The microRNA miR-148a functions as a critical regulator of B cell tolerance and autoimmunity.

Autoreactive B cells have critical roles in a large diversity of autoimmune diseases, but the molecular pathways that control these cells remain poorly understood. We performed an in vivo functional screen of a lymphocyte-expressed microRNA library and identified miR-148a as a potent regulator of B cell tolerance. Elevated miR-148a expression impaired B cell tolerance by promoting the survival of immature B cells after engagement of the B cell antigen receptor by suppressing the expression of the autoimmune suppressor Gadd45α, the tumor suppressor PTEN and the pro-apoptotic protein Bim. Furthermore, increased expression of miR-148a, which occurs frequently in patients with lupus and lupus-prone mice, facilitated the development of lethal autoimmune disease in a mouse model of lupus. Our studies demonstrate a function for miR-148a as a regulator of B cell tolerance and autoimmunity.

PD-L1:CD80 Cis-Heterodimer Triggers the Co-stimulatory Receptor CD28 While Repressing the Inhibitory PD-1 and CTLA-4 Pathways.

Combined immunotherapy targeting the immune checkpoint receptors cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) and programmed cell death 1 (PD-1), or CTLA-4 and the PD-1 ligand (PD-L1) exhibits superior anti-tumor responses compared with single-agent therapy. Here, we examined the molecular basis for this synergy. Using reconstitution assays with fluorescence readouts, we found that PD-L1 and the CTLA-4 ligand CD80 heterodimerize in cis but not trans. Quantitative biochemistry and cell biology assays revealed that PD-L1:CD80 cis-heterodimerization inhibited both PD-L1:PD-1 and CD80:CTLA-4 interactions through distinct mechanisms but preserved the ability of CD80 to activate the T cell co-stimulatory receptor CD28. Furthermore, PD-L1 expression on antigen-presenting cells (APCs) prevented CTLA-4-mediated trans-endocytosis of CD80. Atezolizumab (anti-PD-L1), but not anti-PD-1, reduced cell surface expression of CD80 on APCs, and this effect was negated by co-blockade of CTLA-4 with ipilimumab (anti-CTLA-4). Thus, PD-L1 exerts an immunostimulatory effect by repressing the CTLA-4 axis; this has implications to the synergy of anti-PD-L1 and anti-CTLA-4 combination therapy.

The transcription factor Foxp1 is a critical negative regulator of the differentiation of follicular helper T cells.

CD4(+) follicular helper T cells (T(FH) cells) are essential for germinal center (GC) responses and long-lived antibody responses. Here we report that naive CD4(+) T cells deficient in the transcription factor Foxp1 'preferentially' differentiated into T(FH) cells, which resulted in substantially enhanced GC and antibody responses. We found that Foxp1 used both constitutive Foxp1A and Foxp1D induced by stimulation of the T cell antigen receptor (TCR) to inhibit the generation of T(FH) cells. Mechanistically, Foxp1 directly and negatively regulated interleukin 21 (IL-21); Foxp1 also dampened expression of the costimulatory molecule ICOS and its downstream signaling at early stages of T cell activation, which rendered Foxp1-deficient CD4(+) T cells partially resistant to blockade of the ICOS ligand (ICOSL) during T(FH) cell development. Our findings demonstrate that Foxp1 is a critical negative regulator of T(FH) cell differentiation.

MicroRNAs of the miR-17∼92 family are critical regulators of T(FH) differentiation.

Follicular helper T cells (T(FH) cells) provide critical help to B cells during humoral immune responses. Here we report that mice with T cell-specific deletion of the miR-17∼92 family of microRNAs (miRNAs) had substantially compromised T(FH) differentiation, germinal-center formation and antibody responses and failed to control chronic viral infection. Conversely, mice with T cell-specific expression of a transgene encoding miR-17∼92 spontaneously accumulated T(FH) cells and developed a fatal immunopathology. Mechanistically, the miR-17∼92 family controlled the migration of CD4(+) T cells into B cell follicles by regulating signaling intensity from the inducible costimulator ICOS and kinase PI(3)K by suppressing expression of the phosphatase PHLPP2. Our findings demonstrate an essential role for the miR-17∼92 family in T(FH) differentiation and establish PHLPP2 as an important mediator of their function in this process.

The MicroRNA-183-96-182 Cluster Promotes T Helper 17 Cell Pathogenicity by Negatively Regulating Transcription Factor Foxo1 Expression.

T helper 17 (Th17) cells are key players in autoimmune diseases. However, the roles of non-coding RNAs in Th17 cell development and function are largely unknown. We found that deletion of the endoribonuclease-encoding Dicer1 specifically in Th17 cells protected mice from experimental autoimmune encephalomyelitis. We found that the Dicer1-regulated microRNA (miR)-183-96-182 cluster (miR-183C) was highly expressed in Th17 cells and was induced by cytokine IL-6-STAT3 signaling. miR-183C expression enhanced pathogenic cytokine production from Th17 cells during their development and promoted autoimmunity. Mechanistically, miR-183C in Th17 cells directly repressed expression of the transcription factor Foxo1. Foxo1 negatively regulated the pathogenicity of Th17 cells in part by inhibiting expression of cytokine receptor IL-1R1. These findings indicate that the miR-183C drives Th17 pathogenicity in autoimmune diseases via inhibition of Foxo1 and present promising therapeutic targets.

MicroRNA control in the immune system: basic principles.

MicroRNA (miRNA) control has emerged as a critical regulatory principle in the mammalian immune system. Genetic ablation of the miRNA machinery, as well as loss or deregulation of certain individual miRNAs, severely compromises immune development and response and can lead to immune disorders like autoimmunity and cancer. Although individual miRNAs modulate protein output from hundreds of target genes, they may impact physiological processes by regulating the concentrations of just a few key cellular proteins that may be components of a single or of functionally interrelated pathways in a given cellular context.

Fine particulate matter (PM2.5) induces the stem cell-like properties of hepatocellular carcinoma by activating ROS/Nrf2/Keap1-mediated autophagy.

Exposure to fine particulate matter (PM2.5) has been linked to an increased incidence and mortality of hepatocellular carcinoma (HCC). However, the impact of PM2.5 exposure on HCC progression and the underlying mechanisms remain largely unknown. This study aimed to investigate the effects of PM2.5 exposure on the stem cell-like properties of HCC cells. Our findings indicate that PM2.5 exposure significantly enhances the stemness of HCC cells (p < 0.01). Subsequently, male nude mice were divided into two groups (n = 8/group for tumor-bearing assay, n = 5/group for metastasis assay) for control and PM2.5 exposure. In vivo assays revealed that exposure to PM2.5 promoted the growth, metastasis, and epithelial-mesenchymal transition (EMT) of HCC cells (p < 0.01). Further exploration demonstrated that PM2.5 enhances the stemness of HCC cells by inducing cellular reactive oxygen species (ROS) generation (p < 0.05). Mechanistic investigation indicated that elevated intracellular ROS inhibited kelch-like ECH-associated protein 1 (Keap1) levels, promoting the upregulation and nucleus translocation of NFE2-like bZIP transcription factor 2 (Nrf2). This, in turn, induced autophagy activation, thereby promoting the stemness of HCC cells (p < 0.01). Our present study demonstrates the adverse effects of PM2.5 exposure on HCC development and highlights the mechanism of ROS/Nrf2/Keap1-mediated autophagy. For the first time, we reveal the impact of PM2.5 exposure on the poor prognosis-associated cellular phenotype of HCC and its underlying mechanism, which is expected to provide new theoretical basis for the improvement of public health.

GSK3 Restrains Germinal Center B Cells to Form Plasma Cells.

B cells in the germinal center (GC) are programmed to form plasma cells (PCs) or memory B cells according to signals received by receptors that are translated to carry out appropriate activities of transcription factors. However, the precise mechanism underlying this process to complete the GC reaction is unclear. In this study, we show that both genetic ablation and pharmacological inhibition of glycogen synthase kinase 3 (GSK3) in GC B cells of mice facilitate the cell fate decision toward PC formation, accompanied by acquisition of dark zone B cell properties. Mechanistically, under stimulation with CD40L and IL-21, GSK3 inactivation synergistically induced the transcription factors Foxo1 and c-Myc, leading to increased levels of key transcription factors required for PC differentiation, including IRF4. This GSK3-mediated alteration of transcriptional factors in turn facilitated the dark zone transition and consequent PC fate commitment. Our study thus reveals the upstream master regulator responsible for interpreting external cues in GC B cells to form PCs mediated by key transcription factors.

MiR-150 controls B cell differentiation by targeting the transcription factor c-Myb.

MiR-150 is a microRNA (miRNA) specifically expressed in mature lymphocytes, but not their progenitors. A top predicted target of miR-150 is c-Myb, a transcription factor controlling multiple steps of lymphocyte development. Combining loss- and gain-of-function gene targeting approaches for miR-150 with conditional and partial ablation of c-Myb, we show that miR-150 indeed controls c-Myb expression in vivo in a dose-dependent manner over a narrow range of miRNA and c-Myb concentrations and that this dramatically affects lymphocyte development and response. Our results identify a key transcription factor as a critical target of a stage-specifically expressed miRNA in lymphocytes and suggest that this and perhaps other miRNAs have evolved to control the expression of just a few critical target proteins in particular cellular contexts.

MicroRNA control of B cell tolerance, autoimmunity and cancer.

Since the discovery of the first microRNA (miRNA) in 1993, thousands of miRNAs have been identified in humans and mice and many of them have been shown to control a large variety of cellular processes in different cell types including those composing the immune system. MicroRNAs regulate virtually all aspects of immune cell development, differentiation and function. Studies have shown that these molecules are involved in the maintenance of lymphocyte tolerance and, when dysregulated, promote the development of autoimmune diseases. In this review, we focus on the current knowledge about the roles of miRNAs in B cell tolerance and their contribution to autoimmunity, highlighting additional roles for some of these miRNAs in T cell tolerance. Finally, we will comment on miRNAs that promote both autoimmunity and lymphoma.

Endothelial Scaffolding Protein ENH (Enigma Homolog Protein) Promotes PHLPP2 (Pleckstrin Homology Domain and Leucine-Rich Repeat Protein Phosphatase 2)-Mediated Dephosphorylation of AKT1 and eNOS (Endothelial NO Synthase) Promoting Vascular Remodeling.

OBJECTIVE: A decrease in nitric oxide, leading to vascular smooth muscle cell proliferation, is a common pathological feature of vascular proliferative diseases. Nitric oxide synthesis by eNOS (endothelial nitric oxide synthase) is precisely regulated by protein kinases including AKT1. ENH (enigma homolog protein) is a scaffolding protein for multiple protein kinases, but whether it regulates eNOS activation and vascular remodeling remains unknown. Approach and Results: ENH was upregulated in injured mouse arteries and human atherosclerotic plaques and was associated with coronary artery disease. Neointima formation in carotid arteries, induced by ligation or wire injury, was greatly decreased in endothelium-specific ENH-knockout mice. Vascular ligation reduced AKT and eNOS phosphorylation and nitric oxide production in the endothelium of control but not ENH-knockout mice. ENH was found to interact with AKT1 and its phosphatase PHLPP2 (pleckstrin homology domain and leucine-rich repeat protein phosphatase 2). AKT and eNOS activation were prolonged in VEGF (vascular endothelial growth factor)-induced ENH- or PHLPP2-deficient endothelial cells. Inhibitors of either AKT or eNOS effectively restored ligation-induced neointima formation in ENH-knockout mice. Moreover, endothelium-specific PHLPP2-knockout mice displayed reduced ligation-induced neointima formation. Finally, PHLPP2 was increased in the endothelia of human atherosclerotic plaques and blood cells from patients with coronary artery disease. CONCLUSIONS: ENH forms a complex with AKT1 and its phosphatase PHLPP2 to negatively regulate AKT1 activation in the artery endothelium. AKT1 deactivation, a decrease in nitric oxide generation, and subsequent neointima formation induced by vascular injury are mediated by ENH and PHLPP2. ENH and PHLPP2 are thus new proatherosclerotic factors that could be therapeutically targeted.

Short-term effects of ambient temperature and pollutants on the mortality of respiratory diseases: A time-series analysis in Hefei, China.

BACKGROUND: The air pollution has become an important environmental health problem due to its adverse health effect. The objective of this study was to investigate the effects of ambient temperature and pollutants on mortality of respiratory diseases (RD) in Hefei, China, a typical inland city. METHODS: Nonlinear exposure-response dependencies and delayed effects of urban daily mean temperature (DMT) and pollutants were evaluated by distributed lag non-linear models (DLNM). To further explore this effect, different genders and ages were also examined by stratified analysis. RESULTS: A total of 12876 deaths from RD were collected from January 1, 2014 to December 31, 2018 in Hefei, China. There was a U-shaped correlation between DMT and RD mortality, and the RD mortality rised by 11.6% (95% CI: 2.2-22.0%) when the DMT was 35.8 °C (reference temperature is 20 °C). The results show that risk of death with short-term exposure to elevated concentrations of PM10 and SO2 was not significant. The maximum hysteresis and cumulative relative risk (RR) of RD mortality were 1.012 (95% CI: 1.003 ~ 1.021, lag 0 day) and 1.072 (95% CI: 1.014 ~1.133, lag 10 days) for each 10 µg/m3 augment in NO2; 1.005 (95% CI: 1.001-1.009, lag 0 day) and 1.027 (95% CI: 1.004-1.051, lag 10 days) for each 10 µg/m3 augment in O3; a negative association between CO exposure and the cumulative risk of death was observed (RR = 0.964, 95% CI: 0.935-0.993, lag 07 days). Subgroup analysis showed the effect of high temperatures, NO2, O3 and CO exposure was still statistically significant for the elderly and male. CONCLUSION: The present study found that short-term exposure to high temperature, NO2, O3 and CO were significantly associated with the risk of RD mortality and male as well as elderly are more susceptible to these factors.

The association between gaseous pollutants and non-accidental mortality: a time series study.

To evaluate the effects of gaseous pollutants (SO2, NO2) on non-accidental mortality of residents in Hefei city, we collected non-accidental deaths, air pollutants and meteorological data of Hefei city from 2014 to 2017. After controlling confounding factors with Poisson generalized additive model, we analyzed the relationship between air pollutants and non-accidental mortality and used subgroup analysis to identify susceptible groups. The number of non-accidental deaths during the study period was 42,116, with an average of 28.83 per day. The average concentrations of SO2 and NO2 were 16.08 µg/m3 and 39.10 µg/m3, respectively. In the single-pollutant model, every 10 µg/m3 increase in SO2 and NO2 concentrations was significantly associated with non-accidental mortality, and there was a significant lag effect. SO2 increased the risk of non-accidental death by 4.93% (95% CI: 1.94% ~ 8.00%) at lag0-3. In addition, male, the elderly, non-elderly and low-education people were more susceptible (P < 0.05). NO2 increased the risk of non-accidental death by 2.11% (95% CI: 1.18% ~ 3.05%) at lag0-1 and had an effect on all subgroups (P < 0.05). For every 10 µg/m3 increase in SO2 and NO2, the two-pollutant model showed that the risk of non-accidental death, respectively, increased by 3.34% (95% CI: 0.29% ~ 6.50%) and 1.82% (95% CI: 0.85% ~ 2.79%), suggesting that the effect was weakened. Our study suggested that SO2 and NO2 were associated with non-accidental mortality, and there were lag effects. Therefore, environmental management should be strengthened and health protection education should be carried out for different groups.

The short-term effect of PM2.5/O3 on daily mortality from 2013 to 2018 in Hefei, China.

This research intends to explore the short-term impacts of PM2.5/O3 on daily death in Hefei from 2013 to 2018. Data on daily death of Hefei residents, meteorological factors, and air pollutants were collected from Jan 1, 2013, to Dec 31, 2018. The correlation between PM2.5/O3 and daily death in Hefei during the research period was studied by time series analysis. From 2013 to 2018, there were 61,683 non-accidental deaths, including 27,431 cardiovascular deaths, 5587 respiratory deaths, 20,921 malignant tumor deaths, and 1674 diabetes deaths, in Hefei. Annual mean concentrations of PM2.5, PM10, NO2, SO2, CO, and O3 in Hefei were 66.18, 92.37, 39.75, 15.39, 930, and 79.08 µg m-3, respectively. An increase of 10 µg m-3 in PM2.5 was related with 0.53% (95% CI 0.31-0.75%), 0.93% (95% CI 0.60-1.26%), 0.90% and (95% CI 0.23-1.57%) increase in non-accidental, cardiovascular and respiratory diseases mortality, respectively. The association between ozone and mortality was not significant. In cold seasons, PM2.5 had a stronger effect on the deaths resulting from non-accidental, cardiovascular, and respiratory diseases. The effect of O3 on deaths was not significantly different between the cold season and the warm season. Women and the elders (over 65 years) were at high risk of being affected by PM2.5/O3. Short-term exposure to PM2.5 was positively correlated with increased deaths due to non-accidental, cardiovascular and respiratory diseases in Hefei. Females and elders were more vulnerable to PM2.5/O3 exposure. No significant associations were observed between ozone and deaths from non-accidental, cardiovascular, respiratory, malignant tumors, and diabetes diseases.

Lymphoproliferative disease and autoimmunity in mice with increased miR-17-92 expression in lymphocytes.

The genomic region encoding the miR-17-92 microRNA (miRNA) cluster is often amplified in lymphoma and other cancers, and cancer cells carrying this amplification have higher expression of miRNA in this cluster. Retroviral expression of miR-17-92 accelerates c-Myc-induced lymphoma development, but precisely how higher expression of miR-17-92 promotes lymphomagenesis remains unclear. Here we generated mice with higher expression of miR-17-92 in lymphocytes. These mice developed lymphoproliferative disease and autoimmunity and died prematurely. Lymphocytes from these mice showed more proliferation and less activation-induced cell death. The miR-17-92 miRNA suppressed expression of the tumor suppressor PTEN and the proapoptotic protein Bim. This mechanism probably contributed to the lymphoproliferative disease and autoimmunity of miR-17-92-transgenic mice and contributes to lymphoma development in patients with amplifications of the miR-17-92 coding region.

Differential Sensitivity of Target Genes to Translational Repression by miR-17~92.

MicroRNAs (miRNAs) are thought to exert their functions by modulating the expression of hundreds of target genes and each to a small degree, but it remains unclear how small changes in hundreds of target genes are translated into the specific function of a miRNA. Here, we conducted an integrated analysis of transcriptome and translatome of primary B cells from mutant mice expressing miR-17~92 at three different levels to address this issue. We found that target genes exhibit differential sensitivity to miRNA suppression and that only a small fraction of target genes are actually suppressed by a given concentration of miRNA under physiological conditions. Transgenic expression and deletion of the same miRNA gene regulate largely distinct sets of target genes. miR-17~92 controls target gene expression mainly through translational repression and 5'UTR plays an important role in regulating target gene sensitivity to miRNA suppression. These findings provide molecular insights into a model in which miRNAs exert their specific functions through a small number of key target genes.

miR-17∼92 family clusters control iNKT cell ontogenesis via modulation of TGF-ß signaling.

Invariant natural killer T cells (iNKT) cells are T lymphocytes displaying innate effector functions, acquired through a distinct thymic developmental program regulated by microRNAs (miRNAs). Deleting miRNAs by Dicer ablation (Dicer KO) in thymocytes selectively impairs iNKT cell survival and functional differentiation. To unravel this miRNA-dependent program, we systemically identified transcripts that were differentially expressed between WT and Dicer KO iNKT cells at different differentiation stages and predicted to be targeted by the iNKT cell-specific miRNAs. TGF-ß receptor II (TGF-ßRII), critically implicated in iNKT cell differentiation, was found up-regulated in iNKT Dicer KO cells together with enhanced TGF-ß signaling. miRNA members of the miR-17∼92 family clusters were predicted to target Tgfbr2 mRNA upon iNKT cell development. iNKT cells lacking all three miR-17∼92 family clusters (miR-17∼92, miR-106a∼363, miR-106b∼25) phenocopied both increased TGF-ßRII expression and signaling, and defective effector differentiation, displayed by iNKT Dicer KO cells. Consistently, genetic ablation of TGF-ß signaling in the absence of miRNAs rescued iNKT cell differentiation. These results elucidate the global impact of miRNAs on the iNKT cell developmental program and uncover the targeting of a lineage-specific cytokine signaling by miRNAs as a mechanism regulating innate-like T-cell development and effector differentiation.