MicroRNAs have an important role in bone homeostasis. However, the detailed mechanism of microRNA-mediated intercellular communication between bone cells remains elusive. Here, we report that osteoclasts secrete microRNA-enriched exosomes, by which miR-214 is transferred into osteoblasts to inhibit their function. In a coculture system, inhibition of exosome formation and secretion prevented miR-214 transportation. Exosomes specifically recognized osteoblasts through the interaction between ephrinA2 and EphA2. In osteoclast-specific miR-214 transgenic mice, exosomes were secreted into the serum, and miR-214 and ephrinA2 levels were elevated. Therefore, these exosomes have an inhibitory role in osteoblast activity. miR-214 and ephrinA2 levels in serum exosomes from osteoporotic patients and mice were upregulated substantially. These exosomes may significantly inhibit osteoblast activity. Inhibition of exosome secretion via Rab27a small interfering RNA prevented ovariectomized-induced osteoblast dysfunction in vivo. Taken together, these findings suggest that exosome-mediated transfer of microRNA plays an important role in the regulation of osteoblast activity. Circulating miR-214 in exosomes not only represents a biomarker for bone loss but could selectively regulate osteoblast function.

Abbreviations: BMMs, bone marrow monocytes; M-CSF, macrophage colony stimulating factor; RANKL, receptor activator of nuclear factor-kB ligand; Pten, phosphatase and tensin homolog; OC-TG214, osteoclast specific miR-214 transgenic mice; NFATc1, nuclear factor of activated T-cells cytoplasmic; TRAP, tartrate-resistant acid phosphatase; Dnm3os, Dnm3 opposite strand; WT, wild-type; micro CT, Micro computed tomography; BMD, bone mineral density; BV/TV, ratio of bone volume to tissue volume; Tb.Sp, trabecular spacing; qRT-PCR, quantitative real-time PCR microRNA is necessary for osteoclast differentiation, function and survival. It has been reported that miR-199/214 cluster plays important roles in vertebrate skeletal development and miR-214 inhibits osteoblast function by targeting ATF4. Here, we show that miR-214 is up-regulated during osteoclastogenesis from bone marrow monocytes (BMMs) with macrophage colony stimulating factor (M-CSF) and receptor activator of nuclear factor-kB ligand (RANKL) induction, which indicates that miR-214 plays a critical role in osteoclast differentiation. Overexpression of miR-214 in BMMs promotes osteoclastogenesis, whereas inhibition of miR-214 attenuates it. We further find that miR-214 functions through PI3K/Akt pathway by targeting phosphatase and tensin homolog (Pten). In vivo, osteoclast specific miR-214 transgenic mice (OC-TG214) exhibit down-regulated Pten levels, increased osteoclast activity, and reduced bone mineral density. These results reveal a crucial role of miR-214 in the differentiation of osteoclasts, which will provide a potential therapeutic target for osteoporosis.

Punicalagin (2,3,hexahydroxydiphenoyl-gallagyl-D-glucose and referred to as PUN) is a bioactive ellagitannin isolated from pomegranate, which is widely used for the treatment of inflammatory bowel disease (IBD), diarrhea, and ulcers in Chinese traditional medicine. In this study, we detected the anti-inflammation potentials of PUN in lipopolysaccharide (LPS)-induced macrophages and tried to uncover the underlying mechanism. Results demonstrated that PUN (25, 50, or 100 μM) treatment could significantly decrease the LPS-induced production of nitric oxide), prostaglandin E2 (PGE2), interleukin (IL)-1β, IL-6, and tumor necrosis factor (TNF)-α in RAW264.7 cells. Molecular research showed that PUN inhibited the activation of upstream mediator nuclear factor-κB by suppressing the phosphorylation of IκBα and p65. Results also indicated that PUN could suppress the phosphorylation of mitogen-activated protein kinase including p38, c-Jun N-terminal kinase, and extracellular signal-regulated kinase. In conclusion, we observed that PUN could inhibit LPS-induced inflammation, and it may be a potential choice for the treatment of inflammation diseases.

The bacterial pathogen Legionella pneumophila creates an intracellular niche permissive for its replication by extensively modulating host cell functions using hundreds of effector proteins delivered via its Dot/Icm secretion system 1 . Among these, members of the SidE Users may view, print, copy, and download text and data-mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use:

Reactive oxygen species (ROS) and oxidative stress are thought to play a central role in potentiating macrophage activation, causing excessive inflammation, tissue damage, and sepsis. Recently, we have shown that punicalagin (PUN) exhibits anti-inflammatory activity in LPS-stimulated macrophages. However, the potential antioxidant effects of PUN in macrophages remain unclear. Revealing these effects will help understand the mechanism underlying its ability to inhibit excessive macrophage activation. Hemeoxygenase-1 (HO-1) exhibits antioxidant activity in macrophages. Therefore, we hypothesized that HO-1 is a potential target of PUN and tried to reveal its antioxidant mechanism. Here, PUN treatment increased HO-1 expression together with its upstream mediator nuclear factor-erythroid 2 p45-related factor 2 (Nrf2). However, specific inhibition of Nrf2 by brusatol (a specific Nrf2 inhibitor) dramatically blocked PUN-induced HO-1 expression. Previous research has demonstrated that the PI3K/Akt pathway plays a critical role in modulating Nrf2/HO-1 protein expression as an upstream signaling molecule. Here, LY294002, a specific PI3K/Akt inhibitor, suppressed PUN-induced HO-1 expression and led to ROS accumulation in macrophages. Furthermore, PUN inhibited LPS-induced oxidative stress in macrophages by reducing ROS and NO generation and increasing superoxide dismutase (SOD) 1 mRNA expression. These findings provide new perspectives for novel therapeutic approaches using antioxidant medicines and compounds against oxidative stress and excessive inflammatory diseases including tissue damage, sepsis, and endotoxemic shock.

Highlights d Development of a sensitive and affordable PLpro activity probe d Identification of potential SCoV2 PLpro inhibitors through HTS d Co-crystal structure determination and lead optimization d Characterization of a submicromolar inhibitor of SCoV2

The adaptor CARD9 functions downstream of C-type lectin receptors (CLRs) for the sensing of microbial infection, which leads to responses by the T1 and T17 subsets of helper T cells. The single-nucleotide polymorphism rs4077515 at CARD9 in the human genome, which results in the substitution S12N (CARD9), is associated with several autoimmune diseases. However, the function of CARD9 has remained unknown. Here we generated CARD9 knock-in mice and found that CARD9 facilitated the induction of type 2 immune responses after engagement of CLRs. Mechanistically, CARD9 mediated CLR-induced activation of the non-canonical transcription factor NF-κB subunit RelB, which initiated production of the cytokine IL-5 in alveolar macrophages for the recruitment of eosinophils to drive T2 cell-mediated allergic responses. We identified the homozygous CARD9 mutation encoding S12N in patients with allergic bronchopulmonary aspergillosis and revealed activation of RelB and production of IL-5 in peripheral blood mononuclear cells from these patients. Our study provides genetic and functional evidence demonstrating that CARD9 can turn alveolar macrophages into IL-5-producing cells and facilitates T2 cell-mediated pathologic responses.

As osteoclasts have the central roles in normal bone remodeling, it is ideal to regulate only the osteoclasts performing pathological bone destruction without affecting normal osteoclasts. Based on a hypothesis that pathological osteoclasts form under the pathological microenvironment of the bone tissues, we here set up optimum culture conditions to examine the entity of pathologically activated osteoclasts (PAOCs). Through searching various inflammatory cytokines and their combinations, we found the highest resorbing activity of osteoclasts when osteoclasts were formed in the presence of M-CSF, receptor activator of NF-κB ligand, and IL-1β. We have postulated that these osteoclasts are PAOCs. Analysis using confocal laser microscopy revealed that PAOCs showed extremely high proton secretion detected by the acid-sensitive fluorescence probe Rh-PM and bone resorption activity compared with normal osteoclasts. PAOCs showed unique morphology bearing high thickness and high motility with motile cellular processes in comparison with normal osteoclasts. We further examined the expression of Kindlin-3 and Talin-1, essential molecules for activating integrin β-chains. Although normal osteoclasts express high levels of Kindlin-3 and Talin-1, expression of these molecules was markedly suppressed in PAOCs, suggesting the abnormality in the adhesion property. When whole membrane surface of mature osteoclasts was biotinylated and analyzed, the IL-1β-induced cell surface protein was detected. PAOCs could form a subpopulation of osteoclasts possibly different from normal osteoclasts. PAOC-specific molecules could be an ideal target for regulating pathological bone destruction.