An essential role for calcium flux in phagocytes for apoptotic cell engulfment and the anti-inflammatory response

Abstract

Cells undergo programmed cell death/apoptosis throughout the lifespan of an organism. The subsequent immunologically silent removal of apoptotic cells plays a role in the maintenance of tolerance; defects in corpse clearance have been associated with autoimmune disease. A number of receptors and signaling molecules involved in this process have been identified, but intracellular signaling downstream of corpse recognition is only now being defined. Calcium plays a key role as a second messenger in many cell types, leading to the activation of downstream molecules and eventual transcription of effector genes; however, the role of calcium signaling during apoptotic cell removal is unclear. Here, using studies in cell lines and in the context of a whole organism, we show that apoptotic cell recognition induces both an acute and sustained calcium flux within phagocytes and that the genes required for calcium flux are essential for engulfment. Furthermore, we provide evidence that both the release of calcium from the endoplasmic reticulum and the entry of extracellular calcium through CRAC channels into the phagocytes are important during engulfment. Moreover, knockdown in Caenorhabditis elegans of stim-1 and jph-1, two genes linked to the entry of extracellular calcium into cells, led to increased persistence of apoptotic cells in the nematode. Loss of these genes seemed to affect early signaling events, leading to a decreased enrichment of actin adjacent to the apoptotic cell during corpse removal. We also show that calcium is crucial for the secretion of TGF-b by the phagocytes during the engulfment of apoptotic cells. Taken together, these data point to an earlier unappreciated and evolutionarily conserved role for calcium flux at two distinguishable steps: the formation of the phagocytic cup and the internalization of the apoptotic cell, and the anti-inflammatory signaling induced in phagocytes by contact with apoptotic cells.

Induction of Phagocytosis and Intracellular Signaling by an Inhibitory Channel Catfish Leukocyte Immune-Type Receptor: Evidence for Immunoregulatory Receptor Functional Plasticity in Teleosts

“…IpLITR 2.6b/IpFcRγ-L-mediated phagocytosis of anti-HA mAb-opsonized beads was significantly reduced in the presence of EDTA (2 m M ), suggesting the involvement of extracellular Ca 2+ . This effect is consistent with previous reports of EDTA inhibition of phagocytosis in fibroblasts [42] and murine peritoneal macrophages [43]. Phagocytosis induced by IpLITR 1.1b was refractory to EDTA-induced inhibition and instead showed a slight enhancement of bead internalization in EDTA-treated cells.…”

“…However, the effect of CytoD was more potent for IpLITR 2.6b/IpFcRγ-L-expressing cells. The role of actin polymerization during phagocytosis is well studied [42,47,48]; however, the cellular mechanisms responsible for the differential susceptibility to CytoD observed for IpLITR 2.6b/IpFcRγ-L- and IpLITR 1.1b-induced phagocytosis are unknown. Regardless, these results again suggest that IpLITR 1.1b is capable of promoting immune cell effector responses by controlling signaling events that are likely unique when compared to those induced by the ITAM-encoding IpLITR 2.6b/IpFcRγ-L.…”

Quantitative Proteomics Reveals That Only a Subset of the Endoplasmic Reticulum Contributes to the Phagosome

“…Stim1 was shown to locate to the cortical ER (34), a subdomain of the ER found in the vicinity of the PM. In addition, depletion of extracellular calcium store by metal chelators and ER calcium store by thapsigargin and siRNA against Stim-1 and Orai-1 in Caenorhabditis elegans was shown to affect phagocytosis of apoptotic cells (46). Taken together, those evidence suggest that the fraction of the ER harnessed by the phagosome might include a significant amount of cortical ER.…”

Mitochondrial Fission Promotes the Continued Clearance of Apoptotic Cells by Macrophages

“…However, ATP levels in macrophages were similar in control and Drp1-deficient macrophages under both basal conditions and after the addition of ACs (Figure S4D). We next considered the role of cytoplasmic calcium because (a) cytoplasmic calcium is increased when macrophages encounter ACs, and this process is necessary for efficient AC clearance (Cuttell et al, 2008; Gronski et al, 2009); and (b) in certain settings, cytosolic calcium is decreased by direct transfer of endoplasmic reticulum (ER) calcium to fused mitochondria (de Brito and Scorrano, 2008; Maltecca et al, 2012; Luchsinger et al, 2016). We therefore hypothesized that defective mitochondrial fission causes impairment of AC-induced increase in cytosolic calcium, i.e.…”