Recombinant interleukin-4 enhances Campylobacter jejuni invasion of intestinal pig epithelial cells (IPEC-1)

Microbial Pathogenesis 47 (2009) 38–46

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Recombinant interleukin-4 enhances Campylobacter jejuni invasion of intestinal pig epithelial cells (IPEC-1) G. Parthasarathy 1, L.S. Mansfield* Comparative Enteric Diseases Laboratory, National Food Safety and Toxicology Center, Department of Microbiology and Molecular Genetics, 181 Food Safety Building, Michigan State University, East Lansing, MI 48824, USA

a r t i c l e i n f o

a b s t r a c t

Article history: Received 23 October 2008 Received in revised form 10 March 2009 Accepted 21 April 2009 Available online 3 May 2009

Campylobacter jejuni, a leading cause of bacterial gastroenteritis, has a diverse spectrum of disease expression. Polymicrobial infections may contribute to this, such as Trichuris, which elicits type 2 cytokines (including IL-4) and downregulates type 1 immunity. In previous studies, gnotobiotic piglets infected with C. jejuni and Trichuris suis had bloody diarrhea and marked gastrointestinal pathology, including bacterial invasion into epithelial cells and macrophages. Neonatal swine given these dual infections had elevated IL-4 and IL-10 responses in feces. In the studies reported here, we hypothesized that IL-4 or IL-10 enhances invasion of intestinal pig epithelial cells (IPEC-1) by C. jejuni. 10–14-day-old IPEC-1 cells were pretreated with recombinant IL-4 (rIL-4) or rIL-10 for 5 h and then challenged with C. jejuni. Cells pretreated with rIL-4 were viable and showed approximately 6-fold increases in C. jejuni (but not Escherichia coli DH5a) internalization compared to cells with no pretreatment. Enhanced C. jejuni invasion was rIL-4 dose-dependent and reversed by addition of anti-IL-4 antibody. Preincubation with rIL-10 did not significantly alter C. jejuni internalization. Transepithelial electrical resistance (TEER) was significantly reduced following rIL-4 treatment, but not rIL-10 treatment. After rIL-4 pretreatment and C. jejuni challenge, light microscopy showed vacuolated cells with damaged paracellular junctions. Transmission electron microscopy (TEM) showed multiple internalized bacteria. Most were in the cytoplasm, but some were within or adjacent to vacuoles. We conclude that rIL-4 damages paracellular junctions and alters the physiology of these epithelial cells allowing increased invasion of C. jejuni. Ó 2009 Published by Elsevier Ltd.

Keywords: Interleukin-4 (IL-4) Campylobacter jejuni Epithelial invasion Intestinal Pig Epithelial Cells (IPEC-1) Decreased mucosal barrier function

1. Introduction In the US, diarrheal disease due to Campylobacter jejuni is more prevalent than diarrheal disease due to Salmonella, Shigella, and Escherichia coli O157:H7, with approximately 2.4 million cases annually [1,2]. Campylobacteriosis caused by C. jejuni and Campylobacter coli is a serious illness in humans characterized by a wide range of symptoms including fever, vomiting, abdominal cramps, acute diarrhea with leukocytes and blood in stools and/or non-inflammatory secretory diarrhea [3,4]. Infection with C. jejuni is usually confined to the lower gastrointestinal tract but systemic disease may follow particularly in immunocompromised individuals [3]. Colonic biopsies from infected patients show an acute

Abbreviations: IPEC-1, intestinal pig epithelial cells; rIL-4, recombinant IL-4; rIL10, recombinant IL-10; TEER, transepithelial electrical resistance; TEM, transmission electron microscopy; CM, complete medium. * Corresponding author. Tel.: þ1 517 884 2027; fax: þ1 517 432 2310. E-mail address: mansfi[email protected] (L.S. Mansfield). 1 Present address: 1550, Orleans St, CRBII, Room 176, Johns Hopkins University, Baltimore, MD 21231, USA. 0882-4010/$ – see front matter Ó 2009 Published by Elsevier Ltd. doi:10.1016/j.micpath.2009.04.011

inflammatory response with infiltration of the epithelium and lamina propria with neutrophils and mononuclear cells [5]. Invasive strains of C. jejuni were reported to be more commonly associated with inflammatory diarrhea while non-invasive strains were associated with secretory diarrhea [5,6]. C. jejuni infection with or without diarrheal disease is a documented precondition for autoimmunity leading to several disorders including Guillain Barre´ Syndrome, which occurs in approximately 1/1000 of infected individuals [7]. In animal disease models, where extensive histological studies are possible, C. jejuni has been shown to colonize the mucus overlying the epithelium, mainly in the colonic crypts [8– 11]. Colonic tissues from experimentally infected colostrumdeprived piglets that developed diarrheal disease after C. jejuni inoculation showed ulcerative colitis with denuded villus tips as well as infiltration of the lamina propria and submucosa with neutrophils along with a neutrophilic exudate [8]. Likewise, Macaca nemestrina infected with C. jejuni had bloody diarrhea and colonic pathology with edema, diffuse infiltration of neutrophils, plasma cells, and lymphocytes in the lamina propria and dilated lymphatics with mild to moderate diffuse and perilymphatic infiltration of mononuclear cells in submucosal tissues [11]. Clinical signs and

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pathological lesions produced in C57BL/6 IL-10/ mice mimicked those observed in humans and these other large-animal models with enteritis due to C. jejuni [9,12]. Infected mice developed severe typhlocolitis with mononuclear and neutrophilic exudates in the lamina propria and submucosa and prominent neutrophilic exudates. Challenge with certain pathogenic C. jejuni strains produced marked bloody diarrhea and immunohistochemistry showed the organism in epithelial cells and within the lamina propria. Similar lesions observed in several infected hosts with severe disease demonstrate that invasion of the colonic epithelium is an important part of C. jejuni pathogenesis [13,14]. The pathogenic mechanisms by which Campylobacter species cause disease are under intense scrutiny. Some virulence mechanisms identified include motility, secreted toxins, adhesins, and invasion [13]. Studies have shown that C. jejuni disrupts the absorptive capacity of epithelial cells by the effect of toxins [15,16], by the secretion of virulence proteins through flagella [17,18], through induction of an inflammatory response [19,20] and by invasion [10,12,21–23]. Flagellated, motile strains are more invasive than non-flagellated, non-motile strains that is, in part, regulated by the cheY gene [24]. C. jejuni invasion has been determined to occur by a microtubule-dependent, actin-filament-independent mechanism [25–27]. A C. jejuni invasion antigen (CiaB) has been identified that is secreted into host cells through flagellar Type III secretion machinery that triggers signal transduction events, resulting in Campylobacter uptake [18]. Additionally, cell motility, host–cell interactions, and competence for DNA uptake are affected by production of variant glycolipids and glycoproteins by C. jejuni [28–30]. Despite these advances, more work is needed to understand fully the host factors contributing to C. jejuni invasion. Infection with the helminth Trichuris, which produces robust T helper 2 immune responses, has been associated with exacerbation of campylobacteriosis [10,31–33]. In a swine disease model, C. jejuni acted synergistically with Trichuris suis in the colon of gnotobiotic piglets to produce bloody diarrhea and lesions including infiltration of mononuclear cells and neutrophils, excess mucus secretion, and C. jejuni invasion into epithelial cells and macrophages within the lamina propria similar to campylobacteriosis in humans [10]. Invasion into epithelial cells occurred in undifferentiated crypt and villus tip cells flanking worms in the proximal colon and also in the differentiated follicle associated epithelium of secondary lymphoid structures including Peyer’s patches and lymphoglandular complexes [10,34]. In dually infected pigs, C. jejuni was visualized adherent to epithelial cells, between epithelial cells in the paracellular junctions, at the basolateral surface of epithelial cells, and within and among cells of the underlying lymphoid follicles [10]. In this study, pigs infected with either C. jejuni (1 106 colony forming units [CFU]) or T. suis (3000 infective larvae) alone showed few clinical signs of disease and minimal, more focal pathological lesions. In similar studies with conventionally reared, neonatal piglets, animals that received T. suis (2500 infective larvae) followed 21 days later by C. jejuni (2  108 CFU) had diarrhea, similar lesions and enhanced IL-4 and IL-10 cytokine secretion in feces compared to uninfected controls (Parthasarathy et al., unpublished data). IL-4 and IL-10 are anti-inflammatory cytokines commonly induced during helminth infections. IL-4 has been shown to downregulate IL-1a, IL-1b, TNFa, and nitric oxide in vitro [35–37]. Most importantly, it has also been shown to play a critical role in the enteropathology associated with Trichinella spiralis infection in mice [38], asthma in humans [39], T-cell mediated hepatitis [40] and other diseases [41]. IL-10 is an important pleuripotent immunoregulatory cytokine [42,43]. It stimulates the formation of antigen specific T regulatory (Treg) clones, limits and terminates inflammatory responses and regulates differentiation and proliferation of several immune cells including T cells, B cells, natural

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killer cells, antigen-presenting cells, mast cells, and granulocytes [42,43]. Recently, it has been shown to play a role in early clearance of some pathogens by mediating immunostimulatory activities [42,43]. IL-10 is critical for host resistance and survival during gastrointestinal helminth infections [44]. In this study, we hypothesized that IL-4 or IL-10 induced in response to dual infection enhances differentiated epithelial cell invasion by C. jejuni, which in turn results in pathology. To test this hypothesis, differentiated IPEC-1 cells derived from a neonatal piglet [45] were used to determine the effect of recombinant IL-4 and IL-10 (rIL-4 and rIL-10 respectively) on C. jejuni invasion. In the differentiated state, IPEC-1 cells have a significant microvillus brush border, exhibit distinct apical and basolateral surfaces, and have tight junctions resulting in high electrical resistance across monolayers. The data from this study suggest that sustained physiological levels of IL-4 are likely to play a dominant role in exacerbating pathology in T. suis and C. jejuni infected swine. IL-4 is produced in response to helminths like Trichuris [33] and other pathogens [46]; hence, concomitant infection with a helminth parasite may enhance invasion of the intestinal mucosa by some bacteria. These data suggest that this as a common mucosal mechanism for enhancement of bacterial invasion where enteric helminths are present. 2. Results 2.1. rIL-4 pretreatment increases invasion by C. jejuni rIL-4 pretreatment of IPEC-1 cells caused a significant increase in the number of C. jejuni invading IPEC-1 cells (Fig. 1A). There was a 6–10-fold increase in the number of bacteria that invaded compared to IPEC-1 cells pretreated with medium alone (p < 0.05). Both C. jejuni strains invaded IPEC-1 cells efficiently. There were no statistically significant differences between these strains in their ability to invade based on comparisons of number of intracellular bacteria. rIL-10 pretreatment had no effect on invasion of C. jejuni (Fig. 1A). To determine if rIL-4 treatment was necessary and sufficient for increased invasion of IPEC-1 cells by C. jejuni, cells were incubated with rIL-4 and anti-IL-4 antibody (Fig. 1B). When IPEC-1 cells were pretreated with a mixture of rIL-4 and anti-IL-4 antibody, the number of bacteria that invaded was similar to control levels, while pretreatment with rIL-4 alone showed a significant increase in invasion (p < 0.01). The same results were seen for both C. jejuni strains tested. Pretreatment with anti-IL-4 antibody alone had no effect on invasion (data not shown). rIL-4 pretreatment had a dose-dependent effect on C. jejuni invasion (Fig. 2A). There was a progressive increase in the number of intracellular bacteria with increasing dose of rIL-4 pretreatment. Regression analysis showed a significant dose response effect for both C. jejuni strains (C. jejuni 33292 and C. jejuni 81-176 both at p < 0.05). Increases in bacterial invasion were not observed until 5 h after rIL-4 treatment and were not seen for rIL-10 at any time (Fig. 2B). Transmission electron micrographs of IPEC-1 cells showed bacteria inside cells following rIL-4 pretreatment and gentamicin killing assays (Fig. 3A), but not after medium pretreatment (Fig. 3B). The bacteria were seen predominantly in the cytoplasm just under the plasma membrane or adjacent to vacuoles (Fig. 3). This was the case in almost all the w30 fields analyzed. Bacteria were seen largely near the apical surface of the IPEC-1 cells (Fig. 3A) with a few on the basolateral side (data not shown), and in w93% (28/30) of the cases were free in the cytoplasm. The electron micrographic appearance of intracellular C. jejuni in this study was similar to that observed by Kiehlbauch et al. [47] and showed dense bacterial

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Pretreatment of IPEC-1 for 5 hr Fig. 1. Treatment of IPEC-1 cells with rIL-4 and rIl-10. Results for C. jejuni strain 33292 appear as white bars and for strain 81-176 as black bars. (A) Effect of 5 h pretreatment with rIL-4 (500 pg/well) and rIL-10 (1000 pg/well) on C. jejuni invasion of IPEC-1 cells. Results shown are representative of five to seven independent experiments. All treatments were performed in triplicate for each experiment. Values represent the mean  SEM. Asterisk (*) indicates values significantly different at p < 0.05 when experimental values were compared to medium controls based on Student’s t-test. (B) Effect of anti-rIL-4 antibody on invasion of C. jejuni into rIL-4-treated IPEC-1 cells. 12day-old IPEC-1 cells on transwells were treated with rIL-4 (500 pg/ml), rIL-4 (500 pg/ ml) plus anti-IL-4 antibody (100 ml) or medium alone for 5 h. Gentamicin assay was carried out as described previously. All treatments were performed in triplicate. Results shown are representative of two identical, independent experiments. Values represent the mean  SEM. Asterisk (**) indicates values significantly different at p < 0.01 when experimental values were compared to medium controls using Student’s t-test.

cytoplasm surrounded by an envelope. Coccoid structures also were seen, and were similar to those seen by Mansfield et al. [10] in C. jejuni challenged pigs.

2.2. Bacterial cells contribute to the invasion process To determine if rIL-4 facilitated uptake without active participation by the bacteria, a non-invasive strain of E. coli (DH5a) was used in the invasion assay, both with and without rIL-4 pretreatment of IPEC-1 cells (Fig. 4A). Unlike its effect on C. jejuni, rIL-4 pretreatment had no effect on uptake of E. coli DH5a (p > 0.05). The effect of rIL-4 on C. jejuni adherence to IPEC-1 cells was different from that seen for invasion (Fig. 4B). C. jejuni strain 81-176 adhered equally to IPEC-1 cells with or without pretreatment with rIL-4. The other two bacteria, C. jejuni 33292 and E. coli O157:H43 DEC7A, a pathogenic pig isolate with adherent capabilities, showed

Simultaneous treatment with IL-4 for 3 hr

Simultaneous treatment with IL-10 for 3 hr

Fig. 2. Effects of rIL-4 on C. jejuni invasion of IPEC-1 cells: dose response and time course. Results for C. jejuni strain 33292 appear as white bars and for strain 81-176 as black bars. (A) Dose-dependent effect of rIL-4 on C. jejuni invasion into epithelial cells. The IPEC-1 cells were treated with 100 pg/well, 200 pg/well, 300 pg/well, 400 pg/well and 500 pg/well of rIL-4 for 5 h. Medium alone treatment was used as a control. Gentamicin assay was carried out as described in Materials and methods. All treatments were performed in triplicate. Values for number of intracellular bacteria resulting from each dose were compared using regression analysis. Numbers of intracellular bacteria were proportional to the dose of rIL-4 (p < 0.05). Results shown are representative of two identical experiments. (B) Time dependent effect of rIL-4 on C. jejuni invasion into epithelial cells. The IPEC-1 cells were treated with concurrent addition of bacteria with either 500 pg/well of rIL-4 or 1000 pg/well of IL-10 for 3 h. Medium alone treatment was used as a control. At 3 h time points bacteria were added simultaneously with the cytokines. Gentamicin assay was carried out as described in Materials and methods. All treatments were performed in triplicate. Values for number of intracellular bacteria represent the mean  SEM. Values were not significantly different at p < 0.05 when experimental values were compared to medium controls based on Student’s t-test. Increases in bacterial invasion were not observed until 5 h after rIL-4 pretreatment as seen in Fig. 1A. Increases in bacterial invasion were not observed after rIL-10 pretreatment at any time examined.

a trend toward a decrease in adherence after pretreatment of IPEC1 cells with rIL-4. However, with C. jejuni 33292, most of the cellassociated bacteria were internal, as confirmed by concurrent invasion assay (data not shown). Based on data from the invasion assays and adherence assays, the fraction of C. jejuni cells that invaded expressed as a percentage of total cell-associated bacteria that adhered were w0.2% (strain 81-176) and 0.06% (strain 33292) for medium pretreatment, and 0.8% (strain 81-176) and 1.06% (strain 33292) for rIL-4 pretreatment. 2.3. Epithelial cells are viable, but altered by rIL-4 treatment 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) treatment assays showed that these rIL-4 pretreated cells were viable, and that their viability was not significantly different from that of cells treated with medium alone (Fig. 5A). Also, translocation experiments were performed to determine if there

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Fig. 3. Transmission electron micrographs of IPEC-1 cells pretreated with rIL-4 (A) or medium (B), followed by gentamicin killing invasion assays with C. jejuni strain 33292. Multiple bacteria were seen just beneath the plasma membrane (A, arrows) following rIL-4 pretreatment but not in the medium treated cells (B). Bars represent 1 mm.

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Bacterial Strains Fig. 4. Differential effect of rIL-4 on E. coli DH5a and C. jejuni invasion of and adherence to IPEC-1 cells. (A) Invasion: white bars represent the number of internalized bacteria after pretreatment with media alone for 5 h. Black bars represent the number of internalized bacteria after pretreatment with rIL-4 (500 pg/well) for 5 h. Gentamicin killing assays were carried out as described in Materials and methods. All treatments were performed in triplicate. Results are representative of two identical experiments. Values for internalized bacteria represent the mean  SEM. Asterisk (*) indicates values significantly different at p < 0.05 when experimental values were compared to medium controls using Student’s t-test. (B) Adherence: white bars represent the number of adherent bacteria after no pretreatment. Black bars represent the number of adherent bacteria after pretreatment with rIL-4 (500 pg/well) for 5 h. Adherence assays were carried out as described in Materials and methods. Values of adherent bacteria shown are mean  SEM. Results are representative of two identical, independent experiments. Asterisks (**) and (***) indicate values significantly different at p < 0.01 and p < 0.001, respectively, when experimental values were compared to medium controls using Student’s t-test. C. jejuni 33292 (and E. coli 0157:H43 DEC7A) was not facilitated to adhere based on rIL-4 pretreatment of IPEC-1 cells. However, the Campylobacter strains were internalized at higher levels due to rIL-4 pretreatment.

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Fig. 5. Effects of rIL-4 on cell viability and integrity. (A) Viability: MTT assays to assess cell viability after rIL-4 treatment and medium treatment. Assays were carried out as described in Materials and methods. Values represent the mean  SEM. All treatments were in triplicate and are representative of two independent experiments. Viability in the rIL-4-treated IPEC-1 cells was not significantly different from that of the medium alone treated cells when compared using Student’s t-test. (B) Transepithelial electrical resistance (TEER) across 12-day-old IPEC-1 monolayers before and after treatment with rIL-4, rIL-10, or medium for 5 h. All treatments were in triplicate. Results shown are representative of 3–4 experiments. Values represent mean  SEM. Asterisk (**) indicates values significantly different at p < 0.01 when experimental values were compared to medium controls using Student’s t-test.

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was an increase in the traffic of C. jejuni to the basolateral chamber after rIL-4 pretreatment. Based on CFU observed after culture, the number of bacteria translocated to the basolateral chamber after rIL-4 pretreatment (500 pg/well) for 5 h was only 5 versus none observed in the medium alone pretreated cells (data not shown). In replicate experiments, numbers of C. jejuni transcytosing cells were low with no conclusive difference between groups. rIL-4 treatment had a significant effect on the transelectrical epithelial resistance (TEER) across the IPEC-1 monolayer (Fig. 5B). TEER fell to w50% of its initial values after treatment with rIL-4 (p < 0.01) while cells treated with rIL-10 and medium alone showed no significant decrease in TEER. An SEM image of untreated 12-day-old IPEC-1 cells grown on transwells showed that cells were confluent, and exhibited distinct microvilli on their apical surfaces, typical of differentiated cells (Fig. 6A, 1 and 2. The TEER values for these cells were in the range of w600–800 U cm2). Damage to paracellular junctions after treatment with rIL-4 for 5 h was also corroborated by light microscopy, which showed that IPEC-1 cells no longer adjoined (Fig. 6B, panel C), unlike cells treated with medium alone, which were confluent and closely packed (Fig. 6B, panels A and B). Increased vacuolation also was seen in the cells that received rIL-4 treatment compared to cells that were treated with medium alone (Fig. 6B, panel C). The presence of vacuoles in rIL-4-treated cells was not due to toxicity. 3. Discussion In this study, pretreatment of a differentiated epithelial monolayer with rIL-4 significantly increased invasion by pathogenic strains of C. jejuni compared to those treated with rIL-10 or medium only. Studies to date on host factors that mediate C. jejuni invasion of epithelial cells include signal transduction pathways [48] and host cell surface receptors [49,50] required for binding. Here, we show that a soluble factor produced by host cells can also play a role in C. jejuni invasion. In the swine intestinal epithelial cells used in our study, rIL-4 caused a 6-fold or greater increase in invasion of both pathogenic strains of C. jejuni tested. This effect was seen only when IPEC-1 cells were treated with rIL-4 for more than 3 h; simultaneous addition of both rIL-4 and C. jejuni onto IPEC-1 cells did not enhance invasion even though bacteria were viable (data not shown). This rIL-4-mediated enhancement of bacterial invasion was dose-dependent; increasing concentrations of rIL-4 led to increased internalization of bacteria. The concentrations used in this study were based on the concentrations obtained from feces of

T. suis and C. jejuni infected swine (Parthasarathy and Mansfield unpublished data). The doses were then adjusted conservatively to reflect appropriate concentrations expected locally in the intestinal tract. rIL-10 did not affect C. jejuni invasion of IPEC-1; this fact, and the fact that antibody directed against rIL-4 had a neutralizing effect on rIL-4 enhanced invasion of the C. jejuni strains, demonstrated that the effect was specific to rIL-4. Additional pretreatment studies with other proinflammatory cytokines (IL-1b, IL-8, TNF-a) also showed negative results further supporting this conclusion (data not shown). The results from this study indicate a role for sustained physiological levels of IL-4 in the gut in initiating enhanced invasion by C. jejuni and the subsequent pathological changes in the gastrointestinal epithelium and underlying tissues. Expulsion of the gastrointestinal nematode Trichuris and other GI helminths is mediated by a T helper 2 response involving IL-4 and IL-13, suggesting this as a common mucosal mechanism for enhancement of bacterial invasion. Increased production of these cytokines causes exaggerated goblet cell hyperplasia, mastocytosis and leakiness in the gut leading to expulsion [51,52]. Studies on other disease processes such as asthma in humans [39], T-cell mediated hepatitis [40], and schistosomiasis in murine models [41] provide increasing evidence that IL-4 plays a critical role in development of epithelial pathology. In this study, results support a role for the bacteria in contributing to this rIL-4 facilitated invasion process. The effect of rIL-4 on invasion was observed only with pathogenic isolates of C. jejuni (81-176 and 33292) and not with a laboratory selected, nonpathogenic, non-invasive bacterium like E. coli DH5a. This is not surprising because motility and the presence of functional flagella have been shown to be essential in C. jejuni invasion [53]. Additionally, flagella have been shown to mediate the initial interaction with epithelial cells [54] and are implicated as the agent of transfer for Cia antigens that initiate signal transduction events leading to C. jejuni uptake [18]. In live hosts, we suspect that IL-4 facilitates only certain invasive bacteria and does not influence uptake of noninvasive bacterial commensals. However, more pathogenic and non-pathogenic isolates of enteric bacteria should be tested in challenge studies on differentiated IPEC-1 monolayers before this conclusion can be drawn. These studies should include invasive and non-invasive strains, especially C. jejuni strains that lack essential invasion attributes due to targeted gene knockouts. Also, one must take into account the specific parameters of this IL-4 effect. For example, Hess et al. [55] found no effect of IL-4 pretreatment on internalization of Listeria monocytogenes or Salmonella typhimurium

Fig. 6. IPEC-1 cell microscopy. (6A) Scanning electron micrograph of polarized IPEC-1 cells on transwell membranes. (1) Left panel (a) is overview of cells; bar represents 20 mm. Cells form a continuous monolayer. (b) The apical finger-like microvilli are seen in the right panel; bar represents 1 mm. Micrographs are representative of three samples each. (6B) Light microscopy of polarized IPEC-1 cells on transwell membranes as seen using laser scanning microscopy at 63 aperture through the basolateral surface. (A) Cells before treatment, in medium. (B) Cells after treatment with fresh medium for 5 h. (C) Cells after treatment with rIL-4 (500 pg/well) for 5 h. Bars represent 5 mm. Micrographs are representative of two to three samples analyzed for each panel.

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Fig. 6. (continued).

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into differentiated Caco-2 cells or HT-29 cells. In their study, cells were pretreated with IL-4 for 48 h or 72 h, while the events we describe occurred much earlier. Also, the different results described by Hess et al. could be due to variation in susceptibility of these different cell-lines or to the different bacteria used, or to both [55]. Cytoskeletal changes were apparent in rIL-4-treated IPEC-1 cells challenged with C. jejuni. IPEC-1 is a non-transformed cell-line that when cultured according to this protocol differentiates into polarized monolayers with a microvillus brush border. These cells have tight junctions as evidenced by scanning electron and light microscopy and a high TEER. However, rIL-4 pretreated, challenged cells adopted a spherical shape with vacuolization and wide, irregular paracellular junctions compared to the hexagonal, closely packed shape of the control cells. MTT assays to assess cytotoxicity showed that this increased vacuolation after rIL-4 treatment and C. jejuni challenge was not due to a cytotoxic effect on IPEC-1 cells because there was no significant decrease in viability compared to medium alone treated cells. Based on MTT values these cells were fully viable, but drops in TEER values indicate that junctional complexes were affected. In previous studies with the nematode parasite, Heligmosomoides polygyrus, Shea-Donohue et al. [56] demonstrated increased permeability in mouse intestinal epithelial cells treated with IL-4. They suggested that the classical ‘‘weep and sweep’’ response to worms may be mediated by IL-4 resulting in loosening of junctions between cells so that the worms are no longer tightly embedded and hence can be washed away by the intestinal fluids. Invasion through the paracellular route has been hypothesized as a major component of the pathogenesis of C. jejuni [57]; Bra´s and Ketley suggested a transcellular route in polarized Caco-2 cells [58]. Based on translocation experiments on T84 epithelial monolayers, Monteville and Konkel suggested that C. jejuni invades through the basolateral surface of these cells [49]. In our studies, TEM analysis showed more bacteria just under the apical surface, suggesting that invasion occurred largely through apical surfaces rather than through basolateral surfaces in rIL-4 pretreated IPEC-1 cells. Also, after a 3 h challenge, there was no significant increase in translocation of C. jejuni across IPEC-1 cells to the lower Transwell chamber in pretreated versus medium-only controls suggesting that the apical invasion route predominated. However, nontransformed IPEC-1 cells from swine and T84 cells from humans are different. Also, it is possible that the bacteria may have penetrated the cell basolaterally before reaching the lower chamber and went undetected. It is unlikely that the Transwell membrane impedes C. jejuni entering the lower chamber because Transwells with a 3 mm pore size have been used previously to study C. jejuni transcytosis [58]. It is likely that the impact of this question could be better addressed in vivo in murine models treated with increasing levels of rIL-4 followed by C. jejuni challenge. Finally, in other studies, C. jejuni has been reported to reside intracellularly within vacuoles [59]. In our experiments, although increased vacuolation was seen within the IPEC-1 cells after rIL-4 pretreatment, more bacteria were seen in the cytoplasm and adjacent to vacuoles than within them. However, it is possible that C. jejuni might have escaped the vacuoles, since TEM was not conducted at earlier time points. IL-4 is an anti-inflammatory cytokine strongly induced in response to most helminth infections [60]. Although proinflammatory cytokines like TNFa and IFNg have been traditionally associated with inflammation and pathology [61–63], the role of IL4 as a mediator of pathology has also been documented [64]. In this TCR/ murine model, transferred IFNg/RBHi T cells lead to villus atrophy, goblet cell metaplasia, and a protein losing enteropathy attributable to IL-4 [64]. Previous studies have shown that IL-4 can downregulate TNF-a expression in porcine macrophages by 6 h after exposure and in murine mast cells by 24 h after exposure

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[65,66]. IL-4-mediated downregulation of TNF-a in the mast cells was through STAT-6, a transcriptional activator, and occurred by destabilization of TNF-a mRNA. Recent studies show that alteration of intestinal barrier permeability is also mediated by STAT-6 [67]. In addition, it is generally accepted that STAT-6 activation occurs through IL-4 receptors [64]. Therefore, we propose the following model for the mechanism by which rIL-4 causes enhanced invasion of C. jejuni. rIL-4 binds to a receptor, presumably IL-4Ra, and causes intracellular signal transduction events leading to increased vacuolation and perhaps downregulation of TNF-a or other proinflammatory cytokines, via STAT-6. These events are accompanied by changes to extracellular surfaces marked by breakdown of tight junctions (and differential expression or exposure of C. jejuni host receptors such as fibronectin), facilitating increased invasion by C. jejuni. It should be noted that the IL-4 receptor has a fibronectin domain, which may provide another functional receptor on the apical surface when IL-4 is bound. Further studies are needed to address these hypotheses. Other mechanisms may operate in this model. In studies by Hu and Kopecko C. jejuni invasion has been shown to be a microtubuledependent, actin-filament-independent mechanism [68]. More work is needed to determine whether the IL-4 receptor is necessary and sufficient for mediating this enhanced invasion or whether IL-4 may act directly on some intracellular targets. Alternatively, IL-4 may weaken paracellular junctions by an as yet unknown mechanism leading to the observed damage to this site and possibly enhanced uptake of C. jejuni by the basolateral surface of the epithelial cell. Fully motile C. jejuni are thigmotactic and, thus, may have enhanced ability to navigate the paracellular space damaged by rIL-4. Future experiments will explore the significance of each route in the overall process. 4. Materials and methods 4.1. Bacterial strains and culture C. jejuni strains 33292 (American Type Culture Collection) and 81-176 were used for the invasion studies. Both were initially isolated from humans with enteritis. C. jejuni 81-176 was kindly donated by Dr. Carol Pickett, University of Kentucky. Low passage bacterial colonies were grown for isolation on Bolton Agar supplemented with 5% defibrinated sheep’s blood (Cleveland Scientific, Bath, OH) for a period of 48–72 h. The control strains E. coli DH5a (Life Technologies, Gibco BRL, Gaithersburg, MD) and E. coli O157:H43 DEC7A were grown similarly on nutrient agar (Difco Laboratories, Detroit, MI). An isolated colony was further grown as a lawn on corresponding medium for 20 h. DEC7A was initially isolated from swine and was a kind contribution from Dr. Thomas Whittam, Michigan State University. Bacteria were swabbed from plates and resuspended in RPMI 1640 medium supplemented with phenol red and 5% heat inactivated fetal bovine serum (Invitrogen, Rockville, MD). The optical density (OD560) was adjusted to 0.1 to achieve w108 CFU/ml. Bacterial suspensions were used immediately after preparation. 4.2. Cell culture Intestinal epithelial cells obtained from swine (IPEC-1) were used to elucidate the effect of rIL-4 (or rIL-10) on C. jejuni invasion of epithelial cells. IPEC-1 is a non-immortalized and undifferentiated neonatal small intestinal epithelial cell-line that can be induced to differentiate by growth on porous substrates (TranswellÒ plates, Corning Costar, Corning, NY) for 10–14 days [69,70]. In the differentiated state, IPEC-1 cells have a significant microvillus brush border, exhibit distinct apical and basolateral surfaces, and have tight

junctions resulting in high electrical resistance across monolayers. In previous studies IPEC-1 cells have been used up to 80 passages in in vitro assays [69]. In our study all experiments were confined to passage 54. All media and supplements for cell culture were obtained from Invitrogen, Rockville, MD, unless otherwise stated. The IPEC-1 cells were cultured routinely in DMEM/F-12 medium (Invitrogen, Rockville, MD) supplemented with 5% Fetal Bovine Serum (FBS) and 1% Insulin–Transferrin–Selenium (Invitrogen, Rockville, MD) in cell culture flasks. After 5–6 days of growth, the cells were washed in versene and trypsinized. They were then cultured at a density of 3  105/well on transwell inserts (6.5 mm diameter; 3 mm pore size) that had been previously coated with fibronectin (20 mg/ml; Sigma, St. Louis, MO). Cells were allowed to differentiate for 10–14 days in RPMI 1640 medium containing phenol red and supplemented with 5% Fetal Bovine Serum (FBS). TEER values reflect the integrity of a given monolayer and are also evidentiary of polarization of epithelial cells. TEER across the monolayer was measured to determine confluency and tight junction formation (EVOMX, World Precision Instruments, Sarasota, FL). TEER values between 150 and 600 U cm2 were considered as evidence of a confluent monolayer, and experimental values ranged between those values. 4.3. Gentamicin killing assays 10–12-day-old IPEC-1 cells on transwell membranes were treated apically with either 500 pg of rIL-4 or 1000 pg of rIL-10 in 100 ml of RPMI 1640 supplemented with 5% FBS (complete medium; CM) and incubated at 37  C, 5% CO2. 100 ml of CM alone were included as a negative control. Swine rIL-4, rIL-10, and anti-rIL-4 antibody were obtained from ELISA Kits (Biosource International, Camarillo, CA). After 5 h, the treatment solutions were removed as completely as possible. The 5-h time point was chosen based on previously published studies that determined the time course of effects [55,66]. IPEC-1 cells were not washed in order to avoid damage to the monolayers and were infected apically with 5  107 CFU of bacteria in 100 ml of CM. Invasion was allowed to proceed for 3 h at 37  C, 5% CO2. The bacterial suspension was then removed and adherent bacteria on both sides of the membrane were killed by exposure to 100 mg/ml of gentamicin (Invitrogen, Bethesda, MD) suspended in CM for 1 h. The transwell membranes were excised and the epithelial cells were lysed with a final concentration of 0.1% sodium deoxycholate (Sigma Chemical Co., St. Louis, MO) in 1 PBS. Internalized bacteria were enumerated by spreading on Bolton agar plates. All treatments were performed in triplicate. Subsequently, gentamicin killing assays also were carried out 1) to determine the effect of anti-IL-4 antibody on invasion, 2) to establish the effect of rIL-4 dose on C. jejuni internalization, 3) to establish the effect of time of rIL-4 treatment on C. jejuni internalization, and 4) to determine the effect of rIL-4 pretreatment on non-invasive E. coli DH5a. Also, the number of C. jejuni that translocated to the basolateral chamber after apical placement was determined. IPEC-1 cells were pretreated with either rIL-4 (500 pg/ well) or medium for 5 h, followed by incubation with C. jejuni 81176 for 3 h. Media in the lower chamber were sampled after the 3 h incubation with C. jejuni, plated on Bolton agar and bacterial colonies enumerated. 4.4. Scanning electron and light microscopy Scanning electron microscopy was done on 12-day IPEC-1 cells grown on transwells, to verify their state of differentiation. SEM was carried out at Center for Microscopy, Michigan State University. After 12 days of growth, media was removed and the membranes were excised and fixed in 4% glutaraldehyde in 0.1 M sodium

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phosphate buffer. The specimens were subsequently fixed in 1% osmium tetroxide and dehydrated in a graduated ethanol series. The dehydrated membranes were dried in a critical point dryer (Balzers, Lichtenstein), mounted on aluminum stubs, and sputter coated with 7 nm of gold (Emscope SC500 sputter coater, UK). Visualization of the cells was carried out with a scanning electron microscope (JEOL 6400V, Japan). For scanning light microscopy, 12-day-old IPEC-1 cells on transwells were treated with rIL-4 (500 pg/well) or medium for 5 h. Treatments were removed and the membranes were excised with a sterile scalpel and examined by laser scanning microscopy at 63 aperture (LSM Pascal 5, Zeiss International, Jana, Germany). 4.5. Adherence assays Adherence assays with C. jejuni (strains 81-176 and 33292) were carried out similarly to invasion assays. The adherent strain E. coli O157:H43 DEC 7A was included to determine the effect of rIL-4 on adherence. After pretreatment with 500 pg of rIL-4 in CM or CM alone for 5 h, the treatments were removed and the bacteria (5  107 CFU in CM) were allowed to adhere for 1 h. This time interval allows for bacteria to primarily attach to the surface of cells but not to invade. However, this does not preclude some minimal invasions. The supernatants containing bacteria that did not adhere were then removed and the transwell membranes were excised with a sterile scalpel. The epithelial cells were then lysed as described in the gentamicin assays, and the cell-associated bacteria (mostly surface associated and some that invaded) were enumerated on Bolton agar plates for C. jejuni or nutrient agar plates for E. coli. Invasion assays were conducted for the C. jejuni strains concurrently as internal controls. However, invasion assays were not done for the E. coli strain as 1) it is an adherent strain and its invasion potential is not known and 2) it is used here primarily as positive control for adherence and not an invasion control.

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CO2 in 50 ml volumes. The treatment was removed, and cells were incubated with 0.1% Triton X-100 for 10 min at 37  C and 5% CO2. 100 ml of acidic isopropanol (0.1 N hydrochloric acid in isopropanol) was then added, mixed, and absorbance measured at 570 nm. All treatments were performed in triplicate. 4.8. Data analysis Data are presented as mean  standard error of the mean (SEM). For data presented in Figs. 1A,1B,2B,4A,4B,5A and 5B Student’s t-test was used to demonstrate that the means of the two treatment groups being compared within individual experiments were different. Student’s t-test was performed using Microsoft Excel, unless otherwise stated. p values  0.05 were considered significant. A dose response experiment presented in Fig. 2A was conducted to establish a causal relationship between IL-4 pretreatment of IPEC-1 cells and enhanced invasion by C. jejuni. The independent variable was the concentration of IL-4 applied to the cultured cells; the dependent variable was the number of C. jejuni invading cells post-treatment compared to the controls. Regression analysis was performed using Microsoft Excel. In this analysis confidence was set at the 95% level. Acknowledgements This work was supported by National Institutes of Health grant AI42348-01 and by the Michigan State University Center for Emerging Infectious Diseases. Dr. Parthasarathy’s stipend during the conduct of this work was funded in part by the Michigan State University Graduate School and Michigan State University College of Veterinary Medicine funds provided as a match to federal funds from NIAID, NIH, Department of Health and Human Services, under Contract No. N01-AI-30058. We sincerely thank Dr. Julia Bell for critical review of this article and valuable advice.

4.6. Transmission electron microscopy IPEC-1 cells were subjected to invasion assays as described previously. After the gentamicin treatment, the medium was removed, and the membranes were excised and fixed in 0.1 M phosphate buffer containing 2.5% glutaraldehyde and 2.0% paraformaldehyde (pH 7.4). The membranes were washed three times in 0.1 M phosphate buffer (pH 7.4) for 20 min each. The membranes were then fixed in 2% osmium tetroxide and dehydrated in increasing concentrations of acetone. Dehydrated membranes were subsequently infiltrated with epoxy resin, embedded in silicone molds, polymerized for 2 days at 60  C, and then examined using a transmission electron microscope (JEOL 100CX, Japan). Complete gentamicin killing assays were conducted concurrently as described above as internal controls; bacteria were identified by colony morphology, microscopic examination and colonies enumerated. Seven samples each for rIL-4 pretreatment and medium pretreatment were processed for TEM. For each treatment sample 30 frames were examined at multiple magnifications. Cells were examined for morphological changes and for location of C. jejuni (whether they were adherent, intracellular, intravacuolar or intracytoplasmic). 4.7. (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay MTT assays were performed to determine if rIL-4 had any cytotoxic effects on IPEC-1 cells. Briefly, IPEC-1 cells on transwells pretreated with rIL-4 or medium were incubated with 0.5 mg/ml of MTT (suspended in RPMI medium with 5% FBS), for 3 h at 37  C, 5%

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