PI3K and NADPH oxidase: a class act

In this issue of Blood, Anderson and colleagues show that class III PI3K is essential for CD18-dependent activation of the NADPH oxidase, and production of ROS mediated by this phagocytosis pathway does not require antibody or FcγR for the subsequent synthesis of PtdIns(3)P at phagosome membranes.

Phagocytosis of microorganisms by neutrophils and other phagocytic leukocytes is mediated in part by receptors for opsonins, such as the complement receptor, CD11b/CD18, and/or those specific for antibody Fc (eg, FcγR receptors). Ingestion of microbes triggers generation of reactive oxygen species (ROS), which are important for the antibacterial and antifungal activity of phagocytes. ROS are produced by the NADPH oxidase, a multicomponent enzyme that assembles at the plasma or phagosome membrane. In resting phagocytes, the redox component of NADPH oxidase, flavocytochrome b₅₅₈, resides in the plasma membrane and membranes of granules, whereas other proteins of the enzyme complex, the GTPase-Rac, p47^phox, p67^phox, and p40^phox, are present in the cytosol.¹  During phagocytosis, the cytosolic components translocate to the membrane where they assemble with flavocytochrome b₅₅₈ (composed of gp91^phox and p22^phox) to form the active NADPH oxidase.¹  Translocation of cytosolic components is highly regulated and involves multiple SH3-domain interactions and interaction of phox homology domains (PX) with phosphoinositides.^1-3  Defects in gp91^phox, p22^phox, p47^phox, or p67^phox cause chronic granulomatous disease, a rare hereditary disorder characterized by recurrent bacterial and fungal infections due to the inability of leukocytes to produce ROS.¹  A critical role for p40^phox in NADPH oxidase function has remained elusive, but recent work indicates that p40^phox is essential for optimal killing of bacteria by neutrophils.^1,4 

Phosphatidylinositol 3-kinases (PI3Ks) are important for signal transduction events, leading to the production of ROS following FcγR phagocytosis.^2,3,5-8  class I and class III PI3Ks have unique functional and temporal activities within phagocytic leukocytes and their individual roles in phagocytosis and NADPH oxidase assembly/activation are currently an intense area of investigation. class I PI3Ks, which generate PtdIns(3,4,5)P₃ and PtdIns(3,4)P₂, are necessary for formation of phagosomes.^7,8  Recent studies indicate that the PtdIns(3)P generated by class III PI3K contributes to phagosome maturation, and importantly, the binding of PtdIns(3)P to p40^phox regulates NADPH oxidase activation during phagocytosis.^2,5  Although PI3Ks are known to play a key role in NADPH oxidase activation during FcγR-mediated phagocytosis, there is little or no information bearing on the role of complement- or nonantibody receptors in this process.

A new study by Anderson et al reveals that class III PI3K and PtdIns(3)P are essential for activation of the NADPH oxidase during CD18-mediated phagocytosis. The authors use mice lacking CD18 or antibody to show that ROS production following phagocytosis of serum-opsonized Staphylococcus aureus or Escherichia coli is dependent on serum complement and CD18 rather than antibody and FcγRs. Human or mouse neutrophils treated with wortmannin, a general PI3K inhibitor, had significantly reduced capacity to produce ROS following phagocytosis of opsonized S aureus or E coli. By comparison, inhibitors specific for class I PI3Ks failed to block ROS production under these conditions, and the findings were confirmed by assays with neutrophils from mice genetically deficient in class I or class II PI3Ks in which ROS generation was normal. Collectively, these results indicate that class I or class II PI3Ks do not contribute to production of ROS following complement receptor–mediated phagocytosis. Unlike class I or class II PI3Ks, there is no mouse knockout model for the single eukaryotic class III PI3K (known as Vps34). To circumvent this problem, the authors use an RNA interference approach to demonstrate that class III PI3K regulates accumulation of PtdIns(3)P on bacterial phagosomes and CD18-dependent ROS production in macrophage-like cell lines. These results led to a final series of experiments from which the authors conclude that there are probably additional p40^phox PX-domain–independent roles for PtdIns(3)P in the regulation of phagosomal ROS generation.

The study is the first to provide a direct link between PI3Ks and ROS production following complement receptor–mediated phagocytosis. It is interesting that the phagocytic particle, eg, IgG-opsonized particles versus serum-opsonized bacteria, appears to dictate whether class I or class III PI3K is involved in phagocytosis and subsequent NADPH oxidase activation. A somewhat surprising result is that neutrophil ROS production following phagocytosis of S aureus or E coli is completely independent of antibody and FcγRs. The observation that class III PI3K is important for phagocyte activation following ingestion of bacteria unveils yet another molecule/pathway that may be targeted by pathogenic microorganisms. On the other hand, these data suggest that pharmacologic agents designed to inhibit class I PI3Ks will not likely impact host-mediated killing of bacteria.