Fibrinogen, lipopolysaccharide from Escherichia coli (055:B5; LPS), tunicamycin, thapsigargin, leucine-methyl-ester (LME), staurosporine, hirudin, and Isolectin B₄ from Griffonia simplicifolia-FITC conjugated were from Sigma (Dorset, United Kingdom) and thalidomide was from Tocris Bioscience (Bristol, United Kingdom). Quantikine M TNFα. TGF-β and IL-6 ELISA kits, z-VAD-FMK and z-ATAD-FMK were from R&D Systems (Abingdon, United Kingdom). Caspase 12 (FITC-ATAD-FMK) activity kits were from Promokine (Heidelberg, Germany) and caspase 3/7 (FAM-DEVD-FMK) activity kits were from Millipore (Watford, United Kingdom). Anti-ED1, anti-CD11b and rat IgG isotype controls were from AbD Serotec (Kidlington, United Kingdom), anti-caspase 12 and goat anti-rat-FITC were from Abcam (Cambridge, United Kingdom). Goat anti-rabbit IgG horseradish peroxidase (HRP) and goat anti-mouse IgG HRP were from Autogen Bioclear (Calne, United Kingdom).

All Western blots and PCR analyses were carried out at least three times and those shown are representative. All experiments were performed from at least three separate cell preparations with internal replicates of at least 3 per experiment. Significant differences were estimated using Welch’s two-sided t-test or Student’s un-paired t-test with levels of significance at ^∗∗∗p < 0.001, ^∗∗p < 0.01, and ^∗p < 0.05; p > 0.05 was not significant.

To extend previous findings that FG can induce an inflammatory microglial phenotype, we performed, in parallel, genetic microarray analysis of the well-characterized BV2 mouse microglia cell line, and pro-inflammatory cytokine release from primary rat microglia cultures and enriched neuronal glial cultures (ENGCs). Microarray analysis of BV2 cultures after 6 h of FG exposure identified a significant over-representation, amongst others, of immune system and inflammatory gene upregulation, including tnf, which encodes TNFα (Figure 1A and Table 1). These findings were supported by cytokine release data from rat microglia cultures where primary microglia exposed to FG or lipopolysaccharide (LPS; as a positive control) for 24 h showed significant TNFα secretion (Figure 1B). A number of cytokines and chemokines, many of which are regarded as inflammatory (Owen et al., 2011), were secreted to significance by FG (Figure 1B) including TNFα, MMP-2, IL-6, CXCL2, and CCL2. Whilst many mirrored the release evoked with LPS, Fibulin-3 was secreted by FG stimulation only, whereas IL-1β or IL-1α secretion was observed after LPS treatment, but was absent in FG-treated cultures indicating that FG stimulation of microglia is nuanced compared with LPS stimulation.

In support of previously published data identifying microglial CD11b as a putative receptor for FG (Adams et al., 2007), pre-incubation of primary microglial cultures with a CD11b blocking antibody significantly attenuated the FG-induced TNFα release, but had no effect on LPS-induced secretion (Figure 1C). Given the perceived importance of microglia in mediating the inflammatory effects of FG, and the potential for this inflammation to impact on neuronal integrity, we determined if the microglia present in ENGCs responded in a similar way to isolated primary microglia. The ability to specifically ablate microglia from the ENGCs (Jebelli et al., 2015) allows us to determine any particular dependencies on these cells. In accord with the data for proteome analysis for release of cytokines from primary cultured microglia, exposure of ENGCs to either FG or LPS significantly enhanced the release of TNFα (Figure 1D) and another pro-inflammatory cytokine, interleukin-6 (IL-6; Figure 1E), in a manner similar to that observed in primary microglia cultures. Critically, when microglia were ablated from ENGCs, the LPS- or FG-induced TNFα release (Figure 1C) and IL-6 release (Figure 1D) were significantly attenuated. Further characterization of cytokine release in the ENGCs revealed that transforming growth factor-β1 (TGF-β1) secretion, a cytokine known to have opposing cellular effects to TNFα (Bitzer et al., 2000), was not significantly modulated by either LPS or FG exposure, irrespective of the presence of microglia (Figure 1E), this is possibly due to the dominating expression of this cytokine by the astrocyte population present in the ENGCs (De Groot et al., 1999). Taken together, these data suggest that FG exposure skews microglia toward a pro-inflammatory phenotype.

Confirmation of a FG-mediated induction of a reactive microglial phenotype, as previously indicated (Adams et al., 2007; Piers et al., 2011) was performed by immunocytochemical analysis of characterized activated microglial markers. Control, unstimulated, primary microglial cultures expressed low levels of the activated microglial marker ED-1 (Figure 2Ai), and > 95% of cells stained positive for IB₄ (Supplementary Figure S1), suggesting highly purified cultures, in a down-regulated state. In support of FG-mediated activation, ED1 protein expression was significantly enhanced when compared with non-treated control cultures (Figure 2Aii). Furthermore, FG treatment significantly increased the surface expression of its putative receptor, CD11b (Figures 2Bi,ii), induction of which has previously been associated with a phagocytic microglial phenotype (Adams et al., 2007). These findings provide further support for a shift in microglial activation when the cells are exposed to FG, although interestingly whilst LPS evoked inducible nitric oxide synthase (iNOS) expression in the microglia, FG did not, suggesting a further divergence in activation profiles (Figure 2Aiii).

Whilst it is clear that FG exposure can induce a pro-inflammatory microglial phenotype, it is unclear whether this induced phenotype would have an effect on neuronal viability. To study this, we exposed ENGCs to FG and performed live cell staining with Hoescht/propidium iodide (PI) for total/dead cell analysis, respectively. After 24 h of exposure to FG, the number of PI-positive cells increased significantly when compared with non-treated controls (Figures 3Ai,Aii), which did not increase further after 48 h of exposure (Figure 3Aii). The cleavage of fibrinogen to fibrin by thrombin is a well-defined mechanism in the coagulation cascade (Binnie and Lord, 1993), and thrombin transcripts are present in these cultures when exposed to FG for 48 h (Supplementary Figure S2). Therefore, it was possible that the observed toxicity was due to cleavage of soluble fibrinogen to insoluble fibrin deposits. To clarify this, cultures were co-treated with FG and the thrombin inhibitor hirudin for 24 h. The percentage of PI-positive cells in the FG + hirudin-treated cultures was not significantly different from cultures treated with FG alone (Figure 3B) suggesting soluble FG rather than cleaved fibrin is the cause of the observed reduced neuronal viability. Death was not due to endotoxin contamination of FG since polymyxin B (PMX) treatment to remove endotoxin did not ablate FG-induced death but did ablate LPS induced death (Supplementary Figure S3).

To further characterize the death-signaling pathways induced in the ENGCs by FG exposure, live cell staining for the apoptotic executioner caspase-3/7 was performed, using the fluorescent substrate FAM-DEVD-FMK (Darzynkiewicz et al., 2002). Following 24 h exposure to FG, a significant increase in caspase-3/7 activity was observed, when compared with non-treated control cultures (Figures 3Ci,Cii). Interestingly the most pronounced staining seemed to correlate with cells with large, rounded cytoplasmic morphology, uncharacteristic of neuronal anatomy (white arrow heads, Figure 3Ci), and unlike the prominent staining observed after staurosporine (STS; positive control for the induction of apoptosis) exposure (Figures 3Ci,Cii). These cells fit the description of microglia that we know are present in the ENGCs as confirmed with IB₄ staining and previously published literature (Figure 3Ciii; Piers et al., 2011; Jebelli et al., 2015). Furthermore, previous studies have suggested that microglial activation is dependent on non-apoptotic caspase-3 activity (Shen et al., 2017). Therefore, we wanted to identify whether microglial caspase-3 activity was important in the observed reduction in neuronal viability. Initially, we performed microglial ablation on ENGCs and found that removal of microglia from the cultures significantly attenuated the observed FG-mediated neuronal death (Figure 3D). Interestingly, LPS mediated neuronal death was not significantly different from control levels, suggesting that whilst the ability of FG and LPS to modulate microglial reactivity is similar in terms of cytokine release, the underlying mechanisms of any subsequent neuronal insults differ (Figure 3D).

Further support for the involvement of microglia in FG-mediated neuronal death, specifically, the secreted factors from microglia, was identified by exposing microglia-depleted ENGCs to conditioned medium from primary microglia cultures (MGCM) exposed to FG (Figure 3E). We observed a significant increase in neuronal death when ENGCs were exposed to MGCM from FG-treated microglial cultures when compared to the addition of control MGCM, which could be attenuated by boiling the MGCM prior to its addition to the ENGCs (Figure 3E). Carry-over of FG in MGCM to exert a direct effect is also unlikely since negligible FG was detected in FG-MGCM (Supplementary Figure S4). Finally, we found it was possible to attenuate the effect of MGCM from FG-treated microglia cultures on ENGCs by pre-incubating the microglia cultures with the caspase-3/7 inhibitor z-VAD-FMK (Figure 3F). MG exposed to FG, or LPS, showed caspase-3 activation but no pyknotic nuclei suggesting non-apoptotic activation of caspase-3 (Supplementary Figure S6). Taken together, these data strongly support a microglial-mediated mechanism for the observed increase in FG-induced neurotoxicity, centered on the release of soluble factors, and suggests a possible role for non-apoptotic activation of caspase-3 in microglia within this mechanism, as our previous investigations found no significant increase in PI-positive (i.e., dead cells) nuclei in primary microglial cultures exposed to FG (Piers et al., 2011).

Based on our microarray and cytokine release data, one candidate for a soluble released factor from microglia that may enhance neurotoxicity is TNFα. Studies suggest inflammatory cytokines including TNFα can induce ER stress signaling (Xue et al., 2005; Denis et al., 2010; O’Neill et al., 2013; Dandekar et al., 2015). Cleavage of the ER-located caspase-12 during ER stress triggers downstream apoptotic pathways and is implicated in neurodegenerative disease models associated with BBB dysfunction (Nakagawa et al., 2000). Therefore, we investigated a possible role for TNFα in an ER stress associated mechanism of neurotoxicity. Initially, we found that exposure of ENGCs to FG induced an increase in activated caspase-12 expression as measured by western blotting for the cleaved form of the protein (Figures 4Ai,Aii,Aiii), to levels comparable with the known ER stress inducers, thapsigargin (a specific inhibitor of the sarco-ER calcium-ATPase, SERCA), or tunicamycin (a blocker of protein glycosylation; Nair et al., 2007; Tu). In support of these findings, live cell staining experiments of ENGCs after FG treatment identified significant increases in caspase-12 activity, comparable with levels observed after thapsigargin treatment (Figures 4Bi,Bii). Furthermore, inhibition of caspase-3/7 with z-VAD-FMK, or specific inhibition of TNFα synthesis by thalidomide treatment, was sufficient to attenuate the increased levels of caspase-12 expression (Figures 4Bi,Bii), supporting the hypothesis that a released factor involved in the induction of neurotoxic ER stress signaling was TNFα. In confirmation of ER stress activation, we found CHOP transcripts were also significantly enhanced in microglia exposed to FG (2.5 mg/ml) or LPS (1 μg/ml) for 24 h or Thapsigargin (2μM, as a positive activator of ER stress) (Supplementary Figure S5).

In support of the critical induction of these signaling cascades in the observed FG-mediated neurotoxicity, MGCM from cultures exposed to FG were unable to induce significant neuronal toxicity in ENGCs pre-treated with the caspase-12 specific inhibitor z-ATAD-FMK (Figure 4C). Inhibition of caspase-3/7 also reduced caspase-12 cleavage in ENGCs, suggestive of an upstream mechanism involving microglial activation (Figure 4D). Concomitantly, removal of microglia from ENGCs prevent FG-mediated induction of caspase-12, further supporting a non-cell autonomous mechanism (Figure 4E). Moreover, thalidomide significantly reduced TNFα secretion from microglia treated with FG (Figure 4F). Together, these findings suggest a signaling cascade initiated by FG-mediated microglial activation leading to a non-cell autonomous neurotoxicity via released TNFα and subsequent induction of neuronal ER stress signaling.