Trained Immunity and Reactivity of Macrophages and Endothelial Cells.

Innate immune cells can develop exacerbated immunologic response and long-term inflammatory phenotype following brief exposure to endogenous or exogenous insults, which leads to an altered response towards a second challenge after the return to a nonactivated state. This phenomenon is known as trained immunity (TI). TI is not only important for host defense and vaccine response but also for chronic inflammations such as cardiovascular and metabolic diseases such as atherosclerosis. TI can occur in innate immune cells such as monocytes/macrophages, natural killer cells, endothelial cells (ECs), and nonimmune cells, such as fibroblast. In this brief review, we analyze the significance of TI in ECs, which are also considered as innate immune cells in addition to macrophages. TI can be induced by a variety of stimuli, including lipopolysaccharides, BCG (bacillus Calmette-Guerin), and oxLDL (oxidized low-density lipoprotein), which are defined as risk factors for cardiovascular and metabolic diseases. Furthermore, TI in ECs is functional for inflammation effectiveness and transition to chronic inflammation. Rewiring of cellular metabolism of the trained cells takes place during induction of TI, including increased glycolysis, glutaminolysis, increased accumulation of tricarboxylic acid cycle metabolites and acetyl-coenzyme A production, as well as increased mevalonate synthesis. Subsequently, this leads to epigenetic remodeling, resulting in important changes in chromatin architecture that enables increased gene transcription and enhanced proinflammatory immune response. However, TI pathways and inflammatory pathways are separated to ensure memory stays when inflammation undergoes resolution. Additionally, reactive oxygen species play context-dependent roles in TI. Therefore, TI plays significant roles in EC and macrophage pathology and chronic inflammation. However, further characterization of TI in ECs and macrophages would provide novel insights into cardiovascular disease pathogenesis and new therapeutic targets. Graphic Abstract: A graphic abstract is available for this article.

Vascular Endothelial Cells and Innate Immunity.

In addition to the roles of endothelial cells (ECs) in physiological processes, ECs actively participate in both innate and adaptive immune responses. We previously reported that, in comparison to macrophages, a prototypic innate immune cell type, ECs have many innate immune functions that macrophages carry out, including cytokine secretion, phagocytic function, antigen presentation, pathogen-associated molecular patterns-, and danger-associated molecular patterns-sensing, proinflammatory, immune-enhancing, anti-inflammatory, immunosuppression, migration, heterogeneity, and plasticity. In this highlight, we introduce recent advances published in both ATVB and many other journals: (1) several significant characters classify ECs as novel immune cells not only in infections and allograft transplantation but also in metabolic diseases; (2) several new receptor systems including conditional danger-associated molecular pattern receptors, nonpattern receptors, and homeostasis associated molecular patterns receptors contribute to innate immune functions of ECs; (3) immunometabolism and innate immune memory determine the innate immune functions of ECs; (4) a great induction of the immune checkpoint receptors in ECs during inflammations suggests the immune tolerogenic functions of ECs; and (5) association of immune checkpoint inhibitors with cardiovascular adverse events and cardio-oncology indicates the potential contributions of ECs as innate immune cells.

Caspase-4/11 promotes hyperlipidemia and chronic kidney disease-accelerated vascular inflammation by enhancing trained immunity.

To determine whether hyperlipidemia and chronic kidney disease (CKD) have a synergy in accelerating vascular inflammation via trained immunity (TI), we performed aortic pathological analysis and RNA-Seq of high-fat diet-fed (HFD-fed) 5/6 nephrectomy CKD (HFD+CKD) mice. We made the following findings: (a) HFD+CKD increased aortic cytosolic LPS levels, caspase-11 (CASP11) activation, and 998 gene expressions of TI pathways in the aorta (first-tier TI mechanism); (b) CASP11-/- decreased aortic neointima hyperplasia, aortic recruitment of macrophages, and casp11-gasdermin D-mediated cytokine secretion; (c) CASP11-/- decreased N-terminal gasdermin D (N-GSDMD) membrane expression on aortic endothelial cells and aortic IL-1B levels; (d) LPS transfection into human aortic endothelial cells resulted in CASP4 (human)/CASP11 (mouse) activation and increased N-GSDMD membrane expression; and (e) IL-1B served as the second-tier mechanism underlying HFD+CKD-promoted TI. Taken together, hyperlipidemia and CKD accelerated vascular inflammation by promoting 2-tier trained immunity.

Caspase-11 promotes high-fat diet-induced NAFLD by increasing glycolysis, OXPHOS, and pyroptosis in macrophages.

Introduction: Non-alcoholic fatty liver disease (NAFLD) has a global prevalence of 25% of the population and is a leading cause of cirrhosis and hepatocellular carcinoma. NAFLD ranges from simple steatosis (non-alcoholic fatty liver) to non-alcoholic steatohepatitis (NASH). Hepatic macrophages, specifically Kupffer cells (KCs) and monocyte-derived macrophages, act as key players in the progression of NAFLD. Caspases are a family of endoproteases that provide critical connections to cell regulatory networks that sense disease risk factors, control inflammation, and mediate inflammatory cell death (pyroptosis). Caspase-11 can cleave gasdermin D (GSDMD) to induce pyroptosis and specifically defends against bacterial pathogens that invade the cytosol. However, it's still unknown whether high fat diet (HFD)-facilitated gut microbiota-generated cytoplasmic lipopolysaccharides (LPS) activate caspase-11 and promote NAFLD. Methods: To examine this hypothesis, we performed liver pathological analysis, RNA-seq, FACS, Western blots, Seahorse mitochondrial stress analyses of macrophages and bone marrow transplantation on HFD-induced NAFLD in WT and Casp11-/- mice. Results and Discussion: Our results showed that 1) HFD increases body wight, liver wight, plasma cholesterol levels, liver fat deposition, and NAFLD activity score (NAS score) in wild-type (WT) mice; 2) HFD increases the expression of caspase-11, GSDMD, interleukin-1ß, and guanylate-binding proteins in WT mice; 3) Caspase-11 deficiency decreases fat liver deposition and NAS score; 4) Caspase-11 deficiency decreases bone marrow monocyte-derived macrophage (MDM) pyroptosis (inflammatory cell death) and inflammatory monocyte (IM) surface GSDMD expression; 5) Caspase-11 deficiency re-programs liver transcriptomes and reduces HFD-induced NAFLD; 6) Caspase-11 deficiency decreases extracellular acidification rates (glycolysis) and oxidative phosphorylation (OXPHOS) in inflammatory fatty acid palmitic acid-stimulated macrophages, indicating that caspase-11 significantly contributes to maintain dual fuel bioenergetics-glycolysis and OXPHOS for promoting pyroptosis in macrophages. These results provide novel insights on the roles of the caspase-11-GSDMD pathway in promoting hepatic macrophage inflammation and pyroptosis and novel targets for future therapeutic interventions involving the transition of NAFLD to NASH, hyperlipidemia, type II diabetes, metabolic syndrome, metabolically healthy obesity, atherosclerotic cardiovascular diseases, autoimmune diseases, liver transplantation, and hepatic cancers.

Monocyte Adhesion Assays for Detecting Endothelial Cell Activation in Vascular Inflammation and Atherosclerosis.

Monocyte adhesion assay, a fluorescence-based method, enables the detection and quantification of monocyte adhesion to endothelial cell (EC) monolayers in vitro and measures EC activation. We describe in this chapter a monocyte adhesion assay based on two published papers from our laboratory that can be effectively used in studying the mechanisms of both pro- and anti-inflammatory cytokines in EC activation. Endothelial cell monolayers are cultured and treated with desired drug, cytokines, or other stimuli and incubated with fluorescently labeled monocytes.

Aorta in Pathologies May Function as an Immune Organ by Upregulating Secretomes for Immune and Vascular Cell Activation, Differentiation and Trans-Differentiation-Early Secretomes may Serve as Drivers for Trained Immunity.

To determine whether aorta becomes immune organ in pathologies, we performed transcriptomic analyses of six types of secretomic genes (SGs) in aorta and vascular cells and made the following findings: 1) 53.7% out of 21,306 human protein genes are classified into six secretomes, namely, canonical, caspase 1, caspase 4, exosome, Weibel-Palade body, and autophagy; 2) Atherosclerosis (AS), chronic kidney disease (CKD) and abdominal aortic aneurysm (AAA) modulate six secretomes in aortas; and Middle East Respiratory Syndrome Coronavirus (MERS-CoV, COVID-19 homologous) infected endothelial cells (ECs) and angiotensin-II (Ang-II) treated vascular smooth muscle cells (VSMCs) modulate six secretomes; 3) AS aortas upregulate T and B cell immune SGs; CKD aortas upregulate SGs for cardiac hypertrophy, and hepatic fibrosis; and AAA aorta upregulate SGs for neuromuscular signaling and protein catabolism; 4) Ang-II induced AAA, canonical, caspase 4, and exosome SGs have two expression peaks of high (day 7)-low (day 14)-high (day 28) patterns; 5) Elastase induced AAA aortas have more inflammatory/immune pathways than that of Ang-II induced AAA aortas; 6) Most disease-upregulated cytokines in aorta may be secreted via canonical and exosome secretomes; 7) Canonical and caspase 1 SGs play roles at early MERS-CoV infected ECs whereas caspase 4 and exosome SGs play roles in late/chronic phases; and the early upregulated canonical and caspase 1 SGs may function as drivers for trained immunity (innate immune memory); 8) Venous ECs from arteriovenous fistula (AVF) upregulate SGs in five secretomes; and 9) Increased some of 101 trained immunity genes and decreased trained tolerance regulator IRG1 participate in upregulations of SGs in atherosclerotic, Ang-II induced AAA and CKD aortas, and MERS-CoV infected ECs, but less in SGs upregulated in AVF ECs. IL-1 family cytokines, HIF1α, SET7 and mTOR, ROS regulators NRF2 and NOX2 partially regulate trained immunity genes; and NRF2 plays roles in downregulating SGs more than that of NOX2 in upregulating SGs. These results provide novel insights on the roles of aorta as immune organ in upregulating secretomes and driving immune and vascular cell differentiations in COVID-19, cardiovascular diseases, inflammations, transplantations, autoimmune diseases and cancers.

29 m6A-RNA Methylation (Epitranscriptomic) Regulators Are Regulated in 41 Diseases including Atherosclerosis and Tumors Potentially via ROS Regulation - 102 Transcriptomic Dataset Analyses.

We performed a database mining on 102 transcriptomic datasets for the expressions of 29 m6A-RNA methylation (epitranscriptomic) regulators (m6A-RMRs) in 41 diseases and cancers and made significant findings: (1) a few m6A-RMRs were upregulated; and most m6A-RMRs were downregulated in sepsis, acute respiratory distress syndrome, shock, and trauma; (2) half of 29 m6A-RMRs were downregulated in atherosclerosis; (3) inflammatory bowel disease and rheumatoid arthritis modulated m6A-RMRs more than lupus and psoriasis; (4) some organ failures shared eight upregulated m6A-RMRs; end-stage renal failure (ESRF) downregulated 85% of m6A-RMRs; (5) Middle-East respiratory syndrome coronavirus infections modulated m6A-RMRs the most among viral infections; (6) proinflammatory oxPAPC modulated m6A-RMRs more than DAMP stimulation including LPS and oxLDL; (7) upregulated m6A-RMRs were more than downregulated m6A-RMRs in cancer types; five types of cancers upregulated ≥10 m6A-RMRs; (8) proinflammatory M1 macrophages upregulated seven m6A-RMRs; (9) 86% of m6A-RMRs were differentially expressed in the six clusters of CD4+Foxp3+ immunosuppressive Treg, and 8 out of 12 Treg signatures regulated m6A-RMRs; (10) immune checkpoint receptors TIM3, TIGIT, PD-L2, and CTLA4 modulated m6A-RMRs, and inhibition of CD40 upregulated m6A-RMRs; (11) cytokines and interferons modulated m6A-RMRs; (12) NF-κB and JAK/STAT pathways upregulated more than downregulated m6A-RMRs whereas TP53, PTEN, and APC did the opposite; (13) methionine-homocysteine-methyl cycle enzyme Mthfd1 downregulated more than upregulated m6A-RMRs; (14) m6A writer RBM15 and one m6A eraser FTO, H3K4 methyltransferase MLL1, and DNA methyltransferase, DNMT1, regulated m6A-RMRs; and (15) 40 out of 165 ROS regulators were modulated by m6A eraser FTO and two m6A writers METTL3 and WTAP. Our findings shed new light on the functions of upregulated m6A-RMRs in 41 diseases and cancers, nine cellular and molecular mechanisms, novel therapeutic targets for inflammatory disorders, metabolic cardiovascular diseases, autoimmune diseases, organ failures, and cancers.

Hyperlipidemia May Synergize with Hypomethylation in Establishing Trained Immunity and Promoting Inflammation in NASH and NAFLD.

We performed a panoramic analysis on both human nonalcoholic steatohepatitis (NASH) microarray data and microarray/RNA-seq data from various mouse models of nonalcoholic fatty liver disease NASH/NAFLD with total 4249 genes examined and made the following findings: (i) human NASH and NAFLD mouse models upregulate both cytokines and chemokines; (ii) pathway analysis indicated that human NASH can be classified into metabolic and immune NASH; methionine- and choline-deficient (MCD)+high-fat diet (HFD), glycine N-methyltransferase deficient (GNMT-KO), methionine adenosyltransferase 1A deficient (MAT1A-KO), and HFCD (high-fat-cholesterol diet) can be classified into inflammatory, SAM accumulation, cholesterol/mevalonate, and LXR/RXR-fatty acid ß-oxidation NAFLD, respectively; (iii) canonical and noncanonical inflammasomes play differential roles in the pathogenesis of NASH/NAFLD; (iv) trained immunity (TI) enzymes are significantly upregulated in NASH/NAFLD; HFCD upregulates TI enzymes more than cytokines, chemokines, and inflammasome regulators; (v) the MCD+HFD is a model with the upregulation of proinflammatory cytokines and canonical and noncanonical inflammasomes; however, the HFCD is a model with upregulation of TI enzymes and lipid peroxidation enzymes; and (vi) caspase-11 and caspase-1 act as upstream master regulators, which partially upregulate the expressions of cytokines, chemokines, canonical and noncanonical inflammasome pathway regulators, TI enzymes, and lipid peroxidation enzymes. Our findings provide novel insights on the synergies between hyperlipidemia and hypomethylation in establishing TI and promoting inflammation in NASH and NAFLD progression and novel targets for future therapeutic interventions for NASH and NAFLD, metabolic diseases, transplantation, and cancers.

IL-35 promotes CD4+Foxp3+ Tregs and inhibits atherosclerosis via maintaining CCR5-amplified Treg-suppressive mechanisms.

Tregs play vital roles in suppressing atherogenesis. Pathological conditions reshape Tregs and increase Treg-weakening plasticity. It remains unclear how Tregs preserve their function and how Tregs switch into alternative phenotypes in the environment of atherosclerosis. In this study, we observed a great induction of CD4+Foxp3+ Tregs in the spleen and aorta of ApoE-/- mice, accompanied by a significant increase of plasma IL-35 levels. To determine if IL-35 devotes its role in the rise of Tregs, we generated IL-35 subunit P35-deficient (IL-35P35-deficient) mice on an ApoE-/- background and found Treg reduction in the spleen and aorta compared with ApoE-/- controls. In addition, our RNA sequencing data show the elevation of a set of chemokine receptor transcripts in the ApoE-/- Tregs, and we have validated higher CCR5 expression in ApoE-/- Tregs in the presence of IL-35 than in the absence of IL-35. Furthermore, we observed that CCR5+ Tregs in ApoE-/- have lower Treg-weakening AKT-mTOR signaling, higher expression of inhibitory checkpoint receptors TIGIT and PD-1, and higher expression of IL-10 compared with WT CCR5+ Tregs. In conclusion, IL-35 counteracts hyperlipidemia in maintaining Treg-suppressive function by increasing 3 CCR5-amplified mechanisms, including Treg migration, inhibition of Treg weakening AKT-mTOR signaling, and promotion of TIGIT and PD-1 signaling.

Procaspase-1 patrolled to the nucleus of proatherogenic lipid LPC-activated human aortic endothelial cells induces ROS promoter CYP1B1 and strong inflammation.

To determine the roles of nuclear localization of pro-caspase-1 in human aortic endothelial cells (HAECs) activated by proatherogenic lipid lysophosphatidylcholine (LPC), we examined cytosolic and nuclear localization of pro-caspase-1, identified nuclear export signal (NES) in pro-caspase-1 and sequenced RNAs. We made the following findings: 1) LPC increases nuclear localization of procaspase-1 in HAECs. 2) Nuclear pro-caspase-1 exports back to the cytosol, which is facilitated by a leptomycin B-inhibited mechanism. 3) Increased nuclear localization of pro-caspase-1 by a new NES peptide inhibitor upregulates inflammatory genes in oxidative stress and Th17 pathways; and SUMO activator N106 enhances nuclear localization of pro-caspase-1 and caspase-1 activation (p20) in the nucleus. 4) LPC plus caspase-1 enzymatic inhibitor upregulates inflammatory genes with hypercytokinemia/hyperchemokinemia and interferon pathways, suggesting a novel capsase-1 enzyme-independent inflammatory mechanism. 5) LPC in combination with NES inhibitor and caspase-1 inhibitor upregulate inflammatory gene expression that regulate Th17 activation, endotheli-1 signaling, p38-, and ERK- MAPK pathways. To examine two hallmarks of endothelial activation such as secretomes and membrane protein signaling, LPC plus NES inhibitor upregulate 57 canonical secretomic genes and 76 exosome secretomic genes, respectively, promoting four pathways including Th17, IL-17 promoted cytokines, interferon signaling and cholesterol biosynthesis. LPC with NES inhibitor also promote inflammation via upregulating ROS promoter CYP1B1 and 11 clusters of differentiation (CD) membrane protein pathways. Mechanistically, all the LPC plus NES inhibitor-induced genes are significantly downregulated in CYP1B1-deficient microarray, suggesting that nuclear caspase-1-induced CYP1B1 promotes strong inflammation. These transcriptomic results provide novel insights on the roles of nuclear caspase-1 in sensing DAMPs, inducing ROS promoter CYP1B1 and in regulating a large number of genes that mediate HAEC activation and inflammation. These findings will lead to future development of novel therapeutics for cardiovascular diseases (CVD), inflammations, infections, transplantation, autoimmune disease and cancers. (total words: 284).

Endothelial Immunity Trained by Coronavirus Infections, DAMP Stimulations and Regulated by Anti-Oxidant NRF2 May Contribute to Inflammations, Myelopoiesis, COVID-19 Cytokine Storms and Thromboembolism.

To characterize transcriptomic changes in endothelial cells (ECs) infected by coronaviruses, and stimulated by DAMPs, the expressions of 1311 innate immune regulatomic genes (IGs) were examined in 28 EC microarray datasets with 7 monocyte datasets as controls. We made the following findings: The majority of IGs are upregulated in the first 12 hours post-infection (PI), and maintained until 48 hours PI in human microvascular EC infected by middle east respiratory syndrome-coronavirus (MERS-CoV) (an EC model for COVID-19). The expressions of IGs are modulated in 21 human EC transcriptomic datasets by various PAMPs/DAMPs, including LPS, LPC, shear stress, hyperlipidemia and oxLDL. Upregulation of many IGs such as nucleic acid sensors are shared between ECs infected by MERS-CoV and those stimulated by PAMPs and DAMPs. Human heart EC and mouse aortic EC express all four types of coronavirus receptors such as ANPEP, CEACAM1, ACE2, DPP4 and virus entry facilitator TMPRSS2 (heart EC); most of coronavirus replication-transcription protein complexes are expressed in HMEC, which contribute to viremia, thromboembolism, and cardiovascular comorbidities of COVID-19. ECs have novel trained immunity (TI), in which subsequent inflammation is enhanced. Upregulated proinflammatory cytokines such as TNFα, IL6, CSF1 and CSF3 and TI marker IL-32 as well as TI metabolic enzymes and epigenetic enzymes indicate TI function in HMEC infected by MERS-CoV, which may drive cytokine storms. Upregulated CSF1 and CSF3 demonstrate a novel function of ECs in promoting myelopoiesis. Mechanistically, the ER stress and ROS, together with decreased mitochondrial OXPHOS complexes, facilitate a proinflammatory response and TI. Additionally, an increase of the regulators of mitotic catastrophe cell death, apoptosis, ferroptosis, inflammasomes-driven pyroptosis in ECs infected with MERS-CoV and the upregulation of pro-thrombogenic factors increase thromboembolism potential. Finally, NRF2-suppressed ROS regulate innate immune responses, TI, thrombosis, EC inflammation and death. These transcriptomic results provide novel insights on the roles of ECs in coronavirus infections such as COVID-19, cardiovascular diseases (CVD), inflammation, transplantation, autoimmune disease and cancers.

Novel Knowledge-Based Transcriptomic Profiling of Lipid Lysophosphatidylinositol-Induced Endothelial Cell Activation.

To determine whether pro-inflammatory lipid lysophosphatidylinositols (LPIs) upregulate the expressions of membrane proteins for adhesion/signaling and secretory proteins in human aortic endothelial cell (HAEC) activation, we developed an EC biology knowledge-based transcriptomic formula to profile RNA-Seq data panoramically. We made the following primary findings: first, G protein-coupled receptor 55 (GPR55), the LPI receptor, is expressed in the endothelium of both human and mouse aortas, and is significantly upregulated in hyperlipidemia; second, LPIs upregulate 43 clusters of differentiation (CD) in HAECs, promoting EC activation, innate immune trans-differentiation, and immune/inflammatory responses; 72.1% of LPI-upregulated CDs are not induced in influenza virus-, MERS-CoV virus- and herpes virus-infected human endothelial cells, which hinted the specificity of LPIs in HAEC activation; third, LPIs upregulate six types of 640 secretomic genes (SGs), namely, 216 canonical SGs, 60 caspase-1-gasdermin D (GSDMD) SGs, 117 caspase-4/11-GSDMD SGs, 40 exosome SGs, 179 Human Protein Atlas (HPA)-cytokines, and 28 HPA-chemokines, which make HAECs a large secretory organ for inflammation/immune responses and other functions; fourth, LPIs activate transcriptomic remodeling by upregulating 172 transcription factors (TFs), namely, pro-inflammatory factors NR4A3, FOS, KLF3, and HIF1A; fifth, LPIs upregulate 152 nuclear DNA-encoded mitochondrial (mitoCarta) genes, which alter mitochondrial mechanisms and functions, such as mitochondrial organization, respiration, translation, and transport; sixth, LPIs activate reactive oxygen species (ROS) mechanism by upregulating 18 ROS regulators; finally, utilizing the Cytoscape software, we found that three mechanisms, namely, LPI-upregulated TFs, mitoCarta genes, and ROS regulators, are integrated to promote HAEC activation. Our results provide novel insights into aortic EC activation, formulate an EC biology knowledge-based transcriptomic profile strategy, and identify new targets for the development of therapeutics for cardiovascular diseases, inflammatory conditions, immune diseases, organ transplantation, aging, and cancers.

Ultrasound May Suppress Tumor Growth, Inhibit Inflammation, and Establish Tolerogenesis by Remodeling Innatome via Pathways of ROS, Immune Checkpoints, Cytokines, and Trained Immunity/Tolerance.

BACKGROUND: The immune mechanisms underlying low-intensity ultrasound- (LIUS-) mediated suppression of inflammation and tumorigenesis remain poorly determined. METHODS: We used microarray datasets from the NCBI GEO DataSet repository and conducted comprehensive data-mining analyses, where we examined the gene expression of 1376 innate immune regulators (innatome genes (IGs) in cells treated with LIUS. RESULTS: We made the following findings: (1) LIUS upregulates proinflammatory IGs and downregulates metastasis genes in cancer cells, and LIUS upregulates adaptive immunity pathways but inhibits danger-sensing and inflammation pathways and promote tolerogenic differentiation in bone marrow (BM) cells. (2) LIUS upregulates IGs encoded for proteins localized in the cytoplasm, extracellular space, and others, but downregulates IG proteins localized in nuclear and plasma membranes, and LIUS downregulates phosphatases. (3) LIUS-modulated IGs act partially via several important pathways of reactive oxygen species (ROS), reverse signaling of immune checkpoint receptors B7-H4 and BTNL2, inflammatory cytokines, and static or oscillatory shear stress and heat generation, among which ROS is a dominant mechanism. (4) LIUS upregulates trained immunity enzymes in lymphoma cells and downregulates trained immunity enzymes and presumably establishes trained tolerance in BM cells. (5) LIUS modulates chromatin long-range interactions to differentially regulate IGs expression in cancer cells and noncancer cells. CONCLUSIONS: Our analysis suggests novel molecular mechanisms that are utilized by LIUS to induce tumor suppression and inflammation inhibition. Our findings may lead to development of new treatment protocols for cancers and chronic inflammation.

Canonical Secretomes, Innate Immune Caspase-1-, 4/11-Gasdermin D Non-Canonical Secretomes and Exosomes May Contribute to Maintain Treg-Ness for Treg Immunosuppression, Tissue Repair and Modulate Anti-Tumor Immunity via ROS Pathways.

We performed a transcriptomic analyses using the strategies we pioneered and made the following findings: 1) Normal lymphoid Tregs, diseased kidney Tregs, splenic Tregs from mice with injured muscle have 3, 17 and 3 specific (S-) pathways, respectively; 2) Tumor splenic Tregs share 12 pathways with tumor Tregs; tumor splenic Tregs and tumor Tregs have 11 and 8 S-pathways, respectively; 3) Normal and non-tumor disease Tregs upregulate some of novel 2641 canonical secretomic genes (SGs) with 24 pathways, and tumor Tregs upregulate canonical secretomes with 17 pathways; 4) Normal and non-tumor disease tissue Tregs upregulate some of novel 6560 exosome SGs with 56 exosome SG pathways (ESP), tumor Treg ESP are more focused than other Tregs; 5) Normal, non-tumor diseased Treg and tumor Tregs upregulate some of novel 961 innate immune caspase-1 SGs and 1223 innate immune caspase-4 SGs to fulfill their tissue/SG-specific and shared functions; 6) Most tissue Treg transcriptomes are controlled by Foxp3; and Tumor Tregs had increased Foxp3 non-collaboration genes with ROS and 17 other pathways; 7) Immune checkpoint receptor PD-1 does, but CTLA-4 does not, play significant roles in promoting Treg upregulated genes in normal and non-tumor disease tissue Tregs; and tumor splenic and tumor Tregs have certain CTLA-4-, and PD-1-, non-collaboration transcriptomic changes with innate immune dominant pathways; 8) Tumor Tregs downregulate more immunometabolic and innate immune memory (trained immunity) genes than Tregs from other groups; and 11) ROS significantly regulate Treg transcriptomes; and ROS-suppressed genes are downregulated more in tumor Tregs than Tregs from other groups. Our results have provided novel insights on the roles of Tregs in normal, injuries, regeneration, tumor conditions and some of canonical and innate immune non-canonical secretomes via ROS-regulatory mechanisms and new therapeutic targets for immunosuppression, tissue repair, cardiovascular diseases, chronic kidney disease, autoimmune diseases, transplantation, and cancers.

Organelle Crosstalk Regulators Are Regulated in Diseases, Tumors, and Regulatory T Cells: Novel Classification of Organelle Crosstalk Regulators.

To examine whether the expressions of 260 organelle crosstalk regulators (OCRGs) in 16 functional groups are modulated in 23 diseases and 28 tumors, we performed extensive -omics data mining analyses and made a set of significant findings: (1) the ratios of upregulated vs. downregulated OCRGs are 1:2.8 in acute inflammations, 1:1 in metabolic diseases, 1:1.2 in autoimmune diseases, and 1:3.8 in organ failures; (2) sepsis and trauma-upregulated OCRG groups such as vesicle, mitochondrial (MT) fission, and mitophagy but not others, are termed as the cell crisis-handling OCRGs. Similarly, sepsis and trauma plus organ failures upregulated seven OCRG groups including vesicle, MT fission, mitophagy, sarcoplasmic reticulum-MT, MT fusion, autophagosome-lysosome fusion, and autophagosome/endosome-lysosome fusion, classified as the cell failure-handling OCRGs; (3) suppression of autophagosome-lysosome fusion in endothelial and epithelial cells is required for viral replications, which classify this decreased group as the viral replication-suppressed OCRGs; (4) pro-atherogenic damage-associated molecular patterns (DAMPs) such as oxidized low-density lipoprotein (oxLDL), lipopolysaccharide (LPS), oxidized-1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine (oxPAPC), and interferons (IFNs) totally upregulated 33 OCRGs in endothelial cells (ECs) including vesicle, MT fission, mitophagy, MT fusion, endoplasmic reticulum (ER)-MT contact, ER- plasma membrane (PM) junction, autophagosome/endosome-lysosome fusion, sarcoplasmic reticulum-MT, autophagosome-endosome/lysosome fusion, and ER-Golgi complex (GC) interaction as the 10 EC-activation/inflammation-promoting OCRG groups; (5) the expression of OCRGs is upregulated more than downregulated in regulatory T cells (Tregs) from the lymph nodes, spleen, peripheral blood, intestine, and brown adipose tissue in comparison with that of CD4+CD25- T effector controls; (6) toll-like receptors (TLRs), reactive oxygen species (ROS) regulator nuclear factor erythroid 2-related factor 2 (Nrf2), and inflammasome-activated regulator caspase-1 regulated the expressions of OCRGs in diseases, virus-infected cells, and pro-atherogenic DAMP-treated ECs; (7) OCRG expressions are significantly modulated in all the 28 cancer datasets, and the upregulated OCRGs are correlated with tumor immune infiltrates in some tumors; (8) tumor promoter factor IKK2 and tumor suppressor Tp53 significantly modulate the expressions of OCRGs. Our findings provide novel insights on the roles of upregulated OCRGs in the pathogenesis of inflammatory diseases and cancers, and novel pathways for the future therapeutic interventions for inflammations, sepsis, trauma, organ failures, autoimmune diseases, metabolic cardiovascular diseases (CVDs), and cancers.

Liver Ischemia Reperfusion Injury, Enhanced by Trained Immunity, Is Attenuated in Caspase 1/Caspase 11 Double Gene Knockout Mice.

Ischemia reperfusion injury (IRI) during liver transplantation increases morbidity and contributes to allograft dysfunction. There are no therapeutic strategies to mitigate IRI. We examined a novel hypothesis: caspase 1 and caspase 11 serve as danger-associated molecular pattern (DAMPs) sensors in IRI. By performing microarray analysis and using caspase 1/caspase 11 double-knockout (Casp DKO) mice, we show that the canonical and non-canonical inflammasome regulators are upregulated in mouse liver IRI. Ischemic pre (IPC)- and post-conditioning (IPO) induce upregulation of the canonical and non-canonical inflammasome regulators. Trained immunity (TI) regulators are upregulated in IPC and IPO. Furthermore, caspase 1 is activated during liver IRI, and Casp DKO attenuates liver IRI. Casp DKO maintained normal liver histology via decreased DNA damage. Finally, the decreased TUNEL assay-detected DNA damage is the underlying histopathological and molecular mechanisms of attenuated liver pyroptosis and IRI. In summary, liver IRI induces the upregulation of canonical and non-canonical inflammasomes and TI enzyme pathways. Casp DKO attenuate liver IRI. Development of novel therapeutics targeting caspase 1/caspase 11 and TI may help mitigate injury secondary to IRI. Our findings have provided novel insights on the roles of caspase 1, caspase 11, and inflammasome in sensing IRI derived DAMPs and TI-promoted IRI-induced liver injury.

Approaching Inflammation Paradoxes-Proinflammatory Cytokine Blockages Induce Inflammatory Regulators.

The mechanisms that underlie various inflammation paradoxes, metabolically healthy obesity, and increased inflammations after inflammatory cytokine blockades and deficiencies remain poorly determined. We performed an extensive -omics database mining, determined the expressions of 1367 innate immune regulators in 18 microarrays after deficiencies of 15 proinflammatory cytokines/regulators and eight microarray datasets of patients receiving Mab therapies, and made a set of significant findings: 1) proinflammatory cytokines/regulators suppress the expressions of innate immune regulators; 2) upregulations of innate immune regulators in the deficiencies of IFNγ/IFNγR1, IL-17A, STAT3 and miR155 are more than that after deficiencies of TNFα, IL-1ß, IL-6, IL-18, STAT1, NF-kB, and miR221; 3) IFNγ, IFNγR and IL-17RA inhibit 10, 59 and 39 proinflammatory cytokine/regulator pathways, respectively; in contrast, TNFα, IL-6 and IL-18 each inhibits only four to five pathways; 4) The IFNγ-promoted and -suppressed innate immune regulators have four shared pathways; the IFNγR1-promoted and -suppressed innate immune regulators have 11 shared pathways; and the miR155-promoted and -suppressed innate immune regulators have 13 shared pathways, suggesting negative-feedback mechanisms in their conserved regulatory pathways for innate immune regulators; 5) Deficiencies of proinflammatory cytokine/regulator-suppressed, promoted programs share signaling pathways and increase the likelihood of developing 11 diseases including cardiovascular disease; 6) There are the shared innate immune regulators and pathways between deficiency of TNFα in mice and anti-TNF therapy in clinical patients; 7) Mechanistically, up-regulated reactive oxygen species regulators such as myeloperoxidase caused by suppression of proinflammatory cytokines/regulators can drive the upregulation of suppressed innate immune regulators. Our findings have provided novel insights on various inflammation paradoxes and proinflammatory cytokines regulation of innate immune regulators; and may re-shape new therapeutic strategies for cardiovascular disease and other inflammatory diseases.

End-stage renal disease is different from chronic kidney disease in upregulating ROS-modulated proinflammatory secretome in PBMCs - A novel multiple-hit model for disease progression.

BACKGROUND: The molecular mechanisms underlying chronic kidney disease (CKD) transition to end-stage renal disease (ESRD) and CKD acceleration of cardiovascular and other tissue inflammations remain poorly determined. METHODS: We conducted a comprehensive data analyses on 7 microarray datasets in peripheral blood mononuclear cells (PBMCs) from patients with CKD and ESRD from NCBI-GEO databases, where we examined the expressions of 2641 secretome genes (SG). RESULTS: 1) 86.7% middle class (molecular weight >500 Daltons) uremic toxins (UTs) were encoded by SGs; 2) Upregulation of SGs in PBMCs in patients with ESRD (121 SGs) were significantly higher than that of CKD (44 SGs); 3) Transcriptomic analyses of PBMC secretome had advantages to identify more comprehensive secretome than conventional secretomic analyses; 4) ESRD-induced SGs had strong proinflammatory pathways; 5) Proinflammatory cytokines-based UTs such as IL-1ß and IL-18 promoted ESRD modulation of SGs; 6) ESRD-upregulated co-stimulation receptors CD48 and CD58 increased secretomic upregulation in the PBMCs, which were magnified enormously in tissues; 7) M1-, and M2-macrophage polarization signals contributed to ESRD- and CKD-upregulated SGs; 8) ESRD- and CKD-upregulated SGs contained senescence-promoting regulators by upregulating proinflammatory IGFBP7 and downregulating anti-inflammatory TGF-ß1 and telomere stabilizer SERPINE1/PAI-1; 9) ROS pathways played bigger roles in mediating ESRD-upregulated SGs (11.6%) than that in CKD-upregulated SGs (6.8%), and half of ESRD-upregulated SGs were ROS-independent. CONCLUSIONS: Our analysis suggests novel secretomic upregulation in PBMCs of patients with CKD and ESRD, act synergistically with uremic toxins, to promote inflammation and potential disease progression. Our findings have provided novel insights on PBMC secretome upregulation to promote disease progression and may lead to the identification of new therapeutic targets for novel regimens for CKD, ESRD and their accelerated cardiovascular disease, other inflammations and cancers. (Total words: 279).

Interleukin 35 Delays Hindlimb Ischemia-Induced Angiogenesis Through Regulating ROS-Extracellular Matrix but Spares Later Regenerative Angiogenesis.

Interleukin (IL) 35 is a novel immunosuppressive heterodimeric cytokine in IL-12 family. Whether and how IL-35 regulates ischemia-induced angiogenesis in peripheral artery diseases are unrevealed. To fill this important knowledge gap, we used loss-of-function, gain-of-function, omics data analysis, RNA-Seq, in vivo and in vitro experiments, and we have made the following significant findings: i) IL-35 and its receptor subunit IL-12RB2, but not IL-6ST, are induced in the muscle after hindlimb ischemia (HLI); ii) HLI-induced angiogenesis is improved in Il12rb2-/- mice, in ApoE-/-/Il12rb2-/- mice compared to WT and ApoE-/- controls, respectively, where hyperlipidemia inhibits angiogenesis in vivo and in vitro; iii) IL-35 cytokine injection as a gain-of-function approach delays blood perfusion recovery at day 14 after HLI; iv) IL-35 spares regenerative angiogenesis at the late phase of HLI recovery after day 14 of HLI; v) Transcriptome analysis of endothelial cells (ECs) at 14 days post-HLI reveals a disturbed extracellular matrix re-organization in IL-35-injected mice; vi) IL-35 downregulates three reactive oxygen species (ROS) promoters and upregulates one ROS attenuator, which may functionally mediate IL-35 upregulation of anti-angiogenic extracellular matrix proteins in ECs; and vii) IL-35 inhibits human microvascular EC migration and tube formation in vitro mainly through upregulating anti-angiogenic extracellular matrix-remodeling proteins. These findings provide a novel insight on the future therapeutic potential of IL-35 in suppressing ischemia/inflammation-triggered inflammatory angiogenesis at early phase but sparing regenerative angiogenesis at late phase.

Increased acetylation of H3K14 in the genomic regions that encode trained immunity enzymes in lysophosphatidylcholine-activated human aortic endothelial cells - Novel qualification markers for chronic disease risk factors and conditional DAMPs.

To test our hypothesis that proatherogenic lysophosphatidylcholine (LPC) upregulates trained immunity pathways (TIPs) in human aortic endothelial cells (HAECs), we conducted an intensive analyses on our RNA-Seq data and histone 3 lysine 14 acetylation (H3K14ac)-CHIP-Seq data, both performed on HAEC treated with LPC. Our analysis revealed that: 1) LPC induces upregulation of three TIPs including glycolysis enzymes (GE), mevalonate enzymes (ME), and acetyl-CoA generating enzymes (ACE); 2) LPC induces upregulation of 29% of 31 histone acetyltransferases, three of which acetylate H3K14; 3) LPC induces H3K14 acetylation (H3K14ac) in the genomic DNA that encodes LPC-induced TIP genes (79%) in comparison to that of in LPC-induced effector genes (43%) including ICAM-1; 4) TIP pathways are significantly different from that of EC activation effectors including adhesion molecule ICAM-1; 5) reactive oxygen species generating enzyme NOX2 deficiency decreases, but antioxidant transcription factor Nrf2 deficiency increases, the expressions of a few TIP genes and EC activation effector genes; and 6) LPC induced TIP genes(81%) favor inter-chromosomal long-range interactions (CLRI, trans-chromatin interaction) while LPC induced effector genes (65%) favor intra-chromosomal CLRIs (cis-chromatin interaction). Our findings demonstrated that proatherogenic lipids upregulate TIPs in HAECs, which are a new category of qualification markers for chronic disease risk factors and conditional DAMPs and potential mechanisms for acute inflammation transition to chronic ones. These novel insights may lead to identifications of new cardiovascular risk factors in upregulating TIPs in cardiovascular cells and novel therapeutic targets for the treatment of metabolic cardiovascular diseases, inflammation, and cancers. (total words: 245).