Serine Metabolism Tunes Immune Responses To Promote Oreochromis niloticus Survival upon Edwardsiella tarda Infection

Abstract

Overactive immune response is a critical factor triggering host death upon bacterial infection. However, the mechanism behind the regulation of excessive immune responses is still largely unknown, and the corresponding control and preventive measures are still to be explored. In this study, we find that Nile tilapia, Oreochromis niloticus, that died from Edwardsiella tarda infection had higher levels of immune responses than those that survived. Such immune responses are strongly associated with metabolism that was altered at 6 h postinfection. By gas chromatography-mass spectrometry-based metabolome profiling, we identify glycine, serine, and threonine metabolism as the top three of the most impacted pathways, which were not properly activated in the fish that died. Serine is one of the crucial biomarkers. Exogenous serine can promote O. niloticus survival both as a prophylactic and therapeutic upon E. tarda infection. Our further analysis revealed exogenous serine flux into the glycine, serine, and threonine metabolism and, more importantly, the glutathione metabolism via glycine. The increased glutathione synthesis could downregulate reactive oxygen species. Therefore, these data together suggest that metabolic modulation of immune responses is a potential preventive strategy to control overactive immune responses. IMPORTANCE Bacterial virulence factors are not the only factors responsible for host death. Overactive immune responses, such as cytokine storm, contribute to tissue injury that results in organ failure and ultimately the death of the host. Despite the recent development of anti-inflammation strategies, the way to tune immune responses to an appropriate level is still lacking. We propose that metabolic modulation is a promising approach in tuning immune responses. We find that the metabolomic shift at as early as 6 h postinfection can be predictive of the consequences of infection. Serine is a crucial biomarker whose administration can promote host survival upon bacterial infection either in a prophylactic or therapeutic way. Further analysis demonstrated that exogenous serine promotes the synthesis of glutathione, which downregulates reactive oxygen species to dampen immune responses. Our study exemplifies that the metabolite(s) is a potential therapeutic reagent for overactive immune response during bacterial infection.

Keywords: glutathione; metabolomics; overactive immune response; reactive oxygen species; serine.

FIG 1

Differential immune response of O. niloticus is associated with the consequence of E. tarda infection. (A) Determination of the lethal dose of E. tarda to O. niloticus. O. niloticus fish were infected with either 0.8 × 10⁸, 1.6 × 10⁸, or 3.2 × 10⁸ CFU/fish. Cumulative fish death was monitored for a total of 14 days. (B) qRT-PCR to quantify cytokine expression in head kidneys of control, dying, or surviving fish 48 h postinfection. All of the statistical analyses were performed with Mann-Whitney U test unless otherwise indicated. *, P < 0.05; **, P < 0.01. Error bars represent means ± standard errors of the means (SEM) from at least three biological replicates. All of the experiments were repeated at least three times.

FIG 2

Metabolic shift upon E. tarda infection. (A) Heat map showing relative abundance of metabolites (Wilcoxon P < 0.01) in 5S, 1D, and 1S groups. Scale is shown at bottom, where blue to yellow represents low to high abundance. (B) Z-score plot of differential metabolites based on 5S. Z-score varied between −5.82 and 10.64 for 1S to 5S. Each point represents one metabolite in one technical repeat and is colored by sample types. (C) Z-score plot of differential metabolites based on 5S. Z-score varied between −7.85 and 9.71 for 1D to 5S. Each point represents one metabolite in one technical repeat and is colored by sample type.

FIG 3

Pathway enrichment analysis of differential metabolites. The metabolites of differential abundance were selected and analyzed in MetaboAnalyst to enrich pathways. Nine pathways that had significant difference (P < 0.05) were enriched and sorted by their weights (impact).

FIG 4

Serine was a crucial biomarker. (A) Principal-component analysis of 1D, 1S, and 5S groups. Each dot represented one technical replicate. (B) The model was monitored using permutation tests. (C) The distribution of differential abundance of metabolites’ weight from PLS-DA to samples. Triangles represent metabolites. Potential biomarkers are highlighted in red. (D) Abundance of serine in 1D, 1S, and 5S. Statistical analysis was performed with Mann-Whitney U test. *, P < 0.05; **, P < 0.01. Error bars represented means ± SEM from at least three biological replicates.

FIG 5

Prophylactic effect and therapeutic effects of serine on O. niloticus mortality and E. tarda infection. (A) Percent survival of O. niloticus challenged with E. tarda after serine treatment at the indicated dose. (B) Percent survival of O. niloticus challenged with E. tarda, which received serine treatment at 1, 4, 10, and 20 h postinfection. The mortality was monitored for a total of 14 days, and only 7 days are shown here. All of the experiments were repeated at least three times.

FIG 6

Metabolomic analysis of macrophages/monocytes after serine treatment. (A) Metabolic pathway enrichment analysis of O. niloticus treated with serine. (B) Abundance of metabolites in control and serine treatment.

FIG 7

Serine promotes O. niloticus survival through glutathione synthesis. (A) Quantification of gene expression in control, dying, and surviving groups. (B) Quantification of gene expression in serine-treated group. (C) Quantification of glutathione in control, dying, and surviving groups. (D) Quantification of ROS in control, dying, and survival groups. Statistical analysis was performed with Mann-Whitney U test. *, P < 0.05; **, P < 0.01. Error bars represented means ± SEM from at least three biological replicates. All of the experiments were repeated at least three times.

FIG 8

Glutathione negatively regulates ROS production and downregulates immune response. (A) Glutathione when serine served as the prophylactic metabolite in the control group, serine only group, APR-246 only group, and serine plus APR-246 group. (B) ROS when serine served as prophylactic metabolites in control group, serine only group, APR-246 only group, and serine plus APR-246 group. (C) Glutathione when serines served as therapeutic metabolites in control group, survival group, and serine only group. (D) ROS when serines served as therapeutic metabolites in control group, survival group, and serine only group. (E) Quantification of immune gene expression by qRT-PCR. Statistical analysis was performed with Mann-Whitney U test. *, P < 0.05; **, P < 0.01. Error bars represented means ± SEM from at least three biological replicates. All of the experiments were repeated at least three times.

FIG 9

Proposed model. E. tarda infection causes tilapia death through virulence factors and septic shock-associated oxidative stress. The oxidative stress could be relieved by serine, which fluxes into the glutathione metabolism to reduce ROS production. Glutathione antagonized the ROS, thereby protecting host death from overactive immune responses like ROS.