Mechanism of accumulating CD4+ dendritic cells in the bone marrow and their impact on the de novo differentiation of dendritic cells during polymicrobial sepsis

Als dritthäufigste Todesursache in Deutschland handelt es sich bei der Sepsis um eine lebensbedrohliche Immunkomplikation. Neben schweren Organschäden kommt es zur Entwicklung einer langanhalten Immunsuppression, die das Risiko für nosokomiale Sekundärinfektionen erhöht. Dendritische Zellen (DC) spielen eine wesentliche Rolle in der Immunabwehr bei Infektionen. Sepsis führt zur Differenzierung von dysfunktionalen DC, denen die Fähigkeit fehlt eine effektive TH1-Antwort von T-Zellen auszulösen. Dies wird vermittelt durch die verstärkte Produktion des anti-inflammatorischen Zytokins IL-10, bei meist verminderter IL-12-Produktion. Vorarbeiten wiesen darauf hin, dass bisher nicht charakterisierte CD11c+ Zellen im Knochenmark die DC-Differenzierung beeinflussen können. In dieser Arbeit wurden mit Hilfe des Mausmodells der „Zökalen Ligatur und Punktion“ (CLP) zelluläre Veränderungen des Knochenmarks in der akuten Phase der Sepsis, die zur Entstehung der DC-Dysfunktion führen könnten, untersucht. Bereits 24 Stunden nach CLP akkumulierten aus Monozyten differenzierte Ly6ChiDC (MoDC), CD4+T-Zellen, sowie CD4+DC im Knochenmark. Die, im Verlauf (36 Stunden nach CLP) weiter vermehrte Anzahl CD4+DC assoziierte mit einer Verschiebung der Zytokinbalance der aus dem Knochenmark generierten DC (BMDC) hin zum anti-inflammatorischen IL-10. Die vorhergehende Depletion CD4+DC aus dem Knochenmark hob diese Verschiebung auf. Phänotypisch ähneln die CD4+DC teilweise konventionellen DC aus der Milz aber auch inflammatorischen MoDC. In der frühen Phase der Sepsis (sechs bis zwölf Stunden nach CLP) wurde eine transiente S1P-abhängige Akkumulation CD4+DC in den mesenterialen Lymphknoten, der Milz und im Blut beobachtet. Die Akkumulation CD4+DC im Knochenmark konnte zudem durch systemische Blockade des S1P, sowie MyD88- und teilweise TLR2-abhängig verhindert werden. Diese systemische S1P-Blockade führte jedoch postseptisch zu einer erhöhten Anfälligkeit für die Sekundärinfektion mit P. aeruginosa. Während der akuten Phase der Sepsis ist die Bildung IL-10-produzierender BMDC mit der Akkumulation CD4+DC im Knochenmark assoziiert. Diese wandern vermutlich S1P-abhängig, möglicherweise unter Beteiligung von CD4+T-Zellen und Ly6ChiMoDC, über sekundäre lymphoide Organe und das Blut in das Knochenmark. Weitere Untersuchungen zur Herkunft der CD4+DC und ihrer Wirkung auf die DC-Differenzierung könnten zu Therapieansätzen für die Vermeidung der postseptischen Immunsuppression führen.Sepsis is a life threatening disease that leads to a sustained susceptibility to nosocomial infections due to the development of immunosuppression. The induction of an effective TH1-response by T-cells is impaired by the differentiation of dysfunctional dendritic cells (DC) that predominantly produce the anti-inflammatory cytokine IL-10 and reduced pro-inflammatory IL-12. Recently, we could show, that jet undefined CD11c+ cells inside the bone marrow contribute to the induction of dysfunctional DC. Changes in the cellular composition of the bone marrow, which potentially impact the DC differentiation in vivo, were studied during the acute phase of sepsis (3 to 36 hours) by use of the murine sepsis model cecal ligation and puncture (CLP). The increase of Ly6Chi monocyte derived DC (MoDC) and CD4+T-cells in the bone marrow corresponded to the consecutive accumulation of CD4+DC 24 to 36 hours after CLP. Furthermore, this was associated with the differentiation of predominantly IL-10 producing bone marrow derived DC (BMDC). Previous depletion of CD4+DC inhibited this cytokine shift. In part CD4+DC in the bone marrow shared phenotypic similarities with splenic conventional DC (cDC) as well as inflammatory MoDC. An early (3 to 12 hours after CLP), transient S1P dependent accumulation of CD4+DC was observed in the mesenteric lymph nodes, the spleen and the blood. The appearance of CD4+DC in the bone marrow was abrogated by blockade of S1P receptors and in part dependent on MyD88 and TLR2. Indeed, this systemic blockade of S1P signaling enhanced the susceptibility for an infection with P. aeruginosa. During the acute phase of sepsis the differentiation of predominantly IL-10 producing BMDC is associated with the appearance of CD4+DC in the bone marrow. The accumulation of CD4+DC probably results in the S1P dependent migration trough the blood out of secondary lymphoid organs and could be promoted by CD4+T-cells and Ly6ChiMoDC in the bone marrow. Further studies should focus on the origin of CD4+DC and their impact on DC-Differentiation to investigate protective therapies to avoid post septic immunosuppression

Methylation specific targeting of a chromatin remodeling complex from sponges to humans

DNA cytosine methylation and methyl-cytosine binding domain (MBD) containing proteins are found throughout all vertebrate species studied to date. However, both the presence of DNA methylation and pattern of methylation varies among invertebrate species. Invertebrates generally have only a single MBD protein, MBD2/3, that does not always contain appropriate residues for selectively binding methylated DNA. Therefore, we sought to determine whether sponges, one of the most ancient extant metazoan lineages, possess an MBD2/3 capable of recognizing methylated DNA and recruiting the associated nucleosome remodeling and deacetylase (NuRD) complex. We find that Ephydatia muelleri has genes for each of the NuRD core components including an EmMBD2/3 that selectively binds methylated DNA. NMR analyses reveal a remarkably conserved binding mode, showing almost identical chemical shift changes between binding to methylated and unmethylated CpG dinucleotides. In addition, we find that EmMBD2/3 and EmGATAD2A/B proteins form a coiled-coil interaction known to be critical for the formation of NuRD. Finally, we show that knockdown of EmMBD2/3 expression disrupts normal cellular architecture and development of E. muelleri. These data support a model in which the MBD2/3 methylation-dependent functional role emerged with the earliest multicellular organisms and has been maintained to varying degrees across animal evolution

CD56^bright NK cells are suppressed in <i>Staphylococcus aureus</i>-induced IFN-γ synthesis after invasive surgery.

<p><b>(A-D)</b> PBMC were isolated from “patients 1” 24 h before (-1) and 1 d (+1), 5 d (+5), and 7 d (+7) after surgery and were stained against CD3 and CD56. <b>(A)</b> Gating strategy of CD3^-CD56^{<b>bright</b>} NK cells among total PBMC. The number indicates the percentage of gated cells in the rectangle. <b>(B)</b> Cumulative data on the percentage of gated CD3^-CD56^{<b>bright</b>} NK cells among total PBMC. <b>(C, D)</b> PBMC were cultured in the presence of <i>S</i>. <i>aureus</i> and cells were stained for intracellular IFN-γ. <b>(C)</b> Representative dot plots of intracellular IFN-γ expression in gated CD3^-CD5^{<b>bright</b>} NK cells isolated before and 1 d after injury. Numbers indicate the percentage of IFN-γ-positive (IFN-γ^<b>+</b>) cells. <b>(D)</b> Cumulative data on the kinetics of IFN-γ^<b>+</b> CD3^-CD56^{<b>bright</b>} NK cells upon exposure to <i>S</i>. <i>aureus</i> before and after injuy. <b>(E)</b> CD56^{<b>bright</b>} NK cells were purified from “patients 2” (n = 11) 1 d before and on day +1 after injury and stimulated with IL-12 and IL-18. The release of IFN-γ into the supernatant was quantified. Results are expressed as scatter plots (median, interquartile range). Statistical differences were tested using the Friedman test (B, D) or the Wilcoxon signed rank test (E). *, p<0.05; **, p<0.01; ***, p<0.001 vs. d-1. iso, isotype control antibodies.</p

Visceral surgery interferes with the responsiveness to <i>Staphylococcus aureus</i>.

<p>Peripheral blood from “patients 1” was drawn 1 d before (-1) and 1 d (+1), 5 d (+5), and 7 d (+7) after surgery. PBMC were isolated and stimulated with inactivated <i>S</i>. <i>aureus</i>. The amount of IFN-γ in the supernatant was quantified. Results are expressed as scatter plot (median, interquartile range). Statistical differences were tested using the Wilcoxon signed rank test. n = 20; *, p<0.05 vs. day -1.</p

Patients’ characteristics.

<p>Quantitative variables are expressed as median (25^th-75^th interquartile range), qualitative variables are provided as number/% of total. Significant differences between both groups were tested using Mann-Whitney and Fisher’s exact test, respectively.</p><p>Patients’ characteristics.</p

CD56^bright NK cells express reduced levels of the IL-12Rβ1 chain but increased levels of phosphorylated STAT4 after injury.

<p>PBMC were isolated from “patients 2” 24 h before (d-1) and 1 d (d+1) after injury. PBMC were stained against CD3, CD56, and IL-12Rβ1. CD3^-CD56^{<b>bright</b>} NK cells were gated as shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0130155#pone.0130155.g002" target="_blank">Fig 2A</a>. <b>(A)</b> Representative dot plot of IL-12Rβ1 expression on gated CD56^{<b>bright</b>} NK cells. Numbers indicate the percentage of IL-12Rβ1^<b>+</b> cells. <b>(B)</b> Cumulative data on the percentage of IL-12Rβ1^<b>+</b> cells among CD56^{<b>bright</b>} NK cells before and after injury (n = 20). <b>(C, D)</b> PBMC isolated before (d-1) and 1 d (d+1) after injury were cultured in the absence (med) or presence of <i>S</i>. <i>aureus</i>. Cells were stained against CD3, CD56, and intracellular phosphorylated STAT4 (pSTAT4). <b>(C)</b> Representative histogram of pSTAT4 staining in CD56^{<b>bright</b>} NK cells analyzed before injury. <b>(D)</b> Cumulative data on the percentage of pSTAT4-expressing (pSTAT4^<b>+</b>) cells in gated CD3^-CD56^{<b>bright</b>} NK cells (n = 6). Results are expressed as scatter plot (median, interquartile range) or as box plot (median, interquartile range, range). Statistical differences were tested using the Wilcoxon signed rank test (B) or the Friedman test (D). **, p<0.01; ##, p<0.01. iso, isotype control antibodies.</p

Invasive Surgery Impairs the Regulatory Function of Human CD56^bright Natural Killer Cells in Response to <i>Staphylococcus aureus</i>. Suppression of Interferon-γ Synthesis

<div><p>Major surgery increases the risk for infectious complications due to the development of immunosuppression. CD56^bright NK cells play a key role in the defense against bacterial infections through the release of Interferon (IFN) γ upon stimulation with monocyte-derived Interleukin (IL) 12. We investigated whether invasive visceral surgery interferes with the IFN-γ synthesis of human NK cells in response to <i>Staphylococcus aureus</i>. In a prospective pilot study, peripheral blood mononuclear cells (PBMC) were isolated from 53 patients before and 1 to 7 d after elective visceral surgery. The release of IL-12 and IFN-γ from PBMC upon exposure to <i>S</i>. <i>aureus in vitro</i> was quantified. The expression of the IL-12 receptor β1 chain on the surface, the phosphorylation of signal transducer and activator of transcription (STAT) 4, and the synthesis of IFN-γ on/in individual CD56^bright NK cells were investigated using flow cytometry. The modulatory effect of IL-12 on the <i>S</i>. <i>aureus</i>-induced IFN-γ production in CD56^bright NK cells was analyzed. The IFN-γ secretion from purified CD56^bright NK cells was quantified after stimulation with IL-12 and IL-18. After surgery, CD56^bright NK cells among total PBMC were impaired in the release of IFN-γ for at least 5 d. Likewise, the IL-12-induced release of IFN-γ from purified CD56^bright NK cells was abolished. Upon stimulation with <i>S</i>. <i>aureus</i>, PBMC secreted less IL-12 but supplementation with recombinant IL-12 did not restore the capacity of CD56^bright NK cells to produce IFN-γ. CD56^bright NK cells displayed reduced levels of the IL-12Rβ1 chain whereas the phosphorylation of STAT4, the key transcription factor for the <i>Ifng</i> gene was not diminished. In summary, after invasive visceral surgery, CD56^bright NK cells are impaired in <i>S</i>. <i>aureus</i>-induced IFN-γ production and might contribute to the enhanced susceptibility to opportunistic infections.</p></div

Sphingosine 1-Phosphate- and C-C Chemokine Receptor 2-Dependent Activation of CD4+ Plasmacytoid Dendritic Cells in the Bone Marrow Contributes to Signs of Sepsis-Induced Immunosuppression

Sepsis is the dysregulated response of the host to systemic, mostly bacterial infection, and is associated with an enhanced susceptibility to life-threatening opportunistic infections. During polymicrobial sepsis, dendritic cells (DCs) secrete enhanced levels of interleukin (IL) 10 due to an altered differentiation in the bone marrow and contribute to the development of immunosuppression. We investigated the origin of the altered DC differentiation using murine cecal ligation and puncture (CLP), a model for human polymicrobial sepsis. Bone marrow cells (BMC) were isolated after sham or CLP operation, the cellular composition was analyzed, and bone marrow-derived DCs (BMDCs) were generated in vitro. From 24 h on after CLP, BMC gave rise to BMDC that released enhanced levels of IL-10. In parallel, a population of CD11chiMHCII+CD4+ DCs expanded in the bone marrow in a MyD88-dependent manner. Prior depletion of the CD11chiMHCII+CD4+ DCs from BMC in vitro reversed the increased IL-10 secretion of subsequently differentiating BMDC. The expansion of the CD11chiMHCII+CD4+ DC population in the bone marrow after CLP required the function of sphingosine 1-phosphate receptors and C-C chemokine receptor (CCR) 2, the receptor for C-C chemokine ligand (CCL) 2, but was not associated with monocyte mobilization. CD11chiMHCII+CD4+ DCs were identified as plasmacytoid DCs (pDCs) that had acquired an activated phenotype according to their increased expression of MHC class II and CD86. A redistribution of CD4+ pDCs from MHC class II− to MHC class II+ cells concomitant with enhanced expression of CD11c finally led to the rise in the number of CD11chiMHCII+CD4+ DCs. Enhanced levels of CCL2 were found in the bone marrow of septic mice and the inhibition of CCR2 dampened the expression of CD86 on CD4+ pDCs after CLP in vitro. Depletion of pDCs reversed the bias of splenic DCs toward increased IL-10 synthesis after CLP in vivo. Thus, during polymicrobial sepsis, CD4+ pDCs are activated in the bone marrow and induce functional reprogramming of differentiating BMDC toward an immunosuppressive phenotype