Foxp3-mediated blockage of ryanodine receptor 2 underlies contact-based suppression by regulatory T cells.

The suppression mechanism of Tregs remains an intensely investigated topic. As our focus has shifted toward a model centered on indirect inhibition of DCs, a universally applicable effector mechanism controlled by the transcription factor forkhead box P3 (Foxp3) expression has not been found. Here, we report that Foxp3 blocked the transcription of ER Ca2+-release channel ryanodine receptor 2 (RyR2). Reduced RyR2 shut down basal Ca2+ oscillation in Tregs, which reduced m-calpain activities that are needed for T cells to disengage from DCs, suggesting a persistent blockage of DC antigen presentation. RyR2 deficiency rendered the CD4+ T cell pool immune suppressive and caused it to behave in the same manner as Foxp3+ Tregs in viral infection, asthma, hypersensitivity, colitis, and tumor development. In the absence of Foxp3, Ryr2-deficient CD4+ T cells rescued the systemic autoimmunity associated with scurfy mice. Therefore, Foxp3-mediated Ca2+ signaling inhibition may be a central effector mechanism of Treg immune suppression.

DCLK1 Suppresses Tumor-Specific Cytotoxic T Lymphocyte Function Through Recruitment of MDSCs via the CXCL1-CXCR2 Axis.

BACKGROUND & AIMS: Gastrointestinal cancer stem cell marker doublecortin-like kinase (DCLK1) is strongly associated with poor outcomes in colorectal cancer (CRC). Although DCLK1's regulatory effect on the tumor immune microenvironment has been hypothesized, its mode of action has not been shown previously in vivo, which hampers the potential intervention based on this molecule for clinical practice. METHODS: To define the immunomodulatory mechanisms of DCLK1 in vivo, we generated DCLK1-/- tumor cells by Clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9) and developed subcutaneous and intestinal orthotopic transplantation tumor models. Tumor tissues were harvested and subjected to immunofluorescence staining, flow cytometry analysis of tumor-infiltrating immune cell populations, tumor myeloid-derived suppressor cell (MDSC) sorting by isolation kit and then co-culture with spleen T cells, and RNA sequencing for transcriptomic analysis. RESULTS: We found that DCLK1-/- tumor cells lose their tumorigenicity under immune surveillance. Failed tumor establishment of DCLK1-/- was associated with an increase in infiltration of CD8+ T cells and effector CD4+ T cells, and reduced numbers of MDSCs in the tumor tissue. Furthermore, DCLK1 promoted the up-regulation of C-X-C motif ligand 1, which recruits MDSCs in CRC through chemokine C-X-C motif receptor 2. The ability of in vivo tumor growth of DCLK1-/- tumor cells was rescued by C-X-C motif ligand 1 overexpression. Collectively, we validated that DCLK1 promotes tumor growth in CRC through recruitment of T-cell-suppressive MDSCs. CONCLUSIONS: DCLK1-mediated immune suppression in tumor models allows escaping from the host's antitumor response. Because DCLK1 is one of the most common markers in gastrointestinal tumors, these results identify a precise therapeutic target for related clinical interventions.

Identification and Verification of SLC27A1, PTBP1 and EIF5A With Significantly Altered Expression in Aggressive Pituitary Adenomas.

Background: Aggressive pituitary adenoma encircling the internal carotid artery has a poor clinical prognosis because of a high surgical risk and a high recurrence rate. This seriously affects patients' quality of life and yet there is no effective medical treatment. The European Diagnostic Guidelines have recommended the use of temozolomide (TMZ) for these aggressive pituitary adenomas, but the treatment remission rate has been less than 50%. Methods: In this study, transcriptome sequencing of pituitary tumour tissues and TMZ-treated pituitary tumour cell lines were employed to explore the significance gene expressions affecting the efficacy of TMZ treatment for pituitary tumours. To clarify the roles of these gene expressions, six adult patients with pituitary adenomas treated in Tiantan Hospital from 2015 to 2020 and a pituitary adenoma cell line (Att20 sensitive to TMZ treatment) were analyzed by mRNA transcriptome sequencing. The differentially expressed genes were assayed by analyzing the sequencing results, and the expression level of these genes was further verified by immunohistochemistry. In addition, Ki67, VEGF, and p53 of the tumour tissues were also verified by immunohistochemistry. Results: In tumour tissues, mRNA sequencing showed that PTBP1 and EIF5A were significantly overexpressed in primary pituitary adenomas and SLC27A1 was significantly overexpressed in aggressive pituitary adenomas. Also in the pituitary adenoma cell line (AtT20), SLC27A1 expression levels were suppressed by TMZ treatment. Subsequent immunohistochemistry confirmed the sequencing results. Conclusion: High expression of SLC27A1 and low expression of EIF5A and PTBP1 may be potential indicators to predict the progression of aggressive pituitary adenomas, and patients with high SLC27A1 subtype may be sensitive to TMZ in clinical treatments.

Landscape of Peripheral Blood Mononuclear Cells and Soluble Factors in Severe COVID-19 Patients With Pulmonary Fibrosis Development.

Resulting from severe inflammation and cell destruction, COVID-19 patients could develop pulmonary fibrosis (PF), which remains in the convalescent stage. Nevertheless, how immune response participates in the pathogenesis of PF progression is not well defined. To investigate that question, 12 patients with severe COVID-19 were included in the study. Peripheral mononuclear cell (PBMC) samples were collected shortly after their admission and proceeded for single-cell RNA sequencing (scRNA-seq). After 14 days of discharge, the patients were revisited for chest CT scan. PF index (FI) was computed by AI-assisted CT images. Patients were categorized into FIhi and FIlo based on median of FI. By scRNA-seq analysis, our data demonstrated that frequency of CD4+ activated T cells and Treg cells were approximately 3-fold higher in FIhi patients compared with FIlo ones (p < 0.034 for all). By dissecting the differentially expressed genes, we found an overall downregulation of IFN-responsive genes (STAT1, IRF7, ISG15, ISG20, IFIs, and IFITMs) and S100s alarmins (S100A8, S100A9, S100A12, etc.) in all T-cell clusters, and cytotoxicity-related genes (GZMB, PRF1, and GNLY) in CTLs and γδ T cells in the FIhi cohort, compared with FIlo subjects. The GSEA analysis illustrated decreased expression of genes enriched in IFN signaling, innate immune response, adaptive immune response in T cells, NK cells, and monocytes in FIhi patients compared with FIlo ones. In conclusion, these data indicated that the attenuated IFN-responsive genes and their related signaling pathways could be critical for PF progression in COVID-19 patients.

A multi-tissue transcriptomic landscape of female mice in estrus and diestrus provides clues for precision medicine.

Female reproductive cycle, also known as menstrual cycle or estrous cycle in primate or non-primate mammals, respectively, dominates the reproductive processes in non-pregnant state. However, in addition to reproductive tissues, reproductive cycle could also perform global regulation because the receptors of two major female hormones fluctuating throughout the cycle, estrogen and progesterone, are widely distributed. Therefore, a multi-tissue gene expression landscape is in continuous demand for better understanding the systemic changes during the reproductive cycle but remains largely undefined. Here we delineated a transcriptomic landscape covering 15 tissues of C57BL/6J female mice in two phases of estrous cycle, estrus and diestrus, by RNA-sequencing. Then, a number of genes, pathways, and transcription factors involved in the estrous cycle were revealed. We found the estrous cycle could widely regulate the neuro-functions, immuno-functions, blood coagulation and so on. And behind the transcriptomic alteration between estrus and diestrus, 13 transcription factors may play important roles. Next, bioinformatics modeling with 1,263 manually curated gene signatures of various physiological and pathophysiological states systematically characterized the beneficial/deleterious effects brought by estrus/diestrus on individual tissues. We revealed that the estrous cycle has a significant effect on cardiovascular system (aorta, heart, vein), in which the anti-hypertensive pattern in aorta induced by estrus is one of the most striking findings. Inspired by this point, we validated that two hypotensive drugs, felodipine and acebutolol, could exhibit significantly enhanced efficacy in estrus than diestrus by mouse and rat experiments. Together, this study provides a valuable data resource for investigating reproductive cycle from a transcriptomic perspective, and presents models and clues for investigating precision medicine associated with reproductive cycle.

Comparative analysis of aneurysm subtypes associated genes based on protein-protein interaction network.

The arterial aneurysm refers to localized dilation of blood vessel wall and is common in general population. The majority of aneurysm cases remains asymptomatic until a sudden rupture which is usually fatal and of extremely high mortality (~ 50-60%). Therefore, early diagnosis, prevention and management of aneurysm are in urgent need. Unfortunately, current understanding of disease driver genes of various aneurysm subtypes is still limited, and without appropriate biomarkers and drug targets no specialized drug has been developed for aneurysm treatment. In this research, aneurysm subtypes were analyzed based on protein-protein interaction network to better understand aneurysm pathogenesis. By measuring network-based proximity of aneurysm subtypes, we identified a relevant closest relationship between aortic aneurysm and aortic dissection. An improved random walk method was performed to prioritize candidate driver genes of each aneurysm subtype. Thereafter, transcriptomes of 6 human aneurysm subtypes were collected and differential expression genes were identified to further filter potential driver genes. Functional enrichment of above driver genes indicated a general role of ubiquitination and programmed cell death in aneurysm pathogenesis. Especially, we further observed participation of BCL-2-mediated apoptosis pathway and caspase-1 related pyroptosis in the development of cerebral aneurysm and aneurysmal subarachnoid hemorrhage in corresponding transcriptomes.

Comprehensive Analysis of RNA Expression Correlations between Biofluids and Human Tissues.

In recent years, biofluid has been considered a promising source of non-invasive biomarkers for health monitoring and disease diagnosis. However, the expression consistency between biofluid and human tissue, which is fundamental to RNA biomarker development, has not been fully evaluated. In this study, we collected expression profiles across 53 human tissues and five main biofluid types. Utilizing the above dataset, we uncovered a globally positive correlation pattern between various biofluids (including blood, urine, bile, saliva and stool) and human tissues. However, significantly varied biofluid-tissue similarity levels and tendencies were observed between mRNA and lncRNA. Moreover, a higher correlation was found between biofluid types and their functionally related and anatomically closer tissues. In particular, a highly specific correlation was discovered between urine and the prostate. The biological sex of the donor was also proved to be an important influencing factor in biofluid-tissue correlation. Moreover, genes enriched in basic biological processes were found to display low variability across biofluid types, while genes enriched in catabolism-associated pathways were identified as highly variable.

NK cell-produced IFN-γ regulates cell growth and apoptosis of colorectal cancer by regulating IL-15.

Globally, colorectal cancer (CC) is the third leading cause of mortality associated with cancer. Natural killer (NK) cells are a major class of cells that are responsible for eliminating tumor cells and cytokine production. NK cell-mediated production of interferon gamma (IFN-γ) has antiviral, immunoregulatory and anti-tumor properties. IL-15 is important in linking inflammation with cancer. For instance, IL-15 promotes humoral and cell-mediated immune responses to inhibit tumor growth. IL-15 inhibits colitis-associated colon carcinogenesis by inducing antitumor immunity. However, the effect of NK cell-mediated IFN-γ on IL-15 expression in CC progression remains unknown. mRNA and protein level were detected using reverse transcription-quantitative PCR and western blotting, respectively. IFN-γ concentrations were detected using ELISAs. The cytotoxicity of NK-92 cells on SW480 cells was detected using cytoTox 96® non-radioactive cytotoxicity assays. Cell apoptosis and cell proliferation was detected using flow cytometry and CCK-8 assays, respectively. IL-2 was used for NK-92 stimulation, IL-15 antibodies were used to neutralize IL-15 bioactivity. For the present study, 21 patients with CC and 21 healthy volunteers were enrolled at the First Affiliated Hospital of Xi'an Jiaotong University. IL-15 mRNA and protein expression were significantly lower in NK cells isolated from the CC group compared with healthy volunteer group. IL-2 enhanced the production/secretion of IFN-γ in addition to enhancing NK-92 cell-mediated killing of SW480 cells. Compared with the control group, NK-92 cells treated with IL-2 alone significantly increased cell apoptosis, BAX expression levels as well as phosphorylated (p)-Janus kinase 2 and p-STAT1 protein levels, whilst reducing cell viability and Bcl-2 protein levels in SW480 cells. These observations were not made when treated with IL-2 and polyclonal antibody (pAb) targeting IL-15. Taken together, NK cell-mediated IFN-γ served a pivotal role in CC by regulating IL-15. The effects of IL-2 induced IFN-γ were abolished by pAb IL-15 treatment. The mechanisms of action behind how IFN-γ regulates IL-2 is unclear, and is a promising area for future research.

Inhibition of DCLK1 down-regulates PD-L1 expression through Hippo pathway in human pancreatic cancer.

Immunotherapy is one of the most promising strategies for cancer, compared with traditional treatments. As one of the key emerging immunotherapies, anti-PD-1/PD-L1 treatment has brought survival benefits to many advanced cancer patients. However, in pancreatic cancer, immunotherapy-based approaches have not achieved a favorable clinical effect because of mismatch repair deficiencies. Therefore, the majority of pancreatic tumors are regarded as immune-quiescent tumors and non-responsive to single-checkpoint blockade therapies. Many preclinical and clinical studies suggest that it is still important to clarify the regulatory mechanism of the PD-1/PD-L1 pathway in pancreatic cancer. As a marker of cancer stem cells, DCLK1 has been found to play an important role in the occurrence and development of a plethora of human cancers. Recent researches have revealed that DCLK1 is closely related to EMT process of tumor cells, meanwhile, it could also be used as a biomarker in gastrointestinal tumors to predict the prognoses of patients. However, the role that DCLK1 plays in the immune regulation of tumor microenvironments remains unknown. Therefore, we sought to understand if DCLK1 could positively regulate the expression of PD-L1 in pancreatic cancer cells. Furthermore, we examined if DCLK1 highly correlated with the Hippo pathway through TCGA database analysis. We found that DCLK1 helped regulate the level of PD-L1 expression by affecting the corresponding expression level of yes-associated protein in the Hippo pathway. Collectively, our study identifies DCLK1 as an important regulator of PD-L1 expression in pancreatic tumor and highlights a central role of DCLK1 in the regulation of tumor immunity.

The Role of Circulating CD16+CD56+ Natural Killer Cells in the Screening, Diagnosis, and Staging of Colorectal Cancer before Initial Treatment.

BACKGROUND AND OBJECTIVE: A reliable noninvasive prediction tool for the screening, diagnosis, and/or staging of colorectal cancer (CRC) before surgery is critical for the choice of treatment and prognosis. METHODS: Patients admitted for initial treatment of CRC between January 1, 2015, and December 31, 2018, were retrieved and reviewed. Records of CD16+CD56+ natural killer (NK) cells were analyzed according to the stages of CRC. RESULTS: The number of qualified participants in the healthy, stage I, stage II, stage III, and stage IV CRC patients were 60, 66, 60, 70, and 68, respectively. There was a significant difference in circulating CD16+CD56+ NK cells between the healthy group and the CRC group (p < 0.01), as well as between the healthy group and stage III or IV CRC group (p < 0.01 and 0.001, respectively). The percentage of circulating CD16+CD56+ NK cells in lymphocytes was negatively correlated with the occurrence of CRC. When comparing the pool of stage I and II CRC cases with the pool of stage III and IV CRC cases using circulating CD16+CD56+ NK cells, the area under the Receiver Operating Characteristic curve was 0.878. Using an optimal cutoff value of 15.6%, the OR was 0.06 (0.03, 0.11), p < 0.001, sensitivity was 86.5%, specificity was 72.5%, positive predictive value was 74.2%, and negative predictive value was 85.5%. CONCLUSIONS: Circulating CD16+CD56+ NK cells can be used as a screening and diagnostic/staging tool for CRC.

Circulating CD16+CD56+ nature killer cells indicate the prognosis of colorectal cancer after initial chemotherapy.

As the prognosis of colorectal cancer (CRC) does not always coincide with the pathology and/or surgical findings, a reliable noninvasive prediction tool for the prognosis of CRC is needed. Patients admitted for initial treatment of CRC between January 1, 2015 and December 31, 2015 were retrieved and reviewed. Records of circulating CD16+ CD56+ natural killer (NK) cells were analyzed before and after the initial chemotherapy of FOLFOX plan. Patients were followed up until June 30, 2019. One hundred and twenty-four cases after the FOLFOX chemotherapy were enrolled into this study. There were no significant differences in gender, age, or number of metastasis cases between the survival group and the nonsurvival group (p > 0.05), but significant differences in pre-chemotherapy, post-chemotherapy, and the differences between pre- and post-chemotherapy circulating CD16+ CD56+ NK cells between the survival group and the nonsurvival group (p < 0.01, p < 0.01, and p < 0.05, respectively) were observed. For the prediction of survival and nonsurvival CRC cases, the Areas Under the Curve were 0.626 and 0.759 in the Receiver-Operating Characteristic curves for the pre- and post-chemotherapy circulating CD16+ CD56+NK cells, respectively. Using an optimal cutoff value of 11.8% in post-chemotherapy circulating CD16+CD56+NK cells to differentiate survival and nonsurvival cases, the odds ratio was 0.12 (0.05, 0.27), p < 0.001. The percentages of both pre-chemotherapy and post-chemotherapy circulating CD16+CD56+NK cells were negatively correlated with the prognosis of CRC. The percentage of post-chemotherapy circulating CD16+CD56+NK cells was able to effectively predict the prognosis of CRC cases.

AGD: Aneurysm Gene Database.

An aneurysm is an outward bulge on an arterial wall. Aneurysms are becoming a serious public health concern as the worldwide population ages. Unfortunately, no effective drugs have been developed for aneurysms to date. In addition, aneurysms may be associated with grave prognosis due to conditions such as ruptures and recurrence. Altogether, these factors make earlier aneurysm prevention, diagnosis and intervention strategies even more important. A bioinformatics resource for aneurysm-associated molecules would be helpful for addressing the above issues; however, such a tool is not yet available. In this study, we developed Aneurysm Gene Database (AGD) for the above purpose. AGD contains 1472 aneurysm-gene associations, including 29 types of aneurysms, 967 protein-coding genes, 29 miRNAs, 6 lncRNAs and several other types of molecules. Users can search, browse and download content in AGD. We believe that AGD is a valuable resource that can help us better understand aneurysms and discover novel treatment targets.

PIMT/NCOA6IP Deletion in the Mouse Heart Causes Delayed Cardiomyopathy Attributable to Perturbation in Energy Metabolism.

PIMT/NCOA6IP, a transcriptional coactivator PRIP/NCOA6 binding protein, enhances nuclear receptor transcriptional activity. Germline disruption of PIMT results in early embryonic lethality due to impairment of development around blastocyst and uterine implantation stages. We now generated mice with Cre-mediated cardiac-specific deletion of PIMT (csPIMT-/-) in adult mice. These mice manifest enlargement of heart, with nearly 100% mortality by 7.5 months of age due to dilated cardiomyopathy. Significant reductions in the expression of genes (i) pertaining to mitochondrial respiratory chain complexes I to IV; (ii) calcium cycling cardiac muscle contraction (Atp2a1, Atp2a2, Ryr2); and (iii) nuclear receptor PPAR- regulated genes involved in glucose and fatty acid energy metabolism were found in csPIMT-/- mouse heart. Elevated levels of Nppa and Nppb mRNAs were noted in csPIMT-/- heart indicative of myocardial damage. These hearts revealed increased reparative fibrosis associated with enhanced expression of Tgfß2 and Ctgf. Furthermore, cardiac-specific deletion of PIMT in adult mice, using tamoxifen-inducible Cre-approach (TmcsPIMT-/-), results in the development of cardiomyopathy. Thus, cumulative evidence suggests that PIMT functions in cardiac energy metabolism by interacting with nuclear receptor coactivators and this property could be useful in the management of heart failure.