MAJOR ARTICLE

Immune Profiling of Pregnant Toxoplasma-

Infected US and Colombia Patients Reveals
Surprising Impacts of Infection on Peripheral
Blood Cytokines

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Lena Pernas,1 Raymund Ramirez,2 Tyson H. Holmes,3 José G. Montoya,2,4 and John C. Boothroyd1
1
Department of Microbiology and Immunology, Stanford University School of Medicine; 2Palo Alto Medical Foundation Toxoplasmosis Serology Laboratory;
3
Stanford Center for Human Sleep Research, Department of Psychiatry and Behavioral Sciences, Stanford University School of Medicine, and 4Division of
Infectious Diseases and Geographic Medicine and Department of Medicine, Stanford University School of Medicine, California

In North America (NA) and Europe, the majority of toxoplasmosis cases are benign and generally asymptom-
atic, whereas in South America (SA) toxoplasmosis is associated with much more severe symptoms in adults and
congenitally infected children. The reasons for these differences remain unknown; currently, there is little in-
formation from patients in either region on how the immune system responds to infection with Toxoplasma
gondii. Here, we report the relative abundance of 51 serum cytokines from acute and chronic toxoplasmosis
cohorts of pregnant women from the United States, where approximately one-half of clinical isolates are
Type II, and Colombia, where clinical isolates are generally “atypical” or Type I-like strains. Surprisingly, the
results showed notably lower levels of 23 cytokines in acutely infected patients from the United States, relative to
uninfected US controls. In acutely infected Colombian patients, however, only 8 cytokine levels differed detect-
ably with 4 being lower and 4 higher relative to uninfected controls. Strikingly, there were also differences in the
cytokine profiles of the chronically infected patients relative to uninfected controls in the US cohort. Hence,
Toxoplasma appears to specifically impact levels of circulating cytokines, and our results may partly explain
region-specific differences in the clinical spectrum of toxoplasmosis.
Keywords. acute; chronic; Colombia; congenital, cytokine profile; cytokines; pregnant; toxoplasmosis;
Toxoplasma gondii.

Toxoplasma gondii is an obligate intracellular parasite generally an asymptomatic infection in immunocompe-

with an unparalleled global distribution and mammali-
an host range. This Apicomplexan organism is estimat-
ed to infect about one-third of the world’s human
population [1], with certain geographical regions having
seroprevalence rates as high as 84% [2]. Although
Received 26 November 2013; accepted 13 March 2014; electronically published
23 March 2014.
This work was presented in poster format at the 12th International Congress on
Toxoplasmosis, Oxford, June 2013.
Correspondence: John C. Boothroyd, PhD, Department of Microbiology and
Immunology, Stanford University School of Medicine, Fairchild Bldg, 299 Campus
Drive, Stanford, CA 94305 (john.boothroyd@stanford.edu).
The Journal of Infectious Diseases 2014;210:923–31
© The Author 2014. Published by Oxford University Press on behalf of the Infectious
Diseases Society of America. All rights reserved. For Permissions, please e-mail:
journals.permissions@oup.com.
DOI: 10.1093/infdis/jiu189
tent adults, toxoplasmosis may cause blindness in such
individuals, and it can result in life-threatening disease
in immunocompromised individuals [3–5]. Congenital
Toxoplasma infection can also produce serious disease
with the severity depending on how recently acquired
an infection is and the gestational age of the fetus at
the onset of infection.
After ingestion of food or water contaminated with
Toxoplasma tissue cysts or oocysts, the parasites develop
into rapidly multiplying tachyzoites, which reproduce
within gut tissue of the intermediate host. Tachyzoites
are responsible for establishing the acute phase of infec-
tion during which they disseminate via the bloodstream
to the various tissues in which they proliferate. Al-
though eventually cleared from most organs due to devel-
oping host immunity, surviving tachyzoites differentiate

Immune-profiling Reveals Surprising Impact of Toxoplasma Infection on Peripheral Blood Cytokines • JID 2014:210 (15 September) • 923
into latent bradyzoites, or tissue cyst forms. It is thought that
tissue cysts have a predilection for neural, muscular, and ocular
tissue and persist indefinitely for the life of the host.
Encystation marks the chronic phase of infection, and if a
chronically infected individual becomes immunocompromised,
these tissue cysts serve as a reservoir from which local infections
and further dissemination can develop. Similar to the life-
lasting tissue cysts, Toxoplasma-specific immunoglobulin G
(IgG) levels can also persist for the life of the host [6], likely
due to cycles of reactivation and, perhaps, reinfection. It is pos-
sible to differentiate between acute infections (initiated within
the prior 12 months) and chronic infections (>1 year since
initial infection) by a panel of serological tests [7, 8].
Remarkably, given the ubiquity of this parasite, the majority
of human and animal isolates of Toxoplasma collected to date in
Europe and North America (NA) have been found to be one of
3 genotypes: Types I, II, and III [9]. Interestingly, recent studies
report variation in clinical symptoms by geographic area and
hypothesize that these variations might be associated with par-
asite genotype [10–12]. Type II infections have been recognized
as predominant in North American and European populations
[13], and in these regions, the majority of Toxoplasma infections
are generally asymptomatic [14] and less likely to result in ocu-
lar toxoplasmosis [10]. In contrast, Type I and “atypical” strains
(ie, not Type I, II, or III) predominate in South America (SA)
[15], where toxoplasmosis is associated with much more severe
progression and symptoms [11, 16].
Infections of immunocompetent adults in French Guyana
have even resulted in lethal disseminated disease [17] and in Co-
lombia an increased frequency of active lesions and severity of
symptoms has been reported in ocular cases [18, 19]. A recent
study addressing the role of Toxoplasma strain type in ocular
toxoplasmosis (OT) reported that of the tested OT cases, Co-
lombians were infected with atypical strains and French patients
were uniformly Type II-infected. An analysis of intraocular cy-
tokine levels between these patients revealed increased levels of
interleukin 6 (IL-6) and interleukin 13 (IL-13) and decreased
levels of interferon γ (IFN-γ) and interleukin 17 (IL-17) in
the Colombian cohort [20]. In North America, otherwise
healthy adults with severe, atypical ocular toxoplasmosis were
found to be often infected with Type I or atypical strains but
not the more common Type II strains [21]. To date, however,
no definitive study has been performed to address this question
in large numbers of individuals.
There is also a paucity of data on the effects of acute and
chronic toxoplasmosis on the immune response in infected hu-
mans and whether these responses might differ between indi-
viduals in North America vs South America. To remedy these
gaps in our understanding, we analyzed the relative abundance
of 51 cytokines in peripheral blood sera of acutely and chroni-
cally infected pregnant women from the United States and Co-
lombia. For both cohorts, our results show a difference between
924 • JID 2014:210 (15 September) • Pernas et al
the acutely infected patients relative to uninfected. The nature of
those differences, however, was not the same between the 2 re-
gions, consistent with an impact of Toxoplasma strain type on
the host response to infection. We also saw very surprising ev-
idence of differences within the US cohort between chronically
infected and uninfected individuals. The implications of these
results on the different patient groups are discussed.
METHODS
US Patients
A total of 144 serum samples (Supplementary Table 1) from
human immunodeficiency virus (HIV)-negative, consecutive
pregnant women were collected. At the time the samples were
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obtained, the Toxoplasma-infection status of these women was
unknown, and thus no treatment was administered prior to
sample collection. In sum, 48 (average age 29; range 17–43)
were from uninfected women, 49 were from chronically infected
women (average age 29; range 16–47), and 47 (average age 30;
range 18–43) were from acutely infected women as established
by serological test results performed at the Toxoplasma Serology
Laboratory, Palo Alto Medical Foundation (PAMF-TSL, http://
www.pamf.org/serology/) [7, 8]. Age and state of residence for
each of the patients was collected, but the gestational age of the
fetus was not (it was later determined for those who were found
to be acutely infected but at the time this study was initiated, it
was not considered relevant for the uninfected or chronically in-
fected individuals). Serum was stored at −80°C until analysis.
Comparison of mean age in all US cohorts by unpaired t test
yielded no statistically significant differences. Informed consent
was obtained from all patients.
Colombian Patients
A total of 113 samples (Supplementary Table 2) were obtained
from a previously existing bank of sera isolated from HIV-
negative pregnant Colombian women that was originally
obtained for epidemiological studies aimed at determining the
seroprevalence of T. gondii infection [22, 23]. As with the Amer-
ican cohort, the Toxoplasma-infection status of these women
was unknown when the blood was drawn, and thus no treat-
ment was administered prior to sample collection. From 22
July 2005 to 31 December 2011, 3428 pregnant women from
12 healthcare facilities consented to answer a questionnaire
and allow investigators to take serum samples. Pregnant
women were enrolled at the time they were attending their rou-
tine prenatal care visit. Women who had a suspected or con-
firmed diagnosis of T. gondii infection during pregnancy prior
to being enrolled in the epidemiological study were excluded
from the group studied here. Sera for our study were from 50
consecutive uninfected women (average age 29; range 17–46),
50 consecutive chronically infected women (average age 29;
range 16–43), and 13 consecutive acutely infected women
(average age 27; range 18–36). As for the US patients, gestational
age was not systematically obtained. All sera were initially
placed at 4°C and within 1 week tested for anti-Toxoplasma
IgG and IgM at the Fundacion Valle del Lili (Cali, Colombia)
per the manufacturer’s instructions using a microparticle en-
zyme immunoassay (MEIA) method (Axsym System, Abbott
Laboratories, Chicago, IL) and a bioMerieux VIDAS IgG and
IgM (Marcy l’Etoile, France). The VIDAS IgG reference ranges
are: negative <4 IU/mL, equivocal 4 to <8 IU/mL, positive ≥8
IU/mL; and for IgM: negative <0.55, equivocal 0.55 to <0.65,
positive ≥0.65. The IgM test value is the ratio of the relative
fluorescent value of the sample to that of the calibrator. Based
on these tests, the pregnant women were classified as never in-
fected (negative IgG/negative IgM), chronically infected, that is,
infected more than 12 months ( positive IgG/negative IgM), and
possibly acutely infected ( positive IgG/positive IgM). The re-
mainder of each serum sample was stored at −20°C until
shipped to the United States using a transportation company
that uses a cold-chain system. Serum samples were stored at
PAMF-TSL at −20°C until the day of cytokine testing. Positive
IgG/positive IgM sera underwent confirmatory testing at
PAMF-TSL to determine whether an acute infection had
occurred within 1 year using the same criteria as above for the
US samples. Comparison of mean age in all cohorts by unpaired
t test yielded no statistically significant differences between any
of the Colombian cohorts or between US and Colombian
cohorts.
Serological Testing at PAMF-TSL
Each of the pregnant women in the acute Colombian cohort and
all of the US patients whose sera were used for this study were
tested at the PAMF-TSL. All samples were tested with the T. gon-
dii dye test (measuring primarily IgG antibodies [24]) and IgM
enzyme-linked immunosorbemt assay (ELISA), reported as arbi-
trary units and performed based on in-house standards [25].
Pregnant women were classified into 3 groups according to
their serological test results: (1) uninfected, negative IgG/negative
IgM; (2) chronically infected, positive IgG/negative IgM; (3)
acutely infected, positive IgG/positive IgM. In order to confirm
that the patient had been infected within 1 year prior to the
date of serum sampling, the positive IgG/positive IgM samples
were tested further with T. gondii-specific tests, including the dif-
ferential agglutination or AC/HS test [26], IgG avidity test [3],
IgA antibody test [27], and IgE antibody test [28].
Cytokine Measurement in Serum Samples
Serum samples were analyzed at the Human Immune Monitor-
ing Core (Stanford, CA) by Luminex Human 51-plex kits pur-
chased from Affymetrix (Santa Clara, CA) and used according
to the manufacturer’s recommendations with modifications as
described below. Each sample was measured in duplicate. Plates
were read using a Luminex 200 instrument with a lower bound
Immune-profiling Reveals Surprising Impact of Toxoplasma Infection on Peripheral Blood Cytokines
of 100 beads per sample per cytokine. Luminex measures fluo-
rescent intensities (FIs) of the 51 cytokines and produces a dis-
tribution of typically 200 to 300 FIs. The median FI (MFI) for
each distribution is computed. Because every subject’s sample
was entered in 2 wells, 2 MFIs per cytokine were computed
for each subject. Assay plate layout consisted of 9 standards in
duplicate, 1 blank well and 60 μL duplicates of each serum sam-
ple, each run with a 1:3 dilution. Cytokines assayed were:
CD40L, ENA78, eotaxin, FGF-β, GCSF, GMCSF, CXCL1,
HGF, IFN-α, IFN-β, IFN-γ, IL1-α, IL1-β, IL1R-α, IL-2, IL-4,
IL-5, IL-6, IL-7, IL-8, IL-10, IL12p40, IL12p70, IL-13, IL-15,
IL-17, IL17F, CXCL10, Leptin, LIF, CCL2, CCL7, MCSF,
CXCL9, CCL3, CCL4, NGF, PAI-1, PDGF-β-β, CCL5, RETN,
SCF, FasL, ICAM-1, VCAM1, TGF-α, TGF-β, TNF-α, TNF-β,
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TRAIL, and VEGF.
Statistical Analysis
Comparisons within a country were made on the same plate, so
data were normalized for each feature via centering and scaling
as (Feature – mean (Feature))/SD(Feature). We fit a separate re-
gression model to each feature as the outcome with dummy-
coded (0 = absent, 1 = present) predictor variables for status
(acute, chronic, uninfected) and plate. Regression coefficients
were estimated via generalized maximum entropy [29] using a
set of uniform support points over the interval [−5, 5]. Both raw
and adjusted P-values are reported. All P-value adjustments
were via an adaptive 2-stage linear step-up procedure [30] for
control of false discovery rate (FDR), a method that has been
shown in a comparative study to maintain close control of the
FDR and achieve good statistical power [31].
RESULTS
Cytokine Profiling of Infected US Cohorts
Sera from 47 pregnant US women who were asymptomatic but
acutely infected with Toxoplasma were analyzed and compared
to cytokine profiles from 48 uninfected pregnant females. Acute
infection was defined as one estimated to have commenced
within the prior 12 months as determined by serology tests at
PAMF-TSL. Surprisingly, of the 51 cytokines profiled, 23 were
significantly decreased in the acute cohort relative to the unin-
fected cohort with a stringent P value < .05 at an FDR of 5%
(Table 1, Figure 1A and 2B). These included CXCL1, IL-17,
IL1-α, IL-2, IFN-γ, IL-4, and several chemokines including
CCL5 and CCL7. Macrophage colony stimulating factor
(MCSF) alone was increased in the acutely infected cohort. Dif-
ferences in levels of the remaining 27 cytokines (Supplementary
Table 3) were not statistically significant, including TGF-β
and PDGF-β-β which have previously been demonstrated to
mediate inhibition of intracellular growth of T. gondii [32].
An analysis of the same panel of cytokines using sera from 49
asymptomatic, chronically infected women (ie, >1 year since
• JID 2014:210 (15 September) • 925
Table 1. Average Median Fluorescence Intensity (MFI) Listed per Cytokine for the Uninfected, Acute, and Chronically Infected Pregnant
American Cohorts

Average MFI Uninfected vs Acute Uninfected vs Chronic

Feature Uninfected Acute Chronic FDR P Value Raw P Value FDR P Value Raw P Value
CD40L 439.5 268.3 421.8 .0002 <.0001 .1955 .0345
Eotaxin 93.5 57.0 84.6 <.0001 <.0001 .5594 .3973
FGF-β 80.1 65.2 73.9 .0002 <.0001 .3045 .0847
GCSF 123.7 93.5 109.1 <.0001 <.0001 .1494 .0167
CXCL1 123.8 70.9 131.2 .0304 .0243 .5594 .3297
HGF 620.8 354.7 523.6 <.0001 <.0001 .3045 .1062
IFN-γ 31.9 21.6 28.6 <.0001 <.0001 .3045 .0887
IL-15 41.5 29.8 41.0 .0008 .0004 .6578 .4952
IL-17 92.0 68.2 79.8 <.0001 <.0001 .1441 .0068

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IL1-α 136.7 102.8 125.6 .0001 <.0001 .3045 .0844
IL-2 99.3 70.6 90.6 <.0001 <.0001 .3856 .1663
IL-4 97.4 71.9 90.0 <.0001 <.0001 .3045 .1049
IL-8 2039.8 640.5 657.3 .0275 .0192 .1441 .0113
LIF 55.0 38.7 47.2 <.0001 <.0001 .1441 .0049
CCL7 176.7 139.5 167.0 .0113 .0064 .4225 .2191
MCSF 196.8 284.0 210.0 .0275 .0184 .7025 .5648
CCL4 1835.2 576.7 967.0 .0285 .0209 .1494 .0176
CCL3 1179.6 574.2 887.1 .0129 .0077 .1580 .0217
NGF 25.8 18.6 22.4 <.0001 <.0001 .1441 .0096
CCL5 12 156.9 10 351.5 11 794.6 .0008 .0004 .5594 .3820
RETN 12 293.1 10 616.0 10 650.2 .0191 .0121 .1786 .0280
SCF 98.8 72.5 87.7 .0005 .0002 .3045 .0986
TNF-α 48.2 31.3 46.0 .0287 .0220 .7482 .6455
TRAIL 822.8 513.0 607.7 .0105 .0056 .3856 .1447

N = 48, 47 and 49 for uninfected, acute, and chronic cohorts, respectively. Data shown are only for those cytokines (P < .05, FDR at 5%) that yielded a significant
difference between either pairwise comparison. FDR P value is adjusted to control the FDR as described in methods.
Abbreviations: FDR, false discovery rate; IL, interleukin; TNF, tumor necrosis factor.

initial infection) yielded similar trends: 9 of the 24 cytokines de-
pressed in the acute patients were also depressed in the chronic
patients relative to the uninfected cohort (raw P value < .05), al-
though none of these differences were significant using an FDR
of 5% (Table 1, Figure 1A and 1B).
Cytokine Profiling of Infected Colombian Cohorts
To determine the cytokine profile in peripheral blood from Co-
lombian patients, we compared serum cytokine levels of 13 acutely
infected pregnant Colombian women to a control group of 50 un-
infected pregnant women from this same country. The small
number of acutely infected individuals in the Colombian cohort,
however, meant that large differences between the 2 patient groups
were needed to reach statistical significance (especially given the
naturally large variation in cytokine levels typical between individ-
uals). In contrast to the differences in 24 cytokines between the US
uninfected and acute cohorts, only 8 profiled serum cytokines
were significantly different in Colombian patients with acute toxo-
plasmosis relative to uninfected controls with a P value < .05 at an
FDR of 5%. Four of these, ICAM1, IL1-α, CXCL10, and CXCL9,
were higher on average in the acute patients, whereas CD40L, IL-8,
CCL5, and RETN were observed at lower levels relative to unin-
fected controls (Table 2; Figure 2A and 2B).
Given the small size of the acute Colombian cohort, we ap-
plied a less stringent threshold (FDR at 20%) to see if other cy-
tokines trended in similar directions. We observed that, using
this threshold, CCL7, Trail, and TNF-β were also present at in-
creased levels in acutely infected vs uninfected patients, whereas
CCL4 and HGF were present at decreased levels (Table 2; Fig-
ure 2A and 2B). No differences were observed in the other 38
cytokines assayed (Supplementary Table 3). A similar analysis
of the chronic and uninfected Colombian cohorts, both consist-
ing of 50 individuals, showed no statistically significant differ-
ences between the two groups. These data suggest that, at least
with these cohorts of pregnant Colombian women, acutely in-
fected patients exhibit dramatically different immune profiles in
comparison to uninfected patients, whereas chronic patients are
not detectably different from the uninfected group.

926 • JID 2014:210 (15 September) • Pernas et al

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Figure 1. Cytokine profiling of infected US cohorts. A, Median florescent intensity (MFI) values (y-axis) represent the ratio of the average MFI per cytokine
between acute and uninfected, and chronic and uninfected pregnant US women. All cytokines shown had statistical significance for the acute to uninfected
comparison (P < .05 at FDR 5%). Nine cytokines (denoted by §) also had a raw P value < .05 for the chronic to uninfected comparison, although none were
significant (P < .05) between these 2 groups using the more stringent test of having the FDR set at 5%. Dotted line at 1 represents no difference in MFI
values between uninfected and infected cohorts. B, MFI values (y-axis) reported for all cohorts (x-axis): Uninfected (U), Acute (A), and Chronic (C) for those
cytokines found to be significantly different between uninfected and acutely infected cohorts. Abbreviation: FDR, false discovery rate.

DISCUSSION mediated by elevation of several cytokines, including IL-12,

IFN-γ, IL-6, IL-10, and TNF-α in mice, and CD40L during
Prior studies in vitro or in animal models have shown that pro- human infection [33, 34]. In contrast, we observed that 23 of
tective and inflammatory immune responses to the parasite are 51 cytokines assayed, including IFN-γ, IL1-α, IL-2, TNF-α,

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Figure 2. Cytokine profiling of infected Colombian cohorts. A, MFI values (y-axis) represent the ratio of the average MFI per cytokine between acute and
uninfected, and chronic and uninfected pregnant Colombian women. All cytokines shown had statistical significance for the acute to uninfected comparison
(raw P < .05). Eight cytokines (denoted by *) were also significant (P < .05) using the more stringent test where the FDR is set to 5%. No significant dif-
ferences were detected between the chronic and uninfected cohorts. Dotted line at 1 represents no difference in MFI values between uninfected and
infected cohorts. B, MFI values (y-axis) reported for all cohorts (x-axis): Uninfected (U), Acute (A), and Chronic (C) for those cytokines found to be signifi-
cantly different between uninfected and acutely infected cohorts. Abbreviations: FDR, false discovery rate; MFI, median florescent intensity.

IL-17, CD40L, and IL-4 were lower in the serum of acutely in- and CCL7. In support of these findings, an independent study
fected pregnant US patients relative to uninfected pregnant pa- from Spain revealed that levels of circulating CCL2 were
tients. This was also true of several chemokines, including CCL5 depressed in patients who had active ocular toxoplasmosis

928 • JID 2014:210 (15 September) • Pernas et al

Table 2. Average MFI Listed per Cytokine for the Uninfected, Acute, and Chronically Infected Pregnant Colombian Cohorts

Average MFI Uninfected vs Acute Uninfected vs Chronic

Feature Uninfected Acute Chronic FDR P Value Raw P Value FDR P Value Raw P Value
CD40L 585.0 443.8 633.4 .0199 .0037 .8663 .6282
HGF 671.7 539.0 640.1 .0712 .0199 .8663 .5660
ICAM1 9026.4 12438.8 9887.8 .0038 .0006 .6627 .2988
IL1-α 129.1 243.5 131.8 <.0001 <.0001 .9675 .9245
IL-8 495.1 72.9 496.9 .0033 .0005 .9921 .9839
CXCL10 126.8 728.6 131.9 <.0001 <.0001 .9921 .9921
CCL7 196.5 255.4 214.3 .0514 .0108 .6627 .2560
CXCL9 282.1 640.3 295.9 <.0001 <.0001 .9405 .8181
CCL4 769.0 538.9 805.5 .0642 .0164 .9405 .7437
CCL5 11 817.2 10 274.9 11 639.4 .0006 <.0001 .8663 .5454

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RETN 10 948.7 7629.9 10 279.2 .0005 <.0001 .6918 .3255
TNF-β 108.9 152.7 127.1 .0642 .0164 .6627 .2945
TRAIL 495.0 568.5 527.5 .0895 .0271 .7034 .3586
N = 50, 13, and 50 for uninfected, acute, and chronic cohorts, respectively. Data shown are only for those cytokines that yielded a significant difference (raw P < .05)
between either pairwise comparison.
Abbreviations: FDR, false discovery rate; IL, interleukin; MFI, median fluorescence intensity; TNF, tumor necrosis factor.

when compared to patients with inactive disease or uninfected
controls [35]. Like CCL5 and CCL7, CCL2 is a member of the
β-chemokine family and mediates the recruitment of inflamma-
tory cells to target tissues [36, 37].
Although the results obtained may be affected by the Th2-
polarization reported in pregnant women [38], it is intriguing
that we see a decrease in levels of circulating cytokines in the
acutely infected pregnant American cohort, presumably already
immunosuppressed, relative to uninfected pregnant controls.
These depressed cytokines included GCSF and TNF-α, previ-
ously reported to be often elevated during pregnancy [39] and
leukemia inhibitory factor (LIF), a secreted cytokine with a crit-
ical role in blastocyst development [40], which remained de-
pressed in chronic US patients relative to uninfected controls
(FDR at 20%). The mechanistic basis for the depressed cytokine
levels observed here is not known but could be the result of
down-regulatory pathways activated in an attempt to neutralize
potential immunopathology in various organs including the
placenta.
We observed clear differences in cytokine profiles of the
geographically distinct cohorts (Figure 3). Unlike the systemic
suppression observed in the acutely infected Americans, howev-
er, only 4 cytokines were at significantly (FDR of 5%) lower
levels in the acutely infected pregnant Colombian cohort rela-
tive to uninfected controls. Furthermore, Colombian acute
patients exhibited increased levels of proinflammatory IL1-α
and Trail, a known inducer of apoptosis, both of which were
depressed in serum of US acute patients relative to uninfected
US controls. These differences in cohorts may be attributable
to the infecting Toxoplasma strain type; although we do not
have definitive information on the infecting Toxoplasma strain
in the tested cohorts, Type II Toxoplasma infections have been
shown to dominate in Europe and North America, whereas
“atypical” strains are more commonly found in South America.
A recent study by de-la-Torre et al [20] comparing Toxoplasma
strains and intraocular cytokine levels in OT patients in South
America and Europe found that Colombian OT patients were
infected with various Toxoplasma Types and had lower IFN-γ
and IL-17 and higher IL-6 and IL-13 intraocular levels relative
to French OT patients, who were determined to be uniformly
infected with Type II Toxoplasma. The differing cytokine levels
reported in our work and the de-la-Torre study may reflect
differences observed in the local microenvironment (ie, ocular
fluids) vs the systemic immune response (ie, serum), as previ-
ously reported during inflammation and infection [41, 42].
We cannot, of course, exclude the possibility that confounding
factors could be wholly or partially involved in these regional
differences, including the state of the infecting parasite,
coinfection status, diet, medications, and host genetics. Regard-
less of the mechanism underlying the geographic differences
reported here, our results may provide a partial mechanistic
explanation for why the parasite appears to cause a “clinically be-
nign” acute infection in the vast majority of individuals in the
United States including pregnant woman, whereas it is prone
to cause symptomatic and more aggressive disease in South
America: the immune response to infection in the 2 regions is
very different and this could well impact the resulting pathology.
Interestingly, and very surprisingly, several cytokines includ-
ing GCSF, IL-17, LIF, CCL3, and NGF were depressed (FDR at
20%) in chronically infected US patients relative to uninfected

Immune-profiling Reveals Surprising Impact of Toxoplasma Infection on Peripheral Blood Cytokines • JID 2014:210 (15 September) • 929

Figure 3. Schematic of cytokines significantly dysregulated between US
acute, US chronic, and Colombian acute cohorts in comparison to uninfect-
ed cohorts sampled within the same country. All cytokines significant in
any pair-wise combination (P < .05), either with the more stringent FDR
of 5% (for the US samples) or a raw P value < .05 (for the Colombian co-
hort) were included. Arrows and arrowheads indicate the difference rela-
tive to uninfected controls in the US and Colombian cohorts, respectively.
Abbreviation: FDR, false discovery rate.
controls. This suggests that there may be significant, long-term
effects of a chronic Toxoplasma infection in American pregnant
women who are otherwise asymptomatic. For example, a chron-
ic infection could have profound implications for how the preg-
nant woman’s immune system responds to other infections,
allergens, or vaccines. Chronic immune stimulation has been
shown to alter viral dynamics [43], and while little is known
about the functional significance of steady-state blood cyto-
kines, it is perhaps necessary to revisit the concept of a chronic
infection being “asymptomatic.” Specifically, these results sug-
gest that attention should be paid to minor sequelae that might
occur in children born to even chronically infected mothers.
We know of no reports that such children experience any symp-
toms but such might have been too minor to have been noted
in studies performed to date. In aggregate, our data suggest
that Toxoplasma infection is causing altered levels of several
cytokines in the chronically infected US cohorts, although we
cannot exclude the possibility that these chronically infected
individuals are distinct in some other unknown way compared
to the uninfected group. For example, it could be that extrinsic
factors, like host genetics, coinfection status, lifestyle, etc,
determine whether an individual maintains a measurable titer
of anti-Toxoplasma antibodies for a prolonged period and
that this other factor explains the differences between these
2 groups.
930 • JID 2014:210 (15 September) • Pernas et al
We recognize this study has some limitations. We did not
include nonpregnant women in our cohort, and there could
be an effect of gestational age on the immune response;
hence, findings in this study may not be generalizable to non-
pregnant females or males. Second, although the onset of infec-
tion was estimated for the acute and chronic classifications, we
cannot know the exact date. In addition, the time of day of the
blood draw was not recorded and may impact results because
levels of circulating cytokines are known to be influenced by
circadian rhythms, meals, exercise, and other factors. Within
the United States and Colombia, the patients were sampled
identically, so this is unlikely to explain within-country differ-
ences; however, it could contribute to the between-country dif-
ferences seen here. Similarly, we cannot exclude that differences
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in storage protocols for samples in the 2 countries underlie
some of the region-specific difference, especially the use of
−20°C for the Colombia samples vs −80°C for the US samples.
Cytokines may decay during storage, as a function of both
time and temperature, and this could contribute to some of
the differences seen between regions.
Despite these limitations, this work raises several important
questions. Can knowledge of the cytokine network during
infection enable us to design targeted therapeutic strategies?
There is, in fact, considerable debate about whether to use im-
munosuppressing steroids to control excessive inflammation
during ocular toxoplasmosis in adults or whether such drugs
or treatments are antagonistic to the immune system’s efforts
to control the infection [44]. From what cell types and tissues
are these cytokines being made during infection? By addressing
these questions, we can learn more about what shapes the im-
munological milieu during the initial infection and expansion
of Toxoplasma, and how influencing this milieu can dictate
the progression and pathogenesis of infection in pregnant
women.
Supplementary Data
Supplementary materials are available at The Journal of Infectious Diseases
online (http://jid.oxfordjournals.org/). Supplementary materials consist of
data provided by the author that are published to benefit the reader. The
posted materials are not copyedited. The contents of all supplementary
data are the sole responsibility of the authors. Questions or messages regard-
ing errors should be addressed to the author.
Notes
Acknowledgments. We thank C. Press (Palo Alto Medical Foundation
Toxoplasma Serology), P. Moncada (Fundacion Valle del Lili), and S. Ochoa
for organizing the serum shipments from Colombia, C. Tato for helpful
advice and reading of the manuscript, C. Kwok and Y. Hassenburg-Rosen
(Stanford Human Immune Monitoring Center (HIMC)) for Luminex anal-
ysis, and the staff of the Stanford Institute for Immunity, Transplantation,
and Infection for logistical/administrative support.
Financial support. This work was supported by NIH grant U19
AI-057229 (J. C. B., J. G. M.) and NSF (L. F. P.), L. F. P. was also supported
by a Stanford Graduate Fellowship.
 Potential conflicts of interest. All authors: No reported conflicts.
All authors have submitted the ICMJE Form for Disclosure of Potential
Conflicts of Interest. Conflicts that the editors consider relevant to the con-
tent of the manuscript have been disclosed.
References
1. Montoya JG, Liesenfeld O. Toxoplasmosis. Lancet 2004; 363:1965–76.
2. Bahia-Oliveira LM, Jones JL, Azevedo-Silva J, et al. Highly endemic,
waterborne toxoplasmosis in north Rio de Janeiro state, Brazil. Emerg
Infect Dis 2003; 9:55–62.
3. Montoya JG, Huffman HB, Remington JS. Evaluation of the immuno-
globulin G avidity test for diagnosis of toxoplasmic lymphadenopathy.
J Clin Microbiol 2004; 42:4627–31.
4. Gilbert R, Tan HK, Cliffe S, Guy E, Stanford M. Symptomatic toxoplas-
ma infection due to congenital and postnatally acquired infection. Arch
Dis Child 2006; 91:495–8.
5. Lebech M, Joynson DH, Seitz HM, et al. Classification system and case
definitions of Toxoplasma gondii infection in immunocompetent
pregnant women and their congenitally infected offspring. European
Research Network on Congenital Toxoplasmosis. Eur J Clin Microbiol
Infect Dis 1996; 15:799–805.
6. Montoya JG, Remington JS. Studies on the serodiagnosis of toxoplasmic
lymphadenitis. Clin Infect Dis 1995; 20:781–9.
7. Remington JS, McLeod R, Thuilliez P, Desmonts G. Toxoplasmosis. In:
Remington JS, Klein JO, Wilson CB, Baker C, eds. Infectious diseases of
the fetus and newborn infant. 6th ed. Philadelphia: Elsevier Saunders,
2006.
8. Montoya JG. Laboratory diagnosis of Toxoplasma gondii infection and
toxoplasmosis. J Infect Dis 2002; 185 (Suppl 1):S73–82.
9. Howe DK, Sibley LD. Toxoplasma gondii comprises three clonal
lineages: correlation of parasite genotype with human disease. J Infect
Dis 1995; 172:1561–6.
10. Shobab L, Pleyer U, Johnsen J, et al. Toxoplasma serotype is associated
with development of ocular toxoplasmosis. J Infect Dis 2013; 208:
1520–8.
11. Gilbert RE, Freeman K, Lago EG, et al. Ocular sequelae of congenital
toxoplasmosis in Brazil compared with Europe. PLoS Negl Trop Dis
2008; 2:e277.
12. Mercier A, Devillard S, Ngoubangoye B, et al. Additional haplogroups
of Toxoplasma gondii out of Africa: population structure and mouse-
virulence of strains from Gabon. PLoS Negl Trop Dis 2010; 4:e876.
13. Fuentes I, Rubio JM, Ramirez C, Alvar J. Genotypic characterization of
Toxoplasma gondii strains associated with human toxoplasmosis in
Spain: direct analysis from clinical samples. J Clin Microbiol 2001;
39:1566–70.
14. Furtado JM, Winthrop KL, Butler NJ, Smith JR. Ocular toxoplasmosis I:
parasitology, epidemiology and public health. Clin Experiment Oph-
thalmol 2013; 41:82–94.
15. Lehmann T, Marcet PL, Graham DH, Dahl ER, Dubey JP. Globalization
and the population structure of Toxoplasma gondii. Proc Natl Acad Sci
U S A 2006; 103:11423–8.
16. Glasner PD, Silveira C, Kruszon-Moran D, et al. An unusually high
prevalence of ocular toxoplasmosis in southern Brazil. Am J Ophthal-
mol 1992; 114:136–44.
17. Darde ML, Villena I, Pinon JM, Beguinot I. Severe toxoplasmosis
caused by a Toxoplasma gondii strain with a new isoenzyme type ac-
quired in French Guyana [letter]. J Clin Microbiol 1998; 36:324.
18. de-la-Torre A, Lopez-Castillo CA, Gomez-Marin JE. Incidence and
clinical characteristics in a Colombian cohort of ocular toxoplasmosis.
Eye (London) 2009; 23:1090–3.
19. de-la-Torre A, Lopez-Castillo CA, Rueda JC, et al. Clinical patterns of
uveitis in two ophthalmology centres in Bogota, Colombia. Clin Exper-
iment Ophthalmol 2009; 37:458–66.
20. de-la-Torre A, Sauer A, Pfaff AW, et al. Severe South American ocular
toxoplasmosis is associated with decreased IFN-gamma/Il-17a and
Immune-profiling Reveals Surprising Impact of Toxoplasma Infection on Peripheral Blood Cytokines
increased Il-6/Il-13 intraocular levels. PLoS Negl Trop Dis 2013; 7:
e2541.
21. Grigg ME, Ganatra J, Boothroyd JC, Margolis TP. Unusual abundance
of atypical strains associated with human ocular toxoplasmosis. J Infect
Dis 2001; 184:633–9.
22. Rosso F, Les JT, Agudelo A, et al. Prevalence of infection with Toxoplas-
ma gondii among pregnant women in Cali, Colombia, South America.
Am J Trop Med Hyg 2008; 78:504–8.
23. Ordonez CA, Sanchez AI, Pineda JA, et al. Deferred primary anastomo-
sis versus diversion in patients with severe secondary peritonitis man-
aged with staged laparotomies. World J Surg 2010; 34:169–76.
24. Sabin AB, Feldman HA. Dyes as microchemical indicators of a new
immunity phenomenon affecting a protozoon parasite (Toxoplasma).
Science 1948; 108:660–3.
25. Naot Y, Remington JS. An enzyme-linked immunosorbent assay for
detection of IgM antibodies to Toxoplasma gondii: use for diagnosis
of acute acquired toxoplasmosis. J Infect Dis 1980; 142:757–66.
26. Dannemann BR, Vaughan WC, Thulliez P, Remington JS. Differential
Downloaded from https://academic.oup.com/jid/article/210/6/923/2908544 by guest on 09 May 2023
agglutination test for diagnosis of recently acquired infection with Toxo-
plasma gondii. J Clin Microbiol 1990; 28:1928–33.
27. Stepick-Biek P, Thulliez P, Araujo FG, Remington JS. IgA antibodies for
diagnosis of acute congenital and acquired toxoplasmosis. J Infect Dis
1990; 162:270–3.
28. Wong SY, Hajdu MP, Ramirez R, et al. Role of specific immunoglobulin
E in diagnosis of acute toxoplasma infection and toxoplasmosis. J Clin
Microbiol 1993; 31:2952–9.
29. Benjamini Y, Krieger AM, Yekutieli D. Adaptive linear step-up procedures
that control the false discovery rate. Biometrika 2006; 93:491–507.
30. Kim KI, van de Wiel MA. Effects of dependence in high-dimensional
multiple testing problems. BMC Bioinformatics 2008; 9:114.
31. Golan A, Judge G, Miller D. Maximum Entropy Econometrics. Robust
Estimation with Limited Data. Chichester: John Wiley & Sons Ltd,
1996.
32. Chumpitazi BF, Simon J, Polack B, et al. Human platelet inhibition of
Toxoplasma gondii growth. Clin Exp Immunol 1998; 111:325–33.
33. Dupont CD, Christian DA, Hunter CA. Immune response and immu-
nopathology during toxoplasmosis. Semin Immunopathol 2012; 34:
793–813.
34. Van Grol J, Muniz-Feliciano L, Portillo JA, Bonilha VL, Subauste CS.
CD40 induces anti-Toxoplasma gondii activity in nonhematopoietic
cells dependent on autophagy proteins. Infect Immun 81:2002–11.
35. Rey A, Molins B, Llorenc V, et al. Cytokine profiling reveals decreased
serum levels of CCL2 in active ocular toxoplasmosis. Br J Ophthalmol
2013; 97:1338–42.
36. Moser B, Loetscher P. Lymphocyte traffic control by chemokines. Nat
Immunol 2001; 2:123–8.
37. Zlotnik A, Yoshie O. Chemokines: a new classification system and their
role in immunity. Immunity 2000; 12:121–7.
38. Mor G, Cardenas I, Abrahams V, Guller S. Inflammation and pregnan-
cy: the role of the immune system at the implantation site. Ann N Y
Acad Sci 2011; 1221:80–7.
39. Kraus TA, Sperling RS, Engel SM, et al. Peripheral blood cytokine
profiling during pregnancy and post-partum periods. Am J Reprod
Immunol 2010; 64:411–26.
40. Nachtigall MJ, Kliman HJ, Feinberg RF, et al. The effect of leukemia
inhibitory factor (LIF) on trophoblast differentiation: a potential role
in human implantation. J Clin Endocrinol Metab 1996; 81:801–6.
41. Riche F, Gayat E, Collet C, et al. Local and systemic innate immune
response to secondary human peritonitis. Crit Care 2013; 17:R201.
42. Salinas-Carmona MC, Rosas-Taraco AG, Welsh O. Systemic increased
immune response to Nocardia brasiliensis co-exists with local immunosup-
pressive microenvironment. Antonie Van Leeuwenhoek 2012; 102:473–80.
43. Schwiebert R, Fultz PN. Immune activation and viral burden in acute
disease induced by simian immunodeficiency virus SIVsmmPBj14: cor-
relation between in vitro and in vivo events. J Virol 1994; 68:5538–47.
44. Holland GN. Reconsidering the pathogenesis of ocular toxoplasmosis.
Am J Ophthalmol 1999; 128:502–5.
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