Robin Michael Yates

Macrophages play a pivotal role in clearing debris and microbes from the microenvironment via phagocytosis and orchestrating local inflammation. Although pathogen- and damage-associated molecular patterns (PAMPs and DAMPs) are understood to mostly be released through the synthesis and secretion of soluble mediators, such as cytokines and eicosanoids, it has been recently proposed that macrophages can release previously phagocytosed and processed PAMPs and DAMPs into the local microenvironment via a process termed eructophagy, and that these, in turn, can activate recently recruited leukocytes. Additionally, it has been commonly observed that local macrophages physically interact with other leukocytes, such as neutrophils and T cells, recruited to sites of inflammation. This study demonstrates that eructophagy in macrophages is significantly induced during physical interaction with neutrophils and T cells, which is mediated by ICAM1 on macrophages and lymphocyte function-associated antigen 1 (LFA1) on neutrophils and T cells. Notably, ICAM1 activation alone is sufficient to trigger eructophagy in macrophages and is dependent on Lyn kinase. Through this mechanism, it is proposed that neutrophils and lymphocytes can influence their own activation by interacting with local macrophages containing PAMP-containing phagolysosomes, which subsequently triggers PAMP release into the local microenvironment through eructophagy.

Conventional dendritic cells (cDCs) generate protective cytotoxic T lymphocyte (CTL) responses against extracellular pathogens and tumors. This is achieved through a process known as cross-presentation (XP), and, despite its biological importance, the mechanism(s) driving XP remains unclear. Here, we show that a cDC-specific pore-forming protein called apolipoprotein L 7C (APOL7C) is up-regulated in response to innate immune stimuli and is recruited to phagosomes. Association of APOL7C with phagosomes led to phagosomal rupture and escape of engulfed antigens to the cytosol, where they could be processed via the endogenous MHC class I antigen processing pathway. Accordingly, mice deficient in APOL7C did not efficiently prime CD8 ⁺ T cells in response to immunization with bead-bound and cell-associated antigens. Together, our data indicate the presence of dedicated apolipoproteins that mediate the delivery of phagocytosed proteins to the cytosol of activated cDCs to facilitate XP.

The phagosome is a redox-active organelle. Numerous reductive and oxidative systems play both direct and indirect roles in phagosomal function. With the advent of newer methodologies to study these redox events in live cells, the details of how redox conditions change within the maturing phagosome, how they are regulated, and how they influence other phagosomal functions can be investigated. In this chapter, we detail phagosome-specific, fluorescence-based assays that measure disulfide reduction and the production of reactive oxygen species in live phagocytes such as macrophages and dendritic cells, in real time. Key words Phagosome Lysosome Oxidation Disulfide reduction Redox

The use of Hox-driven conditionally immortalized immune cells has significantly increased in biomedical research over the past 15 years. HoxB8-driven conditionally immortalized myeloid progenitor cells maintain their ability to differentiate into functional macrophages. There are multiple benefits to this conditional immortalization strategy including the ability for unlimited propagation, genetic mutability, primary-like immune cells (macrophages, dendritic cells, and granulocytes) on demand, derivation from variety of mouse strains, and simple cryopreservation and reconstitution. In this chapter, we will discuss how to derive and use these HoxB8-conditionally immortalized myeloid progenitor cells. Key words HoxB8 Conditional immortalization Differentiation Macrophages Phagosomes

The phagolysosome is an antimicrobial and degradative organelle that plays a key role in macrophage-mediated inflammation and homeostasis. Before being presented to the adaptive immune system, phagocytosed proteins must first be processed into immunostimulatory antigens. Until recently, little attention has been given to how other processed PAMPs and DAMPs can stimulate an immune response if they are sequestered in the phagolysosome. Eructophagy is a newly described process in macrophages that releases partially digested immunostimulatory PAMPs and DAMPs extracellularly from the mature phagolysosome to activate vicinal leukocytes. This chapter outlines approaches to observe and quantify eructophagy by simultaneously measuring several phagosomal parameters of individual phagosomes. These methods use specifically designed experimental particles capable of conjugating to multiple reporter/reference fluors in combination with real-time automated fluorescent microscopy. Through the use of high-content image analysis software, each phagosomal parameter can be evaluated quantitatively or semiquantitatively during post-analysis. Key words Phagosome Phagolysosome Macrophage Eructophagy Proteolysis Acidification Secretion

The phagolysosome is an antimicrobial and degradative organelle that plays key roles in macrophage-mediated inflammatory and homeostatic functions. Whereas mature phagolysosomes are known to sequester and degrade their contents into basic nutrients, they were not previously assigned an active role in amplifying inflammation. We have described a novel macrophage process in which partially digested immunostimulatory PAMPs are released extracellularly from the mature phagolysosome via discrete events we term eructophagy. Eructophagy is induced by proinflammatory stimuli, negatively regulated by IL4 and MTOR, and is dependent on key autophagy proteins, including fusion machinery of degradative and secretory autophagy. We propose that macrophages use eructophagy to release processed PAMPs/DAMPs to amplify local inflammation.

Those who study macrophage biology struggle with the decision whether to utilize primary macrophages derived directly from mice or opt for the convenience and genetic tractability of immortalized macrophage‐like cell lines in in vitro studies. Particularly when it comes to studying phagocytosis and phagosomal maturation—a signature cellular process of the macrophage—many commonly used cell lines are not representative of what occurs in primary macrophages. A system developed by Mark Kamps' group, that utilizes conditionally constitutive activity of Hox transcription factors (Hoxb8 and Hoxa9) to immortalize differentiation‐competent myeloid cell progenitors of mice, offers an alternative to the macrophage/macrophage‐like dichotomy. In this resource, we will review the use of Hoxb8 and Hoxa9 as hematopoietic regulators to conditionally immortalize murine hematopoietic progenitor cells which retain their ability to differentiate into many functional immune cell types including macrophages, neutrophils, basophils, osteoclasts, eosinophils, dendritic cells, as well as limited potential for the generation of lymphocytes. We further demonstrate that the use of macrophages derived from Hoxb8/Hoxa9 immortalized progenitors and their similarities to bone marrow‐derived macrophages. To supplement the existing data, mass spectrometry‐based proteomics, flow cytometry, cytology, and in vitro phagosomal assays were conducted on macrophages derived from Hoxb8 immortalized progenitors and compared to bone marrow‐derived macrophages and the macrophage‐like cell line J774. We additionally propose the use of a standardized nomenclature to describe cells derived from the Hoxb8/Hoxa9 system in anticipation of their expanded use in the study of leukocyte cell biology.

Recognition of pathogen-or-damage-associated molecular patterns is critical to inflammation. However, most pathogen-or-damage-associated molecular patterns exist within intact microbes/cells and are typically part of non-diffusible, stable macromolecules that are not optimally immunostimulatory or available for immune detection. Partial digestion of microbes/cells following phagocytosis potentially generates new diffusible pathogen-or-damage-associated molecular patterns, however, our current understanding of phagosomal biology would have these molecules sequestered and destroyed within phagolysosomes. Here, we show the controlled release of partially-digested, soluble material from phagolysosomes of macrophages through transient, iterative fusion-fission events between mature phagolysosomes and the plasma membrane, a process we term eructophagy. Eructophagy is most active in proinflammatory macrophages and further induced by toll like receptor engagement. Eructophagy is mediated by genes encoding proteins required for autophagy and can activate vicinal cells by release of phagolysosomally-processed, partially-digested pathogen associated molecular patterns. We propose that eructophagy allows macrophages to amplify local inflammation through the processing and dissemination of pathogen-or-damage-associated molecular patterns.

The type I interferon (IFN) signaling pathway has important functions in resistance to viral infection, with the downstream induction of interferon stimulated genes (ISG) protecting the host from virus entry, replication and spread. Listeria monocytogenes (Lm), a facultative intracellular foodborne pathogen, can exploit the type I IFN response as part of their pathogenic strategy, but the molecular mechanisms involved remain unclear. Here we show that type I IFN suppresses the antibacterial activity of phagocytes to promote systemic Lm infection. Mechanistically, type I IFN suppresses phagosome maturation and proteolysis of Lm virulence factors ActA and LLO, thereby promoting phagosome escape and cell-to-cell spread; the antiviral protein, IFN-induced transmembrane protein 3 (IFITM3), is required for this type I IFN-mediated alteration. Ifitm3^−/− mice are resistant to systemic infection by Lm, displaying decreased bacterial spread in tissues, and increased immune cell recruitment and pro-inflammatory cytokine signaling. Together, our findings show how an antiviral mechanism in phagocytes can be exploited by bacterial pathogens, and implicate IFITM3 as a potential antimicrobial therapeutic target.