Lymph node (LN) development is a complex process that requires interaction between lymphoid tissue inducer (LTi) cells expressing lymphotoxin α1β2 (LTα1β2) and mesenchymal stromal cells expressing the lymphotoxin receptor (LTβR). In this issue, Vondenhoff et al. (p. 5439) examined the roles of LTβR signaling and TNF-related activation-induced cytokine (TRANCE) expression in the early events of LN organogenesis. The authors first observed TRANCE expression in LN anlagen and went on to determine that LTα was not required for the earliest formation of LTi cell clusters but was required for their maintenance. LTβR triggering was found to control expression of TRANCE, IL-7, and adhesion molecules and to be necessary for the differentiation of mesenchymal cells to stromal organizer cells. LTβR triggering also up-regulated the lymphangiogenic growth factor VEGF-C, suggesting a role for LTβR in angiogenesis during LN development. These data suggest the existence of a positive feedback loop in LN development wherein LTβR signaling induces the differentiation of stromal organizer cells via expression of TRANCE and IL-7, which go on to enhance LTα1β2 expression and the differentiation of mature LTi cells.

The role of NF-κB in inflammatory signaling has been well studied, but how this important transcription factor affects myeloid cell differentiation has remained unclear. Another transcription factor, C/EBPα, is necessary for the proper development of monocytes and granulocytes from myeloid precursors, and NF-κB and C/EBPβ have been shown to cooperatively control the expression of inflammatory molecules. Previous work demonstrated that C/EBPα preferentially binds NF-κB p50 vs NF-κB p65, leading Wang et al. (p. 5757) to examine myelopoiesis in mice lacking NF-κB p50 (p50−/−). They discovered that p50−/− mice had defects in granulopoiesis both in vitro and in vivo, and overall neutrophil numbers were reduced in response to G-CSF. However, in vitro monopoiesis in response to cytokines and LPS was unaffected. p50−/− mice had C/EBPα mRNA and protein levels that were 3-fold less than those in wild-type animals, while PU.1, C/EBPβ, and STAT3 levels were comparable in both mouse strains. Chromatin immunoprecipitation assays demonstrated that NF-κB p50 and C/EBPα bound to the endogenous C/EBPα promoter and that NF-κB p50 was necessary for trans-activating the promoter, thus explaining why p50−/− mice had lower levels of C/EBPα. These data demonstrate that NF-κB signaling is necessary for inflammatory events in mature myeloid cells but also for the stimulation of neutrophil production through a C/EBPα-dependent mechanism.

Interleukin-13 plays important roles in allergy and other Th2-mediated immune responses but is not thought to act directly on T cells. In a recent study, IL-13 was found to negatively regulate Th17 responses in experimental autoimmune encephalomyelitis, leading Newcomb et al. (p. 5317) to take a fresh look at the expression of the IL-13R on T cells. Consistent with previous data, the IL-13-specific receptor chain IL-13Rα1 was expressed in neither naive BALB/c T cells nor in vitro polarized Th1 or Th2 cells. However, IL-13R was functionally expressed in cells polarized to the Th17 lineage. IL-13 administration specifically inhibited expression of IL-17A during both polarization and restimulation of these cells and could be shown to decrease the number of IL-17A-producing cells in these cultures. IL-13-mediated inhibition of IL-17A production was dependent on signaling via STAT6, and IL-13 treatment of Th17 cells led to increased expression of the Th2-associated transcription factor GATA3. Further, IL-13 inhibited the expression of RORγt, a transcription factor necessary for Th17 development. Knowledge that IL-13 can directly inhibit Th17 responses may have important therapeutic implications, as inhibition of IL-13 could lead to the exacerbation of Th17-mediated inflammatory disease.

Regulatory T cells (Treg) and Th17 cells have opposing effects on inflammatory processes yet share some of the same machinery for differentiation. Both cell types are influenced by the TGFβ-induced transcription factor Smad3, which cooperates with NFAT to induce Foxp3 expression and Treg differentiation and with STAT3 to induce RORγt and Th17 differentiation. The IL-1 receptor-associated kinase 1 (IRAK-1) has been shown to phosphorylate STAT3, leading Maitra et al. (p. 5763) to assess this kinase’s role in Th17 and Treg differentiation. Compared with wild-type mice, IRAK-1−/− mice demonstrated an increase in Treg frequency and in Foxp3 expression in CD4+ T cells. The mechanism of enhanced Foxp3 induction in IRAK−/− T cells involved an increase in binding of Smad3 to the Foxp3 promoter via an increase in nuclear NFATc2. In response to TGF-β and IL-6, IRAK−/− T cells showed decreased expression of IL-17 and RORγt compared with wild type that was associated with a decrease in STAT3 phosphorylation and binding to the IL-17 promoter. This reduced IL-17 expression was also observed in vivo and correlated with an amelioration of the inflammatory response in models of septic shock and atherosclerosis. Together, these data define an important role for IRAK-1, which is better known for its role in innate immunity, in modulating the balance between proinflammatory Th17 cells and suppressive Treg cells.

In addition to the production of type I IFNs, the host response to influenza A virus infection involves the induction of apoptosis of infected cells through a mechanism that is not clear. The adaptor protein mitochondrial antiviral signaling protein (MAVS), which is involved in retinoic acid-inducible protein I (RIG-I)-induced antiviral signaling, resides in the mitochondrial outer membrane, suggesting a possible link between antiviral signaling and apoptosis. To begin to clarify the mechanism of virus-induced apoptosis, Öhman et al. (p. 5682) used a subcellular proteomic approach to identify the proteins present in the cytosolic and mitochondrial fractions of human macrophages following influenza A infection. This proteomic analysis demonstrated virus-induced rearrangement of cytoskeletal proteins between the cytosol and mitochondria, including translocation of actin to the mitochondria and actin fragmentation. Components of the RIG-I/MAVS signaling pathway also translocated to the mitochondria upon influenza A infection through a mechanism independent of caspase activation, with RIG-I arriving later than TRIM25, TRADD, actin, and viral proteins. Further analysis of actin involvement in the antiviral response indicated that an intact actin cytoskeleton was required for the induction of antiviral cytokines. Taken together, these data suggest that the actin network regulates antiviral and apoptotic signals, which occur at the mitochondria following influenza A infection.

Inhibition of angiogenesis, which would impair tumor invasion, growth, and metastasis, is an attractive therapeutic goal. To target angiogenesis in Her-2/neu+ breast tumors, Seavey et al. (p. 5537) developed a vaccine consisting of Listeria monocytogenes expressing a chimeric protein that included fragments of fetal liver kinase gene-1 (Flk-1), the mouse homologue of vascular endothelial growth factor receptor 2 (VEGFR2), linked to the adjuvant listeriolysin-O (LLO) (Lm-LLO-Flk-1). By targeting Flk-1 in this way, the authors hoped to inhibit tumor angiogenesis and induce CTL killing of Flk-1-expressing endothelial cells. This vaccine resulted in tumor regression and in some cases eradication with a reduction in microvascular density and the creation of long-lived antitumor immunity. The effectiveness of the vaccine was not due solely to the predicted antivascular effects but also required the induction of epitope spreading to CTL epitopes of the endogenous tumor Ag Her-2/neu. Experimental metastases were also prevented by administration of the Lm-LLO-Flk-1 vaccine, and examination of vaccine toxicity showed no effects on wound healing or pregnancy. This vaccine strategy thus allowed effective targeting of both the tumor vasculature and an endogenous tumor Ag and may open the door to the development of future approaches to tumor therapy or prevention.

B cells in patients with systemic lupus erythematosus (SLE) express lower levels of membrane-localized CD5, which negatively regulates BCR signaling, than those of normal patients. Recent work determined that levels of membrane-bound CD5 are controlled by differential expression of a cytoplasmic isoform (CD5-E1B) and a membrane isoform (CD5-E1A). Garaud et al. (p. 5623) showed by biochemical analysis that the CD5-E1B promoter in B cells from SLE patients was demethylated compared with that in B cells from healthy controls. When B cells were stimulated through the BCR, the differences in methylation became even more pronounced, with CD5-E1B undergoing preferential transcription with a corresponding decrease in CD5-E1A transcription. The authors determined that IL-6, produced at high levels by SLE B cells, was responsible for these differences in methylation. This cytokine decreased the ability of SLE B cells to up-regulate production of the DNA methyltransferase DNMT1. When the B cells of healthy controls were treated with IL-6 or a methylation inhibitor, the CD5-E1B promoter showed the same pattern of demethylation as in SLE B cells. Thus, in SLE B cells, BCR engagement and the presence of IL-6 cause epigenetic modifications that decrease the expression of membrane CD5. This, in turn, can lead to uncontrolled activation of autoreactive B cells in lupus patients.

As indicated by their name, myeloid-derived suppressor cells (MDSC) mediate immunosuppression in many pathogenic conditions, including cancer. The mechanism by which MDSC-mediated suppression occurs has not been completely elucidated, though reactive oxygen species (ROS) have been identified as important players. Corzo et al. (p. 5693) pursued this route of inquiry and examined ROS production by MDSC in seven different models of cancer. The authors discovered that within MDSC, the up-regulation of the NADPH oxidase (NOX2) was responsible for increased ROS production. The NOX2 subunits p47phox and gp91phox were more highly expressed in MDSC from tumor-bearing mice compared with immature myeloid cells in tumor-free mice, and STAT3 was found to control expression of these NOX2 subunits. NOX2 was necessary for maintaining the immature and suppressive phenotype of MDSC; in the absence of NOX2, MDSC were unable to suppress the production of IFN-γ by T cells and quickly acquired the markers of mature macrophages and dendritic cells. This study demonstrates that the ROS produced by MDSC in tumors are due to up-regulation of NOX2, which is thus responsible for maintaining the suppressive milieu of the tumor.

Summaries written by Jennifer Hartt Meyers, Ph.D.