Activation of Hepatic STAT3 Maintains Pulmonary Defense duringEndotoxemia

Kristie L. Hilliard,a,b Eri Allen,b Katrina E. Traber,b Yuri Kim,b,d Gregory A. Wasserman,a,b Matthew R. Jones,b,d

Joseph P. Mizgerd,a,b,c,d Lee J. Quintonb,d,e

Department of Microbiology, Boston University School of Medicine, Boston, Massachusetts, USAa; Pulmonary Center, Boston University School of Medicine, Boston,Massachusetts, USAb; Department of Medicine, Boston University School of Medicine, Boston, Massachusetts, USAc; Department of Biochemistry, Boston UniversitySchool of Medicine, Boston, Massachusetts, USAd; Department of Pathology and Laboratory Medicine, Boston University School of Medicine, Boston, Massachusetts, USAe

Pneumonia and infection-induced sepsis are worldwide public health concerns. Both pathologies elicit systemic inflammationand induce a robust acute-phase response (APR). Although APR activation is well regarded as a hallmark of infection, the directcontributions of liver activation to pulmonary defense during sepsis remain unclear. By targeting STAT3-dependent acute-phasechanges in the liver, we evaluated the role of liver STAT3 activity in promoting host defense in the context of sepsis and pneumo-nia. We employed a two-hit endotoxemia/pneumonia model, whereby administration of 18 h of intraperitoneal lipopolysaccha-ride (LPS; 5 mg/kg of body weight) was followed by intratracheal Escherichia coli (106 CFU) in wild-type mice or those lackinghepatocyte STAT3 (hepSTAT3�/�). Pneumonia alone (without endotoxemia) was effectively controlled in the absence of liverSTAT3. Following endotoxemia and pneumonia, however, hepSTAT3�/� mice, with significantly reduced levels of circulatingand airspace acute-phase proteins, exhibited significantly elevated lung and blood bacterial burdens and mortality. These datasuggested that STAT3-dependent liver responses are necessary to promote host defense. While neither recruited airspace neutro-phils nor lung injury was altered in endotoxemic hepSTAT3�/� mice, alveolar macrophage reactive oxygen species generationwas significantly decreased. Additionally, bronchoalveolar lavage fluid from this group of hepSTAT3�/� mice allowed greaterbacterial growth ex vivo. These results suggest that hepatic STAT3 activation promotes both cellular and humoral lung defenses.Taken together, induction of liver STAT3-dependent gene expression programs is essential to countering the deleterious conse-quences of sepsis on pneumonia susceptibility.

Sepsis is a complex immunopathological syndrome defined bythe systemic inflammatory response to infection and is a lead-

ing contributor to morbidity and mortality in intensive care unitsas evidenced by approximately 750,000 cases per year (2% of allhospital admissions) (1–3). This multifaceted, systemic inflam-matory response can be further complicated by organ dysfunction(severe sepsis) and hypotension (septic shock), all of which lead toa complex, variable syndrome with mortality rates between 30 and50% (4). While pneumonia is the leading cause of sepsis, withabout one-half of all sepsis cases originating as respiratory infec-tions (2), sepsis also greatly increases a patient’s subsequent sus-ceptibility to bacterial pneumonia (5). In fact, 10 to 30% ofmechanically ventilated, septic shock patients develop ventila-tor-associated pneumonia (6). This positive association ex-tends beyond ventilator-related circumstances and has been cor-roborated experimentally by multiple studies demonstratingdeleterious effects of sepsis and/or endotoxemia on pneumoniaoutcomes (7–15). With the rapid increase in prevalence of drug-resistant pathogens and the limited treatment options available,there is a growing need to develop novel pharmaceutical interven-tions and to improve our understanding of the inflammatory pro-cesses involved in both pathologies.

A shared and prominent feature of sepsis, pneumonia, andother inflammatory conditions is the hepatic acute-phase re-sponse (APR) (16–19). Induced by the host defense cytokines tu-mor necrosis factor alpha (TNF-�), interleukin-1 (IL-1), and IL-6(20), the APR is characterized by significant changes in circulatinglevels of acute-phase proteins (APPs) (19, 21, 22). While it is wellappreciated that sepsis can cause pulmonary immunosuppressionand pneumonia susceptibility (5, 7–15), it is unclear whether or

how preexisting liver activation (i.e., sepsis-induced APR) modu-lates subsequent responses to local lung infections.

Signal transducer and activator of transcription-3 (STAT3) isone of two transcription factors (along with NF-�B RelA) re-quired for induction of a strong hepatic APR during pneumonia(23–25). Several lines of evidence implicate beneficial roles forliver-derived APPs during pneumonia (26–29). We have shownthat this lung-liver axis, enabled by both transcription factors, isrequired for maximal protection during pneumonia alone, but thedistinct roles of STAT3 (versus RelA) in this process remain un-clear. Others have linked hepatic STAT3 activity to the APR inmodels of sepsis (30, 31). Given the close association betweenpneumonia, sepsis, STAT3, and the APR, we sought to determinethe direct influence of systemic STAT3-dependent liver activity onsubsequent pneumonia outcomes. Our results demonstrate thesignificance of liver activation during sepsis and that the APRdrives protective networks of gene expression to maximize local

Received 8 April 2015 Returned for modification 6 May 2015Accepted 20 July 2015

Accepted manuscript posted online 27 July 2015

Citation Hilliard KL, Allen E, Traber KE, Kim Y, Wasserman GA, Jones MR, MizgerdJP, Quinton LJ. 2015. Activation of hepatic STAT3 maintains pulmonary defenseduring endotoxemia. Infect Immun 83:4015– 4027. doi:10.1128/IAI.00464-15.

Editor: B. A. McCormick

Address correspondence to Lee J. Quinton, lquinton@bu.edu.

Copyright © 2015, American Society for Microbiology. All Rights Reserved.

doi:10.1128/IAI.00464-15

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defense responses to pulmonary pathogens encountered duringendotoxemia.

MATERIALS AND METHODSMice. Experiments were performed using mice in which hepatocyteSTAT3 was functionally deleted using the Cre-LoxP system. Briefly, micecontaining homozygous floxed alleles for Stat3 (32) were bred with albu-min-driven Cre-recombinase transgenic mice (Alb-Cretg/�/Stat3LoxP/LoxP).Experimental results obtained from hepSTAT3�/� mice were comparedto those from littermate controls lacking the Cre-recombinase transgene(Alb-Cre�/�/Stat3LoxP/LoxP). Male and female mice on a mixed geneticbackground were used between 6 and 12 weeks of age, and each experi-ment was performed at least twice. All animal protocols were approved bythe Boston University Institutional Animal Care and Use Committee.

Experimental endotoxemia and pneumonia. Mice were given an in-traperitoneal (i.p.) injection of 5 mg/kg of body weight of ultrapure lipo-polysaccharide (LPS; InvivoGen) or saline, followed 18 h later by an in-tratracheal (i.t.) infection with 1 � 106 CFU of Escherichia coli (Fig. 1A)(serotype 06:K2:H1; ATCC 19138), as previously described by our labo-ratory (23). For i.t. instillations, mice were anesthetized by i.p. injection ofa mixture of ketamine (50 mg/kg) and xylazine (5 mg/kg), the trachea wasexposed, and a 24-gauge catheter was inserted into the trachea. A 50-�lbolus of saline containing the E. coli was then instilled into the left bron-chus. Mice were euthanized at the indicated time points by isofluraneoverdose, and specific tissues were harvested for measurements detailedbelow.

Bronchoalveolar lavage. Lungs were isolated and bronchoalveolar la-vage fluid (BALF) was collected as previously described at the indicatedtime points (20, 33). Lungs were removed and tethered to a 20-gaugeblunted catheter by way of the trachea. Once secured, the lungs wererepeatedly lavaged 10 times with 1 ml of phosphate-buffered saline (PBS).The cell-free supernatant from the first lavage was aliquoted and stored at�80°C for protein analysis. Pooled cells from all washes were countedusing a hemacytometer, and differential counts were determined aftercytocentrifugation and Diff-Quick (Dade-Behring) staining.

Serum collection. After collection at the indicated time points, bloodwas incubated in MiniCollect Z Serum Separator tubes (Greiner Bio-One)for 30 min at room temperature and then centrifuged for 15 min at1,500 � g and 4°C for serum separation. Serum was aliquoted andstored at �80°C for protein analysis.

Protein measurements. APPs were measured by enzyme-linked im-munosorbent assay (ELISA). Serum amyloid A (SAA) and serum amyloidP (SAP) ELISAs were purchased from Immunology Consultants Labora-tory, Inc. Cytokine protein concentrations were assessed using a Bio-plex200 workstation (Bio-Rad) in conjunction with a Bio-plex cytokine beadarray (Bio-Rad). Included in the panel were IL-1�, IL-6, IL-10, IL-17,granulocyte colony-stimulating factor (G-CSF), granulocyte-macro-phage colony-stimulating factor (GM-CSF), CXCL1, leukemia inhibitoryfactor (LIF), CXCL2, and TNF-�. Total protein concentrations in BALFwere measured using the bicinchoninic acid (BCA) assay (Sigma).

Bacteriology. At 6 and 24 h following E. coli infection, lungs wereisolated and homogenized using a Bullet Blender (Next Advance). Ho-mogenates and heparinized blood were serially diluted in sterile water andplated on 5% sheep blood agar plates (BD Biosciences). After an overnightincubation at 37°C, colonies were counted and expressed as total CFU perlung or per milliliter of blood.

ROS generation. Mice were administered LPS by i.p. injection fol-lowed 18 h later by i.t. E. coli. Six hours after E. coli infection, BALs wereperformed using ice-cold lavage buffer (Hanks’ balanced salt solution[HBSS; Life Technologies, Invitrogen], 2.7 mM EDTA disodium salt so-lution [Sigma-Aldrich], 20 mM HEPES, 100 U/ml penicillin-streptomy-cin [Pen-Strep]). BALF cells were stained for reactive oxygen species(ROS) generation using the CellROX Deep Red Reagent as specified by themanufacturer’s instructions (Life Technologies). Additionally, cell sur-face markers were used to identify airspace neutrophils (CD45�/7AAD�/

Ly6G�/F4/80�) and alveolar macrophages (CD45�/7AAD�/F4/80�/Ly6G�/Autofluorescencehi). The following antibodies were used: CD45-PE/Cy7 clone 30-F11, Ly6G-APC/Cy7 clone 1A8, and F4/80-PEeFluor610 clone CI:A3-1. All antibodies, including 7-amino-actinomycinD (7AAD) viability staining solution, were purchased from Biolegend.Cells were subjected to flow cytometry using the LSRII from BD Biosci-ences and analyzed for ROS generation in each cell type using FlowJo.

pHrodo phagocytosis assay. Phagocytosis was measured using redpHrodo E. coli bioparticles (Life Technologies), which fluoresce only inlow-pH environments (such as the phagolysosomal compartment).pHrodo bioparticles were prepared by suspension in 250 �l of PBS fol-lowed by sonication for 5 min. Mice were given i.p. LPS followed 18 h laterby i.t. E. coli. After 6 h, mice were instilled with a 50-�l bolus of thepHrodo bioparticles, and after another hour, the lungs were lavaged withice-cold lavage buffer (see Fig. 5A). Cells were stained for surface antigensto detect neutrophils and alveolar macrophages as described above.Phagocytosis (phycoerythrin [PE] fluorescence) was examined in eachcell type using FlowJo software.

Cell-based bacterial killing assay. HepSTAT3�/� mutant and wild-type (WT) mice were treated with LPS by i.p. injection, and lungs werelavaged 18 h later with ice-cold lavage buffer. Recovered macrophageswere pelleted by centrifugation at 300 � g for 5 min at 4°C and thenwashed with serum-free, ice-cold RPMI 1640 medium (with 1% penicil-lin-streptomycin). Cells were plated on opaque, tissue culture-treated 96-well plates (Falcon) at a concentration of 100,000 cells/well. After 1 h at37°C and 5% CO2, nonadherent cells were washed away with completeRPMI medium (with 1% penicillin-streptomycin and 10% fetal bovineserum), leaving only the adherent alveolar macrophages. Luminescent,log-phase E. coli cells (strain Xen14; Caliper) were added to the macro-phage cultures at 107 CFU/ml for 1 h in antibiotic-free RPMI medium(with 10% fetal bovine serum). Cells were then washed twice with RPMImedium containing 100 �g/ml gentamicin (with 10% fetal bovine se-rum), which is cell impermeable and thus kills only noninternalized bac-teria. Bacterial luminescence was then measured immediately and hourlyfor the next 4 h using a luminometer. At each time point, baseline cellluminescence was subtracted from each sample, and bacterial killing wasassessed by determining decreases in bacterial luminescence over time.

BALF bacterial growth assay. Luminescent E. coli Xen14 (Caliper)was cultured on blood agar plates overnight at 37°C in 5% CO2. After 18 h,colonies from those plates were then incubated on new plates until thebacteria reached log-phase growth (for around 4 h) at 37°C and 5% CO2.Once in log phase, E. coli was suspended in PBS at 1 � 106 CFU/ml.Cell-free BALF from mutant and wild-type mice infected for 0, 6, or 24 hwith E. coli after LPS injection was aliquoted into a 96-well plate (90�l/well) and then incubated with 10 �l of the bacterial suspension (for astarting concentration of 1 � 105 CFU/ml in each well) while rotating at37°C. Bacterial luminescence (as an indicator of bacterial growth) wasmeasured at the start of and after a 5-hour incubation using a luminom-eter (Turner BioSystems). Growth was calculated as fold increases basedon the starting luminescent values. No viable bacteria were detected in thealiquoted BALF used.

Detection of NETs. In order to quantify neutrophil extracellular trap(NET) release in the BALF, we performed a myeloperoxidase (MPO)-DNA ELISA as previously described (34). A 96-well plate was coated with5 �g/ml of an anti-MPO antibody (rabbit polyclonal, catalogue numberab9535; AbCam) overnight at 4°C, washed with PBS, and then blocked for2 h at room temperature with 5% bovine serum albumin (BSA) in PBS.After washing with PBS, 50 �l cell-free BALF from mutant and WT miceinfected with intrapulmonary E. coli for 0, 6, or 24 h after LPS injectionwas added to the plate and incubated while shaking at room temperaturefor 2 h. After washing with wash buffer (1% BSA and 0.05% Tween inPBS), a peroxidase-labeled anti-DNA monoclonal antibody diluted 1:100in 1% BSA PBS (from the Cell Death Detection ELISAPlus kit, cataloguenumber 11774425001; Roche) was added, and the plate was incubated foranother 2 h. After another wash with washing buffer, 100 �l of 2,2=-

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azinobis(3-ethylbenzthiazolinesulfonic acid) (ABTS) solution (also fromthe Cell Death Detection ELISAPlus kit) was added for 45 min at roomtemperature in the dark. The optical density of the plate at a wavelength of405 nm was recorded.

Statistics. All statistical analyses were done using GraphPad Prism6.0 (GraphPad). CFU data are illustrated as individual values with

medians, whereas the remaining data are shown as means standarderrors of the means (SEM). Two groups were compared using Stu-dent’s t test, while multiple group comparisons (i.e., CFU) were con-ducted using a nonparametric one-way analysis of variance (ANOVA)(Kruskal-Wallis test), followed by Dunn’s test for multiple compari-sons. When applicable, multiple group comparisons were also made

FIG 1 The APR is dependent on liver STAT3 activation during endotoxemia followed by pneumonia. (A) Dual challenge of endotoxemia and pneumonia.WT and mutant mice lacking hepatic STAT3 were pretreated with an intraperitoneal injection of 5 mg/kg LPS. After 18 h, mice were intratracheallyinfected with 1 � 106 CFU of E. coli. Mice were euthanized at 0, 6, or 24 h after E. coli infection. At the indicated time points, serum (B, C) and BALF (D, E)were collected, and SAA and SAP acute-phase protein concentrations were measured using an ELISA. Dashed lines indicate baseline concentrations invehicle-treated, WT mice without pneumonia. *, P 0.05 versus WT mice at the indicated time points as determined by a two-way ANOVA followed bya Holm-Sidak test (n � 3 to 9 per group). (F, G) SAA and SAP concentrations from 24-hour serum and BALF were compared, and a correlation with alinear regression was performed. Each individual point represents a single mouse (both WT and mutant). Pearson r values and P values for eachcorrelation are shown (n � 19).

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using a two-way ANOVA followed by a Holm-Sidak test. Data wereconsidered significant at P values of 0.05 and were marked accord-ingly.

RESULTSThe APR is dependent on liver STAT3 during endotoxemia fol-lowed by pneumonia. In order to determine the effect of STAT3-dependent liver activation in the context of sepsis and pneumonia,we used the Cre-LoxP system to obtain a mouse model of hepato-cyte-specific, functional STAT3 deletion (hepSTAT3�/�). Tomodel the clinical circumstances of sepsis preceding pneumonia,we employed a dual challenge of endotoxemia followed by a bac-terial lung challenge (Fig. 1A). Mutant and WT mice were admin-istered an intraperitoneal injection of either 5 mg/kg of LPS orvehicle (saline). After 18 h, 1 � 106 CFU of E. coli was intratrache-ally instilled into left lung lobes for an additional 0, 6, or 24 h.Gram-negative infections, including E. coli pneumonias, are a ma-jor cause of nosocomial pneumonia (35, 36), which are particu-larly relevant during sepsis, as septic patients have a much greaterrisk of developing enterobacterial, hospital-acquired pneumonias(5, 6). As such, E. coli pneumonias were utilized in this model ofsepsis-induced pneumonia because of its specific relevance to sep-tic patients. Additionally, E. coli pneumonia models induce largeamounts of lung injury necessary to induce plasma protein (andthus APP) extravasation into the airspaces, which is key in under-standing how the liver response to sepsis can directly influencelocal lung defense. Because liver STAT3 activation is required formaximal APR induction (24, 25, 30, 31), we measured the concen-trations of two representative, circulating APPs: serum amyloid A(SAA) and serum amyloid P (SAP). In WT mice, the concentra-tions of both SAA and SAP in serum were induced dramaticallyabove baseline with LPS pretreatment alone (Fig. 1B and C, 0 h).Unlike SAA, SAP concentrations in serum were further increasedby E. coli infection, as there was a significant effect of infectiononly for SAP levels in serum (Fig. 1C). Independent of treatment(LPS and/or E. coli pneumonia), APP concentrations remainedunchanged in mutant mice but were significantly different fromthose of WT mice, indicating that hepatic STAT3 function is nec-essary for a maximal APR.

In order to determine whether an endotoxin-induced (STAT3-dependent) APR could affect the local lung environment, we sam-pled the protein and cellular content of the airspaces by bron-choalveolar lavage. LPS pretreatment alone was insufficient toalter baseline concentrations of airspace SAA and SAP in eithermouse genotype (Fig. 1D and E, 0 h). However, intrapulmonaryinfection with E. coli markedly increased the concentrations ofboth acute-phase proteins in the BALF of WT mice. SAP increasesin BALF were significantly blunted in mutant mice, with a similartrend observed for SAA (Fig. 1E and D, respectively). Thesechanges resembled those in the blood compartment, suggestingthat airspace APP content is a function of plasma extravasationinto pneumonic lungs, especially in the case of SAP. This is furtherevidenced by significant correlations between concentrations ofboth APPs in serum and BALF after 24 h of E. coli infection (Fig. 1Fand G).

Host defense during endotoxemia and pneumonia is com-promised by lack of hepatic STAT3. In order to determine if anendotoxemia-induced hepatic APR affects pulmonary host de-fense and/or inflammation during pneumonia, we measured 6-and 24-h lung and blood bacterial burdens in both genotypes of

mice pretreated with either LPS or vehicle. After 6 h of pneumo-nia, we observed lung bacterial burdens that were similar to theoriginal inoculum (approximately 106 CFU), with no statisticaldifference between any of the groups tested (Fig. 2A). Moreover,bacteremia was not detected in any group (Fig. 2B). At 24 hpostinfection, however, mutant mice pretreated with LPS had sig-nificantly greater lung bacterial burdens than any other group(Fig. 2C), suggesting that STAT3-dependent liver activity is re-quired for local defense in response to preexisting endotoxemia.HepSTAT3�/� mutant mice pretreated with LPS also had signif-icantly increased bacteremia, possibly due to differences in dis-semination and/or systemic clearance (Fig. 2D).

Bacterial killing in the lungs relies on innate immunity, includ-ing that provided by recruited neutrophils and other extravasatedplasma constituents during inflammation (37). In order to deter-mine whether local inflammation was compromised by STAT3deficiency, we measured BALF neutrophils and total protein con-centrations (Fig. 2E and F, respectively). We observed an influx ofneutrophils at 24 h after infection with E. coli in both WT andmutant mice, consistent with an acute pneumonia (Fig. 2E). Ad-ditionally, there were significantly greater numbers of neutrophilsrecruited to the airspaces in mutant mice than in WT mice at 24 hafter infection with E. coli, which was likely secondary to increasedbacterial loads. Total protein concentrations in BALF were alsoincreased due to infection, but no differences were observed be-tween genotypes (Fig. 2F), suggesting changes in serum APP con-centrations and not changes in protein delivery (from blood toairspaces) as the cause of BALF APP differences between geno-types. To reinforce this concept, we performed an additional cor-relation analysis comparing BALF APP concentrations with BALFtotal protein levels. Neither SAA nor SAP levels significantly cor-related with pulmonary edema (for SAA, P � 0.4916, r ��0.1733; for SAP, P � 0.3802, r � �0.2201), suggesting that anychanges in BALF APP content are linked to expression differenceseither systemically or in the lungs themselves (particularly in thecase of mutant mice). Interestingly, 24 h after administration of E.coli in endotoxemic hepSTAT3�/� mice, impaired antibacterialdefense was associated with increased mortality (Fig. 2G). Thesedata suggest that STAT3-dependent liver responses are protectivein the setting of sepsis followed by pneumonia. This response,however, does not appear to be mediated through alveolar neu-trophil recruitment.

Pulmonary and systemic cytokine induction is not reliant onSTAT3-dependent acute-phase changes. As another index oflung and systemic inflammation, cytokine protein concentrationsin BALF and serum were measured (Fig. 3 and 4). We utilized amultiplex bead array to determine the concentrations of 10 cyto-kines, all of which are relevant to pneumonia and/or lung injury(37, 38): IL-1�, IL-6, IL-10, IL-17, G-CSF, GM-CSF, CXCL1,TNF-�, LIF, and CXCL2. We observed several patterns of cyto-kine kinetics in the airspaces, ranging from increases due to E. coliinfection to no change at all, but there were no changes in BALFcytokine concentrations due to genotype (Fig. 3). Serum cytokinechanges were also variable across targets; however, unlike in theBALF, three serum cytokines were significantly changed due to theabsence of liver STAT3: IL-1�, IL-17, and TNF-�. Concentrationsof both IL-17 and TNF-� were significantly greater in mutantmice, consistent with increased bacteremia. Interestingly, IL-1�was significantly decreased in mutant mice after LPS pretreatment(0 h after administration of E. coli), but not during the course of

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FIG 2 Host defense during endotoxemia and pneumonia is compromised by lack of hepatic STAT3. WT and mutant mice were treated for 18 h with intraperitoneal LPSor saline followed by intratracheal E. coli. After 6 (A, B) and 24 (C, D) hours of E. coli infection, lung homogenates (A, C) and blood (B, D) were processed forquantification of viable bacteria. †, P 0.05 between the denoted groups based on a Kruskal-Wallis test followed by Dunn’s multiple-comparison test (n � 5 to 16 pergroup). At the indicated time points, BALF was harvested for determination of recruited neutrophil numbers (E) and total protein concentrations (F). *, P 0.05 versusWT mice at the indicated time points as determined by a two-way ANOVA followed by a Holm-Sidak test (n � 3 to 9 per group). ND, not detected. (G) Survival wasobserved through 24 h of E. coli infection. *, P 0.05 versus all other groups as determined by a Mantel-Cox test (n � 19 to 52 per group).

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FIG 3 Pulmonary cytokine induction is unaffected by hepatic STAT3 deletion. WT and mutant mice were treated for 18 h with intraperitoneal LPS followed byintratracheal E. coli. At the indicated time points after E. coli infection, lungs were lavaged, and BALF cytokine protein concentrations were determined using amultiplex bead array. Dashed lines (some of which overlap the x axis) indicate baseline concentrations in vehicle-treated, WT mice without pneumonia. Therewas no significant overall effect of genotype observed, as determined by a two-way ANOVA (n � 3 to 9 per group).

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FIG 4 Hepatic STAT3 activation has a minimal effect on circulating cytokine concentrations. WT and mutant mice were treated for 18 h with intraperitonealLPS followed by intratracheal E. coli. At the indicated time points after E. coli infection, serum was collected, and cytokine concentrations were measured with amultiplex bead array. Dashed lines (some of which overlap the x axis) indicate baseline concentrations in vehicle-treated, WT mice without pneumonia. ForIL-1�, the asterisk (*) indicates a P value of 0.05 versus WT mice at that time point as determined by a two-way ANOVA followed by a Holm-Sidak test. ForIL-17 and TNF-�, the asterisk (*) indicates a P value of 0.05 for overall effect of genotype as determined by a two-way ANOVA. n � 3 to 9 per group.

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infection, potentially indicating a small defect in systemic innateimmunity. Whether or how this genotype-dependent decrease inIL-1� contributes to the phenotype of this group remains unclear.

The hepatic APR does not modulate phagocytosis in airspacecells during endotoxemia and pneumonia. Hepatic STAT3�/�

mice with a preexistent endotoxemia have increased bacterialburdens both systemically and locally during pneumonia. Neu-trophil recruitment and other inflammatory mediators (i.e.,cytokines) were either unchanged or increased in hep-STAT3�/� mice, suggesting that these aspects of host defenseare uncompromised in mutant mice. To determine if endotoxin-induced liver STAT3 activation affects cellular defenses duringpneumonia, we measured phagocytosis in airspace macrophagesand neutrophils using pHrodo E. coli bioparticles. These biopar-

ticles are conjugated to a phycoerythrin (PE) fluorophore thatfluoresces only in low-pH environments, characteristic of thephagolysosomal compartment. Multiple laboratories have val-idated this system as an effective strategy for discriminatingbetween surface-bound and internalized particles (39–41). Af-ter 18 h of i.p. LPS administration, mutant and WT mice werei.t. infected with E. coli for 6 h, followed by a second i.t. instilla-tion with pHrodo E. coli bioparticles. After 1 h, the lungs werelavaged and cells were analyzed by flow cytometry (Fig. 5A). Air-space macrophages and neutrophils included cells positive forphagocytosis of pHrodo bioparticles (Fig. 5B). Neither macro-phages nor neutrophils, however, exhibited genotype-dependentdifferences in the frequency (Fig. 5C) or magnitude (Fig. 5D) ofphagocytosis. These data suggest that bacterial uptake and

FIG 5 The hepatic APR does not modulate phagocytosis in airspace cells during endotoxemia and pneumonia. (A) WT and mutant mice were treated withintraperitoneal LPS for 18 h. Afterwards, E. coli was instilled intratracheally, followed 6 h later by a second instillation of E. coli pHrodo particles. Lungs werelavaged an hour later, and cells were stained as follows for flow cytometry: neutrophils (CD45�/7AAD�/Ly6G�/F4/80�), alveolar macrophages (CD45�/7AAD�/F4/80�/Ly6G�/Autofluorescencehi). (B) Representative histograms illustrate the percentages of cells positive for pHrodo particle phagocytosis in WT(black line) and mutant (dashed line) mice. Filled curves (gray) represent cells not exposed to pHrodo particles. Summarized data for all mice studied werecalculated to determine the frequency (C) and magnitude (D) of particle ingestion, as determined by the percentage of positive cells and mean fluorescenceintensity, respectively. No significant changes between genotypes were detected, as assessed by Student’s t test (n � 5 or 6 per group).

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phagolysosomal fusion are unlikely to be responsible for impairedbacterial killing in the absence of hepatocyte STAT3 during endo-toxemia and pneumonia.

Maximal ROS generation in airspace macrophages is depen-dent on hepatic STAT3 activation. As an alternative contributorto cellular host defense, ROS generation was measured in airspacecells from both genotypes following 6 h of pneumonia in endo-toxemic mice. Total cells were stained for surface antigens to iden-tify macrophages and neutrophils as described above, and ROSproduction was measured using the CellROX Deep Red Reagentfrom Life Technologies (Fig. 6A). Interestingly, airspace macro-phages from mutant mice had significantly less ROS productionthan those from WT mice (Fig. 6B). A similar trend was apparentwith neutrophils, but this did not reach statistical significance(Fig. 6B). These data connect compromised macrophage ROS

production to impaired pulmonary host defense in endotoxemichepSTAT3�/� mice.

Macrophage intracellular bacterial killing is not impaired byliver STAT3 deletion. As another assessment of cellular host de-fense, we utilized a gentamicin protection assay in which we incu-bated primary alveolar macrophages with luminescent E. coli.Macrophages were originally harvested from mutant or WT mice18 h after treatment with i.p. vehicle or LPS. After an hour to allowfor bacterial uptake, gentamicin, which does not penetrate the cellmembrane, was added to kill any remaining extracellular bacteria,and luminescence was recorded hourly as a measure of bacterialviability. Interestingly, initial bacterial uptake was reduced inmacrophages from either genotype after LPS treatment (Fig. 6C, 0h), consistent with previous reports detailing alveolar macrophagedysfunction after sepsis. Although the uptake of bacteria was com-

FIG 6 Alveolar macrophage reactive oxygen species (ROS) production and airspace bacterial resistance are dependent on the hepatic STAT3 activation. (A) WTand mutant mice were treated for 18 h with intraperitoneal LPS followed by an intratracheal instillation of E. coli. Six hours later, the lungs were lavaged andrecovered cells were stained using the CellROX Deep Red Reagent to determine ROS generation in neutrophils (PMN; CD45�/7AAD�/Ly6G�/F4/80�) andalveolar macrophages (M�; CD45�/7AAD�/F4/80�/Ly6G�/Autofluorescencehi). Representative histograms illustrate the mean fluorescence intensity (MFI) forthe populations positive for ROS generation in WT (black line) and mutant (dashed line) mice. Filled curves (gray) represent cells not exposed to CellROXreagent. (B) ROS generation was quantified in each cell type, and data are shown as the percentage of ROS generation observed in WT mice. §, P 0.05 versusWT as determined by Student’s t test (n � 5 or 6 per group, with each point corresponding to an individual mouse). (C) Mutant and WT mice were treated witheither i.p. vehicle or LPS for 18 h. Lungs were lavaged, and alveolar macrophages were isolated and plated in a 96-well plate. After adhering for 1 h, 1 � 107 CFU/mlof luminescent E. coli were incubated with the macrophages for another hour. Additionally, control, cell-free wells containing only E. coli were included. After 1h, all wells were washed (including the E. coli-only control wells to account for residual bacteria after washing) and then incubated in 100 �g/ml of gentamicinfor 4 h. Luminescence, as a metric of bacterial viability, was recorded at the start and every hour thereafter. The WT/saline group was statistically different fromthe other groups as determined by a two-way ANOVA followed by a Tukey post hoc test for individual comparisons among groups (n � 5 to 9, with each wellcorresponding to a different mouse). Additionally, the experiment was performed on three separate days. *, P 0.05 versus all other groups. §, P 0.05 versusWT/LPS and E. coli � gentamicin. (D) Neutrophil extracellular traps (NETs) were quantified by an MPO-DNA ELISA in the BALF from WT and mutant micepretreated with LPS for 18 h followed by intrapulmonary infection with E. coli for the indicated time periods. A significant overall effect of infection but notgenotype was observed, as determined by a two-way ANOVA (n � 3 to 9 per group, corresponding to BALF used from individual mice infected in at least twoindependent experiments). (E) The same cell-free BALF as that used in panel D was incubated with log-phase luminescent E. coli, rotating at 37°C for 5 h. Bacterialgrowth was calculated as fold increases in luminescence compared to the starting values for each sample. *, P 0.05 versus WT at the specified time point asassessed by a two-way ANOVA followed by a Holm-Sidak test (n � 3 or 4 per group, corresponding to BALF used from 3 or 4 individual mice infected in at leasttwo independent experiments).

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promised in macrophages isolated from LPS-treated mice, no ad-ditional changes were observed due to genotype (Fig. 6C). In fact,luminescence barely exceeded the levels detected in negative-con-trol wells (no macrophages), limiting the opportunity to deter-mine additional effects resulting from liver STAT3 deficiency.

Soluble host defense mediators within the airspaces are de-pendent on the hepatic APR. Neutrophils are immediately re-cruited to the alveolar compartment during early stages of infec-tion to aid in pathogen clearance (42). As an innate defense, inaddition to phagocytosis, neutrophils are equipped to release en-dogenous genomic DNA laced with antimicrobial proteins to ef-fectively trap and lyse invading microbes. These NETs are studdedwith granulocytic proteins, including MPO (43). As a way to de-termine whether NET release was affected by the APR, we mea-sured the relative concentrations of NETs in BALF from WT andmutant mice after endotoxemia and pneumonia (Fig. 6D). As an-ticipated, we observed an overall increase in NET release due topneumonia, and while there is a trend toward decreased NETrelease in mutant mice, this difference did not reach statisticalsignificance (Fig. 6D).

In order to determine whether extracellular products otherthan NETs may contribute to differential bacterial resistance inthe alveolar lining fluid, we developed an assay in which we incu-bated luminescent E. coli (strain Xen14) in cell- and bacteria-freeBALF from endotoxemic and pneumonic WT or mutant mice.Bacterial growth was calculated as fold increases in luminescencecompared to the starting values for each sample. Interestingly,BALF from mutant mice supported bacterial growth significantlymore than did that from WT mice (Fig. 6E), suggesting that theairspace milieu of mutant mice is less resistant to bacterial growth.Whether and how this change in bacterial resistance in the air-spaces relies on differences in the antimicrobial proteome or nu-trient availability of the alveolar lining fluid remains uncertain.

DISCUSSION

The results of this study demonstrate a novel role for the STAT3-dependent liver acute-phase response in driving innate host de-fenses during pneumonia in endotoxemic animals. Using a two-hit model of endotoxemia and intrapulmonary E. coli, weobserved impaired antibacterial defense and higher mortality inmice that were deficient in hepatic STAT3. While several indices ofinflammation (e.g., neutrophilia, edema, and cytokine induction)were largely unaffected by the interruption of hepatic activation,others (e.g., macrophage ROS generation and airway lining fluidcontent) were dependent on hepatic STAT3.

The physiologic and molecular mechanisms by which hepaticinnate responses mediate host defense during sepsis and pneumo-nia have never been elucidated. Several studies, however, haveimplicated an important role for hepatic STAT3 activation duringeither sepsis or pneumonia alone. Alonzi et al. described the ne-cessity of inducible liver STAT3 activation during endotoxemiafor induction of the APR (31). Additionally, Sakamori et al. used ahepatocyte-specific STAT3 knockout mouse to show the impor-tance of this signaling pathway in controlling excessive inflamma-tion during polymicrobial sepsis induced by cecal ligation andpuncture (CLP) (30). In fact, their results for mutant mice duringsepsis alone were consistent with our own, showing decreasedsurvival as well as increases in circulating cytokines; however, theydid not detect changes in blood bacterial burdens. Similarly,Sander et al. demonstrated that liver STAT3-dependent signaling

was also crucial to attenuate mortality, but not host defense, inresponse to CLP through a process facilitated by SAA-dependentmobilization of myeloid-derived suppressor cells (44). The lasttwo studies described above, while notable, were not designed todetermine the degree to which sepsis-induced liver activation (viaSTAT3) calibrates subsequent responses to pneumonia, which is ahighly distinct and clinically relevant scenario.

It is well established that in both septic patients and animalmodels, sepsis results in immunosuppression (45), which isthought to promote secondary infections such as those causingpneumonia (8, 46). A multitude of studies have revealed the det-rimental consequences of sepsis-induced immunosuppression oncritical pneumonia outcomes, including antibacterial defense, al-veolar macrophage function, alveolar neutrophil recruitment,and cytokine production (7, 9, 10, 12–15, 47–51). Our own pro-tocol of endotoxemia followed by pneumonia, however, was notsufficient to recapitulate the circumstances of sepsis-induced im-munosuppression. We observed no effect of endotoxemia aloneon pneumonia outcomes in WT mice, including pulmonary de-fense, lung cytokine expression, and neutrophil recruitment, butrather found that endotoxemia compromised bacterial clearanceonly in mice lacking hepatic STAT3 (Fig. 2C). There are manypossible explanations for this. First, the dose of LPS (5 mg/kg)and/or its type (an ultrapure, Toll-like receptor 4 [TLR4] agonist)may not be sufficient to induce immunosuppression in the settingof our pneumonia protocol. Additionally, the timing of LPS pre-treatment (18 h before E. coli infection) and/or the genetic back-ground of our hepSTAT3�/� mouse strain could also be factors.The lack of observable LPS-induced immunosuppression in WTmice, however, empowered us to more precisely examine the rolesof endotoxin-induced hepatic STAT3 activation on a subsequentlung infection, and this opportunity may have been diminished byoverwhelming immunosuppression due to LPS alone.

Independently, our laboratory and others have reported afunctional role for the APR in pneumonia alone. We have shown,using an APR-null mouse model (lacking both hepatic STAT3 andRelA), that liver activation is required for survival, hepatoprotec-tion, and maximal pulmonary inflammation during an E. colipneumonia (23), as well as systemic defense and opsonophagocy-tosis during pneumococcal pneumonia (24). The common clini-cal observation that sepsis is frequently followed by pneumonia(5, 6, 8) raises the question of whether or how a preexisting liverresponse alters pneumonia susceptibility, for better or for worse.Renckens et al. determined that a preexisting APR induced byturpentine impairs the pulmonary inflammatory response toPseudomonas aeruginosa and Acinetobacter baumannii (52, 53).The model of inducing a preexisting APR via turpentine injectionis very different from our method of inducing the APR throughendotoxemia. Additionally, turpentine’s effects are unlikely to belimited to liver activation. Using our hepatocyte-specific STAT3-null mouse in our model of endotoxemia followed by pneumoniaallowed us, for the first time, to interrogate the role of preexistingliver-specific acute-phase changes on pneumonia susceptibility.This is an important distinction from our earlier studies, whichexamined the global acute-phase changes (driven by both STAT3and RelA) in the setting of pneumonia alone. Moreover, by exam-ining the effects of preexisting STAT3-dependent liver responses,these studies aim to help clarify an important clinical/immuno-logical scenario in which sepsis modifies subsequent immune re-sponses to lung pathogens.

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In association with impaired APP induction, mutant mice pre-treated with LPS had significantly greater bacterial loads in thelungs and blood during pneumonia, implying that local pulmo-nary defenses are particularly affected during endotoxemia in theabsence of an intact liver response. Increased mortality was alsoobserved in this group, suggesting this defect in host defense as apotential cause of mortality. These outcomes were also associatedwith an increase in serum TNF-� that is likely due to greateramounts of circulating bacteria and could also contribute to deathin hepSTAT3�/� mice, as TNF-� can cause septic shock (54). Intrying to determine which aspects of host defense are mediated bythe sepsis-induced APR, we measured pulmonary inflammationand injury. We observed no decrease in neutrophil recruitment,pulmonary cytokine concentrations, or proteinaceous edema be-tween genotypes, suggesting that these characteristic measures ofinflammation were unlikely to contribute to host defense differ-ences in endotoxemic hepSTAT3�/� mice. In fact, the only appar-ent changes in lung cytokine levels (IL-6, G-CSF, and LIF) actuallytrended toward an increase, which we hypothesize to be secondaryto increased bacterial burdens in this experimental group. Overall,the immunosuppression observed in our own study differs fromprevious findings, which typically involve reduced cytokines andinflammation (9, 10).

Phagocytosis and NET production were also equivalent be-tween groups. Regarding the former, however, we acknowledgethe fact that pHrodo E. coli bioparticles (our method of quantify-ing phagocytosis) may not perfectly replicate interactions betweenliving E. coli and the inflammatory milieu (including opsoninssuch as extravasated APPs). Yet we observed extremely efficientuptake using this system (around 40 to 60%) in both cell typesanalyzed, supporting an environment sufficient for comparison ofphagocytic functions. Interestingly, ROS generation was signifi-cantly attenuated in alveolar macrophages from mutant mice,suggesting that the endotoxemia-induced hepatic APR facilitatesat least one fundamental aspect of cell-mediated antimicrobialdefense. We also employed a primary alveolar macrophage-basedbacterial killing assay to determine if differences in ROS produc-tion could manifest as changes in cellular bacterial killing ex vivo.Significantly more bacterial uptake was detected in macrophagesrecovered from vehicle-treated WT mice than in those from LPS-treated mice of either genotype (Fig. 6C). Yet we observed nodifferences in killing with or without hepatocyte STAT3, suggest-ing that either (i) liver STAT3 deficiency (in the setting of endo-toxemia) is insufficient to compromise the antibacterial functionof an otherwise unchallenged (no pneumonia) alveolar macro-phage or (ii) differences in bacterial killing are beyond the detec-tion limit of this experimental system, perhaps due to the smallamounts of bacterial uptake in macrophages from LPS-treatedmice. The immunosuppressive effect of endotoxemia on macro-phage function is consistent with that seen in other studies (11, 49,55). Future investigations are needed to determine whether orhow previously established pathways driving sepsis-inducedpulmonary immunosuppression are mechanistically linked toSTAT3-dependent liver activity.

We also assessed the capacity of alveolar lining fluid to influ-ence bacterial growth after endotoxemia in mice with and withoutSTAT3-dependent liver responses. Indeed, the composition ofsoluble mediators in this niche could have large implications onpathogen resistance at the air-liquid interface. In an effort to un-derstand whether the balance of growth-promoting and growth-

inhibiting soluble factors is dependent on the APR, we incubatedluminescent E. coli with cell- and bacterium-free BALF from mu-tant and WT mice collected at different time points followingendotoxemia and E. coli infection. Interestingly, BALF from mu-tant mice supported E. coli growth significantly more than thatfrom WT mice, suggesting that products downstream of hepaticSTAT3 activation create a less favorable environment for infectionin the airspaces. There have been multiple reports of antimicrobialpolypeptides, including lactoferrin, lysozyme, lipocalins, andbeta-defensins, which are produced in the liver and/or lungs (29,56–58) and could be modulated by the APR either directly orindirectly. Alternatively, increased growth in the BALF from mu-tant mice could be attributable to an altered nutrient pool that ismore supportive of bacterial replication. While our assay does notallow us to discriminate between the bactericidal and bacterio-static components of BALF, this finding, combined with the defectin macrophage ROS generation and increased bacterial burdens,strongly supports the concept that the sepsis-induced hepatic APRis a required component for maintaining pulmonary defense.

While our results identify a critical role for STAT3 activation inthe setting of endotoxemia, the actual mechanisms of LPS-in-duced liver STAT3 activity are not entirely clear. We and othershave shown IL-6 to be both sufficient and necessary for hepaticSTAT3 activation in a variety of contexts (16, 59–61). In responseto LPS, specifically, there is evidence suggesting an important rolefor IL-6, although others have shown evidence of IL-6-indepen-dent LPS-induced liver STAT3 as well. Mechanisms whereby theliver promotes lung defense constitute an even greater knowledgegap. Indeed, liver-derived acute-phase proteins are diverse innumber and function, promoting a wide variety of immunologicalprocesses. Previous studies from our laboratory have implicatedthe APR in the activation of airspace macrophages during pneu-monia (23), and a multitude of APPs have been shown to activatemacrophages (61–64). Also, pentraxins such as SAP and C-reac-tive protein (CRP) engage numerous receptors capable of activat-ing macrophages (and other cells), which can promote ROS gen-eration (65, 66). In addition to this example, which may be linkedto our current ROS results, many other liver-derived proteinslikely integrate to directly and/or indirectly enhance macrophageactivity. Which of these applies to the lung and/or macrophageresponses in our current studies is an avenue of future investiga-tion.

This study puts forth evidence of a novel, immunoprotectiverole for hepatic STAT3 during endotoxemia. Our data suggest thatendotoxemia may initiate both immunosuppressive and immu-noprotective responses, which were effectively balanced in WTmice. In the absence of liver STAT3, however, the immunosup-pressive effects of endotoxemia were revealed, indicating a protec-tive role for STAT3-dependent gene programs. Future insight intothe mechanisms by which sepsis-mediated liver activation is pro-tective during subsequent lung infections will provide valuable,alternative avenues for the treatment and prevention of sepsis andpneumonia.

ACKNOWLEDGMENTS

This work was supported by National Institutes of Health grants R00-HL092956, R01-HL111449, R01-HL079392, and T32-HL007035.

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