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Published online before print August 23, 2007
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From the Departments of Neurobiology and Developmental Sciences* and Infectious Diseases, University of Arkansas for Medical Sciences, Little Rock, Arkansas; and the Department of Microbiology and Immunology, Dartmouth Medical School, Lebanon, New Hampshire
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Abstract |
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Brain abscesses develop in response to a parenchymal infection with pyogenic bacteria, beginning as a localized area of cerebritis and evolving into a suppurative lesion surrounded by a well-vascularized fibrotic capsule.1-3 Brain abscesses are typified by extensive edema and tissue necrosis and tend to localize at white-gray matter junctions where microcirculatory flow is poor.12,13 In addition to the sequential progression from cerebritis to necrosis during brain abscess evolution, the activation of resident glial cells and influx of peripheral leukocytes demonstrate temporal patterns.3,14,15 Specifically, microglial and astrocyte activation is evident immediately following the entry of bacteria into the CNS parenchyma and persists throughout abscess evolution.11,16 Neutrophils are the initial leukocyte subset to infiltrate developing abscesses and are observed as early as 12 hours following bacterial exposure.7,11 Macrophages and T cells are associated with lesions as they progress, with infiltrates generally more pronounced around days 3 and 7, respectively.11,14 Beginning at 7 to 10 days postinfection, a highly vascularized fibrotic wall forms around the necrotic milieu, effectively forming a barrier to contain the infection.11,17,18 It is important to note that the kinetics of cellular activation, influx, and bordering functions represent general time frames, and there is probably overlap between each of these processes during brain abscess evolution.
Although necessary for pathogen containment, excessive inflammation during the early phases of brain abscess development may have deleterious consequences on disease outcome through the induction of exaggerated edematous and/or necrotic responses.3,12,13 Similar detrimental effects have been implicated in the pathophysiology of bacterial meningitis, where the dysregulated production of free radicals, proinflammatory cytokines, and edema lead to significant neuron damage and associated mortality.19,20 Therefore, attenuating glial and peripheral immune cell activation without compromising bacterial containment may result in less damage to normal brain tissue and improvements in cognitive and neurological functions.
One attractive candidate to achieve this balance during the course of brain abscess development is minocycline. Minocycline is a second-generation tetracycline that effectively crosses the blood-brain barrier due to its small (495 d) and lipophilic nature.21-24
In susceptible bacterial strains, minocycline binds to the h34 ribosomal subunit, resulting in a conformational change that effectively interferes with protein synthesis.25,26
Minocycline has been shown to exert neuroprotective effects distinct from its bacteriostatic activity in animal models of cerebral ischemia,27-30
Parkinson’s and Huntington’s disease,31-34
Alzheimer’s disease,35,36
Down’s syndrome,37
amyotrophic lateral sclerosis,38,39
spinal cord injury,40-42
and experimental autoimmune encephalomyelitis, an animal model for multiple sclerosis.43-45
Minocycline is thought to modulate these diseases, in part, by inhibiting the production of potentially cytotoxic molecules by activated microglia.46
For example, minocycline has been shown to inhibit microglial production of NO, interleukin (IL)-1ß, tumor necrosis factor (TNF)-
, and IL-6, which can impact neuron survival.47-49
In addition, minocycline may also protect CNS cells from apoptosis through inhibition of caspase-3 activity.31,41,42
Minocycline has also been shown to down-regulate signal transduction pathways such as nuclear factor-
B and p38 mitogen-activated protein kinase,32,48,50,51
both of which play pivotal roles in controlling the expression of numerous proinflammatory genes. Tetracyclines have also been shown to inhibit production of the proinflammatory molecules TNF-,52,53
NO,54
and matrix metalloproteinases44,55
by leukocytes. However, it is also important to acknowledge the fact that other investigators have reported that minocycline has no impact or, in some cases, exacerbates neuronal injury in several CNS disease models,23,56-60
highlighting the need for further studies in this area to identify specific conditions in which minocycline therapy may exert beneficial effects.
Although a few recent reports have documented favorable effects of minocycline in various viral and parasitic infections,61-64 the consequences of this antimicrobial on modulating inflammatory responses in the context of CNS bacterial infections, such as brain abscess, have not yet been examined. The relative bacteriostatic versus immune modulatory effects of minocycline have been difficult to differentiate; however, in this study we have devised a strategy to discern between these two possibilities by engineering a S. aureus strain that is resistant to the bacteriostatic effects of minocycline. Our findings indicate that an overactive host response to infection can lead to the heightened expression of proinflammatory mediators and subsequent mortality during the initial stages of brain abscess, which can be attenuated by minocycline independently of its antibacterial activity. In addition, minocycline was capable of reducing ongoing inflammation, as both inflammatory mediator expression and brain abscess size were attenuated when minocycline treatment was initiated 3 days following S. aureus infection. Collectively, these results suggest that if not tightly regulated, the inflammatory response that ensues during brain abscess development can exert deleterious effects. Therefore, minocycline may represent an excellent candidate for brain abscess therapy through its ability to attenuate chronic inflammation and reduce the extent of parenchymal tissue damage that is characteristic of infection.
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Materials and Methods |
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The minocycline-sensitive S. aureus strain RN6390 was used as previously described.7-10 A minocycline-resistant isogenic strain of RN6390 was constructed with a tetracycline resistance cassette (tetM), which also confers resistance to minocycline, stably inserted into the bacterial chromosome using the integration vector pCL84, which preferentially inserts into the S. aureus lipase gene.65 Chromosomal insertion of the tetM resistance cassette was confirmed by sequence analysis of chromosomal polymerase chain reaction (PCR) products and Southern blots. To determine whether the insertion of the tetM cassette led to any overt alterations in the S. aureustetM mutant compared with the parental strain RN6390, pulse field gel electrophoresis and sodium dodecyl sulfate-polyacrylamide gel electrophoresis of whole cell lysates and secreted proteins from several independent bacterial colonies were performed.
Minocycline-HCl was purchased from Sigma Chemical Co. (St. Louis, MO), and a 10 mg/ml stock solution was prepared in endotoxin-free H2O and stored in aliquots at –20°C until use. For animal injections, minocycline working stocks were prepared fresh daily in sterile endotoxin-free phosphate-buffered saline (pH 7.4).
Generation of Experimental Brain Abscesses
Brain abscesses were induced in 6- to 8-week-old C57BL/6 mice using a stereotaxic approach as previously described.8,9 For all studies, an equal number of age-matched males and females were used as previous work has established that both genders exhibit qualitatively similar inflammatory profiles following bacterial challenge.7-10 The animal use protocol has been approved by the University of Arkansas for Medical Sciences Institutional Animal Care and Use Committee and is in accord with the National Institutes of Health guidelines for the use of rodents.
For evaluating the potential therapeutic effects of minocycline during brain abscess development, mice were subjected to either one of two treatment paradigms. In the first, the effects of minocycline when administered before S. aureus exposure were examined. For these experiments, mice received once daily intraperitoneal (i.p.) injections of minocycline beginning 1 day before S. aureus infection and continuing until study termination. In the second treatment paradigm, minocycline was evaluated for its ability to modulate ongoing CNS inflammation. For these studies, minocycline was administered 3 days following S. aureus infection, a time point when bacterial burdens have nearly peaked.7,11 After this initial treatment, minocycline injections continued daily until the termination of the study. Minocycline was initially examined at three concentrations (2, 10, and 50 mg/kg/day); however, in subsequent studies one dose was selected (50 mg/kg/day), which was determined to exert optimal effects on the ensuing host CNS antibacterial response. For all experiments, one group of animals received minocycline treatment alone (50 mg/kg/day) to assess any effects of the drug independently of S. aureus infection. Animals receiving 50 mg/kg/day minocycline alone did not demonstrate any overt adverse effects to the drug (ie, no significant alterations in body weight). The doses of minocycline tested are representative of concentrations used in previously published reports of disparate CNS neurodegenerative models,46,66 and although they represent concentrations well above those used in humans, this is offset by the extremely short half-life of minocycline in mice (2 to 3 hours) versus humans (15 to 18 hours).22,66,67
Simultaneous Collection of RNA, Protein, and Quantitation of Viable Bacteria from Brain Abscesses
Brain abscesses were visualized by the stab wound created during injections, sectioned within 2 to 3 mm on all sides, and homogenized in PBS supplemented with protease and RNase inhibitors. Subsequently, RNA, protein, and bacterial titers were obtained as previously described.9 It is not feasible to dissect away necrotic from viable brain tissue while collecting samples for analysis, because these microscopic borders are not easily discernable by the naked eye. Although we do not specify between viable and necrotic tissue when collecting brain abscesses, we propose that this does not introduce artifacts in the results obtained. For example, our results demonstrate that minocycline treatment reduced brain abscess severity as typified by smaller lesions, which translates into a greater amount of surviving tissue. Yet in this case, the expression of several proinflammatory mediators was diminished. If necrotic tissue greatly impacts the results obtained, we would have expected a reduction in cytokine production in brain abscesses of vehicle-treated animals, which was not observed.
Quantitation of Brain Abscess Size
At the indicated time points postinfection, minocycline- and vehicle-treated mice were perfused transcardially with PBS to eliminate leukocytes from the vasculature, whereupon brains were removed and immediately flash-frozen on dry ice. Before cryostat sectioning, brain tissues were embedded in optimal cutting temperature medium, and serial 10-µm sections were made throughout the entire lesioned tissue and stained with hematoxylin and eosin to demarcate abscess margins. Evaluation of serial sections throughout the affected brain parenchyma ensured that the largest abscess cross-sectional area would be identified for comparisons of lesion size. Abscess area (reported as square millimeters) was calculated using the MetaMorph image analysis program (Universal Imaging Corporation, Downingtown, PA).
Evaluation of Tissue Viability Using Nitro Blue Tetrazolium (NBT) Staining
To better demarcate the border between living and nonviable tissue during the early stages of brain abscess development, NBT staining of fresh frozen tissue sections was performed.68 The intensity of staining is dictated by the amount of mitochondrial diaphorase enzymes that were present in viable cells at the time of tissue collection, which metabolize the conversion of NBT into a blue formazan product. The loss of staining is indicative of the degree of cell damage/injury. In brief, 10-µm cryostat sections were fixed for 10 minutes in ice-cold acetone and air-dried. Tissues were subsequently incubated for 40 minutes at room temperature with 200 µl/slide NBT development solution (0.2 mol/L Tris-HCl, pH 7.1, containing 0.25 mg/ml NBT, 2 mg/ml NADH, both from Sigma Chemical Co., and 1 mg/ml MgCl2). To terminate the reaction, slides were rinsed in double distilled water, dehydrated, and coverslipped using Permount mounting medium.
Immunofluorescence Staining of Astrocytes and Microglia/Macrophages
The effects of minocycline on astrocyte and microglia/macrophage activation during brain abscess development were evaluated by immunofluorescence staining using glial fibrillary acidic protein (GFAP) and F4/80, respectively, and confocal microscopy. Brain abscess tissues were collected at the indicated time points following minocycline treatment, and 10-µm cryostat sections were mounted onto SuperFrost Plus slides (Fisher Scientific, Houston, TX), air-dried, and stored at –80°C until use. To initiate staining, slides were equilibrated at room temperature for 15 minutes and fixed in ice-cold methanol. Following numerous rinses in PBS, tissues were incubated with PBS/10% normal donkey serum to minimize nonspecific staining. Brain abscess tissues were incubated with either primary anti-rat GFAP (Zymed, South San Francisco, CA) or F4/80 (Serotec, Raleigh, NC) antibodies overnight at 4°C in a humidified chamber. Following numerous rinses in PBS, tissues were incubated with either a donkey anti-rat IgG fluorescein isothiocyanate conjugate (for GFAP) or biotinylated donkey anti-rat IgG antibody (for F4/80; both from Jackson Immunoresearch, West Grove, PA) for 1 hour at room temperature. F4/80 expression was visualized by the addition of a streptavidin-Alexa Fluor 568 conjugate (Molecular Probes, Eugene, OR). On completion of the staining protocol, slides were coverslipped using the Prolong antifade reagent (Molecular Probes) and sealed using nail polish. Slides were imaged using a Zeiss laser scanning confocal microscope (LSM 510; Carl Zeiss Microimaging Inc., Thornwood, NY), and fluorescein isothiocyanate was excited with a 488-nm argon laser to visualize GFAP immunoreactivity, and Alexa Fluor 568 was excited with a 561-nm diode-pumped solid-state laser to demonstrate F4/80 expression, with images collected using the appropriate emissions. The confocal pinhole was set to obtain an optical section thickness of 1.6 µm. Specific staining of antibodies was confirmed by the absence of fluorescence signal following incubation of brain abscess tissues with secondary antibodies alone (data not shown).
Preparation of Primary Microglia and Astrocyte Cultures
Primary microglia and astrocytes were isolated from neonatal C57BL/6 mice (postnatal days 2 to 4) as previously described.69 The purity of microglia and astrocyte cultures, as determined by immunohistochemical staining using antibodies against CD11b (BD PharMingen, San Jose, CA) and GFAP (Dako Corp., Carpinteria, CA), was routinely greater than 95 and 90%, respectively.
To investigate the effects of minocycline on glial activation, primary microglia and astrocytes were seeded into 96-well plates at 2 x 105 or 1 x 105 cells/well, respectively, and incubated overnight. The following day, cells were treated with various doses of minocycline for 1 hour before stimulation with either heat-inactivated S. aureus [107 colony-forming units (cfu), strain RN6390] or 10 µg/ml peptidoglycan (PGN; InvivoGen, San Diego, CA). Cell-conditioned supernatants were collected at 24 hours following bacterial exposure, whereupon NO and cytokine/chemokine production was evaluated as described below. The doses of bacterial stimuli used were based on our previous studies that established optimal cytokine responses without any evidence of toxicity.70,71
Nitrite Assay
Nitrite (a stable reaction product of NO) levels were determined by adding 50 µl of astrocyte-conditioned culture medium with 50 µl of Griess reagent (0.1% naphthylethylenediamine dihydrochloride, 1% sulfanilamide, and 2.5% phosphoric acid; all from Sigma Chemical Co.) in a 96-well plate. The absorbance at 550 nm was measured on a plate reader (Spectra Max 190; Molecular Devices, Sunnyvale, CA), and nitrite concentrations were calculated using a standard curve with sodium nitrite (NaNO2; Sigma Chemical Co.; level of sensitivity 0.4 µmol/L).
Cell Viability Assays
The effects of minocycline on microglia and astrocyte viability were evaluated using a standard 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium (MTT) (Sigma) assay based on the mitochondrial conversion of MTT into formazan crystals as previously described.72
Protein Extraction and Western Blotting
Protein extracts were prepared from primary microglia or astrocytes as previously described.72,73 The effects of minocycline on glial Toll-like receptor 2 (TLR2) protein expression were evaluated by Western blot analysis. Blots were probed using a goat anti-mouse TLR2 antibody (R&D Systems, Minneapolis, MN) followed by a donkey anti-goat IgG–horseradish peroxidase secondary antibody (Jackson Immunoresearch). Blots were developed using the Immobilon Western substrate (Millipore, Billerica, MA) and visualized by exposure to X-ray film. Subsequently, membranes were stripped and re-probed with a rabbit anti-actin polyclonal antibody (Sigma Chemical Co.) for normalization.
Quantitative Real-Time RT-PCR (qRT-PCR)
Total RNA from brain abscesses was isolated using the TRIzol reagent and treated with DNase1 (both from Invitrogen) before use in qRT-PCR studies. The experimental procedure was performed as previously described.73
IL-1ß, TNF-, and glyceraldehyde-3-phosphate dehydrogenase primers and TAMRA TaqMan probes were designed69,74
and synthesized by Applied Biosystems (ABI, Foster City, CA). An ABI Assays-on-Demand TaqMan kit was used to examine CCL2 expression.
Enzyme-Linked Immunosorbent Assay (ELISA)
Murine TNF-, IL-1ß, and CCL2 (OptiEIA; BD PharMingen) and CXCL2 (MIP-2, DuoSet; R&D Systems) were quantified in brain abscess homogenates using commercial ELISA kits. Results were normalized to the amount of total protein extracted from tissues to correct for differences in sampling size as previously described.8,11
Statistics
Significant differences between experimental groups were determined using the unpaired Student’s t-test at the 95% confidence interval with Sigma Stat (SPSS Science, Chicago, IL). This analysis was determined to be most appropriate since, although we are evaluating changes in proinflammatory mediator expression over time, repeated measurements are not made on the same animal (mice are sacrificed to collect abscess homogenates at each time point), precluding analysis of variance and post hoc analysis of the data.
In this study, we performed a minimum of three independent replicates of each study to confirm the results obtained. The reporting of our results as representative of "x" number of independent experiments was required, because it is difficult to achieve identical bacterial burdens in mice between independent brain abscess studies with distinct bacterial preparations. As a result, the absolute concentrations of the various proinflammatory mediators detected within brain abscesses of minocycline-treated and control mice differed between individual experiments; however, the trends were consistent. This required us to report results from a single experiment where at least four or five mice per time point per group were analyzed and statistical analysis conducted.
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Results |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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Initial experiments established that the S. aureus strain used in our experimental model, RN6390, was sensitive to the antibiotic effects of minocycline (data not shown). To examine whether minocycline affected the acute phase of brain abscess development in this susceptible strain, the antibiotic was administered daily to animals beginning one day before intracerebral S. aureus infection and continuing until termination of the study. Minocycline treatment led to a dose-dependent reduction in bacterial burdens (Figure 1)
that coincided with significant decreases in the expression of several proinflammatory mediators that we have previously determined to be pivotal during the acute stage of brain abscess including IL-1ß, TNF-, and CCL2 (Figure 2)
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The effects of minocycline on proinflammatory mediator expression were probably related to its ability to reduce bacterial burdens and/or modulate signaling pathways important for the induction of cytokines/chemokines including nuclear factor-B or p38 mitogen-activated protein kinase.47,51
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Because we were interested in evaluating the potential anti-inflammatory effects of minocycline in the brain abscess model independently of its antibiotic properties, we generated an isogenic minocycline-resistant strain of S. aureus RN6390 (referred to hereafter as S. aureustetM). It was important to create a minocycline mutant isogenic to the parental strain RN6390 rather than use an unrelated S. aureus isolate that is naturally resistant to minocycline, because we have extensively characterized the host immune response to S. aureus RN6390 in the brain abscess model.7-10,16
To determine whether the insertion of the minocycline resistance cassette tetM resulted in any dramatic changes in phenotype compared with the parental strain RN6390, pulse field gel electrophoresis and sodium dodecyl sulfate-polyacrylamide gel electrophoresis of whole cell lysates and secreted proteins from several independent clones of both the S. aureustetM mutant and parental strains were performed. Pulse field gel electrophoresis revealed the loss of a larger SmaI fragment in the S. aureustetM mutant with the concomitant appearance of two smaller fragments (Figure 3A)
. This finding can be explained by the fact that the pCL84 vector used to insert the tetM gene into the bacterial chromosome contains SmaI sites. Evaluation of whole cell lysates and secreted proteins from both the parental and S. aureustetM strains in vitro revealed that protein expression was nearly identical between the two strains, except for the presence of a faint unique band in supernatants from the latter (Figure 3B)
. Collectively, these results indicate that the introduction of the tetM cassette into the bacterial chromosome did not lead to dramatic alterations in protein expression. In vitro sensitivity of the S. aureustetM strain to minocycline was evaluated, and the minimum inhibitory concentration neared 5 µg/ml (data not shown), in close agreement with the level of resistance previously reported for the tetM gene.75
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. Importantly, these effects were irrespective of the bacteriostatic properties of minocycline, because bacterial burdens between minocycline- and vehicle-treated mice were not significantly different (Figure 4B)
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The improved survival rates of minocycline-treated mice were expected to coincide with reduced levels of select proinflammatory molecules. However, no alterations in IL-1ß, TNF-, CXCL2, or CCL2 expression were observed in minocycline-treated mice at 24 hours following infection (data not shown). Based on its relatively short half-life in the mouse, we considered the possibility that minocycline induced transient changes in inflammatory mediator expression that returned to baseline once therapeutic antibiotic levels had subsided because of the continued presence of high bacterial burdens in the CNS parenchyma. Therefore, we examined the expression of several proinflammatory mediators immediately following S. aureustetM infection (ie, 3, 6, and 12 hours). It is important to note that minocycline was administered immediately before bacteria on the day of infection, such that the amount of drug in the CNS was expected to remain at significant levels, at least during the initial time points examined. Because this analysis occurred at such early intervals postinfection, changes in proinflammatory mediator expression were examined by qRT-PCR, because protein levels had not yet accumulated to concentrations detectable by traditional ELISAs. The expression of several proinflammatory cytokines/chemokines, including IL-1ß and CCL2, was significantly decreased by minocycline in a dose-dependent manner at 3 and 6 hours following S. aureustetM infection (Figure 5, A and B)
. However, these effects were transient, with proinflammatory mediator expression returning to levels observed in vehicle-treated animals within 12 hours postinfection, in agreement with the extremely short half-life of minocycline in mice. Importantly, the observed decreases in proinflammatory mediator expression were independent of the antibiotic activity of minocycline, because pathogen burdens were not statistically different between the various treatment groups (Figure 5C)
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The finding that minocycline was capable of transiently attenuating proinflammatory mediator expression in brain abscesses led us to consider that the degree of tissue injury may be less severe in these animals. The extent of brain abscess involvement was determined by performing hematoxylin and eosin stains on serial tissue sections prepared throughout the entire lesion to identify the largest abscess cross-sectional area. As shown in Figure 6
, minocycline pretreatment led to a significant sparing of brain parenchyma from necrotic damage, as evident by the fact that abscess sizes were dramatically reduced in minocycline-treated mice. To better demarcate the borders between necrotic tissue and viable peri-abscess parenchyma at the early time points examined following S. aureus infection, tissue viability stains with NBT were performed. As shown in Figure 7
, minocycline-treated animals exhibited less necrotic changes compared with vehicle-treated mice.
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; however, minocycline treatment had no effect on either the relative number or degree of astrocyte activation along the abscess margins. We also attempted to evaluate the effects of minocycline on microglial/macrophage activation by F4/80 staining; however, sufficient signal was not observed at these early time points following S. aureus infection (data not shown).
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Although our studies had demonstrated that minocycline attenuated proinflammatory mediator expression and brain abscess size in a pretreatment paradigm, this strategy is not reflective of a plausible clinical approach to modulate infections. Therefore, we next determined whether minocycline could affect established inflammation during brain abscess evolution. In these studies, mice received single daily doses of minocycline beginning at day 3 postinfection. Animals were sacrificed at 1 and 3 days following minocycline treatment for analysis, which corresponded to days 4 and 6 postinfection, respectively. Similar to what was observed in the pretreatment paradigm, minocycline significantly attenuated the expression of several proinflammatory mediators (Figure 9)
as well as brain abscess size (Figure 10)
. The extent of abscess-associated edema observed in minocycline-treated mice was notably reduced compared with vehicle-treated animals and was most dramatic at day 3 following minocycline administration (Figure 9A)
. Interestingly, maximal protection of brain parenchyma was observed when mice received three consecutive daily doses of minocycline, whereas a single dose was not as effective. This result is not unexpected given the fact that at this stage, brain abscesses are typified by a vigorous, progressive inflammatory response, making it difficult for a single minocycline dose to exert any demonstrable effects. Examination of astrocyte and microglial/macrophage activation by immunofluorescence staining with GFAP and F4/80, respectively, did not reveal any significant differences following minocycline treatment, although the relative degree of immunoreactivity of both cell populations in the peri-abscess area progressively increased over time (Figure 11)
, as we have previously demonstrated.11
Collectively, these results indicate that minocycline could represent a potential therapeutic agent even in conditions of pre-existing inflammation to attenuate bystander destruction of surrounding normal brain parenchyma.
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Microglia are the resident innate immune cells in the CNS parenchyma and are uniquely poised to immediately respond to bacterial infections by producing numerous cytokines and chemokines.76-78
The neuroprotective effects of minocycline have been attributed, in part, to its ability to attenuate microglial activation both in vivo and in vitro.31,33,43,48,49
However, the ability of minocycline to modulate microglial responses to a prevalent CNS pathogen, such as S. aureus, has not yet been investigated. Both S. aureus and its cell wall product, PGN, induced microglial activation typified by the release of IL-1ß and CXCL2 (Figure 12)
. Pretreatment of microglia with minocycline for 1 hour before the addition of bacterial stimuli significantly attenuated IL-1ß and CXCL2 expression, although considerable levels of each mediator remained (Figure 12, A and B)
. The reduction in proinflammatory mediator expression following minocycline treatment was not the result of overt cell death, because cell viability assays revealed that minocycline was not toxic to primary microglia at any of the doses examined (Figure 12C)
. Interestingly, the inhibitory effects of minocycline were not universal, as the compound did not have any significant effects in regulating the expression of IL-12 p70 or IL-6 in response to bacterial stimulation (data not shown).
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, respectively). The reduction in astrocytic proinflammatory mediator expression following minocycline treatment was not the result of toxic effects at any of the concentrations examined (Figure 13C)
. Minocycline was not effective at inhibiting microglial or astrocyte activation when administered at either 1 or 6 hours following bacterial exposure (data not shown), which is not unexpected given the relatively modest ability of the compound to affect glial proinflammatory mediator release in the pretreatment dosing paradigm presented.
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TLRs enable the host to recognize microbial motifs that are highly conserved across various classes of microorganisms.81-83
Among the 13 TLR family members identified to date, TLR2 is capable of recognizing lipid-based structural components from a broad range of microbes, including various gram-positive bacteria, fungi, and protozoa.81,84
We elected to focus on evaluating the effects of minocycline on glial TLR2 expression, since we have previously published that this receptor plays a pivotal role in S. aureus and PGN recognition by both microglia and astrocytes, triggering the production of a wide array of inflammatory mediators by activated glia.69,73
Therefore, evaluation of TLR2 expression represents a read-out to imply the degree of "bacterial responsiveness" of glia to infection. To our knowledge, the ability of minocycline to modulate TLR2 expression in activated glia has not yet been examined. TLR2 expression in both microglia and astrocytes was increased following bacterial exposure and was partially suppressed by minocycline (Figure 14, A and B
, respectively).
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Discussion |
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In bacterial meningitis, the CNS inflammatory response that ensues following infection plays a role in disease pathophysiology.19,20
Specifically, numerous cytokines, free radicals, and excitatory amino acids have all been implicated in negatively impacting neuron survival. Although it was possible that if excessive, the immediate antibacterial response to S. aureus in brain abscess might exacerbate disease severity, this possibility remained unclear, since we had previously demonstrated that several inflammatory mediators, including IL-1, TNF-, and CXCL2, were critical during the early phases of infection.7,8
With the generation of a minocycline-resistant S. aureus strain, our results demonstrated that minocycline modulates the acute host response to S. aureus in the CNS parenchyma as evident by the fact that minocycline significantly reduced abscess-associated mortality and improved brain parenchymal survival. Importantly, these effects did not result from differences in bacterial burdens, because both minocycline- and vehicle-treated mice displayed equivalent S. aureus levels. The ability of minocycline to minimize tissue damage may result, in part, from a reduction in cytokine-mediated bystander lysis, because several cytokines/chemokines were attenuated by minocycline in brain abscesses including IL-1ß, TNF-, CXCL2, and CCL2. The ability of minocycline to attenuate IL-1 production may be due to its ability to inhibit caspase-1 activity, as previously reported by others.31,38
These findings indicate that although inflammation is a prerequisite for effective pathogen clearance, this response can become exaggerated and culminate in adverse effects to the host. This dysregulated inflammatory response is thought to be driven, in part, by bacterial cell wall debris and/or DNA associated with the necrotic abscess milieu that serve as further triggers for pattern recognition receptors such as TLRs. In addition, many antibiotics that are used to treat CNS gram-positive infections can enhance the shedding of cell wall antigens, further confounding this issue.85,86
Therefore, we propose that the exaggerated release of proinflammatory mediators such as IL-1ß, TNF-, and CXCL2 may contribute to the nonspecific destruction of surrounding normal brain parenchyma by bystander lysis. In addition, minocycline was capable of reducing the S. aureus-dependent increase in glial TLR2 expression, suggesting that one way minocycline may attenuate inflammation is via interference with bacterial recognition pathways. Collectively, these results indicate that minocycline may serve to balance the ensuing inflammatory response to infection and ensure the development of an optimal antibacterial immune response.
One important question that remains is defining the complete repertoire of neuroprotective mechanisms for minocycline in the experimental brain abscess model, in addition to its ability to modulate proinflammatory mediator expression as demonstrated in this study. However, the accurate identification of affected host responses will require the establishment of an alternative dosing paradigm to overcome obstacles related to the short half-life of minocycline in the mouse (ie, 2 to 3 hours),22,66,67 compounded with the intense inflammation associated with a bacterial infection. For example, in brain abscess, the continued proliferation of bacteria coupled with shedding of cell wall determinants that are potent activators of TLRs83 probably overwhelms the anti-inflammatory effects of minocycline more rapidly compared with a noninfectious inflammatory milieu that is observed in Parkinson’s or Huntington’s disease or following spinal cord injury. Therefore, in brain abscess we envision that a continuous dosing strategy for minocycline would be required to achieve maximal anti-inflammatory effects. We plan to address these issues by delivering a constant supply of minocycline via either slow release pellets or osmotic minipumps. By maintaining therapeutic levels of minocycline in infected animals, we anticipate that potential differences in host responses will be more pronounced, enabling the accurate identification of the pathways affected by minocycline. This was considered to be beyond the scope of this initial report demonstrating the effectiveness of minocycline in the experimental brain abscess model. Notably, we were not able to detect any significant effects of minocycline on astrocyte or microglial/macrophage activation in the peri-abscess area, suggesting that the observed differences in inflammation were not attributable to an overt reduction in glial activation using the dosing paradigms tested in the current study. These results are in agreement with the relatively modest effects of minocycline on microglial and astrocyte activation in vitro, suggesting that mechanisms other than cytokine/chemokine production could play a role in the ability of minocycline to reduce brain abscess size. However, it is important to acknowledge that differences in the degree of astrocyte and/or microglial/macrophage activation could still exist but may not be discernable by merely examining GFAP and F4/80 expression. More detailed studies examining the cytokine expression profiles of these cell populations upon recovery from brain abscesses by fluorescence-activated cell sorting are warranted in the context of a more continuous dosing paradigm as discussed above.
Alternative pathways that may be affected by minocycline to reduce tissue damage during brain abscess development include the drug’s ability to attenuate matrix metalloproteinase expression and activity, inhibit apoptosis, and/or regulate the influx of peripheral immune cells to minimize nonspecific tissue destruction. The ability of minocycline to modulate leukocyte entry into evolving brain abscesses could be due to a number of mechanisms, namely, a direct action on matrix metalloproteinase enzymatic activity and/or matrix metalloproteinase expression,44,55,66 alterations in the levels of adhesion molecules required for leukocyte extravasation across the blood-brain barrier, and/or chemokine expression. On the other hand, minocycline has been reported to act as a free radical scavenger87 and to inhibit the production of neutrophil-attracting molecules from bacteria.88 Finally, in addition to these effector mechanisms, we cannot discount a potential antiapoptotic role for minocycline in reducing parenchymal damage through its ability to inhibit caspase-3 expression. Although we have not yet examined the extent of apoptosis in the brain abscess model, work by others using this model have demonstrated that apoptosis represents a relatively minor component of cell death.14 Cumulatively, the reversal of these pathways that under certain circumstances can be considered detrimental to cell survival could effectively rescue brain parenchyma from excessive destruction during brain abscess evolution.
In addition to its ability to modulate the host response to infection, it is also important to acknowledge that minocycline may also exert direct effects on S. aureus by modulating its virulence factor expression profile. In susceptible strains, minocycline exerts its bacteriostatic activity by binding to bacterial ribosomes and preventing protein expression.25,26 The tetM gene confers resistance to both minocycline and tetracycline and interferes with antibiotic binding to bacterial ribosomes. However, it is possible that minocycline selection may alter the array of virulence factors produced due to direct actions on genes important for bacterial pathogenesis. Although our in vitro analysis of the parental and S. aureustetM strains did not reveal any dramatic differences in protein expression profiles, variations in the face of antibiotic pressure in vivo are still possible. On the other hand, the ability of minocycline to alter the host immune response to S. aureus may lead to adaptive changes in bacterial virulence factor expression as a result of altered selective pressures in the brain. These possibilities could be addressed by performing gene expression profiling microarrays of bacterial RNA recovered from minocycline- or vehicle-treated mice infected with the S. aureustetM strain, studies we are currently exploring in the laboratory.
From a clinical perspective, any candidate immune prophylactic agent should exhibit beneficial effects when administered following the onset of disease. This criterion seemed to be partially satisfied in our present study as demonstrated by the ability of minocycline to attenuate proinflammatory mediator production and brain abscess size when treatment was initiated 3 days following S. aureus infection. Therefore, our data suggest that minocycline may represent an attractive candidate for the therapeutic management of brain abscess for the following reasons. First, the bacteriostatic properties of minocycline effectively reduce CNS bacterial burdens in susceptible strains. Second, in addition to its antimicrobial effects, minocycline can also attenuate pathological CNS inflammation that probably contributes to the expansion of abscess size through bystander damage to surrounding uninfected brain parenchyma. Third, minocycline exhibits excellent blood-brain barrier permeability because of its small and lipophilic nature and, based on these properties, would be expected to reach high therapeutic levels within the abscess milieu, a requisite for any effective antimicrobial therapy for brain abscess.
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Supported by the National Institutes of Health, National Institute of Neurological Disorders and Stroke grant 2R01 NS40730 (to T.K.), and the National Institute of Neurological Disorders and Stroke-supported Core facility at the University of Arkansas for Medical Sciences (grant P30 NS047546). Support for the Digital and Confocal Microscopy Laboratory at the University of Arkansas for Medical Sciences is provided by National Institutes of Health/IDeA Networks of Biomedical Research Excellence P20 RR6460 and National Institutes of Health/National Center for Research Resources S10 RR19395.
Accepted for publication June 19, 2007.
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play a pivotal role in the host immune response in a mouse model of Staphylococcus aureus-induced experimental brain abscess. J Neuropathol Exp Neurol 2004, 63:381-396[Medline]-toxin mutant of Staphylococcus aureus in experimental brain abscesses. Infect Immun 2001, 69:6902-6911
