Staphylococcus aureus suppresses the pentose phosphate pathway in human neutrophils via the adenosine receptor A2aR to enhance intracellular survival.

IMPORTANCE: Staphylococcus aureus is one of the leading causes of antimicrobial-resistant infections whose success as a pathogen is facilitated by its massive array of immune evasion tactics, including intracellular survival within critical immune cells such as neutrophils, the immune system's first line of defense. In this study, we describe a novel pathway by which intracellular S. aureus can suppress the antimicrobial capabilities of human neutrophils by using the anti-inflammatory adenosine receptor, adora2a (A2aR). We show that signaling through A2aR suppresses the pentose phosphate pathway, a metabolic pathway used to fuel the antimicrobial NADPH oxidase complex that generates reactive oxygen species (ROS). As such, neutrophils show enhanced ROS production and reduced intracellular S. aureus when treated with an A2aR inhibitor. Taken together, we identify A2aR as a potential therapeutic target for combatting intracellular S. aureus infection.

Endogenous drivers of altered immune cell metabolism.

Dysregulated metabolism has long been recognized as a feature of many metabolic disorders. However, recent studies demonstrating that metabolic reprogramming occurs in immune cells have led to a growing interest in the relationship between metabolic rewiring and immune-mediated disease pathogeneses. It is clear now that immune cell subsets engage in different metabolic pathways depending on their activation and/or maturation state. As a result, it may be possible to modulate metabolic reprogramming for clinical benefit. In this review, we provide an overview of immune cell metabolism with focus on endogenous drivers of metabolic reprogramming given their link to a number of immune-mediated disorders.

Non-invasive classification of macrophage polarisation by 2P-FLIM and machine learning.

In this study, we utilise fluorescence lifetime imaging of NAD(P)H-based cellular autofluorescence as a non-invasive modality to classify two contrasting states of human macrophages by proxy of their governing metabolic state. Macrophages derived from human blood-circulating monocytes were polarised using established protocols and metabolically challenged using small molecules to validate their responding metabolic actions in extracellular acidification and oxygen consumption. Large field-of-view images of individual polarised macrophages were obtained using fluorescence lifetime imaging microscopy (FLIM). These were challenged in real time with small-molecule perturbations of metabolism during imaging. We uncovered FLIM parameters that are pronounced under the action of carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP), which strongly stratifies the phenotype of polarised human macrophages; however, this performance is impacted by donor variability when analysing the data at a single-cell level. The stratification and parameters emanating from a full field-of-view and single-cell FLIM approach serve as the basis for machine learning models. Applying a random forests model, we identify three strongly governing FLIM parameters, achieving an area under the receiver operating characteristics curve (ROC-AUC) value of 0.944 and out-of-bag (OBB) error rate of 16.67% when classifying human macrophages in a full field-of-view image. To conclude, 2P-FLIM with the integration of machine learning models is showed to be a powerful technique for analysis of both human macrophage metabolism and polarisation at full FoV and single-cell level.

Cholesterol crystals drive metabolic reprogramming and M1 macrophage polarisation in primary human macrophages.

BACKGROUND AND AIMS: Metabolic reprogramming of innate immune cells is emerging as a key player in the progression of a number of chronic diseases, including atherosclerosis, where high rates of glycolysis correlate with plaque instability. This study aimed to investigate if cholesterol crystals, which are key atherosclerosis-associated DAMPs (damage/danger-associated molecular patterns), alter immune cell metabolism and whether this, in turn, impacts on macrophage phenotype and function. METHODS AND RESULTS: Primary human macrophages were treated with cholesterol crystals and expression of M1 (CXCL9, CXCL10) and M2-associated (MRC1, CCL13) macrophage markers, alarmins, and inflammatory cytokines were assessed either by real-time PCR or ELISA. Cholesterol crystal-induced changes in glycolytic markers were determined using real-time PCR and western blotting, while changes in cellular respiration and mitochondrial dynamics were examined via Seahorse analysis, Fluorescence Lifetime Imaging Microscopy (FLIM) and confocal microscopy. Treatment of macrophages with cholesterol crystals upregulated mRNA levels of CXCL9 and CXCL10, while concomitantly downregulating expression of MRC1 and CCL13. Cholesterol crystal--treated macrophages also exhibited a significant shift in metabolism to favour glycolysis, accompanied by the expression of key glycolytic markers GLUT1, Hexokinase 2, HIF1α, GAPDH and PFKFB3. Furthermore, we show that these effects are mediated upstream by the glycolytic enzyme, PKM2, and that direct inhibition of glycolysis or PKM2 nuclear localisation leads to a significant reduction in cholesterol crystal-induced inflammatory readouts. CONCLUSIONS: This study not only provides further insight into how atherosclerosis-associated DAMPs impact on immune cell function, but also highlights metabolic reprogramming as a potential therapeutic target for cholesterol crystal-related inflammation.

The Trypanosoma brucei-Derived Ketoacids, Indole Pyruvate and Hydroxyphenylpyruvate, Induce HO-1 Expression and Suppress Inflammatory Responses in Human Dendritic Cells.

The extracellular parasite and causative agent of African sleeping sickness Trypanosoma brucei (T. brucei) has evolved a number of strategies to avoid immune detection in the host. One recently described mechanism involves the conversion of host-derived amino acids to aromatic ketoacids, which are detected at relatively high concentrations in the bloodstream of infected individuals. These ketoacids have been shown to directly suppress inflammatory responses in murine immune cells, as well as acting as potent inducers of the stress response enzyme, heme oxygenase 1 (HO-1), which has proven anti-inflammatory properties. The aim of this study was to investigate the immunomodulatory properties of the T. brucei-derived ketoacids in primary human immune cells and further examine their potential as a therapy for inflammatory diseases. We report that the T. brucei-derived ketoacids, indole pyruvate (IP) and hydroxyphenylpyruvate (HPP), induce HO-1 expression through Nrf2 activation in human dendritic cells (DC). They also limit DC maturation and suppress the production of pro-inflammatory cytokines, which, in turn, leads to a reduced capacity to differentiate adaptive CD4+ T cells. Furthermore, the ketoacids are capable of modulating DC cellular metabolism and suppressing the inflammatory profile of cells isolated from patients with inflammatory bowel disease. This study therefore not only provides further evidence of the immune-evasion mechanisms employed by T. brucei, but also supports further exploration of this new class of HO-1 inducers as potential therapeutics for the treatment of inflammatory conditions.

Regulation of inflammation by the antioxidant haem oxygenase 1.

Haem oxygenase 1 (HO-1), an inducible enzyme responsible for the breakdown of haem, is primarily considered an antioxidant, and has long been overlooked by immunologists. However, research over the past two decades in particular has demonstrated that HO-1 also exhibits numerous anti-inflammatory properties. These emerging immunomodulatory functions have made HO-1 an appealing target for treatment of diseases characterized by high levels of chronic inflammation. In this Review, we present an introduction to HO-1 for immunologists, including an overview of its roles in iron metabolism and antioxidant defence, and the factors which regulate its expression. We discuss the impact of HO-1 induction in specific immune cell populations and provide new insights into the immunomodulation that accompanies haem catabolism, including its relationship to immunometabolism. Furthermore, we highlight the therapeutic potential of HO-1 induction to treat chronic inflammatory and autoimmune diseases, and the issues faced when trying to translate such therapies to the clinic. Finally, we examine a number of alternative, safer strategies that are under investigation to harness the therapeutic potential of HO-1, including the use of phytochemicals, novel HO-1 inducers and carbon monoxide-based therapies.

Trypanosoma brucei Secreted Aromatic Ketoacids Activate the Nrf2/HO-1 Pathway and Suppress Pro-inflammatory Responses in Primary Murine Glia and Macrophages.

African trypanosomes, such as Trypanosoma brucei (T. brucei), are protozoan parasites of the mammalian vasculature and central nervous system that are best known for causing fatal human sleeping sickness. As exclusively extracellular parasites, trypanosomes are subject to constant challenge from host immune defenses but they have developed very effective strategies to evade and modulate these responses to maintain an infection while simultaneously prolonging host survival. Here we investigate host parasite interactions, especially within the CNS context, which are not well-understood. We demonstrate that T. brucei strongly upregulates the stress response protein, Heme Oxygenase 1 (HO-1), in primary murine glia and macrophages in vitro. Furthermore, using a novel AHADHinT. brucei cell line, we demonstrate that specific aromatic ketoacids secreted by bloodstream forms of T. brucei are potent drivers of HO-1 expression and are capable of inhibiting pro-IL1ß induction in both glia and macrophages. Additionally, we found that these ketoacids significantly reduced IL-6 and TNFα production by glia, but not macrophages. Finally, we present data to support Nrf2 activation as the mechanism of action by which these ketoacids upregulate HO-1 expression and mediate their anti-inflammatory activity. This study therefore reports a novel immune evasion mechanism, whereby T. brucei secretes amino-acid derived metabolites for the purpose of suppressing both the host CNS and peripheral immune response, potentially via induction of the Nrf2/HO-1 pathway.

Plant-Derived Polyphenols Modulate Human Dendritic Cell Metabolism and Immune Function via AMPK-Dependent Induction of Heme Oxygenase-1.

Polyphenols are important immunonutrients which have been investigated in the context of inflammatory and autoimmune disease due to their significant immunosuppressive properties. However, the mechanism of action of many polyphenols is unclear, particularly in human immune cells. The emerging field of immunometabolism has highlighted the significance of metabolic function in the regulation of immune cell activity, yet the effects of polyphenols on immune cell metabolic signaling and function has not been explored. We have investigated the effects of two plant-derived polyphenols, carnosol and curcumin, on the metabolism of primary human dendritic cells (DC). We report that human DC display an increase in glycolysis and spare respiratory capacity in response to LPS stimulation, which was attenuated by both carnosol and curcumin treatment. The regulation of DC metabolism by these polyphenols appeared to be mediated by their activation of the cellular energy sensor, AMP-activated Protein Kinase (AMPK), which resulted in the inhibition of mTOR signaling in LPS-stimulated DC. Previously we have reported that both carnosol and curcumin can regulate the maturation and function of human DC through upregulation of the immunomodulatory enzyme, Heme Oxygenase-1 (HO-1). Here we also demonstrate that the induction of HO-1 by polyphenols in human DC is dependent on their activation of AMPK. Moreover, pharmacological inhibition of AMPK was found to reverse the observed reduction of DC maturation by carnosol and curcumin. This study therefore describes a novel relationship between metabolic signaling via AMPK and HO-1 induction by carnosol and curcumin in human DC, and characterizes the effects of these polyphenols on DC immunometabolism for the first time. These results expand our understanding of the mechanism of action of carnosol and curcumin in human immune cells, and suggest that polyphenol supplementation may be useful to regulate the metabolism and function of immune cells in inflammatory and metabolic disease.

Naturally derived Heme-Oxygenase 1 inducers attenuate inflammatory responses in human dendritic cells and T cells: relevance for psoriasis treatment.

Psoriasis is a chronic autoimmune disease mediated by dysregulated immune responses in dendritic cells (DC) and T cells. The stress-response enzyme heme oxygenase-1 (HO-1) has been described as protective in animal models of psoriasis, however, implementation of HO-1-based therapies is hindered by the lack of clinically-suitable HO-1 inducers. The plant-derived polyphenols, carnosol and curcumin, have been identified as candidate HO-1 inducers however there has been little investigation into their effects on human immune cells. We demonstrate that treatment of human DC with these polyphenols limits DC maturation, reduces pro-inflammatory cytokine production, and prevents induction of allospecific T cell responses, in a manner partially dependent on carbon monoxide (CO). We also characterised their effects in ex-vivo psoriasis PBMC and report that curcumin, but not carnosol, strongly reduces T cell proliferation and cytokine poly-functionality, with reduced expression of psoriatic cytokines IFNγ, IL-17, GM-CSF and IL-22. This study therefore supports reports highlighting the therapeutic potential of curcumin in psoriasis by providing insight into its immunological effects on healthy human DC and psoriasis PBMC. We also demonstrate, for the first time, the anti-inflammatory effects of carnosol in human immune cells.