The transcription factor C/EBPβ is expressed in glia but no direct evidence exists for its involvement in glial activation. In the present study we show that both LPS and LPS+IFNγ upregulate C/EBPβ expression in mixed glial cultures to a similar extent. Both stimuli also induce C/EBPβ binding to proinflammatory gene promoters but this binding is stronger when induced by LPS+IFNγ. Lack of C/EBPβ results in attenuated expression of proinflammatory genes and, again, this effect is more pronounced when glial cells are activated with LPS+IFNγ than when LPS alone is the activating stimulus. Finally, we describe for the first time that neurotoxicity elicited by LPS+IFNγ-treated microglial cells is completely abrogated by lack of C/EBPβ.
In this study we have used mixed glial cultures composed mainly of astrocytes and microglia. This culture system is our model of choice to study glial activation because it allows cross-talk between the two cell types, which is extremely important in glial activation . Working with astrocytes or microglia in isolation may yield misleading results and there are numerous examples of astroglial or microglial responses that are markedly affected by the absence of the other cell type [37–39]. Regarding C/EBPβ, we have previously shown in experiments with mixed glial and astroglial- or microglial-enriched cultures that, upon activation, C/EBPβ is primarily expressed by microglia with a lesser upregulation in astrocytes . This suggests that the data here reported on C/EBPβ in glial activation mainly reflects C/EBPβ changes in microglia although part of the observed effects could be of astroglial origin. However, in the case of the effects of C/EBPβ absence on NOS2 expression and neurotoxicity, the observed effects are clearly microglial, as shown by the microglial localization of NOS2 immunoreactivity and by the use of isolated microglia, respectively.
Most protocols to prepare primary mixed glial cultures from rodents use pools of tissue from several neonates, generally one or two litters. Since C/EBPβ females are sterile  litters of C/EBPβ-null neonates cannot be obtained. Furthermore, approximately 50% of C/EBPβ-null pups die perinatally  which favors the use of late embryos instead of neonates to ensure a maximum number of available C/EBPβ-null mice. Therefore, we established for this study a new protocol of secondary mixed glial cultures by subculturing primary glial cultures prepared from the cerebral cortex of a single E19-E20 embryo. The use of secondary cultures was particularly suitable for this project because we could prepare mixed glial cultures that were very similar to primary cultures in terms of cell density and proportions with a more-than-2-fold higher yield. Besides, the use of siblings eliminates any genetic background effect. Altogether, this makes the use of secondary mixed glial cultures from a single embryo or neonate a useful approach when working with mouse strains of compromised fertility.
LPS is a toll-like receptor 4 agonist that induces marked changes in gene expression in astrocytes and microglia . The combination of LPS, a pathogen factor, with IFNγ, a host factor, potentiates some of the LPS-induced effects . Here we report for the first time a proper comparison between LPS and LPS+IFNγ effects on C/EBPβ and on pro-inflammatory markers in glial cells. We have observed that both LPS and LPS+IFNγ induce similar increases in C/EBPβ mRNA and protein levels as well as in DNA binding. Time-course analyses have revealed that upregulation of the C/EBPβ activating isoforms Full/LAP often precedes upregulation of the inhibitory isoform LIP [21, 24, 42]. When a single time-point is analyzed, as in the present study, the simultaneous increase in activating and inhibitory C/EBPβ isoforms is a common observation. EMSA analysis with supershift experiments showed the presence of C/EBPβ in bands I, II and III. These bands may contain different C/EBPβ isoforms (Full, LAP or LIP) with various post-translational modifications (phosphorylation, SUMOylation or acetylation has been described ). It is likely that some of these bands contain more than one complex (e.g. band II since it is only partially supershifted by anti-C/EBPβ) and that some of these complexes contain other transcription factors, p65-NFκB  and C/EBPδ [45, 46] being two of the most likely candidates to form complexes with C/EBPβ in neuroinflammation. An extensive biochemical analysis would be necessary to characterize the transcriptional C/EBPβ complexes in activated glial cells.
This study shows for the first time in glial cells an analysis of mRNA levels for the pro-inflammatory genes NOS2, IL-1β, IL-6 and TNFα, comparing LPS and LPS+IFNγ as activating stimuli. In this model, IFNγ alone did not trigger any effect (data not shown) whereas LPS and LPS+IFNγ upregulated all four pro-inflammatory genes analyzed. LPS and LPS+IFNγ increased expression of IL-1β, IL-6 and TNFα to the same extent, as reported for macrophages , whereas LPS-induced upregulation of NOS2 was markedly potentiated by cotreatment with IFNγ, in agreement with previous observations in microglia  and macrophages . Even though transcriptional levels of cytokine genes in LPS-treated glial cultures are not modulated by cotreatment with IFNγ, their promoter regions undergo a remodeling of transcriptional complex as proved by qChIP assay. mRNA analysis showed that absence of C/EBPβ does not affect LPS-induced upregulation of the three cytokines, in agreement with absence of C/EBPβ binding to IL-1β, IL-6 or TNFα promoters in LPS-treated glial cultures, as seen by qChIP. Although we cannot exclude the presence of C/EBPβ in other promoter regions, because we focused our promoter analysis on the C/EBPβ consensus sequence most proximal to the translation start site, these data strongly suggest that C/EBPβ does not participate in the LPS-induced expression of these three genes in the present model. It may seem contradictory that strong C/EBPβ binding to IL-1β, IL-6 and TNFα promoters was induced by LPS+IFNγ, but not by LPS alone, whereas the levels of these cytokine mRNAs were similar after treatment with either LPS or LPS+IFNγ. In our opinion, this indicates that different sets of transcription factors act on these promoters after LPS or LPS+IFNγ treatment or, in other words, that there is IFNγ-induced chromatin remodeling on these promoters . This is also suggested by the qPCR data showing that LPS+IFNγ-induced expression of IL-1β is reduced in the absence of C/EBPβ, and that there is also a tendency toward reduced expression of TNFα and IL-6. These data demonstrate for the first time that C/EBPβ plays a role in transactivation of pro-inflammatory cytokine genes in glial cells induced by LPS+IFNγ but not by LPS alone.
In our glial activation model, the NOS2 gene shows a different transcription pattern when compared with the pro-inflammatory cytokines. On the one hand, as mentioned before, LPS-induced NOS2 expression is potentiated by co-treatment with IFNγ. On the other hand, C/EBPβ binding to the NOS2 promoter is already seen after LPS treatment alone and, interestingly, this binding is potentiated by IFNγ treatment. As observed in macrophage cell lines, IFNγ can trigger C/EBPβ phosphorylation, modulating its capacity to form transcriptional complexes with p300  or Med1 . Also, IFNγ can promote C/EBPβ DNA binding activity to IFN-stimulated regulatory elements (ISREs) which we have found tightly associated with C/EBPβ consensus sequences on the mouse NOS2 promoter (unpublished observations). Finally, both LPS- and LPS+IFNγ-induced increases in NOS2 expression are attenuated in the absence of C/EBPβ. These findings suggest that C/EBPβ plays a functional role both in LPS-induced NOS2 expression and in the potentiation of this effect elicited by IFNγ. In accordance with the multiple stage glial activation model , we can hypothesize that LPS alone activates the glia, but that only with a host warning signal, such as IFNγ, are glia totally committed to a hyper-reactive phenotype. We propose that C/EBPβ could trigger this shift through the executive phase of glial activation.
The hypothesis of a pathogenic role for exacerbated glial activation, particularly activation of microglia, is based on the known in vitro neurotoxic effects of activated microglia [53, 54], on the protective effects of anti-inflammatory treatments or genetic modifications in animal models of neurodegenerative disorders [55, 56] and on epidemiological data [57–59]. Since we have shown in this study that C/EBPβ deficiency attenuates expression of potentially neurotoxic pro-inflammatory mediators but not that of anti-inflammatory cytokines, we were interested to test the hypothesis that C/EBPβ plays a key role in the induction of detrimental effects by microglial activation. Reduced neuronal damage after ischemic  or excitotoxic insults  has been observed in C/EBPβ-null mice. Even though C/EBPβ expression has been reported in activated glial cells [22–24], C/EBPβ is known to be also expressed in the adult mouse by neurons  and peripheral cells . Consequently, the neuroprotective effect observed in C/EBPβ-null mice could be mediated by lack of C/EBPβ in any of these cells. We show here that the neurotoxicity elicited by activated wild-type microglial cells co-cultured with wild-type neurons is completely abolished by the absence of C/EBPβ specifically in microglia. This strongly supports a role of C/EBPβ in the regulation of potentially neurotoxic effects of microglia and suggests that the neuroprotective effects of total C/EBPβ absence in vivo [26, 27] are due to microglial C/EBPβ deficiency. Specific microglial C/EBPβ deletion would be very informative to clarify the role of microglial C/EBPβ in neurodegeneration in in vivo models of neurological disease.