Macrophages rely on extracellular serine to suppress aberrant cytokine production

A growing body of evidence indicates that cellular metabolism is involved in immune cell functions, including cytokine production. Serine is a nutritionally non-essential amino acid that can be generated by de novo synthesis and conversion from glycine. Serine contributes to various cellular responses, but the role in inflammatory responses remains poorly understood. Here, we show that macrophages rely on extracellular serine to suppress aberrant cytokine production. Depleting serine from the culture media reduced the cellular serine content in macrophages markedly, suggesting that macrophages depend largely on extracellular serine rather than cellular synthesis. Under serine deprivation, macrophages stimulated with lipopolysaccharide showed aberrant cytokine expression patterns, including a marked reduction of anti-inflammatory interleukin-10 expression and sustained expression of interleukine-6. Transcriptomic and metabolomics analyses revealed that serine deprivation causes mitochondrial dysfunction: reduction in the pyruvate content, the NADH/NAD+ ratio, the oxygen consumption rate, and the mitochondrial production of reactive oxygen species (ROS). We also found the role of mitochondrial ROS in appropriate cytokine production. Thus, our results indicate that cytokine production in macrophages is tightly regulated by the nutritional microenvironment.

Statistical analysis was performed with unpaired t-test. All primers are available in Table S3 Hokkaido System Science Co.,Ltd.

Primers for Q-PCR used in murine samples
Atf4   . 1

) Effect of culture with the serine-and glycine-depleted medium on cellular amino acid content in peritoneal macrophages
Cellular content of amino acids in periMΦs analyzed by CE-TOF MS. PeriMΦs were cultured in Full or ∆SG medium for 24 h, followed by stimulation with LPS (100 ng/ml) for 6 h (n = 3). Cellular contents of serine and glycine are shown in Fig. 1g.
Values are means ± 95% CI. Statistical analysis was performed with unpaired t-test. *p < 0.05; **p < 0.01 Related to Fig. 1.     . 2) Effect of serine and glycine deprivation on cytokine production in macrophages (a) Production of cytokines in BMDMs. BMDMs were cultured in Full or ∆SG medium for 24 h, followed by stimulation with LPS (100 ng/ml) for 24 h (n = 4-6).

(b,c) Gene expression of cytokines in periMΦs (c) and BMDMs (d).
PeriMΦs and BMDMs were cultured in Full or ∆SG medium for 24 h, followed by stimulation with LPS (100 ng/ml) for 8 h and 24 h, respectively (n = 3-4).
(d) Gene expression of cytokines in BMDMs. BMDMs were cultured in Full or ∆SG medium for 24 h, followed by stimulation with LPS (1, 10 ng/ml) for 8 h (n = 4).

SUFFICIENT SERINE
NADH NADH Figure S8. Graphical summary: Potential molecular mechanisms underlying extracellular serine-regulated inflammatory cytokine production In most cell types, there are 3 major pathways providing serine: de novo serine synthesis, uptake of extracellular serine, and conversion from glycine (left). However, the role of serine uptake has been largely unknown. Deletion of serine in the culture media remarkably reduced the cellular serine content in macrophages, indicating that macrophages largely depend on extracellular serine (right). Under serine deprivation, macrophages stimulated with LPS exhibited aberrant cytokine expression patterns, among which expression of anti-inflammatory Il10 was markedly inhibited. In terms of the underlying mechanism, serine deprivation reduced pyruvate transport into mitochondria, impairing mitochondrial production of ROS. Supplementation of IL10 suppressed the sustained expression of Il6. Thus, this study demonstrates that macrophages rely on extracellular serine to suppress aberrant cytokine production.