Heparinized venous blood was obtained from healthy adult volunteers with no history of tularemia in accordance with protocols approved by the Institutional Review Board for Human Subjects at the University of Iowa (201609850 and 200307026) and the University of Missouri (20331322). Neutrophils were isolated using a sequential dextran sedimentation, density-gradient separation with Ficoll-Hypaque (GE Healthcare, Little Chalfont, UK) and hypotonic erythrocyte lysis as previously described (Nauseef, 2007b). Using this method, purity was >95% PMNs. PMNs were suspended in Hank’s balanced salt solution (HBSS) without divalent cations (Fisher Scientific, Hampton, NH) enumerated, and diluted to 2x10⁷/ml. In all cases, replicate experiments were performed using PMNs from different donors.

F. tularensis subspecies holarctica live vaccine strain (LVS) has been previously described and retains key features of fully virulent F. tularensis in human neutrophils in vitro but requires only BSL-2 containment (McCaffrey et al., 2010; Schwartz et al., 2012; Schwartz et al., 2013). Bacteria were inoculated onto Difco cysteine heart agar (BD Biosciences, San Jose, CA) supplemented with 9% defibrinated sheep blood (Remel, Lenexa, KS) (CHAB) and grown for 48 hours at 37°C with humidity in 5% CO₂. Unless otherwise stated, cultures of LVS were started at an OD₆₀₀ of 0.01 in Bacto brain heart infusion (BD Biosciences) (BHI) broth and incubated overnight (14-18 hours), shaking at 200 rpm. Overnight cultures were diluted again and incubated at 37°C in 5% CO₂, shaking at 200 rpm, for 2-4 hours. Mid-exponential growth phase bacteria were harvested and washed once with HBSS containing divalent cations (Fisher Scientific).

Washed bacteria were quantified by measurement of absorbance at 600 nm. Unless otherwise stated, PMNs (5x10⁶/ml) were diluted in HEPES-buffered RPMI-1640 containing L-glutamine and phenol red (Lonza, Walkersville, MD) in the absence of serum and infected with LVS to achieve 20 bacteria/cell as previously described (McCracken et al., 2016). Cultures (1-2 ml each) were incubated in 14 ml polypropylene snap-cap tubes at 37°C with humidity and 5% CO₂ for up to 24 hours.

The Proteome Profiler™ human phospho-MAPK slide array kits (R&D Systems Inc., Minneapolis, MN) were used for detection of 24 different MAPK proteins in PMNs. Whole-cell lysates were prepared according to the manufacturer’s instructions. Briefly, neutrophils were left untreated or were infected with LVS, and 2, 10 or 24 hours later neutrophils were pelleted by centrifugation at 250 g. Cell pellets were resuspended in lysis buffer containing aprotinin, leupeptin, PMSF, AEBSF, levamisole, bestatin, E-64, and pepstatin A (Sigma-Aldrich, St. Louis, MO), supplemented with the Pierce Halt™ phosphatase inhibitor cocktail (sodium fluoride, sodium orthovanadate, sodium pyrophosphate and β-glycerophosphate) (Thermo Fisher Scientific, Waltham, MA). Protein concentrations were determined by performing a Pierce bicinchoninic acid (BCA) protein assay (Thermo Fisher Scientific), according to the manufacturer’s instructions. Array dot blots were processed according to the manufacturer’s protocol.

Neutrophil apoptosis was measured by flow cytometric analysis of phosphatidylserine (PS) externalization using an Accuri C6 flow cytometer (BD Biosciences, Franklin Lakes, NJ). PMNs (5x10⁵) were co-stained with Annexin V-FITC (BioVision, Milpitas, CA) and propidium iodide (PI) (BioVision) in binding buffer (10 mM HEPES pH 7.4, 140 mM NaCl, 2.5mM CaCl₂) according to the manufacturer’s instructions. For each sample, approximately ten thousand events were collected, and the data were analyzed using cFlow software (BD Biosciences). Unless otherwise stated, graphs include Annexin V-positive/PI-negative (early) and Annexin V/PI double-positive (late apoptotic) cells.

Progression to apoptosis was also assessed independently using established methods to quantify nuclear condensation (Schwartz et al., 2012; McCracken et al., 2016; Kinkead et al., 2018). In brief, after incubation at 37°C for 18 hours, control, infected and/or drug-treated neutrophils were attached to glass coverslips by cytocentrifugation and then fixed and stained using with Hema-3 reagents (Fisher Scientific). Coverslips were attached to slides using Permount (Fisher Scientific) and analyzed using a Zeiss Axioplan 2 light microscope (Carl Zeiss Inc., Thornwood, NY). Neutrophils in random fields of view were scored as having healthy nuclei comprised of 3-4 interconnected lobes, or apoptotic, condensed (spherical) nuclei and at least 100 cells/coverslip/sample were assessed (Schwartz et al., 2012; McCracken et al., 2016; Kinkead et al., 2018).

All inhibitors were resuspended from powder in tissue culture-grade dimethyl sulfoxide (DMSO) (Sigma-Aldrich). Neutrophils were pretreated with various inhibitors of survival signaling (Table 1) at 37°C with 5% CO₂ for 30-60 min, as indicated, after which F. tularensis LVS was added to corresponding cultures. Neutrophil apoptosis was quantified after further incubation at 37°C for 4-24 hours, as indicated, using flow cytometry after Annexin V-FITC/PI staining and analysis of nuclear morphology as described above.

To account for possible detrimental effects of drug pretreatment, additional experiments were performed adding inhibitors simultaneously with or shortly after initiation of infection. In addition, to elucidate possible effects of each inhibitor on bacterial viability and fitness, drugs were added to BHI broth and LVS growth was quantified over 18 hours at 37°C by measurement of the OD₆₀₀ (as described above).

As our published data demonstrate that both intracellular and extracellular LVS contribute to apoptosis inhibition (Schwartz et al., 2012; Kinkead et al., 2018) we sought to determine if phagocytosis influenced the effects of signaling inhibitors on apoptosis in our system. To this end, we prevented direct contact between neutrophils and bacteria using 0.4 μm Transwell inserts (Fisher Scientific) as we previously described (Schwartz et al., 2012). After incubation at 37°C for 24 hours, cells were processed for analysis of apoptosis by flow cytometry as described above.

Neutrophils were left untreated or infected with LVS as described above. At the indicated time points, PMNs were fixed in IC fixation buffer (eBioscience, Waltham, MA) for 30 minutes at room temperature. Following fixation, cells were pelleted by centrifugation at 250 g and permeabilized in 100% ice-cold methanol for 30 minutes on ice. After two washes in blocking buffer [Dulbecco’s phosphate-buffered saline (DPBS) without divalent cations (Fisher Scientific) supplemented with 0.5% bovine serum albumin (Sigma-Aldrich)], neutrophils were resuspended in blocking buffer at a concentration of 10⁷ cells/ml. Aliquots of neutrophils (10⁶) were mixed with purified human Fc binding inhibitor and anti-phospho-p38 MAPK (T180/Y182)-APC (clone 4NIT4KK, #17-9078-42) (from eBioscience). After incubation for 30 minutes at room temperature, stained cells were washed twice in blocking buffer and resuspended in DPBS without divalent cations for analysis on an Accuri C6 flow cytometer. Alternatively, cells control and infected neutrophils, or cells stimulated with 1 μM fMLF (Sigma-Aldrich) for 3 minutes in the presence and absence of 100 μM LY294002 (Cell Signaling Technology, Danvers, MA) were fixed and stained to detect active AKT using mouse anti-human phospho-AKT (S473)-APC (clone SDRNR, #17-9715) prior to analysis.

Neutrophils were collected by centrifugation, washed, and then lysed in buffer containing 1% NP-40 along with the following protease and phosphatase inhibitors: aprotinin, leupeptin, AEBSF, PMSF, levimasole, bestatin, E-64, pepstatin A, diisopropylfluorophosphate, vanadate and sodium fluoride (McCracken et al., 2016). Alternatively, washed neutrophils were resuspended in relaxation buffer containing the protease and phosphatase inhibitors listed above and disrupted by nitrogen cavitation followed by separation of nuclei, cytosol and membrane fractions by differential centrifugation (Allen et al., 2005). Protein concentrations of each sample were determined using Pierce BCA assay kits. After boiling in sample buffer, proteins in each sample (5-10 μg) were separated on 4-12% NuPAGE Bis-Tris gradient gels and then transferred to polyvinylidene fluoride membranes by electroblotting. Blocked membranes were probed to detect: phospho-p38 MAPK (T180/Y182) using rabbit polyclonal Ab (pAb) #9211 (Cell Signaling Technology) at 1:500; AKT and phospho-AKT(S473) using rabbit pAb #9272 and #9271, respectively, (Cell Signaling Technology) each at 1:500; p110α using rabbit mAb clone C73F8 #4249T (Cell Signaling Technology) at 1:500; NF-κB p65 using clone D14E12 XP rabbit mAb, #8242 (Cell Signaling Technology) at 1:1,000; IκBα using rabbit mAb clone 44D4, #4812 (Cell Signaling Technology) at 1:1000; phospho-IκBα (S32) rabbit mAb clone 14D4, #2859 (Cell Signaling Technology) at 1:500; and β-actin using rabbit pAb #NB600-503SS (Novus Biologicals, Littleton, CO) at 1:2,000. In each case, bands were detected using horseradish peroxidase-conjugated secondary antibodies at 1:2,000 (Cytiva, Marlborough, MA) and Pierce SuperSignal West Femto chemiluminescence substrate (Thermo Fisher Scientific) followed by Li-COR Odyssey imaging (LI-COR Biosystems, Lincoln, NE).

Data are presented as the mean ± standard error of the mean (SEM) and were analyzed by two-way ANOVA followed by Tukey’s multiple comparisons post-tests. All analyses were performed using Prism version 7or 8 software (GraphPad, San Diego, CA). p-values less than 0.05 were considered statistically significant.

MAPK signaling can be triggered by many stimuli that engage distinct cell surface receptors, including LPS and other TLR agonists, growth factors, and cytokines (Akgul et al., 2001). MEK/ERK MAPK and p38 MAPK pathways can act separately or together to modulate neutrophil apoptosis in response to specific stimuli (Klein et al., 2000; Klein et al., 2001; Geering and Simon, 2011; Tsai et al., 2013; McCracken and Allen, 2014; Cherla et al., 2018). To elucidate possible effects of F. tularensis on activation of MAPK signaling, we initially utilized phospho-MAPK dot blot arrays to survey intermediates in several MAPK cascades. To this end, dot blots were probed with lysates prepared from control neutrophils and their LVS-infected counterparts after 10 hours at 37°C. These data show that infection triggered increased phosphorylation of p38α MAPK and to a lesser extent ERK2 (Figure 1A). The dot blot spot map is shown in Supplementary Figure 1A.

To define the kinetics of p38 MAPK activation we utilized intracellular antigen staining and flow cytometry. (Figures 1B, C). By this assay, phosphorylation and activation of p38 MAPK was low or absent in early infection, peaked at 12 hours post-infection (hpi), and returned to baseline by 24 hpi. Dot blots of lysates prepared at 2 and 20 hours, together with the data in Figure 1A, confirmed this time course (Supplementary Figure 1B).

To examine the possible significance of MAPKs in neutrophil apoptosis inhibition by F. tularensis we utilized the p38 MAPK inhibitor SB203580 and included the related compound SB202474 as a negative control (Tsai et al., 2013). Specifically, neutrophils were left in medium alone or pretreated with 30 μM inhibitor prior to infection with LVS, and apoptosis was quantified 4, 10, 18 and 24 hours later using Annexin V-FITC/PI staining and flow cytometry as we described (McCracken et al., 2016; Kinkead et al., 2018). In all cases, uninfected/untreated cells were included as an additional control. Our data show that inhibition of p38 MAPK markedly accelerated apoptosis of infected PMNs by 18 hpi and completely eliminated the ability of LVS to extend neutrophil lifespan by 24 hpi, whereas the rate of apoptosis of uninfected neutrophils was unchanged (Figures 1D). Inhibitor efficacy was demonstrated by reduced phosphorylation of p38 detected by immunoblotting of cell lysates (Figure 1E). Specificity for p38 MAPK was indicated the lack of effect of the negative control compound, SB202474 (Figure 1D). DMSO, the diluent/vehicle control for all drugs used in this study, was also without effect (Supplementary Figure 2). Moreover, it is standard practice to pretreat neutrophils with inhibitors prior to stimulation or infection, and in this study PMNs were incubated with drugs for 30 or 60 min prior to addition of bacteria (Table 1). However, pretreatment was not essential, as similar results were obtained when the pretreatment step was omitted, and drugs were added simultaneously with initiation of infection or shortly thereafter (data not shown).

The nucleus of healthy human PMNs is comprised of 3-4 interconnected lobes that coalesce and condense into a sphere during apoptosis (Kennedy and DeLeo, 2009; Schwartz et al., 2012). This significant morphological change is a hallmark of apoptotic neutrophils and is readily apparent by light microscopy. Analysis of nuclear morphology at 18 hours confirmed the significant role of p38 MAPK in longevity of LVS-infected PMNs (Figure 1F).

In marked contrast to the data obtained for p38 MAPK, inhibition of MEK1/2 signaling using 50 μM PD98059 did not significantly alter the rate of PMN apoptosis in the presence or absence of LVS (Figure 2), and ERK phosphorylation was not increased above background at other time points examined (Supplementary Figure 1B). Taken together, these data demonstrate that both p38 MAPK and MEK/ERK signaling are activated in infected PMNs, but only p38 MAPK is required for F. tularensis to extend cell lifespan.

Class I PI3Ks catalyze conversion of PI(4,5)P2 to PI(3,4,5)P3 and have been extensively studied (Hawkins et al., 2010; Fortin et al., 2011). Class IA PI3K isoforms (PI3Kα, PI3Kβ, and PI3Kδ) are important for growth and survival of many types of cells and in neutrophils and macrophages play additional roles in regulation of FcγR-mediated phagocytosis, cell adhesion, priming and NADPH oxidase activation. On the other hand, the lone Class IB PI3K isoform (PI3Kγ) is activated by heterotrimeric G-protein coupled receptors (GPCRs) and plays a specific role in regulating neutrophil migration to sites of infection and inflammation.

LY294002 is a competitive, reversible pan-Class I PI3K inhibitor (Vanhaesebroeck et al., 1997; Juss et al., 2012) and we show here that pretreatment of neutrophils with this drug accelerated constitutive apoptosis of control, uninfected PMNs as indicated by Annexin V-FITC/PI staining at 10 and 18 hours (Figure 3A). Apoptosis of LVS-infected cell was also significantly accelerated by LY294002 as indicated by Annexin V-FITC/PI staining at 10, 18 and 24 hpi (Figure 3A) and analysis of nuclear condensation at 18 hpi (Figure 3B). Of note, the role of Class I PI3Ks in Francisella phagocytosis is opsonin-dependent, as uptake of complement-opsonized bacteria is PI3K-dependent whereas uptake of unopsonized bacteria is not (Clemens and Horwitz, 2007; Parsa et al., 2008). Importantly, we have shown that both opsonized and unopsonized LVS delay PMN death and we used unopsonized bacteria in this study to avoid possible confounding effects of LY294002 on infection efficiency (Schwartz et al., 2012; Kinkead et al., 2018).

AKT (also called protein kinase B) is a major Class I PI3K effector. Phosphorylation of AKT on S473 is commonly used as an indicator of PI3K activation and the formyl peptide fMLF is known to activate neutrophils in a PI3K-dependent manner (Wang et al., 2003). To demonstrate LY294002 efficacy, PMNs were stimulated with fMLF for 3 minutes in the presence and absence of this inhibitor. Immunoblotting of cell lysates demonstrated the ability of fMLF to trigger enhanced AKT phosphorylation in PMNs that was sensitive to inhibition by LY294002, whereas total AKT levels and the β-actin loading control were unchanged (Figure 3C). Based on these data, we conclude that PI3K activity is required for delayed apoptosis of F. tularensis-infected neutrophils and also regulates constitutive apoptosis of the uninfected controls.

Our next objective was to determine if a specific Class IA PI3K isoform was required for longevity of F. tularensis-infected neutrophils. To this end, we took advantage of inhibitors that have been designed to target PI3K isoforms individually or in combination (Table 1). Specifically, we pretreated neutrophils with 10 μM HS-173, a specific PI3Kα inhibitor, 20 μM of the PI3Kα/PI3Kδ-selective inhibitor GDC-0941 (also called Pictilisib), 1 μM of the PI3Kδ-selective inhibitors IC-87114 and PIK-294, or 100 μM of the PI3Kβ-selective inhibitor TGX-221 (Juss et al., 2012; Sarker et al., 2015; Setti et al., 2016; Zillikens et al., 2021) and monitored apoptosis over the course of 24 hours in the presence and absence of LVS.

Our data demonstrate that HS-173 had no effect on apoptosis at 4 or 10 hours. Thereafter, apoptosis of the LVS-infected cells was markedly accelerated, whereas death of the uninfected control cells was significantly reduced (Figure 4A). On the other hand, treatment with the selective PI3Kα/PI3Kδ inhibitor GDC-0941 significantly increased apoptosis of both control and LVS-infected PMNs at 10, 18 and 24 hours (Figure 4B). The effect of each drug on cell viability was also assessed by analysis of nuclear morphology at 18 hours (Figures 4C, D), whereas efficacy was demonstrated by their ability to diminish LVS-stimulated membrane translocation of p110α, the catalytic subunit of PI3Kα (Figure 4E). Based on these data, we conclude that that the Class I PI3K isoforms required to prevent apoptosis of control and infected PMNs are distinct. Specifically, our data demonstrate that inhibition of PI3Kα is sufficient to trigger apoptosis after F. tularensis infection. In contrast, inhibition of both PI3Kα and PI3Kδ is required for accelerated apoptosis of the uninfected controls.

In another series of experiments, PMNs were treated with 1 μM IC-87114 or 1 μM PIK-294, both of which preferentially inhibit PI3Kδ but have some activity against PI3Kβ and PI3Kγ. Neither of these drugs altered the rate of apoptosis of control or LVS-infected PMNs (Figures 5A, B). In our final series of PI3K experiments we tested TGX-221, which preferentially inhibits PI3Kβ, but has some activity against PI3Kδ and PI3Kγ. We now show that 100 μM TGX-221 profoundly diminished apoptosis of uninfected PMNs at 18 and 24 hours, yet had no significant effect on LVS-infected cells at any of the time points examined (Figure 5C).

Collectively, our data suggest that Class I PI3Ks play complex roles in PMN survival and death. In particular, we identify PI3Kα activity as critical for survival of neutrophils infected with F. tularensis whereas inhibition of PI3Kβ enhanced survival of the uninfected controls. The data also indicate that simultaneous inhibition of PI3Kα and PI3Kδ, but not specific or selective inhibition of either isoform alone significantly accelerates PMN apoptosis in the absence of infection.

As noted above, AKT is a commonly studied Class I PI3K effector, also known as protein kinase B. There are three isoforms of AKT, and AKT1 and AKT2 are expressed in neutrophils (Chen et al., 2010). To determine if AKT played a role in modulating neutrophil apoptosis, we treated cells with the allosteric pan-AKT inhibitor MK-2206 (Yamaji et al., 2017). In our hands, pretreatment of neutrophils 1 μM MK-2206 had no discernable effect on the rate of neutrophil apoptosis in the presence or absence of LVS as judged by flow cytometry (Figure 6A). As the effects of MK-2206 can be transient (Will et al., 2014; Johnson et al., 2021), we also tested the newer and more potent AKT inhibitor, Afuresertib (Yamaji et al., 2017). Consistent with the aforementioned data, pretreatment with 10 μM Afuresertib had no significant effect on the lifespan of neutrophils infected with LVS, but significantly reduced the rate of apoptosis of the uninfected cells at 18 and 24 hours (Figure 6B). These data were confirmed by analysis of nuclear morphology (data not shown).

Notably, our dot blot arrays did not detect enhanced AKT phosphorylation in resting or infected cells at the examined time points (Figure 1A and Supplementary Figure 1B). Further analysis of cell lysates using conventional immunoblotting suggest that AKT phosphorylation was maintained at a moderate level in LVS-infected cells over 24 hpi yet declined by 12 and 24 hours in the aged, uninfected controls (Figure 6C). Low level AKT phosphorylation in infected PMNs was independently confirmed using flow cytometry as was its diminished phosphorylation in the aged, uninfected controls (data not shown). As expected, AKT phosphorylation was sensitive to inhibition by MK-2206 and Afuresertib (Figure 6D). Considered together, these data suggest that AKT is not an essential regulator of neutrophil apoptosis during F. tularensis infection.

NF-κB plays a key role in neutrophil survival during infection and inflammation by controlling expression of genes that encode important anti-apoptotic regulatory factors (Schwartz et al., 2013). We have shown that several NF-κB target genes are significantly differentially expressed during LVS infection, including BCL2A1, BIRC3, BIRC4, TNFAIP3, CFLAR and CAST, which encode A1, cIAP2, XIAP, A20, FLIP and calpastatin, respectively (Lawrence, 2009). Thus, we predicted that NF-κB activity may be essential in our system for infected cell survival. To test this hypothesis, we utilized NF-κB AI (NF-κB Activation Inhibitor) (Tsai et al., 2017). The data in Figures 7A, B indicate that NF-κB AI significantly accelerated apoptosis of LVS-infected PMNs at all time points examined. In sharp contrast, inhibition of NF-κB did not significantly alter the rate of apoptosis of uninfected PMNs, confirming published data (Dyugovskaya et al., 2011). Immunoblotting of subcellular fractions demonstrated LVS-induced NF-κB activation as indicated by nuclear translocation of the p65 subunit that was coupled to degradation of its inhibitor IκBα (Figure 7C). Proteolysis requires prior phosphorylation of IκBα and this step of the NF-κB activation cascade was inhibited by NF-kB AI, thereby demonstrating its efficacy (Figure 7D). Additionally, a role for NF-kB in infected PMN longevity was independently confirmed using CAPE (Figure 7E) (Armutcu et al., 2015). We therefore conclude that NF-κB is essential for delayed apoptosis of F. tularensis-infected neutrophils.

Data published by us and others demonstrate that F. tularensis rapidly releases bacterial lipoproteins (BLPs) and other unidentified factors that act at a distance to modulate PMN function and lifespan prior to binding and phagocytosis (Moreland et al., 2009; Allen, 2013; Kinkead et al., 2018). For this reason, we sought to determine if phagocytosis was essential for the prosurvival signaling identified in this study. To address this question, direct contact between neutrophils and bacteria was prevented by incubation on the opposite sides of 0.4 μm Transwell filters as previously described (Schwartz et al., 2012; Kinkead et al., 2018). Under these conditions, the p38 MAPK inhibitor SB203580 and the PI3K inhibitors LY294002, HS-173 and GDC-0941 retained their ability to significantly accelerate apoptosis, whereas the effects of NF-κB AI were diminished (p=0.09) (Supplementary Figure 3).

As it is established that viable F. tularensis prolongs neutrophil lifespan whereas killed bacteria do not (Schwartz et al., 2012), we performed additional experiments to determine if any of the drugs used in this study was toxic to LVS. To this end, each agent was added to BHI broth and bacterial growth was quantified over 18 hours at 37°C. By this assay, most drugs were without effect, but NF-κB AI was bacteriostatic (Supplementary Figure 4 and data not shown). To address this limitation, we performed additional experiments using CAPE, a structurally distinct NF-κB inhibitor that has previously been shown not to effect Francisella growth in broth or its ability to be phagocytosed (Santic et al., 2010). The data in Supplementary Figure 4 confirm that CAPE is not toxic and further support the notion that NF-κB is important for infected PMN longevity, as shown in Figure 7.