TNF-α Neutralizing Antibody Research Articles

Cytomegalovirus infection lengthens the cell cycle of granule cell precursors during postnatal cerebellar development.

Human cytomegalovirus (HCMV) infection in infants infected in utero can lead to a variety of neurodevelopmental disorders. However, mechanisms underlying altered neurodevelopment in infected infants remain poorly understood. We have previously described a murine model of congenital HCMV infection in which murine CMV (MCMV) spreads hematogenously and establishes a focal infection in all regions of the brain of newborn mice, including the cerebellum. Infection resulted in disruption of cerebellar cortical development characterized by reduced cerebellar size and foliation. This disruption was associated with altered cell cycle progression of the granule cell precursors (GCPs), which are the progenitors that give rise to granule cells (GCs), the most abundant neurons in the cerebellum. In the current study, we have demonstrated that MCMV infection leads to prolonged GCP cell cycle, premature exit from the cell cycle, and reduced numbers of GCs resulting in cerebellar hypoplasia. Treatment with TNF-α neutralizing antibody partially normalized the cell cycle alterations of GCPs and altered cerebellar morphogenesis induced by MCMV infection. Collectively, our results argue that virus-induced inflammation altered the cell cycle of GCPs resulting in a reduced numbers of GCs and cerebellar cortical hypoplasia, thus providing a potential mechanism for altered neurodevelopment in fetuses infected with HCMV.

Synovial osteoclastogenesis mediated by chondrocyte-secreted TNFα promotes TMJ condylar resorption.

Insufficient occlusal support (IOS) frequently causes subchondral bone absorption in temporomandibular joint osteoarthritis, and the underlying mechanism requires further investigation. An IOS model was established by abrading rat molars. Micro-computed tomography was used to evaluate subchondral bone changes. Osteoclastogenesis of synovium-derived macrophages (SDMs) was confirmed by TRAP staining. Cartilage-specific TNFα depletion was achieved by intra-articular injection of adeno-associated virus carrying shRNA against murine TNFα under control of collagen type II. Invitro, chondrocytes were mechanically compressed and conditioned medium (CM) was collected to detect its ability to induce osteoclastogenesis of SDMs. Synovial osteoclastogenesis and condyle resorption were observed following IOS. TNFα level was elevated in hypertrophic chondrocytes after IOS. Synovial Wnt5a level increased, but Wnt3a level decreased after IOS. Depletion of TNFα in chondrocytes alleviated the synovial osteoclastogenesis and condyle bone resorption. Invitro compression of chondrocytes potentiated TNFα expression and secretion. The CM promoted osteoclastogenesis of SDMs, which were partially prohibited by TNFα neutralizing antibody. Furthermore, inhibition of Wnt3a facilitated osteoclastogenesis, whereas inhibition of Wnt5a partially suppressed osteoclastogenesis, of SDMs cultured in CM. Chondrocyte-secreted TNFα induced by IOS is a critical regulator of synovial osteoclastogenesis and subsequent condylar resorption, partially through non-canonical Wnt5a pathway.

The role of PANoptosis in renal vascular endothelial cells: Implications for trichloroethylene-induced kidney injury

Trichloroethylene (TCE), a widely distributed environmental chemical contaminant, is extensively dispersed throughout the environment. Individuals who are exposed to TCE may manifest occupational medicamentose-like dermatitis due to trichloroethylene (OMDT). Renal impairment typically manifests in the initial phase of OMDT and is intricately linked to the disease progression and patient outcomes. Although recombinant human tumor necrosis factor-α receptor II fusion protein (rh TNFR:Fc) has been employed in the clinical management of OMDT, there was no substantial improvement in renal function observed in patients following one week of treatment. This study primarily examined the mechanism of TNFα- and IFNγ-induced endothelial cells (ECs) PANoptosis in TCE-induced kidney injury and hypothesized that the synergistic effect of TNFα and IFNγ could be the key factor affecting the efficacy of rh TNFR:Fc therapy in OMDT patients. A TCE-sensitized mouse model was utilized in this study to investigate the effects of TNFα and IFNγ neutralizing antibodies on renal vascular endothelial cell PANoptosis. The gene of interferon regulatory factor 1 (IRF1) in human umbilical vein endothelial cells (HUVEC) was silenced by using small interfering RNA (siRNA), and the cells were then treated with TNFα and IFNγ recombinant protein to investigate the mechanism of TNFα combined with IFNγ-induced PANoptosis in HUVEC. The findings indicated that mice sensitized to TCE exhibited increased levels of PANoptosis-related markers in renal endothelial cells, and treatment with TNFα and IFNγ neutralizing antibodies resulted in a significant reduction in PANoptosis and improvement in renal function. In vitro experiments demonstrated that silencing IRF1 could reverse TNFα and IFNγ-induced PANoptosis in endothelial cells. These results suggest that the efficacy of rh TNFR:Fc may be influenced by TNFα and IFNγ-mediated PANoptosis in kidney vascular endothelial cells. The joint application of TNFα and IFNγ neutralizing antibody represented a solid alternative to existing therapeutics.

Chemerin promotes invasion of oral squamous cell carcinoma by stimulating IL-6 and TNF-α production via STAT3 activation.

Elevated levels of adipokine chemerin have been identified in oral squamous cell carcinoma (OSCC) and found to be associated with metastasis to the cervical lymph nodes. The underlying mechanism through which chemerin affects OSCC progression is unclear. The aims of this study were firstly to determine chemerin levels and cytokine concentrations in serum from patients with OSCC and in OSCC cell cultures, and secondly to observe chemerin effects on OSCC cell cytokine secretion, migration, and invasion in vitro. Serum samples were collected from 20 patients diagnosed with OSCC, including groups with (LN+) and without (LN-) cervical lymph node metastasis. A Luminex liquid suspension assay was used to quantify serum concentrations of 27 types of cytokines. Correlations between chemerin and cytokines (i.e., IL-6, IL-15, GM-CSF, RANTES, TNF-α, and VEGF) were analyzed. ELISAs (enzyme-linked immunosorbent assays) were used to determine concentrations of chemerin and selected cytokines in serum and in supernatants of OSCC cell cultures (SCC9 and SCC25 cell lines). OSCC cells were stimulated with human recombinant chemerin, STAT3 inhibitor, or IL-6 together with TNF-α neutralizing antibodies. Phosphorylated STAT3 protein levels were measured with western blot analysis. OSCC cell migration and invasion were investigated with Transwell assays. Compared to the LN- group, OSCC patients with cervical lymph node metastasis had higher levels of IL-6 (P = 0.006), IL-15 (P = 0.020), GM-CSF (P = 0.036), RANTES (P = 0.032), TNF-α (P = 0.005), VEGF (P = 0.006), and chemerin (P = 0.001). Patients' serum chemerin levels correlated directly with IL-6, GM-CSF, TNF-α, and VEGF levels in OSCC patients. Exogenous recombinant chemerin treatment promoted secretion of IL-6 and TNF-α via activation of STAT3 in OSCC cells. Chemerin induced OSCC-cell migration and invasion, and these effects were reduced by IL-6 and TNF-α neutralizing antibodies. Our findings indicate that chemerin may play a role in advancing OSCC progression by increasing production of IL-6 and TNF-α, perhaps via a mechanism involving STAT3 signaling.

TNFα: TNFR1 signaling inhibits maturation and maintains the pro-inflammatory programming of monocyte-derived macrophages in murine chronic granulomatous disease.

Loss of NADPH oxidase activity results in proinflammatory macrophages that contribute to hyperinflammation in Chronic Granulomatous Disease (CGD). Previously, it was shown in a zymosan-induced peritonitis model that gp91phox-/- (CGD) monocyte-derived macrophages (MoMacs) fail to phenotypically mature into pro-resolving MoMacs characteristic of wild type (WT) but retain the ability to do so when placed in the WT milieu. Accordingly, it was hypothesized that soluble factor(s) in the CGD milieu thwart appropriate programming. We sought to identify key constituents using ex vivo culture of peritoneal inflammatory leukocytes and their conditioned media. MoMac phenotyping was performed via flow cytometry, measurement of efferocytic capacity and multiplex analysis of secreted cytokines. Addition of exogenous TNFα, TNFα neutralizing antibody and TNFR1-/- MoMacs were used to study the role of TNFα: TNFR1 signaling in MoMac maturation. More extensive phenotyping defined normal MoMac maturation and demonstrated failure of maturation of CGD MoMacs both ex vivo and in vivo. Protein components, and specifically TNFα, produced and released by CGD neutrophils and MoMacs into conditioned media was identified as critical to preventing maturation. Exogenous addition of TNFα inhibited WT MoMac maturation, and its neutralization allowed maturation of cultured CGD MoMacs. TNFα neutralization also reduced production of IL-1β, IL-6 and CXCL1 by CGD cells though these cytokines played no role in MoMac programming. MoMacs lacking TNFR1 matured more normally in the CGD milieu both ex vivo and following adoptive transfer in vivo. These data lend mechanistic insights into the utility of TNFα blockade in CGD and to other diseases where such therapy has been shown to be beneficial.

Exosomes derived from bladder epithelial cells infected with uropathogenic Escherichia coli increase the severity of urinary tract infections (UTIs) by impairing macrophage function.

Uropathogenic Escherichia coli (UPEC) is the primary causative agent of urinary tract infections (UTIs) in humans. Moreover, as one of the most common bacterial pathogens, UPEC imposes a substantial burden on healthcare systems worldwide. Epithelial cells and macrophages are two major components of the innate immune system, which play critical roles in defending the bladder against UPEC invasion. Yet, the routes of communication between these cells during UTI pathogenesis are still not fully understood. In the present study, we investigated the role of membrane-bound nanovesicles (exosomes) in the communication between bladder epithelial cells and macrophages during UPEC infection, using an array of techniques such as flow cytometry, miRNA profiling, RNA sequencing, and western blotting. Moreover, our in vitro findings were validated in a mouse model of UPEC-induced cystitis. We found that UPEC infection induced the bladder epithelial MB49 cell line to secrete large numbers of exosomes (MB49-U-Exo), which were efficiently absorbed by macrophages both in vivo and in vitro. Assimilation of MB49-U-Exo induced macrophages to produce proinflammatory cytokines, including tumor necrosis factor (TNF)α. Exposure of macrophages to MB49-U-Exo reduced their phagocytic activity (by downregulating the expression of phagocytosis-related genes) and increased their rate of apoptosis. Mechanistically, we showed that MB49-U-Exo were enriched in miR-18a-5p, which induced TNFα expression in macrophages by targeting PTEN and activating the MAPK/JNK signaling pathway. Moreover, administration of the exosome secretion inhibitor GW4869 or a TNFα-neutralizing antibody alleviated UPEC-mediated tissue damage in mice with UPEC-induced cystitis by reducing the bacterial burden of the bladder and dampening the associated inflammatory response. Collectively, these findings suggest that MB49-U-Exo regulate macrophage function in a way that exacerbates UPEC-mediated tissue impairment. Thus, targeting exosomal -release or TNFα signaling during UPEC infection may represent promising non-antibiotic strategies for treating UTIs.

Hypertonicity induces mitochondrial extracellular vesicles (MEVs) that activate TNF-α and β-catenin signaling to promote adipocyte dedifferentiation

BackgroundRecent studies demonstrated that elevated osmolarity could induce adipocyte dedifferentiation, representing an appealing procedure to generate multipotent stem cells. Here we aim to elucidate the molecular mechanisms that underlie osmotic induction of adipocyte reprogramming.MethodsTo induce dedifferentiation, the 3T3-L1 or SVF adipocytes were cultured under the hypertonic pressure in 2% PEG 300 medium. Adipocyte dedifferentiation was monitored by aspect ratio measurement, Oil Red staining and qPCR to examine the morphology, lipid droplets, and specific genes of adipocytes, respectively. The osteogenic and chondrogenic re-differentiation capacities of dedifferentiated adipocytes were also examined. To investigate the mechanisms of the osmotic stress-induced dedifferentiation, extracellular vesicles (EVs) were collected from the reprograming cells, followed by proteomic and functional analyses. In addition, qPCR, ELISA, and TNF-α neutralizing antibody (20 ng/ml) was applied to examine the activation and effects of the TNF-α signaling. Furthermore, we also analyzed the Wnt signaling by assessing the activation of β-catenin and applying BML-284, an agonist of β-catenin.ResultsHypertonic treatment induced dedifferentiation of both 3T3-L1 and the primary stromal vascular fraction (SVF) adipocytes, characterized by morphological and functional changes. Proteomic profiling revealed that hypertonicity induced extracellular vesicles (EVs) containing mitochondrial molecules including NDUFA9 and VDAC. Functionally, the mitochondrial EVs (MEVs) stimulated TNF-α signaling that activates Wnt-β-catenin signaling and adipocyte dedifferentiation. Neutralizing TNF-α inhibited hypertonic dedifferentiation of adipocytes. In addition, direct activation of Wnt-β-catenin signaling using BML-284 could efficiently induce adipocyte dedifferentiation while circumventing the apoptotic effect of the hypertonic treatment.ConclusionsHypertonicity prompts the adipocytes to release MEVs, which in turn enhances the secretion of TNF-α as a pro-inflammatory cytokine during the stress response. Importantly, TNF-α is essential for the activation of the Wnt/β-catenin signaling that drives adipocyte dedifferentiation. A caveat of the hypertonic treatment is apoptosis, which could be circumvented by direct activation of the Wnt/β-catenin signaling using BML-284.

Increased pro-remodeling and pro-thrombotic responses in the left compared to the right atrial appendage: role of low-grade inflammation and the AT1R/NADPH oxidases/SGLT2 pro-oxidant pathway

Abstract Introduction In atrial fibrillation (AF), there is a complex interplay between arrhythmia burden and cardiovascular risk factors leading to left atrial remodeling, an increased risk of stroke and heart failure. Most thrombi get formed in the atrial appendage. Several studies suggested that sodium-glucose cotransporter 2 inhibitors (SGLT2i), besides showing major benefits on heart failure, might lower the risk of incident AF. Therefore, we examined the expression level of SGLT2 in human right and left atrial appendages (RAA, LAA) and determined its role in pro-oxidant, pro-fibrotic and pro-thrombotic responses. Methods Human RAA and LAA were collected from patients undergoing cardiac surgery at a university hospital. The LAA was cut into a proximal part (low stasis, high shear) and a distal part (high stasis, low shear). The level of reactive oxygen species (ROS) was determined using dihydroethidium, mRNA and protein expression levels by RT-qPCR, and Western blot analysis and immunofluorescence, respectively, and the level of fibrosis by Sirius red staining. Results The distal LAA had higher mRNA levels of ICAM-1 and p53, and protein levels of ICAM-1, AT1R, p53, p21 and tissue factor than the proximal LAA. LAA displayed higher mRNA and protein levels of SGLT2 and SGLT1, pro-adhesive, pro-thrombotic, pro-remodeling, pro-fibrotic and senescence markers, and components of the angiotensin system, in addition to IL-1 mRNA levels and p-p65 NF-kB protein levels compared to RAA. These responses were associated with higher levels of oxidative stress, nitrotyrosine and fibrosis. SGLT2 immunofluorescence signals in distal LAA were colocalized with those of CD31, CD68, TNF-α and troponin T. Oxidative stress levels in distal and proximal LAA and RAA were reduced by inhibitors of NADPH oxidases, ACE1 and SGLT2, an AT1R antagonist, and by a TNF-α neutralizing antibody whereas NG-nitro-L-Arginine methyl ester, the non-selective NO synthase inhibitor, inhibited ROS in distal and proximal LAA without affecting RAA. In addition, the level of TNF-α in LAA was positively correlated to the indexed LA volume indicating a link between inflammation and remodeling. Conclusions The distal part of the LAA of patients undergoing heart surgery showed greater impaired endothelial function and senescence associated with more pronounced pro­-oxidant, pro­-remodeling and pro-­inflammatory responses than the proximal part and the RAA. These responses were associated with an increased expression level of SGLT 2 in endothelial cells, cardiomyocytes, and macrophages in areas showing an inflammatory response. Moreover, the pro-oxidant signal was sensitive to inhibitors of the local angiotensin system, NO synthase, SGLT2 and TNF-α. Thus, the AT1R/NADPH oxidases/SGLT2 pathway appears as an interesting target to blunt the stimulatory pro-oxidant signal in cardiac tissues affected by low-grade inflammation that leads to remodeling and fibrosis.

Activation of the TNF-α-Necroptosis Pathway in Parvalbumin-Expressing Interneurons of the Anterior Cingulate Cortex Contributes to Neuropathic Pain.

The hyperexcitability of the anterior cingulate cortex (ACC) has been implicated in the development of chronic pain. As one of the key causes of ACC hyperexcitation, disinhibition of the ACC may be closely related to the dysfunction of inhibitory parvalbumin (PV)-expressing interneurons (PV-INs). However, the molecular mechanism underlying the ACC PV-INs injury remains unclear. The present study demonstrates that spared sciatic nerve injury (SNI) induces an imbalance in the excitation and inhibition (E/I) of the ACC. To test whether tumor necrosis factor-α (TNF-α) upregulation in the ACC after SNI activates necroptosis and participates in PV-INs damage, we performed a differential analysis of transcriptome sequencing using data from neuropathic pain models and found that the expression of genes key to the TNF-α-necroptosis pathway were upregulated. TNF-α immunoreactivity (IR) signals in the ACCs of SNI rats were co-located with p-RIP3- and PV-IR, or p-MLKL- and PV-IR signals. We then systematically detected the expression and cell localization of necroptosis-related proteins, including kinase RIP1, RIP3, MLKL, and their phosphorylated states, in the ACC of SNI rats. Except for RIP1 and MLKL, the levels of these proteins were significantly elevated in the contralateral ACC and mainly expressed in PV-INs. Blocking the ACC TNF-α-necroptosis pathway by microinjecting TNF-α neutralizing antibody or using an siRNA knockdown to block expression of MLKL in the ACC alleviated SNI-induced pain hypersensitivity and inhibited the upregulation of TNF-α and p-MLKL. Targeting TNF-α-triggered necroptosis within ACC PV-INs may help to correct PV-INs injury and E/I imbalance in the ACC in neuropathic pain.

HIF-1α inhibition in macrophages preserves acute liver failure by reducing IL-1β production.

The development of acute liver failure (ALF) is dependent on its local inducer. Inflammation is a high-frequency and critical factor that accelerates hepatocyte death and liver failure. In response to injury stress, the expression of the transcription factor hypoxia-inducible factor-1α (HIF-1α) in macrophages is promoted by both oxygen-dependent and oxygen-independent mechanisms, thus promoting the expression and secretion of the cytokine interleukin-1β (IL-1β). IL-1β further induces hepatocyte apoptosis or necrosis by signaling through the receptor (IL-1R) on hepatocyte. HIF-1α knockout in macrophages or IL-1R knockout in hepatocytes protects against liver failure. However, whether HIF-1α inhibition in macrophages has a protective role in ALF is unclear. In this study, we revealed that the small molecule HIF-1α inhibitor PX-478 inhibits the expression and secretion of IL-1β, but not tumor necrosis factor α (TNFα), in bone marrow-derived macrophages (BMDMs). PX-478 pretreatment alleviates liver injury in LPS/D-GalN-induced ALF mice by decreasing the hepatic inflammatory response. In addition, preventive or therapeutic administration of PX-478 combined with TNFα neutralizing antibody markedly improved LPS/D-GalN-induced ALF. Taken together, our data suggest that PX-478 administration leads to HIF-1α inhibition and decreased IL-1β secretion in macrophages, which represents a promising therapeutic strategy for inflammation-induced ALF.

Crystalline silica-exposed human lung epithelial cells presented enhanced anchorage-independent growth with upregulated expression of BRD4 and EZH2 in autocrine and paracrine manners.

Crystalline silica-induced inflammation possibly facilitates carcinogenesis. Here, we investigated its effect on lung epithelium damage. We prepared conditioned media of immortalized human bronchial epithelial cell lines (hereinafter bronchial cell lines) NL20, BEAS-2B, and 16HBE14o- pre-exposed to crystalline silica (autocrine crystalline silica conditioned medium), a phorbol myristate acetate-differentiated THP-1 macrophage line, and VA13 fibroblast line pre-exposed to crystalline silica (paracrine crystalline silica conditioned medium). As cigarette smoking imposes a combined effect on crystalline silica-induced carcinogenesis, a conditioned medium was also prepared using the tobacco carcinogen benzo[a]pyrene diol epoxide. Crystalline silica-exposed and growth-suppressed bronchial cell lines exhibited enhanced anchorage-independent growth in autocrine crystalline silica and benzo[a]pyrene diol epoxide conditioned medium compared with that in unexposed control conditioned medium. Crystalline silica-exposed nonadherent bronchial cell lines in autocrine crystalline silica and benzo[a]pyrene diol epoxide conditioned medium showed increased expression of cyclin A2, cdc2, and c-Myc, and of epigenetic regulators and enhancers, BRD4 and EZH2. Paracrine crystalline silica and benzo[a]pyrene diol epoxide conditioned medium also accelerated the growth of crystalline silica-exposed nonadherent bronchial cell lines. Culture supernatants of nonadherent NL20 and BEAS-2B in crystalline silica and benzo[a]pyrene diol epoxide conditioned medium had higher EGF concentrations, whereas those of nonadherent 16HBE14o- had higher TNF-α levels. Recombinant human EGF and TNF-α promoted anchorage-independent growth in all lines. Treatment with EGF and TNF-α neutralizing antibodies inhibited cell growth in crystalline silica conditioned medium. Recombinant human TNF-α induced BRD4 and EZH2 expression in nonadherent 16HBE14o-. The expression of γH2AX occasionally increased despite PARP1 upregulation in crystalline silica-exposed nonadherent lines with crystalline silica and benzo[a]pyrene diol epoxide conditioned medium. Collectively, crystalline silica- and benzo[a]pyrene diol epoxide-induced inflammatory microenvironments comprising upregulated EGF or TNF-α expression may promote crystalline silica-damaged nonadherent bronchial cell proliferation and oncogenic protein expression despite occasional γH2AX upregulation. Thus, carcinogenesis may be cooperatively aggravated by crystalline silica-induced inflammation and genotoxicity.

Nupr1 Negatively Regulates Endothelial to Hematopoietic Transition in the Aorta-Gonad-Mesonephros Region.

In the aorta of mid-gestational mouse embryos, a specialized endothelial subpopulation termed hemogenic endothelial cells (HECs) develops into hematopoietic stem and progenitor cells (HSPCs), through a conserved process of endothelial-to-hematopoietic transition (EHT). EHT is tightly controlled by multiple intrinsic and extrinsic mechanisms. Nevertheless, the molecular regulators restraining this process remain poorly understood. Here, it is uncovered that, one of the previously identified HEC signature genes, Nupr1, negatively regulates the EHT process. Nupr1 deletion in endothelial cells results in increased HSPC generation in the aorta-gonad-mesonephros region. Furthermore, single-cell transcriptomics combined with serial functional assays reveals that loss of Nupr1 promotes the EHT process by promoting the specification of hematopoiesis-primed functional HECs and strengthening their subsequent hematopoietic differentiation potential toward HSPCs. This study further finds that the proinflammatory cytokine, tumor necrosis factor α (TNF-α), is significantly upregulated in Nupr1-deficient HECs, and the use of a specific TNF-α neutralizing antibody partially reduces excessive HSPC generation in the explant cultures from Nupr1-deficient embryos. This study identifies a novel negative regulator of EHT and the findings indicate that Nupr1 is a new potential target for future hematopoietic stem cell regeneration research.

Rapamycin regulates osteogenic differentiation through Parkin-mediated mitophagy in rheumatoid arthritis.

Varying degrees of bone destruction and bone loss occur in the development of rheumatoid arthritis (RA). Nevertheless, the mechanism underlying osteoporosis in the development of RA is not completely elucidated. Recent evidence indicates that mitophagy may play a vital role in regulating the differentiation and function of preosteoblast. Parkin is associated with mitophagy and various inflammatory diseases, but the precise role of Parkin in the treatment of osteoporosis in RA is unclear. In the present study, we found that the abnormal bone metabolism of RA is related to the activation of the mechanistic targets of mTORC1 pathway, and chronic inflammation which regulates the differentiation of preosteoblast through mitophagy. In this study, we found that Parkin was upregulated, and the mitochondrion was damaged in tumor necrosis factor alpha (TNF-α) stimulated preosteoblasts. Rapamycin (RAPA, an mTORC1 pathway blocker) upregulation of Parkin-mediated mitophagy tends to attenuate mitochondrial impairment caused by TNF-α in preosteoblasts. Theexperimentinvivo demonstrated that the combination therapy with TNF-α neutralizing antibody and RAPA significantly reduced osteoporosis in AIA mice. Drug inhibition of this pathway can be a potential treatment for osteoporosis in patients with RA.

High-throughput 3D microvessel-on-a-chip model to study defective angiogenesis in systemic sclerosis

In early systemic sclerosis (Scleroderma, SSc), the vasculature is impaired. Although the exact etiology of endothelial cell damage in SSc remains unclear, it is hypothesized that endothelial to mesenchymal transition (EndoMT) plays a key role. To perform physiologically relevant angiogenic studies, we set out to develop an angiogenesis-on-a-chip platform that is suitable for assessing disease parameters that are relevant to SSc and other vasculopathies. In the model, we substituted Fetal Bovine Serum (FBS) with Human Serum without impairing the stability of the culture. We showed that 3D microvessels and angiogenic factor-induced sprouts exposed to key pro-inflammatory and pro-fibrotic cytokines (TNFα and TGFβ) undergo structural alterations consisting of destructive vasculopathy (loss of small vessels). We also showed that these detrimental effects can be prevented by compound-mediated inhibition of TGFβ-ALK5 signaling or addition of a TNFα neutralizing antibody to the 3D cultures. This demonstrates that our in vitro model is suitable for compound testing and identification of new drugs that can protect from microvascular destabilization or regression in disease-mimicking conditions. To support this, we demonstrated that sera obtained from SSc patients can exert an anti-angiogenic effect on the 3D vessel model, opening the doors to screening for potential SSc drugs, enabling direct patient translatability and personalization of drug treatment.

Tumor necrosis factor-α and matrix metalloproteinase-9 cooperatively exacerbate neurovascular degeneration in the neonatal rat retina.

Matrix metalloproteinases (MMPs) and tumor necrosis factor (TNF)-α contribute to the pathogenesis of several ocular diseases. Previous studies have shown that MMP-9 activation plays an important role in capillary degeneration in injured retinas. In this study, we aimed to determine the roles of TNF-α in capillary degeneration and MMP-9 activation in the injured retina. In rats, retinal injury was induced by intravitreal injection of N-methyl-D-aspartic acid (NMDA, 200nmol) at postnatal day 7. We examined (1) the effects of blocking MMP-9 and TNF-α signaling pathway on capillary degeneration, (2) changes in protein levels and distribution of MMP-9 and TNF-α, and (3) the interaction between MMP-9 and TNF-α in regulating the expression level of each protein in retinas of NMDA-injected eyes. Intravitreal injection of GM6001, an MMP inhibitor, or TNF-α neutralizing antibody (anti-TNF-α Ab) attenuated capillary degeneration in retinas of NMDA-injected eyes. Protein levels of TNF-α increased 2h after NMDA injection, whereas those of MMP-9 increased 4h after the injection. Anti-TNF-α Ab suppressed activation of MMP-9 in retinas of NMDA-injected eyes, whereas GM6001 diminished the TNF-α protein expression. Incubation of recombinant TNF-α with supernatants of homogenized retina increased protein levels and activity of MMP-9. These results suggest that TNF-α and MMP-9 collaboratively increase their expression levels in the retina following neurodegeneration, thus leading to retinal capillary degeneration. The cooperative interaction between MMP-9 and TNF-α could be involved in the exacerbation of retinal neurovascular degeneration.

MiR-181a Improved Renal Inflammation by Targeting TNF-α in a Diabetic Nephropathy Animal Model

Introduction: Recent studies have elucidated that miR-181a plays a vital role in various inflammation-associated diseases; however, its role in the pathogenesis of diabetic nephropathy (DN) has not been elucidated clearly. Hence, we investigated the involvement of miR-181a in the pathogenesis of DN in patients and animal models. Methods: miR-181a levels were measured in the kidneys of DN patients and a DN mouse model (db/db mice) by RT-PCR. The interaction of miR-181a with the 3′-UTR of potential target transcripts was investigated using luciferase reporter assays. miR-181a-GFP-AAV and miR-181a inhibitor were used to overexpress or downregulate miR-181a in the kidney of db/db mice, respectively. The expression of inflammatory proteins and target genes was measured in the kidneys of the animals. Results: In DN patients and DN mouse models, expression analyses showed a significant decrease in miR-181a levels in the kidney. In addition, a negative linear correlation between miR-181a and proinflammatory gene (IL-1β and TNF-α) levels was observed in the kidneys of DN patients. Overexpression of miR-181a reduced IL-1β and TNF-α expression, whereas suppression of miR-181a worsened these changes, which was attenuated by combined treatment with TNF-α neutralizing antibody in db/db mice. Moreover, luciferase assays revealed that TNF-α is a direct target of miR-181a. Conclusion: Our data emphasized the anti-inflammatory actions of miR-181a by targeting TNF-α in the context of renal inflammation in DN. Hence, we concluded that miR-181a could be a potential therapeutic option for minimizing renal inflammation in DN.

Targeting TNFα produced by astrocytes expressing amyotrophic lateral sclerosis-linked mutant fused in sarcoma prevents neurodegeneration and motor dysfunction in mice.

Genetic mutations that cause amyotrophic lateral sclerosis (ALS), a progressively lethal motor neuron disease, are commonly found in ubiquitously expressed genes. In addition to direct defects within motor neurons, growing evidence suggests that dysfunction of non‐neuronal cells is also an important driver of disease. Previously, we demonstrated that mutations in DNA/RNA binding protein fused in sarcoma (FUS) induce neurotoxic phenotypes in astrocytes in vitro, via activation of the NF‐κB pathway and release of pro‐inflammatory cytokine TNFα. Here, we developed an intraspinal cord injection model to test whether astrocyte‐specific expression of ALS‐causative FUSR521G variant (mtFUS) causes neuronal damage in vivo. We show that restricted expression of mtFUS in astrocytes is sufficient to induce death of spinal motor neurons leading to motor deficits through upregulation of TNFα. We further demonstrate that TNFα is a key toxic molecule as expression of mtFUS in TNFα knockout animals does not induce pathogenic changes. Accordingly, in mtFUS‐transduced animals, administration of TNFα neutralizing antibodies prevents neurodegeneration and motor dysfunction. Together, these studies strengthen evidence that astrocytes contribute to disease in ALS and establish, for the first time, that FUS‐ALS astrocytes induce pathogenic changes to motor neurons in vivo. Our work identifies TNFα as the critical driver of mtFUS‐astrocytic toxicity and demonstrates therapeutic success of targeting TNFα to attenuate motor neuron dysfunction and death. Ultimately, through defining and subsequently targeting this toxic mechanism, we provide a viable FUS‐ALS specific therapeutic strategy, which may also be applicable to sporadic ALS where FUS activity and cellular localization are frequently perturbed.

Microglia-derived TNF-α inhibiting GABAergic neurons in the anterior lateral bed nucleus of the stria terminalis precipitates visceral hypersensitivity induced by colorectal distension in rats

Irritable bowel syndrome (IBS) is a common and debilitating gastrointestinal disorder that is exacerbated by stress and characterized by abdominal pain. Although microglia in the CNS have been implicated as an important mediator of the stress response, the role of microglia and microglia-GABAergic neuron interactions in the limbic area, most notably BNST, in the development of colorectal hypersensitivity has not been determined. We established a neonatal colorectal distension-induced chronic visceral hyperalgesia model in rats. The results showed that the frequency of spontaneous discharges of alBNST GABAergic neurons and the expression of GAD65/67 were significantly decreased in rats with chronic visceral pain. Moreover, ablation of BNST GABAergic neurons significantly reduced the visceral pain threshold in normal rats. Meanwhile, the number of M1 proinflammatory microglia and the expression of the M1 proinflammatory microglia-derived cytokines IL-6 and TNF-α were increased in the alBNST of rats with chronic visceral pain. Furthermore, alBNST infusion of the microglial inhibitor minocycline or IL-6 and TNF-α neutralizing antibodies significantly increased the visceral pain threshold. The decreased frequency of spontaneous discharges of alBNST GABAergic neurons in rats with chronic visceral pain was mimicked by a bath perfusion of TNF-α, but not IL-6, and was abolished by a perfusion of the microglial inhibitor minocycline. In addition, the alBNST infusion of the microglial inhibitor minocycline upregulated the expression of GAD65/67. Moreover, ablation of BNST GABAergic neurons significantly decreased the visceral pain threshold in normal rats, which was not reversed by a subsequent infusion of the microglial inhibitor minocycline. Our findings revealed this microglia-GABAergic neuron circuit in the alBNST, and this microglia-driven disinhibitory mechanism is essential for brain and gut dysfunction in stressful condition, providing a novel potential target for treating patients with IBS presenting visceral pain that is worsened during episodes of stress.

Met and Cxcr4 cooperate to protect skeletal muscle stem cells against inflammation-induced damage during regeneration.

Acute skeletal muscle injury is followed by an inflammatory response, removal of damaged tissue, and the generation of new muscle fibers by resident muscle stem cells, a process well characterized in murine injury models. Inflammatory cells are needed to remove the debris at the site of injury and provide signals that are beneficial for repair. However, they also release chemokines, reactive oxygen species, as well as enzymes for clearance of damaged cells and fibers, which muscle stem cells have to withstand in order to regenerate the muscle. We show here that MET and CXCR4 cooperate to protect muscle stem cells against the adverse environment encountered during muscle repair. This powerful cyto-protective role was revealed by the genetic ablation of Met and Cxcr4 in muscle stem cells of mice, which resulted in severe apoptosis during early stages of regeneration. TNFα neutralizing antibodies rescued the apoptosis, indicating that TNFα provides crucial cell-death signals during muscle repair that are counteracted by MET and CXCR4. We conclude that muscle stem cells require MET and CXCR4 to protect them against the harsh inflammatory environment encountered in an acute muscle injury.

TNF-α antagonism rescues the effect of ageing on stroke: Perspectives for targeting inflamm-ageing.

AimsEpidemiologic evidence links ischemic stroke to age, yet the mechanisms that underlie the specific and independent effects of age on stroke remain elusive, impeding the development of targeted treatments. This study tested the hypothesis that age directly aggravates stroke outcomes and proposes inflamm‐aging as a mediator and potential therapeutic target.Methods3 months‐ (young) and 18‐20 months‐old (old) mice underwent transient middle cerebral artery occlusion (tMCAO) for 30 minutes followed by 48 hours of reperfusion. Old animals received weekly treatment with the TNF‐α neutralizing antibody adalimumab over 4 weeks before tMCAO in a separate set of experiments. Plasma levels of TNF‐ α were assessed in patients with ischemic stroke and correlated with age and outcome.ResultsOld mice displayed larger stroke size than young ones with increased neuromotor deficit. Immunohistochemical analysis revealed impairment of the blood‐brain barrier in old mice, i.e. increased post‐stroke degradation of endothelial tight junctions and expression of tight junctions‐digesting and neurotoxic matrix metalloproteinases. At baseline, old animals showed a broad modulation of several circulating inflammatory mediators. TNF‐α displayed the highest increase in old animals and its inhibition restored the volume of stroke, neuromotor performance, and survival rates of old mice to the levels observed in young ones. Patients with ischemic stroke showed increased TNF‐α plasma levels which correlated with worsened short‐term neurological outcome as well as with age.ConclusionsThis study identifies TNF‐α as a causative contributor to the deleterious effect of aging on stroke and points to inflamm‐aging as a mechanism of age‐related worsening of stroke outcomes and potential therapeutic target in this context. Thus, this work provides a basis for tailoring novel stroke therapies for the particularly vulnerable elderly population.