TNFR1 KO Research Articles

Abstract 229: TNF Receptor 1 Signaling Is Critically Involved in Mediating Angiotensin II-Induced Cardiac Fibrosis and Dysfunction

Angiotensin-II (Ang-II) plays a key role in the development of cardiomyopathies, as it is associated with many conditions involving heart failure and pathologic hypertrophy. Using a murine model of Ang-II infusion, we found that Ang-II induced the synthesis of monocyte chemoattractant protein 1 (MCP-1) that mediated the uptake of CD34 + /CD45 + monocytic cells into the heart. These precursor cells differentiated into collagen-producing fibroblasts and were responsible for the Ang-II-induced development of reactive fibrosis. Preliminary in vitro data using our monocyte-to-fibroblast differentiation model, suggested that Ang-II required the presence of TNF to induce fibroblast maturation from monocytes. In vivo, they indicated that in mice deficient of both TNF receptors (TNFR1 and TNFR2), Ang-II-induced fibrosis was absent. We now assessed the hypothesis that specific TNFR1 signaling is necessary for Ang-II-mediated cardiac fibrosis. Mice deficient in either TNFR1 (TNFR1-KO) or TNFR2 (TNFR2-KO) were subjected to continuous infusion of Ang-II for 1 to 6 weeks (n=6-8/group). Compared to wild-type, we found that in TNFR1-KO, but not in TNFR2-KO mouse hearts, collagen deposition was attenuated, as was cardiac α-smooth muscle actin protein (a marker for activated fibroblasts). When we isolated viable cardiac fibroblasts and characterized them by flow cytometry, we found that Ang-II infusion in TNFR1-KO, but not in TNFR2-KO, resulted in a marked decrease of CD34 + /CD45 + cells. Quantitative RT-PCR demonstrated a striking reduction of type 1 and 3 collagen, as well of MCP-1 mRNA expression in TNFR1-KO mouse hearts. Further measurements of cardiovascular parameters indicated that TNFR1-KO animals developed lesser Ang-II-mediated LV remodeling, smaller changes in E-linear deceleration times/rates over time, and displayed a lower Tei index (a heart rate independent marker of cardiac function), indicating less stiffness in TNFR1-KO hearts compared to wild-type and TNFR2-KO hearts. The data suggest that Ang-II-dependent cardiac fibrosis requires TNF and its signaling through TNFR1 which enhances the induction of MCP-1 and uptake of monocytic fibroblast precursors that are associated with reactive fibrosis and cardiac remodeling and function.

TNFR2 on non‐haematopoietic cells is required for Foxp3+ Treg‐cell function and disease suppression in EAE

The TNF/TNFR system exerts multiple proinflammatory and immunosuppressive functions in the pathogenesis of chronic inflammation and autoimmunity. In EAE, the experimental model of Multiple Sclerosis (MS), genetic ablation of TNFR2, results in exacerbated immune reactivity and chronic disease course. The underlying mechanism driving this immunosuppressive function of TNFR2 remains unclear. We show here that chronic exacerbated EAE in TNFR2 KO mice is associated with increased Th17-cell responses and reduced numbers of Foxp3(+) Treg cells both in the spinal cord and peripheral lymphoid organs. Treg cells from TNFR2-deficient animals developing EAE show decreased proliferative and suppressive functions, both ex vivo and in vivo, and appear responsible for the exacerbated non-remitting disease, as evidenced by phenotypic rescue following adoptive transfer of Treg cells from WT but not TNFR2(-/-) donors. Reciprocal BM transplantation experiments between WT and TNFR2-deficient mice demonstrated that the capacity of TNFR2 to support Treg-cell expansion and function during EAE is non-intrinsic to Treg or other haematopoietic cells but requires expression of TNFR2 in radiation-resistant cells of the host. These results reveal a previously unsuspected role for non-haematopoietic TNFR2 in modulating Treg-cell expansion and immune suppression during development of autoimmunity and suggest that a similar mechanism may affect chronicity and relapses characterizing human autoimmune disease, including MS.

Characterization of Chronic Cutaneous Lesions from TNF-Receptor-1-Deficient Mice Infected byLeishmania major

Leishmania major-infected TNF receptor 1 deficient (TNFR1 KO) mice resolve parasitism but fail to resolve lesions, while wild-type mice completely heal. We investigated the cell composition, cytokine production, and apoptosis in lesions from L. major-infected TNFR1 KO and wild-type (WT) mice. Chronic lesions from L. major-infected TNFR1 KO mice presented larger number of CD8+ T and Ly6G+ cells. In addition, higher concentrations of mRNA for IFN-γ CCL2 and CCL5, as well as protein, but lower numbers of apoptotic cells, were found in lesions from TNFR1 KO mice than in WT, at late time points of infection. Our studies showed that persistent lesions in L. major-infected TNFR1 KO mice may be mediated by continuous migration of cells to the site of inflammation due to the presence of chemokines and also by lower levels of apoptosis. We suggest that this model has some striking similarities to the mucocutaneous clinical form of leishmaniasis.

Lipopolysaccharide-induced bone resorption is increased in TNF type 2 receptor-deficient mice in vivo

The release of tumor necrosis factor (TNF)-alpha from macrophages upon stimulation of lipopolysaccharide (LPS) is a major etiological factor of inflammatory bone disease and elicits the effects through TNF receptors type 1 and 2. Given the importance of TNF-alpha action on osteoclastic bone resorption, the role of TNF type 2 receptor (TNFR2) on bone resorption has not been elucidated well so far. The purpose of this study is to investigate the role of TNFR2 on LPS-induced inflammatory bone resorption in vivo. LPS at 10 mg/kg (Re 595) was injected subcutaneously on calvariae of wild-type (WT), TNF type 1 receptor (TNFR1)-deficient (KO), and TNFR2 KO mice, killed on day 5 after the LPS injection. The calvarial bone mineral density (BMD) was significantly decreased by LPS compared to the vehicle-injected control in WT mice, but not in TNFR1 KO mice. Interestingly, the decrease of calvarial BMD and the increase of the osteoclast number by LPS in TNFR2 KO mice seemed to be more than those in WT mice. Furthermore, the significant decrease by LPS on the BMD of tibiae, femurs, and lumber vertebrae were observed only in TNFR2 KO mice. Histomorphometric analysis of tibiae showed the significant increases of osteoclast number and surface in the LPS-injected TNFR2 KO mice, and the levels of urinary deoxypyridinoline reflected these increases of bone resorption parameters. The present data indicate that TNFR1 is critical for bone resorption at the site of LPS injection and that TNFR2 might have a protective role on the LPS-induced inflammatory bone resorption process.

Sleep-wake behavior and responses to sleep deprivation of mice lacking both interleukin-1β receptor 1 and tumor necrosis factor-α receptor 1

Data indicate that interleukin (IL)-1β and tumor necrosis factor-α (TNFα) are involved in the regulation of non-rapid eye movement sleep (NREMS). Previous studies demonstrate that mice lacking the IL-1β type 1 receptor spend less time in NREMS during the light period, whereas mice lacking the p55 (type 1) receptor for TNFα spend less time in NREMS during the dark period. To further investigate roles for IL-1β and TNFα in sleep regulation we phenotyped sleep and responses to sleep deprivation of mice lacking both the IL-1β receptor 1 and TNFα receptor 1 (IL-1R1/TNFR1 KO). Male adult mice (IL-1R1/TNFR1 KO, n = 14; B6129SF2/J, n = 14) were surgically instrumented with EEG electrodes and with a thermistor to measure brain temperature. After recovery and adaptation to the recording apparatus, 48 h of undisturbed baseline recordings were obtained. Mice were then subjected to 6 h sleep deprivation at light onset by gentle handling. IL-1R1/TNFR1 KO mice spent less time in NREMS during the last 6 h of the dark period and less time in rapid eye movement sleep (REMS) during the light period. There were no differences between strains in the diurnal timing of delta power during NREMS. However, there were strain differences in the relative power spectra of the NREMS EEG during both the light period and the dark period. In addition, during the light period relative power in the theta frequency band of the REMS EEG differed between strains. After sleep deprivation, control mice exhibited prolonged increases in NREMS and REMS, whereas the duration of the NREMS increase was shorter and there was no increase in REMS of IL-1R1/TNFR1 KO mice. Delta power during NREMS increased in both strains after sleep deprivation, but the increase in delta power during NREMS of IL-1R1/TNFR1 KO mice was of greater magnitude and of longer duration than that observed in control mice. These results provide additional evidence that the IL-1β and TNFα cytokine systems play a role in sleep regulation and in the alterations in sleep that follow prolonged wakefulness.

Abstract 1566: Tumor Necrosis Factor Receptor 2 Signaling Protects Myocardial Function Following Ischemia In Females Via Stat3, Socs3 And Vegf

Introduction: Myocardial ischemia/reperfusion (I/R) increases local TNF production, which contributes to myocardial dysfunction; however, the mechanisms remain incompletely understood. Some investigations have shown that TNF may be protective and others have shown it to be injurious. Our previous studies have shown that females have improved myocardial functional recovery, TNFR1 signaling resistance, and increased SOCS3 expression following acute I/R when compared to males. However, the effect of TNFR2 on gender differences in myocardial functional recovery following I/R is unknown. We hypothesized that TNFR2 mediates myocardial protection from I/R through STAT3, SOCS3 and VEGF in both genders, TNFR2 elicits greater protective signaling in females as compared to males. Methods: Isolated male and female mouse hearts (Langendorff) from TNFR2 knockout (TNFR2 KO), TNFR1/2KO and wild type (WT) (n=3– 6/group) were subjected to 20 minutes of ischemia followed by 60 minutes of reperfusion. Myocardial function (+/−dP/dt) was continuously monitored. After I/R, hearts were analyzed for STAT3, SOCS3 and VEGFa gene expression (RT real-time PCR), STAT3 activation (Western blot). Two-way ANOVA or student’s t-test, p<0.05 statistically significant. Results: TNFR2 deficiency decreased post-ischemic recovery of +/−dP/dt in both genders, but with greater effect on females. This deleterious effect of TNFR2KO was associated with a decrease in gene expression of VEGFa to 38%, STAT3 to 51% and SOCS3 to 36% in female TNFR2KO hearts, but without significant change in males. Additionally, TNFR1/2 KO decreased myocardial function in female hearts, but not in males. Interestingly, TNFR1/2 deficiency increased gene expression of VEGFa by 37%, SOCS3 by 30% in males, but decreased VEGFa by 48%, STAT3 by 45% and SOCS3 by 49% in females. Moreover, STAT3 activation was decreased in female TNFR2KO and TNFR1/2KO hearts. Conclusions: TNFR2 mediates myocardial protection through VEGF, STAT3 and SOCS3 following I/R in both genders, but with a relatively greater benefit of TNFR2 signaling observed in females. These data expand upon gender-based TNF signaling in myocardium, which may in part explain certain clinical differences with important therapeutic implications.

Differential role of tumor necrosis factor receptors in mouse brain inflammatory responses in cryolesion brain injury

Tumor necrosis factor-alpha (TNF-alpha) is one of the mediators dramatically increased after traumatic brain injury that leads to the activation, proliferation, and hypertrophy of mononuclear, phagocytic cells and gliosis. Eventually, TNF-alpha can induce both apoptosis and necrosis via intracellular signaling. This cytokine exerts its functions via interaction with two receptors: type-1 receptor (TNFR1) and type-2 receptor (TNFR2). In this work, the inflammatory response after a freeze injury (cryolesion) in the cortex was studied in wild-type (WT) animals and in mice lacking TNFR1 (TNFR1 KO) or TNFR2 (TNFR2 KO). Lack of TNFR1, but not of TNFR2, significantly decreased the inflammatory response and tissue damage elicited by the cryolesion at both 3 and 7 days postlesion, with decreased gliosis, lower IL-1beta immunostaining, and a reduction of apoptosis markers. Cryolesion produced a clear induction of the proinflammatory cytokines interleukin (IL)-1alpha, IL-1beta, IL-6, and TNF-alpha; this induction was significantly lower in the TNFR1 KO mice. Host response genes (ICAM-1, A20, EB22/5, and GFAP) were also induced by the cryolesion, but to a lesser extent in TNFR1 KO mice. Lack of TNFR1 signaling also affected the expression of apoptosis/cell death-related genes (Fas, Rip, p53), matrix metalloproteinases (MMP3, MMP9, MMP12), and their inhibitors (TIMP1), suggesting a role of TNFR1 in extracellular matrix remodeling after injury. However, GDNF, NGF, and BDNF expression were not affected by TNFR1 deficiency. Overall, these results suggest that TNFR1 is involved in the early establishment of the inflammatory response and that its deficiency causes a decreased inflammatory response and tissue damage following brain injury.

Tumor necrosis factor‐α receptor 1 (p55) knockout only transiently decreases the activation of c‐Jun and does not affect the survival of axotomized dopaminergic nigral neurons

The activation of the c-Jun N-terminal kinases and their substrate transcription factor c-Jun is central to the death of dopaminergic neurons of the substantia nigra pars compacta (SNC) but the underlying signal cascades are poorly understood. We have studied the impact of the p55 tumor necrosis factor-alpha receptor (TNF-R) 1 on the N-terminal phosphorylation of c-Jun and the survival of the dopaminergic SNC neurons after transection of the medial forebrain bundle. The axotomy raised the immunoreactivities of tumor necrosis factor-alpha, p75 TNF-R2 and ED1 (ectodysplasin A) in the substantia nigra equally in wildtype and knockout (ko) mice and of TNF-R1 in wildtype mice. Importantly, TNF-R1 ko significantly reduced the early phosphorylation of c-Jun between 18 h and 3 d post-axotomy but the functional deficiency of TNF-R1 did not affect the survival of the dopaminergic neurons up to day 30. These findings demonstrate that: (i) TNF-R1 is involved in the early cell body response after axon transection; (ii) TNF-R1 operates upstream of c-Jun N-terminal kinase/c-Jun, the central signal system of nerve fiber injury, and (iii) the failure of persistent reduction of activated c-Jun is linked to the failure of protection of dopaminergic SNC neurons by TNF-R1 ko.

Antisense abrogation of DENN expression induces apoptosis of leukemia cells in vitro, causes tumor regression in vivo and alters the transcription of genes involved in apoptosis and the cell cycle

We previously reported that messenger RNA expression of DENN (differentially expressed in normal and neoplastic cells) is considerably higher in cancer cell lines than in normal cells. In our present study, we established that certain cancer cell lines express conspicuously higher levels of the 2 DENN isoforms in contrast to the 2 pro-apoptotic IG20 isoforms. Antisense DENN oligodeoxynucleotide treatment of K36 cells in vitro induced extensive apoptosis, while antisense DENN silencing of K36 tumor-bearing mice caused significant tumor regression in vivo. Compared to wild-type murine embryonic fibroblasts, antisense treatment of NFkappaB and TNFR1 KO cells resulted in markedly more pronounced cell death, whereas antisense-treated TNFalpha and TNFR2 knockouts exhibited less prominent apoptosis. Cell viability and apoptosis were authenticated by flow cytometry, membrane integrity, TUNEL, annexin V assays, histology and electron microscopy. Antisense abrogation of DENN expression culminated in upregulated expression of TNFR2, TRAIL and Fas, but downregulation of TNFalpha, TNFR1 and cyclin D3. Conversely, DENN overexpression stimulated cell proliferation and led to upregulated TRPM2 and cyclin B1, but diminished expression of Fas, TNFR2, TRAIL and Egr-1. The participation of TNFalpha, TNFR1, TNFR2 and Fas in the inhibition of DENN expression was also demonstrated. These data support the anti-apoptotic and cell survival role of DENN, especially in malignant cells, and its interaction with specific genes and proteins involved in the apoptotic and cell cycle pathways.

Protection against murine leukemia virus-induced spongiform myeloencephalopathy in mice overexpressing Bcl-2 but not in mice deficient for interleukin-6, inducible nitric oxide synthetase, ICE, Fas, Fas ligand, or TNF-R1 genes.

Some murine leukemia viruses (MuLVs), among them Cas-Br-E and ts-1 MuLVs, are neurovirulent, inducing spongiform myeloencephalopathy and hind limb paralysis in susceptible mice. It has been shown that the env gene of these viruses harbors the determinant of neurovirulence. It appears that neuronal loss occurs by an indirect mechanism, since the target motor neurons have not been found to be infected. However, the pathogenesis of the disease remains unclear. Several lymphokines, cytokines, and other cellular effectors have been found to be aberrantly expressed in the brains of infected mice, but whether these are required for the development of the neurodegenerative lesions is not known. In an effort to identify the specific effectors which are indeed required for the initiation and/or development of spongiform myeloencephalopathy, we inoculated gene-deficient (knockout [KO]) mice with ts-1 MuLV. We show here that interleukin-6 (IL-6), inducible nitric oxide synthetase (iNOS), ICE, Fas, Fas ligand (FasL), and TNF-R1 KO mice still develop signs of disease. However, transgenic mice overexpressing Bcl-2 in neurons (NSE/Bcl-2) were largely protected from hind limb paralysis and had less-severe spongiform lesions. These results indicate that motor neuron death occurs in this disease at least in part by a Bcl-2-inhibitable pathway not requiring the ICE, iNOS, Fas/FasL, TNF-R1, and IL-6 gene products.

Acute hepatotoxicant exposure induces TNFR-mediated hepatic injury and cytokine/apoptotic gene expression.

Tumor necrosis factor receptor knockout (TNFR KO) mice were used to examine the role of tumor necrosis factor-alpha (TNFalpha) signaling during acute hepatotoxicant exposure. Mice were exposed intraperitoneally (ip) to either vehicle, phosphate-buffered saline (PBS), or dimethylnitrosamine (DMN, 100 mg/kg) for 24 h. Histological evaluation showed that DMN-treated TNFR-2 KO mice had increased liver damage compared to wild type (WT), TNFR-1 KO, or TNFR double KO (DKO) mice. Also, 3 of 8 TNFR-2 KO mice died following DMN treatment, suggesting that hepatic TNFR-2 signaling produces protective responses that counteract TNFR-1-mediated damage. DMN-induced cellular infiltration was absent in TNFR-1-deficient mice, indicating that infiltrating cells do not exacerbate acute hepatotoxic events. In separate experiments, mice were exposed ip to either DMN (5.0 or 100 mg/kg), carbon tetrachloride (CCl4, 0.3 or 1.0 ml/kg), or corresponding PBS/corn oil controls for 6 or 24 h to compare the hepatic mRNA expression of cytokine- and apoptotic-associated genes. Following 24 h of DMN (100 mg/kg) or 6-24 h of CCl4 treatment, hepatic transcripts for TNFalpha, interferon (IFN)-gamma, IL (interleukin)-1RI, and transforming growth factor (TGF)-betaRII were induced. Hepatotoxicant-treated WT and TNFR DKO mice induced liver transcripts for the pro- and anti-apoptotic genes, Bax and Bcl-X(L), respectively, indicating TNF-independent gene activation. The anti-apoptotic gene, Bfl-1, was highly expressed in CCl4-treated, TNFR-positive strains, but minimally expressed in TNFR DKO mice, suggesting that hepatic Bfl-1 is TNF-regulated. Taken together, these data show that acute hepatotoxicant exposure is followed by upregulation of liver cytokine, cytokine receptor, and apoptotic transcripts, and that TNFalpha regulates various aspects of liver inflammation and injury in a TNFR-specific fashion.