Secondary Necrotic Cells Research Articles

Heme and Iron Aggravate Tissue Damage in Sickle Cell Disease By Driving Macrophage Functional Reprogramming through the Coordinated Suppression of Efferocytosis and Mitochondrial Metabolism

Introduction: Sickle cell disease (SCD) is hallmarked by an underlying chronic inflammatory status, which is in part driven by pro-inflammatory macrophages. Macrophages are highly plastic immune cells which integrate and respond to a variety of signals they are exposed to in the surrounding microenvironment. One of these stimuli is represented by heme released upon hemolysis, which acts as damage-associated molecular pattern to stimulate macrophage inflammatory phenotypic switching through TLR4 signaling and ROS production (Vinchi et al., Blood 2016). Heme-induced pro-inflammatory activation program in macrophages promotes sterile inflammation and aggravates hepatic fibrosis in SCD, contributing to the inflammatory landscape and organ damage typically associated with this disease. While previous studies addressed heme ability to induce inflammatory cytokine production in macrophages, how heme alters cell functional properties remains unexplored. Macrophage functions such as immune cell recruitment ability and apoptotic cell clearance are relevant in the context of SCD, where tissue damage and cell apoptosis occur frequently due to vaso-occlusive episodes, hypoxia and ischemic injury. Methods: Here we analyzed macrophage response to apoptotic stimuli in vivo, in mouse models of heme overload and SCD, as well as in vitro, in bone-marrow-derived macrophages (BMDMs), to unveil the impact of hemolysis on macrophage functional properties. Results: Our results demonstrate that, in addition to inflammatory activation, heme severely alters macrophage functional response to apoptotic cell damage by exacerbating their immune cell recruitment ability and impairing their efferocytic capacity. We show that exposure to heme excess drives defective efferocytosis of apoptotic neutrophils in BMDMs and mouse model of heme overload. This is fully recapitulated in SCD mice, where limited efferocytosis contributes to impaired apoptotic cell clearance and exacerbated tissue damage. Mechanistically, altered efferocytosis depends on heme-driven activation of TLR4 signaling pathway and the suppression of the nuclear transcription factor PPARγ and its coactivator PGC1α, which lead to reduced expression of efferocytic receptors and impaired mitochondrial dynamics and functions. This results in limited recognition/engulfment of apoptotic cells and decreased shift to aerobic mitochondrial fatty acid β-oxidation and anti-inflammatory IL-4 and IL-10 release, with consequent inhibition of continual efferocytosis, inflammation resolution and tissue repair. Heme-driven changes are associated with cell metabolic skewing towards glycolysis and pentose phosphate pathway and reduced reliance on mitochondrial fatty acid oxidation and TCA cycle. Macrophages pre-loaded with fatty acids showed decreased oxygen consumption rate and ATP production after heme exposure, indicating that heme-altered mitochondrial dynamics impair lipid catabolism via limited mitochondrial oxidative phosphorylation. Overall, heme induces macrophage phenotypic and functional reprogramming through the coordinated suppression of efferocytosis, via efferocytosis receptor downregulation, and fatty acid β-oxidation, via altered mitochondrial dynamics. We further demonstrate that heme-impaired efferocytosis and tissue damage promote autoimmunity through anti-nuclear antibody production against 'self' secondary necrotic cells, potentially explaining the increasing evidence of autoimmune diseases in SCD patients. Finally, hemopexin-mediated heme scavenging and anti-inflammatory IL-4 treatment improve tissue damage in hemolytic mice through macrophage rewiring. Defective efferocytosis is reproduced in vitro by macrophage exposure to SCD patients' plasma and rescued by heme scavenging via haptoglobin/hemopexin and PPARγ/PGC1α modulation via PPARα agonist or IL-4. Conclusion: Our data indicate that the therapeutic improvement of heme-altered macrophage functional properties via heme scavenging or PPARγ/PGC1α modulation significantly promotes the resolution of inflammation and ameliorates tissue damage associated with SCD pathophysiology. Thus, macrophage functional rewiring offers potentially valuable therapeutic strategies for SCD patients to improve tissue damage resolution upon vaso-occlusive crisis and ischemic events and prevent autoimmune conditions.

Abstract 1636: CLEC-1 is a novel myeloid immune checkpoint for cancer immunotherapy limiting tumor cells phagocytosis and tumor antigen cross-presentation

Abstract Myeloid cells represent one of the most abundant immune cell types in solid tumors that impede myeloid phagocytosis by triggering “don't eat me” and “don't find me” signals. Recent literature demonstrates that C-type lectin receptors (CLRs) are powerful pattern recognition receptors shaping immune cell-mediated tissue damage by positively or negatively regulating myeloid cell functions and hence tumor elimination or evasion. We previously reported that the orphan CLR CLEC-1 expressed by dendritic cells (DCs) and macrophages (MPs) is enhanced by TGFβ and tempers downstream T cells responses. Furthermore, we observed that CLEC-1 is highly expressed by myeloid cells purified from the human tumor microenvironment, in particular, tumor-associated MPs. We evaluated whether CLEC-1 could also be a receptor for DAMPs and influences phagocytosis. We found that CLEC-1 fusion protein binds specifically to secondary necrotic healthy or tumor cells induced by chemotherapy, radiation (UV, X-ray), or culture stress conditions suggesting that ligands of CLEC-1 are generated upon stress and programmed cell death. Importantly, further to promising results in KO CLEC-1 mice, we observed in vivo that CLEC-1 deficient mice, in contrast to wild-type littermates, eradicate MC38 colorectal tumors in combination with cytotoxic and immunogenic chemotherapy. Importantly, disruption of CLEC-1 signaling by Fc-CLEC-1 fusion protein also promotes tumor eradication. We then generated and identified different anti-human CLEC-1 antagonist monoclonal antibodies (mAbs) with the capacity to block CLEC-1/CLEC-1L interaction. We developed innovative antagonist CLEC-1 mAbs which, in contrast to non-antagonist CLEC-1 control mAb, increase the phagocytosis of CLEC-1L-positive human tumor cells by human TGFβ-polarized DCs or MPs. Indeed, TGFβ-polarized DCs phagocytose more efficiently a NSCLC cell line (A549) as well as Rituximab (anti-CD20 mAb)-opsonized Burkitt lymphoma cells (Raji) when CLEC-1 is antagonized by Abs. Similarly, macrophages significantly more efficiently engulfed human tumors in the presence of CLEC-1 antagonist Abs, in particular when tumor cells were opsonized such as Rituximab-opsonized Raji cells, Cetuximab opsonized colon carcinoma cells (DLD-1; EGFR+) or Trastuzumab opsonized mammary carcinoma cells (SK-BR-3; Her2+). Importantly, we observed both in vitro and in vivo that DCs from Clec1a deficient mice cross-present more efficiently dead cell-associated antigens to CD8+ T cells (OT-1). We generated hCLEC-1 knock-in mice and in vivo preclinical evaluation of CLEC-1 blocking mAbs is ongoing. Altogether, these data illustrate that CLEC-1 broadly notably inhibits tumor-cell phagocytosis and synergized with tumor-targeted cytotoxic monoclonal antibodies in both solid and hematological tumors, and hampers DC antigen cross-presentation. Citation Format: Vanessa Gauttier, Sabrina Pengam, Marion Drouin, Javier Saenz, Bérangère Evrard, Kevin Biteau, Caroline Mary, Géraldine Teppaz, Ariane Desselle, Virginie Thépénier, Emmanuelle Wilhelm, Elise Chiffoleau, Nicolas Poirier. CLEC-1 is a novel myeloid immune checkpoint for cancer immunotherapy limiting tumor cells phagocytosis and tumor antigen cross-presentation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1636.

Upregulation of miR-210–5p impairs dead cell clearance by macrophages through the inhibition of Sp1-and HSCARG-dependent NADPH oxidase pathway

The deficiency of dead cell clearance is a prominent pathogenic factor in systemic lupus erythematosus (SLE). In this study, the overexpression of miR-210–5p resulted in the accumulation of secondary necrotic cells (SNECs) in macrophages through the reduction of protein degradation. The upreguation of miR-210–5p inhibited NADPH oxidase (NOX) activation, reactive oxygen species (ROS) generation, and SNEC clearance. miR-210–5p overexpression suppressed Sp1 and HSCARG expression, and the knockdown of SP1 and HSCARG inhibited NOX expression and superoxide production in macrophages. Furthermore, patients with active SLE expressed a higher level of miR-210–5p and lower expression of SP1 and HSCARG in peripheral blood mononuclear cells. In summary, our findings indicate that the upregulation of miR-210–5p increases the accumulation of SNECs through a decrease in the Sp1-and HSCARG-mediated NOX activity and ROS generation in macrophages. Our results also suggest that targeting miR-210–5p may have therapeutic potential for SLE.

Abstract 3259: CLEC-1 is a novel myeloid immune checkpoint for cancer immunotherapy controlling damaged and tumor cells phagocytosis

Abstract Myeloid cells represent one of the most abundant immune cell types in solid tumors and are generally associated with poor clinical outcome. We previously identified the orphan C-type lectin receptors (CLRs) CLEC-1 as over-expressed in situation of established immune tolerance and reported that CLEC-1 expression by dendritic cells (DCs) and macrophages is enhanced by TGFβ and tempers downstream T cells responses. Recent literature demonstrates that tumors could hijack physiological mechanisms of CLRs that normally restrain immune cell-mediated tissue damage to suppress myeloid cell activation and promote tumor immune evasion. Here we report that CLEC-1 is highly expressed by myeloid cells purified from human tumor microenvironment (e.g. ovarian cancer) and significantly more expressed by in vitro generated M2 macrophages compared to monocytes or M1 macrophages. RNAseq public data indicates that Clec1a is highly expressed in XCR1-expressing DC including CD8α+ and CD103+ DCs from lymphoid and peripheral tissues respectively. As these DCs are specialized in cross-presentation of dying/dead cell associated antigens, we evaluated whether CLEC-1 could be a receptor of damaged cells. We found that both human and mouse CLEC-1 fusion protein, but not irrelevant CLRs or negative controls, binds specifically to late apoptotic and secondary necrotic healthy or tumor cells induced by chemotherapy, radiation (UV, X-ray) or culture stress conditions. No binding was observed with viable, primary necrotic or with early apoptotic cells suggesting that CLEC-1 ligand(s) (CLEC-1L) correspond(s) to DAMP modified during the cell death process. Using a CLEC-1 reporter NFAT-lacZ BWZ.36 cell line, we show that this interaction is functional. Importantly, we observed in vivo that dendritic cells from Clec1a deficient mice more efficiently cross-present antigen (OVA-loaded dead cells) to CD8+ T cells (OT-1) and that CLEC-1 deficient mice, but not wild-type, eradicate MC38 colorectal tumors in combination with cytotoxic and immunogenic chemotherapy (eg. cyclophosphamide). Treatment of wild-type mice with a Fc-CLEC-1 fusion protein also synergized with chemotherapy and induced complete response in 37% of mice versus 0% in chemotherapy group. We then generated, screened and identified different anti-human Clec-1 antagonist monoclonal antibodies (mAbs) with the capacity to block the CLEC-1/CLEC-1L interaction. We discovered that antagonist CLEC-1 mAbs, but not non-antagonist CLEC-1 control mAbs, increase the phagocytosis of CLEC-1L-positive human tumor cells by human TGFβ-polarized DCs but also of CLEC-1L-positive opsonized tumor cells (eg: Raji cells opsonized with anti-CD20 Rituximab) by human macrophages. Altogether, these data indicate that tumor cells inhibit myeloid cells phagocytosis through CLEC-1 and that antagonists of the CLEC-1/CLEC-1L novel myeloid checkpoint pathway constitute a novel cancer immunotherapy approach synergistic with chemotherapy. Citation Format: Vanessa Gauttier, Marion Drouin, Javier Saenz, Bérangère Evrard, Caroline Mary, Géraldine Teppaz, Ariane Desalle, Virginie Thépénier, Emmanuelle Wilhelm, Nicolas Poirier, Elise Chiffoleau. CLEC-1 is a novel myeloid immune checkpoint for cancer immunotherapy controlling damaged and tumor cells phagocytosis [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3259.

Abstract 3423: CLEC-1 suppress dendritic cell antigen presentation and is a novel myeloid immune checkpoint target for cancer immunotherapy

Abstract Recent literature demonstrates that tumor hijacks physiological mechanisms of C-type lectin receptors (CLRs) that normally restrain immune cell-mediated tissue damage to suppress myeloid cell activation and promotes immune escape. We previously demonstrated that the orphan CLR CLEC-1 expressed by myeloid cells, is enhanced by the immunosuppressive cytokine TGFβ and tempers downstream CD4+ Th1 and Th17 responses following sterile inflammation. Interestingly, we observed a high CLEC-1 expression by human CD11b+ myeloid cells from ovarian ascites and from in-vitro generated pro-tumoral M2 macrophages. Moreover, open-source data show that Clec1a is highly expressed by XCR1-expressing mouse dendritic cells (DC). As these cDC1 are specialized in cross-presentation of dead cell-associated antigens, we evaluated whether CLEC-1 could be a receptor of damaged cells and regulate anti-tumor immunity. Interestingly, we observed that both human and mouse CLEC-1 bind specifically to secondary necrotic cells. No binding was observed with viable, primary necrotic or with early apoptotic cells suggesting that the ligand(s) of CLEC-1 correspond(s) to Damage-Associated Molecular Pattern(s) induced by the cell death process. Then, to test if CLEC-1 in DCs influences cross-presentation of dead cell-associated antigens, we immunized WT and Clec1a KO mice with OVA-loaded dead cells and co-injected OVA-specific TCR transgenic CD8+ OT-I T cells. Impressively, we observed an increased proliferation of CD8+ T cells in the absence of CLEC-1, demonstrating that CLEC-1 in DCs tempers cross-presentation of dead cell-associated antigens. Interestingly, we observed in a model of subcutaneous MC38 colon carcinoma that the combination of the absence of CLEC-1 with cytotoxic and immunogenic chemotherapy (cyclophosphamide), reduces significantly the tumor growth and cures most of the mice. Similarly, in orthotopic models of mesothelioma (AK7) or hepatocarcinoma (Hepa1.6), we observed a prolongation of survival of Clec1a KO mice. Mechanistically, this was associated with an increase of effector CD69+ and of central memory CD44hi CD62Lhi CCR7hi CD4+ and CD8+ T cells in both liver and spleen. Furthermore, we observed a decrease in myeloid-derived suppressor cells. Besides, macrophages display a more mature phenotype relative to MHC class II expression. In addition, transcriptomics analysis performed in tumor-bearing liver of Clec1a KO mice revealed a profound decrease in CSF1 expression suggesting a defect in immunosuppressive myeloid cell recruitment. Moreover, we observed an increase in CCL17 and CCL7 expression, both known to activate CD8+ T cells through DCs and to enhance anti-tumor immunity. Taken together, these results suggest that CLEC-1 in DCs by acting as sensor of cell damage tempers immune response and represents a novel pharmacological target for cancer immunotherapy. Citation Format: Marion Drouin, Javier Saenz, Bérangère Evrard, Vanessa Gauttier, Géraldine Teppaz, Maria-Dolores Lopez-Robles, Cédric Louvet, Nicolas Poirier, Elise Chiffoleau. CLEC-1 suppress dendritic cell antigen presentation and is a novel myeloid immune checkpoint target for cancer immunotherapy [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3423.

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.

Upregulation of miR‐210‐5p in Systemic Lupus Erythematosus Impairs Silent Clearance of Dead Cell Remnants

Deficiency of dead cell clearance plays a significant contributing factor in the progression of Systemic Lupus Erythematosus (SLE). While several studies support macrophage‐mediated innate immune response for SLE development, the mechanism about “silent” clearance by polarized monocyte/macrophage populations in SLE still needs to be elucidated. By analyzing miRNA level, the expression of miR‐210‐5p from peripheral blood mononuclear cells (PBMCs) in patients with active SLE (n=17) was higher than that in healthy controls (n=16). We further used macrophage‐like THP‐1 as cell models to delineate the status of miR‐210‐5p upregulation on silent clearance and immune response. By miR‐210‐5p mimic transfection, differentiation and phagocytosis analysis, miR‐210‐5p‐overexpression increased accumulation of secondary necrotic cells (SNECs) in THP‐1 cells. ROS production of miR‐210‐5p‐overexpressed macrophage‐like THP‐1 cells was decreased as compared with that in negative controls upon secondary necrotic cells (SNECs) or H2O2 treatment by DCF‐DA staining. Impaired SNECs clearance in miR‐210‐5p‐overexpressed THP‐1 cells was mediated by a decrease in superoxide production through NOX pathway. These findings support the notion that increased miR‐210‐5p in SLE is, in part, due to decrease the NOX‐mediated redox signaling, which affects impaired dead cell clearance and immune response.

NOX2 mediates quiescent handling of dead cell remnants in phagocytes

The phagocyte NADPH oxidase (the NOX2 complex) generates superoxide, the precursor to reactive oxygen species (ROS). ROS possess both antimicrobial and immunoregulatory function. Inactivating mutations in alleles of the NOX2 complex cause chronic granulomatous disease (CGD), characterized by an enhanced susceptibility to infections and autoimmune diseases such as Systemic lupus erythematosus (SLE). The latter is characterized by insufficient removal of dead cells, resulting in an autoimmune response against components of the cell's nucleus when non-cleared apoptotic cells lose their membrane integrity and present autoantigenic molecules in an inflammatory context. Here we aimed to shed light on the role of the NOX2 complex in handling of secondary necrotic cells (SNECs) and associated consequences for inflammation and autoimmunity during lupus.We show that individuals with SLE and CGD display accumulation of SNECs in blood monocytes and neutrophils. In a CGD phenotypic mouse strain (Ncf1** mice) build-up of SNECs in Ly6CHI blood monocytes was connected with a delayed degradation of the phagosomal cargo and accompanied by production of inflammatory mediators. Treatment with H2O2 or activators of ROS-formation reconstituted phagosomal abundance of SNECs to normal levels. Induction of experimental lupus further induced increased antibody-dependent uptake of SNECs into neutrophils. Lupus-primed Ncf1** neutrophils took up more SNECs than wild type neutrophils, whereas SNEC-accumulation in regulatory Ly6C−/LO monocytes was lower in Ncf1**mice. We deduce that the inflammatory rerouting of immune-stimulatory necrotic material into inflammatory phagocyte subsets contributes to the connection between low ROS production by the NOX2 complex and SLE.

Gasdermin E Does Not Limit Apoptotic Cell Disassembly by Promoting Early Onset of Secondary Necrosis in Jurkat T Cells and THP-1 Monocytes.

During the progression of necroptosis and pyroptosis, the plasma membrane will become permeabilized through the activation of mixed lineage kinase domain like pseudokinase (MLKL) or gasdermin D (GSDMD), respectively. Recently, the progression of apoptotic cells into secondary necrotic cells following membrane lysis was shown to be regulated by gasdermin E (GSDME, or DFNA5), a process dependent on caspase 3-mediated cleavage of GSDME. Notably, GSDME was also proposed to negatively regulate the disassembly of apoptotic cells into smaller membrane-bound vesicles known as apoptotic bodies (ApoBDs) by promoting earlier onset of membrane permeabilisation. The presence of a process downstream of caspase 3 that would actively drive cell lysis and limit cell disassembly during apoptosis is somewhat surprising as this could favor the release of proinflammatory intracellular contents and hinder efficient clearance of apoptotic materials. In contrast to the latter studies, we present here that GSDME is not involved in regulating secondary necrosis in human T cells and monocytes, and also unlikely in epithelial cells. Furthermore, GSDME is evidently not a negative regulator of apoptotic cell disassembly in our cell models. Thus, the function of GSDME in regulating membrane permeabilization and cell disassembly during apoptosis may be more limited.

Autoantibodies Recognizing Secondary NEcrotic Cells Promote Neutrophilic Phagocytosis and Identify Patients With Systemic Lupus Erythematosus.

Deficient clearance of apoptotic cells reportedly contributes to the etiopathogenesis of the autoimmune disease systemic lupus erythematosus (SLE). Based on this knowledge, we developed a highly specific and sensitive test for the detection of SLE autoantibodies (AAb) utilizing secondary NEcrotic cell (SNEC)-derived material as a substrate. The goal of the present study was to validate the use of SNEC as an appropriate antigen for the diagnosis of SLE in large cohort of patients. We confirmed the presence of apoptotically modified autoantigens on SNEC (dsDNA, high mobility group box 1 protein, apoptosis-associated chromatin modifications, e.g., histones H3-K27-me3; H2A/H4 AcK8,12,16; and H2B-AcK12). Anti-SNEC AAb were measured in the serum of 155 patients with SLE, 89 normal healthy donors (NHD), and 169 patients with other autoimmune connective tissue diseases employing SNEC-based indirect enzyme-linked immunosorbent assay (SNEC ELISA). We compared the test performance of SNEC ELISA with the routine diagnostic tests dsDNA Farr radioimmunoassay (RIA) and nucleosome-based ELISA (anti-dsDNA-NcX-ELISA). SNEC ELISA distinguished patients with SLE with a specificity of 98.9% and a sensitivity of 70.6% from NHD clearly surpassing RIA and anti-dsDNA-NcX-ELISA. In contrast to the other tests, SNEC ELISA significantly discriminated patients with SLE from patients with rheumatoid arthritis, primary anti-phospholipid syndrome, spondyloarthropathy, psoriatic arthritis, and systemic sclerosis. A positive test result in SNEC ELISA significantly correlated with serological variables and reflected the uptake of opsonized SNEC by neutrophils. This stresses the relevance of SNECs in the pathogenesis of SLE. We conclude that SNEC ELISA allows for the sensitive detection of pathologically relevant AAb, enabling its diagnostic usage. A positive SNEC test reflects the opsonization of cell remnants by AAb, the neutrophil recruitment to tissues, and the enhancement of local and systemic inflammatory responses.

Mer receptor tyrosine kinase signaling prevents TLR-mediated self-ligand sensing and aberrant selection in germinal centers

Abstract Mer receptor tyrosine kinase (Mer) signaling maintains immune tolerance by clearing apoptotic cells and inducing immunoregulatory signaling cascades. We previously showed that Mer-deficient mice on a mixed B6 and 129 background (B6129F2.Mer−/−) have increased germinal center (GC) responses, T cell activation, and apoptotic cell accumulation within GCs. Whereas B6129F2.Mer−/− mice accumulate apoptotic cells in GCs, B6.Mer−/− mice do not accumulate apoptotic cells, resembling B6 mice. Therefore, we used these two models to mechanistically separate the effects of debris released from secondary necrotic cells as a result of apoptotic cell accumulation from those driven by the loss of immune regulation by Mer. Through the study of global and cell-type intrinsic TLR and MyD88 deficiency in B6129F2.Mer−/− mice, we found that GC B cell-intrinsic sensing of self-RNA, but not self-DNA, released from secondary necrotic cells drives self-antigen induced GC responses. While self-ligands drive the magnitude of GC response, the loss of Mer immunoregulatory signaling bolsters antigen presentation and APC-intrinsic TLR activation drives pro-inflammatory cytokine production. Further, the loss of Mer immunoregulatory signaling accelerates the onset of autoimmunity when B6.Mer−/− mice are crossed to autoimmune-prone B6.Sle1b mice (Sle1b.Mer−/−) due to the retention of autoreactive B cells in GCs. Collectively, Mer prevents the cell-intrinsic activation of GC B cells and APCs to regulate the magnitude of GC response and T cell activation, in addition to regulating GC selection events to prevent the development of autoimmunity.

Macrophages transfer antigens to dendritic cells by releasing exosomes containing dead-cell-associated antigens partially through a ceramide-dependent pathway to enhance CD4(+) T-cell responses.

Defects in rapid clearance of apoptotic cells lead to an accumulation of dead cells (late apoptotic or secondary necrotic cells), which results in an aberrant immune response. However, little is known about whether and how macrophages (Mφs) cooperate with dendritic cells (DCs) in the presentation of dead-cell-associated antigens in this process. By transferring high numbers of dead cells to mimic a failure of apoptotic cell clearance in vivo, we found that Mφs and neutrophils were the predominant phagocytes in the uptake of dead cells in the spleen. Moreover, both Mφs and DCs were required for an optimal CD4(+) T-cell response triggered by dead-cell-associated antigens. Importantly, although Mφs alone had a poor capacity for antigen presentation, they could transfer phagocytosed antigens to DCs for potent antigen presentation to enhance T-cell responses. Finally, we found that exosomes released from Mφs acted as a transmitter to convey antigens to DCs partially in a ceramide-dependent manner, since treatment with the neutral sphingomyelinase inhibitor GW4869 and spiroepoxide resulted in a significant reduction of T-cell proliferation in vitro and in vivo. These findings point to a novel pathway of cross-talk between Mφs and DCs, which will be helpful to explain possible mechanisms for autoimmune diseases characterized by increased rates of apoptosis.

Abstract 4037: Enhancement of efferocytosis by rhoA kinase inhibitor following doxorubicin treatment synergistically reduces tumor growth

Abstract Massive dying of tumor cells after chemotherapy may promote chronic inflammation which favors tumor growth by uncleared secondary necrotic cells. Given that defects in apoptotic cell clearance have been linked with various inflammatory diseases and autoimmunity, the prompt removal of apoptotic cells by phagocytes is important for anti-tumor immunity. To improve the anti-tumor effect of doxorubicin, we investigated the role of RhoA-kinase (ROCK) inhibitors on efferocytosis by macrophages, which is important for anti-tumor immunity and tissue regeneration. Treatment of bone marrow-derived macrophages (BMDMs) with ROCK inhibitor increased efferocytosis of dying tumor cells by doxorubicin in vitro. ROCK-blockade after doxorubicin treatment group showed effective inhibition of tumor growth and also T cell-mediated elimination of immunogenic tumors more than the doxorubicin monotherapy group in established tumor models. Furthermore, we also confirmed that macrophage depletion abrogated the therapeutic effect of ROCK inhibitor after doxorubicin treatment on tumor growth. Taken together, our findings indicate that ROCK inhibitor following doxorubicin treatment reduced tumor growth via efferocytosis enhancement. Citation Format: Gihoon Nam, In-San Kim. Enhancement of efferocytosis by rhoA kinase inhibitor following doxorubicin treatment synergistically reduces tumor growth. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4037.

Skewing of peritoneal resident macrophages toward M1-like is involved in enhancement of inflammatory responses induced by secondary necrotic neutrophils in aged mice

Secondary necrotic cells, which are generated if apoptotic cells are incompletely cleared, induce severe inflammatory responses involving MIP-2 production and subsequent neutrophil infiltration. Recently, we showed that the phagocytic capacity of peritoneal resident macrophages from wild type (WT) aged mice as well as SMP30−/− mice fed a VC-limited diet as to secondary necrotic cells was reduced as compared with that in young mice, and that the inflammatory responses induced were stronger than those in young mice, presumably because of the delay in removal of secondary necrotic cells in aged mice. In this study, we investigated why MIP-2 production was increased in aged mice upon injection of secondary necrotic cells and why the phagocytic capacity of peritoneal resident macrophages from aged mice was reduced. When cocultured with secondary necrotic cells, the peritoneal resident macrophages from both types of aged mice significantly produced MIP-2 even in the absence of IFN-γ, whereas MIP-2 production by macrophages from WT young mice required IFN-γ. The peritoneal resident macrophages from both types of aged mice expressed CD40, a M1 macrophage marker, as in the case of M1 macrophages, which were obtained by treatment of macrophages from WT young mice with IFN-γ and LPS. Furthermore, M1 macrophages exhibited less phagocytic capacity as to secondary necrotic cells than non-treated macrophages. These results suggest that the phenotype of peritoneal resident macrophages is skewed toward M1-like in aged mice and that such skewing toward M1-like is involved in enhancement of inflammatory responses induced by secondary necrotic neutrophils in aged mice.

Clearance Deficiency and Cell Death Pathways: A Model for the Pathogenesis of SLE.

Alterations of cell death pathways, including apoptosis and the neutrophil specific kind of death called NETosis, can represent a potential source of autoantigens. Defects in the clearance of apoptotic cells may be responsible for the initiation of systemic autoimmunity in several chronic inflammatory diseases, including systemic lupus erythematosus (SLE). Autoantigens are released mainly from secondary necrotic cells because of a defective clearance of apoptotic cells or an inefficient degradation of DNA-containing neutrophil extracellular traps (NETs). These modified autoantigens are presented by follicular dendritic cells to autoreactive B cells in germinal centers of secondary lymphoid organs. This results in the loss of self-tolerance and production of autoantibodies, a unifying feature of SLE. Immune complexes (IC) are formed from autoantibodies bound to uncleared cellular debris in blood or tissues. Clearance of IC by blood phagocytes, macrophages, and dendritic cells leads to proinflammatory cytokine secretion. In particular, plasmacytoid dendritic cells produce high amounts of interferon-α upon IC uptake, thereby contributing to the interferon signature of patients with SLE. The clearance of antinuclear IC via Fc-gamma receptors is considered a central event in amplifying inflammatory immune responses in SLE. Along with this, the accumulation of cell remnants represents an initiating event of the etiology, while the subsequent generation of autoantibodies against nuclear antigens (including NETs) results in the perpetuation of inflammation and tissue damage in patients with SLE. Here, we discuss the implications of defective clearance of apoptotic cells and NETs in the development of clinical manifestations in SLE.

Sialylation of anti-histone immunoglobulin G autoantibodies determines their capabilities to participate in the clearance of late apoptotic cells.

The Fc portion of immunoglobulin (Ig)G harbours a single glycosylation site. Glycan sialylation is critical for structure and for certain effector functions of IgG. Anti-histone IgG of patients with systemic lupus erythematosus is reportedly responsible for the recruitment of polymorphonuclear cells (PMN) to the clearance of apoptotic cells. Autoantibodies decorating secondary necrotic cells (SNEC) induce proinflammatory responses after activation of blood-borne phagocytes. Analysing the sialylation status of affinity-purified anti-histone IgG in patients with systemic lupus erythematosus (SLE), we demonstrated that the anti-histone IgG was contained preferentially in the non-sialylated fraction. In functional ex-vivo phagocytosis studies, non-sialylated anti-SNEC IgG directed SNEC preferentially into PMN but did not change their cytokine secretion profiles. In contrast, sialylated IgG reduced the phagocytosis by monocytes of SNEC. Moreover, the sialylated anti-SNEC IgG was not simply anti-inflammatory, but switched the cytokine secretion profiles from interleukin (IL)-6/IL-8 to tumour necrosis factor (TNF)-α/IL-1β. Here we describe how different sialylation statuses of IgG autoantibodies contribute to the complex inflammatory network that regulates chronic inflammation.

Suppression of macrophage-mediated phagocytosis of apoptotic cells by soluble β-glucan due to a failure of PKC-βII translocation

If apoptotic cells are not removed efficiently, they may proceed to the stage of secondary necrosis, which would cause inflammation. Therefore, identification of cause(s) and agent(s) for down-modulating phagocytosis of apoptotic cells would help understand the pathologies. In this study we found that macrophage-mediated phagocytosis of apoptotic cells was suppressed by both soluble and particulate β-glucan. This suppression was not observed when secondary necrotic cells were used. The adhesion of apoptotic cells to macrophages was not suppressed by soluble β-glucan, suggesting that soluble β-glucan suppresses phagocytosis at a post-adhesion step. Experiments involving PKC inhibitors suggested that PKC-βII is required for phagocytosis of apoptotic cells but not secondary necrotic ones by macrophages. Translocation of GFP-PKC-βII from the cytoplasm to membranes occurred upon interaction with apoptotic cells but not secondary necrotic ones. Such translocation was inhibited by soluble β-glucan. Overall, this study suggests that suppression of macrophage-mediated phagocytosis of apoptotic cells by soluble β-glucan is due to a failure of PKC-βII translocation.

A subset of patients with systemic lupus erythematosus fails to degrade DNA from multiple clinically relevant sources.

IntroductionPatients with systemic lupus erythematosus (SLE) have a decreased ability to clear cell remnants and multiple deficiencies in the ability to degrade cellular chromatin have been linked to the disease. Since the discovery of neutrophil extracellular traps (NETs), a renewed interest has been sparked in this field of research with multiple studies reporting a decreased ability of patients with SLE to degrade NETs. In this study we extend these findings by investigating the ability of patients with SLE to degrade chromatin from multiple clinically relevant sources.MethodsWe use flow cytometry in combination with NET degradation and DNA zymogram assays to investigate the ability of sera from SLE patients to degrade chromatin from three different sources of DNA such as NETs, apoptotic and necrotic cells. This ability was further associated with clinical manifestations.ResultsWe found that 61 % of the patients had an affected degradation of at least one chromatin source. Further, degradation of NETs correlated with degradation of chromatin from secondary necrotic cells but not with degradation of chromatin from primary necrotic cells. Patients who fail to degrade several forms of DNA more often display anti-nuclear and nephritic involvement whereas this is not observed in patients with decreased ability to degrade chromatin from primary necrotic cells.ConclusionsThe majority of patients with SLE has a decreased ability to degrade chromatin from clinically relevant sources. This decreased ability is further reflected in their clinical presentation.

Attenuated phagocytosis of secondary necrotic neutrophils by macrophages in aged and SMP30 knockout mice.

Secondary necrotic cells generated in vivo induce inflammatory responses; for example, the production of macrophage inflammatory protein-2 (MIP-2) and subsequent infiltration of neutrophils. The aim of the present study was to elucidate the effect of aging on the phagocytosis of secondary necrotic cells and the inflammatory responses by using either wild-type (WT) young mice, WT aged mice or senescence-accelerated mice (SMP30(-/-) mice). The phagocytosis of secondary necrotic neutrophils with resident macrophage from either WT young mice, WT aged mice or SMP30(-/-) mice was examined by coculturing macrophages with secondary necrotic neutrophils in vitro. To investigate the inflammatory response induced by secondary necrotic cells, time-dependent infiltration of neutrophils and production of MIP-2 were determined in the peritoneal cavity on the injection of secondary necrotic cells. The phagocytosis of secondary necrotic cells by macrophages from WT aged and SMP30(-/-) mice was significantly reduced as compared with that by macrophages from WT young mice. On peritoneal injection of secondary necrotic cells, the peak time of neutrophil infiltration was earlier in SMP30(-/-) mice than in WT young mice. The number of neutrophils in SMP30(-/-) mice at the peak time was also greater than that in WT young mice. Our findings showed that the phagocytosis of secondary necrotic cells was attenuated in aged mice and SMP30(-/-) mice, and that the MIP-2 production was enhanced and subsequently neutrophil infiltration was exaggerated on peritoneal injection of secondary necrotic cells into those mice.

The role of dead cell clearance in the etiology and pathogenesis of systemic lupus erythematosus: dendritic cells as potential targets

Overwhelming apoptosis combined with a deficiency in clearing apoptotic cells is thought to be an important etiopathogenic event in the autoimmune disease systemic lupus erythematosus (SLE). Lazy macrophages, complement or DNase I deficiency as well as insufficient natural IgM might be important factors leading to such a clearance deficiency. A defective clearance of apoptotic cells leads to the activation and maturation of plasmacytoid and myeloid dendritic cells (DCs) by material derived from secondary necrotic cells carrying modified autoantigens. This results in the presentation of autoantigens to autoreactive T and B cells in an immunogenic manner, thereby leading to autoantibody production, chronic inflammation and severe tissue damage. Since DC activation and IFN-α production by plasmacytoid dendritic cells play a critical role in the course of SLE pathogenesis, therapeutic intervention to end this vicious cycle might be a promising approach for treating the disease.