ABSTRACT
Infections with Pseudomonas aeruginosa may cause many different diseases. The spectrum of such infections in general includes inflammation and bacterial sepsis. Hospital-acquired pneumonia, naturally resistant to a wide range of antibiotics, is associated with a particularly high mortality rate in mechanically ventilated patients. The pathogenesis of P. aeruginosa is complex and mediated by several virulence factors, as well as cell-associated factors. We have previously demonstrated that stimulation with different bacteria triggers the cytokine response of thymocytes. In this study, we investigated the effect of P. aeruginosa and its different components on the cytokine production of immature and mature immune cells. We found that the induced cytokine pattern in the thymus and the spleen after infections with P. aeruginosa is primarily mediated by lipopolysaccharide (LPS) of the outer cell membrane, but other components of the bacterium can influence the cytokine secretion as well. Stimulation with heat-killed P. aeruginosa and LPS does not influence the amount of cytokine-producing CD4+ T cells but instead suppresses the emergence of Th17 cells. However, stimulation with P. aeruginosa or its components triggers the interleukin-17 (IL-17) response both in thymocytes and in splenocytes. We conclude that infections with P. aeruginosa affect the cytokine secretion of immature and mature cells and that IL-17 and Th17 cells play only a minor role in the development of pathological systemic inflammatory disease conditions during P. aeruginosa infections. Therefore, other inflammatory immune responses must be responsible for septic reactions of the host.
INTRODUCTION
Interleukin-17 (IL-17) is a potent inflammatory cytokine, which was initially reported to be produced mainly by activated memory T cells, also called T helper (Th) 17 cells (1). In addition to induced IL-17-producing T cells, which are primed in the immune periphery during the induction of an antigen-specific T cell response, these IL-17-producing cells are also present in the thymuses of naive wild-type mice (2, 3). In most mice, IL-17-producing thymocytes represent a subpopulation of CD4+ T cells which are able to react immediately on environmental stimuli without further priming phase (2–5). Furthermore, innate immune cells can also produce IL-17 in response to early immune mechanisms (6). Intrinsically, IL-17-producing cells, also referred to as the sentinels of the immune system, play a critical role in several fields of innate and adaptive immunity, including immunity against microbial infections and tumors (6–9). Dysregulated IL-17 production can result in uncontrolled proinflammatory cytokine production and chronic inflammation, promoting tissue damage and leading to autoimmune diseases (10–12). The most commonly used animal model for the human inflammatory demyelinating disease multiple sclerosis is experimental autoimmune encephalomyelitis (EAE) (13).
Systemic bacterial infections and bacterial sepsis are dangerous and potentially lethal disease conditions that occur in response to bacteria or other microbial pathogens (14–16). Pseudomonas aeruginosa is an opportunistic pathogen of immunocompromised humans which typically infects the pulmonary tract, causing bacterial pneumonia (17). Notably, hospital-acquired pneumonia in mechanically ventilated patients is associated with a particularly high mortality rate (18, 19). Moreover, P. aeruginosa can also lead to eye and ear infections, complicated intra-abdominal infections, urinary tract infections, and skin and soft tissue infections (20, 21).
The pathogenesis of P. aeruginosa is mediated both by various adhesins (lipopolysaccharide [LPS] and flagellin) and by secreted compounds (e.g., elastase, exotoxin A, exoenzyme S, and rhamnolipid). Exotoxin A (Exo A), produced during infections with P. aeruginosa, causes disease by inhibiting protein synthesis and interfering with the cellular immune functions of the host (22, 23). In contrast, LPS is the major ingredient of the cell membrane of Gram-negative bacteria and acts as an endotoxin by promoting the secretion of proinflammatory cytokines through cells of the innate immune system (24, 25). N-Acyl-homoserine lactones from P. aeruginosa are signaling molecules produced inside the cell and involved in bacterial quorum sensing to regulate gene expression (26).
We have previously demonstrated that bacteria and their cell wall components influence the cytokine response of thymocytes (27). These are recognized predominantly by Toll-like receptor 2 (TLR2). TLRs in general play an important role in innate immune responses, as well as in the polarization of adaptive immune responses. Activation of an immune cell via TLRs can induce and modify cytokine production by activating downstream kinases and transcriptions factors (28–30). Moreover, stimulation with CpG (TLR9), imiquimod (TLR7), flagellin (TLR5), poly(I·C) (TLR3), or diphtheria toxin can potently coactivate IL-17-producing thymocytes (2, 31, 32). A systematic study on the impact of P. aeruginosa or its compounds on the cytokine response of thymocytes (immature immune cells) and splenocytes (mature immune cells), especially IL-17, is not available thus far. We studied here the influence of P. aeruginosa and different bacterial compounds on inflammatory cytokine production, with a particular focus on IL-17, with respect to their role in autoimmune diseases.
MATERIALS AND METHODS
Animals and EAE induction.Wild-type female C57BL/6J, BALB/c, and Swiss Jim Lambert (SJL)/J mice at age 6 to 8 weeks were purchased from Centre d'Elevage R. Janvier (Le Genest-St-Isle, France) and maintained at the local animal facilities under specific-pathogen-free conditions. All animal experiments were fully approved by the local authorities for animal experimentation. For EAE induction, C57BL/6 mice were immunized subcutaneously with 200 μg of MOG35–55 (myelin oligodendrocyte glycoprotein, amino acids 35 to 55) peptide (Biotrend, Cologne, Germany), and 400 μg of heat-killed Mycobacterium tuberculosis H37RA emulsified in incomplete Freund's adjuvant (Difco, Detroit, MI). On days 0 and 2 after immunization, mice were injected additionally with 200 ng of pertussis toxin (PTx; Sigma-Aldrich, Steinheim, Germany) in phosphate-buffered saline (PBS) intraperitoneally. For the development of paralytic symptoms, mice were assessed daily. The severity of disease was scored and recorded according to the standard scale (1, floppy tail; 2, hind leg weakness; 3, full hind leg paralysis; 4, quadriplegia; and 5, death).
Cell isolation and purification.After sacrificing the animals with isoflurane euthanasia, the thymuses and spleens were prepared. Subsequently, each organ was squeezed using the back of a syringe plunger to obtain single-cell suspensions. To sort out cell clusters, the obtained suspensions were filtered through a 40-μm-pore-size cell strainer (BD Biosciences, Heidelberg, Germany). T cells were purified from splenocytes using a CD4 (L3T4) and CD8a (Ly-2) MACS isolation kit with an OctoMACS separator (MS columns) according to the manufacturer's instructions (Miltenyi Biotech, Bergisch Gladbach, Germany). For negative selection of unlabeled T cells, we used an EasySep mouse CD4+ T cell isolation kit with an EasySep Magnet (Stemcell Technologies). Central nervous system (CNS)-infiltrating lymphocytes were isolated from brains and spinal cords (33). Briefly, the isolated tissue was cut in small pieces and digested with 2.5 mg of Collagenase D (Roche) and 0.1 mg of DNase (Roche) in dissociation buffer (10 mM MgCl2 and 0.9 mM CaCl2 in PBS) for 45 min at 37°C. The resulting tissue homogenates were filtered through a 40-μm-pore-size cell strainer, and leukocytes were enriched using discontinuous 30%/70% Percoll density gradient centrifugation. Bone marrow cells were prepared according to a previously described method (34). Briefly, femurs were removed and flushed with RPMI 1640 medium (Gibco). Collected cells were pelleted, resuspended, and washed twice with PBS. The cells were counted by trypan blue exclusion and plated together with the respective stimulants at the cell numbers indicated in serum-free HL-1 medium (Lonza, Cologne, Germany).
Cell culture.RPMI 1640 medium supplemented with 10% fetal calf serum, 100 U/ml penicillin-streptomycin (P/S), 2 mmol/liter l-glutamine, and 50 μmol/liter 2-mercaptoethanol (Invitrogen, Carlsbad, CA) was used for in vitro cultures. Cell cultures were activated with 1 μg/ml anti-CD3 (clone 145-2C11; BD Pharmingen, San Diego, CA) and 2 μg/ml anti-CD28 or, when measuring the recall responses of antigen-specific cells 15 days after immunization, with MOG35–55 peptide (20 μg/ml). Where indicated, isolated CD4+ T cells were cultured for 5 days under Th17 polarizing conditions: 5 ng/ml recombinant transforming growth factor β (rmTGF-β), 5 ng/ml rmIL-23, 50 ng/ml rmIL-6, 10 μg/ml anti-gamma interferon (IFN-γ), and 10 μg/ml anti-IL-4 (11). For the generation of antigen-presenting cells (APCs), bone marrow cells were cultured for 9 days with 5 ng/ml recombinant granulocyte-macrophage colony-stimulating factor (rmGM-CSF) and 5 ng/ml rmIL-4 (34). The purity of the APCs was confirmed by flow cytometry.
Reagents.The following heat-killed bacteria and bacterial components were utilized in this study: heat-killed Pseudomonas aeruginosa (HKPA; InvivoGen); LPS, lectin (PA-l), exotoxin A, and N-(3-oxodecanoyl)-l-homoserine lactone from P. aeruginosa (Sigma-Aldrich); and curdlan from Alcaligenes faecalis (InvivoGen). Furthermore, we used concanavalin A (ConA) from Canavalia enisformis (Sigma-Aldrich) and alum crystals (InvivoGen). All utilized antigens were solved in sterile endotoxin-free water (InvivoGen), which was also used as control.
Cytokine measurement by ELISpot assay.Enzyme-linked immunospot (ELISpot) assays were essentially performed as described previously (2). Briefly, MultiScreenHTS 96-well filtration plates (Millipore, Schwalbach, Germany) were coated overnight with capture antibodies in sterile PBS. The following coating antibodies were used: IL-1β (B122), IL-6 (MP5-20F3), and IL-17 (TC11-18H10) were used at 2 μg/ml, and gamma interferon (IFN-γ; Ρ46-Α2), IL-2 (JES6-1A12), and IL-4 (11B11) were used at 4 μg/ml. Antibodies were ordered from BD Pharmingen. The plates were blocked with sterile 0.5% PBS–bovine serum albumin (BSA) (PBS/BSA) and washed with sterile PBS. Thymocytes (106 per well), splenocytes (2.5 × 105 or 5 × 105 per well as indicated), and isolated T cells (105 per well) were plated in HL-1 medium (BioWhittaker, Walkersville, MD) containing l-glutamine and P/S in duplicate cultures each. Thereafter, the cells were stimulated with different stimulation reagents and incubated at 37°C and 5% CO2. Plates were washed with PBS before the addition of the detection antibodies (BD Pharmingen) overnight in PBS/BSA. Antibodies against IFN-γ (XMG1.2), IL-1β (Poly5158), IL-2 (JES6-5H4), IL-4 (BVD6-24G2), and IL-6 (MP5-32C11) were used at 2 μg/ml, and antibodies against IL-17 (TC11-8H4) were used at 0.5 μg/ml. After the plates were washed, streptavidin-AP (BD Pharmingen) in PBS/BSA (1:500) was added before the plates were visualized using an AP conjugate substrate kit (Bio-Rad Laboratories, Munich, Germany). Image analysis of ELISpot results was performed with ImmunoSpot analysis software after scanning the plates with an ImmunoSpot analyzer (Cellular Technologies, Cleveland, OH). In brief, digitized images of individual wells of the ELISpot plates were analyzed for cytokine spots, based on a comparison of experimental wells (containing immune cells and stimuli) and control wells (immune cells, no stimuli). After separating spots that touched or partially overlapped, nonspecific “background noise” was gated out by applying spot size and circularity analysis as additional criteria. 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.
FACS analysis.For intracellular staining, cells were fixed and permeabilized using an intracellular fixation and permeabilization buffer set with brefeldin A obtained from eBioscience (San Diego, CA). To stain intracellular transcription factors, we used a FOXP3 Fix/Perm buffer set from BioLegend. To exclude dead cells, we used fixable viability dye eFluor660 (eBioscience). The following fluorescence-activated cell sorting (FACS) antibodies were utilized: fluorescein isothiocyanate (FITC)-labeled anti-CD4, phycoerythrin (PE)-Cy7-labeled anti-CD4, and PE-labeled anti-IL-17 from BD Pharmingen; PE-labeled anti-CD4, allophycocyanin-H7 labeled anti-CD8a, FITC-labeled anti-CD11b, PerCP-Cy5.5-labeled anti-CD11b, allophycocyanin-labeled anti-CD11c, PE-Cy7-labeled anti-CD45, allophycocyanin-labeled anti-F4/80, PE-labeled anti-FcεR1, PE-labeled anti-FOXP3, PE-Cy7-labeled anti-IFN-γ, PE-labeled anti-Ly-6G, PE-labeled anti-Notch 1-3, and allophycocyanin-labeled anti-Notch 4 from BioLegend; and PE-labeled anti-IFN-γ, allophycocyanin-labeled, and anti-RORγt from eBioscience. FACS analysis was performed on a Millipore Guava EasyCyte 8 using guavaSoft software version 2.2.2 or a BD FACSCanto using DIVA software v6.1.3.
Analysis of cell vitality, apoptosis, and proliferation.Vitality response was assessed by using fixable viability dye eFluor780 (eBioscience), which permanently labels dead cells and allows them to be counted. Single-cell suspensions were prepared and treated with different concentrations of Exo A for 24 h. After staining with eFluor780, the cells were analyzed by FACS. Apoptosis was determined by using a Guava Nexin kit (Guava Technologies, Hayward, CA). The assay relies on the translocation of phosphatidylserine to the outer surface of the cell membrane, which is often associated with the beginning of apoptosis. The Nexin assay was performed according to the manufacturer's protocol. Briefly, thymocytes were treated with lectin and concanavalin A (ConA). After 4 h, the cell samples were washed and stained with Guava Nexin reagent, including annexin V-PE and 7-AAD (7-aminoactinomycin D) for 20 min. Annexin V is a phospholipid-binding protein that binds phosphatidylserine on the cell surface, and 7-AAD is a cell impermanent dye for the exclusion of nonviable cells. For caspase detection, we used the Guava caspase assay for caspase 3/7 (Millipore) according to the manufacturer's protocol. To determine cell proliferation, isolated cells were stained with CFSE (carboxyfluorescein succinimidyl ester) and analyzed by FACS.
Statistical analysis.Statistical analyses were performed by analysis of variance with a Dunnett's two-tailed t test (Instat, GraphPad 3.00). The means of antigen-treated cells (±α-CD3) were compared to untreated controls (±α-CD3). Differences at P values of <0.05 were considered statistically significant; P values of <0.01 were considered highly statistically significant.
RESULTS
P. aeruginosa and LPS influence the cytokine secretion of thymocytes and splenocytes.Both stimulation with 106 to 107/ml HKPA and stimulation with 1 to 10 μg/ml LPS led to statistically significantly higher frequencies of IL-6 in thymocytes from C57BL/6 and BALB/c mice (Fig. 1A) compared to controls (endotoxin-free water). Minor higher frequencies of IL-17-producing thymocytes could be detected in all tested mouse species at higher concentrations of HKPA and 1 μg/ml LPS. Additional activation with α-CD3 resulted in statistically significantly higher frequencies of IFN-γ after stimulation with 106 to 107/ml HKPA in C57BL/6 and BALB/c mice and 1 to 10 μg/ml LPS in BALB/c mice compared to α-CD3-activated cells. The IL-6 production was enhanced in all tested mouse species after stimulation with 107/ml HKPA and 1 to 10 μg/ml LPS. Furthermore, we found a dose-dependent increase of IL-17, which resulted in statistically significantly higher frequencies after stimulation with 107/ml HKPA and 1 μg/ml LPS in C57BL/6 and BALB/c mice.
In vitro cytokine response from naive wild-type mice in response to heat-inactivated P. aeruginosa and LPS. The results of ELISpot assays of cytokine-producing thymocytes (A) and splenocytes (B) for the cytokines IFN-γ, IL-6, and IL-17 after stimulation with 105 to 107/ml HKPA and 1 to 10 μg/ml LPS (±α-CD3) for 24 h are shown. Each bar represents the mean of two independent experiments of eight mice ± the standard error of the mean (SEM). *, P < 0.05; **, P < 0.01 (as determined by Dunnett's t test).
In the spleen, statistically significantly higher frequencies could be detected at 107/ml HKPA and 1 μg/ml LPS for IFN-γ and 105 to 107/ml HKPA and 1 to 10 μg/ml LPS for IL-6 in all tested species (Fig. 1B). In addition, higher frequencies of IL-17 could be measured at all tested concentrations, statistically significant at 106 to 107/ml HKPA in BALB/c and SJL mice and 1 μg/ml LPS in BALB/c mice. Additional activation with α-CD3 resulted in statistically significantly higher frequencies of IFN-γ at 106 to 107/ml HKPA and 1 to 10 μg/ml LPS in all tested species, but not in higher frequencies of IL-17. Slightly higher frequencies of IL-6 could be detected after stimulation with lower concentrations of HKPA. Intracellular cytokine staining confirmed that stimulation with HKPA and LPS resulted in larger amounts of IFN-γ- and IL-17-producing CD4− splenocytes (see Fig. S1 in the supplemental material). Activation with α-CD3 caused distinctly higher frequencies of IFN-γ-producing CD4− cells and slightly higher frequencies of IL-17-producing CD4− cells, but not larger amounts of cytokine-producing CD4+ T cells. The intracellular cytokine concentration after stimulation with HKPA and LPS was obviously reduced after 48 h compared to 24 h (see Fig. S1 in the supplemental material).
Furthermore, we showed that IL-17 production of thymocytes after stimulation with HKPA and LPS is dependent on APCs (see Fig. S2 in the supplemental material) and that an additional dose of alum crystals did not influence IL-17 production. Stimulation with HKPA and LPS led to statistically significantly higher responses of IL-1β even without α-CD3 activation. Moreover, stimulation with HKPA increased the proliferation rate of CD4− thymocytes but not of CD4+ thymocytes. In contrast to HKPA, stimulation with LPS did not affect the proliferation rate of CD4− and CD4+ thymocytes but did elevate the proliferation rates of CD45+ and CD45− splenocytes (see Fig. S3 in the supplemental material). Although stimulation with HKPA and LPS decreased the numbers of Notch2+ thymocytes, distinctly larger amounts of Notch2+ cells (especially CD4− cells) could be detected in splenocytes (see Fig. S4 in the supplemental material). In summary, we showed that stimulation with HKPA affects the cytokine secretion of immature and mature immune cells, and this is mainly mediated by the cell wall component LPS.
The virulent factor exotoxin A triggers the cytokine secretion of thymocytes and splenocytes.Stimulation with Exo A resulted in statistically significantly higher frequencies of IL-17 in splenocytes from BALB/c and SJL mice (Fig. 2B). After additional α-CD3 activation Exo A statistically significantly triggers the frequencies of IFN-γ in thymocytes from BALB/c mice (Fig. 2A) and IL-17 in splenocytes from BALB/c and SJL mice (Fig. 2B). All tested concentrations of Exo A did not influence the cell vitality of thymocytes and splenocytes (see Fig. S5 in the supplemental material).
In vitro cytokine response from naive wild-type mice in response to exotoxin A from P. aeruginosa. The results of ELISpot assays of cytokine-producing thymocytes (A) and splenocytes (B) for the cytokines IFN-γ, IL-6, and IL-17 after stimulation with 0 to 100 ng/ml Exo A (±α-CD3) for 24 h are shown. Each bar represents the mean of two independent experiments of eight mice ± SEM. *, P < 0.05; **, P < 0.01 (as determined by Dunnett's t test).
Lectin from P. aeruginosa influences the IL-17 secretion of thymocytes and splenocytes.Stimulation with 10 μg/ml lectin resulted in statistically significantly increased IL-17 secretion in thymocytes from C57BL/6 and BALB/c mice (Fig. 3A) compared to controls (endotoxin-free water). Upon additional activation with α-CD3, higher frequencies of IFN-γ could be detected in thymocytes from BALB/c mice compared to α-CD3-activated cells. In the spleen, stimulation with 1 μg/ml lectin led to statistically significantly increased frequencies of IL-6 in C57BL/6 and BALB/c mice (Fig. 3B). Statistically significantly higher frequencies of IL-17-producing cells could be detected after stimulation with 0.1 to 1 μg/ml lectin in all tested species. Additional activation with α-CD3 resulted only after stimulation with 0.01 and 0.1 μg/ml lectin in statistically significantly higher frequencies of IFN-γ in C57BL/6 mice. Interestingly, stimulation with all tested concentrations of ConA yielded statistically significantly higher IL-17 production in thymocytes (Fig. 3C) and splenocytes (Fig. 3D), even without additional activation with α-CD3. Stimulation with lectin and ConA led to statistically significantly higher secretion of IL-2 and IL-4 (see Fig. S6 in the supplemental material). Furthermore, stimulation with 100 μg/ml curdlan AL, a Dectin-1 receptor agonist, increased IL-17 production of α-CD3-activated thymocytes and led to higher frequencies of IL-17 in splenocytes. In addition, we determined the effect of lectin and ConA on the apoptosis of thymocytes (see Fig. S6 in the supplemental material). Both lectin and ConA increased the number of apoptotic and dead cells and resulted in larger amounts of caspase+ cells.
In vitro cytokine response from naive wild-type mice in response to lectin from P. aeruginosa, curdlan AL, ConA, and homoserine lactone. The results of ELISpot assays of cytokine-producing thymocytes (A) and splenocytes (B) for the cytokines IFN-γ, IL-6, and IL-17 after stimulation with 0.01 to 10 μg/ml lectin from P. aeruginosa are shown. (C and D) IL-17 production of thymocytes (C) and splenocytes (D) after stimulation with 0.1 to 10 μg/ml ConA, 0.1 to 10 μg/ml homoserine lactone, and 10 to 100 μg/ml curdlan AL (±α-CD3) for 24 h. Each bar represents the mean of two independent experiments of eight mice ± the SEM. *, P < 0.05; **, P < 0.01 (as determined by Dunnett's t test).
Acyl-homoserine lactone from P. aeruginosa decreased IL-17 secretion.In contrast to other components of P. aeruginosa, stimulation with acyl-homoserine lactone resulted after α-CD3 activation in statistically significantly lower frequencies of IL-17 at 1 to 10 μg/ml in thymocytes and at 10 μg/ml in splenocytes compared to α-CD3-activated cells (Fig. 3C and D).
Lectins trigger the IL-17 response of CD4+ T cells.Both for thymocytes and for CD4+ and CD8+ T cells, IL-17 secretion was only triggered after further activation with APCs or α-CD3 (Fig. 4A). Statistically significantly higher frequencies of IL-17 could be detected in CD4+ T cells after stimulation with lectin and ConA, as well as in CD8+ T cells, after stimulation with ConA (Fig. 4B), compared to controls (endotoxin-free water). Intracellular cytokine staining showed that stimulation with both lectins and curdlan decreased the number of IFN-γ- and IL-17-producing cells. Stimulation with HKPA, its components, or ConA and curdlan did not increase the number of IFN-γ- and IL-17-producing CD4+ T cells, as well as Th17-polarized CD4+ T cells (Fig. 4C). Furthermore, the influence of HKPA, LPS, and Exo A resulted in distinctly lower frequencies of ROR (related orphan receptor) γt+ CD4+ T cells under Th17 conditions and after stimulation with HKPA and Exo A in clearly higher numbers of FOX (forkhead box) P3+ CD4+ T cells (Fig. 4D).
(A) In vitro IL-17 response of thymocytes and CD4+ and CD8+ T cells after stimulation with HKPA (107/ml) and LPS (1 μg/ml) in combination with 50,000 APCs and α-CD3 (1 μg/ml) for 24 h. Each bar represents the mean of two independent experiments of four mice ± the SEM. (B) IL-17 response of isolated CD4+ and CD8+ T cells after stimulation with lectin (1 μg/ml), ConA (1 μg/ml), and curdlan (100 μg/ml). Each bar represents the mean of three independent experiments of six mice ± the SEM. (C) Flow cytometry after 5 h for IFN-γ and IL-17 in isolated CD4+ T cells and Th17 polarized T cells. The data represent one of three independent experiments. (D) Intracellular staining of FOXP3 and RORγt from Th17 polarized CD4+ T cells after stimulation for 72 h. The data represent one of three independent experiments. *, P < 0.05; **, P < 0.01 (as determined by Dunnett's t test).
Influence on the cytokine secretion of antigen-specific cells from EAE mice.To determine the effect of P. aeruginosa and its components on the cytokine pattern of a primed antigen-specific T cell-mediated immune response, EAE was induced in C57BL/6 mice by MOG35–55/CFA/PTx injection. Stimulation of splenocytes from mice with EAE (EAE mice) with HKPA and LPS resulted in higher responses of IFN-γ, statistically significant after restimulation with MOG35–55 peptide in comparison to controls (PBS and endotoxin-free water) (Fig. 5A). Furthermore, slightly higher responses of IL-17 could be detected both with and without additional stimulation with MOG35–55 peptide. Stimulation with Exo A resulted in no differences compared to splenocytes from wild-type mice either after additional activation with MOG35–55 peptide (Fig. 5A). Stimulation with lectin and ConA resulted in statistically significant higher IFN-γ, IL-6, and IL-17 production. The influence of acyl-homoserine lactone decreased the IFN-γ, IL-6, and IL-17 response of antigen-specific cells after stimulation with MOG35–55 peptide. Moreover, stimulation with HKPA and LPS resulted in larger amounts of IFN-γ-producing CD4+ and CD4− cells from EAE mice but, in contrast to splenocytes from naive mice, not until after 48 h stimulation time (see Fig. S7 in the supplemental material). In addition, the stimulation of CNS-infiltrating cells with HKPA or its components did not result in higher frequencies of IL-17, but bone marrow cells isolated from EAE mice with a high clinical score tend to result in higher frequencies of IL-17 (Fig. 5B). Statistically significantly higher responses of IL-17 could be detected in brain cells after stimulation with ConA. In brief, the cytokine production of MOG-antigen-specific cells was more distinct than in wild-type mice.
In vitro cytokine response of cells from EAE mice in response to HKPA and its components. (A) Results of ELISpot assays of cytokine-producing splenocytes from EAE mice at day 15 of disease for the cytokines IFN-γ, IL-6, and IL-17 after stimulation with HKPA (107/ml), LPS (1 μg/ml), Exo A (1 μg/ml), homoserine lactone (1 μg/ml), lectin (1 μg/ml), ConA (1 μg/ml), and curdlan (100 μg/ml) with or without MOG35–55 peptide (20 μg/ml). Data for four mice tested individually in two independent experiments are shown. (B) IL-17 response of isolated cells from the spinal cords and brains of EAE mice at day 15 of disease after stimulation with 107/ml HKPA with or without MOG35–55 peptide. Data for 12 mice (8 for brain) tested individually in four independent experiments are shown. Each data point indicates an individual animal; the line represents the mean. Symbols represent the clinical score for each mouse. *, P < 0.05; **, P < 0.01 (as determined by Dunnett's t test).
(A) In vitro cytokine response of cells of isolated CD4+ and CD8+ T cells from EAE mice at day 15 of disease after stimulation with HKPA (107/ml), LPS (1 μg/ml), Exo A (1 μg/ml), lectin (1 μg/ml), ConA (1 μg/ml), and curdlan (100 μg/ml) with or without MOG35–55 peptide (20 μg/ml). The data for five mice tested individually in three independent experiments are shown. Measurements were counted in duplicate wells. Each data point indicates an individual animal; the line represents the mean. Symbols represent the clinical score for each mouse. (B) Flow cytometry of proliferation from splenocytes from EAE mice after stimulation with HKPA and LPS with or without α-MOG35–55 peptide for 72 h. Data represent one of three independent experiments with a clinical score of 2.5. (C) Flow cytometry after 5 h for IL-17 in isolated CD4+ T cells from EAE mice and Th17 polarized T cells (MOG-specific Th17 conditions). The data represent one of three independent experiments with a clinical score of 2.5. *, P < 0.05; **, P < 0.01 (as determined by Dunnett's t test).
Furthermore, stimulation with HKPA and LPS (after additional stimulation with MOG35–55 peptide) led to higher frequencies of IL-17 in isolated CD4+ T cells, whereas the influence of lectin and ConA led to statistically significantly increased IL-17 secretion in CD4+ and CD8+ T cells from EAE mice (Fig. 6A). In contrast to CD4+ T cells from naive mice, stimulation with HKPA and lectin resulted in higher frequencies of IL-17-producing CD4+ T cells and Th17-polarized CD4+ T cells from EAE mice (Fig. 6C). Moreover, stimulation with HKPA and LPS elevated the proliferation rates of CD45− splenocytes, but after additional stimulation with MOG35–55 peptide, a slightly lower proliferation rate of CD45− cells could be measured for HKPA (Fig. 6B).
DISCUSSION
Infections with P. aeruginosa, a metabolically versatile bacterium, can cause several opportunistic infections in humans. P. aeruginosa is a nosocomial pathogen. Nosocomial infections are hospital-acquired infections developed during treatment in hospitals or other health care service units (17, 35). Unfortunately, P. aeruginosa has the ability to develop resistance to multiple classes of antibacterial-effective drugs, so that treatment of an infection has become a serious clinical problem (21). Not uncommonly, infections with Gram-negative bacteria (including P. aeruginosa) resulted in systemic inflammatory response syndrome, sepsis, or septic shock (16). These inflammatory diseases are life-threatening conditions that require comprehensive treatment in intensive care units. Apart from hospitals, P. aeruginosa can also prevalent in damp milieus, groceries, and cosmetics and cause disease patterns other than sepsis. The pathogenesis of sepsis or septic shock is not yet completely understood, but the inflammatory changes in the immune system affect organs of the immune periphery, as well as the thymus (14, 36). Currently, there are two different concepts on the clinical dynamometer. The first is a vaccine based on a surface protein of Pseudomonas. The second is a human antibody fragment that should block a virulent factor of the bacterium and repress the inflammation.
The findings are that P. aeruginosa increased the frequencies of IL-1β, IL-6, and IL-17 in the thymus by itself and also enhanced the IFN-γ and IL-17 production of α-CD3-activated thymocytes. In the spleen, higher frequencies of IFN-γ, IL-6, and IL-17 could be measured, whereas additional activation of the T-cell receptor (TCR) resulted in higher frequencies of IFN-γ and IL-6 but not of IL-17. IL-17 secretion by isolated CD4+ and CD8+ T cells seems to be dependent on an additional activation with APCs or directly via TCR with α-CD3. This suggests that the impact of HKPA can trigger the secretion of IL-17 but not influence the amount of IL-17-producing cells. In contrast to this, HKPA increased the number of IFN-γ- and IL-17-producing CD4− splenocytes. Furthermore, HKPA enhanced the proliferation of CD4− cells in the thymus and of CD45+ cells (lymphocytes) and CD45− cells in the spleen. This finding indicates that HKPA can also affect innate immune cells, including dendritic cells and macrophages, which are able to initiate immune responses and trigger (either directly or indirectly) the IL-17 secretion of T cells.
Interestingly, we found a similar cytokine pattern after stimulation with LPS. It seems that cytokine secretion after stimulation with HKPA was mediated by its cell wall components. HKPA is recognized by the immune system via TLR2, whereas LPS is recognized via TLR4. This means that the cytokine response to bacteria is not only mediated through TLR2 activation but also that cell wall components are able to activate other pathways that trigger the cytokine secretion of immature and mature immune cells, which correlates with our previous studies (2, 5, 27, 31, 32). We found that LPS is responsible for the activation of the Notch2+ signaling of mature immune cells. The Notch signaling pathways play an important part in cell-cell communication and control differentiation, proliferation, and apoptotic occurrences in metazoan tissues (37).
Another interesting aspect is that an additional dose of alum crystals did not trigger the IL-1β and IL-17 response. Alum crystals trigger inflammation through the activation of the NALP3 (i.e., NACHT, LRR, and PYD domains containing protein 3) inflammasome (38). Thus, the activation of the TLR2/MyD88/NF-κB pathway represents the first signal sequence and enhances expression of genes for NALP3. The second signal comprising both reactive oxygen species (ROS) and potassium efflux promotes inflammasome activation and enhances the secretion of mature IL-1β (39). In the present study, we demonstrated that the initiation of the first signal via TLR2 was sufficient to trigger IL-1β secretion in the thymus. Previous studies revealed that activation of TLRs resulted in higher ROS production (40, 41). This indicates that the second signal is provided by the cell itself as a result of the TLR2 (or TLR4) activation.
Exotoxin A is an essential pathology factor during infections with P. aeruginosa and belongs, as well as DTx, to the class of AB toxins. The mechanism of Exo A is similar to that of DTx (23, 42). In vivo secretion of Exo A as a proenzyme resulted in an increased apoptosis rate and interference of immune functions of the host (22, 43). Stimulation with Exo A resulted in a slightly higher cytokine response by immature and mature immune cells, whereas no toxic effect could be detected. However, in contrast to humans, rodents are insensitive to AB toxins due to a single amino acid substitution in proHB-EGF (44). Due to the fact that the stimulating effect of Exo A is not mediated by pathogen recognition receptors, we assume that Exo A has an implicit influence on cells of the innate and adaptive immune system that triggers the response of inflammatory cytokines.
Lectin (PA-l), another known virulent factor of P. aeruginosa, strongly influences the IL-17 response of mature and immature immune cells (45). Normally, T cell activation is initiated by the interaction of the TCR with antigenic peptides complexed to major histocompatibility complex class II molecules (46). For our experiments, we partially activated the cells via the CD3/TCR complex. The cytokine response after stimulation with lectin was not dependent on a TCR activation. For further investigations, we tested the herbal lectin ConA from Canavalia enisformis. As a result, we noted a significant IL-2, IL-4, and IL-17 response by mature and immature immune cells and increased IL-17 secretion by isolated CD4+ and CD8+ T cells. The immunostimulatory effect of ConA in mice is mediated by activation of the TCR (47, 48). In contrast to AB toxins and lectin from P. aeruginosa, ConA is a lectin without a sugar residue. This suggests that the immunostimulatory effect of lectin is mediated by TCR activation and that the chemical structure of the compounds is critical for interaction with the TCR. This in turn indicates that the virulent factors Exo A and lectin (PA-I), which are secreted during infections with P. aeruginosa, can contribute to the emergence of septic reactions and other Pseudomonas-mediated diseases.
The recognition of curdlan from Alcaligenes faecalis is mediated by Dectin-1 receptor (49). Because curdlan merely induced higher IL-17 responses in splenocytes, we assume that the stimulatory effect of lectins is not mediated by the Dectin-1 pathway. Generally, the targets of lectins are the sugar-binding sites in the cell membranes of many immune cells. Blockade of this binding site impairs cell communication and leads to toxic effects (50). As expected, lectin and ConA increased the amounts of apoptotic and dead cells. Furthermore, we detected larger amounts of caspase+ cells. Activation of caspase can trigger the production of inflammatory cytokines and activate innate immune mechanisms (51, 52). This suggests that the response of IL-17 in thymocytes after stimulation with lectins could be also triggered by caspase-induced immune activation.
Quorum sensing is a regulatory system that enables bacteria to coordinate their behavior. Recently reported findings describe an inhibiting effect of T-cell differentiation and cytokine production (IFN-γ and IL-4) in splenocytes (26). We found that N-acyl-homoserine lactone from P. aeruginosa inhibited the cytokine secretion of immature and mature immune cells, as well as of antigen-specific cells. Thus, immature immune cells are more sensitive than mature immune cells.
RORγt is typically expressed in Th17 cells, and FOXP3 is a master regulator in the development and function of regulatory T cells (1, 53). Here, we demonstrated that both HKPA and its components inhibit the development of Th17 cells and promote the emergence of regulatory T cells. This indicates that Th17 cells merely play a minor part in the development of disease conditions after P. aeruginosa infections.
IL-17 is crucially involved in the cytokine network as an effector cytokine in EAE (12). The cytokine production of MOG-antigen-specific cells was more distinct than in wild-type mice. Another interesting observation was that the intracellular cytokine concentrations of MOG-specific cells increased over time, whereas the cytokine concentrations of wild-type splenocytes declined. A possible explanation for the higher cytokine response is the active immunization with complete Freund's adjuvant. The included Mycobacterium tuberculosis activates the immune system via TLR and promotes the emergence of CD4+ T cells, which can react immediately against microbial stimuli without a further priming phase.
We conclude (i) that infections with P. aeruginosa affect the cytokine secretion of immature and mature immune cells, (ii) that the immunostimulatory influence is mainly mediated by the cell wall component LPS, but (iii) that also other components of the bacterium can trigger cytokine production. Furthermore, we conclude (iv) that stimulation with P. aeruginosa does not increase the number of IL-17-producing CD4+ T cells and (v) that this stimulation inhibits the development of Th17 cells, but we also conclude (vi) that this stimulation promotes Treg cells. This suggests that IL-17 and Th17 cells merely play a minor role in the development of pathological systemic inflammatory disease conditions during P. aeruginosa infections. Therefore, other inflammatory immune responses must be responsible for the septic reactions of the host.
ACKNOWLEDGMENTS
We thank Zippora Kohne for excellent technical assistance and Felicitas Opdenhövel for critical reading of the manuscript.
FOOTNOTES
- Received 12 July 2015.
- Returned for modification 16 October 2015.
- Accepted 15 February 2016.
- Accepted manuscript posted online 22 February 2016.
Supplemental material for this article may be found at http://dx.doi.org/10.1128/IAI.00905-15.
- Copyright © 2016, American Society for Microbiology. All Rights Reserved.
