Background

Inflammation caused by the spirochete Borrelia burgdorferi is an important factor in the pathogenesis of Lyme neuroborreliosis (LNB) [1]. This form of Lyme disease, which can affect both the central (CNS) and peripheral nervous systems (PNS), manifests in 10–15 % of untreated patients [2]. The invasion of the CNS by B. burgdorferi can lead to increased levels of pro-inflammatory molecules such as IL-6, IL-12, IL-18, and IFN-γ, and the chemokines CXCL8, CCL2, CXCL11, and CXCL13 [3, 4]. Previously, our laboratory demonstrated that interaction of B. burgdorferi with tissue sections isolated from rhesus brain parenchyma and cultured ex vivo induces inflammatory mediators in glial cells, as well as oligodendrocyte and neuronal apoptosis [5]. We also showed that co-culture in vitro of B. burgdorferi with cells isolated from rhesus dorsal root ganglia (chiefly neurons) elicited pro-inflammatory mediators from these cells and caused neuronal apoptosis [6]. Moreover, when neurons of a neuronal cell line were incubated with B. burgdorferi, the neurons died by apoptosis, but only when purified rhesus microglia were also present [7]. Microglia are potent mediators of CNS inflammation [7] as they act as the primary sensors of danger signals or altered microenvironment.

Substance P (SP) is an 11-amino acid neuropeptide and the most abundant member of the tachykinin family of neuropeptides. SP originates from several cellular sources such as neurons, endothelial cells, and immunocytes, and is released by peripheral nerve endings and central terminals of sensory neurons in the CNS [8]. The biological responses to SP are mediated by the neurokinin-1 receptor (NK1R), a G-protein-coupled receptor bearing seven transmembrane domains [9]. Previous studies have shown that SP can synergistically augment B. burgdorferi-induced expression of COX-2 in murine microglia [10], and that endogenous SP/NK1R interactions are required for maximal inflammatory responses to in vivo challenge with bacteria such as Neisseria meningitidis or B. burgdorferi [11]. Furthermore, systemic administration of a specific NK1R antagonist (L703,606) significantly reduced CNS gliosis, demyelination, and associated inflammatory cytokine elevations in murine models of bacterial meningitis [11].

In view of these results, obtained with murine models, we wished to test the effectiveness of this NK1R antagonist (L703,606), in tissues and cells of an animal model that, unlike the mouse, reproduces all of the signs of Lyme disease, including neuroborreliosis [12–14]. We tested if inhibition of SP/NK1R interactions was effective in attenuating inflammatory immune responses and neuronal and glial damage in a non-human primate (NHP) cortical brain explant ex vivo culture model of B. burgdorferi CNS infection, as well as in primary cultures of dorsal root ganglia (DRG) cells from normal adult rhesus macaques, as an in vitro model of PNS infection. The demonstration that inhibition of SP/NK1R interactions ameliorate acute bacterially induced damage in NHP cortical brain tissue and in PNS neurons is a significant step in showing that such an approach could be effective as adjuvant therapy in the context of antibiotic treatment, to limit neuroinflammation and neurologic damage in conditions such as bacterial meningitis.

Methods

Brain tissues

Frontal cortex tissues for ex vivo experiments were collected from seven rhesus macaques (Macaca mulatta) that were slated for euthanasia because they had chronic idiopathic diarrhea or had undergone trauma. Animals were euthanized by a method consistent with the recommendations of the American Veterinary Medical Association’s Panel on Euthanasia.

Incubation of brain slices with B. burgdorferi, and NK1R antagonist treatment

Freshly collected brain tissue was obtained from the frontal cortex immediately after euthanasia. The tissue was sliced into 2-mm sections, and each section was placed in separate wells of 12-well plates. Each well contained 2 mL of RPMI 1640 medium (BioWhittaker, Walkersville, MD) supplemented with 10 % FBS, as previously described [5]. Tissue sections were exposed to medium alone or to medium with added B. burgdorferi strain B31 clone 5A19 spirochetes (1 × 107 bacteria/mL) in the presence or absence of 100 μM NK1R antagonist (L-703,606 oxalate salt hydrate, Sigma-Aldrich, St. Louis, MO). The wells that received NK1R antagonist were pre-treated for 2 h prior to the addition of spirochetes, or medium alone. Incubation at 37 °C for 4 h was allowed to proceed in a humidified 5 % CO2 incubator. At the end of the incubation period, half of the total number of tissue slices was fixed in 2 % paraformaldehyde and cryopreserved as described earlier [6]. The other half was processed to obtain protein lysates, and supernatants from whole sections, as described below.

For tissue protein extraction, a ratio of tissue to CelLytic MT reagent of 1:20 (1 g of tissue/20 mL of reagent) containing Protease Inhibitor Cocktail (Sigma) to a final dilution of 1:400 was added to gentleMACS™ M tubes (Miltenyi BioTec, San Diego, CA). Tissues were lysed in a single run using the Protein 1 setting of a gentleMACS™ Dissociator (Miltenyi BioTec) for 53 s and cooled on ice for 2 min. The lysed tissue was centrifuged at 3273 ×g for 15 min at 4 °C to pellet the tissue debris. The protein-containing lysate was decanted and stored at −80 °C.

Immune mediators in ex vivo culture supernatants and lysates

The concentrations of cytokines and chemokines present in the tissue slice culture supernatants and lysates were quantified using the MILLIPLEX MAP Non-Human Primate Cytokine Magnetic Bead Panel - Premixed 23 Plex, PCYTMG-40 K-PX23 Cytokine-Chemokine Array kit (Millipore, Billerica, MA) following the manufacturer’s instructions.

Immunofluorescence staining for detection of intracytoplasmic immune mediators

For in situ analysis of intracytoplasmic immune mediators, frozen tissue blocks were cryosectioned into 16-μm sections as previously described [6]. Briefly, permeabilization and blocking were performed with 0.1 % Triton X-100-PBS-0.2 % fish-skin gelatin for 30 min, followed by additional blocking incubation with 10 % goat serum-PBS-0.2 % fish-skin gelatin for 1 h. The primary antibodies that were used to label various cell phenotypic markers were anti-human 2′,3′-cyclic nucleotide 3′-phosphodiesterase (CNPase), clone 11-5B mouse IgG1 (Millipore) at 10 μg/mL, anti-human S-100 (Sigma) at 1:500, anti-human neuronal protein NeuN, MAB 377 clone A60, mouse IgG1 (Millipore) at 1:10, anti-human glial fibrillary acidic protein (GFAP), at 1:200, clone G-A-5 purified mouse immunoglobulin conjugated to Cy3 (Sigma), and 2 μg/mL of chicken polyclonal anti-human IBA1 antibody (Aves Labs, Inc., Tigard, OR). Primary antibodies for immune mediators were either anti-human IL-6, mouse IgG2a at 1:1000 (ProSpec, Ness Ziona, Israel), anti-human CCL2 rabbit polyclonal IgG ab7814 at 1:50 (AbCam, Cambridge, MA), or anti-human CXCL8 rabbit polyclonal IgG at 10 μg/mL (RDI, Flanders, NJ). Isotype controls (Sigma) at the concentrations of the respective primary monoclonal antibodies and universal rabbit negative control (Dako Cytomation, Carpinteria, CA) for rabbit polyclonals were also included. Incubation with the primary antibody was followed by secondary antibody staining conjugated to Alexa 488-FITC (green), Alexa 633 (far red), or Alexa 568 (red) (Molecular Probes, Life Technology, Inc. Grand Island, NY). Samples were analyzed on a Leica DMi8 confocal microscope equipped with three lasers (Leica Microsystem, Exton, PA).

Incubation of DRG cell cultures with B. burgdorferi and NK1R antagonist treatment

Chamber slides (two wells) with detachable culture slides were first coated with poly-D lysine (BD Biosciences, Franklin Lakes, NJ) and then laminin (Invitrogen) at a final concentration of 10 μg/mL for a minimum of 2 h before seeding the cells. Before plating the DRG cells, the laminin was removed. DRG were obtained from two adult rhesus macaques at necropsy, minced, trypsinized in 5 mL of 0.25 % trypsin-EDTA (Invitrogen) with 1000 units of DNAse (Sigma), pelleted and seeded at 1 × 105 cells per well. DRG cell cultures were maintained for a period of 6 to 7 days in complete DMEM F12 medium containing 10 % FBS and 1X penicillin/streptomycin (P/S) supplemented with fresh L-glutamine (2 mM), and NGF-7S (50 ng/mL, Invitrogen) (complete medium). The DRG cell culture protocol has been thoroughly described previously [6].

DRG cell cultures were pre-incubated in complete medium as above but without P/S, and treated with 10 μM of NK1R antagonist (L-703,606 oxalate salt hydrate, Sigma) for 2 h at 37 °C, or left untreated. Cultures were then stimulated with live B. burgdorferi at a multiplicity of infection (MOI) of 10:1 at 37 °C for 24 h. After 24 h, culture supernatants were collected and processed for quantification of inflammatory mediators, and cells were fixed and evaluated for apoptosis by the TUNEL assay as described below. Medium controls that were pre-treated and then incubated with the same respective concentrations of NK1R antagonist but without the addition of live B. burgdorferi were also included.

Apoptosis by in situ TUNEL assay

Tissue slides as well as DRG cell culture chamber slides were incubated with anti-NeuN or anti-S-100 antibodies prior to performing the TUNEL assay. Slides were then subjected to the TUNEL-ApopTagPlus fluorescein in situ apoptosis assay (Chemicon, Temecula, CA) as per the manufacturer’s instructions. The percentage of apoptotic neurons in brain sections and DRG cell cultures, or the percentage of apoptotic oligodendrocytes in brain sections, was evaluated by counting at least 500 cells in ten microscope fields, followed by the percentage of cells that showed co-localization of both the TUNEL signal and NeuN or S-100 expression. Cells were counted by viewing slides under a fixed magnification of 40x using Nuance Multispectral Imaging System (CRi, PerkinElmer, Waltham, MA). The identity of the oligodendrocytes that stained with the anti-S-100 antibody was confirmed by their morphology.

Statistical evaluation

The unpaired-two tailed t test was used to evaluate the statistical significance between means of data sets, using Graphpad Prizm software (Graph Pad Software Inc., La Jolla, CA) version 5a. A p value of 0.05 or lower was considered to be statistically significant.

Results

Analysis of SP/NK1R interactions in the ex vivo model of CNS infection

Quantification of pro-inflammatory mediators

Interaction of B. burgdorferi with rhesus macaque brain parenchyma sections cultured ex vivo elicited IL-6, CXCL8, and CCL2 production that was detectable in both lysate and supernatant, as determined by the NHP Cytokine 23 BioPlex Panel. The addition of NK1R antagonist significantly reduced these inflammatory cytokine levels, albeit with animal-to-animal variations (Fig. 1). This pattern of response was seen both in the tissue lysates and tissue section supernatants. It suggests that SP receptor antagonist is able to significantly reduce inflammation derived from the presence of B. burgdorferi within the CNS.

Fig. 1
figure 1

Pro-inflammatory mediator levels in the supernatants and lysates of rhesus macaque frontal cortex sections cultured ex vivo. IL-6 (a), CXCL8 (b), and CCL2 (c) levels were determined by multiplex ELISA in brain tissues that were exposed to B. burgdorferi (Bb), medium alone (Med), B. burgdorferi plus NK1R antagonist (Bb + A), or medium alone plus antagonist (Med + A). The figure shows the arithmetic means ± standard deviations of experimental replicates for each individual animal. **p < 0.001, ***p < 0.0001, ns not significant

Full size image

Quantification of apoptosis

We next evaluated the likely impact of B. burgdorferi-induced inflammation in the induction of apoptosis. As in previous studies [1], we found oligodendrocytes undergoing apoptosis (Fig. 2). Importantly, there was a significant reduction in the amount of B. burgdorferi-induced apoptosis in the presence of NK1R antagonist.

Fig. 2
figure 2

Neural cell apoptosis in rhesus frontal cortex tissue sections cultured ex vivo. a S-100-positive cells (oligodendrocytes, as identified by morphology) and DNA fragmentation detected by the TUNEL assay in green, as visualized by fluorescent microscopy. b Graphical representation of the percent apoptosis as detected by the TUNEL assay in frontal cortex tissue exposed for 4 h to B. burgdorferi (Bb), exposed to B. burgdorferi and treated with NK1R antagonist (Bb + A), in medium alone (Med), and medium with added NK1R antagonist (Med + A) (***p < 0.0001, ns not significant)

Full size image

Neuronal cell death was also evaluated, and overall, the level of neuronal apoptosis was much lower than that seen in oligodendrocytes (less than 2 % in frontal cortex tissues exposed to B. burgdorferi for 4 h). In addition, there were no significant differences in the reduction in the level of B. burgdorferi-induced apoptosis in the presence of NK1R antagonist (data not shown).

Cellular sources of pro-inflammatory mediators

We focused on the cellular sources of IL-6, CXCL8, and CCL2. Interaction of B. burgdorferi with rhesus monkey brain parenchyma sections elicited IL-6 production by several types of glial cells, namely, astrocytes, oligodendrocytes, neurons, and microglia. CXCL8 and CCL2 were not found to be produced by neurons and microglia, respectively, but were present in the other cell types (Table 1 and Fig. 3).

Table 1 Phenotypes of immune mediator producer cells as visualized by confocal microscopy
Full size table
Fig. 3
figure 3

Visualization of IL-6, CXCL8, and CCL2. a IL-6 (green) inside neurons stained red with anti-NeuN antibody. b CCL2 (green) inside neurons that were stained red with anti-NeuN antibody. c IL-6 (green) and astrocytes stained red with anti-GFAP antibody. d CXCL8 (green) and astrocytes stained red. The yellow signal is due to co-localization of the astrocytic marker GFAP (red) and IL-6 or CXCL8 (green) within astrocytes. e CCL2 (green) inside astrocytes stained red. f, g Evidence of IL-6 and CXCL8 (green) in microglia stained with anti-IBA1 antibody (red)

Full size image

Analysis of SP/NK1R interactions in primary cultures of rhesus dorsal root ganglia cells

Quantification of pro-inflammatory mediators

In previous studies, cells that were isolated from rhesus DRG and exposed to live B. burgdorferi in vitro, were shown to produce pro-inflammatory mediators, and DRG neurons were shown to undergo apoptosis [6]. To ascertain the effect of SP/NK1R interactions in cells of the PNS, we set up primary cultures of DRG cells from normal adult rhesus macaques and incubated the cultures with B. burgdorferi (MOI 10:1) for 24 h in the presence or absence of 10 μM NK1R antagonist. Results obtained with the Multiplex cytokine assay from two different biological replicates consistently showed that the interaction of B. burgdorferi with rhesus DRG cells elicited IL-6, CXCL8, CCL2, and VEGF. The addition of NK1R antagonist was able to significantly reduce the level of CCL2. The levels of the other mediators were also reduced, albeit not significantly (Fig. 4).

Fig. 4
figure 4

Pro-inflammatory mediator levels in supernatants from rhesus DRG cultures. IL-6, CXCL8, CCL2, and VEGF levels were detected by Multiplex ELISA in cells exposed to B. burgdorferi (Bb) or medium alone (Med), and treated with NK1R antagonist in the presence (Bb + A) or absence of B. burgdorferi (Med + A). The figure shows the arithmetic means ± standard deviations from two biological replicates. **p < 0.001, ns not significant

Full size image

Quantification of apoptosis

B. burgdorferi-induced apoptosis of sensory neurons in the DRG cultures, and addition of the NK1R antagonist at a concentration of 100 nM significantly reduced the level of neuronal apoptosis (p < 0.001; Fig. 5).

Fig. 5
figure 5

Apoptosis in DRG cell cultures. a Non-apoptotic field of neurons (NeuN-positive cells, red) cultured from rhesus DRG as visualized by fluorescent microscopy. b DNA fragmentation detected by the TUNEL assay (green) as visualized by fluorescent microscopy. c Graphical representation of the percent neuronal apoptosis as quantified by the TUNEL assay in cultured DRG cells exposed for 24 h to B. burgdorferi (Bb), exposed to B. burgdorferi, and treated with NK1R antagonist (Bb + A), in medium alone (Med), and medium plus antagonist (Med + A) (**p < 0.001)

Full size image

Discussion

Recently, using a NHP model of acute LNB, it was demonstrated that inflammation plays a key role in LNB pathogenesis [15]. Moreover, in a murine model, it was shown that SP, which is present throughout the CNS and is the most abundant tachykinin in the brain [16], increases inflammatory mediator production by astrocytes and microglia following exposure to either Neisseria meningitidis or B. burgdorferi [11]. These findings set the scene to investigate the immunoregulatory effects of SP/NK1R interactions in NHP cortical brain tissue and PNS neurons exposed to B. burgdorferi.

We have shown that co-incubation of both CNS tissues and PNS cells with the NK1R antagonist L703,606 attenuates bacterially induced increases in inflammatory cytokine and chemokine production, particularly IL-6, CXCL8, and CCL2 (Figs. 1 and 4), and reduces apoptosis levels of neural cells (Figs. 2 and 5). In addition, using confocal microscopy, we identified cellular sources of these immune mediators (Table 1 and Fig. 3) and confirmed that they are produced by glial cells and neurons [4]. These results suggest that NK1R antagonist treatment is able to reduce downstream pro-inflammatory signaling, thereby indicating that its systemic administration may slow down disease progression.

SP is a potent initiator of neurogenic inflammation in the CNS, an effect that is often followed by alterations in blood-brain barrier permeability and by persistent neurological deficits [10, 17]. The neuroinvasion that manifests in CNS neuroborreliosis depends on the successful translocation of spirochetes across the blood-brain barrier [18] and is concomitant with SP release [10]. We propose that the exacerbation of SP levels may accelerate disease progression, since blockage of NK1R in our ex vivo and in vitro models limits inflammation and improves cell survival. These results agree with the reported effects of NK1R antagonist treatment, which protected dopaminergic neurons, preserved barrier integrity, reduced neuroinflammation, and significantly improved motor function in a rat model of early Parkinson’s disease [19].

Results obtained from the interaction of B. burgdorferi with rhesus DRG cells indicate that activation of NK1R by SP can stimulate the chemokine CCL2. This chemokine plays a fundamental role in inflammation by recruiting inflammatory cells to specific sites [20], and we speculate that stimulation of CCL2 by SP can potentially mediate this recruitment in vivo. In addition, NK1R antagonist modestly reduced IL6, CXCL8, and VEGF production by DRG cells, following the same pattern as with CCL2. However, this phenomenon was not statistically significant (Fig. 4). SP induces a non-apoptotic form of cell death in hippocampal, striatal, and cortical neurons and thus plays an important role in pathological states in which neural cell death occurs [21]. Our results indicate that SP/NK1R interactions can also induce apoptotic cell death, as NK1R antagonist treatment prevented DRG neuronal apoptosis (Fig. 5). Taken together, these data suggest that the NK1R, in addition to modulating inflammation, may be a mediator of cell death in vivo.

We hypothesized that SP contributes to the pathophysiology of neuroborreliosis, and we evaluated this hypothesis with NK1R-expressing CNS tissues. Indeed, our results showed that both secreted and intracellular pro-inflammatory proteins were suppressed in the presence of NK1R antagonist (Fig. 1), albeit with animal-to-animal variation, in our ex vivo culture model of B. burgdorferi CNS infection. We also found an anti-apoptotic effect in oligodendrocytes that was mediated by the NK1R antagonist (Fig. 2), but not in neurons. Oligodendrocytes are vital for the functioning and survival of neurons, and the inflammation and subsequent apoptosis of oligodendrocytes induced by B. burgdorferi could contribute to the pathogenesis of LNB [1].

Previous studies have shown that NK1R signaling plays a role in numerous biological processes, such as the transmission of pain in the spinal cord [22, 23]. Activation of NK1R by SP leads to phosphoinositide hydrolysis, calcium mobilization, and mitogen-activated protein kinase (MAPK) activation [24, 25], and regulates neuroinflammation, neuronal survival, and synaptic activity [21]. Our results agree with this general picture, as they suggest that NK1R/SP interaction contributes to the development of B. burgdorferi-induced inflammation in the CNS and PNS. This interaction thus represents a therapeutic target for neuroinflammation.

From the pertinent literature, it is evident that SP is an important mediator of inflammatory responses and pathological conditions associated with inflammation, including rheumatoid arthritis, multiple sclerosis, inflammatory bowel disease, lymphatic contractility, and pneumococcal meningitis [26–30]. In our present model, inflammation mediated by SP/NK1R likely results from the synergistic effect of this interaction on the neural cell response to B. burgdorferi. We suggest that endogenous SP augments B. burgdorferi-associated pathology within the brain by elevating levels of inflammatory mediators and by promoting cell death probably though activation of the MAPK signaling cascade [31]. We have shown previously that the MEK/ERK pathway is crucial for B. burgdorferi-induced inflammation and P53-mediated apoptosis in oligodendrocytes [32].

The molecular mechanisms leading to inflammatory mediator release from the resident cells of the CNS when exposed to B. burgdorferi are numerous and are currently being investigated in our laboratory. Recently, several studies have shown that multiple receptors and pathways positively and negatively regulate microglial inflammation, resulting in a complex immune network [32–34]. CCL2/CCR2 and MAPK signaling, chiefly MEK/ERK, play a major role in neuroinflammation [32, 35], and our results are consistent with the possibility that blockage of NK1R may act by limiting these signaling cascades. Thus, in addition to antibiotics, treatment with NK1R antagonists could be explored as an adjuvant intervention against LNB.

Conclusions

Our results indicate that NK1R antagonist treatment can attenuate bacterially induced increases of inflammatory mediator production in CNS and PNS cells, particularly, IL-6, CXCL8, and CCL2, as well as apoptosis levels.