Environment tames CGD macrophages

In this issue of Blood, Gibbings et al¹ show that the inflammatory milieu rather than the cell-intrinsic Nox2 deficiency skews monocyte-derived macrophages (MoMacs) into a state of inflammatory hyperresponsiveness and prevents their immunophenotypic maturation in chronic granulomatous disease (CGD).

CGD is a rare primary immunodeficiency of phagocyte function characterized by life-threatening infections, granulomas, and dysregulated inflammatory responses toward a wide array of sterile and nonsterile stimuli/agonists. CGD is genetically heterogenous and caused by null mutations in the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (phox) subunit genes. These null mutations cause the failure of neutrophils and monocytic cells to undergo respiratory burst and generate superoxide (O₂⁻). The membrane-restricted flavocytochrome b₅₅₈, a heterodimer of gp91^phox (Nox2) and p22^phox subunits, is the main catalytic subunit of the NADPH oxidase complex. Approximately two-thirds of CGD cases are X-linked and are due to null mutations in the gene encoding gp91^phox (Nox2). The remaining cases are caused by autosomal recessive defects in the cytosolic p47^phox, p67^phox, and p40^phox subunits.² NADPH oxidase-derived O₂⁻ and downstream reactive oxygen species (ROS) are essential antimicrobial agents. However, they are increasingly recognized to play important modulatory roles in host immune responses, in part by regulating immune effector functions of phagocytes and inflammatory signaling downstream of pattern recognition receptors.^2,3

CGD patients often present with inflammatory complications that are unrelated to infections such as sterile granulomas in hollow organs, colitis, discoid lupus-like lesions, and other autoimmune complications.^2,4 Thus far, studies to understand the molecular and cellular basis of inflammatory complications and in CGD have focused on elucidating specific Nox2/ROS regulated pathways that alter neutrophil or macrophage effector functions in a cell-intrinsic manner. For instance, failure to optimally activate NADPH oxidase delays apoptosis, compromises NETosis, dysregulates ionic fluxes, and leads to excessive degranulation of Nox2 null of CGD neutrophils.³ CGD macrophages also have significant defects in autophagy pathways, have reduced efferocytic capacity, and exhibit delays in phagosomal maturation, consistent with delayed proteolysis of apoptotic cell-derived antigens.^5-8 Besides dysregulation of multiple phagocyte-associated effector responses, CGD neutrophils and macrophages are also hyperactive or hyperresponsive to inflammatory stimuli, often producing more cytokines and chemokines on a per-cell basis compared with cells derived from healthy individuals or wild-type mice. Mechanistically exaggerated responses to pathogen-associated ligands of endogenous ligands released during sterile injury were linked to dysregulated or prolonged activation of redox-sensitive transcription factors (NF-kB, Nrf2) as well as MAP kinases.⁹ Based on these data, it was largely assumed that dysregulated cell-intrinsic inflammatory pathways and immune processes continue to sustain CGD inflammation in vivo, possibly by amplification of inflammatory feedforward loops. However, new findings by Gibbings et al adds another layer of complexity to CGD inflammation by showing that the hyperinflammatory nature of CGD MoMacs can be reversed by the manipulation of their microenvironment, thus showing that extrinsic factors such as the inflammatory milieu foster the dysregulated behavior of CGD macrophages.¹ 

Gibbings et al investigated the maturation programs of newly recruited inflammatory monocytes into mature macrophages using a murine model of zymosan-induced peritonitis. Macrophages are highly plastic cells that dynamically alter their transcriptomes, epigenetic landscapes, and phenotypic profiles in response to environmental signals and local tissue milieu. Their studies show that wild-type MoMacs rapidly transition from an inflammatory state into mature macrophages that exhibit expansion in cell size, downregulation of proinflammatory markers, and upregulation of transcripts involved with resolution of inflammation. In striking contrast, CGD MoMacs were continuously recruited to the inflamed peritoneal cavity and failed to undergo phenotypic maturation and transcriptional reprogramming consistent with the acquisition of resolution properties. They show that CGD MoMacs remained in a migratory state, migrating to the diaphragm where they were found in fibrinogen clots surrounding neutrophil clusters in nascent pyogranulomata. Using mixed chimeras and adoptive transfer experiments, Gibbings et al show that the inflammatory milieu primarily regulated this dysregulated behavior of CGD MoMacs in CGD rather than intrinsic loss of Nox2 activity within the MoMacs themselves. Another important finding of these studies is that even though CGD macrophages have been known to produce more inflammatory cytokines on a per-cell basis, they can be reprogrammed into less inflammatory states by modulating their microenvironment. Overall, these studies show a previously underappreciated role of the inflammatory milieu in sustaining CGD inflammation and hyperresponsiveness of CGD macrophages.