TNF-α Neutralizing Antibody Research Articles

Monoclonal Antibody Requires Immunomodulation for Efficacy Against Acinetobacter baumannii Infection.

Monoclonal antibodies (mAbs) are gaining significant momentum as novel therapeutics for infections caused by antibiotic-resistant bacteria. We evaluated the mechanism by which antibacterial mAb therapy protects against Acinetobacter baumannii infections. Anticapsular mAb enhanced macrophage opsonophagocytosis and rescued mice from lethal infections by harnessing complement, macrophages, and neutrophils; however, the degree of bacterial burden did not correlate with survival. Furthermore, mAb therapy reduced proinflammatory (interleukin-1β [IL-1β], IL-6, tumor necrosis factor-α [TNF-α]) and anti-inflammatory (IL-10) cytokines, which correlated inversely with survival. Although disrupting IL-10 abrogated the survival advantage conferred by the mAb, IL-10-knockout mice treated with mAb could still survive if TNF-α production was suppressed directly (via anti-TNF-α neutralizing antibody) or indirectly (via macrophage depletion). Thus, even for a mAb that enhances microbial clearance via opsonophagocytosis, clinical efficacy required modulation of pro- and anti-inflammatory cytokines. These findings may inform future mAb development targeting bacteria that trigger the sepsis cascade.

Macrophages augment the skeletal muscle proinflammatory response through TNFα following LPS-induced acute lung injury.

Muscle may contribute to the systemic inflammatory environment during critical illness, but leukocyte interaction and cytokine influence on muscle and its response has not been fully explored in this context. Using an in vivo model of intratracheal lipopolysaccharide (IT LPS)-induced acute lung injury, we show that skeletal muscle rapidly responds with expression of proinflammatory genes, which may be explained by migration of LPS into the circulation. Treatment of mature C2C12 myotubes with LPS at a level achieved in the circulation following IT LPS elicited a proinflammatory cytokine expression profile similar to that of in vivo murine muscle following IT LPS. Stimulation with toll-like receptor (TLR) 2 and 3 agonists provoked comparable responses in C2C12 myotubes. Additionally, co-cultures of C2C12 myotubes and bone marrow-derived macrophages (BMDM) identified the capacity of macrophages to increase myotube proinflammatory gene expression, with tumor necrosis factor-α (TNFα) gene and protein expression largely attributable to BMDM. To investigate the contribution of TNFα in the synergy of the co-culture environment, C2C12 myotubes were treated with recombinant TNFα, co-cultures were established using TNF-deficient BMDM, and co-cultures were also depleted of TNFα using antibodies. To determine whether the in vitro observations were relevant in vivo, mice received intramuscular administration of LPS±TNFα or TNFα-neutralizing antibodies and showed that TNFα is both sufficient and necessary to induce synergistic cytokine release from muscle. Taken together, these data demonstrate how skeletal muscle tissue may contribute proinflammatory cytokines following acute endotoxin injury and the potential of leukocytes to augment this response via TNFα secretion.

Extracellular vesicles of Fusobacterium nucleatum compromise intestinal barrier through targeting RIPK1-mediated cell death pathway

ABSTRACT Microbial factors that mediate microbes-host interaction in ulcerative colitis (UC), a chronic disease seriously affecting human health, are not fully known. The emerging oncobacterium Fusobacterium nucleatum (Fn) secretes extracellular vesicles carrying several types of harmful molecules in the intestine which can alter microbes-host interaction, especially the epithelial homeostasis in UC. However, the mechanism is not yet clear. Previously, we isolated EVs by the ultracentrifugation of Fn culture media and characterized them as the potent inducer of pro-inflammatory cytokines. Here, we examined the mechanism in detail. We found that in macrophage/Caco-2 co-cultures, FnEVs significantly promoted epithelial barrier loss and oxidative stress damage, which are related to epithelial necroptosis caused by the activation of receptor-interacting protein kinase 1 (RIPK1) and receptor-interacting protein kinase 3 (RIPK3). Furthermore, FnEVs promoted the migration of RIPK1 and RIPK3 into necrosome in Caco2 cells. Notably, these effects were reversed by TNF-α neutralizing antibody or Necrostatin-1 (Nec-1), a RIPK1 inhibitor. This suggested that FADD-RIPK1-caspase-3 signaling is involved in the process. Moreover, the observed effects were verified in the murine colitis model treated with FnEVs or by adoptive transfer of FnEVs-trained macrophages. In conclusion, we propose that RIPK1-mediated epithelial cell death promotes FnEVs-induced gut barrier disruption in UC and the findings can be used as the basis to further investigate this disease.

Iron-Dependent Apoptosis Causes Embryotoxicity in Inflamed and Obese Pregnancy

Since iron is essential for pregnancy, iron supplements are routinely prescribed to pregnant women without screening for iron deficiency. Most women in developed countries have sufficient iron stores, prompting concerns of the potential risks of iron supplementation in iron-replete women. A signal of potential harm of this practice is the association of maternal iron marker ferritin and pregnancy complications. Since ferritin is also induced by inflammation, it is unclear whether iron excess, inflammation, or their interaction cause fetal injury. We used mouse models to address this question. Two mouse models of maternal iron excess were used and compared to mice with normal iron status. To model dietary iron supplementation, C57BL/6 females were fed high-iron diet (2500-5000ppm iron) for 1 week prior to and during pregnancy. To model genetic iron loading, hepcidin knockout females were used and fed standard chow (185ppm iron). Two models of maternal inflammation were used. Acute systemic maternal inflammation was induced by a single subcutaneous injection of LPS on E8.5 or 15.5. Obesity was used as a model of mild chronic systemic maternal inflammation and was induced by feeding female mice Western diet. We evaluated the maternal, placental, and embryo response to iron excess in LPS- and obesity-induced inflammation at various time points. In the model of acute inflammation, we observed an adverse synergistic effect on embryo development when dams were both iron-loaded and LPS-injected, but not with either condition alone. On E8.5, LPS injection in iron-loaded dams caused embryo loss and encephalic malformations (Fig 1a) and on E15.5, LPS injection in iron-loaded dams caused embryo demise and resorption (Fig 1b). Western blotting and immunostaining showed increased apoptotic marker cleaved caspase-3 protein localized to endothelia in placentas and embryos, only when the dam was both iron-loaded and LPS-injected. Cytokine screen in vitro identified TNFα as the signal that synergized with iron to potentiate apoptosis of endothelial cells. Treatment of iron-loaded dams with TNFα-neutralizing antibody protected embryos from LPS-induced death, demonstrating the embryotoxic role of maternal TNFα in iron-loaded dams. Furthermore, RNA-Seq analysis of placental endothelial cells showed enrichment in oxidative stress pathways with maternal iron loading. Treatment of iron-loaded dams with antioxidant α-tocopherol protected against LPS-induced embryo death, attenuated placental inflammation and cleaved caspase-3 expression, and prevented endothelial apoptosis in the placenta and embryo. These data show that iron loading causes endothelial oxidative stress, which sensitizes endothelial cells to inflammation-induced apoptosis. In the obesity model, iron loading worsened embryotoxicity, causing subcutaneous hemorrhaging and eye malformations (Fig 1c), and potentiated placental cleaved caspase-3 expression. Iron loading increased TNFα levels in maternal serum. Importantly, treatment of obese iron-loaded dams with neutralizing TNFα antibody throughout pregnancy reduced the incidence of embryo eye malformations, confirming the role of TNFα in causing embryo injury in iron-loaded obese pregnancy. In summary, maternal iron excess worsened inflammation-induced embryo injury. Iron-dependent embryotoxicity was mediated by TNFα causing lethal apoptotic damage to embryo and placental endothelium which was prevented by antioxidant therapy. Our models raise the possibility that women exposed to both excessive iron and inflammation could be at risk for preventable pregnancy complications. Figure 1. Embryo outcomes in mouse pregnancies with maternal iron loading and inflammation. Figure 1 Disclosures Ganz: Akebia: Consultancy; Vifor: Consultancy; Ionis Pharmaceuticals: Consultancy; Silarus Therapeutics: Current equity holder in private company; Intrinsic LifeSciences: Current equity holder in private company; Global Blood Therapeutics: Consultancy; Ambys: Consultancy; Gossamer Bio: Consultancy; Astellas: Consultancy; Rockwell: Consultancy; Sierra Oncology: Consultancy; Disc Medicine: Consultancy; American Regent: Consultancy. Nemeth:Intrinsic LifeSciences: Current equity holder in private company; Vifor: Consultancy; Protagonist: Consultancy; Silarus Therapeutics: Current equity holder in private company; Ionis Pharmaceuticals: Consultancy.

Localization of prostaglandin E2 synthases and E-prostanoid receptors in the spinal cord in a rat model of neuropathic pain

Prostaglandin E2 (PGE2) is a lipid mediator which plays a role in the generation of inflammatory and neuropathic pain. In the peripheral nervous system, PGE2 sensitizes nociceptive afferent neurons through E-prostanoid (EP) receptors. In the central nervous system, PGE2 modulates pain sensitivity and contributes to the development of neuropathic pain. However, the distribution of PGE2 and EP receptors in the spinal cord remains unclear. In the present study, we examined the expression of PGE2 synthases (microsomal PGE synthase [mPGES]-1, mPGES-2, and cytosolic PGE synthase [cPGES]) and EP receptors (EP1-4) in a rat model of neuropathic pain.We identified that mPGES-1 mRNA was upregulated in spinal endothelial cells after nerve injury and exhibited co-localization with cyclooxygenase-2 (COX-2). We detected that mPGES-2 mRNA and cPGES mRNA were expressed in spinal neurons and noted that their expression level was not affected by nerve injury. With respect to EP receptors, EP2 mRNA and EP4 mRNA were expressed in spinal neurons in the dorsal horn. EP3 mRNA was expressed in motor neurons, whereas EP1 mRNA was not detected in the spinal cord. Intrathecal injection of tumor necrosis factor alpha (TNFα) upregulated mPGES-1 mRNA in blood vessels in the spinal cord. Intrathecal injection of a TNFα-neutralizing antibody partially inhibited the upregulation of mPGES-1 mRNA after nerve injury.These results indicate that PGE2 is synthesized by COX-2/mPGES-1 in spinal endothelial cells after nerve injury. These results suggest that in neuropathic pain condition, endothelial cell-derived PGE2 may act on EP2 and EP4 receptors on spinal neurons and modulate pain sensitivity.

Lysophosphatidic acid induces tumor necrosis factor-alpha to regulate a pro-inflammatory cytokine network in ovarian cancer.

Epithelial ovarian carcinoma tissues express high levels of tumor necrosis factor-alpha (TNF-α) and other inflammatory cytokines. The underlying mechanism leading to the abnormal TNF-α expression in ovarian cancer remains poorly understood. In the current study, we demonstrated that lysophosphatidic acid (LPA), a lipid mediator present in ascites of ovarian cancer patients, induced expression of TNF-α mRNA and release of TNF-α protein in ovarian cancer cells. LPA also induced expression of interleukin-1β (IL-1β) mRNA but no significant increase in IL-1β protein was detected. LPA enhanced TNF-α mRNA through NF-κB-mediated transcriptional activation. Inactivation of ADAM17, a disintegrin and metalloproteinase, with a specific inhibitor TMI-1 or by shRNA knockdown prevented ovarian cancer cells from releasing TNF-α protein in response to LPA, indicating that LPA-mediated TNF-α production relies on both transcriptional upregulations of the TNF-α gene and the activity of ADAM17, the membrane-associated TNF-α-converting enzyme. Like many other biological responses to LPA, induction of TNF-α by LPA also depended on the transactivation of the epidermal growth factor receptor (EGFR). Interestingly, our results revealed that ADAM17 was also the shedding protease responsible for the transactivation of EGFR by LPA in ovarian cancer cells. To explore the biological outcomes of LPA-induced TNF-α, we examined the effects of a TNF-α neutralizing antibody and recombinant TNF-α soluble receptor on LPA-stimulated expression of pro-tumorigenic cytokines and chemokines overexpressed in ovarian cancer. Blockade of TNF-α signaling significantly reduced the production of IL-8, IL-6, and CXCL1, suggesting a hierarchy of mechanisms contributing to the robust expression of the inflammatory mediators in response to LPA in ovarian cancer cells. In contrast, TNF-α inhibition did not affect LPA-dependent cell proliferation. Taken together, our results establish that the bioactive lipid LPA drives the expression of TNF-α to regulate an inflammatory network in ovarian cancer.

Immunological characterization of HM5023507, an orally active PI3Kδ/γ inhibitor.

Phosphoinositide 3‐kinases, delta (PI3Kδ) and gamma (PI3Kγ) are enriched in immune cells and regulate the development and function of innate and adaptive immunity. Dual PI3Kδγ inhibitors are considered high value targets for their potential to treat a variety of immune‐mediated diseases, but their discovery has been challenging. Here we describe the preclinical pharmacology of HM5023507, an orally active dual inhibitor of δγ isoforms in immune signaling. HM5023507 inhibited PI3Kδ and PI3Kγ isoforms with greater than 100‐fold selectivity against PI3Kα and PI3Kβ in recombinant enzymatic assays and in primary human immune cells with an exquisite selectivity against other targets. HM5023507 attenuated the PI3Kδ/γ signaling in human basophils (IC50: 42/340 nmol/L; selectivity ratio ~1:8). HM5023507 attenuated the activation and function of human B and T cells, Th17 differentiation of CD4 T cells in the blood of healthy donors and rheumatoid arthritis patients, and cytokine and IgG production in human T and B cell cocultures, in vitro. Orally dosed HM5023507 attenuated PI3K δ/γ‐mediated immune signaling in the rat in a dose‐related manner. In addition, HM5023507 inhibited semiestablished collagen‐induced arthritic inflammation in the rats (ED50 of 0.25mg/kg, p.o. BID or 0.5 mg/kg, QD, AUC: 1422 ng/mL*h), improved histopathology‐ and micro‐computed tomography (µCT)‐based indices of joint damage, bone destruction, and attenuated the levels of anti‐collagen antibody, with an overall anti‐inflammatory profile matching that of a TNFα neutralizing antibody. The PI3K δγ inhibitory profile of HM5023507 and its selectivity make it a useful tool to further delineate immunobiology of dual PI3K δγ targeting.

Expression and regulation of macrophage-inducible C-type lectin in human synovial macrophages.

Recent evidence suggests that synovial macrophage activation may be involved in cartilage destruction and pain in osteoarthritis (OA). The macrophage-inducible C-type lectin (Mincle) Clec4e is expressed in macrophages and is regulated in inflammatory conditions. Given that the regulation of Mincle in synovial macrophages has not been elucidated, we investigated the expression and regulation of Mincle in human synovial tissue (ST) harvested from patients with radiographic knee OA during total knee arthroplasty. Immunohistochemical and flow cytometric analyses were used to identify cells with Mincle expression in resected tissues. CD14-positive (CD14+; macrophage-rich cell fraction) and CD14-negative (CD14-; fibroblast-rich cell fraction) cells were extracted from the ST and used to assess MINCLE mRNA expression levels. To determine the role of tumor necrosis factor alpha (TNF-α) in the regulation of MINCLE expression, TNF-α was used to stimulate cultured CD14+ cells. Immunohistochemical staining revealed Mincle-positive cells in the synovial lining layer. Flow cytometric analysis showed that CD45+CD14+ cells were Mincle positive while CD45–/CD14– cells were Mincle negative. MINCLE expression was significantly higher in CD14+ cells than in CD14– cells. Stimulation of cultured CD14+ macrophages with TNF-α significantly increased MINCLE mRNA expression, while stimulation with TNF-α neutralizing antibody significantly decreased expression. That Mincle expression was observed in synovial macrophages and its expression was induced by TNF-α suggests that Mincle might have a key role in synovial inflammation in the osteoarthritic synovium.

A self-assembling peptide hydrogel-based drug co-delivery platform to improve tissue repair after ischemia-reperfusion injury

Ischemia-reperfusion (I/R)-induced organ injury is a serious health problem worldwide, and poor recovery of acute phase injury leads to chronic fibrosis and further organ dysfunction. Thus, a more precise approach to enhance tissue repair is needed. By using a renal I/R model, we aimed to evaluate the role of a hydrogel-based dual-drug delivery platform on promoting tissue repair. An injectable, self-assembling peptide/heparin (SAP/Hep) hydrogel was used to co-deliver TNF-α neutralizing antibody (anti-TNF-α) and hepatocyte growth factor (HGF). The microstructure and controlled release properties of KLD2R/Hep hydrogel were analyzed. The effects of the drug-loaded hydrogel (SAP-drug) on renal injury were evaluated in mice with I/R injury. In vitro, the SAP/Hep hydrogel allowed for a faster release of anti-TNF-α with a sustained release of HGF, and both drugs maintained their bioactivities after release. In vivo, combined anti-TNF-α/HGF showed better renal protective potential than anti-TNF-α or HGF alone. SAP-drug (anti-TNF-α/HGF in SAP hydrogel) treatment reduced the level of serum creatinine (Scr), blood urea nitrogen (BUN), tubular apoptosis, renal inflammatory factors, and macrophage infiltration compared to Free-drug (anti-TNF-α/HGF in solution) or SAP alone. Moreover, the SAP-drug group had better efficacy on promoting tubular cell proliferation and dedifferentiation than SAP or Free-drug alone, and thus reduced chronic renal fibrosis in I/R mice. This study highlighted that SAP could sequentially deliver the two drugs to achieve anti-inflammatory and pro-proliferative effects with one injection and thus is a promising delivery platform for tissue repair. Statement of significanceIschemia-reperfusion (I/R)-induced organ injury is a serious health issue, and delayed tissue repair leads to chronic fibrosis and organ failure. Systemic administration of anti-inflammatory agents or growth factors have shown some benefits on I/R injury, but their therapeutic efficacy was limited by side effects, poor bioavailability, and absent key signals of tissue repair. To address these issues, a hydrogel-based drug co-delivery platform was used to treat I/R injury. This platform could achieve sequential release kinetics with faster rate of anti-TNF-ɑ and slower rate of HGF, and effectively promoted tissue repair by targeting inflammation and proliferation in mice with renal I/R. This nanoscale delivery platform represents a promising strategy for solid organs (heart, liver and kidney) regeneration after I/R

CD8+ T cell/adipocyte inflammatory cross talk and ensuing M1 macrophage polarization are reduced by fish-oil-derived n-3 polyunsaturated fatty acids, in part by a TNF-α-dependent mechanism

Obese visceral adipose tissue (AT) inflammation is driven by adipokine-mediated cross talk between CD8+ T cells and adipocytes, a process mitigated by long-chain (LC) n-3 polyunsaturated fatty acids (PUFA) but underlying mechanisms and ensuing effects on macrophage polarization status are unknown. Using an in vitro co-culture model that recapitulates the degree of CD8+ T cell infiltration reported in obese AT, 3T3-L1 adipocytes were co-cultured for 24 h with purified splenic CD8+ T cells from C57Bl/6 mice consuming either a 10% w/w safflower oil (control, CON) or 7% w/w safflower oil + 3% w/w fish oil (FO) diet for 4 weeks (n=8–10/diet). Co-cultured cells were in direct contact or in a contact-independent condition separated by a Transwell permeable membrane and stimulated with lipopolysaccharide (10 ng/ml) to mimic in vivo obese endotoxin levels. In contact-dependent co-cultures, FO reduced inflammatory (IL-6, TNFα, IFN-γ) and macrophage chemotactic (CCL2, CCL7, CCL3) mRNA expression and/or secreted protein, NF-κB p65 activation, ROS accumulation, NLRP3 inflammasome priming (Nlrp3, Il1β mRNA) and activation (caspase-1 activity) compared to CON (P<.05). The anti-inflammatory action of FO was reproduced by the addition of a TNF-α neutralizing antibody (1 μg/ml) to CON co-cultures (CON/anti-TNF-α), albeit to a lesser degree. Conditioned media from FO and CON/anti-TNF-α co-cultures, in turn, reduced RAW 264.7 macrophage mRNA expression of M1 polarization markers (iNos, Cd11c, Ccr2) and associated inflammatory cytokines (Il6, Tnfα, Il1β) compared to CON. These data suggest that inflammatory CD8+ T cell/adipocyte cross talk is partially attributable to TNF-α signaling, which can be mitigated by LC n-3 PUFA.

Molecular mechanism of tumour necrosis factor alpha regulates hypocretin (orexin) expression, sleep and behaviour.

Hypocretin 1 and hypocretin 2 (orexin A and B) regulate sleep, wakefulness and emotion. Tumour necrosis factor alpha (TNF‐α) is an important neuroinflammation mediator. Here, we examined the effects of TNF‐α treatment on hypocretin expression in vivo and behaviour in mice. TNF‐α decreased hypocretin 1 and hypocretin 2 expression in a dose‐dependent manner in cultured hypothalamic neurons. TNF‐α decreased mRNA stability of prepro‐hypocretin, the single precursor of hypocretin 1 and hypocretin 2. Mice challenged with TNF‐α demonstrated decreased expression of prepro‐hypocretin, hypocretin 1 and hypocretin 2 in hypothalamus. In response to TNF‐α, prepro‐hypocretin mRNA decay was increased in hypothalamus. TNF‐α neutralizing antibody restored the expression of prepro‐hypocretin, hypocretin 1 and hypocretin 2 in vivo in TNF‐α challenged mice, supporting hypocretin system can be impaired by increased TNF‐α through decreasing hypocretin expression. Repeated TNF‐α challenge induced muscle activity during rapid eye movement sleep and sleep fragmentation, but decreased learning, cognition and memory in mice. TNF‐α neutralizing antibody blocked the effects of TNF‐α; in contrast, hypocretin receptor antagonist enhanced the effects of TNF‐α. The data support that TNF‐α is involved in the regulation of hypocretin expression, sleep and cognition. The findings shed some lights on the role of neuroinflammation in neurodegenerative diseases including Alzheimer's disease and Parkinson's disease.

Statement of the expert group on the current practice and prospects for the treatment of complex perirectal fistulas in the course of Crohn's disease.

Perirectal fistulas in the course of Crohn's disease (CD) constitute an important problem in this group of patients. They are observed in a vast majority of patients with involvement through colorectal inflammation. Perirectal fistulas in CD present a great diagnostic and therapeutic challenge due to the intensified clinical symptoms and worse prognosis than in the case of crypt originating fistulas. The condition for implementation of effective treatment of perirectal fistulas in the course of CD is the correct diagnosis, defining the anatomy of fistulas, presence of potential stenoses and inflammation in the gastrointestinal tract. Treatment of these fistulas is difficult and requires close cooperation between the colorectal surgeon and the gastroenterologist. The combination of surgical and pharmacological treatment has higher efficacy compared to surgical treatment or pharmacotherapy alone. In conservative treatment, aminosalicylates and steroids are of minor importance, while chemotherapeutics, antibiotics, and thiopurines find application in daily clinical practice. TNF-α neutralizing antibodies such as infliximab (IFX), adalimumab (ADA) or certolizumab (CER) prove to be the most effective. Surgical treatment may be provided as ad hoc; in this case, drainage procedures are recommended, usually with leaving a loose seton. Planned procedures consist in the excision of fistulas (simple fistulas) or performing more complex procedures, such as advancement flaps or ligation of the intersphincteric fistula tract Surgical measures can be complemented by the use of video technology (video-assisted anal fistula treatment VAAFT) or vacuum therapy. In extreme cases, it may be necessary to create the stoma. Treatment of perirectal fistulas includes adhesives or so-called plugs. High hopes may be associated with the introduction of stem cells into clinical practice, which is the administration of non-hematopoietic multipotent cells to the fistulas to induce the phenomenon of immunomodulation and tissue healing.

Thirty-eight-negative kinase 1 mediates trauma-induced intestinal injury and multi-organ failure.

Dysregulated intestinal epithelial apoptosis initiates gut injury, alters the intestinal barrier, and can facilitate bacterial translocation leading to a systemic inflammatory response syndrome (SIRS) and/or multi-organ dysfunction syndrome (MODS). A variety of gastrointestinal disorders, including inflammatory bowel disease, have been linked to intestinal apoptosis. Similarly, intestinal hyperpermeability and gut failure occur in critically ill patients, putting the gut at the center of SIRS pathology. Regulation of apoptosis and immune-modulatory functions have been ascribed to Thirty-eight-negative kinase 1 (TNK1), whose activity is regulated merely by expression. We investigated the effect of TNK1 on intestinal integrity and its role in MODS. TNK1 expression induced crypt-specific apoptosis, leading to bacterial translocation, subsequent septic shock, and early death. Mechanistically, TNK1 expression in vivo resulted in STAT3 phosphorylation, nuclear translocation of p65, and release of IL-6 and TNF-α. A TNF-α neutralizing antibody partially blocked development of intestinal damage. Conversely, gut-specific deletion of TNK1 protected the intestinal mucosa from experimental colitis and prevented cytokine release in the gut. Finally, TNK1 was found to be deregulated in the gut in murine and porcine trauma models and human inflammatory bowel disease. Thus, TNK1 might be a target during MODS to prevent damage in several organs, notably the gut.

Reduced Nicotinamide Adenine Dinucleotide Phosphate Inhibits MPTP-Induced Neuroinflammation and Neurotoxicity

It is generally believed that oxidative stress and neuroinflammation are implicated in the pathogenesis of Parkinson’s disease (PD). Reduced nicotinamide adenine dinucleotide phosphate (NADPH) has been demonstrated to have potent neuroprotective effects against oxidative stress. In the present research, we investigated if NADPH could offer neuroprotection by inhibiting glia-mediated neuroinflammation induced by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), a mechanism contributing to PD pathogenesis. The current data demonstrated that MPTP/MPP+ increased levels of reactive oxygen species (ROS), activated glial cells, and inflammasome proteins in the substantia nigra (SNpc), in addition to inducing the nuclear translocation of nuclear factor-κB (NF-κB) and phosphorylation of p38 MAPK. These responses were inhibited by supplementation of exogenous NADPH. Moreover, NADPH effectively decreased MPP+-induced excessive production of ROS, p38 phosphorylation and inflammatory protein of Cyclooxygenase2 (COX2) in cultured microglial BV-2 cells in vitro studies. Similarly, the p38 MAPK inhibitor SB203580 suppressed the upregulation of MPP+-induced p38 phosphorylation and COX2 protein levels. Co-culture of neuronal cells with MPP+-primed BV-2 cells increased the levels of tumor necrosis factor-alpha (TNF-α) and induced cell death of neuronal cells. These effects were diminished by TNF-α neutralizing antibody and NADPH. NADPH reduced motor dysfunction and the loss of dopaminergic (DA) cells induced by MPTP. Therefore, the present study demonstrates that NADPH protects DA neurons by inhibiting oxidative stress and glia-mediated neuroinflammation both in vitro and in vivo, thus suggesting a potential of clinical application for PD and other neurodegenerative diseases.

Identification of a NFκB Inhibition Site on the Proximal Promoter Region of Human Organic Anion Transporting Polypeptide 1A2 Coding Gene SLCO1A2.

Organic anion transporting polypeptides (OATPs; gene symbol SLCO) are membrane transporters that mediate the transport of wide ranges of compounds. The expression of different OATP members has been reported in the kidney, liver, placenta, brain, and intestine. Because of their broad substrate spectra and wide distribution within the human body, these transporters have been proposed to play key roles in the influx transport of many oral drugs. Inflammation is known to regulate the expression and functions of many drug-metabolizing enzymes and drug transporters. As a proinflammatory cytokine, tumor necrosis factor-α (TNFα) has been shown to affect the expression of different drug transporters, including OATP family members. In the present study, a putative nuclear factor-κB (NFκB) binding site ranging from -1845 to -1836 was identified at the proximal promoter region of OATP1A2 coding gene SLCO1A2 Electrophoretic mobility shift assays and chromatin immunoprecipitation showed that nuclear extracts from both breast cancer cell MCF7 and liver cancer cell HepG2 interacted with an oligonucleotide probe containing the putative NFκB binding site and that the DNA-protein complexes contained both p65 and p50 subunits of NFκB. Further study revealed that the binding site may be responsible in part for the suppression effect of TNFα toward SLCO1A2 expression because the treatment of TNFα significantly increased. Treatment of TNFα significantly increased formation of the DNA-protein complexes and mutations at essential bases of the putative NFκB binding site abolished responsiveness to the TNFα neutralizing antibody, suggesting that the binding site may be responsible in part for the suppression effect of TNFα towars SLCO1A2 expression.

Activated human B cells stimulate COX-2 expression in follicular dendritic cell-like cells via TNF-α

In spite of the potential importance of cyclooxygenase (COX)-2 expression in the germinal center, its underlying cellular and molecular mechanisms are largely unknown. COX-2 is the key enzyme generating pleiotropic prostaglandins. Based on our previous findings, we hypothesized that lymphocytes would stimulate COX-2 expression in follicular dendritic cell (FDC) by liberating cytokines. In this study, we examined the effect of tonsillar lymphocytes on COX-2 expression in FDC-like cells by immunoblotting. B but not T cells induced COX-2 protein in a time- and dose-dependent manner. Sub-fractionation analysis of B cell subsets revealed that activated but not resting B cells were responsible for the COX-2 induction. Confocal microscopy of frozen tonsils demonstrated that FDCs indeed express COX-2 in situ, in line with the in vitro results. To identify the stimulating molecule, we added neutralizing antibodies to the coculture of FDC-like cells and B cells. COX-2 induction in FDC-like cells was markedly inhibited by TNF-α neutralizing antibody. Finally, the actual production of TNF-α by activated B cells was confirmed by an enzyme immunoassay. The current study implies an unrecognized cellular interaction between FDC and B cells leading to COX-2 expression during immune inflammatory responses.

Tumor Necrosis Factor Alpha Signaling in Trigeminal Ganglion Contributes to Mechanical Hypersensitivity in Masseter Muscle During Temporomandibular Joint Inflammation.

To determine the involvement of tumor necrosis factor alpha (TNFα) signaling in the trigeminal ganglion (TG) in the mechanical hypersensitivity of the masseter muscle during temporomandibular joint (TMJ) inflammation. A total of 55 male Sprague-Dawley rats were used. Following injection of Complete Freund's Adjuvant into the TMJ, the mechanical sensitivities of the masseter muscle and the overlying facial skin were measured. Satellite glial cell (SGC) activation and TNFα expression in the TG were investigated immunohistochemically, and the effects of their inhibition on the mechanical hypersensitivity of the masseter muscle were also examined. Student t test or two-way repeated-measures analysis of variance followed by Bonferroni multiple comparisons test were used for statistical analyses. P < .05 was considered to reflect statistical significance. Mechanical allodynia in the masseter muscle was induced without any inflammatory cell infiltration in the muscle after TMJ inflammation. SGC activation and an increased number of TNFα-immunoreactive cells were induced in the TG following TMJ inflammation. Intra-TG administration of an inhibitor of SGC activity or of TNFα-neutralizing antibody depressed both the increased number of TG cells encircled by activated SGCs and the mechanical hypersensitivity of the masseter following TMJ inflammation. These findings suggest that persistent masseter hypersensitivity associated with TMJ inflammation was mediated by SGC-TG neuron interactions via TNFα signaling in the TG.

IL-17 and TNF-α Are Key Mediators of Moraxella catarrhalis Triggered Exacerbation of Allergic Airway Inflammation.

Alterations of the airway microbiome are often associated with pulmonary diseases. For example, detection of the bacterial pathogen Moraxella catarrhalis in the upper airways is linked with an increased risk to develop or exacerbate asthma. However, the mechanisms by which M. catarrhalis augments allergic airway inflammation (AAI) remain unclear. We here characterized the cellular and soluble mediators of M. catarrhalis triggered excacerbation of AAI in wt and IL-17 deficient as well as in animals treated with TNF-α and IL-6 neutralizing antibodies. We compared the type of inflammatory response in M. catarrhalis infected, house dust mite (HDM)-allergic and animals infected with M. catarrhalis at different time points of HDM sensitization. We found that airway infection of mice with M. catarrhalis triggers a strong inflammatory response with massive neutrophilic infiltrates, high amounts of IL-6 and TNF-α and moderate levels of CD4+ T-cell-derived IFN-γ and IL-17. If bacterial infection occurred during HDM allergen sensitization, the allergic airway response was exacerbated, particularly by the expansion of Th17 cells and increased TNF-α levels. Neutralization of IL-17 or TNF-α but not IL-6 resulted in accelerated clearance of M. catarrhalis and effectively prevented infection-induced exacerbation of AAI. Taken together, our data demonstrate an essential role for TNF-α and IL-17 in infection-triggered exacerbation of AAI.

Haematopoietic TLR4 deletion attenuates perivascular brown adipose tissue inflammation in atherosclerotic mice

AimsTo investigate whether haematopoietic TLR4 deletion attenuates perivascular brown adipose tissue inflammation in atherosclerotic mice. Methods and ResultsExperiments were performed using irradiated LDL receptor-deficient (LDLR−/−) mice with marrow from either TLR4-deficient (TLR4−/−) or age-matched wild-type (WT) mice. After 12 weeks of being fed a high-cholesterol diet, TLR4−/−→LDLR−/− mice developed fewer atherosclerotic lesions in the aorta compared to WT→LDLR−/− mice. This effect was associated with an increase in multilocular lipid droplets and mitochondria in perivascular adipose tissue (PVAT). Immunofluorescence analysis confirmed that there was an increase in capillary density and M2 macrophage infiltration, accompanied by a decrease in tumour necrosis factor (TNF)-α expression in the localized PVAT of TLR4−/−→LDLR−/− mice. In vitro studies indicated that bone marrow-derived macrophages (BMDMs) from WT mice demonstrated an M1-like phenotype and expression of inflammatory cytokines induced by palmitate. These effects were attenuated in BMDMs isolated from TLR4−/− mice. Furthermore, brown adipocytes incubated with conditioned medium (CM) derived from palmitate-treated BMDMs, exhibited larger and more unilocular lipid droplets, and reduced expression of brown adipocyte-specific markers and perilipin-1 compared to those observed in brown adipocytes exposed to CM from palmitate-treated BMDMs of TLR4−/− mice. This decreased potency was primarily due to TNF-α, as demonstrated by the capacity of the TNF-α neutralizing antibody to reverse these effects. ConclusionsThese results suggest that haematopoietic-specific deletion of TLR4 promotes PVAT homeostasis, which is involved in reducing macrophage-induced TNF-α secretion and increasing mitochondrial biogenesis in brown adipocytes.

Impaired expression and function of TLR8 in chronic HBV infection and its association with treatment responses during peg-IFN-α-2a antiviral therapy

Toll-like receptor8 (TLR8) plays an important role in controlling chronic viral infections. However, the role of TLR8 in chronic hepatitisB virus (HBV) infection is poorly understood. In this study, we aimed to investigate the expression and function of TLR8 in peripheral blood mononuclear cells (PBMCs) of chronic hepatitisB (CHB) patients and its alteration during peg-IFN-α-2a therapy. We evaluated TLR8 expression and antiviral function in vitro by real-time RT-PCR and flow cytometry analysis using fresh PBMCs obtained from CHB patients compared to healthy controls. We also employed clinical cohorts to investigate TLR8 expression in response to peg-IFN-α-2a therapy. TLR8 was mainly expressed in monocytes, and simulation with its ligand resulted in high levels of IFN-γ and TNF-α production. Compared with healthy controls, PBMCs obtained from CHB patients displayed reduced levels of TLR8 expression and IFN-γ, TNF-α and IL-12 induction. The exposure of HepG2.2.15 cells to conditioned medium from PBMCs stimulated by ssRNA40 strongly reduced the levels of HBV DNA, HBsAg and HBeAg, whereas the addition of IFN-γ or TNF-α neutralizing antibodies could block the antiviral effect. NK cells and Tcells were the principal IFN-γ-producing lymphocytes after ssRNA40 stimulation, whereas monocytes were the primary source of TNF-α. Analysis of the temporal dynamics showed that patients who achieved a complete response sustained a significant higher level of TLR8 mRNA than those who did not achieve a complete response beginning at week12 of peg-IFN-α-2a therapy. TLR8 expression and function in PBMCs were impaired by chronic HBV infection. Higher TLR8 expression after treatment week12 could potentially predict complete response to peg-IFN-α-2a therapy.