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Departments of
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Medicine and Microbiology and
Pathology, Dartmouth Medical School, Lebanon, NH 03756
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Abstract |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Of the 80 genera in the phylum Microspora, several have been demonstrated in human disease (5). Symptoms due to infection are found in HIV-infected or other immunocompromised individuals (6). Enterocytozoon bieneusi, which is the most common microsporidian observed in AIDS patients, infects enterocytes of the bowel and causes diarrhea (7). Encephalitozoon hellem and Encephalitozoon intestinalis, which are closely related to Encephalitozoon cuniculi, are both reportedly associated with disseminated infection during HIV infection (2). E. cuniculi, which was observed previously in laboratory animals, is considered to be a zoonotic infection (6). Several cases of HIV-infected individuals suffering from the complications due to E. cuniculi infection have been documented recently (8, 9). HIV-infected patients with E. cuniculi infection have presented with renal failure, pneumonitis, sinusitis, keratopathy (10, 11), granulomatous liver necrosis (12), and peritonitis (13). In a recent report, autopsy findings in a patient with AIDS showed disseminated E. cuniculi infection involving the brain (8).
Little is known regarding host immunity to microsporidia, including E. cuniculi. E. cuniculi was the first mammalian microsporidian successfully grown in vitro (14). It infects epithelial and endothelial cells, fibroblasts, and macrophages in wide variety of mammals, including rabbits, rodents carnivores, monkeys, and humans (15). In an experimental model, normal mice infected with E. cuniculi usually express few clinical signs of disease (16). Conversely, immunodeficient hosts such as athymic or SCID mice develop lethal disease after experimental infection (17, 18). These animals reveal numerous microsporidia in visceral and parietal peritoneum as well as in the liver and spleen. Previous studies have shown that T cells are responsible for the prevention of lethal disease. An adoptive transfer of enriched spleen cells from an infected host with normal immunity protects athymic or SCID mice against E. cuniculi challenge (19, 20). However, none of these studies have delineated the role of individual T cell subtypes in immunocompetent E. cuniculi-infected animals. In the present study, we observed significant increases in the CD8+ T cell population during E. cuniculi infection. Mice lacking CD8+ T cells were highly susceptible to infection and developed evidence of infection in several tissues.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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A breeding pair of CD8-/- knockout (KO)3 animals on a C57BL/6 background was kindly provided by Dr. T. W. Mak (Amgen Institute, Ontario, Canada). Animals were bred under approved conditions at the Animal Research Facility at Dartmouth Medical School. CD4-/- and perforin-/- (PF-/-) mice having similar genetic lineage were obtained from The Jackson Laboratory (Bar Harbor, ME). Age- and sex-matched C57BL/6 mice were used as wild-type (wt) controls.
Parasite and infection
A rabbit isolate of E. cuniculi organisms was kindly provided by Dr. Elizabeth Didier of the Tulane Regional Primate Center. The parasites were maintained by continuous passage in a rabbit kidney cell line (RK-13 cells) obtained from the American Type Culture Collection (Manassas, VA). The experimental animals were infected by i.p. injections of 1 x 107 spores.
Histopathology
Tissues from infected PF and CD8 KO animals as well as parental control animals were fixed in 10% buffered formalin and processed for 5-µm histological sections, which were stained with hematoxylin and eosin.
Phenotypic analysis
Following euthanasia, the spleens from infected animals were removed and homogenized in a petri dish. The contaminating RBCs were lysed in RBC lysis buffer (Sigma, St. Louis, MO). Cells were washed and suspended in 3% PBS/BSA. Splenocytes were analyzed for cell phenotype by FACS (Becton Dickinson, Mountain View, CA) using a direct immunofluorescence assay. Cells (1 x 106/ml) were incubated with 1 µg of FITC-labeled anti-CD4+, anti-CD8+, or anti-NK1.1 at a 1/100 dilution (PharMingen, San Diego, CA) in 3% BSA/PBS. After 1 h of incubation at 4°C, the cells were washed several times in buffer, fixed in 1% methanol free formaldehyde, and stored cold for FACS analysis.
Purification of CD8+ T cells
CD8+ T cells from the whole splenocyte population was separated by microbeads (Miltenyi Biotec, Auburn, CA) as described previously (21). The separation procedure was conducted as recommended by the manufacturer. The purity of the separated cells was >95% as determined by FACS analysis. Purified CD8+ T cells were assayed for CD69, CD62 ligand (CD62L), and CD44 expression by direct immunofluorescence. The cells were incubated with FITC-conjugated Ab to CD69, CD62L, or CD44 (PharMingen) and analyzed by FACS as described above.
Cytotoxic assay
A CTL assay was performed to detect the cytotoxic activity of the splenocytes from infected animals. The assay was carried out using a protocol standardized previously in our laboratory (22). Briefly, peritoneal macrophages from thioglycolate-treated mice were collected, washed, and dispensed at a concentration of 5 x 104 cells/well in U-bottom, 96-well plates. After overnight incubation, the cells were infected with 2 x 105 spores of E. cuniculi per well for 48 h. The wells were washed extensively with PBS to clear extracellular parasites. Macrophages were labeled with 51Cr (0.5 µCi/well) for 2 h at 37°C. Macrophages were washed five times with PBS and incubated with cultured spleen cells at various E:T ratios in a final volume of 200 µl of culture medium. The microtiter plates were centrifuged at 200 x g for 3 min and incubated at 37°C for 4 h. Samples (100 µl) were removed and assayed for released cpm by scintillation counting. The percentage of lysis was calculated as follows: ([mean cpm of test sample - mean cpm of spontaneous release]/[mean cpm of maximal release - mean cpm of spontaneous release])/100.
Statistical analysis
Statistical analysis of the data was performed using a two-sampled Student t test (23).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To determine which immune cells were responding to E.
cuniculi infection, a phenotypic analysis of the splenocytes from
infected animals was performed. Spleen cells were isolated at days 10,
17, and 24 postinfection (p.i) and analyzed for the expression of
various immune cell phenotypes. As shown in Table I
, the increase in the absolute numbers
of the CD8+ T cell population became apparent at day
10 p.i. The rise in the CD8+ T cell population was
further enhanced by day 17 p.i. At this timepoint, a >3-fold
increase in the cell type over the uninfected controls was observed.
The CD8+ T cell population remained elevated at day 24
p.i, suggesting that these cells may be involved in a long-term immune
response. The population of NK cells also rose during early infection.
At day 10 p.i., a >2-fold increase in both the percentage and
absolute number of NK cells was observed. However, by day 17
postchallenge, the absolute number of the NK cell population was close
to uninfected controls. There was no significant change in the
CD4+ T cell population during the course of infection.
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The data obtained from the above studies demonstrated an early
induction of the CD8+ T cell response during E.
cuniculi infection. We subsequently determined the earliest
timepoint after infection at which CD8+ T cell activation
begins. For this purpose, purified CD8+ T cells from
infected animals were stained for CD69, which is a surface marker that
is rapidly induced in activated T cells (24). As shown in Table II, a significantly increased percentage
of CD8+ T cells expressing CD69 was observed as early as
day 4 p.i. (21 ± 7) compared with uninfected controls
(6 ± 3) (p = 0.02). CD8+ T
cells from the infected animals continued to show higher CD69
expression at later timepoints. By day 18 p.i., a greater
percentage of CD8+ T cells exhibited increased expression
of CD44 (CD44high), a surface maker present on the
activated cells (p = 0.01). Conversely, low
expression of CD62L (CD62Llow), a surface marker that is
frequently identified with naive or uncommitted T cells, was observed
at day 18 postchallenge.
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Phenotypic analysis of splenocytes from infected animals suggested
an important function for CD8+ T cells during E.
cuniculi infection. The role of individual T cell subtypes in
immunity against the parasite was studied using gene KO animals. The
mice were infected with E. cuniculi spores, and survival was
monitored. All of the CD8-/- mice challenged with the
parasites died between days 15 and 20 p.i. (Fig. 1). Just before death, these mice became
lethargic and developed ascitis. Mice lacking CD4+ T cells
survived the infection until termination of the experiment, similar to
parental wt controls. None of these animals developed clinical signs of
disease (lethargy, ruffled skin, or the development of ascitis).
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CD8+ T cells play an important role in intracellular
infections by their ability to produce protective cytokines or lyse
infected targets (25, 26, 27, 28). The major killing mechanism exhibited by
CD8+ T cells in many intracellular infections is via the PF
pathway (29). To determine whether PF is important in the natural
immunity against E. cuniculi, mice lacking this gene were
infected i.p. with 1 x 107 spores. Mice deficient in
the PF gene succumbed to infection almost at the same time as
CD8-/- animals (Fig. 2).
Similar to CD8-/- mice, these animals developed ascitis
and became inactive. As observed earlier, none of the parental mice
died or developed illness throughout the course of the experiment.
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Tissues from infected KO and parental wt animals were isolated and
subjected to histopathological analysis at day 14 postchallenge.
Infected C57BL/6 parental animals showed scattered evidence of mild
inflammation, most prominently in the liver. Small lymphocytic
collections were noted in the liver, with rare polymorphonuclear
leukocytes (Fig. 3A). Evidence
of hepatocyte necrosis was rare. The morphology of the spleen was
preserved, and there was little indication of inflammation or the
destruction of other organs (Fig. 3D). Very rare mononuclear
cells infected with microsporidia were evident by hematoxylin and eosin
staining, and microsporidial spores were visualized by birefringence
when viewed by polarization microscopy (data not shown).
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C). The spleen showed
areas of acute inflammation and necrosis in the white pulp, with many
enlarged mononuclear cells filled with microsporidia (Fig. 3F). Other organs showed widely scattered areas of less
intense inflammation (data not shown). Microsporidia were not detected
in sites other than the liver and spleen.
Infected mice with the PF-/- genotype showed a pattern
similar to the CD8-/- mice. The parasite load and
inflammatory response were high in both the liver and spleen (Fig. 3, B and E). Other organs were much less affected,
and parasite-infected cells were only evident in areas of necrosis and
inflammation within the liver and spleen.
E. cuniculi-infected mice develop a cytotoxic T cell response
The data obtained from the PF KO animals suggested that a
cytotoxic T cell response in E. cuniculi-infected animals
could be an important component of the protective immune system. To
determine whether this is the case, an in vitro CTL assay was performed
using splenocytes from E. cuniculi-infected animals. Spleen
cells were harvested and cultured in the presence of irradiated spores
for 5 days. The viable cells were isolated by Ficoll and incubated with
infected macrophages at various E:T ratios. Ag-stimulated spleen cells
from animals infected on days 0 and 7 failed to exhibit a cytolytic
effect (Fig. 4A). However,
50–60% target cell lysis was observed with spleen cells from animals
infected 17–24 days earlier (Fig. 4, B and C).
Immune spleen cells were unable to lyse the infected macrophages from
nonsyngeneic BALB/c mice (data not shown). These findings indicate that
the development of an MHC-restricted CTL response may play a role in
the protection of the host against the parasite.
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Although cytolysis is a function of CD8+ T cells,
other cell types, such as NK cells, can also exhibit this property
(30). To verify that this was not the case, mice were infected i.p.
with E. cuniculi as described earlier. At day 17 p.i.,
the splenocytes from the infected animals were collected;
CD8+ T cells were isolated by magnetic separation.
CD8+ T cell-enriched and -depleted fractions were collected
and cultured in vitro in the presence of irradiated parasites and
feeder cells. Nondepleted, whole spleen cells were cultured in the
presence of Ag alone. After 5 days, the cytolytic effect of total
splenocytes and the CD8+ T cell-enriched and
CD8+ T cell-depleted populations was evaluated. Purified
CD8+ T cells exhibited increased CTL activity compared with
the total splenocyte population at all E:T ratios (Fig. 5). On the contrary, the spleen cell
population depleted of CD8+ T cells failed to show any
significant cytolysis of infected targets. Target cell lysis with the
CD8+ T cell depleted population was similar to the
uninfected controls.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The immune response generated during natural E. cuniculi infection has not been well studied. Current literature emphasizes an important role for T cells in protective immunity (18). Although a cellular immune response is reported to be essential for immunity against the parasite, the role of different T cell subtypes in protection against E. cuniculi infection is not well understood. In the present study, we show that CD8+ T cells play an important and perhaps essential role in immunity against this parasite. The protection was shown to be dependent upon a lytic effect of these cells on the infected targets, as mice deficient in the PF gene succumbed to infection almost at the same time as CD8-/- mice. It is possible that in addition to CD8+ T cells, NK cells that are increased during early infection and are known to be abundant in PF (30) also play a role in protective immunity against E. cuniculi. However, a total splenocyte population that has been depleted of CD8+ T cells is unable to lyse the parasite-infected targets. Moreover, the susceptibility of CD8-/- and SCID mice to E. cuniculi infection rules out any major role for these cells in the outcome of infection.
CD8+ T cells are known to play an important role in a wide variety of intracellular infections. These cells have been reported to be critical for many viral, bacterial, and parasitic infections (21, 31, 32, 33). Our studies demonstrate an early activation and substantial increase in the CD8+ T cell population during E. cuniculi infection. Gene KO mice deficient in CD8+ T cells were highly susceptible to infection. Histopathological analysis of the tissues from the infected animals showed disseminated infection in the liver and spleen. In contrast, very few parasites were observed in the organs of infected parental wt mice. Similar to parental C57BL/6 mice, CD4-/- animals were able to resolve E. cuniculi infection. These findings suggest that CD8+ T cell priming during E. cuniculi infection may be independent of CD4+ T cells. The role of CD4+ T cells in the induction of the CD8+ T cell response against viral infections has been described previously (34). Infection with lymphocytic choriomeningitis virus in CD4-/- animals results in reduced CD8+ T cell immunity and viral clearance (35). Conversely, a lack of CD4+ T cells does not affect the CD8+ T cell response in mice during infection with vaccinia virus (36). It is very likely that under certain circumstances, depending upon the type of infection, CD8+ T cells in the absence of CD4+ T cells are primed through alternate redundant mechanisms. Such mechanisms have been reported to exist in some microbial infections (37, 38). The precise role of CD4+ T cells in the induction of a CD8+ T cell response during E. cuniculi infection is being further evaluated.
CD8+ T cell-mediated protection has been shown to occur via
a cytolytic effect on the infected cells, via cytokine production, or
both. During infection with lymphocytic choriomeningitis virus, mice
lacking the PF gene are unable to clear the virus due to a lack of
cytotoxic activity of CD8+ T cells (39). In contrast,
CD8+-dependent immunity against vaccinia virus is largely
mediated by IFN-
production (40). IFN--secreting CD8+
T cells are believed to be important for impeding the chronic
Toxoplasma infection (41). CD8+ CTLs from
Toxoplasma gondii-infected animals exhibit in vitro
cytolytic activity against syngeneic targets (33). However, a lack of
cytolytic capability in the PF-deficient animals did not significantly
alter their protective immunity (42). Similarly, although
CD8+ T cells are crucial for resistance against rodent
malaria (43), the protective immunity is independent of PF-mediated
cytotoxicity (44). The splenocytes from E.
cuniculi-challenged animals were found to exhibit significant
lytic activity against the infected cells. The CD8+ CTLs
generated during the infection mediated the lysis of infected targets.
However, unlike T. gondii and malarial infection, lack of a
PF gene compromised the ability of these mice to withstand E.
cuniculi infection. Similar to mice lacking CD8+ T
cells, tissues from PF-/- mice showed greater parasite
multiplication.
Our data emphasize a critical role for CD8+ CTLs during
E. cuniculi infection. Based on our findings, we postulate
the following: E. cuniculi infection results in early
activation and proliferation of CD8+ CTLs. The Ag-specific
CTLs exhibit cytolytic activity against infected targets. The lack of
cytotoxic CD8+ T cells results in a substantial increase in
parasitic load, which ultimately proves lethal to the host. Ongoing
studies in our laboratory suggest the importance of IFN- during
E. cuniculi infection (our unpublished observations). The
cytokine is known to up-regulate MHC class I molecules, which could
thereby play a role in the induction of CD8+ T cell
immunity (45). IFN- has been reported to be important for the
maintenance of the CD8+ CTL response against bacterial and
viral infections (46, 47). The interactions between CD8+ T
cells and IFN- during E. cuniculi infection are currently
being investigated in our laboratory.
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Imtiaz A. Khan, Department of Medicine, Dartmouth Medical School, HB 7506, One Medical Center Drive, Lebanon, NH 03756. E-mail address:
3 Abbreviations used in this paper: KO, knockout; PF, perforin; wt, wild type; CD62L, CD62 ligand; p.i., postinfection.
Received for publication December 1, 1998. Accepted for publication February 17, 1999.
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References |
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and CD8+ T cells. J. Exp. Med. 180:353. interferon of antiviral cell-mediated immune responses in vivo. J. Virol. 70:1521.[Abstract]
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