Alterations of the volatile metabolome (the collection of volatiles present in secretions and other emanations) that occur in response to inflammation can be detected by conspecifics and chemometric analyses. Using a model system where mouse urinary metabolites are altered by treatment with lipopolysaccharide (found in the outer cell membrane of gram-negative bacteria), we hypothesized that alteration of body odor volatiles will vary according to the pathogen responsible for inducing the inflammation. We tested this hypothesis by treating mice with different immunogens that engage different immune signaling pathways. Results suggest that alterations of body odor volatiles resulting from inflammation do contain detailed information about the type of pathogen that instigated the inflammation and these differences are not merely dependent on the severity of the inflammatory event. These results are encouraging for the future of differential medical diagnosis of febrile diseases by analysis of the volatile metabolome. In particular, our data support the possibility that bacterial infections can be differentiated from viral infections such that antibiotic drug stewardship could be drastically improved by reducing unneeded treatments with antibiotics.

Several studies demonstrate that inflammation affects body odor. Volatile signals associated with inflammation induced by pyrogens like LPS are detectable both by conspecifics and chemical analyses. However, little is known about the mechanisms which translate detection of a foreign molecule or pathogen into a unique body odor, or even how unique that odor may be. Here, we utilized C57BL/6J trained mice to identify the odor of LPS-treated conspecifics to investigate potential pathways between LPS-induced inflammation and changes in body odor, as represented by changes in urine odor. We hypothesized that the change in volatile metabolites could be caused directly by the pro-inflammatory cytokine response mediated by TNF or IL-1β, or by the compensatory anti-inflammatory response mediated by IL-10. We found that trained biosensors generalized learned LPS-associated odors to TNF-induced odors, but not to IL-1β or IL-10-induced odors. Analyses of urine volatiles using headspace gas chromatography revealed distinct profiles of volatile compounds for each treatment. Instrumental discrimination relied on a mixture of compounds, including 2-sec-butyl-4,5-dihydrothiazole, cedrol, nonanal, benzaldehyde, acetic acid, 2-ethyl-1-hexanol, and dehydro-exo-brevicomin. Although interpretation of LDA modeling differed from behavioral testing, it does suggest that treatment with TNF, IL-1β, and LPS can be distinguished by their resultant volatile profiles. These findings indicate there is information found in body odors on the presence of specific cytokines. This result is encouraging for the future of disease diagnosis via analysis of volatiles.

Expression of the inflammatory cytokine TNF is tightly controlled. During endotoxin tolerance, transcription of TNF mRNA is repressed, although not entirely eliminated. Production of TNF cytokine, however, is further controlled by posttranscriptional regulation. In this study, we detail a mechanism of posttranscriptional repression of TNF mRNA by GAPDH binding to the TNF 3' untranslated region. Using RNA immunoprecipitation, we demonstrate that GAPDH-TNF mRNA binding increases when THP-1 monocytes are in a low glycolysis state, and that this binding can be reversed by knocking down GAPDH expression or by increasing glycolysis. We show that reducing glycolysis decreases TNF mRNA association with polysomes. We demonstrate that GAPDH-TNF mRNA binding results in posttranscriptional repression of TNF and that the TNF mRNA 3' untranslated region is sufficient for repression. Finally, after exploring this model in THP-1 cells, we demonstrate this mechanism affects TNF expression in primary human monocytes and macrophages. We conclude that GAPDH-TNF mRNA binding regulates expression of TNF based on cellular metabolic state. We think this mechanism has potentially significant implications for treatment of various immunometabolic conditions, including immune paralysis during septic shock.

Background: Nuclear SIRT1 and SIRT6 switch monocyte energy sources from glycolysis to fatty acid oxidation during sepsis adaptation. Results: Sequential actions of nuclear SIRT1 and RELB differentially induce SIRT3 expression and increase mitochondrial biogenesis during sepsis adaptation. Conclusion: SIRT1 and RELB link nuclear and mitochondrial alterations in bioenergetics during sepsis. Significance: Communication between nuclear and mitochondrial functions may influence sepsis outcomes. We reported that NAD+-dependent SIRT1, RELB, and SIRT6 nuclear proteins in monocytes regulate a switch from the glycolysis-dependent acute inflammatory response to fatty acid oxidation-dependent sepsis adaptation. We also found that disrupting SIRT1 activity during adaptation restores immunometabolic homeostasis and rescues septic mice from death. Here, we show that nuclear SIRT1 guides RELB to differentially induce SIRT3 expression and also increases mitochondrial biogenesis, which alters bioenergetics during sepsis adaptation. We constructed this concept using TLR4-stimulated THP1 human promonocytes, a model that mimics the initiation and adaptation stages of sepsis. Following increased expression, mitochondrial SIRT3 deacetylase activates the rate-limiting tricarboxylic acid cycle (TCA) isocitrate dehydrogenase 2 and superoxide dismutase 2, concomitant with increases in citrate synthase activity. Mitochondrial oxygen consumption rate increases early and decreases during adaptation, parallel with modifications to membrane depolarization, ATP generation, and production of mitochondrial superoxide and whole cell hydrogen peroxide. Evidence of SIRT1-RELB induction of mitochondrial biogenesis included increases in mitochondrial mass, mitochondrial-to-nuclear DNA ratios, and both nuclear and mitochondrial encoded proteins. We confirmed the SIRT-RELB-SIRT3 adaptation link to mitochondrial bioenergetics in both TLR4-stimulated normal and sepsis-adapted human blood monocytes and mouse splenocytes. We also found that SIRT1 inhibition ex vivo reversed the sepsis-induced changes in bioenergetics.

RelB is one of the more unusual members of the NF-kappa B family. This family, arguably the best known group of transcription regulators, regulates an astonishing array of cell types and biological processes. This includes regulation of cell growth, differentiation and death by apoptosis, and the development and function of the innate and adaptive-immune system. RelB is best known for its roles in lymphoid development, DC biology, and noncanonical signaling. Within the last few years, however, surprising functions of RelB have emerged. The N-terminal leucine zipper motif of RelB, a motif unique among the NF-kappa B family, may associate with more diverse DNA sequences than other NF-kappa B members. RelB is capable of direct binding to the AhR that supports the xenobiotic-detoxifying pathway. RelB can regulate the circadian rhythm by directly binding to the BMAL partner of CLOCK. Finally, RelB also couples with bioenergy NAD(+) sensor SIRT1 to integrate acute inflammation with changes in metabolism and mitochondrial bioenergetics. In this review, we will explore these unique aspects of RelB, specifically with regard to its role in immunity.

RelB is one of the more unusual members of the NF‐κB family. This family, arguably the best known group of transcription regulators, regulates an astonishing array of cell types and biological processes. This includes regulation of cell growth, differentiation and death by apoptosis, and the development and function of the innate and adaptive‐immune system. RelB is best known for its roles in lymphoid development, DC biology, and noncanonical signaling. Within the last few years, however, surprising functions of RelB have emerged. The N‐terminal leucine zipper motif of RelB, a motif unique among the NF‐κB family, may associate with more diverse DNA sequences than other NF‐κB members. RelB is capable of direct binding to the AhR that supports the xenobiotic‐detoxifying pathway. RelB can regulate the circadian rhythm by directly binding to the BMAL partner of CLOCK. Finally, RelB also couples with bioenergy NAD+ sensor SIRT1 to integrate acute inflammation with changes in metabolism and mitochondrial bioenergetics. In this review, we will explore these unique aspects of RelB, specifically with regard to its role in immunity.

We review the emerging concept that changes in cellular bioenergetics concomitantly reprogram inflammatory and metabolic responses. The molecular pathways of this integrative process modify innate and adaptive immune reactions associated with inflammation, as well as influencing the physiology of adjacent tissue and organs. The initiating proinflammatory phase of inflammation is anabolic and requires glucose as the primary fuel, whereas the opposing adaptation phase is catabolic and requires fatty acid oxidation. The fuel switch to fatty acid oxidation depends on the sensing of AMP and NAD(+) by AMPK and the SirT family of deacetylases (e.g., SirT1, -6, and -3), respectively, which couple inflammation and metabolism by chromatin and protein reprogramming. The AMP-AMPK/NAD(+)-SirT axis proceeds sequentially during acute systemic inflammation associated with sepsis but ceases during chronic inflammation associated with diabetes, obesity, and atherosclerosis. Rebalancing bioenergetics resolves inflammation. Manipulating cellular bioenergetics is identifying new ways to treat inflammatory and immune diseases. J. Leukoc. Biol. 92: 499-507; 2012.