Adjunctive dexamethasone affects the expression of genes related to inflammation, neurogenesis and apoptosis in infant rat pneumococcal meningitis

Abstract

Streptococcus pneumoniae is the most common pathogen causing non-epidemic bacterial meningitis worldwide. The immune response and inflammatory processes contribute to the pathophysiology. Hence, the anti-inflammatory dexamethasone is advocated as adjuvant treatment although its clinical efficacy remains a question at issue. In experimental models of pneumococcal meningitis, dexamethasone increased neuronal damage in the dentate gyrus. Here, we investigated expressional changes in the hippocampus and cortex at 72 h after infection when dexamethasone was given to infant rats with pneumococcal meningitis. Nursing Wistar rats were intracisternally infected with Streptococcus pneumoniae to induce experimental meningitis or were sham-infected with pyrogen-free saline. Besides antibiotics, animals were either treated with dexamethasone or saline. Expressional changes were assessed by the use of GeneChip® Rat Exon 1.0 ST Arrays and quantitative real-time PCR. Protein levels of brain-derived neurotrophic factor, cytokines and chemokines were evaluated in immunoassays using Luminex xMAP® technology. In infected animals, 213 and 264 genes were significantly regulated by dexamethasone in the hippocampus and cortex respectively. Separately for the cortex and the hippocampus, Gene Ontology analysis identified clusters of biological processes which were assigned to the predefined categories "inflammation", "growth", "apoptosis" and others. Dexamethasone affected the expression of genes and protein levels of chemokines reflecting diminished activation of microglia. Dexamethasone-induced changes of genes related to apoptosis suggest the downregulation of the Akt-survival pathway and the induction of caspase-independent apoptosis. Signalling of pro-neurogenic pathways such as transforming growth factor pathway was reduced by dexamethasone resulting in a lack of pro-survival triggers. The anti-inflammatory properties of dexamethasone were observed on gene and protein level in experimental pneumococcal meningitis. Further dexamethasone-induced expressional changes reflect an increase of pro-apoptotic signals and a decrease of pro-neurogenic processes. The findings may help to identify potential mechanisms leading to apoptosis by dexamethasone in experimental pneumococcal meningitis.

Figure 1. Workflow of the gene expression analysis of the microarray data.

Square textboxes represent the gene lists at the different steps of the workflow and round-edged textboxes represent the different steps performed for data analysis. RMA = Robust Multi-array Average, FU = fluorescence units, id = identifier.

Figure 2. The numbers of significantly regulated genes by comparing the different treatment groups.

C = control animals, D = dexamethasone treatment, S = saline treatment, I = infected animals.

Figure 3. Venn diagrams showing the relation of the number of genes of defined comparisons.

The numbers in the left or right part of the circles represent the number of regulated genes in the given comparison. The number in the intersection of both circles represents the number of genes regulated in both comparisons. The influence of the treatment in infected vs. control animals within the hippocampus (A) and cortex (B) is shown. The intersection shows the number of genes which are regulated by the treatment irrespective of the infection. The influence of the infection in dexamethasone vs. saline treated animals within the hippocampus (C) and cortex (D) is shown. The intersection shows the number of genes regulated by the infection irrespective of the treatment. I = infected animals, D = dexamethasone treatment, S = saline treatment, C = control animals.

Figure 4. The correspondence analysis (COA) of the microarray dataset.

The COA separated in component one (x axis in A and B) the control from the infected animals, in component two (y axis in A) the hippocampus from the cortex samples and in component three (y axis in B) the dexamethasone treated from the saline treated animals. Each square represents an array. H = hippocampus, C = cortex. Equal numbers in the hippocampus and cortex group indicates that these samples derived from the same animal.

Figure 5. Gene Ontology (GO) analysis of biological processes.

Infected and dexamethasone-treated animals were compared with infected and saline-treated animals (ID vs. IS) in the hippocampus (A) and the cortex (B). GO clusters of biological processes were generated by the functional annotation clustering tool of the Database for Annotation, Visualization, and Integrated Discovery (DAVID). These clusters were then assigned to six defined categories.

Figure 6. Gene expression pattern of the quantitative real-time PCR analysis in the hippocampus.

The three experimental groups were compared to control and saline-treated animals. These 14 genes were significantly regulated when comparing infected and dexamethasone-treated animals with infected and saline-treated animals (ID vs. IS). C = control animals, D = dexamethasone treatment, I = infected animals, S = saline treatment.

Figure 7. Western blot and microarray assessment of tyrosine phosphatase, non-receptor type 6 (PTPN6).

The western blot analysis showed increased protein levels in hippocampus and cortex homogenates of infected animals compared to control samples (A). This finding confirms the results of the microarray experiment (B). I = infected animals, S = saline treatment, D = dexamethasone treatment, C = control animals.