Published by the International Society of Protistologists

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Eukaryotic Microbiology

Journal of Eukaryotic Microbiology ISSN 1066-5234

ORIGINAL ARTICLE

IL-18 Cytokine Levels Modulate Innate Immune Responses and Cryptosporidiosis in Mice €rsterc & Jan R. Meada,b Brahmchetna Bedia,b, Nina N. McNairb, Irmgard Fo a Atlanta VA Medical Center, Decatur, Georgia, USA b Department of Pediatrics, Emory University, Atlanta, Georgia, USA c Immunology and Environment, Life and Medical Sciences (LIMES) Institute, University of Bonn, Bonn, Germany

Keywords Antimicrobial peptide; cryptosporidium; immunology; intestinal; opportunistic. Correspondence J.R. Mead, Atlanta VA Medical Center, 1670 Clairmont Road, Decatur, GA 30033, USA Telephone number: +1-404-321-6111, X2545; FAX number: +1-404-728-4847; e-mail: [email protected] For Vish – scientist, colleague, friend. Received: 3 April 2014; revised 9 June 2014; accepted July 11, 2014. doi:10.1111/jeu.12164

ABSTRACT IL-18 is known to play a key role limiting Cryptosporidium parvum infection. In this study, we show that IL-18 depletion in SCID mice significantly exacerbates C. parvum infection, whereas, treatment with recombinant IL-18 (rIL-18), significantly decreases the parasite load, as compared to controls. Increases in serum IFN-c levels as well as the up-regulation of the antimicrobial peptides, cathelicidin antimicrobial peptide and beta defensin 3 (Defb3) were observed in the intestinal mucosa of mice treated with rIL-18. In addition, C. parvum infection significantly increased mRNA expression levels (> 50 fold) of the alpha defensins, Defa3 and 5, respectively. Interestingly, we also found a decrease in mRNA expression of IL-33 (a recently identified cytokine in the same family as IL-18) in the small intestinal tissue from mice treated with rIL18. In comparison, the respective genes were induced by IL-18 depletion. Our findings suggest that IL-18 can mediate its protective effects via different routes such as IFN-c induction or by directly stimulating intestinal epithelial cells to increase antimicrobial activity.

CRYPTOSPORIDIUM parvum is an enteric zoonotic pathogen that infects the epithelial lining of the small intestine causing diarrheal illness in a variety of mammals, including humans. Transmission of infection usually occurs by the fecal-oral route. In healthy individuals, the infection is transient and self-limiting, whereas malnourished infants and immunocompromised hosts, such as AIDS (Acquired Immunodeficiency Syndrome) patients display a prolonged and protracted illness which can be fatal. Currently, no effective therapies are in place for the treatment of chronic disease (Bouzid et al. 2013; Current and Garcia 1991; Current and Reese 1986). Infection with C. parvum induces a local mucosal inflammatory response causing activation of the host intestinal epithelial cells (IECs) and sequestration of phagocytic cells such as macrophages (Takeuchi et al. 2008). IECs act as critical sensors of infection and provide the first line of defense by producing chemokines, antimicrobial peptides (AMPs), and proinflammatory cytokines such as IFN-c, IL-18, and IL-33 (Kagnoff and Eckmann 1997). Both IL-18 and IL-33 belong to the IL-1 family of proinflammatory cytokines. The former cytokine, IL-18 is a key inducer of IFN-c (Dinarello and Fantuzzi 2003) and is expressed by IECs, macrophages and dendritic cells in response to

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infection (Elhofy and Bost 1999; Mastroeni et al. 1999). IL18 is known to play a key role limiting C. parvum infection both in vitro (McDonald et al. 2006) and in vivo (Ehigiator et al. 2007). The latter cytokine, IL-33 has been more recently identified and is localized to epithelial barriers (Smith 2011). Like IL-18, IL-33 can act as a sensor of cellular distress and induce both Th1 and Th2 cytokines (Pastorelli et al. 2013; Schmitz et al. 2005; Smith 2011). Such cytokine-protective effects have been reported to enhance host immune responses against other parasitic organisms (Jones et al. 2010). One of the possible modes of action of IL-18 is the induction of AMPs, which are important evolutionarily conserved innate defense mechanisms (Wang and Wang 2004). Among these are the cathelicidin-related antimicrobial peptide (CRAMP) and the a- and b-defensins (Defa and Defb). While CRAMP and Defb are produced by epithelial cells, Defa is produced mainly by neutrophils and Paneth cells of the small intestine (Kahlenberg and Kaplan 2013; Niyonsaba et al. 2005). We wanted to evaluate the effect of rIL-18 and anti-IL-18 treatments on oocyst shedding, IFN-c, and AMP expression in vivo using an innate mouse model of cryptosporidiosis. It has been shown that depletion of IL-18 in vivo leads to

© 2014 The Author(s) Journal of Eukaryotic Microbiology © 2014 International Society of Protistologists Journal of Eukaryotic Microbiology 2015, 62, 44–50

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increased cryptosporidial infection intensity (Choudhry et al. 2012) and that treatment with rIL-18 of human enterocyte cell lines leads to increased expression of AMPs, cathelicidin-37 (LL-37), and human b-defensin-2 (hBD-2) (human homolog of CRAMP and Defb3, respectively) (Bals et al. 1999; Kahlenberg and Kaplan 2013; McDonald et al. 2006; Nizet et al. 2001). In addition, we also studied the effects of IL-18 depletion and rIL-18 treatment on the expression of adefensins, specifically Defa3 and 5. The expression levels of these genes were noted to be significantly higher than other defensins following infection with Toxoplasma gondii in the small intestinal tissue (Foureau et al. 2010). Moreover, increased expression levels of Defa3 and five persisted to day 5 postinfection with T. gondii, indicating that high expression levels of these genes were sustained at chronic disease states as well (Foureau et al. 2010). We sought to corroborate and extend these studies by evaluating the effect of rIL-18 and anti-IL-18 treatments on oocyst shedding, cytokine, and AMP expression in vivo using an innate mouse model of cryptosporidiosis. Infections in SCID mice are chronic, but initially there is strong resistance to infection due to an intact innate immune system (Barakat et al. 2009; Chen et al. 1993; Mead et al. 1995). In addition, we examined the relative expression of IL-33, a recently identified cytokine that may be important in mucosal response. MATERIALS AND METHODS

500 lg was determined to be the optimal concentration effective for in vivo IL-18 depletion. Mice were then injected intraperitoneally (i.p.) with 500 lg of anti-IL-18 antibody at day 1, 1, and 3. Control mice received rat IgG (Sigma, St. Louis, MO). Mice were administered rIL-18 (MBL, Woburn, MA) daily via i.p. injections at a dose of 0.5 lg. Fecal samples were collected at 7, 10, 14, and 19 d postinfection, n = 8 were used for each experimental group. ELISA Sera samples were collected from mice at 5, 7, 10, and 19 d postinfection and analyzed for the expression of IFNc by ELISA using BD OptEIA kit according to the manufacturer’s instructions (BD Biosciences, San Hose, CA). Quantitative (q)PCR Small intestine samples were harvested and snap frozen in liquid nitrogen at day 19 postinfection. The tissue was homogenized and RNA was extracted using TRIZOL based on the manufacturer’s instructions (Sigma). cDNA was synthesized and qPCR was performed using predesigned and inventoried Taqman MGB primers for the described genes; LL-37, Defb3, Defb1, Defa3, Defa5, IL-33 from Life Technologies (Carlsbad, CA). Glyceraldehyde Phosphate Dehydrogenase (GAPDH) was used as an internal control. Statistics

Animals scid

Female B6.CB17-Prkdc at 6 wk of age were obtained from The Jackson Laboratory (Bar Harbor, ME) and housed under specific pathogen-free conditions at the Veterans’ Affairs Medical Center (Decatur, GA) animal care facility. All experiments were approved by the VAMCInstitutional Animal Care and Use Committee. Preparation of C. parvum inoculum for infection The C. parvum inoculum used for this study was the IOWA bovine isolate and was kindly provided by Dr. Michael Arrowood (Centers for Disease Control and Prevention, Atlanta, GA). Oocysts were generated in neonatal bovine calves, collected in feces and purified through discontinuous sucrose and cesium chloride gradients as previously described (Arrowood et al. 1995). Oocysts were washed free of 2.5% aqueous potassium dichromate (K2Cr2O7, a storage buffer) with phosphate-buffered saline (PBS, pH 7.4) by centrifugation at 18,300 g for 3 min, 3X (Arrowood et al. 1995). The mice were infected by oral gavage using 22 gauge gavage needles. Each mouse was inoculated with 106 oocysts in 0.2 ml of PBS. IL-18 depletion and rIL-18 treatment IL-18 antibody was purified from the supernatant of hybridoma cell line SK113AE-4 (Lochner et al. 2002). Various antibody concentrations were tested prior to the study and

Data are expressed as mean  standard error. Parasite load data were analyzed by Kruskal–Wallis nonparametric test with Tukey–Kramer multiple comparisons test. Significance of parasite load over time with different treatments was established by two-way analysis of variance (ANOVA). Significance of all other studies was determined by oneway ANOVA as well as Kruskal–Wallis nonparametric test with Tukey–Kramer and Dunn’s multiple comparisons test, respectively. Data were analyzed using Prism software (GraphPad Software, Inc., La Jolla, CA). Statistical significance is indicated in the article as *p < 0.05, **p < 0.01, and ***p < 0.001. RESULTS Fecal oocyst shedding in IL-18 depleted and rIL-18-treated SCID mice To evaluate the effect of IL-18 in an innate mouse model of cryptosporidiosis, SCID mice were infected with C. parvum and treated with either irrelevant IgG, anti-IL-18 antibody, or rIL-18. Fecal samples were collected, purified, and then quantified for oocyst load (infection level) at 0, 7, 10, 14, and 19 d postinfection. We found that infections in mice treated with the irrelevant IgG (controls) increased steadily over the 19 d period, Fig. 1A. However, in IL-18 depleted mice infections were significantly higher and oocyst shedding could be

© 2014 The Author(s) Journal of Eukaryotic Microbiology © 2014 International Society of Protistologists Journal of Eukaryotic Microbiology 2015, 62, 44–50

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Figure 1 Effect of anti-IL-18 Ab and rIL-18 treatment on SCID mice infected with Cryptosporidium parvum n. sp. A. Parasite load. B. Serum IFNc levels. Fecal samples were tested for infection at day 10, 14, and 19, postinfection. Oocyst shedding began as early as 7 d in IL-18-depleted mice and were significantly higher as compared to the control group. However, rIL-18 treatment had a protective effect and prevented the parasite load from increasing over the course of the experiment and was significantly lower than the IgG control at dpi 14 and 19. Kruskal–Wallis nonparametric test and Tukey’s multiple comparison posttest was used to determine statistical significance between controls and treatment groups at each time point and two-way ANOVA was used for comparisons between time points, A. Serum IFN-c levels were quantitated at dpi 19. A significant increase was noted in IFN-c levels from mice treated with rIL-18. A small increase was also observed in the infected IL-18-depleted group. Kruskal–Wallis nonparametric test and Dunn’s test for multiple comparisons was performed on the data set, B. Data are representative of two separate experiments. Significance is indicated as (*p < 0.05, **p < 0.01), n = 8 mice/group.

detected easily as early as 7 d postinfection. In contrast, rIL-18 treatment had a protective effect and prevented the parasite load from increasing over the course of the experiment. For example, at 10 d postinfection, rIL18-treated mice were shedding < 20 oocysts/100 ll feces, while in comparison mice treated with anti-IL-18 shed significantly higher number of oocysts (> 75 oocysts/100 ll feces-over a threefold increase). By day 18

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postinfection, rIL-18-treated mice oocyst shedding remained low (< 20 oocysts/100 ll feces) while control mice (> 60 oocysts/100 ll) and mice treated with antiIL-18 (> 130 oocysts/100 ll) continued to increase. Our findings are consistent with previous findings indicating that IL-18 depletion exacerbates C. parvum infection (Choudhry et al. 2012). Data are representative of two separate experiments.

© 2014 The Author(s) Journal of Eukaryotic Microbiology © 2014 International Society of Protistologists Journal of Eukaryotic Microbiology 2015, 62, 44–50

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rIL-18 significantly induces systemic levels of IFN-c

DISCUSSION

We also compared systemic levels of IFN-c among all respective groups at 19 d postinfection. As shown in Fig. 1B, infection alone induced IFN-c levels compared to the control group. There was a slight increase in IFN-c levels in the IL-18 depleted group. However, it was not statistically significant. However, the largest increase in the systemic levels of IFN-c was observed with treatment with rIL-18. IFN-c levels were not detectable in na€ıve SCID mice as well as at earlier time points 5 and 7 d postinfection in any group (data not shown). No significant changes in the gut mRNA expression pertaining to IFN-c mRNA expression were noted with any treatment group (data not shown). Data are representative of two separate experiments.

In this study, we examined the role of IL-18 in modulating oocyst shedding and host cell immune responses in C. parvum infected SCID mice. Our findings clearly indicate that IL-18 depletion significantly exacerbated parasite load as previously reported (Choudhry et al. 2012), whereas treatment with rIL-18 significantly decreased the parasite load in the infected mice. We reported previously that treatment with rIL-18 in IL-12 KO mice, which have only partially impaired adaptive immune system and intact innate responses could result in reduction in infection (Ehigiator et al. 2005). This study as well as others indicates that IL-18 has a significant role in innate immune responses as well. IL-18 acts locally in the gut and also at a systemic level in inducing IFN-c and other immune effectors. An increase in serum IFN-c was noted in infected mice treated with either irrelevant antibody or anti-IL-18 indicating that SCID mice can mount a strong innate response based on nonT-cell sources of IFN-c in spite the absence of IL-18. NonT-cell sources include NK cells and macrophages (Takeuchi et al. 2008). Treatment with rIL-18 significantly increased serum IFN-c levels, which is indicative of a direct systemic effect of IL-18 (Dinarello and Fantuzzi 2003). Locally, IL-18 may be produced by several different cells in the intestinal tract. For example, IECs are known to produce IL-18 in response to infection (McDonald et al. 2006). Although we did not observe any differences in IFN-c mRNA expression (which may be a result of timing) in the intestinal tract, following different treatments, it is probable that IECs were a target of IL-18 cytokine as IL-18 receptor components were detectable in freshly isolated small intestinal epithelia (McDonald et al. 2006). Previous findings have shown that while human LL-37 was increased, no changes in human Defb3 expression were observed in epithelial cells in vitro in response to C. parvum infection (Zaalouk et al. 2004). We observed significant increases in mRNA expression of both the AMPs cathelicidin antimicrobial peptide (CRAMP) and defensin beta 3 (Defb3) in the small intestinal tissues in mice treated with rIL-18. This suggests that IL-18 may also mediate its protective effects by stimulating IECs to increase antimicrobial activity (McDonald et al. 2006). A positive feedback loop between the AMPs and IL-18 induction through the p38, ERK1/2 MAPK signaling pathway in the IEC microenvironment can sustain low infection levels observed in rIL-18 mice (Niyonsaba et al. 2005). AMPs such as CRAMP and Defbs have been shown to mediate their effects via Toll Like Receptor (TLR)-4 of macrophages and dendritic cells as well as IECs, respectively (Vora et al. 2004). Recently, it was shown that apical exosomes containing hBD-2 and LL37, mediated through TLR-4, were generated and released from C. parvum infected monolayer of human cholangiocytic H69 cells (Hu et al. 2013). Overexpression of hBD-2 or LL-37 in H69 cells resulted in decreased infection (Hu et al. 2013) suggesting their importance in mucosal immune responses.

Effect of IL-18 depletion and rIL-18 treatment on the expression of AMPs in the small intestine Because IL-18 has been reported to increase the expression of certain AMPs (McDonald et al. 2006), we examined the expression levels of mouse CRAMP, Defb1,3 and Defa3, 5 after infection and with the different treatment regimes. As shown in Fig. 2A, treatment with rIL-18 significantly increased mRNA expression of CRAMP, as compared to the na€ıve mice and other treatment groups. Likewise, treatment with rIL-18 significantly increased expression of the b-defensin, Defb3. Treatment with antiIL-18 antibody reduced mRNA expression of Defb3, as shown in Fig. 2B. We also observed a slight reduction in Defb3 expression with C. parvum alone. Inconsistent expression of Defb1 was observed in controls as well as other treatment groups (data not shown). We also examined the expression levels of the a-defensins (Defa3 and Defa5) which were reported to increase in response to T. gondii infection. As shown in Fig. 2C and D, infection with C. parvum significantly increased mRNA expression levels > 50 fold of both Defa3 and 5, respectively. In contrast, IL-18 depletion significantly reduced expression levels and rIL-18 treatment further down regulated expression of both genes. Data are representative of 2 separate experiments. mRNA expression of IL-33 in the small intestine after infection and after treatment with anti-IL-18 antibody or rIL-18 IL-33 is in the same family as IL-18 but has not been reported previously in cryptosporidiosis studies. We therefore examined the expression levels of IL-33 in the intestinal tract of infected mice and after treatment with either rIL-18 or anti-IL-18 antibody at 19 d postinfection. As compared to na€ıve SCID mice, the mRNA expression levels of IL-33 increased significantly with infection alone as well as with simultaneous depletion of IL-18. As described in Fig. 3, rIL-18 treatment significantly decreased the expression levels, both in relation to infection alone as well as in comparison with IL-18 depletion. Data are representative of two separate experiments.

© 2014 The Author(s) Journal of Eukaryotic Microbiology © 2014 International Society of Protistologists Journal of Eukaryotic Microbiology 2015, 62, 44–50

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Figure 2 Evaluation of gene expression of antimicrobial peptides (AMPs) in SCID mice infected with Cryptosporidium parvum, as well as treated with anti-IL-18 Ab and rIL-18. A. CRAMP. B. b-defensin 3 (Defb3). C. a-defensin 3 (Defa3). D. a-defensin 5, Defa5. A significant increase in CRAMP mRNA expression was observed in the small intestinal tissues from mice treated with rIL-18, as compared to the IL-18 depleted group, A. Kruskal–Wallis nonparametric test, and Dunn’s test for multiple comparisons was performed on the data set. A significant increase in Defb3 mRNA expression in mice treated with rIL-18, as compared to the IL-18 depleted group, whereas a decrease in expression was noted in infected mice B. A significant increase in Defa3 and Defa5 mRNA expression was observed (> 50 fold increase) in the small intestine of mice infected with C. parvum as compared with the na€ıve mice. IL-18 depletion decreased the expression levels of Defa3 and 5, whereas rIL-18 treatment further decreased expression levels of the respective genes. C, D. Data are representative of two separate experiments. One-way ANOVA and Tukey’s test for multiple comparisons was performed on the data set. Significance is indicated as *p < 0.05, **p < 0.01, ***p < 0.001, n = 8 mice/group.

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© 2014 The Author(s) Journal of Eukaryotic Microbiology © 2014 International Society of Protistologists Journal of Eukaryotic Microbiology 2015, 62, 44–50

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Figure 3 IL-33 mRNA expression in the intestines of infected SCID mice treated with anti-IL-18 Ab and rIL18. A significant decrease in IL33 mRNA expression was observed in the small intestinal tissues from mice treated with rIL-18, as compared to the infected Controls and IL-18 depleted (n = 8 for each treatment group).

The a-defensins 3 and 5 are produced by the IECs as well as Paneth cells. We found marked increases in these peptides following C. parvum infection. Similarly, increases in a-defensins 3 and 5 mRNA expression levels were reported in mice following oral infection with T. gondii and interestingly were found to be regulated in a TLR-9 dependent manner (Foureau et al. 2010). In a contrasting study, decreases in a-defensins following T. gondii infections were attributed to a loss of Paneth cells after an excessive proinflammatory response (Raetz et al. 2013). In our study, treatment with either rIL-18 or anti-IL-18 was accompanied by decreased expression of a-defensins 3 and 5 relative to control mice. Since rIL-18 treatment had a protective effect, it is likely that the suppressed infection levels induced low levels of a-defensins. It is less clear why antiIL-18-treated mice had decreased levels of a-defensins. It is possible that increased infection due to anti-IL-18 treatment resulted in Paneth cell loss and subsequent decrease in a-defensin expression. However, we consider this unlikely since infection and subsequent inflammation is still relatively low at the earlier stages (3 wk postinfection) in this chronic model. More likely, other immunological parameters may have been affected resulting in the suppression of these AMPs. Lastly, we examined the expression levels of the cytokine IL-33. IL-33 is important in the innate mucosal immunity in the lungs and gut and is also an amplifier of mucosal and systemic innate immune responses. Experimental evidence

based on in vitro studies has shown that when cells are damaged by chemical, mechanical as well as infectious agents, IL-33 is released as a danger signal (Smith 2011). We found a significant decrease in mRNA expression of IL-33 and caspase-1 (data not shown) in the small intestine of mice treated with rIL-18, which probably corresponds to a decrease in infection and possibly less tissue damage. On one hand, IL-33 was increased in the anti-IL-18-treated mice (which experience exacerbated oocyst shedding in SCID mice), possibly increasing inflammation and more cell and tissue damage in the intestine. In summary, our findings confirm the importance of IL-18 in immune responses to cryptosporidiosis and as a novel approach to limiting C. parvum infection. It has been shown that anti-Cryptosporidium antibodies fused to LL-37, resulted in a decrease in infection (Imboden et al. 2010). It may be possible that rIL-18 could also be used at an early stage to increase effector molecules such as certain AMPs (e.g. CRAMP and Defb3) and to impede the progression of cryptosporidiosis to a chronic infectious state. ACKNOWLEDGMENTS This study is supported in part by the Department of Veteran’s Affairs (VA Merit Program, grant # BX000983). We thank Mr. Syed Rizvi for excellent technical assistance. We thank Dr. Michael Arrowood (CDC) for the provision C. parvum oocysts for in vitro and in vivo studies.

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© 2014 The Author(s) Journal of Eukaryotic Microbiology © 2014 International Society of Protistologists Journal of Eukaryotic Microbiology 2015, 62, 44–50

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IL-18 cytokine levels modulate innate immune responses and cryptosporidiosis in mice.

IL-18 is known to play a key role limiting Cryptosporidium parvum infection. In this study, we show that IL-18 depletion in SCID mice significantly ex...
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