Receptor sTNFR Research Articles

Therapy Induced Senescence Promote Expression of Death Receptors in Breast Cancer Cells

Chemotherapeutic agents that cause DNA damage also induce cellular senescence known as therapy-induced senescence (TIS). Cells undergoing senescence may exert detrimental effects by promoting tumor progression in healthy cells or supporting metastases in cancer cells due to “senesence-associated secretory phenotype” (SASP), involving secretion of chemokines, cytokines, metalloproteinases, and growth factors. Death receptors belong to the tumor necrosis factor receptor superfamily and implicated in induction of apoptosis via activation of extrinsic pathway. The most recognized death receptors are FAS (CD95), TNFR1 and TRAIL-R1 / 2 (DR4-DR5) etc. and capable of directly inducing apoptosis in the cell. In this study we aim to investigate the expression of cell death receptors in response to TIS of breast cancer cells for their potential use in elimination of senescent cells. Doxorubicin and etoposide were used to induce senescence selectively in MCF7 breast cancer cell line. Senescence induction was confirmed by β-galactosidase staining and cell cycle analysis. Activations of p53, p21, and γ-H2AX and expression levels of cell death receptors (FAS (CD95), TNFR1-2 and DR5 were tested by western blot analysis. Apoptosis was measured by Annexin V/7AAD analysis. Here, we show that chemotherapy agents etoposide and doxorubicin induced senescence by arresting MCF-12A and MCF-7 cells in G1 and G2/M phases of cell cycle., respectively. In addition, Induction of senescence is confirmed by SA-β-gal staining and by activation of g-H2AX, p53 and p21 proteins. Neither etoposide nor doxorubicin induced significant apoptosis in MCF12A or MCF-7 cells. Importantly, TIS increased the protein levels of TNFR1, TNFR2 and DR5 receptors selectively in MCF-7 cells but not in MCF-12A cells. These data suggest that chemotherapy agents induce senescence increased the expression of death receptors in breast cancer cell line MCF-7 thus provide a basis for further investigation of death receptor mediated targeting of senescent cells as potential therapeutic strategy.

A multiscale study on the mechanisms of spatial organization in ligand-receptor interactions on cell surfaces

The binding of cell surface receptors with extracellular ligands triggers distinctive signaling pathways, leading into the corresponding phenotypic variation of cells. It has been found that in many systems, these ligand-receptor complexes can further oligomerize into higher-order structures. This ligand-induced oligomerization of receptors on cell surfaces plays an important role in regulating the functions of cell signaling. The underlying mechanism, however, is not well understood. One typical example is proteins that belong to the tumor necrosis factor receptor (TNFR) superfamily. Using a generic multiscale simulation platform that spans from atomic to subcellular levels, we compared the detailed physical process of ligand-receptor oligomerization for two specific members in the TNFR superfamily: the complex formed between ligand TNFα and receptor TNFR1 versus the complex formed between ligand TNFβ and receptor TNFR2. Interestingly, although these two systems share high similarity on the tertiary and quaternary structural levels, our results indicate that their oligomers are formed with very different dynamic properties and spatial patterns. We demonstrated that the changes of receptor’s conformational fluctuations due to the membrane confinements are closely related to such difference. Consistent to previous experiments, our simulations also showed that TNFR can preassemble into dimers prior to ligand binding, while the introduction of TNF ligands induced higher-order oligomerization due to a multivalent effect. This study, therefore, provides the molecular basis to TNFR oligomerization and reveals new insights to TNFR-mediated signal transduction. Moreover, our multiscale simulation framework serves as a prototype that paves the way to study higher-order assembly of cell surface receptors in many other bio-systems.

Mechanisms of Action of the PGLYRP1/Tag7 Protein in Innate and Acquired Immunity.

One of the promising fields of modern molecular biology is the search for new proteins that regulate the various stages of the immune response and the investigation of the molecular mechanisms of action of these proteins. Such proteins include the multifunctional protein PGLYRP1/Tag7, belonging to the PGRP-S protein family, whose gene was discovered in mice at the Institute of Gene Biology, Russian Academy of Sciences, in 1996. PGLYRP1/Tag7 is classified as a protein of innate immunity; however, it can also participate in the regulation of acquired immunity mechanisms. In this paper, we consider the involvement of PGLYRP1/Tag7 in the triggering of antimicrobial defense mechanisms and formation of subsets of cytotoxic lymphocytes that kill tumor cells. The paper emphasizes that the multifaceted functional activity of Tag7 in the immune response has to do with its ability to interact with various proteins to form stable protein complexes. Hsp70-associated Tag7 can induce the death of tumor cells carrying the TNFR1 receptor. Tag7, associated with the Mts1 (S100A4) protein, can stimulate the migration of innate and adaptive immune cytotoxic lymphocytes to a lesion site. Involvement of Tag7 in the regulation of immunological processes suggests that it may be considered as a promising agent in cancer therapy. These properties of Tag7 were used to develop autologous vaccines that have passed the first and second phases of clinical trials in patients with end-stage melanoma and renal cancer. The C-terminal peptide of Tag7, isolated by limited proteolysis, was shown to protect the cartilage and bone tissue of the ankle joint in mice with induced autoimmune arthritis and may be a promising drug for suppressing the development of inflammatory processes.

Tumor necrosis factor receptor signaling modulates carcinogenesis in a mouse model of breast cancer

Pro-inflammatory conditions have long been associated with mammary carcinogenesis and breast cancer progression. The underlying mechanisms are incompletely understood but signaling of pro-inflammatory cytokine TNFα through its receptors TNFR1 and TNFR2 is a major mediator of inflammation in both obesity and in the response of tissues to radiation, 2 known risk factors for the development of breast cancer. Here, we demonstrated the loss of one TNFR2 allele led to ductal hyperplasia in the mammary gland with increased numbers of mammary epithelial stem cell and terminal end buds. Furthermore, loss of one TNFR2 allele increased the incidence of breast cancer in MMTV-Wnt1 mice and resulted in tumors with a more aggressive phenotype and metastatic potential. The underlying mechanisms include a preferential activation of canonical NF-κB signaling pathway and autocrine production of TNFα. Analysis of the TCGA dataset indicated inferior overall survival for patients with down-regulated TNFR2 expression. These findings unravel the imbalances in TNFR signaling promote the development and progression of breast cancer, indicating that selective agonists of TNFR2 could potentially modulate the risk for breast cancer in high-risk populations.

A systematic review of specific pathways in oral squamous cell carcinoma

Background Oral squamous cell carcinoma (OSCC) eludes all therapeutic modalities because its etiological factors, molecular mechanisms, and pathways of carcinogenesis remain controversial. Objective This systematic review aims to identify and investigate the specific pathways commonly documented in the published research on OSCC. Results The most frequently studied pathways in OSCC, according to the reviewed molecular studies, are p53 tumor suppressor, collective Serine/Threonine Kinases, transcription factor NF-kB, integrins and cell proliferation, vascular endothelial growth factor, Wnt signaling, the PI3K/AKT pathway, cadherins and adhesion of epithelial cells and cyclooxygenase 2 pathways. However, the MEK/ERK pathway is computationally detected as a less frequent oncogenic pathway in OSCC. Advances in Knowledge Although the former pathways have been extensively studied, other pathways are involved in the pathogenesis of OSCC, such as Notch signaling, cell death via Fas or TNFR1 Receptors, and the p38 MAPK pathway, remain understudied in OSCC. Future studies elucidating the contribution of these pathways in the development of OSCC are urgently required.

TNFR1 and the TNFα axis as a targetable mediator of liver injury from stereotactic body radiation therapy

IntroductionRadiation therapy for the management of intrahepatic malignancies can adversely affect liver function. Liver damage has been associated with increased levels of inflammatory cytokines, including tumor necrosis factor alpha (TNFα). We hypothesized that an inflammatory state, characterized by increased soluble TNFα receptor (sTNFR1), mediates sensitivity of the liver to radiation. Materials/MethodsPlasma samples collected during 3 trials of liver radiation for liver malignancies were assayed for sTNFR1 level via enzyme-linked immunosorbent assay (ELISA). Univariate and multivariate logistic regression and longitudinal models were used to characterize associations between liver toxicity (defined as a ≥2-point increase in Child-Pugh [CP] score within 6 months of radiation treatment) and sTNFR1 levels, ALBI score, biocorrected mean liver dose (MLD), age, and baseline laboratory values. ResultsSamples from 78 patients given liver stereotactic body radiation therapy [SBRT] (92%) or hypofractionated radiation were examined. There was a significant association between liver toxicity and sTNFR1 levels, and higher values were associated with increased toxicity over a range of mean liver doses. When ALBI score and biocorrected dose were included in the model with sTNFR1, baseline ALBI score and change in ALBI (ΔALBI) were significantly associated with toxicity, but sTNFR1 was not. Baseline aminotransferase levels also predicted toxicity but not independently of ALBI score. ConclusionsElevated plasma sTNFR1 levels are associated with liver injury after liver radiation, suggesting that elevated inflammatory cytokine activity is a predictor of radiation-induced liver dysfunction. Future studies should determine whether administration of agents that decrease inflammation prior to treatment is warranted.

NF-κB Blockade by NEMO Binding Domain Peptide Ameliorates Inflammation and Neurobehavioral Sequelae After Cranial Radiation Therapy in Juvenile Mice

Cranial radiation therapy (CRT) is a common treatment for pediatric brain tumor patients. However, side effects include significant neurobehavioral dysfunction in survivors. This dysfunction may in part be caused by inflammation, including increased production of tumor necrosis factor alpha (TNFα) and its receptor TNFR1, which can activate the nuclear factor kappa light-chain enhancer of activated B cells (NF-κB). The TNFα blockade abrogates this inflammatory response, although it presents immunologic risks. Thus, modulation of pathway subsets may be preferable. Here, we test whether inhibition of NF-κB activation using an NF-κB essential modulator binding domain (NBD) peptide mitigates CRT-induced neuroinflammation and improves behavioral outcomes. Male C57BL/6J 28-day old mice were randomized to saline (sham), 5 Gy whole-brain CRT, or CRT + NBD-peptide. Brain tissue was collected after 4 hours or 3 months for Western blot or immunohistochemistry. The cortex, corpus callosum (CC), and dentate gyrus were variably imaged for NF-κB-p65, IκBα, proliferation, apoptosis, necroptosis, TNFα, TNFR1, IBA-1, doublecortin, CD11c, and GFAP. Neurobehavioral changes were assessed by open field and elevated plus maze tests 3 months post-CRT. NF-κB expression increased in whole and nuclear fractions 4 hours after CRT and was abrogated by NBD treatment. Cell death increased and proliferation decreased after CRT, including within neuronal progenitors, with some loss mitigated by NBD. Increased levels of TNFα, IBA-1, and GFAP were found in the CC and cortex months after CRT and were limited by NBD. The anti-NF-κB peptide also improved neurobehavioral assessments, yielding improvements in anxiety and exploration. Results suggest a role for NF-κB modulation by NBD peptide in the reduction of neuroinflammation and mitigation of behavioral complications after pediatric radiation therapy.

A novel TNFR2 agonist antibody expands highly potent regulatory T cells.

Regulatory T cells (Treg cells) restrict immune system activity, such as in response to self-antigens, and are switched on by tumor necrosis factor receptor 2 (TNFR2). Therapeutic activation of TNFR2, thereby expanding Treg cells and suppressing immune activity, may be beneficial to patients with various inflammatory diseases. Here, we characterized a new human TNFR2-directed antibody agonist isolated from mice. We found that the antibody agonist expanded the number of Treg cells within cultures of primary human CD4+ T cells from healthy donors and patients with type 1 diabetes or Sézary syndrome. These Treg cells had increased metabolic gene expression and intracellular itaconate concentrations, characteristics associated with maximally suppressive, anti-inflammatory Treg cells. Furthermore, antibody-expanded Treg cells repressed the activity of primary human CD8+ effector T cells (Teff cells). Epitope mapping suggested that the antibody bound to TNFR2 through a natural cross-linking surface and that Treg cell expansion was independent of the antibody Fc region. In addition, Treg cell expansion was not increased by adding either supplemental TNF ligand or a cross-linking reagent, suggesting that the antibody agonist by itself can elicit maximal activity, a notion that was confirmed by increased secretion of soluble TNFR2. Pending in vivo tests, these features indicate that this TNFR2 antibody agonist has the potential to safely and effectively treat various inflammatory disorders.

Patients with obstructive sleep apnea have suppressed levels of soluble cytokine receptors involved in neurodegenerative disease, but normal levels with airways therapy

PurposeObstructive sleep apnea (OSA) results in systemic intermittent hypoxia. By one model, hypoxic stress signaling in OSA patients alters the levels of inflammatory soluble cytokines TNF and IL6, damages the blood brain barrier, and activates microglial targeting of neuronal cell death to increase the risk of neurodegenerative disorders and other diseases. However, it is not yet clear if OSA significantly alters the levels of the soluble isoforms of TNF receptors TNFR1 and TNFR2 and IL6 receptor (IL6R) and co-receptor gp130, which have the potential to modulate TNF and IL6 signaling.MethodsPicogram per milliliter levels of the soluble isoforms of these four cytokine receptors were estimated in OSA patients, in OSA patients receiving airways therapy, and in healthy control subjects. Triplicate samples were examined using Bio-Plex fluorescent bead microfluidic technology. The statistical significance of cytokine data was estimated using the nonparametric Wilcoxon rank-sum test. The clustering of these high-dimensional data was visualized using t-distributed stochastic neighbor embedding (t-SNE).ResultsOSA patients had significant twofold to sevenfold reductions in the soluble serum isoforms of all four cytokine receptors, gp130, IL6R, TNFR1, and TNFR2, as compared with control individuals (p = 1.8 × 10−13 to 4 × 10−8). Relative to untreated OSA patients, airways therapy of OSA patients had significantly higher levels of gp130 (p = 2.8 × 10−13), IL6R (p = 1.1 × 10−9), TNFR1 (p = 2.5 × 10−10), and TNFR2 (p = 5.7 × 10−9), levels indistinguishable from controls (p = 0.29 to 0.95). The data for most airway-treated patients clustered with healthy controls, but the data for a few airway-treated patients clustered with apneic patients.ConclusionsPatients with OSA have aberrantly low levels of four soluble cytokine receptors associated with neurodegenerative disease, gp130, IL6R, TNFR1, and TNFR2. Most OSA patients receiving airways therapy have receptor levels indistinguishable from healthy controls, suggesting a chronic intermittent hypoxia may be one of the factors contributing to low receptor levels in untreated OSA patients.

Modulation of glycosyltransferase ST6Gal-I in gastric cancer-derived organoids disrupts homeostatic epithelial cell turnover

Programmed cell death promotes homeostatic cell turnover in the epithelium but is dysregulated in cancer. The glycosyltransferase ST6Gal-I is known to block homeostatic apoptosis through α2,6-linked sialylation of the death receptor TNFR1 in many cell types. However, its role has not been investigated in gastric epithelial cells or gastric tumorigenesis. We determined that human gastric antral epithelium rarely expressed ST6Gal-I, but the number of ST6Gal-I-expressing epithelial cells increased significantly with advancing premalignancy leading to cancer. The mRNA expression levels of ST6GAL-I and SOX9 in human gastric epithelial cells correlated positively with one another through the premalignancy cascade, indicating that increased epithelial cell expression of ST6Gal-I is associated with premalignant progression. To determine the functional impact of increased ST6Gal-I, we generated human gastric antral organoids from epithelial stem cells and differentiated epithelial monolayers from gastric organoids. Gastric epithelial stem cells strongly expressed ST6Gal-I, suggesting a novel biomarker of stemness. In contrast, organoid-derived epithelial monolayers expressed markedly reduced ST6Gal-I and underwent TNF-induced, caspase-mediated apoptosis, consistent with homeostasis. Conversely, epithelial monolayers generated from gastric cancer stem cells retained high levels of ST6Gal-I and resisted TNF-induced apoptosis, supporting prolonged survival. Protection from TNF-induced apoptosis depended on ST6Gal-I overexpression, because forced ST6Gal-I overexpression in normal gastric stem cell-differentiated monolayers inhibited TNF-induced apoptosis, and cleavage of α2,6-linked sialic acids from gastric cancer organoid-derived monolayers restored susceptibility to TNF-induced apoptosis. These findings implicate up-regulated ST6Gal-I expression in blocking homeostatic epithelial cell apoptosis in gastric cancer pathogenesis, suggesting a mechanism for prolonged epithelioid tumor cell survival.

Tumor Necrosis Factor α Influences Phenotypic Plasticity and Promotes Epigenetic Changes in Human Basal Forebrain Cholinergic Neuroblasts.

TNFα is the main proinflammatory cytokine implicated in the pathogenesis of neurodegenerative disorders, but it also modulates physiological functions in both the developing and adult brain. In this study, we investigated a potential direct role of TNFα in determining phenotypic changes of a recently established cellular model of human basal forebrain cholinergic neuroblasts isolated from the nucleus basalis of Meynert (hfNBMs). Exposing hfNBMs to TNFα reduced the expression of immature markers, such as nestin and β-tubulin III, and inhibited primary cilium formation. On the contrary, TNFα increased the expression of TNFα receptor TNFR2 and the mature neuron marker MAP2, also promoting neurite elongation. Moreover, TNFα affected nerve growth factor receptor expression. We also found that TNFα induced the expression of DNA-methylation enzymes and, accordingly, downregulated genes involved in neuronal development through epigenetic mechanisms, as demonstrated by methylome analysis. In summary, TNFα showed a dual role on hfNBMs phenotypic plasticity, exerting a negative influence on neurogenesis despite a positive effect on differentiation, through mechanisms that remain to be elucidated. Our results help to clarify the complexity of TNFα effects in human neurons and suggest that manipulation of TNFα signaling could provide a potential therapeutic approach against neurodegenerative disorders.

The iRhom2/ADAM17 Axis Attenuates Bacterial Uptake by Phagocytes in a Cell Autonomous Manner.

Uptake of bacteria by phagocytes is a crucial step in innate immune defence. Members of the disintegrin and metalloproteinase (ADAM) family critically control the immune response by limited proteolysis of surface expressed mediator molecules. Here, we investigated the significance of ADAM17 and its regulatory adapter molecule iRhom2 for bacterial uptake by phagocytes. Inhibition of metalloproteinase activity led to increased phagocytosis of pHrodo labelled Gram-negative and -positive bacteria (E. coli and S. aureus, respectively) by human and murine monocytic cell lines or primary phagocytes. Bone marrow-derived macrophages showed enhanced uptake of heat-inactivated and living E. coli when they lacked either ADAM17 or iRhom2 but not upon ADAM10-deficiency. In monocytic THP-1 cells, corresponding short hairpin RNA (shRNA)-mediated knockdown confirmed that ADAM17, but not ADAM10, promoted phagocytosis of E. coli. The augmented bacterial uptake occurred in a cell autonomous manner and was accompanied by increased release of the chemokine CXCL8, less TNFα release and only minimal changes in the surface expression of the receptors TNFR1, TLR6 and CD36. Inhibition experiments indicated that the enhanced bacterial phagocytosis after ADAM17 knockdown was partially dependent on TNFα-activity but not on CXCL8. This novel role of ADAM17 in bacterial uptake needs to be considered in the development of ADAM17 inhibitors as therapeutics.

The expression profile of genes encoding tumor necrosis factor-alpha, interleukin-6 and their receptor in benign adrenal tumors.

In the process of neoplasia, during which benign adrenal tumors are formed, stimulators of new blood vessel growth as well as growth of tumor cells are cytokines, especially tumor necrosis factor-alpha (TNF-α) and interleukin 6 (IL-6). We analyzed the expression profile of genes coding: TNF-α, tumor necrosis factor receptor 1 (TNF-R1), TNF-R2, IL-6, interleukin 6 receptor (IL-6R) in sections of adrenocortical tumor tissue, rated on the Weiss point scale, in patients with clinically diagnosed Conn's and Cushing's syndrome, and the usefulness of determining the examined genes as markers differentiating individual clinical units. There was no correlation between the expression of the examined genes and clinical parameters such as age, BMI or blood pressure, both in the entire study group and in individual subgroups. Elevated expression of the genes coding TNF-α, TNF-R2 and IL-6R was observed, whereas genes encoding TNF-R1 and IL-6 showed relatively low expression. The highest statistically significant differences in the expression of the examined genes were observed between IL-6 and IL-6R. High positive correlation was found in the subgroup of patients with Conn's clinical syndrome, between genes encoding both types of receptors for TNF-α, IL-6 and TNF-R2, TNF-α and IL-6 receptor, and between TNF-R2 and IL6-R receptors, which may suggest the mutual influence of these cytokines and their receptors on their own expression.

Autoantibodies from SLE patients induce programmed cell death in murine fibroblast cells through interaction with TNFR1 receptor

Various pathological processes are known to be associated with the production of IgG autoantibodies, which have high affinity for self-antigens and often cause tissue injury and the development of autoimmune diseases. However, the mechanism of their cytotoxic activity is not clearly understood yet. Here, we have shown that the action of these autoantibodies on cells expressing TNFR1 (the cell surface receptor for TNFα) can cause both caspase-dependent apoptosis and necroptosis of these cells, with suppression of apoptosis resulting in switching to RIP1-dependent necroptosis. Analysis of necroptotic mechanisms has shown that a critical point of necroptosis is phosphorylation of RIP1 and RIP3 kinases, which is followed by the involvement of lysosomes and mitochondria in this process. The induction of cytotoxicity is initiated by the interaction of autoantibodies with TNFR1, and autoantibodies can therefore be regarded as a new functional ligand for this receptor. The innate immunity protein Tag7 (PGLYRP1) described in our recent studies is also a ligand for TNFR1 and competes with autoantibodies for binding with it. Supposedly, the cytotoxic effect of autoantibodies is one of the factors responsible for autoimmune diseases that lead to tissue injury.

Control of Murine Primordial Follicle Growth Activation by IκB/NFκB Signaling.

The transcription factor NFκB has been associated with the timing of menopause in a large human genome-wide association study. Furthermore, preclinical studies demonstrate that loss of Tumor necrosis factor alpha (Tnfα) or its receptor Tnfr2 slows primordial follicle growth activation (PFGA). Although Tnfα:receptor signaling stimulates NFκB and may mechanistically link these findings, very little is known about NFκB signaling in PFGA. Because signaling downstream of Tnfα/Tnfr2 ligand/receptor interaction has not been interrogated as relates to PFGA, we evaluated the expression of key NFκB signaling proteins in primordial and growing follicles, as well as during ovarian aging. We show that key members of the NFκB pathway, including subunits, activating kinases, and inhibitory proteins, are expressed in the murine ovary. Furthermore, the subunits p65 and p50, and the cytosolic inhibitory proteins IκBα and IκBβ, are present in ovarian follicles, including at the primordial stage. Finally, we assessed PFGA in genetically modified mice (AKBI) previously demonstrated to be resistant to inflammatory stress-induced NFκB activation due to overexpression of the NFκB inhibitory protein IκBβ. Consistent with the hypothesis that NFκB plays a key role in PFGA, AKBI mice exhibit slower PGFA than wild-type (WT) controls, and their ovaries contain nearly twice the number of primordial follicles as WT both at early and late reproductive ages. These data provide mechanistic insight on the control of PFGA and suggest that targeting NFκB at the level of IκB proteins may be a tractable route to slowing the rate of PFGA in women faced with early ovarian demise.

Giardia duodenalis induces extrinsic pathway of apoptosis in intestinal epithelial cells through activation of TNFR1 and K63 de-ubiquitination of RIP1 in vitro

Giardia duodenalis is one of main causative agents of diarrhea that affects the health of millions of people on a global scale per year. It has been clear that attachment of G. duodenalis trophozoites to intestinal epithelium cells (IECs) can induce cell death, while the underlying cellular and molecular mechanisms remain to be explored. It was shown in this study that treatment of Caco-2 cells with Giardia trophozoites could result in reduced cell viability. RNA sequencing analysis demonstrated that expressions of many apoptosis-related genes and some deubiquitinase genes displayed marked changes in trophozoite-treated cells. Trophozoites activated the death-signaling receptor TNFR1 on the IEC surface and caspase-3/8 (CASP3/8) signaling pathways in Caco-2 cells. K63 ubiquitination level of RIP1 was reduced upon stimulation with trophozoites, in parallel, the expressions of deubiquitinases CYLD and A20 were increased. The caspase inhibitor Q-VD-OPH could rescue trophozoite-induced cell apoptosis. Likewise, TNFR1, CYLD, and A20 silencing decreased the levels of cleaved CASP3/8 in trophozoite-treated cells and reversed the pro-apoptosis induction effect of trophozoites. These data suggest that Giardia trophozoite stimulation can activate CASP3/8 signaling pathways via activation of TNFR1 and K63 de-ubiquitination of RIP1 caused by up-regulated expressions of CYLD and A20, and promote Caco-2 cell apoptosis. The present study deepens our understanding of the mechanism of interaction between Giardia and IECs.

499-P: Clinical Validation of an AI-Enabled Prognostic Test (KidneyintelX) to Accurately Predict Rapid Kidney Function Decline in Patients with Type 2 Diabetic Kidney Disease

The ability to predict rapid kidney function decline (RKFD) in patients with early stages of type 2 diabetic kidney disease (T2DKD) can improve long-term health outcomes through earlier intervention. Our objective was to develop a robust risk score for patients with early stages of T2DKD. We obtained EHR and plasma from patients with T2DKD (Kidney Disease Improving Global Outcomes [KDIGO] G1-G2, A2-A3 or G3a-G3b, A1-A3) from the Mount Sinai BioMe Biobank and the Penn Medicine Biobank and measured soluble tumor necrosis factor receptor (sTNFR1, sTNFR2) and kidney injury molecule-1 (KIM-1) via a validated quantitative electrochemiluminescent assay. A machine learning model was trained using the EHR/biomarker data to predict RKFD, defined as eGFR decline of ≥5 ml/min/year or ≥40% sustained decline in eGFR or kidney failure within 5 years, and performance was compared to a clinical model and KDIGO risk-matrix. A total of 1146 subjects with a mean age of 63, baseline eGFR 54 ml/min, and median UACR 61 mg/g were randomly divided into 60% training (n=686) and 40% validation (n=460) sets. The median follow-up was 4.3 years and 241 (21%) experienced RKFD. The AUC of KidneyIntelX for RFKD was 0.85 (95% CI 0.84-0.86) in training and 0.77 (95% CI 0.76-0.79) in the validation set vs. AUC of 0.67 (95% CI 0.64-0.70) for a clinical model. Using pre-specified cutoffs from the training set, KidneyIntelX stratified 47%, 37% and 16% of the validation set into low-, intermediate- and high-risk strata, with a PPV of 62% (vs. 41% for KDIGO) in the high-risk group (p <0.001), a net reclassification improvement for events of 41% (p <0.05), and a NPV of 91% in the low-risk group (vs. 85% for KDIGO). KidneyIntelX, a machine learning model combining biomarkers and EHR data, improved prediction of RKFD over standard clinical metrics in patients with T2DKD. Future studies will prospectively evaluate KidneyIntelX in clinical decision-making and impact. Disclosure L. Chan: None. G.N. Nadkarni: Advisory Panel; Self; Renalytix AI. Consultant; Self; AstraZeneca, Reata, Renalytix AI. Stock/Shareholder; Self; Renalytix AI. Other Relationship; Self; Renalytix AI. F. Fleming: Board Member; Self; Renalytix AI plc. F.E. Salem: None. B. Murphy: Board Member; Self; RenalytixAI. M.J. Donovan: Consultant; Self; Renalytix AI plc. S. Coca: Advisory Panel; Self; Renalytix AI plc. Consultant; Self; Bayer Healthcare Pharmaceuticals Inc., CHF Solutions, Relypsa, Inc., Takeda Pharmaceutical Company Limited. Stock/Shareholder; Self; Renalytix AI plc. S.M. Damrauer: Consultant; Spouse/Partner; Rhythm Pharmaceuticals. Research Support; Self; CytoVas LLC, Renalytix AI plc. Funding Renalytix AI plc

Significant Induction of Soluble TNFR2 Compared with TNFR1 in Serum Samples of HIV Patients with or without Antiretroviral Medication.

Tumor necrosis factor and its receptors (sTNFR1 and sTNFR2) have been implicated in many infectious diseases. Identification of the key receptor (sTNFR1 or sTNFR2) which drives the immunopathogenesis of HIV infection is crucial in developing adjunctive therapy for HIV. This study determined the expression levels of sTNFR1 and sTNFR2 in antiretroviral therapy (ART) - experienced and naïve HIV patients. A total of 40 HIV patients comprising 30 with ART and 10 without ART were enrolled from the Pantang Hospital located in the Greater Accra Region of Ghana for data and blood collection. Serum concentrations of sTNFR1 and sTNFR2 were determined by ELISA. Mann- Whitney U test was used to examine differences in serum levels of sTNFR1 and sTNFR2 between patients on ART and ART naïve patients. Wilcoxon Signed-Rank test was performed to determine the difference between sTNFR1 and sTNFR2, and Kruskal Wallis test was conducted to compare the effect of different antiretroviral drugs on the levels of sTNFR1 and sTNFR2. P< 0.05 was considered statistically significant. A Wilcoxon Signed-Ranks Test indicated serum levels of sTNFR2 was statistically significantly higher than sTNFR1 (Z=-5.51; p<0.001). Levels of sTNFR1 and sTNFR2 did not differ by ART status U =91.00 (Z = -1.84), p = 0.065 and U = 131.50 (Z = -0.58, p =0.560), respectively. There were not significant differences in levels of TNFR2 H(2) = 1.86, p=0.395 and sTNFR1 (H (2) = 4.37, p=0.113 across different ART combinations. Compared to sTNFR1, the level of sTNFR2 is significantly increased during HIV infection irrespective of ART status. The high sTNFR2 level is not associated with antiretroviral drugs and may be another potential target for therapeutic development. This is the first study of sTNFRs in African population.

Auto Arginine-GlcNAcylation Is Crucial for Bacterial Pathogens in Regulating Host Cell Death.

Many Gram-negative bacterial pathogens utilize the type III secretion system (T3SS) to inject virulence factors, named effectors, into host cells. These T3SS effectors manipulate host cellular signaling pathways to facilitate bacterial pathogenesis. Death receptor signaling plays an important role in eukaryotic cell death pathways. NleB from enteropathogenic Escherichia coli (EPEC) and SseK1/3 from Salmonella enterica serovar Typhimurium (S. Typhimurium) are T3SS effectors. They are defined as a family of arginine GlcNAc transferase to modify a conserved arginine residue in the death domain (DD) of the death receptor TNFR and their corresponding adaptors to hijack death receptor signaling. Here we identified that these enzymes, NleB, SseK1, and SseK3 could catalyze auto-GlcNAcylation. Residues, including Arg13/53/159/293 in NleB, Arg30/158/339 in SseK1, and Arg153/184/305/335 in SseK3 were identified as the auto-GlcNAcylation sites by mass spectrometry. Mutation of the auto-modification sites of NleB, SseK1, and SseK3 abolished or attenuated the capability of enzyme activity toward their death domain targets during infection. Loss of this ability led to the increased susceptibility of the cells to TNF- or TRAIL-induced cell death during bacterial infection. Overall, our study reveals that the auto-GlcNAcylation of NleB, SseK1, and SseK3 is crucial for their biological activity during infection.

TNF-α orchestrates CD4+ T cell-dependent inflammation via TNFR2 signaling

Abstract CD4+ T helper subsets, in particular Th1 and Th17, produce cytokines that play critical roles in inflammatory diseases. TNF-α, produced largely by innate immune cells, is also potently pro-inflammatory. Here we asked if TNF-α contributes to effector CD4+ Th generation, and found that in the presence of TGF-β TNF-α signals via its non-death receptor TNFR2 to generate Th17 cells expressing inflammatory mediators such as GM-CSF, CXCL3, and IFN-γ. Single cell RNA-Seq of CNS-infiltrating CD4+ T cells in EAE identified an IL-17-positive cluster with an inflammatory gene signature that required TNFR2 expression. Notably, TNFR2-deficient CD4+ T cells produced fewer inflammatory mediators and were relatively nonpathogenic in EAE and colitis. Thus, TNF-α is not just an effector but also an initiator of Th differentiation, raising the possibility that in diseases in which both TNF-α and IL-17 contribute, targeting TNF-α may diminish the generation of pathogenic Th17 cells.