Conserved and Distinct Elements of Phagocytosis in Human and C. elegans

Endocytosis provides the cellular nutrition and homeostasis of organisms, but pathogens often take advantage of this entry point to infect host cells. This is counteracted by phagocytosis that plays a key role in the protection against invading microbes both during the initial engulfment of pathogens and in the clearance of infected cells. Phagocytic cells balance two vital functions: preventing the accumulation of cell corpses to avoid pathological inflammation and autoimmunity, whilst maintaining host defence. In this review, we compare elements of phagocytosis in mammals and the nematode Caenorhabditis elegans. Initial recognition of infection requires different mechanisms. In mammals, pattern recognition receptors bind pathogens directly, whereas activation of the innate immune response in the nematode rather relies on the detection of cellular damage. In contrast, molecules involved in efferocytosis—the engulfment and elimination of dying cells and cell debris—are highly conserved between the two species. Therefore, C. elegans is a powerful model to research mechanisms of the phagocytic machinery. Finally, we show that both mammalian and worm studies help to understand how the two phagocytic functions are interconnected: emerging data suggest the activation of innate immunity as a consequence of defective apoptotic cell clearance.     

Defective Efferocytosis in a Murine Model of Sjögren’s Syndrome Is Mediated by Dysfunctional Mer Tyrosine Kinase Receptor

Sjögren’s syndrome (SjS) is a chronic autoimmune disease primarily involving the exocrine glands in which the involvement of the innate immune system is largely uncharacterized. Mer signaling has been found to be protective in several autoimmune diseases but remains unstudied in SjS. Here, we investigated the role of Mer signaling in SjS. Mer knockout (MerKO) mice were examined for SjS disease criteria. SjS-susceptible (SjS^S) C57BL/6.NOD-Aec1Aec2 mice were assessed for defective Mer signaling outcomes, soluble Mer (sMer) levels, A disintegrin and metalloprotease 17 (ADAM17) activity, and Rac1 activation. In addition, SjS patient plasma samples were evaluated for sMer levels via ELISA, and sMer levels were correlated to disease manifestations. MerKO mice developed submandibular gland (SMG) lymphocytic infiltrates, SMG apoptotic cells, anti-nuclear autoantibodies (ANA), and reduced saliva flow. Mer signaling outcomes were observed to be diminished in SjS^S mice, as evidenced by reduced Rac1 activation in SjS^S mice macrophages in response to apoptotic cells and impaired efferocytosis. Increased sMer was also detected in SjS^S mouse sera, coinciding with higher ADAM17 activity, the enzyme responsible for cleavage and inactivation of Mer. sMer levels were elevated in patient plasma and positively correlated with focus scores, ocular staining scores, rheumatoid factors, and anti-Ro60 levels. Our data indicate that Mer plays a protective role in SjS, similar to other autoimmune diseases. Furthermore, we suggest a series of events where enhanced ADAM17 activity increases Mer inactivation and depresses Mer signaling, thus removing protection against the loss of self-tolerance and the onset of autoimmune disease in SjS^S mice.     

Myeloid ABCG1 Deficiency Enhances Apoptosis and Initiates Efferocytosis in Bronchoalveolar Lavage Cells of Murine Multi-Walled Carbon Nanotube-Induced Granuloma Model

The use of carbon nanotubes has increased in the past few decades. Carbon nanotubes are implicated in the pathogenesis of pulmonary sarcoidosis, a chronic granulomatous inflammatory condition. We developed a murine model of chronic granulomatous inflammation using multiwall carbon nanotubes (MWCNT) to investigate mechanisms of granuloma formation. Using this model, we demonstrated that myeloid deficiency of ATP-binding cassette (ABC) cholesterol transporter (ABCG1) promotes granuloma formation and fibrosis with MWCNT instillation; however, the mechanism remains unclear. Our previous studies showed that MWCNT induced apoptosis in bronchoalveolar lavage (BAL) cells of wild-type (C57BL/6) mice. Given that continual apoptosis causes persistent severe lung inflammation, we hypothesized that ABCG1 deficiency would increase MWCNT-induced apoptosis thereby promoting granulomatous inflammation and fibrosis. To test our hypothesis, we utilized myeloid-specific ABCG1 knockout (ABCG1 KO) mice. Our results demonstrate that MWCNT instillation enhances pulmonary fibrosis in ABCG1 KO mice compared to wild-type controls. Enhanced fibrosis is indicated by increased trichrome staining and transforming growth factor-beta (TGF-β) expression in lungs, together with an increased expression of TGF-β related signaling molecules, interleukin-13 (IL-13) and Smad-3. MWCNT induced more apoptosis in BAL cells of ABCG1 KO mice. Initiation of apoptosis is most likely mediated by the extrinsic pathway since caspase 8 activity and Fas expression are significantly higher in MWCNT instilled ABCG1 KO mice compared to the wild type. In addition, TUNEL staining shows that ABCG1 KO mice instilled with MWCNT have a higher percentage of TUNEL positive BAL cells and more efferocytosis than the WT control. Furthermore, BAL cells of ABCG1 KO mice instilled with MWCNT exhibit an increase in efferocytosis markers, milk fat globule-EGF factor 8 (MFG-E8) and integrin β3. Therefore, our observations suggest that ABCG1 deficiency promotes pulmonary fibrosis by MWCNT, and this effect may be due to an increase in apoptosis and efferocytosis in BAL cells.     

Secretome of Mesenchymal Stem Cells from Consecutive Hypoxic Cultures Promotes Resolution of Lung Inflammation by Reprogramming Anti-Inflammatory Macrophages

The secretome from hypoxia-preconditioned mesenchymal stem cells (MSCs) has been shown to promote resolution of inflammation and alleviate acute lung injury (ALI) through its immunomodulatory function. However, the effects of consecutive hypoxic culture on immunomodulatory function of the MSCs secretome are largely unclarified. Here, we intend to investigate the effects of consecutive hypoxia on therapeutic efficacy of conditioned medium derived from MSCs (MSCs-CM) in alleviating ALI. Human umbilical cord-derived MSCs (UC-MSCs) were consecutively cultured in 21% O₂ (Nor-MSCs) or in 1% O₂ (Hypo-MSCs) from passage 0. Their conditioned medium (Nor-CM and Hypo-CM respectively) was collected and administered into ALI models. Our findings confirmed that Hypo-MSCs exhibited increased proliferation ability and decreased cell senescence compared with Nor-MSCs. Consecutive hypoxia promoted UC-MSCs to secrete immunomodulatory cytokines, such as insulin-like growth factor 1(IGF1), IL10, TNFα-stimulated gene 6(TSG6), TGFβ, and prostaglandin E2 (PGE2). Both Nor-CM and Hypo-CM could effectively limit lung inflammation, promote efferocytosis and modulate anti-inflammatory polarization of lung macrophages in ALI models. Moreover, the effects of Hypo-CM were more potent than Nor-CM. Taken together, our findings indicate that consecutive hypoxic cultures could not only promote both proliferation and quality of UC-MSCs, but also enhance the therapeutic efficacy of their secretome in mitigating lung inflammation by promoting efferocytosis and anti-inflammatory polarization of macrophages.     

Cancer Immunotherapy Based on Cell Membrane-Coated Nanocomposites Augmenting cGAS/STING Activation by Efferocytosis Blockade

Innate immunity triggered by the cGAS/STING pathway has the potential to improve cancer immunotherapy. Previously, the authors reported that double-stranded DNA (dsDNA) released by dying tumor cells can trigger the cGAS/STING pathway. However, owing to efferocytosis, dying tumor cells are engulfed and cleared before the damaged dsDNA is released; hence, immunologic tolerance and immune escape occur. Herein, a cancer-cell-membrane biomimetic nanocomposites that exhibit tumor-immunotherapeutic effects are synthesized by augmenting the cGAS/STING pathway and suppressing efferocytosis. Once internalized by cancer cells, a combined chemo/chemodynamic therapy would be triggered, which damages their nuclear and mitochondrial DNA. Furthermore, the releasing Annexin A5 protein could inhibit efferocytosis effect and promote immunostimulatory secondary necrosis by preventing phosphatidylserine exposure, resulting in the burst release of dsDNA. These dsDNA fragments, as molecular patterns to immunogenic damage, escape from the cancer cells, activate the cGAS/STING pathway, enhance cross-presentation inside dendritic cells, and promote M1-polarization of tumor-associated macrophages. In vivo experiments suggest that the proposed nanocomposite could recruit cytotoxic T-cells and facilitate long-term immunological memory. Moreover, when combined with immune-checkpoint blockades, it could augment the immune response. Therefore, this novel biomimetic nanocomposite is a promising strategy for generating adaptive antitumor immune responses.     

Adjunctive Thymosin Beta-4 Treatment Influences MΦ Effector Cell Function to Improve Disease Outcome in Pseudomonas aeruginosa-Induced Keratitis

Our previous work has shown that topical thymosin beta 4 (Tβ4) as an adjunct to ciprofloxacin treatment reduces inflammatory mediators and inflammatory cell infiltrates (neutrophils/PMN and macrophages/MΦ) while enhancing bacterial killing and wound healing pathway activation in an experimental model of P. aeruginosa-induced keratitis. This study aimed to mechanistically examine how Tβ4 influences MΦ function in particular, leading to reduced inflammation and enhanced host defense following P. aeruginosa-induced infection of the cornea. Flow cytometry was conducted to profile the phenotype of infiltrating MΦ after infection, while generation of reactive nitrogen species and markers of efferocytosis were detected to assess functional activity. In vitro studies were performed utilizing RAW 264.7 cells to verify and extend the in vivo findings. Tβ4 treatment decreases MΦ infiltration and regulates the activation state in response to infected corneas. MΦ functional data demonstrated that the adjunctive Tβ4 treatment group significantly downregulated reactive nitrogen species (RNS) production and efferocytotic activity. In addition, the in vitro studies showed that both Tβ4 alone and adjunctive Tβ4 treatment influenced MΦ cellular function following LPS stimulation. Collectively, these data provide further evidence that adjunctive Tβ4 + ciprofloxacin treatment offers a more efficacious option for treating bacterial keratitis. Not only does the adjunctive therapy address both the infectious pathogen and corneal wound healing response, but it also influences MΦ infiltration, activation, and function, as revealed by the current study.     

Macrophage-Mediated Tumor Cell Phagocytosis: Opportunity for Nanomedicine Intervention

Macrophages are one of the most abundant non-malignant cells in the tumor microenvironment, playing critical roles in mediating tumor immunity. As important innate immune cells, macrophages possess the potential to engulf tumor cells and present tumor-specific antigens for adaptive antitumor immunity induction, leading to growing interest in targeting macrophage phagocytosis for cancer immunotherapy. Nevertheless, live tumor cells have evolved to evade phagocytosis by macrophages via the extensive expression of anti-phagocytic molecules, such as CD47. In addition, macrophages also rapidly recognize and engulf apoptotic cells (efferocytosis) in the tumor microenvironment, which inhibits inflammatory responses and facilitates immune escape of tumor cells. Thus, intervention of macrophage phagocytosis by blocking anti-phagocytic signals on live tumor cells or inhibiting tumor efferocytosis presents a promising strategy for the development of cancer immunotherapies. Here, the regulation of macrophage-mediated tumor cell phagocytosis is first summarized, followed by an overview of strategies targeting macrophage phagocytosis for the development of antitumor therapies. Given the potential off-target effects associated with the administration of traditional therapeutics (for example, monoclonal antibodies and small molecule inhibitors), the opportunity for nanomedicine in macrophage phagocytosis intervention is highlighted.     

Embryonic-Derived Myb− Macrophages Enhance Bacterial Clearance and Improve Survival in Rat Sepsis

Peritoneal resident macrophages play a key role in combating sepsis in the peritoneal cavity. We sought to determine if peritoneal transplantation of embryonic Myb⁻ “peritoneal-like” macrophages attenuate abdominal fecal sepsis. Directed differentiation of rodent pluripotent stem cells (PSCs) was used in factor-defined media to produce embryonic-derived large “peritoneal-like” macrophages (Ed-LPM) that expressed peritoneal macrophage markers and demonstrated phagocytic capacity. Preclinical in vivo studies determined Ed-LPM efficacy in rodent abdominal fecal sepsis with or without Meropenem. Ex vivo studies explored the mechanism and effects of Ed-LPM on host immune cell number and function, including phagocytosis, reactive oxygen species (ROS) production, efferocytosis and apoptosis. Ed-LPM reduced sepsis severity by decreasing bacterial load in the liver, spleen and lungs. Ed-LPM therapy significantly improved animal survival by ~30% and reduced systemic bacterial burden to levels comparable to Meropenem therapy. Ed-LPM therapy decreased peritoneal TNFα while increasing IL-10 concentrations. Ed-LPMs enhanced peritoneal macrophage phagocytosis of bacteria, increased macrophage production of ROS and restored homeostasis via apoptosis and efferocytosis-induced clearance of neutrophils. In conclusion, Ed-LPM reduced systemic sepsis severity, improved survival and reduced bacterial load by enhancing peritoneal macrophage bacterial phagocytosis and killing and clearance of intra-peritoneal neutrophils. Macrophage therapy may be a potential strategy to address sepsis.