eNAMPT neutralization reduces preclinical ARDS severity via rectified NFkB and Akt/mTORC2 signaling

Despite encouraging preclinical data, therapies to reduce ARDS mortality remains a globally unmet need, including during the COVID-19 pandemic. We previously identified extracellular nicotinamide phosphoribosyltransferase (eNAMPT) as a novel damage-associated molecular pattern protein (DAMP) via TLR4 ligation which regulates inflammatory cascade activation. eNAMPT is tightly linked to human ARDS by biomarker and genotyping studies in ARDS subjects. We now hypothesize that an eNAMPT-neutralizing mAb will significantly reduce the severity of ARDS lung inflammatory lung injury in diverse preclinical rat and porcine models. Sprague Dawley rats received eNAMPT mAb intravenously following exposure to intratracheal lipopolysaccharide (LPS) or to a traumatic blast (125 kPa) but prior to initiation of ventilator-induced lung injury (VILI) (4 h). Yucatan minipigs received intravenous eNAMPT mAb 2 h after initiation of septic shock and VILI (12 h). Each rat/porcine ARDS/VILI model was strongly associated with evidence of severe inflammatory lung injury with NFkB pathway activation and marked dysregulation of the Akt/mTORC2 signaling pathway. eNAMPT neutralization dramatically reduced inflammatory indices and the severity of lung injury in each rat/porcine ARDS/VILI model (~ 50% reduction) including reduction in serum lactate, and plasma levels of eNAMPT, IL-6, TNFα and Ang-2. The eNAMPT mAb further rectified NFkB pathway activation and preserved the Akt/mTORC2 signaling pathway. These results strongly support targeting the eNAMPT/TLR4 inflammatory pathway as a potential ARDS strategy to reduce inflammatory lung injury and ARDS mortality.

Comprehensive mechanistic characterization of mQTLs in an Asian population

Identification and characterization of methylation quantitative trait loci (mQTLs) can help elucidate the role of DNA methylation changes as a potential mediator of genetic risk loci. However, mQTLs remain poorly characterized: they have not yet been mapped in the largest ethnic populations, their cell-type specific nature has not been resolved, and the proportion of mQTLs attributed to different molecular mechanisms is unknown. Here we perform the first mQTL-mapping study in a large Han Chinese population, demonstrating that over 80% of mQTLs are shared with those identified in White Caucasians. We further estimate that over 90% of mQTLs are shared between different blood cell-lineages. mQTLs demonstrate a strong enrichment for variants influencing chromatin accessibility. We identify a number of GWAS-linked transcription factor trans-mQTL hotspots associated with eosinophilia, ulcerative colitis and body mass index, and a subset of trans-mQTLs within the NFKB-pathway that may mediate the risk of obesity. In summary, this study significantly expands our understanding of mQTLs and their potential role in mediating disease risk, whilst also contributing the first mQTL-database in an Asian population.

STEM-04. PD-1 INDEPENDENT OF PD-L1 LIGATION PROMOTES GLIOBLASTOMA GROWTH THROUGH THE NFkB PATHWAY

Brain tumor-initiating cells (BTICs) drive glioblastoma growth through not fully understood mechanisms. Here, we found that a proportion of human and murine BTICs expressed programmed cell death protein (PD-1). Gain- or loss-of-function studies revealed that tumor-intrinsic PD-1 promoted proliferation, and self-renewal of BTICs. Mechanistically, site-directed mutagenesis, RNA sequencing and pharmacological inhibitors implicated SHP-2-mediated activation of NFkB downstream of PD-1 in BTICs. Notably, the tumor-intrinsic promoting effects of PD-1 did not require PD-L1 ligation; thus, the therapeutic blocking antibodies inhibiting PD-1/PD-L1 interaction which failed in glioblastoma trials could not overcome the growth advantage of PD-1 in BTICs. Finally, mice with intracranial Pdcd1 over- or underexpressing BTICs had shorter or longer survival, respectively, and this occurred in mice lacking T and B cells. These findings point to a critical role for PD-1 in BTICs and uncover a non-immune resistance mechanism of GBM patients to PD-1 or PD-L1 blocking therapies.

SARS-CoV-2 Nsp14 mediates the effects of viral infection on the host cell transcriptome

To identify functions of SARS-CoV-2 proteins, we performed transcriptomic analyses of cells expressing individual viral proteins. Expression of Nsp14, a protein involved in viral RNA replication, provoked a dramatic remodeling of the transcriptome that strongly resembled that observed following SARS-CoV-2 infection. Moreover, Nsp14 expression altered the splicing of more than 1000 genes and resulted in a dramatic increase in the number of circRNAs, which are linked to innate immunity. These effects were independent of Nsp14 exonuclease activity and the co-factor Nsp10. Activation of the NFkB pathway and increased expression of CXCL8 occurred early upon Nsp14 expression. IMPDH2, which catalyzes the rate-limiting step of guanidine biosynthesis, was identified as a key mediator of the effect. Nsp14 expression caused an increase in GTP cellular levels, and the effect of Nsp14 was strongly decreased in presence of an IMPDH2 inhibitor. Together, our data demonstrate an unknown role for Nsp14 with implications for therapy.

BLNK/BTK: Novel Components in the NF-kappa B Pathways of Endothelial Cells in Diabetic Condition

Peripheral artery disease (PAD) is atherosclerotic occlusion of vessel outside the heart that most commonly affects the lower extremities. The effects of PAD-related ischemia are exacerbated under diabetic hyperglycemic conditions. Under ischemic conditions, an adaptive induction of the NFkB pathways is in vascular endothelial cells is required for recovery. We have recently shown that prolonged exposure of cells to high glucose before ischemia resulted in impairment of the canonical NFkB pathway through decrease in IkBa degradation. However, the signaling pathways involved in hyperglycemia and ischemia mediated effects on the NFkB pathways are not well understood. Since the NFkB signaling pathways propagate through a cascade of phosphorylation events, we used arrays of antibodies to approximately 100 proteins known to participate in the NFkB pathway to identify the changes in their phosphorylation states in human umbilical vein endothelial cells (HUVEC). Cells grown for three days either in culture medium with normal glucose (LG) or high glucose (HG) were subjected to ischemia for 24 hours (LGI and HGI, respectively). Cell lysates were then incubated with the array of antibodies printed on glass slides (Full Moon Biosystems, Sunnyvale, CA) and fluorescent signals were digitally recorded and normalized. The change in protein phosphorylation was calculated by dividing the intensity of the phosphorylated spot by the signal intensity of the corresponding non-phosphorylated spot for each protein. Differential expression between LG and LGI samples, and HG and HGI were calculated by dividing the phosphorylation ratio of the LGI and HGI with that of the LG and HG controls, respectively. A threshold of 1.5-fold increase or decrease was used to determine changes. Compared to the LG, LGI samples had 26 protein sites with increased phosphorylation whereas 36 sites had decreased phosphorylation. Similarly, compared to HG, HGI samples increased phosphorylation of 25 protein sites and decreased phosphorylation of 40 sites. A Venn-diagram analysis of LGI and HGI sites revealed 8 sites with an increase and 12 sites with a decrease in phosphorylation were specific to HGI. Pathway analyses using bioinformatics tools on 65 modulated phosphorylation sites in HGI (represented by 35 genes) suggested involvement of B cell linker/adapter protein (BLNK)/Bruton’s tyrosine kinase (BTK) that are critical for B cell antigen receptor (BCR)-coupled signaling. BTK expression in EC was confirmed by immunoblotting. Inhibition of BTK by a specific inhibitor terreic acid restored IkBa degradation in EC grown in high glucose suggesting a critical role of BLNK/BTK in diabetic ischemia. Thus, we have identified BLNK/BTK as potentially new components of the NFkB pathway in endothelial cells that contributes to the poor recovery during hyperglycemic ischemia.

Role of toll-like receptor 4 on tissue factor regulation in human monocytes

Background Inflammation and coagulation play a pivotal role in the pathogenesis of acute coronary events and an extensive cross-talk links the two systems, whereby inflammation activates coagulation and coagulation affects inflammatory activity. Infact, pro-inflammatory stimuli can induce tissue factor (TF) expression, the principal initiator of the clotting cascade, in circulating monocytes and activate pathways leading to thrombin generation.In turn, TF may bind cellular receptors which may affect the production and release of inflammatory mediators. According to our recent results, proprotein convertase subtilisin/kexin9 (PCSK9) and Gamma-Glutamyltransferase (GGT), two molecules involved in the pathogenesis of cardiovascular disease, are able to up-regulate TF expression in monocytes by activating NFkB pathway but the mechanism and the receptor involved in this biological response is unknown. One plausible possibility is that both molecules bind a Toll-like Receptor (TLR)4 located on membranes of human and cultured monocytes,activating the TLR4/MyD88-NFkB pathway and eventually leading to stimulation of TF expression. Aim To assess whether both molecules are able to bind to TLR4 located on the surface of human monocytes and whether this specific binding involves the TLR4/MyD88-NFkB pathway on TF modulation. Methods THP-1 cells, a human monocytic cell line derived from an acute monocytic leukemia patient, was used as in vitro model. The cells were stimulated with human (h) recombinant (r) PCSK9 (5 μg/ml) and hrGGT (1 μg/ml) or pre-incubated with BAY-117082 (BAY, 10–5M) a NFκB inhibitor, CLI-095 (3x10–6M), a highly Myd88/TLR4 signaling specific inhibitor and LPS-RS (1 μg/ml) a TLR4 antagonist. TF procoagulant activity (PCA), was assessed by 1-stage clotting assay and the results expressed by ρg/mL of active protein. Experimental series were carried out in endotoxin-free conditions, in order to exclude lipopolysaccharide (LPS)-dependent immune responses. Results hrPCSK9 and hrGGT stimulated TF expression (PCA: from 50±20 to 120±20, n=10, p<0.01) and (PCA: from 190±140 to 460±360, n=15, p<0.001) respectively, an effect down-regulated by BAY,a NFκB inhibitor (PCA by hrPCSK9: −71±23%, n=5, p<0.01; PCA by hrGGT: −90±21%, n=7, p<0.001). CLI-095, a TLR4 inhibitor (PCA by hrPCSK9: −86±26%, n=3, p<0.05; and PCA by GGT: −89±10%, n=5, p<0.001).LPS-RS, a TLR4 antagonist, (PCA by hrPCSK9: −74±25%, n=3, p<0.05; PCA by hrGGT: −70±17%, n=5, p<0.001) abolished both PCSK9 and GGT-induced TF expression. Conclusions These data are the first demonstration of a direct role of PCSK9 and GGT as active mediators of inflammatory-based thrombotic diseases. The possible mechanism of action involves recognition of two proteins by TLR4 on monocytes membrane surface, lead to activation of the transcription factor NFκB. Further studies will be needed to better understand the regulatory mechanisms underlying this complex set of biological responses that bind TLR4 modulation and TF expression. Funding Acknowledgement Type of funding source: None

AB0058 CELL-TYPE SPECIFIC REGULATION OF IL-1R SIGNALING BY R835, A DUAL IRAK1/4 INHIBITOR

Background:Interleukin-1 beta (IL-1b) is a key mediator of the inflammatory response and is known to exacerbate damage during chronic disease and acute tissue injury. Through association with the adaptor protein Myd88, interleukin receptor associated kinases (IRAK)1 and 4 initiate signaling downstream of IL-1Rs resulting in the activation of the NFkB and MAPK pathways and the production of proinflammatory cytokines (1). IL-1Rs are broadly expressed across cell types and little is known about differences in signaling between cell types and the role of IRAK1 and IRAK4 kinase activity.Objectives:We have identified a potent and selective IRAK1/4 inhibitor, R835, that substantially suppressed the elevation of LPS (TLR4 agonist)-induced serum cytokines in healthy human volunteers in a recent phase 1 study. The aim of this study was to evaluate the effect of R835 on IL-1R signaling in primary human fibroblasts and endothelial cells.Methods:Human dermal fibroblasts, lung fibroblasts or endothelial cells were stimulated with IL-1b and the effect of R835 on the signaling pathway was evaluated by western blotting. Human dermal fibroblasts were stimulated with different amounts of IL-1b to evaluate both the signaling pathways activated and the cytokines produced. The ability of R835 to inhibit cytokine production induced by high or low amounts of IL-1b in dermal fibroblasts was assessed.Results:In human endothelial cells, inhibition of IRAK1/4 kinases with R835 resulted in a block of IL-1b-induced IRAK4 phosphorylation, IRAK1 degradation and downstream NFkB, p38 and JNK activation. In contrast, in both human primary dermal and lung fibroblasts stimulated with IL-1b, we observed potent inhibition of IRAK4 phosphorylation, IRAK1 degradation, and downstream JNK phosphorylation, but no inhibition of NFkB pathway proteins and only weak inhibition of p38. Upon titration of IL-1b we observed that dermal fibroblasts produced IL-8 and GRO in response to low levels of IL-1b (20pg/ml), and produced additional cytokines including G-CSF and GM-CSF with higher levels of IL-1b (400pg/ml). In the presence of low levels of IL-1b (20pg/ml), we observed a weak activation of NFkB pathway proteins and p38, compared to a very robust NFkB, p38 and additional JNK activation in the presence of higher levels of IL-1b (400pg/ml). Consistent with these results, in dermal fibroblasts, R835 showed little to no inhibition of IL-8 and GRO induced by low levels of IL-1b, but potently inhibited G-CSF and GM-CSF induced by high levels of IL-1b where JNK was activated.Conclusion:This study has elucidated signaling differences between cell types downstream of the IL-1R. In endothelial cells, as in myeloid cells, the kinase activity of IRAK1 and IRAK4 is required for the activation of all downstream signaling. Unexpectedly, in human fibroblasts, IRAK1/4 kinase activity appears to primarily regulate the JNK pathway, and not the NFkB pathway. Concomitant with that, only the cytokines induced by the additional activation of JNK in fibroblasts are regulated by a dual IRAK1/4 inhibitor. Clinically, an IRAK1/4 inhibitor may show select inhibition of IL-1b-induced cytokines depending on the tissue and cell type involved in inflammation.References:[1]Flannery S, Bowie A G. The interleukin-1 receptor-associated kinases: Critical regulators of innate immune signaling. Biochemical Pharmacology, Volume 80, Issue 12, 15 December 2010, Pages 1981-1991.Disclosure of Interests:Sylvia Braselmann Shareholder of: Shareholder of Rigel Pharmaceuticals, Employee of: Employee of Rigel Pharmaceuticals, Ernest Tai Shareholder of: Rigel Pharmaceuticals, Employee of: Rigel Pharmaceuticals, Roy Frances Shareholder of: Rigel Pharmaceuticals, Employee of: Rigel Pharmaceuticals, Chi Young Shareholder of: Rigel Pharmaceuticals, Employee of: Rigel Pharmaceuticals, Vadim Markovtsov Shareholder of: Rigel Pharmaceuticals, Employee of: Rigel Pharmaceuticals, Esteban Masuda Shareholder of: Rigel Pharmaceuticals, Employee of: Rigel Pharmaceuticals, Vanessa Taylor Shareholder of: Rigel Pharmaceuticals, Employee of: Rigel Pharmaceuticals

BMP-9 Modulates the Hepatic Responses to LPS

It was previously shown that Bone Morphogenetic Protein (BMP)-9 is constitutively produced and secreted by hepatic stellate cells (HSC). Upon acute liver damage, BMP-9 expression is transiently down-regulated and blocking BMP-9 under conditions of chronic damage ameliorated liver fibrogenesis in C57BL/6 mice. Thereby, BMP-9 acted as a pro-fibrogenic cytokine in the liver but without directly activating isolated HSC in vitro. Lipopolysaccharide (LPS), an endotoxin derived from the membrane of Gram-negative bacteria in the gut, is known to be essential in the pathogenesis of diverse kinds of liver diseases. The aim of the present project was therefore to investigate how high levels of BMP-9 in the context of LPS signalling might result in enhanced liver damage. For this purpose, we stimulated human liver sinusoidal endothelial cells (LSEC) with LPS and incubated primary human liver myofibroblasts (MF) with the conditioned medium of these cells. We found that LPS led to the secretion of factors from LSEC that upregulate BMP-9 expression in MF. At least one of these BMP-9 enhancing factors was defined to be IL-6. High BMP-9 in turn, especially in combination with LPS stimulation, induced the expression of certain capillarization markers in LSEC and enhanced the LPS-mediated induction of pro-inflammatory cytokines in primary human macrophages. In LSEC, pre-treatment with BMP-9 reduced the LPS-mediated activation of the NfkB pathway, whereas in macrophages, LPS partially inhibited the BMP-9/Smad-1 signaling cascade. In vivo, in mice, BMP-9 led to the enhanced presence of F4/80-positive cells in the liver and it modulated the LPS-mediated regulation of inflammatory mediators. In summary, our data point to BMP-9 being a complex and highly dynamic modulator of hepatic responses to LPS: Initial effects of LPS on LSEC led to the upregulation of BMP-9 in MF but sustained high levels of BMP-9 in turn promote pro-inflammatory reactions of macrophages. Thereby, the spatial and timely fine-tuned presence (or absence) of BMP-9 is needed for efficient wound-healing responses in the liver.

Patients with APECED possess altered NFkB pathway gene expression.

APECED, or autoimmune polyendocrinopathy candidiasis ectodermal dystrophy, is an autoimmune disorder caused by mutations in the autoimmune regulator gene AIRE (1, 2). Though it is known that 42 separate mutations in the AIRE gene can cause APECED (3), it not understood how these mutations lead to the pathology seen in APECED patients, and there are limited systems-level studies of the underlying transcriptional behavior of the immune cells of patients with APECED (4). In this study, we used a dataset (5) to compare the transcriptomes of monocyte-derived dendritic cells (moDCs) from patients with APECED and from control, non-affected patients in order to understand and describe the basic transcriptional nature of cells from patients with APECED. We found that four separate components of the NF-kB signaling pathway were among the genes most differentially expressed by moDCs from APECED patients. These included the NF-kB subunit REL, the NF-kB inhibitor alpha NFKBIA, the NF-kB inhibitor beta NFKBIZ, and the NF-kB inhibitor interacting Ras like 2 gene NKIRAS2. This is the first systems-level analysis of moDCs from patients with APECED to document perturbed gene expression in the NF-kB signaling pathway.