Alarmins MRP8 and MRP14 Induce Stress Tolerance in Phagocytes under Sterile Inflammatory Conditions

Hyporesponsiveness by phagocytes is a well-known phenomenon in sepsis that is frequently induced by low-dose endotoxin stimulation of Toll-like receptor 4 (TLR4) but can also be found under sterile inflammatory conditions. We now demonstrate that the endogenous alarmins MRP8 and MRP14 induce phagocyte hyporesponsiveness via chromatin modifications in a TLR4-dependent manner that results in enhanced survival to septic shock in mice. During sterile inflammation, polytrauma and burn trauma patients initially present with high serum concentrations of myeloid-related proteins (MRPs). Human neonatal phagocytes are primed for hyporesponsiveness by increased peripartal MRP concentrations, which was confirmed in murine neonatal endotoxinemia in wild-type and MRP14(-/-) mice. Our data therefore indicate that alarmin-triggered phagocyte tolerance represents a regulatory mechanism for the susceptibility of neonates during systemic infections and sterile inflammation

C/EBPβ-LAP*/LAP Expression Is Mediated by RSK/eIF4B-Dependent Signalling and Boosted by Increased Protein Stability in Models of Monocytic Differentiation.

The transcription factor C/EBPβ plays a key role in monocytic differentiation and inflammation. Its small isoform LIP is associated with proliferation at early premonocytic developmental stages and regulated via mTOR-dependent signalling. During later stages of (pre)monocytic differentiation there is a considerable increase in the large C/EBPβ isoforms LAP*/LAP which inhibit proliferation thus supporting terminal differentiation. Here, we showed in different models of monocytic differentiation that this dramatic increase in the LAP*/LAP protein and LAP/LIP ratio was accompanied by an only modest/retarded mRNA increase suggesting an important role for (post)translational mechanisms. We found that LAP*/LAP formation was induced via MEK/RSK-dependent cascades, whereas mTOR/S6K1 were not involved. Remarkably, LAP*/LAP expression was dependent on phosphorylated eIF4B, an acceleratory protein of RNA helicase eIF4A. PKR inhibition reduced the expression of eIF4B and C/EBPβ in an eIF2α-independent manner. Furthermore, under our conditions a marked stabilisation of LAP*/LAP protein occurred, accompanied by reduced chymotrypsin-like proteasome/calpain activities and increased calpastatin levels. Our study elucidates new signalling pathways inducing LAP*/LAP expression and indicates new alternative PKR functions in monocytes. The switch from mTOR- to RSK-mediated signalling to orchestrate eIF4B-dependent LAP*/LAP translation, accompanied by increased protein stability but only small mRNA changes, may be a prototypical example for the regulation of protein expression during selected processes of differentiation/proliferation

Highly pathogenic influenza viruses inhibit inflammatory response in monocytes via activation of rar-related orphan receptor RORα

Infections with highly pathogenic avian influenza viruses (HPAIV) in humans lead to systemic disease associated with cytokine storm and multiorgan failure. In this study we aimed to identify the role of monocytes for the host response to HPAIV infection. Using genome-wide microarray analysis, we surprisingly demonstrate a reduced immune response of human monocytes to HPAIV H5N1 compared to human influenza A viruses. In bioinformatic analyses we could reveal a potential role of the Rar-related orphan receptor alpha (RORα) for the gene expression pattern induced by H5N1. RORα is known as an inhibitor of NF-κB signaling. We provide evidence that in monocytes RORα is activated by H5N1, resulting in inhibited NF-κB signaling. Using murine Hoxb8-immortalized RORα⁻/⁻, monocytes rescued NF-κB signaling upon H5N1 infection, confirming the biological relevance of RORα as an H5N1-induced mediator of monocytic immunosuppression. In summary, our study reveals a novel RORα-dependent escape mechanism by which H5N1 prevents an effective inflammatory response of monocytes blocking NF-κB-dependent gene expression

H5N1 virus activates signaling pathways in human endothelial cells resulting in a specific imbalanced inflammatory response

H5N1 influenza virus infections in humans cause a characteristic systemic inflammatory response syndrome; however, the molecular mechanisms are largely unknown. Endothelial cells (ECs) play a pivotal role in hyperdynamic septic diseases. To unravel specific signaling networks activated by H5N1 we used a genome-wide comparative systems biology approach analyzing gene expression in human ECs infected with three different human and avian influenza strains of high and low pathogenicity. Blocking of specific signaling pathways revealed that H5N1 induces an exceptionally NF-κB-dependent gene response in human endothelia. Additionally, the IFN-driven antiviral program in ECs is shown to be dependent on IFN regulatory factor 3 but significantly impaired upon H5N1 infection compared with low pathogenic influenza virus. As additional modulators of this H5N1-specific imbalanced gene response pattern, we identified HMGA1 as a novel transcription factor specifically responsible for the overwhelming proinflammatory but not antiviral response, whereas NFATC4 was found to regulate transcription of specifically H5N1-induced genes. We describe for the first time, to our knowledge, defined signaling patterns specifically activated by H5N1, which, in contrast to low pathogenic influenza viruses, are responsible for an imbalance of an overwhelming proinflammatory and impaired antiviral gene program

C/EBPβ-LAP*/LAP Expression Is Mediated by RSK/eIF4B-Dependent Signalling and Boosted by Increased Protein Stability in Models of Monocytic Differentiation

<div><p>The transcription factor C/EBPβ plays a key role in monocytic differentiation and inflammation. Its small isoform LIP is associated with proliferation at early premonocytic developmental stages and regulated <i>via</i> mTOR-dependent signalling. During later stages of (pre)monocytic differentiation there is a considerable increase in the large C/EBPβ isoforms LAP*/LAP which inhibit proliferation thus supporting terminal differentiation. Here, we showed in different models of monocytic differentiation that this dramatic increase in the LAP*/LAP protein and LAP/LIP ratio was accompanied by an only modest/retarded mRNA increase suggesting an important role for (post)translational mechanisms. We found that LAP*/LAP formation was induced <i>via</i> MEK/RSK-dependent cascades, whereas mTOR/S6K1 were not involved. Remarkably, LAP*/LAP expression was dependent on phosphorylated eIF4B, an acceleratory protein of RNA helicase eIF4A. PKR inhibition reduced the expression of eIF4B and C/EBPβ in an eIF2α-independent manner. Furthermore, under our conditions a marked stabilisation of LAP*/LAP protein occurred, accompanied by reduced chymotrypsin-like proteasome/calpain activities and increased calpastatin levels. Our study elucidates new signalling pathways inducing LAP*/LAP expression and indicates new alternative PKR functions in monocytes. The switch from mTOR- to RSK-mediated signalling to orchestrate eIF4B-dependent LAP*/LAP translation, accompanied by increased protein stability but only small mRNA changes, may be a prototypical example for the regulation of protein expression during selected processes of differentiation/proliferation.</p></div

Expression of LAP*/LAP is regulated by MEK/RSK-dependent signalling pathways.

<p>(A) THP-1 monocytic cells were coincubated with the mTOR inhibitor (mTOR-I) rapamycin (100 nM) and/or 100 nM PMA for 24 h, and the levels of LAP*/LAP were determined (n = 3). (B) THP-1 cells were incubated in the absence or presence of PMA together with increasing doses of S6K1-inhibitor DG2 (S6K1-I), and LAP*/LAP protein amounts were measured (n = 3). Some of the control lanes without S6K1-I have been omitted (thin lines). (C) THP-1 cells were incubated with PMA in the absence/presence of the RSK inhibitor (RSK-I) BI-D1870 (2.5 μM), and LAP*/LAP and LIP levels were evaluated (n = 5). An additional control lane without PMA has been removed (thin line). (D) The LAP/LIP ratio of the experiment shown in C was calculated as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0144338#pone.0144338.g001" target="_blank">Fig 1B</a>. (E) MM-6 cells were incubated with RSK-I and/or PMA, and LAP*/LAP levels were measured (n = 4). (F) THP-1 cells were incubated ± PMA up to 18 h, and the levels of RSK and p-RSK were determined (n = 3). (G) THP-1 cells were incubated with the MEK inhibitor (MEK-I) U0126 (5 μM) or RSK-I and/or PMA for 24 h. The levels of LAP*/LAP, (p-)ERK1/2, (p-)RSK, and (p-)rpS6 were monitored (n = 5; arrows: phosphorylated and unphosphorylated rpS6). (H) HeLa cells were transfected with RSK1 siRNA (I: 50 nM, II: 100 nM) or control siRNA (Co; 100 nM), and following an incubation period of 24 h, the levels of LAP*/LAP and RSK1 were determined (n = 3). (I) THP-1 cells were incubated for 72 h with VitD3 together with increasing doses of RSK-I. Subsequently, LAP*/LAP protein amounts were assessed (n = 3). Lanes showing the effect of RSK-I on unstimulated cells have been removed (thin line).</p

Modest and retarded increase in C/EBPβ mRNA under differentiation-inducing conditions.

<p>(A) Under conditions as shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0144338#pone.0144338.g001" target="_blank">Fig 1A</a>, the expression of C/EBPβ mRNA in THP-1 cells was monitored by qPCR using a C/EBPβ-specific primer pair in the central mRNA section, and GAPDH was used as a housekeeping gene control (mean±SD; n≥6, measured in duplicates). The level of C/EBPβ mRNA in unstimulated cells at 0 h was defined as one (dashed line). (B) Under conditions as described in Fig <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0144338#pone.0144338.g001" target="_blank">1A</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0144338#pone.0144338.g001" target="_blank">1C and 1D</a>, C/EBPβ mRNA expression was measured in monocytic cell lines and analysed as described in A. THP-1 + VitD3, MM-6 + PMA: mean±SD; n = 9 each (duplicates). THP-1 + PMA (5’): qRT-PCR using an additional C/EBPβ-specific primer pair in the 5’ section of the mRNA; mean±SD; n = 4 (duplicates).</p

Dramatic increase in C/EBPβ-LAP*/LAP proteins and LAP/LIP ratio in different models of monocytic differentiation.

<p>(A) THP-1 monocytic cells were incubated with 100 nM PMA up to 48 h, and the levels of the larger C/EBPβ isoforms (LAP*/LAP) and the small isoform LIP were monitored by Western blot analysis using whole cell extracts (loading control: actin; n = 6). (B) LAP*/LAP and LIP levels of a representative experiment were analysed by densitometry. The LAP/LIP ratio was calculated by adding LAP* and LAP and dividing this value by LIP. (C) Monocytic MM-6 cells were incubated with 100 nM PMA up to 24 h, and the levels of LAP*/LAP were measured (loading control: TBP; n = 3). (D) THP-1 cells were incubated with 100 nM VitD3 up to 72 h and the levels of LAP*/LAP were determined (loading control: TBP; n = 6). Please note that a control lane has been removed (indicated by a thin line). (E) Murine bone marrow-derived cells were differentiated to macrophages using 20 ng/ml rmM-CSF up to 7 d and LAP*/LAP were measured at day 3 and day 7 (loading control GAPDH; n = 3). (F) Primary human monocytes were differentiated up to 7 d, and LAP*/LAP levels were assessed at day 0, 1, 3, and 7 (loading control: GAPDH; n = 5).</p