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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online February 7, 2006 as doi:10.1096/fj.05-5234fje. |
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Laboratory of Molecular Endocrinology CHUL Research Center and Department of Anatomy and Physiology, Laval University, Québec, Canada
1Correspondence: E-mail: serge.rivest{at}crchul.ulaval.ca
SPECIFIC AIMS
Regarded as a damaging reaction, innate immune response can either improve or worsen brain outcome after injury. Hence, inflammatory molecules may modulate cell susceptibility or healing events. The aim of this study was to evaluate whether stimulation of CNS immune system affects the mobilization of cells expressing crucial transcription factors and genes encoding components of myelin sheath following acute and more chronic models of demyelination. We also investigated the role of Toll-like receptor 4 (TLR4) activation and NF-
B signaling in a lesion model of surfactant-mediated cytolysis.
PRINCIPAL FINDINGS
1. Lipopolysaccharide (LPS) treatment induces platelet-derived growth factor receptor (PDGFR)-
and modulates early events following acute demyelination
A single intracerebral LPS bolus was used to cause a rapid and transient innate immune reaction in microglia. We verified whether this acute response was able to alter oligodendrocyte progenitor cells (OPCs), which can be identified by the expression of the gene encoding PDGFR-. LPS-induced microglia activation was not associated with extensive demyelination, but instead correlated with a widespread increase in PDGFR- mRNA levels throughout brain parenchyma 2 days after the injection. Since LPS administration changes OPC reactivity, we decided to use an acute model of demyelination through administration of ethidium bromide (EtBr) for studying the impact of innate immunity on mechanisms controlling myelin repair. The remyelination that follows brain lesions is dependent on the recruitment of OPCs and expression of genes controlling differentiation and myelin production, such as Olig1 and Olig2 basic helix-loop-helix (bHLH) transcription factors. We observed that at 2 days post lesion, cells that strongly express Olig1 and Olig2 accumulate at the edge of the lesion. LPS co-infusion switched this pattern to a spread distribution of Olig1- and Olig2-positive cells (Fig. 1
B). To quantify these anatomical changes, a measure of differential optical density (O.D.) in delimitated regions was performed. Values for the "edge effect" and the "spread effect" are defined in Fig. 1A
. We found a significant decrease of the "edge effect" in EtBr/LPS-treated samples compared with those of the EtBr group, while the "spread effect" was increased by LPS co-infusion (Fig. 1C
). Hence, accumulation of Olig1- and Olig2-positive OPCs at the border of the lesion is decreased by LPS.
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2. Brain immunity improves OPC mobilization to demyelinated sites
We next determined whether activation of immune response in the CNS by LPS improved or worsened tissue repair in terms of size of the region to be remyelinated. At 5 days post lesion, EtBr administration caused severe demyelination in regions that no longer expressed transcripts for PDGFR-, Olig1, Olig2, and myelin components (e.g., proteolipid protein (PLP) and myelin oligodendrocyte glycoprotein (MOG), Fig. 2
). LPS-induced microglia activation significantly reduced the size of the area nonpopulated with OPCs as assessed by PDGFR- gene expression. LPS was also able to cause the same effect for Olig1- and Olig2-positive cells (Fig. 2A, B
). PLP is a major constituent of myelin and its reappearance was enhanced by the inflammatory treatment. As for PLP, LPS significantly attenuated the effects of EtBr on MOG-expressing regions (Fig. 2C, D
), implying that microglia activation does indeed lead to a better outcome. In addition, we found that LPS co-infusion resulted in less accumulation of myelin debris and by-products that may delay brain recovery.
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3. Innate immunity-mediated demyelination is permissive to OPC reactivity
We have described previously that MK-801, an antagonist of N-methyl-D-aspartate (NMDA) glutamate receptor subtype, exacerbated microglia response to LPS in the brain of C3H/HeN mice, but not in C3H/HeJ TLR4-deficient animals. Moreover, continuous LPS/MK-801 infusion caused demyelination within 3 days. Using this model we were able to assess the effects of innate immune response on OPC recruitment and reactivity in nondemyelinating (LPS alone) and demyelinating (LPS/MK-801) region showing inflammation. Both LPS/MK-801 and LPS treatments were able to increase PDGFR- transcript levels at 3 and 7 days of treatment, implying that OPC recruitment is actually stimulated by innate immunity independently on the loss in myelin content. Loss in PLP gene expression levels is observed following acute demyelination but surprisingly, we were not able to find sensitive decrease in PLP mRNA levels in the selected time-course. A localized induction was instead detected, especially at 7 days of treatment combining LPS to MK-801, but not in samples treated with saline, LPS, or MK-801 alone. As for PLP, Olig1 mRNA levels were induced specifically by the demyelinating inflammatory treatment. Such association was not as direct as for Olig2, which is also linked to gene transcription in OPCs responding to demyelination.
4. Inflammation increases tissue resistance against surfactant-mediated cell lyses
The experiments with acute and chronic models of demyelination clearly show that innate immunity triggers recruitment of OPCs and accelerates the remyelination process. We next wanted to determine whether such a beneficial effect of inflammation was specific to the mechanisms involved in myelin repair or if it is a more general feature of neuroprotection. The detergent Tween-20 is able to provoke a localized damage when infused into the cerebral tissue. Surprisingly, co-administration of LPS essentially abolished the neurotoxic properties of the cell lysis agent. To discard the possibility that LPS was neutralizing the surfactant via chemical interactions, we used C3H/HeJ mice that are nonresponsive to LPS. While the endotoxin was able to decrease the necrotic area in the control C3H/HeN mice at time 2 days post injection, no difference was found in the brain of TLR4-deficient mice, suggesting that interaction of LPS with its cognate TLR4 and activation of proinflammatory signaling in microglia is the neuroprotective mechanism against Tween-20. Dexamethasone (DEX) is a potent antiinflammatory drug that effectively prevents NF-B activation in the brain and when administered to mice, Tween-20 was highly toxic to the cerebral tissue. This confirms the protective role of innate immunity in this acute model of brain injury.
CONCLUSIONS AND SIGNIFICANCE
Although the immune response is critical for brain protection, microglial cells and proinflammatory signaling have become hallmarks of neurodegenerative processes and the inhibition of different cytokines and chemokines was found to have neuroprotective properties. Nevertheless, microglial reactivity removes cell debris and provides trophic support. For the development of more adequate therapeutic strategies, it is critical to better understand how microglia and proinflammatory signaling modulate the molecular mechanisms involved in brain repair and cellular resistance to insults. Remyelination offers an appropriate model to study the impact of the brain immune response on tissue recovery. This healing process follows the loss of myelin sheaths, a feature of multiple sclerosis (MS) and other demyelinating diseases. In MS patients remyelination may become inefficient with disease progression and accumulation of lesions results in progressive neurological demise. Generation of new oligodendrocytes, the myelinating cells in the CNS, is required for repairing demyelinated axons. While Olig2 bHLH transcription factor is necessary for oligodendrocyte lineage specification during development, Olig1 does not affect the formation of OPCs, but it is critical for remyelination and transcription of myelin-specific genes. Stimulation of brain innate immunity with LPS increased OPC reactivity. This transient inflammatory reaction is associated with changes in the distribution of Olig1- and Olig2-positive cells. The meaning of these changes is not known, however there is evidence that accumulation of OPCs and myelin-producing cells around the lesion may contribute to the formation of tissue scar, which impairs axon growth and proper tissue recovery. Recruitment of remyelinating cells could be improved by preventing the premature differentiation and increasing the level of recruitment signal. In this manner, activation of microglia by LPS may improve brain repair by avoiding precipitated accumulation of differentiating progenitors and inhibitory molecules at the border of the injured site, opposing to what would make difficult for OPCs to migrate and mature into oligodendrocytes far within the core of the lesion. At latter time-points, recruitment of OPCs-expressing Olig1/2 and reappearance of PLP and MOG transcripts is enhanced by LPS co-administration. We propose that stimulation of brain innate immune response improves OPC mobilization to repair tissues from an acute injury. These data may counterbalance a proposed unfavorable role of brain inflammation in demyelinating diseases. The correlation between inflammation and demyelination process observed by many groups advocates for the use of antiinflammatory treatment to prevent greater myelin damage. In spite of this, it has been shown that molecules associated with immune response, such as TNF-, IL-1ß, and LIF enhance remyelination. We now report that LPS-induced microglial activation leads to a more efficient OPC recruitment, enhances expression of key transcription factors linked to remyelination and prevents accumulation of myelin by-products. We also describe that ongoing innate immune activity stimulates OPC response and is permissive to the expression of transcripts associated with myelin repair, such as Olig1 and PLP, at the lesion site (Fig. 3
). Finally, activation of TLR4 and NF-B in microglia increases tissue resistance against highly toxic compounds. The innate immune system, therefore, has powerful neuroprotective properties in the CNS.
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FOOTNOTES
To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.05-5234fje;
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