Roles for NK Cells and an NK Cell-Independent Source of Intestinal Gamma Interferon for Innate Immunity to Cryptosporidium parvum Infection

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1 INFECTION AND IMMUNITY, Nov. 2009, p Vol. 77, No /09/$12.00 doi: /iai Copyright 2009, American Society for Microbiology. All Rights Reserved. Roles for NK Cells and an NK Cell-Independent Source of Intestinal Gamma Interferon for Innate Immunity to Cryptosporidium parvum Infection Farah M. Barakat, 1 Vincent McDonald, 1 * James P. Di Santo, 2 and Daniel S. Korbel 1 Barts and the London School of Medicine and Dentistry, Queen Mary University of London, Institute of Cell and Molecular Science, Centre for Gastroenterology, London, United Kingdom, 1 and Cytokines and Lymphoid Development Unit, Immunology Department, Institut Pasteur, Paris, France 2 Received 3 April 2009/Returned for modification 29 May 2009/Accepted 10 August 2009 A gamma interferon (IFN- )-dependent innate immune response operates against the intestinal parasite Cryptosporidium parvum in T- and B-cell-deficient SCID mice. Although NK cells are a major source of IFN- in innate immunity, their protective role against C. parvum has been unclear. The role of NK cells in innate immunity was investigated using Rag2 mice, which lack T and B cells, and Rag2 c mice, which, in addition, lack NK cells. Adult mice of both knockout lines developed progressive chronic infections; however, on most days the level of oocyst excretion was higher in Rag2 c mice and these animals developed morbidity and died, whereas within the same period the Rag2 mice appeared healthy. Neonatal mice of both mouse lines survived a rapid onset of infection that reached a higher intensity in Rag2 c mice. Significantly, similar levels of intestinal IFN- mrna were expressed in Rag2 and Rag2 c mice. Also, infections in each mouse line were exacerbated by treatment with anti-ifn- neutralizing antibodies. These results support a protective role for NK cells and IFN- in innate immunity against C. parvum. In addition, the study implies that an intestinal cell type other than NK cells may be an important source of IFN- during infection and that NK cells may have an IFN- -independent protective role. Cryptosporidiosis is an infectious diarrheal disease that affects different types of vertebrates, including mammals (3). The etiological agent is the monoxenous protozoan parasite Cryptosporidium, which belongs to the Apicomplexa. One species, Cryptosporidium hominis, may have a predilection for infecting humans, while a morphologically similar parasite, Cryptosporidium parvum, readily infects both cattle and humans (3). The cryptosporidia of mammals invade intestinal epithelial cells, where they multiply asexually to produce merozoites that infect more cells. Eventually, merozoites may undergo differentiation into gamonts that form new oocysts, containing four sporozoites, and the oocysts transmit infection to new hosts by the fecal-oral route. The clinical phase of cryptosporidiosis normally lasts a few days but may persist and become fatal in immunocompromised hosts (2). Studies of protective host immune responses to Cryptosporidium indicate that elimination of infection involves adaptive immunity and, in particular, requires the presence of CD4 T cells. AIDS patients with low CD4 cell counts have shown increased susceptibility to cryptosporidial infection and high rates of morbidity and mortality, while resolution of AIDSassociated infection following anti-human-immunodeficiencyvirus drug treatment coincided with the partial recovery of intestinal CD4 T-cell counts (2, 23). Mice with a CD4 T-cell deficiency were found to be incapable of clearing C. parvum * Corresponding author. Mailing address: Centre for Gastroenterology, Institute of Cell and Molecular Science, Barts and The London School of Medicine and Dentistry, Queen Mary University of London, 4 Newark Street, London E1 2AT, United Kingdom. Phone: Fax: v.mcdonald@qmul.ac.uk. Published ahead of print on 17 August infection (1), and similarly, depletion of these cells from immunocompetent animals with specific antibody increased oocyst production (27). CD4 T cells are also an important source of gamma interferon (IFN- ), and this cytokine plays a key role in the control of infection. Antigen-specific IFN- production by restimulated CD4 T cells from humans who recovered from infection was observed, although cells taken during acute infection were not responsive to antigen (6). IFN- mice or mice administered anti-ifn- neutralizing antibodies had exacerbated infections compared with control animals (18, 27). IFN- activity during C. parvum infection has been associated with a chemokine response by intestinal epithelial cells that attracted both CD4 T cells and macrophages into the lamina propria (10). In addition, IFN- has been shown to have a direct effect on parasite growth by activating epithelial cell antimicrobial killing activity (19). Innate immune responses are also able to limit the reproduction of C. parvum. Immunocompromised adult nude mice (lacking T cells) or SCID mice (lacking T and B cells) developed chronic infections that were controlled for a number of weeks but eventually became progressive and fatal (13, 17, 27). IFN- was important for the initial resistance of these mice, since administration of anti-ifn- neutralizing antibodies to adult or neonatal SCID mice increased susceptibility to infection (14, 28), and repeated antibody treatment resulted in rapid establishment of severe infection (14). In addition, morbidity as a result of parasite reproduction appeared sooner in SCID IFN- mice than in SCID mice (7). NK cells are involved in resistance to intracellular microbial pathogens, including protozoa, and are a major source of IFN- in innate immunity (9). NK cells originate mainly in the bone marrow, from where they migrate to other organs (5, 29). 5044

2 VOL. 77, 2009 NK CELLS AND IFN- IN IMMUNITY TO C. PARVUM 5045 Interleukin-15 (IL-15) is essential for differentiation and subsequent survival of NK cells and can also be important in activation of the cells (5, 9). NK cells are activated by ancillary cells, such as dendritic cells (DCs), by direct contact and by proinflammatory cytokines produced by DCs stimulated by antigen (9). Activated NK cells produce IFN- and other proinflammatory cytokines and may also become cytotoxic against infected cells. The protective role of NK cells in innate immunity to C. parvum is unclear, but some studies imply that these cells may be involved. Human peripheral blood NK cells treated with IL-15 were shown to have cytolytic activity against human intestinal epithelial cell lines infected with C. parvum (4), and intestinal expression of this cytokine has been detected in humans (20). C. parvum infection was found to be more widespread in SCID mice deficient in NK cell cytotoxicity than in SCID mice with normal NK cell function (17). In addition, in vitro studies demonstrated that splenocytes from SCID mice produced IFN- in the presence of cryptosporidial antigens, but if NK cells were depleted, IFN- production did not occur (15). However, attempts to show that NK cells were protective in SCID mice infected with C. parvum have not been successful. In separate studies, treatment of these mice with antiasialo-gm1 antibodies that can deplete NK cells in vivo was shown to have no effect on the course of C. parvum infection (15, 27), and while it has been argued that these antibodies might not have reached the gut in sufficient quantity to be effective, similar antibodies were shown to diminish intestinal NK cell function (30). The aim of the present study was to examine further the role of NK cells and IFN- in the innate immune response to C. parvum. The pattern of infection and immune responses were compared in Rag2 mice, which lack T and B cells, and Rag2 c mice, which, in addition, lack NK cells due to the absence of the c chain component of the IL-15 receptor (5). The results support protective roles for IFN- and NK cells in innate immunity to C. parvum but also indicate that IFN- from a cell type other than NK cells is important for control of infection. MATERIALS AND METHODS Mice. C57BL/6 Rag2 mice and C57BL/6 Rag2 c mice were bred and maintained in filtered cages under specific-pathogen-free conditions. Mice were infected either as neonates (7 days of age) or after weaning (4 to 5 weeks of age), and all procedures performed were licensed by the United Kingdom Home Office. Parasites and infection studies. Purified oocysts of C. parvum in phosphatebuffered saline (PBS), ph 7.2, were obtained from Bunch Grass Farm, Deary, ID, and stored at 4 C. Oocysts were surface sterilized by suspension in 10% (vol/vol) commercial bleach solution (sodium hypochlorite), washed three times in PBS, and enumerated in a Neubauer hemocytometer. Mice were infected by oral gavage, using or oocysts for neonatal animals; older animals received oocysts, as early infection is otherwise hard to detect in these animals (12, 13). Acid-fast stained fecal smears were produced, and the numbers of oocysts in 50 randomly selected microscopic fields (magnification, 1,000) were counted blindly and by at least two observers. Infection was also assessed by histological examination of sections from the ileum, colon, cecum, and liver. Tissues were fixed in formal saline, embedded in paraffin, and stained with hematoxylin and eosin. Antibodies. The H22 hamster anti-mouse IFN- immunoglobulin G monoclonal antibody (R&D Systems) in PBS was employed to neutralize IFN-, and PBS was used as a control, since a nonreactive hamster immunoglobulin G was previously shown to have no effect on parasite development (13, 14). The antibody (100 g in100 l PBS) or PBS alone was administered intraperitoneally immediately before oocyst inoculation. RNA extraction and real-time quantitative PCR. Total RNA from the ileum was isolated using a monophasic solution of phenol and guanidine thiocyanate (Trizol; Invitrogen) according to the manufacturer s instructions, followed by chloroform extraction and isopropanol precipitation. Total RNA was quantified by spectrophotometry, and 3 g RNA was reverse transcribed to cdna at 42 C with 1.5 g of oligo(dt) 15 primer (Promega), 1 mm deoxynucleoside triphosphates, and Moloney murine leukemia virus reverse transcriptase in a volume of 20 l, following the manufacturer s protocol. For real-time quantitative PCR, amplification was achieved with 20 pmol of each oligonucleotide primer (Sigma- Aldrich). The primer sequences were as follows: murine -actin forward, CCT TCC TTC TTG GGT ATG GAA T; murine -actin reverse, GCA CTG TGT TGG CAT AGA GGT (amplicon size, 106 bp); murine IFN- forward, GCC AAG TTT GAG GTC AAC AAC; and murine IFN- reverse, ATC AGC AGC GAC TCC TTT TC (amplicon size, 121 bp). Reaction mixtures were set up to a final volume of 20 l, using a total of 100 ng cdna, 20 pmol of each primer, and 10 l FastStart SYBR green master mix (Roche). Amplification was performed using a Rotor-Gene 3000 instrument (Corbett Research) at least three times with independent cdna samples and in triplicate for each cdna and primer pair. The PCR protocol comprised an initial hold step of 95 C for 10 min, followed by 45 cycles of amplification under the following conditions: denaturation at 95 C for 15 s, annealing at 60 C for 30 s, and elongation at 72 C for 60 s. The comparative threshold method was used for relative quantification ( C T method), where the amount of target gene was normalized to the housekeeping gene -actin and relative to the calibrator (untreated control). Statistical analysis. Mean values standard errors were calculated, and statistical significance was determined using Student s unpaired t test. RESULTS Infection in Rag2 and Rag2 c mice. In order to investigate the protective role of NK cells in innate immunity to C. parvum, studies were made with C57BL/6 Rag2 mice and Rag2 c mice. When mice received oocysts at 4 to 5 weeks of age, similar chronic patterns of infection were obtained for each knockout line (Fig. 1A). During the first week of infection, a sharp peak of oocyst production occurred, and following a temporary remission, there was a gradual buildup in the level of oocyst excretion over subsequent weeks. Importantly, after day 60, as the infection became progressive, the mean numbers of oocysts excreted by Rag2 c mice were frequently significantly higher. In association with this, morbidity in the form of weight loss, listlessness, and sometimes soft fecal pellets appeared in Rag2 c mice from day 91 onwards, and all animals had died by day 140; in contrast, all Rag2 mice appeared healthy up to the latter time (Fig. 1B). Histological studies of Rag2 c mice were performed to confirm that oocyst shedding was a valid means of measuring the infection and that the morbidity shown by these animals was caused by the parasite. Figure 1C shows the colon from a representative Rag2 c mouse at the midpoint of infection, when the animal appeared healthy and 5 oocysts/50 fields were detected in a fecal smear. Parasites were difficult to find in the colon, and there was no evidence of pathology. Figure 1D shows the colon from a representative Rag2 c mouse at the late phase of infection, when the animal showed signs of illness and a fecal smear contained more than 100 oocysts/50 fields. In this case, many crypts contained large numbers of developing parasites, and others were denuded of epithelial cells as a result of heavy infection. Similar observations were made in the terminal ilea and ceca of these animals, and in addition, only in the late stage of infection could parasites also be observed in bile ducts (results not shown). These findings showed correlations between the level

3 5046 BARAKAT ET AL. INFECT. IMMUN. Downloaded from FIG. 1. Effects of C. parvum infection on Rag2 and Rag2 c mice. At 4 to 5 weeks of age, Rag2 and Rag2 c animals were infected by oral gavage with C. parvum oocysts. (A) Microscopic measurements were made of oocyst shedding in Ziehl-Neelsen acid-fast stained fecal smears from mice at different times of infection. The values represent mean numbers of oocysts per 50 fields standard errors for six animals per group (, P 0.02;, P 0.004). (B) Graph showing percentages of mice surviving during infection. (C) Sections of hematoxylinand eosin-stained colons of infected Rag2 c mice taken during the middle (excreting few oocysts) (left) and late (excreting many oocysts and showing morbidity) (right) stages of infection. With a low degree of oocyst shedding, no parasites could be observed in the crypts and there was no sign of pathology. A high degree of oocyst shedding accompanied by morbidity was associated with many crypts infected with large numbers of parasites (arrowheads) and with focal degeneration of the epithelium (arrows). Magnification, 63; bars, 200 m. on April 9, 2019 by guest of oocyst excretion and parasite endogenous development and between morbidity/mortality and histopathology. Flow cytometry experiments were performed to confirm that mice of the Rag2 c colony lacked NK cells. The spleens of Rag2 mice contained substantial numbers of CD49b NK1.1 cells, indicative of the presence of NK cells, whereas these cells were not found in Rag2 c mice (data not shown). Hence, the results suggested that the NK cell deficiency of Rag2 c mice was an important factor in determining their increased susceptibility to C. parvum infection compared to Rag2 mice. Infection in neonatal Rag2 and Rag2 c mice. Infections in the 4- to 5-week-old mice took several weeks to become established, but as immunocompetent neonatal mice are highly susceptible to C. parvum infection (12), it was of interest to compare oocyst shedding in Rag2 and Rag2 c mice infected at 7 days of age. Mice of both knockout lines developed high levels of oocyst production within a few

4 VOL. 77, 2009 NK CELLS AND IFN- IN IMMUNITY TO C. PARVUM 5047 FIG. 2. Course of acute C. parvum infection in neonatal Rag2 and Rag2 c mice. At 7 days of age, Rag2 and Rag2 c animals were infected by oral gavage with C. parvum oocysts, and fecal samples were collected on the days shown. Shed oocysts in fecal smears were stained and counted by light microscopy. The data depicted are mean numbers of oocysts per 50 fields of view. The graph shows mean values for at least eight animals per group (, P 0.05). days (Fig. 2), but significantly, the level of mortality during this period was low. Another important observation was that Rag2 and Rag2 c mice were eventually able to control the infection to the extent that only a few parasites could be detected by day 14 (Fig. 2). For several days, starting around the peak of infection, oocyst production was higher in the Rag c mice. In a similar experiment, mice were infected at 4 days of age, when they might have been more vulnerable to infection than animals at 7 days of age, but similar results were obtained (data not shown). These results show that in the neonatal infection model, parasite reproduction in Rag2 c mice was greater than that in Rag2 mice, a finding that supports a protective role for NK cells in immunity to this parasite. Nevertheless, mice of both knockout lines were able to overcome the early high rate of parasite reproduction and establish a high degree of control. Role of IFN- in recovery of neonatal mice from infection in the absence of NK cells. The ability of the neonatal Rag2 and Rag2 c mice to survive and control C. parvum infection led to a measurement of IFN- mrna expression in these mice, using real-time quantitative PCR (Fig. 3). The expression of IFN- in uninfected mice was poor, but interestingly, there was an upregulation of expression on day 7 postinfection in both Rag2 and Rag2 c mice. Indeed, the level of expression was greater in Rag2 c mice than in Rag2 mice, but the difference between values was not statistically significant. This result indicated that in the innate immune response against C. parvum infection, there appears to be a major cellular source of IFN- other than NK cells. In view of this result, experiments were carried out to determine whether IFN- was involved in the control of infection in Rag2 c and Rag2 mice by administration of anti-ifn- neutralizing antibody prior to oocyst inoculation. Comparing infections in control animals, Rag2 c mice produced larger numbers of oocysts than Rag2 mice did between days 4 and 8, as observed previously (Fig. 4). Importantly, treatment of mice with anti-ifn- resulted in an exacerbation of infection for both knockout lines compared with the respective controls, starting from 2 days before the time of FIG. 3. Intestinal IFN- expression in neonatal Rag2 and Rag2 c mice during acute C. parvum infection. Rag2 and Rag2 c animals were infected by oral gavage with C. parvum oocysts at 7 days of age, and intestinal tissue samples from the small intestine were collected at 7 days postinfection. The amount of intestinal IFN- mrna was quantified by real-time quantitative PCR, using the C T method, with -actin as the reference gene and samples from uninfected animals as the calibrator. The example shown represents four animals per group ( *, P 0.01). maximal oocyst production until 2 days after (days 4 to 8 postinfection). These observations indicated that IFN- played an important part in the ability of both Rag2 and Rag2 c mice to control C. parvum infection. Comparing anti- IFN- -treated mice at the peak of infection (day 6), the level of oocyst production was higher in Rag2 c mice than in Rag2 mice. This observation may suggest that the greater resistance to infection of the antibody-treated Rag2 mice was not dependent on IFN-. DISCUSSION NK cells have a major protective role in the innate immune response to many intracellular pathogens (9). Results from the present study involving Rag2 and Rag2 c mice in- FIG. 4. Effect of IFN- neutralization on course of C. parvum infection in neonatal Rag2 and Rag2 c mice. At 7 days of age, Rag2 and Rag2 c animals were administered (intraperitoneally) either 100 g neutralizing anti-ifn- antibody in PBS or PBS only. Immediately afterwards, the animals received C. parvum oocysts by oral gavage. The data depicted are mean values for 10 animals per group. Significant differences (P 0.05) between mean values for Rag2 control and antibody-treated mice ( *), Rag2 c control and antibody-treated mice (#), and Rag2 antibodytreated and Rag2 c antibody-treated mice ( ) are marked.

5 5048 BARAKAT ET AL. INFECT. IMMUN. dicated that NK cells were an important component of innate immunity against C. parvum. Significantly, in the absence of NK cells, it was evident that another cell type(s) conferred immunity against the parasite, acting at least in part by producing IFN-. Infection of 4- to 5-week-old Rag2 and Rag2 c mice followed a progressive chronic pattern that had previously been observed in adult nude and SCID mice (13, 27). Rag2 c mice had heavier infections than Rag2 mice, as measured by levels of oocyst production and times of survival. These results indicate that NK cells are important in the innate immune response that controls C. parvum infection. However, the ability of Rag2 c mice to sustain resistance to parasite reproduction for numerous weeks indicated that innate immune mechanisms independent of NK cells helped to prolong survival. Since immunocompetent neonatal mice are highly susceptible to C. parvum infection (12), it was of interest to compare the courses of infection in newborn Rag2 and Rag2 c mice. Remarkably, although high levels of infection became established in the newborn mice within a few days, there were few deaths among either Rag2 or Rag2 c mice. Furthermore, the mice of both knockout lines were able to bring infection under control. We also observed that neonatal BALB/c SCID mice developed an acute pattern of infection, although several weeks later there was a resurgence of parasite reproduction leading to mortality (unpublished data). The ability of these young mice to recover may have been due in part to the onset of age-related natural resistance to this infection found in mice and other mammalian species, the basis of which is unclear (24). However, there is clearly also a significant immunological component in host resistance to infection in neonates, since higher levels of infection were obtained in the Rag2 c mice. The increased susceptibility of Rag2 c mice to infection compared to Rag2 mice suggested that NK cells had an important protective role against C. parvum in the neonates. Since it has been established that IFN- is important in innate immunity to this parasite in mice, a study was made of the role of this cytokine in the immunity to infection shown by neonatal Rag2 and Rag2 c mice. Two key observations were made, as follows: first, there was upregulation of IFN- expression in both Rag2 and Rag2 c mice during infection, and second, treatment with anti-ifn- neutralizing antibodies exacerbated infections in each knockout mouse line. These results demonstrated, therefore, that there was a major cellular source of IFN- in the absence of NK cells that contributed to host resistance against the parasite. Further studies with similar mouse lines also lacking the IFN- gene would characterize more definitively the role of this cytokine in innate immunity to the parasite. Studies with bacterial pathogens such as Salmonella suggested that IFN- production by macrophages and neutrophils was important in the early innate immune response to infection that limited bacterial reproduction (8). In pulmonary Chlamydia infection of mice, macrophages capable of expressing IFN- were required for innate immunity in immunocompromised animals, although, in contrast to the findings of the present study, there was no difference in the susceptibility to infection of Rag2 and Rag2 c mice (21). Macrophages have been shown to produce large amounts of IFN- in vitro when stimulated with a combination of IL-12 and IL-18 (22), both of which are expressed during cryptosporidial infection (16, 26). In relation to our findings, other workers recently reported that the transference of peritoneal macrophages from C. parvum-infected mice to immunocompromised mice with a macrophage deficiency allowed the recipients to be resistant to infection with the parasite, and in vitro studies suggested that IFN- from neutrophils might be involved in activation of the macrophages (25). DCs might also have an important protective role in this respect, as they have been found to be a potent source of IFN- when activated by cytokines (11). The findings that Rag2 c mice expressed IFN- as strongly as Rag2 mice and that, after treatment with anti- IFN- antibodies, the infection level in Rag2 mice did not reach the same intensity as that in Rag2 c mice suggested that the protective action of NK cells might be at least partly independent of IFN-. There is evidence suggesting that NK cell cytotoxicity might have a role in immunity to C. parvum infection. One study has shown that human peripheral blood NK cells activated by IL-15 had cytolytic activity against enterocyte cell lines infected with C. parvum and, in addition, infected epithelial cells expressed MICA, a ligand for the NK cell activating receptor NKG2D (4). Also, extraintestinal cryptosporidial infection was observed to be less common in SCID mice than in similar mice that also carried the beige mutation, which causes a deficiency in NK cell cytotoxicity (17). In conclusion, results from this investigation suggest that NK cells in immunocompromised mice are important for maintaining the innate control of C. parvum infection. It was confirmed that IFN- plays a key part in maintaining innate immunity but that a cell type(s) other than NK cells that produces the cytokine is also prominently involved. The data also imply that NK cells may have an important protective role that is independent of IFN- activity. ACKNOWLEDGMENTS Funding for this work was provided by the Biotechnology and Biological Science Research Council of the United Kingdom, by Barts and the London Charitable Foundation (V.M.), and, in addition, by the Institut Pasteur, INSERM, and the Ligue Nationale Contre le Cancer (J.P.D.S.). REFERENCES 1. Aguirre, S. A., P. H. Mason, and L. E. Perryman Susceptibility of major histocompatibility complex (MHC) class I- and MHC class II-deficient mice to Cryptosporidium parvum infection. Infect. Immun. 62: Blanshard, C., A. M. Jackson, D. C. Shanson, N. Francis, and B. G. Gazzard Cryptosporidiosis in HIV-seropositive patients. Q. J. Med. 85: Chen, X. M., J. S. Keithly, C. V. Paya, and N. F. LaRusso Cryptosporidiosis. N. Engl. J. Med. 346: Dann, S. M., H. C. Wang, K. J. Gambarin, J. K. Actor, P. Robinson, D. E. Lewis, S. Caillat-Zucman, and A. C. White, Jr Interleukin-15 activates human natural killer cells to clear the intestinal protozoan Cryptosporidium. J. Infect. 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