Ginsenoside Rb2 Alleviated Atherosclerosis by Inhibiting M1 Macrophages Polarization Induced by MicroRNA-216a

Introduction: Atherosclerosis is a chronic disease characterized by the inflammatory process and lipid depositions. We previously reported that microRNA-216a (miR-216a) can accelerate the progression of atherosclerosis by promoting the polarization of M1 pro-inflammatory phenotype. Ginsenoside Rb2 (Rb2), the major pharmacologically active compound extracted from ginseng, has a high affinity to miR-216a. In this study, we aimed to investigate whether Rb2 can counteract the effect of miR-216a in macrophages to ameliorate atherosclerosis.Methods: The apolipoprotein E deficiency (ApoE−/−) mice model was chronically infected with miR-216a adenovirus via the tail vein and then intraperitoneally injected with Rb2. The plaque lesion area and stability of thoracic aorta were examined. The human myeloid leukemia mononuclear cells (THP-1) or human peripheral blood mononuclear cells (PBMCs) were cultured in vitro, transfected with miR-216a mimics, and treated with Rb2 to explore the mechanisms of Rb2 on the polarization of M1 macrophages, inflammatory process, and lipid accumulation.Results: In the atherosclerotic ApoE−/− mice model, miR-216a greatly increased en face aortic lesion area of the thoracic aorta, lipid accumulation, and M1 macrophages infiltration in plaques, whereas these effects of miR-216a on atherosclerosis burden were significantly alleviated by Rb2 treatment. In the in vitro THP-1 model, the flow cytometry experiment showed that Rb2 treatment inhibited miR-216a–mediated polarization of M1 macrophages characterized by the surface marker CD86 expression but had no effects on M2 polarization characterized by the surface marker CD206 expression. Mechanistically, Rb2 suppressed the miR-216a–mediated inflammatory response through the Smad3/nuclear factor kappa B inhibitor alpha pathway. Moreover, Rb2 reduced the lipid uptake and promoted cholesterol efflux by counteracting the effects of miR-216a in the THP-1–derived foam cells and in the PBMC-derived foam cells under the oxidized low-density lipoproteins.Conclusion: Our findings indicated that Rb2 might be a potential therapeutic molecule for atherosclerosis by attenuating the atherosclerosis plaque lesion, lipid accumulation, and M1 macrophages polarization by targeting miR-216a. Given that accumulation of foam cells in the intima takes place chronically, the role of Rb2 in atherosclerosis progression needs further investigation.

Higher Chondrogenic Potential of Extracellular Vesicles Derived from Mesenchymal Stem Cells Compared to Chondrocytes-EVs In Vitro

The inability of cartilage to self-repair necessitates an effective therapeutic approach to restore damaged tissues. Extracellular vesicles (EVs) are attractive options because of their roles in cellular communication and tissue repair where they regulate the cellular processes of proliferation, differentiation, and recruitment. However, it is a challenge to determine the relevant cell sources for isolation of EVs with high chondrogenic potential. The current study aims to evaluate the chondrogenic potential of EVs derived from chondrocytes (Cho-EV) and mesenchymal stem cells (MSC-EV). The EVs were separately isolated from conditioned media of both rabbit bone marrow MSCs and chondrocyte cultures. The isolated vesicles were assessed in terms of size, morphology, and surface marker expression. The chondrogenic potential of MSCs in the presence of different concentrations of EVs (50, 100, and 150 μg/ml) was evaluated during 21 days, and chondrogenic surface marker expressions were checked by qRT-PCR and histologic assays. The extracted vesicles had a spherical morphology and a size of 44.25 ± 8.89 nm for Cho-EVs and 112.1 ± 10.10 nm for MSC-EVs. Both groups expressed the EV-specific surface markers CD9 and CD81. Higher expression of chondrogenic specified markers, especially collagen type II (COL II), and secretion of glycosaminoglycans (GAGs) and proteoglycans were observed in MSCs treated with 50 and 100 μg/ml MSC-EVs compared to the Cho-EVs. The results from the use of EVs, particularly MSC-EVs, with high chondrogenic ability will provide a basis for developing therapeutic agents for cartilage repair.

Progenitor Cells in Healthy and Osteoarthritic Human Cartilage Have Extensive Culture Expansion Capacity while Retaining Chondrogenic Properties

Objective Articular cartilage-derived progenitor cells (ACPCs) are a potential new cell source for cartilage repair. This study aims to characterize endogenous ACPCs from healthy and osteoarthritic (OA) cartilage, evaluate their potential for cartilage regeneration, and compare this to cartilage formation by chondrocytes. Design ACPCs were isolated from full-thickness healthy and OA human cartilage and separated from the total cell population by clonal growth after differential adhesion to fibronectin. ACPCs were characterized by growth kinetics, multilineage differentiation, and surface marker expression. Chondrogenic redifferentiation of ACPCs was compared with chondrocytes in pellet cultures. Pellets were assessed for cartilage-like matrix production by (immuno)histochemistry, quantitative analyses for glycosaminoglycans and DNA content, and expression of chondrogenic and hypertrophic genes. Results Healthy and OA ACPCs were successfully differentiated toward the adipogenic and chondrogenic lineage, but failed to produce calcified matrix when exposed to osteogenic induction media. Both ACPC populations met the criteria for cell surface marker expression of mesenchymal stromal cells (MSCs). Healthy ACPCs cultured in pellets deposited extracellular matrix containing proteoglycans and type II collagen, devoid of type I collagen. Gene expression of hypertrophic marker type X collagen was lower in healthy ACPC pellets compared with OA pellets. Conclusions This study provides further insight into the ACPC population in healthy and OA human articular cartilage. ACPCs show similarities to MSCs, yet do not produce calcified matrix under well-established osteogenic culture conditions. Due to extensive proliferative potential and chondrogenic capacity, ACPCs show potential for cartilage regeneration and possibly for clinical application, as a promising alternative to MSCs or chondrocytes.

CD63+ and MHC Class I+ Subsets of Extracellular Vesicles Produced by Wild-Type and CD47-Deficient Jurkat T Cells Have Divergent Functional Effects on Endothelial Cell Gene Expression

T cells and endothelial cells engage in bidirectional communication that regulates angiogenesis and T cell transmigration. Extracellular vesicles (EVs) mediate intercellular communication by the transfer of bioactive molecules including RNAs. EVs produced by a given cell type are heterogeneous in their RNA content, but it is unclear how specific EV surface markers relate to their functional effects on target cells. Our previous work established that Jurkat T cell EVs bearing CD63, MHC-I, or CD47 surface markers contain distinct noncoding RNA populations. The present study reveals that CD63+ and MHC-I+ EVs from CD47-deficient Jurkat T cells are enriched in small non-coding RNAs relative to EVs from wild-type Jurkat T cells. CD47-deficient Jurkat T cells secrete more CD63+ and MHC-I+ EVs, but MHC-I+ EVs are selectively taken up more by human umbilical vein endothelial cells. Transcriptomics analysis of endothelial cells treated with CD63+ or MHC-I+ EVs showed surface marker- and CD47-dependent changes in gene expression in the target cells. Gene set enrichment analysis identified CD47-dependent, and surface marker-dependent effects of T cell EVs on VEGF and inflammatory signaling, cell cycle, and lipid and cholesterol metabolism. Thus, subsets of T cell EVs differentially regulate endothelial cell metabolism and inflammatory and angiogenic responses.

The Role of TGFβ1 and Macrophage Differentiation in MSU Crystal-Induced Inflammation

<p>Gout is a painful form of inflammatory arthritis that is caused by the deposition of monosodium urate (MSU) crystals in the joints. MSU crystals trigger a local inflammatory response initiated by resident macrophages followed by a large infiltration of leukocytes. The spontaneous resolution of acute gout is associated with the production of transforming growth factor β1 (TGFβ1). The overall objectives of this thesis were to investigate mechanisms that lead to TGFβ1 production and contribute to the resolution of acute gout, the effect of TGFβ1 on the functional phenotype of differentiated macrophages, and possible changes in surface marker expression by macrophages in response to MSU crystals. To determine macrophage-independent sources of TGFβ1 during the resolution of acute gout and how TGFβ1 production altered MSU crystal-recruited neutrophil functions, neutrophils were purified from MSU crystal-treated mice when levels of TGFβ1 were high. MSU crystal-recruited neutrophils and circulating blood neutrophils were identified as TGFβ1⁺ cells. The mechanism for TGFβ1 production by neutrophils was associated with their ability to phagocytose apoptotic neutrophils. TGFβ1 produced by canibalising neutrophils inhibited both respiratory burst and interleukin-1β (IL-1β) production by MSU crystal-activated neutrophils ex vivo. Importantly, neutrophils from MSU crystal-challenged mice treated with TGFβ1 neutralising antibody in vivo produced elevated levels of superoxide but neutrophil IL-1β production was unaffected. These results show that TGFβ1 produced by canibalising neutrophils can actively suppress neutrophil inflammatory functions and therefore make a significant contribution towards the resolution of gouty inflammation. To investigate the effect of TGFβ1 on macrophage differentiation in vitro, granulocyte macrophage colony-stimulating factor (GM-CSF) bone marrow macrophages (GM-BMMs) and macrophage colony-stimulating factor (M-CSF) bone marrow macrophages (M-BMMs) were generated in the presence of TGFβ1. TGFβ1 was found to drive a hyper-inflammatory GM-BMM phenotype, while contributing to the differentiation of a hypo-inflammatory M-BMM phenotype specifically in response to MSU crystals. Increased IL-1β production by TGFβ1-differentiated GM-BMMs was associated with enhanced NOD like receptor family, pyrin domain-containing 3 (NLRP3) in ammasome activation and caspase 1/caspase 8 interaction, and a down-regulation of receptor-interacting serine/threonine-protein kinase 3 (RIP3) triggered by MSU crystals. At the same, TGFβ1 inhibited antigen-specific T cell proliferation by GM-BMMs. In contrast, TGFβ1-treated M-BMMs down-regulated the expression of active IL-1β that correlated with decreased IL-1β production, and upregulated RIP3 expression in response to MSU crystals. These data indicate that TGFβ1-treated GM-BMMs exhibited a hyper-inflammatory response to MSU crystal stimulation, whereas M-BMMs were found to be hypo-responsive. Macrophages were found to upregulate the surface marker NK1.1, which is primarily expressed on natural killer (NK) cells, and occured as a consequence of phagocytosis. Following phagocytosis of MSU crystals, activated macrophages produced IL-1β and tumour necrosis factor ⍺ (TNF⍺), which triggered the upregulation of NK1.1 expression. Macrophage NK1.1 expression is an activation-driven event specifc to MSU crystals. However, phagocytosis of apoptotic neutrophils also triggered the upregulation of NK1.1 by macrophages, a non-inflammatory event that is characteristic for the resolution of acute inflammation. These findings suggest that macrophages may develop NK cell-like properties initiated by an activation-driven or apoptotic cell clearance mechanism. Taken together, the results of this thesis indicate that canibalising neutrophils self-regulate their inflammatory functions via TGFβ1 and that TGFβ1 drives a hyper-inflammatory GM-BMM phenotype, while shutting down inflammatory functions of M-BMMs. These data highlight a regulatory role for TGFβ1 during acute gouty inflammation.</p>

The Role of TGFβ1 and Macrophage Differentiation in MSU Crystal-Induced Inflammation

<p>Gout is a painful form of inflammatory arthritis that is caused by the deposition of monosodium urate (MSU) crystals in the joints. MSU crystals trigger a local inflammatory response initiated by resident macrophages followed by a large infiltration of leukocytes. The spontaneous resolution of acute gout is associated with the production of transforming growth factor β1 (TGFβ1). The overall objectives of this thesis were to investigate mechanisms that lead to TGFβ1 production and contribute to the resolution of acute gout, the effect of TGFβ1 on the functional phenotype of differentiated macrophages, and possible changes in surface marker expression by macrophages in response to MSU crystals. To determine macrophage-independent sources of TGFβ1 during the resolution of acute gout and how TGFβ1 production altered MSU crystal-recruited neutrophil functions, neutrophils were purified from MSU crystal-treated mice when levels of TGFβ1 were high. MSU crystal-recruited neutrophils and circulating blood neutrophils were identified as TGFβ1⁺ cells. The mechanism for TGFβ1 production by neutrophils was associated with their ability to phagocytose apoptotic neutrophils. TGFβ1 produced by canibalising neutrophils inhibited both respiratory burst and interleukin-1β (IL-1β) production by MSU crystal-activated neutrophils ex vivo. Importantly, neutrophils from MSU crystal-challenged mice treated with TGFβ1 neutralising antibody in vivo produced elevated levels of superoxide but neutrophil IL-1β production was unaffected. These results show that TGFβ1 produced by canibalising neutrophils can actively suppress neutrophil inflammatory functions and therefore make a significant contribution towards the resolution of gouty inflammation. To investigate the effect of TGFβ1 on macrophage differentiation in vitro, granulocyte macrophage colony-stimulating factor (GM-CSF) bone marrow macrophages (GM-BMMs) and macrophage colony-stimulating factor (M-CSF) bone marrow macrophages (M-BMMs) were generated in the presence of TGFβ1. TGFβ1 was found to drive a hyper-inflammatory GM-BMM phenotype, while contributing to the differentiation of a hypo-inflammatory M-BMM phenotype specifically in response to MSU crystals. Increased IL-1β production by TGFβ1-differentiated GM-BMMs was associated with enhanced NOD like receptor family, pyrin domain-containing 3 (NLRP3) in ammasome activation and caspase 1/caspase 8 interaction, and a down-regulation of receptor-interacting serine/threonine-protein kinase 3 (RIP3) triggered by MSU crystals. At the same, TGFβ1 inhibited antigen-specific T cell proliferation by GM-BMMs. In contrast, TGFβ1-treated M-BMMs down-regulated the expression of active IL-1β that correlated with decreased IL-1β production, and upregulated RIP3 expression in response to MSU crystals. These data indicate that TGFβ1-treated GM-BMMs exhibited a hyper-inflammatory response to MSU crystal stimulation, whereas M-BMMs were found to be hypo-responsive. Macrophages were found to upregulate the surface marker NK1.1, which is primarily expressed on natural killer (NK) cells, and occured as a consequence of phagocytosis. Following phagocytosis of MSU crystals, activated macrophages produced IL-1β and tumour necrosis factor ⍺ (TNF⍺), which triggered the upregulation of NK1.1 expression. Macrophage NK1.1 expression is an activation-driven event specifc to MSU crystals. However, phagocytosis of apoptotic neutrophils also triggered the upregulation of NK1.1 by macrophages, a non-inflammatory event that is characteristic for the resolution of acute inflammation. These findings suggest that macrophages may develop NK cell-like properties initiated by an activation-driven or apoptotic cell clearance mechanism. Taken together, the results of this thesis indicate that canibalising neutrophils self-regulate their inflammatory functions via TGFβ1 and that TGFβ1 drives a hyper-inflammatory GM-BMM phenotype, while shutting down inflammatory functions of M-BMMs. These data highlight a regulatory role for TGFβ1 during acute gouty inflammation.</p>

Mechanisms Involved in Type II Macrophage Activation and Effector Functions

<p>Autoimmunities are extremely difficult to treat and involved in their pathogenesis are pro-inflammatory immune responses redirected against one's own tissues. Studies in our lab have shown macrophages that are induced to become type II macrophages protect against an animal model of MS, experimental autoimmune encephalomyelitis (EAE), with protection due to immune deviation. Another way to deviate immune responses away from inflammation is by infection with the parasitic helminth Schistosoma mansoni, which also protects against EAE. The contribution of type II macrophages in this protection is unknown, as are the mechanisms involved in promoting the phenotype induced by type II activation. This project investigates key mechanisms involved in type II activation, while also elucidating the possible effect of schistosome exposure on the induction of this activation state. Using a validated model of type II activation in vitro, we compared the effects of schistosome immune complexes on various macrophage properties such as cytokine, surface marker and enzymatic profiles. This thesis identified that exposure to schistosome complexes induces a macrophage state with characteristics of two distinct activation states (type II and alternative activation), as well as completely novel characteristics. This activation state shows many phenotypic properties associated with immune regulation, and may have important consequences for understanding mechanisms involved in protection against inflammatory illnesses. We also investigated key mechanisms involved in the anti-inflammatory responses induced by type II activation. Cytokine, chemokine and surface marker profiles of macrophages were assessed in response to type II activation in vitro, with the main emphasis on determining the effects of IL-10 and CD40 on the type II activation phenotype and function. This investigation found that type II activated macrophages depend on low levels of CD40/CD40L signalling to polarise Th2 development, as the expression of receptors for Th2-inducing cytokines are significantly impaired in the absence of this interaction. This suggests an important role for the low but maintained levels of CD40 on type II activated macrophages, in aiding the deviation of immune responses, while maintaining Th2 polarization. We also suggest a suppressive role of CD40/CD40L in IL-10 production, which is a novel find. The requirement of new treatments for MS is escalating as more people are affected each year. The impact of MS on the quality of life is severe and long lasting. Having a greater understanding of the mechanisms involved in deviating pro-inflammatory or anti-inflammatory responses will enable the development of much more effective treatments and therapies in the future.</p>

Mechanisms Involved in Type II Macrophage Activation and Effector Functions

<p>Autoimmunities are extremely difficult to treat and involved in their pathogenesis are pro-inflammatory immune responses redirected against one's own tissues. Studies in our lab have shown macrophages that are induced to become type II macrophages protect against an animal model of MS, experimental autoimmune encephalomyelitis (EAE), with protection due to immune deviation. Another way to deviate immune responses away from inflammation is by infection with the parasitic helminth Schistosoma mansoni, which also protects against EAE. The contribution of type II macrophages in this protection is unknown, as are the mechanisms involved in promoting the phenotype induced by type II activation. This project investigates key mechanisms involved in type II activation, while also elucidating the possible effect of schistosome exposure on the induction of this activation state. Using a validated model of type II activation in vitro, we compared the effects of schistosome immune complexes on various macrophage properties such as cytokine, surface marker and enzymatic profiles. This thesis identified that exposure to schistosome complexes induces a macrophage state with characteristics of two distinct activation states (type II and alternative activation), as well as completely novel characteristics. This activation state shows many phenotypic properties associated with immune regulation, and may have important consequences for understanding mechanisms involved in protection against inflammatory illnesses. We also investigated key mechanisms involved in the anti-inflammatory responses induced by type II activation. Cytokine, chemokine and surface marker profiles of macrophages were assessed in response to type II activation in vitro, with the main emphasis on determining the effects of IL-10 and CD40 on the type II activation phenotype and function. This investigation found that type II activated macrophages depend on low levels of CD40/CD40L signalling to polarise Th2 development, as the expression of receptors for Th2-inducing cytokines are significantly impaired in the absence of this interaction. This suggests an important role for the low but maintained levels of CD40 on type II activated macrophages, in aiding the deviation of immune responses, while maintaining Th2 polarization. We also suggest a suppressive role of CD40/CD40L in IL-10 production, which is a novel find. The requirement of new treatments for MS is escalating as more people are affected each year. The impact of MS on the quality of life is severe and long lasting. Having a greater understanding of the mechanisms involved in deviating pro-inflammatory or anti-inflammatory responses will enable the development of much more effective treatments and therapies in the future.</p>

The Therapeutic Modalities of Sickle Cell Disease Reprogram the Platelet Functional-Bioenergetic Profile

BACKGROUND: Sickle cell disease (SCD) is a group of inherited hemoglobinopathies that continues to be highly morbid and lethal. SCD-associated platelet hyperreactivity is a well-recognized contributor to the pathophysiology of the disease via complex interactions with the immune system and endothelium. Aberrant platelet bioenergetics have been implicated as a biological mechanism for SCD-associated platelet hyperreactivity, however, little is known about the impact current medical interventions (e.g., hydroxyurea [HU] and red blood cell [RBC] exchange transfusions) have on the platelet functional-bioenergetic profile. In this study we investigate the effects of hydroxyurea and RBC exchange transfusions on reprograming the platelet functional-bioenergetic profile and provide insight into biological pathways that may be amenable to intervention. METHODS: Platelets from sex-, race-, and aged-matched adult healthy control subjects and adult patients with homozygous SCD (HbSS), actively being treated with hydroxyurea (HU group) or RBC exchange transfusions (RBC exchange transfusion group), were isolated and washed following standard protocols. Platelet activation by flow cytometry was determined at baseline and following activation with thrombin (0.075U/ml) and ADP (1.25uM). Platelet-activated fibrinogen binding site (αIIbβIII), P-selectin, and phosphatidylserine (PS) surface marker expression (as measured by mean fluorescence intensity [MFI]) was determined with PAC-1, P-selectin, and lactadherin antibodies, respectively. The bioenergetic profile of washed platelets was determined by the 24-well format Seahorse extracellular flux analyzer. Statistical analyses were performed using the one-way ANOVA. Correlations were performed by 2-tailed nonparametric Spearman correlations and linear regression analysis with 95% confidence interval (GraphPad Software v9.1.2). Data expressed as mean plus or minus standard error of the mean (SEM). Differences were considered significant at p &lt; 0.05. RESULTS: Platelets from patients in the HU group exhibited increased surface marker expression of αIIbβIII (p = 0.004), P-selectin (p = 0.003), and PS (p = 0.003) at resting conditions when compared to the RBC exchange transfusion group and healthy controls. Additionally, an increase in PS expression was seen in the HU group upon activation with ADP (p = 0.0003). No significant differences were seen in the platelet functional profile after activation with thrombin. The platelet bioenergetic profile in the HU group demonstrated an elevated proton leak (p = 0.03) when compared to the RBC exchange transfusion group. Elevated proton leak in SCD was found to have positive correlation with P-selectin and PS expression (Figure 1). CONCLUSION: While therapeutic interventions have improved overall outcomes in patients with SCD, adverse events continue to be a deterrent to many patients prompting the need for safer, more tolerable, and cost-effective alternatives. We have identified that while HU has little impact on the hyperreactive and procoagulant platelet phenotype in SCD, RBC exchange transfusions appear to mitigate the phenotype and reprogram the bioenergetic profile. Amongst treatment groups, a strong correlation was found between platelet activation markers (i.e., P-selectin and PS) and proton leak, suggesting an interplay between alterations in platelet bioenergetics and SCD-associated platelet hyperreactivity. Further studies are needed to elucidate the metabolic pathways that are responsible for the aberrant platelet functional-bioenergetic profile seen in SCD. These observations are important as targeting the platelet bioenergetic profile via less invasive and toxic therapeutic modalities may be equally efficacious as current interventions. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.