Anti-atherosclerotic effect of tetrahydroxy stilbene glucoside via dual-targeting of hepatic lipid metabolisms and aortic M2 macrophage polarization in ApoE-/- mice.

Tetrahydroxy stilbene glucoside (TSG) is a water-soluble natural product that has shown potential in treating atherosclerosis (AS). However, its underlying mechanisms remain unclear. Here, we demonstrate that an 8-week TSG treatment (100 mg/kg/d) significantly reduces atherosclerotic lesions and alleviates dyslipidemia symptoms in ApoE-/- mice. 1H nuclear magnetic resonance metabolomic analysis reveals differences in both lipid components and water-soluble metabolites in the livers of AS mice compared to control groups, and TSG treatment shifts the metabolic profiles of AS mice towards a normal state. At the transcriptional level, TSG significantly restores the expression of fatty acid metabolism-related genes (Srepb-1c, Fasn, Scd1, Gpat1, Dgat1, Pparα and Cpt1α), and regulates the expression levels of disturbed cholesterol metabolism-related genes (Srebp2, Hmgcr, Ldlr, Acat1, Acat2 and Cyp7a1) associated with lipid metabolism. Furthermore, at the cellular level, TSG remarkably polarizes aortic macrophages to their M2 phenotype. Our data demonstrate that TSG alleviates arthrosclerosis by dual-targeting to hepatic lipid metabolism and aortic M2 macrophage polarization in ApoE-/- mice, with significant implications for translational medicine and the treatment of AS using natural products.

Apoptosis of Dendritic Cells and Autoimmune Disease.

Dendritic cells (DCs), the most efficient antigen-presenting cells (APCs), bridge the innate and adaptive immune systems. As such, the turn-over of DCs is critical during autoimmune responses, and the dysregulation of DC apoptosis could cause severe immune destruction in the host. For example, reduction of immunogenic DCs by increased apoptosis could lead to immune tolerance to pathogen infection that might allow exposure of nuclear autoantigens, whereas reduced apoptosis could result in long-term lymphocyte activation to break the immune tolerance for the development of autoimmune disease. Thus, keeping a balance between survival and apoptosis of DCs is crucial to maintain immune homeostasis. In this review, we summarize the recent development on the factors inducing DC apoptosis and their underlying mechanisms to provide insights into the immunopathogenesis of some autoimmune diseases, which could lead to effective therapeutic interventions in the clinics.

LKB1 Positively Regulates Dendritic Cell-induced T Cell Immunity and Suppresses Tumor Development.

Background: The functions of dendritic cells (DCs) are influenced by their intracellular metabolism, in which liver kinase B1 (LKB1) plays an important role. However, due to the difficulty in isolating the DCs, the roles of LKB1 in DC maturation and functions in tumor settings have been poorly characterized. Objective: To investigate the roles of LKB1 in DC functions including phagocytosis and presentation of antigens, activation, T cell differentiation, and ultimately tumor eradication. Methods: Genetic modification of Lkb1 in the DCs was made by lentiviral transduction, and their impacts on T cell proliferation, differentiation, activity, or B16 melanoma metastasis were examined by flow cytometry, qPCR, or lung tumor nodule counting. Results: LKB1 did not affect antigen uptake and presentation by the DCs, but facilitated the stimulation of T cell proliferation. Interestingly, following T cell activation, Foxp3-expressing regulatory T cells (Treg) were increased (P=0.0267) or decreased (P=0.0195) in mice injected with Lkb1 knockdown DCs or overexpressing DCs, respectively. Further exploration revealed that LKB1 inhibited OX40L (P=0.0385) and CD86 (P=0.0111) expression, and these co-stimulatory molecules enhanced Treg proliferation, and downregulated immune suppressive cytokine IL-10 (P=0.0315). Moreover, we found that the injection of the DCs with limited LKB1 expression before tumor inoculation could reduce their production of granzyme B (P<0.0001) and perforin (P=0.0042) from CD8+T cells, thereby impairing their cytotoxicity and promoting tumor growth. Conclusion: Our data suggest that LKB1 can enhance DC-mediated T cell immunity by restraining Treg development and thereby suppressing tumor growth.

Gain­of­function of IDO in DCs inhibits T cell immunity by metabolically regulating surface molecules and cytokines.

Both tolerogenicity and immunogenicity of dendritic cells (DCs) are regulated by their intracellular metabolism. As a rate-limiting enzyme of tryptophan (Trp) metabolism, indoleamine 2,3-dioxygenase (IDO) is involved in regulating the functions of numerous cell types, including DCs, a subset of which has a high capacity for producing IDO to control over-activated inflammation. To identify the mechanisms of IDO in DCs, stable DC lines with both gain- and reduction-of-function of IDO were established using a recombinant DNA technique. Although the IDO variation did not affect DC survival and migration, it altered Trp metabolism and other features of DCs analyzed by high-performance liquid chromatography and flow cytometry. On the surface of the DCs, IDO inhibited co-stimulatory CD86 but promoted co-inhibitory programmed cell death ligand 1 expression, and suppressed the antigen uptake, which ultimately led to the compromised ability of DCs to activate T cells. Furthermore, IDO also suppressed IL-12 secretion but enhanced that of IL-10 in DCs, which eventually induced T cells into tolerogenic phenotypes by inhibiting the differentiation of Th1 but promoting that of regulatory T cells. Collectively, the findings of the present study demonstrated that IDO is a key molecule for tolerogenic DC induction by metabolically regulating surface molecule and cytokine expression. This conclusion may lead to the targeted development of therapeutic drugs for autoimmune diseases.

Dendritic cell-expressed IDO alleviates atherosclerosis by expanding CD4+CD25+Foxp3+Tregs through IDO-Kyn-AHR axis.

Atherosclerosis is a chronic inflammatory disease, in which immune disorders constitute an essential part of vascular pathogenesis. Accumulating evidence indicates that dendritic cells (DCs) and their tryptophan metabolisms regulate host immune responses. However, the mechanistic involvement of metabolic products from DCs in dysregulating vascular immunity during the development of atherosclerosis is far from clear. Flow cytometry examination showed immune cells were accumulated and gradually increased in the atherosclerotic lesions during the atherosclerosis progression, in which IDO+DCs were enriched. To study the role of DC-expressed IDO in the development of atherosclerosis, we made a stable IDO-overexpressing DC line (IDOoeDCs) by lentiviral infection for adoptive transfer into pro-atherosclerotic mice. Compared with DCs containing empty vector (VectorCtrlDC)-treated group, treatment of IDOoeDCs led to a significant reduction of atherosclerotic lesions in the aorta, with decreased aortic infiltration of Th1 immune cells and reduced vascular inflammation. Importantly, IDOoeDCs increased aortic kynurenine (Kyn) concentration and aryl hydrocarbon receptor (AHR) expression, concomitant with CD4+CD25+Foxp3+Treg expansion in the aortic tissues, which were abrogated by AHR antagonist treatment. These results indicate that DC-expressed IDO reduces atherosclerotic lesions by inducing aortic CD4+CD25+Foxp3+Treg expansion through IDO-Kyn-AHR axis, which may represent a novel possibility for treatment or prevention of atherosclerosis.

CIS controls the functional polarization of GM-CSF-derived macrophages.

The cytokine granulocyte-macrophage-colony stimulating factor (GM-CSF) possesses the capacity to differentiate monocytes into macrophages (MØs) with opposing functions, namely, proinflammatory M1-like MØs and immunosuppressive M2-like MØs. Despite the importance of these opposing biological outcomes, the intrinsic mechanism that regulates the functional polarization of MØs under GM-CSF signaling remains elusive. Here, we showed that GM-CSF-induced MØ polarization resulted in the expression of cytokine-inducible SH2-containing protein (CIS) and that CIS deficiency skewed the differentiation of monocytes toward immunosuppressive M2-like MØs. CIS deficiency resulted in hyperactivation of the JAK-STAT5 signaling pathway, consequently promoting downregulation of the transcription factor Interferon Regulatory Factor 8 (IRF8). Loss- and gain-of-function approaches highlighted IRF8 as a critical regulator of the M1-like polarization program. In vivo, CIS deficiency induced the differentiation of M2-like macrophages, which promoted strong Th2 immune responses characterized by the development of severe experimental asthma. Collectively, our results reveal a CIS-modulated mechanism that clarifies the opposing actions of GM-CSF in MØ differentiation and uncovers the role of GM-CSF in controlling allergic inflammation.

OX40L enhances the immunogenicity of dendritic cells and inhibits tumor metastasis in mice.

A well preserved immune system is a powerful tool to prevent foreign invasion or to suppress internal mutation, which must be tightly controlled by co-stimulatory molecules in different pathophysiological conditions. One such critical molecule is OX40L expressed on activated antigen-presenting cells (APCs). Consistently, its abnormality is associated with various immunological disorders such as autoinflammatory diseases and allergy. However, a comprehensive analysis of the immune-moderating role of OX40L in dendritic cells (DCs), the most powerful APCs to initiate immune responses in vivo, and investigation of its anti-tumor efficacy in the disease setting have not been performed properly. In this study, genetic approaches for both gain-of-function and reduction-of-function were employed to reveal that OX40L was required for the efficient presentation, but not uptake, of antigens by DCs to stimulate CD4+ , as well as CD8+ T cells in vivo. As a result, CD4+ T cells were promoted towards Th1, but inhibited on Treg differentiation, by the LPS-induced OX40L on DCs, which was supported by their altered expression of co-inhibitory receptor, PD-L1. CD8+ T cells, on the other hand, also enhanced their cytotoxicity towards target cells in response to OX40L expression on the DCs transferred in vivo. Finally, in a DC-mediated tumor immunity model, the strong immunogenic roles of OX40L on DCs led to better metastasis inhibition in vivo. Collectively, our results demonstrate that OX40L could serve as a potential target in the DC-based vaccine for enhanced anti-tumor efficacy in vivo.

Tetrahydroxy Stilbene Glucoside Alleviates Ischemic Stroke by Regulating Conformation-Dependent Intracellular Distribution of PKM2 for M2 Macrophage Polarization.

Tetrahydroxy stilbene glucoside (TSG) is a bioactive ingredient with powerful anti-inflammatory and neuroprotective properties. However, the detailed mechanisms concerning the neuroprotective effect of TSG are not fully understood. This study aims to address the molecular mechanism involved in the protective effects of TSG on murine ischemic stroke. We found that TSG meliorated the phenotypes of ischemic stroke in vivo, which was correlated with the increased percentage of infiltrated M2 macrophages in brain after stroke. Mechanistically, TSG regulated macrophage polarization by significantly downregulating the transcriptional levels of M1 marker genes (iNOS and IL-1ß) but upregulating that of the M2 marker genes (arg-1 and IL-4) following lipopolysaccharide/interferon-γ stimulation. Consistently, TSG reversed the metabolic profiling of M1 macrophage toward the M2 status at intracellular energy levels. Surprisingly, the knockdown of an established metabolic enzyme pyruvate kinase M2 (PKM2) that is important for M1 switch in macrophages abolished the promotive effect of TSG on the M2 polarization. Further investigation revealed that TSG markedly downregulated the intracellular ratio of dimer/monomer to the tetramer of PKM2 without affecting its total protein expression, leading to a suppressed nuclear translocation of functioning PKM2 in macrophages for M1 differentiation. Taken together, we identified a novel mechanism for macrophage M2 polarization regulation by a small-molecule chemical that controls the quality (conformation) rather than the quantity (expression) of an intracellular M1-promoting metabolic enzyme, which offers a better understanding of the mechanisms of macrophage plasticity and has serious implication in translational strategies for the treatment of macrophage-mediated neurological diseases with natural bioactive products.

Self-Tolerance of Vascular Tissues Is Broken Down by Vascular Dendritic Cells in Response to Systemic Inflammation to Initiate Regional Autoinflammation.

The correlation of infections with vascular autoinflammatory diseases such as vasculitis and atherosclerosis has been long recognized, and progressive inflammation with the formation of tertiary lymphoid organs in arterial adventitia intensively studied, the immunological basis of the nondiseased vasculatures that predispose to subsequent vascular autoimmunity during inflammation, however, is not well characterized. Here, we investigated the vascular immunity in situ of steady-state C57BL/6 mice and found that healthy vascular tissues contained a comprehensive set of immune cells with relatively higher proportion of innate components than lymphoid organs. Notably, a complete set of dendritic cell (DC) subsets was observed with monocyte-derived DCs (moDCs) constituting a major proportion; this is in contrast to moDCs being considered rare in the steady state. Interestingly, these vascular DCs constitutively expressed more suppressive factors with cDC1 for PD-L1 and moDCs for IL-10; this is concordant with the inhibitive phenotype of T cells in normal vascular tissues. The immunotolerant state of the vascular tissues, however, was readily eroded by systemic inflammation, demonstrated by the upregulation of proinflammatory cytokines and enhanced antigen presentation by vascular DCs to activate both cellular and humoral immunity in situ, which ultimately led to vascular destruction. Different vascular DC subsets elicited selective effects: moDCs were potent cytokine producers and B-cell activators, whereas cDCs, particularly, cDC1, were efficient at presenting antigens to stimulate T cells. Together, we unveil regional immunological features of vascular tissues to explain their dual facets under physiological versus pathological conditions for the better understanding and treatment of cardiovascular autoinflammation.

A myriad of roles of dendritic cells in atherosclerosis.

Atherosclerosis is an inflammatory disease with break-down of homeostatic immune regulation of vascular tissues. As a critical initiator of host immunity, dendritic cells (DCs) have also been identified in the aorta of healthy individuals and atherosclerotic patients, whose roles in regulating arterial inflammation aroused great interest. Accumulating evidence has now pointed to the fundamental roles for DCs in every developmental stage of atherosclerosis due to their myriad of functions in immunity and tolerance induction, ranging from lipid uptake, efferocytosis and antigen presentation to pro- and anti-inflammatory cytokine or chemokine secretion. In this study we provide a timely summary of the published works in this field, and comprehensively discuss both the direct and indirect roles of DCs in atherogenesis. Understanding the pathogenic roles of DCs during the development of atherosclerosis in vascular tissues would certainly help to open therapeutic avenue to the treatment of cardiovascular diseases.

Aryl hydrocarbon receptor connects dysregulated immune cells to atherosclerosis.

As a chronic inflammatory disease with autoimmune components, atherosclerosis is the major cause of cardiovascular morbidity and mortality. Recent studies have revealed that the development of atherosclerosis is strongly linked to the functional activities of aryl hydrocarbon receptor (AHR), a chemical sensor that is also important for the development, maintenance, and function of a variety of immune cells. In this review, we focus on the impact of AHR signaling on the different cell types that are closely related to the atherogenesis, including T cells, B cells, dendritic cells, macrophages, foam cells, and hematopoietic stem cells in the arterial walls, and summarize the latest development on the interplay between this environmental sensor and immune cells in the context of atherosclerosis. Hopefully, elucidation of the role of AHR in atherosclerosis will facilitate the understanding of case variation in disease prevalence and may aid in the development of novel therapies.

Dendritic Cells and T Cells, Partners in Atherogenesis and the Translating Road Ahead.

Atherosclerosis is a chronic process associated with arterial inflammation, the accumulation of lipids, plaque formation in vessel walls, and thrombosis with late mortal complications such as myocardial infarction and ischemic stroke. Immune and inflammatory responses have significant effects on every phase of atherosclerosis. Increasing evidence has shown that both innate and adaptive "arms" of the immune system play important roles in regulating the progression of atherosclerosis. Accumulating evidence suggests that a unique type of innate immune cell, termed dendritic cells (DCs), play an important role as central instigators, whereas adaptive immune cells, called T lymphocytes, are crucial as active executors of the DC immunity in atherogenesis. These two important immune cell types work in pairs to establish pro-atherogenic or atheroprotective immune responses in vascular tissues. Therefore, understanding the role of DCs and T cells in atherosclerosis is extremely important. Here, in this review, we will present a complete overview, based on existing knowledge of these two cell types in the atherosclerotic microenvironment, and discuss some of the novel means of targeting DCs and T cells as therapeutic tactics for the treatment of atherosclerosis.

Suppressed dendritic cell functions by cystatin C lead to compromised immunity in vivo.

Pathogenic microorganisms utilize multiple approaches to break down host immunity in favor of their invasion, of which, cystatin C is one of the soluble factors secreted by parasites reported to affect host immunity in vivo. The cellular targets and mechanisms of action in vivo of cystatin C, however, are far from clear. As professional antigen-presenting cells, dendritic cells (DCs) are first immune cells that contact foreign pathogenic agents or their products to initiate immune responses. We previously reported that cystatin C can regulate the functions of DCs in terms of suppressed CD4+ T cell activation but enhanced Th1/Th17 differentiation via different mechanisms. Here, we further verified these regulatory effects of cystatin C on DCs in vivo. We found that the suppressive role of DC-mediated CD4+ T cell proliferation by cystatin C was partly cell-contact independent and extended to CD8+ T cells in vivo. Although cystatin C-overexpressing DCs trafficked equally as their mock-transduced counterparts, their adoptive transfer suppressed CD8+ T cell immunity and resulted in compromised tumor rejection in both vaccination and treatment regimes. Compared with their role in promoting Th17 differentiation in vivo, cystatin C-transduced DCs had far greater ability to induce T regulatory cells (Tregs), leading to collectively a higher Treg/Th17 ratio in an adoptively transferred disease model, and thus relieved Th17-dependent autoimmunity. Collectively, these data demonstrated strong in vivo evidences for immune regulatory roles of cystatin C in DCs and provided theoretical basis for the application of cystatin C-transduced cell therapy in the treatment or remission of certain autoimmune diseases. (246).

Cystatin C regulates major histocompatibility complex-II-peptide presentation and extracellular signal-regulated kinase-dependent polarizing cytokine production by bone marrow-derived dendritic cells.

Cystatin C is a ubiquitously expressed cysteine protease inhibitor that protects cells from either improper hydrolysis by endogenous proteases or pathogen growth/virulence by exogenous proteases. Although commonly used as a serum biomarker for evaluating renal function, cystatin C is associated with many immunological disorders under various pathophysiological conditions. How cystatin C affects immune cells, especially dendritic cells (DCs), however, is far from clear. In this study, we found that pharmacological treatment with or genetic overexpression of cystatin C in bone marrow-derived DCs (BMDCs) reduced their capacity to stimulate CD4+ T-cell proliferation, despite increased antigen uptake. This reduced capacity corresponded with reduced major histocompatibility complex-II presentation owing to diminished levels of the chaperon H2-DM in BMDCs. Instead of promoting proliferation, cystatin C promoted skewing of T cells toward proinflammatory T-helper (Th)1/Th17 differentiation. This was mediated by augmented extracellular signal-regulated kinase 1/2 mitogen-activated protein kinase phosphorylation in BMDCs, leading to secretion of polarizing cytokines, which in turn led to the Th deviation. Collectively, our study explained the cellular and molecular basis of how this protease inhibitor can regulate immune responses, namely by affecting BMDCs and their cytokine pathway. Our results might open up an avenue for the development of therapeutic agents for the treatment of cystatin C-related immunological diseases.

GM-CSF Quantity Has a Selective Effect on Granulocytic vs. Monocytic Myeloid Development and Function.

GM-CSF promotes myeloid differentiation of cultured bone marrow cells into cells of the granulocytic and monocytic lineage; the latter can further differentiate into monocytes/macrophages and dendritic cells. How GM-CSF selects for these different myeloid fates is unresolved. GM-CSF levels can change either iatrogenically (e.g., augmenting leukopoiesis after radiotherapy) or naturally (e.g., during infection or inflammation) resulting in different immunological outcomes. Therefore, we asked whether the dose of GM-CSF may regulate the development of three types of myeloid cells. Here, we showed that GM-CSF acted as a molecular rheostat where the quantity determined which cell type was favored; moreover, the cellular process by which this was achieved was different for each cell type. Thus, low quantities of GM-CSF promoted the granulocytic lineage, mainly through survival. High quantities promoted the monocytic lineage, mainly through proliferation, whereas moderate quantities promoted moDCs, mainly through differentiation. Finally, we demonstrated that monocytes/macrophages generated with different doses of GM-CSF differed in function. We contend that this selective effect of GM-CSF dose on myeloid differentiation and function should be taken into consideration during pathophysiological states that may alter GM-CSF levels and during GM-CSF agonistic or antagonistic therapy.

Bone marrow-derived inflammatory and steady state DCs are different in both functions and survival.

Dendritic cells (DCs) contain heterogeneous populations, with classical DCs developed at steady state and monocyte-derived DCs mobilized under inflammatory conditions, although their total numbers in vivo are scares. To obtain enough quantity for immunological study or clinical application, we have previously established that bone marrow-derived DCs in the presence of Flt-3L (FL-DCs) or GM-CSF (GM-DCs) in vitro are equivalent to the steady state DCs and inflammatory DCs in vivo respectively. What difference, however, exists between these two most commonly used culture systems in DC functions and survival, and how does it correlate to the division of works by their corresponding counterparts in vivo remain ill-defined. In this study, we found that GM-DCs of inflammatory nature were more phagocytic, potent at inducing Th2 and Th17 differentiation, and had longer survival rate, whereas FL-DCs of steady state characters were stronger T cell activator and better at directing Th1 differentiation. Mechanistically, NO production induced by the LPS-activated GM-DCs could partly explain for their failure to improve T cell proliferation, and the distinct T cell differentiation profiles and viability demonstrated by the two types of DCs were underpinned by their preferential secretion of T cell polarizing cytokines and expression of anti-apoptotic genes. Such disparate functionalities and survival potentials of steady state and inflammatory DCs in vitro fit in well with their respective roles in vivo in particular immunological settings and have serious implications in translational applications.

Inflammatory compound lipopolysaccharide promotes the survival of GM-CSF cultured dendritic cell via PI3 kinase-dependent upregulation of Bcl-x.

As professional antigen-presenting cells, dendritic cells (DCs) initiate and regulate immune responses against inflammation. The invasion of pathogens into the body, however, can in turn cause the change of DCs in both activity and viability, which ultimately affect immune homeostasis. The exact mechanisms that the bacteria utilize to alter the lifespan of DCs, however, are far from clear. In this study, we found that the bacterial wall compound lipopolysaccharide (LPS) can promote the survival of GM-CSF-differentiated DCs (GM-DCs). At molecular levels, we demonstrated that GM-DCs had distinct pattern of mRNA expression for anti-apoptotic BCL-2 family members, of which, Bcl-x increased significantly following LPS stimulation. Interestingly, specific inhibition of BCL-XL protein alone was sufficient to remove the anti-apoptotic effects of LPS on BM-DCs. Further study of the signaling mechanisms revealed that although LPS can activate both Erk MAP kinase and PI3 kinase pathways, only blocking of PI3K abolished both Bcl-x upregulation and the enhanced survival phenotype, suggesting that the PI3K signaling mediated the upregulation of Bcl-x for the LPS-induced pro-survival in GM-DCs. Collectively, this study unveils a molecular mechanism that DCs adapt to maintain innate immunity against the invasion of pathogens.

Involvement of cystatin C in immunity and apoptosis.

As an abundantly expressed cysteine protease inhibitor widely distributed in the organisms, cystatin C is involved in various physiological processes. Due to its relatively small molecular weight and easy detection, cystatin C is commonly used as a measure for glomerular filtration rate. In pathological conditions, however, growing evidences suggest that cystatin C is associated with various immune responses against either exogenous or endogenous antigens, which ultimately result in inflammatory autoimmune diseases or tumor development if not properly controlled. Thus the fluctuation of cystatin C levels might have more clinical implications than a reflection of kidney functions. Here, we summarize the latest development of studies on the pathophysiological functions of cystatin C, with focus on its immune regulatory roles at both cellular and molecular levels including antigen presentation, secretion of cytokines, synthesis of nitric oxide, as well as apoptosis. Finally, we discuss the clinical implications and therapeutic potentials of what this predominantly expressed protease inhibitor can bring to us.

Sterically Protected N2O-Type Benzopyrromethene Boron Complexes from Boronic Acids with Intense Red/Near-Infrared Fluorescence.

A series of N2O-type benzopyrromethene boron complexes have been synthesized from the condensation of pyrrole, formylisoindole, and various boronic acids. These dyes have red/near-infrared absorption, high solution- and solid-state fluorescence, as well as good solubility in a variety of solvents due to the presence of axial-substituted groups that provide steric protection and prevent their possible aggregations. Two water-soluble derivatives with pyridinium ions were developed as near-infrared excitable mitochondrially localizing fluorescent probes.