TNF phase III signalling in tolerant cells is tightly controlled by A20 and CYLD.

Following the acute phase of an inflammatory reaction, a strictly controlled resolution of inflammation is necessary. A dysregulation of this process leads to hyperinflammation, chronic inflammatory disease, or immune paralysis. Different mechanisms participate in the coordinated termination of the inflammatory process, e.g. the expression of antiinflammatory molecules and different forms of tolerance. To better understand the processes which mediate resolution of TNF-dependent inflammation and induce tolerance, it is necessary to characterize the signal transduction quality during TNF long-term (pre)incubation. Within a time frame from 12 to 48h, designated as phase III of the TNF response, we measured an ongoing, constitutive activation of TNFR1/NF-κB-dependent pathways in monocytic cells. Phase III signalling which was also named "constitutive signaling in TNF tolerant cells" induces the expression of low- and high-sensitive target genes including A20 which is differentially regulated by transcriptional and proteolytic events. A20 strictly controls TNF long-term constitutive signalling in an IκB kinase complex- and partially RIP-dependent manner supported by adjuvant ABIN1. In addition, CYLD proteins participate in the regulation of this late-phase signal transduction, whereas downstream molecules such as Bcl3 and p50 are not involved. A20 and CYLD are expressed with different mRNA kinetics resulting in a strong or only a modest increase in protein levels, respectively. The identification of mechanisms which contribute to the termination of inflammation will provide additional diagnostic and therapeutic aspects to specifically diagnose certain aspects of inflammation and specifically modulate them.

S100-alarmin-induced innate immune programming protects newborn infants from sepsis.

The high risk of neonatal death from sepsis is thought to result from impaired responses by innate immune cells; however, the clinical observation of hyperinflammatory courses of neonatal sepsis contradicts this concept. Using transcriptomic, epigenetic and immunological approaches, we demonstrated that high amounts of the perinatal alarmins S100A8 and S100A9 specifically altered MyD88-dependent proinflammatory gene programs. S100 programming prevented hyperinflammatory responses without impairing pathogen defense. TRIF-adaptor-dependent regulatory genes remained unaffected by perinatal S100 programming and responded strongly to lipopolysaccharide, but were barely expressed. Steady-state expression of TRIF-dependent genes increased only gradually during the first year of life in human neonates, shifting immune regulation toward the adult phenotype. Disruption of this critical sequence of transient alarmin programming and subsequent reprogramming of regulatory pathways increased the risk of hyperinflammation and sepsis. Collectively these data suggest that neonates are characterized by a selective, transient microbial unresponsiveness that prevents harmful hyperinflammation in the delicate neonate while allowing for sufficient immunological protection.

In neonates S100A8/S100A9 alarmins prevent the expansion of a specific inflammatory monocyte population promoting septic shock.

The high susceptibility of newborn infants to sepsis is ascribed to an immaturity of the neonatal immune system, but the molecular mechanisms remain unclear. Newborn monocytes massively release the alarmins S100A8/S100A9. In adults, these are major regulators of immunosuppressive myeloid-derived suppressor cells (MDSCs). We investigated whether S100A8/S100A9 cause an expansion of monocytic MDSCs (Mo-MDSCs) in neonates, thereby contributing to an immunocompromised state. Mo-MDSCs have been assigned to CD14+/human leukocyte antigen (HLA)-DR-/low/CD33+ monocytes in humans and to CD11b+/Gr-1int/Ly6G-/Ly6Chi cells in mice. We found monocytes with these phenotypes significantly expanded in their respective newborns. Functionally, however, they did not prove immunosuppressive but rather responded inflammatorily to microbial stimulation. Their expansion did not correlate with high S100A8/S100A9 levels in cord blood. Murine studies revealed an excessive expansion of CD11b+/Gr-1int/Ly6G-/Ly6Chi monocytes in S100A9-/- neonates compared to wild-type neonates. This strong baseline expansion was associated with hyperinflammatory responses during endotoxemia and fatal septic courses. Treating S100A9-/- neonates directly after birth with S100A8/S100A9 alarmins prevented excessive expansion of this inflammatory monocyte population and death from septic shock. Our data suggest that a specific population of inflammatory monocytes promotes fatal courses of sepsis in neonates if its expansion is not regulated by S100A8/S100A9 alarmins.-Heinemann, A. S., Pirr, S., Fehlhaber, B., Mellinger, L., Burgmann, J., Busse, M., Ginzel, M., Friesenhagen, J., von Köckritz-Blickwede, M., Ulas, T., von Kaisenberg, C. S., Roth, J., Vogl, T., Viemann, D. In neonates S100A8/S100A9 alarmins prevent the expansion of a specific inflammatory monocyte population promoting septic shock.

C/EBPß-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/EBPß 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/EBPß 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/EBPß 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.

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.

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.

Highly pathogenic avian influenza viruses inhibit effective immune responses of human blood-derived macrophages.

Systemic infections with HPAIVs, such as H5N1, are characterized by cytokine burst and sepsis. We investigated the role of human monocyte-derived macrophages in these events after infection with different influenza virus strains. Macrophages were infected with low pathogenic H1N1 (PR8) or high pathogenic H7N7 (FPV) and H5N1 (KAN-1) subtypes. Macrophages were found to be nonpermissive for influenza virus propagation. Surprisingly, transcriptome analysis revealed an insufficient innate immune response of macrophages only to HPAIV infections. Induction of inflammatory cytokines, as well as type I IFNs, was significantly attenuated in H5N1- and H7N7-infected cells, contradicting a primary role of macrophages for the cytokine burst. Furthermore, inflammasome activation was impaired significantly in HPAIV-infected macrophages. Interestingly, this finding correlated with a complete suppression of viral protein M2 expression after HPAIV infection, which is known to be involved in influenza viral inflammasome activation. In summary, our data provide first evidences for a strategy of how HPAIVs avoid initial inflammatory responses of macrophages facilitating virus spreading and progression to the systemic stage of disease.

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.