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. Author manuscript; available in PMC: 2021 Jul 1.
Published in final edited form as: Glia. 2020 Feb 14;68(7):1435–1444. doi: 10.1002/glia.23791

Microglial responses after phagocytosis: E. coli bio-particles, but not cell debris or amyloid beta, induce matrix metalloproteinase-9 secretion in cultured rat primary microglial cells

Gen Hamanaka 1, Tomoya Kubo 1, Ryo Ohtomo 1, Hajime Takase 1, Estefania Reyes-Bricio 1, Shuntaro Oribe 3, Noriko Osumi 3, Josephine Lok 1,2, Eng H Lo 1, Ken Arai 1
PMCID: PMC7256913  NIHMSID: NIHMS1588907  PMID: 32057146

Abstract

Upon infection or brain damage, microglia are activated to play roles in immune responses, including phagocytosis and soluble factor release. However, little is known whether the event of phagocytosis could be a trigger for releasing soluble factors from microglia. In this study, we tested if microglia secrete a neurovascular mediator matrix metalloproteinase-9 (MMP-9) after phagocytosis in vitro. Primary microglial cultures were prepared from neonatal rat brains. Cultured microglia phagocytosed E. coli bio-particles within 2 hours after incubation and started to secrete MMP-9 at around 12 hours after the phagocytosis. A TLR4 inhibitor TAK242 suppressed the E. coli-bio-particle-induced MMP-9 secretion. However, TAK242 did not change the engulfment of E. coli bio-particles in microglial cultures. Because lipopolysaccharides (LPS), the major component of the outer membrane of E. coli, also induced MMP-9 secretion in a dose-response manner and because the response was inhibited by TAK242 treatment, we assumed that the LPS-TLR4 pathway, which was activated by adhering to the substance, but not through the engulfing process of phagocytosis, would play a role in releasing MMP-9 from microglia after E. coli bio-particle treatment. To support the finding that the engulfing step would not be a critical trigger for MMP-9 secretion after the event of phagocytosis in microglia, we confirmed that cell debris and amyloid beta were both captured into microglia via phagocytosis, but neither of them induced MMP-9 secretion from microglia. Taken together, these data demonstrate that microglial response in MMP-9 secretion after phagocytosis differs depending on the types of particles/substances that microglia encountered.

Keywords: microglia, phagocytosis, matrix metalloproteinase-9, E. coli, cell debris, amyloid beta

Introduction:

As a sentinel in the brain, microglia constantly monitor the microenvironment, and upon infection or brain damage, they play important roles in immune responses (Chen & Trapp, 2016; Giulian, 1987; Kawanokuchi et al., 2006; Nimmerjahn, Kirchhoff, & Helmchen, 2005; Ransohoff & Cardona, 2010). One of the major roles of microglia is phagocytosis. They scavenge the brain for infectious agents, damaged/dead cells, and amyloid plaques (Ard, Cole, Wei, Mehrle, & Fratkin, 1996; Cai, Hussain, & Yan, 2014; Chen & Trapp, 2016; Davalos et al., 2005; Ludwin, 1990; Ransohoff & Cardona, 2010; Smith, 2001). By removing these harmful substances, microglial cells contribute to brain homeostasis by maintaining a stable microenvironment. In addition, microglia actively release multiple soluble factors, including cytokines, chemokines and reactive oxygen species (ROS), which can be either harmful or beneficial for brain function, depending on the context (J. Lee, Hamanaka, Lo, & Arai, 2019; Nakajima, Kohsaka, Tohyama, & Kurihara, 2003; Ransohoff & Cardona, 2010). Thus far, past studies have extensively examined the cellular and molecular mechanisms of microglial function, such as phagocytosis and production of cytokines and ROS. However, little is known regarding the potential role of phagocytosis as a trigger for microglia release of soluble factors.

Within the neurovascular unit, different types of brain cells maintain brain homeostasis by communicating with each other through the exchange of soluble factors. Matrix metalloproteinases (MMPs) are major mediators for cell-cell or cell-matrix interactions in the neurovascular unit. MMPs comprise a family of zinc endopeptidases, and play an important role in regulating extracellular matrix signaling (Nagase, Visse, & Murphy, 2006). MMPs can degrade extracellular matrix molecules, and modulate axonal growth/regeneration, myelin formation, and vascularization (Verslegers, Lemmens, Van Hove, & Moons, 2013; Yong, 2005). On the other hand, uncontrolled expression or activation of MMPs may result in neurovascular damage, including anoikis-induced neuronal death and blood-brain barrier damage (Lo, 2008; Maki et al., 2013; Moskowitz, Lo, & Iadecola, 2010). Among the MMP superfamily, MMP-9 has been extensively studied as a therapeutic target for several neurological diseases including stroke, multiple sclerosis, Alzheimer’s disease, and cerebral hemorrhage (Avolio et al., 2003; Davis & Senger, 2005; Kook, Seok Hong, Moon, & Mook-Jung, 2013; Nagase et al., 2006; Rosell et al., 2008). Although microglia are known to release MMP-9 under pathological conditions (Gottschall, Yu, & Bing, 1995; Vandooren, Van Damme, & Opdenakker, 2014), no studies have examined if MMP-9 secretion from microglia is related to phagocytic activity. Hence, in this current study, we used primary rat microglial cells to examine whether phagocytosis could be a key trigger to initiate MMP-9 secretion from microglia.

Materials and Methods:

Primary culture of microglia

Microglia were isolated from cultured rat mixed glial cells. Primary rat mixed glial cultures were prepared as described previously with some modifications (Arai & Lo, 2009; Esposito et al., 2018). In brief, cerebral cortices from 1- or 2-day-old Sprague Dawley rats (Charles River Laboratories) were dissected, minced, and digested. Dissociated cells were plated in poly-D-lysine-coated 75 cm2 flask (Thermo Scientific), and maintained in Dulbecco’s Modified Eagle’s medium (DMEM, Gibco) containing 20% heat-inactivated fetal bovine serum (FBS, ATLANTA Biologicals) and 1% of penicillin/streptomycin (P/S, Gibco). The medium was replaced every 3 days until the cells were confluent (〜10 days). To obtain microglia, the flask in which the cells were confluent was shaken for 1 hr on an orbital shaker (218 rpm) at 37C, and the medium was collected. The nonadherent cells in collected media, which constitute microglia, were centrifuged at 1,000 rpm for 5 min, suspended with DMEM/ F12 (Gibco) containing 10% FBS and 1% P/S, and cultured at 37C for 1 day. Then these cultured rat microglial cells were used for experiments. The adherent cells remaining in the flasks after shaking were used for the phagocytosis experiments as a bait consisting of glial cell debris.

Immunocytochemistry

Cells were fixed with 4% paraformaldehyde (PFA) in PBS on ice for 10 min. After washing with PBS with 0.1% Tween-20 (PBST) 5 times at room temperature (RT), cells were treated with 3% BSA in PBST at RT for 1 h. Then, primary antibodies (diluted by PBST containing 0.3% BSA) were added to each well, and the fixed cells were maintained at 4C for overnight. The dilution ratio is as followed; goat anti-Iba1 (1:1000, Abcam) for microglia, rat anti-glial fibrillary acidic protein (GFAP; 1:1000, Thermo Fisher) for astrocyte. After washing with PBST 5 times at RT, secondary antibodies (diluted by PBST containing 0.3% BSA) were added to each well, and the fixed cells were maintained at RT for 1 h. The dilution ratio is as followed; donkey anti-goat IgG H&L (Alexa Fluor 488) (1:1000, Abcam), donkey anti-goat IgG H&L (Alexa 568) (1:1000, Abcam), Rhodamine (TRITC) AffiniPure Donkey Anti-Rat IgG (H+L) (1:1000, Jackson Immuno Research Laboratories, Inc.). After 5 times washing with PBST, the stained cells were treated with Fluoro-Gel II,with DAPI (Electron Microscopy Sciences) at RT for 10 min, and then, cells were examined using a fluorescence microscope (Nikon).

Western blot

Cells were washed with ice-cold PBS 3 times on ice, suspended with 100 μl of 1x NuPAGE LDS sample buffer (Invitrogen) containing NuPAGE Sample Reducing Agent (Invitrogen), and then the cell suspension was boiled at 80C for 10 min. After electrophoresis and transferring to iBlot 2 NC Regular Stacks (Invitorgen), membranes were blocked with 5% skim milk in TBS with 0.1% Tween 20 (Sigma) (TBST) at RT for 1h, and then incubated with primary antibodies (diluted with TBST containing 1% skim milk) at 4C overnight. The dilution ratio is as followed; rabbit anti-platelet-derived growth factor receptor (PDGFR)-α (1:1000, Sigma) for oligodendrocyte precursor cell (OPC), rat anti-GFAP (1:1000, Thermo Fisher) for astrocyte, mouse anti-β-actin (1:10000, Sigma-Aldrich) for loading control. After washing with TBST 5 times at RT, membranes were incubated with secondary antibodies (diluted with TBST containing 1% skim milk). The dilution ratio is as followed; ECL anti-rabbit IgG, horseradish peroxidase linked whole antibody (1:1000, GE Healthcare), anti-rat IgG, horseradish peroxidase linked whole antibody (1:1000, GE Healthcare), anti-mouse IgG, horseradish peroxidase linked whole antibody (1:1000, GE Healthcare). After washing with TBST 5 times at RT, membranes were developed with Pierce ECL Western Blotting Substrate (Thermo Scientific), according to the manufacturer’s protocol.

Preparations of bait materials for phagocytosis assay

Three different kinds of bait were used in this study, i.e. E. coli bioparticle, cell debris from cultured glial cells, and amyloid beta (Aβ). For the E. coli bioparticle bate, pHrodo™ Green E. coli BioParticles Conjugate (Molecular Probes) was purchased and prepared for experiments according to the manufacturer’s protocol. For cell debris and Aβ, the baits were labled with pHrodo™ Red succinimidyl (NHS) ester (Molecular Probes). The pHrodo reagent was diluted with DMSO, according to manufacturer’s protocol, before use. For preparation of cell debris bait, cells which remained at the bottom of the flask after shaking were incubated with 0.25% Trypsin-EDTA (Gibco) at 37C for 10 min, collected into a fresh tube, and then frozen by liquid nitrogen. After thawing by exposing the running water, cell debris were stained with pHrodo™ (10,000x) at RT for 30 min in dark, and washed with PBS twice. For preparation of Aβ bait, amyloid β-protein fragment 25–35 (Sigma-Aldrich) was diluted with PBS with 0.1% acetate acid, and incubated with 37C for 3 days before pHrodo-labeling. The procedure of pHrodo labeling to Aβ25–35 was the same as the one for the cell debris bait.

Phagocytosis assay

Microglia were incubated with E coli bioparticles, cell debris, or amyloid beta, at 37C for 2 h in the regular cell culture conditions. After incubation, microglia were washed 3 times with DMEM/F12 containing 1% P/S. Fluorescent images were taken using a fluorescent microscope, and then, the cells were maintained in DMEM/F12 containing 1% P/S at 37C for 24 h (for gelatin zymography). Fluorescent images were analyzed to measure the percentage of pHrodo-positive cells (e.g. microglia that phagocytosed baits) by operators who did not know the group distribution. For inhibitory experiments, microglia were incubated with a TLR4 inhibitor TAK-242 (Millipore Sigma), a p38 MAPK inhibitor SB 203580 (Millipore Sigma), a MEK inhibitor U0126 (Millipore Sigma), or a JNK inhibitor SP600125 (Millipore Sigma), simultaneously with the E. coli bioparticle bait (the inhibitors started to be pre-treated 1 hour before E. coli bioparticles). These inhibitors were diluted with DMSO, and the final concentration of DMSO in cell culture wells was 0.1%.

Gelatin zymography assay and densitometry

Gelatin zymography assay was carried out as described previously with some modifications (Arai, Lee, & Lo, 2003; Seo et al., 2013; Y. Takahashi et al., 2014). In brief, conditioned media (CM) from microglial cultures, which were treated with baits or LPS (Sigma), were collected, concentrated 20x by using VIVASPIN 500 (10,000 MWCO PES, sartorius), and mixed with an equal volume of 2x Tris-Glycine SDS Sample Buffer (novex). Following electrophoresis on Novex 10% Zymogram Plus (Gelatin) Gel (Invitrogen), gels were incubated with Renaturing Buffer (Invitrogen) at RT for 30 min, Developing Buffer (Invitrogen) at RT for 30 min, and fresh Developing Buffer at 37C for 24 h. Gels were then stained with staining solution (42% (v/v) methanol, 8% (v/v) acetate acid, 0.5%(w/v) coomasie brilliant blue) at RT for 2 h, and de-stained with de-staining solution (40% (v/v) methanol, 10% (v/v) acetate acid) at RT for 30 min. Densitometry of bands on the gels was calculated by Image J by operators who did not know the group distribution. LPS (Sigma)-activated microglia were prepared by incubation with 1 μg/μl of LPS at 37°C for 2h with or without inhibitors, and then, maintained in the DMEM/F12 containing 1% P/S at 37°C for 24 h.

Statistical analysis

Data were expressed as means ± SD. P-value was evaluated by unpaired t-test or One-way ANOVA followed by Tukey test (for multiple comparison), and *P<0.05 was considered significant.

Results:

Purity of microglial culture system

Primary microglial culture system from rat neonatal cortex may get contaminated with other kinds of cells, such as astrocytes and oligodendrocyte lineage cells. Therefore, we first confirmed that our system was reasonably pure to assess microglial function in vitro. Most of the cells in our microglial cell culture system were Iba1-positve cells (96.3% ± 2.3% from 3 independent experiments), and our microglial cultures were not contaminated with GFAP-positive astrocytes (Figure 1). Western blotting also confirmed that our cell culture system did not contain astrocytes and OPCs (Supplementary Figure S1). This is important because astrocytes are also known to play a role in phagocytosis under some conditions (Chung et al., 2013; Morizawa et al., 2017). In addition, under cell culture conditions, it was demonstrated that astrocytes and OPCs could be potent sources of MMP-9 (Arai et al., 2003; Gottschall & Yu, 1995; Seo et al., 2013; Vandooren et al., 2014).

Figure 1. Purity of microglial culture:

Figure 1.

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Immunostaining confirmed that our microglial cultures were positive for Iba1, a microglia marker, but negative for GFAP, an astrocyte marker. The percentage of Iba1-positive cells over DAPI was 96.3 ±2.3% (mean ± SD) from 3 independent experiments. Scale bar = 50 μm.

Microglia demonstrated phagocytotic activity against E. coli and released MMP-9 after exposure to E. coli bioparticles

As a bait for microglia, we first selected E. coli bio-particles which have been widely used in vitro to examine the mechanisms of microglial phagocytosis. More importantly, in biological conditions, microglia would encounter E. coli within the brain in certain diseases, such as bacterial meningitis (Basmaci et al., 2015; Lien et al., 2019; Nielsen et al., 2018). In this study, we labeled E. coli bio-particles with pHrodo, which allowed us to detect E. coli bio-particles in microglial cells by phagocytosis under the fluorescent microscope because pHrodo-labeled materials fluoresce once they are moved into the phagolysosome structure in the cytosol during the phagocytosis process. As expected, after 2 h incubation with E. coli bio-particles, not all but substantial numbers of cultured microglia took in E. coli bio-particles via phagocytosis (Figure 2a-b). While MMP-9 was not detected in the microglial-conditioned media under normal conditions, microglia appeared to secrete MMP-9 at 24 hours after 2-hour-incubation with E. coli bio-particles in a dose dependent manner (Figure 2c). Notably, E. coli-bioparticle-treated microglia started to secrete MMP-9 at around 12 hours after phagocytosis (Figure 2d), confirming that MMP-9 secretion occurred after the event of phagocytosis. The precise mechanism of phagocytotic activity in microglia remains somewhat unknown, however; it has been suggested that toll-like receptors (TLRs)-p38 MAPK signaling may play roles in phagocytosing E. coli (Fu, Shen, Xu, Luo, & Tang, 2014). Therefore, we then examined whether the signaling pathway was also involved in MMP-9 secretion after microglia exposure to E. coli bioparticle treatment in microglia. Gelatin zymography using microglial-conditioned-media showed that this E. coli-bioparticle-induced MMP-9 secretion was at least partly mediated by TLR4 and/or p38 MAP kinase signaling, as a TLR4 inhibitor TAK242 and a p38 MAP kinase inhibitor SB203580 were both effective in suppressing microglial secretion of MMP-9 (Figure 3).

Figure 2. Microglia phagocytosed E. coli bioparticle and secreted MMP-9 after phagocytosing E. coli bioparticle:

Figure 2.

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(a) A representative image for microglia ingesting pHrodo-labeled E. coli bioparticle. The fluorescent images were taken 2 hours after E. coli bioparticle treatment. (b) Not all but substantial numbers of microglia ingested E. coli bioparticles at 2 hours after E. coli bioparticle treatment. Mean ± SD from N=4. (c) E. coli treatment induced MMP-9 secretion from cultured microglial cells. Cultured microglia were treated with E. coli bioparticle for 2 hours, and then the culture media were switched to DMEM/F12 containing 1% P/S without E. coli bioparticles and cells were maintained for 24 hours. MMP-9 levels in the microglial conditioned media were assessed by gelatin zymography. Mean ± SD from N=4. (d) Cultured microglia secreted MMP-9 after phagocytosing E. coli bioparticle. Cultured microglia were treated with E. coli bioparticle for 2 hours, and then the culture media were switched to DMEM/F12 containing 1% P/S without E. coli bioparticles and cells were maintained for 3, 6, 9, 12, or 24 hours. MMP-9 levels in the microglial conditioned media were assessed by gelatin zymography. Mean ± SD from N=4.

Figure 3. E. coli-induced MMP-9 release was mediated by TLR4/p38-MAPK signaling:

Figure 3.

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Cultured microglia were treated with E. coli bioparticle with or without inhibitors for 2 hours, and then the culture media were switched to DMEM/F12 containing 1% P/S without E. coli bioparticles and inhibitors. Thereafter, cells were maintained for 24 hours, and MMP-9 levels in the microglial conditioned media were assessed by gelatin zymography. (a) Representative zymography images for E. coli-induced MMP-9 secretion with a TLR4 inhibitor TAK242 or a p38-MAPK inhibitor SB203580 co-treatment. (b) TAK242 suppressed E. coli-induced MMP-9 secretion in a dose dependent manner. Mean ± SD from N=4. *P<0.05 vs without TAK242. (c) SB203580 suppressed the E. coli-induced MMP-9 secretion in a dose dependent manner. Mean ± SD from N=4. *P<0.05 vs without SB203580.

LPS induced MMP-9 from microglia via TLR4 and/or p38 MAP kinase signaling

Cultured microglia phagocytosed E. coli bio-particles and also secreted MMP-9. However, at least in our system, the percentage of microglia that took in E. coli bio-particles was relatively variable between several independent experiments (Figure 2b), while MMP-9 secretion by E. coli bio-particle stimulation seemed more stable with a trend towards a positive dose-response (Figure 2c). In addition, unexpectedly, the TLR4 inhibitor TAK242 did not suppress microglial phagocytosis of E. coli bio-particles in our microglial culture system (Supplementary Figure S2). These data led to our hypothesis that the step of engulfing substances may not be a critical trigger for microglia in MMP-9 secretion; rather adherence to E. coli bioparticles is a more important stimulus for MMP-9 secretion from microglia. To test this hypothesis, we next examined if LPS, the major component of the outer membrane of E. coli, induces MMP-9 secretion from cultured microglia. Similar to the response after E. coli bio-particle treatment, microglia started to release MMP-9 at a relatively later time point (e.g. at around 9~12 hours) after incubation with LPS (Supplementary Figure S3). More importantly, LPS-induced MMP-9 secretion was suppressed by co-treatment with TAK242 or SB203580 (Figure 4a-c). These data support our idea that the engulfing step of phagocytosis in itself does not mediate MMP-9 secretion from microglia after E. coli treatment.

Figure 4. LPS-induced MMP-9 release was also mediated by TLR4/p38-MAPK signaling:

Figure 4.

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Cultured microglia were treated with LPS with or without inhibitors for 2 hours, and then the culture media were switched to DMEM/F12 containing 1% P/S without LPS and inhibitors. Thereafter, cells were maintained for 24 hours, and MMP-9 levels in the microglial conditioned media were assessed by gelatin zymography. (a) Representative zymography images for LPS-induced MMP-9 secretion with TAK242 or SB203580 co-treatment. (b) TAK242 suppressed LPS-induced MMP-9 secretion in a dose dependent manner. Mean ± SD from N=4. *P<0.05 vs without TAK242. (c) SB203580 suppressed LPS-induced MMP-9 secretion in a dose dependent manner. Mean ± SD from N=4. *P<0.05 vs without SB203580.

Cell debris and amyloid beta were captured into microglia via phagocytosis, but did not induce MMP-9 secretion

To further confirm our hypothesis that the process of engulfing foreign substances is not a sufficient stimulus to induce microglial MMP-9 secretion, we treated cultured microglia with cell debris or amyloid beta, which are both reported to be phagocytosed by microglia under in-vitro and in-vivo conditions (Ard et al., 1996; Chen & Trapp, 2016). With the same approach, cell debris and amyloid beta were labeled with pHrodo before being added to microglial cultures. Fluorescent signals of pHrodo-tagged cell debris or amyloid beta were observed in microglia 2 hours after incubation (Figure 5a-b), confirming that cultured microglia indeed took in those baits via a phagocytosis process. However, neither of these baits induced MMP-9 secretion from microglia up to 24 hours after treatment (Figure 5c). These data further supported the idea that the engulfing step of phagocytosis in itself is not be a key trigger for MMP-9 secretion from microglia.

Figure 5. Microglia phagocytosed cell debris and Aβ25-35, but did not secrete MMP-9 after treatment of cell debris or Aβ25-35:

Figure 5.

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(a) Microglia ingested pHrodo-labeled cell debris. The fluorescent images were taken at 2 hours after the treatment of cell debris. Mean ± SD from N=4. (b) Microglia also ingested pHrodo-labeled Aβ25-35. The fluorescent images were taken at 2 hours after the treatment of Aβ25-35. Mean ± SD from N=4. (c) However, treatment of cell debris or Aβ25-35 did not induce MMP-9 secretion from microglia. Cultured microglia were treated with cell debris or Aβ25-35 for 2 hours. After the culture media were switched to DMEM/F12 containing 1% P/S without cell debris or Aβ25-35, cells were maintained for 24 hours. MMP-9 levels in the microglial conditioned media were assessed by gelatin zymography. Images are representative zymogram gels from 4 independent experiments. Although recombinant MMP-9 (positive control for gelatin zymography) was detected, no MMP-9 secretion was observed in conditioned media from cell-debris-treated or Aβ25-35-treated microglia.

Discussion:

Microglia are resident immune cells in the brain and constantly monitor the microenvironment. Microglia play a critical role in regulating brain homeostasis by removing unnecessary and/or toxic substances via phagocytosis. Even under cell culture conditions, microglia retain the ability to phagocytose, and several studies reported that cultured microglia do ingest several “foreign materials” (Smith, 2001; Wolf, Boddeke, & Kettenmann, 2017). In our own cell culture system, we confirmed that E. coli bio-particles, cell debris from glial cells, and amyloid beta were all captured by cultured microglia via phagocytosis. However, in terms of MMP-9 secretion, microglia differentially responded to those “baits”, i.e. phagocytosis of E. coli bio-particle was associated with MMP-9 secretion, but phagocytosis of cell debris and amyloid beta was not. Together with our data showing that LPS, the major component of the outer membrane of E. coli, produced similar responses in MMP-9 secretion as observed in microglia with E. coli bio-particles treatment, we proposed that the “engulfing” step of phagocytosis itself is not be a critical trigger for MMP-9 secretion, rather adhering to E. coli before initiating the process of eating substance would play an important role in MMP-9 secretion from microglia. These data indicate that depending on substances for phagocytosis, microglia exhibit different responses in secreting soluble factors into the microenvironments after the event of phagocytosis.

One potential importance of this study is that our experimental system has some clinical relevance. All three kinds of baits - E. coli, cell debris, and amyloid beta - used in this study are major substances that are scavenged by microglia in the brain, especially under pathological conditions. E. coli could enter the brain in bacterial meningitis (Basmaci et al., 2015; Lien et al., 2019; Nielsen et al., 2018) and microglia play a critical role in removing the bacteria and helping in fighting the infection. In addition, after brain damage such as stroke, microglia remove cell debris of dead/damaged cells, enabling brain recovery to take place (Ma, Wang, Wang, & Yang, 2017; K. Takahashi, Rochford, & Neumann, 2005). Also, in the brain of Alzheimer’s disease patients, removal of accumulated amyloid plaques by microglia has been considered as a potential therapeutic approach for the disease (Cai et al., 2014; Meyer-Luehmann et al., 2008). Therefore, understanding the responses of microglia to those substances would elucidate the roles of microglia in a wide range of brain diseases.

Another important point in our current study is to demonstrate that upon E. coli stimulation, TLR4 and/or p38 MAP kinase pathways mediate MMP-9 secretion from microglia. MMP-9 is known to have diverse biological effects and participate in brain pathology in a complex way. Under physiological conditions, the level of MMP-9 in brain is low, but after brain damage, multiple types of brain cells, including microglia, secrete MMP-9 and exacerbate acute brain injury (Rosenberg, Estrada, & Dencoff, 1998). On the other hand, during the chronic phase of brain injury (or diseased conditions), MMP-9 may contribute to brain repair/recovery by promoting compensatory angiogenesis/neurogenesis (Andreuzzi et al., 2017; Larsen, Wells, Stallcup, Opdenakker, & Yong, 2003; Vaillant et al., 2003; Zhao, Tejima, & Lo, 2007). Although several studies have reported that microglia secrete MMP-9 (Gottschall et al., 1995; Vandooren et al., 2014), precise mechanisms which regulate MMP-9 production and secretion in microglia remain to be elucidated. Our current study shows that at least in cell culture, the engulfing step of phagocytosis may not be a critical trigger for MMP-9 secretion from microglia. However, it still remains to be determined whether ingesting foreign substances leads to changes in microglial phenotype (or microglial conditions), which may alter the mechanisms of microglial responses in MMP-9 secretion to subsequent stimuli, such as ROS or cytokines, under pathological conditions. Future studies are warranted to more precisely understand the relationship between the two major microglial functions, e.g. phagocytosis and soluble factor release.

We provide a novel insight into the mechanisms as to how microglia respond after encountering foreign materials. In our current study, however, there are some limitations and caveats. First, we focused only on MMP-9 as a secretion factor from microglia. MMP-9 is one of the major neurovascular mediators (Rosell & Lo, 2008; Yang & Rosenberg, 2015; Zhao et al., 2007), and is indeed released by microglia (Gottschall et al., 1995; Vandooren et al., 2014). However, besides MMP-9, microglia can be an important cellular source for multiple factors, such as inflammatory cytokines/chemokines and ROS (Colton & Gilbert, 1987; Giulian, Chen, Ingeman, George, & Noponen, 1989; Wolf et al., 2017; Yao, Keri, Taffs, & Colton, 1992). Although the engulfing step of phagocytosis may not be a critical trigger for MMP-9 release from microglia, it is possible that other soluble factors could be released upon the stimulation of ingesting foreign materials. An approach with array experiments may help expand this research direction in the future. Second, although our microglial cell culture system has been validated for conducting this study, our method for preparing cultured microglia might affect the phenotype of microglia at baseline. It was recently reported that microglial function in vitro may depend on the experimental methods for isolating cells from neonatal rat cortex (Lin, Desai, Wang, Lo, & Xing, 2017). In addition, our microglia in culture have no interaction with other types of brain cells, whereas within the brain microglial function is affected by astrocytes or endothelial cells (Ma et al., 2017; Sola, Casal, Tusell, & Serratosa, 2002; Xing, Li, Deng, Ning, & Lo, 2018). Future studies will be needed to confirm whether the responses observed in this study will be replicated under in-vivo situations. Third, as an intracellular signaling cascade for MMP-9 secretion, we focused on p38 MAPK because it is relatively well accepted that TLRs-p38 MAPK signaling is involved in phagocytosing multiple foreign materials, including E. coli (Fu et al., 2014). However, stimulation of TLRs may lead to the activation of other members of MAPKs (e.g. ERK and JNK), and these two MAPK members are known to be related to MMP-9 secretion in glial cells (Arai et al., 2003; Gorina, Font-Nieves, Marquez-Kisinousky, Santalucia, & Planas, 2011; D. K. Lee et al., 2012). In our own system of microglial cultures, our pilot data suggested that both ERK and JNK pathways participate in MMP-9 secretion after E. coli bioparticle or LPS treatment (Supplementary Figure S4). How the MAPK family members coordinately regulate the mechanism of MMP-9 secretion after the event of phagocytosis in microglia will be an important research topic in future experiments to further understand microglial roles under pathological conditions. And finally, this study used three substrates (e.g. E. coli, cell debris, and amyloid beta) to examine MMP-9 secretion from microglia, but did not examine whether these substrates would promote phenotype change of microglia. Under diseased conditions, depending on the context, microglia would be transformed into either M1- (deleterious) or M2- (protective) microglia (Tang & Le, 2016; Varnum & Ikezu, 2012). These two phenotypes of microglia release different repertories of soluble factors (Olah et al., 2012; Tang & Le, 2016). Whether phagocytosis changes the phenotype of microglia and whether these phenotype changes are influenced by the nature of the ingested substances are intriguing questions that may elucidate the mechanisms that regulate microglial function.

In conclusion, we demonstrate that cultured microglia take in E. coli bio-particles, cell debris of glial cells, and amyloid beta via phagocytosis. Among these substances, only E. coli bio-particles induce MMP-9 secretion from cultured microglia after the event of phagocytosis, presumably through the LPS-TLR4/p38MAPK signaling activated by adhering to the substance before initiating the engulfing process (Figure 6). These data indicate that depending on particles/substances microglia encounters, microglia exhbit different responses in soluble factor release during and/or after phagocytosis. Our current finding would help us understand the complex roles of microglia in maintaining CNS homeostasis.

Figure 6. Summary diagram of this study:

Figure 6.

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In our culture system, cultured rat microglia phagocytose E. coli bioparticle, cell debris, and Aβ25-35. But only microglia that phagocytose E. coli bioparticle secrete MMP-9 after the event of phagocytosis. The MMP-9 secretion by the treatment of E. coli bioparticle may be mediated via TLR4/MAPK signaling pathway that is activated by the adherence to the outer membrane of E. coli. Please see the discussion section for potential limitations/caveats of this study in more detail.

Supplementary Material

Supplementary Figure

Supplementary Figure S1. Cell lysates from microglial cultures were negative for anti-GFAP (astrocyte marker) and anti-PDGFRα (OPC marker) antibodies. Both anti-GFAP and anti-PDGFRα antibodies worked in our western blot system because cell lysates from astrocyte cultures and OPC cultures were positive with anti-GFAP and anti-PDGFRα antibodies, respectively.

Supplementary Figure S2. A TRL4 inhibitor TAK242 did not suppress the microglial phagocytosis of E. coli bioparticle. Cultured microglia were treated with E. coli bioparticle with or without TAK242 for 2 hours, and then the fluorescent images were taken with a fluorescent microscope. The percentage of cells with pHrodo-positive signal was counted in a blinded manner. Mean ± SD from N=4. N.D. indicates “no difference”.

Supplementary Figure S3. Cultured microglia secrete MMP-9 after LPS treatment. Cultured microglia were treated with 1 μg/mL LPS for 2 hours, and then the culture media were switched to DMEM/F12 containing 1% P/S without LPS and cells were maintained for 3, 6, 9, 12, or 24 hours. MMP-9 levels in the microglial conditioned media were assessed by gelatin zymography. Mean ± SD from N=4.

Supplementary Figure S4. Both ERK and JNK signaling pathways are involved in E-coli-bioparticle- or LPS-induced MMP-9 secretion from cultured microglia. (a-b) Cultured microglia were treated with 1 μg/mL E. coli bioparticle or 1 μg/mL LPS with or without 10 μM U0126 for 2 hours. After the culture media were switched to DMEM/F12 containing 1% P/S without E. coli biopaticle (or LPS) and U0126, cells were maintained for 24 hours. MMP-9 levels in the microglial conditioned media were assessed by gelatin zymography. Mean ± SD from N=4. (c-d) Cultured microglia were treated with 1 μg/mL E. coli bioparticle or 1 μg/mL LPS with or without 20 μM SP600125 for 2 hours. After the culture media were switched to DMEM/F12 containing 1% P/S dia without E. coli biopaticle (or LPS) and SP600125, cells were maintained for 24 hours. MMP-9 levels in the microglial conditioned media were assessed by gelatin zymography. Mean ± SD from N=4 for (c) or N=3 for (d).

Main point:

  • microglia phagocytosed E. coli bioparticles, cell debris, and amyloid beta in vitro

  • E. coli bioparticles, but not cell debris or amyloid beta, induced MMP-9 secretion from microglia

Acknowledgements:

Supported in part by National Institutes of Health.

Footnotes

Data Availability Statement: The data that support the findings of this study are openly available in figures at this research manuscript.

Disclosures: none

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supplementary Figure

Supplementary Figure S1. Cell lysates from microglial cultures were negative for anti-GFAP (astrocyte marker) and anti-PDGFRα (OPC marker) antibodies. Both anti-GFAP and anti-PDGFRα antibodies worked in our western blot system because cell lysates from astrocyte cultures and OPC cultures were positive with anti-GFAP and anti-PDGFRα antibodies, respectively.

Supplementary Figure S2. A TRL4 inhibitor TAK242 did not suppress the microglial phagocytosis of E. coli bioparticle. Cultured microglia were treated with E. coli bioparticle with or without TAK242 for 2 hours, and then the fluorescent images were taken with a fluorescent microscope. The percentage of cells with pHrodo-positive signal was counted in a blinded manner. Mean ± SD from N=4. N.D. indicates “no difference”.

Supplementary Figure S3. Cultured microglia secrete MMP-9 after LPS treatment. Cultured microglia were treated with 1 μg/mL LPS for 2 hours, and then the culture media were switched to DMEM/F12 containing 1% P/S without LPS and cells were maintained for 3, 6, 9, 12, or 24 hours. MMP-9 levels in the microglial conditioned media were assessed by gelatin zymography. Mean ± SD from N=4.

Supplementary Figure S4. Both ERK and JNK signaling pathways are involved in E-coli-bioparticle- or LPS-induced MMP-9 secretion from cultured microglia. (a-b) Cultured microglia were treated with 1 μg/mL E. coli bioparticle or 1 μg/mL LPS with or without 10 μM U0126 for 2 hours. After the culture media were switched to DMEM/F12 containing 1% P/S without E. coli biopaticle (or LPS) and U0126, cells were maintained for 24 hours. MMP-9 levels in the microglial conditioned media were assessed by gelatin zymography. Mean ± SD from N=4. (c-d) Cultured microglia were treated with 1 μg/mL E. coli bioparticle or 1 μg/mL LPS with or without 20 μM SP600125 for 2 hours. After the culture media were switched to DMEM/F12 containing 1% P/S dia without E. coli biopaticle (or LPS) and SP600125, cells were maintained for 24 hours. MMP-9 levels in the microglial conditioned media were assessed by gelatin zymography. Mean ± SD from N=4 for (c) or N=3 for (d).

NIHMS1588907-supplement-Supplementary_Figure.docx (299KB, docx)

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