Background Increasing evidences have suggested an important role of microRNAs (miRNAs) in regulating cell death processes including NETosis and apoptosis. Dysregulated expression of miRNAs and increased formation of neutrophil extracellular traps (NETs) and apoptosis participate in autoimmune-mediated diffuse alveolar hemorrhage (DAH), mostly associated with pulmonary capillaritis in systemic lupus erythematosus (SLE) patients. In particular, besides the inhibition of apoptosis, miR-146a can control innate and acquired immune responses, and regulate the toll-like receptor pathway through targeting TRAF6 to reduce the expression of pro-inflammatory cytokines/chemokines like IL-8, a NETosis inducer. Methods Expression of miR-146a, TRAF6 and NETs were examined in peripheral blood neutrophils (PBNs) and lung tissues from SLE-associated DAH patients, and in neutrophils and pristane-induced DAH lung tissues from C57BL/6 mice. To assess NETs formation, we examined NETosis-related DNAs morphology and crucial mediators including protein arginine deiminase 4 and citrullinated Histone 3. Expression of miR-146a and its endogenous RNA SNHG16 were studied in HL-60 promyelocytic cells and MLE-12 alveolar cells during NETosis and apoptosis processes, respectively. MiR-146a-overexpressed and CRISPR-Cas13d-mediated SNHG16-silenced HL-60 cells were investigated for NETosis. MiR-146a-overexpressed MLE-12 cells were analyzed for apoptosis. Pristane-injected mice received intra-pulmonary miR-146a delivery to evaluate therapeutic efficacy in DAH. Results In DAH patients, there were down-regulated miR-146a levels with increased TRAF6 expression and PMA/LPS-induced NETosis in PBNs, and down-regulated miR-146a levels with increased TRAF6, high-mobility group box 1 (HMGB1), IL-8, NETs and apoptosis expression in lung tissues. HMGB1-stimulated mouse neutrophils had down-regulated miR-146a levels with increased TRAF6, IL-8 and NETs expression. PMA-stimulated HL-60 cells had down-regulated miR-146a levels with enhanced NETosis. MiR-146a-overexpressed or SNHG16-silenced HL-60 cells showed reduced NETosis. Apoptotic MLE-12 cells had down-regulated miR-146a expression and increased HMGB1 release, while miR-146a-overexpressed MLE-12 cells showed reduced apoptosis and HMGB1 production. There were down-regulated miR-146a levels with increased TRAF6, HMGB1, IL-8, NETs and apoptosis expression in mouse DAH lung tissues. Intra-pulmonary miR-146a delivery could suppress DAH by reducing TRAF6, IL-8, NETs and apoptosis expression. Conclusions Our results demonstrate firstly down-regulated pulmonary miR-146a levels with increased TRAF6 and IL-8 expression and NETs and apoptosis formation in autoimmune-mediated DAH, and implicate a therapeutic potential of intra-pulmonary miR-146a delivery.

Survival prediction is highly valued in end-of-life care clinical practice, and patient performance status evaluation stands as a predominant component in survival prognostication. While current performance status evaluation tools are limited to their subjective nature, the advent of wearable technology enables continual recordings of patients' activity and has the potential to measure performance status objectively. We hypothesize that wristband actigraphy monitoring devices can predict in-hospital death of end-stage cancer patients during the time of their hospital admissions. The objective of this study was to train and validate a long short-term memory (LSTM) deep-learning prediction model based on activity data of wearable actigraphy devices. The study recruited 60 end-stage cancer patients in a hospice care unit, with 28 deaths and 32 discharged in stable condition at the end of their hospital stay. The standard Karnofsky Performance Status score had an overall prognostic accuracy of 0.83. The LSTM prediction model based on patients' continual actigraphy monitoring had an overall prognostic accuracy of 0.83. Furthermore, the model performance improved with longer input data length up to 48 h. In conclusion, our research suggests the potential feasibility of wristband actigraphy to predict end-of-life admission outcomes in palliative care for end-stage cancer patients.Clinical Trial Registration: The study protocol was registered on ClinicalTrials.gov (ID: NCT04883879).

Systemic lupus erythematosus (SLE) is a systemic autoimmune disease with enhanced NETosis and impaired degradation of neutrophil extracellular traps (NETs). Galectin-3 is a β-galactoside binding protein and is associated with neutrophil functions as well as involved in mediating autoimmune disorders. In this study, we plan to examine the associations of galectin-3 with the pathogenesis of SLE and NETosis. Galectin-3 expression levels were determined in peripheral blood mononuclear cells (PBMCs) of SLE patients for the association with lupus nephritis (LN) or correlation of SLE disease activity index 2000 (SLEDAI-2K). NETosis was observed in human normal and SLE and murine galectin-3 knockout (Gal-3 KO) neutrophils. Gal-3 KO and wild-type (WT) mice induced by pristane were used to evaluate disease signs, including diffuse alveolar haemorrhage (DAH), LN, proteinuria, anti-ribonucleoprotein (RNP) antibody, citrullinated histone 3 (CitH3) levels, and NETosis. Galectin-3 levels are higher in PBMCs of SLE patients compared with normal donors and positively correlated with LN or SLEDAI-2K. Gal-3 KO mice have higher percent survival and lower DAH, LN proteinuria, and anti-RNP antibody levels than WT mice induced by pristane. NETosis and citH3 levels are reduced in Gal-3 KO neutrophils. Furthermore, galectin-3 resides in NETs while human neutrophils undergo NETosis. Galectin-3-associated immune complex deposition can be observed in NETs from spontaneously NETotic cells of SLE patients. In this study, we provide clinical relevance of galectin-3 to the lupus phenotypes and the underlying mechanisms of galectin-3-mediated NETosis for developing novel therapeutic strategies targeting galectin-3 for SLE.

Background: Accelerated cell apoptosis is a crucial pathogenic mechanism in lupus nephritis (LN) with dysregulated expression levels of long noncoding RNAs (lncRNAs). The expression of pro-apoptotic lincRNA-p21 and its competing endogenous RNA target miR-181a were studied in LN patients, human kidney cell and T-lymphocyte lines with CRISPR interference-conducted repression and lentiviral vector-mediated overexpression of lincRNA-p21, and a mouse LN model. Methods: Clinical samples were collected from LN patients with higher disease activity and control subjects including lupus patients without renal involvement and age/sex-matched healthy controls (HCs). The expression of lincRNA-p21, H19 (anti-apoptotic lncRNA) and miR-181a were examined in peripheral blood mononuclear cells (PBMNCs) and urine cells, and analyzed for clinical correlation. Cell lines were treated with doxorubicin (Dox) to induce apoptosis and evaluate for the expression of lincRNA-p21, caspase 3 and p21. LincRNA-p21-silened HEK 293T and Jurkat transfectants were examined for apoptosis and miR-181a expression. LincRNA-p21-overexpressed HK-2 cells were examined for apoptosis and p53-related down-stream molecules levels. Female Balb/C mice were injected with pristane to induce LN, and examined for the expression of anti-DNA, proteinuria, lincRNA-p21, caspase 3 and p21 as well as in situ apoptosis. Results: Up-regulated expression of lincRNA-p21 rather than H19 were identified in PBMNCs from LN patients, positively correlated with disease activity and proteinuria amount. Higher lincRNA-p21 levels were identified in LN CD4+T cells than other subpopulations. LN urine cells had greater lincRNA-p21 levels than HCs. There were lower miR-181a levels in PBMNCs from LN patients, negatively correlated with disease activity. Dox-induced apoptotic cell lines had up-regulated levels of lincRNA-p21, caspase 3 and p21, whereas down-regulated miR-181a expression with decreased TCRζchain and IL-2 levels was identified in Jurkat cells. LincRNA-p21-silenced transfectants displayed reduced apoptosis with up-regulated miR-181a expression. LincRNA-p21-overexpressed HK-2 cells revealed enhanced apoptosis with up-regulated expression of downstream PUMA and Bax molecules. LN mice had in situ apoptosis and progressively increased anti-dsDNA, proteinuria and renal lincRNA-p21 levels with up-regulated expression of caspase 3 and p21.Conclusions: By using clinical samples, human cell lines and a mouse model, we demonstrate up-regulated expression of lincRNA-p21 in LN, implicating a potential activity biomarker and therapeutic target.