We used a systems biological approach to study innate and adaptive responses to influenza vaccination in humans, during 3 consecutive influenza seasons. Healthy adults were vaccinated with inactivated (TIV) or live attenuated (LAIV) influenza vaccines. TIV induced greater antibody titers and enhanced numbers of plasmablasts than LAIV. In TIV vaccinees, early molecular signatures correlated with, and accurately predicted, later antibody titers in two independent trials. Interestingly, the expression of Calcium/calmodulin-dependent kinase IV (CamkIV) at day 3 was inversely correlated with later antibody titers. Vaccination of CamkIV −/− mice with TIV induced enhanced antigen-specific antibody titers, demonstrating an unappreciated role for CaMKIV in the regulation of antibody responses. Thus systems approaches can predict immunogenicity, and reveal new mechanistic insights about vaccines.

The functional interpretation of high throughput metabolomics by mass spectrometry is hindered by the identification of metabolites, a tedious and challenging task. We present a set of computational algorithms which, by leveraging the collective power of metabolic pathways and networks, predict functional activity directly from spectral feature tables without a priori identification of metabolites. The algorithms were experimentally validated on the activation of innate immune cells.

SummaryThe integrated stress response (ISR) is a homeostatic mechanism by which eukaryotic cells sense and respond to stress-inducing signals, such as amino acid starvation. General controlled nonrepressed (GCN2) kinase is a key orchestrator of the ISR, and modulates cellular metabolism in response to amino acid starvation. Here we demonstrate that GCN2 controls intestinal inflammation by suppressing inflammasome activation. Enhanced activation of ISR was observed in intestinal antigen presenting cells (APCs) and epithelial cells during amino acid starvation, or intestinal inflammation. Genetic deletion of GCN2 in CD11c+ APCs or intestinal epithelial cells resulted in enhanced intestinal inflammation and Th17 responses, due to enhanced inflammasome activation and IL-1β production. This was caused by reduced autophagy in GCN2−/− intestinal APCs and epithelial cells, leading to increased reactive oxygen species (ROS), a potent activator of inflammasomes1. Thus, conditional ablation of Atg5 and Atg7 in intestinal APCs resulted in enhanced ROS and Th17 responses. Furthermore, in vivo blockade of ROS and IL-1β resulted in inhibition of Th17 responses and reduced inflammation in GCN2−/− mice. Importantly, acute amino acid starvation suppressed intestinal inflammation via a mechanism dependent on GCN2. These results reveal a mechanism that couples amino acid sensing with control of intestinal inflammation via GCN2.

The yellow fever vaccine YF-17D is one of the most successful vaccines ever developed in humans. Despite its efficacy and widespread use in more than 600 million people, the mechanisms by which it stimulates protective immunity remain poorly understood. Recent studies using systems biology approaches in humans have revealed that YF-17D–induced early expression of general control nonderepressible 2 kinase (GCN2) in the blood strongly correlates with the magnitude of the later CD8+ T cell response. We demonstrate a key role for virus-induced GCN2 activation in programming dendritic cells to initiate autophagy and enhanced antigen presentation to both CD4+ and CD8+ T cells. These results reveal an unappreciated link between virus-induced integrated stress response in dendritic cells and the adaptive immune response.

Although the antimicrobial activity of reactive oxygen species (ROSs) is well defined, the role of ROSs in regulating the immune response of the body is not well understood. We now provide evidence that hydrogen peroxide (H 2 O IntroductionAccumulated damage due to reactive oxygen species (ROSs) has been suggested to occur during aging 1,2 and more clearly demonstrated during the respiratory burst of phagocytes. 3,4 Hydrogen peroxide (H 2 O 2 ), is a primary component of ROSs produced in large amounts by macrophages and granulocytes and is a mediator of innate immunity against the invading pathogens. 5,6 H 2 O 2 is known to function as a second messenger 7,8 and regulates activation of many important transcription factors, such as nuclear factor-B (NF-B) and activator protein-1 (AP-1), 9,10 that control the inducible expression of genes regulating macrophage-effector functions and cytokine signaling. [11][12][13] The macrophage-induced cytokines determine the fate of the subsequent T-cell responses as type 1 14,15 or type 2. 16,17 Thus, the possibility exists that H 2 O 2 may alter the T-cell immune responses by affecting the macrophageeffector responses. The function of ROSs may not be restricted to its antimicrobial activity alone, 18 but ROSs may also play an important role in regulating the immune environment in the body. Such situations may arise during certain pathophysiologic conditions such as tuberculosis. 19,20 We earlier reported that activated macrophages from the Bruton tyrosine kinase (btk)-deficient mice (CBA/N) produced lower levels of ROSs as compared to the wild-type mice (CBA/J). 21 However, in contrast to CBA/J macrophages, the IL-12 induction was significantly higher in macrophages from CBA/N mice with T-cell responses biased toward type 1. 21,22 Although the contribution of btk enzyme in controlling the signal transduction cascades responsible for ROS production independent of IL-12 signal may not be ruled out, it is possible that btk targets the ROS pathway to influence IL-12 production. However, the exact mechanism by which ROSs down-regulate IL-12 production is not well understood. We now provide evidence that H 2 O 2 /ROSs affect the signaling events important for IL-12 production, leading to downregulation of the same in activated macrophages. Materials and methods MiceBALB/c mice were bred and maintained in the animal facility of Indian Immunologicals (IIL; Hyderabad, India). All mice were 6 to 12 weeks old and experimental protocol was approved by the Institutional Review Committee for care and usage of animals of Indian Immunologicals. Macrophage stimulation assayThe peritoneal exudate cells (PECs) were harvested by injecting 4% thioglycolate broth as described elsewhere. 23 The RAW 264.7 macrophages were obtained from NCCS (National Centre for Cell Science, Pune, India) and maintained in Dulbecco minimal essential medium (DMEM; Invitrogen, Grand Island, NY) containing 10% fetal calf serum and antibiotics (DMEM-10). The macrophages were plated at a density of 3 ϫ 10 6 cells/mL and ...

Summary Interleukin‐10 (IL‐10) is known to inhibit IL‐12 production in macrophages primarily at the transcriptional level with the involvement of p50 and p65 nuclear factor‐κB (NF‐κB). We demonstrate that the c‐rel transcription factor also plays a major role in IL‐10‐mediated IL‐12 suppression. Treatment of macrophages with recombinant IL‐10 inhibited nuclear c‐rel levels, whereas addition of neutralizing anti‐IL‐10 antibody up‐regulated both nuclear c‐rel levels and IL‐12 production by macrophages. Decreased nuclear c‐rel was associated with a reduction in phosphorylation of inhibitory kappa B alpha (IκBα) in the cytoplasm, indicating that IL‐10 prevents degradation of IκBα and the subsequent translocation of c‐rel into the nucleus. Treatment with leptomycin B, a known inhibitor of c‐rel at a concentration of 10 nm, when used with anti‐IL‐10 antibody, resulted in reduced expression of IL‐12. In a complementary experiment, in vitro transient expression of p65 NF‐κB could not rescue the inhibitory effect of IL‐10 on IL‐12 production, suggesting that NF‐κB alone was not sufficient to restore IL‐12 production during IL‐10 treatment. However, over‐expression of c‐rel resulted in IL‐12 restoration upon stimulation with lipopolysaccharide plus interferon‐γ during IL‐10 treatment. Our studies highlight the involvement of c‐rel in IL‐10‐mediated IL‐12 regulation.

Ischemia–reperfusion injury is a well-known pathological hallmark associated with diabetic retinopathy, glaucoma, and other related retinopathies that ultimately can lead to visual impairment and vision loss. Retinal ischemia pathogenesis involves a cascade of detrimental events that include energy failure, excitotoxic damage, calcium imbalance, oxidative stress, and eventually cell death. Retina for a long time has been known to be an immune privileged site; however, recent investigations reveal that retina, as well as the central nervous system, elicits immunological responses during various stress cues. Stress condition, such as reperfusion of blood supply post-ischemia results in the sequestration of different immune cells, inflammatory mediators including cytokines, chemokines, etc., to the ischemic region, which in turn facilitates induction of inflammatory conditions in these tissues. The immunological activation during injury or stress per se is beneficial for repair and maintenance of cellular homeostasis, but whether the associated inflammation is good or bad, during ischemia–reperfusion injury, hitherto remains to be explored. Keeping all these notions in mind, the current review tries to address the immune response and host stress response mechanisms involved in ischemia–reperfusion injury with the focus on the retina.

Activation of the amino acid starvation response (AAR) increases lifespan and acute stress resistance as well as regulates inflammation. However, the underlying mechanisms remain unclear. Here, we show that activation of AAR pharmacologically by Halofuginone (HF) significantly inhibits production of the proinflammatory cytokine interleukin 1β (IL-1β) and provides protection from intestinal inflammation in mice. HF inhibits IL-1β through general control nonderepressible 2 kinase (GCN2)–dependent activation of the cytoprotective integrated stress response (ISR) pathway, resulting in rerouting of IL-1β mRNA from translationally active polysomes to inactive ribocluster complexes—such as stress granules (SGs)—via recruitment of RNA-binding proteins (RBPs) T cell–restricted intracellular antigen-1(TIA-1)/TIA-1–related (TIAR), which are further cleared through induction of autophagy. GCN2 ablation resulted in reduced autophagy and SG formation, which is inversely correlated with IL-1β production. Furthermore, HF diminishes inflammasome activation through suppression of reactive oxygen species (ROS) production. Our study unveils a novel mechanism by which IL-1β is regulated by AAR and further suggests that administration of HF might offer an effective therapeutic intervention against inflammatory diseases.