Uncontrolled microglial activation contributes to the pathogenesis of various neurodegenerative diseases. Previous studies have shown that proinflammatory microglia are powered by glycolysis, which relays on high levels of glucose uptake. This study aimed to understand how glucose uptake is facilitated in active microglia and whether microglial activation can be controlled by restricting glucose uptake. Primary murine brain microglia, BV2 cells and the newly established microglial cell line B6M7 were treated with LPS (100 ng/ml) + IFNγ (100 ng/ml) or IL-4 (20 ng/ml) for 24 h. The expression of glucose transporters (GLUTs) was examined by PCR and Western blot. Glucose uptake by microglia was inhibited using the GLUT1-specific inhibitor STF31. The metabolic profiles were tested using the Glycolysis Stress Test and Mito Stress Test Kits using the Seahorse XFe96 Analyser. Inflammatory gene expression was examined by real-time RT-PCR and protein secretion by cytokine beads array. The effect of STF31 on microglial activation and neurodegeneraion was further tested in a mouse model of light-induced retinal degeneration. The mRNA and protein of GLUT1, 3, 4, 5, 6, 8, 9, 10, 12, and 13 were detected in microglia. The expression level of GLUT1 was the highest among all GLUTs detected. LPS + IFNγ treatment further increased GLUT1 expression. STF31 dose-dependently reduced glucose uptake and suppressed Extracellular Acidification Rate (ECAR) in naïve, M(LPS + IFNγ) and M(IL-4) microglia. The treatment also prevented the upregulation of inflammatory cytokines including TNFα, IL-1β, IL-6, and CCL2 in M(LPS + IFNγ) microglia. Interestingly, the Oxygen Consumption Rates (OCR) was increased in M(LPS + IFNγ) microglia but reduced in M(IL-4) microglia by STF31 treatment. Intraperitoneal injection of STF31 reduced light-induced microglial activation and retinal degeneration. Glucose uptake in microglia is facilitated predominately by GLUT1, particularly under inflammatory conditions. Targeting GLUT1 could be an effective approach to control neuroinflammation.

Background Macrophages are tissue resident immune cells important for host defence and homeostasis. During diabetes, macrophages and other innate immune cells are known to have a pro-inflammatory phenotype, which is believed to contribute to the pathogenesis of various diabetic complications. However, diabetic patients are highly susceptible to bacterial infections, and often have impaired wound healing. The molecular mechanism underlying the paradox of macrophage function in diabetes is not fully understood. Recent evidence suggests that macrophage functions are governed by metabolic reprograming. Diabetes is a disorder that affects glucose metabolism; dysregulated macrophage function in diabetes may be related to alterations in their metabolic pathways. In this study, we seek to understand the effect of high glucose exposure on macrophage phenotype and functions. Results Bone marrow cells were cultured in short or long term high glucose and normal glucose medium; the number and phenotype of bone marrow derived macrophages were not affected by long-term high glucose treatment. Short-term high glucose increased the expression of IL-1β. Long-term high glucose increased the expression of IL-1β and TNFα but reduced the expression of IL-12p40 and nitric oxide production in M1 macrophage. The treatment also increased Arg-1 and IL-10 expression in M2 macrophages. Phagocytosis and bactericidal activity was reduced in long-term high glucose treated macrophages and peritoneal macrophages from diabetic mice. Long-term high glucose treatment reduced macrophage glycolytic capacity and glycolytic reserve without affecting mitochondrial ATP production and oxidative respiration. Conclusion Long-term high glucose sensitizes macrophages to cytokine stimulation and reduces phagocytosis and nitric oxide production, which may be related to impaired glycolytic capacity.

The retina, an immune privileged tissue, has specialized immune defense mechanisms against noxious insults that may exist in diseases such as age-related macular degeneration (AMD), diabetic retinopathy (DR), uveoretinitis and glaucoma. The defense system consists of retinal innate immune cells (including microglia, perivascular macrophages, and a small population of dendritic cells) and the complement system. Under normal aging conditions, retinal innate immune cells and the complement system undergo a low-grade activation (parainflammation) which is important for retinal homeostasis. In disease states such as AMD and DR, the parainflammatory response is dysregulated and develops into detrimental chronic inflammation. Complement activation in the retina is an important part of chronic inflammation and may contribute to retinal pathology in these disease states. Here, we review the evidence that supports the role of uncontrolled or dysregulated complement activation in various retinal degenerative and angiogenic conditions. We also discuss current strategies that are used to develop complement-based therapies for retinal diseases such as AMD. The potential benefits of complement inhibition in DR, uveoretinitis and glaucoma are also discussed, as well as the need for further research to better understand the mechanisms of complement-mediated retinal damage in these disease states.

Peritoneal macrophages are widely used in immunological studies. The cells can be collected under non-elicited (resident) or elicited (e.g., with Brewer thioglycollate broth injection) conditions, and their phenotype and functions differ. Recent studies have shown that macrophage phenotype and function are related to their metabolic states, and metabolic reprogramming has been an emerging concept for controlling macrophage function. In this study, we examined the metabolic state of resident and elicited macrophages and investigated how their metabolic state may affect cell function, including phagocytosis. Flow cytometry showed that elicited macrophages expressed higher levels of MHC-II, LFA-1 and CD64 but lower levels of F4/80 compared to naïve resident peritoneal macrophages, suggesting a more mature and active phenotype. Elicited macrophages had significantly higher levels of phagocytic activity compared to that of resident macrophages. Metabolic studies showed that the Extracellular Acidification Rates (ECAR) and Oxygen Consumption Rates (OCR) were both significantly higher in elicited macrophages than those in resident macrophages. The treatment of macrophages with 2-Deoxy-D-glucose suppressed glycolysis and reduced phagocytosis, whereas treatment with oligomycin enhanced glycolysis and increased phagocytosis in elicited macrophages. Naïve resident peritoneal macrophages are less metabolically active compared to elicited macrophages. Elicited macrophages had higher levels of glycolysis and oxidative phosphorylation, which may be related to their increased phagocytic capacity and higher levels of maturation and activation. Further understanding of the molecular links between metabolic pathways and cell function would be crucial to develop strategies to control macrophage function through metabolic reprogramming.

Reactive microglia are commonly seen in retinal degenerative diseases, and neurotoxic microglia responses can contribute to photoreceptor cell death. We and others have previously shown that translocator protein (18 kDa) (TSPO) is highly induced in retinal degenerations and that the selective TSPO ligand XBD173 (AC-5216, emapunil) exerts strong anti-inflammatory effects on microglia in vitro and ex vivo. Here, we investigated whether targeting TSPO with XBD173 has immuno-modulatory and neuroprotective functions in two mouse models of acute retinal degeneration using bright white light exposure. BALB/cJ and Cx3cr1 GFP/+ mice received intraperitoneal injections of 10 mg/kg XBD173 or vehicle for five consecutive days, starting 1 day prior to exposure to either 15,000 lux white light for 1 h or 50,000 lux focal light for 10 min, respectively. The effects of XBD173 treatment on microglia and Müller cell reactivity were analyzed by immuno-stainings of retinal sections and flat mounts, fluorescence-activated cell sorting (FACS) analysis, and mRNA expression of microglia markers using quantitative real-time PCR (qRT-PCR). Optical coherence tomography (OCT), terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) stainings, and morphometric analyses were used to quantify the extent of retinal degeneration and photoreceptor apoptosis. Four days after the mice were challenged with bright white light, a large number of amoeboid-shaped alerted microglia appeared in the degenerating outer retina, which was nearly completely prevented by treatment with XBD173. This treatment also down-regulated the expression of TSPO protein in microglia but did not change the TSPO levels in the retinal pigment epithelium (RPE). RT-PCR analysis showed that the microglia/macrophage markers Cd68 and activated microglia/macrophage whey acidic protein (Amwap) as well as the pro-inflammatory genes Ccl2 and Il6 were reduced after XBD173 treatment. Light-induced degeneration of the outer retina was nearly fully blocked by XBD173 treatment. We further confirmed these findings in an independent mouse model of focal light damage. Retinas of animals receiving XBD173 therapy displayed significantly more ramified non-reactive microglia and more viable arrestin-positive cone photoreceptors than vehicle controls. Targeting TSPO with XBD173 effectively counter-regulates microgliosis and ameliorates light-induced retinal damage, highlighting a new pharmacological concept for the treatment of retinal degenerations.

Infiltrating immune cells including monocytes/macrophages have been implicated in the pathogenesis of neovascular age-related macular degeneration (nAMD). The aim of this study was to investigate the cytokine and chemokine expression and secretion profile of peripheral blood mononuclear cells (PBMCs) from nAMD patients and the relationship between the cytokine/chemokine expression profile and clinical phenotype of nAMD, including macular fibrosis, macular atrophy or the responsiveness to anti-VEGF therapy. One hundred sixty-one nAMD patients and 43 controls were enrolled in this study. nAMD patients were divided into subgroups based on the presence/absence of (1) macular atrophy, (2) macular fibrosis and (3) responsiveness to anti-VEGF therapy; 25–30 ml of peripheral blood were obtained from all participants and 5 ml were used for serum collection, and the remaining were used for PBMC isolation using density gradient centrifugation. Intracellular cytokine expressions by PBMCs following phorbol 12-myristate 13-acetate (PMA) and ionomycin stimulation were examined using flow cytometry. Cytokine productions in lipopolysaccharides (LPS)-or 1% oxygen -treated PBMC were measured using cytometric bead array (CBA) assay. In addition, cytokine and chemokine levels in the serum were also measured by CBA assay. PBMCs from nAMD patients secreted higher levels of IL-8, CCL2 and VEGF, especially following LPS and 1% oxygen stimulation, than those from controls. 60~80% of IL-8 producing cells were CD11b+CD3− monocytes. The percentage of CD11b+CD3− IL-8+ was significantly increased in nAMD patients compared to controls. PBMCs from nAMD patients without macular fibrosis produced the highest levels of IL-8 and CCL2, whilst PBMCs from nAMD patients with macular atrophy produced highest levels of VEGF. In addition, PBMCs from patients who partially responded to anti-VEGF produced higher levels of IL-8 compared to the cells from complete responders. Interestingly, serum level of CCL2 was not increased in nAMD patients although there was a trend of increased IL-8 in nAMD patients. PBMCs, in particular monocytes, may contribute to CNV development in nAMD through secreting elevated levels of IL-8, CCL2 and VEGF after they are recruited to the macula. Apart from VEGF, IL-8 and CCL2 may be additional targets for nAMD management.