Macrophage fumarate hydratase restrains mtRNA-mediated interferon production

Metabolic rewiring underlies the effector functions of macrophages1-3, but the mechanisms involved remain incompletely defined. Here, using unbiased metabolomics and stable isotope-assisted tracing, we show that an inflammatory aspartate-argininosuccinate shunt is induced following lipopolysaccharide stimulation. The shunt, supported by increased argininosuccinate synthase (ASS1) expression, also leads to increased cytosolic fumarate levels and fumarate-mediated protein succination. Pharmacological inhibition and genetic ablation of the tricarboxylic acid cycle enzyme fumarate hydratase (FH) further increases intracellular fumarate levels. Mitochondrial respiration is also suppressed and mitochondrial membrane potential increased. RNA sequencing and proteomics analyses demonstrate that there are strong inflammatory effects resulting from FH inhibition. Notably, acute FH inhibition suppresses interleukin-10 expression, which leads to increased tumour necrosis factor secretion, an effect recapitulated by fumarate esters. Moreover, FH inhibition, but not fumarate esters, increases interferon-β production through mechanisms that are driven by mitochondrial RNA (mtRNA) release and activation of the RNA sensors TLR7, RIG-I and MDA5. This effect is recapitulated endogenously when FH is suppressed following prolonged lipopolysaccharide stimulation. Furthermore, cells from patients with systemic lupus erythematosus also exhibit FH suppression, which indicates a potential pathogenic role for this process in human disease. We therefore identify a protective role for FH in maintaining appropriate macrophage cytokine and interferon responses

Design and synthesis of a stable oxidized phospholipid mimic with specific binding recognition for macrophage scavenger receptors

Macrophage scavenger receptors appear to play a major role in the clearance of oxidized phospholipid (OxPL) products. Discrete peptide-phospholipid conjugates with the phosphatidylcholine head group have been shown to exhibit binding affinity for these receptors. We report the preparation of a water soluble, stable peptide-phospholipid conjugate (9) that possesses the necessary physical properties to enable more detailed study of the role(s) of OxPL in metabolic disease

Design and Synthesis of a Stable Oxidized Phospholipid Mimic with Specific Binding Recognition for Macrophage Scavenger Receptors

Macrophage scavenger receptors appear to play a major role in the clearance of oxidized phospholipid (OxPL) products. Discrete peptide–phospholipid conjugates with the phosphatidylcholine headgroup have been shown to exhibit binding affinity for these receptors. We report the preparation of a water-soluble, stable peptide–phospholipid conjugate (<b>9</b>) that possesses the necessary physical properties to enable more detailed study of the role(s) of OxPL in metabolic disease

eNAMPT/TLR4 inflammatory cascade activation is a key contributor to SLE Lung vasculitis and alveolar hemorrhage

Rationale: Effective therapies to reduce the severity and high mortality of pulmonary vasculitis and diffuse alveolar hemorrhage (DAH) in patients with systemic lupus erythematosus (SLE) is a serious unmet need. We explored whether biologic neutralization of eNAMPT (extracellular nicotinamide phosphoribosyl-transferase), a novel DAMP and Toll-like receptor 4 ligand, represents a viable therapeutic strategy in lupus vasculitis. Methods: Serum was collected from SLE subjects (n = 37) for eNAMPT protein measurements. In the preclinical pristane-induced murine model of lung vasculitis/hemorrhage, C57BL/6 J mice (n = 5–10/group) were treated with PBS, IgG (1 mg/kg), or the eNAMPT-neutralizing ALT-100 mAb (1 mg/kg, IP or subcutaneously (SQ). Lung injury evaluation (Day 10) included histology/immuno-histochemistry, BAL protein/cellularity, tissue biochemistry, RNA sequencing, and plasma biomarker assessment. Results: SLE subjects showed highly significant increases in blood NAMPT mRNA expression and eNAMPT protein levels compared to healthy controls. Preclinical pristane-exposed mice studies showed significantly increased NAMPT lung tissue expression and increased plasma eNAMPT levels accompanied by marked increases in alveolar hemorrhage and lung inflammation (BAL protein, PMNs, activated monocytes). In contrast, ALT-100 mAb-treated mice showed significant attenuation of inflammatory lung injury, alveolar hemorrhage, BAL protein, tissue leukocytes, and plasma inflammatory cytokines (eNAMPT, IL-6, IL-8). Lung RNA sequencing showed pristane-induced activation of inflammatory genes/pathways including NFkB, cytokine/chemokine, IL-1β, and MMP signaling pathways, each rectified in ALT-100 mAb-treated mice. Conclusions: These findings highlight the role of eNAMPT/TLR4-mediated inflammatory signaling in the pathobiology of SLE pulmonary vasculitis and alveolar hemorrhage. Biologic neutralization of this novel DAMP appears to serve as a viable strategy to reduce the severity of SLE lung vasculitis

IL-16/miR-125a axis controls neutrophil recruitment in pristane-induced lung inflammation

Severe lung inflammation and alveolar hemorrhage can be life-threatening in systemic lupus erythematosus (SLE) patients if not treated early and aggressively. Neutrophil influx is the driver key of this pathology, but little is known regarding the molecular events regulating this recruitment. Here, we uncover a role for IL-16/mir-125a in this pathology and show not only that IL-16 is a target for miR-125a but that reduced miR-125a expression in SLE patients associates with lung involvement. Furthermore, in the pristane model of acute "SLE-like" lung inflammation and alveolar hemorrhage, we observed reduced pulmonary miR-125a and enhanced IL-16 expression. Neutrophil infiltration was markedly reduced in the peritoneal lavage of pristane-treated IL-16-deficient mice and elevated following i.n. delivery of IL-16. Moreover, a miR-125a mimic reduced pristane-induced IL-16 expression and neutrophil recruitment and rescued lung pathology. Mechanistically, IL-16 acts directly on the pulmonary epithelium and markedly enhances neutrophil chemoattractant expression both in vitro and in vivo, while the miR-125a mimic can prevent this. Our results reveal a role for miR-125a/IL-16 in regulating lung inflammation and suggest this axis may be a therapeutic target for management of acute lung injury in SLE

IL-16/miR-125a axis controls neutrophil recruitment in pristane-induced lung inflammation

Severe lung inflammation and alveolar hemorrhage can be life-threatening in systemic lupus erythematosus (SLE) patients if not treated early and aggressively. Neutrophil influx is the driver key of this pathology, but little is known regarding the molecular events regulating this recruitment. Here, we uncover a role for IL-16/mir-125a in this pathology and show not only that IL-16 is a target for miR-125a but that reduced miR-125a expression in SLE patients associates with lung involvement. Furthermore, in the pristane model of acute "SLE-like" lung inflammation and alveolar hemorrhage, we observed reduced pulmonary miR-125a and enhanced IL-16 expression. Neutrophil infiltration was markedly reduced in the peritoneal lavage of pristane-treated IL-16-deficient mice and elevated following i.n. delivery of IL-16. Moreover, a miR-125a mimic reduced pristane-induced IL-16 expression and neutrophil recruitment and rescued lung pathology. Mechanistically, IL-16 acts directly on the pulmonary epithelium and markedly enhances neutrophil chemoattractant expression both in vitro and in vivo, while the miR-125a mimic can prevent this. Our results reveal a role for miR-125a/IL-16 in regulating lung inflammation and suggest this axis may be a therapeutic target for management of acute lung injury in SLE