IDH2 deficiency promotes mitochondrial dysfunction and cardiac hypertrophy in mice

Cardiac hypertrophy, a risk factor for heart failure, is associated with enhanced oxidative stress in the mitochondria, resulting from high levels of reactive oxygen species (ROS). The balance between ROS generation and ROS detoxification dictates ROS levels. As such, disruption of these processes results in either increased or decreased levels of ROS. In previous publications, we have demonstrated that one of the primary functions of mitochondrial NADP⁺-dependent isocitrate dehydrogenase (IDH2) is to control the mitochondrial redox balance, and thereby mediate the cellular defense against oxidative damage, via the production of NADPH. To explore the association between IDH2 expression and cardiac function, we measured myocardial hypertrophy, apoptosis, and contractile dysfunction in IDH2 knockout (idh2^−/−) and wild-type (idh2^+/+) mice. As expected, mitochondria from the hearts of knockout mice lacked IDH2 activity and the hearts of IDH2-deficient mice developed accelerated heart failure, increased levels of apoptosis and hypertrophy, and exhibited mitochondrial dysfunction, which was associated with a loss of redox homeostasis. Our results suggest that IDH2 plays an important role in maintaining both baseline mitochondrial function and cardiac contractile function following pressure-overload hypertrophy, by preventing oxidative stress.     

Aminooxyacetic acid attenuates post‐infarct cardiac dysfunction by balancing macrophage polarization through modulating macrophage metabolism in mice

Excessive activation of pro‐inflammatory M1 macrophages following acute myocardial infarction (MI) aggravates adverse cardiac remodelling and heart dysfunction. There are two break points in the tricarboxylic acid cycle of M1 macrophages, and aspartate‐arginosuccinate shunt compensates them. Aminooxyacetic acid (AOAA) is an inhibitor of aspartate aminotransferase in the aspartate‐arginosuccinate shunt. Previous studies showed that manipulating macrophage metabolism may control macrophage polarization and inflammatory response. In this study, we aimed to clarify the effects of AOAA on macrophage metabolism and polarization and heart function after MI. In vitro, AOAA inhibited lactic acid and glycolysis and enhanced ATP levels in classically activated M1 macrophages. Besides, AOAA restrained pro‐inflammatory M1 macrophages and promoted anti‐inflammatory M2 phenotype. In vivo, MI mice were treated with AOAA or saline for three consecutive days. Remarkably, AOAA administration effectively inhibited the proportion of M1 macrophages and boosted M2‐like phenotype, which subsequently attenuated infarct size as well as improved post‐MI cardiac function. Additionally, AOAA attenuated NLRP3‐Caspase1/IL‐1β activation and decreased the release of IL‐6 and TNF‐α pro‐inflammatory cytokines and reciprocally increased IL‐10 anti‐inflammatory cytokine level in both ischaemic myocardium and M1 macrophages. In conclusion, short‐term AOAA treatment significantly improves cardiac function in mice with MI by balancing macrophage polarization through modulating macrophage metabolism and inhibiting NLRP3‐Caspase1/IL‐1β pathway.     

C1q/TNF-related protein-9 promotes macrophage polarization and improves cardiac dysfunction after myocardial infarction

The timely regulation of inflammatory M1 macrophage polarization toward regenerative M2 macrophages suggests the possibility of immunotherapy after myocardial infarction (MI). C1q/TNF-related protein-9 (CTRP9) has anti-inflammatory effects and can ameliorate heart function in mice after long-term myocardial infarction. The role of CTRP9 in macrophage polarization remains completely unclear. This study determined whether CTRP9 can preserve post-MI early cardiac function through the regulation of macrophage polarization. In the present study, an adenovirus-delivered CTRP9 supplement promoted macrophage polarization at Day 3 post MI and improved cardiac function at Day 7 post MI. Pretreatment with gCTRP9 promoted the M1 to M2 polarization transition and attenuated inflammation after lipopolysaccharide + interferon-γ stimulation; the effects were partly abrogated by the adenosine monophosphate kinase (AMPK) inhibitor compound C and were obviously reinforced by pyrrolidine dithiocarbamate, a nuclear factor-κB (NF-κB) inhibitor. Meanwhile, CTPR9 markedly reduced the expression of toll-like receptor 4 (TLR4), myeloid differentiation factor 88 (MyD88), and NF-κB p65 phosphorylation by promoting AMPK phosphorylation in vivo and in vitro. Moreover, the competitive binding of gCTRP9 and LPS to the myeloid differentiation protein 2 (MD2)/TLR4 complex was associated with direct binding to MD2, thereby inhibiting the downstream signaling molecule MyD88. Taken together, we demonstrated that CTRP9 improved post-MI early cardiac function, at least in part, by modulating M1/M2 macrophage polarization, largely via the TLR4/MD2/MyD88 and AMPK-NF-κB pathways.     

Urokinase plasminogen activator receptor promotes macrophage infiltration into the vascular wall of ApoE deficient mice

The urokinase plasminogen activator receptor (uPAR) regulates macrophage adhesion and migration by binding directly to matrix proteins and signaling through integrin complexes. In this study, we examined the role of uPAR on macrophage infiltration into the vascular wall. Stable murine macrophage (Raw264.7) cell lines expressing high levels of human uPAR, human urokinase plasminogen activator (uPA), or both were established using expression vectors driven by the human CD68 promoter. Stimulation with human uPA specifically induced phosphorylation of early response regulated kinase (ERK) in cells expressing human uPAR but not in sham transfected cells. The human uPAR expressing Raw264.7 cells showed increased adhesion to both human uPA and vitronectin (Vn). Raw264.7 cells expressing human uPAR or both human uPAR and uPA, but not uPA alone, were detected in the aortic wall of ApoE(-/-) mice, and no cells were detected in that of age-matched C57BL/6J mice after intravenous infusion of the cells. Blocking of Mac-1/ICAM-1 interaction by anti-alphaM antibody (M1/70) significantly reduced the infiltration of huPAR-expressing Raw264.1 cells into aorta of ApoE(-/-) mice. Treatment of C57BL/6J mice with angiotensin II resulted in infiltration of Raw264.7 cells expressing human uPAR. These data demonstrate that uPAR plays a key role in promoting macrophage infiltration into the arterial wall of ApoE(-/-) mice.     

Loss of G2A promotes macrophage accumulation in atherosclerotic lesions of low density lipoprotein receptor-deficient mice

Lysophosphatidylcholine (LPC) is considered a major proatherogenic component of oxidized low density lipoprotein based on its proinflammatory actions in vitro. LPC stimulates macrophage and T-cell chemotaxis via the G protein-coupled receptor G2A and may thus promote inflammatory cell infiltration during atherosclerotic lesion development. However, G2A also mediates proapoptotic effects of LPC and may therefore promote the death of inflammatory cells within developing lesions. To determine how these effects of LPC modify atherogenesis, we examined atherosclerotic lesion development in G2A-sufficient and G2A-deficient low density lipoprotein receptor knockout mice. Although LPC species capable of activating G2A-dependent responses were increased during lesion development, G2A-deficient mice developed lesions similar in size to those in their G2A-sufficient counterparts. Loss of G2A during atherosclerotic lesion development did not reduce macrophage and T-cell infiltration but instead resulted in increased lesional macrophage content associated with reduced numbers of terminal deoxynucleotidyl transferase-mediated dUTP nick end labeled cells and decreased collagen deposition. These data indicate that the ability of LPC to stimulate macrophage and T-cell chemotaxis via G2A is not manifested in vivo and that G2A-mediated proapoptotic rather than chemotactic action is most penetrant during atherogenesis and may modify the stability of atherosclerotic lesions by promoting macrophage death.     

TREM2 expression promotes liver and peritoneal M2 macrophage polarization in mice infected with Schistosoma japonicum

Schistosomiasis is a tropical parasitic disease that damages the liver and poses a serious threat to human health. Macrophages play a key role in the development of liver granulomas and fibrosis by undergoing polarization from M1 to M2 type during schistosomiasis. Therefore, regulating macrophage polarization is important for controlling pathological changes that occur during this disease. Triggering receptor expressed on myeloid cells 2 (TREM2) expressed on the surface of macrophages, dendritic cells and other immune cells has been shown to play a role in inhibiting inflammatory responses and regulating M2 macrophage polarization, however its role in macrophage polarization in schistosomiasis has not been investigated. In this study, we confirmed that TREM2 expression was upregulated in the livers and peritoneal macrophages of mice infected with Schistosoma japonicum. Moreover, the TREM2 expression trend correlated with the expression of M2 macrophage polarization-related molecules in the liver tissues of S. japonicum-infected mice. Using Trem2^−/− mice, we also showed that Trem2 deletion inhibited Arg1 and Ym1 expression in liver tissues. Trem2 deletion also increased the number of F4/80 + CD86+ cells in peritoneal macrophages of infected mice. In summary, our study suggests that TREM2 may be involved in M2 macrophage polarization during schistosomiasis.     

Deoxyhypusine synthase promotes a pro-inflammatory macrophage phenotype

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IL‐7 suppresses macrophage autophagy and promotes liver pathology in Schistosoma japonicum‐infected mice

In schistosomiasis japonica and mansoni, parasite eggs trapped in host liver elicit severe liver granulomatous inflammation that subsequently leads to periportal fibrosis, portal hypertension, haemorrhage or even death. Macrophages are critical for granuloma formation and the development of liver fibrosis during schistosomiasis. However, whether the aberrant regulation of macrophage autophagy has an effect on the development of liver immunopathology in schistosomiasis remains to be elucidated. In this study, we showed that Schistosoma japonicum (S. japonicum) egg antigen (SEA)‐triggered macrophage autophagy limited the development of pathology in host liver. However, engagement of IL‐7 receptor (IL‐7R/CD127) on macrophages by S. japonicum infection‐induced IL‐7 significantly suppressed SEA‐triggered macrophage autophagy, which led to an enhanced liver pathology. In addition, anti‐IL‐7 neutralizing antibody or anti‐CD127 blocking antibody treatment increased macrophage autophagy and suppressed liver pathology. Finally, we demonstrated that IL‐7 protects macrophage against SEA‐induced autophagy through activation of AMP‐activated protein kinase (AMPK). Our study reveals a novel role for IL‐7 in macrophage autophagy and identifies AMPK as a novel downstream mediator of IL‐7‐IL‐7R signalling and suggests that manipulation of macrophage autophagy by targeting IL‐7‐IL‐7R signalling may have the potential to lead to improved treatment options for liver pathogenesis in schistosomiasis.     

Fucoidan blocks macrophage activation in an inductive phase but promotes macrophage activation in an effector phase

It has been suggested that some polysaccharides play important roles in immune responses. Therefore, we used various types of polysaccharides for analysis of macrophage-mediated tumor cell killing. We report here that fucoidan blocked macrophage activation occurs in an inductive phase but enhanced macrophage activation appears in an effector phase.     

Endothelial dysfunction promotes the transition from compensatory renal hypertrophy to kidney injury after unilateral nephrectomy in mice

Loss of functional nephrons associated with chronic kidney disease induces glomerular hyperfiltration and compensatory renal hypertrophy. We hypothesized that the endothelial nitric oxide synthase (eNOS) [soluble guanylate cyclase (sGC)] protein kinase G (PKG) pathway plays an important role in compensatory renal hypertrophy after unilateral nephrectomy. Analysis of mice subjected to unilateral nephrectomy showed increases in kidney weight-to-body weight and total protein-to-DNA ratios in wild-type but not eNOS knockout (eNOSKO) mice. Serum creatinine and blood urea nitrogen increased after nephrectomy in eNOSKO but not in wild-type mice. Furthermore, Bay 41-2272, an sGC stimulator, induced compensatory renal hypertrophy in eNOSKO mice and rescued renal function. The NO donor S-nitrosoglutathione (GSNO) and Bay 41-2272 stimulated PKG activity and induced phosphorylation of Akt protein in human proximal tubular cells. GSNO also induced phosphorylation of eukaryotic initiation factor 4E-binding protein and ribosomal protein S6. Our results highlight the importance of the eNOS-NO-PKG pathway in compensatory renal hypertrophy and suggest that reduced eNOS-NO bioavailability due to endothelial dysfunction is the underlying mechanism of failure of compensatory hypertrophy and acceleration of progressive renal dysfunction.     

Distribution of Adriamycin in mice bearing mammary adenocarcinoma 16/C

The response of solid mammary adenocarcinoma 16/C to treatment with Adriamycin is highly variable and ranges from growth under treatment to complete regression. Tumour and host factors were evaluated to determine the influence of each on the response. We determined that the concentration of Adriamycin in plasma and tumour was a function of tumour size and treatment history in mice bearing mammary adenocarcinoma 16/C. The plasma concentrations following a single dose of Adriamycin (10 mg/kg) increased in proportion to tumour mass without a concurrent increase in tumour concentration. When mice bearing large tumours (greater than 1.0 g) were treated with a multidose protocol, the plasma concentrations were higher and the tumour concentrations lower following the initial dose than following subsequent doses; in tumour-free mice, prior treatment with Adriamycin did not affect the plasma level achieved after a second dose. The magnitude of the decrease in plasma and increase in tumour concentrations was a function of the initial tumour size and the treatment schedule. The increase in tumour levels represented the sum of residual Adriamycin and drug bound as a result of the dose immediately prior to analysis. At the time of the initial treatment, the Adriamycin was distributed within each tumour in proportion to vascular perfusion. The percent of the tumour mass that was well-perfused appeared to increase with repeated treatments. The results indicate that the plasma concentration of Adriamycin did not necessarily reflect the tumour exposure in the mammary adenocarcinoma 16/C model. In hosts bearing mammary adenocarcinoma 16/C--or, possibly, other tumours that produce similar effects on the host--a low initial dose of Adriamycin might modify the distribution, possibly reduce the toxicity and allow escalation of subsequent doses with increased exposure of the tumour.     

Modelling mitochondrial dysfunction in mice

Understanding mitochondrial role in normal physiology and pathological conditions has proven to be of high importance as mitochondrial dysfunction is connected with a number of disorders as well as some of the most common diseases (e.g. diabetes or Parkinson's disease). Modeling mitochondrial dysfunction has been difficult mainly due to unique features of mitochondrial genetics. Here we discuss some of the most important mouse models generated so far and lessons learned from them.     

Occludin deficiency promotes ethanol-induced disruption of colonic epithelial junctions,gut barrier dysfunction and liver damage in mice

BackgroundDisruption of epithelial tight junctions (TJ), gut barrier dysfunction and endotoxemia play crucial role in the pathogenesis of alcoholic tissue injury. Occludin, a transmembrane protein of TJ, is depleted in colon by alcohol. However, it is unknown whether occludin depletion influences alcoholic gut and liver injury.MethodsWild type (WT) and occludin deficient (Ocln^−/−) mice were fed 1–6% ethanol in Lieber–DeCarli diet. Gut permeability was measured by vascular-to-luminal flux of FITC-inulin. Junctional integrity was analyzed by confocal microscopy. Liver injury was assessed by plasma transaminase, histopathology and triglyceride analyses. The effect of occludin depletion on acetaldehyde-induced TJ disruption was confirmed in Caco-2 cell monolayers.ResultsEthanol feeding significantly reduced body weight gain in Ocln^−/− mice. Ethanol increased inulin permeability in colon of both WT and Ocln^−/− mice, but the effect was 4-fold higher in Ocln^−/− mice. The gross morphology of colonic mucosa was unaltered, but ethanol disrupted the actin cytoskeleton, induced redistribution of occludin, ZO-1, E-cadherin and β-catenin from the junctions and elevated TLR4, which was more severe in Ocln^−/− mice. Occludin knockdown significantly enhanced acetaldehyde-induced TJ disruption and barrier dysfunction in Caco-2 cell monolayers. Ethanol significantly increased liver weight and plasma transaminase activity in Ocln^−/− mice, but not in WT mice. Histological analysis indicated more severe lesions and fat deposition in the liver of ethanol-fed Ocln^−/− mice. Ethanol-induced elevation of liver triglyceride was also higher in Ocln^−/− mice.ConclusionThis study indicates that occludin deficiency increases susceptibility to ethanol-induced colonic mucosal barrier dysfunction and liver damage in mice.     

Human cholesteryl ester transport protein transgene promotes macrophage reverse cholesterol transport in C57BL/6 mice and phospholipid transfer protein gene knockout mice

Journal of Physiology and Biochemistry - Cholesteryl ester transfer protein (CETP) and phospholipid transfer protein (PLTP) belong to the same gene family. Liver-specific expression of CETP...     

Delayed enhanced effects of Adriamycin on the X-irradiation-induced gastrointestinal toxicity in mice

The delayed responses of C3H mice which had been pretreated with various single-dose and two-dose fractionated Adriamycin/X-irradiation protocols were evaluated by stressing the 120-day survivors with either whole-abdomen X-irradiation (LD50/7 assay) or whole-body X-irradiation (crypt colony survival). Pretreatment with Adriamycin alone was as toxic as Adriamycin plus X-irradiation for the animals stressed at 120 days (LD50/7 assay). There was no induced cellular radioresistance (D0) and no apparent increase in crypt size as indicated indirectly by the 10-clone dose at 120 days after completion of treatment. The increased lethality of the X-irradiation-stressed 120-day survivors was most likely a primary gastrointestinal response with little or no contribution from either bone marrow or kidney toxicity. The effect was apparently due to a persistent Adriamycin-induced antiproliferative response at the cellular level but the molecular mechanisms are unknown. Such data suggest caution to our clinical colleagues. Cancer patients treated with high doses of Adriamycin, independent of concomitant X-irradiation, will most likely be moderately to severely compromised in their ability to respond to a stress which requires cellular proliferation, and, based on the murine data, this effect is persistent if, indeed, not permanent.     

Impairment of macrophage migration inhibitory factor synthesis and macrophage migration in protein-malnourished mice

Weanling CD2F1 mice were fed isocaloric diets that were protein sufficient (PS; containing 27% casein) or protein deficient (PD; containing 8% casein). Weight measurements demonstrated that the growth of PD mice was significantly impaired, thus indicating that the PD diet induced protein malnutrition. The cellular immune responsiveness of these mice was assessed from Day 21 to Day 49 of the diet using, as indicators, in vitro production of migration inhibitory factor (MIF) by splenic lymphocytes and MIF responsiveness of peritoneal macrophages. PD lymphocytes, when stimulated with the polyclonal activator concanavalin A, produced significantly less MIF than did PS lymphocytes. The amount of MIF produced by PD lymphocytes, however, increased throughout the study, possibly indicating delayed maturation of MIF synthetic capacity in PD mice. Normal CD2F1 mouse macrophages were used for these assays. MIF responsiveness of PD and PS macrophages was not significantly different when assayed using MIF produced by normal CD2F1 mouse lymphocytes. As compared to that of PS macrophages, the migratory ability of PD macrophages decreased progressively throughout the study. This impaired migratory ability did not interfere with MIF responsiveness of PD macrophages.