Background
Toll-like receptors (TLRs) are expressed in a variety of mammalian immune-related cell types and initiate signals in response to diverse pathogen-associated molecular patterns (PAMPs) [
1]. The signalling pathways activated by the TLRs are broadly classified into MyD88-dependent and -independent pathways [
2]. Upon receptor activation and interaction with MyD88, one or more of the toll/interleukin-1 receptor (TIR)-containing adapter proteins are recruited and MyD88 binds with the TIR domain of the receptor and phosphorylates interleukin-1 receptor-associated kinase 4 (IRAK4), which, in turn, phosphorylates IRAK1. After being phosphorylated, IRAK1 dissociates from MyD88 and interacts with TRAF6. TRAF6 forms a complex that activates downstream signalling of TLRs [
1]. Major downstream signalling pathways activated by TLR engagement include the nuclear factor kappa B (NF-κB) and mitogen-activated protein kinases (MAPKs) such as c-Jun N-terminal kinase (JNK) and p38 MAPK cascades. The MyD88-independent pathway signals through the adaptor TIR-domain-containing adaptor protein inducing interferon (IFN)-β-mediated transcription factor (TRIF) [
1]. TRIF interacts with TRAF3 leading to the recruitment and activation of tumour necrosis factor (TNF) receptor-associated factor-family member-associated NF-κB activator-binding kinase 1 (TBK1) and inhibitor of nuclear factor kappa-B kinase subunit epsilon (IKKε). This culminates in interferon regulatory factor 3 (IRF3) phosphorylation that facilitates IRF3 dimerization and translocation into the nucleus and transcriptional regulation. In relation to cerebral ischemia, it has been shown that disruption of downstream MyD88-independent (TRIF) TLR pathway does not confer protection in
in vitro and
in vivo models of cerebral ischemia [
3]. A variety of endogenous ligands, such as high-mobility group box 1 (HMGB1), also bind to TLR and activate several intracellular inflammatory pathways, including the NF-κB, JNK and p38 MAPK pathways [
4,
5]. The presence of several TLRs has been reported in the brain, both in glial and neuronal cells [
6-
9], and recent studies have reported the pathological roles of TLR2, TLR4 and TLR8 in ischemic stroke-induced brain injury [
7,
9-
12]. Neurons were found to express TLR2, TLR4 and TLR8 under both physiological and pathological conditions, and cortical neuronal cultures from both TLR2 and TLR4 deficient mice were protected against cell death induced by energy deprivation (an
in vitro model of ischemic stroke) when compared with wild type [
7]. Furthermore, we have recently provided evidence that neuronal TLR8 signalling plays a detrimental role by triggering post-stroke inflammation and neuronal cell death [
9].
Intravenous immunoglobulin (IVIg) is an FDA-approved therapeutic modality used for various autoimmune and inflammatory diseases [
13]. Recently, we demonstrated that IVIg treatment significantly reduced brain infarct volume and mortality in an experimental mouse model of ischemic stroke [
14,
15]. IVIg has also been shown to inhibit complement activation, modulate cytokine production, reduce endothelial dysfunction and death, reduce activation and infiltration of leukocytes, as well as inhibit neuronal apoptosis by decreasing the cleavage of caspase-3 in primary cortical neurons subjected to ischemic insults [
14,
15]. It was recently shown that IVIg may modulate TLR9 expression and activation in pathological conditions such as systemic lupus erythematosus (SLE), suggesting a new additional mechanism of IVIg [
16]. IVIg has also been shown to modulate the maturation of TLR-primed peripheral blood monocytes [
17]. In addition, IVIg suppresses TLR4-induced cytokine production induced by lipopolysaccharide (LPS) by inhibiting the NF-κB, JNK and p38 MAPK pathways in human monocytic cells [
18]. IVIg also attenuates multiple cell death pathways by decreasing the phosphorylation of the p65 subunit of NF-κB, JNK, p38 MAPK and c-Jun, in simulated ischemic condition [
15]. However, relatively little is known about the role of IVIg in modulating the expression and activation of TLRs and endogenous ligand-mediated TLR activation following ischemic stroke. Here, we provide the first evidence that IVIg protects neurons by decreasing the expression and activation of TLRs and by suppressing HMGB1-mediated TLR activation. Our findings further support IVIg as a potential therapeutic modality for targeting ischemic stroke-induced neuronal cell death and brain injury.
Methods
Primary cortical neuronal cultures
Dissociated neuron-enriched cell cultures of mouse cerebral cortex were established from day 16 C57BL/6 J mouse embryos, as previously described [
19]. Experiments were performed in 7- to 9-day-old cultures. Approximately 95% of the cells in such cultures were neurons, and the remaining cells were astrocytes.
Glucose deprivation and combined oxygen-glucose deprivation
For glucose deprivation (GD) studies, glucose-free Locke’s buffer containing: 154 mM NaCl, 5.6 mM KCl, 2.3 mM CaCl2, 1 mM MgCl2, 3.6 mM NaHCO3, 5 mM HEPES, pH 7.2, supplemented with gentamicin (5 mg/L) was used. The cultured neurons were incubated in glucose-free Locke’s buffer for up to 24 h. For combined oxygen and glucose deprivation (OGD) studies, neurons were incubated in glucose-free Locke’s buffer in an oxygen-free chamber for 4.5, 6 or 12 h. To observe the effect of IVIg, IVIg (KIOVIG, Baxter) was added at the reported concentrations to the cultures during GD or OGD conditions. The active ingredient in KIOVIG is a human plasma-derived immunoglobulin, concentration of 100 mg/mL (10% w/v), produced from large pools of human plasma by a modified Cohn-Oncley cold ethanol fractionation, yielding an intermediate immunoglobulin G, referred to as precipitate G. Equal concentration of glycine (Sigma Aldrich, St. Louis, MO, USA), an amino acid with no known affinity for any cell receptors, was also included in the experiments as vehicle control for IVIg. To observe the effect of HMGB1, mouse recombinant HMGB1 (Source: E. coli expressed amino acids Met1-Glu215 of mouse HMGB-1 accession # NM_010439; purity: >98%; endotoxin: less than 0.01 ng/μg cytokine) (34-8401, eBioscience, San Diego, CA, USA) was added either alone or simultaneously with IVIg to the OGD-treated neuronal cultures.
Western blot analysis
Protein samples were subjected to sodium dodecyl sulphate-polyacrylamide (7.5% or 10%) gel electrophoresis using a Tris-glycine running buffer (Bio-Rad, Hercules, CA, USA). Gels were then electro-blotted using a semi-dry transfer apparatus (Bio-Rad) in transfer buffer containing 0.025 mol/L Tris base, 0.15 mol/L glycine and 10% (v/v) methanol for 1.5 h at 350 mA onto nitrocellulose membranes (Bio-Rad). The membranes were then incubated in blocking buffer [5% bovine serum albumin (BSA) or 5% non-fat milk in TBS-T (20 mmol/L Tris-HCl, pH 7.5, 137 mmol/L NaCl, 0.2% Tween-20)] for 1 h at room temperature. The membranes were then incubated overnight at 4°C with primary antibodies including those that selectively bind TLR2 (Abcam, Cambridge, UK), TLR4 (Abcam), TLR8 (Abcam), RAGE (Abcam), MyD88 (Abcam), TRAF6 (Abcam), p-JNK (Cell Signaling, Danvers, MA, USA), p-p38 MAPK (Cell Signaling), p-p65 (Cell Signaling), p-c-Jun (Cell Signaling), Bcl-2 (Cell Signaling), cleaved caspase-3 (Cell Signaling), HMGB1 (Cell Signaling) and β-actin (Sigma Aldrich). After washing three times (10 min per wash) with TBS-T, the membranes were incubated with horseradish peroxidase-conjugated secondary antibodies for 1 h at room temperature. The membranes were then washed again with TBS-T and incubated with chemiluminescent substrate for enhanced chemiluminescence (Thermo Fisher Scientific, Waltham, MA, USA) for 5 min. The signals were visualized by exposing the membranes onto X-ray films (Fujifilm Corporation, Minato, Tokyo, Japan).
Middle cerebral artery occlusion and reperfusion
12- to 14-week-old C57BL/6 J male mice were used for
in vivo experiments. The focal cerebral ischemia/reperfusion (I/R) model was similar to that described previously [
20]. Briefly, the mice were anesthetized with isoflurane, a midline incision was made in the neck, and the left external carotid and pterygopalatine arteries were isolated and ligated with 6-0 silk thread. The internal carotid artery (ICA) was occluded at the peripheral site of the bifurcation of the ICA and the pterygopalatine artery using a small clip, and the common carotid artery (CCA) was ligated with 6-0 silk thread. The external carotid artery (ECA) was cut, and a 6-0 nylon monofilament with a blunted tip (0.2 to 0.22 mm) with a coagulator was inserted into the ECA. After the clip at the ICA was removed, the nylon monofilament was advanced into the origin of the middle cerebral artery (MCA) until light resistance was felt. The nylon monofilament and the CCA ligature were removed after 1 h of occlusion to initiate reperfusion. In the sham-operated group, these arteries were visualized but not disturbed. Mice were administered with 1 g/kg body weight of IVIg or vehicle by infusion into the femoral vein (approximately 250 μL) 3 h after the start of reperfusion period. Cerebral blood flow was measured by placing the animal’s head in a fixed frame after it had been anesthetized and prepared for surgery. A craniotomy was performed to access the left middle cerebral artery and was extended to allow positioning of a 0.5-mm Doppler probe (Moor LAB, Moor Instruments, Devon, UK) over the underlying parietal cortex approximately 1 mm posterior to the bregma and 1 mm lateral to the midline. The animals were included in the study if they underwent successful MCA occlusion, defined by an 80% or greater drop in cerebral blood flow seen with laser Doppler flowmetry. The animals were excluded if insertion of the thread resulted in perforation of the vessel wall determined by the presence of sub-arachnoid blood at the time of sacrifice. University of Queensland Animal Care and Use Committee approved all
in vivo experimental procedures.
Statistical analysis
All numerical values are expressed as mean ± SEM. The overall significance of the data was examined by one-way analysis of variance (ANOVA) followed by Newman-Keuls post hoc test to determine group differences. Statistical difference was considered as P < 0.05 throughout the study. Statistical analyses were performed using GraphPad Prism software.
Discussion
IVIg therapy has been shown to be effective in the treatment of various inflammatory and autoimmune disorders [
24]. Several mechanisms have been proposed to explain these beneficial effects of IVIg such as attenuating the activity of complement system, the expression of adhesion molecules, the expression and activation of inflammasome pathways and cytokine production [
14,
25,
26]. IVIg acts also by dampening pro-inflammatory and apoptotic gene expressions [
15,
27]. We and others have previously demonstrated that IVIg treatment reduced brain damage, neurological deficit and mortality in experimental rodent models of stroke [
14,
15,
28]. The current study reveals that a reduction in TLR expression and activation partly accounts for the protective effect of IVIg against neuronal cell death following ischemic stroke. The data indicate that IVIg reduces the expression levels of TLR2, TLR4 and TLR8 and dampens downstream signalling cascades associated with TLRs such as the NF-κB and MAPKs pathways. Furthermore, our data show that IVIg is also targeting HMGB1-mediated TLR signalling in OGD-injured neurons.
Numerous studies have reported pathological roles of TLRs and RAGE in ischemic stroke-induced brain injury [
7,
9,
23]. We previously reported that neurons express several TLRs, and that the levels of TLR2, TLR4 and TLR8 are increased in neurons in response to ischemic conditions [
7,
9]. Neurons from both TLR2 knockout and TLR4-deficient mice were protected against ischemia, and the amount of brain damage and neurological deficits caused by a stroke were significantly lesser in mice deficient in TLR2 or TLR4 compared with control mice [
7]. High concentrations of IVIg prevent neuronal death, but the neuroprotective mechanism is not well established. Recent studies have indicated that IVIg may attenuate the expression and activation of TLR9 in B cells from SLE patients, suggesting a novel additional mechanism of IVIg [
1]. Another recent study showed that IVIg and F(ab)
2 fragments of IVIg that lacks the Fc region inhibit TLR4-mediated activation of dendritic cells [
29]. Our data reveals that treatment with IVIg significantly prevented ischemia-induced increased neuronal expressions of TLR2, TLR4 and TLR8. In addition, we have also found that ischemia-induced increased expressions of TLR adaptor and signalling proteins such as MyD88 and TRAF6 were prevented by IVIg treatment. These expression data indicate that IVIg may exert its beneficial effect by modulating the expression and activation of neuronal TLR2, TLR4 and TLR8.
It is well established that the activation of NF-κB, JNK and p38 MAPK pathways are implicated in TLR2, TLR4 and TLR8 signalling [
2,
30]. Our data show that IVIg treatments significantly attenuated the expression level of the phosphorylated forms of NF-κB p65, JNK, p38 MAPK and c-Jun in neurons following ischemic conditions. It is well known that NF-κB is a regulator of neuronal apoptosis in cerebral ischemia [
31]. Similarly, p38 MAPK, JNK and c-Jun are known to contribute to neuronal cell death following ischemic condition [
32]. Our data has shown that IVIg not only prevented ischemia-induced increased expression levels of TLR2, TLR4 and TLR8 and attenuated its signalling cascades but also ischemia-induced cleavage of apoptotic protease caspase-3.
Our data further suggests that IVIg protects neurons by modulating TLR expression and activation
in vitro are also true for
in vivo. We have previously reported that IVIg treatment reduces brain damage and improves neurological deficits after cerebral I/R injury [
14]. In addition, we have shown that IVIg reduces ischemic stroke-induced infiltration of leukocytes and protects against endothelial dysfunction [
27]. We also found evidence that the neuroprotective effects of IVIg are associated with a significant reduction in the levels of NLRP inflammasome proteins as well as precursors of both IL-1β and IL-18 in a mouse model of focal ischemic stroke [
26]. Here, we show that the increased expression levels of TLR2, TLR4 and TLR8, adaptor protein MyD88, signalling protein TRAF6, TLR downstream signalling proteins such as NF-κB and MAPKs were significantly prevented in IVIg-treated animals compared to control animals following stroke.
The receptor for advanced glycation endproducts (RAGE) is a member of the immunoglobulin superfamily that is located on the plasma membrane and activated by a variety of ligands, including advanced glycation endproducts (AGEs) and HMGB1 [
22,
23]. Similar to the TLRs, ligand binding to RAGE leads to the activation of several intracellular inflammatory pathways, including NF-κB, JNK and p38 MAPK [
33]. We have recently shown that RAGE-deficient mice were protected from ischemic stroke and HMGB1 promotes stroke-induced neuronal cell death by activating signalling cascades associated with RAGE and TLR activation [
23]. Experimental evidence suggests that HMGB1 is released in large amounts into the extracellular space immediately after an ischemic brain insult and subsequently induces an inflammatory reaction via the activation of the TLRs and RAGE in affected brain tissues [
23]. We have also demonstrated that recombinant soluble RAGE (sRAGE), acting as a decoy receptor for HMGB1, significantly reduced the infiltration of immune cells and improved the outcome of injury in mice subjected to I/R as well as protecting cultured neurons against ischemic cell death [
23]. Our current data show that HMGB1 treatment increases TLR2, TLR4 and RAGE expression levels, and IVIg significantly prevented HMGB1-induced increases of these proteins. Similarly, HMGB1-induced MyD88 and TRAF6 expressions were also significantly prevented with IVIg treatment. Furthermore, IVIg significantly prevented HMGB1-induced increases of TLR/RAGE signalling cascade proteins such as NF-κB-p-p65, p-JNK, p-p38 MAPK and p-c-Jun.
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Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
KZL, MB, SM and TVA conceived the study; TVA and SM coordinated the study; KZL carried out ischemic simulations and protein expression studies; TVA performed MCAO experiments; KZL, SM and TVA wrote the manuscript. All authors read and approved the final manuscript.