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Autoimmune and Autoinflammatory Disorders
Betty Diamond
Feinstein Institute for Medical Research, United States
Laurence Morel
University of Florida, United States
Yogesh M. Scindia
Data Availability Statement
Front. Immunol., 13 December 2019 | https://doi.org/10.3389/fimmu.2019.02868
Heme-Oxygenase-1 Is Decreased in Circulating Monocytes and Is Associated With Impaired Phagocytosis and ROS Production in Lupus Nephritis
Loreto Cuitino1, Javiera Obreque1, Patricia Gajardo-Meneses1, Alejandra Villarroel2, Natalia Crisóstomo1, Ignacio F. San Francisco3, Rodrigo A. Valenzuela4, Gonzalo P. Méndez2 and Carolina Llanos1*
1Departamento de Inmunología Clínica y Reumatología, Escuela de Medicina, Pontificia Universidad Católica de Chile, Santiago, Chile
2Departamento de Anatomía Patológica, Escuela de Medicina, Pontificia Universidad Católica de Chile, Santiago, Chile
3Departamento de Urología, Escuela de Medicina, Pontificia Universidad Católica de Chile, Santiago, Chile
4Departamento de Ciencias Químicas y Biológicas, Facultad de Salud, Universidad Bernardo O'Higgins, Santiago, Chile
Lupus nephritis (LN) is one of the most serious manifestations of systemic lupus erythematosus (SLE). Based on studies showing the potential role of heme oxygenase-1 (HO-1), an enzyme that catalyzes the degradation of heme and has anti-inflammatory properties in SLE development, we decided to explore HO-1 in LN. Accordingly, we evaluated HO-1 levels and function in circulating and infiltrating monocytes and neutrophils of LN patients. HO-1 levels were assessed in peripheral monocytes of LN patients and controls by flow cytometry and immunofluorescence microscopy. Phagocytosis and the production of reactive oxygen species (ROS) were evaluated to determine the effect of HO-1 in monocyte function. In addition, renal biopsies with proliferative LN were used to identify HO-1 in infiltrating cells and renal tissue by immunofluorescence and immunohistochemistry. Biopsies of healthy controls (HC) and patients who underwent nephrectomy were included as controls. Circulating pro-inflammatory monocytes and activated neutrophils were increased in LN patients. HO-1 levels were decreased in all subsets of monocytes and in activated neutrophils. LN monocytes showed increased phagocytosis and higher production of ROS than those of HC. When HO-1 was induced, phagocytosis and ROS levels became similar to those of HC. HO-1 was mostly expressed in renal tubular epithelial cells (RTEC). Renal tissue of LN patients showed lower levels of HO-1 than HC, whereas infiltrating immune cells of LN showed lower levels of HO-1 than biopsies of patients who had renal surgery. HO-1 is decreased in circulating monocytes and activated neutrophils of LN patients. HO-1 levels modulate the phagocytosis of LN monocytes and ROS production. HO-1 expression in RTEC might be an attempt of self-protection from inflammation.
Systemic lupus erythematosus (SLE) is a chronic autoimmune disease of unknown etiology that predominantly affects women in childbearing age and is associated with higher rates of mortality than the general population. Although in SLE, inflammation may affect multiple organs, lupus nephritis (LN) remains the focus of attention of clinical and basic scientists given that kidney involvement is a major predictor of morbidity and mortality (1, 2). Indeed, despite efforts to improve clinical outcomes, remission is achieved in only 50–70% of LN patients and 10–20% of them will progress to end-stage renal disease (ESRD) within 5 years of diagnosis. Therefore, the better understanding of the underlying mechanisms of kidney involvement would be a progress in the field that can potentially lead to the development of new therapeutic targets.
Heme oxygenase-1 (HO-1) is an enzyme that catalyzes the degradation of heme and has anti-inflammatory properties. We have previously evaluated HO-1 expression in circulating monocytes of SLE patients, finding that this enzyme is decreased in affected individuals (3) and in an animal model for SLE (4). HO-1 can be induced in human endothelial cells after the release of strong immunogenic cytokines, such as tumor necrosis factor (TNF)-α and interleukin (IL)-1α (5–7) and is required for the regulation of the immune response, with subsequent inhibition of pro-inflammatory signals and recovery of homeostasis (8, 9). Interestingly, when MRL-Faslpr lupus mice are treated with carbon monoxide (CO)—a bystander product and inducer of HO-1 expression—they present lower levels of circulating anti-dsDNA and anti-histone antibodies than untreated mice. In addition, kidneys from CO-treated lupus mice have less number of activated B220+CD4−CD8− T cells than control animals and present delayed renal failure (4). Although it is well-known that, in LN, renal damage is initiated by glomerular deposition of immune complexes (IC) and subsequent complement activation, it has been acknowledged that innate immune cells also play an active role in the propagation of tissue damage. Recent data show that LN patients poorly degrade neutrophil extracellular traps (NETs), which promote the presentation of self-antigens (10). Even more, elegant experiments using a humanized lupus mouse model show that SLE human serum is capable of inducing LN in a process mediated by infiltrating neutrophils recruited by an FcγRIIA-dependent mechanism (11).
On the other hand, monocytes have been less studied than neutrophils but are well-known to participate in SLE pathogenesis. Indeed, expanding data have demonstrated that infiltrating monocytes and macrophages are phenotypically altered in SLE and are associated with both murine and human LN (12–15). In humans, monocytes have been divided into three subtypes based on relative surface expression of lipopolysaccharide (LPS) coreceptor CD14 and FCγIII receptor CD16, "classical" monocytes, "intermediate" monocytes, and "pro-inflammatory" monocytes. Several reports suggest that the primary function of majority of circulating monocytes is phagocytosis (16, 17) of cellular debris, pathogens, and other external agents in a way that does not involve the release of inflammatory mediators; however, it has been observed that monocytes of SLE patients are inefficient in the clearance of apoptotic cells, which is also associated with the production of pro-inflammatory cytokines such as IL-6, TNF-α, IL-10, transforming growth factor (TGF)-β, and interferon (IFN)-α (18–20).
Given our findings in circulating human monocytes and the interesting results obtained after treating lupus mice with CO, we decided to explore whether HO-1 levels of circulating and infiltrating monocytes play a role in the pathogenesis of human LN, with a special focus on the renal interstitium because it has recently been considered an important predictor of renal outcomes in LN patients (21). In addition, we also evaluated HO-1 levels in circulating neutrophils.
This study was approved by the Research Ethics Committee (REC) of Pontificia Universidad Católica de Chile (PUC). All subjects signed PUC-REC-approved consent forms. A total of 15 subjects who met the American College of Rheumatology SLE 1997 criteria and had a renal biopsy showing type III, IV, and/or V LN as per the ISN/RPS classification were included to do peripheral blood and purified monocyte analyses; data of patients are shown in Table 1 (LN-1–LN-15). Fifteen healthy controls (HC) were recruited to analyze peripheral blood samples; demographic data are summarized in Table 2.
Table 1. Clinical and demographic characteristics of patients with lupus nephritis (LN) who donated blood samples.
Table 2. Demographic details of lupus nephritis (LN) patients and healthy controls recruited to obtain blood samples.
Twenty renal biopsies with type III, IV, and/or V LN ISN/RPS classification performed between January 2006 and January 2017 were used for immunofluorescence and immunohistochemistry studies (Table 1, LN-09–LN-28). A summary of histologic characteristics of renal biopsies is shown in Table 3. Biopsies of three patients who underwent surgery and nephrectomy had to be performed as part of the surgical protocol for different reasons, but their kidneys were healthy and were included as HC. Biopsies of eight patients without autoimmune disease who had renal cancer and underwent nephrectomy were included [cancer controls (CC)]. Only tissue that did not present tumor was used for the purpose of this study.
Table 3. Pathological characteristics of lupus nephritis (LN) renal biopsies.
Direct Immunofluorescence Staining for Flow Cytometry [Fluorescence-Activated Cell Sorting (FACS)]
To identify circulating monocytes and neutrophils, samples obtained by peripheral venous puncture, and the total blood was separated in 150 μl. Red blood cells were lysed using ammonium-chloride-potassium lysing buffer (ACK; ThermoFisher Scientific, Waltham, MA, USA) for 5 min at room temperature (RT), then washed and centrifuged. The pellet was resuspended in PBS-2% fetal bovine serum, and cells were counted using TC20 (Biorad, Hercules, CA, USA). To analyze monocytes and neutrophils, flow cytometry exclusion gating strategy was used as previously described (16). Briefly, the staining cells were selected using forward and side scatter characteristics and deleting the doublets (FSC vs. SSC). Monocytes were identified using the following antibodies: CD14-PeCy7, CD16-APC, CD3-Pe, CD19-Pe, CD66b-PerCpCy5.5, CD56-APC-Cy7 (Biolegend, San Diego, CA, USA). Neutrophils were identified using the following antibodies: CD15-APC-Fire750, CD66b-PerCp, CD11b-PE, CD3-FITC, CD19-FITC, CD14-PeCy-7, HLA-DR-APC (Biolegend).
To obtain purified monocytes, peripheral blood mononuclear cells (PBMCs) were isolated by density gradient centrifugation using Ficoll-Paque (GE Healthcare, Chicago, IL, USA) as previously described (22). Then monocytes were purified using Pan Monocyte Isolation Kit, Human (Miltenyi Biotec, Bergisch Gladbach) according to manufacturer's instructions. To identify monocytes, the purified cells (5 × 105 cells) were stained with the following fluorescent conjugated antibodies (Biolegend): CD14-PeCy7 and CD16-APC.
For intracellular detection of HO-1, cells were fixed with fixation/permeabilization solution kit (BD Biosciences, Franklin Lakes, NJ, USA) and stained with an unconjugated rabbit polyclonal antibody (Abcam, Cambridge, MA, USA). After 30 min, cells were incubated with a conjugated secondary antibody (Alexa Fluor 555 or 488; ThermoFisher Scientific). All samples were analyzed in FACS Canto II using FACSDiva software.
Phagocytosis Assay
To determine the phagocytic function of monocytes, FluoSpheres™ Carboxylate-Modified Microspheres (ThermoFisher Scientific) were used (23). The FluoSpheres™ were incubated with 100 μg of purified immunoglobulin G (IgG) for 30 min at 37°C to opsonize the beads. IgGs were purified from serum of a patient with LN who has high titers of anti-DNA antibodies (Table 1, LN-23) or of an HC matched in sex and age with the patient. Purified monocytes from LN patients (Table 1, LN-1–LN-15) or from HC (Table 2) were adjusted to 5 × 105 cells and were incubated with opsonized FluoSpheres™ (ratio of monocytes to beads = 1:5) for 1 h at 37°C to determine the phagocytic function or at 4°C to detect nonspecific binding. Monocytes were washed three times using 1 ml of complete PBS and analyzed in LSRFortessa X20 Flow Cytometer using FACSDiva software (BD Immunocytometry Systems, San Jose, CA, USA).
Determination of Reactive Oxygen Species (ROS)
ROS production was determined in 5 × 105 purified monocytes using CellROX™ Green Flow Cytometry Assay Kit (ThermoFisher Scientific) according to manufacturer's instructions. Briefly, monocytes were incubated with 250 μM of CellROX® reagent for 30 min at 37°C in the dark. To obtain a positive and a negative control for ROS production, monocytes were preincubated with tert-butyl hydroperoxide (TBHP, 250 μM) or N-acetylcysteine (NAC, 1 mM), respectively, for 1 h at 37°C before incubation with CellROX®. During the final 15 min of staining, SYTOX® Red Dead Cell stain solution was added (5 μM) in each sample and mixed gently. Samples were immediately analyzed by flow cytometry.
Induction of HO-1 Using Cobalt Protoporphyrin (CoPP)
Before phagocytosis and/or ROS determination assays, purified monocytes (5 × 105) were treated with 100 μM of CoPP (Frontier Scientific, Logan, UT, USA) for 2 h at 37°C to induce HO-1 expression. Alternatively, to inhibit HO-1 expression, monocytes were treated with 100 μM of tin protoporphyrin (SnPP; Frontier Scientific) using the same protocol as for CoPP treatment. After 2 h, purified monocytes were washed and used for different experiments. CoPP and SnPP were dissolved in 0.1 M NaOH to prepare a stock solution of 10 mM.
Tissues were deparaffinized in xylene and rehydrated using ethanol. The activity of endogenous peroxidase was blocked. For immunohistochemistry staining, immunoperoxidase system (VECTASTAIN PK-6200; Vectors laboratories, Burlingame, CA, USA) was used. Samples were stained with a mouse anti-HO1 (Abcam, Cambridge, MA, USA) monoclonal antibody and analyzed using ImmPACT™ AEC (SK-4205). Finally, samples were counterstained with hematoxylin and blindly evaluated by a pathologist (GM, AV) who determined the H- Score, which includes intensity (0-negative, 1-weak, 2-moderate, and 3-strong) and the percentage of cells that were positive for HO-1 staining (24).
Immunofluorescence in Renal Biopsies
Biopsies were deparaffinized, rehydrated, and stained overnight with conjugated antibodies to identify monocytes (CD14; Abcam), activated neutrophils (CD66b; Biolegend), and macrophages (CD68; ThermoFisher Scientific), together with an unconjugated mouse/rabbit anti-HO-1 monoclonal antibody (Abcam). A secondary conjugated antibody (anti-mouse Alexa Fluor 555 and anti-rabbit Alexa Fluor 488; ThermoFisher Scientific) was also used. For nuclei staining, samples were incubated with DAPI (ThermoFisher Scientific). Images were analyzed using ImageJ software (NIH, Bethesda, MD, USA) to calculate the corrected total cell fluorescence (CTCF) (25).
RNA Extraction and qPCR
Total RNA was extracted from monocytes using TRIzol (ThermoFisher Scientific), and cDNAs were generated from 1 μg RNA using ImProm-II™ Reverse Transcription System (Promega, Fitchburg, WI, USA) according to the manufacturer's protocol. cDNA products were used as template for qPCR experiments performed in a StepOnePlus™ Real-Time PCR system (Applied Biosystems) using Fast SYBR® Green Master Mix (Applied Biosystems). Relative expression of HO-1 was calculated through the ΔΔCt method using glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and 18S genes as reference. Amplification was carried out with 160 nM of the following primers (5′ → 3′): HO-1: forward (fw): CCCCAACGAAAAGCACATCC—reverse (rv): AGACAGCTGCCACATTAGGG; GAPDH: fw: GGTGGTCTCCTCTGACTTCAACA—rv: GTTGCTGTAGCCAAATTCGTTGT; 18S: fw GTA ACC CGT TGA ACC CCA TT—rv: CCA TCC AAT CGG TAG TAG CG. Efficiencies were 98.96% (HO-1), 101.8% (GAPDH), and 103.9% (18S). ΔΔCt was defined as (ΔCt GOI treatment – ΔCt GOI control) – (ΔCt reference treatment – ΔCt reference control) and was calculated automatically by StepOne Software v2.3 (Applied Biosystems).
The results of all groups were expressed as mean ± SD. Statistical analyses were performed with GraphPad Prism 5 software (La Jolla, CA, USA) or SPSS-17 statistical software (SPSS Inc., Chicago, IL, USA). Mann–Whitney non-parametric test was used to compare LN patients with control groups. For statistical analysis between treatments, paired one-way Friedman test was used with Bonferroni post-hoc test over the ranked data. P < 0.05 were considered significant.
Pro-Inflammatory Monocytes and Activated Neutrophils of LN Patients Show Lower Expression of HO-1 Than Those of HC
The expression of HO-1 in peripheral monocytes and neutrophils was evaluated in whole blood of LN patients (Table 1; patients LN-1–LN-15) and HC (Table 2; n = 15). To evaluate monocytes in peripheral blood, neutrophils and lymphocytes were excluded (Figure 1A). LN patients have higher percentage of monocytes than that of HC (Figure 1B). Interestingly, these LN monocytes had lower protein levels of HO-1 than those of HC monocytes (Figures 1C,D). Moreover, recent studies have revealed that peripheral monocytes can be divided based on the level of expression of type IIIa FCγ receptor (CD16) (17, 26), in classical (CD14hiCD16lo), anti-inflammatory (intermediate, CD14hiCD16hi), and pro-inflammatory monocytes (nonclassical, CD14loCD16hi) (Figure 1E). Interestingly, a significant increase of proinflammatory monocytes was observed in LN patients when compared with HC; however, no significant differences were observed when classical and anti-inflammatory monocytes were evaluated in both groups (Figure 1F). HO-1 expression was decreased in all subsets of monocytes, including pro-inflammatory monocytes (Figure 1G). In addition, we analyzed HO-1 expression using MACS kit by real-time PCR (qPCR) (Figure 1H) and immunofluorescence (Figures 1I,J) in purified monocytes of LN patients and HC. We confirmed that HO-1 levels were decreased in LN monocytes compared with those of HC.
Figure 1. Monocytes from peripheral blood of lupus nephritis (LN) patients express lower levels of heme oxygenase (HO)-1 than healthy controls (HC). The proportion of cells and HO-1 expression [mean fluorescence intensity (MFI)] were determined in peripheral blood using flow cytometry. (A) Gating strategy for identification of monocytes showing successive exclusion of neutrophils, natural killer (NK) cells, and B and T cells. (B) Percentage of monocytes circulating. (C) Representative histograms for HO-1 expression in monocytes of LN patients and HC. Red: autofluorescence; light green: LN monocytes; green: HC monocytes. (D) MFI value of HO-1 expression. (E) Representative dot plot to identify the different subpopulations of monocytes. (F) Percentage of each subset's monocytes. (G) Representative MFI value of HO-1 expression in each subset of monocytes. (H) HO-1 expression was quantified in purified monocytes by real-time RT-PCR. (I) Immunofluorescence of HO-1 in purified monocytes. (J) Quantification of HO-1 expression [corrected total cell fluorescence (CTCF)] in purified monocytes. Graphs are showing the mean ± standard deviation. n = 15 LN patients (black bars) and n = 15 HC (white bars). *P < 0.05, **P < 0.01, ***P < 0.001 by Mann–Whitney test.
To evaluate HO-1 expression in peripheral neutrophils, cells CD11b+/CD15+ were identified using exclusion strategy. After deletion of doublets cells from FSC vs. SSC plot, monocytes and lymphocytes were identified and excluded (Figure 2A). No significant differences between LN patients and HC were found in HO-1 expression in peripheral neutrophils (p = 0.5772). However, two different subsets of neutrophils based on the expression of CD66b have been described; thus, CD66b+ cells are activated neutrophils (27). Remarkably, LN patients showed higher percentage of circulating activated neutrophils than HC (Figure 2B), and when HO-1 expression was evaluated in this population, LN-activated neutrophils presented lower levels of this enzyme than HC (Figures 2C,D).
Figure 2. Activated neutrophils of lupus nephritis (LN) patients express lower levels of heme oxygenase (HO)-1 than healthy controls (HC). The proportion of cells and HO-1 expression [mean fluorescence intensity (MFI)] was determined in peripheral blood using flow cytometry. (A) Gating strategy to obtain neutrophils. CD14+ and HLA-DR+ cells were excluded, then CD11b+, CD15+, and CD66b were included. (B) Percentage of activated neutrophils. (C) Representative histograms for HO-1 expression in activated neutrophils of LN patients and HC. Red: autofluorescence; light blue: LN activated neutrophils; blue: HC activated neutrophils. (D) MFI value of HO-1 expression. Graphs are showing the mean ± standard deviation. n = 15 LN patients and n = 15 HC. *P < 0.05 by Mann–Whitney test.
Monocytes of LN Patients Have Different Phagocytic Capabilities Than Monocytes of HC
An important function of monocytes is the phagocytosis of antibody-coated microbes and cellular debris. As a way to evaluate this function, we investigated the ability of purified LN and HC monocytes to phagocytose IgG-opsonized beads. The phagocytic ability was measured by FACS, and the total phagocytosis was calculated as the percentage of cells with engulfed beads. We detected significant differences in the capacity of monocytes to engulf IgG-opsonized beads when comparing monocytes of LN patients with those of HC (Figures 3A,B). Surprisingly, the phagocytosis level was increased in monocytes of LN patients compared with HC, independently of whether the IgGs used to opsonize the beads came from an LN patient or a HC. Therefore, trying to further understand our unpredictable results, we turned our focus to the analysis of the percentage of monocytes that phagocyte different number of beads (Figure 3C). Interestingly, the most important difference was observed in the percentage of monocytes that phagocyte four or more beads, which is higher in LN monocytes regardless of the IgGs used to opsonize the beads (Figures 3D,E). Based on these results, we decided to evaluate ROS production as an additional approach to assess the effector function of monocytes, since it is widely known that ROS production is part of the mechanisms used by monocytes to remove microbes and other threats. Noteworthy, ROS levels at baseline were higher in monocytes of LN patients than in those of HC. Moreover, LN monocytes show at baseline similar levels of ROS to those achieved when HC monocytes were treated with TBHP, a potent ROS inducer (Figure 4).
Figure 3. Monocytes of lupus nephritis (LN) patients show increased phagocytic activity compared with those of healthy controls (HC). (A,B) Phagocytic activity of purified monocytes incubated with opsonized beads using IgG from HC (IgG-HC) or LN patient (IgG-LN). (C) Representative dot plot to identify monocytes phagocyting different numbers of beads. (D,E) Percentage of monocytes that phagocyte four or more beads opsonized with IgG-HC or IgG-LN. Graphs are showing the mean ± standard deviation. n = 15 LN patients and n = 15 HC. ***P < 0.001 by Mann–Whitney test.
Figure 4. Reactive oxygen species (ROS) levels are elevated in monocytes of lupus nephritis (LN) patients. ROS mean intensity of fluorescence (MFI) levels were determined by fluorescence-activated cell sorting (FACS) by using CellROX®Green Reagent. TBHP is a ROS inducer and was used as a positive control. Graphs are showing the mean ± standard deviation. n = 15 LN patients and n = 15 healthy controls (HC). *P < 0.05 by Wilcoxon matched-pairs signed rank test (±TBHP). *P < 0.05, **P < 0.01 by Mann–Whitney test (between groups).
HO-1 Induction Improves the Phagocytic Activity and Decreases ROS Production of LN Monocytes
To evaluate whether increasing the expression of HO-1 modulates ROS production, we treated purified monocytes of LN patients and HC with CoPP, which is known to promote the expression and activity of HO-1 (28, 29). In addition, to inhibit HO-1 induction, SnPP was used as a control (30). We first determined the appropriate concentration of CoPP that would achieve the maximum level of HO-1 induction without compromising cellular viability (Figure 5A). The expression of HO-1 was assessed by qPCR and flow cytometry. As shown in Figures 5B,C, purified monocytes upregulate the expression of HO-1 in response to CoPP. Interestingly, unlike the results previously shown, when monocytes were treated with CoPP, the percentage of LN monocytes that phagocyte IgG-beads decreased regardless of whether the IgG came from HC or LN patient, reaching similar values between LN patients and HC (Figure 6A). In addition, the phagocytic activity of HC monocytes did not change with CoPP treatment. Furthermore, with CoPP treatment, we did not observe differences in the percentage of monocytes that phagocyte four or more beads (Figure 6B), indicating that the induction of HO-1 regulates phagocytosis in monocytes of LN patients. On the other hand, to assess the function of LN monocytes in response to the induction of HO-1 in LN patients, we evaluated ROS production in cells treated with CoPP. Interestingly, HO-1 induction results in a decrease of ROS production in LN and HC (Figure 6C), leading the monocytes of LN patients and the monocytes of HC to produce similar levels of ROS. Noteworthy, these results, including the effect of CoPP in total phagocytosis, phagocytosis of four or more beads, and ROS production, were reversed when treating monocytes with SnPP, an inhibitor of HO-1 (Figure 6). Even in some cases, as in phagocytosis, HC monocytes treated with SnPP phagocytosed even more beads than untreated monocytes from HC (Figure 6).
Figure 5. Cobalt protoporphyrin (CoPP) but not tin protoporphyrin (SnPP) induces heme oxygenase (HO)-1 expression in purified monocytes. (A) HO-1 expression was quantified in purified monocytes from lupus nephritis (LN) patients by real-time RT-PCR with different treatments. (B,C) Purified monocytes from healthy controls (HC) and LN patients were treated with 100 μM CoPP for 2 h at 37°C, and HO-1 expression was evaluated by real-time RT-PCR (B) and fluorescence-activated cell sorting (FACS) (C). *P < 0.05; by Wilcoxon matched-pairs signed rank test (±CoPP).
Figure 6. Heme oxygenase (HO)-1 induction in lupus nephritis (LN) and healthy control (HC) monocytes decreases phagocytic activity and reactive oxygen species (ROS) production. (A) Total phagocytic activity of purified monocytes of LN patients or HC previously treated with cobalt protoporphyrin (CoPP) or tin protoporphyrin (SnPP) to induce or inhibit HO-1 expression, respectively. (B) Percentage of monocytes treated with CoPP or SnPP that phagocyte four or more beads. (C) ROS levels in monocytes incubated with CoPP or SnPP. Graphs are showing the mean ± standard deviation. n = 15 LN patients and n = 15 HC. *P < 0.05, **P < 0.01, ***P < 0.001 by Friedman test (±CoPP ±SnPP).
HO-1 Is Expressed in Renal Tubular Epithelial Cells and Infiltrating Innate Immune Cells of LN Patients
HO-1 expression was evaluated in renal biopsies of LN patients (Table 1; patients LN-09–LN-28; n = 20) and HC (Table 4; n = 3) using immunohistochemistry. Since obtaining renal tissue from HC is extremely difficult, we were able to evaluate HO-1 expression in only three biopsies of subjects who underwent surgery, and a nephrectomy had to be performed as part of the surgical protocol for different reasons, but their kidneys were healthy (HC). Interestingly, we observed that HO-1 was mostly expressed in the cytoplasm of renal tubular epithelial cells (RTEC), whereas other regions of the kidney such as the glomeruli showed limited expression (Figure 7A). Nevertheless, HO-1 expression was significantly higher in HC biopsies than in that in LN (173.33 ± 6.67 vs. 82.45 ± 12.70, p = 0.0313; Figure 7B). However, numbers are small, and although promising, further studies are needed to make definitive conclusions. Therefore, we decided to explore HO-1 expression in healthy tissue of biopsies of patients who had a nephrectomy due to the diagnosis of kidney cancer (CC) (see section Materials and Methods for characteristics and Table 4; n = 8). In this case, HO-1 levels in LN biopsies were lower than those in CC biopsies but did not reach statistical significance (122.5 ± 15.32 vs. 82.45 ± 12.7, p = 0.0658). In addition, we evaluated HO-1 expression in renal biopsies of five patients with antineutrophil cytoplasmic antibodies (ANCA)-associated vasculitis also by immunohistochemistry, and HO-1 expression was lower than that of HC biopsies (173.33 ± 6.67 vs. 43.70 ± 22.33, p = 0.0358) but did not reach statistical significance when compared to that of LN patients (data not shown).
Table 4. Demographic details of lupus nephritis (LN) patients, healthy controls, and cancer control recruited to obtain renal biopsies.
Figure 7. Heme oxygease (HO)-1 expression is lower in infiltrating immune cells than in renal tubular epithelial cells (RTEC) of lupus nephritis (LN) patients. Renal biopsies from LN patients and healthy controls (HC) were analyzed to quantify HO-1 expression. (A) Representative images of immunohistochemistry of LN and HC biopsies. (B) Quantification of H-score in LN and HC kidney biopsies. (C) Representative images of HO-1 expression in renal tubular epithelial cells (RTEC, epithelium) and infiltrating cells (monocytes, macrophages, and activated neutrophils) in LN biopsies detected by immunofluorescence (IF). (D) Quantification of HO-1 expression [corrected total cell fluorescence (CTCF)] in epithelium (Epith) and immune cells (Mo, monocytes; Ma, macrophages; Act. Neu, activated neutrophils). Graphs are showing the mean ± standard deviation. n = 20 LN biopsies and n = 3 HC. *P < 0.05, ***P < 0.001 by Mann–Whitney test (A–C). ***P < 0.001 by Wilcoxon matched-pairs signed rank test (D).
However, when HO-1 was evaluated in infiltrating cells of LN biopsies using immunofluorescence, we found that monocytes, macrophages, and neutrophils showed a low expression of HO-1 compared with the strong expression seen in RTEC (Figures 7C,D). Monocytes, macrophages and neutrophils were identified using specific staining for CD14, CD68, and CD66b, respectively. In addition, infiltrating cells were quantified and normalized to the total area of the biopsy. As expected, the number of infiltrating monocytes, macrophages, and neutrophils in LN biopsies was significantly higher in LN biopsies compared with CC (Figure 8). It is well-known that HO-1 participates in inflammatory processes, increasing its expression as a regulatory mechanism. However, infiltrating cells in LN biopsies showed low levels of HO-1, as well as circulating monocytes and activated neutrophils, which could be fostering tissue damage and disease development.
Figure 8. Lupus nephritis (LN) biopsies show higher number of infiltrating cells than cancer controls (CC). Renal biopsies from LN patients and CC were analyzed to identify infiltrating immune cells by immunofluorescence (IF). Infiltrating monocytes (A), macrophages (B), and activated neutrophils (C) were quantified and normalized per square millimeter. Graphs are showing the mean ± standard deviation. n = 20 LN biopsies and n = 8 CC. *P < 0.05, ***P < 0.001 by Mann–Whitney test.
Monocytes, macrophages, and neutrophils play an important role in human and murine LN (3, 31, 32). We found that innate immune cells of patients with LN show lower expression of HO-1, have impaired functions at baseline, such as increased phagocytosis of opsonized beads and increased ROS production. ROS level and phagocytosis are reduced when HO-1 expression is induced with CoPP. The predominance of CD14loCD16hi subsets of monocytes could be contributing to the pro-inflammatory environment of LN (33). CD16 receptors expressed in monocytes exhibit high-affinity binding to the FC portion of polymeric IgG antibodies and immune complexes (IC). This interaction leads to activation of monocytes/macrophages and triggers several effector functions, such as phagocytosis, antibody-dependent cell-mediated cytotoxicity, release of inflammatory mediators, and clearance of IC (18–20, 33). In fact, it has been shown that CD16+ monocytes and activated neutrophils can exert pro-inflammatory functions secreting TNF-α and interferon (IFN)-γ (32–35), which have been linked to the pathogenesis of LN (32, 36). Serum levels and renal production of these cytokines are increased at the onset of renal disease (32, 34–37), and it has been described in murine models that the use of blocking antibodies against IFN-γ causes a significant delay in disease, stabilizing glomerulonephritis, and significantly decreasing proteinuria, which is associated with improvement in survival (36, 37).
Interestingly, HO-1 has an anti-inflammatory role in immune cells with inhibition of pro-inflammatory signals and recovery of cell homeostasis (38). Various studies have shown that inflamed tissues upregulate the expression of HO-1, which has been proved in gastric ulcers, inflammatory bowel disease, colitis, enteritis, and other diseases (39). The data presented here support that the low expression of HO-1 in monocytes and potentially neutrophils might play an active and relevant role in the pathogenesis of LN, similar to what was observed in multiple sclerosis (MS) (40).
The mechanism responsible for the anti-inflammatory properties of HO-1 is not fully known. However, extensive evidences have demonstrated that the beneficial effects of HO-1 is mediated via the breakdown of heme, a vigorous pro-oxidant molecule and a noxious stimulus that amplifies oxidative insult in several models of injury, and the generation of protective products as carbon monoxide (CO), biliverdin with the subsequent formation of bilirubin, and ferritin via iron release from the heme moiety (41, 42) (Figure 9). On the other hand, has been described that monocyte HO-1 inhibits protease, nitric oxide and pro-inflammatory cytokine production, immune cell recruitment and infiltration (8, 43); induces anti-inflammatory interferon-beta; and inhibits cell chemoattraction (40, 44) by downregulation of chemokine receptor (45, 46). Furthermore, HO-1 may be an important effector of Foxp3-mediated immune suppression (8).
Figure 9. Cytoprotective mechanism of heme oxygenase (HO)-1. Cellular stress and damage release heme that acts as pro-oxidant. Cell injury also induces HO-1 expression through pro-inflammatory signals. HO-1 catabolizes the degradation of heme into free iron (Fe2+), carbon monoxide (CO), and bile pigments (biliverdin), which are converted to bilirubin by the enzymatic action of biliverdin reductase. Bilirubin and CO act as potent antioxidants through NADPH oxidase inhibition and reactive oxygen species (ROS) sequestration. Furthermore, they act as anti-inflammatory, antiapoptotic, and produce vasodilatation. Free iron, a pro-oxidant element, is captured by ferritin, which aids in the cytoprotective mechanisms by reducing ROS generation. (This figure was created using Servier Medical Art templates, which are licensed under a Creative Commons Attribution 3.0 Unported License; https://smart.servier.com).
Disturbances in the phagocytosis of apoptotic cells have been proposed to play a role in the development of autoimmunity, especially in SLE (47–50). Previous works have described defects in the clearance of apoptotic cells in lupus mice and patients (51, 52). In contrast to these reports, our data show an increment in the phagocytosis of monocytes of LN patients. This difference could be explained because most of the published data have evaluated phagocytosis of macrophages that have been in vitro differentiated and our work has been focused on monocytes (51, 53, 54). Furthermore, phagocytosis is an extremely complex process, and no single model can account for the diverse structures and outcomes associated with the ingestion of particles. Nevertheless, independently of the type of ingested cell and the receptors involved, several common stages of the process have been established: recognition, engulfment, phagosome maturation (phagolysosome), and degradation (55). The pathways involved in the uptake of apoptotic cells by either macrophages or non-professional phagocytes seem to be unique and very highly conserved in evolution (efferocytosis) (56, 57). However, there are very few data about the underlying mechanisms that drive apoptotic cell phagosome formation and its consequences for subsequent apoptotic cell digestion. In this study, we evaluated the first step of phagocytosis (engulfed material) and in a short period of time (60 min). Based on our results, LN monocytes do not have problems in this initial step of phagocytosis, but it seems that the whole process works until it reaches a critical amount of engulfed beads, as shown by the increase in monocytes that phagocyte four or more beads in LN patients. The accumulation of engulfed material inside monocytes could lead or be explained by a delay or a defect in the subsequent steps of phagocytosis, resulting in poor clearance of apoptotic cells, but more experiments are needed to confirm this statement. The modulation of the phagocytic capacity represents a potential therapeutic target in the control of disease, since apoptotic cells, when not efficiently cleared, may represent a source of autoantigens that drive the autoimmune response in SLE (47–50). Interestingly, here, we demonstrated that the induction of HO-1 modified the phagocytic activity of LN monocytes to a point that they behave similar to HC monocytes. We can therefore speculate that defects in the clearance of apoptotic cells by monocytes of LN patients could be in part explained by reduced levels of HO-1, which could contribute to the initiation and maintenance of autoimmunity. Future studies designed to investigate the phagocytic pathway would be of interest.
In addition, several studies have shown that high levels of ROS have a role in cellular death in SLE; in fact, elevated ROS production has been associated with increased apoptosis, NETosis, and delayed clearance of apoptotic cells (58, 59). Rokutan et al. (58) showed that there are increased oxygen intermediates in the organs of MRL/lpr mice, whereas reduced serum levels of antioxidants and radical scavengers have been reported in human SLE (60). Indeed, treatment with antioxidants, such as vitamin E, suppressed circulating anti-dsDNA antibody levels and the development of renal disease in MRL/lpr mice (61, 62). Different groups have previously shown increased ROS production by LN neutrophils and monocytes. However, there is some controversy in the literature about their response to oxidative stimuli (63, 64), arguing that differences in methodology and patient selection may explain some of these. Here, we observed that LN monocytes have elevated ROS levels at baseline, but HO-1 induction reduces these levels to those of HC, exerting their cytoprotective function (65).
Other groups have demonstrated that HO-1 is upregulated in proximal tubular cells in response to oxidative stress and exerts cytoprotective and anti-inflammatory effects in severe cases of sublethal cellular injury affecting tubular segments, which includes ischemia, nephrotoxins, cytokines, endotoxin, oxidants, and vasoactive substances (66–70). To the best of our knowledge, our group and Kishimoto et al. (12) first examined the expression of HO-1 in situ in human LN, focusing on different cell types. Our findings show that HO-1 expression is increased in tubular epithelium compared with infiltrating cells and is decreased in the epithelium when compared to that in HC, but no significant differences were observed between the biopsies of patients with LN and CC biopsies of patients with kidney cancer probably because although the examined tissue was not infiltrated by tumor, it was stressed tissue exposed to an antitumor immune response. Strikingly, although we studied only three HC biopsies, our findings are consistent with what is seen in peripheral monocytes, which show a decreased expression of HO-1 in LN patients. Further studies to confirm the results observed in human biopsies are needed. While LN has been always classified as a glomerular disease, an increasing body of evidence suggests that tubulointerstitial injury might have a prominent role (66). Our results may suggest that HO-1 presence in RTEC could be an attempt of the kidney to protect itself from damage, which is perpetrated, among others, by innate immune cells showing a low expression of HO-1. Since LN patients might express lower levels of HO-1 than HC, they could be having less protective mechanisms against the damage inflicted by inflammatory cells. Furthermore, the induction of HO-1 in the tubular epithelium in proteinuric states that occurs in cases of LN cannot be simply ascribed to increased trafficking of albumin per se across the proximal tubule; such expression more likely reflects concomitant injury to tubular epithelial cells occurring pari passu with glomerular disease and/or the pro-inflammatory, pro-oxidant, or other perturbing effects of specific proteins or other species appearing in the urinary space, as it has been demonstrated by some authors (67).
In conclusions, we showed that circulating monocytes and activated neutrophils of LN patients have lower levels of HO-1 than those of HC. Although further experiments to demonstrate that these peripheral cells perform tissue damage in LN are needed, our results suggest that this could be the case. In addition, LN monocytes show increased levels of ROS at baseline, and HO-1 induction is able to return HO-1 levels to normal. Thus, decreased HO-1 expression in circulating and infiltrating monocytes and neutrophils of LN patients might promote a pro-inflammatory environment contributing to renal injury. Moreover, we propose that HO-1 induction might exert a cytoprotective role in renal tissue similar to observed in other models of renal injury (68–70) and might regulate innate immunity in LN.
The raw data supporting the conclusions of this article will be made available upon reasonable request to the corresponding author.
The studies involving human participants were reviewed and approved by Comité Ético Científico Facultad de Medicina Pontificia Universidad Católica de Chile. The patients/participants provided their written informed consent to participate in this study.
LC made substantial contributions to the conception and design of the study and drafted the manuscript, performed experimental procedures, made substantial contributions to the analysis and interpretation of data, reviewed the manuscript critically for important intellectual content, and gave final approval of the version to be published. JO participated in the conception and design of the study, performed the qRT-PCR analysis, participated in the analysis and interpretation of data, and revised the final version of the manuscript. PG-M performed immunofluorescence microscopy and histochemical protocol, made substantial contributions to the analysis and interpretation of data, drafted the manuscript and reviewed the manuscript critically for important intellectual content. AV participated in the conception and design of the study, made substantial contributions to the analysis and interpretation of data and reviewed the manuscript critically for important intellectual content. NC participated in the conception and design of the study, performed phagocytosis experiment, participated in analysis of data, drafted the manuscript and reviewed the manuscript critically for important intellectual content. IS participated in the conception and design of the study, performed part of the renal biopsies, participated in the analysis of data, reviewed the manuscript critically for important intellectual content and revised the final version of the manuscript. RV performed experiments, participated in the analysis and interpretation of data, drafted the manuscript, reviewed the manuscript critically for important intellectual content, and revised the final manuscript. GM participated in the conception and design of the study, made substantial contributions to the analysis and interpretation of data, drafted the manuscript and reviewed the manuscript critically for important intellectual content. CL participated in the conception and design of the study, made substantial contributions to the analysis and interpretation of data, and drafted the manuscript. All authors read and approved the final version of the manuscript.
This work was supported by Comisión Nacional de Ciencia y Tecnología, FONDECYT regular N°1150173, Santiago, Chile.
We would like to thank all SLE patients and HC who participated in this study.
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Keywords: lupus nephritis, monocytes, heme oxygenase-1 (HO-1), phagocytosis, reactive oxygen species (ROS), renal biopsies
Citation: Cuitino L, Obreque J, Gajardo-Meneses P, Villarroel A, Crisóstomo N, San Francisco IF, Valenzuela RA, Méndez GP and Llanos C (2019) Heme-Oxygenase-1 Is Decreased in Circulating Monocytes and Is Associated With Impaired Phagocytosis and ROS Production in Lupus Nephritis. Front. Immunol. 10:2868. doi: 10.3389/fimmu.2019.02868
Received: 30 May 2019; Accepted: 22 November 2019;
Published: 13 December 2019.
Betty Diamond, Feinstein Institute for Medical Research, United States
Yogesh M. Scindia, University of Florida, United States
Laurence Morel, University of Florida, United States
Copyright © 2019 Cuitino, Obreque, Gajardo-Meneses, Villarroel, Crisóstomo, San Francisco, Valenzuela, Méndez and Llanos. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
*Correspondence: Carolina Llanos, cllanos@med.puc.cl


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Soulja Boy Could Win a GRAMMY for Beyonce's "Lemonade" Album
Are you ready to live in a world where Soulja Boy has more GRAMMYs than Nas? That's now a very real possibility.
Nathan Slavik
Our deep dive into the credits for Beyoncé's liner notes turned up some gems, but I was particularly interested in Soulja Boy's appearance because it illustrates just how strange the twists and turns of music industry fate can be. One day Soulja Boy is sitting at home, being a MySpace legend, and the next he's got a shot at a GRAMMY for writing on Beyoncé's album without lifting a finger, or a vocal chord.
To walk through this quickly, "songwriting" is the art of crafting melodies and words for a song. Anyone who writes the words that appear in a song (and isn't a ghostwriter) gets a songwriter credit, which also extends to direct quotes. To use one of my favorite examples, Mac Miller originally said "I ain't a player, I just crush a lot on "The Way," which is clearly a Big Pun quote. But getting approval from Big Pun's estate for the line proved to be a headache, so they asked Miller to shorten the line to just "I'm not a player I just," arguing that certainly no one could claim credit over the phrase "I'm not a player." In the video Miller even makes a point of covering his mouth and not saying the rest of the line.
But when Beyoncé says, "I hop up out the bed and get my swag on / I look in the mirror, say, "What's up?" on "Hold Up" there could clearly and obviously only be one man she's quoting. And so boom, just like that DeAndre Way has a writing credit on what could quite possibly prove to be the biggest album of the year.
Beyond his name in the album credits, that means he gets a cut of the publishing money on the album. If "Hold Up" ends up in a movie or a commercial, which could easily happen considering Beyoncé's star power, he gets paid. And furthermore, it also makes him eligible for a songwriting GRAMMY, which again considering Beyoncé's history - she's the second most awarded female artist of all-time with 20 GRAMMYs - and this album's obvious impact, could easily happen.
Are you ready to live in a world where Soulja Boy (or Swae Lee of Rae Sremmurd, who also has a songwriting credit on Lemonade for "Formation") has more GRAMMYs than Nas? Are you ready for "Turn My Swag On" to indirectly win a GRAMMY? Because that's a world that's quickly approaching.
It goes to show, talent and influence certainly matter - if Soulja Boy hadn't written such a memorable line Beyoncé wouldn't have quoted it, which is the definition of good songwriting - but fate matters too. Soulja Boy planted the seeds for the lemon tree, but the lemonade was completely out of his control. Like a lot of other people on the album and on every big album, he stands to make a lot of money and win awards because his line just happened to pop into an extremely famous person's head while writing a song that just so happened to make an album that just so happened to go on to have blockbuster success.
There's really no other business that operates quite like that, even within entertainment, which is why music is the land of both crushed souls and dreams realized that were so impossible they were never even dreamed at all. So hop up out of bed and turn on your swag on Mr. Way. Destiny chose you for a reason.
By Nathan S, the managing editor of DJBooth and a hip-hop writer. His beard is awesome. This is his Twitter. Image via Instagram.
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The Queen flexes her royal muscle on a surprise release you only need one listen to feel in your bones.
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"You live by the ringtone, you die by the ringtone. Just ask Mims."
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Soulja's mystery "deal" would be worth 20 times Drake's deal with Apple Music, so yeah, consider us more than skeptical.
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A look back at the (basically) dead social site that launched careers ranging from Soulja Boy to Miguel and brought hip-hop into the digital age.
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All it took was Rae Sremmurd, Jay Z and LeBron James for the song to find its way onto Beyoncé's album.
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In 2008 a young Lupe won his first GRAMMY, but it wasn't until three nights ago that he finally held the award and gave his speech.
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We all know The Weeknd is on Bey's new album, but did you know that Outkast, Soulja Boy and many more also earned credits?

redpajama_set_name: RedPajamaCommonCrawl


Boston is the capital city of Massachusetts, and the largest city in New England.
Salon De Beauty first started in Boston, before making a move to South Shore's Weymouth. We still open our doors and welcome residents of Boston to our salon every day; we have had clients since 2003 that have followed us to Weymouth for all of their beauty needs!
Over the years we have earned the nickname, "One Stop Beauty Shop". We have earned this nickname because of all the services we provide to our clients.
Some of these services include: hair styling and hair dying, manicures and pedicures, tanning beds, day spa, massage services, and waxing.
We are also open seven days a week, helping people with the busiest of schedules find time to treat themselves.
For more information about our salon, or to book an appointment, please give us a call at 781-340-4494. We are looking forward to helping you with all your beauty needs.


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Activison hires lobbyists to act on Senate violence bill
By Colin Campbell@ColinCampbellx Sep 9, 2013, 5:15pm EDT
Source The Washington Post | Image ShutterStock
Share All sharing options for: Activison hires lobbyists to act on Senate violence bill
Activision has hired top law firm Akin Gump Strauss Hauer & Feld to lobby as an advocate in the upcoming Senate bill, calling for a study of the impact of violent video games on children, according to a report in The Washington Post.
Lobbying records filed in the last few weeks reveal that lawyers handling the account include Arshi Siddiqui, a one-time senior policy advisor and counsel to former House Speaker Nancy Pelosi, and Ryan Thompson, whose brief includes handling "legislative affairs and crisis management" for tech companies.
The proposed S.134, Violent Content Research Act was introduced in Congress last year by West Virginia Sen. Jay Rockefeller, following the Sandy Hook Elementary School shooting, in which 26 children and teaching staff were murdered by gunman Adam Lanza. Since the shooting, the role of violent video games has come under renewed political scrutiny, with President Obama separately calling for research into the role of media and real world violence.
Akin Gump Strauss Hauer & Feld was founded in 1945 by Richard Gump and Robert Strauss, a former United States ambassador to the Soviet Union and Chairman of the Democratic National Committee in the 1970s. The company, listed as one of the top 20 law firms by The American Lawyer magazine, has offices in 15 cities around the world.
Activision's brands include the valuable Call of Duty first-person shooter franchise. A Polygon request for comment, emailed to Activision earlier today, was not answered.


An easy Keto pancake recipe made from almond flour, but still tastes like a fluffy pancake you'd get from your favorite diner! These are sure to be the best Keto pancakes you ever try, and the whole family will love them too.
Before I fill you in on how to make these Keto Pancakes, I have to tell you this recipe is sponsored by Isopure Protein Powder.
Ok, so now lets get to the good stuff!
Pancakes are one of my absolute favorite foods of all-time. And doing the Keto diet doesn't mean you have to give up pancakes for breakfast. It just means you have to get creative with how you make them. Fortunately, this Almond Flour Keto Pancake Recipe has got you covered.
The best part about this Keto pancake recipe is how easy it is to make. From start to finish, these pancakes should take you no more than 15 minutes. All you have to do is toss all the ingredients into your blender, whizz that thing up, and you're ready to start cooking your pancakes.
Also, you'll only need a few ingredients, like eggs, cottage cheese, and almond flour to make these keto pancakes.
In my opinion, homemade pancakes – let alone Keto pancakes – really don't get much easier than this.
By the way, the second best part about this keto pancake recipe is that each pancake only has 1 gram of net carbs!
Obviously, you'll need a blender for these pancakes.
Definitely pick up a food scale – if you don't have one – to weigh out your ingredients.
You'll also need almond flour, I recommend going with this one.
This was the protein powder I used for my pancakes. It bakes better than any other protein powder I've ever used, check it out if you need some!
The last thing I'll recommend is this 0 carb pancake syrup. I've tried a lot of low carb pancake syrup, and this one is by far the best I've had.
The BEST Keto Pancakes recipe that uses almond flour as a base! This easy low carb pancake recipe is made with eggs, cottage cheese, and of course almond flour, but tastes like a fluffy pancake you'd get at IHOP. Best of all, these Keto Pancakes ONLY HAVE 1 NET CARB a piece!
Also, you can boost the fat content if you'd like by using a higher fat cottage cheese.
All I can say is WOW!!! These pancakes were so good. My husband even ate them, and he's not into "low carb" stuff at all!


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Antonin Pierozzi (Antonin z Florencji), łac. Antoninus Florentinus, wł. Antonino Pierozzi, ur. jako Antonio Pierozzi (ur. 1 marca 1389 we Florencji w Toskanii, zm. 2 maja 1459 w Moncalvo w Piemoncie) – włoski dominikanin (OP), arcybiskup Florencji, święty Kościoła katolickiego.

Życiorys

Antonio urodził się we Florencji w rodzinie Niccolo i Tomasiny Pierozzich. Matka zmarła, gdy miał około pięciu lat. W dorosłym życiu nazywany był Antoninem (Antonino - wł. Antek, Antoś) z powodu swego niskiego wzrostu i wielkiej dobroci.

Wstąpił do Zakonu Kaznodziejskiego w 1405 roku przyjęty doń przez bł. Jana Dominici, który na polecenie bł. Rajmunda z Kapui zajmował się wówczas reformą konwentów dominikańskich. Odbył nowicjat w Kortonie, dalszą formację zakonną odbywał jako pierwszy zakonnik nowo powstałego obserwanckiego konwentu w Fiesole. Tuż po nim formację w tym samym konwencie rozpoczął Jan z Fiesole, który z czasem zyskał przydomek Fra Angelico. W 1413 roku Antonin otrzymał święcenia kapłańskie. Odznaczający się mądrością młody zakonnik od 1418 roku kierował braćmi jako przeor w Kortonie, Fiesole, Neapolu i Rzymie, gdzie został audytorem św. Roty i wykładowcą prawa kanonicznego.

W 1436 roku został przeorem nowo ufundowanego przez Kosmę Medyceusza klasztoru we Florencji, który stał się jednym z ważniejszych ośrodków kultury odrodzenia. Antonin otworzył klasztorną bibliotekę dla wszystkich, nie tylko dla mieszkańców konwentu, czyniąc ją w ten sposób pierwszą publiczną biblioteką w Europie. Także za przeoratu Antonina we florenckim konwencie powstały słynne freski św. Jana z Fiesole. W 1437 roku został wikariuszem generalnym konwentów obserwantów w Italii.

Kiedy w 1438 papież Eugeniusz IV przeniósł sobór z Ferraty do Florencji, Antonin użyczył w klasztorze gościny czterdziestu ojcom soborowym. Konwent św. Marka na czas soboru florenckiego stał się miejscem spotkań papieża ze znaczącymi osobami. Uczestnicy soboru w uznaniu mądrości florenckiego przeora ]nadali mu przydomek "Antonina Doradcy". Kiedy w 1466 roku stolica arcybiskupia we Florencji zaczęła wakować  mieszkańcy miasta wysłali papieżowi Eugeniuszowi IV listę swoich pięciu kandydatów na to stanowisko, ten nie poszedł za oczekiwaniami mieszczan, lecz mianował arcybiskupem Antonina. Jako biskup Antonin wsławił się zwłaszcza uczynkami miłosierdzia oraz założeniem stowarzyszenia "Dobroczyńców św. Marcina". Był prekursorem dzisiejszej opieki społecznej.

Jest autorem wielu dzieł, z których najważniejsze to Summa moralis.

W 1523 papież Hadrian VI zaliczył go w poczet świętych, przez zatwierdzenie kultu.

Jego wspomnienie liturgiczne obchodzone jest 2 maja.

Ciało św. Antonina pozostało w stanie nienaruszonym aż po dziś dzień. Spoczywa w kościele św. Marka we Florencji.

Na obrazach i w rzeźbie przedstawiany jest zazwyczaj w dominikańskim habicie i z paliuszem biskupim. Niekiedy także w stroju biskupim, z pastorałem.

Zobacz też
 kult świętych
 modlitwa za wstawiennictwem świętego
 Antonin (imię)

Przypisy

Bibliografia
 Antonin Pierozzi na DEON.pl (SJ i Wydawnictwo WAM)
 Święty Antoninus na Catholic Encyclopedia

Linki zewnętrzne
 Antonin Pierozzi – dzieła w bibliotece Polona

Włoscy biskupi katoliccy
Antonin Pierozzi
Włoscy dominikanie
Urodzeni w 1389
Zmarli w 1459
Ludzie urodzeni we Florencji

redpajama_set_name: RedPajamaWikipedia


text:
33 Taiwanese hauled back from Croatia charged with telecom scams
China | 30 May 2018 6:52 pm
Thirty-three Taiwanese involved in a cross-border telecom scam ring in Europe that targeted Chinese citizens have been indicted on charges of organized crime and fraud, Miaoli District Prosecutors Office said. They were part of a 91-member fraud gang repatriated from Croatia this year, according to Huang Chen-lun of the Miaoli District Prosecutors Office. Members of the gang was arrested on Janu...
Thirty-three Taiwanese involved in a cross-border telecom scam ring in Europe that targeted Chinese citizens have been indicted on charges of organized crime and fraud, Miaoli District Prosecutors Office said.
They were part of a 91-member fraud gang repatriated from Croatia this year, according to Huang Chen-lun of the Miaoli District Prosecutors Office.
Members of the gang was arrested on January 18 by Croatian police, who found that they had been running similar telecoms fraud scams in Slovenia before moving to Croatia about a month previously, CNA reports.
With the assistance of Taiwanese authorities, the suspects were later repatriated to Taiwan in four different groups.
Since being returned, 89 have been detained for questioning while two pregnant women were released on bail, Huang said.
Prosecutors found that the fraud ring had accrued about NT$230 million (US$7.64 million) in illegal assets in four months by making more than 45,000 scam calls to China.
Acting on information provided by some of the detainees, the gang's leader was arrested during a raid in Taichung in April, during which the suspect, named Tsai, was found to have NT$59 million in illegal assets, according to Miaoli prosecutors.
Yesterday, 33 of the 91 fraud ring members were indicted on charges of fraud and operating as an organized criminal gang, and if found guilty, will be sentenced to 3-10 years in prison, while prosecutors are still investigating the roles of the other suspects.
Other China breaking newsMore news >>
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Tsai Ing-wen to launch hospital project in Caribbean ally St Lucia (07-18 11:04)
Danger from typhoon Danas to Taiwan eases(07-18 11:01)
Man executed in Shaanxi for killings triggered by mother's death (07-17 19:10)
Taiwan shuts schools in outlying islands ahead of storm (07-17 18:14)
Lion King lords over China box office (07-17 13:14)
Taiwan issues storm warning (07-17 13:05)
Taiwan on alert for tropical storm (07-17 11:34)
127 new golf courses halted since 2011(07-16 18:14)
223 inter-provincial drug gang suspects arrested (07-16 17:07)


text:
Die Gösleswand ist ein  hoher Gipfel in der Lasörlinggruppe einer Untergruppe der Venedigergruppe in Osttirol. Markant ist die Südwestwand, an deren Ende () auch das Gipfelkreuz steht. Auf dem höchsten Punkt findet sich eine Panoramatafel, welche Auskunft über die vielen Gipfel der Umgebung gibt. Der ehemals bekannte Mineralienberg ist heute von Sammlern leergesucht.

Anstieg
Der übliche Anstieg erfolgt auf markiertem Steig von der Rote Lenke in ca. 30 Minuten. Er wird oft im Rahmen des Lasörlinghöhenwegs auf der Etappe zwischen Lasörlinghütte und Neuer Reichenberger Hütte bestiegen.

Literatur

Weblinks

Berg in der Venedigergruppe
Geographie (Prägraten am Großvenediger)


The wild environment of the Eastern Cape provides the backdrop for this family-oriented lodge within the malaria-free Shamwari Private Game Reserve. Comprising of nine inter-leading luxury rooms, each room has air conditioning, an en-suite bathroom with separate shower and bathtub, satellite TV and minibar. Outside in the carefully tended grounds there's a swimming pool with a shallow area for children. In fact, children are particularly well looked after here thanks to the "Kids on Safari" programme with a designated children's coordinator who will keep kids entertained, children from the age of four upwards can participate in game drives. Each young explorer also receives a personal gift pack and has access to the kids club and outdoor adventure centre; child minding is also available. Guided 4x4 safaris will take you deep into Big 5 territory, while those seeking a truly immersive experience may like to visit the Wildlife Rehabilitation Centre and Born Free centre on the reserve. When the stars come out, settle down to dine in the firelit boma overlooking the Bushmans River.


text:
Кишинів III () — проміжна залізнична станція Молдовської залізниці на лінії Ревака — Унгени. Розташована в столиці Молдови за 5,6 км від станції Кишинів.

Пасажирське сполучення
Пасажирський рух по станції Кишинів III здійснюється до кінцевих станцій  та Бендери III.

Джерела

Примітки

Посилання

 Розклад руху пасажирських потягів по станції Кишинів III

Станції Молдовської залізниці
Залізничні станції та платформи, збудовані 1871
Залізничні станції за алфавітом


The Village of Old Field maintains a right of way from Old Field Road (South of the Lighthouse) to Old Field Beach (known as Whitehall Beach). Residents are invited to park on the Lighthouse grounds and walk south on Old Field Road to the right of way.
Whitehall Beach offers some of the finest salt water bathing on Long Island. Between April 1 and November 1, no cats, dogs, horses or other domestic animals are allowed on the beach. No open fires or motorized vehicles are allowed at any time. Residents are asked to keep the beach clean and responsibly dispose of all litter.
Kaltenborn Commons is a small park located at the entrance to the Village of Old Field at the intersection of Quaker Path and Old Field Road.
The original fire ring, designed to raise the alarm when fires broke out in the Village of Old Field, can be viewed on Old Field Road near the intersection with Mount Grey Road.
The roads of the Village of Old Field provide both exercise and the opportunity to view the many beautiful vistas that abound in our Village. However, the Village roads are curved and hilly resulting in many blind spots. Therefore, pedestrians and bicyclists are urged to use extreme caution. Pedestrians should walk on the left side of the road (facing oncoming traffic) and fall into single file as vehicles approach. Bicyclists should ride single file on the road (with cars). Please use reflective clothing and lights from dusk till dawn. Helmets are mandatory in Suffolk County for all bicyclists under 18 and highly recommended for all.
The Village of Old Field is surrounded by bodies of water that offer swimming, boating, and fishing opportunities. The main bodies of water are Conscience Bay, The Narrows, the Port Jefferson Harbor, Smithtown Bay, and the Long Island Sound.


text:
Q: How do I migrate a ubuntu server vmware virtual machine with a broken snapshot chain I currently have a ubuntu 8 server virtual machine running within vmware server 2.0 on centos  and need to migrate the virtual machine to a server running vmware vsphere essentials.  However, copying the files to the new server or using vmware converter fails due to a broken snapshot chain.
How do I migrate the virtual machine if there is a broken snapshot chain.

A: It's probably less tiresome to build a new Ubuntu box on the vSphere machine and copy over the data of the broken Ubuntu VM to the new one. What is the old server running? Copying data will save you a lot of time trying to figure out how to rebuild the snapshot chain, when it's not even necessary...

redpajama_set_name: RedPajamaStackExchange


LeeAnne and her partner Dancer.
I believe that dog training should be doable. That everyone in the family — grandparents to grandchildren, and everyone in between — should be capable of meaningful participation in the process. No one needs to be a jock or a genius, although it never hurts to be a little smarter than the dog.
Dog training should be fun for the dog and his trainers. This ensures that everyone will want to do homework, and learning will take place quickly.
Training should be effective and progress visible.


text:
Direct Ship Americas
Factory: Direct Ship Americas
Key Buyers: Fanatics
In January 2019, the university licensee Fanatics, the WRC, and other stakeholders worked together to ensure compliance with university codes of conduct following the closure of Direct Ship Americas (DSA), a factory located in Choloma, Honduras. Fanatics informed the WRC about the closure before it occurred and reported that the factory, which owed its 240 production employees approximately $468,000 in legally mandated compensation, did not possess the resources to pay them. To its credit, the licensee committed to ensuring that the workers would be paid all of the money they were owed. The WRC worked with Fanatics and with its intermediary AGI, the party that ultimately provided the funds owed to workers, to ensure that the payment process was executed smoothly and in the shortest amount of time possible. Most of the DSA workers were paid in early February 2019, just one month after the factory closure, and the remaining workers, who had not come forward in time for the February payment, were paid the following month.
The WRC frequently documents the closure of garment factories, including factories making university logo apparel, in which the employer cannot or will not comply with the law requiring terminal compensation. Under university labor codes, licensees are contractually obligated to ensure that workers are paid in these circumstances and the WRC has achieved substantial and growing success in securing compliance with this obligation, averting severe hardship for tens of thousands of workers and their families. However, it has often required protracted engagement with licensees in a given case to get to a positive resolution. The DSA case is notable because Fanatics informed the WRC of the problem and then recognized and committed to honor its university code obligations from the outset. As a result, the WRC was able to work with Fanatics and its agent to get workers all of their compensation with minimal delay.
Worker Rights Consortium Assessment Direct Ship Americas (Honduras) – April 8, 2019


text:
Microsoft wants you to Teams leave the company and become a app to talk with family and friends
Microsoft Teams has been one of the great "winners"in software with respect to the quarantine for the role they are having in connecting to millions of people during this stage of growth telework. In that sense, Microsoft says more than 44 million people are already using the platform.
However, Microsoft wants it to be much more than an app to talk with co-workersand that transcends the realm of friends and family. The announcement has been made around Office 365, that from 21 April, will be Microsoft 365.
With this name change come two new subscriptions: Microsoft 365 Personal and Microsoft 365 Family. The first will have a cost of 7 euros per month, and the second of 10 euros per month, although you can get a discount by paying annually.
Microsoft Teams, the company's home
The Redmond company has announced at the end of the year, the platform corporate messaging you will receive functions that allow you to connect and organize our digital life with family and friends. For example, through Teams, we will be able to organize a trip, with group chat and video calls, group lists, shared tasks, etc., The new features will arrive in the first place to the mobile version of Teams.
At the family level, we can set calendars, to store secure information such as passwords, Wifi, and of course, send pictures and videos. In terms of location, we can see updates of our contacts, as we have been seeing in other applications.
More than a new instant messaging application, the new Teams it may be worth to integrate attachments, conversations, travel, family events, etc on a same platform (instead of using Trello+WhatsApp)
These changes maybe of concern to people who currently use Microsoft Teams, but the reality is that the tool will remain the same for the companies that already use it in the workplace. What Microsoft is doing is, simply, open the door to your ecosystem of applications out of offices and allows to organize more aspects of our life.
What is certain is that at the time of organizing many of the events that Microsoft assumed that we will be able to with Teams at the end of the year, it is never too comfortable to do with the current tools. While it can be done in groups of WhatsApp or Telegram, at the end there is always that go to other platforms like Trello to do well in terms of organisation of bookings, attachments. In that sense, this is all Microsoft Teams can be very good.
Microsoft Family Safety, a parental control for iOS and Android
In addition to the above-mentioned developments of Microsoft Teams, has been presented
More information | Microsoft
Disney+ is here: try it free for 7 days. After, 6,99 euros a month or 69,99 euros the annual subscription.
PVP in Disney+ 6,99 euro per month (free 7-day trial)


Christmas is a time to relax and enjoy time with your friends and family. I believe that during this time you need to be kind to yourself and allow yourself to relax and not stress about being 100% on point with your nutrition all of the time. Let's face it, you can't really lose weight over the Christmas break but you don't need to go backwards either. You can still maintain your body and not let all of your hard work this year go to waste. I see it every year, where people over indulge then set 'new years' resolutions to lose weight. So they go hard in January and crash diet, then fall off the wagon again and the yo yo dieting continues!
Focus on maintaining the body you already have, that way when the silly season is over you don't have to start from scratch again.
Choose the days you are going to indulge, don't indulge everyday!
Try to drink lower calorie alcoholic beverages like vodka/soda and stay hydrated by drinking loads of water.
Don't go to a party or gathering hungry otherwise you will snack on all the bad snack foods and you won't eat a proper meal.
Try to get protein into all of your meals so that you feel fuller for longer and remember to also eat good fats as they keep you feeling satiated.
Get your exercise done early so that it is done and you don't have time to make excuses or get distracted and then not train at all. Even if is is a walk, just move your body, you will feel better for it!
Lastly, try to take some time out to RELAX and ENJOY the holidays! It's been a big year and it's always good to sit back, reflect, re-focus and take it all in!
Fit, Healthy & Strong is a 6 week program that will teach you all of the tricks and secrets that I know to help you gain lean muscle and burn unwanted body fat. I will teach you how to do this in a way that you will look after your metabolism and protect your lean muscle. This means that you will not be over training, cutting calories or counting anything, you will be eating nutrient dense food and training effectively and with purpose. I want to help you find your inner athlete and base your training goals around how much you are lifting rather than what the number on the scales says. The program comes with a training plan for home and for the gym as well as a HEAP of healthy recipes that taste AMAZING!
CLICK HERE TO JOIN NOW and use the discount code HALFPRICE!
If you answer yes to any of the below then Fit Healthy and Strong is for YOU!!!!
BELOW ARE SOME AMAZING TRANSFORMATIONS THAT HAVE BEEN ACHIEVED BY THE EVERYDAY WOMAN BY FOLLOWING THIS EXACT PROGRAM!
These results happened in as little as 3 weeks into the program!
USUALLY THIS PROGRAM IS $87, BUT I HAVE SLASHED IT DOWN TO JUST AN EASY ONE OFF PAYMENT OF JUST $47 so that you can follow a plan over the Christmas break and NOT let all of your hard work this year go to waste! You have 3 months to complete the program as well so you can do it twice over or take your time with it.
I look forward to helping you achieve your goals, lets make 2018 our best year yet, why wait, START NOW!
HURRY AS THIS PRICE WON'T LAST LONG AND YOU DON'T WANT TO MISS OUT!
This is my own transformation by following this program after I had Leo. I followed my FitMum program and then moved onto my Fit Healthy and Strong Program! I want to show you EXACTLY what I did to flatten and tone my stomach and to get my abs back! If I can do it, you can too!!!


HomeLatest UpdatesLooking for MCN 2018 Program co-chairs!
Looking for MCN 2018 Program co-chairs!
I hope everyone has had the opportunity to Look Back at a great MCN 2017 and 50 years of MCN history in the amazing MCN50 Voices series of interviews, Take Action at their institutions and in their communities, and … Think Ahead about MCN 2018.
The past year has been such an amazing experience as one of the Program Co-Chairs, along with Jennifer Foley and Trish Oxford. With Jennifer and Trish stepping down after their two-year terms have concluded, MCN is looking for two people to take their place and, with me, help plan our annual conference in 2018, then taking the lead for 2019.
It's a lot of work, but you'll be part of a sizable leadership and management team of MCN colleagues, as well as a field full of people who want to speak at, attend, and contribute to MCN 2018.
Finding out just how much I already knew about the museum field—and how much there still is to learn.
Working with the committee of local museum technology professionals in our host city and finding out the amazing work being done there.
Crafting the theme, which starts pretty much right away and gets the Program Committee off to a rollicking start.
Developing Keynote speaker(s) ideas, also with the Program Committee. This year we had some innovative ideas and it was great to see them realized.
Calling for proposals in the spring—watching them trickle and then pour in, sending them out to the Program Committee for review, and then working together, with the help of a few hundred sticky notes, to turn them into a program.
The weekly Co-Chair conference call.
The conference itself, of course, which is a very different experience when you see it come together rather than just presenting.
And yet presenting was still a joy.
Developing the "other format" idea and receiving over two dozen proposals beyond the usual case studies, panels, and presentations.
Commitment: 3-5 hours/week throughout the year, increasing as the conference nears, with milestones in May and September, available full-time during the conference. Available one weekend in late March or early April for a site visit to that year's conference location (paid for by MCN).
Compensation: the Conference Program Co-Chair is a volunteer role and is therefore not compensated; however, MCN does offer complimentary registration to the annual conference during the year(s) you serve.
Location: MCN's Annual Conference is a North American based-conference that supports global involvement and has an emerging international following. The successful candidate is required to attend the Conference in person, as well as participate in regular phone or online meetings.
MCN2018 will take place in Denver, November 13–16, 2018, when the location of MCN2019 will be announced.
Deadline for applying: Extended until end of day, January 7!
Description: MCN is looking for two thoughtful, motivated, and dynamic museum professionals to serve as MCN Conference Program Co-Chairs for a two-year term starting in January 2018. This is an opportunity to help shape a major museum technology conference now and in future years, immerse yourself in cutting edge developments in the sector, broaden your networks on a national level, and to gain experience and professional development in event programming.
The Conference Program Co-Chairs provide leadership for the annual MCN Conference, creating the program through the conception and organization of workshops, panels and presentations in many different formats, experimental programs, keynotes, special events, and innovations not yet imagined. With current Conference Program Chair Rob Weisberg, the newly appointed Conference Program Co-Chairs will work as a team to develop an experiential conference program that serves the evolving needs of the MCN community and then serve as lead Conference Program Co-Chairs for the 2019 conference.
The ideal candidate will be passionate about the intersection of museums and technology and interested in developing an innovative conference program featuring proposals from participants from a wide range of institutions, backgrounds, and perspectives. They will be knowledgeable about MCN and the conference, having attended MCN several times in the previous five years. They will have existing networks within the sector, a strong understanding of the issues confronting museums with regards to technology and the practice of digital, and appreciate the challenges facing their colleagues from many different kinds of institutions and departments in the field. They will be active in the museum or cultural technology community and knowledgeable of trusted sources of information, and will be a proactive self-starter and a calm problem-solver with excellent oral and written communication skills. They will be a creative thinker both about big issues and small details, diplomatic under pressure, and ready to learn and adapt over the course of planning this conference.
About MCN: MCN is a nonprofit organization whose core purpose is to foster innovation and excellence by supporting professionals who seek to transform the way their cultural organizations reach, engage, and educate their audiences using digital technologies. We do this by building a community that attracts, nurtures, inspires and sustains exceptional professionals. Learn more.
For further information and a full overview of responsibilities, email program@mcn.edu.
To apply, please send an email articulating why you think you'd be a good fit for this position, and noting any relevant experience to: program@mcn.edu. Please include a CV or link to your LinkedIn profile.