15 research outputs found
M2 Polarization of Human Macrophages Favors Survival of the Intracellular Pathogen <i>Chlamydia pneumoniae</i>
<div><p>Intracellular pathogens have developed various strategies to escape immunity to enable their survival in host cells, and many bacterial pathogens preferentially reside inside macrophages, using diverse mechanisms to penetrate their defenses and to exploit their high degree of metabolic diversity and plasticity. Here, we characterized the interactions of the intracellular pathogen <i>Chlamydia pneumoniae</i> with polarized human macrophages. Primary human monocytes were pre-differentiated with granulocyte macrophage colony-stimulating factor or macrophage colony-stimulating factor for 7 days to yield M1-like and M2-like macrophages, which were further treated with interferon-γ and lipopolysaccharide or with interleukin-4 for 48 h to obtain fully polarized M1 and M2 macrophages. M1 and M2 cells exhibited distinct morphology with round or spindle-shaped appearance for M1 and M2, respectively, distinct surface marker profiles, as well as different cytokine and chemokine secretion. Macrophage polarization did not influence uptake of <i>C</i>. <i>pneumoniae</i>, since comparable copy numbers of chlamydial DNA were detected in M1 and M2 at 6 h post infection, but an increase in chlamydial DNA over time indicating proliferation was only observed in M2. Accordingly, 72±5% of M2 <i>vs</i>. 48±7% of M1 stained positive for chlamydial lipopolysaccharide, with large perinuclear inclusions in M2 and less clearly bordered inclusions for M1. Viable <i>C</i>. <i>pneumoniae</i> was present in lysates from M2, but not from M1 macrophages. The ability of M1 to restrict chlamydial replication was not observed in M1-like macrophages, since chlamydial load showed an equal increase over time for M1-like and M2-like macrophages. Our findings support the importance of macrophage polarization for the control of intracellular infection, and show that M2 are the preferred survival niche for <i>C</i>. <i>pneumoniae</i>. M1 did not allow for chlamydial proliferation, but failed to completely eliminate chlamydial infection, giving further evidence for the ability of <i>C</i>. <i>pneumoniae</i> to evade cellular defense and to persist in human macrophages.</p></div
Macrophage Polarization.
<p><b>(A)</b> CD14 positive human blood monocytes were treated either with 25 ng/mL GM-CSF or 50 ng/mL M-CSF for 7 days to yield M1-like or M2-like macrophages. M1-like macrophages were activated with GM-CSF, LPS and 50 ng/mL IFN-γ for an additional 48 h to yield M1 macrophages, while M2-like macrophages were treated with M-CSF and IL-4 to yield M2. <b>(B)</b> To examine the impact of macrophage polarization on survival and proliferation of <i>C</i>. <i>pneumoniae</i>, M1 and M2 macrophages were infected and cultured with <i>C</i>. <i>pneumoniae</i> for 24 h. <b>(C)</b> M1-like macrophages were infected with <i>C</i>. <i>pneumoniae</i> and cultured for 48 h in the presence of GM-CSF and IFN-γ, while infected M2-like macrophages were cultured with M-CSF and IL-4. Uninfected macrophages served as control.</p
Morphology and surface marker expression of polarized macrophages.
<p>Monocytes were cultured and polarized as shown in Scheme 1A to generate M1-like, M2-like, M1, and M2 macrophages. Morphology was assessed by light microscopy (panel A, scale bar 100 μm) and by flow cytometry according to forward/side scatter characteristics (panel B). The expression of surface markers was determined by flow cytometry (panel C). Data are expressed as mean ± SD for 3 independent experiments. MFI, mean fluorescence intensity.</p
Cytokine release of M1 and M2 macrophages infected with <i>C</i>. <i>pneumoniae</i>.
<p>Monocytes were cultured and polarized as shown in Scheme 1B and fully polarized M1 and M2 macrophages (4x10<sup>5</sup>/mL each) were infected with <i>C</i>. <i>pneumoniae</i> (4x10<sup>4</sup> IFU) for 24 h. Cytokine concentrations are expressed as mean ± SD for 3 independent experiments.</p
Diminished reaction of <i>PI3Kδ−/−</i> CTLs to allogeneic mixed lymphocytes, but unaltered proliferation and Ca<sup>2+</sup>-response.
<p>A. WT and <i>PI3Kδ−/−</i> splenocytes were CFSE-labeled and cultured in the absence and presence of allogeneic (BALB/c), mitomycin C-treated splenocytes. At the indicated time points, cells were harvested and proliferation of responding CTLs was assessed by flow cytometry. Percentages of proliferating CFSE+CD8+ T cells with and without the stimulus of mixed lymphocytes are illustrated. Proliferating CD8+ T cells were discriminated from undivided T cells by the reduced levels of CFSE in daughter cells. B. WT splenocytes were CFSE-labeled and cultivated in analogy to A. Pharmacological inhibition of PI3Kδ was achieved by treatment with indicated concentrations of CAL-101 during the experimental procedure. DMSO-treatment served as negative control. C. Proliferation of WT and <i>PI3Kδ−/−</i> CTLs in response to aCD3ε treatment was assessed in a CFSE proliferation assay. D. Proliferation of WT and <i>PI3Kδ−/−</i> aCD3-activated T cells was assessed under standard T cell medium conditions (in the presence of IL-2) and after deprivation from IL-2 by performing an [<sup>3</sup>H]-thymidine incorporation assay over 48 hours (with IL-2: WT: 12097±491cpm; versus <i>PI3Kδ−/−</i>: 12413±501cpm; without IL-2: WT: 1392±381cpm; versus <i>PI3Kδ−/−</i>: 1140±160cpm, n = 6, values represent mean±SD). E., F. WT and <i>PI3Kδ−/−</i> splenocytes were stained with 1 µM Indo-1 AM. Ca<sup>2+</sup> flux in response to aCD3ε followed by crosslinking with streptavidin (E) or thapsigargin (F) was measured in CD8+ T cells using flow cytometry. Treatment with ionomycin served as positive control. Three independent experiments were carried out and one representative experiment is shown, respectively.</p
Reduced expression of cytotoxic components in <i>PI3Kδ−/−</i> CTLs.
<p>WT and <i>PI3Kδ−/−</i> splenocytes were activated for 3 days with aCD3ε and cultured in T cell medium. A. mRNA expression of <i>grzmA</i> (WT: 0.097±0.036; versus <i>PI3Kδ−/−</i>: 0.014±0.01, n = 6, <i>p = 0.0003</i>), g<i>rzmB</i> (WT: 1±0.076; versus <i>PI3Kδ−/−</i>: 0.419±0.075, n = 6, <i>p<0.0001</i>) and <i>prf1</i> (WT: 0.0433±0.004; versus <i>PI3Kδ−/−</i>: 0.013±0.003, n = 6, <i>p<0.0001</i>) was quantified by qRT-PCR and normalized to the house-keeping gene g<i>apdh</i>. B. Similarly, under standard culturing conditions mRNA levels of <i>trail</i> (WT: 0.0049±0.0012; versus <i>PI3Kδ−/−</i>: 0.0026±0.0007, n = 4, <i>p = 0.0178</i>) and <i>fasl</i> (WT: 0.0138±0.0015; versus <i>PI3Kδ−/−</i>: 0.0039±0.0001, n = 4, <i>p<0.0001</i>) were measured. C. <i>Ifng</i> mRNA was quantified by qRT-PCR under standard culturing conditions (WT: 0.019±0.0005; versus <i>PI3Kδ−/−</i>: 0.004±0.001, n = 6, <i>p<0.0001</i>) and after stimulation with 5 ng/ml IL-12 for 4 h (WT: 0.241±0.06; versus <i>PI3Kδ−/−</i>: 0.087±0.016, n = 6, <i>p = 0.0002</i>). D. To quantify IFN-γ protein levels, WT and <i>PI3Kδ−/−</i> splenocytes were stimulated with ConA. After 48 h supernatants were harvested and IFN-γ release was measured by ELISA (WT: 6515±2061 pg/ml; versus <i>PI3Kδ−/−</i>: 1359±1147 pg/ml, n = 3, <i>p = 0.0193</i>). IFN-γ release of unstimulated controls of WT and <i>PI3Kδ−/−</i> splenocytes was below detection limit of the assay (<10 pg/ml). Statistics were calculated with an unpaired Student’s <i>t</i>-test, and values represent mean±SD. One out of two independently performed experiments with comparable results is shown.</p
TCR/CD3- and CD59-mediated Ca<sup>2+</sup> signaling are dependent on Lck and LAT.
<p>Cluster distribution of Ca<sup>2+</sup> time traces in differently treated cells upon anti-CD3 and anti-CD59 stimulation. Each color represents the percentage of a certain Ca<sup>2+</sup> time trace cluster in the cell population. (A) Ca<sup>2+</sup> measurements were performed with WT cells transiently transfected with negative control siRNA (siNeg), Lck-specific siRNA (siLck), WT cells treated with 10 µM PP2 (PP2), and Lck-deficient J.CaM1.6 cells. Clusters representing Ca<sup>2+</sup> release patterns are framed in black (89.4±10.1%, 76.5±10.4%, 28.3±39.1%, and 23.4±9.9% upon anti-CD3 stimulation, 36.9±13.2%, 12.7±6.1%, 2.6±2.0%, and 1.2±1.2% upon anti-CD59 stimulation for siNeg, siLck, PP2-treated, and J.CaM1.6 cells, respectively). Mean values from at least two independent experiments, each with three technical replicates, are shown (n ≥ 204 per cell type and condition). (B) Cluster analysis of Ca<sup>2+</sup> time traces in WT cells transiently transfected with negative control siRNA (siNeg), LAT-specific siRNA (siLAT), and LAT-deficient J.CaM2.5 cells. Clusters representing Ca<sup>2+</sup> release patterns are framed in black (89.4±10.1%, 73.5±5.8%, and 3.0±2.3% upon anti-CD3 stimulation, 36.9±13.2%, 13.0±6.6%, and 1.7±1.6% upon anti-CD59 stimulation for siNeg, siLAT, and J.CaM2.5 cells, respectively). Mean values from at least three independent experiments, each with three technical replicates, are shown (n ≥ 208 per cell type and condition). Multiple comparison tests for the fractions showing Ca<sup>2+</sup> release patterns in (A) and (B) were assessed by one-way ANOVA, significances are shown where applicable, ** p < 0.01, *** p < 0.001. (C) Knock-down of target proteins was tested by Western blotting. 48 h after transfection cell lysates from siRNA treated cells were probed with anti-Lck, anti-LAT, and anti-β-actin. J.CaM1.6 cells, J.CaM2.5 cells, and WT cells served as controls.</p
PI3Kδ is indispensable for CTL degranulation.
<p>A. Using the whole cell patch clamp technique, the cellular capacitance of aCD3-activated and in T cell medium cultivated WT and <i>PI3Kδ−/−</i> CTLs was determined. Membrane capacitances before (ctr) and after stimulation with PMA and ionomycin (PMA/iono) at the single cell level are summarized from two representative experiments (WT: ctr: 6.3±1.2pF, PMA/iono: 9±1.8pF, n = 10, <i>p = 0.01</i>; versus <i>PI3Kδ−/−</i>: ctr: 6.7±1.7pF, PMA/iono: 6.6±1.8, n = 9, <i>p = 0.4</i>; paired <i>t</i>-test; values represent mean±SEM). B. Fold increase in membrane capacitance due to stimulation with PMA/iono is calculated (WT: 1.46±0.13, n = 10, <i>p = 0.0064</i>; versus <i>PI3Kδ−/−</i>: 0.94±0.06%, n = 9, <i>p = 0.3</i>). Additionally, WT CTLs were pre-incubated with the PI3Kδ isoform-specific inhibitor IC-87114 (1 µM, 4 µM, 1 hour), DMSO-treatment served as control (DMSO: 2.14±0.18, n = 11, <i>p<0.0001</i>; 1 µM IC-87114: 0.98±0.03, n = 11, <i>p = 0.49</i>; 4 µM IC-87114: 0.98±0.01, n = 9, <i>p = 0.18</i>; values representing mean±SEM, One sample <i>t</i>-test). C. <i>In vitro</i> cultivated WT and <i>PI3Kδ−/−</i> CTLs were challenged with PMA and ionomycin. The percentage of CD107a+ T cells was measured <i>via</i> flow cytometry before and after the stimulus (WT: ctr: 15.5±2.4%, PMA/iono: 41.2±1.3%, <i>p<0.0001</i>; versus <i>PI3Kδ−/−</i>: ctr: 24.6±2.7%, PMA/iono: 29.3±1.7%, <i>p = 0.085</i>; n = 8, paired <i>t</i>-test, values represent mean±SEM). D. Accordingly, the degranulation of WT CTLs upon pharmacological inhibition of PI3Kδ using CAL-101 was tested (ctr: 6±0.7%; +PMA/iono: DMSO: 23.3±2.4%, 0.1 µM: 18.1±2%, 0.5 µM: 16.6±2.1%, 1 µM: 13.6±1.3%, 5 µM: 11.7±1%, n≥6, values represent mean±SEM. One-Way ANOVA revealed p<0.0001, Tukey’s Post-Hoc test was significant <i>p<0.01</i> for 1 µM and highly significant <i>p<0.001</i> for 5 µM compared to DMSO control). E. WT and <i>PI3Kδ−/−</i> CTLs were cultivated in T cell medium and their expression of CD107a was measured <i>via</i> flow cytometry under basal conditions and after electrostimulation or electrostimulation plus the degranulation inhibitor Concanamycin A, respectively (WT: ctr: 11.5±1.2% CD107a+ cells, electrostimulation: 41±3.3% CD107a+ cells, <i>p = 0.0001</i>; versus <i>PI3Kδ−/−</i>: ctr: 11±1.3% CD107a+ cells, electrostimulation: 12±0.8% CD107a+ cells, <i>p = 0.54</i>, paired <i>t</i>-test, values represent mean±SEM). All experiments were performed at least two times independently and summarized in the depicted graphs.</p
Reconstitution of Lck by forced interaction of CD3ζ and Lck facilitates TCR/CD3- but not CD59-mediated Ca<sup>2+</sup> signaling.
<p>(A) Lck expression levels in WT cells, J.CaM1.6 cells and J.CaM1.6 cells expressing mEGFP-tagged Lck fused to CD3ζ (CD3ζ-Lck) were tested by Western blotting, the same blot was reprobed using anti-β-actin as a control. (B) Plasma membrane localization of CD3ζ-Lck-mEGFP in J.CaM1.6 cells was imaged by fluorescence microscopy. CD3ζ-Lck-mEGFP fluorescence and a bright field (BF) image of a transfected J.CaM1.6 cell are shown (scale bars  =  10 µm). (C) Cluster distribution of Ca<sup>2+</sup> time traces in Lck-deficient J.CaM1.6 cells and J.CaM1.6 cells stably expressing mEGFP-tagged Lck fused to CD3ζ (CD3ζ-Lck) is shown upon anti-CD3 or anti-CD59 stimulation. Each color represents the percentage of a certain Ca<sup>2+</sup> time trace cluster in the cell population. Clusters representing Ca<sup>2+</sup> release patterns are framed in black (35.7±4.9% and 71.8±2.3% upon anti-CD3 stimulation, 1.4±2.3% and 1.0±1.3% upon anti-CD59 stimulation for J.CaM1.6 and CD3ζ-Lck cells, respectively). Mean values from three independent experiments, each with three technical replicates, are shown (n ≥ 323 per cell type and condition). Multiple comparison tests for the fractions showing Ca<sup>2+</sup> release patterns were assessed by one-way ANOVA, significances are shown where applicable, ***p < 0.001.</p
CD59-mediated Ca<sup>2+</sup> signaling requires CD3ζ expression.
<p>(A) Cluster distribution of Ca<sup>2+</sup> time traces in WT and TCR<sup>high</sup> cells is shown upon anti-CD3 and anti-CD59 stimulation. Each color represents the percentage of a certain Ca<sup>2+</sup> time trace cluster in the cell population. Clusters representing Ca<sup>2+</sup> release patterns are framed in black (91.4±2.1% and 92.9±3.9% upon anti-CD3 stimulation, 34.4±16.3% and 72.0±16.6% upon anti-CD59 stimulation in WT and TCR<sup>high</sup> cells, respectively). Mean values from two independent experiments, each with three technical replicates are shown (n ≥ 167 per cell type and condition). (B) Cluster distribution of Ca<sup>2+</sup> time traces in WT, TCR<sup>-</sup>, and cells expressing CD8-ζ fusion protein is shown upon anti-CD3 and anti-CD59 stimulation. Each color represents the percentage of a certain Ca<sup>2+</sup> time trace cluster in the cell population. Clusters representing Ca<sup>2+</sup> release patterns are framed in black (90.1±1.4%, 1.9±0.7% and 7.4±2.3% upon anti-CD3 stimulation, 25.2±6.8%, 1.6±0.4% and 13.4±1.6% upon anti-CD59 stimulation for WT, TCR<sup>-</sup>, and CD8-ζ cells, respectively). Mean values from four independent experiments, each with three technical replicates, are shown (n ≥ 343 per cell type and condition). Multiple comparison tests for the fractions showing Ca<sup>2+</sup> release patterns in (A) and (B) were assessed by one-way ANOVA, significances are shown where applicable, * p < 0.05, ** p < 0.01, *** p < 0.001.</p