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Inflammation anergy in human intestinal macrophages is due to Smad-induced IkappaBalpha expression and NF-kappaB inactivation.

ABSTRACT: Human intestinal macrophages contribute to tissue homeostasis in noninflamed mucosa through profound down-regulation of pro-inflammatory cytokine release. Here, we show that this down-regulation extends to Toll-like receptor (TLR)-induced cytokine release, as intestinal macrophages expressed TLR3-TLR9 but did not release cytokines in response to TLR-specific ligands. Likely contributing to this unique functional profile, intestinal macrophages expressed markedly down-regulated adapter proteins MyD88 and Toll interleukin receptor 1 domain-containing adapter-inducing interferon beta, which together mediate all TLR MyD88-dependent and -independent NF-kappaB signaling, did not phosphorylate NF-kappaB p65 or Smad-induced IkappaBalpha, and did not translocate NF-kappaB into the nucleus. Importantly, transforming growth factor-beta released from intestinal extracellular matrix (stroma) induced identical down-regulation in the NF-kappaB signaling and function of blood monocytes, the exclusive source of intestinal macrophages. Our findings implicate stromal transforming growth factor-beta-induced dysregulation of NF-kappaB proteins and Smad signaling in the differentiation of pro-inflammatory blood monocytes into noninflammatory intestinal macrophages.

SUBMITTER: Smythies LE

PROVIDER: S-EPMC2885238 | biostudies-literature | 2010 Jun

REPOSITORIES: biostudies-literature

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Inflammation anergy in human intestinal macrophages is due to Smad-induced IkappaBalpha expression and NF-kappaB inactivation.

Smythies Lesley E LE Shen Ruizhong R Bimczok Diane D Novak Lea L Clements Ronald H RH Eckhoff Devin E DE Bouchard Phillipe P George Michael D MD Hu William K WK Dandekar Satya S Smith Phillip D PD

The Journal of biological chemistry 20100413 25

Human intestinal macrophages contribute to tissue homeostasis in noninflamed mucosa through profound down-regulation of pro-inflammatory cytokine release. Here, we show that this down-regulation extends to Toll-like receptor (TLR)-induced cytokine release, as intestinal macrophages expressed TLR3-TLR9 but did not release cytokines in response to TLR-specific ligands. Likely contributing to this unique functional profile, intestinal macrophages expressed markedly down-regulated adapter proteins M ...[more]

PMID: 20388715

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Project description:AimTo study the effects of IkappaBalpha and its mutants (IkappaBalphaM, IkappaBalpha243N, IkappaBalphaM244C) on NF-kappaB, p53 and their downstream target genes. The relationship of NF-kappaB, p53, and IkappaBalpha was further discussed.MethodspECFP-IkappaBalpha, pECFP-IkappaBalphaM (amino acides 1-317, Ser32, 36A), pECFP-IkappaBalpha243N (amino acides 1-243), pECFP-IkappaBalpha244C (amino acides 244-317), pEYFP-p65 and pp53-DsRed were constructed and transfected to ASTC-alpha-1 cells. Cells were transfected with pECFP-C1 as a control. 30 h after the transfection, location patterns of NF-kappaB, p53 and IkappaBalpha (IkappaBalphaM, IkappaBalpha243N, IkappaBalpha244C) were observed by a laser scanning microscope (LSM510/ConfoCor2, Zeiss). RNA extraction and reverse transcription were performed in cells transfected or co-transfected with different plasmids. Effects of IkappaBalpha and its mutants on the transprition level of NF-kappaB, NF-kappaB downstream target gene TNF-alpha, p53 and p53 downstream target gene Bax were observed by real time QT-PCR. In all experiments beta-actin was reference. Results are expressed as the target/reference ratio of the sample divided by the target/reference ratio of the control. Different transfected cells were incubated with CCK-8 for 2 h in the incubator. Then the absorbance at 450 nm was measured by using a microplate reader.ResultsCells that were transfected with p53-DsRed revealed a predominant nuclear localization. YFP-p65 mainly existed in the cytoplasm. Cells were transfected with CFP-IkappaBalpha, CFP-IkappaBalphaM, and CFP-IkappaBalpha243N respectively and revealed a predominant cytosolic localization. However, cells transfected of CFP-IkappaBalpha244C revealed a predominant nuclear localization. The mRNA levels of p65, TNF-alpha, p53 and Bax in CFP-IkappaBalpha transfected cells did not change significantly, while in YFP-p65/CFP-IkappaBalpha co-transfected cells, IkappaBalpha decreased the transcription of p65 downstream gene TNF-alpha (2.24+/-0.503) compared with the YFP-p65/CFP-C1 co-transfected cells (5.08+/-0.891) (P<0.05). Phosphorylation defective IkappaBalpha (IkappaBalphaM) decreased the transcription levels of all the four genes compared with the control (P<0.05). The N terminus of IkappaBalpha (IkappaBalpha243N) increased the transcription of NF-kappaB (1.84+/-0.176) and TNF-alpha (1.51+/-0.203) a little bit. However, the C terminus of IkappaBalpha (IkappaBalpha244C) increased the transcription of NF-kappaB, TNF-alpha, p53 and Bax significantly (8.29+/-1.662, 14.16+/-2.121, 10.2+/-0.621, 3.72+/-0.346) (P<0.05). The CCK-8 experiment also showed that IkappaBalpha244C and p53 synergistically mediate apoptosis.ConclusionsIkappaBalpha and its mutants (IkappaBalphaM, IkappaBalpha243N, IkappaBalphaM244C) have different effects on NF-kappaB and p53 signaling pathways, according to their different structures. IkappaBalphaM bounds with NF-kappaB and p53 in cytoplasm steadily, and inhibits both of the two signaling pathways. p53 and IkappaBalpha244C may be co-factor in inducing apoptosis. The C terminal of IkappaBalpha enhanced cell death, which suggests that it may be a pro-apoptotic protein existed in cells.

Project description:In the central nervous system (CNS) the transcription factor NF-kappaB is a key regulator of inflammation and secondary injury processes. Following trauma or disease, the expression of NF-kappaB-dependent genes is activated, leading to both protective and detrimental effects on CNS recovery. Here we show that transgenic inactivation of astroglial NF-kappaB in mice (GFAP-IkappaBalpha-dn mice) resulted in dramatic reduction of disease severity and improvement in functional recovery following EAE. This coincided with a higher presence of leukocytes in the cord and brain of transgenic mice at the chronic phase of the disease, when the functional recovery over WT mice was the most significant. We observed that expression of proinflammatory genes in both spinal cord and cerebellum was delayed and reduced, while the loss of neuronal-specific molecules essential for synaptic transmission was limited compared to WT mice. Furthermore, death of retinal ganglion cells in affected retinas was almost abolished, suggesting the activation of neuroprotective mechanisms. Our data indicate that inhibiting NF-kappaB in astrocytes results in neuroprotective effects following EAE, directly implicating astrocytes in the pathophysiology of this disease. Experiment Overall Design: All mice used were 2-4 months old obtained by breeding heterozygous GFAP-IkappaBalpha-dn males with WT females. WT littermates were used as controls. Spinal cords from WT and GFAP-IkappaBalpha-dn transgenic mice (1), naive or induced with experimental autoimmune encephalomyelitis (EAE) at 10, 17 and 80 days post-induction (dpi) were used to isolate RNA. 3 animals were used for each time point. Experiment Overall Design: To identify significantly expressed genes across all replicate arrays we used R software package LIMMA. Each individual array was normalized within the array using global loess normalization. “Between array” normalization was carried out using the “quantile” algorithm also found in the LIMMA package. Differential expression and False Discovery Rate were assessed by using linear model and empirical Bayes moderated F statistics.

Dataset Information

Inflammation anergy in human intestinal macrophages is due to Smad-induced IkappaBalpha expression and NF-kappaB inactivation.

Publications

Inflammation anergy in human intestinal macrophages is due to Smad-induced IkappaBalpha expression and NF-kappaB inactivation.

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