Regulation of cytokine production during phagocytosis of apoptotic cells

Loss of self-tolerance and expansion of auto-reactive lymphocytes are the basis for autoimmunity. Apoptosis and the rapid clearance of apoptotic cells by phagocytes usually occur as coordinated processes that ensure regulated cellularity and stress response with non-pathological outcomes. Defects in clearance of apoptotic ceils would contribute to the generation of self-reactive lymphocytes, which drive autoimmune disorders such as rheumatoid arthritis （RA） and systemic lupus erythematosus （SLE）. The IL-12 family of cytokines （IL-12, IL-23, and IL-27） and IL-10 are produced by phagocytic macrophages and play critical roles in the regulation of antigen-presenting cells （APCs） and effector lymphocytes during an immune response to pathogens. Inappropriate expression of these cytokines and their dysregulated activities have been strongly implicated in the pathogenesis of several autoimmune diseases. The production of pro- and anti-inflammatory cytokines by phagocytic APCs is delicately regulated during the ingestion of apoptotic cells as part of an intrinsic mechanism to prevent inflammatory autoimmune reactions. How apoptotic cell-derived signals regulate cytokine production is poorly understood. A recent study by our group demonstrated that phagocytosis of apoptotic cells by activated macrophages results in strong inhibition of IL-12 p35 gene expression by activating a novel transcription repressor, which we named GC-binding protein （GC-BP）, through tyrosine dephosphorylation. We are also beginning to understand the molecular mechanisms underlying apoptotic cell-triggered production of IL-10 by phagocytes. These studies will help to elucidate some novel immune regulatory mechanisms and explore the regulation of immune responses to autoantigens with potentials to discover new therapeutic targets for the treatment of autoimmune disorders.

Enhanced therapeutic effects of apoptotic cell-conditioned mesenchymal stem cells in lupus-prone MRL/lpr mice

Background::Apoptotic cell-conditioned mesenchymal stem cells (AC-MSCs) exhibit stronger T cell suppressive ability via cyclooxygenase 2 (COX2)/prostaglandin E2 (PGE2);however, whether AC-MSCs exhibit enhanced therapeutic effects on systemic lupus erythematosus (SLE) remains unknown. Methods: Splenocytes from MRL/MPJ-Fas lpr (MRL /lpr) mice were cocultured with AC-MSCs, and the proportion of plasma cells was determined by flow cytometry. MSCs, AC-MSCs, COX2 knockdown MSCs, and COX2 knockdown AC-MSCs were infused into MRL/ lpr mice ( n = 10/group). Survival rates and lupus symptoms, including proteinuria, kidney injury, renal immune complex deposition, and autoantibody production, were assessed. Additionally, the number of plasma cells and serum levels of inflammatory cytokines were measured. Results::The AC-MSCs significantly inhibited plasma cells via PGE2 after 24 h coculture in vitro, whereas MSCs did not. In the MRL /lpr mice, AC-MSC treatment led to a significantly higher survival rate than phosphate-buffered saline (PBS) treatment (90% vs. 50%, p < 0.05). Moreover, AC-MSC infusion decreased urine protein levels as early as 1 week after administration (0.89 ± 0.55 mg/mL vs. 1.59 ± 0.60 mg/mL, p < 0.05, compared with PBS treatment). Administration of both MSCs and AC-MSCs reduced renal immunoglobulin G and complement C3 deposition, whereas COX2 knockdown MSCs and COX2 knockdown AC-MSCs did not. Serum anti-dsDNA antibody levels in AC-MSC-treated mice significantly decreased (0.40 ± 0.25 vs. 0.99 ± 0.58, p < 0.05), compared with PBS treatment, as well as the number of plasma cells in both the spleen ([2.14 ± 1.05] × 10^(6) vs. [8.02 ± 4.01] × 10^(6), p < 0.01) and renal-draining lymph nodes ([0.78 ± 0.68] × 10^(6) vs. [2.49 ± 1.45] × 10^(6), p < 0.05). Additionally, AC-MSCs inhibited the production of inflammatory cytokines, including interleukin-21, tumor necrosis factor-alpha, and monocyte chemoattractant protein-1. Conclusions::AC-MSCs enhanced the therapeutic effects in mice with lupus, which were partially mediated by COX2/PGE2. Therefore, AC preconditioning may be a new strategy for MSC transplantation in the treatment of SLE.

Apoptotic cell administration enhances pancreatic islet engraftment by induction of regulatory T cells and tolerogenic dendritic cells

cell transfer has been found to be able to facilitate engraftment of allograft. However, the underlying mechanisms remain to be fully understood. Here we demonstrate that intravenous administration of donor apoptotic splenocytes can promote pancreatic islet engraftment by inducing generation of tolerogenic dendritic cells （ToI-DCs） and expansion of CD4＋Foxp3＋ regulatory T cells （Tregs）. In vivo clearance of either dendritic cells （DCs） or Tregs prevented the induction of immune tolerance by apoptotic cell administration. Transient elimination of Tregs using anti-CD25, monoclonal antibody （mAb） abrogated the generation of ToI-DCs after administration of apoptotic splenocytes. Reciprocally, depletion of DCs within CD1 lc-DTR mice using diphtheria toxin （DT） prevented the generation of Tregs in the recipients with administration of apoptotic splenocytes. Induction of Tregs by ToI-DCs required direct cell contact between the two cell types, and programmed death 1 ligand （PD-L1） played important role in the Tregs expansion. Apoptotic cell administration failed to induce ToI-DCs in IL-lO-deficient and Smad3-deficient mice, suggesting that IL-10 and transforming growth factor-β （TGF-β） are needed to maintain DCs in the tolerogenic state. Therefore, we demonstrate that ToI-DCs promote the expansion of Tregs via PD-L1 on their surface and reciprocally Tregs facilitate ToI-DCs to maintain transplantation tolerance induced by apoptotic cells via secreting IL-IO and TGF-β.

Surfactant protein A (SP-A) binds to phosphatidylserine and competes with annexin V binding on late apoptotic cells

The role of surfactant protein A(SP-A)in the recognition and clearance of apoptotic cells is well established,but to date,it is still not clear which surface molecules of apoptotic cells are involved in the process.Here we present evidence that phosphatidylserine(PS)is a relevant binding molecule for human SP-A.The binding is Ca^(2+)-dependent and is not inhibited by mannose,suggesting that the sugar-binding site of the carbohydrate recognition domain(CRD)of SP-A is not involved.Flow cytometry studies on apoptotic Jurkat cells revealed apparent inhibition of annexin V binding by increasing concentrations of SP-A in late apoptotic but not early apoptotic cells,and this was consistent for Jurkat cells and neutrophils.Supporting these data,confocal microscopy results show a co-localisation of annexin V and SP-A in late apoptotic but not early apoptotic cells.However,we cannot conclude that this inhibition is exclusively due to the binding of SP-A to PS on the cell surface,as annexin V is not wholly specific for PS and SP-A also interacts with other phospholipids that might become exposed on the apoptotic cell surface.

Electron microscopic observations of apoptotic cells in various etiologies of human cardiovascular diseases

Abstract Objective To observe apoptotic process in various cardiovascular disorders with a particular attention to the ultrastructural morphology of apoptosis in cardiomyocytes, fibroblasts, vascular endothelial cells and smooth muscle cells. Methods Transmission electron microscopic observations of the tissue specimens obtained from endomyocardial biopsies or surgical excisions of left ventricular myocardium or calcified aortic valves were carried out in 50 patients with various cardiovascular diseases. Results The ultrastructural features of apoptosis was consistently observed in cardiomyocytes, fibroblasts, vascular endothelial cells and smooth muscle cells in all diseased tissues. In cardiomyopathies and rheumatic heart diseases apoptosis was commonly observed in the cardiomyocytes. It was often found that fibroblasts underwent apoptosis in calcific aortic valve tissues. Apoptosis of arterial smooth muscle cells was a frequent finding in renal arterial stenosis due to Takayasu's arteritis and fibromuscular dysplasia. Regardless of the cell types, the nuclear hallmarks of apoptosis were identical with minor modifications of the fragmentation of the condensed cells into apoptotic bodies. Conclusions Based on electron microscopic findings, it is suggested that the underlying disease processes determine which type of cells predominantly undergoes apoptotic changes in various cardiovascular disorders. In addition, different cells with similar structural morphology may have common ultrastructural features of apoptosis.

Coupled single-cell and bulk RNA-seq analysis reveals the engulfment role of endothelial cells in atherosclerosis

The clearance of apoptotic cell debris,containing professional phagocytosis and non-professional phagocytosis,is essential for maintaining the homeostasis of healthy tissues.Here,we discovered that endothelial cells could engulf apoptotic cell debris in atherosclerotic plaque.Single-cell RNA sequencing(RNA-seq)has revealed a unique endothelial cell subpopulation in atherosclerosis,which was strongly associated with vascular injury-related pathways.Moreover,integrated analysis of three vascular injury-related RNA-seq datasets showed that the expression of scavenger receptor class B type 1(SR-B1)was up-regulated and specifically enriched in the phagocytosis pathway under vascular injury circumstances.Single-cell RNA-seq and bulk RNA-seq indicate that SR-B1 was highly expressed in a unique endothelial cell subpopulation of mouse aorta and strongly associated with the reorganization of cellular adherent junctions and cytoskeleton which were necessary for phagocytosis.Furthermore,SR-B1 was strongly required for endothelial cells to engulf apoptotic cell debris in atherosclerotic plaque of both mouse and human aorta.Overall,this study demonstrated that apoptotic cell debris could be engulfed by endothelial cells through SR-B1 and associated with the reorganization of cellular adherent junctions and cytoskeleton.

Induction of endoplasmic reticulum-derived oxidative stress by an occult infection related S surface antigen variant

AIM: To investigate the mechanism of endoplasmic reticulum(ER) stress induction by an occult infection related hepatitis B virus S surface antigen(HBsAg)variant.METHODS: We used an HBsAg variant with lower secretion capacity, which was a KD variant from a Korean subject who was occultly infected with the genotype C. We compared the expression profiles of ER stress-related proteins between HuH-7 cells transfected with HBsAg plasmids of a wild-type and a KD variant using Western blot.RESULTS: Confocal microscopy indicated that the KD variant had higher levels of co-localization with ER than the wild-type HBsAg. The KD variant upregulated ER stress-related proteins and induced reactive oxygen species(ROS) compared to the wildtype via an increase in calcium. The KD variant also down-regulated anti-oxidant proteins(HO-1, catalase and SOD) compared to the wild-type, which indicates positive amplification loops of the ER-ROS axis. The KD variant also induced apoptotic cell death via the upregulation of caspase proteins(caspase 6, 9 and 12).Furthermore, the KD variant induced a higher level of nitric oxide than wild-type HBsAg via the up-regulation of the iNOS protein.CONCLUSION: Our data indicate that occult infection related HBsAg variants can lead to ER-derived oxidative stress and liver cell death in HuH-7 cells.

Programming of macrophages by UV-irradiated apoptotic cancer cells inhibits cancer progression and lung metastasis

Apoptotic cell clearance by phagocytes is essential in tissue homeostasis.We demonstrated that conditioned medium(CM)from macrophages exposed to apoptotic cancer cells inhibits the TGFβ1-induced epithelial–mesenchymal transition(EMT),migration,and invasion of cancer cells.Apoptotic 344SQ(ApoSQ)cell-induced PPARγactivity in macrophages increased the levels of PTEN,which was secreted in exosomes.Exosomal PTEN was taken up by recipient lung cancer cells.ApoSQ-exposed CM from PTEN knockdown cells failed to enhance PTEN in 344SQ cells,restore cellular polarity,or exert anti-EMT and anti-invasive effects.The CM that was deficient in PPARγligands,including 15-HETE,lipoxin A4,and 15d-PGJ2,could not reverse the suppression of PPARγactivity or the PTEN increase in 344SQ cells and consequently failed to prevent the EMT process.Moreover,a single injection of ApoSQ cells inhibited lung metastasis in syngeneic immunocompetent mice with enhanced PPARγ/PTEN signaling both in tumorassociated macrophages and in tumor cells.PPARγantagonist GW9662 reversed the signaling by PPARγ/PTEN;the reduction in EMT-activating transcription factors,such as Snai1 and Zeb1;and the antimetastatic effect of the ApoSQ injection.Thus,the injection of apoptotic lung cancer cells may offer a new strategy for the prevention of lung metastasis.

The Effect of n-Hexane on the Gonad Toxicity of Female Mice

Objective To investigate the toxic effects of n-hexane on the Ganod of female mice.Methods n-Hexane was administered to four groups of mice by inhalation at doses of 0,3.0,15.1,and 75.8 mL/m3 respectivelyfor five weeks.Each group consisted of 10 mice,of which half were injected in first with 10 IU of pregnant mare serum gonadotrophin（PMSG） on the 33rd days,and then with 10 IU of human chorionic gonadotrophin（HCG） 48 hrs later.After the treatment,mouse sera were sampled and ovulating hormone（LH）,follicle-stimulating hormone（FSH）,estradiol（E2）,and progesterone（P4） levels were measured by electrochemiluminescence immunoassays（ECLIA）.In each group,the right ovaries of the non-super-ovulated mice were stained with hematoxylin and eosin while ovaries on the left side were prepared with the TUNEL method in order to detect apoptotic cells.Results The duration of the diestrus stage decreased significantly（P0.05） in the 75.8 mL/m3 group.All super-ovulated mice in each treatment group produced fewer eggs than those in the control group（P0.05）.The number of follicles in ovaries in the 75.8 mL/m3 group was smaller compared with the control group（P0.05）.The serum P4 levels in each treatment group were lower than those in the control group（F=6.196,P0.01）.The cell apoptotic rate in the 75.8 mL/m3 group was higher（P0.05）.Conclusion n-Hexane may have directly mediated via alterations hormone secretion and promoted granulosal cell apoptotic,which may be one of the important mechanisms for n-hexane induced mouse ovary impairment.

Reprogramming of cancer-associated fibroblasts by apoptotic cancer cells inhibits lung metastasis via Notch1-WISP-1 signaling

The interplay between apoptotic cancer cells and the tumor microenvironment modulates cancer progression and metastasis.Cancer-associated fibroblasts(CAFs)play a crucial role in promoting these events through paracrine communication.Here,we demonstrate that conditioned medium(CM)from lung CAFs exposed to apoptotic cancer cells suppresses TGF-β1-induced migration and invasion of cancer cells and CAFs.Direct exposure of CAFs to apoptotic 344SQ cells(ApoSQ)inhibited CAF migration and invasion and the expression of CAF activation markers.Enhanced secretion of Wnt‐induced signaling protein 1(WISP-1)by CAFs exposed to ApoSQ was required for these antimigratory and anti-invasive effects.Pharmacological inhibition of Notch1 activation or siRNA-mediated Notch1 silencing prevented WISP-1 production by CAFs and reversed the antimigratory and anti-invasive effects.Enhanced expression of the Notch ligand delta-like protein 1 on the surface of ultraviolet-irradiated apoptotic lung cancer cells triggered Notch1-WISP-1 signaling.Phosphatidylserine receptor brain-specific angiogenesis inhibitor 1(BAI1)-Rac1 signaling,which facilitated efferocytosis by CAFs,participated in crosstalk with Notch1 signaling for optimal production of WISP-1.In addition,a single injection of ApoSQ enhanced WISP-1 production,suppressed the expression of CAF activation markers in isolated Thy1^(+)CAFs,and inhibited lung metastasis in syngeneic immunocompetent mice via Notch1 signaling.Treatment with CM from CAFs exposed to ApoSQ suppressed tumor growth and lung metastasis,whereas treatment with WISP-1-immunodepleted CM from CAFs exposed to ApoSQ reversed the antitumorigenic and antimetastatic effects.Therefore,treatment with CM from CAFs exposed to apoptotic lung cancer cells could be therapeutically applied to suppress CAF activation,thereby preventing cancer progression and metastasis.

The Classical and Regulatory Functions of C1q in Immunity and Autoimmunity

A classical function of Clq is to bind immune complexes and initiate complement activation producing membrane lytic complexes, opsonins and anaphylatoxins. This classical pathway of complement activation is also elicited when Clq binds some other ligands. Besides complement activation, Clq also regulates cell differentiation, adhesion, migration, activation and survival. Clq deficiency is associated with autoimmunity as well as increased susceptibility to infections. In this article, we discuss the basic properties of Clq, its expression, and classical and regulatory functions. Cellular ＆ Molecular Immunology.