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Inhibition of Guanylyl Cyclase Restores Neutrophil Migration and Maintains Bactericidal Activity Increasing Survival in Sepsis

Paula-Neto, Heitor A.*; Alves-Filho, José C.; Souto, Fabricio O.; Spiller, Fernando; Amêndola, Rafael S.*; Freitas, Andressa; Cunha, Fernando Q.; Barja-Fidalgo, Christina*

Author Information
doi: 10.1097/SHK.0b013e3181e37ea8

Abstract

INTRODUCTION

Sepsis is a complex clinical syndrome resulting from a systemic inflammatory response to infection. Septic patients usually present dysfunctions in several physiological systems that, in a substantial number of individuals, can evolve to multiorgan failure and death (1, 2). Cardiovascular dysfunction, for example, is characterized by severe hypotension and, in a number of cases, refractoriness to hypertensive agents (3). In recent years, we and others have described an innate immune dysfunction in animal models of sepsis as well as in human septic subjects (4-13). This is represented by an inability of neutrophils to respond to chemotactic stimuli both in vivo and in vitro. This neutrophil dysfunction correlates well with outcome in septic patients and may contribute to aggravation of sepsis by precluding neutrophil efflux to tissues and an efficient control of the primary infectious focus.

Nitric oxide (NO) is a major contributor to sepsis pathophysiology. NO mediates the cardiovascular dysfunction observed in sepsis through its potent vasodilatory activity (14). Moreover, NO was shown to mediate neutrophil dysfunction in sepsis (4-7). Pharmacological inhibition of NO synthesis or genetic ablation of the inducible NO synthase (iNOS) gene rescues neutrophil functionality in experimental models of sepsis (5-7). Moreover, the unresponsiveness to chemotactic stimuli observed in neutrophils isolated from human septic patients correlates with increased iNOS expression and increased circulating nitrite/nitrate levels (8). However, iNOS-deficient mice or pharmacological inhibition of NO synthesis in animals and human septic patients resulted in increased mortality (5, 7, 15, 16). To our understanding, the limitations of NOS inhibition in sepsis could result at least in part from an interference with the ability of cells to produce NO and kill bacteria to control infection. In fact, in the cecal ligation and puncture (CLP) model of sepsis, iNOS-deficient mice and animals treated with NOS inhibitors presented similar bacterial loads in peritoneal cavity and blood, despite the recovery in neutrophil migratory function (5, 7).

Inhibition of the downstream effector of NO, the enzyme-soluble guanylyl cyclase (sGC) has proven benefit in sepsis and septic shock (17-21). The rationale for the use of sGC inhibitors in sepsis is the recovery of cardiovascular function. However, although the use of both NO or sGC inhibitors results in similar recovery of cardiovascular function in sepsis, only the inhibition of sGC has proved beneficial effects in sepsis outcome (3,15, 17-19, 22). These results suggest that sGC inhibition may have beneficial effects over other physiological systems that could contribute to its observed efficacy. Additionally, the well-recognized dysfunction in neutrophil migration observed in sepsis is NO-dependent, but the involvement of sGC in this process has never been addressed.

Here, we extended our previous studies to investigate the participation of sGC activation in the establishment of neutrophil dysfunction in sepsis. We used an in vitro model of neutrophil stimulation with Toll-like receptor (TLR) ligands to dissect the modulation of neutrophil migratory function by sGC activity. We also used an experimental model of sepsis to confront the effects of iNOS and sGC inhibition in vivo regarding neutrophil function, the effects on infection control, and mice survival.

MATERIALS AND METHODS

Neutrophil isolation and treatment

Blood was collected from human healthy volunteers and transferred to EDTA-containing tubes. Volunteer enrollment and blood handling followed the guidelines of the institution's ethics committee. Informed consent was obtained. Neutrophils were isolated from human venous blood using Percoll (Amersham Biosciences, Pittsburgh, Penn) gradients as described (23). Neutrophil purity and viability were greater than 97% and greater than 99%, respectively, as accessed by differential counts and trypan dye exclusion. Isolated neutrophils (106 cells/mL) were treated with either 1400W, a specific iNOS inhibitor (30 μM); ODQ, a sGC inhibitor (10 μM); KT5823, a specific cGMP-dependent protein kinase (PKG) inhibitor (3 μM); or vehicle (dimethyl sulfoxide 0.01%) for 30 min. Cells were then stimulated with LPS (10 μg/mL) for a further 60 min, washed, and assayed as described below. Where indicated, cells were treated with BAY41-2272, an NO-independent sGC activator (30 μM), for 60 min. Concentrations used were determined in preliminary concentration-effect experiments accessing in vitro neutrophil chemotaxis. In these experiments, we observed no effect of the inhibitors alone or vehicle on neutrophil chemotactic responses to all chemoattractants used (not shown). In selected experiments neutrophils were treated with lipoteichoic acid (LTA, 10 μg/mL) exactly as described for LPS.

In vitro neutrophil chemotaxis

Neutrophil chemotaxis was evaluated using a 48-well Boyden chamber (Neuroprobe Inc, Gaithersburg, Md) using 5-μm-pore-size polyvinylpyrrolidone-free polycarbonate membranes as described (23). Chemoattractants (28 μL) we placed in the bottom chamber and 50 μL of the neutrophil suspension (106 cells/mL) were added to the top chamber. The chambers were then incubated for 1 h at 37°C with 5% CO2, after which membranes were removed, fixed, and stained with Diff-Quick staining kit (Laborclin, Pinhais, Parana, Brazil). The number of neutrophils that have migrated to the lower side of the filter was counted in at least five random fields (1,000× magnification). The results are representative of at least three independent experiments performed in triplicate for each sample and are expressed as mean ± SEM of the number of neutrophils per field. Chemoattractants used were N-formyl-methionyl-leucyl-phenylalanine (fMLP; 10−7M), leukotriene B4 (LTB4; 10−8M), or IL-8 (30 nM). Migration to medium alone (random migration) was used as negative control.

Determination of cGMP intracellular levels

Measurement of cGMP levels was carried out using a commercial kit (GE Healthcare UK Limited, Buckinghamshire, UK). Neutrophils (106 cells) were treated as described, washed, and resuspended in 180 μL RPMI medium. Cells were immediately frozen and kept at −80°C until use. Samples were manipulated as recommended by the manufacturer to determine the total cellular cGMP content.

Flow cytometry

Neutrophils were treated as above and immediately fixed with ice-cold 1% paraformaldehyde in phosphate-buffered saline (PBS). After extensive wash in FACS buffer (2% bovine serum albumin, 5% fetal calf serum, 0.1% sodium azide in PBS), cells were resuspended in 50 μL FACS buffer and incubated with phycoeritrin (PE)-labeled mouse IgG1 anti-human CXCR2 or PE-labeled mouse IgG2a anti-human BLT1 (1:50) for 30 min at 4°C. Cells were then washed in FACS buffer and analyzed using a FACScalibur (BD Biosciences, San Jose, Calif) with help of Cell Quest software. In the CXCR1 expression analysis, the staining protocol was slightly modified. After fixing the cells and washing as described, neutrophils were resuspended in 50 μL FACS buffer and incubated overnight at 4°C with mouse anti-human CXCR1 antibody (1:50; Santa Cruz Biotechnology, Santa Cruz, Calif). After extensive wash in FACS buffer, neutrophils were incubated with secondary fluorescein isothiocyanate-labeled anti-mouse antibody (1:50; Sigma, St. Louis, Mo) for 2 h at 4°C. Stained cells were then washed in FACS buffer, resuspended, and analyzed as described above. Data were analyzed using FCS Express 3.0 for Windows (DeNovo Software, Los Angeles, Calif).

Immunofluorescence

Neutrophils were treated as described above, and G protein-coupled receptor kinase 2 (GRK2) expression was evaluated using a rabbit anti-human GRK2 (1:200) and AlexaFluor 594-conjugated anti-rabbit secondary antibody (Invitrogen, Carlsbad, Calif). Nuclei were counterstained with DAPI. Images were acquired on an epifluorescence microscope (BX-50, Olympus, Center Valley, Pa).

Western blotting

Neutrophils (4 × 106 cells) treated as described were immediately lysed with 80-μL boiling lysis buffer (1% sodium dodecyl sulfate, 1 mM orthovanadate, 10 mM Tris, pH 7.4). Lysates were boiled for 3 min, sonicated, and boiled for a further 2 min. Samples of 5 μL were diluted 1:10 and used for protein quantification by the Bradford method. Lysates were mixed with 20 μL 5× sample buffer, boiled for 3 min, and stored at −20°C until use. Protein samples of 30 μg were separated on a 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to PVDF membranes. GRK2 and β-actin were detected using primary rabbit anti-GRK2 and mouse anti-β-actin antibodies (1:1,000) followed by appropriate horseradish peroxidase-conjugated secondary antibodies (1:10,000). Immunoreactive bands were visualized by ECL. Images were acquired and then analyzed with ImageJ software.

Mice and in vivo procedures

All animal procedures were approved by and conducted following the guidelines of the ethics committee of the Faculdade de Medicina de Ribeirão Preto (FMRP-USP, protocol number 181/2008). C57/BL6 male and female mice, weighing 20 to 25 g, were kept in specific pathogen-free conditions with free access to food and water.

Cecal ligation and puncture

Cecal ligation and puncture was carried out as described previously (10). In brief, mice were anesthetized with tribromoethanol (250 mg/kg); a 1-cm midline incision was made in the abdomen. Cecum was exposed and ligated below the ileocecal junction without causing bowel obstruction. A single puncture was made through the cecum with a 21- or 16-gauge needle to induce sublethal (SL-CLP) or lethal (L-CLP) sepsis, respectively. Peritoneal wall and skin incision were closed, and animals received 1 mL saline subcutaneously.

Pretreatment protocol

Mice were treated with 5 μmol/kg ODQ or 3 mg/kg 1400W by a single s.c. injection 30 min before CLP. Neutrophil migration to the peritoneal cavity, neutrophil sequestration in lungs (as determined by lung myeloperoxidase [MPO] activity), and blood and peritoneal colony-forming unit (CFU) enumeration were carried out 6 h after CLP as described below. In another set of experiments, mice were treated as above, and survival was monitored every 12 h through 72 h after surgery. Doses used were chosen based on our previous report (24). ODQ was diluted in PBS 1% dimethyl sulfoxide, and 1400W was diluted in sterile saline.

Posttreatment protocol

Mice were posttreated with two protocols. In the first, animals were treated with two s.c. injections of 5 μmol/kg ODQ, 3 and 12 h after CLP, and survival was monitored for 72 h after CLP procedure. In the second protocol, animals were treated with three s.c. injections of 5 μmol/kg ODQ at 3, 12, and 24 h after CLP, and survival was monitored for 96 h after CLP procedure.

Neutrophil migration in vivo

Neutrophil migration to the peritoneal cavity was carried out 6 h after CLP as described (10). Peritoneal cavity was washed using 3 mL PBS containing 1 mM EDTA. Total cell counts were performed on hemocytometer, and differential cell counts were carried out on cytocentrifuge smears stained with Diff-Quick staining kit (Laborclin). Results are presented as mean ± SEM of the number of neutrophils per cavity.

Neutrophil apoptosis in vivo

Peritoneal cavities were washed as described above. Collected cells were stained with annexin-V and propidium iodide and analyzed by flow cytometry. Gated neutrophils were considered dead when stained annexin V single positive (apoptotic) or annexin V/propidium iodide double positive (necrotic). Data were analyzed using FCS Express 3.0 for Windows.

Blood and peritoneal colony-forming unit enumeration

Blood and peritoneal CFU enumeration were carried out 6 h after CLP as described (10).

Lung MPO activity

Neutrophil sequestration in lungs was determined by the MPO activity in tissue extracts as described (10), 6 h after CLP. Lungs were perfused with 3 mL of PBS via the right ventricle, removed, and transferred to 1.5-mL tubes containing pH 4.7 buffer (0.1 M NaCl, 0.02 M Na2HPO4, 0.015 M sodium EDTA). Samples were weighted, frozen, and kept at −20°C until use. Upon thawing, tissue was homogenized in buffer and centrifuged at 3,000g for 10 min, and the pellet was subjected to hypotonic lysis (1.5 mL of 0.2% NaCl solution followed 30 s later by addition of an equal volume of a solution containing 1.6% NaCl and 5% glucose). After centrifugation, pellet was resuspended in 0.05 M phosphate buffer (pH 5.4) containing 0.5% hexadecyl-trimethylammonium bromide and rehomogenized. One-milliliter aliquots of the suspension were transferred into 1.5-mL tubes followed by three freeze-thaw cycles. The aliquots were then centrifuged for 15 min at 3,000g before performing the assay. Myeloperoxidase activity in the resuspended pellet was assayed by measuring the change in OD450 nm using tetramethylbenzidine (1.6 mM) and H2O2 (0.5 mM). The reaction was stopped after 30 min of incubation at room temperature, by adding 100 μL of 4 M H2SO4 and was quantified at 450 nm in a spectrophotometer. Neutrophil content was calculated from a standard curve using neutrophils isolated from murine bone marrow. Results are expressed in relative number of neutrophils per milligram of wet tissue.

Determination of cytokine and chemokine levels

Cytokine and chemokine levels in peritoneum and cytokine levels in serum were determined by enzyme-linked immunosorbent assay. Peritoneal cavity was washed with 1.5 mL PBS containing 1 mM EDTA, 6 h after CLP. Blood samples were taken 6 h after CLP. Samples from peritoneal lavage fluid and blood were centrifuged, aliquoted, and stored at −80°C until use. Results are expressed as mean ± SEM of the amount of cytokine or chemokine (in picograms) per cavity (for peritoneal lavage fluid) or per milliliter of blood.

Reagents

Escherichia coli LPS (serotype 0111:B4), Staphylococcus aureus LTA, N-[[3-(aminomethyl)phenyl]methyl]-ethanimidamide dihydrochloride (1400W), rhodamine-conjugated phalloidin, bovine serum albumin, and fMLP were from Sigma. Percoll was from Amersham Biosciences. [1H-(1,2,4)oxadiazolo(4,3-a)quinoxalin-1-one (ODQ), (9S,10R,12R)-2,3,9,10,11,12-hexahydro-10-methoxy-2, 9-dimethyl-1-oxo-9, 12-epoxy-1H-diindolo [1,2,3-fg:3′,2′,1′-kl]pyrrolo [3,4-i][1,6]benzodiazocine-10-carboxylic acid, methyl ester (KT5823) and LTB4 were from Tocris Biosciences (Ellisville, Mo). [5-cyclopropyl-2-[1-(2-fluoro-benzyl)-1H-pyrazolo[3,4-b]pyridine-3-yl]pyrimidin-4-ylamine] (BAY 41-2272) was from Calbiochem (Darmstadt, Germany). IL-8 was from R&D Systems (Minneapolis, Minn). PE-labeled anti-human CXCR2 and BLT1 antibodies were from BD Biosciences. Anti-human GRK2, anti-β-actin, and horseradish peroxidase-conjugated secondary antibodies were from Santa Cruz Biotechnology.

Statistical analysis

Data were analyzed with help of GraphPad Prism 4 for Windows. Statistical significance was tested using ANOVA followed by Bonferroni posttest, except for differences in survival rates, which were tested using GraphPad Prism 4 (GraphPad Software Inc., La Jolla, Calif) built-in survival analysis (log-rank test), and bacterial counts, which were tested using Mann-Whitney U test. P < 0.05 was considered statistically significant.

RESULTS

TLR ligands inhibit human neutrophil chemotaxis through an NO-sGC-PKG-dependent pathway

Diminished neutrophil responsiveness to chemotactic stimulation during sepsis has been previously shown by our group and others in animals as well as human patients (4). This well correlates with sepsis outcome, and experimental data support the involvement of iNOS-derived NO production in this phenomenon (4-8). However, the molecular mechanisms involved are currently unknown. To better access the mechanisms underlying neutrophil unresponsiveness to chemotactic stimulation during severe sepsis, we used a model in which isolated human neutrophils were stimulated ex vivo with LPS, a TLR4 ligand. This stimulation induced a decrease in chemotactic responsiveness to the classic neutrophil chemoattractants IL-8, LTB4, and fMLP (Fig. 1, A-C).

F1-4
Fig. 1:
TLR ligands inhibit human neutrophil chemotaxis through an NO-sGC-PKG-dependent pathway. Neutrophils were treated for 30 min with selective pharmacological inhibitors of iNOS (1400W, 30 μM), sGC (ODQ, 10 μM), or PKG (KT, 3 μM) and stimulated for a further 60 min with LPS (10 μg/mL, panels A-C) or LTA (10 μg/mL, panels D and E). After washing, cells were assayed for chemotaxis in response to IL-8 (30 nM, A), LTB4 (10−8 M, B and D), or fMLP (10−7 M, C and E). Data represent mean ± SEM of the number of neutrophils per 1,000× field counted under optical microscopy. Data derived from three independent experiments. F, Total intracellular cGMP content was accessed in cell extracts of human neutrophils. Data represent mean ± SEM of cGMP content (fmol) per 106 cells in four independent experiments. *Significant differences relative to control neutrophils migrating in response to stimuli.

Treatment of cells with the selective iNOS inhibitor, 1400W, before TLR stimulation, completely restored neutrophil chemotactic responses. This effect was also observed in cells pretreated with ODQ, a selective sGC inhibitor, or KT5823, a selective inhibitor of the sGC downstream target PKG (Fig. 1, A-C). Similar results were observed when we used the TLR2 ligand, LTA. Lipoteichoic acid induced a significant decrease in neutrophil responsiveness to IL-8 and fMLP, which could be restored by pretreatment of cells with pharmacological inhibitors of the components of the NO-sGC-PKG signaling axis (Fig. 1, D and E). Additionally, we measured the total cGMP cellular content in neutrophils stimulated with LPS. As depicted in Figure 1F, LPS induced a marked increase in total intracellular cGMP content, which, as expected, was inhibited by treatment of cells with 1400W or ODQ.

To further characterize the involvement of the NO-sGC-PKG signaling pathway on the impairment of neutrophil chemotaxis, we used the NO-independent sGC activator, BAY41-2272. As shown in Figure 2, A and B, incubation of neutrophils with BAY led to a reduced chemotactic response to IL-8 and fMLP similar to that observed with LPS. Taken together, the data suggest that TLR activation stimulates NO production and activation of the sGC-cGMP-PKG signaling axis, leading to impaired chemotactic responsiveness in human neutrophils.

F2-4
Fig. 2:
sGC activation associates with decreased neutrophil responsiveness to chemotactic stimuli. Neutrophils were incubated for 60 min with the sGC activator BAY41-2272 (BAY) and assayed for chemotactic response to IL-8 (30 nM, A) or fMLP (10−7 M, B). Data represent mean ± SEM of the number of neutrophils per 1,000× field counted under optical microscopy. Data derived from three independent experiments. *Significant differences relative to control neutrophils migrating in response to stimuli.

LPS induces downmodulation of chemotactic receptor expression through an NO-sGC-PKG-dependent pathway

We next tested whether LPS stimulation leads to diminished chemotactic receptor expression in neutrophil membrane. Neutrophils incubated with LPS showed diminished membrane expression of both IL-8 receptors CXCR1 and CXCR2, and the LTB4 receptor, BLT1 (Fig. 3). These effects could be reverted by pretreatment of cells with pharmacological inhibitors of iNOS, sGC, or PKG (Fig. 3). This suggested that at least part of the inhibitory effects of LPS on neutrophil migration could be attributed to a downmodulation of neutrophil chemotactic receptor expression and that this effect is dependent on the NO-sGC-PKG signaling pathway.

F3-4
Fig. 3:
LPS induces downmodulation of chemotactic receptor expression through an NO-sGC-PKG-dependent pathway. Neutrophil membrane expressions of BLT1 (left panels), CXCR1 (middle panels), and CXCR2 (right panels) were accessed by FACS analysis in neutrophils treated as described in Figure 1. Histograms represent one representative of four independent experiments. In all histograms, control cells (black line), cells treated with LPS (dark gray line), and cells treated with inhibitors before LPS treatment (light gray line) are represented. Upper histograms represent cells pretreated with 1400W. Middle histograms represent cells pretreated with ODQ. Lower histograms represent cells pretreated with KT. Graphs are a representation of the FACS data and indicates BLT1 (left), CXCR1 (middle), or CXCR2 (right) mean fluorescence intensity (MFI). Data are mean ± SEM from four independent experiments. *Significantdifference relative to control cells.

LPS upregulates GRK-2 expression through an NO-sGC-PKG-dependent pathway

Chemoattractants signal through G protein-coupled receptors (GPCRs), and their signaling activity is finely regulated by receptor desensitization through the activity of GPCR kinases (GRKs). G protein-coupled receptor kinases mediate GPCR desensitization through receptor phosphorylation and targeting for β-arrestin-dependent internalization, thus turning cells hyporresponsive to subsequent GPCR stimulation (25). Indeed, we recently showed that neutrophils isolated from septic patients presented an increased GRK2 and GRK5 expression (11), and others have shown that in vitro LPS stimulation upregulates GRK2 expression in neutrophils (26). Additionally, CXCR1 and CXCR2 are putative targets for GRK2 (26, 27). This encouraged us to test whether the observed receptor internalization could be explained by an upregulated GRK expression triggered by LPS. Using immunofluorescence and Western blot analysis, we accessed the relative GRK2 expression in human neutrophils stimulated ex vivo with LPS. As shown in Figure 4, LPS stimulation upregulates GRK2 expression in human neutrophils. Pretreatment of cells with 1400W, ODQ, or KT5823 prevented LPS-induced GRK2 upregulation, which may explain the reversal of chemotactic receptor internalization by pretreatment of cells with pharmacological inhibitors of the NO-sGC-PKG signaling pathway.

F4-4
Fig. 4:
LPS upregulates GRK-2 expression through an NO-sGC-PKG-dependent pathway. Upper panel, Immunofluorescence analysis of neutrophils treated as in Figure 1. GRK2 is visualized as red fluorescence (AlexaFluor 594) and nuclei as blue (DAPI). Images show a representative experiment, and graph shows the quantification of three independent experiments. *Significant differences relative to control cells. Lower panel, Western blot analysis of neutrophils treated as in Figure 1. Effect of 1400W (left), ODQ (middle), and KT (right) on the LPS-induced increase in GRK2 expression is presented. One representative experiment of three is presented. Quantification of GRK2 expression relative to the housekeeping marker β-actin is represented in numbers, considering GRK2 expression in control cells as 1. *Significant differences relative to control cells.

Effects of in vivo ODQ treatment in an experimental model of sepsis

As our results pointed to the participation of sGC activation on the NO-dependent impairment in neutrophil chemotactic function, we sought to determine the relative contribution of sGC activation to the establishment of neutrophil dysfunction in vivo. In this regard, we accessed the in vivo effects of GC inhibition in an experimental model of severe sepsis. As shown in Figure 5A, during a controlled, self-limiting infection (SL-CLP), mice presented an important neutrophil influx to the peritoneal cavity 6 h after CLP. In contrast, mice submitted to severe sepsis (L-CLP) presented a substantial decrease in neutrophil migration. This decrease in neutrophil numbers could not be attributed to kinetic differences between SL-CLP and L-CLP groups, because a time-point evaluation of neutrophil migration demonstrated that neutrophil migration in the L-CLP mice did not reach the peak values found in the SL-CLP group in any of the time points analyzed (see Figure, Supplemental Digital Content 1, https://links.lww.com/SHK/A50).

F5-4
Fig. 5:
Effects of iNOS or sGC inhibition in an experimental model of sepsis. A, Absolute neutrophil numbers in peritoneal cavity of mice submitted to SL-CLP, L-CLP, or L-CLP 30 min after treatment with ODQ (5 μmol/kg, s.c.) or 1400W (3 mg/kg, s.c.). Data represent mean ± SEM of the number of neutrophils percavity. *Significant difference relative to SL-CLP group. B, Survival curve for mice submitted to L-CLP 30 min after ODQ or 1400W treatment. Graph shows the percent survival through the 72-h observation period. Mice that have undergone surgery but receiving no punctures (sham) were used as controls. N = 8 for each experimental group. P values are shown for the comparisons between L-CLP and L-CLP/1400W groups, and between L-CLP and L-CLP/ODQ groups. C and D, Bacterial colony-forming unit (CFU) counts in peritoneal cavity (C) and in blood (D) of mice submitted to SL-CLP, L-CLP, or L-CLP 30 min after treatment with ODQ or 1400W. Data represent the log of the CFU count present in peritoneal cavity (C) and blood (D). *Significant difference relative to L-CLP group. N.D. indicates not detected. E, Neutrophil numbers in lung extracts of mice submitted to SL-CLP, L-CLP, or L-CLP 30 min after treatment with ODQ or 1400W. Data represent mean ± SEM of thenumber of neutrophils per milligram of wet tissue. *Significant difference relative to SL-CLP group.

Diminished neutrophil migration in the L-CLP group was prevented by pretreatment of mice with both 1400W and ODQ. Interestingly, mice pretreated with 1400W succumbed to infection in a similar rate than that observed in the untreated group (P = 0.5288, relative to untreated L-CLP mice), whereas mice pretreated with ODQ presented 50% survival (P = 0.0154, relative to untreated L-CLP mice; and P = 0.1140, relative to sham group) (Fig. 5B). Analyzing bacterial infection, we found that animals pretreated with 1400W presented bacterial counts in peritoneum and blood similar to those found in untreated animals (Fig. 5, C and D, respectively). On the other hand, mice treated with ODQ presented significantly less bacteria in the peritoneum and blood (Fig. 5, C and D, respectively). Moreover, neutrophil sequestration in lungs was prevented by ODQ treatment but not 1400W (Fig. 5E). Based on these findings, we conclude that the NO-sGC signaling pathway is operative in vivo and contributes to the establishment of neutrophil migratory dysfunction. Moreover, these findings suggest that the limitations in using NOS inhibitors in sepsis can be, at least in part, attributed to an interference with the ability of neutrophils to control infection, which cannot be compensated for by restoring neutrophil migratory function. In this sense, sGC inhibition preserves the ability of neutrophils to kill bacteria and control infection probably because it does not interfere directly with NO synthesis. These data therefore demonstrate that sGC inhibition restores neutrophil migration without interfering with neutrophil killing function, thus overcoming one important limitation for the use of NOS inhibitors in sepsis.

Effect of ODQ treatment on cytokine production and neutrophil apoptosis in vivo

Diminished neutrophil numbers in peritoneal cavity of L-CLP mice could be attributed to decreased chemokine production or increased neutrophil apoptosis in vivo. To test these possibilities, we measured cytokine and chemokine levels in peritoneum and plasma of mice submitted to CLP. As shown in Figure 6, KC and MIP-2 levels in peritoneal cavity of SL-CLP mice were elevated relative to sham-operated controls. This is in accordance with the observed increase in neutrophil numbers in these mice (Fig. 5A). Mice submitted to L-CLP presented further increased chemokine levels in the peritoneum (Fig. 6, A and B). Despite this increased chemokine levels, neutrophil numbers were significantly lower in these mice (Fig. 5A). Peritoneal and circulating levels of TNF-α and IL-17 were also increased in the L-CLP group (Fig. 6, C-E). These results suggest that impaired neutrophil migration is not a result of decreased chemokine or cytokine production in the L-CLP mice. Furthermore, ODQ treatment of L-CLP mice recovered neutrophil numbers in peritoneum (Fig. 5A) but had no major impact on cytokine or chemokine production (Fig. 6).

F6-4
Fig. 6:
Effects of ODQ treatment on cytokine and chemokine production and neutrophil apoptosis in sepsis. Mice were submitted to L-CLP with or without ODQ pretreatment as described. Cytokines (TNF-α and IL-17) and chemokines (KC and MIP-2) levels were measured 6 h after CLP procedure in peritoneal lavage fluid and plasma by enzyme-linked immunosorbent assay. A-D, Respectively, KC, MIP-2, IL-17, and TNF-α levels in peritoneal cavity of micesubmitted to SL-CLP, L-CLP, or L-CLP with ODQ treatment. E and F, Respectively, IL-17 and TNF-α levels in plasma of mice submitted to SL-CLP, L-CLP, or L-CLP with ODQ treatment. Data are represented as mean ± SEM of the levels of chemokines/cytokines (in picograms) per cavity (A-D) or per milliliter of plasma (E and F). N = 8 for each experimental group. G, Percentage of annexin V-positive neutrophils in peritoneal cavity of mice submitted to SL-CLP, L-CLP, or L-CLP with ODQ treatment as accessed by FACS analysis. Data represent mean ± SEM of the percentage of annexin V-positive neutrophils recovered from the peritoneal cavity 6 h after CLP. *Significant difference relative to SL-CLP group. N = 4 for each experimental group. Sham-operated mice were used as controls. B.L.D. indicates below limit of detection.

We also evaluated neutrophil apoptosis in L-CLP mice. We observed a significant increase in neutrophil apoptosis in L-CLP mice relative to the SL-CLP group (Fig. 6F). In addition, ODQ treatment had no effect on neutrophil apoptosis, but significantly recovered neutrophil numbers in peritoneum. Together, these results suggest that sepsis has a direct impact on neutrophil migratory function, and the diminished neutrophil numbers in L-CLP mice could not be attributed to decreased mediator production or increased neutrophil apoptosis.

Effect of ODQ treatment on mice survival in a model of sepsis

We demonstrated that sGC inhibition in sepsis restored neutrophil migration without impacting neutrophil killing function and that this contributes to an increased survival in mice treated with sGC inhibitors. This encouraged us to test whether sGC inhibition would improve survival of mice after the onset of infection, using a posttreatment protocol consisting of two s.c. injections at 3 and 12 h after CLP. Mice submitted to CLP and treated with vehicle died within 24 to 48 h, a period when ODQ-treated mice showed 50% survival. Survival rates of mice treated with ODQ dropped to 16.7% after 24 h and remained at the end of the observation period, thus reaching no statistical significance (P = 0.1461, relative to untreated L-CLP mice) (Fig. 7A). We then used a second posttreatment protocol consisting of three s.c. injections of ODQ at 3, 12, and 24 h. In this second protocol, survival could be maintained at 40% throughout the observation period (96 h), which was significantly higher than that observed for the untreated group (100% mortality within 48 h, P = 0.0325) (Fig. 7B). We therefore believe that together our results support a detrimental role for sGC activation in sepsis and point to sGC inhibition as a promising potential therapeutic target in sepsis.

F7-4
Fig. 7:
Posttreatment with ODQ improves survival in an experimental model of severe sepsis. A, Survival curve for mice treated with ODQ 3 and 12 h after CLP procedure. ODQ doses were the same as in Figure 5B, and sham mice were used as controls. Graph shows the percent survival through the 72-h observation period. N = 6 for each experimental group. B, Survival curve for mice treated with ODQ 3, 12, and 24 h after CLP procedure. ODQ doses were the same as in A, and sham mice were used as controls. Graph shows the percent survival through the 96-h observation period. N = 10 for each experimental group. P values are shown for the comparisons between L-CLP and L-CLP/ODQ groups.

DISCUSSION

Septic subjects develop dysfunctions on multiple physiological systems including the cardiovascular, respiratory, nervous, and immune systems (1, 2). An innate immune dysfunction observed in sepsis is an inability of neutrophils to respond to chemotactic stimulation in vivo and in vitro (4). This impairs an efficient control of bacterial infection and may contribute to the aggravation of sepsis. The process of neutrophil dysfunction in sepsis is largely dependent on iNOS-derived NO. However, in experimental models, NOS inhibition in vivo resulted in increased mortality, and a recent phase 3 clinical trial using an NO synthase inhibitor resulted in increased mortality of septic patients (5, 15, 16). This limits the use of NOS inhibitors in sepsis therapy. More promising results were observed in the clinics using inhibitors of the downstream effector of NO, the enzyme sGC (17-19). The beneficial effects of sGC inhibitors on the cardiovascular function in sepsis have long been appreciated. However, the participation of sGC on the establishment of neutrophil dysfunction in sepsis and the modulation of this process by sGC inhibitors have never been addressed.

We addressed for the first time the relative contribution of sGC activation to the establishment of neutrophil dysfunction in sepsis. We used a model in which TLR stimulation of human neutrophils leads to decreased neutrophil chemotactic responsiveness to the classic chemotactic mediators IL-8, LTB4, and fMLP. In this model, treatment of human neutrophils with pharmacological inhibitors to different components of the NO-sGC-PKG signaling pathway prevented the effects of LPS on neutrophil responsiveness to chemotactic stimuli. Furthermore, using LTA, another TLR ligand that activates TLR2, we observed similar results, suggesting that the NO-dependent impairment of neutrophil chemotactic function is a common mechanism shared by different TLR ligands and is largely attributed to the activation of the NO-sGC-PKG signaling axis.

Our further study on the mechanisms triggered by LPS on neutrophils demonstrated that LPS stimulation leads to decreased membrane expression of the two IL-8 receptors, CXCR1 and CXCR2, as well as the LTB4 receptor, BLT1. The effect of LPS on CXCR1 and CXCR2 expression has been previously described by others (28, 29), although the mechanisms involved in this process remained unaddressed. Inhibition of the different components of the NO-sGC-PKG signaling pathway recovered CXCR1, CXCR2, and BLT1 membrane expression in neutrophils stimulated with LPS. This suggests that LPS is able to induce heterologous desensitization of neutrophils to IL-8 and LTB4 stimulation through a mechanism involving the activation of the NO-sGC-PKG signaling pathway. We and others have previously demonstrated a crucial role played by CXCR2 and LTB4 receptor in sepsis. Inhibition of CXCR2 or LTB4 receptor signaling rendered animals more susceptible to infection by impairing neutrophil migration to the primary infectious focus (12, 30). As LPS decreased the membrane expression of CXCR1, CXCR2, and BLT1 receptors and decreased neutrophil responsiveness to IL-8 and LTB4, we can suggest that this LPS effect could contribute to worsen the clinical outcome in septic patients.

We have also observed unresponsiveness to fMLP in neutrophils stimulated with LPS. This suggests that LPS and the activation of the NO-sGC-PKG signaling pathway have more profound effects on neutrophil responsiveness to chemotactic stimulation because fMLP engages a different receptor and signals through a different signaling pathway (31). The molecular mechanisms underlying neutrophil unresponsiveness to fMLP were not addressed in the present study and are currently being examined.

Diminished receptor expression in LPS-stimulated neutrophils was paralleled by an increase in GRK2 expression. Because CXCR1 and CXCR2 are targets for GRK2 (26, 27), GRK2-mediated receptor internalization could be a reasonable explanation for the decreased receptor expression in LPS-stimulated cells. Increased GRK2 expression was also blocked by previous treatment of cells with inhibitors of the NO-sGC-PKG pathway. GRK2 upregulation in neutrophils by LPS treatment has been recently reported (26), although the mechanisms involved in this phenomenon were not investigated. More recently, we have shown that, besides LPS, neutrophil stimulation with LTA leads to neutrophil unresponsiveness to chemotactic stimulation that correlates with increased GRK2 expression and decreased CXCR2 membrane expression (13). Based on these findings, it can be proposed that both TLR2 and TLR4 ligands induce NO production and consequent activation of the sGC-cGMP-PKG pathway. This would lead to increased expression of GRK2 and chemotactic receptor internalization, rendering neutrophils unresponsive to chemotactic stimulation.

An alternative mechanism for the decreased receptor expression is LPS-induced receptor shedding. In fact, LPS-induced shedding of CXCR1 and CXCR2 was previously reported by others (32, 33). In any case, the net result of TLR activation in neutrophils would be decreased membrane receptor expression and decreased chemotactic responsiveness. Because we could demonstrate a recovery of receptor expression in neutrophils treated with pharmacological inhibitors to the NO-sGC-PKG signaling pathway, the relevant process (diminished receptor expression), whether due to internalization or shedding, is sGC-dependent. Moreover, upregulation of GRK2 expression would represent a mechanism of long-term desensitization of neutrophils to chemotactic stimuli that could be in place during septic episodes.

Recently, Clements and coworkers (34) reported that NO could inhibit neutrophil chemotaxis through the formation of peroxynitrite. Tyrosine nitration of actin filaments in neutrophils would then impair neutrophil mobility and render cells unable to migrate. This could be happening during sepsis and would argue against our findings as the mechanisms used by NO to impair neutrophil migration would involve the formation of peroxynitrite and not the activation of the NO-sGC-PKG signaling pathway. However, nitration of actin filaments would implicate in a general migratory dysfunction of neutrophils, which is not what is observed in vivo. Neutrophils from septic rats present increased migration to the CCR2 ligands, CCL2 and CCL7 (9). We have observed a similar profile in mice and human septic patients (Souto et al., manuscript in preparation), suggesting that during sepsis neutrophils shift their responsiveness to chemotactic mediators but are still able to migrate. Therefore, the activation of the NO-sGC-PKG signaling pathway is a more likely mechanism by which NO interferes with neutrophil chemotactic function during severe sepsis.

Our in vitro results reveal a point where the beneficial and the detrimental roles of NO in sepsis diverge. NO plays a beneficial role as an important mediator of bacterial killing that is largely dependent on the reactive nature of NO and the attack of cellular components by reactive nitrogen species (35, 36). On the other hand, the detrimental effect of NO on neutrophil responses to chemotactic stimuli seems to depend on the activation the sGC-cGMP-PKG signaling pathway. In addition, the NO-sGC-PKG signaling pathway also contributes significantly to the cardiovascular collapse observed in severe sepsis (20, 22). Therefore, the beneficial role of NO in sepsis is reactive in nature and largely sGC-independent, whereas the detrimental role can be attributed to the activation of the NO-sGC-PKG signaling pathway and thus is mainly sGC-dependent.

Based on these assumptions, we reasoned that interfering with sGC activity would represent a better therapeutic target in sepsis than inhibition of NO synthesis. Soluble guanylyl cyclase inhibition would interfere with the detrimental role of NO but leave NO production and the host's microbicidal activity intact. Intact iNOS-derived NO production may have further benefits besides infection control. In this sense, it was demonstrated, in a model of TNF-induced shock, that the beneficial effects of sGC inhibition required intact iNOS activity (37). Because this model did not involve infection, one may speculate that maintaining iNOS activity while inhibiting its signaling through sGC has beneficial effects over other systems as well.

We compared the effects of iNOS and sGC inhibition in an experimental model of sepsis. We show that inhibition of iNOS activity recovered neutrophil migratory function, but neutrophils were unable to control infection. In contrast, sGC inhibition resulted in recovery of neutrophil migratory function together with efficient control of infection. These different effects of 1400W and ODQ on neutrophil migratory and killing function resulted in opposite outcomes in survival. Whereas ODQ-treated mice presented a significant increase in survival, 1400W-treated animals succumbed infection in a similar kinetic as untreated controls. We therefore reason that the limitations in using NOS inhibitors in sepsis could be attributed, at least in part, to an interference on the ability of neutrophils (and probably other cells of the innate immune system) to produce NO and kill bacteria. In this sense, sGC inhibition could recover neutrophil migratory function without interfering with neutrophil capacity of NO production and bacterial killing. The modulation of neutrophil migration by sGC activity in vivo was recently demonstrated by our group (24). In this study, it was shown that sGC activation by NO inhibits neutrophil migration through the modulation of ICAM-1 expression by endothelium. Our study demonstrates that NO-dependent activation of sGC is able to directly inhibit neutrophil migration. Although the modulation of endothelial ICAM-1 expression by sGC has never been evaluated in the context of sepsis, one may speculate that both mechanisms act synergistically in vivo to inhibit neutrophil migration.

Recent reports suggest that prevention of neutrophil migration in severe sepsis has beneficial effects on survival (38, 39). This is in apparent contrast to our findings, but care must be taken when confronting these data. It has been long appreciated that during sepsis, neutrophils are trapped in lung vasculature, where these cells contribute to tissue damage by release of granule content and reactive species. This trapping process (referred to as neutrophil sequestration) is mechanistically different from the classic neutrophil migration (40, 41). This tissue efflux of neutrophils is not a response to local infection, but a consequence of systemic neutrophil activation. It has been largely demonstrated that neutrophil sequestration contributes significantly to sepsis-induced acute lung injury and liver failure. Therefore, prevention of this process is highly desirable and significantly contributes to sepsis survival.

Therefore, these recent reports are not in contrast to our findings as it may appear. In the present study, we refer to "neutrophil migration" as neutrophil migration to the infectious focus (e.g., peritoneal cavity in CLP). In fact, we demonstrate that ODQ treatment prevents neutrophil sequestration in lungs while restoring neutrophil migration to the peritoneal cavity, which may contribute to the observed increased survival (compare Fig. 5, A and E). In conclusion, neutrophil migration to the infectious focus should be restored, whereas neutrophil recruitment to distal organs (sequestration) must be prevented.

We further tested our hypothesis in a more clinically relevant setting. We treated mice with ODQ 3 and 12 h after the onset of sepsis and found a 50% survival at 24 h after CLP compared with the 100% mortality in the untreated group. After this period, survival decreased to 16.7% and remained throughout the observation period. The lower efficacy seen on posttreatment protocol, relative to the pretreatment protocol, may be explained by a time-dependent effectiveness of sGC inhibition in experimental sepsis, as reported previously (21). In these studies, vascular reactivity was restored only by ODQ when administered 2 or 24 h after LPS injection, a time window not covered by our protocol. We then used a second posttreatment protocol in which three ODQ administrations were used (3, 12, and 24 h). Using this protocol, we could observe a 40% survival in the treated group, whereas untreated mice showed 100% mortality within 24 to 48 h. Taken together, these results strongly support our hypothesis and point to the potential of sGC inhibition as a therapeutic target in sepsis.

This proposition finds clinical support in early studies using the unspecific sGC inhibitor methylene blue (MB) in severe septic subjects. These studies revealed a beneficial outcome for critically ill patients treated with MB as an adjuvant therapy in sepsis. Recently, some groups proposed the consideration of a revisited clinical use of MB in intensive care units (19). Taken together, our data point to sGC inhibition as a promising therapeutic approach in sepsis. However, it remains necessary large clinical trials to definitely demonstrate the efficacy of sGC inhibitors in sepsis and to develop a reproducible protocol for the use of these molecules in the clinics.

The present study shed new light on previous studies by our group and others that describe the involvement of NO on the impairment of neutrophil migration. We show for the first time that the effects of NO on neutrophil migratory function in sepsis are dependent on sGC activation. Our present data also provide, for the first time, cellular and molecular support for the potential clinical benefits of sGC inhibition as a therapy in sepsis. Inhibition of NO synthesis or sGC activity is often based on the roles played by NO in the cardiovascular changes observed in sepsis. Less attention is drawn to the inflammatory/immune roles of NO. The unexpected outcome of NO synthesis inhibition in animal studies and clinical trials may have stemmed, at least in part, from the inhibition of the beneficial role of NO as a microbicidal mechanism used by cells of the innate immune system. Our results suggest that, besides the positive cardiovascular effects of sGC inhibition, this approach could also have a beneficial effect on the immune/inflammatory component of sepsis. Soluble guanylyl cyclase inhibition would then emerge as a potential therapeutic target in sepsis because it could restore neutrophil migration without compromising neutrophil microbicidal activity, thus bypassing the limitations of NO synthesis inhibition.

ACKNOWLEDGMENTS

The authors thank Simone Lima and Marcela Sousa (UERJ) and Walter Turato (FMRP-USP) for their technical assistance with FACS data acquisition. They also thank Prof C. N. Paiva (IMPPG, UFRJ) for discussions and critical reading of the manuscript.

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Keywords:

Nitric oxide; Toll-like receptor; G protein-coupled receptor kinase; protein kinase G; chemotaxis; cyclic GMP

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