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Published in final edited form as: Neurosci Lett. 2010 Jan 5;471(1):10–14. doi: 10.1016/j.neulet.2009.12.076

Toll-like receptor 2 and facial motoneuron survival after facial nerve axotomy

Derek A Wainwright 1,2, Junping Xin 1,2, Nichole A Mesnard 2,3, Virginia M Sanders 4,#, Kathryn J Jones 1,2,#
PMCID: PMC2825370  NIHMSID: NIHMS170980  PMID: 20056129

Abstract

We have previously demonstrated that CD4+ Th2 lymphocytes are required to rescue facial motoneuron (FMN) survival after facial nerve axotomy through interaction with peripheral antigen presenting cells, as well as CNS resident microglia. Furthermore, the innate immune molecule, toll-like receptor 2 (TLR2), has been implicated in the development of Th2-type immune responses and can be activated by intracellular components released by dead or dying cells. The role of TLR2 in the FMN response to axotomy was explored in this study, using a model of facial nerve axotomy at the stylomastoid foramen in the mouse, in which blood-brain-barrier (BBB) permeability does not occur. After facial nerve axotomy, TLR2 mRNA was significantly upregulated in the facial motor nucleus and co-immunofluorescence localized TLR2 to CD68+ microglia, but not GFAP+ astrocytes. Using TLR2-deficient (TLR2−/−) mice, it was determined that TLR2 does not affect FMN survival levels after axotomy. These data contribute to understanding the role of innate immunity after FMN death and may be relevant to motoneuron diseases, such as amyotrophic lateral sclerosis (ALS).

Keywords: neuroprotection, PRR, TLR2, facial motoneuron, CNS

INTRODUCTION

Our lab discovered that facial motoneuron (FMN) survival after facial nerve transection outside the skull is decreased in mice that lack functional adaptive immune systems [32]. Furthermore, it has been demonstrated that T cells infiltrate the mouse facial motor nucleus after peripheral facial nerve injury [16,29]. In support of these findings, our laboratory has shown that Th2 cells develop in the draining cervical lymph node after facial nerve axotomy [41] and rescue FMN survival through interaction with microglia [4,11]. Collectively, these data suggest a mechanism for Th2 cell-mediated rescue of FMN after facial nerve axotomy that depends on microglia.

Microglia are thought to be key mediators of CNS inflammation, since they express major histocompatibility complex II [17], are derived from the hematopoietic lineage [12] and are potent cytokine producers [14]. Furthermore, microglia are considered to be the parenchymal macrophages of the CNS, based on expression of F4/80 [28], CD11b [15] and CD68 [18]. However, in contrast to peripheral macrophages, microglia have decreased CD45 expression [31] and increased F4/80 expression [5].

After mouse facial nerve axotomy, microglia encircle and phagocytize damaged or dying FMN [35]. While the specific signals that perpetuate microglial activation are not fully understood, toll-like receptors may be involved through recognition of molecules released by dead/damaged FMN. In agreement, TLR2 has previously been demonstrated to be upregulated on microglia in denervated zones of the hippocampus after transection of axons in the entorhinal cortex [1]. However, it is unknown if TLR2 is involved in FMN-mediated neuroprotection after facial nerve axotomy, whereby the BBB is not permeabilized [29].

It has been previously shown that mouse immunization with the TLR2 agonist, Pam3Cys, induces the Th2 cytokines, interleukin 5 (IL-5) and IL-13 [30]. Furthermore, in a model of chronic asthma, Th2 cytokine levels are significantly decreased in the lungs of TLR2−/− mice [3]. In agreement, staphylococcus aureus, a bacterium that activates TLR2 [34], causes increased expression of Th2 cytokines, IL-4, IL-5 and IL-13, in a mouse model of allergic conjunctivitis [8]. Thus, TLR2 activation appears to be associated with enhanced Th2-type immune responses.

Based on the ability of TLR2 to induce a Th2-type immune response and the requirement of Th2 lymphocytes for rescuing FMN survival after axotomy, we hypothesized that TLR2 plays a role in FMN survival after peripheral facial nerve axotomy. To investigate this hypothesis, we initially used microarray analysis to analyze TLR2 and TLR4 mRNA levels in the facial motor nucleus at a time point consistent with significant T cell infiltration [29]. Based on microarray analysis, we focused our investigation on TLR2. Co-immunofluorescence revealed that TLR2 localizes to microglia, but not astrocytes, after injury. Furthermore, we used the well established facial nerve axotomy paradigm in conjunction with the TLR2−/− mouse model to determine whether TLR2 plays a role in rescuing FMN survival after injury to the facial nerve. Collectively, the results indicate that TLR2 mRNA expression increases in the facial motor nucleus, localizes to microglia, but does not affect FMN survival after injury.

MATERIALS AND METHODS

Animals and surgical procedures

Seven-week old female wild-type (WT; C57BL/6) and TLR2−/− (C57BL/6 background) mice were obtained from Jackson Laboratories (Bar Harbor, ME) and Taconic (Germantown, NY), respectively. Mice were used at 8 weeks of age in all experiments. All surgical procedures were completed in accordance with National Institutes of Health guidelines on the care and use of laboratory animals for research purposes. Mice were anesthetized with 3% isoflurane for all surgical procedures. Using aseptic techniques, the right facial nerve of each animal was exposed and transected at its exit from the stylomastoid foramen [19].

Microarray experiments

At 7 days post-axotomy (DPA), WT mice (n=4) were euthanized with CO2 and the brains rapidly removed. Coronal sections of brain stem including both facial motor nuclei were collected and tissue punches of the left (uninjured control) and right (axotomized) facial motor nuclei obtained from these sections and pooled (right axotomy vs left control). The tissue punches were placed into 0.65 mL microfuge tubes with 20, 1.4 mm Lysing Matrix D beads (Q-Biogene, Morgan Irvine, CA)/tube, and 80 µL working solution D (4M guanidium thiocyanate, 25mM sodium citrate, .5% sarcosyl, 0.1M β-mercaptoethanol in RNAse-free H2O). RNA was isolated from tissue punches and harvested cells by guanidinium-thiocyanate extraction [6]. Samples were processed at Superarray Bioscience Corporation (Frederick, MD) using the Mouse Chemokines and Receptors Microarray, OMM-022. Fold-changes in gene expression (right axotomy/left control) were calculated for pair-wise comparison using the GEArray Expression Analysis suite. Changes in mRNA levels for select inflammatory genes other than TLR2 and TLR4 are demonstrated in supplementary figure 1.

Real-time PCR experiments

WT mouse brains were removed at 1, 4, 7, 14 and 30 DPA (n=3–6/timepoint) and flash frozen [in 62.5% n-Butyl Bromide (Fisher Scientific, Pittsburgh, PA) + 37.5% 2-methylbutane (Fisher Scientific) surrounded by crushed dry ice]. Additional uninjured animals were also included (n=6). Frozen brains were sectioned on the Leica CM3000 cryostat (Leica, Bannockburn, IL) with a temperature of −24°C and immersed into Tissue Teck O.C.T. Compound (Sakura Finetek USA, Inc., Torrance, CA) at 25 µm intervals and thaw-mounted onto membrane-coated glass slides (Leica Microsystems Inc., Bannockburn, IL). Laser microdissection of control and axotomized facial motor nuclei using the Leica AS LMD (Leica Microsystems Inc.) was accomplished, capturing tissue into 0.5 mL microcentrifuge tube caps with 65 uL Extraction Buffer (PicoPure RNA Isolation kit, Arcturus, Mountain View, CA).

A PicoPure RNA Isolation kit (Arcturus) was used to extract RNA and followed the manufacturer’s instructions. Complementary DNA was generated and used in real-time PCR reactions and amplification was detected with SYBR green fluorescent dye (Applied Biosystems, Carlsbad, CA). Primer sequence information was previously described for TLR2 [7,40] and GAPDH [13]. GAPDH served as the reference gene. For each sample, percent change in TLR2 mRNA levels was calculated using the formula (axotomy/control × 100) − 100%.

Immunofluorescence experiments

At 7 DPA, WT mice were euthanized with CO2 followed by flash freezing [in 62.5% n-Butyl Bromide (Fisher Scientific, Pittsburgh, PA) + 37.5% 2-methylbutane (Fisher Scientific) surrounded by crushed dry ice]. Frozen brains were sectioned on the Leica CM3000 cryostat (Leica, Bannockburn, IL) with a temperature of −24°C and immersed into Tissue Teck O.C.T. Compound (Sakura Finetek USA, Inc., Torrance, CA) at 8 µm intervals and thaw-mounted onto pre-cleaned SuperFrost slides (Fisher Scientific). Sections were post-fixed with 4% paraformaldehyde, blocked for endogenous biotin for 5 min. (1% H2O2 in PBS), and blocked for non-specific staining with 10% bovine serum albumin (A4503; Sigma-Aldrich; Saint Louis, MO) in PBS for 1 hr. Sections were incubated with biotinylated anti-TLR2 (6C2; Ebioscience; San Diego, CA) that, has previously been documented with mouse tissue [1,36,37] and was co-incubated with mouse anti-GFAP alexafluor 488 (131-17719; Invitrogen; Carlsbad, CA) or rat anti-CD68 alexafluor 488 (FA-11; AbD Serotec; Raleigh, NC) in PBS at 4°C for 2 days. Sections were washed extensively and incubated with streptavidin-alexafluor 555 for 1 hr. Following extensive washing, sections were covered with ProLong Gold Antifade reagent (Invitrogen). Finally, no signal was detected for sections processed with the isotype control antibody, rat IgG2b [(Ebioscience) (supplementary Fig. 2)].

Images of antibody-stained sections were captured using the IX70 Fluoview (Olympus; Center Valley, PA) microscope attached to a Retiga 2000R (QImaging; Surrey, BC) CCD camera and image capturing system using Image Pro Plus software (v.6.3; Media Cybernetics; Bethesda, MD). Fluorescent images were captured using the 60× objective, for 600× magnification.

All data are presented as means ± SEM. The results from the experiments were analyzed using GB-STAT School Pak (Dynamic Microsystems, Inc.; Silver Spring, MD). Data were analyzed using the analysis of variance (ANOVA) method, followed by post hoc comparisons using the Newman-Keuls test.

RESULTS

To determine if TLR2 and TLR4 mRNA levels were increased in the facial motor nucleus after facial nerve injury, a preliminary microarray analysis was performed. At 7 DPA, mRNA levels were increased for TLR2 and TLR4, 142 and 4 fold, respectively, in WT mice (Fig. 1). Thus, facial nerve axotomy appears to increase mRNA levels for TLR2 and TLR4 in WT mouse facial motor nucleus.

Figure 1. Toll-like receptor 2 (TLR2) and TLR4 mRNA levels in mouse facial motor nucleus 7 days after facial nerve axotomy.

Figure 1

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mRNA levels are displayed as the fold change between the axotomized side and the uninjured (control) side.

Because TLR2 mRNA changes were so much greater than TLR4, TLR2 mRNA expression was further analyzed in WT mice with real-time PCR (Fig. 2). TLR2 mRNA expression were not significantly different when left and right uninjured facial nuclei were compared. Facial nerve axotomy increased TLR2 mRNA expression at 1, 4, 7, 14 and 30 DPA [194% ± 58%, 481% ± 36%, 893% ± 135%, 1803% ± 38% and 62% ± 38%, (p < 0.01) respectively]. Thus, facial nerve axotomy rapidly induces TLR2 mRNA expression in the WT mouse facial motor nucleus, with a peak at 14 DPA, before significantly decreasing toward baseline levels by 30 DPA.

Figure 2. TLR2 mRNA levels in control or axotomized mouse facial motor nucleus 1, 4, 7, 14 and 30 days post-axotomy (DPA).

Figure 2

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Control TLR2 mRNA levels are displayed as the percent change between the right (uninjured) side and the left (uninjured) control side within each animal. At 1, 4, 7, 14 and 30 DPA, TLR2 mRNA levels are displayed as the percent change between the right (axotomized) side and the left (uninjured) control side within each animal. Bar heights represent means (±SEM). * Denotes significant differences at p ≤ 0.01.

To determine TLR2 cellular localization in the WT injured facial motor nucleus, TLR2 immunofluorescence was performed on brainstem sections from animals 7 DPA (Fig. 3). Co-immunofluorescence for TLR2 with CD68 demonstrated labeling overlap, indicating that TLR2 localizes to microglia in the injured facial motor nucleus. In contrast, co-immunofluorescence for TLR2 with GFAP demonstrated no overlap, showing that TLR2 does not localize to astrocytes in the injured facial motor nucleus. Thus, microglia are reactive for TLR2 in the WT facial motor nucleus after facial nerve axotomy.

Figure 3. TLR2 immunofluorescence in 7 day post-axotomy mouse facial motor nucleus.

Figure 3

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High-power (original magnification, 600×) immunofluorescence photomicrographs of mouse facial motor nucleus immunoreactive for CD68 (green) and TLR2 (red; top row) or GFAP (green) and TLR2 (red; bottom row).

To determine if TLR2 affects FMN survival after injury, a right facial nerve axotomy was performed on WT and TLR2−/− mice. In WT mice, FMN survival levels were 81 ± 5.0%, relative to control values at 4 weeks post-axotomy (Fig. 4). In TLR2−/− mice, FMN survival levels were 79 ± 5.8%, relative to control values at 4 weeks post-axotomy. Thus, TLR2 does not affect FMN survival levels after injury.

Figure 4. Facial motoneuron (FMN) survival levels 4 weeks after facial nerve axotomy in wild-type (WT) and toll-like receptor 2-deficient (TLR2−/−) mice.

Figure 4

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(A) Thionin-stained FMN in control and axotomized facial motor nuclei (original magnification, 100×). (B) Average percent FMN survival after facial nerve injury (±SEM).

DISCUSSION

The mechanism underlying axotomy-induced TLR2 mRNA regulation in the facial motor nucleus after facial nerve axotomy is unknown. It has previously been demonstrated that TLR2 is activated by the intracellular proteins, HSP60 [36], HSP70 [39] and HMGB1 [27]. Co-incidentally, HSP70 mRNA expression is induced in the facial motor nucleus of adult hamsters after facial nerve axotomy [25]. Thus, it is possible that axotomy-induced FMN death results in the release of TLR2 agonists, such as HSP70, causing positive feedback for increased TLR2 mRNA expression. Although the effect of TLR2 on cell recruitment was not determined by this study, it is coincidental that maximum TLR2 mRNA expression occurs at 14 DPA, a time point that is consistent with peak T cell infiltration into the facial motor nucleus [29].

In the present study, we hypothesized that TLR2 plays a role in mouse FMN survival after facial nerve axotomy based on previous studies demonstrating TLR2-induced Th2-type immunity [3,8,30] and that Th2 lymphocytes rescue FMN from axotomy induced death [11]. The data indicate an axotomy-regulated increase in TLR2 mRNA levels in mouse facial motor nucleus coincident with T cell infiltration into the facial motor nucleus [29]. It is important to note that TLR4 mRNA levels slightly increased according to microarray analysis, which may be associated with an ability to recognize select intracellular proteins released by dead or dying cells, such as HSP60 [9]. Regarding TLR2 immunoreactivity, localization to microglia demonstrated by this report corroborates previous results from other laboratories [21,23,26]. Interestingly, our data show that TLR2 does not affect FMN survival after facial nerve axotomy, since there was no difference in FMN survival between TLR2−/− and WT mice.

In a mouse model of sciatic nerve lesion, macrophage recruitment to the sciatic nerve distal stump is decreased in TLR2−/− mice [2]. This may be due to the significant decrease in mRNA levels for the macrophage recruiting chemokine, CCL2, which leads to a delay in axonal regeneration and recovery of locomotor function. In contrast, TLR2 regulates T cell-, but not macrophage-recruitment in the hippocampus after transection of axons in the entorhinal cortex [1]. In TLR2−/− mice, CCL3, CCL4, CXCL2 and CXCL10 mRNA is reduced in denervated hippocampus, while CCL2 mRNA expression is unaffected. Thus, the recruitment of specific cells to the sites of neuronal injury likely depends on location of lesion, the type of neuron being injured and the injury-specific chemokine expression.

In conclusion, the literature supports the hypothesis that TLR2 localizes to microglia, has a Th2 response-promoting effect and is upregulated in areas of neuronal damage. Accordingly, this study confirms that TLR2 mRNA expression is increased in the facial motor nucleus after facial nerve axotomy at times when FMN survival is beginning to be affected [33]. Additionally, this investigation confirms that TLR2 localizes to microglia, but a TLR2-deficiency does not affect FMN survival after injury. However, this study cannot rule out the possibility that prolonged periods of TLR2 over-expression affects neuronal survival during CNS infection [22], malignancy [10] or disease, such as ALS [24]. Interestingly, chimeric mSOD1 (ALS) mice with microglia deficient for a commonly-used adaptor molecule for TLR signal transduction, MyD88, exhibit earlier disease onset and a shorter overall lifespan than mSOD1 mice with WT microglia [20]. Therefore, future goals include further elucidating the role of TLR in the facial motor nucleus after facial nerve axotomy using MyD88-deficient mice, to rule out compensatory TLR signaling after facial nerve axotomy.

Supplementary Material

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Acknowledgements

This work was supported by NIH grant NS40433 (KJJ and VMS) and the Les Turner ALS Foundation (DAW and KJJ).

Footnotes

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Supplementary Materials

01
NIHMS170980-supplement-01.doc (20KB, doc)
02
NIHMS170980-supplement-02.ppt (114KB, ppt)
03
NIHMS170980-supplement-03.ppt (2MB, ppt)

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