FcγR-driven release of IL-6 by macrophages requires NOX2-dependent production of reactive oxygen species

Abstract

Activation of the FcγR via antigen containing immune complexes can lead to the generation of reactive oxygen species, which are potent signal transducing molecules. However, whether ROS contribute to FcγR signaling has not been studied extensively. We set out to elucidate the role of NADPH oxidase-generated ROS in macrophage activation following FcγR engagement using antigen-containing immune complexes. We hypothesized that NOX2 generated ROS is necessary for propagation of downstream FcγR signaling and initiation of the innate immune response. Following exposure of murine bone marrow-derived macrophages (BMDMs) to inactivated Francisella tularensis (iFt)-containing immune complexes, we observed a significant increase in the innate inflammatory cytokine IL-6 at 24 h compared with macrophages treated with Ft LVS-containing immune complexes. Ligation of the FcγR by opsonized Ft also results in significant ROS production. Macrophages lacking the gp91(phox) subunit of NOX2 fail to produce ROS upon FcγR ligation, resulting in decreased Akt phosphorylation and a reduction in the levels of IL-6 compared with wild type macrophages. Similar results were seen following infection of BMDMs with catalase deficient Ft that fail to scavenge hydrogen peroxide. In conclusion, our findings demonstrate that ROS participate in elicitation of an effective innate immune in response to antigen-containing immune complexes through FcγR.

Keywords: Akt; FC Receptors; Francisella tularensis; Macrophages; NADPH Oxidase; Reactive Oxygen Species (ROS).

FIGURE 1.

Loss of gp91 protein attenuates IL-6 production in macrophages. WT and gp91^phox KO BMDMS were stimulated with LVS (A), iFt (B), or bacteria opsonized with 1 μg of monoclonal anti-Ft LPS antibody. Macrophages were also stimulated with LPS, PAM3CSK4, or media alone as positive and negative controls. Supernatants were analyzed for IL-6 at 24 h poststimulation by Lumenix bead array. These data are the result of at least three independent experiments. Error bars represent the S.E. Nonsignificant differences (ns) represent a p value >0.05. Asterisks (*) represent a p value <0.05.

FIGURE 2.

Production of ROS following FcR engagement of bead immune complexes requires NOX2. Wild-type (A) or gp91 KO (B) BMDMs were stimulated with BSA-coated beads or anti-BSA opsonized BSA-coated beads (Beads+mAb). Real-time kinetics of luminol oxidation following stimulation of WT BMDMS (A) and gp91^phox KO BMDMs (B) at 37 °C over the course of 40 min. C, total ROS production in WT and gp91^phox KO BMDMs was calculated by measuring the area under the curve in A and B. D, loss of gp91^phox results in significant drop in IL-6 production following stimulation of BMDMs with Beads+mAb as measured by ELISA. These data show one representative experiment of at least three independent experiments. Error bars represent the S.E. Dashed lines represent luminol assay limit of detection. Asterisks (*) represent a p value <0.05, (***) represents p < 0.001.

FIGURE 3.

Production of ROS following FcR engagement of Ft immune complexes requires NOX2. Wild-type (A and C) or gp91 KO (B and D) BMDMs were stimulated with live or inactivated bacterial immune complexes. The levels of oxidized luminol were measured at 45-s intervals in real-time to detect the production of ROS following Fc Receptor cross linkage. A and B, intensity of light produced from the oxidation of luminol was plotted for each time point for 40 min following binding at 4 °C and incubation at 37 °C in wild-type BMDMs. C and D, total ROS production in WT BMDMs was measured by calculating the area under the curve for each experimental condition. These data are the result of at least three independent experiments. In all panels, error bars represent the S.E. Dashed lines represent luminol assay limit of detection. Asterisks (*) represent a p value <0.05.

FIGURE 4.

Deletion of NOX2 does not inhibit phagocytosis of Ft containing immune complexes. Wild-type or gp91^phox KO BMDMS were incubated with iFtmAb immune complexes. At each time point, macrophages were fixed and stained for extracellularly bound iFtmAb the with anti-IgG_2a-Alexa 488, then permeabilized and stained for intracellular bacteria with anti-IgG_2a-Alexa 594 (left). Cell nuclei were stained with DAPI. Percentage of internalized iFtmAb at the 0 m and 15 m time point were quantified. Data shown represent two independent experiments. Error bars represent the S.E. Nonsignificant differences (ns) represent a p value >0.05.

FIGURE 5.

Blockage of ROS following FcγR cross-linkage reduces Akt activation and IL-6 production in macrophages. WT BMDMs were treated with the NOX2 inhibitor Apocynin prior to and during stimulation with Ft containing immune complexes. Total Production of ROS was measured by luminol assay over the course of the first thirty minutes post stimulation with iFtmAb in the presence of PEG-Catalase (A) or apocynin (C). The effects of catalase (B) and apocynin (D) on the ability of WT BMDMs to produce IL-6 24 h poststimulation were collected and measured by luminex assay Data shown from at least three independent experiments. Error bars represent the S.E. Asterisks represent a p value <0.05 (*) or <0.01 (**), respectively.

FIGURE 6.

Downstream activation of Akt following Fcγ receptor cross linking is reduced in NOX2-deficient macrophages. WT and gp91 KO BMDMs were stimulated with opsonized Ft LVS or iFt for up to one hour. A, at the time points shown above, cells were lysed and levels of phosphorylated Akt at tyrosine residue 308 compared with the level of total cellular Akt were measured by Western blot. Results from one independent and representative experiment are shown in A. B, ratio of phosphorylated Akt to total Akt at 60 m post-stimulation was analyzed by densitometry. Results shown are the average of three independent experiments. Error bars represent the S.E. Asterisks (*) represent a p value <0.05.

FIGURE 7.

Antioxidant-deficient bacteria induce significantly more IL-6 in a NOX2-dependent manner. WT and gp91^phox KO BMDMS were stimulated with Francisella LVS or ΔkatG alone or opsonized with 1 μg monoclonal anti-Ft LPS antibody. A, real-time production of ROS as measured by chemiluminescence following WT BMDM stimulation using LVSmAb or ΔkatGmAb immune complexes. B, total ROS production was quantitated by calculating the area under the curve in A. C, increased levels of ROS are dependent on gp91^phox, as Δgp91^phox BMDMs do not produce a detectable level of ROS following stimulation with ΔkatGmAb immune complexes. ΔkatGmAb immune complexes result in significantly higher levels of total luminol chemiluminescence. D, supernatants were analyzed for IL-6 at 24 h poststimulation by CBA. ΔkatG and ΔkatGmAb elicit significant higher levels of IL-6 in WT BMDMs compared with LVS or LVSmAb infected controls. Data presented in A and C representative figures of at least three independent experiments. Data presented in panels B and D represent at least three independent experiments. Error bars represent the S.E. Dashed lines in A and B represent luminol assay limit of detection. Asterisks (*) represent a p value <0.05.

FIGURE 8.

Treatment of macrophages with the NADPH oxidase inhibitor apocynin reduces production of IL-6. Enhanced production of IL-6 following WT BMDM stimulation with ΔkatGmAb immune complexes in the presence of 250 μm Apocynin. Cell culture supernatants were collected at 24 h poststimulation and IL-6 levels were analyzed by CBA. Data shown are representative of three independent experiments. Asterisk (*) denotes a significant difference of p < 0.05.