Oral Administration of Transforming Growth Factor-β1 (TGF-β1) Protects the Immature Gut from Injury via Smad Protein-dependent Suppression of Epithelial Nuclear Factor κB (NF-κB) Signaling and Proinflammatory Cytokine Production

Inflammatory immune responses play an important role in mucosal homeostasis and gut diseases. Nuclear factor κB (NF-κB), central to the proinflammatory cascade, is activated in necrotizing enterocolitis (NEC), a devastating condition of intestinal injury with extensive inflammation in premature infants. TGF-β is a strong immune suppressor and a factor in breast milk, which has been shown to be protective against NEC. In an NEC animal model, oral administration of the isoform TGF-β1 activated the downstream effector Smad2 in intestine and significantly reduced NEC incidence. In addition, TGF-β1 suppressed NF-κB activation, maintained levels of the NF-κB inhibitor IκBα in the intestinal epithelium, and systemically decreased serum levels of IL-6 and IFN-γ. The immature human fetal intestinal epithelial cell line H4 was used as a reductionistic model of the immature enterocyte to investigate mechanism. TGF-β1 pretreatment inhibited the TNF-α-induced IκBα phosphorylation that targets the IκBα protein for degradation and inhibited NF-κB activation. Chromatin immunoprecipitation (ChIP) assays demonstrated decreased NF-κB binding to the promoters of IL-6, IL-8, and IκBα in response to TNF-α with TGF-β1 pretreatment. These TGF-β1 effects appear to be mediated through the canonical Smad pathway as silencing of the TGF-β central mediator Smad4 resulted in loss of the TGF-β1 effects. Thus, TGF-β1 is capable of eliciting anti-inflammatory effects by inhibiting NF-κB specifically in the intestinal epithelium as well as by decreasing systemic IL-6 and IFN-γ levels. Oral administration of TGF-β1 therefore can potentially be used to protect against gastrointestinal diseases.     

Interleukin-20 Promotes Migration of Bladder Cancer Cells through Extracellular Signal-regulated Kinase (ERK)-mediated MMP-9 Protein Expression Leading to Nuclear Factor (NF-κB) Activation by Inducing the Up-regulation of p21WAF1 Protein Expression

The role of inflammatory cytokine interleukin-20 (IL-20) has not yet been studied in cancer biology. Here, we demonstrated up-regulation of both IL-20 and IL-20R1 in muscle-invasive bladder cancer patients. The expressions of IL-20 and IL-20R1 were observed in bladder cancer 5637 and T-24 cells. We found that IL-20 significantly increased the expression of matrix metalloproteinase (MMP)-9 via binding activity of NF-κB and AP-1 in bladder cancer cells and stimulated the activation of ERK1/2, JNK, p38 MAPK, and JAK-STAT signaling. Among the pathways examined, only ERK1/2 inhibitor U0126 significantly inhibited IL-20-induced migration and invasion. Moreover, siRNA knockdown of IL-20R1 suppressed migration, invasion, ERK1/2 activation, and NF-κB-mediated MMP-9 expression induced by IL-20. Unexpectedly, the cell cycle inhibitor p21^WAF1 was induced by IL-20 treatment without altering cell cycle progression. Blockade of p21^WAF1 function by siRNA reversed migration, invasion, activation of ERK signaling, MMP-9 expression, and activation of NF-κB in IL-20-treated cells. In addition, IL-20 induced the activation of IκB kinase, the degradation and phosphorylation of IκBα, and NF-κB p65 nuclear translocation, which was regulated by ERK1/2. IL-20 stimulated the recruitment of p65 to the MMP-9 promoter region. Finally, the IL-20-induced migration and invasion of cells was confirmed by IL-20 gene transfection and by addition of anti-IL-20 antibody. This is the first report that p21^WAF1 is involved in ERK1/2-mediated MMP-9 expression via increased binding activity of NF-κB, which resulted in the induction of migration in IL-20/IL-20R1 dyad-induced bladder cancer cells. These unexpected results might provide a critical new target for the treatment of bladder cancer.     

IL-1β Promotes Corneal Epithelial Cell Migration by Increasing MMP-9 Expression through NF-κB- and AP-1-Dependent Pathways

Interleukin-1β (IL-1β) plays a critical mediator in the pathogenesis of eye diseases. The implication of IL-1β in inflammatory responses has been shown to be mediated through up-regulation of inflammatory genes, including matrix metalloproteinase-9 (MMP-9). However, the detailed mechanisms of IL-1β-induced MMP-9 expression in Statens Seruminstitut Rabbit Corneal Cells (SIRCs) are largely unclear. Here, we demonstrated that in SIRCs, IL-1β induced MMP-9 promoter activity and mRNA expression associated with an increase in the secretion of pro-MMP-9. IL-1β-induced pro-MMP-9 expression and MMP-9 mRNA levels were attenuated by pretreatment with the inhibitor of MEK1/2 (U0126), JNK1/2 (SP600125), NF-κB (Bay11-7082), or AP-1 (Tanshinone IIA) and transfection with siRNA of p42 or JNK2. Moreover, IL-1β markedly stimulated p42/p44 MAPK and JNK1/2 phosphorylation in SIRCs. In addition, IL-1β also enhanced p42/p44 MAPK translocation from the cytosol into the nucleus. On the other hand, IL-1β induced c-Jun and c-Fos mRNA expression, c-Jun phosphorylation, and AP-1 promoter activity. NF-κB translocation, IκBα degradation, and NF-κB promoter activity were also enhanced by IL-1β. Pretreatment with U0126 or SP600125 inhibited IL-1β-induced AP-1 and NF-κB promoter activity, but not NF-κB translocation from the cytosol into the nucleus. Finally, we established that IL-1β could stimulate SIRCs migration via p42/p44 MAPK-, JNK1/2-, AP-1-, and NF-κB-dependent MMP-9 induction. These results suggested that NF-κB and AP-1 activated by JNK1/2 and p42/p44 MAPK cascade are involved in IL-1β-induced MMP-9 expression in SIRCs.     

TNF and IL-1 exhibit distinct ubiquitin requirements for inducing NEMO–IKK supramolecular structures

Nuclear factor κB (NF-κB) essential modulator (NEMO), a regulatory component of the IκB kinase (IKK) complex, controls NF-κB activation through its interaction with ubiquitin chains. We show here that stimulation with interleukin-1 (IL-1) and TNF induces a rapid and transient recruitment of NEMO into punctate structures that are anchored at the cell periphery. These structures are enriched in activated IKK kinases and ubiquitinated NEMO molecules, which suggests that they serve as organizing centers for the activation of NF-κB. These NEMO-containing structures colocalize with activated TNF receptors but not with activated IL-1 receptors. We investigated the involvement of nondegradative ubiquitination in the formation of these structures, using cells deficient in K63 ubiquitin chains or linear ubiquitin chain assembly complex (LUBAC)-mediated linear ubiquitination. Our results indicate that, unlike TNF, IL-1 requires K63-linked and linear ubiquitin chains to recruit NEMO into higher-order complexes. Thus, different mechanisms are involved in the recruitment of NEMO into supramolecular complexes, which appear to be essential for NF-κB activation.     

Butyrate inhibits IL-1β-induced inflammatory gene expression by suppression of NF-κB activity in pancreatic beta cells

Cytokine-induced beta cell dysfunction is a hallmark of type 2 diabetes (T2D). Chronic exposure of beta cells to inflammatory cytokines affects gene expression and impairs insulin secretion. Thus, identification of anti-inflammatory factors that preserve beta cell function represents an opportunity to prevent or treat T2D. Butyrate is a gut microbial metabolite with anti-inflammatory properties for which we recently showed a role in preventing interleukin-1β (IL-1β)-induced beta cell dysfunction, but how prevention is accomplished is unclear. Here, we investigated the mechanisms by which butyrate exerts anti-inflammatory activity in beta cells. We exposed mouse islets and INS-1E cells to a low dose of IL-1β and/or butyrate and measured expression of inflammatory genes and nitric oxide (NO) production. Additionally, we explored the molecular mechanisms underlying butyrate activity by dissecting the activation of the nuclear factor-κB (NF-κB) pathway. We found that butyrate suppressed IL-1β-induced expression of inflammatory genes, such as Nos2, Cxcl1, and Ptgs2, and reduced NO production. Butyrate did not inhibit IκBα degradation nor NF-κB p65 nuclear translocation. Furthermore, butyrate did not affect binding of NF-κB p65 to target sequences in synthetic DNA but inhibited NF-κB p65 binding and RNA polymerase II recruitment to inflammatory gene promoters in the context of native DNA. We found this was concurrent with increased acetylation of NF-κB p65 and histone H4, suggesting butyrate affects NF-κB activity via inhibition of histone deacetylases. Together, our results show butyrate inhibits IL-1β-induced inflammatory gene expression and NO production through suppression of NF-κB activation and thereby possibly preserves beta cell function.     

Pycnogenol Attenuates the Release of Proinflammatory Cytokines and Expression of Perilipin 2 in Lipopolysaccharide-Stimulated Microglia in Part via Inhibition of NF-κB and AP-1 Activation

Over activation of microglia results in the production of proinflammatory agents that have been implicated in various brain diseases. Pycnogenol is a patented extract from French maritime pine bark (Pinus pinaster Aiton) with strong antioxidant and anti-inflammatory potency. The present study investigated whether pycnogenol may be associated with the production of proinflammatory mediators in lipopolysaccharide-stimulated BV2 (mouse-derived) microglia. It was found that pycnogenol treatment was dose-dependently associated with significantly less release of nitricoxide (NO), TNF-α, IL-6 and IL-1β, and lower levels of intercellular adhesion molecule1 (ICAM-1) and perilipin 2 (PLIN2). Furthermore, this effect was replicated in primary brain microglia. Levels of inducible NO synthase mRNA and protein were attenuated, whereas there was no change in the production of the anti-inflammatory cytokine IL-10. Further evidence indicated that pycnogenol treatment led to the suppression of NF-κB activation through inhibition of p65 translocation into the nucleus and inhibited DNA binding of AP-1, suggesting that these proinflammatory factors are associated with NF-κB and AP-1. We conclude that pycnogenol exerts anti-inflammatory effects through inhibition of the NF-κB and AP-1pathway, and may be useful as a therapeutic agent in the prevention of diseases caused by over activation of microglia.     

The toxin mimic FS48 from the salivary gland of Xenopsylla cheopis functions as a Kv1.3 channel-blocking immunomodulator of T cell activation

The Kv1.3 channel has been widely demonstrated to play crucial roles in the activation and proliferation of T cells, which suggests that selective blockers could serve as potential therapeutics for autoimmune diseases mediated by T cells. We previously described that the toxin mimic FS48 from salivary gland of Xenopsylla cheopis downregulates the secretion of proinflammatory factors by Raw 264.7 cells by blocking the Kv1.3 channel and the subsequent inactivation of the proinflammatory MAPK/NF-κB pathways. However, the effects of FS48 on human T cells and autoimmune diseases are unclear. Here, we described its immunomodulatory effects on human T cells derived from suppression of Kv1.3 channel. Kv1.3 currents in Jurkat T cells were recorded by whole-cell patch-clamp, and Ca²⁺ influx, cell proliferation, and TNF-α and IL-2 secretion were measured using Fluo-4, CCK-8, and ELISA assays, respectively. The in vivo immunosuppressive activity of FS48 was evaluated with a rat DTH model. We found that FS48 reduced Kv1.3 currents in Jurkat T cells in a concentration-dependent manner with an IC₅₀ value of about 1.42 μM. FS48 also significantly suppressed Kv1.3 protein expression, Ca²⁺ influx, MAPK/NF-κB/NFATc1 pathway activation, and TNF-α and IL-2 production in activated Jurkat T cells. Finally, we show that FS48 relieved the DTH response in rats. We therefore conclude that FS48 can block the Kv1.3 channel and inhibit human T cell activation, which most likely contributes to its immunomodulatory actions and highlights the great potential of this evolutionary-guided peptide as a drug template in future studies.