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Open Access

Peer-reviewed

Research Article

Control of Listeria monocytogenes infection requires classical IL-6 signaling in myeloid cells

  • Karsten Lücke,

    Roles Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Validation, Visualization, Writing – original draft, Writing – review & editing

    Affiliation Institute of Immunology, University Medical Centre Hamburg-Eppendorf, Hamburg, Germany

    ⨯
  • Isabell Yan,

    Roles Data curation, Formal analysis, Investigation, Methodology

    Affiliation Institute of Immunology, University Medical Centre Hamburg-Eppendorf, Hamburg, Germany

    ⨯
  • Sonja Krohn,

    Roles Investigation, Methodology

    Affiliation III. Medical Clinic and Polyclinic, University Medical Centre Hamburg-Eppendorf, Hamburg, Germany

    ⨯
  • Annika Volmari,

    Roles Investigation, Methodology

    Affiliation I. Medical Clinic and Polyclinic, University Medical Centre Hamburg-Eppendorf, Hamburg, Germany

    ⨯
  • Stefanie Klinge,

    Roles Investigation, Methodology

    Affiliation Institute of Immunology, University Medical Centre Hamburg-Eppendorf, Hamburg, Germany

    ⨯
  • Joanna Schmid,

    Roles Investigation

    Affiliation Institute of Immunology, University Medical Centre Hamburg-Eppendorf, Hamburg, Germany

    ⨯
  • Valéa Schumacher,

    Roles Investigation

    Affiliation Institute of Immunology, University Medical Centre Hamburg-Eppendorf, Hamburg, Germany

    ⨯
  • Oliver M. Steinmetz,

    Roles Methodology, Resources

    Affiliation III. Medical Clinic and Polyclinic, University Medical Centre Hamburg-Eppendorf, Hamburg, Germany

    ⨯
  • Stefan Rose-John,

    Roles Conceptualization, Resources, Writing – review & editing

    Affiliation Institute for Biochemistry, Medical Faculty, Christian Albrechts University, Kiel, Germany

    ⨯
  • Hans-Willi Mittrücker

    Roles Conceptualization, Data curation, Formal analysis, Funding acquisition, Project administration, Supervision, Validation, Visualization, Writing – original draft, Writing – review & editing

    h.mittruecker@uke.de

    Affiliation Institute of Immunology, University Medical Centre Hamburg-Eppendorf, Hamburg, Germany

    ⨯

Abstract

IL-6 is required for the response of mice against Listeria monocytogenes. Control of infection depends on classical IL-6 signaling via membrane IL-6Rα, but IL-6 target cells and protective mechanisms remain unclear. We used mice with IL-6Rα-deficiency in T cells (Il6rafl/fl×CD4cre) or myeloid cells (Il6rafl/fl×LysMcre) to define the role of these cells in IL-6-mediated protection. Abrogation of IL-6Rα in T cells did not interfere with bacteria control and induction of TH1 and CD8+ T-cell responses. IL-6Rα-deficiency in myeloid cells caused significant defects in listeria control. This defect was not associated with reduced recruitment of granulocytes and inflammatory monocytes, and both cell populations were activated and not impaired in cytokine production. However, IL-6Rα-deficient inflammatory monocytes displayed diminished expression of IL-4Rα and of CD38, a protein required for phagocytosis and innate control of listeria. In vitro studies revealed that IL-4 and IL-6 cooperated in induction of CD38. In listeria-infected mice, phagocytic activity of inflammatory monocytes correlated with CD38 expression levels on cells and inflammatory monocytes of Il6rafl/fl×LysMcre mice were significantly impaired in phagocytosis. In conclusion, we demonstrate that inhibition of classical IL-6 signaling in myeloid cells causes alterations in differentiation and function of these cells, which subsequently prevent effective control of L. monocytogenes.

Citation: Lücke K, Yan I, Krohn S, Volmari A, Klinge S, Schmid J, et al. (2018) Control of Listeria monocytogenes infection requires classical IL-6 signaling in myeloid cells. PLoS ONE 13(8): e0203395. https://doi.org/10.1371/journal.pone.0203395

Editor: Martin E. Rottenberg, Karolinska Institutet, SWEDEN

Received: April 19, 2018; Accepted: August 20, 2018; Published: August 31, 2018

Copyright: © 2018 Lücke et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Data Availability: All relevant data are within the paper and its Supporting Information files.

Funding: This work was supported by Deutsche Forschungsgemeinschaft (dfg.de), grant no.: SFB841, project A3, to HWM; Deutsche Forschungsgemeinschaft (dfg.de) grant no.: SFB841, project C1, to SRJ; Deutsche Forschungsgemeinschaft (dfg.de), grant: Cluster of Excellence ‘Inflammation at Interfaces’, to SRJ. The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Competing interests: The authors have declared that no competing interests exist.

Abbreviations: fMLP, N-formyl-met-leu-phe; Lm, Listeria monocytogenes; LmOVA, Ovalbumin-recombinant Listeria monocytogenes OVA; MMR1, Macrophage mannose receptor 1

Introduction

The cytokine IL-6 has both pro- and anti-inflammatory activities in the immune system [1]. IL-6 controls neutrophil maturation and recruitment to sites of infection [2]. In monocytes, IL-6 regulates differentiation through upregulation of macrophage colony-stimulating factor receptor (M-CSFR) [3]. It also induces IL-4Rα expression and supports formation of alternatively activated macrophages [4, 5]. Furthermore, IL-6 is a central driver of the acute phase response. IL-6 is likewise involved in the adaptive response. IL-6 supports the production of antibodies in B cells either directly or by inducing formation of T follicular helper (TFH) cells [6, 7]. Differentiation of naive CD4+ T cells to TH17 cells strongly depends on IL-6 [8]. In line with its pleiotropic activities, abrogation of IL-6 signaling has substantial effects on the immune system. Diminished IL-6 signaling causes defects in the control of viral, bacterial and parasite infections [9–11]. Conversely, IL-6 signaling is detrimental in inflammatory diseases such as rheumatoid and juvenile arthritis [12] or proliferative disorders like Castleman’s disease [13].

IL-6 signaling is conveyed through the IL-6 receptor. The receptor consists of the 80 kDa membrane-bound IL-6Rα subunit (CD126), which has no intracellular signaling component, and the signal-transducing gp130 subunit. Two receptors interact with two IL-6 proteins to form the hexameric signaling complex [14]. IL-6’s activities in hematopoiesis, glucose metabolism and in the neuroendocrine system rely on classical IL-6 signaling by this complex [15]. Classical signaling is restricted to cells with expression of the membrane-bound IL-6Rα subunit, including hepatocytes and a subset of leukocytes [16]. IL-6Rα is also found as a soluble protein (sIL-6Rα), which can still bind IL-6. These IL-6/sIL-6Rα complexes are able to associate with gp130 subunits on the cell surface and induce IL-6 signaling. Since gp130 is ubiquitously expressed, this IL-6 trans-signaling substantially broadens the range of IL-6 target cells to all cells in the body [17]. In addition to its membrane-bound form, gp130 is constitutively produced as soluble protein which neutralizes IL-6/sIL-6Rα complexes [18]. Therefore, IL-6 trans-signaling only occurs in situations with secretion of IL-6 and extensive shedding of IL-6Rα. Since sgp130 does not interfere with classical IL-6 signaling, IL-6Rα+ cells can still respond to high IL-6 concentrations in the absence of IL-6 trans-signaling. Thus, depending on the concentrations of sIL-6Rα, IL-6 will have distinct effects on the immune system [1]. Recently, a third mode of IL-6 signaling has been described as mechanism to induce TH17 cells in the model of experimental autoimmune encephalomyelitis [19]. In dendritic cells (DC), IL-6 can intracellularly bind to the IL-6Rα resulting in IL-6/IL-6Rα complexes on the DC surface. During DC-mediated T-cell interaction, these complexes interact with gp130 on the T-cell surface and induce IL-6 signaling. As a consequence, this IL-6 cluster signaling does not require IL-6Rα on the T cell [19].

The Gram-positive bacterium Listeria monocytogenes is a human pathogen that causes listeriosis. Risk groups are individuals undergoing immune suppressive treatment and pregnant women where listeria can cause fatal infection of the fetus [20]. Listeria infection is initially controlled by the innate immune system. Rapid mobilization of myeloid cells from the bone marrow and recruitment of these cells to the sites of infection is essential for the restriction of bacterial replication. Due to its intracytosolic habitat, listeria induce strong TH1 and CD8+ T-cell responses, and both T-cell subsets are required for pathogen eradication and provide effective protection to re-infection [21].

We could previously show that classical IL-6 signaling is essential for the early control of L. monocytogenes infection [10], but the target cells and protective mechanisms controlled by IL-6 remained unclear. In the current study, we used mice with IL-6Rα-deficiency restricted to either T cells or myeloid cells to define the role of these cells in IL-6 mediated protection. Abrogation of classical IL-6 signaling in T cells did not interfere with bacteria control or with the induction of specific TH1 and CD8+ T-cell responses. We could, however, detect a defect in TH17-cell differentiation. In contrast, abrogation of classical IL-6 signaling in myeloid cells caused a significant defect in the control of L. monocytogenes. This defect was not associated with reduced accumulation of granulocytes and inflammatory monocytes at sites of infection, and inflammatory monocytes showed an activated phenotype and were not impaired in the production of inflammatory cytokines. However, inflammatory monocytes showed diminished CD38 expression, a protein associated with the microbicidal M1 macrophage phenotype [22], and a significant reduction in their phagocytic activity, which in turn might be the cause for the impaired control of the infection.

Material and methods

Animals

Il6rafl/fl mice (B6.SJL-Il6ratm1.1Drew/J) [23], CD4cre mice (B6.Cg-Tg[Cd4-cre]1Cwi/BfluJ), LysMcre mice (B6.129P2-Lyz2tm1(cre)Ifo/J), Il6KO mice (B6.129S2-Il6tm1Kopf/J) [5], sgp130FcTg mice [24] and wildtype C57BL/6J mice were bred in our facilities. All animals used in this study were on a C57BL/6 background. For all experiments, age and sex matched control mice from the same colony were used. Mouse experiments were carried out in strict accordance with the state guidelines. The protocol was approved by the local ethics committee of the Behörde für Gesundheit und Verbraucherschutz of the City of Hamburg (Permit Number: 81/14). Mice were housed under specific pathogen free conditions in individually ventilated cages with standard food and water ad libitum. During infection experiments, mice were controlled daily and mice with signs of severe disease were euthanized with an O2/CO2 mixture to minimize suffering.

Animal experiments

Mice were infected with wildtype Listeria monocytogenes strain EGD unless stated otherwise. Mice received 2×104 bacteria in 200 μl sterile PBS via the lateral tail vein. Mice were analyzed on day 2 or 3 post-infection (p.i.). For the analysis of primary T-cell responses, mice were i.v. infected with 1×104 ovalbumin-recombinant L. monocytogenes (LmOVA). T-cell responses were characterized on d8 p.i. For the determination of acquired protection, mice were infected with 2×103 Lm and 7 weeks later, reinfected with 1×105 Lm. Bacterial titers were measured two days later. Bacterial inocula were controlled by serial dilution and plating onto tryptic soy broth (TSB) agar plates. Plates were incubated at 37 °C and colony forming units (CFU) were counted the next day. For in vivo phagocytosis analysis, mice were injected with yellow-green fluorescent latex beads diluted 1:25 in PBS (FluoSpheres® Carboxylate-Modified Microspheres, 0.5 μm, Thermo Fisher, Waltham, MA).

For determination of bacterial titers, organs of infected mice were mechanically homogenized in 1 ml 0.1% Triton X-100 in H20 and suspensions were serially diluted. Dilutions were plated on TSB-agar plates and incubated at 37 °C. CFU were counted the next day and bacterial titers in organs were calculated.

Cytokine profile

Organs of naive and infected mice were collected in RIPA Buffer (150 mM NaCl, 1% NP40, 0,1% Triton X-100, 0,1% SDS, 50 mM Tris-HCl, 5 mM EDTA, [pH 8]) supplemented with cOmplete Mini Protease Inhibitor Cocktail (Roche, Rotkreuz, Switzerland). Organs were mechanically homogenized with the gentleMACS dissociator (Miltenyi Biotec, Bergisch Gladbach, Germany). Cytokines were determined using the LegendPLEX mouse inflammation panel (BioLegend, San Diego, CA) according to manufacturer’s instruction.

Whole RNA was obtained by homogenizing tissue samples and extracting RNA using the Nucleospin® RNA Kit (Macherey-Nagel, Düren, Germany). cDNA was transcribed using the high-capacity cDNA reverse transcription kit (Thermo Fisher). Quantitative PCR was performed with the SYBR® Green JumpStart™ Taq ReadyMix™ (Sigma-Aldrich) on a StepOnePlus™ real-time PCR system (Thermo fisher). Results were normalized to 18S RNA using the ΔCT method. qPCR primers: Il1b forward: AAC CTG CTG GTG TGT GAC GTT C, Il1b reverse: CAG CAC GAG GCT TTT TTG TTG T, Il6 forward: TGG GAA ATC GTG GAA ATG AGA, Il6 reverse: AAG TGC ATC ATC GTT GTT CATA CA, Nos2 forward: GCT GTT CTC AGC CCA ACA AT, Nos2 reverse: TTC TGT GCT GTC CCA GTG AG, 18S forward: CAC GGC CGG TAC AGT GAA AC, 18S reverse: AGA GGA GCG AGC GAC CAA A.

Cell isolation

Spleens were mashed through a cell sieve to prepare a single cell suspension. Erythrocytes were lysed by incubation in ammonium chloride potassium (ACK) lysis buffer (155 mM NH4Cl, 10 mM KHCO3, 100 μM EDTA [pH 7.2]). Liver cells were additionally purified using a one-step Easycoll gradient (Biochrom AG, Berlin, Germany) before erythrocyte lysis. Cells were counted with an automated cell counter.

In vitro stimulation of primary murine cells

2×106 cells were incubated in 1ml of standard medium (Iscove’s modified Dulbecco’s medium (IMDM), 5% fetal calf serum, 50 μg/ml gentamicin, 50 μM 2-mercaptoethanol, 200 μM L-glutamine) containing stimulants. T cells were stimulated polyclonally with phorbol-12-myristate-13-acetate (PMA, 50 ng/ml) and ionomycin (1 μM), or antigen-specific with listeriolysin O peptide 189–201 (LLO, 10−5 M, JPT, Berlin, Germany) and ovalbumin peptide 257–264 (OVA, 10−6 M, JPT). For stimulation of monocytes, lipopolysaccharide from E. Coli 055:B5 (LPS, 1 μg/ml, Sigma-Aldrich, St. Louis, MO) was utilized. Cells were incubated for 30 minutes at 37 °C and 5% CO2. Brefeldin A (10 μg/ml) was added to block the Golgi apparatus. After brefeldin A addition, the cells were incubated for additional 210 minutes.

Generation of bone marrow-derived macrophages (BMDM)

Bone marrow was isolated from femur and tibia of naive mice. Cells were counted and resuspended in BMDM medium (Dulbecco’s modified Eagle medium (DMEM), 10% fetal calf serum, 5% horse serum, 10 ng/μl M-CSF (Peprotech, Rocky Hill, NJ), 1 mM HEPES, 100 μM sodium pyruvate, 50 μg/ml gentamicin, 200 μM L-glutamine) at a concentration of 5×105 cells/ml. 10 ml of cell suspension was cultured in a petri dish for 5 days at 37 °C and 5% CO2. Cells were supplemented with 5 ml BMDM medium and incubated for 3 additional days. Cells were mechanically detached from the petri dishes and resuspended in standard medium at a concentration of 5×105 cells/ml. 1 ml of cell suspension per well was seeded onto a 24-well plate. For M1 and M2 polarization, cells were supplemented with either LPS (2 μg/ml, Sigma-Aldrich) and recombinant IFNγ (40 ng/ml) or recombinant IL-4 (40 ng/ml). Cells were additionally incubated with or without recombinant IL-6 (160 ng/ml, Stefan Rose-John, CAU, Kiel) for 24 h. To induce NO production, cells were further stimulated with heat-killed listeria (5×106) for the final 18 h. Supernatant was collected and cells were detached by incubation in PBS with 0.25 mM EDTA and kept on ice until staining. Listeria uptake and control of intracellular replication in BMDM was assessed as described before [25]. NO concentrations were determined using a commercially available kit (Promega, Fitchburg, WI) according to the manufacturer’s instruction. 50 μl undiluted culture supernatant was used for analysis.

Immuno-fluorescent staining for flow cytometry

Non-specific antibody binding was blocked by incubation of cells with rat serum and Fc receptor blocking antibodies (clone 2.4G2, Bio X Cell, West Lebanon, NH). Dead cells were excluded by staining with pacific orange succinimidyl ester (Thermo Fischer). Cells were extracellularly stained with antibodies (S1 Table). For intracellular cytokine staining, cells were fixed in 2% paraformaldehyde and non-specific staining was blocked with rat serum diluted in saponin buffer (PBS, 0.1% BSA, 0.45 g saponin (Sigma-Aldrich)). Intracellular cytokine staining was performed using antibodies diluted in saponin buffer. Stained cells were analyzed with a BD FACS CANTO II (Becton Dickinson) using BD FACS DIVA 8.0 (Becton Dickinson) and Flowjo vX (Flowjo, Ashland, OR). The gating strategy is given in S1A and S1B Fig.

Chemotaxis assay

Chemotaxis index was determined by a transwell assay using 24-well transwell plates with a 5 μm pore size (Costar, Corning, NY). 1×106 spleen cells from infected mice were loaded in standard medium into the top chamber. CCL2 (0.15 ng/μl) or fMLP (N-formyl-met-leu-phe, 12.5 μM) were used as chemotactic agents in the lower chamber. Cells were incubated for 2 h at 37 °C and 5% CO2. Migrated cells were analyzed by flow cytometry.

Immunohistochemistry

Tissue was fixed overnight in 4% buffered formalin and embedded in paraffin. Macrophages and inflammatory monocytes were stained with an antibody specific for F4/80 (BM8, BioLegend) developed with the DAKO EnVision™+ system (Agilent, Santa Clara, CA) and counterstained with Mayer‘s Hematoxylin Solution. 5–15 pictures were taken per slide at 10x magnification. F4/80-positive area was calculated with ImageJ (NIH, Bethesda, MD).

Statistical analysis

Statistical analysis was performed with Prism Software (GraphPad Software, La Jolla, CA). In titers experiments, median values are represented by a line and differences were calculated by Mann-Whitney U test. In all other experiments, bars or lines represent means and error bars represent SEM. Differences were calculated either with student‘s t-test or 2way ANOVA with Bonferroni’s post-test. A P value of <0.05 was considered significant (* P<0.05; ** P<0.01; *** P<0.001).

Results

Classical IL-6 signaling in T cells is not required for the control of Listeria monocytogenes

The cytokine IL-6 has proinflammatory activities and is required for the control of bacterial infection [6, 26]. In a previous study, we could demonstrate that the control of Listeria monocytogenes depends on classical IL-6 signaling but not on IL-6 trans-signaling [10]. To characterize the mechanisms of IL-6-mediated protection and to identify the responsible IL-6 target cells, we used mice with a deficiency of IL-6Rα in CD4+ and CD8+ T cells (Il6rafl/fl×CD4cre mice) or in myeloid cells (Il6rafl/fl×LysMcre mice). In a first set of experiments, we used Il6rafl/fl×CD4cre mice to test the role of classical IL-6 signaling in T cells in the control of L. monocytogenes. Il6rafl/fl×CD4cre mice showed effective reduction of IL-6Rα surface expression on CD4+ and CD8+ T cells (S1C Fig). Il6rafl/fl×CD4cre and control mice (CD4cre) were infected i.v. with 2×104 listeria. Two days p.i., the mice were sacrificed and the bacterial titers in spleen and liver were determined. Abrogation of classic IL-6 signaling in T cells showed no effect on the bacterial burden in spleen and liver (Fig 1A).

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Fig 1. Classical IL-6 signaling in T cells has only limited impact on T-cell differentiation and bacteria control during Listeria monocytogenes infection.

Il6rafl/fl×CD4cre and CD4cre control mice were infected i.v. with 2×104 listeria. Two days p.i., titers in liver and spleen were determined (A). Results for individually analyzed mice and median bars are shown. Significance was determined with the Mann-Whitney U test. Results from two independent experiments were pooled. Mice were i.v. infected with 2×103 and after 7 weeks with 1×105 listeria. Titers were determined two days post reinfection (B). Significance was determined with the Mann-Whitney U test. One representative experiment of two is shown. Il6rafl/fl×CD4cre and control mice were infected i.v. with 1×104 listeria recombinant for ovalbumin. Eight days p.i., spleen cells were isolated and stimulated with LLO189-201 and OVA257-264 (C, D), or ionomycin and PMA (E, F). Intracellular IFNγ, TNFα and IL-17A expression was determined by flow cytometry. (C) Representative dot plots for CD4+ and CD8+ spleen cells from infected mice, with or without peptide stimulation. (D) Frequencies of IFNγ+CD4+ or IFNγ+CD8+ cells from spleens of infected Il6rafl/fl×CD4cre and control mice following in vitro stimulation. (E) Representative dot plots for CD4+ spleen cells from infected mice, with or without polyclonal stimulation. (F) Frequencies of IL-17A+CD4+ cells from spleens of naive and infected Il6rafl/fl×CD4cre and control mice following in vitro stimulation. Bars present mean ± SEM of 4 mice per group. Significance was determined with student’s t-test. One representative experiment of two is shown.

https://doi.org/10.1371/journal.pone.0203395.g001

Next, we analyzed the influence of classical IL-6 signaling in T cells on the specific T-cell responses against L. monocytogenes. Il6rafl/fl×CD4cre and control mice were infected i.v. with 2×104 ovalbumin-recombinant listeria (LmOVA) and sacrificed 8 days p.i. Spleen cells were stimulated with the peptides listeriolysin O189-201 and ovalbumin257-264 which are immunodominant for CD4+ and CD8+ T cells, respectively. Frequencies of IFNγ+ and TNFα+ CD4+ and CD8+ T cells were determined by flow cytometry. Impaired classical IL-6 signaling had no effect on the frequencies of antigen-specific IFNγ or TNFα producing CD4+ or CD8+ T cells (Fig 1C and 1D, and not shown). Consistent with this result, we did not detect significant changes in the specific T-cell response to L. monocytogenes in Il6KO mice and in sgp130FcTg mice, transgenic for a soluble gp130 receptor which neutralizes soluble IL-6Rα/IL-6 complexes and thereby inhibits IL-6 trans-signaling [24] (S2A and S2B Fig).

Control of secondary L. monocytogenes infection depends on T cells. Il6rafl/fl×CD4cre and control mice were infected and after 7 weeks, mice were reinfected with a high dose of L. monocytogenes. Determination of listeria titers in spleen and liver 2 days post reinfection revealed similar control of the secondary infection in both mouse strains (Fig 1B). Thus, classical IL-6 signaling was not required for protective T-cell responses against L. monocytogenes.

Classical IL-6 signaling is responsible for TH17-cell differentiation during infection

TH17-cell differentiation is dependent on IL-6, but the role of classical IL-6 signaling is still controversial. Il6rafl/fl×CD4cre and control mice were infected i.v. with 2×104 LmOVA and sacrificed 8 days p.i. Upon polyclonal stimulation with PMA and ionomycin, we observed significantly reduced frequencies of IL-17A+ CD4+ T cells in naive and infected Il6rafl/fl×CD4cre mice when compared to control mice (Fig 1E and 1F). The same effect was seen after stimulation of spleen cells from Il6KO mice but not from sgp130FcTg mice (S2C and S2D Fig). Taken together, these results show that classical but not IL-6 trans-signaling is responsible for TH17-cell differentiation during L. monocytogenes infection.

Control of Listeria monocytogenes depends on classical IL-6 signaling in myeloid cells

Since IL-6 signaling had no significant effect on the adaptive immune response against L. monocytogenes, we decided to focus on the innate immune response using Il6rafl/fl×LysMcre mice. Inflammatory monocytes showed surface expression of IL-6Rα which was substantially reduced in Il6rafl/fl×LysMcre mice (S1C Fig). In contrast, granulocytes, macrophages and non-classical Ly6Clow monocytes expressed only relatively low levels of surface IL-6Rα and the expression was not further diminished in Il6rafl/fl×LysMcre mice (S1C Fig).

Il6rafl/fl×LysMcre and littermate control mice (Il6rafl/fl) were infected with 2×104 listeria. Two days p.i., bacterial titers in livers and spleens were determined. Titers were significantly increased in livers of Il6rafl/fl×LysMcre mice. Titers in spleens of Il6rafl/fl×LysMcre mice were similar to those of control mice (Fig 2). Higher titers were still detected Il6rafl/fl×LysMcre mice at day 5 p.i. (S3A Fig).

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Fig 2. Control of Listeria monocytogenes depends on classical IL-6 signaling in myeloid cells.

Il6rafl/fl×LysMcre mice and Il6rafl/fl littermate controls were infected i.v. with 2×104 listeria. Two days p.i., titers in livers and spleens were determined. Data was pooled from 4 independent experiments. Results for individually analyzed mice and median bars are shown Significance was determined with the Mann-Whitney U test.

https://doi.org/10.1371/journal.pone.0203395.g002

Il6rafl/fl×LysMcre and control mice were also infected with LmOVA and eight days later, frequencies of T cells responding to the peptides listeriolysin O189-201 and ovalbumin257-264 were determined (S3B Fig). Spleens of both groups of mice showed similar frequencies for IFNγ+ CD8+ T cells. However, we observed significantly higher frequencies of IFNγ+ CD4+ T cells in Il6rafl/fl×LysMcre mice. Thus, absence of classical IL-6 signaling in myeloid cells did not impair but rather enhance the T-cell response to infection.

Effect of myeloid cell-specific IL-6Rα abrogation on the innate immune response

Il6rafl/fl×LysMcre mice and littermate controls were infected i.v. with 2×104 listeria. Mice were sacrificed 2 days p.i. and concentrations of inflammatory cytokines were determined in tissue extracts from spleen and liver (S4A–S4D Fig). In infected mice, we observed an increase in TNFα, IFNγ, IL-1α, GM-CSF, CCL2, IL-6 and IL-1β which was generally more pronounced in spleen compared to liver. However, changes were comparable in Il6rafl/fl×LysMcre and control mice. Other cytokines (IL-12p70, IL-10, IL-17A) did not or only marginally change following infection or were even reduced (IL-23, IFNβ). Interestingly, some cytokines such as IL-12p70, IL-23, IL-17A and IFNβ, were slightly higher expressed in naive and/or infected Il6rafl/fl×LysMcre mice compared to control mice. In summary, infected Il6rafl/fl×LysMcre and control mice showed only minor differences in their cytokine profile.

Hepatic expression of IL-1β, IL-6 and iNOS was assessed on mRNA level (S4E–S4G Fig). Il1b, Nos2 (coding for iNOS) and Il6 were upregulated in infection, however, there was similar upregulation in both mouse strains.

Following L. monocytogenes infection, granulocytes and inflammatory monocytes rapidly accumulate in the infected tissues and particularly inflammatory monocytes are crucial for the control of infection [27, 28]. To test whether abrogated IL-6 signaling in Il6rafl/fl×LysMcre mice resulted in an impaired response of inflammatory monocytes or granulocytes, Il6rafl/fl×LysMcre mice and littermate controls were infected i.v. with 2×104 listeria. Two days p.i., the distribution of various myeloid cell populations in spleen and liver was determined by flow cytometry. We did not detect differences in the total leukocyte numbers as well as in the numbers of granulocytes, inflammatory monocytes, Ly6clow monocytes and macrophages between infected Il6rafl/fl×LysMcre and control mice (Fig 3A, Gating strategy in S1A Fig). Since inflammatory monocytes showed the most pronounced shift in IL-6Rα expression, we focused in subsequent analyses mainly on these cells. In order to study the ability of the inflammatory monocytes to move into the infected organs, the migratory capacity of these cells was directly tested in a chemotaxis assay. Total spleen cells from infected mice were subjected to gradients of CCL2 or fMLP, and the migration of inflammatory monocytes towards high CCL2 and fMLP concentration was determined. The chemokine CCL2 is a ligand of the chemokine receptor CCR2, which plays a major role in mobilization of inflammatory monocytes in response to infection [28]. fMLP is a ligand of the formyl peptide receptor 1 (FPR1), which recognizes formyl methionine-containing bacterial peptides [29, 30]. For both chemotactic factors, we observed no difference in the chemotactic index between inflammatory monocytes from infected Il6rafl/fl×LysMcre and control mice (Fig 3B). Finally, livers and spleens from infected mice were analyzed for the accumulation of F4/80+ cells by immunohistochemistry. We did not observe a significant difference in the F4/80-positive area between Il6rafl/fl×LysMcre mice and littermate controls (Fig 3C and 3D).

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Fig 3. Distribution of inflammatory monocytes during the early phase of listeria infection is unaffected by abrogated classical IL-6 signaling.

Il6rafl/fl×LysMcre and Il6rafl/fl littermate control mice were infected i.v. with 2×104 listeria. Mice were analyzed 2 (A and B) or 3 days (C and D) p.i. (A) Liver and spleen cells were isolated and total leukocyte numbers as well as numbers of macrophages, granulocytes, inflammatory monocytes and Ly6Clow monocytes were determined. Groups contained 6 to 7 mice. Representative results of one of two experiments are shown. (B) Chemotaxis index for inflammatory monocytes was determined by transwell assay. Migration was induced by CCL2 and fMLP. One representative experiment of two with two replicates per group is shown. (C) F4/80 staining of paraffin fixed liver and spleen sections. Representative pictures of liver and spleen from infected mice are shown. (D) Statistical analysis of F4/80-positive areas from liver and spleen sections of infected mice. Groups contained 5 to 7 mice. Bars represent mean ± SEM. Significance was determined by unpaired student’s t-test.

https://doi.org/10.1371/journal.pone.0203395.g003

IL-6 shapes the differentiation of inflammatory monocytes

IL-6 is involved in differentiation and polarization of monocytes and macrophages [4]. Thus, it is possible that deficiency of classical IL-6 signaling in myeloid cells causes altered maturation of inflammatory monocytes during L. monocytogenes infection. Il6rafl/fl×LysMcre mice and littermate controls were infected i.v. with 2×104 listeria. Two days p.i., inflammatory monocytes from spleen and liver were analyzed for the expression of surface proteins associated with activation, differentiation and function (Fig 4A). Flow cytometric analysis revealed higher abundance of CD62L and MHC II on inflammatory monocytes from Il6rafl/fl×LysMcre mice, suggesting a more activated status of these cells [31]. In contrast, the chemokine receptors CCR2 and CX3CR1 were similarly expressed on inflammatory monocytes from infected Il6rafl/fl×LysMcre and control mice. Macrophage mannose receptor 1 (MMR1) and IL-4Rα are upregulated on M2 and CD38 on M1 macrophages [4, 22]. Since IL-6 was described to promote the maturation to M2 macrophages, we analyzed the expression of these proteins on inflammatory monocytes. We observed similar expression of the MMR1 on inflammatory monocytes from infected Il6rafl/fl×LysMcre and control mice. In contrast, surface expression of IL-4Rα and particularly of CD38 was reduced on inflammatory monocytes from Il6rafl/fl×LysMcre mice (Fig 4A and 4B).

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Fig 4. IL-6 affects differentiation of inflammatory monocytes.

Il6rafl/fl×LysMcre and Il6rafl/fl littermate control mice were infected i.v. with 2×104 listeria. Two days p.i., liver and spleen cells from infected mice were isolated and inflammatory monocytes were characterized by flow cytometry (gating strategy is given in S1A Fig). (A) Mean fluorescence intensity (MFI) of different surface proteins. (B) Representative histograms of CD38 expression. (C) Representative dot plots of intracellular TNFα and IL-6 expression in inflammatory monocytes. Spleen cells were stimulated with LPS for 4 h. (D) Frequency of TNFα+ and IL-6+ inflammatory monocytes. (E) MFI of intracellular TNFα and IL-6 staining of inflammatory monocytes. One representative experiment of two with 5 to 7 mice per group is shown. Bars represent mean ± SEM. Significance was determined with student’s t-test.

https://doi.org/10.1371/journal.pone.0203395.g004

Spleen cells were also stimulated with LPS and the production of TNFα and IL-6 by inflammatory monocytes was determined by intracellular cytokine staining. TNFα and IL-6 were produced by similar frequencies of inflammatory monocytes of both mouse strains. However, we observed a higher staining intensity in inflammatory monocytes from Il6rafl/fl×LysMcre mice indicating that individual cells produced more TNFα (Fig 4C–4E).

Overall, inflammatory monocytes from infected Il6rafl/fl×LysMcre mice displayed higher levels of the activation markers MHC II and CD62L and produced more TNFα upon activation. On the other hand, IL-6Rα lacking cells showed reduced surface expression of IL-4Rα but also of CD38, suggesting that absence of classical IL-6 signaling altered their maturation.

Expression of surface markers and cytokines was also assessed on granulocytes (S5A–S5D Fig) and Ly6Clow monocytes (S5E–S5H Fig). Compared to inflammatory monocytes, granulocytes displayed lower expression of all analyzed surface markers. We observed small changes in the expression of CD38, IL-4Rα, CD62L and CX3CR1 in granulocytes from Il6rafl/fl×LysMcre mice. Ly6Clow monocytes from Il6rafl/fl×LysMcre mice had diminished CD38 expression. All other markers did not differ between cells from both mouse lines. Compared to inflammatory monocytes, granulocytes and Ly6Clow monocytes produced less TNFα and IL-6, in terms of both frequencies of positive cells and staining intensity for cytokines. We detected similar expression of TNFα in cells from both mouse strains, but fewer Ly6Clow monocytes produced IL-6.

IL-6 regulates maturation of bone marrow-derived macrophages

To directly determine the impact of IL-6 on the expression on MMR1, IL-4Rα and CD38, BMDM were cultured under M0 (no additional stimulus), M1 (IFNγ + LPS) or M2 (IL-4) polarizing conditions and were subsequently incubated overnight with or without IL-6 (Fig 5A). We observed only low surface expression of CD38 under M0 conditions and expression was slightly increased by IL-6 in macrophages from control mice but not from Il6rafl/fl×LysMcre mice. CD38 was strongly induced under M1 conditions and this induction was independent from IL-6 or the expression of the IL-6Rα. CD38 was also induced under M2 conditions and here, induction required IL-6 and IL-6Rα. Induction of IL-4Rα was observed in cells from control mice under M0 and M2 conditions. Under M1 conditions, macrophages showed slightly enhanced expression, which was independent from IL-6. MMR1 was induced only under M2 conditions and expression was further increased in cells from control mice when treated with IL-6.

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Fig 5. IL-6 regulates maturation of bone marrow-derived macrophages.

(A) BMDM from Il6rafl/fl×LysMcre and Il6rafl/fl control mice were differentiated with IL-4 or LPS + IFNγ and stimulated overnight with IL-6. Surface expression of CD38, IL-4Rα and MMR1 was analyzed by flow cytometry. Histograms and MFI-values are shown. (B) BMDM were stimulated as described before. Heat-killed listeria were added for the final 18 hours. NO2- concentration in supernatants was determined in duplicates. (C) BMDM were infected in vitro with listeria (Strain EGD, MOI 10 1), extracellular bacteria were removed with gentamicin and titers were determined at indicated time points. One representative experiment of two is shown. Bars in (B) represent mean ± SEM and circles in (C) median ± range. Significance was determined with (B) student’s t-test or (C) Mann-Whitney U test.

https://doi.org/10.1371/journal.pone.0203395.g005

BMDM from Il6rafl/fl×LysMcre and control mice were also used to test the role of IL-6 in NO production. M0, M1 and M2 macrophages were stimulated with IL-6 and heat-killed listeria (HKL), and NO in supernatant was determined (Fig 5B). We observed strong NO production by M1 macrophages, which was only marginally increased by HKL. In contrast, M0 and M2 macrophages secreted NO only after HKL stimulation. However, under all conditions NO production was independent from both the expression of the IL-6Rα and the stimulation with IL-6.

Phagocytic potential of inflammatory monocytes is diminished in mice with abrogated classical IL-6 signaling in myeloid cells

Phagocytosis of bacteria is a central mechanism of myeloid cells for the control of bacterial infection. Therefore, we decided to analyze phagocytosis and killing of bacteria by IL-6Rα-deficient myeloid cells. BMDM from Il6rafl/fl×LysMcre and Il6rafl/fl control mice were infected with listeria. Extracellular listeria were killed with gentamicin and the numbers of intracellular listeria were determined at different time points of infection (Fig 5C). BMDM from both mouse strains showed similar listeria uptake. However, at later time points we could recover higher numbers of listeria from BMDM from Il6rafl/fl×LysMcre mice, indicating impaired control of listeria replication in these cells.

To test the phagocytosis in vivo, Il6rafl/fl×LysMcre and littermate control mice were infected i.v. with 2×104 listeria. Two days p.i., mice were injected i.v. with fluorescent latex beads. The mice were sacrificed 1 h later, liver and spleen cells were isolated, and uptake of latex beads by inflammatory monocytes and granulocytes was analyzed by flow cytometry (Fig 6). We could recently demonstrate that CD38-deficiency causes enhanced susceptibility of mice to L. monocytogenes infection, which was largely due to an impaired innate response [25]. Furthermore, it could be shown that abrogation of CD38 signaling can lead to reduced phagocytosis [32]. Therefore, we determined the uptake of beads in CD38+ and CD38- cells. In both mouse strains, CD38+ inflammatory monocytes were more efficient in the uptake of latex beads. Uptake of beads was significantly reduced in CD38+ and CD38- inflammatory monocytes from the liver of Il6rafl/fl×LysMcre mice when compared to corresponding cells from littermate controls. Compared to the liver, we detected less bead-positive cells in the spleen. CD38+ control cells were slightly more efficient in phagocytosis, however, differences did not reach significance in the spleen. Granulocytes were less efficient in phagocytosis of beads, with very little phagocytosis by CD38- granulocytes. For these cells, we detected lower levels of phagocytosis in the spleen.

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Fig 6. Phagocytic potential of inflammatory monocytes is diminished in mice with abrogated classical IL-6 signaling in myeloid cells.

Il6rafl/fl×LysMcre and Il6rafl/fl littermate controls were infected i.v. with 2×104 listeria. Two days p.i., mice were injected i.v. with fluorescently labelled latex beads. Liver and spleen cells were isolated 1 hour after injection. Uptake of latex beads by inflammatory monocytes and neutrophil granulocytes was evaluated by flow cytometry. (A) Representative dot plots (left to right: Il6rafl/fl CD38+, Il6rafl/fl CD38-, Il6rafl/fl×LysMcre CD38+, Il6rafl/fl×LysMcre CD38-) of inflammatory monocytes or neutrophil granulocytes from liver and spleen of infected mice. Numbers give the %-value of latex bead+ cells. (B) Frequencies of latex bead+ CD38+ and CD38- inflammatory monocytes from liver and spleen of infected mice. (C) Frequencies of latex bead+ CD38+ and CD38- neutrophil granulocytes from liver and spleen of infected mice. One representative experiment of three with 4 to 7 mice per group is shown. Mean ± SEM are shown. Significance was determined by 2way ANOVA.

https://doi.org/10.1371/journal.pone.0203395.g006

In conclusion, abrogation of classical IL-6 signaling had a direct effect on the phagocytic activity, particularly in CD38+ inflammatory monocytes. The low expression level of CD38 on inflammatory monocytes from infected Il6rafl/fl×LysMcre mice could further enhance the deficiency in phagocytosis.

Discussion

Infection of mice with L. monocytogenes causes strong induction of IL-6 which is required for effective bacteria control [6, 10, 26]. We could recently demonstrate that this control depends on classical IL-6 signaling but not on IL-6 trans-signaling [10]. In the current study, we aimed at identifying the target cells of IL-6 responsible for listeria control.

Deletion of IL-6Rα in CD4+ and CD8+ T cells did not alter the control of L. monocytogenes since we observed similar bacterial titers in organs of Il6rafl/fl×CD4cre and control mice after primary and secondary infections. Studies on the function of IL-6 in the formation of TH1 cells and CD8+ T cells yielded inconsistent results [9, 33–36]. It could be demonstrated that IL-6 represses TH1-cell responses by induction of TH2 cells and blockade of IL-12 and IFNγ signaling [37, 38]. However, IL-6-deficient mice fail to mount TH1-cell responses to bacterial and fungal infections [35, 39]. Furthermore, IL-6 can prevent Treg-cell mediated suppression of TH1 cells and thereby even supports TH1-cell responses [36, 40]. Absence of IL-6 enhances CD8+ T-cell responses against protozoan infection but has either no effect on or even restricts CD8+ T-cell responses to viral infections [9, 40, 41]. Finally, there is evidence that IL-6 supports the induction of cytotoxic function in CD8+ T cells [42, 43]. In our listeria infection model, Il6rafl/fl×CD4cre and control mice showed similar frequencies of CD4+ and CD8+ T cells responding to stimulation with immunodominant peptides. Although we consistently detect IL-6Rα on naive CD4+ and CD8+ T cells, surface IL-6Rα expression is rapidly lost from these cells upon in vitro stimulation or in vivo following L. monocytogenes infection [10, 44]. Thus, T cells might rely on IL-6 trans-signaling by soluble IL-6Rα/IL-6 complexes or IL-6 cluster-signaling by IL-6Rα+ antigen-presenting cells [18, 43, 45]. However, sgp130FcTg mice with impaired IL-6 trans-signaling and Il6KO mice with a complete lack of IL-6 signaling generated normal CD4+ TH1 and CD8+ T-cell responses to L. monocytogenes indicating that IL-6 in general was not required for the effective formation of listeria-specific T cells. The cause for the apparent lack of IL-6 activity in our model is unclear. L. monocytogenes induces a strong inflammatory response and a wide spectrum of pro-inflammatory cytokines which might compensate for the missing IL-6 signal in T cells. Interestingly, we observed even stronger CD4+ T-cell responses in Il6rafl/fl×LysMcre mice. Enhanced CD4+ T-cell frequencies could reflect a higher bacterial load during induction of the T-cell response. In our experience, particularly the magnitude of the CD4+ T-cell response correlates with the bacterial load (KL and H-WM, unpublished observation). However, we cannot exclude that IL-6Rα-deficient myeloid cells either provide stronger supportive signals or fail to deliver suppressive signals for the T-cell response.

Due to its intracellular replication, control of L. monocytogenes primarily relies on TH1 cells and cytotoxic CD8+ T cells. However, we also detected an increase in frequencies of TH17 cells in spleens of infected mice. Frequencies of IL-17A-secreting TH17 cells were reduced in Il6KO and Il6rafl/fl×CD4cre mice but not in sgp130FcTg mice indicating that in our model, development of TH17 cells was strictly dependent on classical IL-6 signaling which could not be compensated by IL-6 trans-signaling or IL-6 cluster-signaling.

In contrast to Il6rafl/fl×CD4cre mice, Il6rafl/fl×LysMcre mice showed impaired control of L. monocytogenes. Compared to the initial characterizations of Il6KO mice by Kopf et al. [6] and Dalrymple et al. [26], the increase in listeria titers in Il6rafl/fl×LysMcre mice was only modest. However, this result is consistent with our previous study in which Il6KO mice or mice treated with neutralizing anti-IL-6 mAb also show less pronounced increases in listeria titers than those observed in the original studies [10].

It is currently unclear why the deficient control of L. monocytogenes in Il6rafl/fl×LysMcre mice at early time points is restricted to the liver. Analysis of inflammatory cytokines and iNOS in livers of infected mice revealed only marginal differences between Il6rafl/fl×LysMcre and control mice. Neutrophils are recruited to spleen and liver in the early phase of listeria infection. Results on the requirement of neutrophils for the control of L. monocytogenes are conflicting but point to a role primarily in the liver [46, 47]. In our current study, infected Il6rafl/fl×LysMcre and control mice showed similar accumulation of neutrophils in spleen and liver, and cells only marginally differed in all phenotypically and functional analyses. Thus, there is so far no evidence for an impaired granulocyte response in Il6rafl/fl×LysMcre mice.

Early control of L. monocytogenes primarily depends on CCR2-mediated mobilization of inflammatory monocytes from the bone marrow and recruitment of these cells to spleen and liver [27, 28]. We observed normal accumulation of inflammatory monocytes in Il6rafl/fl×LysMcre mice, and migration of these cells towards CCL2 and fMLP was not inhibited. These results recapitulate our results from Il6KO mice, where we observed only minor alterations in inflammatory monocyte and granulocyte accumulation in listeria-infected tissues [10].

Following listeria infection, inflammatory monocytes migrate to sites of infection and mature to pro-inflammatory M1 macrophages. However, under specific conditions, such as the presence of type 2 cytokines, alternatively activated or M2 macrophages can develop. Alternatively activated macrophages participate in helminth infection and allergic inflammations but are also implicated in tissue repair and regeneration [48, 49]. Mauer and colleagues could show that IL-6 induces the expression of the IL-4Rα and thereby promotes development of alternatively activated macrophages [4]. In line with their results, we observed induction of IL-4Rα by IL-6 in BMDM, and inflammatory monocytes from infected Il6rafl/fl×LysMcre mice showed reduced IL-4Rα expression, indicating that they might be less sensitive to IL-4. In the liver, listeria rapidly infect and destroy Kupffer cells, which triggers the release of the alarmin IL-33 from hepatocytes. Interestingly, IL-33 induces the production of IL-4 in basophils and thereby promotes maturation of alternatively activated macrophages. After clearance of the infection, these macrophages replace the destroyed Kupffer cells and restore the integrity of the liver tissue [50]. Thus, IL-4 is available in the listeria-infected liver and can direct differentiation of IL-4Rα+ inflammatory monocytes. Phenotypic and functional characterization of inflammatory monocytes in infected Il6rafl/fl×LysMcre mice revealed a more activated phenotype and enhanced capability to produce TNFα, which would be consistent with an enhanced M1 polarization. However, surface expression of the M2 marker MMR1 was similar and the phagocytic activity was reduced when compared to inflammatory cells from infected control mice. Thus, alterations of IL-6Rα-deficient inflammatory monocytes can only partially be explained by a shift towards an M1 development.

CD38 has been described as an M1 macrophage marker [22] and we observed strong CD38 induction in BMDM cultured with IFNγ and LPS. However, CD38 was also induced by IL-4 and induction depended on IL-6 and IL-6Rα expression on BMDM. Interestingly, CD38 was significantly reduced on inflammatory monocytes from infected Il6rafl/fl×LysMcre mice suggesting that in our model, CD38 expression is mainly driven by IL-6. CD38 has enzymatic activity and can use nicotinamide adenine dinucleotide (NAD) and nicotinamide adenine dinucleotide phosphate (NADP) to generate adenosine diphosphoribose (ADPR), cyclic adenosine diphosphoribose (cADPR) or nicotinic acid adenine dinucleotide phosphate (NAADP). All three products act as second messengers and induce an increase in cytosolic Ca2+ [51, 52]. In neutrophils, monocytes and dendritic cells, CD38 cooperates with chemotactic receptors in the induction of Ca2+ signaling [53]. Cd38KO mice show an ameliorated course of disease in different autoimmune or immune pathology models as well as diminished pathogen control in infection models [52, 54–61], which in most models is associated with reduced accumulation and activation of granulocytes or macrophages. We could demonstrate that Cd38KO mice are highly susceptible to L. monocytogenes [25]. Infected Cd38KO mice present with reduced accumulation of neutrophils and inflammatory monocytes in the spleen, but enhanced recruitment of inflammatory monocytes to the liver [25]. Thus, impaired listeria control cannot simply be explained by altered recruitment of these cells. However, we observed diminished uptake of listeria by CD38-deficient macrophages, which is consistent with a recent report that CD38-deficient macrophages are impaired in phagocytosis [25, 32]. In our current study, CD38- cells were less efficient in phagocytosis of beads. In the liver of infected Il6rafl/fl×LysMcre mice, phagocytosis was significantly reduced in both CD38+ and CD38- cells. Thus, reduced phagocytosis together with diminished CD38 expression could be responsible for the impaired listeria control Il6rafl/fl×LysMcre mice. However, we can currently not exclude that induction of CD38 expression and control of phagocytosis are independent processes regulated by IL-6 in inflammatory monocytes.

Impaired control of L. monocytogenes infection might not be restricted to diminished bacteria uptake but also to defective restriction of intracellular replication, since we could recover higher numbers of listeria from IL-6Rα-deficient BMDM. Macrophages rely on the production of NO to kill intracellular listeria. However, NO production by BMDM was independent from IL-6 and IL-6Rα expression. Therefore, the cause of impaired listeria control in macrophages is currently unclear.

In conclusion, we demonstrate that IL-6-mediated protection against L. monocytogenes depends on classical IL-6 signaling in myeloid cells. Impaired IL-6 signaling is associated with alterations in the differentiation and function of these cells, which subsequently prevent effective control of bacteria.

Supporting information

S1 Fig. Gating strategies and IL-6Rα expression on cells from Il6rafl/fl×CD4cre and Il6rafl/fl×LysMcre mice.

Leukocytes were first defined by granularity (SSC-A) and size (FSC-A). Cell doublets were excluded. Dead cells were excluded by Pacific Orange staining (PacO). (A) Myeloid cells were defined as CD11b+. Granulocytes were identified as Ly6Cint Gr1hi. From cells that were not defined as granulocytes, inflammatory monocytes were characterized as Ly6Chi F4/80int. Ly6Clow monocytes were defined as Ly6Cint and F4/80low and macrophages were defined Ly6Clow and F4/80hi. (B) After the exclusion of dead cells, T cells were defined as CD8+ or CD4+. Dendritic cells were defined as CD11c+ and MHC IIhi. B cells were identified by CD19 expression. (C) CD4+ T cells, CD8+ T cells, B cells, dendritic cells, macrophages, granulocytes, inflammatory monocytes and Ly6Clow monocytes were stained for surface IL-6Rα to determine the efficacy of the cell-specific knockout. Concatamer plots combining indicated cells from different mouse lines are given. From left to right: isotype control, CD4cre, Il6rafl/fl×CD4cre, Il6rafl/fl and Il6rafl/fl×LysMcre. Numbers represent mean fluorescence intensity.

https://doi.org/10.1371/journal.pone.0203395.s001

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S2 Fig. IL-6 signaling does not influence specific T-cell responses against Listeria monocytogenes.

sgp130FcTg, Il6KO and WT mice were infected i.v. with 1×104 listeria recombinant for ovalbumin. Eight days p.i., spleen cells were isolated and stimulated with LLO189-201 and OVA257-264 (A, B), or ionomycin and PMA (C, D). Intracellular IFNγ and IL-17A expression was determined by flow cytometry. Frequencies of IFNγ+ CD4+ and IFNγ+ CD8+ cells from spleens of infected sgp130FcTg (open bars in A, C), Il6KO (open bars in B, D) and wildtype control mice (filled bars in A–D) without (none) or with in vitro stimulation are depicted. Each group contained 4 to 6 mice. Mean ± SEM are shown. Significance was determined by unpaired student’s t-test. One representative experiment of two is shown.

https://doi.org/10.1371/journal.pone.0203395.s002

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S3 Fig. Abrogation of classical IL-6 signaling in myeloid cells impairs listeria control and causes enhanced CD4+ T cell response.

(A) Il6rafl/fl×LysMcre and Il6rafl/fl control mice were infected i.v. with 2×103 listeria. Five days p.i., titers in liver and spleen were determined. Results for individually analyzed mice and median bars are shown. Significance was determined with the Mann-Whitney U test. Results from two independent experiments were pooled. (B) Il6rafl/fl×LysMcre and Il6rafl/fl control mice were infected i.v. with 1×104 listeria recombinant for ovalbumin. Eight days p.i., spleen cells were isolated and stimulated with LLO189-201 and OVA257-264. Intracellular IFNγ expression was determined by flow cytometry. Frequencies of IFNγ+CD4+ or IFNγ+CD8+ spleen cells following in vitro peptide stimulation are shown. Bars represent mean ± SEM of 5 to 6 mice per group. Significance was determined with student’s t-test.

https://doi.org/10.1371/journal.pone.0203395.s003

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S4 Fig. Induction of inflammatory cytokines in Il6rafl/fl×LysMcre mice infected with Listeria monocytogenes.

Il6rafl/fl×LysMcre and Il6rafl/fl littermate control mice were infected i.v. with 2×104 listeria and analyzed d2 p.i. Uninfected mice were included as additional controls. Protein lysates and RNA were isolated from spleen and liver. Cytokine expression in protein lysates of spleen (A, B) and liver (C, D). Expression of Il1b (E), Nos2 (F) and Il6 (G) mRNA was analyzed using qPCR and normalized to 18S RNA using the ΔCT method. Bars represent mean ± SEM and circles represent individually analyzed mice with dashes as medians. Significance was determined by 2way ANOVA.

https://doi.org/10.1371/journal.pone.0203395.s004

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S5 Fig. Phenotype and cytokine expression of granulocytes and Ly6Clow monocytes.

Il6rafl/fl×LysMcre and Il6rafl/fl littermate control mice were infected i.v. with 2×104 listeria. Two days p.i., liver and spleen cells from infected mice were isolated and granulocytes (A-D) and Ly6Clow monocytes (E-H) were characterized by flow cytometry (gating strategy is given in S1A Fig). Mean fluorescence intensity (MFI) of different surface proteins of granulocytes (A, B) and Ly6Clow monocytes (E, F). Spleen cells were stimulated with LPS for 4 h. Frequency of TNFα+ and IL-6+ granulocytes (C) and Ly6Clow monocytes (G). MFI of intracellular TNFα and IL-6 of granulocytes (D) and Ly6Clow monocytes (H). One representative experiment of two with 5 to 7 mice per group is shown. Bars represent mean ± SEM. Significance was determined with student’s t-test.

https://doi.org/10.1371/journal.pone.0203395.s005

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S1 Table. Antibodies used for this study.

Specificity (antigen), conjugated fluorochrome, supplier, clone and ID of each monoclonal antibody used for flow cytometric analyses.

https://doi.org/10.1371/journal.pone.0203395.s006

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