Can motor decline be a modifiable marker of clinical progression in subjective cognitive decline? A national prospective cohort study.

OBJECTIVES: Subjective cognitive decline represents a critical stage for preventing mild cognitive impairment and dementia, but the links between clinical progression in the subjective cognitive decline stage and various motor functions remain inconclusive. This cohort study aimed to elucidate the independent and joint associations between the clinical progression of subjective cognitive decline and motor functions. METHODS: We enrolled 4880 community-dwelling elderly participants from a national cohort and used Cox proportional hazard regression model and restricted cubic spline models to explore the longitudinal associations between motor functions (gait, strength, balance, and endurance) and the clinical progression of subjective cognitive decline. RESULTS: During 5-years follow-up, 1239 participants experienced clinical progression. After adjusting for demographics, vascular burden, body components, and polypharmacy, gait speed [hazard ratios (HRs)= 0.96, 95% confidence interval (CI) 0.94-0.99], chair stand test (HRs=1.02, 95%CI 1.01-1.03), and endurance limitation in jogging 1 kilometer (HRs=1.18, 95%CI 1.04-1.34) were significantly associated with clinical progression. Among all participants, individuals characterized by poor upper- and lower-body strength, as well as those with slow pace and reduced endurance, faced the highest risk of cognitive impairment. CONCLUSIONS: This study emphasizes the potential of gait speed, muscle strength, and endurance as non-cognitive indicators of clinical progression in subjective cognitive decline. Understanding their combined effectiveness may reveal primary physiological mechanisms contributing to the dual decline of motor and cognition.

Aster-B-dependent estradiol synthesis protects female mice from diet-induced obesity.

Aster proteins mediate the nonvesicular transport of cholesterol from the plasma membrane (PM) to the endoplasmic reticulum (ER). However, the importance of nonvesicular sterol movement for physiology and pathophysiology in various tissues is incompletely understood. Here we show that loss of Aster-B leads to diet-induced obesity in female but not in male mice, and that this sex difference is abolished by ovariectomy. We further demonstrate that Aster-B deficiency impairs nonvesicular cholesterol transport from the PM to the ER in ovaries in vivo, leading to hypogonadism and reduced estradiol synthesis. Female Aster-B-deficient mice exhibit reduced locomotor activity and energy expenditure, consistent with established effects of estrogens on systemic metabolism. Administration of exogenous estradiol ameliorates the diet-induced obesity phenotype of Aster-B-deficient female mice. These findings highlight the key role of Aster-B-dependent nonvesicular cholesterol transport in regulating estradiol production and protecting females from obesity.

RNA m6A methylation modulates airway inflammation in allergic asthma via PTX3-dependent macrophage homeostasis.

N6-methyladenosine (m6A), the most prevalent mRNA modification, has an important function in diverse biological processes. However, the involvement of m6A in allergic asthma and macrophage homeostasis remains largely unknown. Here we show that m6A methyltransferases METTL3 is expressed at a low level in monocyte-derived macrophages from childhood allergic asthma patients. Conditional knockout of Mettl3 in myeloid cells enhances Th2 cell response and aggravates allergic airway inflammation by facilitating M2 macrophage activation. Loss and gain functional studies confirm that METTL3 suppresses M2 macrophage activation partly through PI3K/AKT and JAK/STAT6 signaling. Mechanistically, m6A-sequencing shows that loss of METTL3 impairs the m6A-YTHDF3-dependent degradation of PTX3 mRNA, while higher PTX3 expression positively correlates with asthma severity through promoting M2 macrophage activation. Furthermore, the METTL3/YTHDF3-m6A/PTX3 interactions contribute to autophagy maturation in macrophages by modulating STX17 expression. Collectively, this study highlights the function of m6A in regulating macrophage homeostasis and identifies potential targets in controlling allergic asthma.

Twenty-four-color full spectrum flow cytometry panel for minimal residual disease detection in acute myeloid leukemia.

Full spectrum flow cytometry brings a breakthrough for minimal residual disease (MRD) detection in acute myeloid leukemia (AML). We aimed to explore the role of a new panel in MRD detection. We established a 24-color full-spectrum flow cytometry panel. A tube of 24-color antibodies included CD45, CD117, CD34, HLA-DR, CD15, CD64, CD14, CD11c, CD11b, CD13, CD33, CD371, CD7, CD56, CD19, CD4, CD2, CD123, CD200, CD38, CD96, CD71, CD36, and CD9. We discovered that when a tube meets 26 parameters (24 colors), these markers were not only limited to the observation of MRD in AML, but also could be used for fine clustering of bone marrow cells. Mast cells, basophils, myeloid dendritic cells, and plasmacoid dendritic cells were more clearly observed. In addition, immune checkpoint CD96 had the higher expression in CD117+ myeloid naive cells and CD56dimNK cells, while had the lower expression in CD56briNK cells in AML-MRD samples than in normal bone marrow samples. CD200 expression was remarkably enhanced in CD117+ myeloid naive cells, CD4+ T cells, T cells, activated T cells, CD56dimNK cells, and CD56briNK cells in AML-MRD samples. Our results can be used as important basis for auxiliary diagnosis, prognosis judgment, treatment guidance, and immune regulation in AML.

Immune checkpoint blockade induces gut microbiota translocation that augments extraintestinal antitumor immunity.

Gut microbiota, specifically gut bacteria, are critical for effective immune checkpoint blockade therapy (ICT) for cancer. The mechanisms by which gut microbiota augment extraintestinal anticancer immune responses, however, are largely unknown. Here, we find that ICT induces the translocation of specific endogenous gut bacteria into secondary lymphoid organs and subcutaneous melanoma tumors. Mechanistically, ICT induces lymph node remodeling and dendritic cell (DC) activation, which facilitates the translocation of a selective subset of gut bacteria to extraintestinal tissues to promote optimal antitumor T cell responses in both the tumor-draining lymph nodes (TDLNs) and the primary tumor. Antibiotic treatment results in decreased gut microbiota translocation into mesenteric lymph nodes (MLNs) and TDLNs, diminished DC and effector CD8+ T cell responses, and attenuated responses to ICT. Our findings illuminate a key mechanism by which gut microbiota promote extraintestinal anticancer immunity.

30-color full spectrum flow cytometry panel for deep immunophenotyping of T cell subsets in murine tumor tissue.

This 30-color full spectrum flow cytometry panel was developed and optimized for in-depth analysis T cells immunophenotype in tumor microenvironment and peripheral lymphoid organs. The panel presented here first identify the main cell subsets including myeloid cells, B cells, NKT cells, γδ T cells, CD4+ T cells and CD8+ T cells. For CD4+ T cells or CD8+ T cells, the panel includes markers for further characterization by including a selection of activation status(CD44, CD62L, CD69, Ki67, CD127, KLRG1 and CXCR3), costimulatory/co-inhibitory molecules (ICOS, OX-40, PD-1, LAG3, TIM-3, CTLA-4 and TIGIT), pro-inflammatory/anti-inflammatory cytokines (IFN-γ, TNF-α and IL-10) and cytotoxic molecules (Perforin, Granzymes B and CD107a). The panel has been tested on the tumor infiltrating T cells and corresponding spleen T cells in B16-F10 murine melanoma models.

Multidimensional Analysis of Lung Lymph Nodes in a Mouse Model of Allergic Lung Inflammation following PM2.5 and Indeno[1,2,3-cd]pyrene Exposure.

BACKGROUND: Ambient particulate matter with an aerodynamic diameter of ≤2.5 µm (PM2.5) is suggested to act as an adjuvant for allergen-mediated sensitization and recent evidence suggests the importance of T follicular helper (Tfh) cells in allergic diseases. However, the impact of PM2.5 exposure and its absorbed polycyclic aromatic hydrocarbon (PAHs) on Tfh cells and humoral immunity remains unknown. OBJECTIVES: We aimed to explore the impact of environmental PM2.5 and indeno[1,2,3-cd]pyrene (IP), a prominent PAH, as a model, on Tfh cells and the subsequent pulmonary allergic responses. METHODS: PM2.5- or IP-mediated remodeling of cellular composition in lung lymph nodes (LNs) was determined by mass cytometry in a house dust mite (HDM)-induced mouse allergic lung inflammation model. The differentiation and function of Tfh cells in vitro were analyzed by flow cytometry, quantitative reverse transcription polymerase chain reaction, enzyme-linked immunosorbent assay, chromatin immunoprecipitation, immunoprecipitation, and western blot analyses. RESULTS: Mice exposed to PM2.5 during the HDM sensitization period demonstrated immune cell population shifts in lung LNs as compared with those sensitized with HDM alone, with a greater number of differentiated Tfh2 cells, enhanced allergen-induced immunoglobulin E (IgE) response and pulmonary inflammation. Similarly enhanced phenotypes were also found in mice exposed to IP and sensitized with HDM. Further, IP administration was found to induce interleukin-21 (Il21) and Il4 expression and enhance Tfh2 cell differentiation in vitro, a finding which was abrogated in aryl hydrocarbon receptor (AhR)-deficient CD4+ T cells. Moreover, we showed that IP exposure increased the interaction of AhR and cellular musculoaponeurotic fibrosarcoma (c-Maf) and its occupancy on the Il21 and Il4 promoters in differentiated Tfh2 cells. DISCUSSION: These findings suggest that the PM2.5 (IP)-AhR-c-Maf axis in Tfh2 cells was important in allergen sensitization and lung inflammation, thus adding a new dimension in the understanding of Tfh2 cell differentiation and function and providing a basis for establishing the environment-disease causal relationship. https://doi.org/10.1289/EHP11580.

Sensitisation of cancer cells to radiotherapy by serine and glycine starvation.

BACKGROUND: Cellular metabolism is an integral component of cellular adaptation to stress, playing a pivotal role in the resistance of cancer cells to various treatment modalities, including radiotherapy. In response to radiotherapy, cancer cells engage antioxidant and DNA repair mechanisms which mitigate and remove DNA damage, facilitating cancer cell survival. Given the reliance of these resistance mechanisms on amino acid metabolism, we hypothesised that controlling the exogenous availability of the non-essential amino acids serine and glycine would radiosensitise cancer cells. METHODS: We exposed colorectal, breast and pancreatic cancer cell lines/organoids to radiation in vitro and in vivo in the presence and absence of exogenous serine and glycine. We performed phenotypic assays for DNA damage, cell cycle, ROS levels and cell death, combined with a high-resolution untargeted LCMS metabolomics and RNA-Seq. RESULTS: Serine and glycine restriction sensitised a range of cancer cell lines, patient-derived organoids and syngeneic mouse tumour models to radiotherapy. Comprehensive metabolomic and transcriptomic analysis of central carbon metabolism revealed that amino acid restriction impacted not only antioxidant response and nucleotide synthesis but had a marked inhibitory effect on the TCA cycle. CONCLUSION: Dietary restriction of serine and glycine is a viable radio-sensitisation strategy in cancer.

Single-cell RNA-sequencing of zebrafish hair cells reveals novel genes potentially involved in hearing loss.

Hair cells play key roles in hearing and balance, and hair cell loss would result in hearing loss or vestibular dysfunction. Cellular and molecular research in hair cell biology provides us a better understanding of hearing and deafness. Zebrafish, owing to their hair cell-enriched organs, have been widely applied in hair cell-related research worldwide. Similar to mammals, zebrafish have inner ear hair cells. In addition, they also have lateral line neuromast hair cells. These different types of hair cells vary in morphology and function. However, systematic analysis of their molecular characteristics remains lacking. In this study, we analyzed the GFP+ cells isolated from Tg(Brn3c:mGFP) larvae with GFP expression in all hair cells using single-cell RNA-sequencing (scRNA-seq). Three subtypes of hair cells, namely macula hair cell (MHC), crista hair cell (CHC), and neuromast hair cell (NHC), were characterized and validated by whole-mount in situ hybridization analysis of marker genes. The hair cell scRNA-seq data revealed hair cell-specific genes, including hearing loss genes that have been identified in humans and novel genes potentially involved in hair cell formation and function. Two novel genes were discovered to specifically function in NHCs and MHCs, corresponding to their specific expression in NHCs and MHCs. This study allows us to understand the specific genes in hair cell subpopulations of zebrafish, which will shed light on the genetics of both human vestibular and cochlear hair cell function.

Effector memory CD4+ T cells induce damaging innate inflammation and autoimmune pathology by engaging CD40 and TNFR on myeloid cells.

Cytokine storm and sterile inflammation are common features of T cell-mediated autoimmune diseases and T cell-targeted cancer immunotherapies. Although blocking individual cytokines can mitigate some pathology, the upstream mechanisms governing overabundant innate inflammatory cytokine production remain unknown. Here, we have identified a critical signaling node that is engaged by effector memory T cells (TEM) to mobilize a broad proinflammatory program in the innate immune system. Cognate interactions between TEM and myeloid cells led to induction of an inflammatory transcriptional profile that was reminiscent, yet entirely independent, of classical pattern recognition receptor (PRR) activation. This PRR-independent "de novo" inflammation was driven by preexisting TEM engagement of both CD40 and tumor necrosis factor receptor (TNFR) on myeloid cells. Cytokine toxicity and autoimmune pathology could be completely rescued by ablating these pathways genetically or pharmacologically in multiple models of T cell-driven inflammation, indicating that TEM instruction of the innate immune system is a primary driver of associated immunopathology. Thus, we have identified a previously unknown trigger of cytokine storm and autoimmune pathology that is amenable to therapeutic interventions.

Inactivation of AMPK Leads to Attenuation of Antigen Presentation and Immune Evasion in Lung Adenocarcinoma.

PURPOSE: Mutations in STK11 (LKB1) occur in 17% of lung adenocarcinoma (LUAD) and drive a suppressive (cold) tumor immune microenvironment (TIME) and resistance to immunotherapy. The mechanisms underpinning the establishment and maintenance of a cold TIME in LKB1-mutant LUAD remain poorly understood. In this study, we investigated the role of the LKB1 substrate AMPK in immune evasion in human non-small cell lung cancer (NSCLC) and mouse models and explored the mechanisms involved. EXPERIMENTAL DESIGN: We addressed the role of AMPK in immune evasion in NSCLC by correlating AMPK phosphorylation and immune-suppressive signatures and by deleting AMPKα1 (Prkaa1) and AMPKα2 (Prkaa2) in a KrasG12D -driven LUAD. Furthermore, we dissected the molecular mechanisms involved in immune evasion by comparing gene-expression signatures, AMPK activity, and immune infiltration in mouse and human LUAD and gain or loss-of-function experiments with LKB1- or AMPK-deficient cell lines. RESULTS: Inactivation of both AMPKα1 and AMPKα2 together with Kras activation accelerated tumorigenesis and led to tumors with reduced infiltration of CD8+/CD4+ T cells and gene signatures associated with a suppressive TIME. These signatures recapitulate those in Lkb1-deleted murine LUAD and in LKB1-deficient human NSCLC. Interestingly, a similar signature is noted in human NSCLC with low AMPK activity. In mechanistic studies, we find that compromised LKB1 and AMPK activity leads to attenuated antigen presentation in both LUAD mouse models and human NSCLC. CONCLUSIONS: The results provide evidence that the immune evasion noted in LKB1-inactivated lung cancer is due to subsequent inactivation of AMPK and attenuation of antigen presentation.

Hsa_circ_0004287 inhibits macrophage-mediated inflammation in an N6-methyladenosine-dependent manner in atopic dermatitis and psoriasis.

BACKGROUND: Circular RNA (circRNA) has been implicated in various diseases; however, its role in atopic dermatitis (AD) or psoriasis remains unclear. OBJECTIVE: We sought to determine the differential expression profiles of circRNAs in peripheral blood mononuclear cells between healthy controls and AD patients, and explore the mechanisms underlying the effects of circRNAs on the pathogenesis of AD. METHODS: The differential expression profiles of circRNAs were analyzed by circRNA microarray. In vitro function and mechanisms by which circRNAs regulate macrophage-mediated inflammation were detected by reverse transcription quantitative PCR, Western blot analysis, RNA stability assay, immunoprecipitation, ELISA, and methylated RNA immunoprecipitation assay. In vivo roles of circRNAs were determined in 2,4-dinitrochlorobenzene (DNCB)-induced dermatitis and imiquimod (IMQ)-induced psoriasis mouse model. RESULTS: We identified a functional unknown circRNA hsa_circ_0004287 from 88750 circRNAs, which was upregulated in peripheral blood mononuclear cells of both AD and psoriasis patients, and was mainly expressed by macrophages under inflammatory conditions. Hsa_circ_0004287 inhibited M1 macrophage activation in vitro, and macrophage-specific overexpression of hsa_circ_0004287 alleviated skin inflammation in both AD- and psoriasis-like mice. Mechanistically, hsa_circ_0004287 reduced the stability of its host gene metastasis associated lung adenocarcinoma transcript 1 (MALAT1) by competitively binding to IGF2BP3 with MALAT1 in an N6-methyladenosine (m6A)-dependent manner. Lower levels of MALAT1 promoted the ubiquitination degradation of S100A8/S100A9, thereby impeding p38/mitogen-activated protein kinase phosphorylation and macrophage-mediated inflammation. CONCLUSION: hsa_circ_0004287 inhibits M1 macrophage activation in an m6A-dependent manner in AD and psoriasis, and may serve as a general therapeutic candidate for AD and psoriasis.

An immunocompetent rectal cancer model to study radiation therapy.

We describe a mouse model of rectal cancer (RC) involving rapid tumor organoid engraftment via orthotopic transplantation in an immunocompetent setting. This approach uses simple mechanical disruption to allow engraftment, avoiding the use of dextran sulfate sodium. The resulting RC tumors invaded from the mucosal surface and metastasized to distant organs. Histologically, the tumors closely resemble human RC and mirror remodeling of the tumor microenvironment in response to radiation. This murine RC model thus recapitulates key aspects of human RC pathogenesis and presents an accessible approach for more physiologically accurate, preclinical efficacy studies.

Detection of peripheral blood circulating tumor cells in oral squamous cell carcinoma and its clinical significance.

OBJECTIVES: This study aims to investigate the diagnostic value of peripheral blood circulating tumor cells (CTCs) in oral squamous cell carcinoma (OSCC) and its correlation with the clinicopathological features of OSCC. METHODS: Ninety-three patients diagnosed as OSCC in the First Affiliated Hospital of Zhengzhou University from May 2019 to May 2020 were selected as the experimental group, and 20 healthy volunteers were employed as the control group. The CTCs value of peripheral blood of the patients were measured by CTCs detection technology, and its clinical significance was analyzed. RESULTS: The CTCs values in the experimental group were higher than those in the control group, and the difference was statistically significant (P<0.000 1). The CTCs value in the peripheral blood of patients in the experimental group were not correlated with gender, site of onset, and presence or absence of peripheral tissue infiltration (P>0.05), but was correlated with age (P=0.022), tumor T stage (P=0.02), tumor N stage (P=0.007 5), tumor M stage (P=0.013), clinical stage (P=0.029), early or late stage (P=0.022), tumor differentiation degree (P<0.001), and node metastasis (P=0.006 4). The AUC value of CTCs in OSCC diagnosis was 0.925, and the energy efficiency was statistically significant [P=0.000, 95%CI (0.876, 0.974)]. When the CTC value was 8.450 FU/3 mL, the maximum value of the Yoden index was 0.853, and the sensitivity and specificity of OSCC diagnosis were 90.3% and 95.0%, respectively. The AUC value of CTCs in the diagnosis of OSCC metastasis was 0.691, and the energy efficiency was statistically significant [P=0.000, 95%CI (0.580, 0.803)]. When the blood CTC value was 12.250 FU/3 mL, the maximum value of Yoden index was 0.367, the sensitivity was 63.6%, and the specificity was 73.3%. Multivariate regression analysis showed that buccal tumor was negatively correlated with CTCs in patients with OSCC (P=0.001 08), N2 stage (P=0.000 74) and M stage (P=0.026 38). High differentiation (P<0.000 1) and moderate differentiation (P=0.001 5) were negatively correlated with CTCs values in patients with OSCC. CONCLUSIONS: Peripheral blood CTCs has important clinical value for early screening, auxiliary diagnosis, evaluation of metastasis, and determination of malignant degree, progression, and pathological grade of OSCC and a relatively reliable tumor detection indicator.

IRF1 governs the differential interferon-stimulated gene responses in human monocytes and macrophages by regulating chromatin accessibility.

Myeloid lineage cells use TLRs to recognize and respond to diverse microbial ligands. Although unique transcription factors dictate the outcome of specific TLR signaling, whether lineage-specific differences exist to further modulate the quality of TLR-induced inflammation remains unclear. Comprehensive analysis of global gene transcription in human monocytes, monocyte-derived macrophages, and monocyte-derived dendritic cells stimulated with various TLR ligands identifies multiple lineage-specific, TLR-responsive gene programs. Monocytes are hyperresponsive to TLR7/8 stimulation that correlates with the higher expression of the receptors. While macrophages and monocytes express similar levels of TLR4, macrophages, but not monocytes, upregulate interferon-stimulated genes (ISGs) in response to TLR4 stimulation. We find that TLR4 signaling in macrophages uniquely engages transcription factor IRF1, which facilitates the opening of ISG loci for transcription. This study provides a critical mechanistic basis for lineage-specific TLR responses and uncovers IRF1 as a master regulator for the ISG transcriptional program in human macrophages.

lnc-BAZ2B promotes M2 macrophage activation and inflammation in children with asthma through stabilizing BAZ2B pre-mRNA.

BACKGROUND: Dysregulation of long noncoding RNAs (lncRNAs) is associated with a variety of human diseases; however, whether they have a role in childhood asthma is unknown. OBJECTIVE: We sought to determine the differential expression profiles of lncRNAs in PBMCs of children with asthma and the mechanisms underlying the effects of lncRNAs on the pathogenesis of asthma. METHODS: The differential expression profiles of lncRNAs were analyzed by transcriptome microarray. The effects and mechanisms by which lncRNAs influence macrophage activation were detected by real-time quantitative PCR, Western blot, RNase protection assay, and chromatin immunoprecipitation assay. The roles played by lncRNAs in asthma were tested in a cockroach allergen extract (CRE)-induced mouse model. RESULTS: We identified 719 lncRNAs that were differentially expressed in PBMCs of children with asthma, 502 of which were upregulated and 217 were downregulated. An lncRNA of unknown function, lnc-BAZ2B, was dominantly expressed in monocytes and significantly upregulated in children with asthma. lnc-BAZ2B promotes M2 macrophage activation by enhancing BAZ2B expression and exacerbated lung inflammation in an M2 macrophage-associated CRE-induced asthma model. Mechanistically, lnc-BAZ2B promoted the expression of its cis target gene BAZ2B by stabilizing its pre-mRNA. BAZ2B, a reader of H3K14ac modification, enhanced the transcription of IRF4 and promoted M2 macrophage activation. lnc-BAZ2B expression was correlated with that of BAZ2B in PBMCs from children with asthma. Baz2b knockdown could alleviate asthma severity in a CRE-induced asthma model. CONCLUSION: lnc-BAZ2B promotes M2 macrophage activation and inflammation in children with asthma and may serve as a potential therapeutic and diagnostic target in children with asthma.

Hsa_circ_0060450 Negatively Regulates Type I Interferon-Induced Inflammation by Serving as miR-199a-5p Sponge in Type 1 Diabetes Mellitus.

Circular RNAs (circRNAs) constitute a class of covalently circular non-coding RNA molecules formed by 5' and 3' end back-splicing. The rapid development of bioinformatics and large-scale sequencing has led to the identification of functional circRNAs. Despite an overall upward trend, studies focusing on the roles of circRNAs in immune diseases remain relatively scarce. In the present study, we obtained a differential circRNA expression profile based on microarray analysis of peripheral blood mononuclear cells (PBMCs) in children with type 1 diabetes mellitus (T1DM). We characterized one differentially expressed circRNA back-spliced from the MYB Proto-Oncogene Like 2 (MYBL2) gene in patients with T1DM, termed as hsa_circ_0060450. Subsequent assays revealed that hsa_circ_0060450 can serve as the sponge of miR-199a-5p, release its target gene, Src homology 2 (SH2)-containing protein tyrosine phosphatase 2 (SHP2), encoded by the tyrosine-protein phosphatase non-receptor type 11 gene (PTPN11), and further suppress the JAK-STAT signaling pathway triggered by type I interferon (IFN-I) to inhibit macrophage-mediated inflammation, which indicates the important roles of circRNAs in T1DM and represents a promising therapeutic molecule in the treatment of T1DM.

Circular RNA circPPM1F modulates M1 macrophage activation and pancreatic islet inflammation in type 1 diabetes mellitus.

Rationale: Macrophages play critical roles in the pathogenesis of type 1 diabetes mellitus (T1DM). Circular RNAs (circRNAs) are a novel class of endogenous RNAs with covalently closed loop structures, implicated in various disease processes. However, their impact on macrophage activation and T1DM pathogenesis remains elusive. Methods: circRNA expression profiles of peripheral blood mononuclear cells (PBMCs) from T1DM children were determined by whole transcriptome microarray. Bioinformatics, quantitative real-time PCR, Western blot, RNA immunoprecipitation (RIP), cell co-culture, cell proliferation, and cell apoptosis assays were performed to investigate the expression, function, and regulatory mechanisms of circPPM1F in vitro. The regulatory role of circPPM1F in vivo was evaluated in the streptozocin-induced diabetic mouse model. Results: We identified 27 upregulated and 31 downregulated differentially expressed circRNAs in T1DM patients. circPPM1F, a circRNA with unknown function, was dominantly expressed in monocytes and significantly upregulated in T1DM patients. Functionally, circPPM1F promoted lipopolysaccharide (LPS)-induced M1 macrophage activation via enhancement of the NF-κB signaling pathway. Mechanistically, circPPM1F competitively interacted with HuR to impair the translation of protein phosphatase, Mg2+/Mn2+ dependent 1F (PPM1F), thus alleviating the inhibitory effect of PPM1F on the NF-κB pathway. Moreover, eukaryotic initiation factor 4A-III (EIF4A3) and fused in sarcoma (FUS) coordinately regulated circPPM1F expression during M1 macrophage activation. In addition, circPPM1F could exacerbate pancreas injury in the streptozocin-induced diabetic mice by activation of M1 macrophages in vivo. Conclusions:circPPM1F is a novel positive regulator of M1 macrophage activation through the circPPM1F-HuR-PPM1F-NF-κB axis. Overexpression of circPPM1F could promote pancreatic islet injury by enhancing M1 macrophage activation and circPPM1F may serve as a novel potential therapeutic target for T1DM in children.

LKB1 Represses ATOH1 via PDK4 and Energy Metabolism and Regulates Intestinal Stem Cell Fate.

BACKGROUND & AIMS: In addition to the Notch and Wnt signaling pathways, energy metabolism also regulates intestinal stem cell (ISC) function. Tumor suppressor and kinase STK11 (also called LKB1) regulates stem cells and cell metabolism. We investigated whether loss of LKB1 alters ISC homeostasis in mice. METHODS: We deleted LKB1 from ISCs in mice using Lgr5-regulated CRE-ERT2 (Lkb1Lgr5-KO mice) and the traced lineages by using a CRE-dependent TdTomato reporter. Intestinal tissues were collected and analyzed by immunohistochemical and immunofluorescence analyses. We purified ISCs and intestinal progenitors using flow cytometry and performed RNA-sequencing analysis. We measured organoid-forming capacity and ISC percentages using intestinal tissues from Lkb1Lgr5-KO mice. We analyzed human Ls174t cells with knockdown of LKB1 or other proteins by immunoblotting, real-time quantitative polymerase chain reaction, and the Seahorse live-cell metabolic assay. RESULTS: Some intestinal crypts from Lkb1Lgr5-KO mice lost ISCs compared with crypts from control mice. However, most crypts from Lkb1Lgr5-KO mice contained functional ISCs that expressed increased levels of Atoh1 messenger RNA (mRNA), acquired a gene expression signature associated with secretory cells, and generated more cells in the secretory lineage compared with control mice. Knockdown of LKB1 in Ls174t cells induced expression of Atoh1 mRNA and a phenotype of increased mucin production; knockdown of ATOH1 prevented induction of this phenotype. The increased expression of Atoh1 mRNA after LKB1 loss from ISCs or Ls174t cells did not involve Notch or Wnt signaling. Knockdown of pyruvate dehydrogenase kinase 4 (PDK4) or inhibition with dichloroacetate reduced the up-regulation of Atoh1 mRNA after LKB1 knockdown in Ls174t cells. Cells with LKB1 knockdown had a reduced rate of oxygen consumption, which was partially restored by PDK4 inhibition with dichloroacetate. ISCs with knockout of LKB1 increased the expression of PDK4 and had an altered metabolic profile. CONCLUSIONS: LKB1 represses transcription of ATOH1, via PDK4, in ISCs, restricting their differentiation into secretory lineages. These findings provide a connection between metabolism and the fate determination of ISCs.

Transcriptional profiling identifies caspase-1 as a T cell-intrinsic regulator of Th17 differentiation.

Dendritic cells (DCs) are critical for the differentiation of pathogen-specific CD4 T cells. However, to what extent innate cues from DCs dictate transcriptional changes in T cells remains elusive. Here, we used DCs stimulated with specific pathogens to prime CD4 T cells in vitro and found that these T cells express unique transcriptional profiles dictated by the nature of the priming pathogen. More specifically, the transcriptome of in vitro C. rodentium-primed Th17 cells resembled that of Th17 cells primed following infection in vivo but was remarkably distinct from cytokine-polarized Th17 cells. We identified caspase-1 as a unique gene up-regulated only in pathogen-primed Th17 cells and discovered a critical role for T cell-intrinsic caspase-1, independent of inflammasome, in optimal priming of Th17 responses. T cells lacking caspase-1 failed to induce colitis or confer protection against C. rodentium infection due to suboptimal Th17 cell differentiation in vivo. This study underlines the importance of DC-mediated priming in identifying novel regulators of T cell differentiation.