Recruitment of Dendritic Cells Is Responsible for Intestinal Epithelial Damage in the Pathogenesis of Necrotizing Enterocolitis by sakazakii This information is current as of October 2, 2021. Claudia N. Emami, Rahul Mittal, Larry Wang, Henri R. Ford and Nemani V. Prasadarao J Immunol 2011; 186:7067-7079; Prepublished online 6 May 2011;

doi: 10.4049/jimmunol.1100108 Downloaded from http://www.jimmunol.org/content/186/12/7067

Supplementary http://www.jimmunol.org/content/suppl/2011/05/06/jimmunol.110010 Material 8.DC1 http://www.jimmunol.org/ References This article cites 50 articles, 10 of which you can access for free at: http://www.jimmunol.org/content/186/12/7067.full#ref-list-1

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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 © 2011 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Recruitment of Dendritic Cells Is Responsible for Intestinal Epithelial Damage in the Pathogenesis of Necrotizing Enterocolitis by Cronobacter sakazakii

Claudia N. Emami,*,†,1 Rahul Mittal,‡,x,1 Larry Wang,{ Henri R. Ford,*,† and Nemani V. Prasadarao*,†,‡,x

Cronobacter sakazakii is a Gram-negative pathogen associated with the cases of necrotizing enterocolitis (NEC) that result from formula contamination. In a mouse model of NEC, we demonstrate that C. sakazakii infection results in epithelial damage by recruiting greater numbers of dendritic cells (DCs) than macrophages and neutrophils in the gut and suppresses DC maturation, which requires outer membrane protein A (OmpA) expression in C. sakazakii. Pretreatment of intestinal epithelial cell monolayers with supernatant from OmpA+ C. sakazakii/DC culture markedly enhanced membrane permeability and enterocyte apoptosis, whereas OmpA2 C. sakazakii/DC culture supernatant had no effect. Analysis of OmpA+ C. sakazakii/DC coculture supernatant Downloaded from revealed significantly greater TGF-b production compared with the levels produced by OmpA2 C. sakazakii infection. TGF-b levels were elevated in the intestinal tissue of mice infected with OmpA+ C. sakazakii. Cocultures of CaCo-2 cells and DCs in a “double-layer” model followed by infection with OmpA+ C. sakazakii significantly enhanced monolayer leakage by increasing TGF-b production. Elevated levels of inducible NO synthase (iNOS) were also observed in the double-layer infection model, and abrogation of iNOS expression prevented the C. sakazakii-induced CaCo-2 cell monolayer permeability despite the presence of DCs or OmpA+ C. sakazakii/DC supernatant. Blocking TGF-b activity using a neutralizing Ab suppressed iNOS production and http://www.jimmunol.org/ prevented apoptosis and monolayer leakage. Depletion of DCs in newborn mice protected against C. sakazakii-induced NEC, whereas adoptive transfer of DCs rendered the animals susceptible to infection. Therefore, C. sakazakii interaction with DCs in intestine enhances the destruction of the intestinal epithelium and the onset of NEC due to increased TGF-b production. The Journal of Immunology, 2011, 186: 7067–7079.

ecrotizing enterocolitis (NEC) is the most common and breakdown of the intestinal epithelial barrier due to prematurity or lethal gastrointestinal disease of premature neonates (1– ischemic insults followed by translocation of pathogenic

N 3). The incidence of this disease approaches 1 per 1000 (9). Gram-negative bacteria have been cultured from the blood of by guest on October 2, 2021 live births and is associated with a high morbidity and mortality, many septic patients with NEC (10). Cronobacter sakazakii,for- ranging from 10 to 100%, depending on the extent of the intestinal merly known as sakazakii, is an emerging Gram- injury (4–7). In its most severe form, NEC is characterized by full- negative opportunistic pathogen, which is a common contaminant thickness destruction of the intestine accompanied by intestinal of powdered milk and infant formulas (11–14). The number of C. perforation, peritonitis, bacterial invasion, sepsis, and death (8). sakazakii-related infections has substantially increased during the The inciting event in the pathogenesis of NEC is believed to be the past 10 years due to increased use of infant formula (15–17). The U.S. Food and Drug Administration has issued several warnings regarding C. sakazakii infection in newborn infants. Therefore, un- *Department of Surgery, The Saban Research Institute, Children’s Hospital Los Angeles, Los Angeles, CA 90027; †Keck School of Medicine, University of Southern derstanding the pathogenesis of NEC due to C. sakazakii is essential California, Los Angeles, CA 90027; ‡Division of Infectious Diseases, The Saban for developing strategies for prevention or treatment. Our group has Research Institute, Children’s Hospital Los Angeles, Los Angeles, CA 90027; xDe- partment of Pediatrics, The Saban Research Institute, Children’s Hospital Los shown that oral feeding of C. sakazakii to newborn rats under Angeles, Los Angeles, CA 90027; and {Department of Pathology, The Saban Re- hypoxic conditions induces NEC-like inflammation (18). The in- search Institute, Children’s Hospital Los Angeles, Los Angeles, CA 90027 teraction of C. sakazakii with enterocytes induced apoptosis in rat 1C.N.E. and R.M. contributed equally to this work. intestinal epithelial cells (IEC-6) both in vitro and in newborn rats. Received for publication January 13, 2011. Accepted for publication April 3, 2011. C. sakazakii that does not express outer membrane protein A This work was supported by National Institutes of Health Grants AI40567 (to N.V.P.) (OmpA) could not bind to the intestine, indicating that OmpA may and AI4032-30 (to H.R.F.). be playing a role in the interaction of C. sakazakii with enterocytes Address correspondence and reprint requests to Dr. Nemani V. Prasadarao, Division (18). In addition, we have shown that NO, an important second of Infectious Diseases, MS #51, Children’s Hospital Los Angeles and Keck School of Medicine, University of Southern California, 4650 Sunset Boulevard, Los Angeles, messenger and inflammatory mediator, plays a key role in intestinal CA 90027. E-mail address: [email protected] barrier failure seen in NEC (19, 20). Subsequently, we have dem- The online version of this article contains supplemental material. onstrated that the enterocyte injury caused by C. sakazakii appears Abbreviations used in this article: 7-AAD, 7-amino-actinomycin D; BM, bone mar- to be dependent on the production of NO, as inhibition of inducible row; BMDC, bone marrow-derived dendritic cell; DC, dendritic cell; iNOS, inducible NO synthase (iNOS) by small interfering RNA (siRNA) prevented NO synthase; LP, lamina propria; MDC, myeloid dendritic cell; MOI, multiplicity of infection; mr, mouse recombinant; NEC, necrotizing enterocolitis; OmpA, outer apoptosis of IEC-6 cells (19). Although LPS has been shown to membrane protein A; PMN, polymorphonuclear cell; siRNA, small interfering increase iNOS expression in the ileum of mice colonized with RNA; TEER, transepithelial electrical resistance; WT, wild-type. bacteria (21, 22), no studies to date have addressed how OmpA Copyright Ó 2011 by The American Association of Immunologists, Inc. 0022-1767/11/$16.00 interaction with intestine induces NO. www.jimmunol.org/cgi/doi/10.4049/jimmunol.1100108 7068 ROLE OF DENDRITIC CELLS IN C. SAKAZAKII-INDUCED NEC

Dendritic cells (DCs) in the intestine are professional APCs re- of American Type Culture Collection. CaCo-2 cells were seeded in quired for the initiation of cell-mediated immune response (23–27). Transwells and maintained in enterocyte differentiation medium (BD Nonetheless, several pathogens have developed strategies to sup- Biosciences, San Jose, CA) to form monolayers. This medium promotes rapid growth of cells resulting in confluent monolayers of cells within 3–5 press DC maturation so that they can thrive in hostile conditions d. C. sakazakii were transformed with a GFP plasmid as previously de- (28). The stimulation of TLRs in DCs by pathogen-associated mi- scribed and grown in Luria broth containing ampicillin (31). crobial patterns leads to the activation of various inflammatory sig- Isolation of bone marrow-derived and intestinal DCs and of naling pathways and antimicrobial mechanisms (29). Our previous enterocytes studies demonstrated that C. sakazakii interaction with monocyte- derived DCs renders the cells more tolerogenic by increasing the Bone marrow (BM) cells were obtained by flushing the femurs and tibias production of TGF-b and IL-10 (30). We further demonstrated that with DMEM containing 5% FBS and subsequently washed and resuspended in appropriate medium. The BM cells were differentiated into DCs by C. sakazakii interacts with DC-SIGN, present on DCs, to enter and resuspending the cells in RPMI 1640 supplemented with 10% FCS, 2.4 mM 25 subsequently suppress the maturation of DCs. However, the role of L-glutamine (Invitrogen, Carlsbad, CA), 2-mercaptoethanol (5 3 10 M), DCs at the mucosal surfaces in the pathogenesis of NEC is poorly 100 IU/ml penicillin, 100 mg/ml streptomycin, 50 ng/ml mouse recom- understood. We hypothesize that the interaction of DCs with binant (mr) GM-CSF, and 20 ng/ml mrIL-4 (PeproTech, Rocky Hill, NJ). C. sakazakii expressing OmpA contributes to the epithelial barrier On days 1 and 3 post-culture initiation, the contents of each well were divided in half, and the volume of each new well was brought to 2 ml with disruption through secretion of pro- or anti-inflammatory cyto- supplemented DC medium containing mrGM-CSF and mrIL-4. On day kines. Using an in vitro and an in vivo mouse model of NEC, we 6–7, the same procedure was repeated for the final time (32). Subse- demonstrate that robust recruitment of DCs in the lamina propria quently, DCs were sorted by flow cytometry (BD Biosciences, San Jose, (LP) of intestine enhances epithelial cell injury upon infection CA) after staining with anti-CD11c Ab. For isolation of intestinal DCs, + b intestines were harvested from mice, Peyer’s patches removed, and the Downloaded from with OmpA C. sakazakii. TGF- production by DCs infected specimen placed in RPMI 1640 medium. The samples were placed through + with OmpA C. sakazakii promotes increased expression of iNOS a Percoll gradient for separation of enterocytes as described elsewhere from the epithelial cells and thereby enhances apoptosis and (32). CD11c+ F4/802 Gr-12 cells were then sorted using flow cytometry. barrier dysfunction. In agreement with these studies, depletion Flow cytometry of DCs in newborn mice protects the animals from OmpA+ C. sakazakii-induced NEC, whereas adoptive transfer of DCs to DC- Expression of DC maturation markers was detected by staining with ap- depleted mice restores the susceptibility to C. sakazakii infection. propriate fluorochrome-coupled mAbs or isotype-matched mAbs as de- http://www.jimmunol.org/ scribed earlier (30). Cells were analyzed by four-color flow cytometry This is, to our knowledge, the first report to demonstrate the im- using FACSCalibur CellQuest Pro software (BD Biosciences) using CD11c portance of DCs in the pathogenesis of C. sakazakii-induced NEC. as a gating marker, and at least 10,000 events within this gate were col- lected for analysis. Characterization of leukocytes derived from intestines Materials and Methods of newborn mice was done using flow cytometry. Briefly, intestines were Bacterial strains, cells, and reagents removed, transferred to ice-cold Hanks’ buffer/3% FCS, homogenized using a glass potter, and passed through a stainless-steel sieve. The dis- C. sakazakii (strain 51329) and CaCo-2 cells (human colon carcinoma sociated sample was collected by centrifugation and was digested for 60 cells; passage 18) were obtained from and maintained per the instructions min at 37˚C with 1.4 ml of type II collagenase (0.95 U/mg; Sigma) and 104 by guest on October 2, 2021

FIGURE 1. Oral feeding of C. sakazakii to newborn mice recruits greater number of DCs during the onset of NEC. Mice at day 3 were fed orally with 103 CFU OmpA+ C. sakazakii, OmpA2 C. sakazakii, or un- infected. By 96 h after infection with OmpA+ C. sakazakii, 100% of animals died of infection, whereas the OmpA2 C. sakazakii animals survived (A). Mice infected with OmpA+ C. sakazakii showed abdomi- nal distension and dilated and necrotic intestine, but OmpA2 C. sakazakii-fed and control animals did not (B). The intestinal bacterial load was assessed by plating the intestinal tissue homogenates of mice on ampicillin-containing agar (C). The stained sections were scored for severity of disease by a pathologist blinded to specimens. The experiments were repeated three times, and the means of the average scores were plotted (D). Intestines were harvested from mice and subjected to histopathological examination after staining with H&E. Severe epithelial damage, villi destruction (black arrow), and mucosal derangement was seen in the intestines of OmpA+ C. sakazakii-fed mice, but animals infected with OmpA2 C. sakazakii or control showed normal histology (E). Original magnification 310. The data represent mean 6 SD from three independent experiments with at least 15 animals in each group for each experiment. *p , 0.01 by ANOVA, Fisher exact test, and Wilcoxon signed- rank test. OmpA+ CS, OmpA+ C. sakazakii; OmpA2 CS, OmpA2 C. sakazakii. The Journal of Immunology 7069

U DNase I (Sigma) in dissociation buffer (42 mmol/l MgCl2/23 mmol/l mucosal edema, villus core edema, epithelial sloughing/obliteration, neu- CaCl2/50 mmol/l KCI/153 mmol/l NaCl). The digested sample was pel- trophil infiltration, intestinal perforation, and necrosis. In depletion studies, leted and resuspended in PBS. Polymorphonuclear cells (PMNs) were animals received six injections of CD11c Ab (Abcam, Cambridge, MA) in identified using Gr-1 Ab, and macrophages were stained with F4/80 Ab. sterile PBS (50 ml) using i.p. injections starting on day 1. The last injection The presence of bacteria was measured using the GFP expression in the was given prior to infection with C. sakazakii. Depletion was confirmed FITC gate. The expression of iNOS was detected in the intestines of prior to infection using flow cytometry for CD11c expression in liver, infected mice by double staining for E-cadherin and iNOS using appro- spleen, and the intestinal homogenates. Mouse DCs were derived from the priate primary Abs (BD Biosciences) with two separate fluorochrome- BM aspirates from femurs and tibias of mice followed by culturing the coupled secondary Abs. cells in mrIL-4 and mrGM-CSF to differentiate the BM cells into DCs (bone marrow-derived dendritic cells; BMDCs) as described earlier and siRNA transfection then sorted by flow cytometry based on CD11c staining and F4/80 and Gr- 3 6 CaCo-2 cells were grown to 30% confluence in 6-well plates, then trans- 1 negative population. DCs (5 10 ) were injected i.p. into the DC- fected with siRNA for iNOS or control siRNA using Lipofectamine depleted animals 6 h prior to oral infection with bacteria. The presence (Invitrogen) according to the manufacturer’s instructions. The siRNA of DCs was confirmed by flow cytometry with anti-CD11c Ab in the ho- primer sequence used for silencing iNOS expression is 39-CCACCAG- mogenized intestinal tissue. TATGCAATGAAT-59 (Invitrogen). The respective control-siRNA was also purchased from Invitrogen. Cytokine measurements Animal experiments Cytokine levels were measured in CaCo-2 cells as well as in the supernatant from fresh homogenized intestinal tissue after the animals were euthanized The animal studies were approved by the Institutional Animal Care and Use at the end of the experiment by ELISA using commercially available kits Committee of the Saban Research Institute at Children’s Hospital Los (Invitrogen). The tissue was weighed prior to homogenization, and the Angeles and followed National Institutes of Health guidelines for the levels were calculated per gram of intestinal tissue. performance of animal experiments. Time pregnant C57BL/6 mice were Downloaded from obtained from Charles River at E18; after delivery, pups were kept with the Determination of apoptosis mothers and housed at Children’s Hospital Los Angeles. Three-day-old pups were fed 103 CFU OmpA+ C. sakazakii or OmpA2 C. sakazakii in 10 Apoptosis was assessed by TUNEL staining using ApoTag kit (Chemicon, ml sterile PBS orally and then kept with the mother. The control animals Temecula, CA) and annexin-V and propidium iodide staining (BD Bio- + sciences) according to the manufacturers’ instructions. received sterile PBS at day 3. The OmpA C. sakazakii-fed animals suc- cumbed to infection on day 4 postinfection; other pups were euthanized Determination of CaCo-2 cell monolayer integrity at the same time. The animals were carefully monitored for any signs of http://www.jimmunol.org/ disease, and intestinal tissues were collected from these animals at the CaCo-2 cells seeded in Transwells in enterocyte differentiation medium moribund state but not from dead animals. The collected tissues were fixed (BD Biosciences) were cultured for 48 h to reach a resistance of .300 in formalin and stained with H&E. Intestinal sections were graded mi- V×cm2. C. sakazakii strains were added at a multiplicity of infection (MOI) croscopically by a pathologist blinded to groups from grade 0 (normal) to of 10 in Transwells and incubated for varying periods. The monolayer grade 4 (severe) based on pathological manifestations including sub- integrity was determined by measuring transepithelial electrical resistance by guest on October 2, 2021

FIGURE 2. Infection with C. sakazakii results in recruitment of DCs in the intestines. The recruitment of DCs, PMNs, and macrophages was determined in the intestines of infected or control animals and an- alyzed by flow cytometry after staining with Abs to CD11c, Gr-1, and F4/80, respectively (A). The phe- notype of DCs was determined by flow cytometry (B). In separate experiments, DCs isolated from infected mice were examined for the expression of maturation markers by flow cytometry (C). Cryostat sections of intestines were prepared and stained for DCs as well as for nuclei by DAPI. Images were acquired by Zeiss confocal microscope (D). Scale bars, 50 mm. Insets, original magnification 340. The data represent mean 6 SD from three independent experiments with at least 15 animals in each group for each experiment. *p , 0.01 by ANOVA and Fisher exact test. OmpA+ CS, OmpA+ C. sakazakii; OmpA2 CS, OmpA2 C. sakazakii. 7070 ROLE OF DENDRITIC CELLS IN C. SAKAZAKII-INDUCED NEC

FIGURE 3. Pretreatment of CaCo-2 cells with OmpA+ C. sakazakii/DC supernatant compro- mises the tight junction integrity. Confluent mono- layers of CaCo-2 cells grown in Transwell in- serts were infected with OmpA+ C. sakazakii or OmpA2 C. sakazakii, left alone, or pretreated with supernatants from C. sakazakii/DC cocul- tures. TEER (A), HRP (B), and FITC-conjugated dextran leakage (C) was measured as described in Materials and Methods. The results represent mean 6 SD from four separate experiments per- formed in triplicate. The decrease in TEER or Downloaded from increase in the permeability of HRP or FITC- conjugated dextran was statistically significant: *p , 0.001 by ANOVA and Student t test. OmpA+ CS, OmpA+ C. sakazakii; OmpA2 CS, OmpA2 C. sakazakii. http://www.jimmunol.org/ by guest on October 2, 2021

(TEER) and HRP leakage or monolayer permeability to FITC-conjugated Results dextran (4 kDa) as described previously (33, 34). Briefly, FITC-conjugated C. sakazakii induces NEC in newborn mice dextran was added to confluent CaCo-2 monolayers grown on Transwells and then infected with C. sakazakii. The leakage of FITC-conjugated Previous studies from this laboratory have demonstrated that oral dextran in the lower chamber was then determined using a fluorometer. feeding of C. sakazakii to newborn rats under hypoxic conditions The standard curve was calibrated using FITC-conjugated dextran, and the results are expressed in micrograms per milliliter. In some experiments, the induced NEC by day 4 (18, 19). To use knockout mice and to Transwells containing CaCo-2 cells were flipped and seeded with human understand the role of various host and bacterial factors re- myeloid dendritic cells (MDCs), which were isolated as described (30) and sponsible for pathogenesis, it is necessary to establish a mouse cultured for 24 h to form double layers. In addition, C. sakazakii strains were incubated with MDCs for 24 or 48 h, the supernatants were collected, cleared of bacteria by centrifugation, and filtered through an 0.22-mm filter. The absence of bacteria was confirmed by culturing the supernatants on blood agar plates. The supernatants were stored at –70˚C until used in pretreatment experiments. iNOS and NO measurement CaCo-2 cells were infected with C. sakazakii at an MOI of 10 for 4 h; the cells were then washed, fixed in 2% paraformaldehyde, and subsequently permeabilized using Cytoperm solution (BD Biosciences). The cells were stained with fluorochrome conjugated anti-iNOS Ab for 1 h and analyzed using flow cytometry. The supernatants were collected and used to de- termine the production of NO by the Griess method (33). Fluorescence microscopy

Sections of the intestine were cut with a cryostat (Leica, Wetzlar, Germany) + and then stained with tight junction marker ZO-1 (31). Stained tissue FIGURE 4. OmpA C. sakazakii causes tight junction disruption. CaCo-2 + sections were imaged with confocal microscope LSM710 and arranged cells grown on 4-well chamber slides were infected with OmpA C. 2 using Adobe Photoshop 7.0. sakazakii or OmpA C. sakazakii for 4 h and stained for ZO-1. Infection with OmpA+ C. sakazakii resulted in disruption of tight junctions (yellow Statistical analysis arrows), whereas OmpA2 C. sakazakii infection did not affect the integrity Statistical significance was determined by Student t test, ANOVA, Wil- of the tight junctions. The results are representative of five independent 2 coxon signed-rank test, and Fisher exact test. The p values ,0.05 were experiments. OmpA+ CS, OmpA+ C. sakazakii; OmpA2 CS, OmpA C. considered to be statistically significant. sakazakii (original magnification 363). The Journal of Immunology 7071 model of NEC. Therefore, 3-d-old newborn mice were infected OmpA+ C. sakazakii (Fig. 1D). The pathological changes were orally with 103 CFU C. sakazakii expressing GFP. In addition, comparable with those seen in human infants with NEC (de- C. sakazakii lacking OmpA expression was also used to determine rangement of mucosa and villus sloughing) (Fig. 1E). In contrast, the role of OmpA in the induction of NEC. The animals fed intestinal sections from OmpA2 C. sakazakii-infected animals OmpA+ C. sakazakii succumbed to infection by 72–96 h post- showed no such injury. The average pathology scores were sig- inoculation, whereas those fed OmpA2 C. sakazakii survived nificantly greater in mice infected with OmpA+ C. sakazakii similar to the uninfected control mice (Fig. 1A). The mice fed compared with those in mice infected with OmpA2 C. sakazakii OmpA+ C. sakazakii develop morphological signs of NEC in- (p , 0.001 by Wilcoxon signed-rank test). cluding intestinal dilation and bowel wall discoloration as early C. sakazakii has been shown to manipulate the maturation of as 48 h postinfection (Fig. 1B). The infected animals appeared DCs by entering via DC-SIGN (30). DCs are important APCs in sick with abdominal distention and decreased activity. OmpA2 the gut, which sample pathogenic luminal bacteria. To assess C. sakazakii-infected mice behaved like uninfected control mice whether DCs recruit to the LP as a result of OmpA+ C. sakazakii until euthanized. Next, we examined the binding of OmpA+ and infection, intestinal DCs as well as PMNs and macrophages were OmpA2 C. sakazakii to the mouse intestine by plating the in- isolated from digested intestinal tissue after separation by Percoll testinal tissue homogenates on agar containing antibiotic. As gradient. Flow cytometric analysis revealed that higher numbers shown in Fig. 1C, OmpA+ C. sakazakii bound to the intestine in of DCs were recruited to the LP in animals fed OmpA+ C. significantly greater numbers compared with OmpA2 C. sakazakii sakazakii compared with uninfected controls based on CD11c at 72 h postinfection. Of note, we observed that the binding of staining whereas the number of PMNs and macrophages increased OmpA2 C. sakazakii was similar to that of OmpA+ C. sakazakii only slightly (p , 0.001 by ANOVA and Fisher exact test, Fig. up to 12 h postinfection (data not shown). Scoring of intestinal 2A). Notably, the number of DCs recruited to the LP was similar in Downloaded from sections from the infected mice after staining with H&E by a pa- OmpA2 C. sakazakii-fed animals, indicating that the recruitment thologist blinded to specimens revealed severe epithelial injury as is not dependent on OmpA expression. Thirty-one percent of the well as mucosal sloughing and villi destruction in mice fed immunocytes in the intestines of OmpA+ C. sakazakii-fed mice were http://www.jimmunol.org/

FIGURE 5. Increased apoptosis in by guest on October 2, 2021 CaCo-2 cells pretreated with OmpA+ C. sakazakii/DC supernatant. Confluent monolayers of CaCo-2 cells were pre- conditioned with C. sakazakii/DC super- natants for 24 h and then infected with C. sakazakii or left uninfected. The mono- layers were assessed for apoptosis by TUNEL staining using an ApoTag kit. Bacteria (green), apoptotic cells (red), and nuclei (blue) (A). Original magnifi- cation 320; insets, original magnifica- tion 363. The number of apoptotic nu- clei per total nuclei was counted and graphed (B). In separate experiments, the cells were also stained with annexin- V/7-AAD and examined by flow cy- tometry (C). The error bars represent mean 6 SD of five independent experi- ments performed in triplicate. *p , 0.01 by ANOVA and Student t test. CS, C. sakazakii; OmpA+ CS, OmpA+ C. saka- zakii; OmpA2 CS, OmpA2 C. sakazakii. 7072 ROLE OF DENDRITIC CELLS IN C. SAKAZAKII-INDUCED NEC

CD11c+ compared with less than 1% in the gut of PBS-fed control were grown in Transwell inserts and at a baseline of .300 V×cm2 animals. The intestinal DCs recruited to the LP were CD11c/CD103 were infected with OmpA+ C. sakazakii or OmpA2 C. sakazakii positive and F4/80/Gr-1 negative as shown by flow cytometry (Fig. for varying periods. The TEER and the permeability of mono- + 2B). However, we observed that infection with OmpA C. sakazakii layers using the HRP or FITC–dextran leakage method were suppresses the expression of maturation markers (CD40, MHC class 2 measured (33, 34). Because the baseline TEER for various mon- II, and CD86) in the intestinal DCs, whereas the OmpA C. saka- olayers in different sets of experiments vary, the results are zakii-infected intestinal DCs express these markers (Fig. 2C). Con- expressed as percentage of TEER. At 1 h postinfection, OmpA+ C. focal microscopy of the intestinal tissue from mice infected with 2 sakazakii interaction with CaCo-2 cells induced a 20% decrease OmpA+ C. sakazakii or OmpA C. sakazakii upon staining with V× 2 V× 2 CD11c Ab also revealed significant recruitment of DCs to the LP in in TEER from a baseline of 316 cm to 253 cm , whereas the TEER decreased by .60% after 4 h postinfection (Fig. 3A). agreement with our flow cytometry data (Fig. 2D). Collectively, these 2 results suggest that OmpA expression in C. sakazakii is critical for OmpA C. sakazakii induced only a 15% decrease in TEER 2 efficient binding to the intestine and that OmpA+ C. sakazakii can across the monolayers even after 4 h postinfection (329 V×cm at survive intrinsic DC defenses by suppressing DC maturation. In baseline versus 277 V×cm2 at 4 h). In agreement with the TEER 2 contrast, OmpA C. sakazakii, despite showing some initial binding, data, HRP leakage across CaCo-2 monolayers was gradually in- could not survive in the intestine. creased with OmpA+ C. sakazakii infection up to 4 h post- + infection, whereas a minor leakage of HRP was observed with Preconditioning of CaCo-2 cells with OmpA C. sakazakii/DC 2 supernatant compromises the monolayer permeability OmpA C. sakazakii (Fig. 3B). To examine further the monolayer

permeability in response to C. sakazakii infection, we used FITC- Downloaded from Because the disruption of the intestinal barrier is an important conjugated dextran, which provides a finer measurement of feature of the pathogenesis of NEC, we hypothesize that OmpA+ C. sakazakii interaction with DCs might contribute to the patho- paracellular flux and help in assessing tight junction integrity genesis of NEC by disrupting the epithelial barrier. To test this independent of cell viability. On par with our HRP data, we ob- notion, we first examined the effect of C. sakazakii interaction on served significant leakage of FITC-conjugated dextran across + the permeability of CaCo-2 cell monolayers, which make very CaCo-2 monolayers infected with OmpA C. sakazakii (Fig. 3C). good tight junctions compared with IEC-6 cells, which were used In contrast, very minimal leakage of FITC-conjugated dextran was http://www.jimmunol.org/ in previous studies from this laboratory (18, 19). CaCo-2 cells observed in OmpA2 C. sakazakii infected CaCo-2 monolayers. by guest on October 2, 2021

FIGURE 6. OmpA+ C. sakazakii causes tight junction disruption prior to apoptosis. In separate experiments, CaCo-2 cells were pretreated with apoptosis inhibitor ZVAD (10 mM) and then infected with C. sakazakii. TEER (A), HRP (B), and FITC-conjugated dextran leakage (C) was then measured. Apoptosis was measured in these cells after treatment with ZVAD by staining with annexin-V/7-AAD and examination by flow cyto- metry (D). The results represent mean 6 SD from five separate experiments performed in triplicate. The decrease in TEER or increase in the perme- ability of HRP or FITC-conjugated dextran be- tween ZVAD-treated and untreated cells was not statistically significant. *p , 0.01, **p . 0.05 by ANOVA and Student t test. OmpA+ CS, OmpA+ C. sakazakii; OmpA2 CS, OmpA2 C. sakazakii. The Journal of Immunology 7073

To examine further whether any soluble factors released into the C. sakazakii infection has been demonstrated to induce NO in culture supernatants are responsible for the permeability of CaCo-2 IEC-6 cells by triggering the activation of iNOS, which is shown cell monolayers, supernatants obtained from the bacteria/DC in- to be responsible for apoptosis of the cells (19). Therefore, to ex- fection experiments after clearing the bacteria were used to treat amine whether OmpA+ C. sakazakii/DC supernatant increases CaCo-2 cells. The monolayer permeability was assessed in the the production of NO, and thus causes an increased apoptosis of presence or absence of bacteria. The MDCs were isolated and CaCo-2 cells, the cells were treated with the supernatants in the cultured from human donor monocytes as a surrogate for our presence or absence of C. sakazakii. The expression of iNOS and in vitro model to match CaCo-2 monolayers. These MDCs were the production of NO were then determined by flow cytometry and cultured with OmpA+ C. sakazakii or OmpA2 C. sakazakii for 24 Griess reagent, respectively (33) As predicted, CaCo-2 cells treated h, and the supernatants from the cocultures were collected after with OmpA+ C. sakazakii/DC supernatant prior to infection with centrifugation and filtered through an 0.22-mm filter to remove C. sakazakii showed higher iNOS expression, and enhanced pro- bacteria. The CaCo-2 monolayers were pretreated with the duction of NO by 2.5- and 6.0-fold, respectively, compared with supernatants for 24 h prior to infection with C. sakazakii, and those of infected cells alone (Supplemental Fig. 1A,1B). In con- TEER, HRP, as well as FITC-conjugated dextran leakage was trast, treatment with OmpA2 C. sakazakii/DC supernatant had no measured. Pretreatment with supernatant from OmpA+ C. saka- effect on the production of NO. Taken together, these results sug- zakii/DC coculture resulted in significantly faster decline (.60%) gest that OmpA+ C. sakazakii/DC supernatants contain soluble of TEER by 2 h compared with that of untreated monolayers or factors that promote tight junction disruption of CaCo-2 cells pretreated monolayers in the absence of bacteria (Fig. 3A). In contrast, the supernatant from OmpA2 C. sakazakii/DC coculture induced no such decline. The HRP leakage across the monolayer Downloaded from was also significantly increased after pretreatment with OmpA+ C. sakazakii/DC supernatant (Fig. 3B). Similar results were also observed with FITC-conjugated dextran assay (Fig. 3C). In agreement with permeability data, CaCo-2 cells infected with OmpA+ C. sakazakii demonstrated the disruption of ZO-1 staining at the periphery of the cells compared with the staining in un- http://www.jimmunol.org/ treated or OmpA2 C. sakazakii-treated cells (Fig. 4). These results suggest that the interaction of OmpA+ C. sakazakii with DCs produces factors that synergistically promote the disruption of tight junctions in enterocytes. OmpA+ C. sakazakii/DC supernatant pretreatment increases apoptosis of CaCo-2 cells We have previously demonstrated that C. sakazakii induced apo- by guest on October 2, 2021 ptosis by 6 h postinfection in IEC-6 cells (18, 19). To examine whether the presence of soluble factors in DC/C. sakazakii super- natants enhance enterocyte apoptosis, CaCo-2 cells were pretreated with OmpA+ C. sakazakii/DC supernatant, and the number of ap- optotic cells was determined by TUNEL staining as well as by annexin-V/7-amino-actinomycin D (7-AAD) method. Pretreatment with OmpA+ C. sakazakii/DC supernatant for 24 h prior to in- cubation with OmpA+ C. sakazakii significantly increased enter- ocyte apoptosis compared with pretreatment with OmpA2 C. sakazakii/DC supernatant or untreated cells (p , 0.001 by ANOVA test) (Fig. 5A). In addition, pretreatment with the supernatants alone did not induce apoptosis in CaCo-2 cells. Quantitative deter- mination of apoptotic cells revealed that pretreatment of CaCo-2 cells with OmpA+ C. sakazakii/DC supernatant increased apoptosis by 4-fold compared with OmpA2 C. sakazakii/DC supernatant and by 2-fold compared with OmpA+ C. sakazakii alone (Fig. 5B). Similar results were obtained with annexin-V/7-AAD and propi- dium iodide staining (Fig. 5C and data not shown). Our hypothesis is that tight junction disruption occurs first followed by apoptosis of cells in response to C. sakazakii infection. To examine this hypothesis, we pretreated CaCo-2 cells with apoptosis inhibitor ZVAD and then infected with C. sakazakii. We observed decrease FIGURE 7. OmpA+ C. sakazakii induces the production of TGF-b both in TEER with increase in postinfection time similar to untreated + 2 infected cells (Fig. 6A). In agreement, the leakage of HRP and in vitro and in vivo. Fresh intestinal tissue from OmpA and OmpA C. sakazakii was homogenized, and the levels of TGF-b were determined FITC-conjugated dextran remained increased with ZVAD pre- + by ELISA (A). In separate experiments, intestinal DCs (B) and BMDCs (C) treatment similar to that of OmpA C. sakazakii-infected cells were infected with OmpA+ and OmpA2 C. sakazakii and production of (Fig. 6B,6C). However, there was a significant decrease in apo- TGF-b was examined in the supernatants by ELISA. The data represent ptosis of CaCo-2 cells after ZVAD pretreatment despite infection means 6 SD and are representative of three separate experiments from five + with OmpA C. sakazakii (Fig. 6D). These results indicate that animals in each experiment. The increase or decrease in cytokine levels is changes in tight junction occur prior to the onset of apoptosis. statistically significant: *p , 0.001 by ANOVA. 7074 ROLE OF DENDRITIC CELLS IN C. SAKAZAKII-INDUCED NEC followed by apoptosis of cells by increasing the production of in- duction of IL-10 in MDCs, therefore we also used neutralizing ducible NO. Abs to IL-10 along with TGF-b (30). CaCo-2 cells express both TGF-b and IL-10 receptors (35, 36). The TGF-b–neutralizing Ab TGF-b production significantly increases postinfection with + binds all three isoforms of the protein. The cells were pretreated OmpA C. sakazakii both in vitro and in vivo with the corresponding neutralizing Ab for 1 h prior to infection We previously demonstrated that the interaction of OmpA+ C. with bacteria for varying periods. Blocking of IL-10 did not affect sakazakii with myeloid DCs in vitro results in increased levels of the OmpA+ C. sakazakii-induced enterocyte apoptosis, whereas TGF-b production (30). In agreement, a significant rise in TGF-b neutralizing TGF-b abrogated OmpA+ C. sakazakii-induced ap- levels was observed in the intestines of infected mice upon in- optosis (Fig. 8A). Also, monolayer permeability and electrical fection with OmpA+ C. sakazakii compared with the levels in- resistance decreased significantly upon infection with OmpA+ duced by OmpA2 C. sakazakii infection (Fig. 7A). These results C.sakazakii despite neutralizing IL-10. However, pretreatment were also confirmed by RT-PCR using total RNA extracted from with TGF-b–neutralizing Ab prevented the effects of OmpA+ C. the mucosal scrapings of intestines (data not shown). Similarly, sakazakii on TEER and monolayer permeability (Fig. 8B–D). BMDCs and intestinal DCs infected with OmpA+ C. sakazakii Addition of TGF-b or IL-10 alone in the absence of bacteria did in vitro showed higher levels of TGF-b consistent with what was not induce any apoptosis or barrier permeability (data not shown). observed in MDCs (Fig. 7B,7C). We believe that the significant These results confirm that TGF-b expression exacerbates C. rise in the levels of TGF-b is a result of the higher recruitment of sakazakii-induced injury in enterocytes and is the putative culprit DCs promoted by the OmpA+ C. sakazakii. These results suggest in the DC/C. sakazakii coculture supernatant that causes an in- that OmpA+ C. sakazakii invades, survives within, and suppresses crease in OmpA+ C. sakazakii-induced epithelial cell monolayer the maturation of intestinal DCs similar to that of myeloid-derived permeability and apoptosis. To determine whether the change in Downloaded from DCs for which OmpA expression is critical. the levels of TGF-b has any effect on increased iNOS expression observed with OmpA+ C. sakazakii infection, flow cytometry was b + Blocking of TGF- with a neutralizing Ab prevents OmpA C. performed. The production of NO in the culture medium in the sakazakii-induced epithelial monolayer damage and apoptosis presence of neutralizing Abs was determined using the Griess To delineate the role of TGF-b in OmpA+ C. sakazakii infection, method. Notably, blocking TGF-b prevented the upregulation of we performed the experiments using CaCo-2 monolayers in the iNOS expression in CaCo-2 cells despite infection with OmpA+ C. http://www.jimmunol.org/ presence of TGF-b–neutralizing Ab. Our previous studies have sakazakii (Supplemental Fig. 2A,2B). In agreement with the iNOS shown that OmpA+ C. sakazakii also induces significant pro- expression, NO production also was significantly decreased in by guest on October 2, 2021

FIGURE 8. Blocking of TGF-b ac- tivity by a neutralizing Ab prevents OmpA+ C. sakazakii-induced CaCo-2 monolayer integrity and apoptosis, whereas blocking IL-10 had no effect. Confluent monolayers of CaCo-2 cells were pretreated with neutralizing Abs to TGF-b (against all three isoforms) or IL-10 for 1 h prior to infection with OmpA+ C. sakazakii or OmpA2 C. sakazakii. Blocking of TGF-b pre- vented OmpA+ C. sakazakii-induced ap- optosis as measured by annexin-V/7- AADstainingfollowedbyflowcytom- etry (A). CaCo-2 monolayers grown to confluence on Transwells were pretreated with neutralizing Abs, then TEER (B), HRP (C), and FITC-conjugated dextran leakage (D) was determined at various time periods. Neutralizing TGF-b activ- ity blocked OmpA+ C. sakazakii-induced monolayer dysfunction, whereas neutra- lizing IL-10 had no effect. *p , 0.001 by ANOVA. OmpA+ CS, OmpA+ C. saka- zakii;OmpA2 CS, OmpA2 C. sakazakii. The Journal of Immunology 7075 cells pretreated with anti–TGF-b Ab. By contrast, anti–IL-10 Ab cocultures at 4 h postinfection; p , 0.01 by ANOVA) (Fig. 9B). treatment did not prevent the increase in iNOS expression. These Similar rise in values of FITC-conjugated dextran was also ob- results suggest that TGF-b secreted by CaCo-2 cells upon OmpA+ served (Fig. 9C). OmpA2 C. sakazakii could not induce such C. sakazakii infection could be responsible for enhancing the tight rapid changes in TEER and permeability despite the presence of junction disruption and increasing apoptosis of these cells by in- DCs. The iNOS expression and amount of NO released into the ducing NO production. medium in cocultures was robustly increased in the presence of DCs (Supplemental Fig. 2C,2D). In a follow-up experiments, DCs Presence of DCs exacerbates the effect of C. sakazakii-induced were collected from the double layers at 4 h postinfection, and epithelial monolayer permeability in a “double-layer” model their maturation marker profile was measured by flow cytometry To mimic the presence of DCs in the LP of intestine, we used using Abs to MHC class II, CD40, and CD86. As previously a “double-layer” model as described previously (32) and exam- demonstrated, the DCs infected with OmpA+ C. sakazakii showed ined OmpA+ C. sakazakii-induced epithelial barrier injury. CaCo- suppression of these markers in the presence of OmpA+ C. 2 cells were cocultured to form monolayers on Transwells, and sakazakii. However, OmpA2 C. sakazakii-infected as well as the then DCs were seeded on the opposite side of the Transwells to LPS-treated double layers showed maturation of DCs in the bot- form “double layers”. Bacteria were added in the top chamber at tom layer with significant rise in the levels of these markers (Fig. an MOI of 10 (cell to bacteria ratio 1:10) and incubated for var- 9D). No bacteria were observed in the bottom chamber in these ious time points. The presence of DCs resulted in a rapid decline experiments, indicating that the suppression of DC maturation is in TEER in the double layers after 1 h of incubation with bacteria not due to overspill of the bacteria from the top chamber. (Fig. 9A). TEER dropped from 60% at 1 h postinfection to 30% by To confirm the role of iNOS in CaCo-2 cell monolayer integrity 4 h postinfection in the presence of OmpA+ C. sakazakii. The during C. sakazakii infection, the cells were transfected with siRNA Downloaded from steep decline in TEER is mirrored by an increase in permeability to iNOS. These iNOS siRNA-transfected CaCo-2 cells form mon- as demonstrated by rapid rise in HRP leakage through the mon- olayers similar to untransfected cells. However, iNOS siRNA- olayers (∼400 pg/ml without DCs versus ∼750 pg/ml with DC transfected CaCo-2 cells infected with OmpA+ C. sakazakii do not http://www.jimmunol.org/ by guest on October 2, 2021 FIGURE 9. Presence of DCs exacer- bates the permeability of CaCo-2 cells, and silencing of iNOS expression in CaCo-2 cells prevents the monolayer in- tegrity. CaCo-2 cells were cocultured with DCs as “double layers” in Trans- wells as described in Materials and Methods. Cells were infected with OmpA+ or OmpA2 C. sakazakii and incubated for varying periods. TEER (A), HRP (B), and FITC-conjugated dextran leakage (C) was determined. The DCs were collected in a separate set of experiments from the double layers at the end of the experiment, and maturation markers were measured using flow cytometry. LPS was used as a control in one of the Transwells (D). The results represent mean 6 SD from four separate experiments performed in tripli- cate. *p , 0.05 by ANOVA and Student t test. OmpA+ CS, OmpA+ C. sakazakii; OmpA2 CS, OmpA2 C. sakazakii. 7076 ROLE OF DENDRITIC CELLS IN C. SAKAZAKII-INDUCED NEC show increase in expression of iNOS and therefore NO production anti-CD11c Ab prior to infection with the OmpA+ C. sakazakii on compared with untransfected cells (Fig. 10A,10B). The mono- day 3. We found that CD11c and control Ab when used at con- layer permeability and HRP leakage was significantly reduced centrations of 5 mg were not toxic in mice. We observed no dif- when the CaCo-2 cells were transfected with siRNA to iNOS ferences in the number of neutrophils, macrophages, monocytes, compared with that of untransfected cells (Fig. 10C–E). In addi- or CD4 and CD8 T cells between CD11c or isotype-matched tion, iNOS expression was significantly elevated in enterocytes treated animals and untreated animals at these concentrations in upon OmpA+ C. sakazakii infection. Therefore, increased TGF-b various tissues (data not shown). DC depletion was confirmed in expression in DCs postinfection with OmpA+ C. sakazakii primes the intestine prior to infection as well as postinfection using anti- the enterocytes leading to an increase in iNOS expression and thus CD11c Ab by flow cytometry (Supplemental Fig. 4A). As pre- more apoptosis. CaCo-2 cells also produce TGF-b upon infection dicted, 100% of the DC-depleted mice survived up to day 7 with OmpA+ C. sakazakii but the quantities are not as high as the postinfection, whereas control Ab-treated/infected and wild-type levels produced by the DCs (Supplemental Fig. 3). Therefore, the (WT)/infected mice succumbed to infection within 96 h post- presence of DCs exacerbates the response to bacteria, and in- infection (Fig. 11A). The intestinal bacterial load was significantly creased production of TGF-b by DCs appears to be involved in the lower in DC-depleted/infected mice (log 1.23 CFU/g tissue) observed barrier injury. compared with that of WT/infected (log 5.67 CFU/g tissue) or control Ab-treated/C. sakazakii-infected (log 5.51 CFU/g tissue, Depletion of DCs in newborn mice protects the animals from , + p 0.01 by ANOVA) mice (Fig. 11B). Histopathological exam- OmpA C. sakazakii-induced NEC ination revealed that the DC-depleted animals showed normal To substantiate the requirement of DCs for disease severity in the mucosa, whereas severe infiltration of neutrophils along with de- pathogenesis of NEC, DCs were depleted in newborn mice by struction of villus was observed in WT/infected and control Ab- Downloaded from injecting anti-CD11c Ab at day 1 after birth. Control animals treated/infected mice (Fig. 11C). The average pathology score was received isotype-matched Ab. Animals received six injections of 0.5 in DC-depleted/infected mice, whereas it was 3.6, 3.85, and http://www.jimmunol.org/ by guest on October 2, 2021

FIGURE 10. Silencing of iNOS pre- vents OmpA+ C. sakazakii-mediated mono- layer permeability. CaCo-2 cells were transfected with siRNA to iNOS or con- trol siRNA and then cocultured with DCs. There was no increase in expression of iNOS (A) and hence NO production (B) in iNOS siRNA-transfected CaCo-2 cells infected with C. sakazakii. TEER (C), HRP (D), and FITC-conjugated dextran leakage (E) were then measured. The error bars represent SD of four in- dividual experiments performed in tripli- cate. *p , 0.001 by Student t test and ANOVA. OmpA+ CS, OmpA+ C. saka- zakii; OmpA2 CS, OmpA2 C. sakazakii. The Journal of Immunology 7077

FIGURE 11. Depletion of DCs protects newborn mice from OmpA+ C. sakazakii- induced NEC. DCs were depleted in new- born mice by injecting anti-CD11c Ab and then infected with C. sakazakii as described in Materials and Methods.In some experiments, DC-depleted mice were used for adoptive transfer of DCs and then infected. DC-depleted animals survived OmpA+ C. sakazakii infection, whereas the adoptive transfer of DCs resulted in similar mortality as that of WT or control Ab-injected animals infected with OmpA+ C. sakazakii (A). The bacterial load in the intestines of mice from experiments de- scribed in A were determined by plating the tissue homogenates and expressed as log CFU per gram of intestinal tissue (B). Histopathological examination of intes- tines from DC-depleted mice demonstrated intact mucosal architecture. Meanwhile, the Downloaded from animals that received DCs by adoptive transfer demonstrate villus destruction and inflammation similar to WT/infected con- trols (C). H&E, original magnification 310. The pathology scores in these animals are comparable with their histological findings

(D). The intestinal epithelial cells from http://www.jimmunol.org/ the animals were isolated by Percoll as described in Materials and Methods. The cells were stained with E-cadherin and annexin-V/7-AAD. The cells obtained from DC-repleted/infected animals show similar percentage of apoptosis as WT/ infected or control Ab-treated and infected mice, whereas DC/depleted epithelial cells showed only basal-level apoptosis (E). The data represent mean 6 SD from three in- by guest on October 2, 2021 dependent experiments and at least 15 animals in each experiment. *p , 0.01 by ANOVA, Student t test, and Wilcoxon signed-rank test. CS, C. sakazakii.

3.5 in WT/infected, DC-repleted/infected, or control Ab-treated/ replenishment of DCs in the gut of these adoptively transferred infected animals, respectively (Fig. 11D, p , 0.001 by Wilcoxon mice based on CD11c staining (Supplemental Fig. 4A). DC- signed-rank test). Control Ab injection did not affect the course of depleted mice replenished with DCs succumbed to infection infection, and the animals histologically were similar to WT/ within 96 h postinfection similar to WT/infected mice (Fig. 11A). infected controls (Fig. 11C,11D). On par with these findings, Notably, there was no statistical difference in intestinal bacterial enterocytes isolated from DC-depleted mice showed very little load of DC-replenished and WT/infected mice (Fig. 11B). His- apoptosis (3%), whereas more than 50% of enterocytes were ap- topathological examination of intestinal tissues revealed severe optotic in WT or control Ab-treated animals as measured by villus destruction and recruitment of inflammatory cells and hence annexin-V/7-AAD staining (Fig. 11E, p , 0.001 by ANOVA). comparable severity scores as WT/infected mice (Fig. 11C,11D). We next examined the expression of iNOS and production of NO In agreement with these findings, severe apoptosis of enterocytes by enterocytes in infected mice. Enterocytes from DC-depleted was also seen in DC-replenished mice assessed by annexin-V/7- mice showed significant decrease in the expression of iNOS, AAD staining (Fig. 11E). The percentage of apoptotic cells was whereas those from WT or control Ab-treated animals showed comparable with that of WT/infected animals. Higher expression upregulation of iNOS (Supplemental Fig. 4B). In agreement with of iNOS and TGF-b production was also demonstrated in the these results, intestinal tissues of DC-depleted mice showed very intestines of DC-reconstituted mice (Supplemental Fig. 4B,4C). + low levels of TGF-b (Supplemental Fig. 4C). These findings These findings clearly indicate that OmpA C. sakazakii infection suggest that C. sakazakii exploits DCs to cause intestinal injury by and intestinal injury in animals is a result of manipulation of DC upregulating the expression of iNOS protein and local NO pro- function and recruitment by this bacterium. duction in enterocytes. To confirm the deleterious role of DCs in C. sakazakii-mediated intestinal insult, mouse DCs were adop- Discussion tively transferred to DC-depleted mice by i.p. injection before In this study, we have reported three novel observations regarding infecting with OmpA+ C. sakazakii. Flow cytometry confirmed the pathogenesis of C. sakazakii-induced NEC: 1) DC recruitment 7078 ROLE OF DENDRITIC CELLS IN C. SAKAZAKII-INDUCED NEC to LP upon infection with C. sakazakii is responsible for the in- The level of IL-10 produced by DCs upon C. sakazakii infection testinal barrier dysfunction; 2) TGF-b production by DCs en- did not affect epithelial apoptosis or barrier dysfunction. There- hances tight junction disruption and apoptosis of enterocytes; and fore, the mechanism of C. sakazakii-induced epithelial injury 3) OmpA expression in C. sakazakii is important for these path- might be distinctly tied to the expression and activity of TGF-b ological changes observed in the onset of NEC. In addition, we produced by a larger number of recruited DCs. The specific have demonstrated for the first time, to our knowledge, that C. mechanism of this interaction is currently being elucidated. In sakazakii can induce NEC in mice under normal conditions. The addition, although TGF-b can be produced by macrophages re- recruited DCs send dendrites outside the epithelium to sample cruited upon C. sakazakii infection, depletion of these cells led environmental microorganisms without compromising epithelial to a higher number of mucosal DCs being recruited upon infection barrier function. It has been shown that DCs express tight junction and an exaggerated response to C. sakazakii infection (data not molecules such as ZO-1, occludins, and claudins, thus the in- shown). Substantiating the role of DCs in C. sakazakii-induced tegrity of the epithelial barrier should be preserved due to their NEC, depletion of DCs in the newborn pups protected against recruitment (37). In contrast, our studies demonstrate that DC OmpA+ C. sakazakii-induced injury. Furthermore, adoptive recruitment disturbed the tight junctions both in the intestines of transfer of DCs in DC-depleted animals resulted in reinfection, newborn mice in vivo and in a double-layer model in vitro. demonstrating that DCs are both necessary and sufficient for C. Therefore, the disruption of tight junction integrity could be due to sakazakii-induced NEC in these animals. the interaction of C. sakazakii with DCs, which may modulate the Host–bacteria interactions play an important role in the patho- expression of tight junction molecules. Alternatively, the cyto- genesis of NEC. Bacteria-derived products activate TLR on in- kines or chemokines secreted by either epithelial cells and/or DCs testinal epithelial cells resulting in activation of the inflammatory modulate the function of the epithelial barrier. In agreement with cascade that invariably leads to NEC (47). Of note, C. sakazakii Downloaded from the latter concept, C. sakazakii interaction with DCs induced that does not express OmpA could not elicit the production of NO greater levels of cytokines such as IL-10 and TGF-b. Our data from intestinal epithelial cells despite the presence of LPS on its demonstrate that TGF-b induces tight junction disruption in the surface. Therefore, OmpA may bind a receptor on enterocytes to presence of C. sakazakii as well as apoptosis of enterocytes. Al- induce iNOS activation. It is possible that the OmpA receptor in though we did not observe bacteria traversing across the porous enterocytes could be modulated by TGF-b produced by both DCs membrane used in Transwell experiments, C. sakazakii could still and enterocytes. Because OmpA is present in all Cronobacter http://www.jimmunol.org/ modulate the function of DCs. Therefore, the interaction of C. strains, it is possible that other virulence factors such as type VI sakazakii with DC dendrites appears to be sufficient to suppress secretion pathways, a newly described mechanism for protein their activation. The presence of PMNs and macrophages does not transport across the membrane of Gram-negative bacteria that can appear to play a role in the C. sakazakii infection in vivo as our enhance the apoptotic machinery in epithelial cells, may be studies demonstrated that the absence of PMNs or macrophages expressed upon the interaction of OmpA with enterocytes (48–50). accelerates the course of infection (C.N. Emami, R. Mittal, L. Wang, In summary, we have demonstrated a novel observation that H.R. Ford, and N.V. Prasadarao, unpublished observations). This OmpA+–C. sakazakii interaction with DCs exacerbates the pro- is, to our knowledge, the first report to show that TGF-b causes duction of NO in enterocytes and induces intestinal epithelial cell by guest on October 2, 2021 damage to the epithelial barrier in the presence of a bacterial monolayer leakage and apoptosis. This interaction involves a sig- pathogen in the intestine. nificant increase in the levels of anti-inflammatory cytokines TGF- A critical mediator of the inflammatory response associated with b and IL-10. However, the presence of TGF-b seems to be re- the pathogenesis of NEC is NO (38). High quantities of NO exert quired for upregulation of iNOS and the ensuing epithelial injury. cytopathic effects on the intestine (39). Previous studies from our Identification of receptors on enterocytes to which OmpA of C. laboratory have shown that feeding C. sakazakii-contaminated sakazakii binds and/or inhibition of C. sakazakii interaction with formula to newborn rat pups causes severe pathological changes DCs can lead to the development of novel therapeutic strategies similar to NEC by inducing apoptosis of enterocytes under hyp- for preventing C. sakazakii-induced NEC. oxic conditions (18, 19). Attachment of C. sakazakii to IEC-6 cells increased iNOS expression and NO production, which is shown to Acknowledgments be responsible for causing enterocyte apoptosis. Although it was We thank Nancy Smiley for assistance in animal breeding. We also thank shown that release of bacterial LPS leads to prevention of enter- Esteban Fernandez of the Cellular Imaging Core at Children’s Hospital Los ocyte migration through activation of RhoA in a SHP-2–depen- Angeles. dent manner, we demonstrate that despite having similar amounts of LPS as those of OmpA+ C. sakazakii, OmpA2 C. sakazakii did Disclosures not induce NEC (40, 41). It is noteworthy that anti-inflammatory The authors have no financial conflicts of interest. cytokines IL-10 and TGF-b traditionally exhibit an inverse re- lationship with regard to iNOS activation (42–45). Nonetheless, References + our studies demonstrate that the supernatant from OmpA C. 1. Henry, M. C., and R. L. Moss. 2009. Necrotizing enterocolitis. Annu. Rev. Med. sakazakii/DCs, which contains a significantly high level of TGF- 60: 111–124. 2. Pellegrini, M., N. Lagrasta, C. Garcı`a Garcı`a, J. Campos Serna, E. Zicari, and b, augments NO production, which in turn induces apoptosis and G. 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