Hop-derived fraction rich in beta acids and prenylflavonoids regulates the inflammatory response in dendritic cells differently from quercetin: unveiling metabolic changes by mass spectrometry-based metabolomics.

Dendritic cells (DCs) represent a heterogeneous family of immune cells that link innate and adaptive immunity and their activation is linked to metabolic changes that are essential to support their activity and function. Hence, targeting the metabolism of DCs represents an opportunity to modify the inflammatory and immune response. Among the natural matrices, Humulus lupulus (Hop) compounds have recently been shown to exhibit immunomodulatory and anti-inflammatory activity. This study aimed to evaluate the ability of specific Hop fractions to modulate DCs metabolism after stimulation with lipopolysaccharide (LPS) by an untargeted metabolomics approach and compare their effect with flavonol quercetin. Following liquid chromatography-based fractionation, three fractions (A, B, and C) were obtained and tested. Cytokine and gene expression were evaluated using ELISA and qPCR, respectively, while the untargeted metabolomics analysis was performed using a combined HILIC-HRMS and DI-FT-ICR approach. The HOP C fraction and quercetin could both reduce the production of several inflammatory cytokines such as IL-6, IL-1α, IL-1ß, and TNF, but differently from quercetin, the HOP C mechanism is independent of extracellular iron-sequestration and showed significant upregulation of the Nrf2/Nqo1 pathway and Ap-1 compared to quercetin. The untargeted analysis revealed the modulation of several key pathways linked to pro-inflammatory and glycolytic phenotypes. In particular, HOP C treatment could modulate the oxidative step of the pentose phosphate pathway (PPP) and reduce the inflammatory mediator succinate, citrulline, and purine-pyrimidine metabolism, differently from quercetin. These results highlight the potential anti-inflammatory mechanism of specific Hop-derived compounds in restoring the dysregulated metabolism in DCs, which can be used in preventive or adjuvant therapies to suppress the undesirable inflammatory response.

Succinate Produced by Intestinal Microbes Promotes Specification of Tuft Cells to Suppress Ileal Inflammation.

BACKGROUND & AIMS: Countries endemic for parasitic infestations have a lower incidence of Crohn's disease (CD) than nonendemic countries, and there have been anecdotal reports of the beneficial effects of helminths in CD patients. Tuft cells in the small intestine sense and direct the immune response against eukaryotic parasites. We investigated the activities of tuft cells in patients with CD and mouse models of intestinal inflammation. METHODS: We used microscopy to quantify tuft cells in intestinal specimens from patients with ileal CD (n = 19), healthy individuals (n = 14), and TNFΔARE/+ mice, which develop Crohn's-like ileitis. We performed single-cell RNA sequencing, mass spectrometry, and microbiome profiling of intestinal tissues from wild-type and Atoh1-knockout mice, which have expansion of tuft cells, to study interactions between microbes and tuft cell populations. We assessed microbe dependence of tuft cell populations using microbiome depletion, organoids, and microbe transplant experiments. We used multiplex imaging and cytokine assays to assess alterations in inflammatory response following expansion of tuft cells with succinate administration in TNFΔARE/+ and anti-CD3E CD mouse models. RESULTS: Inflamed ileal tissues from patients and mice had reduced numbers of tuft cells, compared with healthy individuals or wild-type mice. Expansion of tuft cells was associated with increased expression of genes that regulate the tricarboxylic acid cycle, which resulted from microbe production of the metabolite succinate. Experiments in which we manipulated the intestinal microbiota of mice revealed the existence of an ATOH1-independent population of tuft cells that was sensitive to metabolites produced by microbes. Administration of succinate to mice expanded tuft cells and reduced intestinal inflammation in TNFΔARE/+ mice and anti-CD3E-treated mice, increased GATA3+ cells and type 2 cytokines (IL22, IL25, IL13), and decreased RORGT+ cells and type 17 cytokines (IL23) in a tuft cell-dependent manner. CONCLUSIONS: We found that tuft cell expansion reduced chronic intestinal inflammation in mice. Strategies to expand tuft cells might be developed for treatment of CD.

Cinnamaldehyde suppresses NLRP3 derived IL-1ß via activating succinate/HIF-1 in rheumatoid arthritis rats.

Cinnamaldehyde (CA) is an essential component of cinnamon (Cinnamomum cassia Presland), which is often used as a flavoring condiment in beverages, pastries, perfumes, etc. Cinnamon is also used as herbal medicine in China and Southeast Asia to treat rheumatoid arthritis. However, the molecular mechanism is unclear. In this study, we aim to investigate its anti-inflammatory effects against Rheumatoid arthritis (RA) using activated macrophages (Raw246.7) in vitro and adjuvant arthritis rats (AA) in vivo. The results demonstrated that CA significantly reduced synovial inflammation in AA rats, possibly due to suppression of the expressions of pro-inflammatory cytokines, especially the IL-1ß. Further investigation found that CA also suppressed the activity of HIF-1α by inhibiting the accumulation of succinate in cytoplasm. As we know, the reduction of HIF-1α nucleation slows down IL-1ß production, because HIF-1α activates the expression of NLRP3, which is involved in the assembly of inflammasome and processing of IL-1ß. In addition, CA also inhibited the expression of the succinate receptor GPR91, which in turn inhibited the activation of HIF-1α. In conclusions, our results suggested that CA might be a potential therapeutic compound to relieve rheumatoid arthritis progress by suppressing IL-1ß through modulating succinate/HIF-1α axis and inhibition of NLRP3.

Cytokine-like Roles for Metabolites in Immunity.

Metabolites have functions in the immune system independent of their conventional roles as sources or intermediates in biosynthesis and bioenergetics. We are still in the pioneering phase of gathering information about the functions of specific metabolites in immunoregulation. In this review, we cover succinate, itaconate, α-ketoglutarate, and lactate as examples. Each of these metabolites has a different story of how their immunoregulatory functions were discovered and how their roles in the complex process of inflammation were revealed. Parallels and interactions are emerging between metabolites and cytokines, well-known immunoregulators. We depict molecular mechanisms by which metabolites prime cellular and often physiological changes focusing on intra- and extra-cellular activities and signaling pathways. Possible therapeutic opportunities for immune and inflammatory diseases are emerging.

SUCNR1 controls an anti-inflammatory program in macrophages to regulate the metabolic response to obesity.

Succinate is a signaling metabolite sensed extracellularly by succinate receptor 1 (SUNCR1). The accumulation of succinate in macrophages is known to activate a pro-inflammatory program; however, the contribution of SUCNR1 to macrophage phenotype and function has remained unclear. Here we found that activation of SUCNR1 had a critical role in the anti-inflammatory responses in macrophages. Myeloid-specific deficiency in SUCNR1 promoted a local pro-inflammatory phenotype, disrupted glucose homeostasis in mice fed a normal chow diet, exacerbated the metabolic consequences of diet-induced obesity and impaired adipose-tissue browning in response to cold exposure. Activation of SUCNR1 promoted an anti-inflammatory phenotype in macrophages and boosted the response of these cells to type 2 cytokines, including interleukin-4. Succinate decreased the expression of inflammatory markers in adipose tissue from lean human subjects but not that from obese subjects, who had lower expression of SUCNR1 in adipose-tissue-resident macrophages. Our findings highlight the importance of succinate-SUCNR1 signaling in determining macrophage polarization and assign a role to succinate in limiting inflammation.

Quantitative proteome and lysine succinylome analyses provide insights into metabolic regulation in breast cancer.

BACKGROUND: Breast cancer, the most common invasive cancer and cause of cancer-related death in women worldwide, is a multifactorial, complex disease, and many molecular players and mechanisms underlying the complexity of its clinical behavior remain unknown. METHODS: To explore the molecular features of breast cancer, quantitative proteome and succinylome analyses in breast cancer were extensively studied using quantitative proteomics techniques, anti-succinyl lysine antibody-based affinity enrichment, and high-resolution LC-MS/MS analysis. RESULTS: Our study is the first to detect the regulation of lysine succinylation in breast cancer progression. We identified a novel mechanism by which the pentose phosphate pathway and the endoplasmic reticulum protein processing pathway might be regulated via lysine succinylation in their core enzymes. CONCLUSIONS: These results expand our understanding of tumorigenesis mechanisms and provide a basis for further characterization of the pathophysiological roles in breast cancer progression, laying a foundation for innovative and novel breast cancer drugs and therapies.

Biochemistry of proinflammatory macrophage activation.

In the last decade, metabolism has been recognized as a major determinant of immunological processes. During an inflammatory response, macrophages undergo striking changes in their metabolism. This metabolic reprogramming is governed by a complex interplay between metabolic enzymes and metabolites of different pathways and represents the basis for proper macrophage function. It is now evident that these changes go far beyond the well-known Warburg effect and the perturbation of metabolic targets is being investigated as a means to treat infections and auto-immune diseases. In the present review, we will aim to provide an overview of the metabolic responses during proinflammatory macrophage activation and show how these changes modulate the immune response.

Intermediates of Metabolism: From Bystanders to Signalling Molecules.

The integration of biochemistry into immune cell biology has contributed immensely to our understanding of immune cell function and the associated pathologies. So far, most studies have focused on the regulation of metabolic pathways during an immune response and their contribution to its success. More recently, novel signalling functions of metabolic intermediates are being discovered that might play important roles in the regulation of immunity. Here we describe the three long-known small metabolites lactate, acetyl-CoA, and succinate in the context of immunometabolic signalling. Functions of these ubiquitous molecules are largely dependent on their intra- and extracellular concentrations as well as their subcompartmental localisation. Importantly, the signalling functions of these metabolic intermediates extend beyond self-regulatory roles and include cell-to-cell communication and sensing of microenvironmental conditions to elicit stress responses and cellular adaptation.

Unsaturated compounds induce up-regulation of CD86 on dendritic cells in the in vitro sensitization assay LCSA.

Unsaturated compounds are known to cause false-positive reactions in the local lymph node assay (LLNA) but not in the guinea pig maximization test. We have tested a panel of substances (succinic acid, undecylenic acid, 1-octyn-3-ol, fumaric acid, maleic acid, linoleic acid, oleic acid, alpha-linolenic acid, squalene, and arachidonic acid) in the loose-fit coculture-based sensitization assay (LCSA) to evaluate whether unspecific activation of dendritic cells is a confounder for sensitization testing in vitro. Eight out of 10 tested substances caused significant up-regulation of CD86 on dendritic cells cocultured with keratinocytes and would have been classified as sensitizers; only succinic acid was tested negative, and squalene had to be excluded from data analysis due to poor solubility in cell culture medium. Based on human data, only undecylenic acid can be considered a true sensitizer. The true sensitizing potential of 1-octyn-3-ol is uncertain. Fumaric acid and its isomer maleic acid are not known as sensitizers, but their esters are contact allergens. A group of 18- to 20-carbon chain unsaturated fatty acids (linoleic acid, oleic acid, alpha-linolenic acid, and arachidonic acid) elicited the strongest reaction in vitro. This is possibly due to the formation of pro-inflammatory lipid mediators in the cell culture causing nonspecific activation of dendritic cells. In conclusion, both the LLNA and the LCSA seem to provide false-positive results for unsaturated fatty acids. The inclusion of T cells in dendritic cell-based in vitro sensitization assays may help to eliminate false-positive results due to nonspecific dendritic cell activation. This would lead to more accurate prediction of sensitizers, which is paramount for consumer health protection and occupational safety.