Structure-function analysis of NF - kB1 p105

NF-kB transcription factors regulate the expression of genes that promote immunity, inflammation and cell survival. The p105 precursor protein of NF-kB1 p50 acts as an NF-kB inhibitory protein (IkB), retaining associated NF-kB/Rel subunits inactive in the cytoplasm of cells. The pro-inflammatory cytokine tumor necrosis factor- [alpha] (TNF[alpha]) and bacterial lipopolysacharide (LPS) stimulate p105 proteolysis via IkB kinase (IKK) mediated phoshorylation of p105. Subsequent ubiquitination of p105 precedes its degradation by the proteasome, releasing associated NF-kB/Rel subunits to translocate to the nucleus. In this study, it is demonstrated that p105 binds to IKK via the death domain (DD) motif in its C-terminus. Furthermore, the p105 DD is required for the efficient phosphorylation and proteolysis of p105 induced by IKK or in response to TNF stimulation. These data suggest that the p105 DD is a docking domain that couples p105 to signal-induced proteolysis. The MAP 3-kinase tumor progression locus-2 (TPL-2), which is required for the activation of MEK1/2 MAP 2-kinases in response to LPS in macrophages, associates stoichiometrically with p105. Here, it is demonstrated that TPL-2 stability relies on its high affinity association with p105 through two interaction sites. While the TPL-2 C- terminus binds to residues 497-538 of p105, the p105 DD interacts with the TPL-2 kinase domain. Binding to the p105 DD inhibits TPL-2 MEK kinase activity, but concomitant interaction of the TPL-2 C-terminus with residues 497-538 of p105 is required for efficient TPL-2 inhibition by p105. Consequently, the C-terminally truncated form of TPL-2 is insensitive to p105 regulation in vivo, which may explain why such a mutation is oncogenic. These data indicate that in addition to its role as a precursor for p50 and a cytoplasmic inhibitor of NF-kB, p105 is a negative regulator of TPL-2

Intranasal Bifidobacterium longum protects against viral-induced lung inflammation and injury in a murine model of lethal influenza infection

Background: Prophylactic strategies are urgently needed for prevention of severe inflammatory responses to respiratory viral infections. Bacterial-host interactions may modify the immune response to viral infections. Methods: We examined the contribution of Intranasal administration of two different Bifidobacterium longum strains or its isolated cell wall in controlling viral induced inflammation using a murine model of influenza infection. We monitored mortality and morbidity over a 10-day period and viral load, differential broncho alveolar lavage (BAL) fluid inflammatory cell counts, Lung tissue histology, BAL and serum cytokines, markers of vascular damage and cell death were quantified. Findings: Intranasal administration of Bifidobacterium longum35624® or its isolated cell wall prior to virus inoculation significantly reduced viral load within the lungs and significantly improved survival. Reduced viral load was associated with reduced lung injury as suggested by cell death and vascular leakage markers, a shift from neutrophil to macrophage recruitment, reduced inflammatory cytokine levels (including IL-6), reduced type 1 and 2 interferon levels, but increased levels of interferon-λ and surfactant protein D. These protective effects were maintained when the bifidobacterial cell wall preparation was administered 24 h after viral inoculation. The protective effects were also observed for the Bifidobacterium longumPB-VIR™ strain. Interpretation: Exposure to these bifidobacterial strains protect against the inflammatory sequelae and damage associated with uncontrolled viral replication within the lung. Funding: This work has been funded, in part, by a research grant from GlaxoSmithKline, PrecisionBiotics Group Ltd., Swiss National Science Foundation grants (project numbers CRSII3_154488, 310030_144219, 310030_127356 and 310030_144219) and Christine Kühne - Center for Allergy Research and Education (CK-CARE). Keywords: Influenza; Interferon; Prevention; Probiotic

Influenza-induced monocyte-derived alveolar macrophages confer prolonged antibacterial protection

Respiratory infections occur throughout life but how this shapes the lung immune system through time is unclear. Wack and colleagues show that a previous influenza infection recruits monocytes to the lung, which then assume an alveolar macrophage-like phenotype and mediate long-term antibacterial protection. Despite the prevalence and clinical importance of influenza, its long-term effect on lung immunity is unclear. Here we describe that following viral clearance and clinical recovery, at 1 month after infection with influenza, mice are better protected from Streptococcus pneumoniae infection due to a population of monocyte-derived alveolar macrophages (AMs) that produce increased interleukin-6. Influenza-induced monocyte-derived AMs have a surface phenotype similar to resident AMs but display a unique functional, transcriptional and epigenetic profile that is distinct from resident AMs. In contrast, influenza-experienced resident AMs remain largely similar to naive AMs. Thus, influenza changes the composition of the AM population to provide prolonged antibacterial protection. Monocyte-derived AMs persist over time but lose their protective profile. Our results help to understand how transient respiratory infections, a common occurrence in human life, can constantly alter lung immunity by contributing monocyte-derived, recruited cells to the AM population

IFNλ is a potent anti‐influenza therapeutic without the inflammatory side effects of IFNα treatment

Abstract Influenza A virus (IAV)‐induced severe disease is characterized by infected lung epithelia, robust inflammatory responses and acute lung injury. Since type I interferon (IFNαβ) and type III interferon (IFNλ) are potent antiviral cytokines with immunomodulatory potential, we assessed their efficacy as IAV treatments. IFNλ treatment of IAV‐infected Mx1‐positive mice lowered viral load and protected from disease. IFNα treatment also restricted IAV replication but exacerbated disease. IFNα treatment increased pulmonary proinflammatory cytokine secretion, innate cell recruitment and epithelial cell death, unlike IFNλ‐treatment. IFNλ lacked the direct stimulatory activity of IFNα on immune cells. In epithelia, both IFNs induced antiviral genes but no inflammatory cytokines. Similarly, human airway epithelia responded to both IFNα and IFNλ by induction of antiviral genes but not of cytokines, while hPBMCs responded only to IFNα. The restriction of both IFNλ responsiveness and productive IAV replication to pulmonary epithelia allows IFNλ to limit IAV spread through antiviral gene induction in relevant cells without overstimulating the immune system and driving immunopathology. We propose IFNλ as a non‐inflammatory and hence superior treatment option for human IAV infection

Corrected Super-Resolution Microscopy Enables Nanoscale Imaging of Autofluorescent Lung Macrophages

Observing the cell surface and underlying cytoskeleton at nanoscale resolution using super-resolution microscopy has enabled many insights into cell signaling and function. However, the nanoscale dynamics of tissue-specific immune cells have been relatively little studied. Tissue macrophages, for example, are highly autofluorescent, severely limiting the utility of light microscopy. Here, we report a correction technique to remove autofluorescent noise from stochastic optical reconstruction microscopy (STORM) data sets. Simulations and analysis of experimental data identified a moving median filter as an accurate and robust correction technique, which is widely applicable across challenging biological samples. Here, we used this method to visualize lung macrophages activated through Fc receptors by antibody-coated glass slides. Accurate, nanoscale quantification of macrophage morphology revealed that activation induced the formation of cellular protrusions tipped with MHC class I protein. These data are consistent with a role for lung macrophage protrusions in antigen presentation. Moreover, the tetraspanin protein CD81, known to mark extracellular vesicles, appeared in ring-shaped structures (mean diameter 93 ± 50 nm) at the surface of activated lung macrophages. Thus, a moving median filter correction technique allowed us to quantitatively analyze extracellular secretions and membrane structure in tissue-derived immune cells

Transcriptomic analyses reveal anti-viral responses of epithelial cells and multiple immune cell types in HRV infected human lung tissue

Background: Human rhinovirus (HRV) is a main cause of airway infections and a major risk factor of exacerbations in asthma and COPD. Investigation of HRV pathogenesis has been hampered by the lack of complex in vitro models that closely represent the human disease. The aim of the study was to characterize the immune response of viable human lung tissue to ex vivo HRV infection using Precision-Cut Lung Slices (PCLS). Method: Human PCLS containing airways were inoculated with HRV1B, UV‐inactivated HRV, medium or HRV in the presence of 3C protease inhibitor Rupintrivir. At day 1 and day 3 post infection (p.i.) tissue vitality, viral load and cytokine release were measured and transcriptomic analyses upon RNA isolation from PCLS were performed. Results: HRV infection of human PCLS induced no strong cytopathic effect as indicated by intact tissue viability. The transcriptomic analyses revealed that HRV infection of PCLS induced 5977 and 4322 gene expression changes at day 1 or day 3 p.i., respectively. These gene signatures were indicative of interferon signalling, epithelial cell differentiation, lymphocyte regulation, antigen presentation and NK cell cytotoxicity. Rupintrivir downregulated about one third of the HRV upregulated genes. These data were confirmed by increased protein levels of pro‐inflammatory and anti‐viral cytokines induced by HRV, e.g. TNF‐α and IFNα2a, which were also diminished by Rupintrivir. Conclusion: In conclusion, ex vivo infection of human lung tissue with HRV induced a strong antiviral and pro‐inflammatory immune response. The observed gene expression profile revealed involvement of epithelial but also multiple immune cells. This enables us to study HRV induced immune responses in the human lung microenvironment

Inhibition of BET recruitment to chromatin as an effective treatment for MLL-fusion leukaemia

Recurrent chromosomal translocations involving the mixed lineage leukaemia (MLL) gene initiate aggressive forms of leukaemia, which are often refractory to conventional therapies1. Many MLL-fusion partners are members of the super elongation complex (SEC), a critical regulator of transcriptional elongation, suggesting that aberrant control of this process has an important role in leukaemia induction2, 3. Here we use a global proteomic strategy to demonstrate that MLL fusions, as part of SEC2, 3 and the polymerase-associated factor complex (PAFc)4, 5, are associated with the BET family of acetyl-lysine recognizing, chromatin ‘adaptor’ proteins. These data provided the basis for therapeutic intervention in MLL-fusion leukaemia, via the displacement of the BET family of proteins from chromatin. We show that a novel small molecule inhibitor of the BET family, GSK1210151A (I-BET151), has profound efficacy against human and murine MLL-fusion leukaemic cell lines, through the induction of early cell cycle arrest and apoptosis. I-BET151 treatment in two human leukaemia cell lines with different MLL fusions alters the expression of a common set of genes whose function may account for these phenotypic changes. The mode of action of I-BET151 is, at least in part, due to the inhibition of transcription at key genes (BCL2, C-MYC and CDK6) through the displacement of BRD3/4, PAFc and SEC components from chromatin. In vivo studies indicate that I-BET151 has significant therapeutic value, providing survival benefit in two distinct mouse models of murine MLL–AF9 and human MLL–AF4 leukaemia. Finally, the efficacy of I-BET151 against human leukaemia stem cells is demonstrated, providing further evidence of its potent therapeutic potential. These findings establish the displacement of BET proteins from chromatin as a promising epigenetic therapy for these aggressive leukaemias

Inhibition of BET recruitment to chromatin as an effective treatment for MLL-fusion leukaemia

Recurrent chromosomal translocations involving the mixed lineage leukaemia (MLL) gene initiate aggressive forms of leukaemia, which are often refractory to conventional therapies1. Many MLL-fusion partners are members of the super elongation complex (SEC), a critical regulator of transcriptional elongation, suggesting that aberrant control of this process has an important role in leukaemia induction2, 3. Here we use a global proteomic strategy to demonstrate that MLL fusions, as part of SEC2, 3 and the polymerase-associated factor complex (PAFc)4, 5, are associated with the BET family of acetyl-lysine recognizing, chromatin ‘adaptor’ proteins. These data provided the basis for therapeutic intervention in MLL-fusion leukaemia, via the displacement of the BET family of proteins from chromatin. We show that a novel small molecule inhibitor of the BET family, GSK1210151A (I-BET151), has profound efficacy against human and murine MLL-fusion leukaemic cell lines, through the induction of early cell cycle arrest and apoptosis. I-BET151 treatment in two human leukaemia cell lines with different MLL fusions alters the expression of a common set of genes whose function may account for these phenotypic changes. The mode of action of I-BET151 is, at least in part, due to the inhibition of transcription at key genes (BCL2, C-MYC and CDK6) through the displacement of BRD3/4, PAFc and SEC components from chromatin. In vivo studies indicate that I-BET151 has significant therapeutic value, providing survival benefit in two distinct mouse models of murine MLL–AF9 and human MLL–AF4 leukaemia. Finally, the efficacy of I-BET151 against human leukaemia stem cells is demonstrated, providing further evidence of its potent therapeutic potential. These findings establish the displacement of BET proteins from chromatin as a promising epigenetic therapy for these aggressive leukaemias