TNFR1 KO Mice Research Articles

Targeting dendritic cell-specific TNFR2 improves skin and joint inflammation by inhibiting IL-12/ IFN-γ pathways in a mouse model of psoriatic arthritis.

Psoriasis (PsO) and Psoriatic arthritis (PsA) are immune-mediated inflammatory diseases affecting the skin and joints. Approximately, 30% of patients with PsO develop PsA over time with both conditions being associated with elevated tumor necrosis factor-alpha (TNF-α) expression. TNF-α mediates its effect through two membrane receptors, TNFR1 and TNFR2. While current TNF-α-neutralizing agents, targeting both TNFR1 and TNFR2 receptors, constitute the primary treatment for psoriatic diseases, their long-term use is limited due to an increase in opportunistic infections, tuberculosis reactivation and malignancies likely attributed to TNFR1 inactivation. Recent findings suggest a pivotal role of TNFR2 in psoriatic disease, as evidenced by its amelioration in global TNFR2-knockout (TNFR2KO) mice, but not in TNFR1KO mice. The diminished disease phenotype in TNFR2KO mice is accompanied by a decrease in DC populations. However, the specific contribution of TNFR2 in dendritic cells (DCs) remains unclear. Here, utilizing a mannan-oligosaccharide (MOS)-induced PsA model, we demonstrate a significant reduction in PsA-like skin scaling and joint inflammation in dendritic cell-specific TNFR2 knockout mice (DC-TNFR2KO). Notably, MOS treatment in control mice (TNFR2 fl/fl) led to an increase in conventional type 1 dendritic cells (cDC1) population in the spleen, a response inhibited in DC-TNFR2KO mice. Furthermore, DC-TNFR2KO mice exhibited reduced levels of interleukin-12 (IL-12), a Th1 cell activator, as well as diminished Th1 cells, and interferon-gamma (IFN-γ) levels in the serum compared to controls following MOS stimulation. In summary, our study provides compelling evidence supporting the role of TNFR2 in promoting PsA-like inflammation through cDC1/Th1 activation pathways.

Tumor Necrosis Factor-α Receptor 1 Mediates Borna Disease Virus 1-Induced Changes in Peroxisomal and Mitochondrial Dynamics in Neurons.

Borna disease virus 1 (BoDV1) causes a persistent infection in the mammalian brain. Peroxisomes and mitochondria play essential roles in the cellular antiviral immune response, but the effect of BoDV1 infection on peroxisomal and mitochondrial dynamics and their respective antioxidant capacities is still not clear. Using different mouse lines-i.e., tumor necrosis factor-α transgenic (TNFTg; to pro-inflammatory status), TNF receptor-1 knockout (TNFR1ko), and TNFR2ko mice in comparison to wild-type (Wt) mice-we analyzed the abundances of both organelles and their main antioxidant enzymes, catalase and superoxide dismutase 2 (SOD2), in neurons of the hippocampal, cerebral, and cerebellar cortices. In TNFTg mice, a strong increase in mitochondrial (6.9-fold) and SOD2 (12.1-fold) abundances was detected; meanwhile, peroxisomal abundance increased slightly (1.5-fold), but that of catalase decreased (2.9-fold). After BoDV1 infection, a strong decrease in mitochondrial (2.1-6.5-fold), SOD2 (2.7-9.1-fold), and catalase (2.7-10.3-fold) abundances, but a slight increase in peroxisomes (1.3-1.6-fold), were detected in Wt and TNFR2ko mice, whereas no changes occurred in TNFR1ko mice. Our data suggest that the TNF system plays a crucial role in the biogenesis of both subcellular organelles. Moreover, TNFR1 signaling mediated the changes in peroxisomal and mitochondrial dynamics after BoDV1 infection, highlighting new mechanisms by which BoDV1 may achieve immune evasion and viral persistence.

Subchronic exposure to 1,2-naphthoquinone induces adipose tissue inflammation and changes the energy homeostasis of mice, partially due to TNFR1 and TLR4

Air pollution affects energy homeostasis detrimentally. Yet, knowledge of how each isolated pollutant can impact energy metabolism remains incomplete. The present study was designed to investigate the distinct effects of 1,2-naphthoquinone (1,2-NQ) on energy metabolism since this pollutant increases at the same rate as diesel combustion. In particular, we aimed to determine in vivo effects of subchronic exposure to 1,2-NQ on metabolic and inflammatory parameters of wild-type mice (WT) and to explore the involvement of tumor necrosis factor receptor 1 (TNFR1) and toll-like receptor 4 (TLR4) in this process. Males WT, TNFR1KO, and TLR4KO mice at eight weeks of age received 1,2-NQ or vehicle via nebulization five days a week for 17 weeks. In WT mice, 1,2-NQ slightly decreased the body mass compared to vehicle-WT. This effect was likely due to a mild food intake reduction and increased energy expenditure (EE) observed after six weeks of exposure. After nine weeks of exposure, we observed higher fasting blood glucose and impaired glucose tolerance, whereas insulin sensitivity was slightly improved compared to vehicle-WT. After 17 weeks of 1,2-NQ exposure, WT mice displayed an increased percentage of M1 and a decreased (p = 0.057) percentage of M2 macrophages in adipose tissue. The deletion of TNFR1 and TLR4 abolished most of the metabolic impacts caused by 1,2-NQ exposure, except for the EE and insulin sensitivity, which remained high in these mice under 1,2-NQ exposure. Our study demonstrates for the first time that subchronic exposure to 1,2-NQ affects energy metabolism in vivo. Although 1,2-NQ increased EE and slightly reduced feeding and body mass, the WT mice displayed higher inflammation in adipose tissue and impaired fasting blood glucose and glucose tolerance. Thus, in vivo subchronic exposure to 1,2-NQ is harmful, and TNFR1 and TLR4 are partially involved in these outcomes.

TNFR1 Mediated Apoptosis Is Protective against Mycobacterium avium in Mice

Mycobacterium avium is an intracellular proliferating pathogen that causes chronic refractory respiratory infection. Although apoptosis induced by M. avium has been reported in vitro, the role of apoptosis against M. avium infection in vivo remains unclear. Here, we investigated the role of apoptosis in mouse models of M. avium infection. Tumor necrosis factor receptor-1 knockout mice (TNFR1-KO) andTNFR2-KO micewere used. M. avium (1 × 107 cfu/body) was administered intratracheally to mice. Apoptosis in lungs was detected by terminal deoxynucleotidyl transferase mediated dUTP nick end labeling and lung histology as well as cell death detection kits using BAL fluids. TNFR1-KO mice were susceptible to M. avium infection compared with TNFR2-KO and wild type mice based on the bacterial number and lung histology. Higher numbers of apoptotic cells were detected in the lungs of TNFR2-KO and wild-type mice were compared with TNFR1-KO mice. The inhalation of Z-VAD-FMK deteriorated M. avium infection compared with vehicle-inhaled controls. Overexpression of Iκ-B alpha by adenovirus vector attenuated M. avium infection. Our study showed apoptosis had an important role in innate immunity against M. avium in mice. The induction of apoptosis in M. avium-infected cells might be a new strategy to control M. avium infection.

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.

Angiotensin II-induced renal angiotensinogen formation is enhanced in mice lacking tumor necrosis factor-alpha type 1 receptor.

In hypertension induced by angiotensin II (AngII) administration with high salt (HS) intake, intrarenal angiotensinogen (AGT) and tumor necrosis factor‐alpha (TNF‐α) levels increase. However, TNF‐α has been shown to suppress AGT formation in cultured renal proximal tubular cells. We examined the hypothesis that elevated AngII levels during HS intake reduces TNF‐α receptor type 1 (TNFR1) activity in the kidneys, thus facilitating increased intrarenal AGT formation. The responses to HS diet (4% NaCl) with chronic infusion of AngII (25 ng/min) via implanted minipump for 4 weeks were assessed in wild‐type (WT) and knockout (KO) mice lacking TNFR1 or TNFR2 receptors. Blood pressure was measured by tail‐cuff plethysmography, and 24‐h urine samples were collected using metabolic cages prior to start (0 day) and at the end of 2nd and 4th week periods. The urinary excretion rate of AGT (uAGT; marker for intrarenal AGT) was measured using ELISA. HS +AngII treatment for 4 weeks increased mean arterial pressure (MAP) in all strains of mice. However, the increase in MAP in TNFR1KO (77 ± 2 to 115 ± 3 mmHg; n = 7) was significantly greater (p < 0.01) than in WT (76 ± 1 to 102 ± 2 mmHg; n = 7) or in TNFR2KO (78 ± 2 to 99 ± 5 mmHg; n = 6). The increase in uAGT at 4th week was also greater (p < 0.05) in TNFR1KO mice (6 ± 2 to 167 ± 75 ng/24 h) than that in WT (6 ± 3 to 46 ± 16 ng/24 h) or in TNFR2KO mice (8 ± 7 to 65 ± 44 ng/24 h). The results indicate that TNFR1 exerts a protective role by mitigating intrarenal AGT formation induced by elevated AngII and HS intake.

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.

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.

Attenuated Salmonella VNP20009 mutant (ΔhtrA) is a promising candidate for bacteria-mediated tumour therapy in hosts with TNFR1 deficiency.

VNP20009 is attenuated Salmonella with a good safety widely used for tumour-targeting bacterial therapies. Little is known about its toxicity in hosts with low immunity. This study is the first systemically comparative analysis of their toxicity of VNP20009 and its mutant ΔhtrA in TNFR1-KO mice. Research on toxicity of tumour-targeting Salmonella in mice with immunodeficiency can facilitate the optimization of bacterial therapies with reduced adverse effects in future clinical trials.

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-α/TNFR1 Signaling is Required for the Full Expression of Acute and Chronic Itch in Mice via Peripheral and Central Mechanisms.

Increasing evidence suggests that cytokines and chemokines play crucial roles in chronic itch. In the present study, we evaluated the roles of tumor necrosis factor-alpha (TNF-α) and its receptors TNF receptor subtype-1 (TNFR1) and TNFR2 in acute and chronic itch in mice. Compared to wild-type (WT) mice, TNFR1-knockout (TNFR1-KO) and TNFR1/R2 double-KO (DKO), but not TNFR2-KO mice, exhibited reduced acute itch induced by compound 48/80 and chloroquine (CQ). Application of the TNF-synthesis inhibitor thalidomide and the TNF-α antagonist etanercept dose-dependently suppressed acute itch. Intradermal injection of TNF-α was not sufficient to evoke scratching, but potentiated itch induced by compound 48/80, but not CQ. In addition, compound 48/80 induced TNF-α mRNA expression in the skin, while CQ induced its expression in the dorsal root ganglia (DRG) and spinal cord. Furthermore, chronic itch induced by dry skin was reduced by administration of thalidomide and etanercept and in TNFR1/R2 DKO mice. Dry skin induced TNF-α expression in the skin, DRG, and spinal cord and TNFR1 expression only in the spinal cord. Thus, our findings suggest that TNF-α/TNFR1 signaling is required for the full expression of acute and chronic itch via peripheral and central mechanisms, and targeting TNFR1 may be beneficial for chronic itch treatment.

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.

Abstract 76: Effects of Long-term Angiotensin-II Infusion on Cardiac and Renal Fibrosis are Blunted in TNFR1-deficient Mice

Background: Brief systemic infusion of Angiotensin-II (Ang-II) to wild-type (WT) mice initiates the development of cardiac interstitial fibrosis. Genetic deletion of tumor necrosis factor receptor 1 (TNFR1) obviates this development and concurrently inhibits Ang-II-induced cardiac remodeling and dysfunction. We now investigated long-term effects of Ang-II on the heart, kidney, and cardiorenal function. Methods: WT and TNFR1-KO mice were infused with 1.5 ug/kg/min Ang-II for 1 and 6 weeks (no uninephrectomy or high-salt diet). Heart, kidney, and serum were isolated and evaluated by histology, cytometry, qPCR, and ELISA techniques. Cardiac function was determined by 2D-echocardiography, systolic blood pressure by tail-cuff plethysmography. Results: Brief infusion of Ang-II to WT mice did not evoke a fibrotic response in the kidney. However, after 6 weeks, WT kidneys developed minimal, but significant interstitial collagen deposition which was supported by upregulation of collagen-I, collagen-III, and alpha-smooth muscle actin gene activation. This fibrotic development was associated with the appearance of myeloid fibroblast precursors, pro-inflammatory M1 and pro-fibrotic M2 cells, and myofibroblasts. Transcriptional expression of pro-inflammatory and pro-fibrotic genes was also increased. These changes were not seen in Ang-II-infused TNFR1-KO kidneys. In WT hearts, despite the disappearance of myeloid cells, cardiac fibrosis persisted throughout the 6-week infusion. WT hearts developed clear evidence of accelerated cardiac hypertrophy and remodeling associated with impaired systolic function. Again, these changes were not seen in Ang-II-infused TNFR1-KO hearts. By contrast, both WT and TNFR1-KO mice responded identically with similar elevations of systolic blood pressure, and serum blood urea nitrogen and creatinine levels. Conclusions: Ang-II-infusion induced an immediate fibrotic response in the heart while fibrosis in the kidney developed slowly. The cardiac fibrosis was accompanied by progressive adverse remodeling and worsening of function over time. TNFR1-KO mice were protected from the Ang-II-induced cardiac and renal fibrosis, despite similar increases in blood pressure and renal dysfunction.

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.

Abstract 215: Angiotensin-II-induced Cardiac Remodeling is Reduced in TNFR1-deficient Mice Despite Increased Blood Pressure

Background: Infusion of angiotensin-II (Ang-II) to wild-type (WT) mice results in hypertension, development of interstitial cardiac fibrosis and hypertrophy, and deterioration of myocardial function. We previously showed that after 1 week of Ang-II infusion, these effects were absent in mice deficient in tumor necrosis factor receptor 1 (TNFR1). We now investigated long-term effects of Ang-II infusion. Methods: WT and TNFR1-KO mice were infused with Ang-II for 6 weeks. Systolic blood pressure (SBP) was measured by tail-cuff plethysmography; cardiac function by 2D-echocardiography and Doppler ultrasound. Hearts were analyzed for collagen deposition (histology) and expression of fibrosis- and hypertrophy- related genes (quantitative PCR). Results: In WT mice, SBP increased within 7 days and remained elevated at 6 weeks (152±4 mmHg); cardiac fibrosis developed after 1 week and persisted at 6 weeks (6.2±1.1% collagen area). By contrast, in TNFR1-KO mice, SBP at 7 days was low, but increased by 6 weeks (144±4 mmHg), whereas cardiac fibrosis was absent at 1 week and did not significantly increase by 6 weeks (2.5±0.5%). In support of these data, collagen I and collagen III mRNA expression at 6 weeks were upregulated in WT (2.9±0.6 and 4.1±0.8 -fold over sham), but not in TNFR1-KO hearts (1.3±0.1 and 1.8±0.2). In both mouse groups, cardiac hypertrophy and cardiac dysfunction developed over time, however, these changes were less prominent in TNFR1-KO mice: at 6 weeks, the heart-weight to body-weight ratio in WT was 6.7±0.4, in TNFR1-KO mice 5.5±0.2; the changes in anterior and posterior wall thicknesses in WT were 44±12% and 32±15%, in TNFR1-KO mice 19±8% and 17±10%; the change in ejection fraction in WT was -67±12%, in TNFR1-KO mice -39±5%; and the change in Tei-index (myocardial performance) in WT was 18±9%, in TNFR1-KO -1±7%. Also, hypertrophy-related atrial natriuretic peptide (ANP) and beta-myosin heavy chain (b-MHC) mRNA were upregulated in WT (4.3±0.9 and 4.3±0.6 -fold over sham), but less in TNFR1-KO hearts (2.6±0.5 and 2.4±0.5). Conclusion: Despite a significant increase in blood pressure over 6 weeks of Ang-II infusion, TNFR1-KO mice developed less cardiac fibrosis and hypertrophy and had better cardiac function compared to WT mice.

Abstract 75: TNF Receptor 1 Signaling: a Mechanistic Link between Cardiac Inflammation and Fibrosis

Background: We previously showed in several models of cardiac hypertrophy and failure that, in the absence of cell death, cardiac interstitial fibrosis was mediated by the uptake of monocyte-derived fibroblast precursor cells; fibrosis and concurrent cardiac remodeling were blocked by deletion of monocyte chemoattractant protein-1 (MCP-1), as well as of tumor necrosis factor receptor-1 (TNFR1). We now investigated the cellular origin, kinetics of uptake, and subpopulation of fibroblast precursors in the angiotensin-II (Ang-II)-challenged heart. Methods: Mice with genetic deletion of TNFR1 (TNFR1-KO mice) were irradiated and rescued with bone marrow from wild-type (WT) mice, and were subjected to continuous infusion of Ang-II for 7 days. Flow cytometry was performed on isolated cells, immunostaining on perfusion-fixed tissue, quantitative PCR on whole heart mRNA isolations. Results: In WT mice, Ang-II induced the early uptake of monocytes which became M1 macrophages (CD86+CD45+) in an M1/Th1 environment. Monocytes entering the heart after 2-3 days polarized to M2 macrophages (CD301+CD45+) in an M2/Th2 milieu with concurrent appearance of collagen-producing CD301+ fibroblasts. M1 cells produced TNF, whereas M2 cells did not; both expressed TNFR1. Ang-II-exposed TNFR1-KO hearts showed similar cardiac infiltration of M1 cells, but the amount of M2 cells was significantly lower. They also had reduced expression of M1 and M2 cytokines, but not of Th1 and Th2 interleukins. Transplantation of WT bone marrow to TNFR1-KO mice before Ang-II exposure restored cardiac fibrosis, uptake of M2 cells, and expression of cytokines to levels observed in Ang-II-exposed WT animals. Many TNFR1+ cells in chimeric TNFR1-KO/WT were committed to the fibroblast lineage. Conclusion: Our data show that monocytic fibroblast precursor cells originated in the bone marrow, and that signaling through TNFR1 was required for their uptake and maturation into collagen-producing M2/fibroblasts that mediated the development of cardiac fibrosis in response to Ang-II. They also suggest a mechanistic link between inflammation and fibrosis, i.e. pro-inflammatory, M1-produced TNF initiates pro-fibrotic M2/fibroblast maturation via TNFR1 signaling.

Abstract 264: TNFR2-Bmx Signaling in Cardiac Stem Cells and Cardiac Repair

While cytokine TNF via TNFR1 induces inflammation and apoptosis, it through its second receptor TNFR2 induces cell survival and migration by activating bone marrow non-receptor tyrosine kinase Bmx. Since Bmx has been implicated in self-renewal of stem cells, we hypothesize that TNF via TNFR2 activates Bmx in cardiac stem cells (CSCs) to mediate cardiac repair. We show that in human cardiac tissue affected by ischemia heart disease (IHD), TNFR2 is expressed on intrinsic CSCs, identified as c-kit(+) /CD45(-) /VEGFR2(-) interstitial round cells, which are activated as determined by entry to cell cycle and expression of Lin-28. Wild-type mouse heart organ cultures subjected to hypoxic conditions both increase cardiac TNF expression and show induced TNFR2 and Lin-28 expression in c-kit(+) CSCs that have entered cell cycle. These CSC responses are enhanced by exogenous TNF. TNFR2(-/-) mouse heart organ cultures subjected to hypoxia increase cardiac TNF but fail to induce CSC activation. Similarly, c-kit(+) CSCs isolated from mouse hearts exposed to hypoxia or TNF show induction of Lin-28, TNFR2, cell cycle entry, and cardiogenic marker, α-sarcomeric actin (α-SA), responses more pronounced by hypoxia in combination with TNF. Knockdown of Lin-28 by siRNA results in reduced levels of TNFR2 expression, cell cycle entry, and diminished expression of α-SA (references: Stem Cells 2013;31:1881-1892). In the present study, we detect the c-kit(+)Lin28(+) CSCs populations in a mouse coronary artery ligation ischemic model. Furthermore, the c-kit(+) CSCs are reduced in TNFR2-KO and Bmx-KO mice. Mechanistically, we show a crosstalk between the TNFR2-Bmx and the c-Kit signaling pathways to mediate CSC proliferation, survival and migration. These observations suggest that TNFR2-Bmx signaling in c-kit(+) CSCs induces cardiac repair, providing a potential strategy to stimulate cardiac regeneration by TNFR2-specific agonists.