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This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process which may lead to differences between this version and the Version of Record. Please cite this article as an 'Accepted Article', doi: 10.1111/imm.12438

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Received Date : 08-Apr-2014

Revised Date : 15-Dec-2014

Accepted Date : 23-Dec-2014

Article type : Original Article

STAT-1 plays a critical role in control of

Trypanosoma cruzi infection

Manjusha M. Kulkarni 2,3#, Sanjay Varikuti 4#, Cesar Terrazas 4, Jennifer L. Kimble 4, Abhay Satoskar

2,3,4* and Bradford S. McGwire 1,2,3 *

1Division of Infectious Diseases, 2Center for Microbial Interface Biology, 3Department of Microbial

Infection and Immunity, and Department of Pathology 4

The Ohio State University Medical Center, Columbus, Ohio, USA

Running title: STAT-1 controls T. cruzi infection

Key words: STAT-1, Trypanosoma cruzi, infection, immune response

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Abbreviations: CpG, cytosine-phosphate-guanine; DMEM, Dulbecco’s modified-Eagles medium;

ELISA, enzyme-linked immunosorbent assay; IL, interleukin; IFN-γ, interferon gamma; KO, knock

out; STAT, signal transducer and activator of transcription; Th, T-cell helper; LPS,

lipopolysaccharide; WT, wild-type; TNF, tumor necrosis factor

# Authors contributed equally and share first authorship

*Corresponding authors: Bradford S. McGwire, MD, PhD, Doan Hall N1145, 410 W. 10th Ave.

Columbus, Ohio 43210. Email: [email protected]; Tel: 614-292-3226; Fax: 614-292-9616;

Abhay Satoskar, MD, PhD, M417 Starling-Loving Hall, 320 10th Ave. Columbus, Ohio 43210. Email:

[email protected]; Tel. 614-366-3417; Fax. (614) 292-7072.

ABSTRACT

The control of Trypanosoma cruzi infection is related to IFN-γ activation leading to intracellular

clearance of parasites. The transcription factor STAT (signal transducer and activator of

transcription) -1 is a key mediator of IFN-γ intracellular signaling and knock-out of this protein

leads to susceptibility to several intracellular microbes. To determine the role of STAT-1 in host

susceptibility to T. cruzi infection we compared the survival, parasite loads and balance of IFN-

γ and IL-10 responses between WT and STAT-1KO mice. We found that the lack of STAT-1 resulted

in a more robust infection, leading to higher levels of blood and tissue parasites and markedly

reduced survival. In addition, infected STAT-1KO mice had higher systemic levels of both IFN-γ and

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IL-10 suggesting that the absence of STAT-1 leads to a disequilibrium of pro- and anti-

inflammatory cytokines. Analysis of spleen cells indicates that CD4, CD8 generate IFN-γ and NK

cells express IL-13 in STAT-1KO animals. The production of IL-17 is particularly enhanced in the

absence STAT-1 expression yet did not reduce mortality. Overall these results indicate that STAT-1

is important for the control of T. cruzi infection in mice.

INTRODUCTION

The parasitic protozoan Trypanosoma cruzi has a complex life cycle wherein it alternately infects

insect and mammalian hosts. Infection of mammals is initiated by deposition of infective

metacyclic trypomastigotes present in insect urine and feces into the bite wound. Parasites then

infect both non-phagocytic and phagocytic cells causing acute infection. Internalized parasites

transform into intracellular amastigotes where they replicate and thereafter transform into

motile trypomastigotes and exit infected host cells. Dissemination of these parasites occurs

throughout the body in the blood and lymphatic systems lead to infection of multiple cell types.

Chronic infection leads to disease in the cardiovascular (cardiomyopathy) and gastrointestinal

(megaorgan syndromes) pathology in about 30% of infected patients. Host control of T. cruzi

infection is not well understood but probably involves the expression of interferon-gamma (IFN-γ)

and its action on target cells which initiate the production of anti-parasitic nitric oxide in both

non-phagocytic and phagocytic cells 1.

The transduction of signals from engagement of IFN-γ receptors is mediated by JAK (Janus Kinase)

phosphorylation of STAT-1 (signal transducers and activators of transcription) which translocates

to the nucleus to act on the target promoters leading to downstream gene activation which is

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important for the production pro-inflammatory responses responsible for microbial control 2-5.

Studies using STAT-1 knockout mice have shown that they are more susceptible to viral infections

and other microbial agents such as Listeria monocytogenes and intracellular protozoa Leishmania

and Toxoplasma 6-8. The microbial susceptibility in these mice are thought to be due to impaired

IFN-γ responsiveness leading to defective nitric oxide production and impaired T-cell responses.

STAT-1 expression may also be important for the regulation of anti-inflammatory IL-10 during

microbial infection9.

Although IFN-γ probably plays a central role in controlling T. cruzi infection as does the

development of a Th1 response, the roles of the balance in Th1/Th2 responses in the outcome of

infection are unknown10-12. The role of STAT-1 in control of T. cruzi infection has not previously

been reported although IL-17, which undergoes STAT-1-dependent regulation13, has recently been

reported to be important for controlling the inflammation and mortality associated with T. cruzi

infection 14, 15. Here we show that STAT-1KO leads to enhanced susceptibility to T. cruzi infection

and dissemination of parasites and death. The elevated levels of systemic IFN-γ and IL-10 in

infected STAT-1KO mice suggest dysregulation of opposing pro- and anti-inflammatory cytokine

production in the absence STAT-1. IL-17 is elevated in the absence of STAT-1 which coincides with

the neutrophilic inflammation of the spleen and heart pointing to reciprocal regulation of IFN-γ

and IL-17 in the control of T. cruzi infection.

MATERIALS AND METHODS

Parasites- Trypanosoma cruzi strains (Brazil) were routinely cultivated as epimastigotes in liver-

digested neutralized-tryptone medium supplemented with 10% heat inactivated fetal bovine

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serum (HIFBS) and 20 μg ml-1 hemin. Late stationary phase cells were used to infect the HCF9 line

of rat heart myoblasts which were routinely grown in DMEM supplemented with 10% heat-

inactivated bovine serum. Tissue culture trypomastigotes emerging from these infected cultures

were collected, enumerated and diluted to the appropriate density prior to use in infection

studies.

Mouse infection- Six- to 8-wk-old STAT-1−/− female Balb/c mice were generated as described

previously 8. Wild-type (WT) female Balb/c mice were purchased from The Jackson Laboratory

(Bar Harbor, ME, USA). All mice were kept in specific pathogen-free facilities at Ohio State

University according to institutional guidelines. Mice were infected intraperitoneally with 105

tissue-culture trypomastigotes in 100 μL of DMEM. Mice were observed up to 30 days post-

infection. Mice that were determined to be significantly ill but had not yet succumbed to infection

were subjected to euthanasia and processed for analysis. Tissue from animals was processed for

parasite outgrowth explantation by mincing of tissue and placing in LDNT and incubating cultures

at 25º C and observed daily by light microscopy. In cultures where motile parasites first appeared,

the average number of parasites in at least 10 high-powered fields was counted. For

histopathologic analysis organs were fixed in formalin prior to sectioning and staining.

In vivo antibody injections -WT and STAT-1KO mice infected with T. cruzi were treated with either

200μg rat anti IL-10 monoclonal antibody (BioXcell, West Lebanon, NH) or rat IgG isotype control

(Sigma Aldrich) at day 1, 3, 5, 8 and 12 post-infection via intra-peritoneal route.

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Serum and intracellular cytokine analysis- Serum was collected from animals at indicated times

post-infection and the levels of TNF-α, IFN-γ, IL-4, IL-6, IL-10, IL-12 and IL-13 were measured by

ELISA. All reagents for cytokine ELISA were purchased from BioLegend Inc. (San Diego, CA, USA).

For intracellular staining, cells were stimulated with 50ng/ml PMA, 1ug/ml of ionomycin and

stained for extracellular markers, fixed with 2% para-formaldehyde, permeabilized, and stained

with anti-IFN-¥ and IL-13 antibodies( Biolegend). Cells were acquired through Fluorescence

Activated Cell Sorter (FACS, BD biosciences, San Jose, CA, USA). Analysis was performed with

FlowJo software (Tree Star Inc, Ashland, OR, USA).

Statistical analysis-An unpaired Student’s t test was used to determine the statistical significance

of differences observed in the cytokine profiles. The significance of differences in survival

determined using the Kaplan-Meier method. Values of P < 0.05 were considered significant.

RESULTS

STAT-1KO mice are more susceptible to T. cruzi infection than wild-type animals. Three

independent infection experiments (4-6 mice in each group) were performed to compare the

differences in survival of wild-type (WT) and STAT-1KO mice infected with Brazil strain T. cruzi. In

each experiment the 100% of the STAT-1KO mice became sick (developed ruffled fur, slow

mobility, huddled and had decreased food and water intake) or died between 13-18 days post-

infection (Fig 1). Whereas the infected WT control mice remained healthy throughout the 30 day

observation period. Infected STAT-1KO and matched WT mice were sacrificed at 15 days post-

infection and tissue was analyzed histopathologically and by parasite outgrowth assays. Analysis

of cardiac tissue from STAT-1KO mice showed that it was heavily infected with parasites in nests of

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pseudocysts (Fig. 2). This was typical, being found in 75% of the analyzed hearts. The pseudocysts

were found in different areas of the myocardium and in some areas we found pseudocysts also

infiltrated with predominantly neutrophils (see Fig. 2, panel B). We did not find evidence of

infiltrating parasites or pseudocysts in WT mice at 15 days post-infection. The spleens of all

parasite-infected STAT-1KO mice were between 3-5 times the weights of those of WT mice.

Parasite growth explant analysis of blood, heart and splenic tissue showed that all tissues were

heavily parasitized as compared to infected WT animals (Table 1). Parasites grew from the blood

of the majority of animals from the STAT-1KO group but none from the WT animals. Similarly,

parasites grew from cardiac and spleen tissue of the majority of the STAT-1KO mice but not the WT

groups.

Systemic IL-10 and IFN-γ dysregulation occurs in T. cruzi-infected STAT-1KO mice. Since STAT-1

expression is important for the transduction of IFN-γ signals during inflammation which are

important for macrophage activation and intracellular killing, we measured systemic IFN-γ levels

in the serum of infected animals at days 5, 10 and 15 (Fig. 3) Interestingly, we found that the

levels of IFN-γ in infected STAT-1KO mice were ~100-1000-fold higher on days 10- and 15-post-

infection compared with WT mice. This suggests that during uncontrolled infection there is hyper-

IFN-γ response that is ineffective at inducing intracellular killing in mice with absent STAT-1

expression. The systemic levels of IL-4 and IL-12 were not statistically different between groups,

and we found no detectable TNF-α or IL-6 in either group. The levels of the anti-inflammatory

cytokine IL-10, were approximately 3- to 9-fold higher at 10-15 days post-infection, respectively,

compared to WT animals. IL-10 blockade has been shown to protect mice from T. cruzi infection,

presumably increasing IFN-γ mediated signaling16. We tested whether neutralization of IL-10 could

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alter the diminished survival of parasite infected STAT-1KO mice. Mice were treated with daily

administrations of IL-10 antibody, or isotype control antibody, through the course of parasite

infection. Despite IL-10 neutralization, the infected STAT-1KO mice had decreased overall survival

relative to infected WT mice. Thus neutralization of IL-10 using antibody therapy had no effect on

changes in survival.

CD4 and CD8 cells contribute to IFN-γ and IL-13 production in infected STAT-1 KO mice. The lack of

STAT-1 expression leads to IFN-γ hyper-production, thus we wanted to determine which cells are

responsible for this. Intracellular cytokine analysis flow cytometric analysis revealed that CD4 and

CD8 cells both contributed to the production of IFN-γ in STAT-1KO mice (Fig 4) whereas NK cells did

not appear to contribute to the increase of production of IFN-γ in the STAT-1KO over that of the

WT mice (Fig. 4). IL-13 expression appeared to be variably expressed in splenic at different times

post-infection in NK, CD4 and CD8 cells in both WT and STAT-1KO mice.

Antigen-specific IL-17 is robust early in infection but diminishes in STAT-1KO infected mice. IL-17

production is increased in deficient IFN-γ signaling secondary to STAT-1 ablation 13. In addition, IL-

17 expression is important in reducing mortality and inflammation during T. cruzi infection 14, 15.

Thus, we compared the IL-17 responses in splenic cells in T. cruzi-infected WT and STAT-1KO

animals and found that the STAT-1KO mice had increased levels of Ag-induced IL-17 expression

over those of WT mice, especially prevalent at days 5 and 10 post-infection. CD3-induction of IL-

17 was present in both WT and STAT-1KO animals indicating that they were both able to respond

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to TCR-engagement, but the day 5 and 10 post-infected STAT-1KO animals had especially robust

induction of IL-17 in response to CD3.

DISCUSSION

These results indicate that the expression of STAT-1 is important to the control of T. cruzi

replication and dissemination in the infected mice. The uncontrolled replication and

dissemination of parasites in the knock-out strain leads to death in the acute stages of infection

and dissemination includes deposition of parasites within the myocardium, a chief site of

pathology in T. cruzi infection. The heavy parasitemia in these animals is evidenced by both the

finding of viable parasites in the blood of most infected animals as well as the heavy deposition in

the spleens.

It has been shown that the induction of IL-10 by LPS or E. coli infection occurs independent of

STAT-1 expression, and that subsequent repression of IL-10 are dependent on STAT-19. Similarly

the production of IL-10 by dendritic cells exposed to LPS or CpG is suppressed by IFN-γ in a STAT-1

dependent manner17. The presence of IL-10 has been shown to promote T. cruzi infection in the

C57BL/6 mouse strain and that depletion of IL-10 using IL-10 antibodies is protective 16, 18. This it

thought to be related to the IL-10 mediated interference of IFN-γ macrophage activation. In the

absence of STAT-1, IFN-γ mediated activation cannot occur, and the elevation of IL-10 does not

function as an anti-inflammatory cytokine in the absence of STAT-1. The elevation of IFN-γ and IL-

10 may be a reflection of exaggerated Th1 and Th2 responses during infection as a result of the

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absence of STAT-1. We tested the role of IL-10 as reason for diminished survival in STAT-1KO mice

using anti-IL-10 administration throughout the infection. Neutralization of IL-10 in these animals

did not alter the diminished survival. Thus IL-10 expression, in the absence of STAT-1 expression,

does not appear to be responsible for our observations.

The role of IL-13 in T. cruzi infection in mice is not well described. This IL-4-like cytokine is

implicated in allergic responses and parasitic nematode expulsion and is produced by Th-2 cells,

basophils and eosinophils. IL-13 expression is important for the development of murine

cutaneous disease by several Leishmania species where it plays a role in parasite persistence

during Th2 responses19, 20. Our data indicates that it is produced systemically in the early phase of

T. cruzi infection in wild-type mice, but is ablated in the absence of STAT-1 expression. The effects

of IL-13 and IL-4 are mediated through the activation of both STAT-1 and STAT-6 in a variety of cell

types 21 yet we do not understand the relationship between the lack of STAT-1 and the lack of

systemic IL-13 provoked during parasite infection. Perhaps the absence of STAT-1 affects the

production of IL-13 by cells in response to parasite exposure. We found that CD4+, CD8+ and NK

cells were responsible for IL-13 production in the spleen of both WT and STAT-1KO presenting

higher IL-13 production in STAT-1KO mice at day 10. Despite this, the systemic levels of IL-13 were

higher in WT animals, suggesting that IL-13 is produced predominantly from a non-splenic source

in these animals, or, less likely, that in the absence of STAT-1 IL-13 secretion is inhibited despite

the presence of demonstrable intracellular protein.

IL-17 expression is important for the control of T. cruzi infection and is thought to be

predominantly generated by B cells in response to exposure by parasite trans-sialidase 14,

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although it is classically secreted by Th17 helper cells. Although not entirely elucidated, IL-

17 is important for host resistant to T. cruzi infection. However, IL-17 production can be

down-regulated by STAT-1 13. This can explain the higher levels of IL-17 found in STAT-1KO

infected mice, and also the neutrophilic inflammation in the heart and spleen.

Interestingly, during T. cruzi infection, IL-17RA is important for recruitment of IL-10

producing neutrophils which in turn suppresses inflammation and reduces mortality15.

Our results found that splenic Ag-driven IL-17 was up-regulated in infected STAT-1KO

animals as early as day 5 post-infection and remained substantially elevated through day

10, whereas WT animals had nearly undetectable IL-17 responses. We measured the

abundance of Ly6G+, Ly6c+ neutrophils in the spleens of uninfected and T. cruzi-infected

WT and STAT-1KO animals and found that at baseline, STAT-1KO animals had ~2-fold more

resident neutrophils than WT mice, and upon infection both increased 2-3 fold above this

baseline with the number of neutrophils in the STAT-1KO animals being 1.4-fold higher

than in WT animals. This supports the idea that IL-17 drives neutrophilic response;

however we found this neutrophilic response was only robust in the absence of STAT-1

expression yet ineffective at controlling parasite load and preventing premature death

relative to WT animals. Further experiments are necessary to elucidate the phenotype of

neutrophils recruited in STAT-1KO mice.

Overall this study demonstrates that STAT-1 is critical for the control of T. cruzi infection and also

suggests that STAT-1 plays a role in the maintenance of cytokine systemic levels, particularly IFN-

γ, IL-10 and IL-13, during infection. The absence of STAT-1 has pleotrophic effects causing the

production of and response to multiple cytokines. Further detailed studies are necessary to

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understand the cell-type and organ-specific origin of these effects with respect to T. cruzi

infection.

ACKNOWLEDGEMENTS: MK, SV, CT, JLK performed experiments and analyzed data; AS, designed

experiments, analyzed data and edited manuscript; BM, designed experiments, analyzed data,

wrote and edited manuscript. The work in this paper was supported by grants from the American

Heart Association (SDG #0735241N to BSM). Work in the Satoskar lab is supported by grants from

the National Institutes of Health.

Disclosures: The authors have no conflicts of interest to report.

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Table 1. Parasite burden in tissue of T. cruzi infected micea

Parasite burden in infected tissue

Group Blood Spleen Heart

WT 0 (6/6) 1+ (1/6) 0 (6/6)

STAT-1KO 1+ (4/6) 3+ (5/6) 2+ (4/6)

a Equal amounts indicated tissues for each animal was harvested and explanted into

parasite growth medium and incubated at 25◦ C and observed microscopically for 7 days.

The amount of parasites first emerging during culture was quantified as: 1+, average of 1-

3 parasites per high power field (hpf) per mg of tissue or 100 mL of blood; 2+, 4-6 per hfp;

3+, 7 or greater parasites per hpf. The number of animals for which parasites we

observed in tissue is indicated on the parentheses. This is a representative experiment of

of three experiments with similar results.

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FIGURE LEGENDS

Fig .1. Kaplan-Meier survival plot of T. cruzi-infected WT and STAT-1KO mice. Mice were infected

intraperitoneally with 10,000 Brazil strain tissue-culture derived trypomastigotes of T. cruzi.

Survival of animals was determined over the course of 30 days post-infection. STAT-1KO animals

succumbed to infection between 13-18 days post infection whereas WT animals survived. Blood

and tissue from mice was analyzed histopathologically, by parasite outgrowth explantation and

cytokine analysis (see Figs 2, 3 and Table 1). This shows a representative experiment of 3

experiments with similar results.

Fig. 2. Histopathologic analysis of heart tissue from infected mice. STAT-1 (panels a-c) and WT

(panel d) mice infected with Brazil strain T. cruzi tissue culture trypomastigotes were sacrificed at

15 days post-infection. Cardiac tissue from STAT-1 mice showed numerous areas of parasite

pseudocyst formation (indicated by arrows) with areas of associated inflammation, particularly of

neutrophils (see panel b), whereas tissue from WT mice did not show evidence of disease. 100X

(panel a) and 40x (panels b-c) magnification are shown.

Fig. 3. Measurement of serum cytokines from T. cruzi-infected WT and STAT-1KO mice. The

serum obtained from the indicated infected mice from post-infection day 5, 10 and 15 were

analyzed by ELISA using cytokine specific antisera. The mean values ±SE (n= 3-4 animals per

group) are shown. * P values are ≤ 0.05 compared to the corresponding values in the opposing

group.

Fig. 4. Analysis of splenic cells subsets for production of IFN-γ and IL-13. Spleen cells harvested

from parasite-infected WT and STAT-1KO animals were analyzed by FACS for co-expression of

CD4-, CD8- and NK-associated surface markers and intracellular staining of the indicated cytokines

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(n= 4-5 animals per group were

y-axis. * P values are ≤ 0.05 com

Fig. 5. Analysis of IL-17 and neu

from spleens of parasite infected

indicated times post-infection; b

and Ly6c/Ly6c (lower) from WT

0.05 compared to the correspon

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analyzed). The % of the total population of cells are

mpared to the corresponding values in the opposing

trophils in spleens of infected animals. a) The IL-17

d animals in response to T. cruzi antigen (Ag) and CD

b) FACS analysis of spleen cells for expression of CD1

and STATKO animals at 15 days post-infection. *** P

nding values in the opposing group.

e shown on the

group.

7 production

D3 from

11b (upper)

values are ≤

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