Macrophages are innate immune cells that adopt diverse activation states in response to their microenvironment. Editing macrophage activation to dampen inflammatory diseases by promoting the repolarization of inflammatory (M1) macrophages to anti-inflammatory (M2) macrophages is of high interest. Here, we find that mouse and human M1 macrophages fail to convert into M2 cells upon IL-4 exposure in vitro and in vivo. In sharp contrast, M2 macrophages are more plastic and readily repolarized into an inflammatory M1 state. We identify M1-associated inhibition of mitochondrial oxidative phosphorylation as the factor responsible for preventing M1→M2 repolarization. Inhibiting nitric oxide production, a key effector molecule in M1 cells, dampens the decline in mitochondrial function to improve metabolic and phenotypic reprogramming to M2 macrophages. Thus, inflammatory macrophage activation blunts oxidative phosphorylation, thereby preventing repolarization. Therapeutically restoring mitochondrial function might be useful to improve the reprogramming of inflammatory macrophages into anti-inflammatory cells to control disease.

Cxcl18b is a chemokine found in zebrafish and in other piscine and amphibian species. Cxcl18b is a reliable inflammatory marker; however, its function is yet to be elucidated. Here, we found that Cxcl18b is chemotactic towards neutrophils, similarly to Cxcl8a/Interleukin-8, the best characterised neutrophil chemoattractant in humans and teleosts. Like Cxcl8a, Cxcl18b-dependent recruitment required the chemokine receptor Cxcr2, while it was unaffected by depletion of the other two neutrophil receptors cxcr1 and cxcr4b. To visualise cxcl18b induction, we generated a Tg(cxcl18b:eGFP) reporter line. The transgene is induced locally upon bacterial infection with the fish pathogen Mycobacterium marinum, but strikingly is not directly expressed by infected cells. Instead, cxcl18b is induced by non-phagocytic uninfected cells that compose the stroma of the granulomas, typical inflammatory lesions formed upon mycobacterial infections. Together, these results suggest that Cxcl18b might be an important contributor to neutrophil chemotaxis in the inflammatory microenvironment and indicate that the zebrafish model could be explored to further investigate in vivo the biological relevance of different Cxcl8-like chemokine lineages.

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Abstract Background Liver kinase B1 (LKB1) has been studied extensively as a tumor suppressor gene (Stk11) in the context of cancer. We hypothesized that myeloid LKB1 plays a role in innate immunity during pneumonia. Methods Mice deficient for LKB1 in myeloid cells (LysM-cre x Stk11fl/fl ) or neutrophils (Mrp8-cre x Stk11fl/fl) were infected with Klebsiellapneumoniae via the airways. LysM-cre x Stk11fl/fl mice were also intranasally challenged with lipopolysaccharide (LPS). Results Myeloid, but not neutrophil LKB1 deficient mice had increased bacterial loads in lungs from 6 to 40 hours after infection as compared to control mice, pointing at a role for LKB1 in macrophages. Myeloid LKB1 deficiency was associated with reduced cytokine release into the airways upon local LPS instillation. The number of classical (SiglecFhighCD11bneg) alveolar macrophages (AMs) was reduced by approximately 50% in the lungs of myeloid LKB1 deficient mice, which was not caused by increased cell death or reduced proliferation. Instead, myeloid LKB1 deficient mice had AMs with a ‘non-classical’ (SiglecFlowCD11bpos) phenotype. AMs did not upregulate glycolysis in response to LPS, irrespective of LKB1 presence. Conclusion Myeloid LKB1 is important for local host defense during Klebsiella pneumonia by maintaining adequate AM numbers in the lung.

Background: The nature and timing of the host immune response during infections remain uncertain and most knowledge is derived from critically ill sepsis patients. We aimed to test the hypothesis that community-acquired pneumonia (CAP) is associated with concurrent immune suppression and systemic inflammation. Methods: Blood was collected from 79 CAP patients within 24 h after hospitalization and 1 month after discharge; 42 age-and sex-matched subjects without acute infection served as controls. Blood leukocytes were stimulated with lipopolysaccharide (LPS) or Klebsiella pneumoniae, and cytokines were measured in supernatants. Fifteen plasma biomarkers reflective of key host response pathways were compared between CAP patients with the strongest immune suppression (lowest 25% blood leukocyte tumor necrosis factor (TNF)-α production in response to LPS) and those with the least immune suppression (highest 25% of LPS-induced TNF-α production). Results: Blood leukocytes of CAP patients (relative to control subjects) showed a reduced capacity to release TNF-α, interleukin (IL)-1β, IL-6 and IL-10 upon stimulation with LPS or K. pneumoniae, with a concurrently enhanced ability to release the antiinflammatory mediator IL-1 receptor antagonist, irrespective of the presence of sepsis (18.9% of cases). Low (relative to high) TNF-α producers displayed higher plasma levels of biomarkers reflecting systemic inflammation, neutrophil degranulation, endothelial cell activation, a disturbed vascular barrier function and coagulation activation. Conclusion: CAP replicates a common feature of immune suppression in sepsis. The coexistence of immune suppression and hyperinflammation in CAP argues against the theory of two distinct phases during the host response to sepsis.

Background The plasticity of monocytes enables them to exert multiple roles during an immune response, including promoting immune tolerance. How monocytes alter their functions to convey immune tolerance in the context of lower respiratory tract infections in humans is not well understood. Here, we sought to identify epigenetic and transcriptomic features of cytokine production capacity in circulating monocytes during community-acquired pneumonia (CAP). Methods Circulating CD14+ monocytes were obtained from the blood of CAP patients included in a longitudinal, observational cohort study, on hospitalization (acute stage, n=75), and from the same patients after a 1-month follow-up (recovery stage, n=56). Age and sex-matched non-infectious participants were included as controls (n=41). Ex vivo cytokine production after lipopolysaccharide (LPS) exposure was assessed by multiplex assay. Transcriptomes of circulating monocytes were generated by RNA-sequencing, and DNA methylation levels in the same monocytes were measured by reduced representation bisulfite sequencing. Data were integrated by fitting projection-to-latent-structure models, and signatures derived by partial least squares discrimination. Results Monocytes captured during the acute stage exhibited impaired TNF, IL-1β, IL-6, and IL-10 production after ex vivo stimulation with LPS, relative to controls. IL-6 production was not resolved in recovery monocytes. Multivariate analysis of RNA-sequencing data identified 2938 significantly altered RNA transcripts in acute-stage monocytes (fold expression ≤−1.5 or ≥1.5; adjusted p ≤ 0.01), relative to controls. Comparing DNA methylation levels in circulating monocytes of CAP patients to controls revealed minimal differences, specifically in DNAse hypersensitive sites (HS) of acute-stage monocytes. Data integration identified a cholesterol biosynthesis gene signature and DNAse HS axis of IL-1β and IL-10 production (R2 =0.51). Conclusions Circulating monocytes obtained from CAP patients during the acute stage exhibited impaired cytokine production capacities, indicative of reprogramming to a state of immune tolerance, which was not fully resolved after 1 month. Our split-sample study showed that 51% of the immune tolerance phenotype can be explained, at least in part, by coordinated shifts in cholesterol biosynthesis gene expression and DNAse HS methylation levels. A multi-scale model identified an epigenetic and transcriptomic signature of immune tolerance in monocytes, with implications for future interventions in immunosuppression. Trial registration NCT number NCT02928367

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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-1b (proinflammatory) and more IL-10 (antiinflammatory) 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 [ 13 C]-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.