CCAAT/enhancer-binding protein delta (C/EBPδ) is a member of the C/EBP family of transcription factors. According to the current paradigm, C/EBPδ potentiates cytokine production and modulates macrophage function thereby enhancing the inflammatory response. Remarkably, however, C/EBPδ deficiency does not consistently lead to a reduction in Lipopolysaccharide (LPS)-induced cytokine production by macrophages. Here, we address this apparent discrepancy and show that the effect of C/EBPδ on cytokine production and macrophage function depends on both the macrophage subtype and the LPS concentration used. Using CRISPR-Cas generated macrophages in which the transactivation domain of C/EBPδ was deleted from the endogenous locus (ΔTAD macrophages), we next show that the context-dependent role of C/EBPδ in macrophage biology relies on compensatory transcriptional activity in the absence of C/EBPδ. We extend these findings by revealing a large discrepancy between transcriptional programs in C/EBPδ knock-out and C/EBPδ transactivation dead (ΔTAD) macrophages implying that compensatory mechanisms do not specifically modify C/EBPδ-dependent inflammatory responses but affect overall macrophage biology. Overall, these data imply that knock-out approaches are not suited for identifying the genuine transcriptional program regulated by C/EBPδ, and we suggest that this phenomenon applies for transcription factor families in general.

Most macrophages generate energy to mount an inflammatory cytokine response by increased glucose metabolism through intracellular glycolysis. Previous studies have suggested that alveolar macrophages (AMs), which reside in a glucose-poor natural environment, are less capable to utilize glycolysis and instead rely on other substrates to fuel oxidative phosphorylation (OXPHOS) for energy supply. At present, it is not known whether AMs are capable to use glucose metabolism to produce cytokines when other metabolic options are blocked. Here, we studied human AMs retrieved by bronchoalveolar lavage from healthy subjects, and examined their glucose metabolism in response to activation by the gram-negative bacterial component lipopolysaccharide (LPS) ex vivo. The immunological and metabolic responses of AMs were compared to those of cultured blood monocyte-derived macrophages (MDMs) from the same subjects. LPS stimulation enhanced cytokine release by both AMs and MDMs, which was associated with increased lactate release by MDMs (reflecting glycolysis), but not by AMs. In agreement, LPS induced higher mRNA expression of multiple glycolytic regulators in MDMs, but not in AMs. Flux analyses of [¹³C]-glucose revealed no differences in [¹³C]-incorporation in glucose metabolism intermediates in AMs. Inhibition of OXPHOS by oligomycin strongly reduced LPS-induced cytokine production by AMs, but not by MDMs. Collectively, these results indicate that human AMs, in contrast to MDMs, do not use glucose metabolism during LPS-induced activation and fully rely on OXPHOS for cytokine production.

Gut microbiota influence the severity of pneumonia by producing metabolites that enhance systemic and pulmonary immune responses. Preclinical studies suggested that gut microbiota-derived indoles have protective effects against numerous diseases, including influenza and abdominal infections. However, the precise role of tryptophan metabolites during pneumonia is unknown. Here, we perform translational analyses in a large general-population cohort (n = 13,464), critically ill patients with severe community-acquired pneumonia (CAP; n = 158; NCT01905033), a randomized human intervention trial on antibiotic-mediated microbiota modulation (NCT03051698), and mice to investigate the effects of tryptophan metabolites, specifically indole-3-acetic acid (IAA), on pneumonia. In the population-based cohort, baseline IAA is associated with a higher risk of future hospital admission for pneumonia (cause-specific hazard ratio 1.15, 95% confidence interval 1.09-1.22 p < 0.0001). In patients with severe CAP higher levels of IAA are associated with increased mortality, independent from potential confounders (hazard ratio 1.30 per log2 increase, 95% confidence interval 1.02-1.68, p = 0.037). In a mouse model of bacterial pneumonia, IAA supplementation aggravates pulmonary damage while reducing systemic dissemination, which is mediated by the aryl hydrocarbon receptor (AhR) and increased release of reactive oxygen species from neutrophils. In summary, these findings from general population and severe pneumonia cohorts, and murine pneumonia experiments, show that the gut microbiota-derived tryptophan metabolite IAA affects pneumonia, suggesting that various indoles may have diverging, context-dependent effects.

In a mouse model of Escherichia coli sepsis characterized by a primary peritoneal infection with 10(4) E. coli and a gradually growing bacterial load, we here show that the early cytokine response and antibacterial defense are dominated by TLR4 via a cooperative action of MyD88 and Trif. Although MyD88(-/-) mice succumbed earlier than WT mice in this E. coli peritonitis model, Trif(-/-) mice displayed a small but significant survival advantage. Despite a large early deficit in antimicrobial defense, TLR4(-/-) mice showed an unaltered survival with normal neutrophil attraction to the peritoneal cavity and normal or even elevated late cytokine release. TLR2 compensated for the lack of TLR4 because TLR2(-/-)/TLR4(-/-) mice did show decreased neutrophil attraction and increased mortality compared with WT mice. Nearly normal early peritoneal TNFα production and lack of early counterregulating systemic levels of the chemoattractant KC were associated with normal peritoneal neutrophil attraction in TLR4(-/-) mice. Late stage increased TNF, IL-1β, IFN-β, and typical IFN-γ production in TLR4(-/-) mice prompted us to evaluate expression of the negative feedback regulator SOCS-1. Lack of early hepatic SOCS-1 expression in TLR4(-/-) mice explained the late innate production of IFN-γ by the liver in TLR4(-/-) mice in this low dose E. coli peritonitis model. In contrast, early TLR4-induced IFN-γ production is described as a hallmark in high dose E. coli peritonitis models. The present study displays how the kinetics of pro- and anti-inflammatory mechanisms are regulated by TLRs during peritonitis by a gradually growing E. coli load and how these kinetics may affect outcome.

Circulating nonadherent monocytes can migrate to extravascular sites by a process that involves adherence. Alterations in intracellular metabolism shape the immunological phenotype of phagocytes upon activation. To determine the effect of adherence on their metabolic and functional response human monocytes were stimulated with LPS under nonadherent and adherent conditions. Adherent monocytes (relative to nonadherent monocytes) produced less TNF and IL-1β (proinflammatory) and more IL-10 (anti-inflammatory) upon LPS stimulation and had an increased capacity to phagocytose and produce reactive oxygen species. RNA sequencing analysis confirmed that adherence modified the LPS-induced response of monocytes, reducing expression of proinflammatory genes involved in TLR signaling and increasing induction of genes involved in pathogen elimination. Adherence resulted in an increased glycolytic response as indicated by lactate release, gene set enrichment, and [¹³C]-glucose flux analysis. To determine the role of glycolysis in LPS-induced immune responses, this pathway was inhibited by glucose deprivation or the glucose analogue 2-deoxy-d-glucose (2DG). Although both interventions equally inhibited glycolysis, only 2DG influenced monocyte functions, inhibiting expression of genes involved in TLR signaling and pathogen elimination, as well as cytokine release. 2DG, but not glucose deprivation, reduced expression of genes involved in oxidative phosphorylation. Inhibition of oxidative phosphorylation affected TNF and IL-10 release in a similar way as 2DG. Collectively, these data suggest that adherence may modify the metabolic and immunological profile of monocytes and that inhibition of glycolysis and oxidative phosphorylation, but not inhibition of glycolysis alone, has a profound effect on immune functions of monocytes exposed to LPS. Key Points Nonadherent monocytes produce more TNF and IL-1β upon LPS stimulation. Adherent, but not nonadherent, monocytes induce glycolysis upon stimulation. Treatment with 2DG, but not glucose deprivation, influences monocyte functions.

Tet methylcytosine dioxygenase 2 (Tet2) mediates demethylation of DNA. We here sought to determine the expression and function of Tet2 in macrophages upon exposure to lipopolysaccharide (LPS), and in the host response to LPS induced lung and peritoneal inflammation, and during Escherichia (E.) coli induced peritonitis. LPS induced Tet2 expression in mouse macrophages and human monocytes in vitro, as well as in human alveolar macrophages after bronchial instillation in vivo. Bone marrow-derived macrophages from myeloid Tet2 deficient (Tet2^fl/flLysM^Cre) mice displayed enhanced production of IL-1β, IL-6 and CXCL1 upon stimulation with several Toll-like receptor agonists; similar results were obtained with LPS stimulated alveolar and peritoneal macrophages. Histone deacetylation was involved in the effect of Tet2 on IL-6 production, whilst methylation at the Il6 promoter was not altered by Tet2 deficiency. Tet2^fl/flLysM^Cre mice showed higher IL-6 and TNF levels in bronchoalveolar and peritoneal lavage fluid after intranasal and intraperitoneal LPS administration, respectively, whilst other inflammatory responses were unaltered. E. coli induced stronger production of IL-1β and IL-6 by Tet2 deficient peritoneal macrophages but not in peritoneal lavage fluid of Tet2^fl/flLysM^Cre mice after in vivo intraperitoneal infection. Tet2^fl/flLysM^Cre mice displayed enhanced bacterial growth during E. coli peritonitis, which was associated with a reduced capacity of Tet2^fl/flLysM^Cre peritoneal macrophages to inhibit the growth of E. coli in vitro. Collectively, these data suggest that Tet2 is involved in the regulation of macrophage functions triggered by LPS and during E. coli infection.