NF-κB-dependent Luciferase Activation and Quantification of Gene Expression in Salmonella Infected Tissue Culture Cells.

The dimeric transcription factor NF-κB regulates many cellular response pathways, including inflammatory pathways by inducing the expression of various cytokines and chemokines. NF-κB is constitutively expressed and is sequestered in the cytosol by the inhibitory protein nuclear factor of kappa light polypeptide gene enhancer in B cells inhibitor, alpha (IκBα). Activation of NF-κB requires the degradation of IκBα, which then exposes a nuclear localization signal on NF-κB and promotes its trafficking to the nucleus. Once in the nucleus, NF-κB binds to the promotor region of NF-κB target genes such as interleukin 6 (IL-6) and IL-23, to promote their expression. The activation of NF-κB occurs independently of transcription or translation. Therefore, the activation state of NF-κB must be measured either by quantifying NF-κB specifically in the nucleus, or by quantifying expression of NF-κB target genes. In this protocol, cells stably transfected with an NF-κB::luciferase reporter construct are assayed for NF-κB activation using in vitro tissue culture techniques. These cells are infected with Salmonella Typhimurium to activate NF-κB, which traffics to the nucleus and binds to κB sites in the promoter region of luciferase, inducing its expression. Cells are lysed and analyzed with the luciferase assay system. The amount of luciferase produced by the cells correlates with the intensity of the luminescence signal, which is detected by a plate reader. The luminescence signal generated by this procedure provides a quick and highly sensitive method by which to assess NF-κB activation under a range of conditions. This protocol also utilizes quantitative reverse transcription PCR (RT-qPCR) to detect relative mRNA levels that are indicative of gene expression.

NOD1 and NOD2: Beyond Peptidoglycan Sensing.

NOD1 and NOD2 are pattern recognition receptors of the innate immune system with well-established roles in sensing fragments of bacterial peptidoglycan. In addition to their role as microbial sensors, recent evidence indicates that nucleotide-binding oligomerization domains (NODs) can also recognize a broader array of danger signals. Indeed, recent work has expanded the roles of NOD1 and NOD2 to encompass not only sensing of infections with viruses and parasites but also perceiving perturbations of cellular processes such as regulation of the actin cytoskeleton and maintenance of endoplasmic reticulum homeostasis. This review will comment on recent progress and point out emerging questions in these areas.

Invasive behavior of Campylobacter jejuni in immunosuppressed chicken.

Campylobacter jejuni is a predominant cause of gastroenteritis in humans but rather harmless in chickens. The basis of this difference is unknown. We investigated the effect of the chicken immune defense on the behavior of C. jejuni using glucocorticoid (GC)-treated and mock-treated 17-day old Ross 308 chicken bearing in mind that GCs have immunosuppressive effects and dampen the innate immune response. The effect of GC administration on the behavior of C. jejuni was compared with that on infection with Salmonella Enteritidis to address possible microbe-associated differences. Our results revealed that GC treatment fastened the intestinal colonization of C. jejuni (p < 0.001) and enhanced its dissemination to the liver (p = 0.007). The effect of GC on intestinal colonization of S. Enteritidis was less pronounced (p = 0.033) but GC did speed up the spread of this pathogen to the liver (p < 0.001). Cytokine transcript analysis showed an up to 30-fold reduction in baseline levels of IL-8 mRNA in the cecal (but not spleen) tissue at Day 1 after GC treatment (p < 0.005). Challenge with C. jejuni strongly increased intestinal IL-8, IL-6, and iNOS transcript levels in the non-GC treated animals but not in the GC-treated birds (P < 0.005). In vitro assays with chicken macrophages showed that GC dampened the TLR agonist- and C. jejuni induced-inflammatory gene transcription and production of nitric oxide (P < 0.005). Together, the results support the hypothesis that C. jejuni has the intrinsic ability to invade chicken tissue and that an effective innate immune response may limit its invasive behavior.

NOD1 and NOD2 signalling links ER stress with inflammation.

Endoplasmic reticulum (ER) stress is a major contributor to inflammatory diseases, such as Crohn disease and type 2 diabetes. ER stress induces the unfolded protein response, which involves activation of three transmembrane receptors, ATF6, PERK and IRE1α. Once activated, IRE1α recruits TRAF2 to the ER membrane to initiate inflammatory responses via the NF-κB pathway. Inflammation is commonly triggered when pattern recognition receptors (PRRs), such as Toll-like receptors or nucleotide-binding oligomerization domain (NOD)-like receptors, detect tissue damage or microbial infection. However, it is not clear which PRRs have a major role in inducing inflammation during ER stress. Here we show that NOD1 and NOD2, two members of the NOD-like receptor family of PRRs, are important mediators of ER-stress-induced inflammation in mouse and human cells. The ER stress inducers thapsigargin and dithiothreitol trigger production of the pro-inflammatory cytokine IL-6 in a NOD1/2-dependent fashion. Inflammation and IL-6 production triggered by infection with Brucella abortus, which induces ER stress by injecting the type IV secretion system effector protein VceC into host cells, is TRAF2, NOD1/2 and RIP2-dependent and can be reduced by treatment with the ER stress inhibitor tauroursodeoxycholate or an IRE1α kinase inhibitor. The association of NOD1 and NOD2 with pro-inflammatory responses induced by the IRE1α/TRAF2 signalling pathway provides a novel link between innate immunity and ER-stress-induced inflammation.