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Depletion of dendritic cells (DC) in secondary lymphoid organs is a hallmark of sepsis-induced immune dysfunction. In this setting, we investigated if Toll-like receptor (TLR)-dependent signaling might modulate the maturation process and the survival of DC. Using a model of sublethal polymicrobial sepsis induced by cecal ligation and puncture, we investigated the quantitative and functional features of spleen DC in wild-type, TLR2−/−, TLR4−/−, and TLR2−/− TLR4−/− mice. By 24 h, a decrease in the relative percentage of CD11chigh spleen DC occurred in wild-type mice but was prevented in TLR2−/−, TLR4−/−, and TLR2−/− TLR4−/− mice. In wild-type mice, sepsis dramatically affected both CD11c+ CD8α+ and CD11c+ CD8α− subsets. In all three types of knockout mice studied, the CD11c+ CD8α+ subset followed a depletion pattern similar to that for wild-type mice. In contrast, the loss of CD11c+ CD8α− cells was attenuated in TLR2−/− and TLR4−/− mice and completely prevented in TLR2−/− TLR4−/− mice. Accordingly, apoptosis of spleen DC was increased in septic wild-type mice and inhibited in knockout mice. In addition we characterized the functional features of spleen DC obtained from septic mice. As shown by increased expression of major histocompatibility complex class II and CD86, polymicrobial sepsis induced maturation of DC, with subsequent increased capacity to prime T lymphocytes, similarly in wild-type and knockout mice. In response to CpG DNA stimulation, production of interleukin-12 was equally impaired in DC obtained from wild-type and knockout septic mice. In conclusion, although dispensable for the DC maturation process, TLR2 and TLR4 are involved in the mechanisms leading to depletion of spleen DC following polymicrobial sepsis.
Upon microbial infection, the innate immune response depends on early recognition of pathogen-associated molecular patterns (PAMP) that constitute the molecular signature of microorganisms. Mammalian Toll-like receptors (TLR) play an important role in the host defense against bacteria, as they readily recognize the distinct PAMP presented by the invading microorganism in order to orchestrate the activation of phagocytic cells and the elimination of pathogens (1). TLR2, TLR4, TLR5, and TLR9 specifically contribute to host defense against bacteria. Stimulation of these receptors activates myeloid differentiation primary response protein 88 (MyD88)-dependent signaling pathways that contribute to the characteristic inflammatory response characterized by the release of a range of proinflammatory cytokines, such as interleukin-1 (IL-1), IL-8, IL-12, alpha interferon (IFN-α), and IFN-γ.
In addition to the response aimed at pathogen eradication, antigen-presenting cells such as dendritic cells (DC) upregulate adhesion molecules and costimulatory molecules and initiate the development of adaptive immune responses after microbial invasion. Present in an immature state in peripheral tissues as sentinels to detect invading pathogens, DC are key players in the organization of the innate immune response. Upon activation through pattern recognition receptors such as TLR, DC migrate into secondary lymphoid organs, mature, and upregulate the expression of surface molecules such as CD80, CD86, and major histocompatibility complex class II (MHC-II). Within lymph nodes, DC activate T cells and provide signals that determine the direction of T-cell polarization in order to drive their differentiation into Th-1 cells, Th-2 cells, T regulatory cells, or IL-17-producing T cells (Th-17). In several studies, purified or synthetic TLR ligands have been shown to induce maturation of DC. However, during infection the immune system is not challenged by a single ligand but rather by multiple pathogens that comprise several PAMP and activate multiple receptors. Variability in the activation patterns of TLR-dependent signaling pathways has been shown to influence the magnitude of the inflammatory phase of the innate immune response and the efficiency of host defense mechanisms (28).
This initial proinflammatory response is often followed by a complex immune suppression that may compromise the eradication of the primary infection and favor the emergence of secondary infections (2, 4). Several experimental observations indicate that quantitative and functional abnormalities of DC also contribute to the advent of sepsis-induced immune dysfunction. Indeed a depletion of DC in secondary lymphoid organs has been reported at the early phase (24 h) of polymicrobial sepsis in mice (7, 27). The important impact of DC loss was strongly suggested by the increased mortality of DC-depleted mice subjected to polymicrobial sepsis (26). Patients who died from infection similarly exhibited a profound loss of spleen DC (13). Accordingly, circulating DC counts were decreased in patients during the first 24 h of septic shock, low DC counts being correlated with disease severity and predicting a fatal outcome (10). Altogether, animal and human data suggest that systemic DC depletion is an early event in the course of sepsis that may impair the host defense mechanisms. In addition, we recently reported that polymicrobial sepsis induces persistent functional abnormalities of bone marrow-derived DC and increases long-term susceptibility to secondary Pseudomonas aeruginosa pneumonia (23). The molecular mechanisms that contribute to DC depletion and to the development of cellular functional abnormalities remain to be elucidated, and the specific contribution of TLR signaling to development of sepsis-induced immune dysfunction is poorly understood. We hypothesized that variations in TLR-dependent signaling might modulate the maturation process and/or survival of DC after polymicrobial sepsis. To test this hypothesis, we assessed the early (24-h) quantitative and functional features of spleen DC in mice deficient for TLR2, TLR4, or both TLR2 and TLR4 (TLR2−/− TLR4−/−) in a model of sublethal polymicrobial sepsis.
Female C57BL/6J mice, 8 to 12 weeks old, were purchased from Charles River Laboratories. TLR2−/− and TLR4−/− mice were obtained from S. Akira (Osaka University, Osaka, Japan) and were backcrossed eight times with C57BL/6J mice to ensure similar genetic backgrounds. Double-knockout TLR2−/− TLR4−/− mice were generated by breeding TLR2−/− and TLR4−/− mice. Female BALB/c mice (also purchased from Charles River Laboratories) were used for purification of allogeneic T lymphocytes. All animals were maintained in the pathogen-free animal facility of the Cochin Institute. Experiments were conducted in accordance with Cochin Institute guidelines in compliance with European animal welfare regulations.
Cecal ligation and puncture (CLP) were performed as follows: after a midline incision (<1 cm), the cecum was exposed, ligatured at its external third, and punctured through and through with a 21-gauge needle. The incision was sutured in layers, and animals were resuscitated with an intraperitoneal injection of 1 ml saline. Controls were sham-operation mice, i.e., mice undergoing abdominal surgery that involved only exposition of the cecum without CLP. Six hours following surgery and then every 12 h for 3 days, mice received an intraperitoneal injection of antibiotics (imipenem-cilastatin [Tienam]; Merck Sharp & Dohme; 25 mg/kg of body weight in 0.5 ml saline). Survival was monitored every 12 h for up to 10 days after induction of sepsis.
Spleens were dispersed in collagenase D (Roche; 400 IU/ml at 37°C) for 30 min and then filtered through a cell strainer. After red blood cell lysis by an ammonium chloride solution, the percentage of CD11chigh DC in the single-cell suspension was assessed by flow cytometry.
DC were separated with a cycle of positive selection by means of an immunomagnetic procedure (magnetically activated cell separation CD11c isolation kit; Mylteni Biotech). The purity of the positive fraction was generally >80%, as confirmed by CD11c staining analyzed by flow cytometry. Repartition of CD11c+ CD8α+ and CD11c+ CD8α− subsets was assessed through flow cytometry analysis.
Fluorescent antibodies (CD11c, CD8α, MHC-II, and CD86) and Fc blocker antibody (anti-CD16/CD32) were purchased from Becton Dickinson. Cell suspensions (2 × 105 cells) were first incubated with Fc blocker antibody for 5 min and then stained with fluorescent antibodies for 30 min at 4°C. Acquisition was performed on a FACSCalibur flow cytometer (Becton Dickinson).
Two different assays were used to quantify apoptosis. Freshly CD11c-purified spleen DC were double stained with fluorescein isothiocyanate-coupled annexin-V and propidium iodide (PI) (Miltenyi Biotech). Analysis was performed on a FACSCalibur flow cytometer. Early and late apoptotic cells were identified as annexin-V+ PI− and annexin-V+ PI+, respectively. DC apoptosis was also assessed through the intracellular detection of cleaved (activated) caspase-3 by an Alexa Fluor 488-conjugated antibody (Cell Signaling).
T lymphocytes were isolated from lymph nodes of BALB/c mice using a T-cell negative isolation kit (Dynal Biotech ASA, Oslo, Norway), which provides a highly purified (>95%) CD3-positive fraction. T lymphocytes (2 × 105) were cultured for 72 h in round-bottom 96-well plates in the presence of spleen DC (T-lymphocyte/DC ratios, 40:1, 20:1, and 10:1; in triplicate). Proliferation of T cells was assessed by measuring the incorporation of [3H]thymidine after a 6-hour pulse with 1 μCi per well.
Purified spleen DC were cultured for 24 h at a concentration of 2 × 106 DC per ml in RPMI 1640 medium supplemented with 10% fetal calf serum, 10 mM HEPES, 10 mM glutamine, and 1% penicillin and streptomycin, in the presence of CpG at 1 μg/ml (ODN 1826; InvivoGen). IL-12p40, IL-12p70, and IL-10 concentrations in cell culture supernatants were quantified by enzyme-linked immunosorbent assay according to the manufacturer's instructions (R&D Systems).
Continuous variables were represented as means ± standard deviations (SD) and compared using the Student t test. Survival curves were obtained by using the Kaplan-Meier method and compared by the log rank test. P values <0.05 indicated statistically significant differences.
To investigate quantitative changes in spleen DC, we induced sublethal polymicrobial sepsis by CLP followed by a short antibiotic course. The majority of wild-type and knockout animals survived. TLR4−/− and TLR2−/− TLR4−/− mice displayed higher survival rates of 90% and 85%, respectively, than the crude survival rates of 60% and 65% observed in wild-type and TLR2−/− mice, respectively (Fig. (Fig.1).1). All sham-operation mice survived (data not shown). In order to evaluate the influence of TLR2- and TLR4-dependent signaling pathways on sepsis-induced early depletion of DC, we assessed the number of spleen DC by determining the proportion of CD11chigh cells in a single-cell suspension of splenocytes from these four mouse strains (Fig. 2A and B). Whereas the percentage of CD11chigh cells was significantly decreased 24 h after CLP in wild-type mice, it remained similar to that for sham-operation animals in all three knockout mouse strains, suggesting that TLR signaling contributes to the mechanisms involved in the early depletion of DC.
To assess whether polymicrobial sepsis affects the repartition of spleen lymphoid and myeloid DC, we purified CD11c+ spleen DC obtained from sham-operation mice and mice subjected to CLP (CLP mice) at day 1 and we assessed the distribution of CD11c+ CD8α+ and CD11c+ CD8α− subsets. In wild-type mice, polymicrobial sepsis induced a marked relative depletion of the lymphoid CD11c+ CD8α+ subset (Fig. (Fig.3A).3A). However, actual numbers of CD11c+ CD8α− and CD11c+ CD8α+ cells revealed that both DC subsets were depleted in the process (Fig. (Fig.3B),3B), thereby suggesting that both lymphoid and myeloid DC subsets accounted for sepsis-induced depletion of spleen DC. In TLR2−/−, TLR4−/− and TLR2−/− TLR4−/− mice, the CD11c+ CD8α+ subset followed similar depletion patterns (Fig. 3A and B). In contrast, the loss of the CD11c+ CD8α− cells was attenuated in TLR2−/− and TLR4−/− mice and completely prevented in TLR2−/− TLR4−/− mice (Fig. (Fig.3B).3B). These data suggest that TLR2 and TLR4 deficiencies do not prevent the loss of lymphoid CD11c+ CD8α+ cells but might additively contribute to the early depletion of myeloid CD11c+ CD8α− DC.
Apoptosis has been identified as the main mechanism leading to the depletion of spleen DC in models of lethal sepsis. To test whether apoptosis contributes to depletion of spleen DC in our model, freshly purified spleen DC were obtained from sham-operation and CLP mice 18 h after surgery and were double stained with annexin-V and PI. As shown in Fig. Fig.4A,4A, sepsis induced marked apoptosis of spleen DC in wild-type mice (32.6% annexin-V-positive cells versus 16.6% in sham-operation mice). In contrast, spleen DC obtained from septic TLR2−/−, TLR4−/−, and TLR2−/− TLR4−/− mice exhibited only minimal apoptosis comparable to that exhibited by sham-operation animals. Consistent results were obtained by using an alternative apoptosis assay through detection of cleaved (activated) caspase-3 (Fig. (Fig.4B).4B). These data suggest that TLR2 and TLR4 are involved in the control of apoptosis of spleen DC during sublethal polymicrobial sepsis.
Maturation of DC leads to increased expression of MHC-II and costimulatory molecules, as well as to the production of cytokines with subsequent ability to prime T lymphocytes and direct the adaptive immune response. As TLR-dependent signaling pathways are critical in the maturation process of DC, we hypothesized that TLR2−/− and TLR4−/− animals might exhibit distinct DC maturation patterns. To test this hypothesis, we characterized the functional features of spleen DC obtained from wild-type and knockout septic mice 1 day after CLP. As revealed by increased expression of MHC-II and of the costimulatory molecule CD86, polymicrobial sepsis induced maturation of spleen DC (Fig. (Fig.5A).5A). Consistent with the high expression of MHC-II and CD86, spleen DC obtained from either wild-type or TLR2−/− mice 1 day after CLP exhibited increased ability to prime T lymphocytes in allogeneic mixed lymphocyte reactions (Fig. (Fig.5B).5B). Similar results were obtained for TLR4−/− and TLR2−/− TLR4−/− mice (data not shown).
To further investigate the function of spleen DC, we also measured cytokine release after TLR9 stimulation by CpG DNA in spleen DC obtained from wild-type, TLR2−/−, TLR4−/−, and TLR2−/− TLR4−/− mice. Regardless of TLR deficiency, CpG-induced production of IL-12p40 or of the bioactive IL-12p70 was decreased in DC obtained from mice 1 day after CLP, while production of IL-10 was sustained (Fig. (Fig.6).6). Altogether these results suggest that TLR2 and TLR4 deficiencies do not compromise the early maturation of DC following polymicrobial sepsis.
Depletion of DC in secondary lymphoid organs is a hallmark of sepsis-induced immune suppression. Whereas targeted disruption of TLR2 and/or TLR4 genes does not alter the maturation process of spleen DC following polymicrobial sepsis, we report that TLR signaling contributes to the early depletion of DC. Indeed, the decrease of the CD11c+ CD8α− subset is attenuated in single-knockout mice and inhibited in TLR2−/− TLR4−/− mice, whereas TLR2 and TLR4 deficiencies had no impact on the CD11c+ CD8α+ subset. Although the actual numbers of purified CD11c+ CD8α− and CD11c+ CD8α+ cells do not strictly correlate with the inhibition of the relative decrease of CD11chigh splenocytes in knockout septic mice, our results nevertheless suggest that TLR2 and TLR4 are involved in the homeostasis of spleen DC in the early stage of polymicrobial sepsis. In the same way, the contribution of TLR9 to sepsis-induced depletion of DC has also been recently suggested by Plitas et al. (24). Whereas polymicrobial sepsis decreased the number of peritoneal and spleen DC in wild-type mice, TLR9−/− mice exhibited increased DC counts following sepsis. Altogether, these data highlight the importance of TLR-dependent signaling in the early loss of DC. The depletion of spleen DC has been shown to be transient in surviving mice, which progressively recover normal DC counts within 3 weeks after induction of sepsis (30). Recent data indicate that TLR signaling also contributes to the late immunologic consequences of sepsis. Indeed, prolonged intra-abdominal infection induces spleen enlargement through a MyD88-dependent dramatic expansion of an immature GR-1+ CD11b+ myelomonocytic population, which likely accounts for the relative decrease of other cell types (5).
Apoptosis has been proposed as one of the primary mechanisms of the depletion of DC that occurs during the early stage of sepsis and preferentially affects the CD11c+ CD8α+ subset in this setting (7, 27). Accordingly, we report that DC apoptosis is increased 18 h after CLP in wild-type mice and likely accounts for the early depletion of spleen DC. Consistent with the attenuation of spleen DC depletion, the apoptotic rate for CLP knockout mice is comparable to that for sham-operation animals, thereby suggesting that TLR2 and TLR4 signaling is involved in the control of DC survival in sepsis. In the setting of polymicrobial sepsis, the mechanisms leading to DC apoptosis remain unclear. Besides direct cell stimulation by PAMP (9), the systemic inflammatory response in itself could also trigger apoptosis, as similar patterns of spleen DC apoptosis and depletion have been observed in noninfectious acute inflammatory conditions (16). Impaired interactions between DC and other immune cells might also affect DC survival. Indeed, survival of mature DC depends upon the cellular interactions with T lymphocytes involving RANK/TRANCE and CD40/CD40L (19). The concomitant loss of lymphocytes in secondary lymphoid organs could thereby contribute to sepsis-induced depletion of DC (7). Our results are consistent with recent data suggesting that the MyD88-dependent pathway is involved in the control of immune cell homeostasis during sepsis. MyD88 deficiency decreased T- and B-lymphocyte apoptosis in lymphoid organs from mice subjected to CLP (22). Hence, increased T-cell survival could contribute to the prevention of sepsis-induced early depletion of DC.
TLR signaling has been shown to influence maturation of DC through MyD88-dependent (11, 18) and -independent (6, 15) pathways. TLR2 exclusively signals through MyD88, whereas TLR4 can activate both MyD88-dependent and MyD88-independent pathways. In our study, neither TLR2 nor TLR4 deficiencies impacted the maturation of DC or the IL-12/IL-10 imbalance. Since preliminary studies from our laboratory showed that MyD88 deficiency did not alter the sepsis-induced maturation of spleen DC (unpublished observations), it is likely that a MyD88-independent pathway contributed to the intact basal and stimulated expression of costimulatory molecules. Assessment of the maturation of DC in the TIR domain-containing adaptor inducing IFN-β (TRIF)-deficient mice certainly would definitely delineate the respective contributions of MyD88-dependent and TRIF-dependent pathways during polymicrobial sepsis. In addition, maturation of DC also leads to increased production of cytokines with subsequent ability to prime T lymphocytes. DC consistently display impaired capacity to release IL-12 during sepsis (8), thereby shifting the Th-cell pattern to a predominantly Th-2 response (3). The decreased release of IL-12 during the early stage of sepsis might be related to the relative depletion of the IL-12-producing CD11c+ CD8α+ DC subset that preferentially primes Th-1 differentiation (17) and to the development of transient tolerance of TLR agonists (31). Most importantly, the long-term suppression of IL-12 production by spleen DC has been recently linked to an epigenetic mechanism during polymicrobial sepsis (30). In our study, TLR2 and TLR4 deficiencies did not prevent the impairment in DC-derived release of IL-12, suggesting that TLR2- and TLR4-dependent signaling pathways do not contribute to the epigenetic changes observed in polymicrobial sepsis.
Upon infection, pathogen recognition by TLR promotes a potent inflammatory response, which is required for pathogen clearance but which can contribute to host tissue damage and lead to septic shock. TLR2- and TLR4-deficient mice are susceptible to various monobacterial infections, but both TLR2 and TLR4 seem dispensable for host defense against polymicrobial sepsis induced by colon ascendens stent peritonitis (29). In the current model of sublethal CLP, which mimics a frequent clinical condition, the majority of wild-type, TLR2−/−, TLR4−/−, and TLR2−/− TLR4−/− mice survived the insult. Interestingly enough, mortality was reduced in TLR4−/− and TLR2−/− TLR4−/− mice compared to wild-type and TLR2−/− littermates, indicating that TLR4-mediated signals might contribute to mortality. Whereas TLR4 has long been recognized as the primary lipopolysaccharide receptor, a growing body of evidence suggests that TLR4 also has endogenous ligands. Indeed, recent data indicate that alarmins such as high mobility group box 1 (HMGB1), a critical endogenous mediator of lethality in murine models of endotoxemia and polymicrobial sepsis (14, 25), also signal through members of the TLR family such as TLR4. HMGB1 binding of TLR4 results in NF-κB upregulation (20, 21), thus making it likely that HMGB1 stimulation of TLR4 can lead to cytokine release, tissue injury, and death. Of note, we could not observe any clear association between outcomes and the intensity of DC defects. However, the control of bacterial burden by a short course of broad-spectrum antibiotics may have impacted outcome differences related to host response. Moreover, although DC have been critically involved in the different phases of sepsis in order to mount an efficient host response against polymicrobial sepsis or secondary infections, the prognostic value of DC depletion per se has not been clearly established in animal models. This is in contrast with lymphocyte apoptosis, for example, which has been unquestionably associated with a worse outcome in sepsis (12). Hence, it is likely that impairment of the main functions of DC (e.g., impaired production of IL-12) also accounts for immune dysfunction in sepsis.
In conclusion, sublethal polymicrobial sepsis is associated with a transient depletion and functional abnormalities of DC. Although TLR control the magnitude of the inflammatory response and are critically involved in the DC maturation process, we report that TLR2 and TLR4 deficiencies do not compromise the maturation but may prevent the early loss of spleen DC. Whether TLR signaling actually contributes to the development of sepsis-induced immune suppression and to the subsequent susceptibility to secondary infections remains to be established.
This study was supported by a grant from the Fonds d'Etude et de Recherche du Corps Médical of the Assistance Publique-Hôpitaux de Paris.
There are no conflicts of interest.
Editor: F. C. Fang
Published ahead of print on 5 October 2009.