Pin1 maintains the effector program of pathogenic Th17 cells in autoimmune neuroinflammation.

Th17 cells mediated immune response is the basis of a variety of autoimmune diseases, including multiple sclerosis and its mouse model of immune aspects, experimental autoimmune encephalomyelitis (EAE). The gene network that drives both the development of Th17 and the expression of its effector program is dependent on the transcription factor RORγt. In this report, we showed that Peptidylprolyl Cis/Trans Isomerase, NIMA-Interacting 1 (Pin1) formed a complex with RORγt, and enhanced its transactivation activity, thus sustained the expression of the effector genes as well as RORγt in the EAE-pathogenic Th17 cells. We first found out that PIN1 was highly expressed in the samples from patients of multiple sclerosis, and the expression of Pin1 by the infiltrating lymphocytes in the central nerve system of EAE mice was elevated as well. An array of experiments with transgenic mouse models, cellular and molecular assays was included in the study to elucidate the role of Pin1 in the pathology of EAE. It turned out that Pin1 promoted the activation and maintained the effector program of EAE-pathogenic Th17 cells in the inflammation foci, but had little effect on the priming of Th17 cells in the draining lymph nodes. Mechanistically, Pin1 stabilized the phosphorylation of STAT3 induced by proinflammatory stimuli, and interacted with STAT3 in the nucleus of Th17 cells, which resulted in the increased expression of Rorc. Moreover, Pin1 formed a complex with RORγt, and enhanced the transactivation of RORγt to the +11 kb enhancer of Rorc, which enforced and maintained the expression of both Rorc and the effector program of pathogenic Th17 cells in EAE. Finally, the inhibition of Pin1, by genetic knockdown or by small molecule inhibitor, deceased the population of Th17 cells and the neuroinflammation, and alleviated the symptoms of EAE. These findings suggest that Pin1 is a potential therapeutic target for MS and other autoimmune inflammatory diseases.

Anlotinib attenuates experimental autoimmune encephalomyelitis mice model of multiple sclerosis via modulating the differentiation of Th17 and Treg cells.

BACKGROUND: In multiple sclerosis (MS), the imbalance between T helper (Th)-17 cells and regulatory T (Treg) cells are critical in autoimmune central nervous system (CNS) inflammation and demyelination. Experimental autoimmune encephalomyelitis (EAE) is an established mouse MS model and simulates MS at diverse levels. OBJECTIVES: This study aims at investigating the impact of anlotinib on the clinical severity of EAE and CD4+ T cell differentiation. MATERIALS AND METHODS: EAE-induced mice were treated with water (control) or 6 mg/kg anlotinib by gavage daily. At the peak of EAE, histopathological examination and flow cytometry analysis of CNS-infiltrating CD4+ T cells were performed. In vitro differentiation of CD4+ T cells under different conditions was detected by flow cytometry and quantitative real-time PCR. Finally, the impacts of anlotinib on the phosphorylation of signal transducer and activator of transcription 3 (STAT3) and the transcription levels of key genes involved in Th17 and Treg differentiation were tested. RESULTS: Anlotinib attenuated the clinical severity of EAE and changed the frequencies of CNS-infiltrating CD4+ T cell subsets. Anlotinib inhibited the differentiation of Th17 cells in vitro, decreased the phosphorylation of STAT3, and reduced the expression of Rorc. Anlotinib promoted the differentiation of Treg cells and upregulated the expression levels of CD39 and CD73. DISCUSSION AND CONCLUSIONS: Anlotinib alleviated the symptoms of EAE via inhibiting the Th17 cell differentiation and promoting Treg cell differentiation. Our study provides new opportunities for the exploitation of anlotinib as a therapeutic agent for the treatment of MS.

Parthenolide regulates oxidative stress-induced mitophagy and suppresses apoptosis through p53 signaling pathway in C2C12 myoblasts.

Muscle redox disturbances and oxidative stress have emerged as a common pathogenetic mechanism and potential therapeutic intervention in some muscle diseases. Parthenolide (PTL), a sesquiterpene lactone found in large amounts in the leaves of feverfew, possesses anti-inflammatory, anti-migraine, and anticancer properties. Although PTL was reported to alleviate cancer cachexia and improve skeletal muscle characteristics in a cancer cachexia model, its actions on oxidative stress-induced damage in C2C12 myoblasts have not been reported and the regulatory mechanisms have not yet been defined. In our study, PTL attenuated H2 O2 -induced growth inhibition and morphological changes. Furthermore, PTL exhibited scavenging activity against reactive oxygen species and protected C2C12 cells from apoptosis in response to H2 O2 . Meanwhile, PTL suppressed collapse of the mitochondrial membrane potential, thereby contributing to normalizing H2 O2 -induced autophagy flux and mitophagy, correlating with inhibiting degradation of mitochondrial marker protein TIM23, the increase in LC3-II expression and the reduction of mitochondria DNA. Besides its protective effect on mitochondria, PTL also prevented H2 O2 -induced lysosomes damage in C2C12 cells. In addition, the phosphorylation of p53, cathepsin B, and Bax/Bcl-2 protein levels, and the translocation of Bax from the cytosol to mitochondria induced by H2 O2 in C2C12 cells was significantly reduced by PTL. In conclusion, PTL modulates oxidative stress-induced mitophagy and protects C2C12 myoblasts against apoptosis, suggesting a potential protective effect against oxidative stress-associated skeletal muscle diseases.

Structural basis for DNA recognition by STAT6.

STAT6 participates in classical IL-4/IL-13 signaling and stimulator of interferon genes-mediated antiviral innate immune responses. Aberrations in STAT6-mediated signaling are linked to development of asthma and diseases of the immune system. In addition, STAT6 remains constitutively active in multiple types of cancer. Therefore, targeting STAT6 is an attractive proposition for treating related diseases. Although a lot is known about the role of STAT6 in transcriptional regulation, molecular details on how STAT6 recognizes and binds specific segments of DNA to exert its function are not clearly understood. Here, we report the crystal structures of a homodimer of phosphorylated STAT6 core fragment (STAT6CF) alone and bound with the N3 and N4 DNA binding site. Analysis of the structures reveals that STAT6 undergoes a dramatic conformational change on DNA binding, which was further validated by performing molecular dynamics simulation studies and small angle X-ray scattering analysis. Our data show that a larger angle at the intersection where the two protomers of STAT meet and the presence of a unique residue, H415, in the DNA-binding domain play important roles in discrimination of the N4 site DNA from the N3 site by STAT6. H415N mutation of STAT6CF decreased affinity of the protein for the N4 site DNA, but increased its affinity for N3 site DNA, both in vitro and in vivo. Results of our structure-function studies on STAT6 shed light on mechanism of DNA recognition by STATs in general and explain the reasons underlying STAT6's preference for N4 site DNA over N3.

MicroRNA-181c promotes Th17 cell differentiation and mediates experimental autoimmune encephalomyelitis.

Among T helper (Th) cell subsets differentiated from naive CD4+ T cells, IL-17-producing Th17 cells are closely associated with the pathogenesis of autoimmune diseases, including multiple sclerosis (MS) and the MS animal model, experimental autoimmune encephalomyelitis (EAE). The modulation of Th17 differentiation offers a potential avenue for treatment. Although a series of microRNAs (miRNAs) that modulate autoimmune disease development have been reported, further studies on miRNA roles in Th17 differentiation and MS pathogenesis are still warranted. Here, we demonstrated that mice with miR-181c knockdown presented with delayed EAE and slowed disease progression, along with a decreased Th17 cell population. We also found that miR-181c was a Th17 cell-associated miRNA and that Smad7, a negative regulator of TGF-ß signaling, was a potential target of miR-181c. miR-181c knockdown rendered T cells less sensitive to TGF-ß-induced Smad2/3, enhancing the expression of IL-2 which has been reported to inhibit Th17 cell differentiation. Moreover, through the analysis of published miRNA expression profiles from the Gene Expression Omnibus database, increased miR-181c levels were found in peripheral blood from MS patients. Our results identified a novel miRNA that promotes Th17 cell differentiation and autoimmunity, thus miR-181c may serve as a potential treatment target in patients with MS.

miR-142-5p regulates tumor cell PD-L1 expression and enhances anti-tumor immunity.

Cancer immunotherapy has many great achievements in recent years. One of the most promising cancer immunotherapies is PD-1/PD-L1 pathway blockade. miRNAs (MicroRNAs) belongs to small noncoding RNA and can regulate gene expression by binding to the 3'UTR. Many miRNAs can inhibit cancer growth by regulating the PD-L1 expression in cancer cells. Herein, we firstly found that PD-L1 could be the target of miR-142-5p by using bioinformatics methods, then we conduct luciferase activity assay, RT-PCR and western blot experiments to demonstrate that miR-142-5p can regulate PD-L1 expression by binding to its 3'UTR. And in vivo experiments certified that miR-142-5p overexpression can inhibit pancreatic cancer growth. Flow cytometry and RT-PCR experiment demonstrated that miR-142-5p overexpression on tumor cells inhibits the expression of PD-L1 on tumor cells which result in the increase of CD4+ T lymphocytes and CD8+ T lymphocytes, the decrease of PD-1+ T lymphocytes and increase of IFN-γ and TNF-α. So, miR-142-5p overexpression can enhance anti-tumor immunity by blocking PD-L1/PD-1 pathway. Our results identify a novel mechanism by which PD-L1 is regulated by miR-142-5p and overexpression of miR-142-5p could enhance the anti-tumor immunity.

IL-23 Promotes Myocardial I/R Injury by Increasing the Inflammatory Responses and Oxidative Stress Reactions.

BACKGROUND/AIMS: Inflammation and oxidative stress play an important role in myocardial ischemia and reperfusion (I/R) injury. We hypothesized that IL-23, a pro-inflammatory cytokine, could promote myocardial I/R injury by increasing the inflammatory response and oxidative stress. METHODS: Male Sprague-Dawley rats were randomly assigned into sham operated control (SO) group, ischemia and reperfusion (I/R) group, (IL-23 + I/R) group and (anti-IL-23 + I/R) group. At 4 h after reperfusion, the serum concentration of lactate dehydrogenase (LDH), creatine kinase (CK) and the tissue MDA concentration and SOD activity were measured. The infarcte size was measured by TTC staining. Apoptosis in heart sections were measured by TUNEL staining. The expression of HMGB1 and IL-17A were detected by Western Blotting and the expression of TNF-α and IL-6 were detected by Elisa. RESULTS: After 4 h reperfusion, compared with the I/R group, IL-23 significantly increased the infarct size, the apoptosis of cardiomyocytes and the levels of LDH and CK (all P < 0.05). Meanwhile, IL-23 significantly increased the expression of eIL-17A, TNF-α and IL-6 and enhanced both the increase of the MDA level and the decrease of the SOD level induced by I/R (all P<0.05). IL-23 had no effect on the expression of HMGB1 (p > 0.05). All these effects were abolished by anti-IL-23 administration. CONCLUSION: The present study suggested that IL-23 may promote myocardial I/R injury by increasing the inflammatory responses and oxidative stress reaction.

Mesenchymal stem cells and their secreted molecules predominantly ameliorate fulminant hepatic failure and chronic liver fibrosis in mice respectively.

BACKGROUND: Orthotopic liver transplantation is the only effective treatment for liver failure but limited with shortage of available donor organs. Recent studies show promising results of mesenchymal stem cells (MSCs)-based therapies. METHODS: We systematically investigate the therapeutic effects of MSCs or MSC-conditioned medium (MSC-CM) in ameliorating fulminant hepatic failure (FHF) and chronic liver fibrosis in mice. In addition, extensive flow cytometry analysis of spleens from vehicle and MSC- and MSC-CM-treated mice was applied to reveal the alteration of inflammatory state. RESULTS: In FHF model, MSCs treatment reduced remarkably the death incidents; the analysis of gross histopathology showed that control livers were soft and shrunken with extensive extravasated blood, which was gradually reduced at later time points, while MSC-treated livers showed gross pathological changes, even 24 h after MSC infusion, and hematoxylin and eosin staining revealed dramatical hepatocellular death with cytoplasmic vacuolization suppressed by MSCs treatment; flow cytometry analysis of total lymphocytes showed that macrophages (F4/80) infiltrated into control livers more than MSC-treated livers; by contrast, MSC-CM partially ameliorates FHF. In chronic liver injury model, MSC and MSC-CM both suppressed fibrogenesis and necroinflammatory, and the later was better; activation of hepatic stellate cells (α-SMA) was inhibited; glycogen synthesis and storage (indicated by periodic acid-Schiff -staining) was improved; liver regeneration (Ki67) was promoted while liver apoptosis (TUNEL) was reduced. In the in vitro, MSCs promote macrophage line RAW264.7 apoptosis and MSC-CM promotes apoptosis and inhibits proliferation of HSC line LX-2. We also found that MSCs and MSC-CM could improve spleen; MSC-CM increased levels of Th2 and Treg cells, and reduced levels of Th17 cells, whereas levels of Th1 cells were unchanged; comparatively, MSC treatment did not affect Th17 and Treg cells and only slightly alters inflammatory state; MSC and MSC-CM treatment both substantially down-regulated macrophages in the spleens. CONCLUSION: Both MSCs and MSC-CM exert therapeutic effects by acting on various key cells during the pathogenesis of FHF and chronic fibrosis, stimulating hepatocyte proliferation and suppressing apoptosis, down-regulating infiltrating macrophages, converting CD4(+) T lymphocyte system into an anti-inflammatory state, and facilitating hepatic stellate cell death.

Comprehensive quantifications of tumour microenvironment to predict the responsiveness to immunotherapy and prognosis for paediatric neuroblastomas.

Treatment strategies for paediatric neuroblastoma as well as many other cancers are limited by the unfavourable tumour microenvironment (TME). In this study, the TMEs of neuroblastoma were grouped by their genetic signatures into four distinct subtypes: immune enriched, immune desert, non-proliferative and fibrotic. An Immune Score and a Proliferation Score were constructed based on the molecular features of the subtypes to quantify the immune microenvironment or malignancy degree of cancer cells in neuroblastoma, respectively. The Immune Score correlated with a patient's response to immunotherapy; the Proliferation Score was an independent prognostic biomarker for neuroblastoma and proved to be more accurate than the existing clinical predictors. This double scoring system was further validated and the conserved molecular pattern associated with immune landscape and malignancy degree was confirmed. Axitinib and BI-2536 were confirmed as candidate drugs for neuroblastoma by the double scoring system. Both in vivo and in vitro experiments demonstrated that axitinib-induced pyroptosis of neuroblastoma cells activated anti-tumour immunity and inhibited tumour growth; BI-2536 induced cell cycle arrest at the S phase in neuroblastoma cells. The comprehensive double scoring system of neuroblastoma may predict prognosis and screen for therapeutic strategies which could provide personalized treatments.

The multifunctional human p100 protein 'hooks' methylated ligands.

The human p100 protein is a vital transcription regulator that increases gene transcription by forming a physical bridge between promoter-specific activators and the basal transcription machinery. Here we demonstrate that the tudor and SN (TSN) domain of p100 interacts with U small nuclear ribonucleoprotein (snRNP) complexes, suggesting a role for p100 in the processing of precursor messenger RNA. We determined the crystal structure of the p100 TSN domain to delineate the molecular basis of p100's proposed functions. The interdigitated structure resembles a hook, with a hinge controlling the movement and orientation of the hook. Our studies suggest that a conserved aromatic cage hooks methyl groups of snRNPs and anchors p100 to the spliceosome. These structural insights partly explain the distinct roles of p100 in transcription and splicing.

Using a Gene Network of Pyroptosis to Quantify the Responses to Immunotherapy and Prognosis for Neuroblastoma Patients.

Background: Pyroptosis, as an inflammatory form of cell death, is involved in many physiological and pathological processes. Neuroblastoma is the most common extra-cranial solid tumor in children. In this study, the relationship between pyroptosis and tumor microenvironment in neuroblastoma was systematically studied. Methods: We integrated four datasets of neuroblastomas. Through robust clustering of the mRNA expression profiles of 24 pyroptosis-related genes, a total of three pyroptosis patterns were identified. We then constructed a novel scoring method named as pyroscore to quantify the level of pyroptosis in neuroblastoma. Multi-omics data and single-cell RNA sequencing were used to accurately and comprehensively evaluate the effectiveness of pyroscore. Clinical data sets were used to evaluate the use of pyroscore to predict the responsiveness of immune checkpoint treatment. Results: High pyroscore was associated with good prognosis, immune activation, and increased response to checkpoint blockade immunotherapy. Multivariate Cox analysis revealed that the pyroscore was an independent prognostic biomarker and could increase the accuracy of clinical prediction models. Etoposide, a drug picked up by our analysis, could increase the sensitivity of neuroblastoma cells to pyroptosis. External verification using four cohorts of patients who had received immunotherapy showed that high pyroscore was significantly associated with immunotherapy treatment benefit. Conclusions: Taken together, this study revealed that pyroptosis-related gene network could quantify the response of neuroblastoma to immune checkpoint blockade therapy and prognosis, and it may be helpful for clinical practitioners to choose treatment strategies for neuroblastoma patients.

Pin1 Promotes NLRP3 Inflammasome Activation by Phosphorylation of p38 MAPK Pathway in Septic Shock.

Pin1 is the only known peptidyl-prolyl cis-trans isomerase (PPIase) that can specifically recognize and isomerize the phosphorylated Serine/Threonine-Proline (pSer/Thr-Pro) motif, change the conformation of proteins through protein phosphorylation, thus regulate various cellular processes in the body. Pin1 plays an important role in cancer, Alzheimer's disease, and autoimmune diseases. However, the specific mechanism of Pin1 regulation in LPS-induced septic shock is unclear. Here, we found that lack of Pin1 reduced shock mortality and organ damage in mice, and NLRP3 inflammasome activation also was reduced in this process. We further confirmed that Pin1 can affect the expression of NLRP3, ASC, Caspase1, and this process can be regulated through the p38 MAPK pathway. We analyzed that p38 MAPK signaling pathway was highly expressed in septic shock and showed a positive correlation with Pin1 in the Gene Expression Omnibus database. We found that Pin1 could affect the phosphorylation of p38 MAPK, have no obvious difference in extracellular signal-regulated kinases (ERK) and Jun-amino-terminal kinase (JNK) signaling. We further found that Pin1 and p-p38 MAPK interacted, but not directly. In addition, Pin1 deficiency inhibited the cleavage of gasdermin D (GSDMD) and promoted the death of macrophages with LPS treatment, and reduced secretion of inflammatory cytokines including IL-1ß and IL-18. In general, our results suggest that Pin1 regulates the NLRP3 inflammasome activation by p38 MAPK signaling pathway in macrophages. Thus, Pin1 may be a potential target for the treatment of inflammatory diseases such as septic shock.

Loss of fragile site-associated tumor suppressor promotes antitumor immunity via macrophage polarization.

Common fragile sites (CFSs) are specific breakage-prone genomic regions and are present frequently in cancer cells. The (E2-independent) E3 ubiquitin-conjugating enzyme FATS (fragile site-associated tumor suppressor) has antitumor activity in cancer cells, but the function of FATS in immune cells is unknown. Here, we report a function of FATS in tumor development via regulation of tumor immunity. Fats-/- mice show reduced subcutaneous B16 melanoma and H7 pancreatic tumor growth compared with WT controls. The reduced tumor growth in Fats-/- mice is macrophage dependent and is associated with a phenotypic shift of macrophages within the tumor from tumor-promoting M2-like to antitumor M1-like macrophages. In addition, FATS deficiency promotes M1 polarization by stimulating and prolonging NF-κB activation by disrupting NF-κB/IκBα negative feedback loops and indirectly enhances both CD4+ T helper type 1 (Th1) and cytotoxic T lymphocyte (CTL) adaptive immune responses to promote tumor regression. Notably, transfer of Fats-/- macrophages protects mice against B16 melanoma. Together, these data suggest that FATS functions as an immune regulator and is a potential target in cancer immunotherapy.

Effect of temperature treatment on fruit quality and immunoregulation of Satsuma (Citrus unshiu Marc.) during storage.

Satsuma (Citrus unshiu Marc.) is rich in high levels of nutrients and popular for its unique flavor, but the consumption of satsuma is limited by some adverse reactions in human body. Previous studies have mainly focused on the effects of storage temperature on the postharvest quality of satsumas, and little attention has paid to the effect of postharvest satsumas on human body immunoregulation. The purpose of this study was to explore the differences in fruit quality, and the effect of satsuma fruits stored at different temperatures on human health. Satsumas stored at low temperature (5.8°C, LT) and room temperature (23 ± 2°C, RT) for 60 days were sampled every 10 days to measure the fruit quality. Sixty volunteers were recruited for the oral stimulation experiment of satsumas, and then the effect of satsumas on human health was examined through the immunoregulation of RAW 264.7 macrophages. The results showed that compared with RT treatment, LT treatment could delay the degradation of satsuma fruit quality. Both the results of the volunteer experiment and cell experiment indicated that postharvest temperature treatments could reduce the adverse effects of satsuma fruits on human body. These findings indicated that 10-day storage at room temperature plus subsequent storage at low temperature was the optimal treatment to maintain fruit quality and functional components of postharvest satsumas. This study provides useful information on satsuma consumption and research work from the perspective of immunoregulation evaluation.

Upregulation of microRNA-451 attenuates myocardial I/R injury by suppressing HMGB1.

Both MicroRNAs and HMGB1 took part in pathological process of myocardial I/R injury though several signaling pathways. We hypothesized that mircoRNA451 (miR-451), a group of small non-coding RNAs, could improve this injury by inhibiting HMGB1. Male SD rats were randomly distributed into 5 groups and subjected to I/R process. After 24 hours of reperfusion injury, the serum content of CK and LDH, the content of MDA in tissue and activity of SOD were detected; The infarcted areas were defined by TTC staining and Evans Blue; TUNEL staining and cleaved-Caspase 3 were used to test apoptosis; HMGB1 was detected by real-time fluorescence quantitative PCR and Western Blotting. Compared with the I/R and I/R+Ad-GFP group, upregulation of miR-451 could reduce the infarcted areas, cardiomyocytes apoptosis index, expression of cleaved-caspase 3 and content of CK and LDH significantly(P<0.05); Meanwhile, upregulation of miR-451 could also obviously inhibit HMGB1, the increase of MDA and the decrease of SOD (P<0.05). So this study revealed that upregulation of miR-451 could prevent myocardial I/R injury by suppressing HMGB1.

Endoplasmic Reticulum Stress Regulates Cardiomyocyte Apoptosis in Myocardial Fibrosis Development via PERK-Mediated Autophagy.

Endoplasmic reticulum stress (ERS) is involved in a variety of diseases. Recently, it was found that ERS induces not only apoptosis but also autophagy. Previous studies showed that inhibition of autophagy alleviates cell injury. The purpose of our study was to investigate the involvement of the R-like ER kinase (PERK) in ERS-induced autophagy in H9c2 cardiomyoblasts. To address this aim, therefore, H9c2 cells were treated with PERK agonist and inhibitor after establishment of rapamycin-induced ERS models in H9c2 cardiomyoblasts. Transmission electron microscopy and immunofluorescence staining were used to detect degrees of ERS-induced autophagy, apoptosis and myocardial fibrosis. Western blotting was employed to detect the levels of total and phosphorylated PERK, light chain 3 (LC3), P62, Caspase3, Bcl2 and Bax. Immunofluorescence staining was used to assess α-SMA density. TGF-ß induced H9c2 cardiomyoblasts time-dependently upregulated col I, col III, FN, and LC3 expressions, PERK phosphorylation and α-SMA density, and downregulated P62 level compared with control cells. Treatment with PERK agonist and inhibitor respectively increased and decreased LC3 expression, conversely in P62 level, which is consistent with effect of ERS agonists and inhibitors. And a PERK inhibitor upregulated the expressions of Caspase3 and Bax, and downregulated Bcl2 level, which developed H9c2 cardiomyoblasts. Moreover, siRNA-mediated knockdown of PERK reduced ERS mediated autophagy activity and increased cells apoptosis. On the other hand, elevated autophagy activity could downregulated PERK level. Our finding showed that PERK activity mediates upregulation of ERS-induced autophagy and regulation of cardiomyocyte apoptosis in H9c2 cardiomyoblasts.

Restoration of miR-340 controls pancreatic cancer cell CD47 expression to promote macrophage phagocytosis and enhance antitumor immunity.

BACKGROUND: Immune checkpoint blockade has emerged as a potential cancer immunotherapy. The "don't eat me" signal CD47 in cancer cells binds signal regulatory protein-α on macrophages and prevents their phagocytosis. The role of miR-340 in pancreatic ductal adenocarcinoma (PDAC), especially in tumor immunity, has not been explored. Here, we examined the clinical and biological relevance of miR-340 and the molecular pathways regulated by miR-340 in PDAC. METHODS: CD47 and miR-340 expression and the relationship with cancer patient survival were analyzed by bioinformatics. The mechanism of miR-340 action was explored through bioinformatics, luciferase reporter, qRT-PCR and western blot analyses. The effects of miR-340 on cancer cells were analyzed in terms of apoptosis, proliferation, migration and phagocytosis by macrophages. In vivo tumorigenesis was studied in orthotopic and subcutaneous models, and immune cells from the peripheral and tumor immune microenvironments were analyzed by flow cytometry. Depletion of macrophages was used to verify the role of macrophages in impacting the function of miR-340 in tumor progression. RESULTS: miR-340 directly regulates and inversely correlates with CD47, and it predicts patient survival in PDAC. The restoration of miR-340 expression in pancreatic cancer cells was sufficient to downregulate CD47 and promote phagocytosis of macrophages, further inhibiting tumor growth. The overexpression of miR-340 promoted macrophages to become M1-like phenotype polarized in peripheral and tumor immune microenvironments and increased T cells, especially CD8+ T cells, contributing to the antitumor effect of miR-340. CONCLUSIONS: miR-340 is a key regulator of phagocytosis and antitumor immunity, and it could offer a new opportunity for immunotherapy for PDAC.

lincRNA-Cox2 regulates NLRP3 inflammasome and autophagy mediated neuroinflammation.

Inflammasome activation plays key roles in host defense, but also contributes to the pathogenesis of auto-inflammatory, and neurodegenerative diseases. As autophagy is connected with both the innate and adaptive immune systems, autophagic dysfunction is also closely related to inflammation, infection, and neurodegeneration. Here we identify that lincRNA-Cox2, previously known as a mediator of both the activation and repression of immune genes expression in innate immune cells, could bind NF-κB p65 and promote its nuclear translocation and transcription, modulating the expression of inflammasome sensor NLRP3 and adaptor ASC. Knockdown of lincRNA-Cox2 inhibited the inflammasome activation and prevented the lincRNA-Cox2-triggered caspase-1 activation, leading to decreased IL-1ß secretion and weakened TIR-domain-containing adapter-inducing interferon-ß (TRIF) cleavage, thereby enhancing TRIF-mediated autophagy. Elucidation of the link between lincRNA-Cox2 and the inflammasome-autophagy crosstalk in macrophage and microglia reveals a role for lncRNAs in activation of NLRP3 inflammasome and autophagy, and provides new opportunities for therapeutic intervention in neuroinflammation-dependent diseases.

Mir223 restrains autophagy and promotes CNS inflammation by targeting ATG16L1.

Microglia are innate immune cells in the central nervous system (CNS), that supplies neurons with key factors for executing autophagosomal/lysosomal functions. Macroautophagy/autophagy is a cellular catabolic process that maintains cell balance in response to stress-related stimulation. Abnormal autophagy occurs with many pathologies, such as cancer, and autoimmune and neurodegenerative diseases. Hence, clarification of the mechanisms of autophagy regulation is of utmost importance. Recently, researchers presented microRNAs (miRNAs) as novel and potent modulators of autophagic activity. Here, we found that Mir223 deficiency significantly ameliorated CNS inflammation, demyelination and the clinical symptoms of experimental autoimmune encephalomyelitis (EAE) and increased resting microglia and autophagy in brain microglial cells. In contrast, the autophagy inhibitor 3-methylademine (3-MA) aggravated the clinical symptoms of EAE in wild-type (WT) and Mir223-deficienct mice. Furthermore, it was confirmed that Mir223 deficiency in mice increased the protein expression of ATG16L1 (autophagy related 16-like 1 [S. cerevisiae]) and LC3-II in bone marrow-derived macrophage cells compared with cells from WT mice. Indeed, the cellular level of Atg16l1 was decreased in BV2 cells upon Mir223 overexpression and increased following the introduction of antagomirs. We also showed that the 3' UTR of Atg16l1 contained functional Mir223-responsive sequences and that overexpression of ATG16L1 returned autophagy to normal levels even in the presence of Mir223 mimics. Collectively, these data indicate that Mir223 is a novel and important regulator of autophagy and that Atg16l1 is a Mir223 target in this process, which may have implications for improving our understanding of the neuroinflammatory process of EAE. Abbreviations: 3-MA: 3-methylademine; ACTB/ß-actin: actin, beta; ATG: autophagy related; ATG16L1: autophagy related 16-like 1 (S. cerevisiae); BECN1: beclin 1, autophagy related; CNR2: cannabinoid receptor 2 (macrophage); CNS: central nervous system; CQ: chloroquine; EAE: experimental autoimmune encephalomyelitis; FOXO3: forkhead box O3; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; H&E: hematoxylin and eosin; ITGAM: integrin alpha M; LPS: lipoplysaccharide; MAP1LC3/LC3: microtubule-associated protein 1 light chain 3; miRNAs: microRNAs; MS: multiple sclerosis; PPARG: peroxisome proliferator activated receptor gamma; PTPRC: protein tyrosine phosphatase, receptor type, C; RA: rheumatoid arthritis; SQSTM1: sequestosome 1; TB: tuberculosis; TIMM23: translocase of inner mitochondrial membrane 23; TLR: toll-like receptor.

miR-21 promotes NLRP3 inflammasome activation to mediate pyroptosis and endotoxic shock.

miR-21 is aberrantly expressed, and plays a role in various types of tumors and many other diseases. However, the mechanism of miR-21 in LPS-induced septic shock is still unclear. In this study, we investigated the mechanism of miR-21 in LPS-induced pyroptosis and septic shock. Here, we show that miR-21 deficiency inhibited NLRP3, ASC, and caspase-1 expression, as well as inflammasome activation in myeloid cells from both mice and humans. We found that the NF-κB pathway was regulated by miR-21, and that A20 was a direct target of miR-21. Furthermore, miR-21 deficiency inhibited the ASC pyroptosome, which restrained caspase-1 activation and GSDMD cleavage, thereby preventing LPS-induced pyroptosis and septic shock. miR-21 deficiency resulted in an increase in A20, which led to decreased IL-1ß production and caspase-1 activation. Caspase-1-mediated GSDMD cleavage was consequently decreased, which prevented pyroptosis in LPS-induced sepsis in mice. Our results demonstrate that miR-21 is a critical positive regulator of the NF-κB pathway and NLRP3 inflammasomes in pyroptosis and septic shock via A20. In addition, by analyzing published miRNA expression profiles in the Gene Expression Omnibus database, we found that the miR-21 levels in peripheral blood from patients with septic shock were elevated. Thus, miR-21 may serve as a potential treatment target in patients with septic shock.