Upregulation Of Anti-inflammatory Genes Research Articles

Initial therapeutic evidence of a borosilicate bioactive glass (BSG) and Fe3O4 magnetic nanoparticle scaffold on implant-associated Staphylococcal aureus bone infection

Implant-associated Staphylococcus aureus (S. aureus) osteomyelitis is a severe challenge in orthopedics. While antibiotic-loaded bone cement is a standardized therapeutic approach for S. aureus osteomyelitis, it falls short in eradicating Staphylococcus abscess communities (SACs) and bacteria within osteocyte-lacuna canalicular network (OLCN) and repairing bone defects. To address limitations, we developed a borosilicate bioactive glass (BSG) combined with ferroferric oxide (Fe3O4) magnetic scaffold to enhance antibacterial efficacy and bone repair capabilities. We conducted comprehensive assessments of the osteoinductive, immunomodulatory, antibacterial properties, and thermal response of this scaffold, with or without an alternating magnetic field (AMF). Utilizing a well-established implant-related S. aureus tibial infection rabbit model, we evaluated its antibacterial performance in vivo. RNA transcriptome sequencing demonstrated that BSG + 5%Fe3O4 enhanced the immune response to bacteria and promoted osteogenic differentiation and mineralization of MSCs. Notably, BSG + 5%Fe3O4 upregulated gene expression of NOD-like receptor and TNF pathway in MSCs, alongside increased the expression of osteogenic factors (RUNX2, ALP and OCN) in vitro. Flow cytometry on macrophage exhibited a polarization effect towards M2, accompanied by upregulation of anti-inflammatory genes (TGF-β1 and IL-1Ra) and downregulation of pro-inflammatory genes (IL-6 and IL-1β) among macrophages. In vivo CT imaging revealed the absence of osteolysis and periosteal response in rabbits treated with BSG + 5%Fe3O4 + AMF at 42 days. Histological analysis indicated complete controls of SACs and bacteria within OLCN by day 42, along with new bone formation, signifying effective control of S. aureus osteomyelitis. Further investigations will focus on the in vivo biosafety and biological mechanism of this scaffold within infectious microenvironment.

Maillard Reaction-Derived S-Doped Carbon Dots Promotes Downregulation of PPARγ, C/EBPα, and SREBP-1 Genes In-Vitro.

Carbon nanodots (CDs) are commonly found in food products and have attracted significant attention from food scientists. There is a high probability of CD exposure in humans, but its impacts on health are unclear. Therefore, health effects associated with CD consumption should be investigated. In this study, we attempted to create a model system of the Maillard reaction between cystine and glucose using a simple cooking approach. The CDs (CG-CDs) were isolated from cystine-glucose-based Maillard reaction products and characterized using fluorescence spectroscopy, X-ray diffractometer (XRD), and transmission electron microscope (TEM). Furthermore, human mesenchymal stem cells (hMCs) were used as a model to unravel the CDs' cytotoxic properties. The physiochemical assessment revealed that CG-CDs emit excitation-dependent fluorescence and possess a circular shape with sizes ranging from 2 to 13 nm. CG-CDs are predominantly composed of carbon, oxygen, and sulfur. The results of the cytotoxicity evaluation indicate good biocompatibility, where no severe toxicity was observed in hMCs up to 400 μg/mL. The DPPH assay demonstrated that CDs exert potent antioxidant abilities. The qPCR analysis revealed that CDs promote the downregulation of the key regulatory genes, PPARγ, C/EBPα, SREBP-1, and HMGCR, coupled with the upregulation of anti-inflammatory genes. Our findings suggested that, along with their excellent biocompatibility, CG-CDs may offer positive health outcomes by modulating critical genes involved in lipogenesis, homeostasis, and obesity pathogenesis.

Microglia/macrophage polarization regulates spontaneous remyelination in intermittent cuprizone model of demyelination

Central nervous system (CNS) lesions can repeatedly be de-and remyelinated during demyelinating diseases such as multiple sclerosis (MS). Here, we designed an intermittent demyelination model by 0.3 % Cuprizone feeding in C57/BL6 mice followed by two weeks recovery. Histochemical staining of luxol fast blue (LFB) was used for study of remyelination, detection of glial and endothelial cells was performed by immunohistochemistry staining for the following antibodies: anti Olig2 for oligodendrocyte progenitor cells, anti APC for mature oligodendrocytes, anti GFAP for astrocytes, and anti Iba-1 for microglia/macrophages, anti iNOS for M1 microglia/macrophage phenotype, anti TREM-2 for M2 microglia/macrophage phenotype and anti CD31 for endothelial cells. Also, real-time polymerase chain reaction was performed for assessment of the expression of the targeted genes. LFB staining results showed enhanced remyelination in the intermittent cuprizone (INTRCPZ) group, which was accompanied by improved motor function, increased mature oligodendrocyte cells, and reduction of astrogliosis and microgliosis. Moreover, switching from M1 to M2 polarity increased in the INTRCPZ group that was in association with downregulation of pro-inflammatory and upregulation of anti-inflammatory genes. Finally, evaluation of microvascular changes revealed a remarkable decrease in the endothelial cells in the cuprizone (CPZ) group which recovered in the INTERCPZ group. The outcomes demonstrate enhanced myelin content during recovery in the intermittent demyelination model which is in association with reshaping macrophage polarity and modification of glial and endothelial cells.

The Protective Role of TREM2 in the Heterogenous Population of Macrophages during Post-Myocardial Infarction Inflammation.

Advances in interventions after myocardial infarction (MI) have dramatically increased survival, but MI remains the leading cause of heart failure due to maladaptive ventricular remodeling following ischemic damage. Inflammation is crucial in both the initial response to ischemia and subsequent wound healing in the myocardium. To date, preclinical and clinical efforts have been made to elucidate the deleterious effects of immune cells contributing to ventricular remodeling and to identify therapeutic molecular targets. The conventional concept classifies macrophages or monocytes into dichotomous populations, while recent studies support their diverse subpopulations and spatiotemporal dynamicity. The single-cell and spatial transcriptomic landscapes of macrophages in infarcted hearts successfully revealed the heterogeneity of cell types and their subpopulations post-MI. Among them, subsets of Trem2hi macrophages were identified that were recruited to infarcted myocardial tissue in the subacute phase of MI. The upregulation of anti-inflammatory genes was observed in Trem2hi macrophages, and an in vivo injection of soluble Trem2 during the subacute phase of MI significantly improved myocardial function and the remodeling of infarcted mice hearts, suggesting the potential therapeutic role of Trem2 in LV remodeling. Further investigation of the reparative role of Trem2 in LV remodeling would provide novel therapeutic targets for MI.

Insulin‐like growth factor 1 induces a reparative neutrophil phenotype

Cardiovascular diseases are the leading cause of death in western societies and neutrophils were long thought to play a detrimental role in ischaemic heart disease. But recent data showed that neutrophils also play an important role in cardiac remodelling and can exhibit an anti‐inflammatory N2‐phenotype or a pro‐inflammatory N1 phenotype. Previous experiments showed that short‐term treatment with insulin‐like growth factor (IGF1) preserves cardiac function after myocardial infarction (MI). This effect was mediated by myeloid cells. Therefore, it was the aim to analyse to what extent and how IGF1 modulates neutrophil function.Murine bone marrow neutrophils were isolated by density gradient centrifugation and were left untreated or polarized for 4 hours in vitrowith IL‐4 (20 ng/ml) or IGF1 (10 ng/ml), Insulin (10 ng/ml) or LPS/IFN‐γ (10 ng/ml / 2 ng/ml).Transcript expression analysis and qRT‐PCR were performed to analyse to what extent transcriptional changes were triggered by growth factor and cytokine induced polarization after 4 hours. These analyses revealed that IL‐4, and interestingly also IGF1, induces a reparative N2‐like neutrophil phenotype, characterised by the induction of Arg1, Retnlα and Chi3l3.Surprisingly, Insulin, despite the high homology to IGF1, did not induce an antiinflammatory phenotype. LPS/IFN‐y treatment induced upregulation of proinflammatory genes (TNFα, IL12a, iNOS). As expected, RNAseq analysis showed, that IL‐4 treatment upregulated genes involved in the IL‐4 pathway. Surprisingly, IGF1 treatment induced almost the same pathways as IL‐4. Therefore, the canonical IL‐4 signaling pathway Jak‐Stat was analysed. This revealed that IL‐4, and also IGF1, induced phosphorylation of Stat6. Additionally, Jak1/2/3/Tyk2 was inhibited with pharmacological inhibitors (InSolution Jak Inhibitor I, Ruxolitinib, BMS‐911543). Inhibition of all Jaks prevented IL‐4, and also IGF1, induced upregulation of N2 marker genes. Specific inhibition of Jak2 showed that IL‐4‐ and IGF1‐induced polarization is most likely dependent on Jak2 activation, as N2 marker gene expression was reduced. Western blot analysis as well revealed that Stat6 phosphorylation was reduced after Jak2 inhibition and Proximity ligation assay proved an interaction of the IGF1R and Jak2. Regarding the missing effect of insulin also the canonical insulin/IGF1 pathway, as for example the Akt and Ras‐Raf‐Erk pathway, were analysed. By using Tie2 Akt1 and Tie2 Akt2 KO mice, we showed the independency of IL‐4 and IGF1 induced polarization on those Akt isoforms, as upregulation of antiinflammatory genes was not reduced.Additionally, the effect of different polarizers on the formation of neutrophil extracellular traps, an inflammatory mechanism often found in heart failure, was analysed. This revealed that both, IL‐4 and IGF1, are able to reduce the formation of NETs, whereas LPS/IFN‐y treatment induces the formation of NETs.In conclusion, these findings indicate that IGF1 is able to polarize neutrophils to a reparative N2‐like phenotype most likely via the non‐canonical Jak2‐Stat6 axis, comparable to that induced by the classical M2/N2 polarizer IL‐4. Additionally, IGF1 is able to reverse proinflammatory mechanisms.

Liposomal delivery of azithromycin enhances its immunotherapeutic efficacy and reduces toxicity in myocardial infarction

A growing body of evidence shows that altering the inflammatory response by alternative macrophage polarization is protective against complications related to acute myocardial infarction (MI). We have previously shown that oral azithromycin (AZM), initiated prior to MI, reduces inflammation and its negative sequelae on the myocardium. Here, we investigated the immunomodulatory role of a liposomal AZM formulation (L-AZM) in a clinically relevant model to enhance its therapeutic potency and avoid off-target effects. L-AZM (40 or 10 mg/kg, IV) was administered immediately post-MI and compared to free AZM (F-AZM). L-AZM reduced cardiac toxicity and associated mortality by 50% in mice. We observed a significant shift favoring reparatory/anti-inflammatory macrophages with L-AZM formulation. L-AZM use resulted in a remarkable decrease in cardiac inflammatory neutrophils and the infiltration of inflammatory monocytes. Immune cell modulation was associated with the downregulation of pro-inflammatory genes and the upregulation of anti-inflammatory genes. The immunomodulatory effects of L-AZM were associated with a reduction in cardiac cell death and scar size as well as enhanced angiogenesis. Overall, L-AZM use enhanced cardiac recovery and survival after MI. Importantly, L-AZM was protective from F-AZM cardiac off-target effects. We demonstrate that the liposomal formulation of AZM enhances the drug’s efficacy and safety in an animal model of acute myocardial injury. This is the first study to establish the immunomodulatory properties of liposomal AZM formulations. Our findings strongly support clinical trials using L-AZM as a novel and clinically relevant therapeutic target to improve cardiac recovery and reduce heart failure post-MI in humans.

Heart rate reduction with ivabradine in the early phase of atherosclerosis is protective in the endothelium of ApoE-deficient mice.

Ivabradine, a heart rate reducing agent, protects the vascular system by unidentified mechanisms. We sought to determine the effects of the treatment with ivabradine, started before plaque formation, on early transcriptional changes and endothelium lesions in regions of aorta subjected to disturbed blood flow. Six week-old apolipoprotein E-deficient (ApoE-/-) mice, fed a low-fat diet, were treated with ivabradine to determine the effect on transcriptional changes (2-and 4-week treatment) and on lesions formation (19-week treatment) in the endothelium of the aortic arch. Microarrays analysis (60k probes) of endothelium-enriched RNA was carried out to detect changes in gene expression induced by treatment. Endothelium damage was assessed by en-face immunofluorescence staining for vascular endothelial (VE) cadherin. According to microarray analysis, 930 transcripts were affected by the treatment. We found downregulation of pro-apoptotic and pro-inflammatory genes, the majority of which are nuclear factor-κB (NF-κB)-and/or angiotensin II-regulated genes, and upregulation of anti-inflammatory genes. Many shear stress-responsive genes were affected by the treatment and the MAPK, Notch signalling and sterol metabolic processes were among the most significantly affected pathways. Consistently, we observed increased levels of Hes5, a Notch target gene, together with a reduction of endothelium damage, in the lower aortic arch of treated- compared with untreated- mice. We concluded that an early treatment with ivabradine protected the endothelium of the aortic arch of ApoE-/- mice. Activation of the Notch signalling could be part of the mechanism underlying this protection.

Abstract 21281: Cardiosphere-Derived Cell and Mesenchymal Stem Cell Extracellular Vesicles Contain Distinct RNA Cargo

Introduction: Cardiosphere-derived cells (CDCs) possess regenerative, immunomodulatory and cardioprotective characteristics when delivered to the heart post-myocardial infarction (MI). CDCs derived from different donors possess variable regenerative potency in a MI model. We previously demonstrated that human CDC-derived extracellular vesicles (CDC-EVs) recapitulate the effects of CDCs in acute and chronic in vivo MI models. We are testing the hypothesis that a distinct cargo profile defines the functional efficacy between CDC-EVs and mesenchymal stem cell-derived EVs (MSC-EVs). Also, we assessed the immunomodulatory role of CDC-EVs derived from potent and non-potent CDCs in comparison with MSC-EVs. Methods: CDCs or MSCs were cultured in serum-free medium for 15 days and the conditioned media was concentrated by ultrafiltration to isolate EVs. The miRNeasy Serum/Plasma kit was used to extract total RNA from EVs followed by small RNA sequencing (NextSeq 500, Illumina) (CDC-EVs, n =12; MSC-EVs, n =4). We performed an in vitro and in vivo macrophage assay to assess the immunomodulatory role of EVs. Results: RNA sequencing analysis of CDC-EVs and MSC-EVs revealed a greater overall abundance of Y-RNA fragments and miRNA in CDC-EVs. Examining the specific Y-RNA classes, we found that MSC-EVs contained an expression profile with lower Y4 (p<0.05) but higher Y5 (p<0.05) Y-RNA fragments. Four miRNAs (miR-146a, miR-151, miR-409, miR-423) were highly enriched in CDC-EVs while miR-10a and miR-4792 were more abundant in MSC-EVs. EVs from potent CDCs showed a stronger upregulation of anti-inflammatory genes in activated macrophages ( in vitro ) and prevented accumulation of macrophages ( in vivo ) when compared with EVs obtained from non-potent CDCs or MSCs. Conclusions: We demonstrated that CDC-EVs contain a unique RNA cargo set that can differentiate CDC-EVs from MSC-EVs. The higher presence of Y-RNA in CDC-EVs as compared to MSC-EVs could be responsible for the ability of CDCs to regulate stem cell activation and tissue regeneration. The highly enriched panel of miRNAs observed in CDC-EVs and the ability to reduce the inflammatory reaction by macrophages, in contrast to MSC-EVs, suggest that they may support some of the functional benefits observed post-MI.

Simultaneously Monitoring Immune Response and Microbial Infections during Pregnancy through Plasma cfRNA Sequencing.

Plasma cell-free RNA (cfRNA) encompasses a broad spectrum of RNA species that can be derived from both human cells and microbes. Because cfRNA is fragmented and of low concentration, it has been challenging to profile its transcriptome using standard RNA-seq methods. We assessed several recently developed RNA-seq methods on cfRNA samples. We then analyzed the dynamic changes of both the human transcriptome and the microbiome of plasma during pregnancy from 60 women. cfRNA reflects a well-orchestrated immune modulation during pregnancy: an up-regulation of antiinflammatory genes and an increased abundance of antimicrobial genes. We observed that the plasma microbiome remained relatively stable during pregnancy. The bacteria Ureaplasma shows an increased prevalence and increased abundance at postpartum, which is likely to be associated with postpartum infection. We demonstrated that cfRNA-seq can be used to monitor viral infections. We detected a number of human pathogens in our patients, including an undiagnosed patient with a high load of human parvovirus B19 virus (B19V), which is known to be a potential cause of complications in pregnancy. Plasma cfRNA-seq demonstrates the potential to simultaneously monitor immune response and microbial infections during pregnancy.

Pancreastatin-dependent inflammatory signaling mediates obesity-induced insulin resistance.

Chromogranin A knockout (Chga-KO) mice exhibit enhanced insulin sensitivity despite obesity. Here, we probed the role of the chromogranin A-derived peptide pancreastatin (PST: CHGA(273-301)) by investigating the effect of diet-induced obesity (DIO) on insulin sensitivity of these mice. We found that on a high-fat diet (HFD), Chga-KO mice (KO-DIO) remain more insulin sensitive than wild-type DIO (WT-DIO) mice. Concomitant with this phenotype is enhanced Akt and AMPK signaling in muscle and white adipose tissue (WAT) as well as increased FoxO1 phosphorylation and expression of mature Srebp-1c in liver and downregulation of the hepatic gluconeogenic genes, Pepck and G6pase. KO-DIO mice also exhibited downregulation of cytokines and proinflammatory genes and upregulation of anti-inflammatory genes in WAT, and peritoneal macrophages from KO mice displayed similarly reduced proinflammatory gene expression. The insulin-sensitive, anti-inflammatory phenotype of KO-DIO mice is masked by supplementing PST. Conversely, a PST variant peptide PSTv1 (PST-NΔ3: CHGA(276-301)), lacking PST activity, simulated the KO phenotype by sensitizing WT-DIO mice to insulin. In summary, the reduced inflammation due to PST deficiency prevented the development of insulin resistance in KO-DIO mice. Thus, obesity manifests insulin resistance only in the presence of PST, and in its absence obesity is dissociated from insulin resistance.

Limb Ischemia Protects Against Contrast-Induced Nephropathy

Contrast-induced acute kidney injury (CI-AKI), or nephropathy (CIN), is frequently diagnosed in the setting of coronary angiography. The incidence varies in the literature partly related to the differences in diagnostic criteria used for CIN. In a recently published article in Circulation , Maioli et al1 used a definition of an increase of ≥0.5 mg/dL over baseline serum creatinine within 3 days of the administration of contrast medium and found an incidence of 12.1% among 1490 patients who had a baseline estimated creatinine clearance of 4-fold in patients who had mild AKI (≥0.3 mg/L or …

Consequences of Intermittent PTH Treatment on Myeloma Growth, Bone Disease and Molecular Profiling of Whole Myelomatous Bone

AbstractAbstract 4063Intermittent PTH (iPTH) treatment is bone anabolic and has been successfully used for treatment of osteoporosis. The aims of the study were to investigate the effects of iPTH-induced bone formation on myeloma progression and unravel molecular mechanisms associated with these effects, using the SCID-rab and SCID-hu models. Using our reported procedure (Xin et al., BJH 2007), we established a novel myeloma cell line, Hg, capable of sequential passaging in experimental models. Hg cells have similar gene expression profiling (GEP) as the original patient's plasma cells, are classified in the MMSET subtype and express DKK1. SCID-hu or SCID-rab (8-10 hosts/group in each model) mice engrafted with Hg myeloma cells were subcutaneously treated with saline or iPTH (80 ug/kg/day) for 4 weeks. Overall, whereas BMD of the myelomatous bones in saline-treated hosts was similarly reduced in SCID-rab and SCID-hu mice by 14±5%, it was increased by 10±2% in iPTH-treated hosts (p<0.002 saline vs. iPTH-treated hosts). Histologic and histomorphometric analyses revealed increased bone formation parameters and no effect on number of osteoclasts. The bone anabolic effect of iPTH was associated with reduced myeloma growth by >50% (p<0.03) assessed by measurement of human immunoglobulin level in mice sera and histologically. Treatment with iPTH also increased BMD and attenuated myeloma growth in SCID-rab mice engrafted with myeloma cells from 10 patients. We found by qRT-PCR that type-1 PTH receptor was not expressed by myeloma cells and that PTH had no direct effect on in vitro growth of myeloma cells indicating that PTH anti-myeloma effect is indirectly mediated through modulation of the BM microenvironment. To shed light on molecular mechanisms associated with iPTH effects, human GEP and qRT-PCR validation of selected genes were preformed on whole myelomatous human bones from Hg-bearing SCID-hu mice treated with saline or iPTH for 4 weeks (5 hosts/group). Mice were sacrificed 2 hours after the last injection. Treatment with iPTH upregulated 343 genes and downregulated 410 genes ('2 folds, p<0.05) in myelomatous bones. There was a remarkable alteration in expression of genes associated with cAMP (e.g. RGS1/2 upregulation) and Wnt (e.g. LRP4 upregulation; DKK1 downregulation) signaling, upregulation of growth factors and receptors involved in bone remodeling (e.g. FGFR1/2, FDGFA, FDGFRA, TGFB, TGFBR1), and increased expression of osteoblast markers (e.g. osteocalcin, RUNX2). Interestingly, although iPTH induced upregulation of RANKL there was a reduction in expression of osteoclastic genes (e.g. ACP5/TRAP, NFATC1), probably due to increased osteoblast numbers, downregulation of inflammatory genes (e.g. AIF1) and upregulation of anti-inflammatory genes (e.g. TNFAIP6, CXCL14). Moreover, iPTH reduced expression of typical myeloma associated genes (e.g. CD38, WHSC1, IRF4) and had no effect on expression of myeloma growth factors such as IL6 and IGF1. Expression of certain documented anti-myeloma factors was upregulated by iPTH (e.g. decorin). We conclude that iPTH-induced bone formation in myelomatous bones is mediated by activation of multiple signaling pathways involved in osteoblastogenesis, and attenuated bone resorption and myeloma growth through mechanisms involving increased production of anti-myeloma factors by osteoblasts and minimizing inflammatory conditions induced by myeloma. Treatment with iPTH may be a promising approach for myeloma bone disease and tumor progression. Disclosures:No relevant conflicts of interest to declare.

Gene profiling reveals increased expression of uteroglobin and other anti-inflammatory genes in glucocorticoid-treated nasal polyps

Treatment with local glucocorticoids (GCs) decreases symptoms and the size of nasal polyps. This might depend on the downregulation of proinflammatory genes, as well as the upregulation of anti-inflammatory genes. We sought to identify GC-regulated anti-inflammatory genes in nasal polyps. Affymetrix DNA microarrays were used to analyze the expression of 22,283 genes in 4 nasal polyps before and after local treatment with fluticasone (400 microg/d). Expression of uteroglobin and mammaglobin B was analyzed with real-time PCR in 6 nasal polyps and in nasal biopsy specimens from 6 healthy control subjects. Two hundred three genes had changed in expression in treated polyps, and 139 had known functions: 54 genes were downregulated, and 85 were upregulated. Genes associated with inflammation constituted the largest single functional group. These genes affected key steps in inflammation (eg, immunoglobulin production; antigen processing and presentation; and the chemoattraction and activation of granulocytes, T cells, and B cells). Several proinflammatory genes were downregulated. In contrast, some anti-inflammatory genes were upregulated. The gene that increased most in terms of expression was uteroglobin. This was confirmed with real-time PCR. By contrast, expression of uteroglobin was lower in untreated polyps than in healthy nasal mucosa. Immunohistochemical investigation showed staining of uteroglobin in the epithelium and in seromucous glands in control subjects and in nasal polyps. Upregulation of anti-inflammatory genes, such as uteroglobin, might contribute to the effects of local treatment with GCs in nasal polyps.