The nuclear factor-κB (NF-κB) signaling pathway is one of the best understood immune-related pathways thanks to almost four decades of intense research. NF-κB signaling is activated by numerous discrete stimuli and is a master regulator of the inflammatory response to pathogens and cancerous cells, as well as a key regulator of autoimmune diseases. In this regard, the role of NF-κB signaling in immunity is not unlike that of the macrophage. The dynamics by which NF-κB proteins shuttle between the cytoplasm and the nucleus to initiate transcription have been studied rigorously in fibroblasts and other non-hematopoietic cells, but many questions remain as to how current models of NF-κB signaling and dynamics can be translated to innate immune cells such as macrophages. In this review, we will present recent research on the dynamics of NF-κB signaling and focus especially on how these dynamics vary in different cell types, while discussing why these characteristics may be important. We will end by looking ahead to how new techniques and technologies should allow us to analyze these signaling processes with greater clarity, bringing us closer to a more complete understanding of inflammatory transcription factor dynamics and how different cellular contexts might allow for appropriate control of innate immune responses.

Regulation of N -methyl- d -aspartate (NMDA) receptor activity by kinases and phosphatases contributes to the modulation of synaptic transmission. Targeting of these enzymes near the substrate is proposed to enhance phosphorylation-dependent modulation. Yotiao, an NMDA receptor–associated protein, bound the type I protein phosphatase (PP1) and the adenosine 3′,5′-monophosphate (cAMP)–dependent protein kinase (PKA) holoenzyme. Anchored PP1 was active, limiting channel activity, whereas PKA activation overcame constitutive PP1 activity and conferred rapid enhancement of NMDA receptor currents. Hence, yotiao is a scaffold protein that physically attaches PP1 and PKA to NMDA receptors to regulate channel activity.

Regulation of intracellular cyclic adenosine 3 ′,5 ′-monophosphate (cAMP) is integral in mediating cell growth, cell differentiation, and immune responses in hematopoietic cells. To facilitate studies of cAMP regulation we developed a BRET (bioluminescence resonance energy transfer) sensor for cAMP, CAMYEL (cAMP sensor using YFP-Epac-RLuc), which can quantitatively and rapidly monitor intracellular concentrations of cAMP in vivo. This sensor was used to characterize three distinct pathways for modulation of cAMP synthesis stimulated by presumed G_s-dependent receptors for isoproterenol and prostaglandin E₂. Whereas two ligands, uridine 5 ′-diphosphate and complement C5a, appear to use known mechanisms for augmentation of cAMP via G_q/calcium and G_i, the action of sphingosine 1-phosphate (S1P) is novel. In these cells, S1P, a biologically active lysophospholipid, greatly enhances increases in intracellular cAMP triggered by the ligands for G_s-coupled receptors while having only a minimal effect by itself. The enhancement of cAMP by S1P is resistant to pertussis toxin and independent of intracellular calcium. Studies with RNAi and chemical perturbations demonstrate that the effect of S1P is mediated by the S1P₂ receptor and the heterotrimeric G₁₃ protein. Thus in these macrophage cells, all four major classes of G proteins can regulate intracellular cAMP.

RNAi is proving to be a powerful experimental tool for the functional annotation of mammalian genomes. The full potential of this technology will be realized through development of approaches permitting regulated manipulation of endogenous gene expression with coordinated reexpression of exogenous transgenes. We describe the development of a lentiviral vector platform, pSLIK (single lentivector for inducible knockdown), which permits tetracycline-regulated expression of microRNA-like short hairpin RNAs from a single viral infection of any naïve cell system. In mouse embryonic fibroblasts, the pSLIK platform was used to conditionally deplete the expression of the heterotrimeric G proteins Gα12 and Gα13 both singly and in combination, demonstrating the Gα13 dependence of serum response element-mediated transcription. In RAW264.7 macrophages, regulated knockdown of Gβ2 correlated with a reduced Ca ²⁺ response to C5a. Insertion of a GFP transgene upstream of the Gβ2 microRNA-like short hairpin RNA allowed concomitant reexpression of a heterologous mRNA during tetracycline-dependent target gene knockdown, significantly enhancing the experimental applicability of the pSLIK system.

Compartmentalization of protein kinases with substrates is a mechanism that may promote specificity of intracellular phosphorylation events. We have cloned a low‐molecular weight A‐kinase Anchoring Protein, called AKAP18, which targets the cAMP‐dependent protein kinase (PKA) to the plasma membrane, and permits functional coupling to the L‐type calcium channel. Membrane anchoring is mediated by the first 10 amino acids of AKAP18, and involves residues Gly1, Cys4 and Cys5 which are lipid‐modified through myristoylation and dual palmitoylation, respectively. Transient transfection of AKAP18 into HEK‐293 cells expressing the cardiac L‐type Ca2+ channel promoted a 34 9% increase in cAMP‐responsive Ca2+ currents. In contrast, a targeting‐deficient mutant of AKAP18 had no effect on Ca2+ currents in response to the application of a cAMP analog. Further studies demonstrate that AKAP18 facilitates GLP‐1‐mediated insulin secretion in a pancreatic β cell line (RINm5F), suggesting that membrane anchoring of the kinase participates in physiologically relevant cAMP‐responsive events that may involve ion channel activation.

Monocytes play an important part in the clearance of pathogens during infection. Yasmine Belkaid and her colleagues now report that inflammatory monocytes can also have a regulatory role. Specifically, these cells can mitigate the tissue damage induced by neutrophils during infection of mice with Toxoplasma gondii by releasing prostaglandin E2 in response to commensal bacteria. The commensal flora can promote both immunity to pathogens and mucosal inflammation. How commensal-driven inflammation is regulated in the context of infection remains poorly understood. Here, we show that during acute mucosal infection of mice with Toxoplasma gondii, inflammatory monocytes acquire a tissue-specific regulatory phenotype associated with production of the lipid mediator prostaglandin E2 (PGE2). Notably, in response to commensals, inflammatory monocytes can directly inhibit neutrophil activation in a PGE2-dependent manner. Further, in the absence of inflammatory monocytes, mice develop severe neutrophil-mediated pathology in response to pathogen challenge that can be controlled by PGE2 analog treatment. Complementing these findings, inhibition of PGE2 led to enhanced neutrophil activation and host mortality after infection. These data demonstrate a previously unappreciated dual action of inflammatory monocytes in controlling pathogen expansion while limiting commensal-mediated damage to the gut. Collectively, our results place inflammatory monocyte–derived PGE2 at the center of a commensal-driven regulatory loop required to control host-commensal dialog during pathogen-induced inflammation.