TNFR1 Signaling Research Articles

Impaired exploration induced by type 1 diabetes is related to locomotor activity rather than a reduction in motivation

Type 1 diabetes mellitus (T1D) is associated with cognitive impairments in humans. A well-established animal model of T1D is induced through the administration of streptozotocin (STZ), a glucose analog that induces pancreatic β-cell death, resulting in hyperglycemia and cognitive impairment linked to neuroinflammation and oxidative stress. Tumor necrosis factor (TNF)-α, a key inflammatory mediator, is elevated in the central nervous system (CNS) of diabetic animals. In this study, we utilized TNFR1 knockout mice to investigate the role of TNFR1 signaling in short-term T1D-related cognitive impairment. Our findings showed that diabetic animals did not develop cognitive damage within the first 2 weeks of T1D but exhibited reduced exploration in all behavioral tests. Our findings suggest that this reduction in exploration was attributable to motor impairment, as there was no reduction in motivated novelty-seeking behavior. Additionally, deletion of TNFR1 signaling attenuated gait speed impairment in diabetic mice, but did not affect other motor-related or exploratory behaviors.

Towards understanding the role of nanomedicine in targeting TNFR2 in rheumatoid arthritis.

Rheumatoid arthritis (RA) is a chronic autoimmune disease characterized by inflammation of the synovium and progressive joint destruction which significantly affects both quality of life and socioeconomic status. Admittedly, various treatments are available, but they are usually accompanied by various side effects, from mild to severe, and potentially with adverse events. Tumour necrosis factor-alpha (TNF-α) plays a crucial role in the pathophysiology of RA. It promotes inflammatory, apoptosis and necroptosis via TNF receptor-1 (TNFR1) but elicit anti-inflammatory effects via TNFR2. Herein, targeting TNFR2 has gained attention in RA studies. Understanding the role of nanomedicine in modulating TNFR2 signalling may be the instrument in development of RA therapies. Nanotechnology has made a significant progress in treating various conditions of diseases since its inception. Due to this, nanomedicine has emerged as a promising therapeutics approach for RA. Recent studies have demonstrated the potential of nanomedicine in RA theranostics, combining therapy and diagnostics for improved treatment outcomes. Owing to the challenges and advancements in the field of nanotechnology, nanoparticles are seen as an applicable candidate in the treatment of RA. In this review, we provide an overview of the role of nanomedicine in targeting TNFR2 for the treatment of RA and highlight the limitations of current therapies as well as the potential of nanocarriers with controlled drug release and active targeting abilities.

Differential signalling requirements for RIPK1-dependent pyroptosis in neutrophils and macrophages

TLR4 and TNFR1 signalling promotes potent proinflammatory signal transduction events, thus, are often hijacked by pathogenic microorganisms. We recently reported that myeloid cells retaliate Yersinia blockade of TAK1/IKK signalling by triggering RIPK1-dependent caspase-8 activation that promotes downstream GSDMD and GSDME-mediated pyroptosis in macrophages and neutrophils respectively. However, the upstream signalling events for RIPK1 activation in these cells are not well defined. Here, we demonstrate that unlike in macrophages, RIPK1-driven pyroptosis and cytokine priming in neutrophils are driven through TNFR1 signalling, while TLR4-TRIF signalling is dispensable. Furthermore, we demonstrate that activation of RIPK1-dependent pyroptosis in neutrophils during Yersinia infection requires IFN-γ priming, which serves to induce surface TNFR1 expression and amplify soluble TNF secretion. In contrast, macrophages utilise both TNFR1 and TLR4-TRIF signalling to trigger cell death, but only require TRIF but not autocrine TNFR1 for cytokine production. Together, these data highlight the emerging theme of cell type-specific regulation in cell death and immune signalling in myeloid cells.

TNFR1-mediated senescence and lack of TNFR2-signaling limit human intervertebral disc cell repair in back pain conditions.

TNFR1 signaling drives cells towards senesce and muted inflammatory response in painful intervertebral disc degeneration, while limited TNFR2 signaling may limit disc cell repair responses.

The RIPK1 death domain restrains ZBP1- and TRIF-mediated cell death and inflammation

RIPK1 is a multi-functional kinase that regulates cell death and inflammation and has been implicated in the pathogenesis of inflammatory diseases. RIPK1 acts in a kinase-dependent and kinase-independent manner to promote or suppress apoptosis and necroptosis, but the underlying mechanisms remain poorly understood. Here, we show that a mutation (R588E) disrupting the RIPK1 death domain (DD) caused perinatal lethality induced by ZBP1-mediated necroptosis. Additionally, these mice developed postnatal inflammatory pathology, which was mediated by necroptosis-independent TNFR1, TRADD, and TRIF signaling, partially requiring RIPK3. Our biochemical mechanistic studies revealed that ZBP1- and TRIF-mediated activation of RIPK3 required RIPK1 kinase activity in wild-type cells but not in Ripk1R588E/R588E cells, suggesting that DD-dependent oligomerization of RIPK1 and its interaction with FADD determine the mechanisms of RIPK3 activation by ZBP1 and TRIF. Collectively, these findings revealed a critical physiological role of DD-dependent RIPK1 signaling that is important for the regulation of tissue homeostasis and inflammation.

N4BP1 functions as a dimerization-dependent linear ubiquitin reader which regulates TNF signalling

Signalling through TNFR1 modulates proinflammatory gene transcription and programmed cell death, and its impairment causes autoimmune diseases and cancer. NEDD4-binding protein 1 (N4BP1) is a critical suppressor of proinflammatory cytokine production that acts as a regulator of innate immune signalling and inflammation. However, our current understanding about the molecular properties that enable N4BP1 to exert its suppressive potential remain limited. Here, we show that N4BP1 is a novel linear ubiquitin reader that negatively regulates NFκB signalling by its unique dimerization-dependent ubiquitin-binding module that we named LUBIN. Dimeric N4BP1 strategically positions two non-selective ubiquitin-binding domains to ensure preferential recognition of linear ubiquitin. Under proinflammatory conditions, N4BP1 is recruited to the nascent TNFR1 signalling complex, where it regulates duration of proinflammatory signalling in LUBIN-dependent manner. N4BP1 deficiency accelerates TNFα-induced cell death by increasing complex II assembly. Under proapoptotic conditions, caspase-8 mediates proteolytic processing of N4BP1, resulting in rapid degradation of N4BP1 by the 26 S proteasome, and acceleration of apoptosis. In summary, our findings demonstrate that N4BP1 dimerization creates a novel type of ubiquitin reader that selectively recognises linear ubiquitin which enables the timely and coordinated regulation of TNFR1-mediated inflammation and cell death.

TAK1 inhibition leads to RIPK1-dependent apoptosis in immune-activated cancers.

Poor survival and lack of treatment response in glioblastoma (GBM) is attributed to the persistence of glioma stem cells (GSCs). To identify novel therapeutic approaches, we performed CRISPR/Cas9 knockout screens and discovered TGFβ activated kinase (TAK1) as a selective survival factor in a significant fraction of GSCs. Loss of TAK1 kinase activity results in RIPK1-dependent apoptosis via Caspase-8/FADD complex activation, dependent on autocrine TNFα ligand production and constitutive TNFR signaling. We identify a transcriptional signature associated with immune activation and the mesenchymal GBM subtype to be a characteristic of cancer cells sensitive to TAK1 perturbation and employ this signature to accurately predict sensitivity to the TAK1 kinase inhibitor HS-276. In addition, exposure to pro-inflammatory cytokines IFNγ and TNFα can sensitize resistant GSCs to TAK1 inhibition. Our findings reveal dependency on TAK1 kinase activity as a novel vulnerability in immune-activated cancers, including mesenchymal GBMs that can be exploited therapeutically.

Tumor Necrosis Factor Receptor-2 Signals Clear-Cell Renal Carcinoma Proliferation via Phosphorylated 4E Binding Protein-1 and Mitochondrial Gene Translation

Clear cell renal cell carcinoma (ccRCC), a tubular epithelial malignancy, secretes tumor necrosis factor (TNF) which signals ccRCC cells in an autocrine manner via two cell surface receptors, TNFR1 and TNFR2, to activate shared and distinct signalling pathways. Selective ligation of TNFR2 was shown to drive cell cycle entry of malignant cells via a signalling pathway involving Etk, VEGFR2, PI3K, Akt, pSer727-Stat3 and mTOR. In this study, phosphorylated-4EBP1 serine 65 (pSer65-4EBP1) is identified as a downstream target of this TNFR2 signalling pathway. pSer65-4EBP1 expression is significantly elevated relative to total 4EBP1 in ccRCC tissue compared to normal kidneys, signal intensity increasing with malignant grade. Selective ligation of TNFR2 with the mutein R2TNF increases pSer65-4EBP1 expression in organ cultures that co-localises with internalised TNFR2 in mitochondria and increases expression of mitochondrially-encoded COX (cytochrome c oxidase subunit) Cox1, as well as nuclear-encoded Cox4/5b subunits. Pharmacological inhibition of mTOR reduces both R2TNF-mediated phosphorylation of 4EBP1 and cell cycle activation in tumor cells while increasing cell death. These results signify the importance of pSer65-4EBP1 in mediating TNFR2-driven cell-cycle entry in tumor cells in ccRCC and implicate a novel relationship between the TNFR2/pSer65-4EBP1/COX axis and mitochondrial function.

TNFRSF1B Signaling Blockade Protects Airway Epithelial Cells from Oxidative Stress.

Progressive respiratory airway destruction due to unresolved inflammation induced by periodic infectious exacerbation episodes is a hallmark of cystic fibrosis (CF) lung pathology. To clear bacteria, neutrophils release high amounts of reactive oxygen species (ROS), which inflict collateral damage to the neighboring epithelial cells causing oxidative stress. A former genome-wide small interfering RNA (siRNA) screening in CF submucosal gland cells, instrumental for mucociliary clearance, proposed tumor necrosis factor receptor superfamily member 1B (TNFRSF1B; TNFR2) as a potential hit involved in oxidative stress susceptibility. Here, we demonstrate the relevance of TNFRSF1B transcript knock-down for epithelial cell protection under strong oxidative stress conditions. Moreover, a blockade of TNFR signaling through its ligand lymphotoxin-α (LTA), overexpressed in airway epithelial cells under oxidative stress conditions, using the anti-tumor necrosis factor (TNF) biologic etanercept significantly increased the viability of these cells from a toxic oxidizing agent. Furthermore, bioinformatic analyses considering our previous RNA interference (RNAi) screening output highlight the relevance of TNFRSF1B and of other genes within the TNF pathway leading to epithelial cell death. Thus, the inhibition of the LTα3-TNFR2 axis could represent a useful therapeutic strategy to protect the respiratory airway epithelial lining from the oxidative stress challenge because of recurrent infection/inflammation cycles faced by CF patients.

Immunophenotypic and Gene Expression Analyses of the Inflammatory Microenvironment in High-Grade Oral Epithelial Dysplasia and Oral Lichen Planus

BackgroundOral lichen planus (OLP) and oral epithelial dysplasia (OED) present diagnostic challenges due to clinical and histologic overlap. This study explores the immune microenvironment in OED, hypothesizing that immune signatures could aid in diagnostic differentiation and predict malignant transformation.MethodsTissue samples from OED and OLP cases were analyzed using immunofluorescence/immunohistochemistry (IF/IHC) for CD4, CD8, CD163/STAT1, and PD-1/PDL-1 expression. RNA-sequencing was performed on the samples, and data was subjected to CIBERSORTx analysis for immune cell composition. Gene Ontology analysis on the immune differentially expressed genes was also conducted.ResultsIn OED, CD8 + T-cells infiltrated dysplastic epithelium, correlating with dysplasia severity. CD4 + lymphocytes increased in the basal layer. STAT1/CD163 + macrophages correlated with CD4 + intraepithelial distribution. PD-1/PDL-1 expression varied. IF/IHC analysis revealed differential immune cell composition between OED and OLP. RNA-sequencing identified upregulated genes associated with cytotoxic response and immunosurveillance in OED. Downregulated genes were linked to signaling, immune cell recruitment, and tumor suppression.ConclusionsThe immune microenvironment distinguishes OED and OLP, suggesting diagnostic potential. Upregulated genes indicate cytotoxic immune response in OED. Downregulation of TRADD, CX3CL1, and ILI24 implies dysregulation in TNFR1 signaling, immune recruitment, and tumor suppression. This study contributes to the foundation for understanding immune interactions in OED and OLP, offering insights into future objective diagnostic avenues.

Effects of multi-walled carbon nanotubes on gene and microRNA expression in human hepatocarcinoma HepG2 cells.

The usage of multi-walled carbon nanotubes (MWCNT) has increased exponentially in the past years, but, potential toxicity mechanisms are not clear. We studied the transcriptomic alterations induced by one multi-walled carbon nanotube (MWCNT) and its -OH and -COOH functionalized derivatives in human HepG2 cells. We showed that all three MWCNT treatments induced alterations in stress-related signaling pathways, inflammation-related signaling pathways, cholesterol synthesis pathways, proliferation-related pathways, senescence-related pathways and cancer-related pathways. In stress-related pathways, the acute phase response was induced in all three MWCNTs and all doses treated and ranked high. Other stress-related pathways were also related to the oxidative-induced signaling pathways, such as NRF-2 mediated oxidative stress response, hepatic fibrosis/Stella cell activation, iNOS signaling, and Hif1α signaling. Many inflammation-related pathways were altered, such as IL-8, IL-6, TNFR1, TNFR2, and NF-κB signaling pathways. These results were consistent with our previous results with exposures to the same three multi-walled carbon nanotubes in human lung BEAS-2B and also with results in mice and rats. From the microRNA target filter analysis, TXNIP & miR-128-3p interaction was present in all three MWCNT treatments, and maybe important for the induction of oxidative stress. CXCL-8 & miR-146-5p and Wee1 & miR-128-3p were only present in the cells treated with the parent and the OH-functionalized MWCNTs. These mRNA-miRNA interactions were involved in oxidative stress, inflammation, cell cycle, cholesterol biosynthesis and cancer related pathways. Target filter analysis also showed altered liver hyperplasia/hyperproliferation and hepatic cancer pathways. In short, target filter analysis complemented the transcriptomic analysis and pointed to specific gene/microRNA interactions that can help inform mechanism of action. Moreover, our study showed that the signaling pathways altered in HepG2 cells correlated well with the toxicity and carcinogenicity observed in vivo, indicating that HepG2 may be a good in vitro predictive model for MWCNT toxicity studies.

251 (PB-067) Poster - XIAP overexpressing Inflammatory Breast Cancer Patients have high Infiltration of immunosuppressive subsets and increased TNFR1 signaling targetable with Birinapant, a pan- IAP antagonist

251 (PB-067) Poster - XIAP overexpressing Inflammatory Breast Cancer Patients have high Infiltration of immunosuppressive subsets and increased TNFR1 signaling targetable with Birinapant, a pan- IAP antagonist

TNFR1 signaling is positively regulated by Jak-2 and c-Src via tyrosine phosphorylation.

Tumor necrosis factor alpha (TNFα, a.k.a. TNF) is a pleiotropic cytokine that exerts most of its effects through type 1 TNF receptor (TNFR1). Following TNF binding, TNFR1 recruits TRADD (tumor necrosis factor receptor type 1-associated DEATH domain). This interaction triggers formation of signalosome complexes which have been claimed to induce apoptosis (via downstream caspase activations), inflammation (via NF-kappaB) and stress pathways (JNK & p38). However, the mechanism underlying TNF-induced ERK and AKT activation is not completely revealed. TNFR1 is known to constitutively bind c-Src and JAK2, and these enzymes were previously demonstrated to modulate TNF signaling. Therefore, we hypothesized that TNFR1 could be tyrosine phosphorylated by JAK2 and/or c-Src and TNF-induced ERK and Akt activation may be mediated by this phosphorylation. Site-directed mutagenesis (SDM) was performed to substitute the two putative Tyrosine phosphorylation sites on TNFR1 (Y360 and Y401) with alanine (A) or with aspartic acid (D), to inhibit or mimic constitutive phosphorylation, respectively. In 293T cells transfected with mutated or wild type TNFR1, ERK and Akt activations were determined by western blot. TNFR1 interaction with c-Src, JAK2, p85 and Grb2 was examined by co-IP. NF-kB activation was measured by luciferase assay, while proliferation was measured by MTT and apoptosis was evaluated by colorimetric caspase 8/3 assays. For determination of necrosis rates, cellular DNA fragmentation ELISA was performed. In this report, we show that TNFR1 is phosphorylated by JAK2 tyrosine kinase at Y401 and by c-Src at Y360 and Y401. Phosphorylation of Y360 and Y401 augments the interaction of Grb2 and PI3Kp85 with TNFR1. We also demonstrate that phosphomimetic mutations of Y360D and Y401D enhance ERK and Akt activation. TNFR1 is tyrosine phosphorylated by both c-Src and JAK2, triggering a "noncanonical" pathway, that activates ERK and Akt.

A TNF-IL-1 circuit controls Yersinia within intestinal pyogranulomas.

Tumor necrosis factor (TNF) is a pleiotropic inflammatory cytokine that mediates antimicrobial defense and granuloma formation in response to infection by numerous pathogens. We previously reported that Yersinia pseudotuberculosis colonizes the intestinal mucosa and induces the recruitment of neutrophils and inflammatory monocytes into organized immune structures termed pyogranulomas (PG) that control Yersinia infection. Inflammatory monocytes are essential for the control and clearance of Yersinia within intestinal PG, but how monocytes mediate Yersinia restriction is poorly understood. Here, we demonstrate that TNF signaling in monocytes is required for bacterial containment following enteric Yersinia infection. We further show that monocyte-intrinsic TNFR1 signaling drives the production of monocyte-derived interleukin-1 (IL-1), which signals through IL-1 receptors on non-hematopoietic cells to enable PG-mediated control of intestinal Yersinia infection. Altogether, our work reveals a monocyte-intrinsic TNF-IL-1 collaborative inflammatory circuit that restricts intestinal Yersinia infection.

TNFα: TNFR1 signaling inhibits maturation and maintains the pro-inflammatory programming of monocyte-derived macrophages in murine chronic granulomatous disease.

Loss of NADPH oxidase activity results in proinflammatory macrophages that contribute to hyperinflammation in Chronic Granulomatous Disease (CGD). Previously, it was shown in a zymosan-induced peritonitis model that gp91phox-/- (CGD) monocyte-derived macrophages (MoMacs) fail to phenotypically mature into pro-resolving MoMacs characteristic of wild type (WT) but retain the ability to do so when placed in the WT milieu. Accordingly, it was hypothesized that soluble factor(s) in the CGD milieu thwart appropriate programming. We sought to identify key constituents using ex vivo culture of peritoneal inflammatory leukocytes and their conditioned media. MoMac phenotyping was performed via flow cytometry, measurement of efferocytic capacity and multiplex analysis of secreted cytokines. Addition of exogenous TNFα, TNFα neutralizing antibody and TNFR1-/- MoMacs were used to study the role of TNFα: TNFR1 signaling in MoMac maturation. More extensive phenotyping defined normal MoMac maturation and demonstrated failure of maturation of CGD MoMacs both ex vivo and in vivo. Protein components, and specifically TNFα, produced and released by CGD neutrophils and MoMacs into conditioned media was identified as critical to preventing maturation. Exogenous addition of TNFα inhibited WT MoMac maturation, and its neutralization allowed maturation of cultured CGD MoMacs. TNFα neutralization also reduced production of IL-1β, IL-6 and CXCL1 by CGD cells though these cytokines played no role in MoMac programming. MoMacs lacking TNFR1 matured more normally in the CGD milieu both ex vivo and following adoptive transfer in vivo. These data lend mechanistic insights into the utility of TNFα blockade in CGD and to other diseases where such therapy has been shown to be beneficial.

Tumor Necrosis Factor-α Receptor 1 Mediates Borna Disease Virus 1-Induced Changes in Peroxisomal and Mitochondrial Dynamics in Neurons.

Borna disease virus 1 (BoDV1) causes a persistent infection in the mammalian brain. Peroxisomes and mitochondria play essential roles in the cellular antiviral immune response, but the effect of BoDV1 infection on peroxisomal and mitochondrial dynamics and their respective antioxidant capacities is still not clear. Using different mouse lines-i.e., tumor necrosis factor-α transgenic (TNFTg; to pro-inflammatory status), TNF receptor-1 knockout (TNFR1ko), and TNFR2ko mice in comparison to wild-type (Wt) mice-we analyzed the abundances of both organelles and their main antioxidant enzymes, catalase and superoxide dismutase 2 (SOD2), in neurons of the hippocampal, cerebral, and cerebellar cortices. In TNFTg mice, a strong increase in mitochondrial (6.9-fold) and SOD2 (12.1-fold) abundances was detected; meanwhile, peroxisomal abundance increased slightly (1.5-fold), but that of catalase decreased (2.9-fold). After BoDV1 infection, a strong decrease in mitochondrial (2.1-6.5-fold), SOD2 (2.7-9.1-fold), and catalase (2.7-10.3-fold) abundances, but a slight increase in peroxisomes (1.3-1.6-fold), were detected in Wt and TNFR2ko mice, whereas no changes occurred in TNFR1ko mice. Our data suggest that the TNF system plays a crucial role in the biogenesis of both subcellular organelles. Moreover, TNFR1 signaling mediated the changes in peroxisomal and mitochondrial dynamics after BoDV1 infection, highlighting new mechanisms by which BoDV1 may achieve immune evasion and viral persistence.

Involvement of the tumour necrosis factor receptor system in glioblastoma cell death induced by palbociclib-heptamethine cyanine dye conjugate

Glioblastoma is the most common and aggressive primary brain tumour in adults. The development of anti-brain cancer agents are challenged by the blood-brain barrier and the resistance conferred by the local tumour microenvironment. Heptamethine cyanine dyes (HMCDs) are a class of near-infrared fluorescence compounds that have recently emerged as promising agents for drug delivery. We conjugated palbociclib, a cyclin-dependent kinase (CDK) 4/6 inhibitor, to an HMCD, MHI-148, and conducted drug activity analysis on primary patient-derived glioblastoma cell lines. In addition to the expected cytostatic activity, our in vitro studies revealed that palbociclib-MHI-148 conjugate resulted in an almost 100-fold increase in cytotoxicity compared to palbociclib alone. This shift of palbociclib from cytostatic to cytotoxic when conjugated to MHI-148 was due to increased DNA damage, as indicated by an increase in γH2AX foci, followed by an increased expression of key extrinsic apoptosis genes, including TP53, TNFR1, TRAIL, FADD and caspase 8. In addition, we observed a time-dependent increase in the cell surface expression of TNFR1, consistent with an observed increase in the secretion TNFα, followed by TNFR1 endocytosis at 48 h. The treatment of patient GBM cells with the palbociclib-MHI-148 conjugate prevented TNFα-induced NFκB translocation, suggesting conjugate-induced TNFR1 signalling favoured the TNFR1-mediated apoptotic response rather than the pro-inflammatory response pathway. Notably, pharmacological inhibition of endocytosis of TNFR1, and siRNA-knockdown of TNFR1 reversed the palbociclib-MHI-148-induced cell death. These results show a novel susceptibility of glioblastoma cells to TNFR1-dependent apoptosis, dependent on inhibition of canonical NFκB signalling using our previously reported palbociclib-HMCD conjugate.9u5DmcuTyYJ7m7Hnyrq7sRVideo

Atsttrin regulates osteoblastogenesis and osteoclastogenesis through the TNFR pathway

Osteoporosis is a systemic metabolic bone disorder for which inflammatory cytokines play an important role. To develop new osteoporosis treatments, strategies for improving the microenvironment for osteoblast and osteoclast balance are needed. Tumor necrosis factor-α (TNF-α) plays an important role in the initiation and development of osteoporosis. Atsttrin is an engineered protein derived from the growth factor, progranulin (PGRN). The present study investigates whether Atsttrin affects osteoclast formation and osteoblast formation. Here we show Atsttrin inhibits TNF-α-induced osteoclastogenesis and inflammation. Further mechanistic investigation indicates Atsttrin inhibits TNF-α-induced osteoclastogenesis through the TNFR1 signaling pathway. Moreover, Atsttrin rescues TNF-α-mediated inhibition of osteoblastogenesis via the TNFR1 pathway. Importantly, the present study indicates that while Atsttrin cannot directly induce osteoblastogenesis, it can significantly enhance osteoblastogenesis through TNFR2-Akt-Erk1/2 signaling. These results suggest that Atsttrin treatment could potentially be a strategy for maintaining proper bone homeostasis by regulating the osteoclast/osteoblast balance. Additionally, these results provide new insights for other bone metabolism-related diseases.

Synergy between Iron Deficiency and Inflammation Adversely Affects Pregnancy

Iron is essential for maternal and fetal health during pregnancy. In many developing countries, nutritional deficiencies are common, with anemia affecting upwards of 60% of pregnancies. In addition, infection and inflammation, which are known to be detrimental to pregnancy, are also very prevalent in these regions. Even in high-resource countries, approximately 22% of pregnant women are anemic, and obesity and diabetes are common causes of maternal inflammation. Thus, iron deficiency and inflammation commonly co-exist. In our studies, we noted in mice a striking synergistic adverse interaction between maternal iron deficiency and inflammation that resulted in embryotoxicity, not observed in either condition alone. Considering the high global prevalence of concomitant iron deficiency and inflammation in pregnant women, the issue has outstanding translational importance. We induced iron deficiency in mice by feeding females an iron-poor diet (4ppm) for 1-3 weeks prior to mating and for the duration of pregnancy. This resulted in development of iron deficiency and anemia in both mothers and embryos by term [embryonic day (E)18.5] and placental iron concentrations decreased by half, compared to pregnancies fed control diet (100ppm iron). To model acute systemic maternal inflammation, pregnant females received a single moderate dose of LPS (E. coli serotype O55:B5, 0.5μg/g bodyweight subcutaneously), either in the “2nd” (E8.5) or “3rd” (E15.5) trimester. Embryos and placentas from the E8.5 LPS treatment were collected at E18.5, while those from the E15.5 LPS treatment were collected after 24h. Consistent with other reports, LPS injection alone resulted in some adverse pregnancy outcomes: injection on E8.5 in iron replete dams caused abortion in 22% of dams (total n=9 dams) and at E15.5 caused preterm birth in 50% of dams (n=8) but no embryo malformations or resorption were noted. In contrast, when pregnant dams were both iron-depleted and LPS-injected, we observed a striking synergistic effect resulting in embryo death and resorption in all E8.5 treated pregnancies (n=3 dams). In E15.5-treated pregnancies, 60% of embryos were dead and resorbing (n=42 embryos from 5 dams). This dramatic interaction between iron deficiency and inflammation has not been reported previously. Our data suggest the mechanism appears to be, at least in part, related to activation of the TNFα/TNF-R1 pathway and apoptosis of placental endothelial cells. To test the contribution of the TNFα/TNF-R1 pathway to adverse synergy between iron deficiency and inflammation, we generated endothelial TNF-R1 knockout (KO) mice using two different Cre drivers ( Tnfsfr1 f/f;VEC-Cre + or Tnfsfr1 f/f;Tie2-Cre +). Tnfsfr1 f/f and KO females were iron-depleted and mated with Tnfsfr1 f/fmales. Pregnant dams received a single subcutaneous dose of LPS at E15.5 and pregnancy outcomes were assessed after 24h. Embryos from KO dams were protected from LPS-induced embryotoxicity (30% death, n=7 dams) compared to embryos from WT dams (70% death, n=3 dams). Surprisingly, embryo genotype did not play a role in adverse synergy. Our data suggest that maternal TNFR signaling facilitates adverse interactions between iron deficiency and inflammation during pregnancy, which results in embryo death. These studies are ongoing and our results could provide a mechanistic explanation for known adverse outcomes in complicated human pregnancies, and eventually help inform interventions to optimize outcomes in such pregnancies

Systemic treatment with a selective TNFR2 agonist alters the central and peripheral immune responses and transiently improves functional outcome after experimental ischemic stroke

Ischemic stroke often leaves survivors with permanent disabilities and therapies aimed at limiting detrimental inflammation and improving functional outcome are still needed. Tumor necrosis factor (TNF) levels increase rapidly after ischemic stroke, and while signaling through TNF receptor 1 (TNFR1) is primarily detrimental, TNFR2 signaling mainly has protective functions. We therefore investigated how systemic stimulation of TNFR2 with the TNFR2 agonist NewSTAR2 affects ischemic stroke in mice. We found that NewSTAR2 treatment induced changes in peripheral immune cell numbers and transiently affected microglial numbers and neuroinflammation. However, this was not sufficient to improve long-term functional outcome after stroke in mice.