Background
Inflammation is the body’s attempt at self-protection to remove harmful stimuli, including damaged cells, irritants and pathogens—and initiate the healing process. Activation of the innate immune system is an integral aspect of the response to inflammation. When prolonged, however, inflammation can become adversarial. Inflammation and neurological diseases are intimately connected, with ever-growing evidence pointing to its being a key feature in the pathobiology of neuropathic pain, chronic neurodegenerative diseases, brain ischemia, spinal cord injury, traumatic brain injury, some neuropsychiatric disorders [
1‐
5] and possibly even autism spectrum disorder [
6]. Microglia, the resident macrophage population in the CNS—along with astrocytes—constitute principal players in neuroinflammatory responses [
7‐
13].
The innate immune system relies on a set of germ-line encoded receptors that recognize conserved molecular patterns found only in microorganisms. This large family of so-called pattern recognition receptors includes the Toll-like receptors (TLRs), considered as crucial environmental-sensing molecular motifs for pathogen-associated molecular patterns, which are linked with microbial pathogens or cell stress, as well as danger-associated molecular patterns released during cell damage [
14‐
17]. Rodent microglia express mRNA for all of the recently identified TLRs, TLR1-9, used for recognition of bacterial and viral molecular patterns, whereas other neural cells (e.g. astrocytes) express a more limited TLR repertoire [
15,
18]. TLR signalling pathways have been implicated in neurodegenerative disorders [
19], including motor neuron disease [
20], as well as in pathological pain [
21‐
24].
Recent studies indicate that microglia-free astrocytes, unlike microglia, are unresponsive to TLR engagement in terms of pro-inflammatory molecule output [
7,
8,
25]. Further, several of these reports have suggested that microglial cells express a heightened responsivity to TLR agonists when cultured in the presence of astrocytes [
7,
8]. While the literature contains numerous examples of astrocyte response to TLR activation, these cell preparations more often than not contain up to 5 % contaminating microglia (e.g. [
26]), which can confound the effects observed [
27‐
29]. However, whether or not these observations extend also to the ability of glia TLRs to regulate their own expression, or that of other TLR subtypes, remains to be fully explored. Given that multiple TLRs may be activated in neuropathological settings, the up- or down-regulation of one TLR consequent to engagement of another may have important disease implications. In order to address this question, we interrogated purified cortical microglia and astrocytes for their capacity to express TLR2, TLR3 and TLR4 mRNA and protein upon treatment with either the cognate ligand or that of one of the other TLRs. Our results show a complex pattern of TLR regulation in microglia and, in addition, the capacity of nominally microglia-free astrocytes to both express and respond to TLR agonists in a nuclear factor-κB (NF-κB)-dependent manner.
Methods
Tissue culture media, antibiotics, fetal calf serum (FCS) and NP40 cell lysis buffer (10×) were purchased from Life Technologies (San Giuliano Milanese, Italy); lipopolysaccaride (LPS) (Ultra-Pure LPS-EB from E. coli 0111:B4 strain), zymosan, Pam3CSK4 (VacciGrade™) and polyinosinic-polycytidylic acid (poly(I:C)) (high molecular weight) were from InvivoGen (Cayla-Invivogen Europe, Toulouse, France); BD CytoFix/CytoPerm and CytoFix were from BD Biosciences (SACCO srl, Cadorago (CO), Italy); Ro-106-9920 (Tocris-Cookson, Space Import–export srl, Milan, Italy); poly-l-lysine hydrobromide (mol wt 70,000–150,000), papain, DNase I (bovine pancreas), trypsin inhibitor, l-leucyl-l-leucine methyl ester (L-LME), SB202190, protease inhibitor cocktail, Pefabloc® SC (100 mM), LPS from E. coli 026:B6 (<5 % protein impurities), polymyxin B and all other biochemicals were purchased from Sigma-Aldrich (Milan, Italy) unless noted otherwise; Falcon tissue culture plasticware was purchased from BD Biosciences. Sterilin petri plastic dishes (10 cm Ø) were obtained from Sarstedt (Verona, Italy).
Cell culture
Mixed glial cell cultures from cortex were prepared from postnatal day 1–2 rat pups (strain: CD) as described previously [
30]. Experiments were performed in accordance with the National Institutes of Health guidelines for the care and use of laboratory animals and those of the Italian Ministry of Health (D.L. 116/92), and were approved by the Institutional Animal Care and Use Committee. Tissue dissociates were plated in 75-cm
2 poly-
l-lysine-coated tissue culture flasks at a density of 1.5 brains per flask and grown in high-glucose Dulbecco’s modified Eagle’s medium (DMEM) with 2 mM glutamine, 100 units/ml penicillin/50 μg/ml streptomycin, 50 μg/ml gentamicin and 10 % (vol/vol) FCS. Culture medium was changed after 24 h. Upon reaching confluency (approximately 7 days later), microglia were dislodged by shaking the flasks at 200 rpm for 1 h (37 °C). The culture supernatants enriched in microglia were transferred to plastic Petri dishes (Sterilin) and incubated for 45 min at 37 °C (5 % CO
2, 95 % air) to allow differential adhesion of microglia. Adherent microglia (>99.9 % purity) were mechanically scraped into culture medium and replated in this medium on poly-
l-lysine-coated 96-well microwell culture plates or 24-well multiwall plates (50,000 and 250,000 cells per well, respectively). The remaining cell monolayers were highly enriched in astrocytes (<5 % microglia, flow cytometry using cell type-specific antibodies). For some experiments, the astrocyte monolayers were depleted of residual microglia using a 60-min exposure (50 mM) to the lysosomotropic agent L-LME [
27], as described previously [
7,
8]. Astrocyte plating densities were the same as used for microglia.
Culture treatments
Microglia or astrocyte cultures were treated with one of the following TLR ligands: 100 ng/ml LPS-EB Ultra-Pure ('LPS') (a selective agonist for TLR4 with no LPS-independent activity); 10 μg/ml zymosan (TLR2 agonist) [
31]; 50 μg/ml poly(I:C) (a synthetic analogue of double-stranded RNA which activates TLR3) [
32]. Agonists were added in DMEM + 10 % FCS. Cells were treated for 6 or 24 h for gene expression and flow cytometry (FCM) analysis.
FCM
Monolayers of purified cortical microglia and astrocytes (±L-LME treatment) were washed with phosphate-buffered saline (PBS), scraped into PBS and the cells pelleted by centrifugation (200g, 5 min). Samples were fixed with BD CytoFix or fixed/permeabilized with BD CytoFix/CytoPerm at 4 °C for 20 min, depending on whether the antigen of interest was located on the cell surface or intracellularly. Purified microglia and enriched astrocytes were immunophenotypically characterized by FCM using the following primary antibodies against rat markers: Alexa Fluor® 647 mouse anti-glial fibrillary acidic protein (GFAP) (Cell Signaling Technology Europe, Leiden, The Netherlands); rabbit anti-ionized calcium-binding adapter molecule 1 (Iba1) (Wako, Richmond, VA, USA); rabbit anti-TLR2 (sc-10739) polyclonal antibody raised against amino acids 180–354 of TLR2 of human origin (Santa Cruz Biotechnology, Heidelberg, Germany); rabbit anti-rat TLR3 (sc-28999) polyclonal antibody raised against amino acids 26–325 mapping within an N-terminal extracellular domain of TLR4 of mouse origin (Santa Cruz); rabbit anti-rat TLR4 (sc-30002) polyclonal antibody raised against amino acids 339–638 mapping within an N-terminal extracellular domain of TLR4 of mouse origin (Santa Cruz); AlexaFluor® 647 mouse monoclonal antibody IgG1 isotype control (Cell Signaling); AlexaFluor®488 anti-rabbit or anti-mouse secondary antibodies (II Ab) (Life Technologies). For staining, 200,000 cells were incubated with the selected antibody in PBS containing 0.5 % bovine serum albumin (BSA) (Sigma-Aldrich) for 15 min at room temperature. For indirect labelling, the samples were washed with 0.5 % BSA solution and then stained with appropriate secondary antibody. In parallel, samples labelled with isotype or secondary conjugated antibodies were prepared as negative controls. Data were acquired using FACSCanto II Flow cytometer (BD Biosciences) and then analysed with Summit 4.3 (DAKO-Beckman Coulter) and FACSDiva v6.1.3 (BD Biosciences) softwares. The expression of GFAP and Iba1 was reported as geometric mean fluorescent intensity (MFI) ± standard deviation (SD) and percentage of positive cells while data of TLRs were expressed as the ratio of relative MFI (rMFI) values derived from resting (Ctr + 10 % FBS) and primed (+TLR agonist) cultures for each TLR normalized to its II AB-matched negative control (relative MFI). Assuming that a ratio equal to 1 was observed in the case of undetectable difference in primed cells compared to resting samples, values greater or less than 1 were indicative of an increase or decrease in TLR expression, respectively, in samples treated with TLR agonists.
Quantitative Real-Time-PCR (qRT-PCR)
Total RNA was extracted from cells using the ReliaPrep™ RNA Cell Miniprep System (Promega), according to the manufacturer’s instructions. RNA integrity and quantity were determined by RNA 6000 Nano assay in an Agilent BioAnalyser. Samples were reverse transcribed with Superscript III reverse transcriptase (Life Technologies). The RT-PCR reaction was performed as described previously using a MX 3000P (Stratagene) [
7]. Primer sequences are listed in Table
1. Amounts of each gene product were calculated using linear regression analysis from standard curves, demonstrating amplification efficiencies ranging from 90 to 100 %. Dissociation curves were generated for each primer pair showing single product amplification. In the figures, the term ‘fold-increase’ is defined as the cDNA ratio between target gene and reference gene (GAPDH) normalized to untreated control.
Table 1
Primer pairs used in this study
GAPDH | GAPDH For | 5′-CAAGGTCATCCATGACAACTTTG-3′ |
GAPDH Rev | 5′-GGGCCATCCACAGTCTTCTG-3′ |
IL-1ß | IL-1ß For | 5′-TGTGGCAGCTACCTATGTCT-3′ |
IL-1ß Rev | 5′-GGGAACATCACACACTAGCA-3′ |
TNF-α | TNF-a For | 5′-CATCTTCTCAAAACTCGAGTGACAA-3′ |
TNF-a Rev | 5′-TGGGAGTAGATAAGGTACAGCCC-3′ |
IL-6 | IL-6 For | 5′-TCACAGAAGGAGTGGCTAAGG-3′ |
IL-6 Rev | 5′-GCTTAGGCATAGCACACTAGG-3′ |
TLR2 | TLR2 For | 5′-TCCATGTCCTGGTTGACTGG-3′ |
TLR2 Rev | 5′-AGGAGAAGGGCACAGCAGAC-3′ |
TLR4 | TLR4 For | 5′-GATTGCTCAGACATGGCAGTTTC-3′ |
TLR4 Rev | 5′-CACTCGAGGTAGGTGTTTCTGCTAA-3′ |
TLR3 | TLR3 For | 5′-TGAAAACTACGGCGATGCAG-3′ |
TLR3 Rev | 5′-AGGCAGTTTTACTTCCCCGA-3′ |
Western blots
Cell monolayers were washed with cold PBS and to each well was then added 40 μl of lysis solution (890 μl NP40 cell lysis buffer (Life Technologies, 100 μl protease inhibitor cocktail (Sigma-Aldrich), 10 μl of 0.1 M Pefabloc SC (Fluka)). After 30 min on ice, the lysates were collected and cleared by centrifugation at 13,000 rpm for 10 min (Microfuge® 22R centrifuge, Beckman Coulter). The supernatants were retained and stored at −80 °C. Protein content of lysates was determined using the BCA Protein Assay Reagent Kit (Pierce) following the manufacturer’s protocol. Protein samples (10 μg) were separated on a Mini-PROTEAN® Precast Gel (Biorad) with a 4–15 % gradient for 90 min at 140 V. Proteins were electrophoretically transferred onto polyvinylidene difluoride (Merck Millipore) membranes overnight at 4 °C at 25 V. Membranes were then blocked with 3 % BSA and incubated overnight at 4 °C with one of the following primary antibodies: mouse monoclonal against β-actin (working dilution 1:25000; Sigma-Aldrich); rabbit polyclonal against rat TLR4 (working dilution 1:300; Santa Cruz Biotechnology); rabbit polyclonal against rat CD14 (working dilution 1:200; Santa Cruz Biotechnology); rabbit polyclonal against rat MD2 (working dilution 1:1000; Abcam®). The membranes were then washed and incubated 1 h with the appropriate secondary antibody (goat anti-rabbit IgG horseradish peroxidase-conjugated or goat anti-mouse IgG horseradish peroxidase-conjugated, Merck Millipore) diluted 1:4000. Blots were developed using an enhanced chemiluminescence substrate (Sigma-Aldrich) and immunoreactivity visualized utilizing the VersaDoc Imaging System. Protein expression was normalized to β-actin for band density quantification.
Immunofluorescence
Enriched astrocytes were seeded on poly-l-lysine-coated 12-mm diameter cover glasses (Menzel-Gläser, Menzel GmbH, Germany) placed in the wells of a 12-well plate (500,000 cells per well) and allowed to adhere overnight. The next day, the cells were treated with 50 mM L-LME for 1 h and allowed to recover for 1 day in L-LME-free medium. Cells were fixed with 4 % paraformaldehyde (Sigma-Aldrich), at 4 °C for 30 min. After fixation, cells were washed 3 × 10 min in PBS, pH 7.4. Cells were then permeabilized and blocked with PBS/0.05 % Triton X-100/10 % normal goat serum for 1 h at room temperature, after which time they were incubated overnight with one of the following primary antibodies: mouse monoclonal anti-GFAP antibody (1:400 dilution, Sigma-Aldrich), rabbit polyclonal anti-TLR4 antibody (1:200 dilution, Santa Cruz), or LPS conjugated with Alexa Fluor® 488 (1:200 dilution, Wako, Japan). Cells were washed 3 × 10 min with PBS and subsequently incubated for 1 h at room temperature with Alexa Fluor® 488 goat anti-rabbit IgG or Alexa Fluor® 555 goat anti-mouse IgG secondary antibody (1:500). Nuclei where visualized by incubating for 2 min with DAPI (Boehringer Mannheim, Germany). Cover glasses were mounted onto glass slides using Fluoromount-G (Southern Biotech, USA), and images were acquired on a Leica DMI4000 B microscope equipped for immunofluorescence (Leica Microsystems GmbH, Wetzlar, Germany) using a Leica DFC 480 digital camera (Leica Microsystems GmbH, Wetzlar, Germany).
Cytokine ELISA assays
Cells were stimulated to release pro-inflammatory mediators in medium containing TLR ligand as indicated in the respective figure legend. Cell supernatants were collected and stored at −20 °C until the day of assay. Cell lysates were prepared as described previously [
8]. Interleukin-1β (IL-1β), IL-6 and tumor necrosis factor-α (TNF-α) released into the culture medium (and IL-1β in cell lysates) were analysed using commercially available ELISA kits according to the manufacturer’s instructions (Antigenix America, Huntington Station, NY, USA). Standards with known amounts of IL-1β, IL-6 or TNF-α were used to convert values into absolute concentrations of cytokine in pg/culture well.
Statistical analysis
Data are given as mean ± SEM. Statistical analyses to determine group differences were performed by one-way analysis of variance, followed by Dunnett’s or Bonferroni’s post-hoc test for comparisons involving more than two data groups. Significance was taken at p < 0.05.
Discussion
Toll-like receptors play a fundamental role in recognizing pathogens and initiating an innate immune response to protect the host. However, sterile inflammation can result when TLRs, in the process of detecting tissue damage bind endogenous ligands released by stressed or injured cells [
45]. Not only immune system-related cells but also CNS neuronal and non-neuronal cell types (including microglia and astrocytes) express and respond to TLR ligation [
15,
18,
45,
46]. For microglia, these behaviours include the output of pro-inflammatory cytokines following stimulation with TLR2/3/4 agonists ([
7,
8,
18,
47], among others). The present study describes a novel response of microglia to TLR ligands, namely, the ability of these agents to induce homologous and heterologous changes in both TLR gene expression and cell surface TLR2/4 or intracellular TLR3 expression. Further, nominally microglia-free astrocytes, which do not respond to TLR agonists in terms of pro-inflammatory mediator production [
7,
8,
25], remained responsive to TLR ligand challenge in terms of altered TLR alterations, as for microglia.
TLR activation is receiving increasing attention as being implicated in neurodegenerative disorders [
19,
20], as well as in inflammatory pain and neuropathic pain [
21‐
24,
46,
48]. Primary cultures of CNS-derived microglia and astrocytes have found widespread application for investigating the molecular events underlying TLR actions. However, the cell preparative methodologies currently used, while capable of yielding quite pure populations of microglia, result in astrocytes which generally contain a minor subset (up to 5 %) of contaminating microglia [
26,
28,
49,
50]. A number of recent studies have highlighted the need for caution in interpreting observations made with such astrocyte cultures [
7,
8,
25,
28,
29]. Our observations confirm that L-LME-mediated eradication of the residual microglia effectively negated astrocyte responses to TLR2/3/4 ligands in terms of IL-1β, IL-6 and TNF-α gene induction. Yet, purified astrocytes appeared to express TLR4, CD14 and MD2 by Western blot, and confocal microscopy revealed co-expression of GFAP and TLR4 in these cells. Moreover, fluorescently conjugated LPS underwent binding and transport in astrocytes and co-localized with GFAP. Thus, astrocytes have the capability to recognize this TLR4 ligand.
The observation that TLR agonists are capable of influencing gene expression not only for their cognate receptor but also for other TLRs, in both microglia and astrocytes, was somewhat unexpected. The TLR2/3/4 agonists examined (LPS, zymosan and poly(I:C), respectively) decreased TLR2 and TLR4 mRNA, while LPS and zymosan (but not poly(I:C) decreased TLR3 transcript levels at 24 h only. Apart from a report describing that cell wall components derived from Gram-negative bacteria induce TLR2 gene in the CNS [
51], other studies dealing with TLR ligand effects on cognate receptor expression have generally been carried out in non-neural cell types, such as human aortic smooth muscle cells [
52], cardiomyoblasts [
53] and dendritic cells [
54] and TLR2. In Laflamme et al. [
51], peptidoglycan, a TLR2 ligand derived from Gram-negative bacteria failed to alter TLR2 expression. Olson and Miller [
18] observed an increased expression of TLR2 and TLR4 (but not TLR3) mRNA in LPS (5 μg/ml)-treated mouse microglia after 7 h. Several other reports have examined the effects of TLR ligands on TLR expression in glia, although differences are to be found with the present data. Such differences include the use of astrocytes less pure than ours [
39,
55,
56], lack of comparison with microglia [
56], the use of a microglial cell line instead of primary cells [
57], lack of analysis by flow cytometry [
56,
57] and use of LPS only and not other TLR ligands [
56]. As such, even a small percentage of contaminating microglia could account for the expression of TLRs observed in astrocyte cultures. Further, it is well-documented that established protocols for isolating LPS result in the co-purification of varying amounts of endotoxin protein(s) such as lipoproteins [
58,
59] (addressed at length in [
58] and references within). As these contaminants possess potent bioactivity, assigning cellular responses to the LPS component of a particular preparation may be confounded by the presence of these contaminants which could be responsible for the TLR2-mediated signalling observed upon LPS stimulation. The relatively high concentrations (5–25 μg/ml) of LPS used in several of the above studies [
55,
56], together with a lack of information as to LPS purity needs to be considered when interpreting the data. We also performed an experiment comparing the biological activity LPS from
E. coli 026:B6 (Sigma) with LPS-EB Ultra-Pure (InvivoGen) in the presence of polymyxin B, which competes with LPS for binding to (and activation of) TLR4. LPS (InvivoGen) activity in terms of IL-1β release and nitric oxide production from rat cortical microglia, used at 1000 ng/ml, was fully blocked by polymyxin B—in contrast to LPS from Sigma (Additional file
1: Figure S4). Therefore, the Sigma LPS contains LPS-independent activity.
The endogenous TLR4 ligand myeloid-related protein 8 induced IL-1β in astrocytes, although cells were only 95 % pure [
60]. Surface expression of TLR2 and TLR4, and intracellular expression of TLR3, was consistently reduced in microglia by LPS and poly(I:C). On the other hand, changes induced by zymosan were more variable. Astrocyte responses to LPS, zymosan and poly(I:C) were substantially like those of microglia, with all three agonists down-regulating TLR4 transcripts, while up-regulating TLR2. In contradistinction to microglia, these TLR ligands up-regulated TLR3 mRNA in astrocytes. In general, the relative levels of TLR mRNA were lower in purified (L-LME-treated) astrocytes. While the latter observation is likely to reflect removal of contaminating microglia, the behaviour of TLR3 mRNA in astrocytes contrasts with a lack of change in this TLR for LPS- and poly(I:C)-treated microglia. Conceivably, astrocytes may be more responsive in the presence of microglia, or the small numbers of residual microglia could behave differently in the presence of astrocytes [
7,
8,
18,
29]. Further studies will be needed to shed more light on this question. Although outside the scope of the present study, it will be interesting to explore possible developmental considerations regarding microglia and astrocyte sensitivity to TLR stimulation.
Ligand-regulated TLR expression is likely to be a complex process, especially when cross-TLR expression is involved. Indeed, changes in TLR expression, as determined by qRT-PCR and FCM did not agree with each other in several instances. A discrepancy between qPCR and corresponding protein expression has already been reported [
39] and could be justified considering two different regulatory levels of TLR signalling. On the one hand, activation of MyD88-dependent and MyD88-independent pathways after agonist stimulation promotes increased expression of TLR target genes [
39,
61]. Up-regulation of TLR2 mRNA is considered consequent to the activation of pro-inflammatory signalling [
62], as TLR2 is a target gene of NF-κB. On the other hand, the level of TLR membrane expression is subject to regulation in order to tune cellular responsiveness to bacterial, fungal and viral insults and to reduce/increase the activation of microglia and astrocytes. Such expression may differ for a given cell type as a function of species, maturation and activation state, and is known to be modified by positive and negative feedback mechanisms [
39,
63], inhibition of translational machinery [
64], negative regulation by pro-inflammatory cytokines [
65] and endocytic pathways [
66]. After bacterial insults, astrocytes and microglia are spared from hypersensitivity through LPS-mediated endocytosis of TLRs or negative regulatory mechanisms [
63]. In L-LME-treated astrocytes stimulated with Ultra-Pure LPS, membrane TLR2 expression was unchanged from 1 to 6 h but down-regulated at 24 h. In contrast, TLR4 expression was unaffected. Prior exposure to LPS reportedly induces a transient state of endotoxin tolerance (cell refractoriness) to LPS re-stimulation which correlates with reduced cell surface expression of the LPS receptor complex (TLR4/MD-2) or with other inhibitory mechanisms downstream of TLR4/MD-2. In the present study, analysis of TLRs on glial cells suggested that purified astrocytes retain their surveillance activity against a specific stimulus without changing expression of the related TLR (i.e. TLR4 for astrocytes stimulated with LPS), but rather induce tolerance to other stimuli by decreasing expression of their corresponding receptor.
It is important to keep in mind that the aim of this study was to define cell responsiveness to TLR ligands; thus, we used flow cytometry analysis on non-permabilized samples to detect plasma membrane-exposed TLR2 and TLR4. Different types of information are derived from FCM, immunofluorescence and Western blot. FCM permits one to quantify the expression of target proteins using a defined cell number, whereas immunofluorescence allows for the detection of TLR4 distribution together with GFAP in one region of interest of limited area. Moreover, Western blot analysis was performed using 10 μg total proteins from each sample (corresponding to 250,000 cells—far greater than the 10,000 cells routinely used for FCM analysis). In order to detect and better quantify minimal changes in the expression of TLRs upon treatment with LPS, poly(I:C) or zymosan, FCM was preferred to Western blotting because the former is a rapid and reliable method offering high sensitivity of detection compared to alternative methods [
67]. Due to this higher sensitivity in detecting minimal differences among samples and cells of heterogenous samples, FCM is used instead of Western blotting at the clinical level as an elective method for microbiological studies [
68], immunophenotyping of haematological disorders [
69] and monitoring of HIV-infected patients [
70]. Moreover, in several studies on TLR signalling during the immune response of microglia and astrocytes performed using FCM combined with qPCR, FCM data were not confirmed by Western blot [
39,
55,
71], perhaps as a consequence of differences in sample preparation which maintains conformational epitope(s) in FCM but results in its destruction in Western blot. Flow cytometry as well allows one to analyse target markers considering a defined number of cells and to correlate the expression of TLRs with the physical parameters (cell size and complexity) of each glial cell subset.
Activation of the p38 MAPK/NF-κB signalling cascade by ligand engagement of TLR2 and TLR4 is central to the production of pro-inflammatory cytokines such as TNF-α and a number of interleukin family members. Interestingly, an established NF-κB inhibitor was efficacious in blocking LPS-induced up-regulation of TLR2 mRNA in both purified microglia and astrocytes. The p65 subunit of NF-κB is a substrate for p38 [
72], and the p38 inhibitor SB-202190 was also efficacious in substantially blocking the LPS effects on TLR2 (and TNF-α) gene expression in both cell populations (Marinelli and Skaper, unpublished observations). A more complete elucidation of the molecular components involved in this action awaits further studies.
TLR2 and TLR4 activation in particular, in concert with microglia and astrocytes, comprise key elements in the initiation and maintenance of neuropathic pain [
48,
73‐
76]. The finding that both homologous (zymosan) and heterologous (LPS, poly(I:C)) TLR ligands are capable of regulating TLR2 gene expression may have important implications in understanding the relative contributions of different TLRs in neurological disorders associated with neuroinflammation.
Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
CM participated in the design of the study and carried out the molecular biological experiments and their statistical analysis, immunocytochemistry and the FACS analysis; RDL participated in the design of the study, performed the FACS analysis and data interpretation; LF helped to prepare the cell cultures and treatments and carried out the ELISA assays; TB performed the Western blots and FACS analysis; MTC and MZ participated in the design of the study and data interpretation; SDS conceived the study, participated in its design and coordination and helped to draft the manuscript; PG participated in the study design and helped to draft the manuscript. All authors read and approved the final manuscript.