Anti-inflammatory Gene Expression Research Articles

Estradiol treatment increases M2-like visceral adipose tissue macrophages in obese ovariectomized mice regardless of its anorectic action

ABSTRACT Estradiol (E2) treatment has been known to induce changes in food intake, energy expenditure, and weight gain. However, its direct effects on adipose tissue macrophages (ATM) in vivo are not fully understood. Thus, we aimed to explore this aspect at cellular and molecular levels in ovariectomized obese mice. We examined the changes in ATMs after eight weeks of a high-fat diet (HFD) in male, female, and ovariectomized (OVX) mice. After eight weeks, osmotic pumps were inserted into OVX mice to provide two weeks of E2 treatment. We additionally set up a vehicle Pair-Fed (PF) control group that supplied the same amount of HFD consumed by the E2-treated group. We then investigated the in vivo phenotypic changes of visceral adipose tissue (VAT) macrophages. The percentage of M1-like ATMs decreased by the anorectic effect of E2, while M2-like ATMs increased regardless of the anorexia. E2 treatment increased the expression of anti-inflammatory genes but decreased pro-inflammatory genes in VAT. Monocyte recruitment and local proliferation contributed to M2-like ATMs. Furthermore, M2-like phenotypes were induced by E2 treatment in human macrophages. E2 treatment increases M2-like macrophages and improves the tissue milieu of VAT regardless of the anorectic reaction of E2.

Nature-inspired allomelanin nanomedicine alleviates cardiac ischemia/reperfusion injury via scavenging free radicals and ameliorating myocardial microenvironment

Myocardial ischemia/reperfusion (I/R) injury is the resultantly pathological heart damage from the restoration of blood supply through percutaneous coronary intervention. Herein, inspired by naturally occurring antioxidative allomelanin derived from Fungi, a distinct self-assembly synthesis strategy is ingeniously leveraged to engineer allomelanin nanoparticles (AMNPs) for improving therapeutic efficacy of myocardial I/R injury. These PEGylated AMNPs@PEG nanomedicines feature favorable capability in scavenging intracellular free radicals, inhibiting mitochondrial membrane potential depolarization, and improving cell survival. Attributing to the augmented vascular permeability during myocardial I/R injury, AMNPs@PEG could target and accumulate at the damaged cardiac sites for photoacoustic visualization. Importantly, AMNPs@PEG could achieve the macrophage polarization from M1 to M2 subtype and inhibit neutrophil recruitment, accompanied by the reduced expression of pro-inflammatory genes and the elevated expression of anti-inflammatory genes, respectively, which lead to a considerable reduction in myocardial infarction size and an obvious improvement in cardiac function after I/R injury. This study elucidates that the engineered AMNPs nanomedicines could not only be used as an antioxidative and anti-inflammatory nanoplatform for effective therapy of myocardial I/R injury, but also provide an alternative therapeutic option for other reactive oxygen species-related diseases.

Contrasting effects of sleep fragmentation and angiotensin-II treatment upon pro-inflammatory responses of mice

Disordered sleep promotes inflammation in brain and peripheral tissues, but the mechanisms that regulate these responses are poorly understood. One hypothesis is that activation of the sympathetic nervous system (SNS) from sleep loss elevates blood pressure to promote vascular sheer stress leading to inflammation. As catecholamines produced from SNS activation can directly regulate inflammation, we pharmacologically altered blood pressure using an alternative approach-manipulation of the renin-angiotensin system (RAS). Male C57BL6/J mice were treated with angiotensin or captopril to elevate and reduce blood pressure, respectively and then exposed to 24-h of sleep fragmentation (SF) or allowed to sleep (control). Pro- and anti-inflammatory cytokine gene expression and as endothelial adhesion gene expression as well as serum glucocorticoids (corticosterone) were measured. RAS manipulation elevated cytokines and endothelial adhesion expression in heart and aorta while SF increased cytokine expression in peripheral tissues, but not brain. However, there were interactive effects of angiotensin-II and SF upon cytokine gene expression in hippocampus and hypothalamus, but not prefrontal cortex. SF, but not RAS manipulation, elevated serum corticosterone concentration. These findings highlight the contrasting effects of RAS manipulation and SF, implying that inflammation from SF is acting on different pathways that are largely independent of RAS manipulation.

Deacetylepoxyazadiradione Derived from Epoxyazadiradione of Neem (Azadirachta indica A. Juss) Fruits Mitigates LPS-Induced Oxidative Stress and Inflammation in Zebrafish Larvae.

Reactive oxygen species (ROS) produced by cell metabolism have a duplex role in oxidation and inflammation reactions which involve cell damage or repair responses. Excess ROS production has detrimental effects on the survival of cells. We examined the protective effect of a semi-natural compound NF2 (deacetylepoxyazadiradione), for its protective activity against free radical-mediated stress and inflammatory response to lipopolysaccharide (LPS) using zebrafish larvae. Preliminary antioxidant assays indicated an increase in scavenging of free radicals from NF2 than NF1 (Epoxyazadiradione) in a concentration-dependent manner. Cell cytotoxicity was determined using rat myoblast cell lines (L6), and more than 95 % of cell viability was obtained. Zebrafish developmental toxicity test indicated that NF2 is not toxic even at 150 μM. The percentage of ROS, lipid peroxidation, nitric oxide and apoptosis were reduced significantly in NF2 treated LPS-stressed zebrafish larvae. The reduced number of employed macrophages on NF2 treatment was observed in neutral red dye-marked macrophage localization images. Relative expression of antioxidant genes in zebrafish larvae after treatment with NF2 is significantly increased. The RT-PCR quantification of antioxidant and anti-inflammatory gene expression indicated decreased relative folds of pro-inflammatory cytokines, iNOS and increased relative folds of mitochondrial antioxidant genes (GR, GST and GPx) in LPS stressed zebrafish larvae after treatment with NF2. From the overall obtained results, it can be concluded that NF2 reduced the oxidative stress and inflammatory response by scavenging free radicals caused by LPS.

Mechanism of bisphosphonate-related osteonecrosis of the jaw (BRONJ) revealed by targeted removal of legacy bisphosphonate from jawbone using competing inert hydroxymethylene diphosphonate.

Bisphosphonate-related osteonecrosis of the jaw (BRONJ) presents as a morbid jawbone lesion in patients exposed to a nitrogen-containing bisphosphonate (N-BP). Although it is rare, BRONJ has caused apprehension among patients and healthcare providers and decreased acceptance of this antiresorptive drug class to treat osteoporosis and metastatic osteolysis. We report here a novel method to elucidate the pathological mechanism of BRONJ by the selective removal of legacy N-BP from the jawbone using an intra-oral application of hydroxymethylene diphosphonate (HMDP) formulated in liposome-based deformable nanoscale vesicles (DNV). After maxillary tooth extraction, zoledronate-treated mice developed delayed gingival wound closure, delayed tooth extraction socket healing and increased jawbone osteonecrosis consistent with human BRONJ lesions. Single cell RNA sequencing of mouse gingival cells revealed oral barrier immune dysregulation and unresolved proinflammatory reaction. HMDP-DNV topical applications to nascent mouse BRONJ lesions resulted in accelerated gingival wound closure and bone socket healing as well as attenuation of osteonecrosis development. The gingival single cell RNA sequencing demonstrated resolution of chronic inflammation by increased anti-inflammatory signature gene expression of lymphocytes and myeloid-derived suppressor cells. This study suggests that BRONJ pathology is related to N-BP levels in jawbones and demonstrates the potential of HMDP-DNV as an effective BRONJ therapy.

Biosynthesis of silver nanoparticles with antibacterial, antioxidant, anti-inflammatory properties and their burn wound healing efficacy.

The current study aims to develop a novel burn wound ointment consisting of sheep’s tail ointment loaded with AgNP. The AgNP in the ointment serves as an antibacterial, antioxidant and anti-inflammatory agent. The AgNP was developed via the biological method with the assistance of the medicinal plant Rhodiola rosea. The characterization of AgNP was assessed using UV-Vis spectroscopy, FTIR, Zeta Potential, XRD, PCCS, SEM, and EDX techniques. The formation of AgNP was confirmed by UV-Vis spectrum at the absorbance of ∼430 nm, and the biomolecules responsible for reducing and capping the AgNP were characterized by FTIR analysis. The stability of AgNP was determined with Zeta potential, which revealed a highly stable colloidal solution with a surface charge of −68.38 ± 3.4 mV. The synthesized AgNP had a face-centered cubic structure with a crystallite size of 23 nm and average grain size of 67.5 nm. The SEM image showed a fairly monodisperse 20 nm-sized spherical-shaped AgNP. The synthesized AgNP contained high purity of the silver, and a low concentration of AgNP inhibited both Gram-positive and Gram-negative bacteria. Moreover, the scavenging activity of AgNP was investigated using DPPH and H2O2 scavenging assay, and the results revealed a dose-dependent antioxidant activity with the highest activity at a concentration of 450 μg/ml. Finally, the burn wound healing effect was evaluated by applying the AgNP-loaded ointment to the wound site of BALB/c mice. The in-vivo studies confirmed that AgNP-loaded ointment reduced the wound size, decreased the epidermis layer, and lowered mast cell migration compared to untreated burn wounds. And the synthesized AgNP regulated both pro-inflammatory and anti-inflammatory gene expression, thereby promoting burn wound closure on BALB/c mice. The developed AgNP-loaded ointment has the potential to be applied in the biomedical field.

Folic Acid: Sources, Chemistry, Absorption, Metabolism, Beneficial Effects on Poultry Performance and Health.

Recently, there has been an increasing interest in the study of the effects of folic acid (FA) on poultry because it was observed that FA could overcome problems in poultry health while improving its performance. FA, or folate, is a water-soluble B vitamin essential in poultry, so FA intake must be available in the feed. Sources of FA in feed come from plants or animals, and animal sources have relatively more stable FA. The ingested FA will be absorbed in the intestinal lumen and transported into the liver through the blood vessels. Therefore, FA has a positive effect on the performance and health status of poultry. The effect of FA on poultry performance is to increase reproductive tract development, FA content in eggs, hatchability, weight gain, average initial body weight, feed intake, relative growth rate, chick body weight, breast fillet percentage, and reduce FCR and white striping score. At the same time, the effect on poultry health influences antioxidant activities, thyroid hormones, blood biochemicals, anti-inflammatory gene expressions, and immune responses. The present review deals with FA sources, chemistry, absorption, metabolism, effects on performance, and poultry health, which are based on valid basic information.

TBK1-mTOR Signaling Attenuates Obesity-Linked Hyperglycemia and Insulin Resistance.

The innate immune kinase TBK1 (TANK-binding kinase 1) responds to microbial-derived signals to initiate responses against viral and bacterial pathogens. More recent work implicates TBK1 in metabolism and tumorigenesis. The kinase mTOR (mechanistic target of rapamycin) integrates diverse environmental cues to control fundamental cellular processes. Our prior work demonstrated in cells that TBK1 phosphorylates mTOR (on S2159) to increase mTORC1 and mTORC2 catalytic activity and signaling. Here we investigate a role for TBK1-mTOR signaling in control of glucose metabolism in vivo. We find that mice with diet-induced obesity (DIO) but not lean mice bearing a whole-body "TBK1-resistant" Mtor S2159A knock-in allele (MtorA/A) display exacerbated hyperglycemia and systemic insulin resistance with no change in energy balance. Mechanistically, Mtor S2159A knock-in in DIO mice reduces mTORC1 and mTORC2 signaling in response to insulin and innate immune agonists, reduces anti-inflammatory gene expression in adipose tissue, and blunts anti-inflammatory macrophage M2 polarization, phenotypes shared by mice with tissue-specific inactivation of TBK1 or mTOR complexes. Tissues from DIO mice display elevated TBK1 activity and mTOR S2159 phosphorylation relative to lean mice. We propose a model whereby obesity-associated signals increase TBK1 activity and mTOR phosphorylation, which boost mTORC1 and mTORC2 signaling in parallel to the insulin pathway, thereby attenuating insulin resistance to improve glycemic control during diet-induced obesity.

Autophagy-induced mesenchymal stem cell-derived extracellular vesicles ameliorated renal fibrosis in an in vitro model.

Chronic and progressive damage to the kidney by inflammatory processes, may lead to an increase in the extracellular matrix production, a condition known as renal fibrosis. The current study aims to evaluate if the extracellular vesicles (EVs) derived from autophagic adipose-derived mesenchymal stem cells (ADMSCs) can reduce the inflammation and extracellular matrix accumulation in damaged kidney tissue. Autophagy was induced in ADMSCs using 2µM concentration curcumin and was confirmed by evaluating LC3B, ATG7, and Beclin1 using real-time polymerase chain reaction (PCR) and Western blot. An in vitro renal fibrotic model was established in HEK-293 cells exposed to H2O2 (0.8mM) for 24 and 72 hours. The fibrotic model was confirmed through evaluation of collagen I, transforming growth factor-beta 1 (TGF-β1), E-cadherin, and vimentin genes expression using real-time PCR, collagen I protein by ELISA. After induction of fibrosis for 24 and 72 hours, the HEK cells were treated with NEVs (non-autophagy EVs) (50µM) or AEVs (autophagy EVs) (50µM) at 48, 96, and 124 hours, and then the samples were collected at 72 and 148 hours. Expression of collagen I, TGF-β1, E-cadherin, and vimentin Genes was evaluated via RT-PCR, and protein levels of IL1, TNF-α, IL4, IL10 using ELISA. Induction of autophagy using curcumin (2µM) for 24 hours significantly increased LC3B, Beclin1, and ATG7 in the ADMSCs. Upregulation in anti-fibrotic (E-cadherin) and anti-inflammatory (IL4, IL10) gene expression was significantly different in the fibrotic model treated by AEVs compared to NEVs. Also, the downregulation of fibrotic (TGF-β1, vimentin, collagen I) and pro-inflammatory (IL1, TNFα) gene expression was significantly different in AEVs compared with those treated by NEVs. Our findings suggest that AEVs can be considered as a therapeutic modality for renal fibrosis in the future.

Resveratrol treatment modulates several antioxidant and anti-inflammatory genes expression and ameliorated oxidative stress mediated fibrosis in the kidneys of high-fat diet-fed rats

ObjectiveResveratrol is a polyphenolic compound that possesses strong antioxidant and anti-inflammatory activities. This study evaluated the effects of resveratrol on oxidative stress, fibrosis and multiple genes regulation in the kidneys of high fat (HF) diet-fed rats. MethodsWistar rats were fed with HF diet for eight weeks. These rats were also treated with resveratrol for eight weeks. Finally, kidney tissue samples were isolated from all sacrificed rats. The histological changes, creatinine and uric acid levels, oxidative stress parameters such as malondialdehyde (MDA), nitric oxide, and advanced oxidation protein product (AOPP) levels were analyzed. The antioxidant enzymes such as catalase, superoxide dismutase (SOD) activities and reduced glutathione (GSH) levels; gene expression of inflammatory and fibrosis-related genes namely, inducible nitric oxide synthase (iNOS), interleukin-6 (IL-6), tumor necrosis factor-alpha (TNF-α), transforming growth factor beta1 (TGF-β1), and collagen-1 were assessed. Moreover, gene expression of oxidative stress-related genes such as nuclear factor erythroid 2–related factor 2(Nrf-2), SOD, catalase, and glutathione reductase, were also assessed. ResultsHF diet-fed rats showed increased creatinine and uric acid levels in plasma which were lowered by resveratrol treatment. The study findings also revealed that resveratrol counterbalanced the oxidative stress and prevented the expression of the inflammatory genes; restored the catalase and SOD activities followed by the up-regulation of antioxidant genes expression in the kidneys of HF diet-fed rats. HF diet caused the Nrf-2 down-regulation followed by the decreased expression of HO-1 and HO-2 genes, which was restored by resveratrol treatment. Moreover, the histological assessment showed lipotoxicity and increased fibrosis in the kidneys of HF diet-fed rats. Resveratrol prevented the kidney fibrosis probably by limiting oxidative stress, inflammation, and down-regulating TGF-β1 mediated signaling pathway. ConclusionIn conclusion, resveratrol treatment showed beneficial effects in preventing oxidative stress and fibrosis in the kidneys of HF diet-fed rats probably by modulating the gene expression of oxidative stress and inflammation related factors and enzymes.

Antagonism of miR-148a attenuates atherosclerosis progression in APOBTGApobec-/-Ldlr+/- mice: A brief report

ObjectivemiR-148a-3p (miR-148a) is a hepatic and immune-enriched microRNA (miRNA) that regulates macrophage-related lipoprotein metabolism, cholesterol homeostasis, and inflammation. The contribution of miR-148a-3p to the progression of atherosclerosis is unknown. In this study, we determined whether miR-148a silencing mitigated atherogenesis in APOBTGApobec-/-Ldlr+/- mice. MethodsAPOBTGApobec-/-Ldlr+/- mice were fed a typical Western-style diet for 22 weeks and injected with a nontargeting locked nucleic acid (LNA; LNA control) or miR-148a LNA (LNA 148a) for the last 10 weeks. At the end of the treatment, the mice were sacrificed, and circulating lipids, hepatic gene expression, and atherosclerotic lesions were analyzed. ResultsExamination of atherosclerotic lesions revealed a significant reduction in plaque size, with marked remodeling of the lesions toward a more stable phenotype. Mechanistically, miR-148a levels influenced macrophage cholesterol efflux and the inflammatory response. Suppression of miR-148a in murine primary macrophages decreased mRNA levels of proinflammatory M1-like markers (Nos2, Il6, Cox2, and Tnf) and increased the expression of anti-inflammatory genes (Arg1, Retlna, and Mrc1). ConclusionsTherapeutic silencing of miR148a mitigated the progression of atherosclerosis and promoted plaque stability. The antiatherogenic effect of miR-148a antisense therapy is likely mediated by the anti-inflammatory effects observed in macrophages treated with miR-148 LNA and independent of significant changes in circulating low-density lipoprotein cholesterol (LDL-C) and high-density lipoprotein cholesterol (HDL-C).

Better intestine health in fast‐growing bullfrogs ( Lithobates catesbeianus ) fed a soybean meal‐based diet than slow‐growing bullfrogs

This study examined the differences in intestinal health between fast- and slow-growing bullfrogs (Lithobates catesbeianus) fed a soybean meal (SM)-based diet. A total of 108 froglets (26.14 ± 0.21 g) were randomly distributed into three tanks at a density of 36 froglets per tank and fed a SM-based diet for 8 weeks. At the end of the feeding period, four heaviest and four lightest bullfrogs from each tank were captured and considered as fast-growing (FG) and slow-growing (SG) groups. The FG group had significantly higher weight gain than the SG group (p < 0.05). A basic intestinal tissue structure integrity with higher values of jejunal diameter, villus numbers, villus thickness and muscular thickness was observed in the FG group compared with the SG group (p < 0.05). Moreover, lower activity of serum diamine oxidase was detected in FG group than the SG group, which indicated better gut barrier function of froglets in FG group (p < 0.05). However, no significant differences were found in the mRNA expression level of tight junction proteins including occludin and zo-1 in the jejunum, and serum D-lactate concentration between the two groups (p > 0.05). Significantly lower pH values in corpora ventriculi and ileum and higher jejunal protease and amylase activities were found in the FG group than SG group (p < 0.05). Relatively higher expression of anti-inflammatory genes and lower expression of pro-inflammatory genes were found in the FG group; however, the differences were not significant. The abundances of potential probiotics including Akkermansia, Lactobacillus and Prevotellaceae_UCG_001 were higher in the intestinal microflora of FG group compared with the SG group. In conclusion, these findings suggested that higher growth of FG bullfrogs fed the SM-based diet could be attributed to their better intestinal morphological structure and barrier function compared with the SG bullfrogs.

Using adenovirus-mediated gene transfer to study the effect of myeloperoxidase on plasma lipid levels, HDL structure and functionality in mice expressing human apoA-I forms

Apolipoprotein A-I (apoA-I), the main protein component of High-Density Lipoprotein (HDL), is modified in plasma and the arterial wall by various enzymes. Myeloperoxidase (MPO), a leukocyte-derived peroxidase, is highly expressed during inflammation and associates with HDL reducing its functionality and contributing to atherosclerosis. In the present study we sought to explore further the effect of MPO on HDL structure and functionality in vivo using adenovirus-mediated gene transfer of human MPO combined with human apoA-I forms containing substitutions at MPO-sensitive sites or wild type apoA-I. We found that overexpression of MPO in mice significantly increased plasma apoA-I and HDL levels without affecting the expression of genes involved in HDL biogenesis or catabolism in the liver. Overexpression of MPO in the liver reduced the expression of pro-inflammatory genes and increased or did not affect the expression of anti-inflammatory genes suggesting that MPO had no toxic effects in this organ. In the plasma of mice overexpressing MPO, no significant alterations in HDL size or electrophoretic mobility was observed with the exception of mice expressing apoA-I (M148A) which showed enriched pre-β relative to α HDL particles, suggesting that the apoA-I (M148A) mutation may interfere with HDL remodelling. Overexpression of MPO was associated with reduced anti-oxidant capacity of HDL particles in all mice. Interestingly, HDL particles bearing apoA-I (Y192A) showed enhanced ABCA1-dependent cholesterol efflux from macrophages which was not affected by MPO and these mice had reduced levels of LDL-c. These findings provide new insights on the role of specific amino acid residues of apoA-I in HDL structure and function following modification by MPO. This knowledge may facilitate the development of novel therapies based on improved HDL forms for patients with chronic diseases that are characterized by dysfunctional HDL.

Protective Effects of Cinnamaldehyde on the Oxidative Stress, Inflammatory Response, and Apoptosis in the Hepatocytes of Salmonella Gallinarum-Challenged Young Chicks.

The development of novel therapeutics to treat multidrug-resistant pathogenic infections like Salmonella gallinarum is the need of the hour. Salmonella infection causes typhoid fever, jaundice, and Salmonella hepatitis resulting in severe liver injury. Natural compounds have been proved beneficial for the treatment of these bacterial infections. The beneficial roles of cinnamaldehyde due to its antibacterial, anti-inflammatory, and antioxidative properties have been determined by many researchers. However, alleviation of liver damage caused by S. gallinarum infection to young chicks by cinnamaldehyde remains largely unknown. Therefore, this study was performed to identify the effects of cinnamaldehyde on ameliorating liver damage in young chicks. Young chicks were intraperitoneally infected with S. gallinarum and treated with cinnamaldehyde orally. Liver and serum parameters were investigated by qRT-PCR, ELISA kits, biochemistry kits, flow cytometry, JC-1 dye experiment, and transcriptome analysis. We found that ROS, cytochrome c, mitochondrial membrane potential (Ψm), caspase-3 activity, ATP production, hepatic CFU, ALT, and AST, which were initially increased by Salmonella infection, significantly (P < 0.05) decreased by cinnamaldehyde treatment at 1, 3, and 5 days postinfection (DPI). In addition, S. gallinarum infection significantly increased proinflammatory gene expression (IL-1β, IL-6, IL-12, NF-κB, TNF-α, and MyD-88) and decreased the expression of anti-inflammatory genes (IL-8, IL-10, and iNOS); however, cinnamaldehyde reverted these effects at 1, 3, and 5 DPI. Transcriptome analysis showed that S. gallinarum modulates certain genes of the AMPK-mTOR pathway for its survival and replication, and these pathway modulations were reversed by cinnamaldehyde treatment. We concluded that cinnamaldehyde ameliorates inflammation and apoptosis by suppressing NF-Kβ/caspase-3 pathway and reverts the metabolic changes caused by S. gallinarum infection via modulating the AMPK-mTOR pathway. Furthermore, cinnamaldehyde has antibacterial, anti-inflammatory, antioxidative, and antiapoptotic properties against S. gallinarum-challenged young chicks and can be a candidate novel drug to treat salmonellosis in poultry production.

Human IL-17 and TNF-α Additively or Synergistically Regulate the Expression of Proinflammatory Genes, Coagulation-Related Genes, and Tight Junction Genes in Porcine Aortic Endothelial Cells.

Immune rejection is the major limitation for porcine xenograft survival in primate recipients. Proinflammatory cytokines play important roles in immune rejection and have been found to mediate the pathological effects in various clinical and experimental transplantation trials. IL-17 and TNF-α play critical pathological roles in immune disorders, such as psoriasis and rheumatoid arthritis. However, the pathological roles of human IL-17 (hIL-17) and human TNF-α (hTNF-α) in xenotransplantation remain unclear. Here we found that hIL-17 and hTNF-α additively or synergistically regulate the expression of 697 genes in porcine aortic endothelial cells (PAECs). Overall, 415 genes were found to be synergistically regulated, while 282 genes were found to be additively regulated. Among these, 315 genes were upregulated and 382 genes were downregulated in PAECs. Furthermore, we found that hIL-17 and hTNF-α additively or synergistically induced the expression of various proinflammatory cytokines and chemokines (e.g., IL1α, IL6, and CXCL8) and decreased the expression of certain anti-inflammatory genes (e.g., IL10). Moreover, hIL-17 plus hTNF-α increased the expression of IL1R1 and IL6ST, receptors for IL1 and IL6, respectively, and decreased anti-inflammatory gene receptor expression (IL10R). hIL-17 and hTNF-α synergistically or additively induced CXCL8 and CCL2 expression and consequently promoted primary human neutrophil and human leukemia monocytic cell migration, respectively. In addition, hIL-17 and hTNF-α induced pro-coagulation gene (SERPINB2 and F3) expression and decreased anti-coagulation gene (TFPI, THBS1, and THBD) expression. Additionally, hIL-17 and hTNF-α synergistically decreased occludin expression and consequently promoted human antibody-mediated complement-dependent cytotoxicity. Interestingly, hTNF-α increased swine leukocyte antigen (SLA) class I expression; however, hIL-17 decreased TNF-α-mediated SLA-I upregulation. We concluded that hIL-17 and hTNF-α likely promote the inflammatory response, coagulation cascade, and xenoantibody-mediated cell injury. Thus, blockade of hIL-17 and hTNF-α together might be beneficial for xenograft survival in recipients.

Effects of Different Radiation Sources on the Performance of Collagen-Based Corneal Repair Materials and Macrophage Polarization

Owing to the lack of donor corneas, there is an urgent need for suitable corneal substitutes. As the main component of the corneal stroma, collagen has great advantages as a corneal repair material. If there are microorganisms such as bacteria in the corneal repair material, it may induce postoperative infection, causing the failure of corneal transplantation. Therefore, irradiation, as a common sterilization method, is often used to control the microorganisms in the material. However, it has not been reported which type of radiation source and what doses can sterilize more effectively without affecting the properties of collagen-based corneal repair materials (CCRMs) and have a positive impact on macrophage polarization. In this study, three different radiation sources of ultraviolet, cobalt-60, and electron beam at four different doses of 2, 5, 8, and 10 kGy were used to irradiate CCRMs. The swelling, stretching, transmittance, and degradation of the irradiated CCRMs were characterized, and the proliferation of human corneal epithelial cells on the irradiated CCRMs was characterized using the CCK8 kit. The results showed that low dose (<5 kGy) of radiation had little effect on the performance of CCRMs. Three irradiation methods with less influence were selected for the further study on RAW264.7 macrophage polarization. The results indicated that CCRMs treated with UV could downregulate the expression of pro-inflammatory related genes and upregulate the expression of anti-inflammatory genes in macrophages, which indicated that UV irradiation is a beneficial process for the preparation of CCRMs.

Abstract 2162: Inhibition of GPR65 counteracts low pH induced immunosuppressive polarization of macrophages: In vitro and in vivo characterization of potent, selective and orally bioavailable small molecule GPR65 antagonists

Abstract The acidic tumour microenvironment (TME) and the abundance of tumour associated macrophages (TAMs) are key features of solid tumours that drive immune suppression, support tumour growth and limit the efficacy of approved therapies. We identified the pH sensing GPCR, GPR65, as a key determinant of low pH induced immune suppression in human cancers, particularly in TAMs, based on the following observations: 1) cancer patients who are homozygous for a hypomorphic coding variant in GPR65 (I231L) show a statistically significant increase in survival and altered expression of key immune system genes compared to other genotypes; 2) single cell RNA sequencing (scRNAseq) data from multiple solid tumors show that GPR65 and downstream pathway genes are ubiquitously expressed in myeloid and other innate immune cells in human cancers; and 3) low pH acting via GPR65 profoundly alters gene expression in human macrophages in vitro, bringing about a pronounced suppression of proinflammatory cytokines and a marked upregulation of tissue repair genes. These findings identify GPR65 as a novel innate immune check point, and suggest that GPR65 inhibition could be highly beneficial in cancer. Indeed, previous work has shown that genetic ablation of the GPR65 signaling pathway in B16.F10 tumour bearing mice upregulates immunostimulatory genes in TAMs and significantly reduces tumor growth1. Pathios has identified potent and selective antagonists of human GPR65 with excellent oral bioavailability and pharmacokinetics. In line with their potencies in recombinant cell systems, lead molecules are able to inhibit low pH induced cAMP elevations in primary human macrophages with IC50 values in the single digit nM range. In macrophages subjected to acidic conditions, the inhibitory effects on cAMP are accompanied by a reduction in the expression of anti inflammatory and tissue repair genes, and the enhancement of immunostimulatory genes. In particular, GPR65 inhibition counteracts the pronounced low pH induced suppression of key Type I/II interferon (IFN) response genes and chemokines such as CXCL9, CXCL10 and CXCL11. Following oral administration in tumour bearing mice, lead compounds up-regulate the expression of anti-tumor immune response genes at systemic exposures that significantly suppress GPR65 signalling in vitro. Results from tumour growth inhibition studies in mice with our GPR65 inhibitors will be also be presented. In summary, ‘Macrophage Conditioning’ via GPR65 inhibition holds substantial promise as a novel immunoncology strategy to counteract the immunosuppressive effects of the acidic TME on TAMs, and could be deployed either as monotherapy or in combination with T cell checkpoint inhibitors or other standard of care treatments. 1Nat. Immunol. 19:1319 Citation Format: Barbara Cipriani, David Miller, Alan Naylor, Gavin Milne, Barbara Young, Rupert Satchell, Suorav Sarkar, Zoe Smith, Rhoanne McPherson, Anastasia Nika, Preeti Singh, Toszka Bohn, Tobias Bopp, Tom McCarthy, Stuart Hughes. Inhibition of GPR65 counteracts low pH induced immunosuppressive polarization of macrophages: In vitro and in vivo characterization of potent, selective and orally bioavailable small molecule GPR65 antagonists [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2162.

Quercetin-Coating Promotes Osteogenic Differentiation, Osseointegration and Anti-Inflammatory Properties of Nano-Topographic Modificated 3D-Printed Ti6Al4V Implant.

The capabilities of osseointegration and anti-inflammatory properties are of equal significance to the bio-inert titanium implant surface. Quercetin has proved its capacities of activating anti-inflammation through macrophage modulation and promoting osteogenic differentiation. Herein, we fabricated quercetin-coating on nano-topographic modificated 3D-printed Ti6Al4V implant surface. Subsequently the biological cells responses in vitro, anti-inflammatory and osseointegration performance in vivo were evaluated. In vitro studies indicated that quercetin-coating can enhance the adhesion and osteogenic differentiation of rBMSCs, while modulating the polarization of macrophages from M1 to M2 phase and improving the anti-inflammatory and vascular gene expression. Moreover, quercetin-loaded implants reduced the level of peri-implant inflammation and ameliorated new bone formation and rapid osseoinegration in vivo. Quercetin-coating might provide a feasible and favorable scheme for endowing 3D-printed titanium alloy implant surface with enhanced the rapid osseointegration and anti-inflammatory properties.

Exploration of molecular mechanisms responsible for anti-inflammatory and anti-angiogenic attributes of methanolic extract of Viola betonicifolia

Uncontrolled inflammation plays a central role in the pathogenesis of various diseases. Currently available anti-inflammatory agents on prolonged use may lead to ulcers or thrombus formation. The present study was designed to evaluate the anti-inflammatory, anti-arthritic and anti-angiogenic potentials of methanol extract of Viola betonicifolia using battery of in vivo models. Methanol extract of Viola betonicifolia (Vb.Me) was prepared through maceration. High performance liquid chromatography (HPLC) and gas chromatography mass spectrometery (GC-MS) were performed to identify bioactive compounds present in Vb.Me. In vivo safety profile of Vb.Me was evaluated following OECD 425 acute toxicity guidelines. Anti-inflammatory potential of Vb.Me at three different dose levels was evaluated in in vivo acute (carrageenan and, histamine-induced paw oedema), sub-chronic (cotton pellet-induced granuloma) and chronic (Complete Freund's adjuvant-induced arthritis) models. Blood and paws samples were collected to study effects of Vb.Me treatment on the expression of various pro-and anti-inflammatory genes (RT-PCR)and to study the histopathological changes at tissue levels. Effects of Vb.Me on neovasculature development were studied in ex-ovo chicken chorioallantoic membrane (CAM) assay. Quercetin and n-hexadecanoic were identified as one of the major bioactive molecules in HPLC and GC-MS analysis of Vb.Me. Toxicity data revealed that Vb.Me was safe for administration up to thedose of 2000mg/kg. Findings of inflammatory models showed that Vb.Me produced time and dose-dependent effects. 500mg/kg Vb.Me showed significantly (p < 0.05) better effects as compared with 125 and 250mg/kg. 500mg/kg Vb.Me also showed comparable anti-inflammatory effects with indomethacin in both acute and chronic models respectively. RT-PCR data exhibited significant (p < 0.05) down-regulation of IL-6, IL-1ß, NF-kß, TNF-α and COX-2 genes with simultaneous up-regulation of IL-4 and IL-10 genes in the blood samples of animals treated with 500mg/kg of Vb.Me and 10mg/kg of indomethacinrespectively. CAM assay data revealed arrest of microvessel outgrowth in Vb.Me-treated eggs. Altogether, findings of the current study indicate that Vb.Me exerts in vivo anti-inflammatory and anti-angiogenic effects through regulation of expression of various pro-and anti-inflammatory genes. Synergist actions of various bioactive molecules in Vb.Me are proposed to be responsible for these attributes. However, further studies to standardize the extract and evaluation of its potential in various inflammation-induced diseases are warranted.

Polarization of human iPSC-derived macrophages directs their immunological response to secondary pro-inflammatory stimuli

Macrophages can be found in various tissues and play an important role in organ function by sensing and eradicating pathogens, regulating immune responses and contributing to tissue homeostasis and repair. Nowadays, increasing numbers of macrophage-based cell therapies are entering (pre-) clinical studies e.g. for the treatment of liver cirrhosis. Given limited availability of suitable donors as well as problems with variability in quantities and qualities of human monocytes that can be derived from apheresis, induced pluripotent stem cells (iPSC) offer an attractive source of therapeutic macrophages. However, considering the diverse functions, activation stages and overall plasticity of macrophages, further knowledge about (i) the potential to induce different activation stages in iPSC-derived macrophages (iPSC-Mac) as well as (ii) the stability of these phenotypes upon additional external stimuli is of high relevance. We here demonstrate that iPSC-Mac produced in a scalable differentiation platform can be polarized into defined pro- (M1) and anti-inflammatory (M2) activation stages characterized by specific surface marker expression, cytokine secretion and whole transcriptome analysis, similarly to peripheral blood-derived macrophages. Even more importantly, we show that differentially polarized iPSC-Mac maintained key characteristics of their activation status upon a subsequent inflammatory trigger. Interferon (IFN) γ polarized, M1-iPSC-Mac demonstrated an enhanced inflammatory response after additional lipopolysaccharide (LPS) stimulation, whereas Interleukin (IL)-4 stimulated M2a iPSC-Mac and IL-10/TGFβ primed M2c iPSC-Mac showed a reduced activation upon LPS treatment and maintained expression of anti-inflammatory genes. Together, our data demonstrate that defined polarized iPSC-Mac subsets can be generated. Moreover, these cells maintain key characteristics of their activation profile upon a subsequent inflammatory trigger. Thus, the use of stably polarized iPSC-Mac has the potential to further improve the applicability and efficacy of macrophage-based therapies.