Phagocytes play a critical role in host defense by producing reactive oxygen species against invading microorganisms. One of the most important enzymes in producing microbicidal oxidants is the superoxide (O2 2)-generating NADPH oxidase.1 The NADPH oxidase is a multicomponent enzyme complex whose redox center is a membrane-associated flavocytochrome b 558 heterodimer composed of gp91phox and p22phox. In addition, three cytosolic oxidase subunits, p47phox, p67phox, and a low molecular weight GTP binding protein Rac, are required for high level production of O2 2. In resting phagocytes, the dormant oxidase is unassembled. However, upon phagocyte activation, the active oxidase complex is rapidly formed by translocation of the cytosolic oxidase components to the plasma membrane via interactions with the cytochrome.2 Subsequently, electrons are transferred from cytosolic NADPH to molecular oxygen (O2) at the external face of the membrane to generate superoxide anion .
In activated neutrophils, a membrane-bound NADPH-dependent oxidase generates high levels of superoxide, a process traditionally called "respiratory burst". In unstimulated neutrophils, components of the NADPH oxidase complex are separated in cytosol (p40phox, p47phox, p67phox, Rac2) and membrane compartments (flavocytochrome b558, Rap1A). During phagocytosis, the cytosolic components translocate to the plasma and/or phagosome membrane and associate with flavocytochrome b558, a transmembrane heterodimer comprised of gp91phox (Nox2) and p22phox, thereby forming the active oxidase. The oxidase transfers electrons from cytosolic NADPH to intraphagosomal molecular oxygen, thus producing superoxide. Superoxide anion is short-lived and dismutates rapidly to hydrogen peroxide and forms other secondary products, such as hypochlorous acid, hydroxyl radical, and singlet oxygen, which are effective microbicidal compounds .
Briefly, deletion of gp91phox results in enhancement of composite asthma phenotype in mouse. Double deletion of gp91phoix and MMp12, a critical enzyme for phagocyte associated inflammation results in no alteration of the phenotype generated in the single deletion of gp91phox-/-. Overall inflammation is robust. Both naïve and allergen challenged KO mice show statistically significant exacerbation of Th2 inflammation. All the classical features of a robust acute allergenic asthma phenotype are significantly amplified in the KO mice showing that NADPH oxidase, which is essentially an extra-cellualr matrix associated enzyme expressed by phagocytes, which by definition may denote a non-specific inflammatory response or at the best an inflammatory phenomenon associated with lung remodeling in chronic asthma, surprisingly may also have a regulatory role in the sensitization or priming stage. This is no more apparent than in the level of circulating and tissue bound progenitors which show a clear mobilization denoted by the dearth of stem as well as mature cells in bone marrow. Because of the absence of gp91phox, the cells seem to have less of an adherence to the stroma and migrate out of their niche. The cellular traffic out of bone marrow (CFU in femur was assessed) also shows a more robust lympho- and eosinophilopoiesis. Does gp91phox have any known role in hematopoiesis or granulocytopoiesis?  says that NADPH essentially increases neutrophil and eosinophil synthesis and migration to inflammatory tissues. If gp91phox is imagined to have a regulatory role in development of the asthma phenmotype, MMP12 seems to have a similar if not synergistic effect. These molecules are however not necessary for migration, but are critical for PMA induced proliferation and MCP-1 induced chemotaxis. The overall Th2 response was enhanced possibly due to a lack of control over T cell: APC cross-talk in the KO mice as shown by MLR.
The results described above indicate that gp91phox-/- mice respond to OVA in a more exaggerated fashion compared to WT post-OVA, in terms of total number of cells migrated to lung (1.8 folds) and BALf (1.7 folds), although total number of cells in bone marrow (obtained from two femurs) and that in circulating peripheral blood were comparable (Table 1). Expressed as a fraction of circulating cells recruited into lungs and BALf in response to allergic immune response, both knockout mice seem to show similar trends. Cell subsets for which recruitment index is more than 1, indicate a cumulative effect where both cells from circulation as well as resident cells normally present in the pulmonary milieu in surveillance, seem to be equally important. Recruitment of B cells, monocytes, neutrophils and basophils are increased in lungs of both knockout mice compared to post-OVA wildtype while that of T cells, neutrophils and basophils in BALf are increased in the knockout vs. the OVA-treated wildtype (Table 1). In gp91phox-/- progenitors in BM decrease by 28.8% while in double knockout, it decreases by 27.9% compared to the values in WT post-OVA. In lung, the decrease in progenitor number was 1.8-fold and 1.5-fold respectively. NOX2 and DKO mice both show spontaneous pulmonary inflammation. In naïve mouse, this may account for the decrease in cell number in femur and lungs (due to rapid and uncontrolled mobilization out of the niche and out of the parenchyma into the interstitium respectively. Spleen, because of this constant and uncontrolled inflammatory background naturally becomes inflamed and shows an increase in size and cell number. Under treatment with allergen, this scenario gets further aggravated. Femur (bone marrow) has been known to be the main pool of progenitors for granulopoiesis (PMNs and eosinophils). Cells from the lung parenchyma, on the other hand, need to migrate into the interstitium for Th2 category inflammation in the lung tissue. These may account for dearth of progenitors and mature cells in these tissues while spleen registered a higher than normal cell number anyway in these mice and under a heightened condition of inflammatory exacerbations, shows a higher reserve despite this limitation.
My work [17–22] in conjunction with other work in the public domain using genetic knockout models of mice in a preclinical set up to study molecular roles of deleted molecules in the pathogenesis of acute allergic asthma has revealed the following. The alternately delicate and robust interplay of sensitization-priming vs. fully activated state of cells play a crucial role in the final translation of the pathogenesis status of the animal. In humans Chronic granulomatous disease (CGD) where is a group of inherited disorders in which phagocytes are unable to generate superoxide due to genetic defects in any 1 of 4 essential NADPH oxidase components. Mutations in the X-linked gene for gp91phox, the large subunit of the flavocytochrome b558 heterodimer, account for the majority of CGD. Patients with CGD develop recurrent, often life-threatening bacterial and fungal infections due to impaired microbicidal oxidant generation by the patient's phagocytes. ROS are also important in inducing in schemia-induced lung angiogenesis.  gp91phox, expressed in CHO (Chinese hamster ovary) cells, functions as a voltage-dependent proton channel.  gp91phox was also found to regulate adaptive immune response at the level of both T cells and APCs .
Activation of NADPH oxidase represents an essential mechanism of defense against pathogens. Dendritic cells (DC) are phagocytic cells specialized in Ag presentation rather than in bacteria killing. Human monocyte-derived DC were found to express the NADPH oxidase components and to release superoxide anions in response to phorbol esters and phagocytic agonists. The NADPH oxidase components p47phox and gp91phox were down-regulated during monocyte differentiation to DC, and maturation of DC with pathogen-derived molecules, known to activate TLRs, increased p47phox and gp91phox expression and enhanced superoxide anions release. Similar results were obtained with plasmacytoid DC following maturation with influenza virus. In contrast, activation of DC by immune stimuli (CD40 ligand) did not regulate NADPH oxidase components or respiratory burst. NADPH oxidase-derived oxygen radicals did not play any role in DC differentiation, maturation, cytokine production, and induction of T cell proliferation, as based on the normal function of DC generated from chronic granulomatous disease patients and the use of an oxygen radical scavenger. However, NADPH oxidase activation was required for DC killing of intracellular Escherichia coli. It is likely that the selective regulation of oxygen radicals production by pathogen-activated DC may function to limit pathogen dissemination during DC trafficking to secondary lymphoid tissues . Reactive oxygen species (ROS) play an important role in regulating vascular tone and intracellular signaling .
In summary, the work presenetd in this communication describes in detail, the phenotype expressed in two genotype knockout mice, gp91phox-/- and gp91phox, MMP-12 double knockout. The data obtained indicates clearly the following:
All aspects of acute allergic asthma are manifested in an exaggerated manner in the knockout mice vs. the WT, viz. cell recruitment is more, inflammatory cell recruitment,, specifically myeloid cells in increased; T cells are also recruited not only in greated numbers but are also directly involved in the disease pathogenesis by clearly increasing TH2 cytokines, antigen-specific IgG and hypertrophy and hyperplasia in goblet cells of the lung. So these molecules seem to have regulatory roles in (a) cell priming, (b) direct cell activation, and (c) cell recruitment (which may include synthesis, mobilization and homing out of their niches) and by extrapolation, dendritic cells, phagocytes (macrophage and neutrophils) and T cells in the afferent arm of the disease pathology and eosinophils, B cells as denoted by Ig sequestration and probably endothelial and smooth muscle cells of the lung as well, in the efferent arm (responsible for exacerbations) may be defined as components under direct regulation of these two molecules (gp91phox and MMP-12).
While gp91phox and MMP12 deletion affects the mature cell polulation thus, the progenitor numbers are also similarly affected. This possibly alludes to greated synthesis in bone marrow or adult tissue reserves as well as enhanced mobilization. This is an important finding as it provides a critical link between the actual increase in cell number and a decreased adhesion to stroma thereby setting the stage of more efficient mobilization. While gp91phox and MMP12 interaction have been known to orchestrate ROS activity and tissue damage during inflammtion, there was no information regarding the upstream implication between this manifestation of innate immunity and the actual mechanistic phenomenon encouraging cells to actually migrate in larger numbers and more quickly to tissue spaces. Whether this also aids specific tissue homing is unknown at the present time and needs more work in this model.
The function of gp91phox may be primary in these regulatory cascade and MMP-12 may come in later. However, based on the data presented in this study, they seem to have synergistic or at best redundant, but definitely not additive roles.