TNF-α Knockout Mice Research Articles

Simvastatin Protects Hepatocytes From Apoptosis by Suppressing the TNF-α/Caspase-3 Signaling Pathway in Mice With Burn Injury

To investigate the liver cellular apoptosis in response to burn injury and find out if statin treatment can ameliorate this process. The hypothesis is that statin may modulate apoptosis-related gene expression and thereby reduce hepatocytic apoptosis after burn injury. Mice were subjected to 30% full-thickness burn injury and then treated either with or without simvastatin. The livers were harvested for histological assessment and determinations of gene expression. To investigate the mechanism involved, tumor necrosis factor (TNF)-α and caspase-3 knockout (KO) mice were also used to evaluate the effects of burn injury and simvastatin treatment on burn-induced liver injury. The effects of simvastatin on TNF-α and caspase-3 expressions were also evaluated in cultured mouse hepatocytes. Burn injury induced significant liver damage, which was indicated by striking levels of apoptosis. Simvastatin reduced the apoptotic index in the livers of mice with burn injury and this effect could be abrogated by TNF-α or caspase-3 inhibitors. Simvastatin also decreased burn-induced TNF-α and caspase-3 expression in the liver. TNF-α and caspase-3 KO mice demonstrated lower levels of apoptotic hepatocytes in response to burn, and simvastatin did not further decrease hepatocyte apoptosis in either strain of KO mice. An in vitro study demonstrated that simvastatin suppresses TNF-α and caspase-3 expression in primary cultures of mouse hepatocytes. Simvastatin reduces mouse hepatocyte apoptosis by suppressing expression of the TNF-α/caspase-3 pathway.

Abstract WP262: Therapeutically Targeting TNFa-S1P Signaling Restores Microvascular Reactivity after Experimental Subarachnoid Hemorrhage

Background: Subarachnoid hemorrhage (SAH) is characterized by an initial hemorrhagic and ischemic brain injury followed by delayed macro- and microvascular constriction. Large-artery vasospasm and enhanced microcirculatory myogenic tone may contribute to delayed cerebral ischemia. Although this implies that therapeutic interventions must specifically correct the SAH-induced myogenic tone enhancement, current therapeutic approaches non-selectively interfere with vasoconstriction and risk disrupting cerebral autoregulation. This may explain why most interventions do not improve clinical outcome. This study identifies the molecular basis for exacerbated cerebrovascular constriction and validates new targets for SAH treatment. Methods: Wild-type, tumor necrosis factor α (TNFα) knockout, sphingosine-1-kinase (Sphk1) knockout and inducible, smooth muscle cell-targeted TNFα knockout mice were used. SAH was created by injection of 80 μl of arterial blood into the prechiasmatic cistern. Myogenic tone in the olfactory artery was assessed with a myograph system. Standard procedures for fluorescent immunolocalization, Western blotting and assessment of apoptosis were used. Results: SAH increased myogenic tone and vascular wall TNFα expression, without enhancing overall vascular contractility in response to phenylephrine. Knockout of TNFα globally or smooth muscle cell-specifically prevented SAH-induced increased myogenic tone. Inhibition of TNFα-shedding (TAPI, 50 μmol/L) or receptor-binding (etanercept, 10 mg/ml) eliminated SAH-mediated myogenic tone augmentation. Cystic fibrosis transmembrane regulator (CFTR) protein expression was down-regulated in cerebral arteries after SAH, which was abolished by antagonism of TNFα. Genetic mouse models confirmed that S1P signaling mediates the myogenic tone augmentation in SAH. Finally, disrupting TNFα signaling attenuated neuronal apoptosis in SAH animals. Conclusion: We identify a novel smooth muscle cell autocrine/paracrine signaling network that augments myogenic tone in SAH. It links TNFα, CFTR and sphingosine-1-phosphate (S1P) signaling. Targeting TNFα and the S1P 2 receptor subtype are potential therapeutic options to improve clinical outcome in SAH.

Attenuated EAN in TNF-α Deficient Mice Is Associated with an Altered Balance of M1/M2 Macrophages

The role of tumor necrosis factor (TNF)-α and its receptors in neuroautoimmune and neuroinflammatory diseases has been controversial. On the basis of our previous studies, we hereby aimed to further clarify TNF-α’s mechanism of action and to explore the potential role of TNF-α receptor (TNFR)1 as a therapeutic target in experimental autoimmune neuritis (EAN). EAN was induced by immunization with P0 peptide 180–199 in TNF-α knockout (KO) mice and anti-TNFR1 antibodies were used to treat EAN. Particularly, the effects of TNF-α deficiency and TNFR1 blockade on macrophage functions were investigated. The onset of EAN in TNF-α KO mice was markedly later than that in wild type (WT) mice. From day 14 post immunization, the clinical signs of TNF-α KO mice were significantly milder than those of their WT counterparts. Further, we showed that the clinical severity of WT mice treated with anti-TNFR1 antibodies was less severe than that of the control WT mice receiving PBS. Nevertheless, no difference with regard to the clinical signs of EAN or inflammatory infiltration in cauda equina was seen between TNF-α KO and WT mice with EAN after blockade of TNFR1. Although TNF-α deficiency did not alter the proliferation of lymphocytes in response to either antigenic or mitogenic stimuli, it down-regulated the production of interleukin (IL)-12 and nitric oxide (NO), and enhanced the production of IL-10 in macrophages. Increased ratio of regulatory T cells (Tregs) and reduced production of interferon (IFN)-γ in cauda equina infiltrating cells, and elevated levels of IgG2b antibodies against P0 peptide 180–199 in sera were found in TNF-α KO mice with EAN. In conclusion, TNF-α deficiency attenuates EAN via altering the M1/M2 balance of macrophages.

Abstract 1339: The role of TNF-α in PGE2-induced tumorigenesis in gastric cancer mouse model

Abstract Accumulating evidence has indicated that chronic inflammation is associated with cancer development. Mouse genetic studies have shown that the proinflammatory mediator, PGE2, plays an essential role in gastrointestinal tumorigenesis. Moreover, the proinflammatory cytokine TNF-α also plays a key role in tumorigenesis, including colitis-associated colon cancer. However, the relationship between PGE2 and TNF-α in tumorigenesis has not yet been fully investigated. We previously constructed a gastric tumor mouse model, Gan mice, that recapitulates human intestinal-type gastric cancer, in which both the PGE2 pathway and Wnt signaling are activated simultaneously in the stomach by transgenic expression of the COX-2, mPGES-1, and Wnt1 genes. The induction of the PGE2 pathway triggers an inflammatory response in the Gan mouse stomach, and the expression of proinflammatory cytokines and chemokines is induced in gastric tumor tissues. We herein examined the role of TNF-α in the PGE2-induced inflammation-associated gastric tumorigenesis by crossing Gan mice with TNF-α knockout mice. Importantly, the gastric tumorigenesis in the Gan TNF-α (-/-) compound mice was significantly suppressed in comparison to the control Gan mice. Moreover, the β-catenin level decreased significantly in the gastric tumors of Gan TNF-α (-/-) mice, suggesting a role for TNF-α in the promotion of Wnt signaling. Interestingly, inflammatory cells, including macrophages and CD4+ T cells, still infiltrated into the Gan TNF-α (-/-) mouse tumors, and the expression levels of IL-1α and IL-6 were almost the same as those in the control Gan mouse tumors. Accordingly, it is possible that TNF-α is an important tumor-promoting factor for gastric tumorigenesis, although TNF-α is not essential for PGE2-induced inflammation in the stomach. Notably, macrophages in the Gan TNF-α (-/-) mouse tumors lost their expression of the M2 marker CD206. It has been suggested that macrophages polarized to the M2(-like) type are important for tissue remodeling and tumorigenesis. It is therefore possible that TNF-α is required for macrophage polarization to a pro-tumorigenic status, which may be important for Wnt activation and gastric tumor promotion. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1339. doi:1538-7445.AM2012-1339

Involvement of histamine receptors in SAPK/JNK phosphorylation

Histamine is a mediator of inflammation in allergic disease and asthma. Stress activated protein kinases/c-jun N-terminal kinases (SAPK/JNK) are involved in asthma. This study examined the role of histamine receptors on the phosphorylation of SAPK/JNK in splenocytes. C57BL/6 mice splenocytes were treated with histamine (10−4M to 10−11M), and its selective receptor agonists, phorbol 12 myristate 13-acetate (PMA) was used as a positive control, and phosphorylation of SAPK/JNK was determined. Histamine (10−4M–10−8M) inhibited phosphorylation of SAPK/JNK. H1R agonist betahistine (10−5M) decreased SAPK/JNK phosphorylation and H2R agonist amthamine (10−5M) did not show any significant effect. However, H3R agonist methimepip (10−6M) and H4R agonist 4-methyl histamine (10−6M), increased SAPK/JNK phosphorylation.We used TNFα knockout mice to determine if histamine regulated SAPK/JNK phosphorylation via TNFα. While the effects of histamine and H1 agonists were similar to that of wild type mice in inhibiting the phosphorylation of SAPK/JNK, the effects of H3 and H4 agonists differed in TNFα knockout mice splenocytes. Activation of H3 receptors decreased SAPK/JNK phosphorylation in TNFα knockout mice, as opposed to an increase in wild type mice, whereas H4 agonist did not show any significant effect on the phosphorylation of SAPK/JNK. This data showed that histamine acting through H4 receptors caused the phosphorylation of SAPK/JNK via TNFα. The role of H4 receptors in pro-inflammatory response is intriguing.

Homeostatic plasticity mechanisms are required for juvenile, but not adult, ocular dominance plasticity.

Ocular dominance (OD) plasticity in the visual cortex is a classic model system for understanding developmental plasticity, but the visual cortex also shows plasticity in adulthood. Whether the plasticity mechanisms are similar or different at the two ages is not clear. Several plasticity mechanisms operate during development, including homeostatic plasticity, which acts to maintain the total excitatory drive to a neuron. In agreement with this idea, we found that an often-studied substrain of C57BL/6 mice, C57BL/6JOlaHsd (6JOla), lacks both the homeostatic component of OD plasticity as assessed by intrinsic signal imaging and synaptic scaling of mEPSC amplitudes after a short period of dark exposure during the critical period, whereas another substrain, C57BL/6J (6J), exhibits both plasticity processes. However, in adult mice, OD plasticity was identical in the 6JOla and 6J substrains, suggesting that adult plasticity occurs by a different mechanism. Consistent with this interpretation, adult OD plasticity was normal in TNFα knockout mice, which are known to lack juvenile synaptic scaling and the homeostatic component of OD plasticity, but was absent in adult α-calcium/calmodulin-dependent protein kinase II;T286A (αCaMKII(T286A)) mice, which have a point mutation that prevents autophosphorylation of αCaMKII. We conclude that increased responsiveness to open-eye stimulation after monocular deprivation during the critical period is a homeostatic process that depends mechanistically on synaptic scaling during the critical period, whereas in adult mice it is mediated by a different mechanism that requires αCaMKII autophosphorylation. Thus, our study reveals a transition between homeostatic and long-term potentiation-like plasticity mechanisms with increasing age.

Glial-Derived Prodegenerative Signaling in the Drosophila Neuromuscular System

We provide evidence for a prodegenerative, glial-derived signaling framework in the Drosophila neuromuscular system that includes caspase and mitochondria-dependent signaling. We demonstrate that Drosophila TNF-α (eiger) is expressed in a subset of peripheral glia, and the TNF-α receptor (TNFR), Wengen, is expressed in motoneurons. NMJ degeneration caused by disruption of the spectrin/ankyrin skeleton is suppressed by an eiger mutation or by eiger knockdown within a subset of peripheral glia. Loss of wengen in motoneurons causes a similar suppression providing evidence for glial-derived prodegenerative TNF-α signaling. Neither JNK nor NFκβ is required for prodegenerative signaling. However, we provide evidence for the involvement of both an initiator and effector caspase, Dronc and Dcp-1, and mitochondrial-dependent signaling. Mutations that deplete the axon and nerve terminal of mitochondria suppress degeneration as do mutations in Drosophila Bcl-2 (debcl), a mitochondria-associated protein, and Apaf-1 (dark), which links mitochondrial signaling with caspase activity in other systems.

Chronic Inflammatory Cardiomyopathy of Interferon γ–Overexpressing Transgenic Mice Is Mediated by Tumor Necrosis Factor-α

We recently described a model of inflammatory cardiomyopathy in interferon (IFN)-γ overexpressing transgenic mice stably circulating IFN-γ in the serum referred to as SAP--IFN-γ mice. SAP-IFN-γ transgenic mice show cardiac infiltration by mononuclear leukocytes, culminating in dilated cardiomyopathy characterized by an increase of left ventricular end diastolic diameter and reduction of fractional shortening. We hypothesized that the pathological mechanism underlying SAP-IFN-γ cardiomyopathy might be mediated by (auto)immune processes or tumor necrosis factor (TNF)-α synthesis from IFN-γ-activated macrophages. To verify these hypotheses, we crossed SAP-IFN-γ transgenic mice with immunodeficient Rag1(-/-) or TNF-α(-/-) knockout mice and analyzed the cardiac phenotype of the resulting double-mutant offspring. Immunodeficient Rag1(-/-) SAP-IFN-γ mice had a decreased impaired life span and intensive cardiac inflammatory reactions, showing that the cardiotoxic IFN-γ effect operative in SAP-IFN-γ mice was not mediated by an adaptive immune mechanism. SAP-IFN-γ TNF-α(-/-) hearts showed virtually no histopathological alterations, a significant reduction of cardiac infiltration by CD11c(+) dendritic cells and F4/80(+) macrophages, almost complete normalization of cardiac troponin T levels in serum and of left ventricular end diastolic diameter and fractional shortening, and a dramatic increase of life span, compared with SAP-IFN-γ transgenic controls. Thus, myocarditis and cardiomyopathy developing in IFN-γ-overexpressing transgenic mice is, to a significant degree, mediated by TNF-α. TNF-α-mediated cardiotoxicity in SAP-IFN-γ transgenic mice is independent of changes of apoptosis.

Induction of Thymic Stromal Lymphopoietin Production by Xylene and Exacerbation of Picryl Chloride-Induced Allergic Inflammation in Mice

Background: Some chemical compounds in the environment worsen allergic inflammation. In this study, we examined whether organic solvents induce the production of thymic stromal lymphopoietin (TSLP) which elicits Th2-type immune responses. Methods: Organic solvents were painted on the earlobes of BALB/c mice. The expression of TSLP in the ear was determined by ELISA. Results: Xylene and toluene, but not chloroform or ethyl acetate, induced the expression of mRNA for TSLP in the earlobe tissue. Among the aromatic compounds, xylene, especially m-xylene, and trimethylbenzene caused apparent TSLP production. The level of TSLP in the xylene-treated earlobes reached a maximum at 24 h, and TSLP was expressed in epithelial tissues. Production of TSLP was unaffected in mast cell-deficient W/W^v mice but apparently diminished in TNF-α knockout mice and IL-4 receptor knockout mice. Repeated painting of xylene for 7 days induced an increase in the weight of cervical lymph nodes and expression of OX40 ligand, both of which were inhibited in TSLP receptor knockout mice. Xylene promoted the picryl chloride-induced thickening of the ear and IL-4 production, which were reversed in TSLP receptor knockout mice. Conclusion: Xylene induced TSLP production, resulting in an exacerbation of allergic inflammation. Thus, xylene might be a good tool for examining the roles of TSLP in eliciting allergy in experimental animals.

Regulation of ERK2 phosphorylation by histamine in splenocytes

Histamine is implicated in allergic disease and asthma and ERK1/2 is involved in allergic inflammation including Th2 differentiation and proliferation. This study was designed to study the effects of histamine on ERK1/2 phosphorylation in splenocytes. C57/BL6 splenocytes were treated with different concentrations of histamine (10−4 to 10−11 M). Histamine (10−4 M) increased ERK2 phosphorylation. There was, however, no significant effect seen at other concentrations (10−11 to 10−6 M). Surprisingly, H1 receptor agonist β-histine (10−5 M), H2 agonist amthamine (10−5 M), H3 agonist methimepip (10−6 M), and H4 agonist 4-methyl histamine (10−6 M), all increased ERK2 phosphorylation. H1R antagonist pyrilamine (10−6 M), H2R antagonist ranitidine (10−5 M), H3/H4R antagonist thioperamide (10−6 M), and H3R antagonist clobenpropit (10−5 M) inhibited histamine-mediated ERK2 phosphorylation suggesting that all four histamine receptor subtypes played some role in this phosphorylation. Because tumor necrosis factor-α (TNF-α) causes phosphorylation of ERK1/2, we investigated whether histamine acted via secretion of TNF-α to affect ERK1/2 phosphorylation. As a consequence, TNF-α knockout mice were used and we found that there was inhibition of ERK1 and ERK2 phosphorylation by H2, H3, and H4 agonists. This was in contrast to the wild-type splenocytes where histamine augmented the phosphorylation of ERK2 via H2, H3, and H4 receptors. In TNF-α knockout mice histamine did not affect the phosphorylation of ERK2 via H1 receptors. The results suggested that histamine indirectly caused the ERK2 phosphorylation via its effects on the secretion of TNF-α and these effects were mediated via H1, H2, H3, and H4 receptors.

Tumor necrosis factor α knockout increases fertility of mice

Tumor necrosis factor α (TNFα) acts through two receptors, TNFα receptor| (TNFR|) and TNFα‖ (TNFR‖). Tumor necrosis factor α receptor| knockout mice had early senescence and poor fertility, whereas TNFR‖ knockout mice had reproductive performance not different from wild type (WT) mice. In the present study, TNFα knockout mice were used to study the roles of TNFα in female reproduction. The TNFα−/− mice had similar vaginal opening time (PD 27.6 ± 1.8 vs PD 27.7 ± 1.9, respectively, P > 0.05) and exogenous gonadotropin primed TNFα−/− mice shed more ova (28.9 ± 3.75 vs 9.8 ± 0.51, respectively, P = 0.001) compared with WT controls. At 2 mo of age, in 21 d, TNFα−/− mice had more estrous cycles than WT counterparts (6.0 ± 0.25 vs 4.0 ± 0.28, respectively, P < 0.05). Tumor necrosis factor α mutation also influenced ovarian follicular development; TNFα−/− mice had approximately a two-fold larger follicle pool in the early neonatal period (6087 ± 508.15 vs 3440 ± 261.91, respectively, P = 0.004), whereas TNFα knockout affected growth of primordial follicles to the antral stage as well. Moreover, TNFα−/− mice gave birth to 21% more pups than control mice during a 12 mo breeding period (37.38 ± 3.69 vs 22.38 ± 3.53, respectively, P = 0.03). At 1 y of age, the follicular reserve in TNFα−/− mice was more than that in WT mice. These physiological differences in TNFα−/− mice were associated with increased proliferation of granulosa cells and decreased apoptosis of oocytes. This was apparently the first demonstration that in the TNFα−/− mouse model, multiple parameters of ovarian function were altered, and that lack of TNFα increased fertility in mice.

The role of tumor necrosis factor-α for interleukin-10 production by murine dendritic cells

In the present study, we examined the role of tumor necrosis factor (TNF) in interleukin (IL)-10 production by dendritic cells (DCs) using bone-marrow derived DCs from wild type (WT) and TNF-α knockout (TNF-α −/−) mice. Toll-like receptor (TLR) stimulation induced substantial level of IL-10 production by WT DCs, but significantly low level of IL-10 production by TNF-α −/− DCs. In contrast, no significant difference was detected in IL-12 p40 production between WT and TNF-α −/− DCs. Addition of TNF-α during TLR stimulation recovered the impaired ability of TNF-α −/− DCs for IL-10 production. This recovery appeared to be associated with an activation of extracellular signal-regulated kinase, p38 mitogen-activated protein kinase, and phosphatidylinositol 3-kinase/Akt following the TNF-α addition. Blocking these kinases significantly inhibited IL-10 production by TNF-α −/− DCs stimulated with TLR ligands plus TNF-α. Thus, TNF-α may be a key molecule to regulate the balance between anti-inflammatory versus inflammatory cytokine production in DCs.

Inhibition of lipopolysaccharide-induced inflammatory responses by piperine

Piperine, a main component of Piper longum Linn. and Piper nigrum Linn., is a plant alkaloid with a long history of medical use. Piperine exhibits anti-inflammatory activity; however, the underlying mechanism remains unknown. We examined the effects of piperine on lipopolysaccharide (LPS)-induced inflammatory responses. Administration of piperine inhibited LPS-induced endotoxin shock, leukocyte accumulation and the production of tumor necrosis factor-α (TNF-α), but not of interleukin (IL)-1β and IL-6. In peritoneal macrophages, piperine inhibited LPS/poly (I:C)/CpG-ODN-induced TNF-α production. Piperine also inhibited LPS-induced endotoxin shock in TNF-α knockout (KO) mice. To clarify the inhibitory mechanism of LPS-induced endotoxin shock, type 1 interferon (IFN) mRNA expression was determined. Piperine inhibited LPS-induced expression of type 1 IFN mRNA. Piperine inhibited the levels of interferon regulatory factor (IRF)-1 and IRF-7 mRNA, and the phosphorylation and nuclear translocation of IRF-3. Piperine also reduced activation of signal transducer and activator of transcription (STAT)-1. In addition, activation of STAT-1 was inhibited in IFN-α/β-treated cells by piperine. These results suggest that piperine inhibits LPS-induced endotoxin shock through inhibition of type 1 IFN production.

Estrogen receptor–β activated apoptosis in benign hyperplasia and cancer of the prostate is androgen independent and TNFα mediated

Prostate cancer (PCa) and benign prostatic hyperplasia (BPH) are androgen-dependent diseases commonly treated by inhibiting androgen action. However, androgen ablation or castration fail to target androgen-independent cells implicated in disease etiology and recurrence. Mechanistically different to castration, this study shows beneficial proapoptotic actions of estrogen receptor-beta (ERbeta) in BPH and PCa. ERbeta agonist induces apoptosis in prostatic stromal, luminal and castrate-resistant basal epithelial cells of estrogen-deficient aromatase knock-out mice. This occurs via extrinsic (caspase-8) pathways, without reducing serum hormones, and perturbs the regenerative capacity of the epithelium. TNFalpha knock-out mice fail to respond to ERbeta agonist, demonstrating the requirement for TNFalpha signaling. In human tissues, ERbeta agonist induces apoptosis in stroma and epithelium of xenografted BPH specimens, including in the CD133(+) enriched putative stem/progenitor cells isolated from BPH-1 cells in vitro. In PCa, ERbeta causes apoptosis in Gleason Grade 7 xenografted tissues and androgen-independent cells lines (PC3 and DU145) via caspase-8. These data provide evidence of the beneficial effects of ERbeta agonist on epithelium and stroma of BPH, as well as androgen-independent tumor cells implicated in recurrent disease. Our data are indicative of the therapeutic potential of ERbeta agonist for treatment of PCa and/or BPH with or without androgen withdrawal.

A Pivotal Role of Endothelial-Specific NF-κB Signaling in the Pathogenesis of Septic Shock and Septic Vascular Dysfunction

Although the role of NF-kappaB in the pathogenesis of sepsis and septic shock has been extensively studied, little is known about the causative contribution of endothelial-intrinsic NF-kappaB to these pathological processes. In this study, we used transgenic (TG) mice (on FVB genetic background) that conditionally overexpress the NF-kappaB inhibitor, mutant I-kappaBalpha, selectively on endothelium and their transgene-negative littermates (wild type (WT)) to define the causative role of endothelial-specific NF-kappaB signaling in septic shock and septic vascular dysfunction. In WT mice, LPS challenge caused systemic hypotension, a significantly blunted vasoconstrictor response to norepinephrine, and an impaired endothelium-dependent vasodilator response to acetylcholine, concomitant with a markedly increased aortic inducible NO synthase expression, significantly elevated plasma and aortic levels of nitrite/nitrate, increased aortic TNF-alpha expression, and decreased aortic endothelial NO synthase (eNOS) expression. In TG mice whose endothelial NF-kappaB was selectively blocked, LPS caused significantly less hypotension and no impairments in vasoconstrictor and endothelium-dependent vasodilator responses, associated with significantly reduced aortic inducible NO synthase expression, decreased plasma and aortic levels of nitrite/nitrate, reduced aortic TNF-alpha expression, and increased aortic eNOS expression. TNF-alpha knockout mice prevented LPS-induced eNOS down-regulation. WT mice subjected to cecal ligation and puncture showed significant systemic hypotension, which was prevented in TG mice. Our data show that selective blockade of endothelial-intrinsic NF-kappaB pathway is sufficient to abrogate the cascades of molecular events that lead to septic shock and septic vascular dysfunction, demonstrating a pivotal role of endothelial-specific NF-kappaB signaling in the pathogenesis of septic shock and septic vascular dysfunction.

TNF-α plays a role in hepatocyte apoptosis in Niemann-Pick type C liver disease

Niemann-Pick type C (NPC) is a fatal autosomal recessive lysosomal storage disease clinically characterized by neurodegeneration and liver disease. Heterogeneous mutations in the NPC1 and NPC2 genes cause impaired egress of free cholesterol from lysosomes, leading to accumulation of cholesterol and glycosphingolipids. Key features of NPC liver disease include hepatic apoptosis, inflammation, and fibrosis. It is unclear what signaling events regulate these disease processes in NPC. We hypothesize that tumor necrosis factor alpha (TNF-alpha), which is involved in both proinflammatory and apoptotic signaling cascades, is a key mediator of inflammation, apoptosis, and fibrosis in NPC liver disease. In this study, we evaluated the role of TNF-alpha signaling in NPC liver disease by utilizing NPC1-specific antisense oligonucleotides to knock down NPC1 expression in control and TNF-alpha knockout mice. In the absence of TNF-alpha, NPC1 knockdown produced liver disease with significantly less inflammation, apoptosis, and fibrosis.

TNFα is required for cholestasis-induced liver fibrosis in the mouse

TNFα, a mediator of hepatotoxicity in several animal models, is elevated in acute and chronic liver diseases. Therefore, we investigated whether hepatic injury and fibrosis due to bile duct ligation (BDL) would be reduced in TNFα knockout mice (TNFα−/−). Survival after BDL was 60% in wild-type mice (TNFα+/+) and 90% in TNFα−/− mice. Body weight loss and liver to body weight ratios were reduced in TNFα−/− mice compared to TNFα+/+ mice. Following BDL, serum alanine transaminases (ALT) levels were elevated in TNFα+/+ mice (268.6 ± 28.2 U/L) compared to TNFα−/− mice (105.9 U/L ± 24.4). TNFα−/− mice revealed lower hepatic collagen expression and less liver fibrosis in the histology. Further, α-smooth muscle actin, an indicator for activated myofibroblasts, and TGF-β mRNA, a profibrogenic cytokine, were markedly reduced in TNFα−/− mice compared to TNFα+/+ mice. Thus, our data indicate that TNFα induces hepatotoxicity and promotes fibrogenesis in the BDL model.

Reduced testicular steroidogenesis in tumor necrosis factor-α knockout mice

We previously demonstrated that the expression of Mullerian inhibiting substance (MIS) in Sertoli cells is downregulated by tumor necrosis factor alpha (TNF-α), which is secreted by meiotic germ cells, in mouse testes. Several studies have reported that MIS that is secreted by Sertoli cells inhibits steroidogenesis and, thus, the synthesis of testosterone in testicular Leydig cells. Here, we demonstrate that in TNF-α knockout testes, which show high levels of MIS, steroidogenesis is decreased compared to that in wild-type testes. The levels of testosterone and the mRNA levels of steroidogenesis-related genes were significantly lower after puberty in TNF-α knockout testes than in wild-type testes. Furthermore, the number of sperm was reduced in TNF-α knockout mice. Histological analysis revealed that spermatogenesis is also delayed in TNF-α knockout testes. In conclusion, TNF-α knockout mice show reduced testicular steroidogenesis, which is likely due to the high level of testicular MIS compared to that seen in wild-type mice.

TLR2 Expression in Astrocytes Is Induced by TNF-α- and NF-κB-Dependent Pathways

Astrocytes participate in CNS innate immune responses as evident by their ability to produce a wide array of inflammatory mediators upon exposure to diverse stimuli. Although we have established that astrocytes use TLR2 to signal inflammatory mediator production in response to Staphylococcus aureus, a common etiological agent of CNS infections, the signal transduction pathways triggered by this pathogen and how TLR2 expression is regulated remain undefined. Three disparate inhibitors that block distinct steps in the NF-kappaB pathway, namely SC-514, BAY 11-7082, and caffeic acid phenethyl ester, attenuated NO, TNF-alpha, and CXCL2 release from S. aureus-activated astrocytes. Among these proinflammatory mediators, autocrine/paracrine TNF-alpha was pivotal for augmenting TLR2 expression, since receptor levels were not elevated in astrocytes isolated from TNF-alpha knockout mice upon bacterial exposure. Since TLR2 is critical for signaling astrocytic cytokine production in response to S. aureus, we evaluated the effect of TNF-alpha loss on proinflammatory mediator release. Interestingly, among the molecules assayed, only NO production was significantly attenuated in TNF-alpha knockout astrocytes compared with wild-type cells. Similar results were obtained following LPS treatment, suggesting that TNF-alpha is an important regulator of astrocytic TLR2 expression and NO release in response to diverse microbial stimuli. In addition, NF-kappaB inhibitors attenuated TNF-alpha-induced TLR2 expression in astrocytes. Overall, this study suggests that two important anti-bacterial effector molecules, TLR2 and NO, are regulated, in part, by NF-kappaB-dependent autocrine/paracrine effects of TNF-alpha in astrocytes.

Tumor Necrosis Factor-α Develops Late Anaphylactic Reaction through Cytosolic Phospholipase A2 Activation

Background: We have recently reported that tumor necrosis factor (TNF)-α plays an important role in the development of a late anaphylactic reaction, but the downstream pathway beyond TNF-α remains unclear. Objective: It was the aim of this study to examine whether TNF-α induces late-phase anaphylaxis via the activation of cytosolic phospholipase A₂ (cPLA₂). Methods: Using a murine model of active systemic anaphylaxis to penicillin V, the induction of the late phase of anaphylaxis was quantified by measuring the increase in hematocrit value as well as the plasma level of platelet-activating factor in TNF-α knockout mice. Phosphorylation of mitogen-activated protein kinases (MAPKs) and cPLA₂ was measured by immunoprecipitation. cPLA₂ activity was assessed by using 1-stearoyl-2-[1-¹⁴C] arachidonyl-sn-glycero-3-phosphocholine as the substrate. Results: Phosphorylation and enzymatic activity of cPLA₂, and phosphorylation of the 3 known MAPKs, i.e. p38, extracellular signal-regulated kinase 1/2 (ERK1/2) and c-Jun NH2-terminal kinase, were markedly increased in a TNF-α-dependent way in the lungs of mice undergoing anaphylaxis. A specific cPLA₂ inhibitor significantly attenuated the late anaphylactic symptoms. Either p38 or an ERK inhibitor significantly attenuated not only cPLA₂ phosphorylation and activity, but also the late-phase anaphylaxis. Conclusion: TNF-α-induces cPLA₂ activation through the pathway involving p38 MAPK and ERK activation and appears to be the key mechanism leading to the development of late-phase anaphylaxis.