Embed Size (px)
Citation preview
UvA-DARE is a service provided by the library of the University of Amsterdam (http://dare.uva.nl)
UvA-DARE (Digital Academic Repository)
Keeping an eye on the brain of perinatally HIV-infected children
Blokhuis, C.
Link to publication
Citation for published version (APA):Blokhuis, C. (2017). Keeping an eye on the brain of perinatally HIV-infected children.
General rightsIt is not permitted to download or to forward/distribute the text or part of it without the consent of the author(s) and/or copyright holder(s),other than for strictly personal, individual use, unless the work is under an open content license (like Creative Commons).
Disclaimer/Complaints regulationsIf you believe that digital publication of certain material infringes any of your rights or (privacy) interests, please let the Library know, statingyour reasons. In case of a legitimate complaint, the Library will make the material inaccessible and/or remove it from the website. Please Askthe Library: https://uba.uva.nl/en/contact, or a letter to: Library of the University of Amsterdam, Secretariat, Singel 425, 1012 WP Amsterdam,The Netherlands. You will be contacted as soon as possible.
Download date: 12 Jan 2020
Inflammatory and Neuronal
Biomarkers Associated With
Retinal Thinning in Pediatric HIV
07
Charlotte Blokhuis *
Susanne Doeleman *
Sophie Cohen
Nazli Demirkaya
Henriëtte Scherpbier
Neeltje A. Kootstra
Jens Kuhle
Charlotte E. Teunissen
Frank D. Verbraak
Dasja Pajkrt
* Equal contributors
Submitted
145biomarkers of retinal thinning in pediatric hiv
abstract
PURPOSE - The pathophysiology of neuroretinal thinning in
children with human immunodeficiency virus (HIV) is poorly
understood. The current study aimed to assess whether
neuroretinal thinning in clinically stable perinatally HIV-
infected children was associated with biomarkers of immune
activation, inflammation, and neuronal damage.
METHODS - Inflammation-associated and neuronal damage
markers were measured in blood and cerebrospinal fluid
(CSF) of HIV-infected children aged 8-18 years. Using mixed-
effects regression analyses, we assessed associations
between these biomarkers and neuroretinal layer thickness,
as measured with Spectral-Domain Optical Coherence
Tomography.
RESULTS - Thirty-two HIV-infected children (median age
13.6 years, 50% male) were included. Higher plasma levels
of interleukin-6, monocyte chemoattractant protein-1, and
soluble intercellular adhesion molecule-1 were associated
with lower foveal inner plexiform layer thickness (coef= -4.40,
P<.001; coef= -9.67, P=.047; coef= -10.48, P =.042, respectively).
Plasma interleukin-6 was also associated with reduced
foveal ganglion cell layer thickness (coef= -2.49, P =.010).
Increased CSF total Tau levels associated with reduced outer
nuclear layer and inner segments thickness (foveal: coef=-
19.3, P-value=.029; pericentral: coef=-18.09, P-value=.006) and
pericentral total retinal thickness (coef=-28.2, P-value=.017).
CONCLUSIONS - Neuroretinal thinning was associated with
inflammation-associated and neuronal injury biomarkers
in a cohort of cART treated perinatally HIV-infected children.
These findings suggest that ongoing immune activation,
inflammation, and neuronal injury occur in parallel with
retinal thinning in pediatric HIV, and could be involved in its
pathogenesis.
147biomarkers of retinal thinning in pediatric hiv
INTRODUCTION
Human immunodeficiency virus (HIV) infection has been associated with retinal structural abnormalities
and subtle visual impairments in perinatally infected children, despite adequate viral suppression and
absence of ocular opportunistic infections.1–3 As the retina contains neuronal tissue, the pathogenesis of
retinal structural damage in HIV infection may have similarities with central nervous system (CNS) damage in
HIV. This hypothesis is supported by the previously detected association between retinal thinning and white
matter microstructural injury in HIV patients.4
The pathogenesis of retinal and cerebral abnormalities in paediatric HIV is poorly understood,
and may not only involve direct injury by HIV and/or antiretroviral therapy (cART), but also chronic
immune activation, inflammation, and microvasculopathy.5,6 Several biomarkers associated with systemic
inflammation are increased in plasma of HIV-infected children, including C-reactive protein (CRP), monocyte
chemoattractant protein (MCP-1), soluble CD14 (sCD14), soluble intercellular cell adhesion molecule-1
(sICAM-1), and soluble vascular cell adhesion molecule-1 (sVCAM-1)7–11. Limited evidence from studies in
HIV-infected adults suggests that immune activation and inflammation correlate with biochemical and
neuroimaging markers of neuronal injury, including elevated cerebrospinal fluid (CSF) levels of neurofilament
light chain (NFL), poorer white matter microstructural integrity, and altered magnetic resonance spectroscopy
neurometabolites.12–14 Further, inflammation-associated markers MCP-1 and interleukin-6 (IL-6) in plasma
have previously been shown to impair blood-retinal barrier (BRB) function, leading to increased vulnerability
of the retina to potential insults by HIV and/or inflammation.15
Neuronal injury may subsequently lead to release of neuronal cytoskeleton components, such
as neurofilaments and Tau protein. Increased levels of these markers were associated with HIV-associated
neurocognitive disorders (HAND) in adults.16,17 Release and aggregation of these proteins have also been
linked to several forms of non-HIV-associated retinal pathology18,19, prompting us to study these markers in
the context of HIV-related retinal thinning.
The current study aimed to gain insight in the pathogenesis of retinal thinning in clinically stable
perinatally HIV-infected children. We hypothesized that immune activation, inflammation, and neuronal
148 chapter 07
injury may be related to retinal thinning. To investigate this, we assessed whether Spectral-Domain Optical
Coherence Tomography (SD-OCT)-measured retinal thickness (RT) was associated with plasma and CSF
markers of immune activation, inflammation, and neuronal injury in a cohort of perinatally HIV-infected
children, of whom most were on long-time suppressive cART.
METHODS
This study is part of the NOVICE study, an interdisciplinary observational cross-sectional case-control
study, assessing neurological, cognitive and ophthalmic performance in perinatally HIV-1-infected children
as compared to uninfected controls matched for age, sex, ethnicity, and socio-economic status (SES) in
The Netherlands (Dutch Trial Registry ID NTR4074).20 This study adhered to the tenets of the Declaration of
Helsinki and informed consent was obtained from all parents and from children aged 12 and above. The
research was approved by the investigational review board at the Academic Medical Center in Amsterdam.
Study Participants
The NOVICE cohort consists of perinatally HIV-1-infected children between 8 and 18 years old, recruited from
the Amsterdam Medical Center in Amsterdam between December 2012 and January 2014.20 Exclusion criteria
for participation were traumatic brain injury, (history of) intracerebral neoplasms, chronic HIV-unrelated
neurological disease, psychological disorders and absence of biomarker data. Additional ophthalmic
exclusion criteria were visual acuity below 0.1 on the logMAR chart, intraocular pressure above 21 mm Hg,
high refractive errors (spherical equivalent [SE] exceeding >+5.5 or <−8.5 D), significant media opacities, and
a history of ocular surgery or disease.3
SD-OCT and Retinal Layer Segmentation
Thickness of individual retinal layers was assessed using SD-OCT (Topcon 3D OCT-100; Topcon, Inc., Paramus,
NJ, USA) as described previously.3 In short, OCT images were obtained using 3D macular and disc volume
scan protocols. Low-quality images with a Topcon image quality factor (QF) <60 were excluded. Individual
neuroretinal layers were automatically segmented from 3D macular volumes using the Iowa Reference
Algorithm21 that enables computations of individual retinal layer thickness for each of the nine macular
regions as defined by the Early Treatment of Diabetic Retinopathy Study (EDTRS) (see Supplemental Figure
1). For the current study, we included the total foveal and pericentral RT, as well as the thickness of retinal
layers that contain neuronal tissue, most of which were affected by thinning in our cohort3: the ganglion cell
layer (GCL), inner plexiform layer (IPL), inner nuclear layer (INL), and outer nuclear layer + inner segments
(ONL+IS).
149biomarkers of retinal thinning in pediatric hiv
Biomarker analysis
Blood samples were collected from all participants using venipuncture. CSF was obtained from a subset of
HIV-infected children.22 Samples were centrifuged within two hours at 1700×g for ten minutes, after which
the supernatant was transferred into a polypropylene tube (Sarstedt, Numbrecht, Germany) and stored at
-80°C until biomarker analysis.
We selected the following biomarkers representing HIV-associated immune activation,
inflammation, and vascular endothelial activation that were previously found to be upregulated in our
cohort10,11 and/or in other HIV-infected populations7–9,23: CRP, IL-6, interleukin-15 (IL-15), interferon-gamma
(IFN-γ), interferon-gamma inducible protein 10 (IP-10), MCP-1, sCD14, sICAM-1, and sVCAM-1. These
inflammation-associated biomarkers were quantified in plasma and CSF using Meso Scale Discovery, a highly
sensitive electrochemiluminescence-based immunoassay, according to the manufacturer’s instructions,24
except sCD14 which was analyzed using an enzyme-linked immunosorbent assay ELISA (R&D systems,
Minneapolis, Minnesota).
Neuronal damage marker NFL was quantified in CSF using an (ELISA; Uman Diagnostics, Umea,
Sweden) and in serum using an in-house developed test for the Meso Scale Discovery platform, using the
same antibodies as in the Uman ELISA.25 CSF NFH was measured using an in-house developed Luminex
assay26 and CSF total Tau (tTau) was measured using the Innotest (Fujirebio, Gent, Belgium).27
Statistical Analysis
Statistical analyses were performed using Stata Statistical Software, release 13 (StataCorp LP,
College Station, TX, USA). We assessed associations between inflammation-associated and neuronal damage
biomarkers (independent parameters) and thickness of selected retinal layers (dependent parameters) using
mixed-effects multivariable linear regression. This regression model was chosen to take correlation between
right and left eyes within participants into account. Biomarker levels that were reported as below or above
the range of quantification were imputed using the lower or upper limit of quantification, respectively. All
biomarkers were transformed using base-10 log transformation. Variables that did not approach a normal
distribution were dichotomized using median splits. All analyses were adjusted for age, sex, and spherical
equivalent (SE). Analyses were not corrected for OCT QF, because median QF was high in both groups
(median QF: HIV-infected = 85.07; controls = 85.17; P-value = .80). In line with the explorative nature of this
study, we did not adjust for multiple comparisons, and results should be interpreted accordingly.
150 chapter 07
RESULTS
Participants
The NOVICE cohort consisted of 36 HIV-infected children. For the current study, three HIV-infected children
were excluded from OCT (two without consent to OCT examination; one with a history of cytomegalovirus
retinitis in both eyes), and one participant was excluded due to unavailability of laboratory data. We excluded
two left eyes from two patients from the analysis due to the presence of congenital toxoplasmosis lesions
and uveitis, respectively; for these participants, we used only measurements from the right eye.3
A total of 32 clinically stable HIV-infected children (50% male, median age 13.6 [IQR 11.8–15.9]
years) were included in the current study. Demographical and clinical characteristics of included participants
are presented in Table 1. At the time of study assessment, 27 (84%) participants had been using cART for a
median duration of 10.7 (IQR 5.3–13.7) years, of whom all but one (96%) were virologically suppressed in
blood and CSF. The median CD4+ T-cell count at study inclusion was 770*106/L (IQR 580-970), corresponding
to a Z-score of -0.1 (IQR -0.3–0.2), indicating minimal deviation from the age-adjusted norm for uninfected
children. CSF was available of a subgroup of 24 participants, which were younger than children of whom no
CSF was available (CSF: 12.8 years; no CSF: 15.6 years, P-value=.03; data not shown), but did not differ from
the other HIV-infected participants in terms of demographical and clinical characteristics.
Associations between biomarkers and retinal thickness
Associations between inflammation-associated biomarkers and RT are displayed in Table 2. Higher systemic
levels of IL-6, MCP-1, and sICAM1 were associated with foveal IPL thinning (IL-6: coef= -4.40, P-value=<.001;
MCP-1: coef= -9.67, P-value=.047; sICAM1: coef= -10.48, P-value=.042). Further, higher plasma IL-6 levels were
associated with thinning of the foveal GCL (coef= -2.49, P-value=.010). CSF levels of these biomarkers were
not associated with RT.
Among neuronal damage markers (Table 3), higher CSF tTau was significantly associated with lower
pericentral total RT (coef= -28.2, P-value=.017), and with a thinner ONL+IS in both the foveal (coef= -19.3,
P-value=.029) and pericentral region (coef= -18.1, P-value=.006). In addition, we observed trends towards
association between higher CSF NFH levels and thinning of several retinal layers, including the foveal total
RT (coef= -37.4, P-value=.071), foveal GCL (coef= -6.3, P-value=.070), foveal ONL+IS (coef= -21.3, P-value=.080),
and pericentral INL (coef= -5.8, P-value=.068).
151biomarkers of retinal thinning in pediatric hiv
DEMOGRAPHIC CHARACTERISTICS HIV-INFECTED CHILDREN (N=32)
Sex (male) 16 (50)
Age 13.6 (11.8 – 15.9)
Ethnicity Black 24 (75)
Mixed black 5 (16)
Other 3 (9)
HIV- AND CART RELATED CHARACTERISTICS
CLINICAL
Age at HIV diagnosis (y) 2.4 (0.7 – 4.9)
CDC stage N/A 9 (28)
B 15 (47)
C 8 (25)
HIV-encephalopathy 2 (6)
CD4+ T-CELL COUNTS AND HIV VIRAL LOAD
Peak HIV viral load (log copies/mL) † 5.58 (5.06 – 5.96)
Nadir CD4+ T-cell count † *106/L 445 (270-570)
Z-score -0.7 (-1.4 – 0.4)
Viral suppression at study inclusion 26 (81)
CD4+ T-cell count at study inclusion *106/L 770 (580 – 970)
Z-score -0.1 (-0.3 – 0.2)
Time with CD4+ T-cell count <500*106/L (m) 1.05 (0 – 37.6)
CART
Age at cART initiation (y) † 2.6 (1.2 – 6.2)
Prescribed cART at study inclusion 27 (84)
Duration cART use (y) † 10.7 (5.3 – 13.7)
TABLE 1. PARTICIPANT CHARACTERISTICS
Demographic, HIV- and cART related characteristics of 32 included participants, displayed as median (IQR) or N(%). Abbreviations:
HIV=Human Immunodeficiency Virus, y = years, m = months, cART=combination antiretroviral therapy, CDC= Centers for Disease Control
and Prevention stage (N/A=no or minimal symptoms; B=moderate symptoms; C=severe symptoms or acquired immunodeficiency
syndrome) Footnotes: † historical data was incomplete for peak HIV viral load (n=28; 4 missing), nadir CD4 T-cell counts (n=30; 2
missing), and cART initiation and duration (n=29; 3 missing).
152 chapter 07
TOTA
L RT
GC
LIP
LIN
LO
NL+
IS
Fove
alPe
ricen
tral
Fove
alPe
ricen
tral
Fove
alPe
ricen
tral
Fove
alPe
ricen
tral
Fove
alPe
ricen
tral
PLA
SMA
(N=3
2)
CRP
-4.0
6 (.4
0)-0
.36
(.93)
-0.7
5 (.3
5)0.
46 (.
74)
-1.5
2 (.0
9)-0
.85
(.33)
-0.6
6 (.4
7)0.
37 (.
68)
0.67
(.83
)1.
20 (.
60)
IL-6
† - 9
.56
(.11)
3.83
(.48
)-2
.49
(.010
*)1.
06 (.
55)
-4.4
0 (<
.001
*)-1
.00
(.38)
-1.7
8 (.1
2)1.
12 (.
33)
-1.8
1 (.6
4)1.
66 (.
58)
IL-15
-2
6.32
(.18
)-1
3.07
(.45
)-5
.81
(.07)
-1.1
9 (.8
4)-7
.11
(.06)
-2.4
2 (.5
1)-3
.06
(.42)
3.87
(.29
)-2
.79
(.83)
-6.3
2 (.5
1)
IFN-
γ 8.
58 (.
31)
6.79
(.35
)1.
27 (.
37)
1.10
(.65
)-1
.17
(.48)
-1.0
1 (.5
1)0.
10 (.
95)
1.41
(.37
)6.
15 (.
24)
4.56
(.25
)
IP-1
0 2.
11 (.
83)
1.39
(.87
)0.
74 (.
66)
-0.6
3 (.8
3)-1
.71
(.38)
0.35
(.85
)0.
51 (.
79)
0.12
(.95
)1.
16 (.
85)
2.22
(.64
)
MCP
-1
11.2
7 (.6
7)12
.16
(.59)
0.50
(.91
)-6
.23
(.40)
-9.6
7 (.0
47*)
-8.2
4 (.0
7)-5
.79
(.24)
3.45
(.48
)25
.58
(.11)
22.1
5 (.0
6)
sCD1
4 -9
.42
(.43)
-13.
39 (.
19)
0.84
(.68
)-1
.98
(.56)
-0.9
6 (.6
8)-0
.40
(.85)
-0.2
3 (.9
2)-0
.03
(.99)
-3.9
9 (.5
9)-4
.95
(.38)
sVCA
M-1
-1
4.63
(.54
)-9
.42
(.65)
-0.4
2 (.9
2)1.
48 (.
83)
-0.8
9 (.8
5)-1
.39
(.75)
2.21
(.62
)-3
.27
(.46)
-8.1
0 (.5
8)0.
23 (.
98)
sICA
M-1
-1
4.11
(.61
)9.
80 (.
69)
-5.6
1 (.2
2)3.
75 (.
64)
-10.
48 (.
042*
)-7
.68
(.12)
-5.0
8 (.3
3)1.
51 (.
77)
7.13
(.68
)18
.40
(.15)
CSF
(N=2
4)‡
CRP
-5.5
2 (.4
5)0.
36 (.
95)
-1.3
0 (.2
8)1.
33 (.
48)
-1.7
5 (.2
3)-1
.43
(.22)
-0.9
7 (.4
7)0.
07 (.
95)
-0.2
2 (.9
6)1.
75 (.
60)
IL-6
5.91
(.72
)-2
.96
(.82)
-0.4
5 (.8
7)-3
.12
(.46)
-0.2
5 (.9
4)-3
.03
(.25)
-0.8
5 (.7
8)1.
51 (.
55)
9.36
(.32
)6.
98 (.
34)
IL-15
†-5
.52
(.54)
-7.0
9 (.3
1)-0
.22
(.88)
-3.3
1 (.1
4)0.
69 (.
67)
0.34
(.82
)-0
.08
(.96)
-0.4
8 (.7
3)-7
.45
(.13)
-3.8
6 (.3
0)
IFN-
γ †
-0.4
6 (.9
6)1.
46 (.
84)
-0.5
7 (.7
0)-1
.34
(.56)
-1.0
9 (.5
4)0.
41 (.
77)
-1.0
8 (.5
1)0.
13 (.
93)
2.38
(.64
)2.
54 (.
52)
IP-1
0 †
-6.6
0 (.4
6)4.
43 (.
54)
-2.1
9 (.1
3)0.
40 (.
87)
-1.9
6 (.2
7)-2
.17
(.12)
-2.2
8 (.1
6)-0
.12
(.93)
3.65
(.49
)7.
23 (.
06)
MCP
-1 †
-2.3
2 (.7
8)-7
.52
(.25)
0.03
(.98
)-1
.01
(.65)
0.85
(.62
)0.
31 (.
82)
1.30
(.40
)-0
.93
(.47)
-4.4
2 (.3
7)-5
.00
(.18)
sCD1
4 ‡
-1.8
4 (.8
6)-1
.17
(.89)
-0.1
7 (.9
2)1.
59 (.
55)
1.62
(.43
)-1
.52
(.38)
0.08
(.97
)-0
.22
(.89)
-1.1
4 (.8
5)0.
07 (.
99)
sVCA
M-1
-1
8.21
(.32
)-6
.82
(.65)
-2.9
0 (.3
4)3.
11 (.
52)
-2.0
2 (.5
9)-2
.04
(.50)
-0.3
7 (.9
2)0.
23 (.
94)
-5.5
5 (.6
1)-3
.76
(.66)
sICA
M-1
-34.
58 (.
16)
-4.4
6 (.8
3)-7
.20
(.07)
5.74
(.38
)-8
.99
(.07)
-6.2
3 (.1
1)-5
.75
(.21)
0.61
(.88
)-2
.48
(.87)
4.43
(.70
)
TAB
LE 2
. CO
RREL
ATIO
NS
BET
WEE
N IN
FLAM
MO
RY M
ARKE
RS A
ND
RET
INAL
TH
ICKN
ESS
IN H
IV-I
NFE
CTED
CH
ILD
REN
153biomarkers of retinal thinning in pediatric hiv
Tabl
e 2
(abo
ve).
This
tabl
e sh
ows
the
resu
lts o
f the
mix
ed e
ffect
s re
gres
sion
ana
lyse
s, e
valu
atin
g as
soci
atio
ns b
etw
een
retin
al la
yers
and
infla
mm
atio
n-as
soci
ated
bio
mar
kers
. Acc
ordi
ng t
o
dist
ribut
ion,
var
iabl
es a
re lo
g pg
/ml,
or d
icho
tom
ized
usi
ng a
med
ian
split
whe
re s
peci
fied.
Dat
a ar
e pr
esen
ted
as c
oeffi
cien
t (P-
valu
e). A
bbre
viat
ions
: CRP
= C
-rea
ctiv
e pr
otei
n; IL
= in
terle
ukin
;
MCP
-1 =
mon
ocyt
e ch
emoa
ttra
ctan
t pro
tein
; IFN
γ =
inte
rfero
n ga
mm
a; IP
-10
= in
terfe
ron-
gam
ma-
indu
cibl
e pr
otei
n 10
; sVC
AM-1
= s
olub
le v
ascu
lar c
ell a
dhes
ion
mol
ecul
e-1;
sIC
AM-1
= s
olub
le
inte
rcel
lula
r adh
esio
n m
olec
ule-
1; R
T =
retin
al th
ickn
ess;
GCL
= g
angl
ion
cell
laye
r; IP
L =
inne
r ple
xifo
rm la
yer;
INL
= in
ner n
ucle
ar la
yer;
ON
L+IS
= o
uter
nuc
lear
laye
r + in
ner s
egm
ents
. Foo
tnot
es:
*P v
alue
<0.
05. † v
aria
ble
dich
otom
ized
usi
ng m
edia
n sp
lit. ‡ n
=24
for s
CD14
and
n=2
2 fo
r all
othe
r bio
mar
kers
.
TOTA
L RT
GC
LIP
LIN
LO
NL+
IS
Fove
alPe
ricen
tral
Fove
alPe
ricen
tral
Fove
alPe
ricen
tral
Fove
alPe
ricen
tral
Fove
alPe
ricen
tral
SERU
M
NFL
†7.
15 (.
24)
3.88
(.47
)0.
80 (.
44)
-0.0
1 (>
.99)
1.63
(.17
)1.
13 (.
31)
0.27
(.82
)0.
95 (.
41)
3.55
(.36
)2.
43 (.
41)
CSF
‡
NFL
† -1
.15
(.89)
4.03
(.55
)-0
.83
(.56)
1.93
(.38
)-0
.11
(.95)
-0.3
8 (.7
9)-0
.60
(.70)
-0.2
4 (.8
6)1.
08 (.
83)
1.45
(.71
)
NFH
-37.
37 (.
07)
-18.
63 (.
28)
-6.2
7 (.0
7)-4
.19
(.46)
-3.0
2 (.5
0)-2
.13
(.55)
-3.8
6 (.3
4)-5
.80
(.07)
-21.
27 (.
08)
-10.
67 (.
28)
tTau
-9
.16
(.58)
-28.
22 (.
017*
)0.
76 (.
78)
-4.3
8 (.3
0)4.
81 (.
14)
-3.0
9 (.2
4)1.
98 (.
52)
-3.5
2 (.1
5)-1
9.30
(.02
9*)
-18.
09 (.
006*
)
TAB
LE 3
. CO
RREL
ATIO
NS
BET
WEE
N N
EURO
NAL
DAM
AGE
MAR
KERS
AN
D R
ETIN
AL T
HIC
KNES
S IN
HIV
-IN
FECT
ED C
HIL
DRE
N
This
tabl
e sh
ows
the
resu
lts o
f the
mix
ed e
ffect
s re
gres
sion
ana
lyse
s, e
valu
atin
g as
soci
atio
ns b
etw
een
retin
al la
yers
and
neu
rona
l dam
age
mar
kers
in s
erum
and
CSF
. Dat
a ar
e pr
esen
ted
as
coeff
icie
nt (P
-val
ue).
Abbr
evia
tions
: NFL
= n
euro
filam
ent l
ight
cha
in; N
FH =
neu
rofil
amen
t hea
vy c
hain
; tTa
u =
tota
l Tau
; RT
= re
tinal
thic
knes
s; G
CL =
gan
glio
n ce
ll la
yer;
IPL
= in
ner p
lexi
form
laye
r;
INL
= in
ner n
ucle
ar la
yer;
ON
L+IS
= o
uter
nuc
lear
laye
r + in
ner s
egm
ents
; CSF
= c
ereb
rosp
inal
flui
d. F
ootn
otes
: *P-
valu
e <.
05. †
var
iabl
e di
chot
omiz
ed u
sing
med
ian
split
. ‡ n
=20
for N
FL a
nd n
=22
for a
ll ot
her b
iom
arke
rs
154 chapter 07
DISCUSSION
In this study, we explored whether biomarkers of immune activation, inflammation, and neuronal
injury were associated with retinal thinning in perinatally HIV-infected children. Increased systemic levels of
inflammation-associated biomarkers IL-6, MCP-1, and sICAM-1, and of CSF neuronal damage marker tTau,
were associated with retinal thinning. In addition, CSF NFH showed trends towards association with thinning
of several retinal layers, primarily in the foveal region. These findings could indicate that immune activation,
inflammation, vascular endothelial activation, and neuronal injury occur in parallel with retinal thinning,
and that they may play a role in the pathogenesis of retinal thinning in pediatric HIV.
A possible mechanism by which increased peripheral blood levels of inflammation-associated
markers IL-6, MCP-1, and sICAM-1 could contribute to the pathogenesis of retinal thinning is impairment
of retinal barrier function. Both MCP-1 and IL-6 have been shown to impair tight junctions in the BRB,
which may render the retina more vulnerable to viral particles and pro-inflammatory cytokines.15 Barrier
dysfunction has also been described in human brain microvascular endothelial cells of the BBB, where HIV
has been shown to upregulate IL-6, MCP-1, and sICAM-1, facilitating adhesion and migration of monocytes
into the brain.28,29
We found no associations between retinal thinning and CSF inflammation-associated biomarkers.
We hypothesize that this may be explained by the relatively short half-life of most cytokines and the larger
distance between the retina and CSF as compared to blood.30 Additionally, immune responses may differ
between the two compartments, which is supported by the limited concordance between systemic and
intrathecal levels of these markers in our cohort.10 With the sparsity of pediatric data on CSF biomarkers,
further studies are needed to confirm our findings, and to elucidate how plasma and CSF immune
activation and inflammation relate to each other and to HIV-associated neuroretinal injury over time. We
previously found that the pathogenesis of neuroretinal thinning may share common features with that of
microstructural white matter injury in HIV-infected children.4 Thus, investigating systemic and intrathecal
inflammation-associated biomarkers in relation to white matter microstructure may also contribute to our
understanding of the pathogenesis of both cerebral and retinal injury in pediatric HIV. Increased tTau levels
were associated with reduced thickness of the foveal and pericentral ONL+IS and pericentral total RT. Tau
is a neuronal cytoskeletal protein that, when distorted, causes neurodegeneration via various pathways, for
instance by accumulation in oligomers and tangles in hyperphosphorylated form.31 In HIV-infected adults,
elevated tTau levels were associated with HAND severity, indicating a potential role in HIV-related neuronal
injury.32,33 Studies evaluating Tau in the retina are scarce in the field of HIV, but tauopathy has been shown
to contribute to retinal injury in various other ocular and neurodegenerative diseases, including glaucoma
and Alzheimer’s disease.19,31 Additionally, in a mouse model of retinal degeneration, Tau deposits were
associated with degeneration of photoreceptors, with a concomitant reduction in thickness of the inner
and outer nuclear layers, the latter of which contains photoreceptor cell bodies.34 As the ONL+IS showed
the most prominent thinning in our cohort3, it is valuable to further investigate the localization and different
forms of Tau proteins to clarify their exact role in HIV-associated retinal pathology.
155biomarkers of retinal thinning in pediatric hiv
While associations between neuronal damage marker NFH in CNS and retinal thinning were not
statistically significant, several trends towards association were found. While this does not provide evidence
that release or accumulation of NFH is involved in to retinal pathology, it may be a marker worth further
investigation. In support of such a relation, a study in untreated HIV-infected adults showed that higher
CSF NFH levels were associated with increased monocyte activation and impaired processing speed,
suggesting NFH may indeed be a marker of neuronal injury in HIV infection.35 Studies specifically addressing
the relevance of NFH in retinal injury associated with HIV or other neurodegenerative diseases are scarce,
hindering comparison of our results with other findings. A recent study showed that NFH measured
in the vitreous body of the eye was increased for up to two years after retinal pathology (such as retinal
detachment), confirming that NFH reflects neuronal degeneration in the retina.36 This technique could be
useful to study the potential role of NFH in HIV, although ethical considerations considerably limit its use.
Our study does not provide evidence that NFL plays a role in neuroretinal injury in pediatric HIV, as no
associations were found between retinal thinning and increased blood or CSF levels of NFL. While CSF NFL
has been consistently associated with severity of HIV disease and HAND in adults16,37, the relevance of NFL
in pediatric HIV-associated CNS injury remains less clear due to lack of CSF data in pediatric populations.
While we are the first to explore a potential role for immune activation, inflammation and neuronal
injury in the pathogenesis of retinal alterations in perinatally HIV-infected children on long-term cART, our
study was subject to some limitations. Our small sample size for the regression analyses may have led to
insufficient power to detect relevant correlations. Since the large majority of children was cART treated, we
cannot differentiate between effects of HIV and different antiretroviral drugs, each of which may have specific
contributions to immune activation, vascular dysfunction, and neurotoxicity in chronic HIV-infection.5,6 Due
to the cross-sectional design, we are unable to draw conclusions about cause and effect of the observed
associations. Ideally, pathogenesis of retinal changes is investigated by detailing the histopathology of
retinal changes and evaluating the presence of biomarkers in the vitreous humor of the eye. In the current
setting, biomarkers in CSF and plasma were the best available proxy for the presence of ocular immune
activation, inflammation and neuronal damage in HIV-infected children. It is unknown to which degree the
measured neuronal damage markers are derived from retinal (versus cerebral) neurons, or which form of
these proteins are associated with retinal injury. For instance, functions of Tau differ between isoforms, and
between axonal, nuclear, synaptic, or non-neuronal localization.38 It was recently established that Tau can
additionally promote aggregation via exosomes in CSF.39 Phosphorylation of Tau and NFH further modulates
their properties.35,38 Given that the clinical effects of Tau and NFH proteins are dependent on their different
forms and localizations, future research is needed to clarify which pathways contribute to HIV-associated
retinal damage.
In conclusion, retinal thinning was associated with systemic inflammation-associated markers
IL-6, MCP-1, and sICAM-1, and CSF neuronal damage marker tTau, in a cohort of long-term clinically and
virally controlled HIV-infected children. These findings point to immune activation, endothelial dysfunction,
and neuronal injury as potential contributors to the pathogenesis of retinal thinning. To further test this
hypothesis, the relationship between inflammation-associated biomarkers, BRB and retinal integrity, and
neuronal injury could be investigated on a microstructural level, using immunohistochemical analysis or
in vivo localization imaging techniques. Longitudinal studies are needed to evaluate how inflammation-
156 chapter 07
associated and neuronal biomarkers relate to the clinical course and severity of retinal deficits over
time. Considering the association between retinal and microstructural white matter injury, this could not
only contribute to our understanding of the pathogenesis of retinal thinning, but also of cerebral and
neurocognitive deficits in pediatric HIV. These are essential steps towards better treatment and prevention
of cerebral and retinal injury in treated HIV-infected children now surviving into adulthood.
ACKNOWLEDGEMENTS
The authors thank all participants and their legal guardians, ms. A.M. Weijsenfeld and ms. A. van der Plas for
their help with recruiting participants.
FUNDING
This work was supported by Bayer Healthcare’s Global Ophthalmology Awards Program 2012, the Emma
Foundation (grant number 11.001), Stichting Mitialto, ViiV Healthcare, AIDS Fonds, and Dr. C.J. Vaillantfonds.
These funding bodies had no role in the design or conduct of the study, nor in the analysis or interpretation
of the results.
Disclosures
J. Kuhle: Novartis, Protagen AG (C); Swiss MS Society, Biogen, Novartis, Roche, Genzyme, Merck Serono,
Novartis (R); ECTRIMS Research Fellowship Programme, University of Basel, Swiss MS Society, Swiss
National Research Foundation, Bayer (Schweiz) AG, Genzyme, Novartis (F). C.E. Teunissen: Fujirebio, Roche
(S); ADxNeurosciences (F); Janssen Prevention Center, Boehringer, EIP Farma, Roche, Probiodrug (R). F.D.
Verbraak: Bayer (C, R), Novartis, IDxDR (C). The other authors have no conflicts of interest to disclose.
157biomarkers of retinal thinning in pediatric hiv
1. Moschos MM, Mostrou G, Psimenidou E, Spoulou V,
Theodoridou M. Objective analysis of retinal function in HIV-
positive children without retinitis using optical coherence
tomography. Ocul Immunol Inflamm. 2007;15(4):319-23.
2. Moschos MM, Margetis I, Markopoulos I, Moschos MN. Optical
coherence tomography and multifocal electroretinogram
study in human immunodeficiency virus-positive children
without infectious retinitis. Clin Exp Optom. 2011;94(3):291-
295.
3. Demirkaya N, Cohen S, Wit FWNM, et al. Retinal Structure
and Function in Perinatally HIV-Infected and cART-Treated
Children: A Matched Case-Control Study. Invest Ophthalmol
Vis Sci. 2015;56(6):3945-3954.
4. Blokhuis C, Demirkaya N, Cohen S, et al. The eye as a window
to the brain: Neuroretinal thickness is associated with
microstructural white matter injury in HIV-infected children.
Investig Ophthalmol Vis Sci. 2016;57(8):3864-3871.
5. Blokhuis C, Kootstra NA, Caan MW, Pajkrt D.
Neurodevelopmental delay in pediatric HIV/AIDS: current
perspectives. Neurobehav HIV Med. 2016;7(1):1-13.
6. Demirkaya N, Wit F, Schlingemann R, Verbraak F. Neuroretinal
Degeneration in HIV Patients Without Opportunistic
Ocular Infections in the cART Era. AIDS Patient Care STDS.
2015;29(10):519-532.
7. Miller TL, Borkowsky W, Dimeglio L a., et al. Metabolic
abnormalities and viral replication are associated with
biomarkers of vascular dysfunction in HIV-infected children.
HIV Med. 2012;13(5):264-275.
8. Sainz T, Diaz L, Navarro ML, et al. Cardiovascular biomarkers
in vertically HIV-infected children without metabolic
abnormalities. Atherosclerosis. 2014;233(2):410-414.
9. Ross AC, O’Riordan MA, Storer N, Dogra V, McComsey GA.
Heightened inflammation is linked to carotid intima-media
thickness and endothelial activation in HIV-infected children.
Atherosclerosis. 2010;211(2):492-498.
10. Blokhuis C, Cohen S, Scherpbier HJ, et al. Elevated systemic
IL-15, IFNγ, IP-10, and MCP-1 do not correlate with intrathecal
inflammation in cART-treated perinatally HIV-infected
children. Poster Presented at: 10th Netherlands Conference
on HIV Pathogenesis, Treatment and Prevention; November
22, 2016; Amsterdam, the Netherlands.
11. Blokhuis C, Zanten M van, Cohen S, et al. Endothelial
activation and inflammation associated with microstructural
white matter injury and poor visuomotor integration in HIV-
infected children. Poster Presented at: 10th Netherlands
Conference on HIV Pathogenesis, Treatment and Prevention;
Amsterdam, the Netherlands.
12. Peluso MJ, Meyerhoff DJ, Price RW, et al. Cerebrospinal fluid
and neuroimaging biomarker abnormalities suggest early
neurological injury in a subset of individuals during primary
HIV infection. J Infect Dis. 2013;207(11):1703-1712.
13. Anderson AM, Harezlak J, Bharti A, et al. Plasma and
cerebrospinal fluid biomarkers predict cerebral injury in
HIV-infected individuals on stable combination antiretroviral
therapy. JAIDS J Acquir Immune Defic Syndr. 2015;69(1):29-
35.
14. Vera JH, Guo Q, Cole JH, et al. Neuroinflammation in treated
HIV-positive individuals. Neurology. 2016;86(15):1425-1432.
15. Tan S, Duan H, Xun T, et al. HIV-1 impairs human retinal
pigment epithelial barrier function: possible association
with the pathogenesis of HIV-associated retinopathy. Lab
Invest. 2014;94(7):777-87.
16. Gisslén M, Price RW, Andreasson U, et al. Plasma
Concentration of the Neurofilament Light Protein (NFL) is a
Biomarker of CNS Injury in HIV Infection: A Cross-Sectional
Study. EBioMedicine. 2016;3:135-140.
17. Peterson J, Gisslen M, Zetterberg H, et al. Cerebrospinal
Fluid (CSF) Neuronal Biomarkers across the Spectrum of
HIV Infection: Hierarchy of Injury and Detection. PLoS One.
2014;9(12):e116081.
18. Geiger K, Howes E, Gallina M, Huang XJ, Travis GH, Sarvetnick
N. Transgenic mice expressing IFN-gamma in the retina
develop inflammation of the eye and photoreceptor loss.
Invest Ophthalmol Vis Sci. 1994;35(6):2667-2681.
19. Frost S, Martins RN, Kanagasingam Y. Ocular biomarkers for
early detection of Alzheimer’s disease. J Alzheimer’s Dis.
2010;22(1):1-16.
20. Cohen S, Stege JA, Geurtsen GJ, et al. Poorer Cognitive
Performance in Perinatally HIV-Infected Children Versus
Healthy Socioeconomically Matched Controls. Clin Infect
Dis. 2015;60(7):1111-1119.
21. Garvin MK, Abramoff MD, Kardon R, Russell SR, Wu X,
reference list
158 chapter 07
Sonka M. Intraretinal layer segmentation of macular optical
coherence tomography images using optimal 3-D graph
search. IEEE Trans Med Imaging. 2008;27(10):1495-1505.
22. Van Dalen YW, Blokhuis C, Cohen S, et al. Neurometabolite
Alterations Associated With Cognitive Performance in
Perinatally HIV-Infected Children. Medicine (Baltimore).
2016;95(12):e3093.
23. Dolan SE, Hadigan C, Killilea KM, et al. Increased
cardiovascular disease risk indices in HIV-infected women. J
Acquir Immune Defic Syndr. 2005;39(1):44-54.
24. Malekzadeh A, Twaalfhoven H, Wijnstok NJ, Killestein J,
Blankenstein MA, Teunissen CE. Comparison of multiplex
platforms for cytokine assessments and their potential
use for biomarker profiling in multiple sclerosis. Cytokine.
2017;91:145-152.
25. Gaiottino J, Norgren N, Dobson R, et al. Increased
Neurofilament Light Chain Blood Levels in
Neurodegenerative Neurological Diseases. Reindl M, ed.
PLoS One. 2013;8(9):1-9.
26. Koel-Simmelink MJA, Vennegoor A, Killestein J, et al.
The impact of pre-analytical variables on the stability of
neurofilament proteins in CSF, determined by a novel
validated SinglePlex Luminex assay and ELISA. J Immunol
Methods. 2013;402(1-2):43-49.
27. Van Der Flier WM, Pijnenburg YA, Prins N, et al. Optimizing
patient care and research: The Amsterdam dementia cohort.
J Alzheimer’s Dis. 2014;41(1):313-327.
28. Pu H, Tian J, Flora G, et al. HIV-1 Tat protein upregulates
inflammatory mediators and induces monocyte invasion
into the brain. Mol Cell Neurosci. 2003;24(1):224-37.
29. Yang B, Akhter S, Chaudhuri A, Kanmogne GD. HIV-1 gp120
induces cytokine expression, leukocyte adhesion, and
transmigration across the blood-brain barrier: modulatory
effects of STAT1 signaling. Microvasc Res. 2009;77(2):212-9.
30. Bocci V. Interleukins. Clin Pharmacokinet. 1991;21(4):274-
284.
31. Ho W-L, Leung Y, Tsang AW-T, So K-F, Chiu K, Chang RC-
C. Review: tauopathy in the retina and optic nerve: does
it shadow pathological changes in the brain? Mol Vis.
2012;18:2700-10.
32. Brew BJ, Pemberton L, Blennow K, Wallin a, Hagberg L. CSF
amyloid beta42 and tau levels correlate with AIDS dementia
complex. Neurology. 2005;65(9):1490-1492.
33. Steinbrink F, Evers S, Buerke B, et al. Cognitive impairment
in HIV infection is associated with MRI and CSF pattern of
neurodegeneration. Eur J Neurol. 2013;20(3):420-428.
34. Cronin T, Raffelsberger W, Lee-Rivera I, et al. The disruption
of the rod-derived cone viability gene leads to photoreceptor
dysfunction and susceptibility to oxidative stress. Cell Death
Differ. 2010;17(7):1199-210.
35. McGuire JL, Gill AJ, Douglas SD, Kolson D. Central and
peripheral markers of neurodegeneration and monocyte
activation in HIV-associated neurocognitive disorders. J
Neurovirol. 2015;21(4):439-448.
36. Petzold A, Junemann A, Rejdak K, et al. A novel biomarker for
retinal degeneration: Vitreous body neurofilament proteins.
J Neural Transm. 2009;116(12):1601-1606.
37. Krut JJ, Mellberg T, Price RW, et al. Biomarker evidence of
axonal injury in neuroasymptomatic HIV-1 patients. PLoS
One. 2014;9(2):e88591.
38. Buée L, Bussière T, Buée-Scherrer V, Delacourte A, Hof
PR. Tau protein isoforms, phosphorylation and role in
neurodegenerative disorders. Brain Res Rev. 2000;33(1):95-
130.
39. Wang Y, Balaji V, Kaniyappan S, et al. The release and
trans-synaptic transmission of Tau via exosomes. Mol
Neurodegener. 2017;12(1):5.
