NADPH oxidase activation regulates apoptotic neutrophil clearance by murine macrophages

Abstract

The phagocyte reduced NAD phosphate (NADPH) oxidase generates superoxide, the precursor to reactive oxygen species (ROS) that has both antimicrobial and immunoregulatory functions. Inactivating mutations in NADPH oxidase alleles cause chronic granulomatous disease (CGD), characterized by enhanced susceptibility to life-threatening microbial infections and inflammatory disorders; hypomorphic NADPH oxidase alleles are associated with autoimmunity. Impaired apoptotic cell (AC) clearance is implicated as an important contributing factor in chronic inflammation and autoimmunity, but the role of NADPH oxidase-derived ROS in this process is incompletely understood. Here, we demonstrate that phagocytosis of AC (efferocytosis) potently activated NADPH oxidase in mouse peritoneal exudate macrophages (PEMs). ROS generation was dependent on macrophage CD11b, Toll-like receptor 2 (TLR2), TLR4, and myeloid differentiation primary response 88 (MyD88), and was also regulated by phosphatidylinositol 3-phosphate binding to the p40 oxidase subunit. Maturation of efferosomes containing apoptotic neutrophils was significantly delayed in CGD PEMs, including acidification and acquisition of proteolytic activity, and was associated with slower digestion of apoptotic neutrophil proteins. Treatment of wild-type macrophages with the vacuolar-type H+ ATPase inhibitor bafilomycin also delayed proteolysis within efferosomes, showing that luminal acidification was essential for efficient digestion of efferosome proteins. Finally, cross-presentation of AC-associated antigens by CGD PEMs to CD8 T cells was increased. These studies unravel a key role for the NADPH oxidase in the disposal of ACs by inflammatory macrophages. The oxidants generated promote efferosome maturation and acidification that facilitate the degradation of ingested ACs.

Inflammatory consequences of inherited disorders affecting neutrophil function

“…9,68 Electron transfer across the membrane by the leukocyte oxidase also has electrogenic effects that can increase neutrophil intracellular calcium levels, 69 as well as alter the pH within phagosomes and the activity of digestive proteases. 13,70 Thus, NADPH oxidase ROS can have multiple effects (Figure 2), including damaging oxidation that aids microbial killing, but it can be injurious to host tissue if oxidant production is not appropriately regulated or confined. Triggering of NOX2 ROS production has coevolved with activation of other immune responses, and it exerts immunoregulatory effects that balance immune activation (Figure 3).…”

“…Activated human CGD monocytes display increased production of IL-1a and IL-1b, 45-47 with similar findings in mouse macrophages and bone marrow-derived dendritic cells. 70,75 The underlying mechanism is not understood. One proposal is that enhanced IL-1 production and/or release in CGD reflects reduced activity of autophagy pathways, 45 but this is unresolved.…”

The roles of NADPH oxidase in modulating neutrophil effector responses

“…PEMs from gp91 phox − / − mice that lack the capacity to generate ROS, exhibited significant delays in the clearance of ingested apoptotic cells. Cross presentation of apoptotic cells associated antigens to CD8 T cells was enhanced leading to CD8 T cell clonal expansion . Ingestion of apoptotic neutrophils is also an important resolution signal and reprograms macrophages from a pro‐inflammatory (M1) phenotype to an anti‐inflammatory or pro‐resolving (M2) and augments the generation of pro‐resolving mediators.…”

“…For instance, CGD patients are highly susceptible to sterile inflammatory complications, such as granulomatous inflammation in the genitourinary and gastrointestinal tract, and discoid mucocutaneous skin lesions. Further, mice lacking NADPH oxidase activity (due to deletion of Cybb [encoding gp91 phox ]) exhibited hyper‐inflammatory responses characterized by elevated levels of pro‐inflammatory cytokines and excessive neutrophilic recruitment in multiple models of sterile or microbe‐elicited challenge . Interestingly, hyper‐inflammation in these mice was observed even in the absence of active infection.…”

Chronic granulomatous disease 2018: advances in pathophysiology and clinical management

“…Chronic granulomatous inflammation may compromise vital organs and account for additional morbidity. Apoptosis of neutrophils (Sanford et al 2006) and their clearance by macrophages are delayed in CGD (Bagaitkar et al 2018). Defective clearance of dying cells leads to excess generation of proinflammatory cytokines (e.g., IL-1 beta and TNF alpha) and autoinflammation ( Figure 5).…”