id: MODULE:efferocytosis_induced_macrophage_proliferation title: Efferocytosis-induced macrophage proliferation description: >- After a macrophage engulfs an apoptotic cell, it can divide. Two signals converge to make it do so. Lysosomal DNase II digests the engulfed cell's DNA in the phagolysosome, and the resulting oligonucleotides activate DNA-PKcs, then mTORC2 and AKT. Separately, GAS6 bound to the apoptotic cell activates the MERTK receptor tyrosine kinase and ERK1/2. Both routes raise MYC, which induces the transcription factor BHLHE40. BHLHE40 represses the anti-proliferative factor c-MAF, and the macrophage enters the cell cycle. The daughter cells keep clearing apoptotic cells, so the programme expands the pool of pro-resolving macrophages at sites of injury. status: DRAFT scope: ABSTRACT references: - id: PMID:34784501 title: Efferocytosis induces macrophage proliferation to help resolve tissue injury. reference_review: relevance: HIGH correctness: VERIFIED review_notes: >- PubMed-verified; full text cached and read in full. Defines the programme and its controls in mouse BMDMs, dexamethasone-treated thymus and atherosclerosis regression. The mechanism rests mainly on siRNA, with Rictor deletion in bone marrow and nanoparticle siRNA in vivo. evidence: - source_id: PMID:34784501 title: Efferocytosis induces macrophage proliferation to help resolve tissue injury. statement: >- The programme is triggered by digestion of the engulfed apoptotic cell, not by phagocytosis as such. Red cells, which have no DNA, are engulfed without effect, while red cells loaded with DNA trigger proliferation. supporting_text: However, the macrophages that ingested IgG-RBCs did not show this response even though the uptake was similar to that of ACs - source_id: PMID:34784501 title: Efferocytosis induces macrophage proliferation to help resolve tissue injury. statement: DNA loaded into red cells is enough to trigger proliferation, and this depends on DNase II. supporting_text: The DNA-loaded IgG-RBCs were able to stimulate macrophage EdU incorporation compared with unloaded IgG-RBCs, and this proliferative response was blunted in macrophages treated with siDnase2a - source_id: PMID:34784501 title: Efferocytosis induces macrophage proliferation to help resolve tissue injury. statement: >- Blocking phagolysosomal degradation does not block CSF1-driven proliferation, so the effect is specific to the cargo route. supporting_text: As an important control, we showed that CSF1-induced macrophage proliferation, which should not involve phagolysosomes, was not blocked by bafilomycin notes: >- Boundary: this module starts with apoptotic-cell DNA in the phagolysosome and with GAS6-bound apoptotic cells at the macrophage surface, and ends with macrophage proliferation. Engulfment, phagosome maturation and the rest of cargo digestion belong to the phagocytosis modules (MODULE:phagocytic_engulfment, MODULE:phagosome_maturation). RUBCN and the V-ATPase are needed for this programme only because they are needed for phagolysosome formation, so they are not participants here. GO has no term for this programme. The closest is GO:0061517 macrophage proliferation, used as the module concept. The term "efferocytosis-induced macrophage proliferation" (is_a GO:0061517, preceded_by GO:0043277) has been proposed to the GO editors in go-ontology#32748. Taxon scope: the programme is shown in mouse and its proliferative phenotype in human monocyte-derived macrophages. DNase II and RICTOR are conserved across animals. An OpenScientist phylogenetic profile (file:human/PRKDC/PRKDC-hypotheses/prkdc-animal-distribution/openscientist.md) found DNA-PKcs (PTHR11139:SF68) ancestral to animals, present from sponges and cnidarians through most insects, and lost in Drosophila melanogaster and C. elegans, which keep the related PIKK kinases. All the components of the DNA-sensing route are therefore present in most animals, but the route has only been tested in mouse (see knowledge gaps). module: id: efferocytosis_induced_macrophage_proliferation label: Efferocytosis-induced macrophage proliferation module_type: SIGNALING_PATHWAY concepts: - preferred_term: macrophage proliferation term: id: GO:0061517 label: macrophage proliferation notes: >- Output process. A specific term, efferocytosis-induced macrophage proliferation, has been proposed to GO (go-ontology#32748). context: taxa: - preferred_term: Mammalia term: id: NCBITaxon:40674 label: Mammalia notes: Shown in mouse (mechanism) and in human monocyte-derived macrophages (phenotype). cell_types: - preferred_term: macrophage term: id: CL:0000235 label: macrophage parts: - order: 1 role: cargo DNA digestion node: id: cargo_dna_digestion label: Digestion of apoptotic-cell DNA in the phagolysosome module_type: BIOLOGICAL_PROCESS description: >- Lysosomal DNase II cuts the engulfed cell's DNA into oligonucleotides. This step makes the signal, so the programme needs phagolysosome formation as well as DNase II itself. concepts: - preferred_term: DNA catabolic process term: id: GO:0006308 label: DNA catabolic process annotons: - id: dnase2_cargo_dna label: DNase II digests apoptotic-cell DNA participant: selector_type: FAMILY family: preferred_term: DNase II alpha term: id: PANTHER:PTHR10858:SF9 label: DEOXYRIBONUCLEASE-2-ALPHA representative_members: - preferred_term: Dnase2 (mouse) term: id: UniProtKB:P56542 label: Deoxyribonuclease-2-alpha - preferred_term: DNASE2 (human) term: id: UniProtKB:O00115 label: Deoxyribonuclease-2-alpha function: preferred_term: deoxyribonuclease II activity term: id: GO:0004531 label: deoxyribonuclease II activity locations: - preferred_term: phagolysosome term: id: GO:0032010 label: phagolysosome role_description: >- Makes the oligonucleotide signal from engulfed DNA. Its knockdown blocks proliferation after efferocytosis but not uptake, and blocks the response to DNA-loaded red cells. evidence: - source_id: PMID:34784501 title: Efferocytosis induces macrophage proliferation to help resolve tissue injury. statement: The proliferative signal comes from phagolysosomal hydrolysis of apoptotic-cell DNA. supporting_text: We found that nucleotides derived from the phagolysosomal hydrolysis of AC-DNA during efferocytosis triggers a signaling pathway that promotes the proliferation of pro-resolving macrophages. - order: 2 role: oligonucleotide-triggered kinase signalling node: id: dna_pk_mtorc2_signaling label: DNA-PKcs, mTORC2 and AKT signalling module_type: SIGNALING_PATHWAY description: >- Cargo-derived oligonucleotides activate DNA-PKcs, which acts upstream of mTORC2. mTORC2 phosphorylates AKT on Ser473, and AKT signalling raises MYC. Phosphorylation of DNA-PKcs after efferocytosis depends on DNase II. concepts: - preferred_term: TORC2 signaling term: id: GO:0038203 label: TORC2 signaling annotons: - id: dna_pkcs_sensor label: DNA-PKcs activated by cargo oligonucleotides participant: selector_type: FAMILY family: preferred_term: DNA-PK catalytic subunit term: id: PANTHER:PTHR11139:SF68 label: DNA-DEPENDENT PROTEIN KINASE CATALYTIC SUBUNIT representative_members: - preferred_term: Prkdc (mouse) term: id: UniProtKB:P97313 label: DNA-dependent protein kinase catalytic subunit - preferred_term: PRKDC (human) term: id: UniProtKB:P78527 label: DNA-dependent protein kinase catalytic subunit ancestral_nodes: - preferred_term: PAINT node PTN000124197 term: id: PANTHER:PTN000124197 label: PTN000124197 description: >- Root PAINT IBD node of PTHR11139 propagating F:GO:0004674 protein serine/threonine kinase activity, with PRKDC (P78527) and mouse Prkdc (MGI:MGI:104779) among the seeds. It supports the parent kinase activity, not the DNA-dependent activity asserted here. evidence: - source_id: GO_REF:0000033 statement: IBD propagation of protein serine/threonine kinase activity from PAINT node PTN000124197 in PTHR11139. function: preferred_term: DNA-dependent protein kinase activity term: id: GO:0004677 label: DNA-dependent protein kinase activity role_description: >- Proposed sensor of the cargo oligonucleotides. Its knockdown blocks proliferation and AKT Ser473 phosphorylation. Direct binding of the oligonucleotides was not shown. evidence: - source_id: PMID:34784501 title: Efferocytosis induces macrophage proliferation to help resolve tissue injury. statement: DNA-PKcs is activated after efferocytosis, and this needs DNase II. supporting_text: Further, phosphorylation of DNA-PKcs, which is a marker of its activation, was increased in AC+ versus AC− macrophages, and this increase was completely abrogated by siDnase2a - id: mtorc2_kinase label: mTORC2 phosphorylates AKT participant: selector_type: PROTEIN_COMPLEX protein_complex: preferred_term: TORC2 complex term: id: GO:0031932 label: TORC2 complex active_units: - label: RICTOR participant: selector_type: FAMILY family: preferred_term: RICTOR (AVO3) term: id: PANTHER:PTHR13298:SF11 label: RAPAMYCIN-INSENSITIVE COMPANION OF MTOR representative_members: - preferred_term: Rictor (mouse) term: id: UniProtKB:Q6QI06 label: Rapamycin-insensitive companion of mTOR - preferred_term: RICTOR (human) term: id: UniProtKB:Q6R327 label: Rapamycin-insensitive companion of mTOR - preferred_term: TSC11/AVO3 (S. cerevisiae) term: id: UniProtKB:P40061 label: Target of rapamycin complex 2 subunit TSC11 - label: MTOR kinase participant: selector_type: FAMILY family: preferred_term: MTOR term: id: PANTHER:PTHR11139:SF9 label: SERINE_THREONINE-PROTEIN KINASE MTOR representative_members: - preferred_term: MTOR (human) term: id: UniProtKB:P42345 label: Serine/threonine-protein kinase mTOR ancestral_nodes: - preferred_term: PAINT node PTN000124328 term: id: PANTHER:PTN000124328 label: PTN000124328 description: >- PAINT IBD node in PTHR11139 propagating P:GO:0038203 TORC2 signaling, with MTOR (P42345) among the seeds. evidence: - source_id: GO_REF:0000033 statement: IBD propagation of TORC2 signaling from PAINT node PTN000124328 in PTHR11139. function: preferred_term: protein serine/threonine kinase activity term: id: GO:0004674 label: protein serine/threonine kinase activity processes: - preferred_term: TORC2 signaling term: id: GO:0038203 label: TORC2 signaling role_description: >- Relays the DNA-PKcs signal to AKT. Rictor knockdown blocks efferocytosis-induced proliferation but not CSF1-induced proliferation; Rictor deletion in bone marrow blocks proliferation of efferocytosing macrophages in the thymus. evidence: - source_id: PMID:34784501 title: Efferocytosis induces macrophage proliferation to help resolve tissue injury. statement: The authors place DNA-PKcs upstream of mTORC2 and AKT in this programme. supporting_text: "These combined data support a role for the following pathway in EIMP: Dnase2a-hydrolyzed AC-DNA → DNA-PKcs → mTORC2 → phospho-Akt → proliferation." - id: akt_kinase label: AKT downstream of mTORC2 participant: selector_type: FAMILY family: preferred_term: AKT kinase term: id: PANTHER:PTHR24356:SF171 label: RAC-ALPHA SERINE_THREONINE-PROTEIN KINASE representative_members: - preferred_term: AKT1 (human) term: id: UniProtKB:P31749 label: RAC-alpha serine/threonine-protein kinase - preferred_term: Akt1 (mouse) term: id: UniProtKB:P31750 label: RAC-alpha serine/threonine-protein kinase notes: >- Grounded on PANTHER's own classification (PTHR24356:SF171). UniProt's PANTHER cross-reference places AKT1 in PTHR24351. function: preferred_term: protein serine/threonine kinase activity term: id: GO:0004674 label: protein serine/threonine kinase activity role_description: >- Its Ser473 phosphorylation is the readout of mTORC2 activity here. Its requirement rests on a pan-AKT inhibitor, and the isoform involved is not known. notes: AKT1 is shown as an orienting member only; the paper does not resolve the isoform. - order: 3 role: receptor arm node: id: gas6_mertk_erk_arm label: GAS6, MERTK and ERK1/2 signalling module_type: SIGNALING_PATHWAY description: >- GAS6 bound to the apoptotic cell activates MERTK, likely before the cell is engulfed, and MERTK activates ERK1/2. This arm does not need phagolysosomal degradation and adds to the DNA-sensing arm in raising MYC. concepts: - preferred_term: ERK1 and ERK2 cascade term: id: GO:0070371 label: ERK1 and ERK2 cascade annotons: - id: gas6_ligand label: GAS6 bridges the apoptotic cell to MERTK participant: selector_type: FAMILY family: preferred_term: GAS6 term: id: PANTHER:PTHR24040:SF14 label: GROWTH ARREST-SPECIFIC PROTEIN 6 representative_members: - preferred_term: Gas6 (mouse) term: id: UniProtKB:Q61592 label: Growth arrest-specific protein 6 - preferred_term: GAS6 (human) term: id: UniProtKB:Q14393 label: Growth arrest-specific protein 6 function: preferred_term: receptor ligand activity term: id: GO:0048018 label: receptor ligand activity locations: - preferred_term: extracellular region term: id: GO:0005576 label: extracellular region role_description: Activates MERTK. Added GAS6 is enough to induce proliferation without degradation. - id: mertk_receptor label: MERTK receptor tyrosine kinase participant: selector_type: FAMILY family: preferred_term: MERTK term: id: PANTHER:PTHR24416:SF257 label: TYROSINE-PROTEIN KINASE MER representative_members: - preferred_term: Mertk (mouse) term: id: UniProtKB:Q60805 label: Tyrosine-protein kinase Mer - preferred_term: MERTK (human) term: id: UniProtKB:Q12866 label: Tyrosine-protein kinase Mer function: preferred_term: transmembrane receptor protein tyrosine kinase activity term: id: GO:0004714 label: transmembrane receptor protein tyrosine kinase activity locations: - preferred_term: plasma membrane term: id: GO:0005886 label: plasma membrane role_description: >- Signals to ERK1/2 and MYC. Myeloid Mertk deletion blunts MYC induction in macrophages that have engulfed an apoptotic cell. MERTK also helps engulfment itself, so the analysis was gated on cells that had engulfed. evidence: - source_id: PMID:34784501 title: Efferocytosis induces macrophage proliferation to help resolve tissue injury. statement: The MERTK route acts alongside the phagolysosomal route. supporting_text: "Thus, two separate pathways contribute to EIMP: a pathway requiring degradation of ACs in phagolysosomes and a pathway downstream of MerTK, which likely becomes activated by AC-bound Gas6 prior to AC engulfment." - id: erk_kinase label: ERK1/2 downstream of MERTK participant: selector_type: FAMILY family: preferred_term: ERK1/ERK2 term: id: PANTHER:PTHR24055 label: MITOGEN-ACTIVATED PROTEIN KINASE representative_members: - preferred_term: Mapk3 (mouse) term: id: UniProtKB:Q63844 label: Mitogen-activated protein kinase 3 - preferred_term: MAPK1 (human) term: id: UniProtKB:P28482 label: Mitogen-activated protein kinase 1 function: preferred_term: MAP kinase activity term: id: GO:0004707 label: MAP kinase activity role_description: >- Required for MYC induction by the MERTK arm. The evidence is a MEK inhibitor, so ERK1 and ERK2 are not separated. - order: 4 role: transcriptional switch node: id: myc_bhlhe40_switch label: MYC and BHLHE40 relieve c-MAF restraint module_type: REGULATORY_STEP description: >- The two arms together raise MYC to a level that induces BHLHE40. BHLHE40 represses c-MAF, a transcription factor that holds macrophages out of the cell cycle, and proliferation follows. annotons: - id: myc_tf label: MYC induces BHLHE40 participant: selector_type: FAMILY family: preferred_term: MYC term: id: PANTHER:PTHR45851 label: MYC PROTO-ONCOGENE representative_members: - preferred_term: Myc (mouse) term: id: UniProtKB:P01108 label: Myc proto-oncogene protein - preferred_term: MYC (human) term: id: UniProtKB:P01106 label: Myc proto-oncogene protein ancestral_nodes: - preferred_term: PAINT node PTN001691821 term: id: PANTHER:PTN001691821 label: PTN001691821 description: >- PAINT IBD node in PTHR45851 propagating F:GO:0000981 DNA-binding transcription factor activity, RNA polymerase II-specific, with MYC (P01106) among the seeds. evidence: - source_id: GO_REF:0000033 statement: IBD propagation of RNA polymerase II-specific DNA-binding transcription factor activity from PAINT node PTN001691821 in PTHR45851. function: preferred_term: DNA-binding transcription factor activity, RNA polymerase II-specific term: id: GO:0000981 label: DNA-binding transcription factor activity, RNA polymerase II-specific locations: - preferred_term: nucleus term: id: GO:0005634 label: nucleus role_description: Point where the two arms converge. Myc knockdown blocks proliferation and BHLHE40 induction. evidence: - source_id: PMID:34784501 title: Efferocytosis induces macrophage proliferation to help resolve tissue injury. statement: Both arms converge on MYC, which is needed for the programme. supporting_text: Thus, the phagolysosomal and MerTK pathways, via mTORC2 and ERK1/2, respectively, combine to increase Myc in efferocytosing macrophages, which is necessary for EIMP. - id: bhlhe40_repressor label: BHLHE40 represses c-MAF participant: selector_type: FAMILY family: preferred_term: BHLHE40 (DEC1) term: id: PANTHER:PTHR10985:SF3 label: CLASS E BASIC HELIX-LOOP-HELIX PROTEIN 40 representative_members: - preferred_term: Bhlhe40 (mouse) term: id: UniProtKB:O35185 label: Class E basic helix-loop-helix protein 40 - preferred_term: BHLHE40 (human) term: id: UniProtKB:O14503 label: Class E basic helix-loop-helix protein 40 ancestral_nodes: - preferred_term: PAINT node PTN004213585 term: id: PANTHER:PTN004213585 label: PTN004213585 description: >- Eukaryote-level PAINT IBD node in PTHR10985 propagating F:GO:0000981 and P:GO:0000122 negative regulation of transcription by RNA polymerase II, with BHLHE40 (O14503) among the seeds. evidence: - source_id: GO_REF:0000033 statement: IBD propagation of RNA polymerase II-specific DNA-binding transcription factor activity from PAINT node PTN004213585 in PTHR10985. function: preferred_term: DNA-binding transcription repressor activity, RNA polymerase II-specific term: id: GO:0001227 label: DNA-binding transcription repressor activity, RNA polymerase II-specific locations: - preferred_term: nucleus term: id: GO:0005634 label: nucleus role_description: >- Its knockdown completely blocks proliferation after efferocytosis but not after CSF1, so it is the programme-specific step. evidence: - source_id: PMID:34784501 title: Efferocytosis induces macrophage proliferation to help resolve tissue injury. statement: BHLHE40 drives proliferation by repressing c-MAF. supporting_text: Bhlhe40 then represses the cell-cycling-inhibitor c-Maf to drive proliferation - id: cmaf_brake label: c-MAF anti-proliferative factor (repressed) participant: selector_type: FAMILY family: preferred_term: c-MAF term: id: PANTHER:PTHR10129:SF9 label: TRANSCRIPTION FACTOR MAF representative_members: - preferred_term: Maf (mouse) term: id: UniProtKB:P54843 label: Transcription factor Maf - preferred_term: MAF (human) term: id: UniProtKB:O75444 label: Transcription factor Maf function: preferred_term: DNA-binding transcription factor activity, RNA polymerase II-specific term: id: GO:0000981 label: DNA-binding transcription factor activity, RNA polymerase II-specific locations: - preferred_term: nucleus term: id: GO:0005634 label: nucleus role_description: >- Holds macrophages out of the cycle. It falls after efferocytosis, and its overexpression blocks the programme. notes: >- The overexpression construct is called a "Maf1 plasmid" in the paper's methods. MAF1 is an unrelated gene, so this is probably a naming slip for c-Maf, but it is unverified. connections: - source: cargo_dna_digestion target: dna_pk_mtorc2_signaling connection_type: PROVIDES_INPUT_FOR description: DNase II-generated oligonucleotides activate DNA-PKcs. - source: dna_pkcs_sensor target: mtorc2_kinase connection_type: POSITIVELY_REGULATES description: DNA-PKcs acts upstream of mTORC2; whether directly is not shown. - source: mtorc2_kinase target: akt_kinase connection_type: POSITIVELY_REGULATES description: mTORC2 phosphorylates AKT on Ser473. - source: gas6_ligand target: mertk_receptor connection_type: POSITIVELY_REGULATES - source: mertk_receptor target: erk_kinase connection_type: POSITIVELY_REGULATES - source: akt_kinase target: myc_tf connection_type: POSITIVELY_REGULATES description: Raises Myc mRNA and protein. - source: erk_kinase target: myc_tf connection_type: POSITIVELY_REGULATES - source: myc_tf target: bhlhe40_repressor connection_type: POSITIVELY_REGULATES description: MYC induces Bhlhe40 transcription. - source: bhlhe40_repressor target: cmaf_brake connection_type: NEGATIVELY_REGULATES description: BHLHE40 represses c-Maf. knowledge_gaps: - gap_statement: >- How cargo-derived oligonucleotides from the phagolysosome reach and activate DNA-PKcs is unknown. boundary: >- DNA-PKcs phosphorylation after efferocytosis depends on DNase II, and DNA-PKcs knockdown blocks the programme, but direct binding of the oligonucleotides and the route out of the phagolysosome were not shown. gap_kind: - BIOLOGY dark_aspect: MF_DARK status: OPEN significance: The sensor step is inferred, so the DNA-PKcs annoton rests on a requirement, not a mechanism. resolution: >- Test binding of labelled cargo oligonucleotides to DNA-PK in efferocytosing macrophages, use a kinase-dead DNA-PKcs, and test whether the Ku subunits are needed. provenance: - reference_id: PMID:34784501 supporting_text: Further, phosphorylation of DNA-PKcs, which is a marker of its activation, was increased in AC+ versus AC− macrophages, and this increase was completely abrogated by siDnase2a - gap_statement: >- Whether efferocytosis drives phagocyte proliferation outside mammals is unknown. boundary: >- The programme is shown in mouse. DNase II, DNA-PKcs and RICTOR are present in most animals, but DNA-PKcs was lost in Drosophila and C. elegans, so any equivalent programme in those models would need a different sensor. gap_kind: - BIOLOGY dark_aspect: BP_DARK status: OPEN significance: Limits the taxonomic scope of the module. resolution: >- Test for DNase II- and DNA-PKcs-dependent proliferation of corpse-engulfing phagocytes in zebrafish and in an invertebrate that keeps DNA-PKcs (for example mosquito hemocytes), and compare with Drosophila, which lacks it. provenance: - reference_id: PMID:34784501 supporting_text: We found that nucleotides derived from the phagolysosomal hydrolysis of AC-DNA during efferocytosis triggers a signaling pathway that promotes the proliferation of pro-resolving macrophages.