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Basic Science Aspects

FTY720-Induced Lymphopenia Does Not Aggravate Mortality in a Murine Model of Polymicrobial Abdominal Sepsis

Enderes, Jana; van der Linde, Julia; Müller, Jan; Tran, Bich-Thu; von Bernstorff, Wolfram; Heidecke, Claus-Dieter; Schulze, Tobias

Author Information
doi: 10.1097/SHK.0000000000000739

Abstract

INTRODUCTION

Estimates suggest that there are up to 19 million incidences of sepsis per year worldwide (1). In industrialized countries mortality due to septic shock has dropped from over 80% 30 years ago to a present level of 20% to 30% (2, 3). However, the incidence of sepsis and septic shock is increasing. This is due to an aging population, an increase of chronic diseases, increasing numbers of invasive procedures, and an increase of patients taking immunosuppressive medication or with immunocompromising diseases. In surgical intensive care units, abdominal sepsis is a frequent cause of severe sepsis and septic shock leading to mortality rates between 32.4% and 42.3% (4). The cellular mechanisms leading to and perpetuating the septic cascade have been extensively examined. The role of the innate immune system in sepsis has long been the main focus of scientific interest. Recent experimental work has convincingly shown that cells of the adaptive immune system not only have an impact on the long-term outcome of septic patients but also intervene at early stages of sepsis (5, 6).

FTY720 is an immunosuppressive molecule acting fundamentally different from other immunosuppressive drugs: while the latter mostly impact on activation, proliferation, expansion or cytokine production of lymphocytes, FTY720 profoundly remodels lymphocyte trafficking by inhibiting lymphocyte egress from primary and secondary lymphatic organs (7, 8). FTY720 binds to four out of five S1P receptors (S1P1, S1P3–S1P5) (9). It acts as a functional antagonist of S1P1 by inducing its internalisation. By blocking signalling via S1P1, FTY720 induces lymphocyte sequestration in secondary lymphatic organs and in the thymus via an exit block. FTY720 has also been shown to modify cellular infiltrate at the inflammatory site (10).

In 2010, FTY720 was approved by the Food and Drug administration for the treatment of the chronic relapsing form of multiple sclerosis in the United States. Multiple sclerosis is a chronic inflammatory autoimmune disease of the central nervous system affecting one to two in 1,000 people of the white population in Western Europe. During clinical trials evaluating FTY720 treatment in multiple sclerosis patients there were deaths due to infectious complications (11). Although total infectious complications were rare an increased incidence of lower respiratory tract infections was observed in the FTY720 group (12). FTY720 has recently been shown to have beneficial effects on cardiac dysfunction in a murine model of polymicrobial sepsis (13). Also, in other forms of shock, i.e., trauma and hemorrhagic shock, FTY720 action seemed to limit a multi-organ dysfunction syndrome (14). However, the impact of concomitant FTY720 medication on sepsis mortality has not yet been explored. Given the recent introduction of FTY720 for the treatment of multiple sclerosis, visceral surgeons will be confronted with an increasing number of patients on FTY720 treatment presenting for elective or emergency surgery. Therefore, the risk of FTY720-treated patients to experience more severe courses of abdominal sepsis than immunocompetent patients needs to be further evaluated.

In the present work, the impact of FTY720 on immune cell populations, cytokine kinetics, and the clinical course of abdominal sepsis was investigated in the colon ascendens stent peritonitis (CASP) model (15). We show that although FTY720 induces profound changes in the composition of immune cell populations in various immune compartments, sepsis mortality remained unchanged under pre-existing FTY720 medication.

MATERIALS AND METHODS

Mice

Specific-pathogen-free female C57BL/6 mice, 12 weeks of age and weight of 20 g to 25 g, were used for all experiments. Mice were obtained from Charles River Laboratories, Germany, and kept for 2 weeks in our animal facility to recover from transportation. Animal experiments were performed according to the German Protection of Animals Act (TierSchG); permission was obtained from the veterinary government authority (7221.3-1.1-057/11, LALLF, Rostock, Germany).

FTY720

A stock solution of FTY720 (Cayman Chemical, Ann Arbor, Mich) was prepared at a concentration of 10 mg/mL FTY720 in ethanol. This stock solution was then diluted with phosphate-buffered saline (PBS). A first dose of FTY720 of 0.5 mg/kg KG was intraperitoneally administered 24 h prior to surgery, followed by a second dose of FTY720 of 0.5 mg/kg KG directly after surgery.

Colon ascendens stent peritonitis surgery

CASP surgery was performed as previously described (15). Briefly, mice were anaesthetized and placed in a supine position. A median laparotomy was performed and the caecum was identified. A plastic stent (16 G; BD Biosciences, Heidelberg, Germany) was inserted into the antimesenteric wall of the cecum 1.0 cm distal to the ileocaecal valve and fixed using a suture (Mariderm black 7/0 USP suture, Catgut GmbH, Markneukirchen, Germany). Stool was milked out and fluid resuscitation was performed by intraperitoneal administration of 0.5 mL of sterile saline solution. The abdominal wall was closed by a single-layer continuous suture (Polyester white 4/0 USP suture, Catgut GmbH).

In the FTY720 and Placebo group CASP surgery was performed as described above, whereas in the Sham group a stent was sutured to the colonic wall without previous puncture of the intestinal wall (Sham surgery).

Fluorescence activated cell sorting (FACS) analysis

FACS analysis was performed in the blood, the peritoneal lavage fluid, the mesenteric lymph nodes, and the spleen.

Sample preparation. Blood was harvested by retroorbital puncture. Peritoneal lavage was performed by installation of 3.0 mL sterile saline solution in the abdominal cavity, which was subsequently completely aspirated. Single cell suspensions of mesenteric lymph nodes and spleen were obtained by mechanical dissociation using a 40 μm cell strainer (BD Biosciences).

Sample staining. Peritoneal lavage fluid, mesenteric lymph nodes, and spleens: Single cell solutions were blocked with FcR-block (anti-CD16/32, BioLegend GmbH, Fell, Germany). The myeloid cell staining was composed of FITC-labeled anti-Gr1 (BioLegend), PE-labeled SiglecF (BD Biosciences), PerCp Cy5.5-labeled CD11b (BD Biosciences), PE Cy7-labeled F4/80 (eBioscience, Frankfurt am Main, Germany), APC-labeled MHCII (Miltenyi Biotec, Bergisch Gladbach, Germany), APC-Cy7-labeled CD45 (BioLegend), and Pacific blue-labeled Ly6G (BioLegend) to determine eosinophil, neutrophil, monocyte, and macrophage numbers within the peritoneal lavage fluid. The lymphocyte staining contained FITC-labeled CD4 (BioLegend), PE-labeled CD19 (BD Biosciences), PerCp Cy5.5-labeled CD3e (eBioscience), PE-Cy7-labeled CD8a (BioLegend), APC-labeled Ly6G (Miltenyi Biotec), APC-Cy7-labeled CD45 (BioLegend), and V450-labeled CD11b (BD Biosciences) to determine CD4+ and CD8+ T cell numbers as well as B-cell numbers within the peritoneal lavage, mesenteric lymph nodes, and spleens. Absolute cell counts of the peritoneal lavage and the spleens were determined using BD Trucount tubes (BD Biosciences) according to the manufacturer's recommendations.

Peripheral blood: lymphocyte staining was performed in whole blood with the antibody combination described above. Erythrocytes were subsequently lysed using lysis buffer (155 mM NH4Cl, 10 mM KHCO3, 0.1 mM EDTA ad 1 L of distilled water).

Measurement: Flow cytometry was conducted using FACSCanto II flow cytometer (Becton Dickinson, Franklin Lakes, NJ). Data were analyzed using the FlowJo 7.6 software.

Cytokine analysis

Cytokine determination was performed in plasma and peritoneal lavage fluid as well as in homogenates from lung and liver. Lung and liver were harvested and homogenized using a Precellys24 tissue homogenizer. Organ homogenates were obtained using a membrane dissolving solution (1 mM EDTA, 0.25%w/v 3-((cholamidopropyl)-dimethylamino) propansulfate (CHAPS), 0.5% v/v Tween 20, 1 mM 4-(2-aminoethyl) benzenesulfonyl fluoride hydrochloride (PEFA-Bloc), 1 mM phenylmethylsulfonyl fluoride (PMSF), and one tablet Complete protease inhibitor in 25 mL PBS). Cytokines were analyzed using the Cytometric Bead Array “Mouse Inflammation Kit” (BD Biosciences) as well as the Mouse TH1/TH2/TH17/TH22 13plex Kit FlowCytomix (eBioscience) following the manufacturer's instructions.

Bacteriology

For detection of the bacterial load in liver and lung, organs were removed 12 h after surgical treatment and placed in 5 mL ice-cold sterile saline solution (0.9% NaCl). The organs were homogenized using a Precellys24 tissue homogenizer. Homogenates were plated at different dilutions on blood-containing agar (Columbus 5% SB; BD Biosciences) and incubated for 18 h at 37°C. The number of colony-forming units (cfu) was related to 1 mg organ weight. Whole blood was collected in EDTA-containing vials (Becton Dickinson, Heidelberg, Germany), 10 μL was plated on blood-containing agar at different dilutions and incubated for 18 h at 37°C. The number of colony-forming units (cfu) was related to 1 mL of blood.

Impact of FTY720 on survival after CASP

To assess the impact of FTY720-induced immunosuppression on survival after polymicrobial abdominal sepsis mice were pretreated with FTY720 or placebo 24 h before induction of CASP to efficiently induce peripheral lymphopenia and lymphocyte sequestration to secondary lymphoid organs (Supplementary Figure S1, http://links.lww.com/SHK/A461). For partial compensation of diminished pathogen clearance due to hampered T-cell function two more groups treated with a combination of FTY720/broad spectrum antibiotics or placebo/broad spectrum antibiotics were added to the experimental setting.

Five different experimental groups were generated: Placebo (30 mice), FTY720 (30 mice), Placebo + broad spectrum antibiotics (30 mice), FTY720 + broad spectrum antibiotics (30 mice), Sham (30 mice).

FTY720 treatment as well as the appropriate controls was performed as described above. CASP surgery was performed on both Placebo and FTY720 groups, Sham surgery was performed on the Sham group as described above. Antibiotics were administered by intraperitoneal injection: Ceftriaxone (25 mg/kg body weight) and metronidazole (12.5 mg/kg body weight). Antibiotic therapy was initiated 1 h after surgery followed by further administrations every 12 h until time of death or time of censoring (192 h = 8 days), respectively.

Survival was monitored every 2 h during the first 24 h and every 4 h thereafter. The respective times of death or times of censoring were documented, respectively.

Statistical analysis

Statistical analysis was performed using Graph Pad Prism Version 6.0 for Windows software (GraphPad Software, San Diego, Calif). The data (except for the survival curves) were reported as means ± SD.

Statistical differences were assessed using Mann–Whitney–(Wilcoxon) U test, repeated measures two-way ANOVA followed by Bonferroni post-test and ordinary one-way ANOVA followed by Tukey post-test. Survival data were analyzed using Kaplan–Meier survival curves and the log rank test. Data were considered statistically significant at P < 0.05.

RESULTS

Cell dynamics and cytokine levels after CASP induction

CASP is one of the most frequently employed murine models for polymicrobial abdominal sepsis. However, the dynamic changes in cell populations of immune cells in various immune compartments during the development of abdominal sepsis have not yet been described in this model. In order to characterize the sepsis-induced changes of cell populations within the abdominal cavity, the mesenteric lymph nodes, the spleen and the peripheral blood, FACS analyses of these organs were performed during the initial 24 h of abdominal sepsis.

Peripheral blood

CASP mice developed peripheral leukopenia 24 h after peritonitis induction. B cells were most severely affected, showing a 66% reduction within the peripheral blood at 24 h after CASP induction (P = 0.0007). However, CD4+ and CD8+ T-cell numbers did not show any significant changes during the initial 24 h after peritonitis induction (CD4+: P = 0.8570; CD8+: P = 0.0942). After an initial increase at 6 h, myeloid cells and neutrophil granulocytes showed slightly diminished cell numbers after 24 h. However, the latter changes did not reach statistical significance (myeloid cells: P = 0.7668, neutrophils: P = 0.9960) (Fig. 1).

Fig. 1
Fig. 1:
Cell dynamics in the peripheral blood after CASP induction.Leukocytes (A), B cells (B), CD4+ T cells (C), CD8+ T cells (D), myeloid cells (CD45+ CD11b+) (E), and neutrophils (CD45+ CD11b+ SiglecF Gr1hi Ly6Ghi) (F) were analyzed at different time points (t = 6, 12, 18, and 24 h) after CASP induction. Untreated mice did not receive any surgical intervention (t = 0 h) (n = 3). Data were reported as means ± SD. CASP indicates colon ascendens stent peritonitis. *P < 0.05, ***P < 0.001.

Peritoneal cavity

Absolute cell numbers within the peritoneal cavity increased during the initial 24 h after CASP induction (P = 0.0372). However, individual cell populations showed different dynamics (Fig. 2). While B cells, CD4+, and CD8+ T cells as well as macrophages decreased rapidly after sepsis induction to reach their nadir after 6 h, neutrophil numbers already started a rapid and massive increase after 6 h and continued to rise up to 12 h after sepsis induction. After 6 h, all lymphocyte lineages showed a slow but steady increase within the peritoneal fluid. F4/80low MHCIIhigh and F4/80high MHCIIlow macrophages, which were present at low numbers in the peritoneal cavity of healthy mice, also rapidly decreased after sepsis induction to reach a minimum after 6 h. In contrast to lymphocyte numbers already starting to increase 12 h after sepsis induction, the increase in macrophage numbers occurred later and with a lower slope. While lymphocyte numbers reached presepsis levels 24 h after CASP, both F4/80low MHCIIhigh and F4/80high MHCIIlow macrophages did not attain their preseptic base levels. Interestingly, monocytes—almost absent in the peritoneal cavity of nonseptic mice—showed a clear increase 24 h after sepsis induction (P = 0.0342).

Fig. 2
Fig. 2:
Cell dynamics within the peritoneal lavage fluid after CASP induction.Leukocytes (A), B cells (B), CD4+ T cells (C), CD8+ T cells (D), neutrophils (CD45+ CD11b+ SiglecF Gr1hi Ly6Ghi) (E), monocytes (CD45+ CD11b+ SiglecF Gr1hi Ly6Gint) (F), F4/80lowMHCIIhigh macrophages (G), and F4/80highMHCIIlow macrophages (H) were analyzed at different time points (t = 6, 12, 18, and 24 h) after CASP induction. Untreated mice did not receive any surgical intervention (t = 0 h) (n = 3). Data were reported as means ± SD. CASP indicates colon ascendens stent peritonitis. *P < 0.05, **P < 0.01, ***P < 0.001.

Mesenteric lymph nodes

The percentages of B cells, CD4+, and CD8+ T cells in single cell suspensions of mesenteric lymph nodes did not show any quantitative changes. However, the small fraction of myeloid cells showed a relative reduction of 50% 6, 12, and 18 h postoperatively (p.op.). However, after 24 h, cell numbers returned to the preoperative level (Supplementary Figure 2, http://links.lww.com/SHK/A462).

Spleen

After sepsis induction, neither the CD4+ T-cell fraction nor the B-cell fraction of the spleens showed quantitative changes. In contrast, the small splenic CD8+ T-cell fraction was significantly increased after 6 h (50% increase; P = 0.0082) and subsequently returned to preoperative levels 24 h after sepsis induction. Contrary to CD8+ T cells, myeloid cells showed a clear reduction 6 h after CASP (P = 0.0100), and returned to the initial value 24 h p.op. (Supplementary Figure 3, http://links.lww.com/SHK/A463).

Cytokine development after CASP induction

The kinetics of cytokine levels were assessed within the peritoneal cavity and plasma after CASP induction (Supplementary Figure 4, http://links.lww.com/SHK/A464). In the peritoneal cavity, the levels of the pro-inflammatory cytokines lL-6 and TNFα already showed an early massive increase 6 h after CASP, remained constant at increased levels until 24 h p.op., and showed a clear decrease after 36 h p.op. The development of MCP-1 levels showed similar characteristics. Interestingly, the immunoregulatory cytokine IL-10 showed similar kinetics to the pro-inflammatory cytokines IL-6 and TNFα, rapidly rising to 2493.6 pg/mL at 6 h and reaching maximum levels at 24 h (5474.7 pg/mL). At 36 h, IL-10 levels were clearly decreased compared with maximum levels at 24 h p.op. In contrast to the aforementioned cytokines, both IL-12 and INFγ showed a less steep increase. IL-12 reached its maximum level at 6 h p.op. In contrast, INFγ reached its plateau value at 12 h p.op. Neither cytokine showed a significant decrease until 36 h p.op.

In plasma, the cytokines IL-6, IL-10, MCP-1, and TNFα showed kinetics comparable to those of the peritoneal compartment. IL-12 as well as INFγ levels did not increase but undulated around the preoperative levels.

Impact of FTY720 on cellular dynamics and cytokine levels during CASP

Based on the results of the previously described experiments, the time point 12 h p.op. was chosen to analyze the impact of FTY720 treatment on cell dynamics after CASP induction in various immune compartments.

Peripheral blood

As expected, B cells, CD4+ T cells, and CD8+ T cells were significantly reduced in FTY720-treated animals 12 h after CASP induction (B cells: P = 0.0048; CD4+: P < 0.0001; CD8+: P < 0.0001). CD4+ T-cell populations were reduced by 80.9%, while CD8+ T-cell numbers were reduced by 81.53%. B cells were less affected, showing a reduction of only 41.2% (Fig. 3).

Fig. 3
Fig. 3:
Impact of FTY720 on cellular dynamics in the blood after CASP induction.CASP surgery was performed on mice treated with FTY720 or Placebo. 12 h after surgery absolute cell counts of leukocytes (A), B cells (B), CD4+ T cells (C), and CD8+ T cells (D) were analyzed (n = 16). Data were reported as means ± SD. CASP indicates colon ascendens stent peritonitis. **P < 0.01, ****P < 0.0001.

Peritoneal cavity

Within the peritoneal cavity FTY720 administration before CASP induction did not result in significant quantitative differences in cell numbers of the lymphoid lineage, neutrophils, monocytes, and F4/80high MHCIIlow macrophages (B cells: P = 0.5658; CD4+: P = 0.4865; CD8+: P = 0.4491; neutrophils: P = 0.8328; monocytes: P = 0.3164; F4/80high MHCIIlow macrophages: P = 0.0792) (Fig. 4). Interestingly, the number of F4/80low MHCIIhigh macrophages was significantly increased in FTY720-treated animals (P = 0.0374).

Fig. 4
Fig. 4:
Impact of FTY720 on cellular dynamics in the peritoneal lavage fluid after CASP induction.CASP surgery was performed on mice treated with FTY720 or Placebo. 12 h after surgery absolute cell counts of leukocytes (A), B cells (B), CD4+ T cells (C), CD8+ T cells (D), neutrophils (CD45+ CD11b+ SiglecF-Gr1hi Ly6Ghi) (E), monocytes (CD45+ CD11b+ SiglecF Gr1hi Ly6Gint) (F), F4/80lowMHCIIhigh macrophages (G), and F4/80highMHCIIlow macrophages (H) were analyzed (n = 16). Data were reported as means ± SD. CASP indicates colon ascendens stent peritonitis. *P < 0.05.

Mesenteric lymph nodes

In mesenteric lymph nodes no relevant differences in the proportion of B cells, CD4+, and CD8+ T cells were revealed by flow cytometric analyses (data not shown).

Spleen

In spleens, FTY720 administration before CASP induction induced reduced cell numbers of CD4+ und CD8+ cells (CD4+: P = 0.0156; CD8+: P = 0.0374) (Fig. 5).

Fig. 5
Fig. 5:
Impact of FTY720 on cellular dynamics in the spleen after CASP induction.CASP surgery was performed on mice treated with FTY720 or Placebo. 12 h after surgery absolute cell counts of leukocytes (A), lymphocytes (B), B cells (C), CD4+ T cells (D), and CD8+ T cells (E) were analyzed (n = 16). Data were reported as means ± SD. CASP indicates colon ascendens stent peritonitis. *P < 0.05.

Cytokine levels

Levels of pro- and anti-inflammatory cytokines (IL-1α, IL-2, IL-4, IL-5, IL-6, IL-10, IL-13, IL-17, IL21, IL-22, Interferon Gamma, and TNF alpha) were measured in plasma, peritoneal lavage fluid, liver, and lung. No differences were seen between FTY720-treated and untreated animals 12 h after CASP (Supplementary Figure S5, http://links.lww.com/SHK/A465).

Impact of FTY720 on bacterial load in blood, lung, and liver

Since FTY720 was shown to influence dissemination of intracellular bacteria and clearance of enteropathogenic noninvasive bacteria, we performed bacteriological analysis of blood, lung, and liver 12 h after CASP induction. Bacterial load showed no significant differences between the FTY720 and the control group either in the blood or in the distant organs lung and liver (Fig. 6).

Fig. 6
Fig. 6:
Impact of FTY720 on bacterial load during CASP.Mice were treated with FTY720 or Placebo. Bacterial count was analyzed 12 h after CASP induction in blood (A), lung (B), and liver (C) (n = 5). CASP indicates colon ascendens stent peritonitis. Data were reported as means ± SD and considered statistically significant at P ≤ 0.05.

Impact of FTY720 on survival after CASP

CASP-induced sepsis resulted in similar survival in FTY720 and placebo pretreated mice. No animal survived longer than 37 h, and survival curves were not different in both groups (Fig. 7). Antibiotic treatment resulted in significantly prolonged survival in both experimental groups. After 192 h, there was a 7% survival in the FTY720/broad spectrum antibiotics as well as in the placebo/broad spectrum antibiotics group. However, there were not any statistically significant differences between survival curves of both experimental groups (survival FTY720/placebo: P = 0.6309; survival FTY720+AB/placebo+AB: P = 0.8998).

Fig. 7
Fig. 7:
Impact of FTY720 on survival after CASP.CASP surgery was performed on mice treated with either FTY720, Placebo, FTY720/broad spectrum antibiotics (FTY720+AB) or Placebo/broad spectrum antibiotics (Placebo+AB). Control mice underwent Sham surgery (n = 15). Survival was monitored over 192 h. CASP indicates colon ascendens stent peritonitis. Data were analyzed using the log rank test and considered statistically significant at P < 0.05.

DISCUSSION

Immunosuppression was identified as a risk factor for the development of community-acquired sepsis (16), as well as for the development of sepsis after trauma (17). Furthermore, presence of immunosuppression prior to the onset of sepsis is a risk factor for the development of septic shock and an increased 28-days mortality in septic patients (18). In the latter study, the odds ratio for death within 28 days after sepsis onset was 1.85 in patients with any kind of T lymphocyte suppression (18). Interestingly, a clinical study from 1979 on postoperative sepsis identified low preoperative lymphocyte counts as a significant risk factor for the development of postoperative septic complications (19). Also, HIV-infected patients with low peripheral CD4+ T-cell counts have a higher risk to develop postoperative sepsis (20).

FTY720 is a novel immunosuppressive drug with a unique mode of action. Its functional antagonism to S1P1-mediated signalling induces a profound inhibition of T-cell egress from secondary lymphoid organs. This leads to a peripheral lymphopenia and an inhibition of T effector cell migration to the site of inflammation. Although an increasing number of patients have been treated with this drug since its introduction in the treatment of chronic relapsing multiple sclerosis, the effects of FTY720 on the clinical course of sepsis of various origins have not yet been assessed.

In the present study, we could show that FTY720-induced lymphopenia did not result in increased mortality in a murine CASP model of polymicrobial abdominal sepsis.

Other investigators have shown that administration of immunosuppressive drugs to mice with CASP resulted in severely increased mortality (21). The drugs tested were tacrolimus, glucocorticoids, and mycophenolate mofetil (MMF). Combinations of immunosuppressive and antibiotic treatment significantly prolonged survival in this study by Assfalg et al. Interestingly, neither prolongation of survival nor reduced mortality was seen in our study after combining FTY720 and antibiotics compared to a placebo group treated with antibiotics only. However, the mechanism of action of the drugs used by Assfalg et al. is fundamentally different from that of FTY720. Tacrolimus causes a profound inhibition of IL-2 production and thus inhibits T-cell activation and proliferation (22). MMF supresses both B and T-cell function by inhibiting the type II isoform of adenosine desaminase (AD) preferentially found in T and B cells. Suppression of AD activity in T cells results in decreased T-cell proliferation and increased apoptosis of activated T cells (23). MMF blocks B-cell proliferation and antibody production. In contrast to Tacrolimus and MMF, corticosteroids exert their action on a much broader range of cells both of the innate and the adaptive immune system. Glucocorticoid action on antigen-presenting cells and on T cells favors the development of a Th2 polarized immune response and hampers the activation of proinflammatory cytokine genes (for review see (24)). In contrast, FTY720-induced immunosuppression does not result from the inhibition of T-cell proliferation, survival, or cytokine production but from T-cell sequestration in secondary lymphatic organs. Effector cell exit and migration to the site of infection is blocked.

In the experimental setting of Assfalg et al., combined treatment with tacrolimus and antibiotics increased sepsis lethality compared with controls treated with antibiotics only. This is in contrast to the unchanged mortality of animals receiving FTY720 and antibiotic treatment seen in our experimental setting. However, although both molecules act on T cells, the intrinsic T-cell functions affected are different. While tacrolimus-induced inhibition of T-cell proliferation and activation seems to contribute to a reduced early survival after polymicrobial sepsis, blockage of T-cell migration and the consecutive presence of peripheral lymphopenia did not influence survival under our experimental conditions.

Double or triple immunosuppressive treatment with tacrolimus, MMF and glucocorticosteroids combined with antibiotic treatment resulted in reduced mortality under the experimental conditions used by Assfalg et al. (25). IL-22 levels were significantly reduced in spleens of immunosuppressed animals, and there was experimental evidence for increased neutrophil activation and antimicrobial activity. In septic patients, elevated IL-22 levels have been shown in a postoperative setting (25). In animal models of septic shock, elevated IL-22 levels were found in blood and distal compartments, and blocking IL-22 action resulted in decreased bacterial burden (26). FTY720 has been shown to reduce IL-22 expression in human T-cells ex vivo(27). Under our experimental conditions FTY720 did not reduce IL-22 levels in mice with polymicrobial abdominal sepsis. Although IL-22 levels were slightly lower in the blood and lung of FTY720-treated animals, the differences did not reach statistical significance. Thus, in polymicrobial abdominal sepsis the FTY720-induced decrease of IL-22 expression described in vitro did not translate into a clinically significant survival advantage.

In our model FTY720 resulted in significantly reduced lymphocyte numbers in the peripheral blood after sepsis induction. The role of T-lymphocytes in early stages of septic shock is still being debated. Given the delay in the development of an adaptive immune response, T cells have been thought to intervene in later phases of sepsis. In recent years, T-lymphocytes have been shown to respond to bacterial products in an antigen-non-specific manner due to the engagement of Toll-like receptors (TLR) (28). In an infectious disease model, in which viral infection led to stimulation of TLR3 resulting in an early cytokine storm and early death, both CD4+ and CD8+ T cells tempered the initial innate immune response of non-lymphoid cells. The inhibition was mediated by cell-cell contact and was MHC-dependent. The same group reported that LPS-stimulated cytokine production by non-T-cells was controlled by T-cells ex vivo(29). These results clearly show that T-lymphocytes dispose of the molecular armament to intervene at early stages of septic disease. Murine sepsis models using various lymphocyte-deficient mouse strains or different methods of lymphocyte depletion revealed conflicting data on the impact of lymphocytes on sepsis survival. While some investigators reported no influence of lymphocyte deficiency on mortality in septic mice, reduced survival in Rag-1−/− and nude mice was shown in various polymicrobial sepsis models by others. Our results clearly showed that peripheral lymphopenia during the infectious challenge as well as during the early stages of abdominal sepsis did not affect sepsis mortality.

FTY720-induced lymphopenia did not influence the bacterial load found in blood and liver as well as in the lung early after CASP induction. This corresponds to findings of Murphy et al. (30), who found similar cfu numbers of Citrobacter rodentium in the spleen of FTY720- and placebo-treated mice 8 days after mucosal infection. However, in the latter study, pathogen clearance was delayed in the FTY720 group during long-term follow-up and splenic bacterial load was significantly higher 14 days after infection with this noninvasive enteropathogenic bacteria. Thus, we cannot exclude that in a less severe model of polymicrobial sepsis, FTY720-induced attenuation of the adaptive immune response may lead to a higher bacterial load in the long-term follow-up. Further studies on this issue in a less severe sepsis model are required.

In a recent study, Coldewey et al. (13) were able to show that FTY720 at a dose of 0.1 mg/kg attenuated sepsis-induced cardiac dysfunction in a murine sepsis model. The influence of this dose on the immune homeostasis was not reported in the study of Coldewey et al. (13). In our study, sepsis-induced organ dysfunction was not assessed. Thus, it is not certain whether positive effects of FTY720 on sepsis-induced cardiac dysfunction also occur to the same extent at a dose of 0.5 mg/kg used in our study. Moreover, positive effects of FTY720 treatment in sepsis, such as improved cardiac function as shown by Coldewey et al. (13) for murine models of polymicrobial sepsis and systemic inflammation may have been masked in our study due to the severity of the CASP model and the high early mortality rate.

Although the CASP model of abdominal sepsis has been extensively characterized with respect to macroscopic findings within the peritoneal cavity, microscopic findings within the small intestine as well as the liver, the bacterial load, and the levels of selected cytokines (15), a detailed chronological study of quantitative changes in cell populations and cytokine levels in various immune compartments has not yet been performed. The present study provides data to fill this lacuna. In our work, we present for the first time a detailed description of these quantitative changes during the first 24 h after CASP induction. These descriptions provide a global picture of the redistribution of immune cells during the onset of abdominal sepsis.

In summary, our work demonstrated that the inhibition of T-cell migration and the induction of peripheral lymphopenia did not have an impact on survival in our model of severe murine sepsis. Also, the presence of reduced T- and B-cell numbers in the peripheral blood during a septic challenge did not negatively affect bacterial load and sepsis mortality in our model of severe abdominal sepsis. However, further experiments in sepsis models with less severe forms of abdominal sepsis and lower mortality are required to assess the impact on FTY720 in later stages of the abdominal sepsis. Our results suggest that concurrent FTY720 treatment does not increase the risk for more severe clinical courses of abdominal sepsis after surgery.

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Keywords:

FTY720; immunosuppression; lymphopenia; polymicrobial abdominal sepsis; sphingosine-1-phosphate

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