IFN-γ targets macrophage-mediated immune responses toward Staphylococcus aureus

IFN-γ-regulated cellular processes may potentiate a beneficial innate immune response in staphylococcal infection.

Abstract

Infections, especially with Staphylococcus aureus (SA), commonly cause morbidity and mortality in patients with chronic granulomatous disease (CGD), a condition characterized by a defective phagocyte oxidase. IFN-γ reduces the frequency and consequences of infection in CGD by mechanisms that remain unknown. As IFN-γ promotes bacterial killing, efferocytosis of effete polymorphonuclear neutrophils (PMN), and cytokine production in macrophages—the same macrophage effector functions that are impaired in response to SA—we hypothesized that IFN-γ may reverse these defects and thereby, augment macrophage control of SA during infection. IFN-γ primed activation of the NADPH oxidase in a time-dependent manner, enhanced killing of ingested SA independent of any effects on phagocytosis, and increased binding of SA-laden neutrophils (PMN-SA) to macrophages. However, IFN-γ did not increase the percentage of apoptotic PMN or PMN-SA internalized by macrophages. Under conditions in which viable SA were eliminated, PMN-SA primed the inflammasome for subsequent activation by silica but did not induce IL-1β production by macrophages. IFN-γ enhanced IL-6 production in response to SA or PMN-SA but did not increase inflammasome activation in response to either agonist. In summary, IFN-γ augmented direct killing of SA by macrophages, promoted engagement of PMN-SA, and enhanced macrophage-mediated cytokine responses that could collectively augment control of SA infection. Together, these findings support the hypothesis that IFN-γ improves responsiveness of macrophages to SA and provides insights into the mechanism of the clinical benefits of IFN-γ.

Introduction

Effective host defense against microbial infection relies on the coordinated responses of phagocytes, both PMN and macrophages, and soluble factors to kill and degrade potential pathogens and to restore homeostasis. By underscoring the critical contribution of phagocytes to optimal antimicrobial defense, individuals with an inadequate number of circulating normal PMN incur frequent and severe infections. For example, life-threatening infection complicates CGD, an inherited disorder of the NADPH oxidase, rendering phagocytes unable to generate reactive oxygen species. Patients with CGD also exhibit exuberant granulomatous inflammation (reviewed in refs. [1, 2]), thereby amplifying the adverse clinical sequelae of infection. Efforts to reduce the risk of infection in individuals with CGD include prophylactic antibiotics to decrease the load of endogenous organisms and IFN-γ to augment inherent host defenses [3]. Based on the results of an international clinical trial demonstrating a reduction in the prevalence and duration of infection in treated patients [4], IFN-γ has become part of standard care for patients with CGD, although the molecular basis for its salutary action remains unknown.

Interactions between SA, a common human pathogen that produces frequent and especially serious disease in CGD patients [3], and human macrophages do not favor the host: ingested SA survive within macrophages [5, 6] and prompt phagocyte cell death [6–9]. We recently reported that PMN fail to kill all of the ingested inoculum of pathogenic SA [10] and that macrophages bind but do not efferocytose PMN-SA [11]. Furthermore, macrophages challenged with PMN-SA release less proinflammatory cytokines than do those that ingest SA alone [11]. Taken together, our data suggest that the inability of PMN to kill ingested SA and the inefficient clearance of PMN-SA perpetuate infection and promote local inflammation, two features that characterize human infection with SA [12, 13].

In light of both experimental data and clinical observations that demonstrate shortcomings in phagocyte defenses against SA and given the therapeutic benefits of prophylactic IFN-γ in patients with CGD, we tested the hypothesis that IFN-γ improves defects in macrophage killing of SA, efferocytosis of PMN-SA, and cytokine production, thereby contributing to better control of SA infection and more efficient restoration of homeostasis.

MATERIALS AND METHODS

Reagents, antibodies, and cells

All reagents were purchased from Thermo Fisher Scientific (Waltham, MA, USA) unless otherwise indicated. Heparin (APP Pharmaceuticals, Schaumburg, IL, USA), clinical-grade Dextran T500 (Pharmacosmos A/S, Holbaek, Denmark), Ficoll-Hypaque Plus (GE Healthcare, Piscataway, NJ, USA), and sterile endotoxin-free H₂O and 0.9% sterile endotoxin-free sodium chloride (Baxter, Deerfield, IL, USA) were used for cell isolation. HBSS, with and without divalent cations, Dulbecco’s PBS, and HEPES were obtained from Mediatech (Manassas, VA, USA). RPMI 1640 was purchased from Lonza (Hopkinton, MN, USA). Twenty-five percent HSA was purchased from Talecris Biotherapeutics (Raleigh, NC, USA). Hyclone FBS and TrypLE Express recombinant-dissociating enzyme were purchased from Thermo Fisher Scientific and heat-inactivated for 30 min at 56°C. PMA and HRP were purchased from Sigma-Aldrich (St. Louis, MO, USA). Amplex Red and CTFR 7-hydroxy-9H-(1,3-dichloro-9,9-dimethylacridin-2-one)-succinimidyl ester (Thermo Fisher Scientific) were reconstituted to 50 or 2 mM, respectively, in DMSO and stored at −20°C. Anti-human CD15-PE and anti-human CD14-PC5.5 (Beckman Coulter, Brea, CA, USA) were used for phagocytosis assays. Pharmaceutical-grade recombinant IFN-γ (Actimmune) was provided by Horizon Pharma (Lake Forest, IL, USA). Gentamicin was obtained from Thermo Fisher Scientific, ultrapure LPS from Escherichia coli O111:B4 was obtained from InvivoGen (San Diego, CA, USA), and silica was obtained from U.S. Silica (Berkeley Springs, WV). Recombinant protein and antibodies against IL-1β and IL-6 ELISAs were purchased from Affymetrix (San Diego, CA, USA).

PMN and PBMC isolation

Written consent was obtained from each volunteer in accordance with a protocol approved by the Institutional Review Board for Human Subjects at the University of Iowa (Iowa City, IA, USA). PMN and PBMCs were obtained as described in Nauseef [14]. Heparinized blood was collected from healthy donors, and leukocytes were collected following sedimentation with 3% Dextran and separation on Ficoll Hypaque gradient. Mononuclear cells were collected from the middle layer of the gradient and washed with twice with RPMI. The cell pellet containing PMN was subjected to hypotonic lysis to remove RBCs, and PMN were resuspended in HBSS without divalent cations.

Staphylococcal culture

The community-associated methicillin-resistant SA strain USA300 LAC (referred to as SA throughout) was obtained from Dr. Alex Horswill (Department of Microbiology, University of Iowa). SA was grown in TSB (BD Biosciences, San Jose, CA, USA) overnight at 37°C at 180 rpm. Bacteria were then diluted to an OD₅₅₀ of 0.05 in TSB and grown to early logarithmic phase (OD₅₅₀ of 0.5–1.0). Bacteria were then suspended in HBSS with divalent cations containing 20 mM HEPES. For all experiments, SA were opsonized in 10% PHS while tumbling for 20 min at 37°C.

Macrophage activation

Macrophages were obtained as previously described [15]. In brief, monocytes from PBMCs matured to macrophages after 6–8 days culture in Teflon jars with RPMI 1640 containing 20% autologous serum. Macrophages adhered to glass or plastic overnight in RPMI 1640 containing 10% PHS, and nonadherent cells were washed away with warm RPMI the following day. Unless otherwise indicated, macrophages were incubated with or without 420 U/ml IFN-γ in RPMI 1640 containing 10% PHS for 3–4 days.

Amplex Red assay

Macrophages (2 × 10⁵/well) were plated in 96-well plastic tissue-culture plates in the absence or presence of added IFN-γ. After treatment with or without IFN-γ for 0–4 days, macrophages were washed and treated with 100 ng/ml PMA or buffer. The A₅₅₀ was measured over 120 min in a reaction containing macrophages, 500 μM Amplex Red, and 10 U/ml HRP [16]. The A₅₅₀ was measured, and the nanomoles of H₂O₂ produced by macrophages was calculated from a standard curve A₅₅₀ and H₂O₂.

Killing assay

Macrophages adhered to a 24-well dish for ≥2 h at 37°C were washed and cultured in buffer, with or without added IFN-γ. Macrophages were fed SA (1:1 MOI), and phagocytosis was synchronized by centrifuging bacteria at 416 g for 5 min at 4°C. Macrophages were allowed to ingest bacteria for 30 min and were then washed extensively with warm RPMI. At indicated time points, macrophages were lysed in pH 11 H₂O, and bacterial viability was determined by subsequent plating of serial dilutions on tryptic soy agar plates overnight and enumeration of CFUs [17].

Uptake assay

Macrophage uptake was assessed as described in Greenlee-Wacker et al. [11]. In brief, macrophages were adhered to Nunc Lab Tek chambers for ≥2 h at 37°C, washed, and then cultured in buffer, with or without added IFN-γ. Apoptotic PMN were generated by culturing freshly isolated cells in RPMI containing 10% FBS at 37°C in 5% CO₂ for 18–24 h. Freshly isolated and apoptotic PMN were stained with CTFR at a 1:1000 dilution for 20 min at 37°C, washed, and resuspended in HBSS containing 0.1% HSA. PMN were left in buffer or fed opsonized SA (1:1 MOI) and tumbled for 10 min at 37°C. After 10 min, extracellular bacteria were removed by centrifugation at 162 g for 5 min. PMN were resuspended in HBSS²⁺ containing 20 mM HEPES and 1% HSA and tumbled for an additional hour. After 1 h, PMN, apoptotic PMN, and PMN-SA were opsonized with 10% PHS in RPMI containing 5 mM MgCl₂ (PB), washed, resuspended in PB, and fed to macrophages at a 20:1 ratio for 60 min. Macrophages were washed gently with PBS to remove uningested PMN, and macrophages were collected with TrypLE Express. Leukocytes were stained with CD15-PE and CD14-PC5.5 and analyzed by flow cytometry. Macrophages were defined as the CD14^high side-scatter^high population. Whereas CTFR stains all PMN, CD15 recognize bound but not ingested PMN, thus providing a method to discriminate between binding and ingestion of PMN-SA. Percent binding was calculated as the number of CD15⁺CTFR⁺ macrophages/total number of macrophages × 100, and percent internalization was calculated as the number of CD15⁻CTFR⁺ macrophages/total number of macrophages × 100.

Cytokine analysis

Macrophages were adhered to a 24-well dish for ≥2 h at 37°C, washed, and cultured in buffer, with or without added IFN-γ. PMN-SA were generated as described previously but without CTFR staining, added to freshly washed macrophages at a 5:1 ratio, and spun at 416 g for 5 min at 4°C to synchronize phagocytosis of PMN-SA by macrophages. After 1 h, macrophages were washed 3 times with RPMI to remove unbound PMN-SA. Fresh RPMI containing 10% PHS, supplemented with or without 50 ng/ml LPS and/or 10 μg/ml gentamicin, was then added to macrophages. Following 4 h of stimulation of macrophages with LPS, 500 μg/ml silica was added to a set of wells. Supernatants were removed, and lysates were collected 20 h after stimulation with PMN-SA. IL-6 and IL-1β in supernatants were quantified by ELISA.

RESULTS

IFN-γ activation enhances staphylococcal killing in human macrophages

IFN-γ enhances NADPH oxidase activity and antimicrobial capacity of macrophages against several different pathogens [18–21]. Therefore, we investigated the effect of IFN-γ treatment on killing of SA by human macrophages. With the reasoning that augmentation of the NADPH oxidase would be one mechanism by which antimicrobial activity could be promoted, we first confirmed that IFN-γ activation induces a time-dependent increase in PMA-stimulated extracellular oxidant production by macrophages [21, 22]. IFN-γ alone did not stimulate macrophage H₂O₂ production (data not shown) but primed macrophages for subsequent agonist-triggered NADPH oxidase activity in a time-dependent manner (Fig. 1). Macrophages treated with 420 U/ml IFN-γ for 3 days produced 1.8- to 3.1-fold more H₂O₂ in response to PMA than did PMA-stimulated control macrophages. Likewise, macrophages treated with 420 U/ml IFN-γ for 4 days exhibited higher production of H₂O₂ in response to PMA than did control macrophages (2.1- to 11.2-fold; Fig. 1). Exposure of macrophages to IFN-γ at 4200 U/ml did not enhance PMA-dependent H₂O₂ production over that achieved by treatment with 420 U/ml IFN-γ (data not shown). Based on these data, we elected to treat human macrophages with 420 U/ml IFN-γ for 3–4 days in our experimental system to determine if IFN-γ-enhanced oxidase activity improved macrophage-mediated responses to SA.

Figure 1. IFN-γ priming of NADPH oxidase activity of macrophages is time dependent.

Macrophages were plated on day 7 and treated for 1–4 days (day 7–10) with 420 U/ml IFN-γ and stimulated with PMA on day 11. Control cells were plated on day 7 and stimulated with PMA on day 11. The change in A₅₅₀ from 0 to 120 min was measured, and nanomoles of H₂O₂ produced by PMA-stimulated macrophages were calculated by using a standard curve of H₂O₂. Bars represent the average of 6 experiments ± sem. P values were determined using a repeated-measures one-way ANOVA and Dunnett's post-test. *P < 0.05 vs. control; ***P < 0.0001 vs. control.

To determine if IFN-γ altered phagocytosis and staphylococcal killing, we measured recovery of viable organisms from macrophages fed SA. To evaluate phagocytosis, recovery of viable SA from control and IFN-γ-treated macrophages was assessed after 30 min of phagocytosis (t = 0). Recovery of equivalent numbers of viable SA from control and IFN-γ-treated macrophages at t = 0 suggested that there was no difference in phagocytosis. However, 1.5 h after phagocytosis, there was a 47.3% ± 10.5% reduction in the number of SA recovered from IFN-γ-treated macrophages compared with that recovered from control macrophages (Fig. 2). Compared with SA recovery at 1.5 h, the number of SA recovered at 3 h from IFN-γ-activated macrophages had more than doubled, but total bacterial numbers remained lower than in control macrophages (Fig. 2). Together, these data suggest that IFN-γ-treated macrophages compromised but did not terminate growth of ingested SA.

Figure 2. IFN-γ enhances macrophage killing of SA.

Macrophages were treated with buffer, without (control, white bars) or with (black bars) added IFN-γ for 3 days, and then assayed for their ability to kill SA. Macrophages from a single donor were fed SA at a 1:1 MOI for 30 min. At indicated time points, macrophages were lysed in pH 11 H₂O, bacteria recovered, and CFU enumerated. Bars indicate the average CFU/ml recovered from duplicate wells from 4 separate experiments ± sem. P values were determined using one-way ANOVA and Newman-Keuls post-test. *P < 0.05 vs. control at 90 min; #P < 0.05 vs. control at 180 min; ^P < 0.05 vs. IFN-γ at 180 min.

IFN-γ activation enhances cell–cell interaction between macrophages and PMN-SA

Overnight IFN-γ treatment of macrophages enhances efferocytosis of opsonized apoptotic Jurkat T cells [23]. Consequently, we reasoned that IFN-γ may similarly enhance the internalization of PMN-SA by macrophages. As expected, few freshly isolated PMN bound to or were ingested by control macrophages (Fig. 3). In contrast, macrophages readily ingested apoptotic PMN, and IFN-γ treatment did not increase efferocytosis of apoptotic PMN (Fig. 3). Compared with apoptotic PMN, PMN-SA bound similarly to control macrophages, but few PMN-SA were ingested. IFN-γ treatment of macrophages enhanced binding of PMN-SA but did not increase macrophages internalization of PMN-SA (Fig. 3). These data suggest that IFN-γ augmented cell–cell interactions between macrophages and PMN-SA but did not stimulate efferocytosis of apoptotic PMN or PMN-SA.

Figure 3. IFN-γ treatment of macrophages enhances binding but not internalization of PMN-SA.

Control macrophages (white bars) or macrophages treated for 4 days with 420 U/ml IFN-γ (black bars) were fed freshly isolated PMN, apoptotic PMN, or PMN-SA for 60 min. Percent binding (A) and percent internalization (B) were measured by flow cytometry. Bars indicate the average of 4 experiments ± sem (n = 4). P values were determined using a two-way ANOVA and Bonferroni’s post-test. *P < 0.05 IFN-γ vs. control.

PMN-SA modulate subsequent inflammasome activation by macrophages

Although IL-1β is required for optimal neutrophil responses in murine models of staphylococcal infection [24], its role in human anti-staphylococcal responses is not fully characterized. IL-1β and IL-6 production is blunted in human macrophages treated with PMN-SA vs. SA alone [11], but it is unclear if the observed decrease in cytokine production resulted from the failure of PMN-SA to activate macrophages or from active inhibition of cytokine production. Consequently, we sought to distinguish between these two potential causes for the depressed cytokine production by macrophages in response to PMN-SA in advance of studies directed at IFN-γ. Το that end, we examined the effects of PMN-SA exposure on the capacity of macrophages to respond to subsequent stimulation with established agonists. In these experiments, macrophage stimulation extended over 20 h, an interval during which PMN-SA lyse and release viable SA, which in turn, will directly prompt cytokine production by macrophages. To eliminate any contribution to macrophage cytokine production by viable extracellular SA, we modified our experimental design by adding the antibiotic gentamicin after 1 h of stimulation to kill any escaped SA. We recognized that gentamicin enters eukaryotic cells [25–27]; however, PMN-SA lysis still occurs when gentamicin kills extracellular and to some extent, intracellular bacteria [28].

Agonist-triggered production of IL-1β is a two-step process that first requires synthesis of pro-IL-1β to prime the inflammasome for subsequent activation and concomitant processing of pro-IL-1β to the mature cytokine. LPS and silica—agents that prime and activate the NLRP3 inflammasome, respectively—stimulated IL-1β production by macrophages (P < 0.05, paired Student’s t test), and gentamicin did not alter this response, demonstrating that gentamicin alone did not alter IL-1β production (Fig. 4A). Although PMN-SA elicited IL-1β release from macrophages, gentamicin inhibited the response, suggesting that viable extracellular SA from lysed PMN-SA, not PMN-SA per se, were responsible for inflammasome activation. These data suggest that the production of IL-1β by macrophages exposed to PMN-SA was indirect and secondary to escaped, viable SA.

Figure 4. PMN-SA modulate macrophage NLRP3 inflammasome activation.

Macrophages were treated with buffer (RPMI with 10% PHS) or fed PMN-SA in buffer for 1 h. After 1 h, cells were washed, and fresh buffer, with or without gentamicin (Gent), was added. Macrophages were then treated with buffer or 50 ng/ml LPS for 4 h, followed by 500 μg/ml silica for 15 h. Supernatants were analyzed for IL-1β (A) or IL-6 (B) production by ELISA. Bars represent the mean cytokine production from at least 3 experiments ± sem. P values were determined using a two-way ANOVA and Bonferroni’s post-test; **P < 0.01. (C) Lysates were analyzed for IL-1β processing by immunoblot and imaged using the Odyssey imaging system (LI-COR, Lincoln, NE, USA). Shown is a representative experiment of 6. Pro-IL-1β signal (sum of pixel values in a given area) was quantified using LI-COR software. Mϕ, Macrophage. (D) Bars represent the average of 6 experiments ± sem. P values were determined using a one-way ANOVA and Dunnett’s post-test. *P < 0.05; ***P < 0.0001 vs. control. (E) Supernatants were analyzed for IL-1β production by ELISA. Macrophages were treated as described in A and B with the inclusion of gentamicin in the media. Bars represent the mean cytokine production from at least 3 experiments ± sem. P values were determined using a repeated-measures one-way ANOVA and Tukey’s post-test. ^P < 0.05 vs. control; #P < 0.05 vs. LPS; ‡P < 0.05 vs. PMN-SA; **P < 0.01 vs. PMN-SA + LPS + silica.

Unexpectedly, pretreatment with PMN-SA tended to increase IL-1β production by macrophages subsequently stimulated with LPS + silica when compared with responses of macrophages treated with LPS + silica alone (Fig. 4A). Gentamicin did not significantly inhibit the PMN-SA-dependent augmented response to LPS and silica, suggesting that viable bacteria are not relevant to the response. As viable bacteria were not required to elicit an enhanced response to LPS and silica, PMN-SA might directly contribute to macrophage NLRP3 inflammasome priming.

As production of IL-6 correlates with synthesis of pro-IL-1β and is used clinically as a surrogate marker for IL-1β [29, 30], IL-6 production by macrophages was assessed. Compared with unstimulated macrophages, macrophages treated with PMN-SA for 20 h produced more IL-6 (P < 0.05, paired Student’s t test). Gentamicin did not affect the amount of IL-6 produced by LPS-stimulated macrophages, demonstrating that gentamicin did not directly alter macrophage cytokine production (Fig. 4B). Importantly, macrophages stimulated with PMN-SA in the absence or presence of gentamicin produced equivalent amounts of IL-6. Taken together, these data demonstrate that PMN-SA alone, not viable SA that escaped from PMN-SA, mediated IL-6 production by macrophages and might contribute to inflammasome priming.

To test further our conclusion that PMN-SA primed inflammasome activation in macrophages, we probed lysates from macrophages, cultured with or without PMN-SA, for the presence of pro-IL-1β. As positive controls, we treated macrophages with LPS and LPS + silica, conditions known to enhance expression of pro-IL-1β and IL-1β processing in macrophages, respectively. We detected pro-IL-1β in lysates from macrophages under all of the conditions where IL-6 was detectable by ELISA, including PMN-SA (Fig. 4C and D). In addition, immunoblots demonstrated caspase-1-dependent processing of pro-IL-1β to the mature 17-kDa fragment under conditions where silica was present (Fig. 4C). In the immunoblot of IL-1β, a doublet of PMN-related bands flanked the 17-kDa fragment (Fig. 4C). To confirm that IL-1β processing occurred, we quantitated IL-1β release. Treatment of macrophages with LPS + silica, but not with media or LPS alone, stimulated release of IL-1β. In contrast to LPS + silica, PMN-SA failed to induce significant IL-1β production by macrophages. To determine if PMN-SA exposure primed macrophages for inflammasome activation, silica was added following pretreatment with PMN-SA. Addition of silica caused a 11.3-fold increase in IL-1β production compared with PMN-SA pretreatment alone (Fig. 4E), suggesting that PMN-SA directly prime the NLRP3 inflammasome. Moreover, PMN-SA pretreatment and LPS-priming had an additive effect on IL-1β production following stimulation with silica. Taken together, these data suggest that PMN-SA enhances both macrophage IL-6 production and inflammasome priming.

IFN-γ activation augments macrophage IL-6 production in response to SA and PMN-SA

As IFN-γ increases macrophage responsiveness to TLR ligands and cytokines [31–34] and binding of PMN-SA (Fig. 3), we investigated the effect of IFN-γ treatment on proinflammatory cytokine production by macrophages in response to various agonists. IFN-γ treatment alone did not trigger IL-6 or IL-1β production from macrophages, whereas phagocytosis of SA by macrophages induced production of IL-6 and IL-1β (Fig. 5). In response to SA, IFN-γ-treated macrophages produced 4-fold more IL-6 than did untreated macrophages. As previously demonstrated, macrophages fed PMN-SA produced less IL-6 than did macrophages fed SA alone (1.6 ± 0.7 vs. 4.3 ± 0.8 ng/ml, respectively; P < 0.01, paired Student’s t test). Treatment of macrophages with IFN-γ enhanced IL-6 production by macrophages fed PMN-SA from 1.6 ± 0.7 to 8.2 ± 3.1 ng/ml (Fig. 5A). Although these data were not significant in a 2-way ANOVA, it is worth noting that IFN-γ restored macrophage IL-6 levels in response to PMN-SA to that of macrophages fed SA alone (8.2 ± 3.1 vs. 4.3 ± 0.8 ng/ml), suggesting that the blunted production of IL-6 in response to PMN-SA could be overcome with IFN-γ. Despite the heightened levels of IL-6 produced by IFN-γ-treated macrophages, the levels of IL-1β produced by macrophages remained unchanged in control and IFN-γ-treated macrophages after treatment with SA, PMN-SA, or LPS and silica (Fig. 5B). Together, these data suggest that IFN-γ enhanced IL-6 production without prompting excessive IL-1β production.

Figure 5. IFN-γ augments macrophage production of IL-6 but not IL-1β.

Macrophages were treated with buffer [no treatment (NT); white bars] or buffer containing 420 U/ml IFN-γ (black bars) for 4 days. Macrophages were then treated with buffer or fed SA or PMN-SA for 1 h. Uningested cells were washed away, fresh buffer with gentamicin added, and macrophages incubated for 20 h. Alternatively, macrophages were treated with 50 ng/ml LPS for 4 h, followed by 500 μg/ml silica for 15 h. Supernatants were analyzed for IL-6 (A) and IL-1β (B) production by ELISA. Bars represent the mean cytokine from 3 experiments ± sem. P values were determined using a two-way ANOVA and Bonferroni’s post-test. ***P < 0.001.

DISCUSSION

The failure of phagocytes to kill and eliminate SA culminates in both persistent infection and amplification of local inflammation. PMN retaining viable SA lyse in a Necrostatin-1-dependent manner, thereby releasing not only living SA but also potentially noxious intracellular components that fuel additional tissue damage. Likewise, macrophages fail to kill SA and ultimately die from excessive SA burden [5–9]. Although SA causes disease in apparently healthy individuals, infection occurs more frequently and with more extreme consequences in patients with CGD. IFN-γ reduces the risk and severity of pyogenic infections in individuals with CGD, although the cellular basis for the observed protection is unknown. As IFN-γ can regulate several macrophage functions (reviewed in ref. [35]), we tested the hypothesis that IFN-γ treatment could correct the innate pathways that are typically derailed in SA infection, namely killing of SA, efferocytosis of PMN-SA, and production of cytokines. We provide evidence that IFN-γ treatment of macrophages enhanced the ability of macrophages to kill SA and augmented production of IL-6 in response to SA.

Multiple elements of phagocyte responses to SA contribute to the overall reaction to infection. Although, IFN-γ does not increase staphylococcal killing by PMN from CGD patients [4], IFN-γ-dependent, cell-mediated killing of SA has been shown in other cell types, including mast cells, monocytes, and endothelial cells [36–38]. Here, we show that under IFN-γ treatment conditions that prime the NADPH oxidase, IFN-γ enhanced the capacity of human macrophages to kill SA (Figs. 1 and 2). Engagement of the IFN-γR activates Jak1 and Stat1 signaling pathways within 15–30 min of IFN-γ treatment to induce the first wave of transcription of responsive genes [35, 39]. In contrast to these rapid transcriptional responses, activity of the NADPH oxidase in human macrophages peaks between 3 and 4 days of IFN-γ treatment (Fig. 1 and refs. [21, 22]). Enhanced bacteriostatic activity against SA by macrophages in the presence of IFN-γ may reflect the combined effects of established IFN-γ-mediated responses, including priming of the phagocyte NADPH oxidase, altered maturation of phagosomes, or induction of vitamin D-dependent antimicrobial peptides [40–43]. IFN-γ enhanced bacteriostatic activity in human macrophages in the first 90 min after phagocytosis, and it is plausible that a reduction in bacterial load could benefit host anti-staphylococcal responses. For example, leukocytes recruited to the site of infection will encounter fewer bacteria, and beneficial innate immune responses will be enhanced by the action of cytokines, including IL-6, chemokines, and other mediators produced during the acute-phase inflammation.

In addition to examining the modulation of phagocyte responses directly to SA, we reasoned that IFN-γ might alter how macrophages condition the local setting of the infection, with respect to both interactions with PMN-SA and to cytokine production. IFN-γ enhanced the binding of PMN-SA to macrophages but did not increase internalization of PMN-SA or apoptotic PMN. Fernandez-Boyanapalli et al. [23] demonstrated that treatment of macrophages with 4200 U/ml IFN-γ for 24 h enhances uptake of apoptotic Jurkat T cells by IFN-γ-primed macrophages. In contrast, treatment of macrophages with IFN-γ with 4200 U/ml for 24 h or 420 U/ml for 4 days (our standard conditions) had no effect on internalization of effete PMN (Fig. 3, and data not shown), suggesting that modulation of efferocytosis by IFN-γ depends on the target cell being engulfed by macrophages. PMN fed heat-killed SA also bound to macrophages without triggering significant engulfment (Supplemental Fig. 1); however, PMN fed heat-killed SA do not lyse [28]. We hypothesize that PMN-SA exacerbates inflammation in two complementary ways: by resisting engulfment by macrophages and by lysing and releasing DAMPs. Consistent with this hypothesis, we found that SA-driven PMN lysis contributes to proinflammatory cytokine production.

Despite inefficient engulfment by macrophages, PMN-SA stimulated IL-6 production and enhanced macrophage responsiveness to activation of the NLRP3 inflammasome (Fig. 4). Killing of extracellular SA with gentamicin did not affect IL-6 production (Fig. 4B) but diminished IL-1β production from macrophages fed PMN-SA (Fig. 4A). These data illustrate that viable organisms released from lysed PMN directly activated inflammasomes and support the notion that escaped SA could contribute to rampant inflammation. Moreover, PMN-SA potentiated subsequent activation by the prototypical NLRP3 inflammasome activator silica in the absence of viable extracellular bacteria (Fig. 4C–E). It is likely that in the latter example, DAMPs and PAMPs released from lysed PMN-SA contributed to macrophage IL-6 production and pro-IL-1β synthesis (Fig. 4A). Candidates include the SA cell wall component lipoteichoic acid, host-derived migration inhibitory factor-related protein 8/14 or S100 calcium-binding protein A12, or the SA pore-forming toxin Panton-Valentine leukocidin, each of which can stimulate IL-1β production in monocytes [8]. The precise contributions of DAMPs and PAMPs to IL-6 and IL-1β production remain unidentified.

However, the discordant effects of IFN-γ on production of IL-6 and IL-1β may suggest a nuanced modulation of the inflammatory response. IL-6 plays important roles both in innate and adaptive immunity. In the context of innate responses, IL-6 supports the initial wave of PMN recruitment, promotes the subsequent switch to monocyte recruitment, and then enhances PMN apoptosis, thereby augmenting the resolution of neutrophilic inflammation. IL-6 also promotes antibody production by B cells, recruits T cells, and in the presence of TGF-β, skews naïve T cells toward Th17 differentiation [44]. Given the clinical picture of SA infection and predisposition of CGD patients toward unwanted inflammatory reactions, limiting excessive neutrophilic infiltration while promoting a beneficial Th17 adaptive immune response could decrease the severity and frequency of staphylococcal infections and may account for the salutary effects of IFN-γ in the context of CGD.

In line with our observations that IFN-γ can augment selective macrophage responses pertinent to host defense, other groups have shown that IFN-γ provides protection in murine SA infections. IFN-γ reduces mortality in a mouse model of sepsis [45], and endogenous IFN-γ from stimulated NK cells enhances the ability of macrophages to protect mice in an SA pneumonia model [46]. Whereas murine phagocytes rely more heavily on IFN-γ-inducible NO production than do human cells [47], we suggest that the ability of IFN-γ to enhance oxidase function, SA killing, and cytokine production contributes to improved host defense against staphylococcal infection.

In summary, IFN-γ improved several elements of the phagocyte response to SA. IFN-γ treatment of human macrophages accelerated initial killing of SA (Fig. 2) and heightened IL-6 production in response to SA without concomitantly prompting additional IL-1β production (Fig. 5). IFN-γ augmented binding, but not internalization, of PMN-SA and restored IL-6 cytokine production to the same level as that of control macrophages that engulfed SA (Figs. 3 and 5). The modulation of these pathways could explain how IFN-γ activation of human macrophages augments host resistance to SA infection when administered prophylactically to individuals with CGD. Our current efforts are directed at investigating how IFN-γ-treated macrophages from CGD patients respond to stimulation with SA and PMN-SA. Together, these data will provide insights into the therapeutic potential of IFN-γ in CGD and perhaps in other disorders of phagocyte function.

AUTHORSHIP

M.C.G.-W. executed experiments, and M.C.G.-W. and W.M.N. conceptualized the study, designed experiments, interpreted data, and wrote the manuscript.

Glossary

A₅₅₀

absorbance at 550 nm

CGD

chronic granulomatous disease

CTFR

CellTrace Far Red

DAMP

danger-associated molecular pattern

HSA

human serum albumin

MOI

multiplicity of infection

NLRP3

nucleotide-binding oligomerization domain-leucine-rich repeats containing pyrin domain 3

PAMP

pathogen-associated molecular pattern

PB

phagocytosis buffer

PHS

pooled human serum

PMN

polymorphonuclear leukocyte(s), neutrophil(s)

SA

Staphylococcus aureus

TSB

tryptic soy broth

DISCLOSURES

M.C.G.-W. received salary support from Horizon Pharma.