Hox-driven conditional immortalization of myeloid and lymphoid progenitors: Uses, advantages, and future potential.

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.

Extracellular cathepsin Z signals through the α5 integrin and augments NLRP3 inflammasome activation.

Respiratory silicosis is a preventable occupational disease that develops secondary to the aspiration of crystalline silicon dioxide (silica) into the lungs, activation of the NLRP3 inflammasome, and IL-1ß production. Cathepsin Z has been associated with the development of inflammation and IL-1ß production; however, the mechanism of how cathepsin Z leads to IL-1ß production is unknown. Here, the requirement for cathepsin Z in silicosis was determined using WT mice and mice deficient in cathepsin Z. The activation of the NLRP3 inflammasome in macrophages was studied using WT and cathepsin Z-deficient bone marrow-derived murine dendritic cells and the human monocytic cell line THP-1. The cells were activated with silica, and IL-1ß release was determined using enzyme-linked immunosorbent assay or IL-1ß bioassays. The relative contribution of the active domain or integrin-binding domain of cathepsin Z was studied using recombinant cathepsin Z constructs and the α5 integrin neutralizing antibody. We report that the lysosomal cysteine protease cathepsin Z potentiates the development of inflammation associated with respiratory silicosis by augmenting NLRP3 inflammasome-derived IL-1ß expression in response to silica. The secreted cathepsin Z functions nonproteolytically via the internal integrin-binding domain to impact caspase-1 activation and the production of active IL-1ß through integrin α5 without affecting the transcription levels of NLRP3 inflammasome components. This work reveals a regulatory pathway for the NLRP3 inflammasome that occurs in an outside-in fashion and provides a link between extracellular cathepsin Z and inflammation. Furthermore, it reveals a level of NLRP3 inflammasome regulation that has previously only been found downstream of extracellular pathogens.