Acute bacterial meningitis (BM) is a severe and potentially life-threatening infection of the central nervous system (CNS). Currently, there is no specific treatment to prevent or reverse hippocampal damage caused by BM. Streptococcus pneumoniae is the leading etiological agent of community-acquired BM in children under 5 years old, and the elderly worldwide. Despite the availability of effective antibiotics, pneumococcal meningitis (PM) still carries significant morbidity and mortality rates (1). One of the most critical complications of BM is hippocampal damage, which can lead to severe cognitive deficits in survivors. The outcome of PM depends on complex interactions between the bacterial pathogenicity factors and the host response, including the activation of various immune signaling pathways (2).

Recent studies have shown that epigenetic modifications play a critical role in regulating the host response to PM. Epigenetic modifications refer to changes in gene expression mediated by non-coding RNA, DNA methylation, and histone modifications that do not involve alterations in the DNA sequence. Specifically, it has been demonstrated that global changes in the miRNome (3) and a decrease in global DNA methylation (4) occur in the hippocampus during PM. These alterations can impact genome stability and the expression of genes associated with neuroinflammation, oxidative stress, and the equilibrium between neurogenesis and neuronal death. As a result, epigenetic mechanisms can influence the infection’s outcome and contribute to the development of cognitive deficits in survivors. No prior research has explored the role of histone markings in modulating the host response to BM. Understanding the epigenetic regulation of the host response to PM has significant implications for the development of new therapies. Targeting specific epigenetic modifications may provide a novel approach to modulating the host response to infection, improving patient outcomes, and reducing the risk of long-term sequelae.

We have previously demonstrated that vitamin B12, a cofactor of the enzyme methionine synthase in the sulfur amino acid pathway, acts as an epidrug by increasing the production of S-adenosyl-methionine, the universal donor of methyl, in the hippocampus of infant rats with PM. By this mechanism, adjuvant therapy with B12 down-regulated the inflammatory genes Interleukin-1 beta (Il1b), C-C Motif Chemokine Receptor 2 (Ccr2) and C-C Motif Chemokine Ligand 3 (Ccl3) and largely attenuated the apoptotic death of progenitor cells and postmitotic neurons in the hippocampus dentate gyrus (4). Here, the safe profile of vitamin B12 and its positive effect on the hippocampal transcriptome of infant rats with PM are described, and evidence is provided that B12 also modulates methyl-dependent histone modifications.

The pathophysiology of PM involves a dynamic interplay between bacterial virulence factors and the host immune response, resulting in neuroinflammation and, ultimately, neurological dysfunction. The presence of the bacteria in the CSF initiates an inflammatory response, primarily mediated by microglia and perivascular and meningeal macrophages, which subsequently release inflammatory mediators such as cytokines and chemokines. These proinflammatory factors promote further inflammation and oxidative stress, leading to the disruption of the BBB, the pleocytosis of peripheral leukocytes, and the influx of excitatory amino acids into the CNS (2). This cascade of events triggers apoptosis in both progenitor cells and postmitotic neurons, which are scattered throughout the inner granular layer of the dentate gyrus in the hippocampus (16). This detrimental condition causes extensive damage to the hippocampal formation, a brain region which plays a crucial role in the consolidation of new memories and spatial navigation. Hippocampal apoptosis was associated with abnormal neuropsychological test results in animals after PM (17, 18). Besides, studies have shown that patients who have survive PM often experience long-term cognitive deficits, including problems with memory, attention, executive function, and cognitive performance (2, 19–21). Currently, PM is treated with antibiotics and corticosteroids, but these treatments do not prevent hippocampal damage (17, 18).

B12 vitamers are essential cofactors for key enzymes in energy metabolism and the sulfur amino acid pathway. They also play a beneficial role in neuroinflammatory conditions by scavenging reactive oxygen species (ROS), maintaining appropriate H₂S levels, preventing the harmful effects of excess homocysteine, and reducing N-methyl-D-aspartate (NMDA) receptor-mediated excitotoxicity in CNS cells. Furthermore, B12 modulates chromatin remodeling mechanisms that down-regulate critical genes in neuroinflammation (22). It has been previously reported that adjuvant therapy with vitamin B12 mitigates apoptotic damage to the hippocampus and down-regulates some key inflammatory genes, namely Il1b, Ccr2, and Ccl3, in infant rats with PM (4). Specifically, vitamin B12 has been found to down-regulate the expression of Ccl3 by enhancing the methylation of specific cytosine residues in its promoter region. Despite the fact that therapeutic administration of B12 has been shown to increase global DNA methylation in the hippocampus of healthy infant rats (4), the present study demonstrated that in the absence of infection, the vitamin regulates only a limited number of genes, which are expected to have an overall positive effect on hippocampal function ( Additional File 1 ). Therefore, the findings of the present study provide support for the expected safe profile of B12.

PM is known to broadly modify the hippocampal transcriptome, leading to the activation of several pathways functionally related to the major components of the physiopathology of this disease, including microglia activation, oxidative stress, BBB disruption, leukocyte extravasation into the CNS, neuroinflammation, and cell death, among others. Furthermore, PM also inhibits some anti-inflammatory and cell survival pathways (23, 24). The findings of the present study support and improve this previous knowledge. Of particular interest in the scope of this study is the outcome of adjuvant therapy with B12 in the canonical pathways most affected by PM, as the vitamin effectively reversed these disease effects ( Figure 2B , Additional File 3 ).

Pivotal canonical pathways involved in pneumococci detection in the CNS and signaling to trigger the innate immune response, namely Role of PRRs in Recognition of Bacteria and Viruses, Toll-like Receptor signaling, Inflammasome pathway, NF-kB Signaling, and TREM1 Signaling, are activated by PM, and inhibited by B12 ( Figure 2B , Additional File 3 ).

The first line of defense against invading pneumococci in the CNS is constituted by the innate immune system, which is activated by the recognition of conserved microbial structures or pathogen-associated molecular patterns (PAMPs) by antigen presenting cells (APC) expressing pattern-recognition receptors (PRRs) (25). Among these APCs there are blood cells, which are predominantly found in the meninges, choroid plexus, and perivascular space, as well as astrocytes and microglial cells that reside within the brain parenchyma (26). Transmembrane Toll-like receptors (TLRs) 2, 4, and 9, and cytosolic Nucleotide-Binding Oligomerization Domain 2 (NOD2) receptors are critical PRRs responsible for APC detection of S. pneumoniae in the CNS (27–29). All TLR as well as NOD2 signaling pathways ultimately result in the activation of Nuclear Factor Kappa B (NF-kB) (28, 30). This transcriptional factor activates several genes that contribute to the pathophysiology of BM, such as IL-1β, Tumor Necrosis Factor (TNF)-α, Interleukin 6 (IL-6), Interleukin 8 (IL-8), Macrophage Inflammatory Protein-1 Alpha (MIP-1α), inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX)-2, and Intercellular Adhesion Molecule-1 (ICAM-1) (31). Additionally, the Triggering Receptor Expressed On Myeloid Cells (TREM) protein receptor family is emerging as a crucial regulator of various cellular functions, including inflammation amplification. Evidence indicates that activation of TREM-1 through danger- and pathogen-associated molecular patterns (DAMPs and PAMPs) can lead to the production of cytokines (32). TREM-1 signaling has already been associated with host defense during the early stages of infection with highly pathogenic Streptococcus suis (33). Inflammasome-mediated recognition of the pneumococcus also contributes to the host innate immune response. Indeed, the NALP3 inflammasome regulates Caspase-1 activation and subsequent secretion of both IL-1β and Interleukin 18 (IL-18) (34). NK cells stimulated by IL-18 produce the pro-inflammatory cytokine Interferon Gamma (IFNG), which is a pivotal driver of neuropathology and behavioral sequelae in experimental PM. IFNG modulates a range of processes, including myeloid recruitment and activation, as well as inhibition of bacterial clearance (35). Although the levels of IFNG are elevated in both the CSF of patients with PM (36, 37) and in the brain tissue of rats (38) with the disease, the exact role of this cytokine in PM remains unclear. The present study identified IFNG as the upstream regulator of several critical genes regulated by PM. It is noteworthy that B12 significantly mitigated the impact of the infection on these IFNG-regulated genes. Furthermore, B12 counteracted the PM-induced alterations in the expression of multiple genes regulated by other predicted upstream regulatory molecules ( Figure 2C ).

In addition to counteracting the effects of PM in the initial processes of pathogen recognition and innate immunity activation, B12 also inhibits disease effects in canonical pathways that directly regulate the major component of neuroinflammation (Neuroinflammation Signaling Pathway, IL6 Signaling, Integrin Signaling, and Leukocyte Extravasation Signaling, Production of NO and ROS in macrophages, and Fcy Receptor-mediated Phagocytosis in Macrophages and Monocytes) ( Figure 2B , Additional File 3 ). Neuroinflammation is defined as an inflammatory response within the brain or spinal cord (39). This response is mediated by the production of cytokines, chemokines, ROS, reactive nitrogen species (RNS), and secondary messengers produced by microglia, astrocytes, endothelial cells, and peripherally derived immune cells.

The activation of NF-kB through PRRs stimulates the production of early-phase cytokines, IL-1β, TNF, and IL-6 (40, 41). These cytokines induce the up-regulation of several adhesion factors on the vascular endothelium, leading to the influx of leukocytes, primarily neutrophils, into the infected site, mainly within the first few hours of infection (42–45). However, in PM, it appears that IL-6 is not directly associated with promoting pleocytosis in the CSF but rather with brain edema, BBB disruption, and increased intracranial pressure (46). Leukocytes cross the BBB by binding to selectins and rolling across the endothelium (45). Integrins up-regulated on the vascular endothelium facilitate the binding of leukocytes and subsequent BBB migration (26).

During PM, endothelial cells and neutrophils produce RNS such as nitric oxide (NO), catalyzed by endothelial nitric oxide synthase (eNOS) and iNOS, respectively (47, 48). Neuronal nitric oxide synthase (nNOS) appears to play a minor role in BM (49). Moreover, in neutrophils, macrophages, and endothelial cells, NADPH oxidase induces the production of ROS, such as superoxide (O₂ ^-), in response to infection (50). Although neutrophils produce a higher amount of ROS compared to macrophages, the latter cells produce a greater quantity of RNS than the former (51). S. pneumoniae generates hydrogen peroxide, which reacts with NO to produce peroxynitrite (ONOO^-). This highly reactive compound can cause lipid peroxidation and destabilization of cell membranes, DNA damage, and subsequent activation of the DNA repair enzyme poly (ADP-ribose) polymerase (PARP) leading to cellular energy collapse and death (48, 50). Moreover, ROS and RNS have been identified as mediators of BBB breakdown (52).

Phagocytosis is a host cell endocytic response to particulate matter like bacteria. The process of phagocytosis is complex and comprises several events like particle binding, receptor clustering, actin nucleation, pseudopod extension, membrane recycling, and phagosome closure. The Fc gamma receptors (FcγR; subtypes FcγR1A, FcγRIIA and FcγRIIIA) of the immunoglobulin superfamily are the best-characterized receptors for phagocytosis in avidly phagocytic cells of the hematopoietic lineage, like macrophages, neutrophils, and microglia (53, 54).

The findings discussed above are in line with the results of a previous study in which B12 was shown to down-regulate proinflammatory genes that are up-regulated by PM by promoting the hypermethylation of their promoter regions (4). However, B12 also activated the anti-inflammatory canonical pathways PPAR Signaling and LXR/RXR Activation, which are inhibited by PM.

The peroxisome proliferator-activated receptors (PPARs) are a group of nuclear receptor proteins that regulate the transcription of genes involved in energy production, lipid metabolism, and inflammation. The PPAR family includes PPARα and PPARγ. Upon activation by their ligands, these two receptors down-regulate the production of pro inflammatory cytokines such as TNF, IL-6, and Il-1β. In the case of PPARα, these effects are due to its capacity to inhibit NF-κB signaling pathway (55).

The Liver X receptors (LXRs) are a group of nuclear receptor proteins that play a significant role in the regulation of lipid metabolism and cholesterol homeostasis, including the conversion of cholesterol to bile acids. LXRs have also been shown to modulate immune and inflammatory responses, particularly in macrophages. LXRs act as ligand-dependent transcription factors that form heterodimers with the retinoid X receptor (RXR), which then bind to LXR-responsive elements (LXREs) in DNA to promote the expression of target genes. Interestingly, ligand activation of LXRs not only activates transcription of target genes, but also inhibits transcription from promoters of certain genes of proinflammatory cytokines that do not contain LXREs, a phenomenon referred to as trans-repression (56). Due to their cholesterol sensing and anti-inflammatory activities, LXRs are considered as integrators of metabolic and inflammatory signaling (57).

The anticipated anti-neuroinflammatory and neuroprotective effects of B12, as predicted through transcriptome analysis in infected animals treated with the vitamin, were successfully confirmed at the histological level ( Figures 4 and 1 , respectively). The anti-inflammatory effect of B12 is further supported by the deconvolution analysis of the RNA-Seq data, suggesting that B12 reduces the infiltration of neutrophils into the CNS in infected animals. These computational findings also align with prior research, which demonstrated that PM triggers the infiltration of neutrophils, and to a lesser extent, monocytes, into the CNS (58). The suggested increase in the B cell fraction within the inflammatory infiltrate in the CNS due to B12, regardless of the infection, warrants further investigation. It is important to note that these results should be interpreted cautiously since the reference dataset does not include microglial cells.

In infected animals, adjuvant therapy with B12 leads to the up-regulation of another interesting gene, Npas4 ( Supplementary Material 2 , Figure 3 ). Npas4 is a transcription factor with pronounced expression in the brain, including the hippocampus, where it plays a pivotal role in regulating the formation and maintenance of inhibitory synapses in response to excitatory synaptic activity (59, 60). By this mechanism, Npas4 may act as a neuroprotective factor in the context of PM, where the influx of excitatory amino acids into the CNS can induce neuronal excitotoxicity (61). A previous work demonstrated that administering B12 to infant rats with PM increases methyl bioavailability and DNA methylation in the hippocampus (4). However, the observed expression pattern of Npas4, as further validated by RT-qPCR ( Figure 3 ), cannot be attributed to an increase in its promoter methylation induced by B12. To explore potential explanations for the positive regulation of genes like Npas4 by B12, histone markings were assessed by immunohistochemistry in hippocampal sections. The up-regulation of Npas4 in PM animals treated with B12 indeed correlates with the expected effect of the increased epigenetic mark H3k4me4 observed within the granular layer of the hippocampal dentate gyrus of animals in this particular group ( Figure 5 ). H3k4me3 is associated with the active transcriptional state of chromatin, while H3k9me3 is linked to transcriptionally inactive heterochromatin (62). Thus, the results presented herein suggest that changes in histone methylation patterns may work in concert with DNA methylation, ultimately contributing to a positive balance in protecting progenitor cells and postmitotic neurons in the hippocampal dentate gyrus during PM, as depicted in Figure 1 .

It is conceivable that the favorable impact of therapeutic vitamin B12 in PM may also be partially mediated by its direct effects in counteracting oxidative stress, preventing excitotoxicity, and other related mechanisms. This study was limited by its sole focus on exploring the impact of the vitamin on epigenetic and transcriptional regulation.

In conclusion, adjuvant therapy with B12 modulates the hippocampal transcriptional signature induced by PM in a way that is consistent with the mitigation of several aspects of the innate immune response activated by the disease. These aspects include the recognition of pathogens by immune system cells, signaling via NF-kB, production of pro-inflammatory cytokines, migration of peripheral leukocytes into the CNS, and production of ROS and RNS. B12 also activates anti-inflammatory pathways inhibited by PM. Consequently, B12 attenuates neuroinflammation and apoptotic cells death in the hippocampal dentate gyrus. The effects of B12 at the transcriptional level are mediated not only through the previously demonstrated DNA hypermethylation but also by alterations in histone methylation patterns. Notably, no adverse effects of B12 were predicted or observed, reinforcing the well-known safety profile of this epidrug. These findings support the proposition of a clinical trial to evaluate the potential of B12 as an adjuvant therapy to mitigate hippocampal damage associated with neuroinflammation in PM.

The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found below: https://www.ncbi.nlm.nih.gov/sra/PRJNA966183.

The animal study was approved by Ethics Committee on the Care and Use of Laboratory Animals (CEUA-FIOCRUZ, protocol LW-23/17). The study was conducted in accordance with the local legislation and institutional requirements.

LC - performed most of the experiments, analyzed and interpreted data, and wrote the manuscript. MO – contributed to the experiments, analyzed and interpreted data, and critically reviewed the manuscript. KQ - contributed to the experimental infection and immunohistochemistry, and reviewed the manuscript. AA - contributed to the immunohistochemistry, and reviewed the manuscript. AS - made substantial contributions to the NGS experiments; GF - made substantial contributions to the Bioinformatic analysis, and reviewed the manuscript. CC - made substantial contributions to the histological and immunohistochemical analysis, analyzed and interpreted data, and critically reviewed the manuscript. RC - designed and oversaw the study, analyzed and interpreted data, and wrote the manuscript. All authors contributed to the article and approved the submitted version.