M-CSF Mediates Host Defense During Bacterial Pneumonia by Promoting

M-CSF Mediates Host Defense during Bacterial Pneumonia by Promoting the Survival of Lung and Liver Mononuclear Phagocytes This information is current as of September 23, 2021. Alexandra Bettina, Zhimin Zhang, Kathryn Michels, R. Elaine Cagnina, Isaah S. Vincent, Marie D. Burdick, Alexandra Kadl and Borna Mehrad J Immunol published online 4 May 2016 http://www.jimmunol.org/content/early/2016/05/03/jimmun Downloaded from ol.1600306 http://www.jimmunol.org/ Why The JI? Submit online.

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2016 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published May 4, 2016, doi:10.4049/jimmunol.1600306 The Journal of Immunology

M-CSF Mediates Host Defense during Bacterial Pneumonia by Promoting the Survival of Lung and Liver Mononuclear Phagocytes

Alexandra Bettina,* Zhimin Zhang,† Kathryn Michels,* R. Elaine Cagnina,† Isaah S. Vincent,‡ Marie D. Burdick,† Alexandra Kadl,†,x and Borna Mehrad*,†,{

Gram-negative bacterial pneumonia is a common and dangerous infection with diminishing treatment options due to increasing antibiotic resistance among causal pathogens. The mononuclear phagocyte system is a heterogeneous group of leukocytes com- posed of tissue-resident macrophages, dendritic cells, and monocyte-derived cells that are critical in defense against pneumonia, but mechanisms that regulate their maintenance and function during infection are poorly deﬁned. M-CSF has myriad effects on

mononuclear phagocytes but its role in pneumonia is unknown. We therefore tested the hypothesis that M-CSF is required for Downloaded from mononuclear phagocyte-mediated host defenses during bacterial pneumonia in a murine model of infection. Genetic deletion or immunoneutralization of M-CSF resulted in reduced survival, increased bacterial burden, and greater lung injury. M-CSF was necessary for the expansion of lung mononuclear phagocytes during infection but did not affect the number of bone marrow or blood monocytes, proliferation of precursors, or recruitment of leukocytes to the lungs. In contrast, M-CSF was essential to survival and antimicrobial functions of both lung and liver mononuclear phagocytes during pneumonia, and its absence resulted

in bacterial dissemination to the liver and hepatic necrosis. We conclude that M-CSF is critical to host defenses against bacterial http://www.jimmunol.org/ pneumonia by mediating survival and antimicrobial functions of mononuclear phagocytes in the lungs and liver. The Journal of Immunology, 2016, 196: 000–000.

neumonia caused by aerobic Gram-negative bacilli is yolk sac–derived and self-renewing tissue-resident macro- among the most common and dangerous nosocomial phages, Flt3 ligand–dependent pre–dendritic cell (DC)-derived P infections and an important cause of death, prolonged cells, and monocytes and their progeny (3). The latter lineage, hospital stay, and increased healthcare costs. Gram-negative bacilli derived from circulating monocytes that express high levels of the colonize the upper aerodigestive tract as part of the normal response surface glycoprotein Ly6C in mice, can differentiate into diverse to acute illness and cause pneumonia when introduced into the populations of mononuclear phagocytes depending on environ- by guest on September 23, 2021 lower respiratory tract by microaspiration (1, 2). The progressive mental cues: under homeostatic conditions, mouse Ly6Chi mono- rise in antibiotic resistance among the causative organisms in cytes terminally differentiate into endothelial-patrolling Ly6Clo recent decades has markedly diminished the available treatment counterparts in the blood and alsogiverisetosomeshort-lived options for these infections, lending new impetus to mechanistic populations of tissue macrophages. In response to tissue injury, studies that may inform the development of better therapeutic Ly6Chi monocytes extravasate into the damaged tissue and differen- strategies. tiate into inflammatory macrophages, repopulate some tissue-resident The mononuclear phagocyte system encompasses heteroge- macrophage populations, and differentiate into monocyte-derived neous populations of cells from three distinct lineages, namely inflammatory DC that are phenotypically similar to, but tran- scriptionally distinct from, CD11b+ pre-DC–derived conven- *Department of Microbiology, Immunology, and Cancer Biology, University of Vir- tional DC (4). The mouse lung, in particular, contains at least five ginia, Charlottesville, VA 22908; †Division of Pulmonary and Critical Care, Department of Medicine, University of Virginia, Charlottesville, VA 22908; ‡Division of Nephrol- populations of mononuclear phagocytes under homeostatic con- ogy, Department of Medicine, University of Virginia, Charlottesville, VA 22908; xDe- ditions, namely the tissue-resident and long-lived alveolar mac- partment of Pharmacology, University of Virginia, Charlottesville, VA 22908; and { rophages, short-lived monocyte-derived interstitial macrophages, Beirne B. Carter Center for Immunology, University of Virginia, Charlottesville, VA + + 22908 CD11b conventional DC, CD103 airway DC, and plasmacytoid hi lo ORCID: 0000-0001-6285-574X (A.K.). DC (5). In the context of injury, Ly6C and Ly6C monocytes, Received for publication February 22, 2016. Accepted for publication April 4, 2016. monocyte-derived DC, and macrophages enter the lungs and res- This work was supported by National Institutes of Health Grants HL098329 and ident cells display accelerated efflux and death, comprising a AI117397. complex and dynamic network. A.B., M.D.B., A.K., and B.M. conceived and designed experiments; A.B., Z.Z., K.M., M-CSF was the first cytokine described for its induction of R.E.C., I.S.V., M.D.B., A.K., and B.M. performed experiments; A.B., I.S.V., and A.K. macrophage colonies from bone marrow progenitors in soft agar analyzed data; I.S.V. and A.K. contributed reagents and analysis tools; A.B. and B.M. wrote the paper; and all authors revised and approved the text. (6, 7) and is a key regulator of the development and homeostasis Address correspondence and reprint requests to Dr. Borna Mehrad, University of the mononuclear phagocyte system (8–10). In different exper- of Virginia, P.O. Box 800546, Charlottesville, VA 22908. E-mail address: mehrad@ imental conditions, M-CSF has been shown to mediate diverse virginia.edu functions in the differentiation, proliferation, survival, and effector Abbreviations used in this article: 7-AAD, 7-aminoactinomycin D; DC, dendritic functions of multiple cell types from different mononuclear phago- cell. cyte lineages (11). In addition, thesolereceptorforM-CSF,CSF1R, Copyright Ó 2016 by The American Association of Immunologists, Inc. 0022-1767/16/$30.00 or CD115 is shared with a structurally unrelated ligand, IL-34 (12).

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1600306 2 M-CSF IN BACTERIAL PNEUMONIA

M-CSF and IL-34 have distinct tissue expression patterns and, at (clone X54-5/7.1), anti-CD103–allophycocyanin (clone 2E7), anti-CD115 least in the development of osteoclasts, microglia, and skin Lang- (clone AFS98), anti-CD117-FITC (clone 2B8), anti-CD135–BV421 (clone erhans cells, have nonredundant roles (13). Most recently, protein A2F10.1), anti-F4/80–Pacific Blue (clone BM8), anti-F4/80-PE (clone T45- z 2342), Fc block (anti-CD16/CD32), anti–I-A/I-E–FITC or AmCyan (clone tyrosine phosphatase- was described as a second receptor for IL-34 M5/114.15.12), Ki-67–PE and isotype control (clones B56 and MOPC-21), (14, 15). anti-Ly6C–PE or Pacific Blue (clone HK1.4), anti-Ly6G–PE-Cy7 (clone Evidence from multiple experimental systems support a critical 1A8), anti-NK1.1–PE-Cy7 (clone PK136), and anti–plasmacytoid DC role for mononuclear phagocytes in host defenses but the mech- Ag-1–allophycocyanin (clone 129c1). In some experiments, cells were labeled, fixed, and permeabilized using a commercial kit (Cytofix/Cytoperm; anisms that control the development, homing, and survival of these BD Biosciences) before staining for intracellular Ags. Data were acquired cells during specific infections are incompletely defined. In the on an FACS Canto II instrument using BD FACSDiva software (version context of bacterial pneumonia, monocytes are recruited to the 8.0; BD Biosciences) and analyzed using FlowJo software (version 8.8.6; lungs via a CCR2-dependent mechanism, and both recruited and Tree Star, Ashland, OR). Leukocyte subsets were identified as previously resident mononuclear phagocytes mediate host defenses by direct described (4, 17, 33) with minor modifications, as depicted in the figures. k The absolute number of each cell type was determined as the product of killing of bacteria and NF- B–mediated recruitment of other the percentage of the cell type and the total number of cells in the leukocytes (16–20). In this context, we previously defined the sample, as determined on an automated cell counter (Countess; Invitrogen, dynamic changes in the various mononuclear phagocyte pop- Carlsbad, CA). ulations during experimental Gram-negative pneumonia and ELISA and assays for alanine transaminase, aspartate reported that CCR2 was necessary not only to the recruitment of aminotransferase, urea nitrogen, and lactate monocyte-derived lineage cells but, unexpectedly, also controlled the number and phenotype of alveolar macrophages and all DC ELISAs for M-CSF and IL-34 were performed according to the manu- Downloaded from facturer’s instructions (R&D Systems, Minneapolis, MN). Albumin ELISA populations in this infection (17). Little is known about the role of was performed using 5 mg/ml goat anti-mouse albumin (A90-134A), 0.025 the M-CSF/IL-34 biologic axis in the context of infection, and mg/ml goat-HRP detection (A90-134P), mouse reference serum (rs10-101) many of the published studies in the field predate current under- (Bethyl Laboratories; Montgomery, TX), and 1-Step Ultra TMB-ELISA standing of the lineages of mononuclear phagocytes (21–25). To (Thermo Fisher Scientific). Plasma alanine transaminase, aspartate ami- our knowledge, this mechanism has not been investigated in notransferase, and urea nitrogen were measured using commercial kits (Liquid ALT Reagent Set and Liquid AST Reagent Set; Pointe Scientific, pulmonary infections to date. We therefore sought to define the Canton, MI; and DetectX Urea Nitrogen Detection Kit; Arbor Assays, Ann http://www.jimmunol.org/ role of this biological axis in pneumonia by testing the hypothesis Arbor, MI). Plasma lactate was measured using a commercial device that M-CSF is required for mononuclear phagocyte-mediated host (Lactate Plus Meter; Nova Biomedical, Waltham, MA). defense during Klebsiella pneumonia. In vitro studies Bone marrow cells were cultured as described to generate macrophages Materials and Methods (34). In viability assays, macrophages were washed and resuspended in 3 5 Animals and in vivo procedures DMEM with 10% FCS without antibiotics, and 1 10 cells were cultured overnight in opaque flat-bottom 96-well tissue-culture plates (BD Falcon, We used a previously characterized model of experimental bacterial pneu- Franklin Lakes, NJ). Cells were then incubated with or without 5 ng monia (26, 27). C57BL/6 and op/+ mice, purchased (The Jackson Laboratory, M-CSF for 2 h before the addition of K. pneumoniae. After 150 min, plates by guest on September 23, 2021 Bar Harbor, ME), were bred and maintained under pathogen-free conditions were washed with sterile HBSS, and viability was measured using a and in compliance with institutional animal care regulations. Age- and sex- commercial kit (LIVE/DEAD viability/cytotoxicity kit for mammalian matched6-to8-wk-oldmicewereusedinallexperiments.Klebsiella cells; Invitrogen Molecular Probes, Eugene, OR). For phagocytosis assays, pneumoniae strain 43816 (American Type Culture Collection, Manassas, VA) bacteria were labeled with a pH-sensitive cyanine dye that acquires fluo- was grown overnight at 37˚C to midlog phase in tryptic soy broth. Bacteria rescence in acidic environments (35, 36): 1 3 105 macrophages were seeded were administered intratracheally as previously described (17). Unless other- into round-bottom tissue culture–treated plates (Costar, Corning, NY) and in- wise noted, all mice received i.p. injections of 100 mg/100 ml anti–M-CSF– cubated with or without M-CSF as above. Midlog phase K. pneumoniae cells neutralizing Ab (clone 5A1), anti-CSF1R–neutralizing Ab (clone AFS98), were suspended at 1 3 109/ml in sterile saline, fixed in 5 ml 70% ethanol for IgG1 isotype control (clone HRPN), or IgG2A isotype control (clone 2A3) 5 min, washed, and resuspended in sterile HBSS; CypHer5E dye (GE Health- beginning 2 h before intratracheal inoculation and then every 24 h through care Life Sciences, Pittsburgh, PA) was added at a final concentration of 5 mmol day 2 postinfection (all from BioXCell, West Lebanon, NH). In some ex- before incubation at room temperature for 30 min. Bacteria were then washed, periments, mice received three i.p. injections of 2 mg BrdU (BD Biosciences, and 1 3 108 bacteria were added to wells containing macrophages. Plates were SanJose,CA)in0.2mlPBS,every3huntil 2 h before bacterial inoculation, centrifuged for 30 s to achieve cell contact and incubated at 37˚C and 5% CO2 as described (28). At designated time points, mice were euthanized with an for 30 min, then placed on ice, washed in cold HBSS, and analyzed immediately injection of ketamine and xylazine. by flow cytometry. Positive gating was set using an unstained control. Tissue harvest Statistical analysis Blood was collected in heparinized syringes from the right ventricle. Lung Data were analyzed using Prism statistical software (version 5.0d; and liver vasculature were perfused with 3 ml PBS with 2 mM EDTA before GraphPad Software, San Diego, CA). Data from survival experiments were excision. Blood and homogenized lungs and right liver lobes were serially analyzed using the log-rank test. Values between two groups over multiple diluted and cultured on blood agar plates for quantification of bacterial time points were compared with two-way ANOVA. Comparisons between content. Bronchoalveolar lavage was performed as previously described two groups at a single time were performed using the nonparametric Mann– (26). Histology was performed on lungs and left liver lobes as previously Whitney U test. Comparisons between multiple groups at a single time described (26, 29). In other experiments, whole lungs, left liver lobes, and point were performed using the Kruskal–Wallis nonparametric test with plasma were processed for ELISAs as described (27). Dunn comparison posttest. Comparison of paired samples receiving different treatments was made using Wilcoxon matched-pairs signed-rank test Flow cytometry or two-way repeated-measures ANOVA. The p values ,0.05 were con- sidered statistically significant. Flow cytometry was performed as previously described (26, 30, 31). Cell suspensions of whole lungs, median liver lobes, bone marrow, and peripheral blood were prepared as previously described (27, 29, 32). The following reagents were used to label cells for flow cytometry (from BD Biosciences, Results San Jose, CA; eBioscience, San Diego, CA; or BioLegend, San Diego, CA): Role of M-CSF during pulmonary infection with 7-aminoactinomycin D (7-AAD), anti-CD3–PE-Cy7 (clone 17A2), anti- K. pneumoniae CD11b–allophycocyanin-Cy7 (clone M1/70), anti-CD11c–PE-Cy7 (clone HL3), anti-CD19–PE-Cy7 (clone 1D3), anti-CD24–allophycocyanin (clone We began by measuring the concentration of M-CSF and IL-34 protein M1/69), anti-CD45–PerCP or AmCyan (clone 30-F11), anti-CD64–BV421 in whole-lung homogenates during experimental pneumonia induced The Journal of Immunology 3 by K. pneumoniae. Both ligands were detectable in uninfected lungs. As compared with animals challenged with intratracheal saline vehicle, the concentration of M-CSF increased ∼2-fold as the infection progressed but the concentration of IL-34 did not change significantly (Fig. 1A, 1B). In contrast, the concentration of plasma M-CSF protein remained undetectable in plasma throughout the time course, suggesting that M-CSF was produced locally in the lungs during infection. Given these findings, we focused our studies on the role of M-CSF during Klebsiella pneumonia. Downloaded from http://www.jimmunol.org/

FIGURE 2. CSF1R is required for host defense in pneumonia. Con- centration of circulating monocytes (A) and lung mononuclear phagocytes (B) in uninfected mice after administration of daily CSF1R Ab or isotype control for 7 d using the gating strategy depicted in Fig. 3A; n = 4 to 5 per group (*p , 0.05, Mann–Whitney). (C) Outcome of pneumonia in animals treated with anti-CSFR1 or isotype control Ab; n = 23–24 per group from two experiments (**p , 0.01, log-rank test). (D) Bacterial burden in lungs by guest on September 23, 2021 of animals with pneumonia treated with anti-CSFR1 or isotype control Ab on day 3 of infection. Data from two experiments. Samples with no recoverable bacteria are depicted as 1 CFU. *p , 0.05, Mann–Whitney. Mw, macrophages.

To assess the contribution of M-CSF during bacterial pneumonia, we next examined the outcome of animals deficient in M-CSF during infection. Mice homozygous for an inactivating mutation in the M-CSF locus (op/op) had notably increased mortality during experimental pneumonia as compared with littermate controls, with no animals surviving beyond 2 d; this was associated with increased incidence and severity of bacteremia on the first day of the infection (Fig. 1C, 1D). The role of M-CSF during development of the mononuclear phagocyte system results in a number of well- documented phenotypic abnormalities in op/op mice, including FIGURE 1. M-CSF level increases in the lungs of mice with pneumonia and is required for host defense. Time series of mean 6 SEM of lung reduced number of alveolar macrophages in juvenile, but not adult, M-CSF (A) and IL-34 (B) protein in mice infected with K. pneumoniae; animals and spontaneous production of matrix metalloproteinases n = 5 to 6 per time point per group from two experiments. Time 0 repre- by mutant alveolar macrophages (37). To discriminate between the sents uninfected animals. ****p , 0.0001, two-way ANOVA. (C) Out- role of M-CSF induction during bacterial pneumonia and its role come of pneumonia in op/op mice and littermate controls. n = 9 to 10 per during development, we tested the effect of immunoneutralization group from two experiments (*p , 0.05, log-rank test). (D) Bacterial of M-CSF starting at the onset of the infection. Wild-type mice burden in lungs and blood of infected op/op mice and littermate controls receiving anti–M-CSF–neutralizing Ab had increased mortality, on day 1 of infection. Each data point represents one animal, and horizontal increased lung bacterial burden, and markedly increased incidence , lines indicate medians. Data from two experiments (*p 0.05, Mann– and severity of bacteremia as compared with animals receiving an Whitney). (E) Outcome of pneumonia in animals treated with anti–M-CSF or isotype control Ab (Fig. 1E, 1F). We found similar results with isotype control Ab; n = 23 to 24 per group from two experiments (**p , 0.01, log-rank test). (F) Bacterial burden in lungs and blood of animals with antagonism of CSF1R during the infection. The administration of a pneumonia on day 3 of infection in animals treated with anti–M-CSF or neutralizing CSF1R Ab to uninfected animals did not influence the hi isotype control Ab. n = 17–19 per group; data shown are representative of number of blood Ly6C monocytes or lung mononuclear phago- lo two experiments (**p , 0.01, Mann–Whitney). In (D)and(F), samples with cytes but lead to a reduction in blood Ly6C monocytes (Fig. 2A, no recoverable bacteria are depicted as 1 CFU on the logarithmic scale. 2B), consistent with prior reports that, in steady state, M-CSF 4 M-CSF IN BACTERIAL PNEUMONIA

FIGURE 3. M-CSF is required for the maintenance of lung mononuclear phagocytes during pneumonia. (A) Representative ﬂow cytometry plots showing gating strategy for lung leukocytes. Downloaded from Cells were enumerated in single-cell suspensions from whole lung. (A–J) Time-series of mean 6 SEM of indicated lung leukocyte populations; n = 20–24 per time point per group from four experiments. Time 0 represents uninfected animals. *p , 0.05, **p , 0.01, ***p , 0.001, ****p , 0.0001, two-way ANOVA. cDC, conventional CD11b+ http://www.jimmunol.org/ DCs; FSC, forward light scatter; Mw, macrophages; MHC II, MHC class II; PDCA1, plasmacytoid DC Ag-1; SSC, side scatter. by guest on September 23, 2021

mediates differentiation of blood Ly6Chi monocytes to their Ly6Clo but not other DC subsets or neutrophils (Fig. 3E–J), similar to our counterparts (38, 39). In the context of infection, administration of prior report in mice with CCR2 deficiency (17). anti-CSF1R Ab resulted in a similar increase in mortality and To understand how the M-CSF–CSF1R axis contributes to the bacterial burden as noted with M-CSF neutralization (Fig. 2C, 2D). numbers of lung mononuclear phagocytes during Klebsiella pneu- These data indicate that the M-CSF–CSF1R interaction is necessary monia, we reasoned that the observed reduction in lung monocyte- for host defenses during bacterial pneumonia independent of their derived cells, and possibly alveolar macrophages and conventional role in development. CD11b+ DC, may be attributable to impairments in monocytopoeisis, trafficking between the bone marrow, blood, and lung compartments, Mechanism of M-CSF–mediated host defense during or survival of leukocytes that have arrived in the lungs. We found that pneumonia neutralization of M-CSF had no effect on the concentration of Ly6Chi To begin to address how M-CSF mediates its beneficial effects and Ly6Clo monocyte numbers in bone marrow or blood (Fig. 4A, during bacterial pneumonia, we examined the effect of M-CSF 4B). Consistent with this, we found no difference in the proliferation neutralization on lung mononuclear phagocyte subsets in the of bone marrow macrophage-DC progenitor or the recently described lungs in the context of pneumonia (Fig. 3A). M-CSF neutralization committed-monocyte progenitor (40) after neutralization of M-CSF resulted in a significant reduction in the numbers of cells of (Fig. 4C, 4D). These data indicate that M-CSF is dispensable for the monocyte lineage, namely Ly6Chi and Ly6Clo monocytes, generation of monocytes from progenitor cells in the bone marrow monocyte-derived macrophages, and monocyte-derived inflam- during bacterial pneumonia. matory DC, in the lungs of mice during pneumonia (Fig. 3B–D). M-CSF can be chemotactic for mononuclear phagocytes (41, In addition, M-CSF neutralization caused a reduction in the 42). To assess for this possibility, we pulsed uninfected mice with number of alveolar macrophages and conventional CD11b+ DC, the thymidine analog, BrdU, thereby labeling populations of The Journal of Immunology 5

FIGURE 4. M-CSF is dispensable for maintenance of bone marrow and blood monocytes and progenitor proliferation during pneumonia. (A and B) Time series of mean 6 SEM of indicated leukocyte populations in the blood and bone marrow. n = 10–12 from two experiments. (C) Representative flow plots of CD45+ bone marrow cells, showing gating strategy of bone marrow to identify progenitor cells. (D) Time series of mean 6 SEM of percent Ki67+ bone marrow monocyte progenitors. n = 6–12 per group per time point from two experiments. Time 0 represents uninfected animals. Downloaded from proliferating cells that were in S-phase at the time of the pulse and also assessed the survival of bone marrow–derived macrophages then quantified the number of labeled leukocytes in various coincubated with K. pneumoniae. As compared with cells incubated compartments after the onset of infection. As expected, we found with bacteria in the presence of M-CSF, monocyte-derived macro- fewer BrdU-positive bone marrow neutrophils and a marked in- phages incubated with bacteria in the absence of M-CSF had reduced crease in labeled neutrophils in the blood and lung of infected survival (Fig. 6B). In addition, the surviving monocyte-derived http://www.jimmunol.org/ mice compared with uninfected controls that was independent of macrophages had impaired phagocytosis of K. pneumoniae in the M-CSF neutralization (Fig. 5A–C). M-CSF neutralization also did absence of M-CSF (Fig. 6C), suggesting that M-CSF promotes the not influence the numbers of labeled Ly6Chi monocytes in the survival of mononuclear phagocytes during infection and also bone marrow, blood, or lungs during infection (Fig. 5A–C), sug- contributes to the antimicrobial functions of these cells inde- gesting that M-CSF is also dispensable for the recruitment of lung pendent of its effect on the number of leukocytes. monocyte-derived cells during pneumonia. We next assessed the role of M-CSF on survival of mononuclear Role of M-CSF in controlling liver injury during bacterial phagocytes during infection, because the M-CSF–CSF1R axis can pneumonia by guest on September 23, 2021 mediate survival of mononuclear phagocytes under homeostatic In the course of the above studies, we noticed that M-CSF neutral- conditions (43). We addressed this question by measuring the ization during pneumonia was associated with gross visual evidence proportion of dead cells in the bronchoalveolar lavage fluid of of liver injury. To investigate this, we assessed the effect of M-CSF infected animals to minimize the artifact of ex vivo cell death that neutralization on the histology of lung and liver during experimental inevitably occurs during processing of samples. M-CSF neutrali- pneumonia. The lung histology did not differ between pneumonic zation resulted in a significant increase in the proportion of dead mice treated with anti–M-CSF and isotype controls; in contrast, Ly6Chi monocytes in the lungs of infected animals (Fig. 6A), M-CSF neutralization during pneumonia resulted in large areas of potentially implicating M-CSF as a survival factor for lung sharply demarcated hepatocellular necrosis, associated with monocyte-derived cells during pneumonia. To directly examine the prominent hepatocyte microvesicles (Fig. 7A). Consistent with role of M-CSF in mediating monocyte survival during infection, we this, neutralization of M-CSF resulted in elevations in plasma

FIGURE 5. M-CSF is dispensable to the inﬂux of mononuclear phagocytes during pneumonia. Mean 6 SEM of BrdU-positive leukocyte populations in the bone marrow (A), blood (B), and lungs (C). n = 6–12 per group per time point from two experiments. *p , 0.05, ***p , 0.001, one-way ANOVA with Dunn posttest. 6 M-CSF IN BACTERIAL PNEUMONIA

in the bronchoalveolar lavage fluid (Fig. 8A). In contrast to its effect on the liver, however, M-CSF neutralization did not affect renal function, as measured by the concentration of blood urea nitrogen, nor cause significant end-organ hypoperfusion, as quantified by plasma lactate concentration (Fig. 8B, 8C), suggesting that M-CSF neutralization results in specific injury to the liver in addition to the lungs. In order to investigate the contribution of M-CSF in the liver during bacterial pneumonia, we assessed the liver levels of M-CSF protein, because the plasma concentrations of M-CSF were un- FIGURE 6. M-CSF is required for the survival and function of mononuclear phagocytes during pneumonia. (A) Percentage of 7-AAD+ lung detectable during the infection. The liver contained high concen- Ly6Chi monocytes on day 3 of infection. n = 6 per group from two ex- trations of M-CSF protein at baseline that did not change periments (*p , 0.05, Mann–Whitney). (B) Effect of M-CSF on viability significantly during the infection (Fig. 9A). We therefore tested the of bone marrow–derived macrophages incubated with or without Klebsi- hypothesis that, similar to our findings in the lungs, liver M-CSF ella in vitro, as measured in relative fluorescence units (RFU). Each data mediates the survival of liver mononuclear phagocytes during the point represents an independent biological replicate paired to its respective infection, leading to better clearance of bacteria from the liver. control (**p , 0.01, two-way repeated-measures ANOVA). (C) Effect of Consistent with this and analogous to the lungs, we found that M-CSF on phagocytosis of CypHer5E-labeled Klebsiella by bone marrow– liver monocyte-derived macrophages and F4/80+CD11b2 resident derived macrophages. Each data point represents an independent biological Kupffer cells were reduced in number with M-CSF neutralization Downloaded from replicate paired to its respective control (*p , 0.05, Wilcoxon matched- during pneumonia (Fig. 9B, 9C). Also similar to our findings in pairs signed-rank test). the lungs, M-CSF neutralization resulted in increased death of liver Ly6Chi monocytes during pneumonia (Fig. 9D). Taken to- concentrations of aspartate transaminase and alanine transami- gether, these data indicate that M-CSF is also required for nase during pneumonia (Fig. 7B). Because M-CSF neutralization mononuclear phagocyte-mediated defense in the liver during resulted in increased incidence and extent of bacteremia (Fig. 1), bacterial pneumonia. http://www.jimmunol.org/ we next assessed whether the observed hepatocellular injury was associated with increased hepatic bacterial content after the liver Discussion had been perfused to displace its blood content. We found that, The mononuclear phagocyte system has been implicated in host in mice with pneumonia, M-CSF neutralization resulted in in- defense against bacterial pneumonia (16, 17, 44, 45), but the creased liver bacterial burden (Fig. 7C), potentially explaining mechanisms that generate and maintain these cells during the the increased liver injury. To assess whether the hepatocellular infection have not been defined. In the current study, we report injury is merely a reflection of multiorgan dysfunction in the that M-CSF, but not IL-34, is induced in the lung during the in- context of sepsis, we also quantified other parameters of end- fection. M-CSF was essential to the survival of the host as a organ dysfunction. As expected, M-CSF neutralization resulted critical survival signal for mononuclear phagocytes in infected tis- by guest on September 23, 2021 in increased lung injury, as measured by albumin concentration sues, but appeared to be dispensable for the generation, recruitment,

FIGURE 7. M-CSF is required for controlling bacterial dissemination and secondary liver damage during pneumonia. (A) H&E stain of lung (top panels) and liver sections (middle and bottom panels) on day 3 of infection in animals treated with anti–M-CSF or isotype control Ab. Top and bottom panels, Scale bars 40 mm, original magnification 3400; middle panels, scale bars 200 mm, original magnification 340. Representative data from three mice per group. Top panels show alveolar infiltration by acute inflammatory cells. Middle panels show areas of necrosis with anti–M-CSF treatment (bright pink areas). Bottom panels show central hepatic veins and surrounding lobule; the hepatocytes in mice treated with anti–M-CSF show prominent microvesicles. (B) Time series of mean 6 SEM of plasma aspartate aminotransferase (AST) and alanine aminotransferase (ALT) during pneumonia, as measured in IUs per liter. n = 10–12 from two experiments. *p , 0.05, **p , 0.01, two-way ANOVA. (C) Bacterial burden in the right lobe of the liver in mice treated with anti–M-CSF or isotype control Ab. Each data point represents one animal, and horizontal lines indicate medians; samples with no recoverable bacteria are depicted as 1 CFU on the logarithmic scale. Data from two experiments (**p , 0.01, Mann–Whitney). The Journal of Immunology 7

An unexpected finding in our study was the role of M-CSF in protecting the liver from injury during experimental pneumonia. The propensity of Gram-negative bacteria, specifically Klebsiella, to infect the liver is recognized clinically (60). In the context of experimental pneumonia, the liver is responsible for a protective acute-phase response, but is also susceptible to TNF-mediated injury (61–63). M-CSF has recently been shown to mediate proliferation of liver macrophages and recruitment of monocytes to the injured liver (64). Our data add to this literature, indicating FIGURE 8. M-CSF neutralization causes lung injury but not renal that endogenous production of M-CSF during pneumonia medi- failure or tissue hypoperfusion during pneumonia. Time series of mean 6 ates the accumulation of both monocyte-derived macrophages and SEM of bronchoalveolar lavage (BAL) albumin concentration (A), blood Kupffer cells and protects the liver from disseminated infection. urea nitrogen (BUN; B), and plasma lactate (C) in mice with pneumonia, The present work has several implications for future research. treated with anti–M-CSF or isotype Ab. n = 8–12 per group per time point First, our data suggest that M-CSF is required not only for main- from two experiments. Time 0 represents uninfected animals. *p , 0.05, taining the number of monocyte-derived cells, but also alveolar two-way ANOVA. macrophages and CD24+ [and thus pre-DC–derived (4)] CD11b+ DC in the lungs as well as Kupffer cells in the liver. This finding is and differentiation of mononuclear phagocytes during the infection. reminiscent of our prior report in CCR2-deficient mice (17) and

The absence of M-CSF thus resulted in reduced numbers of both suggests either that Ly6Chi monocytes repopulate other lineages of Downloaded from monocyte-derived cells and resident macrophage populations in the lungs and the liver, resulting in increased bacterial burden and injury to these organs and increased incidence and extent of bacteremia. M-CSF is produced by many cell types, including mononuclear phagocytes themselves, and has been documented to have a mul-

titude of effects on mononuclear phagocytes in different experi- http://www.jimmunol.org/ mental conditions, including development during embryogenesis, monocytopoeisis in adulthood, proliferation, differentiation, che- motaxis, and survival (as reviewed in Ref. 11). M-CSF has not been studied extensively in the context of infections, but has been associated with host defense in intracellular infections caused by Listeria monocytogenes (22, 46) and influences macrophage phenotype in experimental tuberculosis and schistosomiasis (47, 48). We report that M-CSF appeared to be dispensable for the proliferation and recruitment of monocytes and their progenitors during pneumonia, by guest on September 23, 2021 similar to recent findings in sterile peritonitis (39). Our studies did not directly examine the role of M-CSF in the differentiation of mononuclear phagocytes in the lungs in vivo, but the intact recruitment of monocytes to the lungs, together with the role of M-CSF in maintaining the numbers of both lung monocytes and monocyte-derived cells during infection indicates that the differentiation of monocytes into other mononuclear phagocytes during pneumonia remained intact despite M-CSF neutralization. In contrast, M-CSF was essential in maintaining lung and liver monocytic phagocyte populations by promoting their survival. M-CSF has been shown to be critical to survival of mononuclear phagocytes during development and under homeostatic conditions (49–52), but, to our knowledge, has not been shown to mediate mononuclear phagocyte survival in the setting of infection or inflammation. In this context, macrophage survival is a known regulatory factor in the context of infection: alveolar macrophage apoptosis, for example, is both antimicrobial and anti-inflammatory during pneumococcal pneumonia (53, 54), whereas macrophage necroptosis induces lung damage during Gram-negative and S. aureus pneumonia (55, 56). In addition, we found that M-CSF mediates enhanced phagocytosis FIGURE 9. M-CSF is required for the maintenance of mononuclear of bacteria by surviving mononuclear phagocytes. In this context, phagocytes in the liver during pneumonia. (A) Time series of mean 6 SEM macrophages from op/op animals have been shown to have impaired of M-CSF protein in the left liver lobe in mice with pneumonia. n = 6–16 phagocytosis against bacteria but not parasites (57, 58), but it is per time point per group from two experiments. (B) Representative flow C difficult to differentiate whether any effect in op/op macrophages is cytometry plots showing gating strategy for Kupffer cells. ( ) Time series of mean 6 SEM of indicated mononuclear phagocytes in the median attributable to developmental abnormalities of the cells or lack of liver lobe. Monocyte-derived macrophages were identified as depicted exposure to M-CSF during acute infection. Similar to our findings, in Fig. 3A. n = 10–12 per group per time point from two independent however, M-CSF results in enhanced phagocytosis of fungal conidia experiments. ***p , 0.001, two-way ANOVA. (D) 7-AAD–positive Ly6Chi by human monocyte-derived macrophages in vitro (59). Taken to- liver monocytes on day 3 of infection; data from two independent experi- gether, these data suggest that locally produced M-CSF can enhance ments (*p , 0.05, Mann–Whitney). Time 0 represents uninfected animals. antimicrobial properties of mononuclear phagocytes in target organs. Mw, macrophages; SSC, side scatter. 8 M-CSF IN BACTERIAL PNEUMONIA mononuclear phagocytes in the acute setting, as has been shown 21. Bernier, T., R. Halter, D. Pau, S. Rittinghausen, and A. Emmendo¨rffer. 2001. M-CSF transgenic mice: role of M-CSF in infection and autoimmunity. Exp. previously (9, 65), or that M-CSF is necessary to the maintenance of Toxicol. Pathol. 53: 165–173. all three lineages of mononuclear phagocytes in the setting of acute 22. Gregory, S. H., E. J. Wing, D. J. Tweardy, R. K. Shadduck, and H. S. Lin. 1992. injury. Second, we found M-CSF to have a marked role in the ac- Primary listerial infections are exacerbated in mice administered neutralizing lo antibody to macrophage colony-stimulating factor. J. Immunol. 149: 188–193. cumulation of Ly6C monocytes in the lungs, but any role of these 23. Doyle, A. G., W. J. Halliday, C. J. Barnett, T. L. Dunn, and D. A. Hume. 1992. cells in the setting of acute infection remains undefined. Third, our Effect of recombinant human macrophage colony-stimulating factor 1 on immu- work documents the critical role of M-CSF during bacterial pneu- nopathology of experimental brucellosis in mice. Infect. Immun. 60: 1465–1472. 24. Kayashima, S., S. Tsuru, N. Shinomiya, Y. Katsura, K. Motoyoshi, monia but does not preclude an additional contribution for IL-34 or M. Rokutanda, and N. Nagata. 1991. Effects of macrophage colony-stimulating its second receptor, protein tyrosine phosphatase-z, in the context of factor on reduction of viable bacteria and survival of mice during Listeria pneumonia. Finally, our findings may be relevant to efforts to inhibit monocytogenes infection: characteristics of monocyte subpopulations. Infect. Immun. 59: 4677–4680. M-CSF signaling as a therapeutic strategy in cancer (66), because 25. Cenci, E., A. Bartocci, P. Puccetti, S. Mocci, E. R. Stanley, and F. 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