Toll-like Receptor Signaling Rescues Fungicidal Activity in Syk-deficient Neutrophils

Abstract

An impaired neutrophil response to pathogenic fungi puts patients at risk for fungal infections with a high risk of morbidity and mortality. Acquired neutrophil dysfunction in the setting of iatrogenic immune modulators can include the inhibition of critical kinases such as spleen tyrosine kinase (Syk). In this study, we utilized an established system of conditionally immortalized mouse neutrophil progenitors to investigate the ability to augment Syk-deficient neutrophil function against Candida albicans with Toll-Like Receptor (TLR) agonist signaling. LPS, a known immunomodulatory molecule derived from gram negative bacteria, was capable of rescuing effector functions of Syk-deficient neutrophils, which are known to have poor fungicidal activity against Candida species. LPS priming of Syk-deficient mouse neutrophils demonstrate partial rescue of fungicidal activity including phagocytosis, degranulation and neutrophil swarming, but not reactive oxygen species (ROS) production against C. albicans, in part, due to c-Fos activation. Similarly, LPS priming of human neutrophil rescues fungicidal activity in the presence of pharmacologic inhibition of Syk and Bruton’s tyrosine kinase (Btk), both critical kinases innate immune response to fungi. In vivo, neutropenic mice were reconstituted with WT or Syk deficient neutrophils and challenged with intraperitoneal C. albicans. In this model, LPS improved WT neutrophil homing to the fungal challenge, while Syk deficient neutrophils did not persist in vivo, speaking to its crucial role on in vivo persistence. Taken together, we identify TLR signaling as an alternate activation pathway capable of partially restoring neutrophil effector function against Candida in a Syk-independent manner.

Introduction

Invasive fungal disease, and in particular invasive candidiasis (IC), affects people on a global scale, with over 700,000 cases of IC annually¹. Of the Candida spp., C. albicans is the most common and accounts for ~50% of the clinical isolates, depending on the geographic area^2,3. Development of IC may be attributed to intrinsic patient risk factors including diabetes mellitus, inflammatory bowel diseases, increased age, or sepsis as well as iatrogenic risk factors that increase the risk of IC include use of empiric antibiotics, central venous catheters, solid organ or stem cell transplant, chronic corticosteroid usage and long-term hospital or intensive care unit stays^2,4–6. Considering the substantial mortality rate of IC^6,7, patient risk prediction and risk reduction is a critical and evolving challenge.

C. albicans in healthy individuals can exist as a commensal organism on the skin, the oral, gastrointestinal and/or vaginal microbiota. Risk factors associated with invasive Candida infection tend to coincide with alterations in the host immune system. The innate immune system, composed primarily of myeloid cells capable of rapid pathogen engagement, are the first cell types to interact with C. albicans. The most abundant of these innate cells are neutrophils, which provide first line protection against many infections, including IC. Neutropenia is a major risk factor for IC, demonstrating the crucial role neutrophils play in the prevention of candidemia and other fungal infections^8–12. In addition, several primary immune deficiencies linked to impaired neutrophil killing function, such as NADPH oxidase and CARD9 deficiency, often result in recurrent fungal infections^13,14.

Neutrophils play a central part in executing an early and effective response to fungal infections. This process involves the initial recognition of C. albicans through the use host pattern recognition receptors (PRRs) and binding of pathogen-associated molecular patterns (PAMPs). β- glucan, a fungal cell wall carbohydrate, is one of the several PAMPs recognized by neutrophils, principally through the lectin receptor, Dectin-1¹⁵. Additional receptors known to contribute to the detection of C. albicans include Toll-like receptors (TLRs), nucleotide-binding oligomerization domain (NOD)- like receptors, retinoid-inducible gene 1 protein (RIG1)-like receptors as well as the complement receptor^16–19.

The binding of C. albicans carbohydrate cell wall antigens through lectin receptors promotes an intracellular protein signaling cascade predominantly through spleen tyrosine kinase (Syk), an integral step in the initiation of intracellular signaling and cell stimulation²⁰. This activation leads to downstream effector functions including phagocytosis of C. albicans, reactive oxygen species production (ROS), degranulation, cytokine production and expulsion of neutrophil extracellular traps (NETs)^19,21. Our previous work demonstrated a critical dependence on Syk in mouse neutrophils for a well-coordinated response to C. albicans²².

Small molecule kinase inhibitors (SMKIs) targeting Syk, Btk and other tyrosine kinases are a family of emerging therapeutics efficacious in the treatment of a broad spectrum of diseases including lymphomas, leukemias and immune thrombocytopenia, however, paired with these positive disease outcomes have been an increased rate of invasive fungal infections^23–26.

We sought to define the interplay of Syk-dependent and Syk-independent alternate signaling pathways capable of enhancing neutrophil fungicidal activity in the face of genetic Syk deletion or use of SMKIs. In this study, we investigated if TLR signaling is capable of augmenting Syk-deficient neutrophil function. As a model, we utilize LPS, which is recognized by TLR4-myeloid differentiation primary response gene 88 (MyD88) signaling, with a resulting downstream response leading to NF-kB activation and proinflammatory gene expression^27–29. Our results demonstrate partial restoration of Syk-deficient neutrophil function through TLR signaling, highlighting the existence of alternate pathways able to circumvent crucial tyrosine kinases such as Syk in neutrophil responses to the human fungal pathogen, C. albicans.

Materials and Methods

Reagents

LPS (InvivoGen, San Diego, CA) was used at a concentration of 400 ng/mL. CytochalasinD (Sigma-Aldrich, St. Louis, MO) was used at a concentration of 20 nM. For assays utilizing a TLR panel of ligands for TLR1 through TLR9 activation was purchased from InvivoGen and used as suggested by the manufacturer. R406, a selective Syk inhibitor (Selleck Chemical, Houston, TX) was used at a concentration of 20 μM. Ibrutinib, a Bruton’s tyrosine kinase (Btk) inhibitor (Cayman Chemical, Ann Harbor, MI) was used at a concentration of 1 μM. Nonidet P-40 (NP-40) was purchased from American Bioanalytical (Natick, MA). Flow cytometry staining buffer (FACS buffer) was composed of 2% heat inactivated Fetal Bovine Serum (FBS, Gibco, Dun Laoghaire, Ireland) and 1mM EDTA (Gibco) in Phosphate Buffer solution (PBS) (Corning, Corning, NY). 10% Formalin was purchased through Sigma-Aldrich. To culture C. albicans, Yeast Extract-Peptone-Dextrose broth (YPD) was used, composed of 1% yeast extract (Acros Organics, Fair Lawn, NJ), 2% peptone (BD Biosciences, San Jose, CA) and 2% dextrose (Sigma-Aldrich). Complete RPMI (cRPMI) was made up of RPMI (Corning) with 2 mM L-glutamine (Gibco), 10% heat-inactivated FBS and 1% penicillin-streptomycin. RPMI-MOPS contained RPMI 1640 with 2% glucose (Sigma-Aldrich) and 0.165M morpholinepropanesulfonic acid (MOPS, Thermo Fisher) buffered to pH 7. Beta-estradiol positive (E2+) media is composed of complete RPMI plus Stem Cell Factor (SCF) added as a conditioned media from a SCF-overexpressing Chinese hamster ovary (CHO) cells to a final of 1% with 0.5 mM b-estradiol (Sigma-Aldrich). Swarming media is composed of IMDM plus 20% FBS. Alexa Fluor 647 or 405 succinimidyl ester (AF647/AF405) was purchased through Thermo Fisher for labeling of C. albicans. Anti-CD11b-phycoerythrin antibody (CD11b-PE) and anti-CD45.2-Alexa Fluor 647 (CD45.2-AF647) were purchased through Biolegend, (San Diego, CA). Hoechst 33342 stain and Sytox Green were both purchased from Thermo Fisher.

Candida albicans preparation

SC5314 wild-type C. albicans was purchased from the ATCC (Manassas, VA). FarRed Fluorescent Protein-expressing SC5314 (iRFP C. albicans) was a gift from Robert Wheeler (University of Maine, Orono, ME)³⁰. SC5314 was cultured in YPD broth overnight at 30°C on a shaking platform, washed, and resuspended in PBS and then counted using a Luna Automated Fluorescence Cell Counter (VitaScientific, Beltsville, MD). C. albicans hyphae or pseudo hyphae (roughly 2x the length of a yeast body) were produced by incubating the washed yeast in cRPMI for up to two hours at 37°C, 5% CO₂. Heat killed C. albicans are generated by incubating C. albicans in PBS in a heat block at 95°C for 10 minutes. C. albicans were fluorescently labeled with AF647 or AF405 at 1ng/mL for 30 minutes in the dark at RT while shaking and then washed and resuspended in PBS.

Primary human Neutrophils

Healthy donors were consented under a Massachusetts General Hospital Institutional Review Board approved protocol and whole blood was drawn the same day of experiments. In brief, whole blood was drawn into an EDTA-coated blood tube (BD Biosciences) and centrifuged at 1500xg for 15 min at RT. The top plasma layer was aspirated, and the buffy coat was subject to neutrophil isolation by negative selection using the EasySep Direct Human Neutrophil Isolation Kit (STEMCELL Technologies, Seattle, WA). To confirm the purity of neutrophils at the end of the isolation process, Wright-Giemsa (Thermo Fisher) staining was performed as previously reported and viabilities were verified via live/dead staining with Acridine Orange/Propidium Iodine (AO/PI) staining and counting on the LUNA Automated Fluorescent Cell Counter at a 1:10 dilution. Both were reported at or above 87%^31,32.

Murine neutrophil progenitor cell lines

Wild-type (WT) and Syk deficient (Syk KO) conditionally immortalized neutrophil progenitor cell lines were established and characterized as previously described²². In brief, bone marrow cells from Cas9 transgenic mice³³ were transduced using a retrovirus encoding ER-Hoxb8 allowing conditional immortalization of granulocyte-macrophage progenitors (GMP)³⁴. In the presence of beta-estradiol, GMPs remain in an undifferentiated and self-renewing state. Upon removal of beta-estradiol, the ER-Hoxb8 is no longer active in the nucleus and the cells undergo synchronous differentiation from GMP into mature neutrophils³⁵. Assays of mature neutrophils were completed in cRPMI unless otherwise noted.

Candida albicans killing assay

Mature neutrophils were plated in a clear flat-bottom 96-well plate (Corning) at a concentration of 1e5 cells per well. Ibrutinib, R406 or DMSO control were added to each well and incubated at 37°C, 5% CO₂ for 30 minutes. Then, LPS or dH2O control was added to each well and incubated at 37°C, 5% CO₂ for an additional 30 minutes. Live C. albicans was then co-cultured with neutrophils at a multiplicity of infection (MOI) 2. A serial dilution series of C. albicans only was plated to establish a standard curve. Following this incubation, 1% NP-40 containing 10mM Tris HCl, 150mM sodium chloride, and 5 mM magnesium chloride at pH 7.5 was added for complete lysis of the neutrophils. Proceeding lysis, MOPS-RPMI and 10% PrestoBlue (Thermo Fisher) was added to each well and read continuously on an SpectraMax i3x reader (Molecular Devices, San Jose, CA) at wave lengths of 560 and 590 nm every hour for 18 hours at 30°C. The inflection point of each well reading was determined, and amount of live yeast in each well was determined using the yeast standard curve. Percent killing was determined using the following equation [(1-(# of C. albicans in well with neutrophils/# of C. albicans alone)) *100] based on control wells containing C. albicans alone as maximal growth and compared to WT plus vehicle.

Neutrophil gelatinase-associated lipocalin (NGAL)

Neutrophils were plated in a clear flat bottom 96-well plate (Corning) at 1e5 cells per well with LPS or vehicle control and incubated at 37°C, 5% CO₂ for 10 minutes. Live C. albicans were then added at a MOI of 10 and co-incubated for 2 hours at 37°C, 5% CO₂. Supernatant was collected and diluted to 1:2048 in reagent diluent (R&D Systems, Minneapolis, MN). Diluted supernatants were applied to a Mouse Lipocalin-2/NGAL DuoSet ELISA and run according to the manufacturer’s protocol to determine the concentration of NGAL. The ELISA was analyzed on a SpectraMax i3x reader at 650nM. NGAL concentrations were determined by comparison to a NGAL standard curve and expressed as picograms/mL (pg/mL).

Reactive oxygen species production

In a white walled flat bottom 96-well plate (Corning), 1e5 neutrophils were added to each well followed by a given TLR agonist or its respective vehicle control. The plate was then incubated at 37°C, 5% CO₂ for 30 minutes. After, heat killed C. albicans hyphae or vehicle control was added to each well at a ratio of 20 to 1 (C. albicans to neutrophil), followed by Lucigenin (Thermo Fisher) at a final concentration of 15 μM in DMSO. The plate was read in a SpectraMax i3x with an analysis of luminescence at wavelength 578nM every 5 minutes for 4 hours at 37°C. Data is expressed as a sum of all readings over time in arbitrary units (AU) and compared to WT plus LPS plus HK C. albicans.

Phagocytosis assay

Neutrophils were added to microcentrifuge tubes at 5e6 per mL in cRPMI along with LPS, cytochalasin D or vehicle control and incubated for 30 minutes at 37°C, 5% CO₂ followed by the addition of HK C. albicans hyphae labeled with AF647 at a ratio of 10:1 [Pathogen to neutrophils] and co incubated for 4.5 hours at 37°C, 5% CO₂. Following co-incubation, the media was washed out and the samples were fixed in 10% formalin and stained for CD11b-PE in FACS buffer at 1:50 for 30 minutes in the dark on ice. Flow cytometry was performed on a BD FACSCelesta (BD Biosciences). Percent phagocytosis was determined by gating on CD11b positive neutrophils and determining the percentage of those events that are double positive CD11b+/AF647+, which represent Candida bound neutrophils. Percent double positive events were calculated using FlowJo 10 software (BD Biosciences).

Western blotting

Mouse neutrophils were incubated in a microcentrifuge tube at 2.5e6 per mL in cRPMI along with LPS (400 ng/mL) and/or HK C. albicans hyphae at a ratio of 10:1 [pathogen to neutrophils] for 40 minutes at 37°C, 5% CO₂. Cells were then collected and lysed with Laemmli sample Buffer (Bio-Rad; Hercules, CA) containing protease inhibitors (cOmplete Mini; Roche Diagnostics, Indianapolis, IN), reducing agent (NuPAGE Sample Reducing Agent; Thermo Fisher) and phosphatase inhibitors (sodium orthovanadate; New England Biolabs, Ipswich, MA). Proteins were resolved by SDS-PAGE, transferred onto polyvinylidene difluoride (PVDF) membrane, which were blocked with PBS-0.1% Tween (PBS-T)-5% bovine serum albumin (Sigma-Aldrich). Phosphorylated c-Fos levels were probed using rabbit mAb D82C12 anti-phospho-c-Fos (Cell Signaling; Danvers, MA) (1:1,000) and total c-Fos levels were probed using rabbit mAb 9F6 anti-c-Fos (Cell Signaling). For β-actin, mouse mAb AC-15 anti-β-actin (Sigma-Aldrich) (1:200,000) was used to confirm equal loading.

Neutrophil Swarming Assay

Neutrophils were added to microcentrifuge tubes at 5e5 cells per mL followed by Hoechst staining at 1:500 for 10 minutes at 37°C, 5% CO₂. The cells were washed with PBS and resuspended in swarming media. R406, ibrutinib or vehicle control were added, and the cells were pre-incubated at 37°C, 5% CO₂ for 30 minutes, followed by the addition of LPS or vehicle control and again, incubated at 37°C, 5% CO₂ for an additional 30 minutes. Sytox Green was then added to a final concentration of 0.5 μM in swarming media. Swarming experiments and analysis were performed as previously described³⁶. In brief, sixteen-well ProPlate wells (Grace Bio-labs, Bend OR) were bound to glass slides with printed poly-l-lysine/ZETAG arrays. A solution of C. albicans yeast was added for 10 minutes to bind to the slides and then, subsequently washed to remove unbound pathogen. The neutrophils from the microcentrifuge tubes were then transferred to the appropriate well and time-lapse imaging of selected swarming targets was performed using an automated Nikon TiE microscope (Minato City, Tokyo, Japan) with a 10X Plan Fluor Ph1 DLL (numerical aperture [NA] = 0.3) lens. The area of fungal growth was determined by manually outlining the perimeter of fungal growth for each fungal cluster after 16 hours using FIJI (ImageJ v2.0.0-rc 59/1.52p, NIH) software.

In vivo homing to Candida albicans

B6.SJL-Ptprc^a Pepc^b/BoyJ (Pep Boy) mice (The Jackson Laboratory, Bar Harbor, ME) were cared for and housed in accordance with the Institutional Animal Care and Use Committee (IACUC) guidelines and prior approval for experiments was obtained. Mice were lethally irradiated with a total of 900 centi-gray through X-ray irradiation in two equally fractionated doses 4–6 hours apart. Mice rested for one day followed by retro-orbital injection of either WT or Syk KO GMP cells in PBS (total of 40E6 cells over 2 transfusions). Mice were then housed for a subsequent 4 days to allow the GMP cells to fully mature into neutrophils. On the fourth day after cell transfusions the mice were challenged with either PBS, LPS, heat killed C. albicans plus PBS or heat killed C. albicans plus LPS intraperitoneally. C. albicans were prior labelled with succinimidylester-AF405. After five hours, mice were euthanized, and peritoneal lavage was collected along with bone marrow. Samples were immediately fixed in 10% formalin, then washed and stored in FACS buffer. Cells were stained with CD11b-PE and CD45.2-AF647 and subject to flow analysis. Adoptively transferred WT and Syk KO cells were identified by CD45.2 labelling in the CD45.1 recipient mice. Percent of neutrophils in lavage were determined by gating on CD45.2 and CD11b double positive events and percent transfused cells in the bone marrow were determined by gating on CD45.2 positive cells.

Statistics

All statistical analyses for normality and significance were performed using Graphpad Prism 9 software (San Diego, CA). A p-value of <0.05 was considered significant.

Results

LPS improves the fungicidal capacity of Syk deficient or inhibited murine neutrophils

We investigated the capacity to prime mouse neutrophils towards the killing of C. albicans (Fig 1A). Without LPS priming, WT neutrophils kill significantly more C. albicans than Syk KO neutrophils. When WT and Syk KO neutrophils are primed with LPS, the fungicidal capacity of neutrophils is significantly improved with Syk KO neutrophil levels reaching that of WT neutrophil. We then investigated the ability for LPS to rescue WT neutrophils from drug inhibition of Syk using R406 (Fig1B). R406 alone results in complete loss of neutrophil killing capacity of C. albicans. However, LPS was able to partially rescue WT neutrophil fungicial activity in the presence of Syk.

Figure 1. LPS improves fungicidal capacity of Syk deficient or inhibited mouse neutrophils against C. albicans.

(A) Percent killing of C. albicans at a MOI of 2 after 2.5 hours by WT and Syk deficient neutrophils pre-incubated with LPS or matching vehicle control. (B) Percent killing of C. albicans by WT neutrophils at a MOI of 2 after 3 hours pre-incubated with 10 μM Syk inhibitor R406 or the matching vehicle control followed by 400ng/mL LPS or matching vehicle control. Data are represented as mean ± SEM. *p<0.05, **p<0.01, ***p<0.0001. Data analyzed by one-way ANOVA with Tukey’s posttest and data is representative of 3 independent experiments.

LPS priming improves phagocytosis, degranulation and swarming but not ROS production of Syk deficient neutrophils

Having observed the clear improvement in killing of C. albicans, we sought to determine which of the neutrophil’s effector mechanisms were improved following LPS priming. Using a flow cytometry-based phagocytosis assay, we gated on live, CD11b positive cells to select for mature neutrophils (Figure 2A) and determined the percentage that were physically bound to AF647 labeled heat killed (HK) C. albicans. As a control, we utilized the same gating strategy with C. albicans alone to demonstrate there are no events in the “neutrophil bound C. albicans” gate confirming that these events represent double positive neutrophil/C. albicans cells. We next determined the percent phagocytosis of HK C. albicans by WT and Syk KO neutrophils. We found Syk KO neutrophils are severely impaired in their ability to bind HK C. albicans (Fig 2B). Priming WT and Syk KO neutrophils with LPS significantly improved phagocytosis in both cell types, while cytochalasin D, an actin polymerization inhibitor, acted as a potent suppressor of phagocytosis.

Figure 2. LPS priming improves phagocytosis, degranulation and swarming but not ROS production of Syk deficient Neutrophils.

(A) Flow Cytometry gating scheme to determine the percent of mouse neutrophils that have phagocytosed heat killed C. albicans. FSC: forward scatter, SSC: side scatter, CD11b-PE: Anti-CD11b antibody conjugated to phycoerythrin, AF647-SE: Alexa Fluor 647-Succimydyl ester bound to heat killed C. albicans pseudo hyphae. (B) Percent phagocytosis of heat killed C. albicans by WT or Syk deficient neutrophils after 5 hours co-incubation, primed with 400ng/mL LPS, matching vehicle control or 20nM CytoD. CytoD: Cytochalasin D. (C) NGAL release in pg/mL by WT and Syk deficient neutrophils stimulated for 2.5 hours with 400ng/mL LPS, matching vehicle control, C. albicans at a MOI of 10 plus LPS or C. albicans plus matching vehicle control. NGAL: Neutrophil Gelatinase-associated Lipocalin (D) Area of fungal growth by C. albicans clusters on fabricated swarming arrays with or without WT or Syk deficient neutrophils, pre-incubated with 400ng/mL LPS or matching vehicle. Data presented as the growth of clusters measured after 700 minutes and normalized to C. albicans only controls. (E) ROS production by WT or Syk deficient neutrophils stimulated for 4 hours with 400ng/mL LPS, matching vehicle control, C. albicans at a MOI of 10 plus LPS or C. albicans plus matching vehicle control. Data represented as percent ROS production of WT neutrophils stimulated with LPS plus C. albicans. Data are represented as mean ± SEM. *p<0.05, **p<0.01, ***p<0.0001 and analyzed by one-way ANOVA with Tukey’s posttest. Data representative of 3 independent experiments.

We then quantified the ability of WT and Syk KO neutrophils to degranulate via measurement of NGAL release into supernatant following live C. albicans stimulation. Compared to their resting state, Syk KO neutrophil had impaired NGAL degranulation compared to WT, although NGAL release significantly augmented when stimulated with LPS, C. albicans or both stimuli (Fig 2C).

Next, we investigated the ability for these cells to engage in a coordinated multi-step response to C. albicans as determined by swarming function of WT and Syk KO neutrophils primed with LPS (Fig 2D). WT neutrophils significantly suppress fungal growth compared to C. albicans alone or Syk KO neutrophils plus C. albicans. LPS priming did not improve WT neutrophil’s ability to suppress fungal growth and while LPS priming of Syk KO neutrophils did enhance control of fungal growth, the effect was not statistically significant.

Lastly, we determined ROS production of WT and Syk KO neutrophils in response to HK C. albicans hyphae +/− LPS (Fig 2E). LPS did not boost ROS production in WT or Syk KO neutrophils, though the response was more robust following stimulation of WT neutrophils with the combination of HK C. albicans hyphae +/− LPS. Syk KO neutrophils ROS production was not significantly improved with either HK C. albicans hyphae or hyphae in combination with LPS.

Specific TLR receptor pathway activation, improves candidacidal killing by Syk deficient neutrophils

Given the results of TLR4 signaling by LPS, we examined whether other common TLR receptor agonists could improve the response of Syk KO neutrophils against C. albicans. Percent killing by Syk KO neutrophils was improved following priming with ligands to TLR 1/2, 2, 4, 5 and 6/2 (Fig 3A) but conversely, ligation of the intracellular receptors, TLR 3, 7 and 9, decreased Syk KO neutrophil killing.

Figure 3. Specific TLR receptor pathway activation improves candidacidal killing by Syk deficient neutrophils.

(A) Percent killing of C. albicans at a MOI of 2 after 2 hours co-incubation with WT or Syk deficient neutrophils pre-incubated with select TLR agonist or their matching vehicle control. Data is represented as percent of killing by WT plus LPS vehicle control. Both WT and Syk KO plus LPS or vehicle control data pooled from 4 independent experiments. Syk KO plus agonist 1–3 and 5–9 data pooled from 2 independent experiments. (C and D) ROS production by WT or Syk deficient neutrophils after 4 hours in the presence of a select TLR agonist, vehicle control, heat killed C. albicans hyphae at a MOI of 20 plus select TLR agonist or heat killed C. albicans hyphae plus vehicle control. Data represented as percent of ROS production by WT neutrophil stimulated with LPS and heat killed C. albicans hyphae and shown as mean ± SEM. *p<0.05, **p<0.01, ***p<0.0001. Data analyzed by one-way ANOVA with Tukey’s posttest and representative of 2 independent experiments.

We also investigated the ability of TLR ligation to augment ROS production from Syk KO neutrophils (Fig 3B and 3C). Values were based off percent ROS production of WT plus LPS plus C. albicans. WT neutrophils did not produce significantly more ROS when primed with LPS, however HK C. albicans boosted production compared to LPS alone and both stimulants together improved production above HK C. albicans alone. Across all TLR ligands, there was not a significant improvement in ROS production by Syk KO neutrophils in response to LPS, HK C. albicans or LPS plus HK C. albicans compared to vehicle.

LPS modulates killing of human neutrophils against C. albicans in the setting of kinase inhibitors

We also sought to determine if human neutrophil fungicidal activity is augmented in the presence of LPS. Primary human neutrophils treated with inhibitors of Syk (R406) or Btk (ibrutinib) were primed with LPS and then challenged with C. albicans (Fig 4A). LPS augmented human neutrophil killing against C. albicans whereas ibrutinib significantly decreased the killing capacity of human neutrophils. LPS, however, was able to rescue the inhibitory effect of ibrutinib on fungifical activity of neutrophils to those similar to control neutrophils. R406 also significantly decreased human neutrophil killing and in a similar process, LPS priming was able to partially restore fungicidal activity against C. albicans.

Figure 4. LPS modulates killing of human neutrophils against C. albicans in the setting of kinase inhibitors.

(A) Percent killing of C. albicans at a MOI of 4 after 3 hours by healthy donor human neutrophils pre-incubated with 1 μM Btk inhibitor Ibrutinib, 10μM Syk inhibitor R406 or their respective matching vehicle controls followed by pre-incubation with 400ng/mL LPS. (B) Microscopy images of swarming slides showing bright field images, iRFP C. albicans, and Hoechst-stained neutrophils. (C). Area of fungal growth by C. albicans clusters on fabricated swarming arrays with or without human neutrophils, pre-incubated with Syk inhibitor, R406 at 10μM, followed by 400ng/mL LPS or matching vehicle. Data presented as the growth of clusters measured after 700 minutes and normalized to C. albicans only control. Data are represented as mean ± SEM *p<0.05, **p<0.01, ***p<0.0001 and analyzed by one-way ANOVA with Tukey’s posttest. Data representative of 3 independent experiments.

Additionally, we analyzed the ability of human neutrophils to restrict C. albicans area of fungal growth through swarming in the setting of Syk inhibition. Bright field and fluorescence microscopy images were taken at designated time points of wells containing either C. albicans alone, C. albicans plus neutrophil, C. albicans plus neutrophil and R406, or C. albicans plus neutrophil plus R406 and LPS. In order to visualize iRFP C. albicans and neutrophil nuclei through Hoescht staining (Fig 4B). Analyzing the area of fungal growth when normalized to C. albicans alone, the presence of R406 causes severe limitation of neutrophils ability to restrict the fungal growth of C. albicans compared to vehicle control. However, when the neutrophils are primed with LPS prior to Syk inhibition, there is a partial restoration of ability to suppress fungal growth compared to control. As a control, we tested if LPS priming alone had influence on the suppression of fungal growth versus vehicle control, which showed that there was no LPS effect on neutrophil function.

WT but not Syk deficient transfused neutrophil progenitors persist in the bone marrow and mature into neutrophils

The ER-Hoxb8 GMP progenitors were transfused into recipient mice following conditioning with radiation³⁷. The transfused progenitor’s home to the bone marrow of the recipient where they undergo normal maturation to terminally differentiated neutrophils. This provides a model system in which to study the in vivo activity of WT versus Syk deficient neutrophil function.

To investigate the capacity for engraftment, differentiation, and homing to the site of a HK C. albicans challenge, we transfused WT and Syk deficient GMP cells (CD45.2) into ablated CD45.1 mice. After 4 days, mice were challenged with an intraperitoneal HK C. albicans, and after 5 hours the mice were euthanized for analysis of their bone marrow and intraperitoneal lavage.

We gated on live transfused cells as marked by CD45.2 expression, which were present in all mice (Fig 5A). There was a significantly larger percent of transfused WT cells in the bone marrow compared to Syk KO. Furthermore, the percent of mature neutrophils in the bone marrow, as defined by being CD45.2 and CD11b double positive, was significantly higher in recipients of WT cells compared to Syk KO (Fig 5C) suggesting that Syk may play a role in GMP persistence in bone marrow.

Figure 5. WT but not Syk deficient transfused neutrophil progenitors persist and mature in the bone marrow.

(A) Gating scheme of isolated mouse bone marrow 4 days post transfusion of WT or Syk deficient neutrophil progenitors and following intraperitoneal challenge. Singlets were gated on and then analyzed by CD11b-PE and CD45.2-AF647. CD45.2 positive events indicate the transfused neutrophil progenitors and CD45.2/CD11b double positive events indicate mature neutrophils. FSC: forward scatter, SSC: side scatter, CD11b-PE: Anti-CD11b antibody conjugated to phycoerythrin, CD45.2-AF647: Anti-CD45.2 antibody conjugated to Alexa Fluor 647. (B) Percent CD45.2 positive events in the bone marrow. (C) Of those CD45.2 positive events, the percent CD11b positive events were determined to indicate mature neutrophils in the bone marrow. Data are represented as mean ± SEM, n=3/4. *p<0.05, **p<0.01, ***p<0.0001 and analyzed by two-tailed unpaired t test with Welch’s correction.) Data representative of 2 independent experiments.

Neutrophils lacking Syk are incapable of homing to an intraperitoneal C. albicans challenge

We also examined if the transfused cells could home to the site of an infection following an intraperitoneal challenge with HK C. albicans. Following peritoneal lavanage, the number of CD45.2 and CD11b double positive cells in the lavage was determine and we found a statistically significant increase in recruited neutrophil in mice that received WT GMPs that were challenged with HK C. albicans plus LPS compared to unchallenged mice (Fig 6). There was no significant change in WT GMP recipient mice that received vehicle control, LPS or HK C. albicans plus vehicle. Amongst the groups that were transfused with Syk KO GMPs, there was not a significant difference in mice that received vehicle control, LPS, HK C. albicans with or without LPS compared to no challenge.

Figure 6. WT but not Syk deficient neutrophils home to an intraperitoneal Candida challenge and LPS boosts this homing.

(A) Gating scheme of isolated lavage after 5-hour intraperitoneal challenge of 1ug LPS, vehicle control, 100e6 heat killed C. albicans plus 1ug LPS, 100e6 heat killed C. albicans plus vehicle control or no intraperitoneal challenge at all. Singlets were gated on and then analyzed by CD11b-PE and CD45.2-AF647. CD45.2 positive events indicate the transfused neutrophil progenitors and CD45.2/CD11b double positive events indicate mature neutrophils. FSC: forward scatter, SSC: side scatter, CD11b-PE: Anti-CD11b antibody conjugated to phycoerythrin, CD45.2-AF647: Anti-CD45.2 antibody conjugated to Alexa Fluor 647 (B) Total number of CD45.2/CD11b double positive events from the lavage fluid post intraperitoneal challenge. Data are represented as mean ± SEM, n=3/4. *p<0.05, **p<0.01, ***p<0.0001 and analyzed by one-way ANOVA with Tukey’s posttest. Data representative of 2 independent experiments.

Syk-independent c-Fos activation in WT and Syk KO neutrophils following Candida and LPS stimulation.

To investigate potential alternate pathways of neutrophil activation capable of circumventing loss of Syk, we analyzed c-Fos activation in response to LPS and HK C. albicans hyphae through western blot analysis. WT neutrophils showed minimal levels of phosphorylated c-Fos which became more pronounced following stimulation with LPS or LPS plus HK C. albicans hyphae. Syk KO neutrophils likewise did not show robust levels of c-Fos phosphorylation at rest or when stimulated LPS or HK C. albicans hyphae, however prominent activation was detected in the presence of both LPS and C. albicans suggesting activation through a Syk-independent alternate pathway (Supplemental Figure 1). As a control, LPS plus HK C. albicans hyphae alone revealed no detectable levels of phosphorylated c-Fos. Despite even protein loading as shown by β-actin, fluctuations in total c-Fos were seen across the conditions. WT neutrophils at rest, Syk KO neutrophils at rest and Syk KO neutrophils stimulated with HK C. albicans hyphae appeared to have minimal levels of c-Fos, while the other conditions yielded robust levels of total c-Fos. These data suggest the total available pool of c-Fos, as well as the fraction that results in a phosphorylated and activated state is dynamic depending on stimulation condition.

Discussion

Neutrophils rely on a critical set of receptors for initial recognition of invasive fungal pathogens and subsequent activation of a fungicidal response. Many of these receptors belong to the lectin family, which rely on Syk for intracellular signaling and cellular activation. We have previously demonstrated an essential reliance on Syk for neutrophil response against Candida spp²². In this study, we sought to determine if alternate pathways exist in Syk deficient neutrophils capable of restoring function against C. albicans. Given TLR expression on neutrophils, we examined TLR agonists as potential alternative activation signaling pathways. Previous data supports the augmentation of neutrophil fungicidal activity through TLR4 recognition of LPS³⁸. Interestingly, TLR4 also has the capacity to recognize C. albicans through the recognition of cell wall O- mannan³⁹ and to trigger downstream Syk activation, further complicating potential mechanisms of Syk independent activation through TLRs⁴⁰.

Here, we present data that supports our hypothesis of TLR mediated rescue of neutrophil function in the setting of Syk signaling deficits, both in mouse and human neutrophils. This improvement was seen across many, but not all, effector functions including phagocytosis, degranulation, ROS production and neutrophil swarming (Fig 7). For example, ROS production was not significantly improved in the Syk KO neutrophils when responding to C. albicans in the setting of LPS priming, which corroborates other published work that LPS is not a potent activator of ROS in mouse or human neutrophils^41,42. This finding was confirmed to likely be a common feature of TLR signaling, as no members of a TLR agonist panel were able to significantly boost ROS production in Syk KO neutrophils. On the other hand, depending on the TLR receptor activated, there appear to be a differences in neutrophil augmentation. Syk KO neutrophil killing of C. albicans was improved by priming with PAM3CSK4, heat killed Listeria monocytogenes (HKLM) and FSL-1, ligands to the TLR receptors 1/2, 2 and 6/2, respectively. PAM3CSK4 has been previously reported to induce a primed human neutrophil state including shedding of L-selectin and induction of NADPH oxidase activity in, which may account for the increase fungicidal activity seen in our mouse neutrophils⁴³. Interestingly, HKLM has previously been shown to boost candidacidal activity of peritoneal cells as well as evade destruction through activation of macrophage mitophagy^44,45. In part, low mitochondria density in neutrophils may play a role in suppressing HKLM evasion. Additionally, work has been done to show that agonists to TLR 1/2, 2/6 and 4 improve macrophage phagocytosis of zymosan particles, whereas agonist to TLR 3 and 5 do not⁴⁶. Taken together our data suggests that specific TLR activation can influence downstream neutrophil effector functions with improved fungicidal activity.

Figure 7. Classical Versus Alternative Pathway to Neutrophil Effector Function in Response to C. albicans and/or LPS.

Schematic of neutrophil signaling in response to C. albicans. Classical pathway (left panel) in response to the ligand β-glucan and Candida. Binding to Dectin-1 leads to Syk phosphorylation and activation leading to downstream effector functions of including killing, phagocytosis, degranulation, ROS production and swarming. In Syk impairment (righ panel), there is a loss of downstream signaling, however binding of LPS from bacteria by TLR leads to activation of through an alternate pathway leading to killing, phagocytosis, degranulation, and swarming. Illustration by Nicole Wolf, MS, ©2021. (nicolewolf@nicolewolfart.com). Printed with Permission.

LPS could restore specific neutrophil functions including phagocytosis and swarming. Syk KO neutrophils without LPS demonstrate nearly complete absence of response, however following LPS priming, the ability to phagocytose and swarm against C. albicans was significantly improved. One possible explanation is the differential expression of PRRs following LPS priming such as upregulation of CD11b expression^47,48 known to have the capacity to bind fungal cell wall components including β-glucan⁴⁹, although CD11b relies, in part, on Syk for subsequent signalling and cellular activation⁵⁶. The exact molecular mechanism that defines rescue of Syk deficient neutrophils through a LPS-dependent pathway will require additional investigation.

As with mouse neutrophil, human neutrophil function is also linked to Syk activity. R406, a small molecule inhibitor of Syk, severely impairs human neutrophil capacity to kill C. albicans and limits neutrophil swarming. Using R406 and ibrutinib, a Btk inhibitor, which also plays a critical role in neutrophil response to invasive fungal and bacterial infections^26,50, we examined if LPS was able to rescue human neutrophil dysfunction. Our data show a restoration of human neutrophil function in the setting of kinase inhibition suggesting that these alternate activation pathways appear to be conserved in both mouse and human neutrophils.

We next asked if restoration of function might be replicated in vivo. Following bone marrow ablation, WT and Syk-deficient GMPs were transfused in irradiated mouse recipients. Interestingly, there was a stark difference in the proportion of WT versus Syk-deficient transfused cells that could home and persist in the bone marrow. WT GMP cells composed 85–90% of the bone marrow cells analyzed whereas less than 15% Syk KO GMP cells were found, raising the possibility of a critical role of Syk in GMP engraftment, persistence, or survival in the bone marrow. Syk has been implicated in general hematopoiesis⁵¹ and our previous work showed Syk protein levels increase from the GMP to neutrophil developmental pathway²². Despite not being an abundant kinase in early GMP stages, it does raise the possibility of Syk playing a role in the bone marrow microenvironment as GMPs mature into neutrophils. The influence of Syk on GMP appears to be a in vivo phenomenon specific to the bone marrow microenvironment as previous data indicate the Syk-deficient neutrophils differentiate and mature in vitro without loss of viability, granule protein production or expression of surface markers²². The impact of Syk loss may also include the possible imparirment of neutrophil homing to sites of inflammation given the contribution of Syk to molecular machinery responsible for adhesion, rolling, and extravasation from circulation. Interestingly, LPS dramatically improved the recruitment of WT neutrophils to peritoneal fungal inflammation consistent with other studies using LPS priming of tissue resident macrophages in antifungal responses⁵³. These Syk-independent pathways highlight possible therapeutic avenues in patients for more precision based immune augmentation.

The mechanisms of improvement seen following LPS priming in the setting of Syk-deficiency remains unclear. One possibility is crosstalk or feedthrough between TLR4 and Dectin-1 signaling pathways. We investigated possible downstream targets that could act as a bridge. In macrophages activation downstream of Syk and TLR4 involves c-Fos dependent cytokine release⁵⁴. Further investigation identifys c-Fos activation in response to C. albicans infections in human epithelial cells⁵⁵. Our western blot analysis showed there to be differential levels of c-Fos phosphorylation when WT and Syk-deficient neutrophils were stimulated with LPS, C. albicans or both. Though the total levels of c-Fos fluctuated as well, there was increased c-Fos phosphorylation with LPS and C. albicans compared to either stimulus alone. This seemingly synergistic effect may shed insight on downstream activating proteins of TLR4 and Dectin-1 signaling, although further investigation is required to further define the molecular mechanism of this cross talk pathway.

These data show that loss or inhibition of essential kinases that significantly impair neutrophil response to C. albicans can be partially rescued through priming with LPS. Syk-deficient mouse neutrophil effector functions are significantly diminished, however LPS priming can provide a robust, augmented response to some, but not all of these, suggesting specificity to the restoration. Specifically, phagocytosis, degranulation, swarming, and overall fungicidal activity were all improved. On the other hand, ROS production was not improved, and further studies are needed to define where this signaling potentiation may be occurring and if it is dependent on activator chaperones downstream of Syk. Despite differences between human and mouse neutrophils, including diminished myeloperoxidase levels and absent defensins^52,, LPS rescue of neutrophil function also restored killing and swarming of impaired human neutrophils in the setting of small molecular inhibitors to Syk and Btk. As more kinase inhibitors are approved and brought to market to treat a broad spectrum of human diseases, these results provide insight into how we may address neutrophil dysfunction in the face of these drug interactions and improve outcomes for patients with invasive fungal infections.

Key Points.

1. Spleen tyrosine kinase is critical for neutrophil response to Candida albicans

2. LPS is capable of rescuing fungicidal activity in Syk deficient neutrophils

3. Restoration of fungicidal activity is, in part, due to activation of c-Fos