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Jiu 189
Jiu 189
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.
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 the acutely infected patients relative to uninfected. The nature of
tissue cysts have a predilection for neural, muscular, and ocular those differences, however, was not the same between the 2 re-
tissue and persist indefinitely for the life of the host. gions, consistent with an impact of Toxoplasma strain type on
Encystation marks the chronic phase of infection, and if a the host response to infection. We also saw very surprising ev-
chronically infected individual becomes immunocompromised, idence of differences within the US cohort between chronically
these tissue cysts serve as a reservoir from which local infections infected and uninfected individuals. The implications of these
and further dissemination can develop. Similar to the life- results on the different patient groups are discussed.
lasting tissue cysts, Toxoplasma-specific immunoglobulin G
(IgG) levels can also persist for the life of the host [6], likely METHODS
due to cycles of reactivation and, perhaps, reinfection. It is pos-
sible to differentiate between acute infections (initiated within US Patients
the prior 12 months) and chronic infections (>1 year since A total of 144 serum samples (Supplementary Table 1) from
initial infection) by a panel of serological tests [7, 8]. human immunodeficiency virus (HIV)-negative, consecutive
Remarkably, given the ubiquity of this parasite, the majority pregnant women were collected. At the time the samples were
Immune-profiling Reveals Surprising Impact of Toxoplasma Infection on Peripheral Blood Cytokines • 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
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
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- FDR of 5%. Four of these, ICAM1, IL1-α, CXCL10, and CXCL9,
pressed in the acute patients were also depressed in the chronic were higher on average in the acute patients, whereas CD40L, IL-8,
patients relative to the uninfected cohort (raw P value < .05), al- CCL5, and RETN were observed at lower levels relative to unin-
though none of these differences were significant using an FDR fected controls (Table 2; Figure 2A and 2B).
of 5% (Table 1, Figure 1A and 1B). Given the small size of the acute Colombian cohort, we ap-
plied a less stringent threshold (FDR at 20%) to see if other cy-
Cytokine Profiling of Infected Colombian Cohorts tokines trended in similar directions. We observed that, using
To determine the cytokine profile in peripheral blood from Co- this threshold, CCL7, Trail, and TNF-β were also present at in-
lombian patients, we compared serum cytokine levels of 13 acutely creased levels in acutely infected vs uninfected patients, whereas
infected pregnant Colombian women to a control group of 50 un- CCL4 and HGF were present at decreased levels (Table 2; Fig-
infected pregnant women from this same country. The small ure 2A and 2B). No differences were observed in the other 38
number of acutely infected individuals in the Colombian cohort, cytokines assayed (Supplementary Table 3). A similar analysis
however, meant that large differences between the 2 patient groups of the chronic and uninfected Colombian cohorts, both consist-
were needed to reach statistical significance (especially given the ing of 50 individuals, showed no statistically significant differ-
naturally large variation in cytokine levels typical between individ- ences between the two groups. These data suggest that, at least
uals). In contrast to the differences in 24 cytokines between the US with these cohorts of pregnant Colombian women, acutely in-
uninfected and acute cohorts, only 8 profiled serum cytokines fected patients exhibit dramatically different immune profiles in
were significantly different in Colombian patients with acute toxo- comparison to uninfected patients, whereas chronic patients are
plasmosis relative to uninfected controls with a P value < .05 at an not detectably different from the uninfected group.
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.
Immune-profiling Reveals Surprising Impact of Toxoplasma Infection on Peripheral Blood Cytokines • JID 2014:210 (15 September) • 927
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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
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
when compared to patients with inactive disease or uninfected have definitive information on the infecting Toxoplasma strain
controls [35]. Like CCL5 and CCL7, CCL2 is a member of the in the tested cohorts, Type II Toxoplasma infections have been
β-chemokine family and mediates the recruitment of inflamma- shown to dominate in Europe and North America, whereas
tory cells to target tissues [36, 37]. “atypical” strains are more commonly found in South America.
Although the results obtained may be affected by the Th2- A recent study by de-la-Torre et al [20] comparing Toxoplasma
polarization reported in pregnant women [38], it is intriguing strains and intraocular cytokine levels in OT patients in South
that we see a decrease in levels of circulating cytokines in the America and Europe found that Colombian OT patients were
acutely infected pregnant American cohort, presumably already infected with various Toxoplasma Types and had lower IFN-γ
immunosuppressed, relative to uninfected pregnant controls. and IL-17 and higher IL-6 and IL-13 intraocular levels relative
These depressed cytokines included GCSF and TNF-α, previ- to French OT patients, who were determined to be uniformly
ously reported to be often elevated during pregnancy [39] and infected with Type II Toxoplasma. The differing cytokine levels
leukemia inhibitory factor (LIF), a secreted cytokine with a crit- reported in our work and the de-la-Torre study may reflect
ical role in blastocyst development [40], which remained de- differences observed in the local microenvironment (ie, ocular
pressed in chronic US patients relative to uninfected controls fluids) vs the systemic immune response (ie, serum), as previ-
(FDR at 20%). The mechanistic basis for the depressed cytokine ously reported during inflammation and infection [41, 42].
levels observed here is not known but could be the result of We cannot, of course, exclude the possibility that confounding
down-regulatory pathways activated in an attempt to neutralize factors could be wholly or partially involved in these regional
potential immunopathology in various organs including the differences, including the state of the infecting parasite,
placenta. coinfection status, diet, medications, and host genetics. Regard-
We observed clear differences in cytokine profiles of the less of the mechanism underlying the geographic differences
geographically distinct cohorts (Figure 3). Unlike the systemic reported here, our results may provide a partial mechanistic
suppression observed in the acutely infected Americans, howev- explanation for why the parasite appears to cause a “clinically be-
er, only 4 cytokines were at significantly (FDR of 5%) lower nign” acute infection in the vast majority of individuals in the
levels in the acutely infected pregnant Colombian cohort rela- United States including pregnant woman, whereas it is prone
tive to uninfected controls. Furthermore, Colombian acute to cause symptomatic and more aggressive disease in South
patients exhibited increased levels of proinflammatory IL1-α America: the immune response to infection in the 2 regions is
and Trail, a known inducer of apoptosis, both of which were very different and this could well impact the resulting pathology.
depressed in serum of US acute patients relative to uninfected Interestingly, and very surprisingly, several cytokines includ-
US controls. These differences in cohorts may be attributable ing GCSF, IL-17, LIF, CCL3, and NGF were depressed (FDR at
to the infecting Toxoplasma strain type; although we do not 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
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
Immune-profiling Reveals Surprising Impact of Toxoplasma Infection on Peripheral Blood Cytokines • JID 2014:210 (15 September) • 931