Francisella novicida inhibits spontaneous apoptosis and extends human neutrophil lifespan

F. novicida acts via the intrinsic and extrinsic pathways to prolong neutrophil viability by a mechanism independent of opsonins and phagocytosis.

Abstract

Francisella novicida is a Gram-negative bacterium that is closely related to the highly virulent facultative intracellular pathogen, Francisella tularensis. Data published by us and others demonstrate that F. tularensis virulence correlates directly with its ability to impair constitutive apoptosis and extend human neutrophil lifespan. In contrast, F. novicida is attenuated in humans, and the mechanisms that account for this are incompletely defined. Our published data demonstrate that F. novicida binds natural IgG that is present in normal human serum, which in turn, elicits NADPH oxidase activation that does not occur in response to F. tularensis. As it is established that phagocytosis and oxidant production markedly accelerate neutrophil death, we predicted that F. novicida may influence the neutrophil lifespan in an opsonin-dependent manner. To test this hypothesis, we quantified bacterial uptake, phosphatidylserine (PS) externalization, and changes in nuclear morphology, as well as the kinetics of procaspase-3, -8, and -9 processing and activation. To our surprise, we discovered that F. novicida not only failed to accelerate neutrophil death but also diminished and delayed apoptosis in a dose-dependent, but opsonin-independent, manner. In keeping with this, studies of conditioned media (CM) showed that neutrophil longevity could be uncoupled from phagocytosis and that F. novicida stimulated neutrophil secretion of CXCL8. Taken together, the results of this study reveal shared and unique aspects of the mechanisms used by Francisella species to manipulate neutrophil lifespan and as such, advance understanding of cell death regulation during infection.

Introduction

PMNs (neutrophils) are the most abundant leukocyte in the circulation of humans and are rapidly recruited to sites of infection. As a crucial effector of the innate immune system, neutrophils are the first line of defense against invading bacterial and fungal pathogens. Upon recruitment, these immune cells phagocytose and subsequently kill microbes using a combination of ROS and cytotoxic granule proteins, including antimicrobial peptides and proteolytic enzymes [1, 2]. Neutrophils have an inherently short lifespan and undergo constitutive (spontaneous) apoptosis, generally within 24 h following release into circulation [3, 4]. However, the rate of neutrophil apoptosis can be influenced by a number of factors, including cytokines, PAMPs, and phagocytosis [5]. Notably, phagocytosis of most microbes and subsequent ROS production significantly accelerate neutrophil apoptosis and promote clearance of these cells by tissue macrophages [6]. It is particularly important that neutrophils undergo apoptosis and are cleared in a timely manner to limit leakage of cytotoxic and inflammatory debris that can damage host tissue and exacerbate inflammation [7].

F. tularensis is a Gram-negative, facultative intracellular bacterium and the etiologic agent of the highly infectious, zoonotic disease tularemia [8]. As few as 10 organisms can culminate in severe disease with fatality rates as high as 60% if infections are left untreated [9, 10]. Two subsp. of F. tularensis, —subsp. tularensis (type A) and subsp. holarctica (type B)—cause disease in humans. Although these organisms differ in geographic distribution, virulence, biochemical characteristics, and growth requirements, their genomes are nearly identical [11–14].

F. tularensis has evolved a number of strategies to modulate effectors of the innate immune response, which allows this organism to cause severe disease at low inocula. These include an atypical LPS with low bioactivity; resistance to complement-mediated lysis; a capacity to infect multiple cell types, including macrophages, neutrophils, dendritic cells, and epithelial cells; disruption of oxidative host defense via inhibition of NADPH oxidase assembly and activity; and an ability to replicate to high density in host cell cytosol subsequent to phagosome escape [15, 16]. Furthermore, we have shown that both type A and type B F. tularensis strains interfere with the major pathways of apoptosis in neutrophils and significantly extend the cell lifespan, demonstrating another innate immune evasion strategy of this pathogen [16–19].

The contribution of neutrophils to tularemia pathogenesis has been highlighted in a number of studies. Aerosol infection with virulent type B F. tularensis in nonhuman primate models provided evidence that neutrophils function in tissue destruction and inflammation [20, 21]. Moreover, numerous studies have demonstrated that infected animals present with gradual obstruction of airways by immune cells, bacteria, and necrotic debris. In keeping with these data, flow cytometric analysis of infected cells in the lung demonstrate that neutrophils comprise the majority of F. tularensis-infected cells in the lung by day 3 postinfection with either type A or type B strains [22]. These findings indicate that neutrophil antimicrobial and proresolving functions are uncontrolled in the context of tularemia and ultimately contribute to rapid disease advancement. Moreover, recent studies have shown that the blocking of neutrophil migration into the lungs of F. tularensis-infected mice decreases bacterial burden and significantly enhances host survival [23]. Collectively, these observations support the concept that neutrophils are unable to eliminate F. tularensis and ultimately contribute to inflammatory tissue damage and failure to resolve infection [24, 25].

In 1950, a bacterium was isolated from a salt-water sample collected in Utah that morphologically resembled F. tularensis. However, following extensive characterization, including biochemical and virulence studies, this bacterium was classified as a separate species of the genus Francisella [26, 27]. F. novicida has since been found in other sources of brackish water but does not appear to infect animals or arthropod vectors in nature [28]. Unlike F. tularensis, which is endemic in many regions of the world, there are only 15 documented cases of human infection with F. novicida, several of which have been linked to near-drowning incidents. Of the 14 cases for which data are available, 12 occurred in persons who had significant underlying health problems or were immunocompromised, and the two infections of healthy individuals were notable only for lymphadenopathy, which was not accompanied by fever or other signs of illness [28, 29].

As the genomes of F. novicida and F. tularensis are almost identical, yet the virulence and pathogenicity of these organisms are incongruous, there is significant interest in the identification and characterization of their shared and distinct features [28]. Studies of isolated phagocytes and animal models demonstrate that F. novicida exhibits only some of the same virulence attributes and mechanisms as F. tularensis. In particular, F. novicida is markedly more proinflammatory, as indicated by analyses of TLR signaling, proinflammatory cytokine production, and inflammasome activation, which significantly diminish bacterial replication in macrophages and favor host defense [30–32]. In addition, we have shown that these organisms are opsonized by different mechanisms upon incubation in 50% normal human serum, with natural IgG binding to F. novicida eliciting neutrophil NADPH oxidase activation that does not occur following natural IgM binding to F. tularensis [16, 33–36]. As oxidants generated during the respiratory burst typically accelerate neutrophil apoptosis, we hypothesized that F. novicida may affect the neutrophil lifespan in an opsonin-dependent manner [16, 17, 19, 33, 34, 37]. The data presented here show that we disproved our hypothesis and in so doing, identified both conserved and incongruent molecular mechanisms that allow both of these Francisella species to extend the human neutrophil lifespan significantly.

MATERIALS AND METHODS

Neutrophil isolation

Heparinized venous blood was obtained from healthy adult volunteers with no history of tularemia, in accordance with a protocol approved by the Institutional Review Board for Human Subjects at the University of Iowa. PMNs were isolated using dextran sedimentation, subsequent Ficoll-Hypaque density gradient separation, and hypotonic erythrocyte lysis, as previously described [38]. PMNs were suspended in HBSS without divalent cations, enumerated, and diluted to 2 × 10⁷ PMNs/ml. With the use of this method, PMN purity was routinely >95%, with eosinophils as the major contaminant. Replicate experiments were performed using PMNs from different donors.

Bacterial strains and growth conditions

F. novicida strain U112 was obtained from Dr. Colin Manoil (University of Washington, Seattle, WA, USA) and has been described previously [39]. U112 was inoculated onto cysteine heart agar plates supplemented with 9% defibrinated sheep blood and grown for 24 h at 37°C in 5% CO₂. Liquid cultures of U112 were started at an OD₆₀₀ = 0.005 in brain heart infusion broth and incubated overnight with shaking at 200 rpm. Overnight cultures were diluted to an OD₆₀₀ = 0.200 in broth and incubated at 37°C, with shaking at 200 rpm for 2–4 h. Midexponential phase bacteria were harvested and washed twice with HBSS containing divalent cations. Cultures of sGFP-expressing U112 contained 25 μg/ml spectinomycin to ensure retention of the associated plasmid. Selected experiments included sGFP-expressing F. tularensis LVS that was cultivated and in a similar manner [19, 36].

Infection of neutrophils

Our current and previous studies of neutrophil apoptosis use a serum-free infection model to avoid possible confounding effects of growth factors and other serum components on neutrophil lifespan [17, 40]. PMNs were diluted to 5 × 10⁶/ml in HEPES-buffered RPMI 1640 containing l-glutamine and phenol red (Lonza, Walkersville, MD, USA). Unless otherwise indicated, infections were performed using unopsonized F. novicida at an MOI of 200:1. Cultures (1–2 ml each) were incubated in 14 ml polypropylene tubes at 37°C with 5% CO₂ for 0–48 h. Selected experiments included U112 that were killed by incubation in 10% formalin before washing and addition to PMNs [33] or live bacteria that were opsonized for 30 min at 37°C in 50% pooled human nonimmune serum that was prepared as we described previously [36]. All bacteria were washed twice with HBSS containing divalent cations and quantified by measurement of the absorbance at 600 nm.

Detection of PS externalization

PS externalization was determined by flow cytometric analysis using AV-FITC, according to the manufacturer’s instructions (BioVision, Milpitas, CA, USA). PI (BioVision) staining was included to differentiate early apoptotic from late apoptotic/necrotic PMNs. PMNs (5 × 10⁵) were costained with AV-FITC and PI in binding buffer (10 mM HEPES, pH 7.4, 140 mM NaCl, 2.5 mM CaCl₂) before analysis using the BD Accuri C6 Cytometer (San Jose, CA, USA). Approximately 10,000 events were collected for each sample, and the data were analyzed using the BD Accuri CFlow software.

Quantitation of infection efficiency by flow cytometry

PMNs were infected with sGFP-expressing F. novicida, as described above. At 0, 6, 12, and 24 hpi, neutrophils were diluted 1:1 in cold PBS and analyzed by flow cytometry using our established methods [36]. In brief, events were gated to include only PMNs based on forward- and side-scatter properties. Approximately 10,000 gated events were collected, and data were analyzed using both BD Accuri CFlow and FlowJo (Tree Star, Ashland, OR, USA) software. sGFP-positive PMNs were determined by comparison with uninfected controls. PMNs that had ingested U112-sGFP were distinguished from PMNs with only surface-attached U112 sGFP by normalizing to PMNs incubated with bacteria at 4°C (which prevents phagocytosis). An infection index was calculated using the following equation: (% positive PMNs × MFI at 37°C) − (% positive cells × MFI at 4°C) [36].

Quantitation of bacterial binding and uptake by fluorescence microscopy

To analyze bacterial binding and uptake at the level of single cells, PMNs in suspension were infected with opsonized or unopsonized sGFP-expressing F. novicida for 60 min at 37°C and then cytocentrifuged onto acid-washed coverslips or plated onto coverslips that had been precoated with polylysine. Attached PMNs were fixed with 10% formalin, permeabilized with cold acetone:methanol (1:1), washed once with PBS, and blocked in PBS containing 0.5 mg/ml NaN₃, 5 mg/ml BSA, and 10% horse serum, as we described previously [36, 41]. Fixed and permeabilized cells were stained with DAPI (University of Iowa Central Microscopy Research Facility, Iowa City, IA, USA) to label PMN nuclei or with mAb specific for CD16 (Clone 3G8) at 1:100 dilution (BioLegend, San Diego, CA, USA) and Dylight 549-conjugated goat anti-mouse F(ab′)₂ secondary antibodies (Jackson ImmunoResearch Laboratories, West Grove, PA, USA) at 1:500 dilution to label PMN plasma membranes. Where noted, binding and uptake of U112 were directly compared with neutrophils that were infected with opsonized or unopsonized sGFP-expressing F. tularensis LVS.

For synchronized phagocytosis assays, PMNs were plated onto acid-washed glass coverslips in 35 mm tissue-culture dishes that had been precoated with 10% serum [41]. Opsonized or unopsonized U112 were added to each dish at an MOI of 25:1 in 2 ml serum-free RPMI 1640. Dishes were centrifuged at 600 g for 4 min at 15°C to synchronize bacterial binding and then transferred to the tissue-culture incubator to initiate phagocytosis [41]. After 15 min at 37°C, the medium was aspirated, monolayers were washed once with HBSS, and cells were fixed and processed for fluorescence microscopy as described above. Cells were analyzed and photographed using an Axioplan 2 photomicroscope (Carl Zeiss Microscopy, Thornwood, NY, USA). Each experiment was performed in triplicate, and at least 100 infected cells per coverslip were examined.

Simultaneous analysis of infection and PS externalization

PMNs were left untreated or were infected with sGFP-expressing U112 for 6, 12, 18, or 24 h at 37°C, stained with AV-APC (BioLegend), and then processed for flow cytometry as described above.

NADPH oxidase activity

Generation of neutrophil ROS was assessed using the luminol assay and by NBT staining, as described previously [33, 35, 42].

Fas-induced apoptosis

PMNs were left untreated or infected with F. novicida for 1 h before the addition of 500 ng/ml mouse anti-Fas IgM (human-activating Clone CH11; Millipore, Darmstadt, Germany). Neutrophil apoptosis was measured by assessing PS externalization using flow cytometry at 6 h following Fas cross-linking.

Preparation of neutrophil lysates for immunoblotting

PMNs (5 × 10⁶) were left untreated, treated with 1 μM STS (Sigma-Aldrich, St. Louis, MO, USA) or 500 ng/ml anti-Fas IgM, or infected with F. novicida, as described above. At the indicated time points, cell lysates were prepared, as described previously [11, 17, 19, 40]. In brief, neutrophils were pelleted and then resuspended in ice-cold protease inhibitor cocktail (TBS containing aprotinin, leupeptin, PMSF, 4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride, levamisole, bestatin, E-64, and pepstatin A; Sigma-Aldrich), supplemented with the Halt phosphatase inhibitor cocktail (sodium fluoride, sodium orthovanadate, sodium pyrophosphate, and β-glycerophosphate; Thermo Fisher Scientific, Waltham, MA, USA), and incubated on ice for 10 min. Neutrophils were then lysed with 1% Nonidet P-40 and clarified by centrifugation. Protein concentrations were determined using the Pierce BCA Protein Assay Kit (Thermo Fisher Scientific). Proteins (5–15 μg/lane) were separated using NuPAGE 4–12% Bis-Tris gradient gels and subsequently transferred to polyvinylidene fluoride membranes by electroblotting. Western blot analyses for processing of caspase-3, -8, and -9 were performed using mouse anti-caspase-3 mAb (BioVision), mouse anti-caspase-8 (1C12) mAb (Cell Signaling Technology, Danvers, MA, USA), and rabbit anti-cleaved caspase-9 mAb (BioVision). Mouse anti-actin mAb was used as a loading control (BioLegend). Bands were detected, using HRP-conjugated secondary antibodies and SuperSignal West Femto chemiluminescent substrate, on the Odyssey Fc Imaging System (LI-COR, Lincoln, NE, USA).

Caspase activity assays

PMNs were left untreated, infected with F. novicida, or treated with anti-Fas IgM or STS in HEPES-buffered RPMI 1640 without phenol red. The activities of caspase isoforms were measured using Caspase-Glo 3/7, 8, and 9 Assay Kits (Promega, Madison, WI, USA), in accordance with the manufacturer’s instructions. In each case, enzyme activity was assessed by quantifying the luminescence generated upon cleavage of the respective proluminescent caspase isoform-specific tetrapeptide substrate: Asp-Glu-Val-Asp (caspase-3), Leu-Glu-Thr-Asp (caspase-8), or Leu-Glu-His-Asp (caspase-9). In brief, PMNs (5 × 10⁴) were transferred to a white-walled 96-well microplate in triplicate. Equal volumes of the Caspase-Glo reagent, containing a respective tetrapeptide, were added to the wells, mixed thoroughly, and incubated with shaking at room temperature for 45 min. Luminescence was detected using a NOVOstar luminometer (BMG Labtech, Cary, NC, USA). Caspase activities were expressed as relative mean luminescence intensity after normalization to the maximum (peak) response obtained with STS or anti-Fas IgM.

Preparation of CM

CM were prepared from 5 × 10⁶/ml uninfected PMNs (PMN-CM), U112-iPMNs (iPMN-CM), or 1 × 10⁹/ml bacteria alone (U112-CM) after incubation in HEPES-buffered RPMI 1640 containing l-glutamine at 37°C with 5% CO₂ for 24 h. After centrifugation to pellet cells and/or bacteria, the supernatants were sterilized using 0.45 μm syringe filters. CM sterility was confirmed by plating for CFUs.

Assessment of nuclear morphology

PMNs were cytocentrifuged onto acid-washed coverslips, as described above, and then fixed and stained using the Fisher HealthCare PROTOCOL Hema 3 staining kit (Fisher Scientific, Pittsburgh, PA, USA). PMNs were examined using light microscopy, and cells with condensed nuclei (spherical or bilobed) were scored as apoptotic [7, 17]. For each replicate, >300 PMNs per coverslip were counted independently by 2 individuals.

Quantitation of CXCL8 and PGE₂ secretion by ELISA

At the indicated time points, supernatants from cultures of control PMNs or cells that were infected with U112, opsonized U112, or fk U112 were collected and assayed for the presence of CXCL8 or PGE₂ using the human CXCL8 or PGE₂ ELISA kits (both from R&D Systems, Minneapolis, MN, USA), according to the manufacturer’s instructions. Absorbance was measured using a SpectraMax Plus (Molecular Devices, Sunnyvale, CA, USA).

Effects of CXCL8 on apoptosis

Neutrophils were treated with 100 ng/ml recombinant human CXCL8 (BioLegend) for 12 or 18 h, and apoptosis was measured using AV-FITC/PI staining and flow cytometry as described above. Where noted, PMNs were treated with 1 μM Reparixin or 1 μM SB225002 (both from Cayman Chemical, Ann Arbor, MI, USA) in the presence or absence of iPMN-CM, and apoptosis was quantified by flow cytometry.

Statistical analyses

Data from experiments with 1 control and 1 experimental sample were analyzed using a paired Student’s t test. Experiments with 1 control group and 2 or more experimental groups were analyzed by one-way ANOVA (single time point) or two-way ANOVA (time course), followed by Dunnett’s, Tukey’s, or Sidak’s multiple comparisons post-tests, as indicated in the figure legends. All analyses were performed using GraphPad Prism version 6.0 or 7 software. P < 0.05 was considered statistically significant.

RESULTS

F. novicida prolongs human neutrophil lifespan

Neutrophils are inherently short-lived leukocytes that are programmed to undergo constitutive apoptosis after ∼24 h, and cell death is typically accelerated further by phagocytosis [6, 43]. One of the early changes detectable in apoptotic cells is the translocation of PS from the inner leaflet to the outer leaflet of the plasma membrane [44]. To determine possible effects of F. novicida on neutrophil apoptosis, we quantified the rate and extent of PS externalization using AV-FITC, which binds PS in a calcium-dependent manner and can be quantified by flow cytometry [17]. The data in Fig. 1A demonstrate that as compared with the uninfected controls, F. novicida did not accelerate PMN death. Conversely, PMN apoptosis was significantly diminished and delayed, as 75.1 ± 4.0% of control PMNs were apoptotic (AV⁺/PI⁻) at 24 h, whereas only 38.7 ± 6.2% of PMNs infected with F. novicida were apoptotic at that time point (P = 0.0368, n = 3). Furthermore, we costained neutrophils with PI to distinguish early apoptotic PMNs from those that had progressed to late apoptosis/secondary necrosis. Compared with uninfected controls, infection with F. novicida also decreased the number of cells that had lost plasma membrane integrity, as 10.2 ± 0.8% of control PMNs were AV⁺/PI⁺ compared with only 3.7 ± 0.8% of U112-infected neutrophils. Hence, infection with F. novicida delayed neutrophil apoptosis and correspondingly, inhibited progression to secondary necrosis (Fig. 1B). Additional experiments demonstrated that the effects of F. novicida were dose dependent, with efficacy apparent at the lowest MOI tested (5:1) and maximal inhibition achieved at MOIs of 25:1 or greater (Fig. 1C). These data indicate that unopsonized F. novicida inhibits neutrophil apoptosis in a dose-dependent manner.

Figure 1. F. novicida delays spontaneous neutrophil apoptosis in a dose-dependent, opsonin-independent manner.

Unless otherwise noted, PMNs were left uninfected or infected with unopsonized U112 for the indicated amounts of time. (A) Pooled flow cytometric data indicate the percentage of early apoptotic (AV⁺/PI⁻) and late apoptotic (AV⁺/PI⁺) control (uninfected) and U112 iPMNs at 6, 12, and 24 h. Data are the means ± sem of 3 independent experiments, and P values were calculated by two-way repeated-measures ANOVA with Sidak’s multiple comparisons test. **P < 0.01. (B) Representative dot plots show the extent of AV-FITC and PI staining of uninfected and U112 iPMNs at 6, 12, and 24 h. (C) PMNs were infected with increasing doses of F. novicida, and AV-positive cells were quantified at 24 hpi. Data shown are the means ± sem of 5 independent experiments. (D) PMNs were left untreated or were infected with unopsonized or normal serum-opsonized (ops) F. novicida at high (200:1) and a low (25:1) MOIs. Apoptosis was quantified by flow cytometry after AV-FITC/PI staining at 24 h. Data are the means ± sem of 2 independent experiments. Where not visible, error bars are smaller than symbols.

Differential activation of neutrophils by opsonized and unopsonized F. novicida

Both F. tularensis and F. novicida resist complement-mediated lysis, and the viability of LVS and U112 is not diminished by opsonization with 50% normal human serum [45, 46]. However, these organisms are opsonized by different mechanisms, and opsonized U112 triggers a respiratory burst, whereas opsonized F. tularensis, including LVS, does not [33–36]. We extended these studies here and directly compared the ability of opsonized and unopsonized U112 and LVS to trigger NADPH oxidase activation. The rate and extent of ROS production were assessed first using the highly sensitive luminol assay. The data in Supplemental Fig. 1A confirm the ability of opsonized U112 to trigger rapid and robust ROS production. In contrast, responses to unopsonized U112 and either opsonized or unopsonized LVS were very low and barely exceeded chemiluminescence of the uninfected controls. NBT staining is a valuable tool for analysis of superoxide production at the level of individual phagosomes using light microscopy [33, 35]. By this assay, ∼80% of phagosomes containing opsonized U112 contained blue formazan deposits by 60 min after infection, and a representative image is shown in Supplemental Fig. 1B. Notably, NBT staining also highlights the established, normal heterogeneity in superoxide production, apparent between cells and among phagosomes within individual cells, which has been documented in neutrophils and macrophages by us and others [35, 47].

Both opsonized and unopsonized F. novicida delay neutrophil apoptosis

Phagocytosis and ROS production typically accelerates neutrophil apoptosis, and like constitutive PMN turnover, PICD is also tightly regulated [6, 7, 48]. Based on this paradigm, we hypothesized that F. novicida would influence the neutrophil lifespan in an opsonin-dependent manner. In marked contrast, we found that the opsonization state of F. novicida did not affect its ability to extend the neutrophil lifespan (Fig. 1D), as the data obtained for opsonized and unopsonized U112 were nearly identical at both MOIs tested.

Quantitation of bacterial binding, phagocytosis, and infection efficiency

Next, we considered the possibility that neutrophils may be infected to different extents by opsonized and unopsonized F. novicida, perhaps leading to subpopulations of cells within each suspension that differ with respect to lifespan or activation state. In initial experiments, we infected PMNs with sGFP-expressing U112 and quantified fluorescent cells at 6 h intervals using flow cytometry [36]. These data suggest that the number of sGFP-positive neutrophils increased progressively over the first several hours of infection (Fig. 2A). Although this assay has the advantage of being high throughput, it cannot distinguish surface attached and intracellular bacteria. Thus, in a second series of experiments, we used fluorescence microscopy to define infection more precisely via single-cell analysis. PMNs were infected in parallel with opsonized or unopsonized sGFP-expressing bacteria for 60 min before plating on coverslips or fixation; counterstaining with DAPI or antibodies to CD16 (to label PMN nuclei or plasma membranes, respectively); and analyzing using phase-contrast and fluorescence optics. Representative images of neutrophils infected with F. novicida are shown in Fig. 2B, and images of cells infected with F. tularensis are shown in Supplemental Fig. 2. These data revealed differences in these 2 organisms, as phagocytosis of cell-associated LVS was more dependent on opsonization compared with U112. For further analysis of U112 infection, we quantified the percentage of infected neutrophils, the bacterial load per infected cell, and the percentage of cell-associated bacteria that were phagocytosed (Fig. 2C). These data demonstrate that during the first hour of infection, opsonization significantly increased both the percentage of PMNs that were infected (53.2 ± 4 vs. 87.5 ± 4.5%, P < 0.001, n = 4) and the bacterial load per infected cell (3.0 ± 0.3 vs. 15.0 ± 4.7, P < 0.001, n = 4), yet in both cases, >90% of all cell-associated bacteria were intracellular. To confirm these results, we also quantified bacterial binding and uptake using a synchronized phagocytosis assay. Rapid bacterial binding of U112 (MOI 25:1) to adherent PMNs was induced by centrifugation (4 min, 600 g, 15°C), followed by a 15 min incubation at 37°C to induce phagocytosis [33]. Under these conditions, 81 ± 4% and 95 ± 2% of neutrophils were infected (Fig. 2D), despite the 8-fold decrease in MOI, and differences between opsonized and unopsonized bacteria were diminished but not ablated. Collectively, these data indicate that opsonins specifically enhance U112 binding to neutrophils under our standard assay conditions and that regardless of opsonization status, nearly all cell-associated F. novicida were readily engulfed, whereas LVS were not.

Figure 2. Quantitation of phagocytosis by flow cytometry and microscopy.

(A) Flow cytometry histograms show the extent of neutrophil infection with U112-sGFP at 0, 6, 12, 18, and 24 hpi. Data are from 1 experiment representative of 3 independent determinations. (B) Fluorescence microscopy images show the extent of neutrophil infection with unopsonized and opsonized U112 sGFP. At 60 min postinfection, cells were attached to coverslips, fixed, stained with DAPI, and then analyzed by fluorescence microscopy. Bacteria are shown in green (GFP fluorescence) as separate images and merged with DAPI fluorescence and phase-contrast optics. Arrows and arrowheads indicate surface-associated (uningested) and free extracellular bacteria, respectively. Unmarked bacteria are intracellular (ingested). (C) In additional experiments, neutrophils infected with opsonized or unopsonized (Unops.) U112 sGFP for 60 min were stained to detect CD16 in the plasma membrane to distinguish better attached and ingested bacteria. The percentage of infected PMNs, number of bacteria per infected cell, and percentage of ingested (intracellular) cell-associated bacteria were scored. Data are the means ± sem from 4 independent experiments. ***P < 0.001 by Student’s t test. (D) Synchronized phagocytosis of opsonized and unopsonized U112 sGFP by adherent PMNs. Bacteria were added to PMN monolayers on glass coverslips at an MOI of 25:1. Bacterial binding and uptake were synchronized by centrifugation (600 g, 4 min, 15°C), followed by incubation at 37°C for 15 min [33, 41]. Samples were processed for microscopy and analyzed as in C. Data are the means ± sem from 3 experiments. *P < 0.05 by Student’s t test.

F. novicida delays apoptosis of all neutrophils in the population to a similar extent

The data shown in Fig. 2A and C show that PMN suspensions contained a mixture of infected and uninfected cells during the first few hours after addition of U112. To determine if these cell subpopulations underwent apoptosis at similar or distinct rates, we used AV-APC and U112-sGFP for simultaneous detection of PS externalization and cell-associated bacteria using flow cytometry and included uninfected neutrophils as controls (Fig. 3A). These data demonstrate that both infected and uninfected neutrophils in the population progressed to apoptosis at a similar rate (e.g., 20 and 25% AV⁺ at 24 hpi) and remained healthy longer than the uninfected controls. The data in Figs. 2A and 3A also document a 10-fold increase in total PMN-associated sGFP fluorescence at 18−24 hpi that was quantified and presented as an overall infection index in Fig. 3B. These data suggest that as with LVS [17, 33], U112 may escape the phagosome and replicate ∼10-fold in neutrophil cytosol.

Figure 3. Simultaneous analysis of phagocytosis and PS externalization.

(A) Neutrophils were left untreated or were infected with U112 sGFP for 6, 12, 18, or 24 h and analyzed by flow cytometry after staining with AV-APC. Data shown are from 1 experiment that is representative of 3 independent determinations. (B) PMNs were infected with U112 sGFP for 0, 6, 12, 18, and 24 h. Infection was quantified by flow cytometry, and an overall infection index was calculated, as described in the Materials and Methods. Data are the means ± sem of 3 independent experiments.

F. novicida diminishes and delays procaspase-3 processing and inhibits caspase-3 activity in neutrophils

To gain insight into the mechanisms used by F. novicida to extend the PMN lifespan, we determined its effects on core apoptosis pathway components. Caspase-3 is the major effector caspase in human neutrophils and is primarily responsible for the cleavage of cellular substrates leading to the biochemical and morphologic changes that are characteristic of apoptotic cells. These changes include PS externalization, nuclear condensation, and DNA fragmentation [44]. Caspase-3 is synthesized as an inactive 35 kDa proenzyme that is processed into its mature/active (19–17 kDa) form following upstream initiator caspase activation and signaling. We used immunoblotting to determine the time course of procaspase-3 processing during F. novicida infection, and neutrophils infected with bacteria were directly compared with untreated controls, as well as PMNs that were treated with STS. STS is a protein kinase inhibitor with multiple targets that ultimately leads to robust activation of caspase-3 and is used in many studies to trigger rapid apoptosis via the intrinsic pathway [17, 19, 49].

Trace amounts of mature caspase-3 were apparent in the lysates of untreated control PMNs at 6 h and increased progressively thereafter, leading to nearly complete disappearance of the proenzyme by 24 h (Fig. 4A). As expected [19], procaspase-3 processing was accelerated by STS and was complete or nearly complete by 6 h after treatment (Fig. 4A). In contrast, procaspase-3 processing was diminished and delayed by U112 and was minimal even at 24 hpi (Fig. 4A).

Figure 4. Caspase-3 processing and activity are impaired by F. novicida.

PMNs were left untreated, infected with U112, or treated with 1 μM STS or 500 ng/ml anti-Fas IgM, as indicated. (A) Whole-cell lysates were collected from PMNs at 6, 12, and 24 hpi. Immunoblots show the rate and extent of procaspase-3 (35 kDa) processing to its mature, active (19, 17 kDa) form. Actin was used as the loading control. Data shown are from 1 experiment that is representative of 3 independent determinations. (B) Caspase-3 activity was measured at the indicated times points using a caspase-3/7-specific proluminescent substrate. Data are the means ± sem of 3 independent experiments performed in triplicate. RLU, Relative light units. P values were calculated using 2-way ANOVA analysis and Tukey’s multiple comparisons post-test. *P < 0.05 and **P < 0.01 versus the uninfected, untreated controls; #P < 0.05, ##P < 0.01, and ###P < 0.001 versus PMNs infected with U112.

Although procaspase-3 processing is essential for apoptosis, the catalytic activity of the mature enzyme can be regulated by cytosolic factors, such as the X-linked inhibitor of apoptosis protein [50]. Thus, we used a luminescence assay to quantify caspase-3 activity in control and U112-infected neutrophils and included cells treated with STS or anti-Fas IgM as additional positive controls. As shown in Fig. 4B, caspase-3 activity increased gradually over the course of 24 h in uninfected neutrophils, consistent with the immunoblotting data in Fig. 4A, confirming published results [17]. Fas cross-linking triggers activation of the extrinsic apoptosis pathway [51], and we demonstrated previously [17] and show again here (Fig. 4B) that caspase-3 activity peaks ∼12 h after treatment of PMNs with anti-Fas IgM or STS and then declines concurrently with cell death. In contrast, caspase-3 activity was barely detectable 12 h after infection with U112 and remained low at 24 h (Fig. 4B), consistent with the immunoblotting data shown in Fig. 4A. As caspase-3 is essential for apoptosis, the ability of U112 to extend the neutrophil lifespan is, at least in part, a result of its ability to inhibit processing and activation of this critical enzyme.

F. novicida inhibits both processing and activation of initiator caspases in the extrinsic and intrinsic apoptosis pathways

Having established that F. novicida impairs activation of caspase-3, we sought to determine if upstream initiator caspases of the extrinsic or intrinsic apoptosis pathways were similarly affected [52]. The cross-linking of surface death receptors, including Fas, initiates extrinsic pathway signaling and processing of procaspase-8 via assembly of the death-inducing signaling complex [37, 51]. Within this complex, procaspase-8 (56 kDa) is cleaved into 2 intermediate forms (p43/41) and further processed into mature caspase-8 (p18).

We used anti-Fas IgM to activate the extrinsic apoptosis pathway in PMNs and analyzed both procaspase-8 processing (Fig. 5A) and enzyme activity (Fig. 5B). Mature caspase-8 was not detected in freshly isolated PMNs (Fig. 5A). However, procaspase-8 processing was apparent within 6 h of Fas cross-linking, as indicated by the presence of both intermediate and mature forms of the enzyme in PMN lysates (Fig. 5A), and enzyme activity peaked by 12 h (Fig. 5B), confirming published data [17]. A slower rate of procaspase-8 processing and activation was apparent in control PMNs undergoing constitutive apoptosis, and this was reduced further by infection with U112 (Fig. 5A and B). Thus, whereas procaspase-8 was nearly undetectable in control and anti-Fas-treated cells by 24 h, it remained abundant following F. novicida infection, and levels of the intermediate and mature forms were reduced (Fig. 5A). Of note, the band marked with an asterisk (see Fig. 5A) that migrates just above the intermediate forms of caspase-8 is an F. novicida protein that cross-reacts with the anti-caspase-8 antibody but is currently unidentifiable using bioinformatics approaches (data not shown).

Figure 5. F. novicida significantly inhibits processing and activation of initiator caspases-8 and -9.

PMNs were left untreated, infected with U112, or treated with 1 μM STS or 500 ng/ml anti-Fas IgM, as indicated. (A) Immunoblots from 1 of 3 independent experiments show the extent of full-length procaspase-8 (56 kDa) processing to its intermediate (43, 41 kDa) and mature, active (18 kDa) forms. The asterisk (*) indicates a nonspecific cross-reacting band of bacterial origin. Actin is shown as a loading control. (B) Immunoblots show active caspase-9 (37 kDa) and actin in PMN lysates and are representative of 3 independent experiments. (C and D) Caspase-8 (C) and caspase-9 (D) activities were measured at 0, 12, and 24 hpi using caspase-8- or caspase-9-specific proluminescent substrates. In each case, data are the means ± sem of 3 independent experiments performed in triplicate. RLU, Relative light units. P values were calculated using two-way ANOVA and Tukey’s multiple comparisons post-test. *P < 0.05, **P < 0.01, and ***P < 0.001 versus uninfected, untreated controls; #P < 0.05, ##P < 0.01, ###P < 0.001, and ####P < 0.0001 versus U112 infected PMNs.

The initiator caspase of the intrinsic pathway is caspase-9, and this pathway is central to constitutive neutrophil apoptosis [52, 53]. The processing of procaspase-9 requires formation of the apoptosome, which is comprised of cytochrome c and Apaf-1. Following loss of outer mitochondrial membrane potential from various upstream proapoptotic cues, cytochrome c is released from the mitochondrial intermembrane space and binds Apaf-1, and together, these molecules recruit and process procaspase-9 to its active, 37 kDa form [37]. As noted above, STS leads to selective activation of the intrinsic apoptosis pathway, and data obtained using an antibody specific for mature, active caspase-9 indicate that the 37 kDa form of this enzyme was present within 6 h of STS addition (Fig. 5C), whereas caspase-9 activity peaked at 12 h (Fig. 5D). During constitutive PMN apoptosis, caspase-9 processing and activation were apparent by 12 h and increased further by 24 h (Fig. 5C and D). In marked contrast, processing and activation of this enzyme at 12 h were nearly ablated by U112 infection and remained lower than the controls at 24 h (Fig. 5C and D). These data show that F. novicida acts at multiple points upstream of executioner caspase-3 to diminish and delay PMN apoptosis and demonstrate that both the intrinsic and extrinsic pathways are altered by this bacterium.

F. novicida significantly inhibits Fas-mediated apoptosis

We probed the effects of F. novicida on the extrinsic pathway in more detail by testing its ability to counteract the effects of Fas cross-linking. To this end, neutrophils were left untreated or infected with F. novicida for 1 h before treatment with anti-Fas IgM, and 6 h later, apoptotic neutrophils were quantified by AV-FITC/PI staining and flow cytometry. In agreement with the data shown above, 61.0 ± 6.6% of neutrophils subjected to Fas cross-linking had externalized PS by 6 h compared with only 17.2 ± 5.8% of the controls and 9.6 ± 5.5% of cells infected with U112 (Fig. 6). However, prior infection with U112 reduced the ability of Fas cross-linking to induce apoptosis by ∼24% (Fig. 6; P < 0.05, n = 5). These data indicate that F. novicida can overcome the effects of a specific proapoptotic stimulus and as such, can partially inhibit the extrinsic apoptosis pathway.

Figure 6. F. novicida partially blocks Fas-induced apoptosis.

Control PMNs and cells that had been infected with U112 for 1 h were treated with anti-Fas IgM (500 ng/ml) for an additional 6 h, at which point, apoptosis was assessed by flow cytometry after AV staining. Data are the means ± sem of 5 independent experiments. P values were calculated using two-way ANOVA and Sidak’s multiple comparisons post-test. *P < 0.05 and ***P < 0.001; #P < 0.05 for Fas-cross-linked PMNs in the presence and absence of F. novicida infection; ns, not significant versus uninfected, untreated control.

Media conditioned by F. novicida-infected neutrophils prolong the cell lifespan

The data in Fig. 3A suggest that apoptosis inhibition does not require direct infection with F. novicida and could also be conferred by factors that are secreted or released into the culture medium. To address this question, we generated 3 types of CM. PMN-CM was prepared from cultures of neutrophils only, iPMN-CM was prepared from cultures of U112-infected neutrophils, and U112-CM was prepared from F. novicida cultured in RPMI 1640 in the absence of neutrophils. CM were processed as indicated in the Materials and Methods and then tested on PMNs from an autologous donor using the AV-FITC/PI flow cytometry assay, with infected neutrophils and untreated neutrophils included as positive and negative controls, respectively. After 24 h (83.1 ± 3.4% of the control), untreated PMNs had progressed to apoptosis compared with 37.9 ± 5.0% of neutrophils that were directly infected with U112 (Fig. 7A; P < 0.0001, n = 7). Although less potent than direct infection, iPMN-CM also had a statistically significant effect on apoptosis, as only 50.9 ± 7.8% of treated PMNs were AV positive at 24 h (P < 0.01, n = 7). A slight reduction in apoptosis was also apparent in cells treated with CM that were conditioned by host cells or bacteria alone (67.8 ± 3.4% and 62.2 ± 7.7% AV positive, respectively), but these data did not reach statistical significance.

Figure 7. F. novicida-mediated neutrophil apoptosis inhibition does not require direct infection.

The ability of media that had been conditioned by neutrophils alone (PMN-CM), infected neutrophils (iPMN-CM), or bacteria alone (U112-CM) to modulate PMN apoptosis was quantified by AV-FITC staining and flow cytometry (A) and by analysis of nuclear morphology (B and C). In each case, untreated PMNs and cells infected with U112 were used as controls. (A) Data are the means ± sem of 7 independent experiments, and P values were calculated using one-way ANOVA analysis and Dunnett’s multiple comparisons post-test. **P < 0.01 and ****P < 0.0001; ns, not significant when compared with untreated controls. (B) The percentage of PMNs with apoptotic nuclei was quantified using light microscopy. Data are the means ± sem of 4 independent experiments. P values were calculated using one-way ANOVA analysis and Dunnett’s multiple comparisons post-test. *P < 0.05, ***P < 0.001, and ****P < 0.0001 when compared with untreated controls. (C) Representative images show PMN nuclear morphology. Boxes 1–5 indicate control PMNs, U112 iPMNs, and cells exposed to PMN-CM, iPMN-CM, or U112-CM for 24 h, respectively. Arrowheads indicate cells with normal, segmented nuclei. Arrows indicate cells with condensed, apoptotic nuclei.

Neutrophil apoptosis can also be quantified by analysis of nuclear morphology. The nucleus of a healthy, mature human neutrophil is segmented and is comprised of 3–4 interconnected nuclear lobes. During apoptosis, these lobes coalesce and condense, and for this reason, neutrophils that contain small spherical or bilobed nuclei are defined as having an apoptotic morphology [7, 17, 54]. To assess further the extent to which each type of CM affects neutrophil apoptosis, we examined this late event in the pathway. The number of neutrophils with apoptotic nuclei at 24 h was quantified in control, U112-infected, or CM-treated cultures using Hema 3 staining and light microscopy, with pooled data shown in Fig. 7B and representative images shown in Fig. 7C. These data confirm that the uninfected, untreated control PMNs undergo apoptosis over the course of 24 h in culture, as 58.3 ± 7.2% of these cells exhibited an apoptotic nuclear morphology compared with only 8.8 ± 1.5% of neutrophils infected with F. novicida (Fig. 7B, P < 0.0001, n = 4; and Fig. 7C, compare panels 1 and 2). By this assay, all 3 CM significantly diminished apoptosis, albeit to a lesser extent than direct infection, and once again, the iPMN-CM was more effective than media conditioned by host cells or U112 alone (Fig. 7B and C). Taken together, these data indicate that PMN lifespan can be extended in the absence of direct infection with F. novicida. In addition, the effects of iPMN-CM suggest that apoptosis inhibition is likely complex and may result from the combined effects of shed or released bacterial factors and factors that are secreted by the PMNs themselves.

F. novicida induces CXCL8 secretion by neutrophils

Chlamydia pneumoniae and Anaplasma phagocytophilum are obligate intracellular pathogens of neutrophils that induce secretion of CXCL8 (formerly called IL-8) as a principal mechanism to delay PMN apoptosis [55, 56]. Gavrilin et al. [57], have shown that F. novicida up-regulates CXCL8 mRNA in human monocytes and elicits CXCL8 secretion from these cells over 24 h postinfection, but whether this CXCL8 up-regulation and secretion also occur in PMNs is unknown. Therefore, we collected supernatants from cultures of control and U112-infected neutrophils over the course of 48 h and quantified CXCL8 secretion by ELISA. Low levels of this cytokine were present in the medium of uninfected neutrophils and barely exceeded the level of detection of the assay at all time points examined (Fig. 8A). In contrast, U112 stimulated a significant increase in CXCL8 secretion by neutrophils that was apparent by 6 h and increased progressively to 48 h, the latest time point examined (Fig. 8A).

Figure 8. F. novicida-infected neutrophils secrete the antiapoptotic cytokine CXCL8.

(A) PMNs were left untreated or were infected with U112. Supernatants were collected at the indicated time points, and CXCL8 secretion was quantified by ELISA. Data are the means ± sem of 3 independent experiments, and P values were calculated using a two-way ANOVA analysis and Sidak’s multiple comparisons post-test. *P < 0.05 and ****P < 0.0001. (B) Comparison of CXCL8 secretion induced by U112, opsonized U112, and fk U112. Data are the means ± sem of 3 independent experiments. (C) The ability of U112, opsonized (ops) U112, and fk U112 to delay PMN apoptosis was assayed in parallel using AV-FITC staining and flow cytometry at 24 and 48 h. Data are the means ± sem of 3 independent experiments. (D) Effect of human recombinant (r)CXCL8 on PMN apoptosis assayed at 12 and 18 h. Data are the means ± sem of 4 experiments. *P < 0.05 by Student’s t test.

Additional experiments showed that fk and opsonized U112 also elicited CXCL8 secretion (Fig. 8B) and extended PMN lifespan (Fig. 8C). In contrast, neither live, killed, or opsonized U112 elicited secretion of PGE₂ (data not shown). Recombinant human CXCL8 elicited transient apoptosis suppression that was statistically significant at 12 h and waned thereafter (Fig. 8D). Published data indicate that CXCL8 binds with high affinity to both CXCR1 and CXCR2 and that the latter receptor is responsible for apoptosis inhibition by this cytokine [58]. To determine if CXCL8 was essential in our system, neutrophils were treated with the CXCR1 antagonist Reparixin and/or the CXCR2 antagonist SB225002 at 1 μM final concentration in the presence and absence of iPMN-CM, and apoptosis was assayed by flow cytometry (Supplemental Fig. 3). In our hands, Reparixin had no apparent effect on PMN apoptosis. To our surprise, SB225002, alone or in combination with Reparixin, favored survival of the uninfected control PMNs yet increased apoptosis in the presence of iPMN-CM. What accounts for these paradoxical results is unknown, but it is worth noting that several other targets of SB225002 were recently identified [59]. Taken together, our data suggest that CXCL8 contributes to, but is not essential for, apoptosis inhibition by F. novicida.

DISCUSSION

A successful immune response to invading pathogens is contingent on efficient recruitment of neutrophils to the site of infection, as well as the antimicrobial responses of these cells upon arrival. Under normal circumstances, phagocytosis accelerates neutrophil apoptosis, which aids in both pathogen killing and neutrophil clearance [48]. Subsequent removal of apoptotic neutrophils and their cargo by macrophages is crucial, as this process prevents release of proinflammatory and cytotoxic cell content and debris and down-regulates proinflammatory responses [60–62]. Defects in neutrophil turnover markedly impair the ability of the host immune system to resolve infection and restore homeostasis and are indicative of an aberrant inflammatory response that enhances host tissue destruction [24].

The first report describing the discovery of F. novicida strain U112 demonstrated that this organism was antigenically distinct from F. tularensis despite the ≥97.7% nucleotide identity of the 2 genomes [28]. Subsequent studies demonstrated that the F. novicida genome encodes more complete metabolic pathways and possesses significantly fewer pseudogenes and insertion sequences than F. tularensis [28]. All of these data are in agreement with the notion that F. tularensis evolved as a host-adapted organism, maintaining specific growth requirements consistent with an intracellular niche, whereas F. novicida evolved as an environmental organism with added pressure to retain genes involved in metabolism and restriction/modification systems yet conserving the ability to infect similar cell types in vitro and following direct inoculation into laboratory mice.

Alveolar macrophages are the first cell type that is infected following introduction of Francisella into the lungs of mice, but neutrophils account for at least one-half of the infected cells in this locale by day 3 of infection [22]. For both F. tularensis and F. novicida, strategies that prevent neutrophil migration into the lungs markedly reduce tissue destruction and increase host survival, even though the mechanisms driving PMN influx are distinct [23, 63, 64]. These data definitively demonstrate that neutrophils play a central role in tissue destruction and perturbation of the inflammatory response following Francisella infection.

Invasion of host cells is critical for virulence of intracellular pathogens, and it is established that mechanism of entry influences microbe fate [65]. Relevant here is the ability of FcR ligation to enhance antimicrobial defense, including the respiratory burst [65]. In addition, phagocytosis typically accelerates PMN apoptosis via activation of the PICD pathway, which is driven, in part, by NADPH oxidase-derived ROS [37, 48, 66]. We have shown that both opsonized and unopsonized F. tularensis strains impair NADPH oxidase assembly and activation and significantly prolong PMN lifespan [17, 35, 37]. As the ability of F. novicida to elicit a respiratory burst in neutrophils is IgG dependent [34] (Supplemental Fig. 2A), we hypothesized that apoptosis may be influenced in an opsonin-dependent manner. Nevertheless, the results of our initial experiments demonstrated that the ability of F. novicida to extend the PMN lifespan was not only dose dependent (Fig. 1C) but also appeared to be opsonin independent (Fig. 1D).

To validate the latter conclusion, we needed to account for the fact that phagocytosis of F. novicida is relatively inefficient [16, 34, 45, 46], potentially resulting in subpopulations of infected and uninfected cells that progressed to apoptosis at different rates. We quantified bacterial binding and uptake using fluorescence microscopy, and the data demonstrate that 53% of neutrophils were infected within 1 h of exposure to unopsonized U112 (Fig. 2C). Opsonization increased both the percentage of infected PMNs and the bacterial load per cell but did not influence phagocytosis per se, as nearly all cell-associated bacteria were ingested regardless of opsonization status (Fig. 2B and C). Although PMN suspensions contained a similar number of infected and uninfected cells, 1 h after addition of unopsonized U112, simultaneous analysis of bacterial load and PS externalization demonstrated that all neutrophils in the population progressed to apoptosis at a similar rate (Fig. 3A). These results are consistent with the ability of CM to delay apoptosis (Fig. 5) and the ability of F. novicida to delay apoptosis at very low MOI when only a small fraction of cells may be infected (Fig. 1C).

The flow cytometry data also show that bacterial load, measured as sGFP fluorescence, continued to increase over 24 h (Figs. 2A and 3). This likely reflects continued phagocytosis during the first few hours of infection, with subsequent phagosome escape and bacterial replication in the cytosol accounting for the increased fluorescence that occurs between 12 and 24 h after infection, as we have shown for LVS [17]. As such, it is attractive to predict that extracellular, phagosomal, and cytosolic F. novicida may play distinct and synergistic roles in modulation of PMN lifespan.

Single-cell analysis demonstrated that suspensions of neutrophils infected with opsonized U112 also behaved in a uniform manner, as 88% of cells were infected by 1 h (Fig. 2C), and at least 80% of phagosomes were formazan positive (Supplemental Fig. 1B), demonstrating local superoxide accumulation. Thus, although some heterogeneity of NBT staining intensity at the level of individual phagosomes is expected [35, 47], a distinct NBT-negative PMN subset was not detected. Despite ROS production, the lifespan of these PMNs was indistinguishable from neutrophils that were infected with unopsonized bacteria (Figs. 1D and 8C). Thus, although opsonization with IgG is sufficient for neutrophil activation by F. novicida, this does not ablate the ability of this organism to delay apoptosis.

We demonstrated previously that both type A and type B strains F. tularensis inhibit human neutrophil apoptosis by interfering with the extrinsic and intrinsic apoptotic pathways [17–19]. In this study, we extended our studies to F. novicida and present multiple lines of evidence to demonstrate that this bacterium also significantly delays PMN apoptosis as indicated by analyses of PS externalization (Fig. 1) and nuclear morphology (Fig. 7), as well as processing and activation of intrinsic and extrinsic pathway caspases (Figs. 4 and 5). Moreover, F. novicida partially blocked the ability of anti-Fas IgM to trigger apoptosis (Fig. 6), indicating a capacity to inhibit significantly a robust and specific pro-death stimulus. Delayed apoptosis of cells infected with opsonized U112, despite NADPH oxidase activation and ROS production, suggests that the PICD pathway is also impaired.

Regulation of apoptosis is complex, and our data indicate that although the effects of F. novicida on the PMN lifespan were most efficient under conditions that support direct infection, apoptosis was also significantly delayed by factors in iPMN-CM but not by media that were conditioned by bacteria or PMNs alone (Fig. 7). These data suggest that neutrophil lifespan may be influenced by complex cross-talk between bacteria and host, with factors secreted or released by extracellular bacteria acting on neutrophils and potentially triggering prosurvival signaling and changes in gene expression that are enhanced and reinforced by intracellular bacteria within phagosomes and perhaps in the cytosol. In this regard, one factor of interest in iPMN-CM is CXCL8 (Fig. 8A). A variety of pathogens that modulate neutrophil apoptosis induce secretion of CXCL8 as their preferred mechanism of neutrophil apoptosis inhibition [55, 56, 67–69]. Our data showing that F. novicida-infected neutrophils secrete CXCL8 are significant, as they indicate a distinct mechanism used by F. novicida to delay neutrophil apoptosis that is not shared with F. tularensis [17]. CXCL8 binds CXCR1 and CXCR2 with high affinity, and although ligation of either receptor can elicit chemotaxis, anti-apoptosis signaling is driven specifically by ligation of CXCR2 [58]. The data in Fig. 8D confirm the ability of CXCL8 to delay neutrophil death, but the limited magnitude and short duration of this response also suggest that this chemokine does not entirely account for apoptosis inhibition by iPMN-CM or direct infection with F. novicida. Whether growth-related oncogene α, which can also influence PMN lifespan via CXCR2 ligation, is relevant in our system remains to be determined [58]. Signals downstream of CXCR2 that are linked to cell survival include class I PI3K, specifically PI3Kγ, which may synergize with PI3Kβ and PI3Kδ activation downstream of TLR2, CR3, and/or FcR, as one mechanism to sustain neutrophil viability during F. novicida infection [70, 71]. PGE₂ is secreted by F. tularensis-infected murine macrophages, and in other systems has been shown to delay neutrophil apoptosis via a cAMP-dependent mechanism [72, 73]. Although we did not detect PGE₂ in supernatants of F. novicida-infected neutrophils, this PG may influence neutrophil lifespan at sites of infection where multiple cells types are present.

Identification and characterization of the bacterial factors that mediate neutrophil apoptosis inhibition during Francisella infection are a focus of current research in our laboratory. During infection with C. pneumoniae, LPS is responsible for inducing CXCL8 secretion, but the corresponding mechanism used by A. phagocytophilum is unknown [55, 56, 69]. F. tularensis LPS does not influence PMN apoptosis [17]. Preliminary data suggest that this is also true for F. novicida and that neither LPS nor other bacterial lipids are contributory and implicate instead other outer membrane components that may differ in abundance or potency in F. tularensis and F. novicida [Kinkead, Barker, and Allen, unpublished data].

Typically, apoptotic neutrophils are rapidly cleared by macrophages via efferocytosis [24]. Defects in this process favor neutrophil progression to secondary necrosis and exacerbate tissue destruction during infectious and inflammatory disease. To our knowledge, neutrophil efferocytosis has not been studied in the context of Francisella infection. However, the profound accumulation of neutrophils and necrotic debris in the airways of infected nonhuman primates and other animals suggests that efferocytosis may be impaired, and in vitro studies have shown that the ability of F. novicida-infected macrophages to engulf necrotic cell debris is diminished [16, 74]. On the other hand, apoptotic neutrophils can also act as Trojan horses for infection of macrophages, as has been shown for Yersinia pestis [75], but whether this occurs during Francisella infection is unknown. Thus, much remains to be determined regarding the fate of Francisella-infected neutrophils and their effects on macrophages and disease progression.

Considered together, the available data suggest that opsonization with natural IgG is one factor that contributes to the inability of F. novicida to cause human disease. Inasmuch as opsonization with IgG is sufficient for NADPH oxidase activation and ROS production, the ability of the absent in melanoma 2 inflammasome to curtail F. novicida growth in macrophages may be critical, and IL-1β released during macrophages pyroptosis is another factor that may contribute to prolonged neutrophil survival [32]. Thus, in the context of F. novicida infection, it is conceivable that delayed neutrophil apoptosis favors phagocytic killing of bacteria released by dying macrophages.

In summary, human neutrophils are short lived and inherently programmed to undergo constitutive apoptosis ∼24 h after release into the circulation. The results of this study demonstrate the ability of F. novicida to diminish and delay activation of caspase-3, -8, and -9 and markedly extend PMN lifespan, as indicated by quantitation of PS externalization and changes in nuclear morphology. As the effects of F. novicida are dose dependent yet can be uncoupled from phagocytosis and opsonization, our data suggest that this organism may be able to act at a distance to influence neutrophil function as soon as these cells enter the lungs or other infected tissues, a property that is shared with F. tularensis [17, 76]. At the same time, our current and published data identify shared and distinct aspects of the underlying molecular mechanisms, thereby highlighting the complexity of neutrophil apoptosis regulation and informing understanding of Francisella pathogenicity (refs. [17, 18] and this study). Additional studies are needed to determine the receptors that mediate uptake of unopsonized Francisella, define further how delayed apoptosis is achieved, elucidate the fate of infected neutrophils, and explore the potential use of lifespan-modulating bacterial factors as vaccine or therapeutic targets for prevention and treatment of tularemia.

AUTHORSHIP

L.C.K., D.C.F., and L-A.H.A. designed and performed experiments and analyzed data. L.C.K. wrote the first draft of the manuscript, with editing and revisions provided by L-A.H.A.

Glossary

Apaf-1

apoptotic protease-activating factor-1

APC

allophycocyanin

AV

Annexin V

CM

conditioned medium

fk

formalin-killed

hpi

hours postinfection

iPMN

infected polymorphonuclear leukocyte

LVS

live vaccine strain

MOI

multiplicity of infection

OD₆₀₀

OD wavelength of 600 nm

PI

propidium iodide

PICD

phagocytosis-induced cell death

PMN

polymorphonuclear leukocyte

PS

phosphatidylserine

ROS

reactive oxygen species

sGFP

superfolder GFP

STS

staurosporine

subsp.

subspecies

DISCLOSURES

The authors declare no conflicts of interest.