Regulation of Monocytes/Macrophages by the Renin-Angiotensin System in Diabetic Nephropathy: State of the Art and Results of a Pilot Study.

Diabetic nephropathy (DN) is characterized by albuminuria, loss of renal function, renal fibrosis and infiltration of macrophages originating from peripheral monocytes inside kidneys. DN is also associated with intrarenal overactivation of the renin-angiotensin system (RAS), an enzymatic cascade which is expressed and controlled at the cell and/or tissue levels. All members of the RAS are present in the kidneys and most of them are also expressed in monocytes/macrophages. This review focuses on the control of monocyte recruitment and the modulation of macrophage polarization by the RAS in the context of DN. The local RAS favors the adhesion of monocytes on renal endothelial cells and increases the production of monocyte chemotactic protein-1 and of osteopontin in tubular cells, driving monocytes into the kidneys. There, proinflammatory cytokines and the RAS promote the differentiation of macrophages into the M1 proinflammatory phenotype, largely contributing to renal lesions of DN. Finally, resolution of the inflammatory process is associated with a phenotype switch of macrophages into the M2 anti-inflammatory subset, which protects against DN. The pharmacologic interruption of the RAS reduces albuminuria, improves the trajectory of the renal function, decreases macrophage infiltration in the kidneys and promotes the switch of the macrophage phenotype from M1 to M2.

Gene Doping with Peroxisome-Proliferator-Activated Receptor Beta/Delta Agonists Alters Immunity but Exercise Training Mitigates the Detection of Effects in Blood Samples.

Synthetic ligands of peroxisome-proliferator-activated receptor beta/delta (PPARß/δ) are being used as performance-enhancing drugs by athletes. Since we previously showed that PPARß/δ activation affects T cell biology, we wanted to investigate whether a specific blood T cell signature could be employed as a method to detect the use of PPARß/δ agonists. We analyzed in primary human T cells the in vitro effect of PPARß/δ activation on fatty acid oxidation (FAO) and on their differentiation into regulatory T cells (Tregs). Furthermore, we conducted studies in mice assigned to groups according to an 8-week exercise training program and/or a 6-week treatment with 3 mg/kg/day of GW0742, a PPARß/δ agonist, in order to (1) determine the immune impact of the treatment on secondary lymphoid organs and to (2) validate a blood signature. Our results show that PPARß/δ activation increases FAO potential in human and mouse T cells and mouse secondary lymphoid organs. This was accompanied by increased Treg polarization of human primary T cells. Moreover, Treg prevalence in mouse lymph nodes was increased when PPARß/δ activation was combined with exercise training. Lastly, PPARß/δ activation increased FAO potential in mouse blood T cells. Unfortunately, this signature was masked by training in mice. In conclusion, beyond the fact that it is unlikely that this signature could be used as a doping-control strategy, our results suggest that the use of PPARß/δ agonists could have potential detrimental immune effects that may not be detectable in blood samples.

Control of Muscle Fibro-Adipogenic Progenitors by Myogenic Lineage is Altered in Aging and Duchenne Muscular Dystrophy.

BACKGROUND/AIMS: Fibro-adipogenic progenitors (FAPs), a muscle-resident stem cell population, have recently emerged as important actors of muscle regeneration by interacting with myogenic progenitors (MPs) to promote the formation of new muscle fibers. However, FAPs are also considered as main contributors of intramuscular fibrotic and fat depositions, resulting in a poor quality of muscles and a defective regeneration in aging and Duchenne Muscular Dystrophy disease (DMD). Therefore, the understanding of the control of FAP fate is an important aspect of muscle repair and homeostasis, but little is known in humans. We wondered the extent to which human FAP proliferation, adipogenesis and fibrogenesis can be regulated by human myogenic progenitors (MPs) in physiological and pathological contexts. METHODS: FAPs and MPs were isolated from skeletal muscles of healthy young or old donors and DMD patients. FAP/MP contact co-cultures and conditioned-media from undifferentiated MPs or differentiated myotubes were assessed on both proliferation and fibro-adipogenic differentiation of FAPs. RESULTS: We showed that soluble molecules released by MPs activate the phosphoinositide 3-kinase (PI3Kinase)/Akt pathway in FAPs, resulting in the stimulation of FAP proliferation. FAP differentiation was regulated by MP-derived myotubes through the secretion of pro-fibrogenic factors and anti-adipogenic factors. Importantly, the regulation of FAP adipogenic and fibrogenic fates by myotubes was found to be mediated by Smad2 phosphorylation and the gene expression of glioma-associated oncogene homolog 1 (GLI1). Surprisingly, the regulations of proliferation and differentiation were disrupted for FAPs and MPs derived from aged individuals and patients with DMD. CONCLUSION: Our results highlight a novel crosstalk between FAPs and the myogenic lineage in humans that could be crucial in the formation of adipocyte and myofibroblast accumulation in dystrophic and aged skeletal muscle.

Transforming growth factor ß neutralization finely tunes macrophage phenotype in elastase-induced abdominal aortic aneurysm and is associated with an increase of arginase 1 expression in the aorta.

OBJECTIVE: Macrophages play a critical role in the initiation and progression of abdominal aortic aneurysm (AAA) and are classically distinguished into M1 "proinflammatory" and M2 "anti-inflammatory" macrophages. Topical application of elastase associated with transforming growth factor ß (TGF-ß) systemic neutralization reproduces the main pathologic features of human AAA, offering a new model to investigate their role. The aim of this study was to investigate whether macrophages contribute to the expression of canonical M1/M2 markers in the aorta in the AAA model induced by elastase and systemic blockade of TGF-ß and whether blocking of TGF-ß activity affects macrophage phenotype and the expression of the M2 marker arginase 1 (ARG1). METHODS: C57Bl/6J male mice (6-8 weeks old) were randomly assigned to three experimental groups: mice that had local application of heat-inactivated elastase or elastase and mice that had elastase application and received injection of anti-TGF-ß (elastase + anti-TGF-ß group). Monocyte-macrophage depletion was achieved in the elastase + anti-TGF-ß group using liposome clodronate. Macrophage phenotype was characterized by quantitative polymerase chain reaction, flow cytometry, and immunohistochemistry. Human infrarenal AAA tissues (n = 10) were obtained to analyze ARG1 expression. RESULTS: Analysis of gene expression in the infrarenal aortic wall revealed that after 14 days, no significant difference for the expression of CCL2, NOS2, and Ym1/2 was observed in the elastase group compared with the elastase + anti-TGF-ß group, whereas the expression of ARG1, interleukin (IL) 1ß, and IL-6 was significantly increased. Macrophage depletion in the elastase + anti-TGF-ß group led to a significant decrease of IL-1ß, IL-6, ARG1, and Ym1/2 gene expression. Immunofluorescent staining confirmed that TGF-ß neutralization significantly enhanced ARG1 protein expression in the aneurysmal tissue. Flow cytometry analysis revealed an increase of macrophages expressing ARG1 in the aorta of mice treated with elastase + anti-TGF-ß compared with the elastase group, and their proportion increased with aneurysmal dilation. In humans, ARG1 protein expression was increased in aneurysmal tissues compared with controls, and positive cells were mainly found in the adventitia. CONCLUSIONS: TGF-ß neutralization finely tunes macrophage phenotype in elastase-induced AAA and leads to an increase in ARG1 gene and protein expression in the aortic wall. Even if further studies are required to elucidate its role in AAA development, ARG1 could represent a new prognostic or therapeutic target in aneurysmal disease.

JAK2 inhibition has different therapeutic effects according to myeloproliferative neoplasm development in mice.

JAK2 inhibition therapy is used to treat patients suffering from myeloproliferative neoplasms (MPN). Conflicting data on this therapy are reported possibly linked to the types of inhibitors or disease type. Therefore, we decided to compare in mice the effect of a JAK2 inhibitor, Fedratinib, in MPN models of increasing severity: polycythemia vera (PV), post-PV myelofibrosis (PPMF) and rapid post-essential thrombocythemia MF (PTMF). The models were generated through JAK2 activation by the JAK2(V617F) mutation or MPL constant stimulation. JAK2 inhibition induced a correction of splenomegaly, leucocytosis and microcytosis in all three MPN models. However, the effects on fibrosis, osteosclerosis, granulocytosis, erythropoiesis or platelet counts varied according to the disease severity stage. Strikingly, complete blockade of fibrosis and osteosclerosis was observed in the PPMF model, linked to correction of MK hyper/dysplasia, but not in the PTMF model, suggesting that MF development may also become JAK2-independent. Interestingly, we originally found a decreased in the JAK2(V617F) allele burden in progenitor cells from the spleen but not in other cell types. Overall, this study shows that JAK2 inhibition has different effects according to disease phenotypes and can (i) normalize platelet counts, (ii) prevent the development of marrow fibrosis/osteosclerosis at an early stage and (iii) reduce splenomegaly through blockage of stem cell mobilization in the spleen.

An exploratory human study investigating the influence of type 2 diabetes on macrophage phenotype after myocardial infarction.

Background: Myocardial infarction (MI) is the primary cause of death in subjects with type 2 diabetes (T2D) and their in-hospital mortality after MI is still elevated compared with those without T2D. Therefore, it is of crucial importance to identify possible mechanisms of worse clinical outcomes and mortality in T2D subjects. Monocyte/macrophage-mediated immune response plays an important role in heart remodelling to limit functional deterioration after MI. Indeed, first pro-inflammatory macrophages digest damaged tissue, then anti-inflammatory macrophages become prevalent and promote tissue repair. Here, we hypothesize that the worse clinical outcomes in patients with T2D could be the consequence of a defective or a delayed polarization of macrophages toward an anti-inflammatory phenotype. Methods and results: In an exploratory human study, circulating monocytes from male patients with or without T2D at different time-points after MI were in vitro differentiated toward pro- or anti-inflammatory macrophages. The results of this pilot study suggest that the phenotype of circulating monocytes, as well as the pro- and anti-inflammatory macrophage polarization, or the kinetics of the pro- and anti-inflammatory polarization, is not influenced by T2D. Conclusion: Further studies will be necessary to understand the real contribution of macrophages after MI in humans.

Diabetes-Induced Changes in Macrophage Biology Might Lead to Reduced Risk for Abdominal Aortic Aneurysm Development.

Type 2 diabetes patients are less likely to develop an abdominal aortic aneurysm (AAA). Since macrophages play a crucial role in AAA development, we hypothesized that this decrease in AAA risk in diabetic patients might be due to diabetes-induced changes in macrophage biology. To test this hypothesis, we treated primary macrophages obtained from healthy human volunteers with serum from non-diabetic vs. diabetic AAA patients and observed differences in extracellular acidification and the expression of genes involved in glycolysis and lipid oxidation. These results suggest an increase in metabolism in macrophages treated with serum from diabetic AAA patients. Since serum samples used did not differ in glucose content, these changes are not likely to be caused by differences in glycemia. Macrophage functions have been shown to be linked to their metabolism. In line with this, our data suggest that this increase in macrophage metabolism is accompanied by a shift towards an anti-inflammatory state. Together, these results support a model where diabetes-induced changes in metabolism in macrophages might lead to a reduced risk for AAA development.

The FibromiR miR-214-3p Is Upregulated in Duchenne Muscular Dystrophy and Promotes Differentiation of Human Fibro-Adipogenic Muscle Progenitors.

Fibrosis is a deleterious invasion of tissues associated with many pathological conditions, such as Duchenne muscular dystrophy (DMD) for which no cure is at present available for its prevention or its treatment. Fibro-adipogenic progenitors (FAPs) are resident cells in the human skeletal muscle and can differentiate into myofibroblasts, which represent the key cell population responsible for fibrosis. In this study, we delineated the pool of microRNAs (miRNAs) that are specifically modulated by TGFß1 in FAPs versus myogenic progenitors (MPs) by a global miRNome analysis. A subset of candidates, including several "FibromiRs", was found differentially expressed between FAPs and MPs and was also deregulated in DMD versus healthy biopsies. Among them, the expression of the TGFß1-induced miR-199a~214 cluster was strongly correlated with the fibrotic score in DMD biopsies. Loss-of-function experiments in FAPs indicated that a miR-214-3p inhibitor efficiently blocked expression of fibrogenic markers in both basal conditions and following TGFß1 stimulation. We found that FGFR1 is a functional target of miR-214-3p, preventing the signaling of the anti-fibrotic FGF2 pathway during FAP fibrogenesis. Overall, our work demonstrates that the « FibromiR ¼ miR-214-3p is a key activator of FAP fibrogenesis by modulating the FGF2/FGFR1/TGFß axis, opening new avenues for the treatment of DMD.

Differential micro-RNA expression in diabetic patients with abdominal aortic aneurysm.

OBJECTIVES: The potential implication of micro-RNAs (miRs) in the negative association between diabetes and abdominal aortic aneurysm (AAA) has so far never been addressed. The aim of this study was to compare miR expression between diabetic and non-diabetic patients with AAA. METHODS: Ten diabetic patients were prospectively included and compared to 10 age- and sex-matched non-diabetic patients with infrarenal AAA. A profiling analysis of 752 human miRs was performed from peripheral blood mononuclear cells (PBMCs) using miRCURY LNA Universal RT microRNA PCR (Exiqon- Qiagen®). miR that showed significant differential expression (P < 0.05) were selected and further analyzed in the entire cohort in sera, plasma and aneurysmal aortic tissues. RESULTS: Four miRs were significantly differentially expressed in PBMCs of diabetic patients compared to non-diabetics: 3 were upregulated (miR-144-3p, 20a-5p and 188-3p) and 1 downregulated (miR-548k). miR-144-3p and miR-548k were also increased in aneurysmal tissue and miR-20a-5p was increased in serum. The expression of miR-20a-5p in PBMCs was correlated with fructosamine concentration (r = 0.62, p = 0.006). CONCLUSIONS: Even if further studies are required to determine their direct role in AAA, these miRs could represent interesting new targets.

Investigation of Plasma Inflammatory Profile in Diabetic Patients With Abdominal Aortic Aneurysm: A Pilot Study.

INTRODUCTION:: Clinical studies have unraveled a negative association between diabetes and abdominal aortic aneurysm (AAA), but the mechanisms involved are still poorly understood. The aim of this study was to determine whether diabetic patients with AAA had a distinct plasma inflammatory profile compared to nondiabetic patients. METHODS:: Plasma samples were obtained from 10 diabetic patients with AAA and 10 nondiabetic patients with AAA. The relative protein expression of 92 inflammatory-related human protein biomarkers was assessed by proximity extension assay technology using Proseek Multiplex Inflammation I kit (Olink). RESULTS:: Clinical characteristics were similar in diabetic patients with AAA compared to nondiabetic patients with AAA, the median ages being 67 and 73 years, respectively ( P = .61). The AAA diameters were, respectively, 50 and 49 mm ( P = .72). Among the 92 markers screened, 67 (72.8%) were detected in all samples. Diabetic patients had significantly lower protein expression of C-C motif chemokine 19 (CCL19) and C-C motif chemokine 23 (CCL23; 542.3 vs 980.3, P = .01 and 1236 vs 1406, P = .04, respectively). They tended to have higher expression of tumor necrosis factor ligand superfamily member 14 (TNFSF14) compared to controls (14.6 vs 10.8, P = .05). CONCLUSION:: Diabetic patients with AAA differentially expressed CCL19, CCL23 and TNFSF14 in plasma compared to nondiabetic patients with AAA. Further studies are required to determine whether the markers identified could play a role in the negative association between diabetes and AAA pathogenesis.

Association of abdominal aortic aneurysm diameter with insulin resistance index.

INTRODUCTION: Epidemiological studies have highlighted a negative association between diabetes and abdominal aortic aneurysm (AAA). The aim of this study was to investigate the association between insulin resistance and AAA size. MATERIALS AND METHODS: This prospective cross sectional monocentric study analysed fasting blood samples from 55 patients with AAA eligible for surgical repair. They were divided into 2 groups according to the median AAA diameter: diameter < 50 mm (N = 28) and diameter > 50 mm (N = 27). The median ages were respectively 73 years (62 - 79) and 72 years (67 - 81). Glucose and fructosamine concentrations were determined by spectrophotometry; insulin and C-peptide using chemiluminescent technology. Homeostasis model assessment 2 calculator was used to estimate insulin resistance index (HOMA2 IR). RESULTS: There was no significant difference for fasting glucose concentration between the groups (6.1 vs. 5.9 mmol/L, P = 0.825). C-peptide and insulin concentrations, as well as HOMA2 IR index were significantly higher in patients with AAA > 50 mm (0.82 vs. 0.54 nmol/L, P = 0.012; 9 vs. 5 mU/L, P = 0.019 and 1.72 vs. 1.26, P = 0.028, respectively). No linear correlation was identified between AAA diameter and HOMA2 IR. Fructosamine concentration was lower in patients with AAA > 50 mm (225.5 vs. 251 µmol/L, P = 0.005) and negatively correlated with AAA diameter (r = - 0.54, P < 0.001). CONCLUSION: This study evidenced an association between AAA diameter and insulin resistance. Further studies are required to determine a causal link between insulin resistance and AAA development.

Instructive role of M-CSF on commitment of bipotent myeloid cells involves ERK-dependent positive and negative signaling.

M-CSF and G-CSF are instructive cytokines that specifically induce differentiation of bipotent myeloid progenitors into macrophages and granulocytes, respectively. Through morphology and colony assay studies, flow cytometry analysis of specific markers, and expression of myeloid transcription factors, we show here that the Eger/Fms cell line is composed of cells whose differentiation fate is instructed by M-CSF and G-CSF, thus representing a good in vitro model of myeloid bipotent progenitors. Consistent with the essential role of ERK1/2 during macrophage differentiation and defects of macrophagic differentiation in native ERK1(-/-) progenitors, ERK signaling is strongly activated in Eger/Fms cells upon M-CSF-induced macrophagic differentiation but only to a very small extent during G-CSF-induced granulocytic differentiation. Previous in vivo studies indicated a key role of Fli-1 in myeloid differentiation and demonstrated its weak expression during macrophagic differentiation with a strong expression during granulocytic differentiation. Here, we demonstrated that this effect could be mediated by a differential regulation of protein kinase Cδ (PKCd) on Fli-1 expression in response to M-CSF and G-CSF. With the use of knockdown of PKCd by small interfering RNA, we demonstrated that M-CSF activates PKCd, which in turn, inhibits Fli-1 expression and granulocytic differentiation. Finally, we studied the connection between ERK and PKCd and showed that in the presence of the MEK inhibitor U0126, PKCd expression is decreased, and Fli-1 expression is increased in response to M-CSF. Altogether, we demonstrated that in bipotent myeloid cells, M-CSF promotes macrophagic over granulocytic differentiation by inducing ERK activation but also PKCd expression, which in turn, down-regulates Fli-1 expression and prevents granulocytic differentiation.