TNFR1 KO Research Articles

The Therapeutic Effect of Tacrolimus in a Mouse Psoriatic Model is Associated with the Induction of Myeloid-derived Suppressor Cells.

Topical administration of Tacrolimus (TAC) is efective in the treatment of psoriasis in human patients and in mouse models. Previously, we showed that, though promoting the proliferative expansion of CD4+Foxp3+ regulatory T cells (Tregs), TNFR2 was protective in mouse psoriasis model. We thus examined the role of TNFR2 signal in the efect of TAC in the treatment of mouse psoriasis. To this end, psoriasis was induced in WT, or TNFR1 KO, or TNFR2 KO mice, and the psoriatic mice were treated with or without IMQ. The results showed that TAC treatment potently inhibited the development of psoriasis in WT and TNFR1 KO mice, but not in TNFR2 KO mice. However, the treatment of TAC failed to induce the expansion of Tregs in psoriatic mice. In addition to playing a decisive role in the activation of Tregs, TNFR2 stimulates the generation and activation of myeloid-derived suppressor cells (MDSCs). This led us to found that the topical treatment with TAC markedly increased the number of MDSCs in the spleen of WT and TNFR1 KO mice, but not in TNFR2 KO mice. Consequently, TAC potently decreased serum levels of IL-17A, INF-γ, and TNF and their mRNA levels in the inflamed skin lesion. Therefore, our study for the first time found that the therapeutic efect of TAC in psoriasis is associated with the expansion of MDSCs in a TNFR2-dependent manner.

Absence of Tumor Necrosis Factor Receptor 1 Inhibits Osteoclast Activity in Apical Dental Resorption Caused by Endodontic Infection in Mice

IntroductionThe aim of this study was to evaluate osteoclastogenesis and dental resorption resulting from endodontic infection in wild-type (WT) and tumor necrosis factor receptor 1 genetically deficient (TNFR1 KO) mice. MethodsAfter approval by the ethics committee on the use of animals, 40 mice were distributed into 2 experimental groups based on time periods: 14 days (n = 10 WT mice and n = 10 TNFR1 KO mice) and 42 days (n = 10 WT mice and n = 10 TNFR1 KO mice). After these periods, morphometric analysis was performed using bright field and fluorescence microscopy and tartrate-resistant acid phosphatase histoenzymology to identify osteoclasts. One-way analysis of variance followed by the Tukey post hoc test was used for the statistical analysis (α = 0.05). ResultsWT mice in the 42-day period had a greater apical dental resorption in the distal root of the first molar than TNFR1 KO mice (P < .05). On the other hand, TNFR1 KO mice showed a smaller number of osteoclasts on the dental surface than WT mice (P < .05). ConclusionsWT mice with apical periodontitis had more extensive apical dental resorptions and a larger number of osteoclasts on the tooth surface than TNFR1 KO mice.

Topical Application of Tetrandrine Nanoemulsion Promotes the Expansion of CD4+Foxp3+ Regulatory T Cells and Alleviates Imiquimod-Induced Psoriasis in Mice.

There is compelling evidence that CD4+Foxp3+ regulatory T cells (Tregs) are indispensable in the inhibition of autoimmune inflammatory responses, including psoriasis. Recently, we showed that systemically treatment with tetrandrine (TET), a two-pore channel inhibitor identified from the Chinese herb Stephania tetrandra S. Moor, could promote the proliferative expansion of Tregs in mice through stimulation of TNF-TNFR2 interaction. We thus hypothesized that topical administration of TET might also expand Tregs and consequently inhibit psoriasis. To this end, we developed a TET nanoemulsion and examined its effect on the expansion of Tregs after topical administration on mouse psoriasis induced by imiquimod. The result of our experiment showed that topical treatment with TET nanoemulsion markedly increased the proportion and number of Tregs in the spleen, as well as TNFR2 and Ki-67 expression by Tregs, in WT and TNFR1 KO mice, but not in TNFR2 KO mice. Consequently, TET nanoemulsion potently inhibited IL-17-expressing cells in the spleen and lymph nodes of imiquimod-treated WT mice, accompanied by decreased serum levels of IL-17A, INF-γ, and TNF and their mRNA levels in the flamed lesion. Importantly, TET nanoemulsion treatment markedly inhibited the development of psoriasis-like disease in WT and TNFR1 KO mice but not in TNFR2 KO mice. Therefore, our study indicates that the topical administration of TET could also stimulate the expansion of Tregs through the TNF-TNFR2 pathway. This effect of TET and its analogs may be useful in the treatment of inflammatory skin diseases such as psoriasis.

TNFR2 Is a Crucial Hub Controlling Mesenchymal Stem Cell Biological and Functional Properties.

Mesenchymal stem cells (MSCs) have drawn lots of attention as gold standard stem cells in fundamental and clinical researches during the last 20 years. Due to their tissue and vascular repair capacities, MSCs have been used to treat a variety of degenerative disorders. Moreover, MSCs are able to modulate immune cells’ functions, particularly T cells while inducing regulatory T cells (iTregs). MSCs are very sensitive to inflammatory signals. Their biological functions could remarkably vary after exposure to different pro-inflammatory cytokines, notably TNFα. In this article, we have explored the importance of TNFR2 expression in a series of MSCs’ biological and functional properties. Thus, MSCs from wild-type (WT) and TNFR2 knockout (TNFR2 KO) mice were isolated and underwent several ex vivo experiments to investigate the biological significance of TNFR2 molecule in MSC main functions. Hampering in TNFR2 signaling resulted in reduced MSC colony-forming units and proliferation rate and diminished the expression of all MSC characteristic markers such as stem cell antigen-1 (Sca1), CD90, CD105, CD44, and CD73. TNFR2 KO-MSCs produced more pro-inflammatory cytokines like TNFα, IFNγ, and IL-6 and less anti-inflammatory mediators such as IL-10, TGFβ, and NO and induced Tregs with less suppressive effect. Furthermore, the TNFR2 blockade remarkably decreased MSC regenerative functions such as wound healing, complex tube formation, and endothelial pro-angiogenic support. Therefore, our results reveal the TNFα–TNFR2 axis as a crucial regulator of MSC immunological and regenerative functions.

Tumor Necrosis Factor Receptor-1 (p55) Deficiency Attenuates Tumor Growth and Intratumoral Angiogenesis and Stimulates CD8+ T Cell Function in Melanoma.

The role of tumor necrosis factor-α (TNF-α) in shaping the tumor microenvironment is ambiguous. Consistent with its uncertain role in melanoma, TNF-α plays a dual role, either acting as a cytotoxic cytokine or favoring a tumorigenic inflammatory microenvironment. TNF-α signals via two cognate receptors, namely TNFR1 (p55) and TNFR2 (p75), which mediate divergent biological activities. Here, we analyzed the impact of TNFR1 deficiency in tumor progression in the B16.F1 melanoma model. Tumors developed in mice lacking TNFR1 (TNFR1 knock-out; KO) were smaller and displayed lower proliferation compared to their wild type (WT) counterpart. Moreover, TNFR1 KO mice showed reduced tumor angiogenesis. Although no evidence of spontaneous metastases was observed, conditioned media obtained from TNFR1 KO tumors increased tumor cell migration. Whereas the analysis of tumor-associated immune cell infiltrates showed similar frequency of total and M2-polarized tumor-associated macrophages (TAMs), the percentage of CD8+ T cells was augmented in TNFR1 KO tumors. Indeed, functional ex vivo assays demonstrated that CD8+ T cells obtained from TNFR1KO mice displayed an increased cytotoxic function. Thus, lack of TNFR1 attenuates melanoma growth by modulating tumor cell proliferation, migration, angiogenesis and CD8+ T cell accumulation and activation, suggesting that interruption of TNF-TNFR1 signaling may contribute to control tumor burden.

TNF\u03b1/TNFR2 signaling pathway: an active immune checkpoint for mesenchymal stem cell immunoregulatory function

BackgroundIn addition to their multilineage potential, mesenchymal stem cells (MSCs) have a broad range of functions from tissue regeneration to immunomodulation. MSCs have the ability to modulate the immune response and change the progression of different inflammatory and autoimmune disorders. However, there are still many challenges to overcome before their widespread clinical administration including the mechanisms behind their immunoregulatory function. MSCs inhibit effector T cells and other immune cells, while inducing regulatory T cells (T regs), thus, reducing directly and indirectly the production of pro-inflammatory cytokines. TNF/TNFR signaling plays a dual role: while the interaction of TNFα with TNFR1 mediates pro-inflammatory effects and cell death, its interaction with TNFR2 mediates anti-inflammatory effects and cell survival. Many immunosuppressive cells like T regs, regulatory B cells (B regs), endothelial progenitor cells (EPCs), and myeloid-derived suppressor cells (MDSCs) express TNFR2, and this is directly related to their immunosuppression efficiency. In this article, we investigated the role of the TNFα/TNFR2 immune checkpoint signaling pathway in the immunomodulatory capacities of MSCs.MethodsCo-cultures of MSCs from wild-type (WT) and TNFR2 knocked-out (TNFR2 KO) mice with T cells (WT and TNFα KO) were performed under various experimental conditions.ResultsWe demonstrate that TNFR2 is a key regulatory molecule which is strongly involved in the immunomodulatory properties of MSCs. This includes their ability to suppress T cell proliferation, activation, and pro-inflammatory cytokine production, in addition to their capacity to induce active T regs.ConclusionsOur results reveal for the first time the importance of the TNFα/TNFR2 axis as an active immune checkpoint regulating MSC immunological functions.

Abstract P3-03-07: TNFR-signaling in breast cancer development

Abstract Introduction: Breast cancer (BC) is the 2nd leading cause of cancer death among women in the US. Known risk factors for the development of BC include thoracic radiotherapy during puberty and obesity with the latter turning into a nation-wide health crisis. The underlying mechanisms that lead to increased BC incidences in these two scenarios are incompletely understood. However, exposure to ionizing radiation (IR) or obesity generate pro-inflammatory conditions that have been linked to mammary carcinogenesis. As inflammation involves signaling through TNF receptors (TNFR), we have therefore employed crosses between mice that spontaneously develop BC (MMTV-Wnt1) and TNFR KO mice to study the impact of TNFR downstream signaling on BC development. Material and Methods: MMTV-Wnt1 were crossed with TNFR1 or TNFR2 KO mice to explore the BC incidence during the first year post-partum in both genders. Mammary gland area and ductal outgrowth were evaluated by whole mount histological staining with 6-week-old female (end of puberty) and 14-week-old mice (adult). Tumor cells were harvested from tumor-developed MMTV-Wnt1 and MMTV-Wnt1 x TNFR2 KO female mice, the population of tumor initiating cells (EpCAMlow/CD49fhi) was analyzed by FACS. Vasculature formation (CD31), cell proliferation (Ki67) and immune system (CD4, CD8, F4/80) were evaluated within tumors by immunohistochemistry staining. Tumor cells were digested out from primary tumors and cultured in spheres condition. In-vitro sphere formation assay was performed w/o IR or TNFα treatment. All quantitative results were treated by ANOVA and Bonferroni analysis, upon verification of normal data distribution with α value at 0.05. Results: TNFR2KO drastically accelerates BC in female animals but not in males. In contrary, TNFR1 KOhad no effect on female BC development but completely prevented BC in males. TNFR2 KO led to elongation of mammary gland duct system, expansion of mammary epithelial stem cell pool and increase of BC initiating cells, as well as more vasculatures and proliferating cells in developed tumors. T-cellsand macrophage content in established tumors don’t correlate with differences in BC incidence between the crosses. MMTV-Wnt1 x TNFR2 KO mammospheres were more resistant to IR compared to Wnt1 x TNFR1 KO and MMTV-Wnt1. Pro-inflammatory TNFα ligand increased the sphere formation in a dose-dependent manner within Wnt1 x TNFR1 KO and MMTV-Wnt1 spheres, but not in Wnt1 x TNFR2 KO ones. Conclusion: Heterozygous TNFR2 KO in female MMTV-Wnt1 Tg mice increases the BC incidence, along with more resistance to IR, suggesting the downstream of TNFR2 may potentially suppress breast carcinogenesis. Also TNFR2 KO leads to more mammary gland branching and expansion of epithelial stem cells during development, which imply the potential use of TNFR2 agonist in thoracic radiotherapy and obese scenarios to reduce the BC incidence. Citation Format: Ling He, Kruttika Bhat, Paul Medina, Claudia Alli, Mohammad Saki, Fei Cheng, Frank Pajonk. TNFR-signaling in breast cancer development [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P3-03-07.

Tumor necrosis factor receptor II (TNFR2) promotes the growth of mouse CT26 colon cancer

Abstract TNFR2 is expressed by several tumor cells including colon cancer, Hodgkin lymphoma, myeloma, renal carcinoma and ovarian cancer. Recently, TNFR2 is becoming appreciated as an oncogene. However, the exact role of TNFR2 on the tumor development remains to be fully understood. In this study, we examined the tumorigenesis and growth of mouse CT26 colon cancer cell lines with genetically modified TNFR2 expression in syngeneic Balb/c mice. We used CRISPR/Cas9- induced loss-of-function approaches to obtain stable TNFR2 KO CT26 cell line and gain-of-function techniques to obtain TNFR2 overexpressing CT26 cell line. WT, TNFR2 KO and TNFR2 overexpressing CT26 cells were inoculated s.c. into the mice. Although TNFR2 overexpressing CT26 tumor grew markedly faster than TNFR2 KO CT26 cells, there was no difference in the initial tumorigenesis, Intriguingly, all TNFR2 KO CT26 tumors completely regressed 2 weeks after inoculation. The proportion of Foxp3+ Tregs, as well as their TNFR2 expression, in the draining LNs was markedly higher in mice bearing TNFR2 overexpressing CT26 tumor. The levels of soluble TNFR2 in the peripheral blood of CT26 tumor bearing mice were ~3 folds higher than that in tumor free mice, which is comparable with the elevated levels of soluble TNFR2 in human tumor patients. In mice bearing TNFR2 overexpressing CT26 tumors, the levels of soluble TNFR2 was &amp;gt;4 folds higher than that in mice bearing WT CT26 tumors. In vitro, exogenous TNF was able to up-regulate TNFR2 surface expression on CT26 tumor cells. Taken together, our data suggest that TNF-TNFR2 signaling pathway may play a major role in tumor growth and tumor immune evasion, and thus may represent an important therapeutic target.

Tetrandrine (TET) up-regulated mTNF expression on dendritic cells and consequently induces TNFR2 mediated proliferation of Tregs

Abstract Tetrandrine (TET) is a purified chemical compound with well-established anti-inflammatory and immunosuppressive properties. The underlying mechanism of its immunosuppressive effect remains incompletely understood. It is also reported that membrane-bound (mTNF) is more effective than soluble TNF to expand Tregs through TNFR2. Therefore, we examined the effect of TET on TNF expression on dendritic cells (DCs) and its effect on Tregs in wildtype and TNFR2 KO mice. We found that, TET didn’t directly expand Tregs in vitro. But TET increased mTNF expression on DC2.4 cells and human monocyte-derived DCs. In addition, these effects of TET weren’t due to LPS contamination. Intraperitoneal injection of TET into wildtype mice increased the number of Tregs by ~1.5-fold compared with control mice. TNFR2 expression was up-regulated on Tregs rather than Teffs by TET treatment. However, the number of Tregs wasn’t changed by TET in TNFR2 KO mice. Taken together, our study revealed that TET promoted mTNF expression on DCs and consequently expanded Tregs via mTNF-TNFR2 signaling. Therefore, this represents a novel mechanism underlying the anti-inflammatory effect of TET.

TNF Receptor 2 on CD8+ T cells and TNF receptor 1 on non-CD8+ T cells Contribute Significantly to Chlamydia pneumoniae-Induced Atherosclerosis

Abstract Chlamydia pneumoniae is an important cause of community-acquired pneumonia and has been associated with exacerbation of atherosclerosis. A role of TNF-α in Chlamydia pneumoniae (Cpn) induced atherosclerosis has been reported previously; however, the relative contribution of the two cognate receptors of TNF-α, TNF receptor 1 (TNFR1) and TNFR2 had not been studied. In the current study, C57BL/6J (WT) mice, TNFR1, TNFR2 and TNFR1/2 DKO mice were infected intranasally on days 0, 14 and 28 with 1 × 107 IFU of Cpn (AR39) and were fed a high fat (HF) diet throughout the experiment. One group of WT mice were mock infected with PBS as a control. All Cpn-infected KO mice displayed serum anti-Cpn antibody response, splenic cellular Cpn-specific IFN-γ and TNF-α response, and serum total cholesterol levels comparable to infected WT animals. On day 100 following infection, Cpn infected TNFR1, TNFR2 and TNFR1/2 DKO mice displayed significantly reduced atherosclerotic pathology compared to infected WT mice, and at levels comparable to mock-infected WT animals., suggesting that TNFR1 and TNFR2 play a significant role in Cpn-induced atherosclerosis. We demonstrated recently that CD8+ T cells induce Cpn-mediated atherosclerosis; therefore, we further investigated the role of TNFR1 and TNFR2 on CD8+ T cells in pathogenesis. TNFR2 KO mice, not TNFR1 KO mice, replete with WT CD8+ T cells displayed enhancement of atherosclerotic pathology to levels observed in Cpn-infected WT animals. Collectively, these results demonstrate that TNFR2 on CD8+ T cells and TNFR1 on non-CD8+ T cells contribute significantly to Chlamydia pneumoniae induced atherosclerosis.

Innate myeloid cell TNFR1 mediates first line defence against primary Mycobacterium tuberculosis infection.

TNF is crucial for controlling Mycobacterium tuberculosis infection and understanding how will help immunomodulating the host response. Here we assessed the contribution of TNFR1 pathway from innate myeloid versus T cells. We first established the prominent role of TNFR1 in haematopoietic cells for controlling M. tuberculosis in TNFR1 KO chimera mice. Further, absence of TNFR1 specifically on myeloid cells (M-TNFR1 KO) recapitulated the uncontrolled M. tuberculosis infection seen in fully TNFR1 deficient mice, with increased bacterial burden, exacerbated lung inflammation, and rapid death. Pulmonary IL-12p40 over-expression was attributed to a prominent CD11b+ Gr1high cell population in infected M-TNFR1 KO mice. By contrast, absence of TNFR1 on T-cells did not compromise the control of M. tuberculosis infection over 6-months. Thus, the protective TNF/TNFR1 pathway essential for controlling primary M. tuberculosis infection depends on innate macrophage and neutrophil myeloid cells, while TNFR1 pathway in T cells is dispensable.

GBM Derived Gangliosides Induce T Cell Apoptosis through Activation of the Caspase Cascade Involving Both the Extrinsic and the Intrinsic Pathway.

Previously we demonstrated that human glioblastoma cell lines induce apoptosis in peripheral blood T cells through partial involvement of secreted gangliosides. Here we show that GBM-derived gangliosides induce apoptosis through involvement of the TNF receptor and activation of the caspase cascade. Culturing T lymphocytes with GBM cell line derived gangliosides (10-20μg/ml) demonstrated increased ROS production as early as 18 hrs as indicated by increased uptake of the dye H2DCFDA while western blotting demonstrated mitochondrial damage as evident by cleavage of Bid to t-Bid and by the release of cytochrome-c into the cytosol. Within 48-72 hrs apoptosis was evident by nuclear blebbing, trypan blue positivity and annexinV/7AAD staining. GBM-ganglioside induced activation of the effector caspase-3 along with both initiator caspases (-9 and -8) in T cells while both the caspase-8 and -9 inhibitors were equally effective in blocking apoptosis (60% protection) confirming the role of caspases in the apoptotic process. Ganglioside-induced T cell apoptosis did not involve production of TNF-α since anti-human TNFα antibody was unable to protect T cells from nuclear blebbing and subsequent cell death. However, confocal microscopy demonstrated co-localization of GM2 ganglioside with the TNF receptor and co-immunoprecipitation experiments showed recruitment of death domains FADD and TRADD with the TNF receptor post ganglioside treatment, suggesting direct interaction of gangliosides with the TNF receptor. Further confirmation of the interaction between GM2 and TNFR1 was obtained from confocal microscopy data with wild type and TNFR1 KO (TALEN mediated) Jurkat cells, which clearly demonstrated co-localization of GM2 and TNFR1 in the wild type cells but not in the TNFR1 KO clones. Thus, GBM-ganglioside can mediate T cell apoptosis by interacting with the TNF receptor followed by activation of both the extrinsic and the intrinsic pathway of caspases.

Doxycycline Attenuated Mycobacterium avium Induced Inflammation in Mice

Mycobacterium avium causes chronic and progressive respiratory infection. A therapeutic regimen including clarithromycin, rifampin and ethambutol has been commonly employed, however, the effect of such antibacterial therapy is often unsatisfactory. Doxycycline is an antibiotic known to have immuno-modulating effects as well as antibacterial activity. In this study, we investigated the effect of doxycycline administration on M. avium infection in mice. The administration of doxycycline attenuated lung inflammation caused by M. avium according to the results from a histology analysis and the number of inflammatory cells from BAL fluids. Moreover, doxycycline improved the survival rate in TNF-R1 KO mice infected with M. avium. However, doxycycline did not affect the colony number of M. avium in the lungs. These results suggest that doxycycline may have protective effects against M. avium induced inflammation in mice. The effects of doxycycline may be due to its biological effect apart from its antimicrobial function.

Tumor Necrosis Factor (TNF) Receptor Superfamily Member 1b on CD8+ T Cells and TNF Receptor Superfamily Member 1a on Non-CD8+ T Cells Contribute Significantly to Upper Genital Tract Pathology Following Chlamydial Infection.

We demonstrated previously that tumor necrosis factor α (TNF-α)-producing Chlamydia-specific CD8(+) T cells cause oviduct pathological sequelae. In the current study, we used wild-type C57BL/6J (WT) mice with a deficiency in genes encoding TNF receptor superfamily member 1a (TNFR1; TNFR1 knockout [KO] mice), TNF receptor superfamily member 1b (TNFR2; TNFR2 KO mice), and both TNFR1 and TNFR2 (TNFR1/2 double KO [DKO] mice) and mix-match adoptive transfers of CD8(+) T cells to study chlamydial pathogenesis. TNFR1 KO, TNFR2 KO, and TNFR1/2 DKO mice displayed comparable clearance of primary or secondary genital Chlamydia muridarum infection but significantly reduced oviduct pathology, compared with WT animals. The Chlamydia-specific total cellular cytokine response in splenic and draining lymph nodes and the antibody response in serum were comparable between the WT and KO animals. However, CD8(+) T cells from TNFR2 KO mice displayed significantly reduced activation (CD11a expression and cytokine production), compared with TNFR1 KO or WT animals. Repletion of TNFR2 KO mice with WT CD8(+) T cells but not with TNFR2 KO CD8(+) T cells and repletion of TNFR1 KO mice with either WT or TNFR1 KO CD8(+) T cells restored oviduct pathology to WT levels in both KO groups. Collectively, these results demonstrate that TNFR2-bearing CD8(+) T cells and TNFR1-bearing non-CD8(+) T cells contribute significantly to oviduct pathology following genital chlamydial infection.

TNF receptor 2 on CD8 T cells and TNF receptor 1 on non-CD8 T cells mediate upper genital tract pathology following primary genital Chlamydia muridarum infection (MUC7P.764)

Abstract We recently demonstrated that pathogenesis in the upper genital tract (UGT) following primary vaginal Chlamydia muridarum infection in mice is dependent upon TNF-alpha production and antigen-specific CD8 T cells. In this study, we evaluated the contribution of TNF receptor 1 (TNFR1) and TNFR2 to UGT pathogenesis following primary intravaginal C. muridarum infection (5X104 IFU/mouse), using depo-provera ® treated mice deficient in TNFR1 (TNFR1 KO), TNFR2 (TNFR2 KO), or TNFR1 and 2 (TNFR1/2 DKO), and the corresponding C57BL/6J wild type (WT) mice. All groups (n=8) of mice displayed comparable chlamydial clearance, antigen-specific cytokine response, and serum anti-chlamydial antibody following primary bacterial inoculation. On day 80 after primary inoculation, TNFR1 KO, TNFR2 KO, and TNFR1/2 DKO mice displayed significantly reduced oviduct pathology (hydrosalpinx), when compared to WT mice, suggesting that both TNFR1 and TNFR2 contribute significantly to oviduct pathology. Repletion of wild type or TNFR1 KO CD8 T cells did not enhance pathology significantly in TNFR1 KO mice. On the other hand, repletion of WT, but not TNFR2 KO, CD8 T cells enhanced oviduct pathology to WT levels in TNFR2 KO mice. Collectively, these results suggest that TNFR2 on CD8 T cells and TNFR1 on non-CD8 T cells mediate UGT pathology following primary genital chlamydial infection.

Modulation of mammalian circadian rhythms by tumor necrosis factor-α

Systemic low doses of the endotoxin lipopolysaccharide (LPS, 100 µg/kg) administered during the early night induce phase-delays of locomotor activity rhythms in mice. Our aim was to evaluate the role of tumor necrosis factor (Tnf)-alpha and its receptor 1/p55 (Tnfr1) in the modulation of LPS-induced circadian effects on the suprachiasmatic nucleus (SCN). We observed that Tnfr1-defective mice (Tnfr1 KO), although exhibiting similar circadian behavior and light response to that of control mice, did not show LPS-induced phase-delays of locomotor activity rhythms, nor LPS-induced cFos and Per2 expression in the SCN and Per1 expression in the paraventricular hypothalamic nucleus (PVN) as compared to wild-type (WT) mice. We also analyzed Tnfr1 expression in the SCN of WT mice, peaking during the early night, when LPS has a circadian effect. Peripheral inoculation of LPS induced an increase in cytokine/chemokine levels (Tnf, Il-6 and Ccl2) in the SCN and in the PVN. In conclusion, in this study, we show that LPS-induced circadian responses are mediated by Tnf. Our results also suggest that this cytokine stimulates the SCN after LPS peripheral inoculation; and the time-related effect of LPS (i.e. phase shifts elicited only at early night) might depend on the increased levels of Tnfr1 expression. We also confirmed that LPS modulates clock gene expression in the SCN and PVN in WT but not in Tnfr1 KO mice.Highlights: We demonstrate a fundamental role for Tnf and its receptor in circadian modulation by immune stimuli at the level of the SCN biological clock.

TNF-α-mediated JNK activation in the dorsal root ganglion neurons contributes to Bortezomib-induced peripheral neuropathy

Bortezomib (BTZ) is a frequently used chemotherapeutic drug for the treatment of refractory multiple myeloma and hematological neoplasms. The mechanism by which the administration of BTZ leads to painful peripheral neuropathy remains unclear. In the present study, we first determined that the administration of BTZ upregulated the expression of TNF-α and phosphorylated JNK1/2 in the dorsal root ganglion (DRG) of rat. Furthermore, the TNF-α synthesis inhibitor thalidomide significantly blocked the activation of both isoforms JNK1 and JNK2 in the DRG and attenuated mechanical allodynia following BTZ treatment. Knockout of the expression of TNF-α receptor TNFR1 (TNFR1 KO mice) or TNFR2 (TNFR2 KO mice) inhibited JNK1 and JNK2 activation and decreased mechanical allodynia induced by BTZ. These results suggest that upregulated TNF-α expression may activate JNK signaling via TNFR1 or TNFR2 to mediate mechanical allodynia following BTZ treatment.

TNF-alpha receptors 1 and 2 contribute to the development of upper genital tract pathology following primary genital chlamydia muridarum infection in mice (P3200)

Abstract Chlamydia is the leading cause of sexually transmitted disease worldwide and causes serious pathologies in the upper reproductive tract. In this study, we evaluated the contribution of TNF receptor 1 (TNFR1) and TNFR2 to upper genital tract pathogenesis following primary intravaginal chlamydial infection in mice deficient in TNFR1 (TNFR1 KO), TNFR2 (TNFR2 KO), or TNFR1 and 2 (TNFR1/2 DKO), and the corresponding C57BL/6J wild type mice. All groups (n=8) of mice displayed comparable chlamydial clearance following primary bacterial inoculation. The production of IFN-gamma, IL-5, TNF-alpha, and IL-1 beta in the genital tracts, draining iliac lymph nodes, and spleens on day 9 following inoculation were comparable in all the groups. Serum anti-Chlamydia total Ab, IgG2c, and IgG1 response on days 14 and 60 following primary inoculation were comparable in all the groups. On day 80 after primary incoluation, TNFR1 KO, TNFR2 KO, and TNFR1/2 DKO mice displayed significantly reduced hydrosalpinx (6 %, 44%, and 0% oviducts, respectively), when compared to C57BL/6J mice (75% oviducts). Anti-Chlamydia antibody response and bacterial clearance following secondary intravaginal C. muridarum infection was comparable in all groups of mice. Collectively, these results suggest that TNFR1 and TNFR2 do not contribute significantly to chlamydial clearance, but both TNFR1 and TNFR2, with a dominant role for TNFR1, contribute to the UGT pathology following primary genital chlamydial infection.

Abstract 1438: TNF-TNFR interactions influence tumor growth and metastasis by manipulating regulatory T cell homeostasis.

Abstract Tumor necrosis factor (TNF) signals via two cell surface receptors, TNFR1 and TNFR2 and is involved in a number of physiological processes including the regulation of immune cell homeostasis and function. Initially identified to cause tumor necrosis, a number of recent studies showed TNF to drive inflammation-induced cancer and metastasis. We employed two syngeneic in vivo bioluminescence imaging (BLI)-based mouse tumor models in conjunction with exogenous TNF treatment, TNF/TNFR knockout (ko) mice and bone marrow chimeras to determine the role of TNF-TNFR interactions in tumor growth and metastasis. 8-12 week-old female C57BL/6 mice were injected i.v. with luciferase-expressing B16F10 melanoma cells or orthotopically with luciferase-expressing Panc02 pancreatic cancer cells. We assessed the growth of B16F10 lung metastases and Panc02 primary pancreatic tumors using BLI. Finally, tumor-bearing organs were imaged ex vivo and analyzed by flow cytometry, immunofluorescence microscopy, and qRT-PCR. In the B16F10 model, treatment of mice with exogenous TNF markedly enhanced tumor metastasis resulting in elevated in vivo luciferase signals and increased lung metastases numbers. TNF-induced metastasis correlated with an accumulation of regulatory T cells (Tregs) in the lungs in vivo and we found TNF to induce Tregs in vitro in a TNFR2-dependent manner. This receptor was significantly higher expressed on Tregs than on effector CD4 T cells. The depletion of Tregs in genetically engineered mice attenuated B16 metastasis and ablated the pro-metastatic effect of TNF-treatment. Loss of either TNF or TNFR2 on immune cells in vivo reduced both metastasis and the number of Tregs. In the pancreatic tumor model, neither the loss of TNF nor TNFR2 changed tumor growth compared to wildtype mice. Loss of TNFR1 strongly promoted tumor growth and increased the infiltration of CD4 T cells and Tregs into the tumor tissue. Influx of CD8 T-cells into the tumors inversely correlated with the numbers of Tregs. Expression of activation markers on tumor infiltrating T cells was the same in TNFR1 ko and wildtype mice, as was mRNA-expression of immune suppressive genes. Loss of TNFR1 resulted in up-regulation of IL-4 expression within Panc02 tumors which suggests changes in TH1/TH2 immunity to be involved in tumor immunity in this model. Enhanced pancreatic tumor growth observed in TNFR1 ko mice may furthermore reflect reduced activity of other anti-tumoral acting cell types that are stimulated via TNFR1. We suggest that TNF activates TNFR2 on Tregs and thereby contributes to tumor-associated immune suppression in the B16F10 lung metastasis model. The induction of suppressive Tregs could be seen as a feedback-loop in which TNF down-regulates its own pro-inflammatory functions and eventually ablates immune control of tumors and enhances tumor growth and metastasis. Citation Format: Martin R. Chopra, Marlene Biehl, Carina A. Bäuerlein, Christian Brede, Ana-Laura Jordan Garotte, Sabrina Kraus, Simone S. Riedel, Katharina Mattenheimer, Miriam Ritz, Victoria Schäfer, Anja Mottok, Hermann Einsele, Harald Wajant, Andreas Beilhack. TNF-TNFR interactions influence tumor growth and metastasis by manipulating regulatory T cell homeostasis. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1438. doi:10.1158/1538-7445.AM2013-1438

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.