Microbiota triggers STING-type I IFN-dependent monocyte reprogramming of the tumor microenvironment.

The tumor microenvironment (TME) influences cancer progression and therapy response. Therefore, understanding what regulates the TME immune compartment is vital. Here we show that microbiota signals program mononuclear phagocytes in the TME toward immunostimulatory monocytes and dendritic cells (DCs). Single-cell RNA sequencing revealed that absence of microbiota skews the TME toward pro-tumorigenic macrophages. Mechanistically, we show that microbiota-derived stimulator of interferon genes (STING) agonists induce type I interferon (IFN-I) production by intratumoral monocytes to regulate macrophage polarization and natural killer (NK) cell-DC crosstalk. Microbiota modulation with a high-fiber diet triggered the intratumoral IFN-I-NK cell-DC axis and improved the efficacy of immune checkpoint blockade (ICB). We validated our findings in individuals with melanoma treated with ICB and showed that the predicted intratumoral IFN-I and immune compositional differences between responder and non-responder individuals can be transferred by fecal microbiota transplantation. Our study uncovers a mechanistic link between the microbiota and the innate TME that can be harnessed to improve cancer therapies.

Amyloid Cascade into Clarity.

In a recent issue of Nature, Sevigny et al. (2016) report findings from a phase 1b clinical trial of aducanumab (a monoclonal antibody targeting misfolded amyloid-ß peptides), revitalizing the "amyloid cascade hypothesis" and bringing mononuclear phagocytes center stage in the treatment of Alzheimer's disease.

Caspase-8 Acts in a Non-enzymatic Role as a Scaffold for Assembly of a Pro-inflammatory "FADDosome" Complex upon TRAIL Stimulation.

TRAIL is a potent inducer of apoptosis and has been studied almost exclusively in this context. However, TRAIL can also induce NFκB-dependent expression of multiple pro-inflammatory cytokines and chemokines. Surprisingly, whereas inhibition of caspase activity blocked TRAIL-induced apoptosis, but not cytokine production, knock down or deletion of caspase-8 suppressed both outcomes, suggesting that caspase-8 participates in TRAIL-induced inflammatory signaling in a scaffold role. Consistent with this, introduction of a catalytically inactive caspase-8 mutant into CASP-8 null cells restored TRAIL-induced cytokine production, but not cell death. Furthermore, affinity precipitation of the native TRAIL receptor complex revealed that pro-caspase-8 was required for recruitment of RIPK1, via FADD, to promote NFκB activation and pro-inflammatory cytokine production downstream. Thus, caspase-8 can serve in two distinct roles in response to TRAIL receptor engagement, as a scaffold for assembly of a Caspase-8-FADD-RIPK1 "FADDosome" complex, leading to NFκB-dependent inflammation, or as a protease that promotes apoptosis.

Early antitumor activity of oral Langerhans cells is compromised by a carcinogen.

Early diagnosis of oral squamous cell carcinoma (OSCC) remains an unmet clinical need. Therefore, elucidating the initial events of OSCC preceding tumor development could benefit OSCC prognosis. Here, we define the Langerhans cells (LCs) of the tongue and demonstrate that LCs protect the epithelium from carcinogen-induced OSCC by rapidly priming αßT cells capable of eliminating γH2AX+ epithelial cells, whereas γδT and natural killer cells are dispensable. The carcinogen, however, dysregulates the epithelial resident mononuclear phagocytes, reducing LC frequencies, while dendritic cells (DCs), macrophages, and plasmacytoid DCs (pDCs) populate the epithelium. Single-cell RNA-sequencing analysis indicates that these newly differentiated cells display an immunosuppressive phenotype accompanied by an expansion of T regulatory (Treg) cells. Accumulation of the Treg cells was regulated, in part, by pDCs and precedes the formation of visible tumors. This suggests LCs play an early protective role during OSCC, yet the capacity of the carcinogen to dysregulate the differentiation of mononuclear phagocytes facilitates oral carcinogenesis.

Mononuclear phagocyte morphological response to chemoattractants is dependent on geranylgeranyl pyrophosphate.

Statins are used to treat hypercholesterolemia and function by inhibiting the production of the rate-limiting metabolite mevalonate. As such, statin treatment not only inhibits de novo synthesis of cholesterol but also isoprenoids that are involved in prenylation, the posttranslational lipid modification of proteins. The immunomodulatory effects of statins are broad and often conflicting. Previous work demonstrated that statins increased survival and inhibited myeloid cell trafficking in a murine model of sepsis, but the exact mechanisms underlying this phenomenon were unclear. Herein, we investigated the role of prenylation in chemoattractant responses. We found that simvastatin treatment abolished chemoattractant responses induced by stimulation by C5a and FMLP. The inhibitory effect of simvastatin treatment was unaffected by the addition of either farnesyl pyrophosphate (FPP) or squalene but was reversed by restoring geranylgeranyl pyrophosphate (GGPP). Treatment with prenyltransferase inhibitors showed that the chemoattractant response to both chemoattractants was dependent on geranylgeranylation. Proteomic analysis of C15AlkOPP-prenylated proteins identified several geranylgeranylated proteins involved in chemoattractant responses, including RHOA, RAC1, CDC42, and GNG2. Chemoattractant responses in THP-1 human macrophages were also geranylgeranylation dependent. These studies provide data that help clarify paradoxical findings on the immunomodulatory effects of statins. Furthermore, they establish the role of geranylgeranylation in mediating the morphological response to chemoattractant C5a.NEW & NOTEWORTHY The immunomodulatory effect of prenylation is ill-defined. We investigated the role of prenylation on the chemoattractant response to C5a. Simvastatin treatment inhibits the cytoskeletal remodeling associated with a chemotactic response. We showed that the chemoattractant response to C5a was dependent on geranylgeranylation, and proteomic analysis identified several geranylgeranylated proteins that are involved in C5a receptor signaling and cytoskeletal remodeling. Furthermore, they establish the role of geranylgeranylation in mediating the response to chemoattractant C5a.

LRRK2 and Rab10 coordinate macropinocytosis to mediate immunological responses in phagocytes.

Genetic variation in LRRK2 associates with the susceptibility to Parkinson's disease, Crohn's disease, and mycobacteria infection. High expression of LRRK2 and its substrate Rab10 occurs in phagocytic cells in the immune system. In mouse and human primary macrophages, dendritic cells, and microglia-like cells, we find that Rab10 specifically regulates a specialized form of endocytosis known as macropinocytosis, without affecting phagocytosis or clathrin-mediated endocytosis. LRRK2 phosphorylates cytoplasmic PI(3,4,5)P3-positive GTP-Rab10, before EEA1 and Rab5 recruitment to early macropinosomes occurs. Macropinosome cargo in macrophages includes CCR5, CD11b, and MHCII, and LRRK2-phosphorylation of Rab10 potently blocks EHBP1L1-mediated recycling tubules and cargo turnover. EHBP1L1 overexpression competitively inhibits LRRK2-phosphorylation of Rab10, mimicking the effects of LRRK2 kinase inhibition in promoting cargo recycling. Both Rab10 knockdown and LRRK2 kinase inhibition potently suppress the maturation of macropinosome-derived CCR5-loaded signaling endosomes that are critical for CCL5-induced immunological responses that include Akt activation and chemotaxis. These data support a novel signaling axis in the endolysosomal system whereby LRRK2-mediated Rab10 phosphorylation stalls vesicle fast recycling to promote PI3K-Akt immunological responses.

Modulation of immune cell reactivity with cis-binding Siglec agonists.

Inflammatory pathologies caused by phagocytes lead to numerous debilitating conditions, including chronic pain and blindness due to age-related macular degeneration. Many members of the sialic acid-binding immunoglobulin-like lectin (Siglec) family are immunoinhibitory receptors whose agonism is an attractive approach for antiinflammatory therapy. Here, we show that synthetic lipid-conjugated glycopolypeptides can insert into cell membranes and engage Siglec receptors in cis, leading to inhibitory signaling. Specifically, we construct a cis-binding agonist of Siglec-9 and show that it modulates mitogen-activated protein kinase (MAPK) signaling in reporter cell lines, immortalized macrophage and microglial cell lines, and primary human macrophages. Thus, these cis-binding agonists of Siglecs present a method for therapeutic suppression of immune cell reactivity.

Pioglitazone Reverses Alcohol-Induced Alveolar Macrophage Phagocytic Dysfunction.

Alcohol use disorders (AUD) increase susceptibility to respiratory infections by 2- to 4-fold in part because of impaired alveolar macrophage (AM) immune function. Alcohol causes AM oxidative stress, diminishing AM phagocytic capacity and clearance of microbes from the alveolar space. Alcohol increases AM NADPH oxidases (Noxes), primary sources of AM oxidative stress, and reduces peroxisome proliferator-activated receptor γ (PPARγ) expression, a critical regulator of AM immune function. To investigate the underlying mechanisms of these alcohol-induced AM derangements, we hypothesized that alcohol stimulates CCAAT/enhancer-binding protein ß (C/EBPß) to suppress Nox-related microRNAs (miRs), thereby enhancing AM Nox expression, oxidative stress, and phagocytic dysfunction. Furthermore, we postulated that pharmacologic PPARγ activation with pioglitazone would inhibit C/EBPß and attenuate alcohol-induced AM dysfunction. AM isolated from human AUD subjects or otherwise healthy control subjects were examined. Compared with control AM, alcohol activated AM C/EBPß, decreased Nox1-related miR-1264 and Nox2-related miR-107, and increased Nox1, Nox2, and Nox4 expression and activity. These alcohol-induced AM derangements were abrogated by inhibition of C/EBPß, overexpression of miR-1264 or miR-107, or pioglitazone treatment. These findings define novel molecular mechanisms of alcohol-induced AM dysfunction mediated by C/EBPß and Nox-related miRs that are amenable to therapeutic targeting with PPARγ ligands. These results demonstrate that PPARγ ligands provide a novel and rapidly translatable strategy to mitigate susceptibility to respiratory infections and related morbidity in individuals with AUD.

Macrophage hypophagia as a mechanism of innate immune exhaustion in mAb-induced cell clearance.

Macrophage antibody (Ab)-dependent cellular phagocytosis (ADCP) is a major cytotoxic mechanism for both therapeutic unconjugated monoclonal Abs (mAbs) such as rituximab and Ab-induced hemolytic anemia and immune thrombocytopenia. Here, we studied the mechanisms controlling the rate and capacity of macrophages to carry out ADCP in settings of high target/effector cell ratios, such as those seen in patients with circulating tumor burden in leukemic phase disease. Using quantitative live-cell imaging of primary human and mouse macrophages, we found that, upon initial challenge with mAb-opsonized lymphocytes, macrophages underwent a brief burst (<1 hour) of rapid phagocytosis, which was then invariably followed by a sharp reduction in phagocytic activity that could persist for days. This previously unknown refractory period of ADCP, or hypophagia, was observed in all macrophage, mAb, and target cell conditions tested in vitro and was also seen in vivo in Kupffer cells from mice induced to undergo successive rounds of αCD20 mAb-dependent clearance of circulating B cells. Importantly, hypophagia had no effect on Ab-independent phagocytosis and did not alter macrophage viability. In mechanistic studies, we found that the rapid loss of activating Fc receptors from the surface and their subsequent proteolytic degradation were the primary mechanisms responsible for the loss of ADCP activity in hypophagia. These data suggest hypophagia is a critical limiting step in macrophage-mediated clearance of cells via ADCP, and understanding such limitations to innate immune system cytotoxic capacity will aid in the development of mAb regimens that could optimize ADCP and improve patient outcome.

Enhanced translation expands the endo-lysosome size and promotes antigen presentation during phagocyte activation.

The mechanisms that govern organelle adaptation and remodelling remain poorly defined. The endo-lysosomal system degrades cargo from various routes, including endocytosis, phagocytosis, and autophagy. For phagocytes, endosomes and lysosomes (endo-lysosomes) are kingpin organelles because they are essential to kill pathogens and process and present antigens. During phagocyte activation, endo-lysosomes undergo a morphological transformation, going from a collection of dozens of globular structures to a tubular network in a process that requires the phosphatidylinositol-3-kinase-AKT-mechanistic target of rapamycin (mTOR) signalling pathway. Here, we show that the endo-lysosomal system undergoes an expansion in volume and holding capacity during phagocyte activation within 2 h of lipopolysaccharides (LPS) stimulation. Endo-lysosomal expansion was paralleled by an increase in lysosomal protein levels, but this was unexpectedly largely independent of the transcription factor EB (TFEB) and transcription factor E3 (TFE3), which are known to scale up lysosome biogenesis. Instead, we demonstrate a hitherto unappreciated mechanism of acute organelle expansion via mTOR Complex 1 (mTORC1)-dependent increase in translation, which appears to be mediated by both S6Ks and 4E-BPs. Moreover, we show that stimulation of RAW 264.7 macrophage cell line with LPS alters translation of a subset but not all of mRNAs encoding endo-lysosomal proteins, thereby suggesting that endo-lysosome expansion is accompanied by functional remodelling. Importantly, mTORC1-dependent increase in translation activity was necessary for efficient and rapid antigen presentation by dendritic cells. Collectively, we identified a previously unknown and functionally relevant mechanism for endo-lysosome expansion that relies on mTORC1-dependent translation to stimulate endo-lysosome biogenesis in response to an infection signal.

Functional characterization of adult human trabecular meshwork stem cells.

We earlier identified native human trabecular meshwork stem cells (TMSCs) based on two-parameters- high ABCG2 expression and high nucleus to cytoplasmic ratio. The TMSCs also expressed p75 and AnkyrinG. Based on the high expression of ABCG2 and p75, the TMSCs were identified to be located in the Schwalbe's line region of the TM. In continuation, the current study aimed at elucidating the functional characteristics of human TMSCs. Upon culturing, only a small proportion of TM cells (0.96 ± 0.21% in <30 years) expressing stem cell markers ABCG2 and p75 adhered to the culture dish. This proportion significantly reduced with ageing (0.32 ± 0.23% in 30-60 years and 0.35 ± 0.04% in >60 years). Characterization of the primary TM cultures identified 7.00 ± 1.80% of stem cells with label retaining property. Further, cultured cells had the ability to form TM spheres (0.82 ± 0.23%) which consisted of high ABCG2 and p75 positive cells. Upon dexamethasone induction, 86.00 ± 14.87% and 64.60 ± 7.24% of the cells derived from the TM spheres expressed myocilin and exhibited cross linked actin networks respectively, indicating differentiation of the TMSCs in the sphere to TM cells. In addition, the sphere derived TM cells also possessed phagocytic potential (13.28 ± 3.30%) equivalent to primary TM cells (16.33 ± 4.04%) which was evident upon internalization of zymosan particles. In conclusion, this study has established that a proportion of cultured TM cells had the label retaining property as well as sphere forming ability of adult stem cells. Thus, these results confirm the presence of adult stem cells in the human TM that might be responsible for the maintenance of tissue homeostasis.

Chemical depletion of phagocytic immune cells in Anopheles gambiae reveals dual roles of mosquito hemocytes in anti-Plasmodium immunity.

Mosquito immunity is composed of both cellular and humoral factors that provide protection from invading pathogens. Immune cells known as hemocytes, have been intricately associated with phagocytosis and innate immune signaling. However, the lack of genetic tools has limited hemocyte study despite their importance in mosquito anti-Plasmodium immunity. To address these limitations, we employ the use of a chemical-based treatment to deplete phagocytic immune cells in Anopheles gambiae, demonstrating the role of phagocytes in complement recognition and prophenoloxidase production that limit the ookinete and oocyst stages of malaria parasite development, respectively. Through these experiments, we also define specific subtypes of phagocytic immune cells in An. gambiae, providing insights beyond the morphological characteristics that traditionally define mosquito hemocyte populations. Together, this study represents a significant advancement in our understanding of the roles of mosquito phagocytes in mosquito vector competence and demonstrates the utility of clodronate liposomes as an important tool in the study of invertebrate immunity.

D-series Resolvins activate Phospholipase D in phagocytes during inflammation and resolution.

A successful acute inflammatory response results in the elimination of infectious agents by neutrophils and monocytes, followed by resolution and repair through tissue-resident and recruited macrophages. Resolvins (D-series and E-series) are pro-resolving lipid mediators involved in resolution and tissue repair, whose intracellular signaling remains of interest. Here, we report that D-series resolvins (RvD1- RvD5) activate phospholipase D (PLD), a ubiquitously expressed membrane lipase enzyme activity in modulating phagocyte functions. The mechanism for PLD-mediated actions of Resolvin-D5 (RvD5) in polarizing macrophages (M1-like toward M2-like) was found to be two-pronged: (a) RvD5 inhibits post-transcriptional modifications, by miRs and 3'exonucleases that process PLD2 mRNA, thus increasing PLD2 expression and activity; and (b) RvD5 enhances PLD2-S6Kinase signaling required for membrane expansion and efferocytosis. In an in vivo model of second organ reflow injury, we found that RvD5 did not reduce lung neutrophil myeloperoxidase levels in PLD2-/- mice compared to WT and PLD1-/- mice, confirming a novel role of PLD2 as the isoform in RvD5-mediated resolution processes. These results demonstrate that RvD5-PLD2 are attractive targets for therapeutic interventions in vascular inflammation such as ischemia-reperfusion injury and cardiovascular diseases.

Incorporating Gangliosides into PEGylated Cationic Liposomes that Complexed DNA Attenuates Anti-PEG Antibody Production but Not Anti-DNA Antibody Production in Mice.

Gangliosides (glycosphingolipids) reduce antibody production by inhibiting B-cell receptor (BCR) signaling. We have shown that a copresentation of gangliosides and polyethylene glycol (PEG) on the same liposomes suppresses anti-PEG IgM production in mice. In addition, we recently observed that pDNA incorporated in PEGylated cationic liposomes (PCLs) induces anti-DNA IgM, which could be a hurdle to the development of efficient gene delivery systems. Therefore, the focus of this study was to determine if the copresentation of gangliosides and DNA on the same PCL would suppress antibody production against DNA. PCLs including DNA induced both anti-PEG IgM production and anti-DNA IgM production. The extent of anti-PEG and anti-DNA IgM production was likely dependent on the immunogenicity of the complexed DNA. Treatment of clodronate-containing liposomes, which causes a depletion of phagocytic cells, suppressed anti-PEG IgM production from PCLs that did not include DNA but failed to suppress anti-PEG IgM production from PCLs that complexed DNA (PCLD). Both anti-PEG IgM and anti-DNA IgM was induced in T-cell-deficient nude mice as well as in normal mice following treatment with PCLs and PCLD, respectively. These results indicate that phagocytic cells contribute to anti-PEG IgM production but not to anti-DNA IgM production, while T-cells do not contribute to any form of antibody production. The copresentation of gangliosides and DNA significantly reduced anti-PEG IgM production but unfortunately did not reduce anti-DNA IgM production. It appears that the immunosuppressive effect of gangliosides, presumably via the CD22 signaling pathway, is limited only to anti-PEG immunity.

Zymosan enhances in vitro phagocyte function and the immune response of mice infected with Paracoccidioides brasiliensis.

Paracoccidioides brasiliensis is the major etiologic agent of Paracoccidioidomycosis (PCM), the most frequent human deep mycosis in Latin America. It is proposed that masking of ß-glucan in P. brasiliensis cell wall is a critical virulence factor that contributes to the development of a chronic disease characterized by a long period of treatment, which is usually toxic. In this context, the search for immunomodulatory agents for therapeutic purposes is highly desirable. One strategy is to use pattern recognition receptors (PRRs) ligands to stimulate the immune response mediated by phagocytes. Here, we sought to evaluate if Zymosan, a ß-glucan-containing ligand of the PRRs Dectin-1/TLR-2, would enhance phagocyte function and the immune response of mice challenged with P. brasiliensis. Dendritic cells (DCs) infected with P. brasiliensis and treated with Zymosan showed improved secretion of several proinflammatory cytokines and expression of maturation markers. In addition, when cocultured with splenic lymphocytes, these cells induced the production of a potential protective type 1 and 17 cytokine patterns. In macrophages, Zymosan ensued a significant fungicidal activity associated with nitric oxide production and phagolysosome acidification. Importantly, we observed a protective effect of Zymosan-primed DCs delivered intranasally in experimental pulmonary PCM. Overall, our findings support the potential use of ß-glucan-containing compounds such as Zymosan as an alternative or complementary antifungal therapy. LAY SUMMARY: We report for the first time that Paracoccidioides brasiliensis-infected phagocytes treated with Zymosan (cell wall extract from bakers' yeast) show enhanced cytokine production, maturation, and fungal killing. Also, Zymosan-primed phagocytes induce a protective immune response in infected mice.

Efficient antisense inhibition reveals microRNA-155 to restrain a late-myeloid inflammatory programme in primary human phagocytes.

A persisting obstacle in human immunology is that blood-derived leukocytes are notoriously difficult to manipulate at the RNA level. Therefore, our knowledge about immune-regulatory RNA-networks is largely based on tumour cell-line and rodent knockout models, which do not fully mimic human leukocyte biology. Here, we exploit straightforward cell penetrating peptide (CPP) chemistry to enable efficient loss-of-function phenotyping of regulatory RNAs in primary human blood-derived cells. The classical CPP octaarginine (R8) enabled antisense peptide-nucleic-acid (PNA) oligomer delivery into nearly 100% of human blood-derived macrophages without apparent cytotoxicity even up to micromolar concentrations. In a proof-of-principle experiment, we successfully de-repressed the global microRNA-155 regulome in primary human macrophages using a PNA-R8 oligomer, which phenocopies a CRISPR-Cas9 induced gene knockout. Interestingly, although it is often believed that fairly high concentrations (µM) are needed to achieve antisense activity, our PNA-R8 was effective at 200 nM. RNA-seq characterized microRNA-155 as a broad-acting riboregulator, feedback restraining a late myeloid differentiation-induced pro-inflammatory network, comprising MyD88-signalling and ubiquitin-proteasome components. Our results highlight the important role of the microRNA machinery in fine-control of blood-derived human phagocyte immunity and open the door for further studies on regulatory RNAs in difficult-to-transfect primary human immune cells.

Rac1-dependent phagocytosis of apoptotic cells by oral squamous cell carcinoma cells: A possible driving force for tumor progression.

Apoptotic cell death frequently occurs in human cancer tissues including oral squamous cell carcinoma (SCC), wherein apoptotic tumor cells are phagocytosed not only by macrophages but also by neighboring tumor cells. We previously reported that the engulfment of apoptotic SCC cells by neighboring SCC cells frequently occurs at the invading front. Therefore, we hypothesized that the phagocytosis of these apoptotic cells by tumor cells contributes to disease progression. Herein, using cultured oral SCC cells, we aimed to confirm whether tumor cells actually phagocytose apoptotic cells and to examine whether cellular activities are regulated by the phagocytosis of apoptotic cells. Co-culture experiments showed that living cells could ingest apoptotic cells into phagolysosomes. NSC23766, an inhibitor of Rac1, which is a key regulator of phagocytic cup formation in professional phagocytes, dramatically suppressed the phagocytosis of apoptotic cells by living cells. Additionally, cell migration and the secretion of DKK1, a tumor-promoting protein, were enhanced by co-culture with apoptotic cells, whereas NSC23766 inhibited these effects. These results show that tumor cells can actively phagocytose apoptotic neighbors in a Rac1-dependent manner and that such activity increases their migration. The regulation of apoptotic cell phagocytosis thus represents new directions for therapeutic intervention for oral cancer.

Phagocytic elimination of synapses by microglia during sleep.

Synaptic strength reduces during sleep, but the underlying mechanisms of this process are unclear. This study showed reduction of synaptic proteins in rat prefrontal cortex (PFC) at AM7 or Zeitgeber Time (ZT0), when the light phase or sleeping period for rats started. At this time point, microglia were weakly activated, displaying larger and more granular somata with increased CD11b expression compared with those at ZT12, as revealed by flow cytometry. Expression of opsonins, such as complements or MFG-E8, matrix metalloproteinases, and microglial markers at ZT0 were increased compared with that at ZT12. Microglia at ZT0 phagocytosed synapses, as revealed by immunohistochemical staining. Immunoblotting detected more synapsin I in the isolated microglia at ZT0 than at ZT12. Complement C3- or MFG-E8-bound synapses were the most abundant at ZT0, some of which were phagocytosed by microglia. Systemic administration of synthetic glucocorticoid dexamethasone reduced microglial size, granularity and CD11b expression at ZT0, resembling microglia at ZT12, and increased synaptic proteins and decreased the sleeping period. Noradrenaline (NA) suppressed glutamate-induced phagocytosis in primary cultured microglia. Systemic administration of the brain monoamine-depleting agent reserpine decreased NA content and synapsin I expression in PFC, and increased expression of microglia markers, C3 and MFG-E8, while increasing the sleeping period. A NA precursor l-threo-dihydroxyphenylserine abolished the reserpine-induced changes. These results suggest that microglia may eliminate presumably weak synapses during every sleep phase. The circadian changes in concentrations of circulating glucocorticoids and brain NA might be correlated with the circadian changes of microglial phenotypes and synaptic strength.

Increased ILC3s associated with higher levels of IL-1ß aggravates inflammatory arthritis in mice lacking phagocytic NADPH oxidase.

The role of redox regulation in immune-mediated arthritis has been previously described. However, the relationship between innate immune cells, including innate lymphoid cells (ILCs) and phagocyte-derived ROS, in this process remains unclear. Here, we characterize ILCs and measure the IL-1 family cytokines along with other cytokines relevant to ILC functions and development in serum-induced arthritic joints in wild type and phagocytic NADPH oxidase (NOX2)-deficient Ncf1-/- mice. We found more severe serum-induced joint inflammation and increased NCR+ ILC3s in inflamed joints of Ncf1-/- mice. Furthermore, in vitro stimulation with IL-1ß on Tbet+ ILC1s from joints facilitated their differentiation into ROR-γt+ ILC3s. Moreover, treatment with IL-1 antagonists effectively lowered the proportions of NCR+ ILC3s and IL-17A producing ILC3s in Ncf1-/- arthritic mice and ameliorated the joint inflammation. These results suggest that NOX2 is an essential regulator of ILC transdifferentiation and may mediate this process in a redox-dependent manner through IL-1ß production in the inflammatory joint. Our findings shed important light on the role of ILCs in the initiation and progression in tissue inflammation and delineate a novel innate immune cell-mediated pathogenic mechanism through which redox regulation may determine the direction of immune responses in joints.

Phagomimetic action of antibiotics: Revisited. How do antibiotics know where to go?

Phagocytic cells know exactly where an infection is by following chemotactic signals. The phagocytosis of bacteria results in a 'respiratory burst' in which superoxide radicals are released. We have previously compared the release of reactive oxygen species (ROS) by antibiotics, during electron transfer reactions, to this event. Antibiotics in their normal bacterial environment, and ROS, are both increasingly implicated in purposeful signalling functions, rather than their more widely known roles in bacterial killing and molecular damage. Here, we extend our comparison between antibiotics and phagocytic cells to propose that antibiotics actively accumulate at a site of pathogen infection or tumour growth. A common link being virulent cellular growth. When this occurs, new proteins are secreted, aberrant iron acquisition takes place, and lipocalins are released. Each provide a mechanism by which antibiotics can bind, and be retained, at an active site of pathogen infection or tumour growth.