The Citrus Flavone Nobiletin Reduces Pro-Inflammatory and Pro-Labour Mediators in Fetal Membranes and Myometrium: Implications for Preterm Birth

Spontaneous preterm birth is the leading cause of infant death and of neurological disabilities in survivors. A significant proportion of spontaneous preterm births are associated with infection. Infection activates inflammation which induces a cascade of events that leads to myometrial contractions and rupture of fetal membranes. In non-gestational tissues, the citrus flavone nobiletin has been shown to exert potent anti-inflammatory properties. Thus, in this study, we sought to determine the effect of nobiletin on pro-inflammatory mediators in human fetal membranes and myometrium. Human fetal membranes and myometrium were treated with bacterial endotoxin lipopolysaccharide (LPS) in the absence or presence of nobiletin. In addition, the effect of nobiletin in fetal membranes taken from spontaneous preterm deliveries with and without infection (i.e. histological chorioamnionitis) was also examined. In human fetal membranes and myometrium, nobiletin significantly decreased LPS-stimulated expression of pro-inflammatory cytokines (TNF-α, IL-1β, IL-6 and IL-8) and MMP-9 expression and pro-MMP-9 secretion. Additionally, nobiletin significantly decreased COX-2 expression and subsequent prostaglandin (PG) E₂ production. Notably, nobiletin was also able to reduce the expression and production of pro-inflammatory cytokines and MMP-9 in fetal membranes taken from women after spontaneous preterm birth. In conclusion, our study demonstrates that nobiletin can reduce infection-induced pro-inflammatory mediators in human fetal membranes and myometrium. These in vitro studies further support the increasing volume and quality of evidence that high fruit and vegetable intake in pregnancy is associated with a decreased risk of adverse pregnancy outcomes.     

Alkaline Phosphatase Protects Lipopolysaccharide-Induced Early Pregnancy Defects in Mice

Excessive cytokine inflammatory response due to chronic or superphysiological level of microbial infection during pregnancy leads to pregnancy complications such as early pregnancy defects/loss and preterm birth. Bacterial toxin lipopolysaccharide (LPS), long recognized as a potent proinflammatory mediator, has been identified as a risk factor for pregnancy complications. Alkaline phosphatase (AP) isozymes have been shown to detoxify LPS by dephosphorylation. In this study, we examined the role of alkaline phosphatase (AP) in mitigating LPS-induced early pregnancy complications in mice. We found that 1) the uterus prior to implantation and implantation sites following embryo implantation produce LPS recognition and dephosphorylation molecules TLR4 and tissue non-specific AP (TNAP) isozyme, respectively; 2) uterine TNAP isozyme dephosphorylates LPS at its sites of production; 3) while LPS administration following embryo implantation elicits proinflammatory cytokine mRNA levels at the embryo implantation sites (EISs) and causes early pregnancy loss, dephosphorylated LPS neither triggers proinflammatory cytokine mRNA levels at the EISs nor induces pregnancy complications; 4) AP isozyme supplementation to accelerate LPS detoxification attenuates LPS-induced pregnancy complications following embryo implantation. These findings suggest that a LPS dephosphorylation strategy using AP isozyme may have a unique therapeutic potential to mitigate LPS- or Gram-negative bacteria-induced pregnancy complications in at-risk women.     

Critical paracrine interactions between TNF-alpha and IL-10 regulate lipopolysaccharide-stimulated human choriodecidual cytokine and prostaglandin E2 production

Increased production of PGs by gestational membranes is believed to be a principal initiator of term and preterm labor. Intrauterine infection is associated with an inflammatory response in the choriodecidua characterized by elevated production of cytokines and PGs. The precise physiological significance of enhanced choriodecidual cytokine production in the mechanism of preterm labor remains uncertain. These studies were undertaken to dissect the roles and regulation of endogenous cytokines in regulating PG production by human choriodecidua. We used LPS treatment of human choriodecidual explants as our model system. In choriodecidual explant cultures, LPS (5 microg/ml) induced a rapid increase in TNF-alpha production, peaking at 4 h. In contrast, IL-10, IL-1beta, and PGE2 production rates peaked 8, 12, and 24 h, respectively, after LPS stimulation. Immunoneutralization studies indicated that TNF-alpha was a primary regulator of IL-1beta, IL-10, and PGE2 production, while IL-1beta stimulated only PGE2 production. Neutralization of endogenous IL-10 resulted in increased TNF-alpha and PGE2 production. IL-10 treatment markedly decreased TNF-alpha and IL-1beta production, but had no effect on PGE2 production. Taken together, these results demonstrate that the effects of LPS on choriodecidual cytokine and PG production are modulated by both positive and negative feedback loops. In the setting of an infection of the intrauterine, TNF-alpha may be a potential target for treatment intervention; IL-10 could be one such therapeutic.     

Differential regulation of gelatinase A and B and TIMP-1 and -2 by TNFalpha and HIV virions in astrocytes

Changes in the fine balance between matrix metalloproteinases and their tissue inhibitors, which drives extracellular matrix turnover, may be critical to central nervous system inflammation in HIV infection as well as in neurotoxicity. Although they do not produce virus when infected by HIV, astrocytes may be directly affected by the virion, because some viral proteins are known to transduce signaling in brain cells and are also sensitive to the major proinflammatory cytokine TNFalpha. We therefore studied the effects of HIV and TNFalpha on MMP-2, MMP-9 and their inhibitors, TIMP-1 and TIMP-2, in astrocytes, by zymography and ELISA, respectively, or by RT-PCR for both of them. HIV slightly increased the production of pro-MMP-2 and pro-MMP-9 by astrocytes, in a dose-dependent manner. TNFalpha strongly induced pro-MMP-9. TIMP-1 and TIMP-2 levels were affected only slightly, if at all, by HIV and TNFalpha. Thus, astrocyte/HIV contact may lead to extracellular matrix activation, which may be strongly amplified by the inflammatory response. Our data strongly suggest that, besides their physiological production of MMP-2, astrocytes would be a major source of MMP-9 in the inflamed brain.     

PECAM-1 ligation negatively regulates TLR4 signaling in macrophages

Uncontrolled TLR4 signaling may induce excessive production of proinflammatory cytokines and lead to harmful inflammation; therefore, negative regulation of TLR4 signaling attracts much attention now. PECAM-1, a member of Ig-ITIM family, can mediate inhibitory signals in T cells and B cells. However, the role and the mechanisms of PECAM-1 in the regulation of TLR4-mediated LPS response in macrophages remain unclear. In this study, we demonstrate that PECAM-1 ligation with CD38-Fc fusion protein negatively regulates LPS-induced proinflammatory cytokine TNF-alpha, IL-6, and IFN-beta production by inhibiting JNK, NF-kappaB, and IFN regulatory factor 3 activation in macrophages. In addition, PECAM-1 ligation-recruited Src homology region 2 domain-containing phosphatase 1 (SHP-1) and Src homology region 2 domain-containing phosphatase 2 (SHP-2) may be involved in the inhibitory effect of PECAM-1 on TLR4 signaling. Consistently, silencing of PECAM-1 enhances the macrophage response to LPS stimulation. Taken together with the data that PECAM-1 is constitutively expressed in macrophages and its expression is up-regulated by LPS stimulation, PECAM-1 might function as a feedback negative regulator of LPS inflammatory response in macrophages. This study may provide a potential target for intervention of inflammatory diseases.     

Cytokine release from placental endothelial cells, a process associated with preterm labour in the absence of intrauterine infection

There is currently a great deal of interest in the role that cytokines may play in the processes mediating preterm as well as normal term labour. In case of preterm delivery a cause-effect relationship between infection, uncontrollable preterm labour, and increased uterine cytokine concentrations is widely accepted, but there is considerable information that increased uterine cytokine release is also a condition in normal term labour and preterm labour not due to infection. Thereby, the exact cellular sources of cytokine production have not yet been identified. In the present study, the authors used immunohistochemical analysis to localize interleukin 1beta (IL-1beta) interleukin 6 (IL-6) and tumour necrosis factor alpha (TNF-alpha) immunoreactivity within trophoblastic villi and fetal membranes. In the absence of chorioamnionitis, uncontrollable preterm labour, and also normal term labour was associated with strong immunoreactivity for IL-1beta and IL-6 in the endothelial cells within trophoblastic villi. In contrast, preterm delivery accompanied by histologically confirmed chorioamnionitis, was not associated with increased expression of cytokine antigens within endothelial cells of the fetal vascular system, but strong cytokine activity was found in polymorphonuclear cells infiltrating the amniochorionic membranes. Therefore, the data suggest two well-defined subgroups among patients delivering preterm. Thereby, increased uterine cytokine concentrations may be realized in both groups, but the cellular sources of cytokine production may be different.     

Essential role for IL-10 in resistance to lipopolysaccharide-induced preterm labor in mice

IL-10 is highly expressed in the uterus and placenta and is implicated in controlling inflammation-induced pathologies of pregnancy. To investigate the role of IL-10 in regulating preterm labor, the response of IL-10 null mutant mice to low-dose LPS in late gestation was evaluated. When IL-10 null mutant C57BL/6 (IL-10(-/-)) and control (IL-10(+/+)) mice were administered LPS on day 17 of pregnancy, the dose of LPS required to elicit 50% preterm fetal loss was 10-fold lower in IL-10(-/-) mice than in IL-10(+/+) mice. Surviving fetuses in IL-10(-/-) mice exhibited fetal growth restriction at lower doses of LPS than IL-10(+/+) mice. Marked elevation of LPS-induced immunoactive TNF-alpha and IL-6 was evident in the serum, uterus, and placenta of IL-10(-/-) mice, and TNF-alpha and IL-6 mRNA expression was elevated in the uterus and placenta, but not the fetus. Serum IL-1alpha, IFN-gamma, and IL-12p40 were increased and soluble TNFRII was diminished in the absence of IL-10, with these changes also reflected in the gestational tissues. Administration of rIL-10 to IL-10(-/-) mice attenuated proinflammatory cytokine synthesis and alleviated their increased susceptibility to preterm loss. Exogenous IL-10 also protected IL-10(+/+) mice from fetal loss. These data show that IL-10 modulates resistance to inflammatory stimuli by down-regulating proinflammatory cytokines in the uterus and placenta. Abundance of endogenous IL-10 in gestational tissues is therefore identified as a critical determinant of resistance to preterm labor, and IL-10 may provide a useful therapeutic agent in this common condition.     

Maternal LPS induces cytokines in the amniotic fluid and corticotropin releasing hormone in the fetal rat brain

Perinatal infections are a risk factor for fetal neurological pathologies, including cerebral palsy and schizophrenia. Cytokines that are produced as part of the inflammatory response are proposed to partially mediate the neurological injury. This study investigated the effects of intraperitoneal injections of lipopolysaccharide (LPS) to pregnant rats on the production of cytokines and stress markers in the fetal environment. Gestation day 18 pregnant rats were treated with LPS (100 microg/kg body wt i.p.), and maternal serum, amniotic fluid, placenta, chorioamnion, and fetal brain were harvested at 1, 6, 12, and 24 h posttreatment to assay for LPS-induced changes in cytokine protein (ELISA) and mRNA (real-time RT-PCR) levels. We observed induction of proinflammatory cytokines interleukin (IL)-1 beta, IL-6, and tumor necrosis factor-alpha (TNF-alpha) as well as the anti-inflammatory cytokine IL-10 in the maternal serum within 6 h of LPS exposure. Similarly, proinflammatory cytokines were induced in the amniotic fluid in response to LPS; however, no significant induction of IL-10 was observed in the amniotic fluid. LPS-induced mRNA changes included upregulation of the stress-related peptide corticotropin-releasing factor in the fetal whole brain, TNF-alpha, IL-6, and IL-10 in the chorioamnion, and TNF-alpha, IL-1 beta, and IL-6 in the placenta. These findings suggest that maternal infections may lead to an unbalanced inflammatory reaction in the fetal environment that activates the fetal stress axis.     

Elastin receptor (spliced galactosidase) occupancy by elastin peptides counteracts proinflammatory cytokine expression in lipopolysaccharide-stimulated human monocytes through NF-kappaB down-regulation

In inflammatory diseases, strong release of elastinolytic proteases results in elastin fiber degradation generating elastin peptides (EPs). Chemotactic activity for inflammatory cells was, among wide range of properties, the former identified biological activity exerted by EPs. Recently, we demonstrated the ability of EPs to favor a Th1 cytokine (IL-2, IFN-gamma) cell response in lymphocytes and to regulate IL-1beta expression in melanoma cells. We hypothesized that EPs might also influence inflammatory cell properties by regulating cytokine expression by these cells. Therefore, we investigated the influence of EPs on inflammatory cytokine synthesis by human monocytes. We evidenced that EPs down-regulated both at the mRNA and protein levels the proinflammatory TNF-alpha, IL-1beta, and IL-6 expression in LPS-activated monocytes. Such negative feedback loop could be accounted solely for EP-mediated effects on proinflammatory cytokine production because EPs did not affect anti-inflammatory IL-10 or TGF-beta secretion by LPS-activated monocytes. Furthermore, we demonstrated that EP effect on proinflammatory cytokine expression by LPS-stimulated monocytes could not be due either to a decrease of LPS receptor expression or to an alteration of LPS binding to its receptor. The inhibitory effects of EPs on cytokine expression were found to be mediated by receptor (spliced galactosidase) occupancy, as being suppressed by lactose, and to be associated with the decrease of NF-kappaB-DNA complex formation. As a whole, these results demonstrated that EP/spliced galactosidase interaction on human monocytes down-regulated NF-kappaB-dependent proinflammatory cytokine expression and pointed out the critical role of EPs in the regulation of inflammatory response.     

The role of prostaglandins in the mechanism of lipopolysaccharide-induced proMMP9 secretion from human placenta and fetal membrane cells

The abnormal degradation of the extracellular matrix by matrix metalloproteinases (MMPs) in the fetal membranes has been proposed as a central event in preterm premature rupture of the membranes (pPROM). Prostaglandins (PGs) are thought to increase the risk of preterm premature rupture of the fetal membranes by causing matrix degradation. The aim of this study was to assess the mediating role of PGs on lipopolysaccharide (LPS)-induced MMP9 secretion in vitro. ELISA, zymography, and Western blotting were performed on cells and medium from cultures of purified chorion trophoblasts (CTs) and syncytiotrophoblasts (STs) from the human placenta and fetal membranes treated with LPS, meloxicam, (a selective prostaglandin-endoperoxide synthase 2 [PTGS2, previously known as cyclooxygenase 2] inhibitor), or replacement PGE(2) or PGF(2alpha). LPS significantly (P < 0.01) increased proMMP9 secretion and prostaglandin E(2) (PGE(2)) output by cultured CTs and STs, but there was no effect on tissue inhibitor of matrix metalloproteinase 1 (TIMP1) secretion. In these cells, meloxicam significantly blocked LPS-induced proMMP9 secretion and PGE(2) output (P < 0.01). Exogenous PGE(2) and PGF(2alpha) significantly reversed the reduction in proMMP9 secretion caused by meloxicam in a dose-dependent manner (P < 0.01). The expression of PTGS2 protein in CTs and STs was increased dramatically after LPS treatment, but there was no significant effect on the expression of PTGS1 (previously known as cyclooxygenase 1), membrane-associated prostaglandin E synthases (membrane-associated PTGES, previously known as mPGES) 1 and 2, or cytosolic prostaglandin E synthase (cytosolic PTGES, previously knows as cPGES) proteins. Our results suggest that PGs may mediate the selective increase in MMP9 after exposure of trophoblast cells to LPS. There was no effect of LPS on TIMP1. Understanding this relationship may help in developing strategies for the prevention and management of pPROM and preterm labor.     

Production of matrix metalloproteinase-9 in lipopolysaccharide-stimulated human amnion occurs through an autocrine and paracrine proinflammatory cytokine-dependent system

The objective of this study was to determine the presence of autocrine/paracrine regulation of matrix metalloproteinase-9 (MMP-9) expression mediated by proinflammatory cytokines in human fetal membranes. Fetal membranes obtained from women who underwent cesarean delivery before labor were manually separated into amnion and chorion layers and maintained in culture. These explants were stimulated with tumor necrosis factor alpha (TNFalpha), interleukin-1beta (IL-1beta), and either lipopolysaccharide (LPS) alone or LPS with anti-TNFalpha or anti-IL-1beta-neutralizing antibodies. Levels of proMMP-9 in culture media were evaluated by zymography. Enzyme-linked immunosorbant assay was performed to measure the quantity of IL-1beta, TNFalpha, and tissue inhibitor of matrix metalloproteinases-1 (TIMP-1) after LPS stimulation. ProMMP-9 activity was upregulated after stimulation of the amnion by LPS, TNFalpha, and IL-1beta. The increased activity of proMMP-9 resulting from LPS stimulation in the amnion was blocked by the addition of TNFalpha neutralizing antibody but not with anti-IL-1beta. No significant effect of LPS, TNFalpha, or IL-1beta on proMMP-9 expression was observed in the chorion; however, the chorion produced both cytokines when stimulated with LPS. In contrast, TIMP-1 levels remained unchanged in all cultures incubated in the presence of LPS. Therefore, these data indicate that proMMP-9 is produced by the amnion but not the chorion in response to LPS. Because anti-TNFalpha-neutralizing antibody inhibits proMMP-9 activity in the amnion, TNFalpha appears to upregulate proMMP-9 production by the amnion in an autocrine fashion. Meanwhile, TNFalpha and IL-1beta produced by the chorion may upregulate amnionic proMMP-9 production in a paracrine manner.     

IL-6 trans-signaling modulates TLR4-dependent inflammatory responses via STAT3

Innate immune responses triggered by the prototypical inflammatory stimulus LPS are mediated by TLR4 and involve the coordinated production of a multitude of inflammatory mediators, especially IL-6, which signals via the shared IL-6 cytokine family receptor subunit gp130. However, the exact role of IL-6, which can elicit either proinflammatory or anti-inflammatory responses, in the pathogenesis of TLR4-driven inflammatory disorders, as well as the identity of signaling pathways activated by IL-6 in a proinflammatory state, remain unclear. To define the contribution of gp130 signaling events to TLR4-driven inflammatory responses, we combined genetic and therapeutic approaches based on a series of gp130(F/F) knock-in mutant mice displaying hyperactivated IL-6-dependent JAK/STAT signaling in an experimental model of LPS/TLR4-mediated septic shock. The gp130(F/F) mice were markedly hypersensitive to LPS, which was associated with the specific upregulated production of IL-6, but not TNF-α. In gp130(F/F) mice, either genetic ablation of IL-6, Ab-mediated inhibition of IL-6R signaling or therapeutic blockade of IL-6 trans-signaling completely protected mice from LPS hypersensitivity. Furthermore, genetic reduction of STAT3 activity in gp130(F/F):Stat3(+/-) mice alleviated LPS hypersensitivity and reduced LPS-induced IL-6 production. Additional genetic approaches demonstrated that the TLR4/Mal pathway contributed to LPS hypersensitivity and increased IL-6 production in gp130(F/F) mice. Collectively, these data demonstrate for the first time, to our knowledge, that IL-6 trans-signaling via STAT3 is a critical modulator of LPS-driven proinflammatory responses through cross-talk regulation of the TLR4/Mal signaling pathway, and potentially implicate cross-talk between JAK/STAT and TLR pathways as a broader mechanism that regulates the severity of the host inflammatory response.     

Modulation of matrix metalloproteinase production from human lung fibroblasts by type 4 phosphodiesterase inhibitors

Over-expression of matrix metalloproteinases by lung fibroblasts has been blamed for much of the tissue destruction associated with airway inflammation. Because cyclic AMP is known to regulate fibroblast proliferation, as well as cytokine and extracellular matrix protein production, the current study was designed to evaluate the ability of three selective phosphodiesterase (PDE) type 4 inhibitors, rolipram, cilomilast and CI-1044, to inhibit extracellular matrix degradation. Using zymography and ELISA, we found that pro-MMP-2 release was enhanced following 24 h treatment of human lung fibroblast (MRC-5) with TGF-beta1 (10 ng/ml) or TNF-alpha (10 ng/ml), whereas PMA (0.02 microM) had no effect. One hour of pre-incubation with PDE4 inhibitors (10 microM) induced an inhibition of TNF-alpha-stimulated pro-MMP-2 release. Zymography and immunoblotting revealed that fibroblasts cultured with PMA or TNF-alpha released increased amounts of pro-MMP-1, whereas TGF-beta1 had no effect. Incubation with CI-1044 or cilomilast significantly prevented the TNF-alpha increase in pro-MMP-1. These results suggest that PDE4 inhibitors are effective in inhibiting the pro-MMP-2 and pro-MMP-1 secretion induced by TNF-alpha and might underline a potential therapeutic benefit of selective PDE4 inhibitors in lung diseases associated with abnormal tissue remodelling.     

Docosahexaenoic acid prevents lipopolysaccharide-induced cytokine production in microglial cells by inhibiting lipopolysaccharide receptor presentation but not its membrane subdomain localization

Recognition of lipopolysaccharide (LPS), the endotoxin of gram-negative bacteria, by microglia occurs through its binding to specific receptors, cluster of differentiation 14 and toll-like receptor-4. LPS binding to these receptors triggers the synthesis of proinflammatory cytokines that coordinate the brain innate immune response to protect the CNS of the infection. Docosahexaenoic acid (DHA), a n -3 polyunsaturated fatty acid highly incorporated in the brain, is a potent immunomodulator. In this study, we investigated whether DHA modulates LPS receptor localization and, as a consequence, LPS-induced signaling pathway and proinflammatory cytokine production. We demonstrated that DHA, when added exogenously, is specifically enriched in membrane phospholipids, but not in raft lipids of microglial cells. DHA incorporation in membrane impaired surface presentation of LPS receptors cluster of differentiation 14 and toll-like receptor-4, but not their membrane subdomain localization. LPS-induced nuclear factor kappa B activation was inhibited by DHA, hence, LPS-induced proinflammatory cytokine synthesis of interleukin-1β and tumor necrosis factor α was strongly attenuated. We suggest that DHA is highly anti-inflammatory by targeting LPS receptor surface location, therefore reducing LPS action on microglia. This effect represents a new insight by which DHA modulates in the brain the expression of proinflammatory cytokines in response to bacterial product.     

Matrix Metalloproteinase-3 (MMP-3) Is an Endogenous Activator of the MMP-9 Secreted by Placental Leukocytes: Implication in Human Labor

Background

The activity of matrix degrading enzymes plays a leading role in the rupture of the fetal membranes under normal and pathological human labor, and matrix metalloproteinase-9 (MMP-9) it is considered a biomarker of this event. To gain further insight into local MMP-9 origin and activation, in this study we analyzed the contribution of human placental leukocytes to MMP-9 secretion and explored the local mechanisms of the pro-enzyme activation.

Methods

Placental blood leukocytes were obtained from women at term gestation without labor and maintained in culture up to 72 h. MMP-9 activity in the culture supernatants was determined by zymography and using a specific substrate. The presence of a potential pro-MMP-9 activator in the culture supernatants was monitored using a recombinant biotin-labeled human pro-MMP-9. To characterize the endogenous pro-MMP-9 activator, MMP-1, -3, -7 and -9 were measured by multiplex assay in the supernatants, and an inhibition assay of MMP-9 activation was performed using an anti-human MMP-3 and a specific MMP-3 inhibitor. Finally, production of MMP-9 and MMP-3 in placental leukocytes obtained from term pregnancies with and without labor was assessed by immunofluorescence.

Results

Placental leukocytes spontaneously secreted pro-MMP-9 after 24 h of culture, increasing significantly at 48 h (P≤0.05), when the active form of MMP-9 was detected. Culture supernatants activated the recombinant pro-MMP-9 showing that placental leukocytes secrete the activator. A significant increase in MMP-3 secretion by placental leukocytes was observed since 48 h in culture (P≤0.05) and up to 72 h (P≤0.001), when concentration reached its maximum value. Specific activity of MMP-9 decreased significantly (P≤0.005) when an anti-MMP-3 antibody or a specific MMP-3 inhibitor were added to the culture media. Placental leukocytes from term labor produced more MMP-9 and MMP-3 compared to term non-labor cells.

Conclusions

In this work we confirm that placental leukocytes from human term pregnancies are able to secrete large amounts of MMP-9, and that the production of the enzyme it is enhanced by labor. We also demonstrate for the first time that endogenous MMP-3 plays a major role in MMP-9 activation process. These findings support the contribution of placental leukocytes to create the collagenolytic microenvironment that induces the rupture of the fetal membranes during human labor.     

Caspase inhibition prolongs inflammation by promoting a signaling complex with activated RIPK1

Activation of inflammation by lipopolysaccharide (LPS) is an important innate immune response. Here we investigated the contribution of caspases to the LPS-mediated inflammatory response and discovered distinctive temporal roles of RIPK1 in mediating proinflammatory cytokine production when caspases are inhibited. We propose a biphasic model that differentiates the role of RIPK1 in early versus late phase. The early production of proinflammation cytokines stimulated by LPS with caspase inhibition is mediated by the NF-κB pathway that requires the scaffold function of RIPK1 but is kinase independent. Autocrine production of TNFα in the late phase promotes the formation of a novel TNFR1-associated complex with activated RIPK1, FADD, caspase-8, and key mediators of NF-κB signaling. The production of proinflammatory cytokines in the late phase can be blocked by RIPK1 kinase inhibitor Nec-1s. Our study demonstrates a mechanism by which the activation of RIPK1 promotes its own scaffold function to regulate the NF-κB–mediated proinflammatory cytokine production that is negatively regulated by caspases to restrain inflammatory signaling.