Tumor Necrosis Factor Response Research Articles

143 Individual Vagus Nerve Sensory Neurons Respond Distinctly to Specific Pro-inflammatory Cytokines

INTRODUCTION: The peripheral nervous system (PNS) communicates with the immune system in response to injury or infection. In the case of inflammation, sensory signals travel up to the brain via the vagus nerve, which is a major pathway for neuro-immune communication. While our previous work showed that systemic cytokine administration was associated with specific patterns of vagus nerve activity, we did not know how individual vagal sensory neurons encoded this immune information. Here, we use invivo calcium imaging to monitor the neural activity of individual vagal sensory neurons in response to specific inflammatory mediators. METHODS: Using mice that express the calcium sensor GCaMP6f in glutamatergic neurons, we imaged neurons of the nodose ganglion in-vivo using a 1-photon miniature microscope (Miniscope). During imaging, the inflammatory mediator tumor necrosis factor (TNF) and interleukin 1β (IL-1β) were applied directly to the vagus nerve. The fluorescence data were analyzed with Python-based calcium imaging analysis (CaImAn) software and customized pipelines to output the neural activity as the normalized change in fluorescence (DFF) individually detected neurons. RESULTS: Our results reveal that specific vagal sensory neurons respond differentially to specific immune mediators. The average amplitude, and integral of TNF-responsive neurons were significantly higher than IL-1β-responsive cells (TNF vs IL-1β, p < 0.01). This result suggests that TNF application triggers higher firing rates for a more prolonged time in TNF-responsive neurons compared to IL-1β specific responses. Neuronal responses to IL-1β had a significantly higher number of peaks when compared to TNF responses (IL-1β vs TNF, p < 0.05). CONCLUSIONS: Our findings demonstrate that individual vagal sensory neurons encode information about distinct inflammatory stimuli and provide insight into the neural sensing of inflammation. Further investigation into neuro-immune signaling may identify novel neural targets for the treatment of inflammatory disorders.

Cell state transition analysis identifies interventions that improve control of Mycobacterium tuberculosis infection by susceptible macrophages.

Understanding cell state transitions and purposefully controlling them to improve therapies is a longstanding challenge in biological research and medicine. Here, we identify a transcriptional signature that distinguishes activated macrophages from the tuberculosis (TB) susceptible and resistant mice. We then apply the cSTAR (cell state transition assessment and regulation) approach to data from screening-by-RNA sequencing to identify chemical perturbations that shift the transcriptional state of tumor necrosis factor (TNF)-activated TB-susceptible macrophages toward that of TB-resistant cells, i.e., prevents their aberrant activation without suppressing beneficial TNF responses. Last, we demonstrate that the compounds identified with this approach enhance the resistance of the TB-susceptible mouse macrophages to virulent Mycobacterium tuberculosis.

Eucalcemia during lipopolysaccharide challenge in postpartum dairy cows: I. Clinical, inflammatory, and metabolic response

Hypocalcemia induced by immune activation is a conserved response across mammalian species; however, administration of Ca is discouraged in other species as it is associated with increased morbidity and mortality. Early postpartum cows experience a decrease in circulating Ca concentration following acute inflammation. Corrective Ca therapy during the transition period, particularly in dairy cows experiencing acute disease, is common practice. However, the effect of Ca administration on the inflammatory response during acute immune activation is unknown. Our objective was to compare the clinical, inflammatory, and metabolic response to an intravenous (IV) lipopolysaccharide (LPS) challenge between postpartum cows infused, or not, with IV Ca to maintain eucalcemia. Cows (n = 14, 8 ± 1 d in milk) were enrolled in a matched-pair randomized controlled design to receive IV Ca (IVCa) or sterile 0.9% NaCl (CTRL) during an IV LPS challenge (0.040 or 0.045 µg of LPS/kg of body weight over 1 h). Ionized Ca (iCa) was monitored cow-side, and IV Ca infusion was adjusted in a eucalcemic clamp for 12 h following the start of LPS infusion. Cows were monitored during the 24 h following challenge and serial blood samples were collected to quantify concentrations of glucose, β-hydroxybutyrate, nonesterified fatty acids, urea nitrogen, cytokines, acute-phase proteins, and cortisol. Blood iCa concentration decreased to 0.87 ± 0.03 mM in CTRL during challenge, and by design, iCa concentration was maintained within 3% of baseline in IVCa. Body temperature, heart rate, and respiratory rate were monitored for 24 h following the start of challenge and did not differ between groups. A treatment × time interaction was identified such that serum cortisol concentrations increased in both groups at 2 h but decreased to a greater extent at 6 h in IVCa compared with CTRL. Rumination time (min/h) over the first 12 h following challenge was greater in IVCa, but total rumination time in the 24 h following challenge did not differ from CTRL. Serum glucose and nonesterified fatty acid concentrations decreased, and β-hydroxybutyrate and urea nitrogen concentrations increased over time, but did not differ between groups. Acute leukopenia occurred in both groups at 4 h before leukocytosis was observed at 24 h with total white blood cell counts returning to baseline within 72 h. Plasma concentrations of tumor necrosis factor (TNF) and interleukin-10 (IL-10) increased within 1 h following the start of challenge and did not differ between groups. Serum haptoglobin and serum amyloid A concentrations increased within the 24 h following challenge and were elevated through 72 h but did not differ between groups. Eucalcemia during the acute systemic inflammatory response did not alter the TNF or IL-10 cytokine response, or the acute-phase protein SAA and haptoglobin response in this LPS challenge model; however, eucalcemia was associated with a more rapid decline in cortisol response and greater rumination time in the first 12 h following challenge. We did not find evidence that eucalcemia exacerbated the inflammatory response in early postpartum cows, but Ca administration may alter the clinical response to acute systemic inflammation.

Pneumolysin suppresses the initial macrophage pro-inflammatory response to Streptococcuspneumoniae.

Published data for the Streptococcus pneumoniae virulence factor Pneumolysin (Ply) show contradictory effects on the host inflammatory response to infection. Ply has been shown to activate the inflammasome, but also can bind to MRC-1 resulting in suppression of dendritic cell inflammatory responses. We have used an in vitro infection model of human monocyte-derived macrophages (MDM), and a mouse model of pneumonia to clarify whether pro- or anti-inflammatory effects dominate the effects of Ply on the initial macrophage inflammatory response to S. pneumoniae, and the consequences during early lung infection. We found that infection with S. pneumoniae expressing Ply suppressed tumour necrosis factor (TNF) and interleukin-6 production by MDMs compared to cells infected with ply-deficient S. pneumoniae. This effect was independent of bacterial effects on cell death. Transcriptional analysis demonstrated S. pneumoniae expressing Ply caused a qualitatively similar but quantitatively lower MDM transcriptional response to S. pneumoniae compared to ply-deficient S. pneumoniae, with reduced expression of TNF and type I IFN inducible genes. Reduction of the MDM inflammatory response was prevented by inhibition of SOCS1. In the early lung infection mouse model, the TNF response to ply-deficient S. pneumoniae was enhanced and bacterial clearance increased compared to infection with wild-type S. pneumoniae. Overall, these data show Ply inhibits the initial macrophage inflammatory response to S. pneumoniae, probably mediated through SOCS1, and this was associated with improved immune evasion during early lung infection.

Vagus Nerve Sensory Neurons Respond Distinctly to Specific Inflammatory Mediators

The peripheral nervous system communicates with the immune system in response to injury or infection. In the case of inflammation, sensory signals travel up to the brain via the vagus nerve, which is a major pathway for neuro‐immune communication. While previous work from our group showed that cytokines administered systemically were associated with specific patterns of vagus nerve activity, we did not know how individual vagal sensory neurons encoded this immune information.Here, we use in vivo calcium imaging to monitor the neural activity of individual vagal sensory neurons in response to specific inflammatory mediators. Using mice that express the calcium sensor GCaMP6f in glutamatergic neurons, we imaged neurons of the nodose ganglion in situ using a 1‐photon miniature microscope (Miniscope). During imaging, the inflammatory mediators tumor necrosis factor (TNF) and interleukin 1β (IL‐1β) were applied directly to the vagus nerve. The raw fluorescence data was analyzed with a Python‐based calcium imaging analysis (CaImAn) software and a customized pipelines to output the neural activity (change in fluorescence: ∆F) of individually detected neurons. An average of 69±16.3 neurons were recorded per 1 hour imaging session.Our results reveal that specific vagal sensory neurons respond to differentially to specific immune mediators. The average amplitude, integral, and delay of TNF‐responsive neurons was significantly higher than IL‐1β‐responsive cells (TNF vs IL‐1β, p < 0.01). This result suggests that TNF application triggers responses later and with higher firing rates in responsive neurons compared to IL‐1β. Neuronal responses to IL‐1β had a significantly higher number of peaks when compared to TNF responses (IL‐1β vs TNF, p < 0.05). When compared to responses for the TRPV1‐specific agonist capsaicin, the cytokine responses were found to have significantly lower number of peaks (capsaicin vs IL‐1β, p < 0.05), which suggests that immune mediators produce different neural activity than canonical activators. Taken together, our findings demonstrate that individual vagal sensory neurons encode information about distinct inflammatory stimuli and provide insight into the neural sensing of inflammation. Further investigation into this type of neuro‐immune signaling may identify novel neural targets for the treatment of inflammatory disorders.

Early IFN-α signatures and persistent dysfunction are distinguishing features of NK cells in severe COVID-19

Longitudinal analyses of the innate immune system, including the earliest time points, are essential to understand the immunopathogenesis and clinical course of coronavirus disease (COVID-19). Here, we performed a detailed characterization of natural killer (NK) cells in 205 patients (403 samples; days 2 to 41 after symptom onset) from four independent cohorts using single-cell transcriptomics and proteomics together with functional studies. We found elevated interferon (IFN)-α plasma levels in early severe COVD-19 alongside increased NK cell expression of IFN-stimulated genes (ISGs) and genes involved in IFN-α signaling, while upregulation of tumor necrosis factor (TNF)-induced genes was observed in moderate diseases. NK cells exert anti-SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) activity but are functionally impaired in severe COVID-19. Further, NK cell dysfunction may be relevant for the development of fibrotic lung disease in severe COVID-19, as NK cells exhibited impaired anti-fibrotic activity. Our study indicates preferential IFN-α and TNF responses in severe and moderate COVID-19, respectively, and associates a prolonged IFN-α-induced NK cell response with poorer disease outcome.

Anti-Interleukin-10 Unleashes Transcriptional Response to Leishmanial Antigens in Visceral Leishmaniasis Patients.

Visceral leishmaniasis (VL; Leishmania donovani) cases produce interferon-γ and tumor necrosis factor in response to soluble leishmanial antigen (SLA) in whole-blood assays. Using transcriptional profiling, we demonstrate the impact of interleukin-10 (IL-10), a cytokine implicated in VL, on this response. SLA stimulation identified 28 differentially expressed genes (DEGs), 17/28 in a single network with TNF as hub. SLA plus anti-IL-10 produced 454 DEGs, 292 in a single network with TNF, IFNG, NFKBIA, IL6, and IL1B as hubs in concert with a remarkable chemokine/cytokine storm. Our data demonstrate the singular effect of IL-10 as a potent immune modulator in VL.

The Cytokine TNF Promotes Transcription Factor SREBP Activity and Binding to Inflammatory Genes to Activate Macrophages and Limit Tissue Repair

Cytokine tumor necrosis factor (TNF)-mediated macrophage polarization is important for inflammatory disease pathogenesis, but the mechanisms regulating polarization are not clear. We performed transcriptomic and epigenomic analysis of the TNF response in primary human macrophages and revealed late-phase activation of SREBP2, the master regulator of cholesterol biosynthesis genes. TNF stimulation extended the genomic profile of SREBP2 occupancy to include binding to and activation of inflammatory and interferon response genes independently of its functions in sterol metabolism. Genetic ablation of SREBP function shifted the balance of macrophage polarization from an inflammatory to a reparative phenotype in peritonitis and skin wound healing models. Genetic ablation of SREBP activity in myeloid cells or topical pharmacological inhibition of SREBP improved skin wound healing under homeostatic and chronic inflammatory conditions. Our results identify a function and mechanism of action for SREBPs in augmenting TNF-induced macrophage activation and inflammation and open therapeutic avenues for promoting wound repair.

Mitochondria Are a Subset of Extracellular Vesicles Released by Activated Monocytes and Induce Type I IFN and TNF Responses in Endothelial Cells.

Extracellular vesicles, including microvesicles, are increasingly recognized as important mediators in cardiovascular disease. The cargo and surface proteins they carry are considered to define their biological activity, including their inflammatory properties. Monocyte to endothelial cell signaling is a prerequisite for the propagation of inflammatory responses. However, the contribution of microvesicles in this process is poorly understood. To elucidate the mechanisms by which microvesicles derived from activated monocytic cells exert inflammatory effects on endothelial cells. LPS (lipopolysaccharide)-stimulated monocytic cells release free mitochondria and microvesicles with mitochondrial content as demonstrated by flow cytometry, quantitative polymerase chain reaction, Western Blot, and transmission electron microscopy. Using RNAseq analysis and quantitative reverse transcription-polymerase chain reaction, we demonstrated that both mitochondria directly isolated from and microvesicles released by LPS-activated monocytic cells, as well as circulating microvesicles isolated from volunteers receiving low-dose LPS-injections, induce type I IFN (interferon), and TNF (tumor necrosis factor) responses in endothelial cells. Depletion of free mitochondria significantly reduced the ability of these microvesicles to induce type I IFN and TNF-dependent genes. We identified mitochondria-associated TNFα and RNA from stressed mitochondria as major inducers of these responses. Finally, we demonstrated that the proinflammatory potential of microvesicles and directly isolated mitochondria were drastically reduced when they were derived from monocytic cells with nonrespiring mitochondria or monocytic cells cultured in the presence of pyruvate or the mitochondrial reactive oxygen species scavenger MitoTEMPO. Mitochondria and mitochondria embedded in microvesicles constitute a major subset of extracellular vesicles released by activated monocytes, and their proinflammatory activity on endothelial cells is determined by the activation status of their parental cells. Thus, mitochondria may represent critical intercellular mediators in cardiovascular disease and other inflammatory settings associated with type I IFN and TNF signaling.

Immune Differentiation Regulator p100 Tunes NF-κB Responses to TNF.

Tumor necrosis factor (TNF) is a pleiotropic cytokine whose primary physiological function involves coordinating inflammatory and adaptive immune responses. However, uncontrolled TNF signaling causes aberrant inflammation and has been implicated in several human ailments. Therefore, an understanding of the molecular mechanisms underlying dynamical and gene controls of TNF signaling bear significance for human health. As such, TNF engages the canonical nuclear factor kappa B (NF-κB) pathway to activate RelA:p50 heterodimers, which induce expression of specific immune response genes. Brief and chronic TNF stimulation produces transient and long-lasting NF-κB activities, respectively. Negative feedback regulators of the canonical pathway, including IκBα, are thought to ensure transient RelA:p50 responses to short-lived TNF signals. The non-canonical NF-κB pathway mediates RelB activity during immune differentiation involving p100. We uncovered an unexpected role of p100 in TNF signaling. Brief TNF stimulation of p100-deficient cells triggered an additional late NF-κB activity consisting of RelB:p50 heterodimers, which modified the TNF-induced gene-expression program. In p100-deficient cells subjected to brief TNF stimulation, RelB:p50 not only sustained the expression of a subset of RelA-target immune response genes but also activated additional genes that were not normally induced by TNF in WT mouse embryonic fibroblasts (MEFs) and were related to immune differentiation and metabolic processes. Despite this RelB-mediated distinct gene control, however, RelA and RelB bound to mostly overlapping chromatin sites in p100-deficient cells. Repeated TNF pulses strengthened this RelB:p50 activity, which was supported by NF-κB-driven RelB synthesis. Finally, brief TNF stimulation elicited late-acting expressions of NF-κB target pro-survival genes in p100-deficient myeloma cells. In sum, our study suggests that the immune-differentiation regulator p100 enforces specificity of TNF signaling and that varied p100 levels may provide for modifying TNF responses in diverse physiological and pathological settings.

Secreted and intracellular cytokines are complementary measures for human monocytes treated with Toll-like receptor agonists

Cytokine production by human peripheral blood mononuclear cells including monocytes, is frequently assessed by measuring secreted cytokines using enzyme linked immunosorbent assay (ELISA), whereby the total concentration of one cytokine of interest is obtained without information regarding the cell type responsible for making the cytokine. Cytokines can be retained inside the cell using protein transport inhibitors. Subsequent analysis by flow cytometry not only identifies the cell type producing the cytokine but can semi-quantitate the amount of cytokine produced by measuring the geometric mean fluorescence intensity (gMFI) and is amenable to analyzing more than one protein associated with the same cell (multiplexing). We hypothesized that a more comprehensive and biologically meaningful cytokine profile could be acquired by measuring both secreted and the retained intracellular cytokines in parallel cultures of magnetic-sorted CD14+ monocytes. Peripheral monocytes were isolated from 18 healthy donors and treated with standardized molecules that stimulate cytokine production; Toll-like receptor (TLR)4 agonist (lipopolysaccharide, LPS) or TLR7/8 agonist (R848). Pro-inflammatory cytokines (interleukin (IL)-6, IL-8 and tumour necrosis factor (TNF)) secreted into the culture medium were measured by ELISA. Parallel cultures were treated with LPS and R848 in the presence of brefeldin A (protein transport inhibitor) and the accumulated intracellular cytokines measured by flow cytometry. Each cytokine (IL-6/IL-8/TNF) gave a unique general pattern when secreted versus intracellular cytokine measurements (frequency and gMFI) were plotted to determine correlation. For monocytes treated with the TLR4 agonist, secreted IL-8 correlated with the frequency of IL-8 positive cells (R = 0.559, p = .016) and not with the amount (gMFI) of IL-8 per cell. In contrast, monocytes treated with the TLR7/8 agonist showed no correlation of secreted IL-8 with the frequency of IL-8 positive cells, but with this treatment secreted IL-6 was correlated with an increase in the frequency of IL-6 positive cells (R = 0.501, p = .034). TNF secretion from monocytes treated with either the TLR4 or TLR7/8 agonist did not correlate with the frequency or gMFI of TNF positive cells. However, there were significant correlations between the TLR4 and TLR7/8 induced TNF response (secreted and gMFI). We conclude that there are fundamental differences in secreted and intracellular IL-6/IL-8/TNF production after monocytes are treated with TLR agonists. Furthermore, secreted and intracellular cytokine analyses are complementary measures that should be used in parallel to explore inflammatory response and cytokine biology.

Tumour necrosis factor and interleukin 10 in blood pressure regulation in spontaneously hypertensive and normotensive rats

IntroductionA growing body of evidence indicates that brain cytokines are involved in the control of the cardiovascular system. Tumour necrosis factor (TNF) is an archetypal cytokine, which exerts its proinflammatory actions via type 1 receptor (TNFR1). Interleukin 10 (IL-10) plays a critical anti-inflammatory role by binding to its receptor (IL-10Ra). The orchestrated inflammatory response is largely dependent on an intricate balance between proinflammatory and anti-inflammatory cytokines and expression of their receptors. AimIn the study we evaluated the expression of the cytokines and their receptors in the brains of spontaneously hypertensive (SH) and normotensive Wistar-Kyoto (WKY) rats, and how the cytokines affect arterial blood pressure. MethodsIn SH and WKY rats we recorded systolic blood pressure with tail cuff method and measured concentration of TNF, IL-10, TNFR1, and IL-10Ra in the serum, the brainstem, and the hypothalamus; we also measured serum concentrations of copeptin, a surrogate of vasopressin release, angiotensin II and norepinephrine. We immunostained brainstem sections for TNFR1, IL-10Ra, neurons, astrocytes and microglia for confocal imaging. In urethane anaesthetized SH and WKY rats, we invasively recorded blood pressure response to intracerebroventricular (IVC) infusion of TNF or IL-10. We also pharmacologically evaluated baroreflex with phenylephrine and chemoreflex with cyanide in SH and WKY rats. ResultsCompared to WKY rats, SH rats had: (1) higher blood pressure; (2) blunted baroreflex and augmented peripheral chemoreflex; (3) greater pressor response to ICV infused TNF and greater hypotensive response to ICV infused IL-10; (4) higher concentration of TNF in the ventral and dorsal aspects of the medulla oblongata; (5) higher expression of TNFR1 in the dorsal medulla; (6) higher concentration of IL-10 in both aspects of the medulla; (7) lower expression of IL-10Ra in the dorsal medulla. Confocal imaging showed co-localization of TNFR1 and IL-10Ra with neurons, astrocytes and microglia in both SH and WKY rats. The concentration of the cytokines and their receptors were significantly higher in the brain than in the serum. There were no significant differences in the concentration of the cytokines and their receptors in the hypothalamic region and in the serum between SH and WKY rats. Serum concentrations of norepinephrine, angiotensin II and copeptin were similar between SH and WKY rats. ConclusionsTaken together, these findings suggest the presence of a potent milieu for effective TNF signalling in the brainstem, which is associated with the hypertensive phenotype and enhanced hemodynamic response to intrabrain administration of the cytokines. In addition, we hypothesize that the increased IL-10 concentration in the brainstem is a compensatory mechanism for the upregulated TNF system.

Tumor Necrosis Factor dynamically regulates the mRNA stabilome in rheumatoid arthritis fibroblast-like synoviocytes.

During rheumatoid arthritis (RA), Tumor Necrosis Factor (TNF) activates fibroblast-like synoviocytes (FLS) inducing in a temporal order a constellation of genes, which perpetuate synovial inflammation. Although the molecular mechanisms regulating TNF-induced transcription are well characterized, little is known about the impact of mRNA stability on gene expression and the impact of TNF on decay rates of mRNA transcripts in FLS. To address these issues we performed RNA sequencing and genome-wide analysis of the mRNA stabilome in RA FLS. We found that TNF induces a biphasic gene expression program: initially, the inducible transcriptome consists primarily of unstable transcripts but progressively switches and becomes dominated by very stable transcripts. This temporal switch is due to: a) TNF-induced prolonged stabilization of previously unstable transcripts that enables progressive transcript accumulation over days and b) sustained expression and late induction of very stable transcripts. TNF-induced mRNA stabilization in RA FLS occurs during the late phase of TNF response, is MAPK-dependent, and involves several genes with pathogenic potential such as IL6, CXCL1, CXCL3, CXCL8/IL8, CCL2, and PTGS2. These results provide the first insights into genome-wide regulation of mRNA stability in RA FLS and highlight the potential contribution of dynamic regulation of the mRNA stabilome by TNF to chronic synovitis.

TNF up-regulates ST3GAL4 and sialyl-Lewisx expression in lung epithelial cells through an intronic ATF2-responsive element.

We have previously shown that tumor necrosis factor (TNF) induced the up-regulation of the sialyltransferase gene ST3GAL4 (α2,3-sialyltransferase gene) BX transcript through mitogen- and stress-activated kinase 1/2 (MSK1/2), extracellular signal-regulated kinase (ERK) and p38 mitogen-activated protein kinase (MAPK) signaling pathways. This up-regulation resulted in sialyl-Lewisx (sLex) overexpression on high-molecular-weight glycoproteins in inflamed airway epithelium and increased the adhesion of Pseudomonas aeruginosa PAO1 and PAK strains to lung epithelial cells. In the present study, we describe a TNF-responsive element in an intronic region of the ST3GAL4 gene, whose TNF-dependent activity is repressed by ERK/p38 and MSK1/2 inhibitors. This TNF-responsive element contains potential binding sites for ETS1 and ATF2 transcription factors related to TNF signaling. We also show that ATF2 is involved in TNF responsiveness, as well as in TNF-induced ST3GAL4 BX transcript and sLex overexpression in A549 lung epithelial cells. Moreover, we show that TNF induces the binding of ATF2 to the TNF-responsive element. Altogether, these data suggest that ATF2 could be a potential target to prevent inflammation-induced P. aeruginosa binding in the lung of patients suffering from lung diseases such as chronic bronchitis or cystic fibrosis.

Non-canonical NF\u03baB mutations reinforce pro-survival TNF response in multiple myeloma through an autoregulatory RelB:p50 NF\u03baB pathway

Environmental drug resistance constitutes a serious impediment for therapeutic intervention in multiple myeloma. Tumor-promoting cytokines, such as tumor necrosis factor (TNF), induce nuclear factor-κB (NFκB)- driven expression of pro-survival factors, which confer resistance in myeloma cells to apoptotic insults from TNF-related apoptosis-inducing ligand (TRAIL) and other chemotherapeutic drugs. It is thought that RelA:p50 dimer, activated from IκBα-inhibited complex in response to TNF-induced canonical NFκB signal, mediates the pro-survival NFκB function in cancerous cells. Myeloma cells additionally acquire gain-of-function mutations in the non-canonical NFκB module, which induces partial proteolysis of p100 into p52 to promote RelB:p52/NFκB activation from p100-inhibited complex during immune cell differentiation. However, role of non-canonical NFκB signaling in the drug resistance in multiple myeloma remains unclear. Here we report that myeloma-associated non-canonical aberrations reinforce pro-survival TNF signaling in producing a protracted TRAIL-refractory state. These mutations did not act through a typical p52 NFκB complex, but completely degraded p100 to reposition RelB under IκBα control, whose degradation during TNF signaling induced an early RelB:p50 containing NFκB activity. More so, autoregulatory RelB synthesis prolonged this TNF-induced RelB:p50 activity in myeloma cells harboring non-canonical mutations. Intriguingly, TNF-activated RelB:p50 dimer was both necessary and sufficient, and RelA was not required, for NFκB-dependent pro-survival gene expressions and suppression of apoptosis. Indeed, high RelB mRNA expressions in myeloma patients correlated with the augmented level of pro-survival factors and resistance to therapeutic intervention. In sum, we provide evidence that cancer-associated mutations perpetuate TNF-induced pro-survival NFκB activity through autoregulatory RelB control and thereby exacerbate environmental drug resistance in multiple myeloma.

The Physiological Role of Tumor Necrosis Factor in Human Immunity and Its Potential Implications in Spinal Manipulative Therapy: A Narrative Literature Review

ObjectiveAlthough tumor necrosis factor (TNF) is a well-known inflammatory cytokine in the pathological development of various human diseases, its physiological roles are not widely understood nor appreciated. The molecular mechanisms underlying spinal manipulation therapy (SMT) remain elusive. The relationship between TNF and SMT is unclear. Thus, we performed this literature review to better understand TNF physiology and its potential relationship with SMT, and we propose a novel mechanism by which SMT may achieve clinical benefits by using certain beneficial features of TNF. MethodsWe searched several databases for relevant articles published between 1975 and 2015 and then reexamined the studies from current immunophysiological perspectives. ResultsThe history and recent progresses in TNF physiology research were explored. Conflicting reports on the relationship between TNF and SMT were identified. Based on the newly discovered interaction between TNF and regulatory T cells, we proposed a putative biphasic TNF response to SMT, which may resolve the conflicts in the reported observations and interpretations. ConclusionThe current literature about TNF informed our discussion of new physiological roles for TNF, which may help to better understand the physiological effects of SMT.

Interferon γ and Tumor Necrosis Factor Are Not Essential Parameters of CD4+ T-Cell Responses for Vaccine Control of Tuberculosis.

Mycobacterium tuberculosis infects one third of the world's population and causes >8 million cases of tuberculosis annually. New vaccines are necessary to control the spread of tuberculosis. T cells, interferon γ (IFN-γ), and tumor necrosis factor (TNF) are necessary to control M. tuberculosis infection in both humans and unvaccinated experimental animal models. However, the immune responses necessary for vaccine efficacy against M. tuberculosis have not been defined. The multifunctional activity of T-helper type 1 (TH1) cells that simultaneously produce IFN-γ and TNF has been proposed as a candidate mechanism of vaccine efficacy. We used a mouse model of T-cell transfer and aerosolized M. tuberculosis infection to assess the contributions of TNF, IFN-γ, and inducible nitric oxide synthase (iNOS) to vaccine efficacy. CD4(+) T cells were necessary and sufficient to transfer protection against aerosolized M. tuberculosis, but neither CD4(+) T cell-produced TNF nor host cell responsiveness to IFN-γ were necessary. Transfer of Tnf(-/-) CD4(+) T cells from vaccinated donors to Ifngr(-/-) recipients was also sufficient to confer protection. Activation of iNOS to produce reactive nitrogen species was not necessary for vaccine efficacy. Induction of TH1 cells that coexpress IFN-γ and TNF is not a requirement for vaccine efficacy against M. tuberculosis, despite these cytokines being essential for control of M. tuberculosis in nonvaccinated animals.

Infliximab therapy increases the frequency of circulating CD16(+) monocytes and modifies macrophage cytokine response to bacterial infection.

Crohn's disease (CD) has been correlated with altered macrophage response to microorganisms. Considering the efficacy of infliximab treatment on CD remission, we investigated infliximab effects on circulating monocyte subsets and on macrophage cytokine response to bacteria. Human peripheral blood monocyte-derived macrophages were obtained from CD patients, treated or not with infliximab. Macrophages were infected with Escherichia coli, Enterococcus faecalis, Mycobacterium avium subsp. paratuberculosis (MAP) or M. avium subsp avium, and cytokine levels [tumour necrosis factor (TNF) and interleukin (IL)-10] were evaluated at different time-points. To evaluate infliximab-dependent effects on monocyte subsets, we studied CD14 and CD16 expression by peripheral blood monocytes before and after different infliximab administrations. We also investigated TNF secretion by macrophages obtained from CD16(+) and CD16(-) monocytes and the frequency of TNF(+) cells among CD16(+) and CD16(-) monocyte-derived macrophages from CD patients. Infliximab treatment resulted in elevated TNF and IL-10 macrophage response to bacteria. An infliximab-dependent increase in the frequency of circulating CD16(+) monocytes (particularly the CD14(++) CD16(+) subset) was also observed (before infliximab: 4·65 ± 0·58%; after three administrations: 10·68 ± 2·23%). In response to MAP infection, macrophages obtained from CD16(+) monocytes were higher TNF producers and CD16(+) macrophages from infliximab-treated CD patients showed increased frequency of TNF(+) cells. In conclusion, infliximab treatment increased the TNF production of CD macrophages in response to bacteria, which seemed to depend upon enrichment of CD16(+) circulating monocytes, particularly of the CD14(++) CD16(+) subset. Infliximab treatment of CD patients also resulted in increased macrophage IL-10 production in response to bacteria, suggesting an infliximab-induced shift to M2 macrophages.

A High-Content RNAi Screen Identifies Ubiquitin Modifiers That Regulate TNF-Dependent Nuclear Accumulation of NF-κB.

The mammalian tumor necrosis factor (TNF) cytokine is a central mediator of inflammatory events. Recent studies revealed a number of complex and sophisticated interactions between the TNF pathway and the enzymatic activities encoded by ubiquitin ligases and deubiquitylation enzymes. However, very little is known about the identity of the ubiquitin pathway members that control the extent of ubiquitylation in TNF responses. To address this deficit, we conducted an unbiased, high-content screen of the human ubiquitin pathway for gene products that control defining features of the cellular response to TNF. In particular, we sought to identify ubiquitin modifying enzymes that alter the ability of TNF to regulate the nuclear accumulation of nuclear factor kappa B. In this screen, we identified and validated several novel regulators of the TNF pathway. We believe these regulators constitute potential targets for pharmacological interventions that manipulate TNF-dependent inflammation.

Endogenous aryl hydrocarbon receptor promotes basal and inducible expression of tumor necrosis factor target genes in MCF-7 cancer cells

The aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor that upon activation by the toxicant 2,3,7,8 tetrachlorodibenzo-p-dioxin (TCDD) stimulates gene expression and toxicity. AHR is also important for normal mouse physiology and may play a role in cancer progression in the absence of environmental toxicants. The objective of this report was to identify AHR-dependent genes (ADGs) whose expression is regulated by AHR in the absence of toxicants. RNA-Seq analysis revealed that AHR regulated the expression of over 600 genes at an FDR<10% in MCF-7 breast cancer cells upon knockdown with short interfering RNA. Pathway analysis revealed that a significant number of ADGs were components of TCDD and tumor necrosis factor (TNF) pathways. We also demonstrated that siRNA knockdown of AHR modulated TNF induction of MNSOD and cytotoxicity in MCF-7 cells. Collectively, the major new findings of this report are: (1) endogenous AHR promotes the expression of xenobiotic metabolizing enzymes even in the absence of toxicants and drugs, (2) AHR by modulating the basal expression of a large fraction of TNF target genes may prime them for TNF stimulation and (3) AHR is required for TNF induction of MNSOD and the cellular response to cytotoxicity in MCF-7 cells. This latter result provides a potentially new role for AHR in MCF-7 cancer progression as a mediator of TNF and antioxidant responses.