The aim of this work was to elucidate the effect of T. gondii infection on the metabolism of mouse spleen, a key organ in regulating immune response against T. gondii infection. LC-MS/MS-based metabolomics and multivariate statistical analyses revealed new metabolic changes in the spleen of T. gondii-infected mice compared with uninfected mice and identified metabolic signatures that differentiated between the acute infection and chronic infection.
Stage-specific metabolic signatures
A total of 389 differential ions were screened based on using VIP scores (VIP > 1) of individual metabolites obtained from the PLS-DA model. We succeeded to identify 259 of the 389 differential ions, which highlights the challenges associated with the identification of metabolites. We used heatmaps to present the results of these differential ions between different mouse groups. The heatmaps showed a clear difference between acutely infected group and the control group (Fig. 3c, Additional file 2: Figure S2c), but chronically infected group was not fully distinct from the control group (Fig. 3d, Additional file 2: Figure S2d). In the positive and negative modes, two mice in the chronically infected group were clustered with mice in the control group, which could be attributed to inter-individual variations among chronically infected mouse group. Also, converging metabolic responses between these two chronically infected and control mice is probably due to homeostatic recovery that might have occurred as infection progressed to the chronic phase. Interestingly, we identified 132 significantly altered metabolites, 23 from acutely infected vs control and 109 from chronically infected vs control mice (Additional file 4: Table S2, Additional file 5: Table S3), suggesting that as infection progressed the number of differential metabolites increased. This result disagrees with previous metabolomic profiling of serum [12] and brain [13] of T. gondii-infected mice where the most predominant metabolic changes, compared with control mice, happened at an early stage of infection. These distinct temporal metabolic patterns between metabolomics studies can be attributed to organ-specific metabolomics organization (i.e. different repertoire of small molecules present in different organs). Also, the metabolomic response to infection of a major peripheral immune organ such as spleen, is expected to be different from that of the serum [12] or brain [13].
There were 12 shared differential metabolites between the acute and chronic infection phases (Table 2, Fig. 4). 4,4-Dimethyl-5alpha-cholesta-8,14,24-trien-3beta-ol was significantly upregulated and ubiquinone-8 was significantly downregulated, suggesting that these two metabolites might play important roles during T. gondii infection. The upregulation of 4,4-Dimethyl-5alpha-cholesta-8,14,24-trien-3beta-ol during both acute and chronic phases might be related to the regulatory effect of this molecule on host cell meiosis [24]. Expression patterns of AA were distinct between the acute and chronic infection phases (i.e. infection stage-specific). As shown in Additional file 4: Table S2 and Additional file 5: Table S3, in the two infection phases, most of the identified differential metabolites were lipids, which are essential for the biogenesis of cell and parasite membranes in order to ensure parasite’s survival and replication within host cells [25].
We chose spleen in this study because of its importance in immune surveillance during infection. Spleen comprises two morphologically and functionally distinct regions (red pulp and white pulp) and contains multiple subsets of specialized myeloid and dendritic cells. However, a major drawback of splenic tissue is its complex and dynamic cellular heterogeneity especially in response to infection. In the present study, T. gondii infection induced spatial alterations in the composition of splenic cellular populations and splenomegaly was a relatively sub-fatal complication of infection. These changes could have a profound effect on the metabolome of this tissue and consequently make it hard to dissociate the direct effect of infection on metabolite expression patterns from the contribution of the anatomical abnormalities associated with infection. Therefore, it is possible that changes we observed in the levels of metabolites is a consequence of the differential contributions of cellular populations of spleen from acute, chronic and control mice rather than the result of actual changes in the metabolic activity in spleen cells triggered by T. gondii infection per se. Precise mechanisms, however, remain unclear and the specific contributions of the metabolites of the red and white pulps to the alterations in the levels of metabolites observed during T. gondii infection remains to be clarified.
The impact on host defense mechanisms
In the acute phase, the level of corticosterone was markedly increased with log2 fold change of 3.049. Hormones are very important signaling molecules in mammals and are fundamental for their metabolic and immune homeostasis [26]. Corticosterone is vital in the metabolism of carbohydrates, fatty acids and amino acids. The markedly altered corticosterone might reflect variations in the energy metabolism in the spleen of infected mice, which might be triggered by illness-related anorexia. Ubiquinol of the oxidative phosphorylation pathway was probably upregulated to supply energy needed by the body to balance its metabolic status during the latent phase of infection. The anti-fungal molecule gambieric acids A [27] and the antibiotic molecules, neamine and difloxacin (INN), were also significantly upregulated. Additionally, AA, Phorbol and (5Z,7E,9E,14Z,17Z)-Eicosapentaenoate, which act as inflammatory mediators, were upregulated. It is likely that spleen defends against T. gondii infection not only by specific immune processes, but also by eliciting different metabolic reactions as part of the innate immunity to limit the infection.
Toxoplasma gondii infection disturbs eicosanoid metabolism
The altered metabolic pathways identified involved primary bile acid biosynthesis, steroid biosynthesis and arachidonic acid metabolism. Steroid hormones are involved in a variety of physiological processes; relevant to T. gondii pathogenesis are the immunoregulatory and anti-inflammatory effects of steroids, which can influence the host immune responses to infection [28]. Our results also revealed AA metabolism, the main precursor of eicosanoid hormones, as the most significantly affected metabolic pathway by T. gondii during acute and chronic infection. At 11 dpi, the level of AA decreased, indicating down-regulation of AA metabolism during acute infection. By contrast, at 30 dpi the level of AA was significantly upregulated in the chronic phase (Fig. 5c). The major inflammatory mediators, LTA4, 14,15-HETE, and 15-deoxy-Δ12,14-PGJ2 [29,30,31], were also up-regulated. In response to an inflammatory stimulus, AA, the main polyunsaturated fatty acid present in the phospholipid of cell membranes, is released and metabolized to a series of eicosanoids, including the inflammatory leukotrienes and prostanoids (e.g. prostaglandins, prostacyclins and thromboxanes) [32]. AA and its eicosanoid metabolites play an important role in the regulation of many cellular processes, such as cell survival, angiogenesis, chemotaxis, mitogenesis, apoptosis and migration [33, 34]. Elevated level of AA in the supernatant of T. gondii-infected cultured J774A.1 cells was assumed to be triggered by increased Phospholipase A production in order to release AA via decomposing the host cell membrane phospholipids, thus promoting the parasites invasion by increasing the host cell membrane permeability and fluidity [35]. Likewise, T. gondii was shown to increase AA concentration and agglutination of microfilaments in phagocytic host cells to accelerate the parasite’s invasion [35].