Introduction
Bacterial meningitis remains an important cause of mortality and morbidity worldwide, despite the implementation of vaccination strategies, effective antibiotic therapy and adjunctive dexamethasone treatment [
1,
2]. Pneumococcal meningitis is the most common and most severe form of meningitis: 16% to 35% of the patients die and up to 50% of survivors suffer from long-term sequelae, including hearing loss, cognitive impairment, and focal neurological deficits [
3,
4]. The pathophysiology of bacterial meningitis is characterized by a strong inflammatory response in the subarachnoid space, and this host inflammatory response seems to cause adverse events during bacterial meningitis [
5,
6]. The severity of the inflammatory response largely has been shown to determine the prognosis in experimental pneumococcal meningitis [
7] and novel therapeutic agents may thus target the inflammatory response. However, the inflammatory response during meningitis is complex and has only been partially elucidated.
CAAT/enhancer-binding protein δ (C/EBPδ), a member of the C/EBP family of transcription factors that is upregulated during the acute phase response, recently emerged as an essential player in the inflammatory response to bacterial infections [
8]. C/EBPδ levels rapidly increase after pro-inflammatory stimuli such as lipopolysaccharide (LPS), interleukin (IL)-1, IL-6, interferon γ (IFN-γ), and tumor necrosis factor-α (TNF-α) [
9-
11]. Reciprocally, C/EBPδ enhances cytokine production and C/EBPδ-induced inflammation contributes to elimination of bacteria during infectious disease. C/EBPδ was shown to limit bacterial dissemination and prolong survival during a lethal model of
Escherichia coli-induced peritonitis and during
Klebsiella pneumoniae-induced pulmonary infection [
12,
13]. During
Streptococcus pneumoniae-induced pulmonary infection, however, C/EBPδ exaggerated bacterial dissemination and wild-type mice succumbed earlier to the disease as compared to C/EBPδ
−/− mice [
14]. Furthermore, C/EBPδ did not affect disease progression during non-lethal models of
E. coli-induced peritonitis or urinary tract infection [
12,
15]. C/EBPδ therefore seems to play a complex and potential dual role during infectious disease most likely depending on the causing pathogen, the severity of the infection and the infection site. In the present study, we assessed the role of C/EBPδ in experimental pneumococcal meningitis.
Discussion
This study shows that C/EBPδ expression in the brain is induced during pneumococcal meningitis and that C/EBPδ contributes to bacterial colonization of the brain and dissemination to distant organs during disease progression. Although C/EBPδ potentiates the pro-inflammatory response during meningitis, it does not modify clinical parameters of disease severity nor does it affect mortality.
The observed increase in C/EBPδ expression during meningitis is in line with increased C/EBPδ expression in alternative infectious disease models. C/EBPδ expression was previously shown to be increased during peritonitis [
12], pneumonia [
14,
13], and urinary tract infection [
15]. However, the consequence of increased C/EBPδ expression in infectious disease seems not to be straightforward as it correlates with increased survival in some models and with decreased survival in others. During
E. coli-induced peritonitis, C/EBPδ limits bacterial growth and C/EBPδ
−/− mice succumb due to the infection at an earlier time point as compared to wild-type mice [
12]. Similar to these findings, increased C/EBPδ expression during
K. pneumoniae-induced pulmonary infection was found to contribute to improved survival as evident from reduced survival of C/EBPδ
−/− mice [
13]. In contrast, increased C/EBPδ expression during urinary tract infection did not affect disease progression nor outcome [
15]. C/EBPδ expression also contributed to bacterial outgrowth and dissemination leading to prolonged survival of C/EBPδ
−/− mice during
S. pneumoniae-induced pulmonary infection [
14]. This is in line with our results showing that bacterial dissemination was hampered in C/EBPδ
−/− mice as compared to wild-type mice during pneumococcal meningitis leading to a lower bacterial burden in blood and peripheral organs. C/EBPδ-driven bacterial dissemination during meningitis did, however, not modify clinical parameters of meningitis and did not affect overall survival, whereas C/EBPδ-driven dissemination during pneumonia was detrimental. These data underscore the fact that C/EBPδ-driven dissemination plays a divergent role during infectious disease leading to different outcome of the disease.
In our study, we confirm the established role of C/EBPδ as activator of the inflammatory response, reflected by the reduction in IL-10 and KC levels in the brain and IL-6, IL-10, KC, and MIP-2 in plasma during pneumococcal meningitis. The reduced levels of inflammatory mediators in C/EBPδ
−/− mice during meningitis may be a direct consequence of C/EBPδ-driven transcriptional activity but may also be a mere reflection of the bacterial burden. Indeed, the extent of inflammation closely follows the bacterial burden during experimental pneumococcal pneumonia [
20,
21], although such a correlation has not been established for pneumococcal meningitis yet. Here, we show for the first time that during meningitis cytokine and chemokine levels also positively correlate with the bacterial burden in the brain of wild-type mice (id est IL-6, TNF-α, IL-1β, IL-10, KC, and MIP-2). However, such a correlation is not observed for IL-6, IL-10, and KC in C/EBPδ
−/− mice, which suggests that C/EBPδ directly drives transcriptional activity of these inflammatory mediators during meningitis. This is in line with previous reports showing that C/EBPδ is involved in the regulation of IL-6 [
12,
22] and IL-10 [
23] after LPS stimulation, although C/EBPδ suppresses IL-10 expression in dendritic cells of the central nervous system in an animal model of multiple sclerosis [
24]. Moreover, TNF-α and MIP-2 levels are not dependent on C/EBPδ during pneumococcal meningitis, although these cytokines have been shown to be regulated by C/EBPδ during LPS-induced models of acute lung injury [
22] and endotoxemia [
25]. We currently have no proper explanation for these seeming contradictive results, although the role of C/EBPδ in the inflammatory response might strongly depend on the underlying disease.
Our results show that the C/EPBδ-mediated inflammatory response is not a major mechanism explaining poor outcome in pneumococcal meningitis. We did not find differences in survival nor clinical parameters between wild-type and C/EBPδ
−/− mice. Apparently, the lower bacterial burden in combination with the lower inflammatory response in C/EBPδ
−/− did not affect clinical parameters of meningitis. In a previous study, reduced bacterial loads did correlate with improved survival during
S. pneumoniae-induced meningitis. Formyl peptide receptor (FPR)-1 or -2 deficient mice had higher bacterial titers than wild-type mice after pneumococcal meningitis, and wild-type mice lived significantly longer than both knockout strains [
26]. Although we do not have a clear explanation for these different results at the moment, gender differences (male mice in the FPR study and female mice in our study) and/or used pathogens (type 2
vs. type 3
S. pneumoniae in the FPF and our study, respectively) may explain at least part of the observed differences. A possible explanation for the discrepancy between clinical outcome and decreased bacterial burden and lower inflammatory response observed in this study could be that C/EBPδ also plays a crucial role in neuroprotective processes. Indeed, levels of IL-10, an important anti-inflammatory mediator with a protective function in pneumococcal meningitis [
7], are significantly decreased in C/EBPδ
−/− mice as compared to wild-type mice. Although reduced IL-10 levels may thus explain the lack of effect on clinical parameters, alternative, currently unknown, neuroprotective properties of C/EBPδ in the brain cannot be excluded.
Our study has several limitations. First, we only studied a single dose of a single serotype pneumococcus. As the virulence of different pneumococcal serotypes varies widely, experimental meningitis due to other serotypes may yield different results. Furthermore, using different inoculum sizes may show more subtle differences. However, our model uses the most common serotype found in patients with pneumococcal meningitis, and the used inoculation size results in a mortality similar to that observed in patients [
17,
4]. Therefore, we feel that our model provides sufficient information to conclude there is a lack of effect on survival of C/EBPδ. Second, we did not treat our mice with antibiotic therapy, and therefore our model does not reflect the clinical situation. However, the aim of our study was to further study the role of C/EBPδ in the pathophysiology of pneumococcal meningitis, which is best evaluated in untreated models.
In conclusion, we show that C/EBPδ expression increases in the brain of mice during meningitis and that C/EBPδ−/− expression contributes to bacterial growth and/or dissemination and induces the inflammatory response. C/EBPδ expression did not affect clinical parameters nor did it change survival suggesting that C/EBPδ plays complex role in S. pneumoniae-induced meningitis.
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Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
The work presented here was carried out in collaboration between all authors. Experimental procedures were carried out by MVS and JWD with technical assistance of SRH. Histological assessment of slides was performed by JYL and MG. The manuscript was drafted by MVS and JWD and discussed and edited by MCB, DvdB, and CAS. All authors have read and approved the final version of the manuscript.