Reduced inflammatory state promotes reinnervation of endometriotic-like lesions in TNFRp55 deficient mice.

Endometriosis is a chronic gynecological disease, characterized by growth of endometrial tissue in ectopic sites due to alteration of peritoneal homeostasis and deregulation of apoptosis. Here we have examined whether TNFRp55 deficiency modulates the pro-inflammatory state and the reinnervation of endometriotic-like lesions in mice. Two-month-old female C57BL/6 mice, eight wild type (WT) and eight TNFRp55-/- (KO) were used in the study. Endometriotic-like lesions were induced experimentally. The right uterine horn was removed from the animal, divided longitudinally, cut in three square pieces and sutured to the intestine mesentery. After 4 weeks, the lesions and the peritoneal fluid were collected. The level of TNFα in the peritoneal fluid was evaluated by enzyme-linked immunosorbent assay (EIA). The expressions of COX2, GRα and GRß were evaluated in the lesions by western blot and immunohistochemistry. ß-III TUBULIN, BDNF and NGF protein concentrations were evaluated in the lesions by western blot. Gene expression of Pgp 9.5, SP and Th was analyzed by RT-PCR, whereas relative concentrations of TRKA, NTRp75, phosphorylated NFκB (pNFκB) and total NFκB in lesions were measured by EIA. Compared with the WT group, the KO mice showed lower TNFα levels in the peritoneal fluid and lower numbers of COX2 immunoreactive cells along with increased expression of GRα, ß-III TUBULIN, Pgp 9.5, SP, Th, BDNF, NGF, NTRp75 and pNFκB in the lesions. Future histological studies will be necessary to confirm the sensory/sympathetic imbalance in the endometriotic-like lesions of the KO mice. Our results suggest that a reduced inflammatory state promotes reinnervation of endometriotic-like lesions in TNFRp55-/- mice. Chronic deregulation of TNF receptors can have serious consequences for women with advanced endometriosis.

Endometriosis progression in tumor necrosis factor receptor p55-deficient mice: Impact on oxidative/nitrosative stress and metallomic profile.

The present study was conducted to investigate whether the deficiency of tumor necrosis factor receptor p55 (TNFRp55) modulates oxidative/nitrosative stress and metallomic profile into the peritoneal cavity during the experimental endometriosis progression in mice. Female C57BL/6 mice, wild-type (WT) and TNFRp55 knockout (KO) of two months were used. Endometriosis was induced experimentally by autotransplanting three pieces of the right uterine horn to the intestinal mesentery. After four weeks, endometriotic-like lesions and peritoneal lavage fluid were collected. The obtained peritoneal fluid was analyzed for nitrite levels using the Griess method and trace elements concentrations by ICP-MS. Both endometriotic-like lesions and cells isolated from peritoneal lavage were analyzed for the following oxidative/nitrosative stress markers: inducible nitric oxide synthase (iNOS) expression by Western Blot; total antioxidant capacity (TAC), the activity of two antioxidant enzymes (CAT and GPX) and thiobarbituric acid-reactive substances (TBARS) concentration, by spectrophotometric method; and protein carbonyl content and nitrotyrosine presence by ELISA. In comparison to WT group, KO mice exhibited larger lesion volume; higher levels of nitrite, copper (Cu) and strontium (Sr) in the peritoneal fluid; increased TAC, CAT, and GPX in peritoneal lavage cells; decreased concentration of TBARS in lesions and protein carbonyl in peritoneal lavage cells. Significant positive correlations between Cu and lesion volume, Sr and lesion volume, and Cu and Sr were obtained. Our results suggest that the TNFRp55 deficiency increases antioxidant protection and promotes high Cu-Sr concentrations in the peritoneal cavity, which favors the progression of experimental endometriosis.

TNFRp55 deficiency promotes the development of ectopic endometriotic-like lesions in mice.

Endometriosis is an inflammatory disease depending on estradiol, with TNF-α being one of the most representative cytokines involved in its pathogenesis. TNF-α acts through its bond to the TNFRp55 and TNFRp75 membrane receptors. The aim of this study was to analyze the effect of the TNFRp55 deficiency on the development of ectopic endometriotic-like lesions. Endometriosis was induced surgically in mice of the C57BL/6 strain, wild type (WT) and TNFRp55-/- (KO). After four weeks, the peritoneal fluid was collected and the lesions were counted, measured with a caliper, removed, weighed, fixed or kept at -80°C. We evaluated the cell proliferation by proliferating cell nuclear antigen (PCNA) immunohistochemistry and apoptosis by TUNEL technique in the ectopic lesions. MMP-2 and MMP-9 activities (factors involved in invasiveness) were measured by zymography in the peritoneal fluid; estradiol and progesterone levels were measured by radioimmunoassay in the lesions and in the peritoneal fluid. We found that in KO animals the mean number of lesions established per mouse, the lesion volume, weight and cell proliferation increased and apoptosis decreased. In addition, the activity of MMP-2 and the estradiol level increased, whereas the progesterone level was not significantly modified. In conclusion, the deficiency of TNFRp55 promoted the establishment and development of endometriosis through an increase in the lesion size and high levels of estradiol which correlate with an increase in the MMP-2 activity. This is evidence of the possible association of the deregulation of the TNFRp55 expression and the survival of the endometriotic tissue in ectopic sites.

Impact of tumor necrosis factor receptor p55 deficiency in susceptibility of C57BL/6 mice to infection with Leishmania (Leishmania) amazonensis.

Tumor necrosis factor (TNF) is involved in host resistance to several intracellular pathogens. Although the critical role of TNF receptor (TNFR)p55 in Leishmania (Leishmania) major infection has been demonstrated, the impact of TNFRp55 deficiency on L. (L.) amazonensis infection has not been explored. L. (L.) amazonensis-infected TNFRp55(-/-) mice failed to resolve lesions, whereas C57BL/6 wild-type mice completely healed. The susceptibility of the TNFRp55(-/-) mice was characterized by higher lesion size and histopathological damage in comparison with the wild-type mice. A marked increased of the splenic index was observed in the TNFRp55(-/-) mice after 15 weeks infection. These results show that in the absence of TNFRp55, L. (L.) amazonensis-infected knockout mice fail to resolve lesions, whereas wild-type mice completely heal.

Soluble TNFRp75 regulates host protective immunity against Mycobacterium tuberculosis.

Development of host protective immunity against Mycobacterium tuberculosis infection is critically dependent on the inflammatory cytokine TNF. TNF signals through 2 receptors, TNFRp55 and TNFRp75; however, the role of TNFRp75-dependent signaling in immune regulation is poorly defined. Here we found that mice lacking TNFRp75 exhibit greater control of M. tuberculosis infection compared with WT mice. TNFRp75-/- mice developed effective bactericidal granulomas and demonstrated increased pulmonary recruitment of activated DCs. Moreover, IL-12p40-dependent migration of DCs to lung draining LNs of infected TNFRp75-/- mice was substantially higher than that observed in WT M. tuberculosis-infected animals and was associated with enhanced frequencies of activated M. tuberculosis-specific IFN-γ-expressing CD4+ T cells. In WT mice, TNFRp75 shedding correlated with markedly reduced bioactive TNF levels and IL-12p40 expression. Neutralization of TNFRp75 in M. tuberculosis-infected WT BM-derived DCs (BMDCs) increased production of bioactive TNF and IL-12p40 to a level equivalent to that produced by TNFRp75-/- BMDCs. Addition of exogenous TNFRp75 to TNFRp75-/- BMDCs infected with M. tuberculosis decreased IL-12p40 synthesis, demonstrating that TNFRp75 shedding regulates DC activation. These data indicate that TNFRp75 shedding downmodulates protective immune function and reduces host resistance and survival; therefore, targeting TNFRp75 may be beneficial for improving disease outcome.

TNFRp55 controls regulatory T cell responses in Yersinia-induced reactive arthritis.

In addition to its well-known pro-inflammatory effects, tumor necrosis factor (TNF) displays anti-inflammatory activities through mechanisms poorly understood. Previously, we reported the development of severe chronic Yersinia enterocolitica-induced reactive arthritis (ReA) in mice lacking the TNF receptor (TNFR)p55. As regulatory T (T(reg)) cells limit chronic inflammation, here we aim to investigate the expansion and function of CD4(+)CD25(+)FoxP3(+) T(reg) cells in the ReA animal model. The number of T(reg) cells as well as the FoxP3 mRNA expression and interleukin (IL)-10 levels were significantly decreased in joint regional lymph nodes (RLNs) of TNFRp55(-/-) mice vs wild-type (WT) mice at the arthritis onset. However, at chronic phase of arthritis, the number of T(reg) cell in TNFRp55(-/-) was similar to WT mice. To explore the in vivo function of T(reg) cells at this chronic phase in WT and TNFRp55-deficient mice, we adoptively transferred CD4(+) T cells from TNFRp55-deficient mice of day 21, into naïve WT or TNFRp55(-/-) mice. When knockout mice were used as recipients we observed higher delayed-type hypersensitivity (DTH) responses and joint inflammation after heat-killed Yersinia (HKY) stimulation. Accordingly, we found higher levels of IL-17, interferon (IFN)-γ, IL-6, transforming growth factor (TGF)-ß1 and IL-12/23p40 and lower IL-10 levels in RLN of paws challenged with HKY in TNFRp55(-/-) recipient mice. In addition, we found that CD4(+) T cells from TNFRp55(-/-) mice controlled antigen-specific IL-12/23(p40) production in recipient WT mice. Our results show that TNFRp55 controls the induction and function of T(reg) cells through differential regulation of cytokine production, suggesting a novel molecular target for immune intervention in ReA.

Brucella abortus-infected macrophages modulate T lymphocytes to promote osteoclastogenesis via IL-17.

The pathogenic mechanisms of bone loss caused by Brucella species have not been completely deciphered. Although T lymphocytes (LTs) are considered important to control infection, the mechanism of Brucella-induced T-cell responses to immunopathological features is not known. We present in vitro and in vivo evidence showing that Brucella abortus-induced inflammatory response leads to the activation of LTs, which further promote osteoclastogenesis. Pre-activated murine LTs treated with culture supernatant from macrophages infected with B. abortus induced bone marrow-derived monocytes (BMMs) to undergo osteoclastogenesis. Furthermore, osteoclastogenesis was mediated by CD4(+) T cells. Although B. abortus-activated T cells actively secreted the pro-osteoclastogenic cytokines RANKL and IL-17, osteoclastogenesis depended on IL-17, because osteoclast generation induced by Brucella-activated T cells was completely abrogated when these cells were cultured with BMMs from IL-17 receptor knockout mice. Neutralization experiments indicated that IL-6, generated by Brucella infection, induced the production of pro-osteoclastogenic IL-17 from LTs. By using BMMs from tumor necrosis factor receptor p55 knockout mice, we also demonstrated that IL-17 indirectly induced osteoclastogenesis through the induction of tumor necrosis factor-α from osteoclast precursors. Finally, extensive and widespread osteoclastogenesis was observed in the knee joints of mice injected with Brucella-activated T cells. Our results indicate that activated T cells, elicited by B. abortus-infected macrophages and influenced by the inflammatory milieu, promote the generation of osteoclasts, leading to bone loss.

Cross-platform array screening identifies COL1A2, THBS1, TNFRSF10D and UCHL1 as genes frequently silenced by methylation in melanoma.

Epigenetic regulation of tumor suppressor genes (TSGs) has been shown to play a central role in melanomagenesis. By integrating gene expression and methylation array analysis we identified novel candidate genes frequently methylated in melanoma. We validated the methylation status of the most promising genes using highly sensitive Sequenom Epityper assays in a large panel of melanoma cell lines and resected melanomas, and compared the findings with those from cultured melanocytes. We found transcript levels of UCHL1, COL1A2, THBS1 and TNFRSF10D were inversely correlated with promoter methylation. For THBS1 and UCHL1 the effect of this methylation on expression was confirmed at the protein level. Identification of these candidate TSGs and future research designed to understand how their silencing is related to melanoma development will increase our understanding of the etiology of this cancer and may provide tools for its early diagnosis.

Chronic OVA allergen challenged TNF p55/p75 receptor deficient mice have reduced airway remodeling.

The role of tumor necrosis factor-α (TNF-α) in contributing to allergen induced airway remodeling in asthma is unknown. In this study we have utilized a mouse model of chronic OVA allergen induced airway remodeling to determine whether TNF p55/p75 receptor deficient mice (abbreviated TNF-R KO) had reduced levels of airway remodeling. Chronic OVA challenged WT mice had significantly increased levels of lung eosinophilic inflammation as well as features of airway remodeling including increased peribronchial fibrosis, thickness of the peribronchial smooth muscle layer, mucus expression, and deposition of extracellular matrix proteins. In contrast, TNF-R KO mice had significantly reduced levels of major basic protein positive peribronchial eosinophils and significantly reduced peribronchial fibrosis assessed by quantitating the area of peribronchial trichrome staining and total lung collagen. In addition, TNF-R KO mice had significantly reduced thickness of the peribronchial smooth muscle layer, area of peribronchial α-smooth muscle actin immunostaining, and levels of the extracellular matrix protein fibronectin. There was a non-significant trend for reduced mucus expression in TNF-R KO mice. Levels of peribronchial cells immunostaining positive for TGF-ß1 were significantly reduced in TNF-R KO mice suggesting that reduced levels of TGF-ß1 expression in TNF-R KO mice may contribute to reduced airway remodeling. Overall, this study suggests an important role for TNF-α in contributing to many features of allergen induced airway remodeling including changes in levels of peribronchial smooth muscle, subepithelial fibrosis, and deposition of extracellular matrix.

TNFR1-dependent regulation of myeloid cell function in experimental autoimmune uveoretinitis.

Experimental autoimmune uveoretinitis is an autoimmune disease induced in mice, which involves the infiltration of CD11b(+) macrophages and CD4(+) T cells into the normally immune-privileged retina. Damage is produced in the target organ following the activation of Th1 and Th17 T cells and by the release of cytotoxic mediators such as NO by activated macrophages. The majority of immune cells infiltrating into the retina are CD11b(+) myeloid cells, but, despite the presence of these APCs, relatively limited numbers of T cells are observed in the retina during the disease course. These T cells do not proliferate when leukocytes are isolated from the retina and restimulated in vitro, although they do produce both IFN-gamma and IL-17. T cell proliferation was restored by depleting the myeloid cells from the cultures and furthermore those isolated myeloid cells were able to regulate the proliferation of other T cells. The ability of macrophages to regulate proliferation depends on activation by T cell-produced IFN-gamma and autocrine TNF-alpha signaling in the myeloid cells via TNFR1. In the absence of TNFR1 signaling, relative T cell expansion in the retina is increased, indicating that regulatory myeloid cells may also act in vivo. However, TNFR1 signaling is also required for macrophages, but not T cells, to migrate into the target organ. Thus, in TNFR1 knock out mice, the amplification of autoimmunity is limited, leading to resistance to experimental autoimmune uveoretinitis induction.

Tumor necrosis factor alpha is a proximal mediator of synergistic hepatotoxicity from trovafloxacin/lipopolysaccharide coexposure.

The use of trovafloxacin (TVX), a fluoroquinolone antibiotic, was severely restricted because of an association of TVX therapy with idiosyncratic hepatotoxicity in patients. The mechanisms underlying idiosyncratic toxicity are unknown; however, one hypothesis is that an inflammatory stress can render an individual sensitive to the drug. Previously, we reported that treatment of mice with TVX and lipopolysaccharide (LPS) induced tumor necrosis factor (TNF) alpha-dependent liver injury, whereas TVX or LPS treatment alone was nontoxic. The goal of this study was to elucidate the role of TNFalpha in TVX/LPS-induced liver injury. TNF receptor (TNFR) 1 p55(-/-) and TNFR2 (p75(-/-)) mice were protected from hepatotoxicity caused by TVX/LPS coexposure, suggesting that TVX/LPS-induced liver injury requires both TNF receptors. TNFalpha inhibition using etanercept significantly reduced the TVX/LPS-induced increases in the plasma concentrations of several cytokines around the time of onset of liver injury. However, despite the reduction in chemokines, etanercept treatment did not affect the TVX/LPS-induced hepatic accumulation of neutrophils. In addition, etanercept treatment attenuated TVX/LPS induction of plasminogen activator inhibitor-1, and this was associated with a reduction in hepatic fibrin deposition. Mice treated with TVX and a nontoxic dose of TNFalpha also developed liver injury. In summary, TNFalpha acts through p55 and p75 receptors to precipitate an innocuous inflammatory cascade. TVX enhances this cascade, converting it into one that results in hepatocellular injury.

Effect of traumatic brain injury on cognitive function in mice lacking p55 and p75 tumor necrosis factor receptors.

BACKGROUND: Tumor necrosis factor (TNF)-alpha has been suggested to play both a deleterious and beneficial role in neurobehavioral dysfunction and recovery following traumatic brain injury (TBI). The goal of this study was to evaluate the specific role of tumor necrosis factor (TNF) receptors p55 and p75 in mediating cognitive outcome following controlled cortical impact (CCI) brain injury by comparing post-traumatic cognitive function in mice with genetically engineered deletion of the gene for either p55 (-/-) or p75 (-/-) receptors. METHOD: Male C57B1/6 mice (WT, n=29), and mice genetically engineered to delete p55 TNF (p55 (-/-), n=8) or p75 TNF (p75 (-/-), n=23) receptors were used. They were anesthetized with intraperitoneal (i.p.) administration of sodium pentobarbital (65 mg/kg) and subjected to CCI brain injury of moderate severity. Sham-injured control mice were anesthetized and surgically prepared similarly but they received no impact. Assessment of mRNA expression of inflammatory, proapoptotic and antiapoptotic genes was done by real time-polymerase chain reaction (RT-PCR). Cognitive outcome was evaluated at 4 weeks postinjury using the Morris water maze (MWM). FINDINGS: mRNA expression of inflammatory, proapoptotic and antiapoptotic genes prior to TBI did not reveal any baseline difference between p55 and p75 (-/-) mice. WT mice showed greater baseline expression of inflammatory genes. The learning ability of p55 (-/-) brain-injured mice was significantly better than that observed in p75 (-/-) brain-injured mice (p < 0.05). Cognitive learning in WT control mice fell between the p55 (-/-) and p75 (-/-) mice. CONCLUSIONS: These data suggest that TNF-alpha may both exacerbate cognitive dysfunction via p55 receptor and attenuate it via p75 receptor.

Apoptosis-inducing factor of a cytotoxic T cell line: involvement of a secretory phospholipase A2.

Natural killer (NK) cells and cytotoxic T lymphocytes (CTLs) kill target cells by the granule-exocytosis pathway and by the engagement of molecules belonging to the tumor necrosis factor family. The involvement of secretory phospholipase A(2) (sPLA(2)) in the cytotoxic process has been proposed in NK cells. However, its molecular identity and intracellular localization remain unknown, and its mechanism of action is poorly understood. Here, we have readdressed this issue by studying the cytotoxic activity of whole cell extracts of a CTL line. We observed that inactivation of the perforin-granzyme pathway at 37 degrees C in the presence of 1 mM Ca(2+) enhanced the ability of CTL extracts to induce apoptosis. This potentiation of cell death was Ca(2+)-dependent, thermo-resistant, and inhibited by 4-bromophenacyl bromide and scalaradial (two inhibitors of sPLA(2)). The involvement of an sPLA(2) was confirmed by blocking the pro-apoptotic activity of the Ca(2+)-treated cell extract with an anti-sPLA(2) polyclonal antibody. By cell fractionation assays, we showed that the pro-apoptotic sPLA(2) was localized in the cytoplasmic fraction but not in perforin-rich granules or plasma membrane fractions. Western blotting analysis revealed the presence of four distinct bands of 56, 29.5, 21, and 15 kDa. The highest molecular weight band was consistent with the expression of a group III sPLA2. Taken together, these data indicate that an apoptosis-inducing sPLA(2) is expressed in the cytosol of a CTL cell line and suggest that it plays an effector role in CTL-mediated cytotoxicity.