Lipopolysaccharide and Curcumin Co-Stimulation Potentiates Olfactory Ensheathing Cell Phagocytosis Via Enhancing Their Activation
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The gradual deterioration following central nervous system (CNS) injuries or neurodegenerative disorders is usually accompanied by infiltration of degenerated and apoptotic neural tissue debris. A rapid and efficient clearance of these deteriorated cell products is of pivotal importance in creating a permissive environment for regeneration of those damaged neurons. Our recent report revealed that the phagocytic activity of olfactory ensheathing cells (OECs) can make a substantial contribution to neuronal growth in such a hostile environment. However, little is known about how to further increase the ability of OECs in phagocytosing deleterious products. Here, we used an in vitro model of primary cells to investigate the effects of lipopolysaccharide (LPS) and curcumin (CCM) co-stimulation on phagocytic activity of OECs and the possible underlying mechanisms. Our results showed that co-stimulation using LPS and CCM can significantly enhance the activation of OECs, displaying a remarkable up-regulation in chemokine (C-X-C motif) ligand 1, chemokine (C-X-C motif) ligand 2, tumor necrosis factor-α, and Toll-like receptor 4, increased OEC proliferative activity, and improved phagocytic capacity compared with normal and LPS- or CCM-treated OECs. More importantly, this potentiated phagocytosis activity greatly facilitated neuronal growth under hostile culture conditions. Moreover, the up-regulation of transglutaminase-2 and phosphatidylserine receptor in OECs activated by LPS and CCM co-stimulation are likely responsible for mechanisms underlying the observed cellular events, because cystamine (a specific inhibitor of transglutaminase-2) and neutrophil elastase (a cleavage enzyme of phosphatidylserine receptor) can effectively abrogate all the positive effects of OECs, including phagocytic capacity and promotive effects on neuronal growth. This study provides an alternative strategy for the repair of traumatic nerve injury and neurologic diseases with the application of OECs in combination with LPS and CCM.
KeywordsOlfactory ensheathing cells Cell activation Lipopolysaccharide Curcumin Phagocytosis Neuron growth
Olfactory ensheathing cells (OECs) have hitherto been regarded as a leading potential candidate in cell transplantation therapies for nerve repair. Compelling evidence indicates that OECs exhibit a range of valuable cell attributes, including 1) active stimulation of neuronal survival and axon regeneration by secretion of numerous neurotrophic growth factors, neurite-promoting guidance molecules, and physical substrates; and 2) critical aspects of tissue repair by structural remodeling and support, enhancement of antigenic stimuli, immune phagocytosis, and penetration of astrocytic glial scar tissue [1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11]. Based on these unique properties, OECs could be a special cell type with a strong comprehensive potential for proregeneration. Nevertheless, an increasing number of controversial issues related to the proregeneration capabilities of OECs are debated, as several studies were abortive by using OEC transplantation therapy for central nervous system (CNS) injuries [12, 13]. These studies revealed that the inability of damaged neurons to regrow is likely to be attributed to transplanted OECs that either did not support or failed to guide neural regeneration . With advances in regenerative medicine and neurobiology, this view is facing a great challenge. It is well known that in CNS injuries or neurodegenerative disorders, numerous neural cells hardly elude apoptotic fate to distinct damages. Consequently, this results in formation of an extrinsic inhibitory environment within CNS lesions, in which various growth inhibitory factors, for example interleukin-1β, chemokine (C-X-C motif) ligand (CXCL)8, Nogo, oligodendrocyte-myelin glycoprotein, reactive oxygen species, and repulsive guidance molecule α are present and seriously inhibit neuronal survival and axonal regrowth [14, 15, 16, 17, 18, 19, 20, 21]. Of note, the majority of these harmful molecules directly originate from degenerating or dying neurons and apoptotic neural debris [8, 9, 22]. Thus, rapid and efficient clearance of these deteriorated cell products is essential in creating a microenvironment beneficial for neuron survival and axonal regeneration following CNS damages or degeneration. More importantly, efficient removal of these degenerated tissues/cells also avoids the diffusion of damaging degradation products into tissue surroundings within CNS lesions, which could further exacerbate nerve damage or axonal degeneration . Therefore, establishing an ideal, rapid, and effective strategy for the removal of degenerated cell debris for neuron survival, axonal regeneration, and subsequent neural function recovery is crucial. Based on our recent studies and some previous reports by other groups [8, 9, 23, 24, 25, 26], increasing attention has been paid to OEC-mediated phagocytosis in CNS injury and degeneration, owing to their unique attributes. Thus, it is likely that OECs are potential candidates for therapeutic treatments.
Regardless of the phagocytic function of OECs in creating a microenvironment suitable for axonal regeneration, it is still unknown how to improve effectively OEC phagocytic activity without triggering undesirable consequences. Therefore, it is essential to develop an ideal, simple, and effective approach for the activation of OECs that contributes to the clearance of degenerated cell debris.
Lipopolysaccharide (LPS) is the major component of the outer membrane of Gram-negative bacteria; it also increases the negative charge of the cell membrane and helps stabilize the overall membrane structure [27, 28]. Importantly, LPS elicits a strong response from the normal animal immune system and promotes the secretion of proinflammatory cytokines by binding the CD14/Toll-like receptor 4 (TLR4)/Myeloid differentiation factor 2 receptor complex in many cell types [29, 30]. A growing body of evidence indicates that OECs can be activated by LPS, resulting in cellular stress response, release of cytokines, production of reactive oxygen species, and upregulation of phosphatidylserine receptor (PSR), which is involved in mediating the engulfment of apoptotic cell debris [9, 31, 32]. Curcumin (CCM) has been used as a traditional medicine in far-eastern countries such as China and India for centuries . Several research groups have shown anti-inflammatory effects of CCM in gastrointestinal diseases and cognitive disorders, as well as beneficial outcomes in psychiatric and anticancer settings [34, 35, 36, 37, 38, 39]. Moreover, CCM can also exert distinct beneficial effects in antiproliferation, antioxidation, and antiapoptosis in a wide variety of cells [40, 41, 42]. In addition to the reported benefits, CCM is also known to possess neuroprotective properties [43, 44]. Strikingly, the biological effects of CCM are mainly dosage dependent and cell-type specific [45, 46, 47]. For instance, at high concentrations (>40 μM), CCM induces cell apoptosis in diverse cancer cells , whereas low doses of CCM (0.1–0.5 μM) can stimulate cell proliferation in neural progenitor cells . Within the injured spinal cord, CCM has been shown to exert anti-inflammatory and antioxidative effects at concentrations of 40–60 mg/kg . Excitingly, several recent reports have demonstrated that CCM can improve OEC proliferation, migration, morphologic changes, and phagocytic activity . In spite of growing interest in the investigation of the activation characteristics of OECs, particularly in the engulfment of apoptotic or degenerated cell debris, caused by CCM or other molecules, the strategies that further potentiate the beneficial behavior of OECs in the rapid clearance of degenerated cell debris is still unknown. However, whether activated OECs with phagocytic capacity are able to promote the growth of neurons in the CNS is unclear. More importantly, mechanisms underlying the cellular events are not entirely understood.
In the current study, we present an efficient induction approach, together with molecular and cellular evidence demonstrating that co-stimulation of OECs with LPS and CCM potentiates the phagocytic activity of OECs by effectively triggering their activation. Three significant breakthroughs were observed. First, the combination of the 2 molecules can effectively induce the activation of OECs without diminishing 1) its beneficial attributes, and 2) elevation of undesirable effects. Second, the activation of OECs by the combination of LPS and CCM can efficiently promote neuronal growth under hostile conditions. Third, transglutaminase-2 (TG2) and PSR may be involved in the cellular event. Importantly, OECs activated by LPS and CCM to clear degraded neural debris may open new avenues towards a potential therapy to repair an injured CNS or in the treatment of neurodegenerative diseases.
Materials and Methods
Dulbecco's modified Eagle's medium (DMEM)/F12, fetal bovine serum (FBS), trypsin, DMEM, G5 supplement, B27 supplement, Neurabasal medium, Hanks’ balanced salt solution (HBSS), collagenase, and normal goat serum were purchased from Gibco (Carlsbad, CA, USA); LPS, BrdU, CCM, forskolin, penicillin G, streptomycin, glutamine, poly-L-lysine (PLL), 3-(4,5)-dimethylthiazol-2-yl)-2,5-dihenyltetrazolium bromide (MTT), ethylenediaminetetraacetic acid, HEPES, cystamine (Cyst), and bovine serum albumin were purchased from Sigma-Aldrich (St. Louis, MO, USA); neutrophil elastase (NE) was from R&D (Minneapolis, MN, USA); anti-γ-tubulin and anti-Tuj-1 antibody, antiglial fibrillary acidic protein (GFAP) antibody, anti-S100 antibody, and anti-p75 antibody were purchased from Abcam (Cambridge, MA, USA); anti-CXCL1, anti-CXCL2, antitumor necrosis factor (TNF)-α, and anti-TLR4 antibody were purchased from Santa Cruz (Santa Cruz, CA, USA); TG2 and PSR antibodies were from Cell Signaling (Danvers, MA, USA). The RNA extract kit was purchased from Takara (Shiga, Japan); Revert Aid First strand cDNA synthesis kit was purchased from Thermo Scientific (Rockford, IL, USA); QPCR kit was purchased from Invitrogen (Shanghai, China); 4’,6-diamidino-2-phenylindole and a bicinchoninic acid kit was from Qiagen (Hilden, Germany); a chemiluminescence Western blotting kit was purchased from Plus (Roche, Mannheim, Germany); Fluor488-conjugated goat anti-mouse IgG, and Fluor594-conjugated donkey antirabbit were purchased from Molecular Probes (Eugene, OR, USA). Thirty-five-millimeter dishes, cell culture plates, plastic coverslips, and flasks were all purchased from Corning (Corning, NY, USA).
Primary OEC and Neuron Culture
All procedures conducted on animals were approved by the Animal Experimentation Ethics Committee of Xi’an Jiaotong University and were consistent with the China Code of Practice for the Care and Use of Animals for Scientific Purposes. Primary OECs were prepared from mouse olfactory bulbs at 2.5 months of age and further purified as described previously , with minor modifications. Briefly, the outer nerve fiber and granular layers of olfactory bulbs were dissected from male mice and rinsed twice in HBSS without Ca2+/Mg2+. Then, tissues were minced using an iris scissor and digested using a solution composed of 0.25 % trypsin and 0.2 % dispase II for 25 min at 37 °C. The digested tissue was triturated, and filtered through an 80-μm metal mesh. The cell suspension was resuspended in DMEM/F12 with 10 % FBS and plated on PLL-coated flasks and cultured at 37 °C in a humidified incubator at 5 % CO2. Half the media were changed every 3 days. When confluence of OECs reached 85 %, cell purification was conducted by differential cell adhesiveness. The purified cell suspension was collected and reseeded onto 25-cm2 PLL-coated culture flasks and incubated with 10 % FBS-containing DMEM/F12 supplemented with 2 μM forskolin for 2 days. Subsequently, cells were maintained in DMEM/F12 media containing 1 % G5 supplement for further experiments.
As for neuron cultures, primary cultures of spinal cord neurons were prepared from embryonic 12- to 14-day-old red fluorescent protein (RFP) transgenic or normal mice pursuant to the procedure as previously described by Yang et al. [8, 51]. Briefly, the spinal cords were dissected and washed with ice-cold HBSS without Ca2+ and Mg2+. Subsequently, tissues were trypsinized with 2 mL 0.125 % (w/v) trypsin at 37 °C for 25 min before mechanical trituration. The dissociated cells were then collected by centrifugation and diluted to an approximate initial plating density of 2 × 105 cells/cm2 with Neurabasal medium supplemented with 2 % B27, and plated into either 35-mm Petri dishes or coverslips coated with PLL. Cultures were maintained in an incubator at 37 °C in a humidified atmosphere of 5 % CO2. The cultures were maintained and prepared for further experiments.
Treatment of OECs
To examine if the combination of LPS and CCM contributes to the beneficial activation of OECs, OECs were reseeded on coverslips, 35-mm dishes, and 96-well plates, and subsequently divided into the following groups: 1) normal OECs; 2) OECs treated with 1 μg/ml LPS; 3) OECs treated with 1 μM curcumin; 4) OECs treated with 1 μg/ml LPS and 1 μM curcumin; 4) OECs pretreated with Cyst (an inhibitor of TG2) and NE (a cleavage enzyme of PSR) prior to constant co-stimulation with 1 μg/ml LPS and 1 μM curcumin. After 1, 2, and 3 days, cells were processed for the following experiments.
Preparation of Degenerative Neuronal Debris
Neural debris was prepared from RFP mice. Apoptotic neuronal bodies and axon debris was harvested by scratching with a cell blade and further trituration, according to our previously described methods , resulting in fragmented debris consisting of microtubules and neurofilaments. Degenerative neuronal debris was stored at −80 °C until required for use.
For determination of OEC activation, MTT assay was used to assess the proliferative capacity of OECs. In brief, OECs were plated in 96-well plates and treated for 1, 2, and 3 days. MTT, 5 mg/ml, was added directly to cultures. After 4 h incubation at 37 °C, the medium from each well was gently removed by aspiration and 200 μl dimethylsulfoxide was added to each well followed by incubation and shaking for 10 s, to dissolve the insoluble formazan. Subsequently, OD values were measured in a microplate reader at 490 nm to determine the number of viable cells at indicated time points. All data presented herein were obtained from 3 independent experiments.
BrdU Incorporation Assay
The increase in the proliferative ability of OECs is an important feature of their activation. To assess the proliferation of OECs treated under different conditions, BrdU (5 μM) was added to the culture medium for 18 h and cells were cultured for 1, 2, and 3 days. After removal of the supernatant, cells were fixed with 4 % paraformaldehyde, and treated with 2 N hydrochloric acid for 30 min at 37 °C to denature DNA for further immunostaining. Subsequently, immunostaining procedures were performed as previously described .
Real-Time Polymerase Chain Reaction
Real-time polymerase chain reaction primers
Differently treated OECs were lysed in RIPA buffer for 30 min on ice and crude extracts were sonicated to shear DNA. Protein samples were harvested by centrifugation at 12,000 × g and clarified lysates were measured using a bicinchoninic acid protein assay. The supernatant was collected and Western blots performed according to the protocol as described previously . The following antibodies were used: CXCL1, CXCL2, p75, TLR4, TNF-α, TG2, and PSR. β-Actin was used as an internal control. After washes in phosphate-buffered saline (PBS), immunoblots were visualized using enhanced chemiluminescence. Densitometric analysis of bands was repeated 4 times and integrated densitometry value (IDV) was calculated.
Cell-cycle evaluation was determined by flow cytometry (ProfileII, Coulter, Brea, CA, USA) according to a previously described protocol [52, 53]. Briefly, after OECs were treated under different conditions for the 3 abovementioned time points, cells were trypsinized with 0.125 % trypsin and 0.02 % ethylenediaminetetraacetic acid, collected, and washed twice in ice-cold PBS. Single-cell suspension was achieved by gentle trituration up and down, and filtration through an 80-μm nylon mesh before analysis. Cells were then transferred to 1.5-ml Eppendorf tubes and fixed with 70 % ethanol at 4 °C overnight. Subsequently, cells were incubated with 10 μl RNase A (5 mg/ml) at 37 °C for 30 min, and cellular DNA was stained with propidium iodide (100 mg/ml) for 30 min. Cells were washed in PFN (PBS buffer supplemented with 10 % FCS and 0.02 % sodium azide) 3 times and resuspended in PFN buffer prior to flow cytometry analysis. The proliferation index (PI) was calculated using the following equation: PI (%) = (S + G2/M)/ (G0/G1 + S + G2/M) 100 %.
To determine if LPS and CCM synergistically strengthen OEC phagocytic capacity, OECs were seeded in 35-mm culture dishes prior to pretreatment with LPS at 1 μg/ml and 1 μM CCM for 12 h, and incubated with the abovementioned degeneration debris for 1, 3, and 5 days. In parallel, pretreatment of OECs with 100 μM Cyst and 100 ng/ml NE prior to exposure to LPS and CCM was conducted to further substantiate the hypothesis. The volume of cell degeneration debris administrated into OEC cultures was measured, and the ability of OECs to engulf the degenerated cell debris was assessed by a phagocytic index, according to our previously described protocol . The results were obtained from 3 independent experiments.
Distinct Treatment of Neurons
To determine if OECs treated with the combination of LPS and CCM actively contribute to neuron growth under degenerated neuron debris culture conditions, neurons cultured on coverslips were divided as follows: 1) normal neurons co-cultured with OECs; 2) normal neurons co-cultured with OECs supplemented with degenerative neuronal debris; 3) normal neurons co-cultured with OECs exposed to LPS and CCM supplemented with degenerative neuronal debris; 4) normal neurons co-cultured with OECs exposed to LPS supplemented with degenerative neuronal debris; 5) normal neurons co-cultured with OECs exposed to CCM-supplemented degenerative neural debris; 6) normal neurons treated with LPS; 7) normal neurons treated with CCM; 8) normal neurons pretreated with Cyst and NE prior to exposure to LPS and CCM; 9) normal neurons treated with LPS and CCM in the presence or absence of degenerated neural debris; 10) normal neurons treated with degenerative neuronal debris alone. Cells from all groups were maintained at 37 °C in a humidified 5 % CO2 atmosphere for 3 days. Notably, OECs were pretreated with the LPS + CCM, LPS alone, or CCM alone for 12 h prior to co-culture with neurons. Concomitantly, cells were processed for following different examinations.
To determine the survival of neurons under the different treatments mentioned in the previous section, all Tuj-1-positive cells, regardless of their phenotypes, were counted as described previously . Cell count was performed by a person blinded to the experimental setting. From each coverslip 15 randomly chosen fields were counted. In all analyses, data represent the mean ± SEM of 4 independent experiments. Each performed experiment was comprised of 4 coverslips and the obtained result considered as the neuron survival index.
All cells of all groups and treatments were fixed with 4 % paraformaldehyde for 15–20 min, treated with 0.01 % Triton X-100 (for verification of OEC identity, not treated with Triton X-100), and blocked with 3 % normal donkey serum in 0.01 M PBS at room temperature (RT) for 30 min. The following primary antibodies were used: rabbit anti-Tuj-1 (1:400); mouse anti-p75 (1:200); goat anti-GFAP (1:500); mouse anti-S100 (1:300); mouse anti-OX42 (1:200); rabbit anti-TG2 (1:400); rabbit anti-PSR (1:200). Incubation of primary antibodies was carried out at 4 °C overnight. After washing 3 times in PBS, cells were incubated with corresponding fluorescence-conjugated secondary antibodies (Alexa Fluor 488 donkey antirabbit IgG antibody for Tuj-1, TG2 and PSR, 1:400 dilution; Alexa Fluor 488-conjugated goat antimouse IgG for p75, 1:400 dilution; Alexa Fluor 594 donkey antigoat IgG antibody for GFAP, 1:800 dilution; Alexa Fluor 594 donkey antimouse IgG antibody for S100 and OX42, 1:500) for 1 h at RT. Thereafter, counter staining using 4,6-diamidino-2-phenylindole (2 μg/ml) at RT for 15 min was performed and fluorescence evaluated using a confocal epifluorescence microscope (Leica, Wetzlar, Germany). Immunofluorescence was performed in triplicate and representative images were captured.
All data are expressed as mean ± SD or mean ± SEM. Statistical significance between groups was determined by 1-way analysis of variance, followed by Scheffe’s post hoc analysis. A p-value < 0.05 was considered statistically significant.
Characterization and Identification of OECs and Neurons
Enhancement of OEC Activation Through Co-stimulation by LPS and CCM
LPS and CCM Synergistically Potentiate the Proliferation of OECs
The Effects of LPS and CCM on the Phagocytic Ability of OECs Targeting Degenerated Neural Debris
The Effects of OEC Activation by LPS and CCM on Neuron Growth
To validate directly the response of neurons to OECs pretreated with the combination of CCM + LPS in the presence or absence of degenerated neural debris, we performed co-culture of neuronal cells with OECs, and determined the effects of the activated OECs on neuron outgrowth by means of Tuj-1 and p75 immunostaining. As shown in Fig. 5b, the combination of CCM + LPS remarkably promoted neuron (green, Tuj-1-positive) survival in the presence of degenerated neural debris, and almost all surviving cells were characteristically exuberant with pronounced longer neurite extensions compared with the corresponding control. Adversely, in the absence of CCM and LPS, a relative decrease in Tuj-1-positive cells was found in the visual field, and these cells exhibited few processes with poor arborization. However, a small amount of scattered residual debris around the positive cells was seen. As for normal neurons co-cultured with OECs exposed to CCM and LPS, there were no observable differences in neurite extension except for the number of surviving neurons when compared with the group of degenerated neural debris. Strikingly, there was a significant difference in neuron survival and neurite outgrowth between normal neurons in the presence and absence of CCM and LPS.
Furthermore, enhancement of neuronal survival and neurite outgrowth by activated OECs (induced by the combination of LPS + CMM) was quantitatively analyzed by cell count and neurite assays. As shown in Fig. 5c, after culture for 3 days under the aforementioned conditions, the presence of degenerated cell debris suppressed neuron survival remarkably. This was evidenced by a significant decrease in living cells compared with the normal cultured neurons (p < 0.01). Unfortunately, the administration of LPS alone, CCM alone, or their combination (without OECs) did not remarkably alleviate the suppression from the degenerated cell debris. When co-cultured with OECs, neuron survival was significantly improved compared with the corresponding controls. Notably, the combination of LPS and CCM markedly enhanced neuron survival. Statistical analysis revealed that there was a significant difference when compared with the groups of LPS alone and CCM alone in the presence or absence of the degenerated neuron debris (p < 0.01 and p < 0.001, respectively).
Besides neuronal survival, neurite length was also measured. As shown in Fig. 5d, average neurite lengths (μm) from the different groups were 520 ± 55 for the normal group; 310 ± 64.3 for the debris group; 297 ± 64.8 for the debris + LPS group; 320.5 ± 75.3 for the debris + CCM group; 350 ± 45.2 for the debris + LPS/CCM group; 590 ± 64.2 for the debris + CCM/OEC group; 570.4 ± 35.3 for the debris + LPS/OEC group; 698.3 ± 86.2 for the LPS/OEC group; 730.5 ± 183.2 for the CCM/OEC group; 955 ± 73.5 for the debris + LPS/CCM/OEC group; and 1105.2 ± 45.8 for the LPS/CCM/OEC group. Statistical analysis showed that degenerated neural debris can significantly inhibit neurite outgrowth and arborization, and the addition of LPS alone or CCM alone did not attenuate the inhibitory effect. When LPS alone or CCM alone was administered to the co-culture with OECs, it seemed that the inhibitory effects on neurite outgrowth were ameliorated, displaying longer neurite extensions and more process arborizations. As expected, a significant difference was found when compared with the normal and control groups. As for the combination of LPS and CCM in co-culture with OECs groups, LPS and CCM alone can remarkably promote neurite outgrowth and branching, and attenuate the inhibitory effects caused by degraded cell debris. Statistically, there was a significant difference compared with LPS-alone and CCM-alone groups.
The Possible Mechanism Underlying the Enhancement of Neuronal Growth Mediated by Activated OECs
Effect of CCM + LPS on Expression of TG2 and PSR in OECs
Disruption of TG2 and PSR Suppresses CCM- and LPS-Mediated OEC Activation
Currently, OECs are considered as attractive candidates for cell-based therapies for CNS injuries and neurodegenerative diseases. However, their proregeneration remains controversial. Some reports show no therapeutic efficacy for CNS insults, while a tremendous body of research states that OECs only mediate limited regeneration and functional recovery. Different laboratories show disparate results, ranging from little to extensive functional recovery [12, 13, 66]. Nonetheless, little is known about transplanted OECs in the damaged CNS sites. This is based on the fact that severe insults to CNS usually result in the presence of an unfavorable or hostile microenvironment in the injured zone. This hostile environment not only prevents neural regeneration, but also has a direct impact on transplanted OEC bioactivities, including survival, migration, proliferation, secretion, and phagocytosis . In general, different lesion models induce different degrees of tissue damage and inflammation. The hostile and inhibitory environment arising from certain acute CNS damage may lead to the progressive death of numerous transplanted OECs, finally resulting in abortive or unsatisfactory neuroregeneration outcomes. Therefore, seeking an ideal and effective strategy for strengthening and releasing the therapeutic potential of OECs for eliciting neural regeneration by cell-based transplantation is of pivotal importance. Until now, little is known about enhancing the potential of OECs in creating a permissive environment as early as possible to repair CNS injuries and disorders.
In this study, we present in vitro data emphasizing the potential efficacy of a combination of LPS and CCM in inducing activation of OECs. Our results demonstrated convincingly that OECs can be significantly activated in conjunction with LPS and CCM. These activated OECs express a set of relative genes and proteins that define cell activation (CXCL1, CXCL2, TLR4, and TNFα) [9, 31], and are endowed with enhanced proliferation and phagocytic capacity. More importantly, activated OECs can efficiently promote neural survival and neurite outgrowth in the presence of degenerated neuronal debris. In comparison with other treatment conditions (LPS alone, CCM alone), OECs treated with a combination of LPS and the CCM acquired the most pronounced capacity to promote neuronal growth. Moreover, the elevated beneficial activation of OECs may be intimately associated with upregulation of TG2 and PSR. Therefore, the present therapeutic approach using activated OECs could be a promising option for potential cell therapies targeting acute CNS injury and neurodegenerative diseases.
As CNS injuries or neurodegenerative disorders occur, a significant proportion of neurons will undergo a complicated degeneration process, resulting in large amounts of harmful substances, for example cell debris and inhibitory molecules, in the injury lesions [8, 9, 17]. Therefore, rapid and efficient clearance of degenerated products is required for neuronal survival and neurite growth. Our recent study has shown that OECs have the capacity to ingest and engulf degenerated nerve tissue debris, resulting in neuron growth . Despite this, formation of a local hostile environment after CNS damage does not usually support the survival of OECs, even though OECs have strong adaptability. Thus, they could not be used to promote neural regeneration. Fortunately, the proliferation and phagocytosis capacity of OECs can be markedly strengthened after activation. This can be used to remedy progressive OEC death and further deterioration of the microenvironment within injured neural tissue, but is dependent on continuous proliferation of endogenous OECs to further replenish the loss of transplanted OECs without the need for secondary transplantation. On the basis of these elucidations, we wished to induce OEC activation with a combination of LPS and CCM. Our data proved that the combined treatment can effectively induce the activation of OECs, indicating that this protocol is of particular interest for neural repair therapies.
It was reported previously that LPS can lead to functional activation of OECs [9, 68], by improving their phagocytic capacity. These findings are in line with our recent reports, except that no distinct promotion of neuron survival and neurite outgrowth in co-culture with LPS-treated OECs was found in our experiments. Although rapid and efficient clearance of degenerated cell debris is of importance for neural regeneration, limited and slow removal of debris after CNS injury seems less supportive. Retention of degenerated cell debris can trigger a serious of inflammatory cascades, which are harmful to cells as a result of the release of oxygen free radicals and neurotoxic enzymes [21, 69, 70]. Therefore, OEC proliferation, motility, and migration are crucial for creating a favorable environment for both nerve regeneration and restoration. CCM has been shown to stimulate the proliferation of neural progenitors . More recently, another report has demonstrated that CCM selectively regulates the activation of extracellular-regulated kinase and p38 mitogen-activated protein kinase pathways to elicit OEC proliferation and migration . In our study, we found that CCM effectively improved OEC proliferation and phagocytic capacity in presence of LPS. Intriguingly, further exploration revealed that OEC activation induced by the combination of LPS and CCM remarkably promoted neuron survival and neurite outgrowth under hostile conditions (delivering a considerable amount of degenerated cell debris). This positive effect may associate with a combination of OEC proliferation, motility, and migration. First, OEC proliferation results in an increase in cell numbers, which effectively decreases the amount of debris in culture, replenishes the loss of OECs in the hostile environment, and elevates total secretion of neurotrophic molecules. Second, motility and migration of OECs may be improved , which further supports better phagocytosis. Notwithstanding, LPS alone or CCM alone seems to weakly promote OEC activation, resulting in moderate levels of proliferation and phagocytosis. Of note, these promotive effects are inferior to the combination of LPS and CCM, suggesting that LPS and CCM might exert a synergetic effect in inducing OEC activation.
In the present study, LPS was used to enhance the activation of OECs, which may lead to some risks in future clinical translation because LPS can cause neuroinflammation by triggering activation of microglial cells in vivo. The neuroinflammation may further exacerbate neuronal damage. Nevertheless, the neuroinflammation arising from activating microglial cells in vivo must be dependent on high-dose LPS stimulation, while the low dose of LPS used in our study could hardly evoke effective activation of microglial cells in vivo. In comparison, the rapid removal of degenerated debris by activated OECs may be more important in improving the microenvironment, reducing inflammatory injury and further promoting neuronal survival and regeneration. Even though LPS at 1 μM may cause slight activation of microglia in vivo and further result in neuroinflammation at a low level, the inflammation may only result in negligible negative effect on neurons. In addition, CCM has a significant anti-inflammatory effect [42, 43], which may offset the hazard caused by the LPS-induced minor inflammatory reaction.
In order to further substantiate our hypothesis, TG2 and PSR expression in OECs treated with LPS and CCM were investigated in several assays. The results revealed that the combination of LPS and CCM elevated TG2 and PSR remarkably, both in regard to gene transcription and protein expression levels, suggesting that the combination of LPS and CCM truly elicits OEC activation because TG2 and PSR, as either mediator or effector in their downstream signaling, were activated. In line with our results, it has been demonstrated that TG2 plays an important role in activating an intracellular signaling cascade and receptor signaling that are tightly associated with cell proliferation and motility [54, 58, 61]. Moreover, TG2 can mediate NF-κB activation, resulting in Akt activation [57, 58]. It is well-known that NF-κB activation can facilitate cell survival and cell proliferation [44, 46, 61, 71]. Recent studies have demonstrated that upregulation of TG2 in epithelial cells results in constructive activation of focal adhesion kinase, Akt, NF-κB, RhoA, and mitogen-activated protein kinase, contributing to cell adhesion, migration, and invasion [53, 54, 56, 72, 73]. Accordingly, the possible underlying mechanism of OEC activation and subsequent promotion of neuron growth is associated with upregulation of TG2 caused by synergistic stimulation by LPS and CCM. Meantime, TG2 upregulation is a likely explanation of the abovementioned cellular events. Strikingly, our data also revealed that PSR, a critical mediator in phagocytosis, was significantly upregulated at the mRNA and protein levels. Normally, PSR is displayed on the outer of membrane leaflet as a so-called “eat-me” signal . When apoptosis and necrosis occur, cell surface-exposed PSR usually serves as a single and direct signal for recognition/uptake by phagocytic cells [62, 63]. Increased PSR expression levels are indicative of increased phagocytic activity [9, 65]. As expected, we also observed that LPS and CCM can significantly increase the expression of PSR when compared with that induced by LPS alone, CCM alone, or normal control, further emphasizing the synergistic effects of LPS and CCM on OEC activation and phagocytosis. On the basis of our present data, as well as on several previous reports, upregulation of TG2 and PSR may be involved in LPS- and CCM-mediated activation of OECs and the promoting effects on neurons. Still, it is unclear whether the signaling of TG2 and PSR is truly responsible for possible mechanisms underlying the special cell event. In order to elucidate the issue, Cyst (a specific inhibitor of TG2) and NE (a cleavage enzyme of PSR ) were administered into OEC cultures/neuron and OEC co-cultures pretreated with LPS and CCM. Our results revealed that disruption of TG2 and PSR biofunctions effectively abrogated OEC activation and the further promotive effects on neurons, implying that the signaling of TG2 and PRS is involved in LPS- and CCM-mediated cellular events. Although the precise mechanisms responsible for the activation of OECs induced by LPS and CCM and subsequent promotive effects on neuron growth remain unclear, a possible mechanism modulated by LPS and CCM eliciting TG2 and PRS upregulation by OECs could account for the intricate event.
We conclude that the combination of LPS and CCM can remarkably potentiate activation of OECs, resulting in a strengthened phagocytic capacity of OECs with regard to degenerated debris, which, in turn, efficiently promotes neuron survival and neurite outgrowth. Furthermore, we also unravel the possible molecular mechanisms responsible for the cascade event. This study is likely to provide a novel strategy for autologous OEC-based transplantation in CNS injury and a variety of neurological disorders. Furthermore, we clarify controversial issues regarding OEC biofunctions in proregeneration.
This work was supported by the Natural Science Foundation of China (grants: 81371411, 81472098, 81571208, 81670846, and YJ2014001). The authors confirm that there has been no financial support for this research that could have influenced its outcome.
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