Myeloid cell diversification during regenerative inflammation: Lessons from skeletal muscle.

Understanding the mechanisms of tissue and organ regeneration in adult animals and humans is of great interest from a basic biology as well as a medical, therapeutical point of view. It is increasingly clear that the relatively limited ability to regenerate tissues and organs in mammals as oppose to lower vertebrates is the consequence of evolutionary trade-offs and changes during development and aging. Thus, the coordinated interaction of the immune system, particularly the innate part of it, and the injured, degenerated parenchymal tissues such as skeletal muscle, liver, lung, or kidney shape physiological and also pathological processes. In this review, we provide an overview of how morphologically and functionally complete (ad integrum) regeneration is achieved using skeletal muscle as a model. We will review recent advances about the differentiation, activation, and subtype specification of circulating monocyte to resolution or repair-type macrophages during the process we term regenerative inflammation, resulting in complete restoration of skeletal muscle in murine models of toxin-induced injury.

Defining the proteomic landscape of cultured macrophages and their polarization continuum.

Macrophages have previously been characterized based on phenotypical and functional differences into suggested simplified subtypes of MØ, M1, M2a and M2c. These macrophage subtypes can be generated in a well-established primary monocyte culture model that produces cells expressing accepted subtype surface markers. To determine how these subtypes retain functional similarities and better understand their formation, we generated all four subtypes from the same donors. Comparative whole-cell proteomics confirmed that four distinct macrophage subtypes could be induced from the same donor material, with > 50% of 5435 identified proteins being significantly altered in abundance between subtypes. Functional assessment highlighted that these distinct protein expression profiles are primed to enable specific cell functions, indicating that this shifting proteome is predictive of meaningful changes in cell characteristics. Importantly, the 2552 proteins remained consistent in abundance across all macrophage subtypes examined, demonstrating maintenance of a stable core proteome that likely enables swift polarity changes. We next explored the cross-polarization capabilities of preactivated M1 macrophages treated with dexamethasone. Importantly, these treated cells undergo a partial repolarization toward the M2c surface markers but still retain the M1 functional phenotype. Our investigation of polarized macrophage subtypes therefore provides evidence of a sliding scale of macrophage functionality, with these data sets providing a valuable benchmark resource for further studies of macrophage polarity, with relevance for cell therapy development and drug discovery.

Divergent effect of Birinapant, and BV6 SMAC mimetic on TNFα induced NF-κB signaling and cell viability in activated hepatic stellate cells.

BACKGROUND: Tumor necrosis factor-α (TNFα) is a pleiotropic cytokine involved in nuclear factor kappa B (NF-κB) mediated cell survival as well as cell death. High serum TNFα levels correlate with liver fibrosis and enhance hepatic stellate cell (HSC) viability. However, the regulatory role of cellular inhibitor of apoptosis-1/2 (cIAP1/2) during TNFα induced NF-κB signaling in activated HSCs is largely unknown. METHOD AND RESULTS: Activated HSCs were treated with cIAP1/2 inhbitiors i.e., SMAC mimetic BV6, and Birinapant in the presence of TNFα and macrophage conditioned media. TNFα cytokine increased cIAP2 expression and enhanced cell viability through the canonical NF-κB signaling in activated HSCs. cIAP2 inhibition via BV6 decreased the TNFα induced canonical NF-κB signaling, and reduced cell viability in activated HSCs. SMAC mimetic, Birinapant alone did not affect the cell viability but treatment of TNFα sensitized HSCs with Birinapant induced cell death. While BV6 mediated cIAP2 ablation was able to decrease the TNFα induced canonical NF-κB signaling, this effect was not observed with Birinapant treatment. Secreted TNFα from M1 polarized macrophages sensitized activated HSCs to BV6 or Birinapant mediated cell death. However, M2 polarized macrophage conditioned medium rescued the activated HSCs from BV6 mediated cytotoxicity. CONCLUSION: In this study, we describe the regulatory role of cIAP2 in TNFα induced NF-κB signaling in activated HSCs. Targeting cIAP2 may be a promising approach for liver fibrosis treatment via modulating NF-κB signaling in activated HSCs.

M2b Macrophages Regulate Cardiac Fibroblast Activation and Alleviate Cardiac Fibrosis After Reperfusion Injury.

BACKGROUND: Macrophages play an important role in the development of cardiac fibrosis. However, the roles of different macrophage subtypes in cardiac fibroblast (CF) activation and cardiac fibrosis are unknown.Methods and Results:Bone marrow-derived macrophages (BMDMs) were treated with different stimuli to induce differentiation into M1, M2a, M2b, and M2c macrophage subtypes. CFs were co-cultured with different subtypes of macrophages or cultured with macrophage supernatants. Results revealed that M2b macrophages significantly suppressed the proliferation and migration of CFs, the expression of fibrosis-related proteins (collagen I [COL-1] and α-smooth muscle actin [α-SMA]), and differentiation into cardiac myofibroblasts (MFs). The opposite effects were observed with M2a macrophages. A rat model of cardiac ischemia/reperfusion (I/R) injury was used to determine the effect of M2b macrophages transplantation. After cardiac I/R injury, transplantation of M2b macrophages improved cardiac function and reduced cardiac fibrosis. The effect of macrophage subtypes on p-ERK, ERK, p-p38, and p38 phosphorylation was examined by Western blotting. The results showed that M2b macrophages significantly inhibited the mitogen-activated protein kinase (MAPK) signaling pathway. CONCLUSIONS: These study results demonstrate for the first time that different subtypes of macrophages have different roles in regulating CF activation. M2b macrophages inhibit CF activation, and thus can be considered anti-fibrotic macrophages. M2a macrophages promote CF activation, and thus are pro-fibrotic macrophages.

Inflammatory response under zinc deficiency is exacerbated by dysfunction of the T helper type 2 lymphocyte-M2 macrophage pathway.

Nutritional zinc deficiency leads to immune dysfunction and aggravates inflammation. However, the underlying mechanism remains unknown. In this study, the relationship between macrophage subtypes (M1 and M2) and helper T lymphocytes (Th1 and Th2) was investigated using the spleen from rats fed zinc-deficient or standard diet. In experiment I, 5-week-old male Sprague-Dawley rats were fed a zinc-deficient diet (without zinc additives) or a standard diet (containing 0·01% zinc) for 6 weeks. In experiment II, the rats were divided into four groups: one group was fed a standard diet for 6 weeks; two groups were fed zinc-deficient diets and were injected three times a week with either saline or interleukin-4 (IL-4) (zinc-deficient/IL-4 i.p.); a fourth group (zinc-deficient/standard) was fed a zinc-deficient diet for 6 weeks followed by a standard diet for 4 weeks. In experiment I; GATA-binding protein 3 (GATA-3) protein level, M2 macrophage, CD3+  CD8+ cells, and IL-4/IL-13-positive cells significantly decreased in the spleens of the zinc-deficient group. Additionally, IL-1ß and macrophage inflammatory protein-1α (MIP-1α) mRNA levels significantly increased in the splenic macrophages of the zinc-deficient group. In experiment II; M2 macrophages, CD3+  CD8+ cells, IL-4/IL-13-positive cells, and GATA-3 protein levels significantly increased in the spleens of the zinc-deficient/IL-4 i.p. and zinc-deficient/standard groups. Furthermore, IL-1ß and MIP-1α mRNA levels decreased in the splenic macrophages of the zinc-deficient/IL-4 i.p. and zinc-deficient/standard groups. Zinc deficiency-induced aggravated inflammation is related to Th2 lymphocytes and followed by the association with loss of GATA-3, IL-4 and anti-inflammatory M2 macrophages. Importantly, IL-4 injection or zinc supplementation can reverse the effects of zinc deficiency on immune function.

Anti-inflammatory and immunomodulatory mechanisms of atorvastatin in a murine model of traumatic brain injury.

BACKGROUND: Neuroinflammation is an important secondary injury mechanism that has dual beneficial and detrimental roles in the pathophysiology of traumatic brain injury (TBI). Compelling data indicate that statins, a group of lipid-lowering drugs, also have extensive immunomodulatory and anti-inflammatory properties. Among statins, atorvastatin has been demonstrated as a neuroprotective agent in experimental TBI; however, there is a lack of evidence regarding its effects on neuroinflammation during the acute phase of TBI. The current study aimed to evaluate the effects of atorvastatin therapy on modulating the immune reaction, and to explore the possible involvement of peripheral leukocyte invasion and microglia/macrophage polarization in the acute period post-TBI. METHODS: C57BL/6 mice were subjected to TBI using a controlled cortical impact (CCI) device. Either atorvastatin or vehicle saline was administered orally starting 1 h post-TBI for three consecutive days. Short-term neurological deficits were evaluated using the modified neurological severity score (mNSS) and Rota-rod. Brain-invading leukocyte subpopulations were analyzed by flow cytometry and immunohistochemistry. Pro- and anti-inflammatory cytokines and chemokines were examined using enzyme-linked immunosorbent assay (ELISA). Markers of classically activated (M1) and alternatively activated (M2) microglia/macrophages were then determined by quantitative real-time PCR (qRT-PCR) and flow cytometry. Neuronal apoptosis was identified by double staining of terminal deoxynucleotidyl transferase-dUTP nick end labeling (TUNEL) staining and immunofluorescence labeling for neuronal nuclei (NeuN). RESULTS: Acute treatment with atorvastatin at doses of 1 mg/kg/day significantly reduced neuronal apoptosis and improved behavioral deficits. Invasions of T cells, neutrophils and natural killer (NK) cells were attenuated profoundly after atorvastatin therapy, as was the production of pro-inflammatory cytokines (IFN-γ and IL-6) and chemokines (RANTES and IP-10). Notably, atorvastatin treatment significantly increased the proportion of regulatory T cells (Tregs) in both the peripheral spleen and brain, and at the same time, increased their main effector cytokines IL-10 and TGF-ß1. We also found that atorvastatin significantly attenuated total microglia/macrophage activation but augmented the M2/M1 ratio by both inhibiting M1 polarization and enhancing M2 polarization. CONCLUSIONS: Our data demonstrated that acute atorvastatin administration could modulate post-TBI neuroinflammation effectively, via a mechanism that involves altering peripheral leukocyte invasion and the alternative polarization of microglia/macrophages.

Survivin inhibition ameliorates liver fibrosis by inducing hepatic stellate cell senescence and depleting hepatic macrophage population.

Persistent activation of hepatic stellate cells (HSCs) in the injured liver leads to the progression of liver injury from fibrosis to detrimental cirrhosis. In a previous study, we have shown that survivin protein is upregulated during the early activation of HSCs, which triggers the onset of liver fibrosis. However, the therapeutic potential of targeting survivin in a fully established fibrotic liver needs to be investigated. In this study, we chemically induced hepatic fibrosis in mice using carbon tetrachloride (CCl4) for 6 weeks, which was followed by treatment with a survivin suppressant (YM155). We also evaluated survivin expression in fibrotic human liver tissues, primary HSCs, and HSC cell line by histological analysis. αSMA+ HSCs in human and mice fibrotic liver tissues showed enhanced survivin expression, whereas the hepatocytes and quiescent (qHSCs) displayed minimal expression. Alternatively, activated M2 macrophage subtype induced survivin expression in HSCs through the TGF-ß-TGF-ß receptor-I/II signaling. Inhibition of survivin in HSCs promoted cell cycle arrest and senescence, which eventually suppressed their activation. In vivo, YM155 treatment increased the expression of cell senescence makers in HSCs around fibrotic septa such as p53, p21, and ß-galactosidase. YM155 treatment in vivo also reduced the hepatic macrophage population and inflammatory cytokine expression in the liver. In conclusion, downregulation of survivin in the fibrotic liver decreases HSC activation by inducing cellular senescence and modulating macrophage cytokine expression that collectively ameliorates liver fibrosis.

Mesenchymal Stromal Cells Regulate M1/M2 Macrophage Polarization in Mice with Immune Thrombocytopenia.

Mesenchymal stromal cells have shown promising effects in the treatment of immune thrombocytopenia. However, the underlying mechanisms are not fully understood. In this study, we investigated the therapeutic effects of human bone marrow mesenchymal stromal cells (hBMSCs) and analyzed their unique role in regulating the M1/M2 macrophage ratio. We established a passive immune thrombocytopenia (ITP) mouse model and showed that there was a significant M1/M2 imbalance in ITP model mice by assessing the M1/M2 ratios in the liver, spleen, and bone marrow; we observed excessive activation of M1 cells and decreased M2 cell numbers in vivo. We have shown that systemic infusion of hBMSCs effectively elevated platelet levels after disease onset. Further analysis revealed that hBMSCs treatment significantly suppressed the number of proinflammatory M1 macrophages and enhanced the number of anti-inflammatory M2 macrophages; in addition, the levels of proinflammatory factors, such as interleukin-1ß (IL-1ß) and tumor necrosis factor-α (TNF-α), were significantly decreased in vivo, while the levels of the anti-inflammatory factor interleukin-10 (IL-10) were increased. In conclusion, our data suggest that hBMSCs treatment can effectively increase platelet counts, and the mechanism is related to the induction of macrophage polarization toward the anti-inflammatory M2 phenotype and the decrease in proinflammatory cytokine production, which together ameliorate innate immune disorders.

Effect of intravenous transplantation of hUCB-MSCs on M1/M2 subtype conversion in monocyte/macrophages of AMI mice.

BACKGROUND: The transplantation of stem cells is effective in the treatment of acute myocardial infarction (AMI). But the mechanisms of stem cell transplantation for the treatment of AMI have not been clearly confirmed. This article is to compare cardiac function, myocardial fibrosis, inflammatory cell infiltration, apoptotic index, and M1 macrophage to M2 macrophage ratios 4 weeks after hUCB-MSCs transplantation in Mice with AMI. METHODS: Mice model of AMI was divided into two groups randomly. Human umbilical cord blood mesenchymal stem cells (hUCB-MSCs) were transplanted to the intervention group via mice tail vein, and saline solution was used for the control group. Masson staining calculated the proportion of the remaining myocardium and collagen volume fraction (CVF) in the infarct area, flow cytometry and immunofluorescence staining to analyze M1 / M2 subtype and its ratio were performed. Serum IL-6 and galectin-3 levels were determined using ELISA. RESULTS: The results showed that heart function of the intervention group was significantly better than that of the control group, and the degree of fibrosis and inflammation in the intervention group were lower than those in the control group. The ratio of monocyte M1/M2 in peripheral blood, spleen and myocardial tissue in hUCB-MSCs transplantation was significantly lower than that of the control group. But there is no significant difference in the apoptotic index between two groups. The ELISA results showed that serum IL-6 (97.98 ± 5.94 pg/ml) and galectin-3 levels (69.94 ± 5.11 ng/ml) were lower in the intervention group than in the control group (IL-6: 118.2 ± 5.03 pg/ml; galectin-3: 83.14 ± 2.76 ng / ml). However, compared with the control group, only IL-6 showed a significant decrease in the intervention group (p = 0.032). CONCLUSION: Intravenous transplantation of hUCB-MSCs can reduce inflammatory response by stimulating the conversion of intracardiac and extracardiac macrophage subtype M1 / M2, decrease the serum IL-6 and galectin-3 levels, improve cardiac function and protect the infarcted myocardium.

Effect of Galectin-9/ Tim-3 pathway on the polarization of M1/M2 subtype in murine macrophages induced by lipopolysaccharide / 中华危重病急救医学

Objective To investigate the meaning and molecular mechanisms of Galectin-9/ T-cell immunoglobulin mucin-3 (Tim-3) pathway on lipopolysaccharide (LPS) induced murine macrophage M1/M2 subtype polarization.Methods The murine peritoneal macrophages RAW264.7 were cultured in vitro until the cells had matured with 80％-90％ fusion rate. ① The cells were cultured in serum-free medium and treated with 0 (blank control), 0.01, 0.1, 1, 10 and 100 mg/L LPS for 24 hours. Real-time fluorescence quantitative reverse transcription-polymerase chain reaction (RT-qPCR) or Western Blot was used to determine the expressions of M1 macrophage markers such as interleukin-6 (IL-6), inducible nitric oxide synthase (iNOS) and M2 macrophage markers such as arginase-1 (Arg-1), leukocyte differentiation antigen 206 (CD206), as well as Tim-3 and Galectin-9 in the cells. ② The other mice peritoneal macrophages were divided into blank control group (cultured in serum-free DMEM medium for 24 hours), LPS treatment group (cultured in serum-free DMEM medium containing 0.1 mg/L LPS for 24 hours) and α-lactose pretreatment group (pretreated with serum-free DMEM containing 40μmol/L Galectin-9 signal antagonist 1 hour before LPS stimulation). Over closed Galectin-9 signal was used to verify the role of Galectin-9 in macrophage M1/M2 subtype polarization.Results ① After stimulation with low concentrations of LPS (0.01 mg/L, 0.1 mg/L) for 24 hours, the expression of M1 markers was only slightly increased such as iNOS mRNA or not significantly changed such as IL-6 mRNA in macrophages, while the expressions of M2 markers such as Arg-1 mRNA and CD206 mRNA were significantly increased and peaked at LPS concentrations of 0.1 mg/L and 0.01 mg/L [compared with blank control group:Arg-1 mRNA (2-ΔΔCt) was 1.85±0.07 vs. 1.00±0.02, CD206 mRNA (2-ΔΔCt) was 2.03±0.11 vs.1.00±0.05, both P < 0.01]. With the increase of LPS concentration, the expressions of IL-6 mRNA and iNOS mRNA continued to increase, while the expressions of Arg-1 mRNA and CD206 mRNA were gradually decreased, and the macrophage M1/M2 subtype polarization status changed. At the same time, the level of Tim-3 protein in macrophages was significantly up-regulated after stimulation with 0.01 mg/L LPS as compared with that of blank control group (Tim-3/GAPDH:0.84±0.04 vs. 0.69±0.02,P < 0.01), peaked at LPS concentrations of 0.1 mg/L, and then decreased with increasing LPS concentration. The intracellular Galectin-9 and supernatant secreted Galectin-9 (s-Galectin-9) protein levels showed no significant change after stimulation with low concentrations of LPS (0.01 mg/L, 0.1 mg/L), while then gradually decreased with the increase of LPS concentration. ② Compared with blank control group, the mRNA expressions of M1 marker iNOS and M2 markers Arg-1 and CD206 were significantly increased in LPS treatment group, but IL-6 mRNA level was not changed significantly. The mRNA levels of IL-6 and iNOS were further elevated after pretreatment with α-lactose as compared with that of the LPS treatment group [IL-6 mRNA (2-ΔΔCt): 1.44±0.02 vs. 1.14±0.11, iNOS mRNA (2-ΔΔCt):2.45±0.04 vs. 2.01±0.08, bothP < 0.01], while the mRNA levels of Arg-1 and CD206 were significantly decreased [Arg-1 mRNA (2-ΔΔCt): 0.75±0.01 vs. 1.85±0.02, CD206 mRNA (2-ΔΔCt): 0.58±0.02 vs. 2.03±0.14, bothP < 0.01]. Meanwhile, the blocking of Galectin-9 signaling could also reduce the extracellular s-Galectin-9 (compared with LPS treatment group: s-Galectin-9/GAPDH was 0.10±0.01 vs. 0.23±0.02,P < 0.01), down-regulated the expressions of Tim-3 and Galectin-9 (Tim-3/GAPDH: 0.28±0.01 vs. 0.43±0.01, Galectin-9/GAPDH: 0.21±0.01 vs. 0.43±0.01, bothP < 0.01).Conclusions LPS regulates macrophage M1/M2 subtype polarization via Galectin-9/ Tim-3 signaling pathway. Low-doses of LPS can limit the development of inflammation by accommodating the expression and secretion of Galectin-9 to polarize macrophages to M2. High-doses of LPS promotes the development of inflammation by down-regulating the expression and secretion of Galectin-9 to polarize macrophages to M1.