Moderate Increase of Indoxyl Sulfate Promotes Monocyte Transition into Profibrotic Macrophages

Objective

The uremic toxin Indoxyl-3-sulphate (IS), a ligand of Aryl hydrocarbon Receptor (AhR), raises in blood during early renal dysfunction as a consequence of tubular damage, which may be present even when eGFR is normal or only moderately reduced, and promotes cardiovascular damage and monocyte-macrophage activation. We previously found that patients with abdominal aortic aneurysms (AAAs) have higher CD14⁺CD16⁺ monocyte frequency and prevalence of moderate chronic kidney disease (CKD) than age-matched control subjects. Here we aimed to evaluate the IS levels in plasma from AAA patients and to investigate in vitro the effects of IS concentrations corresponding to mild-to-moderate CKD on monocyte polarization and macrophage differentiation.

Methods

Free IS plasma levels, monocyte subsets and laboratory parameters were evaluated on blood from AAA patients and eGFR-matched controls. THP-1 monocytes, treated with IS 1, 10, 20 μM were evaluated for CD163 expression, AhR signaling and then induced to differentiate into macrophages by PMA. Their phenotype was evaluated both at the stage of semi-differentiated and fully differentiated macrophages. AAA and control sera were similarly used to treat THP-1 monocytes and the resulting macrophage phenotype was analyzed.

Results

IS plasma concentration correlated positively with CD14⁺CD16⁺ monocytes and was increased in AAA patients. In THP-1 cells, IS promoted CD163 expression and transition to macrophages with hallmarks of classical (IL-6, CCL2, COX2) and alternative phenotype (IL-10, PPARγ, TGF-β, TIMP-1), via AhR/Nrf2 activation. Analogously, AAA sera induced differentiation of macrophages with enhanced IL-6, MCP1, TGF-β, PPARγ and TIMP-1 expression.

Conclusion

IS skews monocyte differentiation toward low-inflammatory, profibrotic macrophages and may contribute to sustain chronic inflammation and maladaptive vascular remodeling.     

Activation of Olfactory Receptors on Mouse Pulmonary Macrophages Promotes Monocyte Chemotactic Protein-1 Production

Background

Emerging evidence suggests that non-olfactory tissues and cells can express olfactory receptors (ORs), however, the exact function of ectopic OR expression remains unknown. We have previously shown in mouse models that a unique cooperation between interferon-γ (IFN-γ) and lipopolysaccharide (LPS) drives the activation of pulmonary macrophages and leads to the induction of pathogenic responses in the respiratory tract. Further, through gene array studies, we have shown that activation of macrophages by these molecules results in the selective expression of a number of ORs. In this study, we validated the expression of these ORs in mouse airway and pulmonary macrophages in response to IFN-γ and LPS (γ/LPS) stimulation, and further explored the effect of odorant stimulation on macrophage function.

Methodology/Principal Findings

OR expression in airway and pulmonary macrophages in response to IFN-γ, LPS or γ/LPS treatments was assessed by microarray and validated by q-PCR. OR expression (e.g. OR622) on macrophages was confirmed by visualization in immunofluoresence assays. Functional responses to odorants were assessed by quantifying inflammatory cytokine and chemokine expression using q-PCR and cell migration was assessed by a modified Boyden chamber migration assay. Our results demonstrate that eight ORs are expressed at basal levels in both airway and pulmonary macrophages, and that γ/LPS stimulation cooperatively increased this expression. Pulmonary macrophages exposed to the combined treatment of γ/LPS+octanal (an odorant) exhibited a 3-fold increase in MCP-1 protein production, compared to cells treated with γ/LPS alone. Supernatants from γ/LPS+octanal exposed macrophages also increased macrophage migration in vitro.

Conclusions/Significance

Eight different ORs are expressed at basal levels in pulmonary macrophages and expression is upregulated by the synergistic action of γ/LPS. Octanal stimulation further increased MCP-1 production and the motility of macrophages. Our results suggest that ORs may mediate macrophage function by regulating MCP-1 production and cell migration.     

IL-10 Acts As a Developmental Switch Guiding Monocyte Differentiation to Macrophages during a Murine Peritoneal Infection

The peritoneal wash of BALB/c or C57BL/6 mice contains two populations of macrophages that differ in their level of expression of MHC class II (MHC II). Although both populations efficiently phagocytose bacteria in vivo, only the MHC II(lo) population is effective at phagocytosing apoptotic cells in vivo and only the MHC II(hi) population is effective at presenting Ag to T cells in vitro. Soon after induction of a peritoneal infection both of these macrophage populations are lost from the peritoneal wash fraction. Blood monocytes then enter the inflamed peritoneum and develop into new peritoneal macrophages. Whether these monocytes develop into MHC II(lo) or into MHC II(hi) macrophages is crucially dependent on the cytokine IL-10, which is transiently elevated in the peritoneal wash during the early phase of infection. Monocytes from CD45.1 animals transferred early in infection when the IL-10 concentration is high into congenic CD45.2 recipients develop into the MHC II(lo) macrophage population. Monocytes transferred later, when the IL-10 concentration has fallen, develop into the MHC II(hi) population. In infected IL-10-deficient animals monocytes fail to develop into the MHC II(lo) population but can be induced to do so by exogenous application of IL-10. Finally, high numbers of wild-type monocytes injected into IL-10R1-deficient animals develop into MHC II(lo) macrophages and were able by a bystander effect to induce the differentiation of the endogenous monocytes to the same fate.     

TSLP Promotes Induction of Th2 Differentiation but Is Not Necessary during Established Allergen-Induced Pulmonary Disease

Thymic stromal lymphopoietin (TSLP) has been implicated in the development of allergic inflammation by promoting Th2-type responses and has become a potential therapeutic target. Using in vitro T cell differentiation cultures we were able to validate that TSLP played a more critical role in the early development of Th2 immune responses with less significant enhancement of already developed Th2 responses. Adoptive transfer of naive DO11.10 ovalbumin-specific T cells followed by airway exposure to ovalbumin showed an early impairment of Th2 immune response in TSLP−/− mice compared to wild type mice during the development of a Th2 response. In contrast, transfer of already differentiated Th2 cells into TSLP−/− mice did not change lung pathology or Th2 cytokine production upon ovalbumin challenge compared to transfer into wild type mice. An allergen-induced Th2 airway model demonstrated that there was only a difference in gob5 expression (a mucus-associated gene) between wild type and TSLP−/− mice. Furthermore, when allergic animals with established disease were treated with a neutralizing anti-TSLP antibody there was no change in airway hyperreponsiveness (AHR) or Th2 cytokine production compared to the control antibody treated animals, whereas a change in gob5 gene expression was also observed similar to the TSLP−/− mouse studies. In contrast, when animals were treated with anti-TSLP during the initial stages of allergen sensitization there was a significant change in Th2 cytokines during the final allergen challenge. Collectively, these studies suggest that in mice TSLP has an important role during the early development of Th2 immune responses, whereas its role at later stages of allergic disease may not be as critical for maintaining the Th2-driven allergic disease.     

Impaired Cholesterol Efflux in Senescent Macrophages Promotes Age-Related Macular Degeneration

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Highlights? Macrophage senescence impairs cholesterol efflux and promotes macular degeneration ? Senescent macrophages polarize to a proangiogenic, disease-promoting phenotype ? Macrophage cholesterol efflux is regulated by miR33 and its target ABCA1 ? Age-related decrease in macrophage cholesterol efflux is therapeutically reversible     

干细胞诱导分化为胰岛素分泌细胞的研究现状

胰腺或胰岛细胞移植是目前治疗Ⅰ型糖尿病和部分Ⅱ型糖尿病效果最理想的方法,但因来源组织短缺及需要终生服用免疫抑制剂等问题限制了它的广泛应用.利用胰腺或胰腺外的多能干细胞产生胰岛样细胞有望克服上述问题而用于治疗糖尿病.本文就将干细胞诱导分化为胰岛样细胞中所用的重要的转录因子和可溶性诱导因子及其作用以及胰岛素分泌细胞的来源做一综述.     

注射紫杉醇的小鼠骨髓细胞体外分化巨噬细胞增强

目的研究小鼠腹腔注射紫杉醇对体外骨髓细胞诱导分化巨噬细胞的影响。方法小鼠连续5d腹腔注射紫杉醇,无菌制备骨髓细胞,用含巨噬细胞集落刺激因子（M-CSF）的RPMI1640培养液培养骨髓细胞,通过流式细胞仪对其诱导分化的巨噬细胞表面分子、吞噬功能进行分析。结果紫杉醇明显降低小鼠骨髓细胞数量,但骨髓细胞体外诱导分化成巨噬细胞的数量明显增加;F4/80^＋巨噬细胞中CD80、CD14表面分子表达升高,而I-A^d表达降低;紫杉醇处理组诱导分化的巨噬细胞吞噬鸡红细胞的能力提高。结论结果提示紫杉醇可能具有调节巨噬细胞表面分子的表达和吞噬功能。     

Heme-Mediated Induction of CXCL10 and Depletion of CD34+ Progenitor Cells Is Toll-Like Receptor 4 Dependent

Plasmodium falciparum infection can cause microvascular dysfunction, cerebral encephalopathy and death if untreated. We have previously shown that high concentrations of free heme, and C-X-C motif chemokine 10 (CXCL10) in sera of malaria patients induce apoptosis in microvascular endothelial and neuronal cells contributing to vascular dysfunction, blood-brain barrier (BBB) damage and mortality. Endothelial progenitor cells (EPC) are microvascular endothelial cell precursors partly responsible for repair and regeneration of damaged BBB endothelium. Studies have shown that EPC’s are depleted in severe malaria patients, but the mechanisms mediating this phenomenon are unknown. Toll-like receptors recognize a wide variety of pathogen-associated molecular patterns generated by pathogens such as bacteria and parasites. We tested the hypothesis that EPC depletion during malaria pathogenesis is a function of heme-induced apoptosis mediated by CXCL10 induction and toll-like receptor (TLR) activation. Heme and CXCL10 concentrations in plasma obtained from malaria patients were elevated compared with non-malaria subjects. EPC numbers were significantly decreased in malaria patients (P < 0.02) and TLR4 expression was significantly elevated in vivo. These findings were confirmed in EPC precursors in vitro; where it was determined that heme-induced apoptosis and CXCL10 expression was TLR4-mediated. We conclude that increased serum heme mediates depletion of EPC during malaria pathogenesis.     

S100A6通过招募和活化巨噬细胞促进血管形成*

目的:探讨S100A6经由巨噬细胞介导的促血管生成作用及机制。方法:(1)用重组蛋白 GST-hS100A6处理巨噬细胞后:①收集上清制备条件培养基(简称A6-Mφ-CM),并用之重悬人脐静脉内皮细胞(HUVEC),用体外血管形成试验检测各处理因素对血管形成的影响;②分别用实时荧光定量PCR和Western blot检测巨噬细胞的M2型标志物CD163及促血管形成因子CCL2、IL-6、VEGFA的mRNA和蛋白质水平,以及JAK2和STAT3的蛋白质及其磷酸化水平;③用Transwell迁移试验检测巨噬细胞迁移能力的变化。(2)使用JAK2抑制剂(XL019)预处理巨噬细胞后再加GST-hS100A6处理,检测S100A6促巨噬细胞迁移作用的变化。以重组蛋白GST为实验对照。 结果:(1)A6-Mφ-CM组的血管分支数和血管分支长度既明显高于GST-Mφ-CM组(P值均小于0.05),也明显高于GST-hS100A6直接处理的HUVEC组(P<0.001,P<0.01),提示S100A6处理后的巨噬细胞具有促进血管形成的作用;(2)GST-hS100A6处理后的巨噬细胞中,CD163、CCL2、IL-6、VEGFA的mRNA和蛋白质水平明显高于GST组(P值均小于0.05),提示S100A6诱导巨噬细胞向促血管表型(pro-angiogenic phenotype)转化;(3)GST-hS100A6处理后,巨噬细胞的迁移数是GST组的1.4倍(P<0.01),提示S100A6具有招募巨噬细胞的作用;(4)GST-hS100A6处理组巨噬细胞的JAK2和STAT3的蛋白质及其磷酸化水平都明显高于GST组(P值均小于0.05),而JAK2抑制剂XL019可部分抑制S100A6促进巨噬细胞迁移的作用(P<0.01),提示S100A6促进巨噬细胞迁移作用机制涉及JAK2/STAT3信号通路的激活。 结论:微环境中的S100A6可通过招募巨噬细胞并进一步诱导其向促血管表型转化,进而促进新生血管形成;其招募巨噬细胞的机制涉及JAK2/STAT3信号通路的激活。     

The Long Pentraxin PTX3 Promotes Fibrocyte Differentiation

Monocyte-derived, fibroblast-like cells called fibrocytes are associated with fibrotic lesions. The plasma protein serum amyloid P component (SAP; also known as pentraxin-2, PTX2) inhibits fibrocyte differentiation in vitro, and injections of SAP inhibit fibrosis in vivo. SAP is a member of the pentraxin family of proteins that includes C-reactive protein (CRP; PTX1) and pentraxin-3 (PTX3). All three pentraxins are associated with fibrosis, but only SAP and CRP have been studied for their effects on fibrocyte differentiation. We find that compared to SAP and CRP, PTX3 promotes human and murine fibrocyte differentiation. The effect of PTX3 is dependent on FcγRI. In competition studies, the fibrocyte-inhibitory activity of SAP is dominant over PTX3. Binding competition studies indicate that SAP and PTX3 bind human FcγRI at different sites. In murine models of lung fibrosis, PTX3 is present in fibrotic areas, and the PTX3 distribution is associated with collagen deposition. In lung tissue from pulmonary fibrosis patients, PTX3 has a widespread distribution, both in unaffected tissue and in fibrotic lesions, whereas SAP is restricted to areas adjacent to vessels, and absent from fibrotic areas. These data suggest that the relative levels of SAP and PTX3 present at sites of fibrosis may have a significant effect on the ability of monocytes to differentiate into fibrocytes.     

mTORC1 Signaling Promotes Osteoblast Differentiation from Preosteoblasts

Preosteoblasts are precursor cells that are committed to the osteoblast lineage. Differentiation of these cells to mature osteoblasts is regulated by the extracellular factors and environmental cues. Recent studies have implicated mTOR signaling in the regulation of osteoblast differentiation. However, mTOR exists in two distinct protein complexes (mTORC1 and mTORC2), and the specific role of mTORC1 in regulating the progression of preosteoblasts to mature osteoblastis still unclear. In this study, we first deleted Raptor, a unique and essential component of mTORC1, in primary calvarial cells. Deletion of Raptor resulted in loss of mTORC1 but an increase in mTORC2 signaling without overtly affecting autophagy. Under the osteogenic culture condition, Raptor-deficient cells exhibited a decrease in matrix synthesis and mineralization. qPCR analyses revealed that deletion of Raptor reduced the expression of late-stage markers for osteoblast differentiation (Bglap, Ibsp, and Col1a), while slightly increasing early osteoblast markers (Runx2, Sp7, and Alpl). Consistent with the findings in vitro, genetic ablation of Raptor in osterix-expressing cells led to osteopenia in mice. Together, our findings have identified a specific role for mTORC1 in the transition from preosteoblasts to mature osteoblasts.     

Histone Deacetylase Inhibitor Valproic Acid Promotes the Differentiation of Human Induced Pluripotent Stem Cells into Hepatocyte-Like Cells

In this study, we aimed to elucidate the effects and mechanism of action of valproic acid on hepatic differentiation from human induced pluripotent stem cell-derived hepatic progenitor cells. Human induced pluripotent stem cells were differentiated into endodermal cells in the presence of activin A and then into hepatic progenitor cells using dimethyl sulfoxide. Hepatic progenitor cells were matured in the presence of hepatocyte growth factor, oncostatin M, and dexamethasone with valproic acid that was added during the maturation process. After 25 days of differentiation, cells expressed hepatic marker genes and drug-metabolizing enzymes and exhibited drug-metabolizing enzyme activities. These expression levels and activities were increased by treatment with valproic acid, the timing and duration of which were important parameters to promote differentiation from human induced pluripotent stem cell-derived hepatic progenitor cells into hepatocytes. Valproic acid inhibited histone deacetylase activity during differentiation of human induced pluripotent stem cells, and other histone deacetylase inhibitors also enhanced differentiation into hepatocytes. In conclusion, histone deacetylase inhibitors such as valproic acid can be used to promote hepatic differentiation from human induced pluripotent stem cell-derived hepatic progenitor cells.     

Repression of SIRT1 Promotes the Differentiation of Mouse Induced Pluripotent Stem Cells into Neural Stem Cells

The use of transplanting functional neural stem cells (NSCs) derived from induced pluripotent stem cells (iPSCs) has increased for the treatment of brain diseases. As such, it is important to understand the molecular mechanisms that promote NSCs differentiation of iPSCs for future NSC-based therapies. Sirtuin 1 (SIRT1), a NAD⁺-dependent protein deacetylase, has attracted significant attention over the past decade due to its prominent role in processes including organ development, longevity, and cancer. However, it remains unclear whether SIRT1 plays a role in the differentiation of mouse iPSCs toward NSCs. In this study, we produced NSCs from mouse iPSCs using serum-free medium supplemented with retinoic acid. We then assessed changes in the expression of SIRT1 and microRNA-34a, which regulates SIRT1 expression. Moreover, we used a SIRT1 inhibitor to investigate the role of SIRT1 in NSCs differentiation of iPSCs. Data revealed that the expression of SIRT1 decreased, whereas miRNAs-34a increased, during this process. In addition, the inhibition of SIRT1 enhanced the generation of NSCs and mature neurocytes. This suggests that SIRT1 negatively regulated the differentiation of mouse iPSCs into NSCs, and that this process may be regulated by miRNA-34a.     

IL-17A Promotes Pulmonary B-1a Cell Differentiation via Induction of Blimp-1 Expression during Influenza Virus Infection

B-1 cells play a critical role in early protection during influenza infections by producing natural IgM antibodies. However, the underlying mechanisms involved in regulating this process are largely unknown. Here we found that during influenza infection pleural cavity B-1a cells rapidly infiltrated lungs, where they underwent plasmacytic differentiation with enhanced IgM production. This process was promoted by IL-17A signaling via induction of Blimp-1 expression and NF-κB activation in B-1a cells. Deficiency of IL-17A led to severely impaired B-1a-derived antibody production in the respiratory tract, resulting in a deficiency in viral clearance. Transfer of B-1a-derived natural antibodies rescued Il17a ^-/- mice from otherwise lethal infections. Together, we identify a critical function of IL-17A in promoting the plasmacytic differentiation of B-1a cells. Our findings provide new insights into the mechanisms underlying the regulation of pulmonary B-1a cell response against influenza infection.     

The Nocardia Rubra Cell Wall Skeleton Regulates Macrophages and Promotes Wound Healing

The Nocardia rubra cell wall skeleton (Nr-CWS) is an immunomodulator used clinically for its ability to modulate the body’s immune function. Macrophages are an important hub of the immune response during wound healing. In this study, we hypothesized that a Nr-CWS could modulate macrophage physiological activities, polarize macrophages toward M2, and promote wound healing. Through in vivo experiments, we made two full-thickness excisional wounds on the backs of mice; one was treated with a Nr-CWS, and the other was treated with saline. We photographed and recorded the wound change every other day. We observed the histopathological examination and collagen deposition using H&E and Masson staining, then analyzed the macrophage surface markers using immunofluorescence. Through in vitro experiments, we studied the effect of the Nr-CWS on RAW264.7 cells through CCK8, transwell, flow cytometry, western blot, immunofluorescence, and ELISA. We found that the Nr-CWS can enhance the proliferation, migration, and phagocytosis of macrophages. In addition, it can promote the recruitment of macrophages on the wound surface, polarize macrophages to M2, and increase the expression of pro-healing cytokines. Ultimately, the Nr-CWS accelerated wound healing.     

Diesel Exhaust Particle Exposure In Vitro Alters Monocyte Differentiation and Function

Air pollution by diesel exhaust particles is associated with elevated mortality and increased hospital admissions in individuals with respiratory diseases such as asthma and chronic obstructive pulmonary disease. During active inflammation monocytes are recruited to the airways and can replace resident alveolar macrophages. We therefore investigated whether chronic fourteen day exposure to low concentrations of diesel exhaust particles can alter the phenotype and function of monocytes from healthy individuals and those with chronic obstructive pulmonary disease. Monocytes were purified from the blood of healthy individuals and people with a diagnosis of chronic obstructive pulmonary disease. Monocyte-derived macrophages were generated in the presence or absence of diesel exhaust particles and their phenotypes studied through investigation of their lifespan, cytokine generation in response to Toll like receptor agonists and heat killed bacteria, and expression of surface markers. Chronic fourteen day exposure of monocyte-derived macrophages to concentrations of diesel exhaust particles >10 µg/ml caused mitochondrial and lysosomal dysfunction, and a gradual loss of cells over time both in healthy and chronic obstructive pulmonary disease individuals. Chronic exposure to lower concentrations of diesel exhaust particles impaired CXCL8 cytokine responses to lipopolysaccharide and heat killed E. coli, and this phenotype was associated with a reduction in CD14 and CD11b expression. Chronic diesel exhaust particle exposure may therefore alter both numbers and function of lung macrophages differentiating from locally recruited monocytes in the lungs of healthy people and patients with chronic obstructive pulmonary disease.