sign in sign up
<<
Home , Macrophage

The Journal of

Acute-Phase Deaths from Murine Polymicrobial Are Characterized by Innate Immune Suppression Rather Than Exhaustion

Evan L. Chiswick,* Juan R. Mella,† John Bernardo,‡ and Daniel G. Remick*

Sepsis, a leading cause of death in the United States, has poorly understood mechanisms of mortality. To address this, our model of cecal ligation and puncture (CLP) induced sepsis stratifies mice as predicted to Live (Live-P) or Die (Die-P) based on plasma IL-6. Six hours post-CLP, both Live-P and Die-P groups have equivalent peritoneal bacterial colony forming units and recruitment of . By 24 h, however, Die-P mice have increased bacterial burden, despite increased recruitment, suggesting Die-P phagocytes have impaired bacterial killing. Peritoneal cells were used to study multiple bactericidal processes: bacterial killing, reactive species (ROS) generation, and . Total phagocytosis and intraphagosomal processes were deter- mined with triple-labeled , covalently labeled with ROS- and pH-sensitive probes, and an ROS/pH-insensitive probe for normalization. Although similar proportions of Live-P and Die-P phagocytes responded to exogenous stimuli, Die-P phagocytes showed marked deficits in all parameters measured, thus suggesting immunosuppression rather than exhaustion. This contradicts the prevailing sepsis paradigm that acute-phase sepsis deaths (<5 d) result from excessive inflammation, whereas chronic-phase deaths (>5 d) are characterized by insufficient inflammation and immunosuppression. These data suggest that suppression of cellular innate in sepsis occurs within the first 6 h. The Journal of Immunology, 2015, 195: 3793–3802.

epsis is an immune-mediated immune disorder costing so- teases (6). Similarly, macrophages and inflammatory ciety approximately $24 billion annually (1), and claiming phagocytize microbes and process them in a manner similar to S .200,000 lives per year (2). Despite causing a similar endosomal cargo, ultimately fusing with and digesting number of lives lost as cancer, there are few treatment options the via pH-sensitive proteases (7). As the first responders available. Currently, patients receive aggressive treatment that in an , they are central to the initiation of sepsis. includes fluid resuscitation, vasopressors, and broad spectrum Cecal ligation and puncture (CLP) induced peritonitis in (3). Despite these interventions, postmortem studies outbred mice produces a spectrum of inflammation ranging from revealed the majority of patients still had infectious foci present a rampant inflammatory response that is thought to become (4), thus suggesting a deficit in bacterial clearance. dysregulated, leading to immune paralysis, bacterial overgrowth, and macrophages comprise the phagocytic arm of and death (8). At the other end of the spectrum is a tightly theinnateimmunesystemlargely responsible for eradicating regulated response that clears without inducing host a bacterial infection. Following infection, tissue macrophages damage. Studies have shown that altering leukocyte recruitment engage the and secrete distress factors to recruit (9) or enhancing leukocyte function results in decreased bac- neutrophils and inflammatory monocytes. Neutrophils help to terial burden (10), increased organ perfusion (11, 12), and ul- neutralize the infection by secreting neutrophil extracellular timately increased survival. However, the majority of these traps (NETs) (5) and/or by phagocytizing microbes and expos- studies compare sham-operated animals to CLP operated. Al- ing them to reactive oxygen species (ROS) and cationic pro- though this approach helps define the host response to sepsis, it may be less appropriate for delineating the mechanisms of sepsis *Department of Pathology and Laboratory Medicine, Boston University School of mortality. Medicine, Boston, MA 02118; †Department of , Boston University Medical Center, Boston, MA 02118; and ‡Division of Pulmonary, , and Critical Care Previousstudieshaveshownthatwithin6hofCLPinmice, Medicine, Boston University Medical Center, Boston, MA 02118 circulating biomarkers can be used to accurately predict mortality Received for publication April 14, 2015. Accepted for publication August 3, 2015. during the acute phase of sepsis (13). This powerful capability This work was supported in part by National Institutes of Health Grants enables stratified interventions to assess efficacy (14, 15), and it T32AI007309, T32HL007501, R01GM97320, T32GM86308, and R21AI112887. provides a window of time in which to observe the detrimental E.L.C. performed experiments and analyzed data; J.R.M. cultured bacteria from divergence between survivors and nonsurvivors. This approach and prepared data; J.B. provided equipment, reagents, technical expertise, and intellectual support; and E.L.C. and D.G.R. conceived the study and revealed that similar peritoneal bacterial CFUs and wrote the manuscript. recruitment occurs 6 h after CLP. However by 24 h, nonsurvivors Address correspondence and reprint requests to Dr. Daniel G. Remick, Department of recruited significantly more phagocytes, yet they also showed Pathology and Laboratory Medicine, Boston University School of Medicine, 670 Albany Street, Room 407, Boston, MA 02118. E-mail address: [email protected] significantly increased bacterial burden (16). We hypothesized that phagocytes from nonsurvivors were The online version of this article contains supplemental material. impaired in their bacterial killing capacity. If the cell function was Abbreviations used in this article: CLP, cecal ligation and puncture; DHR-123, dihy- drorhodamine 1,2,3; Fsc, forward light scatter; MDSC, myeloid-derived suppressor reduced, the host attempted to correct by increasing the numbers cell; NET, neutrophil extracellular trap; NOX, NADPH oxidase; OpH–E. coli, opson- of phagocytic cells. The current study examined whether reduced ized pHrodo–labeled E. coli; ROS, reactive oxygen species; TB, trypan blue. cellular killing of bacteria occurs in mice predicted to die and Copyright Ó 2015 by The American Association of Immunologists, Inc. 0022-1767/15/$25.00 define the mechanisms of this impairment. www.jimmunol.org/cgi/doi/10.4049/jimmunol.1500874 3794 ACUTE-PHASE SEPSIS DEATHS FROM IMMUNOSUPPRESSION

Materials and Methods Basal ROS and ROS burst kinetics Animals Peritoneal cells were added to white-opaque microplate wells in duplicate at 3 6 m Female ICR (CD-1) mice (Harlan-Sprague Dawley, South Easton, MA) 24– a concentration of 4 10 /ml. Luminol was present at 20 M. Cells were 28 g were used for all studies. Mice were received and acclimated to our placed in a temperature-controlled fluorescent plate reader (Tecan Infinite housing room for at least 96 h prior to surgery and cared for as described m1000) and warmed to 37˚C for 5 min. Stimulus was then added (100 nM PMA or 20:1 bacteria:cell), and chemiluminescence was measured every previously (16). The experiments were approved by the Boston University ∼ Animal Care and Use Committee. 10 s (0.5-s integration time) followed by mechanical shaking. 3 Sepsis model 3 -Labeled bacteria preparation Labeling was performed as published previously (23). Briefly, 2 3 1010/ml Cecal ligation and puncture was performed as first described (17) with heat-killed (80˚C at 1 h) HB101 E. coli were sequentially labeled with minor modifications (16, 18). Approximately two-thirds the length of the 500 mM dihydrodichlorofluorescein-succinimidyl ester to detect ROS, cecum from the distal tip was ligated and then double punctured longi- 50 mM pHrodo-SE to detect pH changes, and 159 mM Alexa Fluor-350-SE tudinally with 16-gauge needle. Mice were resuscitated with 1 ml saline to calibrate DCF/pHrodo fluorescence (Life Technologies). Labeling was (37˚C) with buprenorphine (0.05 mg/kg) for pain management (one in- performed in PBS (pH 9), degassed, and purged with N to minimize auto jection every 12 h for 2 d). Antibiotics (25mg/kg imipenem) were ad- 2 oxidation of DCF. Bacteria (∼1.5 3 1010/ml) were opsonized with 1/10th ministered 2 h post-CLP and then once every 12 h for 5 d. volume of normal mouse plasma (heparinized) and anti–E. coli Abs m Sampling (50 g/ml) (Life Technologies). The use of particles covalently labeled with multiple fluorescent indicators/substrates provides temporal, spatial, sampling was performed initially at 6 and 24 h to generate IL-6 and calibrated information for phagocytosis and phagosomal events that receiver operator characteristic curves and discrimination levels for pre- are not readily available with most cell permeable dyes. These approaches dicting survival. Blood (20 ml) was collected by facial vein puncture and have been validated in previous publications (24, 25). diluted 1/10 in PBS containing 3.38 mM EDTA tripotassium salt. Blood was centrifuged for 5 min at 1000 3 g, and the plasma was frozen at 220˚C. Phagocytosis and phagosomal ROS/acidification of For mice that were sacrificed, blood was collected from the retro-orbital 33-labeled bacteria venous plexus under anesthesia (ketamine/xylazine), followed by eutha- Peritoneal cells were loaded into polypropylene cryovials at 4 3 106/ml. nasia via cervical dislocation. Plasma collected at 6 and/or 24 h was an- ∼ alyzed for IL-6 concentrations by ELISA as described previously (19). While on ice, 150 bacteria/cell were added to cells. Tubes were trans- ferred to a water bath at 37˚C, placed on a stir plate, and agitated at lowest The peritoneal cavity was lavaged as described previously (16). Cell 3 pellets were resuspended in 2 ml wash buffer (PBS + 0.5% BSA) and 100 speed for 30 min with 7 2-mm micro stir bars. For controls, samples ml retained for total cell counts and cytospins. The remaining cell volume were also incubated on ice for 30 min. Following incubation, cells were was underlain with 2 ml 30% sucrose (w/v) in PBS and centrifuged for kept on ice until data acquisition with an LSRII flow cytometer (BD 8 min at 450 3 g to remove extracellular bacteria (20). The supernatant Biosciences). containing bacteria was aspirated, and the cell pellet was resuspended in Ice controls were acquired first. Approximately 30,000 events were 3 ml wash buffer. One milliliter of 100% isotonic Percoll in PBS was collected to measure attachment. Then, trypan blue (TB) was added to quench extracellular fluorescence (0.25% final concentration) and incubated added to the cell volume to generate a 25% Percoll solution. This was ∼ underlain with 1 ml Histopaque 1.119 g/ml and then centrifuged for 25 min on ice for 1 min, and then, 30,000 gated events were collected. Cells that at 500 3 g. The viable leukocytes were located at the Percoll Histopaque were allowed to phagocytize were only acquired with TB present. Non- debris cells were gated on by Fsc/side scatter (of light). Doublets were interface. Light debris was aspirated from the 25% Percoll layer, dead cells + were removed from the pellet by pipette (21), and the remaining viable removed by Fsc-h versus Fsc-w. Phagocytosis gates were constructed for cells were washed once, followed by resuspension in HBSS containing each sample based off its own ice control (no phagocytosis occurs). Ca2+ and Mg2+ + 1% BSA. Cells were enumerated with a Beckman- DCF, pHrodo, and Alexa Fluor 350 fluorescence was excited by 488-, Coulter particle counter model ZF (Coulter electronics, Hialeah, FL). 561-, and 355-nm lasers, respectively, and their emission was collected by (wavelength/band pass) 530/15, 590/10, and 450/50 filters, respectively. It Peritoneal bacterial quantification was important to use a custom 590/10 filter for pHrodo because TB autofluoresces when bound to protein and its emission begins at ∼615 nm. One hundred microliters of the first milliliter of lavage fluid was serially Data were analyzed with Flowjo (Tree Star). Derived parameters diluted with HBSS and cultured on 5% sheep blood agar plates (Fisher (i.e., calibrated fluorescence) were constructed so that DCF or pHrodo Scientific) for 24 h at 37˚C in anaerobic or aerobic conditions, followed by fluorescence for each event was divided by the fluorescence of Alexa 350 for CFU enumeration. that event. The ROS index was calculated by dividing the DCF/Alexa ratio of the cell by the DCF/Alexa ratio of the bacteria. Similar calculations were Bacterial killing by peritoneal cells performed for the acidification index. Streptomycin-resistant Escherichia coli (E. coli strain HB101) were grown Statistical analysis to log phase in tryptic soy broth containing streptomycin. Bacteria were opsonized 20 min at 37˚C with normal mouse plasma, added to peritoneal Statistics were performed using Prism 5 software (Graphpad Software, San cells (4 3 106/ml) at a 5:1 microbe:cell, and then incubated for 1 h at 37˚C. Diego, CA). For comparison between two groups, an unpaired Student t test Killing was stopped by lysis with H2O (pH 11) (22). Serial dilutions were was used. For survival analysis, a Kaplan–Meier curve and log-rank sur- cultured overnight on streptomycin-infused tryptic soy agar plates to vival were performed with a 95% confidence interval. All values were prevent contamination. The percentage of bacteria killed = (CFU sample + expressed as mean 6 SE. bacteria)/(bacteria alone).

Neutrophil and macrophage ROS burst Results Characterization of CLP-induced sepsis: similar beginning, ROS was measured by the conversion of nonfluorescent dihydrorhodamine dissimilar fate 1,2,3 (DHR-123) to fluorescent R-123. Cells (4 3 106/ml) were loaded with 2 mM DHR-123 and stimulated with opsonized heat-killed E. coli (20:1 To study the mechanisms of mortality, our laboratory uses a murine bacteria:cell) or 100 nM PMA for 30 min at 37˚C, then chilled on ice, model of peritoneal sepsis induced by CLP. This model, which followed by extracellular marker staining for flow cytometry. The ROS includes fluid resuscitation and treatment, produces burst was calculated by the percentage increase of R-123 (geometric mean ∼ fluorescence intensity) of stimulated cells over unstimulated cells. Cellular 50% mortality within the first 5 d of sepsis (acute phase). Mice FcRs were blocked with Fc Block (BD Biosciences). Cell viability was were subjected to CLP, a small sample of blood was collected at 6 determined with Sytox Blue stain (Life Technologies), added 5 min before and 24 h post-CLP, and the mice were followed for survival. acquisition. The following Abs were used: CD11b (clone M170), CD19 Monitoring plasma IL-6 levels of survivors/non-survivors shows (1d3), CD3e (145-2C11), Ly6G (1A8), Gr-1 (RB6-8C5), F480 (Ci-A13), and Ly6C (HK1.4). Only Sytox Blue negative events (Live Cells) were that mice that succumb to sepsis have significantly elevated IL-6 used for gating. Doublet discrimination was performed by forward light 24 h post-CLP (Fig. 1A) as well as at 6 h post-CLP (data not scatter (Fsc)-A versus Fsc-H. shown). Receiver operator characteristic curves were used to The Journal of Immunology 3795

FIGURE 1. Phagocyte dysfunction precedes death from CLP-induced sepsis. CLP was performed to induce ∼50% mortality in ICR CD1- outbred mice. Plasma was collected at 24 h post-CLP, and animals were monitored for survival. The mice who died in the first 5 d had significantly higher plasma levels of IL-6 (A). ROC curve analysis was used to generate IL-6 values predictive for mortality. None of the mice with IL-6 . 3.13 ng/ml survived (B). Peritoneal bacterial loads and cellular recruitment between Live-predicted/Die-predicted mice were compared following euthanasia at 6 or 24 h post-CLP. Total peritoneal cells (C) and bacterial CFUs (D). *p , 0.05, ***p , 0.0001 between the indicated groups. generate IL-6 discrimination values to stratify mice as predicted to toneal cells were carefully processed to remove excess bacteria and live (Live-P) or predicted to die (Die-P), as described previously dead cells, and the cells were then used in a bacterial killing assay. (13). This discrimination value accurately predicted survival This assay assessed the ability of the peritoneal cells to kill ex- (Fig. 1B). Using the discrimination value, mice were sacrificed in ogenous E. coli. Interestingly, at 6 h post-CLP when cellular re- subsequent experiments at 6 or 24 h post-CLP and posthumously cruitment and bacterial burden were similar, differences in cellular stratified into Die-P and Live-P. function were already present. When bacterial killing was exam- As reported previously by this laboratory, Die-P mice have ined using live E. coli, Die-P mice showed a marked reduction in similar numbers of cells in their peritoneum as compared with Live- bacteria killed compared with Live-P mice within 6 h post-CLP P mice at 6 h post-CLP (Fig. 1C). Similarly, there is no difference and this persisted through 24 h (Fig. 2). Previous studies have in the number of bacteria within the peritoneal cavity at 6 h post- shown that increased bacterial burden correlates with mortality CLP (Fig. 1D) (16). This demonstrates that our CLP model is (26–28) and that source control (removal of infectious foci) consistent, but more importantly, that both Live-P and Die-P increases survival (13). In conjunction with those studies, the groups were subjected to a similar initial bacterial insult with current data strongly suggest that the reduced bacterial killing a similar initial cellular response. Despite a nearly identical 6-h within the first 6 h of sepsis leads to subsequent mortality. response, by 24 h post-CLP Die-P, mice have increased peritoneal Mechanisms of reduced bacterial killing cells (Fig. 1C) and bacteria (Fig. 1D). The divergence from similar phagocyte recruitment and bacterial burden at 6 h post-CLP to To determine why Die-P peritoneal phagocytes kill fewer bacteria increased phagocytes and bacterial numbers at 24 h post-CLP than Live-P, bactericidal mechanisms were further examined. While suggests an early defect in phagocyte function in Die-P mice. multiple mechanisms are important in the clearance of pathogens, ROS generation is integral to the microbial killing process, as evi- Die-P phagocytes show impaired bactericidal activity denced by humans with chronic granulomatous disease, a condition To test the idea that impaired phagocyte function was responsible where patients are prone to recurrent bacterial infections because of for the increased bacterial burden observed in Die-P mice, peri- a mutation that renders the ROS producing NADPH oxidase (NOX) 3796 ACUTE-PHASE SEPSIS DEATHS FROM IMMUNOSUPPRESSION

Neutrophils were defined as CD11b+, LY6G Hi and monocytes/ macrophages were defined as CD11b+, LY6G lo/neg (Supplemental Fig. 1) (29, 30). Although the analyzed macrophage population expressed the classical macrophage marker F4\80, its vari- able level of expression in Die-P mice made it unsuitable for gating purposes (Supplemental Fig. 1G). Recent work has also shown that express F4\80, and inflammatory con- ditions induced by thioglyocollate result in decreased F4\80 expression by macrophages (31). This is likely due in part to newly recruited monocytes/macrophages that express little F4\80 (32). Distinguishing bactericidal activity between neutrophils and macrophages showed that both populations likely contributed to the impaired bacterial killing observed in Die-P peritoneal cells. Following incubation with OpH–E. coli, there is a marked increase in ROS production measured by R-1,2,3 fluorescence in Live-P neutrophils, whereas the histograms for stimulated/unstimulated FIGURE 2. Die-P mice exhibit impaired bacterial killing of E. coli at 6 Die-P neutrophils are virtually superimposable (Fig. 3A). Die-P and 24 h post-CLP. E. coli were incubated with or without peritoneal neutrophils produce significantly less ROS than their Live-P cells for 1 h and then plated overnight for CFU counts. Data from at least counterparts in response to OpH–E. coli within 6 h after induc- three independent experiments. n = 7–13/group at 6 h, 7–8/group at 24 h, tion of sepsis (Fig. 3B, left panel), and this deficiency persists , where N represents an individual mouse. ***p 0.0001 comparing the through 24 h post-CLP. The use of OpH–E. coli as a stimulus is indicated groups. physiologically relevant; it measures how a cell would likely re- spond in vivo when encountering cecal bacteria. This process complex inoperable. To assess the ROS burst, peritoneal cells were leads to ROS production subsequent to cell surface receptor li- loaded with DHR-1,2,3, stimulated with opsonized pHrodo–labeled gation and signaling. However, if there were decreased receptor E. coli (OpH–E. coli) or PMA, and analyzed by flow cytometry. expression, desensitization, or other means of blunted receptor Phagocytes consist primarily of neutrophils and macrophages. signaling, the ROS burst may fail to occur despite the ROS ma- To determine whether impairments in either cell type were re- chinery (e.g., NOX) remaining fully functional. To determine sponsible for the decreased bacterial killing, both neutrophil and whether the differences in ROS production were due to impaired macrophage function were distinguished through the use of cell- pattern recognition receptor signaling, or a decreased capacity to specific markers. generate ROS, PMA was used to stimulate the cells. PMA, as

FIGURE 3. Die-P peritoneal phagocytes have decreased ROS burst. (A) Live-P and Die-P peritoneal cells loaded with DHR-1,2,3 were incubated with OpH–E. coli or PMA to delineate physiological-response ROS or cellular ROS capacity via flow cytometry. Histograms show representative staining of PMN incubated with (red histogram) or without OpH–E. coli (blue). (B and C) Bar graphs show results from three independent experiments (n = 4–7 mice per group. (D and E) Representative ROS Kinetics for unstimulated (basal) (D) or Opsonized–E. coli (E) stimulated cells for Live-P and Die-P mice, as measured by Luminol based chemiluminescence (relative light unit [RLU]). (F) Bar graphs show the area under the curve (AUC) calculated for samples in unstimulated and stimulated conditions. (n = 3 mice/group). For all graphs, black filled symbols/bars represent Die-P mice and open symbols/bars represent Live-P mice. *p , 0.05, **p , 0.01, ***p , 0.001 between LIVE-P and DIE-P. The Journal of Immunology 3797 a nonspecific activator of ROS production, bypasses the require- harvested from the peritoneums of mice with extreme peritonitis. ment of pattern recognition receptor signaling for NOX activation. Although these cells are already stimulated, there were signifi- Following PMA stimulation, Die-P neutrophils were shown to be cantly different responses at 6 h post-CLP, a time at which bac- capable of generating ROS, albeit significantly less so than Live-P terial loads (i.e., preassay stimulation) were similar (Fig. 1D). A at both time points (Fig. 3B, right panel). Similar differences in caveat to end-point measurements for ROS lies in that our anal- ROS production in response to either stimuli were also evident in ysis, and many others’ (34–37), employ normalization to an macrophages (Fig. 3C). unstimulated control that is producing basal ROS. Because these Diminished ROS generation alone does not explain decreased cells were procured from inflamed tissue, it may be that Die-P bacterial killing because macrophages with defective NOX and phagocytes appear to generate less ROS than Live-P phagocytes in inducible NO synthase (iNOS) are as bactericidal as their response to stimuli because the Die-P cells are already generating NOX/inducible NO synthase competent counterparts (33). This maximal ROS (i.e., hyperstimulated). If the Die-P mice were occurs because, following internalization, the acidifies hyperstimulated, it would be difficult to detect any increase in and enables pH-sensitive antimicrobial products to destroy the ROS upon further stimulation. To determine whether Die-P cells phagosome’s microbial cargo. Because pHrodo is a pH-sensitive were hyperstimulated, a chemiluminescence-based kinetic assay fluorophore, the term pHrodocytosis was used to reflect the fact was used to measure ROS. Basal ROS data show that Die-P that differences in fluorescence could be due to differences in the peritoneal cells produce less ROS prior to stimulation, thus ar- number of attached and/or internalized bacteria or due to differ- guing against the hyperstimulation hypothesis (Fig. 3D). Similar ences in pH. Comparing pHrodo (OpH–E. coli) fluorescence to what was observed with the flow cytometric ROS measure- showed decreased pHrodocytosis for neutrophils and macrophages ments, opsonized–E. coli (Fig. 3E) resulted in less ROS produc- at both time points (data not shown). tion in Die-P cells. The sum of the cellular response to either Interestingly, Die-P phagocytes produce less ROS in response to opsonized–E. coli or PMA was calculated as the total area under OpH–E. coli stimulation compared with the unstimulated state the curve (Fig. 3E, 3F). Importantly, luminol chemiluminescence (Fig. 3A, right histogram, 3B, 3C, black bars). It is important to shows that Die-P cells do generate more ROS from opsonized– note that unstimulated control cells are not naive cells; they were E. coli as compared with basal conditions, unlike what was observed

FIGURE 4. Die-P phagocytes are suppressed in phagocytosis. 33-Labeled E. coli were incubated with peritoneal cells for 30 min. Gating for Phagocyto- sis+ events: (A) Ice control (left), 37˚C incubation (right). TB was used to quench extracellular fluorescence. The percentage of Peritoneal cells from Live- P and Die-P mice that phagocytize bac- teria at 6 h (B) and 24 h post-CLP (C), and the overall amount of bacteria in- ternalized at 6 h (D) and 24 h (E). Data collected from three to four independent experiments. n = 4/group for 6 h, n =5– 7/group for 24 h. *p , 0.05, ***p , 0.0001 Live-P versus Die-P. 3798 ACUTE-PHASE SEPSIS DEATHS FROM IMMUNOSUPPRESSION by flow cytometry (Fig. 3B, 3C, leftmost panels). It was deter- may exist between groups, but the Die-P response is significantly mined that the increased fluorescence seen in Die-P cells prior weaker than Live-P (i.e., suppressed cells). to exogenous stimulation (Fig. 3A) was due to increased auto- To address this, a saturating dose of labeled bacteria was used fluorescence (data not shown). Importantly, luminol chemilu- to stimulate the peritoneal phagocytes. A saturating dose is de- minescence is not confounded by cellular autofluorescence. fined as the number of bacteria per cell needed to stimulate the Furthermore, whereas opsonized–E. coli incubation produces a maximum number of cells to phagocytize the bacteria. This minuscule ROS burst in Die-P cells, PMA stimulation results in presumably occurs due to ligation of all available receptors, as a marked increase in ROS production (Fig. 3F). This suggests that suggested by a study in human PMNs which showed that despite although Die-P cells retain the capacity to respond more vigor- equal binding of ligand by all cells, only a subset of cells will ously to bacteria with ROS, they fail to do so. Taken together, actually respond (i.e., phagocytize) (38). In conjunction with TB these data suggest that Die-P cells are not hyperstimulated but to quench extracellular fluorescence, cells that have internalized may instead be exhausted or suppressed, resulting in decreased bacteria can be discriminated from those that have not by bacterial killing (Fig. 2), increased bacterial burden, and increased comparing cells incubated with bacteria on ice versus 37˚C mortality (Fig. 1). (Fig. 4A). Using a saturating dose, similar proportions of cells phagocy- Die-P phagocytes are immunosuppressed tized the bacteria at both 6 h (Fig. 4B) and 24 h (Fig. 4C). However, The preceding data suggest that Die-P phagocytes are impaired Die-P cells phagocytized significantly less bacteria overall than compared with Live-P, but the data do not indicate the mechanism. Live-P at both the early and later time points (Fig. 4D, 4E). De- Die-P cells may exhibit impaired bactericidal activity because creased phagocytosis could be due to decreased attachment of the fewer cells have the capacity to respond to stimuli (i.e., exhausted bacteria to the cell surface. However, this does not seem likely as cells). Alternatively, a similar fraction of stimuli-responsive cells both groups exhibit similar levels of bacterial fluorescence when

FIGURE 5. Gating for neutrophils and macrophages based on phagocytic activity. 33-Labeled E. coli were incubated with peritoneal cells for 30 min and TB added to quench extracellular fluorescence. (A) Con- tour plots for Phagocytosis+ events. DCF and pHrodo fluorescence of phagocytosis+ events (B) and normalization of DCF and pHrodo fluorescence to Alexa 350 fluores- cence (C). (D) Cytospin preparations of sorted high ROS and low ROS events reveal them to be neutrophils and macrophages, respectively. Original magnification 350. (E) The overall amount of internalized bac- teria for neutrophils and macrophages was calculated via its Alexa 350 fluorescence. n = 4 at 6 h and n = 5–7 at 24 h from three independent experiments. *p , 0.05, **p , 0.01, ***p , 0.001 Live-P versus Die-P. The Journal of Immunology 3799 incubated on ice and without addition of TB quenching agent increased ROS (39), whereas macrophages have more phagosomal (data not shown). These data support the differences observed in acidification (40) and an increased capacity for phagocytosis (41). bacterial killing and suggest that Die-P peritoneal cells are sup- To determine whether the decreased phagocytosis of Die-P cells pressed in their function but are not exhausted because a similar was due to decreased phagocytosis by either macrophages or percentage of cells do phagocytize, albeit significantly less than neutrophils, the peritoneal phagocytosis data (Fig. 4D, 4E) were re- seen in Live-P. examined by first stratifying the cell populations based on high or low phagosomal ROS production (Fig. 5C). This approach showed Quantifying phagosomal responses that both Die-P neutrophils and macrophages are suppressed in Intraphagosomal processes were examined to further define the their ability to phagocytize within 6 h post-CLP, and this continues mechanism of suppression and ascertain whether bacteria were still through 24 h (Fig. 5E), thus suggesting why Die-P cells kill fewer exposed to the caustic microbicidal environment following inter- bacteria than Live-P. nalization. This was accomplished with a recently developed Similar to their impaired ability to phagocytize (Figs. 4, 5), Die- technique in which E. coli were triple labeled with Dichloro- P peritoneal phagocytes also show decreased phagosomal matu- fluorescein (ROS sensor), pHrodo ( fusion, i.e., ration (i.e., ROS/acidification) as compared with Live-P. acidification), and Alexa Fluor 350 (ROS/pH insensitive). A specific gating strategy was used to determine the relative oxi- Differences in ROS production and phagosome acidification dation and/or acidification of the . For all phagocy- Although significant differences in PMN phagocytosis between tizing cells (Fig. 5A), DCF/pHrodo fluorescence (Fig. 5B) was Live-P and Die-P existed by 6 h (Fig 5E), there were not significant normalized to Alexa Fluor 350 fluorescence. In this manner, DCF/ differences in the generation of phagosomal ROS, the hallmark of pHrodo fluorescence can be attributed to the amount of oxidation/ PMN bacterial killing (Fig. 6A). However, by 24 h, there were acidification and not to differences in the amount of labeled significant differences in phagosomal ROS (Fig. 6B), with Live-P bacteria within the cell. This approach identified two distinct demonstrating an increased burst as compared with 6 h, whereas populations of high and low ROS cells (Fig. 5C). Furthermore, Die-P PMNs did not show this improvement. Although less pro- cell sorting of high ROS and low ROS cells revealed the cells to be nounced in PMNs, phagosomal acidification and fusion with PMNs or macrophages, respectively (Fig. 5D). This agrees with lysosomes contributes significantly to bacterial killing (42). Sim- previous literature that showed the proclivity of PMNs to produce ilar to PMN ROS, no significant differences in PMN phagosomal

FIGURE 6. Die-P peritoneal phagocytes are suppressed in phagosomal maturation. 33-Labeled E. coli were in- cubated with peritoneal cells for 30 min and TB added to quench extracellular fluorescence. PMN phagosomal ROS was calculated by calibrating DCF (ROS sensitive) fluo- rescence to Alexa 350 fluorescence (ROS/pH insensitive) at 6 h (A) and 24 h post-CLP (B). PMN phagosomal acidification was calculated by normalizing pHrodo fluo- rescence to Alexa 350 at 6 h (C) and 24 h (D). Macrophage phagosome acidification at 6 h (E) and 24 h (F). n = 5–7 from three independent experiments. *p , 0.05, **p , 0.01, ***p , 0.001 Live-P versus Die-P. 3800 ACUTE-PHASE SEPSIS DEATHS FROM IMMUNOSUPPRESSION acidification existed within 6 h post-CLP (Fig. 6C), but significant Others have shown that treatments affecting differences did emerge by 24 h (Fig. 6D). In contrast to Die-P and function result in increased survival (53, 54). Although these PMNs, macrophage phagosomal acidification was significantly studies did not stratify by survival likelihood, it is interesting to decreased within 6 h post-CLP (Fig. 6E) and this continued speculate that impaired lymphocyte function or death drives the through 24hr (Fig. 6F). phagocyte dysfunction observed in our model. Certainly, the re- Taken together, these data strongly support that Die-P phago- port by Rauch et al. (52) detailing GM-CSF–producing B cells cytes are suppressed in their ability to internalize bacteria and to supports this. Also not addressed is the role endotoxin tolerance create the caustic conditions necessary to kill internalized bac- may have in phagocytic suppression. The endotoxin tolerance teria. This suppression was evident within 6 h after the onset of phenotype of monocytes/macrophages is one of epigenetic re- sepsis. This in turn explains why despite increased phagocyte modeling (55) and decreased production with increased recruitment, Die-P mice fail to eliminate their infection and phagocytosis (56). It is plausible that Live-P phagocytes experi- subsequently die. ence similar rewiring that promotes bacterial clearance along with decreased cytokine production (i.e., IL-6). Discussion Another limitation to this study is that only peritoneal cells The most important finding in this study is that despite receiving were evaluated for performance. It would be interesting to know equivalent inoculums and recruiting similar numbers of phago- whether Die-P phagocytes become impaired upon their arrival to cytes in response to the inoculum, Die-P peritoneal phagocytes the peritoneum or whether their impairment exists prior to their are impaired in their ability to kill bacteria. These cellular defects arrival. Myeloid-derived suppressor cells (MDSCs) may play were present in the very first hours of sepsis and became pro- a role if the phagocytes are suppressed prior to recruitment to gressively worse. Less obvious but of equal concern is that the site of infection. Delano et al. showed that CLP results without proper stratification of mice according to their likelihood in suppressed ROS burst by monocytic and of survival (i.e., mild versus severe sepsis), considerable vari- marrow neutrophils, suggesting that phagocytic suppression is ability may confound studies that compare sham operated mice to systemic. It will be important to determine to what extent sys- septic mice. For example, our laboratory has published that cell temic alterations are implicated in a survivor/non-survivor function, renal function, bacteria, and differ signifi- model of sepsis. MDSCs have been shown to improve or hin- cantly between survivors and nonsurvivors (13, 43). Our labo- der survival from CLP, depending on the maturity of the MDSCs ratory has also published that high IL-6 levels merely correlate (57, 58). Unfortunately, the phenotypic markers for MDSCs with, but do not mediate mortality from CLP (44). These studies (e.g.Cd11b+,Gr-1+) are insufficiently specific to readily dis- suggest that stratification would be of considerable benefit in tinguish them from the peritoneal neutrophils or macrophages/ human studies where heterogeneity in individuals and their re- monocytesinthisstudy. sponses are more pronounced. In further support of stratification There is considerable evidence that suggests that overstimulation and our findings of decreased bacterial killing preceding death, of cells with contradictory stimuli (e.g., pro- and antiapoptosis Danikas et al. (45) showed phagocytic activity as prognostic for agents) can result in a terminally nonresponsive state, affection- outcome in human sepsis. ately termed zombie cells (59). Along similar lines, Die-P mice Although numerous studies have argued for and against organ demonstrate a significantly larger surge in pro and anti-inflam- injury preceding death from sepsis (43, 46, 47), this study matory mediators in their peritoneum and plasma (16), potentially strongly suggests that early phagocytic impairment (within 6 h) creating the conditions for terminally nonresponsive cells that ultimately leads to uncontrolled microbial growth and death. This render them incapable of defending the host. In light of this, occurs even with the use of effective broad spectrum antibiotics. It perhaps the most hopeful finding of this study is that Die-P cells may be that failure to contain the initial infection allows the in- are functionally intact and equally capable of responding to bac- tense inflammatory reaction to ripple throughout the organism, teria, although they are deficient in both the amount they can leading to organ injury. The successful eradication of microbial phagocytize and how they process their phagosomal cargo. Be- pathogens requires recruitment of myeloid cells to the site of in- cause they are responsive, phagocytes would be attractive targets fection (48, 49). To that end, other laboratories have shown that for functional modulators, such as antagonists of adenosine increasing or inhibiting neutrophil recruitment following CLP receptors or programmed death receptor-1, both of which have (50, 51) affects bacterial burden and animal survival. Our labo- been suggested to increase phagocyte function and improve sur- ratory has shown that augmented recruitment of neutrophils also vival from sepsis (15, 60). increases survival (16). However, that study and this one dem- onstrate that the mice that are predicted to die do not fail to recruit Acknowledgments neutrophils or monocytes/macrophages as compared with the mice We thank Elizabeth R. Simons for her intellectual and technical support. predicted to live. In fact, Die-P mice recruit more cells by 24 h We also thank the Boston University Medical Center Core post-CLP. Similar findings were recently reported that used CLP Facility for its support. to produce ∼60% mortality in control mice (52) and demonstrated that without GM-CSF–producing B cells, CLP resulted in 100% Disclosures mortality. Most importantly, as it relates to this study, the The authors have no financial conflicts of interest. GM-CSF2/2 mice recruited significantly more neutrophils to the peritoneum yet still had increased bacterial burden, increased in- flammatory mediators, and decreased phagocytosis, as observed References in our model. Taken all together, this suggests that although 1. Lagu, T., M. B. Rothberg, M. S. Shieh, P. S. Pekow, J. S. Steingrub, and phagocyte recruitment is important, their functional status is more P. K. Lindenauer. 2012. Hospitalizations, costs, and outcomes of severe sepsis in the United States 2003 to 2007. Crit. Care Med. 40: 754–761. important. 2. Martin, G. S., D. M. Mannino, S. Eaton, and M. Moss. 2003. The epidemiology Although this study strongly argues for impaired phagocyte of sepsis in the United States from 1979 through 2000. N. Engl. J. Med. 348: 1546–1554. function as contributing to death, it does not address the role 3. Dellinger, R. P., M. M. Levy, A. Rhodes, D. Annane, H. Gerlach, S. M. Opal, of , which are fundamental to an immune response. J. E. Sevransky, C. L. Sprung, I. S. Douglas, R. Jaeschke, et al. 2013. Surviving The Journal of Immunology 3801

sepsis campaign: international guidelines for management of severe sepsis and 31. Misharin, A. V., R. Saber, and H. Perlman. 2012. contamination of : 2012. Crit. Care Med. 41: 580–637. thioglycollate-elicited peritoneal macrophage cultures skews the functional 4. Torgersen, C., P. Moser, G. Luckner, V. Mayr, S. Jochberger, W. R. Hasibeder, readouts of in vitro assays. J. Leukoc. Biol. 92: 325–331. and M. W. Dunser.€ 2009. Macroscopic postmortem findings in 235 surgical in- 32. Ghosn, E. E., A. A. Cassado, G. R. Govoni, T. Fukuhara, Y. Yang, D. M. Monack, tensive care patients with sepsis. Anesth. Analg. 108: 1841–1847. K. R. Bortoluci, S. R. Almeida, L. A. Herzenberg, and L. A. Herzenberg. 2010. 5. Urban, C. F., D. Ermert, M. Schmid, U. Abu-Abed, C. Goosmann, W. Nacken, Two physically, functionally, and developmentally distinct peritoneal macro- V. Brinkmann, P. R. Jungblut, and A. Zychlinsky. 2009. Neutrophil extracellular phage subsets. Proc. Natl. Acad. Sci. USA 107: 2568–2573. traps contain , a cytosolic protein complex involved in host defense 33. Fairbairn, I. P., C. B. Stober, D. S. Kumararatne, and D. A. Lammas. 2001. ATP- against Candida albicans. PLoS Pathog. 5: e1000639. mediated killing of intracellular mycobacteria by macrophages is a P2X(7)- 6. Segal, B. H., M. J. Grimm, A. N. Khan, W. Han, and T. S. Blackwell. 2012. dependent process inducing bacterial death by phagosome- fusion. J. Regulation of innate immunity by NADPH oxidase. Free Radic. Biol. Med. 53: Immunol. 167: 3300–3307. 72–80. 34. O’Gorman, M. R., and V. Corrochano. 1995. Rapid whole-blood flow cytometry 7. Vieira, O. V., R. J. Botelho, and S. Grinstein. 2002. Phagosome maturation: assay for diagnosis of chronic granulomatous disease. Clin. Diagn. Lab. aging gracefully. Biochem. J. 366: 689–704. Immunol. 2: 227–232. 8. Xiao, H., J. Siddiqui, and D. G. Remick. 2006. Mechanisms of mortality in early 35. Elloumi, H. Z., and S. M. Holland. 2014. Diagnostic assays for chronic and late sepsis. Infect. Immun. 74: 5227–5235. granulomatous disease and other neutrophil disorders. Methods Mol. Biol. 9.Ocuin,L.M.,Z.M.Bamboat,V.P.Balachandran,M.J.Cavnar,H.Obaid, 1124: 517–535. G. Plitas, and R. P. DeMatteo. 2011. Neutrophil IL-10 suppresses peritoneal 36. Alvarez-Larra´n, A., T. Toll, S. Rives, and J. Estella. 2005. Assessment of neu- inflammatory monocytes during polymicrobial sepsis. J. Leukoc. Biol. 89: trophil activation in whole blood by flow cytometry. Clin. Lab. Haematol. 27: 423–432. 41–46. 10.Huang,X.,F.Venet,Y.L.Wang,A.Lepape,Z.Yuan,Y.Chen,R.Swan, 37. Delano, M. J., T. Thayer, S. Gabrilovich, K. M. Kelly-Scumpia, R. D. Winfield, H. Kherouf, G. Monneret, C. S. Chung, and A. Ayala. 2009. PD-1 expression P. O. Scumpia, A. G. Cuenca, E. Warner, S. M. Wallet, M. A. Wallet, et al. 2011. by macrophages plays a pathologic role in altering microbial clearance and Sepsis induces early alterations in innate immunity that impact mortality to theinnateinflammatoryresponsetosepsis.Proc. Natl. Acad. Sci. USA 106: secondary infection. J. Immunol. 186: 195–202. 6303–6308. 38. Brunkhorst, B. A., K. G. Lazzari, G. Strohmeier, G. Weil, and E. R. Simons. 11. Oliveira, R. P., I. Velasco, F. G. Soriano, and G. Friedman. 2002. Clinical review: 1991. Calcium changes in -stimulated human neutrophils. Si- hypertonic saline resuscitation in sepsis. Crit. Care 6: 418–423. multaneous measurement of receptor occupancy and activation reveals full 12. Theobaldo, M. C., F. Llimona, R. C. Petroni, E. C. Rios, I. T. Velasco, and population stimulus binding but subpopulation activation. J. Biol. Chem. 266: F. G. Soriano. 2013. Hypertonic saline solution drives neutrophil from bystander 13035–13043. organ to infectious site in polymicrobial sepsis: a cecal ligation and puncture 39. Nathan, C., and M. U. Shiloh. 2000. Reactive oxygen and nitrogen intermediates model. PLoS One 8: e74369. in the relationship between mammalian hosts and microbial pathogens. Proc. 13. Remick, D. G., G. R. Bolgos, J. Siddiqui, J. Shin, and J. A. Nemzek. 2002. Six at Natl. Acad. Sci. USA 97: 8841–8848. six: -6 measured 6 h after the initiation of sepsis predicts mortality 40. Nordenfelt, P., and H. Tapper. 2011. Phagosome dynamics during phagocytosis over 3 days. Shock 17: 463–467. by neutrophils. J. Leukoc. Biol. 90: 271–284. 14. Osuchowski, M. F., J. Connett, K. Welch, J. Granger, and D. G. Remick. 2009. 41. Dale, D. C., L. Boxer, and W. C. Liles. 2008. The phagocytes: neutrophils and Stratification is the key: inflammatory biomarkers accurately direct immuno- monocytes. Blood 112: 935–945. modulatory therapy in experimental sepsis. Crit. Care Med. 37: 1567–1573. 42. Beertsen, W., M. Willenborg, V. Everts, A. Zirogianni, R. Podschun, 15. Belikoff, B. G., S. Hatfield, P. Georgiev, A. Ohta, D. Lukashev, J. A. Buras, B. Schro¨der, E. L. Eskelinen, and P. Saftig. 2008. Impaired phagosomal matu- D. G. Remick, and M. Sitkovsky. 2011. A2B adenosine receptor blockade ration in neutrophils leads to periodontitis in lysosomal-associated membrane enhances macrophage-mediated bacterial phagocytosis and improves poly- protein-2 knockout mice. J. Immunol. 180: 475–482. microbial sepsis survival in mice. J. Immunol. 186: 2444–2453. 43. Craciun, F. L., K. N. Iskander, E. L. Chiswick, D. M. Stepien, J. M. Henderson, 16. Craciun, F. L., E. R. Schuller, and D. G. Remick. 2010. Early enhanced local and D. G. Remick. 2014. Early murine polymicrobial sepsis predominantly neutrophil recruitment in peritonitis-induced sepsis improves bacterial clearance causes renal injury. Shock 41: 97–103. and survival. J. Immunol. 185: 6930–6938. 44. Remick, D. G., G. Bolgos, S. Copeland, and J. Siddiqui. 2005. Role of 17. Wichterman, K. A., A. E. Baue, and I. H. Chaudry. 1980. Sepsis and septic shock— interleukin-6 in mortality from and physiologic response to sepsis. Infect. a review of laboratory models and a proposal. J. Surg. Res. 29: 189–201. Immun. 73: 2751–2757. 18. Ebong, S. J., D. R. Call, G. Bolgos, D. E. Newcomb, J. I. Granger, M. O’Reilly, 45. Danikas, D. D., M. Karakantza, G. L. Theodorou, G. C. Sakellaropoulos, and and D. G. Remick. 1999. Immunopathologic responses to non-lethal sepsis. C. A. Gogos. 2008. Prognostic value of phagocytic activity of neutrophils and Shock 12: 118–126. monocytes in sepsis. Correlation to CD64 and CD14 expression. Clin. 19. Nemzek, J. A., J. Siddiqui, and D. G. Remick. 2001. Development and opti- Exp. Immunol. 154: 87–97. mization of cytokine ELISAs using commercial pairs. J. Immunol. 46. Iskander, K. N., F. L. Craciun, D. M. Stepien, E. R. Duffy, J. Kim, R. Moitra, Methods 255: 149–157. L. J. Vaickus, M. F. Osuchowski, and D. G. Remick. 2013. Cecal ligation and 20. Campbell, P. A., B. P. Canono, and D. A. Drevets. 2001. Measurement of bac- puncture-induced murine sepsis does not cause injury. Crit. Care Med. 41: terial ingestion and killing by macrophages. Curr. Protoc. Immunol. Chapter 14: 159–170. Unit 14.16. 47. Doerschug, K. C., L. S. Powers, M. M. Monick, P. S. Thorne, and 21. Zhou, C., J. Wu, J. Borillo, L. Torres, J. McMahon, and Y. H. Lou. 2009. G. W. Hunninghake. 2004. Antibiotics delay but do not prevent bacteremia Potential roles of a special CD8 aa+ cell population and CC and lung injury in murine sepsis. Crit. Care Med. 32: 489–494. -expressed chemokine in ovulation related inflammation. J. Immunol. 48. Dunay, I. R., R. A. Damatta, B. Fux, R. Presti, S. Greco, M. Colonna, and 182: 596–603. L. D. Sibley. 2008. Gr1+ inflammatory monocytes are required for mucosal re- 22. Decleva, E., R. Menegazzi, S. Busetto, P. Patriarca, and P. Dri. 2006. Common sistance to the pathogen Toxoplasma gondii. Immunity 29: 306–317. methodology is inadequate for studies on the microbicidal activity of neu- 49. Belaaouaj, A., R. McCarthy, M. Baumann, Z. Gao, T. J. Ley, S. N. Abraham, and trophils. J. Leukoc. Biol. 79: 87–94. S. D. Shapiro. 1998. Mice lacking neutrophil elastase reveal impaired host de- 23. Simons, E. R. 2010. Measurement of phagocytosis and of the phagosomal en- fense against gram negative bacterial sepsis. Nat. Med. 4: 615–618. vironment in polymorphonuclear phagocytes by flow cytometry. Curr. Protoc. 50. Alves-Filho, J. C., F. Soˆnego, F. O. Souto, A. Freitas, W. A. Verri, Jr., Cytom. Chapter 9: Unit9 31. M. Auxiliadora-Martins, A. Basile-Filho, A. N. McKenzie, D. Xu, F. Q. Cunha, 24. Yates, R. M., and D. G. Russell. 2005. Phagosome maturation proceeds inde- and F. Y. Liew. 2010. Interleukin-33 attenuates sepsis by enhancing neutrophil pendently of stimulation of toll-like receptors 2 and 4. Immunity 23: 409–417. influx to the site of infection. Nat. Med. 16: 708–712. 25. Bernardo, J., H. J. Long, and E. R. Simons. 2010. Initial cytoplasmic and 51. Delano, M. J., K. M. Kelly-Scumpia, T. C. Thayer, R. D. Winfield, phagosomal consequences of human neutrophil exposure to Staphylococcus P. O. Scumpia, A. G. Cuenca, P. B. Harrington, K. A. O’Malley, E. Warner, epidermidis. Cytometry A 77: 243‑252. S. Gabrilovich, et al. 2011. Neutrophil mobilization from the 26. Cso´ka, B., Z. H. Ne´meth, P. Mukhopadhyay, Z. Spolarics, M. Rajesh, S. Federici, during polymicrobial sepsis is dependent on CXCL12 signaling. J. Immunol. E. A. Deitch, S. Ba´tkai, P. Pacher, and G. Hasko´. 2009. CB2 cannabinoid 187: 911–918. receptors contribute to bacterial invasion and mortality in polymicrobial sepsis. 52. Rauch, P. J., A. Chudnovskiy, C. S. Robbins, G. F. Weber, M. Etzrodt, PLoS One 4: e6409. I. Hilgendorf, E. Tiglao, J. L. Figueiredo, Y. Iwamoto, I. Theurl, et al. 2012. 27. Hotchkiss, R. S., K. C. Chang, P. E. Swanson, K. W. Tinsley, J. J. Hui, Innate response activator B cells protect against microbial sepsis. Science 335: P. Klender, S. Xanthoudakis, S. Roy, C. Black, E. Grimm, et al. 2000. Caspase 597–601. inhibitors improve survival in sepsis: a critical role of the lymphocyte. Nat. 53. Inoue, S., J. Unsinger, C. G. Davis, J. T. Muenzer, T. A. Ferguson, K. Chang, Immunol. 1: 496–501. D. F. Osborne, A. T. Clark, C. M. Coopersmith, J. E. McDunn, and 28. Walker, W. E., A. T. Bozzi, and D. R. Goldstein. 2012. IRF3 contributes to sepsis R. S. Hotchkiss. 2010. IL-15 prevents apoptosis, reverses innate and adaptive pathogenesis in the mouse cecal ligation and puncture model. J. Leukoc. Biol. immune dysfunction, and improves survival in sepsis. J. Immunol. 184: 1401– 92: 1261–1268. 1409. 29. Rose, S., A. Misharin, and H. Perlman. 2012. A novel Ly6C/Ly6G-based 54. Unsinger, J., M. McGlynn, K. R. Kasten, A. S. Hoekzema, E. Watanabe, strategy to analyze the mouse splenic myeloid compartment. Cytometry A 81: J. T. Muenzer, J. S. McDonough, J. Tschoep, T. A. Ferguson, J. E. McDunn, et al. 343‑350. 2010. IL-7 promotes viability, trafficking, and functionality and improves 30. Narni-Mancinelli, E., S. M. Soudja, K. Crozat, M. Dalod, P. Gounon, survival in sepsis. J. Immunol. 184: 3768‑3779. F. Geissmann, and G. Lauvau. 2011. Inflammatory monocytes and neutrophils 55. Saeed, S., J. Quintin, H. H. Kerstens, N. A. Rao, A. Aghajanirefah, F. Matarese, are licensed to kill during memory responses in vivo. PLoS Pathog. 7: e1002457. S. C. Cheng, J. Ratter, K. Berentsen, M. A. van der Ent, et al. 2014. Epigenetic 3802 ACUTE-PHASE SEPSIS DEATHS FROM IMMUNOSUPPRESSION

programming of -to-macrophage differentiation and trained innate 58. Derive, M., Y. Bouazza, C. Alauzet, and S. Gibot. 2012. Myeloid-derived sup- immunity. Science 345: 1251086. pressor cells control microbial sepsis. Intensive Care Med. 38: 1040–1049. 56. Biswas, S. K., and E. Lopez-Collazo. 2009. Endotoxin tolerance: new 59. Hotchkiss, R. S., A. Strasser, J. E. McDunn, and P. E. Swanson. 2009. Cell death. mechanisms, molecules and clinical significance. Trends Immunol. 30: 475– N. Engl. J. Med. 361: 1570–1583. 487. 60. Zhang, Y., Y. Zhou, J. Lou, J. Li, L. Bo, K. Zhu, X. Wan, X. Deng, and Z. Cai. 57. Brudecki, L., D. A. Ferguson, C. E. McCall, and M. El Gazzar. 2012. Myeloid- 2010. PD-L1 blockade improves survival in experimental sepsis by in- derived suppressor cells evolve during sepsis and can enhance or attenuate the hibiting lymphocyte apoptosis and reversing monocyte dysfunction. Crit. Care 14: systemic inflammatory response. Infect. Immun. 80: 2026–2034. R220.