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*
Department of Microbiology and Immunology, University of Maryland, School of Medicine, Baltimore, MD 21201; and
Malaria Program, Naval Medical Research Center, Silver Spring, MD 20910
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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T cells
and NK1.1int cells in immunized animal models. In contrast,
there is little information concerning responses in a naive host. Here
we report that several host gene expressions in the liver, spleen, and
kidney of BALB/c mice are altered during the liver stage of
Plasmodium yoelii infection. Really interesting new gene
3 (Ring3), semaphorin subclass 4 member G, glutamylcysteine synthetase,
and p45 NF erythroid 2 were all up-regulated 24 h after infection
with P. yoelii. Semaphorin subclass 4 member G
expression was elevated in the kidney, whereas Ring3 was elevated in
both spleen and kidney. The expression of TNF-
(TNF- and IFN-)
were down-regulated in all three tissues tested except in infected
spleen where IFN- was elevated. P. yoelii-related
host gene changes were compared with those in Toxoplasma
gondii-infected livers. Ring3 expression increased 5-fold over
control values, whereas expression of the other transcripts remained
unchanged. TNF- and IFN- expressions were increased in the
Toxoplasma-infected livers. The uniform increase of
Ring3 expression in both Plasmodium- and
Toxoplasma-infected livers suggests an innate immune
response against parasitic infections, whereas the other gene
expression changes are consistent with Plasmodium
parasite-specific responses. Taken together, these changes suggest the
immune responses to P. yoelii infection are both
parasite and organ specific. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Host immune responses pertaining to the erythrocytic stage have been widely studied and are believed to be primarily humoral by nature because mature erythrocytes do not express appreciable levels of the MHC. Immunologically naive children can be temporarily protected from infection by the administration of immune sera obtained from adults who were repeatedly exposed to Plasmodium falciparum (1). These Abs, which specifically target erythrocytic stage Ags, are believed to inhibit further cell invasion and cytoadherence between the infected and the uninfected erythrocytes, and mediate Ab-dependent cytotoxic activity. When compared with the erythrocytic infection, relatively less is known about the host responses that occur during the liver stage of the infection.
Although a significant amount of work has been done to assess the immune response at the liver stage to irradiated sporozoites and subunit vaccines (2, 3), much less effort has been spent studying early immune responses in the naive hosts.
Several studies have looked at cellular infiltrates such as the role of
T cells (4) and Kupffer cells. Indirect evidence
suggests that Kupffer cells have the capacity to eliminate sporozoites.
The relative number of liver stage parasites rose significantly when
Kupffer cells were eliminated in vivo (5). But, little
information exists about the molecular changes that may occur in the
liver in response to infection.
As part of a project to identify plasmodial genes that were expressed
during the liver stage, differential display analysis was undertaken on
P. yoelii-infected livers. In addition to the identification
of parasite genes that were expressed during the liver stage
(6), mouse gene sequences were also amplified. Herein, we
report the expression of several host transcripts, namely really
interesting new gene 3 (Ring3),3 semaphorin
subclass 4 member G (sema 4g), p45 NF erythroid 2 (p45 NF E2),
glutamylcysteine synthetase (GCS), IFN-, and TNF-. Furthermore,
their expression appears to be regulated in a tissue- and
parasite-specific manner during the liver stage of P. yoelii
infection.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Female BALB/c mice, 6–8 wk old, were purchased from Charles River Breeding Laboratories (Wilmington, MA). The animals were cared for and used strictly in accordance with the University of Maryland, Baltimore Institutional Animal Care and Use Committee as well as the Public Health Services guidelines (Committee on Care and Use of Laboratory Animals, National Institutes of Health, Bethesda, MD).
Isolation of sporozoites and infection of mice
Anopheles stephensi mosquitoes were fed on gametocytemic mice and maintained thereafter at 24°C and 75% humidity for 14 days. The infected mosquitoes were sugar fed, and their parasite burden was monitored by examining five mosquito midguts for oocysts. Sporozoites were isolated from these mosquitoes as previously described (6, 7). A sporozoite dose-response RT-PCR was conducted to determine the optimum sporozoite dose to detect parasite-specific stage-specific mRNA (8). Sporozoites were then resuspended in 500 µl of M199/5% FCS and injected via tail vein at a concentration of 2 x 106 sporozoites per animal. Sham-infected animals were injected with material isolated from an equal number of uninfected mosquitoes.
Differential display (DD)3
Sham-infected (UL) and P.
yoelii-infected (IL) livers were harvested
from the mice 24 h post P. yoelii infection. The livers
were processed for the extraction of total RNA using TRIzol reagent
(Life Technologies, Gaithersburg, MD). Two micrograms of total RNA from
sham-infected and infected livers were used in the DD reaction as
previously described (6). Gene-specific primers were
synthesized based on the sequences of the DD clones. Table I
shows the mouse transcripts, their
gene-specific primer sequences, and the corresponding annealing
temperatures in the PCR step. PCR began with an initial denaturing step
of 94°C for 3 min followed by 94°C for 30 s, "x"°C (see
Table I) for each individual transcript for 45 s; 72°C for
30 s for a total of 30 cycles, and a final extension step of
72°C for 10 min.
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Two micrograms of total RNA was used from the
UL treatment group to set up a RT using random
hexamers 50 ng/µl and 200 U of Superscript II RTase (Life
Technologies). PCR was conducted with the gene-specific primers (shown
in Table I
) and [32P]deoxyribocytidine
(Amersham Pharmacia Biotech, Piscataway, NJ) at a concentration of 0.1
µCi/µl. A 5-µl sample of each reaction was removed after every
five PCR cycles. These resulting PCR products were electrophoresed on a
10% Tris-buffered EDTA polyacrylamide gel, and the exponential portion
of the amplification curve was plotted for each transcript. Briefly,
the total counts from each amplification were measured with a
phosphoimager (Molecular Dynamics, Sunnyvale, CA), and the values that
corresponded to half the maximal amplification were determined
graphically. Fig. 1 shows an example of the PCR exponential curve of
several host gene transcripts plotted to determine the cycle number
under set conditions that would correspond to the 50th percentile of
the amplification. Quantitative RT-PCR was subsequently set up using 2
µg of total RNA from UL and
IL groups. cDNA was then pooled into a PCR master
mix, which was then aliquoted and amplified with gene-specific primers
for a predetermined number of cycles (Table II). Individual transcript values were
normalized to those of 
-actin for both UL and
IL groups, and intra-assay variation was
controlled by expressing values as a percentage (%) of the control
UL values.
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The ME 49 strain of T. gondii was passaged as cysts in BALB/c mice. Twenty cysts of ME 49 strain were then injected i.p. per animal. Livers were harvested 24 h postinfection, and total RNA was extracted as described above.
Statistical analysis
Results were expressed as the mean ± SEM with n = 6 for all the Plasmodium infections. Student’s t test was used to compare differences between control and treatment groups, and the differences were assessed by a one-way ANOVA. The level of significance was set at p = 0.05.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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on freshly synthesized
UL and IL cDNA. The
amplification results demonstrated that these transcripts were
constitutively expressed in UL, although
quantitative expression levels were not determined (data not
shown).
To measure changes in gene expression of these transcripts via a
semiquantitative RT-PCR, cycle titration RT-PCR was first performed to
determine the exponential portion of the amplification curve for each
transcript (9). Fig. 1 shows
the plotted exponential curves of -actin, sema 4g, and TNF- in
the UL cDNA. The PCR cycle number that
corresponded to half the maximal amplification was determined for each
gene product from the liver, spleen, and kidney tissue samples and is
shown in Table II. cDNA from UL and
IL was subsequently amplified for the appropriate
number of cycles using gene-specific primers for Ring3, sema 4g, GCS,
p45 NF E2, IFN-, and TNF-. Transcript values (measured as counts
of [32P]deoxyribocytidine incorporation) were
then normalized to those of -actin for each tissue tested. Because
IFN- has been shown to participate in the inhibition of liver stage
parasites in immunized animals (10), and TNF-
expression has been shown to be inversely correlated with that of GCS
(11), transcript levels of these two cytokines were also
measured.
There was a 2-fold increase in expression levels of sema 4g and p45 NF
E2 compared with that in UL cDNA, whereas a 3-
and 4-fold increase in GCS and Ring3 expression levels were detected in
the IL cDNA. On the contrary, IFN- and TNF-
levels were both down-regulated in IL cDNA.
IFN- expression was decreased by 68%, whereas that of TNF- was
decreased by 64% relative to that of the UL
levels (Fig. 2).
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30%, whereas sema 4g, GCS, and p45 NF E2 were not
detected. Splenic IFN- expression from parasite-infected mice, was
10 times higher than in the UL sample. However,
TNF- expression level decreased by 30% (Fig. 3). In the kidney, Ring3 and sema 4g
expression was 6 and 4 times greater than that in the
UL, respectively, whereas p45 NF E2 and IFN-
were not detected. Lastly, TNF- was down-regulated by 44% in the
kidney (Fig. 4).
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and TNF- levels increased by 6- and 40-fold, respectively, to
those of the controls (Fig. 5).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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T cells,
CD4-CD8-NK1.1+TCR-int,
were up-regulated during the acute blood stage infection, they also
reported that these NK1.1+ cells were able to
inhibit parasite growth inside hepatocytes, and their killing effect
was reversed by anti-CD3 Ab (15). Evidence strongly
suggests that specific host responses occur during liver stage
infection and may represent the attempt by the host to control the
infection. This study further suggests that there are at least several
host transcripts whose expression pattern is altered due to
Plasmodium liver infection. Ring3 is localized to the MHC class II region in all species studied and, thus, it may participate in MHC presentation or gene expression (16). Ring3 is constitutively expressed in the liver, testis, lung, heart, and kidney (17). Limited functional analysis has revealed that Ring3 may be a serine/threonine kinase localized exclusively in the nucleus; however, a recent article by Platt et al. suggests that Ring3 is not a kinase but appears to recruit an as yet unidentified serine/threonine protein kinase into its complex (18). Expression of Ring3 is elevated upon cell proliferation and in patients with acute and chronic lymphocytic leukemia (19). Currently, the genomic sequence of the mouse Ring3 has been completed but the exact physiological function of Ring3 and its potential of being a kinase remain unknown. Although the significance of the increase of Ring3 expression in livers, spleens, and kidneys in P. yoelii-infected mice is equally unclear, its association with MHC class II suggests that it may be involved in the immune response of the host. The up-regulation of Ring3 expression in both Plasmodium-infected liver and kidney was much more dramatic than that in the infected spleen. Further investigation into the basic function of Ring3 is required to explain this phenomenon. One possibility could be that the liver and the kidney were simply more responsive than the spleen to changes resulting from the Plasmodium infection. In addition, the up-regulation of Ring3 was also observed in T. gondii-infected liver, suggesting that the increased level of Ring3 mRNA may be a generic and systemic host response to parasitic infection.
Sema 4g expression was similarly elevated in livers and kidneys but not
expressed in spleens of P. yoelii-infected mice. This was in
agreement with the recently published work by Li et al., which also
found sema 4g to be expressed only in liver, kidney, and brain
(19). Interestingly, infection with T. gondii
did not produce a change in the expression of sema 4g mRNA. Thus, the
increase of sema 4g expression appeared to be parasite specific.
However, the up-regulation of sema 4g expression in the infected livers
was not statistically significant; therefore, we concluded that the
up-regulation of sema 4g expression level appeared to be kidney
specific. The semaphorin family contains secreted and transmembrane
signal proteins that function in the nervous, cardiovascular, and
immune systems. Sema 4g was first isolated in the mouse
(20) where the protein contains semaphorin, a single
putative Ig-like transmembrane, and cytoplasmic domains. Sema 4g
belongs to semaphorin subclass 4, which is expressed in high levels in
lymphoid tissues, unlike other members of the semaphorin family
(21). Therefore, immune cells expressing sema 4g may be
involved in the P. yoelii liver infection. McKenna et al.
(4) recently reported that T cells participate in
the early response to the exoerythrocytic stage of the malarial
infection. It is reasonable to suggest that other immune cells may be
involved as well. Thus, the increased sema 4g expression could reflect
the expression of the gene in activated immune cells within the kidney
vasculature.
GCS initiates the synthesis of glutathione, a major cellular
antioxidant. Glutathione is involved in regulating the redox status of
cells (22). GCS is a heterodimer made up of a catalytic (H
chain) and a regulatory (L chain) subunit. Changes in GCS activity can
result from regulation at either the transcriptional or
posttranslational level, affecting only the heavy subunit or both of
the subunits. Conditions such as drug resistance, hormonal influences,
oxidative stress, and treatment with antioxidants have all been shown
to affect GCS expression and activity (22). Manna et al.
(11) reported that the overexpression of GCS blocked the
effect of TNF- on NF-
B activation, cytoplasmic I-B
degradation, nuclear translocation of p65, NF-B-dependent gene
transcription, and, most importantly, TNF--mediated cytotoxicity and
caspase 3 activation. The simultaneous up-regulation in GCS expression
and down-regulation of IFN- and TNF- in P.
yoelii-infected livers are consistent with the successful
development of mature parasites during this stage of malarial
infection. Type 1 cytokine responses, which include these two
cytokines, play a crucial role in the clearing of the exoerythrocytic
stage of the malaria parasites (10, 23). The diminished
expression levels of both cytokines could potentially promote parasite
survival in the liver. NF-B-dependent gene transcription includes
ICAM-1, VCAM-1, and E-selectin. The down-regulation of
NF-B-dependent gene transcription due to the overexpression of GCS
(11) may potentially contribute to the lack of neutrophil
aggregation in the P. yoelii-infected liver
(24) because these adhesion molecules are involved in the
recruitment of neutrophils to sites of infection.
As expected, both IFN- and TNF- transcript levels were
substantially elevated in Toxoplasma-infected livers.
Experimentally, one of the hallmark characteristics of Toxoplasmosis is
the elevated levels of IFN- and TNF-, which prevent uncontrolled
parasite growth and, consequently, host mortality (25).
These cytokines induce the production of NO, which is responsible for
the elimination of the parasites. Toxoplasma-infected livers
frequently appear discolored and display occasional foci of
inflammatory cells that are mediated by IFN- and TNF-. The
pathologic responses generated in Toxoplasma-infected livers
do not occur in Plasmodium-infected livers, so it might be
expected that IFN- and TNF- expression would not be up-regulated
in a Plasmodium-infected mouse.
IFN- expression levels in the spleen were elevated, whereas the
TNF- levels were reduced. This was unexpected because these
cytokines are usually coordinately expressed. This discordance cannot
currently be explained, but may be due to the differential responses of
tissues to P. yoelii infection. Alternatively, because a
large dose of P. yoelii sporozoites were inoculated into
each animal, the possibility exists that some sporozoites may be
cleared by the spleen, thus generating an immune response independent
to the liver.
p45 NF E2 is a member of the basic leucine zipper family of dimeric transcription factors. It consists of a widely expressed 18-kDa subunit and a tissue-restricted 45-kDa subunit (26). p45 NF E2 is known to function in the regulation of globin gene transcription and platelet production. Its expression level is reduced in apoptotic cells, although its binding capability to DNA is not altered (27). The up-regulation of p45 NF E2 in Plasmodium-infected livers but not in T. gondii livers is suggestive of an underlying anti-apoptotic and adaptive mechanism of infection inherent to P. yoelii.
Clearly, host responses generated as a result of Plasmodium infection exist (28, 29, 30, 31, 32). For instance, GPI is a potent Plasmodium glycolipid toxin that not only induces IL-1 production by macrophages during the blood stage of the infection, but also regulates glucose metabolism in adipocytes, resulting in profound hypoglycemia (28). It is postulated that GPI may be responsible for pleiotropic effects on a variety of host cells by substituting for the endogenous GPI second messenger signaling pathway of the host (29). Therefore, it is possible that the differential regulation of the reported host genes in P. yoelii-infected livers may be caused by parasite-derived material that affects the host locally or systemically. Further studies are needed to determine how Plasmodium actively influences intracellular processes that ultimately control gene transcription within infected cells of other tissues. Nevertheless, the changes in expression levels of many host gene products may facilitate parasite development and promote the survival of the parasite.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Audrey O. T. Lau, University of California at Los Angeles Department of Microbiology and Immunology, 1602 Molecular Sciences Building, 405 Hilgard Avenue, Los Angeles, CA 90095-1489.
3 Differentially displayed bands were detected exclusively in the IL samples.
4 Abbreviations used in this paper: Ring3, Really Interesting New Gene 3; GCS, glutamylcysteine synthetase; p45 NF E2, p45 NF erythroid 2; sema 4g, semaphorin subclass 4 member G; DD, differential(ly) display(ed); UL, sham-infected livers; IL, 24 h P. yoelii-infected livers.
5 Abbreviations used in this paper: Ring3, really interesting new gene 3; GCS, glutamylcysteine synthetase; p45 NF E2, p45 NF erythroid 2; sema 4g, semaphorin subclass 4 member G; DD, differential(ly) display(ed); UL, sham-infected livers; IL, 24 h P. yoelii-infected livers.
Received for publication August 25, 2000. Accepted for publication November 13, 2000.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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T cells are a component of early immunity against preerythrocytic malaria parasites. Infect. Immun. 68:2224. inhibits development of Plasmodium berghei exoerythrocytic stages in hepatocytes by an L-arginine dependent effector mechanism. J. Immunol. 146:3971.[Abstract]
-glutamylcysteine synthetase suppresses tumor necrosis factor-induced apoptosis and activation of nuclear transcription factor- B and activator protein-1. Oncogene 18:4371.[Medline]
and early death in C57BL/6 mice. J. Immunol. 163:2089. and TNF- expression in macrophages by protein tyrosine kinase mediated signal transduction. Biochem. Biophys. Res. Commun. 205:984.[Medline]
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