Nascent Phagosomes Research Articles

Phagosome Isolation with Magnetic Beads and Purification of Toll-Like Receptor (TLR) Complexes in Phagosomes.

Phagocytosis is a central process by which macrophage cells internalize and eliminate microbes as well as apoptotic cells. The nascent phagosome undergoes a complex maturation process involving sequential fusion with endosomal compartments. The endosomal TLRs, including TLR3, -7, -8, and -9, play a critical role in innate immunity by sensing bacterial or viral nucleic acids and are preferentially transported to the phagosomal membrane of innate immune cells upon activation. Therefore, phagosome isolation is helpful for studies on pathogenic invasion and the functions of phagosome proteins, including endosomal TLRs.

Timing of Phagosome Maturation Depends on Their Transport Switching from Actin to Microtubule Tracks.

Phagosomes, specialized membrane compartments responsible for digesting internalized pathogens, undergo sequential dynamic and biochemical changes as they mature from nascent phagosomes to degradative phagolysosomes. Maturation of phagosomes depends on their transport along actin filaments and microtubules. However, the specific quantitative relationship between the biochemical transformation and transport dynamics remains poorly characterized. The autonomous nature of phagosomes, moving and maturing at different rates, makes understanding this relationship challenging. Addressing this challenge, in this study we engineered particle sensors to image and quantify single phagosomes' maturation. We found that as phagosomes move from the actin cortex to microtubule tracks, the timing of their actin-to-microtubule transition governs the duration of the early phagosome stage before acquiring degradative capacities. Prolonged entrapment of phagosomes in the actin cortex extends the early phagosome stage by delaying the dissociation of early endosome markers and phagosome acidification. Conversely, a shortened transition from actin- to microtubule-based movements causes the opposite effect on phagosome maturation. These results suggest that the actin- and microtubule-based transport of phagosomes functions like a "clock" to coordinate the timing of biochemical events during phagosome maturation, which is crucial for effective pathogen degradation.

An In Vitro System to Analyze Generation and Degradation of Phagosomal Phosphatidylinositol Phosphates.

Phagosomes are formed when phagocytic cells take up large particles, and they develop into phagolysosomes where the particles are degraded. The transformation of nascent phagosomes into phagolysosomes is a complex multi-step process, and the precise timing of these steps depends at least in part on phosphatidylinositol phosphates (PIPs). Some such-called "intracellular pathogens" are not delivered to microbicidal phagolysosomes and manipulate the PIP composition of the phagosomes they reside in. Studying the dynamic changes of the PIP composition of inert-particle phagosomes will help to understand why the pathogens' manipulations reprogram phagosome maturation.We here describe a method to detect and to follow generation and degradation of PIPs on purified phagosomes. To this end, phagosomes formed around inert latex beads are purified from J774E macrophages and incubated in vitro with PIP-binding protein domains or PIP-binding antibodies. Binding of such PIP sensors to phagosomes indicates presence of the cognate PIP and is quantified by immunofluorescence microscopy. When phagosomes are incubated with PIP sensors and ATP at a physiological temperature, the generation and degradation of PIPs can be followed, and PIP-metabolizing enzymes can be identified using specific inhibitory agents.

Endoplasmic reticulum-Phagosome contact sites from the cradle to the grave.

Phagocytosis is a key component of the innate immune system used to ingest apoptotic cells and microorganisms for their destruction and recycling of macromolecules and the presentation of antigens to adaptive immune system cells. The newly formed vacuole or nascent phagosome undergoes a maturation process reminiscent of the classical endocytic maturation process, reaching a highly degradative phagolysosome stage before its tubulovesicular breakdown into lysosomes. The process is highly regulated and can be disrupted by various pathogenic organisms. The exchange of proteins, lipids, and other metabolites between organelles, including maturing phagosomes, is enabled by two processes, vesicular and non-vesicular transport at membrane contact sites (MCS). For decades the specific role(s) of the endoplasmic reticulum (ER) in phagocytosis has been the subject of much debate. In parallel, the last two decades have seen a burst in research on the numerous roles of ER contact sites and resident proteins in all aspects of organelle biology. Here, in this minireview, we describe ER-phagosome contact sites' functions from the early stages of particle engulfment to the phagolysosome dissolution into lysosomes. We also discuss several aspects of ER-phagosome contact sites that remain to be explored.

PtdIns(3,4)P2, Lamellipodin, and VASP coordinate actin dynamics during phagocytosis in macrophages.

Phosphoinositides are pivotal regulators of vesicular traffic and signaling during phagocytosis. Phagosome formation, the initial step of the process, is characterized by local membrane remodeling and reorganization of the actin cytoskeleton that leads to formation of the pseudopods that drive particle engulfment. Using genetically encoded fluorescent probes, we found that upon particle engagement a localized pool of PtdIns(3,4)P2 is generated by the sequential activities of class I phosphoinositide 3-kinases and phosphoinositide 5-phosphatases. Depletion of this locally generated pool of PtdIns(3,4)P2 blocks pseudopod progression and ultimately phagocytosis. We show that the PtdIns(3,4)P2 effector Lamellipodin (Lpd) is recruited to nascent phagosomes by PtdIns(3,4)P2. Furthermore, we show that silencing of Lpd inhibits phagocytosis and produces aberrant pseudopodia with disorganized actin filaments. Finally, vasodilator-stimulated phosphoprotein (VASP) was identified as a key actin-regulatory protein mediating phagosome formation downstream of Lpd. Mechanistically, our findings imply that a pathway involving PtdIns(3,4)P2, Lpd, and VASP mediates phagocytosis at the stage of particle engulfment.

Phosphatidylinositol-4-kinase 2α enhances phagosomal TLR signaling and MHC class II presentation in mouse dendritic cells

Toll like receptor (TLR) recruitment to bacteria-containing phagosomes and subsequent TLR signaling in dendritic cells (DCs) are essential to initiate anti-microbial immune responses, but the mechanisms underlying TLR trafficking to phagosomes are poorly characterized. We previously showed that the endosomal adaptor protein-3 (AP-3) optimizes TLR4 and MyD88 recruitment to phagosomes and favors phagosomal TLR signaling to proinflammatory cytokine secretion, membrane tubulation and MHC-II presentation. However, AP-3 functions indirectly in these activities. We hypothesized that this reflects AP-3’s role in endolysosomal protein sorting, and that defining direct targets of AP-3 sorting will elucidate new membrane pathways controlling TLR signaling and anti-bacterial immune responses. We have identified that one such AP-3 effector is phosphatidylinositol (PI)-4-kinase 2α (PI4K2α), which generates PI4P, a lipid reported to engage the TLR adaptor TIRAP to recruit MyD88 and TIRAP to membranes. We show that PI4K2α is recruited to nascent LPS-bead phagosomes in wild-type (WT) bone marrow-derived (BM) DCs and that its recruitment is reduced in AP-3-deficient pearl BMDCs, both by immunofluorescence microscopy and immunoblotting of isolated phagosomes. Moreover, both PI4K2α-GFP and PI4P co-localize with LPS/OVA-texas red bead phagosomes 2 hours after uptake in WT DCs and on phagotubules. The formation of tubules from phagosomes in BMDCs is impaired upon knock-down of PI4K2α but not PI4K2β. This correlates with reduced MHC-II presentation of particulate Ealpha antigen and impaired T cell activation. Additionally, after LPS priming, IL-1β secretion induced by multiple stimuli is reduced in BMDCs by PI4K2α knock-down compared to controls, suggesting that PI4K2α is necessary for optimal inflammasome activity, likely by impacting either priming (via TLR signaling) or NLR stimulation (by increasing membrane surface via tubulation). In summary, PI4K2α is a novel regulator of phagosomal signaling required for TLR recruitment to phagosomes and downstream signaling pathways in DCs.

The sodium proton exchanger NHE9 regulates phagosome maturation and bactericidal activity in macrophages

Acidification of phagosomes is essential for the bactericidal activity of macrophages. Targeting machinery that regulates pH within the phagosomes is a prominent strategy employed by various pathogens that have emerged as major threats to public health. Nascent phagosomes acquire the machinery for pH regulation through a graded maturation process involving fusion with endolysosomes. Meticulous coordination between proton pumping and leakage mechanisms is crucial for maintaining optimal pH within the phagosome. However, relative to mechanisms involved in acidifying the phagosome lumen, little is known about proton leakage pathways in this organelle. Sodium proton transporter NHE9 is a known proton leakage pathway located on the endosomes. As phagosomes acquire proteins through fusions with endosomes during maturation, NHE9 seemed a promising candidate for regulating proton fluxes on the phagosome. Here, using genetic and biophysical approaches, we show NHE9 is an important proton leakage pathway associated with the maturing phagosome. NHE9 is highly expressed in immune cells, specifically macrophages; however, NHE9 expression is strongly downregulated upon bacterial infection. We show that compensatory ectopic NHE9 expression hinders the directed motion of phagosomes along microtubules and promotes early detachment from the microtubule tracks. As a result, these phagosomes have shorter run lengths and are not successful in reaching the lysosome. In accordance with this observation, we demonstrate that NHE9 expression levels negatively correlate with bacterial survival. Together, our findings show that NHE9 regulates lumenal pH to affect phagosome maturation, and consequently, microbicidal activity in macrophages.

Mapping Sphingolipid Metabolism Pathways during Phagosomal Maturation.

Phagocytosis is an important physiological process, which, in higher organisms, is a means of fighting infections and clearing cellular debris. During phagocytosis, detrimental foreign particles (e.g. pathogens and apoptotic cells) are engulfed by phagocytes (e.g. macrophages), enclosed in membrane-bound vesicles called phagosomes, and transported to the lysosome for eventual detoxification. During this well-choreographed process, the nascent phagosome (also called early phagosome, EP) undergoes a series of spatiotemporally regulated changes in its protein and lipid composition and matures into a late phagosome (LP), which subsequently fuses with the lysosomal membrane to form the phagolysosome. While several elegant proteomic studies have identified the role of unique proteins during phagosomal maturation, the corresponding lipidomic studies are sparse. Recently, we reported a comparative lipidomic analysis between EPs and LPs and showed that ceramides are enriched on the LPs. Further, we found that this ceramide accumulation on LPs was orchestrated by ceramide synthase 2, inhibition of which hampers phagosomal maturation. Following up on this study, here, using biochemical assays, we first show that the increased ceramidase activity on EPs also significantly contributes to the accumulation of ceramides on LPs. Next, leveraging lipidomics, we show that de novo ceramide synthesis does not significantly contribute to the ceramide accumulation on LPs, while concomitant to increased ceramides, glucosylceramides are substantially elevated on LPs. We validate this interesting finding using biochemical assays and show that LPs indeed have heightened glucosylceramide synthase activity. Taken together, our studies provide interesting insights and possible new roles of sphingolipid metabolism during phagosomal maturation.

Structural and functional study of Legionella pneumophila effector RavA.

Legionella pneumophila is an intracellular pathogen that causes Legionnaire's disease in humans. This bacterium can be found in freshwater environments as a free-living organism, but it is also an intracellular parasite of protozoa. Human infection occurs when inhaled aerosolized pathogen comes into contact with the alveolar mucosa and replicates in alveolar macrophages. Legionella enters the host cell by phagocytosis and redirects the Legionella-containing phagosomes from the phagocytic maturation pathway. These nascent phagosomes fuse with ER-derived secretory vesicles and membranes forming the Legionella-containing vacuole. Legionella subverts many host cellular processes by secreting over 300 effector proteins into the host cell via the Dot/Icm type IV secretion system. The cellular function for many Dot/Icm effectors is still unknown. Here, we present a structural and functional study of L. pneumophila effector RavA (Lpg0008). Structural analysis revealed that the RavA consists of four ~85 residue long α-helical domains with similar folds, which show only a low level of structural similarity to other protein domains. The ~90 residues long C-terminal segment is predicted to be natively unfolded. We show that during L. pneumophila infection of human cells, RavA localizes to the Golgi apparatus and to the plasma membrane. The same localization is observed when RavA is expressed in human cells. The localization signal resides within the C-terminal sequence C409 WTSFCGLF417 . Yeast-two-hybrid screen using RavA as bait identified RAB11A as a potential binding partner. RavA is present in L. pneumophila strains but only distant homologs are found in other Legionella species, where the number of repeats varies.

Better Together: Current Insights Into Phagosome-Lysosome Fusion.

Following phagocytosis, the nascent phagosome undergoes maturation to become a phagolysosome with an acidic, hydrolytic, and often oxidative lumen that can efficiently kill and digest engulfed microbes, cells, and debris. The fusion of phagosomes with lysosomes is a principal driver of phagosomal maturation and is targeted by several adapted intracellular pathogens. Impairment of this process has significant consequences for microbial infection, tissue inflammation, the onset of adaptive immunity, and disease. Given the importance of phagosome-lysosome fusion to phagocyte function and the many virulence factors that target it, it is unsurprising that multiple molecular pathways have evolved to mediate this essential process. While the full range of these pathways has yet to be fully characterized, several pathways involving proteins such as members of the Rab GTPases, tethering factors and SNAREs have been identified. Here, we summarize the current state of knowledge to clarify the ambiguities in the field and construct a more comprehensive phagolysosome formation model. Lastly, we discuss how other cellular pathways help support phagolysosome biogenesis and, consequently, phagocyte function.

NAADP‐regulated two‐pore channels drive phagocytosis through endo‐lysosomal Ca2+ nanodomains, calcineurin and dynamin

Macrophages clear pathogens by phagocytosis and lysosomes that fuse with phagosomes are traditionally regarded as to a source of membranes and luminal degradative enzymes. Here, we reveal that endo‐lysosomes act as platforms for a new phagocytic signalling pathway in which FcγR activation recruits the second messenger NAADP and thereby promotes the opening of Ca2+‐permeable two‐pore channels (TPCs). Remarkably, phagocytosis is driven by these local endo‐lysosomal Ca2+ nanodomains rather than global cytoplasmic or ER Ca2+ signals. Motile endolysosomes contact nascent phagosomes to promote phagocytosis, whereas endo‐lysosome immobilization prevents it. We show that TPC‐released Ca2+ rapidly activates calcineurin, which in turn dephosphorylates and activates the GTPase dynamin‐2. Finally, we find that different endo‐lysosomal Ca2+ channels play diverse roles, with TPCs providing a universal phagocytic signal for a wide range of particles and TRPML1 being only required for phagocytosis of large targets.

The Role of Membrane Surface Charge in Phagocytosis.

The formation and maturation of phagosomes are accompanied by acute changes in lipid metabolism. Phosphoinositides, in particular, undergo extensive modification as part of the signaling sequence that drives cytoskeletal and membrane remodeling. Because the phosphoinositides provide much of the anionic charge of the cytosolic leaflet of the plasmalemma and phagosomal membrane, the metabolic changes associated with signaling result in marked changes of the surface charge. Here we summarize the pathways involved in lipid remodeling during phagocytosis, the resultant alterations in the surface charge of the nascent and maturing phagosomes, and the consequent effects on the association of proteins attached to the membrane by electrostatic means.

Phagosome-endoplasmic reticulum contacts: Kissing and not running.

The role of the endoplasmic reticulum (ER) in phagocytosis has been the subject of debate for over a decade. Proteomic determinations and dynamic microscopy of live cells led to conflicting conclusions. Recent insights into the existence of a variety of membrane contact sites (MCS) may help reconcile the seemingly disparate views. Specifically, earlier results can be rationalized considering that the ER forms specialized MCS with nascent and maturing phagosomes, without undergoing fusion. The composition and function of documented ER-to-phagosome contact sites is described. In addition, we speculate about the possible existence of additional phagosomal contact sites, based on available knowledge of interactions between the ER and other endocytic compartments. The interaction between phagosomes and the ER has been the subject of debate. Earlier observations that led to the suggestion that the ER fuses with the phagosomal membrane can now be explained in the light of recent evidence that intimate contacts form between the two organelles.

Phagolysosome resolution requires contacts with the endoplasmic reticulum and phosphatidylinositol-4-phosphate signalling.

Phosphoinositides have a pivotal role in the maturation of nascent phagosomes into microbicidal phagolysosomes. Following degradation of their contents, mature phagolysosomes undergo resolution, a process that remains largely uninvestigated. Here we studied the role of phosphoinositides in phagolysosome resolution. Phosphatidylinositol-4-phosphate (PtdIns(4)P), which is abundant in maturing phagolysosomes, was depleted as they tubulated and resorbed. Depletion was caused, in part, by transfer of phagolysosomal PtdIns(4)P to the endoplasmic reticulum, a process mediated by oxysterol-binding protein-related protein 1L (ORP1L), a RAB7 effector. ORP1L formed discrete tethers between the phagolysosome and the endoplasmic reticulum, resulting in distinct regions with alternating PtdIns(4)P depletion and enrichment. Tubules emerged from PtdIns(4)P-rich regions, where ADP-ribosylation factor-like protein 8B (ARL8B) and SifA- and kinesin-interacting protein/pleckstrin homology domain-containing family M member 2 (SKIP/PLEKHM2) accumulated. SKIP binds preferentially to monophosphorylated phosphoinositides, of which PtdIns(4)P is most abundant in phagolysosomes, contributing to their tubulation. Accordingly, premature hydrolysis of PtdIns(4)P impaired SKIP recruitment and phagosome resolution. Thus, resolution involves phosphoinositides and tethering of phagolysosomes to the endoplasmic reticulum.

Dynamic Podosome-Like Structures in Nascent Phagosomes Are Coordinated by Phosphoinositides

Phagocytosis, the engulfment of particulate matter, requires the coordinated polymerization of F-actin; however, the nature and dynamics of the F-actin structures generated during the process are incompletely defined. Using super-resolution microscopy, we observed the formation of podosome-like structures during Fc receptor-mediated phagocytosis. Unlike conventional podosomes, these structures are short lived and vectorial, expanding radially from the sites where phagocytic targets are initially engaged. The expanding ring of podosome-like structures requires the localized formation of PtdIns(3,4,5)P3. Concomitantly, the initial podosome-like structures disappear from the center of the phagocytic cup, enabling membrane bending around the target. This coordinated disappearance is mediated by localized hydrolysis of PtdIns(4,5)P2 at the center of the cup. Interference reflection microscopy revealed that the podosome-like structures attach tightly to the target, facilitating the progressive engagement and activation of phagocytic receptors, creating a diffusion barrier and serving as support for the extension of exploratory lamellipodia that probe the target surface.

Regulation of Phospholipase D by Arf6 during FcγR-Mediated Phagocytosis.

Phagocytosis is an essential element of the immune response, assuring the elimination of pathogens, cellular debris, and apoptotic and tumoral cells. Activation of phagocytosis by the FcγR stimulates phospholipase D (PLD) activity and triggers the production of phosphatidic acid (PA) at the plasma membrane of macrophages, but the regulatory mechanisms involved are still not clearly understood. In this study, we examined the role of the small GTPase Arf6 in the activation of the PLD isoforms during FcγR-mediated phagocytosis. In RAW 264.7 macrophage cells, expressed Arf6-GFP partially colocalized with PLD1-hemagglutinin on intracellular membrane-bound vesicles and with PLD2-hemagglutinin at the plasma membrane. Both PLD isoforms were found to interact with Arf6 during FcγR-mediated phagocytosis as seen by immunoprecipitation experiments. In macrophages stimulated for phagocytosis, Arf6 was observed to be associated with nascent phagosomes. RNA interference knockdown of Arf6 reduced the amount of active Arf6 associated with phagosomes, revealed by the MT2-GFP probe that specifically binds to Arf6-GTP. Arf6 silencing concomitantly decreased PLD activity as well as the levels of PA found on phagosomes and phagocytic sites as shown with the PA probe Spo20p-GFP. Altogether, our results indicate that Arf6 is involved in the regulation of PLD activity and PA synthesis required for efficient phagocytosis.

The small GTPase RAB-35 defines a third pathway that is required for the recognition and degradation of apoptotic cells.

In metazoans, apoptotic cells are swiftly engulfed by phagocytes and degraded inside phagosomes. Multiple small GTPases in the Rab family are known to function in phagosome maturation by regulating vesicle trafficking. We discovered rab-35 as a new gene important for apoptotic cell clearance from a genetic screen targeting putative Rab GTPases in Caenorhabditis elegans. We further identified TBC-10 as a putative GTPase-activating protein (GAP), and FLCN-1 and RME-4 as two putative Guanine Nucleotide Exchange Factors (GEFs), for RAB-35. We found that RAB-35 was required for the efficient incorporation of early endosomes to phagosomes and for the timely degradation of apoptotic cell corpses. More specifically, RAB-35 promotes two essential events that initiate phagosome maturation: the switch of phagosomal membrane phosphatidylinositol species from PtdIns(4,5)P2 to PtdIns(3)P, and the recruitment of the small GTPase RAB-5 to phagosomal surfaces. These functions of RAB-35 were previously unknown. Remarkably, although the phagocytic receptor CED-1 regulates these same events, RAB-35 and CED-1 appear to function independently. Upstream of degradation, RAB-35 also facilitates the recognition of apoptotic cells independently of the known CED-1 and CED-5 pathways. RAB-35 localizes to extending pseudopods and is further enriched on nascent phagosomes, consistent with its dual roles in regulating apoptotic cell-recognition and phagosome maturation. Epistasis analyses indicate that rab-35 acts in parallel to both of the canonical ced-1/6/7 and ced-2/5/10/12 clearance pathways. We propose that RAB-35 acts as a robustness factor, defining a novel pathway that aids these canonical pathways in both the recognition and degradation of apoptotic cells.

Legionella Effector RavD Binds Host Phosphoinositide‐3‐phosphate and Contributes to Lysosomal Avoidance

Subversion of phosphoinositide lipids is a key strategy that bacterial pathogens employ to survive within hosts. Due to the importance of phosphoinositides in phagocytosis, bacterial pathogens that infect innate immune cells manipulate these lipids to alter phagosome maturation with the purpose of converting the nascent phagosome into a replication‐permissive niche. The intracellular bacterial pathogen, Legionella pneumophila, the causative agent of a severe pneumonia known as Legionnaires' disease, infects alveolar macrophages where it proliferates within the safety of a vacuole. Upon phagocytosis, L. pneumophila hijacks the nascent phagosome and remodels the cytosolic façade of the phagosomal membrane by stimulating fusion with vesicles derived from the endoplasmic reticulum. This process relies on the function of bacterial effector proteins translocated directly into the host cell via a specialized secretion system. A number of L. pneumophila effector proteins have been shown to bind phosphoinositides either for the purpose of manipulating their metabolism or as a means of targeting specific host membrane compartments. Our preliminary data indicate that many more phosphoinositide‐binding effectors remain to be discovered. Here, we show that the L. pneumophila effector, RavD, is a novel phosphoinositide‐3‐phosphate (PI3P) binding‐protein. We establish that RavD is found on the Legionella‐containing vacuole (LCV) at early stages of infection and harbors its PI3P binding domain within the C terminal region of the protein. In the absence of the ravD gene, the LCV accumulates lysosomal markers at 2 hours post‐infection, consistent with the notion that RavD function is necessary to arrest phagosome maturation.Support or Funding InformationThis work was supported by the University of Delaware Research Foundation.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

PALLD Regulates Phagocytosis by Enabling Timely Actin Polymerization and Depolymerization.

PALLD is an actin cross-linker supporting cellular mechanical tension. However, its involvement in the regulation of phagocytosis, a cellular activity essential for innate immunity and physiological tissue turnover, is unclear. We report that PALLD is highly induced along with all-trans-retinoic acid-induced maturation of myeloid leukemia cells, to promote Ig- or complement-opsonized phagocytosis. PALLD mechanistically facilitates phagocytic receptor clustering by regulating actin polymerization and c-Src dynamic activation during particle binding and early phagosome formation. PALLD is also required at the nascent phagosome to recruit phosphatase oculocerebrorenal syndrome of Lowe, which regulates phosphatidylinositol-4,5-bisphosphate hydrolysis and actin depolymerization to complete phagosome closure. Collectively, our results show a new function for PALLD as a crucial regulator of the early phase of phagocytosis by elaborating dynamic actin polymerization and depolymerization.

Involvement of the p62/NRF2 signal transduction pathway on erythrophagocytosis

Erythrophagocytosis, the phagocytic removal of damaged red blood cells (RBC), and subsequent phagolysosome biogenesis are important processes in iron/heme metabolism and homeostasis. Phagolysosome biogenesis implies the interaction of nascent phagosomes with endocytic compartments and also autophagy effectors. Here, we report that besides recruitment of microtubule-associated protein-1-light chain 3 (LC3), additional autophagy machinery such as sequestosome 1 (p62) is also acquired by single-membrane phagosomes at very early stages of the phagocytic process and that its acquisition is very important to the outcome of the process. In bone marrow-derived macrophages (BMDM) silenced for p62, RBC degradation is inhibited. P62, is also required for nuclear translocation and activation of the transcription factor Nuclear factor E2-related Factor 2 (NRF2) during erythrophagocytosis. Deletion of the Nrf2 allele reduces p62 expression and compromises RBC degradation. In conclusion, we reveal that erythrophagocytosis relies on an interplay between p62 and NRF2, potentially acting as protective mechanism to maintain reactive oxygen species at basal levels and preserve macrophage homeostasis.