Structured Abstract Introduction Monocytes circulate in the bloodstream for up to 3–5 days. Concomitantly, immunological imprinting of either tolerance (immunosuppression) or trained immunity (innate immune memory) determines the functional fate of monocytes and monocyte-derived macrophages, as observed after infection or vaccination. Methods Purified circulating monocytes from healthy volunteers were differentiated under the homeostatic M-CSF concentrations present in human serum. During the first 24 hours, trained immunity was induced by β-glucan (BG) priming, while post-sepsis immunoparalysis was mimicked by exposure to LPS, generating endotoxin-induced tolerance. Epigenomic profiling of the histone marks H3K4me1, H3K4me3 and H3K27ac, DNase I accessibility and RNA sequencing were performed at both the start of the experiment (ex vivo monocytes) and at the end of the six days of in vitro culture (macrophages). Results Compared to monocytes (Mo), naïve macrophages (Mf) display a remodeled metabolic enzyme repertoire and attenuated innate inflammatory pathways; most likely necessary to generate functional tissue macrophages. Epigenetic profiling uncovered ~8000 dynamic regions associated with ~11000 DNase I hypersensitive sites. Changes in histone acetylation identified most dynamic events. Furthermore, these regions of differential histone marks displayed some degree of DNase I accessibility that was already present in monocytes. H3K4me1 mark increased in parallel with de novo H3K27ac deposition at distal regulatory regions; H3K4me1 mark remained even after the loss of H3K27ac, marking decommissioned regulatory elements. β-glucan priming specifically induced ~3000 distal regulatory elements, whereas LPS-tolerization uniquely induced H3K27ac at ~500 distal regulatory regions. At the transcriptional level, we identified co-regulated gene modules during monocyte to macrophage differentiation, as well as discordant modules between trained and tolerized cells. These indicate that training likely involves an increased expression of modules expressed in naïve macrophages, including genes that code for metabolic enzymes. On the other hand, endotoxin tolerance involves gene modules that are more active in monocytes than in naïve macrophages. About 12% of known human transcription factors display variation in expression during macrophage differentiation, training and tolerance. We also observed transcription factor motifs in DNase I hypersensitive sites at condition-specific dynamic epigenomic regions, implying that specific transcription factors are required for trained and tolerized macrophage epigenetic and transcriptional programs. Finally, our analyses and functional validation indicate that the inhibition of cAMP generation blocked trained immunity in vitro and during an in vivo model of lethal C. albicans infection, abolishing the protective effects of trained immunity. Discussion We documented the importance of epigenetic regulation of the immunological pathways underlying monocyte-to-macrophage differenti...

Microbes or danger signals trigger inflammasome sensors, which induce polymerization of the adapter ASC and assembly of an ASC speck. ASC specks recruit and activate caspase-1, which induces IL-1β cytokine maturation and pyroptotic cell death. Here we show that after pyroptosis ASC specks accumulate in the extracellular space, where they promote further IL-1β maturation. In addition, phagocytosis of ASC specks induces lysosomal damage, nucleation of soluble ASC as well as caspase-1 and IL-1β activation in the recipient cell. ASC specks appear in bodily fluids from inflamed tissues and autoantibodies against ASC specks develop in patients and animals with autoimmune pathologies. Together, these findings reveal extracellular functions of ASC specks and a novel form of cell-to-cell communication.

Microbial stimuli such as lipopolysaccharide (LPS) induce robust metabolic rewiring in immune cells known as the Warburg effect. It is unknown whether this increase in glycolysis and decrease in oxidative phosphorylation (OXPHOS) is a general characteristic of monocytes that have encountered a pathogen. Using CD14 monocytes from healthy donors, we demonstrated that most microbial stimuli increased glycolysis, but that only stimulation of Toll-like receptor (TLR) 4 with LPS led to a decrease in OXPHOS. Instead, activation of other TLRs, such as TLR2 activation by Pam3CysSK4 (P3C), increased oxygen consumption and mitochondrial enzyme activity. Transcriptome and metabolome analysis of monocytes stimulated with P3C versus LPS confirmed the divergent metabolic responses between both stimuli, and revealed significant differences in the tricarboxylic acid cycle, OXPHOS and lipid metabolism pathways following stimulation of monocytes with P3C versus LPS. At a functional level, pharmacological inhibition of complex I of the mitochondrial electron transport chain diminished cytokine production and phagocytosis in P3C- but not LPS-stimulated monocytes. Thus, unlike LPS, complex microbial stimuli and the TLR2 ligand P3C induce a specific pattern of metabolic rewiring that involves upregulation of both glycolysis and OXPHOS, which enables activation of host defence mechanisms such as cytokine production and phagocytosis.

Lactate, the end product of anaerobic glycolysis, is produced in high amounts by innate immune cells during inflammatory activation. Although immunomodulating effects of lactate have been reported, evidence from human studies is scarce. Here we show that expression of genes involved in lactate metabolism and transport is modulated in human immune cells during infection and upon inflammatory activation with TLR ligands in vitro, indicating an important role for lactate metabolism in inflammation. Extracellular lactate induces metabolic reprogramming in innate immune cells, as evidenced by reduced glycolytic and increased oxidative rates of monocytes immediately after exposure to lactate. A short-term infusion of lactate in humans in vivo increased ex vivo glucose consumption of PBMCs, but effects on metabolic rates and cytokine production were limited. Interestingly, long-term treatment with lactate ex vivo, reflecting pathophysiological conditions in local microenvironments such as tumor or adipose tissue, significantly modulated cytokine production with predominantly anti-inflammatory effects. We found time- and stimuli-dependent effects of extracellular lactate on cytokine production, further emphasizing the complex interplay between metabolism and immune cell function. Together, our findings reveal lactate as a modulator of immune cell metabolism which translates to reduced inflammation and may ultimately function as a negative feedback signal to prevent excessive inflammatory responses.

Monocytes are innate immune cells that play a pivotal role in antifungal immunity, but little is known regarding the cellular metabolic events that regulate their function during infection. Using complementary transcriptomic and immunological studies in human primary monocytes, we show that activation of monocytes by Candida albicans yeast and hyphae was accompanied by metabolic rewiring induced through C-type lectin-signaling pathways. We describe that the innate immune responses against Candida yeast are energy-demanding processes that lead to the mobilization of intracellular metabolite pools and require induction of glucose metabolism, oxidative phosphorylation and glutaminolysis, while responses to hyphae primarily rely on glycolysis. Experimental models of systemic candidiasis models validated a central role for glucose metabolism in anti-Candida immunity, as the impairment of glycolysis led to increased susceptibility in mice. Collectively, these data highlight the importance of understanding the complex network of metabolic responses triggered during infections, and unveil new potential targets for therapeutic approaches against fungal diseases.

Background Metformin, the most widely administered diabetes drug, has been proposed as a candidate adjunctive host-directed therapy for tuberculosis, but little is known about its effects on human host responses to Mycobacterium tuberculosis . Methods We investigated in vitro and in vivo effects of metformin in humans. Results Metformin added to peripheral blood mononuclear cells from healthy volunteers enhanced in vitro cellular metabolism while inhibiting the mammalian target of rapamycin targets p70S6K and 4EBP1, with decreased cytokine production and cellular proliferation and increased phagocytosis activity. Metformin administered to healthy human volunteers led to significant downregulation of genes involved in oxidative phosphorylation, mammalian target of rapamycin signaling, and type I interferon response pathways, particularly following stimulation with M. tuberculosis , and upregulation of genes involved in phagocytosis and reactive oxygen species production was increased. These in vivo effects were accompanied by a metformin-induced shift in myeloid cells from classical to nonclassical monocytes. At a functional level, metformin lowered ex vivo production of tumor necrosis factor α, interferon γ , and interleukin 1β but increased phagocytosis activity and reactive oxygen species production. Conclusion Metformin has a range of potentially beneficial effects on cellular metabolism, immune function, and gene transcription involved in innate host responses to M. tuberculosis.

Severe hypoglycemic events have been associated with increased cardiovascular mortality in patients with diabetes, which may be explained by hypoglycemia-induced inflammation. We used ex vivo stimulations of peripheral blood mononuclear cells (PBMCs) and monocytes obtained during hyperinsulinemic-euglycemic (5.0 mmol/L)-hypoglycemic (2.6 mmol/L) clamps in 11 healthy participants, 10 patients with type 1 diabetes and normal awareness of hypoglycemia (NAH), and 10 patients with type 1 diabetes and impaired awareness (IAH) to test whether the composition and inflammatory function of immune cells adapt to a more proinflammatory state after hypoglycemia. Hypoglycemia increased leukocyte numbers in healthy control participants and patients with NAH but not in patients with IAH. Leukocytosis strongly correlated with the adrenaline response to hypoglycemia. Ex vivo, PBMCs and monocytes displayed a more robust cytokine response to microbial stimulation after hypoglycemia compared with euglycemia, although it was less pronounced in patients with IAH. Of note, hypoglycemia increased the expression of markers of demargination and inflammation in PBMCs. We conclude that hypoglycemia promotes mobilization of specific leukocyte subsets from the marginal pool and induces proinflammatory functional changes in immune cells. Inflammatory responses were less pronounced in IAH, indicating that counterregulatory hormone responses are key modulators of hypoglycemia-induced proinflammatory effects. Hypoglycemia-induced proinflammatory changes may promote a sustained inflammatory state.

Sodium butyrate is well-known for its immune-modulatory properties. Studies until now only focused on the in vitro effects of butyrate or assessed local effects in the gut upon butyrate administration. In this trial, we studied the systemic anti-inflammatory effects induced by sodium butyrate supplementation in humans. Nine healthy (Lean) and ten obese (metabolic syndrome group, MetSyn) males were given 4 grams sodium butyrate daily for 4 weeks. PBMCs were isolated before and after supplementation for direct stimulation experiments and induction of trained immunity by oxidized low-density lipoprotein (oxLDL), β-glucan, or Bacillus Calmette-Guérin vaccine (BCG). Butyrate supplementation moderately affected some of the cytokine responses in the MetSyn group. In the direct stimulation setup, effects of butyrate supplementation were limited. Interestingly, butyrate supplementation decreased oxLDL-induced trained immunity in the MetSyn group for LPS-induced IL-6 responses and Pam3CSK4-induced TNF-α responses. Induction of trained immunity by β-glucan was decreased by butyrate in the MetSyn group for Pam3CSK4-induced IL-10 production. In this study, while having only limited effects on the direct stimulation of cytokine production, butyrate supplementation significantly affected trained immunity in monocytes of obese individuals with metabolic complications. Therefore, oral butyrate supplementation may be beneficial in reducing the overall inflammatory status of circulating monocytes in patients with metabolic syndrome.