Early Response of Mucosal Epithelial Cells during Infection Chia-Hsin Ju, Annapoorani Chockalingam and Cynthia A. Leifer This information is current as of September 27, 2021. J Immunol 2009; 183:7420-7427; Prepublished online 16 November 2009; doi: 10.4049/jimmunol.0900640 http://www.jimmunol.org/content/183/11/7420 Downloaded from

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2009 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Early Response of Mucosal Epithelial Cells during Toxoplasma gondii Infection

Chia-Hsin Ju, Annapoorani Chockalingam, and Cynthia A. Leifer1

The innate immune response of mucosal epithelial cells during pathogen invasion plays a central role in immune regulation in the gut. Toxoplasma gondii is a protozoan intracellular parasite that is usually transmitted through oral infection. Although much of the information on immunity to T. gondii has come from i.p. infection models, more recent studies have revealed the importance of studying immunity following infection through the natural peroral route. Oral infection studies have identified many of the key players in the intestinal response; however, they have relied on responses detected days to weeks following infection. Much less is known about how the gut epithelial layer senses and reacts during initial contact with the pathogen. Given the importance of epithelial cells during pathogen invasion, this study uses an in vitro approach to isolate the key players and examine the early

response of intestinal epithelial cells during infection by T. gondii. We show that human intestinal epithelial cells infected with T. Downloaded from gondii elicit rapid MAPK phosphorylation, NF-␬B nuclear translocation, and secretion of IL-8. Both ERK1/2 activation and IL-8 secretion responses were shown to be MyD88 dependent and TLR2 was identified to be involved in the recognition of the parasite regardless of the parasite genotype. Furthermore, we were able to identify additional T. gondii-regulated genes in the infected cells using a pathway-focused array. Together, our findings suggest that intestinal epithelial cells were able to recognize T. gondii during infection, and the outcome is important for modulating intestinal immune responses. The Journal of Immunology, 2009, 183:

7420–7427. http://www.jimmunol.org/

single layer of intestinal epithelial cells that line the cell-induced protection (13, 14), whereas IFN-␥ plays a major role mucosal surface must prevent the entry of exogenous in protection through CD8ϩ T cells (14, 15). A Ags, allow absorption of essential nutrients, and yet ini- TLRs are innate immune receptors that directly recognize mi- tiate effective and appropriate immune responses when pathogens crobial structures and initiate an inflammatory response. All TLRs, are present (1). defenses at mucosal surfaces include the se- except TLR3, use the adapter molecule MyD88 to initiate the sig- cretion of IgA, defensins, and and . IgA and naling cascade. MyD88-deficient mice are highly susceptible to T. defensins prevent bacterial adherence and contribute to pathogen gondii infection due to a failure to produce IL-12 (16). Multiple elimination, whereas cytokines and chemokines participate in gut TLRs have been linked to protective immunity against T. gondii by guest on September 27, 2021 homeostasis as well as the recruitment of immune cells during infection. In mice, TLR11 expressed by dendritic cells (DCs)2 is infection. Epithelial cells express several innate immune receptors, required for the secretion of IL-12 in response to stimulation with including nucleotide oligomerization domain proteins and TLRs the T. gondii protein profilin (17, 18). TLR2-deficient mice show that participate in initiating the immune response (2). However, increased susceptibility with high-dose i.p. infection (19). Oral in- activation is tightly controlled to prevent pathology due to mucosal fection of mice results in intestinal inflammation, ileitis, in wild- inflammation (2–6). Upon encounter with pathogenic , ep- type mice but not in mice deficient in TLR9 or TLR4 (20, 21). This ithelial cells elicit a potent response that shapes the ensuing im- suggests that TLR9 and TLR4 may play much more important mune response (7, 8). roles in initiating immunity to T. gondii at the mucosal surface. Toxoplasma gondii is an orally acquired apicomplexan proto- The role of TLRs in human cell recognition of T. gondii infection zoan parasite (9). Human infections are usually asymptomatic, but is much less well studied. Human TLR2 can respond to GPIs from reactivation of chronic infection in immunosuppressed individuals T. gondii, but the role of TLRs during live infection of human results in toxoplasmic encephalitis (10, 11). Serological surveys cells, especially in the gut, has not been studied. have estimated that one-third of the world’s population has been T. gondii can infect the gut mucosa by direct invasion of epi- exposed to this parasite (12). However, there is no vaccine and thelial cells in the small intestine (22). Therefore, epithelial cells therapeutic treatment regimens have significant side effects. T. may respond directly to T. gondii infection and initiate early local gondii infections are controlled primarily by T . IL-12 mucosal immune responses. This is supported by in vitro RNase and TNF-␣ are critical cytokines for stimulating Th1 CD4ϩ T protection studies using an immortalized mouse small intestinal enterocyte cell line, which demonstrated that the chemokines MCP-1, MIP-1, and eotaxin were induced upon infection (23). In the present study, we have investigated whether human intestinal Department of Microbiology and Immunology, College of Veterinary Medicine, Cor- nell University, Ithaca, NY 14853 epithelial cells respond directly to infection with T. gondii and by Received for publication February 26, 2009. Accepted for publication October what mechanism this recognition occurs. 2, 2009. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 2 Abbreviations used in this paper: DC, dendritic cell; shRNA, short hairpin RNA; 1 Address correspondence and reprint requests to Dr. Cynthia A. Leifer, Department STAg, soluble Toxoplasma Ag; WM, wortmannin; YFP, yellow fluorescent protein. of Microbiology and Immunology, College of Veterinary Medicine, Cornell Univer- sity, Ithaca, NY 14853-6401. E-mail address: [email protected] Copyright © 2009 by The American Association of Immunologists, Inc. 0022-1767/09/$2.00 www.jimmunol.org/cgi/doi/10.4049/jimmunol.0900640 The Journal of Immunology 7421

Materials and Methods GCGCAGTGTGG-3Ј; CCL20 forward, 5Ј-AGTTTGCTCCTGGCTG Ј Ј Cell culture and parasites CTTTGATG-3 ; CCL20 reverse, 5 -CTGCCGTGTGAAGCCCACA ATAAA-3Ј; CCL15 forward, 5Ј-TTGGATCCCAGGCCCAGTTCATAA- Henle 407 (human embryonic intestinal epithelial cells), HEK293 (human 3Ј; CCL15 reverse, 5Ј-AGCAGTCAGCAGCAAAGTGAAAGC-3Ј; embryonic kidney cells), and HS27 (human foreskin fibroblasts) cells were CCL24 forward, 5Ј-ATGCCTCAAGGCAGGAGTGATCTT-3Ј; CCL24 maintained in DMEM supplemented with 2 mM L-glutamine, 50 U/ml reverse, 5Ј-TCTTCATGTACCTCTGGACCCACT-3Ј; MyD88 forward, penicillin, 50 ␮g/ml streptomycin, 10 mM HEPES, 1 mM sodium pyru- 5Ј-AGATGATCCGGCAACTGGAACAGA-3Ј; and MyD88 reverse, vate, and 10% low endotoxin FBS. T. gondii tachyzoite strains were main- AGTCACATTCCTTGCTCTGCAGGT. TLR and GAPDH primers were tained by serial 2-day passages on HS27 monolayers in DMEM. All cell used as previously described (25, 26). cultures and parasites were routinely checked for mycoplasma by a high sensitivity PCR-ELISA based Mycoplasma detection kit (Roche). Luciferase reporter assays In vitro infections HEK293 cells were plated at 1 ϫ 104 cells per well in 96-well plates. Cells were transfected using TransIT transfection reagent (Mirus Bio) and a total T. gondii tachyzoites were added to Henle 407 cells and briefly centrifuged of 200 ng of DNA per well consisting of human TLR2 plasmids and (400 ϫ g for 1 min) to initiate parasite and cell contact. At time points NF-␬B or IL-8 luciferase reporters. Cells were stimulated with TLR2 li- indicated in each figure legend, supernatants or cell lysates were collected gand or infected with T. gondii tachyzoites, lysed in reporter lysis buffer for further analysis. In some experiments, 50 ng/ml wortmannin (WM) was (Promega), and assayed for luciferase (Promega) activity. NF-␬B activity added 2 h prior to infection. was calculated and processed by Microsoft Excel.

In vivo passage of T. gondii tachyzoites Gene array analysis ϫ 4 To pass the parasite through mice, 5 10 RH tachyzoites were i.p. in- The commercial pathway-focused oligonucleotide microarrays (OHS-011; jected into C57BL/6 mice. After 3 days of infection, the parasites were Oligo GEArray human inflammatory cytokines and receptors microarray) Downloaded from collected from the peritoneal fluid and washed once with HBSS. The par- were purchased from SuperArray Bioscience. The array analyses were per- asites were then expanded by one additional passage in HS27 fibroblasts formed using a chemiluminescence-based detection system according to prior to stimulation. the manufacturer’s instructions. Images of the array were developed on Antibodies x-ray films. Image data sets were scanned and analyzed using ScanAlyze (Eisen Lab), and Microsoft Excel software. Background adjustment was Abs specific to total and phosphorylated forms of ERK1/2, p38, and PKB performed by subtracting the lowest measured value on the membrane

(Akt) were from Technology. The anti-MyD88 polyclonal from the values of all genes. The signals from the expression of each gene http://www.jimmunol.org/ Ab was from Alexis Biotechnology. The NF-␬B p65 Ab was from Santa on the array were normalized against the signal from the internal house- Cruz Biotechnology, and Alexa Fluor 594 was from Molecular Probes keeping gene GAPDH to obtain the processed data sets. Fold changes were (Invitrogen). calculated as the normalized ratio of average experimental processed data sets divided by the average medium control processed data sets. Thresholds NF-␬B translocation assay were set to select for genes up-regulated 2-fold or more. The original array data were deposited in National Center for Biotechnology Information For immunofluorescence analyses, Henle 407 cells were plated at a density Gene Expression Omnibus database under accession no. GSE18085. of 2–3 ϫ 105 cells per well on sterile coverslips placed in a 24-well plate. Cell monolayers were infected with T. gondii RH-yellow fluorescent pro- ELISA tein (YFP) tachyzoites and then fixed with 3% paraformaldehyde in PBS for 20 min at room temperature. Fixed cells were permeabilized with 0.1% Production of IL-8 was measured with the human CXCL8/IL-8 DuoSet by guest on September 27, 2021 Triton X-100 in TBS (TBS-TX) for 15 min and blocked in 1% BSA in ELISA development kits (R&D Systems) according to the manufacturer’s TBS-TX for 20 min. The cells were then stained with primary rabbit anti- instructions. NF-␬B p65 (1/1,000) followed by conjugated secondary Ab goat anti- rabbit IgG Alexa Fluor 594. Nuclei were stained using DAPI (1/10,000). Statistical analysis Confocal images were taken with a Leica laser scanning confocal micro- scope using a 63ϫ lens. Contrast and brightness of individual channels Minitab 15 statistical software (Minitab) was used for Student’s t tests. A Ͻ were adjusted linearly in Photoshop (Adobe). For Western blot analyses, value of p 0.05 was considered significant. Henle 407 cells were plated at a density of 1.5 ϫ 106 cells per well in 6-well plate and infected with T. gondii RH for the indicated times. Cy- Results toplasmic and nuclear proteins were isolated according to previous proto- Intestinal epithelial cells respond to T. gondii infection in vitro cols (24) and blotted for p65 (1/500). Oral infection with T. gondii results in ileitis in C57BL/6 mice RNA interference mediated by a robust Th1 type of response (23). CD4ϩ T cells SureSilencing human MyD88 short hairpin RNA (shRNA), TLR2 and synergize with intestinal epithelial cells to drive the secretion of TLR9 shRNA, and control plasmids were purchased from SuperArray Bio- Th1 type cytokines and various chemokines. The response of in- science. Henle 407 cells were transfected using TransIT transfection re- testinal epithelial cells directly to the parasite during the early agent (Mirus Bio) according to manufacturer’s protocols. Transfected cells were selected with neomycin (1.0 mg/ml) for 14 days, and antibiotic-re- stage of infection in mice, and especially in humans, remains un- sistant individual colonies were isolated for further analysis and maintained clear. To determine whether human intestinal epithelial cells re- in the presence of neomycin. For transient transfections, Henle 407 cells spond directly to T. gondii, we incubated T. gondii tachyzoites (RH were transfected with shRNA plasmids by electroporation. Cells were used strain) with human intestinal epithelial cell lines. Consistent with 48–96 h post-transfection. other studies on the large intestine, colon cell lines did not activate RNA extraction and PCR analysis MAPKs in response to T. gondii infection even though they were permissive for infection and parasite replication (data not shown). Total RNA of Henle 407 cells infected with T. gondii was extracted using the RNeasy mini kit (Qiagen). Reverse transcription of the RNA (1 ␮g) T. gondii also infected the human small intestinal epithelial cell was performed using ImProm-II reverse transcription system (Promega). line Henle 407 (supplemental Fig. 1).3 The kinetics of infected PCR was performed in 25 ␮l of a reaction mixture containing 1 ␮lofthe Henle 407 cells showed increased parasite invasion during the first reverse-transcribed RNA. The final PCR products were electrophoresed on few hours, followed by parasite replication after8hasindicated by 2% agarose gels and visualized using UV light illumination after ethidium bromide staining. Real-time PCR was performed in the Applied Biosys- the appearance of rosettes (supplemental Fig. 1, arrows). Unlike tems 7500 real-time PCR system according to the manufacturer’s instruc- colon cell lines, T. gondii infection of Henle 407 cells induced tions (Applied Biosystems). The reaction was performed using the Power phosphorylation of both ERK1/2 and p38 MAPKs by 15 min SYBR Green PCR master mix. GAPDH was used as an internal control for each sample. The primers used were as follows: IL-8 forward, 5Ј-AGCCT TCCTGATTTCTGCAGCTCT-3Ј; IL-8 reverse, 5Ј-AATTTCTGTGTTG 3 The online version of this article contains supplemental material. 7422 EPITHELIAL CELL RESPONSE TO T. gondii IS MEDIATED BY TLR2

transgenic parasites expressing YFP allowed the visualization of in- fected cells. Staining for p65 NF-␬B demonstrated that infected, but not uninfected, cells translocated NF-␬B to the nucleus (Fig. 1D). Early immune defense against T. gondii involves the recruitment of several innate immune cell populations, including , , DCs, and eosinophils (27–30). Neutrophils, re- cruited by IL-8 (MIP-2 in mice), are often the first cell type re- cruited to the area of infection and provide the initial source of IL-12 that triggers Th1 T cell-mediated immunity (27, 31). There- fore, we next tested whether epithelial cells could participate in the recruitment of neutrophils by secreting IL-8 in response to T. gon- dii infection. Henle 407 cells produced significant levels of IL-8 at 12 h after infection with T. gondii (Fig. 1B). IL-8 levels continued to rise up to 24 h postinfection. Taken together, these data demon- FIGURE 1. Intestinal epithelial cells respond to T. gondii infection. A, strate that human small intestinal epithelial cells respond directly to Henle 407 cells were infected with RH tachyzoites (parasite to cell ratio, infection with T. gondii tachyzoites by activating MAPK and NF-␬B 6:1) for the indicated times. Whole cell lysates were collected for immu- signaling cascades as well as by producing chemokines that actively noblotting with Abs against total and phosphorylated (p-) forms of ERK1/2 participate in the innate immune response to T. gondii.

and p38 MAPK. B, IL-8 levels in infected culture supernatants were col- Downloaded from lected at the indicated time points (p.i., postinfection) and assayed by ELISA (triplicate assays; error bar, SD). Experiments were performed T. gondii-infected intestinal epithelial cells express several p Ͻ 0.05, compared with IL-8 levels of inflammatory genes ,ء ;three times with similar results uninfected cells. C and D, Henle 407 cells were infected with RH (C)or Oral infection studies have demonstrated an increase in several RH-YFP (D) parasites for the indicated times. NF-␬B p65 localization was cytokines and chemokines in response to T. gondii infection; how- visualized by immunoblotting of cytosolic (Cy) and nuclear (Nu) extracts ever, the mixed population of the intestinal mucosa did not allow (C) or by immunofluorescent staining (D) for anti-NF-␬B p65 (red). http://www.jimmunol.org/ for the determination of the relative role that specific cell popula- tions play in the production of these mediators. To address the role (Fig. 1A). ERK1/2 and p38 phosphorylation decreased at 30 min of intestinal epithelial cells to modulate the cytokine environment and was reduced nearly to baseline by 45 min. early following infection, we used specific pathway arrays to iden- NF-␬B regulates proinflammatory and antiapoptotic genes in tify cytokines and chemokines induced 4 h after exposure to T. response to pathogens. To determine whether T. gondii infection of gondii. At this time point most cells have become infected, but the Henle 407 cells results in activation of NF-␬B, we infected the parasites have not replicated. Consistent with previous studies on cells for various times with RH tachyzoites and immunoblotted late responses, infected epithelial cells expressed higher levels of cytosolic and nuclear extracts for p65 NF-␬B. Translocation of proinflammatory chemokines, including MIP-1␣/CCL3, MIP-2/ by guest on September 27, 2021 NF-␬B to the nucleus was observed as early as 15 min, peaked at 60 CXCL2, RANTES/CCL5, MCP-2/CCL8, MCP-3/CCL7, and IFN- min, and was still detected 2 h postinfection (Fig. 1C). Infection with ␥-inducible protein-10 (Table I). The induced cytokines and

Table I. Pathway-specific microarrays revealed up-regulation of several T. gondii-induced inflammatory cytokines, chemokines, and receptorsa

Cytokines and Cytokine Receptors Chemokines and Receptors

Gene GenBank no. Fold change Gene GenBank no. Fold change

Il3 NM_000588 10.5 Ccl3 NM_002983 3.9 Il8 NM_000584 2.3 Ccl5 NM_002985 4.7 Il9 NM_000590 2.5 Ccl7 NM_006273 2.7 Il11 NM_000641 2.2 Ccl8 NM_005623 3.2 Il12a NM_000882 2.3 Ccl11 NM_002986 5.9 Il18 NM_001562 8.5 Ccl13 NM_005408 3.8 Il1rn NM_000577 2.9 Ccl15 NM_032965 6.2 Il1r2 NM_004633 2.1 Ccl20 NM_004591 5.8 Il8rb NM_001557 2.9 Ccl23 NM_005064 4.5 Il13ra2 NM_000640 2.1 Ccl24 NM_002991 11.1 Il15ra NM_002189 3.9 Cxcl2 NM_002089 2.1 Il18r1 NM_003855 7.9 Cxcl3 NM_002090 3.6 Mif NM_002415 3.6 Cxcl10 NM_001565 5.6 Tnf NM_000594 2.9 Cxcl13 NM_006419 2.4 Cd40lgb NM_000074 2.9 Ccr2 NM_000648 2.5 Cebpbb NM_005194 3.4 Ccr3 NM_001837 2.6 Crpb NM_000567 2.4 Ccr6 NM_004367 2.4 Edg3b NM_005226 6.7 Ccr7 NM_001838 3.1 Rps27ab NM_002954 2.8 Ccr9 NM_006641 6.3 Spp1b NM_000582 4.4 Blr1 NM_001716 2.8 Pf4 NM_002619 2.5

a Henle 407 cells were infected with RH parasites and total RNA was collected at 4 h postinfection. RNA from infected and uninfected cells was compared using a pathway-focused oligonucleotide microarray containing 113 genes involved in the inflammatory response. Genes that were up-regulated 2-fold or more after infection were shown. b Non-cytokine gene. The Journal of Immunology 7423

FIGURE 2. Cytokine and chemokine induction by T. gondii infection. Human IL-8, CCL15, CCL20, and CCL24 gene transcript levels were mea- sured by real-time PCR analysis 4 h post-T. gondii infection (p.i.). The data Downloaded from are normalized to GAPDH and compared against uninfected expression levels (mRNA REL %, relative expression levels).

FIGURE 3. T. gondii-induced MAPK activation and IL-8 secretion in chemokines included ones that induce (IL-8 intestinal epithelial cells is PI3K signaling independent. A, Henle 407 cells were preincubated for 2 h with (ϩ) or without (Ϫ) the PI3K inhibitor WM and MIP-2/CXCL2), homing of mucosal DCs (CCL20), and mi- http://www.jimmunol.org/ gration of DCs to sites of infection (MIP-1␣/CCL3 and RANTES/ (50 ng/ml) followed by infection with T. gondii (parasite to cell ratio, 6:1). CCL5) (27, 32). Of note, IL-18, which enhances IL-12 mediated At the indicated time points, cell lysates were collected for immunoblotting immune responses to T. gondii, was highly induced in infected with Akt and ERK1/2 (p-, phosphorylated form). The blot was reprobed for total ERK1/2. B, Henle 407 cells were pretreated with or without WM and epithelial cells (33–35). By PCR analysis, there was little or no infected with T. gondii as described above. IL-8 levels in infected culture IL-8 mRNA but a low level of IL-18 mRNA in uninfected cells. supernatants were collected at the indicated time points and assayed by p Ͻ 0.05, compared with IL-8 ,ء ;Four hours after infection both IL-8 and IL-18 mRNA were up- ELISA (triplicate assays; error bar, SD regulated, supporting our array data (supplemental Fig. 2). Real- levels of uninfected cells). C, Henle 407 cells were pretreated with or time PCR analysis confirmed the up-regulation of several neutro- without WM and infected with T. gondii as described above. Analysis of phil and chemoattractants, including IL-8, CCL15, IL-8 and GAPDH transcripts was performed by real-time PCR. Each sam- by guest on September 27, 2021 CCL20, and CCL24 upon T. gondii infection (Fig. 2). Together, ple was normalized to internal GAPDH expression levels (IL-8 mRNA these data demonstrate that human intestinal epithelial cells induce REL %, relative expression levels). chemotactic and inflammatory mediators capable of modulating the local immune response early (minutes to hours) after infection. nonhematopoietic cells express TLR9, and experiments using bone Epithelial cell response to T. gondii is PI3K independent marrow chimeras suggest that both compartments are critical for T. gondii infection of mouse macrophages induces MAPK and the host response. However, it is unclear whether nonhematopoi- protein kinase B (also known as Akt) activation through a Gi- etic cells, such as epithelial cells, respond directly to T. gondii dependent PI3K signaling pathway (36). Similarly as in macro- through TLR9 or whether the response is secondary to commensal phages, phosphorylation of Akt occurred in Henle 407 cells 60 min bacterial leak into the lamina propria following damage to the after T. gondii infection (Fig. 3A). The PI3K inhibitor WM com- epithelium. To determine whether TLR9 or other TLRs play a role pletely blocked T. gondii-induced phosphorylation of Akt but only in the early response of epithelial cells to T. gondii, we depleted slightly reduced the phosphorylation of ERK1/2 and p38 at 15 min MyD88 from Henle 407 cells by stable expression of a shRNA and had no detectable effect on ERK1/2 at 60 min (Fig. 3A). Con- against MyD88. All TLRs, except TLR3, are dependent on MyD88 stitutive production of IL-8 was dramatically inhibited by treat- for signal transduction. Control shRNA-transfected Henle 407 ment with WM (Fig. 3B; compared with no infection controls). cells expressed similar levels of MyD88 protein as nontransfected However, T. gondii infection of WM-treated cells still resulted in cells. However, cells transfected with the MyD88 shRNA ex- an induction of IL-8 secretion with similar fold induction as that of pressed significantly less MyD88 protein, confirming the effect of untreated cells (Fig. 3B). Real-time PCR analysis demonstrated the RNA interference-mediated knockdown (Fig. 4A). Upon T. that IL-8 mRNA up-regulation was not affected by WM in infected gondii infection, cells deficient in MyD88 had a significantly re- or uninfected cells (Fig. 3C). This suggests that although the duced level of ERK1/2 phosphorylation and a slightly reduced steady-state production of IL-8, but not mRNA production, is de- level of p38 phosphorylation, compared with control cells (Fig. 4B pendent on PI3K, T. gondii-induced up-regulation is independent and data not shown). Furthermore, MyD88-deficient cells failed to of PI3K. induce IL-8 upon exposure to T. gondii (Fig. 4C). MyD88 is also an adapter protein for IL-1 and IL-18. Because IL-18 is induced Epithelial cell response to T. gondii infection is MyD88 upon T. gondii infection and contributes to small intestinal pathol- dependent ogy in C57BL/6 mice (33, 34), it remains possible that the MyD88 TLR9 has been implicated in the host response to oral infection dependence is via IL-18 signaling. However, in Henle 407 cells the with T. gondii because TLR9-deficient mice fail to develop ileitis bioactive forms of both IL-18 and caspase-1 were not detected that is observed in wild-type mice (21). Both hematopoietic and until 6 h post-T. gondii infection (C. Ju, unpublished observation). 7424 EPITHELIAL CELL RESPONSE TO T. gondii IS MEDIATED BY TLR2

FIGURE 5. TLR genes are expressed in Henle 407 cells. A, Total RNA was collected from Henle 407 cells, reverse transcribed to cDNA, and then FIGURE 4. T. gondii-induced MAPK activation and IL-8 secretion is amplified for human TLR or GAPDH by PCR. Genomic DNA (gDNA) MyD88 dependent. A, Henle 407 cells were either untransfected (lane 1), was used as a positive control. B, Henle 407 cells were treated with 10 transfected with control shRNA plasmids (lane 2), or transfected with ng/ml PMA as a positive control or TLR1/2, TLR4, TLR2/6, and TLR9 MyD88 shRNA plasmids carrying the MyD88 RNA interference sequence ligands (1 ␮g/ml Pam3Cys, 100 ng/ml LPS, 1 ␮g/ml Malp-2, and 5 ␮g/ml (lane 3). Cell lysates from the stably transfected cell lines were collected CpG, respectively). Total cell lysates were collected and immunoblotted Downloaded from for immunoblotting with Abs against MyD88 or ␤-tubulin. B, Activation of for total and phosphorylated (p-) forms of ERK1/2 and p38. ERK1/2 in T. gondii-infected MyD88 knockout Henle 407 cells. Control Henle 407 cells (lanes 1), control shRNA cells (lanes 2), and MyD88 shRNA cells (lanes 3) were infected with RH tachyzoites (parasite to cell ratio, 6:1) at the indicated time points. Cell lysates were collected for induction. Therefore, the response is almost entirely MyD88 de- immunoblotting with Abs against total and phosphorylated (p-) forms of pendent but only partially dependent on TLR2. Attempts to com-

ERK1/2. C, Control Henle 407 (lane 1), control shRNA (lane 2), and bine TLR2 with other TLRs, including TLR4 and TLR9, in our http://www.jimmunol.org/ MyD88 shRNA (lane 3) cells were infected with T. gondii (parasite to cell HEK293-based stimulation assay did not result in further increases 6:1). Supernatants from each sample were collected for IL-8 ELISA anal- in NF-␬B activation (data not shown). TLR2 was required for IL-8 p Ͻ 0.05, compared with IL-8 production because its expression in HEK293 cells was sufficient ,ء ;ysis (triplicate assays; error bar, SD levels of uninfected cells). to permit induction of an IL-8 regulated luciferase reporter (Fig. 6E). Pretreatment with the ERK1/2 inhibitor U0126 inhibited IL-8 luciferase reporter activity. These data demonstrate that TLR2- This suggested that although the IL-18 mRNA level is regulated induced IL-8 production was through the ERK1/2 pathway (Fig. after infection, the post-translational cleavage to generate the bio- 6E). In our system, TLR9 was neither necessary nor sufficient for active form of IL-18 did not occur until later. Therefore, MyD88 epithelial cell response to T. gondii infection (supplemental Fig. 3 by guest on September 27, 2021 plays a critical role in the response of Henle 407 small intestine and Fig. 6A). Taken together, T. gondii induced IL-8 is dependent epithelial cells to T. gondii infection, most likely through a TLR. on ERK1/2, MyD88, and TLR2.

T. gondii activates TLR2 on intestinal epithelial cells Genotype of T. gondii does not influence epithelial cell response Similarly as in other studies on primary human small intestine T. gondii strains have been classified into three clonal lineages that cells, Henle 407 cells expressed most of the TLRs except TLR8 differ in their pathogenicity in mouse models (38). Several strains (37) (Fig. 5A). Stimulation with TLR ligands or PMA as a control of each clonal lineage were tested for their ability to induce epi- induced phosphorylation of ERK1/2 and p38 within 15 min thelial cell response and activate TLR2. Henle 407 cells phosphor- (TLR1/2, Pam3Cys; TLR2/6, Malp-2; TLR3, poly(I:C); TLR4, ylated ERK1/2 in response to all strains from each of the three LPS; TLR5, flagellin; TLR7, loxoribine; and TLR9, CpG DNA) lineages (Fig. 7A and data not shown; type I: RH and GT; type II: (Fig. 5B and data not shown). To specifically identify which hu- PT-G, CC, and DEG; and type III: VEG). The various strains man TLR was involved in T. gondii recognition, we reconstituted differed dramatically in their capacity to induce phosphorylation of HEK293 cells with each human TLR independently and measured p38, but there was no correlation with genotype (Fig. 7A). All the activation of NF-␬B following stimulation with positive con- strains induced NF-␬B activation through TLR2 (Fig. 7B and data trol ligands, soluble Toxoplasma Ag (STAg) or live T. gondii in- not shown). The type I RH strain had a lower capacity to induce fection using a luciferase reporter. Although each positive control NF-␬B when compared with another type I strain, GT-1 (data not ligand stimulated NF-␬B activation in the respective TLR express- shown). RH tachyzoites grown in cell cultures have reduced vir- ing cells, only TLR2 expression was permissive for NF-␬B re- ulence when compared with those passaged in mice (39). Simi- sponse to live T. gondii infection (Fig. 6A). STAg failed to activate larly, we observed that RH tachyzoites passaged in mice induced NF-␬B, suggesting that live infection was required. significantly more TLR2-dependent NF-␬B activation than those To confirm the role of TLR2 in Henle 407 cell response to T. passaged through fibroblasts in vitro (data not shown). The ability gondii infection, we knocked down TLR2 expression using of live T. gondii to activate cellular responses through TLR2 was shRNA. Transient transfection with TLR2 shRNA depleted the not unique to human cells, because cells transfected with mouse mRNA relative expression levels to 6% of wild-type levels (Fig. TLR2 responded similarly (data not shown). Together, we con- 6B). Knockdown of TLR2 reduced the activation of ERK1/2, p38, clude that TLR2-dependent activation of signaling cascades by T. and IL-8 induction in response to T. gondii infection (Fig. 6, C and gondii is not genotype dependent. D). Transient transfection of MyD88 shRNA gave similar results compared with those of stably transfected cells (Fig. 6, C and D, Discussion compared with Fig. 4). TLR2 knockdown in Henle 407 cells does In the present study, we evaluated the initial cellular responses of not completely block the response of MAPK activation and IL-8 human intestinal epithelial cells to T. gondii infection. This type of The Journal of Immunology 7425

FIGURE 6. Human TLR2 (hTLR2) is involved in the recognition of T. gondii. A, HEK293 cells were trans- fected with different human TLRs and a NF-␬B luciferase reporter plasmid. The transfected HEK293 cells were stimulated with TLR ligands or STAg or infected with live RH tachyzoites. Cell lysates were collected and as- sayed for luciferase activity (triplicate assays; error bar, SD). B–D, Henle 407 cells were transiently transfected with shRNA plasmids carrying TLR2 (lanes marked 2), MyD88 RNA inter- fering sequences (lanes marked M), or no DNA control (lanes marked C). B, TLR2 and MyD88 mRNA levels as measured by real-time PCR. C, To- tal cell lysates from cells infected Downloaded from with RH tachyzoites (parasite to cell ratio, 6:1) for the indicated time points were immunoblotted with Abs against total and phosphorylated (p-) forms of ERK1/2 and p38. D, IL-8 levels from cells in B as measured by http://www.jimmunol.org/ real-time PCR following4hofinfec- tion with T. gondii RH tachyzoites. E, HEK293 cells were transfected with TLR2 and an IL-8 promoter-lucif- erase reporter construct. Luciferase activity from cells left in medium alone (control) or infected with tachyzoites (RH strain) is shown. Cells were pretreated with ERK1/2 inhibitor U0126 or DMSO as a sol- by guest on September 27, 2021 vent control before infection (tripli- cate assays; error bar, SD).

study is critical for identifying the very early innate immune re- host defense and are one of the first cells recruited to the site of sponses to parasitic infection of the intestinal mucosa. Using an in infection. They play a key role in the recruitment and activation of vitro model in which an isolated cell type is directly exposed to the macrophages and DCs (27, 31, 42). Therefore, our findings that the infectious agent offers the advantage over mixed cell populations human small intestine epithelial cell line Henle 407 responds di- in identifying the response of a specific cell type. Most studies on rectly to T. gondii infection suggests that an in vivo epithelial the immune response to T. gondii infection have used a peritoneal response would modulate the local inflammatory environment to challenge model for this orally acquired pathogen. More recently, initiate host defense against infection. the importance of studying the natural route of infection has re- This series of studies also elucidates the molecular mechanism vealed the importance of epithelial cell response in influencing the for epithelial response to T. gondii infection. We show that, sim- outcome of the local and systemic immune response (40, 41). By ilarly as macrophages and DCs, epithelial cells activate the MAPK examining the response of the cells most likely to first encounter pathway. The inability of MyD88-deficient epithelial cells to se- the pathogen, we can begin to uncover the early responses that may crete IL-8 and activate ERK1/2 during infection suggests that limit or induce the spread of T. gondii to other tissues such as TLRs play a critical role in initiation of the mucosal inflammatory muscle and brain, where a persistent infection results. Therefore, it process. Human TLR2 responds to live T. gondii infection in our is highly relevant to study the response of these cells during T. heterologous reconstitution assay. However, knocking down TLR2 gondii infection, which occurs both locally in the intestine and with shRNAs in intestinal epithelial cells only partially reduced the systemically. activation of ERK1/2. Thus, although TLR2 contributes to the ep- Very little is known about the human intestinal response to T. ithelial response to T. gondii infection, there is likely an interaction gondii infection; therefore, a major finding of this study is that with additional TLRs or other receptors that we could not detect in human small intestinal epithelial cells respond directly to T. gondii our assay. In fact, preliminary examination of the dependence of within minutes by activating signaling cascades. The neutrophil several cytokines and chemokines on MyD88 and TLR2 by using chemoattractant IL-8 is up-regulated both at the protein and shRNA knockdown revealed a complex pattern. Although several mRNA level within hours. Several additional cytokines and che- genes were TLR2 dependent (IL-8, CCL10, CCL15), a few were mokines are also up-regulated at the mRNA level within 4 h. Dur- TLR2 independent and MyD88 dependent (CCL5 (RANTES) and ing mouse infections with T. gondii, neutrophils are critical for CCL11). IL-18 and CCL20 did not depend on either MyD88 or 7426 EPITHELIAL CELL RESPONSE TO T. gondii IS MEDIATED BY TLR2

effect on the ability of epithelial cells to respond to infection. T. gondii infection of macrophages fails to induce NF-␬B nuclear translocation and, in fact, inhibits activation in response to TLR ligands such as LPS (47, 48). In epithelial cells, NF-␬B translo- cation to the nucleus was not impaired. Similar results in infected murine embryonic fibroblasts have been reported (49, 50). Both ERK1/2 and p38 kinases were activated by T. gondii infection of epithelial cells; however, activation was almost entirely dependent on MyD88 and only partially dependent on TLR2. Additional in- nate immune receptors or other recognition mechanisms present in the intestinal epithelial cells may cooperate to regulate the immune response of T. gondii infection. These other receptors are unlikely to be TLRs, because attempts to coexpress other TLRs with TLR2 did not enhance the response in our HEK293 based stimulation assay. There are three clonal lineages of T. gondii that differ in their ability to induce cytokines and virulence. Low virulence type II parasites show higher induction levels of IL-12p40, IL-10, IL-1␤,

and IL-6, whereas high virulence type I parasites attract more neu- Downloaded from trophils during infection (51, 52). We predicted that the ability to activate TLR2 might correlate with lower virulence due to an in- creased activation of the immune response, especially because FIGURE 7. MAPK activation and TLR2-dependent response to T. gon- NF-␬B activation and cytokine secretion in immune cells correlate dii is not strain type specific. A, Henle 407 cells were infected with, GT-1 with genotype (53). However, all types elicited ERK1/2 and p38 T. gondii (type I), CC, DEG (type II), and VEG (type III) strains for the MAPK activation through TLR2. Although the strains varied in the http://www.jimmunol.org/ indicated time points. Total cell lysates were collected for immunoblotting level of activation, there was no correlation with genotype. Viru- with Abs against total and phosphorylated (p-) forms of ERK1/2 and p38. lence and cytokine induction differences among strains are not due B, HEK293 cells were transfected with human TLR2 and an NF-␬B lu- to an ability to activate epithelial cells via TLR2. ciferase reporter plasmid. The transfected HEK293 cells were stimulated with the TLR2 ligand Pam3Cys or different T. gondii tachyzoite strains. In summary, this study demonstrates that human intestinal ep- Cell lysates were collected at 18 h and assayed for luciferase activity (trip- ithelial cells respond directly to T. gondii infection via MyD88- .p Ͻ 0.05, compared with NF-␬B activity of and TLR2-driven ERK1/2 kinase and NF-␬B signaling pathways ,ء ;licate assays; error bar, SD uninfected controls). An interesting question for future studies is how epithelial cells crosstalk and influence immune cells during infection. Our pre- liminary data point to a complex pattern of cytokine regulation in by guest on September 27, 2021 TLR2 (data not shown). TLR4 and TLR9 are candidates for work- which TLR2 is important but not the whole story. Understanding ing in concert with TLR2 for the production of cytokines and che- the local immune response against pathogens in the intestine will mokines, because mice deficient in these TLRs have reduced in- provide insight into the development of intestinal disorders, mech- testinal pathology during oral T. gondii infection. Furthermore, anisms for enhancing immune response to infection, or targets for TLR9 in either hematopoietic or nonhematopoietic compartments vaccine development. is important for efficient T cell responses to oral infection (21). However, in our in vitro system knocking down TLR9 in Henle Acknowledgments 407 cells or reconstituting TLR9 in HEK293 cells does not affect We thank Jitender P. Dubey and Eric Denkers for providing the parasites. the MAPK or elicit a NF-␬B response to T. gondii. Commensal We gratefully thank He´le`ne Marquis and Eric Denkers for providing in- bacteria are present in the intestine and are capable of activating tellectual discussions on this manuscript. TLR9. Therefore, it is likely that the TLR9-dependent pathology induced during oral infection with T. gondii is secondary to epi- Disclosures thelial damage and recognition by TLR9 of bacteria that penetrate The authors have no financial conflict of interest. the epithelial barrier. References Ligands for TLR2 include lipopeptides, lipoproteins, and GPIs. 1. Shao, L., D. Serrano, and L. Mayer. 2001. The role of epithelial cells in immune GPI-anchored proteins are abundant on the surface of T. gondii regulation in the gut. Semin. Immunol. 13: 163–176. tachyzoites, and GPIs from Trypanosoma cruzi and 2. Sanderson, I. R., and W. A. Walker. 2007. TLRs in the gut. I. 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