Protection against the Early Acute Phase of Cryptosporidium parvum Infection Conferred by Interleukin-4 Induced Expression of T Helper 1 Cytokines

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1 MAJOR ARTICLE Protection against the Early Acute Phase of Cryptosporidium parvum Infection Conferred by Interleukin-4 Induced Expression of T Helper 1 Cytokines Stuart A. C. McDonald, 1 John E. O Grady, 2 Mona Bajaj-Elliott, 1,a Clare A. Notley, 1,a James Alexander, 2 Frank Brombacher, 3 and Vincent McDonald 1 1 Department of Adult and Paediatric Gastroenterology, Digestive Diseases Research Centre, Barts and the London School of Medicine, London, and 2 Department of Immunology, University of Strathclyde, Glasgow, United Kingdom; 3 Department of Immunology, Medical School, University of Cape Town, Cape Town, South Africa Immunity to Cryptosporidium parvum infection involves a T helper (Th) 1 response with interferon (IFN) g and interleukin (IL) 12 activity, but the role of Th2 cytokines, such as IL-4, is unclear. Around the peak of infection, production of oocysts in IL-4 deficient and IL-4 receptor a deficient neonatal BALB/c mice was greater than that in wild-type (wt) mice. Susceptibility to infection was increased or decreased, respectively, in wt mice treated with anti IL-4 neutralizing antibodies or recombinant IL-4. Excretion of oocysts by IFNg deficient mice was unaffected by treatment with anti IL-4, indicating that IL-4 stimulated IFN-g activity. Early during infection, wt mice had increased intestinal expression of IFN-g and IL-12 mrna, compared with IL-4 deficient mice. Intestinal IL-4 was detected by Western blotting in wt mice 24 h after infection but not in uninfected control mice. These findings suggest that, early during C. parvum infection of BALB/c mice, there is production of IL-4 that promotes Th1-mediated immunity. Cryptosporidiosis is an infectious diarrheal disease of humans and animals that is normally transient but may be chronic and severe in immunocompromised or malnourished hosts. The agent of infection is the intracellular protozoan parasite Cryptosporidium parvum, which reproduces in the apical portion of gut epithelial cells and is transmitted in a fecal-oral manner by oocysts [1]. In the immune response to C. parvum infection, Received 23 January 2004; accepted 12 March 2004; electronically published 22 July Presented in part: Digestive Disease Week, American Gastroenterological Association, May 2003, Orlando, FL (abstract T1747). Financial support: Wellcome Trust (grants to V.M., M.B.-E., and F.B.). a Present affiliations: Department of Infectious Diseases and Microbiology, Institute of Child Health, University College London, London, United Kingdom (M.B.- E.); Sir Alexander Fleming Building, South Kensington Campus, Imperial College Medical School, London, United Kingdom (C.A.N.). Reprints or correspondence: Dr. Vincent McDonald, Barts and the London School of Medicine, Dept. of Adult and Paediatric Gastroenterology, Digestive Diseases Research Centre, Turner St., London E1 2AD, UK (v.mcdonald@qmul.ac.uk). The Journal of Infectious Diseases 2004; 190: by the Infectious Diseases Society of America. All rights reserved /2004/ $15.00 CD4 + T cells play an important protective role [2 10]. In contrast, there appears to be only a limited or no requirement for CD8 + T cells and antibody in control of infection [7 10]. Interferon (IFN) g activity is associated with a partially protective innate immunity against the parasite and with the adaptive immune response that eliminates the infection [7, 11 14]. IFN-g has been shown to inhibit reproduction of C. parvum in epithelial cell lines [15], and intraepithelial lymphocytes at the site of infection may be an important source of the cytokine [16]. Interleukin (IL) 12, which induces production of IFN-g by NK cells and T cells, also appears to be involved in immunity to C. parvum, because mice with impaired IL-12 function are more susceptible to infection [13, 17]. The requirement for both IL-12 and IFN-g in host resistance implies that a Th1 cell mediated response is important [18]. The Th2 antibody mediated response is characterized by production of IL-4, IL-5, IL-10, and IL-13 [18]; it is recognized that Th2 activity may exacerbate intra- IL-4 in Immunity to C. parvum JID 2004:190 (1 September) 1019

2 cellular infections [19, 20]. However, with C. parvum infection, there is evidence that Th2 cytokines may play a protective role. In mice, reproduction of parasites increased in adult BALB/c mice after treatment with anti IL-5 neutralizing antibodies [21]. In humans with HIV infection, increased expression of IL-4 mrna was associated with recovery from C. parvum infection after antiretroviral therapy [22]. In a study of adult C57BL/6 mice, IL-4 deficient (IL-4 / ) mice had increased levels of excretion of oocysts, but only during the recovery phase of infection [23]. Hence, both the Th1 and Th2 pathways may provide significant elements of the protective immune response against C. parvum, although possibly during different stages of the infection [23]. The present study used BALB/c mice, which, compared with C57BL/6 mice, often preferentially develop Th2 responses [19, 20] and, when infected with C. parvum, are less dependent on IFN-g for control of infection [24]. Accordingly, the hypothesis was that the investigation of C. parvum infection in BALB/c mice would reveal a novel function for IL-4 in the immune response to this parasite. The neonatal mouse model of infection was used [25], because neonatal mice are significantly more susceptible to infection than are adult mice. In the experiments performed, a study was made of the effect of IL-4 deficiency, as a result of IL-4 or IL-4 receptor a (IL-4Ra) gene deletion or treatment with anti IL-4 neutralizing antibodies, on susceptibility to C. parvum infection. In addition, intestinal expression of IL-4, IFN-g, IL-12, and IL-18 was compared in wild-type (wt) and IL-4 deficient mice during infection. The results indicate that there is a protective role for IL-4 early during infection and that one mechanism of action may involve enhancement of intestinal expression of IL-12 and IFN-g. MATERIALS AND METHODS Animals. The procedures performed with mice in the present study were licensed by the United Kingdom Home Office. The neonatal mouse model of C. parvum infection was used [25]. BALB/c IL-4Ra deficient (IL-4Ra / ) [26], IL-4 / [27], SCID [16], IFN-g deficient (IFN-g / ; GKO mice originally from Jackson Laboratory), and wt mice were bred and maintained under pathogen-free conditions. Reagents. Murine recombinant IL-4 (ril-4) was obtained in lyophilized form and was reconstituted as recommended by the supplier (Peprotech). The rat anti mouse IL-4 IgG neutralizing monoclonal antibody (MAb) 11B11 was provided by the National Cancer Institute. Other reagents were obtained from Sigma-Aldrich. Parasites and infection studies. Purified C. parvum oocysts of the Moredun (genotype 2) isolate were obtained from Moredun Scientific and were stored at 4 C. In preliminary experiments, neonatal BALB/c mice were infected with doses of oocysts, with no significant differences in either the number of shed oocysts or in the duration of infection (data not shown). Routinely, therefore, groups of mice, either 4 or 7 days of age, were infected orally with oocysts, and, on various days after infection, the level of infection was measured in 5 13 mice from each group. Colonic contents were smeared thinly onto microscope slides, dried, fixed with methanol, and acid-fast stained by use of the Ziehl-Neelsen method for detection of oocysts. The smears were examined microscopically (magnification, 1000), and the numbers of oocysts in 50 high-power fields (HPFs) were counted. RNA extraction and semiquantitative reverse-transcriptase (RT) polymerase chain reaction (PCR). RT-PCR was performed on gut tissue snap-frozen in liquid nitrogen immediately after excision. Total cellular RNA was isolated by homogenizing tissues in TRIzol (Invitrogen) and incubating them for 5 min at room temperature. RNA was extracted by adding chloroform (Sigma-Aldrich), followed by centrifugation for 15 min at 12,000 g at 4 C. RNA was then precipitated with isopropanol and centrifuged for 10 min at 12,000 g. The pellet was washed with 70% ethanol and resuspended in 50 ml of water. Total RNA concentration was determined by spectrophotometric analysis. Total RNA (1 mg) was primed with 500 ng of oligo(dt) (Amersham Biosciences) for 10 min at 70 C and then chilled on ice. This mixture was then made up to a 20-mL reaction volume containing 50 mmol/l Tris (ph 8.3), 75 mmol/l KCl, 3 mmol/l MgCl 2, 3 mmol/l dithiothreitol, 10 mmol/l dntp mix (Amersham Biosciences), and 100 U of RT (Moloney murine leukemia virus; Promega). The mixture was incubated for 45 min at 42 C, and the reaction was terminated by heat inactivation for 10 min at 70 C. PCR amplification was conducted routinely in 50-mL reaction volumes (10 mmol/l Tris [ph 9.0], 50 mmol/l KCl, 1.5 mmol/l MgCl 2, 200 mmol/l dntps, 50 pmol of 5 and 3 primers, as described elsewhere [28], and 0.5 UofTaq polymerase [Amersham Biosciences]). The reaction mixture was subjected to 35 amplification cycles (within the linear phase of PCR amplification) of denaturation for 45 s at 94 C, annealing for 45 s at 58 C, and extension for 75 s at 72 C. After amplification, PCR products were analyzed on 1% agarose gels, and bands were visualized by ethidium-bromide staining. Band intensities were quantified by densitometry (Eastman Kodak). Cytokine PCR band intensities were normalized by comparison with those of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and were expressed as the number of cytokine mrna units per 1000 GAPDH mrna units. Immunoprecipitation and Western blotting. In brief, frozen ileum was homogenized in 1 ml of protein extraction buffer (10 mmol/l Tris [ph 7.4], 1% SDS, 0.5% Triton X-100, and complete protease inhibitor cocktail [Roche]). Nonspecific binding was blocked by the addition of 0.25 mg of normal goat IgG (Sigma-Aldrich) and 20 ml of 25% (vol/vol) protein G 1020 JID 2004:190 (1 September) McDonald et al.

3 Figure 1. Comparison of excretion of oocysts in neonatal BALB/c wildtype (wt), interleukin (IL) 4 deficient (IL-4 / ), and IL-4 receptor a deficient (IL-4Ra / ) mice infected with Cryptosporidium parvum. Acid-fast stained oocysts in fecal smears were counted in 50 random high-power fields (HPFs). On day 7 after infection, wt mice produced fewer oocysts than did IL-4 / and IL4Ra / mice. On day 10 after infection, wt mice produced fewer oocysts than did IL-4Ra / mice. Data are mean SEM ( n p 12 mice/group). * P!.05; ** P!.01 (Student s t test). sepharose beads (Sigma-Aldrich) to each ileal homogenate, and then the homogenates were incubated for 30 min at 4 C while being shaken. Blocking beads were removed by centrifugation (1000 g). Protein G sepharose beads (5 ml) coated with 10 mg of goat anti mouse IL-4 IgG (Santa Cruz Biotechnology) were added to each ileal homogenate and were incubated overnight at 4 C while being shaken. Beads were collected, washed 3 times in PBS, added to boiling SDS-PAGE loading buffer (50 mmol/ L Tris [ph 6.8], 2% SDS, 0.1% bromophenol blue, 10% glycerol, and 1% b-mercaptoethanol), and boiled for a further 2 min. Immunoprecipitates were run through a 12% polyacrylamide- SDS gel and were transferred onto an epichemiluminescence (ECL) compatible nitrocellulose membrane (Amersham Biosciences). Membranes were probed with goat anti mouse IL-4 IgG (0.2 mg/ml) overnight at 4 C and then with horseradish peroxidase conjugated donkey anti goat IgG (0.1 mg/ml; Santa Cruz Biotechnology) for 1 h at room temperature. Blots were then developed using ECL reagent (Amersham Biosciences). Statistical analyses. Each experiment was performed at least twice, and data (expressed as mean SE) were analyzed by use of Student s t test. RESULTS Increased susceptibility of IL-4 5/5 and IL-4Ra 5/5 BALB/c mice to C. parvum infection. C. parvum infection in neonatal BALB/c mice, as measured by production of oocysts, shows an acute pattern [2, 29]; in our experiments, infection was measured at key points established in preliminary experiments with wt mice. In a comparison of infections in wt, IL-4 /, and IL- 4Ra / mice (figure 1), similar small numbers of oocysts were present in both wt and IL-4 / mice on day 4 after infection, when infection usually first becomes patent. At that time, the mean number of oocysts in the IL-4Ra / mice was higher than that in the other groups, but the difference was not significant. The highest levels of infection were observed on day 7 after infection, but the numbers of oocysts produced in the IL-4 / and IL-4Ra / mice were greater than those in wt mice by a factor of 3 ( P!.05 and P!.01, respectively, Student s t test). No diarrhea was observed in any group of mice at the peak of infection. By day 10 after infection, the parasite burdens had decreased, and, although oocyst counts in IL-4 / and wt mice were similar, the oocyst count in IL-4Ra / mice was still higher than that in wt mice ( P!.05). By day 14, infection was undetectable in most mice from all 3 groups, and no parasites were seen on day 21. There were no significant differences in levels of production of oocysts between IL-4 / and IL-4Ra / mice at any of these times. These results were reproducible, although, in one experiment, substantially higher numbers of oocysts were already present in IL-4 / mice, compared with wt mice, on day 4 after infection (data not shown). These findings indicate that loss of IL-4 function in neonatal BALB/ c mice allowed increased reproduction of C. parvum in the early acute phase but did not prolong the convalescent phase of infection. Protective role for IL-4 in wt mice. To further investigate the role of IL-4 in immunity, reproduction of oocysts was examined in mice administered a single subcutaneous (sc) injection of 100 mg of rat anti mouse IL-4 IgG neutralizing MAb 11B11 at the start of infection (day 0) or on day 4 after infection. Previous studies have shown that a single dose of this antibody at this concentration per kilogram of body weight has Figure 2. Effect of treatment with interleukin (IL) 4 neutralizing antibodies on Cryptosporidium parvum development in neonatal BALB/c mice. Mice were injected subcutaneously with PBS (Control) or 100 mg of rat anti mouse IL-4 monoclonal antibody 11B11 in PBS on day 0 or day 4 after infection. Production of oocysts was measured on day 7 after infection. The level of excretion of oocysts was increased when anti IL- 4 was administered on day 0 but not when it was administered on day 4. Data are mean SEM ( n p 8 mice/group). HPFs, high-power fields. IL-4 in Immunity to C. parvum JID 2004:190 (1 September) 1021

4 Figure 3. Effect of treatment with murine recombinant (r) interleukin (IL) 4 on Cryptosporidium parvum development in neonatal BALB/c mice. Mice were injected subcutaneously with PBS (Control) or 1.0 mg of ril- 4 in PBS on day 0 ( n p 13 mice; A) or day 4 ( n p 8 mice; B) after infection. Production of oocysts was measured on day 7 after infection. Treatment with exogenous cytokine increased resistance to infection when administered on day 0 but not when administered on day 4. Data are mean SEM. HPFs, high-power fields. a potent inhibitory effect on IL-4 activity in mice [30]. When antibodies were administered on day 0, production of oocysts on day 7 after infection was greater than that in the control mice by a factor 13 (figure 2; P!.005, Student s t test). However, when anti IL-4 antibodies were administered on day 4 after infection, similar oocyst counts were obtained in the antibody-treated and the control mice on day 7 after infection. By use of a different approach, infections were examined in wt mice injected sc with 1.0 mg of murine ril-4 in PBS or PBS alone, on either day 0 or day 4 after infection. When the cytokine was administered on day 0, the level of production of oocysts on day 7 after infection (figure 3A) was substantially reduced, compared with that in control mice ( P!.005, Student s t test). However, when introduced on day 4 after infection, treatment with exogenous cytokine had no effect on the subsequent oocyst burden on day 7 after infection (figure 3B). These results corroborate findings that implicate IL-4 in the induction of early immune effector mechanisms against C. parvum in BALB/c mice. Increased intestinal expression of IL-4 early during C. parvum infection of wt mice. Experiments were performed to measure the effect of the early stage of infection in wt mice on expression of IL-4, measured by Western blotting with a polyclonal anti IL-4 antibody (SC-1260; Santa Cruz Biotechnology) after immunoprecipitation of the cytokine from tissue homogenates (see Materials and Methods). Expression of IL-4 protein in infected wt mice was detected 24 and 48 h after infection (figure 4). However, in infected IL-4 / mice and in uninfected control wt mice, no IL-4 was detected at these times. IFN-g dependent IL-4 mediated protection against infection. Although IFN-g is important in the early immune response to C. parvum [11 14], there are also IFN-g independent mechanisms, since adult BALB/c IFN-g / mice survive an acute C. parvum infection [24]. Hence, the role of IL-4 in IFNg independent immune responses in neonatal BALB/c IFNg / mice was studied. The pattern of infection in these neonates was similar to that described elsewhere for adult BALB/ c IFN-g / mice [24]: an early acute phase was followed by a low-level chronic infection, although the pups, unlike adults, often developed diarrhea early during infection (data not shown). To examine the involvement of IL-4 in the early control of the acute phase, anti IL-4 MAb was administered to IFN-g / mice on day 0, and reproduction of parasites was quantified on days 4 and 7 after infection. Figure 5 shows that anti IL-4 did not influence the level of infection. This result suggests that, in BALB/c mice, IL-4 is not involved in IFN-g independent mechanisms of immunity and that the protective role of IL-4 is IFN-g dependent. Control of C. parvum infection has been associated with increased expression of IFN-g mrna and protein in the intestine [14, 29]. Thus, the intestinal expression of IFN-g mrna from BALB/c wt and IL-4 / mice was measured at key times of infection. No expression of IFN-g mrna was observed in Figure 4. Immunoprecipitation and Western blotting of interleukin (IL) 4 from ileal homogenates of BALB/c mice obtained 24 and 48 h after infection with Cryptosporidium parvum. Uninfected, age-matched wildtype mice (2 samples at each time point) and infected IL-4 deficient (IL- 4 / ) mice (at 48 h after infection) were used as controls. Bacterially expressed (and hence unglycosylated) murine recombinant IL-4 was used as a positive control. IL-4 was expressed in the ileum 24 and 48 h after infection, but not in age-matched, uninfected or infected IL-4 / mice. Data are a representative sample of 2 experiments JID 2004:190 (1 September) McDonald et al.

5 was no significant difference between levels of excretion of oocysts in IL-4 / and IL-4Ra / mice. The early protective role for IL-4 was confirmed by the finding that treatment of wt mice with anti IL-4 neutralizing MAb or ril-4 increased or decreased, respectively, production of oocysts if administered Figure 5. Effect of treatment of with anti interleukin (IL) 4 neutralizing antibodies on Cryptosporidium parvum development in neonatal BALB/c interferon-g deficient mice. Mice were injected subcutaneously with PBS (Control) or 100 mg of rat anti mouse IL-4 monoclonal antibody 11B11 in PBS before infection. Production of oocysts was measured on days 4 and 7 after infection. Treatment with antibodies had no effect on the level of infection. Data are mean SEM ( n p 8 mice/group). HPFs, high-power fields; NS, not significant. uninfected control mice at any stage of infection (data not shown). IFN-g mrna was detected in all mice in both groups at 4, 7, 10, and 14 days after infection, and the kinetics of levels of expression were similar to the patterns of production of oocysts (figure 6A); however, the level of expression was significantly lower in the IL-4 / mice on days 4 and 7 after infection ( P!.05 and P!.01, respectively, Student s t test). An investigation was also made of the possibility that expression of IFN-g was reduced in IL-4 / mice because of reduced expression of either IL-12 or IL-18, both of which are important in the activation of production of IFN-g. IL-12 mrna was not detected in uninfected neonatal mice but was expressed during the infection at levels that, as with IFN-g, reflected the oocyst burden (figure 6B). Expression of IL-12 mrna was significantly reduced in IL-4 / mice on days 4 and 7 after infection, compared with infected wt mice ( P!.05, Student s t test). In contrast to IL-12, expression of IL-18 mrna was detected in uninfected wt and IL-4 / mice, and infection did not affect expression in either group (data not shown). These results suggest that IL-4 activity in promoting early control of C. parvum infection in BALB/c mice involves stimulation of expression of IFN-g and IL-12 in the intestine. DISCUSSION The present study of neonatal IL-4 / or IL-4Ra / BALB/c mice has demonstrated an important defensive role for IL-4 at an early stage of C. parvum infection. IL-4Ra / mice lack both IL-4 and IL-13 functions [26], suggesting that there might also be a protective role for IL-13 against C. parvum; however, there Figure 6. Expression of intestinal interferon (IFN) g (A) and interleukin (IL) 12 (B) mrna in neonatal wild-type (wt) and IL-4 deficient (IL-4 / ) mice after Cryptosporidium parvum infection. Expression of IFN-g and IL- 12 mrna was significantly reduced in the intestines of IL-4 / mice on days 4 and 7 after infection, compared with infected wt mice (polymerase chain reaction product photographs at day 7 are shown below graphs). This correlates with increased susceptibility of IL-4 / mice to C. parvum infection. Neither IFN-g nor IL-12 mrna was detected in uninfected IL- 4 / or wt mice (data not shown). Data are mean + SEM ( n p 5 mice/ group). GAPDH, glyceraldehyde-3-phosphate dehydrogenase. IL-4 in Immunity to C. parvum JID 2004:190 (1 September) 1023

6 just before, but not 4 days after, infection. In a similar study with adult C57BL/6 mice, however, IL-4 promoted resistance to infection only during convalescence [23]. The discrepancies between the findings of these 2 studies may be related to genetic differences between the mouse strains, age of infected mice, or the parasite isolates used. Expression of IL-4 protein was detected as early as 24 h after infection but was not detected in uninfected mice. During human or murine C. parvum infection, IL-4 protein or mrna has been detected at various stages [31, 32, 33], but the present study is the first to show production very early during infection. Of interest, IL-4 mrna was not detected late during infection of calves, but the appearance of C. parvum p23 antigen-specific IgG1, which is up-regulated by IL-4, suggested that the cytokine was expressed earlier [34]. IFN-g independent mechanisms are potentially important in immunity to C. parvum in BALB/c mice [24]. In the present study, neonatal BALB/c IFN-g / mice, like adult mice [24], overcame the early acute infection. However, although treatment of wt mice with anti IL-4 had increased early production of oocysts in wt mice, the same treatment had no effect in IFNg / mice. This result is in agreement with those of other researchers, who observed no increased resistance to infection after treatment of adult IFN-g / mice with ril-4 [35]. Our findings, therefore, do not support a role for IL-4 in IFN-g independent immune mechanisms but implicate involvement in promoting IFN-g dependent immunity. IFN-g and IL-12 play an important part in a partially protective innate immunity against C. parvum in SCID mice [11 13, 36]. Treatment of neonatal BALB/c SCID mice with anti IL-4, however, had no effect on early excretion of oocysts (data not shown), suggesting that the protective effect of IL-4 involved adaptive immunity. In both mice and humans, T cell mediated elimination of C. parvum requires the expression of IFN-g [13, 14, 22, 29], and production of IFN-g early during infection involves IL-12 activity [13]. In our experiments, the levels of intestinal IFN-g and IL-12 mrna were greater in wt mice than in IL-4 / mice early during infection, suggesting that IL-4 either directly supports expression of IFN-g or does so indirectly by up-regulating IL-12. The role of IL-18 in C. parvum infection is not known, but similar high levels of expression of IL-18 mrna were obtained in uninfected wt and IL-4 / mice, and infection did not alter the level of expression. These observations suggest that expression of IL-18 mrna in the neonatal mouse gut is constitutive and is not influenced by IL-4 or by C. parvum infection. The source of this IL-18 mrna is unknown, but epithelial cells, dendritic cells, and macrophages are capable of producing the cytokine [37]. A better insight into the part played by IL-18 in immunity to this parasite might be gained by studies with IL-18 deficient mice and by measurements of mature IL-18 protein. Because IL-4 is important in sustaining the Th2 pathway, it is paradoxical that it might be involved in the induction of a Th1 response. There is increasing evidence, however, that IL-4 can intensify Th1 responses or amplify IFN-g mediated effector mechanisms. Treatment with IL-4 can exacerbate Th1- mediated diseases [38], and the cytokine may enhance development of Th1 responses by inducing chemokine production [30], increasing production of IL-12 by dendritic cells [39], or promoting production of IFN-g by maturing Th1 lymphocytes [40]. However, in other murine infections that induce Th1 immunity (e.g., Candida albicans or Toxoplasma gondii), variable effects of IL-4 deficiency on susceptibility to infection and on expression of IFN-g have been reported elsewhere [41, 42]. 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