TNF mRNA Research Articles

Probiotic use decreases intestinal inflammation and increases bone density in healthy male but not female mice

Intestinal inflammation (ie: inflammatory bowel disease, IBD) can cause osteoporosis. Probiotics, beneficial microorganisms, are currently being examined to reduce gut inflammation and treat IBD. Here, we tested if treating healthy male and female mice (moved from pathogen free to standard housing) with L. reuteri (a probiotic with anti‐TNF activity) could affect intestinal and bone health. Adult mice were given L. reuteri orally by gavage 3X/week for four weeks. L. reuteri suppressed basal intestinal TNF mRNA levels in male mice, but interestingly not female mice. Microcomputed tomography identified that L. reuteri intake increased trabecular bone parameters (mineral density, volume, trabecular number, etc..) in the distal femur metaphyseal region as well as in the lumbar vertebrae in male mice. In contrast, female mice did not display a bone response. Cortical bone parameters did not change in either gender. Histomorphometry and serum makers indicate increased osteoblast bone formation in L. reuteri treated male mice. Taken together our studies indicate that responses to oral probiotic use are gender dependent; only males display increased osteoblast activity and enhanced bone volume.

Antagonism of the histamine H4 receptor reduces LPS-induced TNF production in vivo

ObjectiveAntagonism of the histamine H4 receptor (H4R) has been shown to be anti-inflammatory in a number of preclinical disease models, however the exact mechanisms behind this are still being uncovered. In vitro, the receptor interacts with TLR and impacts inflammatory mediator production from a number of different cell types. Here it is shown that this interaction also occurs in vivo.Materials and methodsWild-type and H4R deficient BALB/c mice received an i.p. injection of LPS in PBS in conjunction with p.o. JNJ 7777120 or JNJ 28307474 (H4R antagonists). Two hours later blood was collected and TNF was measured.ResultsTwo different H4R antagonists inhibited LPS-induced TNF production in mice and this production was also reduced in H4R-deficient mice. The TNF mRNA analysis showed that the major source of the cytokine was the liver and not blood, and that the H4R antagonist only reduced the expression levels in the liver. Depletion or inactivation of macrophages reduced the TNF levels and eliminated the H4R sensitivity. Treatment with an H4R antagonist also reduced LPS-induced liver injury and blocked LPS-enhanced lung inflammation in mice.ConclusionThe data support an interaction between H4R and TLR activation in vivo that can drive inflammatory responses.Electronic supplementary materialThe online version of this article (doi:10.1007/s00011-013-0612-5) contains supplementary material, which is available to authorized users.

Histidine supplementation improves insulin resistance through suppressed inflammation in obese women with the metabolic syndrome: a randomised controlled trial

Increased inflammation and oxidative stress are associated with insulin resistance (IR) and metabolic disorders. Serum histidine levels are lower and are negatively associated with inflammation and oxidative stress in obese women. The objective of this study was to evaluate the efficacy of histidine supplementation on IR, inflammation, oxidative stress and metabolic disorders in obese women with the metabolic syndrome (MetS). A total of 100 obese women aged 33-51 years with BMI ≥ 28 kg/m² and diagnosed with MetS were included following a health examination in the community hospital in this randomised, double-blinded, placebo-controlled trial. Participants were allocated to interventions by an investigator using sequentially numbered sealed envelopes and received 4 g/day histidine (n = 50) or identical placebo (n = 50) for 12 weeks. Participants then attended the same clinic every 2 weeks for scheduled interviews and to count tablets returned. Serum histidine, HOMA-IR, BMI, waist circumference, fat mass, serum NEFA, and variables connected to inflammation and oxidative stress were measured at baseline and 12 weeks. Participants, examining physicians and investigators assessing the outcomes were blinded to group assignment. In addition, the inflammatory mechanisms of histidine were also explored in adipocytes. At 12 weeks, a total of 92 participants completed this trail. Compared with the placebo group (n = 47), histidine supplementation significantly decreased HOMA-IR (-1.09 [95% CI -1.49, -0.68]), BMI (-0.86 kg/m² [95% CI -1.55, -0.17]), waist circumference (-2.86 cm [95% CI -3.86, -1.86]), fat mass (-2.71 kg [95% CI -3.69, -1.73]), serum NEFA (-173.26 μmol/l [95% CI -208.57, -137.94]), serum inflammatory cytokines (TNF-α, -3.96 pg/ml [95% CI -5.29, -2.62]; IL-6, -2.15 pg/ml [95% CI -2.52, -1.78]), oxidative stress (superoxide dismutase, 17.84 U/ml [95% CI 15.03, 20.65]; glutathione peroxidase, 13.71 nmol/ml [95% CI 9.65, 17.78]) and increased serum histidine and adiponectin by 18.23 μmol/l [95% CI 11.74, 24.71] and 2.02 ng/ml [95% CI 0.60, 3.44] in histidine supplementation group (n = 45), respectively. There were significant correlations between changes in serum histidine and changes of IR and its risk factors. No side effects were observed during the intervention. In vitro study indicated that histidine suppresses IL6 and TNF mRNA expression and nuclear factor kappa-B (NF-κB) protein production in palmitic acid-induced adipocytes in a dose-dependent manner, and these changes were diminished by an inhibitor of NF-κB. Histidine supplementation could improve IR, reduce BMI, fat mass and NEFA and suppress inflammation and oxidative stress in obese women with MetS; histidine could improve IR through suppressed pro-inflammatory cytokine expression, possibly by the NF-κB pathway, in adipocytes.

Antiandrogen flutamide affects folliculogenesis during fetal development in pigs

Androgen deficiency during prenatal development may affect the expression of genes involved in the folliculogenesis regulation. In order to study the effect of antiandrogen on fetal ovarian development, pregnant gilts were injected with flutamide (for 7 days, 50 mg/kg bodyweight per day) or corn oil (control groups) starting on gestation days 43 (GD50), 83 (GD90), or 101 (GD108). The obtained fetal ovaries were fixed for histology and immunohistochemistry or frozen for real-time PCR. Morphological evaluation, TUNEL assay, and expression of selected factors (Ki-67, GATA binding transcription factor 4 (GATA4), E-Cadherin and tumor necrosis factor a (TNFa)) were performed. On GD90 and GD108, ovaries following flutamide administration showed a higher number of egg nests and lower number off ollicles than those in respective control groups. An increased mRNA and protein expression of Ki-67 was observed in flutamide-treated groups compared with controls on GD50 and GD108 but decreased expression was found on GD90. In comparison to control groups a higher percentage of TUNEL-positive cells was shown after flutamide exposure on GD50 and GD90 and a lower percentage of apoptotic cells was observed on GD108. These data were consistent with changes in TNF (TNFa) mRNA expression, which increased on GD90 and decreased on GD108. E-cadherin mRNA and protein expression was upregulated on GD50 and downregulated on GD90 and GD108. In conclusion diminished androgen action in porcine fetal ovaries during mid- and late gestation leads to changes in the expression of genes crucial for follicle formation. Consequently, delayed folliculogenesis was observed on GD90 and GD108. It seems however that androgens exhibit diverse biological effects depending on the gestational period.

Methyl-β-cyclodextrin alters adipokine gene expression and glucose metabolism in swine adipose tissue

This study was designed to determine whether methyl-β-cyclodextrin (MCD) can substitute for albumin in incubation medium for neonatal swine adipose tissue explants, or whether MCD affects metabolism and cytokine expression. Subcutaneous adipose tissue explants (100 ± 10 mg) were prepared from 21-day-old pigs. Explants were incubated in medium 199 supplemented with 25 mM HEPES, 1.0 nM insulin at 37°C. The medium also contained bovine serum albumin (BSA) or MCD at 0%, 0.05%, 0.1%, 0.2% or 0.3%. Tissue explants were treated with these media for 1 h and then switched to the same basal incubation medium containing 0.05% BSA. Explants were removed from basal medium at 2 or 8 h of incubation, and real-time PCR was performed to assess expression of tumor necrosis α (TNF) and interleukin 6 (IL6), acetyl CoA carboxylase (ACAC) and fatty acid synthase (FASN). Alternatively, rates of 14C-glucose oxidation and lipogenesis were monitored ± insulin (100 nM), following MCD treatment. Incubation with BSA had minimal effects on gene expression or adipose tissue metabolism, only producing a doubling in TNF mRNA abundance (P < 0.01). Treatment with MCD increased TNF mRNA abundance by eightfold (P < 0.009), whereas IL6 gene expression increased a 100-fold (P < 0.001) with a suppression in ACAC and FASN expression (P < 0.01). This was paralleled by MCD inhibition of insulin-stimulated glucose oxidation and lipogenesis (P < 0.001). Addition of a TNF antibody to the incubation medium alleviated this inhibition of insulin-stimulated glucose metabolism by ∼30% (P < 0.05).

Homing of Antigen-Presenting Cells in Head Kidney and Spleen – Salmon Head Kidney Hosts Diverse APC Types

Lymph nodes and spleen are major organs where mammalian antigen-presenting cells (APCs) initiate and orchestrate Ag-specific immune responses. Unlike mammals, teleosts lack lymph nodes and an interesting question is whether alternative organs may serve as sites for antigen presentation in teleosts. In the current study, fluorescent ovalbumin (Ova) and CpG oligonucleotides (ODNs) injected intra-abdominally were detected in significant numbers of salmon head kidney (HK) MHCII+ cells over a period of 2 weeks while in spleen the percentage of these was transient and declined from day 1 post injection. In vitro studies further shed light on the properties of the diverse MHCII+ cell types found in HK. The ultrastructure of a subpopulation of MHCII+ cells with a high capacity to endocytose and process Ova indicated that these were able to perform constitutive macropinocytosis. Upon stimulation with CpG ODNs these cells upregulated CD86 and gave very high levels of TNF mRNA indicating that these are professional APCs, related to macrophages and dendritic cells (DCs). A subpopulation of HK granulocytes expressed high levels of surface MHCII and upon CpG stimulation upregulated most of the tested APC marker genes. Although these granulocytes expressed TNF weakly, they had relatively high basal levels of IL-1β mRNA and the CpG stimulation upregulated IL-1β, along with its signaling and decoy receptors, to the highest levels as compared to other HK cell types. Interestingly, the high expression of IL-1β mRNA in the granulocytes correlated with a high autophagy flux as demonstrated by LC3-II conversion. Autophagy has recently been found to be implicated in IL-1β processing and secretion and the presented data suggests that granulocytes of salmon, and perhaps other teleost species, may serve as a valuable model to study the involvement of autophagy in regulation of the vertebrate immune response.

NKCC2A and NFAT5 regulate renal TNF production induced by hypertonic NaCl intake

Pathways that contribute to TNF production by the kidney are not well defined. Mice given 1% NaCl in the drinking water for 3 days exhibited a 2.5-fold increase in urinary, but not plasma, TNF levels compared with mice given tap water. Since furosemide attenuated the increase in TNF levels, we hypothesized that hypertonic NaCl intake increases renal TNF production by a pathway involving the Na(+)-K(+)-2Cl(-) cotransporter (NKCC2). A 2.5-fold increase in NKCC2A mRNA accumulation was observed in medullary thick ascending limb (mTAL) tubules from mice given 1% NaCl; a concomitant 2-fold increase in nuclear factor of activated T cells 5 (NFAT5) mRNA and protein expression was observed in the outer medulla. Urinary TNF levels were reduced in mice given 1% NaCl after an intrarenal injection of a lentivirus construct designed to specifically knockdown NKCC2A (EGFP-N2A-ex4); plasma levels of TNF did not change after injection of EGFP-N2A-ex4. Intrarenal injection of EGFP-N2A-ex4 also inhibited the increase of NFAT5 mRNA abundance in the outer medulla of mice given 1% NaCl. TNF production by primary cultures of mTAL cells increased approximately sixfold in response to an increase in osmolality to 400 mosmol/kgH2O produced with NaCl and was inhibited in cells transiently transfected with a dnNFAT5 construct. Transduction of cells with EGFP-N2A-ex4 also prevented increases in TNF mRNA and protein production in response to high NaCl concentration and reduced transcriptional activity of a NFAT5 promoter construct. Since NKCC2A expression is restricted to the TAL, NKCC2A-dependent activation of NFAT5 is part of a pathway by which the TAL produces TNF in response to hypertonic NaCl intake.

Astrocytic Fas ligand expression is required to induce T‐cell apoptosis and recovery from experimental autoimmune encephalomyelitis

In T-cell-mediated autoimmune diseases of the CNS, apoptosis of Fas(+) T cells by FasL contributes to resolution of disease. However, the apoptosis-inducing cell population still remains to be identified. To address the role of astrocytic FasL in the regulation of T-cell apoptosis in experimental autoimmune encephalomyelitis, we immunized C57BL/6 glial fibrillary acid protein (GFAP)-Cre FasL(fl/fl) mice selectively lacking FasL in astrocytes with MOG(35-55) peptide. GFAP-Cre FasL(fl/fl) mice were unable to resolve EAE and suffered from persisting demyelination and paralysis, while FasL(fl/fl) control mice recovered. In contrast to FasL(fl/fl) mice, GFAP-Cre FasL(fl/fl) mice failed to induce apoptosis of Fas(+) activated CD4(+) T cells and to increase numbers of Foxp3(+) Treg cells beyond day 15 post immunization, the time point of maximal clinical disease in control mice. The persistence of activated and GM-CSF-producing CD4(+) T cells in GFAP-Cre FasL(fl/fl) mice also resulted in an increased IL-17, IFN-γ, TNF, and GM-CSF mRNA expression in the CNS. In vitro, FasL(+) but not FasL(-) astrocytes induced caspase-3 expression and apoptosis of activated T cells. In conclusion, FasL expression of astrocytes plays an important role in the control and elimination of autoimmune T cells from the CNS, thereby determining recovery from EAE.

Inhibitory effect of opiates on LPS mediated release of TNF and IL-8

Most patients with advanced cancer experience severe pain and are often treated with opiates. Cancer patients are especially susceptible to opportunistic infections due to treatment with immunosuppressive and cytostatic drugs. Since opiates have been demonstrated to have immunomodulatory effects, it is of clinical importance to evaluate potential differences between commonly used opiates with regard to their effect on the immune system. The aim of this study was to evaluate the effect of morphine, tramadol, fentanyl and ketobemidone on the functioning of the immune system with special reference to TNF and IL-8 release. Method. U-937 cells were preincubated with different concentrations of opioids followed by stimulation with LPS 100 μg/ml for three hours. The effect of opioids on the levels of cytokine mRNA was studied using RT-PCR. Erk and Akt phosphorylation was also measured by Western blot. Results. All opioids with the exception of fentanyl were capable of inhibiting TNF release from U-937 cells. Morphine had no effect on IL-8 release but the effect of other opiates was almost the same as the effect on TNF. All opioids with the exception of fentanyl were capable of inhibiting production of mRNA for TNF and IL-8. The observed effects of opiates were not always reversible by naloxone, suggesting that the effects might be mediated by other receptors or through a non-receptor mediated direct effect. Although preliminary evidence suggests the involvement of Erk and Akt pathways, further studies are needed to unravel the intracellular pathways involved in mediating the effects of opiates. Our data suggests that the order of potency with regard to inhibition of cytokine release is as follows: tramadol > ketobemidone > morphine > fentanyl. Conclusion. Further studies are needed to understand the clinical implications of the observed immunosuppressive effects of tramadol and ketobemidone and to improve opioid treatment strategies in patients with cancer.

Relative overexpression of membrane-bound versus soluble TNF in spondyloarthritis

Background Macrophages and their pro-inflammatory cytokines, including TNF, are pivotal mediators of chronic synovitis in Rheumatoid Arthritis (RA) and Spondyloarthritis (SpA). Despite similar levels of synovial macrophage infiltration and clinical responses to TNF-blockade, SpA is characterized by more pronounced infiltration with alternatively activated macrophages and ongoing osteoproliferation. Here, we investigated whether these differences were related to differential expression and/or function of TNF between both diseases. Methods Expression of membrane-bound TNF (mTNF) and soluble TNF (sTNF) was measured in IFN-g, IL-4 or IL-10 polarized macrophages obtained from healthy donors. Expression of TNF and its receptors was also measured in synovial fluid (SF) and synovial tissue (ST) of actively inflamed knee joints. Mice transgenically overexpressing mTNF were evaluated for spondylitis and arthritis. Results The expression of mTNF was increased in IL-4 and IL-10 polarized macrophages compared to IFN-g polarized macrophages. Moreover, secretion of sTNF was impaired in both alternatively polarized macrophages, indicating a shift from sTNF to mTNF. In line with these data, the sTNF SF levels were lower in SpA compared to RA (p=0.01) despite similar TNF mRNA levels in ST. This was not related to altered expression of TNF receptors as both TNF-R1 and TNF-R2 were similarly expressed in ST. mRNA levels of TACE, the enzyme responsible for the cleavage of TNF, were also similar between SpA and RA ST. To investigate the relevance of overexpressed mTNF in SpA pathophysiology, we characterized mTNF transgenic mice. As previously described, these mice develop a moderate arthritis, resulting in deformation of paws and loss of grip. Histological, the joints were characterized by synovitis, lymphoid aggregates and osteoproliferation. Besides arthritis, all mice spontaneously developed spondylitis as evidenced by a crinkled tail, a hunchback and histological pathophysiology. Conclusions Membrane-bound TNF is relatively overexpressed by alternatively polarized macrophages in SpA synovitis and leads to an axial and peripheral SpA phenotype in transgenic mice.

Diagnostic and Prognostic Utility of Measuring Tumor Necrosis Factor in the Peripheral Circulation of Patients With Immune-Mediated Sensorineural Hearing Loss

To characterize levels of tumor necrosis factor (TNF; formerly known as tumor necrosis factor α), a well-established proinflammatory cytokine, in patients with immune-mediated sensorineural hearing loss (IM-SNHL) and to determine the role of this cytokine in identifying steroid-responsive hearing loss. Prospective case-control study. Tertiary care academic medical center. A total of 11 control subjects and 85 patients with clinical and audiometric characteristics of IM-SNHL (autoimmune inner ear disease and sudden SNHL combined) treated with corticosteroids were enrolled in the study. Patients were categorized as steroid responders (n = 47) and steroid nonresponders (n = 38). Peripheral venous blood was used to determine the total amount of plasma TNF by enzyme-linked immunosorbent assay. Peripheral blood mononuclear cells (PBMCs) were isolated and treated with in vitro dexamethasone. Treated and untreated PBMCs were then analyzed for release of soluble TNF protein into conditioned supernatants as well as expression of TNF messenger RNA (mRNA). Mean plasma levels of TNF, unstimulated and dexamethasone-stimulated PBMC-secreted levels of TNF, and TNF mRNA levels in unstimulated and dexamethasone-stimulated PBMCs. Steroid nonresponders had the highest mean baseline plasma levels of TNF compared with steroid responders and control subjects (27.6, 24.1, and 14.4 pg/mL, respectively) (P = .03). For patients with IM-SNHL with a high baseline plasma levels of TNF (>14.4 pg/mL), the mean TNF secreted by PBMCs was 59.1 pg/mL, which decreased to 7.2 pg/mL with in vitro dexamethasone stimulation in the responder group, while the mean TNF secreted by PBMCs was 11.2 pg/mL, which slightly increased to 11.7 pg/mL with in vitro dexamethasone stimulation in the nonresponder group (P = .04). The level of TNF can be used as both a diagnostic and prognostic cytokine for IM-SNHL. For patients presenting with a sudden change in hearing threshold, a high baseline plasma TNF from the peripheral circulation is supportive of the diagnosis if it is greater than 18.8 pg/mL, with a positive predictive value higher than 97%. In addition, this study demonstrates that for patients with IM-SNHL and high plasma levels of TNF, their clinical response to oral glucocorticoids can be predicted by their in vitro PBMC response to dexamethasone. This algorithm may further guide optimal medical treatment and possibly avoid the deleterious adverse effects of administering glucocorticoids to those patients who would not benefit from their effect.

Effect of cytokines and ovarian steroids on equine endometrial function: an in vitro study

Regulation of immune-endocrine interactions in the equine endometrium is not fully understood. The aims of the present study were to: (1) investigate the presence of tumour necrosis factor alpha (TNF), interferon gamma (IFNG), Fas ligand (FASLG) and their receptors in the mare endometrium throughout the oestrous cycle; and (2) assess endometrial secretory function (prostaglandins), angiogenic activity and cell viability in response to TNF, oestradiol (E2), progesterone (P4) and oxytocin (OXT). Transcription of TNF and FASLG mRNA increased during the early and late luteal phase (LP), whereas IFNG mRNA increased in late LP. Transcription of the mRNA of both TNF receptors was highest in the mid-LP. All cytokines and receptors were expressed in surface and glandular epithelium, as well as in the stroma. Expression of TNF and its receptor TNFRSF1A increased during the follicular phase (FP) and mid-LP. IFNG was expressed in the mid-LP, whereas its receptor IFNR1 was expressed in the in mid- and late LP. The highest expression of FASLG and FAS occurred during the late LP. OXT increased the secretion of prostaglandin (PG) E2 and PGF2α in the FP and mid-LP. In the mid-LP, E2 and P4+E2 stimulated PGF2α secretion, whereas TNF and P4 increased cell viability. All treatments, with the exception of P4, increased nitric oxide and angiogenic activity in both phases. The coordinated action of cytokines and ovarian hormones may regulate secretory, angiogenic and proliferative functions in the equine endometrium.

Opsonization of malaria-infected erythrocytes activates the inflammasome and enhances inflammatory cytokine secretion by human macrophages

BackgroundAntibody opsonization of Plasmodium falciparum-infected erythrocytes (IE) plays a crucial role in anti-malarial immunity by promoting clearance of blood-stage infection by monocytes and macrophages. The effects of phagocytosis of opsonized IE on macrophage pro-inflammatory cytokine responses are poorly understood.MethodsPhagocytic clearance, cytokine response and intracellular signalling were measured using IFN-γ-primed human monocyte-derived macrophages (MDM) incubated with opsonized and unopsonized trophozoite-stage CS2 IE, a chondroitin sulphate-binding malaria strain. Cytokine secretion was measured by bead array or ELISA, mRNA using quantitative PCR, and activation of NF-κB by Western blot and electrophoretic mobility shift assay. Data were analysed using the Mann–Whitney U test or the Wilcoxon signed rank test as appropriate.ResultsUnopsonized CS2 IE were not phagocytosed whereas IE opsonized with pooled patient immune serum (PPS) were (Phagocytic index (PI)=18.4, [SE 0.38] n=3). Unopsonized and opsonized IE induced expression of TNF, IL-1β and IL-6 mRNA by MDM and activated NF-κB to a similar extent. Unopsonized IE induced secretion of IL-6 (median= 622 pg/ml [IQR=1,250-240], n=9) but no IL-1β or TNF, whereas PPS-opsonized IE induced secretion of IL-1β (18.6 pg/mL [34.2-14.4]) and TNF (113 pg/ml [421–17.0]) and increased IL-6 secretion (2,195 pg/ml [4,658-1,095]). Opsonized, but not unopsonized, CS2 IE activated caspase-1 cleavage and enzymatic activity in MDM showing that Fc receptor-mediated phagocytosis activates the inflammasome. MDM attached to IgG-coated surfaces however secreted IL-1β in response to unopsonized IE, suggesting that internalization of IE is not absolutely required to activate the inflammasome and stimulate IL-1β secretion.ConclusionsIt is concluded that IL-6 secretion from MDM in response to CS2 IE does not require phagocytosis, whereas secretion of TNF and IL-1β is dependent on Fcγ receptor-mediated phagocytosis; for IL-1β, this occurs by activation of the inflammasome. The data presented in this paper show that generating antibody responses to blood-stage malaria parasites is potentially beneficial both in reducing parasitaemia via Fcγ receptor-dependent macrophage phagocytosis and in generating a robust pro-inflammatory response.

Oral Administration of AVX-470m, A Novel Anti-TNF Antibody, Suppresses Inflammation in a Murine Model of Inflammatory Bowel Disease

Purpose: AVX-470m is an oral polyclonal bovine anti-TNF antibody in development for IBD. The aim of the current study was to evaluate the effects of AVX-470m in modulation of pro-inflammatory cytokines and inflammatory cells in dextran sulfate sodium (DSS)-induced colitis. Methods: Colitis was induced in C57BL/6 mice by exposing the mice to 3% DSS in drinking water from Day 0 to Day 5. Animals were treated with saline, prednisolone (1.5 mg/kg), AVX-470m (0.5-5 mg/mouse) and control immunoglobulin (Ig) (1.5 mg/mouse), on Days 6 to 19, twice a day by oral gavage. Animals were sacrificed on day 19 and the colon tissues were collected. Tissues were analyzed by qPCR and immunohistochemistry (IHC). Total RNA from frozen colon samples was extracted and tested for the level of expression of TNF, IL-1β, IL-6, IL-12p40, KC, MMP-9, sICAM-1, TNF-R1, TNF-R2, and COX-2 using Taqman kits. Paraffin embedded colon tissue sections were analyzed by IHC to detect expression of TNF, IL-1β, IL-6, IL-12, IL-17, IFN-γ, CD3, CD68 and myeloperoxidase (MPO). Results: Oral administration of AVX-470m significantly reduced disease severity by endoscopy, colon weight, colon length and colon pathology in this model. Induction of colitis with DSS resulted in increased mRNA expression in colon tissues of all markers tested. Likewise, analysis of protein expression by IHC showed all tested markers to be up-regulated, reflecting increased inflammation and an increased colonic infiltration of T cells, macrophages and neutrophils. Treatment with AVX-470m, but not control Ig, resulted in statistically significant decreased expression of mRNA for TNF, IL-1β, IL-6, IL-12p40 and MMP-9. In addition, statistically significant decreased expression of TNF, IL-1β, IL-6, IL-12, IL-17, IFN-γ, CD68 and MPO was seen by IHC. No effect on T cell infiltrates (CD3 expression) was observed. Analysis of the distribution of AVX-470m in tissue revealed local deposition of the antibody in the colon, with little if any distribution into the systemic circulation. Conclusion: AVX-470m reduced colonic expression of TNF-mediated pathways, including mRNA and protein levels of inflammatory mediators and cell markers, in a murine model of IBD. These results support the therapeutic potential of an oral anti-TNF antibody for treatment of IBD, acting by inhibition of TNF-mediated inflammation pathways in the GI tract in the absence of immunosuppressive systemic exposure. Disclosure: Dr. Bhol - Employee, Stockholder/Ownership interest, Avaxia Biologics, Inc. Supported by NIH Grant 2R44DK083810-02. Mr. Keane - Employee, Stockholder/Ownership interest, Avaxia Biologics, Inc. Supported by NIH Grant 2R44DK083810-02. Ms. Lemos - Employee, Stockholder/Ownership interest, Avaxia Biologics, Inc. Supported by NIH Grant 2R44DK083810-02. Dr. Fox - Employee, Board Member, Stockholder/Ownership interest, Avaxia Biologics, Inc. Supported by NIH Grant 2R44DK083810-02. Dr. Tracey - Employee, Stockholder/Ownership interest, Avaxia Biologics, Inc. Supported by NIH Grant 2R44DK083810-02.

The p38/MK2-Driven Exchange between Tristetraprolin and HuR Regulates AU–Rich Element–Dependent Translation

TNF expression of macrophages is under stringent translational control that depends on the p38 MAPK/MK2 pathway and the AU–rich element (ARE) in the TNF mRNA. Here, we elucidate the molecular mechanism of phosphorylation-regulated translation of TNF. We demonstrate that translation of the TNF-precursor at the ER requires expression of the ARE–binding and -stabilizing factor human antigen R (HuR) together with either activity of the p38 MAPK/MK2 pathway or the absence of the ARE-binding and -destabilizing factor tristetraprolin (TTP). We show that phosphorylation of TTP by MK2 decreases its affinity to the ARE, inhibits its ability to replace HuR, and permits HuR-mediated initiation of translation of TNF mRNA. Since translation of TTP's own mRNA is also regulated by this mechanism, an intrinsic feedback control of the inflammatory response is ensured. The phosphorylation-regulated TTP/HuR exchange at target mRNAs provides a reversible switch between unstable/non-translatable and stable/efficiently translated mRNAs.

Theiler's murine encephalomyelitis virus induced phenotype switch of microglia in vitro

The present in vitro study aimed to define the involvement of astrocytes and microglia in the initial inflammatory response of Theiler's murine encephalomyelitis (TME), a virus-induced mouse model of multiple sclerosis, and whether intralesional microglia exert pro- (M1) or anti-inflammatory (M2) effects following TME virus (TMEV) infection. Therefore astrocytes and microglia were purified from neonatal murine brains and inoculated either with TMEV or mock-solution. Gene expression of IL-1, IL-2, IL-10, IL-12, TNF, TNF receptors (TNFR1, TNFR2), TGFβ1, IFNγ and transcription factors NF-κB (p50, p65) and AP-1 (c-jun, c-fos) were quantified using RT-qPCR at 6, 48, and 240h post infection (hpi). In addition, IL-1, IL-10, IL-12, TNF and TGFβ1 mRNA transcripts were investigated at 168 hpi in TMEV- and mock-infected SJL/J mice. Overall in vitro astrocytes showed a significant higher amount of viral RNA compared to microglia. In addition, TMEV-infected astrocytes showed higher numbers of IL-1, IL-12 and TNF transcripts at 48 hpi. In microglia high IL-10 and low IL-12 mRNA levels were detected at 48 hpi, while the opposite was the case at 240 hpi. In addition, TNF mRNA was increased in microglia at 240 hpi. In addition, the observed up-regulation of IL-1, IL-12 and IL-10 in the early phase of TME in vivo substantiates the relevance of these cytokines during the disease induction.Summarized data indicate that TMEV infection of microglia induces a switch from the anti-inflammatory (M2) during the early phase to the pro-inflammatory (M1) phenotype in the later phase of the infection. The simultaneous expression of TNF and its receptors by both cell types might generate autocrine feedback loops possibly associated with pro-inflammatory actions of astrocytes via TNFR1.

Life or Death Decisions in the Corpus Luteum

The corpus luteum (CL) is an ephemeral endocrine organ. During its lifespan, it undergoes a period of extremely rapid growth that involves hypertrophy, proliferation and differentiation of the steroidogenic cells, as well as extensive angiogenesis. The growth phase is followed by a period in which remodelling of the tissue ceases, but it engages in unparalleled production of steroids, resulting in extraordinarily high metabolic activity within the tissue. It is during this stage that a critical juncture occurs. In the non-fertile cycle, uterine release of prostaglandin (PG)F(2α) initiates a cascade of events that result in rapid loss of steroidogenesis and destruction of the luteal tissue. Alternatively, if a viable embryo is present, signals are produced that result in rescue of the CL. This review article summarizes the major concepts related to the fate of the CL, with particular focus on recent insights into the mechanisms associated with the ability of PGF(2α) to bring about complete luteolysis. It has become clear that the achievement of luteolysis depends on repeated exposure to PGF(2α) and involves coordinated actions of heterogeneous cell types within the CL. Together, these components of the process bring about not only the loss in progesterone production, but also the rapid demise of the structure itself.

Increasing TNF levels solely in the rat hippocampus produces persistent pain-like symptoms

The manifestation of chronic, neuropathic pain includes elevated levels of the cytokine tumor necrosis factor–alpha (TNF). Previously, we have shown that the hippocampus, an area of the brain most notable for its role in learning and memory formation, plays a fundamental role in pain sensation. Using an animal model of peripheral neuropathic pain, we have demonstrated that intracerebroventricular infusion of a TNF antibody adjacent to the hippocampus completely alleviated pain. Furthermore, intracerebroventricular infusion of rTNF adjacent to the hippocampus induced pain behavior in naïve animals similar to that expressed during a model of neuropathic pain. These data support our premise that enhanced production of hippocampal-TNF is integral in pain sensation. In the present study, TNF gene expression was induced exclusively in the hippocampus, eliciting increased local bioactive TNF levels, and animals were assessed for pain behaviors. Male Sprague-Dawley rats received stereotaxic injection of gold nanorod (GNR)-complexed cDNA (control or TNF) plasmids (nanoplasmidexes), and pain responses (i.e., thermal hyperalgesia and mechanical allodynia) were measured. Animals receiving hippocampal microinjection of TNF nanoplasmidexes developed thermal hyperalgesia bilaterally. Sensitivity to mechanical stimulation also developed bilaterally in the rat hind paws. In support of these behavioral findings, immunoreactive staining for TNF, bioactive levels of TNF, and levels of TNF mRNA per polymerase chain reaction analysis were assessed in several brain regions and found to be increased only in the hippocampus. These findings indicate that the specific elevation of TNF in the hippocampus is not a consequence of pain, but in fact induces these behaviors/symptoms.

The Protein Zfand5 Binds and Stabilizes mRNAs with AU-rich Elements in Their 3′-Untranslated Regions

AU-rich elements (AREs) in the 3'-UTR of unstable transcripts play a vital role in the regulation of many inflammatory mediators. To identify novel ARE-dependent gene regulators, we screened a human leukocyte cDNA library for candidates that enhanced the activity of a luciferase reporter bearing the ARE sequence from TNF (ARE(TNF)). Among 171 hits, we focused on Zfand5 (zinc finger, AN1-type domain 5), a 23-kDa protein containing two zinc finger domains. Zfand5 expression was induced in macrophages in response to IFNγ and Toll-like receptor ligands. Knockdown of Zfand5 in macrophages decreased expression of ARE class II transcripts TNF and COX2, whereas overexpression stabilized TNF mRNA by suppressing deadenylation. Zfand5 specifically bound to ARE(TNF) mRNA and competed with tristetraprolin, a protein known to bind and destabilize class II ARE-containing RNAs. Truncation studies indicated that both zinc fingers of Zfand5 contributed to its mRNA-stabilizing function. These findings add Zfand5 to the growing list of RNA-binding proteins and suggest that Zfand5 can enhance ARE-containing mRNA stability by competing with tristetraprolin for mRNA binding.

Proteomic and pathway analyses reveal a network of inflammatory genes associated with differences in skin tumor promotion susceptibility in DBA/2 and C57BL/6 mice

Genetic susceptibility to two-stage skin carcinogenesis is known to vary significantly among different stocks and strains of mice. In an effort to identify specific protein changes or altered signaling pathways associated with skin tumor promotion susceptibility, a proteomic approach was used to examine and identify proteins that were differentially expressed in epidermis between promotion-sensitive DBA/2 and promotion-resistant C57BL/6 mice following treatment with 12-O-tetradecanoylphorbol-13-acetate (TPA). We identified 19 differentially expressed proteins of which 5 were the calcium-binding proteins annexin A1, parvalbumin α, S100A8, S100A9, and S100A11. Further analyses revealed that S100A8 and S100A9 protein levels were also similarly differentially upregulated in epidermis of DBA/2 versus C57BL/6 mice following topical treatment with two other skin tumor promoters, okadaic acid and chrysarobin. Pathway analysis of all 19 identified proteins from the present study suggested that these proteins were components of several networks that included inflammation-associated proteins known to be involved in skin tumor promotion (e.g. TNF-α, NFκB). Follow-up studies revealed that Tnf, Nfkb1, Il22, Il1b, Cxcl1, Cxcl2 and Cxcl5 mRNAs were highly expressed in epidermis of DBA/2 compared with C57BL/6 mice at 24h following treatment with TPA. Furthermore, NFκB (p65) was also highly activated at the same time point (as measured by phosphorylation at ser276) in epidermis of DBA/2 mice compared with C57BL/6 mice. Taken together, the present data suggest that differential expression of genes involved in inflammatory pathways in epidermis may play a key role in genetic differences in susceptibility to skin tumor promotion in DBA/2 and C57BL/6 mice.