Mika Nagasaki

CD8 + effector T cells contribute to macrophage recruitment and adipose tissue inflammation in obesity

Inflammation is increasingly regarded as a key process underlying metabolic diseases in obese individuals. In particular, obese adipose tissue shows features characteristic of active local inflammation. At present, however, little is known about the sequence of events that comprises the inflammatory cascade or the mechanism by which inflammation develops. We found that large numbers of CD8+ effector T cells infiltrated obese epididymal adipose tissue in mice fed a high-fat diet, whereas the numbers of CD4+ helper and regulatory T cells were diminished. The infiltration by CD8+ T cells preceded the accumulation of macrophages, and immunological and genetic depletion of CD8+ T cells lowered macrophage infiltration and adipose tissue inflammation and ameliorated systemic insulin resistance. Conversely, adoptive transfer of CD8+ T cells to CD8-deficient mice aggravated adipose inflammation. Coculture and other in vitro experiments revealed a vicious cycle of interactions between CD8+ T cells, macrophages and adipose tissue. Our findings suggest that obese adipose tissue activates CD8+ T cells, which, in turn, promote the recruitment and activation of macrophages in this tissue. These results support the notion that CD8+ T cells have an essential role in the initiation and propagation of adipose inflammation.

Adipogenesis in Obesity Requires Close Interplay Between Differentiating Adipocytes, Stromal Cells, and Blood Vessels

OBJECTIVE—The expansion of adipose tissue mass seen in obesity involves both hyperplasia and hypertrophy of adipocytes. However, little is known about how adipocytes, adipocyte precursors, blood vessels, and stromal cells interact with one another to achieve adipogenesis. RESEARCH DESIGN AND METHODS—We have developed a confocal microscopy-based method of three-dimensional visualization of intact living adipose tissue that enabled us to simultaneously evaluate angiogenesis and adipogenesis in db/db mice. RESULTS—We found that adipocyte differentiation takes place within cell clusters (which we designated adipogenic/angiogenic cell clusters) that contain multiple cell types, including endothelial cells and stromal cells that express CD34 and CD68 and bind lectin. There were close spatial and temporal interrelationships between blood vessel formation and adipogenesis, and the sprouting of new blood vessels from preexisting vasculature was coupled to adipocyte differentiation. CD34+ CD68+ lectin-binding cells could clearly be distinguished from CD34− CD68+ macrophages, which were scattered in the stroma and did not bind lectin. Adipogenic/angiogenic cell clusters can morphologically and immunohistochemically be distinguished from crown-like structures frequently seen in the late stages of adipose tissue obesity. Administration of anti–vascular endothelial growth factor (VEGF) antibodies inhibited not only angiogenesis but also the formation of adipogenic/angiogenic cell clusters, indicating that the coupling of adipogenesis and angiogenesis is essential for differentiation of adipocytes in obesity and that VEGF is a key mediator of that process. CONCLUSIONS—Living tissue imaging techniques provide novel evidence of the dynamic interactions between differentiating adipocytes, stromal cells, and angiogenesis in living obese adipose tissue.

In vivo imaging in mice reveals local cell dynamics and inflammation in obese adipose tissue

To assess physiological and pathophysiological events that involve dynamic interplay between multiple cell types, real-time, in vivo analysis is necessary. We developed a technique based on confocal laser microscopy that enabled us to analyze and compare the 3-dimensional structures, cellular dynamics, and vascular function within mouse lean and obese adipose tissue in vivo with high spatiotemporal resolution. We found increased leukocyte-EC-platelet interaction in the microcirculation of obese visceral adipose tissue in ob/ob and high-fat diet-induced obese mice. These changes were indicative of activation of the leukocyte adhesion cascade, a hallmark of inflammation. Local platelet activation in obese adipose tissue was indicated by increased P-selectin expression and formation of monocyte-platelet conjugates. We observed upregulated expression of adhesion molecules on macrophages and ECs in obese visceral adipose tissue, suggesting that interactions between these cells contribute to local activation of inflammatory processes. Furthermore, administration of anti-ICAM-1 antibody normalized the cell dynamics seen in obese visceral fat. This imaging technique to analyze the complex cellular interplay within obese adipose tissue allowed us to show that visceral adipose tissue obesity is an inflammatory disease. In addition, this technique may prove to be a valuable tool to evaluate potential therapeutic interventions.

Adipose Natural Regulatory B Cells Negatively Control Adipose Tissue Inflammation

Distinct B cell populations, designated regulatory B (Breg) cells, are known to restrain immune responses associated with autoimmune diseases. Additionally, obesity is known to induce local inflammation within adipose tissue that contributes to systemic metabolic abnormalities, but the underlying mechanisms that modulate adipose inflammation remain poorly understood. We identified Breg cells that produce interleukin-10 constitutively within adipose tissue. B cell-specific Il10 deletion enhanced adipose inflammation and insulin resistance in diet-induced obese mice, whereas adoptive transfer of adipose tissue Breg cells ameliorated those effects. Adipose environmental factors, including CXCL12 and free fatty acids, support Breg cell function, and Breg cell fraction and function were reduced in adipose tissue from obese mice and humans. Our findings indicate that adipose tissue Breg cells are a naturally occurring regulatory B cell subset that maintains homeostasis within adipose tissue and that Breg cell dysfunction contributes pivotally to the progression of adipose tissue inflammation in obesity.

IL-1α induces thrombopoiesis through megakaryocyte rupture in response to acute platelet needs

An alternative pathway triggering enhanced platelet release from bone marrow megakaryocytes via a rupture-based mechanism is regulated by IL-1α in response to acute platelet requirements.

In vivo imaging visualizes discoid platelet aggregations without endothelium disruption and implicates contribution of inflammatory cytokine and integrin signaling

The mechanism by which thrombotic vessel occlusion occurs independently of plaque development or endothelial cell (EC) disruption remains unclear, largely because of an inability to visualize the formation of thrombus, especially at the single-platelet level in real time. Here we demonstrate that rapidly developing thrombi composed of discoid platelets can be induced in the mesenteric capillaries, arterioles, and large-sized arteries of living mice, enabling characterization of the kinetics of thrombosis initiation and the multicellular interrelationships during thrombus development. Platelet aggregation without EC disruption was triggered by reactive oxygen species (ROS) photochemically induced by moderate power laser irradiation. The inflammatory cytokines TNF-α and IL-1 could be key components of the EC response, acting through regulation of VWF mobilization to the cell surface. Thrombus formation was then initiated by the binding of platelet GPIbα to endothelial VWF in our model, and this effect was inhibited by the ROS scavenger N-acetylcysteine. Actin linker talin-dependent activation of alphaIIb-beta3 integrin or Rac1 in platelets was required for late-phase thrombus stability. Our novel imaging technology illustrates the molecular mechanism underlying inflammation-based thrombus formation by discoid platelets on undisrupted ECs and suggests control of ROS could be a useful therapeutic target for the prevention of thrombotic diseases.

Responses of single-ventricular myocytes to dynamic axial stretching.

Mechano-electrical feedback (MEF) has mainly been studied in isolated single cardiomyocytes using the microelectrode and micropipette techniques, but information regarding its dynamic aspects at the cellular level is limited due to the technical difficulties associated with manipulating single cells and maintaining stable attachment of these devices. To overcome such difficulties, we have combined two experimental methods, namely a carbon fiber technique to hold single myocytes and a ratiometric fluorescence measurement technique to monitor Ca2+ transients or membrane potentials. Following an overview of the experimental technique for stretching myocytes, the results for single rat ventricular myocytes under axial stretching are presented. Ca2+ transients were influenced by the loading conditions and involvement of myofilaments was suspected in regulatory mechanism. Membrane potential measurements during dynamic axial stretching revealed that the action potential duration was prolonged when the stretch was applied during the late phase of twitch contraction, and that depolarization of the resting membrane potential depended on the phase, amplitude and speed of the applied stretch. The amplitude may also modulate the ion selectivity of stretch-activated channels. This combination of the carbon fiber technique with fluorescence measurement could represent a powerful tool for clarifying MEF at the cellular level.

ENPP2 contributes to adipose tissue expansion and insulin resistance in diet-induced obesity.

Body weight is tightly regulated by food intake and energy dissipation, and obesity is related to decreased energy expenditure (EE). Herein, we show that nucleotide pyrophosphatase/phosphodiesterase 2 (ENPP2, autotaxin) is an adipose-derived, secreted enzyme that controls adipose expansion, brown adipose tissue (BAT) function, and EE. In mice, Enpp2 was highly expressed in visceral white adipose tissue and BAT and is downregulated in hypertrophied adipocytes/adipose tissue. Enpp2+/− mice and adipocyte-specific Enpp2 knockout mice fed a high-fat diet showed smaller body weight gains and less insulin resistance than control mice fed the same diet. BAT was functionally more active and EE was increased in Enpp2-deficient mice. In humans, ENPP2 expression in subcutaneous fat and ENPP2 levels in serum were reduced in obese subjects. Taken together, our results establish ENPP2 as an adipose-derived, secreted enzyme that regulates adipose obesity and systemic metabolism. They also suggest ENPP2 could be a useful therapeutic target for the treatment of metabolic disease.

Identification of Ganglioside GM3 Molecular Species in Human Serum Associated with Risk Factors of Metabolic Syndrome.

Serum GM3 molecular species were quantified in 125 Japanese residents using tandem mass spectrometry multiple reaction monitoring. Individuals were categorized by the presence or absence of metabolic disease risk factors including visceral fat accumulation, hyperglycemia and dyslipidemia. A total of 23 GM3 molecular species were measured, of these, eight were found to be significantly elevated in individuals with visceral fat accumulation and metabolic disease, defined as the presence of hyperglycemia and dyslipidemia. All of the GM3 molecular species were composed of the sphingoid base sphingosine (d18:1 (Δ4)) and, interestingly, six of the eight elevated GM3 molecular species contained a hydroxylated ceramide moiety. The hydroxylated GM3 species were, in order of decreasing abundance, d18:1-h24:0 ≈ d18:1-h24:1 > d18:1-h22:0 » d18:1-h20:0 > d18:1-h21:0 > d18:1-h18:1. Univariate and multiple linear regression analyses were conducted using a number of clinical health variables associated with obesity, type 2 diabetes, metabolic disease, atherosclerosis and hypertension. GM3(d18:1-h24:1) was identified as the best candidate for metabolic screening, proving to be significantly correlated with intima-media thickness, used for the detection of atherosclerotic disease in humans, and a number of metabolic disease risk factors including autotaxin, LDL-c and homeostatic model assessment insulin resistance (HOMA-IR).

Cardiac rehabilitation decreases plasma pentraxin 3 in patients with cardiovascular diseases

Background: Inflammatory markers such as serum C-reactive protein (CRP), serum amyloid A (SAA), and plasma pentraxin 3 (PTX3), which belong to the pentraxin superfamily, increase due to various inflammatory diseases. Some studies demonstrated that serum CRP and SAA are predictors of cardiovascular diseases, and cardiac rehabilitation (CR) induces anti-inflammatory effects. In the present study, we investigated the effects of CR on pentraxins (serum CRP, SAA, and plasma PTX3) in patients with cardiovascular diseases. Methods: Fifty patients with cardiovascular diseases [61 ± 13 (mean ± SD) years old, male/female 44/6] participated. Each subject performed CR using aerobic bicycle exercise two or three times per week for 3–6 months. We measured resting serum high-sensitivity CRP (hsCRP), SAA, and plasma PTX3 before and 3 and 6 months after CR, and compared them with VO2peak determined using a standard increment cycle ergometer protocol, B-type natriuretic peptide (BNP), and other biochemical data such as HbA1c. Results: There was a significant positive correlation between hsCRP and SAA (r = 0.92, p < 0.001), but no relations between these parameters and PTX3. Plasma PTX3 significantly decreased time dependently during CR (at baseline 3.2 ± 2.0 ng/ml, at 3 months 2.3 ± 0.8 ng/ml, at 6 months 2.1 ± 0.7 ng/ml; all p < 0.05). Serum hsCRP tended to decrease, but not statistically significantly. At baseline, plasma PTX3 was negatively correlated with the percentage of the predicted values of VO2peak and positively correlated with BNP. CR significantly increased the percentage of the predicted values of VO2peak and decreased BNP. Conclusions: Plasma PTX3, an inflammatory marker, which was quite different from CRP and SAA, decreased during cardiac rehabilitation with an improvement of exercise capacity in patients with cardiovascular diseases.