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.

Objective: Antimicrobial resistance is an emerging problem and multi-drug resistant (MDR) Klebsiella pneumoniae (K. pneumoniae) represents an enormous risk of failing therapy in hospital-acquired pneumonia. The current study aimed to determine the immunomodulatory effect of topical flagellin in addition to antibiotic treatment during respiratory infection evoked by hypervirulent antibiotic-susceptible and antibiotic-resistant K. pneumoniae in mice.Methods: C57BL6 mice were inoculated intranasally with hypervirulent K. pneumoniae (K2:O1) which was either antibiotic-susceptible or multi-drug resistant. Six hours after infection, mice were treated with antibiotics intraperitoneally and flagellin or vehicle intranasally. Mice were sacrificed 24 hours after infection. Samples were analyzed for bacterial loads and for inflammatory and coagulation markers.Results: Flagellin therapy induced neutrophil influx in the lung during antibiotic-treated pneumonia evoked by either antibiotic-susceptible or -resistant K. pneumoniae. The pulmonary neutrophil response was matched by elevated levels of neutrophil-attracting chemokines, neutrophil degranulation products, and local coagulation activation. The combined therapy of effective antibiotics and flagellin did not impact K. pneumoniae outgrowth in the lung, but decreased bacterial counts in distant organs. Neutrophil depletion abrogated the flagellin-mediated effect on bacterial dissemination and local coagulation responses.Conclusion: Topical flagellin administration as an adjunctive to antibiotic treatment augments neutrophil responses during pneumonia evoked by MDR-K. pneumoniae, thereby reducing bacterial dissemination to distant organs.

Pneumonia caused by multi-drug-resistant Klebsiella pneumoniae (MDR-Kpneu) poses a major public health threat, especially to immunocompromised or hospitalized patients. This study aimed to determine the immunostimulatory effect of the Toll-like receptor 5 ligand flagellin on primary human lung epithelial cells during infection with MDR-Kpneu. Human bronchial epithelial (HBE) cells, grown on an air–liquid interface, were inoculated with MDR-Kpneu on the apical side and treated during ongoing infection with antibiotics (meropenem) and/or flagellin on the basolateral and apical side, respectively; the antimicrobial and inflammatory effects of flagellin were determined in the presence or absence of meropenem. In the absence of meropenem, flagellin treatment of MDR-Kpneu-infected HBE cells increased the expression of antibacterial defense genes and the secretion of chemokines; moreover, supernatants of flagellin-exposed HBE cells activated blood neutrophils and monocytes. However, in the presence of meropenem, flagellin did not augment these responses compared to meropenem alone. Flagellin did not impact the outgrowth of MDR-Kpneu. Flagellin enhances antimicrobial gene expression and chemokine release by the MDR-Kpneu-infected primary human bronchial epithelium, which is associated with the release of mediators that activate neutrophils and monocytes. Topical flagellin therapy may have potential to boost immune responses in the lung during pneumonia.

Introduction: Alveolar macrophages (AMs) play an essential role in maintaining homeostasis in the lung and in innate immunity for host defense. To fuel inflammatory responses, AMs do not rely on glycolysis, but require oxidative phosphorylation. However, which nutrients AMs use to fuel their energy demand during inflammatory responses, is still unknown. The present study aimed to determine the contribution of three key metabolic pathways; fatty acid oxidation, glutaminolysis, and glycogenolysis, to the inflammatory response of AMs. Methods: Primary AMs were isolated from healthy human volunteers and stimulated with lipopolysaccharide (LPS). After 24 hours, cells were subjected to analyses of metabolic flux, expression of genes involved in these metabolic pathways, and inflammatory cytokine secretion in the presence of metabolic inhibitors. Results: The results of our study show that human AMs display expression of genes involved in fatty acid and glutamine metabolism and are capable of metabolizing oleic acid and glutamine during homeostasis, but do not use these metabolites to fuel the production of inflammatory cytokines. We demonstrate that AMs, while residing in a glucose-deprived environment, contain glycogen and use glycogenolysis to fuel inflammatory cytokine secretion, as reflected by reduced TNF, IL-1β and IL-6 levels in supernatant of LPS-stimulated AMs treated with the glycogenolysis inhibitor CP316819. Moreover, AMs display marked expression of genes involved in glycogenesis, including FBP1 and GYS. Conclusions: Taken together, these results indicate that primary human AMs are equipped to use different nutrients to fuel their metabolic demands. Moreover, our findings suggest that glycogenolysis is critical for the inflammatory response of AMs.

Objective: The airway epithelium provides a first line of defense against pathogens by release of antimicrobial factors and neutrophil-attracting chemokines. Pseudomonas (P.) aeruginosa, a Gram-negative bacterium that expresses flagellin as an important virulence factor, is a common cause of injurious airway inflammation. The aim of our study was to determine the contribution of flagellin to the inflammatory, antimicrobial, and metabolic responses of the airway epithelium to P. aeruginosa. Furthermore, as we previously showed that targeting mTOR limited the glycolytic and inflammatory response induced by flagellin, we assessed the effect of rapamycin on human bronchial epithelial (HBE) cells stimulated with flagellated and non-flagellated P. aeruginosa. Methods: Primary pseudostratified HBE cells, cultured on an air-liquid-interface, were treated on the basolateral side with medium, vehicle or rapamycin, exposed on the apical side with flagellated or flagellin-deficient P. aeruginosa, and analyzed for their inflammatory, antimicrobial, and glycolytic responses. Results: Flagellin augmented the P. aeruginosa-induced expression of antimicrobial factors and secretion of chemokines by HBE cells but did not further increase the glycolytic response. Treatment of HBE cells with rapamycin inhibited mTOR activation in general and flagellin-augmented mTOR activation in particular, but did not affect the glycolytic response. Rapamycin, however, diminished the flagellin-augmented inflammatory and antimicrobial response induced by Pseudomonas. Conclusions: These results demonstrate that flagellin is a significant factor that augments the inflammatory and antimicrobial response of human airway epithelial cells upon exposure to P. aeruginosa and suggest that mTOR inhibition by rapamycin in the airway epithelium diminishes these exaggerated responses.

Background: Antimicrobial resistance is an emerging problem and multi-drug resistant (MDR) Klebsiella pneumoniae represents an enormous risk of failing therapy in nosocomial pneumonia. Aim of the study To determine the immunomodulatory effect of topical flagellin in addition to antibiotic treatment during pneumosepsis evoked by hypervirulent Klebsiella pneumoniae in mice. Methods: C57BL6 mice were inoculated intranasally with hypervirulent Klebsiella pneumoniae (K2:O1) which were either antibiotic susceptible (Kpneu) or MDR (MDR-Kpneu). Subsequently, mice were treated with antibiotics intraperitoneally and flagellin or vehicle intranasally 6 hours after infection. Mice were sacrificed 24 hours after infection. Samples were analysed for bacterial loads and inflammatory markers. Results: Flagellin therapy induced neutrophil influx in the lung during antibiotic-treated pneumonia evoked by either Kpneu or MDR-Kpneu. The pulmonary neutrophil response was accompanied by elevated levels of myeloperoxidase, elastase, and neutrophil-attracting chemokines. The combined therapy of effective antibiotics (ceftriaxone for Kpneu; colistin for MDR-Kpneu) and flagellin reduced Klebsiella loads in distant organs, but did not impact bacterial counts in the lung. This flagellin-mediated effect was diminished when neutrophils were depleted during infection. Conclusions: Topical flagellin administration as an adjunctive to antibiotic treatment induced neutrophil recruitment and activation during (MDR-)Klebsiella pneumonia, thereby reducing bacterial dissemination to distant organs.