Diversity of Interferon γ and Granulocyte-Macrophage Colony-Stimulating Factor in Restoring Immune Dysfunction of Dendritic Cells and Macrophages During Polymicrobial Sepsis
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The development of immunosuppression during polymicrobial sepsis is associated with the failure of dendritic cells (DC) to promote the polarization of T helper (Th) cells toward a protective Th1 type. The aim of the study was to test potential immunomodulatory approaches to restore the capacity of splenic DC to secrete interleukin (IL) 12 that represents the key cytokine in Th1 cell polarization. Murine polymicrobial sepsis was induced by cecal ligation and puncture (CLP). Splenic DC were isolated at different time points after CLP or sham operation, and stimulated with bacterial components in the presence or absence of neutralizing anti-IL-10 antibodies, murine interferon (IFN) γ, and/or granulocyte macrophage colony-stimulating factor (GM-CSF). DC from septic mice showed an impaired capacity to release the pro-inflammatory and Th1-promoting cytokines tumor necrosis factor α, IFN-γ, and IL-12 in response to bacterial stimuli, but secreted IL-10. Endogenous IL-10 was not responsible for the impaired IL-12 secretion. Up to 6 h after CLP, the combined treatment of DC from septic mice with IFN-γ and GM-CSF increased the secretion of IL-12. Later, DC from septic mice responded to IFN-γ and GM-CSF with increased expression of the co-stimulatory molecule CD86, while IL-12 secretion was no more enhanced. In contrast, splenic macrophages from septic mice during late sepsis responded to GM-CSF with increased cytokine release. Thus, therapy of sepsis with IFN-γ/GM-CSF might be sufficient to restore the activity of macrophages, but fails to restore DC function adequate for the development of a protective Th1-like immune response.
Sepsis is associated with the failure of the host to develop an effective immune response against invading microorganisms due to immunosuppression of unknown genesis. The consequence is unrestricted spreading of bacteria that may lead to multiorgan failure and death (1). During sepsis, monocytes/macrophages fail to secrete tumor-necrosis factor (TNF) a in response to in vitro stimulation with lipopolysaccharide (LPS) (2,3). Moreover, the proliferative capacity, as well as the secretion of the T helper (Th) type 1-asso-ciated cytokines interleukin (IL) 2 and interferon (IFN) γ from T-lymphocytes, is impaired (2,4, 5, 6). Therefore, therapies that modulate this cellular dysfunction might have beneficial effects on the outcome of sepsis.
An effective immune response against bacterial infections requires the development of a Th1 response that is associated with the release of IFN-γ. Antigen-specific T cells are activated by antigen-presenting cells (APC) through the interaction of co-stimulatory molecules such as CD40 and CD86 on the APC, with their respective ligands CD40 ligand (CD40L) and CD28/CTLA-4, respectively, on T cells. Dendritic cells (DC) are the most potent APC due to their high expression of major histocompatibility complex (MHC) and co-stimulatory molecules that are upregulated during DC maturation triggered by microbial agents (7). Stimulated DC secrete a distinct pattern of cytokines that is decisive for the type of subsequent Th cell differentiation. IL-12 is a heterodimeric cytokine and promotes the polarization of naïve Th cells toward Th1. Microbial stimuli such as immunostimulatory oligonucleotides (CpG) or LPS are recognized by Toll-like receptors (TLR) and are potent inducers of DC-derived IL-12. LPS requires additional ligation of CD40 on the surface of DC for optimal induction of IL-12 synthesis (8,9). In contrast, DC-derived IL-10 favors the development of Th2 cells, but suppresses the development of a Th1 response through inhibition of IL-12 secretion (10). Neutralization of endogenous IL-10 results in increased IL-12 secretion in response to LPS or CpG (11,12).
There is an increasing body of evidence that DC are involved in the pathomechanisms leading to sepsis-associated immune dysfunction. DC are beneficial at least during the early phase of sepsis development because depletion of DC in vivo before onset of disease results in increased mortality (13). Later, apoptosis of DC takes place in various lymphoid and non-lymphoid tissues in septic patients (14) as well as in septic mice (15, 16, 17), and the extent of DC loss in septic patients correlates with poor outcome (18). We have recently shown that, in mice, splenic DC rapidly increase their expression of CD40 and CD86 after induction of sepsis, but simultaneously develop a dysfunction that is characterized by an impaired capacity to secrete IL-12 in response to bacterial stimuli and to drive Th cell proliferation (19).
The relevance of DC dysfunction in disease development has been clearly shown by Benjamim et al. who applied competent DC from naïve mice into post-septic mice and thereby reduced infection-induced mortality (20). IL-12 is essential for survival of sepsis because it promotes the release of IFN-γ that in turn supports the clearance of the infection through enhanced microbicidal activity of the innate immune system (21). The aim of the present study was to find immunomodulatory approaches to restore the suppressed capacity of splenic DC to secrete IL-12 during murine polymicrobial sepsis induced by cecal ligation and puncture (CLP). Selected approaches were the neutralization of endogenous IL-10 and treatment with the immunomodulatory substances IFN-γ and/or GM-CSF.
Materials and Methods
Cecal Ligation and Puncture
Female BALB/c mice (Harlan Winkelmann, Borchen, Germany) were 8–10 weeks old and had access to standard rodent food and water ad libitum. All animal procedures were carried out following institutional guidelines at the Medical Faculty, University of Duisburg-Essen, Germany. Polymicrobial sepsis was induced by CLP using a 17-gauge needle as described previously (19). Under these conditions, the mortality was 20% within 24 h. Sham animals underwent a laparotomy without ligation and puncture of the cecum.
Culture Medium and Reagents
Very low endotoxin medium VLE RPMI 1640 (Biochrom, Berlin, Germany) containing 10% heat-inactivated FCS (Sigma, Taufkirchen, Germany), 10 mM HEPES (Biochrom), 2 mM L-Glutamine (Biochrom), 0.06 mg/mL Penicillin (Sigma), 0.02 mg/mL Gentamicin G (Sigma), and 0.05 mM 2-ME (Sigma) was used as culture medium throughout all experiments. Murine recombinant GM-CSF, IFN-γ, CD40L, neutralizing anti-IL-10 antibodies (clone JES052A5), and the respective rat IgG1 isotype control were purchased from R&D Systems, Wiesbaden, Germany. Synthetic phosphorothioated CpG 1668 oligonucleotides (22) were purchased from Qiagen, Köln, Germany. All these reagents were free of detectable LPS contaminations as tested using Limulus Amebocyte Assay (Biowhittaker, Walkersville, MD, USA). LPS (E. coli 026:B6) was obtained from Sigma.
Preparation and Culture of Total Spleen Cells
At different time points after CLP or sham operation, spleens were removed and single cell suspensions were prepared through collagenase digestion using 0.02 U/mL Blendzyme 2 (Roche, Grenzach-Wyhlen, Germany) at 37°C for 18 min. Spleens were minced through a cell strainer (70 µm diameter) and red blood cells were lysed using ammonium chloride. For flow cytometric analyses of DC, total spleen cells were cultured in 48-well plates (Nunc, Wiesbaden, Germany; 106 cells/well in 300 µL culture medium). In case of macrophage analyses, total spleen cells were kept in 24-well “low attachment” plates (Corning, Schiphol-Rijk, The Netherlands; 2.5 × 106/well in 500 µL culture medium) to enable detachment of adherent cells. CD86 or CD40 expression on DC was analyzed using total spleen cells cultured in the presence or absence of 10 ng/mL GM-CSF, 10 ng/mL IFN-γ, 5 µg/mL CpG, or 100 ng/mL LPS for 18 h. Stimulation of total spleen cells with 10 ng/mL LPS for 8 h in the absence or presence of 10 ng/mL GM-CSF or with 5 µg/mL CpG for 18 h was used for the examination of intracellular levels of TNF-α and IL-12p40, respectively. Unstimulated cells served as negative controls. All cultures were set up in triplicate and were pooled before flow cytometric analyses.
Purification of Splenic DC
Splenic DC were purified from freshly isolated total spleen cells using CD11c Microbeads (Miltenyi Biotech, Moenchengladbach, Germany) and magnetic cell sorting (MACS, Miltenyi Biotech) and were cultured as described previously (19). Purity was generally 85% to 90% as confirmed by CD11c staining and flow cytometry. Purified splenic DC were cultured with 5 µg/mL CpG or 100 ng/mL LPS + 2.5 µg/mL CD40L. In some experiments, purified splenic DC were stimulated with CpG in combination with medium, GM-CSF (10 ng/mL), IFN-γ (10 ng/mL), IFN-γ + GM-CSF, anti-IL-10 antibodies (10 µg/mL), or the respective rat IgG1 isotype control (10 µg/mL). Previous experiments have shown that 10 µg/mL anti-IL-10 antibodies were sufficient to neutralize the activity of 1 ng/mL IL-10 (data not shown). All cultures were set up in triplicate. After 18 h, supernatants were analyzed for the presence of IL-12p70, TNF-α, IFN-γ, or IL-10 using the cytometric bead array (CBA) Mouse Inflammation Kit (BD Biosciences, Heidelberg, Germany) or ELISA (ebioscience, NatuTec, Frankfurt, Germany).
Total spleen cells were sequentially incubated with total mouse IgG (100 µg/mL; Sigma) to block unspecific binding, and with an antibody mixture (all antibodies from BD Biosciences) containing anti-CD11c-allophycocyanin (APC; clone HL3) either in combination with biotinylated anti-IFN-γ receptor (clone GR20), with anti-CD86-phycoerythrin (PE; clone GL1), or with anti-CD40-fluorescein isothiocyanate (FITC; clone 3/23). Streptavidin-PE was added in a third step to cells labeled with the biotin-conjugated antibody. Appropriate isotype controls were used for all stainings. For intracellular IL-12p40 or TNF-α staining, monensin (GolgiStop 0.66 µL/mL, BD Biosciences) was added to the cells during the last 6 h of culture. After surface staining using APC-labeled anti-CD11c or anti-F4/80 (clone BM8; ebioscience) antibodies, cells were fixed and permeabilized using Cytofix/Cytoperm (BD Biosciences) for 20 min at RT. Thereafter, intracellular TNF-α or IL-12p40 were stained using anti-TNF-α-PE (clone MP6-XT22), anti-IL-12p40-PE (clone C15.6) antibodies, or the respective isotype control antibody (all from BD Biosciences). Cells were washed with permeabilization buffer (BD Biosciences) and were resuspended in Cell Wash. All data were acquired using a FACScalibur (BD Biosciences). Living cells were selected according to forward and side scatter properties. DC were gated as CD11c-positive cells, macrophages were gated as F4/80-positive cells. The fluorescence intensity value that was exceeded by less than 2% of the cells upon isotype control staining was defined as the threshold for specific staining. According to this threshold, the percentage of positive cells and the corresponding mean fluorescence intensity (MFI) of the gated cells were determined.
Cytokine Profile and CD40 Expression of Splenic DC During Sepsis
To analyze the cytokine expression of DC that were not separated from other splenic cell populations, total spleen cells were prepared 3 and 24 h after CLP or sham operation, and were stimulated with CpG. DC were stained for intracellular IL-12p40 that represents the regulated subunit of the IL-12 heterodimer in combination with CD11c on the cell surface. At both time points, the percentage of IL-12p40-positive DC from sham mice strongly increased upon stimulation with CpG in comparison to unstimulated cells (Figure 2B). By 3 h after CLP, the percentage of IL-12p40-positive DC increased upon stimulation with CpG, but to a lesser extend than it was found for DC from sham mice. In contrast, upon CpG-stimulation of total spleen cells prepared 24 h after CLP, the percentage of IL-12p40-positive DC remained on the level obtained for unstimulated DC (Figure 2B). Thus, the impaired secretion of CpG-induced IL-12 from DC correlates with a strong reduction of the number of DC that express IL-12p40.
The completely contrary effects were seen in terms of IL-10 production. From 16 h after CLP, DC from septic mice released IL-10 even in the absence of any stimulus and further enhanced IL-10 secretion upon stimulation with CpG (Figure 2A). In contrast, the secretion of IL-10 in response to LPS + CD40L did not change significantly within the first 16 h after CLP.
Decreased IL-12 Release of DC During Sepsis is Not Caused by Endogenous IL-10 Production
Influence of IFN-γ and GM-CSF on DC-Derived IL-12 Secretion
To investigate whether the suppressed capacity of DC to secrete IL-12 during sepsis can be restored, splenic DC were isolated 3, 6, and 24 h after CLP or sham operation, and were stimulated with CpG either in the absence or presence of GM-CSF, IFN-γ, or a combination of both. GM-CSF, IFN-γ, and IFN-γ + GM-CSF enhanced the CpG-induced secretion of IL-12 from DC of sham mice with IFN-γ + GM-CSF being superior to the individual cytokines (Figure 1). DC from septic mice isolated 3 h after CLP, showed a slightly reduced IL-12 secretion upon stimulation with CpG alone. GM-CSF marginally increased the IL-12 secretion of CpG-stimulated DC from septic mice (Figure 1A). IL-12 levels of DC stimulated in the presence of IFN-γ or of the combination of IFN-γ and GM-CSF, exceeded even the IL-12 level of CpG-stimulated DC from sham mice (Figure 1A). However, DC from septic mice 6 h after CLP had lost the responsiveness to IFN-γ and to GM-CSF. At this time point, the IL-12 production of DC from septic mice was enhanced only through a combined treatment with IFN-γ + GM-CSF, however it did not reach IL-12 levels released by CpG-stimulated DC from sham mice (Figure 1B). At 24 h after CLP, DC from septic mice completely failed to secrete IL-12 and additionally were absolutely unresponsive to any immunomodulator (Figure 1C). IFN-γ or GM-CSF alone did not induce the release of IL-12 from DC of sham or septic mice (data not shown). Thus, during sepsis, DC responded to IFN-γ and/or GM-CSF with increased IL-12 secretion only during a short time frame early after induction of sepsis.
Decreased IFN-γ Receptor Expression on Splenic DC after CLP
Influence of IFN-γ and GM-CSF on the Expression of CD86 on DC
Diverse Responsiveness of Macrophages and DC to GM-CSF During Sepsis
The present study shows that during murine polymicrobial sepsis, splenic DC rapidly lose their capacity to respond to bacterial components with the release of pro-inflammatory and Th1-promoting cytokines. The impaired capacity of DC to secrete IL-12 was not mediated by the parallel rise of endogenous IL-10 production. Moreover, attempts to restore the suppressed release of IL-12 through treatment with the immunomodulatory cytokines IFN-γ and GM-CSF showed that DC responded to these mediators only during the very early phase after induction of sepsis. Downregulation of cytokine receptors does not seem to be responsible for this insensitivity of DC to IFN-γ and GM-CSF because DC still responded to these cytokines with increased expression of CD86 even at later time points during sepsis. In contrast, macrophages that similarly showed a sepsis-associated reduced responsiveness to bacterial products could be reactivated through GM-CSF.
DC play a decisive role in the interaction between the innate and the adaptive immune system due to their cytokine secretion pattern. We show here that DC dysfunction characterized by the reduced secretion of IL-12, IFN-γ, and TNF-α in response to bacterial stimuli becomes visible as soon as 3 h after induction of sepsis (Figure 2A). Among these three cytokines, IL-12 represents the most relevant one with regard to its contribution to the development of Th1 responses. This state of abnormal responsiveness of DC to bacterial stimuli is not restricted to the acute phase of sepsis as we describe here, but has also been found even after resolution of the disease—at which time it contributes to an enhanced susceptibility to secondary infections (20). The mechanisms that underlie the impaired capacity of DC from septic mice to secrete Th1-promoting cytokines are not clear so far. The finding that DC from septic mice increase the expression of CD40 on the surface at time points when IL-12 secretion is already reduced (for example, 3 and 6 h after CLP; Figure 3), argues against a defect in the recognition of CpG or LPS through their receptors. We can exclude that endogenous IL-10 is involved neither at early time points when IL-12 secretion is not yet completely blocked (Figure 4), nor at later time points during sepsis when the DC-derived IL-10 secretion increases (19). Sepsis is not associated with a general dysfunction of DC. The finding that DC in the peritoneal cavity that represents the site of sepsis initiation are able to secrete IL-12 (24) suggests that DC in the spleen are modulated through factors, so far unknown, prior or in parallel to their contact with spreading bacteria. Such potential mediators are transforming growth factor β prostaglandin E2, IL-10, but also catecholamines, such as norepinephrine, that are produced in the peritoneal cavity, in the gut, or in the liver, and might reach the DC in the spleen via circulation (3,25,26). All these factors are known to suppress DC-derived IL-12 production (27, 28, 29, 30).
During acute sepsis, the presence of IL-12 is required for the polarization of Th cells toward Th1 and for the release of IFN-γ that stimulate the bactericidal activity of phagocytes (21). Whether DC are the cellular source of the indispensable IL-12 has not been determined in that report. Strategies that increase the levels of IL-12 at the site of infection, either through application of DC from naïve mice or through adenoviral transfection, lead to an improved immune response against microorganisms (20,31). There-fore, it is assumed that treatment regimens increasing the DC-derived levels of IL-12 at the site of DC/T cell interaction during sepsis result in an improved bacterial clearance and, possibly, outcome.
As possible candidates to restore the capacity of DC from septic mice to release IL-12, we analyzed the immunomodulatory cytokines GM-CSF and IFN-γ. DC from sham mice responded to both agents with increased expression of the co-stimulatory molecule CD86 and enhanced secretion of IL-12 and TNF-α upon challenge with bacterial stimuli (Figures 1,6,7). GM-CSF and IFN-γ and, most effectively, a combination of both substances, increased the release of IL-12 from DC of septic mice only within the first 6 h after induction of sepsis (Figure 1). At 24 h after sepsis, DC were irreversibly changed and became refractory to both immunostimulatory substances in terms of IL-12 secretion (Figure 1). A downregulation of the IFN-γ-receptor might contribute, at least in part, to the unresponsiveness of DC to IFN-γ during the later phase of sepsis (Figure 5B). However, the missing responsiveness of DC to GM-CSF and IFN-γ during sepsis is restricted to the modulation of cytokine secretion, because the expression of the co-stimulatory molecule CD86 was enhanced upon treatment with either or both substances even 24 h after sepsis (Figure 6). This fact argues against a receptor-dependent mechanism for the failure of GM-CSF and IFN-γ to restore DC-derived IL-12 secretion, but rather indicates changes in the IFN-γ and GM-CSF receptors downstream signaling pathways in DC during sepsis.
IFN-γ signals via Janus kinase (JAK)-mediated phosphorylation of transcription factors termed signal transducer and activation of transcription (STAT) 1. Additional STAT1 activation can occur through mitogen-activated protein kinases (MAPK) that are also involved in TLR signaling (32). This cross-signaling between STAT1 and MAPK is supposed to account for the amplifying effect of IFN-γ on TLR ligand-induced gene activation. Similarly, increased MAPK activation is involved in the GM-CSF-mediated increase of TNF-α secretion upon stimulation with LPS (33,34). The majority of reports on GM-CSF- or IFN-γ-mediated signaling pathways has been performed with macrophages. Whether IFN-γ and GM-CSF-induced signaling pathways in splenic DC equal those of macrophages is not clear. The finding that macrophages maintain their responsiveness to GM-CSF during sepsis (Figure 7) argues against identical signaling events in macrophages and DC. This assumption is supported by a previous report that GM-CSF induces a diverse pattern of activated STAT molecules in DC and macrophages (35). Thus, a disturbance of the JAK/STAT and/or MAPK pathway might be responsible for the impaired sensitivity of DC to IFN-γ and GM-CSF during sepsis.
The present study shows that LPS-stimulated macrophages from septic mice increased the secretion of TNF-α upon exposure to GM-CSF and, thus, behaved like monocytes from septic patients (2). Ex vivo studies showing that GM-CSF and IFN-γ stimulated increased HLA-DR expression on monocytes from septic patients and enhanced LPS-induced TNF-α secretion prompted several clinical trials using GM-CSF or IFN-γ in sepsis therapy (36, 37, 38, 39). However, treatment of trauma patients with IFN-γ showed only minor effects on the rate of infections and did not reduce mortality significantly, despite an IFN-γ-mediated increased HLA-DR expression on monocytes in all studies (40,41). In other trials, GM-CSF did not show any benefit in terms of postoperative septic complications (42) or survival, nevertheless it led to a faster clearance of the infection (43). Additionally, GM-CSF given 3 h after CLP had no protective effect on septic rats (44). The reasons why such immunomodulatory therapies did not accomplish previous expectations are not clear. Considering the requirement of intact DC function in the development of a protective immune response against the infection, the unresponsiveness of DC to GM-CSF and IFN-γ during sepsis might explain the partial failure of IFN-γ and GM-CSF in sepsis therapy. To verify this hypothesis, further studies that correlate the responsiveness of DC to GM-CSF and IFN-γ with disease development after treatment with these cytokines must be performed in the same animal model.
A novel approach for immunotherapy of pancreatitis associated with the development of immunosuppression similar to sepsis is the application of a combination of GM-CSF and IFN-γ. Ex vivo treatment of monocytes from patients with acute pancreatitis with GM-CSF plus IFN-γ normalized both the suppressed HLA-DR expression and the impaired capacity to secrete TNF-α up to normal levels while each cytokine alone failed to elevate these immune functions (45). Similarly, the combination of GM-CSF and IFN-γ was the most effective in restoring IL-12 production of DC from septic mice in our model (Figure 1). The combination was still effective when the single substances failed to exert any effects (Figure 1B). Under this view, we expect that an efficient treatment of sepsis with the combination of GM-CSF and IFN-γ must be initiated within the first 6 h after the appearance of bacteria. The mechanisms that underlie the synergism of GM-CSF and IFN-γ have not yet been elucidated. Signaling molecules of the MAPK pathway are activated by both cytokines and therefore might amplify each other (32, 33, 34). Further studies are required to address this issue.
In summary, the development of immunosuppression during sepsis is associated with an impaired capacity of splenic DC and macrophages to release Th1-promoting cytokines upon stimulation with bacterial products. GM-CSF can restore the impaired cytokine secretion of macrophages, whereas DC are insensitive to GM-CSF and IFN-γ in this regard. Future studies should aim to understand the cause of DC dysfunction during sepsis and to find strategies that restore the unique features of DC as major players in the induction of protective Th1 cell responses.
This work is supported by DFG grant FL-353/2-1 (to Stefanie B Flohé). We are grateful to Michaela Bak for excellent technical assistance and to Ernst Kreuzfelder and to Bärbel Nyadu for support in flow cytometry.