The Presumptive Phosphatidylserine Receptor Is Dispensable for Innate Anti-inflammatory Recognition and Clearance of Apoptotic Cells

Abstract Background NK-4 has been used to promote wound healing since the early-1950s; however, the mechanism of action of NK-4 is unknown. In this study, we examined whether NK-4 exerts a regulatory effect on macrophages, which play multiple roles during wound healing from the initial inflammatory phase until the tissue regeneration phase. Results NK-4 treatment of THP-1 macrophages induced morphological features characteristic of classically-activated M1 macrophages, an inflammatory cytokine profile, and increased expression of the M1 macrophage-associated molecules CD38 and CD86. Interestingly, NK-4 augmented TNF-α production by THP-1 macrophages in combination with LPS, Pam3CSK4, or poly(I:C). Furthermore, NK-4 treatment enhanced THP-1 macrophage phagocytosis of latex beads. These results indicate that NK-4 drives macrophage polarization toward an inflammatory M1-like phenotype with increased phagocytic activity. Efferocytosis is a crucial event for resolution of the inflammatory phase in wound healing. NK-4-treated THP-1 macrophages co-cultured with apoptotic Jurkat E6.1 (Apo-J) cells switched from an M1-like phenotype to an M2-like phenotype, as seen in the inverted ratio of TNF-α to IL-10 produced in response to LPS. We identified two separate mechanisms that are involved in this phenotypic switch. First, recognition of phosphatidylserine molecules on Apo-J cells by THP-1 macrophages downregulates TNF-α production. Second, phagocytosis of Apo-J cells by THP-1 macrophages and activation of PI3K/Akt signaling pathway upregulates IL-10 production. Conclusion It is postulated that the phenotypic switch from a proinflammatory M1-like phenotype to an anti-inflammatory M2-like phenotype is dysregulated due to impaired efferocytosis of apoptotic neutrophils at the wound site. Our results demonstrate that NK-4 improves phagocytosis of apoptotic cells, suggesting its potential as a therapeutic strategy to resolve sustained inflammation in chronic wounds.

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Mesenchymal Stem Cells (MSCs) Coculture Protects [Ca2+]i Orchestrated Oxidant Mediated Damage in Differentiated Neurons In Vitro

Cells ◽

10.3390/cells7120250 ◽

2018 ◽

Vol 7 (12) ◽

pp. 250 ◽

Cited By ~ 9

Author(s):

Adel Alhazzani ◽

Prasanna Rajagopalan ◽

Zaher Albarqi ◽

Anantharam Devaraj ◽

Mohamed Hessian Mohamed ◽

...

Keyword(s):

Cell Death ◽

Transforming Growth Factor ◽

Neuronal Cells ◽

Neuronal Cell Death ◽

Neuronal Cell ◽

Apoptotic Cells ◽

Sequence Of Events ◽

Cox 2 ◽

Anti Inflammatory

Cell-therapy modalities using mesenchymal stem (MSCs) in experimental strokes are being investigated due to the role of MSCs in neuroprotection and regeneration. It is necessary to know the sequence of events that occur during stress and how MSCs complement the rescue of neuronal cell death mediated by [Ca2+]i and reactive oxygen species (ROS). In the current study, SH-SY5Y-differentiated neuronal cells were subjected to in vitro cerebral ischemia-like stress and were experimentally rescued from cell death using an MSCs/neuronal cell coculture model. Neuronal cell death was characterized by the induction of proinflammatory tumor necrosis factor (TNF)-α, interleukin (IL)-1β and -12, up to 35-fold with corresponding downregulation of anti-inflammatory cytokine transforming growth factor (TGF)-β, IL-6 and -10 by approximately 1 to 7 fold. Increased intracellular calcium [Ca2+]i and ROS clearly reaffirmed oxidative stress-mediated apoptosis, while upregulation of nuclear factor NF-B and cyclo-oxygenase (COX)-2 expressions, along with ~41% accumulation of early and late phase apoptotic cells, confirmed ischemic stress-mediated cell death. Stressed neuronal cells were rescued from death when cocultured with MSCs via increased expression of anti-inflammatory cytokines (TGF-β, 17%; IL-6, 4%; and IL-10, 13%), significantly downregulated NF-B and proinflammatory COX-2 expression. Further accumulation of early and late apoptotic cells was diminished to 23%, while corresponding cell death decreased from 40% to 17%. Low superoxide dismutase 1 (SOD1) expression at the mRNA level was rescued by MSCs coculture, while no significant changes were observed with catalase (CAT) and glutathione peroxidase (GPx). Interestingly, increased serotonin release into the culture supernatant was proportionate to the elevated [Ca2+]i and corresponding ROS, which were later rescued by the MSCs coculture to near normalcy. Taken together, all of these results primarily support MSCs-mediated modulation of stressed neuronal cell survival in vitro.

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Immunosuppression via adenosine receptor activation by adenosine monophosphate released from apoptotic cells

eLife ◽

10.7554/elife.02172 ◽

2014 ◽

Vol 3 ◽

Cited By ~ 40

Author(s):

Hiroshi Yamaguchi ◽

Toshihiko Maruyama ◽

Yoshihiro Urade ◽

Shigekazu Nagata

Keyword(s):

Adenosine Receptor ◽

Molecular Mechanisms ◽

Apoptotic Cell ◽

Adenosine Monophosphate ◽

Receptor Activation ◽

Death Process ◽

Apoptotic Cells ◽

Dependent Manner ◽

A2a Adenosine Receptor ◽

Anti Inflammatory

Apoptosis is coupled with recruitment of macrophages for engulfment of dead cells, and with compensatory proliferation of neighboring cells. Yet, this death process is silent, and it does not cause inflammation. The molecular mechanisms underlying anti-inflammatory nature of the apoptotic process remains poorly understood. In this study, we found that the culture supernatant of apoptotic cells activated the macrophages to express anti-inflammatory genes such as Nr4a and Thbs1. A high level of AMP accumulated in the apoptotic cell supernatant in a Pannexin1-dependent manner. A nucleotidase inhibitor and A2a adenosine receptor antagonist inhibited the apoptotic supernatant-induced gene expression, suggesting AMP was metabolized to adenosine by an ecto-5’-nucleotidase expressed on macrophages, to activate the macrophage A2a adenosine receptor. Intraperitoneal injection of zymosan into Adora2a- or Panx1-deficient mice produced high, sustained levels of inflammatory mediators in the peritoneal lavage. These results indicated that AMP from apoptotic cells suppresses inflammation as a ‘calm down’ signal.

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Persistence of apoptotic cells without autoimmune disease or inflammation in CD14−/− mice

The Journal of Cell Biology ◽

10.1083/jcb.200410057 ◽

2004 ◽

Vol 167 (6) ◽

pp. 1161-1170 ◽

Cited By ~ 96

Author(s):

Andrew Devitt ◽

Kate G. Parker ◽

Carol Anne Ogden ◽

Ceri Oldreive ◽

Michael F. Clay ◽

...

Keyword(s):

Autoimmune Disease ◽

Apoptotic Cell ◽

Apoptotic Cells ◽

Autoantibody Production ◽

Apoptotic Cell Clearance ◽

Cell Clearance ◽

Surface Receptor ◽

Anti Inflammatory

Interaction of macrophages with apoptotic cells involves multiple steps including recognition, tethering, phagocytosis, and anti-inflammatory macrophage responses. Defective apoptotic cell clearance is associated with pathogenesis of autoimmune disease. CD14 is a surface receptor that functions in vitro in the removal of apoptotic cells by human and murine macrophages, but its mechanism of action has not been defined. Here, we demonstrate that CD14 functions as a macrophage tethering receptor for apoptotic cells. Significantly, CD14−/− macrophages in vivo are defective in clearing apoptotic cells in multiple tissues, suggesting a broad role for CD14 in the clearance process. However, the resultant persistence of apoptotic cells does not lead to inflammation or increased autoantibody production, most likely because, as we show, CD14−/− macrophages retain the ability to generate anti-inflammatory signals in response to apoptotic cells. We conclude that CD14 plays a broad tethering role in apoptotic cell clearance in vivo and that apoptotic cells can persist in the absence of proinflammatory consequences.

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Defective fetal liver erythropoiesis and T lymphopoiesis in mice lacking the phosphatidylserine receptor

Blood ◽

10.1182/blood-2003-09-3245 ◽

2004 ◽

Vol 103 (9) ◽

pp. 3362-3364 ◽

Cited By ~ 78

Author(s):

Yuya Kunisaki ◽

Sadahiko Masuko ◽

Mayuko Noda ◽

Ayumi Inayoshi ◽

Terukazu Sanui ◽

...

Keyword(s):

Inflammatory Responses ◽

Apoptotic Cell ◽

Fetal Liver ◽

Lymphoid Cells ◽

Cell Uptake ◽

Developmental Defect ◽

Apoptotic Cells ◽

Phosphatidylserine Receptor ◽

T Lymphopoiesis

Abstract Clearance of apoptotic cells by macrophages is considered important for prevention of inflammatory responses leading to tissue damage. The phosphatidylserine receptor (PSR), which specifically binds to phosphatidylserine (PS) exposed on the surface of apoptotic cells, mediates uptake of apoptotic cells in vitro, yet the physiologic relevance of PSR remains unknown. This issue was addressed by generating PSR-deficient (PSR-/-) mice. PSR-/- mice exhibited severe anemia and died during the perinatal period. In the PSR-/- fetal livers, erythroid differentiation was blocked at an early erythroblast stage. In addition, PSR-/- embryos exhibited thymus atrophy owing to a developmental defect of T-lymphoid cells. Clearance of apoptotic cells by macrophages was impaired in both liver and thymus of PSR-/- embryos. However, this did not induce up-regulation of inflammatory cytokines. These results indicate that during embryonic development, PSR-mediated apoptotic cell uptake is required for definitive erythropoiesis and T lymphopoiesis, independently of the prevention of inflammatory responses. (Blood. 2004;103:3362-3364)

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