TNF-α Infusion Research Articles

18F-FDG PET/CT Imaging Biomarkers for CAR-T Cell Therapy Outcomes in Large-B-Cell Lymphoma

Introduction: CAR-T cell therapy has transformed the care of patients with large-B-cell lymphoma (LBCL), but disease relapse and toxicities remain clinical challenges, while effective quantitative biomarkers are lacking. Our aim was to determine the role of 18F-FDG PET/CT imaging features as biomarkers in LBCL treated with CD19 CAR-T cell therapy. Methods: 18F-FDG PET/CT scans performed before CAR-T for LBCL were analyzed retrospectively by a board certified radiologist. Metabolic tumor volume (MTV), SUVmax, total lesion glycolysis (TLG), presence of bulky disease, and 106 radiomic features per lesion were recorded using the PET/CT and PyRadiomics plugins for FIJI. Laboratory values including IL-6, IL-10, TNF-α, LDH, ferritin, fibrinogen, and D-dimer pre CAR-T infusion were recorded. Response to CAR-T cell therapy was defined according to Lugano criteria. Cytokine release syndrome (CRS) and neurotoxicity were graded according to ASTCT guidelines. Progression-free survival (PFS) and overall survival (OS) were measured from time of CAR-T cell infusion; PFS events were death, relapse, and disease progression. Association between PET features, markers of inflammation and outcomes were studied with Spearman rank correlations, Cox, and logistic regression models. Multivariable generalized estimating equation models were applied to evaluate differential expression of radiomic features between responders and non-responders, accounting for the dependency structure among ROIs from the same individual. Results: A total of 180 LBCL patients (median age 66 years) treated with autologous CD19 CAR-T (93 axi-cel, 52 tisa-cel, 35 liso-cel) were included. Two time points were considered: 1) pre-apheresis scans (n=161; median SUVmax 19, MTV 56 mL, TLG 427 SUV*mL), and 2) pre-infusion, i.e., latest disease evaluation before CAR-T infusion including post-apheresis/post-bridging, as well as pre-apheresis scans for patients without additional imaging afterwards (n=180; median SUVmax 15, MTV 44 mL, TLG 338 SUV*mL), with a total of 341 PET/CT scans evaluated. At lymphodepletion, MTV was significantly correlated with multiple elevated inflammatory laboratory markers; at latest assessment, all PET and inflammatory markers showed significant correlations. MTV at last assessment before infusion was associated with increased risk of grade ≥ 2 CRS (OR 1.08 [95%CI 1.01-1.20], P=0.031), but not ICANS, in a multivariable logistic regression model adjusted for age, pre-lymphodepletion LDH, CAR-T product, bridging and SUVmax. A higher SUVmax at latest assessment was associated with a lower likelihood of non-complete response (CR) (OR 0.58 [0.41-0.80], P<0.001), while MTV lacked such an association after multivariable model. Increased MTV both before apheresis and at last disease assessment before infusion, but not SUVmax, were associated with decreased PFS (HR 1.11 [1.05-1.17] and HR 1.04 [1.02-1.07], respectively), in a multivariable adjustment. A similar pattern was observed for OS. Notably, the combination of MTV and LDH enabled clear separation of high and low PFS risk groups (P<0.001, Figure 1). Finally, when considering over 100 granular pre-infusion radiomic features ( Figure 2), which are not routinely evaluated in CAR-T patients, multiple novel determinants of CR were discovered, including gray level dependence, run length matrix, and shape-based features (P<0.05). Conclusions: To the best of our knowledge, this represents the most extensive analysis of quantitative 18F-FDG PET/CT features within an LBCL cohort undergoing CAR-T treatment. Our findings indicate that standard PET features (MTV and SUVmax) predict both toxicity and efficacy of CAR-T cell therapy, while also showing correlations with systemic inflammation. Additionally, we present, for the first time, the relevance of granular radiomic features in assessing the probability of treatment success. If confirmed, PET features could serve as effective biomarkers in CAR-T patients and have the potential to guide interventions in high-risk populations.

A comparison of rat models that best mimic immune-driven preeclampsia in humans.

Preeclampsia (PE), a hypertensive pregnancy disorder, can originate from varied etiology. Placenta malperfusion has long been considered the primary cause of PE. However, we and others have showed that this disorder can also result from heightened inflammation at the maternal-fetal interface. To advance our understanding of this understudied PE subtype, it is important to establish validated rodent models to study the pathophysiology and test therapies. We evaluated three previously described approaches to induce inflammation-mediated PE-like features in pregnant rats: 1) Tumor necrosis factor-α (TNF-α) infusion via osmotic pump from gestational day (GD) 14-19 at 50ng/day/animal; 2) Polyinosinic:polycytidylic acid (Poly I:C) intraperitoneal (IP) injections from GD 10-18 (alternate days) at 10mg/kg/day/animal; and, 3) Lipopolysaccharide (LPS) IP injections from GD 13-18 at 20ug-70ug/kg/day per animal. Maternal blood pressure was measured by tail-cuff. Upon sacrifice, fetal and placenta weights were recorded. Placenta histomorphology was assessed using H&E sections. Placenta inflammation was determined by quantifying TNF-α levels and inflammatory gene expression. Placenta metabolic and mitochondrial health were determined by measuring mitochondrial respiration rates and placenta NAD+/NADH content. Of the three rodent models tested, we found that Poly I:C and LPS decreased both fetal weight and survival; and correlated with a reduction in region specific placenta growth. As the least effective model characterized, TNF-α treatment resulted in a subtle decrease in fetal/placenta weight and placenta mitochondrial respiration. Only the LPS model was able to induce maternal hypertension and exhibited pronounced placenta metabolic and mitochondrial dysfunction, common features of PE. Thus, the rat LPS model was most effective for recapitulating features observed in cases of human inflammatory PE. Future mechanistic and/or therapeutic intervention studies focuses on this distinct PE patient population may benefit from the employment of this rodent model of PE.

Development of a Predictive Model for Cytokine Release Syndrome to Inform Risk Stratification and CRS Management Following Immunotherapy

Development of a Predictive Model for Cytokine Release Syndrome to Inform Risk Stratification and CRS Management Following Immunotherapy

The TNF-α/TNFR2 Pathway: Targeting a Brake to Release the Anti-tumor Immune Response.

Newly discovered anti-cancer immunotherapies, such as immune checkpoint inhibitors and chimeric antigen receptor T cells, focus on spurring the anti-tumor effector T cell (Teff) response. Although such strategies have already demonstrated a sustained beneficial effect in certain malignancies, a substantial proportion of treated patients does not respond. CD4+FOXP3+ regulatory T cells (Tregs), a suppressive subset of T cells, can impair anti-tumor responses and reduce the efficacy of currently available immunotherapies. An alternative view that has emerged over the last decade proposes to tackle this immune brake by targeting the suppressive action of Tregs on the anti-tumoral response. It was recently demonstrated that the tumor necrosis factor alpha (TNF-α) tumor necrosis factor receptor 2 (TNFR2) is critical for the phenotypic stabilization and suppressive function of human and mouse Tregs. The broad non-specific effects of TNF-α infusion in patients initially led clinicians to abandon this signaling pathway as first-line therapy against neoplasms. Previously unrecognized, TNFR2 has emerged recently as a legitimate target for anti-cancer immune checkpoint therapy. Considering the accumulation of pre-clinical data on the role of TNFR2 and clinical reports of TNFR2+ Tregs and tumor cells in cancer patients, it is now clear that a TNFR2-centered approach could be a viable strategy, once again making the TNF-α pathway a promising anti-cancer target. Here, we review the role of the TNFR2 signaling pathway in tolerance and the equilibrium of T cell responses and its connections with oncogenesis. We analyze recent discoveries concerning the targeting of TNFR2 in cancer, as well as the advantages, limitations, and perspectives of such a strategy.

Macrophage-Specific Toll Like Receptor 9 (TLR9) Causes Corpus Cavernosum Dysfunction in Mice Fed a High Fat Diet

BackgroundErectile dysfunction (ED) has been shown to be related with inflammatory markers in humans. Chronic infusion of TNF-α caused ED in mice while TNF-α knockout mice exhibited improvement in the relaxation of the corpus cavernosum (CC). AimSince obesity triggers an inflammatory process, we aimed to investigate the hypothesis that in obesity, Toll-like receptor 9 (TLR9) activation leads to increased TNF-α levels and impairment in CC reactivity. MethodsFour-week old male C57BL6 (WT) and TLR9 mutant (TLR9MUT) mice were fed a standard chow or high fat diet (HFD) for 12 weeks. Body weight and nonfasting blood glucose were analyzed. Contractile and relaxation responses of the CC were evaluated by electrical field stimulation and concentration response curves to phenylephrine and acetylcholine. Protein expression of nNOS, TNF-α, TNF-R1, TLR9 and MyD88 were measured by western blot. Plasma levels of TNF-α were measured by ELISA. OutcomeIn obesity, impaired cavernosal relaxation is associated with the activation of the innate immune system, by increasing the production of TNF-α through the activation of TLR9 in the macrophages. ResultsAfter 12 weeks of HFD both WT and TLR9MUT mice had increased body weight and nonfasting blood glucose compared to standard chow. In the CC, acetylcholine-induced relaxation was not changed. A trend to increased contraction to phenylephrine and KCl was seen in WT HFD only. electrical field stimulation-induced relaxation of the CC was decreased in WT HFD as well as nNOS expression in the CC of WT HFD, but not in TLR9MUT HFD. In the CC, protein expression of TLR9 and MyD88 was similar in all groups. While circulating levels of TNF-α presented only a trend to increase in mice fed HFD, the CC expression of TNF-α was increased only in WT HFD mice. Clinical TranslationThe innate immune system can be a target for the treatment of erectile complications in obesity. Strengths and LimitationsThis is the first study demonstrating that activation of TLR9 expressed in macrophages leads to impaired cavernosal relaxation. The main limitation of the study is the lack of understanding about the source/expression of the macrophages in the cavernous tissue. Further, herein, the experiments were performed only in isolated cavernous tissue (in vitro), thus the lack of knowledge on how the TLR9 modulates the in vivo response of the erectile tissue is another limitation of this study. ConclusionOur findings indicate that CC dysfunction observed in obesity is at least in part mediated by the production of TNF-α upon activation of TLR9 expressed in the macrophages. Priviero F, Calmasini F, Dela Justina V, et al. Macrophage-Specific Toll Like Receptor 9 (TLR9) Causes Corpus Cavernosum Dysfunction in Mice Fed a High Fat Diet. J Sex Med 2021;18:723-731.

Diffusible Tumor Necrosis Factor-Alpha (TNF-α) Promotes Noise-Induced Parvalbumin-Positive (PV+) Neuron Loss and Auditory Processing Impairments.

Neuroinflammation has been implicated in noise-induced auditory processing disorder and tinnitus. Certain non-auditory neurological disorders can also increase the levels of proinflammatory cytokines in the brain. To investigate the impact of increased brain proinflammatory cytokine levels on the central auditory pathway, we infused recombinant TNF-α into the right lateral cerebral ventricle, and examined auditory processing and cytoarchitecture of the auditory cortex. Microglial deramification was observed in the auditory cortex of mice that had received both TNF-α infusion and exposure to an 86-dB noise, but not in mice that had received either TNF-α infusion or noise exposure alone. In addition, we observed reduced cortical PV+ neuron density and impaired performances in gap detection and prepulse inhibition (PPI) only in mice that received both TNF-α infusion and the noise exposure. These results suggest that disease-related increase in brain proinflammatory cytokine release could be a risk factor for noise-induced auditory processing disorder and tinnitus.

Abstract P147: Attenuation Of Diuretic And Natriuretic Responses To Acute Saline Volume Expansion In Mice Pre-treated With Tumor Necrosis Factor-alpha (TNFα) Inhibitor, Etanercept.

High salt intake induces an immune response that activates the mononuclear phagocyte system (MPS) cells to produce TNFα. We have previously demonstrated that intravenous infusion of TNFα in mice induces diuresis and natriuresis by inhibiting renal tubular sodium reabsorption. We hypothesize that the intravenous saline infusion can also induce the production of TNFα from MPS cells and such increase in TNFα influences saline induced natriuresis in the kidney. To examine this hypothesis, we measured the changes in TNFα levels in plasma and in urinary excretion rate (U TNFα V) during intravenous infusion of isotonic saline (0.9% NaCl), first at euvolemic condition (3 μL/min for 60 min; Baseline period) and then at an enhanced infusion rate (12 μL/min for 90 min; Volume expansion period) in anesthetized mice (n=5). Renal blood flow (RBF) and glomerular filtration rate (GFR) were measured by PAH and inulin clearances respectively. TNFα concentration in plasma and urine samples were determined using ELISA kit (Ebioscience, Woburn, MA). TNFα level in plasma collected during baseline period was undetectable, however, TNFα was increased to 3.7±1.3 pg/mL during volume expansion (VE) period. Baseline U TNFα V level was 0.01±0.002 pg/min/g (kidney wt), which was increased to 0.11±0.03 pg/min/g (P<0.05) during VE period. VE increased urine flow (V, 5.4±0.5 to 22.0±5.1 μL/min/g; P<0.05) and sodium excretion (U Na V; 0.54±0.19 to 4.84 ±1.16 μmol/min/g; P<0.05) without significant changes in RBF (7.1±1.0 to 6.0±1.8 mL/min/g) and GFR (1.18±0.11 to 0.94±0.28 mL/min/g). Interestingly, the diuretic and natriuretic responses to VE were markedly attenuated in mice (n=5) pretreated with a TNFα inhibitor, etanercept (ETN; 0.5 mg/kg intraperitoneally once daily for 3 days prior to the experiment day). VE induced changes in ETN treated mice are as follows: V, 5.9±0.65 to 7.9 ±1.6 μL/min/g; U Na V, 0.43±0.6 to 1.1±0.28 μmol/min/g; RBF, 5.4±0.4 to 6.6±0.9 mL/min/g and GFR, 0.87±0.09 to 0.98±0.13 mL/min/g. These findings demonstrate for the first time that an intravenous saline volume infusion resulted an increase in TNFα level in plasma and in urine. These results strongly suggest a physiological role for TNFα in regulating renal excretory function during saline volume expansion.

Attenuating ischemia/reperfusion injury in rat cardiac transplantation by intracoronary infusion with siRNA cocktail solution.

Tumor necrosis factor-α (TNF-α), caspase-8, and complement component 5a receptor (C5aR) are known to play a crucial role in the myocardial ischemia/reperfusion (I/R) injury in cardiac transplantation. We hypothesized that the intracoronary infusion of TNF-α, caspase-8, and C5aR small interfering RNAs (siRNA) would protect cardiac allograft function and improve graft survival from I/R injury-induced organ failure. I/R injury of cardiac allograft was induced by syngeneic rat cardiac transplantation, in which the transplanted hearts were infused with saline or different amounts of siRNA cocktail solution targeting TNF-α, caspase-8, and C5aR via coronary arteries, and subsequently subjected to 18 h of preservation at 4°C in histidine–tryptophan–ketoglutarate (HTK) solution. The effects of siRNA cocktail solution on prolonged cold I/R injury were determined by assessing graft survival, histopathological changes, myeloperoxidase (MPO) activity, and malondialdehyde (MDA) concentration. The perfused siRNA cocktail solution successfully knocked down the expression of TNF-α, caspase-8, and C5aR in vitro and in vivo. Approximately 91.7% of control hearts that underwent 18 h of cold ischemia ceased their function after transplantation; however, 87.5% of cardiac allografts from the highest dose siRNA cocktail solution-pretreated hearts survived >14 days and exhibited minimal histological changes, with minimal cellular infiltration, interstitial edema, and inflammation and maximal reduced MPO activity and MDA concentration in the cardiac allograft. We demonstrated the feasibility and efficiency of infusion of TNF-α, caspase-8, and C5aR siRNA via the intracoronary route as a promising strategy for gene silencing against I/R injury in cardiac transplantation.

Isotonic saline infusion increases plasma and urinary levels of tumor necrosis factor‐alpha (TNFα) in mice; evidence for a physiological role of this cytokine in regulating renal function during saline volume expansion.

Although TNFα is considered to play its role in many pathological conditions, the physiological role of this cytokine in regulating many organs’ function including the kidney is increasingly recognized in recent days. Chronic high salt (HS) intake in diet induces an immune response that activates the mononuclear phagocyte system (MPS) cells to release TNFα that appears in the circulation in its soluble form (sTNFα). It has been shown that HS (4% NaCl) diet alone for 2 weeks increased MPS cell infiltration in the renal tissue in mice and this is associated with increases in the circulating sTNFα level in the plasma (Singh et al‐2013). It was also demonstrated that 1% salt added to the drinking water for 3 days in mice increases the urinary excretion rate of sTNFα indicating that its release in the kidney from the infiltrated MPS cells occur even at the early stage of HS intake (Hao et al‐2013). We have previously demonstrated that intravenous infusion of recombinant TNFα in mice induces natriuretic response by inhibiting tubular sodium reabsorption (Shahid et al‐2008; Majid‐2011). We hypothesize that the intravenous saline infusion can also induce the production of sTNFα from activated MPS cells due to an increase in salt content in immune tissues and such increase in sTNF influences saline induced natriuretic response in the kidney. To examine this hypothesis, we measured the changes in sTNFα levels in plasma and urinary excretion rate (UTNFαV) during intravenous infusion of isotonic saline (0.9% NaCl), first at euvolemic conditions (3 μL/min for 60 min; Baseline period) and then at an enhanced infusion rate (12 μL/min for 90 min; saline volume infusion period) in anesthetized mice (n=5). The concentration of sTNFα in plasma and urine samples were determined using ELISA kit (Ebioscience, Woburn, MA) for measuring this cytokine. Baseline level of plasma sTNFα was undetectable, however, the level was increased to 3.7±1.3 pg/mL during saline volume infusion period. Baseline UTNFαV level was 0.01±0.002 pg/min/g of kidney wt, which was increased to 0.11±0.03 pg/min/g (P<0.05) during volume infusion period. In another group of mice (n=5) pretreated with a TNFα inhibitor, etanercept (0.5 mg/kg intraperitoneally once daily for 3 days prior to the experiment day), it was observed that this increase in UTNFαV during saline volume infusion period was markedly attenuated (0.003±0.002 to 0.006±0.004 pg/min/g; P=n.s.). The usual natriuretic response (0.5±0.2 to 3.8 ±1.1 μmol/min/g; P<0.05) to saline volume infusion observed in control mice was seen markedly attenuated (0.4±0.1 to 0.8±0.3 μmol/min/g; P=n.s.) in etanercept pretreated mice. These findings demonstrate for the first time that an intravenous saline volume infusion resulted an increase in sTNFα level in plasma and in urine. These results strongly suggest a physiological natriuretic role for sTNFα in regulating renal excretory function during saline volume expansion.

Tumor necrosis factor-α impairs cerebral blood flow in pregnant rats: role of vascular β-epithelial Na+ channel.

Preeclampsia is a pregnancy-related disorder characterized by hypertension, vascular dysfunction and an increase in circulating inflammatory factors including the cytokine, tumor necrosis factor-α (TNF-α). Studies have shown that placental ischemia is associated with 1) increased circulating TNF-α, 2) attenuated pressure-induced cerebral vascular tone, and 3) suppression of β-epithelial Na+ channel (βENaC) protein in cerebral vessels. In addition to its role in epithelial Na+ and water transport, βENaC is an essential signaling element in transduction of pressure-induced (aka "myogenic") constriction, a critical mechanism of blood flow autoregulation. While cytokines inhibit expression of certain ENaC proteins in epithelial tissue, it is unknown if the increased circulating TNF-α associated with placental ischemia mediates the loss of cerebrovascular βENaC and cerebral blood flow regulation. Therefore, the purpose of this study was to test the hypothesis that increasing plasma TNF-α in normal pregnant rats reduces cerebrovascular βENaC expression and impairs cerebral blood flow (CBF) regulation. In vivo TNF-α infusion (200 ng/day, 5 days) inhibited cerebrovascular expression of βENaC and impaired CBF regulation in pregnant rats. To determine the direct effects of TNF-α and underlying pathways mediating vascular smooth muscle cell βENaC reduction, we exposed cultured VSMCs (A10 cell line) to TNF-α (1-100 ng/mL) for 16-24 h. TNF-α reduced βENaC protein expression in a concentration-dependent fashion from 0.1 to 100 ng/mL, without affecting cell death. To assess the role of canonical MAPK signaling in this response, VSMCs were treated with p38MAPK or c-Jun kinase (JNK) inhibitors in the presence of TNF-α. We found that both p38MAPK and JNK blockade prevented TNF-α-mediated βENaC protein suppression. These data provide evidence that disorders associated with increased circulating TNF-α could lead to impaired cerebrovascular regulation, possibly due to reduced βENaC-mediated vascular function.NEW & NOTEWORTHY This manuscript identifies TNF-α as a possible placental-derived cytokine that could be involved in declining cerebrovascular health observed in preeclampsia. We found that infusion of TNF-α during pregnancy impaired cerebral blood flow control in rats at high arterial pressures. We further discovered that cerebrovascular β-epithelial sodium channel (βENaC) protein, a degenerin protein involved in mechanotransduction, was reduced by TNF-α in pregnant rats, indicating a potential link between impaired blood flow and this myogenic player. We next examined this effect in vitro using a rat vascular smooth muscle cell line. TNF-α reduced βENaC through canonical MAPK-signaling pathways and was not dependent on cell death. This study demonstrates the pejorative effects of TNF-α on cerebrovascular function during pregnancy and warrants future investigations to study the role of cytokines on vascular function during pregnancy.

Tacrolimus restores the high- and low-pressure baroreflex control of renal sympathetic nerve activity in cisplatin-induced renal injury rats.

What is the central question of this study? Does immunosuppression restore the baroreflex control of renal sympathetic nerve activity (RSNA) in an animal model of kidney injury? What is the main finding and its importance? Tacrolimus, a calcineurin inhibitor, restored the high- and low-pressure baroreflex control of RSNA following cisplatin-induced renal injury. Cisplatin administration causes depression of renal haemodynamic and excretory function and is associated with renal sympatho-excitation and loss of baroreflex regulation of renal sympathetic nerve activity (RSNA). This study investigated whether administration of the immunosuppressant tacrolimus in this cisplatin-mediated renal injury model could restore, or the acute intra-renal infusion of tumour necrosis factor α (TNF-α) could blunt, the high- or low-pressure baroreflex control of RSNA. Groups of control and cisplatin-treated (5mgkg-1 , i.p. on day 0) rats received either saline or tacrolimus (0.25mgkg-1 day-1 , i.p.) for 7days prior to study. Rats were anaesthetised and prepared for measurement of mean arterial pressure (MAP), heart rate (HR) and RSNA. Baroreflex gain curves were generated and the degree of renal sympatho-inhibition determined (area under the curve (AUC) reported as %RSNAmin) during acute volume expansion. Intrarenal TNF-α infusion (0.3µgkg-1 h-1 ) in control rats decreased baroreflex gain by 32% (P<0.05) compared to intra-renal saline infusion. In the cisplatin group (MAP: 98±14mmHg; HR: 391±24beatsmin-1 ), the baroreflex gain for RSNA was 39% (P<0.05) lower than that for the control group (MAP: 91±7mmHg; HR: 382±29beatsmin-1 ). In cisplatin-treated rats given daily tacrolimus (MAP: 84±12mmHg; HR: 357±30beatsmin-1 ), the baroreflex gain and renal sympatho-inhibition (AUC, 2440±1071vs. 635±498%min) were restored to normal values. These findings provide evidence for the view that cisplatin administration initiates an injury involving inflammation which may contribute to the deranged baroreflex regulation of RSNA. This phenomenon appears mediated in part via the renal innervation.

Effect of placental growth factor in models of experimental pre-eclampsia and trophoblast invasion.

Placental growth factor (PlGF) is decreased in early gestation of pregnant women who subsequently develop pre-eclampsia. In this study, pre-emptive treatment with PlGF to prevent pre-eclampsia was evaluated in an in vivo rodent model of experimental pre-eclampsia (EPE) induced by TNF-α and in an in vitro model of human first-trimester trophoblast invasion. Pregnant C57/BL6 mice were treated with recombinant mouse placental growth factor-2 (rmPlGF-2) 100μg/kg/day IP from gestational day (gd) 10. Animals had EPE induced by continuous TNF-α infusion on gd 13 and were subject to either continuous blood pressure monitoring by radiotelemetry throughout pregnancy or live placenta T2 -weighted magnetic resonance imaging (MRI) to demonstrate placental function on gd 17. There was no difference in BP (P>.99), proteinuria (P=.9) or T2 values on MRI (P=.9) between control and rmPlGF-2-treated animals. On gd 13, animals treated with rmPlGF-2 demonstrated increased placenta PlGF (P=.01) and Toll-like receptor-3 (P=.03) mRNA expression as compared with controls. Fluorescent-labelled human uterine microvascular endothelial cells and HTR8/SVNeo cells were co-cultured on Matrigel™ and treated with recombinant human PlGF (rhPlGF) (10ng/mL) and/or TNF-α (0.5ng/mL). Trophoblast integration into endothelial networks was reduced by added TNF-α (P=.006), as was rhPlGF concentration in conditioned media (P<.0001). Cell integration was not ameliorated by addition of rhPlGF (P>.9). Although TNF-α-induced EPE was not reversed with pre-emptive rmPlGF-2, a further trial of pre-emptive rhPlGF in vivo is required to determine whether the absence of effect of rhPlGF demonstrated in vitro precludes PlGF as a preventative therapy for pre-eclampsia.

Neuronal NMDAR Currents of the Hippocampus and Learning Performance in Autoimmune Anti-NMDAR Encephalitis and Involvement of TNF-α and IL-6.

Among autoimmune encephalitis, patients with anti-N-methyl D- aspartate receptor (NMDAR) encephalitis typically present epileptic seizures, memory deficits and psychiatric symptoms. However, the signal mechanisms leading to the functional disorders of autoantibodies are largely unclear. In this study, anti-NMDAR antibody was administered into dentate gyri against the NR1 subunit of the NMDAR. The purpose of the study examined the effects of pro-inflammatory tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) on neuronal NMDAR currents of the hippocampus in rats with anti-NMDAR encephalitis and we further determined the role played by TNF-α and IL-6 in modulating learning performance. In results, we observed a decrease in amplitude of the NMDAR-mediated excitatory postsynaptic currents (NMDAR-EPSCs) in the hippocampal neurons of animals treated with anti-NMDAR. In those rats with anti-NMDAR, we also observed impaired learning performance in the Morris water maze and spatial working memory test. Of note, cerebral infusion of TNF-α and IL-6 worsened NMDAR-EPSCs and this was accompanied with exaggeration of impaired learning performance. In conclusion, our findings suggest that the role played by neuroinflammation in exacerbating the memory impairment found in animals treated with anti-NMDAR. Anti-inflammation is a potential target in improving the memory impairment induced by anti-NMDA encephalitis.

Neuroinflammation mediates noise-induced synaptic imbalance and tinnitus in rodent models.

Hearing loss is a major risk factor for tinnitus, hyperacusis, and central auditory processing disorder. Although recent studies indicate that hearing loss causes neuroinflammation in the auditory pathway, the mechanisms underlying hearing loss–related pathologies are still poorly understood. We examined neuroinflammation in the auditory cortex following noise-induced hearing loss (NIHL) and its role in tinnitus in rodent models. Our results indicate that NIHL is associated with elevated expression of proinflammatory cytokines and microglial activation—two defining features of neuroinflammatory responses—in the primary auditory cortex (AI). Genetic knockout of tumor necrosis factor alpha (TNF-α) or pharmacologically blocking TNF-α expression prevented neuroinflammation and ameliorated the behavioral phenotype associated with tinnitus in mice with NIHL. Conversely, infusion of TNF-α into AI resulted in behavioral signs of tinnitus in both wild-type and TNF-α knockout mice with normal hearing. Pharmacological depletion of microglia also prevented tinnitus in mice with NIHL. At the synaptic level, the frequency of miniature excitatory synaptic currents (mEPSCs) increased and that of miniature inhibitory synaptic currents (mIPSCs) decreased in AI pyramidal neurons in animals with NIHL. This excitatory-to-inhibitory synaptic imbalance was completely prevented by pharmacological blockade of TNF-α expression. These results implicate neuroinflammation as a therapeutic target for treating tinnitus and other hearing loss–related disorders.

Macrophage TNF-α licenses donor T cells in murine bone marrow failure and can be implicated in human aplastic anemia

AbstractInterferon-γ (IFN-γ) and tumor necrosis factor-α (TNF-α) have been implicated historically in the immune pathophysiology of aplastic anemia (AA) and other bone marrow (BM) failure syndromes. We recently defined the essential roles of IFN-γ produced by donor T cells and the IFN-γ receptor in the host in murine immune-mediated BM failure models. TNF-α has been assumed to function similarly to IFN-γ. We used our murine models and mice genetically deficient in TNF-α or TNF-α receptors (TNF-αRs) to establish an analogous mechanism. Unexpectedly, infusion of TNF-α−/− donor lymph node (LN) cells into CByB6F1 recipients or injection of FVB LN cells into TNF-αR−/− recipients both induced BM failure, with concurrent marked increases in plasma IFN-γ and TNF-α levels. Surprisingly, in TNF-α−/− recipients, BM damage was attenuated, suggesting that TNF-α of host origin was essential for immune destruction of hematopoiesis. Depletion of host macrophages before LN injection reduced T-cell IFN-γ levels and reduced BM damage, whereas injection of recombinant TNF-α into FVB-LN cell-infused TNF-α−/− recipients increased T-cell IFN-γ expression and accelerated BM damage. Furthermore, infusion of TNF-αR−/− donor LN cells into CByB6F1 recipients reduced BM T-cell infiltration, suppressed T-cell IFN-γ production, and alleviated BM destruction. Thus, TNF-α from host macrophages and TNF-αR expressed on donor effector T cells were critical in the pathogenesis of murine immune-mediated BM failure, acting by modulation of IFN-γ secretion. In AA patients, TNF-α–producing macrophages in the BM were more frequent than in healthy controls, suggesting the involvement of this cytokine and these cells in human disease.

214. TNFα impairs cerebral blood flow autoregulation during pregnancy

Background Cerebral complications are a leading cause of morbidity and mortality in preeclamptic pregnancies, accounting for approximately 40% of all preeclampsia/eclampsia related deaths. However, the underlying pathophysiological mechanisms remain unclear. Given the central role of the ischemic placenta in driving preeclampsia, we hypothesized that circulating factors released from the ischemic placenta directly impair cerebral vascular function and blood brain barrier permeability. Specifically, the role of TNFα, an inflammatory cytokine increased in the placenta and circulation of preeclamptic pregnancies, was examined. Methods Pregnant Sprague Dawley Rats were infused with vehicle or TNFα (200 ng/day i.p, from gestational day 14 to 19). Evan’s blue extravasation was measured in the cerebral cortex as an indicator of blood brain barrier (BBB) permeability, and brain water content was assessed as an indicator of cerebral edema at the time of euthanasia. In a separate experimental group, rats were infused with vehicle or TNFα in order to examine the impact on cerebral blood flow (CBF) autoregulation during pregnancy. CBF was measured by laser Doppler flowmetry under anesthesia. Rats were intubated and ventilated in order to maintain constant blood gases, and mean arterial pressure was elevated step-wise from 100-190 mmHg by infusion of phenylephrine to determine changes in CBF. Results Infusion of TNFα in pregnant rats caused a significant 10 mmHg increase in arterial pressure (n = 12, p Conclusions These data suggest that increasing TNFα to levels observed during preeclampsia results in cerebral vascular changes that may mechanistically underlie the increased risk for encephalopathy.

Quantification of placental change in mouse models of preeclampsia using magnetic resonance microscopy.

Abnormal development of the placenta is postulated to be central to the aetiology of preeclampsia. This study investigates changes in placental histopathology in mouse models of preeclampsia compared to the morphology using magnetic resonance microscopy (MRM) (11.7 T) of intact ex vivo tissue followed by 3D analysis of the image data. Here, C57BL/6JArc pregnant mice were subject to either normal pregnancy (n=3), or to one of two experimental models of preeclampsia; TNF-α infusion (n=3) or reduced uterine perfusion pressure (RUPP) (n=3). Placental tissue was collected at gestational day (gd) 17, fixed in formalin and incubated with Magnavist™ contrast agent, and high resolution images (50 μm × 50 μm × 50 μm voxels) obtained by magnetic resonance imaging at 11.74 T. Visual segmentation into placental subregions and three dimensional (3D) reconstruction followed by volume analysis was performed with Amira™ 3D analysis software. The significance of differences between treatment groups in total and regional volumes was assessed. In a single placenta the volumes measure by standard histology were compared. Three placentas from each animal were imaged, segmented into anatomical regions and 3D reconstructions generated. Total placental volume, labyrinth and decidual volume were not significantly different between groups. The junctional zone volume was found to be significantly larger in the RUPP animals (18.5±1.5 mm3) compared to TNF-α infused animals (15.8±1.5) or control animals (15.0±0.7, P<0.01). However, the decidual/junctional zone volume was smaller in the TNF-a compared to control animals (P<0.05). Placental structural change in experimental models of preeclampsia is able to be visualized and quantified using MRM and 3-D analysis. These techniques could prove to be a powerful tool in examining changes in placental morphology.

Understanding Angiotensin II Type 1 Receptor Signaling in Vascular Pathophysiology.

Angiotensin II is the most important endocrine ligand in the renin–angiotensin system (RAS), contributing to the development of several cardiovascular diseases including hypertension.1 Angiotensin II mediates its signal transduction and functions via the angiotensin II receptors.2 Historically, the presence of 2 subtypes of angiotensin II receptors were pharmacologically recognized based on the sensitivity to the first orally active nonpeptide angiotensin II receptor antagonist, losartan. The losartan-sensitive receptor was termed AT1 (angiotensin II receptor type 1) receptor. It was assumed to be a heterotrimeric GPCR (G protein–coupled receptor) because it generates inositol triphosphate and diacylglycerol, leading to intracellular Ca2+ elevation and protein kinase C activation, respectively. Most known physiological and pathophysiological functions of angiotensin II, including stimulation of vasoconstriction and salt and water reabsorption, are mediated through the AT1 receptor. The losartan-insensitive receptor was termed AT2 receptor, whereas its G protein coupling remains unclear.1,3,4 In 1991, 2 research groups in the United States independently isolated cDNA (termed AGTR1) encoding the mammalian AT1 receptor.5,6 Subsequently, rat AT2 receptor cDNA (AGTR2) was cloned in 1993.7,8 This pioneer work revealed complete amino acid sequences of the angiotensin II receptor subtypes belonging to the 7-transmembrane GPCR superfamily. In the early 1990s, several studies reported that AT1 receptor elicits tyrosine phosphorylation of multiple proteins and activation of MAPK (mitogen-activated protein kinase; eg, p42/p44 MAPK)/ERK (extracellular signal–regulated kinase; eg, ERK1/2) in various cell types including vascular smooth muscle cells (VSMC). The early 1990s also saw the establishment of the concept that angiotensin II via the AT1 receptor has a direct action on cardiac myocytes, fibroblasts, and VSMCs causing hypertrophic and fibrotic cardiovascular remodeling.9,10 The cardiovascular remodeling caused by angiotensin II seemed to be …

Placental ischemia-induced increases in brain water content and cerebrovascular permeability: role of TNF-α.

Cerebrovascular complications and increased risk of encephalopathies are characteristic of preeclampsia and contribute to 40% of preeclampsia/eclampsia-related deaths. Circulating tumor necrosis factor-α (TNF-α) is elevated in preeclamptic women, and infusion of TNF-α into pregnant rats mimics characteristics of preeclampsia. While this suggests that TNF-α has a mechanistic role to promote preeclampsia, the impact of TNF-α on the cerebral vasculature during pregnancy remains unclear. We tested the hypothesis that TNF-α contributes to cerebrovascular abnormalities during placental ischemia by first infusing TNF-α in pregnant rats (200 ng/day ip, from gestational day 14 to 19) at levels to mimic those reported in preeclamptic women. TNF-α increased mean arterial pressure (MAP, P < 0.05) and brain water content in the anterior cerebrum (P < 0.05); however, TNF-α infusion had no effect on blood-brain barrier (BBB) permeability in the anterior cerebrum or posterior cerebrum. We then assessed the role of endogenous TNF-α in mediating these abnormalities in a model of placental ischemia induced by reducing uterine perfusion pressure followed by treatment with the soluble TNF-α receptor (etanercept, 0.8 mg/kg sc) on gestational day 18. Etanercept reduced placental ischemia-mediated increases in MAP, anterior brain water content (P < 0.05), and BBB permeability (202 ± 44% in placental ischemic rats to 101 ± 28% of normal pregnant rats). Our results indicate that TNF-α mechanistically contributes to cerebral edema by increasing BBB permeability and is an underlying factor in the development of cerebrovascular abnormalities associated with preeclampsia complicated by placental ischemia.

Heme oxygenase induction attenuates TNF-α-induced hypertension in pregnant rodents.

Pre-eclampsia is a hypertensive disorder of pregnancy initiated by placental insufficiency and chronic ischemia. In response, several pathways activated in the placenta are responsible for the maternal syndrome, including increased production of the anti-angiogenic protein, sFlt-1, and inflammatory cytokines, especially tumor necrosis factor-alpha (TNF-α). Previous studies have demonstrated that heme oxygenase (HO) induction can block TNF-α pathways in vitro and attenuate placental ischemia-induced sFlt-1 in vivo. Here, we investigated whether HO-1 induction could attenuate TNF-α-induced hypertension in pregnant rats. In response to TNF-α infusion (100 ng/day i.p.), maternal mean arterial pressure (MAP) increased vs. control animals (104 ± 3 vs. 119 ± 3 mmHg). HO-1 induction had no effect in control animals, but significantly decreased MAP in TNF-α-infused animals (108 ± 2 mmHg). Placental vascular endothelial growth factor (VEGF) was decreased in response to TNF-α infusion (92 ± 4 vs. 76 ± 2 pg/mg). Placental sFlt-1 was increased by TNF-α infusion (758 ± 45 vs. 936 ± 46 pg/mg, p < 0.05), which trended to normalization by HO-1 induction (779 ± 98 pg/mg). In contrast, HO-1 induction had no significant effect on placental VEGF in TNF-α-infused animals. Taken together, these data suggest that one of the key mechanisms by which HO exerts cytoprotective actions in the placenta during inflammation due to chronic ischemia is through suppression of sFlt-1. Further work elucidating the bioactive metabolites of HO-1 in innate inflammatory responses to placental ischemia is warranted.