Intestinal lymphatic transport of Smilax china L. pectic polysaccharide via Peyer's patches and its uptake and transport mechanisms in mononuclear phagocytes.

Recently, the intestinal lymphatic transport based on Peyer's patches (PPs) is emerging as a promising absorption pathway for natural polysaccharides. Herein, the aim of this study is to investigate the PP-based oral absorption of a pectic polysaccharide from Smilax china L. (SCLP), as well as its uptake and transport mechanisms in related immune cells. Taking advantages of the traceability of fluorescently labeled SCLP, we confirmed that SCLP could be absorbed into PPs and captured by their mononuclear phagocytes (dendritic cells and macrophages) following oral administration. Subsequently, the systematic in vitro study suggested that the endocytic mechanisms of SCLP by model mononuclear phagocytes (BMDCs and RAW264.7 cells) mainly involved caveolae-mediated endocytosis, macropinocytosis and phagocytosis. More importantly, SCLP directly binds and interacts with toll-like receptor 2 (TLR2) and galectin 3 (Gal-3) receptor, and was taken up by mononuclear phagocytes in receptor-mediated manner. After internalization, SCLP was intracellularly transported primarily through endolysosomal pathway and ultimately localized in lysosomes. In summary, this work reveals novel information and perspectives about the in vivo fate of SCLP, which will contribute to further research and utilization of SCLP and other pectic polysaccharides.

Phosphatidylserine and/or Sialic Acid Modified Liposomes Increase Uptake by Tumor-associated Macrophages and Enhance the Anti-tumor Effect.

DOX liposomes have better therapeutic effects and lower toxic side effects. The targeting ability of liposomes is one of the key factors affecting the therapeutic effect of DOX liposomes. This study developed two types of targeted liposomes. Sialic acid (SA)-modified liposomes were designed to target the highly expressed Siglec-1 receptor on tumor-associated macrophages surface. Phosphatidylserine (PS)-modified liposomes were designed to promote phagocytosis by monocyte-derived macrophages through PS apoptotic signaling. In order to assess and compare the therapeutic potential of different targeted pathways in the context of anti-tumor treatment, we compared four phosphatidylserine membrane materials (DOPS, DSPS, DPPS and DMPS) and found that liposomes prepared using DOPS as material could significantly improve the uptake ability of RAW264.7 cells for DOX liposomes. On this basis, normal DOX liposomes (CL-DOX) and SA-modified DOX liposomes (SAL-DOX), PS-modified DOX liposomes (PS-CL-DOX), SA and PS co-modified DOX liposomes (PS-SAL-DOX) were prepared. The anti-tumor cells function of each liposome on S180 and RAW264.7 in vitro was investigated, and it was found that SA on the surface of liposomes can increase the inhibitory effect. In vivo efficacy results exhibited that SAL-DOX and PS-CL-DOX were superior to other groups in terms of ability to inhibit tumor growth and tumor inhibition index, among which SAL-DOX had the best anti-tumor effect. Moreover, SAL-DOX group mice had high expression of IFN-γ as well as IL-12 factors, which could significantly inhibit mice tumor growth, improve the immune microenvironment of the tumor site, and have excellent targeted delivery potential.

Efferocytosis in dendritic cells: an overlooked immunoregulatory process.

Efferocytosis, the process of engulfing and removing apoptotic cells, plays an essential role in preserving tissue health and averting undue inflammation. While macrophages are primarily known for this task, dendritic cells (DCs) also play a significant role. This review delves into the unique contributions of various DC subsets to efferocytosis, highlighting the distinctions in how DCs and macrophages recognize and handle apoptotic cells. It further explores how efferocytosis influences DC maturation, thereby affecting immune tolerance. This underscores the pivotal role of DCs in orchestrating immune responses and sustaining immune equilibrium, providing new insights into their function in immune regulation.

HMMR triggers immune evasion of hepatocellular carcinoma by inactivation of phagocyte killing.

Hepatocellular carcinoma (HCC) acquires an immunosuppressive microenvironment, leading to unbeneficial therapeutic outcomes. Hyaluronan-mediated motility receptor (HMMR) plays a crucial role in tumor progression. Here, we found that aberrant expression of HMMR could be a predictive biomarker for the immune suppressive microenvironment of HCC, but the mechanism remains unclear. We established an HMMR-/- liver cancer mouse model to elucidate the HMMR-mediated mechanism of the dysregulated "don't eat me" signal. HMMR knockout inhibited liver cancer growth and induced phagocytosis. HMMRhigh liver cancer cells escaped from phagocytosis via sustaining CD47 signaling. Patients with HMMRhighCD47high expression showed a worse prognosis than those with HMMRlowCD47low expression. HMMR formed a complex with FAK/SRC in the cytoplasm to activate NF-κB signaling, which could be independent of membrane interaction with CD44. Notably, targeting HMMR could enhance anti-PD-1 treatment efficiency by recruiting CD8+ T cells. Overall, our data revealed a regulatory mechanism of the "don't eat me" signal and knockdown of HMMR for enhancing anti-PD-1 treatment.

Stiffness sensing via Piezo1 enhances macrophage efferocytosis and promotes the resolution of liver fibrosis.

Tissue stiffening is a predominant feature of fibrotic disorders, but the response of macrophages to changes in tissue stiffness and cellular context in fibrotic diseases remains unclear. Here, we found that the mechanosensitive ion channel Piezo1 was up-regulated in hepatic fibrosis. Macrophages lacking Piezo1 showed sustained inflammation and impaired spontaneous resolution of early liver fibrosis. Further analysis revealed an impairment of clearance of apoptotic cells by macrophages in the fibrotic liver. Macrophages showed enhanced efferocytosis when cultured on rigid substrates but not soft ones, suggesting stiffness-dependent efferocytosis of macrophages required Piezo1 activation. Besides, Piezo1 was involved in the efficient acidification of the engulfed cargo in the phagolysosomes and affected the subsequent expression of anti-inflammation genes after efferocytosis. Pharmacological activation of Piezo1 increased the efferocytosis capacity of macrophages and accelerated the resolution of inflammation and fibrosis. Our study supports the antifibrotic role of Piezo1-mediated mechanical sensation in liver fibrosis, suggesting that targeting PIEZO1 to enhance macrophage efferocytosis could induce fibrosis regression.

Combining SiRPα decoy-coengineered T cells and antibodies augments macrophage-mediated phagocytosis of tumor cells.

The adoptive transfer of T cell receptor-engineered (TCR-engineered) T cells (ACT) targeting the HLA-A2-restricted cancer-testis epitope NY-ESO-1157-165 (A2/NY) has yielded favorable clinical responses against several cancers. Two approaches to improve ACT are TCR affinity optimization and T cell coengineering to express immunomodulatory molecules that can exploit endogenous immunity. By computational design we previously developed a panel of binding-enhanced A2/NY-TCRs including A97L, which augmented the in vitro function of gene-modified T cells as compared with WT. Here, we demonstrated higher persistence and improved tumor control by A97L-T cells. In order to harness macrophages in tumors, we further coengineered A97L-T cells to secrete a high-affinity signal regulatory protein α (SiRPα) decoy (CV1) that blocks CD47. While CV1-Fc-coengineered A97L-T cells mediated significantly better control of tumor outgrowth and survival in Winn assays, in subcutaneous xenograft models the T cells, coated by CV1-Fc, were depleted. Importantly, there was no phagocytosis of CV1 monomer-coengineered T cells by human macrophages. Moreover, avelumab and cetuximab enhanced macrophage-mediated phagocytosis of tumor cells in vitro in the presence of CV1 and improved tumor control upon coadministration with A97L-T cells. Taken together, our study indicates important clinical promise for harnessing macrophages by combining CV1-coengineered TCR-T cells with targeted antibodies to direct phagocytosis against tumor cells.

Macrophage depletion overcomes human hematopoietic cell engraftment failure in zebrafish embryo.

Zebrafish is widely adopted as a grafting model for studying human development and diseases. Current zebrafish xenotransplantations are performed using embryo recipients, as the adaptive immune system, responsible for host versus graft rejection, only reaches maturity at juvenile stage. However, transplanted primary human hematopoietic stem/progenitor cells (HSC) rapidly disappear even in zebrafish embryos, suggesting that another barrier to transplantation exists before the onset of adaptive immunity. Here, using a labelled macrophage zebrafish line, we demonstrated that engraftment of human HSC induces a massive recruitment of macrophages which rapidly phagocyte transplanted cells. Macrophages depletion, by chemical or pharmacological treatments, significantly improved the uptake and survival of transplanted cells, demonstrating the crucial implication of these innate immune cells for the successful engraftment of human cells in zebrafish. Beyond identifying the reasons for human hematopoietic cell engraftment failure, this work images the fate of human cells in real time over several days in macrophage-depleted zebrafish embryos.

Extracellular vesicles containing MFGE8 from colorectal cancer facilitate macrophage efferocytosis.

BACKGROUND: Colorectal cancer (CRC) commonly exhibits tolerance to cisplatin treatment, but the underlying mechanisms remain unclear. Within the tumor microenvironment, macrophages play a role in resisting the cytotoxic effects of chemotherapy by engaging in efferocytosis to clear apoptotic cells induced by chemotherapeutic agents. The involvement of extracellular vesicles (EVs), an intercellular communicator within the tumor microenvironment, in regulating the efferocytosis for the promotion of drug resistance has not been thoroughly investigated. METHODS: We constructed GFP fluorescent-expressing CRC cell lines (including GFP-CT26 and GFP-MC38) to detect macrophage efferocytosis through flow cytometric analysis. We isolated and purified CRC-secreted EVs using a multi-step ultracentrifugation method and identified them through electron microscopy and nanoflow cytometry. Proteomic analysis was conducted to identify the protein molecules carried by CRC-EVs. MFGE8 knockout CRC cell lines were constructed using CRISPR-Cas9, and their effects were validated through in vitro and in vivo experiments using Western blotting, immunofluorescence, and flow cytometric analysis, confirming that these EVs activate the macrophage αvß3-Src-FAK-STAT3 signaling pathway, thereby promoting efferocytosis. RESULTS: In this study, we found that CRC-derived EVs (CRC-EVs) enhanced macrophage efferocytosis of cisplatin-induced apoptotic CRC cells. Analysis of The Cancer Genome Atlas (TCGA) database revealed a high expression of the efferocytosis-associated gene MFGE8 in CRC patients, suggesting a poorer prognosis. Additionally, mass spectrometry-based proteomic analysis identified a high abundance of MFGE8 protein in CRC-EVs. Utilizing CRISPR-Cas9 gene edition system, we generated MFGE8-knockout CRC cells, demonstrating that their EVs fail to upregulate macrophage efferocytosis in vitro and in vivo. Furthermore, we demonstrated that MFGE8 in CRC-EVs stimulated macrophage efferocytosis by increasing the expression of αvß3 on the cell surface, thereby activating the intracellular Src-FAK-STAT3 signaling pathway. CONCLUSIONS: Therefore, this study highlighted a mechanism in CRC-EVs carrying MFGE8 activated the macrophage efferocytosis. This activation promoted the clearance of cisplatin-induced apoptotic CRC cells, contributing to CRC resistance against cisplatin. These findings provide novel insights into the potential synergistic application of chemotherapy drugs, EVs inhibitors, and efferocytosis antagonists for CRC treatment.

Structural Characterization of Polygonatum Cyrtonema Polysaccharide and Its Immunomodulatory Effects on Macrophages.

A neutral Polygonatum cyrtonema polysaccharide (NPCP) was isolated and purified from Polygonatum cyrtonema by various chromatographic techniques, including DEAE-52 and Sephadex-G100 chromatography. The structure of NPCP was characterized by HPLC, HPGPC, GC-MS, FT-IR, NMR, and SEM. Results showed that NPCP is composed of glucose (55.4%) and galactose (44.6%) with a molecular weight of 3.2 kDa, and the sugar chain of NPCP was →1)-α-D-Glc-(4→1)-ß-D-Gal-(3→. In vitro bioactivity experiments demonstrated that NPCP significantly enhanced macrophages proliferation and phagocytosis while inhibiting the M1 polarization induced by LPS as well as the M2 polarization induced by IL-4 and IL-13 in macrophages. Additionally, NPCP suppressed the secretion of IL-6 and TNF-α in both M1 and M2 cells but promoted the secretion of IL-10. These results suggest that NPCP could serve as an immunomodulatory agent with potential applications in anti-inflammatory therapy.

Killer Function of Circulating Neutrophils in Relation to Cytokines in Uterine Myoma and Endometrial Cancer.

The study presents the killer functions of circulating neutrophils: myeloperoxidase activity, the ability to generate ROS, phagocytic activity, receptor status, NETosis, as well as the level of cytokines IL-2, IL-4, IL-6, IL-17A, and IL-18, granulocyte CSF, monocyte chemotactic protein 1, and neutrophil elastase in the serum of patients with uterine myoma and endometrial cancer (FIGO stages I-III). The phagocytic ability of neutrophils in uterine myoma was influenced by serum levels of granulocyte CSF and IL-2 in 54% of the total variance. The degranulation ability of neutrophils in endometrial cancer was determined by circulating IL-18 in 50% of the total variance. In uterine myoma, 66% of the total variance in neutrophil myeloperoxidase activity was explained by a model dependent on blood levels of IL-17A, IL-6, and IL-4. The risk of endometrial cancer increases when elevated levels of monocyte chemotactic protein 1 in circulating neutrophils are associated with reduced ability to capture particles via extracellular traps (96% probability).

Resolvin E1 improves efferocytosis and rescues severe aplastic anemia in mice.

Severe aplastic anemia (SAA) is a rare, fatal disease characterized by severe cytopenias and loss of hematopoietic stem cells (HSCs). Immune-mediated destruction and inflammation are known drivers of SAA, however, the underlying mechanisms driving persistent inflammation are unknown. Current treatments for SAA rely on immunosuppressive therapies or HSC transplantation, however, these treatments are not always effective. Using an established mouse model of SAA, we observed a significant increase in apoptotic cells within the bone marrow (BM) and impaired efferocytosis in SAA mice, relative to radiation controls. Single-cell transcriptomic analysis revealed heterogeneity among BM monocytes and unique populations emerged during SAA characterized by increased inflammatory signatures and significantly increased expression of Sirpa and Cd47. CD47, a "don't eat me" signal, was increased on both live and apoptotic BM cells, concurrent with markedly increased expression of signal regulatory protein alpha (SIRPα) on monocytes. Functionally, SIRPα blockade improved cell clearance and reduced accumulation of CD47-positive apoptotic cells. Lipidomic analysis revealed a reduction in the precursors of specialized pro-resolving lipid mediators (SPMs) and increased prostaglandins in the BM during SAA, indicative of impaired inflammation resolution. Specifically, 18-HEPE, a precursor of E-series resolvins, was significantly reduced in SAA-induced mice relative to radiation controls. Treatment of SAA mice with Resolvin E1 (RvE1) improved efferocytic function, BM cellularity, platelet output, and survival. Our data suggest that impaired efferocytosis and inflammation resolution contributes to SAA progression and demonstrate that SPMs, such as RvE1, offer new and/or complementary treatments for SAA that do not rely on immune suppression.

CD5L as a promising biological therapeutic for treating sepsis.

Sepsis results from systemic, dysregulated inflammatory responses to infection, culminating in multiple organ failure. Here, we demonstrate the utility of CD5L for treating experimental sepsis caused by cecal ligation and puncture (CLP). We show that CD5L's important features include its ability to enhance neutrophil recruitment and activation by increasing circulating levels of CXCL1, and to promote neutrophil phagocytosis. CD5L-deficient mice exhibit impaired neutrophil recruitment and compromised bacterial control, rendering them susceptible to attenuated CLP. CD5L-/- peritoneal cells from mice subjected to medium-grade CLP exhibit a heightened pro-inflammatory transcriptional profile, reflecting a loss of control of the immune response to the infection. Intravenous administration of recombinant CD5L (rCD5L) in immunocompetent C57BL/6 wild-type (WT) mice significantly ameliorates measures of disease in the setting of high-grade CLP-induced sepsis. Furthermore, rCD5L lowers endotoxin and damage-associated molecular pattern (DAMP) levels, and protects WT mice from LPS-induced endotoxic shock. These findings warrant the investigation of rCD5L as a possible treatment for sepsis in humans.

Transcriptomic profiling and regulatory pathways of cardiac resident macrophages in aging.

Cardiovascular diseases are an array of age-related disorders, and accumulating evidence suggests a link between cardiac resident macrophages (CRMs) and the age-related disorders. However, how does CRMs alter with aging remains elusive. In the present study, aged mice (20 months old) have been employed to check for their cardiac structural and functional alterations, and the changes in the proportion of CRM subsets as well, followed by sorting of CRMs, including C-C Motif Chemokine Receptor 2 (CCR2)+ and CCR2- CRMs, which were subjected to Smart-Seq. Integrated analysis of the Smart-Seq data with three publicly available single-cell RNA-seq datasets revealed that inflammatory genes were drastic upregulated for both CCR2+ and CCR2- CRMs with aging, but genes germane to wound healing were downregulated for CCR2- CRMs, suggesting the differential functions of these two subsets. More importantly, inflammatory genes involved in damage sensing, complement cascades, and phagocytosis were largely upregulated in CCR2- CRMs, implying the imbalance of inflammatory response upon aging. Our work provides a comprehensive framework and transcriptional resource for assessing the impact of aging on CRMs with a potential for further understanding cardiac aging.

Efferocytosis by macrophages in physiological and pathological conditions: regulatory pathways and molecular mechanisms.

Efferocytosis is defined as the highly effective phagocytic removal of apoptotic cells (ACs) by professional or non-professional phagocytes. Tissue-resident professional phagocytes ("efferocytes"), such as macrophages, have high phagocytic capacity and are crucial to resolve inflammation and aid in homeostasis. Recently, numerous exciting discoveries have revealed divergent (and even diametrically opposite) findings regarding metabolic immune reprogramming associated with efferocytosis by macrophages. In this review, we highlight the key metabolites involved in the three phases of efferocytosis and immune reprogramming of macrophages under physiological and pathological conditions. The next decade is expected to yield further breakthroughs in the regulatory pathways and molecular mechanisms connecting immunological outcomes to metabolic cues as well as avenues for "personalized" therapeutic intervention.

Molecular characteristics of Echinococcus multilocularis FABP1 and its regulatory functions on murine macrophages.

Fatty acid binding proteins (FABPs) have emerged as attractive vaccination candidates for several platyhelminth species. To explore the physiological functions of Echinococcus multilocularis (E. multilocularis) FABP, the molecular characteristics of EmFABP1 were analyzed by online software, and the regulatory roles of rEmFABP1 protein in murine macrophages were further investigated. The emfabp1 gene encodes 133 amino acids with the characteristic ß-barrel shape of the cytoplasmic FABP family. Natural EmFABP1 protein is predominantly expressed in protoscoleces tegument and germinal layer cells and is also detected in cyst fluid and exosomes of E. multilocularis. rEmFABP1 protein demonstrated a notable suppression of phagocytic activity and nitric oxide production in murine macrophages. Additionally, the protein was observed to promote apoptosis and regulate cytokine expression in macrophages. These findings suggested that E. multilocularis FABP1 is critical in modifying macrophage physiological processes and that this protein may have immunomodulatory roles during infection.

Molecular incompatibility between pig CD200 and human CD200 receptor in in vitro xenogeneic immune responses.

Overexpression of human CD200 (hCD200) in porcine endothelial cells (PECs) has been reported to suppress xenogeneic immune responses of human macrophages against porcine endothelial cells. The current study aimed to address whether the above-mentioned beneficial effect of hCD200 is mediated by overcoming the molecular incompatibility between porcine CD200 (pCD200) and hCD200 receptor or simply by increasing the expression levels of CD200 without any molecular incompatibility across the two species. We overexpressed hCD200 or pCD200 using lentiviral vectors with V5 marker in porcine endothelial cells and compared their suppressive activity against U937-derived human macrophage-like cells (hMCs) and primary macrophages. In xenogeneic coculture of porcine endothelial cells and human macrophage-like cells or macrophages, hCD200-porcine endothelial cells suppressed phagocytosis and cytotoxicity of human macrophages to a greater extent than pCD200-porcine endothelial cells. Secretion of tumor necrosis factor-α, interleukin-1ß, and monocyte chemoattractant protein-1 from human macrophages and expression of M1 phenotypes (inducible nitric oxide synthase, dectin-1, and CD86) were also suppressed by hCD200 to a greater extent than pCD200. Furthermore, in signal transduction downstream of CD200 receptor, hCD200 induced Dok2 phosphorylation and suppressed IκB phosphorylation to a greater extent than pCD200. The above data supported the possibility of a significant molecular incompatibility between pCD200 and human CD200 receptor, suggesting that the beneficial effects of hCD200 overexpression in porcine endothelial cells could be mediated by overcoming the molecular incompatibility across the species barrier rather than by simple overexpression effects of CD200.

Glioblastoma Phagocytic Cell Death: Balancing the Opportunities for Therapeutic Manipulation.

Macrophages and microglia are professional phagocytes that sense and migrate toward "eat-me" signals. The role of phagocytic cells is to maintain homeostasis by engulfing senescent or apoptotic cells, debris, and abnormally aggregated macromolecules. Usually, dying cells send out "find-me" signals, facilitating the recruitment of phagocytes. Healthy cells can also promote or inhibit the phagocytosis phenomenon of macrophages and microglia by tuning the balance between "eat-me" and "don't-eat-me" signals at different stages in their lifespan, while the "don't-eat-me" signals are often hijacked by tumor cells as a mechanism of immune evasion. Using a combination of bioinformatic analysis and spatial profiling, we delineate the balance of the "don't-eat-me" CD47/SIRPα and "eat-me" CALR/STC1 ligand-receptor interactions to guide therapeutic strategies that are being developed for glioblastoma sequestered in the central nervous system (CNS).

Interleukin-33 ameliorates perioperative neurocognitive disorders by modulating microglial state.

Perioperative neurocognitive disorders (PND) are cognitive dysfunctions that usually occur in elderly patients after anesthesia and surgery. Microglial overactivation is a key underlying mechanism. Interleukin-33 (IL-33) is a member of the IL-1 family that orchestrates microglial function. In the present study, we explored how IL-33, which regulates microglia, contributes to cognitive improvement in a male mouse model of PND. An exploratory laparotomy was performed to establish a PND model. The expression levels of IL-33 and its receptor ST2 were evaluated using Western blot. IL-33/ST2 secretion, microglial density, morphology, phagocytosis of synapse, and proliferation, and dystrophic microglia were assessed using immunofluorescence. Synaptic plasticity was measured using Golgi staining and long-term potentiation. The Morris water maze and open field test were used to evaluate cognitive function and anxiety. Hippocampal expression of IL-33 and ST2 were elevated on postoperative day 3. We confirmed that IL-33 was secreted by astrocytes and neurons, whereas ST2 mainly colocalized with microglia. IL-33 treatment induced microgliosis after anesthesia and surgery. These microglia had larger soma sizes and shorter and fragmented branches. Compared to the Surgery group, IL-33 treatment reduced the synaptic phagocytosis of microglia and increased microglial proliferation and dystrophic microglia. IL-33 treatment also reversed the impaired synaptic plasticity and cognitive function caused by anesthesia and surgery. In conclusion, these results indicate that IL-33 plays a key role in regulating microglial state and synaptic phagocytosis in a PND mouse model. IL-33 treatment has a therapeutic potential for improving cognitive dysfunction in PND.

In the Eyes of the Beholder-New Mertk Knockout Mouse and Re-Evaluation of Phagocytosis versus Anti-Inflammatory Functions of MERTK.

Greg Lemke's laboratory was one of the pioneers of research into the TAM family of receptor tyrosine kinases (RTKs). Not only was Tyro3 cloned in his laboratory, but his group also extensively studied mice knocked out for individual or various combinations of the TAM RTKs Tyro3, Axl, and Mertk. Here we primarily focus on one of the paralogs-MERTK. We provide a historical perspective on rodent models of loss of Mertk function and their association with retinal degeneration and blindness. We describe later studies employing mouse genetics and the generation of newer knockout models that point out incongruencies with the inference that loss of MERTK-dependent phagocytosis is sufficient for severe, early-onset photoreceptor degeneration in mice. This discussion is meant to raise awareness with regards to the limitations of the original Mertk knockout mouse model generated using 129 derived embryonic stem cells and carrying 129 derived alleles and the role of these alleles in modifying Mertk knockout phenotypes or even displaying Mertk-independent phenotypes. We also suggest molecular approaches that can further Greg Lemke's scintillating legacy of dissecting the molecular functions of MERTK-a protein that has been described to function in phagocytosis as well as in the negative regulation of inflammation.

Transient caspase-mediated activation of caspase-activated DNase causes DNA damage required for phagocytic macrophage differentiation.

Phagocytic macrophages are crucial for innate immunity and tissue homeostasis. Most tissue-resident macrophages develop from embryonic precursors that populate every organ before birth to lifelong self-renew. However, the mechanisms for versatile macrophage differentiation remain unknown. Here, we use in vivo genetic and cell biological analysis of the Drosophila larval hematopoietic organ, the lymph gland that produces macrophages. We show that the developmentally regulated transient activation of caspase-activated DNase (CAD)-mediated DNA strand breaks in intermediate progenitors is essential for macrophage differentiation. Insulin receptor-mediated PI3K/Akt signaling regulates the apoptosis signal-regulating kinase 1 (Ask1)/c-Jun kinase (JNK) axis to control sublethal levels of caspase activation, causing DNA strand breaks during macrophage development. Furthermore, caspase activity is also required for embryonic-origin macrophage development and efficient phagocytosis. Our study provides insights into developmental signaling and CAD-mediated DNA strand breaks associated with multifunctional and heterogeneous macrophage differentiation.