| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
* Unidad de Biomedicina, FES-Iztacala and
Instituto de Investigaciones Biomédicas, Universidad Nacional Autónoma de México (UNAM), Mexico City, Mexico;
Department of Medicine, Yale University School of Medicine, New Haven, Connecticut, USA;
Department of Pathology, Seth G. S. Medical College and KEM Hospital, Mumbai, India; and
¶ Department of Pathology, The Ohio State University Medical Center, and
|| Department of Microbiology, The Ohio State University College of Medicine, Columbus, Ohio, USA
1Correspondence: M.R.-S., Unidad de Biomedicina, FES-Iztacala, UNAM. Av. de los Barrios #1, Los Reyes Iztacala, 54090 Tlalnepantla, Edo. de México. Mexico. E-mail: rodriguezm{at}campus.iztacala.unam.mx; or A.R.S., Department of Microbiology, The Ohio State University, Columbus, Ohio, USA. E-mail: satoskar.2{at}osu.edu
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
than WT BMDCs after stimulation with soluble Toxoplasma antigen (STAg). Similar observations were made in CD11c+ low-density cells isolated from the spleens of MIF–/– mice challenged with STAg. MIF–/– C57BL/6 mice succumbed to ME49 infection faster than their WT counterparts. C57BL/6 mice that succumbed to infection with the ME49 strain produced less MIF than resistant BALB/c mice similarly infected. Interestingly, an analysis of brains from patients with cerebral toxoplasmosis showed low levels of MIF expression. Together, these findings demonstrate that MIF plays a critical role in mediating host resistance against T. Gondii.—Flores, M., Saavedra, R., Bautista, R., Viedma, R., Tenorio, E. P., Leng, L., Sánchez, Y., Juárez, I., Satoskar, A. A., Shenoy, A. S., Terrazas, L. I., Bucala, R., Barbi J., Satoskar, A. R., Rodriguez-Sosa, M. Macrophage migration inhibitory factor (MIF) is critical for the host resistance against Toxoplasma gondii.
Key Words: proinflammatory cytokines • innate immunity • cerebral toxoplasmosis
|
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
, and IFN-
. These cytokines are critical for controlling parasite growth in the brain and other organs, but they also induce immunopathology and tissue damage responsible for mortality (1
2
3
4)
.
Macrophage migration inhibitory factor (MIF) is a pleiotropic cytokine that is produced by a variety of cells, including activated macrophages and T cells (5
6
7)
. MIF inhibits the anti-inflammatory effects of corticosteroids and plays a critical role in the pathogenesis of sepsis (8
9
10
11)
. High levels of MIF are also observed in patients with autoimmune inflammatory diseases such as rheumatoid arthritis (12)
and chronic colitis (13)
. Experimental studies using MIF-neutralizing antibodies (Abs) as well as MIF–/– mice show that this cytokine is involved in pathogenesis of such inflammatory diseases as collagen-induced arthritis, immunologically induced kidney diseases, and colitis (14)
.
MIF plays a critical role in determining the outcome of infections caused by a variety of pathogens, including bacteria (15
16
17)
, parasites (18
19
20
21)
, and viruses (22
, 23)
. The role of MIF in parasitic infections appears complex. MIF is critical for the host defense against Leishmania major (21)
, Trypanosoma cruzi (20)
, Taenia crassiceps (19)
and Schistosoma japonicum (24)
by enhancing the microbicidal activity of the innate response. However, MIF’s proinflammatory action may promote the pathogenesis of Plasmodium infection by inhibiting erythropoiesis and promoting anemia (25
, 26)
.
To investigate the role of MIF in toxoplasmosis, we analyzed the course of T. gondii infection in MIF–/– mice backcrossed to either the Th2-prone (BALB/c) or the Th1-prone (C57BL/6) genetic backgrounds. MIF–/– BALB/c mice succumbed significantly faster than wild-type (WT) mice after challenge with virulent RH as well as moderately virulent M49 strains of T. gondii. The increased susceptibility of MIF–/– BALB/c mice to T. gondii was associated with a poor induction of early inflammatory response, increased parasite loads, and impaired production of IL-12 and TNF- by the dendritic cells (DCs). MIF–/– C57BL/6 mice also were highly susceptible to the ME49 strain of T. gondii and succumbed to infection faster than the WT counterparts. In the infected brains of patients who died of toxoplasmosis, the expression of MIF was low compared with the brains of patients who died from fungal meningitis. These findings indicate that MIF plays a critical role in mediating protection against T. gondii infection by regulating early inflammatory responses.
|
MATERIALS AND METHODS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
and backcrossed for more than 10 generations to a BALB/c genetic background. In some experiments, female C57BL/6 mice (Harlan, Mexico City, Mexico) and MIF–/– in C57BL/6 genetic background were used.
Parasites and experimental infections
Tachyzoites of the virulent RH were maintained in vitro by infection of human foreskin fibroblast and biweekly passage in 10% fetal bovine serum/Dulbecco modified Eagle medium (DMEM; Invitrogen, Carlsbad, CA, USA) containing penicillin (100 U/ml), and streptomycin (100 µg/ml; Life Technologies, Inc. BLR, Grand Island, NY, USA). Cysts of the avirulent ME49 strain were harvested from the brains of C57BL/6 mice that had been inoculated with 20 cysts i.p. 1–2 months before. For experimental infections, BALB/c and MIF–/– (BALB/c background) received either 100 tachyzoites of the RH strain or 40 ME49 cysts or PBS i.p. Control inoculations with normal brains failed to elicit detectable inflammatory responses or significant increase in cytokine levels. Soluble Toxoplasma antigen (STAg) was prepared as described previously (27)
.
In vivo analysis of acute and chronic infection
Acute tachyzoite growth was assessed using cytospin analysis of peritoneal exudate cells (PECs) 5 days after infection. Samples were prepared from 5 x 105 PECs in a cytospin (Hermle, Wehingen, Germany) set for 7 min at 1000 rpm. Slide preparations were fixed in absolute methanol and stained with Wright-Giemsa stain (Sigma-Aldrich, St. Louis, MO, USA). Analysis of the number of infected cells was performed on 300–400 cells using an oil immersion objective (x100; Sigma).
To assess chronic disease progression, mice were killed by CO2. Brain and liver tissue were removed aseptically. Brain tissue was homogenized in 2 ml of PBS. The total number of cysts was determined by counting the cysts in a 10 µl suspension and multiplying by 200. Parallel semiquantification of parasite-specific sequences of DNA was performed on the same brain samples in order to confirm the microscopic findings. Livers were fixed in a solution that contained 10% formalin, 70% ethanol, and 5% acetic acid (all from Sigma-Aldrich). Sagittal sections of livers (5 µm thick) were stained with hematoxylin and eosin (Sigma-Aldrich).
Cytokine measurement
IL-12p70, TNF-, IL-1β, IL-18, IL-4, IL-10, and IFN- levels in sera or culture supernatant were measured by ELISA, using commercial kits purchased from Peprotech (Rocky Hill, NJ, USA).
Splenic low-density (LOD) cell purification
Mice were injected i.p. with STAg at 25 µg/mouse and 18 h later; spleens were removed, and splenic low-density DCs were isolated as recently described (28)
.
Flow cytometry
PECs from infected mice (40 cysts of ME49 strain/mouse), BMDCs stimulated with 2.5 µg/ml of STAg or lipopolysaccharide (LPS; 0.5 µg/ml) for 18 h, or LOD cells from STAg-injected mice were isolated, washed, first incubated with anti-CD16/CD32 (Biolegend, San Diego, CA, USA) for 30 min at 4°C, and then suspended in 3% BSA-PBS. Phenotypic analysis of PECs, LOD cells, or DCs was conducted using direct immunofluorescent staining and FACS analysis. Cells (1x106/ml) were incubated with fluorescein isothiocyanate- and/or phycoerythrin (PE) -labeled Abs F4/80, CCR5, IFN-R, TNF-R, Toll-like receptor (TLR) -4, TLR-2, or CD11c (all from Biolegend) at 4°C for 30 min. After incubation, cells were washed several times in buffer, fixed in 1% paraformaldehyde (Sigma-Aldrich), and stored at 4°C in darkness before analysis using a FACS Calibur and CellQuest software (Becton Dickinson, Franklin Lakes, NJ, USA).
Semiquantitation of parasite burden
Brains from T. gondii-infected animals were collected at days 10, 20, and 30 postinfection (p.i.). DNA was extracted from tissues using the Qiamp tissue kit (Qiagen, Chatsworth, CA, USA), and 100, 25, and 12 ng of each sample was analyzed by polymerase chain reaction (PCR). Amplification of parasite DNA was performed using primers specific for a 35-fold repetitive sequence of the Toxoplasma B1 gene (5'-GGAACTGCATCCGTTCATGAG-3' and 5'-TCTTTAAAGCTTCGTGGT C-3'), which is found in all known parasite strains (29)
. A 134-bp competitive internal standard containing the same primer template sequences as the 194-bp B1 PCR fragment was also synthesized (30)
.
Cytokine PCR
BMDCs were stimulated with 2.5 µg/ml of STAg for 18 h, and RNA from the culture samples was collected using Trizol (Invitrogen) according to the manufacturer’s instructions. Reverse transcription was performed using Moloney murine leukemia virus reverse transcriptase (Invitrogen) and random hexamer primers (Promega, Madison, WI, USA). Levels of mRNA for IL-12p40, IL-12p35, IL-12p19, and MIF (mRNA obtained from brains) were measured by quantitative real-time PCR using the PQRS quantitative method. The tissues from uninfected mice were used to establish a baseline value of 1.0, against which the level of message for cytokine in the test mice was quantitated.
Immunostaining of MIF
Formalin-fixed paraffin-embedded sections of brains from 5 patients who died from cerebral toxoplasmosis were stained using a rabbit polyclonal anti-MIF Ab raised to pure, recombinant human MIF (25)
. All 5 patients died at KEM Hospital (Mumbai, India), and were males aged 60–70 yr. Sections from a normal brain and from a patient who died from fungal encephalomyelitis were used as negative and positive controls, respectively. Nonspecific background staining was blocked by treating the sections with mouse antiserum. After blocking, sections were incubated overnight with anti-MIF Ab (1:1000) in PBS 1% BSA at 4°C. The slides were washed and incubated with anti-rabbit immunoglobulin-horseradish peroxidase (Dako; 1:500; Biolegend) in 1% BSA PBS for 30 min at room temperature, washed, and treated with diaminobenzidine substrate (Sigma-Aldrich) for 5 min. The slides were counterstained with hematoxyline (Sigma-Aldrich).
Statistical analysis
Data are expressed as means ± SD. The statistical significance of differences in mean values was determined using Student’s t test. Survival data are presented as a Kaplan-Meier survival curve and analyzed with log-rank test. Differences of at least P < 0.05 are considered significant.
|
RESULTS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
). Next, we examined the course of T. gondii infection in MIF–/– BALB/c mice after challenge with the moderately virulent strain of T. gondii ME49. MIF–/– BALB/c mice challenged i.p. with 40 cysts rapidly showed clinical signs for the disease that were sustained for 7 days, whereas WT mice showed light symptoms. By day 10 p.i., MIF–/– mice started to lose weight and showed piloerection and prostrated behavior (Supplemental Fig. 1). ME49-infected MIF–/– mice began to succumb as early as day 10 p.i., reaching 90% mortality by day 30 p.i.. In contrast, WT mice survived until day 70 and did not show any other symptoms related to the infection (Fig. 1B
). MIF–/– mice had significantly more parasitized macrophages in their peritonea than WT mice early after i.p. challenge with ME49 (Fig. 2A
) and also contained significantly more T. gondii cysts in their brains on days 10 and 30 p.i. (Fig. 2B
, P=0.0012). To further confirm our microscopic findings, we quantified the level of T. gondii DNA in the brains by semiquantitative PCR as well as brain sections of both groups of mice. The level of parasite DNA in the infected brains of WT and MIF–/– mice correlated with the numbers of cysts, confirming higher parasite burden in the brains of MIF–/– mice (Fig. 2C
). Furthermore, hematoxylin-eosin-stained sections on day 10 p.i. showed that cysts were already formed in the brains of MIF–/– but not WT mice at this point. Taken together, these data suggest that MIF is critical for the host defense against T. gondii and that increased mortality in MIF–/– mice is at least in part caused by their inability to restrict parasite replication.
|
|
MIF–/– mice develop significant liver pathology after T. gondii infection
Because of the remarkable difference in mortality between infected MIF–/– and control mice after infection with ME49, we performed histopathological analysis of livers at day 10 p.i. Infected livers from MIF–/– mice showed significant inflammation associated with small granulomas as well as a more remarkable reactive hepatitis (Fig. 3A
) and scattered hepatic inflammatory nodules. In contrast, inflammatory changes in livers of infected WT mice were less profound (Fig. 3A
). To further quantify the extent of the liver damage, we measured levels of transaminase enzymes (ALT and AST) in sera of both groups of mice 2 wk after infection. Both MIF–/– and WT mice showed a significant increase in transaminases in their sera after infection, but the levels of both enzymes were significantly higher in ME-49-infected MIF–/– mice compared with their WT counterparts (Fig. 3B, C
). These data suggest that T. gondii infection induces a severe inflammatory and functional hepatic damage in MIF–/– mice.
|
Impaired early proinflammatory cytokine and nitric oxide production during infection of MIF–/– mice
Several studies have shown that MIF regulates production of proinflammatory cytokines such as IL-12 and TNF- by macrophages in infectious as well as noninfectious diseases (31)
. We therefore measured levels of proinflammatory cytokines in sera from WT and MIF–/– mice on days 0, 1, 2, 3, 5, and 15 after T. gondii infection. After infection with the RH strain, MIF–/– mice produced significantly less IL-12, IL-1β, TNF-, IL-18, and IFN- than WT controls at all the time points examined (Fig. 4A-E
). Similar differences were observed in the production of proinflammatory cytokines as well as nitric oxide (NO) between WT and MIF–/– mice infected with ME49 strain with the exception of IL-18, which was produced in comparable amounts in WT and MIF–/– mice (Fig. 5A-D
). Interestingly, MIF–/– mice that survived T. gondii infection eventually produced IFN- at a level comparable to WT mice. It is noteworthy that anti-inflammatory cytokines such as IL-4 and IL-10 were produced in comparable levels in both MIF–/–- and MIF+/+-infected mice (data not shown; Figs. 4F
and 5E
).
|
|
Altered expression of macrophage markers and impaired responses in DCs from MIF–/– mice
It is well known that macrophages and DCs are very important innate cells that respond promptly to T. gondii infection as well as to its soluble antigen, STAg. Moreover, it is known that CCR5 is one of the main receptors for STAg involved in triggering the early production of IL-12 and TNF- (32)
. To determine whether MIF deficiency phenotypically and/or functionally alters either or both these cell populations, we analyzed the expression of CCR5 on PECs recruited 5 days after i.p. challenge with ME49 or RH strains. In addition, we compared cytokine production in DCs isolated from WT and MIF–/– mice in response to STAg in vitro and in vivo. Both ME49-infected and RH-infected MIF–/– mice contained significantly fewer macrophages (F4/80+) expressing CCR5 in their peritonea compared with WT mice (Fig. 6
). To determine whether the lack of MIF alters the function of DCs, we first compared cytokine production by BMDCs from WT and MIF–/– in response to stimulation with STAg in vitro. BMDCs from MIF–/– mice displayed significantly lower transcripts for IL-12p35, IL-12p40, and Il-12p19 and also produced less IL-1β and TNF- in response to STAg than BMDCs from WT mice (Fig. 7A-B
). Similar results were obtained using LPS as stimulus (data not shown). To determine whether MIF deficiency alters DC function in vivo, we measured spontaneous IL-12 and TNF- production by CD11c+ DCs isolated from the spleens of WT and MIF–/– mice 18 h after i.p. challenge with STAg or T. gondii. The absence of MIF did not alter the number (percentage) of DCs (CD11c+) in spleens (Fig. 7C
); however, spontaneous IL-12 and TNF- production by CD11c+ LOD cells was significantly diminished in MIF–/– mice in both cases, with STAg injection (Fig. 7C
) and in T. gondii infection (data not shown).
|
|
Expression of TNF-R, IFN-R, and TLR-4, but not TLR-2, is impaired in T. gondii-infected MIF–/– mice
MIF has been shown to regulate the expression of receptors such as IL-1R and p55 TNFR1 on immune cells (33)
. Hence, we analyzed by flow cytometry the levels of TNF-R, IFN-R, TLR-4, and TLR-2 on PECs isolated from ME49-infected WT and MIF–/– mice. ME49-infected MIF–/– mice displayed at least 30% lower expression of IFN-R, TLR-4, and TNF-R, mainly associated with F4/80+ cells (Fig. 6)
. In contrast, expression of TLR-2, a molecule that may be involved in T. gondii recognition, was unaltered. Similarly, splenocytes (CD4+) from the infected MIF–/– mice showed lower expression of IFN-R compared with splenocytes from WT-infected mice (Supplemental Fig. 2). Thus, it is possible that cells from MIF–/– mice have a lesser chance to rapidly respond to T. gondii infection, given that TNF- and IFN- are involved in NO production, a main molecule related to protection during T. gondii infection. Indeed, sera levels of NO were significantly inhibited in MIF–/– mice during infection (Fig. 5F
).
Susceptible C57BL/6 mice produce less MIF than resistant BALB/c mice after T. gondii ME49 infection
Several studies have shown that Th1-prone C57BL/6 mice, but not Th2-prone BALB/c mice, are highly susceptible to the ME49 strain of T. gondii and rapidly succumb to infection. We therefore measured MIF in sera from ME49-infected C57BL/6 and BALB/c mice. In addition, we compared mRNA levels of MIF in infected brains from both these strains by real-time reverse transcriptase-PCR (RT-PCR). On day 6 p.i., ME49-infeced C57BL/6 mice contained significantly less MIF in their sera compared with BALB/c mice (Fig. 8A
). Furthermore, the brains from ME49-infected C57BL/6 mice displayed significantly lower levels of MIF mRNA compared with those from BALB/c mice (Fig. 8B
). These findings suggest that impaired production of MIF in C57BL/6 mice may contribute to their increased susceptibility to ME49.
|
MIF is expressed in low levels in the infected brains from the patients who died of encephalitic toxoplasmosis
Finally, to link our data with human toxoplasmosis, we performed immunohistochemistry assays for MIF in specimens of brain from patients who died of either encephalitic toxoplasmosis or cryptococcal encephalitis without AIDS. Figure 9
shows the high intense and extensive reactivity to MIF in brains with cryptococcal fungal infection; in contrast, in brains with toxoplasmosis, almost no MIF reactivity was observed.
|
|
DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
The proinflammatory cytokines IL-1β, IL-12, TNF-, and IFN- play an important role in mediating host resistance against T. gondii (34
, 35)
, and MIF has been shown to promote IL-12 and TNF- production in macrophages as well as enhance their microbicidal activity by increasing production of NO (18
, 31)
. In the present study, RH-infected MIF–/– BALB/c mice produced significantly less IL-1β, IL-12, IL-18, TNF-, and IFN- during the early phase of infection. These differences are associated with the infection processes, given that uninfected WT and MIF–/– mice displayed close levels of proinflammatory cytokines. Similar differences were also noted in the levels of IL-1β, IL-12, TNF-, IFN-, and NO in sera from MIF–/– and WT BALB/c mice infected with ME49 during early infection, although ME49-infected MIF–/– mice that survived the infection eventually produced as much proinflammatory cytokines and NO as WT mice (data not shown). Furthermore, the deficient production of proinflammatory cytokines in ME49-infected MIF–/– mice was also associated with a significant increase in the number of cysts in their brains. These results indicated that MIF is critical for the efficient induction of early but not late inflammatory mediator response after T. gondii infection. In addition, they suggest that MIF may mediate resistance against acute T. gondii infection either by directly enhancing macrophage microbicidal activity, by regulating early proinflammatory cytokine production, or even by up-regulating cytokine receptor expression such as TNF-R and IFN-R, all required for control of the intracellular pathogen.
Previous studies have reported that lethal toxoplasmosis in CD1 or C57BL/6 mice after type I or type II strain infection is associated with significant liver damage and with marked elevation in the serum levels of IL-18 and IFN- (36)
. Mordue et al. (37)
showed that neutralization of IL-18 in RH-infected CD1 mice prolonged their survival, suggesting that IL-18 is involved in mediating pathology during toxoplasmosis. In the present study, WT BALB/c mice showed a significant increase in serum levels of IL-18 and IFN- after both RH and ME49 infection and developed mild to moderate hepatic pathology. In contrast, T. gondii-infected MIF–/– BALB/c mice produced less IL-18 and IFN- than WT mice but developed more severe liver damage than the latter, which was associated with an increase in levels of liver enzymes AST and ALT, suggesting that increased liver inflammation is caused by tissue damage from uncontrolled parasite growth rather than an increased inflammatory response. Thus, IL-18 and IFN- are apparently not involved in mediating inflammatory hepatic damage and lethality during acute type I or type II T. gondii infection in MIF–/– BALB/c mice. Because MIF–/– BALB/c mice produced less natural killer (NK) cell-activating IL-12 and IL-18, impaired IFN- production during early infection in these mice can be explained by an inadequate activation of NK cells (38)
. WT and MIF–/– mice, however, contained comparable numbers of DX5+ NK cells in their peritonea early after ME49 challenge, as measured by flow cytometry (data not shown). Also, administration of recombinant IL-18 to ME49-infected MIF–/– mice restored IFN- production (data not shown). It is therefore unlikely that impaired IFN- production in T. gondii-infected MIF–/– mice is the result of an intrinsic defect in NK cell activity. This is perhaps not surprising since a previous study has shown that MIF inhibits rather than augments NK cell function (39)
.
MyD88-dependent TLR signaling pathway is indispensable for immunity against T. gondii (40
, 41)
. Both TLR2 and TLR4 also participate in host defense against T. gondii, and their activation by T. gondii GPIs is required for optimal TNF- production by macrophages (42
43
44)
. MIF has been shown to induce production of proinflammatory cytokines in macrophages by regulating their responsiveness to LPS by modulating TLR4 expression (45)
. Furthermore, Toh et al. (33)
have shown that endogenous MIF is required for efficient expression of p55 TNFR1 and IL-1R. In the present study, PECs from MIF–/– mice expressed significantly less TLR4, IFN-R, TNFR1, and CCR5 on macrophages compared with WT mice, but levels of TLR2 were comparable in both. Expression of IFN-R was also lower on CD4+ and CD8+ T cells from ME49-infected MIF–/– mice (Supplemental Fig. 2). Thus, low expression of TLR4, IFN-R, and TNFR1 on immune cells may contribute to impaired proinflammatory cytokine responses, whereas low levels of CCR5 on MIF–/– macrophages may reduce their migration to the site of infection as well as the ability to directly and promptly respond to T. gondii infection, given that CCR5 is critically involved in responding to T. gondii antigens (32)
. Both these defects can contribute to increased susceptibility of MIF–/– mice to T. gondii.
The DCs play a critical role in development of acquired immunity against T. gondii (28)
. They function as antigen-presenting cells and also as the main producers of IL-12 and TNF- in response to T. gondii antigens (46)
. IL-12 induces early IFN- production in NK cells and facilitates subsequent Th1 development whereas TNF- is involved in mediating resistance to acute and chronic T. gondii infections. MIF has been shown to activate antigen-presenting DCs and induce their production of proinflammatory cytokines (47)
. We found that BMDCs from MIF–/– mice produced significantly less IL-1β, IL-12, and TNF- than WT BMDCs after in vitro stimulation with STAg. Furthermore, CD11c+ LOD cells isolated from the spleens of STAg-challenged or ME49-infected MIF–/– mice also produced less IL-12 and TNF- than WT mice. These data agree with recent observations suggesting that CD11+ LOD cells are mainly involved in the early IL-12 production after STAg challenge in vivo (28)
. Collectively, these results indicate that MIF is required for efficient production of proinflammatory cytokines from the DCs during T. gondii infection. They also suggest that impaired proinflammatory cytokine responses in T. gondii-infected MIF–/– mice are at least in part the result of reduced production of these cytokines in MIF-deficient DCs.
Both T cells and B cells have been shown to play important roles in adaptive immunity against T. gondii (48)
. MIF controls T-cell activation (49)
and also plays a role in maintaining mature B-cell populations. MIF deficiency, however, seems to have minimal effect on B-cell and T-cell responses during T. gondii infection because ME49-infected WT and MIF–/– displayed comparable titers of T. gondii-specific Th1-associated IgG2a and Th2-associated IgG1 Abs (Supplemental Fig. 3). In addition, spleen cells from WT and MIF–/– mice proliferated similarly in response to STAg (data not shown). These data are consistent with previous reports on other parasitic models, which found that parasite-specific T-cell responses are not altered in MIF–/– mice (19
, 21)
.
It has been well established that type II strains of T. gondii are prevalent in animals and are usually associated with T. gondii infection in humans. Infections caused by T. gondii type II strains are asymptomatic and nonlethal in immunocompetent humans. Similarly, genetically resistant mice such as BALB/c also develop a nonlethal infection when infected with type II strains such as ME49. Because our results in the present study showed that MIF was critical for mediating immunity and reducing lethality in BALB/c mice after ME49 infection, we hypothesized that MIF may be involved in preventing lethal toxoplasma encephalitis (TE) in humans infected with T. gondii. We therefore analyzed expression of MIF in infected brains of patients who died from TE by immunohistochemistry and compared the results with patients who died from fungal infection of the brain. The brains from TE patients contained fewer MIF-expressing inflammatory cells compared with those from the patients who died from fungal infection, which expressed high levels of MIF. These findings suggest that MIF may play a role in preventing TE and controlling T. gondii infection in humans. This possibility is further supported by the evidence that the impaired production of MIF in patients with AIDS is associated with rapid development of tuberculosis (50)
, therefore another opportunistic infection disease such as TE may also be accelerated. Indeed, a recent report showed that MIF production is enhanced on human placenta after STAg stimulation, which may imply MIF as an early defense mechanism against mother-child transmission of toxoplasmosis (51)
. The exact mechanism by which MIF may prevent TE is not clear, however, and is under investigation in our laboratory. Activated microglia has been shown to inhibit T. gondii growth in the brain via an NO-dependent mechanism (52)
, and it is likely that MIF is required for optimal microglia activation and NO production. Indeed, NO production has been largely known to be significantly diminished in MIF–/– mice (53)
, and as we show here, systemic levels of NO (in sera) were significantly diminished in MIF–/– T. gondii-infected mice. In addition to the NO-mediated mechanism, Yap et al. (54)
recently suggested that TNF-dependent resistance of genetically susceptible C57BL/6 mice against TE involves an effector function distinct from NOS2 activation. In the present study, levels of mRNA for TNF- in the brain were not analyzed, but low production of TNF- in T. gondii-infected MIF–/– mice suggests that a protective role of MIF in prevention of TE may, at least in part, be the result of its ability to regulate TNF- production.
In conclusion, our findings show that endogenous MIF plays a critical role in controlling T. gondii infection regardless of the host genetic background or parasite strain. Furthermore, they indicate that MIF mediates immunity against T. gondii by regulating innate proinflammatory cytokine responses as well as DC function. Impaired expression of MIF in the brains of patients who died from TE suggests that MIF is also likely to be involved in conferring resistance to human toxoplasmosis.
|
ACKNOWLEDGMENTS |
|---|
Received for publication April 21, 2008. Accepted for publication June 5, 2008.
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
, and the chemokines macrophage-inflammatory protein-1 and -1 β in response to Toxoplasma gondii antigens. J. Immunol. 162,7369-7375+ dendritic cells. Nat. Immunol. 1,83-87[CrossRef][Medline]: the major mediator of resistance against Toxoplasma gondii. Science 240,516-518-interferon. J. Med. Microbiol. 52,869-876This article has been cited by other articles:
|
|
 |
|
  I. Assuncao-Miranda, F. A. Amaral, F. A. Bozza, C. T. Fagundes, L. P. Sousa, D. G. Souza, P. Pacheco, G. Barbosa-Lima, R. N. Gomes, P. T. Bozza, et al. Contribution of macrophage migration inhibitory factor to the pathogenesis of dengue virus infection FASEB J, January 1, 2010; 24(1): 218 - 228. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
12 mice/group. B) Survival rate of WT and MIF–/– mice infected with 40 cysts of T. gondii strain ME49. Experiment is representative of 3 separate experiments; 5 mice/group.
