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The NLRP3 inflammasome plays a central role in antimicrobial defense as well as in the context of sterile inflammatory conditions. NLRP3 activity is governed by two independent signals: the first signal primes NLRP3, rendering it responsive to the second signal, which then triggers inflammasome formation. Our understanding of how NLRP3 priming contributes to inflammasome activation remains limited. Here, we show that IKKβ, a kinase activated during priming, induces recruitment of NLRP3 to phosphatidylinositol-4-phosphate (PI4P), a phospholipid enriched on the trans-Golgi network. NEK7, a mitotic spindle kinase that had previously been thought to be indispensable for NLRP3 activation, was redundant for inflammasome formation when IKKβ recruited NLRP3 to PI4P. Studying iPSC-derived human macrophages revealed that the IKKβ-mediated NEK7-independent pathway constitutes the predominant NLRP3 priming mechanism in human myeloid cells. Our results suggest that PI4P binding represents a primed state into which NLRP3 is brought by IKKβ activity.

Despite considerable recent advances in understanding and treating many other cancers, malignant brain tumors remain associated with low survival or severe long-term sequelae. Limited progress, including development of immunotherapies, relates in part to difficulties in accurately reproducing brain microenvironment with current preclinical models. The cellular interactions among resident microglia, recruited tumor-associated macrophages, stromal cells, glial cells, neurons, and cancer cells and how they affect tumor growth or behavior are emerging, yet many questions remain. The role of the blood-brain barrier, extracellular matrix components, and heterogeneity among tumor types and within different regions of a single tumor further complicate the matter. Here, we focus on brain microenvironment features impacted by tumor biology. We also discuss limits of current preclinical models and how complementary models, such as humanized animals and organoids, will allow deeper mechanistic insights on cancer biology, allowing for more efficient testing of therapeutic strategies, including immunotherapy, for brain cancers.

Macrophages are immune cells with important roles in tissue homeostasis, inflammation and pathologies. Hence, macrophage populations represent promising targets for modern medicine. Exploiting the potential of macrophage-targeted therapies will require a thorough understanding of the mechanisms controlling their development, specialization and maintenance throughout their lifespan. Macrophages have been studied in vitro for many years, but recent advances in the field of macrophage biology have called into question the validity of traditional approaches. New models, such as recent innovations in generating macrophages from induced pluripotent stem cells (iPSCs), must take into account the impact of heterogeneity in the origin and tissue-specific functions of macrophages. Here, we discuss these protocols and argue for a better understanding of the type of macrophages made in vitro; we also encourage recognition of the importance of tissue identity of macrophages, which cannot be recapitulated by cytokine-dependent protocols. We suggest that a two-step model — in which iPSC-derived macrophages are first generated based on their ontogeny and then conditioned by their tissue-specific environment — offers immense potential for generating biologically relevant macrophages for future studies. Can macrophages derived from induced pluripotent stem cells (iPSCs) be used to effectively model in vivo tissue macrophages? Here, the authors consider the pros and cons of the different protocols for generating macrophages from iPSCs and suggest that a two-step model, based on ontogeny and tissue-specific microenvironment, enables the generation of biologically relevant macrophages.

The innate immune system senses RNA viruses by pattern recognition receptors (PRRs) and protects the host from virus infection. PRRs mediate the production of immune modulatory factors and direct the elimination of RNA viruses. Here, we show a unique PRR that mediates antiviral response. Tetrachlorodibenzo-p-dioxin (TCDD)-inducible poly(ADP ribose) polymerase (TIPARP), a Cysteine3 Histidine (CCCH)-type zinc finger-containing protein, binds to Sindbis virus (SINV) RNA via its zinc finger domain and recruits an exosome to induce viral RNA degradation. TIPARP typically localizes in the nucleus, but it accumulates in the cytoplasm after SINV infection, allowing targeting of cytoplasmic SINV RNA. Redistribution of TIPARP is induced by reactive oxygen species (ROS)-dependent oxidization of the nuclear pore that affects cytoplasmic-nuclear transport. BCL2-associated X protein (BAX) and BCL2 antagonist/killer 1 (BAK1), B-cell leukemia/lymphoma 2 (BCL2) family members, mediate mitochondrial damage to generate ROS after SINV infection. Thus, TIPARP is a viral RNA-sensing PRR that mediates antiviral responses triggered by BAX- and BAK1-dependent mitochondrial damage.

Macrophages are immune cells with important roles in tissue homeostasis, inflammation and pathologies. Hence, macrophage populations represent promising targets for modern medicine. Exploiting the potential of macrophage-targeted therapies will require a thorough understanding of the mechanisms controlling their development, specialization and maintenance throughout their lifespan. Macrophages have been studied in vitro for many years, but recent advances in the field of macrophage biology have called into question the validity of traditional approaches. New models, such as recent innovations in generating macrophages from induced pluripotent stem cells (iPSCs), must take into account the impact of heterogeneity in the origin and tissue-specific functions of macrophages. Here, we discuss these protocols and argue for a better understanding of the type of macrophages made in vitro; we also encourage recognition of the importance of tissue identity of macrophages, which cannot be recapitulated by cytokine-dependent protocols. We suggest that a two-step model - in which iPSC-derived macrophages are first generated based on their ontogeny and then conditioned by their tissue-specific environment - offers immense potential for generating biologically relevant macrophages for future studies.

With its adaptor protein apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC), Nod-like receptor family, pyrin domain containing 3 (NLRP3) forms the inflammasome and mediates inflammatory innate immune responses. Development of an anti-inflammatory drug targeting the NLRP3-inflammasome is urgently required because its aberrant activation often causes inflammatory diseases, including gout. We show that resveratrol, a natural polyphenol in grapes and wine, is a safe and effective phytochemical that inhibits NLRP3-inflammasome activation. Resveratrol inhibits the accumulation of acetylated α-tubulin caused by mitochondrial damage in macrophages stimulated with inducers of the NLRP3-inflammasome. Consequently, resveratrol inhibits the acetylated-α-tubulin-mediated spatial arrangement of mitochondria and their subsequent contact with the endoplasmic reticulum (ER), causing insufficient assembly of ASC on the mitochondria and NLRP3 on the ER. These findings indicate that resveratrol targets the generation of an optimal site for the assembly of NLRP3 and ASC, thus inhibiting NLRP3-inflammasome activation. Therefore, resveratrol could be an effective medication for the treatment of NLRP3-related inflammatory diseases.

Accumulating evidence indicates that type I interferon (IFN) mediates the host protective response to RNA viruses. However, the anti-viral effector molecules involved in this response have not been fully identified. Here, we show that zinc-finger anti-viral protein (ZAP), an IFN-inducible gene, plays a critical role in the elimination of Sindbis virus (SINV) in vitro and in vivo. The loss of ZAP greatly enhances the replication of SINV but does not inhibit type I IFN production in primary mouse embryonic fibroblasts (MEFs). ZAP binds and destabilizes SINV RNA, thereby suppressing the replication of SINV. Type I IFN fails to suppress SINV replication in ZAP-deficient MEFs, whereas the ectopic expression of ZAP is sufficient to suppress the replication of SINV in MEFs lacking the expression of type I IFN and the IFN-inducible genes. ZAP-deficient mice are highly susceptible to SINV infection, although they produce sufficient amounts of type I IFN. Therefore, ZAP is an RNA-sensing anti-viral effector molecule that mediates the type-I-IFN-dependent host defense against SINV.

TNFR-associated factor family member–associated NF-κB activator (TANK)–binding kinase 1 (TBK1) is critical for the activation of IFN regulatory factor 3 and type I IFN production upon virus infection. A set of TBK1-binding proteins, 5-azacytidine–induced gene 2 (AZI2; also known as NAP1), TANK, and TBK1-binding protein 1 (TBKBP1), have also been implicated in the production of type I IFNs. Among them, TANK was found to be dispensable for the responses against virus infection. However, physiological roles of AZI2 and TBKBP1 have yet to be clarified. In this study, we found that none of these TBK1-binding proteins is critical for type I IFN production in mice. In contrast, AZI2, but not TBKBP1, is critical for the differentiation of conventional dendritic cells (cDCs) from bone marrow cells in response to GM-CSF. AZI2 controls GM-CSF–induced cell cycling of bone marrow cells via TBK1. GM-CSF–derived DCs from AZI2-deficient mice show severe defects in cytokine production and T cell activation both in vitro and in vivo. Reciprocally, overexpression of AZI2 results in efficient generation of cDCs, and the cells show enhanced T cell activation in response to Ag stimulation. Taken together, AZI2 expression is critical for the generation of cDCs by GM-CSF and can potentially be used to increase the efficiency of immunization by cDCs.

SummaryRIG-I-like receptors (RLRs) sense virus-derived RNA or polyinosinic-polycytidylic acid (poly IC) to exert antiviral immune responses. Here, we examine the mechanisms underlying the adjuvant effects of poly IC. Poly IC was taken up by dendritic cells (DCs), and it induced lysosomal destabilization, which, in turn, activated an RLR-dependent signaling pathway. Upon poly IC stimulation, cathepsin D was released into the cytoplasm from the lysosome to interact with IPS-1, an adaptor molecule for RLRs. This interaction facilitated cathepsin D cleavage of caspase 8 and the activation of the transcription factor NF-κB, resulting in enhanced cytokine production. Further recruitment of the kinase RIP-1 to this complex initiated the necroptosis of a small number of DCs. HMGB1 released by dying cells enhanced IFN-β production in concert with poly IC. Collectively, these findings suggest that cathepsin D-triggered, IPS-1-dependent necroptosis is a mechanism that propagates the adjuvant efficacy of poly IC.

In the original Figure 1, an arrow was mistakenly added between the fetal liver monocytes and the short-term and long-term HSCs. This arrow has now been removed.

Summary Tissue macrophages arise during embryogenesis from yolk-sac (YS) progenitors that give rise to primitive YS macrophages. Until recently, it has been impossible to isolate or derive sufficient numbers of YS-derived macrophages for further study, but data now suggest that induced pluripotent stem cells (iPSCs) can be driven to undergo a process reminiscent of YS-hematopoiesis in vitro. We asked whether iPSC-derived primitive macrophages (iMacs) can terminally differentiate into specialized macrophages with the help of growth factors and organ-specific cues. Co-culturing human or murine iMacs with iPSC-derived neurons promoted differentiation into microglia-like cells in vitro. Furthermore, murine iMacs differentiated in vivo into microglia after injection into the brain and into functional alveolar macrophages after engraftment in the lung. Finally, iPSCs from a patient with familial Mediterranean fever differentiated into iMacs with pro-inflammatory characteristics, mimicking the disease phenotype. Altogether, iMacs constitute a source of tissue-resident macrophage precursors that can be used for biological, pathophysiological, and therapeutic studies.

When host cells are infected by an RNA virus, pattern-recognition receptors (PRRs) recognize the viral RNA and induce the antiviral innate immunity. Toll-like receptor 7 (TLR7) detects the genomic RNA of incoming murine leukemia virus (MLV) in endosomes and mediates the antiviral response. However, the RNA-sensing PRR that recognizes the MLV in the cytosol is not fully understood. Here, we definitively demonstrate that zinc-finger antiviral protein (ZAP) acts as a cytosolic RNA sensor, inducing the degradation of the MLV transcripts by the exosome, an RNA degradation system, on RNA granules. Although the retinoic acid inducible gene I (RIG-I)–like receptors (RLRs) RIG-I and melanoma differentiation-associated protein 5 detect various RNA viruses in the cytosol and induce the type I IFN-dependent antiviral response, RLR loss does not alter the replication efficiency of MLV. In sharp contrast, the loss of ZAP greatly enhances the replication efficiency of MLV. ZAP localizes to RNA granules, where the processing-body and stress-granule proteins assemble. ZAP induces the recruitment of the MLV transcripts and exosome components to the RNA granules. The CCCH-type zinc-finger domains of ZAP, which are RNA-binding motifs, mediate its localization to RNA granules and MLV transcripts degradation by the exosome. Although ZAP was known as a regulator of RIG-I signaling in a human cell line, ZAP deficiency does not affect the RIG-I–dependent production of type I IFN in mouse cells. Thus, ZAP is a unique member of the cytosolic RNA-sensing PRR family that targets and eliminates intracellular RNA viruses independently of TLR and RLR family members.

NLRP3 forms an inflammasome with its adaptor ASC, and its excessive activation can cause inflammatory diseases. However, little is known about the mechanisms that control assembly of the inflammasome complex. Here we show that microtubules mediated assembly of the NLRP3 inflammasome. Inducers of the NLRP3 inflammasome caused aberrant mitochondrial homeostasis to diminish the concentration of the coenzyme NAD(+), which in turn inactivated the NAD(+)-dependent α-tubulin deacetylase sirtuin 2; this resulted in the accumulation of acetylated α-tubulin. Acetylated α-tubulin mediated the dynein-dependent transport of mitochondria and subsequent apposition of ASC on mitochondria to NLRP3 on the endoplasmic reticulum. Therefore, in addition to direct activation of NLRP3, the creation of optimal sites for signal transduction by microtubules is required for activation of the entire NLRP3 inflammasome.

Dopaminergic neurons in the substantia nigra pars compacta modulate complex motor control. Nigral dopaminergic neurons exhibit three different firing patterns in vivo: a pacemaker mode, a random mode, and a burst mode. These firing patterns are closely related to motor control. However, the changes in the proportion of the firing patterns with respect to age have not been fully established. To clarify the age-dependent changes in the proportion of dopaminergic firing patterns, we used single unit extracellular recordings in male F344/N rats. We observed that, with age, the distribution of the spikes fired by dopaminergic neurons shifts from pacemaker to random mode, and then from random to burst mode. These results suggest that the age-dependent changes in the proportion of nigral dopaminergic firing patterns may have an effect on motor function.

Dopaminergic neurons in the substantia nigra pars compacta modulate complex motor control. Nigral dopaminergic neurons exhibit three different firing patterns in vivo: a pacemaker mode, a random mode, and a burst mode. These firing patterns are closely related to motor control. However, the changes in the proportion of the firing patterns with respect to age have not been fully established. To clarify the age-dependent changes in the proportion of dopaminergic firing patterns, we used single unit extracellular recordings in male F344/N rats. We observed that, with age, the distribution of the spikes fired by dopaminergic neurons shifts from pacemaker to random mode, and then from random to burst mode. These results suggest that the age-dependent changes in the proportion of nigral dopaminergic firing patterns may have an effect on motor function.

We investigated the Raman, nuclear magnetic resonance (NMR), and transport characteristics of 13C-enriched single-walled carbon nanotubes (13CNTs). Systematic Raman spectroscopy measurements show that the 13C content of 13CNT can be controlled by the 13C source. Full width at half maximum of Raman spectra show a broad peak around 50% of 13C while G/D ratio remains almost constant. 13C NMR measurements indicate that the spin-lattice relaxation consists of two mechanisms and spin-spin relaxation time is of order of sub-millisecond. In transport measurement, we demonstrate that 13CNT shows quantum-dot features at low temperatures.