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Extracellular Superoxide Dismutase Enhances Recruitment of Immature Neutrophils to the Liver Timothy J. Break,a* Alexandra R. Witter,a Mohanalaxmi Indramohan,a* Mark E. Mummert,a,b Ladislav Dory,c

Rance E. Berga

a

Department of Cell Biology and Immunology, University of North Texas Health Science Center, Fort Worth, Texas, USA ; Mental Sciences Institute, University of North Texas Health Science Center, Fort Worth, Texas, USAb; Institute for Cardiovascular and Metabolic Diseases, University of North Texas Health Science Center, Fort Worth, Texas, USAc

Listeria monocytogenes is a Gram-positive intracellular pathogen that causes spontaneous abortion in pregnant women, as well as septicemia, meningitis, and gastroenteritis, primarily in immunocompromised individuals. Although L. monocytogenes can usually be effectively treated with antibiotics, there is still around a 25% mortality rate with individuals who develop clinical listeriosis. Neutrophils are innate immune cells required for the clearance of pathogenic organisms, including L. monocytogenes. The diverse roles of neutrophils during both infectious and noninfectious inflammation have recently gained much attention. However, the impact of reactive oxygen species, and the enzymes that control their production, on neutrophil recruitment and function is not well understood. Using congenic mice with varying levels of extracellular superoxide dismutase (ecSOD) activity, we have recently shown that the presence of ecSOD decreases clearance of L. monocytogenes while increasing the recruitment of neutrophils that are not protective in the liver. The data presented here show that ecSOD activity does not lead to a cell-intrinsic increase in neutrophil-homing potential or a decrease in protection against L. monocytogenes. Instead, ecSOD activity enhances the production of neutrophil-attracting factors and protects hyaluronic acid (HA) from damage. Furthermore, neutrophils from the livers of ecSOD-expressing mice have decreased intracellular and surface-bound myeloperoxidase, are less capable of killing phagocytosed L. monocytogenes, and have decreased oxidative burst. Collectively, our data reveal that ecSOD activity modulates neutrophil recruitment and function in a cell-extrinsic fashion, highlighting the importance of the enzyme in protecting tissues from oxidative damage.

L

isteria monocytogenes is a Gram-positive bacterium that can be found in the soil, fruits, vegetables, meats, and dairy products. Ingestion of L. monocytogenes-contaminated foods can cause spontaneous abortions in pregnant women, as well as septicemia, meningitis, and gastroenteritis, primarily in immunocompromised individuals (1). Surprisingly, clinical listeriosis results in an ⬃25% mortality rate despite the use of antibiotics (1), which makes understanding the immune response against the pathogen important. Furthermore, L. monocytogenes is a widely used model pathogen to study and understand host-pathogen interactions due to its ease of use and manipulation. Neutrophils are short-lived immune cells that differentiate in the bone marrow from the common myeloid progenitor (2). Under normal homeostatic conditions, most neutrophils are retained in the bone marrow until inflammation leads to signaling for their egress. One of the mechanisms by which neutrophils are recruited out of the bone marrow and into tissues is through the increased production of CXCL1 and CXCL2 (3, 4), which are both induced by the interleukin 23 (IL-23)/IL-17 pathway (5, 6). During lipopolysaccharide (LPS)-induced hepatic injury, hyaluronic acid (HA)-CD44 interactions facilitate neutrophil adherence to the endothelium and extravasation into the liver (7). Furthermore, injection of CXCL2 leads to a large influx of neutrophils at the site of injection, which is dependent on HA-CD44 interactions (8). However, there is still much to be learned about the mechanisms by which neutrophils are recruited to tissues under homeostatic conditions and during inflammation or infection. It was originally believed that neutrophils were solely proinflammatory cells that cleared infectious pathogens, but it is becoming increasingly obvious that neutrophils are also involved in tissue repair, modulating immune responses, and shaping the

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ability of the host to respond to subsequent infections (9). Some of the main ways in which neutrophils are thought to exert their proinflammatory actions are through phagocytosis and oxidativeburst-dependent killing of pathogens, though they also produce proinflammatory mediators, including tumor necrosis factor alpha (TNF-␣), to enhance immune responses. In terms of oxidative burst, neutrophils lacking components of the NADPH oxidase are impaired in the ability to kill pathogens (10, 11). Furthermore, patients with chronic granulomatous disease, which is due to a lack of functional NADPH oxidase, are highly compromised in their ability to clear infections (11). Neutrophils are also known to produce and store many different inflammatory medi-

Received 19 July 2016 Returned for modification 2 August 2016 Accepted 31 August 2016 Accepted manuscript posted online 6 September 2016 Citation Break TJ, Witter AR, Indramohan M, Mummert ME, Dory L, Berg RE. 2016. Extracellular superoxide dismutase enhances recruitment of immature neutrophils to the liver. Infect Immun 84:3302–3312. doi:10.1128/IAI.00603-16. Editor: C. R. Roy, Yale University School of Medicine Address correspondence to Rance E. Berg, [email protected]. * Present address: Timothy J. Break, Fungal Pathogenesis Unit, Laboratory of Clinical Infectious Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, USA; Mohanalaxmi Indramohan, Department of Medicine, Rheumatology Division, Northwestern University, Chicago, Illinois, USA. T.J.B. and A.R.W. contributed equally to this article. Supplemental material for this article may be found at http://dx.doi.org/10.1128 /IAI.00603-16. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

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ecSOD Enhances Immature Neutrophil Recruitment

ators, including myeloperoxidase (MPO), which is important for clearance of pathogenic microorganisms in both humans and mice (12–14). Supporting the anti-inflammatory or tissue repair mechanisms of neutrophils, several studies have established that neutrophils can suppress immune responses against Trypanosoma cruzi, Candida albicans, and other infections (15, 16). During infection with methicillin-resistant Staphylococcus aureus, three different subsets of neutrophils—naive neutrophils, proinflammatory neutrophils, and anti-inflammatory neutrophils—that differentially secrete pro- or anti-inflammatory cytokines were identified (17). The production of reactive oxygen species (ROS) by immune cells is critical for protection against pathogens but can also result in extensive tissue damage. ROS can directly damage the extracellular matrix (ECM), thus degrading HA and heparan sulfate (18). Extracellular superoxide dismutase (ecSOD) is an extracellular antioxidant enzyme that converts superoxide (O2˙⫺) into hydrogen peroxide (H2O2), which can be further converted into water and free oxygen. Due to its location, ecSOD is the major regulator of ROS in the ECM and is necessary for protecting the ECM from oxidative damage. Furthermore, ecSOD protects against oxidative fragmentation of HA in models of asbestos-induced lung injury and cigarette smoke-induced emphysema. The mechanism of protection is through direct binding of ecSOD to HA and other ECM components, resulting in reduced O2˙⫺ concentrations and decreased oxidative damage to the ECM (18, 19). Interestingly, ecSOD has also been shown to decrease neutrophil recruitment to the lung during noninfectious inflammatory insults (20–22), and exogenous SOD can induce neutrophil apoptosis (23). Our laboratory has recently found that ecSOD activity is detrimental to the host during infection with L. monocytogenes. It was also demonstrated that ecSOD activity enhances neutrophil recruitment to the liver both prior to and during L. monocytogenes infection (24). This observation is counterintuitive, as ecSOD activity increases the susceptibility of mice to L. monocytogenes infection and neutrophils are known to protect against L. monocytogenes infection (25, 26). Although we have previously demonstrated that ecSOD enhances the recruitment of neutrophils into the liver while decreasing their ability to protect against infection, the current study provides mechanistic insight into these phenomena. Interestingly, our novel data establish that ecSOD activity enhances the production of neutrophil-recruiting chemokines and protects the ECM from damage. EcSOD activity also leads to the recruitment of functionally impaired neutrophils into the liver. Importantly, these ecSOD-mediated effects are not intrinsic to the neutrophils, but instead, are influenced by the tissue microenvironment. MATERIALS AND METHODS Mice and L. monocytogenes infections. EcSOD HI and ecSOD WT mice were generated as previously described (27) and express high and wildtype (WT) levels of ecSOD, respectively. The ecSOD knockout (KO) mice were originally provided by Cheryl L. Fattman (University of Pittsburg). All of the mice have been backcrossed to the C57BL/6 background at least eight times. The mice were age and gender matched and were used between 7 and 12 weeks of age. All the studies were performed in compliance with the U.S. Department of Health and Human Services Guide for the Care and Use of Laboratory Animals and the guidelines of the Institutional Animal Care and Use Committee at the University of North Texas Health Science Center. L. monocytogenes 10403 serotype 1 was maintained on brain heart

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infusion (BHI) agar plates (BD Bacto, Sparks, MD). The virulence of the strain was maintained by periodic passage through C57BL/6 mice. In order to infect mice, log-phase cultures of L. monocytogenes grown in BHI broth were harvested, washed twice, and diluted in phosphate-buffered saline (PBS). The mice were intravenously infected with L. monocytogenes at a dose of 1 ⫻ 104 CFU. Isolation of cells and preparation of organs. Liver and blood leukocytes were obtained as previously described, and red blood cells were lysed with Tris ammonium chloride (28). Bone marrow cells were obtained by flushing femurs and tibiae with Hanks balanced salt solution (HBSS), 2% fetal bovine serum (FBS), 2 mM EDTA through a 70-␮m nylon cell strainer (BD Falcon). Red blood cells were lysed using a 0.2% solution of NaCl in double-distilled H2O (ddH2O) for 20 s, followed by quenching with 1.6% NaCl in ddH2O (adapted from reference 29). Liver homogenates were prepared by homogenizing whole livers in ice-cold PBS containing 0.01% Triton X-100, followed by centrifugation at 10,000 ⫻ g for 30 min and collection of supernatants for analysis. For bone homogenates, tibiae and femurs were flash frozen with liquid nitrogen and ground with a mortar and pestle to a powder. The bone powder was reconstituted with 1 ml of 1⫻ PBS with protease inhibitors (EMD Millipore Chemicals). Samples were spun at 5,000 ⫻ g for 2 min, and the supernatants were collected for analysis. For serum, blood was obtained from the retroorbital plexus of mice, placed on ice for ⬃8 h, and centrifuged at 18,000 ⫻ g for 30 min. Quantitative real-time RT-PCR array. EcSOD HI and ecSOD KO mice were infected with L. monocytogenes for 1 day, and mRNA was isolated from the liver using a TRIzol-based isolation technique. The quantity of the mRNA was determined using a Nanodrop ND-1000 spectrophotometer. The mRNA was then converted to cDNA using an RT2 First Strand kit (SA Biosciences). An Inflammatory Response and Autoimmunity PCR array (SA Biosciences) was used to measure the mRNA content for 84 genes associated with autoimmunity and inflammation. The realtime reverse transcription (RT)-PCR array was run using an Applied Biosystems StepOnePlus 96-well RT-PCR machine. The data were analyzed using software obtained from SA Biosciences. Flow cytometry and ELISA. To perform cell surface staining, the following antibodies were used: anti-Ly6G fluorescein isothiocyanate (FITC) (1A8) and anti-CD11b phycoerythrin (PE)-Cy7 (M1/70) (BD Biosciences, San Diego, CA); anti-CD44 FITC (1M7), anti-CD16/CD32 (no. 93), and streptavidin-PE (eBioscience, San Diego, CA); anti-Ly6G PE and PE-Cy7 (1A8) and anti-CD18 PE (M18/2) (BioLegend, San Diego, CA); anti-CD11b PE-Texas Red (TR) (M1/70.15) and streptavidin PE-TR (Invitrogen, Grand Island, NY); anti-MPO FITC or anti-MPO biotin (8F4) (Hycult Biotech, Plymouth Meeting, PA); and HA-fluorescein (generated by Mark Mummert). Cells were incubated at 4°C for 15 to 30 min with saturating amounts of antibody and anti-CD16/CD32, to block Fc receptors, in fluorescence-activated cell sorter (FACS) buffer. Biotinylated antibodies were detected by incubating the cells with streptavidin PE or streptavidin PE-TR at 4°C for 20 min. The cells were then fixed with 1% paraformaldehyde. For intracellular staining, cells were fixed and permeabilized using a kit from BD Biosciences. Next, the cells were incubated with anti-MPO FITC for 20 min at 4°C. To determine the efficiency of neutrophil binding to HA, blood, liver, and bone marrow cells were incubated with HA-fluorescein for 30 min, stained with cell surface markers, and analyzed by flow cytometry. A Beckman Coulter FC500 flow cytometer was used to collect data, and CXP or Kaluza software was used to analyze the data. Enzyme-linked immunosorbent assay (ELISA) kits were purchased from R&D systems (Minneapolis, MN) for HA, CXCL1, CXCL2, and granulocyte colony-stimulating factor (G-CSF). A Biotek EL808 spectrophotometer was used to collect data. Cell sorting for intracellular L. monocytogenes. For determination of the quantity of L. monocytogenes inside neutrophils, ⬃1 ⫻ 105 neutrophils (Ly6G⫹ CD11b⫹) were sorted from the liver using a BD Influx cell sorter. The sorted neutrophils were lysed with 0.1% Triton X-100 in ddH2O and

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plated to determine the number of CFU. The data are presented as the total number of L. monocytogenes CFU per 1 ⫻ 104 sorted neutrophils. Transfer studies. To investigate neutrophil recruitment, bone marrow cells were harvested from ecSOD HI and ecSOD KO mice and labeled with either 1 ␮M carboxyfluorescein succinimidyl ester (CFSE) (Invitrogen) or 5 ␮M eFluor 670 (eBioscience). The labeled bone marrow cells (4 ⫻ 106 per dye) from ecSOD HI and ecSOD KO mice were mixed at a ratio of 1:1 and intravenously (i.v.) injected into recipient ecSOD WT mice that were left uninfected or infected 1 day prior to injection. After ⬃18 h, splenocytes and liver leukocytes were isolated. The percentage of neutrophils labeled with CFSE or eFluor 670 was determined via flow cytometry. The percent recovery was calculated as follows: (total number of labeled neutrophils recovered/total number of labeled neutrophils injected) ⫻ 100. To investigate cell-intrinsic versus -extrinsic protection of neutrophils against L. monocytogenes infection, a neutrophil transfer study was performed. Three sets of ecSOD WT mice were infected with L. monocytogenes. One day postinfection (p.i.), neutrophils from the bone marrow of uninfected ecSOD HI and ecSOD KO mice were isolated using a Histopaque gradient (Sigma) (29). One set of the infected ecSOD WT mice received neutrophils (3.5 ⫻ 106) from ecSOD HI mice, one set received neutrophils (3.5 ⫻ 106) from ecSOD KO mice, and the third set received no neutrophils. The numbers of CFU were determined to assess the bacterial burdens in the spleens and livers of the three sets of ecSOD WT mice at day 3 post-L. monocytogenes infection (2 days post-neutrophil transfer). Measurement of phagocytosis and oxidative burst. Liver leukocytes (1 ⫻ 105) were added to a 96-well flat-bottom tissue culture plate and incubated at 37°C for 30 min in Dulbecco’s modified Eagle’s medium (DMEM). Next, pHrodo Escherichia coli bioparticle conjugates (Life Technologies), which fluoresce as the pH decreases from neutral to acidic, were prepared according to the manufacturer’s protocol and coated with mouse serum (1:10) for 10 min at room temperature. The cells were spun down, the supernatants were removed, and the pHrodo mixture was added to the wells. The cells were incubated at 37°C for 1 h and stained for Ly6G and CD11b for flow cytometric analysis. Hydroethidine (HE) was used to measure O2˙⫺ production (an indicator of oxidative burst). Liver leukocytes (2.5 ⫻ 105) were cultured in the presence of HE (16 ␮M) with heat-killed L. monocytogenes (HKLM) stimulation in a 96-well round-bottom plate at 37°C with 5% CO2. After 3 h, the cells were stained for Ly6G and CD11b for flow cytometric analysis. Statistical analyses. One- or two-way analysis of variance (ANOVA) was performed on the data to determine the statistical significance. Bonferroni t tests or Newman Keuls t tests were used for post hoc analyses. A P value of less than 0.05 was considered significant in all cases.

RESULTS

EcSOD-mediated recruitment of neutrophils to the liver is not cell intrinsic. We have previously shown that ecSOD enhances the recruitment of neutrophils to the liver in mice prior to and during L. monocytogenes infection (24). However, the mechanism by which ecSOD enhances neutrophil recruitment is not understood. Due to the fact that ecSOD activity increases the recruitment of neutrophils to the liver in the absence of infection, it is possible that there is a cell-intrinsic component to this ecSOD-enhanced neutrophil recruitment. To test this idea, a cotransfer experiment with eFluor 670- or CFSE-labeled bone marrow cells from ecSOD HI and ecSOD KO mice was performed (Fig. 1A shows a diagram of the experimental setup). An equivalent percentage of bone marrow neutrophils isolated from either ecSOD HI or ecSOD KO mice was coinjected into the ecSOD WT mice (Fig. 1B). It was found that neutrophils from the ecSOD HI and ecSOD KO mice were equally recruited to the livers of uninfected ecSOD WT mice (Fig. 1C). Additionally, the percentages of recovery of neutrophils (based upon the total number injected versus the number recov-

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ered) were also equivalent between the ecSOD HI and ecSOD KO mice (Fig. 1D). To investigate whether a cell-intrinsic difference in neutrophil recruitment exists during infection with L. monocytogenes, recovery was also determined at day 1 p.i., since all three groups of mice have equivalent numbers of CFU at this time point. When bone marrow cells were transferred into ecSOD WT recipients that had been infected with L. monocytogenes 1 day prior to transfer, there were similar rates of recovery of ecSOD HI and ecSOD KO neutrophils (Fig. 1D). Therefore, these data indicate that the ecSOD-dependent regulation of neutrophil recruitment into the liver is not cell intrinsic but depends on the in vivo environment both prior to and during L. monocytogenes infection. EcSOD activity protects the ECM from degradation in both the absence and presence of L. monocytogenes infection. Because ecSOD did not confer a neutrophil-intrinsic effect on recruitment, the impact of ecSOD activity on the in vivo environment was investigated. The interaction between CD44 and HA is vital for the recruitment of neutrophils, especially into the liver (7). CD44-HA interactions are also required for the ability of exogenous CXCL2 to recruit neutrophils in vivo (8). Furthermore, there is evidence to suggest that CD44-HA interactions are important for the trafficking of stem cells in the bone marrow (30), though the impact of CD44-HA interactions on neutrophil recruitment out of the bone marrow is not known. Consequently, these CD44-HA interactions could play a large role in the ability of neutrophils to migrate out of the bone marrow and into the liver. Several approaches were taken to determine if ecSOD activity had an impact on CD44-HA interactions. First, it is possible that ecSOD activity could influence the expression of CD44 on neutrophils or impact the ability of these neutrophils to effectively bind to HA. Interestingly, no large differences existed in the neutrophil-specific expression of CD44, as the percentages of neutrophils expressing CD44 and the mean fluorescence intensities (MFI) of CD44 were similar in all three groups of mice in the liver, blood, and bone marrow both prior to and during L. monocytogenes infection (see Fig. S1A to D in the supplemental material). Additionally, to determine whether ecSOD impacts the ability of neutrophils to bind to HA, neutrophils were incubated with fluorescein-labeled HA and the percentage of neutrophils that bound the HA-fluorescein conjugate was determined via flow cytometry. Interestingly, ecSOD does not alter the percentage of neutrophils from the liver, blood, or bone marrow that bind HA or the amount of HA bound by the neutrophils (as assessed by MFI) (see Fig. S1E to H in the supplemental material), decreasing the likelihood of either of these mechanisms accounting for the ecSOD-dependent neutrophil recruitment. Second, it is possible that ecSOD activity could impact the integrity of the HA itself. It has been shown that ecSOD activity protects tissues during noninfectious inflammation in the lung (20–22, 31–35). However, whether ecSOD activity protects the ECM from degradation in the basal state or during infection-induced inflammation has not been thoroughly investigated. To determine the impact of ecSOD activity on the protection of the ECM from degradation, the concentrations of HA in liver and bone homogenates were determined. Interestingly, ecSOD activity is positively correlated with high basal levels of HA in the liver (Fig. 2A) and bone (Fig. 2B). Furthermore, in agreement with the data from uninfected mice, it was found that ecSOD HI mice have the highest concentrations of HA in the liver (Fig. 2C) and bone (Fig. 2D) at day 1 p.i. These data indicate that ecSOD activity protects the ECM components from degradation

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ecSOD Enhances Immature Neutrophil Recruitment

FIG 1 EcSOD-mediated recruitment of neutrophils to the liver is not cell intrinsic. (A) Bone marrow cells from uninfected ecSOD HI and ecSOD KO mice were isolated and labeled with CFSE or eFluor 670. The labeled cells were then injected into uninfected ecSOD WT mice or ecSOD WT mice infected with L. monocytogenes for 1 day. (B and C) Flow cytometry was used to determine the percentages of labeled neutrophils (Ly6G⫹ CD11b⫹) prior to transfer (B) and in the liver 18 h posttransfer (C). (D) The percentages of neutrophils that were recovered, based on the number injected, were determined in the spleen and liver. Two-way ANOVA was unable to detect significant differences between groups. The data are combined from two independent experiments with reciprocal labeling of ecSOD HI and ecSOD KO bone marrow cells. All the data are expressed as means and standard errors of the mean (SEM) (n ⫽ 8 or 9/group).

both prior to and during L. monocytogenes infection, which likely impacts the ability of neutrophils to extravasate out of the bone marrow and be recruited to the liver via CD44-HA interactions. EcSOD activity enhances the production of neutrophil-attracting chemokines. Although CD44-HA interactions are necessary for proper recruitment of neutrophils into the inflamed liver, chemokines are also essential for neutrophil migration, particularly during infection. To explore the role of ecSOD on chemokine expression, an RT-PCR array was performed that included many chemokines known to attract neutrophils. At day 1 p.i., in the ecSOD KO liver, there were 23 genes with decreased mRNA content and 3 genes with increased mRNA content compared with the ecSOD HI liver (see Fig. S2A and B in the supplemental material for scatter plots). Interestingly, the majority of the genes that were regulated more than 2-fold during L. monocytogenes infection are associated with the recruitment of inflammatory cells. In fact, the mRNA contents of the chemokines CCL2, CCL3, CCL8, CCL11, CCL12, CCL17, CCL20, CXCL2, CXCL3, and CXCL5; the chemokine receptors CCR4, CXCR1, and CXCR4; and the p19 subunit of the cytokine IL-23 were increased in the livers of ecSOD HI mice (see Fig. S2A in the supplemental material). Additionally, a greater-than-200-fold downregulation of lymphotoxin alpha (LTA) mRNA in ecSOD KO mouse livers was observed compared to

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ecSOD HI mice. However, an ELISA performed to determine protein concentrations of LTA indicated no differences between the ecSOD congenic mice (data not shown). Two important neutrophil-attracting chemokines are CXCL1 and CXCL2. Interestingly, CXCL1 and CXCL2 mRNA contents were increased by ⬃1.5-fold (data not shown) and ⬃6-fold (see Fig. S2A in the supplemental material), respectively, in the ecSOD HI mice. Therefore, protein levels of CXCL1 and CXCL2 were measured in the sera and livers of ecSOD HI, ecSOD WT, and ecSOD KO mice to confirm the results of the RT-PCR array. One day p.i., ecSOD KO mice have the lowest production of CXCL1 and CXCL2 in the serum and liver during L. monocytogenes infection (Fig. 3A to D). G-CSF has been shown to induce the production of CXCL1 and CXCL2 (36, 37). Therefore, the concentration of G-CSF was measured in the serum. EcSOD KO mice had decreased levels of G-CSF in the circulation compared to the other groups of mice at day 1 p.i. (Fig. 3E). These results suggest that ecSOD activity may enhance the recruitment of neutrophils into the circulation via the production of CXCL1, CXCL2, and G-CSF. EcSOD-mediated neutrophil protection in the liver is not cell intrinsic. We have previously demonstrated that neutrophils from the livers of ecSOD HI mice are unable to provide protection against L. monocytogenes, while those from ecSOD KO and ecSOD

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FIG 2 EcSOD activity protects the ECM from degradation. EcSOD HI, ecSOD WT, and ecSOD KO mice were left uninfected (A and B) or infected with L. monocytogenes for 1 day (C and D). The hyaluronan content was measured in liver (A and C) and bone (B and D) homogenates. One-way ANOVA detected significant differences between groups. The asterisks indicate that the groups differ significantly (*, P ⬍ 0.05; **, P ⬍ 0.01). The data shown in panels A, B, and D are from single experiments (n ⫽ 3 to 5/group), while the data shown in panel C are combined from two independent experiments (n ⫽ 6 to 8/group). All the data are expressed as means and SEM.

WT mice are protective (24). Figure 1 shows that the differences in neutrophil recruitment to the liver are not cell intrinsic but are, instead, dependent on the environment. However, the functional status of these neutrophils could vary based on ecSOD expression. Therefore, in an effort to determine if the differences in the abilities of neutrophils to protect against L. monocytogenes are cell intrinsic, neutrophils were purified from the bone marrow of ecSOD HI and KO mice and injected into ecSOD WT mice that had been infected with L. monocytogenes for 1 day. Three days p.i., the numbers of CFU in the spleen and liver were determined (Fig. 4A shows a diagram of the experimental setup). As expected, there was no significant protection offered by neutrophils in the spleen, as it has been found that neutrophils play a limited role against L. monocytogenes infection in the spleen at this dose (25). Infected ecSOD WT mice that received transferred neutrophils had decreased bacterial burdens compared to infected control ecSOD WT mice that did not receive additional neutrophils, suggesting that the transferred neutrophils were still functional and able to protect against L. monocytogenes in the liver. Interestingly, it was found that transferred neutrophils from either ecSOD HI or ecSOD KO mice were able to confer similar protection (⬃l-logunit decrease in CFU) in the livers of L. monocytogenes-infected ecSOD WT mice (Fig. 4B). This is in contrast to what we observed in the L. monocytogenes-infected ecSOD HI mice, in which there was a decrease in numbers of CFU upon depletion of neutrophils, indicating that the neutrophils in the ecSOD HI liver are not protective (24). This strongly suggests that there is not a cell-intrinsic defect in the ability of ecSOD HI neutrophils to protect against L. monocytogenes. Instead, alterations that occur in the tissues due to varying levels of ecSOD activity influence the protective ability of neutrophils in the liver during infection.

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EcSOD activity decreases intracellular and cell membranebound MPO in neutrophils. Despite the plasticity of neutrophils displayed in the neutrophil transfer experiments shown in Fig. 4, the effects of ecSOD in the environment on neutrophil function required further investigation. Depletion of neutrophils from the three groups of ecSOD congenic mice showed that neutrophils in the livers of ecSOD HI mice are not beneficial, while neutrophils in the livers of ecSOD KO mice are hyperfunctional, producing more TNF-␣ (24). MPO is produced during early stages of neutrophil development and stored in the primary granules of mature neutrophils. Upon activation of the neutrophil, degranulation occurs and MPO is released into the phagosome to convert H2O2 into hypochlorous acid, an essential component of bacterial killing (38, 39). Neutrophil-specific MPO production was measured to further evaluate the effects of ecSOD on liver neutrophil function. As expected, it was found that ⬃100% of the neutrophils express intracellular MPO in all three groups of mice (data not shown). Importantly, however, neutrophils from ecSOD KO mice have a higher MFI for MPO than neutrophils from ecSOD HI mice both in an uninfected state (Fig. 5A to C shows representative histograms, and Fig. 5D shows a graph) and at day 1 p.i. (Fig. 5E). These data show that ecSOD activity decreases the intracellular concentration of MPO in neutrophils, suggesting that the neutrophils from the ecSOD KO mice are more mature and activated. Interestingly, it has also been shown that human neutrophils release MPO, which binds to surface-expressed CD11b and CD18, allowing intracellular signaling to occur. This, in turn, activates the neutrophils to become more functional, as measured by increased degranulation and oxidative burst (40). There was no difference in neutrophil-specific expression of CD11b prior to or during L. monocytogenes infection (see Fig. S3A, B, and C in the

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FIG 3 EcSOD activity increases the production of neutrophil-attracting chemokines. EcSOD HI, ecSOD WT, and ecSOD KO mice were infected for 1 day with L. monocytogenes. The concentrations of CXCL1 (A and B), CXCL2 (C and D), and G-CSF (E) were determined by ELISA in the sera (A, C, and E) or liver homogenates (B and D). One-way ANOVA detected significant differences between groups. The asterisks indicate that the groups differ significantly (*, P ⬍ 0.05; **, P ⬍ 0.01). The data in panels B and E are representative of two independent experiments (n ⫽ 3 to 5/group), while the data in panels A, C, and D are combined from two independent experiments (n ⫽ 7 to 10/group). All the data are expressed as means and SEM.

supplemental material). Additionally, there were no differences in neutrophil-specific expression of CD18 prior to or during L. monocytogenes infection in the livers of ecSOD congenic mice (see Fig. S3D and E in the supplemental material). However, there was a greater percentage of liver neutrophils in the ecSOD KO mice than of neutrophils from ecSOD HI mice that had surface-bound MPO both in an uninfected state (Fig. 5I) and during infection with L. monocytogenes for 1 day (Fig. 5F to H shows representative histograms, and J shows a graph). These data indicate that ecSOD activity decreases neutrophil expression of intracellular and extracellular MPO and that neutrophils from the ecSOD KO mice are more activated and better able to contain an L. monocytogenes infection. EcSOD activity slightly increases the ability of liver neutrophils to undergo phagocytosis. Neutrophils take up pathogens via phagocytosis; however, the ability of L. monocytogenes to effectively infect cells depends on uptake via a vesicle, upon which L. monocytogenes escapes from the phagocytic vesicle and replicates within the cytoplasm (41). To measure the phagocytic potential of neutrophils from ecSOD congenic mice, pHrodo E. coli bioparticle conjugates, which fluoresce only when there is a drop in pH,

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were used. There was no difference between the groups of mice in the ability of liver neutrophils to take up the pHrodo E. coli bioparticle conjugates, as ⬃100% of the neutrophils were positive for the fluorescent signal (data not shown). However, there was a slight increase in the phagocytic potential of neutrophils from ecSOD HI mice, as measured by MFI (Fig. 6A). This is counterintuitive, which led us to speculate that there may be a defect in the ability of neutrophils from the ecSOD HI mice to effectively eradicate the bacteria once phagocytosed. EcSOD activity increases the number of intracellular L. monocytogenes CFU in neutrophils in vivo. To determine if ecSOD activity was limiting the ability of neutrophils to kill intracellular L. monocytogenes, liver neutrophils were sorted from mice infected with L. monocytogenes for 1 day or 3 days, lysed, and plated to determine the numbers of cell-associated CFU. Studies performed after infection with L. monocytogenes for 1 day resulted in CFU numbers below the limit of detection. However, at day 3 postinfection, it was found that neutrophils from ecSOD HI mice were associated with a higher number of L. monocytogenes CFU than ecSOD KO neutrophils, which were not associated with any viable L. monocytogenes (Fig. 6B). These data suggest that ecSOD

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FIG 4 EcSOD-mediated neutrophil protection in the liver is not cell intrinsic. (A) Neutrophils from the bone marrow of uninfected ecSOD HI and ecSOD KO mice were isolated using a Histopaque gradient. The neutrophils (3.5 ⫻ 106) were then injected into ecSOD WT mice that had been infected with L. monocytogenes 1 day prior to the transfer. (B) CFU were used to determine the bacterial burdens in the spleens and livers of ecSOD WT mice at day 3 after L. monocytogenes infection. Two-way ANOVA detected significant differences between groups. The asterisks indicate that the groups differ significantly (*, P ⬍ 0.05; **, P ⬍ 0.01). These data are representative of two independent experiments. All the data are expressed as means and SEM (n ⫽ 3 to 5/group).

activity hampers the ability of neutrophils to effectively kill phagocytosed L. monocytogenes. EcSOD activity decreases the ability of neutrophils to undergo oxidative burst. Oxidative burst is one of the main ways in which neutrophils kill pathogens, including L. monocytogenes (42). Furthermore, it has been shown that the binding of MPO to surface CD11b and CD18 on neutrophils increases the ability of neutrophils to undergo oxidative burst (40). HE, a redox-sensitive dye (43), used in combination with flow cytometric analysis indicated that liver neutrophils from uninfected ecSOD KO mice were better able to undergo oxidative burst than neutrophils from ecSOD HI mice (Fig. 6C to E shows representative histograms, and Fig. 6F shows a graph). These data suggest that ecSOD activity decreases the ability of neutrophils to effectively undergo oxidative burst. DISCUSSION

Our data show that ecSOD activity induces the recruitment of neutrophils by enhancing the production of neutrophil-attracting chemokines and by protecting the ECM from degradation, not via a neutrophil-intrinsic mechanism. Perhaps due to more intact ECM providing increased binding sites for adhesion molecules and chemokines, ecSOD activity may facilitate the premature release of immature neutrophils from the bone marrow and increased recruitment of functionally impaired neutrophils into the

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liver. In the presence of ecSOD, neutrophils express less MPO and are less able to generate oxidative burst, indicating decreased functionality and maturity. Interestingly, even though ecSOD activity slightly enhances the ability of neutrophils to phagocytose bioparticles, it decreases the ability of the neutrophils to effectively kill L. monocytogenes, likely due to the observed decrease in oxidative burst. It is likely that a mechanism by which ecSOD activity enhances the recruitment of neutrophils into the liver is due to the differences in HA content in the liver, as CD44-HA interactions are essential for neutrophil recruitment into the organ (7). Furthermore, binding interactions between chemokines and the ECM are very important for neutrophil recruitment. Positively charged chemokines will bind to the negatively charged sulfates on heparan sulfate or other glycosaminoglycans that are part of the ECM of endothelial cells. Once these chemokines are bound to the ECM, neutrophils cross the endothelial lining through binding of chemokine receptors and the necessary adhesion molecules (4). Therefore, since ecSOD activity protects against degradation of the ECM, this would increase the binding sites available for chemokines. Intriguingly, we have yet to discover a mechanism by which ecSOD activity increases the production of neutrophil-attracting chemokines. It has been shown that H2O2, the product of ecSOD, can enhance the production of IL-17 (44), though it is not known if H2O2 can increase downstream effector chemokines like

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FIG 5 EcSOD activity decreases neutrophil-specific intracellular and surface-bound MPO. EcSOD HI, ecSOD WT, and ecSOD KO mice were left uninfected (A to D and I) or infected with L. monocytogenes for 1 day (E to H and J). (D and E) Liver neutrophils were gated based on their expression of Ly6G and CD11b. The MFI of neutrophils that stained positive for intracellular MPO was determined. (I and J) The percentage of neutrophils that stained positive for surface-bound MPO was also determined. (A to C) Representative histograms of intracellular MPO staining are shown for uninfected neutrophils. (F to H) Representative histograms of surface-bound MPO staining are shown for neutrophils from mice infected with L. monocytogenes for 1 day. One-way ANOVA detected significant differences between groups. The asterisks indicate that the groups differ significantly (*, P ⬍ 0.05; **, P ⬍ 0.01; ***, P ⬍ 0.001). These data are representative of two independent experiments. All the data are expressed as means and SEM (n ⫽ 4 or 5/group).

CXCL1 and CXCL2. Nonetheless, it is possible that the increased production of chemokines in the ecSOD HI mice is due to the increased availability of H2O2. Furthermore, our data demonstrate that there is not an ecSOD-dependent cell-intrinsic defect in the ability of neutrophils to be recruited to the liver. Therefore, our results suggest that the environment is the most important factor in mediating ecSOD-dependent neutrophil recruitment to the liver. Our data showing that ecSOD activity enhances the recruitment of neutrophils to the liver contrast with studies regarding ecSOD activity in the lung. In several noninfectious inflammatory lung disease models (aerosolized LPS, oil fly ash, hemorrhage, hyperoxia, cigarette smoke, and asbestos) it has been shown that ecSOD decreases neutrophil recruitment, potentially by decreasing overall inflammatory responses and cytokine/chemokine pro-

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duction (18, 20–22, 45, 46). Similar to the lung inflammatory models, our current and previous studies (24) demonstrate that ecSOD activity decreases inflammatory responses. Differences in the amounts of ecSOD produced, the nature of the organ studied, and the nature of the inflammation could explain differences in the role of ecSOD in neutrophil recruitment. Interestingly, in a lung E. coli infection model, it was recently shown that ecSOD augments bacterial killing and reduces the lung bacterial burden, although this effect was attributed to alterations in macrophage, not neutrophil, function in the ecSOD KO mice (47). Further studies are required to elucidate the roles of ecSOD in different organs under different inflammatory and infectious conditions. It has been shown that actin rearrangement, which is regulated by ROS, is essential for the movement of immune cells. It has also been recently established that ROS are necessary for the glutathio-

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FIG 6 EcSOD activity slightly increases phagocytosis of neutrophils while decreasing intracellular L. monocytogenes killing and oxidative burst. EcSOD HI, ecSOD WT, and ecSOD KO mice were left uninfected (A and C to F) or infected with L. monocytogenes (LM) for 3 days (B). (A) Liver leukocytes were cultured in the presence of serum-opsonized pHrodo E. coli bioparticle conjugates for 1 h to determine phagocytic potential. The MFI of pHrodo⫹ neutrophils was determined by flow cytometry. (B) Neutrophils (⬃1 ⫻ 105) were sorted from mice infected with L. monocytogenes for 3 days, lysed, and plated on BHI plates, and the number of intracellular L. monocytogenes CFU per 1 ⫻ 104 neutrophils was determined. (C to F) Liver leukocytes (2.5 ⫻ 105) were cultured in the presence of hydroethidine and HKLM for 3 h. (F) The percentages of neutrophils that stained positive for hydroethidine were determined by flow cytometry. (C to E) Representative histograms of hydroethidine staining are shown. One-way ANOVA detected significant differences between groups. The asterisks indicate that the groups differ significantly (*, P ⬍ 0.05; **, P ⬍ 0.01). The data are representative of two independent experiments. All the data are expressed as means and SEM (n ⫽ 3 to 5/group).

nylation of globular actin (G-actin). However, the G-actin needs to be deglutathionylated in order to be polymerized into filamentous actin (F-actin) (48). The actin rearrangement is necessary, not only for recruitment of neutrophils, but also for the phagocytosis of pathogens (49). By altering the relative levels of ROS, ecSOD may lead to an alteration in the ability of neutrophils to respond to a stimulus. The present data indicate that ecSOD activity increases the ability of neutrophils to be recruited to the liver and to phagocytose pHrodo bioparticles. However, our previous data demonstrated that neutrophils from ecSOD HI mice did not effectively colocalize with L. monocytogenes lesions in the liver (24). Therefore, it is possible that actin dynamics are altered by ecSOD. The decreased oxidative burst observed in neutrophils from ecSOD HI mice may suggest that ecSOD is being taken up during phagocytosis, as has been observed previously in other cell types (50, 51). If this were occurring, the availability of ecSOD in the

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phagosome would allow the rapid conversion of O2˙⫺ into H2O2 when the neutrophil is undergoing oxidative burst. As a result, the neutrophils from the ecSOD HI mice would be less able to effectively kill L. monocytogenes. Indeed, the data shown here indicate that ecSOD HI neutrophils are less able to undergo oxidative burst, suggesting that the internalization of ecSOD is a possible mechanism. However, this has not been thoroughly studied and deserves further investigation. Polymorphisms in the ecSOD gene have been linked to several diseases in humans, including type 2 diabetes, acute lung injury, ischemic heart disease, chronic obstructive pulmonary disease, and preeclampsia (52–56). However, the impact of ecSOD on the susceptibility of the human population to pathogenic infections has not been studied. Furthermore, the functional ability of neutrophils from populations with ecSOD polymorphisms has not been assessed. One might expect that loss of ecSOD activity would actually increase the functional ability of neutrophils and confer

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greater protection against a pathogen while also increasing tissue and organ damage due to oxidative stress. However, the side effects that are possible upon neutralization of ecSOD might be acceptable if the therapy protects a patient from an otherwise lethal infection. Future studies are required to dissect the beneficial versus protective effects of ecSOD, or lack thereof, during infectious and inflammatory diseases in humans.

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ACKNOWLEDGMENTS We thank Herenia Armenta, Mariano Aufiero, and Jessica M. F. Hall for technical assistance. Flow cytometry was performed in the Flow Cytometry and Laser Capture Microdissection Core Facility at the University of North Texas Health Science Center. This work was supported by a grant from NIH/NIAID (AI109630) to R.E.B., a BioLegend grant to R.E.B. and T.J.B., Intramural seed grants from UNTHSC to R.E.B. and T.J.B., a Sigma Xi Grant-In-Aid of Research grant to T.J.B., and an AAI Careers in Immunology fellowship to R.E.B. and A.R.W.

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