Microglial activation by microbial neuraminidase through TLR2 and TLR4 receptors

TLR2 (B6.129-Tlr2^tm1kir/J) and TLR4 (B6.B10ScN-Tlr4^lps-del/JthJ)-deficient mice (TLR2^-/- and TLR4^-/- respectively) were breed by The Jackson Laboratory and purchased through Charles River Laboratories (Lyon, France). The wild-type strain used as control was C57BL/6 J. These animals were maintained in the animal house at Universidad de Málaga, under a 12 h light/dark cycle, at 23 °C and 60% humidity, with food and water available ad libitum. Animal care and handling were performed according to the guidelines established by Spanish legislation (RD 53/2013) and the European Union regulation (2010/63/EU). All procedures performed were approved by the ethics committee of Universidad de Málaga (Comité Ético de Experimentación de la Universidad de Málaga; reference 2012-0013). All efforts were made to minimize the number of animals used and their suffering.

An acute and sterile neuroinflammatory process was generated in mice by a single injection of the enzyme NA within the right lateral ventricle of the brain [27, 28]. Sham rodents were injected with 0.9% sterile saline. Animals were sacrificed at different times after the injection. Intracerebroventricular (ICV) injection procedure was performed as previously described in rats [29], but in this case, fine-tuned for mice. Briefly, the animals were anesthetized and positioned in a stereotaxic frame. A scalp incision along the sagittal midline was performed to access the skull and the bone was perforated with a drill in the following coordinates: 0.1 mm posterior and 0.9 mm lateral from Bregma [33]. NA from Clostridium perfringens (Sigma-Aldrich, N3001) dissolved in 0.9% sterile saline was administered by a single injection 2.0 mm below the dura mater with the aid of a pump; the amount of NA injected was 75 mU in 1 μL, perfused during 5 min at a rate of 0.2 μL/min. The animals were sacrificed at different times post-injection and their brains were used for RNA extraction or for immunohistochemistry (6 and 24 h, respectively).

Microglial cells were isolated according to Saura’s method [34]. The mix cell cultures were obtained from 3 to 5-day-old mice sacrificed by decapitation. The brains were dissected out and meninges eliminated. Cortical brain tissue was disrupted in the presence of 0.25% trypsin solution plus 1 mM of ethylenediaminetetraacetic acid (EDTA) in Hank’s Balanced Salt Solution (HBSS; Sigma). After mechanical and chemical dissociation, cells were harvested by centrifugation (300×g, 10 min) and seeded in Dulbecco’s Modified Eagle Medium/Nutrient Mixture F-12 (DMEM-F12; Gibco), supplemented with 10% fetal bovine serum (FBS, Sigma-Aldrich) and 1% penicillin/streptomycin (Sigma-Aldrich) at a density of 250,000 cells/mL, and cultured at 37 °C in humidified 5% CO₂. The medium was replaced every 5 days; confluence was achieved after 2 weeks. After maintaining the culture in confluence for 1–2 weeks, microglial cells were isolated by the following mild trypsinization method. The medium was removed and the mixed culture carefully washed; then it was treated with 0.25% trypsin solution plus 1 mM EDTA in HBSS during 1 h at 37 °C. During this incubation, the main monolayer detaches like a sheet, leaving exposed the microglia underneath which is adhered to the plate bottom. The purity of these microglial cultures was checked by immunocytochemistry and usually was about 95%. The average yield with this method was about 5000 cells/well in 24 multiwell plates and about 20,000 cells/well in 12 multiwell plates (Sigma-Aldrich, TPP tissue culture plates). Microglial cultures were obtained from wild type, TLR2^-/- and TLR4^-/- mouse strains. When required, microglial cultures were activated by the addition of [1] the synthetic triacylated lipoprotein Pam3CSK4 (P3C; InvivoGen, 12A10-MM; 2 μg/mL), which is an agonist of TLR2 [2]; ultrapure (a highly purified fraction from E. coli) lipopolysaccharide (LPS; InvivoGen, 13I06-MM; 5 μg/mL), which is a specific ligand of TLR4; or [3] NA (Sigma-Aldrich, N3001; 50 mU/mL), whose effect on microglial cells is under investigation herein.

The sialidase activity of NA was eliminated by either a heat treatment or with inhibitors. For heat-inactivation, NA (50 mU/mL) was incubated at 60 °C during 20 min. The sialidase inhibitors used were oseltamivir phosphate (Sigma-Aldrich, 100 μg/mL) or N-acetyl-2,3-dehydro-2-deoxyneuraminic acid (NADNA; Sigma-Aldrich, 250 μg/mL).

The inactivated/inhibited NA was used to stimulate cultured microglial cells. Native or inactivated/inhibited NA was added to microglial cells isolated from WT mice and was kept for 24 h. Microglia were then harvested for RNA isolation and quantitative PCR (qPCR). The stimulation was evaluated by the expression of the cytokines IL-1β and TNFα.

Free-floating vibratome sections were first treated, to quench endogenous peroxidase, with 10% methanol and 3% hydrogen peroxide in PBS during 45 min. After washings with PBS, nonspecific binding sites were saturated with PBT solution (0.3 % bovine serum albumin, 0.3% Triton X-100 in PBS pH 7.3). Primary antibodies were rabbit anti-rat IBA1 (Wako), and goat anti-rat IL-1β (R&D Systems), both diluted 1:500 in PBT solution. The primary antibodies were incubated overnight at 4 °C. The following morning the sections were washed with PBS and incubated with the appropriate biotinylated secondary antibody (goat anti-rabbit, Pierce; or rabbit anti-goat, Vector) diluted 1:1000 in PBT, at room temperature for 1.5 h. The avidin-biotin-complex amplification system (ABC; 1:250 dilution; Thermo Fisher Scientific) was used afterwards (at room temperature, 45 min) to detect the secondary biotinylated antibodies. The peroxidase activity was revealed with 0.05% diaminobenzidine and 0.03% hydrogen peroxide in PBS for 10 min. After thorough washes, the sections were then mounted onto gelatin-coated slides, air-dried, dehydrated in graded ethanol, cleared in xylene, and coverslipped with Eukitt mounting medium.

Animals were anesthetized and transcardially perfused with heparinized 0.9% saline. The brains were then extracted and properly dissected under RNase-free conditions. Immediately after dissection, tissue samples were immersed in RNA later (Sigma-Aldrich) and kept overnight at 4 °C. The next day, this solution was removed and the tissue pieces placed at – 80 °C for storage until RNA isolation.

Total RNA from the hypothalamic region was isolated using TRIzol reagent (Invitrogen). One milliliter of reagent was added to about 100 mg of tissue, and RNA was isolated following manufacturer’s instructions. Finally, the RNA was resuspended in a volume of ≤ 50 μL of RNase-free water.

When RNA was isolated from microglia cultures, the culture medium was removed, the cells washed with cold PBS, and TRIzol reagent was added to the well (0.5–1 mL, depending on the type of culture plate), to have about 20 × 10³ cells/mL of TRIzol. Cells were homogenized by passing TRIzol solution through a pipette several times. RNA isolation proceeded as indicated by the manufacturer, adjusting the volume of reagents when required (0.5 mL starting volume of TRIzol).

The concentration of RNA was measured in a NanoDrop microvolume spectrophotometer (NanoDrop 1000, Thermo Fisher Scientific). The A_260/280 ratio of the isolated RNA was usually about 1.8.

Before preparing the reverse transcription (RT) reaction, RNA samples were diluted with RNase-free water in order to bring them to a similar range of RNA concentration. cDNA synthesis from isolated total RNA was performed using the SuperScript^TM III First-Strand Synthesis (Invitrogen). The reaction mix was prepared according to the manufacturer’s protocol, and RNA was added to have ≈ 500 ng in a final volume of 20 μL. The RT reaction was carried out in a thermocycler (MasterCycler Gradient, Eppendorf). In these conditions, the resulting cDNA concentration should be equivalent to 25 ng RNA/μL. cDNA was stored at – 20 °C

Real-time PCR was used to quantify specific mRNAs represented in cDNA samples. The hot start reaction mix FastStart Essential DNA Green Master (Roche), based on SYBR Green I fluorescence dye, was used for this purpose. PCR reactions were prepared following manufacturer’s instructions. Forward and reverse primers were used at a final concentration of 0.4 μM and cDNA at 10 ng (amount established after preliminary trials). The qPCR reaction was carried out in a LightCycler 96 Instrument (Roche). The information obtained (amplification curves, melting curves and crossing points, CP, or cycle threshold, Ct) for each transcript was processed using the software provided with the LightCycler equipment.

To estimate the PCR efficiency (E), serial dilutions of the cDNA samples were amplified, and E is calculated according to the equation E = 10^[-1/slope] [35]. Relative quantification was based on the level of expression of a target gene relative to the level of expression of a reference gene [36], which was glyceraldehyde 3-phosphate dehydrogenase (GAPDH).

Primers to target the mRNA of genes related to inflammation (Table 1) were designed using the program Primer3 (https://​primer3.​org/​). Target genes sequences were obtained from the Genbank NCBI Reference Sequence (https://​www.​ncbi.​nlm.​nih.​gov/​).

Images of immunostained tissue were acquired for cell quantification purposes; sometimes cells were counted directly under the microscope. Photographs were systematically taken from the brain areas of interest, using a digital camera (Nikon Digital Sight, DS.Fi1) coupled to an optical microscope (Leica, model DMLB) using NIS Elements software F2.20 (Nikon).

Wild-type, TLR2^-/- and TLR4^-/- mice were ICV injected with NA, and sacrificed 24 h later. Brain sections were subjected to immunolabeling with selected markers: i) IBA1, which labels microglia and peripheral, perivascular, and meningeal macrophages; ii) IL-1β, a pro-inflammatory cytokine expressed in microglia which indicates its pro-inflammatory activation.

Mice injected with saline showed a homogeneous distribution of microglial cells in the septofimbria (Fig. 1a–c) and the hypothalamus (Fig. 1d–f). Microglia presented a moderate IBA1 staining and a ramified morphology. No apparent differences were observed between saline-injected WT (Fig. 1a, d), TLR2^-/- (Fig. 1b, e), and TLR4^-/- (Fig. 1c, f) mice.

IBA1 staining considerably increased after NA injection in the three strains, both in the septofimbria (Fig. 1g–i) and in the hypothalamus (Fig. 1j–l), indicating the activation of microglial cells. Not only IBA1 staining increased but also cells appeared partially de-ramified and with a larger cell body. This morphological change was more evident in the septofimbria (compare Fig. 1a and g) than in the hypothalamus (compare Fig. 1d and j). The morphological change of microglia induced by NA has been previously reported in both brain regions [30]. Both TLR2^-/- mice (Fig. 1h, k) and TLR4^-/- mice (Fig. 1i, l) showed a similar response to NA to that observed in WT mice (Fig. 1g, j), that is, IBA1 overexpression, enlargement of the cell body, and some thickening and shortening of ramifications.

IBA1-positive cell counts were performed in the septofimbria (close to the ICV injection site; Fig. 1m) and in the hypothalamus (farther form the injection site; Fig. 1n). In both locations, the number of IBA1-positive cells increased after NA-injection compared to saline-injected animals. However, such increase occurred only in WT and TLR2^-/- animals (P < 0.001 vs corresponding saline), but not in TLR4^-/- mice, where cell counts were similar to those of the saline-treated group.

In microglial cells, IL-1β expression is associated with a proinflammatory state. This cytokine is overexpressed in microglial cells few hours after NA stimulation [30]. The number of IL-1β positive cells provides valuable information about the inflammation degree coming about in the tissue. While saline-treated animals were essentially devoid of IL-1β positive cells (not shown) 24 h after ICV injection, NA administration resulted in the emergence of IL-1β positive cells in various brain locations, particularly in periventricular areas including the septofimbria (Fig. 2a–c) and the hypothalamus (Fig. 2d–f). The intensity of the IL-1β immunostaining was higher in WT mice (Fig. 2a, d), more moderate in TLR2^-/- mice (Fig. 2b, e), and quite faint in TLR4^-/- mice (Fig. 2c, f). These observations were supported by IL-1β-positive cell counts, as NA injection resulted in increased IL-1β cell counts in the three strains when compared with their respective saline controls (Fig. 2g–h). While in WT mice, NA provoked a remarkable increase in the number of IL-1β-positive cells (P < 0.001 vs saline WT), a similar although milder increase was observed in TLR2^-/- mice (P < 0.001 vs NA WT). In TLR4^-/- mice, NA also induced expression of IL-1β in some cells, but in this case in a more restricted population than in WT or TLR2^-/- (P < 0.001 vs NA WT or NA TLR2^-/-).

The NA-induced inflammatory reaction was further analyzed by quantifying the mRNA levels of pro-inflammatory cytokines by qPCR. Wild type, TLR2^-/-, and TLR4^-/- mice were saline or NA injected and sacrificed 6 h later. The hypothalamus was dissected out for total RNA isolation and qPCR. This brain region was preferred for this study because it is surrounding the third ventricle, the route of the cerebrospinal fluid flow just after the injection of NA within the lateral ventricle, and also distant enough from the injection site as to be devoid of injection damage. NA induced a significant increase of IL-1β expression (Fig. 3a) in the hypothalamus of WT and TLR2^-/- mice compared to saline-injected animals (P < 0.001 and P < 0.005 respectively), but not in TLR4^-/- mice. Similarly, TNFα levels (Fig. 3b) were higher after NA-injection in WT (P < 0.001 vs saline WT) and in TLR2^-/- (P < 0.005 vs saline TLR2^-/-) mice, but not in TLR4^-/- animals. Interleukin-6 (IL-6) mRNA levels behaved in a similar way (Fig. 3c), presenting increased values in WT (P < 0.001 vs saline WT) and in TLR2^-/- mice (P < 0.005 vs saline TLR2^-/-) injected with NA, but not in TLR4^-/- animals. Therefore, in TLR4^-/- animals, no differences were observed between NA-treated and saline-injected animals for any of the cytokines analyzed. Also, when comparing WT and TLR2^-/- strains both under NA stimulation, the overexpression of IL-1β, TNFα, and IL-6 was milder in TLR2^-/- mice than in WT ones (P < 0.05 vs NA WT for the three cytokines).

To find out whether microglial cells can initiate by themselves the inflammatory response provoked by NA, and also to elucidate if NA acts through TLR2 and/or TLR4 receptors, microglia were isolated from WT, TLR2^-/-, and TLR4^-/- mice to obtain cultures of microglial cells. These primary microglial cultures were stimulated with NA or with specific ligands for TLR2 and TLR4: P3C, a synthetic triacylated lipoprotein which specifically activates TLR2, and ultrapure LPS, which is a specific ligand for TLR4. Control cultures were added saline. Six hours after NA, P3C, or LPS addition, total RNA was isolated and the expression of the pro-inflammatory cytokines IL-1β, TNFα, and IL-6 was quantified by qPCR (Fig. 4).

Two-way ANOVA pointed out significant differences in the expression levels of IL-1β (F = 5.9; df = 11, 32; P < 0.001), TNFα (F = 4.7; df = 11, 32; P < 0.001), and IL-6 (F = 12.8; df = 11, 32; P < 0.001) between different treatments. The expression of the three pro-inflammatory cytokines in WT microglia cultures increased after treatment with LPS, P3C, and NA compared to saline-treated cultures (Fig. 4). This result demonstrates that, along with LPS and P3C, NA is also able to directly activate microglial cells.

Microglia isolated from TLR2^-/- responded with an increased expression of IL-1β (Fig. 4a), TNFα (Fig. 4b), and IL-6 (Fig. 4c) after LPS and NA treatments but, as expected, no response was observed after treatment with the TLR2 agonist P3C.

Conversely, TLR4^-/- microglial cells did not show an increase in the expression of any cytokine after LPS stimulation, as was expected since LPS is a TLR4 agonist (Fig. 4). Interestingly, and similarly to LPS, NA was not able either to induce a response in TLR4^-/- microglial cells. It is noteworthy to mention that TLR4^-/- microglia was activated by the TLR2 agonist P3C, indicating that microglial cells are capable of initiating an immune response independent of TLR4. Of note, the IL-1β and TNFα responses of TLR2^-/- microglia to LPS were not as that of WT microglia, but slightly milder. Because the dose of LPS used was relatively high, in this experimental condition, WT microglia could have been activated not only by LPS binding to TLR4 but additionally by contaminants present in the LPS preparations acting through other receptors. This could explain the higher activation level of WT microglia compared to TLR2^-/- microglia. A similar explanation could be applied to the lower TNFα response of TLR4^-/- microglia to P3C, as this agonist was also used at a relatively high dose.

Therefore, these results point that NA can directly activate microglial cells, probably acting through the receptor TLR4.

The above results demonstrate that NA per se is able to activate microglial cells. However, the question remains if this happens thanks to its recognition as a PAMP by pattern recognition receptors of the host, or if its sialidase activity is involved in the microglial cell activation process. To address this issue, cultures of WT microglial cells were exposed to native NA, heat-inactivated NA, and native NA with sialidase inhibitors, namely oseltamivir phosphate and NADNA. Microglial cultures were exposed to these conditions for 24 h and then processed for qPCR quantification of IL-1β and TNFα expression.

Prior to the experiment, the efficiency of the heat inactivation and the effective dose of the inhibitors were checked by lectin histochemistry, using a sialic acid–specific lectin such as SNA (Fig. 5a–e). This test was performed on brain sections containing ependyma, whose surface is rich in sialic acid. While the ependymal surface was SNA-positive in non-treated sections (Fig. 5a), it turned SNA-negative after incubation of the sections with NA (Fig. 5b), indicating the removal of sialic acid by the sialidase activity of NA. Heat inactivation of NA (Fig. 5c), or the presence of oseltamivir (Fig. 5d) or NADNA (Fig. 5e), abolished the sialidase activity of NA, as demonstrated by the binding of SNA to the ependymal surface.

As expected, native NA was able to stimulate the overexpression of both IL-1β (Fig. 5f) and TNFα (Fig. 5g) in cultured microglia (P < 0.001 vs control without NA, for both genes). However, such capacity was greatly diminished after heat inactivation of NA, as well as when adding oseltamivir or NADNA along with native NA to the culture (Fig. 5f, g). Still, a marginal activation of microglia by these inactive/inhibited forms of NA could be observed (compare NA + Q, NA + oseltamivir, and NA + NADNA with any NA–condition).

Therefore, these results indicate that it is the sialidase activity of NA, and not so much the NA protein itself, that induces the activation of microglia.