Mitochondrial Fission Promotes the Continued Clearance of Apoptotic Cells by Macrophages.

Clearance of apoptotic cells (ACs) by phagocytes (efferocytosis) prevents post-apoptotic necrosis and dampens inflammation. Defective efferocytosis drives important diseases, including atherosclerosis. For efficient efferocytosis, phagocytes must be able to internalize multiple ACs. We show here that uptake of multiple ACs by macrophages requires dynamin-related protein 1 (Drp1)-mediated mitochondrial fission, which is triggered by AC uptake. When mitochondrial fission is disabled, AC-induced increase in cytosolic calcium is blunted owing to mitochondrial calcium sequestration, and calcium-dependent phagosome formation around secondarily encountered ACs is impaired. These defects can be corrected by silencing the mitochondrial calcium uniporter (MCU). Mice lacking myeloid Drp1 showed defective efferocytosis and its pathologic consequences in the thymus after dexamethasone treatment and in advanced atherosclerotic lesions in fat-fed Ldlr-/- mice. Thus, mitochondrial fission in response to AC uptake is a critical process that enables macrophages to clear multiple ACs and to avoid the pathologic consequences of defective efferocytosis in vivo.

Different bacterial cargo in apoptotic cells drive distinct macrophage phenotypes.

The removal of dead cells (efferocytosis) contributes to the resolution of the infection and preservation of the tissue. Depending on the environment milieu, macrophages may show inflammatory (M1) or anti-inflammatory (M2) phenotypes. Inflammatory leukocytes are recruited during infection, followed by the accumulation of infected and non-infected apoptotic cells (AC). Efferocytosis of non-infected AC promotes TGF-ß, IL-10, and PGE2 production and the polarization of anti-inflammatory macrophages. These M2 macrophages acquire an efficient ability to remove apoptotic cells that are involved in tissue repair and resolution of inflammation. On the other hand, the impact of efferocytosis of infected apoptotic cells on macrophage activation profile remains unknown. Here, we are showing that the efferocytosis of gram-positive Streptococcus pneumoniae-AC (Sp-AC) or gram-negative Klebsiella pneumoniae-AC (Kp-AC) promotes distinct gene expression and cytokine signature in macrophages. Whereas the efferocytosis of Kp-AC triggered a predominant M1 phenotype in vitro and in vivo, the efferocytosis of Sp-AC promoted a mixed M1/M2 activation in vitro and in vivo in a model of allergic asthma. Together, these findings suggest that the nature of the pathogen and antigen load into AC may have different impacts on inducing macrophage polarization.

Investigations of cellular copper metabolism in ovarian cancer cells using a ratiometric fluorescent copper dye.

Imbalances in metal homeostasis have been implicated in the progression and drug response of cancer cells. Understanding these changes will enable identification of new treatment regimes and precision medicine approaches to cancer treatment. In particular, there has been considerable interest in the interplay between copper homeostasis and response to platinum-based chemotherapeutic agents. Here, we have studied differences in the Cu uptake and distributions in the ovarian cancer cell line, A2780, and its cisplatin resistant form, A2780.CisR, by measuring total Cu content and the bioavailable Cu pool. Atomic absorption spectroscopy (AAS) revealed a lower total Cu uptake in A2780.CisR compared to A2780 cells. Conversely, live-cell confocal microscopy studies with the ratiometric Cu(I)-sensitive fluorescent dye, InCCu1, revealed higher relative cellular content of labile Cu in A2780.CisR cells compared with A2780 cells. These results demonstrate that Cu trafficking, homeostasis and speciation are different in the Pt-sensitive and resistant cells and may be associated with the predominance of different phenotypes for A2780 (epithelial) and A2780.CisR (mesenchymal) cells.

Phagocytosing differentiated cell-fragments is a novel mechanism for controlling somatic stem cell differentiation within a short time frame.

Stem cells undergo cytokine-driven differentiation, but this process often takes longer than several weeks to complete. A novel mechanism for somatic stem cell differentiation via phagocytosing 'model cells' (apoptotic differentiated cells) was found to require only a short time frame. Pluripotent-like Muse cells, multipotent mesenchymal stem cells (MSCs), and neural stem cells (NSCs) phagocytosed apoptotic differentiated cells via different phagocytic receptor subsets than macrophages. The phagocytosed-differentiated cell-derived contents (e.g., transcription factors) were quickly released into the cytoplasm, translocated into the nucleus, and bound to promoter regions of the stem cell genomes. Within 24 ~ 36 h, the cells expressed lineage-specific markers corresponding to the phagocytosed-differentiated cells, both in vitro and in vivo. At 1 week, the gene expression profiles were similar to those of the authentic differentiated cells and expressed functional markers. Differentiation was limited to the inherent potential of each cell line: triploblastic-, adipogenic-/chondrogenic-, and neural-lineages in Muse cells, MSCs, and NSCs, respectively. Disruption of phagocytosis, either by phagocytic receptor inhibition via small interfering RNA or annexin V treatment, impeded differentiation in vitro and in vivo. Together, our findings uncovered a simple mechanism by which differentiation-directing factors are directly transferred to somatic stem cells by phagocytosing apoptotic differentiated cells to trigger their rapid differentiation into the target cell lineage.

Molecular analysis of Annexin expression in cancer.

BACKGROUND: Uptake of apoptotic cells induces a tolerogenic phenotype in phagocytes and promotes peripheral tolerance. The highly conserved Annexin core domain, present in all members of the Annexin family, becomes exposed on the apoptotic cell-surface and triggers tolerogenic signalling in phagocytes via the Dectin-1 receptor. Consequently, Annexins exposed on tumour cells upon cell death are expected to induce tolerance towards tumour antigens, inhibiting tumour rejection. METHODS: Expression analysis for all Annexin family members was conducted in cancer cell lines of diverse origins. Presentation of Annexins on the cell surface during apoptosis of cancer cell lines was investigated using surface washes and immunoblotting. Expression data from the GEO database was analysed to compare Annexin levels between malignant and healthy tissue. RESULTS: Six Annexins at least were consistently detected on mRNA and protein level for each investigated cell line. AnxA1, AnxA2 and AnxA5 constituted the major part of total Annexin expression. All expressed Annexins translocated to the cell surface upon apoptosis induction in all cell lines. Human expression data indicate a correlation between immune infiltration and overall Annexin expression in malignant compared to healthy tissue. CONCLUSIONS: This study is the first comprehensive analysis of expression, distribution and presentation of Annexins in cancer.

Photoprotective effect of solid lipid nanoparticles of rutin against UVB radiation damage on skin biopsies and tissue-engineered skin.

Solid lipid nanoparticles (SLNs) containing rutin were prepared to enhance their photochemopreventive effect on the skin. SLNs were produced by the hot melt microemulsion technique. Two 3D skin models: ex vivo skin explants and 3D tissue engineering skin were used to evaluate the photochemopreventive effect of topical formulations containing rutin SLNs, against ultraviolet B (UVB) radiation, inducing sunburn cells, caspase-3, cyclobutane pyrimidine dimers, lipid peroxidation, and metalloproteinase formation. The rutin SLNs presented average size of 74.22 ± 2.77 nm, polydispersion index of 0.16 ± 0.04, encapsulation efficiency of 98.90 ± 0.25%, and zeta potential of -53.0 ± 1.61 mV. The rutin SLNs were able to efficiently protect against UVB induced in the analysed parameters in both skin models. Furthermore, the rutin SLNs inhibited lipid peroxidation and metalloproteinase formation. These results support the use of rutin SLNs as skin photochemopreventive agents for topical application to the skin.

Roles of Calreticulin in Protein Folding, Immunity, Calcium Signaling and Cell Transformation.

The endoplasmic reticulum (ER) is an organelle that mediates the proper folding and assembly of proteins destined for the cell surface, the extracellular space and subcellular compartments such as the lysosomes. The ER contains a wide range of molecular chaperones to handle the folding requirements of a diverse set of proteins that traffic through this compartment. The lectin-like chaperones calreticulin and calnexin are an important class of structurally-related chaperones relevant for the folding and assembly of many N-linked glycoproteins. Despite the conserved mechanism of action of these two chaperones in nascent protein recognition and folding, calreticulin has unique functions in cellular calcium signaling and in the immune response. The ER-related functions of calreticulin in the assembly of major histocompatibility complex (MHC) class I molecules are well-studied and provide many insights into the modes of substrate and co-chaperone recognition by calreticulin. Calreticulin is also detectable on the cell surface under some conditions, where it induces the phagocytosis of apoptotic cells. Furthermore, mutations of calreticulin induce cell transformation in myeloproliferative neoplasms (MPN). Studies of the functions of the mutant calreticulin in cell transformation and immunity have provided many insights into the normal biology of calreticulin, which are discussed.

The B cell response to both protein and nucleic acid antigens displayed on apoptotic cells are dependent on endosomal pattern recognition receptors.

In systemic autoimmune diseases such as systemic lupus erythematosus (SLE), B cell tolerance is lost and there is a production of autoantibodies that drive pathology. The specificities of these antibodies are towards a wide range of autoantigens including proteins such as serum factors including cytokines as well as towards nucleic acids and modified glycolipids. It is known that endosomal pattern recognition receptors are involved in specific responses but if they drive specificity towards a specific group of autoantigens is not known. Here, we used syngeneic apoptotic cells alone to break B cell tolerance and investigated the antibody response in Unc93b1 mutant mice that lack signalling from the TLR3, TLR7 and TLR9 receptors. We found that specific B cell responses known from patients with SLE including antibodies towards Ro-52/60, La, cardiolipin as well as DNA were all significantly lower in the knockout mice. Thus, we found that endosomal TLR receptors were involved in break of tolerance and drive B cell responses for protein, nucleic acid and modified lipid antigens. This pinpoints these receptors as key drivers for the full range of antibody driven pathology in SLE and suggests that targeting of endosomal TLR driven responses will quench all B cell driven autoreactivity.

Efferocytosis and the Story of "Find Me," "Eat Me," and "Don't Eat Me" Signaling in the Tumor Microenvironment.

The process of efferocytosis involves removal of dying or dead cells by phagocytosis. Another term "efferosome" is used which means a fluid-filled membrane vesicle which engulfs dead cells. The process of efferocytosis works in coordination with apoptosis because before the contents of apoptotic cells are bleached out, they are engulfed by efferosomes. Thus, the microenvironment is not polluted with toxic enzymes and oxidants. A defect in the apoptotic cell clearance may participate in autoimmunity and chronic inflammation for homeostasis and proper tissue development, for which removal of dead cells is essential. This also protects from chronic inflammation and autoimmunity. In different tumor types and other diseases, efferocytosis has been studied extensively and potential pathways identified. A few of the intermediates in different pathways, which create aggressive and tolerogenic tumor microenvironment, might be considered for therapeutic or interventional purposes. Since the key players in efferocytosis are macrophages and dendritic cells, development of antigen-dependent antitumor immunity is affected by efferocytosis. The literature analysis suggests that efferocytosis is an underappreciated immune checkpoint, perhaps one that might be therapeutically targeted in the setting of cancer. The current status of efferocytosis and its role in tumor microenvironment is discussed in this article.

Intestinal host defense outcome is dictated by PGE2 production during efferocytosis of infected cells.

Inflammatory responses are terminated by the clearance of dead cells, a process termed efferocytosis. A consequence of efferocytosis is the synthesis of the antiinflammatory mediators TGF-ß, PGE2, and IL-10; however, the efferocytosis of infected cells favors Th17 responses by eliciting the synthesis of TGF-ß, IL-6, and IL-23. Recently, we showed that the efferocytosis of apoptotic Escherichia coli-infected macrophages by dendritic cells triggers PGE2 production in addition to pro-Th17 cytokine expression. We therefore examined the role of PGE2 during Th17 differentiation and intestinal pathology. The efferocytosis of apoptotic E. coli-infected cells by dendritic cells promoted high levels of PGE2, which impaired IL-1R expression via the EP4-PKA pathway in T cells and consequently inhibited Th17 differentiation. The outcome of murine intestinal Citrobacter rodentium infection was dependent on the EP4 receptor. Infected mice treated with EP4 antagonist showed enhanced intestinal defense against C. rodentium compared with infected mice treated with vehicle control. Those results suggest that EP4 signaling during infectious colitis could be targeted as a way to enhance Th17 immunity and host defense.

C-reactive protein exacerbates epithelial-mesenchymal transition through Wnt/ß-catenin and ERK signaling in streptozocin-induced diabetic nephropathy.

Previous studies have reported the pathogenic role of C-reactive protein (CRP) during diabetic kidney disease (DKD) in human CRP transgenic and Crp-/- mice. However, because humans and mice have inverse acute phase expression patterns of CRP and serum amyloid P component, this could lead to the inaccurate evaluation of CRP function with the above-mentioned CRP transgenic mouse. But different from mice, rats have the same acute phase protein expression pattern as human, which might avoid this problem and be a better choice for CRP function studies. To dispel this doubt and accurately define the role of CRP during diabetic nephropathy, we created the first Crp-/- rat model, which we treated with streptozocin to induce DKD for in vivo studies. Moreover, an established cell line (human kidney 2) was used to further investigate the pathologic mechanisms of CRP. We found that CRP promotes epithelial-mesenchymal transition (EMT) through Wnt/ß-catenin and ERK1/2 signaling, which are dependent on CRP binding to FcγRII on apoptotic cells. By promoting EMT, CRP was demonstrated to accelerate the development of DKD. We thus present convincing evidence demonstrating CRP as a therapeutic target for DKD treatment.-Zhang, L., Shen, Z.-Y., Wang, K., Li, W., Shi, J.-M., Osoro, E. K., Ullah, N., Zhou, Y., Ji, S.-R. C-reactive protein exacerbates epithelial-mesenchymal transition through Wnt/ß-catenin and ERK signaling in streptozocin-induced diabetic nephropathy.

Effect of Cryopreservation on Autologous Chimeric Antigen Receptor T Cell Characteristics.

As clinical applications for chimeric antigen receptor T cell (CART) therapy extend beyond early phase trials, commercial manufacture incorporating cryopreservation steps becomes a logistical necessity. The effect of cryopreservation on CART characteristics is unclear. We retrospectively evaluated the effect of cryopreservation on product release criteria and in vivo characteristics in 158 autologous CART products from 6 single-center clinical trials. Further, from 3 healthy donor manufacturing runs, we prospectively identified differentially expressed cell surface markers and gene signatures among fresh versus cryopreserved CARTs. Within 2 days of culture initiation, cell viability of the starting fraction (peripheral blood mononuclear cells [PBMNCs]) decreased significantly in the cryo-thawed arm compared to the fresh arm. Despite this, PBMNC cryopreservation did not affect final CART fold expansion, transduction efficiency, CD3%, or CD4:CD8 ratios. In vivo CART persistence and clinical responses did not differ among fresh and cryopreserved final products. In healthy donors, compared to fresh CARTs, early apoptotic cell-surface markers were significantly elevated in cryo-thawed CARTs. Cryo-thawed CARTs also demonstrated significantly elevated expression of mitochondrial dysfunction, apoptosis signaling, and cell cycle damage pathways. Cryopreservation during CART manufacture is a viable strategy, based on standard product release parameters. The clinical impact of cryopreservation-related subtle micro-cellular damage needs further study.

Apoptosis and secondary necrosis of neutrophils and monocytes in the immunopathogenesis of rheumatoid arthritis: a cohort study.

Rheumatoid arthritis (RA) is a progressive chronic inflammatory and autoimmune joint disease. Neutrophils and monocytes are the main target cells of innate immune defense that modulate the course of inflammatory rheumatic diseases. Dysfunctional phagocytosis is a common feature in RA. The aim of this study was to evaluate the diagnostic value of apoptotic changes in neutrophils and monocytes and their relationship with rheumatoid activity measured by the DAS28 score. We used the APOLECT flow cytometric assay for evaluating primary necrotic, apoptotic, and secondary necrotic neutrophils and monocytes determination in RA patients compared with healthy controls. The apoptotic granulocytes were greater in RA patients compared to healthy controls (0.76 ± 0.15% vs. 0.58 ± 0.17%, P < 0.05). The percentage of primary necrotic granulocytes was significantly elevated in RA patients compared to healthy controls (3.84 ± 0.5% vs. 1.96 ± 0.33%). No significant difference was noted for primary necrotic monocytes. The number of secondary necrotic granulocytes and monocytes was high in RA patients (0.94 ± 0.15% vs. 0.4 ± 0.06% and 4.83 ± 1.06% vs. 1.8 ± 0.33%, respectively). The obtained results suggest that neutrophils and monocytes undergo apoptotic modifications which are accompanied by secondary necrotic cells formation in RA. These shifts may lead to autoantigen accumulation that results in the progressive course RA.

Investigation of Dextran-Coated Superparamagnetic Nanoparticles for Targeted Vinblastine Controlled Release, Delivery, Apoptosis Induction, and Gene Expression in Pancreatic Cancer Cells.

In the current study, the surface of superparamagnetic iron oxide (SPION) was coated with dextran (DEX), and conjugated with folic acid (FA), to enhance the targeted delivery and uptake of vinblastine (VBL) in PANC-1 pancreatic cancer cells. Numerous analyses were performed to validate the prepared FA-DEX-VBL-SPION, such as field emission scanning transmission electron microscopy, high-resolution transmission electron microscopy, dynamic light scattering (DLS), Zeta Potential, Fourier transform infrared spectroscopy, and vibrating sample magnetometry (VSM). The delivery system capacity was evaluated by loading and release experiments. Moreover, in vitro biological studies, including a cytotoxicity study, cellular uptake assessment, apoptosis analysis, and real-time PCR, were carried out. The results revealed that the obtained nanocarrier was spherical with a suitable dispersion and without visible aggregation. Its average size, polydispersity, and zeta were 74 ± 13 nm, 0.080, and -45 mV, respectively. This dual functional nanocarrier also exhibited low cytotoxicity and a high apoptosis induction potential for successful VBL co-delivery. Real-time quantitative PCR analysis demonstrated the activation of caspase-3, NF-1, PDL-1, and H-ras inhibition, in PANC-1 cells treated with the FA-VBL-DEX-SPION nanostructure. Close inspection of the obtained data proved that the FA-VBL-DEX-SPION nanostructure possesses a noteworthy chemo-preventive effect on pancreatic cancer cells through the inhibition of cell proliferation and induction of apoptosis.

Apoptotic cell recognition receptors and scavenger receptors.

Phosphatidylserine recognition receptors are a highly diverse set of receptors grouped by their ability to recognize the 'eat-me' signal phosphatidylserine on apoptotic cells. Most of the phosphatidylserine recognition receptors dampen inflammation by inducing the production of anti-inflammatory mediators during the phagocytosis of apoptotic corpses. However, many phosphatidylserine receptors are also capable of recognizing other ligands, with some receptors being categorized as scavenger receptors. It is now appreciated that these receptors can elicit different downstream events for particular ligands. Therefore, how phosphatidylserine recognition receptors mediate specific signals during recognition of apoptotic cells versus other ligands, and how this might help regulate the inflammatory state of a tissue is an important question that is not fully understood. Here, we revisit the work on signaling downstream of the phosphatidylserine recognition receptor BAI1, and evaluate how these and other signaling modules mediate signaling downstream from other receptors, including Stabilin-2, MerTK, and αvß5. We also propose the concept that phosphatidylserine recognition receptors could be viewed as a subset of scavenger receptors that are capable of eliciting anti-inflammatory responses to apoptotic cells.

Effects and mechanisms of scalp acupuncture on learning and memory in mice with lead poisoning.

OBJECTIVE: To study the effects and mechanism of scalp acupuncture on learning and memory ability in mice with lead poisoning. METHODS: From March 2018 to December 2018, 30 Kunming mice were randomly divided equally into the control group and the intervention group after intraperitoneal injection of lead acetate The intervention group received scalp acupuncture on the first day of the model establishment; the model group only received conventional feeding without treatment. At the same time, a control group of 15 rats was given the intraperitoneal injections of normal saline for 8 consecutive days, and only after routine feeding, no treatment was given. Determination of lead in blood was detected by Graphite Furnace Atomic Absorption Spectrometry, the Morris water maze test was used to detect the learning and memory function of mice, hydroxylamine colorimetric method was used to measure acetylcholinesterase (AChE) activity, and TUNEL staining was used to detect the apoptotic cells in the hippocampus. RESULTS: The results showed that the blood lead level of the model group (231.42±12.53µg/L) was significantly higher than that of the control group (20.43±4.62µg/L) (P<0.05); and there was no significant difference in blood lead content between the intervention group (228.12±5.21µg/L) and the model group. The Morris water maze test showed that from the fourth day of the orientation navigation experiment, the escape latency of the model group (22.2±4.10s) was longer than that of the control group (13.64±2.93s) (P<0.05); besides, from the third day, the escape latency of mice in the intervention group (13.52±9.18s) was significantly shortened compared with the model group (19.95±3.52s). In the space exploration experiment, in terms of passing through the platform, the distance (1.57±0.49m) and time (15.54±3.72s) of mice in the model group were longer than that of mice in the control group (0.73±0.44m, 3.24±2.24s) (P<0.05), the distance (0.41±0.28m) and time (3.0±1.93s) of mice in the intervention group were shorter than that of mice in the model group, and the difference was statistically significant (P<0.05). The apoptosis rate of hippocampus in the model group (8.79±0.37%) was significantly higher than that in the control group (3.56±0.44%) (P<0.05), and the apoptosis rate of hippocampus in the intervention group (4.36±0.12%0 was significantly lower than that in the model group (P<0.05). The expression of AchE in the model group (0.5±0.13U/ug) was significantly higher than that in the control group (0.23±0.04U/ug), but there was no significant difference in the AChE activity between the intervention group and the model group. CONCLUSIONS: In conclusion, scalp acupuncture can improve the learning and memory ability of mice with lead poisoning, and the decrease of hippocampal apoptotic cells may be a possible mechanism for the improvement of learning and memory function.

Downregulated Rac1 promotes apoptosis and inhibits the clearance of apoptotic cells in airway epithelial cells, which may be associated with airway hyper-responsiveness in asthma.

The accumulation of airway apoptotic cells may be an important factor causing airway hyper-responsiveness (AHR). Whether the apoptotic cells can be promptly removed is related to the occurrence and course of asthma. In recent years, studies have shown that Rac1 is involved in many cellular biological activities including the formation and elimination of apoptotic cells. In this study, based on the analysis of airway local cells and related factors in asthmatic mice, we evaluated the expression of Rac1 in airway epithelial cells or phagocytes and analysed its relationship with the incidence of apoptosis or scavenging of apoptotic cells. Our data showed that the expression level of Rac1 in asthmatic mice decreased significantly, while the expression of IL-33 increased obviously. The airway epithelial cell line was stimulated by curcumin at 50 µmol/L for 24-48 hours; more than 50% of the cells were apoptotic, and of which, about 20% were late apoptosis. Rac1 inhibitor (NSC23766) can enhance the apoptosis effect. In addition, the ability of phagocytosis and migration in the epithelial cells or macrophages was increased following the application of Rac1 inhibitors or specific siRNA in a dose-dependent manner, and the expression level of IL-33 was simultaneously increased after blocking Rac1. It is suggested that the down regulation of Rac1 in asthma may contribute to the apoptosis of airway epithelial cells and affect the clearance of apoptotic cells, which will lead to the aggregation of the apoptotic cells in the respiratory tract and participate in AHR.

CED-10-WASP-Arp2/3 signaling axis regulates apoptotic cell corpse engulfment in C. elegans.

Efficient clearance of apoptotic cells is essential for tissue homeostasis in metazoans. Genetic studies in Caenorhabditis elegans have identified signaling cascades that activate CED-10/Rac1 GTPase and promote actin cytoskeletal rearrangement during apoptotic cell engulfment. However, the molecular connection between CED-10 activation and actin reorganization remains elusive. Here, we provide evidence that CED-10 binds to the Arp2/3 nucleation promoting factor WASP; CED-10 recruits WASP and Arp2/3 to apoptotic cell corpses in the phagocytes. The loss of WASP and Arp2/3 impaired cell corpse engulfment. Furthermore, we uncover that a WASP-activating factor SEM-5/GRB2 functions in the phagocytes to promote cell corpse clearance. Together, our results suggest CED-10 reorganizes the actin cytoskeleton by recruiting the WASP-Arp2/3 actin nucleation factors during apoptotic cell engulfment.

A STAT6 Inhibitor AS1517499 Reduces Preventive Effects of Apoptotic Cell Instillation on Bleomycin-Induced Lung Fibrosis by Suppressing PPARγ.

BACKGROUND/AIMS: The signal transducer and activator of transcription 6 (STAT6) transcription factor mediates PPARγ-regulated gene expression in macrophages. However, it remains largely unknown how proximal membrane signaling events initiated by apoptotic cell recognition upregulate PPARγ expression and activate the lung homeostatic program. METHODS: The STAT6 inhibitor AS1517499 was used to determine the role of STAT6 in mediating PPARγ activity, anti-inflammatory effects, and anti-fibrotic effects induced by apoptotic cell instillation after bleomycin treatment into C57BL/6 mice. Bronchoalveolar lavage fluid, alveolar macrophages and lungs were harvested at days 2, 7, and 14 and then analyzed by real-time PCR, immunoblotting, ELISA, immunocytochemistry and immunohistochemistry assays. RESULTS: Our data demonstrate that apoptotic cell instillation after bleomycin results in prolonged enhancement of STAT6 phosphorylation in alveolar macrophages and lung. Co-administration of the STAT6 inhibitor, AS1517499, reversed the enhanced PPARγ expression and activity induced by apoptotic cell instillation after bleomycin treatment. By reducing the expression of PPARγ target genes, including CD36, macrophage mannose receptor, and arginase 1, AS1517499 inhibited efferocytosis and restored pro-inflammatory cytokine expression, neutrophil recruitment, protein levels, hydroxyproline content, and expression of fibrosis markers, including type 1 collagen α2, fibronectin, and α-smooth muscle actin. STAT6 inhibition reversed the expression profile of hepatocyte growth factor and interleukin-10. CONCLUSION: These results indicate that prolonged STAT6 activation following one-time apoptotic cell instillation facilitates continuous PPARγ activation, resulting in the resolution of bleomycin-induced lung inflammation and fibrosis.

Combination of anti-early apoptotic cell autoantibodies and anti-SSA autoantibodies in lupus nephritis.

The aim of this study was to investigate the association of anti-early apoptotic cell autoantibodies, anti-SSA, and anti-SSB with clinical features of lupus nephritis (LN). Multiparameter flow cytometry was used to determine early apoptotic cells and for measuring the simultaneous binding of annexin V, 7-AAD, and IgG from LN patients (n = 39). The association between clinical features of LN and autoantibodies against early apoptotic cells, and between the autoantibodies and anti-SSA and anti-SSB, were further investigated. Thirteen LN patients (33.3 %) were positive for autoantibodies against early apoptotic cells. The prevalence of anti-SSA and anti-SSB were similar in patients with anti-early apoptotic cell autoantibodies and those without (anti-SSA: 9/13 versus 15/26; anti-SSB: 3/13 versus 4/26). Anti-early apoptotic cell antibody-positive patients had lower C3 levels (0.34 ± 0.22) than the antibody-negative patients (0.47 ± 0.17, p = 0.059); and significantly higher anti-dsDNA levels (502.99 ± 275.48 versus 214.13 ± 229.29, p = 0.001). In univariate logistic regression analysis, the presence of anti-early apoptotic cell antibody could predict poor short-term prognosis (HR 7.500, 95 % CI: 1.210 - 46.504, p = 0.030), while patients who were double positive for anti-SSA and anti-early apoptotic cell antibody had significantly increased risk of poor short-term outcome (HR 17.500, 95 % CI: 2.500 - 122.500, p = 0.004). The combination of anti-early apoptotic cell autoantibodies and anti-SSA might be of predictive value in LN.