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Antimicrobial Agents and Chemotherapy, May 2006, p. 1798-1804, Vol. 50, No. 5
0066-4804/06/$08.00+0     doi:10.1128/AAC.50.5.1798-1804.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Minocycline Impedes African Trypanosome Invasion of the Brain in a Murine Model

Willias Masocha,^1* Martin E. Rottenberg,² and Krister Kristensson¹

Department of Neuroscience,¹ Microbiology and Tumor Biology Center, Karolinska Institutet, SE-171 77 Stockholm, Sweden²

Received 12 October 2005/ Returned for modification 28 November 2005/ Accepted 17 February 2006

Top Abstract Introduction Materials and Methods Results Discussion References

 
Passage of Trypanosoma brucei across the blood-brain barrier (BBB) is a hallmark of late-stage human African trypanosomiasis. In the present study we found that daily administration of minocycline, a tetracycline antibiotic, impedes the penetration of leukocytes and trypanosomes into the brain parenchyma of T. brucei brucei-infected C57BL/6 mice. The trypanosome-induced astrocytic and microglial reactions were reduced in the minocycline-treated mice, as were the levels in the brain of transcripts encoding adhesion molecules intercellular adhesion molecule 1 (ICAM-1) and endothelial-leukocyte adhesion molecule 1 (E-selectin); the inflammatory cytokines tumor necrosis factor alpha, interleukin-1 (IL-1), IL-1ß, IL-6, and gamma interferon; and matrix metalloprotease 3 (MMP-3), MMP-8, and MMP-12. Loss of weight occurring during infection with T. b. brucei was not observed after treatment of the mice with minocycline; these mice also survived longer than nontreated mice. Invasion of trypanosomes and leukocytes into the brain parenchyma most likely triggered the loss of weight and death of infected animals, since minocycline did not affect the growth of T. b. brucei either in vitro or in vivo or the levels of the transcripts encoding the cytokines and MMPs in the spleen. In conclusion, our data show that T. b. brucei invasion of the brain is related to that of leukocytes and that minocycline can ameliorate the disease in trypanosome-infected mice.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Human African trypanosomiasis (HAT) or sleeping sickness is caused by the inoculation of subspecies of Trypanosoma brucei by infected tsetse flies. T. brucei gambiense, which occurs in West and Central Africa, causes a chronic form of the disease, whereas T. brucei rhodesiense, which is found in East Africa, causes an acute variant of the disease. Clinically, the disease is divided into two stages: the early stage, in which the parasites invade the hemolymphatic system, and the late meningo-encephalitic stage, with severe signs of nervous system involvement (3, 12). If not treated, the infection is lethal. Neuropathologically, the changes are characterized by an infiltration of inflammatory cells in the brain, which is most prominent in the white matter (leukencephalitis), accompanied by a marked activation of astrocytes and microglia (1, 11).

In a mouse model of the disease, parasites penetrate the BBB at a late stage and can enter the brain parenchyma with preserved tight junction proteins in the cerebral vessels (16). The penetration of trypanosomes across the BBB shows certain similarities to that of leukocytes since endothelial basement membranes, which form part of the BBB, containing laminin 8 are permissive for both T-cell and T. brucei brucei transmigration, whereas those containing laminin 10 are restrictive (15, 23). Minocycline, a second-generation tetracycline antibiotic with multiple biological effects distinct from its antimicrobial actions, has been demonstrated to reduce the number of leukocytes invading the central nervous system (CNS) parenchyma in experimental allergic encephalitis (EAE) (2, 19), and this effect contributes to its therapeutic activity against the disease. The drug also reduces the expression or activity of molecules associated with leukocyte transmigration from the blood vessels into an inflammatory site, such as adhesion molecules (8), cytokines and chemokines and their receptors (10), and matrix metalloproteases (MMPs) (2, 19), which can degrade components of the extracellular matrix and basement membranes.

We investigated the effect of minocycline on the brain parenchyma invasion by T. b. brucei in a murine model of the infection. We report that treatment with minocycline reduced penetration of the parasite, as well as CD45⁺ leukocytes, into the brain parenchyma, prevented weight loss, and prolonged the survival of infected mice. This was paralleled by reduced levels of adhesion molecules, cytokines, and MMP transcripts, as well as reduced microglia and astrocyte activation, in the brain.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Mice, parasites, infection, and minocycline treatment. All experiments were conducted according to protocols that received institutional approval and authorization by the local animal ethical committee (Stockholms Norra Djurförsöksetiska Nämnd, project N451/03). Efforts were made to minimize animal numbers and the suffering of the animals used. C57BL/6 mice were used and were supplied by the breeding unit at the Microbiology and Tumor Biology Center, Karolinska Institutet, Stockholm, Sweden, and were kept with food and water ad libitum under specific-pathogen-free conditions.

Mice (8 to 12 weeks old) were infected by intraperitoneal (i.p.) injection with 2,000 to 3,000 parasites of a pleomorphic stabilate of T. b. brucei, AnTat 1.1E, derived from stabilate EATRO 1125 (passaged in C57BL/6 mice; obtained from N. van Meirveinne, Laboratory of Serology, Institute of Tropical Medicine "Prince Leopold," Antwerp, Belgium). Parasites were diluted in 60 mM phosphate-buffered saline (PBS) containing 40 mM glucose. Mice were treated i.p. daily with minocycline (Sigma, Steinheim, Germany) or its vehicle (PBS), commencing on the day of T. b. brucei inoculation. Control infected mice were injected with 200 µl of PBS daily, while the minocycline-treated infected mice received 50 mg of the drug/kg twice a day for the first 2 days and once daily for the next 5 days, followed by 25 mg/kg for the subsequent days until the animals were sacrificed. These doses were chosen based on those reported to reduce T-cell invasion of the CNS during EAE (19). Animals were weighed and checked daily for signs of disease. Blood samples were taken from the tip of the tail during the course of infection to assess parasitemia by using the Herbert and Lumsden chart (5).

In vitro studies. T. b. brucei, freshly isolated from infected C57BL/6 mice, were separated by DEAE-cellulose chromatography and incubated in HMI-9 medium at 37°C and 5% CO₂ as described previously (6, 13). The number of mobile trypanosomes was counted by using a Neubauer hemacytometer after 24, 48, and 72 h in the presence or in the absence of minocycline (covering a range of 1 to 20 µg/ml). The concentration range of minocycline used for the in vitro experiments was chosen taking into consideration that the doses of minocycline used in vivo produced a peak concentration of 7 µg/ml in plasma in rats (2, 19).

Immunohistochemistry. Mice were deeply anesthetized with isoflurane and sacrificed by decapitation, and the brains were dissected at different times after infection for immunohistochemistry. To examine the passage of trypanosomes across the BBB, sections at a level of the lateral ventricles containing the choroid plexus and the septal nuclei were cut, mounted, fixed, and immunostained with anti-AnTat 1.1 VSG (1:5,000; kindly provided by N. van Meirveinne) and goat polyclonal anti-glucose transporter 1 (1:40; GLUT-1; Santa Cruz Biotechnology, Santa Cruz, CA) as described previously (15). Additional sections were incubated with anti-glial fibrillary protein antibodies (anti-GFAP; 1:100; Dako, Glostrup, Denmark) to immunostain astrocytes. For the labeling of microglia, another set of sections was fixed in methanol for 10 min at –20°C and incubated with 2% H₂O₂ in methanol for 10 min at room temperature, rinsed in PBS, and further incubated with biotinylated tomato lectin (1:50, 20 µg/ml; Sigma, St. Louis, MO) overnight at 4°C. The tomato lectin binding was detected and visualized by using the Vectastain ABC and DAB peroxidase substrate kits as instructed by the manufacturer (Vector Laboratories, Burlingame, CA).

To examine the presence of leukocytes in the brain parenchyma, the sections were fixed in methanol for 10 min at –20°C and rinsed in PBS prior to immunohistochemical processing. The sections were immunostained with rat anti-mouse CD45 (1:50; BD Biosciences Pharmingen, San Diego, CA) and goat anti-GLUT-1 as described above. Sections were examined and analyzed by using a Nikon fluorescence microscope. Photomicrographs were taken with a Zeiss AxioCam digital camera.

The number of VSG-immunopositive T. b. brucei in five ocular fields (viewed through 10x ocular and 20x objective lenses) from the cortex and corpus callosum on either side of the midline was determined and in four fields from the septal nucleus in four animals from each postinfection (p.i.) time point. The parasites were divided into two groups according to their relationship with the vessels; intravascular or extravascular. CD45⁺ leukocytes were counted in a manner similar to that described for the parasites.

Real-time reverse transcription-PCR. Gene transcripts of several adhesion molecules, proinflammatory cytokines, MMPs, and tissue inhibitors of metalloproteases (TIMPs) (Tables 1, 2 and 3) were quantified in the brains of minocycline-treated and PBS-treated uninfected and infected mice by real-time PCR. Total RNA was extracted from half of the fresh frozen brains and reverse transcribed, and the transcripts levels were quantified on an ABI Prism 7000 sequence detection system (Applied Biosystems) as described previously (15). The sequences of the primers used are listed in Tables1 2 and 3. Tenfold dilutions of a cDNA sample were amplified to control amplification efficiency for each primer pair. Thereafter, the threshold cycle (C_T; i.e., the fractional cycle number at which the fluorescence passes the fixed threshold) values were obtained for all cDNA samples. The amount of transcripts of individual animal samples (n = 4 per group) was normalized to cyclophilin (C_T). The relative amount of target gene transcripts was calculated by using the 2^–CT method as described previously (14). These values were then used to calculate the means and standard errors of the mean (SEM) of the relative expression of the target gene mRNA in the brains of uninfected and infected mice.

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TABLE 1. PCR primer sequences of cyclophilin, adhesion molecules, and cytokines

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TABLE 2. PCR primer sequences of TIMPs and MMPs

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TABLE 3. PCR primer sequences of MMPs

Statistical analyses were performed by using the software GraphPad Prism version 3.00 (GraphPad Software, Inc.). For weight changes and parasitemia comparisons, two-way analysis of variance (ANOVA), followed by Bonferroni post-tests, was performed. One-way ANOVA, followed by a Newman-Keuls multiple comparison test, was used to compare the transcript levels of adhesion molecules, cytokines, and MMPs. The densities of trypanosomes and CD45⁺ cells in the brain parenchyma were compared by using the Mann-Whitney U test. The differences were considered significant when the P value was <0.05. The results in the text and figures are expressed as the means ± the SEM.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Course of infection. Trypanosomes were observed in the blood of the mice 3 to 5 days after i.p. inoculation with 2 x 10³ T. b. brucei (Fig. 1A). Similar parasitemia were observed in PBS-treated and minocycline-treated infected mice (Fig. 1A). In line with this observation, minocycline did not show toxic effects on T. b. brucei in vitro since there were no significant differences in the number of mobile trypanosomes cultured axenically for 24, 48, and 72 h in the presence of 1 to 20 µg of minocycline/ml or medium alone (Fig. 1B).

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FIG. 1. (A) Levels of parasitemia in minocycline-treated and PBS-treated mice at different time points p.i. Each point represents the mean ± the SEM of the values obtained from 11 to 16 animals. (B) Effect of minocycline on the viability of T. b. brucei in vitro. T. b. brucei organisms were incubated with the indicated minocycline concentrations for 24, 48, and 72 h at 37°C and 5% CO2. Each point represents the mean ± the SEM of the values obtained from three independent experiments which were done in duplicate. Only motile cells were counted. (C) Body weight in uninfected and infected minocycline-treated or PBS-treated mice. Body weight is expressed as a percentage change of the preinfection weight at day 0. Each point represents the mean ± the SEM of the values obtained from 4 to 16 animals. Statistically significant differences in comparison with uninfected control animals were determined (*, P < 0.05; **, P < 0.01 [two-way ANOVA]).

There was a gradual continuous increase in body weight of uninfected mice during a 35-day observation period, which was not affected by minocycline treatment. Nontreated, infected mice showed decreased body weights from about 20 days p.i. In contrast, minocycline-treated, infected mice gained weight as the uninfected controls did (Fig. 1C). Nontreated, infected mice were sacrificed between days 30 to 35 p.i., before they became moribund. On the other hand, infected mice treated with minocycline for 30 days p.i. did not show signs of disease around that time period (30 to 35 days p.i.) and were sacrificed between days 40 and 45 p.i., before becoming moribund.

Minocycline reduces the number of trypanosomes invading the brain parenchyma. In order to assess the effect of minocycline on the parasite invasion of the brain parenchyma, double immunolabeling with antibodies to T. b. brucei and glucose transporter 1 (GLUT-1), a marker of cerebral blood vessel endothelial cells, was performed on brain sections. Daily treatment with minocycline reduced the density of T. b. brucei in the brain parenchyma in mice examined both at 20 and 30 days p.i. (Fig. 2A). In nontreated mice several parasites were seen extravascularly in the brain parenchyma (Fig. 2B), whereas in the minocycline-treated mice the parasites were mainly localized in the lumens of the blood vessels (Fig. 2C). There was no conspicuous cuffing of parasites around blood vessels in either nontreated or minocycline-treated mice as we previously described in IFN--, IFN- receptor-, and RAG-deficient mice (15).

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FIG. 2. Effects of minocycline treatment on trypanosome invasion into the brain parenchyma. (A) Reduction in density of parasites in the parenchyma of minocycline-treated compared to PBS-treated mice at 20 and 30 days p.i. Each bar represents the mean ± the SEM of the values obtained from five to six animals. *, P < 0.05 (Mann-Whitney U test). (B and C) Double immunofluorescence labeling of trypanosomes (red) and cerebral endothelial cells (green) at 30 days p.i. in PBS (B)- and minocycline (C)-treated mice. Note the presence of extravascular parasites in PBS-treated and intravascular parasites in minocycline-treated mice. Bar, 25 µm.

Minocycline reduces the T-cell infiltration and glial reaction during T. b. brucei infection. To characterize the effect of minocycline on the T. b. brucei infection-induced brain inflammation, the density of CD45⁺ leukocytes in the brain parenchyma, as well as the activation of astrocytes and microglia, were evaluated. The number of CD45⁺ cells in the brain parenchyma of minocycline-treated mice was reduced compared to that of nontreated, infected mice both at days 20 and 30 p.i. (Fig. 3). In the nontreated, T. b. brucei-infected mice intense tomato lectin immunoreactivity appeared in mainly hypertrophic ramified microglia that was most pronounced in the white matter and hypothalamic periventricular areas (Fig. 4A and data not shown), and this immunoreactivity was markedly reduced in the minocycline-treated mice (Fig. 4B). In the nontreated, infected mice there was increased GFAP immunoreactivity of astrocytes, particularly prominent in the white matter, septum, and hypothalamic periventricular regions at both 20 and 30 days p.i (Fig. 4C and data not shown). This immunoreactivity was reduced in the minocycline-treated, infected mice (Fig. 4D).

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FIG. 3. Reduction in density of CD45+ cells in the parenchyma of minocycline-treated compared to PBS-treated mice at 20 and 30 days p.i. Each bar represents the mean ± the SEM of the values obtained from five to six animals. *, P < 0.05 (Mann-Whitney U test).

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FIG. 4. Glia reaction at 30 days p.i from PBS-treated (A and C) and minocycline-treated (B and D) mice. Sections stained immunohistochemically for tomato lectin, a microglia marker, show hypertrophic ramified microglia in PBS-treated mice (A) but not in minocycline-treated mice (B) in the corpus callosum. GFAP immunoreactivity in astrocytes is prominent in PBS-treated mice (C) and is reduced in minocycline-treated mice (D) in the septal nuclei. Bar, 50 µm.

Effect of minocycline on levels of transcripts encoding adhesion molecules, cytokines, and MMPs in the brain and spleen. The levels of mRNAs encoding intercellular adhesion molecule 1 (ICAM-1) and endothelial-leukocyte adhesion molecule 1 (E-selectin) were increased in the brains of infected animals and were reduced by minocycline treatment (Fig. 5A and B), whereas those of vascular cell adhesion molecule 1 (VCAM-1) and platelet endothelial cell adhesion molecule 1 (PECAM-1) were less, or not, affected by the infection (data not shown). Brains from trypanosome-infected mice showed significant increase in the levels of MMP-3, -8, and -12 mRNA titers compared to uninfected controls, whereas levels of MMP-1b, -2, -7, -9, -11, -13, -14, and -19 and TIMP-1 and -2 mRNA were unaltered, and MMP-10 was below the detection limit (Fig. 5C and D and data not shown). Minocycline treatment reduced the infection-induced elevation of MMP-3, -8, and -12 mRNA levels in the brain at 30 but not at 20 days p.i. (Fig. 5C and D and data not shown). The levels of tumor necrosis factor alpha (TNF-), gamma interferon (IFN-), interleukin-1 (IL-1), IL-1ß, and IL-6 mRNAs were also increased in the brains of infected mice and reduced by minocycline treatment (Fig. 6 and data not shown).

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FIG. 5. Relative expression of ICAM-1 (A), E-selectin (B), MMP-3 (C), and MMP-12 (D) mRNA in the brains of uninfected control, infected PBS-treated, and infected minocycline-treated (mino) mice. Each bar represents the mean ± the SEM of the values obtained from four animals. Statistically significant differences in comparison with uninfected animals (*, P < 0.05; **, P < 0.01) and between PBS-treated and minocycline-treated infected mice sacrificed at the same time point p.i. (#, P < 0.05; ##, P < 0.01) (one-way ANOVA).

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FIG. 6. Relative expression of TNF- (A), IFN- (B), IL-1 (C), and IL-1ß (D) mRNA in the brains of uninfected control, infected PBS-treated and infected minocycline-treated (mino) mice. Each bar represents the mean ± the SEM of the values obtained from four animals. Statistically significant differences in comparison with uninfected animals (*, P < 0.05; **, P < 0.01) and between PBS-treated and minocycline-treated infected mice sacrificed at the same time point p.i. (#, P < 0.05; ##, P < 0.01) (one-way ANOVA).

Spleens from infected and uninfected mice showed similar expression of mRNAs encoding ICAM-1 and E-selectin. Elevated TNF-, IFN-, IL-1, IL-1ß, and IL-6 mRNA levels in the spleens from mice at 20 and 30 days p.i. were not altered by minocycline treatment (data not shown). Increased levels of MMP-8 and -9 transcripts were detected in spleens from infected mice at day 20 and 30 p.i., whereas the levels of MMP-2, -3, -11, and -12 were unaltered compared to uninfected controls. Minocycline treatment did not reduce the elevated MMPs transcript levels in the spleen (data not shown).

Top Abstract Introduction Materials and Methods Results Discussion References

 
We have previously reported that the penetration of trypanosomes across the basement membranes of the BBB shows similarities to that of leukocytes (15). In the present study, minocycline reduced the invasion of the brain parenchyma not only by leukocytes (CD45⁺ cells) but also by trypanosomes. This suggests that neuroinvasion of trypanosomes is related to that of leukocytes, whereby the latter may pave the way for the former. The transmigration of leukocytes from the blood into the brain parenchyma takes place in an integrated sequence of events, including rolling and activation of leukocytes, adhesion to the vascular endothelium, diapedesis across the endothelium, and ultimately penetration through the endothelial and astrocytic basement membranes (23, 27). Several molecules, including cell adhesion molecules, cytokines, chemokines, and MMPs, play a role during this multistep process (22, 27). Minocycline with its pleiotropic effects might impede the leukocyte transmigration into the CNS by regulating any of these molecules.

We observed that the infection-induced expression of the adhesion molecules ICAM-1 and E-selectin transcripts in the brains of T. b. brucei-infected mice was reduced by minocycline treatment, which also possibly curbed the brain invasion of the parasites and leukocytes by reducing the expression of these molecules. Minocycline has been reported to downregulate the expression of ICAM-1 and reduce the number of infiltrating cells during ischemic renal injury, as well as to impair leukocyte chemotaxis (8).

The reduction of cytokine expression in the brain by minocycline treatment could partly be due to the reduced influx of cytokine-producing leukocytes into the brain, since the levels of cytokine expression in spleen cells were not affected by the treatment. In line with this, no increase in IFN- transcripts was observed in the brains of T. b. brucei-infected RAG-1^–/– mice, which lack B and T cells, suggesting that lymphocytes are the major source of this proinflammatory cytokine in the brains of infected wild-type mice (15). In addition, a direct inhibitory effect by minocycline on activation of astrocytes and microglia has been described (9, 24), and this could also have contributed to the reduced cytokine expression in the brain.

Minocycline reduced the increased levels of MMP-3, -8, and -12 transcripts in infected mice brains at 30 days p.i. This reduction could also reflect a suppressed influx of leukocytes into the brain. Minocycline abrogated MMP-2 expression in the CNS of rats with EAE and, in parallel, suppressed T-cell recruitment into the CNS (19). However, at 20 days p.i., where minocycline treatment reduced the numbers of leukocytes and trypanosomes in the brain parenchyma, there was no significant increase in MMP transcript expression in the brains, thus ruling out a role of MMP transcript regulation on the protective effect conferred by minocycline treatment. A direct inhibitory effect of minocycline on MMP enzymatic activity has, however, been described (2, 18, 21) and could play a role in the altered outcome of brain infection in minocycline treated mice.

The infected minocycline-treated mice did not lose weight as the nontreated mice did, in spite of the fact that the two groups of animals showed similar levels of parasitemia and transcripts for inflammatory molecules in the spleen. Probably, the reduction of infection-induced expression of TNF-, IL-1, IL-1ß, IL-6, and IFN- in the brains of treated mice mediates the protective effect of minocycline. These proinflammatory cytokines have been demonstrated to be involved in weight loss associated with chronic infections, sepsis, and cancer (7, 25). Furthermore, intracerebroventricular infusion of IL-1 receptor antagonist or a combination of IL-1 receptor antagonist and soluble type-1 receptor of TNF restores or prevents the loss of weight in rats caused by infection with T. b. brucei (20). Thus, these findings suggest that the loss of weight and mortality caused by the T. b. brucei is due to brain involvement rather than to systemic effects of the infection.

The present study demonstrates that treatment with minocycline reduces T. b. brucei invasion of the brain parenchyma, ameliorates CNS inflammatory parameters associated with the infection, prevents weight loss, and prolongs survival of the animals.

Minocycline and other tetracycline antibiotics have been used in combination therapy against other parasitic diseases such as malaria (17, 26, 28) and toxoplasmic encephalitis (4). Our findings may therefore be of relevance for the development of treatment strategies with minocycline as a supplement to trypanocidal drugs used in the treatment of the disease.

 
W.M. is the recipient of an IBRO fellowship. This study was supported by grants from the Swedish Research Council (no. 4480), the Swedish International Development Cooperation, the World Health Organization, and EC-FP6-2004-DEV-3032324.

 
* Corresponding author. Mailing address: Department of Neuroscience, Retzius väg 8, Karolinska Institutet, SE-171 77 Stockholm, Sweden. Phone: 46-8-524-878-10. Fax: 46-8-32-53-25. E-mail: willias.masocha{at}ki.se.

Top Abstract Introduction Materials and Methods Results Discussion References

 

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Antimicrobial Agents and Chemotherapy, May 2006, p. 1798-1804, Vol. 50, No. 5
0066-4804/06/$08.00+0     doi:10.1128/AAC.50.5.1798-1804.2006
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