Intercrypt sentinel macrophages tune antibacterial NF-κB responses in gut epithelial cells via TNF.
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ABSTRACT:
SUBMITTER: Hausmann A
PROVIDER: S-EPMC8480669 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description: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.
| S-EPMC6514058 | biostudies-literature
Project description:The mammalian innate immune system senses many bacterial stimuli through the toll-like receptor (TLR) family. Activation of the TLR4 receptor by bacterial lipopolysaccharide (LPS) is the most widely studied TLR pathway due to its central role in host responses to gram-negative bacterial infection and its contribution to endotoxemia and sepsis. Here we describe a genome-wide siRNA screen to identify genes regulating the mouse macrophage TNF-α and NF-κB responses to LPS. We include a secondary validation screen conducted with six independent siRNAs per gene to facilitate removal of off-target screen hits. We also provide microarray data from the same LPS-treated macrophage cells to facilitate downstream data analysis. These data provide a resource for analyzing gene function in the predominant pathway driving inflammatory signaling and cytokine expression in mouse macrophages.
| S-EPMC5332015 | biostudies-literature
Project description:Macrophages are sentinel immune cells that survey the tissue microenvironment, releasing cytokines in response to both exogenous insults and endogenous events such as tumorigenesis. Macrophages mediate tumor surveillance and therapy-induced tumor regression; however, tumor-associated macrophages (TAM) and their products may also promote tumor progression. Whereas NF-κB is prominent in macrophage-initiated inflammatory responses, little is known about the role of p53 in macrophage responses to environmental challenge, including chemotherapy or in TAMs. Here, we report that NF-κB and p53, which generally have opposing effects in cancer cells, coregulate induction of proinflammatory genes in primary human monocytes and macrophages. Using Nutlin-3 as a tool, we demonstrate that p53 and NF-κB rapidly and highly induce interleukin (IL)-6 by binding to its promoter. Transcriptome analysis revealed global p53/NF-κB co-regulation of immune response genes, including several chemokines, which effectively induced human neutrophil migration. In addition, we show that p53, activated by tumor cell paracrine factors, induces high basal levels of macrophage IL-6 in a TAM model system [tumor-conditioned macrophages (TCM)]. Compared with normal macrophages, TCMs exhibited higher p53 levels, enhanced p53 binding to the IL-6 promoter, and reduced IL-6 levels upon p53 inhibition. Taken together, we describe a mechanism by which human macrophages integrate signals through p53 and NF-κB to drive proinflammatory cytokine induction. Our results implicate a novel role for macrophage p53 in conditioning the tumor microenvironment and suggest a potential mechanism by which p53-activating chemotherapeutics, acting upon p53-sufficient macrophages and precursor monocytes, may indirectly impact tumors lacking functional p53.
| S-EPMC3994464 | biostudies-literature
Project description:Enterotoxigenic Escherichia coli (ETEC) causes childhood diarrhea in developing countries. ETEC strains produce the heat-labile enterotoxin (LT) and/or heat-stable enterotoxins (ST) and encode a diverse set of colonization factors used for adherence to intestinal epithelial cells. We previously found that ETEC secretes a heat-stable protein we designated as ETEC Secreted Factor (ESF) that inhibits the extent of NF-κB activation normally induced by tumor necrosis factor alpha (TNF). Here we fractionated ETEC supernatants using fast protein liquid chromatography (FPLC) and determined that ETEC flagellin was necessary and sufficient to protect IκBα from degradation in response to TNF stimulation. These data suggest a potentially novel mechanism by which ETEC may evade the host innate immune response by down-regulating NF-κB-dependent host responses.
| S-EPMC5488652 | biostudies-literature
Project description:Unbalanced tumor necrosis factor (TNF)-α production is associated with pathogenesis of a variety of human diseases. However, the molecular pathways maintaining TNF-α homeostasis remain elusive. Here, we report that NF-κB/p65-DICER-miRs axis negatively regulates TNF-α production. We demonstrated that NF-κB bound to DICER promoter and transcriptionally regulated DICER expression. In addition, the NF-κB/DICER signaling suppresses TNF-α expression by generating mature forms of miR-125b and miR-130a which negatively regulate TNF-α mRNA. Furthermore, we showed that the hepatocyte-specific depletion of Dicer in mice resulted in TNF-α overproduction and sensitized the mice to endotoxin, which could be corrected by administration of miR-125b mimics. These data suggest that NF-κB/p65-DICER-miRs axis involved in maintaining of TNF-α homeostasis, and injection of miR-125b as a potential therapeutic method for septic shock.
| S-EPMC4582149 | biostudies-literature
Project description:The mammalian innate immune system senses many bacterial stimuli through the toll-like receptor (TLR) family. Activation of the TLR4 receptor by bacterial lipopolysaccharide (LPS) is the most widely studied TLR pathway due to its central role in host responses to gram-negative bacterial infection and its contribution to endotoxemia and sepsis. Here we describe a genome-wide siRNA screen to identify genes regulating the mouse macrophage TNF-α and NF-κB responses to LPS. We include a secondary validation screen conducted with six independent siRNAs per gene to facilitate removal of off-target screen hits. We also provide microarray data from the same LPS-treated macrophage cells to facilitate downstream data analysis. These data provide a resource for analyzing gene function in the predominant pathway driving inflammatory signaling and cytokine expression in mouse macrophages.
2017-02-23 | GSE83825 | GEO
Project description:The intracellular survival of Mycobacterium tuberculosis (Mtb) has a central role in the pathogenesis of tuberculosis. Mtb Rv2346c is a member of 6-kDa early secreted antigenic target family of proteins, which are known to inhibit the host immune responses to promote bacillary persistence in macrophages. However, the mechanism through which Rv2346c participates in Mtb pathogenesis is unclear. In the present study, recombinant Rv2346c protein was synthesized and used to treat Bacillus Calmette-Guérin (BCG)-infected macrophages. The results showed that Rv2346c inhibited the proliferation of BCG-infected macrophages and enhanced the survival of BCG in macrophages. Tumor necrosis factor-α (TNF-α) and interleukin (IL)-6 were upregulated during BCG infection but downregulated by Rv2346c. Additional experiments showed that nuclear transcription factor-κB (NF-κB) in BCG-infected macrophages induced the production of TNF-α and IL-6. In addition, miR-155 and miR-99b had a suppressive effect on NF-κB, and the expression of these miRNAs was promoted by p38. Furthermore, Rv2346c was shown to decrease the activation of NF-κB, whereas it enhanced the phosphorylation of p38 and the expression of miR-155 and miR-99b. The function of Rv2346c was also verified in Mtb-infected mice. The results showed that Rv2346c increased the observed bacterial load and lung injury and downregulated TNF-α and IL-6 in vivo. Overall, our results reveal that Rv2346c enhances mycobacterial survival in macrophages via inhibiting the production of TNF-α and IL-6 in a p38/miRNA/NF-κB pathway-dependent manner, suggesting that Rv2346c acts as a crucial virulence factor in Mtb infection and has potential use as a target for anti-tuberculosis therapy.
| S-EPMC6145905 | biostudies-other
Project description:PurposeDelayed wound healing is an intractable complex of diabetes and substance P (SP) is proved to benefit wound healing, whose functioning mechanism remains elusive. This study aims at revealing whether the influence of SP on diabetic wound healing is dependent on inflammatory responses, particularly NF-κB.MethodsSkin fibroblasts of genetically diabetic mice were co-cultured with bone marrow-derived macrophages, and treated with SP, SP + L703,606 (a neurokinin-1 receptor antagonist), or SP + MG132 (an inhibitor of NF-κB). For macrophages, their migration ability was assessed by Transwell experiments, and their M2 polarization was analyzed by flow cytometry and markers for M2 phenotype. Pro-inflammatory factors in the supernatant were detected by enzyme-linked immunosorbent assay. In fibroblasts, the transcription levels of the four pro-inflammatory factors and the protein levels of NF-κB regulators like inhibitor of NF-κB alpha (IκBα) and IκB kinases (IKKs) were monitored by real-time quantitative PCR and western blot, respectively.ResultsSP could significantly induce migration to fibroblasts (P<0.01), M2 polarization (P<0.001) and pro-inflammatory factor concentration (P<0.01) in the co-culture system. It also promotes the transcription process of pro-inflammatory factors in fibroblasts (P<0.01), and induce activation of IKKα/β and phosphorylation of IκBα, which caused NF-κB activation. All these effects were reversed if NF-κB was inhibited.ConclusionThe promoting effects of SP on diabetic wound healing was dependent on enhanced inflammatory responses, especially the activation of NF-κB. This study provided evidence for the potential usage of SP in accelerating diabetic wound healing.
| S-EPMC4891430 | biostudies-literature
Project description:The NF-κB signaling pathway is crucial during development and inflammatory processes. We have previously shown that NF-κB activation induces dedifferentiation of astrocytes into neural progenitor cells (NPCs). Here, we provide evidence that the NF-κB pathway plays also a fundamental role during the differentiation of NPCs into astrocytes. First, we show that the NF-κB pathway is essential to initiate astrocytic differentiation as its early inhibition induces NPC apoptosis and impedes their differentiation. Second, we demonstrate that persistent NF-κB activation affects NPC-derived astrocyte differentiation. Tumor necrosis factor (TNF)-treated NPCs show NF-κB activation, maintain their multipotential and proliferation properties, display persistent expression of immature markers and inhibit astrocyte markers. Third, we analyze the effect of NF-κB activation on the main known astrocytic differentiation pathways, such as NOTCH and JAK-STAT. Our findings suggest that the NF-κB pathway plays a dual fundamental role during NPC differentiation into astrocytes: it promotes astrocyte specification, but its persistent activation impedes their differentiation.
| S-EPMC8068246 | biostudies-literature
Project description:Background and aimsTumor necrosis factor (TNF) is a major pathogenic effector and a therapeutic target in inflammatory bowel disease (IBD), yet the basis for TNF-induced intestinal epithelial cell (IEC) death is unknown, because TNF does not kill normal IECs. Here, we investigated how chronic nuclear factor (NF)- κB activation, which occurs in human IBD, promotes TNF-dependent IEC death in mice.MethodsHuman IBD specimens were stained for p65 and cleaved caspase-3. C57BL/6 mice with constitutively active IKKβ in IEC (Ikkβ(EE)IEC), Ripk1D138N/D138N knockin mice, and Ripk3-/- mice were injected with TNF or lipopolysaccharide. Enteroids were also isolated from these mice and challenged with TNF with or without RIPK1 and RIPK3 inhibitors or butylated hydroxyanisole. Ripoptosome-mediated caspase-8 activation was assessed by immunoprecipitation.ResultsNF-κB activation in human IBD correlated with appearance of cleaved caspase-3. Congruently, unlike normal mouse IECs that are TNF-resistant, IECs in Ikkβ(EE)IEC mice and enteroids were susceptible to TNF-dependent apoptosis, which depended on the protein kinase function of RIPK1. Constitutively active IKKβ facilitated ripoptosome formation, a RIPK1 signaling complex that mediates caspase-8 activation by TNF. Butylated hydroxyanisole treatment and RIPK1 inhibitors attenuated TNF-induced and ripoptosome-mediated caspase-8 activation and IEC death in vitro and in vivo.ConclusionsContrary to common expectations, chronic NF-κB activation induced intestinal crypt apoptosis after TNF stimulation, resulting in severe mucosal erosion. RIPK1 kinase inhibitors selectively inhibited TNF destructive properties while preserving its survival and proliferative properties, which do not require RIPK1 kinase activity. RIPK1 kinase inhibition could be a potential treatment for IBD.
| S-EPMC6957844 | biostudies-literature