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

A gamma interferon (IFN-γ)-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-γ 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^−/− γ_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^−/− γ_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^−/− γ_c^−/− mice. Significantly, similar levels of intestinal IFN-γ mRNA were expressed in Rag2^−/− and Rag2^−/− γ_c^−/− mice. Also, infections in each mouse line were exacerbated by treatment with anti-IFN-γ neutralizing antibodies. These results support a protective role for NK cells and IFN-γ 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-γ during infection and that NK cells may have an IFN-γ-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 AIDS-associated infection following anti-human-immunodeficiency-virus 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 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-γ), and this cytokine plays a key role in the control of infection. Antigen-specific IFN-γ 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-γ^−/− mice or mice administered anti-IFN-γ neutralizing antibodies had exacerbated infections compared with control animals (18, 27). IFN-γ 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-γ 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-γ was important for the initial resistance of these mice, since administration of anti-IFN-γ 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-γ^−/− 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-γ in innate immunity (9). NK cells originate mainly in the bone marrow, from where they migrate to other organs (5, 29). 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-γ 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-γ in the presence of cryptosporidial antigens, but if NK cells were depleted, IFN-γ 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 anti-asialo-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-γ 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^−/− γ_c^−/− mice, which, in addition, lack NK cells due to the absence of the γ_c chain component of the IL-15 receptor (5). The results support protective roles for IFN-γ and NK cells in innate immunity to C. parvum but also indicate that IFN-γ from a cell type other than NK cells is important for control of infection.