Vitamin D receptor (VDR) plays an essential role in gastrointestinal inflammation. Most investigations have focused on the immune response; however, how bacteria regulate VDR and how VDR modulates the nuclear factor (NF)-B pathway in intestinal epithelial cells remain unexplored. This study investigated the effects of VDR ablation on NF-B activation in intestinal epithelia and the role of enteric bacteria on VDR expression. We found that VDR ؊/؊ mice exhibited a pro-inflammatory bias. After Salmonella infection, VDR ؊/؊ mice had increased bacterial burden and mortality. Serum interleukin-6 in noninfected VDR ؉/؉ mice was undetectable, but was easily detectable in VDR ؊/؊ mice. NF-B p65 formed a complex with VDR in noninfected wild-type mouse intestine. In contrast, deletion of VDR abolished VDR/P65 binding. P65 nuclear translocation occurred in colonic epithelial cells of untreated VDR ؊/؊ mice. VDR deletion also elevated NF-B activity in intestinal epithelia. VDR was localized to the surface epithelia of germ-free mice, but to crypt epithelial cells in conventionalized mice. VDR expression, distribution, transcriptional activity, and target genes were regulated by Salmonella stimulation, independent of 1,25-dihydroxyvitamin D3. Our study demonstrates that commensal and pathogenic bacteria directly regulate colonic epithelial VDR expression and location in vivo. VDR negatively regulates bacterial-induced intestinal NF-B activation and attenuates response to infection. Therefore, VDR is an important contributor to intestinal homeostasis and host protection from bacterial invasion and infection.

1,25-Dihydroxyvitamin D [1,25(OH)2D3] is known to have anti-inflammatory activity; however, the molecular mechanism remains poorly defined. Here we show that the nuclear vitamin D receptor (VDR) is directly involved in the regulation of NF-κB activation, a pathway essential for inflammatory response. In mouse embryonic fibroblasts (MEFs) derived from VDR−/− mice, the basal level of κB inhibitor (IκB) α protein was markedly decreased compared with VDR+/− MEFs; however, degradation of IκBα and its phosphorylation in response to TNF-α treatment or Salmonella infection were not altered in VDR−/− cells, neither were the levels of IκB kinase-α and IκB kinase-β proteins. Consistent with IκBα reduction, p65 accumulation in the nucleus was markedly increased in unstimulated VDR−/− cells. In addition, the physical interaction between VDR and p65 was absent in VDR−/− MEFs, which may free p65 and increase its activity. Consequently, these alterations combined led to a marked increase in nuclear p65 DNA binding and NF-κB transcriptional activity; consistently, induction of IL-6 by TNF-α or IL-1β was much more robust in VDR−/− than in VDR+/− cells, indicating that VDR−/− cells are more susceptible to inflammatory stimulation. Therefore, cells lacking VDR appear to be more proinflammatory due to the intrinsic high NF-κB activity. The reduction of IκBα in VDR−/− MEFs may be partially explained by the lack of VDR-mediated stabilization of IκBα by 1,25(OH)2D3. This is supported by the observation that IκBα degradation induced by TNF-α was inhibited by 1,25(OH)2D3 in VDR+/− cells, but not in VDR−/− cells. Taken together, these data suggest that VDR plays an inhibitory role in the regulation of NF-κB activation.

Wild-type (WT) Salmonella typhimurium causes acute intestinal inflammation by activating the nuclear factor kappa B (NF-kB) pathway. Interestingly, WT Salmonella infection also causes degradation of b-catenin, a regulator of cellular proliferation. Regulation of b-catenin and the inhibitor of NF-kB, IkBa, is strikingly similar, involving phosphorylation at identical sites, ubiquitination by the same E3 ligase, and subsequent proteasomal degradation. However, how b-catenin directly regulates the NF-kB pathway during bacteria-induced inflammation in vivo is unknown. Using streptomycinpretreated mice challenged with Salmonella, we demonstrated that WT Salmonella stimulated b-catenin degradation and decreased the physical association between NF-kB and b-catenin. Accordingly, WT Salmonella infection decreased the expression of c-myc, a b-catenin-regulated target gene, and increased the levels of IL-6 and TNF-a, the NF-kB-regulated target genes. Bacterial infection directly stimulated phosphorylation of b-catenin, both in vivo and in vitro. Closer examination revealed that glycogen synthase kinase 3b (GSK-3b) kinase activity was increased in response to WT Salmonella, whereas non-virulent Salmonella had no effect. siRNA of GSK-3b was able to stabilize IkBa in response to WT Salmonella. Pretreatment for 24 h with LiCl, an inhibitor of GSK-3b, reduced WT Salmonella induced IL-8 secretion. Additionally, cells expressing constitutively active b-catenin showed IkBa stabilization and inhibition of NF-kB activity not only after WT Salmonella infection but also after commensal bacteria (Escherichia coli F18) and TNF-a treatment. This study suggests a new role for b-catenin as a negative regulator of inflammation. Bacteria pathogenicity requires overcoming or altering many very effective host defense mechanisms, 1 including the activation of nuclear factor kappa B (NF-kB). 2-4 Bacterial invasion of intestinal epithelial cells (IECs) stimulates NF-kB and, interestingly, degradation of b-catenin, 5 a potent transcriptional factor responsible for cellular proliferation and differentiation. It is notable that regulation of b-catenin and the inhibitor of NF-kB, IkBa, are strikingly similar, involving phosphorylation of the same N-terminal serine sequence sites, ubiquitination by the same E3 ligase complex, and subsequent proteasomal degradation. 6,7 To date, no publications have reported on the physiological significance of b-catenin's potential inter-relationship with the NF-kB inflammatory pathway after bacterial infection in vivo.In this study, therefore, we investigated the role of b-catenin in modulating the proinflammatory response mediated by NF-kB subsequent to Salmonella infection in vivo. We examined the possibility that b-catenin functions as a negative regulator of NF-kB, much in the same way as IkBa, through physical interaction with NF-kB. Additionally, we established the role of glycogen synthase kinase 3b (GSK-3b), as the negative regulator of b-catenin's stability and subsequent reactivity with NF-kB in both in vit...

Introduction-Dermatomyositis (DM) is an autoimmune disease involving muscle and skin. Perifascicular atrophy (PFA) of myofibers is a specific and characteristic DM pathological lesion. Interferon-stimulated gene 15 (ISG15) is a ubiquitin-like modifier with a poorly understood immunological role.

Salmonella Typhimurium is a major cause of human gastroenteritis. The Salmonella type III secretory system secretes virulence proteins, called effectors. Effectors are responsible for the alteration of tight junction (TJ) structure and function in intestinal epithelial cells. AvrA is a newly described bacterial effector found in Salmonella. We report here that AvrA expression stabilizes cell permeability and tight junctions in intestinal epithelial cells. Cells colonized with an AvrA-deficient bacterial strain (AvrA−) displayed decreased cell permeability, disruption of TJs, and an increased inflammatory response. Western blot data showed that TJ proteins, such as ZO-1, claudin-1, decreased after AvrA- colonization for only 1 hour. In contrast, cells colonized with AvrA-sufficient bacteria maintained cell permeability with stabilized TJ structure. This difference was confirmed in vivo. Fluorescent tracer studies showed increased fluorescence in the blood of mice infected with AvrA- compared to those infected with the AvrA-sufficient strains. AvrA- disrupted TJ structure and function and increased inflammation in vivo, compared to the AvrA- sufficient strain. Additionally, AvrA overexpression increased TJ protein expression when transfected into colonic epithelial cells. An intriguing aspect of this study is that AvrA stabilized TJs, even though the other TTSS proteins, SopB, SopE, and SopE2, are known to disrupt TJs. AvrA may play a role in stabilizing TJs and balancing the opposing action of other bacterial effectors. Our findings indicate an important role for the bacterial effector AvrA in regulation of intestinal epithelial cell TJs during inflammation. The role of AvrA represents a highly refined bacterial strategy that helps the bacteria survive in the host and dampen the inflammatory response.

Elevated blood IFNβ protein concentration is associated with DM. Systemic and local production of IFNβ might contribute to, but may not fully explain, the marked overproduction of type 1 IFN-inducible transcripts and proteins seen in DM muscle and blood.

We have used mouse embryonic fibroblasts (MEFs) derived from VDR(+/-) and VDR(-/-) mice to determine whether the nuclear vitamin D receptor (VDR) is directly involved in the regulation of NF-kappaB activation. We found that the basal IkappaBalpha protein level was markedly decreased in VDR(-/-) MEFs compared to VDR(+/-) MEFs; however, degradation of IkappaBalpha and its phosphorylation were not altered in VDR(-/-) cells, neither were the levels of IKKalpha and IKKbeta proteins. Consistently, p65 nuclear translocation was increased in unstimulated VDR(-/-) cells. The physical interaction between VDR and p65 was absent in VDR(-/-) MEFs, which may free p65 and increase its activity. Consequently, these alterations combined led to a marked increase in NF-kappaB transcriptional activity. Consistently, induction of IL-6 by TNFalpha or IL-1beta was much more robust in VDR(-/-) than in VDR(+/-) cells, indicating that VDR(-/-) cells are more susceptible to inflammatory stimulation. Therefore, fibroblasts lacking VDR appear to be more pro-inflammatory due to the intrinsic high NF-kappaB activity. The reduction of IkappaBalpha in VDR(-/-) MEFs may be partially explained by the lack of VDR-mediated stabilization of IkappaBalpha by 1,25(OH)(2)D(3). These data suggest that VDR plays an inhibitory role in the regulation of NF-kappaB activation.

To determine the potential consequences of plasmacytoid dendritic cell (pDC) accumulation in tissue sites observed in several autoimmune diseases, we measured type 1 interferon production from circulating human pDCs as a function of pDC concentration. The effects of interferon-alpha and blockade of the type 1 interferon receptor (IFNAR) on human pDC type 1 interferon and interferoninducible transcription and protein production were measured. Human pDCs became far more efficient producers of interferon-alpha at concentrations beyond those normally present in blood, through an IFNAR-dependent mechanism. Extracellular interferon-alpha increased pDC production of type 1 interferons. The accumulation of pDCs in diseased tissue sites allows marked non-linear amplification of type 1 interferon production locally. The role of the IFNAR-dependent mechanism of interferon production by human pDCs is greater than previously suggested. IFNAR blockade has potential for diminishing type 1 interferon production by all human cells.