SummaryThe molecular basis of signal-dependent transcriptional activation has been extensively studied in macrophage polarization, but our understanding remains limited regarding the molecular determinants of repression. Here we show that IL-4-activated STAT6 transcription factor is required for the direct transcriptional repression of a large number of genes during in vitro and in vivo alternative macrophage polarization. Repression results in decreased lineage-determining transcription factor, p300, and RNA polymerase II binding followed by reduced enhancer RNA expression, H3K27 acetylation, and chromatin accessibility. The repressor function of STAT6 is HDAC3 dependent on a subset of IL-4-repressed genes. In addition, STAT6-repressed enhancers show extensive overlap with the NF-κB p65 cistrome and exhibit decreased responsiveness to lipopolysaccharide after IL-4 stimulus on a subset of genes. As a consequence, macrophages exhibit diminished inflammasome activation, decreased IL-1β production, and pyroptosis. Thus, the IL-4-STAT6 signaling pathway establishes an alternative polarization-specific epigenenomic signature resulting in dampened macrophage responsiveness to inflammatory stimuli.

a b s t r a c tAloe vera has been used in traditional herbal medicine as an immunomodulatory agent inducing antiinflammatory effects. However, its role on the IL-1␤ inflammatory cytokine production has not been studied. IL-1␤ production is strictly regulated both at transcriptional and posttranslational levels through the activity of Nlrp3 inflammasome. In this study we aimed to determine the effect of Aloe vera on the molecular mechanisms of Nlrp3 inflammasome-mediated IL-1␤ production in LPS-activated human THP-1 cells and monocyte-derived macrophages. Our results show that Aloe vera significantly reduced IL-8, TNF␣, IL-6 and IL-1␤ cytokine production in a dose dependent manner. The inhibitory effect was substantially more pronounced in the primary cells. We found that Aloe vera inhibited the expression of pro-IL-1␤, Nlrp3, caspase-1 as well as that of the P2X7 receptor in the LPS-induced primary macrophages. Furthermore, LPS-induced activation of signaling pathways like NF-B, p38, JNK and ERK were inhibited by Aloe vera in these cells.Altogether, we show for the first time that Aloe vera-mediated strong reduction of IL-1␤ appears to be the consequence of the reduced expression of both pro-IL-1␤ as well as Nlrp3 inflammasome components via suppressing specific signal transduction pathways. Furthermore, we show that the expression of the ATP sensor P2X7 receptor is also downregulated by Aloe vera that could also contribute to the attenuated IL-1␤ cytokine secretion. These results may provide a new therapeutic approach to regulate inflammasome-mediated responses.

Mitochondrial reactive oxygen species (mtROS) generated continuously under physiological conditions have recently emerged as critical players in the regulation of immune signaling pathways. In this study we have investigated the regulation of antiviral signaling by increased mtROS production in plasmacytoid dendritic cells (pDCs), which, as major producers of type I interferons (IFN), are the key coordinators of antiviral immunity. The early phase of type I IFN production in pDCs is mediated by endosomal Toll-like receptors (TLRs), whereas the late phase of IFN response can also be triggered by cytosolic retinoic acid-inducible gene-I (RIG-I), expression of which is induced upon TLR stimulation. Therefore, pDCs provide an ideal model to study the impact of elevated mtROS on the antiviral signaling pathways initiated by receptors with distinct subcellular localization. We found that elevated level of mtROS alone did not change the phenotype and the baseline cytokine profile of resting pDCs. Nevertheless increased mtROS levels in pDCs lowered the TLR9-induced secretion of pro-inflammatory mediators slightly, whereas reduced type I IFN production markedly via blocking phosphorylation of interferon regulatory factor 7 (IRF7), the key transcription factor of the TLR9 signaling pathway. The TLR9-induced expression of RIG-I in pDCs was also negatively regulated by enhanced mtROS production. On the contrary, elevated mtROS significantly augmented the RIG-I-stimulated expression of type I IFNs, as well as the expression of mitochondrial antiviral-signaling (MAVS) protein and the phosphorylation of Akt and IRF3 that are essential components of RIG-I signaling. Collectively, our data suggest that increased mtROS exert diverse immunoregulatory functions in pDCs both in the early and late phase of type I IFN responses depending on which type of viral sensing pathway is stimulated.

IL-1b is a "master" cytokine regulating a wide variety of physiologic and immunologic processes. The most frequently studied models for NLRP3 inflammasomemediated IL-1b production are the macrophages; however, depending on their microenvironment, they can develop into functionally different cells. Several protocols have been developed to model the diversity of these cells in vitro. Here, we report for the first time, to our knowledge, a comparative study about the dynamics and molecular mechanisms of NLRP3 inflammasome priming and activation in LPS-stimulated, human, monocytederived GM-or M-macrophages, differentiated in the presence of GM-CSF or M-CSF, respectively. Our results show that IL-1b production by LPS-stimulated M-macrophages is a rapid and short event that requires ATP supplementation and is attenuated, in part, by the presence of IL-10, which reduces Akt signaling. However, IL-1b production by GMmacrophages develops gradually, and these cells produce IL-1b, even in the absence of ATP supplementation, because of the constitutively active caspase-1 enzyme. We show that the membranebound ectonucleotidases have an important regulatory role on the IL-1b secretion in GM-macrophages. Furthermore, we provide evidence that adenosine treatment enhances LPS-primed IL-1b secretion by GM-macrophages, but not by M-macrophages. These results show that, because of the different activation status and expression levels of the NLRP3 inflammasome components, as well as the signaling activity of the pathways, the two subtypes of macrophages respond very differently to the same stimuli. For this reason, the molecular composition of the microenvironment that shapes macrophage development should be considered when research or therapeutic methods are planned to control IL-1b production.

Besides its well-known psychoactive effects, caffeine has a broad range of actions. It regulates several physiological mechanisms as well as modulates both native and adaptive immune responses by various ways. Although caffeine is assumed to be a negative regulator of inflammation, the effect on the secretion of pro- and anti-inflammatory cytokines is highly controversial. Macrophages are major mediators of inflammatory responses; however, the various subpopulations develop different effects ranging from the initiation to the resolution of inflammation. Here we report a comparative analysis of the effect of caffeine on two subpopulations of human monocyte-derived macrophages differentiated in the presence of macrophage colony-stimulating factor (M-CSF) or granulocyte-macrophage colony-stimulating factor (GM-CSF), resulting in M-MΦs and GM-MΦs, respectively. We showed that although TNF-α secretion was downregulated in both LPS-activated MΦ subtypes by caffeine, the secretion of IL-8, IL-6, and IL-1β as well as the expression of Nod-like receptors was enhanced in M-MΦs, while it did not change in GM-MΦs. We showed that caffeine (1) altered adenosine receptor expression, (2) changed Akt/AMPK/mTOR signaling pathways, and (3) inhibited STAT1/IL-10 signaling axis in M-MΦs. We hypothesized that these alterations play an important modulatory role in the upregulation of NLRP3 inflammasome-mediated IL-1β secretion in LPS-activated M-MΦs following caffeine treatment.

SummaryRagweed pollen extract (RWE) possesses intrinsic NADPH oxidase activity that induces oxidative stress by initiating the production of intracellular reactive oxygen species (ROS). The ROS are important contributors to the manifestation of allergic inflammation; furthermore, concomitant exposure to an allergen and an endotoxin trigger a stronger inflammatory response. One of the main pro-inflammatory cytokines produced in inflammatory responses is interleukin-1b (IL-1b), and its production is associated with caspase-1-containing inflammasome complexes. Intracellular ROS have been implicated in NLRP3 inflammasome-mediated IL-1b production, therefore, we aimed to study whether RWE influences the function of NLRP3 inflammasome. Here we describe that, in the presence of NADPH, RWE significantly elevates lipopolysaccharide-induced IL-1b production of THP-1 cells as well as human primary macrophages and dendritic cells. We also demonstrate that increased IL-1b production is mediated through NLRP3 inflammasome in THP-1 macrophages. We provide evidence that RWE elevates cytosolic ROS level in these cells, and ROS inhibitors abolish IL-1b production. Furthermore, we show that RWE enhances lipopolysaccharide-induced gene transcription/expression of pro-IL-1b and key components of the inflammasome via a ROS-dependent mechanism.

The development of long-lived immune memory cells against pathogens is critical for the success of vaccines to establish protection against future infections. However, the mechanisms governing the long-term survival of immune memory cells remain to be elucidated. In this article, we show that the maintenance mitochondrial homeostasis by autophagy is critical for restricting metabolic functions to protect IgG memory B cell survival. Knockout of mitochondrial autophagy genes, Nix and Bnip3, leads to mitochondrial accumulation and increases in oxidative phosphorylation and fatty acid synthesis, resulting in the loss of IgG+ memory B cells in mice. Inhibiting fatty acid synthesis or silencing necroptosis gene Ripk3 rescued Nix−/−Bnip3−/− IgG memory B cells, indicating that mitochondrial autophagy is important for limiting metabolic functions to prevent cell death. Our results suggest a critical role for mitochondrial autophagy in the maintenance of immunological memory by protecting the metabolic quiescence and longevity of memory B cells.

The reservoirs of the HIV display cellular properties resembling long‐lived immune memory cells that could be exploited for viral clearance. Our interest in developing a cure for HIV stems from the studies of immunologic memory against infections. We and others have found that long‐lived immune memory cells employ prosurvival autophagy and antiapoptotic mechanisms to protect their longevity. Here, we describe the rationale for the development of an approach to clear HIV‐1 by selective elimination of host cells harboring replication‐competent HIV (SECH). While reactivation of HIV‐1 in the host cells with latency reversing agents (LRAs) induces viral gene expression leading to cell death, LRAs also simultaneously up‐regulate prosurvival antiapoptotic molecules and autophagy. Mechanistically, transcription factors that promote HIV‐1 LTR‐directed gene expression, such as NF‐κB, AP‐1, and Hif‐1α, can also enhance the expression of cellular genes essential for cell survival and metabolic regulation, including Bcl‐xL, Mcl‐1, and autophagy genes. In the SECH approach, we inhibit the prosurvival antiapoptotic molecules and autophagy induced by LRAs, thereby allowing maximum killing of host cells by the induced HIV‐1 proteins. SECH treatments cleared HIV‐1 infections in humanized mice in vivo and in HIV‐1 patient PBMCs ex vivo. SECH also cleared infections by the SIV in rhesus macaque PBMCs ex vivo. Research efforts are underway to improve the efficacy and safety of SECH and to facilitate the development of SECH as a therapeutic approach for treating people with HIV.