Nuclear Factor of Activated T Cells Regulates Neutrophil Recruitment, Systemic Inflammation, and T-Cell Dysfunction in Abdominal Sepsis

Abstract

The signaling mechanisms regulating neutrophil recruitment, systemic inflammation, and T-cell dysfunction in polymicrobial sepsis are not clear. This study explored the potential involvement of the calcium/calcineurin-dependent transcription factor, nuclear factor of activated T cells (NFAT), in abdominal sepsis. Cecal ligation and puncture (CLP) triggered NFAT-dependent transcriptional activity in the lung, spleen, liver, and aorta in NFAT-luciferase reporter mice. Treatment with the NFAT inhibitor A-285222 prior to CLP completely prevented sepsis-induced NFAT activation in all these organs. Inhibition of NFAT activity reduced sepsis-induced formation of CXCL1, CXCL2, and CXCL5 chemokines and edema as well as neutrophil infiltration in the lung. Notably, NFAT inhibition efficiently reduced the CLP-evoked increases in HMBG1, interleukin 6 (IL-6), and CXCL5 levels in plasma. Moreover, administration of A-285222 restored sepsis-induced T-cell dysfunction, as evidenced by markedly decreased apoptosis and restored proliferative capacity of CD4 T cells. Along these lines, treatment with A-285222 restored gamma interferon (IFN-γ) and IL-4 levels in the spleen, which were markedly reduced in septic mice. CLP-induced formation of regulatory T cells (CD4⁺ CD25⁺ Foxp3⁺) in the spleen was also abolished in A-285222-treated animals. All together, these novel findings suggest that NFAT is a powerful regulator of pathological inflammation and T-cell immune dysfunction in abdominal sepsis. Thus, our data suggest that NFAT signaling might be a useful target to protect against respiratory failure and immunosuppression in patients with sepsis.

INTRODUCTION

Abdominal sepsis is a major cause of mortality in intensive care units in spite of significant investigational efforts (1, 2). The septic insult triggers two distinct responses of the immune system. On one hand, intestinal perforation and contamination of the abdominal cavity with bacterial antigens and toxins provoke local formation of proinflammatory substances, which, in turn, can translocate into the circulation and cause a systemic inflammatory response syndrome (SIRS). SIRS is associated with organ damage, and it is widely held that the lung is the most vulnerable and critical target organ in patients with sepsis (3). Convincing data have shown that neutrophil recruitment is a rate-limiting step in septic lung injury (4). On the other hand, SIRS is followed by a compensatory anti-inflammatory response syndrome (CARS), in which the immune cells become incapable of mounting appropriate host-defense reactions against microbes. CARS is characterized by decreased ability of macrophages to present antigens and T-cell apoptosis as well as induction of regulatory T cells which, together, compromise the antibacterial responses of the host (5–7). Infectious complications are an insidious component in septic patients with CARS (8). However, the signaling pathways underlying pulmonary infiltration of neutrophils and T-cell dysfunction in abdominal sepsis remain elusive.

Extracellular stress signals trigger intracellular signaling cascades converging on specific transcription factors, which control gene expression and formation of proinflammatory substances. Cytosolic calcium is a major determinant of immune cell activation (9). One key target of calcium in eukaryotic cells is calcineurin, a unique calcium/calmodulin-activated serine/threonine protein phosphatase, playing a key function in several cellular processes and calcium-dependent signal transduction pathways (10, 11). Calcineurin is effectively inhibited by immunosuppressant drugs, such as FK506, used for preventing transplant rejection (12). Interestingly, a recent study reported that FK506 protects against endotoxin-induced toxicity (13). However, calcineurin inhibition, due to its ability to engage a broad range of substrates and binding partners (10, 14), is associated with serious side effects and may not be suitable to use in patients with sepsis (15). Alternatively, we hypothesized that inhibition of downstream targets of calcineurin signaling might be a more useful way to attenuate pulmonary accumulation of neutrophils and T-cell dysfunction in abdominal sepsis. One important downstream target of calcineurin is the family of four nuclear factor of activated T cells (NFATc1-c4) transcription factors, which are heavily phosphorylated and cytosolic under basal conditions but able to translocate to the nucleus upon stimulation and dephosphorylation by calcineurin (16). NFAT activation initiates a cascade of transcriptional events involved in physiological and pathological processes (17–19). NFAT was originally described as a transcriptional activator of cytokine and immunoregulatory genes in T cells (20, 21) but is now known to play a role in several cell types outside the immune system (17). However, the role of NFAT signaling in the response to systemic bacterial infections has not been investigated. Thus, the potential role of NFAT in the pathophysiology of abdominal sepsis remains elusive.

Based on the considerations given above, the aim of this study was to investigate whether NFAT plays a role in pro- and anti-inflammatory components of the host response in abdominal sepsis. For this purpose, we used a model based on cecal ligation and puncture (CLP) to induce sepsis in mice.

MATERIALS AND METHODS

Animals.

All experimental procedures in this study were conducted in accordance with approved ethical permission by the Regional Ethical Committee for Animal Experimentation at Lund University, Sweden. Phenotypically normal adult FVB/N 9×-NFAT-luciferase reporter mice (NFAT-luc) were used. NFAT-luc mice express nine copies of an NFAT binding site from the interleukin 4 (IL-4) promoter (5′-TGGAAAATT-3′) positioned 5′ to a minimal promoter from the α-myosin heavy chain gene (−164 to +16) and inserted upstream of a luciferase reporter gene (22). Mice were housed in an animal facility with 12-h/12-h light-dark cycle at 22°C and fed a normal laboratory diet and water ad libitum. Mice were anesthetized with 7.5 mg of ketamine hydrochloride (Hoffman-La Roche, Basel, Switzerland) and 2.5 mg of xylazine (Janssen Pharmaceutica, Beerse, Belgium) per 100 g body weight intraperitoneally (i.p.).

Experimental protocol of sepsis.

Polymicrobial sepsis in mice was induced by a cecal ligation and puncture (CLP) procedure as previously described in detail (23). Under anesthesia, animals underwent a midline incision to identify and exteriorize the cecum, which was filled with feces by milking stool backwards from the ascending colon, and 75% of the cecum was ligated with a 5-0 silk suture. The cecum was soaked with phosphate-buffered saline (PBS) (pH 7.4) and was then double punctured with a 21-gauge needle on the antimesenteric border. A small amount of bowel contents was extruded, and the cecum was returned into the peritoneal cavity and the abdomen was closed in two layers. One milliliter of PBS mixed with buprenorphine hydrochloride (0.05 mg/kg; Schering-Plough Corporation, New Jersey, USA) was administered subcutaneously (s.c.) for resuscitation and pain control. Sham animals underwent the identical laparotomy and resuscitation procedures, but the cecum was neither ligated nor punctured. Animals were randomized into four groups: sham-operated mice pretreated with (i) vehicle (PBS) or (ii) the NFAT blocker A-285222 (0.15 mg/kg body weight, administered i.p. twice daily for 7 consecutive days and in the morning of operation) and CLP mice pretreated with (iii) vehicle (PBS) or (iv) A-285222. Treatment with A-285222 was well tolerated and performed according to a previously established protocol (24). A-285222 was kindly provided by Abbott Laboratories. Experiments were terminated 24 h after CLP or sham procedure, at which point mice were reanesthetized, blood was obtained from the vena cava and centrifuged, and plasma was frozen at −20°C. Mice were euthanized and the left lung was ligated and excised for edema measurement. Bronchoalveolar lavage fluid (BALF) was collected from the right lung to quantify neutrophils, after which the lung was excised, one lobe was fixed in formaldehyde for histology, and another piece of lung, together with half of the spleen, liver, and aorta, were dissected for luciferase measurements. The remaining lung tissue was weighed, snap-frozen in liquid nitrogen, and stored at −80°C for later enzyme-linked immunosorbent assay (ELISA) as described below.

Luciferase reporter assay.

Luciferase activity was measured as previously described in tissue homogenates from lung, spleen, liver, and aorta in NFAT-luc mice from each group as specified in the text (25). Optical density was measured using a Tecan Infinite M200 instrument (Tecan Nordic AB, Mölndal, Sweden), and data are expressed as relative luciferase units (RLU) per microgram of protein.

Systemic leukocyte counts.

Blood was collected from the tail vein 24 h after CLP or sham procedure and mixed with Turk's solution (Merck, Damnstadt, Germany) in a 1:20 dilution. Leukocytes were identified as monomorphonuclear (MNL) and polymorphonuclear (PMNL) leukocytes in a Burker chamber (23).

BALF.

Animals were placed supine and the trachea was exposed by dissection. An angiocatheter was inserted into the trachea. Bronchoalveolar lavage fluid (BALF) was collected by five washes with 1 ml of PBS containing 5 mM EDTA. The numbers of MNL and PMNL cells were counted in a Burker chamber after being stained with Turk's solution (Merck) (23).

Lung edema.

The left lung was excised and weighed. The tissue was then dried at 60°C for 72 h and reweighed. The change in the ratio of wet weight to dry weight was used as an indicator of lung edema formation (23).

ELISA.

CXCL1, CXCL2, and CXCL5 levels in lung tissue were analyzed by using double antibody Quantikine ELISA kits (R&D Systems, Europe, Abingdon, Oxon, United Kingdom) using recombinant murine CXCL1, CXCL2, and CXCL5 as standards. Blood samples were collected from the vena cava (1:10 acid citrate dextrose) and centrifuged at 18,800 × g for 10 min at 4°C and stored at −20°C until use. ELISA kits were also used to quantify plasma levels of IL-6 (R&D Systems) and high-mobility group box 1 (HMGB1) (Chondrex, Redmond, WA, USA) and CXCL5 (R&D Systems) according to the manufacturers' instructions (23, 26).

Histology.

Lung samples were fixed in 4% formaldehyde phosphate buffer overnight and then dehydrated and paraffin embedded. Sections (6 μm) were stained with hematoxylin and eosin as previously described (23). Lung injury was quantified in a blinded manner by adoption of a modified scoring system as described previously (27–29), including size of alveolar spaces, thickness of alveolar septa, alveolar hemorrhage, and neutrophil infiltration graded on a 0 (absent) to 4 (extensive) scale.

Isolation of splenocytes.

Half of the spleen was excised for cell culture and flow cytometric analysis 24 h after CLP induction or sham procedure. Single splenocyte suspension was prepared under sterile conditions by pressing the spleens through a 40-μm-pore-size cell strainer (BD Falcon, Becton, Dickinson, Mountain View, CA, USA). Erythrocytes were removed using red blood cell ACK lysing buffer (Invitrogen, Carlsbad, CA, USA). Cells were washed and resuspended with Click's medium (Sigma-Aldrich, Stockholm, Sweden) supplemented with 10% (vol/vol) fetal bovine serum, penicillin (100 unit/ml), and streptomycin (0.1 mg/ml) (Sigma-Aldrich). The same medium was used in all experiment described below. Splenocytes were quantified in a Burker chamber after staining with Turk's solution (Merck) (26).

Cytokine formation in splenocytes.

Isolated splenocytes were loaded at 1.0 × 10⁶ cells/well in 48-well plates precoated with anti-CD3ε antibody (5 μg/well, IgG, clone 145-2C11) and in the presence of soluble anti-CD28 antibody (5 μg/well, IgG, clone 37.51) at 37°C in a humidified chamber with 5% CO₂ for 24 h. Levels of gamma interferon (IFN-γ) and IL-4 in the culture medium were detected by ELISA kits (R&D Systems) according to the manufacturer's instructions (26). All antibodies used were purchased from eBioscience (San Diego, CA, USA) unless indicated.

T-cell apoptosis.

To evaluate apoptosis of CD4 T cells, splenocytes were fixed and stained by a APO-BRDU kit, which labels DNA strand breaks by BrdUTP according to the manufacturer's instruction (Phoenix Flow Systems, San Diego, CA, USA). Allophycocyanin (APC)-conjugated anti-CD4 antibody (IgG2b, κ, clone GK1.5) was used to indicate CD4 T cells. Splenocytes were acquired by a FACSCalibur flow cytometer (Becton, Dickinson, Mountain View, CA, USA) and analyzed with Cell-Quest Pro software (BD Bioscience, San Jose, CA, USA) (26).

T-cell proliferation.

Isolated splenocytes were stained with carboxyfluorescein diacetate succinimidyl ester (CFSE; 5 μM; Sigma-Aldrich) and incubated at 1.5 × 10⁶ cells/well in 150 μl CLICK's medium in 96-well plates precoated with or without anti-CD3ε antibody (5 μg/ml, IgG, clone 145-2C11) and in the presence or absence of soluble anti-CD28 antibody (2 μg/ml, IgG, clone 37.51) at 37°C in a humidified chamber with 5% CO₂ for 72 h. For analysis of cell proliferation, splenocytes were stained with APC-conjugated anti-CD4 antibody (IgG2b, κ, clone GK1.5) and propidium iodide (PI) (Phoenix Flow Systems). Flow cytometric analysis was performed on a FACSCalibur flow cytometer, and PI-negative cells were gated to exclude dead cells.

Regulatory T-cell analysis.

Splenocytes were stained with fluorescein isothiocyanate (FITC)-conjugated anti-CD4 (Rat IgG2a, κ, clone RM4-5), APC-conjugated anti-CD25 (Rat IgG1, λ, clone PC61.5), and phycoerythrin (PE)-conjugated anti-Foxp3 (Rat IgG2a, κ, clone FJK-16S) antibodies. Flow cytometric analysis was performed on a FACSCalibur flow cytometer.

Bacterial cultures.

The number of CFU was determined in blood, the peritoneal cavity, as well as lung and spleen. Briefly, 24 h after CLP, blood was taken from the interior vena cava, and the peritoneal lavage fluid was obtained with 2 ml cold, sterile PBS. Lung and spleen were harvested and homogenized under aseptic conditions. Subsequently, serial diluted blood and peritoneal lavage fluid, as well as organ homogenates, were plated on Trypticase soy agar II with 5% sheep blood (Becton, Dickinson GmbH, Heidelberg, Germany) and incubated under aerobic conditions at 37°C for 24 h. The numbers of bacterial colonies were then counted and specified as CFU/ml blood or peritoneal lavage fluid and CFU/g tissue.

Statistics.

Data are presented as mean values ± standard errors of the means (SEM). Statistical evaluations were performed using a Mann-Whitney rank sum test for comparing sham and vehicle versus CLP and vehicle as well as CLP and vehicle versus CLP and A-285222. P values of <0.05 were considered significant, and n represents the total number of mice in each group.

RESULTS

NFAT-dependent transcriptional activity in abdominal sepsis.

Induction of abdominal sepsis by CLP in transgenic NFAT-luc reporter mice resulted in enhanced luciferase activity in the lung, spleen, liver, and aorta (Fig. 1; *, P < 0.05 versus sham-operated vehicle; n = 5). Treatment with the NFAT inhibitor A-285222 blocked the CLP-induced NFAT activation in all studied organs, showing that A-285222 is an effective inhibitor of NFAT transcriptional activity (Fig. 1; #, P < 0.05 versus CLP vehicle; n = 5). A-285222 prevents NFAT nuclear accumulation, without affecting NF-κB or AP-1 activation or calcineurin phosphatase activity (30).

FIG 1.

Luciferase activity (RLU/μg protein) in lung (a), spleen (b), liver (c), and aorta (d) of NFAT-luc mice. Mice were pretreated with A-285222 or vehicle (Veh) i.p. twice daily for 7 consecutive days and in the morning before sham operation (white bars) or induction of CLP (black bars). Samples were obtained 24 h after sham operation or CLP induction. Data represent means ± SEM. All experiments were repeated five times (n = 5 mice). *, P < 0.05 versus sham-operated vehicle; #, P < 0.05 versus CLP vehicle.

Neutrophil recruitment and septic lung injury.

Cellular analysis of BALF showed that the number of neutrophils in the bronchoalveolar space increased by 4.5-fold 24 h after induction of CLP (Fig. 2a; *, P < 0.05 versus sham-operated vehicle; n = 5). Notably, inhibition of NFAT reduced CLP-induced infiltration of neutrophils into the alveolar compartment from 118.4 × 10³ ± 16.2 × 10³ to 66.4 × 10³ ± 5.2 × 10³ cells, corresponding to a 54% decrease in neutrophil recruitment (Fig. 2a; #, P < 0.05 versus CLP vehicle; n = 5). CLP caused significant pulmonary damage, characterized by lung edema formation, i.e., wet/dry ratio increased from 4.6 ± 0.1 to 5.2 ± 0.1 (Fig. 2b; *, P < 0.05 versus sham-operated vehicle; n = 5). Note that baseline values of the wet/dry ratio in sham-operated vehicle mice represent normal levels in healthy animals, and only the increase in the wet/dry ratio represents actual edema formation. Administration of A-285222 reduced the CLP-induced lung wet/dry ratio to 4.8 ± 0.1, corresponding to an 82% reduction in lung edema (Fig. 2b; #, P < 0.05 versus CLP vehicle; n = 5). Moreover, CLP caused severe destruction of the pulmonary tissue structure characterized by extensive edema of the interstitial tissue and massive infiltration of neutrophils (Fig. 2c), whereas morphological examination revealed normal microarchitecture in lungs of sham-operated animals (Fig. 2c). NFAT inhibition markedly decreased CLP-induced destruction of the tissue architecture and reduced neutrophil accumulation in the lung (Fig. 2c). Quantification of the morphological changes revealed that CLP increased the lung injury score, and administration of A-285222 significantly reduced the lung injury score in CLP animals (Fig. 2d). CLP induction in mice clearly resulted in neutropenia at 24 h, and this was prevented by inhibition of NFAT (Table 1).

FIG 2.

NFAT regulates CLP-induced neutrophil recruitment and tissue injury in the lung. Number of BALF neutrophils (a) and edema formation in the lung (b) 24 h after sham operation or CLP induction. Mice were pretreated with A-285222 or vehicle (Veh) i.p. twice daily for 7 consecutive days and in the morning before sham operation (white bars) or induction of CLP (black bars). Representative H&E sections of the lung (c) and corresponding lung histology scores (d). Scale bar indicates 100 μm. Data represent means ± SEM. All experiments were repeated five times (n = 5 mice). *, P < 0.05 versus sham-operated vehicle; #, P < 0.05 versus CLP vehicle.

TABLE 1.

Systemic leukocyte differential counts^a

Treatment	Mean cell count ± SEM (×106)
	MNL	PMNL	Total
Vehicle	3.3 ± 0.7	1.0 ± 0.2	4.3 ± 0.8
A-285222	3.6 ± 1.4	0.9 ± 0.2	4.5 ± 1.6
Vehicle and CLP	2.6 ± 0.3	0.5 ± 0.1b	3.2 ± 0.3
A-285222 and CLP	3.1 ± 0.8	1.7 ± 0.4c	4.8 ± 1.1

^aBlood was collected from vehicle- and A-285222 (0.15 mg/kg)-treated animals exposed to CLP for 24 h as well as from sham-operated mice. Cells were identified as monomorphonuclear (MNL) and polymorphonuclear (PMNL) cells. Data represent means ± SEM, ×10⁶ cells/ml. All experiments were repeated five times (n = 5 mice).

^bP < 0.05 versus sham-operated vehicle.

^cP < 0.05 versus CLP vehicle.

CXC chemokine formation in the lung.

CXC chemokines, such as CXCL1 and CXCL2, are known to coordinate neutrophil trafficking in the lung. Levels of CXCL1 and CXCL2 were low but detectable in sham animals (Fig. 3a and b). It was found that formation of CXC chemokines in the lung was greatly enhanced in CLP mice (Fig. 3a and b; *, P < 0.05 versus sham-operated vehicle; n = 5). Notably, we observed that NFAT inhibition decreased CLP-provoked production of CXCL1 by 62% and CXCL2 by 81% in the lung (Fig. 3a and b; #, P < 0.05 versus CLP vehicle; n = 5). In addition, CLP increased the CXCL5 levels in the lung (Fig. 3c; *, P < 0.05 versus sham-operated vehicle; n = 5), and inhibition of NFAT reduced the lung levels of CXCL5 by 59% in septic animals (Fig. 3c; #, P < 0.05 versus CLP vehicle; n = 5).

FIG 3.

NFAT regulates CXC chemokine formation in the lung. Mice were pretreated with A-285222 or vehicle (Veh) i.p. twice daily for 7 consecutive days and in the morning before sham operation (white bars) or induction of CLP (black bars). ELISA was used to quantify the levels of CXCL1 (a), CXCL2 (b), and CXCL5 (c) in the lung of mice 24 h after sham operation or CLP induction. Data represent means ± SEM. All experiments were repeated five times (n = 5 mice). *, P < 0.05 versus sham-operated vehicle; #, P < 0.05 versus CLP vehicle.

Systemic levels of HMGB1 and IL-6.

Plasma levels of HMGB1 in control animals were low (Fig. 4a; n = 5). It was found that CLP increased plasma levels of HMGB1 by 13-fold, from 1.6 ± 0.7 ng/ml up to 23.4 ± 3.0 ng/ml (Fig. 4a; *, P < 0.05 versus sham-operated vehicle; n = 5). Treatment with A-285222 reduced CLP-induced formation of HMGB1 to 3.1 ± 0.8 ng/ml (Fig. 4a; #, P < 0.05 versus CLP vehicle; n = 5). In addition, we observed that the plasma levels of IL-6 were markedly increased in septic compared to sham mice (Fig. 4b; *, P < 0.05 versus sham-operated vehicle; n = 5). Notably, inhibition of NFAT decreased plasma levels of IL-6 from 177.7 ± 24.2 ng/ml to 11.2 ± 5.4 ng/ml in septic animals (Fig. 4b; #, P < 0.05 versus CLP vehicle; n = 5). A similar pattern was observed for plasma levels of CXCL5 (Fig. 4c). Thus, NFAT inhibition reduced CLP-induced plasma levels of HMGB1 by 93%, IL-6 by 94%, and CXCL5 by 54%.

FIG 4.

NFAT controls systemic levels of HMGB1, IL-6, and CXCL5. Mice were pretreated with A-285222 or vehicle (Veh) i.p. twice daily for 7 consecutive days and in the morning before sham operation (white bars) or induction of CLP (black bars). Levels of HMGB1 (a), IL-6 (b), and CXCL5 (c) in plasma were determined 24 h after sham operation or CLP induction by ELISA. Data represent means ± SEM. All experiments were repeated five times (n = 5 mice). *, P < 0.05 versus sham-operated vehicle; #, P < 0.05 versus CLP vehicle.

T-cell apoptosis, proliferation, and cytokine formation.

A significant increase in CD4 T-cell apoptosis in the spleen was observed after CLP (Fig. 5). The percentage of apoptotic CD4 T cells was 1.5% in sham and increased to 12.4% in CLP animals (Fig. 5b; *, P < 0.05 versus sham-operated vehicle; n = 5). Administration of A-285222 reduced the percentage of CD4 T-cell apoptosis to 2.2%, corresponding to a 93% decrease (Fig. 5b; #, P < 0.05 versus CLP vehicle; n = 5). Using PI as a marker of necrotic cells, we found that the number of living splenocytes was decreased in CLP animals (Fig. 6a; *, P < 0.05 versus sham-operated vehicle; n = 5). This was almost completely prevented by treatment with A-285222 (Fig. 6a; #, P < 0.05 versus CLP vehicle; n = 5). Moreover, CLP decreased the percentage of PI⁻ CD4 T cells of all CD4 T cells (Fig. 6c; *, P < 0.05 versus sham-operated vehicle; n = 5). NFAT inhibition prevented both the decrease in PI⁻ CD4 T cells of all splenocytes (Fig. 6b; #, P < 0.05 versus CLP vehicle; n = 5) as well as the decrease in the number of PI⁻ CD4 T cells of all CD4 T cells (Fig. 6c; #, P < 0.05 versus CLP vehicle; n = 5). Flow cytometry showed that the percentage of nondividing CD4 T cells was 19.8% in sham-operated vehicle animals (Fig. 6e). CLP increased the percentage of nondividing CD4 T cells to 69.5% (Fig. 6e; *, P < 0.05 versus sham-operated vehicle; n = 5), and this was prevented by treatment with A-285222, after which levels were 21.4%, corresponding to a 97% reduction (Fig. 6e; #, P < 0.05 versus CLP vehicle; n = 5). Generation of IFN-γ is of key importance to T-cell-dependent immunity (31). Stimulation of splenocytes from CLP mice with anti-CD3ε and anti-CD28 antibodies resulted in lower IFN-γ production than that observed in sham-operated vehicle mice (38.9 pg/ml versus 166.4 pg/ml; Fig. 7a; *, P < 0.05 versus sham-operated vehicle; n = 5), corresponding to approximately a 77% reduction. This reduced IFN-γ was limited by treatment with the NFAT blocker (Fig. 7a; #, P < 0.05 versus CLP vehicle; n = 5). CLP also reduced IL-4 formation in splenocytes by more than 59% (Fig. 7b; *, P < 0.05 versus sham-operated vehicle; n = 5), which was attenuated by treatment with A-285222 (Fig. 7b; #, P < 0.05 versus CLP vehicle; n = 5).

FIG 5.

NFAT regulates CLP-induced CD4 T-cell apoptosis. Mice were pretreated with A-285222 or vehicle (Veh) i.p. twice daily for 7 consecutive days and in the morning before sham operation (white bars) or induction of CLP (black bars). Apoptosis was determined 24 h after sham operation or CLP induction by measuring labeling of DNA strand breaks with BrdUTP as described in Materials and Methods. (a) Representative dot plots of splenocytes from the CD4⁺ gate; (b) aggregate data on apoptosis in CD4 T-cell in the spleen. Data represent means ± SEM. All experiments were repeated five times (n = 5 mice). *, P < 0.05 versus sham-operated vehicle; #, P < 0.05 versus CLP vehicle.

FIG 6.

NFAT regulates CLP-induced hypoproliferation of CD4 T cells. Mice were pretreated with A-285222 or vehicle (Veh) i.p. twice daily for 7 consecutive days and in the morning before sham operation (white bars) or induction of CLP (black bars). Splenocytes were isolated and stained with propidium iodine (PI), carboxyfluorescein diacetate succinimidyl ester (CFSE), and an anti-CD4 antibody. Cell division of CFSE-labeled splenocytes was stimulated with anti-CD3ε and anti-CD28 antibodies and determined by flow cytometry as described in Materials and Methods. (a) A representative dot plot showing splenocytes and aggregate data on PI-negative (PI⁻) splenocytes. (b) A representative dot plot showing splenocytes stained with PI and an anti-CD4 antibody and aggregate data on the percentage of PI⁻ CD4 T cells of all splenocytes. (c) A representative dot plot showing splenocytes stained with PI and an anti-CD4 antibody and aggregate data on the percentage of PI⁻ CD4 T cells of all CD4 T cells. (d) Representative histograms of CFSE profiles of CD4 T cells. Gray line indicates negative-control cells. (e) The line graph shows the percentages of viable CD4 T cells according to the number of divisions. Data represent means ± SEM. All experiments were repeated five times (n = 5 mice). *, P < 0.05 versus sham-operated vehicle; #, P < 0.05 versus CLP vehicle.

FIG 7.

NFAT regulates splenocyte formation of IFN-γ and IL-4 in CLP mice. Mice were pretreated with A-285222 or vehicle (Veh) i.p. twice daily for 7 consecutive days and in the morning before sham operation (white bars) or induction of CLP (black bars). Splenocytes were harvested 24 h after sham operation or CLP induction. Levels of IFN-γ (a) and IL-4 (b) formation in splenocytes were determined 24 h after incubation with anti-CD3ε and anti-CD28 antibodies by ELISA. Data represent means ± SEM. All experiments were repeated five times (n = 5 mice). *, P < 0.05 versus sham-operated vehicle; #, P < 0.05 versus CLP vehicle.

Regulatory T cells.

Regulatory T cells (CD4⁺ CD25⁺ Foxp3⁺) are known to impair immune responses (32). In the present study, it was observed that CLP increased the percentage of regulatory T cells in the spleen by 54% (Fig. 8b; *, P < 0.05 versus sham-operated vehicle; n = 5). Inhibition of NFAT activity reduced the percentage of regulatory T cells to 10.5% in CLP mice, which is similar to levels in sham-operated animals (Fig. 8b; #, P < 0.05 versus CLP vehicle; n = 5).

FIG 8.

NFAT regulates CLP-induced expansion of regulatory T cells. Mice were pretreated with A-285222 or vehicle (Veh) i.p. twice daily for 7 consecutive days and in the morning before sham operation (white bars) or induction of CLP (black bars). The percentage of regulatory T cells (CD4⁺ CD25⁺ Foxp3⁺) in the spleen was determined 24 h after sham operation or CLP induction by flow cytometry. (a) Representative dot plots from the CD4⁺ gate. (b) Aggregate data on the percentages of regulatory T cells in the spleen. Data represent means ± SEM. All experiments were repeated five times (n = 5 mice). *, P < 0.05 versus sham-operated vehicle; #, P < 0.05 versus CLP vehicle.

Systemic bacteremia.

CLP markedly increased the number of bacteria in blood, peritoneal cavity, spleen, and lung (Fig. 9; *, P < 0.05 versus sham-operated vehicle; n = 5). Treatment with A-285222 had, within the time frame investigated here (24 h), no significant impact on the number of bacteria in the septic animals (Fig. 9; P > 0.05 versus CLP vehicle; n = 5).

FIG 9.

Bacterial counts were quantified in blood (a), peritoneal cavity (b), lung (c), and spleen (d) as described in Materials and Methods. Mice were pretreated with A-285222 or vehicle (Veh) i.p. twice daily for 7 consecutive days and in the morning before sham operation (white bars) or induction of CLP (black bars). Samples were obtained 24 h after sham operation or CLP induction. Data represent means ± SEM. All experiments were repeated five times (n = 5 mice). *, P < 0.05 versus sham-operated vehicle.

DISCUSSION

Management of septic patients with infectious complications poses a major challenge to clinicians due to the limited therapeutic options. Systemic inflammation and immune suppression are insidious aspects of the host reaction to severe infections or major trauma. Our novel findings show for the first time that NFAT is an important regulator of pulmonary accumulation of neutrophils, systemic inflammation, and T-cell dysfunction in abdominal sepsis.

It is well understood that activation of the host innate immune system is a critical step in sepsis, causing lung dysfunction and impaired gaseous exchange (33). However, the signaling pathways regulating neutrophil infiltration and lung injury in polymicrobial sepsis remain elusive. NFAT activity is generally considered to control aspects of tissue development during embryogenesis, including vasculogenesis, axonal outgrowth, muscle and bone formation, as well as maturation of the immune system (34–38). However, a growing body of literature also implicates NFAT signaling in inflammatory processes, such as atherosclerosis (39), autoimmune diseases (40), and acute pancreatitis (24). Here, we show that in vivo administration of the NFAT inhibitor (A-285222) readily blocked sepsis-induced NFAT-dependent transcriptional activity not only in the lung but also in the spleen, liver, and aorta, suggesting not only that NFAT is activated in sepsis but also that A-285222 is an effective inhibitor of NFAT.

Moreover, we demonstrate that A-285222 markedly reduced pulmonary edema and tissue damage in abdominal sepsis. It is well known that depletion of neutrophils or targeting specific adhesion molecules critical in the extravasation process of neutrophils protects against septic lung injury, showing that neutrophil accumulation is a fundamental component in sepsis (23, 41). In the present study, we could document that inhibition of NFAT by administration of A-285222 decreased sepsis-induced neutrophil infiltration in the bronchoalveolar space by 54%, indicating that A-285222 effectively inhibits neutrophil accumulation in septic lung damage. This is the first study to show that NFAT plays a key role in regulating neutrophil trafficking. In general, tissue navigation of leukocytes at sites of inflammation is orchestrated by secreted chemokines (42). Neutrophils are particularly activated and attracted by CXC chemokines, comprising CXCL1, CXCL2, and CXCL5 in mice. In the present study, we found that administration of A-285222 reduced sepsis-induced formation of CXCL1, CXCL2, and CXCL5 in the lung by more than 62%, 81%, and 59%, respectively, suggesting that NFAT activity is a key regulator of CXC chemokine production in septic lung damage. In addition, this observation may help to explain the inhibitory effect of A-285222 on CLP-induced neutrophil infiltration in the lung. Interestingly, some evidence in the literature suggests that cyclosporine and FK506, two calcineurin inhibitors, can reduce neutrophil responses and protect against endotoxemia and acute lung injury (43, 44). Considering that NFAT activity is regulated by calcineurin (45), our findings might help explain these inhibitory effects of calcineurin inhibitors on endotoxemia and pulmonary injury. Collectively, our data suggest a pathological role for the calcium/calcineurin-NFAT signaling axis in the development of septic lung damage similar to that proposed for the development of cardiac hypertrophy (22), diabetes-induced vascular inflammation (46), atherosclerosis (39, 47), and acute pancreatitis (24). HMGB1 and IL-6 are potent proinflammatory cytokines and markers of systemic inflammation in endotoxemia and sepsis (48, 49). In line with previous studies, we observed that CLP caused a clear-cut increase in the plasma levels of HMGB1 and IL-6. Notably, A-285222 treatment reduced HMGB1 and IL-6 levels in the plasma by more than 93% and 94%, respectively, in septic mice, indicating that NFAT is a potent regulator of systemic inflammation in sepsis. IL-6 has previously been described as an NFAT target gene (25, 50), but the finding that NFAT regulates HMGB1 is new. High levels of HMGB1 have recently been shown to prevent NFAT activation in T cells (51, 52), suggesting that it may act as an endogenous negative regulator of NFAT in sepsis in an attempt to limit the damage. Similar examples of endogenous regulators of calcineurin/NFAT have been demonstrated in the past, such as DSCR1 (53). The effects of NFAT on HMGB1 and IL-6 might have significant implications in other systemic inflammatory diseases, such as severe acute pancreatitis. Our present results constitute the first evidence that NFAT plays an important role in systemic inflammation, pulmonary recruitment of neutrophils, and tissue damage in abdominal sepsis. In this context, it should be noted that one should be cautious when targeting key transcription factors, such as NFAT, in patients due to potential side effects. However, the strength of our approach is that we are using very low doses of A-285222, sufficient to block sepsis-induced parameters but without effect on basal NFAT-dependent transcriptional activity, as shown in Fig. 1.

Sepsis not only causes a systemic proinflammatory phase but also an anti-inflammatory phase characterized by T-cell dysfunction. During this anti-inflammatory phase of sepsis, infectious complications constitute the major cause of mortality in septic patients (1). Herein, we observed that A-285222 decreased both CD4 T-cell apoptosis and increased the proliferative response of CD4 T cells in septic mice, indicating that inhibition of NFAT signaling protects T cells in the course of polymicrobial sepsis, which, in turn, might optimize host defenses against microbial invasions. Herein, we found that inhibition of NFAT activity also inhibited sepsis-induced suppression of IFN-γ and IL-4 production in splenocytes, which could help to raise effective antibacterial responses over time. However, in this study we found no impact of NFAT inhibition on bacterial burden in blood, peritoneal cavity, lung, and spleen 24 h after CLP induction. In this context, it is important to note that NFAT has been well established as a promoter of T-cell maturation and differentiation (54). At first glance, our findings showing that inhibition of NFAT protects T-cell survival and cytokine formation might appear counterintuitive. However, we also observed that NFAT inhibition had no effect on T-cell apoptosis and proliferation in sham animals but only in septic animals.

Knowing that T-cell dysfunction is a consequence of the overwhelming systemic inflammatory response, we conclude that the T-cell protective effects of inhibiting NFAT signaling are secondary and due mainly to the attenuated proinflammatory response in septic mice. Another phenomenon during the anti-inflammatory phase of sepsis is the induction of regulatory T cells. These cells are known to be powerful regulators of T-cell-mediated immune responses (55). In the present study, it was found that the number of regulatory T cells was clearly increased in the spleen of septic animals. Administration of A-285222 markedly decreased the CLP-triggered induction of regulatory T cells, indicating that NFAT signaling mediates regulatory T-cell induction during the compensatory anti-inflammatory phase. It is concluded that inhibition of NFAT activity controls T-cell function by restoring the number of CD4 T cells and their cytokine formation capacity and by antagonizing the induction of regulatory T cells. In this context, it is interesting to note that a previous study reported that HMGB1 inhibition reduces cancer cell-induced generation of regulatory T cells (56). Whether HMGB1 might be involved in the induction of regulatory T cells in polymicrobial sepsis remains to be elucidated, but such a link might shed light on the connection between NFAT activity on one hand and induction of regulatory T cells on the other hand in polymicrobial sepsis.

A schematic representation of the proposed model for the effects of NFAT signaling inhibition on CLP-triggered responses is shown in Fig. 10. In summary, our findings document for the first time that NFAT signaling is a key feature in abdominal sepsis. We demonstrated that sepsis is associated with enhanced NFAT transcriptional activity in the lung as well as in the spleen, liver, and aorta and that pharmacological inhibition of NFAT signaling decreased sepsis-induced NFAT-dependent transcriptional activity, the formation of CXC chemokines, and neutrophil infiltration, as well as edema and tissue damage in the lung. Circulating levels of plasma HMGB1, IL-6, and CXCL5 are also enhanced by CLP, evidencing a systemic inflammatory response in this model. We also showed that inhibition of NFAT activity clearly reduced sepsis-triggered formation of these cytokines in plasma. Sepsis also caused CD4 T-cell dysfunction, as shown by increased splenocyte apoptosis, decreased splenocyte proliferative capacity, and concomitant decreased IFN-γ and IL-4 production. Inhibition of NFAT restored CD4 T-cell function and also antagonized the induction of regulatory T cells in septic mice. Thus, NFAT signaling plays an important role in abdominal sepsis and might be a useful target in order to attenuate pathological inflammation and improve immune function in patients with abdominal sepsis.

FIG 10.

Proposed model for the effects of NFAT signaling inhibition on CLP-triggered responses. CLP activates NFAT-dependent transcriptional activity in lung, spleen, liver, and aorta (1). In the lung, CLP increases CXCL1, CXCL2, and CXCL5 levels (2) and neutrophil infiltration, which contributes to pulmonary edema (3). Circulating levels of IL-6, HMGB1, and CXCL5 are also enhanced by CLP, evidence of a systemic inflammatory response. This in turn results in CD4 T-cell dysfunction and reduced IFN-γ and IL-4 levels (4) and induction of regulatory T cells (T-reg) (5). Treatment with the NFAT blocker A-285222 not only inhibited NFAT-dependent transcriptional activity and all CLP-induced events (1 to 3) but also restored CD4 T-cell function (via combined effects on T-cell apoptosis, proliferation, and cytokine production) and antagonized the induction of T-regs (4, 5). The effects of A-285222 on 4 and 5 may be secondary to the attenuation of systemic inflammation, nevertheless critical for the regulation of antimicrobial responses during the later anti-inflammatory phase of sepsis.