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Keywords:

  • animal models/studies – mice/rats;
  • CD4 T cells (T helper; Th0, Th1, Th2, Th3, Th17);
  • inflammation/inflammatory mediators including eicosanoids;
  • T cells;
  • thymus

Summary

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Disclosure
  9. References

Proinflammatory cytokines are essential mediators of the immunopathology associated with microbial sepsis. The fungal cell wall component zymosan and bacterial DNA are well-studied experimental tools for investigating these processes, simulating the presence of fungal or bacterial infection. Cells of the immune periphery, but also immune cells in the thymus, are affected essentially by the presence of microbes or their immune stimuli in sepsis. For this reason, we investigated the cytokine pattern present in the spleen (containing mature immune cells) and the thymus (containing immature immune cells) upon exposure to zymosan and Escherichia coli DNA. To study the role of T cell activation status, we investigated ex-vivo cultures with and without αCD3 stimulation for changes in their cytokine secretion pattern as measured by cytokine enzyme-linked immunospot (ELISPOT) and flow cytometry analysis. We found that both substances strongly co-stimulate αCD3-induced interferon (IFN)-γ and interleukin (IL)-6 secretion in the thymus and in the spleen, but stimulate IL-17 production only moderately. Moreover, zymosan increases PLP peptide (PLPp)-specific IFN-γ and IL-6 production in experimental autoimmune encephalomyelitis (EAE) induced in Swiss Jim Lambert (SJL)/J mice, confirming that T cell activation status is crucial for the cytokines secreted by an immune cell population encountering a microbial pathogen or immunostimulating parts of it.


Introduction

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Disclosure
  9. References

Sepsis and septic shock, dangerous and potentially lethal disease conditions, are an inflammatory cascade in response to bacteria or other microbial pathogens [1-3]. These inflammatory changes in the immune system affect organs of the immune periphery as well as the thymus [4, 5]. Proinflammatory cytokines (e.g. interferon (IFN)-γ or interleukin (IL)-6) play an important role and are induced by microbial agents, or parts thereof. These are recognized predominantly by Toll-like receptors (TLRs), and have immunostimulatory properties. Activation of an immune cell via TLRs can induce and modify cytokine production after activating downstream kinases and transcription factors [6, 7].

In this study we investigated the influence of two different TLR ligands, which are used in experimental models to monitor conditions of sepsis and septic shock on pro- and anti-inflammatory cytokine production in the thymus (harbouring immature immune cells) and in the spleen, containing mature immune cells and representing the immune periphery [8]. These are zymosan and bacterial DNA from Escherichia coli K12. Zymosan, an extract from the cell wall of the yeast Saccharomyces cerevisiae, is recognized by TLR-2 and has properties of a potent immune stimulant with the capability of activating adaptive immune responses [9]. Unmethylated cytosine–phosphate–guanine (CpG) dinucleotides in endotoxin-free bacterial DNA sequences from E. coli K12 are recognized by TLR-9 [10]. Plasmacytoid dendritic cells (DCs) and B cells express TLR-9 and produce T helper type 1 (Th1)-like proinflammatory cytokines [11].

IL-17-producing T cells play an important role in the immune response against microbial pathogens [12]. In general, experimental studies have focused on understanding the activity of IL-17-producing T cells, which are differentiated from naive T cells in the peripheral immune system. We and others, however, have demonstrated that IL-17-producing T cells are also present in the thymus of naive wild-type mice, and can be co-activated by microbial stimuli [13-15]. Given a strong affection of the thymus in microbial sepsis, resulting in proinflammatory cytokine production, apoptosis and thymic atrophy, in this study the effect of zymosan and E. coli K12 DNA was analysed with an ex-vivo model on murine splenocyte (mature immune cell) and thymocyte (immature immune cell) cytokine production, with a particular focus on cytokine IL-17 in comparison with IL-6 and IFN-γ [4, 5].

Materials and methods

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Disclosure
  9. References

Animals, antigens and treatments

Wild-type female C57.BL/6 mice and Swiss Jim Lambert (SJL)/J mice aged 6–8 weeks were obtained from Janvier (Le Genest Saint Isle, France), and maintained at the local animal facilities under special pathogen-free conditions. All animal experiments were approved by the local government authorities. SJL/J mice were immunized subcutaneously with 1 mg/ml PLP peptide (PLPp) amino acids139–151 (Biotrend, Cologne, Germany) in 0·5 mg/ml complete Freund's adjuvant (CFA; Difco, Detroit, MI, USA). On days 0 and 2 after immunization, mice were injected additionally with 0·4 μg/ml pertussis toxin (PTX; Sigma-Aldrich, St Louis, MO, USA) in phosphate-buffered saline (PBS) intraperitoneally. For development of paralytic symptoms, mice were assessed daily. The severity of disease was recorded according to the standard scale: grade 1, floppy tail; grade 2, hind leg weakness; grade 3, full hind leg paralysis; grade 4, quadriplegia; grade 5, death.

Cell preparation from the spleen and thymus

Animals were euthanized with isoflurane and spleen and thymus were removed under sterile conditions. Subsequently, single cell suspensions were prepared and the cells were counted by trypan blue exclusion and plated at the indicated cell density in serum-free HL-1 medium (Lonza, Cologne, Germany).

Cytokine enzyme-linked immunospot (ELISPOT) assays

ELISPOT assays were performed essentially as described previously [13]. Briefly, MultiScreenHTS plates (Millipore, Schwalbach, Germany) were coated overnight with the capture antibodies in sterile PBS. All antibodies were obtained from BD Pharmingen (San Diego, CA, USA). The following coating antibodies were used: R46-A2 at 4 μg/ml for IFN-γ, TRFK5 at 2 μg/ml for IL-5, MP5-20F3 at 2 μg/ml for IL-6 and TC11-18H10 at 2 μg/ml for IL-17. The plates were blocked for 1 h in sterile 0·5% PBS/bovine serum albumin (BSA) and washed three times with sterile PBS. Splenocytes (5 × 105 or 106 per well as indicated) and thymocytes (106 per well) were plated in HL-1 medium containing 1% glutamine. Cells were stimulated with anti-mouse CD3 antibody 145-2C11 (αCD3) (BD Pharmingen) at 1 μg/ml, zymosan at different concentrations (0·01 μg/ml, 0·1 μg/ml, 1 μg/ml, 10 μg/ml and 100 μg/ml) (InvivoGen, San Diego, CA, USA), endotoxin-free bacterial DNA from E. coli K12 at different concentrations (0·0025 μg/ml, 0·025 μg/ml, 0·25 μg/ml, 2·5 μg/ml, 25 μg/ml) (InvivoGen) and zymosan and E. coli DNA separately in combination with αCD3, 20 μg/ml PLPp or PLPp in combination with 0·1 μg/ml zymosan. After incubation for 20 or 48 h (PLPp-specific IL-5) at 37°C, 5% CO2, plates were washed six times with PBS and detection antibodies (BD Pharmingen) were added overnight. XMG1·2-biotin was used for IFN-γ, TRFK4 for IL-5, biotinylated MP5-32C11 for IL-6 and biotinylated TC11-8H4·1 for IL-17. The plate-bound secondary antibody was then visualized by adding streptavidin–alkaline phosphatase 1:1000 (SAV-AP; Dako, Glostrup, Denmark) and 5-bromo-4-chloro-3-indolyl-phosphate in conjunction with nitro blue tetrazolium (NBT/BCIP) substrate (Bio-Rad, Munich, Germany).

ELISPOT image analysis

Image analysis of ELISPOT assays was performed with ImmunoSpotTM Analysis Software after scanning the plates with an ImmunoSpotTM Analyzer (CTL Europe, Bonn, Germany). In brief, digitized images of individual wells of the ELISPOT plates were analyzed for cytokine spots, based on the comparison of experimental wells (containing immune cells and stimuli) and control wells (immune cells, no stimuli). After separating spots that touched or partially overlapped, non-specific ‘background noise’ was gated out by applying spot size and circularity analysis as additional criteria. Then, spots that fell within the accepted criteria were highlighted and counted. Single wells which could not be enumerated, because of confluence phenomena, were assessed by using the highest numbers of cytokine-producing cells which could be counted regularly in other wells in the same assay as an approximated estimate.

Flow cytometry analysis

Single cell suspensions of thymocytes and splenocytes were prepared; 2·5 × 106 cells (splenocytes after erythrocyte lysis) were plated in HL-1 medium containing 1% glutamine and stimulated with 1 μg/ml αCD3 in the presence of 1 μg/ml zymosan or endotoxin-free bacterial DNA from 0·25 μg/ml E. coli K12 for 24 h. For the last 4 h of culture, BD GolgiStop (BD Pharmingen) was added. After fixable viability dye eFluor®660 (eBioscience, San Diego, CA, USA) staining, cells were fixed and permeabilized using a BD Cytofix/Cytoperm™ Plus fixation/permeabilization kit (with BD GolgiStop™ protein transport inhibitor containing monensin) from BD Pharmingen. Intracellular staining was performed with phycoerythrin-cyanin7 (PE-Cy7)-labelled IFN-γ (clone XMG1·2) and fluorescein isothiocyanate (FITC)-labelled IL-6 (clone MP5-20F3). FACS analysis was performed on a FACS Guava EasyCyteTM 8 (Millipore) using GuavaSoftTM software version 2.2.2.

Statistical analysis

For statistical analysis, one-way analysis of variance (anova) for multiple group analyses and Dunnett's post-hoc test or the two-sided t-test for comparison of two groups were used (GraphPad Prism; GraphPad Software, San Diego, CA, USA). Differences at P < 0·05 (*) were considered statistically significant, P < 0·01 (**) highly significant and P < 0·001 (***) very highly significant.

Results

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Disclosure
  9. References

Zymosan co-stimulates αCD3-induced proinflammatory cytokine production in the spleen and thymus

To study the influence of the TLR-2 activator zymosan on pro- and anti-inflammatory cytokine production (IFN-γ, IL-5, IL-6 and IL-17) in the spleen as well as in the thymus, we tested different concentrations of zymosan in combination with the stimulatory αCD3 antibody. αCD3 stimulation alone induced low frequencies of IL-5-producing cells in the thymus, IFN-γ-producing cells in the spleen and IL-17-producing cells in both immune organs (Fig. 1b). In response to the combination of αCD3 and zymosan, a strong elevation of IFN-γ- and IL-6-producing cells could be detected from low to high concentrations, both in spleen and thymus. A significant increase in IFN-γ and IL-6 production resulted after co-stimulation with 1 μg/ml to 100 μg/ml zymosan. αCD3-induced IL-17 production was enhanced moderately by additional zymosan stimulation in both organs, but was only increased significantly after the addition of 1 μg/ml zymosan in the spleen. Regarding the low frequencies of αCD3-induced IL-5-producing cells, only the addition of 0·1 μg/ml zymosan to thymocytes had a significant effect. Without any stimulation, or stimulation with zymosan alone, no relevant frequencies of cytokine-producing cells were present in both organs, except for IL-6-producing cells. After stimulation with 10 μg/ml zymosan in the thymus and 100 μg/ml zymosan in the spleen, a significant increase in IL-6 production resulted.

figure

Figure 1. Cytokine response of thymocytes and splenocytes from naive mice in response to zymosan and αCD3 stimulation for the cytokines indicated as measured by enzyme-linked immunospot (ELISPOT) assays (a–b) and intracellular fluorescence activated cell scan (FACS) analysis (c). (a–b) Thymocytes and splenocytes of eight female C57.BL/6 mice were isolated and single cell suspensions were prepared in two independent experiments, as described in Materials and methods. Cells were plated at a density of 1 000 000 cells per well. (a) ELISPOT wells of interleukin (IL)-17-producing cells of one representative mouse and (b) frequencies of cytokine-producing cells for the cytokines interferon (IFN)-γ, IL-5, IL-6 and IL-17 after zymosan stimulation (0, 0·01, 0·1, 1, 10 and 100 μg/ml) are shown. Each bar represents the mean of two independent experiments of eight mice in total (four mice per experiment) ± standard error of the mean. (c) Flow cytometry (intracellular cytokine staining) for IFN-γ and IL-6 in thymocytes and splenocytes stimulated with αCD3 in the presence of 1 μg/ml zymosan. Cell populations were gated on lymphocytes and living cells. Numbers in quadrants indicate percentage of positive cells. Data represent one of two independent experiments. *P < 0·05; **P < 0·01; ***P < 0·001.

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To confirm the findings of the co-stimulating effects on IFN-γ and IL-6 obtained by ELISPOT, intracellular cytokine staining with FACS was performed (Fig. 1c). In the thymus and spleen, co-stimulating effects on IFN-γ could be detected after the addition of 1 μg/ml zymosan, but not production of IL-6. In comparison to thymocytes, more than twice as many IFN-γ- and IL-6-producing cells could be detected in splenocytes by ELISPOT as well as FACS analysis.

Endotoxin-free bacterial DNA from E. coli K12 co-stimulates αCD3-induced proinflammatory cytokine production in the spleen and thymus

To study the influence of the TLR-9 ligand E. coli K12 DNA on pro- and anti-inflammatory cytokine production (IFN-γ, IL-5, IL-6 and IL-17) in the spleen and thymus, we tested different concentrations of E. coli K12 DNA in combination with the stimulatory αCD3 antibody. As shown in Fig. 2, αCD3 stimulation resulted in a significant increase of IFN-γ production in the spleen and IFN-γ and IL-17 production in both organs and (low) IL-5 production in the spleen. Upon co-stimulation, αCD3-induced IFN-γ and IL-6 production was enhanced significantly in the thymus by 25 μg/ml and also IL-6 by 2·5 μg/ml E. coli K12 DNA. αCD3-induced IFN-γ secretion also appeared to be stimulated by 2·5 μg/ml E. coli DNA, but this effect was not significant. No significant increase of IL-17 or IL-5 production could be detected. In the spleen, only IL-6 producing cells were affected significantly by 25 μg/ml to 2·5 μg/ml E. coli DNA. No relevant induction of cytokine-producing cells was detected after stimulation with E. coli K12 DNA alone, except for IL-6 by 25 μg/ml DNA in splenocytes. To support the ELISPOT findings of the αCD3/E. coli K12 DNA costimulating effect on IFN-γ and IL-6 secretion, intracellular cytokine staining with FACS was performed (Fig. 1c).

figure

Figure 2. Cytokine response of thymocytes and splenocytes from naive mice in response to endotoxin-free bacterial DNA from Escherichia coli K12 and αCD3 stimulation for the cytokines indicated as measured by enzyme-linked immunospot (ELISPOT) assay (a–b) and intracellular fluorescence activated cell scan (FACS) analysis (c). (a–b) Thymocytes and splenocytes of four female C57.BL/6 mice in one experiment were isolated and single cell suspensions were prepared as described in Materials and methods. Cells were plated at a density of 1 000 000 cells per well. (a) ELISPOT wells of interleukin (IL)-17-producing cells of one representative mouse and (b) frequencies of cytokine-producing cells for the cytokines interferon (IFN)-γ, IL-5, IL-6 and IL-17 after DNA-stimulation (0, 0·0025, 0·025, 0·25, 2·5 and 25 μg/ml DNA) are shown. Each bar represents the mean of one experiment ± standard error of the mean. (c) Flow cytometry (intracellular cytokine staining) for IFN-γ and IL-6 in thymocytes and splenocytes stimulated with αCD3 in the presence of 0·25 μg/ml E. coliK12 DNA. Cell populations were gated on lymphocytes and living cells. Numbers in quadrants indicate percentage of positive cells. Data represent one of two independent experiments. *P < 0·05; **P < 0·01; ***P < 0·001.

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In the thymus and spleen, augmenting effects on IFN-γ could be detected after co-stimulation with 0·25 μg/ml E. coli K12 DNA. No production of IL-6 resulted. In both organs more than twice as many IFN-γ- and IL-6-producing cells could be detected by ELISPOT as well as FACS analysis after αCD3/zymosan co-stimulation in comparison to αCD3 single stimulation.

Zymosan co-stimulates PLPp-specific IL-6 production in PLPp-induced EAE in SJL/J mice

To determine the effect of zymosan on the cytokine pattern of a primed antigen-specific T cell-mediated immune response, EAE was induced in female SJL/J mice with PLPp/CFA/PTX injection, as described in Materials and methods. ELISPOT assays with splenocytes were performed on day 14 after immunization and single cell suspensions were stimulated with PLPp, zymosan or both agents in combination. EAE disease course (a) and the frequencies of cytokine-producing cells (b) are depicted in Fig 3. Variations in PLPp-specific cytokine responses between immunized mice were detectable, but a similar trend in proinflammatory cytokine production can be seen: PLPp-induced production of IL-6 is triggered significantly by zymosan, whereas PLPp-induced IL-5 production is reduced. IFN-γ- and IL-17-producing cells are also enhanced after co-stimulation, but not significantly. Zymosan alone leads to low cytokine production, which is not significant.

figure

Figure 3. (a) Clinical disease course of experimental autoimmune encephalomyelitis (EAE) induced in female Swiss Jim Lambert (SJL/J) mice by immunization with PLP peptide/complete Freund's adjuvant/pertussis toxin (PLPp/CFA/PTX), as described in Materials and methods. Symbols represent the mean clinical EAE score of 4 mice in total investigated in one experiment ± standard error of the mean. (b) Frequencies of cytokine-producing cells for the cytokines indicated as measured by enzyme-linked immunospot (ELISPOT). Cytokine production was assessed in the spleen 14 days after immunization with PLPp/CFA/PTX. Splenocytes were stimulated with PLPp, zymosan or both agents in combination as indicated. Data of one experiment with four mice (1–4) are displayed. Each symbol shows one animal tested in duplicate wells, bars represent the mean. *P < 0·05.

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Discussion

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Disclosure
  9. References

In the immune system, T cells play an important role in the initiation of immune responses against infectious agents [16]. Ligands of TLRs, sensors of microbial factors, can modulate T cell function by enhancing proliferation and production of cytokines [17]. Microbial pathogens, or parts thereof, therefore have the ability to induce (over) production of proinflammatory cytokines which can give rise to systemic inflammatory response syndrome (SIRS), sepsis or septic shock [1]. For this reason, the purpose of this study was to investigate the influence of two well-characterized microbial stimulants, zymosan and endotoxin-free bacterial DNA from E. coli K12, on mature and immature immune cell activation by analysing pro- and anti-inflammatory cytokine production. In our experiments, thymocytes and splenocytes were activated using a monoclonal activating αCD3 antibody, exposed in parallel to the microbial stimulus. Using a very simple model, which we have used previously, this represents the presence of the microbial stimulus at the site of T cell activation [13, 18]. To investigate its immunological effect alone in contrast to its co-stimulating effect, we also studied the effect of the respective microbial stimulant on thymocytes and splenocytes alone without simultaneous activation via the T cell receptor complex.

The main findings are that zymosan potentiates αCD3-induced production of the proinflammatory cytokines IFN-γ and IL-6, but does not affect production of the anti-inflammatory cytokine IL-5, in both the spleen and the thymus. For the proinflammatory cytokines, there is a dose-dependent enhancement of production in both organs with different maxima. Production of IFN-γ and IL-6 rises with increasing concentrations of zymosan from 1 μg/ml to 100 μg/ml. In contrast, production of IL-17 appears to decrease, but not significantly. Only 1 μg/ml zymosan results in a significant co-stimulating effect on αCD3-induced IL-17 production in the spleen. Zymosan alone induces significant IL-6 production in the thymus at 10 μg/ml and also at 100 μg/ml in the spleen, but not production of the other cytokines. Intracellular staining supports the co-stimulating effect of zymosan on IFN-γ secretion in thymus and in the spleen detected by ELISPOT. In contrast to IFN-γ-producing cells, IL-6-producing cells triggered by αCD3/zymosan co-stimulation are not T cells.

Zymosan activates the signalling cascade by binding to TLR-2 and Dectin-1 [19]. Pattern recognition receptors (PRRs) such as TLRs and Dectin-1 are expressed in many cell types, mainly innate immune cells such as DCs and macrophages [16]. Zymosan-dependent responses by DCs and macrophages lead to secretion of inflammatory cytokines, including TNF-α, IL-6, IL-10, IL-12 and IL-23 [20, 21]. Our findings, of significant secretion of IL-6 in the spleen and thymus (which in a large majority belong to the non-T cell compartment), support these results. The cytokines induced by zymosan can also induce the production of T cell subsets via different signalling pathways [16]. IL-6-producing cells were triggered by zymosan alone both in thymocytes and splenocytes without any relevant IL-17 production. This dissociated effect suggests that the augmenting effect on IL-17 production exerted by 1 μg/ml zymosan in the spleen is unlikely to be mediated via IL-6. IL-6 is a requirement for the activation of IL-17-producing T cells via induction of the transcription factor signal transducer and activator of transcription-3 (STAT-3), but in vivo its action can also be compensated by other cytokines, such as IL-21 [22, 23]. Zymosan binding also leads to IL-23 secretion, which promotes Th17 differentiation [16]. Moreover, our experiments detected a significant increase of IFN-γ production after co-stimulation with zymosan, both in ELISPOT and intracellular cytokine staining. IL-12 induces Th1 differentiation, which is reflected in Th1 cytokine production of IFN-γ [24]. IFN-γ might inhibit IL-17 production at higher concentrations by activating the transcription factor STAT-1 [22].

Moreover, we investigated the effect of zymosan on the antigen-specific cytokine production of a T cell population which has already undergone priming to an (auto-)antigen. For this purpose, we used acute EAE, an animal model of the human disease multiple sclerosis (MS). In this study we tested the co-stimulating effect of zymosan on the PLPp-specific cytokine signature in PLPp-induced acute EAE in SJL/J mice. In parallel to the results obtained with splenocytes from naive C57.BL/6 mice, we did not detect a cytokine-inducing effect of zymosan by itself, but a co-stimulating effect on proinflammatory cytokine production. There was an overall trend, but only the increase of IL-6 production was significant. In comparison to cells of unsensitized animals, no significant costimulating effect on IFN-γ could be detected. Recently reported findings describe an inhibiting zymosan effect on T cells in EAE in vivo caused by differentiation of regulatory T cells, which is dose- and time-dependent and shows down-regulation of IFN-γ and IL-17, as well as up-regulation of IL-5 [25]. In light of this study, our results support not only a dose-dependent immune cell activation by zymosan, but also the relationship between zymosan-induced immune cell activation and the importance of T cell activation status during encounter with a microbial stimulus.

E. coli K12 DNA had a slightly different effect on immune cell cytokine production in comparison with zymosan. Similar to zymosan, no cytokine-producing cells were induced by E. coli K12 DNA itself besides IL-6-producing cells. In contrast to zymosan, however, IL-6 secretion could be detected only in the spleen at the highest concentration used. This might be explained by a specific activating effect on macrophages exerted by zymosan, but which might not be achieved in such a pronounced way by E. coli K12 DNA [26, 27]. No co-stimulatory effect on αCD3-induced IL-5 production and no relevant effect on IL-17 production were detectable in both thymocytes and splenocytes. In contrast, in parallel to zymosan, E. coli K12 DNA potentiated αCD3-induced IFN-γ and IL-6 production in the thymus and spleen. The subtle qualitative and quantitative differences between zymosan and E. coli K12 DNA could be explained by the fact that they activate immune cells via different TLRs [28]. Each TLR has a different intracellular signalling process and recruits a specific set of additional adaptor molecules which lead to activation of different transduction pathways and, finally, a specific cytokine secretion pattern. TLR-2 (zymosan) and TLR-9 (E. coli DNA), therefore, diverge not only in their cellular localization, but also in the manner of activating these down-stream processes [28].

Looking at the difference between immature immune cells (thymocytes) and mature immune cells in the immune periphery (splenocytes), for all cytokines and stimulation conditions a quantitative (showing higher frequencies of cytokine-producing cells in splenocytes than thymocytes), but not a qualitative alteration could be detected. IFN-γ and IL-6, but not IL-17, are the predominantly triggered cytokines. This supports the idea that IL-17 might not be the main destructive inflammatory mediator driving the pathological process leading to multiple organ failure in bacterial or fungal sepsis. This observation is in line with the recently reported finding that in a zymosan-induced inflammatory fungal model, IL-17 did not play a crucial role in severe inflammation, which finally leads to organ failure [29].

In conclusion, our data indicate that (i) both zymosan and bacterial DNA affect mainly cells of the innate immune system such as macrophages and DCs, both in mature and immature immune cell populations; (ii) IFN-γ and IL-6, but not IL-17, are the cytokines triggered predominantly; and (iii) there are no qualitative differences between the effect of zymosan and bacterial DNA on immature versus mature immune cells, respectively.

Acknowledgements

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Disclosure
  9. References

H.H.H. was supported by grants from the Deutsche Forschungsgemeinschaft (Ho 4392/1-1), of the Strategischer Forschungsfonds der Universität Düsseldorf and the Deutsche Multiple Sklerose Gesellschaft. We thank Zippora Kohne for excellent technical assistance.

Disclosure

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Disclosure
  9. References

The authors declare that they have no conflicts of interest.

References

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Disclosure
  9. References