Efferocytosis in multisystem diseases (Review).

Efferocytosis, the phagocytosis of apoptotic cells performed by both specialized phagocytes (such as macrophages) and non­specialized phagocytes (such as epithelial cells), is involved in tissue repair and homeostasis. Effective efferocytosis prevents secondary necrosis, terminates inflammatory responses, promotes self­tolerance and activates pro­resolving pathways to maintain homeostasis. When efferocytosis is impaired, apoptotic cells that could not be cleared in time aggregate, resulting in the necrosis of apoptotic cells and release of pro­inflammatory factors. In addition, defective efferocytosis inhibits the intracellular cholesterol reverse transportation pathways, which may lead to atherosclerosis, lung damage, non­alcoholic fatty liver disease and neurodegenerative diseases. The uncleared apoptotic cells can also release autoantigens, which can cause autoimmune diseases. Cancer cells escape from phagocytosis via efferocytosis. Therefore, new treatment strategies for diseases related to defective efferocytosis are proposed. This review illustrated the mechanisms of efferocytosis in multisystem diseases and organismal homeostasis and the pathophysiological consequences of defective efferocytosis. Several drugs and treatments available to enhance efferocytosis are also mentioned in the review, serving as new evidence for clinical application.

C-Reactive Protein Enhances IgG-Mediated Cellular Destruction Through IgG-Fc Receptors in vitro.

Antibody-mediated blood disorders ensue after auto- or alloimmunization against blood cell antigens, resulting in cytopenia. Although the mechanisms of cell destruction are the same as in immunotherapies targeting tumor cells, many factors are still unknown. Antibody titers, for example, often do not strictly correlate with clinical outcome. Previously, we found C-reactive protein (CRP) levels to be elevated in thrombocytopenic patients, correlating with thrombocyte counts, and bleeding severity. Functionally, CRP amplified antibody-mediated phagocytosis of thrombocytes by phagocytes. To investigate whether CRP is a general enhancer of IgG-mediated target cell destruction, we extensively studied the effect of CRP on in vitro IgG-Fc receptor (FcγR)-mediated cell destruction: through respiratory burst, phagocytosis, and cellular cytotoxicity by a variety of effector cells. We now demonstrate that CRP also enhances IgG-mediated effector functions toward opsonized erythrocytes, in particular by activated neutrophils. We performed a first-of-a-kind profiling of CRP binding to all human FcγRs and IgA-Fc receptor I (FcαRI) using a surface plasmon resonance array. CRP bound these receptors with relative affinities of FcγRIa = FcγRIIa/b = FcγRIIIa > FcγRIIIb = FcαRI. Furthermore, FcγR blocking (in particular FcγRIa) abrogated CRP's ability to amplify IgG-mediated neutrophil effector functions toward opsonized erythrocytes. Finally, we observed that CRP also amplified killing of breast-cancer tumor cell line SKBR3 by neutrophils through anti-Her2 (trastuzumab). Altogether, we provide for the first time evidence for the involvement of specific CRP-FcγR interactions in the exacerbation of in vitro IgG-mediated cellular destruction; a trait that should be further evaluated as potential therapeutic target e.g., for tumor eradication.

Simple propagation method for resident macrophages by co-culture and subculture, and their isolation from various organs.

BACKGROUND: Resident macrophages (Mø) originating from yolk sac Mø and/or foetal monocytes colonise tissues/organs during embryonic development. They persist into adulthood by self-renewal at a steady state, independent of adult monocyte inputs, except for those in the intestines and dermis. Thus, many resident Mø can be propagated in vitro under optimal conditions; however, there are no specific in vitro culture methods available for the propagation of resident Mø from diverse tissues/organs. RESULTS: We provided a simple method for propagating resident Mø derived from the liver, spleen, lung, and brain of ICR male mice by co-culture and subculture along with the propagation of other stromal cells of the respective organs in standard culture media and successfully demonstrated the propagation of resident Mø colonising these organs. We also proposed a simple method for segregating Mø from stromal cells according to their adhesive property on bacteriological Petri dishes, which enabled the collection of more than 97.6% of the resident Mø from each organ. Expression analyses of conventional Mø markers by flow cytometry showed similar expression patterns among the Mø collected from the organs. CONCLUSION: This is the first study to clearly provide a practical Mø propagation method applicable to resident Mø of diverse tissues and organs. Thus, this novel practical Mø propagation method can offer broad applications for the use of resident Mø of diverse tissues and organs.

p16INK4A-expressing mesenchymal stromal cells restore the senescence-clearance-regeneration sequence that is impaired in chronic muscle inflammation.

BACKGROUND: The therapeutic benefits of mesenchymal stromal cells (MSCs) include treatment of chronic inflammation. However, given the short-lived engraftment of these cells in vivo, their therapeutic efficacy remains mysterious. Transient induction of cellular senescence contributes to activation of immune cells, which promotes clearance of damaged cells during tissue remodelling. This may occur in tissue-resident mesenchymal progenitor cells during regeneration. Elucidation of the role of senescence in tissue-resident mesenchymal progenitor cells during regeneration would provide insight into the profile of therapeutic MSCs for treatment of chronic inflammatory disease. METHODS: We evaluated multipotent mesenchymal progenitor cells, termed fibro/adipogenic progenitors (FAPs), and immune cells in acute muscle injury (AMI) model mice and mice with myosin-induced experimental autoimmune myositis, a model of chronic inflammatory myopathy (CIM). Human bone marrow MSCs were optimised for the treatment of CIM using placental extract. FINDING: FAPs in AMI transiently expressed p16INK4A on days 1 and 2 after injury and recruited phagocytic immune cells, whereas in CIM, p16INK4A expression in FAPs was low. Cellular senescence occurs during the natural maturation of the placenta. Therefore, we used human placental extract to induce p16INK4A expression in therapeutic human bone marrow MSCs in culture. Treatment of CIM with p16INK4A-expressing MSCs promoted tissue remodelling by transiently increasing the abundance of engrafted MSCs, inducing cellular senescence in innate FAPs, and recruiting phagocytic immune cells. INTERPRETATION: MSCs may exert their effect by remodelling the chronic inflammatory environment via senescence-related regenerative processes.

Immune Checkpoint Inhibition Overcomes ADCP-Induced Immunosuppression by Macrophages.

Antibody-dependent cellular cytotoxicity (ADCC) and antibody-dependent cellular phagocytosis (ADCP) critically contribute to the efficacy of anti-tumor therapeutic antibodies. We report here an unexpected finding that macrophages after ADCP inhibit NK cell-mediated ADCC and T cell-mediated cytotoxicity in breast cancers and lymphomas. Mechanistically, AIM2 is recruited to the phagosomes by FcγR signaling following ADCP and activated by sensing the phagocytosed tumor DNAs through the disrupted phagosomal membrane, which subsequently upregulates PD-L1 and IDO and causes immunosuppression. Combined treatment with anti-HER2 antibody and inhibitors of PD-L1 and IDO enhances anti-tumor immunity and anti-HER2 therapeutic efficacy in mouse models. Furthermore, neoadjuvant trastuzumab therapy significantly upregulates PD-L1 and IDO in the tumor-associated macrophages (TAMs) of HER2+ breast cancer patients, correlating with poor trastuzumab response. Collectively, our findings unveil a deleterious role of ADCP macrophages in cancer immunosuppression and suggest that therapeutic antibody plus immune checkpoint blockade may provide synergistic effects in cancer treatment.

slan+ Monocytes and Macrophages Mediate CD20-Dependent B-cell Lymphoma Elimination via ADCC and ADCP.

Terminal tissue differentiation and function of slan+ monocytes in cancer is largely unexplored. Our recent studies demonstrated that slan+ monocytes differentiate into a distinct subset of dendritic cells (DC) in human tonsils and that slan+ cells colonize metastatic carcinoma-draining lymph nodes. Herein, we report by retrospective analysis of multi-institutional cohorts that slan+ cells infiltrate various types of non-Hodgkin lymphomas (NHL), particularly the diffuse large B-cell lymphoma (DLBCL) group, including the most aggressive, nodal and extranodal, forms. Nodal slan+ cells displayed features of either immature DC or macrophages, in the latter case ingesting tumor cells and apoptotic bodies. We also found in patients with DLBCL that peripheral blood slan+ monocytes, but not CD14+ monocytes, increased in number and displayed highly efficient rituximab-mediated antibody-dependent cellular cytotoxicity, almost equivalent to that exerted by NK cells. Notably, slan+ monocytes cultured in conditioned medium from nodal DLBCL (DCM) acquired a macrophage-like phenotype, retained CD16 expression, and became very efficient in rituximab-mediated antibody-dependent cellular phagocytosis (ADCP). Macrophages derived from DCM-treated CD14+ monocytes performed very efficient rituximab-mediated ADCP, however, using different FcγRs from those used by slan+ macrophages. Our observations shed new light on the complexity of the immune microenvironment of DLBCL and demonstrate plasticity of slan+ monocytes homing to cancer tissues. Altogether, data identify slan+ monocytes and macrophages as prominent effectors of antibody-mediated tumor cell targeting in patients with DLBCL.Significance: slan+ monocytes differentiate into macrophages that function as prominent effectors of antibody-mediated tumor cell targeting in lymphoma.Graphical Abstract: http://cancerres.aacrjournals.org/content/canres/78/13/3544/F1.large.jpg Cancer Res; 78(13); 3544-59. ©2018 AACR.

Dead cell phagocytosis and innate immune checkpoint.

The human body loses several billions of cells daily. When cells die in vivo, the corpse of each dead cell is immediately cleared. Specifically, dead cells are efficiently recognized and cleared by multiple types of neighboring phagocytes. Early research on cell death focused more on molecular mechanisms of cell death regulation while the cellular corpses were merely considered cellular debris. However, it has come to light that various biological stimuli following cell death are important for immune regulation. Clearance of normal dead cells occurs silently in immune tolerance. Exogenous or mutated antigens of malignant or infected cells can initiate adaptive immunity, thereby inducing immunogenicity by adjuvant signals. Several pathogens and cancer cells have strategies to limit the adjuvant signals and escape immune surveillance. In this review, we present an overview of the mechanisms of dead cell clearance and its immune regulations. [BMB Reports 2017; 50(10): 496-503].

Efferocytosis is impaired in Gaucher macrophages.

Gaucher disease, the inherited deficiency of lysosomal glucocerebrosidase, is characterized by the presence of glucosylceramide-laden macrophages resulting from impaired digestion of aged erythrocytes or apoptotic leukocytes. Studies of macrophages from patients with type 1 Gaucher disease with genotypes N370S/N370S, N370S/L444P or N370S/c.84dupG revealed that Gaucher macrophages have impaired efferocytosis resulting from reduced levels of p67phox and Rab7. The decreased Rab7 expression leads to impaired fusion of phagosomes with lysosomes. Moreover, there is defective translocation of p67phox to phagosomes, resulting in reduced intracellular production of reactive oxygen species. These factors contribute to defective deposition and clearance of apoptotic cells in phagolysosomes, which may have an impact on the inflammatory response and contribute to the organomegaly and inflammation seen in patients with Gaucher disease.

Uncoupling complement C1s activation from C1q binding in apoptotic cell phagocytosis and immunosuppressive capacity.

Complement activation contributes to inflammation in many diseases, yet it also supports physiologic apoptotic cells (AC) clearance and its downstream immunosuppressive effects. The roles of individual complement components in AC phagocytosis have been difficult to dissect with artificially depleted sera. Using human in vitro systems and the novel antibody complement C1s inhibitor TNT003, we uncoupled the role of the enzymatic activation of the classical pathway from the opsonizing role of C1q in mediating a) the phagocytosis of early and late AC, and b) the immunosuppressive capacity of early AC. We found that C1s inhibition had a small impact on the physiologic clearance of early AC, leaving their immunosuppressive properties entirely unaffected, while mainly inhibiting the phagocytosis of late apoptotic/secondary necrotic cells. Our data suggest that C1s inhibition may represent a valuable therapeutic strategy to control classical pathway activation without causing significant AC accumulation in diseases without defects in AC phagocytosis.

Molecular Pathways: Activating T Cells after Cancer Cell Phagocytosis from Blockade of CD47 "Don't Eat Me" Signals.

Recent advances with immunotherapy agents for the treatment of cancer have provided remarkable, and in some cases, curative results. Our laboratory has identified CD47 as an important "don't eat me" signal expressed on malignant cells. Blockade of the CD47:SIRP-α axis between tumor cells and innate immune cells (monocytes, macrophages, and dendritic cells) increases tumor cell phagocytosis in both solid tumors (including, but not limited to, bladder, breast, colon, lung, and pancreatic) and hematologic malignancies. These phagocytic innate cells are also professional antigen-presenting cells (APC), providing a link from innate to adaptive antitumor immunity. Preliminary studies have demonstrated that APCs present antigens from phagocytosed tumor cells, causing T-cell activation. Therefore, agents that block the CD47:SIRP-α engagement are attractive therapeutic targets as a monotherapy or in combination with additional immune-modulating agents for activating antitumor T cells in vivo.

The matricellular protein CCN1 mediates neutrophil efferocytosis in cutaneous wound healing.

Neutrophil infiltration constitutes the first step in wound healing, although their timely clearance by macrophage engulfment, or efferocytosis, is critical for efficient tissue repair. However, the specific mechanism for neutrophil clearance in wound healing remains undefined. Here we uncover a key role for CCN1 in neutrophil efferocytosis by acting as a bridging molecule that binds phosphatidylserine, the 'eat-me' signal on apoptotic cells and integrins αvß3/αvß5 in macrophages to trigger efferocytosis. Both knockin mice expressing a mutant CCN1 that is unable to bind αvß3/αvß5 and mice with Ccn1 knockdown are defective in neutrophil efferocytosis, resulting in exuberant neutrophil accumulation and delayed healing. Treatment of wounds with CCN1 accelerates neutrophil clearance in both Ccn1 knockin mice and diabetic Lepr(db/db) mice, which suffer from neutrophil persistence and impaired healing. These findings establish CCN1 as a critical opsonin in skin injury and suggest a therapeutic potential for CCN1 in certain types of non-healing wounds.

Anti-C1q autoantibodies from active lupus nephritis patients could inhibit the clearance of apoptotic cells and complement classical pathway activation mediated by C1q in vitro.

Anti-C1q antibodies are prevalent in patients with active lupus nephritis and were found to be closely associated with renal involvement and predictive for a flare of nephritis. However, the pathogenesis of anti-C1q antibodies involved in human lupus nephritis remains unclear. C1q, which plays a key role in apoptotic cell and immune complex removal, is a very important functional molecule in the pathogenesis of SLE. The aim of this study was to investigate the influence of anti-C1q autoantibodies from active lupus nephritis patients on the bio-functions of C1q in vitro. We purified IgG autoantibodies against C1q from lupus nephritis patients, and found that they could recognize C1q bound on early apoptotic cells at 30 µg/ml, and could significantly decrease the phagocytosis by macrophages of early apoptotic cells opsonized by 50 µg/ml C1q in comparison with normal IgG. Levels of circulating immune complexes of the ten patients were measured by a circulating immune complexes (CIC)-C1q Enzyme Immunoassay Kit. Anti-C1q autoantibodies affinity purified by microtiter plates could significantly inhibit the deposition of C3c on CIC-C1q in a dose dependent manner in comparison with IgG from 10 healthy blood donors. The binding of opsonized immune complexes to RBCs was significantly inhibited by anti-C1q autoantibodies purified by microtiter plates in a dose dependent manner. Our observations suggest that serum anti-C1q autoantibodies from active lupus nephritis patients could interfere with some biological function of C1q in vitro.

Monocytes from Sjögren's syndrome patients display increased vasoactive intestinal peptide receptor 2 expression and impaired apoptotic cell phagocytosis.

Sjögren's syndrome (SS) is a chronic autoimmune disease characterized by salivary and lacrimal gland dysfunction. Clinical observations and results from animal models of SS support the role of aberrant epithelial cell apoptosis and immune homeostasis loss in the glands as triggering factors for the autoimmune response. Vasoactive intestinal peptide (VIP) promotes potent anti-inflammatory effects in several inflammatory and autoimmune disease models, including the non-obese diabetic (NOD) mouse model of SS. With the knowledge that VIP modulates monocyte function through vasoactive intestinal peptide receptors (VPAC) and that immune homeostasis maintenance depends strongly upon a rapid and immunosuppressant apoptotic cell clearance by monocytes/macrophages, in this study we explored VPAC expression on monocytes from primary SS (pSS) patients and the ability of VIP to modulate apoptotic cell phagocytic function and cytokine profile. Monocytes isolated from individual pSS patients showed an increased expression of VPAC2 subtype of VIP receptors, absent in monocytes from control subjects, with no changes in VPAC1 expression. VPAC2 receptor expression could be induced further with lipopolysaccharide (LPS) in pSS monocytes and VIP inhibited the effect. Moreover, monocytes from pSS patients showed an impaired phagocytosis of apoptotic epithelial cells, as evidenced by reduced engulfment ability and the failure to promote an immunosuppressant cytokine profile. However, VIP neither modulated monocyte/macrophage phagocytic function nor did it reverse their inflammatory profile. We conclude that monocytes from pSS patients express high levels of VPAC2 and display a deficient clearance of apoptotic cells that is not modulated by VIP.

Pyroptotic death storms and cytopenia.

For over two decades, we have embraced the cytokine storm theory to explain sepsis, severe sepsis and septic shock. The failure of numerous large-scale clinical trials, which aimed to treat sepsis by neutralizing inflammatory cytokines and LPS, indicates that alternative pathophysiological mechanisms are likely to account for sepsis and the associated immune suppression in patients with severe infection. Recent insights that extricate pyroptotic death from inflammatory cytokine production in vivo have highlighted a need to investigate the consequences of apoptotic and non-apoptotic death in contributing to cytopenia and immune suppression. In this review, we will focus on the biochemical and cellular mechanisms controlling pyroptosis, a Caspase-1/11 dependent form of cell death during infection.

Epstein-Barr virus present in T cells or B cells shows differential effects on hemophagocytic symptoms associated with outcome in T-cell lymphomas.

Non-specific peripheral (PTCL-NOS) and angioimmunoblastic T-cell lymphoma (AITL) frequently show Epstein-Barr virus (EBV) expression in lymphoma or bystander B cells. However, whether EBV localization affects clinicopathologic features is unclear. We correlated EBV localization with clinicopathologic findings in PTCL-NOS (n = 63) and AITL (n = 26). PTCL-NOS showed EBV+ in 41%, with 22% in lymphoma T cells (T-EBV) and 19% in bystander B cells (B-EBV), and more EBV+ cells in T-EBV cases (39.3% vs. 11.8%, p = 0.003). Compared to B-EBV cases, T-EBV PTCL-NOS had higher rates of type II EBV latency (p = 0.003), leukopenia (p = 0.020) and hemophagocytosis (p = 0.061), which predicted a poor outcome (p < 0.001). In contrast, 88% of AITLs were EBV+, exclusively in B cells. EBV+ cases showed lower rates of hemophagocytosis (p = 0.006), but this was insignificant for prognosis. Therefore, hemophagocytic symptoms in PTCL-NOS are much more tightly associated with T-EBV and carry poor prognoses. In contrast, hemophagocytosis in AITL is correlated with EBV-, but is not significant for outcome.

[Erythrophagocytosis: a new diagnostic marker of immunologic myocardial damage in ischemic heart disease].

Using data of epidemiological and clinical examination of women of reproductive and menopausal age we studied the processes of erythrophagocytosis (EF) in 46 women with ischemic heart disease (IHD) aged 20-59 years in comparison with a group of healthy individuals. We found that women with IHD had almost 10-fold increase of EF compared with healthy individuals. Therefore determination of EF could be used as laboratory test for detecting autoimmune component of IHD. We also found associations between identified immunological abnormalities and dyslipidemia, in particular elevation of low density lipoprotein cholesterol level which was more pronounced during menopause. EF can serve as an immunological marker of IHD in women.

Subcutaneous panniculitis-like T-cell lymphoma (SPTCL) with hemophagocytosis (HPS): successful treatment using high-dose chemotherapy (BFM-NHL & ALL-90) and autologous peripheral blood stem cell transplantation.

Subcutaneous panniculitis-like T-cell lymphoma (SPTCL) is a rare form of non-Hodgkin lymphoma, in which lymphoma cells infiltrate preferentially into subcutaneous adipose tissue. Although various treatment trials for SPTCL have been attempted, no standardized therapy has been established. Here, we report a case of α/ß(+) T-cell-phenotype SPTCL (SPTCL-AB) with hemophagocytosis (HPS) in a 14-year-old girl, who presented with low-grade fever, general fatigue and chest swelling. Laboratory examinations revealed leukocytopenia, and bone marrow aspiration cytology showed HPS. The diagnosis of SPTCL-AB was made by biopsy on the basis of thickened subcutaneous tissue in the chest wall. Following high-dose chemotherapy (HDT) of BFM-NHL & ALL-90, autologous peripheral blood stem cell transplantation (auto-PBSCT) was performed. The patient responded to the treatment and has remained asymptomatic for 2 years. Our results suggest that a combination of HDT of BFM-NHL & ALL-90 and auto-SCT treatment is effective for SPTCL associated with HPS.

Innate immune reactions in locally limited tonsillar cancer.

Tonsillar cancers often present as locally limited tumors but with cervical metastases. When the primary tumors of tonsillar cancers with cervical metastases are as small as clinically occult, the clinical features are diagnosed as primary-unknown cervical metastases. However, little is known as to why small tonsillar cancers establish cervical metastases. The aim of this study was to investigate a possibility that innate immune reactions might suppress the growth of tumors arising in the palatine tonsils, because the palatine tonsils contain various immune effector cells. Infiltration of natural killer (NK) cells and macrophages, which are major innate immune cells, in surgically removed tumors from patients with locally limited tonsillar cancers and tongue cancers was immunohistochemically studied by using anti-CD57 and anti-CD68 antibodies. Phagocytosis of the tumor cells by macrophages was also studied by dual immunofluorescence labeling. The number of infiltrating CD57+ NK cells and CD68+ macrophages was significantly increased in locally limited tonsillar cancers in comparison to normal tonsils and tongue cancers. The phagocytosis of tumor cells by CD68+ macrophages was observed significantly more frequently in tonsillar cancers than in tongue cancers. These results indicated that the innate immune reactions were more strongly induced in locally limited tonsillar cancers than in tongue cancers, and might therefore suppress the growth of primary tumors in palatine tonsils. The innate immune reactions against cancers in palatine tonsils were suggested to be one of the possible etiologies for the developing of primary-unknown cervical metastases.