Jietang Mai

Targeting mitochondrial reactive oxygen species as novel therapy for inflammatory diseases and cancers

There are multiple sources of reactive oxygen species (ROS) in the cell. As a major site of ROS production, mitochondria have drawn considerable interest because it was recently discovered that mitochondrial ROS (mtROS) directly stimulate the production of proinflammatory cytokines and pathological conditions as diverse as malignancies, autoimmune diseases, and cardiovascular diseases all share common phenotype of increased mtROS production above basal levels. Several excellent reviews on this topic have been published, but ever-changing new discoveries mandated a more up-to-date and comprehensive review on this topic. Therefore, we update recent understanding of how mitochondria generate and regulate the production of mtROS and the function of mtROS both in physiological and pathological conditions. In addition, we describe newly developed methods to probe or scavenge mtROS and compare these methods in detail. Thorough understanding of this topic and the application of mtROS-targeting drugs in the research is significant towards development of better therapies to combat inflammatory diseases and inflammatory malignancies.

IL-35 is a novel responsive anti-inflammatory cytokine--a new system of categorizing anti-inflammatory cytokines.

It remains unknown whether newly identified anti-inflammatory/immunosuppressive cytokine interleukin-35 (IL-35) is different from other anti-inflammatory cytokines such as IL-10 and transforming growth factor (TGF)-β in terms of inhibition of inflammation initiation and suppression of full-blown inflammation. Using experimental database mining and statistical analysis methods we developed, we examined the tissue expression profiles and regulatory mechanisms of IL-35 in comparison to other anti-inflammatory cytokines. Our results suggest that in contrast to TGF-β, IL-35 is not constitutively expressed in human tissues but it is inducible in response to inflammatory stimuli. We also provide structural evidence that AU-rich element (ARE) binding proteins and microRNAs target IL-35 subunit transcripts, by which IL-35 may achieve non-constitutive expression status. Furthermore, we propose a new system to categorize anti-inflammatory cytokines into two groups: (1) the house-keeping cytokines, such as TGF-β, inhibit the initiation of inflammation whereas (2) the responsive cytokines including IL-35 suppress inflammation in full-blown stage. Our in-depth analyses of molecular events that regulate the production of IL-35 as well as the new categorization system of anti-inflammatory cytokines are important for the design of new strategies of immune therapies.

Inflammasomes are differentially expressed in cardiovascular and other tissues.

To determine the expression of components in Toll-like receptors (TLRs)/Nod-like receptors (NLRs)/inflammasome/caspase-1/interleukin (IL-1)-β pathway, we examined the expression profiles of those genes by analyzing the data from expression sequence tag cDNA cloning and sequencing. We made several important findings: firstly, among 11 tissues examined, vascular tissues and heart express fewer types of TLRs and NLRs than immune and defense tissues including blood, lymph nodes, thymus and trachea; secondly, brain, lymph nodes and thymus do not express proinflammatory cytokines IL-1β and IL-18 constitutively, suggesting that these two cytokines need to be upregulated in the tissues; and thirdly, based on the expression data of three characterized inflammasomes (NALP1, NALP3 and IPAF inflammasome), the examined tissues can be classified into three tiers: the first tier tissues including brain, placenta, blood and thymus express inflammasome(s) in constitutive status; the second tier tissues have inflammasome(s) in nearly-ready expression status (with the requirement of upregulation of one component); the third tier tissues, like heart and bone marrow, require upregulation of at least two components in order to assemble functional inflammasomes. Our original model of three-tier expression of inflammasomes would suggest a new concept of tissue inflammation privilege, and provides an insight to the differences among tissues in initiating acute inflammation in response to stimuli.

An evolving new paradigm: endothelial cells – conditional innate immune cells

Endothelial cells (ECs) are a heterogeneous population that fulfills many physiological processes. ECs also actively participate in both innate and adaptive immune responses. ECs are one of the first cell types to detect foreign pathogens and endogenous metabolite-related danger signals in the bloodstream, in which ECs function as danger signal sensors. Treatment with lipopolysaccharide activates ECs, causing the production of pro-inflammatory cytokines and chemokines, which amplify the immune response by recruiting immune cells. Thus, ECs function as immune/inflammation effectors and immune cell mobilizers. ECs also induce cytokine production by immune cells, in which ECs function as immune regulators either by activating or suppressing immune cell function. In addition, under certain conditions, ECs can serve as antigen presenting cells (antigen presenters) by expressing both MHC I and II molecules and presenting endothelial antigens to T cells. These facts along with the new concept of endothelial plasticity suggest that ECs are dynamic cells that respond to extracellular environmental changes and play a meaningful role in immune system function. Based on these novel EC functions, we propose a new paradigm that ECs are conditional innate immune cells. This paradigm provides a novel insight into the functions of ECs in inflammatory/immune pathologies.

Regulatory T cells and Atherosclerosis.

Atherosclerosis is a chronic autoimmune inflammatory disease. The involvement of both innate and adaptive immune responses in the pathogenesis of the disease has been well recognized. Tregs are an essential part of the immune system and have indispensable functions in maintaining immune system homeostasis, mediating peripheral tolerance, preventing autoimmune diseases, and suppressing inflammatory and proatherogenic immune response. Tregs carry out their immunosuppressive functions via several mechansims. One of the well-documented suppressive mechanisms of Tregs is the secretion of anti-inflammatory cytokines including IL-10, TGF-β, and IL-35. Studies have found that IL-10 and TGF-β have atheroprotective properties. In addition, Tregs can suppress the activity of proatherogenic effector T cells, suggesting an atheroprotective role. In fact, fewer Tregs are found in atherogenic ApoE-/- mice comparing to wild-type mice, suggesting an uncontrolled balance between weakened Tregs and effector T cells in atherogenesis. Some clinical studies of autoimmune diseases also suggest that decreased Tregs numbers are associated with increased disease activity. The importance of Tregs in many autoimmune diseases and experimental atherosclerosis has been established in in vivo and in vitro studies. However, the roles of Tregs in atherosclerosis in the clinical setting remains to be further characterized.

Early Hyperlipidemia Promotes Endothelial Activation via a Caspase-1-Sirtuin 1 Pathway

Objective— The role of receptors for endogenous metabolic danger signals–associated molecular patterns has been characterized recently as bridging innate immune sensory systems for danger signals–associated molecular patterns to initiation of inflammation in bone marrow–derived cells, such as macrophages. However, it remains unknown whether endothelial cells (ECs), the cell type with the largest numbers and the first vessel cell type exposed to circulating danger signals–associated molecular patterns in the blood, can sense hyperlipidemia. This report determined whether caspase-1 plays a role in ECs in sensing hyperlipidemia and promoting EC activation. Approach and Results— Using biochemical, immunologic, pathological, and bone marrow transplantation methods together with the generation of new apoplipoprotein E (ApoE)−/−/caspase-1−/− double knockout mice, we made the following observations: (1) early hyperlipidemia induced caspase-1 activation in ApoE−/− mouse aorta; (2) caspase-1−/−/ApoE−/− mice attenuated early atherosclerosis; (3) caspase-1−/−/ApoE−/− mice had decreased aortic expression of proinflammatory cytokines and attenuated aortic monocyte recruitment; and (4) caspase-1−/−/ApoE−/− mice had decreased EC activation, including reduced adhesion molecule expression and cytokine secretion. Mechanistically, oxidized lipids activated caspase-1 and promoted pyroptosis in ECs by a reactive oxygen species mechanism. Caspase-1 inhibition resulted in accumulation of sirtuin 1 in the ApoE−/− aorta, and sirtuin 1 inhibited caspase-1 upregulated genes via activator protein-1 pathway. Conclusions— Our results demonstrate for the first time that early hyperlipidemia promotes EC activation before monocyte recruitment via a caspase-1–sirtuin 1–activator protein-1 pathway, which provides an important insight into the development of novel therapeutics for blocking caspase-1 activation as early intervention of metabolic cardiovascular diseases and inflammations.

Hyperhomocysteinemia and Hyperglycemia Induce and Potentiate Endothelial Dysfunction via μ-Calpain Activation

Plasma homocysteine (Hcy) levels are positively correlated with cardiovascular mortality in diabetes. However, the joint effect of hyperhomocysteinemia (HHcy) and hyperglycemia (HG) on endothelial dysfunction (ED) and the underlying mechanisms have not been studied. Mild (22 µmol/L) and moderate (88 µmol/L) HHcy were induced in cystathionine β-synthase wild-type (Cbs+/+) and heterozygous-deficient (Cbs−/+) mice by a high-methionine (HM) diet. HG was induced by consecutive injection of streptozotocin. We found that HG worsened HHcy and elevated Hcy levels to 53 and 173 µmol/L in Cbs+/+ and Cbs−/+ mice fed an HM diet, respectively. Both mild and moderate HHcy aggravated HG-impaired endothelium-dependent vascular relaxation to acetylcholine, which was completely abolished by endothelial nitric oxide synthase (eNOS) inhibitor NG-nitro-L-arginine methyl ester. HHcy potentiated HG-induced calpain activation in aortic endothelial cells isolated from Cbs mice. Calpain inhibitors rescued HHcy- and HHcy/HG-induced ED in vivo and ex vivo. Moderate HHcy- and HG-induced μ-calpain activation was potentiated by a combination of HHcy and HG in the mouse aorta. μ-Calpain small interfering RNA (μ-calpsiRNA) prevented HHcy/HG-induced ED in the mouse aorta and calpain activation in human aortic endothelial cells (HAECs) treated with DL-Hcy (500 µmol/L) and d-glucose (25 mmol) for 48 h. In addition, HHcy accelerated HG-induced superoxide production as determined by dihydroethidium and 3-nitrotyrosin staining and urinary 8-isoprostane/creatinine assay. Antioxidants rescued HHcy/HG-induced ED in mouse aortas and calpain activation in cultured HAECs. Finally, HHcy potentiated HG-suppressed nitric oxide production and eNOS activity in HAECs, which were prevented by calpain inhibitors or μ-calpsiRNA. HHcy aggravated HG-increased phosphorylation of eNOS at threonine 497/495 (eNOS-pThr497/495) in the mouse aorta and HAECs. HHcy/HG-induced eNOS-pThr497/495 was reversed by µ-calpsiRNA and adenoviral transduced dominant negative protein kinase C (PKC)β2 in HAECs. HHcy and HG induced ED, which was potentiated by the combination of HHcy and HG via μ-calpain/PKCβ2 activation–induced eNOS-pThr497/495 and eNOS inactivation.

Pathological conditions re-shape physiological Tregs into pathological Tregs

CD4+FOXP3+ regulatory T cells (Tregs) are a subset of CD4 T cells that play an essential role in maintaining peripheral immune tolerance, controlling acute and chronic inflammation, allergy, autoimmune diseases, and anti-cancer immune responses. Over the past 20 years, a significant progress has been made since Tregs were first characterized in 1995. Many concepts and principles regarding Tregs generation, phenotypic features, subsets (tTregs, pTregs, iTregs, and iTreg35), tissue specificity (central Tregs, effector Tregs, and tissue resident Tregs), homeostasis (highly dynamic and apoptotic), regulation of Tregs by receptors for PAMPs and DAMPs, Treg plasticity (re-differentiation to other CD4 T helper cell subsets, Th1, Th2, Tfh, and Th17), and epigenetic regulation of Tregs phenotypes and functions have been innovated. In this concise review, we want to briefly analyze these eight new progresses in the study of Tregs. We have also proposed for the first time a novel concept that “physiological Tregs” have been re-shaped into “pathological Tregs” in various pathological environments. Continuing of the improvement in our understanding on this important cellular component about the immune tolerance and immune suppression would lead to the future development of novel therapeutics approaches for acute and chronic inflammatory diseases, allergy, allogeneic transplantation-related immunity, sepsis, autoimmune diseases, and cancers.

MicroRNA-155 Deficiency Leads to Decreased Atherosclerosis, Increased White Adipose Tissue Obesity, and Non-alcoholic Fatty Liver Disease A NOVEL MOUSE MODEL OF OBESITY PARADOX

Obesity paradox (OP) describes a widely observed clinical finding of improved cardiovascular fitness and survival in some overweight or obese patients. The molecular mechanisms underlying OP remain enigmatic partly due to a lack of animal models mirroring OP in patients. Using apolipoprotein E knock-out (apoE−/−) mice on a high fat (HF) diet as an atherosclerotic obesity model, we demonstrated 1) microRNA-155 (miRNA-155, miR-155) is significantly up-regulated in the aortas of apoE−/− mice, and miR-155 deficiency in apoE−/− mice inhibits atherosclerosis; 2) apoE−/−/miR-155−/− (double knock-out (DKO)) mice show HF diet-induced obesity, adipocyte hypertrophy, and present with non-alcoholic fatty liver disease; 3) DKO mice demonstrate HF diet-induced elevations of plasma leptin, resistin, fed-state and fasting insulin and increased expression of adipogenic transcription factors but lack glucose intolerance and insulin resistance. Our results are the first to present an OP model using DKO mice with features of decreased atherosclerosis, increased obesity, and non-alcoholic fatty liver disease. Our findings suggest the mechanistic role of reduced miR-155 expression in OP and present a new OP working model based on a single miRNA deficiency in diet-induced obese atherogenic mice. Furthermore, our results serve as a breakthrough in understanding the potential mechanism underlying OP and provide a new biomarker and novel therapeutic target for OP-related metabolic diseases.

Interleukin-17A Promotes Aortic Endothelial Cell Activation via Transcriptionally and Post-translationally Activating p38 Mitogen-activated Protein Kinase (MAPK) Pathway.

Interleukin-17 (IL-17)-secreting T helper 17 cells were recently identified as a CD4+ T helper subset and implicated in various inflammatory and autoimmune diseases. The issues of whether and by what mechanism hyperlipidemic stress induces IL-17A to activate aortic endothelial cells (ECs) and enhance monocyte adhesion remained largely unknown. Using biochemical, immunological, microarray, experimental data mining analysis, and pathological approaches focused on primary human and mouse aortic ECs (HAECs and MAECs) and our newly generated apolipoprotein E (ApoE)−/−/IL-17A−/− mice, we report the following new findings. 1) The hyperlipidemia stimulus oxidized low density lipoprotein up-regulated IL-17 receptor(s) in HAECs and MAECs. 2) IL-17A activated HAECs and increased human monocyte adhesion in vitro. 3) A deficiency of IL-17A reduced leukocyte adhesion to endothelium in vivo. 3) IL-17A activated HAECs and MAECs via up-regulation of proinflammatory cytokines IL-6, granulocyte-macrophage colony-stimulating factor (GM-CSF), chemokine CXC motif ligand 1 (CXCL1), and CXCL2. 4) IL-17A activated ECs specifically via the p38 mitogen-activated protein kinases (MAPK) pathway; the inhibition of p38 MAPK in ECs attenuated IL-17A-mediated activation by ameliorating the expression of the aforementioned proinflammatory cytokines, chemokines, and EC adhesion molecules including intercellular adhesion molecule 1. Taken together, our results demonstrate for the first time that IL-17A activates aortic ECs specifically via p38 MAPK pathway.