TLR-dependency of Poke weed mitogen (PWM)-induced cell stimulation.

<p><b>A: B cell proliferation.</b> Human CD19+ peripheral blood B cells were stained with CFSE and stimulated with 1 or 10 µg/ml PWM in the presence or absence of 5 µg/ml <i>Staphylococcus aureus</i> protein A (SpA) or 5 µg/ml anti-human Ig F(ab′)₂ fragments (aIg) as B cell receptor stimuli, with SpA or aIg alone, or left unstimulated. After 5 days B cells were harvested, stained with anti-CD20 and live gated cells were analyzed for CFSE dilution. The percentages of live gated cells (upper right corner) and proliferating cells (left) are indicated in each diagram. The diagrams depict the results from one representative experiment of n = 3. <b>B: TNF-induction.</b> Human PBMC were stimulated for four hours with or without PWM, TLR2 ligands FSL-1R (FSL) and Pam₃CSK₄ (P3), phosphorothioate-modified DNA ODN CpG 2006 (CpG) and 2006 GC (GC) and LPS in the presence of Brefeldin A (BfA) or with LPS in the absence of BfA. Subsequently, intracellular staining was performed with an anti-human TNF mAb and TNF expression was analyzed by flow cytometry. The results show a summary of the data obtained in n = 4 experiments. Mean values of anti-TNF mean fluorescence intensities are provided ± SEM. <b>C: Antagonization with Polymyxin B.</b> CFSE-stained human CD19+ B cells were stimulated with PWM in the presence and absence of polymyxin B. Proliferation was assessed by flow cytometric analysis of CFSE dilution on day 5. The results obtained in two representative donors of n = 3 are shown. <b>D: MyD88-dependency of B cell stimulation.</b> B220+ B cells were isolated from the spleens of MyD88^−/− mice and their wild type counterparts. B cells were stimulated with CpG, P3, PWM and PMA/Ionomycin (P/I) and harvested after 72 hours and a 18 hour pulse with ³H-thymidine. The diagram shows the average values in counts per minute (cpm) of n = 4 experiments ± SEM. <b>E: TLR-dependency.</b> Wild type, TLR2−/− and TLR9−/− B cells were isolated from murine spleen with anti-CD19 microbeads, labelled with CFSE and stimulated with TLR2 ligands Pam₃CSK₄ (P3) and FSL-1R (FSL), PWM (10 µg/ml), TLR9 ligand CpG ODN 1668 or 1668 GC control ODN. After 4 days B cell proliferation (CFSE dilution) was quantified by flow cytometry. The diagram depicts the mean fluorescence intensity (MFI) for CFSE in live gated cells as mean value ± SEM from n = 4 experiments. Note that low MFI corresponds to strong proliferation while high MFI values reveal absence of proliferation.</p

Synergistic effects of TLR ligands with poke weed lectin.

<p><b>A: Effect of TLR2 blockage on human B cell proliferation in response to PWM.</b> CD19+ human peripheral blood B cells were labelled with CFSE, preincubated with anti-TLR2 mAb or the corresponding isotype control (murine IgG_1κ), and stimulated with Pam₃CSK₄ or PWM (10 µg/ml) ± SpA (5 µg/ml) for 5 days. Proliferation was assessed by CFSE dilution. The gate denotes the percentage of live gated proliferating B cells as depicted. One representative experiment of n = 3 is shown. <b>B: Synergistic effects of PWM and immunostimulatory DNA.</b> Human B cells were stained with CFSE for assessment of B cell proliferation. Stimulation was performed with CpG ODN 2006 (PTO), DNAse- or mock (reaction buffer only)-treated PWM, 2006 GC (PTO) or CpG 2006 (PO) and combinations thereof as indicated. The graphs show the results from one representative experiment from n≥3. The percentage of proliferating B cells is indicated in the graphs.</p

Detection of TLR ligands in PWM preparations.

<p><b>A:</b> Human B cells were stimulated with different lots of PWM. Proliferation rates were quantified by 3H-thymidine incorporation (cpm = counts per minute). The diagram shows the mean values ± SEM from n = 3 experiments. <b>B:</b> LAL-assay was performed to quantify the LPS content in PWM Lot. A–D. The diagram depicts the mean values ± standard deviation obtained by testing in quadruplicates. <b>C:</b> TLR2 activity was assessed by measuring IL-8 concentrations in the supernatants of pTLR2-transfected or non-transfected HEK293 cells stimulated with different lots of PWM (left) or Pam₃CSK₄ (right) at the concentrations indicated. One representative experiment performed in triplicates of n = 2 is shown.</p

Contribution of the lectin component.

<p><b>A:</b> Human B cell proliferation in response to PWM (Lot. B and C) in the presence or absence of N,N-di-acetylchitobiose or N,N,N-tri-acetylchitotriose was assessed by ³H-thymidine incorporation given in counts per minute (cpm). The mean values ± SEM from one representative experiment performed in triplicates of n = 2 is shown. <b>B:</b> Human CD19+ B cells were left unstimulated or stimulated with 0.25 µM GpC PTO ODN (GC) and/or 10 µg/ml of PWM, 5 µg/ml SpA or 10 µg/ml anti-Ig (aIg) for 72 hours. Proliferation was quantified by ³H-thymidine incorporation. The diagram shows the means from n = 4 experiments ± SEM. The values obtained were normalized to GC = 1 (710±280 cpm = mean ± SEM). <b>C:</b> Human CD19+ B cells were stained with CFSE and stimulated with highly purified PWM (Lot. D) or <i>Lycopersicon esculentum</i> lectin (LEA) with or without GpC PTO ODN (GC). Proliferation was quantified by CFSE dilution on day 4. The percentage of proliferating cells is provided in each dot plot. The results from two independent donors of n = 4 are shown.</p

Contribution of TLR ligands to B cell activation.

<p><b>A: Stimulation of human B cells with TLR ligands.</b> CFSE-labelled CD19+ human B cells were stimulated with or without TLR ligands (TLR2 ligand MALP-2, TLR4 ligand LPS (0.1 and 1 µg/ml) or TLR9 ligand CpG ODN 2006 (CpG)) and/or BCR stimuli (5 µg/ml SpA or anti-Ig (aIg)). After 5 days cells were harvested, stained with anti-CD20 and analyzed by flow cytometry. The graphs depict cell survival (percentage of live gated cells; upper right angle) and cell proliferation (CFSE dilution; % proliferating cells of live gated cells (left) where indicated). The experiment shown is representative of n≥3 experiments. <b>B–D: Assessment of TLR2 activity in PWM preparations.</b> HEK293 cells were transfected with pTLR2 or lipofectamine alone (Lf). <b>B:</b> non-transfected and pTLR2-transfected HEK293 cells were stained for TLR2 expression with anti-TLR2 mAb or the respective isotype control as indicated. <b>C:</b> HEK293 cells transfected with pTLR2 or Lf only were stimulated with Pam₃CSK₄ (P3) or PWM (10 µg/ml). After 24 hours cellular supernatants were collected and analyzed for IL-8 secretion. One representative experiment of n≥3 experiments is shown. <b>D:</b> HEK293 cells transfected with pTLR2 were pretreated with anti-TLR2 mAb or the isotype control before stimulation with Pam₃CSK₄ (P3) or PWM (10 µg/ml). IL-8 was quantified in the 24 hour supernatants. <b>E: 16S rDNA PCR.</b> DNA isolation and PCR amplification of bacterial 16S ribosomal DNA from DNA from <i>E. coli</i> (EC), a negative clinical specimen (NC), a positive clinical sample (CS) and the PWM preparation (PW) or the water control (H₂O), with an expected PCR fragment size of approximately 900 bp.</p

Data_Sheet_1_The Interplay of Notch Signaling and STAT3 in TLR-Activated Human Primary Monocytes.pdf

<p>The highly conserved Notch signaling pathway essentially participates in immunity through regulation of developmental processes and immune cell activity. In the adaptive immune system, the impact of the Notch cascade in T and B differentiation is well studied. In contrast, the function, and regulation of Notch signaling in the myeloid lineage during infection is poorly understood. Here we show that TLR signaling, triggered through LPS stimulation or in vitro infection with various Gram-negative and -positive bacteria, stimulates Notch receptor ligand Delta-like 1 (DLL1) expression and Notch signaling in human blood-derived monocytes. TLR activation induces DLL1 indirectly, through stimulated cytokine expression and autocrine cytokine receptor-mediated signal transducer and activator of transcription 3 (STAT3). Furthermore, we reveal a positive feedback loop between Notch signaling and Janus kinase (JAK)/STAT3 pathway during in vitro infection that involves Notch-boosted IL-6. Inhibition of Notch signaling by γ-secretase inhibitor DAPT impairs TLR4-stimulated accumulation of NF-κB subunits p65 in the nucleus and subsequently reduces LPS- and infection-mediated IL-6 production. The reduced IL-6 release correlates with a diminished STAT3 phosphorylation and reduced expression of STAT3-dependent target gene programmed death-ligand 1 (PD-L1). Corroborating recombinant soluble DLL1 and Notch activator oxaliplatin stimulate STAT3 phosphorylation and expression of immune-suppressive PD-L1. Therefore we propose a bidirectional interaction between Notch signaling and STAT3 that stabilizes activation of the transcription factor and supports STAT3-dependent remodeling of myeloid cells toward an immuno-suppressive phenotype. In summary, the study provides new insights into the complex network of Notch regulation in myeloid cells during in vitro infection. Moreover, it points to a participation of Notch in stabilizing TLR-mediated STAT3 activation and STAT3-mediated modulation of myeloid functional phenotype through induction of immune-suppressive PD-L1.</p

table_1_Hsa-miR-99b/let-7e/miR-125a Cluster Regulates Pathogen Recognition Receptor-Stimulated Suppressive Antigen-Presenting Cells.PDF

<p>Antigen-presenting cells (APCs) regulate the balance of our immune response toward microbes. Whereas immunogenic APCs boost inflammation and activate lymphocytes, the highly plastic cells can switch into a tolerogenic/suppressive phenotype that dampens and resolves the response. Thereby the initially mediated inflammation seems to prime the switch of APCs while the strength of activation determines the grade of the suppressive phenotype. Recently, we showed that pathogen recognition receptor-mediated pro-inflammatory cytokines reprogram differentiating human blood monocytes in vitro toward an immunosuppressive phenotype through prolonged activation of signal transducer and activator of transcription (STAT) 3. The TLR7/8 ligand R848 (Resiquimod) triggers the high release of cytokines from GM-CSF/IL-4-treated monocytes. These cytokines subsequently upregulate T cell suppressive factors, such as programmed death-ligand 1 (PD-L1) and indolamin-2,3-dioxygenase (IDO) through cytokine receptor-mediated STAT3 activation. Here, we reveal an essential role for the microRNA (miR, miRNA) hsa-miR-99b/let-7e/miR-125a cluster in stabilizing the suppressive phenotype of R848-stimulated APCs on different levels. On the one hand, the miR cluster boosts R848-stimulated cytokine production through regulation of MAPkinase inhibitor Tribbles pseudokinase 2, thereby enhancing cytokine-stimulated activation of STAT3. One the other hand, the STAT3 inhibitor suppressor of cytokine signaling-1 is targeted by the miR cluster, stabilizing the STAT3-induced expression of immunosuppressive factors PD-L1 and IDO. Finally, hsa-miR-99b/let-7e/miR-125a cluster regulates generation of the suppressive tryptophan (Trp) metabolite kynurenine by targeting the tryptophanyl-tRNA synthetase WARS, the direct competitor of IDO in terms of availability of Trp. In summary, our results reveal the hsa-miR-99b/let-7e/miR-125a cluster as an important player in the concerted combination of mechanisms that stabilizes STAT3 activity and thus regulate R848-stimulated suppressive APCs.</p

data_sheet_1_Hsa-miR-99b/let-7e/miR-125a Cluster Regulates Pathogen Recognition Receptor-Stimulated Suppressive Antigen-Presenting Cells.PDF

<p>Antigen-presenting cells (APCs) regulate the balance of our immune response toward microbes. Whereas immunogenic APCs boost inflammation and activate lymphocytes, the highly plastic cells can switch into a tolerogenic/suppressive phenotype that dampens and resolves the response. Thereby the initially mediated inflammation seems to prime the switch of APCs while the strength of activation determines the grade of the suppressive phenotype. Recently, we showed that pathogen recognition receptor-mediated pro-inflammatory cytokines reprogram differentiating human blood monocytes in vitro toward an immunosuppressive phenotype through prolonged activation of signal transducer and activator of transcription (STAT) 3. The TLR7/8 ligand R848 (Resiquimod) triggers the high release of cytokines from GM-CSF/IL-4-treated monocytes. These cytokines subsequently upregulate T cell suppressive factors, such as programmed death-ligand 1 (PD-L1) and indolamin-2,3-dioxygenase (IDO) through cytokine receptor-mediated STAT3 activation. Here, we reveal an essential role for the microRNA (miR, miRNA) hsa-miR-99b/let-7e/miR-125a cluster in stabilizing the suppressive phenotype of R848-stimulated APCs on different levels. On the one hand, the miR cluster boosts R848-stimulated cytokine production through regulation of MAPkinase inhibitor Tribbles pseudokinase 2, thereby enhancing cytokine-stimulated activation of STAT3. One the other hand, the STAT3 inhibitor suppressor of cytokine signaling-1 is targeted by the miR cluster, stabilizing the STAT3-induced expression of immunosuppressive factors PD-L1 and IDO. Finally, hsa-miR-99b/let-7e/miR-125a cluster regulates generation of the suppressive tryptophan (Trp) metabolite kynurenine by targeting the tryptophanyl-tRNA synthetase WARS, the direct competitor of IDO in terms of availability of Trp. In summary, our results reveal the hsa-miR-99b/let-7e/miR-125a cluster as an important player in the concerted combination of mechanisms that stabilizes STAT3 activity and thus regulate R848-stimulated suppressive APCs.</p

SCV formation can be associated with reduced TLR2-activity.

<p><b>A,C and D:</b> HEK293 cells were transfected with or without TLR2 cDNA (100 ng/well in <b>A+D</b> or 200 ng/well in <b>C</b>) and stimulated with <i>S. aureus</i> strains for 18–20 hours. IL-8 secretion was quantified as indicator of TLR2 activation. All diagrams show the results obtained in three independent experiments given as mean values ± SD (standard deviation). <b>A:</b> Stimulation of HEK293 cells w/o pTLR2 with <i>S. aureus</i> SH1000 and its isogenic thymidine-auxotrophic mutant SCV SH1000 <i>ΔthyA</i>; *p(SH1000:<i>ΔthyA) = </i>0.049. <b>B:</b> Comparison of SpA expression in SH1000 (TLR2^high) and its isogenic SCV SH1000 <i>ΔthyA</i> (TLR2^low). Left: Western blot analysis of SpA expression in bacterial lysates using anti-SpA mAb and anti-murine IgG-HRP as secondary antibody. Right: Control blot incubated with human serum and biotinylated anti-human IgG+streptavidin-HRP. One representative experiment of n = 3 experiment is shown. <b>C:</b> HEK293 cells w/o TLR2 were stimulated with SA113 or its mutants <i>ΔhemB</i> and Δ<i>sdh</i> (grown on Mueller Hinton (MH) or Columbia agar (CA)) or with 1 µg/ml of the TLR2 ligand Pam₃CSK₄ (P3) or left unstimulated (<b>−</b>). <b>D:</b> Stimulation with five different clinical <i>S. aureus</i> SCV (isolate source: CF = cystic fibrosis, INV = invasive) and SA113. (<b>−</b>) = unstimulated.</p

The TLR2-stimulatory capacity varies among <i>S. aureus</i> isolates.

<p>HEK293 cells transfected with or without TLR2 plasmid (100 ng/well) were stimulated with either cystic fibrosis (CF) or invasive (INV) <i>S. aureus</i> isolates. IL-8 production was quantified 24 hours after stimulation. <b>A:</b> TLR2-activity was tested in 53 CF (left) and 53 INV (right) isolates. The dots provide the values obtained for single strains, the bars indicate the means. No significant difference was observed when comparing CF to INV isolates. <b>B:</b> The diagram shows one representative experiment with 16 CF (left, grey) and 16 INV (right, black) isolates. The origins of the isolates with absent to low TLR2-activity (arrows) are indicated in the graph. (<b>−</b>) refers to unstimulated cells.</p