Noriaki Kawanishi

Exercise training attenuates hepatic inflammation, fibrosis and macrophage infiltration during diet induced-obesity in mice

Nonalcoholic steatohepatitis, which is considered the hepatic event in metabolic syndrome, was recently associated with the innate immune system. Although regular exercise reduces hepatic injury markers like serum alanine aminotransferase (ALT) levels, the mechanisms regulating the effects of exercise on steatohepatitis are unclear. This study aimed to clarify whether exercise training suppresses hepatic injury, inflammation, and fibrosis by suppressing macrophage infiltration. Male C57BL/6J (4-week old) mice were randomly divided into four groups: normal diet (ND) control (n=7), ND exercise (n=5), high-fat diet and high-fructose water (HFF) control (n=11), and HFF exercise (n=11) groups. Mice were fed the ND or HFF from 4 to 20 weeks of age. The exercise groups were trained on a motorized treadmill for 60 min/day, five times/week. The nonalcoholic fatty liver disease (NAFLD) activity score and plasma ALT activity, indicators of liver injury, were increased in HFF control mice but were attenuated in HFF exercise mice. Hepatic inflammation, indicated by hepatic tumor necrosis factor (TNF)-α levels and hepatic resident macrophage infiltration, was significantly lower in HFF exercise mice than in HFF control mice. Hepatic fibrosis markers (histological hepatic fibrosis detected by Sirius red and α-smooth muscle actin staining and tissue inhibitor of matrix metalloproteinase-1 mRNA) were attenuated in HFF exercise mice compared with HFF control mice. These results suggest that exercise training reduces hepatic inflammation, injury, and fibrosis by suppressing macrophage infiltration.

Mechanism of cell death by 5‐aminolevulinic acid‐based photodynamic action and its enhancement by ferrochelatase inhibitors in human histiocytic lymphoma cell line U937

Photodynamic therapy (PDT) for tumors is based on the tumor‐selective accumulation of a photosensitizer, protoporphyrin IX (PpIX), followed by irradiation with visible light. However, the molecular mechanism of cell death caused by PDT has not been fully elucidated. The 5‐aminolevulinic acid (ALA)‐based photodynamic action (PDA) was dependent on the accumulation of PpIX, the level of which decreased rapidly by eliminating ALA from the incubation medium in human histiocytic lymphoma U937 cells. PDA induced apoptosis characterized by lipid peroxidation, increase in Bak and Bax/Bcl‐xL, decrease in Bid, membrane depolarization, cytochrome c release, caspase‐3 activation, phosphatidylserine (PS) externalization. PDT‐induced cell death seemed to occur predominantly via apoptosis through distribution of PpIX in mitochondria. These cell death events were enhanced by ferrochelatase inhibitors. These results indicated that ALA‐based‐PDA induced apoptotic cell death through a mitochondrial pathway and that ferrochelatase inhibitors might enhanced the effect of PDT for tumors even at low concentrations of ALA. Copyright

Exercise attenuates M1 macrophages and CD8+ T cells in the adipose tissue of obese mice.

PURPOSE Obesity is associated with adipose tissue inflammation, which has been attributed to changes in the number and types of leukocytes in adipose tissue. Exercise training is thought to be important for the reduction of adipose tissue inflammation, but the mechanisms by which this may occur are incompletely understood. Here, we evaluated the effect of exercise training on several inflammation-associated changes in adipose tissue, including infiltration of inflammatory macrophages and T cells. METHODS Four-week-old male C57BL/6J mice were randomly assigned to four groups that received a normal diet (ND) plus sedentary (n = 8), an ND plus exercise training (n = 8), a high-fat diet (HFD) plus sedentary (n = 12), and an HFD plus exercise training (n = 12). Mice were fed the ND or the HFD from 4 to 20 wk of age. Mice in the exercise groups ran on a treadmill for 60 min·d, 5 d·wk over the same points. RESULTS Mice fed the HFD had increased numbers of macrophage clusters in adipose tissue, which were reduced by exercise training. Similarly, adipose tissue from the HFD sedentary mice contained higher levels of tumor necrosis factor α mRNA and increased numbers of CD11c inflammatory macrophages and CD8 T cells than adipose tissue from the ND mice, and those were also lowered by exercise training. The mRNA levels of monocyte chemoattractant proteins 1 and 2 and macrophage inflammatory proteins 1α and 1β in adipose tissue were lower in the HFD exercise mice than those in the HFD sedentary mice. CONCLUSIONS The results suggest that exercise training reduces adipose tissue inflammation by suppressing infiltration of inflammatory macrophages and CD8 T cells.

Curcumin attenuates oxidative stress following downhill running-induced muscle damage

Downhill running causes muscle damage, and induces oxidative stress and inflammatory reaction. Recently, it is shown that curcumin possesses anti-oxidant and anti-inflammatory potentials. Interestingly, curcumin reduces inflammatory cytokine concentrations in skeletal muscle after downhill running of mice. However, it is not known whether curcumin affects oxidative stress after downhill running-induced muscle damage. Therefore, the purpose of this study was to investigate the effects of curcumin on oxidative stress following downhill running induced-muscle damage. We also investigated whether curcumin affects macrophage infiltration via chemokines such as MCP-1 and CXCL14. Male C57BL/6 mice were divided into four groups; rest, rest plus curcumin, downhill running, or downhill running plus curcumin. Downhill running mice ran at 22 m/min, -15% grade on the treadmill for 150 min. Curcumin (3mg) was administered in oral administration immediately after downhill running. Hydrogen peroxide concentration and NADPH-oxidase mRNA expression in the downhill running mice were significantly higher than those in the rest mice, but these variables were significantly attenuated by curcumin administration in downhill running mice. In addition, mRNA expression levels of MCP-1, CXCL14 and F4/80 reflecting presence of macrophages in the downhill running mice were significantly higher than those in the rest mice. However, MCP-1 and F4/80 mRNA expression levels were significantly attenuated by curcumin administration in downhill running mice. Curcumin may attenuate oxidative stress following downhill running-induced muscle damage.

Exhaustive Exercise Reduces Tumor Necrosis Factor-α Production in Response to Lipopolysaccharide in Mice

Objective: Stressful exercise reduces the plasma pro-inflammatory cytokine concentration in response to lipopolysaccharide (LPS). The aim of this study was to clarify the mechanism of exhaustive exercise-induced suppression of the plasma tumor necrosis factor (TNF)-α concentration in response to LPS. Methods: Male C3H/HeN mice (n = 66) were randomized to treadmill running to exhaustion (Ex) or a sedentary (Non-Ex) condition. Monocytes and splenic macrophages were collected from some animals, and other animals were injected with LPS (1 mg/kg) immediately after the exercise. The liver, lung and spleen tissues in the mice were removed 30 min after the LPS injection for determination of TNF-α mRNA expression. Blood and tissue samples were collected for determination of TNF-α and TNF receptors (TNFR) 1 h after the LPS injection. Results: Although there was a significant suppression in LPS-induced plasma TNF-α in the Ex mice when compared to the Non-Ex mice (p < 0.01), soluble TNFR in plasma was not affected by the exercise. There was no change in cell-surface expression of Toll-like receptor 4 (TLR4) and in LPS-induced TNF-α mRNA expression and TNFR content in tissues between the Ex and Non-Ex groups. Interestingly, TNF-α contents in the liver, lung and spleen of the Ex mice were significantly lower than those of the Non-Ex group (p < 0.01, p < 0.01 and p < 0.05, respectively). Conclusion: These data suggest that exhaustive exercise-induced suppression of the plasma TNF-α concentration despite LPS stimulation might depend on translation of TNF-α in tissues.

Neutrophil Depletion Attenuates Muscle Injury after Exhaustive Exercise.

PURPOSE The infiltration of macrophages in skeletal muscle during exhaustive exercise promotes inflammation, myofiber lesion, and muscle injury. Although neutrophils upregulate macrophage infiltration in skeletal muscles during exercise, the role of neutrophils in promoting muscle injury after exhaustive exercise remains unclear. In this study, we investigated the effects of preexercise neutrophil depletion with antineutrophil antibody treatment on muscle injury, inflammation, and macrophage infiltration after exhaustive exercise. METHODS Male C57BL/6J mice were randomly assigned to four groups, namely, sedentary with control antibody (n = 10), sedentary with antineutrophil antibody (n = 10), exhaustive exercise with control antibody (n = 10), and exhaustive exercise with antineutrophil antibody (n = 10). The mice were given intraperitoneal injection of the antineutrophil antibody (anti-Ly-6G, clone 1A8) or the control antibody (anti-Ly-6G, clone 2A3), and remained inactive or performed exhaustive exercise on a treadmill 48 h after the injection. Twenty-four hours after the exhaustive exercise, the gastrocnemius muscles were removed for histological and polymerase chain reaction (PCR) analyses. Infiltration of neutrophils and macrophages was evaluated with Ly-6G and F4/80 immunohistochemistry staining procedures. Muscle fiber injury was detected based on the number of IgG staining fiber. The mRNA expression levels of proinflammatory cytokines and chemokines were evaluated with real-time reverse transcription PCR. RESULTS Exhaustive exercise increased neutrophil infiltration into the gastrocnemius muscle substantially by 3.1-fold and caused muscle injury, but these effects were markedly suppressed by preexercise treatment with antineutrophil antibody (neutrophil infiltration, 0.42-fold, and muscle injury, 0.18-fold). Treatment with antineutrophil antibody also decreased macrophage infiltration (0.44-fold) and mRNA expression of tumor necrosis factor-α (0.55-fold) and interleukin-6 (0.51-fold) in the skeletal muscle after exhaustive exercise. CONCLUSION These results suggest that neutrophils contribute to exacerbating muscle injury by regulating inflammation through the induction of macrophage infiltration.

Exercise training attenuates adipose tissue fibrosis in diet-induced obese mice

Tissue fibrosis, such as that which occurs in obesity, is associated with chronic inflammatory diseases. Although regular exercise reduces adipose tissue inflammation, the mechanisms regulating the effects of exercise on adipose tissue fibrosis are unclear. This study aimed to clarify whether exercise training attenuates adipose tissue fibrosis with consequent reduction of extracellular matrix including collagens. Male C57BL/6J (4-week old) mice were randomly assigned to four groups that received a normal diet (ND) plus sedentary (n=8), an ND plus exercise training (n=8), a high-fat diet (HFD) plus sedentary (n=12), and an HFD plus exercise training (n=12). Mice were fed the ND or HFD from 4 to 20 weeks of age. The exercise groups were trained on a motorized treadmill for 60 min/day, 5 times/week over the same period. Histological hepatic fibrosis detected by Sirius red and α-smooth muscle actin staining were attenuated in HFD exercise mice compared with HFD sedentary mice. mRNA levels of transforming growth factor-β and tissue inhibitors of metalloproteinase-1, major regulators of tissue fibrosis, were increased in HFD sedentary mice but were attenuated in HFD exercise mice. Similarly, adipose tissue from the HFD sedentary mice contained higher macrophages than adipose tissue from the ND mice, and this was also lowered by exercise training. These findings suggest that exercise training may be effective for attenuating adipose tissue inflammation in obesity.

Exercise training attenuates neutrophil infiltration and elastase expression in adipose tissue of high‐fat‐diet‐induced obese mice

The innate immune system is associated with the development of local inflammation. Neutrophils play an essential role in the development of the adipose tissue (AT) inflammation associated with obesity by producing elastase, which can promote the activation and infiltration of macrophages. Exercise training attenuates AT inflammation via suppression of macrophage infiltration. However, the mechanisms driving this phenomenon remains to be elucidated. Here, we evaluated the effects of exercise training on the infiltration of neutrophils and elastase expression in an obese mouse model. Four‐week‐old male C57BL/6J mice were randomly assigned to one of three groups that either received a normal diet (ND) plus sedentary activity (n = 15), a high‐fat diet (HFD) plus sedentary activity (n = 15), or a HFD plus exercise training (n = 15). Mice were fed the ND or HFD from the age of 4 weeks until 20 weeks. Mice in the exercise group ran on a treadmill for 60 min/day, 5 days/week over the same experimental period. Mice fed with the HFD had increased content of macrophages in the AT and increased inflammatory cytokine mRNA levels, which were reduced by exercise training. Similarly, AT from the HFD sedentary mice contained more neutrophils than AT from the ND mice, and the amount of neutrophils in this tissue in HFD‐fed mice was lowered by exercise training. The mRNA levels of neutrophil elastase in AT were lower in the HFD exercise‐trained mice than those in the HFD sedentary mice. These results suggest that exercise training plays a critical role in reducing macrophage infiltration and AT inflammation by regulating the infiltration of neutrophils.

Macrophage depletion by clodronate liposome attenuates muscle injury and inflammation following exhaustive exercise

Exhaustive exercise promotes muscle injury, including myofiber lesions; however, its exact mechanism has not yet been elucidated. In this study, we tested the hypothesis that macrophage depletion by pretreatment with clodronate liposomes alters muscle injury and inflammation following exhaustive exercise. Male C57BL/6J mice were divided into four groups: rest plus control liposome (n=8), rest plus clodronate liposome (n=8), exhaustive exercise plus control liposome (n=8), and exhaustive exercise plus clodronate liposome (n=8). Mice were treated with clodronate liposome or control liposome for 48 h before undergoing exhaustive exercise on a treadmill. Twenty-four hours after exhaustive exercise, the gastrocnemius muscles were removed for histological and PCR analyses. Exhaustive exercise increased the number of macrophages in the muscle; however, clodronate liposome treatment reduced this infiltration. Although exhaustive exercise resulted in an increase in injured myofibers, clodronate liposome treatment following exhaustive exercise reduced the injured myofibers. Clodronate liposome treatment also decreased the mRNA expression levels of inflammatory cytokines (TNF-α, IL-1β, and IL-6) in the skeletal muscle after exhaustive exercise. These results suggest that macrophages play a critical role in increasing muscle injury by regulating inflammation.

Exhaustive exercise increases the TNF-α production in response to flagellin via the upregulation of toll-like receptor 5 in the large intestine in mice

Although intense exercise may induce temporary immune depression, it is unclear whether exercise stimulates tumor necrosis factor-alpha (TNF-α) production in response to flagella protein flagellin (FG), which binds to toll-like receptor 5 (TLR5) and induces the production of pro-inflammatory cytokines. Male C3H/HeN mice were administered FG (1mg/kg, i.v.) after exhaustive exercise (EX), and the plasma TNF-α concentrations were examined. The production of TNF-α and the TLR5 expression in both RAW264 and Caco2 cells were measured under FG conditions in vitro. Although the plasma TNF-α concentrations were observed to significantly increase in both the EX and non-EX (N-EX) mice (p<0.01, respectively) following FG injection, the TNF-α levels in the EX mice were significantly higher than those observed in the N-EX mice (p<0.01). Epinephrine (Ep) treatment accelerated the FG-induced TNF-α production and TLR5 expression on the Caco2, but not RAW264 cells. Interestingly, a high Ep-induced TLR5 expression was observed on the Caco2 cell surface, which was inhibited by an inhibitor of phosphoinositide3-kinase (PI3K), Ly294002, as well as a β-adrenergic blocker, propranolol. In addition, the EX-induced TNF-α production observed in response to FG was also attenuated by pretreatment with propranolol. Our findings suggest that exhaustive exercise upregulates the production of TNF-α in response to FG via a high expression of TLR5 on the intestinal cell surface following the stimulation of β-adrenergic receptors with exercise.