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Keywords:

  • Innate immune response;
  • In situ hybridization histochemistry;
  • Inflammation;
  • Lipopolysaccharide;
  • Pro-inflammatory cytokine

Abstract

  1. Top of page
  2. Abstract
  3. 1 Introduction
  4. 2 Results
  5. 3 Discussion
  6. 4 Materials and methods
  7. Acknowledgements

In this study we investigated whether induction of toll-like receptor 2 (TLR2) amplifies the effect of a cell wall component derived from gram-positive bacteria, namely peptidoglycan (PGN). Mice received a first systemic lipopolysaccharide (LPS) injection to pre-induce TLR2 in various regions of the brain, and 6 h later, a second administration of either LPS or PGN. The data show a robust transcriptional activation of TLR2, TNF-α and monocyte chemotactic protein-1 (MCP-1) in microglial cells of mice challenged twice with LPS, whereas PGN essentially abolished this response. TLR4 plays a critical role in this process, because C3H/HeJ mice no longer responded to LPS but exhibited a normal reaction to PGN. Conversely, a robust signal for genes encoding innate immune proteinswas found in the brain of TLR2-deficient mice challenged with LPS. However, the second LPS bolus failed to trigger TNF-α and IL-12 in TLR2-deficient mice, while the same treatment caused a strong induction of these genes in the cerebral tissue of wild-type littermates. The present data provide evidence that cooperation exists between TLR4 and TLR2. While TLR4 is absolutely necessary to engage the innate immune response in the brain, TLR2 participates in the regulation of genes encoding TNF-α and IL-12 during severe endotoxemia. Such collaboration between TLR4 and TLR2 may be determinant for the transfer from the innate to the adaptive immunity within the CNS of infected animals.

Abbreviations:
PAMP:

Pathogen-associated molecular pattern

PGN:

Peptidoglycan

LTA:

Lipoteichoic acid

TLR:

Toll-like receptor

CVO:

Circumventricular organs

chp:

Choroid plexus

MCP-1:

Monocyte chemotactic protein-1

IκBα:

Inhibitory factor kappa B alpha

1 Introduction

  1. Top of page
  2. Abstract
  3. 1 Introduction
  4. 2 Results
  5. 3 Discussion
  6. 4 Materials and methods
  7. Acknowledgements

The presence of infection is recognized by receptors for specific elements called the pathogen-associated molecular patterns (PAMP), which are produced by microorganisms, such as bacteria, fungi and yeast 1. Recognition of these PAMP by myeloid cells is the first step of a complex inflammatory reaction that characterizes innate immune responses. The endotoxin lipopolysaccharide (LPS), a major component of the outer membranes of gram-negative bacteria, is the best-known target of innate recognition and induces a robust inflammatory response by phagocytic cells 2. Peptidoglycan (PGN) and lipoteichoic acid (LTA) from gram-positive bacteria are other common structural patterns that are recognized by host germ-line-encoded receptors and inducestimulation of the nuclear factor kappa B (NF-κB) signaling pathways and cytokine synthesis.

Cell wall components from gram-negative and gram-positive bacteria stimulate cytokine production by circulating monocytes/neutrophils and tissue macrophages. This process occurs via a recentlyclarified signaling cascade. Toll-like receptors (TLR) initiate the pro-inflammatory signal transduction pathways because they act as key molecules that recognize the major PAMP produced by bacteria and viruses (for a review, see 3). While TLR2 recognizes PAMP produced by gram-positive bacterial cell wall components, TLR4 is critical for the recognition of LPS. Flagellin, the principal element of bacterial flagella, is recognized by TLR5; and TLR9 is required for the inflammatory response triggered by bacterial DNA. TLR3 induces an innate immune response to double-stranded RNA viruses. The broad spectrum of components recognized by these receptors suggests a complex pattern of recognition that is also defined by cooperation between TLR.

Dimerization of the cytoplasmic domain of TLR2 does not induce cytokine production in macrophages, whereas similar dimerization of the TLR4 cytoplasmic domain is associated with pro-inflammatory signaling 4. Cytoplasmic domains of TLR2 can form functional pairs with TLR6 and TLR1 to lead to signal transduction and cytokine gene expression 4. TLR7 and TLR8 can independently confer responsiveness to the same antiviral compound R-848, pointing to a possible redundancy between these two receptors 5. All the TLR trigger signaling pathways that are similar to those activated by IL-1. Indeed, the type 1 IL-1 receptor (IL-1R1) has the same Toll/IL-1R homology domain capable of interacting with myeloid differentiation factor 88 (MyD88), an adaptor protein that is recruited upon activation of IL-1R1 or TLR 6. This leads to NF-κB signaling and transcriptional activation of numerous pro-inflammatory genes encoding cytokines, chemokines, proteins of the complement system, enzymes (iNOS, COX-2), adhesion molecules and receptors. Collectively, these molecules engage and control the innate immune response, which is essential for eliminating pathogens and preparing for transfer to the more specific, acquired immune response.

Although surprising, there is an innate immune system in the brain that is under control of a wide variety of molecules. It is now clear that gram-negative cell wall components stimulate pro-inflammatory signal transduction pathways by interacting with TLR4 expressed on the surface of immune cells. This interaction between LPS and TLR4 also takes place in the circumventricular organs (CVO) and choroid plexus (chp), which enables intracellular signaling and then rapid transcription of pro-inflammatory cytokines first within these organs and thereafter throughout the brain parenchyma during severe endotoxemia 7. Interestingly, a strong and transient increase in the expression of the gene encoding TLR2 was found in the brain of LPS-challenged mice 8. Surprisingly, PGN, LTA or a combination of both components failed to modulate TLR2 expression 8. These data provided strong evidence that, while TLR2 may be the recognizing receptor for gram-positive bacteria, transcriptional activation occurs in the brain only in response to gram-negative elements.

However, it is possible that pre-induction of TLR2 by cell wall component derived from gram-negative bacteria is a prerequisite for the action of PGN on the cerebral tissue. Therefore, the present study was accomplished to determine whether pretreatment with LPS has the ability to increase the effects of PGN on the transcription of genes involved in the innate immune response in the CNS. Unexpectedly, PGN failed to activate the innate immune response in the brain of mice presensitized with LPS. Actually, it interfered with the ability of the endotoxin to stimulate and maintain expression of genes encoding pro-inflammatory proteins in the CNS. Mutant mice were therefore used to investigate the respective role of TLR2 and TLR4 in these events.

2 Results

  1. Top of page
  2. Abstract
  3. 1 Introduction
  4. 2 Results
  5. 3 Discussion
  6. 4 Materials and methods
  7. Acknowledgements

2.1 Regulation of TLR2 gene expression in the CNS

As previously reported 8, a significant increase in TLR2 mRNA levels takes place in the chp as soon as 30 min post LPS injection (Fig. 1). The hybridization signal gradually increased in this structure to reach a maximal level between 90 min and 6 h after the single injection of the endotoxin. At that time, the message was also strong in cells lining the blood vessels and the leptomeninges. The signal declined slowly in these structures and returned to basal levels from 12 to 24 h post LPS treatment. A second wave of induction was found in the parenchymal brain 1 day after the i.p. LPS administration. Although TLR2 mRNA seemed to return to basal levels after 12 h, numerous small scattered isolated cells exhibited an intense hybridization signal in the parenchymal brain surrounding the chp (Fig. 1, bottom right). Induction of TLR2 takes place essentially within cells of myeloid origin, which are immunoreactive for iba1 8.

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Figure 1. Time-related induction of the gene encoding TLR2 in the brain of LPS-challenged mice. These dark-field photomicrographs of nuclear emulsion-dipped coronal sections depict a localized positive hybridization signal (agglomeration of silver grains) in the subfornical organ (SFO) and choroid plexus (Chp) of the vehicle-injected animal (Veh). The signal gradually increased in these structures to reach a maximal level between 90 min and 6 h after the i.p. LPS bolus and declined slowly thereafter. At 90 min post LPS administration, the message became strong in cells lining the cerebro-microvasculature. A second response wave of TLR2-expressing cells was found in the brain parenchyma 24 h after the endotoxin challenge, and the mRNA signal co-localized with iba1, used here as a marker of microglial cells (right bottom). Arrowheads: double-labeled cells. bv: blood vessels. Scale bar = 30 μm.

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2.2 Effects of pretreatment with LPS

In contrast to LPS, PGN and LTA, either alone or combined together, failed to trigger TLR2 in the CNS 8. We therefore tested the hypothesis that pre-induction of TLR2 by LPS was necessary for gram-positive cell wall components to stimulate the innate immune response in the CNS. Surprisingly, injection of PGN essentially abolished the effects of LPS on TLR2, TNF-α and monocyte chemotactic protein-1 (MCP-1) gene expression in microglial cells across the brain parenchyma. Indeed, while the signal for all these transcripts was strong 30 h after the first systemic LPS administration (Fig. 2, LPS-Saline), TLR2 and TNF-α mRNA levels decreased by more than 90% in response to PGN that was injected 6 h following the gram-negative cell wall component (Fig. 2, LPS-PGN). A similar phenomenon occurred for the chemokine MCP-1 in the brain parenchyma, but the message remained positive in few regions of the CNS.

These data indicate that despite up-regulation of TLR2 by LPS, PGN is able to interfere with the ability of the endotoxin to maintain expression of pro-inflammatory genes in the CNS. In contrast, animals that received two injections of LPS still exhibited a strong transcriptional activation of these genes in microglial cells across the cerebral parenchymal tissue (Fig. 2, middle column).

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Figure 2. Effects of LPS or PGN on the innate immune response in the brain of mice that were pretreated with LPS. Mice received a first injection with LPS 6 h before the second systemic bolus with the cell wall component derived from gram-negative or gram-positive bacteria. Mice were killed 24 h after the second injection with either LPS or PGN. The insets depict the coronal sections taken from the X-ray film. The dark-field photomicrographs were taken from the same sections, but dipped into nuclear emulsion. While the second LPS injection maintained TLR2, TNF-α and MCP-1 gene expression in the brain parenchyma of mice pretreated with the endotoxin, PGN significantly abolished the second response wave of these genes in the brain parenchyma. Data are means ± SEM for a minimum of four mice per group. *p<0.001 vs. LPS/saline-treated group. Representative expression level in the brain parenchyma was evaluated in three different rostro-caudal levels between the dorsal third and lateral ventricles. Please see the region delineated by the rectangle in the inset of the top left panel. (A) Between Bregma –0.22 and –0.34. (B) Between Bregma –0.94 and –1.06. (C) Between Bregma –1.46 and –1.58. Scale bar for the dark-field photomicrograph = 500 μm.

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2.3 Role of TLR4 in LPS-induced gene expression in the CNS

The mutant C3H/HeJ mice and their control littermates were used to verify the role TLR4 in the regulation of genes encoding pro-inflammatory proteins in the CNS by circulating cell wall components. While a single bolus of LPS provoked a strong transcriptional activation of TLR2, TNF-α, CD14 and MCP-1 mRNA in the CVO, leptomeninges, cells lining the microcapillaries and parenchymal elements of C3H/HeN mice, the gram-negative cell wall component failed to increase expression of these genes in C3H/HeJ mice (Fig. 3). Only constitutive expression levels of TLR2 and CD14 were found in the CNS of these animals that did not respond at all to either one or two injections of LPS.

Like our previous report 8, a low dose of PGN (1 mg/kg) did not increase TLR2 and cytokine gene expression in the brain of both C3H/HeN and C3H/HeJ mice (data not shown), but a single injection with a high dose of PGN (25 mg/kg) was able to stimulate both CD14 and MCP-1 transcripts in cells lining the endothelium of the brain capillaries (Fig. 4). Although few positive cells were found at 6 h post injection, the hybridization signal for CD14 and MCP-1 mRNA increased 24 h after PGN administration (Fig. 4, bottom panels). The signal remained associated with cells of the blood-brain barrier (BBB) and not within parenchymal elements of the brain. This contrasts with LPS, which is able to increase expression of these genes throughout the cerebral tissues of C3H/HeN mice (Fig. 3).

These data support previous studies that C3H/HeJ mice respond to gram-positive cell wall components, but not to a TLR4 ligand. The response to PGN was nevertheless very modest and localized to cells lining the BBB. This was also the case for the gene encoding inhibitory factor kappa B alpha (IκBα) following a single i.m. turpentine insult. This model of systemic inflammatory insult is known to increase NF-κB in the endothelium of the brain capillaries, and both C3H/HeN and C3H/HeJ exhibited similar expression pattern for IκBα mRNA (Fig. 5). Therefore, C3H/HeJ mice are resistant to LPS, but not to PGN and a systemic model of sterile localized inflammation.

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Figure 3. The endotoxin LPS failed to activate the innate immune response in the brain of C3H/HeJ mice that bear a loss of function in the tlr4 gene. On the contrary, a single systemic LPS injection caused a profound transcriptional activation of the gene encoding TLR2, TNF-α, CD14 and MCP-1 in the cerebral tissue of C3H/HeN mice. These dark-field photomicrographs were taken from coronal sections hybridized with radioactive cRNA probes and dipped into nuclear emulsion milk. These images are representative examples of the expression pattern for each transcript at 6 and 24 h post LPS injection.

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Figure 4. Effects of a cell wall component derived from gram-positive bacteria on gene expression in the CNS of C3H/HeN and C3H/HeJ mice. PGN was injected i.p. at a dose of 25 mg/kg and mice were killed 6 and 24 h thereafter. Such high dose failed to trigger TLR2 and TNF-α gene transcription in the brain of both mouse strains, but increased CD14 and MCP-1 mRNA expression in cells lining the endothelium of the brain capillaries. Indeed, the blood vessels and microcapillaries exhibited positive hybridization for both CD14 and MCP-1 transcripts in response to PGN, especially at time 24 h. Scare bar = 250 μm.

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Figure 5. TLR4-mutant mice are sensitive to a systemic model of sterile inflammation. De-novo expression of IκBα is a reliable index of NF-κB activity in the brain of immune-challenged animals 7, 20, 22, 23. I.m. turpentine administration is a model of sterile and localized inflammatory insult that provokes a robust swelling at the site of injection. A selective expression of IκBα was detected along the cerebral endothelium of both C3H/HeN and C3H/HeJ mice 24 h after the systemic treatment, which indicates that mice that bear a loss of function of the tlr4 gene are resistant to LPS, but not to other models of systemic inflammation. Scale bar = 50 μm.

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2.4 TLR2 is involved in adapting the cerebral response to LPS

A single bolus of LPS caused a strong and similar induction of inflammatory genes in the brain of TLR2-deficient mice and their wild-type littermates (Fig. 6). The median eminence and the other CVO of both mouse strains exhibited a localized pattern of TNF-α-expressing cells 6 h after the single LPS administration (Fig. 6, top panels). The other transcripts were found in cells lining the endothelium of the brain blood vessels and across the parenchyma of various regions, such as the hypothalamic area (IκBα), cerebral cortex (MCP-1) and amygdaloid complex (CD14) of LPS-injected wild-type and TLR2-deficient mice.

As previously observed in C3H/HeN mice, a second bolus of LPS maintained the expression levels of specific transcripts in wild-type animals, but not in TLR2-deficient mice. The hybridization signal for both IL-12 and TNF-α mRNA essentially vanished 24 h after the second injection with LPS (Fig. 7). Although numerous small-scattered cells were found in the brain of wild-type mice, very few IL-12- and TNF-α-expressing cells were detected within the cerebral tissue of TLR2-deficient animals that received two injections of the cell wall component derived from gram-negative bacteria. Of interest, the other transcripts analyzed in the present study were still expressed in the brain of TLR2-deficient mice challenged twice with the endotoxin (data not shown).

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Figure 6. Effect of LPS on the pro-inflammatory signaling events in the brain of TLR2-deficient mice. These dark-field photomicrographs were taken from coronal sections hybridized with different cRNA probes and dipped into NTB2 emulsion milk (Kodak). Both TLR2-deficient and wild-type groups of mice exhibited a similar induction pattern for the gene encoding TNF-α, IκBα, MCP-1 and CD14 in various regions of the brain 6 h after a single LPS injection. Except for TNF-α, which was not associated with blood vessels (bv), the other induced transcripts were found in both vascular and parenchymal elements of the cerebral tissue. ME: median eminence. Scale bar = 500 μm.

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Figure 7. Expression of IL-12 and TNF-α is profoundly altered in the brain of TLR2-deficient mice challenged twice with the endotoxin LPS. TLR2-deficient mice (KO) and their wild-type littermates (WT) received a first i.p. LPS injection, and 6 h later a second bolus with the endotoxin. Mice were killed 24 h after the last set of injection. This treatment caused a strong transcriptional activation of both IL-12 and TNF genes in the brain of wild-type animals, but not in TLR2-mutant mice. These dark-field photomicrographs were taken from coronal sections of both strains of mice and are representative examples of the hybridization signals in the fimbria adjacent to the subfornical organ for IL-12 mRNA and cortex/amygdaloid complex for TNF-α. The number of positive cells in these regions was significantly lower in mutant mice than in their wild-type littermates. Actually, very few cells remained positive in the brain of TLR2-deficient mice challenged twice with the cell wall component derived from gram-negative bacteria. Data are means ± SEM for a minimum of four mice per group. *p<0.001 vs. wild-type mice. Scale bar = 500 μm.

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3 Discussion

  1. Top of page
  2. Abstract
  3. 1 Introduction
  4. 2 Results
  5. 3 Discussion
  6. 4 Materials and methods
  7. Acknowledgements

The present set of data provides evidence that LPS causes a strong transcriptional activation of TLR2 and numerous genes involved in the control of the innate immune response. Pre-induction of TLR2 did not enable PGN to increase expression of genes encoding cytokines, chemokines and receptors in the cerebral tissue. On the contrary, systemic PGN administration largely abolished the effect of LPS on TLR2, TNF-α and MCP-1 gene expression in microglial cells across the brain parenchyma. This phenomenon depends on TLR4, because C3H/HeJ mice no longer responded to LPS. These mutant mice, however, exhibited a localized immune reaction in cells lining the endothelium of the brain capillaries following i.p. PGN injection and i.m. turpentine insult. Although TLR2 is not involved in the profound transcriptional activation of genes encoding pro-inflammatory proteins in response to a single injection with LPS, it plays a critical role in maintaining TNF-α and IL-12 gene expression in the brain of animals that received two subsequent boluses of LPS. These data indicate that TLR4 is necessary for LPS to trigger expression of TLR2 and other immune-related genes in the CNS, while TLR2 is involved in the second wave of TNF-α and IL-12 expression that takes place in the brain during endotoxemia. Induction of TLR2 may be a determinant mechanism to stimulate microglial IL-12 production, which plays a critical role in the transfer from the innate to adaptive immune response.

Although LPS is able to cause a robust and transient expression of a wide variety of genes involved in the innate immune reactions, cell wall component from gram-positive bacteria are very poor stimuli to activate microglial cells 8. The poor potency of PGN to activate transcriptional activation in the brain is in agreement with many in vitro studies showing that it takes up to a thousand times more of PGN than LPS to obtain a similar response 9. We hypothesized that pre-induction of TLR2 by a single bolus of LPS might increase the effects of PGN, because TLR2 is rapidly induced in the CVO and the microvasculature. Circulating PGN may therefore have a direct access to the cells that exhibit up-regulation of its cognate receptor and trigger NF-κB signaling cascade and gene expression. Surprisingly, such process did not enable PGN to stimulate the innate immune response in the CNS, but on the contrary abolished the influence of the primary LPS injection. Indeed, a second injection with the endotoxin caused a pro-longed induction of TLR2, TNF-α and MCP-1 as well as numerous other pro-inflammatory genes in microglial cells across the brain parenchyma. Animals that received PGN instead of LPS after the first bolus of LPS exhibited a much lower response than those treated twice with the gram-negative cell wall component. Actually, the hybridization signal for TLR2 and TNF-α was barely detectable 24 h after the systemic PGN administration, which clearly indicates that the cell wall component derived from gram-positive bacteria repressed rather than exacerbated the innate immune response in the CNS of LPS-challenged mice.

The mechanisms involved in such down-regulation of genes encoding immune molecules remain to be determined, but the data that C3H/HeJ mice no longer respond to LPS point in direction to this receptor. C3H/HeJ mice bear a loss of function in the tlr4 gene, and these animals are totally resistant to LPS 10. This is also the case in the brain, because the endotoxin injected either once or twice did not activate microglial cells in the CNS of C3H/HeJ mice. Since PGN is able to decrease the activity of microglial cells in response to LPS, it is possible to reach the conclusion that cooperation or interference between TLR4 and TLR2 exists in the cerebral tissue. TLR4 is, however, not required for PGN to activate pro-inflammatory signal transduction pathways, because C3H/HeJ and C3H/HeN mice respond similarly to PGN and other cell wall components derived from gram-positive bacteria 11. Induction of IκBα was also found in cells lining the endothelium of the brain capillaries of these mice after an i.m. turpentine injection, which indicates that C3H/HeJ mice are not resistant to other models of systemic insults. It is proposed here that LPS signals through TLR4 expressed on the surface of monocytic cells in the CVO and chp. This leads to an expression wave of genes encoding innate immune proteins that is further increased by a subsequent injection with a ligand for TLR4 and, in contrast, decreased in presence of a ligand that binds TLR2.

It is possible that Toll-interacting protein (Tollip) participates in the effects of PGN to down-regulate the innate immune response in the CNS of LPS-challenged mice. The adaptor protein Tollip is an important constituent of the IL-1R signaling pathway that associates directly with TLR2 and TLR4 and plays an inhibitory role in TLR-mediated cell activation 12. Moreover, Tollip co-immunoprecipitates with TLR2 and TLR4, and overexpression of Tollip inhibits NF-κB activation in response to TLR2 and TLR4 signaling 13, 14. Inhibition by Tollip is mediated by the ability of this protein to suppress the activity of IL-1R-associated kinase (IRAK) upon TLR activation 12. Whether association between IRAK and Tollip is accelerated in the cerebral tissue of mice that received LPS and then PGN has yet to be determined, but such mechanism remains a promising avenue for future investigations. Indeed, Tollip is an induced molecule that inhibits pro-inflammatory signal transduction pathways, and its overexpression results in impaired NF-κB activation 14. A similar role of Tollip is also expected in the brain of mice challenged twice with LPS, because both NF-κB and activator protein-1 are activated by LPS and PGN, and both are dose-dependently inhibited by Tollip 12. However, we did not find such profound attenuation in microglial cells of mice challenged twice with LPS, and Tollip may have a limited potential to prevent pro-inflammatory signaling in this model.

Dimerization of the cytoplasmic domain of TLR2 does not induce cytokine production in macrophages, whereas similar dimerization of the TLR4 cytoplasmic domain is associated with pro-inflammatory signaling 4. It has indeed been observed that the cytoplasmic domain of TLR2 can form functional pairs with TLR6 and TLR1 to lead to signal transduction and cytokine gene expression 4. Such necessary combinatorial repertoire may also be involved in the alteration of microglial activity by PGN, although we have yet to find expression patterns of the other members of this family of innate receptors in the CNS. TLR2 was originally proposed to be the receptor for PAMP produced by gram-negative bacteria, but this was an artifact caused by the preparation of LPS that contained low concentrations of highly bioactive contaminants 15. It is now clear that gram-negative cell wall components stimulate pro-inflammatory signal transduction pathways by interacting with TLR4 expressed on the surface of immune cells. However, TLR2-deficient mice challenged with two subsequent boluses of LPS exhibited a much lower response than their wild-type littermates. A single injection of LPS caused a similar expression pattern of genes encoding TNF-α, IκBα, MCP-1 and CD14 in the brain of both TLR2-deficient and wild-type animals, but clear differences took place for TNF-α and IL-12 transcripts in response to the double treatment with the endotoxin. These data indicate that TLR2 is not responsible to trigger transcription of pro-inflammatory genes in the cerebral tissue, but is involved in the TNF-α and IL-12 production during severe endotoxemia.

Except for its recognized role to act as the signaling receptor for lipoproteins, gram-positive, spirochetal and fungal cell wall components, little is known about TLR2 in mammalian cells, and the physiological relevance of the strong de-novo expression has yet to be clarified. This is especially the case for the brain, as we are still at the embryonic stage of the functional aspects of TLR biology and, so far, no known endogenous ligand binds to these receptors. Why then is a receptor present without ligand? It is possible that external stimuli and insults induce TLR2 gene expression to prepare the cells to rapidly engage the innate immune response and eliminate additional foreign pathogens. This association between external insults and increase in TLR2 may be particularly important in the cerebral tissue to protect the neuronal elements in case of invasion due to a BBB breakdown or cerebral infection. Once induced, this receptor may enable recruitment of MyD88 and other proteins involved in the NF-κB signaling cascade despite the lack of specific ligands.

In this regard, TLR2 plays a critical role in maintaining the transcriptional activation of TNF-α and IL-12p40 mRNA in microglial cells of the brain parenchyma of mice challenged with two consecutive boluses of LPS. This treatment was in reality able to increase these transcripts in the brain of wild-type mice, but not in that of TLR2-deficient animals. It is interesting that these animals showed a normal response to a single bolus of LPS, but not following two LPS administrations that normally cause a severe and long-lasting expression of immune-related genes in the CNS. While a modest increase in IL-12 mRNA levels was detected in the brain of mice killed after a single LPS injection, a strong hybridization signal was found for this cytokine in the CNS of CD1 (data not shown) and wild-type mice 24 h after the second LPS injection. This indicates that such treatment provokes a severe innate immune response in the cerebral tissue that is greatly attenuated in TLR2-deficient mice. Although surprising, these results are in agreement with another study that showed induction of IL-12 in a TLR2-dependent manner. Indeed, pretreatment with anti-TLR2 monoclonal antibody inhibited IL-12 production from human dendritic cells exposed to bacterial lipoproteins or LPS 16.

IL-12 is a key cytokine involved in the transfer between the innate and adaptive immunity. Macrophage-derived IL-12 stimulates the differentiation of a subset of T lymphocytes (CD4+) into T helper 1 (Th-1) cells, which produce IFN-γ. These activated Th-1 cells are actually believed to play a critical role in multiple sclerosis, especially during the demyelinating episodes (for a review, see 17). Of interest is that the clinical course of experimental autoimmune encephalomyelitis is associated with a profound and sustained transcriptional activation of the gene encoding TLR2 in the mouse CNS 18. Although this study does not provide direct evidence that TLR2 is responsible for the transfer from the innate to adaptive immune response, it is tempting to propose that this receptor plays a critical role in activating the APC of the brain, namely microglia. The fact that these cells no longer express IL-12 mRNA in TLR2-deficient mice during severe endotoxemia clearly support this concept. Although the link between the innate immune reaction and acquired immunity has been better clarified during the past few years, numerous questions immediately arise as to whether a defect in this fine interplay is a direct cause of autoimmune diseases taking place in systemic structures as well as the CNS.

In conclusion, the present set of data indicates that TLR4 is necessary for initiating the cerebral immune response to LPS, which can be rapidly abolished by PGN in a TLR4-dependent manner. On the other hand, TLR2 is involved in the second wave of IL-12 and TNF-α expression by microglial cells of the mouse CNS during severe endotoxemia. The prolonged induction of TLR2 is a determinant mechanism to stimulate microglial IL-12 production, which plays a critical role in the transfer from the innate to adaptive immune response. There is an elegant cooperation between TLR4 and TLR2 in the brain of immune-challenged mice. This interaction is likely to play a critical role in the control of the subsequent events and to have profound consequences for the CNS during infection with gram-negative and gram-positive bacteria. The fact that TLR2-deficient mice are highly susceptible to cerebral infection and meningitis supports this concept 19.

4 Materials and methods

  1. Top of page
  2. Abstract
  3. 1 Introduction
  4. 2 Results
  5. 3 Discussion
  6. 4 Materials and methods
  7. Acknowledgements

4.1 Animals

Adult male C3H/HeN mice (Charles River Canada, St. Constant, Canada; ∼22–25 g b.w.) and C3H/HeJ (Jax Mice, Jackson Laboratory, Bar Harbor, ME; 6 weeks of age, ∼22–25 g b.w.) were acclimated to standard laboratory conditions (14-h light, 10-h dark cycle; lights on at 06:00 and off at 20:00) with free access to mouse chow and water. Animal breeding and experiments were conducted according to Canadian Council on Animal Care guidelines, as administered by the Laval University Animal Care Committee. A total of 108 mice (54 C3H/HeN and 54 C3H/HeJ) were assigned to different treatments and post-injection times (6 and 24 h). Paired vehicle-treated mice were also killed at corresponding times after the injection.

TLR2-deficient mice and their wild-type littermates were generated as previously described 19. Briefly, TLR2–/– mice were backcrossed for six generations on a C57BL/6 background and were kept under specific pathogen-free conditions in the animal house of the Department of Research, University Hospital Basel, Switzerland, according to the regulations of the Swiss veterinary law. Seventeen wild-type C57BL/6 and 17 TLR2–/– mice were used in the present study.

4.2 Experimental protocols

On the day of the experiment (∼08:30 in the morning), the endotoxin LPS (1 mg/kg; from Escherichia coli, serotype 055:B5; Sigma, L2880, lot 31KH4120) or the vehicle solution (100 μl sterile pyrogen-free 0.9% saline) was injected into the mouse peritoneal cavity. A second i.p. injection with either LPS (1 mg/kg), PGN (1 or 25 mg/kg; from Staphylococcus aureus; Fluka, 77140, lot 13239/1 31500) or the vehicle solution was performed 6 h later. Another group of animals was injected into the left thigh muscle with 50 μl/100 g b.w. of turpentine (Spectrum Chemical Mfg. Corp., TU 109, CAS 8006–64–2, Gandena, CA) or 0.9% of sterile pyrogen-free saline. This experimental model of sterile inflammation (i.e. an inflammatory response developing in the absence of any microbial stimulus) induces a local tissue damage that is responsible for the development of a systemic acute-phase response.

Six or 24 h after the last systemic injections, animals were deeply anesthetized via an i.p. injection of a mixture of ketamine hydrochloride (91 mg/ml) and xylazine (9 mg/ml) and then rapidly transcardially perfused with 0.9% saline, followed by 4% paraformaldehyde in 0.1 M borax buffer (pH 9.5 at 4°C). Brains were rapidly removed from the skulls, postfixed for 2–8 days and then placed in a solution containing 10% sucrose diluted in 4% paraformaldehyde-borax buffer overnight at 4°C. The frozen brains were mounted on a microtome (Reichert-Jung, Cambridge Instruments Company, Deerfield, IL) and cut into 20-μm coronal sections from the olfactory bulb to the end of the medulla. The slices were collected in a cold cryoprotectant solution (0.05 M sodium phosphate buffer pH 7.3, 30% ethylene glycol, 20% glycerol) and stored at –20°C.

4.3 cRNA probe preparation and in situ hybridization histochemistry

Plasmids were linearized and the sense and antisense riboprobes synthesized with the appropriate RNA polymerase as described in the Table 1. Radioactive cRNA copies were synthesized by incubation of 250 ng linearized plasmid in 6 mM MgCl2, 40 mM Tris (pH 7.9), 2 mM spermidine, 10 mM NaCl, 10 mM dithiothreitol (DTT), 0.2 mM ATP/GTP/CTP, 100 μCi of [α-35S]UTP (NEG 039H, Dupont NEN), 20 U RNAsin (Promega, Madison, WI) and 10 U of either T7, SP6 or T3 RNA polymerase for 60 min at 37°C (see Table 1). Unincorporated nucleotides were removed using ammonium acetate precipitation method; 100 μl of DNase solution (1 μl DNase, 5 μl of 5 mg/ml tRNA, 94 μl of 10 mM Tris/10 mM MgCl2) was added, and 10 min later, a phenol-chloroform extraction was performed. The cRNA was precipitated with 80 μl of 5 M ammonium acetate and 500 μl of 100% ethanol for 20 min on dry ice. The pellet was dried and resuspended in 50 μl of 10 mM Tris/1 mM EDTA. A probe containing 107 cpm was mixed into 1 ml of hybridization solution [500 μl formamide, 60 μl 5 M NaCl, 10 μl 1 M Tris (pH 8.0), 2 μl 0.5 M EDTA (pH 8.0), 50 μl 20× Denhart's solution, 200 μl 50% dextran sulfate, 50 μl 10 mg/ml tRNA, 10 μl 1 M DTT, (118 μl DEPC water – volume of probe used)]. This solution was mixed and heated for 10 min at 65°C before being spotted on slides. Hybridization histochemical localization of TLR2, TNF-α, MCP-1, IκBα, CD14 and IL-12 mRNA was carried out on every twelfth section of the whole rostro-caudal extent of each brain using 35S-labeled cRNA probes as described previously 7, 20, 21.

Table 1. Plasmids and enzymes used for the synthesis of the cRNA probes
PlasmidVectorInsertAntisense probeSense probeSource
  1. a) The DNA fragment of 2.278 kb corresponding to the almost complete coding sequence (2.355 kb) of the reported mouse TLR2 mRNA (nucleotides 307–2661, GenBank accession no. AF185284) wasamplified by PCR from a cDNA macrophage B10R cell line library using a pair of 23-bp oligonucleotide primers complementary to nucleotides 323–345 (5′-GGCTCTTCTGGATCTTGGTGGCC-3′) and 2579–2601 (5′-GGGCCACTCCAGGTAGGTCTTGG-3′).

Mouse TLR2PCR blunt II2.248 kbEcoR V/SP6Spe I/T7PCR amplificationa)
Mouse TNF αbluescript SK++1.3 kbPst I/T3BamH I/T7Dr. D. Radzioch, McGill U. Montréal, Canada
Mouse MCP-1pGEM-1578 bpBamH I/T7Sac I/SP6Dr. S. C. Williams, Texas Tech U., Lubbock, TX
Mouse CD14pRc/CMV1.5 kbApa I/T7Hind III/SP6Dr. R. Landmann, Basel, Switzerland
Mouse IL-12p40pCL-NEO1.05 kbXho I/T3Not I/T7Dr. K. Pahan U. Nebraska, Lincoln, NE
Mouse IκBαbluescript SK++1.114 kbBamH I/T7Hind III/T3Dr. A. Asrael Inst. Pasteur, Paris, France

4.4 Quantitative analysis

Quantitative analysis of hybridization signal was carried out on X-ray films (Biomax, Kodak) over different regions of the brain as previously described 21, 22. Data are reported as mean optical density values (± SEM) and statistical analysis was performed by analysis of variance (ANOVA) for each transcript, followed by a Bonferroni/Dunn test procedure as post-hoc comparisons with Statview software (version 4.01, Macintosh). The number of positive cells was also measured in specific regions of the brain using an Olympus BX60 microscope under dark-field illumination.

Acknowledgements

  1. Top of page
  2. Abstract
  3. 1 Introduction
  4. 2 Results
  5. 3 Discussion
  6. 4 Materials and methods
  7. Acknowledgements

This Research was supported by the Canadian Institutes of Health Research [CIHR; the former Medical Research Council of Canada (MRCC)]. Serge Rivest is an MRCC Scientist and holds a Canadian Research Chair in Neuroimmunology. We thank Dr. A. Israel (Institut Pasteur, Paris, France) for the mouse IκBα cDNA, Dr. D. Radzioch (McGill University, Montréal, Canada) for the plasmid containing the mouse TNF-α cDNA, Dr. K. Pahan (University of Nebraska, Lincoln, NE, USA) for the mouse IL-12p40 cDNA and Dr. S.C. Williams (Texas Tech University, Lubbock, TX, USA) for the mouse MCP-1 plasmid.

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