] (pg/mL) p β

Adjusted R2 (model)

Globus pallidus IL-1β 0.0016 -0.40 0.41

Putamen IL-1β 0.0035 -0.36 0.43

Cerebral white matter

IL-8 0.0036 -0.30 0.60tPAI-1 0.0345 -0.23 0.56sE_Selectin 0.0345 -0.22 0.56

Cortical gray matter tPAI-1 0.0446 -0.23 0.50

Nucleus accumbens

SAP 0.0276 -0.31 0.27MPO 0.0446 -0.28 0.25

Cytokine concentrations and regional volumes

No volume-cytokine associations with p < 0.001

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Region (mm3)

Cytokine ratio p β Adjusted R2 (model)

White matter (cerebral)

MPO/IL-10 0.0006 -0.36 0.64IL-1β/IL-10 0.0008 -0.36 0.63sE-Selectin/IL-10 0.0009 -0.34 0.63IL-8/IL-10 0.0012 -0.34 0.62VEGF/IL-10 0.0013 -0.34 0.62TNF-α/IL-10 0.0018 -0.33 0.62NT_ProBNP/IL-10 0.0052 -0.30 0.60IL-6/IL-10 0.0053 -0.29 0.60MCP-1/IL-10 0.0070 -0.29 0.59MMP-9/IL-10 0.0113 -0.28 0.58

Cytokine ratios and volumes: white matter

In bold: p < 0.001

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Region (mm3) Cytokine ratio p β Adjusted

R2 (model)

Thalamus

MPO/IL-10 0.0047 -0.36 0.45IL-1β/IL-10 0.0048 -0.38 0.45VEGF/IL-10 0.0056 -0.35 0.44 TNF-α/IL-10 0.0061 -0.34 0.44MCP-1/IL-10 0.0097 -0.33 0.43sE-Selectin/IL-10 0.0136 -0.31 0.42IL-8/IL-10 0.0181 -0.30 0.41MMP-9/IL-10 0.0114 -0.32 0.43 IFN-γ/IL-10 0.0381 -0.27 0.39

Cytokine ratios and volumes: thalamus

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Cytokine concentrations correlated inversely with composite NP z-scores

i.Executive function

ii.Psychomotor speed

iii.Learning and memory MCP-1 and NPZlrn_mem: β = -0.37, p=0.02

SAP and NPZef: β = -0.45, p=0.002 MPO and NPZef: β = -0.34, p=0.03

VEGF and NPZpm: β = -0.41, p=0.007 IL-6 and NPZpm: β = -0.31, p=0.043

Global and composite NP z-scores did not relate to cytokine ratios

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Summary

High ratios of multiple pro-inflammatory circulating cytokines to IL-10 decreased white matter, thalamus

volume

Most absolute cytokine concentrations did not relate to regional brain volumes (though IL-1β, IL-8 showed volumetric associations with p<0.01)

Elevated cytokine concentrations (not ratios) neurocognitive impairment

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Study limitations

Cross-sectional

No HIV-negative controls

Relatively small N

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Conclusions

High circulating cytokine ratios to IL-10 (particularly with co-occurrence of elevated IL-1β or IL-8) may mark brain atrophy in HIV patients on cART

An inflammatory environment characterized by inadequate IL-10 response to elevated pro-inflammatory cytokines may be a key factor in HIV neuropathogenesis and persistence of HAND.

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Additional points

i. IL-1β/IL-10, etc. may reflect the immune status of chronically HIV-infected patients

ii. Pro- to anti-inflammatory cytokine ratios should be studied further as potential markers of disease severity that can supplement traditional assessment

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Grant support: NIH grants R01HL095135, U54RR026136, U54MD007584

Acknowledgments: Sincere thanks to our patients and staff for making this study possible.

Thank you

John A. Burns School of Medicine, University of Hawai’i at Manoa

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Points for discussion

a) Why do multiple cytokine ratios over IL-10 correlate with regional brain volumes but not with NP scores?

b) Any other anti-inflammatory cytokines that should be examined (in pro- to anti-inflammatory ratios)?

c) Can circulating cytokine ratios be used early in disease to identify patients at higher risk of brain atrophy or neurocognitive problems?