Macrophage Inflammatory Cytokine Research Articles

Density dependent regulation of inflammatory responses in macrophages.

Macrophage distribution density is tightly regulated within the body, yet the importance of macrophage crowding during in vitro culture is largely unstudied. Using a human induced pluripotent stem cell (iPSC)-derived macrophage model of tissue resident macrophages, we characterize how increasing macrophage culture density changes their morphology and phenotype before and after inflammatory stimulation. In particular, density drives changes in macrophage inflammatory cytokine and chemokine secretion in both resting and activated states. This density regulated inflammatory state is also evident in blood monocyte derived-macrophages, the human monocytic THP-1 immortalized cell line, and iPSC-derived microglia. Density-dependent changes appear to be driven by a transferable soluble factor, yet the precise mechanism remains unknown. Our findings highlight cell plating density as an important but frequently overlooked consideration of in vitro macrophage research relevant to a variety of fields ranging from basic macrophage cell biology to disease studies.

GPR183 antagonism reduces macrophage infiltration in influenza and SARS-CoV-2 infection.

Severe viral respiratory infections are often characterised by extensive myeloid cell infiltration and activation and persistent lung tissue injury. However, the immunological mechanisms driving excessive inflammation in the lung remain poorly understood. To identify the mechanisms that drive immune cell recruitment in the lung during viral respiratory infections and identify novel drug targets to reduce inflammation and disease severity. Preclinical murine models of influenza A virus and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. Oxidised cholesterols and the oxysterol-sensing receptor GPR183 were identified as drivers of monocyte/macrophage infiltration to the lung during influenza A virus (IAV) and SARS-CoV-2 infection. Both IAV and SARS-CoV-2 infection upregulated the enzymes cholesterol 25-hydroxylase (CH25H) and cytochrome P450 family 7 subfamily member B1 (CYP7B1) in the lung, resulting in local production of the oxidised cholesterols 25-hydroxycholesterol (25-OHC) and 7α,25-dihydroxycholesterol (7α,25-OHC). Loss-of-function mutation of Gpr183 or treatment with a GPR183 antagonist reduced macrophage infiltration and inflammatory cytokine production in the lungs of IAV- or SARS-CoV-2-infected mice. The GPR183 antagonist significantly attenuated the severity of SARS-CoV-2 infection and viral loads. Analysis of single-cell RNA-sequencing data on bronchoalveolar lavage samples from healthy controls and COVID-19 patients with moderate and severe disease revealed that CH25H, CYP7B1 and GPR183 are significantly upregulated in macrophages during COVID-19. This study demonstrates that oxysterols drive inflammation in the lung via GPR183 and provides the first preclinical evidence for the therapeutic benefit of targeting GPR183 during severe viral respiratory infections.

Identification of therapeutic targets in a murine model of severe exertional heat stroke.

Exertional heat stroke (EHS) is a potentially lethal condition resulting from high core body temperatures (TC) in combination with a systemic inflammatory response syndrome (SIRS) with varying degrees of severity across victims, and limited understanding of the underlying mechanism(s). We established a mouse model of severe EHS to identify mechanisms of hyperthermia/inflammation that may be responsible for organ damage. Mice were forced to run on a motorized wheel in a 37.5°C chamber until loss of consciousness and were either removed immediately (exertional heat injury or EHI; TCMax = 42.4 ± 0.2°C) or remained in the chamber an additional 20 min (EHS; TCMax = 42.5 ± 0.4°C). Exercise control mice (ExC) experienced identical procedures to EHS at 25°C. At 3 h post-EHS, there was evidence for an immune/inflammatory response as elevated blood chemokine [interferon γ-induced protein 10 (IP-10), keratinocytes-derived chemokine (KC), macrophage inflammatory proteins (MIP-1α), MIP-1β, MIP-2] and cytokine [granulocyte colony-stimulating factor (G-CSF), interleukins (IL-10), IL-6] levels peaked and were highest in EHS mice compared with EHI and ExC mice. Immunoblotting of organs susceptible to EHS damage indicated that several kinases were sensitive to stress associated with heat/inflammation and exercise; specifically, phosphorylation of liver c-Jun NH2-terminal kinase (JNK) at threonine 183/tyrosine 185 immediately (0 h) postheating related to heat illness severity. We have established a mouse EHS model, and JNK [or its downstream target(s)] could underlie EHS symptomatology, allowing the identification of molecular pathways or countermeasure targets to mitigate heat illness severity, enable complete recovery, and decrease overall EHS-related fatalities.

P17.06: Escape From Macrophage-Mediated Rejection by Human Surfactant Protein (SP)-A

Introduction: Macrophage-mediated xenogeneic rejection is one of the important immunological obstructions that need to be overcome. Because macrophage is one of the main sources of proinflammatory cytokines and the fact that proinflammatory cytokines orchestrate a variety of inflammatory responses, the regulation of macrophages could result in solving the problem of xenogeneic rejection. We recently reported that membrane-type surfactant D (SP-D) on swine endothelial cells (SECs) suppresses macrophage-mediated rejection. Similar to SP-D, the carbohydrate recognition domain of surfactant protein-A (SP-A) induces inhibitory signals in effector cells. In this study, we examined the suppressive effect of SP-A on macrophage-mediated xenogeneic rejection. Methods: Naïve SEC and SP-A-transfected SEC (SEC/SP-A) were co-cultured with THP-1 cells and cytotoxicity was evaluated. To investigate the effect on phagocytosis, human macrophages were co-cultured with SEC or SEC/SP-A, and the extent of phagocytosis and production of reactive oxygen species (ROS) were assessed by flow cytometry. The mRNA expression of inflammatory cytokines in macrophages was determined using RT-PCR. In addition, THP-1 cell with a luciferase reporter gene driven by an NF-kB response element (THP-1 Luc NF-kB) cells was used to verify their effects on the NF-κB transcription factors. Results: The cytotoxicity of SEC/SP-A was significantly reduced compared to those of naïve SEC (40.7 vs 22.9%, p=0.025). The phagocytosis by macrophages against SEC was also significantly suppressed by SP-A on SEC (75.8 vs 33.0%, n=5, p=0.0010). Co-culture of human macrophages with SEC/SP-A decreased ROS production (%MFI: 99.9 vs 79.5, p=0.013). The mRNA expression of TNFα was reduced in macrophages (1.160 vs 0.009, p=0.0022), whereas that of IL-10 was increased (1.357 vs 10.20, p=0.0007). The balance between iNOS and Arg-1 was significantly suppressed by CL-SPA (1.002 vs 0.4792, p = 0.0027), indicating that CL-SPA on porcine cells inhibits the differentiation of peripheral blood monocytes into inflammatory M1 macrophages. The NF-κB transcription was decreased in THP-1 Luc NF-kB which was co-cultured with SEC/SP-A compared to that in THP-1 Luc NF-kB with SEC (RLU: 1160 vs 983.8, p=0.0258). Conclusion: The ectopic expression of human SP-A in porcine cells represents an attractive method for suppressing macrophage-mediated cytotoxicity. To further investigate the effects of SP-A on xenogeneic innate immune response in more detail, in vivo studies should be performed in the future.

Mycobacterium tuberculosis Rv2387 Facilitates Mycobacterial Survival by Silencing TLR2/p38/JNK Signaling.

Mycobacterium tuberculosis (Mtb) can evade antimicrobial immunity and persist within macrophages by interfering with multiple host cellular functions through its virulence factors, causing latent tuberculosis. The Rv2387 protein has been identified as a putative effector that potentially participates in Mtb pathogenicity. To explore the role of the Rv2387 protein in host–mycobacteria interactions, we established recombinant M. smegmatis strains and RAW264.7 cell lines that stably express the Rv2387 protein. We found that this protein suppresses mycobacteria infection-induced macrophage apoptosis by inactivating caspase-3/-8, thus facilitating the intracellular survival of mycobacteria. In addition, Rv2387 inhibits the production of inflammatory cytokines in macrophages by specifically suppressing TLR2-dependent stimulation of p38 and JNK MAPK pathways. Moreover, we further determined that the Rv2387 protein conferred a growth advantage over recombinant M. smegmatis and suppressed the inflammatory response in a mouse infection model. Overall, these data suggested that Rv2387 facilitates mycobacteria to escape host immunity and might be an essential virulence factor in Mtb.

Activation of Inflammation by MCF-7 Cells-Derived Small Extracellular Vesicles (sEV): Comparison of Three Different Isolation Methods of sEV.

Small extracellular vesicles (sEV) containing proteins and RNAs play important roles as intercellular signal mediators. A critical issue is that there are multiple methods to prepare sEV fractions. The purpose of this study was to examine whether cancer cell-derived sEV fractions prepared by different isolation methods show similar responses for the induction of inflammatory cytokines in macrophages. sEV fractions from the conditioned medium of MCF-7 cells were prepared by ultracentrifugation (UC), the MagCapture Exosome Isolation Kit PS (PS), or the ExoQuick-TC kit (EQ). The mRNA levels of inflammatory cytokines in differentiated THP-1 cells treated with the sEV fractions were evaluated. The yields of sEV fractions obtained from 1 mL conditioned medium by UC, PS, or EQ were 3.2×108 particles (0.27 μg protein), 12.8×108 particles (0.87 μg protein) and 23.5 ×108 particles (4.50 μg protein), respectively. The average particle sizes in the UC, PS, and EQ fractions were 184.8 ± 1.8 nm, 157.8 ± 1.3 nm and 165.8 ± 1.1 nm, respectively. CD9 and CD81, markers of sEV, were most highly detected in the PS fraction, followed by the EQ and UC fractions. These results suggest that PS gave sEV with relatively high purity, and many protein contaminants appear to be included in the EQ fraction. The mRNA levels of inflammatory cytokines in THP-1 macrophages were most prominently increased by treatment with the UC fraction, followed by the EQ and PS fractions, suggesting that contaminants rather than sEV may largely induce an inflammatory response. The isolation method affects the evaluation of sEV function.

Suppression of xenogeneic innate immune response by a membrane-type human surfactant protein-A.

Macrophage-mediated xenogeneic rejection is a major immunological obstacle. We recently reported that membrane-type surfactant protein-D (SP-D) on swine endothelial cells (SECs) suppressed macrophage-mediated rejection. Similar to SP-D, the carbohydrate recognition domain of surfactant protein-A (SP-A) can induce inhibitory signals in effector cells. The present study aimed to examine the suppressive effect of SP-A on macrophage-mediated xenogeneic rejection. Naive SECs and SPA-transfected SECs (SEC/SP-A) were co-cultured with THP-1 cells and cytotoxicity was evaluated. To investigate the effect of SP-A on phagocytosis, human macrophages were co-cultured with SEC or SEC/SP-A, and the extent of phagocytosis and production of reactive oxygen species were assessed via flow cytometry. The mRNA expression levels of inflammatory cytokines in macrophages were determined using reverse transcription-PCR. Additionally, the effects of THP-1-Lucia NF-κB cells on transcription factors were evaluated. The cytotoxicity and phagocytosis of SEC/SP-A were significantly decreased compared with those of naive SEC. Furthermore, the co-culture of human macrophages with SEC/SP-A decreased reactive oxygen species production, and the mRNA expression levels of TNFα were decreased in macrophages, whereas those of IL-10 were increased. In addition, NF-κB transcription was decreased in SEC/SP-A compared with that in SEC. In conclusion, the ectopic expression of human SP-A in porcine cells represents an attractive method for suppressing macrophage-mediated cytotoxicity.

Slc6a13 Deficiency Attenuates Pasteurella multocida Infection-Induced Inflammation via Glycine-Inflammasome Signaling

We have previously demonstrated that Slc6a13-deficient (Slc6a13−/−; KO) mice are resistant to P. multocida infection, which might be in connection with macrophage-mediated inflammation; however, the specific metabolic mechanism is still enigmatic. Here we reproduce the less sensitive to P. multocida infection in overall survival assays as well as reduced bacterial loads, tissue lesions, and inflammation of lungs in KO mice. The transcriptome sequencing analysis of wild-type (WT) and KO mice shows a large number of differentially expressed genes that are enriched in amino acid metabolism by functional analysis. Of note, glycine levels are substantially increased in the lungs of KO mice with or without P. multocida infection in comparison to the WT controls. Interestingly, exogenous glycine supplementation alleviates P. multocida infection-induced inflammation. Mechanistically, glycine reduces the production of inflammatory cytokines in macrophages by blocking the activation of inflammasome (NALP1, NLRP3, NLRC4, AIM2, and Caspase-1). Together, Slc6a13 deficiency attenuates P. multocida infection through lessening the excessive inflammatory responses of macrophages involving glycine-inflammasome signaling.

RANKL elevation activates the NIK/NF-κB pathway, inducing obesity in ovariectomized mice.

Menopausal women are susceptible to visceral obesity, which increases the risk of metabolic disorders. However, the mechanisms of menopause-induced visceral fat accumulation are not fully understood. Circulating levels of receptor activator of nuclear factor-kappa B (NF-κB) ligand (RANKL) are elevated in an animal model of menopause. RANKL, a multifunctional cytokine, activates the NF-κB pathway, which serves as a pivotal mediator of inflammatory responses. Here, we investigated whether RANKL-induced non-canonical NF-κB pathway activation induces inflammation and lipid accumulation in adipose tissues. RANKL induced Tnfa expression via the non-canonical NF-κB pathway in bone marrow cells. We therefore analyzed aly/aly mice, in which the non-canonical NF-κB pathway is not activated, owing to an inactive form of NF-κB-inducing kinase. A postmenopausal obesity model was generated by ovariectomy and subsequent high-fat and high-sucrose diet feeding. In aly/aly mice with postmenopausal obesity, serum RANKL levels were elevated, and hepatic lipid accumulation and adipocyte hypertrophy were suppressed, resulting in reduced macrophage infiltration and inflammatory cytokine mRNA expression in visceral adipose tissue. Furthermore, aly/aly mice showed protection from glucose intolerance and insulin resistance, which were observed in ovariectomized WT obese mice. These findings indicate that non-canonical NF-κB pathway activation via serum RANKL elevation contributes to postmenopausal obesity.

Inhibition of Trimethylamine N-Oxide Attenuates Neointimal Formation Through Reduction of Inflammasome and Oxidative Stress in a Mouse Model of Carotid Artery Ligation.

Aims: Trimethylamine-N-oxide (TMAO) is a metabolite generated from dietary choline, betaine, and l-carnitine, after their oxidization in the liver. TMAO has been identified as a novel independent risk factor for atherosclerosis through the induction of vascular inflammation. However, the effect of TMAO on neointimal formation in response to vascular injury remains unclear. Results: This study was conducted using a murine model of acutely disturbed flow-induced atherosclerosis induced by partial carotid artery ligation. 3,3-Dimethyl-1-butanol (DMB) was used to reduce TMAO concentrations. Wild-type mice were divided into four groups [regular diet, high-TMAO diet, high-choline diet, and high-choline diet+DMB] to investigate the effects of TMAO elevation and its inhibition by DMB. Mice fed high-TMAO and high-choline diets had significantly enhanced neointimal hyperplasia and advanced plaques, elevated arterial elastin fragmentation, increased macrophage infiltration and inflammatory cytokine secretion, and enhanced activation of nuclear factor (NF)-κB, the NLRP3 inflammasome, and endoplasmic reticulum (ER) stress relative to the control group. Mice fed high-choline diets with DMB treatment exhibited attenuated flow-induced atherosclerosis, inflammasome expression, ER stress, and reactive oxygen species expression. Human aortic smooth muscle cells (HASMCs) were used to investigate the mechanism of TMAO-induced injury. The HASMCs were treated with TMAO with or without an ER stress inhibitor to determine whether inhibition of ER stress modulates the TMAO-induced inflammatory response. Innovation: This study demonstrates that TMAO regulates vascular remodeling via ER stress. Conclusion: Our findings demonstrate that TMAO elevation promotes disturbed flow-induced atherosclerosis and that DMB administration mitigates vascular remodeling, suggesting a rationale for a TMAO-targeted strategy for the treatment of atherosclerosis. Antioxid. Redox Signal. 38, 215-233.

The sodium-glucose co-transporter 2 inhibitor tofogliflozin suppresses atherosclerosis through glucose lowering in ApoE-deficient mice with streptozotocin-induced diabetes.

Epidemiological and animal studies have revealed that sodium‐glucose cotransporter 2 (SGLT2) inhibitors suppress cardiovascular events in subjects with type 2 diabetes and atherosclerosis in animal models of diabetes. However, it still remains unclear if the anti‐atherosclerotic effect of SGLT2 inhibitors is entirely dependent on their glucose‐lowering effect. Tofogliflozin, a highly specific SGLT2 inhibitor, was administrated to apolipoprotein‐E‐deficient (ApoEKO) with streptozotocin (STZ)‐induced diabetes and nondiabetic ApoEKO mice. After 6 weeks, samples were collected to investigate the histological changes and peritoneal macrophage inflammatory cytokine levels. Tofogliflozin suppressed atherosclerosis in the diabetic ApoEKO mice. The atherosclerosis lesion areas and accumulation of macrophages in these areas were reduced by tofogliflozin treatment. The expression levels of interleukin (IL)‐1β and IL‐6 in the peritoneal macrophages were significantly suppressed in the tofogliflozin‐treated diabetic ApoEKO mice. Tofogliflozin treatment failed to inhibit atherosclerosis in the nondiabetic ApoEKO mice. No significant difference in the anti‐atherosclerotic effects of insulin and tofogliflozin was observed between diabetic ApoEKO mice with equivalent degrees of glycemic control achieved with the two treatments. Insulin treatment significantly reduced the IL‐1β and IL‐6 expression levels in the peritoneal macrophages of the diabetic ApoEKO mice. Significant decrease of the LPS‐stimulated IL‐1β concentrations was also observed in the conditioned medium of the peritoneal macrophages collected from insulin‐ and tofogliflozin‐treated diabetic ApoEKO mice. These results suggest that tofogliflozin suppresses atherosclerosis by improving glucose intolerance associated with inhibition of inflammation. Tofogliflozin suppresses atherosclerosis in ApoEKO mice with STZ‐induced diabetes via its glucose‐lowering effect.

Cholesterol crystals drive metabolic reprogramming and M1 macrophage polarisation in primary human macrophages

Background and aimsMetabolic reprogramming of innate immune cells is emerging as a key player in the progression of a number of chronic diseases, including atherosclerosis, where high rates of glycolysis correlate with plaque instability. This study aimed to investigate if cholesterol crystals, which are key atherosclerosis-associated DAMPs (damage/danger-associated molecular patterns), alter immune cell metabolism and whether this, in turn, impacts on macrophage phenotype and function. Methods and ResultsPrimary human macrophages were treated with cholesterol crystals and expression of M1 (CXCL9, CXCL10) and M2-associated (MRC1, CCL13) macrophage markers, alarmins, and inflammatory cytokines were assessed either by real-time PCR or ELISA. Cholesterol crystal-induced changes in glycolytic markers were determined using real-time PCR and western blotting, while changes in cellular respiration and mitochondrial dynamics were examined via Seahorse analysis, Fluorescence Lifetime Imaging Microscopy (FLIM) and confocal microscopy. Treatment of macrophages with cholesterol crystals upregulated mRNA levels of CXCL9 and CXCL10, while concomitantly downregulating expression of MRC1 and CCL13. Cholesterol crystal--treated macrophages also exhibited a significant shift in metabolism to favour glycolysis, accompanied by the expression of key glycolytic markers GLUT1, Hexokinase 2, HIF1α, GAPDH and PFKFB3. Furthermore, we show that these effects are mediated upstream by the glycolytic enzyme, PKM2, and that direct inhibition of glycolysis or PKM2 nuclear localisation leads to a significant reduction in cholesterol crystal-induced inflammatory readouts. ConclusionsThis study not only provides further insight into how atherosclerosis-associated DAMPs impact on immune cell function, but also highlights metabolic reprogramming as a potential therapeutic target for cholesterol crystal-related inflammation.

3,5,3'-Triiodothyronine-Loaded Liposomes Inhibit Hepatocarcinogenesis Via Inflammation-Associated Macrophages.

BackgroundHepatocellular carcinoma (HCC) is inflammation-related cancer. Persistent inflammatory injury of the liver is an important factor mediating the occurrence and development of liver cancer. Hepatic macrophages play an important role in the inflammatory microenvironment, which mediates tumor immune escape, tumor growth, and metastasis. Previous studies have suggested that L-3,5,3-triiodothyronine (T3) can regulate inflammation; however, its use is associated with serious cardiac side effects, and its role in hepatocarcinogenesis remains unclear. In this study, we aimed to develop an effective T3 delivery system with reduced cardiac toxicity and to explore its effects on HCC occurrence.MethodsT3 liposomes (T3-lipo) were prepared using the thin-film hydration method, and their characteristics, including particle size, polydispersity index, zeta potential, encapsulation efficiency, drug loading, drug release, and stability, were evaluated in vitro. We assessed the effect of T3-lipo on hepatocarcinogenesis in diethylnitrosamine (DEN)–induced primary HCC in rats and examined the biodistribution of T3 and T3-lipo by high-performance liquid chromatography–mass spectrometry. Furthermore, we explored the potential molecular mechanism of T3-lipo in hepatocarcinogenesis by immunohistochemistry and immunofluorescence analyses, Bio-Plex assays, real-time polymerase chain reaction analysis, and Western blotting assays.ResultsCompared with T3, T3-lipo had an enhanced inhibitory effect on hepatocarcinogenesis and reduced cardiac side effects in DEN-induced primary HCC in rats. Mechanistically, T3-lipo were absorbed by hepatic macrophages and regulated the secretion of inflammatory cytokines in macrophages by inhibiting inflammatory signaling pathways.ConclusionsT3-lipo may suppress hepatocarcinogenesis by regulating the inflammatory microenvironment in the liver and reduce the cardiac side effects meanwhile.

MED1 Deficiency in Macrophages Aggravates Isoproterenol-Induced Cardiac Fibrosis in Mice

Mediator 1 (MED1), a key subunit of the mediator complex, interacts with various nuclear receptors and functions in lipid metabolism and energy homeostasis. Dilated cardiomyopathy-related ventricular dilatation and heart failure have been reported in mice with cardiomyocyte-specific Med1 deficiency. However, the contribution of macrophage-specific MED1 in cardiac remodeling remains unclear. In this study, macrophage-specific Med1 knockout (Med1ΔMac) mice were generated and exposed to isoproterenol (ISO) to induce cardiac fibrosis; these mice showed aggravated cardiac fibrosis compared with Med1fl/fl mice. The levels of expression of marker genes for myofibroblast transdifferentiation [α-smooth muscle actin (SMA)] and of profibrotic genes, including Col1a1, Col3a1, Postn, Mmp2, Timp1, and Fn1, were significantly increased in the cardiac tissues of Med1ΔMac mice with ISO-induced myocardial fibrosis. In particular, the transforming growth factor (TGF)-β-Smad2/3 signaling pathway was activated. In bone marrow-derived and peritoneal macrophages, Med1 deficiency was also associated with elevated levels of expression of proinflammatory genes, including Il6, Tnfa, and Il1b. These findings indicate that macrophage-specific MED1 deficiency may aggravate ISO-induced cardiac fibrosis via the regulation of the TGF-β-SMAD2/3 pathway, and the underlying mechanism may involve MED1 deficiency triggering the activation of inflammatory cytokines in macrophages, which in turn may stimulate phenotypic switch of cardiac fibroblasts and accelerate cardiac fibrosis. Thus, MED1 is a potential therapeutic target for cardiac fibrosis.

RANKL Impairs the TLR4 Pathway by Increasing TRAF6 and RANK Interaction in Macrophages.

High serum levels of osteoprotegerin (OPG) are found in patients with obesity, type 2 diabetes, sepsis, or septic shock and are associated with a high mortality rate in stroke. The primary known function of OPG is to bind to the receptor activator of NF-κB ligand (RANKL), and by doing so, it inhibits the binding between RANKL and its receptor (RANK). TLR4 signaling in macrophages involves TRAF6 recruitment and contributes to low-grade chronic inflammation in adipose tissue. LPS is a classical activator of the TLR4 pathway and induces the expression of inflammatory cytokines in macrophages. We have previously observed that in the presence of RANKL, there is no LPS-induced activation of TLR4 in macrophages. In this study, we investigated the crosstalk between RANK and TLR4 pathways in macrophages stimulated with both RANKL and LPS to unveil the role of OPG in inflammatory processes. We found that RANKL inhibits TLR4 activation by binding to RANK, promoting the binding between TRAF6 and RANK, lowering TLR4 activation and the expression of proinflammatory mediators. Furthermore, high OPG levels aggravate inflammation by inhibiting RANKL. Our findings elect RANKL as a candidate for drug development as a way to mitigate the impact of obesity-induced inflammation in patients.

Mild inflammation persists in the glenohumeral joint of patients with shoulder instability: Cross-sectional study

ObjectiveApproximately two-thirds of patients with history of shoulder dislocation may develop osteoarthritis (OA) of the glenohumeral joint. However, the biochemical mechanisms underlying the association between dislocation and OA are largely unknown. This study aimed to investigate macrophage markers and inflammatory cytokine expression associated with shoulder instability (SI) in comparison to rotator cuff tears (RCTs). DesignThis study included 30 patients with SI and 30 patients with RCTs. Synovial membrane samples were harvested from the rotator interval during the arthroscopic anatomical repair for both groups. The localization of tumor necrosis factor-α (TNF-α), interleukin (IL)-1β, and cluster of differentiation (CD) 68 in synovial membranes was determined by immunohistochemistry. Transcript-level expressions of the inflammatory cytokines (TNFA and IL1B) and macrophage markers pan-CD68 and -M1 (CD80 and CD86) were quantified. CD80 and CD86 expression in macrophages from the SI group was confirmed using flow cytometry. ResultsTNF-α, IL-1β, and CD68 were expressed in the synovial lining layer of the synovial tissue in both groups. In addition, the mRNA expressions of TNFA, IL1B, CD68, and CD80 were significantly higher in the SI group compared to the RCT group (P ​= ​0.012, 0.014, 0.022, 0.003, respectively). In samples from the SI group, 96.3% of CD68+/CD14+ macrophages were CD86-positive, whereas 2.5% of CD68+/CD14+/CD86+ cells were CD80-positive. ConclusionsPatients with SI had higher mRNA levels of TNFA, IL1B, CD68, and CD80 than those with RCTs. These findings may partially explain the biochemical mechanism underlying the frequent development and progression of osteoarthritis in patients with SI.

Beneficial Activities of Alisma orientale Extract in a Western Diet-Induced Murine Non-Alcoholic Steatohepatitis and Related Fibrosis Model via Regulation of the Hepatic Adiponectin and Farnesoid X Receptor Pathways.

The hepatic adiponectin and farnesoid X receptor (FXR) signaling pathways play multiple roles in modulating lipid and glucose metabolism, reducing hepatic inflammation and fibrosis, and altering various metabolic targets for the management of non-alcoholic fatty liver disease (NAFLD). Alisma orientale (AO, Ze xie in Chinese and Taeksa in Korean) is an herbal plant whose tubers are enriched with triterpenoids, which have been reported to exhibit various bioactive properties associated with NAFLD. Here, the present study provides a preclinical evaluation of the biological functions and related signaling pathways of AO extract for the treatment of NAFLD in a Western diet (WD)-induced mouse model. The findings showed that AO extract significantly reversed serum markers (liver function, lipid profile, and glucose) and improved histological features in the liver sections of mice fed WD for 52 weeks. In addition, it also reduced hepatic expression of fibrogenic markers in liver tissue and decreased the extent of collagen-positive areas, as well as inhibited F4/80 macrophage aggregation and inflammatory cytokine secretion. The activation of adiponectin and FXR expression in hepatic tissue may be a major mechanistic signaling cascade supporting the promising role of AO in NAFLD pharmacotherapy. Collectively, our results demonstrated that AO extract improves non-alcoholic steatohepatitis (NASH) resolution, particularly with respect to NASH-related fibrosis, along with the regulation of liver enzymes, postprandial hyperglycemia, hyperlipidemia, and weight loss, probably through the modulation of the hepatic adiponectin and FXR pathways.

Inhibition of protein kinase C alpha attenuates lipopolysaccharide-triggered acute lung injury by alleviating the hyperinflammatory response and oxidative stress.

BackgroundThe currently available treatment methods are ineffective in reducing mortality or improving outcomes in acute lung injury (ALI). The activation of protein kinase C alpha (PKCα) has recently been implicated in ALI development. We explored the potential therapeutic outcomes of PKCα inhibition in cases of ALI and to elucidate the related mechanisms.MethodsIndexes of lung inflammation and injury were examined in lipopolysaccharide (LPS)-treated C57BL/6J mice (male) and macrophages after pretreatment with a PKCα inhibitor. Tissues were collected to assess lung injury by hematoxylin and eosin (H&E) staining. Bronchoalveolar lavage fluid was used to measure the pulmonary edema, hyperinflammatory response, and oxidative stress by bicinchoninic acid (BCA) method and enzyme-linked immunosorbent assay (ELISA). We tested the effect of PKCα inhibition on LPS-induced proliferation, cytotoxicity, oxidative damage, and the release of inflammatory cytokines in macrophages using the Cell Counting Kit-8 (CCK-8) and lactate dehydrogenase (LDH) cytotoxicity assay kit, flow cytometry, quantitative reverse-transcription polymerase chain reaction (qRT-PCR), and ELISA. The nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) signaling pathway related proteins were detected by Western blot, immunohistochemistry (IHC), and immunofluorescence staining.ResultsWe observed that LPS upregulated PKCα phosphorylation, induced a hyperinflammatory response, and caused lung injury. However, PKCα inhibition effectively attenuated the changes caused by LPS. Moreover, we confirmed that inhibiting PKCα weakened the activity of the NF-κB pathway under LPS-induced ALI. These findings indicated that inhibition of PKCα is protective against LPS-induced hyperinflammatory response in ALI, this effect is likely to attributed to the downregulation of NF-κB signaling pathways.ConclusionsThe results showed that PKCα inhibition could attenuate ALI which may closely related to its anti-inflammatory and anti-oxidative effects.

Improving dexamethasone drug loading and efficacy in treating arthritis through a lipophilic prodrug entrapped into PLGA-PEG nanoparticles.

Targeted delivery of dexamethasone to inflamed tissues using nanoparticles is much-needed to improve its efficacy while reducing side effects. To drastically improve dexamethasone loading and prevent burst release once injected intravenously, a lipophilic prodrug dexamethasone palmitate (DXP) was encapsulated into poly(DL-lactide-co-glycolide)-polyethylene glycol (PLGA-PEG) nanoparticles (NPs). DXP-loaded PLGA-PEG NPs (DXP-NPs) of about 150 nm with a drug loading as high as 7.5% exhibited low hemolytic profile and cytotoxicity. DXP-NPs were able to inhibit the LPS-induced release of inflammatory cytokines in macrophages. After an intravenous injection to mice, dexamethasone (DXM) pharmacokinetic profile was also significantly improved. The concentration of DXM in the plasma of healthy mice remained high up to 18 h, much longer than the commercial soluble drug dexamethasone phosphate (DSP). Biodistribution studies showed lower DXM concentrations in the liver, kidneys, and lungs when DXP-NPs were administered as compared with the soluble drug. Histology analysis revealed an improvement in the knee structure and reduction of cell infiltration in animals treated with the encapsulated DXP compared with the soluble DSP or non-treated animals. In summary, the encapsulation of a lipidic prodrug of dexamethasone into PLGA-PEG NPs appears as a promising strategy to improve the pharmacological profile and reduce joint inflammation in a murine model of rheumatoid arthritis.Graphical abstract Supplementary informationThe online version contains supplementary material available at 10.1007/s13346-021-01112-3.

BET protein inhibitor apabetalone represses Porphyromonas gingivalis LPS-induced macrophage M1 polarization via regulating miR-130a/STAT3 axis

Periodontitis is a frequent chronic inflammatory disorder destroying periodontium. Recent studies have revealed the role of bromodomain and extraterminal domain inhibitor (BETi) and microRNA (miR)-130a in regulating macrophage polarization and pro-inflammatory response. However, little is known about whether apabetalone (a novel BETi) and miR-130a are correlated with chronic inflammatory state in periodontitis by regulating macrophage polarization. Here murine RAW264.7 macrophages were applied as an in vitro inflammatory model. After treatment with Porphyromonas gingivalis-derived lipopolysaccharide (Pg LPS) and apabetalone, the expression of macrophage M1 polarization markers and inflammatory cytokines was assessed using real-time PCR, western blot, and enzyme-linked immuno sorbent assay (ELISA). MiR-130a level was assessed using real-time PCR, and the target gene was identified using dual luciferase reporter assay. We demonstrated that apabetalone repressed Pg LPS-induced macrophage M1 polarization in a dose-dependent manner, as evidenced by decreased expression of inducible nitric oxide synthase (iNOS), CD86, and pro-inflammatory cytokines, and increased expression of Arg-1 and CD206. Mechanistically, Pg LPS increased miR-130a expression in macrophages, whereas apabetalone treatment repressed the effect. Functionally, forced expression of miR-130a promoted macrophage M1 polarization, and signal transducer and activator of transcription (STAT)-3 was the direct target gene of miR-130a in the process. Taken together, apabetalone decreases Pg LPS-induced macrophage M1 polarization via regulating miR-130a-3p/STAT3 axis, and may be a promising target for the clinical management of periodontitis.