ANT2 functions as a translocon for mitochondrial cross-membrane translocation of RNAs.

Bidirectional transcription of mammalian mitochondrial DNA generates overlapping transcripts that are capable of forming double-stranded RNA (dsRNA) structures. Release of mitochondrial dsRNA into the cytosol activates the dsRNA-sensing immune signaling, which is a defense mechanism against microbial and viral attack and possibly cancer, but could cause autoimmune diseases when unchecked. A better understanding of the process is vital in therapeutic application of this defense mechanism and treatment of cognate human diseases. In addition to exporting dsRNAs, mitochondria also export and import a variety of non-coding RNAs. However, little is known about how these RNAs are transported across mitochondrial membranes. Here we provide direct evidence showing that adenine nucleotide translocase-2 (ANT2) functions as a mammalian RNA translocon in the mitochondrial inner membrane, independent of its ADP/ATP translocase activity. We also show that mitochondrial dsRNA efflux through ANT2 triggers innate immunity. Inhibiting this process alleviates inflammation in vivo, providing a potential therapeutic approach for treating autoimmune diseases.

Dichotomous roles of RIPK3 in regulating the IFN response and NLRP3 inflammasome in human monocytes.

Regulation of the profile and magnitude of toll-like receptor (TLR) responses is important for effective host defense against infections while minimizing inflammatory toxicity. The chemokine CXCL4 regulates the TLR8 response to amplify inflammatory gene and inflammasome activation while attenuating the interferon (IFN) response in primary monocytes. In this study, we describe an unexpected role for the kinase RIPK3 in suppressing the CXCL4 + TLR8-induced IFN response and providing signal 2 to activate the NLRP3 inflammasome and interleukin (IL)-1 production in primary human monocytes. RIPK3 also amplifies induction of inflammatory genes such as TNF, IL6, and IL1B while suppressing IL12B. Mechanistically, RIPK3 inhibits STAT1 activation and activates PI3K-Akt-dependent and XBP1- and NRF2-mediated stress responses to regulate downstream genes in a dichotomous manner. These findings identify new functions for RIPK3 in modulating TLR responses and provide potential mechanisms by which RIPK3 plays roles in inflammatory diseases and suggest targeting RIPK3 and XBP1- and NRF2-mediated stress responses as therapeutic strategies to suppress inflammation while preserving the IFN response for host defense.

Spatio-temporal analysis of the sources and transformations of anthropogenic nitrogen in a highly degraded coastal basin in Southeast China.

Nitrogen transport from terrestrial to aquatic environments could cause water quality deterioration and eutrophication. By sampling in the high- and low-flow periods in a highly disturbed coastal basin of Southeast China, hydrochemical characteristics, nitrate stable isotope composition, estimation of potential nitrogen source input fluxes, and the Bayesian mixing model were combined to determine the sources and transformation of nitrogen. Nitrate was the main form of nitrogen. Nitrification, nitrate assimilation, and NH4+ volatilization were the main nitrogen transformation processes, whereas denitrification was limited due to the high flow rate and unsuitable physicochemical properties. For both sampling periods, non-point source pollution from the upper to the middle reaches was the main source of nitrogen, especially in the high-flow period. In addition to synthetic fertilizer, atmospheric deposition and sewage and manure input were also major nitrate sources in the low-flow period. Hydrological condition was the main factor determining nitrate transformation in this coastal basin, despite the high degree of urbanization and the high volume of sewage discharge in the middle to the lower reaches. The findings of this study highlight that the control of agricultural non-point contamination sources is essential to pollution and eutrophication alleviation, especially for watersheds that receive high amounts of annual precipitation.

Bone marrow Adipoq-lineage progenitors are a major cellular source of M-CSF that dominates bone marrow macrophage development, osteoclastogenesis, and bone mass.

M-CSF is a critical growth factor for myeloid lineage cells, including monocytes, macrophages, and osteoclasts. Tissue-resident macrophages in most organs rely on local M-CSF. However, it is unclear what specific cells in the bone marrow produce M-CSF to maintain myeloid homeostasis. Here, we found that Adipoq-lineage progenitors but not mature adipocytes in bone marrow or in peripheral adipose tissue, are a major cellular source of M-CSF, with these Adipoq-lineage progenitors producing M-CSF at levels much higher than those produced by osteoblast lineage cells. The Adipoq-lineage progenitors with high CSF1 expression also exist in human bone marrow. Deficiency of M-CSF in bone marrow Adipoq-lineage progenitors drastically reduces the generation of bone marrow macrophages and osteoclasts, leading to severe osteopetrosis in mice. Furthermore, the osteoporosis in ovariectomized mice can be significantly alleviated by the absence of M-CSF in bone marrow Adipoq-lineage progenitors. Our findings identify bone marrow Adipoq-lineage progenitors as a major cellular source of M-CSF in bone marrow and reveal their crucial contribution to bone marrow macrophage development, osteoclastogenesis, bone homeostasis, and pathological bone loss.

CXCL4 synergizes with TLR8 for TBK1-IRF5 activation, epigenomic remodeling and inflammatory response in human monocytes.

Regulation of endosomal Toll-like receptor (TLR) responses by the chemokine CXCL4 is implicated in inflammatory and fibrotic diseases, with CXCL4 proposed to potentiate TLR responses by binding to nucleic acid TLR ligands and facilitating their endosomal delivery. Here we report that in human monocytes/macrophages, CXCL4 initiates signaling cascades and downstream epigenomic reprogramming that change the profile of the TLR8 response by selectively amplifying inflammatory gene transcription and interleukin (IL)-1ß production, while partially attenuating the interferon response. Mechanistically, costimulation by CXCL4 and TLR8 synergistically activates TBK1 and IKKε, repurposes these kinases towards an inflammatory response via coupling with IRF5, and activates the NLRP3 inflammasome. CXCL4 signaling, in a cooperative and synergistic manner with TLR8, induces chromatin remodeling and activates de novo enhancers associated with inflammatory genes. Our findings thus identify new regulatory mechanisms of TLR responses relevant for cytokine storm, and suggest targeting the TBK1-IKKε-IRF5 axis may be beneficial in inflammatory diseases.

Determining the origin and fate of nitrate in the Nanyang Basin, Central China, using environmental isotopes and the Bayesian mixing model.

Identifying sources of nitrate contamination has been a long-term challenge in areas with different land uses. We investigated the biogeochemical processes and quantified the contribution of potential nitrate sources in the Nanyang Basin, the source area of the South to North Water Diversion Project in China. Hydrogeochemical characteristics, the dual-isotope method (δ15N-NO3- and δ18O-NO3-), and the Bayesian mixing model (SIAR) were combined. The results for 160 samples indicated that mean nitrate concentrations of residential area (162.83 mg L-1) and farmland (75.71 mg L-1) were higher compared with those of surface water (16.15 mg L-1) and forest (36.25 mg L-1). Hydrochemical facies and molar ratios of major ions indicated that the natural environment was greatly impacted by anthropogenic activities. Nitrification, ammonium volatilization, and mixing effects were the dominant processes in nitrogen transformation. The contributions of different sources to nitrate contamination were 45.41%, 35.81%, 17.87%, and 0.91% for sewage and manure, soil organic nitrogen, synthetic fertilizer, and atmospheric deposition, respectively. Undeveloped infrastructure and sewage disposal in rural areas were the main causes of nitrate contamination. Our results provide a theoretical basis for the development of measures to guarantee long-term water supply of the South to North Water Diversion Project.

The Cytokine TNF Promotes Transcription Factor SREBP Activity and Binding to Inflammatory Genes to Activate Macrophages and Limit Tissue Repair.

Cytokine tumor necrosis factor (TNF)-mediated macrophage polarization is important for inflammatory disease pathogenesis, but the mechanisms regulating polarization are not clear. We performed transcriptomic and epigenomic analysis of the TNF response in primary human macrophages and revealed late-phase activation of SREBP2, the master regulator of cholesterol biosynthesis genes. TNF stimulation extended the genomic profile of SREBP2 occupancy to include binding to and activation of inflammatory and interferon response genes independently of its functions in sterol metabolism. Genetic ablation of SREBP function shifted the balance of macrophage polarization from an inflammatory to a reparative phenotype in peritonitis and skin wound healing models. Genetic ablation of SREBP activity in myeloid cells or topical pharmacological inhibition of SREBP improved skin wound healing under homeostatic and chronic inflammatory conditions. Our results identify a function and mechanism of action for SREBPs in augmenting TNF-induced macrophage activation and inflammation and open therapeutic avenues for promoting wound repair.

TNF-induced inflammatory genes escape repression in fibroblast-like synoviocytes: transcriptomic and epigenomic analysis.

OBJECTIVE: We investigated genome-wide changes in gene expression and chromatin remodelling induced by tumour necrosis factor (TNF) in fibroblast-like synoviocytes (FLS) and macrophages to better understand the contribution of FLS to the pathogenesis of rheumatoid arthritis (RA). METHODS: FLS were purified from patients with RA and CD14+ human monocyte-derived macrophages were obtained from healthy donors. RNA-sequencing, histone 3 lysine 27 acetylation (H3K27ac), chromatin immunoprecipitation-sequencing (ChIP-seq) and assay for transposable accessible chromatin by high throughput sequencing (ATAC-seq) were performed in control and TNF-stimulated cells. RESULTS: We discovered 280 TNF-inducible arthritogenic genes which are transiently expressed and subsequently repressed in macrophages, but in RA, FLS are expressed with prolonged kinetics that parallel the unremitting kinetics of RA synovitis. 80 out of these 280 fibroblast-sustained genes (FSGs) that escape repression in FLS relative to macrophages were desensitised (tolerised) in macrophages. Epigenomic analysis revealed persistent H3K27 acetylation and increased chromatin accessibility in regulatory elements associated with FSGs in TNF-stimulated FLS. The accessible regulatory elements of FSGs were enriched in binding motifs for nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), interferon-regulatory factors (IRFs) and activating protein-1 (AP-1). Inhibition of bromodomain and extra-terminal motif (BET) proteins, which interact with histone acetylation, suppressed sustained induction of FSGs by TNF. CONCLUSION: Our genome-wide analysis has identified the escape of genes from transcriptional repression in FLS as a novel mechanism potentially contributing to the chronic unremitting synovitis observed in RA. Our finding that TNF induces sustained chromatin activation in regulatory elements of the genes that escape repression in RA FLS suggests that altering or targeting chromatin states in FLS (eg, with inhibitors of BET proteins) is an attractive therapeutic strategy.

Hypoxia-Sensitive COMMD1 Integrates Signaling and Cellular Metabolism in Human Macrophages and Suppresses Osteoclastogenesis.

Hypoxia augments inflammatory responses and osteoclastogenesis by incompletely understood mechanisms. We identified COMMD1 as a cell-intrinsic negative regulator of osteoclastogenesis that is suppressed by hypoxia. In human macrophages, COMMD1 restrained induction of NF-κB signaling and a transcription factor E2F1-dependent metabolic pathway by the cytokine RANKL. Downregulation of COMMD1 protein expression by hypoxia augmented RANKL-induced expression of inflammatory and E2F1 target genes and downstream osteoclastogenesis. E2F1 targets included glycolysis and metabolic genes including CKB that enabled cells to meet metabolic demands in challenging environments, as well as inflammatory cytokine-driven target genes. Expression quantitative trait locus analysis linked increased COMMD1 expression with decreased bone erosion in rheumatoid arthritis. Myeloid deletion of Commd1 resulted in increased osteoclastogenesis in arthritis and inflammatory osteolysis models. These results identify COMMD1 and an E2F-metabolic pathway as key regulators of osteoclastogenic responses under pathological inflammatory conditions and provide a mechanism by which hypoxia augments inflammation and bone destruction.

Control of lupus nephritis by changes of gut microbiota.

BACKGROUND: Systemic lupus erythematosus, characterized by persistent inflammation, is a complex autoimmune disorder with no known cure. Immunosuppressants used in treatment put patients at a higher risk of infections. New knowledge of disease modulators, such as symbiotic bacteria, can enable fine-tuning of parts of the immune system, rather than suppressing it altogether. RESULTS: Dysbiosis of gut microbiota promotes autoimmune disorders that damage extraintestinal organs. Here we report a role of gut microbiota in the pathogenesis of renal dysfunction in lupus. Using a classical model of lupus nephritis, MRL/lpr, we found a marked depletion of Lactobacillales in the gut microbiota. Increasing Lactobacillales in the gut improved renal function of these mice and prolonged their survival. We used a mixture of 5 Lactobacillus strains (Lactobacillus oris, Lactobacillus rhamnosus, Lactobacillus reuteri, Lactobacillus johnsonii, and Lactobacillus gasseri), but L. reuteri and an uncultured Lactobacillus sp. accounted for most of the observed effects. Further studies revealed that MRL/lpr mice possessed a "leaky" gut, which was reversed by increased Lactobacillus colonization. Lactobacillus treatment contributed to an anti-inflammatory environment by decreasing IL-6 and increasing IL-10 production in the gut. In the circulation, Lactobacillus treatment increased IL-10 and decreased IgG2a that is considered to be a major immune deposit in the kidney of MRL/lpr mice. Inside the kidney, Lactobacillus treatment also skewed the Treg-Th17 balance towards a Treg phenotype. These beneficial effects were present in female and castrated male mice, but not in intact males, suggesting that the gut microbiota controls lupus nephritis in a sex hormone-dependent manner. CONCLUSIONS: This work demonstrates essential mechanisms on how changes of the gut microbiota regulate lupus-associated immune responses in mice. Future studies are warranted to determine if these results can be replicated in human subjects.

Tollip Deficiency Alters Atherosclerosis and Steatosis by Disrupting Lipophagy.

BACKGROUND: Compromised lipophagy with unknown mechanisms may be critically involved in the intracellular accumulation of lipids, contributing to elevated atherosclerosis and liver steatosis. We hypothesize that toll-interacting protein (Tollip), a key innate immune molecule involved in the fusion of autolysosome, may play a significant role in lipophagy and modulate lipid accumulation during the pathogenesis of atherosclerosis and liver steatosis. METHODS AND RESULTS: By comparing mice fed with either a Western high-fat diet or a regular chow diet, we observed that both atherosclerosis and liver steatosis were aggravated in apolipoprotein E-deficient (ApoE-/-)/Tollip-/- mice as compared with ApoE-/- mice. Through electron microscopy analyses, we observed compromised fusion of lipid droplets with lysosomes within aortic macrophages as well as liver hepatocytes from ApoE-/-/Tollip-/- mice as compared with ApoE-/- mice. As a molecular indicator for disrupted lysosome fusion, the levels of p62 were significantly elevated in aortic and liver tissues from ApoE-/-/Tollip-/- mice. Molecules involved in facilitating lipophagy completion such as Ras-related protein 7 and gamma-aminobutyric acid receptor-associated protein were reduced in ApoE-/-/Tollip-/- mice as compared with ApoE-/- mice. Intriguingly, ApoE-/-/Tollip-/- mice had reduced circulating levels of inflammatory cytokines such as tumor necrosis factor-α and increased levels of transforming growth factor-ß. The reduced inflammation due to Tollip deficiency is consistent with a stable atherosclerotic plaque phenotype with increased levels of plaque collagen and smooth muscle cells in ApoE-/-/Tollip-/- mice. CONCLUSIONS: Tollip deficiency selectively leads to enlarged yet stable atherosclerotic plaques, increased circulating lipids, liver steatosis, and reduced inflammation. Compromised lipophagy and reduced expression of inflammatory mediators due to Tollip deficiency may be the underlying causes. Our data suggest that lipid accumulation and inflammation may be intertwined yet independent processes during the progression of atherosclerosis and steatosis.

Toll-interacting protein deficiency promotes neurodegeneration via impeding autophagy completion in high-fat diet-fed ApoE-/- mouse model.

The excessive accumulation of specific cellular proteins or autophagic vacuoles (AVs) within neurons is a pathologic hallmark of neurodegenerative diseases. Constitutive autophagy in neurons prevents abnormal intracellular protein aggregation and is critical for maintaining cell survival. Since our previous study showed that Toll-interacting protein (Tollip)-deficient macrophages had constitutive disruption of endosome-lysosome fusion, we hypothesize that Tollip deficiency may also promote neuron death via blockage of autophagy completion. Indeed, we observed significantly higher levels of neuron death in the brain regions of cerebral cortex, hippocampus, and cerebellum from ApoE-/-/Tollip-/- mice as compared to ApoE-/- mice fed with high fat diet (HFD). We further documented diminished density of neurons and increased ratios of TUNEL positive cells in the hippocampus of ApoE-/-/Tollip-/- mice. The ultrastructural electron microscopy analyses revealed neuron cell shrinkage as well as loss of intracellular structure in brain tissues from ApoE-/-/Tollip-/- mice. There was dramatic accumulation of autophagosomes in the cytoplasm, elevated accumulation of ß-amyloid and α-synuclein, and increased levels of p62 and Parkin in the brain tissues from ApoE-/-/Tollip-/- mice as compared to ApoE-/- mice. Our data suggest that Tollip may play a crucial role in sustaining neuron health by facilitating the completion of autophagy, and that Tollip-deficiency may accelerate neuron death related to neurological disease such as Alzheimer's disease.

Molecular Mechanisms That Underlie the Dynamic Adaptation of Innate Monocyte Memory to Varying Stimulant Strength of TLR Ligands.

In adaptation to rising stimulant strength, innate monocytes can be dynamically programed to preferentially express either pro- or anti-inflammatory mediators. Such dynamic innate adaptation or programing may bear profound relevance in host health and disease. However, molecular mechanisms that govern innate adaptation to varying strength of stimulants are not well understood. Using lipopolysaccharide (LPS), the model stimulant of toll-like-receptor 4 (TLR4), we reported that the expressions of pro-inflammatory mediators are preferentially sustained in monocytes adapted by lower doses of LPS, and suppressed/tolerized in monocytes adapted by higher doses of LPS. Mechanistically, monocytes adapted by super-low dose LPS exhibited higher levels of transcription factor, interferon regulatory factor 5 (IRF5), and reduced levels of transcriptional modulator B lymphocyte-induced maturation protein-1 (Blimp-1). Intriguingly, the inflammatory monocyte adaptation by super-low dose LPS is dependent upon TRAM/TRIF but not MyD88. Similar to LPS, we also observed biphasic inflammatory adaptation and tolerance in monocytes challenged with varying dosages of TLR7 agonist. In sharp contrast, rising doses of TLR3 agonist preferentially caused inflammatory adaptation without inducing tolerance. At the molecular level, the differential regulation of IRF5 and Blimp-1 coincides with unique monocyte adaptation dynamics by TLR4/7 and TLR3 agonists. Our study provides novel clue toward the understanding of monocyte adaptation and memory toward distinct TLR ligands.

The persistence of low-grade inflammatory monocytes contributes to aggravated atherosclerosis.

Sustained low-grade inflammation mediated by non-resolving inflammatory monocytes has long been suspected in the pathogenesis of atherosclerosis; however, the molecular mechanisms responsible for the sustainment of non-resolving inflammatory monocytes during atherosclerosis are poorly understood. Here we observe that subclinical endotoxemia, often seen in humans with chronic inflammation, aggravates murine atherosclerosis through programming monocytes into a non-resolving inflammatory state with elevated Ly6C, CCR5, MCP-1 and reduced SR-B1. The sustainment of inflammatory monocytes is due to the disruption of homeostatic tolerance through the elevation of miR-24 and reduction of the key negative-feedback regulator IRAK-M. miR-24 reduces the levels of Smad4 required for the expression of IRAK-M and also downregulates key lipid-processing molecule SR-B1. IRAK-M deficiency in turn leads to elevated miR-24 levels, sustains disruption of monocyte homeostasis and aggravates atherosclerosis. Our data define an integrated feedback circuit in monocytes and its disruption may lead to non-resolving low-grade inflammation conducive to atherosclerosis.

Deficiency in Toll-interacting protein (Tollip) skews inflamed yet incompetent innate leukocytes in vivo during DSS-induced septic colitis.

Functionally compromised neutrophils contribute to adverse clinical outcomes in patients with severe inflammation and injury such as colitis and sepsis. However, the ontogeny of dysfunctional neutrophil during septic colitis remain poorly understood. We report that the dysfunctional neutrophil may be derived by the suppression of Toll-interacting-protein (Tollip). We observed that Tollip deficient neutrophils had compromised migratory capacity toward bacterial product fMLF due to reduced activity of AKT and reduction of FPR2, reduced potential to generate bacterial-killing neutrophil extra-cellular trap (NET), and compromised bacterial killing activity. On the other hand, Tollip deficient neutrophils had elevated levels of CCR5, responsible for their homing to sterile inflamed tissues. The inflamed and incompetent neutrophil phenotype was also observed in vivo in Tollip deficient mice subjected to DSS-induced colitis. We observed that TUDCA, a compound capable of restoring Tollip cellular function, can potently alleviate the severity of DSS-induced colitis. In humans, we observed significantly reduced Tollip levels in peripheral blood collected from human colitis patients as compared to blood samples from healthy donors. Collectively, our data reveal a novel mechanism in Tollip alteration that underlies the inflamed and incompetent polarization of neutrophils leading to severe outcomes of colitis.

Tissue-resident dendritic cells and diseases involving dendritic cell malfunction.

Dendritic cells (DCs) control immune responses and are central to the development of immune memory and tolerance. DCs initiate and orchestrate immune responses in a manner that depends on signals they receive from microbes and cellular environment. Although DCs consist mainly of bone marrow-derived and resident populations, a third tissue-derived population resides the spleen and lymph nodes (LNs), different subsets of tissue-derived DCs have been identified in the blood, spleen, lymph nodes, skin, lung, liver, gut and kidney to maintain the tolerance and control immune responses. Tissue-resident DCs express different receptors for microbe-associated molecular patterns (MAMPs) and damage-associated molecular patterns (DAMPs), which were activated to promote the production of pro- or anti-inflammatory cytokines. Malfunction of DCs contributes to diseases such as autoimmunity, allergy, and cancer. It is therefore important to update the knowledge about resident DC subsets and diseases associated with DC malfunction.

Subclinical-Dose Endotoxin Sustains Low-Grade Inflammation and Exacerbates Steatohepatitis in High-Fat Diet-Fed Mice.

Subclinical circulating bacterial endotoxin LPS has been implicated as an important cofactor in the development and progression of nonalcoholic steatohepatitis, but the underlying mechanisms remain unclear. In this study, we demonstrated that 4-wk injection with superlow-dose LPS significantly promoted neutrophil infiltration and accelerated nonalcoholic steatohepatitis progression, including exacerbated macrovesicular steatosis, inflammation, and hepatocyte ballooning in high-fat diet-fed apolipoprotein E knockout mice. This effect could sustain for a month after stoppage of LPS injection. LPS also significantly increased numbers of apoptotic nuclei in hepatocytes and expressions of proapoptotic regulators. Moreover, LPS sustained the low-grade activation of p38 MAPK and inhibited the expression of the upstream MAPK phosphatase 7. By applying selective inhibitors, we demonstrated that the activation of p38 MAPKs is required for neutrophil migration induced by superlow-dose LPS in vitro. Together, these data suggest that superlow-dose LPS may sustain the low-grade activation of p38 MAPKs and neutrophil infiltration, leading to the exacerbation of steatohepatitis.

Dynamic modulation of innate immunity programming and memory.

Recent progress harkens back to the old theme of immune memory, except this time in the area of innate immunity, to which traditional paradigm only prescribes a rudimentary first-line defense function with no memory. However, both in vitro and in vivo studies reveal that innate leukocytes may adopt distinct activation states such as priming, tolerance, and exhaustion, depending upon the history of prior challenges. The dynamic programming and potential memory of innate leukocytes may have far-reaching consequences in health and disease. This review aims to provide some salient features of innate programing and memory, patho-physiological consequences, underlying mechanisms, and current pressing issues.