Enhanced glucose uptake and a switch to glycolysis are key traits of M1 macrophages, whereas enhanced fatty acid oxidation and oxidative phosphorylation are the main metabolic characteristics of M2 macrophages. Recent studies challenge this traditional view, indicating that glycolysis may also be critically important for M2 macrophage differentiation, based on experiments with 2-DG. Here we confirm the inhibitory effect of 2-DG on glycolysis, but also demonstrate that 2-DG impairs oxidative phosphorylation and significantly reduces C-labeled Krebs cycle metabolites and intracellular ATP levels. These metabolic derangements were associated with reduced JAK-STAT6 pathway activity and M2 differentiation marker expression. While glucose deprivation and glucose substitution with galactose effectively suppressed glycolytic activity, there was no effective suppression of oxidative phosphorylation, intracellular ATP levels, STAT6 phosphorylation, and M2 differentiation marker expression. These data indicate that glycolytic stimulation is not required for M2 macrophage differentiation as long as oxidative phosphorylation remains active.

Classical activation of M1 macrophages with lipopolysaccharide (LPS) is associated with a metabolic switch from oxidative phosphorylation to glycolysis. However, the generalizability of such metabolic remodeling to other modes of M1 macrophage stimulation, e.g. type II interferons (IFNs) such as IFNγ, has remained unknown as has the functional significance of aerobic glycolysis during macrophage activation. Here we demonstrate that IFNγ induces a rapid activation of aerobic glycolysis followed by a reduction in oxidative phosphorylation in M1 macrophages. Elevated glycolytic flux sustains cell viability and inflammatory activity, while limiting reliance on mitochondrial oxidative metabolism. Adenosine triphosphate (ATP) distributed by aerobic glycolysis is critical for sustaining IFN-γ triggered JAK (Janus tyrosine kinase)-STAT-1 (Signal Transducer and Activator of Transcription 1) signaling with phosphorylation of the transcription factor STAT-1 as its signature trait. Inhibition of aerobic glycolysis not only blocks the M1 phenotype and pro-inflammatory cytokine/chemokine production in murine macrophages and also human monocytes/macrophages. These findings extend on the potential functional role of immuno-metabolism from LPS- to IFNγ-linked diseases such as atherosclerosis and autoimmune disease.

No abstract

The in vitro differentiation and immunosuppressive capacity of mesenchymal stem cells (MSCs) derived from synovial fluid (SF-MSCs) and bone marrow extract (BM-MSCs) in an isogenic background of minipigs were comparatively analyzed in a collagen-induced arthritis (CIA) mouse model of rheumatoid arthritis (RA). The proliferation capacity and expression of pluripotent transcription factors (Oct3/4 and Sox2) were significantly (P<0.05) higher in SF-MSCs than in BM-MSCs. The differentiation capacity of SF-MSCs into adipocytes, osteocytes and neurocytes was significantly (P<0.05) lower than that of BM-MSCs, and the differentiation capacity of SF-MSCs into chondrocytes was significantly (P<0.05) higher than that of BM-MSCs. Systemic injection of BM- and SF-MSCs significantly (P<0.05) ameliorated the clinical symptoms of CIA mice, with SF-MSCs having significantly (P<0.05) higher clinical and histopathological recovery scores than BM-MSCs. Furthermore, the immunosuppressive properties of SF-MSCs in CIA mice were associated with increased levels of the anti-inflammatory cytokine interleukin (IL)-10, and decreased levels of the pro-inflammatory cytokine IL-1β and osteoclast-related sRANKL. In conclusion, SF-MSCs exhibited eminent pluripotency and differentiation capacity into chondrocytes, addition to substantial in vivo immunosuppressive capacity by elevating IL-10 and reducing IL-1β levels in CIA mice.

Mesenchymal stromal/stem cells (MSCs) demonstrate immunomodulation capacity that has been implicated in the reduction of graft-versus-host disease. Accordingly, we herein investigated the capacity of MSCs derived from several tissue sources to modulate both proinflammatory (interferon [IFN] γ and tumor necrosis factor [TNF] α) and immunosuppressive cytokines (transforming growth factor [TGF] β and interleukin [IL] 10) employing xenogeneic human MSC-mixed lymphocyte reaction (MLR) test. Bone marrow-derived MSCs showed higher self-renewal capacity with relatively slow proliferation rate in contrast to adipose-derived MSCs which displayed higher proliferation rate. Except for the lipoprotein gene, there were no marked changes in osteogenesis- and adipogenesis-related genes following in vitro differentiation; however, the histological marker analysis revealed that adipose MSCs could be differentiated into both adipose and bone tissue. TGFβ and IL10 were detected in adipose MSCs and bone marrow MSCs, respectively. However, skin-derived MSCs expressed both IFNγ and IL10, which may render them sensitive to immunomodulation. The xenogeneic human MLR test revealed that MSCs had a partial immunomodulation capacity, as proliferation of activated and resting peripheral blood mononuclear cells was not affected, but this did not differ among MSC sources. MSCs were not tumorigenic when introduced into immunodeficient mice. We concluded that the characteristics of MSCs are tissue source-dependent and their in vivo application requires more in-depth investigation regarding their precise immunomodulation capacities.

The identification of stable reference genes is a prerequisite for ensuring accurate validation of gene expression, yet too little is known about stable reference genes of porcine MSCs. The present study was, therefore, conducted to assess the stability of reference genes in porcine MSCs derived from bone marrow (BMSCs), adipose (AMSCs), and skin (SMSCs) with their in vitro differentiated cells into mesenchymal lineages such as adipocytes, osteocytes, and chondrocytes. Twelve commonly used reference genes were investigated for their threshold cycle (Ct) values by qRT-PCR. The Ct values of candidate reference genes were analyzed by geNorm software to clarify stable expression regardless of experimental conditions. Thus, Pearson's correlation was applied to determine correlation between the three most stable reference genes (NF3) and optimal number of reference genes (NFopt). In assessment of stability of reference gene across experimental conditions by geNorm analysis, undifferentiated MSCs and each differentiated status into mesenchymal lineages showed slightly different results but similar patterns about more or less stable rankings. Furthermore, Pearson's correlation revealed high correlation (r > 0.9) between NF3 and NFopt. Overall, the present study showed that HMBS, YWHAZ, SDHA, and TBP are suitable reference genes for qRT-PCR in porcine MSCs.

The aim of this study was to examine the effects of human umbilical cord blood-derived CD34-positive endothelial progenitor cells (CD34+ EPCs) on osteoblastic differentiation of cultured human periosteal-derived osteoblasts (POs). CD34+ cells from human umbilical cord blood were sorted to purify more EPCs in characterization. These sorted cells showed CD31, VE-cadherin, and KDR expression as well as CD34 expression and formed typical tubes in Matrigel. These sorted cells were referred to as human cord blood-derived CD34+ EPCs. In in vivo bone formation using a miniature pig model, the newly formed bone was clearly examined in defects filled with polydioxanone/pluronic F127 (PDO/Pluronic F127) scaffolds containing either human umbilical cord blood-derived CD34+ EPCs and POs or human umbilical vein endothelial cells (HUVEC) and POs; however, the new bone had the greatest density in the defect treated with CD34+ EPCs and POs. Osteoblastic phenotypes of cultured human POs using ALP activity and von Kossa staining were also more clearly found in CD34+ EPC-conditioned medium than CD34-negative (CD34-) cell-conditioned medium, whereas HUVEC-conditioned medium had an intermediate effect. PCR array for common cytokines and growth factors showed that the secretion of interleukin (IL)-1β was significantly higher in CD34+ EPCs than in HUVEC, followed by level in CD34- cells. In addition, IL-1β also potently and dose dependently increased ALP activity and mineralization of POs in culture. These results suggest that human umbilical cord blood-derived CD34+ EPCs stimulates osteoblastic differentiation of cultured human POs. The functional role of human umbilical cord blood-derived CD34+ EPCs in increasing the osteogenic phenotypes of cultured human POs may depend on IL-1β secreted from human umbilical cord blood-derived CD34+ EPCs.

No abstract