Expression Of TNFRSF1B Research Articles

Regulation of plasma soluble receptors of TNF and IL-1 in patients with COVID-19 differs from that observed in sepsis

IL-1α/β and TNF are closely linked to the pathology of severe COVID-19 and sepsis. The soluble forms of their receptors, functioning as decoy receptors, exhibit inhibitory effects. However, little is known about their regulation in severe bacterial and viral infections, which we aimed to investigate in this study. The circulating soluble receptors of TNF (sTNFR1 and sTNFR2) and IL-1α/β (sIL-1R1, sIL-1R2) were evaluated in the plasma of patients with COVID-19, severe bacterial infections, and sepsis and compared with healthy controls. Additionally, IL1R1, IL1R2, TNFRSF1A, and TNFRSF1B expression was evaluated at the single cell level in PBMCs derived from COVID-19 or sepsis patients. Plasma concentrations of sIL-1R1, sTNFR1, and sTNFR2 were significantly higher in COVID-19 patients compared to healthy subjects. Notably, sIL-1R1 levels were particularly elevated in ICU COVID-19 patients, and transcriptome analysis indicated heightened IL1R1 expression in PBMCs from severe COVID-19 patients. In severe bacterial infections, only sTNFR1 and sTNFR2 exhibited increased levels compared to healthy controls. Sepsis patients had decreased sIL-1R1 plasma concentrations but elevated sIL-1R2, sTNFR1, and sTNFR2 levels compared to healthy individuals, reflecting the heightened expression due to the increased numbers of monocytes present in sepsis. Finally, elevated concentrations of sIL-1R2, sTNFR1, and sTNFR2 were moderately associated with reduced 28-day survival in sepsis patients. Our study reveals distinct regulation of plasma concentrations of soluble IL-1 receptors in COVID-19 and sepsis. Moreover, soluble TNF receptors 1 and 2 consistently rise in all conditions and show a positive correlation with disease severity in sepsis.

TNFRSF1B Gene Variants in Clinicopathological Aspects and Prognosis of Patients with Cutaneous Melanoma.

Regulatory T lymphocytes play a critical role in immune regulation and are involved in the aberrant cell elimination by facilitating tumor necrosis factor connection to the TNFR2 receptor, encoded by the TNFRSF1B polymorphic gene. We aimed to examine the effects of single nucleotide variants TNFRSF1B c.587T>G, c.*188A>G, c.*215C>T, and c.*922C>T on the clinicopathological characteristics and survival of cutaneous melanoma (CM) patients. Patients were genotyped using RT-PCR. TNFRSF1B levels were measured using qPCR. Luciferase reporter assay evaluated the interaction of miR-96 and miR-1271 with the 3'-UTR of TNFRSF1B. The c.587TT genotype was more common in patients younger than 54 years old than in older patients. Patients with c.*922CT or TT, c.587TG or GG + c.*922CT or TT genotypes, as well as those with the haplotype TATT, presented a higher risk of tumor progression and death due to the disease effects. Individuals with the c.*922TT genotype had a higher TNFRSF1B expression than those with the CC genotype. miR-1271 had less efficient binding with the 3'-UTR of the T allele when compared with the C allele of the SNV c.*922C>T. Our findings, for the first time, demonstrate that TNFRSF1B c.587T>G and c.*922C>T variants can serve as independent prognostic factors in CM patients.

Galactosylceramide Upregulates the Expression of the BCL2 Gene and Downregulates the Expression of TNFRSF1B and TNFRSF9 Genes, Acting as an Anti-Apoptotic Molecule in Breast Cancer Cells.

Galactosylceramide (GalCer) increases the resistance of breast cancer cells to doxorubicin, paclitaxel, and cisplatin by acting as an anti-apoptotic molecule. GalCer was found to specifically downregulate the levels of the pro-apoptotic TNFRSF1B and TNFRSF9 genes and upregulate the levels of the anti-apoptotic BCL2 gene, suggesting that this glycosphingolipid regulates their expression at the transcriptional level. Consistent with this hypothesis, MDA-MB-231 and MCF7 breast cancer cells with high levels of GalCer showed lower activity of the TNFRSF1B and TNFRSF9 promoters than cells lacking GalCer. In contrast, the activity of the BCL2 promoter was higher in MCF7 cells overproducing GalCer than in MCF7 cells without GalCer. However, no difference in BCL2 promoter activity was observed between MDA-MB-231 cells with high and no GalCer content. Instead, we found that high levels of GalCer increased the stability of Bcl-2 mRNA. Subsequent studies showed that breast cancer cells with high levels of GalCer are characterized by significantly lower expression of P53. Importantly, inhibition of P53 expression by siRNA in MCF7 and MDA-MB-231 cells lacking GalCer resulted in decreased expression and promoter activity of the TNFRS1B and TNFRSF9 genes. On the other hand, increased expression and promoter activity of the BCL2 gene was found in such MCF7 cells, and increased stability of Bcl-2 transcripts was observed in such MDA-MB-231 cells. Taken together, these data strongly suggest that the regulatory protein that simultaneously increases the expression of the TNFRSF1B and TNFRSF9 genes and decreases the expression of the BCL2 gene and the stability of Bcl-2 transcripts is most likely P53, the expression of which is GalCer dependent.

Single-cell transcriptomics identify TNFRSF1B as a novel T-cell exhaustion marker for ovarian cancer.

Ovarian cancer (OC) patients routinely show poor immunotherapeutic response due to the complex tumour microenvironment (TME). It is urgent to explore new immunotherapeutic markers. Through the single-cell RNA sequencing (scRNA-seq) analyses on high-grade serous OC (HGSOC), moderate severity borderline tumour and matched normal ovary, we identified a novel exhausted T cells subpopulation that related to poor prognosis in OC. Histological staining, multiple immunofluorescences, and flow cytometry were applied to validate some results from scRNA-seq. Furthermore, a tumour-bearing mice model was constructed to investigate the effects of TNFRSF1B treatment on tumour growth in vivo. Highly immunosuppressive TME in HGSOC is displayed compared to moderate severity borderline tumour and matched normal ovary. Subsequently, a novel exhausted subpopulation of CD8+ TNFRSF1B+ T cells is identified, which is associated with poor survival. In vitro experiments demonstrate that TNFRSF1B is specifically upregulated on activated CD8+ T cells and suppressed interferon-γ secretion. The expression of TNFRSF1B on CD8+ T cells is closely related to OC clinical malignancy and is a marker of poor prognosis through 140 OC patients' verification. In addition, the blockade of TNFRSF1B inhibits tumour growth via profoundly remodeling the immune microenvironment in the OC mouse model. Our transcriptomic results analyzed by scRNA-seq delineate a high-resolution snapshot of the entire tumour ecosystem of OC TME. The major applications of our findings were an exhausted subpopulation of CD8+ TNFRSF1B+ T cells for predicting OC patient prognosis and the potential therapeutic value of TNFRSF1B. These findings demonstrated the clinical value of TNFRSF1B as a potential immunotherapy target and extended our understanding of factors contributing to immunotherapy failure in OC.

TNFR1 and TNFR2, Which Link NF-κB Activation, Drive Lung Cancer Progression, Cell Dedifferentiation, and Metastasis.

TNFR1 and TNFR2, encoded by TNFRSF1A and TNFRSF1B, respectively, are the most well-characterized members among the TNFR superfamily. TNFR1 is expressed in most cell types, while TNFR2 has been reported to be preferentially expressed in leukocytes. Lung cancer remains the leading cause of cancer mortality worldwide but TNFRs' activities in lung cancer development have not been fully evaluated. Recently, overexpressed TNFR1 was reported in a large proportion of human lung squamous cell carcinomas. Increased TNFR1 coupled with increased UBCH10 caused lung SCC cell dedifferentiation with epithelial-mesenchymal transition features and the metastasis in a combined spontaneous lung SCC and TNFR1 transgenic mouse model. UBCH10, an E2 ubiquitin-conjugating enzyme that is an oncogene, increased Sox2, c-Myc, Twist1, and Bcl2 levels. Increased TNFR1 upregulated UBCH10 expression by activating c-Rel and p65 NF-κB. Lung SCC patients overexpressing TNFRSF1A and one of these target genes died early compared to lung SCC patients expressing lower levels of these genes. Recently, we also revealed that TNFR2 was required for lung adenocarcinoma progression, delivering a signaling pathway of TNF/TNFR2/NF-κB-c-Rel, in which macrophage-produced ROS and TNF converted CD4 T cells to Foxp3 Treg cells, generating an immunosuppressive tumor microenvironment and promoting lung ADC progression. In human lung ADC cohorts, TNFRSF1B expression was highly correlated with TNF, FOXP3, and CD4 expression. Of note, TNF stimulated the activities of TNFR1 and TNFR2, two membrane-binding receptors, which accelerate tumorigenesis through diverse mechanisms. This review focuses on these new findings regarding the roles of TNFR1 and TNFR2 in lung SCC and ADC development in humans and mice, and highlights the potential therapeutic targets of human lung cancers.

TNFα Induces LGR5+ Stem Cell Dysfunction In Patients With Crohn's Disease.

Background & AimsTumor necrosis factor alpha (TNFα) is considered a major tissue damage-promoting effector in Crohn’s disease (CD) pathogenesis. Patient-derived intestinal organoid (enteroid) recapitulates the disease-specific characteristics of the intestinal epithelium. This study aimed to evaluate the intestinal epithelial responses to TNFα in enteroids derived from healthy controls and compare them with those of CD patient-derived enteroids.MethodsHuman enteroids derived from patients with CD and controls were treated with TNFα (30 ng/mL), and cell viability and gene expression patterns were evaluated.ResultsTNFα induced MLKL-mediated necroptotic cell death, which was more pronounced in CD patient-derived enteroids than in control enteroids. Immunohistochemistry and RNA sequencing revealed that treatment with TNFα caused expansion of the intestinal stem cell (ISC) populations. However, expanded ISC subpopulations differed in control and CD patient-derived enteroids, with LGR5+ active ISCs in control enteroids and reserve ISCs, such as BMI1+ cells, in CD patient-derived enteroids. In single-cell RNA sequencing, LGR5+ ISC-enriched cell cluster showed strong expression of TNFRSF1B (TNFR2) and cyclooxygenase-prostaglandin E2 (PGE2) activation. In TNFα-treated CD patient-derived enteroids, exogenous PGE2 (10 nmol/L) induced the expansion of the LGR5+ ISC population and improved organoid-forming efficiency, viability, and wound healing.ConclusionsTNFα increases necroptosis of differentiated cells and induces the expansion of LGR5+ ISCs. In CD patient-derived enteroids, TNFα causes LGR5+ stem cell dysfunction (expansion failure), and exogenous PGE2 treatment restored the functions of LGR5+ stem cells. Therefore, PGE2 can be used to promote mucosal healing in patients with CD.

Polymorphisms in TNF Receptor Superfamily 1B (TNFRSF1B:rs3397) are Linked to Mycobacterium avium paratuberculosis Infection and Osteoporosis in Rheumatoid Arthritis.

We previously discovered that single nucleotide polymorphisms (SNPs) in PTPN2/22 (T-cell negative-regulators) occur in 78% of rheumatoid arthritis (RA), along with Mycobacterium avium paratuberculosis (MAP) infection in 33% of patients. In Crohn’s disease, we reported that SNPs in TNFα and receptors (TNFRSF1A/TNFRSF1B) benefited intracellular MAP-survival, increased infection, and elevated inflammatory response mimicking the poor response to anti-TNFα treatment in some patients. Here, we studied the frequency and effects of SNPs in TNFα/TNFRSF1A/TNFRSF1B in RA including gene expression, MAP infection, and osteoporosis marker levels in blood (54 RA and 48 healthy controls). TNFα:rs1800629 (GA) was detected in 19/48 (40%) RA and 8/54 (15%) controls (p-value < 0.05, odds ratio (OR) = 3.6, 95% CI: 1.37–9.54). TNFRS1B:rs3397 (CT) was detected in 21/48 (44%) RA and 10/54 (19%) controls (p-value < 0.05, OR = 4.43, 95% CI: 1.73–11.33). In RA, rs3397 downregulated TNFRSF1B expression (CC > CT (0.34 ± 0.14) and CC > TT (0.27 ± 0.12)), compared to wildtype CC (0.51 ± 0.17), p-value < 0.05. MAP DNA was detected significantly in 17/48 (35.4%) RA compared to 11/54 (20.4%) controls (p-value < 0.05, OR = 2.14, 95% CI: 1.12–5.20). The average osteocalcin level was significantly lower (p-value < 0.05) in RA (2.70 ± 0.87 ng/mL), RA + MAP (0.60 ± 0.31 ng/mL), RA + TNFRSF1B:rs3397 (TT) (0.67 ± 0.35 ng/mL), compared to the healthy control (5.31 ± 1.39 ng/mL), and MAP-free RA (3.85 ± 1.31 ng/mL). Overall, rs3397 appears to downregulate TNFRSF1B, increase MAP infection, worsen inflammation, and cause osteocalcin deficiency and possibly osteoporosis in RA.

CRISPR Screens Identify Mechanisms of Natural Killer Cell Evasion across Blood Cancers

Natural killer (NK) cells have been suggested to control progression and relapse in several hematological malignancies. Enhancing NK cell reactivity represents an attractive approach to improve treatment responses. However, mechanisms enabling evasion of hematologic cancer cells from NK cells are incompletely understood. To identify cancer cell-intrinsic factors enabling resistance to NK cell cytotoxicity, we conducted genome-wide CRISPR screens in a range of hematological malignancies. Cas9-expressing cancer cells from diverse hematological malignancies, including acute and chronic myeloid leukemia (AML and CML), multiple myeloma (MM), diffuse large B-cell lymphoma (DLBCL), and B cell acute lymphoblastic leukemia were infected with a genome-scale lentiviral sgRNA library and exposed to primary human NK cells. Genes essential for NK cell cytotoxicity were discovered from surviving cancer cells which were enriched with gene knockouts inducing reduced sensitivity to NK cell killing. Additional data from a gain-of-function screen using a genome-scale CRISPR activation system were generated using the MM.1S myeloma cell line. Results from the functional genomic screens were integrated with transcriptomic data from &amp;gt; 9,000 patients across hematological cancers, as well as multi-omics data from AML and DLBCL, and both public and in-house single-cell RNA-sequencing data from AML patients. Knockout of genes encoding components involved in antigen presentation (B2M, HLA-A, HLA-C, HLA-E, TAP1, TAP2) sensitized multiple blood cancer cell lines to NK cell cytotoxicity, consistent with the inhibitory effect of MHC I on NK cells. Furthermore, knockout of interferon and JAK-STAT signaling components sensitized cancer cells to NK cell lysis, suggesting that MHC class I induction in response to interferon promotes NK cell resistance across cancer types. Interestingly, genes and pathways whose disruption conferred resistance for NK cell-mediated lysis exhibited partial overlap but also substantial variability across blood cancer types. Knockout of NCR3LG1 (B7-H6, ligand for the NKp30 activating NK cell receptor), resulted in resistance of K562 CML cells to NK cell cytotoxicity. In contrast, disruption of genes encoding apoptotic mediators (FADD, PMAIP1, BID) and TRAIL receptors (TNFRSF10B) conferred resistance to NK cell cytotoxicity in SUDHL4 DLBCL cells. The same pathways were identified in the MM cell line MM.1S, in which knockout of FAS, CASP2, and CASP8 as well as the TRAIL receptor TNFRSF10A induced NK cell resistance. Furthermore, loss of CD48, a ligand of the non-MHC binding receptor CD244 on NK cell surface, resulted in resistance and a genome-scale CRISPR gain-of-function screen in the same cell line showed sensitization upon CD48 overexpression. A CRISPR screen in the AML cell line MOLM14 identified disruption of TNFRSF1B encoding TNFR2 as strongly conferring NK cell resistance. Interestingly, TNFRSF1B overexpression sensitized the MM cell line MM.1S to NK cell treatment in the gain-of-function screen. Integration with genomic data from patients with hematological malignancies revealed selective expression of TNFRSF1B in AML. Within AML, TNFRSF1B expression was enriched in myelomonocytic and monocytic subtypes as well as in MLL-rearranged cases represented by the MOLM14 cell line. Further dissection at the single-cell level revealed increased expression of TNFRSF1B with differentiation of AML cells along the monocytic lineage. Consistently, the less differentiated MOLM13 cell line established from the same patient as MOLM14 was resistant to NK cell killing, suggesting that a less differentiated phenotype of AML cells confers resistance to NK cell cytotoxicity through lack of TNFRSF1B expression. Our data suggest that different lineages of hematological malignancies may be susceptible to NK cells through distinct mechanisms. In some cases, this heterogeneity is governed by lineage-specific expression of susceptibility genes, such as TNFRSF1B in monocytic AML. Particularly, our data identify a mechanism of NK cell evasion in less differentiated AML cells, suggesting potential for enhancing immune clearance of AML cells through differentiating therapies. figure Disclosures Lee: Kiadis Pharma: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents &amp; Royalties, Research Funding. Mitsiades:Takeda: Other: employment of a relative ; Ionis Pharmaceuticals: Honoraria; Fate Therapeutics: Honoraria; Arch Oncology: Research Funding; Sanofi: Research Funding; Karyopharm: Research Funding; Abbvie: Research Funding; TEVA: Research Funding; EMD Serono: Research Funding; Janssen/Johnson &amp; Johnson: Research Funding. Mustjoki:BMS: Honoraria, Research Funding; Novartis: Research Funding; Pfizer: Research Funding.

Postprandial inflammatory responses after oral glucose, lipid and protein challenges: Influence of obesity, sex and polycystic ovary syndrome

Most evidence linking the polycystic ovary syndrome (PCOS) with chronic low-grade inflammation has been obtained in the fasting state. We have studied the postprandial inflammatory response to oral glucose, lipid and protein challenges and the possible influences of obesity, sex and PCOS on these responses. On alternate days, we submitted 17 women with PCOS (9 non-obese, 8 obese), 17 control women (9 non-obese, 8 obese) and 19 control men (10 non-obese, 9 obese) to isocaloric (300Kcal) oral macronutrient loads. We assayed serum for TNF-α, IL-6, IL-18, IL-10, pentraxin-3 and galectin-3 concentrations and leukocytes for expression of TNF, IL6, IL10 and their receptors TNFRSF1B, IL6R and IL10RA. Circulating IL-6 levels decreased after glucose and protein ingestion but slightly increased after oral lipid intake. Leukocyte IL6 expression did not change after the ingestion of any macronutrient yet IL6R expression increased during all macronutrient challenges, the largest increase being observed after glucose ingestion. Serum TNF-α similarly decreased during either macronutrient load, whereas TNF expression increased after macronutrient ingestion, the highest increase observed after oral glucose. TNFRSF1B expression also increased after glucose intake but not after lipid or protein ingestion. No global effect of obesity or group on postprandial circulating IL-6, TNF-α, or IL6, IL6R, TNF and TNFRSF1B expression was found. Circulating IL-18 concentrations decreased during all oral challenges, whereas in case of galectin-3 and pentraxin-3 only the protein load caused a reduction in its concentrations. Of the genes studied here, IL10 showed the largest increase in expression throughout all the postprandial curves, particularly after glucose. Obesity blunted the increase in IL10 expression. IL10RA expression decreased after glucose ingestion but remained unchanged during lipid and protein loads. Glucose ingestion, as opposed to lipid and protein intake, results into the largest increase in leukocyte gene expression of inflammatory mediators. The expression of the anti-inflammatory cytokine IL10 was the largest observed here, suggesting a compensatory mechanisms against postprandial inflammation that may be blunted in obesity. However, these responses did not translate into the circulating concentrations of these inflammatory mediators during the immediate postprandial phase.

MiR-125a-5p promotes osteoclastogenesis by targeting TNFRSF1B

AimTo investigate the dysregulation of microRNAs (miRNAs) during the differentiation of osteoclasts and the precise roles of miR-125a-5p in the differentiation of osteoclasts.MethodsThe cell model of RAW 264.7 osteoclast precursor cell differentiation induced by RANKL plus M-CSF stimulation was established. During the early stage of osteoclast differentiation, miRNA expression profiles were detected using the biochip technique and analyzed by cluster analysis. TargetScan, miRTarBase and miRDB database analysis was applied to find the key target genes of miR-125a-5p. A dual luciferase experiment was conducted to identify the direct target of miR-125a-5p. MiR-125a-5p mimic transfection and anti-miR-125-5p treatment were conducted to verify the role of miR-125q-5p in osteoclast differentiation. The levels of triiodothyronine receptor auxiliary protein (TRAP), matrix metallopeptidase 2 (MMP-2), MMP-9 and cathepsin K were analyzed by qRT-PCR and western blot assay. The expression levels of MMP-2 and MMP-9 were determined using western blotting and immunofluorescence assay. The migration and invasion of RAW 264.7 cells were assessed by wound healing and Transwell invasion assays. The proliferation of RAW 264.7 osteoclast precursor cells was detected using MTT assay.ResultsThere were 44 microRNAs differently expressed during the differentiation of RAW 264.7 osteoclast precursor cells into osteoclasts, 35 of which were up-regulated and 9 were down-regulated. By luciferase reporter assay, it was confirmed that the TNF receptor superfamily member 1B gene (TNFRSF1B) was the target gene of miR-125a-5p. Up-regulation of miR-125a-5p inhibited TNFRSF1B protein expression and promoted osteoclast differentiation whereas down-regulation of miR-125a-5p caused completely opposite results.ConclusionsIn conclusion, overexpression of miR-125a-5p suppresses the expression of TNFRSF1B and promotes osteoclast differentiation. These results reveal the crucial role of miR-125a-5p in the differentiation of osteoclasts.

Gene expression comparison reveals distinct basal expression of HOX members and differential TNF-induced response between brain- and spinal cord-derived microvascular endothelial cells.

BackgroundThe heterogeneity of endothelial cell types underlies their remarkable ability to sub-specialize and provide specific requirements for a given vascular bed. Here, we compared rat microvascular endothelial cells (MECs) derived from the brain and spinal cord in both basal and inflammatory conditions.MethodsWe used whole rat genome microarrays to compare, at different time points, basal and TNF-α-induced gene expression of rat MECs from in vitro models of the blood-brain barrier (BBB) and blood-spinal cord barrier (BSCB). Validation at both messenger RNA (mRNA) and protein levels was performed on freshly extracted microvessels (MVs) from the brain and spinal cord (BMVs and SCMVs, respectively), as these were considered the closest in vivo tissues to cultured MECs.ResultsMost of the genes encoding adhesion/tight junction molecules and known endothelial markers were similarly expressed in brain and spinal cord MECs (BMECs and SCMECs, respectively). However, one striking finding was the higher expression of several Hox genes, which encode transcription factors involved in positional identity. The differential expression of Hoxa9 and Hoxb7 at the mRNA levels as well as protein levels was confirmed in BMVs and SCMVs. Although the TNF-α response was in general higher in BMECs than in SCMECs at 12 h, the opposite was observed at 48 h. Furthermore, we found that expression of Tnfrsf1a and Tnfrsf1b encoding the TNF receptor super-family member 1a/TNFR1 and 1b/TNFR2, respectively, were constitutively higher in BMVs compared to SCMVs. However, only Tnfrsf1b was induced in SCMECs in response to TNF-α at 24 and 48 h.ConclusionsOur results support a role for HOX members in defining the positional identities of MECs in vivo. Our data also suggest that the delayed transcriptional activation upon TNF-α treatment in SCMECs results from the requirement of the TNF-induced expression of Tnfrsf1b. In contrast, its high basal expression in BMECs might be sufficient to confer an immediate and efficient TNF-α response.Electronic supplementary materialThe online version of this article (doi:10.1186/s12974-016-0749-6) contains supplementary material, which is available to authorized users.

Deletion of angiotensin-converting enzyme 2 exacerbates renal inflammation and injury in apolipoprotein E-deficient mice through modulation of the nephrin and TNF-alpha-TNFRSF1A signaling.

BackgroundThe renin-angiotensin system (RAS) has been implicated in atherosclerotic lesions and progression to chronic kidney diseases. We examined regulatory roles of angiotensin-converting enzyme 2 (ACE2) in the apolipoprotein E (ApoE) knockout (KO) kidneys.MethodsThe 3-month-old wild-type, ApoEKO, ACE2KO and ApoE/ACE2 double-KO (DKO) mice in a C57BL/6 background were used. The ApoEKO mice were randomized to daily deliver either Ang II (1.5 mg/kg) and/or human recombinant ACE2 (rhACE2; 2 mg/kg) for 2 weeks. We examined changes in pro-inflammatory cytokines, renal ultrastructure, and pathological signaling in mouse kidneys.ResultsDownregulation of ACE2 and nephrin levels was observed in ApoEKO kidneys. Genetic ACE2 deletion resulted in modest elevations in systolic blood pressure levels and Ang II type 1 receptor expression and reduced nephrin expression in kidneys of the ApoE/ACE2 DKO mice with a decrease in renal Ang-(1-7) levels. These changes were linked with marked increases in renal superoxide generation, NADPH oxidase (NOX) 4 and proinflammatory factors levels, including interleukin (IL)-1beta, IL-6, IL-17A, RANTES, ICAM-1, Tumor necrosis factor-alpha (TNF-alpha) and TNFRSF1A. Renal dysfunction and ultrastructure injury were aggravated in the ApoE/ACE2 DKO mice and Ang II-infused ApoEKO mice with increased plasma levels of creatinine, blood urea nitrogen and enhanced levels of Ang II in plasma and kidneys. The Ang II-mediated reductions of renal ACE2 and nephrin levels in ApoEKO mice were remarkably rescued by rhACE2 supplementation, along with augmentation of renal Ang-(1-7) levels. More importantly, rhACE2 treatment significantly reversed Ang II-induced renal inflammation, superoxide generation, kidney dysfunction and adverse renal injury in ApoEKO mice with suppression of the NOX4 and TNF-alpha-TNFRSF1A signaling. However, rhACE2 had no effect on renal NOX2 and TNFRSF1B expression and circulating lipid levels.ConclusionsACE2 deficiency exacerbates kidney inflammation, oxidative stress and adverse renal injury in the ApoE-mutant mice through modulation of the nephrin, NOX4 and TNF-alpha-TNFRSF1A signaling. While rhACE2 supplementation alleviates inflammation, renal dysfunction and glomerulus injury in the ApoE-mutant mice associated with upregulations of Ang-(1-7) levels and nephrin expression and suppression of the TNF-alpha-TNFRSF1A signaling. Strategies aimed at enhancing the ACE2/Ang-(1-7) actions may have important therapeutic potential for atherosclerotic renal injury and kidney diseases.

Abstract 1490: Impact of bone marrow microenvironment on mRNA expression of genes in the apoptotic pathway in CLL cells

Abstract Chronic lymphocytic leukemia (CLL) is thought to arise due to dysregulated apoptosis rather than uncontrolled proliferation. Recent reports revealed that CLL cells accumulate due to both an intrinsic defect in apoptosis and extrinsic signals delivered by the microenvironment. Although several entities of bone marrow stroma cell (MSC) have been unveiled, the critical components involved in CLL survival remain obscure. In the present study, we used low density microarray analysis to identify the essential apoptotic genes (n=93) that are involved in cell survival. Primary cells obtained from CLL patients (n=12) were co-cultured with MSC (Nktert, human stromal cell line that mimics bone marrow microenvironment) in a ratio of 100:1. At the end of the incubation (12hr, d1, and d3), the expression profile of 93 genes as well as the percent apoptosis were evaluated. When CLL lymphocytes were co-cultured on MSC for 6 days, the viability of CLL cells, measured by Annexin/PI staining, increased by 6-27% at d1-d6 (n=9, p&amp;lt;0.001) compared to time matched controls. Next, we investigated the effect of MSC on global transcription. There was a 1.7+0.5 (d1, n=13) and 2.5+1.0 (d3, n=7) fold increase in global RNA synthesis as measured by [3H]uridine incorporation (p&amp;lt;0.001). Examination of mRNA profile of 93 genes showed that among the BCL2 family, the mRNA levels of the pro-apoptotic members such as BCL2L13, BAK1 and BIK decreased, while mRNA levels of anti-apoptotic, BCL2, BFL-1, BCL-XL, increased at d1 (1.46+0.56, p=0.015; 1.68+0.67, p=0.005; 2.17+1.46, p=0.018; respectively). Among caspase recruitment domain targets, pro-apoptotic APAF1 mRNA level significantly decreased at d1 but NALP1 mRNA level increased significantly (1.53+0.42, p=0.001), suggesting this gene may have alternate function in apoptosis pathway. In addition, NFKB-REL complex family members demonstrated decrease with MSC; in addition, member of IKB family, inhibitor of NFKB complex, such as NFKBIA increased significantly (1.56+0.7, p=0.018) at d1. Furthermore, in tumor necrosis factor receptor super family, TNFRSF1B expression significantly decreased (d1,0.74+0.21, p=0.001; d3,0.75+0.18, p=0.005), while TNFRSF10A and TNFRSF21 mRNA expression levels significantly increased (1.85+0.72, p=0.002; 2.81+2.08, p=0.016) at d1 but returned to baseline at d3. Additionally, 4 out of 6 DED domain containing pro-apoptotic proteins, (FADD, TRADD-adaptor proteins interacting with TNFRSF proteins, DEDD, DEDD2) demonstrated increase in mRNA levels compared to control. Moreover, mRNA levels of anti-apoptotic FLIP, which inhibits Caspase8 and Caspase10, increased significantly (1.50+0.59, p=0.013) after incubation with MSC. In summary, our data shows that MSC promoted survival to CLL lymphocytes is associated with transcriptional regulation of several genes including TNFRSF and BCL2 anti-apoptotic family members. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1490. doi:1538-7445.AM2012-1490

Expression of Nerve Growth Factor and Its Receptors NTRK1 and TNFRSF1B Is Regulated by Estrogen and Progesterone in the Uteri of Golden Hamsters1

Experiments were conducted using female golden hamsters to identify the presence of nerve growth factor (NGF) and its receptors NTRK1 and TNFRSF1B in the uteri of female animals and regulation on their expression by estrogen and progesterone. NGF and its receptor NTRK1 were immunolocalized to luminal epithelial cells, glandular cells, and stromal cells. TNFRSF1B was immunolocalized in luminal epithelial and glandular cells, with no staining found in stromal cells of the uterine horns of normal cyclic golden hamsters. Strong immunostaining of NGF and its receptors NTRK1 and TNFRSF1B was observed in uteri on the day of proestrus as compared to the other stages of the estrous cycle. Results of immunoblot analysis of NGF revealed that there was a positive correlation between uterine NGF expression and plasma concentrations of estradiol-17beta. To clarify the effects of estrogen and progesterone on NGF, NTRK1, and TNFRSF1B expression, adult female golden hamsters were ovariectomized and treated with estradiol-17beta and/or progesterone. Immunoblot analysis and immunohistochemistry indicated that estradiol-17beta stimulated expression of NGF and its two receptors in the uterus. Treatment with progesterone also increased NGF and NTRK1 expression in the uterus. However, no additive effect of these steroids on expression of NGF and its receptors was observed. Changes in uterine weights induced by estradiol-17beta and/or progesterone showed the same profile with that of NGF, suggesting that a proliferative act of NGF may be involved in uterine growth. These results suggest that NGF may play important roles in action of steroids on uterine function.